{ "PMC5173035": { "annotations": [ { "sid": 0, "sent": "Biochemical and structural characterization of a DNA N6-adenine methyltransferase from Helicobacter pylori", "section": "TITLE", "ner": [ [ 0, 43, "Biochemical and structural characterization", "experimental_method" ], [ 49, 81, "DNA N6-adenine methyltransferase", "protein_type" ], [ 87, 106, "Helicobacter pylori", "species" ] ] }, { "sid": 1, "sent": "DNA N6-methyladenine modification plays an important role in regulating a variety of biological functions in bacteria.", "section": "ABSTRACT", "ner": [ [ 0, 20, "DNA N6-methyladenine", "ptm" ], [ 109, 117, "bacteria", "taxonomy_domain" ] ] }, { "sid": 2, "sent": "However, the mechanism of sequence-specific recognition in N6-methyladenine modification remains elusive.", "section": "ABSTRACT", "ner": [ [ 59, 75, "N6-methyladenine", "ptm" ] ] }, { "sid": 3, "sent": "M1.HpyAVI, a DNA N6-adenine methyltransferase from Helicobacter pylori, shows more promiscuous substrate specificity than other enzymes.", "section": "ABSTRACT", "ner": [ [ 0, 9, "M1.HpyAVI", "protein" ], [ 13, 45, "DNA N6-adenine methyltransferase", "protein_type" ], [ 51, 70, "Helicobacter pylori", "species" ] ] }, { "sid": 4, "sent": "Here, we present the crystal structures of cofactor-free and AdoMet-bound structures of this enzyme, which were determined at resolutions of 3.0 \u00c5 and 3.1 \u00c5, respectively.", "section": "ABSTRACT", "ner": [ [ 21, 39, "crystal structures", "evidence" ], [ 43, 56, "cofactor-free", "protein_state" ], [ 61, 73, "AdoMet-bound", "protein_state" ], [ 74, 84, "structures", "evidence" ] ] }, { "sid": 5, "sent": "The core structure of M1.HpyAVI resembles the canonical AdoMet-dependent MTase fold, while the putative DNA binding regions considerably differ from those of the other MTases, which may account for the substrate promiscuity of this enzyme.", "section": "ABSTRACT", "ner": [ [ 22, 31, "M1.HpyAVI", "protein" ], [ 56, 78, "AdoMet-dependent MTase", "protein_type" ], [ 104, 123, "DNA binding regions", "site" ], [ 168, 174, "MTases", "protein_type" ] ] }, { "sid": 6, "sent": "Site-directed mutagenesis experiments identified residues D29 and E216 as crucial amino acids for cofactor binding and the methyl transfer activity of the enzyme, while P41, located in a highly flexible loop, playing a determinant role for substrate specificity.", "section": "ABSTRACT", "ner": [ [ 0, 25, "Site-directed mutagenesis", "experimental_method" ], [ 58, 61, "D29", "residue_name_number" ], [ 66, 70, "E216", "residue_name_number" ], [ 123, 129, "methyl", "chemical" ], [ 169, 172, "P41", "residue_name_number" ], [ 187, 202, "highly flexible", "protein_state" ], [ 203, 207, "loop", "structure_element" ] ] }, { "sid": 7, "sent": "Taken together, our data revealed the structural basis underlying DNA N6-adenine methyltransferase substrate promiscuity.", "section": "ABSTRACT", "ner": [ [ 66, 98, "DNA N6-adenine methyltransferase", "protein_type" ] ] }, { "sid": 8, "sent": "DNA methylation is a common form of modification on nucleic acids occurring in both prokaryotes and eukaryotes.", "section": "INTRO", "ner": [ [ 0, 15, "DNA methylation", "ptm" ], [ 84, 95, "prokaryotes", "taxonomy_domain" ], [ 100, 110, "eukaryotes", "taxonomy_domain" ] ] }, { "sid": 9, "sent": "Such a modification creates a signature motif recognized by DNA-interacting proteins and functions as a mechanism to regulate gene expression.", "section": "INTRO", "ner": [ [ 60, 63, "DNA", "chemical" ] ] }, { "sid": 10, "sent": "DNA methylation is mediated by DNA methyltransferases (MTases), which catalyze the transfer of a methyl group from S-adenosyl-L- methionine (AdoMet) to a given position of a particular DNA base within a specific DNA sequence.", "section": "INTRO", "ner": [ [ 0, 15, "DNA methylation", "ptm" ], [ 31, 53, "DNA methyltransferases", "protein_type" ], [ 55, 61, "MTases", "protein_type" ], [ 97, 103, "methyl", "chemical" ], [ 115, 139, "S-adenosyl-L- methionine", "chemical" ], [ 141, 147, "AdoMet", "chemical" ], [ 185, 188, "DNA", "chemical" ], [ 212, 215, "DNA", "chemical" ] ] }, { "sid": 11, "sent": "Three classes of DNA MTases have been identified to transfer a methyl group to different positions of DNA bases.", "section": "INTRO", "ner": [ [ 17, 27, "DNA MTases", "protein_type" ], [ 63, 69, "methyl", "chemical" ], [ 102, 105, "DNA", "chemical" ] ] }, { "sid": 12, "sent": "C5-cytosine MTases, for example, methylate C5 of cytosine (m5C).", "section": "INTRO", "ner": [ [ 0, 18, "C5-cytosine MTases", "protein_type" ], [ 49, 57, "cytosine", "residue_name" ], [ 59, 62, "m5C", "ptm" ] ] }, { "sid": 13, "sent": "In eukaryotes, m5C plays an important role in gene expression, chromatin organization, genome maintenance and parental imprinting, and is involved in a variety of human diseases including cancer.", "section": "INTRO", "ner": [ [ 3, 13, "eukaryotes", "taxonomy_domain" ], [ 15, 18, "m5C", "ptm" ], [ 163, 168, "human", "species" ] ] }, { "sid": 14, "sent": "By contrast, the functions of the prokaryotic DNA cytosine MTase remain unknown.", "section": "INTRO", "ner": [ [ 34, 45, "prokaryotic", "taxonomy_domain" ], [ 46, 64, "DNA cytosine MTase", "protein_type" ] ] }, { "sid": 15, "sent": "N4-cytosine MTases, which are frequently present in thermophilic or mesophilic bacteria, transfer a methyl group to the exocyclic amino group of cytosine (4mC).", "section": "INTRO", "ner": [ [ 0, 18, "N4-cytosine MTases", "protein_type" ], [ 52, 64, "thermophilic", "taxonomy_domain" ], [ 68, 78, "mesophilic", "taxonomy_domain" ], [ 79, 87, "bacteria", "taxonomy_domain" ], [ 100, 106, "methyl", "chemical" ], [ 145, 153, "cytosine", "residue_name" ], [ 155, 158, "4mC", "ptm" ] ] }, { "sid": 16, "sent": "N4 methylation seems to be primarily a component of bacterial immune system against invasion by foreign DNA, such as conjugative plasmids and bacteriophages.", "section": "INTRO", "ner": [ [ 0, 14, "N4 methylation", "ptm" ], [ 52, 61, "bacterial", "taxonomy_domain" ], [ 104, 107, "DNA", "chemical" ], [ 142, 156, "bacteriophages", "taxonomy_domain" ] ] }, { "sid": 17, "sent": "The third group, N6-adenine MTases methylate the exocyclic amino groups of adenine (6mA), which exists in prokaryotes as a signal for genome defense, DNA replication and repair, regulation of gene expression, control of transposition and host-pathogen interactions.", "section": "INTRO", "ner": [ [ 17, 34, "N6-adenine MTases", "protein_type" ], [ 75, 82, "adenine", "residue_name" ], [ 84, 87, "6mA", "ptm" ], [ 106, 117, "prokaryotes", "taxonomy_domain" ], [ 150, 153, "DNA", "chemical" ] ] }, { "sid": 18, "sent": "Recent studies utilizing new sequencing approaches have showed the existence of 6mA in several eukaryotic species.", "section": "INTRO", "ner": [ [ 80, 83, "6mA", "ptm" ], [ 95, 105, "eukaryotic", "taxonomy_domain" ] ] }, { "sid": 19, "sent": "DNA 6mA modification is associated with important biological processes including nucleosome distribution close to the transcription start sites in Chlamydomonas, carrying heritable epigenetic information in C.elegans or controlling development of Drosophila.", "section": "INTRO", "ner": [ [ 0, 3, "DNA", "chemical" ], [ 4, 7, "6mA", "ptm" ], [ 147, 160, "Chlamydomonas", "taxonomy_domain" ], [ 207, 216, "C.elegans", "species" ], [ 247, 257, "Drosophila", "taxonomy_domain" ] ] }, { "sid": 20, "sent": "All the three types of methylation exist in prokaryotes, and most DNA MTases are components of the restriction-modification (R-M) systems.", "section": "INTRO", "ner": [ [ 23, 34, "methylation", "ptm" ], [ 44, 55, "prokaryotes", "taxonomy_domain" ], [ 66, 76, "DNA MTases", "protein_type" ] ] }, { "sid": 21, "sent": "\u201cR\u201d stands for a restriction endonuclease cleaving specific DNA sequences, while \u201cM\u201d symbolizes a modification methyltransferase rendering these sequences resistant to cleavage.", "section": "INTRO", "ner": [ [ 17, 41, "restriction endonuclease", "protein_type" ], [ 60, 63, "DNA", "chemical" ], [ 98, 128, "modification methyltransferase", "protein_type" ] ] }, { "sid": 22, "sent": "The cooperation of these two enzymes provides a defensive mechanism to protect bacteria from infection by bacteriophages.", "section": "INTRO", "ner": [ [ 79, 87, "bacteria", "taxonomy_domain" ], [ 106, 120, "bacteriophages", "taxonomy_domain" ] ] }, { "sid": 23, "sent": "The R-M systems are classified into three types based on specific structural features, position of DNA cleavage and cofactor requirements.", "section": "INTRO", "ner": [ [ 99, 102, "DNA", "chemical" ] ] }, { "sid": 24, "sent": "In types I and III, the DNA adenine or cytosine methyltransferase is part of a multi-subunit enzyme that catalyzes both restriction and modification.", "section": "INTRO", "ner": [ [ 24, 65, "DNA adenine or cytosine methyltransferase", "protein_type" ] ] }, { "sid": 25, "sent": "By contrast, two separate enzymes exist in type II systems, where a restriction endonuclease and a DNA adenine or cytosine methyltransferase recognize the same targets.", "section": "INTRO", "ner": [ [ 68, 92, "restriction endonuclease", "protein_type" ], [ 99, 140, "DNA adenine or cytosine methyltransferase", "protein_type" ] ] }, { "sid": 26, "sent": "To date, a number of bacterial DNA MTases have been structurally characterized, covering enzymes from all the three classes.", "section": "INTRO", "ner": [ [ 21, 30, "bacterial", "taxonomy_domain" ], [ 31, 41, "DNA MTases", "protein_type" ], [ 52, 78, "structurally characterized", "experimental_method" ] ] }, { "sid": 27, "sent": "All these MTases exhibit high similarity in their overall architectures, which are generally folded into two domains: a conserved larger catalytic domain comprising an active site for methyl transfer and a site for AdoMet-binding, and a smaller target (DNA)-recognition domain (TRD) containing variable regions implicated in sequence-specific DNA recognition and the infiltration of the DNA to flip the target base.", "section": "INTRO", "ner": [ [ 10, 16, "MTases", "protein_type" ], [ 120, 129, "conserved", "protein_state" ], [ 137, 153, "catalytic domain", "structure_element" ], [ 168, 179, "active site", "site" ], [ 184, 190, "methyl", "chemical" ], [ 215, 221, "AdoMet", "chemical" ], [ 245, 276, "target (DNA)-recognition domain", "structure_element" ], [ 278, 281, "TRD", "structure_element" ], [ 343, 346, "DNA", "chemical" ], [ 387, 390, "DNA", "chemical" ] ] }, { "sid": 28, "sent": "Conserved amino acid motifs have been identified from reported structures, including ten motifs (I-X) in cytosine MTases and nine motifs (I-VIII and X) in adenine MTases, all of which are arranged in an almost constant order.", "section": "INTRO", "ner": [ [ 0, 9, "Conserved", "protein_state" ], [ 63, 73, "structures", "evidence" ], [ 97, 100, "I-X", "structure_element" ], [ 105, 120, "cytosine MTases", "protein_type" ], [ 138, 144, "I-VIII", "structure_element" ], [ 149, 150, "X", "structure_element" ], [ 155, 169, "adenine MTases", "protein_type" ] ] }, { "sid": 29, "sent": "According to the linear arrangement of three conserved domains, exocyclic amino MTases are subdivided into six groups (namely \u03b1, \u03b2, \u03b3, \u03b6, \u03b4 and \u03b5).", "section": "INTRO", "ner": [ [ 45, 54, "conserved", "protein_state" ], [ 64, 86, "exocyclic amino MTases", "protein_type" ], [ 126, 127, "\u03b1", "protein_type" ], [ 129, 130, "\u03b2", "protein_type" ], [ 132, 133, "\u03b3", "protein_type" ], [ 135, 136, "\u03b6", "protein_type" ], [ 138, 139, "\u03b4", "protein_type" ], [ 144, 145, "\u03b5", "protein_type" ] ] }, { "sid": 30, "sent": "N6-adenine and N4-cytosine MTases, in particular, are closely related by sharing common structural features.", "section": "RESULTS", "ner": [ [ 0, 33, "N6-adenine and N4-cytosine MTases", "protein_type" ], [ 0, 33, "N6-adenine and N4-cytosine MTases", "protein_type" ] ] }, { "sid": 31, "sent": "Despite the considerable similarity among bacterial MTases, some differences were observed among the enzymes from various species.", "section": "INTRO", "ner": [ [ 42, 51, "bacterial", "taxonomy_domain" ], [ 52, 58, "MTases", "protein_type" ] ] }, { "sid": 32, "sent": "For example, the structural regions of MTases beyond the catalytic domain are rather variable, such as the C-terminal domain of M.TaqI, the extended arm of M.MboIIA and M.RsrI, the helix bundle of EcoDam, and so on.", "section": "INTRO", "ner": [ [ 39, 45, "MTases", "protein_type" ], [ 57, 73, "catalytic domain", "structure_element" ], [ 107, 124, "C-terminal domain", "structure_element" ], [ 128, 134, "M.TaqI", "protein" ], [ 156, 164, "M.MboIIA", "protein" ], [ 169, 175, "M.RsrI", "protein" ], [ 181, 193, "helix bundle", "structure_element" ], [ 197, 203, "EcoDam", "protein" ] ] }, { "sid": 33, "sent": "DNA methylation is thought to influence bacterial virulence.", "section": "INTRO", "ner": [ [ 0, 15, "DNA methylation", "ptm" ], [ 40, 49, "bacterial", "taxonomy_domain" ] ] }, { "sid": 34, "sent": "DNA adenine methyltransferase has been shown to play a crucial role in colonization of deep tissue sites in Salmonella typhimurium and Aeromonas hydrophila.", "section": "INTRO", "ner": [ [ 0, 29, "DNA adenine methyltransferase", "protein_type" ], [ 108, 130, "Salmonella typhimurium", "species" ], [ 135, 155, "Aeromonas hydrophila", "species" ] ] }, { "sid": 35, "sent": "Importantly, DNA adenine methylation is a global regulator of genes expressed during infection and inhibitors of DNA adenine methylation are likely to have a broad antimicrobial action.", "section": "INTRO", "ner": [ [ 13, 36, "DNA adenine methylation", "ptm" ], [ 113, 136, "DNA adenine methylation", "ptm" ] ] }, { "sid": 36, "sent": "Dam was considered a promising target for antimicrobial drug development.", "section": "INTRO", "ner": [ [ 0, 3, "Dam", "protein_type" ] ] }, { "sid": 37, "sent": "Helicobacter pylori is a Gram-negative bacterium that persistently colonizes in human stomach worldwide.", "section": "INTRO", "ner": [ [ 0, 19, "Helicobacter pylori", "species" ], [ 25, 48, "Gram-negative bacterium", "taxonomy_domain" ], [ 80, 85, "human", "species" ] ] }, { "sid": 38, "sent": "H. pylori is involved in 90% of all gastric malignancies, infecting nearly 50% of the world's population and is the most crucial etiologic agent for gastric adenocarcinoma.", "section": "INTRO", "ner": [ [ 0, 9, "H. pylori", "species" ] ] }, { "sid": 39, "sent": "H. pylori strains possess a few R-M systems like other bacteria to function as defensive systems.", "section": "INTRO", "ner": [ [ 0, 9, "H. pylori", "species" ], [ 55, 63, "bacteria", "taxonomy_domain" ] ] }, { "sid": 40, "sent": "H. pylori 26695, for example, has 23 R-M systems.", "section": "INTRO", "ner": [ [ 0, 15, "H. pylori 26695", "species" ] ] }, { "sid": 41, "sent": "Methyltransferases were suggested to be involved in H. pylori pathogenicity.", "section": "INTRO", "ner": [ [ 0, 18, "Methyltransferases", "protein_type" ], [ 52, 61, "H. pylori", "species" ] ] }, { "sid": 42, "sent": "M1.HpyAVI is a DNA adenine MTase that belongs to the type II R-M system.", "section": "INTRO", "ner": [ [ 0, 9, "M1.HpyAVI", "protein" ], [ 15, 32, "DNA adenine MTase", "protein_type" ] ] }, { "sid": 43, "sent": "This system contains two DNA MTases named M1.HpyAVI and M2.HpyAVI, and a putative restriction enzyme.", "section": "INTRO", "ner": [ [ 25, 35, "DNA MTases", "protein_type" ], [ 42, 51, "M1.HpyAVI", "protein" ], [ 56, 65, "M2.HpyAVI", "protein" ], [ 82, 100, "restriction enzyme", "protein_type" ] ] }, { "sid": 44, "sent": "M1.HpyAVI encoded by ORF hp0050 is an N6-adenine methyltransferase belonging to the \u03b2-class MTase.", "section": "INTRO", "ner": [ [ 0, 9, "M1.HpyAVI", "protein" ], [ 25, 31, "hp0050", "gene" ], [ 38, 66, "N6-adenine methyltransferase", "protein_type" ], [ 84, 97, "\u03b2-class MTase", "protein_type" ] ] }, { "sid": 45, "sent": "It has been reported recently that this enzyme recognizes the sequence of 5\u2032-GAGG-3\u2032, 5\u2032-GGAG-3\u2032 or 5\u2032-GAAG-3\u2032 and methylates adenines in these sequences.", "section": "INTRO", "ner": [ [ 74, 85, "5\u2032-GAGG-3\u2032,", "chemical" ], [ 86, 96, "5\u2032-GGAG-3\u2032", "chemical" ], [ 100, 110, "5\u2032-GAAG-3\u2032", "chemical" ], [ 126, 134, "adenines", "residue_name" ] ] }, { "sid": 46, "sent": "Given that methylation of two adjacent adenines on the same strand have never been observed for other N6-adenine MTases, the methylation activity on 5\u2032-GAAG-3\u2032 seems to be a unique feature of M1.HpyAVI, compared with the homologs from other strains of H.pylori which is able to methylate only 5\u2032-GAGG-3\u2032. The structural basis and the catalytic mechanism underlying such a distinct activity are not well understood due to the lack of an available 3D structure of this enzyme.", "section": "INTRO", "ner": [ [ 11, 22, "methylation", "ptm" ], [ 39, 47, "adenines", "residue_name" ], [ 102, 119, "N6-adenine MTases", "protein_type" ], [ 125, 136, "methylation", "ptm" ], [ 149, 159, "5\u2032-GAAG-3\u2032", "chemical" ], [ 192, 201, "M1.HpyAVI", "protein" ], [ 252, 260, "H.pylori", "species" ], [ 293, 303, "5\u2032-GAGG-3\u2032", "chemical" ], [ 449, 458, "structure", "evidence" ] ] }, { "sid": 47, "sent": "Here, we report the crystal structure of M1.HpyAVI from H. pylori 26695, which is the first determined N6-adenine MTase structure in H. pylori.", "section": "INTRO", "ner": [ [ 20, 37, "crystal structure", "evidence" ], [ 41, 50, "M1.HpyAVI", "protein" ], [ 56, 71, "H. pylori 26695", "species" ], [ 103, 119, "N6-adenine MTase", "protein_type" ], [ 120, 129, "structure", "evidence" ], [ 133, 142, "H. pylori", "species" ] ] }, { "sid": 48, "sent": "The structure reveals a similar architecture as the canonical fold of homologous proteins, but displays several differences in the loop regions and TRD.", "section": "INTRO", "ner": [ [ 4, 13, "structure", "evidence" ], [ 131, 135, "loop", "structure_element" ], [ 148, 151, "TRD", "structure_element" ] ] }, { "sid": 49, "sent": "Based on structural and biochemical analyses, we then identified two conserved amino acids, D29 at the catalytic site and E216 close to the C-terminus, as crucial residues for cofactor binding and methyltransferase activity of M1.HpyAVI.", "section": "INTRO", "ner": [ [ 9, 44, "structural and biochemical analyses", "experimental_method" ], [ 69, 78, "conserved", "protein_state" ], [ 92, 95, "D29", "residue_name_number" ], [ 103, 117, "catalytic site", "site" ], [ 122, 126, "E216", "residue_name_number" ], [ 197, 214, "methyltransferase", "protein_type" ], [ 227, 236, "M1.HpyAVI", "protein" ] ] }, { "sid": 50, "sent": "In addition, a non-conserved amino acid, P41, seems to play a key role in substrate recognition.", "section": "INTRO", "ner": [ [ 15, 28, "non-conserved", "protein_state" ], [ 41, 44, "P41", "residue_name_number" ] ] }, { "sid": 51, "sent": "Overall structure", "section": "RESULTS", "ner": [ [ 8, 17, "structure", "evidence" ] ] }, { "sid": 52, "sent": "Recombinant full-length M1.HpyAVI was produced as a soluble protein in Escherichia coli, but was quite unstable and tended to aggregate in low salt environment.", "section": "RESULTS", "ner": [ [ 12, 23, "full-length", "protein_state" ], [ 24, 33, "M1.HpyAVI", "protein" ], [ 71, 87, "Escherichia coli", "species" ] ] }, { "sid": 53, "sent": "The protein, however, remained fully soluble in a buffer containing higher concentration of sodium chloride (>300 mM), which prompted that M1.HpyAVI is likely a halophilic protein.", "section": "RESULTS", "ner": [ [ 92, 107, "sodium chloride", "chemical" ], [ 139, 148, "M1.HpyAVI", "protein" ], [ 161, 171, "halophilic", "protein_state" ] ] }, { "sid": 54, "sent": "The cofactor-free and AdoMet-bound proteins were crystallized at different conditions.", "section": "RESULTS", "ner": [ [ 4, 17, "cofactor-free", "protein_state" ], [ 22, 34, "AdoMet-bound", "protein_state" ], [ 49, 61, "crystallized", "experimental_method" ] ] }, { "sid": 55, "sent": "Both structures were determined by means of molecular replacement, and refined to 3.0 \u00c5 and 3.1 \u00c5, respectively.", "section": "RESULTS", "ner": [ [ 5, 15, "structures", "evidence" ], [ 44, 65, "molecular replacement", "experimental_method" ] ] }, { "sid": 56, "sent": "Statistics of X-ray data collection and structure refinement were summarized in Table 1.", "section": "RESULTS", "ner": [ [ 14, 35, "X-ray data collection", "experimental_method" ], [ 40, 60, "structure refinement", "experimental_method" ] ] }, { "sid": 57, "sent": "Data collection and structure refinement statistics of M1.HpyAVI", "section": "TABLE", "ner": [ [ 20, 51, "structure refinement statistics", "evidence" ], [ 55, 64, "M1.HpyAVI", "protein" ] ] }, { "sid": 58, "sent": "\tM1.HpyAVI\tM1.HpyAVI-AdoMet complex\t \tData collection\t\t\t \tWavelength (\u00c5)\t1.0000\t0.97772\t \tSpace group\tP43212\tP65\t \tUnit-cell parameters (\u00c5, \u02da)\ta = b = 69.73, c = 532.75\u03b1 = \u03b2 = \u03b3 = 90\ta = b = 135.60, c = 265.15\u03b1 = \u03b2 = 90, \u03b3 = 120\t \tResolution range (\u00c5) a\t49.09-3.00 (3.09-3.00)\t48.91-3.10 (3.18-3.10)\t \tUnique reflections a\t27243\t49833\t \tMultiplicity a\t3.7 (3.8)\t5.6 (4.0)\t \tCompleteness (%)a\t98.7 (98.9)\t99.7 (97.8)\t \tMean I/\u03b4 (I) a\t12.1 (3.4)\t14.0 (1.9)\t \tSolvent content (%)\t58.67\t61.96\t \tRmergea\t0.073 (0.378)\t0.106 (0.769)\t \tStructure refinement\t\t\t \tRwork\t0.251\t0.221\t \tRfree\t0.308\t0.276\t \tR.m.s.d., bond lengths (\u00c5)\t0.007\t0.007\t \tR.m.s.d., bond angles (\u02da)\t1.408\t1.651\t \tRamachandran plot\t\t\t \tFavoured region (%)\t89.44\t91.44\t \tAllowed region (%)\t9.58\t7.11\t \tOutliers (%)\t0.99\t1.45\t \t", "section": "TABLE", "ner": [ [ 1, 10, "M1.HpyAVI", "protein" ], [ 11, 27, "M1.HpyAVI-AdoMet", "complex_assembly" ], [ 594, 601, "R.m.s.d", "evidence" ], [ 635, 642, "R.m.s.d", "evidence" ] ] }, { "sid": 59, "sent": "Four and eight protein monomers resided in the asymmetric units of the two crystal structures.", "section": "RESULTS", "ner": [ [ 23, 31, "monomers", "oligomeric_state" ], [ 75, 93, "crystal structures", "evidence" ] ] }, { "sid": 60, "sent": "Some amino acids, particularly those within two loops (residues 32-61 and 152-172) in both structures, were poorly defined in electron density and had to be omitted from the refined models.", "section": "RESULTS", "ner": [ [ 48, 53, "loops", "structure_element" ], [ 64, 69, "32-61", "residue_range" ], [ 74, 81, "152-172", "residue_range" ], [ 91, 101, "structures", "evidence" ], [ 126, 142, "electron density", "evidence" ] ] }, { "sid": 61, "sent": "The two structures are very similar to each other (Figure 1) and could be well overlaid with an RMSD of 0.76 \u00c5 on 191 C\u03b1 atoms.", "section": "RESULTS", "ner": [ [ 8, 18, "structures", "evidence" ], [ 96, 100, "RMSD", "evidence" ] ] }, { "sid": 62, "sent": "The overall architecture of M1.HpyAVI revealed in these structures resembles the AdoMet-dependent MTase fold in which a twisted seven-stranded \u03b2-sheet flanked by six \u03b1-helices forms the structural core.", "section": "RESULTS", "ner": [ [ 28, 37, "M1.HpyAVI", "protein" ], [ 56, 66, "structures", "evidence" ], [ 81, 103, "AdoMet-dependent MTase", "protein_type" ], [ 143, 150, "\u03b2-sheet", "structure_element" ], [ 166, 175, "\u03b1-helices", "structure_element" ] ] }, { "sid": 63, "sent": "Like the reported structures of the larger domain of MTases, three helices (\u03b1A, \u03b1B and \u03b1Z) are located at one face of the central \u03b2-sheet, while the other three \u03b1D, \u03b1E and \u03b1C sit at the other side.", "section": "RESULTS", "ner": [ [ 18, 28, "structures", "evidence" ], [ 53, 59, "MTases", "protein_type" ], [ 67, 74, "helices", "structure_element" ], [ 76, 78, "\u03b1A", "structure_element" ], [ 80, 82, "\u03b1B", "structure_element" ], [ 87, 89, "\u03b1Z", "structure_element" ], [ 130, 137, "\u03b2-sheet", "structure_element" ], [ 161, 163, "\u03b1D", "structure_element" ], [ 165, 167, "\u03b1E", "structure_element" ], [ 172, 174, "\u03b1C", "structure_element" ] ] }, { "sid": 64, "sent": "All these conserved structural motifs form a typical \u03b1/\u03b2 Rossmann fold.", "section": "RESULTS", "ner": [ [ 10, 19, "conserved", "protein_state" ], [ 53, 70, "\u03b1/\u03b2 Rossmann fold", "structure_element" ] ] }, { "sid": 65, "sent": "The catalytic motif DPPY lies in a loop connecting \u03b1D and \u03b24, and the cofactor AdoMet binds in a neighboring cavity.", "section": "RESULTS", "ner": [ [ 4, 19, "catalytic motif", "structure_element" ], [ 20, 24, "DPPY", "structure_element" ], [ 35, 39, "loop", "structure_element" ], [ 51, 53, "\u03b1D", "structure_element" ], [ 58, 60, "\u03b24", "structure_element" ], [ 79, 85, "AdoMet", "chemical" ], [ 109, 115, "cavity", "site" ] ] }, { "sid": 66, "sent": "The loop (residues 136-166) located between \u03b27 and \u03b1Z corresponds to a highly diverse region in other MTases that is involved in target DNA recognition.", "section": "RESULTS", "ner": [ [ 4, 8, "loop", "structure_element" ], [ 19, 26, "136-166", "residue_range" ], [ 44, 46, "\u03b27", "structure_element" ], [ 51, 53, "\u03b1Z", "structure_element" ], [ 71, 85, "highly diverse", "protein_state" ], [ 102, 108, "MTases", "protein_type" ], [ 136, 139, "DNA", "chemical" ] ] }, { "sid": 67, "sent": "The hairpin loop (residues 101-133) bridging \u03b26 and \u03b27, which is proposed to bind DNA in the minor groove, displays a similar conformation as those observed in M.MboIIA, M.RsrI and M.pvuII.", "section": "RESULTS", "ner": [ [ 4, 16, "hairpin loop", "structure_element" ], [ 27, 34, "101-133", "residue_range" ], [ 45, 47, "\u03b26", "structure_element" ], [ 52, 54, "\u03b27", "structure_element" ], [ 82, 85, "DNA", "chemical" ], [ 93, 105, "minor groove", "structure_element" ], [ 160, 168, "M.MboIIA", "protein" ], [ 170, 176, "M.RsrI", "protein" ], [ 181, 188, "M.pvuII", "protein" ] ] }, { "sid": 68, "sent": "The missing loop (residues 33-58) in the structure of M1.HpyAVI corresponds to loop I in M.TaqI, which was also invisible in a structure without DNA.", "section": "RESULTS", "ner": [ [ 4, 11, "missing", "protein_state" ], [ 12, 16, "loop", "structure_element" ], [ 27, 32, "33-58", "residue_range" ], [ 41, 50, "structure", "evidence" ], [ 54, 63, "M1.HpyAVI", "protein" ], [ 79, 85, "loop I", "structure_element" ], [ 89, 95, "M.TaqI", "protein" ], [ 127, 136, "structure", "evidence" ], [ 137, 148, "without DNA", "protein_state" ] ] }, { "sid": 69, "sent": "This loop, however, was well ordered in an M.TaqI-DNA complex structure and was shown to play a crucial role in DNA methylation by contacting the flipping adenine and recognizing specific DNA sequence.", "section": "RESULTS", "ner": [ [ 5, 9, "loop", "structure_element" ], [ 24, 36, "well ordered", "protein_state" ], [ 43, 71, "M.TaqI-DNA complex structure", "evidence" ], [ 112, 127, "DNA methylation", "ptm" ], [ 155, 162, "adenine", "residue_name" ], [ 188, 191, "DNA", "chemical" ] ] }, { "sid": 70, "sent": "Overall structure of M1.HpyAVI", "section": "FIG", "ner": [ [ 8, 17, "structure", "evidence" ], [ 21, 30, "M1.HpyAVI", "protein" ] ] }, { "sid": 71, "sent": "A. Free form B. AdoMet-bound form.", "section": "FIG", "ner": [ [ 3, 7, "Free", "protein_state" ], [ 16, 28, "AdoMet-bound", "protein_state" ] ] }, { "sid": 72, "sent": "Ribbon diagram of M1.HpyAVI resembles an \u201cAdoMet-dependent MTase fold\u201d, a mixed seven-stranded \u03b2-sheet flanked by six \u03b1-helices, \u03b1A, \u03b1B, \u03b1Z on one side and \u03b1D, \u03b1E, \u03b1C on the other side, the cofactor AdoMet is bound in a cavity near the conserved enzyme activity motif DPPY.", "section": "FIG", "ner": [ [ 18, 27, "M1.HpyAVI", "protein" ], [ 42, 64, "AdoMet-dependent MTase", "protein_type" ], [ 95, 102, "\u03b2-sheet", "structure_element" ], [ 118, 127, "\u03b1-helices", "structure_element" ], [ 129, 131, "\u03b1A", "structure_element" ], [ 133, 135, "\u03b1B", "structure_element" ], [ 137, 139, "\u03b1Z", "structure_element" ], [ 156, 158, "\u03b1D", "structure_element" ], [ 160, 162, "\u03b1E", "structure_element" ], [ 164, 166, "\u03b1C", "structure_element" ], [ 199, 205, "AdoMet", "chemical" ], [ 209, 217, "bound in", "protein_state" ], [ 220, 226, "cavity", "site" ], [ 236, 245, "conserved", "protein_state" ], [ 268, 272, "DPPY", "structure_element" ] ] }, { "sid": 73, "sent": "The \u03b1-helices and \u03b2-strands are labelled and numbered according to the commonly numbering rule for the known MTases.", "section": "FIG", "ner": [ [ 4, 13, "\u03b1-helices", "structure_element" ], [ 18, 27, "\u03b2-strands", "structure_element" ], [ 109, 115, "MTases", "protein_type" ] ] }, { "sid": 74, "sent": "The AdoMet molecule is shown in green.", "section": "FIG", "ner": [ [ 4, 10, "AdoMet", "chemical" ] ] }, { "sid": 75, "sent": "Dimeric state of M1.HpyAVI in crystal and solution", "section": "RESULTS", "ner": [ [ 0, 7, "Dimeric", "oligomeric_state" ], [ 17, 26, "M1.HpyAVI", "protein" ], [ 30, 37, "crystal", "evidence" ], [ 42, 50, "solution", "experimental_method" ] ] }, { "sid": 76, "sent": "Previous studies showed that some DNA MTases, e.g. M.BamHI and M.EcoRI, exist as monomer in solution, in agreement with the fact that a DNA substrate for a typical MTase is hemimethylated and therefore needs only a single methylation event to convert it into a fully methylated state.", "section": "RESULTS", "ner": [ [ 34, 44, "DNA MTases", "protein_type" ], [ 51, 58, "M.BamHI", "protein" ], [ 63, 70, "M.EcoRI", "protein" ], [ 81, 88, "monomer", "oligomeric_state" ], [ 136, 139, "DNA", "chemical" ], [ 164, 169, "MTase", "protein_type" ], [ 173, 187, "hemimethylated", "protein_state" ], [ 222, 233, "methylation", "ptm" ], [ 261, 277, "fully methylated", "protein_state" ] ] }, { "sid": 77, "sent": "Increasing number of dimeric DNA MTases, however, has been identified from later studies.", "section": "RESULTS", "ner": [ [ 21, 28, "dimeric", "oligomeric_state" ], [ 29, 39, "DNA MTases", "protein_type" ] ] }, { "sid": 78, "sent": "For instance, M.DpnII, M.RsrI, M.KpnI, and M.MboIIA have been found as dimers in solution.", "section": "RESULTS", "ner": [ [ 14, 21, "M.DpnII", "protein" ], [ 23, 29, "M.RsrI", "protein" ], [ 31, 37, "M.KpnI", "protein" ], [ 43, 51, "M.MboIIA", "protein" ], [ 71, 77, "dimers", "oligomeric_state" ] ] }, { "sid": 79, "sent": "In addition, several MTases including M.MboIIA, M.RsrI and TTH0409 form tightly associated dimers in crystal structures.", "section": "RESULTS", "ner": [ [ 21, 27, "MTases", "protein_type" ], [ 38, 46, "M.MboIIA", "protein" ], [ 48, 54, "M.RsrI", "protein" ], [ 59, 66, "TTH0409", "protein" ], [ 91, 97, "dimers", "oligomeric_state" ], [ 101, 119, "crystal structures", "evidence" ] ] }, { "sid": 80, "sent": "Nonetheless, some DNA MTases such as M.CcrMI and the Bacillus amyloliquefaciens MTase dissociate from dimer into monomer upon DNA-binding.", "section": "RESULTS", "ner": [ [ 18, 28, "DNA MTases", "protein_type" ], [ 37, 44, "M.CcrMI", "protein" ], [ 53, 79, "Bacillus amyloliquefaciens", "species" ], [ 80, 85, "MTase", "protein_type" ], [ 102, 107, "dimer", "oligomeric_state" ], [ 113, 120, "monomer", "oligomeric_state" ], [ 126, 129, "DNA", "chemical" ] ] }, { "sid": 81, "sent": "According to the arrangement of the three conserved domains, M1.HpyAVI belongs to the \u03b2-subgroup, in which a conserved motif NXXTX9\u221211AXRXFSXXHX4WX6\u22129 YXFXLX3RX9\u221226NPX1\u22126NVWX29\u221234A has been identified at the dimerization interface in crystal structures.", "section": "RESULTS", "ner": [ [ 42, 51, "conserved", "protein_state" ], [ 61, 70, "M1.HpyAVI", "protein" ], [ 86, 96, "\u03b2-subgroup", "protein_type" ], [ 109, 118, "conserved", "protein_state" ], [ 125, 180, "NXXTX9\u221211AXRXFSXXHX4WX6\u22129 YXFXLX3RX9\u221226NPX1\u22126NVWX29\u221234A", "structure_element" ], [ 208, 230, "dimerization interface", "site" ], [ 234, 252, "crystal structures", "evidence" ] ] }, { "sid": 82, "sent": "Most of conserved amino acids within that motif are present in the sequence of M1.HpyAVI (Figure 2A), implying dimerization of this protein.", "section": "RESULTS", "ner": [ [ 8, 17, "conserved", "protein_state" ], [ 79, 88, "M1.HpyAVI", "protein" ], [ 111, 123, "dimerization", "oligomeric_state" ] ] }, { "sid": 83, "sent": "In agreement, a dimer of M1.HpyAVI was observed in our crystal structures with the two monomers related by a two-fold axis (Figure 2B and 2C).", "section": "RESULTS", "ner": [ [ 16, 21, "dimer", "oligomeric_state" ], [ 25, 34, "M1.HpyAVI", "protein" ], [ 55, 73, "crystal structures", "evidence" ], [ 87, 95, "monomers", "oligomeric_state" ] ] }, { "sid": 84, "sent": "An area of ~1900 \u00c52 was buried at the dimeric interface, taking up ca 17% of the total area.", "section": "RESULTS", "ner": [ [ 38, 55, "dimeric interface", "site" ] ] }, { "sid": 85, "sent": "The dimeric architecture was greatly stabilized by hydrogen bonds and salt bridges formed among residues R86, D93 and E96.", "section": "RESULTS", "ner": [ [ 4, 11, "dimeric", "oligomeric_state" ], [ 51, 65, "hydrogen bonds", "bond_interaction" ], [ 70, 82, "salt bridges", "bond_interaction" ], [ 105, 108, "R86", "residue_name_number" ], [ 110, 113, "D93", "residue_name_number" ], [ 118, 121, "E96", "residue_name_number" ] ] }, { "sid": 86, "sent": "In addition, comparison of the dimer structure of M1.HpyAVI with some other \u03b2-class MTases (M1.MboIIA, M.RsrI and TTHA0409) suggested that the M1.HpyAVI dimer organized in a similar form as others (Figure S3).", "section": "RESULTS", "ner": [ [ 31, 36, "dimer", "oligomeric_state" ], [ 37, 46, "structure", "evidence" ], [ 50, 59, "M1.HpyAVI", "protein" ], [ 76, 90, "\u03b2-class MTases", "protein_type" ], [ 92, 101, "M1.MboIIA", "protein" ], [ 103, 109, "M.RsrI", "protein" ], [ 114, 122, "TTHA0409", "protein" ], [ 143, 152, "M1.HpyAVI", "protein" ], [ 153, 158, "dimer", "oligomeric_state" ] ] }, { "sid": 87, "sent": "M1.HpyAVI exists as dimer in crystal and solution", "section": "FIG", "ner": [ [ 0, 9, "M1.HpyAVI", "protein" ], [ 20, 25, "dimer", "oligomeric_state" ], [ 29, 36, "crystal", "evidence" ] ] }, { "sid": 88, "sent": "A. A conserved interface area of \u03b2-class MTases is defined in M1.HpyAVI.", "section": "FIG", "ner": [ [ 5, 14, "conserved", "protein_state" ], [ 15, 29, "interface area", "site" ], [ 33, 47, "\u03b2-class MTases", "protein_type" ], [ 62, 71, "M1.HpyAVI", "protein" ] ] }, { "sid": 89, "sent": "Residues that involved are signed in red color; Dimerization of free-form M1.HpyAVI B. and cofactor-bound M1.HpyAVI C. The two monomers are marked in green and blue, AdoMet molecules are marked in magenta.", "section": "FIG", "ner": [ [ 48, 60, "Dimerization", "oligomeric_state" ], [ 64, 68, "free", "protein_state" ], [ 74, 83, "M1.HpyAVI", "protein" ], [ 91, 105, "cofactor-bound", "protein_state" ], [ 106, 115, "M1.HpyAVI", "protein" ], [ 127, 135, "monomers", "oligomeric_state" ], [ 166, 172, "AdoMet", "chemical" ] ] }, { "sid": 90, "sent": "D. Gel-filtration analysis revealed that M1.HpyAVI exist as a dimer in solution.", "section": "FIG", "ner": [ [ 3, 26, "Gel-filtration analysis", "experimental_method" ], [ 41, 50, "M1.HpyAVI", "protein" ], [ 62, 67, "dimer", "oligomeric_state" ] ] }, { "sid": 91, "sent": "FPLC system coupled to a Superdex 75 10/300 column.", "section": "FIG", "ner": [ [ 0, 4, "FPLC", "experimental_method" ] ] }, { "sid": 92, "sent": "Elution profiles at 280 nm (blue) and 260 nm (red) are: different concentration (0.05, 0.1, 0.2, 0.5 mg/ml) of M1.HpyAVI protein.", "section": "FIG", "ner": [ [ 0, 16, "Elution profiles", "evidence" ], [ 111, 120, "M1.HpyAVI", "protein" ] ] }, { "sid": 93, "sent": "To probe the oligomeric form of M1.HpyAVI in solution, different concentrations of purified enzyme was loaded onto a Superdex 75 10/300 column.", "section": "RESULTS", "ner": [ [ 32, 41, "M1.HpyAVI", "protein" ] ] }, { "sid": 94, "sent": "The protein was eluted at ~10 ml regardless of the protein concentrations, corresponding to a dimeric molecular mass of 54 kDa (Figure 2D).", "section": "RESULTS", "ner": [ [ 94, 101, "dimeric", "oligomeric_state" ], [ 102, 116, "molecular mass", "evidence" ] ] }, { "sid": 95, "sent": "Our results clearly showed that M1.HpyAVI forms a dimer in both crystal and solution as other \u03b2-class MTases, which however disagrees with a previous investigation using dynamic light scattering (DLS) measurement and gel-filtration chromatography, suggesting that M1.HpyAVI is taking a monomeric state in solution.", "section": "RESULTS", "ner": [ [ 32, 41, "M1.HpyAVI", "protein" ], [ 50, 55, "dimer", "oligomeric_state" ], [ 64, 71, "crystal", "evidence" ], [ 94, 108, "\u03b2-class MTases", "protein_type" ], [ 170, 194, "dynamic light scattering", "experimental_method" ], [ 196, 199, "DLS", "experimental_method" ], [ 217, 246, "gel-filtration chromatography", "experimental_method" ], [ 264, 273, "M1.HpyAVI", "protein" ], [ 286, 295, "monomeric", "oligomeric_state" ] ] }, { "sid": 96, "sent": "This variance might be caused by an addition of 100 mM arginine before cell lysis to keep protein solubility and also by later replacement of arginine with 30% glycerol by dialysis.", "section": "RESULTS", "ner": [ [ 55, 63, "arginine", "chemical" ], [ 142, 150, "arginine", "chemical" ], [ 160, 168, "glycerol", "chemical" ] ] }, { "sid": 97, "sent": "Structure comparisons", "section": "RESULTS", "ner": [ [ 0, 21, "Structure comparisons", "experimental_method" ] ] }, { "sid": 98, "sent": "As a \u03b2-class N6 adenine MTase, the M1.HpyAVI structure displayed a good similarity with M.MboIIA (PDB ID 1G60) and M.RsrI (PDB ID 1NW7), which are falling into the same subgroup.", "section": "RESULTS", "ner": [ [ 5, 29, "\u03b2-class N6 adenine MTase", "protein_type" ], [ 35, 44, "M1.HpyAVI", "protein" ], [ 45, 54, "structure", "evidence" ], [ 88, 96, "M.MboIIA", "protein" ], [ 115, 121, "M.RsrI", "protein" ] ] }, { "sid": 99, "sent": "Superimposition of M1.HpyAVI onto them gave RMSDs of 1.63 \u00c5 and 1.9 \u00c5 on 168 and 190 C\u03b1 atoms, respectively.", "section": "RESULTS", "ner": [ [ 0, 15, "Superimposition", "experimental_method" ], [ 19, 28, "M1.HpyAVI", "protein" ], [ 44, 49, "RMSDs", "evidence" ] ] }, { "sid": 100, "sent": "The most striking structural difference was found to locate on the TRD region (residues 133-163 in M1.HpyAVI) (Figure 3A\u20133C), where the secondary structures vary among these structures.", "section": "RESULTS", "ner": [ [ 67, 70, "TRD", "structure_element" ], [ 88, 95, "133-163", "residue_range" ], [ 99, 108, "M1.HpyAVI", "protein" ] ] }, { "sid": 101, "sent": "By comparison with the other two enzymes that possess protruding arms containing several \u03b1-helices and/or \u03b2-strands, the TRD of M1.HpyAVI is much shorter in length (Figure S1), wrapping more closely around the structural core and lacking apparent secondary structures.", "section": "RESULTS", "ner": [ [ 89, 98, "\u03b1-helices", "structure_element" ], [ 106, 115, "\u03b2-strands", "structure_element" ], [ 121, 124, "TRD", "structure_element" ], [ 128, 137, "M1.HpyAVI", "protein" ], [ 230, 237, "lacking", "protein_state" ] ] }, { "sid": 102, "sent": "Given the proposed role of the TRD for DNA interaction at the major groove, some differences of DNA recognition mode can be expected.", "section": "RESULTS", "ner": [ [ 31, 34, "TRD", "structure_element" ], [ 39, 42, "DNA", "chemical" ], [ 62, 74, "major groove", "structure_element" ], [ 96, 99, "DNA", "chemical" ] ] }, { "sid": 103, "sent": "Another difference locates at the highly flexible loop between \u03b24 and \u03b1D (residues 33-58) of M1.HpyAVI, which was invisible in our structures but present in the structures of M.MboIIA and M.RsrI. Sequence alignment revealed that this region of M1.HpyAVI was longer than its counterparts by 13 and 16 amino acids respectively, which likely renders the H. pylori enzyme more flexible.", "section": "RESULTS", "ner": [ [ 34, 49, "highly flexible", "protein_state" ], [ 50, 54, "loop", "structure_element" ], [ 63, 65, "\u03b24", "structure_element" ], [ 70, 72, "\u03b1D", "structure_element" ], [ 83, 88, "33-58", "residue_range" ], [ 93, 102, "M1.HpyAVI", "protein" ], [ 131, 141, "structures", "evidence" ], [ 161, 171, "structures", "evidence" ], [ 175, 183, "M.MboIIA", "protein" ], [ 188, 194, "M.RsrI", "protein" ], [ 196, 214, "Sequence alignment", "experimental_method" ], [ 244, 253, "M1.HpyAVI", "protein" ], [ 351, 360, "H. pylori", "species" ], [ 373, 381, "flexible", "protein_state" ] ] }, { "sid": 104, "sent": "Structural comparisons between M1.HpyAVI and other DNA MTases", "section": "FIG", "ner": [ [ 0, 22, "Structural comparisons", "experimental_method" ], [ 31, 40, "M1.HpyAVI", "protein" ], [ 51, 61, "DNA MTases", "protein_type" ] ] }, { "sid": 105, "sent": "A. M1.HpyAVI; B. M.MboIIA; C. M.RsrI; D. TTHA0409; E. DpnM; F. M.TaqI. M1.HpyAVI possesses only a long disorder TRD region, compared with the structure-rich TRD of M.MboIIA, M.RsrI and TTHA0409, or the extra DNA-binding domain of DpnM and M.TaqI. The core structure is in cyan; TRD of M1.HpyAVI, M.MboIIA, M.RsrI and TTHA0409 is in red; The region between \u03b24 and \u03b1D of M.MboIIA and M.RsrI is in green; DNA-binding domain of DpnM is in magenta; The C-terminal domain of M.TaqI is in orange.", "section": "FIG", "ner": [ [ 3, 12, "M1.HpyAVI", "protein" ], [ 17, 25, "M.MboIIA", "protein" ], [ 30, 36, "M.RsrI", "protein" ], [ 41, 49, "TTHA0409", "protein" ], [ 54, 58, "DpnM", "protein" ], [ 63, 69, "M.TaqI", "protein" ], [ 71, 80, "M1.HpyAVI", "protein" ], [ 98, 111, "long disorder", "protein_state" ], [ 112, 115, "TRD", "structure_element" ], [ 142, 156, "structure-rich", "protein_state" ], [ 157, 160, "TRD", "structure_element" ], [ 164, 172, "M.MboIIA", "protein" ], [ 174, 180, "M.RsrI", "protein" ], [ 185, 193, "TTHA0409", "protein" ], [ 208, 226, "DNA-binding domain", "structure_element" ], [ 230, 234, "DpnM", "protein" ], [ 239, 245, "M.TaqI", "protein" ], [ 278, 281, "TRD", "structure_element" ], [ 285, 294, "M1.HpyAVI", "protein" ], [ 296, 304, "M.MboIIA", "protein" ], [ 306, 312, "M.RsrI", "protein" ], [ 317, 325, "TTHA0409", "protein" ], [ 356, 358, "\u03b24", "structure_element" ], [ 363, 365, "\u03b1D", "structure_element" ], [ 369, 377, "M.MboIIA", "protein" ], [ 382, 388, "M.RsrI", "protein" ], [ 402, 420, "DNA-binding domain", "structure_element" ], [ 424, 428, "DpnM", "protein" ], [ 448, 465, "C-terminal domain", "structure_element" ], [ 469, 475, "M.TaqI", "protein" ] ] }, { "sid": 106, "sent": "Structural comparison between M1.HpyAVI and a putative \u03b2-class N4 cytosine MTase named TTHA0409 (PDB ID 2ZIF) showed a good similarity as well, giving an RMSD of 1.73 \u00c5 on 164 C\u03b1 atoms (Figure 3D).", "section": "RESULTS", "ner": [ [ 0, 21, "Structural comparison", "experimental_method" ], [ 30, 39, "M1.HpyAVI", "protein" ], [ 55, 80, "\u03b2-class N4 cytosine MTase", "protein_type" ], [ 87, 95, "TTHA0409", "protein" ], [ 154, 158, "RMSD", "evidence" ] ] }, { "sid": 107, "sent": "Exactly like the above comparison, the most significant difference exists in the TRD, where the structures vary in terms of length and presence of \u03b1-helices (Figure S1).", "section": "RESULTS", "ner": [ [ 81, 84, "TRD", "structure_element" ], [ 96, 106, "structures", "evidence" ], [ 147, 156, "\u03b1-helices", "structure_element" ] ] }, { "sid": 108, "sent": "M1.HpyAVI displayed a considerable structural dissimilarity in comparison with N6-adenine MTases from other subgroups including the \u03b1-class DpnM (PDB ID 2DPM) and the \u03b3-class M.TaqI (PDB ID 2ADM).", "section": "RESULTS", "ner": [ [ 0, 9, "M1.HpyAVI", "protein" ], [ 79, 96, "N6-adenine MTases", "protein_type" ], [ 132, 139, "\u03b1-class", "protein_type" ], [ 140, 144, "DpnM", "protein" ], [ 167, 174, "\u03b3-class", "protein_type" ], [ 175, 181, "M.TaqI", "protein" ] ] }, { "sid": 109, "sent": "Both comparisons gave RMSDs above 3.0 \u00c5 (Figure 3E and 3F).", "section": "RESULTS", "ner": [ [ 22, 27, "RMSDs", "evidence" ] ] }, { "sid": 110, "sent": "These two enzymes lack a counterpart loop present in the TRD of M1.HpyAVI, but instead rely on an extra domain for DNA binding and sequence recognition.", "section": "RESULTS", "ner": [ [ 18, 22, "lack", "protein_state" ], [ 25, 41, "counterpart loop", "structure_element" ], [ 57, 60, "TRD", "structure_element" ], [ 64, 73, "M1.HpyAVI", "protein" ], [ 115, 118, "DNA", "chemical" ] ] }, { "sid": 111, "sent": "Collectively, M1.HpyAVI possesses a long disordered TRD, which is in sharp contrast to the secondary structure-rich TRD in other \u03b2-class N6 adenine or N4 cytosine MTases or the extra DNA binding domain present in DNA MTases from other subgroups.", "section": "RESULTS", "ner": [ [ 14, 23, "M1.HpyAVI", "protein" ], [ 36, 51, "long disordered", "protein_state" ], [ 52, 55, "TRD", "structure_element" ], [ 91, 115, "secondary structure-rich", "protein_state" ], [ 116, 119, "TRD", "structure_element" ], [ 129, 169, "\u03b2-class N6 adenine or N4 cytosine MTases", "protein_type" ], [ 213, 223, "DNA MTases", "protein_type" ] ] }, { "sid": 112, "sent": "This striking difference may be a significant determinant of the wider substrate spectrum of this H. pylori enzyme.", "section": "RESULTS", "ner": [ [ 98, 107, "H. pylori", "species" ] ] }, { "sid": 113, "sent": "AdoMet-binding pocket", "section": "RESULTS", "ner": [ [ 0, 21, "AdoMet-binding pocket", "site" ] ] }, { "sid": 114, "sent": "The cofactor binding pocket of M1.HpyAVI is surrounded by residues 7-9, 29-31, 165-167, 216-218 and 221 (Figure 4A), which are conserved among most of DNA MTases.", "section": "RESULTS", "ner": [ [ 4, 27, "cofactor binding pocket", "site" ], [ 31, 40, "M1.HpyAVI", "protein" ], [ 67, 70, "7-9", "residue_range" ], [ 72, 77, "29-31", "residue_range" ], [ 79, 86, "165-167", "residue_range" ], [ 88, 95, "216-218", "residue_range" ], [ 100, 103, "221", "residue_number" ], [ 127, 136, "conserved", "protein_state" ], [ 151, 161, "DNA MTases", "protein_type" ] ] }, { "sid": 115, "sent": "A hydrogen bond between D29 in the catalytic motif DPPY and the amino group of bound AdoMet is preserved as other MTase structures.", "section": "RESULTS", "ner": [ [ 2, 15, "hydrogen bond", "bond_interaction" ], [ 24, 27, "D29", "residue_name_number" ], [ 35, 50, "catalytic motif", "structure_element" ], [ 51, 55, "DPPY", "structure_element" ], [ 79, 84, "bound", "protein_state" ], [ 85, 91, "AdoMet", "chemical" ], [ 114, 119, "MTase", "protein_type" ], [ 120, 130, "structures", "evidence" ] ] }, { "sid": 116, "sent": "Residues D8 and A9 from hydrogen-bonds with N6 and N1 of the purine ring, respectively, and E216 also locates at hydrogen bonding distance with O2\u2032 and O3\u2032 of the ribose.", "section": "RESULTS", "ner": [ [ 9, 11, "D8", "residue_name_number" ], [ 16, 18, "A9", "residue_name_number" ], [ 24, 38, "hydrogen-bonds", "bond_interaction" ], [ 61, 67, "purine", "chemical" ], [ 92, 96, "E216", "residue_name_number" ], [ 113, 129, "hydrogen bonding", "bond_interaction" ], [ 163, 169, "ribose", "chemical" ] ] }, { "sid": 117, "sent": "In addition, H168, T200 and S198 contact the terminal carboxyl of AdoMet.", "section": "RESULTS", "ner": [ [ 13, 17, "H168", "residue_name_number" ], [ 19, 23, "T200", "residue_name_number" ], [ 28, 32, "S198", "residue_name_number" ], [ 66, 72, "AdoMet", "chemical" ] ] }, { "sid": 118, "sent": "Superposition of M1.HpyAVI with the five structures shown in Figure 3 reveals that the orientation of cofactor is rather conserved except for M.TaqI (Figure 4B).", "section": "RESULTS", "ner": [ [ 0, 13, "Superposition", "experimental_method" ], [ 17, 26, "M1.HpyAVI", "protein" ], [ 41, 51, "structures", "evidence" ], [ 114, 130, "rather conserved", "protein_state" ], [ 142, 148, "M.TaqI", "protein" ] ] }, { "sid": 119, "sent": "The different conformation of the bound cofactor observed in M.TaqI might be attributable to the absence of corresponding residues of the conserved AdoMet-binding motif FXGXG in that structure.", "section": "RESULTS", "ner": [ [ 34, 39, "bound", "protein_state" ], [ 61, 67, "M.TaqI", "protein" ], [ 97, 107, "absence of", "protein_state" ], [ 138, 147, "conserved", "protein_state" ], [ 148, 154, "AdoMet", "chemical" ], [ 169, 174, "FXGXG", "structure_element" ], [ 183, 192, "structure", "evidence" ] ] }, { "sid": 120, "sent": "Structural and biochemical analyses define two conserved residues D29 and E216 to be the key sites for AdoMet binding", "section": "FIG", "ner": [ [ 0, 35, "Structural and biochemical analyses", "experimental_method" ], [ 47, 56, "conserved", "protein_state" ], [ 66, 69, "D29", "residue_name_number" ], [ 74, 78, "E216", "residue_name_number" ], [ 103, 109, "AdoMet", "chemical" ] ] }, { "sid": 121, "sent": "A. The cofactor-binding cavity of M1.HpyAVI.", "section": "FIG", "ner": [ [ 7, 30, "cofactor-binding cavity", "site" ], [ 34, 43, "M1.HpyAVI", "protein" ] ] }, { "sid": 122, "sent": "Residues (yellow) that form direct hydrogen bonds with AdoMet (green) are indicated, distance of the hydrogen bond is marked.", "section": "FIG", "ner": [ [ 35, 49, "hydrogen bonds", "bond_interaction" ], [ 55, 61, "AdoMet", "chemical" ], [ 101, 114, "hydrogen bond", "bond_interaction" ] ] }, { "sid": 123, "sent": "B. Superposition of AdoMet in the structures of M1.HpyAVI (green), DpnM (yellow) and M.TaqI (orange).", "section": "FIG", "ner": [ [ 3, 16, "Superposition", "experimental_method" ], [ 20, 26, "AdoMet", "chemical" ], [ 34, 44, "structures", "evidence" ], [ 48, 57, "M1.HpyAVI", "protein" ], [ 67, 71, "DpnM", "protein" ], [ 85, 91, "M.TaqI", "protein" ] ] }, { "sid": 124, "sent": "The AdoMet terminal carboxyl of M.TaqI reveals different orientations.", "section": "FIG", "ner": [ [ 4, 10, "AdoMet", "chemical" ], [ 32, 38, "M.TaqI", "protein" ] ] }, { "sid": 125, "sent": "C. Cofactor binding affinity of wt-/mutants M1.HpyAVI proteins analyzed by microscale thermophoresis (MST).", "section": "FIG", "ner": [ [ 3, 28, "Cofactor binding affinity", "evidence" ], [ 32, 34, "wt", "protein_state" ], [ 36, 43, "mutants", "protein_state" ], [ 44, 53, "M1.HpyAVI", "protein" ], [ 75, 100, "microscale thermophoresis", "experimental_method" ], [ 102, 105, "MST", "experimental_method" ] ] }, { "sid": 126, "sent": "The binding affinity was determined between fluorescently labelled M1.HpyAVI protein and unlabeled AdoMet.", "section": "FIG", "ner": [ [ 4, 20, "binding affinity", "evidence" ], [ 67, 76, "M1.HpyAVI", "protein" ], [ 89, 98, "unlabeled", "protein_state" ], [ 99, 105, "AdoMet", "chemical" ] ] }, { "sid": 127, "sent": "AdoMet (15 nM to 1 mM) was titrated into a fixed concentration of M1.HpyAVI wt/mutant proteins (800 nM).", "section": "FIG", "ner": [ [ 0, 6, "AdoMet", "chemical" ], [ 27, 35, "titrated", "experimental_method" ], [ 66, 75, "M1.HpyAVI", "protein" ], [ 76, 78, "wt", "protein_state" ], [ 79, 85, "mutant", "protein_state" ] ] }, { "sid": 128, "sent": "The dissociation constant (KD) is yielded according to the law of mass action from the isotherm derived of the raw data: M1.HpyAVI-wt: 41 \u00b1 6 \u03bcM; M1.HpyAVI-D8A :212 \u00b1 11 \u03bcM; M1.HpyAVI-D29A : 0 \u03bcM; M1.HpyAVI-H168A : 471 \u00b1 51 \u03bcM; M1.HpyAVI-S198A : 242 \u00b1 32 \u03bcM; M1.HpyAVI-T200A : 252 \u00b1 28 \u03bcM; M1.HpyAVI-E216A : 0 \u03bcM. Standard for three replicates is indicated.", "section": "FIG", "ner": [ [ 4, 25, "dissociation constant", "evidence" ], [ 27, 29, "KD", "evidence" ], [ 87, 95, "isotherm", "evidence" ], [ 121, 130, "M1.HpyAVI", "protein" ], [ 131, 133, "wt", "protein_state" ], [ 146, 159, "M1.HpyAVI-D8A", "mutant" ], [ 174, 188, "M1.HpyAVI-D29A", "mutant" ], [ 197, 212, "M1.HpyAVI-H168A", "mutant" ], [ 228, 243, "M1.HpyAVI-S198A", "mutant" ], [ 259, 274, "M1.HpyAVI-T200A", "mutant" ], [ 290, 305, "M1.HpyAVI-E216A", "mutant" ] ] }, { "sid": 129, "sent": "D. DNA methyltransferase activity of wide type protein and the mutants is quantified using radioactive assay.", "section": "FIG", "ner": [ [ 3, 24, "DNA methyltransferase", "protein_type" ], [ 37, 46, "wide type", "protein_state" ], [ 63, 70, "mutants", "protein_state" ], [ 91, 108, "radioactive assay", "experimental_method" ] ] }, { "sid": 130, "sent": "[3H]-methyl transferred to duplex DNA containing 5\u2032-GAGG-3\u2032 was quantified by Beckman LS6500 for 10 min, experiments were repeated for three times and data were corrected by subtraction of the background.", "section": "FIG", "ner": [ [ 0, 11, "[3H]-methyl", "chemical" ], [ 34, 37, "DNA", "chemical" ], [ 49, 59, "5\u2032-GAGG-3\u2032", "chemical" ] ] }, { "sid": 131, "sent": "E. Superposition of M1.HpyAVI (green) with M.MboIIA (cyan) and M.RsrI (magenta).", "section": "FIG", "ner": [ [ 3, 16, "Superposition", "experimental_method" ], [ 20, 29, "M1.HpyAVI", "protein" ], [ 43, 51, "M.MboIIA", "protein" ], [ 63, 69, "M.RsrI", "protein" ] ] }, { "sid": 132, "sent": "Residues D29 and E216 are conserved through all the DNA MTases mentioned in Figure 3 (not shown in Figure 4).", "section": "FIG", "ner": [ [ 9, 12, "D29", "residue_name_number" ], [ 17, 21, "E216", "residue_name_number" ], [ 26, 35, "conserved", "protein_state" ], [ 52, 62, "DNA MTases", "protein_type" ] ] }, { "sid": 133, "sent": "To confirm the key residues for ligand binding, we prepared a series of single mutants by replacing D8, D29, H168, S198, T200, E216 with alanine and investigated their ligand binding affinity using microscale thermophoresis (MST) assay.", "section": "RESULTS", "ner": [ [ 72, 86, "single mutants", "experimental_method" ], [ 90, 99, "replacing", "experimental_method" ], [ 100, 102, "D8", "residue_name_number" ], [ 104, 107, "D29", "residue_name_number" ], [ 109, 113, "H168", "residue_name_number" ], [ 115, 119, "S198", "residue_name_number" ], [ 121, 125, "T200", "residue_name_number" ], [ 127, 131, "E216", "residue_name_number" ], [ 137, 144, "alanine", "residue_name" ], [ 168, 191, "ligand binding affinity", "evidence" ], [ 198, 223, "microscale thermophoresis", "experimental_method" ], [ 225, 228, "MST", "experimental_method" ] ] }, { "sid": 134, "sent": "As shown in Figure 4C, by contrast to the wild type enzyme, most mutants displayed variable reduction of KD value, among them the D29A and E216A mutants displayed no protein-AdoMet affinity at all.", "section": "RESULTS", "ner": [ [ 42, 51, "wild type", "protein_state" ], [ 65, 72, "mutants", "protein_state" ], [ 105, 107, "KD", "evidence" ], [ 130, 134, "D29A", "mutant" ], [ 139, 144, "E216A", "mutant" ], [ 145, 152, "mutants", "protein_state" ], [ 166, 189, "protein-AdoMet affinity", "evidence" ] ] }, { "sid": 135, "sent": "The results suggested that the hydrogen bonds formed by D29 and E216 with AdoMet were most crucial interactions for cofactor binding.", "section": "RESULTS", "ner": [ [ 31, 45, "hydrogen bonds", "bond_interaction" ], [ 56, 59, "D29", "residue_name_number" ], [ 64, 68, "E216", "residue_name_number" ], [ 74, 80, "AdoMet", "chemical" ] ] }, { "sid": 136, "sent": "Mutation of the two residues may directly prevent the methyl transfer reaction of M1.HpyAVI.", "section": "RESULTS", "ner": [ [ 0, 8, "Mutation", "experimental_method" ], [ 54, 60, "methyl", "chemical" ], [ 82, 91, "M1.HpyAVI", "protein" ] ] }, { "sid": 137, "sent": "The importance of D29 is preserved because it belongs to the catalytic active site DPPY, but the residue E216 has not been fully investigated even being a conserved amino acid throughout MTases (Figure 4E).", "section": "RESULTS", "ner": [ [ 18, 21, "D29", "residue_name_number" ], [ 61, 82, "catalytic active site", "site" ], [ 83, 87, "DPPY", "structure_element" ], [ 105, 109, "E216", "residue_name_number" ], [ 155, 164, "conserved", "protein_state" ], [ 165, 175, "amino acid", "chemical" ], [ 187, 193, "MTases", "protein_type" ] ] }, { "sid": 138, "sent": "E216 is the last residue of \u03b22, which contacts the two hydroxyls of the ribose of AdoMet.", "section": "RESULTS", "ner": [ [ 0, 4, "E216", "residue_name_number" ], [ 28, 30, "\u03b22", "structure_element" ], [ 72, 78, "ribose", "chemical" ], [ 82, 88, "AdoMet", "chemical" ] ] }, { "sid": 139, "sent": "Replacement of this residue by alanine completely abolishes the key hydrogen bonds for AdoMet-binding, and very likely blocks the methyl transfer reaction.", "section": "RESULTS", "ner": [ [ 0, 11, "Replacement", "experimental_method" ], [ 31, 38, "alanine", "residue_name" ], [ 68, 82, "hydrogen bonds", "bond_interaction" ], [ 87, 93, "AdoMet", "chemical" ], [ 130, 136, "methyl", "chemical" ] ] }, { "sid": 140, "sent": "To confirm this notion, [3H]AdoMet radiological assay was applied to quantify the methyl transfer activity of the mutants.", "section": "RESULTS", "ner": [ [ 24, 53, "[3H]AdoMet radiological assay", "experimental_method" ], [ 82, 88, "methyl", "chemical" ], [ 114, 121, "mutants", "protein_state" ] ] }, { "sid": 141, "sent": "As shown in Figure 4D, the result of radiological assay agreed well with the MST measurement.", "section": "RESULTS", "ner": [ [ 37, 55, "radiological assay", "experimental_method" ], [ 77, 80, "MST", "experimental_method" ] ] }, { "sid": 142, "sent": "The D29A and E216A mutants showed little or no methyl transfer activity, while other mutants exhibited reduced methyltransferase activity.", "section": "RESULTS", "ner": [ [ 4, 8, "D29A", "mutant" ], [ 13, 18, "E216A", "mutant" ], [ 19, 26, "mutants", "protein_state" ], [ 47, 53, "methyl", "chemical" ], [ 85, 92, "mutants", "protein_state" ], [ 111, 128, "methyltransferase", "protein_type" ] ] }, { "sid": 143, "sent": "As mentioned previously, FXGXG is a conserved AdoMet-binding motif of DNA MTases.", "section": "RESULTS", "ner": [ [ 25, 30, "FXGXG", "structure_element" ], [ 36, 45, "conserved", "protein_state" ], [ 46, 52, "AdoMet", "chemical" ], [ 70, 80, "DNA MTases", "protein_type" ] ] }, { "sid": 144, "sent": "We also made mutants of \u201cFMGSG\u201d to alanine for every amino acid, and found that the F195A mutant was insoluble probably due to decreasing the local hydrophobicity upon this mutation.", "section": "RESULTS", "ner": [ [ 13, 20, "mutants", "protein_state" ], [ 25, 30, "FMGSG", "structure_element" ], [ 35, 42, "alanine", "residue_name" ], [ 53, 63, "amino acid", "chemical" ], [ 84, 89, "F195A", "mutant" ], [ 90, 96, "mutant", "protein_state" ] ] }, { "sid": 145, "sent": "We subsequently investigated the ligand binding affinity and methyl transfer reaction of the other mutants using MST and a radiological assay.", "section": "RESULTS", "ner": [ [ 33, 56, "ligand binding affinity", "evidence" ], [ 61, 67, "methyl", "chemical" ], [ 99, 106, "mutants", "protein_state" ], [ 113, 116, "MST", "experimental_method" ], [ 123, 141, "radiological assay", "experimental_method" ] ] }, { "sid": 146, "sent": "We found that G197 played a crucial role in AdoMet-binding, while mutagenesis of M196 and G199 did not influence cofactor binding and catalytic activity (Figure S2A and B).", "section": "RESULTS", "ner": [ [ 14, 18, "G197", "residue_name_number" ], [ 44, 50, "AdoMet", "chemical" ], [ 66, 77, "mutagenesis", "experimental_method" ], [ 81, 85, "M196", "residue_name_number" ], [ 90, 94, "G199", "residue_name_number" ] ] }, { "sid": 147, "sent": "G197 is a conserved residue throughout the DNA MTases, and replacing by alanine at this site likely change the local conformation of cofactor-binding pocket.", "section": "RESULTS", "ner": [ [ 0, 4, "G197", "residue_name_number" ], [ 10, 19, "conserved", "protein_state" ], [ 43, 53, "DNA MTases", "protein_type" ], [ 59, 68, "replacing", "experimental_method" ], [ 72, 79, "alanine", "residue_name" ], [ 133, 156, "cofactor-binding pocket", "site" ] ] }, { "sid": 148, "sent": "Mutagenesis on this glycine residue in M.EcoKI or M.EcoP15I also abolished the AdoMet-binding activity.", "section": "RESULTS", "ner": [ [ 0, 11, "Mutagenesis", "experimental_method" ], [ 20, 27, "glycine", "residue_name" ], [ 39, 46, "M.EcoKI", "protein" ], [ 50, 59, "M.EcoP15I", "protein" ], [ 79, 85, "AdoMet", "chemical" ] ] }, { "sid": 149, "sent": "Although mutational study could not tell the role of F195 in ligand binding due to the insolubility of the F195A mutant, structural analysis suggested the importance of this residue in AdoMet-binding.", "section": "RESULTS", "ner": [ [ 9, 25, "mutational study", "experimental_method" ], [ 53, 57, "F195", "residue_name_number" ], [ 107, 112, "F195A", "mutant" ], [ 113, 119, "mutant", "protein_state" ], [ 121, 140, "structural analysis", "experimental_method" ], [ 185, 191, "AdoMet", "chemical" ] ] }, { "sid": 150, "sent": "The phenyl ring of F195 forms a perpendicular \u03c0-stacking interaction with the purine ring of AdoMet, which stabilizes the orientation of AdoMet bound in the pocket of M1.HpyAVI (Figure S2C).", "section": "RESULTS", "ner": [ [ 19, 23, "F195", "residue_name_number" ], [ 46, 68, "\u03c0-stacking interaction", "bond_interaction" ], [ 93, 99, "AdoMet", "chemical" ], [ 137, 143, "AdoMet", "chemical" ], [ 144, 152, "bound in", "protein_state" ], [ 157, 163, "pocket", "site" ], [ 167, 176, "M1.HpyAVI", "protein" ] ] }, { "sid": 151, "sent": "In a separate scenario, mutagenesis of this residue in M.EcoRV has been proven to play an important role in AdoMet binding.", "section": "RESULTS", "ner": [ [ 24, 35, "mutagenesis", "experimental_method" ], [ 55, 62, "M.EcoRV", "protein" ], [ 108, 114, "AdoMet", "chemical" ] ] }, { "sid": 152, "sent": "Potential DNA-binding sites", "section": "RESULTS", "ner": [ [ 10, 27, "DNA-binding sites", "site" ] ] }, { "sid": 153, "sent": "The putative DNA binding region of M1.HpyAVI involves the hairpin loop (residue 101-133), the TRD (residues 136-166), and a highly flexible loop (residues 33-58).", "section": "RESULTS", "ner": [ [ 13, 31, "DNA binding region", "site" ], [ 35, 44, "M1.HpyAVI", "protein" ], [ 58, 70, "hairpin loop", "structure_element" ], [ 80, 87, "101-133", "residue_range" ], [ 94, 97, "TRD", "structure_element" ], [ 108, 115, "136-166", "residue_range" ], [ 124, 139, "highly flexible", "protein_state" ], [ 140, 144, "loop", "structure_element" ], [ 155, 160, "33-58", "residue_range" ] ] }, { "sid": 154, "sent": "The hairpin loop between \u03b26 and \u03b27 strands that carries a conserved HRRY sequence signature in the middle is proposed to insert into the minor groove of the bound DNA.", "section": "RESULTS", "ner": [ [ 4, 16, "hairpin loop", "structure_element" ], [ 25, 27, "\u03b26", "structure_element" ], [ 32, 34, "\u03b27", "structure_element" ], [ 58, 67, "conserved", "protein_state" ], [ 68, 72, "HRRY", "structure_element" ], [ 137, 149, "minor groove", "structure_element" ], [ 157, 162, "bound", "protein_state" ], [ 163, 166, "DNA", "chemical" ] ] }, { "sid": 155, "sent": "As aforementioned, the TRD of M1.HpyAVI shows striking difference from the other DNA MTases, and the relaxed specificity of substrate recognition may be at least partially attributable to the disordered TRD.", "section": "RESULTS", "ner": [ [ 23, 26, "TRD", "structure_element" ], [ 30, 39, "M1.HpyAVI", "protein" ], [ 81, 91, "DNA MTases", "protein_type" ], [ 192, 202, "disordered", "protein_state" ], [ 203, 206, "TRD", "structure_element" ] ] }, { "sid": 156, "sent": "In addition, the highly flexible loop immediately following the DPPY motif in M1.HpyAVI was poorly defined in electron density, exactly like the corresponding loops in the AdoMet-bound structures of M.PvuII, DpnM or M.TaqI that were invisible either.", "section": "RESULTS", "ner": [ [ 17, 32, "highly flexible", "protein_state" ], [ 33, 37, "loop", "structure_element" ], [ 64, 68, "DPPY", "structure_element" ], [ 78, 87, "M1.HpyAVI", "protein" ], [ 110, 126, "electron density", "evidence" ], [ 159, 164, "loops", "structure_element" ], [ 172, 184, "AdoMet-bound", "protein_state" ], [ 185, 195, "structures", "evidence" ], [ 199, 206, "M.PvuII", "protein" ], [ 208, 212, "DpnM", "protein" ], [ 216, 222, "M.TaqI", "protein" ] ] }, { "sid": 157, "sent": "This loop, however, was largely stabilized upon DNA binding, as observed in the protein-DNA complex structures of M.TaqI (PDB ID 2IBS), M.HhaI (PDB ID 1MHT) and M.HaeIII (PDB ID 1DCT).", "section": "RESULTS", "ner": [ [ 5, 9, "loop", "structure_element" ], [ 48, 51, "DNA", "chemical" ], [ 80, 110, "protein-DNA complex structures", "evidence" ], [ 114, 120, "M.TaqI", "protein" ], [ 136, 142, "M.HhaI", "protein" ], [ 161, 169, "M.HaeIII", "protein" ] ] }, { "sid": 158, "sent": "The well-ordered loop in those structures directly contacts the flipping adenine and forms hydrogen bond with neighboring bases.", "section": "RESULTS", "ner": [ [ 4, 16, "well-ordered", "protein_state" ], [ 17, 21, "loop", "structure_element" ], [ 31, 41, "structures", "evidence" ], [ 73, 80, "adenine", "residue_name" ], [ 91, 104, "hydrogen bond", "bond_interaction" ] ] }, { "sid": 159, "sent": "These observations implied that the corresponding loop in other MTases, e.g. M1.HpyAVI, is likely responsible for reducing sequence recognition specificity and thus plays crucial roles in catalysis.", "section": "RESULTS", "ner": [ [ 50, 54, "loop", "structure_element" ], [ 64, 70, "MTases", "protein_type" ], [ 77, 86, "M1.HpyAVI", "protein" ] ] }, { "sid": 160, "sent": "Previous research suggested that M1.HpyAVI from strain 26695 was the first N6 adenine MTase that can methylate the adenine of 5\u2032-GAGG-3\u2032/5\u2032-GGAG-3\u2032 or both two adenines of 5\u2032-GAAG-3\u2032, compared with the homologs from other strains that can methylate only one adenine of 5\u2032-GAGG-3\u2032. To answer why M1.HpyAVI displayed a wider specificity for DNA recognition, we randomly choose fifty of M1.HpyAVI sequences from hundreds of H. pylori strains for multiple sequence alignment.", "section": "RESULTS", "ner": [ [ 33, 42, "M1.HpyAVI", "protein" ], [ 75, 91, "N6 adenine MTase", "protein_type" ], [ 115, 122, "adenine", "residue_name" ], [ 126, 136, "5\u2032-GAGG-3\u2032", "chemical" ], [ 137, 147, "5\u2032-GGAG-3\u2032", "chemical" ], [ 160, 168, "adenines", "residue_name" ], [ 172, 182, "5\u2032-GAAG-3\u2032", "chemical" ], [ 258, 265, "adenine", "residue_name" ], [ 269, 279, "5\u2032-GAGG-3\u2032", "chemical" ], [ 295, 304, "M1.HpyAVI", "protein" ], [ 339, 342, "DNA", "chemical" ], [ 384, 393, "M1.HpyAVI", "protein" ], [ 421, 430, "H. pylori", "species" ], [ 443, 470, "multiple sequence alignment", "experimental_method" ] ] }, { "sid": 161, "sent": "Based on sequence comparison and structural analysis, four residues including P41, N111, K165 and T166 were selected and replaced by serine, threonine, threonine and valine, respectively (Figure 5A).", "section": "RESULTS", "ner": [ [ 9, 28, "sequence comparison", "experimental_method" ], [ 33, 52, "structural analysis", "experimental_method" ], [ 78, 81, "P41", "residue_name_number" ], [ 83, 87, "N111", "residue_name_number" ], [ 89, 93, "K165", "residue_name_number" ], [ 98, 102, "T166", "residue_name_number" ], [ 121, 129, "replaced", "experimental_method" ], [ 133, 139, "serine", "residue_name" ], [ 141, 150, "threonine", "residue_name" ], [ 152, 161, "threonine", "residue_name" ], [ 166, 172, "valine", "residue_name" ] ] }, { "sid": 162, "sent": "Then, a [3H]AdoMet radiological assay was applied to quantify the methyl transfer activity of the wide type protein and the mutants.", "section": "RESULTS", "ner": [ [ 8, 37, "[3H]AdoMet radiological assay", "experimental_method" ], [ 66, 72, "methyl", "chemical" ], [ 98, 107, "wide type", "protein_state" ], [ 124, 131, "mutants", "protein_state" ] ] }, { "sid": 163, "sent": "As shown in Figure 5, when the substrate DNA contains 5\u2032-GAGG-3\u2032 or 5\u2032-GAAG-3\u2032, all the mutants showed no apparent difference of methyl transfer activity compared to the wt-M1.HpyAVI; but when the recognition sequence was 5\u2032-GGAG-3\u2032, the methyl transfer activity of the P41S mutant was significantly reduced compared to the wild type M1.HpyAVI.", "section": "RESULTS", "ner": [ [ 41, 44, "DNA", "chemical" ], [ 54, 64, "5\u2032-GAGG-3\u2032", "chemical" ], [ 68, 79, "5\u2032-GAAG-3\u2032,", "chemical" ], [ 88, 95, "mutants", "protein_state" ], [ 129, 135, "methyl", "chemical" ], [ 170, 172, "wt", "protein_state" ], [ 173, 182, "M1.HpyAVI", "protein" ], [ 222, 233, "5\u2032-GGAG-3\u2032,", "chemical" ], [ 238, 244, "methyl", "chemical" ], [ 270, 274, "P41S", "mutant" ], [ 275, 281, "mutant", "protein_state" ], [ 324, 333, "wild type", "protein_state" ], [ 334, 343, "M1.HpyAVI", "protein" ] ] }, { "sid": 164, "sent": "Sequence alignment, structural analysis and radioactive methyl transfer activity define the key residue for wider substrate specificity of M1.HpyAVI", "section": "FIG", "ner": [ [ 0, 18, "Sequence alignment", "experimental_method" ], [ 20, 39, "structural analysis", "experimental_method" ], [ 44, 80, "radioactive methyl transfer activity", "experimental_method" ], [ 139, 148, "M1.HpyAVI", "protein" ] ] }, { "sid": 165, "sent": "A. Sequence alignment of M1.HpyAVI from 50 H. pylori strains including 26695 revealed several variant residues.", "section": "FIG", "ner": [ [ 3, 21, "Sequence alignment", "experimental_method" ], [ 25, 34, "M1.HpyAVI", "protein" ], [ 43, 52, "H. pylori", "species" ] ] }, { "sid": 166, "sent": "Residues P41, N111, K165 and T166 of M1.HpyAVI from strain 26695 were chosen based on structural analysis and sequence alignment (shown in red arrow).", "section": "FIG", "ner": [ [ 9, 12, "P41", "residue_name_number" ], [ 14, 18, "N111", "residue_name_number" ], [ 20, 24, "K165", "residue_name_number" ], [ 29, 33, "T166", "residue_name_number" ], [ 37, 46, "M1.HpyAVI", "protein" ], [ 59, 64, "26695", "species" ], [ 86, 105, "structural analysis", "experimental_method" ], [ 110, 128, "sequence alignment", "experimental_method" ] ] }, { "sid": 167, "sent": "Amino-acid conservation is depicted using WebLogo (Crooks et al, 2004).", "section": "FIG", "ner": [ [ 42, 49, "WebLogo", "experimental_method" ] ] }, { "sid": 168, "sent": "B., C., D. Methyl transfer reactions were performed using wt-M1.HpyAVI, M1.HpyAVI-P41S, M1.HpyAVI-N111T, and M1.HpyAVI-K165R T166V, respectively.", "section": "FIG", "ner": [ [ 11, 17, "Methyl", "chemical" ], [ 58, 60, "wt", "protein_state" ], [ 61, 70, "M1.HpyAVI", "protein" ], [ 72, 86, "M1.HpyAVI-P41S", "mutant" ], [ 88, 103, "M1.HpyAVI-N111T", "mutant" ], [ 109, 130, "M1.HpyAVI-K165R T166V", "mutant" ] ] }, { "sid": 169, "sent": "Radioactivity incorporated into the duplex DNA containing 5\u2032-GAGG-3\u2032, 5\u2032-GAAG-3\u2032 or 5\u2032-GGAG-3\u2032 was quantified by Beckman LS6500 for 10 min.", "section": "FIG", "ner": [ [ 43, 46, "DNA", "chemical" ], [ 58, 68, "5\u2032-GAGG-3\u2032", "chemical" ], [ 70, 80, "5\u2032-GAAG-3\u2032", "chemical" ], [ 84, 94, "5\u2032-GGAG-3\u2032", "chemical" ] ] }, { "sid": 170, "sent": "Our experimental data identified P41 as a key residue determining the recognition of GGAG of M1.HpyAVI.", "section": "RESULTS", "ner": [ [ 33, 36, "P41", "residue_name_number" ], [ 85, 89, "GGAG", "structure_element" ], [ 93, 102, "M1.HpyAVI", "protein" ] ] }, { "sid": 171, "sent": "This amino acid locates in the highly flexible loop between residues 33 and 58, which is involved in DNA binding and substrate recognition as shown above.", "section": "RESULTS", "ner": [ [ 31, 46, "highly flexible", "protein_state" ], [ 47, 51, "loop", "structure_element" ], [ 69, 78, "33 and 58", "residue_range" ], [ 101, 104, "DNA", "chemical" ] ] }, { "sid": 172, "sent": "Replacement by serine at this position definitely changes the local conformation and hydrophobicity, and probably some structural properties of the whole loop, which may in turn result in reduced specificity for sequence recognition of the enzyme from strain 26695.", "section": "RESULTS", "ner": [ [ 0, 11, "Replacement", "experimental_method" ], [ 15, 21, "serine", "residue_name" ], [ 154, 158, "loop", "structure_element" ], [ 259, 264, "26695", "species" ] ] }, { "sid": 173, "sent": "Although the DNA-bound structure of previous investigation on a \u03b3-class N6-adenine MTase revealed that the target adenine was rotated out of DNA helix, details of the methyl transfer process were still unclear.", "section": "DISCUSS", "ner": [ [ 13, 22, "DNA-bound", "protein_state" ], [ 23, 32, "structure", "evidence" ], [ 64, 88, "\u03b3-class N6-adenine MTase", "protein_type" ], [ 114, 121, "adenine", "residue_name" ], [ 141, 144, "DNA", "chemical" ], [ 167, 173, "methyl", "chemical" ] ] }, { "sid": 174, "sent": "Additionally, recent studies reported the importance of N6-methyladenine in some eukaryotic species, but until now there has not been any N6-adenine MTases being identified in eukaryotes.", "section": "DISCUSS", "ner": [ [ 56, 72, "N6-methyladenine", "ptm" ], [ 81, 91, "eukaryotic", "taxonomy_domain" ], [ 138, 155, "N6-adenine MTases", "protein_type" ], [ 176, 186, "eukaryotes", "taxonomy_domain" ] ] }, { "sid": 175, "sent": "Biochemical and structural characterization of M1.HpyAVI provides a new model for uncovering the methyl transfer mechanism and for investigating the N6-methyladenine in eukaryotes.", "section": "DISCUSS", "ner": [ [ 0, 43, "Biochemical and structural characterization", "experimental_method" ], [ 47, 56, "M1.HpyAVI", "protein" ], [ 97, 103, "methyl", "chemical" ], [ 149, 165, "N6-methyladenine", "ptm" ], [ 169, 179, "eukaryotes", "taxonomy_domain" ] ] }, { "sid": 176, "sent": "Oligomeric state of DNA MTases was long accepted as monomer, but our study indicated here that M1.HpyAVI exists as a dimer both in crystal and solution.", "section": "DISCUSS", "ner": [ [ 20, 30, "DNA MTases", "protein_type" ], [ 52, 59, "monomer", "oligomeric_state" ], [ 95, 104, "M1.HpyAVI", "protein" ], [ 117, 122, "dimer", "oligomeric_state" ], [ 131, 138, "crystal", "evidence" ] ] }, { "sid": 177, "sent": "Interestingly, some other \u03b2-class DNA exocyclic MTases showed similar oligomeric state in crystal and in solution, indicating that dimer may be the functional state shared by a subgroup of DNA MTases.", "section": "DISCUSS", "ner": [ [ 26, 54, "\u03b2-class DNA exocyclic MTases", "protein_type" ], [ 90, 97, "crystal", "evidence" ], [ 131, 136, "dimer", "oligomeric_state" ], [ 189, 199, "DNA MTases", "protein_type" ] ] }, { "sid": 178, "sent": "The highly flexible region (residues 33-58) and TRD (residues 133-163) of M1.HpyAVI are supposed to interact with DNA at minor and major grooves, respectively.", "section": "DISCUSS", "ner": [ [ 4, 19, "highly flexible", "protein_state" ], [ 37, 42, "33-58", "residue_range" ], [ 48, 51, "TRD", "structure_element" ], [ 62, 69, "133-163", "residue_range" ], [ 74, 83, "M1.HpyAVI", "protein" ], [ 114, 117, "DNA", "chemical" ], [ 121, 144, "minor and major grooves", "structure_element" ] ] }, { "sid": 179, "sent": "And residue P41 might be a key residue partially determining the substrate spectrum of M1.HpyAVI.", "section": "DISCUSS", "ner": [ [ 12, 15, "P41", "residue_name_number" ], [ 87, 96, "M1.HpyAVI", "protein" ] ] }, { "sid": 180, "sent": "The missing loop between residues 33 and 58 may need DNA binding so as to form a stable conformation, which is similar to the condition of M.TaqI. Crystallization of M1.HpyAVI-DNA complex warrants future investigations, with the purpose of revealing the mechanism behind the wider substrate specificity of this enzyme.", "section": "DISCUSS", "ner": [ [ 4, 11, "missing", "protein_state" ], [ 12, 16, "loop", "structure_element" ], [ 34, 43, "33 and 58", "residue_range" ], [ 53, 56, "DNA", "chemical" ], [ 81, 87, "stable", "protein_state" ], [ 139, 145, "M.TaqI", "protein" ], [ 147, 162, "Crystallization", "experimental_method" ], [ 166, 179, "M1.HpyAVI-DNA", "complex_assembly" ] ] }, { "sid": 181, "sent": "DNA methylation plays an important role in bacterial pathogenicity.", "section": "DISCUSS", "ner": [ [ 0, 15, "DNA methylation", "ptm" ], [ 43, 52, "bacterial", "taxonomy_domain" ] ] }, { "sid": 182, "sent": "DNA adenine methylation was known to regulate the expression of some virulence genes in bacteria including H.pylori.", "section": "DISCUSS", "ner": [ [ 0, 23, "DNA adenine methylation", "ptm" ], [ 88, 96, "bacteria", "taxonomy_domain" ], [ 107, 115, "H.pylori", "species" ] ] }, { "sid": 183, "sent": "Inhibitors of DNA adenine methylation may have a broad antimicrobial action by targeting DNA adenine methyltransferase.", "section": "DISCUSS", "ner": [ [ 14, 37, "DNA adenine methylation", "ptm" ], [ 89, 118, "DNA adenine methyltransferase", "protein_type" ] ] }, { "sid": 184, "sent": "As an important biological modification, DNA methylation directly influences bacterial survival.", "section": "DISCUSS", "ner": [ [ 41, 56, "DNA methylation", "ptm" ], [ 77, 86, "bacterial", "taxonomy_domain" ] ] }, { "sid": 185, "sent": "Knockout of M1.HpyAVI largely prevents the growth of H. pylori.", "section": "DISCUSS", "ner": [ [ 0, 11, "Knockout of", "experimental_method" ], [ 12, 21, "M1.HpyAVI", "protein" ], [ 53, 62, "H. pylori", "species" ] ] }, { "sid": 186, "sent": "Importantly, H. pylori is involved in 90% of all gastric malignancies.", "section": "DISCUSS", "ner": [ [ 13, 22, "H. pylori", "species" ] ] }, { "sid": 187, "sent": "Appropriate antibiotic regimens could successfully cure gastric diseases caused by H.pylori infection.", "section": "DISCUSS", "ner": [ [ 83, 91, "H.pylori", "species" ] ] }, { "sid": 188, "sent": "However, eradication of H. pylori infection remains a big challenge for the significantly increasing prevalence of its resistance to antibiotics.", "section": "DISCUSS", "ner": [ [ 24, 33, "H. pylori", "species" ] ] }, { "sid": 189, "sent": "The development of new drugs targeting adenine MTases such as M1.HpyAVI offers a new opportunity for inhibition of H. pylori infection.", "section": "DISCUSS", "ner": [ [ 39, 53, "adenine MTases", "protein_type" ], [ 62, 71, "M1.HpyAVI", "protein" ], [ 115, 124, "H. pylori", "species" ] ] }, { "sid": 190, "sent": "Residues that play crucial roles for catalytic activity like D29 or E216 may influence the H.pylori survival.", "section": "DISCUSS", "ner": [ [ 61, 64, "D29", "residue_name_number" ], [ 68, 72, "E216", "residue_name_number" ], [ 91, 99, "H.pylori", "species" ] ] }, { "sid": 191, "sent": "Small molecules targeting these highly conserved residues are likely to emerge less drug resistance.", "section": "DISCUSS", "ner": [ [ 32, 48, "highly conserved", "protein_state" ] ] }, { "sid": 192, "sent": "In summary, the structure of M1.HpyAVI is featured with a disordered TRD and a key residue P41that located in the putative DNA binding region that may associate with the wider substrate specificity.", "section": "DISCUSS", "ner": [ [ 16, 25, "structure", "evidence" ], [ 29, 38, "M1.HpyAVI", "protein" ], [ 58, 68, "disordered", "protein_state" ], [ 69, 72, "TRD", "structure_element" ], [ 91, 94, "P41", "residue_name_number" ], [ 123, 141, "DNA binding region", "site" ] ] }, { "sid": 193, "sent": "Residues D29 and E216 were identified to play a crucial role in cofactor binding.", "section": "DISCUSS", "ner": [ [ 9, 12, "D29", "residue_name_number" ], [ 17, 21, "E216", "residue_name_number" ] ] }, { "sid": 194, "sent": "As the first crystal structure of N6-adenine MTase in H.pylori, this model may shed light on design of new antibiotics to interfere the growth and pathogenesis of H.pylori in human.", "section": "DISCUSS", "ner": [ [ 13, 30, "crystal structure", "evidence" ], [ 34, 50, "N6-adenine MTase", "protein_type" ], [ 54, 62, "H.pylori", "species" ], [ 163, 171, "H.pylori", "species" ], [ 175, 180, "human", "species" ] ] } ] }, "PMC4993997": { "annotations": [ { "sid": 0, "sent": "Structure and function of human Naa60 (NatF), a Golgi-localized bi-functional acetyltransferase", "section": "TITLE", "ner": [ [ 26, 31, "human", "species" ], [ 32, 37, "Naa60", "protein" ], [ 39, 43, "NatF", "complex_assembly" ], [ 78, 95, "acetyltransferase", "protein_type" ] ] }, { "sid": 1, "sent": "N-terminal acetylation (Nt-acetylation), carried out by N-terminal acetyltransferases (NATs), is a conserved and primary modification of nascent peptide chains.", "section": "ABSTRACT", "ner": [ [ 0, 22, "N-terminal acetylation", "ptm" ], [ 24, 38, "Nt-acetylation", "ptm" ], [ 56, 85, "N-terminal acetyltransferases", "protein_type" ], [ 87, 91, "NATs", "protein_type" ], [ 145, 152, "peptide", "chemical" ] ] }, { "sid": 2, "sent": "Naa60 (also named NatF) is a recently identified NAT found only in multicellular eukaryotes.", "section": "ABSTRACT", "ner": [ [ 0, 5, "Naa60", "protein" ], [ 18, 22, "NatF", "complex_assembly" ], [ 49, 52, "NAT", "protein_type" ], [ 67, 91, "multicellular eukaryotes", "taxonomy_domain" ] ] }, { "sid": 3, "sent": "This protein was shown to locate on the Golgi apparatus and mainly catalyze the Nt-acetylation of transmembrane proteins, and it also harbors lysine N\u03b5-acetyltransferase (KAT) activity to catalyze the acetylation of lysine \u03b5-amine.", "section": "ABSTRACT", "ner": [ [ 80, 94, "Nt-acetylation", "ptm" ], [ 142, 169, "lysine N\u03b5-acetyltransferase", "protein_type" ], [ 171, 174, "KAT", "protein_type" ], [ 201, 212, "acetylation", "ptm" ], [ 216, 222, "lysine", "residue_name" ] ] }, { "sid": 4, "sent": "Here, we report the crystal structures of human Naa60 (hNaa60) in complex with Acetyl-Coenzyme A (Ac-CoA) or Coenzyme A (CoA).", "section": "ABSTRACT", "ner": [ [ 20, 38, "crystal structures", "evidence" ], [ 42, 47, "human", "species" ], [ 48, 53, "Naa60", "protein" ], [ 55, 61, "hNaa60", "protein" ], [ 63, 78, "in complex with", "protein_state" ], [ 79, 96, "Acetyl-Coenzyme A", "chemical" ], [ 98, 104, "Ac-CoA", "chemical" ], [ 109, 119, "Coenzyme A", "chemical" ], [ 121, 124, "CoA", "chemical" ] ] }, { "sid": 5, "sent": "The hNaa60 protein contains an amphipathic helix following its GNAT domain that may contribute to Golgi localization of hNaa60, and the \u03b27-\u03b28 hairpin adopted different conformations in the hNaa60(1-242) and hNaa60(1-199) crystal structures.", "section": "ABSTRACT", "ner": [ [ 4, 10, "hNaa60", "protein" ], [ 31, 48, "amphipathic helix", "structure_element" ], [ 63, 74, "GNAT domain", "structure_element" ], [ 120, 126, "hNaa60", "protein" ], [ 136, 149, "\u03b27-\u03b28 hairpin", "structure_element" ], [ 189, 195, "hNaa60", "protein" ], [ 196, 201, "1-242", "residue_range" ], [ 207, 220, "hNaa60(1-199)", "mutant" ], [ 221, 239, "crystal structures", "evidence" ] ] }, { "sid": 6, "sent": "Remarkably, we found that the side-chain of Phe 34 can influence the position of the coenzyme, indicating a new regulatory mechanism involving enzyme, co-factor and substrates interactions.", "section": "ABSTRACT", "ner": [ [ 44, 50, "Phe 34", "residue_name_number" ], [ 85, 93, "coenzyme", "chemical" ] ] }, { "sid": 7, "sent": "Moreover, structural comparison and biochemical studies indicated that Tyr 97 and His 138 are key residues for catalytic reaction and that a non-conserved \u03b23-\u03b24 long loop participates in the regulation of hNaa60 activity.", "section": "ABSTRACT", "ner": [ [ 10, 55, "structural comparison and biochemical studies", "experimental_method" ], [ 71, 77, "Tyr 97", "residue_name_number" ], [ 82, 89, "His 138", "residue_name_number" ], [ 141, 154, "non-conserved", "protein_state" ], [ 155, 170, "\u03b23-\u03b24 long loop", "structure_element" ], [ 205, 211, "hNaa60", "protein" ] ] }, { "sid": 8, "sent": "Acetylation is one of the most ubiquitous modifications that plays a vital role in many biological processes, such as transcriptional regulation, protein-protein interaction, enzyme activity, protein stability, antibiotic resistance, biological rhythm and so on.", "section": "INTRO", "ner": [ [ 0, 11, "Acetylation", "ptm" ] ] }, { "sid": 9, "sent": "Protein acetylation can be grouped into lysine N\u03b5-acetylation and peptide N-terminal acetylation (Nt-acetylation).", "section": "INTRO", "ner": [ [ 8, 19, "acetylation", "ptm" ], [ 40, 61, "lysine N\u03b5-acetylation", "ptm" ], [ 66, 73, "peptide", "chemical" ], [ 74, 96, "N-terminal acetylation", "ptm" ], [ 98, 112, "Nt-acetylation", "ptm" ] ] }, { "sid": 10, "sent": "Generally, N\u03b5-acetylation refers to the transfer of an acetyl group from an acetyl coenzyme A (Ac-CoA) to the \u03b5-amino group of lysine.", "section": "INTRO", "ner": [ [ 11, 25, "N\u03b5-acetylation", "ptm" ], [ 55, 61, "acetyl", "chemical" ], [ 76, 93, "acetyl coenzyme A", "chemical" ], [ 95, 101, "Ac-CoA", "chemical" ], [ 127, 133, "lysine", "residue_name" ] ] }, { "sid": 11, "sent": "This kind of modification is catalyzed by lysine acetyltransferases (KATs), some of which are named histone acetyltransferases (HATs) because early studies focused mostly on the post-transcriptional acetylation of histones.", "section": "INTRO", "ner": [ [ 42, 67, "lysine acetyltransferases", "protein_type" ], [ 69, 73, "KATs", "protein_type" ], [ 100, 126, "histone acetyltransferases", "protein_type" ], [ 128, 132, "HATs", "protein_type" ], [ 199, 210, "acetylation", "ptm" ], [ 214, 222, "histones", "protein_type" ] ] }, { "sid": 12, "sent": "Despite the prominent accomplishments in the field regarding N\u03b5-acetylation by KATs for over 50 years, the significance of the more evolutionarily conserved Nt-acetylation is still inconclusive.", "section": "INTRO", "ner": [ [ 61, 75, "N\u03b5-acetylation", "ptm" ], [ 79, 83, "KATs", "protein_type" ], [ 157, 171, "Nt-acetylation", "ptm" ] ] }, { "sid": 13, "sent": "Nt-acetylation is an abundant and evolutionarily conserved modification occurring in bacteria, archaea and eukaryotes.", "section": "INTRO", "ner": [ [ 0, 14, "Nt-acetylation", "ptm" ], [ 85, 93, "bacteria", "taxonomy_domain" ], [ 95, 102, "archaea", "taxonomy_domain" ], [ 107, 117, "eukaryotes", "taxonomy_domain" ] ] }, { "sid": 14, "sent": "It is estimated that about 80\u201390% of soluble human proteins and 50\u201370% of yeast proteins are subjected to Nt-acetylation, where an acetyl moiety is transferred from Ac-CoA to the \u03b1-amino group of the first residue.", "section": "INTRO", "ner": [ [ 45, 50, "human", "species" ], [ 74, 79, "yeast", "taxonomy_domain" ], [ 106, 120, "Nt-acetylation", "ptm" ], [ 131, 137, "acetyl", "chemical" ], [ 165, 171, "Ac-CoA", "chemical" ] ] }, { "sid": 15, "sent": "Recently Nt-acetylome expands the Nt-acetylation to transmembrane proteins.", "section": "INTRO", "ner": [ [ 34, 48, "Nt-acetylation", "ptm" ] ] }, { "sid": 16, "sent": "Unlike N\u03b5-acetylation that can be eliminated by deacetylases, Nt-acetylation is considered irreversible since no corresponding deacetylase is found to date.", "section": "INTRO", "ner": [ [ 7, 21, "N\u03b5-acetylation", "ptm" ], [ 48, 60, "deacetylases", "protein_type" ], [ 62, 76, "Nt-acetylation", "ptm" ], [ 91, 103, "irreversible", "protein_state" ], [ 127, 138, "deacetylase", "protein_type" ] ] }, { "sid": 17, "sent": "Although Nt-acetylation has been regarded as a co-translational modification traditionally, there is evidence that post-translational Nt-acetylation exists.", "section": "INTRO", "ner": [ [ 9, 23, "Nt-acetylation", "ptm" ], [ 134, 148, "Nt-acetylation", "ptm" ] ] }, { "sid": 18, "sent": "During the past decades, a large number of Nt-acetylome researches have shed light on the functional roles of Nt-acetylation, including protein degradation, subcellular localization, protein-protein interaction, protein-membrane interaction, plant development, stress-response and protein stability.", "section": "INTRO", "ner": [ [ 110, 124, "Nt-acetylation", "ptm" ], [ 242, 247, "plant", "taxonomy_domain" ] ] }, { "sid": 19, "sent": "The Nt-acetylation is carried out by N-terminal acetyltransferases (NATs) that belong to the GNAT superfamily.", "section": "INTRO", "ner": [ [ 4, 18, "Nt-acetylation", "ptm" ], [ 37, 66, "N-terminal acetyltransferases", "protein_type" ], [ 68, 72, "NATs", "protein_type" ], [ 93, 109, "GNAT superfamily", "protein_type" ] ] }, { "sid": 20, "sent": "To date, six NATs (NatA/B/C/D/E/F) have been identified in eukaryotes.", "section": "INTRO", "ner": [ [ 13, 17, "NATs", "protein_type" ], [ 19, 23, "NatA", "complex_assembly" ], [ 24, 25, "B", "complex_assembly" ], [ 26, 27, "C", "complex_assembly" ], [ 28, 29, "D", "complex_assembly" ], [ 30, 31, "E", "complex_assembly" ], [ 32, 33, "F", "complex_assembly" ], [ 59, 69, "eukaryotes", "taxonomy_domain" ] ] }, { "sid": 21, "sent": "About 40 percent of Nt-acetylation of soluble proteins in cells is catalyzed by NatA complex which is composed of the catalytic subunit Naa10p and the auxiliary subunit Naa15p.", "section": "INTRO", "ner": [ [ 20, 34, "Nt-acetylation", "ptm" ], [ 80, 84, "NatA", "complex_assembly" ], [ 136, 142, "Naa10p", "protein" ], [ 169, 175, "Naa15p", "protein" ] ] }, { "sid": 22, "sent": "NatE was found to physically interact with the NatA complex without any observation of impact on NatA-activity.", "section": "INTRO", "ner": [ [ 0, 4, "NatE", "complex_assembly" ], [ 47, 51, "NatA", "complex_assembly" ], [ 97, 101, "NatA", "complex_assembly" ] ] }, { "sid": 23, "sent": "Two other multimeric complexes of NATs are NatB and NatC which contain the catalytic subunits Naa20 and Naa30 and the auxiliary subunits Naa25 and Naa35/Naa38, respectively.", "section": "INTRO", "ner": [ [ 34, 38, "NATs", "protein_type" ], [ 43, 47, "NatB", "complex_assembly" ], [ 52, 56, "NatC", "complex_assembly" ], [ 94, 99, "Naa20", "protein" ], [ 104, 109, "Naa30", "protein" ], [ 137, 142, "Naa25", "protein" ], [ 147, 152, "Naa35", "protein" ], [ 153, 158, "Naa38", "protein" ] ] }, { "sid": 24, "sent": "Furthermore, only the catalytic subunits Naa40 and Naa60 were found for NatD and NatF, respectively.", "section": "INTRO", "ner": [ [ 41, 46, "Naa40", "protein" ], [ 51, 56, "Naa60", "protein" ], [ 72, 76, "NatD", "complex_assembly" ], [ 81, 85, "NatF", "complex_assembly" ] ] }, { "sid": 25, "sent": "Besides Nt-acetylation, accumulating reports have proposed N\u03b5-acetylation carried out by NATs.", "section": "INTRO", "ner": [ [ 8, 22, "Nt-acetylation", "ptm" ], [ 59, 73, "N\u03b5-acetylation", "ptm" ], [ 89, 93, "NATs", "protein_type" ] ] }, { "sid": 26, "sent": "There is an evolutionary increasing in the degree of Nt-acetylation between yeast and human which could partly be explained by the contribution of NatF. As the first N-terminal acetyltransferase discovered on an organelle, NatF, encoded by NAA60 and also named as Histone acetyltransferase type B protein 4 (HAT4), Naa60 or N-acetyltransferase 15 (NAT15), is the youngest member of the NAT family.", "section": "INTRO", "ner": [ [ 53, 67, "Nt-acetylation", "ptm" ], [ 76, 81, "yeast", "taxonomy_domain" ], [ 86, 91, "human", "species" ], [ 147, 151, "NatF", "complex_assembly" ], [ 166, 194, "N-terminal acetyltransferase", "protein_type" ], [ 223, 227, "NatF", "complex_assembly" ], [ 240, 245, "NAA60", "protein" ], [ 264, 306, "Histone acetyltransferase type B protein 4", "protein" ], [ 308, 312, "HAT4", "protein" ], [ 315, 320, "Naa60", "protein" ], [ 324, 346, "N-acetyltransferase 15", "protein" ], [ 348, 353, "NAT15", "protein" ], [ 386, 389, "NAT", "protein_type" ] ] }, { "sid": 27, "sent": "Unlike other NATs that are highly conserved among lower and higher eukaryotes, NatF only exists in higher eukaryotes.", "section": "INTRO", "ner": [ [ 13, 17, "NATs", "protein_type" ], [ 27, 43, "highly conserved", "protein_state" ], [ 50, 55, "lower", "taxonomy_domain" ], [ 60, 77, "higher eukaryotes", "taxonomy_domain" ], [ 79, 83, "NatF", "complex_assembly" ], [ 99, 116, "higher eukaryotes", "taxonomy_domain" ] ] }, { "sid": 28, "sent": "Subsequent researches indicated that NatF displays its catalytic ability with both Nt-acetylation and lysine N\u03b5-acetylation.", "section": "INTRO", "ner": [ [ 37, 41, "NatF", "complex_assembly" ], [ 83, 97, "Nt-acetylation", "ptm" ], [ 102, 123, "lysine N\u03b5-acetylation", "ptm" ] ] }, { "sid": 29, "sent": "As an N-terminal acetyltransferase, NatF can specifically catalyze acetylation of the N-terminal \u03b1-amine of most transmembrane proteins and has substrate preference towards proteins with Met-Lys-, Met-Val-, Met-Ala- and Met-Met-N-termini, thus partially overlapping substrate selectivity with NatC and NatE. On the other hand, NatF, with its lysine acetyltransferase activity, mediates the lysine acetylation of free histone H4, including H4K20, H4K79 and H4K91.", "section": "INTRO", "ner": [ [ 6, 34, "N-terminal acetyltransferase", "protein_type" ], [ 36, 40, "NatF", "complex_assembly" ], [ 67, 78, "acetylation", "ptm" ], [ 187, 195, "Met-Lys-", "structure_element" ], [ 197, 205, "Met-Val-", "structure_element" ], [ 207, 215, "Met-Ala-", "structure_element" ], [ 220, 228, "Met-Met-", "structure_element" ], [ 293, 297, "NatC", "complex_assembly" ], [ 302, 306, "NatE", "complex_assembly" ], [ 327, 331, "NatF", "complex_assembly" ], [ 342, 366, "lysine acetyltransferase", "protein_type" ], [ 390, 408, "lysine acetylation", "ptm" ], [ 417, 424, "histone", "protein_type" ], [ 425, 427, "H4", "protein_type" ], [ 439, 441, "H4", "protein_type" ], [ 441, 444, "K20", "residue_name_number" ], [ 446, 448, "H4", "protein_type" ], [ 448, 451, "K79", "residue_name_number" ], [ 456, 458, "H4", "protein_type" ], [ 458, 461, "K91", "residue_name_number" ] ] }, { "sid": 30, "sent": "Another important feature of NatF is that this protein is anchored on the Golgi apparatus through its C-terminal membrane-integrating region and takes part in the maintaining of Golgi integrity.", "section": "INTRO", "ner": [ [ 29, 33, "NatF", "complex_assembly" ], [ 113, 140, "membrane-integrating region", "structure_element" ] ] }, { "sid": 31, "sent": "With its unique intracellular organellar localization and substrate selectivity, NatF appears to provide more evolutionary information among the NAT family members.", "section": "INTRO", "ner": [ [ 81, 85, "NatF", "complex_assembly" ], [ 145, 148, "NAT", "protein_type" ] ] }, { "sid": 32, "sent": "It was recently found that NatF facilitates nucleosomes assembly and that NAA60 knockdown in MCF7-cell inhibits cell proliferation, sensitizes cells to DNA damage and induces cell apoptosis.", "section": "INTRO", "ner": [ [ 27, 31, "NatF", "complex_assembly" ], [ 44, 55, "nucleosomes", "complex_assembly" ], [ 74, 79, "NAA60", "protein" ] ] }, { "sid": 33, "sent": "In Drosophila cells, NAA60 knockdown induces chromosomal segregation defects during anaphase including lagging chromosomes and chromosomal bridges.", "section": "INTRO", "ner": [ [ 3, 13, "Drosophila", "taxonomy_domain" ], [ 21, 26, "NAA60", "protein" ] ] }, { "sid": 34, "sent": "Much recent attention has also been focused on the requirement of NatF for regulation of organellar structure.", "section": "INTRO", "ner": [ [ 66, 70, "NatF", "complex_assembly" ] ] }, { "sid": 35, "sent": "In HeLa cells, NAA60 knockdown causes Golgi apparatus fragmentation which can be rescued by overexpression Naa60.", "section": "INTRO", "ner": [ [ 15, 20, "NAA60", "protein" ], [ 92, 106, "overexpression", "experimental_method" ], [ 107, 112, "Naa60", "protein" ] ] }, { "sid": 36, "sent": "The systematic investigation of publicly available microarray data showed that NATs share distinct tissue-specific expression patterns in Drosophila and NatF shows a higher expression level in central nervous system of Drosophila.", "section": "INTRO", "ner": [ [ 79, 83, "NATs", "protein_type" ], [ 138, 148, "Drosophila", "taxonomy_domain" ], [ 153, 157, "NatF", "complex_assembly" ], [ 219, 229, "Drosophila", "taxonomy_domain" ] ] }, { "sid": 37, "sent": "In this study, we solved the structures of human Naa60 (NatF) in complex with coenzyme.", "section": "INTRO", "ner": [ [ 18, 24, "solved", "experimental_method" ], [ 29, 39, "structures", "evidence" ], [ 43, 48, "human", "species" ], [ 49, 54, "Naa60", "protein" ], [ 56, 60, "NatF", "complex_assembly" ], [ 62, 77, "in complex with", "protein_state" ], [ 78, 86, "coenzyme", "chemical" ] ] }, { "sid": 38, "sent": "The hNaa60 protein contains a unique amphipathic \u03b1-helix (\u03b15) following its GNAT domain that might account for the Golgi localization of this protein.", "section": "INTRO", "ner": [ [ 4, 10, "hNaa60", "protein" ], [ 37, 56, "amphipathic \u03b1-helix", "structure_element" ], [ 58, 60, "\u03b15", "structure_element" ], [ 76, 87, "GNAT domain", "structure_element" ] ] }, { "sid": 39, "sent": "Crystal structures showed that the \u03b27-\u03b28 hairpin rotated about 50 degrees upon removing the C-terminal region of the protein and this movement substantially changed the geometry of the substrate-binding pocket.", "section": "INTRO", "ner": [ [ 0, 18, "Crystal structures", "evidence" ], [ 35, 48, "\u03b27-\u03b28 hairpin", "structure_element" ], [ 92, 109, "C-terminal region", "structure_element" ], [ 185, 209, "substrate-binding pocket", "site" ] ] }, { "sid": 40, "sent": "Remarkably, we find that Phe 34 may participate in the proper positioning of the coenzyme for the transfer reaction to occur.", "section": "INTRO", "ner": [ [ 25, 31, "Phe 34", "residue_name_number" ], [ 81, 89, "coenzyme", "chemical" ] ] }, { "sid": 41, "sent": "Further structure comparison and biochemical studies also identified other key structural elements essential for the enzyme activity of Naa60.", "section": "INTRO", "ner": [ [ 8, 28, "structure comparison", "experimental_method" ], [ 33, 52, "biochemical studies", "experimental_method" ], [ 136, 141, "Naa60", "protein" ] ] }, { "sid": 42, "sent": "Overall structure of hNaa60", "section": "RESULTS", "ner": [ [ 8, 17, "structure", "evidence" ], [ 21, 27, "hNaa60", "protein" ] ] }, { "sid": 43, "sent": "In the effort to prepare the protein for structural studies, we tried a large number of hNaa60 constructs but all failed due to heavy precipitation or aggregation.", "section": "RESULTS", "ner": [ [ 88, 94, "hNaa60", "protein" ] ] }, { "sid": 44, "sent": "Sequence alignment of Naa60 from different species revealed a Glu-Glu-Arg (EER) versus Val-Val-Pro (VVP) sequence difference near the N-terminus of the protein in Xenopus Laevis versus Homo sapiens (Fig. 1A).", "section": "RESULTS", "ner": [ [ 0, 18, "Sequence alignment", "experimental_method" ], [ 22, 27, "Naa60", "protein" ], [ 62, 73, "Glu-Glu-Arg", "structure_element" ], [ 75, 78, "EER", "structure_element" ], [ 87, 98, "Val-Val-Pro", "structure_element" ], [ 100, 103, "VVP", "structure_element" ], [ 163, 177, "Xenopus Laevis", "species" ], [ 185, 197, "Homo sapiens", "species" ] ] }, { "sid": 45, "sent": "Considering that terminal residues may lack higher-order structure and hydrophobic residues in this region may expose to solvent and hence cause protein aggregation, we mutated residues 4\u20136 from VVP to EER for the purpose of improving solubility of this protein.", "section": "RESULTS", "ner": [ [ 169, 176, "mutated", "experimental_method" ], [ 186, 189, "4\u20136", "residue_range" ], [ 195, 205, "VVP to EER", "mutant" ] ] }, { "sid": 46, "sent": "According to previous studies, this N-terminal region should not interfere with hNaa60\u2019s Golgi localization.", "section": "RESULTS", "ner": [ [ 80, 86, "hNaa60", "protein" ] ] }, { "sid": 47, "sent": "We tried many hNaa60 constructs with the three-residues mutation but only the truncated variant 1-199 and the full-length protein behaved well.", "section": "RESULTS", "ner": [ [ 14, 20, "hNaa60", "protein" ], [ 56, 64, "mutation", "experimental_method" ], [ 78, 87, "truncated", "protein_state" ], [ 96, 101, "1-199", "residue_range" ], [ 110, 121, "full-length", "protein_state" ] ] }, { "sid": 48, "sent": "We obtained the crystal of the truncated variant 1-199 in complex with CoA first, and after extensive trials we got the crystal of the full-length protein (spanning residues 1-242) in complex with Ac-CoA (Fig. 1B,C).", "section": "RESULTS", "ner": [ [ 16, 23, "crystal", "evidence" ], [ 31, 40, "truncated", "protein_state" ], [ 49, 54, "1-199", "residue_range" ], [ 55, 70, "in complex with", "protein_state" ], [ 71, 74, "CoA", "chemical" ], [ 120, 127, "crystal", "evidence" ], [ 135, 146, "full-length", "protein_state" ], [ 174, 179, "1-242", "residue_range" ], [ 181, 196, "in complex with", "protein_state" ], [ 197, 203, "Ac-CoA", "chemical" ] ] }, { "sid": 49, "sent": "Hereafter, all deletions or point mutants of hNaa60 we describe here are with the EER mutation.", "section": "RESULTS", "ner": [ [ 34, 41, "mutants", "protein_state" ], [ 45, 51, "hNaa60", "protein" ], [ 82, 85, "EER", "structure_element" ], [ 86, 94, "mutation", "experimental_method" ] ] }, { "sid": 50, "sent": "The crystal structures of hNaa60(1-242)/Ac-CoA and hNaa60(1-199)/CoA were determined by molecular replacement and refined to 1.38\u2009\u00c5 and 1.60\u2009\u00c5 resolution, respectively (Table 1).", "section": "RESULTS", "ner": [ [ 4, 22, "crystal structures", "evidence" ], [ 26, 46, "hNaa60(1-242)/Ac-CoA", "complex_assembly" ], [ 51, 68, "hNaa60(1-199)/CoA", "complex_assembly" ], [ 88, 109, "molecular replacement", "experimental_method" ] ] }, { "sid": 51, "sent": "The electron density maps were of sufficient quality to trace residues 1-211 of hNaa60(1-242) and residues 5-199 of hNaa60(1-199).", "section": "RESULTS", "ner": [ [ 4, 25, "electron density maps", "evidence" ], [ 71, 76, "1-211", "residue_range" ], [ 80, 86, "hNaa60", "protein" ], [ 87, 92, "1-242", "residue_range" ], [ 107, 112, "5-199", "residue_range" ], [ 116, 129, "hNaa60(1-199)", "mutant" ] ] }, { "sid": 52, "sent": "The structure of hNaa60 protein contains a central domain exhibiting a classic GCN5-related N-acetyltransferase (GNAT) folding, along with the extended N- and C-terminal regions (Fig. 1B,C).", "section": "RESULTS", "ner": [ [ 4, 13, "structure", "evidence" ], [ 17, 23, "hNaa60", "protein" ], [ 43, 57, "central domain", "structure_element" ], [ 79, 111, "GCN5-related N-acetyltransferase", "protein_type" ], [ 113, 117, "GNAT", "protein_type" ], [ 143, 151, "extended", "protein_state" ], [ 152, 177, "N- and C-terminal regions", "structure_element" ] ] }, { "sid": 53, "sent": "The central domain includes nine \u03b2 strands (\u03b21-\u03b29) and four \u03b1-helixes (\u03b11-\u03b14) and is highly similar to the known hNaa50p and other reported NATs (Fig. 1D).", "section": "RESULTS", "ner": [ [ 4, 18, "central domain", "structure_element" ], [ 33, 42, "\u03b2 strands", "structure_element" ], [ 44, 49, "\u03b21-\u03b29", "structure_element" ], [ 60, 69, "\u03b1-helixes", "structure_element" ], [ 71, 76, "\u03b11-\u03b14", "structure_element" ], [ 85, 99, "highly similar", "protein_state" ], [ 113, 120, "hNaa50p", "protein" ], [ 140, 144, "NATs", "protein_type" ] ] }, { "sid": 54, "sent": "However, in hNaa60, there is an extra 20-residue loop between \u03b23 and \u03b24 that forms a small subdomain with well-defined 3D structure (Fig. 1B\u2013D).", "section": "RESULTS", "ner": [ [ 12, 18, "hNaa60", "protein" ], [ 32, 53, "extra 20-residue loop", "structure_element" ], [ 62, 64, "\u03b23", "structure_element" ], [ 69, 71, "\u03b24", "structure_element" ], [ 85, 100, "small subdomain", "structure_element" ] ] }, { "sid": 55, "sent": "Furthermore, the \u03b27-\u03b28 strands form an approximately antiparallel \u03b2-hairpin structure remarkably different from that in hNaa50p (Fig. 1D).", "section": "RESULTS", "ner": [ [ 17, 30, "\u03b27-\u03b28 strands", "structure_element" ], [ 39, 85, "approximately antiparallel \u03b2-hairpin structure", "structure_element" ], [ 120, 127, "hNaa50p", "protein" ] ] }, { "sid": 56, "sent": "The N- and C-terminal regions form helical structures (\u03b10 and \u03b15) stretching out from the central GCN5-domain (Fig. 1C).", "section": "RESULTS", "ner": [ [ 4, 29, "N- and C-terminal regions", "structure_element" ], [ 35, 53, "helical structures", "structure_element" ], [ 55, 57, "\u03b10", "structure_element" ], [ 62, 64, "\u03b15", "structure_element" ], [ 98, 109, "GCN5-domain", "structure_element" ] ] }, { "sid": 57, "sent": "Interestingly, we found that the catalytic activity of hNaa60(1-242) is much lower than that of hNaa60(1-199) (Figure S1), indicating that residues 200\u2013242 may have some auto-inhibitory effect on the activity of the enzyme.", "section": "RESULTS", "ner": [ [ 55, 61, "hNaa60", "protein" ], [ 62, 67, "1-242", "residue_range" ], [ 96, 109, "hNaa60(1-199)", "mutant" ], [ 148, 155, "200\u2013242", "residue_range" ] ] }, { "sid": 58, "sent": "However, since this region was not visible in the hNaa60(1-242) crystal structure, we do not yet understand how this happens.", "section": "RESULTS", "ner": [ [ 50, 56, "hNaa60", "protein" ], [ 57, 62, "1-242", "residue_range" ], [ 64, 81, "crystal structure", "evidence" ] ] }, { "sid": 59, "sent": "Another possibility is that since hNaa60 is localized on Golgi apparatus, the observed low activity of the full-length hNaa60 might be related to lack of Golgi localization of the enzyme in our in vitro studies.", "section": "RESULTS", "ner": [ [ 34, 40, "hNaa60", "protein" ], [ 107, 118, "full-length", "protein_state" ], [ 119, 125, "hNaa60", "protein" ] ] }, { "sid": 60, "sent": "For the convenience of studying the kinetics of mutants, the mutagenesis studies described hereafter were all based on hNaa60 (1-199).", "section": "RESULTS", "ner": [ [ 48, 55, "mutants", "protein_state" ], [ 61, 80, "mutagenesis studies", "experimental_method" ], [ 119, 133, "hNaa60 (1-199)", "mutant" ] ] }, { "sid": 61, "sent": "An amphipathic \u03b1-helix in the C-terminal region may contribute to Golgi localization of hNaa60", "section": "RESULTS", "ner": [ [ 3, 22, "amphipathic \u03b1-helix", "structure_element" ], [ 30, 47, "C-terminal region", "structure_element" ], [ 88, 94, "hNaa60", "protein" ] ] }, { "sid": 62, "sent": "There is one hNaa60 molecule in the asymmetric unit in the hNaa60(1-242)/Ac-CoA structure.", "section": "RESULTS", "ner": [ [ 13, 19, "hNaa60", "protein" ], [ 59, 79, "hNaa60(1-242)/Ac-CoA", "complex_assembly" ], [ 80, 89, "structure", "evidence" ] ] }, { "sid": 63, "sent": "The C-terminal region extended from the GCN5-domain forms an amphipathic helix (\u03b15) and interacts with a molecule in a neighbor asymmetric unit through hydrophobic interactions between \u03b15-helix and a hydrophobic groove between the N-terminal \u03b21 and \u03b23 strands of the neighbor molecule (Fig. 2A).", "section": "RESULTS", "ner": [ [ 4, 21, "C-terminal region", "structure_element" ], [ 40, 51, "GCN5-domain", "structure_element" ], [ 61, 78, "amphipathic helix", "structure_element" ], [ 80, 82, "\u03b15", "structure_element" ], [ 152, 176, "hydrophobic interactions", "bond_interaction" ], [ 185, 193, "\u03b15-helix", "structure_element" ], [ 200, 218, "hydrophobic groove", "site" ], [ 242, 244, "\u03b21", "structure_element" ], [ 249, 259, "\u03b23 strands", "structure_element" ] ] }, { "sid": 64, "sent": "The C-terminal extension following \u03b15-helix forms a \u03b2-turn that wraps around and interacts with the neighbor protein molecule through hydrophobic interactions, too.", "section": "RESULTS", "ner": [ [ 4, 24, "C-terminal extension", "structure_element" ], [ 35, 43, "\u03b15-helix", "structure_element" ], [ 52, 58, "\u03b2-turn", "structure_element" ], [ 134, 158, "hydrophobic interactions", "bond_interaction" ] ] }, { "sid": 65, "sent": "In the hNaa60(1-199)/CoA structure, a part of the \u03b15-helix is deleted due to truncation of the C-terminal region (Fig. 1B).", "section": "RESULTS", "ner": [ [ 7, 24, "hNaa60(1-199)/CoA", "complex_assembly" ], [ 25, 34, "structure", "evidence" ], [ 50, 58, "\u03b15-helix", "structure_element" ], [ 95, 112, "C-terminal region", "structure_element" ] ] }, { "sid": 66, "sent": "Interestingly, the remaining residues in \u03b15-helix still form an amphipathic helix although the hydrophobic interaction with the N-terminal hydrophobic groove of a neighbor molecule is abolished and the helix is largely exposed in solvent due to different crystal packing (Fig. 2B).", "section": "RESULTS", "ner": [ [ 41, 49, "\u03b15-helix", "structure_element" ], [ 64, 81, "amphipathic helix", "structure_element" ], [ 95, 118, "hydrophobic interaction", "bond_interaction" ], [ 139, 157, "hydrophobic groove", "site" ], [ 202, 207, "helix", "structure_element" ], [ 255, 270, "crystal packing", "evidence" ] ] }, { "sid": 67, "sent": "A recent research showed that residues 182\u2013216 are important for the localization of hNaa60 on Golgi. According to our structure, the solvent-exposed amphipathic helix (\u03b15) formed by residues 190-202 with an array of hydrophobic residues located on one side (Ile 190, Leu 191, Ile 194, Leu 197 and Leu 201) and hydrophilic residues on the other side (Fig. S2) might account for interaction between hNaa60 and Golgi membrane, as it is a typical structure accounting for membrane association through immersing into the lipid bi-layer with its hydrophobic side as was observed with KalSec14, Atg3, PB1-F2 etc.", "section": "RESULTS", "ner": [ [ 39, 46, "182\u2013216", "residue_range" ], [ 85, 91, "hNaa60", "protein" ], [ 119, 128, "structure", "evidence" ], [ 134, 149, "solvent-exposed", "protein_state" ], [ 150, 167, "amphipathic helix", "structure_element" ], [ 169, 171, "\u03b15", "structure_element" ], [ 192, 199, "190-202", "residue_range" ], [ 259, 266, "Ile 190", "residue_name_number" ], [ 268, 275, "Leu 191", "residue_name_number" ], [ 277, 284, "Ile 194", "residue_name_number" ], [ 286, 293, "Leu 197", "residue_name_number" ], [ 298, 305, "Leu 201", "residue_name_number" ], [ 398, 404, "hNaa60", "protein" ], [ 579, 587, "KalSec14", "protein" ], [ 589, 593, "Atg3", "protein" ], [ 595, 601, "PB1-F2", "protein" ] ] }, { "sid": 68, "sent": "The \u03b27-\u03b28 hairpin showed alternative conformations in the hNaa60 crystal structures", "section": "RESULTS", "ner": [ [ 4, 17, "\u03b27-\u03b28 hairpin", "structure_element" ], [ 58, 64, "hNaa60", "protein" ], [ 65, 83, "crystal structures", "evidence" ] ] }, { "sid": 69, "sent": "Superposition of hNaa60(1-242)/Ac-CoA, hNaa60(1-199)/CoA and hNaa50/CoA/peptide (PDB 3TFY) revealed considerable difference in the \u03b27-\u03b28 hairpin region despite the overall stability and similarity of the GNAT domain (Fig. 1D).", "section": "RESULTS", "ner": [ [ 0, 13, "Superposition", "experimental_method" ], [ 17, 37, "hNaa60(1-242)/Ac-CoA", "complex_assembly" ], [ 39, 56, "hNaa60(1-199)/CoA", "complex_assembly" ], [ 61, 79, "hNaa50/CoA/peptide", "complex_assembly" ], [ 131, 144, "\u03b27-\u03b28 hairpin", "structure_element" ], [ 204, 215, "GNAT domain", "structure_element" ] ] }, { "sid": 70, "sent": "In hNaa60(1-242), the \u03b27-\u03b28 hairpin is located in close proximity to the \u03b11-\u03b12 loop, creating a more compact substrate binding site than that in hNaa50, where this region adopts a more flexible loop conformation (\u03b26-\u03b27 loop).", "section": "RESULTS", "ner": [ [ 3, 9, "hNaa60", "protein" ], [ 10, 15, "1-242", "residue_range" ], [ 22, 35, "\u03b27-\u03b28 hairpin", "structure_element" ], [ 73, 83, "\u03b11-\u03b12 loop", "structure_element" ], [ 109, 131, "substrate binding site", "site" ], [ 145, 151, "hNaa50", "protein" ], [ 185, 193, "flexible", "protein_state" ], [ 194, 198, "loop", "structure_element" ], [ 213, 223, "\u03b26-\u03b27 loop", "structure_element" ] ] }, { "sid": 71, "sent": "Upon removing the C-terminal region of hNaa60, we observed that hNaa60 (1-199) molecules pack in a different way involving the \u03b27-\u03b28 hairpin in the crystal, leading to about 50 degree rotation of the hairpin which moves away from the \u03b11-\u03b12 loop (Figs 1D and 2C).", "section": "RESULTS", "ner": [ [ 5, 13, "removing", "experimental_method" ], [ 18, 35, "C-terminal region", "structure_element" ], [ 39, 45, "hNaa60", "protein" ], [ 64, 78, "hNaa60 (1-199)", "mutant" ], [ 127, 140, "\u03b27-\u03b28 hairpin", "structure_element" ], [ 148, 155, "crystal", "evidence" ], [ 200, 207, "hairpin", "structure_element" ], [ 234, 244, "\u03b11-\u03b12 loop", "structure_element" ] ] }, { "sid": 72, "sent": "This conformational change substantially altered the geometry of the substrate binding site, which could potentially change the way in which the substrate accesses the active site of the enzyme.", "section": "RESULTS", "ner": [ [ 69, 91, "substrate binding site", "site" ], [ 168, 179, "active site", "site" ] ] }, { "sid": 73, "sent": "In hNaa60(1-242), the \u03b27-\u03b28 hairpin covers the active site in a way similar to that observed in hNaa50, presumably leaving only one way for the substrate to access the active site, i.e. to enter from the opposite end into the same tunnel where Ac-CoA/CoA binds (Fig. 2D), which may accommodate access of a NAT substrate only.", "section": "RESULTS", "ner": [ [ 3, 9, "hNaa60", "protein" ], [ 10, 15, "1-242", "residue_range" ], [ 22, 35, "\u03b27-\u03b28 hairpin", "structure_element" ], [ 47, 58, "active site", "site" ], [ 96, 102, "hNaa50", "protein" ], [ 168, 179, "active site", "site" ], [ 231, 237, "tunnel", "site" ], [ 244, 250, "Ac-CoA", "chemical" ], [ 251, 254, "CoA", "chemical" ], [ 306, 309, "NAT", "protein_type" ] ] }, { "sid": 74, "sent": "KAT activity of hNaa60 toward histone H4 has been noted in previous study, and our enzyme kinetic data also indicated that hNaa60 can acetylate H3-H4 tetramer in vitro (Figure S3).", "section": "RESULTS", "ner": [ [ 0, 3, "KAT", "protein_type" ], [ 16, 22, "hNaa60", "protein" ], [ 30, 37, "histone", "protein_type" ], [ 38, 40, "H4", "protein_type" ], [ 83, 102, "enzyme kinetic data", "evidence" ], [ 123, 129, "hNaa60", "protein" ], [ 144, 149, "H3-H4", "complex_assembly" ], [ 150, 158, "tetramer", "oligomeric_state" ] ] }, { "sid": 75, "sent": "Furthermore, we analyzed the acetylation status of histone H3-H4 tetramer using mass spectrometry and observed that multiple lysine residues in the protein showed significantly increased acetylation level and changed acetylation profile upon treatment with hNaa60(1-199) (Figure S4).", "section": "RESULTS", "ner": [ [ 29, 40, "acetylation", "ptm" ], [ 51, 58, "histone", "protein_type" ], [ 59, 64, "H3-H4", "complex_assembly" ], [ 65, 73, "tetramer", "oligomeric_state" ], [ 80, 97, "mass spectrometry", "experimental_method" ], [ 125, 131, "lysine", "residue_name" ], [ 187, 198, "acetylation", "ptm" ], [ 217, 228, "acetylation", "ptm" ], [ 257, 270, "hNaa60(1-199)", "mutant" ] ] }, { "sid": 76, "sent": "We also conducted liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis on a synthetic peptide (NH2-MKGKEEKEGGAR-COOH) after treatment with hNaa60(1-199), and the data confirmed that both the N-terminal \u03b1-amine and lysine side-chain \u03b5-amine were robustly acetylated after the treatment (Table S1).", "section": "RESULTS", "ner": [ [ 18, 64, "liquid chromatography-tandem mass spectrometry", "experimental_method" ], [ 66, 74, "LC/MS/MS", "experimental_method" ], [ 100, 107, "peptide", "chemical" ], [ 109, 130, "NH2-MKGKEEKEGGAR-COOH", "chemical" ], [ 153, 166, "hNaa60(1-199)", "mutant" ], [ 228, 234, "lysine", "residue_name" ], [ 268, 278, "acetylated", "protein_state" ] ] }, { "sid": 77, "sent": "Recent structural investigation of other NATs proposed that the \u03b26-\u03b27 loop, corresponding to the \u03b27-\u03b28 hairpin in hNaa60, and the \u03b11-\u03b12 loop flanking the substrate-binding site of NATs, prevent the lysine side-chain of the KAT substrates from inserting into the active site.", "section": "RESULTS", "ner": [ [ 7, 31, "structural investigation", "experimental_method" ], [ 41, 45, "NATs", "protein_type" ], [ 64, 74, "\u03b26-\u03b27 loop", "structure_element" ], [ 97, 110, "\u03b27-\u03b28 hairpin", "structure_element" ], [ 114, 120, "hNaa60", "protein" ], [ 130, 140, "\u03b11-\u03b12 loop", "structure_element" ], [ 154, 176, "substrate-binding site", "site" ], [ 180, 184, "NATs", "protein_type" ], [ 198, 204, "lysine", "residue_name" ], [ 223, 226, "KAT", "protein_type" ], [ 262, 273, "active site", "site" ] ] }, { "sid": 78, "sent": "Indeed, superposition of hNaa60(1-242) structure on that of Hat1p, a typical KAT, in complex with a histone H4 peptide revealed obvious overlapping/clashing of the H4 peptide (a KAT substrate) with the \u03b27-\u03b28 hairpin of hNaa60(1-242) (Fig. 2D).", "section": "RESULTS", "ner": [ [ 8, 21, "superposition", "experimental_method" ], [ 25, 31, "hNaa60", "protein" ], [ 32, 37, "1-242", "residue_range" ], [ 39, 48, "structure", "evidence" ], [ 60, 65, "Hat1p", "protein" ], [ 77, 80, "KAT", "protein_type" ], [ 82, 97, "in complex with", "protein_state" ], [ 100, 107, "histone", "protein_type" ], [ 108, 110, "H4", "protein_type" ], [ 111, 118, "peptide", "chemical" ], [ 164, 166, "H4", "protein_type" ], [ 167, 174, "peptide", "chemical" ], [ 178, 181, "KAT", "protein_type" ], [ 202, 215, "\u03b27-\u03b28 hairpin", "structure_element" ], [ 219, 225, "hNaa60", "protein" ], [ 226, 231, "1-242", "residue_range" ] ] }, { "sid": 79, "sent": "Interestingly, in the hNaa60(1-199) crystal structure, the displaced \u03b27-\u03b28 hairpin opened a second way for the substrate to access the active center that would readily accommodate the binding of the H4 peptide (Fig. 2E), thus implied a potential explanation for KAT activity of this enzyme from a structural biological view.", "section": "RESULTS", "ner": [ [ 22, 35, "hNaa60(1-199)", "mutant" ], [ 36, 53, "crystal structure", "evidence" ], [ 69, 82, "\u03b27-\u03b28 hairpin", "structure_element" ], [ 135, 148, "active center", "site" ], [ 199, 201, "H4", "protein_type" ], [ 202, 209, "peptide", "chemical" ], [ 262, 265, "KAT", "protein_type" ] ] }, { "sid": 80, "sent": "However, since hNaa60(1-242) and hNaa60(1-199) were crystallized in different crystal forms, the observed conformational change of the \u03b27-\u03b28 hairpin may simply be an artifact related to the different crystal packing.", "section": "RESULTS", "ner": [ [ 15, 21, "hNaa60", "protein" ], [ 22, 27, "1-242", "residue_range" ], [ 33, 39, "hNaa60", "protein" ], [ 52, 64, "crystallized", "experimental_method" ], [ 78, 91, "crystal forms", "evidence" ], [ 135, 148, "\u03b27-\u03b28 hairpin", "structure_element" ], [ 200, 215, "crystal packing", "evidence" ] ] }, { "sid": 81, "sent": "Whether the KAT substrates bind to the \u03b27-\u03b28 hairpin displaced conformation of the enzyme needs to be verified by further structural and functional studies.", "section": "RESULTS", "ner": [ [ 12, 15, "KAT", "protein_type" ], [ 39, 52, "\u03b27-\u03b28 hairpin", "structure_element" ], [ 122, 155, "structural and functional studies", "experimental_method" ] ] }, { "sid": 82, "sent": "Phe 34 facilitates proper positioning of the cofactor for acetyl-transfer", "section": "RESULTS", "ner": [ [ 0, 6, "Phe 34", "residue_name_number" ], [ 58, 64, "acetyl", "chemical" ] ] }, { "sid": 83, "sent": "The electron density of Phe 34 side-chain is well defined in the hNaa60(1-242)/Ac-CoA structure, but becomes invisible in the hNaa60(1-199)/CoA structure, indicating displacement of the Phe 34 side-chain in the latter (Fig. 3A,B).", "section": "RESULTS", "ner": [ [ 4, 20, "electron density", "evidence" ], [ 24, 30, "Phe 34", "residue_name_number" ], [ 65, 85, "hNaa60(1-242)/Ac-CoA", "complex_assembly" ], [ 86, 95, "structure", "evidence" ], [ 126, 143, "hNaa60(1-199)/CoA", "complex_assembly" ], [ 144, 153, "structure", "evidence" ], [ 186, 192, "Phe 34", "residue_name_number" ] ] }, { "sid": 84, "sent": "A solvent-derived malonate molecule is found beside Phe 34 and the ethanethioate moiety of Ac-CoA in the high-resolution hNaa60(1-242)/Ac-CoA structure (Fig. 3A).", "section": "RESULTS", "ner": [ [ 18, 26, "malonate", "chemical" ], [ 52, 58, "Phe 34", "residue_name_number" ], [ 67, 80, "ethanethioate", "chemical" ], [ 91, 97, "Ac-CoA", "chemical" ], [ 121, 141, "hNaa60(1-242)/Ac-CoA", "complex_assembly" ], [ 142, 151, "structure", "evidence" ] ] }, { "sid": 85, "sent": "Superposition of this structure on that of hNaa50p/CoA/peptide shows that the malonate molecule overlaps well on the N-terminal methionine of the substrate peptide and residue Phe 34 in hNaa60 overlaps well on Phe 27 in hNaa50 (Fig. 4A).", "section": "RESULTS", "ner": [ [ 0, 13, "Superposition", "experimental_method" ], [ 22, 31, "structure", "evidence" ], [ 43, 62, "hNaa50p/CoA/peptide", "complex_assembly" ], [ 78, 86, "malonate", "chemical" ], [ 128, 138, "methionine", "residue_name" ], [ 156, 163, "peptide", "chemical" ], [ 176, 182, "Phe 34", "residue_name_number" ], [ 186, 192, "hNaa60", "protein" ], [ 210, 216, "Phe 27", "residue_name_number" ], [ 220, 226, "hNaa50", "protein" ] ] }, { "sid": 86, "sent": "Interestingly, in the structure of hNaa60(1-199)/CoA, the terminal thiol of CoA adopts alternative conformations.", "section": "RESULTS", "ner": [ [ 22, 31, "structure", "evidence" ], [ 35, 52, "hNaa60(1-199)/CoA", "complex_assembly" ], [ 76, 79, "CoA", "chemical" ] ] }, { "sid": 87, "sent": "One is to approach the substrate amine (as indicated by the superimposed hNaa50/CoA/peptide structure), similar to the terminal ethanethioate of Ac-CoA in the structure of hNaa60(1-242)/Ac-CoA; the other is to approach the \u03b11-\u03b12 loop and away from the substrate amine (Fig. 3B).", "section": "RESULTS", "ner": [ [ 33, 38, "amine", "chemical" ], [ 60, 72, "superimposed", "experimental_method" ], [ 73, 91, "hNaa50/CoA/peptide", "complex_assembly" ], [ 92, 101, "structure", "evidence" ], [ 128, 141, "ethanethioate", "chemical" ], [ 145, 151, "Ac-CoA", "chemical" ], [ 159, 168, "structure", "evidence" ], [ 172, 192, "hNaa60(1-242)/Ac-CoA", "complex_assembly" ], [ 223, 233, "\u03b11-\u03b12 loop", "structure_element" ] ] }, { "sid": 88, "sent": "To rule out the possibility that the electron density we define as the alternative conformation of the thiol terminus is residual electron density of the displaced side-chain of Phe 34, we solved the crystal structure of hNaa60(1-199) F34A/CoA. The structure of this mutant is highly similar to hNaa60(1-199)/CoA and there is essentially the same electron density corresponding to the alternative conformation of the thiol (Fig. 3C).", "section": "RESULTS", "ner": [ [ 37, 53, "electron density", "evidence" ], [ 130, 146, "electron density", "evidence" ], [ 178, 184, "Phe 34", "residue_name_number" ], [ 189, 195, "solved", "experimental_method" ], [ 200, 217, "crystal structure", "evidence" ], [ 221, 243, "hNaa60(1-199) F34A/CoA", "complex_assembly" ], [ 249, 258, "structure", "evidence" ], [ 267, 273, "mutant", "protein_state" ], [ 295, 312, "hNaa60(1-199)/CoA", "complex_assembly" ], [ 347, 363, "electron density", "evidence" ] ] }, { "sid": 89, "sent": "Phe 27 in hNaa50p (equivalent to Phe 34 in hNaa60) has been implicated to facilitate the binding of N-terminal methionine of the substrate peptide through hydrophobic interaction.", "section": "RESULTS", "ner": [ [ 0, 6, "Phe 27", "residue_name_number" ], [ 10, 17, "hNaa50p", "protein" ], [ 33, 39, "Phe 34", "residue_name_number" ], [ 43, 49, "hNaa60", "protein" ], [ 111, 121, "methionine", "residue_name" ], [ 139, 146, "peptide", "chemical" ], [ 155, 178, "hydrophobic interaction", "bond_interaction" ] ] }, { "sid": 90, "sent": "However, in the hNaa60/Ac-CoA structure, a hydrophilic malonate molecule is found at the same location where the N-terminal methionine should bind as is indicated by the superposition (Fig. 3A), suggesting that Phe 34 may accommodate binding of hydrophilic substrate, too.", "section": "RESULTS", "ner": [ [ 16, 29, "hNaa60/Ac-CoA", "complex_assembly" ], [ 30, 39, "structure", "evidence" ], [ 55, 63, "malonate", "chemical" ], [ 124, 134, "methionine", "residue_name" ], [ 170, 183, "superposition", "experimental_method" ], [ 211, 217, "Phe 34", "residue_name_number" ] ] }, { "sid": 91, "sent": "Moreover, orientation of Phe 34 side-chain seems to be co-related to positioning of the terminus of the co-enzyme and important for placing it at a location in close proximity to the substrate amine.", "section": "RESULTS", "ner": [ [ 25, 31, "Phe 34", "residue_name_number" ] ] }, { "sid": 92, "sent": "We hypothesize that if Phe 34 only works to facilitate the binding of the hydrophobic N-terminal Met residue, to mutate it from Phe to Ala would not abolish the catalytic activity of this enzyme, while if Phe 34 also plays an essential role to position the ethanethioate moiety of Ac-CoA, the mutation would be expected to abrogate the activity of the enzyme.", "section": "RESULTS", "ner": [ [ 23, 29, "Phe 34", "residue_name_number" ], [ 97, 100, "Met", "residue_name" ], [ 113, 119, "mutate", "experimental_method" ], [ 128, 131, "Phe", "residue_name" ], [ 135, 138, "Ala", "residue_name" ], [ 205, 211, "Phe 34", "residue_name_number" ], [ 257, 270, "ethanethioate", "chemical" ], [ 281, 287, "Ac-CoA", "chemical" ], [ 293, 301, "mutation", "experimental_method" ] ] }, { "sid": 93, "sent": "Indeed, our enzyme kinetic data showed that hNaa60(1-199) F34A mutant showed no detectable activity (Fig. 5A).", "section": "RESULTS", "ner": [ [ 12, 31, "enzyme kinetic data", "evidence" ], [ 44, 57, "hNaa60(1-199)", "mutant" ], [ 58, 62, "F34A", "mutant" ], [ 63, 69, "mutant", "protein_state" ] ] }, { "sid": 94, "sent": "In order to rule out the possibility that the observed loss of activity may be related to bad folding of the mutant protein, we studied the circular dichroism (CD) spectrum of the protein (Fig. 5B) and determined its crystal structure (Fig. 3C).", "section": "RESULTS", "ner": [ [ 109, 115, "mutant", "protein_state" ], [ 140, 158, "circular dichroism", "experimental_method" ], [ 160, 162, "CD", "experimental_method" ], [ 164, 172, "spectrum", "evidence" ], [ 217, 234, "crystal structure", "evidence" ] ] }, { "sid": 95, "sent": "Both studies proved that the F34A mutant protein is well-folded.", "section": "RESULTS", "ner": [ [ 29, 33, "F34A", "mutant" ], [ 34, 40, "mutant", "protein_state" ], [ 52, 63, "well-folded", "protein_state" ] ] }, { "sid": 96, "sent": "Many studies have addressed the crucial effect of \u03b11-\u03b12 loop on catalysis, showing that some residues located in this area are involved in the binding of substrates.", "section": "RESULTS", "ner": [ [ 50, 60, "\u03b11-\u03b12 loop", "structure_element" ] ] }, { "sid": 97, "sent": "We propose that Phe 34 may play a dual role both in interacting with the peptide substrate (recognition) and in positioning of the ethanethioate moiety of Ac-CoA to the right location to facilitate acetyl-transfer.", "section": "RESULTS", "ner": [ [ 16, 22, "Phe 34", "residue_name_number" ], [ 73, 80, "peptide", "chemical" ], [ 131, 144, "ethanethioate", "chemical" ], [ 155, 161, "Ac-CoA", "chemical" ], [ 198, 204, "acetyl", "chemical" ] ] }, { "sid": 98, "sent": "Structural basis for hNaa60 substrate binding", "section": "RESULTS", "ner": [ [ 21, 27, "hNaa60", "protein" ] ] }, { "sid": 99, "sent": "Several studies have demonstrated that the substrate specificities of hNaa60 and hNaa50 are highly overlapped.", "section": "RESULTS", "ner": [ [ 70, 76, "hNaa60", "protein" ], [ 81, 87, "hNaa50", "protein" ] ] }, { "sid": 100, "sent": "The structure of hNaa50p/CoA/peptide provides detailed information about the position of substrate N-terminal residues in the active site of hNaa50.", "section": "RESULTS", "ner": [ [ 4, 13, "structure", "evidence" ], [ 17, 36, "hNaa50p/CoA/peptide", "complex_assembly" ], [ 126, 137, "active site", "site" ], [ 141, 147, "hNaa50", "protein" ] ] }, { "sid": 101, "sent": "Comparing the active site of hNaa60(1-242)/Ac-CoA with hNaa50p/CoA/peptide revealed that key catalytic and substrate binding residues are highly conserved in both proteins (Fig. 4A).", "section": "RESULTS", "ner": [ [ 14, 25, "active site", "site" ], [ 29, 49, "hNaa60(1-242)/Ac-CoA", "complex_assembly" ], [ 55, 74, "hNaa50p/CoA/peptide", "complex_assembly" ], [ 93, 133, "catalytic and substrate binding residues", "site" ], [ 138, 154, "highly conserved", "protein_state" ] ] }, { "sid": 102, "sent": "With respect to catalysis, hNaa50p has been shown to employ residues Tyr 73 and His 112 to abstract proton from the \u03b1-amino group from the substrate\u2019s first residue through a well-ordered water.", "section": "RESULTS", "ner": [ [ 27, 34, "hNaa50p", "protein" ], [ 69, 75, "Tyr 73", "residue_name_number" ], [ 80, 87, "His 112", "residue_name_number" ], [ 175, 187, "well-ordered", "protein_state" ], [ 188, 193, "water", "chemical" ] ] }, { "sid": 103, "sent": "A well-ordered water was also found between Tyr 97 and His 138 in hNaa60 (1-199)/CoA and hNaa60 (1-242)/Ac-CoA (Fig. 4B).", "section": "RESULTS", "ner": [ [ 2, 14, "well-ordered", "protein_state" ], [ 15, 20, "water", "chemical" ], [ 44, 50, "Tyr 97", "residue_name_number" ], [ 55, 62, "His 138", "residue_name_number" ], [ 66, 84, "hNaa60 (1-199)/CoA", "complex_assembly" ], [ 89, 110, "hNaa60 (1-242)/Ac-CoA", "complex_assembly" ] ] }, { "sid": 104, "sent": "To determine the function of Tyr 97 and His 138 in hNaa60 catalysis, we mutated these residues to alanine and phenylalanine, respectively, and confirmed that all these mutants used in our kinetic assays are well-folded by CD spectra (Fig. 5B).", "section": "RESULTS", "ner": [ [ 29, 35, "Tyr 97", "residue_name_number" ], [ 40, 47, "His 138", "residue_name_number" ], [ 51, 57, "hNaa60", "protein" ], [ 72, 79, "mutated", "experimental_method" ], [ 98, 105, "alanine", "residue_name" ], [ 110, 123, "phenylalanine", "residue_name" ], [ 168, 175, "mutants", "protein_state" ], [ 188, 202, "kinetic assays", "experimental_method" ], [ 207, 218, "well-folded", "protein_state" ], [ 222, 224, "CD", "experimental_method" ], [ 225, 232, "spectra", "evidence" ] ] }, { "sid": 105, "sent": "Purity of all proteins were also analyzed by SDS-PAGE (Figure S5).", "section": "RESULTS", "ner": [ [ 45, 53, "SDS-PAGE", "experimental_method" ] ] }, { "sid": 106, "sent": "As show in Fig. 5A, the mutants Y97A, Y97F, H138A and H138F abolished the activity of hNaa60.", "section": "RESULTS", "ner": [ [ 24, 31, "mutants", "protein_state" ], [ 32, 36, "Y97A", "mutant" ], [ 38, 42, "Y97F", "mutant" ], [ 44, 49, "H138A", "mutant" ], [ 54, 59, "H138F", "mutant" ], [ 60, 82, "abolished the activity", "protein_state" ], [ 86, 92, "hNaa60", "protein" ] ] }, { "sid": 107, "sent": "In contrast, to mutate the nearby solvent exposed residue Glu 37 to Ala (E37A) has little impact on the activity of hNaa60 (Figs 4B and 5A).", "section": "RESULTS", "ner": [ [ 16, 22, "mutate", "experimental_method" ], [ 34, 49, "solvent exposed", "protein_state" ], [ 58, 64, "Glu 37", "residue_name_number" ], [ 68, 71, "Ala", "residue_name" ], [ 73, 77, "E37A", "mutant" ], [ 116, 122, "hNaa60", "protein" ] ] }, { "sid": 108, "sent": "In conclusion, the structural and functional studies indicate that hNaa60 applies the same two base mechanism through Tyr 97, His 138 and a well-ordered water as was described for hNaa50.", "section": "RESULTS", "ner": [ [ 19, 52, "structural and functional studies", "experimental_method" ], [ 67, 73, "hNaa60", "protein" ], [ 118, 124, "Tyr 97", "residue_name_number" ], [ 126, 133, "His 138", "residue_name_number" ], [ 140, 152, "well-ordered", "protein_state" ], [ 153, 158, "water", "chemical" ], [ 180, 186, "hNaa50", "protein" ] ] }, { "sid": 109, "sent": "The malonate molecule observed in the hNaa60(1-242)/Ac-CoA crystal structure may be indicative of the substrate binding position of hNaa60 since it is located in the active site and overlaps the N-terminal Met of the substrate peptide in the superposition with the hNaa50p/CoA/peptide structure (Fig. 4A).", "section": "RESULTS", "ner": [ [ 4, 12, "malonate", "chemical" ], [ 38, 58, "hNaa60(1-242)/Ac-CoA", "complex_assembly" ], [ 59, 76, "crystal structure", "evidence" ], [ 132, 138, "hNaa60", "protein" ], [ 166, 177, "active site", "site" ], [ 206, 209, "Met", "residue_name" ], [ 227, 234, "peptide", "chemical" ], [ 242, 255, "superposition", "experimental_method" ], [ 265, 284, "hNaa50p/CoA/peptide", "complex_assembly" ], [ 285, 294, "structure", "evidence" ] ] }, { "sid": 110, "sent": "Residues Tyr 38, Asn 143 and Tyr 165 are located around the malonate and interact with it through direct hydrogen bonds or water bridge (Fig. 4C).", "section": "RESULTS", "ner": [ [ 9, 15, "Tyr 38", "residue_name_number" ], [ 17, 24, "Asn 143", "residue_name_number" ], [ 29, 36, "Tyr 165", "residue_name_number" ], [ 60, 68, "malonate", "chemical" ], [ 105, 119, "hydrogen bonds", "bond_interaction" ], [ 123, 135, "water bridge", "bond_interaction" ] ] }, { "sid": 111, "sent": "Although malonate is negatively charged, which is different from that of lysine \u03b5-amine or peptide N-terminal amine, similar hydrophilic interactions may take place when substrate amine presents in the same position, since Tyr 38, Asn 143 and Tyr 165 are not positively or negatively charged.", "section": "RESULTS", "ner": [ [ 9, 17, "malonate", "chemical" ], [ 73, 79, "lysine", "residue_name" ], [ 91, 98, "peptide", "chemical" ], [ 125, 149, "hydrophilic interactions", "bond_interaction" ], [ 223, 229, "Tyr 38", "residue_name_number" ], [ 231, 238, "Asn 143", "residue_name_number" ], [ 243, 250, "Tyr 165", "residue_name_number" ] ] }, { "sid": 112, "sent": "In agreement with this hypothesis, it was found that the Y38A, N143A and Y165A mutants all showed remarkably reduced activities as compared to WT, implying that these residues may be critical for substrate binding (Figs 4C and 5A).", "section": "RESULTS", "ner": [ [ 57, 61, "Y38A", "mutant" ], [ 63, 68, "N143A", "mutant" ], [ 73, 78, "Y165A", "mutant" ], [ 79, 86, "mutants", "protein_state" ], [ 143, 145, "WT", "protein_state" ] ] }, { "sid": 113, "sent": "The \u03b23-\u03b24 loop participates in the regulation of hNaa60-activity", "section": "RESULTS", "ner": [ [ 4, 14, "\u03b23-\u03b24 loop", "structure_element" ], [ 49, 55, "hNaa60", "protein" ] ] }, { "sid": 114, "sent": "Residues between \u03b23 and \u03b24 of hNaa60 form a unique 20-residue long loop (residues 73\u201392) that is a short turn in many other NAT members (Fig. 1D).", "section": "RESULTS", "ner": [ [ 17, 19, "\u03b23", "structure_element" ], [ 24, 26, "\u03b24", "structure_element" ], [ 30, 36, "hNaa60", "protein" ], [ 51, 71, "20-residue long loop", "structure_element" ], [ 82, 87, "73\u201392", "residue_range" ], [ 99, 109, "short turn", "structure_element" ], [ 124, 127, "NAT", "protein_type" ] ] }, { "sid": 115, "sent": "Previous study indicated that auto-acetylation of hNaa60K79 could influence the activity of hNaa60; however, we were not able to determine if Lys 79 is acetylated in our crystal structures due to poor quality of the electron density of Lys 79 side-chain.", "section": "RESULTS", "ner": [ [ 30, 46, "auto-acetylation", "ptm" ], [ 50, 56, "hNaa60", "protein" ], [ 56, 59, "K79", "residue_name_number" ], [ 92, 98, "hNaa60", "protein" ], [ 142, 148, "Lys 79", "residue_name_number" ], [ 152, 162, "acetylated", "protein_state" ], [ 170, 188, "crystal structures", "evidence" ], [ 216, 232, "electron density", "evidence" ], [ 236, 242, "Lys 79", "residue_name_number" ] ] }, { "sid": 116, "sent": "We therefore used mass spectrometry to analyze if Lys 79 was acetylated in our bacterially purified proteins, and observed no modification on this residue (Figure S6).", "section": "RESULTS", "ner": [ [ 18, 35, "mass spectrometry", "experimental_method" ], [ 50, 56, "Lys 79", "residue_name_number" ], [ 61, 71, "acetylated", "protein_state" ] ] }, { "sid": 117, "sent": "To assess the impact of hNaa60K79 auto-acetylation, we studied the kinetics of K79R and K79Q mutants which mimic the un-acetylated and acetylated form of Lys 79, respectively.", "section": "RESULTS", "ner": [ [ 24, 30, "hNaa60", "protein" ], [ 30, 33, "K79", "residue_name_number" ], [ 34, 50, "auto-acetylation", "ptm" ], [ 79, 83, "K79R", "mutant" ], [ 88, 92, "K79Q", "mutant" ], [ 93, 100, "mutants", "protein_state" ], [ 117, 130, "un-acetylated", "protein_state" ], [ 135, 145, "acetylated", "protein_state" ], [ 154, 160, "Lys 79", "residue_name_number" ] ] }, { "sid": 118, "sent": "Interestingly, both K79R and K79Q mutants led to an increase in the catalytic activity of hNaa60, while K79A mutant led to modest decrease of the activity (Fig. 5A).", "section": "RESULTS", "ner": [ [ 20, 24, "K79R", "mutant" ], [ 29, 33, "K79Q", "mutant" ], [ 34, 41, "mutants", "protein_state" ], [ 90, 96, "hNaa60", "protein" ], [ 104, 108, "K79A", "mutant" ], [ 109, 115, "mutant", "protein_state" ] ] }, { "sid": 119, "sent": "These data indicate that the acetylation of Lys 79 is not required for optimal catalytic activity of hNaa60 in vitro.", "section": "RESULTS", "ner": [ [ 29, 40, "acetylation", "ptm" ], [ 44, 50, "Lys 79", "residue_name_number" ], [ 101, 107, "hNaa60", "protein" ] ] }, { "sid": 120, "sent": "It is noted that the \u03b23-\u03b24 loop of hNaa60 acts like a door leaf to partly cover the substrate-binding pathway.", "section": "RESULTS", "ner": [ [ 21, 31, "\u03b23-\u03b24 loop", "structure_element" ], [ 35, 41, "hNaa60", "protein" ], [ 84, 109, "substrate-binding pathway", "site" ] ] }, { "sid": 121, "sent": "We hence hypothesize that the \u03b23-\u03b24 loop may interfere with the access of the peptide substrates and that the solvent-exposing Lys 79 may play a potential role to remove the door leaf when it hovers in solvent (Fig. 4D).", "section": "RESULTS", "ner": [ [ 30, 40, "\u03b23-\u03b24 loop", "structure_element" ], [ 78, 85, "peptide", "chemical" ], [ 110, 126, "solvent-exposing", "protein_state" ], [ 127, 133, "Lys 79", "residue_name_number" ] ] }, { "sid": 122, "sent": "Acidic residues Glu 80, Asp 81 and Asp 83 interact with His 138, His 159 and His 158 to maintain the conformation of the \u03b23-\u03b24 loop, thus contribute to control the substrate binding (Fig. 4D).", "section": "RESULTS", "ner": [ [ 16, 22, "Glu 80", "residue_name_number" ], [ 24, 30, "Asp 81", "residue_name_number" ], [ 35, 41, "Asp 83", "residue_name_number" ], [ 56, 63, "His 138", "residue_name_number" ], [ 65, 72, "His 159", "residue_name_number" ], [ 77, 84, "His 158", "residue_name_number" ], [ 121, 131, "\u03b23-\u03b24 loop", "structure_element" ] ] }, { "sid": 123, "sent": "To verify this hypothesis, we mutated Glu 80, Asp 81 and Asp 83 to Ala respectively.", "section": "RESULTS", "ner": [ [ 30, 37, "mutated", "experimental_method" ], [ 38, 44, "Glu 80", "residue_name_number" ], [ 46, 52, "Asp 81", "residue_name_number" ], [ 57, 63, "Asp 83", "residue_name_number" ], [ 67, 70, "Ala", "residue_name" ] ] }, { "sid": 124, "sent": "In line with our hypothesis, E80A, D81A and D83A mutants exhibit at least 2-fold increase in hNaa60-activity (Fig. 5A).", "section": "RESULTS", "ner": [ [ 29, 33, "E80A", "mutant" ], [ 35, 39, "D81A", "mutant" ], [ 44, 48, "D83A", "mutant" ], [ 49, 56, "mutants", "protein_state" ], [ 93, 99, "hNaa60", "protein" ] ] }, { "sid": 125, "sent": "Interestingly, the structure of an ancestral NAT from S. solfataricus also exhibits a 10-residue long extension between \u03b23 and \u03b24, and the structure and biochemical studies showed that the extension of SsNat has the ability to stabilize structure of the active site and potentiate SsNat-activity.", "section": "RESULTS", "ner": [ [ 19, 28, "structure", "evidence" ], [ 45, 48, "NAT", "protein_type" ], [ 54, 69, "S. solfataricus", "species" ], [ 86, 111, "10-residue long extension", "structure_element" ], [ 120, 122, "\u03b23", "structure_element" ], [ 127, 129, "\u03b24", "structure_element" ], [ 139, 172, "structure and biochemical studies", "experimental_method" ], [ 189, 198, "extension", "structure_element" ], [ 202, 207, "SsNat", "protein" ], [ 254, 265, "active site", "site" ], [ 281, 286, "SsNat", "protein" ] ] }, { "sid": 126, "sent": "Nt-acetylation, which is carried out by the NAT family acetyltransferases, is an ancient and essential modification of proteins.", "section": "DISCUSS", "ner": [ [ 0, 14, "Nt-acetylation", "ptm" ], [ 44, 73, "NAT family acetyltransferases", "protein_type" ] ] }, { "sid": 127, "sent": "Although many NATs are highly conserved from lower to higher eukaryotes and the substrate bias of them appears to be partially overlapped, there is a significant increase in the overall level of N-terminal acetylation from lower to higher eukaryotes.", "section": "DISCUSS", "ner": [ [ 14, 18, "NATs", "protein_type" ], [ 23, 39, "highly conserved", "protein_state" ], [ 45, 50, "lower", "taxonomy_domain" ], [ 54, 71, "higher eukaryotes", "taxonomy_domain" ], [ 195, 217, "N-terminal acetylation", "ptm" ], [ 223, 228, "lower", "taxonomy_domain" ], [ 232, 249, "higher eukaryotes", "taxonomy_domain" ] ] }, { "sid": 128, "sent": "In this study we provide structural insights into Naa60 found only in multicellular eukaryotes.", "section": "DISCUSS", "ner": [ [ 50, 55, "Naa60", "protein" ], [ 70, 94, "multicellular eukaryotes", "taxonomy_domain" ] ] }, { "sid": 129, "sent": "The N-terminus of hNaa60 harbors three hydrophobic residues (VVP) that makes it very difficult to express and purify the protein.", "section": "DISCUSS", "ner": [ [ 18, 24, "hNaa60", "protein" ], [ 61, 64, "VVP", "structure_element" ] ] }, { "sid": 130, "sent": "This problem was solved by replacing residues 4\u20136 from VVP to EER that are found in Naa60 from Xenopus Laevis.", "section": "DISCUSS", "ner": [ [ 27, 36, "replacing", "experimental_method" ], [ 46, 49, "4\u20136", "residue_range" ], [ 55, 58, "VVP", "structure_element" ], [ 62, 65, "EER", "structure_element" ], [ 84, 89, "Naa60", "protein" ], [ 95, 109, "Xenopus Laevis", "species" ] ] }, { "sid": 131, "sent": "Since Naa60 from human and from Xenopus Laevis are highly homologous (Fig. 1A), we speculate that these two proteins should have the same biological function.", "section": "DISCUSS", "ner": [ [ 6, 11, "Naa60", "protein" ], [ 17, 22, "human", "species" ], [ 32, 46, "Xenopus Laevis", "species" ], [ 51, 68, "highly homologous", "protein_state" ] ] }, { "sid": 132, "sent": "Therefore it is deduced that the VVP to EER replacement on the N-terminus of hNaa60 may not interfere with its function.", "section": "DISCUSS", "ner": [ [ 33, 43, "VVP to EER", "mutant" ], [ 44, 55, "replacement", "experimental_method" ], [ 77, 83, "hNaa60", "protein" ] ] }, { "sid": 133, "sent": "However, in the hNaa60(1-242) structure the N-terminus adopts an \u03b1-helical structure which will probably be kinked if residue 6 is proline (Fig. 1C), and in the hNaa60(1-199) structure the N-terminus adopts a different semi-helical structure (Fig. 1B) likely due to different crystal packing.", "section": "DISCUSS", "ner": [ [ 16, 22, "hNaa60", "protein" ], [ 23, 28, "1-242", "residue_range" ], [ 30, 39, "structure", "evidence" ], [ 65, 84, "\u03b1-helical structure", "structure_element" ], [ 126, 127, "6", "residue_number" ], [ 131, 138, "proline", "residue_name" ], [ 161, 174, "hNaa60(1-199)", "mutant" ], [ 175, 184, "structure", "evidence" ], [ 219, 241, "semi-helical structure", "structure_element" ], [ 276, 291, "crystal packing", "evidence" ] ] }, { "sid": 134, "sent": "Hence it is not clear if the N-terminal end of wild-type hNaa60 is an \u03b1-helix, and what roles the hydrophobic residues 4\u20136 play in structure and function of wild-type hNaa60.", "section": "DISCUSS", "ner": [ [ 47, 56, "wild-type", "protein_state" ], [ 57, 63, "hNaa60", "protein" ], [ 70, 77, "\u03b1-helix", "structure_element" ], [ 119, 122, "4\u20136", "residue_range" ], [ 157, 166, "wild-type", "protein_state" ], [ 167, 173, "hNaa60", "protein" ] ] }, { "sid": 135, "sent": "In addition to the three-residue mutation (VVP to EER), we also tried many other hNaa60 constructs, but only the full-length protein and the truncated variant 1-199 behaved well.", "section": "DISCUSS", "ner": [ [ 33, 41, "mutation", "experimental_method" ], [ 43, 46, "VVP", "structure_element" ], [ 50, 53, "EER", "structure_element" ], [ 81, 87, "hNaa60", "protein" ], [ 113, 124, "full-length", "protein_state" ], [ 141, 150, "truncated", "protein_state" ], [ 159, 164, "1-199", "residue_range" ] ] }, { "sid": 136, "sent": "The finding that the catalytic activity of hNaa60(1-242) is much lower than that of hNaa60(1-199) is intriguing.", "section": "DISCUSS", "ner": [ [ 43, 49, "hNaa60", "protein" ], [ 50, 55, "1-242", "residue_range" ], [ 84, 97, "hNaa60(1-199)", "mutant" ] ] }, { "sid": 137, "sent": "We speculate that low activity of the full-length hNaa60 might be related to lack of Golgi localization of the enzyme in our in vitro studies or there remains some undiscovered auto-inhibitory regulation in the full-length protein.", "section": "DISCUSS", "ner": [ [ 38, 49, "full-length", "protein_state" ], [ 50, 56, "hNaa60", "protein" ], [ 211, 222, "full-length", "protein_state" ] ] }, { "sid": 138, "sent": "The hNaa60 protein was proven to be localized on Golgi apparatus.", "section": "DISCUSS", "ner": [ [ 4, 10, "hNaa60", "protein" ] ] }, { "sid": 139, "sent": "Aksnes and colleagues predicted putative transmembrane domains and two putative sites of S-palmitoylation, by bioinformatics means, to account for Golgi localization of the protein.", "section": "DISCUSS", "ner": [ [ 41, 62, "transmembrane domains", "structure_element" ], [ 89, 105, "S-palmitoylation", "ptm" ] ] }, { "sid": 140, "sent": "They then mutated all five cysteine residues of hNaa60\u2019s to serine, including the two putative S-palmitoylation sites.", "section": "DISCUSS", "ner": [ [ 10, 17, "mutated", "experimental_method" ], [ 27, 35, "cysteine", "residue_name" ], [ 48, 54, "hNaa60", "protein" ], [ 60, 66, "serine", "residue_name" ], [ 95, 117, "S-palmitoylation sites", "site" ] ] }, { "sid": 141, "sent": "However, these mutations did not abolish Naa60 membrane localization, indicating that S-palmitoylation is unlikely to (solely) account for targeting hNaa60 on Golgi.", "section": "DISCUSS", "ner": [ [ 15, 24, "mutations", "experimental_method" ], [ 41, 46, "Naa60", "protein" ], [ 86, 102, "S-palmitoylation", "ptm" ], [ 149, 155, "hNaa60", "protein" ] ] }, { "sid": 142, "sent": "Furthermore, adding residues 217\u2013242 of hNaa60 (containing residues 217\u2013236, one of the putative transmembrane domains) to the C terminus of eGFP were not sufficient to localize the protein on Golgi apparatus, while eGFP-hNaa60182-242 was sufficient to, suggesting that residues 182\u2013216 are important for Golgi localization of hNaa60.", "section": "DISCUSS", "ner": [ [ 13, 19, "adding", "experimental_method" ], [ 29, 36, "217\u2013242", "residue_range" ], [ 40, 46, "hNaa60", "protein" ], [ 68, 75, "217\u2013236", "residue_range" ], [ 97, 118, "transmembrane domains", "structure_element" ], [ 141, 145, "eGFP", "experimental_method" ], [ 216, 220, "eGFP", "experimental_method" ], [ 221, 234, "hNaa60182-242", "mutant" ], [ 279, 286, "182\u2013216", "residue_range" ], [ 327, 333, "hNaa60", "protein" ] ] }, { "sid": 143, "sent": "We found that residues 190\u2013202 formed an amphipathic helix with an array of hydrophobic residues located on one side.", "section": "DISCUSS", "ner": [ [ 23, 30, "190\u2013202", "residue_range" ], [ 41, 58, "amphipathic helix", "structure_element" ] ] }, { "sid": 144, "sent": "This observation is reminiscent of the protein/membrane interaction through amphipathic helices in the cases of KalSec14, Atg3, PB1-F2 etc.", "section": "DISCUSS", "ner": [ [ 76, 95, "amphipathic helices", "structure_element" ], [ 112, 120, "KalSec14", "protein" ], [ 122, 126, "Atg3", "protein" ], [ 128, 134, "PB1-F2", "protein" ] ] }, { "sid": 145, "sent": "In this model an amphipathic helix can immerse its hydrophobic side into the lipid bilayer through hydrophobic interactions.", "section": "DISCUSS", "ner": [ [ 17, 34, "amphipathic helix", "structure_element" ], [ 99, 123, "hydrophobic interactions", "bond_interaction" ] ] }, { "sid": 146, "sent": "Therefore we propose that the amphipathic helix \u03b15 may contribute to Golgi localization of hNaa60.", "section": "DISCUSS", "ner": [ [ 30, 47, "amphipathic helix", "structure_element" ], [ 48, 50, "\u03b15", "structure_element" ], [ 91, 97, "hNaa60", "protein" ] ] }, { "sid": 147, "sent": "Previous studies indicated that members of NAT family are bi-functional NAT and KAT enzymes.", "section": "DISCUSS", "ner": [ [ 43, 46, "NAT", "protein_type" ], [ 72, 75, "NAT", "protein_type" ], [ 80, 83, "KAT", "protein_type" ] ] }, { "sid": 148, "sent": "However, known structures of NATs do not well support this hypothesis, since the \u03b26-\u03b27 hairpin/loop of most of NATs is involved in the formation of a tunnel-like substrate-binding site with the \u03b11-\u03b12 loop, which would be good for the NAT but not KAT activity of the enzyme.", "section": "DISCUSS", "ner": [ [ 15, 25, "structures", "evidence" ], [ 29, 33, "NATs", "protein_type" ], [ 81, 94, "\u03b26-\u03b27 hairpin", "structure_element" ], [ 95, 99, "loop", "structure_element" ], [ 111, 115, "NATs", "protein_type" ], [ 150, 184, "tunnel-like substrate-binding site", "site" ], [ 194, 204, "\u03b11-\u03b12 loop", "structure_element" ], [ 234, 237, "NAT", "protein_type" ], [ 246, 249, "KAT", "protein_type" ] ] }, { "sid": 149, "sent": "Kinetic studies have been conducted to compare the NAT and KAT activity of hNaa50 in vitro, and indicate that the NAT activity of Naa50 is much higher than KAT activity.", "section": "DISCUSS", "ner": [ [ 0, 15, "Kinetic studies", "experimental_method" ], [ 51, 54, "NAT", "protein_type" ], [ 59, 62, "KAT", "protein_type" ], [ 75, 81, "hNaa50", "protein" ], [ 114, 117, "NAT", "protein_type" ], [ 130, 135, "Naa50", "protein" ], [ 156, 159, "KAT", "protein_type" ] ] }, { "sid": 150, "sent": "However, the substrate used in this study for assessing KAT activity was a small peptide which could not really mimic the 3D structure of a folded protein substrate in vivo.", "section": "DISCUSS", "ner": [ [ 56, 59, "KAT", "protein_type" ], [ 81, 88, "peptide", "chemical" ], [ 122, 134, "3D structure", "evidence" ], [ 140, 146, "folded", "protein_state" ] ] }, { "sid": 151, "sent": "Our mass spectrometry data indicated that there were robust acetylation of histone H3-H4 tetramer lysines and both N-terminal acetylation and lysine acetylation of the peptide used in the activity assay, thus confirmed the KAT activity of this enzyme in vitro.", "section": "DISCUSS", "ner": [ [ 4, 21, "mass spectrometry", "experimental_method" ], [ 22, 26, "data", "evidence" ], [ 60, 71, "acetylation", "ptm" ], [ 75, 82, "histone", "protein_type" ], [ 83, 88, "H3-H4", "complex_assembly" ], [ 89, 97, "tetramer", "oligomeric_state" ], [ 98, 105, "lysines", "residue_name" ], [ 115, 137, "N-terminal acetylation", "ptm" ], [ 142, 160, "lysine acetylation", "ptm" ], [ 168, 175, "peptide", "chemical" ], [ 188, 202, "activity assay", "experimental_method" ], [ 223, 226, "KAT", "protein_type" ] ] }, { "sid": 152, "sent": "Conformational change of the \u03b27-\u03b28 hairpin (corresponding to the \u03b26-\u03b27 loop of other NATs) is noted in our structures (Figs 1D and 2C), which might provide an explanation to the NAT/KAT dual-activity in a structural biological view, but we were unable to rule out the possibility that the observed conformational change of this hairpin might be an artifact related to crystal packing or truncation of the C-terminal end of the protein.", "section": "DISCUSS", "ner": [ [ 29, 42, "\u03b27-\u03b28 hairpin", "structure_element" ], [ 65, 75, "\u03b26-\u03b27 loop", "structure_element" ], [ 85, 89, "NATs", "protein_type" ], [ 107, 117, "structures", "evidence" ], [ 178, 181, "NAT", "protein_type" ], [ 182, 185, "KAT", "protein_type" ], [ 328, 335, "hairpin", "structure_element" ], [ 368, 383, "crystal packing", "evidence" ] ] }, { "sid": 153, "sent": "Further studies are therefore needed to reveal the mechanism for the KAT activity of this enzyme.", "section": "DISCUSS", "ner": [ [ 69, 72, "KAT", "protein_type" ] ] }, { "sid": 154, "sent": "In early years, researchers found adjustment of GCN5 histone acetyltransferase structure when it binds CoA molecule.", "section": "DISCUSS", "ner": [ [ 48, 78, "GCN5 histone acetyltransferase", "protein_type" ], [ 79, 88, "structure", "evidence" ], [ 103, 106, "CoA", "chemical" ] ] }, { "sid": 155, "sent": "The complexed form of NatA is more suitable for catalytic activation, since the \u03b11-\u03b12 loop undergoes a conformation change to participate in the formation of substrate-binding site when the auxiliary subunit Naa15 interacts with Naa10 (the catalytic subunit of NatA).", "section": "DISCUSS", "ner": [ [ 4, 13, "complexed", "protein_state" ], [ 22, 26, "NatA", "complex_assembly" ], [ 80, 90, "\u03b11-\u03b12 loop", "structure_element" ], [ 158, 180, "substrate-binding site", "site" ], [ 208, 213, "Naa15", "protein" ], [ 229, 234, "Naa10", "protein" ], [ 240, 249, "catalytic", "protein_state" ], [ 250, 257, "subunit", "structure_element" ], [ 261, 265, "NatA", "complex_assembly" ] ] }, { "sid": 156, "sent": "In the structure of hNaa50/CoA/peptide, Phe 27 in the \u03b11-\u03b12 loop appears to make hydrophobic interaction with the N-terminal Met of substrate peptide.", "section": "DISCUSS", "ner": [ [ 7, 16, "structure", "evidence" ], [ 20, 38, "hNaa50/CoA/peptide", "complex_assembly" ], [ 40, 46, "Phe 27", "residue_name_number" ], [ 54, 64, "\u03b11-\u03b12 loop", "structure_element" ], [ 81, 104, "hydrophobic interaction", "bond_interaction" ], [ 125, 128, "Met", "residue_name" ], [ 142, 149, "peptide", "chemical" ] ] }, { "sid": 157, "sent": "However, the hNaa60(1-242)/Ac-CoA crystal structure indicated that its counterpart in hNaa60, Phe 34, could also accommodate the binding of a hydrophilic malonate that occupied the substrate binding site although it maintained the same conformation as that observed in hNaa50.", "section": "DISCUSS", "ner": [ [ 13, 33, "hNaa60(1-242)/Ac-CoA", "complex_assembly" ], [ 34, 51, "crystal structure", "evidence" ], [ 86, 92, "hNaa60", "protein" ], [ 94, 100, "Phe 34", "residue_name_number" ], [ 154, 162, "malonate", "chemical" ], [ 181, 203, "substrate binding site", "site" ], [ 269, 275, "hNaa50", "protein" ] ] }, { "sid": 158, "sent": "Interestingly, the terminal thiol of CoA adopted alternative conformations in the structure of hNaa60(1-199)/CoA. One was to approach the substrate amine; the other was to approach the \u03b11-\u03b12 loop and away from the substrate amine.", "section": "DISCUSS", "ner": [ [ 28, 33, "thiol", "chemical" ], [ 37, 40, "CoA", "chemical" ], [ 82, 91, "structure", "evidence" ], [ 95, 112, "hNaa60(1-199)/CoA", "complex_assembly" ], [ 185, 195, "\u03b11-\u03b12 loop", "structure_element" ] ] }, { "sid": 159, "sent": "Same alternative conformations of CoA were observed in the hNaa60(1-199)(F34A) crystal structure, and our kinetic data showed that the F34A mutation abolished the activity of the enzyme.", "section": "DISCUSS", "ner": [ [ 34, 37, "CoA", "chemical" ], [ 59, 78, "hNaa60(1-199)(F34A)", "mutant" ], [ 79, 96, "crystal structure", "evidence" ], [ 106, 118, "kinetic data", "evidence" ], [ 135, 139, "F34A", "mutant" ], [ 140, 148, "mutation", "experimental_method" ] ] }, { "sid": 160, "sent": "Taken together, our data indicated that Phe 34 in hNaa60 may play a role in placing co-enzyme at the right location to facilitate the acetyl-transfer.", "section": "DISCUSS", "ner": [ [ 40, 46, "Phe 34", "residue_name_number" ], [ 50, 56, "hNaa60", "protein" ], [ 134, 140, "acetyl", "chemical" ] ] }, { "sid": 161, "sent": "However, these data did not rule out that possibility that Phe 34 may coordinate the binding of the N-terminal Met through hydrophobic interaction as was proposed by previous studies.", "section": "DISCUSS", "ner": [ [ 59, 65, "Phe 34", "residue_name_number" ], [ 111, 114, "Met", "residue_name" ], [ 123, 146, "hydrophobic interaction", "bond_interaction" ] ] }, { "sid": 162, "sent": "Furthermore, we showed that hNaa60 adopts the classical two base mechanism to catalyze acetyl-transfer.", "section": "DISCUSS", "ner": [ [ 28, 34, "hNaa60", "protein" ], [ 87, 93, "acetyl", "chemical" ] ] }, { "sid": 163, "sent": "Although sequence identity between hNaa60 and hNaa50 is low, key residues in the active site of both enzymes are highly conserved.", "section": "DISCUSS", "ner": [ [ 35, 41, "hNaa60", "protein" ], [ 46, 52, "hNaa50", "protein" ], [ 81, 92, "active site", "site" ], [ 113, 129, "highly conserved", "protein_state" ] ] }, { "sid": 164, "sent": "This can reasonably explain the high overlapping substrates specificities between hNaa60 and hNaa50.", "section": "DISCUSS", "ner": [ [ 82, 88, "hNaa60", "protein" ], [ 93, 99, "hNaa50", "protein" ] ] }, { "sid": 165, "sent": "Another structural feature of hNaa60 that distinguishes it from other NATs is the \u03b23-\u03b24 long loop which appears to inhibit the catalytic activity of hNaa60.", "section": "DISCUSS", "ner": [ [ 30, 36, "hNaa60", "protein" ], [ 70, 74, "NATs", "protein_type" ], [ 82, 97, "\u03b23-\u03b24 long loop", "structure_element" ], [ 149, 155, "hNaa60", "protein" ] ] }, { "sid": 166, "sent": "However, this loop also seems to stabilize the whole hNaa60 structure, because deletion mutations of this region led to protein precipitation and aggregation (Figure S7).", "section": "DISCUSS", "ner": [ [ 14, 18, "loop", "structure_element" ], [ 53, 59, "hNaa60", "protein" ], [ 60, 69, "structure", "evidence" ], [ 79, 97, "deletion mutations", "experimental_method" ] ] }, { "sid": 167, "sent": "A previous study suggested that the auto-acetylation of Lys 79 was important for hNaa60-activity, whereas the point mutation K79R did not decrease the activity of hNaa60 in our study.", "section": "DISCUSS", "ner": [ [ 36, 52, "auto-acetylation", "ptm" ], [ 56, 62, "Lys 79", "residue_name_number" ], [ 81, 87, "hNaa60", "protein" ], [ 110, 124, "point mutation", "experimental_method" ], [ 125, 129, "K79R", "mutant" ], [ 163, 169, "hNaa60", "protein" ] ] }, { "sid": 168, "sent": "Meanwhile, no electron density of acetyl group was found on Lys 79 in our structures and mass spectrometry analysis.", "section": "DISCUSS", "ner": [ [ 14, 30, "electron density", "evidence" ], [ 34, 40, "acetyl", "chemical" ], [ 60, 66, "Lys 79", "residue_name_number" ], [ 74, 84, "structures", "evidence" ], [ 89, 106, "mass spectrometry", "experimental_method" ] ] }, { "sid": 169, "sent": "Hence, it appears that the auto-acetylation of hNaa60 is not an essential modification for its activity for the protein we used here.", "section": "DISCUSS", "ner": [ [ 27, 43, "auto-acetylation", "ptm" ], [ 47, 53, "hNaa60", "protein" ] ] }, { "sid": 170, "sent": "As for the reason why K79R in Yang\u2019s previous studies reduced the activity of the enzyme, but in our studies it didn\u2019t, we suspect that the stability of this mutant may play some role.", "section": "DISCUSS", "ner": [ [ 22, 26, "K79R", "mutant" ], [ 158, 164, "mutant", "protein_state" ] ] }, { "sid": 171, "sent": "K79R is less stable than the wild-type enzyme as was judged by its poorer gel-filtration behavior and tendency to precipitate.", "section": "DISCUSS", "ner": [ [ 0, 4, "K79R", "mutant" ], [ 13, 19, "stable", "protein_state" ], [ 29, 38, "wild-type", "protein_state" ], [ 74, 88, "gel-filtration", "experimental_method" ] ] }, { "sid": 172, "sent": "In our studies we have paid special attention and carefully handled this protein to ensure that we did get enough of the protein in good condition for kinetic assays.", "section": "DISCUSS", "ner": [ [ 151, 165, "kinetic assays", "experimental_method" ] ] }, { "sid": 173, "sent": "The intracellular environment is more complicated than our in vitro assay and the substrate specificity of hNaa60 most focuses on transmembrane proteins.", "section": "DISCUSS", "ner": [ [ 107, 113, "hNaa60", "protein" ] ] }, { "sid": 174, "sent": "The interaction between hNaa60 and its substrates may involve the protein-membrane interaction which would further increase the complexity.", "section": "DISCUSS", "ner": [ [ 24, 30, "hNaa60", "protein" ] ] }, { "sid": 175, "sent": "It is not clear if the structure of hNaa60 is different in vivo or if other potential partner proteins may help to regulate its activity.", "section": "DISCUSS", "ner": [ [ 23, 32, "structure", "evidence" ], [ 36, 42, "hNaa60", "protein" ] ] }, { "sid": 176, "sent": "Nevertheless, our study may be an inspiration for further studies on the functions and regulation of this youngest member of the NAT family.", "section": "DISCUSS", "ner": [ [ 129, 132, "NAT", "protein_type" ] ] }, { "sid": 177, "sent": "Overall structure of Naa60.", "section": "FIG", "ner": [ [ 8, 17, "structure", "evidence" ], [ 21, 26, "Naa60", "protein" ] ] }, { "sid": 178, "sent": "(A) Sequence alignment of Naa60 (NatF, HAT4) from different species including Homo sapiens (Homo), Bos mutus (Bos), Salmo salar (Salmo) and Xenopus (Silurana) tropicalis (Xenopus).", "section": "FIG", "ner": [ [ 4, 22, "Sequence alignment", "experimental_method" ], [ 26, 31, "Naa60", "protein" ], [ 33, 37, "NatF", "complex_assembly" ], [ 39, 43, "HAT4", "protein" ], [ 78, 90, "Homo sapiens", "species" ], [ 92, 96, "Homo", "species" ], [ 99, 108, "Bos mutus", "species" ], [ 110, 113, "Bos", "species" ], [ 116, 127, "Salmo salar", "species" ], [ 129, 134, "Salmo", "species" ], [ 140, 147, "Xenopus", "species" ], [ 149, 157, "Silurana", "species" ], [ 159, 169, "tropicalis", "species" ], [ 171, 178, "Xenopus", "species" ] ] }, { "sid": 179, "sent": "Alignment was generated using NPS@ and ESPript.3.0 (http://espript.ibcp.fr/ESPript/ESPript/).", "section": "FIG", "ner": [ [ 0, 9, "Alignment", "experimental_method" ] ] }, { "sid": 180, "sent": "Residues 4\u20136 are highlighted in red box.", "section": "FIG", "ner": [ [ 9, 12, "4\u20136", "residue_range" ] ] }, { "sid": 181, "sent": "(B) The structure of hNaa60(1-199)/CoA complex is shown as a yellow cartoon model.", "section": "FIG", "ner": [ [ 8, 17, "structure", "evidence" ], [ 21, 38, "hNaa60(1-199)/CoA", "complex_assembly" ] ] }, { "sid": 182, "sent": "The CoA molecule is shown as sticks. (C) The structure of hNaa60(1-242)/Ac-CoA complex is presented as a cartoon model in cyan.", "section": "FIG", "ner": [ [ 4, 7, "CoA", "chemical" ], [ 45, 54, "structure", "evidence" ], [ 58, 78, "hNaa60(1-242)/Ac-CoA", "complex_assembly" ] ] }, { "sid": 183, "sent": "The Ac-CoA and malonate molecules are shown as cyan and purple sticks, respectively.", "section": "FIG", "ner": [ [ 4, 10, "Ac-CoA", "chemical" ], [ 15, 23, "malonate", "chemical" ] ] }, { "sid": 184, "sent": "The secondary structures are labeled starting with \u03b10. (D) Superposition of hNaa60(1-242) (cyan), hNaa60(1-199) (yellow) and hNaa50 (pink, PDB 3TFY).", "section": "FIG", "ner": [ [ 51, 53, "\u03b10", "structure_element" ], [ 59, 72, "Superposition", "experimental_method" ], [ 76, 82, "hNaa60", "protein" ], [ 83, 88, "1-242", "residue_range" ], [ 98, 111, "hNaa60(1-199)", "mutant" ], [ 125, 131, "hNaa50", "protein" ] ] }, { "sid": 185, "sent": "The Ac-CoA of hNaa60(1-242)/Ac-CoA complex is represented as cyan sticks.", "section": "FIG", "ner": [ [ 4, 10, "Ac-CoA", "chemical" ], [ 14, 34, "hNaa60(1-242)/Ac-CoA", "complex_assembly" ] ] }, { "sid": 186, "sent": "Amphipathicity of the \u03b15 helix and alternative conformations of the \u03b27-\u03b28 hairpin.", "section": "FIG", "ner": [ [ 0, 14, "Amphipathicity", "protein_state" ], [ 22, 30, "\u03b15 helix", "structure_element" ], [ 68, 81, "\u03b27-\u03b28 hairpin", "structure_element" ] ] }, { "sid": 187, "sent": "(A) The \u03b15 helix of hNaa60(1-242) in one asymmetric unit (slate) interacts with another hNaa60 molecule in a neighboring asymmetric unit (cyan).", "section": "FIG", "ner": [ [ 8, 16, "\u03b15 helix", "structure_element" ], [ 20, 26, "hNaa60", "protein" ], [ 27, 32, "1-242", "residue_range" ], [ 88, 94, "hNaa60", "protein" ] ] }, { "sid": 188, "sent": "Side-chains of hydrophobic residues on \u03b15 helix and the neighboring molecule participating in the interaction are shown as yellow and green sticks, respectively. (B) The \u03b15 helix of hNaa60(1-199) in one asymmetric unit (yellow) interacts with another hNaa60 molecule in the neighboring asymmetric units (green).", "section": "FIG", "ner": [ [ 39, 47, "\u03b15 helix", "structure_element" ], [ 170, 178, "\u03b15 helix", "structure_element" ], [ 182, 195, "hNaa60(1-199)", "mutant" ], [ 251, 257, "hNaa60", "protein" ] ] }, { "sid": 189, "sent": "Side-chains of hydrophobic residues on \u03b15 helix and the neighboring molecule (green) participating in the interaction are shown as yellow and green sticks, respectively.", "section": "FIG", "ner": [ [ 39, 47, "\u03b15 helix", "structure_element" ] ] }, { "sid": 190, "sent": "The third molecule (pink) does not directly interact with the \u03b15 helix.", "section": "FIG", "ner": [ [ 62, 70, "\u03b15 helix", "structure_element" ] ] }, { "sid": 191, "sent": "(C) Superposition of hNaa60(1-199) (yellow) and hNaa60(1-242) (cyan) showing conformational change of the \u03b27-\u03b28 hairpin in these two structures. (D,E) Superposition of Hat1p/H4 (gray, drawn from PDB 4PSW) with hNaa60(1-242) (cyan, D) or hNaa60(1-199) (yellow, E).", "section": "FIG", "ner": [ [ 4, 17, "Superposition", "experimental_method" ], [ 21, 34, "hNaa60(1-199)", "mutant" ], [ 48, 54, "hNaa60", "protein" ], [ 55, 60, "1-242", "residue_range" ], [ 106, 119, "\u03b27-\u03b28 hairpin", "structure_element" ], [ 133, 143, "structures", "evidence" ], [ 151, 164, "Superposition", "experimental_method" ], [ 168, 173, "Hat1p", "protein" ], [ 174, 176, "H4", "protein_type" ], [ 210, 216, "hNaa60", "protein" ], [ 217, 222, "1-242", "residue_range" ], [ 237, 250, "hNaa60(1-199)", "mutant" ] ] }, { "sid": 192, "sent": "The histone H4 peptide (a KAT substrate) bound to Hat1p is shown in purple (D,E), while the peptide bound to hNaa50 (a NAT substrate, drawn from PDB 3TFY) is shown in orange (Nt-peptide) after superimposing hNaa50 (not shown in figure) on hNaa60 (D).", "section": "FIG", "ner": [ [ 4, 11, "histone", "protein_type" ], [ 12, 14, "H4", "protein_type" ], [ 15, 22, "peptide", "chemical" ], [ 26, 29, "KAT", "protein_type" ], [ 41, 49, "bound to", "protein_state" ], [ 50, 55, "Hat1p", "protein" ], [ 92, 99, "peptide", "chemical" ], [ 100, 108, "bound to", "protein_state" ], [ 109, 115, "hNaa50", "protein" ], [ 119, 122, "NAT", "protein_type" ], [ 175, 185, "Nt-peptide", "chemical" ], [ 193, 206, "superimposing", "experimental_method" ], [ 207, 213, "hNaa50", "protein" ], [ 239, 245, "hNaa60", "protein" ] ] }, { "sid": 193, "sent": "The \u03b1-amine of the NAT substrate and \u03b5-amine of the KAT substrate (along with the lysine side-chain) subject to acetylation are shown as sticks.", "section": "FIG", "ner": [ [ 19, 22, "NAT", "protein_type" ], [ 52, 55, "KAT", "protein_type" ], [ 82, 88, "lysine", "residue_name" ], [ 112, 123, "acetylation", "ptm" ] ] }, { "sid": 194, "sent": "Electron density map of the active site.", "section": "FIG", "ner": [ [ 0, 20, "Electron density map", "evidence" ], [ 28, 39, "active site", "site" ] ] }, { "sid": 195, "sent": "The 2Fo-Fc maps contoured at 1.0\u03c3 are shown for hNaa60(1-242)/Ac-CoA (A), hNaa60(1-199)/CoA (B) and hNaa60(1-199) F34A/CoA (C).", "section": "FIG", "ner": [ [ 4, 15, "2Fo-Fc maps", "evidence" ], [ 48, 68, "hNaa60(1-242)/Ac-CoA", "complex_assembly" ], [ 74, 91, "hNaa60(1-199)/CoA", "complex_assembly" ], [ 100, 122, "hNaa60(1-199) F34A/CoA", "complex_assembly" ] ] }, { "sid": 196, "sent": "The putative substrate peptide binding site is indicated by the peptide (shown as pink sticks) from the hNaa50/CoA/peptide complex structure after superimposing hNaa50 on the hNaa60 structures determined in this study.", "section": "FIG", "ner": [ [ 13, 43, "substrate peptide binding site", "site" ], [ 64, 71, "peptide", "chemical" ], [ 104, 122, "hNaa50/CoA/peptide", "complex_assembly" ], [ 131, 140, "structure", "evidence" ], [ 147, 160, "superimposing", "experimental_method" ], [ 161, 167, "hNaa50", "protein" ], [ 175, 181, "hNaa60", "protein" ], [ 182, 192, "structures", "evidence" ] ] }, { "sid": 197, "sent": "The black arrow indicates the \u03b1-amine of the first Met (M1) (all panels).", "section": "FIG", "ner": [ [ 45, 54, "first Met", "residue_name_number" ], [ 56, 58, "M1", "residue_name_number" ] ] }, { "sid": 198, "sent": "The purple arrow indicates the acetyl moiety of Ac-CoA (A).", "section": "FIG", "ner": [ [ 31, 37, "acetyl", "chemical" ], [ 48, 54, "Ac-CoA", "chemical" ] ] }, { "sid": 199, "sent": "The red arrow indicates the alternative conformation of the thiol moiety of the co-enzyme when Phe 34 side-chain is displaced (B) or mutated to Ala (C).", "section": "FIG", "ner": [ [ 95, 101, "Phe 34", "residue_name_number" ], [ 133, 140, "mutated", "experimental_method" ], [ 144, 147, "Ala", "residue_name" ] ] }, { "sid": 200, "sent": "Structural basis for hNaa60 catalytic activity.", "section": "FIG", "ner": [ [ 21, 27, "hNaa60", "protein" ] ] }, { "sid": 201, "sent": "(A) Superposition of hNaa60 active site (cyan) on that of hNaa50 (pink, PDB 3TFY).", "section": "FIG", "ner": [ [ 4, 17, "Superposition", "experimental_method" ], [ 21, 27, "hNaa60", "protein" ], [ 28, 39, "active site", "site" ], [ 58, 64, "hNaa50", "protein" ] ] }, { "sid": 202, "sent": "Side-chains of key catalytic and substrate-binding residues are highlighted as sticks.", "section": "FIG", "ner": [ [ 19, 59, "catalytic and substrate-binding residues", "site" ] ] }, { "sid": 203, "sent": "The malonate molecule in the hNaa60(1-242)/Ac-CoA structure and the peptide in the hNaa50/CoA/peptide structure are shown as purple and yellow sticks respectively. (B) A close view of the active site of hNaa60.", "section": "FIG", "ner": [ [ 4, 12, "malonate", "chemical" ], [ 29, 49, "hNaa60(1-242)/Ac-CoA", "complex_assembly" ], [ 50, 59, "structure", "evidence" ], [ 68, 75, "peptide", "chemical" ], [ 83, 101, "hNaa50/CoA/peptide", "complex_assembly" ], [ 102, 111, "structure", "evidence" ], [ 188, 199, "active site", "site" ], [ 203, 209, "hNaa60", "protein" ] ] }, { "sid": 204, "sent": "Residues Glu 37, Tyr 97 and His 138 in hNaa60 (cyan) and corresponding residues (Tyr 73 and His 112) in hNaa50 (pink) as well as the side-chain of corresponding residues (Glu 24, His 72 and His 111) in complexed formed hNaa10p (warmpink) are highlighted as sticks.", "section": "FIG", "ner": [ [ 9, 15, "Glu 37", "residue_name_number" ], [ 17, 23, "Tyr 97", "residue_name_number" ], [ 28, 35, "His 138", "residue_name_number" ], [ 39, 45, "hNaa60", "protein" ], [ 81, 87, "Tyr 73", "residue_name_number" ], [ 92, 99, "His 112", "residue_name_number" ], [ 104, 110, "hNaa50", "protein" ], [ 171, 177, "Glu 24", "residue_name_number" ], [ 179, 185, "His 72", "residue_name_number" ], [ 190, 197, "His 111", "residue_name_number" ], [ 202, 211, "complexed", "protein_state" ], [ 219, 226, "hNaa10p", "protein" ] ] }, { "sid": 205, "sent": "The water molecules participating in catalysis in the hNaa60 and hNaa50 structures are showed as green and red spheres, separately. (C) The interaction between the malonate molecule and surrounding residues observed in the hNaa60(1-242)/Ac-CoA structure.", "section": "FIG", "ner": [ [ 4, 9, "water", "chemical" ], [ 54, 60, "hNaa60", "protein" ], [ 65, 71, "hNaa50", "protein" ], [ 72, 82, "structures", "evidence" ], [ 164, 172, "malonate", "chemical" ], [ 223, 243, "hNaa60(1-242)/Ac-CoA", "complex_assembly" ], [ 244, 253, "structure", "evidence" ] ] }, { "sid": 206, "sent": "The yellow dotted lines indicate the hydrogen bonds. (D) A zoomed view of \u03b23-\u03b24 loop of hNaa60.", "section": "FIG", "ner": [ [ 37, 51, "hydrogen bonds", "bond_interaction" ], [ 74, 84, "\u03b23-\u03b24 loop", "structure_element" ], [ 88, 94, "hNaa60", "protein" ] ] }, { "sid": 207, "sent": "Key residues discussed in the text (cyan), the malonate (purple) and Ac-CoA (gray) are shown as sticks.", "section": "FIG", "ner": [ [ 47, 55, "malonate", "chemical" ], [ 69, 75, "Ac-CoA", "chemical" ] ] }, { "sid": 208, "sent": "The yellow dotted lines indicate the salt bridges.", "section": "FIG", "ner": [ [ 37, 49, "salt bridges", "bond_interaction" ] ] }, { "sid": 209, "sent": "Catalytic activity of hNaa60 and mutant proteins.", "section": "FIG", "ner": [ [ 22, 28, "hNaa60", "protein" ], [ 33, 39, "mutant", "protein_state" ] ] }, { "sid": 210, "sent": "(A) Catalytic efficiency (shown as kcat/Km values) of hNaa60 (1-199) WT and mutants.", "section": "FIG", "ner": [ [ 4, 24, "Catalytic efficiency", "evidence" ], [ 35, 39, "kcat", "evidence" ], [ 40, 42, "Km", "evidence" ], [ 54, 68, "hNaa60 (1-199)", "mutant" ], [ 69, 71, "WT", "protein_state" ], [ 76, 83, "mutants", "protein_state" ] ] }, { "sid": 211, "sent": "(B) CD spectra of wild-type and mutant proteins from 250\u2009nm to 190\u2009nm.", "section": "FIG", "ner": [ [ 4, 6, "CD", "experimental_method" ], [ 7, 14, "spectra", "evidence" ], [ 18, 27, "wild-type", "protein_state" ], [ 32, 38, "mutant", "protein_state" ] ] }, { "sid": 212, "sent": "The sample concentration was 4.5\u2009\u03bcM in 20\u2009mM Tris, pH 8.0, 150\u2009mM NaCl, 1% glycerol and 1\u2009mM TCEP at room temperature.", "section": "FIG", "ner": [ [ 93, 97, "TCEP", "chemical" ] ] }, { "sid": 213, "sent": "Data collection and refinement statistics.", "section": "TABLE", "ner": [ [ 0, 41, "Data collection and refinement statistics", "evidence" ] ] }, { "sid": 214, "sent": "Structure and PDB ID\thNaa60(1-242)/Ac-CoA 5HGZ\thNaa60(1-199)/CoA 5HH0\thNaa60(1-199)F34A/CoA 5HH1\t \tData collection*\t \t\u2003Space group\tP212121\tP21212\tP21212\t \tCell dimensions\t \t\u2003a, b, c (\u00c5)\t53.3, 57.4, 68.8\t67.8, 73.8, 43.2\t66.7, 74.0, 43.5\t \t\u2003\u03b1,\u03b2,\u03b3 (\u00b0)\t90.0, 90.0, 90.0\t90.0, 90.0, 90.0\t90.0, 90.0, 90.0\t \tResolution (\u00c5)\t50\u20131.38 (1.42\u20131.38)\t50\u20131.60 (1.66\u20131.60)\t50\u20131.80 (1.86\u20131.80)\t \tRp.i.m.(%)**\t3.0 (34.4)\t2.1 (32.5)\t2.6 (47.8)\t \tI/\u03c3\t21.5 (2.0)\t31.8 (2.0)\t28.0 (2.4)\t \tCompleteness (%)\t99.8 (99.1)\t99.6 (98.5)\t99.9 (99.7)\t \tRedundancy\t6.9 (5.0)\t6.9 (6.2)\t6.3 (5.9)\t \tRefinement\t \t\u2003Resolution (\u00c5)\t25.81\u20131.38\t33.55\u20131.60\t43.52\u20131.80\t \t\u2003No. reflections\t43660\t28588\t20490\t \t\u2003Rwork/Rfree\t0.182/0.192\t0.181/0.184\t0.189/0.209\t \tNo. atoms\t \t\u2003Protein\t1717\t1576\t1566\t \t\u2003Ligand/ion\t116\t96\t96\t \t\u2003Water\t289\t258\t168\t \tB-factors\t \t\u2003Protein\t23.8\t32.0\t37.4\t \t\u2003Ligand/ion\t22.2\t34.6\t43.7\t \t\u2003Water\t35.1\t46.4\t49.1\t \tR.m.s.", "section": "TABLE", "ner": [ [ 21, 41, "hNaa60(1-242)/Ac-CoA", "complex_assembly" ], [ 47, 64, "hNaa60(1-199)/CoA", "complex_assembly" ], [ 70, 91, "hNaa60(1-199)F34A/CoA", "complex_assembly" ], [ 780, 785, "Water", "chemical" ], [ 868, 873, "Water", "chemical" ] ] }, { "sid": 215, "sent": "One crystal was used for each data set.", "section": "TABLE", "ner": [ [ 4, 11, "crystal", "evidence" ] ] }, { "sid": 216, "sent": "**Rp.i.m., a redundancy-independent R factor was used to evaluate the diffraction data quality as was proposed by Evans.", "section": "TABLE", "ner": [ [ 36, 44, "R factor", "evidence" ], [ 70, 86, "diffraction data", "evidence" ] ] } ] }, "PMC5014086": { "annotations": [ { "sid": 0, "sent": "Structure of the Dual-Mode Wnt Regulator Kremen1 and Insight into Ternary Complex Formation with LRP6 and Dickkopf", "section": "TITLE", "ner": [ [ 0, 9, "Structure", "evidence" ], [ 27, 30, "Wnt", "protein_type" ], [ 41, 48, "Kremen1", "protein" ], [ 97, 101, "LRP6", "protein" ], [ 106, 114, "Dickkopf", "protein_type" ] ] }, { "sid": 1, "sent": "Kremen 1 and 2 have been identified as co-receptors for Dickkopf (Dkk) proteins, hallmark secreted antagonists of canonical Wnt signaling.", "section": "ABSTRACT", "ner": [ [ 0, 14, "Kremen 1 and 2", "protein_type" ], [ 39, 51, "co-receptors", "protein_type" ], [ 56, 64, "Dickkopf", "protein_type" ], [ 66, 69, "Dkk", "protein_type" ], [ 124, 127, "Wnt", "protein_type" ] ] }, { "sid": 2, "sent": "We present here three crystal structures of the ectodomain of human Kremen1 (KRM1ECD) at resolutions between 1.9 and 3.2\u00a0\u00c5. KRM1ECD emerges as a rigid molecule with tight interactions stabilizing a triangular arrangement of its Kringle, WSC, and CUB structural domains.", "section": "ABSTRACT", "ner": [ [ 22, 40, "crystal structures", "evidence" ], [ 48, 58, "ectodomain", "structure_element" ], [ 62, 67, "human", "species" ], [ 68, 75, "Kremen1", "protein" ], [ 77, 81, "KRM1", "protein" ], [ 81, 84, "ECD", "structure_element" ], [ 124, 128, "KRM1", "protein" ], [ 128, 131, "ECD", "structure_element" ], [ 198, 220, "triangular arrangement", "protein_state" ], [ 228, 235, "Kringle", "structure_element" ], [ 237, 240, "WSC", "structure_element" ], [ 246, 249, "CUB", "structure_element" ] ] }, { "sid": 3, "sent": "The structures reveal an unpredicted homology of the WSC domain to hepatocyte growth factor.", "section": "ABSTRACT", "ner": [ [ 4, 14, "structures", "evidence" ], [ 53, 56, "WSC", "structure_element" ], [ 67, 91, "hepatocyte growth factor", "protein_type" ] ] }, { "sid": 4, "sent": "We further report the general architecture of the ternary complex formed by the Wnt co-receptor Lrp5/6, Dkk, and Krm, determined from a low-resolution complex crystal structure between \u03b2-propeller/EGF repeats (PE) 3 and 4 of the Wnt co-receptor LRP6 (LRP6PE3PE4), the cysteine-rich domain 2 (CRD2) of DKK1, and KRM1ECD.", "section": "ABSTRACT", "ner": [ [ 80, 83, "Wnt", "protein_type" ], [ 84, 95, "co-receptor", "protein_type" ], [ 96, 102, "Lrp5/6", "protein_type" ], [ 104, 107, "Dkk", "protein_type" ], [ 113, 116, "Krm", "protein_type" ], [ 159, 176, "crystal structure", "evidence" ], [ 185, 221, "\u03b2-propeller/EGF repeats (PE) 3 and 4", "structure_element" ], [ 229, 232, "Wnt", "protein_type" ], [ 233, 244, "co-receptor", "protein_type" ], [ 245, 249, "LRP6", "protein" ], [ 251, 255, "LRP6", "protein" ], [ 255, 261, "PE3PE4", "structure_element" ], [ 268, 290, "cysteine-rich domain 2", "structure_element" ], [ 292, 296, "CRD2", "structure_element" ], [ 301, 305, "DKK1", "protein" ], [ 311, 315, "KRM1", "protein" ], [ 315, 318, "ECD", "structure_element" ] ] }, { "sid": 5, "sent": "DKK1CRD2 is sandwiched between LRP6PE3 and KRM1Kringle-WSC.", "section": "ABSTRACT", "ner": [ [ 0, 4, "DKK1", "protein" ], [ 4, 8, "CRD2", "structure_element" ], [ 31, 35, "LRP6", "protein" ], [ 35, 38, "PE3", "structure_element" ], [ 43, 47, "KRM1", "protein" ], [ 47, 58, "Kringle-WSC", "structure_element" ] ] }, { "sid": 6, "sent": "Modeling studies supported by surface plasmon resonance suggest a direct interaction site between Krm1CUB and Lrp6PE2.", "section": "ABSTRACT", "ner": [ [ 0, 8, "Modeling", "experimental_method" ], [ 30, 55, "surface plasmon resonance", "experimental_method" ], [ 73, 89, "interaction site", "site" ], [ 98, 102, "Krm1", "protein" ], [ 102, 105, "CUB", "structure_element" ], [ 110, 114, "Lrp6", "protein" ], [ 114, 117, "PE2", "structure_element" ] ] }, { "sid": 7, "sent": "The structure of the KREMEN 1 ectodomain is solved from three crystal forms", "section": "ABSTRACT", "ner": [ [ 4, 13, "structure", "evidence" ], [ 21, 29, "KREMEN 1", "protein" ], [ 30, 40, "ectodomain", "structure_element" ], [ 44, 50, "solved", "experimental_method" ], [ 62, 75, "crystal forms", "evidence" ] ] }, { "sid": 8, "sent": "Kringle, WSC, and CUB subdomains interact tightly to form a single structural unit", "section": "ABSTRACT", "ner": [ [ 0, 7, "Kringle", "structure_element" ], [ 9, 12, "WSC", "structure_element" ], [ 18, 21, "CUB", "structure_element" ] ] }, { "sid": 9, "sent": "The interface to DKKs is formed from the Kringle and WSC domains", "section": "ABSTRACT", "ner": [ [ 4, 13, "interface", "site" ], [ 17, 21, "DKKs", "protein_type" ], [ 41, 48, "Kringle", "structure_element" ], [ 53, 56, "WSC", "structure_element" ] ] }, { "sid": 10, "sent": "The CUB domain is found to interact directly with LRP6PE1PE2", "section": "ABSTRACT", "ner": [ [ 4, 7, "CUB", "structure_element" ], [ 50, 54, "LRP6", "protein" ], [ 54, 60, "PE1PE2", "structure_element" ] ] }, { "sid": 11, "sent": "Zebisch et\u00a0al. describe the ectodomain structure of KREMEN 1, a receptor for Wnt antagonists of the DKK family.", "section": "ABSTRACT", "ner": [ [ 28, 38, "ectodomain", "structure_element" ], [ 39, 48, "structure", "evidence" ], [ 52, 60, "KREMEN 1", "protein" ], [ 64, 72, "receptor", "protein_type" ], [ 77, 80, "Wnt", "protein_type" ], [ 100, 103, "DKK", "protein_type" ] ] }, { "sid": 12, "sent": "Apo structures and a complex with functional fragments of DKK1 and LRP6 shed light on the function of this dual-mode regulator of Wnt signaling.", "section": "ABSTRACT", "ner": [ [ 0, 3, "Apo", "protein_state" ], [ 4, 14, "structures", "evidence" ], [ 21, 33, "complex with", "protein_state" ], [ 34, 54, "functional fragments", "protein_state" ], [ 58, 62, "DKK1", "protein" ], [ 67, 71, "LRP6", "protein" ], [ 130, 133, "Wnt", "protein_type" ] ] }, { "sid": 13, "sent": "Signaling by Wnt morphogens is renowned for its fundamental roles in embryonic development, tissue homeostasis, and stem cell maintenance.", "section": "INTRO", "ner": [ [ 13, 16, "Wnt", "protein_type" ] ] }, { "sid": 14, "sent": "Due to these functions, generation, delivery, and interpretation of Wnt signals are all heavily regulated in the animal body.", "section": "INTRO", "ner": [ [ 68, 71, "Wnt", "protein_type" ] ] }, { "sid": 15, "sent": "Vertebrate Dickkopf proteins (Dkk1, 2, and 4) are one of many secreted antagonists of Wnt and function by blocking access to the Wnt co-receptor LRP5/6.", "section": "INTRO", "ner": [ [ 0, 10, "Vertebrate", "taxonomy_domain" ], [ 11, 19, "Dickkopf", "protein_type" ], [ 30, 34, "Dkk1", "protein_type" ], [ 36, 37, "2", "protein_type" ], [ 43, 44, "4", "protein_type" ], [ 86, 89, "Wnt", "protein_type" ], [ 129, 132, "Wnt", "protein_type" ], [ 133, 144, "co-receptor", "protein_type" ], [ 145, 151, "LRP5/6", "protein" ] ] }, { "sid": 16, "sent": "Kremen proteins (Krm1 and Krm2) have been identified as additional high-affinity transmembrane receptors for Dkk.", "section": "INTRO", "ner": [ [ 0, 6, "Kremen", "protein_type" ], [ 17, 21, "Krm1", "protein_type" ], [ 26, 30, "Krm2", "protein_type" ], [ 81, 104, "transmembrane receptors", "protein_type" ], [ 109, 112, "Dkk", "protein_type" ] ] }, { "sid": 17, "sent": "Krm and Dkk synergize in Wnt inhibition during Xenopus embryogenesis to regulate anterior-posterior patterning.", "section": "INTRO", "ner": [ [ 0, 3, "Krm", "protein_type" ], [ 8, 11, "Dkk", "protein_type" ], [ 25, 28, "Wnt", "protein_type" ], [ 47, 54, "Xenopus", "taxonomy_domain" ] ] }, { "sid": 18, "sent": "Mechanistically it is thought that, in the presence of Dkk, Krm forms a ternary complex with Lrp6, which is then rapidly endocytosed.", "section": "INTRO", "ner": [ [ 43, 54, "presence of", "protein_state" ], [ 55, 58, "Dkk", "protein_type" ], [ 60, 63, "Krm", "protein_type" ], [ 80, 92, "complex with", "protein_state" ], [ 93, 97, "Lrp6", "protein_type" ] ] }, { "sid": 19, "sent": "This amplifies the intrinsic Wnt antagonistic activity of Dkk by efficiently depleting the cell surface of the Wnt co-receptor.", "section": "INTRO", "ner": [ [ 29, 32, "Wnt", "protein_type" ], [ 58, 61, "Dkk", "protein_type" ], [ 111, 114, "Wnt", "protein_type" ], [ 115, 126, "co-receptor", "protein_type" ] ] }, { "sid": 20, "sent": "In accordance with this, Krm1\u2212/\u2212 and Krm2\u2212/\u2212 double knockout mice show a high bone mass phenotype typical of increased Wnt signaling, as well as growth of ectopic forelimb digits.", "section": "INTRO", "ner": [ [ 25, 29, "Krm1", "protein_type" ], [ 37, 41, "Krm2", "protein_type" ], [ 45, 60, "double knockout", "experimental_method" ], [ 61, 65, "mice", "taxonomy_domain" ], [ 119, 122, "Wnt", "protein_type" ] ] }, { "sid": 21, "sent": "Growth of ectopic digits is further enhanced upon additional loss of dkk expression.", "section": "INTRO", "ner": [ [ 69, 72, "dkk", "protein_type" ] ] }, { "sid": 22, "sent": "The Wnt antagonistic activity of Krm1 is also linked to its importance for correct thymus epithelium formation in mice.", "section": "INTRO", "ner": [ [ 4, 7, "Wnt", "protein_type" ], [ 33, 37, "Krm1", "protein_type" ], [ 114, 118, "mice", "taxonomy_domain" ] ] }, { "sid": 23, "sent": "The importance of intact KRM1 for normal human development and health is highlighted by the recent finding that a homozygous mutation in the ectodomain of KRM1 leads to severe ectodermal dysplasia including oligodontia.", "section": "INTRO", "ner": [ [ 18, 24, "intact", "protein_state" ], [ 25, 29, "KRM1", "protein" ], [ 41, 46, "human", "species" ], [ 141, 151, "ectodomain", "structure_element" ], [ 155, 159, "KRM1", "protein" ] ] }, { "sid": 24, "sent": "Interestingly, the Wnt antagonistic activity of Krm is context dependent, and Krm proteins are actually dual-mode Wnt regulators.", "section": "INTRO", "ner": [ [ 19, 22, "Wnt", "protein_type" ], [ 48, 51, "Krm", "protein_type" ], [ 78, 81, "Krm", "protein_type" ], [ 114, 117, "Wnt", "protein_type" ] ] }, { "sid": 25, "sent": "In the absence of Dkk, Krm1 and 2 change their function from inhibition to enhancement of Lrp6-mediated signaling.", "section": "INTRO", "ner": [ [ 7, 17, "absence of", "protein_state" ], [ 18, 21, "Dkk", "protein_type" ], [ 23, 27, "Krm1", "protein_type" ], [ 32, 33, "2", "protein_type" ], [ 90, 94, "Lrp6", "protein_type" ] ] }, { "sid": 26, "sent": "By direct binding to Lrp6 via the ectodomains, Krm proteins promote Lrp6 cell-surface localization and hence increase receptor availability.", "section": "INTRO", "ner": [ [ 21, 25, "Lrp6", "protein_type" ], [ 34, 45, "ectodomains", "structure_element" ], [ 47, 50, "Krm", "protein_type" ], [ 68, 72, "Lrp6", "protein_type" ] ] }, { "sid": 27, "sent": "Further increasing the complexity of Krm functionality, it was recently found that Krm1 (but not Krm2) can also act independently of LRP5/6 and Wnt as a dependence receptor, triggering apoptosis unless bound to Dkk.", "section": "INTRO", "ner": [ [ 37, 40, "Krm", "protein_type" ], [ 83, 87, "Krm1", "protein_type" ], [ 97, 101, "Krm2", "protein_type" ], [ 133, 139, "LRP5/6", "protein" ], [ 144, 147, "Wnt", "protein_type" ], [ 202, 210, "bound to", "protein_state" ], [ 211, 214, "Dkk", "protein_type" ] ] }, { "sid": 28, "sent": "Structurally, Krm1 and 2 are type I transmembrane proteins with a 40\u00a0kDa ectodomain and a flexible cytoplasmic tail consisting of 60\u201375 residues.", "section": "INTRO", "ner": [ [ 14, 18, "Krm1", "protein_type" ], [ 23, 24, "2", "protein_type" ], [ 29, 58, "type I transmembrane proteins", "protein_type" ], [ 73, 83, "ectodomain", "structure_element" ], [ 90, 98, "flexible", "protein_state" ], [ 99, 115, "cytoplasmic tail", "structure_element" ], [ 130, 132, "60", "residue_range" ], [ 133, 135, "75", "residue_range" ] ] }, { "sid": 29, "sent": "The ectodomain consists of three similarly sized structural domains of around 10\u00a0kDa each: the N-terminal Kringle domain (KR) is followed by a WSC domain of unknown fold.", "section": "INTRO", "ner": [ [ 4, 14, "ectodomain", "structure_element" ], [ 106, 113, "Kringle", "structure_element" ], [ 122, 124, "KR", "structure_element" ], [ 143, 146, "WSC", "structure_element" ] ] }, { "sid": 30, "sent": "The third structural domain is a CUB domain.", "section": "INTRO", "ner": [ [ 33, 36, "CUB", "structure_element" ] ] }, { "sid": 31, "sent": "An approximately 70-residue linker connects the CUB domain to the transmembrane span.", "section": "INTRO", "ner": [ [ 3, 27, "approximately 70-residue", "residue_range" ], [ 28, 34, "linker", "structure_element" ], [ 48, 51, "CUB", "structure_element" ], [ 66, 84, "transmembrane span", "structure_element" ] ] }, { "sid": 32, "sent": "An intact KR-WSC-CUB domain triplet and membrane attachment is required for Wnt antagonism.", "section": "INTRO", "ner": [ [ 3, 9, "intact", "protein_state" ], [ 10, 20, "KR-WSC-CUB", "structure_element" ], [ 76, 79, "Wnt", "protein_type" ] ] }, { "sid": 33, "sent": "The transmembrane span and cytoplasmic tail can be replaced with a GPI linker without impact on Wnt antagonism.", "section": "INTRO", "ner": [ [ 4, 22, "transmembrane span", "structure_element" ], [ 27, 43, "cytoplasmic tail", "structure_element" ], [ 67, 70, "GPI", "structure_element" ], [ 71, 77, "linker", "structure_element" ], [ 96, 99, "Wnt", "protein_type" ] ] }, { "sid": 34, "sent": "The structures presented here reveal the unknown fold of the WSC domain and the tight interactions of all three domains.", "section": "INTRO", "ner": [ [ 4, 14, "structures", "evidence" ], [ 61, 64, "WSC", "structure_element" ] ] }, { "sid": 35, "sent": "We further succeeded in determination of a low-resolution LRP6PE3PE4-DKK1CRD2-KRM1ECD complex, defining the architecture of the Wnt inhibitory complex that leads to Lrp6 cell-surface depletion.", "section": "INTRO", "ner": [ [ 58, 85, "LRP6PE3PE4-DKK1CRD2-KRM1ECD", "complex_assembly" ], [ 128, 131, "Wnt", "protein_type" ], [ 132, 150, "inhibitory complex", "complex_assembly" ], [ 165, 169, "Lrp6", "protein" ] ] }, { "sid": 36, "sent": "The recombinant production of the extracellular domain of Krm for structural studies proved challenging (see Experimental Procedures).", "section": "RESULTS", "ner": [ [ 34, 54, "extracellular domain", "structure_element" ], [ 58, 61, "Krm", "protein_type" ], [ 66, 84, "structural studies", "experimental_method" ] ] }, { "sid": 37, "sent": "We succeeded in purifying KRM1ECD complexes with DKK1fl, DKK1Linker-CRD2, and DKK1CRD2 that were monodisperse and stable in gel filtration, hence indicating at least micromolar affinity (data not shown).", "section": "RESULTS", "ner": [ [ 26, 30, "KRM1", "protein" ], [ 30, 33, "ECD", "structure_element" ], [ 34, 48, "complexes with", "protein_state" ], [ 49, 55, "DKK1fl", "protein" ], [ 57, 61, "DKK1", "protein" ], [ 61, 72, "Linker-CRD2", "structure_element" ], [ 78, 82, "DKK1", "protein" ], [ 82, 86, "CRD2", "structure_element" ], [ 124, 138, "gel filtration", "experimental_method" ] ] }, { "sid": 38, "sent": "Several crystal forms were obtained from these complexes, however, crystals always contained only KRM1 protein.", "section": "RESULTS", "ner": [ [ 8, 21, "crystal forms", "evidence" ], [ 67, 75, "crystals", "evidence" ], [ 98, 102, "KRM1", "protein" ] ] }, { "sid": 39, "sent": "We solved the structure of KRM1ECD in three crystal forms at 1.9, 2.8, and 3.2\u00a0\u00c5 resolution (Table 1).", "section": "RESULTS", "ner": [ [ 3, 9, "solved", "experimental_method" ], [ 14, 23, "structure", "evidence" ], [ 27, 31, "KRM1", "protein" ], [ 31, 34, "ECD", "structure_element" ] ] }, { "sid": 40, "sent": "The high-resolution structure is a near full-length model (Figure\u00a01).", "section": "RESULTS", "ner": [ [ 20, 29, "structure", "evidence" ], [ 40, 51, "full-length", "protein_state" ] ] }, { "sid": 41, "sent": "The small, flexible, and charged 98AEHED102 loop could only be modeled in a slightly lower resolution structure and in crystal form III.", "section": "RESULTS", "ner": [ [ 4, 9, "small", "protein_state" ], [ 11, 19, "flexible", "protein_state" ], [ 25, 32, "charged", "protein_state" ], [ 33, 48, "98AEHED102 loop", "structure_element" ], [ 102, 111, "structure", "evidence" ] ] }, { "sid": 42, "sent": "The KR, WSC, and CUB are arranged in a roughly triangular fashion with tight interactions between all three domains.", "section": "RESULTS", "ner": [ [ 4, 6, "KR", "structure_element" ], [ 8, 11, "WSC", "structure_element" ], [ 17, 20, "CUB", "structure_element" ] ] }, { "sid": 43, "sent": "The KR domain, which bears two of the four glycosylation sites, contains the canonical three disulfide bridges (C32-C114, C55-C95, C84-C109) and, like other Kringle domains, is low in secondary structure elements.", "section": "RESULTS", "ner": [ [ 4, 6, "KR", "structure_element" ], [ 43, 62, "glycosylation sites", "site" ], [ 93, 110, "disulfide bridges", "ptm" ], [ 112, 115, "C32", "residue_name_number" ], [ 116, 120, "C114", "residue_name_number" ], [ 122, 125, "C55", "residue_name_number" ], [ 126, 129, "C95", "residue_name_number" ], [ 131, 134, "C84", "residue_name_number" ], [ 135, 139, "C109", "residue_name_number" ], [ 157, 164, "Kringle", "structure_element" ] ] }, { "sid": 44, "sent": "The structurally most similar Kringle domain is that of human plasminogen (PDB: 1PKR) with an root-mean-square deviation (RMSD) of 1.7\u00a0\u00c5 for 73 aligned C\u03b1 (Figure\u00a01B).", "section": "RESULTS", "ner": [ [ 30, 37, "Kringle", "structure_element" ], [ 56, 61, "human", "species" ], [ 62, 73, "plasminogen", "protein" ], [ 94, 120, "root-mean-square deviation", "evidence" ], [ 122, 126, "RMSD", "evidence" ] ] }, { "sid": 45, "sent": "The KRM1 structure reveals the fold of the WSC domain for the first time.", "section": "RESULTS", "ner": [ [ 4, 8, "KRM1", "protein" ], [ 9, 18, "structure", "evidence" ], [ 43, 46, "WSC", "structure_element" ] ] }, { "sid": 46, "sent": "The structure is best described as a sandwich of a \u03b21-\u03b25-\u03b23-\u03b24-\u03b22 antiparallel \u03b2 sheet and a single \u03b1 helix.", "section": "RESULTS", "ner": [ [ 4, 13, "structure", "evidence" ], [ 37, 45, "sandwich", "structure_element" ], [ 51, 86, "\u03b21-\u03b25-\u03b23-\u03b24-\u03b22 antiparallel \u03b2 sheet", "structure_element" ], [ 100, 107, "\u03b1 helix", "structure_element" ] ] }, { "sid": 47, "sent": "The structure is also rich in loops and is stabilized by four disulfide bridges (C122-C186, C147-C167, C151-C169, C190-C198).", "section": "RESULTS", "ner": [ [ 4, 13, "structure", "evidence" ], [ 30, 35, "loops", "structure_element" ], [ 62, 79, "disulfide bridges", "ptm" ], [ 81, 85, "C122", "residue_name_number" ], [ 86, 90, "C186", "residue_name_number" ], [ 92, 96, "C147", "residue_name_number" ], [ 97, 101, "C167", "residue_name_number" ], [ 103, 107, "C151", "residue_name_number" ], [ 108, 112, "C169", "residue_name_number" ], [ 114, 118, "C190", "residue_name_number" ], [ 119, 123, "C198", "residue_name_number" ] ] }, { "sid": 48, "sent": "Using the PDBeFold server, we detected a surprising yet significant homology to PAN module domains.", "section": "RESULTS", "ner": [ [ 10, 25, "PDBeFold server", "experimental_method" ], [ 80, 98, "PAN module domains", "structure_element" ] ] }, { "sid": 49, "sent": "The closest structural relative is hepatocyte growth factor (HGF, PDB: 1GP9), which superposes with an RMSD of 2.3\u00a0\u00c5 for 58 aligned C\u03b1 (Figure\u00a01B).", "section": "RESULTS", "ner": [ [ 35, 59, "hepatocyte growth factor", "protein_type" ], [ 61, 64, "HGF", "protein_type" ], [ 84, 94, "superposes", "experimental_method" ], [ 103, 107, "RMSD", "evidence" ] ] }, { "sid": 50, "sent": "The CUB domain bears two glycosylation sites.", "section": "RESULTS", "ner": [ [ 4, 7, "CUB", "structure_element" ], [ 25, 44, "glycosylation sites", "site" ] ] }, { "sid": 51, "sent": "Although present, the quality of the electron density around N217 did not allow modeling of the sugar moiety.", "section": "RESULTS", "ner": [ [ 37, 53, "electron density", "evidence" ], [ 61, 65, "N217", "residue_name_number" ] ] }, { "sid": 52, "sent": "In crystal form I, a calcium ion is present at the canonical position coordinated by the carboxylates of D263, D266 (bidentate), and D306, as well as the carbonyl of N309 and a water molecule.", "section": "RESULTS", "ner": [ [ 3, 17, "crystal form I", "evidence" ], [ 21, 28, "calcium", "chemical" ], [ 70, 84, "coordinated by", "bond_interaction" ], [ 105, 109, "D263", "residue_name_number" ], [ 111, 115, "D266", "residue_name_number" ], [ 133, 137, "D306", "residue_name_number" ], [ 166, 170, "N309", "residue_name_number" ], [ 177, 182, "water", "chemical" ] ] }, { "sid": 53, "sent": "The coordination sphere deviates significantly from perfectly octahedral (not shown).", "section": "RESULTS", "ner": [ [ 4, 23, "coordination sphere", "site" ] ] }, { "sid": 54, "sent": "This might result in the site having a low affinity and may explain why calcium is not present in the two low-resolution crystal forms.", "section": "RESULTS", "ner": [ [ 72, 79, "calcium", "chemical" ], [ 121, 134, "crystal forms", "evidence" ] ] }, { "sid": 55, "sent": "Loss of calcium has led to loop rearrangements and partial disorder in these crystal forms.", "section": "RESULTS", "ner": [ [ 0, 7, "Loss of", "protein_state" ], [ 8, 15, "calcium", "chemical" ], [ 27, 31, "loop", "structure_element" ], [ 77, 90, "crystal forms", "evidence" ] ] }, { "sid": 56, "sent": "The closest structural relative is the CUB_C domain of Tsg-6 (PDB: 2WNO), which superposes with KRMCUB with an RMSD of 1.6\u00a0\u00c5 for 104 C\u03b1 (Figure\u00a01B).", "section": "RESULTS", "ner": [ [ 39, 44, "CUB_C", "structure_element" ], [ 55, 60, "Tsg-6", "protein" ], [ 80, 90, "superposes", "experimental_method" ], [ 96, 99, "KRM", "protein" ], [ 99, 102, "CUB", "structure_element" ], [ 111, 115, "RMSD", "evidence" ] ] }, { "sid": 57, "sent": "A superposition of the three KRM1 structures reveals no major structural differences (Figure\u00a01C) as anticipated from the plethora of interactions between the three domains.", "section": "RESULTS", "ner": [ [ 2, 15, "superposition", "experimental_method" ], [ 29, 33, "KRM1", "protein" ], [ 34, 44, "structures", "evidence" ] ] }, { "sid": 58, "sent": "Minor differences are caused by the collapse of the Ca2+ binding site in crystal forms II and III and loop flexibility in the KR domain.", "section": "RESULTS", "ner": [ [ 52, 69, "Ca2+ binding site", "site" ], [ 73, 97, "crystal forms II and III", "evidence" ], [ 102, 106, "loop", "structure_element" ], [ 126, 128, "KR", "structure_element" ] ] }, { "sid": 59, "sent": "The F207S mutation recently found to cause ectodermal dysplasia in Palestinian families maps to the hydrophobic core of the protein at the interface of the three subdomains (Figure\u00a01A).", "section": "RESULTS", "ner": [ [ 4, 9, "F207S", "mutant" ], [ 100, 116, "hydrophobic core", "site" ], [ 139, 148, "interface", "site" ] ] }, { "sid": 60, "sent": "Such a mutation is bound to severely destabilize the protein structure of KRM1, leading to disturbance of its Wnt antagonistic, Wnt stimulatory, and Wnt independent activity.", "section": "RESULTS", "ner": [ [ 19, 27, "bound to", "protein_state" ], [ 74, 78, "KRM1", "protein" ], [ 110, 113, "Wnt", "protein_type" ], [ 128, 131, "Wnt", "protein_type" ], [ 149, 152, "Wnt", "protein_type" ] ] }, { "sid": 61, "sent": "Co-crystallization of LRP6PE3PE4 with DKK1CRD2, and LRP6PE1 with an N-terminal peptide of DKK1 has provided valuable structural insight into direct Wnt inhibition by Dkk ligands.", "section": "RESULTS", "ner": [ [ 0, 18, "Co-crystallization", "experimental_method" ], [ 22, 26, "LRP6", "protein" ], [ 26, 32, "PE3PE4", "structure_element" ], [ 38, 42, "DKK1", "protein" ], [ 42, 46, "CRD2", "structure_element" ], [ 52, 56, "LRP6", "protein" ], [ 56, 59, "PE1", "structure_element" ], [ 90, 94, "DKK1", "protein" ], [ 148, 151, "Wnt", "protein_type" ], [ 166, 169, "Dkk", "protein_type" ] ] }, { "sid": 62, "sent": "One face of the rather flat DKK1CRD2 fragment binds to the third \u03b2 propeller of LRP6.", "section": "RESULTS", "ner": [ [ 23, 27, "flat", "protein_state" ], [ 28, 32, "DKK1", "protein" ], [ 32, 36, "CRD2", "structure_element" ], [ 46, 54, "binds to", "protein_state" ], [ 59, 76, "third \u03b2 propeller", "structure_element" ], [ 80, 84, "LRP6", "protein" ] ] }, { "sid": 63, "sent": "Mutational analyses further implied that the LRP6PE3-averted face of DKK1CRD2 bears the Krm binding site, hence suggesting how Dkk can recruit both receptors into a ternary complex.", "section": "RESULTS", "ner": [ [ 0, 19, "Mutational analyses", "experimental_method" ], [ 45, 49, "LRP6", "protein" ], [ 49, 52, "PE3", "structure_element" ], [ 69, 73, "DKK1", "protein" ], [ 73, 77, "CRD2", "structure_element" ], [ 88, 104, "Krm binding site", "site" ], [ 127, 130, "Dkk", "protein_type" ], [ 148, 157, "receptors", "protein_type" ] ] }, { "sid": 64, "sent": "To obtain direct insight into ternary complex formation by Lrp5/6, Dkk, and Krm, we subjected an LRP6PE3PE4-DKK1fl-KRM1ECD complex to crystallization trials.", "section": "RESULTS", "ner": [ [ 59, 65, "Lrp5/6", "protein_type" ], [ 67, 70, "Dkk", "protein_type" ], [ 76, 79, "Krm", "protein_type" ], [ 97, 122, "LRP6PE3PE4-DKK1fl-KRM1ECD", "complex_assembly" ], [ 134, 156, "crystallization trials", "experimental_method" ] ] }, { "sid": 65, "sent": "Diffraction data collected from the resulting crystals were highly anisotropic with diffraction extending in the best directions to 3.5\u00a0\u00c5 and 3.7\u00a0\u00c5 but only to 6.4\u00a0\u00c5 in the third direction.", "section": "RESULTS", "ner": [ [ 0, 16, "Diffraction data", "evidence" ], [ 46, 54, "crystals", "evidence" ] ] }, { "sid": 66, "sent": "Despite the lack of high-resolution diffraction, the general architecture of the ternary complex is revealed (Figure\u00a02A).", "section": "RESULTS", "ner": [ [ 36, 47, "diffraction", "evidence" ] ] }, { "sid": 67, "sent": "DKK1CRD2 binds to the top face of the LRP6 PE3 \u03b2 propeller as described earlier for the binary complex.", "section": "RESULTS", "ner": [ [ 0, 4, "DKK1", "protein" ], [ 4, 8, "CRD2", "structure_element" ], [ 9, 17, "binds to", "protein_state" ], [ 38, 42, "LRP6", "protein" ], [ 43, 46, "PE3", "structure_element" ], [ 47, 58, "\u03b2 propeller", "structure_element" ] ] }, { "sid": 68, "sent": "KRM1ECD does indeed bind on the opposite side of DKK1CRD2 with only its KR and WSC domains engaged in binding (Figure\u00a02A).", "section": "RESULTS", "ner": [ [ 0, 4, "KRM1", "protein" ], [ 4, 7, "ECD", "structure_element" ], [ 20, 27, "bind on", "protein_state" ], [ 49, 53, "DKK1", "protein" ], [ 53, 57, "CRD2", "structure_element" ], [ 72, 74, "KR", "structure_element" ], [ 79, 82, "WSC", "structure_element" ] ] }, { "sid": 69, "sent": "Although present in the complex subjected to crystallization, we observe no density that could correspond to CRD1 or the domain linker\u00a0(L).", "section": "RESULTS", "ner": [ [ 45, 60, "crystallization", "experimental_method" ], [ 76, 83, "density", "evidence" ], [ 109, 113, "CRD1", "structure_element" ], [ 121, 134, "domain linker", "structure_element" ], [ 136, 137, "L", "structure_element" ] ] }, { "sid": 70, "sent": "We confirm that the CRD2 of DKK1 is required and sufficient for binding to KRM1: In surface plasmon resonance (SPR), we measured low micromolar affinity between full-length DKK1 and immobilized KRM1ECD (Figure\u00a02B).", "section": "RESULTS", "ner": [ [ 20, 24, "CRD2", "structure_element" ], [ 28, 32, "DKK1", "protein" ], [ 75, 79, "KRM1", "protein" ], [ 84, 109, "surface plasmon resonance", "experimental_method" ], [ 111, 114, "SPR", "experimental_method" ], [ 144, 152, "affinity", "evidence" ], [ 161, 172, "full-length", "protein_state" ], [ 173, 177, "DKK1", "protein" ], [ 194, 198, "KRM1", "protein" ], [ 198, 201, "ECD", "structure_element" ] ] }, { "sid": 71, "sent": "A SUMO fusion of DKK1L-CRD2 displayed a similar (slightly higher) affinity.", "section": "RESULTS", "ner": [ [ 2, 13, "SUMO fusion", "experimental_method" ], [ 17, 27, "DKK1L-CRD2", "structure_element" ], [ 66, 74, "affinity", "evidence" ] ] }, { "sid": 72, "sent": "In contrast, a SUMO fusion of DKK1CRD1-L did not display binding for concentrations tested up to 325\u00a0\u03bcM (Figure\u00a02B).", "section": "RESULTS", "ner": [ [ 15, 26, "SUMO fusion", "experimental_method" ], [ 30, 40, "DKK1CRD1-L", "structure_element" ] ] }, { "sid": 73, "sent": "Overall, the DKK1-KRM1 interface is characterized by a large number of polar interactions but only few hydrophobic contacts (Figure\u00a02C).", "section": "RESULTS", "ner": [ [ 13, 32, "DKK1-KRM1 interface", "site" ], [ 71, 89, "polar interactions", "bond_interaction" ], [ 103, 123, "hydrophobic contacts", "bond_interaction" ] ] }, { "sid": 74, "sent": "The crystal structure gives an explanation for DKK1 loss-of-binding mutations identified previously: R191 of DKK1 forms a double salt bridge to D125 and E162 of KRM1 (Figure\u00a02C).", "section": "RESULTS", "ner": [ [ 4, 21, "crystal structure", "evidence" ], [ 47, 51, "DKK1", "protein" ], [ 101, 105, "R191", "residue_name_number" ], [ 109, 113, "DKK1", "protein" ], [ 129, 140, "salt bridge", "bond_interaction" ], [ 144, 148, "D125", "residue_name_number" ], [ 153, 157, "E162", "residue_name_number" ], [ 161, 165, "KRM1", "protein" ] ] }, { "sid": 75, "sent": "A charge reversal as in the mouse Dkk1 (mDkk1) R197E variant would severely disrupt the binding.", "section": "RESULTS", "ner": [ [ 2, 17, "charge reversal", "experimental_method" ], [ 28, 33, "mouse", "taxonomy_domain" ], [ 34, 38, "Dkk1", "protein" ], [ 40, 45, "mDkk1", "protein" ], [ 47, 52, "R197E", "mutant" ] ] }, { "sid": 76, "sent": "Similarly, the K226 side chain of DKK1, which points to a small hydrophobic pocket on the surface of KRM1 formed by Y108, W94, and W106, forms salt bridges with the side chains of KRM1 D88 and D90.", "section": "RESULTS", "ner": [ [ 15, 19, "K226", "residue_name_number" ], [ 34, 38, "DKK1", "protein" ], [ 64, 82, "hydrophobic pocket", "site" ], [ 101, 105, "KRM1", "protein" ], [ 116, 120, "Y108", "residue_name_number" ], [ 122, 125, "W94", "residue_name_number" ], [ 131, 135, "W106", "residue_name_number" ], [ 143, 155, "salt bridges", "bond_interaction" ], [ 180, 184, "KRM1", "protein" ], [ 185, 188, "D88", "residue_name_number" ], [ 193, 196, "D90", "residue_name_number" ] ] }, { "sid": 77, "sent": "Again, a charge reversal as shown before for mDkk1 K232E would be incompatible with binding.", "section": "RESULTS", "ner": [ [ 9, 24, "charge reversal", "experimental_method" ], [ 45, 50, "mDkk1", "protein" ], [ 51, 56, "K232E", "mutant" ] ] }, { "sid": 78, "sent": "The side chain of DKK1 S192 was also predicted to be involved in Krm binding.", "section": "RESULTS", "ner": [ [ 18, 22, "DKK1", "protein" ], [ 23, 27, "S192", "residue_name_number" ], [ 65, 68, "Krm", "protein_type" ] ] }, { "sid": 79, "sent": "Indeed, we found (Figure\u00a02C) that the side chain of D201 of KRM1 forms two hydrogen bonds to the side-chain hydroxyl and the backbone amide of S192 (mouse, S198).", "section": "RESULTS", "ner": [ [ 52, 56, "D201", "residue_name_number" ], [ 60, 64, "KRM1", "protein" ], [ 75, 89, "hydrogen bonds", "bond_interaction" ], [ 143, 147, "S192", "residue_name_number" ], [ 149, 154, "mouse", "taxonomy_domain" ], [ 156, 160, "S198", "residue_name_number" ] ] }, { "sid": 80, "sent": "Additional polar interactions are formed between the N140, S163, and Y165 side chains of KRM1 and DKK1 backbone carbonyls of W206, L190, and C189, respectively.", "section": "RESULTS", "ner": [ [ 11, 29, "polar interactions", "bond_interaction" ], [ 53, 57, "N140", "residue_name_number" ], [ 59, 63, "S163", "residue_name_number" ], [ 69, 73, "Y165", "residue_name_number" ], [ 89, 93, "KRM1", "protein" ], [ 98, 102, "DKK1", "protein" ], [ 125, 129, "W206", "residue_name_number" ], [ 131, 135, "L190", "residue_name_number" ], [ 141, 145, "C189", "residue_name_number" ] ] }, { "sid": 81, "sent": "The carbonyl of DKK1 R224 is hydrogen bonded to Y105 and W106 of KRM1.", "section": "RESULTS", "ner": [ [ 16, 20, "DKK1", "protein" ], [ 21, 25, "R224", "residue_name_number" ], [ 29, 44, "hydrogen bonded", "bond_interaction" ], [ 48, 52, "Y105", "residue_name_number" ], [ 57, 61, "W106", "residue_name_number" ], [ 65, 69, "KRM1", "protein" ] ] }, { "sid": 82, "sent": "We suspect that the Dkk charge reversal mutations performed in the murine background and shown to\u00a0diminish Krm binding K211E and R203E (mouse K217E and\u00a0R209E) do so likely indirectly by disruption\u00a0of the Dkk fold.", "section": "RESULTS", "ner": [ [ 20, 23, "Dkk", "protein_type" ], [ 24, 49, "charge reversal mutations", "experimental_method" ], [ 67, 73, "murine", "taxonomy_domain" ], [ 107, 110, "Krm", "protein_type" ], [ 119, 124, "K211E", "mutant" ], [ 129, 134, "R203E", "mutant" ], [ 136, 141, "mouse", "taxonomy_domain" ], [ 142, 147, "K217E", "mutant" ], [ 152, 157, "R209E", "mutant" ], [ 204, 207, "Dkk", "protein_type" ] ] }, { "sid": 83, "sent": "We further validated the DKK1 binding site\u00a0on\u00a0KRM1 by introducing glycosylation sites at the KR (90DVS92\u2192NVS) and WSC (189VCF191\u2192NCS) domains pointing toward DKK (Figures 2A and 2D).", "section": "RESULTS", "ner": [ [ 25, 42, "DKK1 binding site", "site" ], [ 46, 50, "KRM1", "protein" ], [ 54, 65, "introducing", "experimental_method" ], [ 66, 85, "glycosylation sites", "site" ], [ 93, 95, "KR", "structure_element" ], [ 97, 108, "90DVS92\u2192NVS", "mutant" ], [ 114, 117, "WSC", "structure_element" ], [ 119, 132, "189VCF191\u2192NCS", "mutant" ], [ 158, 161, "DKK", "protein" ] ] }, { "sid": 84, "sent": "Introduction of N-linked glycans in protein-protein-binding sites is an established way of disrupting protein-binding interfaces.", "section": "RESULTS", "ner": [ [ 16, 32, "N-linked glycans", "ptm" ], [ 36, 65, "protein-protein-binding sites", "site" ], [ 102, 128, "protein-binding interfaces", "site" ] ] }, { "sid": 85, "sent": "Both ectodomain mutants were secreted comparably with the wild-type, indicating correct folding, but failed to achieve any detectable binding in SPR using full-length DKK1 as analyte.", "section": "RESULTS", "ner": [ [ 5, 15, "ectodomain", "structure_element" ], [ 16, 23, "mutants", "protein_state" ], [ 58, 67, "wild-type", "protein_state" ], [ 145, 148, "SPR", "experimental_method" ], [ 155, 166, "full-length", "protein_state" ], [ 167, 171, "DKK1", "protein" ] ] }, { "sid": 86, "sent": "In contrast, a mutant carrying an additional N-glycan outside the interface at the CUB domain (309NQA311\u2192NQS), was wild-type-like in DKK1 binding (Figure\u00a02D).", "section": "RESULTS", "ner": [ [ 15, 21, "mutant", "protein_state" ], [ 45, 53, "N-glycan", "ptm" ], [ 66, 75, "interface", "site" ], [ 83, 86, "CUB", "structure_element" ], [ 95, 108, "309NQA311\u2192NQS", "mutant" ], [ 115, 124, "wild-type", "protein_state" ], [ 133, 137, "DKK1", "protein" ] ] }, { "sid": 87, "sent": "Identification of a Direct LRP6-KRM1 Binding Site", "section": "RESULTS", "ner": [ [ 27, 49, "LRP6-KRM1 Binding Site", "site" ] ] }, { "sid": 88, "sent": "The LRP6PE3PE4-DKK1CRD2-KRM1ECD complex structure reveals no direct interactions between KRM1 and LRP6.", "section": "RESULTS", "ner": [ [ 4, 31, "LRP6PE3PE4-DKK1CRD2-KRM1ECD", "complex_assembly" ], [ 40, 49, "structure", "evidence" ], [ 89, 93, "KRM1", "protein" ], [ 98, 102, "LRP6", "protein" ] ] }, { "sid": 89, "sent": "We constructed in\u00a0silico a ternary complex with a close to full-length LRP6 ectodomain (PE1PE2PE3PE4 horse shoe) similar to but without refinement against electron microscopy (EM) or small-angle X-ray scattering data.", "section": "RESULTS", "ner": [ [ 35, 47, "complex with", "protein_state" ], [ 59, 70, "full-length", "protein_state" ], [ 71, 75, "LRP6", "protein" ], [ 76, 86, "ectodomain", "structure_element" ], [ 88, 100, "PE1PE2PE3PE4", "structure_element" ], [ 101, 111, "horse shoe", "structure_element" ], [ 155, 174, "electron microscopy", "experimental_method" ], [ 176, 178, "EM", "experimental_method" ], [ 183, 211, "small-angle X-ray scattering", "experimental_method" ] ] }, { "sid": 90, "sent": "An auxiliary PE3PE4 fragment was superimposed via PE4 onto PE3 of the crystal structure, and the LRP6PE1PE2 structure was superimposed via PE2 onto PE3 of this auxiliary fragment (Figure\u00a03A).", "section": "RESULTS", "ner": [ [ 13, 19, "PE3PE4", "structure_element" ], [ 33, 45, "superimposed", "experimental_method" ], [ 50, 53, "PE4", "structure_element" ], [ 59, 62, "PE3", "structure_element" ], [ 70, 87, "crystal structure", "evidence" ], [ 97, 101, "LRP6", "protein" ], [ 101, 107, "PE1PE2", "structure_element" ], [ 108, 117, "structure", "evidence" ], [ 122, 134, "superimposed", "experimental_method" ], [ 139, 142, "PE2", "structure_element" ], [ 148, 151, "PE3", "structure_element" ] ] }, { "sid": 91, "sent": "For this crude approximation of a true ternary complex, we noted very close proximity between the Ca2+-binding region of KRM1 and the top face of the PE2 \u03b2 propeller of LRP6.", "section": "RESULTS", "ner": [ [ 98, 117, "Ca2+-binding region", "site" ], [ 121, 125, "KRM1", "protein" ], [ 150, 153, "PE2", "structure_element" ], [ 154, 165, "\u03b2 propeller", "structure_element" ], [ 169, 173, "LRP6", "protein" ] ] }, { "sid": 92, "sent": "The solvent-exposed residues R307, I308, and N309 of the central Ca2+-binding \u03b2 connection loop of KRM1 would be almost ideally positioned for binding to this face, which is commonly used as a binding site on \u03b2 propellers.", "section": "RESULTS", "ner": [ [ 4, 19, "solvent-exposed", "protein_state" ], [ 29, 33, "R307", "residue_name_number" ], [ 35, 39, "I308", "residue_name_number" ], [ 45, 49, "N309", "residue_name_number" ], [ 65, 95, "Ca2+-binding \u03b2 connection loop", "structure_element" ], [ 99, 103, "KRM1", "protein" ], [ 193, 205, "binding site", "site" ], [ 209, 221, "\u03b2 propellers", "structure_element" ] ] }, { "sid": 93, "sent": "Peptides containing arginine/lysine, isoleucine, and asparagine (consensus sequence N-X-I-(G)-R/K) are also employed by DKK1 and SOST to bind to LRP6 (albeit to propeller 1; Figure\u00a03B).", "section": "RESULTS", "ner": [ [ 20, 28, "arginine", "residue_name" ], [ 29, 35, "lysine", "residue_name" ], [ 37, 47, "isoleucine", "residue_name" ], [ 53, 63, "asparagine", "residue_name" ], [ 84, 97, "N-X-I-(G)-R/K", "structure_element" ], [ 120, 124, "DKK1", "protein" ], [ 129, 133, "SOST", "protein" ], [ 145, 149, "LRP6", "protein" ], [ 161, 172, "propeller 1", "structure_element" ] ] }, { "sid": 94, "sent": "To support the hypothesis that KRM1CUB binds to LRP6PE2, we used SPR and compared binding of the wild-type and the GlycoCUB mutant of KRM1ECD (bearing an N-glycosylation site at N309) with a purified LRP6PE1PE2 fragment.", "section": "RESULTS", "ner": [ [ 31, 35, "KRM1", "protein" ], [ 35, 38, "CUB", "structure_element" ], [ 39, 47, "binds to", "protein_state" ], [ 48, 52, "LRP6", "protein" ], [ 52, 55, "PE2", "structure_element" ], [ 65, 68, "SPR", "experimental_method" ], [ 97, 106, "wild-type", "protein_state" ], [ 115, 130, "GlycoCUB mutant", "protein_state" ], [ 134, 138, "KRM1", "protein" ], [ 138, 141, "ECD", "structure_element" ], [ 154, 174, "N-glycosylation site", "site" ], [ 178, 182, "N309", "residue_name_number" ], [ 200, 204, "LRP6", "protein" ], [ 204, 210, "PE1PE2", "structure_element" ] ] }, { "sid": 95, "sent": "Indeed, we found that in the absence of Dkk, KRM1ECD bound with considerable affinity to LRP6PE1PE2 (Figure\u00a03C).", "section": "RESULTS", "ner": [ [ 29, 39, "absence of", "protein_state" ], [ 40, 43, "Dkk", "protein_type" ], [ 45, 49, "KRM1", "protein" ], [ 49, 52, "ECD", "structure_element" ], [ 53, 58, "bound", "protein_state" ], [ 86, 88, "to", "protein_state" ], [ 89, 93, "LRP6", "protein" ], [ 93, 99, "PE1PE2", "structure_element" ] ] }, { "sid": 96, "sent": "In contrast, no saturable binding was observed between KRM1 and LRP6PE3PE4.", "section": "RESULTS", "ner": [ [ 55, 59, "KRM1", "protein" ], [ 64, 68, "LRP6", "protein" ], [ 68, 74, "PE3PE4", "structure_element" ] ] }, { "sid": 97, "sent": "Introduction of an N-glycosylation site at N309 in KRM1ECD abolished LRP6PE1PE2 binding (Figure\u00a03C), while binding to DKK1 was unaffected (Figure\u00a02D).", "section": "RESULTS", "ner": [ [ 0, 15, "Introduction of", "experimental_method" ], [ 19, 39, "N-glycosylation site", "site" ], [ 43, 47, "N309", "residue_name_number" ], [ 51, 55, "KRM1", "protein" ], [ 55, 58, "ECD", "structure_element" ], [ 69, 73, "LRP6", "protein" ], [ 73, 79, "PE1PE2", "structure_element" ], [ 118, 122, "DKK1", "protein" ] ] }, { "sid": 98, "sent": "We conclude that the predicted binding site between KRM1CUB and LRP6PE2 is a strong candidate for mediating the direct Lrp6-Krm interaction, which is thought to increase Wnt responsiveness by stabilizing Lrp6 at the cell surface.", "section": "RESULTS", "ner": [ [ 31, 43, "binding site", "site" ], [ 52, 56, "KRM1", "protein" ], [ 56, 59, "CUB", "structure_element" ], [ 64, 68, "LRP6", "protein" ], [ 68, 71, "PE2", "structure_element" ], [ 119, 127, "Lrp6-Krm", "complex_assembly" ], [ 170, 173, "Wnt", "protein_type" ], [ 204, 208, "Lrp6", "protein" ] ] }, { "sid": 99, "sent": "Further experiments are required to pinpoint the exact binding site.", "section": "RESULTS", "ner": [ [ 55, 67, "binding site", "site" ] ] }, { "sid": 100, "sent": "Although LRP6PE1 appears somewhat out of reach in the modeled ternary complex, it cannot be excluded as the Krm binding site in the ternary complex and LRP6-Krm binary complex.", "section": "RESULTS", "ner": [ [ 9, 13, "LRP6", "protein" ], [ 13, 16, "PE1", "structure_element" ], [ 108, 124, "Krm binding site", "site" ], [ 152, 160, "LRP6-Krm", "complex_assembly" ] ] }, { "sid": 101, "sent": "The presence of DKK may govern which propeller (PE1 versus PE2) of LRP6 is available for Krm binding.", "section": "RESULTS", "ner": [ [ 4, 15, "presence of", "protein_state" ], [ 16, 19, "DKK", "protein" ], [ 37, 46, "propeller", "structure_element" ], [ 48, 51, "PE1", "structure_element" ], [ 59, 62, "PE2", "structure_element" ], [ 67, 71, "LRP6", "protein" ], [ 89, 92, "Krm", "protein_type" ] ] }, { "sid": 102, "sent": "Apparent binding across the proposed KRM1CUB-LRP6PE2 interface is expected to be higher once Krm is also cross-linked to LRP6PE3 via DKK1CRD2 (Figure\u00a03D).", "section": "RESULTS", "ner": [ [ 37, 62, "KRM1CUB-LRP6PE2 interface", "site" ], [ 93, 96, "Krm", "protein_type" ], [ 121, 125, "LRP6", "protein" ], [ 125, 128, "PE3", "structure_element" ], [ 133, 137, "DKK1", "protein" ], [ 137, 141, "CRD2", "structure_element" ] ] }, { "sid": 103, "sent": "Low-resolution negative-stain EM and small-angle X-ray scattering studies of LRP6PE1PE2PE3PE4, in isolation and in complex with Dkk1, plus negative-stain EM of full-length LRP6 ectodomain, have indicated curved, platform-like conformations but also potential flexibility between PE2 and PE3.", "section": "RESULTS", "ner": [ [ 15, 32, "negative-stain EM", "experimental_method" ], [ 37, 65, "small-angle X-ray scattering", "experimental_method" ], [ 77, 81, "LRP6", "protein" ], [ 81, 93, "PE1PE2PE3PE4", "structure_element" ], [ 95, 107, "in isolation", "protein_state" ], [ 112, 127, "in complex with", "protein_state" ], [ 128, 132, "Dkk1", "protein_type" ], [ 139, 156, "negative-stain EM", "experimental_method" ], [ 160, 171, "full-length", "protein_state" ], [ 172, 176, "LRP6", "protein" ], [ 177, 187, "ectodomain", "structure_element" ], [ 204, 210, "curved", "protein_state" ], [ 212, 225, "platform-like", "protein_state" ], [ 279, 282, "PE2", "structure_element" ], [ 287, 290, "PE3", "structure_element" ] ] }, { "sid": 104, "sent": "It is therefore possible that the interplay of Krm and Dkk binding can promote changes in LRP6 ectodomain conformation with functional consequences; however, such ideas await investigation.", "section": "RESULTS", "ner": [ [ 47, 50, "Krm", "protein_type" ], [ 55, 58, "Dkk", "protein_type" ], [ 90, 94, "LRP6", "protein" ], [ 95, 105, "ectodomain", "structure_element" ] ] }, { "sid": 105, "sent": "Taken together, the structural and biophysical studies we report here extend our mechanistic understanding of Wnt signal regulation.", "section": "RESULTS", "ner": [ [ 20, 54, "structural and biophysical studies", "experimental_method" ], [ 110, 113, "Wnt", "protein_type" ] ] }, { "sid": 106, "sent": "We describe the ectodomain structure of the dual Wnt regulator Krm1, providing an explanation for the detrimental effect on health and development of a homozygous KRM1 mutation.", "section": "RESULTS", "ner": [ [ 16, 26, "ectodomain", "structure_element" ], [ 27, 36, "structure", "evidence" ], [ 49, 52, "Wnt", "protein_type" ], [ 63, 67, "Krm1", "protein_type" ], [ 163, 167, "KRM1", "protein" ] ] }, { "sid": 107, "sent": "We also reveal the interaction mode of Krm-Dkk and the architecture of the ternary complex formed by Lrp5/6, Dkk, and Krm.", "section": "RESULTS", "ner": [ [ 39, 46, "Krm-Dkk", "complex_assembly" ], [ 101, 107, "Lrp5/6", "protein_type" ], [ 109, 112, "Dkk", "protein_type" ], [ 118, 121, "Krm", "protein_type" ] ] }, { "sid": 108, "sent": "Furthermore, the ternary crystal structure has guided in\u00a0silico and biophysical analyses to suggest a direct LRP6-KRM1 interaction site.", "section": "RESULTS", "ner": [ [ 25, 42, "crystal structure", "evidence" ], [ 54, 88, "in\u00a0silico and biophysical analyses", "experimental_method" ], [ 109, 135, "LRP6-KRM1 interaction site", "site" ] ] }, { "sid": 109, "sent": "Our findings provide a solid foundation for additional studies to probe how ternary complex formation triggers internalization, whereas Krm binding in the absence of Dkk stabilizes the Wnt co-receptor at the cell surface.", "section": "RESULTS", "ner": [ [ 136, 139, "Krm", "protein_type" ], [ 155, 165, "absence of", "protein_state" ], [ 166, 169, "Dkk", "protein_type" ], [ 185, 188, "Wnt", "protein_type" ], [ 189, 200, "co-receptor", "protein_type" ] ] }, { "sid": 110, "sent": "Structure of Unliganded KRM1ECD", "section": "FIG", "ner": [ [ 0, 9, "Structure", "evidence" ], [ 13, 23, "Unliganded", "protein_state" ], [ 24, 28, "KRM1", "protein" ], [ 28, 31, "ECD", "structure_element" ] ] }, { "sid": 111, "sent": "(A) The KRM1ECD fold (crystal form I) colored blue to red from the N to C terminus.", "section": "FIG", "ner": [ [ 8, 12, "KRM1", "protein" ], [ 12, 15, "ECD", "structure_element" ], [ 22, 36, "crystal form I", "evidence" ] ] }, { "sid": 112, "sent": "Cysteines as ball and sticks, glycosylation sites as sticks.", "section": "FIG", "ner": [ [ 0, 9, "Cysteines", "residue_name" ], [ 30, 49, "glycosylation sites", "site" ] ] }, { "sid": 113, "sent": "The bound calcium is shown as a gray sphere.", "section": "FIG", "ner": [ [ 10, 17, "calcium", "chemical" ] ] }, { "sid": 114, "sent": "The site of the\u00a0F207S mutation associated with ectodermal dysplasia in humans is shown as mesh.", "section": "FIG", "ner": [ [ 16, 21, "F207S", "mutant" ], [ 71, 77, "humans", "species" ] ] }, { "sid": 115, "sent": "(B) Superposition of the three KRM1ECD subdomains (solid) with their next structurally characterized homologs (half transparent).", "section": "FIG", "ner": [ [ 4, 17, "Superposition", "experimental_method" ], [ 31, 35, "KRM1", "protein" ], [ 35, 38, "ECD", "structure_element" ] ] }, { "sid": 116, "sent": "(C) Superposition of KRM1ECD from the three crystal forms.", "section": "FIG", "ner": [ [ 4, 17, "Superposition", "experimental_method" ], [ 21, 25, "KRM1", "protein" ], [ 25, 28, "ECD", "structure_element" ], [ 44, 57, "crystal forms", "evidence" ] ] }, { "sid": 117, "sent": "Alignment scores for each pairing are indicated on the dashed triangle.", "section": "FIG", "ner": [ [ 0, 16, "Alignment scores", "evidence" ] ] }, { "sid": 118, "sent": "(A) The structure of the ternary LRP6PE3PE4-DKK1CRD2-KRM1ECD complex.", "section": "FIG", "ner": [ [ 8, 17, "structure", "evidence" ], [ 33, 60, "LRP6PE3PE4-DKK1CRD2-KRM1ECD", "complex_assembly" ] ] }, { "sid": 119, "sent": "DKK1 (orange) is sandwiched between the PE3 module of LRP6 (blue) and the KR-WSC domain pair of KRM1 (green).", "section": "FIG", "ner": [ [ 0, 4, "DKK1", "protein" ], [ 40, 43, "PE3", "structure_element" ], [ 54, 58, "LRP6", "protein" ], [ 74, 80, "KR-WSC", "structure_element" ], [ 96, 100, "KRM1", "protein" ] ] }, { "sid": 120, "sent": "Colored symbols indicate introduced N-glycan attachment sites (see D).", "section": "FIG", "ner": [ [ 36, 61, "N-glycan attachment sites", "site" ] ] }, { "sid": 121, "sent": "(B) SPR data comparing binding of full-length DKK1 and SUMO fusions of DKK1 truncations for binding to immobilized wild-type KRM1ECD.", "section": "FIG", "ner": [ [ 4, 7, "SPR", "experimental_method" ], [ 34, 45, "full-length", "protein_state" ], [ 46, 50, "DKK1", "protein" ], [ 55, 67, "SUMO fusions", "experimental_method" ], [ 71, 75, "DKK1", "protein" ], [ 115, 124, "wild-type", "protein_state" ], [ 125, 129, "KRM1", "protein" ], [ 129, 132, "ECD", "structure_element" ] ] }, { "sid": 122, "sent": "(C) Close-up view of the DKK1CRD2-KRM1ECD interface.", "section": "FIG", "ner": [ [ 25, 51, "DKK1CRD2-KRM1ECD interface", "site" ] ] }, { "sid": 123, "sent": "Residues involved in interface formation are shown as sticks; those mentioned in the text are labeled.", "section": "FIG", "ner": [ [ 21, 30, "interface", "site" ] ] }, { "sid": 124, "sent": "Salt bridges are in pink and hydrogen bonds in black.", "section": "FIG", "ner": [ [ 0, 12, "Salt bridges", "bond_interaction" ], [ 29, 43, "hydrogen bonds", "bond_interaction" ] ] }, { "sid": 125, "sent": "(D) SPR binding data comparing DKK1 analyte binding with wild-type KRM1ECD and three variants bearing engineered glycosylation sites on the KR and WSC domains (green and blue pointing to DKK1) and on the CUB domain (orange).", "section": "FIG", "ner": [ [ 4, 7, "SPR", "experimental_method" ], [ 8, 20, "binding data", "evidence" ], [ 31, 35, "DKK1", "protein" ], [ 57, 66, "wild-type", "protein_state" ], [ 67, 71, "KRM1", "protein" ], [ 71, 74, "ECD", "structure_element" ], [ 102, 112, "engineered", "protein_state" ], [ 113, 132, "glycosylation sites", "site" ], [ 140, 142, "KR", "structure_element" ], [ 147, 150, "WSC", "structure_element" ], [ 187, 191, "DKK1", "protein" ], [ 204, 207, "CUB", "structure_element" ] ] }, { "sid": 126, "sent": "LRP6-KRM1 Direct Interaction and Summary", "section": "FIG", "ner": [ [ 0, 9, "LRP6-KRM1", "complex_assembly" ] ] }, { "sid": 127, "sent": "(A) In a construction of a ternary complex with all four \u03b2 propellers of LRP6 intact, the CUB domain points via its Ca2+-binding region toward the top face of the second \u03b2 propeller.", "section": "FIG", "ner": [ [ 35, 47, "complex with", "protein_state" ], [ 57, 69, "\u03b2 propellers", "structure_element" ], [ 73, 77, "LRP6", "protein" ], [ 78, 84, "intact", "protein_state" ], [ 90, 93, "CUB", "structure_element" ], [ 116, 135, "Ca2+-binding region", "site" ], [ 163, 181, "second \u03b2 propeller", "structure_element" ] ] }, { "sid": 128, "sent": "(B) Close-up view of the potential interaction site.", "section": "FIG", "ner": [ [ 35, 51, "interaction site", "site" ] ] }, { "sid": 129, "sent": "In addition, LRP6PE2 has been superimposed with DKK1 (yellow) and SOST (pink) peptide complexes of LRP6PE1.", "section": "FIG", "ner": [ [ 13, 17, "LRP6", "protein" ], [ 17, 20, "PE2", "structure_element" ], [ 30, 42, "superimposed", "experimental_method" ], [ 48, 52, "DKK1", "protein" ], [ 66, 70, "SOST", "protein" ], [ 99, 103, "LRP6", "protein" ], [ 103, 106, "PE1", "structure_element" ] ] }, { "sid": 130, "sent": "(C) SPR measurements comparing LRP6PE1PE2 binding with wild-type KRM1ECD and the GlycoCUB mutant bearing an N-glycan at N309.", "section": "FIG", "ner": [ [ 4, 20, "SPR measurements", "experimental_method" ], [ 31, 35, "LRP6", "protein" ], [ 35, 41, "PE1PE2", "structure_element" ], [ 55, 64, "wild-type", "protein_state" ], [ 65, 69, "KRM1", "protein" ], [ 69, 72, "ECD", "structure_element" ], [ 81, 96, "GlycoCUB mutant", "protein_state" ], [ 108, 116, "N-glycan", "ptm" ], [ 120, 124, "N309", "residue_name_number" ] ] }, { "sid": 131, "sent": "(D) Schematic representation of structural and biophysical findings and their implications for Wnt-dependent (left, middle) and independent (right) signaling.", "section": "FIG", "ner": [ [ 95, 98, "Wnt", "protein_type" ] ] }, { "sid": 132, "sent": "Conformational differences in the depictions of LRP6 are included purely for ease of representation.", "section": "FIG", "ner": [ [ 48, 52, "LRP6", "protein" ] ] }, { "sid": 133, "sent": "Diffraction and Refinement Statistics", "section": "TABLE", "ner": [ [ 0, 37, "Diffraction and Refinement Statistics", "evidence" ] ] }, { "sid": 134, "sent": "\tKRM1ECD\tKRM1ECD\tKRM1ECD\tKRM1ECD\tLRP6PE3PE4-DKKCRD2-KRM1ECD\t \tCrystal form\tI\tI\tII\tIII\tI\t \tX-ray source\tDiamond i04\tDiamond i03\tDiamond i03\tDiamond i04\tDiamond i04\t \tWavelength (\u00c5)\t0.9793\t0.9700\t0.9700\t0.9795\t0.9795\t \tSpace group\tP3121\tP3121\tP43\tP41212\tC2221\t \tUnit cell a/\u03b1 (\u00c5/\u00b0)\t50.9/90\t50.5/90\t65.8/90\t67.8/90\t86.9/90\t \tb/\u03b2 (\u00c5/\u00b0)\t50.9/90\t50.5/90\t65.8/90\t67.8/90\t100.1/90\t \tc/\u03b3 (\u00c5/\u00b0)\t188.4/120\t187.4/120\t75.0/90\t198.2/90\t270.7/90\t \tWilson B factor (\u00c52)\t31\t41\t76\t77\tNA\t \tResolution range (\u00c5)\t47.10\u20131.90 (1.95\u20131.90)\t62.47\u20132.10 (2.16\u20132.10)\t75.00\u20132.80 (2.99\u20132.80)\t67.80\u20133.20 (3.42\u20133.20)\t67.68\u20133.50 (7.16\u20136.40, 3.92\u20133.50)\t \tUnique reflections\t23,300 (1,524)\t17,089 (1,428)\t7,964 (1,448)\t8,171 (1,343)\t8,070 (723, 645)\t \tAverage multiplicity\t9.1 (9.2)\t5.2 (5.3)\t3.7 (3.7)\t22.7 (12.6)\t3.8 (3.5, 4.4)\t \tCompleteness (%)\t99.8 (98.5)\t100 (100)\t99.8 (100)\t98.8 (93.4)\t51.6 (98.5, 14.1)\t \t\t11.4 (1.7)\t12.0 (1.7)\t14.9 (1.5)\t13.1 (1.9)\t4.6 (4.1, 2.2)\t \tRmerge (%)\t14.8 (158.3)\t9.3 (98.0)\t6.2 (98.9)\t29.8 (142.2)\t44.9 (40.5, 114.2)\t \tRpim (%)\t15.7 (55.3)\t10.3 (109.0)\t3.7 (53.8)\t6.3 (40.0)\t24.7 (23.9, 59.9)\t \t\t \tRefinement\t \t\t \tRwork (%)\t17.9\t18.4\t21.6\t20.2\t32.1\t \tRfree (%)\t22.7\t23.2\t30.7\t27.1\t35.5\t \t\t \tNo. of Non-Hydrogen Atoms\t \t\t \tProtein\t2,260\t2,301\t2,102\t2,305\t7,730\t \tN-glycans\t42\t42\t28\t28\t0\t \tWater\t79\t54\t0\t2\t0\t \tLigands\t6\t6\t2\t5\t0\t \t\t \tAverage B factor (\u00c52)\t \t\t \tProtein\t63\t65\t108\t84\t\u2013\t \tN-glycans\t35\t46\t102\t18\t\u2013\t \tWater\t68\t85\t\u2013\t75\t\u2013\t \tLigands\t36\t47\t91\t75\t66\t \t\t \tRMSD from Ideality\t \t\t \tBond lengths (\u00c5)\t0.020\t0.016\t0.019\t0.016\t0.004\t \tBond angles (\u00b0)\t2.050\t1.748\t1.952\t1.796\t0.770\t \t\t \tRamachandran Plot\t \t\t \tFavored (%)\t96.8\t95.5\t96.9\t94.9\t92.3\t \tAllowed (%)\t99.7\t100.0\t100.0\t99.7\t99.8\t \tNumber of outliers\t1\t0\t0\t1\t2\t \tPDB code\t5FWS\t5FWT\t5FWU\t5FWV\t5FWW\t \t", "section": "TABLE", "ner": [ [ 1, 5, "KRM1", "protein" ], [ 5, 8, "ECD", "structure_element" ], [ 9, 13, "KRM1", "protein" ], [ 13, 16, "ECD", "structure_element" ], [ 17, 21, "KRM1", "protein" ], [ 21, 24, "ECD", "structure_element" ], [ 25, 29, "KRM1", "protein" ], [ 29, 32, "ECD", "structure_element" ], [ 33, 59, "LRP6PE3PE4-DKKCRD2-KRM1ECD", "complex_assembly" ], [ 1295, 1300, "Water", "chemical" ], [ 1417, 1422, "Water", "chemical" ], [ 1466, 1470, "RMSD", "evidence" ] ] }, { "sid": 135, "sent": "An additional shell given for the ternary complex corresponds to the last shell with near-complete diffraction data.", "section": "TABLE", "ner": [ [ 99, 115, "diffraction data", "evidence" ] ] } ] }, "PMC5063996": { "annotations": [ { "sid": 0, "sent": "The Mechanism by Which Arabinoxylanases Can Recognize Highly Decorated Xylans*", "section": "TITLE", "ner": [ [ 23, 39, "Arabinoxylanases", "protein_type" ], [ 54, 70, "Highly Decorated", "protein_state" ], [ 71, 77, "Xylans", "chemical" ] ] }, { "sid": 1, "sent": "The enzymatic degradation of plant cell walls is an important biological process of increasing environmental and industrial significance.", "section": "ABSTRACT", "ner": [ [ 29, 34, "plant", "taxonomy_domain" ] ] }, { "sid": 2, "sent": "Xylan, a major component of the plant cell wall, consists of a backbone of \u03b2-1,4-xylose (Xylp) units that are often decorated with arabinofuranose (Araf) side chains.", "section": "ABSTRACT", "ner": [ [ 0, 5, "Xylan", "chemical" ], [ 32, 37, "plant", "taxonomy_domain" ], [ 75, 87, "\u03b2-1,4-xylose", "chemical" ], [ 89, 93, "Xylp", "chemical" ], [ 131, 146, "arabinofuranose", "chemical" ], [ 148, 152, "Araf", "chemical" ] ] }, { "sid": 3, "sent": "A large penta-modular enzyme, CtXyl5A, was shown previously to specifically target arabinoxylans.", "section": "ABSTRACT", "ner": [ [ 8, 28, "penta-modular enzyme", "protein_type" ], [ 30, 37, "CtXyl5A", "protein" ], [ 83, 96, "arabinoxylans", "chemical" ] ] }, { "sid": 4, "sent": "Here we report the crystal structure of the arabinoxylanase and the enzyme in complex with ligands.", "section": "ABSTRACT", "ner": [ [ 19, 36, "crystal structure", "evidence" ], [ 44, 59, "arabinoxylanase", "protein_type" ], [ 75, 90, "in complex with", "protein_state" ], [ 91, 98, "ligands", "chemical" ] ] }, { "sid": 5, "sent": "The data showed that four of the protein modules adopt a rigid structure, which stabilizes the catalytic domain.", "section": "ABSTRACT", "ner": [ [ 95, 111, "catalytic domain", "structure_element" ] ] }, { "sid": 6, "sent": "The C-terminal non-catalytic carbohydrate binding module could not be observed in the crystal structure, suggesting positional flexibility.", "section": "ABSTRACT", "ner": [ [ 15, 56, "non-catalytic carbohydrate binding module", "structure_element" ], [ 86, 103, "crystal structure", "evidence" ] ] }, { "sid": 7, "sent": "The structure of the enzyme in complex with Xylp-\u03b2-1,4-Xylp-\u03b2-1,4-Xylp-[\u03b1-1,3-Araf]-\u03b2-1,4-Xylp showed that the Araf decoration linked O3 to the xylose in the active site is located in the pocket (\u22122* subsite) that abuts onto the catalytic center.", "section": "ABSTRACT", "ner": [ [ 4, 13, "structure", "evidence" ], [ 28, 43, "in complex with", "protein_state" ], [ 44, 94, "Xylp-\u03b2-1,4-Xylp-\u03b2-1,4-Xylp-[\u03b1-1,3-Araf]-\u03b2-1,4-Xylp", "chemical" ], [ 111, 115, "Araf", "chemical" ], [ 144, 150, "xylose", "chemical" ], [ 158, 169, "active site", "site" ], [ 188, 194, "pocket", "site" ], [ 196, 207, "\u22122* subsite", "site" ], [ 229, 245, "catalytic center", "site" ] ] }, { "sid": 8, "sent": "The \u22122* subsite can also bind to Xylp and Arap, explaining why the enzyme can utilize xylose and arabinose as specificity determinants.", "section": "ABSTRACT", "ner": [ [ 4, 15, "\u22122* subsite", "site" ], [ 33, 37, "Xylp", "chemical" ], [ 42, 46, "Arap", "chemical" ], [ 86, 92, "xylose", "chemical" ], [ 97, 106, "arabinose", "chemical" ] ] }, { "sid": 9, "sent": "Alanine substitution of Glu68, Tyr92, or Asn139, which interact with arabinose and xylose side chains at the \u22122* subsite, abrogates catalytic activity.", "section": "ABSTRACT", "ner": [ [ 0, 20, "Alanine substitution", "experimental_method" ], [ 24, 29, "Glu68", "residue_name_number" ], [ 31, 36, "Tyr92", "residue_name_number" ], [ 41, 47, "Asn139", "residue_name_number" ], [ 69, 78, "arabinose", "chemical" ], [ 83, 89, "xylose", "chemical" ], [ 109, 120, "\u22122* subsite", "site" ] ] }, { "sid": 10, "sent": "Distal to the active site, the xylan backbone makes limited apolar contacts with the enzyme, and the hydroxyls are solvent-exposed.", "section": "ABSTRACT", "ner": [ [ 14, 25, "active site", "site" ], [ 31, 36, "xylan", "chemical" ], [ 115, 130, "solvent-exposed", "protein_state" ] ] }, { "sid": 11, "sent": "This explains why CtXyl5A is capable of hydrolyzing xylans that are extensively decorated and that are recalcitrant to classic endo-xylanase attack.", "section": "ABSTRACT", "ner": [ [ 18, 25, "CtXyl5A", "protein" ], [ 52, 58, "xylans", "chemical" ], [ 127, 140, "endo-xylanase", "protein_type" ] ] }, { "sid": 12, "sent": "The plant cell wall is an important biological substrate.", "section": "INTRO", "ner": [ [ 4, 9, "plant", "taxonomy_domain" ] ] }, { "sid": 13, "sent": "This complex composite structure is depolymerized by microorganisms that occupy important highly competitive ecological niches, whereas the process makes an important contribution to the carbon cycle.", "section": "INTRO", "ner": [ [ 53, 67, "microorganisms", "taxonomy_domain" ] ] }, { "sid": 14, "sent": "Given that the plant cell wall is the most abundant source of renewable organic carbon on the planet, this macromolecular substrate has substantial industrial potential.", "section": "INTRO", "ner": [ [ 15, 20, "plant", "taxonomy_domain" ] ] }, { "sid": 15, "sent": "An example of the chemical complexity of the plant cell wall is provided by xylan, which is the major hemicellulosic component.", "section": "INTRO", "ner": [ [ 45, 50, "plant", "taxonomy_domain" ], [ 76, 81, "xylan", "chemical" ] ] }, { "sid": 16, "sent": "This polysaccharide comprises a backbone of \u03b2-1,4-d-xylose residues in their pyranose configuration (Xylp) that are decorated at O2 with 4-O-methyl-d-glucuronic acid (GlcA) and at O2 and/or O3 with \u03b1-l-arabinofuranose (Araf) residues, whereas the polysaccharide can also be extensively acetylated.", "section": "INTRO", "ner": [ [ 5, 19, "polysaccharide", "chemical" ], [ 44, 58, "\u03b2-1,4-d-xylose", "chemical" ], [ 77, 85, "pyranose", "chemical" ], [ 101, 105, "Xylp", "chemical" ], [ 137, 165, "4-O-methyl-d-glucuronic acid", "chemical" ], [ 167, 171, "GlcA", "chemical" ], [ 198, 217, "\u03b1-l-arabinofuranose", "chemical" ], [ 219, 223, "Araf", "chemical" ], [ 247, 261, "polysaccharide", "chemical" ] ] }, { "sid": 17, "sent": "In addition, the Araf side chain decorations can also be esterified to ferulic acid that, in some species, provide a chemical link between hemicellulose and lignin.", "section": "INTRO", "ner": [ [ 17, 21, "Araf", "chemical" ], [ 71, 83, "ferulic acid", "chemical" ], [ 139, 152, "hemicellulose", "chemical" ], [ 157, 163, "lignin", "chemical" ] ] }, { "sid": 18, "sent": "The precise structure of xylans varies between plant species, in particular in different tissues and during cellular differentiation.", "section": "INTRO", "ner": [ [ 25, 31, "xylans", "chemical" ], [ 47, 52, "plant", "taxonomy_domain" ] ] }, { "sid": 19, "sent": "In specialized plant tissues, such as the outer layer of cereal grains, xylans are extremely complex, and side chains may comprise a range of other sugars including l- and d-galactose and \u03b2- and \u03b1-Xylp units.", "section": "INTRO", "ner": [ [ 15, 20, "plant", "taxonomy_domain" ], [ 57, 63, "cereal", "taxonomy_domain" ], [ 72, 78, "xylans", "chemical" ], [ 148, 154, "sugars", "chemical" ], [ 165, 183, "l- and d-galactose", "chemical" ], [ 188, 201, "\u03b2- and \u03b1-Xylp", "chemical" ] ] }, { "sid": 20, "sent": "Indeed, in these cereal brans, xylans have very few backbone Xylp units that are undecorated, and the side chains can contain up to six sugars.", "section": "INTRO", "ner": [ [ 17, 23, "cereal", "taxonomy_domain" ], [ 31, 37, "xylans", "chemical" ], [ 61, 65, "Xylp", "chemical" ], [ 136, 142, "sugars", "chemical" ] ] }, { "sid": 21, "sent": "Reflecting the chemical and physical complexity of the plant cell wall, microorganisms that utilize these composite structures express a large number of polysaccharide-degrading enzymes, primarily glycoside hydrolases, but also polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases.", "section": "INTRO", "ner": [ [ 55, 60, "plant", "taxonomy_domain" ], [ 72, 86, "microorganisms", "taxonomy_domain" ], [ 153, 185, "polysaccharide-degrading enzymes", "protein_type" ], [ 197, 217, "glycoside hydrolases", "protein_type" ], [ 228, 249, "polysaccharide lyases", "protein_type" ], [ 251, 273, "carbohydrate esterases", "protein_type" ], [ 279, 314, "lytic polysaccharide monooxygenases", "protein_type" ] ] }, { "sid": 22, "sent": "These carbohydrate active enzymes are grouped into sequence-based families in the CAZy database.", "section": "INTRO", "ner": [ [ 6, 33, "carbohydrate active enzymes", "protein_type" ] ] }, { "sid": 23, "sent": "With respect to xylan degradation, the backbone of simple xylans is hydrolyzed by endo-acting xylanases, the majority of which are located in glycoside hydrolase (GH)5 families GH10 and GH11, although they are also present in GH8.", "section": "INTRO", "ner": [ [ 16, 21, "xylan", "chemical" ], [ 58, 64, "xylans", "chemical" ], [ 82, 103, "endo-acting xylanases", "protein_type" ], [ 142, 161, "glycoside hydrolase", "protein_type" ], [ 163, 165, "GH", "protein_type" ], [ 166, 167, "5", "protein_type" ], [ 177, 181, "GH10", "protein_type" ], [ 186, 190, "GH11", "protein_type" ], [ 226, 229, "GH8", "protein_type" ] ] }, { "sid": 24, "sent": "The extensive decoration of the xylan backbone generally restricts the capacity of these enzymes to attack the polysaccharide prior to removal of the side chains by a range of \u03b1-glucuronidases, \u03b1-arabinofuranosidases, and esterases.", "section": "INTRO", "ner": [ [ 32, 37, "xylan", "chemical" ], [ 111, 125, "polysaccharide", "chemical" ], [ 176, 192, "\u03b1-glucuronidases", "protein_type" ], [ 194, 216, "\u03b1-arabinofuranosidases", "protein_type" ], [ 222, 231, "esterases", "protein_type" ] ] }, { "sid": 25, "sent": "Two xylanases, however, utilize the side chains as essential specificity determinants and thus target decorated forms of the hemicellulose.", "section": "INTRO", "ner": [ [ 4, 13, "xylanases", "protein_type" ], [ 125, 138, "hemicellulose", "chemical" ] ] }, { "sid": 26, "sent": "The GH30 glucuronoxylanases require the Xylp bound at the \u22122 to contain a GlcA side chain (the scissile bond targeted by glycoside hydrolases is between subsites \u22121 and +1, and subsites that extend toward the non-reducing and reducing ends of the substrate are assigned increasing negative and positive numbers, respectively).", "section": "INTRO", "ner": [ [ 4, 8, "GH30", "protein_type" ], [ 9, 27, "glucuronoxylanases", "protein_type" ], [ 40, 44, "Xylp", "chemical" ], [ 45, 53, "bound at", "protein_state" ], [ 58, 60, "\u22122", "site" ], [ 74, 78, "GlcA", "chemical" ], [ 121, 141, "glycoside hydrolases", "protein_type" ], [ 153, 171, "subsites \u22121 and +1", "site" ], [ 177, 185, "subsites", "site" ] ] }, { "sid": 27, "sent": "The GH5 arabinoxylanase (CtXyl5A) derived from Clostridium thermocellum displays an absolute requirement for xylans that contain Araf side chains.", "section": "INTRO", "ner": [ [ 4, 7, "GH5", "protein_type" ], [ 8, 23, "arabinoxylanase", "protein_type" ], [ 25, 32, "CtXyl5A", "protein" ], [ 47, 71, "Clostridium thermocellum", "species" ], [ 109, 115, "xylans", "chemical" ], [ 129, 133, "Araf", "chemical" ] ] }, { "sid": 28, "sent": "In this enzyme, the key specificity determinant is the Araf appended to O3 of the Xylp bound in the active site (\u22121 subsite).", "section": "INTRO", "ner": [ [ 55, 59, "Araf", "chemical" ], [ 82, 86, "Xylp", "chemical" ], [ 87, 95, "bound in", "protein_state" ], [ 100, 111, "active site", "site" ], [ 113, 123, "\u22121 subsite", "site" ] ] }, { "sid": 29, "sent": "The reaction products generated from arabinoxylans, however, suggest that Araf can be accommodated at subsites distal to the active site.", "section": "INTRO", "ner": [ [ 37, 50, "arabinoxylans", "chemical" ], [ 74, 78, "Araf", "chemical" ], [ 102, 110, "subsites", "site" ], [ 125, 136, "active site", "site" ] ] }, { "sid": 30, "sent": "CtXyl5A is a multimodular enzyme containing, in addition to the GH5 catalytic module (CtGH5); three non-catalytic carbohydrate binding modules (CBMs) belonging to families 6 (CtCBM6), 13 (CtCBM13), and 62 (CtCBM62); fibronectin type 3 (Fn3) domain; and a C-terminal dockerin domain Fig. 1.", "section": "INTRO", "ner": [ [ 0, 7, "CtXyl5A", "protein" ], [ 64, 67, "GH5", "protein_type" ], [ 68, 84, "catalytic module", "structure_element" ], [ 86, 91, "CtGH5", "structure_element" ], [ 100, 142, "non-catalytic carbohydrate binding modules", "structure_element" ], [ 144, 148, "CBMs", "structure_element" ], [ 172, 173, "6", "protein_type" ], [ 175, 181, "CtCBM6", "structure_element" ], [ 184, 186, "13", "protein_type" ], [ 188, 195, "CtCBM13", "structure_element" ], [ 202, 204, "62", "protein_type" ], [ 206, 213, "CtCBM62", "structure_element" ], [ 216, 234, "fibronectin type 3", "protein_type" ], [ 236, 239, "Fn3", "structure_element" ], [ 266, 274, "dockerin", "structure_element" ] ] }, { "sid": 31, "sent": "Previous studies of Fn3 domains have indicated that they might function as ligand-binding modules, as a compact form of peptide linkers or spacers between other domains, as cellulose-disrupting modules, or as proteins that help large enzyme complexes remain soluble.", "section": "INTRO", "ner": [ [ 20, 23, "Fn3", "structure_element" ], [ 75, 97, "ligand-binding modules", "structure_element" ], [ 173, 201, "cellulose-disrupting modules", "structure_element" ] ] }, { "sid": 32, "sent": "The dockerin domain recruits the enzyme into the cellulosome, a multienzyme plant cell wall degrading complex presented on the surface of C. thermocellum.", "section": "INTRO", "ner": [ [ 4, 12, "dockerin", "structure_element" ], [ 49, 60, "cellulosome", "complex_assembly" ], [ 76, 81, "plant", "taxonomy_domain" ], [ 138, 153, "C. thermocellum", "species" ] ] }, { "sid": 33, "sent": "CtCBM6 stabilizes CtGH5, and CtCBM62 binds to d-galactopyranose and l-arabinopyranose.", "section": "INTRO", "ner": [ [ 0, 6, "CtCBM6", "structure_element" ], [ 18, 23, "CtGH5", "structure_element" ], [ 29, 36, "CtCBM62", "structure_element" ], [ 46, 63, "d-galactopyranose", "chemical" ], [ 68, 85, "l-arabinopyranose", "chemical" ] ] }, { "sid": 34, "sent": "The function of the CtCBM13 and Fn3 modules remains unclear.", "section": "INTRO", "ner": [ [ 20, 27, "CtCBM13", "structure_element" ], [ 32, 35, "Fn3", "structure_element" ] ] }, { "sid": 35, "sent": "This report exploits the crystal structure of mature CtXyl5A lacking its C-terminal dockerin domain (CtXyl5A-Doc), and the enzyme in complex with ligands, to explore the mechanism of substrate specificity.", "section": "INTRO", "ner": [ [ 25, 42, "crystal structure", "evidence" ], [ 46, 52, "mature", "protein_state" ], [ 53, 60, "CtXyl5A", "protein" ], [ 61, 68, "lacking", "protein_state" ], [ 84, 92, "dockerin", "structure_element" ], [ 101, 112, "CtXyl5A-Doc", "mutant" ], [ 130, 145, "in complex with", "protein_state" ], [ 146, 153, "ligands", "chemical" ] ] }, { "sid": 36, "sent": "The data show that the plasticity in substrate recognition enables the enzyme to hydrolyze highly complex xylans that are not accessible to classical GH10 and GH11 endo-xylanases.", "section": "INTRO", "ner": [ [ 106, 112, "xylans", "chemical" ], [ 150, 154, "GH10", "protein_type" ], [ 159, 163, "GH11", "protein_type" ], [ 164, 178, "endo-xylanases", "protein_type" ] ] }, { "sid": 37, "sent": "Molecular architecture of GH5_34 enzymes.", "section": "FIG", "ner": [ [ 26, 32, "GH5_34", "protein_type" ] ] }, { "sid": 38, "sent": "Modules prefaced by GH, CBM, or CE are modules in the indicated glycoside hydrolase, carbohydrate binding module, or carbohydrate esterase families, respectively.", "section": "FIG", "ner": [ [ 20, 22, "GH", "structure_element" ], [ 24, 27, "CBM", "structure_element" ], [ 32, 34, "CE", "structure_element" ], [ 64, 83, "glycoside hydrolase", "protein_type" ], [ 85, 112, "carbohydrate binding module", "structure_element" ], [ 117, 138, "carbohydrate esterase", "protein_type" ] ] }, { "sid": 39, "sent": "Laminin_3_G domain belongs to the concanavalin A lectin superfamily, and FN3 denotes a fibronectin type 3 domain.", "section": "FIG", "ner": [ [ 0, 11, "Laminin_3_G", "structure_element" ], [ 34, 67, "concanavalin A lectin superfamily", "protein_type" ], [ 73, 76, "FN3", "structure_element" ], [ 87, 112, "fibronectin type 3 domain", "structure_element" ] ] }, { "sid": 40, "sent": "Segments labeled D are dockerin domains.", "section": "FIG", "ner": [ [ 23, 31, "dockerin", "structure_element" ] ] }, { "sid": 41, "sent": "Substrate Specificity of CtXyl5A", "section": "RESULTS", "ner": [ [ 25, 32, "CtXyl5A", "protein" ] ] }, { "sid": 42, "sent": "Previous studies showed that CtXyl5A is an arabinoxylan-specific xylanase that generates xylooligosaccharides with an arabinose linked O3 to the reducing end xylose.", "section": "RESULTS", "ner": [ [ 29, 36, "CtXyl5A", "protein" ], [ 43, 73, "arabinoxylan-specific xylanase", "protein_type" ], [ 89, 109, "xylooligosaccharides", "chemical" ], [ 118, 127, "arabinose", "chemical" ], [ 158, 164, "xylose", "chemical" ] ] }, { "sid": 43, "sent": "The enzyme is active against both wheat and rye arabinoxylans (abbreviated as WAX and RAX, respectively).", "section": "RESULTS", "ner": [ [ 34, 39, "wheat", "taxonomy_domain" ], [ 44, 47, "rye", "taxonomy_domain" ], [ 48, 61, "arabinoxylans", "chemical" ], [ 78, 81, "WAX", "chemical" ], [ 86, 89, "RAX", "chemical" ] ] }, { "sid": 44, "sent": "It was proposed that arabinose decorations make productive interactions with a pocket (\u22122*) that is abutted onto the active site or \u22121 subsite.", "section": "RESULTS", "ner": [ [ 21, 30, "arabinose", "chemical" ], [ 79, 85, "pocket", "site" ], [ 87, 90, "\u22122*", "site" ], [ 117, 128, "active site", "site" ], [ 132, 142, "\u22121 subsite", "site" ] ] }, { "sid": 45, "sent": "Arabinose side chains of the other backbone xylose units in the oligosaccharides generated by CtXyl5A were essentially random.", "section": "RESULTS", "ner": [ [ 0, 9, "Arabinose", "chemical" ], [ 44, 50, "xylose", "chemical" ], [ 64, 80, "oligosaccharides", "chemical" ], [ 94, 101, "CtXyl5A", "protein" ] ] }, { "sid": 46, "sent": "These data suggest that O3, and possibly O2, on the xylose residues at subsites distal to the active site and \u22122* pocket are solvent-exposed, implying that the enzyme can access highly decorated xylans.", "section": "RESULTS", "ner": [ [ 52, 58, "xylose", "chemical" ], [ 71, 79, "subsites", "site" ], [ 94, 105, "active site", "site" ], [ 110, 120, "\u22122* pocket", "site" ], [ 125, 140, "solvent-exposed", "protein_state" ], [ 195, 201, "xylans", "chemical" ] ] }, { "sid": 47, "sent": "To test this hypothesis, the activity of CtXyl5A against xylans from cereal brans was assessed.", "section": "RESULTS", "ner": [ [ 41, 48, "CtXyl5A", "protein" ], [ 57, 63, "xylans", "chemical" ], [ 69, 75, "cereal", "taxonomy_domain" ] ] }, { "sid": 48, "sent": "CtXyl5a was incubated with a range of xylans for 16 h at 60 \u00b0C, and the limit products were visualized by TLC.", "section": "RESULTS", "ner": [ [ 0, 7, "CtXyl5a", "protein" ], [ 12, 21, "incubated", "experimental_method" ], [ 38, 44, "xylans", "chemical" ], [ 106, 109, "TLC", "experimental_method" ] ] }, { "sid": 49, "sent": "These xylans are highly decorated not only with Araf and GlcA units but also with l-Gal, d-Gal, and d-Xyl.", "section": "RESULTS", "ner": [ [ 6, 12, "xylans", "chemical" ], [ 48, 52, "Araf", "chemical" ], [ 57, 61, "GlcA", "chemical" ], [ 82, 87, "l-Gal", "chemical" ], [ 89, 94, "d-Gal", "chemical" ], [ 100, 105, "d-Xyl", "chemical" ] ] }, { "sid": 50, "sent": "Indeed, very few xylose units in the backbone of bran xylans lack side chains.", "section": "RESULTS", "ner": [ [ 17, 23, "xylose", "chemical" ], [ 54, 60, "xylans", "chemical" ] ] }, { "sid": 51, "sent": "The data presented in Table 1 showed that CtXyl5A was active against corn bran xylan (CX).", "section": "RESULTS", "ner": [ [ 42, 49, "CtXyl5A", "protein" ], [ 69, 73, "corn", "taxonomy_domain" ], [ 79, 84, "xylan", "chemical" ], [ 86, 88, "CX", "chemical" ] ] }, { "sid": 52, "sent": "In contrast typical endo-xylanases from GH10 and GH11 were unable to attack CX, reflecting the lack of undecorated xylose units in the backbone (the active site of these enzymes can only bind to non-substituted xylose residues).", "section": "RESULTS", "ner": [ [ 20, 34, "endo-xylanases", "protein_type" ], [ 40, 44, "GH10", "protein_type" ], [ 49, 53, "GH11", "protein_type" ], [ 76, 78, "CX", "chemical" ], [ 95, 102, "lack of", "protein_state" ], [ 115, 121, "xylose", "chemical" ], [ 149, 160, "active site", "site" ], [ 187, 194, "bind to", "protein_state" ], [ 211, 217, "xylose", "chemical" ] ] }, { "sid": 53, "sent": "The limit products generated by CtXyl5A from CX consisted of an extensive range of oligosaccharides.", "section": "RESULTS", "ner": [ [ 32, 39, "CtXyl5A", "protein" ], [ 45, 47, "CX", "chemical" ], [ 83, 99, "oligosaccharides", "chemical" ] ] }, { "sid": 54, "sent": "These data support the view that in subsites out with the active site the O2 and O3 groups of the bound xylose units are solvent-exposed and will thus tolerate decoration.", "section": "RESULTS", "ner": [ [ 36, 44, "subsites", "site" ], [ 58, 69, "active site", "site" ], [ 104, 110, "xylose", "chemical" ], [ 121, 136, "solvent-exposed", "protein_state" ] ] }, { "sid": 55, "sent": "Kinetics of GH5_34 arabinoxylanases", "section": "TABLE", "ner": [ [ 0, 8, "Kinetics", "evidence" ], [ 12, 18, "GH5_34", "protein_type" ], [ 19, 35, "arabinoxylanases", "protein_type" ] ] }, { "sid": 56, "sent": "Enzyme\tVariant\tkcat/Km\t \tWAX\tRAX\tCX\t \t\t\tmin\u22121mg\u22121ml\t \tCtXyl5A\tCtGH5-CBM6-CBM13-Fn3-CBM62\t800\tND\t460\t \tCtXyl5A\tCtGH5-CBM6-CBM13-Fn3\t1,232\tND\t659\t \tCtXyl5A\tCtGH5-CBM6-CBM13\t1,307\tND\t620\t \tCtXyl5A\tCtGH5-CBM6\t488\tND\t102\t \tCtXyl5A\tCtGH5-CBM6: E68A\tNA\tNA\tNA\t \tCtXyl5A\tCtGH5-CBM6: Y92A\tNA\tNA\tNA\t \tCtXyl5A\tCtGH5-CBM6: N135A\t260\tND\tND\t \tCtXyl5A\tCtGH5-CBM6: N139A\tNA\tNA\tNA\t \tAcGH5\tWild type\t628\t1,641\t289\t \tGpGH5\tWild type\t2,600\t9,986\t314\t \tVbGH5\tWild type\tND\tND\tND\t \tVbGH5\tD45A\t102\t203\t23\t \t", "section": "TABLE", "ner": [ [ 15, 19, "kcat", "evidence" ], [ 20, 22, "Km", "evidence" ], [ 25, 28, "WAX", "chemical" ], [ 29, 32, "RAX", "chemical" ], [ 33, 35, "CX", "chemical" ], [ 54, 61, "CtXyl5A", "protein" ], [ 62, 88, "CtGH5-CBM6-CBM13-Fn3-CBM62", "structure_element" ], [ 102, 109, "CtXyl5A", "protein" ], [ 110, 130, "CtGH5-CBM6-CBM13-Fn3", "structure_element" ], [ 146, 153, "CtXyl5A", "protein" ], [ 154, 170, "CtGH5-CBM6-CBM13", "structure_element" ], [ 186, 193, "CtXyl5A", "protein" ], [ 194, 204, "CtGH5-CBM6", "structure_element" ], [ 218, 225, "CtXyl5A", "protein" ], [ 226, 236, "CtGH5-CBM6", "structure_element" ], [ 238, 242, "E68A", "mutant" ], [ 254, 261, "CtXyl5A", "protein" ], [ 262, 272, "CtGH5-CBM6", "structure_element" ], [ 274, 278, "Y92A", "mutant" ], [ 290, 297, "CtXyl5A", "protein" ], [ 298, 308, "CtGH5-CBM6", "structure_element" ], [ 310, 315, "N135A", "mutant" ], [ 328, 335, "CtXyl5A", "protein" ], [ 336, 346, "CtGH5-CBM6", "structure_element" ], [ 348, 353, "N139A", "mutant" ], [ 365, 370, "AcGH5", "protein" ], [ 371, 380, "Wild type", "protein_state" ], [ 397, 402, "GpGH5", "protein" ], [ 403, 412, "Wild type", "protein_state" ], [ 431, 436, "VbGH5", "protein" ], [ 437, 446, "Wild type", "protein_state" ], [ 458, 463, "VbGH5", "protein" ], [ 464, 468, "D45A", "mutant" ] ] }, { "sid": 57, "sent": "To explore whether substrate bound only at \u22122* and \u22121 in the negative subsites was hydrolyzed by CtXyl5A, the limit products of CX digested by the arabinoxylanase were subjected to size exclusion chromatography using a Bio-Gel P-2, and the smallest oligosaccharides (largest elution volume) were chosen for further study.", "section": "RESULTS", "ner": [ [ 29, 42, "bound only at", "protein_state" ], [ 43, 46, "\u22122*", "site" ], [ 51, 53, "\u22121", "site" ], [ 61, 78, "negative subsites", "site" ], [ 97, 104, "CtXyl5A", "protein" ], [ 128, 130, "CX", "chemical" ], [ 147, 162, "arabinoxylanase", "protein_type" ], [ 181, 210, "size exclusion chromatography", "experimental_method" ], [ 249, 265, "oligosaccharides", "chemical" ] ] }, { "sid": 58, "sent": "HPAEC analysis of the smallest oligosaccharide fraction (pool 4) contained two species with retention times of 14.0 min (oligosaccharide 1) and 20.8 min (oligosaccharide 2) (Fig. 2).", "section": "RESULTS", "ner": [ [ 0, 5, "HPAEC", "experimental_method" ], [ 31, 46, "oligosaccharide", "chemical" ], [ 121, 136, "oligosaccharide", "chemical" ], [ 154, 169, "oligosaccharide", "chemical" ] ] }, { "sid": 59, "sent": "Positive mode electrospray mass spectrometry showed that pool 4 contained exclusively molecular ions with a m/z = 305 [M + Na]+, which corresponds to a pentose-pentose disaccharide (molecular mass = 282 Da) as a sodium ion adduct, whereas a dimer of the disaccharide with a sodium adduct (m/z = 587 [2M+Na]+) was also evident.", "section": "RESULTS", "ner": [ [ 0, 44, "Positive mode electrospray mass spectrometry", "experimental_method" ], [ 152, 159, "pentose", "chemical" ], [ 160, 167, "pentose", "chemical" ], [ 168, 180, "disaccharide", "chemical" ], [ 254, 266, "disaccharide", "chemical" ] ] }, { "sid": 60, "sent": "The monosaccharide composition of pool 4 determined by TFA hydrolysis contained xylose and arabinose in a 3:1 ratio.", "section": "RESULTS", "ner": [ [ 55, 69, "TFA hydrolysis", "experimental_method" ], [ 80, 86, "xylose", "chemical" ], [ 91, 100, "arabinose", "chemical" ] ] }, { "sid": 61, "sent": "This suggests that the two oligosaccharides consist of two disaccharides: one consisting of two xylose residues and the other consisting of an arabinose linked to a xylose.", "section": "RESULTS", "ner": [ [ 27, 43, "oligosaccharides", "chemical" ], [ 59, 72, "disaccharides", "chemical" ], [ 96, 102, "xylose", "chemical" ], [ 143, 152, "arabinose", "chemical" ], [ 165, 171, "xylose", "chemical" ] ] }, { "sid": 62, "sent": "Treatment of pool 4 with the nonspecific arabinofuranosidase, CjAbf51A, resulted in the loss of oligosaccharide 2 and the production of both xylose and arabinose, indicative of a disaccharide of xylose and arabinose.", "section": "RESULTS", "ner": [ [ 29, 60, "nonspecific arabinofuranosidase", "protein_type" ], [ 62, 70, "CjAbf51A", "protein" ], [ 96, 111, "oligosaccharide", "chemical" ], [ 141, 147, "xylose", "chemical" ], [ 152, 161, "arabinose", "chemical" ], [ 179, 191, "disaccharide", "chemical" ], [ 195, 201, "xylose", "chemical" ], [ 206, 215, "arabinose", "chemical" ] ] }, { "sid": 63, "sent": "Incubation of pool 4 with a \u03b2-1,3-xylosidase (XynB) converted oligosaccharide 1 into xylose, demonstrating that this molecule is the disaccharide \u03b2-1,3-xylobiose.", "section": "RESULTS", "ner": [ [ 28, 44, "\u03b2-1,3-xylosidase", "protein_type" ], [ 46, 50, "XynB", "protein" ], [ 62, 77, "oligosaccharide", "chemical" ], [ 85, 91, "xylose", "chemical" ], [ 133, 145, "disaccharide", "chemical" ], [ 146, 161, "\u03b2-1,3-xylobiose", "chemical" ] ] }, { "sid": 64, "sent": "This view is supported by the inability of a \u03b2-1,4-specific xylosidase to hydrolyze oligosaccharide 1 or oligosaccharide 2 (data not shown).", "section": "RESULTS", "ner": [ [ 45, 70, "\u03b2-1,4-specific xylosidase", "protein_type" ], [ 84, 99, "oligosaccharide", "chemical" ], [ 105, 120, "oligosaccharide", "chemical" ] ] }, { "sid": 65, "sent": "The crucial importance of occupancy of the \u22122* pocket for catalytic competence is illustrated by the inability of the enzyme to hydrolyze linear \u03b2-1,4-xylooligosaccharides.", "section": "RESULTS", "ner": [ [ 43, 53, "\u22122* pocket", "site" ], [ 145, 171, "\u03b2-1,4-xylooligosaccharides", "chemical" ] ] }, { "sid": 66, "sent": "The generation of Araf-Xylp and Xyl-\u03b2-1,3-Xyl as reaction products demonstrates that occupancy of the \u22122 subsite is not essential for catalytic activity, which is in contrast to all endo-acting xylanases where this subsite plays a critical role in enzyme activity.", "section": "RESULTS", "ner": [ [ 18, 27, "Araf-Xylp", "chemical" ], [ 32, 45, "Xyl-\u03b2-1,3-Xyl", "chemical" ], [ 102, 112, "\u22122 subsite", "site" ], [ 182, 203, "endo-acting xylanases", "protein_type" ], [ 215, 222, "subsite", "site" ] ] }, { "sid": 67, "sent": "Indeed, the data demonstrate that \u22122* plays a more important role in productive substrate binding than the \u22122 subsite.", "section": "RESULTS", "ner": [ [ 34, 37, "\u22122*", "site" ], [ 107, 117, "\u22122 subsite", "site" ] ] }, { "sid": 68, "sent": "Unfortunately, the inability to generate highly purified (Xyl-\u03b2-1,4)n-[\u03b2-1,3-Xyl/Ara]-Xyl oligosaccharides from arabinoxylans prevented the precise binding energies at the negative subsites to be determined.", "section": "RESULTS", "ner": [ [ 57, 89, "(Xyl-\u03b2-1,4)n-[\u03b2-1,3-Xyl/Ara]-Xyl", "chemical" ], [ 90, 106, "oligosaccharides", "chemical" ], [ 112, 125, "arabinoxylans", "chemical" ] ] }, { "sid": 69, "sent": "Identification of the disaccharide reaction products generated from CX.", "section": "FIG", "ner": [ [ 22, 34, "disaccharide", "chemical" ], [ 68, 70, "CX", "chemical" ] ] }, { "sid": 70, "sent": "The smallest reaction products were purified by size exclusion chromatography and analyzed by HPAEC (A) and positive mode ESI-MS (B), respectively.", "section": "FIG", "ner": [ [ 48, 77, "size exclusion chromatography", "experimental_method" ], [ 94, 99, "HPAEC", "experimental_method" ], [ 122, 128, "ESI-MS", "experimental_method" ] ] }, { "sid": 71, "sent": "The samples were treated with a nonspecific arabinofuranosidase (CjAbf51A) and a GH3 xylosidase (XynB) that targeted \u03b2-1,3-xylosidic bonds.", "section": "FIG", "ner": [ [ 32, 63, "nonspecific arabinofuranosidase", "protein_type" ], [ 65, 73, "CjAbf51A", "protein" ], [ 81, 95, "GH3 xylosidase", "protein_type" ], [ 97, 101, "XynB", "protein" ] ] }, { "sid": 72, "sent": "X, xylose; A, arabinose.", "section": "FIG", "ner": [ [ 3, 9, "xylose", "chemical" ], [ 14, 23, "arabinose", "chemical" ] ] }, { "sid": 73, "sent": "The m/z = 305 species denotes a pentose disaccharide as a sodium adduct [M + Na]+, whereas the m/z = 587 signal corresponds to an ESI-MS dimer of the pentose disaccharide also as a sodium adduct [2M + Na]+.", "section": "FIG", "ner": [ [ 32, 39, "pentose", "chemical" ], [ 40, 52, "disaccharide", "chemical" ], [ 130, 136, "ESI-MS", "experimental_method" ], [ 150, 157, "pentose", "chemical" ], [ 158, 170, "disaccharide", "chemical" ] ] }, { "sid": 74, "sent": "Crystal Structure of the Catalytic Module of CtXyl5A in Complex with Ligands", "section": "RESULTS", "ner": [ [ 0, 17, "Crystal Structure", "evidence" ], [ 25, 41, "Catalytic Module", "structure_element" ], [ 45, 52, "CtXyl5A", "protein" ], [ 53, 68, "in Complex with", "protein_state" ], [ 69, 76, "Ligands", "chemical" ] ] }, { "sid": 75, "sent": "To understand the structural basis for the biochemical properties of CtXyl5A, the crystal structure of the enzyme with ligands that occupy the substrate binding cleft and the critical \u22122* subsite were sought.", "section": "RESULTS", "ner": [ [ 69, 76, "CtXyl5A", "protein" ], [ 82, 99, "crystal structure", "evidence" ], [ 143, 166, "substrate binding cleft", "site" ], [ 184, 195, "\u22122* subsite", "site" ] ] }, { "sid": 76, "sent": "The data presented in Fig. 3A show the structure of the CtXyl5A derivative CtGH5-CtCBM6 in complex with arabinose bound in the \u22122* pocket.", "section": "RESULTS", "ner": [ [ 39, 48, "structure", "evidence" ], [ 56, 63, "CtXyl5A", "protein" ], [ 75, 87, "CtGH5-CtCBM6", "structure_element" ], [ 88, 103, "in complex with", "protein_state" ], [ 104, 113, "arabinose", "chemical" ], [ 114, 122, "bound in", "protein_state" ], [ 127, 137, "\u22122* pocket", "site" ] ] }, { "sid": 77, "sent": "Interestingly, the bound arabinose was in the pyranose conformation rather than in its furanose form found in arabinoxylans.", "section": "RESULTS", "ner": [ [ 19, 24, "bound", "protein_state" ], [ 25, 34, "arabinose", "chemical" ], [ 46, 54, "pyranose", "chemical" ], [ 87, 95, "furanose", "chemical" ], [ 110, 123, "arabinoxylans", "chemical" ] ] }, { "sid": 78, "sent": "O1 was facing toward the active site \u22121 subsite, indicative of the bound arabinose being in the right orientation to be linked to the xylan backbone via an \u03b1-1,3 linkage.", "section": "RESULTS", "ner": [ [ 25, 36, "active site", "site" ], [ 37, 47, "\u22121 subsite", "site" ], [ 67, 72, "bound", "protein_state" ], [ 73, 82, "arabinose", "chemical" ], [ 134, 139, "xylan", "chemical" ] ] }, { "sid": 79, "sent": "As discussed on below, the axial O4 of the Arap did not interact with the \u22122* subsite, suggesting that the pocket might be capable of binding a xylose molecule.", "section": "RESULTS", "ner": [ [ 43, 47, "Arap", "chemical" ], [ 74, 85, "\u22122* subsite", "site" ], [ 107, 113, "pocket", "site" ], [ 144, 150, "xylose", "chemical" ] ] }, { "sid": 80, "sent": "Indeed, soaking apo crystals with xylose showed that the pentose sugar also bound in the \u22122* subsite in its pyranose conformation (Fig. 3B).", "section": "RESULTS", "ner": [ [ 8, 15, "soaking", "experimental_method" ], [ 16, 19, "apo", "protein_state" ], [ 20, 28, "crystals", "evidence" ], [ 34, 40, "xylose", "chemical" ], [ 57, 64, "pentose", "chemical" ], [ 65, 70, "sugar", "chemical" ], [ 76, 84, "bound in", "protein_state" ], [ 89, 100, "\u22122* subsite", "site" ], [ 108, 116, "pyranose", "chemical" ] ] }, { "sid": 81, "sent": "These crystal structures support the biochemical data presented above showing that the enzyme generated \u03b2-1,3-xylobiose from CX, which would require the disaccharide to bind at the \u22121 and \u22122* subsites.", "section": "RESULTS", "ner": [ [ 6, 24, "crystal structures", "evidence" ], [ 104, 119, "\u03b2-1,3-xylobiose", "chemical" ], [ 125, 127, "CX", "chemical" ], [ 153, 165, "disaccharide", "chemical" ], [ 181, 200, "\u22121 and \u22122* subsites", "site" ] ] }, { "sid": 82, "sent": "A third product complex was generated by co-crystallizing the nucleophile inactive mutant CtGH5E279S-CtCBM6 with a WAX-derived oligosaccharide (Fig. 3C).", "section": "RESULTS", "ner": [ [ 41, 57, "co-crystallizing", "experimental_method" ], [ 62, 82, "nucleophile inactive", "protein_state" ], [ 83, 89, "mutant", "protein_state" ], [ 90, 100, "CtGH5E279S", "mutant" ], [ 101, 107, "CtCBM6", "structure_element" ], [ 115, 118, "WAX", "chemical" ], [ 127, 142, "oligosaccharide", "chemical" ] ] }, { "sid": 83, "sent": "The data revealed a pentasaccharide bound to the enzyme, comprising \u03b2-1,4-xylotetraose with an Araf linked \u03b1-1,3 to the reducing end xylose.", "section": "RESULTS", "ner": [ [ 20, 35, "pentasaccharide", "chemical" ], [ 36, 44, "bound to", "protein_state" ], [ 68, 86, "\u03b2-1,4-xylotetraose", "chemical" ], [ 95, 99, "Araf", "chemical" ], [ 133, 139, "xylose", "chemical" ] ] }, { "sid": 84, "sent": "The xylotetraose was positioned in subsites \u22121 to \u22124 and the Araf in the \u22122* pocket.", "section": "RESULTS", "ner": [ [ 4, 16, "xylotetraose", "chemical" ], [ 35, 52, "subsites \u22121 to \u22124", "site" ], [ 61, 65, "Araf", "chemical" ], [ 73, 83, "\u22122* pocket", "site" ] ] }, { "sid": 85, "sent": "Analysis of the three structures showed that O1, O2, O3, and the endocyclic oxygen occupied identical positions in the Arap, Araf, and Xylp ligands bound in the \u22122* subsite and thus made identical interactions with the pocket.", "section": "RESULTS", "ner": [ [ 22, 32, "structures", "evidence" ], [ 119, 123, "Arap", "chemical" ], [ 125, 129, "Araf", "chemical" ], [ 135, 139, "Xylp", "chemical" ], [ 148, 156, "bound in", "protein_state" ], [ 161, 172, "\u22122* subsite", "site" ], [ 219, 225, "pocket", "site" ] ] }, { "sid": 86, "sent": "O1 makes a polar contact with N\u03b42 of Asn139, O2 is within hydrogen bonding distance with O\u03b41 of Asn139 and the backbone N of Asn135, and O3 interacts with the N of Gly136 and O\u03f52 of Glu68.", "section": "RESULTS", "ner": [ [ 11, 24, "polar contact", "bond_interaction" ], [ 37, 43, "Asn139", "residue_name_number" ], [ 58, 74, "hydrogen bonding", "bond_interaction" ], [ 96, 102, "Asn139", "residue_name_number" ], [ 125, 131, "Asn135", "residue_name_number" ], [ 164, 170, "Gly136", "residue_name_number" ], [ 182, 187, "Glu68", "residue_name_number" ] ] }, { "sid": 87, "sent": "Although O4 of Arap does not make a direct interaction with the enzyme, O4 and O5 of Xylp and Araf, respectively, form hydrogen bonds with O\u03f51 of Glu68.", "section": "RESULTS", "ner": [ [ 15, 19, "Arap", "chemical" ], [ 85, 89, "Xylp", "chemical" ], [ 94, 98, "Araf", "chemical" ], [ 119, 133, "hydrogen bonds", "bond_interaction" ], [ 146, 151, "Glu68", "residue_name_number" ] ] }, { "sid": 88, "sent": "Finally Tyr92 makes apolar parallel interactions with the pyranose or furanose rings of the three sugars.", "section": "RESULTS", "ner": [ [ 8, 13, "Tyr92", "residue_name_number" ], [ 27, 48, "parallel interactions", "bond_interaction" ], [ 58, 66, "pyranose", "chemical" ], [ 70, 78, "furanose", "chemical" ] ] }, { "sid": 89, "sent": "Representation of the residues involved in the ligands recognition at the \u22122* subsite.", "section": "FIG", "ner": [ [ 74, 85, "\u22122* subsite", "site" ] ] }, { "sid": 90, "sent": "Interacting residues are represented as stick in blue, and the catalytic residues and the mutated glutamate (into a serine) are in magenta.", "section": "FIG", "ner": [ [ 63, 81, "catalytic residues", "site" ], [ 90, 97, "mutated", "experimental_method" ], [ 98, 107, "glutamate", "residue_name" ], [ 116, 122, "serine", "residue_name" ] ] }, { "sid": 91, "sent": "A, CtGH5-CBM6 in complex with an arabinopyranose.", "section": "FIG", "ner": [ [ 3, 13, "CtGH5-CBM6", "structure_element" ], [ 14, 29, "in complex with", "protein_state" ], [ 33, 48, "arabinopyranose", "chemical" ] ] }, { "sid": 92, "sent": "B, CtGH5-CBM6 in complex with a xylopyranose.", "section": "FIG", "ner": [ [ 3, 13, "CtGH5-CBM6", "structure_element" ], [ 14, 29, "in complex with", "protein_state" ], [ 32, 44, "xylopyranose", "chemical" ] ] }, { "sid": 93, "sent": "C, CtGH5E279S-CBM6 in complex with a pentasaccharide (\u03b21,4-xylotetraose with an l-Araf linked \u03b11,3 to the reducing end xylose).", "section": "FIG", "ner": [ [ 3, 13, "CtGH5E279S", "mutant" ], [ 14, 18, "CBM6", "structure_element" ], [ 19, 34, "in complex with", "protein_state" ], [ 37, 52, "pentasaccharide", "chemical" ], [ 54, 71, "\u03b21,4-xylotetraose", "chemical" ], [ 80, 86, "l-Araf", "chemical" ], [ 119, 125, "xylose", "chemical" ] ] }, { "sid": 94, "sent": "The xylan backbone is shown transparently for more clarity.", "section": "FIG", "ner": [ [ 4, 9, "xylan", "chemical" ] ] }, { "sid": 95, "sent": "Densities shown in blue are RefMac maximum-likelihood \u03c3A-weighted 2Fo \u2212 Fc at 1.5 \u03c3.", "section": "FIG", "ner": [ [ 0, 9, "Densities", "evidence" ], [ 35, 83, "maximum-likelihood \u03c3A-weighted 2Fo \u2212 Fc at 1.5 \u03c3", "evidence" ] ] }, { "sid": 96, "sent": "The importance of the interactions between the ligands and the side chains of the residues in the \u22122* pocket were evaluated by alanine substitution of these amino acids.", "section": "RESULTS", "ner": [ [ 98, 108, "\u22122* pocket", "site" ], [ 127, 147, "alanine substitution", "experimental_method" ] ] }, { "sid": 97, "sent": "The mutants E68A, Y92A, and N139A were all inactive (Table 1), demonstrating the importance of the interactions of these residues with the substrate and reinforcing the critical role the \u22122* subsite plays in the activity of the enzyme.", "section": "RESULTS", "ner": [ [ 4, 11, "mutants", "protein_state" ], [ 12, 16, "E68A", "mutant" ], [ 18, 22, "Y92A", "mutant" ], [ 28, 33, "N139A", "mutant" ], [ 43, 51, "inactive", "protein_state" ], [ 187, 198, "\u22122* subsite", "site" ] ] }, { "sid": 98, "sent": "N135A retained wild type activity because the O2 of the sugars interacts with the backbone N of Asn135 and not with the side chain.", "section": "RESULTS", "ner": [ [ 0, 5, "N135A", "mutant" ], [ 15, 24, "wild type", "protein_state" ], [ 96, 102, "Asn135", "residue_name_number" ] ] }, { "sid": 99, "sent": "Because the hydroxyls of Xylp or Araf in the \u22122* pocket are not solvent-exposed, the active site of the arabinoxylanase can only bind to xylose residues that contain a single xylose or arabinose O3 decoration.", "section": "RESULTS", "ner": [ [ 25, 29, "Xylp", "chemical" ], [ 33, 37, "Araf", "chemical" ], [ 45, 55, "\u22122* pocket", "site" ], [ 64, 79, "solvent-exposed", "protein_state" ], [ 85, 96, "active site", "site" ], [ 104, 119, "arabinoxylanase", "protein_type" ], [ 137, 143, "xylose", "chemical" ], [ 175, 181, "xylose", "chemical" ], [ 185, 194, "arabinose", "chemical" ] ] }, { "sid": 100, "sent": "This may explain why the kcat/Km for CtXyl5A against WAX was 2-fold higher than against CX (Table 1).", "section": "RESULTS", "ner": [ [ 25, 29, "kcat", "evidence" ], [ 30, 32, "Km", "evidence" ], [ 37, 44, "CtXyl5A", "protein" ], [ 53, 56, "WAX", "chemical" ], [ 88, 90, "CX", "chemical" ] ] }, { "sid": 101, "sent": "WAX is likely to have a higher concentration of single Araf decorations compared with CX and thus contain more substrate available to the arabinoxylanase.", "section": "RESULTS", "ner": [ [ 0, 3, "WAX", "chemical" ], [ 55, 59, "Araf", "chemical" ], [ 86, 88, "CX", "chemical" ], [ 138, 153, "arabinoxylanase", "protein_type" ] ] }, { "sid": 102, "sent": "In the active site of CtXyl5A the \u03b1-d-Xylp, which is in its relaxed 4C1 conformation, makes the following interactions with the enzyme (Fig. 4, A\u2013C): O1 hydrogen bonds with the N\u03b41 of His253 and O\u03f52 of Glu171 (catalytic acid-base) and makes a possible weak polar contact with the OH of Tyr255 and O\u03b3 of Ser279 (mutation of the catalytic nucleophile); O2 hydrogen bonds with N\u03b42 of Asn170 and OH of Tyr92.", "section": "RESULTS", "ner": [ [ 7, 18, "active site", "site" ], [ 22, 29, "CtXyl5A", "protein" ], [ 34, 42, "\u03b1-d-Xylp", "chemical" ], [ 153, 167, "hydrogen bonds", "bond_interaction" ], [ 184, 190, "His253", "residue_name_number" ], [ 202, 208, "Glu171", "residue_name_number" ], [ 257, 270, "polar contact", "bond_interaction" ], [ 286, 292, "Tyr255", "residue_name_number" ], [ 303, 309, "Ser279", "residue_name_number" ], [ 354, 368, "hydrogen bonds", "bond_interaction" ], [ 381, 387, "Asn170", "residue_name_number" ], [ 398, 403, "Tyr92", "residue_name_number" ] ] }, { "sid": 103, "sent": "O3 (O1 of the Araf at the \u22122* subsite) makes a polar contact with N\u03b42 of Asn139; the endocyclic oxygen hydrogens bonds with the OH of Tyr255.", "section": "RESULTS", "ner": [ [ 14, 18, "Araf", "chemical" ], [ 26, 37, "\u22122* subsite", "site" ], [ 47, 60, "polar contact", "bond_interaction" ], [ 73, 79, "Asn139", "residue_name_number" ], [ 103, 118, "hydrogens bonds", "bond_interaction" ], [ 134, 140, "Tyr255", "residue_name_number" ] ] }, { "sid": 104, "sent": "The Xylp in the active site makes strong parallel apolar interactions with Phe310.", "section": "RESULTS", "ner": [ [ 4, 8, "Xylp", "chemical" ], [ 16, 27, "active site", "site" ], [ 41, 69, "parallel apolar interactions", "bond_interaction" ], [ 75, 81, "Phe310", "residue_name_number" ] ] }, { "sid": 105, "sent": "Substrate recognition in the active site is conserved between CtXyl5A and the closest GH5 structural homolog, the endoglucanase BaCel5A (PDB code 1qi2) as noted previously.", "section": "RESULTS", "ner": [ [ 29, 40, "active site", "site" ], [ 44, 53, "conserved", "protein_state" ], [ 62, 69, "CtXyl5A", "protein" ], [ 86, 89, "GH5", "protein_type" ], [ 114, 127, "endoglucanase", "protein_type" ], [ 128, 135, "BaCel5A", "protein" ] ] }, { "sid": 106, "sent": "Comparison of the ligand recognition at the distal negative subsites between CtGH5E279S-CBM6, the cellulase BaCel5A, and the xylanase GH10.", "section": "FIG", "ner": [ [ 51, 68, "negative subsites", "site" ], [ 77, 87, "CtGH5E279S", "mutant" ], [ 88, 92, "CBM6", "structure_element" ], [ 98, 107, "cellulase", "protein_type" ], [ 108, 115, "BaCel5A", "protein" ], [ 125, 133, "xylanase", "protein_type" ], [ 134, 138, "GH10", "protein_type" ] ] }, { "sid": 107, "sent": " A\u2013C show CtGH5E279S-CBM6 is in complex with a pentasaccharide (\u03b21,4-xylotetraose with an l-Araf linked \u03b11,3 to the reducing end xylose).", "section": "FIG", "ner": [ [ 10, 20, "CtGH5E279S", "mutant" ], [ 29, 44, "in complex with", "protein_state" ], [ 47, 62, "pentasaccharide", "chemical" ], [ 64, 81, "\u03b21,4-xylotetraose", "chemical" ], [ 90, 96, "l-Araf", "chemical" ], [ 129, 135, "xylose", "chemical" ] ] }, { "sid": 108, "sent": "A, Poseview representation highlighting the hydrogen bonding and the hydrophobic interactions that occur in the negative subsites.", "section": "FIG", "ner": [ [ 44, 60, "hydrogen bonding", "bond_interaction" ], [ 69, 93, "hydrophobic interactions", "bond_interaction" ], [ 112, 129, "negative subsites", "site" ] ] }, { "sid": 109, "sent": "C, density of the ligand shown in blue is RefMac maximum-likelihood \u03c3A-weighted 2Fo \u2212 Fc at 1.5 \u03c3.", "section": "FIG", "ner": [ [ 3, 10, "density", "evidence" ], [ 49, 97, "maximum-likelihood \u03c3A-weighted 2Fo \u2212 Fc at 1.5 \u03c3", "evidence" ] ] }, { "sid": 110, "sent": "D and E display BaCel5A in complex with deoxy-2-fluoro-\u03b2-d-cellotrioside (PDB code 1qi2), and F and G show CmXyn10B in complex with a xylotriose (PDB code 1uqy).", "section": "FIG", "ner": [ [ 16, 23, "BaCel5A", "protein" ], [ 24, 39, "in complex with", "protein_state" ], [ 40, 72, "deoxy-2-fluoro-\u03b2-d-cellotrioside", "chemical" ], [ 107, 115, "CmXyn10B", "protein" ], [ 116, 131, "in complex with", "protein_state" ], [ 134, 144, "xylotriose", "chemical" ] ] }, { "sid": 111, "sent": "B, D, and F are surface representations (CtGH5E279S-CBM6 in gray, BaCel5A in cyan, and the xylanase GH10 in light brown).", "section": "FIG", "ner": [ [ 41, 51, "CtGH5E279S", "mutant" ], [ 66, 73, "BaCel5A", "protein" ], [ 91, 99, "xylanase", "protein_type" ], [ 100, 104, "GH10", "protein_type" ] ] }, { "sid": 112, "sent": "The black dashes represent the hydrogen bonds.", "section": "FIG", "ner": [ [ 31, 45, "hydrogen bonds", "bond_interaction" ], [ 31, 45, "hydrogen bonds", "bond_interaction" ] ] }, { "sid": 113, "sent": "The capacity of CtXyl5A to act on the highly decorated xylan CX indicates that O3 and possibly O2 of the backbone Xylp units are solvent-exposed.", "section": "RESULTS", "ner": [ [ 16, 23, "CtXyl5A", "protein" ], [ 55, 60, "xylan", "chemical" ], [ 61, 63, "CX", "chemical" ], [ 114, 118, "Xylp", "chemical" ], [ 129, 144, "solvent-exposed", "protein_state" ] ] }, { "sid": 114, "sent": "This is consistent with the interaction of the xylotetraose backbone with the enzyme distal to the active site.", "section": "RESULTS", "ner": [ [ 47, 59, "xylotetraose", "chemical" ], [ 99, 110, "active site", "site" ] ] }, { "sid": 115, "sent": "A surface representation of the enzyme (Fig. 4B) shows that O3 and O2 of xylose units at subsites \u22122 to \u22124 are solvent-exposed and are thus available for decoration.", "section": "RESULTS", "ner": [ [ 73, 79, "xylose", "chemical" ], [ 89, 106, "subsites \u22122 to \u22124", "site" ], [ 111, 126, "solvent-exposed", "protein_state" ] ] }, { "sid": 116, "sent": "Indeed, these pyranose sugars make very weak apolar interactions with the arabinoxylanase.", "section": "RESULTS", "ner": [ [ 14, 22, "pyranose", "chemical" ], [ 23, 29, "sugars", "chemical" ], [ 45, 64, "apolar interactions", "bond_interaction" ], [ 74, 89, "arabinoxylanase", "protein_type" ] ] }, { "sid": 117, "sent": "At \u22122, Xylp makes planar apolar interactions with the Araf bound to the \u22122* subsite (Fig. 4C).", "section": "RESULTS", "ner": [ [ 3, 5, "\u22122", "site" ], [ 7, 11, "Xylp", "chemical" ], [ 18, 44, "planar apolar interactions", "bond_interaction" ], [ 54, 58, "Araf", "chemical" ], [ 59, 67, "bound to", "protein_state" ], [ 72, 83, "\u22122* subsite", "site" ] ] }, { "sid": 118, "sent": "Xylp at subsites \u22122 and \u22123, respectively, make weak hydrophobic contact with Val318, the \u22123 Xylp makes planar apolar interactions with Ala137, whereas the xylose at \u22124 forms parallel apolar contacts with Trp69.", "section": "RESULTS", "ner": [ [ 0, 4, "Xylp", "chemical" ], [ 8, 26, "subsites \u22122 and \u22123", "site" ], [ 52, 71, "hydrophobic contact", "bond_interaction" ], [ 77, 83, "Val318", "residue_name_number" ], [ 89, 91, "\u22123", "site" ], [ 92, 96, "Xylp", "chemical" ], [ 103, 129, "planar apolar interactions", "bond_interaction" ], [ 135, 141, "Ala137", "residue_name_number" ], [ 155, 161, "xylose", "chemical" ], [ 165, 167, "\u22124", "site" ], [ 174, 198, "parallel apolar contacts", "bond_interaction" ], [ 204, 209, "Trp69", "residue_name_number" ] ] }, { "sid": 119, "sent": "Comparison of the distal negative subsites of CtXyl5A with BaCel5A and a typical GH10 xylanase (CmXyn10B, PDB code 1uqy) highlights the paucity of interactions between the arabinoxylanase and its substrate out with the active site (Fig. 4).", "section": "RESULTS", "ner": [ [ 25, 42, "negative subsites", "site" ], [ 46, 53, "CtXyl5A", "protein" ], [ 59, 66, "BaCel5A", "protein" ], [ 81, 85, "GH10", "protein_type" ], [ 86, 94, "xylanase", "protein_type" ], [ 96, 104, "CmXyn10B", "protein" ], [ 172, 187, "arabinoxylanase", "protein_type" ], [ 219, 230, "active site", "site" ] ] }, { "sid": 120, "sent": "Thus, the cellulase contains three negative subsites and the sugars bound in the \u22122 and \u22123 subsites make a total of 9 polar interactions with the enzyme (Fig. 4, D and E).", "section": "RESULTS", "ner": [ [ 10, 19, "cellulase", "protein_type" ], [ 35, 52, "negative subsites", "site" ], [ 61, 67, "sugars", "chemical" ], [ 68, 76, "bound in", "protein_state" ], [ 81, 99, "\u22122 and \u22123 subsites", "site" ], [ 118, 136, "polar interactions", "bond_interaction" ] ] }, { "sid": 121, "sent": "The GH10 xylanase also contains a \u22122 subsite that, similar to the cellulase, makes numerous interactions with the substrate (Fig. 4, F and G).", "section": "RESULTS", "ner": [ [ 4, 8, "GH10", "protein_type" ], [ 9, 17, "xylanase", "protein_type" ], [ 34, 44, "\u22122 subsite", "site" ], [ 66, 75, "cellulase", "protein_type" ] ] }, { "sid": 122, "sent": "The Influence of the Modular Architecture of CtXyl5A on Catalytic Activity", "section": "RESULTS", "ner": [ [ 45, 52, "CtXyl5A", "protein" ] ] }, { "sid": 123, "sent": "CtXyl5A, in addition to its catalytic module, contains three CBMs (CtCBM6, CtCBM13, and CtCBM62) and a fibronectin domain (CtFn3).", "section": "RESULTS", "ner": [ [ 0, 7, "CtXyl5A", "protein" ], [ 28, 44, "catalytic module", "structure_element" ], [ 61, 65, "CBMs", "structure_element" ], [ 67, 73, "CtCBM6", "structure_element" ], [ 75, 82, "CtCBM13", "structure_element" ], [ 88, 95, "CtCBM62", "structure_element" ], [ 103, 121, "fibronectin domain", "structure_element" ], [ 123, 128, "CtFn3", "structure_element" ] ] }, { "sid": 124, "sent": "A previous study showed that although the CBM6 bound in an exo-mode to xylo- and cellulooligosaccharides, the primary role of this module was to stabilize the structure of the GH5 catalytic module.", "section": "RESULTS", "ner": [ [ 42, 46, "CBM6", "structure_element" ], [ 47, 55, "bound in", "protein_state" ], [ 59, 67, "exo-mode", "protein_state" ], [ 71, 104, "xylo- and cellulooligosaccharides", "chemical" ], [ 176, 179, "GH5", "protein_type" ], [ 180, 196, "catalytic module", "structure_element" ] ] }, { "sid": 125, "sent": "To explore the contribution of the other non-catalytic modules to CtXyl5A function, the activity of a series of truncated derivatives of the arabinoxylanase were assessed.", "section": "RESULTS", "ner": [ [ 41, 62, "non-catalytic modules", "structure_element" ], [ 66, 73, "CtXyl5A", "protein" ], [ 112, 121, "truncated", "protein_state" ], [ 141, 156, "arabinoxylanase", "protein_type" ] ] }, { "sid": 126, "sent": "The data in Table 1 show that removal of CtCBM62 caused a modest increase in activity against both WAX and CX, whereas deletion of the Fn3 domain had no further impact on catalytic performance.", "section": "RESULTS", "ner": [ [ 30, 40, "removal of", "experimental_method" ], [ 41, 48, "CtCBM62", "structure_element" ], [ 99, 102, "WAX", "chemical" ], [ 107, 109, "CX", "chemical" ], [ 119, 130, "deletion of", "experimental_method" ], [ 135, 138, "Fn3", "structure_element" ] ] }, { "sid": 127, "sent": "Truncation of CtCBM13, however, caused a 4\u20135-fold reduction in activity against both substrates.", "section": "RESULTS", "ner": [ [ 0, 10, "Truncation", "experimental_method" ], [ 14, 21, "CtCBM13", "structure_element" ] ] }, { "sid": 128, "sent": "Members of CBM13 have been shown to bind to xylans, mannose, and galactose residues in complex glycans, hinting that the function of CtCBM13 is to increase the proximity of substrate to the catalytic module of CtXyl5A.", "section": "RESULTS", "ner": [ [ 11, 16, "CBM13", "structure_element" ], [ 44, 50, "xylans", "chemical" ], [ 52, 59, "mannose", "chemical" ], [ 65, 74, "galactose", "chemical" ], [ 87, 102, "complex glycans", "chemical" ], [ 133, 140, "CtCBM13", "structure_element" ], [ 190, 206, "catalytic module", "structure_element" ], [ 210, 217, "CtXyl5A", "protein" ] ] }, { "sid": 129, "sent": "Binding studies, however, showed that CtCBM13 displayed no affinity for a range of relevant glycans including WAX, CX, xylose, mannose, galactose, and birchwood xylan (BX) (data not shown).", "section": "RESULTS", "ner": [ [ 0, 15, "Binding studies", "experimental_method" ], [ 38, 45, "CtCBM13", "structure_element" ], [ 92, 99, "glycans", "chemical" ], [ 110, 113, "WAX", "chemical" ], [ 115, 117, "CX", "chemical" ], [ 119, 125, "xylose", "chemical" ], [ 127, 134, "mannose", "chemical" ], [ 136, 145, "galactose", "chemical" ], [ 151, 166, "birchwood xylan", "chemical" ], [ 168, 170, "BX", "chemical" ] ] }, { "sid": 130, "sent": "It would appear, therefore, that CtCBM13 makes a structural contribution to the function of CtXyl5A.", "section": "RESULTS", "ner": [ [ 33, 40, "CtCBM13", "structure_element" ], [ 92, 99, "CtXyl5A", "protein" ] ] }, { "sid": 131, "sent": "Crystal Structure of CtXyl5A-D", "section": "RESULTS", "ner": [ [ 0, 17, "Crystal Structure", "evidence" ], [ 21, 30, "CtXyl5A-D", "mutant" ] ] }, { "sid": 132, "sent": "To explore further the role of the non-catalytic modules in CtXyl5A the crystal structure of CtXyl5A extending from CtGH5 to CtCBM62 was sought.", "section": "RESULTS", "ner": [ [ 35, 56, "non-catalytic modules", "structure_element" ], [ 60, 67, "CtXyl5A", "protein" ], [ 72, 89, "crystal structure", "evidence" ], [ 93, 100, "CtXyl5A", "protein" ], [ 116, 121, "CtGH5", "structure_element" ], [ 125, 132, "CtCBM62", "structure_element" ] ] }, { "sid": 133, "sent": "To obtain a construct that could potentially be crystallized, the protein was generated without the C-terminal dockerin domain because it is known to be unstable and prone to cleavage.", "section": "RESULTS", "ner": [ [ 48, 60, "crystallized", "experimental_method" ], [ 88, 95, "without", "protein_state" ], [ 111, 119, "dockerin", "structure_element" ] ] }, { "sid": 134, "sent": "Using this construct (CtXyl5A-D) the crystal structure of the arabinoxylanase was determined by molecular replacement to a resolution of 2.64 \u212b with Rwork and Rfree at 23.7% and 27.8%, respectively.", "section": "RESULTS", "ner": [ [ 22, 31, "CtXyl5A-D", "mutant" ], [ 37, 54, "crystal structure", "evidence" ], [ 62, 77, "arabinoxylanase", "protein_type" ], [ 96, 117, "molecular replacement", "experimental_method" ], [ 149, 154, "Rwork", "evidence" ], [ 159, 164, "Rfree", "evidence" ] ] }, { "sid": 135, "sent": "The structure comprises a continuous polypeptide extending from Ala36 to Trp742 displaying four modules GH5-CBM6-CBM13-Fn3.", "section": "RESULTS", "ner": [ [ 4, 13, "structure", "evidence" ], [ 64, 79, "Ala36 to Trp742", "residue_range" ], [ 104, 122, "GH5-CBM6-CBM13-Fn3", "structure_element" ] ] }, { "sid": 136, "sent": "Although there was some electron density for CtCBM62, it was not sufficient to confidently build the module (Fig. 5).", "section": "RESULTS", "ner": [ [ 24, 40, "electron density", "evidence" ], [ 45, 52, "CtCBM62", "structure_element" ] ] }, { "sid": 137, "sent": "Further investigation of the crystal packing revealed a large solvent channel adjacent to the area the CBM62 occupies.", "section": "RESULTS", "ner": [ [ 29, 44, "crystal packing", "evidence" ], [ 62, 77, "solvent channel", "site" ], [ 103, 108, "CBM62", "structure_element" ] ] }, { "sid": 138, "sent": "We postulate that the reason for the poor electron density is due to the CtCBM62 being mobile compared with the rest of the protein.", "section": "RESULTS", "ner": [ [ 42, 58, "electron density", "evidence" ], [ 73, 80, "CtCBM62", "structure_element" ], [ 87, 93, "mobile", "protein_state" ] ] }, { "sid": 139, "sent": "The structures of CtGH5 and CtCBM6 have been described previously.", "section": "RESULTS", "ner": [ [ 4, 14, "structures", "evidence" ], [ 18, 23, "CtGH5", "structure_element" ], [ 28, 34, "CtCBM6", "structure_element" ] ] }, { "sid": 140, "sent": "Surface representation of the tetra-modular arabinoxylanase and zoom view on the CtGH5 loop.", "section": "FIG", "ner": [ [ 44, 59, "arabinoxylanase", "protein_type" ], [ 81, 86, "CtGH5", "structure_element" ], [ 87, 91, "loop", "structure_element" ] ] }, { "sid": 141, "sent": "The blue module is the CtGH5 catalytic domain, the green module corresponds to the CtCBM6, the yellow module is the CtCBM13, and the salmon module is the fibronectin domain.", "section": "FIG", "ner": [ [ 23, 28, "CtGH5", "structure_element" ], [ 29, 45, "catalytic domain", "structure_element" ], [ 83, 89, "CtCBM6", "structure_element" ], [ 116, 123, "CtCBM13", "structure_element" ], [ 154, 172, "fibronectin domain", "structure_element" ] ] }, { "sid": 142, "sent": "The CtGH5 loop is stabilized between the CtCBM6 and the CtCBM13 modules.", "section": "FIG", "ner": [ [ 4, 9, "CtGH5", "structure_element" ], [ 10, 14, "loop", "structure_element" ], [ 41, 47, "CtCBM6", "structure_element" ], [ 56, 63, "CtCBM13", "structure_element" ] ] }, { "sid": 143, "sent": "CtCBM13 extends from Gly567 to Pro648.", "section": "RESULTS", "ner": [ [ 0, 7, "CtCBM13", "structure_element" ], [ 21, 37, "Gly567 to Pro648", "residue_range" ] ] }, { "sid": 144, "sent": "Typical of CBM13 proteins CtCBM13 displays a \u03b2-trefoil fold comprising the canonical pseudo 3-fold symmetry with a 3-fold repeating unit of 40\u201350 amino acid residues characteristic of the Ricin superfamily.", "section": "RESULTS", "ner": [ [ 11, 16, "CBM13", "protein_type" ], [ 26, 33, "CtCBM13", "structure_element" ], [ 45, 59, "\u03b2-trefoil fold", "structure_element" ], [ 115, 136, "3-fold repeating unit", "structure_element" ], [ 140, 156, "40\u201350 amino acid", "residue_range" ], [ 188, 205, "Ricin superfamily", "protein_type" ] ] }, { "sid": 145, "sent": "Each repeat contains two pairs of antiparallel \u03b2-strands.", "section": "RESULTS", "ner": [ [ 5, 11, "repeat", "structure_element" ], [ 34, 56, "antiparallel \u03b2-strands", "structure_element" ] ] }, { "sid": 146, "sent": "A Dali search revealed structural homologs from the CBM13 family with an root mean square deviation less than 2.0 \u212b and sequence identities of less than 20% that include the functionally relevant homologs C. thermocellum exo-\u03b2-1,3-galactanase (PDB code 3vsz), Streptomyces avermitilis \u03b2-l-arabinopyranosidase (PDB code 3a21), Streptomyces lividans xylanase 10A (PDB code, 1mc9), and Streptomyces olivaceoviridis E-86 xylanase 10A (PDB code 1v6v).", "section": "RESULTS", "ner": [ [ 2, 13, "Dali search", "experimental_method" ], [ 52, 57, "CBM13", "protein_type" ], [ 73, 99, "root mean square deviation", "evidence" ], [ 205, 220, "C. thermocellum", "species" ], [ 221, 242, "exo-\u03b2-1,3-galactanase", "protein_type" ], [ 260, 284, "Streptomyces avermitilis", "species" ], [ 285, 308, "\u03b2-l-arabinopyranosidase", "protein_type" ], [ 326, 347, "Streptomyces lividans", "species" ], [ 348, 360, "xylanase 10A", "protein" ], [ 383, 416, "Streptomyces olivaceoviridis E-86", "species" ], [ 417, 429, "xylanase 10A", "protein" ] ] }, { "sid": 147, "sent": "The Fn3 module displays a typical \u03b2-sandwich fold with the two sheets comprising, primarily, three antiparallel strands in the order \u03b21-\u03b22-\u03b25 in \u03b2-sheet 1 and \u03b24-\u03b23-\u03b26 in \u03b2-sheet 2.", "section": "RESULTS", "ner": [ [ 4, 7, "Fn3", "structure_element" ], [ 34, 49, "\u03b2-sandwich fold", "structure_element" ], [ 63, 69, "sheets", "structure_element" ], [ 99, 119, "antiparallel strands", "structure_element" ], [ 133, 141, "\u03b21-\u03b22-\u03b25", "structure_element" ], [ 145, 154, "\u03b2-sheet 1", "structure_element" ], [ 159, 167, "\u03b24-\u03b23-\u03b26", "structure_element" ], [ 171, 180, "\u03b2-sheet 2", "structure_element" ] ] }, { "sid": 148, "sent": "Although \u03b2-sheet 2 presents a cleft-like topology, typical of endo-binding CBMs, the surface lacks aromatic residues that play a key role in ligand recognition, and in the context of the full-length enzyme, the cleft abuts into CtCBM13 and thus would not be able to accommodate an extended polysaccharide chain (see below).", "section": "RESULTS", "ner": [ [ 9, 18, "\u03b2-sheet 2", "structure_element" ], [ 30, 35, "cleft", "site" ], [ 62, 79, "endo-binding CBMs", "protein_type" ], [ 187, 198, "full-length", "protein_state" ], [ 199, 205, "enzyme", "protein" ], [ 211, 216, "cleft", "site" ], [ 228, 235, "CtCBM13", "structure_element" ], [ 290, 304, "polysaccharide", "chemical" ] ] }, { "sid": 149, "sent": "In the structure of CtXyl5A-D, the four modules form a three-leaf clover-like structure (Fig. 5).", "section": "RESULTS", "ner": [ [ 7, 16, "structure", "evidence" ], [ 20, 29, "CtXyl5A-D", "mutant" ], [ 40, 47, "modules", "structure_element" ] ] }, { "sid": 150, "sent": "Between the interfaces of CtGH5-CBM6-CBM13 there are a number of interactions that maintain the modules in a fixed position relative to each other.", "section": "RESULTS", "ner": [ [ 12, 22, "interfaces", "site" ], [ 26, 42, "CtGH5-CBM6-CBM13", "structure_element" ] ] }, { "sid": 151, "sent": "The interaction of CtGH5 and CtCBM6, which buries a substantial apolar solvent-exposed surface of the two modules, has been described previously.", "section": "RESULTS", "ner": [ [ 19, 24, "CtGH5", "structure_element" ], [ 29, 35, "CtCBM6", "structure_element" ], [ 64, 94, "apolar solvent-exposed surface", "site" ] ] }, { "sid": 152, "sent": "The polar interactions between these two modules comprise 14 hydrogen bonds and 5 salt bridges.", "section": "RESULTS", "ner": [ [ 4, 22, "polar interactions", "bond_interaction" ], [ 61, 75, "hydrogen bonds", "bond_interaction" ], [ 82, 94, "salt bridges", "bond_interaction" ] ] }, { "sid": 153, "sent": "The apolar and polar interactions between these two modules likely explaining why they do not fold independently compared with other glycoside hydrolases that contain CBMs.", "section": "RESULTS", "ner": [ [ 4, 33, "apolar and polar interactions", "bond_interaction" ], [ 133, 153, "glycoside hydrolases", "protein_type" ], [ 167, 171, "CBMs", "structure_element" ] ] }, { "sid": 154, "sent": "CtCBM13 acts as the central domain, which interacts with CtGH5, CtCBM6, and CtFn3 via 2, 5, and 4 hydrogen bonds, respectively, burying a surface area of \u223c450, 350, and 500 \u212b2, respectively, to form a compact heterotetramer.", "section": "RESULTS", "ner": [ [ 0, 7, "CtCBM13", "structure_element" ], [ 20, 34, "central domain", "structure_element" ], [ 42, 56, "interacts with", "protein_state" ], [ 57, 62, "CtGH5", "structure_element" ], [ 64, 70, "CtCBM6", "structure_element" ], [ 76, 81, "CtFn3", "structure_element" ], [ 98, 112, "hydrogen bonds", "bond_interaction" ], [ 201, 208, "compact", "protein_state" ], [ 209, 223, "heterotetramer", "oligomeric_state" ] ] }, { "sid": 155, "sent": "With respect to the CtCBM6-CBM13 interface, the linker (SPISTGTIP) between the two modules, extending from Ser514 to Pro522, adopts a fixed conformation.", "section": "RESULTS", "ner": [ [ 20, 42, "CtCBM6-CBM13 interface", "site" ], [ 48, 54, "linker", "structure_element" ], [ 56, 65, "SPISTGTIP", "structure_element" ], [ 83, 90, "modules", "structure_element" ], [ 107, 113, "Ser514", "residue_name_number" ], [ 117, 123, "Pro522", "residue_name_number" ], [ 134, 152, "fixed conformation", "protein_state" ] ] }, { "sid": 156, "sent": "Such sequences are normally extremely flexible; however, the two Ile residues make extensive apolar contacts within the linker and with the two CBMs, leading to conformational stabilization.", "section": "RESULTS", "ner": [ [ 65, 68, "Ile", "residue_name" ], [ 93, 108, "apolar contacts", "bond_interaction" ], [ 120, 126, "linker", "structure_element" ], [ 144, 148, "CBMs", "structure_element" ] ] }, { "sid": 157, "sent": "The interactions between CtGH5 and the two CBMs, which are mediated by the tip of the loop between \u03b2-7 and \u03b1-7 (loop 7) of CtGH5, not only stabilize the trimodular clover-like structure but also make a contribution to catalytic function.", "section": "RESULTS", "ner": [ [ 25, 30, "CtGH5", "structure_element" ], [ 43, 47, "CBMs", "structure_element" ], [ 86, 90, "loop", "structure_element" ], [ 99, 102, "\u03b2-7", "structure_element" ], [ 107, 110, "\u03b1-7", "structure_element" ], [ 112, 118, "loop 7", "structure_element" ], [ 123, 128, "CtGH5", "structure_element" ], [ 153, 170, "trimodular clover", "structure_element" ] ] }, { "sid": 158, "sent": "Central to the interactions between the three modules is Trp285, which is intercalated between the two CBMs.", "section": "RESULTS", "ner": [ [ 46, 53, "modules", "structure_element" ], [ 57, 63, "Trp285", "residue_name_number" ], [ 74, 94, "intercalated between", "bond_interaction" ], [ 103, 107, "CBMs", "structure_element" ] ] }, { "sid": 159, "sent": "The N\u03f5 of this aromatic residue makes hydrogen bonds with the backbone carbonyl of Val615 and Gly616 in CtCBM13, and the indole ring makes several apolar contacts with CtCBM6 (Pro440, Phe489, Gly491, and Ala492) (Fig. 5).", "section": "RESULTS", "ner": [ [ 38, 52, "hydrogen bonds", "bond_interaction" ], [ 83, 89, "Val615", "residue_name_number" ], [ 94, 100, "Gly616", "residue_name_number" ], [ 104, 111, "CtCBM13", "structure_element" ], [ 147, 162, "apolar contacts", "bond_interaction" ], [ 168, 174, "CtCBM6", "structure_element" ], [ 176, 182, "Pro440", "residue_name_number" ], [ 184, 190, "Phe489", "residue_name_number" ], [ 192, 198, "Gly491", "residue_name_number" ], [ 204, 210, "Ala492", "residue_name_number" ] ] }, { "sid": 160, "sent": "Indeed, loop 7 is completely disordered in the truncated derivative of CtXyl5A comprising CtGH5 and CtCBM6, demonstrating that the interactions with CtCBM13 stabilize the conformation of this loop.", "section": "RESULTS", "ner": [ [ 8, 14, "loop 7", "structure_element" ], [ 18, 39, "completely disordered", "protein_state" ], [ 47, 56, "truncated", "protein_state" ], [ 71, 78, "CtXyl5A", "protein" ], [ 90, 95, "CtGH5", "structure_element" ], [ 100, 106, "CtCBM6", "structure_element" ], [ 149, 156, "CtCBM13", "structure_element" ], [ 192, 196, "loop", "structure_element" ] ] }, { "sid": 161, "sent": "Although the tip of loop 7 does not directly contribute to the topology of the active site, it is only \u223c12 \u212b from the catalytic nucleophile Glu279.", "section": "RESULTS", "ner": [ [ 20, 26, "loop 7", "structure_element" ], [ 79, 90, "active site", "site" ], [ 140, 146, "Glu279", "residue_name_number" ] ] }, { "sid": 162, "sent": "Thus, any perturbation of the loop (through the removal of CtCBM13) is likely to influence the electrostatic and apolar environment of the catalytic apparatus, which could explain the reduction in activity associated with the deletion of CtCBM13.", "section": "RESULTS", "ner": [ [ 30, 34, "loop", "structure_element" ], [ 48, 55, "removal", "experimental_method" ], [ 59, 66, "CtCBM13", "structure_element" ], [ 226, 234, "deletion", "experimental_method" ], [ 238, 245, "CtCBM13", "structure_element" ] ] }, { "sid": 163, "sent": "Similar to the interactions between CtCBM6 and CtCBM13, there are extensive hydrophobic interactions between CtCBM13 and CtFn3, resulting in very little flexibility between these modules.", "section": "RESULTS", "ner": [ [ 36, 42, "CtCBM6", "structure_element" ], [ 47, 54, "CtCBM13", "structure_element" ], [ 76, 100, "hydrophobic interactions", "bond_interaction" ], [ 109, 116, "CtCBM13", "structure_element" ], [ 121, 126, "CtFn3", "structure_element" ], [ 179, 186, "modules", "structure_element" ] ] }, { "sid": 164, "sent": "As stated above, the absence of CtCBM62 in the structure suggests that the module can adopt multiple positions with respect to the rest of the protein.", "section": "RESULTS", "ner": [ [ 21, 31, "absence of", "protein_state" ], [ 32, 39, "CtCBM62", "structure_element" ], [ 47, 56, "structure", "evidence" ], [ 75, 81, "module", "structure_element" ] ] }, { "sid": 165, "sent": "The CtCBM62, by binding to its ligands (d-Galp and l-Arap) in plant cell walls, may be able to recruit the enzyme onto its target substrate.", "section": "RESULTS", "ner": [ [ 4, 11, "CtCBM62", "structure_element" ], [ 16, 26, "binding to", "protein_state" ], [ 40, 46, "d-Galp", "chemical" ], [ 51, 57, "l-Arap", "chemical" ], [ 62, 67, "plant", "taxonomy_domain" ] ] }, { "sid": 166, "sent": "Xylans are not generally thought to contain such sugars.", "section": "RESULTS", "ner": [ [ 0, 6, "Xylans", "chemical" ], [ 49, 55, "sugars", "chemical" ] ] }, { "sid": 167, "sent": "d-Galp, however, has been detected in xylans in the outer layer of cereal grains and in eucalyptus trees, which are substrates used by CtXyl5A.", "section": "RESULTS", "ner": [ [ 0, 6, "d-Galp", "chemical" ], [ 38, 44, "xylans", "chemical" ], [ 67, 73, "cereal", "taxonomy_domain" ], [ 88, 104, "eucalyptus trees", "taxonomy_domain" ], [ 135, 142, "CtXyl5A", "protein" ] ] }, { "sid": 168, "sent": "Thus, CtCBM62 may direct the enzyme to particularly complex xylans containing d-Galp at the non-reducing termini of the side chains, consistent with the open substrate binding cleft of the arabinoxylanase that is optimized to bind highly decorated forms of the hemicellulose.", "section": "RESULTS", "ner": [ [ 6, 13, "CtCBM62", "structure_element" ], [ 60, 66, "xylans", "chemical" ], [ 78, 84, "d-Galp", "chemical" ], [ 153, 157, "open", "protein_state" ], [ 158, 181, "substrate binding cleft", "site" ], [ 189, 204, "arabinoxylanase", "protein_type" ], [ 261, 274, "hemicellulose", "chemical" ] ] }, { "sid": 169, "sent": "In general CBMs have little influence on enzyme activity against soluble substrates but have a significant impact on glycans within plant cell walls.", "section": "RESULTS", "ner": [ [ 11, 15, "CBMs", "structure_element" ], [ 117, 124, "glycans", "chemical" ], [ 132, 137, "plant", "taxonomy_domain" ] ] }, { "sid": 170, "sent": "Thus, the role of CBM62 will likely only be evident against insoluble composite substrates.", "section": "RESULTS", "ner": [ [ 18, 23, "CBM62", "structure_element" ] ] }, { "sid": 171, "sent": "Exploring GH5 Subfamily 34", "section": "RESULTS", "ner": [ [ 10, 26, "GH5 Subfamily 34", "protein_type" ] ] }, { "sid": 172, "sent": "CtXyl5A is a member of a seven-protein subfamily of GH5, GH5_34.", "section": "RESULTS", "ner": [ [ 0, 7, "CtXyl5A", "protein" ], [ 52, 55, "GH5", "protein_type" ], [ 57, 63, "GH5_34", "protein_type" ] ] }, { "sid": 173, "sent": "Four of these proteins are distinct, whereas the other three members are essentially identical (derived from different strains of C. thermocellum).", "section": "RESULTS", "ner": [ [ 130, 145, "C. thermocellum", "species" ] ] }, { "sid": 174, "sent": "To investigate further the substrate specificity within this subfamily, recombinant forms of three members of GH5_34 that were distinct from CtXyl5A were generated.", "section": "RESULTS", "ner": [ [ 110, 116, "GH5_34", "protein_type" ], [ 141, 148, "CtXyl5A", "protein" ] ] }, { "sid": 175, "sent": "AcGH5 has a similar molecular architecture to CtXyl5A with the exception of an additional carbohydrate esterase family 6 module at the C terminus (Fig. 1).", "section": "RESULTS", "ner": [ [ 0, 5, "AcGH5", "protein" ], [ 46, 53, "CtXyl5A", "protein" ], [ 90, 127, "carbohydrate esterase family 6 module", "structure_element" ] ] }, { "sid": 176, "sent": "The GH5_34 from Verrucomicrobiae bacterium, VbGH5, contains the GH5-CBM6-CBM13 core structure, but the C-terminal Fn3-CBM62-dockerin modules, present in CtXyl5A, are replaced with a Laminin_3_G domain, which, by analogy to homologous domains in other proteins that have affinity for carbohydrates, may display a glycan binding function.", "section": "RESULTS", "ner": [ [ 4, 10, "GH5_34", "protein_type" ], [ 16, 32, "Verrucomicrobiae", "taxonomy_domain" ], [ 33, 42, "bacterium", "taxonomy_domain" ], [ 44, 49, "VbGH5", "protein" ], [ 64, 78, "GH5-CBM6-CBM13", "structure_element" ], [ 114, 132, "Fn3-CBM62-dockerin", "structure_element" ], [ 153, 160, "CtXyl5A", "protein" ], [ 182, 200, "Laminin_3_G domain", "structure_element" ], [ 283, 296, "carbohydrates", "chemical" ], [ 312, 318, "glycan", "chemical" ] ] }, { "sid": 177, "sent": "The Verrucomicobiae enzyme also has an N-terminal GH43 subfamily 10 (GH43_10) catalytic module.", "section": "RESULTS", "ner": [ [ 4, 19, "Verrucomicobiae", "taxonomy_domain" ], [ 50, 67, "GH43 subfamily 10", "protein_type" ], [ 69, 76, "GH43_10", "protein_type" ], [ 78, 94, "catalytic module", "structure_element" ] ] }, { "sid": 178, "sent": "The fungal GH5_34, GpGH5, unlike the two bacterial homologs, comprises a single GH5 catalytic module lacking all of the other accessory modules (Fig. 1).", "section": "RESULTS", "ner": [ [ 4, 10, "fungal", "taxonomy_domain" ], [ 11, 17, "GH5_34", "protein_type" ], [ 19, 24, "GpGH5", "protein" ], [ 41, 50, "bacterial", "taxonomy_domain" ], [ 80, 83, "GH5", "protein_type" ], [ 84, 100, "catalytic module", "structure_element" ] ] }, { "sid": 179, "sent": "GpGh5 is particularly interesting as Gonapodya prolifera is the only fungus of the several hundred fungal genomes that encodes a GH5_34 enzyme.", "section": "RESULTS", "ner": [ [ 0, 5, "GpGh5", "protein" ], [ 37, 56, "Gonapodya prolifera", "species" ], [ 69, 75, "fungus", "taxonomy_domain" ], [ 99, 105, "fungal", "taxonomy_domain" ], [ 129, 135, "GH5_34", "protein_type" ] ] }, { "sid": 180, "sent": "In fact there are four potential GH5_34 sequences in the G. prolifera genome, all of which show high sequence homology to Clostridium GH5_34 sequences.", "section": "RESULTS", "ner": [ [ 33, 39, "GH5_34", "protein_type" ], [ 57, 69, "G. prolifera", "species" ], [ 122, 133, "Clostridium", "taxonomy_domain" ], [ 134, 140, "GH5_34", "protein_type" ] ] }, { "sid": 181, "sent": "G. prolifera and Clostridium occupy similar environments, suggesting that the GpGH5_34 gene was acquired from a Clostridium species, which was followed by duplication of the gene in the fungal genome.", "section": "RESULTS", "ner": [ [ 0, 12, "G. prolifera", "species" ], [ 17, 28, "Clostridium", "taxonomy_domain" ], [ 78, 86, "GpGH5_34", "protein" ], [ 112, 123, "Clostridium", "taxonomy_domain" ], [ 186, 192, "fungal", "taxonomy_domain" ] ] }, { "sid": 182, "sent": "The sequence identity of the GH5_34 catalytic modules with CtXyl5A ranged from 55 to 80% (supplemental Fig. S1).", "section": "RESULTS", "ner": [ [ 29, 35, "GH5_34", "protein_type" ], [ 36, 53, "catalytic modules", "structure_element" ], [ 59, 66, "CtXyl5A", "protein" ] ] }, { "sid": 183, "sent": "All the GH5_34 enzymes were active on the arabinoxylans RAX, WAX, and CX but displayed no activity on BX (Table 1 and Fig. 6) and are thus defined as arabinoxylanases.", "section": "RESULTS", "ner": [ [ 8, 14, "GH5_34", "protein_type" ], [ 42, 55, "arabinoxylans", "chemical" ], [ 56, 59, "RAX", "chemical" ], [ 61, 64, "WAX", "chemical" ], [ 70, 72, "CX", "chemical" ], [ 102, 104, "BX", "chemical" ], [ 150, 166, "arabinoxylanases", "protein_type" ] ] }, { "sid": 184, "sent": "The limit products generated by CtXyl5A, AcGH5, and GpGH5 comprised a range of oligosaccharides with some high molecular weight material.", "section": "RESULTS", "ner": [ [ 32, 39, "CtXyl5A", "protein" ], [ 41, 46, "AcGH5", "protein" ], [ 52, 57, "GpGH5", "protein" ], [ 79, 95, "oligosaccharides", "chemical" ] ] }, { "sid": 185, "sent": "The oligosaccharides with low degrees of polymerization were absent in the VbGH5 reaction products.", "section": "RESULTS", "ner": [ [ 4, 20, "oligosaccharides", "chemical" ], [ 75, 80, "VbGH5", "protein" ] ] }, { "sid": 186, "sent": "However, the enzyme generated a large amount of arabinose, which was not produced by the other arabinoxylanases.", "section": "RESULTS", "ner": [ [ 48, 57, "arabinose", "chemical" ], [ 95, 111, "arabinoxylanases", "protein_type" ] ] }, { "sid": 187, "sent": "Given that GH43_10 is predominantly an arabinofuranosidase subfamily of GH43, the arabinose generated by VbGH5 is likely mediated by the N-terminal catalytic module (see below).", "section": "RESULTS", "ner": [ [ 11, 18, "GH43_10", "protein_type" ], [ 39, 58, "arabinofuranosidase", "protein_type" ], [ 72, 76, "GH43", "protein_type" ], [ 82, 91, "arabinose", "chemical" ], [ 105, 110, "VbGH5", "protein" ], [ 148, 164, "catalytic module", "structure_element" ] ] }, { "sid": 188, "sent": "Kinetic analysis showed that AcGH5 displayed similar activity to CtXyl5A against both WAX and RAX and was 2-fold less active against CX.", "section": "RESULTS", "ner": [ [ 29, 34, "AcGH5", "protein" ], [ 65, 72, "CtXyl5A", "protein" ], [ 86, 89, "WAX", "chemical" ], [ 94, 97, "RAX", "chemical" ], [ 133, 135, "CX", "chemical" ] ] }, { "sid": 189, "sent": "When initially measuring the activity of wild type VbGH5 against the different substrates, no clear data could be obtained, regardless of the concentration of enzyme used the reaction appeared to cease after a few minutes.", "section": "RESULTS", "ner": [ [ 41, 50, "wild type", "protein_state" ], [ 51, 56, "VbGH5", "protein" ] ] }, { "sid": 190, "sent": "We hypothesized that the N-terminal GH43_10 rapidly removed single arabinose decorations from the arabinoxylans depleting the substrate available to the arabinoxylanase, explaining why this activity was short lived.", "section": "RESULTS", "ner": [ [ 36, 43, "GH43_10", "protein_type" ], [ 67, 76, "arabinose", "chemical" ], [ 98, 111, "arabinoxylans", "chemical" ], [ 153, 168, "arabinoxylanase", "protein_type" ] ] }, { "sid": 191, "sent": "To test this hypothesis, the conserved catalytic base (Asp45) of the GH43_10 module of VbGH5 was substituted with alanine, which is predicted to inactivate this catalytic module.", "section": "RESULTS", "ner": [ [ 29, 38, "conserved", "protein_state" ], [ 55, 60, "Asp45", "residue_name_number" ], [ 69, 76, "GH43_10", "structure_element" ], [ 87, 92, "VbGH5", "protein" ], [ 97, 113, "substituted with", "experimental_method" ], [ 114, 121, "alanine", "residue_name" ], [ 161, 177, "catalytic module", "structure_element" ] ] }, { "sid": 192, "sent": "The D45A mutant did not produce arabinose consistent with the arabinofuranosidase activity displayed by the GH43_10 module in the wild type enzyme (Fig. 6).", "section": "RESULTS", "ner": [ [ 4, 8, "D45A", "mutant" ], [ 9, 15, "mutant", "protein_state" ], [ 32, 41, "arabinose", "chemical" ], [ 62, 81, "arabinofuranosidase", "protein_type" ], [ 108, 115, "GH43_10", "structure_element" ], [ 130, 139, "wild type", "protein_state" ] ] }, { "sid": 193, "sent": "The kinetics of the GH5_34 arabinoxylanase catalytic module was now measurable, and activities were determined to be between \u223c6- and 10-fold lower than that of CtXyl5A.", "section": "RESULTS", "ner": [ [ 4, 12, "kinetics", "evidence" ], [ 20, 26, "GH5_34", "protein_type" ], [ 27, 42, "arabinoxylanase", "protein_type" ], [ 43, 59, "catalytic module", "structure_element" ], [ 160, 167, "CtXyl5A", "protein" ] ] }, { "sid": 194, "sent": "Interestingly, the fungal arabinoxylanase displays the highest activities against WAX and RAX, \u223c4- and 6-fold higher, respectively, than CtXyl5A; however, there is very little difference in the activity between the eukaryotic and prokaryotic enzymes against CX.", "section": "RESULTS", "ner": [ [ 19, 25, "fungal", "taxonomy_domain" ], [ 26, 41, "arabinoxylanase", "protein_type" ], [ 82, 85, "WAX", "chemical" ], [ 90, 93, "RAX", "chemical" ], [ 137, 144, "CtXyl5A", "protein" ], [ 215, 225, "eukaryotic", "taxonomy_domain" ], [ 230, 241, "prokaryotic", "taxonomy_domain" ], [ 258, 260, "CX", "chemical" ] ] }, { "sid": 195, "sent": "Attempts to express individual modules of a variety of truncations of AcGH5 and VbGH5 were unsuccessful.", "section": "RESULTS", "ner": [ [ 70, 75, "AcGH5", "protein" ], [ 80, 85, "VbGH5", "protein" ] ] }, { "sid": 196, "sent": "This may indicate that the individual modules can only fold correctly when incorporated into the full-length enzyme, demonstrating the importance of intermodule interactions to maintain the structural integrity of these enzymes.", "section": "RESULTS", "ner": [ [ 97, 108, "full-length", "protein_state" ] ] }, { "sid": 197, "sent": "Products profile generated of GH5_34 enzymes.", "section": "FIG", "ner": [ [ 30, 36, "GH5_34", "protein_type" ] ] }, { "sid": 198, "sent": "The enzymes at 1 \u03bcm were incubated with the four different xylans at 1% in 50 mm sodium phosphate buffer for 16 h at 37 \u00b0C (GpGH5, VbGH5, and AcGH5) or 60 \u00b0C.", "section": "FIG", "ner": [ [ 25, 34, "incubated", "experimental_method" ], [ 59, 65, "xylans", "chemical" ], [ 124, 129, "GpGH5", "protein" ], [ 131, 136, "VbGH5", "protein" ], [ 142, 147, "AcGH5", "protein" ] ] }, { "sid": 199, "sent": "The limit products were separated by TLC.", "section": "FIG", "ner": [ [ 37, 40, "TLC", "experimental_method" ] ] }, { "sid": 200, "sent": "The xylooligosaccharide standards (X) are indicated by their degrees of polymerization.", "section": "FIG", "ner": [ [ 4, 23, "xylooligosaccharide", "chemical" ] ] }, { "sid": 201, "sent": "A characteristic feature of enzymes that attack the plant cell wall is their complex molecular architecture.", "section": "DISCUSS", "ner": [ [ 52, 57, "plant", "taxonomy_domain" ] ] }, { "sid": 202, "sent": "The CBMs in these enzymes generally play a role in substrate targeting and are appended to the catalytic modules through flexible linker sequences.", "section": "DISCUSS", "ner": [ [ 4, 8, "CBMs", "structure_element" ], [ 95, 112, "catalytic modules", "structure_element" ], [ 121, 146, "flexible linker sequences", "structure_element" ] ] }, { "sid": 203, "sent": "CtXyl5A provides a rare visualization of the structure of multiple modules within a single enzyme.", "section": "DISCUSS", "ner": [ [ 0, 7, "CtXyl5A", "protein" ], [ 45, 54, "structure", "evidence" ] ] }, { "sid": 204, "sent": "The central feature of these data is the structural role played by two of the CBMs, CtCBM6 and CtCBM13, in maintaining the active conformation of the catalytic module, CtGH5.", "section": "DISCUSS", "ner": [ [ 78, 82, "CBMs", "structure_element" ], [ 84, 90, "CtCBM6", "structure_element" ], [ 95, 102, "CtCBM13", "structure_element" ], [ 123, 129, "active", "protein_state" ], [ 150, 166, "catalytic module", "structure_element" ], [ 168, 173, "CtGH5", "structure_element" ] ] }, { "sid": 205, "sent": "The crystallographic data described here are supported by biochemical data showing either that these two modules do not bind to glycans (CtCBM13) or that the recognition of the non-reducing end of xylan or cellulose chains (CtCBM6) is unlikely to be biologically significant.", "section": "DISCUSS", "ner": [ [ 4, 25, "crystallographic data", "evidence" ], [ 128, 135, "glycans", "chemical" ], [ 137, 144, "CtCBM13", "structure_element" ], [ 197, 202, "xylan", "chemical" ], [ 206, 215, "cellulose", "chemical" ], [ 224, 230, "CtCBM6", "structure_element" ] ] }, { "sid": 206, "sent": "It should be emphasized, however, that glycan binding and substrate targeting may only be evident in the full-length enzyme acting on highly complex structures such as the plant cell wall, as observed recently by a CBM46 module in the Bacillus xyloglucanase/mixed linked glucanase BhCel5B.", "section": "DISCUSS", "ner": [ [ 39, 45, "glycan", "chemical" ], [ 105, 116, "full-length", "protein_state" ], [ 172, 177, "plant", "taxonomy_domain" ], [ 215, 220, "CBM46", "structure_element" ], [ 235, 243, "Bacillus", "taxonomy_domain" ], [ 244, 257, "xyloglucanase", "protein_type" ], [ 258, 280, "mixed linked glucanase", "protein_type" ], [ 281, 288, "BhCel5B", "protein" ] ] }, { "sid": 207, "sent": "CtXyl5A is a member of GH5 that contains 6644 members.", "section": "DISCUSS", "ner": [ [ 0, 7, "CtXyl5A", "protein" ], [ 23, 26, "GH5", "protein_type" ] ] }, { "sid": 208, "sent": "CtXyl5A is a member of subfamily GH5_34.", "section": "DISCUSS", "ner": [ [ 0, 7, "CtXyl5A", "protein" ], [ 33, 39, "GH5_34", "protein_type" ] ] }, { "sid": 209, "sent": "Despite differences in sequence identity all of the homologs were shown to be arabinoxylanases.", "section": "DISCUSS", "ner": [ [ 78, 94, "arabinoxylanases", "protein_type" ] ] }, { "sid": 210, "sent": "Consistent with the conserved substrate specificity, all members of GH5_34 contained the specificity determinants Glu68, Tyr92, and Asn139, which make critical interactions with the xylose or arabinose in the \u22122* subsite, which are 1,3-linked to the xylose positioned in the active site.", "section": "DISCUSS", "ner": [ [ 68, 74, "GH5_34", "protein_type" ], [ 89, 113, "specificity determinants", "site" ], [ 114, 119, "Glu68", "residue_name_number" ], [ 121, 126, "Tyr92", "residue_name_number" ], [ 132, 138, "Asn139", "residue_name_number" ], [ 182, 188, "xylose", "chemical" ], [ 192, 201, "arabinose", "chemical" ], [ 209, 220, "\u22122* subsite", "site" ], [ 250, 256, "xylose", "chemical" ], [ 275, 286, "active site", "site" ] ] }, { "sid": 211, "sent": "The presence of a CBM62 in CtXyl5A and AcGH5 suggests that these enzymes target highly complex xylans that contain d-galactose in their side chains.", "section": "DISCUSS", "ner": [ [ 18, 23, "CBM62", "structure_element" ], [ 27, 34, "CtXyl5A", "protein" ], [ 39, 44, "AcGH5", "protein" ], [ 95, 101, "xylans", "chemical" ], [ 115, 126, "d-galactose", "chemical" ] ] }, { "sid": 212, "sent": "The absence of a \u201cnon-structural\u201d CBM in GpGH5 may indicate that this arabinoxylanase is designed to target simpler arabinoxylans present in the endosperm of cereals.", "section": "DISCUSS", "ner": [ [ 4, 14, "absence of", "protein_state" ], [ 34, 37, "CBM", "structure_element" ], [ 41, 46, "GpGH5", "protein" ], [ 70, 85, "arabinoxylanase", "protein_type" ], [ 116, 129, "arabinoxylans", "chemical" ], [ 158, 165, "cereals", "taxonomy_domain" ] ] }, { "sid": 213, "sent": "Although the characterization of all members of GH5_34 suggests that this subfamily is monospecific, differences in specificity are observed in other subfamilies of GHs including GH43 and GH5.", "section": "DISCUSS", "ner": [ [ 48, 54, "GH5_34", "protein_type" ], [ 165, 168, "GHs", "protein_type" ], [ 179, 183, "GH43", "protein_type" ], [ 188, 191, "GH5", "protein_type" ] ] }, { "sid": 214, "sent": "Thus, as new members of GH5_34 are identified from genomic sequence data and subsequently characterized, the specificity of this family may require reinterpretation.", "section": "DISCUSS", "ner": [ [ 24, 30, "GH5_34", "protein_type" ] ] }, { "sid": 215, "sent": "An intriguing feature of VbGH5 is that the limited products generated by this enzymes are much larger than those produced by the other arabinoxylanases.", "section": "DISCUSS", "ner": [ [ 25, 30, "VbGH5", "protein" ], [ 135, 151, "arabinoxylanases", "protein_type" ] ] }, { "sid": 216, "sent": "This suggests that although arabinose decorations contribute to enzyme specificity (VbGH5 is not active on xylans lacking arabinose side chains), the enzyme requires other specificity determinants that occur less frequently in arabinoxylans.", "section": "DISCUSS", "ner": [ [ 28, 37, "arabinose", "chemical" ], [ 84, 89, "VbGH5", "protein" ], [ 107, 113, "xylans", "chemical" ], [ 122, 131, "arabinose", "chemical" ], [ 227, 240, "arabinoxylans", "chemical" ] ] }, { "sid": 217, "sent": "This has some resonance with a recently described GH98 xylanase that also exploits specificity determinants that occur infrequently and are only evident in highly complex xylans (e.g. CX).", "section": "DISCUSS", "ner": [ [ 50, 54, "GH98", "protein_type" ], [ 55, 63, "xylanase", "protein_type" ], [ 171, 177, "xylans", "chemical" ], [ 184, 186, "CX", "chemical" ] ] }, { "sid": 218, "sent": "To conclude, this study provides the molecular basis for the specificity displayed by arabinoxylanases.", "section": "DISCUSS", "ner": [ [ 86, 102, "arabinoxylanases", "protein_type" ] ] }, { "sid": 219, "sent": "Substrate specificity is dominated by the pocket that binds single arabinose or xylose side chains.", "section": "DISCUSS", "ner": [ [ 42, 48, "pocket", "site" ], [ 67, 76, "arabinose", "chemical" ], [ 80, 86, "xylose", "chemical" ] ] }, { "sid": 220, "sent": "The open xylan binding cleft explains why the enzyme is able to attack highly decorated forms of the hemicellulose.", "section": "DISCUSS", "ner": [ [ 4, 8, "open", "protein_state" ], [ 9, 28, "xylan binding cleft", "site" ], [ 101, 114, "hemicellulose", "chemical" ] ] }, { "sid": 221, "sent": "It is also evident that appending additional catalytic modules and CBMs onto the core components of these enzymes generates bespoke arabinoxylanases with activities optimized for specific functions.", "section": "DISCUSS", "ner": [ [ 45, 62, "catalytic modules", "structure_element" ], [ 67, 71, "CBMs", "structure_element" ], [ 132, 148, "arabinoxylanases", "protein_type" ] ] }, { "sid": 222, "sent": "The specificities of the arabinoxylanases described here are distinct from the classical endo-xylanases and thus have the potential to contribute to the toolbox of biocatalysts required by industries that exploit the plant cell wall as a sustainable substrate.", "section": "DISCUSS", "ner": [ [ 25, 41, "arabinoxylanases", "protein_type" ], [ 89, 103, "endo-xylanases", "protein_type" ], [ 217, 222, "plant", "taxonomy_domain" ] ] }, { "sid": 223, "sent": "Data collection and refinement statistics", "section": "TABLE", "ner": [ [ 0, 41, "Data collection and refinement statistics", "evidence" ] ] }, { "sid": 224, "sent": "\tCtXyl5A-D\tGH5-CBM6-Arap\tGH5-CBM6-Xylp\tGH5-CBM6- (Araf-Xylp4)\t \tData collection\t\t\t\t\t \t\u2003\u2003\u2003\u2003Source\tESRF-ID14-1\tDiamond I04\u20131\tDiamond I24\tDiamond I02\t \t\u2003\u2003\u2003\u2003Wavelength (\u00c5)\t0.9334\t0.9173\t0.9772\t0.9791\t \t\u2003\u2003\u2003\u2003Space group\tP21212\tP212121\tP212121\tP212121\t \t\u2003\u2003\u2003\u2003Cell dimensions\t\t\t\t\t \t\u2003\u2003\u2003\u2003\u2003\u2003\u2003\u2003a, b, c (\u00c5)\t147.4, 191.7, 50.7\t67.1, 72.4, 109.1\t67.9, 72.5, 109.5\t76.3, 123.2, 125.4\t \t\u2003\u2003\u2003\u2003\u2003\u2003\u2003\u2003\u03b1, \u03b2, \u03b3 (\u00b0)\t90, 90, 90\t90, 90, 90\t90, 90, 90\t90, 90, 90\t \t\u2003\u2003\u2003\u2003No. of measured reflections\t244,475 (29,324)\t224,842 (11,281)\t152,004 (4,996)\t463,237 (23,068)\t \t\u2003\u2003\u2003\u2003No. of independent reflections\t42246 (5,920)\t63,523 (3,175)\t42,716 (2,334)\t140,288 (6,879)\t \t\u2003\u2003\u2003\u2003Resolution (\u00c5)\t50.70\u20132.64 (2.78\u20132.64)\t44.85\u20131.65 (1.68\u20131.65)\t45.16\u20131.90 (1.94\u20131.90)\t48.43\u20131.65 (1.68\u20131.65)\t \t\u2003\u2003\u2003\u2003Rmerge (%)\t16.5 (69.5)\t6.7 (65.1)\t2.8 (8.4)\t5.7 (74.9)\t \t\u2003\u2003\u2003\u2003CC1/2\t0.985 (0.478)\t0.998 (0.594)\t0.999 (0.982)\t0.998 (0.484)\t \t\u2003\u2003\u2003\u2003I/\u03c3I\t8.0 (2.0)\t13 (1.6)\t26.6 (8.0)\t11.2 (1.6)\t \t\u2003\u2003\u2003\u2003Completeness (%)\t98.5 (96.4)\t98.5 (99.4)\t98.6 (85.0)\t98.8 (99.4)\t \t\u2003\u2003\u2003\u2003Redundancy\t5.8 (5.0)\t3.5 (3.6)\t3.6 (2.1)\t3.3 (3.4)\t \t\t \tRefinement\t\t\t\t\t \t\u2003\u2003\u2003\u2003Rwork/Rfree\t23.7/27.8\t12.2/17.0\t12.9/16.1\t14.5/19.9\t \t\u2003\u2003\u2003\u2003No. atoms\t\t\t\t\t \t\u2003\u2003\u2003\u2003\u2003\u2003\u2003\u2003Protein\t5446\t3790\t3729\t7333\t \t\u2003\u2003\u2003\u2003\u2003\u2003\u2003\u2003Ligand\t19\t20\t20\t92\t \t\u2003\u2003\u2003\u2003\u2003\u2003\u2003\u2003Water\t227\t579\t601\t923\t \t\u2003\u2003\u2003\u2003B-factors\t\t\t\t\t \t\u2003\u2003\u2003\u2003\u2003\u2003\u2003\u2003Protein\t41.6\t17.8\t15.8\t21.0\t \t\u2003\u2003\u2003\u2003\u2003\u2003\u2003\u2003Ligand\t65.0\t19.4\t24.2\t39.5\t \t\u2003\u2003\u2003\u2003\u2003\u2003\u2003\u2003Water\t35.4\t38.5\t32.2\t37.6\t \t\u2003\u2003\u2003\u2003R.m.s deviations\t\t\t\t\t \t\u2003\u2003\u2003\u2003\u2003\u2003\u2003\u2003Bond lengths (\u00c5)\t0.008\t0.015\t0.012\t0.012\t \t\u2003\u2003\u2003\u2003\u2003\u2003\u2003\u2003Bond angles (\u00b0)\t1.233\t1.502\t1.624\t1.554\t \t\u2003\u2003\u2003\u2003Protein Data Bank code\t5G56\t5LA0\t5LA1\t2LA2\t \t", "section": "TABLE", "ner": [ [ 1, 10, "CtXyl5A-D", "mutant" ], [ 11, 24, "GH5-CBM6-Arap", "complex_assembly" ], [ 25, 38, "GH5-CBM6-Xylp", "complex_assembly" ], [ 39, 61, "GH5-CBM6- (Araf-Xylp4)", "complex_assembly" ], [ 1079, 1084, "Rwork", "evidence" ], [ 1085, 1090, "Rfree", "evidence" ] ] }, { "sid": 225, "sent": "GH", "section": "SUPPL", "ner": [ [ 0, 2, "GH", "protein_type" ] ] }, { "sid": 226, "sent": "glycoside hydrolase", "section": "SUPPL", "ner": [ [ 0, 19, "glycoside hydrolase", "protein_type" ] ] }, { "sid": 227, "sent": "CtXyl5A", "section": "SUPPL", "ner": [ [ 0, 7, "CtXyl5A", "protein" ] ] }, { "sid": 228, "sent": "C. thermocellum arabinoxylanase", "section": "SUPPL", "ner": [ [ 0, 15, "C. thermocellum", "species" ], [ 16, 31, "arabinoxylanase", "protein_type" ] ] }, { "sid": 229, "sent": "CBM", "section": "SUPPL", "ner": [ [ 0, 3, "CBM", "structure_element" ] ] }, { "sid": 230, "sent": "non-catalytic carbohydrate binding module", "section": "SUPPL", "ner": [ [ 0, 41, "non-catalytic carbohydrate binding module", "structure_element" ] ] }, { "sid": 231, "sent": "Fn", "section": "SUPPL", "ner": [ [ 0, 2, "Fn", "protein_type" ] ] }, { "sid": 232, "sent": "fibronectin", "section": "SUPPL", "ner": [ [ 0, 11, "fibronectin", "protein_type" ] ] }, { "sid": 233, "sent": "WAX", "section": "SUPPL", "ner": [ [ 0, 3, "WAX", "chemical" ] ] }, { "sid": 234, "sent": "wheat arabinoxylan", "section": "SUPPL", "ner": [ [ 0, 5, "wheat", "taxonomy_domain" ], [ 6, 18, "arabinoxylan", "chemical" ] ] }, { "sid": 235, "sent": "RAX", "section": "SUPPL", "ner": [ [ 0, 3, "RAX", "chemical" ] ] }, { "sid": 236, "sent": "rye arabinoxylan", "section": "SUPPL", "ner": [ [ 0, 3, "rye", "taxonomy_domain" ], [ 4, 16, "arabinoxylan", "chemical" ] ] }, { "sid": 237, "sent": "CX", "section": "SUPPL", "ner": [ [ 0, 2, "CX", "chemical" ] ] }, { "sid": 238, "sent": "corn bran xylan", "section": "SUPPL", "ner": [ [ 0, 4, "corn", "taxonomy_domain" ], [ 10, 15, "xylan", "chemical" ] ] }, { "sid": 239, "sent": "HPAEC", "section": "SUPPL", "ner": [ [ 0, 5, "HPAEC", "experimental_method" ] ] }, { "sid": 240, "sent": "high performance anion exchange chromatography", "section": "SUPPL", "ner": [ [ 0, 46, "high performance anion exchange chromatography", "experimental_method" ] ] }, { "sid": 241, "sent": "birchwood xylan", "section": "SUPPL", "ner": [ [ 0, 9, "birchwood", "taxonomy_domain" ], [ 10, 15, "xylan", "chemical" ] ] }, { "sid": 242, "sent": "electrospray ionization.", "section": "SUPPL", "ner": [ [ 0, 23, "electrospray ionization", "experimental_method" ] ] } ] }, "PMC4980666": { "annotations": [ { "sid": 0, "sent": "N-acylhydrazone inhibitors of influenza virus PA endonuclease with versatile metal binding modes", "section": "TITLE", "ner": [ [ 0, 15, "N-acylhydrazone", "chemical" ], [ 30, 39, "influenza", "taxonomy_domain" ], [ 40, 45, "virus", "taxonomy_domain" ], [ 46, 48, "PA", "protein" ], [ 49, 61, "endonuclease", "protein_type" ] ] }, { "sid": 1, "sent": "Influenza virus PA endonuclease has recently emerged as an attractive target for the development of novel antiviral therapeutics.", "section": "ABSTRACT", "ner": [ [ 0, 9, "Influenza", "taxonomy_domain" ], [ 10, 15, "virus", "taxonomy_domain" ], [ 16, 18, "PA", "protein" ], [ 19, 31, "endonuclease", "protein_type" ] ] }, { "sid": 2, "sent": "This is an enzyme with divalent metal ion(s) (Mg2+ or Mn2+) in its catalytic site: chelation of these metal cofactors is an attractive strategy to inhibit enzymatic activity.", "section": "ABSTRACT", "ner": [ [ 46, 50, "Mg2+", "chemical" ], [ 54, 58, "Mn2+", "chemical" ], [ 67, 81, "catalytic site", "site" ], [ 83, 92, "chelation", "bond_interaction" ] ] }, { "sid": 3, "sent": "Here we report the activity of a series of N-acylhydrazones in an enzymatic assay with PA-Nter endonuclease, as well as in cell-based influenza vRNP reconstitution and virus yield assays.", "section": "ABSTRACT", "ner": [ [ 43, 59, "N-acylhydrazones", "chemical" ], [ 66, 81, "enzymatic assay", "experimental_method" ], [ 87, 89, "PA", "protein" ], [ 90, 94, "Nter", "structure_element" ], [ 95, 107, "endonuclease", "protein_type" ], [ 123, 163, "cell-based influenza vRNP reconstitution", "experimental_method" ], [ 168, 186, "virus yield assays", "experimental_method" ] ] }, { "sid": 4, "sent": "Several N-acylhydrazones were found to have promising anti-influenza activity in the low micromolar concentration range and good selectivity.", "section": "ABSTRACT", "ner": [ [ 8, 24, "N-acylhydrazones", "chemical" ], [ 59, 68, "influenza", "taxonomy_domain" ] ] }, { "sid": 5, "sent": "Computational docking studies are carried on to investigate the key features that determine inhibition of the endonuclease enzyme by N-acylhydrazones.", "section": "ABSTRACT", "ner": [ [ 0, 29, "Computational docking studies", "experimental_method" ], [ 110, 122, "endonuclease", "protein_type" ], [ 133, 149, "N-acylhydrazones", "chemical" ] ] }, { "sid": 6, "sent": "Moreover, we here describe the crystal structure of PA-Nter in complex with one of the most active inhibitors, revealing its interactions within the protein\u2019s active site.", "section": "ABSTRACT", "ner": [ [ 31, 48, "crystal structure", "evidence" ], [ 52, 54, "PA", "protein" ], [ 55, 59, "Nter", "structure_element" ], [ 60, 75, "in complex with", "protein_state" ], [ 159, 170, "active site", "site" ] ] }, { "sid": 7, "sent": "Influenza virus is an enveloped virus with a segmented negative-oriented single-stranded RNA genome, belonging to the Orthomyxoviridae.", "section": "INTRO", "ner": [ [ 0, 9, "Influenza", "taxonomy_domain" ], [ 10, 15, "virus", "taxonomy_domain" ], [ 22, 37, "enveloped virus", "taxonomy_domain" ], [ 55, 92, "negative-oriented single-stranded RNA", "chemical" ], [ 118, 134, "Orthomyxoviridae", "taxonomy_domain" ] ] }, { "sid": 8, "sent": "Seasonal influenza A and B viruses affect each year approximately 5\u201310% of the adult and 20\u201330% of the paediatric population, and there is a permanent risk of sudden influenza pandemics, such as the notorious \u2018Spanish flu\u2019 in 1918 and the swine-origin H1N1 pandemic in 2009.", "section": "INTRO", "ner": [ [ 9, 20, "influenza A", "taxonomy_domain" ], [ 25, 26, "B", "taxonomy_domain" ], [ 27, 34, "viruses", "taxonomy_domain" ], [ 166, 175, "influenza", "taxonomy_domain" ], [ 252, 256, "H1N1", "species" ] ] }, { "sid": 9, "sent": "Two classes of anti-influenza virus drugs are available, acting on the viral M2 ion-channel (amantadine and rimantadine) or on the viral neuraminidase (zanamivir and oseltamivir).", "section": "INTRO", "ner": [ [ 20, 29, "influenza", "taxonomy_domain" ], [ 30, 35, "virus", "taxonomy_domain" ], [ 71, 76, "viral", "taxonomy_domain" ], [ 77, 91, "M2 ion-channel", "protein_type" ], [ 93, 103, "amantadine", "chemical" ], [ 108, 119, "rimantadine", "chemical" ], [ 131, 136, "viral", "taxonomy_domain" ], [ 137, 150, "neuraminidase", "protein_type" ], [ 152, 161, "zanamivir", "chemical" ], [ 166, 177, "oseltamivir", "chemical" ] ] }, { "sid": 10, "sent": "The M2 inhibitors have limited clinical utility due to their central nervous system side effects and widespread resistance, as in the case of the 2009 pandemic H1N1 virus; resistance is also a growing concern for oseltamivir.", "section": "INTRO", "ner": [ [ 4, 6, "M2", "protein_type" ], [ 160, 164, "H1N1", "species" ], [ 165, 170, "virus", "taxonomy_domain" ], [ 213, 224, "oseltamivir", "chemical" ] ] }, { "sid": 11, "sent": "The influenza virus polymerase complex is composed of three subunits: PB1, PB2 and PA.", "section": "INTRO", "ner": [ [ 4, 13, "influenza", "taxonomy_domain" ], [ 14, 19, "virus", "taxonomy_domain" ], [ 20, 30, "polymerase", "protein_type" ], [ 70, 73, "PB1", "protein" ], [ 75, 78, "PB2", "protein" ], [ 83, 85, "PA", "protein" ] ] }, { "sid": 12, "sent": "The PA subunit performs the \u2018cap-snatching\u2019 endonuclease reaction, the PB2 subunit is responsible for initial binding of the capped RNAs, while the actual RNA synthesis is performed by the PB1 protein.", "section": "INTRO", "ner": [ [ 4, 6, "PA", "protein" ], [ 7, 14, "subunit", "structure_element" ], [ 44, 56, "endonuclease", "protein_type" ], [ 71, 74, "PB2", "protein" ], [ 75, 82, "subunit", "structure_element" ], [ 125, 136, "capped RNAs", "chemical" ], [ 155, 158, "RNA", "chemical" ], [ 189, 192, "PB1", "protein" ] ] }, { "sid": 13, "sent": "Given its crucial role in the viral life cycle, the influenza virus polymerase is widely recognized as a superior target for antiviral drug development and, in particular, inhibition of the PA endonuclease has deserved much attention in recent years.", "section": "INTRO", "ner": [ [ 30, 35, "viral", "taxonomy_domain" ], [ 52, 61, "influenza", "taxonomy_domain" ], [ 62, 67, "virus", "taxonomy_domain" ], [ 68, 78, "polymerase", "protein_type" ], [ 190, 192, "PA", "protein" ], [ 193, 205, "endonuclease", "protein_type" ] ] }, { "sid": 14, "sent": "The endonuclease catalytic site resides in the N-terminal domain of PA (PA-Nter; residues 1~195).", "section": "INTRO", "ner": [ [ 4, 16, "endonuclease", "protein_type" ], [ 17, 31, "catalytic site", "site" ], [ 47, 64, "N-terminal domain", "structure_element" ], [ 68, 70, "PA", "protein" ], [ 72, 74, "PA", "protein" ], [ 75, 79, "Nter", "structure_element" ], [ 90, 95, "1~195", "residue_range" ] ] }, { "sid": 15, "sent": "It comprises a histidine (His41) and a cluster of three strictly conserved acidic residues (Glu80, Asp108, Glu119), which coordinate (together with Ile120) one, two, or three manganese or magnesium ions.", "section": "INTRO", "ner": [ [ 15, 24, "histidine", "residue_name" ], [ 26, 31, "His41", "residue_name_number" ], [ 56, 74, "strictly conserved", "protein_state" ], [ 75, 81, "acidic", "protein_state" ], [ 92, 97, "Glu80", "residue_name_number" ], [ 99, 105, "Asp108", "residue_name_number" ], [ 107, 113, "Glu119", "residue_name_number" ], [ 122, 132, "coordinate", "bond_interaction" ], [ 148, 154, "Ile120", "residue_name_number" ], [ 175, 184, "manganese", "chemical" ], [ 188, 197, "magnesium", "chemical" ] ] }, { "sid": 16, "sent": "Since the intracellular concentration of Mg2+ is at least 1000-fold higher than that of Mn2+, magnesium may be more biologically relevant.", "section": "INTRO", "ner": [ [ 41, 45, "Mg2+", "chemical" ], [ 88, 93, "Mn2+,", "chemical" ], [ 94, 103, "magnesium", "chemical" ] ] }, { "sid": 17, "sent": "A controversy about number and type of metal ions exists also for the active site of HIV-1 integrase.", "section": "INTRO", "ner": [ [ 70, 81, "active site", "site" ], [ 85, 90, "HIV-1", "species" ], [ 91, 100, "integrase", "protein_type" ] ] }, { "sid": 18, "sent": "HIV-1 integrase inhibitors are a paradigm for the innovative drug concept that is based on coordination with the metal cofactor(s) of viral enzymes: similarly, several PA-binding agents with metal-chelating properties have been identified as influenza endonuclease inhibitors (Fig. 1), including 2,4-dioxobutanoic acid derivatives, flutimide and its derivatives, 2-hydroxyphenyl amide derivatives, as well as tetramic acids, 5-hydroxypyrimidin-4-one derivatives, marchantins and green tea catechins, like epigallocatechin-3-gallate (EGCG, Fig. 1).", "section": "INTRO", "ner": [ [ 0, 5, "HIV-1", "species" ], [ 6, 15, "integrase", "protein_type" ], [ 113, 118, "metal", "chemical" ], [ 134, 139, "viral", "taxonomy_domain" ], [ 168, 170, "PA", "protein" ], [ 242, 251, "influenza", "taxonomy_domain" ], [ 252, 264, "endonuclease", "protein_type" ], [ 296, 318, "2,4-dioxobutanoic acid", "chemical" ], [ 332, 341, "flutimide", "chemical" ], [ 363, 384, "2-hydroxyphenyl amide", "chemical" ], [ 409, 423, "tetramic acids", "chemical" ], [ 425, 449, "5-hydroxypyrimidin-4-one", "chemical" ], [ 463, 474, "marchantins", "chemical" ], [ 479, 488, "green tea", "taxonomy_domain" ], [ 489, 498, "catechins", "chemical" ], [ 505, 531, "epigallocatechin-3-gallate", "chemical" ], [ 533, 537, "EGCG", "chemical" ] ] }, { "sid": 19, "sent": "In recent years, we focused our research on chemical scaffolds that are able to chelate metal ions of PA-Nter, resulting in inhibition of influenza virus replication.", "section": "INTRO", "ner": [ [ 102, 104, "PA", "protein" ], [ 105, 109, "Nter", "structure_element" ], [ 138, 147, "influenza", "taxonomy_domain" ], [ 148, 153, "virus", "taxonomy_domain" ] ] }, { "sid": 20, "sent": "N-acylhydrazones represent an appealing class of chelating ligands with a broad spectrum of biological activities, such as activity against HIV, hepatitis A, vaccinia and influenza virus.", "section": "INTRO", "ner": [ [ 0, 16, "N-acylhydrazones", "chemical" ], [ 80, 88, "spectrum", "evidence" ], [ 140, 143, "HIV", "taxonomy_domain" ], [ 145, 156, "hepatitis A", "taxonomy_domain" ], [ 158, 166, "vaccinia", "taxonomy_domain" ], [ 171, 180, "influenza", "taxonomy_domain" ], [ 181, 186, "virus", "taxonomy_domain" ] ] }, { "sid": 21, "sent": "In the present work, we report the biological activity of a series of N-acylhydrazones (Fig. 2), as determined in an enzymatic assay with PA-Nter endonuclease as well as in cell-based influenza viral ribonucleoprotein (vRNP) reconstitution and virus yield assays.", "section": "INTRO", "ner": [ [ 70, 86, "N-acylhydrazones", "chemical" ], [ 117, 132, "enzymatic assay", "experimental_method" ], [ 138, 140, "PA", "protein" ], [ 141, 145, "Nter", "structure_element" ], [ 146, 158, "endonuclease", "protein_type" ], [ 173, 239, "cell-based influenza viral ribonucleoprotein (vRNP) reconstitution", "experimental_method" ], [ 244, 262, "virus yield assays", "experimental_method" ] ] }, { "sid": 22, "sent": "Several N-acylhydrazones were found to have promising anti-influenza activity with 50% effective concentration values (EC50) in the range of 3\u201320\u2009\u03bcM and good selectivity (Table 1 and Fig. 3).", "section": "INTRO", "ner": [ [ 8, 24, "N-acylhydrazones", "chemical" ], [ 59, 68, "influenza", "taxonomy_domain" ], [ 83, 110, "50% effective concentration", "evidence" ], [ 119, 123, "EC50", "evidence" ] ] }, { "sid": 23, "sent": "Computational docking studies of two candidate ligands in the PA-Nter active site gave information about the features that could determine inhibition of endonuclease activity.", "section": "INTRO", "ner": [ [ 0, 29, "Computational docking studies", "experimental_method" ], [ 62, 64, "PA", "protein" ], [ 65, 69, "Nter", "structure_element" ], [ 70, 81, "active site", "site" ], [ 153, 165, "endonuclease", "protein_type" ] ] }, { "sid": 24, "sent": "Moreover, we describe the X-ray crystal structure of PA-Nter in complex with one of the most active inhibitors.", "section": "INTRO", "ner": [ [ 26, 49, "X-ray crystal structure", "evidence" ], [ 53, 55, "PA", "protein" ], [ 56, 60, "Nter", "structure_element" ], [ 61, 76, "in complex with", "protein_state" ] ] }, { "sid": 25, "sent": "N-acylhydrazones 1\u201327 (Fig. 2) were prepared in high yields by following literature methods (Fig. 2A); they were characterized by spectroscopic tools, mass spectrometry and elemental analysis.", "section": "RESULTS", "ner": [ [ 0, 16, "N-acylhydrazones", "chemical" ], [ 17, 21, "1\u201327", "chemical" ], [ 151, 168, "mass spectrometry", "experimental_method" ], [ 173, 191, "elemental analysis", "experimental_method" ] ] }, { "sid": 26, "sent": "Even if isomerism around the C\u2009=\u2009N bond is possible, 1\u201327 are present in the E form in solution, as evidenced by the chemical shift values of the HC\u2009=\u2009N and NH protons in the 1H-NMR spectrum.", "section": "RESULTS", "ner": [ [ 53, 57, "1\u201327", "chemical" ], [ 175, 181, "1H-NMR", "experimental_method" ], [ 182, 190, "spectrum", "evidence" ] ] }, { "sid": 27, "sent": "Exceptions are represented by the alkyl-derivatives 3 and 4 (2:1 and 5:3 E:Z ratio, respectively).", "section": "RESULTS", "ner": [ [ 52, 53, "3", "chemical" ], [ 58, 59, "4", "chemical" ] ] }, { "sid": 28, "sent": "If R\u2019 (Fig. 2A) is a 2-hydroxy substituted phenyl ring, the corresponding acylhydrazones can coordinate one or, depending on denticity, two metal centers (modes A and B in Fig. 4).", "section": "RESULTS", "ner": [ [ 74, 88, "acylhydrazones", "chemical" ], [ 93, 103, "coordinate", "bond_interaction" ] ] }, { "sid": 29, "sent": "Starting from N\u2019-(2,3-dihydroxybenzylidene)-semicarbazide (1) and its methoxy-analogue (2), we modified the acylhydrazonic substituent R\u201d (3\u20138, 18, 19, Fig. 2A).", "section": "RESULTS", "ner": [ [ 14, 57, "N\u2019-(2,3-dihydroxybenzylidene)-semicarbazide", "chemical" ], [ 59, 60, "1", "chemical" ], [ 88, 89, "2", "chemical" ], [ 139, 142, "3\u20138", "chemical" ], [ 144, 146, "18", "chemical" ], [ 148, 150, "19", "chemical" ] ] }, { "sid": 30, "sent": "In 18 and 19, also the gallic moiety can be involved in the chelation of the metal cofactors (mode C, Fig. 4).", "section": "RESULTS", "ner": [ [ 3, 5, "18", "chemical" ], [ 10, 12, "19", "chemical" ], [ 23, 29, "gallic", "chemical" ], [ 60, 69, "chelation", "bond_interaction" ] ] }, { "sid": 31, "sent": "In order to investigate the role of hydroxyl substituents 9\u201311, 13\u201317, 20\u201323 and 27 were also synthesized.", "section": "RESULTS", "ner": [ [ 58, 62, "9\u201311", "chemical" ], [ 64, 69, "13\u201317", "chemical" ], [ 71, 76, "20\u201323", "chemical" ], [ 81, 83, "27", "chemical" ] ] }, { "sid": 32, "sent": "Compound 12 was synthesized in order to confirm the crucial influence of the gallic moiety.", "section": "RESULTS", "ner": [ [ 9, 11, "12", "chemical" ], [ 77, 83, "gallic", "chemical" ] ] }, { "sid": 33, "sent": "Finally, 26 was here considered, because it is an inhibitor of HIV RNase H, another enzyme with two magnesium ions in its active site.", "section": "RESULTS", "ner": [ [ 9, 11, "26", "chemical" ], [ 63, 66, "HIV", "taxonomy_domain" ], [ 67, 74, "RNase H", "protein" ], [ 100, 109, "magnesium", "chemical" ], [ 122, 133, "active site", "site" ] ] }, { "sid": 34, "sent": "Since the inhibitory activity of the N-acylhydrazones could be related to chelation of the divalent metal cofactor(s) in the influenza PA-Nter active site, we investigated the coordination properties of one model ligand (i.e. 19, H2L) towards Mg2+.", "section": "RESULTS", "ner": [ [ 37, 53, "N-acylhydrazones", "chemical" ], [ 74, 83, "chelation", "bond_interaction" ], [ 100, 105, "metal", "chemical" ], [ 125, 134, "influenza", "taxonomy_domain" ], [ 135, 137, "PA", "protein" ], [ 138, 142, "Nter", "structure_element" ], [ 143, 154, "active site", "site" ], [ 226, 228, "19", "chemical" ], [ 230, 233, "H2L", "chemical" ], [ 243, 247, "Mg2+", "chemical" ] ] }, { "sid": 35, "sent": "Different reaction conditions were used (1:1 and 1:2 metal to ligand ratio, up to 4 equivalents of triethylamine), but in any case the same chemical species Mg(HL)2\u22194H2O was recovered and conveniently characterized.", "section": "RESULTS", "ner": [ [ 99, 112, "triethylamine", "chemical" ], [ 157, 169, "Mg(HL)2\u22194H2O", "chemical" ] ] }, { "sid": 36, "sent": "The use of a coordinating solvent as d6-DMSO causes partial decoordination of the ligand, but the 1H-NMR spectrum in MeOD, instead, shows only the signals attributable to the complex.", "section": "RESULTS", "ner": [ [ 37, 44, "d6-DMSO", "chemical" ], [ 98, 104, "1H-NMR", "experimental_method" ], [ 105, 113, "spectrum", "evidence" ] ] }, { "sid": 37, "sent": "In the 13C-NMR spectrum, the signal of the C\u2009=\u2009O quaternary carbon is practically unaffected by complexation, suggesting that the C\u2009=\u2009O group is weakly involved in the coordination to the metal ion.", "section": "RESULTS", "ner": [ [ 7, 14, "13C-NMR", "experimental_method" ], [ 15, 23, "spectrum", "evidence" ] ] }, { "sid": 38, "sent": "This is confirmed, in the IR spectrum, by the shift of about 20\u2009cm\u22121 of the C\u2009=\u2009O absorption, while a shift of 30\u201350\u2009cm\u22121 is expected when the carbonylic oxygen is tightly bound to the metal ion.", "section": "RESULTS", "ner": [ [ 26, 28, "IR", "experimental_method" ], [ 29, 37, "spectrum", "evidence" ] ] }, { "sid": 39, "sent": "ESI-mass spectra and elemental analysis confirmed the formula Mg(HL)2\u22194H2O.", "section": "RESULTS", "ner": [ [ 0, 8, "ESI-mass", "experimental_method" ], [ 9, 16, "spectra", "evidence" ], [ 21, 39, "elemental analysis", "experimental_method" ], [ 62, 74, "Mg(HL)2\u22194H2O", "chemical" ] ] }, { "sid": 40, "sent": "The interaction between the N-acylhydrazone ligands and the magnesium cation was investigated also by means of UV-visible spectroscopy (UV-visible titrations of 23 and 19 with increasing amount of Mg(CH3COO)2 are shown in Figure S1).", "section": "RESULTS", "ner": [ [ 28, 43, "N-acylhydrazone", "chemical" ], [ 60, 69, "magnesium", "chemical" ], [ 111, 134, "UV-visible spectroscopy", "experimental_method" ], [ 136, 157, "UV-visible titrations", "experimental_method" ], [ 161, 163, "23", "chemical" ], [ 168, 170, "19", "chemical" ], [ 176, 193, "increasing amount", "experimental_method" ], [ 197, 208, "Mg(CH3COO)2", "chemical" ] ] }, { "sid": 41, "sent": "The spectrum of 19 includes a band at 313\u2009nm assignable to n-\u03c0* transitions of the C\u2009=\u2009N and C\u2009=\u2009O groups.", "section": "RESULTS", "ner": [ [ 4, 12, "spectrum", "evidence" ], [ 16, 18, "19", "chemical" ] ] }, { "sid": 42, "sent": "By adding increasing equivalents of Mg(CH3COO)2, the absorption around 400\u2009nm increases, and a new band appears with a maximum at 397\u2009nm.", "section": "RESULTS", "ner": [ [ 36, 47, "Mg(CH3COO)2", "chemical" ] ] }, { "sid": 43, "sent": "When the same experiment was performed with 23, a different behavior was observed.", "section": "RESULTS", "ner": [ [ 44, 46, "23", "chemical" ] ] }, { "sid": 44, "sent": "Increasing concentration of Mg2+, in fact, caused a diminution in the maximum absorption, an isosbestic point is visible at about 345\u2009nm, but a new band at 400\u2009nm does not appear.", "section": "RESULTS", "ner": [ [ 28, 33, "Mg2+,", "chemical" ] ] }, { "sid": 45, "sent": "Ligands 19 and 23 coordinate the Mg2+ ions in different ways: 19 chelates the metal ion by using the deprotonated salicyl oxygen and the iminic nitrogen, while for 23, the gallic moiety is supposed to be involved (Fig. 4A,B versus C), leading to different, less extensive, modifications of the UV spectrum.", "section": "RESULTS", "ner": [ [ 8, 10, "19", "chemical" ], [ 15, 17, "23", "chemical" ], [ 18, 28, "coordinate", "bond_interaction" ], [ 33, 37, "Mg2+", "chemical" ], [ 62, 64, "19", "chemical" ], [ 164, 166, "23", "chemical" ], [ 294, 296, "UV", "experimental_method" ], [ 297, 305, "spectrum", "evidence" ] ] }, { "sid": 46, "sent": "Inhibition of the PA-Nter enzyme", "section": "RESULTS", "ner": [ [ 18, 20, "PA", "protein" ], [ 21, 25, "Nter", "structure_element" ] ] }, { "sid": 47, "sent": "All the compounds were tested for their ability to inhibit the influenza endonuclease in an enzymatic plasmid-based assay with recombinant PA-Nter, as well as in cell-based influenza methods (i.e. virus yield and vRNP reconstitution assays).", "section": "RESULTS", "ner": [ [ 63, 72, "influenza", "taxonomy_domain" ], [ 73, 85, "endonuclease", "protein_type" ], [ 92, 121, "enzymatic plasmid-based assay", "experimental_method" ], [ 139, 141, "PA", "protein" ], [ 142, 146, "Nter", "structure_element" ], [ 162, 190, "cell-based influenza methods", "experimental_method" ], [ 197, 239, "virus yield and vRNP reconstitution assays", "experimental_method" ] ] }, { "sid": 48, "sent": "The results are shown in Table 1 and summarized in Fig. 3 to visualize the structure-activity relationships; Figure S2 shows the dose-response curves for three representative compounds (i.e. 10, 13 and 23) in either the PA-enzyme or vRNP reconstitution assay.", "section": "RESULTS", "ner": [ [ 129, 149, "dose-response curves", "evidence" ], [ 191, 193, "10", "chemical" ], [ 195, 197, "13", "chemical" ], [ 202, 204, "23", "chemical" ], [ 220, 258, "PA-enzyme or vRNP reconstitution assay", "experimental_method" ] ] }, { "sid": 49, "sent": "The moderate activity (IC50\u2009=\u200924\u2009\u03bcM) of N\u2019-2,3-dihydroxybenzylidene semicarbazide (1) was completely lost when the NH2 moiety was replaced by a hydrophobic heptyl chain (3), but it is less affected when a phenyl or a 2-hydroxyphenyl is present (5 and 7, IC50\u2009=\u200984 and 54\u2009\u03bcM, respectively).", "section": "RESULTS", "ner": [ [ 23, 27, "IC50", "evidence" ], [ 40, 81, "N\u2019-2,3-dihydroxybenzylidene semicarbazide", "chemical" ], [ 83, 84, "1", "chemical" ], [ 170, 171, "3", "chemical" ], [ 245, 246, "5", "chemical" ], [ 251, 252, "7", "chemical" ], [ 254, 258, "IC50", "evidence" ] ] }, { "sid": 50, "sent": "When the hydroxyl in position 3 on R1 (2,3-dihydroxybenzylidene) was replaced by a methoxy group (2-hydroxy-3-methoxybenzylidene), the activity disappeared (compounds 2, 4, 6 and 8).", "section": "RESULTS", "ner": [ [ 39, 63, "2,3-dihydroxybenzylidene", "chemical" ], [ 98, 128, "2-hydroxy-3-methoxybenzylidene", "chemical" ], [ 167, 168, "2", "chemical" ], [ 170, 171, "4", "chemical" ], [ 173, 174, "6", "chemical" ], [ 179, 180, "8", "chemical" ] ] }, { "sid": 51, "sent": "The activity is unaffected (IC50 values ranging from 45 to 75\u2009\u03bcM) when going from two hydroxyls in R1 (7) to compounds with three hydroxyls (i.e. 9, 10 and 11).", "section": "RESULTS", "ner": [ [ 28, 32, "IC50", "evidence" ], [ 103, 104, "7", "chemical" ], [ 146, 147, "9", "chemical" ], [ 149, 151, "10", "chemical" ], [ 156, 158, "11", "chemical" ] ] }, { "sid": 52, "sent": "Similarly, 11 (R1\u2009=\u20093,4,5-trihydroxyphenyl, R2\u2009=\u20092-hydroxyphenyl) had comparable activity as 27 (R1\u2009=\u20093,4,5-trihydroxyphenyl, R2\u2009=\u2009NH2).", "section": "RESULTS", "ner": [ [ 11, 13, "11", "chemical" ], [ 93, 95, "27", "chemical" ] ] }, { "sid": 53, "sent": "Within the series carrying a 2-hydroxyphenyl R2 group, the activity of 11 is particularly intriguing.", "section": "RESULTS", "ner": [ [ 71, 73, "11", "chemical" ] ] }, { "sid": 54, "sent": "11 does not have the possibility to chelate in a tridentate ONO fashion (mode A in Fig. 4), but it can coordinate two cations by means of its three OH groups in R1 (mode C, Fig. 4).", "section": "RESULTS", "ner": [ [ 0, 2, "11", "chemical" ], [ 103, 113, "coordinate", "bond_interaction" ] ] }, { "sid": 55, "sent": "Note that a similar chelating mode was observed in a crystal structure, solved by Cusack and coworkers, of PA-Nter endonuclease in complex with the inhibitor EGCG.", "section": "RESULTS", "ner": [ [ 53, 70, "crystal structure", "evidence" ], [ 107, 109, "PA", "protein" ], [ 110, 114, "Nter", "structure_element" ], [ 115, 127, "endonuclease", "protein_type" ], [ 128, 143, "in complex with", "protein_state" ], [ 158, 162, "EGCG", "chemical" ] ] }, { "sid": 56, "sent": "The PA-Nter inhibitory activity strongly depends on the number and position of hydroxyl substituents in R1 and R2: this is clearly highlighted by the data obtained with compounds 13\u201323, in which R2 is a 3,4,5-trihydroxyphenyl (gallic) group, the most active scaffold in our series.", "section": "RESULTS", "ner": [ [ 4, 6, "PA", "protein" ], [ 7, 11, "Nter", "structure_element" ], [ 179, 184, "13\u201323", "chemical" ] ] }, { "sid": 57, "sent": "The analogue carrying an unsubstituted aromatic ring as R1 (compound 13) had moderate activity (IC50\u2009=\u200969\u2009\u03bcM).", "section": "RESULTS", "ner": [ [ 69, 71, "13", "chemical" ], [ 96, 100, "IC50", "evidence" ] ] }, { "sid": 58, "sent": "When one OH was added at position 2 of the R1 ring (14), the activity was lost.", "section": "RESULTS", "ner": [ [ 52, 54, "14", "chemical" ] ] }, { "sid": 59, "sent": "Adding a second OH substituent at position 5 resulted in strong activity (compound 15, IC50\u2009=\u20099\u2009\u03bcM); medium activity for a 3-OH (18; IC50\u2009=\u200983\u2009\u03bcM), and marginal activity when the second OH is at position 4 (17, IC50\u2009\u2265\u2009370\u2009\u03bcM).", "section": "RESULTS", "ner": [ [ 83, 85, "15", "chemical" ], [ 87, 91, "IC50", "evidence" ], [ 129, 131, "18", "chemical" ], [ 133, 137, "IC50", "evidence" ], [ 207, 209, "17", "chemical" ], [ 211, 215, "IC50", "evidence" ] ] }, { "sid": 60, "sent": "The addition of a 3-methoxy group (19) abolished all inhibitory activity.", "section": "RESULTS", "ner": [ [ 35, 37, "19", "chemical" ] ] }, { "sid": 61, "sent": "This cannot be related to variations in the chelating features displayed by the R1 moiety, since compounds 14\u201319 all have, in theory, the capacity to chelate one metal ion through the ortho-OH and iminic nitrogen (mode A in Fig. 4).", "section": "RESULTS", "ner": [ [ 107, 112, "14\u201319", "chemical" ] ] }, { "sid": 62, "sent": "Moreover, compound 18 can, in principle, chelate the two M2+ ions in the active site according to mode B (Fig. 4), yet it (IC50\u2009=\u200983\u2009\u03bcM) has nine-fold lower activity than 15, that does not possess this two-metal chelating feature.", "section": "RESULTS", "ner": [ [ 19, 21, "18", "chemical" ], [ 57, 60, "M2+", "chemical" ], [ 73, 84, "active site", "site" ], [ 123, 127, "IC50", "evidence" ], [ 171, 173, "15", "chemical" ] ] }, { "sid": 63, "sent": "Therefore, we hypothesized that the inhibitory activity of the series containing the gallic moiety is determined by: (i) the capacity of the moiety R2 to chelate two metal ions in the active site of the enzyme, according to mode C (Fig. 4); and (ii) the presence and position of one or more hydroxyl substituents in R1, which may possibly result in ligand-protein interactions (e.g. through hydrogen bonds).", "section": "RESULTS", "ner": [ [ 184, 195, "active site", "site" ], [ 391, 405, "hydrogen bonds", "bond_interaction" ] ] }, { "sid": 64, "sent": "This assumption was supported by molecular docking calculations and X-ray analysis of inhibitor 23 in complex with PA-Nter (vide infra).", "section": "RESULTS", "ner": [ [ 33, 63, "molecular docking calculations", "experimental_method" ], [ 68, 82, "X-ray analysis", "experimental_method" ], [ 96, 98, "23", "chemical" ], [ 99, 114, "in complex with", "protein_state" ], [ 115, 117, "PA", "protein" ], [ 118, 122, "Nter", "structure_element" ] ] }, { "sid": 65, "sent": "Substitution of the 5-hydroxyl in 15 by a methoxy group (16) causes a dramatic drop in activity (IC50\u2009=\u20099 and 454\u2009\u03bcM for 15 and 16, respectively).", "section": "RESULTS", "ner": [ [ 34, 36, "15", "chemical" ], [ 57, 59, "16", "chemical" ], [ 97, 101, "IC50", "evidence" ], [ 121, 123, "15", "chemical" ], [ 128, 130, "16", "chemical" ] ] }, { "sid": 66, "sent": "In particular, all the compounds with a trihydroxylated phenyl group as R1 (i.e. 20, 21, 22 and 23) were able to inhibit PA-Nter quite potently.", "section": "RESULTS", "ner": [ [ 81, 83, "20", "chemical" ], [ 85, 87, "21", "chemical" ], [ 89, 91, "22", "chemical" ], [ 96, 98, "23", "chemical" ], [ 121, 123, "PA", "protein" ], [ 124, 128, "Nter", "structure_element" ] ] }, { "sid": 67, "sent": "The lowest IC50 values were obtained for 21 and 23 (IC50\u2009=\u200913 and 7\u2009\u03bcM, respectively), which both have one of their three hydroxyl groups at position 5.", "section": "RESULTS", "ner": [ [ 11, 15, "IC50", "evidence" ], [ 41, 43, "21", "chemical" ], [ 48, 50, "23", "chemical" ], [ 52, 56, "IC50", "evidence" ] ] }, { "sid": 68, "sent": "The most active compound in this series was 23, which lacks the hydroxyl group at position 2 of R1, further confirming that this function is undesirable or even detrimental for inhibitory activity against PA-Nter, as already noticed above for 14.", "section": "RESULTS", "ner": [ [ 44, 46, "23", "chemical" ], [ 205, 207, "PA", "protein" ], [ 208, 212, "Nter", "structure_element" ], [ 243, 245, "14", "chemical" ] ] }, { "sid": 69, "sent": "Consistent with a crucial role of the R2 gallic moiety in metal chelation, the strong activity of 15 was completely lost in its 3,4,5-trimethoxy analogue 12.", "section": "RESULTS", "ner": [ [ 64, 73, "chelation", "bond_interaction" ], [ 98, 100, "15", "chemical" ], [ 154, 156, "12", "chemical" ] ] }, { "sid": 70, "sent": "On the other hand, the R2 gallic containing compounds displayed moderate activity (IC50 values around 40\u2009\u03bcM) when R1 was absent (i.e. the 3,4,5-trihydroxybenzohydrazide 28, Fig. 2), or composed of an extended ring system (26) or a pyrrole ring (25).", "section": "RESULTS", "ner": [ [ 83, 87, "IC50", "evidence" ], [ 138, 168, "3,4,5-trihydroxybenzohydrazide", "chemical" ], [ 169, 171, "28", "chemical" ], [ 222, 224, "26", "chemical" ], [ 245, 247, "25", "chemical" ] ] }, { "sid": 71, "sent": "Still lower activity was seen with the pyridine analogue 24.", "section": "RESULTS", "ner": [ [ 57, 59, "24", "chemical" ] ] }, { "sid": 72, "sent": "Evidently, the 3,4,5-trihydroxybenzyl moiety at R2 is fundamental but not sufficient to ensure potent PA-Nter endonuclease inhibition, since the interactions of R1 with the amino acid side chains of the protein appear crucial in modulating activity.", "section": "RESULTS", "ner": [ [ 102, 104, "PA", "protein" ], [ 105, 109, "Nter", "structure_element" ], [ 110, 122, "endonuclease", "protein_type" ] ] }, { "sid": 73, "sent": "Inhibition of vRNP activity or virus replication in cells", "section": "RESULTS", "ner": [ [ 14, 18, "vRNP", "complex_assembly" ], [ 31, 36, "virus", "taxonomy_domain" ] ] }, { "sid": 74, "sent": "To determine the anti-influenza virus activity of compounds 1\u201328 in cell culture, we performed an influenza vRNP reconstitution assay in human embryonic kidney 293\u2009T (HEK293T) cells, then subjected the active compounds (i.e. EC50\u2009<\u2009100\u2009\u03bcM) to a virus yield assay in influenza virus-infected Madin-Darby canine kidney (MDCK) cells (Table 1 and Fig. 3).", "section": "RESULTS", "ner": [ [ 22, 31, "influenza", "taxonomy_domain" ], [ 32, 37, "virus", "taxonomy_domain" ], [ 60, 64, "1\u201328", "chemical" ], [ 98, 133, "influenza vRNP reconstitution assay", "experimental_method" ], [ 137, 142, "human", "species" ], [ 225, 229, "EC50", "evidence" ], [ 245, 262, "virus yield assay", "experimental_method" ], [ 266, 275, "influenza", "taxonomy_domain" ], [ 276, 281, "virus", "taxonomy_domain" ] ] }, { "sid": 75, "sent": "For some N-acylhydrazone compounds, we observed quite potent and selective activity in the vRNP reconstitution assay.", "section": "RESULTS", "ner": [ [ 9, 24, "N-acylhydrazone", "chemical" ], [ 91, 116, "vRNP reconstitution assay", "experimental_method" ] ] }, { "sid": 76, "sent": "This indicates that they are able to inhibit viral RNA synthesis and suggests that they could be classified as original PA inhibitors.", "section": "RESULTS", "ner": [ [ 45, 50, "viral", "taxonomy_domain" ], [ 51, 54, "RNA", "chemical" ], [ 120, 122, "PA", "protein" ] ] }, { "sid": 77, "sent": "Values for EC50 (vRNP) or EC90 (virus yield) in the range of 0.4\u201318\u2009\u03bcM were obtained for compounds 15 and 20\u201323, which all carry a 3,4,5-trihydroxyphenyl as R2, and possess either two (15) or three (20\u201323) hydroxyl substituents in the R1 moiety.", "section": "RESULTS", "ner": [ [ 11, 15, "EC50", "evidence" ], [ 17, 21, "vRNP", "complex_assembly" ], [ 26, 30, "EC90", "evidence" ], [ 32, 37, "virus", "taxonomy_domain" ], [ 99, 101, "15", "chemical" ], [ 106, 111, "20\u201323", "chemical" ], [ 185, 187, "15", "chemical" ], [ 199, 201, "20", "chemical" ], [ 202, 204, "23", "chemical" ] ] }, { "sid": 78, "sent": "As in the enzymatic PA-Nter assays, the compounds having R2 as a gallic moiety (Fig. 3: 21, 22 and 23) showed slightly higher activity than the compounds carrying a 2-hydroxyl R2 group (9, 10 and 11); 10 and 22 have substantially the same EC50 in the vRNP reconstitution assay in HEK293T cells.", "section": "RESULTS", "ner": [ [ 10, 34, "enzymatic PA-Nter assays", "experimental_method" ], [ 88, 90, "21", "chemical" ], [ 92, 94, "22", "chemical" ], [ 99, 101, "23", "chemical" ], [ 186, 187, "9", "chemical" ], [ 189, 191, "10", "chemical" ], [ 196, 198, "11", "chemical" ], [ 201, 203, "10", "chemical" ], [ 208, 210, "22", "chemical" ], [ 239, 243, "EC50", "evidence" ], [ 251, 276, "vRNP reconstitution assay", "experimental_method" ] ] }, { "sid": 79, "sent": "The hydrazide 28 displayed weak (virus yield) to moderate (vRNP reconstitution) activity, albeit less than the most active molecules in the 3,4,5-trihydroxyphenyl series (i.e. 18 and 21\u201323).", "section": "RESULTS", "ner": [ [ 4, 13, "hydrazide", "chemical" ], [ 14, 16, "28", "chemical" ], [ 33, 38, "virus", "taxonomy_domain" ], [ 59, 78, "vRNP reconstitution", "experimental_method" ], [ 176, 178, "18", "chemical" ], [ 183, 188, "21\u201323", "chemical" ] ] }, { "sid": 80, "sent": "Even if there are no data indicating that the compounds reported in the paper are subject to hydrolysis, the activity of 28 could raise the concern that for some N-acylhydrazones the antiviral activity in cell culture may be related to their intracellular hydrolysis.", "section": "RESULTS", "ner": [ [ 121, 123, "28", "chemical" ], [ 162, 178, "N-acylhydrazones", "chemical" ] ] }, { "sid": 81, "sent": "However, this is unlikely, since the antiviral potency showed large differences (i.e. EC50 values between 0.42 and 29\u2009\u03bcM) for compounds with the same R2 but different R1 groups, meaning that R1 does play a role in modulating the antiviral effect.", "section": "RESULTS", "ner": [ [ 86, 90, "EC50", "evidence" ] ] }, { "sid": 82, "sent": "Most compounds carrying as R1 a 2,3-dihydroxybenzylidene (i.e. 3, 5 and 7) or 2-hydroxy-3-methoxybenzylidene moiety (i.e. 4, 6 and 8) showed relatively high cytotoxicity in the vRNP assay, with CC50 values below 50\u2009\u03bcM and a selectivity index (ratio of CC50 to EC50) below 8.", "section": "RESULTS", "ner": [ [ 32, 56, "2,3-dihydroxybenzylidene", "chemical" ], [ 63, 64, "3", "chemical" ], [ 66, 67, "5", "chemical" ], [ 72, 73, "7", "chemical" ], [ 78, 108, "2-hydroxy-3-methoxybenzylidene", "chemical" ], [ 122, 123, "4", "chemical" ], [ 125, 126, "6", "chemical" ], [ 131, 132, "8", "chemical" ], [ 177, 187, "vRNP assay", "experimental_method" ], [ 194, 198, "CC50", "evidence" ], [ 224, 241, "selectivity index", "evidence" ], [ 252, 256, "CC50", "evidence" ], [ 260, 264, "EC50", "evidence" ] ] }, { "sid": 83, "sent": "Two notable exceptions are 18 and 19 (containing a 2,3-dihydroxybenzylidene or 2-hydroxy-3-methoxybenzylidene R1, respectively) which were not cytotoxic at 200\u2009\u03bcM and displayed favorable antiviral selectivity.", "section": "RESULTS", "ner": [ [ 27, 29, "18", "chemical" ], [ 34, 36, "19", "chemical" ], [ 51, 75, "2,3-dihydroxybenzylidene", "chemical" ], [ 79, 109, "2-hydroxy-3-methoxybenzylidene", "chemical" ] ] }, { "sid": 84, "sent": "Some N-acylhydrazone compounds were devoid of activity in the enzymatic assay, yet showed good to moderate efficacy in cell culture (e.g. 14 and 19, having EC50 values of 2.2 and 7.1\u2009\u03bcM, respectively).", "section": "RESULTS", "ner": [ [ 5, 20, "N-acylhydrazone", "chemical" ], [ 62, 77, "enzymatic assay", "experimental_method" ], [ 138, 140, "14", "chemical" ], [ 145, 147, "19", "chemical" ], [ 156, 160, "EC50", "evidence" ] ] }, { "sid": 85, "sent": "For most of the active compounds (i.e. 9, 11, 13, 15\u201321, 23, 24 and 26) a fair correlation was seen for the two cell-based assays, since the EC50 values obtained in the vRNP assay were maximum 5-fold different from the EC90 values in the virus yield assay.", "section": "RESULTS", "ner": [ [ 39, 40, "9", "chemical" ], [ 42, 44, "11", "chemical" ], [ 46, 48, "13", "chemical" ], [ 50, 55, "15\u201321", "chemical" ], [ 57, 59, "23", "chemical" ], [ 61, 63, "24", "chemical" ], [ 68, 70, "26", "chemical" ], [ 112, 129, "cell-based assays", "experimental_method" ], [ 141, 145, "EC50", "evidence" ], [ 169, 179, "vRNP assay", "experimental_method" ], [ 219, 223, "EC90", "evidence" ], [ 238, 255, "virus yield assay", "experimental_method" ] ] }, { "sid": 86, "sent": "On the other hand, this difference was 8-fold or more for 7, 10, 14, 22, 25 and 28.", "section": "RESULTS", "ner": [ [ 58, 59, "7", "chemical" ], [ 61, 63, "10", "chemical" ], [ 65, 67, "14", "chemical" ], [ 69, 71, "22", "chemical" ], [ 73, 75, "25", "chemical" ], [ 80, 82, "28", "chemical" ] ] }, { "sid": 87, "sent": "Some N-acylhydrazone compounds showed good to moderate efficacy in the vRNP assay (e.g. 14 and 19, having EC50 values of 2.3 and 5.7\u2009\u03bcM, respectively), yet were devoid of activity in the enzymatic assay.", "section": "RESULTS", "ner": [ [ 5, 20, "N-acylhydrazone", "chemical" ], [ 71, 81, "vRNP assay", "experimental_method" ], [ 88, 90, "14", "chemical" ], [ 95, 97, "19", "chemical" ], [ 106, 110, "EC50", "evidence" ], [ 187, 202, "enzymatic assay", "experimental_method" ] ] }, { "sid": 88, "sent": "This observation suggests that they may inhibit the viral polymerase in an endonuclease-independent manner.", "section": "RESULTS", "ner": [ [ 52, 57, "viral", "taxonomy_domain" ], [ 58, 68, "polymerase", "protein_type" ], [ 75, 87, "endonuclease", "protein_type" ] ] }, { "sid": 89, "sent": "To achieve a clear insight into the antiviral profile of the N-acylhydrazones, specific mechanistic experiments are currently ongoing in our laboratory, in which we are analyzing in full depth their effects on virus entry, polymerase-dependent RNA synthesis or the late stage (maturation and release) of the virus replication cycle.", "section": "RESULTS", "ner": [ [ 61, 77, "N-acylhydrazones", "chemical" ], [ 210, 215, "virus", "taxonomy_domain" ], [ 223, 233, "polymerase", "protein_type" ], [ 244, 247, "RNA", "chemical" ], [ 308, 313, "virus", "taxonomy_domain" ] ] }, { "sid": 90, "sent": "Docking studies", "section": "RESULTS", "ner": [ [ 0, 15, "Docking studies", "experimental_method" ] ] }, { "sid": 91, "sent": "In order to explore the possible binding mode of the synthesized compounds, docking simulations by GOLD program were performed by using the structural coordinates (PDB code 4AWM) for the PA-Nter endonuclease in complex with EGCG.", "section": "RESULTS", "ner": [ [ 76, 95, "docking simulations", "experimental_method" ], [ 99, 111, "GOLD program", "experimental_method" ], [ 187, 189, "PA", "protein" ], [ 190, 194, "Nter", "structure_element" ], [ 195, 207, "endonuclease", "protein_type" ], [ 208, 223, "in complex with", "protein_state" ], [ 224, 228, "EGCG", "chemical" ] ] }, { "sid": 92, "sent": "Considering that the position of the side-chains of some residues changes depending on which pocket the ligand is occupying, we superimposed some X-ray structures of complexes between PA-Nter endonuclease and known active ligands.", "section": "RESULTS", "ner": [ [ 128, 140, "superimposed", "experimental_method" ], [ 146, 162, "X-ray structures", "evidence" ], [ 184, 186, "PA", "protein" ], [ 187, 191, "Nter", "structure_element" ], [ 192, 204, "endonuclease", "protein_type" ] ] }, { "sid": 93, "sent": "It was observed that the side-chain of amino acid Tyr24 shows greater movement than the other residues and for this reason we considered it as a flexible residue during the docking procedure.", "section": "RESULTS", "ner": [ [ 50, 55, "Tyr24", "residue_name_number" ], [ 145, 153, "flexible", "protein_state" ], [ 173, 190, "docking procedure", "experimental_method" ] ] }, { "sid": 94, "sent": "First, test docking calculations, using EGCG, L-742,001 and 2-(4-(1H-tetrazol-5-yl)phenyl)-5-hydroxypyrimidin-4(3H)-one (Fig. 1), were carried out to compare experimental and predicted binding modes and validate docking procedure.", "section": "RESULTS", "ner": [ [ 7, 32, "test docking calculations", "experimental_method" ], [ 40, 44, "EGCG", "chemical" ], [ 46, 55, "L-742,001", "chemical" ], [ 60, 119, "2-(4-(1H-tetrazol-5-yl)phenyl)-5-hydroxypyrimidin-4(3H)-one", "chemical" ], [ 212, 229, "docking procedure", "experimental_method" ] ] }, { "sid": 95, "sent": "Their best docking poses agreed well with the experimental binding modes (rmsd values of 0.8, 1.2 and 0.7, respectively).", "section": "RESULTS", "ner": [ [ 74, 78, "rmsd", "evidence" ] ] }, { "sid": 96, "sent": "Next, docking of several N-acylhydrazones was performed and this generated a number of possible binding conformations, highlighting that the active site cavity of the PA endonuclease is quite spacious, as already demonstrated by crystallographic studies, and confirming the ability of this scaffold to chelate the two M2+ ions in different ways (Mode A-C in Fig. 4).", "section": "RESULTS", "ner": [ [ 6, 13, "docking", "experimental_method" ], [ 25, 41, "N-acylhydrazones", "chemical" ], [ 141, 159, "active site cavity", "site" ], [ 167, 169, "PA", "protein" ], [ 170, 182, "endonuclease", "protein_type" ], [ 229, 253, "crystallographic studies", "experimental_method" ], [ 318, 321, "M2+", "chemical" ] ] }, { "sid": 97, "sent": "Figure 5 displays the first (panel A) and second (panel B) GOLD cluster docked solutions for compound 23.", "section": "RESULTS", "ner": [ [ 59, 78, "GOLD cluster docked", "experimental_method" ], [ 102, 104, "23", "chemical" ] ] }, { "sid": 98, "sent": "These two complex structures represent the largest clusters with similar fitness values (59.20 and 58.65, respectively).", "section": "RESULTS", "ner": [ [ 18, 28, "structures", "evidence" ] ] }, { "sid": 99, "sent": "In both cases, 23 appears able to coordinate the two M2+ ions in the active site through the three contiguous OH groups (Fig. 5).", "section": "RESULTS", "ner": [ [ 15, 17, "23", "chemical" ], [ 34, 44, "coordinate", "bond_interaction" ], [ 53, 56, "M2+", "chemical" ], [ 69, 80, "active site", "site" ] ] }, { "sid": 100, "sent": "In addition, 23 was predicted to form two hydrogen bonding interactions, i.e. with the catalytic Lys134 on the one side and Glu26 on the other side.", "section": "RESULTS", "ner": [ [ 13, 15, "23", "chemical" ], [ 42, 71, "hydrogen bonding interactions", "bond_interaction" ], [ 87, 96, "catalytic", "protein_state" ], [ 97, 103, "Lys134", "residue_name_number" ], [ 124, 129, "Glu26", "residue_name_number" ] ] }, { "sid": 101, "sent": "Furthermore, in these two different binding modes, 23 forms \u03c0\u2013\u03c0 interactions with the aromatic ring of Tyr24, in a fashion similar to that described for other endonuclease inhibitors, i.e. EGCG and L-742,001.", "section": "RESULTS", "ner": [ [ 51, 53, "23", "chemical" ], [ 60, 76, "\u03c0\u2013\u03c0 interactions", "bond_interaction" ], [ 103, 108, "Tyr24", "residue_name_number" ], [ 159, 171, "endonuclease", "protein_type" ], [ 189, 193, "EGCG", "chemical" ], [ 198, 207, "L-742,001", "chemical" ] ] }, { "sid": 102, "sent": "The best docked conformation for compound 15 (Fig. 6, fitness value 68.56), which has an activity slightly lower than 23, reveals a different role for the gallic moiety.", "section": "RESULTS", "ner": [ [ 42, 44, "15", "chemical" ], [ 54, 67, "fitness value", "evidence" ] ] }, { "sid": 103, "sent": "The ligand seems to form two hydrogen bonding interactions with Tyr130 as well as a cation\u2013\u03c0 interaction with Lys134.", "section": "RESULTS", "ner": [ [ 29, 58, "hydrogen bonding interactions", "bond_interaction" ], [ 64, 70, "Tyr130", "residue_name_number" ], [ 84, 104, "cation\u2013\u03c0 interaction", "bond_interaction" ], [ 110, 116, "Lys134", "residue_name_number" ] ] }, { "sid": 104, "sent": "Tyr130 lies in a pocket that also contains Arg124, a residue that was proposed to have a crucial role in binding of the RNA substrate.", "section": "RESULTS", "ner": [ [ 0, 6, "Tyr130", "residue_name_number" ], [ 17, 23, "pocket", "site" ], [ 43, 49, "Arg124", "residue_name_number" ], [ 120, 123, "RNA", "chemical" ] ] }, { "sid": 105, "sent": "Compound 15 appears further stabilized by hydrogen bonding interactions between two hydroxyl groups and Arg82 and Asp108.", "section": "RESULTS", "ner": [ [ 9, 11, "15", "chemical" ], [ 42, 71, "hydrogen bonding interactions", "bond_interaction" ], [ 104, 109, "Arg82", "residue_name_number" ], [ 114, 120, "Asp108", "residue_name_number" ] ] }, { "sid": 106, "sent": "In this case, chelation of the two M2+ ions is carried out by involving the imine group (mode A in Fig. 4).", "section": "RESULTS", "ner": [ [ 14, 23, "chelation", "bond_interaction" ], [ 35, 38, "M2+", "chemical" ] ] }, { "sid": 107, "sent": "It is important to highlight that compounds 23 and 15, although in different ways, both are able to chelate the metal cofactors and to establish interactions with highly conserved aminoacids (Tyr24, Glu26, Arg124, Tyr130 and Lys134) that are very important for both endonuclease activity and transcription in vitro.", "section": "RESULTS", "ner": [ [ 44, 46, "23", "chemical" ], [ 51, 53, "15", "chemical" ], [ 163, 179, "highly conserved", "protein_state" ], [ 192, 197, "Tyr24", "residue_name_number" ], [ 199, 204, "Glu26", "residue_name_number" ], [ 206, 212, "Arg124", "residue_name_number" ], [ 214, 220, "Tyr130", "residue_name_number" ], [ 225, 231, "Lys134", "residue_name_number" ], [ 266, 278, "endonuclease", "protein_type" ] ] }, { "sid": 108, "sent": "The crucial role of such interactions is underlined by the differences in activity between 15 (IC50\u2009=\u20099.0\u2009\u03bcM) and 19 (>500\u2009\u03bcM): their coordinating features are similar, since both coordinate to the divalent metal ion through the phenolic oxygen, the iminic nitrogen and the carbonylic oxygen (mode A in Fig. 4), but the biological activity could be related to their different ability to engage interactions with the protein environment.", "section": "RESULTS", "ner": [ [ 91, 93, "15", "chemical" ], [ 95, 99, "IC50", "evidence" ], [ 114, 116, "19", "chemical" ], [ 180, 190, "coordinate", "bond_interaction" ] ] }, { "sid": 109, "sent": "Crystallographic Studies", "section": "RESULTS", "ner": [ [ 0, 24, "Crystallographic Studies", "experimental_method" ] ] }, { "sid": 110, "sent": "Attempts were made to co-crystallize PA-Nter with 15, 20, 21 and 23 in one to four molar excess.", "section": "RESULTS", "ner": [ [ 22, 36, "co-crystallize", "experimental_method" ], [ 37, 39, "PA", "protein" ], [ 40, 44, "Nter", "structure_element" ], [ 50, 52, "15", "chemical" ], [ 54, 56, "20", "chemical" ], [ 58, 60, "21", "chemical" ], [ 65, 67, "23", "chemical" ] ] }, { "sid": 111, "sent": "While crystals appeared and diffracted well, upon data processing, no or very little electron density for the inhibitors was observed.", "section": "RESULTS", "ner": [ [ 6, 14, "crystals", "evidence" ], [ 85, 101, "electron density", "evidence" ] ] }, { "sid": 112, "sent": "Attempts to soak apo crystals in crystallization solution containing 5 mM inhibitor overnight also did not result in substantial electron density for the inhibitor.", "section": "RESULTS", "ner": [ [ 17, 20, "apo", "protein_state" ], [ 21, 29, "crystals", "evidence" ], [ 129, 145, "electron density", "evidence" ] ] }, { "sid": 113, "sent": "As a last resort, dry powder of the inhibitor was sprinkled over the crystallization drop containing apo crystals and left over night.", "section": "RESULTS", "ner": [ [ 101, 104, "apo", "protein_state" ], [ 105, 113, "crystals", "evidence" ] ] }, { "sid": 114, "sent": "This experiment was successful for compound 23, the crystals diffracted to 2.15 \u00c5 and diffraction data were collected (PDB ID 5EGA).", "section": "RESULTS", "ner": [ [ 44, 46, "23", "chemical" ], [ 52, 60, "crystals", "evidence" ] ] }, { "sid": 115, "sent": "The refined structure shows unambiguous electron density for the inhibitor (Table S1 and Fig. 7).", "section": "RESULTS", "ner": [ [ 12, 21, "structure", "evidence" ], [ 40, 56, "electron density", "evidence" ] ] }, { "sid": 116, "sent": "The complex structure confirms one of the two binding modes predicted by the docking simulations (Fig. 5, panel B).", "section": "RESULTS", "ner": [ [ 4, 21, "complex structure", "evidence" ], [ 77, 96, "docking simulations", "experimental_method" ] ] }, { "sid": 117, "sent": "The galloyl moiety chelates the manganese ions, while the trihydroxyphenyl group stacks against the Tyr24 side chain.", "section": "RESULTS", "ner": [ [ 32, 41, "manganese", "chemical" ], [ 100, 105, "Tyr24", "residue_name_number" ] ] }, { "sid": 118, "sent": "It is interesting to note that two of these hydroxyl groups are in position to form hydrogen bonds with the side chain of Glu26 and Lys34 (Fig. 7).", "section": "RESULTS", "ner": [ [ 84, 98, "hydrogen bonds", "bond_interaction" ], [ 122, 127, "Glu26", "residue_name_number" ], [ 132, 137, "Lys34", "residue_name_number" ] ] }, { "sid": 119, "sent": "These interactions suggest that other functional groups, e.g. halogens, could be used in place of the hydroxyl groups for better interactions with Glu26 and Lys34 side chains, and the inhibitory potency of these compounds could be further improved.", "section": "RESULTS", "ner": [ [ 147, 152, "Glu26", "residue_name_number" ], [ 157, 162, "Lys34", "residue_name_number" ] ] }, { "sid": 120, "sent": "The development of new agents for the treatment of influenza infection that exert their action by inhibition of the endonuclease activity of influenza RNA-dependent RNA polymerase is a strategy that recently is gaining a lot of interest.", "section": "CONCL", "ner": [ [ 51, 60, "influenza", "taxonomy_domain" ], [ 116, 128, "endonuclease", "protein_type" ], [ 141, 150, "influenza", "taxonomy_domain" ], [ 151, 179, "RNA-dependent RNA polymerase", "protein_type" ] ] }, { "sid": 121, "sent": "The results here presented add the N-acylhydrazone scaffold to the library of the chelating molecules with potent antiviral activity (EC90\u2009<\u20095 \u03bcM, virus yield assay in influenza virus-infected MDCK cells).", "section": "CONCL", "ner": [ [ 35, 50, "N-acylhydrazone", "chemical" ], [ 134, 138, "EC90", "evidence" ], [ 147, 164, "virus yield assay", "experimental_method" ], [ 168, 177, "influenza", "taxonomy_domain" ], [ 178, 183, "virus", "taxonomy_domain" ] ] }, { "sid": 122, "sent": "The structure of the N-acylhydrazone 23 co-crystallized with PA-Nter is important not only because confirms that the polyhydroxypheyl group efficiently coordinates two metal ions in the active site of the enzyme, but also because highlights the importance of the (flexible) inhibitor backbone in order to engage effective interactions with crucial aminoacids of the protein.", "section": "CONCL", "ner": [ [ 4, 13, "structure", "evidence" ], [ 21, 36, "N-acylhydrazone", "chemical" ], [ 37, 39, "23", "chemical" ], [ 40, 55, "co-crystallized", "experimental_method" ], [ 61, 63, "PA", "protein" ], [ 64, 68, "Nter", "structure_element" ], [ 152, 163, "coordinates", "bond_interaction" ], [ 168, 173, "metal", "chemical" ], [ 186, 197, "active site", "site" ] ] }, { "sid": 123, "sent": "Inhibition of the endonuclease activity of influenza RNA-dependent RNA polymerase could represent another example, after carbonic anhydrase, histone deacetylase, and HIV-1 integrase, of metal binding as a successful strategy in drug design.", "section": "CONCL", "ner": [ [ 18, 30, "endonuclease", "protein_type" ], [ 43, 52, "influenza", "taxonomy_domain" ], [ 53, 81, "RNA-dependent RNA polymerase", "protein_type" ], [ 121, 139, "carbonic anhydrase", "protein_type" ], [ 141, 160, "histone deacetylase", "protein_type" ], [ 166, 171, "HIV-1", "species" ], [ 172, 181, "integrase", "protein_type" ], [ 186, 191, "metal", "chemical" ] ] }, { "sid": 124, "sent": "The ligand and water molecules were discarded and the hydrogens were added to the protein by Discovery Studio 2.5.", "section": "METHODS", "ner": [ [ 15, 20, "water", "chemical" ] ] }, { "sid": 125, "sent": "One microgram of recombinant PA-Nter (residues 1\u2013217 from the PA protein of influenza virus strain A/X-31) was incubated with 1 \u03bcg (16.7 nM) of single-stranded circular DNA plasmid M13mp18 (Bayou Biolabs, Metairie, Louisiana) in the presence of the test compounds and at a final volume of 25 \u03bcL. The assay buffer contained 50 mM Tris-HCl pH 8, 100 mM NaCl, 10 mM \u03b2-mercaptoethanol and 1 mM MnCl2.", "section": "METHODS", "ner": [ [ 233, 244, "presence of", "protein_state" ] ] }, { "sid": 126, "sent": "After incubation at 37\u2009\u00b0C for 24 h in the presence of serial dilutions of the test compounds, the ONE-Glo luciferase assay system (Promega, Madison, WI) was used to determine luciferase activity.", "section": "METHODS", "ner": [ [ 42, 53, "presence of", "protein_state" ] ] }, { "sid": 127, "sent": "The compound concentration values causing a 2-log10 (EC99) and a 1-log10 (EC90) reduction in viral RNA (vRNA) copy number at 24 h p.i., as compared to the virus control receiving no compound, were calculated by interpolation from data of at least three experiments.", "section": "METHODS", "ner": [ [ 53, 57, "EC99", "evidence" ], [ 74, 78, "EC90", "evidence" ] ] }, { "sid": 128, "sent": "A PAN construct (PAN\u0394Loop) with a loop (residues 51\u201372) deleted and replaced with GGS from A/California/04/2009 H1N1 strain was used for the crystallographic studies.", "section": "METHODS", "ner": [ [ 17, 25, "PAN\u0394Loop", "mutant" ] ] }, { "sid": 129, "sent": "The apo structure of PAN\u0394Loop (PDB ID: 5DES) was used as starting model for molecular replacement.", "section": "METHODS", "ner": [ [ 21, 29, "PAN\u0394Loop", "mutant" ] ] }, { "sid": 130, "sent": "Chemical structures of some prototype inhibitors of influenza virus endonuclease.", "section": "FIG", "ner": [ [ 52, 61, "influenza", "taxonomy_domain" ], [ 62, 67, "virus", "taxonomy_domain" ], [ 68, 80, "endonuclease", "protein_type" ] ] }, { "sid": 131, "sent": "Inhibitor activity in enzymatic assays (IC50,\u2009\u03bcM) as reported in: aref., bref., cref., dref..", "section": "FIG", "ner": [ [ 22, 38, "enzymatic assays", "experimental_method" ], [ 40, 44, "IC50", "evidence" ] ] }, { "sid": 132, "sent": "General synthesis for N-acylhydrazones 1\u201327 and hydrazides 28 and 29 (A).", "section": "FIG", "ner": [ [ 22, 38, "N-acylhydrazones", "chemical" ], [ 39, 43, "1\u201327", "chemical" ], [ 48, 58, "hydrazides", "chemical" ], [ 59, 61, "28", "chemical" ], [ 66, 68, "29", "chemical" ] ] }, { "sid": 133, "sent": "Chemical structures of compounds 1\u201327 (B).", "section": "FIG", "ner": [ [ 33, 37, "1\u201327", "chemical" ] ] }, { "sid": 134, "sent": "Overview of the structure-activity relationship for compounds 1\u201327.", "section": "FIG", "ner": [ [ 62, 66, "1\u201327", "chemical" ] ] }, { "sid": 135, "sent": "Scheme of possible binding modes of the studied N-acylhydrazones.", "section": "FIG", "ner": [ [ 48, 64, "N-acylhydrazones", "chemical" ] ] }, { "sid": 136, "sent": "First (A) and second (B) GOLD cluster docked solutions of compound 23 (orange and cyan, respectively) in complex with PA endonuclease.", "section": "FIG", "ner": [ [ 25, 44, "GOLD cluster docked", "experimental_method" ], [ 67, 69, "23", "chemical" ], [ 102, 117, "in complex with", "protein_state" ], [ 118, 120, "PA", "protein" ], [ 121, 133, "endonuclease", "protein_type" ] ] }, { "sid": 137, "sent": "Key residues of the pocket are presented using PyMOL [ http://www.pymol.org] and LIGPLUS [Laskowski, R. A.; Swindells, M. B. Journal of chemical information and modeling 2011, 51, 2778].", "section": "FIG", "ner": [ [ 20, 26, "pocket", "site" ], [ 81, 88, "LIGPLUS", "experimental_method" ], [ 20, 26, "pocket", "site" ], [ 81, 88, "LIGPLUS", "experimental_method" ] ] }, { "sid": 138, "sent": "Hydrogen bonds are illustrated by dotted lines, while the divalent metal ions are shown as purple spheres.", "section": "FIG", "ner": [ [ 0, 14, "Hydrogen bonds", "bond_interaction" ] ] }, { "sid": 139, "sent": "Schematic drawings of the interactions of the first (C) and second (D) GOLD cluster docked solutions generated using LIGPLUS.", "section": "FIG", "ner": [ [ 71, 90, "GOLD cluster docked", "experimental_method" ], [ 117, 124, "LIGPLUS", "experimental_method" ] ] }, { "sid": 140, "sent": "Dashed lines are hydrogen bonds and \u2018eyelashes\u2019 show residues involved in hydrophobic interactions.", "section": "FIG", "ner": [ [ 17, 31, "hydrogen bonds", "bond_interaction" ], [ 74, 98, "hydrophobic interactions", "bond_interaction" ], [ 17, 31, "hydrogen bonds", "bond_interaction" ], [ 74, 98, "hydrophobic interactions", "bond_interaction" ] ] }, { "sid": 141, "sent": "(A) Binding mode of compound 15 (orange) in complex with PA endonuclease.", "section": "FIG", "ner": [ [ 29, 31, "15", "chemical" ], [ 41, 56, "in complex with", "protein_state" ], [ 57, 59, "PA", "protein" ], [ 60, 72, "endonuclease", "protein_type" ] ] }, { "sid": 142, "sent": "Hydrogen bonds are illustrated by dotted lines while the divalent metal ions are shown as purple spheres.", "section": "FIG", "ner": [ [ 0, 14, "Hydrogen bonds", "bond_interaction" ] ] }, { "sid": 143, "sent": "(B) Schematic drawing of the interactions of compound 15 generated using LIGPLUS.", "section": "FIG", "ner": [ [ 54, 56, "15", "chemical" ], [ 73, 80, "LIGPLUS", "experimental_method" ] ] }, { "sid": 144, "sent": "Crystal structure of PAN\u0394Loop in complex with compound 23.", "section": "FIG", "ner": [ [ 0, 17, "Crystal structure", "evidence" ], [ 21, 29, "PAN\u0394Loop", "mutant" ], [ 30, 45, "in complex with", "protein_state" ], [ 55, 57, "23", "chemical" ] ] }, { "sid": 145, "sent": "Active site residues are shown in sticks with green carbons, manganese atoms are shown as purple spheres and water molecules as red spheres.", "section": "FIG", "ner": [ [ 0, 11, "Active site", "site" ], [ 61, 70, "manganese", "chemical" ], [ 109, 114, "water", "chemical" ] ] }, { "sid": 146, "sent": "Compound 23 is shown in sticks with yellow carbons.", "section": "FIG", "ner": [ [ 9, 11, "23", "chemical" ] ] }, { "sid": 147, "sent": "2Fo-Fc electron density map contoured at 1\u03c3 is shown as blue mesh.", "section": "FIG", "ner": [ [ 0, 27, "2Fo-Fc electron density map", "evidence" ] ] }, { "sid": 148, "sent": "Hydrogen bonds and metal coordination are shown with dotted lines.", "section": "FIG", "ner": [ [ 0, 14, "Hydrogen bonds", "bond_interaction" ], [ 19, 37, "metal coordination", "bond_interaction" ] ] }, { "sid": 149, "sent": "The H-bond distances from the side chain carboxyl group of Glu26 to p-OH and m-OH of the trihydroxyphenyl group of the inhibitor are 2.7 \u00c5 and 3.0 \u00c5, respectively.", "section": "FIG", "ner": [ [ 4, 10, "H-bond", "bond_interaction" ], [ 59, 64, "Glu26", "residue_name_number" ] ] }, { "sid": 150, "sent": "The H-bond distance from the side chain of Lys34 to p-OH of the trihydroxyphenyl group is 3.6 \u00c5. The H-bond distance to the water molecule from m-OH of the galloyl moiety is 3.0 \u00c5, which in turn is H-bonded to the side chain of Tyr130 with a distance of 2.7 \u00c5. Crystal structure has been deposited in the RCSB Protein Data Bank with PDB ID: 5EGA.", "section": "FIG", "ner": [ [ 4, 10, "H-bond", "bond_interaction" ], [ 43, 48, "Lys34", "residue_name_number" ], [ 101, 107, "H-bond", "bond_interaction" ], [ 124, 129, "water", "chemical" ], [ 198, 206, "H-bonded", "bond_interaction" ], [ 228, 234, "Tyr130", "residue_name_number" ], [ 261, 278, "Crystal structure", "evidence" ] ] }, { "sid": 151, "sent": "Inhibitory activity of the N-acylhydrazones 1\u201327 and hydrazide 28 in the enzymatic assay with influenza virus PA-Nter endonuclease, or in cellular influenza virus assays.", "section": "TABLE", "ner": [ [ 27, 43, "N-acylhydrazones", "chemical" ], [ 44, 48, "1\u201327", "chemical" ], [ 53, 62, "hydrazide", "chemical" ], [ 63, 65, "28", "chemical" ], [ 73, 88, "enzymatic assay", "experimental_method" ], [ 94, 103, "influenza", "taxonomy_domain" ], [ 104, 109, "virus", "taxonomy_domain" ], [ 110, 112, "PA", "protein" ], [ 113, 117, "Nter", "structure_element" ], [ 118, 130, "endonuclease", "protein_type" ], [ 138, 169, "cellular influenza virus assays", "experimental_method" ] ] }, { "sid": 152, "sent": "Compound\tEnzyme assay with PA-Ntera\tVirus yield assay in influenza virus-infected MDCK cellsb\tvRNP reconstitution assay in HEK293T cellsc\t \tAntiviral activity\tCytotoxicity\tSId\tActivity\tCytotoxicity\t \tIC50\tEC99\tEC90\tCC50\tEC50\tCC50\t \t(1)\t24\tNDf\tND\tND\t\u00a0\t107\t>200\t \t(2)\t>500\tND\tND\tND\t\u00a0\t>100\t>200\t \t(3)\t>500\tND\tND\t>200\t\u00a0\t5.9\t48\t \t(4)\t>500\tND\tND\t>200\t\u00a0\t6.3\t33\t \t(5)\t67\t>25\t>25\t\u2265146\t\u00a0\t2.6\t10\t \t(6)\t>500\t>50\t>50\t>200\t\u00a0\t15\t14\t \t(7)\t54\t172\t100\t>200\t>2.0\t3.2\t8.9\t \t(8)\t>500\t>12.5\t>12.5\t>200\t\u00a0\t1.9\t15\t \t(9)\t34\t16\t5.3\t>200\t>38\t5.5\t>200\t \t(10)\t68\t14\t8.5\t111\t>13\t0.40\t132\t \t(11)\t45\t30\t12\t>200\t>17\t5.6\t>200\t \t(12)\t>500\t>12.5\t>12.5\t>200\t\u00a0\t20\t39\t \t(13)\t69\t71\t34\t>200\t>5.9\t6.3\t>200\t \t(14)\t>500\t63\t37\t>200\t>5.4\t2.3\t>200\t \t(15)\t8.9\t18\t7.5\t\u2265172\t\u226523\t14\t>200\t \t(16)\t454\t67\t28\t>200\t>7.1\t5.2\t>200\t \t(17)\t482\t21\t8.1\t>200\t>25\t7.1\t>200\t \t(18)\t83\t6.2\t2.2\t>200\t>91\t3.3\t>200\t \t(19)\t>500\t53\t26\t>200\t>7.7\t5.7\t>200\t \t(20)\t18\t35\t11\t>200\t>18\t2.2\t>200\t \t(21)\t13\t8.3\t3.6\t>200\t>56\t2.5\t>200\t \t(22)\t75\t7.4\t3.4\t>200\t>59\t0.42\t>200\t \t(23)\t8.7\t11\t3.5\t>200\t>57\t3.1\t>200\t \t(24)\t131\t58\t26\t>200\t>7.7\t25\t>200\t \t(25)\t40\t132\t70\t>200\t>2.9\t4.1\t>200\t \t(26)\t30\t36\t13\t>200\t>15\t5.5\t>200\t \t(27)\t36\tND\tND\tND\t\u00a0\t21\t>200\t \t(28)\t40\t158\t85\t>200\t>2.4\t7.2\t>200\t \tDPBAe\t5.3\tND\tND\tND\t\u00a0\tND\tND\t \tRibavirin\tND\t13\t8.5\t>200\t>24\t9.4\t>200\t \t", "section": "TABLE", "ner": [ [ 0, 21, "Compound\tEnzyme assay", "experimental_method" ], [ 27, 29, "PA", "protein" ], [ 36, 53, "Virus yield assay", "experimental_method" ], [ 57, 66, "influenza", "taxonomy_domain" ], [ 67, 72, "virus", "taxonomy_domain" ], [ 94, 119, "vRNP reconstitution assay", "experimental_method" ], [ 200, 204, "IC50", "evidence" ], [ 205, 209, "EC99", "evidence" ], [ 210, 214, "EC90", "evidence" ], [ 215, 219, "CC50", "evidence" ], [ 220, 224, "EC50", "evidence" ], [ 225, 229, "CC50", "evidence" ] ] }, { "sid": 153, "sent": "aRecombinant PA-Nter was incubated with the ssDNA plasmid substrate, a Mn2+-containing buffer and test compounds.", "section": "TABLE", "ner": [ [ 13, 15, "PA", "protein" ], [ 16, 20, "Nter", "structure_element" ], [ 25, 34, "incubated", "experimental_method" ], [ 44, 49, "ssDNA", "chemical" ], [ 71, 75, "Mn2+", "chemical" ] ] }, { "sid": 154, "sent": "The IC50 represents the compound concentration (in \u03bcM) required to obtain 50% inhibition of cleavage, calculated by nonlinear least-squares regression analysis (using GraphPad Prism software) of the results from 2\u20134 independent experiments.", "section": "TABLE", "ner": [ [ 4, 8, "IC50", "evidence" ], [ 116, 159, "nonlinear least-squares regression analysis", "experimental_method" ] ] }, { "sid": 155, "sent": "bMDCK cells were infected with influenza A virus (strain A/PR/8/34) and incubated with the compounds during 24 h. The virus yield in the supernatant was assessed by real-time qPCR.", "section": "TABLE", "ner": [ [ 31, 42, "influenza A", "taxonomy_domain" ], [ 43, 48, "virus", "taxonomy_domain" ], [ 118, 123, "virus", "taxonomy_domain" ], [ 165, 179, "real-time qPCR", "experimental_method" ] ] }, { "sid": 156, "sent": "The EC99 and EC90 values represent the compound concentrations (in \u03bcM) producing a 2-log10 or 1-log10 reduction in virus titer, respectively, determined in 2\u20133 independent experiments.", "section": "TABLE", "ner": [ [ 4, 8, "EC99", "evidence" ], [ 13, 17, "EC90", "evidence" ], [ 115, 120, "virus", "taxonomy_domain" ] ] }, { "sid": 157, "sent": "The cytotoxicity, assessed in uninfected MDCK cells, was expressed as the CC50 value (50% cytotoxic concentration, determined with the MTS cell viability assay, in \u03bcM).", "section": "TABLE", "ner": [ [ 74, 78, "CC50", "evidence" ], [ 135, 159, "MTS cell viability assay", "experimental_method" ] ] }, { "sid": 158, "sent": "cHEK293T cells were co-transfected with the four vRNP-reconstituting plasmids and the luciferase reporter plasmid in the presence of the test compounds.", "section": "TABLE", "ner": [ [ 20, 34, "co-transfected", "experimental_method" ], [ 49, 53, "vRNP", "complex_assembly" ], [ 121, 132, "presence of", "protein_state" ] ] }, { "sid": 159, "sent": "The EC50 represents the compound concentration (in \u03bcM) producing 50% reduction in vRNP-driven firefly reporter signal, estimated at 24\u2009h after transfection.", "section": "TABLE", "ner": [ [ 4, 8, "EC50", "evidence" ], [ 82, 86, "vRNP", "complex_assembly" ] ] }, { "sid": 160, "sent": "The EC50 value was derived from data from 2\u20134 independent experiments, by nonlinear least-squares regression analysis (using GraphPad Prism software).", "section": "TABLE", "ner": [ [ 4, 8, "EC50", "evidence" ], [ 74, 117, "nonlinear least-squares regression analysis", "experimental_method" ] ] }, { "sid": 161, "sent": "The CC50 (in \u03bcM), i.e. the 50% cytotoxic concentration, was determined in untransfected HEK293T cells by MTS cell viability assay.", "section": "TABLE", "ner": [ [ 4, 8, "CC50", "evidence" ], [ 105, 129, "MTS cell viability assay", "experimental_method" ] ] }, { "sid": 162, "sent": "dSI, selectivity index, defined as the ratio between the CC50 and EC90.", "section": "TABLE", "ner": [ [ 0, 3, "dSI", "evidence" ], [ 5, 22, "selectivity index", "evidence" ], [ 57, 61, "CC50", "evidence" ], [ 66, 70, "EC90", "evidence" ] ] }, { "sid": 163, "sent": "eDPBA, 2,4-dioxo-4-phenylbutanoic acid.", "section": "TABLE", "ner": [ [ 0, 5, "eDPBA", "chemical" ], [ 7, 38, "2,4-dioxo-4-phenylbutanoic acid", "chemical" ] ] } ] }, "PMC4817029": { "annotations": [ { "sid": 0, "sent": "Molecular characterization of a family 5 glycoside hydrolase suggests an induced-fit enzymatic mechanism", "section": "TITLE", "ner": [ [ 32, 60, "family 5 glycoside hydrolase", "protein_type" ] ] }, { "sid": 1, "sent": "Glycoside hydrolases (GHs) play fundamental roles in the decomposition of lignocellulosic biomaterials.", "section": "ABSTRACT", "ner": [ [ 0, 20, "Glycoside hydrolases", "protein_type" ], [ 22, 25, "GHs", "protein_type" ] ] }, { "sid": 2, "sent": "Here, we report the full-length structure of a cellulase from Bacillus licheniformis (BlCel5B), a member of the GH5 subfamily 4 that is entirely dependent on its two ancillary modules (Ig-like module and CBM46) for catalytic activity.", "section": "ABSTRACT", "ner": [ [ 20, 31, "full-length", "protein_state" ], [ 32, 41, "structure", "evidence" ], [ 47, 56, "cellulase", "protein_type" ], [ 62, 84, "Bacillus licheniformis", "species" ], [ 86, 93, "BlCel5B", "protein" ], [ 112, 127, "GH5 subfamily 4", "protein_type" ], [ 166, 183, "ancillary modules", "structure_element" ], [ 185, 199, "Ig-like module", "structure_element" ], [ 204, 209, "CBM46", "structure_element" ] ] }, { "sid": 3, "sent": "Using X-ray crystallography, small-angle X-ray scattering and molecular dynamics simulations, we propose that the C-terminal CBM46 caps the distal N-terminal catalytic domain (CD) to establish a fully functional active site via a combination of large-scale multidomain conformational selection and induced-fit mechanisms.", "section": "ABSTRACT", "ner": [ [ 6, 27, "X-ray crystallography", "experimental_method" ], [ 29, 57, "small-angle X-ray scattering", "experimental_method" ], [ 62, 92, "molecular dynamics simulations", "experimental_method" ], [ 125, 130, "CBM46", "structure_element" ], [ 158, 174, "catalytic domain", "structure_element" ], [ 176, 178, "CD", "structure_element" ], [ 195, 211, "fully functional", "protein_state" ], [ 212, 223, "active site", "site" ] ] }, { "sid": 4, "sent": "The Ig-like module is pivoting the packing and unpacking motions of CBM46 relative to CD in the assembly of the binding subsite.", "section": "ABSTRACT", "ner": [ [ 4, 18, "Ig-like module", "structure_element" ], [ 68, 73, "CBM46", "structure_element" ], [ 86, 88, "CD", "structure_element" ], [ 112, 127, "binding subsite", "site" ] ] }, { "sid": 5, "sent": "This is the first example of a multidomain GH relying on large amplitude motions of the CBM46 for assembly of the catalytically competent form of the enzyme.", "section": "ABSTRACT", "ner": [ [ 43, 45, "GH", "protein_type" ], [ 88, 93, "CBM46", "structure_element" ], [ 114, 137, "catalytically competent", "protein_state" ] ] }, { "sid": 6, "sent": "Plant biomass-the most abundant source of carbohydrates on Earth-is primarily composed of cellulose microfibrils surrounded by a hydrated heteropolymeric matrix of hemicellulose and lignin.", "section": "INTRO", "ner": [ [ 0, 5, "Plant", "taxonomy_domain" ], [ 42, 55, "carbohydrates", "chemical" ], [ 90, 99, "cellulose", "chemical" ], [ 164, 177, "hemicellulose", "chemical" ], [ 182, 188, "lignin", "chemical" ] ] }, { "sid": 7, "sent": "Plant biomass may be subjected to thermo-chemical pretreatments and enzymatic reactions to produce soluble fermentable sugars.", "section": "INTRO", "ner": [ [ 0, 5, "Plant", "taxonomy_domain" ], [ 119, 125, "sugars", "chemical" ] ] }, { "sid": 8, "sent": "The canonical model of hydrolytic degradation of cellulose requires at least three classes of enzymes.", "section": "INTRO", "ner": [ [ 49, 58, "cellulose", "chemical" ] ] }, { "sid": 9, "sent": "Cellobiohydrolases (CBHs) processively cleave the glycosidic bonds at the reducing and non-reducing ends of cellulose chains in crystalline regions to produce cellobiose.", "section": "INTRO", "ner": [ [ 0, 18, "Cellobiohydrolases", "protein_type" ], [ 20, 24, "CBHs", "protein_type" ], [ 108, 117, "cellulose", "chemical" ], [ 159, 169, "cellobiose", "chemical" ] ] }, { "sid": 10, "sent": "Endoglucanases (EGs) introduce random cuts in the amorphous regions of cellulose and create new chain extremities for CBH attack; thus, these enzymes act synergistically.", "section": "INTRO", "ner": [ [ 0, 14, "Endoglucanases", "protein_type" ], [ 16, 19, "EGs", "protein_type" ], [ 71, 80, "cellulose", "chemical" ], [ 118, 121, "CBH", "protein_type" ] ] }, { "sid": 11, "sent": "The released cellobiose molecules are then enzymatically converted into glucose by \u03b2-glucosidases.", "section": "INTRO", "ner": [ [ 13, 23, "cellobiose", "chemical" ], [ 72, 79, "glucose", "chemical" ], [ 83, 97, "\u03b2-glucosidases", "protein_type" ] ] }, { "sid": 12, "sent": "The molecular architecture of glycoside hydrolases (GHs) frequently consists of a catalytic domain (CD), where hydrolysis occurs, and one or more ancillary modules (AMs), which are usually connected by less structured linkers.", "section": "INTRO", "ner": [ [ 30, 50, "glycoside hydrolases", "protein_type" ], [ 52, 55, "GHs", "protein_type" ], [ 82, 98, "catalytic domain", "structure_element" ], [ 100, 102, "CD", "structure_element" ], [ 146, 163, "ancillary modules", "structure_element" ], [ 165, 168, "AMs", "structure_element" ], [ 202, 217, "less structured", "protein_state" ], [ 218, 225, "linkers", "structure_element" ] ] }, { "sid": 13, "sent": "The most common type of AMs are carbohydrate-binding modules (CBMs), which are able to recognize and bind specific carbohydrate chains.", "section": "INTRO", "ner": [ [ 24, 27, "AMs", "structure_element" ], [ 32, 60, "carbohydrate-binding modules", "structure_element" ], [ 62, 66, "CBMs", "structure_element" ], [ 115, 127, "carbohydrate", "chemical" ] ] }, { "sid": 14, "sent": "Generally distinct and independent structural domains, the CBMs facilitate carbohydrate hydrolysis by increasing the local concentration of enzymes at the surface of insoluble substrates, thereby targeting the CD component to its cognate ligands.", "section": "INTRO", "ner": [ [ 59, 63, "CBMs", "structure_element" ], [ 75, 87, "carbohydrate", "chemical" ], [ 210, 212, "CD", "structure_element" ] ] }, { "sid": 15, "sent": "CBMs might also disrupt the crystalline structure of cellulose microfibrils, although the underlying mechanism remains poorly understood.", "section": "INTRO", "ner": [ [ 0, 4, "CBMs", "structure_element" ], [ 53, 62, "cellulose", "chemical" ] ] }, { "sid": 16, "sent": "Thus, CBMs enhance the accessibility of CDs to carbohydrate chains to improve enzymatic activity, making them important candidates for the development of effective biomass-degrading enzymes in industrial settings.", "section": "INTRO", "ner": [ [ 6, 10, "CBMs", "structure_element" ], [ 40, 43, "CDs", "structure_element" ], [ 47, 59, "carbohydrate", "chemical" ] ] }, { "sid": 17, "sent": "Although there are examples of active GHs that lack AMs, the majority of the enzymes depend on AMs for activity.", "section": "INTRO", "ner": [ [ 31, 37, "active", "protein_state" ], [ 38, 41, "GHs", "protein_type" ], [ 47, 51, "lack", "protein_state" ], [ 52, 55, "AMs", "structure_element" ], [ 95, 98, "AMs", "structure_element" ] ] }, { "sid": 18, "sent": "In several cases, CBMs were shown to extend and complement the CD substrate-binding site in multimodular carbohydrate-active enzymes, such as endo/exocellulase E4 from Thermobifida fusca, chitinase B from Serratia marcescens, a starch phosphatase from Arabidopsis thaliana and a GH5 subfamily 4 (GH5_4) endoglucanase from Bacillus halodurans (BhCel5B).", "section": "INTRO", "ner": [ [ 18, 22, "CBMs", "structure_element" ], [ 63, 65, "CD", "structure_element" ], [ 66, 88, "substrate-binding site", "site" ], [ 105, 132, "carbohydrate-active enzymes", "protein_type" ], [ 142, 159, "endo/exocellulase", "protein_type" ], [ 160, 162, "E4", "protein" ], [ 168, 186, "Thermobifida fusca", "species" ], [ 188, 199, "chitinase B", "protein" ], [ 205, 224, "Serratia marcescens", "species" ], [ 228, 246, "starch phosphatase", "protein_type" ], [ 252, 272, "Arabidopsis thaliana", "species" ], [ 279, 294, "GH5 subfamily 4", "protein_type" ], [ 296, 301, "GH5_4", "protein_type" ], [ 303, 316, "endoglucanase", "protein_type" ], [ 322, 341, "Bacillus halodurans", "species" ], [ 343, 350, "BhCel5B", "protein" ] ] }, { "sid": 19, "sent": "A pioneer work of Sakon et al. revealed that rigid structural extension of the GH9 CD by a type C CBM3 imprints a processive mode of action to this endoglucanase.", "section": "INTRO", "ner": [ [ 79, 82, "GH9", "protein_type" ], [ 83, 85, "CD", "structure_element" ], [ 91, 102, "type C CBM3", "structure_element" ], [ 148, 161, "endoglucanase", "protein_type" ] ] }, { "sid": 20, "sent": "Further publications showed that CBM-based structural extensions of the active site are important for substrate engagement and recognition.", "section": "INTRO", "ner": [ [ 33, 36, "CBM", "structure_element" ], [ 72, 83, "active site", "site" ] ] }, { "sid": 21, "sent": "Recently, Venditto et al. reported the X-ray structure of the tri-modular GH5_4 endoglucanase from Bacillus halodurans (31% sequence identity to BlCel5B), with the CBM46 extension of the active site appended to the CD via an immunoglobulin (Ig)-like module.", "section": "INTRO", "ner": [ [ 39, 54, "X-ray structure", "evidence" ], [ 62, 73, "tri-modular", "structure_element" ], [ 74, 79, "GH5_4", "protein_type" ], [ 80, 93, "endoglucanase", "protein_type" ], [ 99, 118, "Bacillus halodurans", "species" ], [ 145, 152, "BlCel5B", "protein" ], [ 164, 169, "CBM46", "structure_element" ], [ 187, 198, "active site", "site" ], [ 215, 217, "CD", "structure_element" ], [ 225, 256, "immunoglobulin (Ig)-like module", "structure_element" ] ] }, { "sid": 22, "sent": "Removal of the CBM46 caused a ~60-fold reduction of the activity of the enzyme against \u03b2-glucans, but showed little or no effect against xyloglucan hydrolysis.", "section": "INTRO", "ner": [ [ 0, 10, "Removal of", "experimental_method" ], [ 15, 20, "CBM46", "structure_element" ], [ 87, 96, "\u03b2-glucans", "chemical" ], [ 137, 147, "xyloglucan", "chemical" ] ] }, { "sid": 23, "sent": "Moreover, the CBM46 mediated a significant increase in the BhCel5B activity in plant cell wall settings.", "section": "INTRO", "ner": [ [ 14, 19, "CBM46", "structure_element" ], [ 59, 66, "BhCel5B", "protein" ], [ 79, 84, "plant", "taxonomy_domain" ] ] }, { "sid": 24, "sent": "Modeling of cellotriose in the negative subsites of the active site of BhCel5B demonstrated the structural conservation of the -1 position, but provided little information about direct interactions between CBM46 and the substrate.", "section": "INTRO", "ner": [ [ 0, 8, "Modeling", "experimental_method" ], [ 12, 23, "cellotriose", "chemical" ], [ 31, 48, "negative subsites", "site" ], [ 56, 67, "active site", "site" ], [ 71, 78, "BhCel5B", "protein" ], [ 96, 119, "structural conservation", "protein_state" ], [ 127, 129, "-1", "residue_number" ], [ 206, 211, "CBM46", "structure_element" ] ] }, { "sid": 25, "sent": "It was speculated that \u03b2-1,3 kink of the \u03b2-glucan might allow the ligand to reach for the CBM46, whereas pure \u03b2-1,4 linkages in the backbone of xyloglucan chains would restrict binding to the CD, thus explaining the lack of influence of the CBM46 on the enzymatic activity of BhCel5B against xyloglucans in solution.", "section": "INTRO", "ner": [ [ 41, 49, "\u03b2-glucan", "chemical" ], [ 90, 95, "CBM46", "structure_element" ], [ 144, 154, "xyloglucan", "chemical" ], [ 192, 194, "CD", "structure_element" ], [ 241, 246, "CBM46", "structure_element" ], [ 276, 283, "BhCel5B", "protein" ], [ 292, 303, "xyloglucans", "chemical" ] ] }, { "sid": 26, "sent": "It was also argued that the CBM46 could potentialize the activity by driving BhCel5B towards xyloglucan-rich regions in the context of the plant cell walls, but no large-scale conformational adjustments of the AMs have been shown to occur or suggested to take part in the enzymatic activity.", "section": "INTRO", "ner": [ [ 28, 33, "CBM46", "structure_element" ], [ 77, 84, "BhCel5B", "protein" ], [ 93, 116, "xyloglucan-rich regions", "structure_element" ], [ 139, 144, "plant", "taxonomy_domain" ], [ 210, 213, "AMs", "structure_element" ] ] }, { "sid": 27, "sent": "Although initially introduced as contradictory theories, these two limiting cases can be unified considering the flux description concept or the extended conformational selection model.", "section": "INTRO", "ner": [ [ 145, 153, "extended", "protein_state" ] ] }, { "sid": 28, "sent": "While local ligand-induced conformational adjustments have been reported for carbohydrate-active enzymes, cognate ligands recognition and hydrolysis mediated by a large-scale conformational mobility of distinct domains in multidomain settings is uncommon for endoglucanases.", "section": "INTRO", "ner": [ [ 77, 104, "carbohydrate-active enzymes", "protein_type" ], [ 259, 273, "endoglucanases", "protein_type" ] ] }, { "sid": 29, "sent": "Here, we report the crystal structure of a full-length GH5_4 enzyme from Bacillus licheniformis (BlCel5B) that exhibits two AMs (Ig-like module and CBM46) appended to the CD.", "section": "INTRO", "ner": [ [ 20, 37, "crystal structure", "evidence" ], [ 43, 54, "full-length", "protein_state" ], [ 55, 60, "GH5_4", "protein_type" ], [ 73, 95, "Bacillus licheniformis", "species" ], [ 97, 104, "BlCel5B", "protein" ], [ 124, 127, "AMs", "structure_element" ], [ 129, 143, "Ig-like module", "structure_element" ], [ 148, 153, "CBM46", "structure_element" ], [ 171, 173, "CD", "structure_element" ] ] }, { "sid": 30, "sent": "We structurally and functionally characterize the enzyme using a combination of protein crystallography, small-angle X-ray scattering (SAXS), molecular dynamics computer simulations and site-directed mutagenesis, and show that the AMs and their conformational mobility are essential for the enzymatic activity of BlCel5B.", "section": "INTRO", "ner": [ [ 3, 45, "structurally and functionally characterize", "experimental_method" ], [ 80, 103, "protein crystallography", "experimental_method" ], [ 105, 133, "small-angle X-ray scattering", "experimental_method" ], [ 135, 139, "SAXS", "experimental_method" ], [ 142, 181, "molecular dynamics computer simulations", "experimental_method" ], [ 186, 211, "site-directed mutagenesis", "experimental_method" ], [ 231, 234, "AMs", "structure_element" ], [ 313, 320, "BlCel5B", "protein" ] ] }, { "sid": 31, "sent": "We find that the large-scale conformational adjustments of the distal CBM46 mediated by the Ig-like hinge domain are crucial in active-site assembly for optimal substrate binding and hydrolysis.", "section": "INTRO", "ner": [ [ 70, 75, "CBM46", "structure_element" ], [ 92, 112, "Ig-like hinge domain", "structure_element" ], [ 128, 139, "active-site", "site" ] ] }, { "sid": 32, "sent": "We propose that the BlCel5B conformational selection/induced-fit mechanism of hydrolysis represents a novel paradigm that applies to several GH5_4 members and, possibly, to a number of other multidomain GHs.", "section": "INTRO", "ner": [ [ 20, 27, "BlCel5B", "protein" ], [ 141, 146, "GH5_4", "protein_type" ], [ 203, 206, "GHs", "protein_type" ] ] }, { "sid": 33, "sent": "BlCel5B Crystal Structure", "section": "RESULTS", "ner": [ [ 0, 7, "BlCel5B", "protein" ], [ 8, 25, "Crystal Structure", "evidence" ] ] }, { "sid": 34, "sent": "BlCel5B crystals in the substrate-free form and complexed with cellopentaose (C5) were obtained and diffracted to 1.7\u2009\u00c5 and 1.75\u2009\u00c5 resolutions, respectively (Supplementary Table 1).", "section": "RESULTS", "ner": [ [ 0, 7, "BlCel5B", "protein" ], [ 8, 16, "crystals", "evidence" ], [ 24, 38, "substrate-free", "protein_state" ], [ 48, 62, "complexed with", "protein_state" ], [ 63, 76, "cellopentaose", "chemical" ], [ 78, 80, "C5", "chemical" ] ] }, { "sid": 35, "sent": "The substrate-free and complexed structures exhibited no substantial conformational differences (with the exception of the substrate).", "section": "RESULTS", "ner": [ [ 4, 18, "substrate-free", "protein_state" ], [ 23, 32, "complexed", "protein_state" ], [ 33, 43, "structures", "evidence" ] ] }, { "sid": 36, "sent": "Because of minor variations in the loops located distal to the substrate-binding site, a root mean squared deviation (rmsd) of 0.33\u2009\u00c5 between the complexed and substrate-free structures was observed.", "section": "RESULTS", "ner": [ [ 35, 40, "loops", "structure_element" ], [ 63, 85, "substrate-binding site", "site" ], [ 89, 116, "root mean squared deviation", "evidence" ], [ 118, 122, "rmsd", "evidence" ], [ 146, 155, "complexed", "protein_state" ], [ 160, 174, "substrate-free", "protein_state" ], [ 175, 185, "structures", "evidence" ] ] }, { "sid": 37, "sent": "A single protein chain occupies the asymmetric unit, and most of the residues were built, with the exception of the first 17 residues and those in the loop between L398 and P405 due to weak electron density.", "section": "RESULTS", "ner": [ [ 116, 133, "first 17 residues", "residue_range" ], [ 151, 155, "loop", "structure_element" ], [ 164, 168, "L398", "residue_name_number" ], [ 173, 177, "P405", "residue_name_number" ], [ 190, 206, "electron density", "evidence" ] ] }, { "sid": 38, "sent": "The BlCel5B structure comprises three distinct domains: an N-terminal CD (residues 18 to 330), an Ig-like module (residues 335 to 428) and a family 46 CBM (residues 432 to 533) (Fig. 1A,B).", "section": "RESULTS", "ner": [ [ 4, 11, "BlCel5B", "protein" ], [ 12, 21, "structure", "evidence" ], [ 70, 72, "CD", "structure_element" ], [ 83, 92, "18 to 330", "residue_range" ], [ 98, 112, "Ig-like module", "structure_element" ], [ 123, 133, "335 to 428", "residue_range" ], [ 141, 154, "family 46 CBM", "structure_element" ], [ 165, 175, "432 to 533", "residue_range" ] ] }, { "sid": 39, "sent": "Similarly to other members of the GH5 family, the CD of BlCel5B has a typical TIM barrel fold with eight inner \u03b2-strands and eight outer \u03b1 helices that are interconnected by loops and three short \u03b1 helices.", "section": "RESULTS", "ner": [ [ 34, 37, "GH5", "protein_type" ], [ 50, 52, "CD", "structure_element" ], [ 56, 63, "BlCel5B", "protein" ], [ 78, 93, "TIM barrel fold", "structure_element" ], [ 111, 120, "\u03b2-strands", "structure_element" ], [ 137, 146, "\u03b1 helices", "structure_element" ], [ 174, 179, "loops", "structure_element" ], [ 196, 205, "\u03b1 helices", "structure_element" ] ] }, { "sid": 40, "sent": "Very short linkers, D429-D430-P431 and V331-P332-N333-A334, connect the CBM46 to the Ig-like module and the Ig-like module to the CD, respectively.", "section": "RESULTS", "ner": [ [ 11, 18, "linkers", "structure_element" ], [ 20, 34, "D429-D430-P431", "structure_element" ], [ 39, 58, "V331-P332-N333-A334", "structure_element" ], [ 72, 77, "CBM46", "structure_element" ], [ 85, 99, "Ig-like module", "structure_element" ], [ 108, 122, "Ig-like module", "structure_element" ], [ 130, 132, "CD", "structure_element" ] ] }, { "sid": 41, "sent": "Both Ig-like module and CBM46 have a \u03b2-sandwich fold composed of two \u03b2-sheets of four and three antiparallel \u03b2-strands interconnected by loops and a short \u03b1 helix between strands \u03b23 and \u03b24 (Fig. 1C).", "section": "RESULTS", "ner": [ [ 5, 19, "Ig-like module", "structure_element" ], [ 24, 29, "CBM46", "structure_element" ], [ 37, 52, "\u03b2-sandwich fold", "structure_element" ], [ 69, 77, "\u03b2-sheets", "structure_element" ], [ 96, 118, "antiparallel \u03b2-strands", "structure_element" ], [ 137, 142, "loops", "structure_element" ], [ 155, 162, "\u03b1 helix", "structure_element" ], [ 171, 178, "strands", "structure_element" ], [ 179, 181, "\u03b23", "structure_element" ], [ 186, 188, "\u03b24", "structure_element" ] ] }, { "sid": 42, "sent": "A structural comparison between the Ig-like module and the CBM46 using the Dali server yielded an rmsd of 2.3\u2009\u00c5 and a Z-score of 10.2.", "section": "RESULTS", "ner": [ [ 2, 23, "structural comparison", "experimental_method" ], [ 36, 50, "Ig-like module", "structure_element" ], [ 59, 64, "CBM46", "structure_element" ], [ 75, 86, "Dali server", "experimental_method" ], [ 98, 102, "rmsd", "evidence" ], [ 118, 125, "Z-score", "evidence" ] ] }, { "sid": 43, "sent": "A structure-based search performed using the same server showed that the Ig-like module is similar to the Ig-like module from a recently solved crystal structure of a tri-modular GH5_4 enzyme from Bacillus halodurans, BhCel5B, with rmsd\u2009=\u20091.3\u2009\u00c5 and Z-score\u2009=\u200915.3.", "section": "RESULTS", "ner": [ [ 2, 24, "structure-based search", "experimental_method" ], [ 73, 87, "Ig-like module", "structure_element" ], [ 106, 120, "Ig-like module", "structure_element" ], [ 137, 143, "solved", "experimental_method" ], [ 144, 161, "crystal structure", "evidence" ], [ 167, 178, "tri-modular", "structure_element" ], [ 179, 184, "GH5_4", "protein_type" ], [ 197, 216, "Bacillus halodurans", "species" ], [ 218, 225, "BhCel5B", "protein" ], [ 232, 236, "rmsd", "evidence" ], [ 249, 256, "Z-score", "evidence" ] ] }, { "sid": 44, "sent": "The CBM46 from BhCel5B is the most structurally similar to BlCel5B CBM46, with rmsd\u2009=\u20091.6\u2009\u00c5 and Z-score\u2009=\u200912.4.", "section": "RESULTS", "ner": [ [ 4, 9, "CBM46", "structure_element" ], [ 15, 22, "BhCel5B", "protein" ], [ 59, 66, "BlCel5B", "protein" ], [ 67, 72, "CBM46", "structure_element" ], [ 79, 83, "rmsd", "evidence" ], [ 96, 103, "Z-score", "evidence" ] ] }, { "sid": 45, "sent": "The sequence identity relative to BhCel5B, however, is low (28% for Ig-like and 25% for CBM46).", "section": "RESULTS", "ner": [ [ 34, 41, "BhCel5B", "protein" ], [ 68, 75, "Ig-like", "structure_element" ], [ 88, 93, "CBM46", "structure_element" ] ] }, { "sid": 46, "sent": "The Ig-like module, adjacent to the CD, contains only one tyrosine (Y367) exposed to solvent and no tryptophan residues.", "section": "RESULTS", "ner": [ [ 4, 18, "Ig-like module", "structure_element" ], [ 36, 38, "CD", "structure_element" ], [ 58, 66, "tyrosine", "residue_name" ], [ 68, 72, "Y367", "residue_name_number" ], [ 100, 110, "tryptophan", "residue_name" ] ] }, { "sid": 47, "sent": "Because aromatic residues play a major role in glucose recognition, this observation suggests that substrate binding may not be the primary function of Ig-like module.", "section": "RESULTS", "ner": [ [ 47, 54, "glucose", "chemical" ], [ 152, 166, "Ig-like module", "structure_element" ] ] }, { "sid": 48, "sent": "In contrast, the CBM46 has three tryptophan residues, two of which face the CD substrate binding site (Fig. 1A), indicating that it may be actively engaged in the carbohydrate binding.", "section": "RESULTS", "ner": [ [ 17, 22, "CBM46", "structure_element" ], [ 33, 43, "tryptophan", "residue_name" ], [ 76, 78, "CD", "structure_element" ], [ 79, 101, "substrate binding site", "site" ], [ 163, 175, "carbohydrate", "chemical" ] ] }, { "sid": 49, "sent": "Electron density maps clearly reveal the presence of a cellotetraose (C4) and not a soaked cellopentaose (C5) in the CD negative substrate-binding subsites (Fig. 1D), indicating that BlCel5B is catalytically active in the crystal state and able to cleave a C5 molecule.", "section": "RESULTS", "ner": [ [ 0, 21, "Electron density maps", "evidence" ], [ 41, 52, "presence of", "protein_state" ], [ 55, 68, "cellotetraose", "chemical" ], [ 70, 72, "C4", "chemical" ], [ 91, 104, "cellopentaose", "chemical" ], [ 106, 108, "C5", "chemical" ], [ 117, 119, "CD", "structure_element" ], [ 120, 155, "negative substrate-binding subsites", "site" ], [ 183, 190, "BlCel5B", "protein" ], [ 194, 214, "catalytically active", "protein_state" ], [ 257, 259, "C5", "chemical" ] ] }, { "sid": 50, "sent": "The lack of electron density verifies the absence of the fifth glucose moiety from the soaked C5, and a closer inspection of the structure confirmed that the presence of a fifth glucose unit would be sterically hindered by the catalytic residues on the reducing end and by residue R234 of a symmetry-related enzyme molecule on the non-reducing end.", "section": "RESULTS", "ner": [ [ 4, 28, "lack of electron density", "evidence" ], [ 42, 52, "absence of", "protein_state" ], [ 57, 62, "fifth", "residue_number" ], [ 63, 70, "glucose", "chemical" ], [ 94, 96, "C5", "chemical" ], [ 129, 138, "structure", "evidence" ], [ 158, 169, "presence of", "protein_state" ], [ 172, 177, "fifth", "residue_number" ], [ 178, 185, "glucose", "chemical" ], [ 227, 245, "catalytic residues", "site" ], [ 281, 285, "R234", "residue_name_number" ] ] }, { "sid": 51, "sent": "The ability of BlCel5B to cleave C5 into glucose and C4 molecules in solution was demonstrated by enzymatic product profile mass spectrometry analysis (Fig. 2A).", "section": "RESULTS", "ner": [ [ 15, 22, "BlCel5B", "protein" ], [ 33, 35, "C5", "chemical" ], [ 41, 48, "glucose", "chemical" ], [ 53, 55, "C4", "chemical" ], [ 98, 141, "enzymatic product profile mass spectrometry", "experimental_method" ] ] }, { "sid": 52, "sent": "The C4 oligomer in the BlCel5B binding site is coordinated by hydrogen bonds to residues N36, H113, H114, N158, W301, and N303 and by a CH-\u03c0 interaction with residue W47 (Fig. 1D).", "section": "RESULTS", "ner": [ [ 4, 6, "C4", "chemical" ], [ 23, 30, "BlCel5B", "protein" ], [ 31, 43, "binding site", "site" ], [ 47, 58, "coordinated", "bond_interaction" ], [ 62, 76, "hydrogen bonds", "bond_interaction" ], [ 89, 92, "N36", "residue_name_number" ], [ 94, 98, "H113", "residue_name_number" ], [ 100, 104, "H114", "residue_name_number" ], [ 106, 110, "N158", "residue_name_number" ], [ 112, 116, "W301", "residue_name_number" ], [ 122, 126, "N303", "residue_name_number" ], [ 136, 152, "CH-\u03c0 interaction", "bond_interaction" ], [ 166, 169, "W47", "residue_name_number" ] ] }, { "sid": 53, "sent": "These residues belong to the CD and are conserved in the GH5 family.", "section": "RESULTS", "ner": [ [ 29, 31, "CD", "structure_element" ], [ 40, 49, "conserved", "protein_state" ], [ 57, 60, "GH5", "protein_type" ] ] }, { "sid": 54, "sent": "BlCel5B enzymatic activity", "section": "RESULTS", "ner": [ [ 0, 7, "BlCel5B", "protein" ] ] }, { "sid": 55, "sent": "BlCel5B exhibits optimum activity toward carboxymethylcellulose (CMC; 8.7\u2009U/mg) at a pH of 4.0 and 55\u2009\u00b0C and retains approximately half of its maximum activity at 80\u2009\u00b0C, demonstrating considerable thermal stability (Fig. 2B,C).", "section": "RESULTS", "ner": [ [ 0, 7, "BlCel5B", "protein" ], [ 41, 63, "carboxymethylcellulose", "chemical" ], [ 65, 68, "CMC", "chemical" ] ] }, { "sid": 56, "sent": "BlCel5B is also active on \u03b2-glucan (34\u2009U/mg), lichenan (17.8\u2009U/mg) and xyloglucan (15.7\u2009U/mg) substrates (Table 1), whereas no activity was detected on galactomannan, rye arabinoxylan, 1,4-\u03b2-mannan or the insoluble substrate Azo-Avicel.", "section": "RESULTS", "ner": [ [ 0, 7, "BlCel5B", "protein" ], [ 16, 22, "active", "protein_state" ], [ 26, 34, "\u03b2-glucan", "chemical" ], [ 46, 54, "lichenan", "chemical" ], [ 71, 81, "xyloglucan", "chemical" ], [ 152, 165, "galactomannan", "chemical" ], [ 167, 170, "rye", "taxonomy_domain" ], [ 171, 183, "arabinoxylan", "chemical" ], [ 185, 197, "1,4-\u03b2-mannan", "chemical" ], [ 225, 235, "Azo-Avicel", "chemical" ] ] }, { "sid": 57, "sent": "Kinetic parameters were calculated assuming Michaelis-Menten behavior with CMC as substrate: KM\u2009=\u20091.78\u2009g L\u22121 and Vmax\u2009=\u20091.41\u2009\u00d7\u200910\u22124 g s\u22121 mg protein\u22121 (Fig. 2D).", "section": "RESULTS", "ner": [ [ 44, 69, "Michaelis-Menten behavior", "experimental_method" ], [ 75, 78, "CMC", "chemical" ], [ 93, 95, "KM", "evidence" ], [ 113, 117, "Vmax", "evidence" ] ] }, { "sid": 58, "sent": "Although BlCel5B is not a highly active enzyme against one specific substrate as compared to others GH5_4, it has the advantage of being active against different substrates with \u03b2-1,3 and/or \u03b2-1,4 glycosidic linkages.", "section": "RESULTS", "ner": [ [ 9, 16, "BlCel5B", "protein" ], [ 33, 39, "active", "protein_state" ], [ 100, 105, "GH5_4", "protein_type" ], [ 137, 143, "active", "protein_state" ] ] }, { "sid": 59, "sent": "To understand the importance of the ancillary modules for BlCel5B activity, enzymatic assays were carried out using four enzyme mutants: a CBM46 deletion (\u0394CBM46) and an Ig-like\u2009+\u2009CBM46 deletion (\u0394Ig-CBM46) as well as point mutations of the CBM46 inner surface residues W479A and W481A.", "section": "RESULTS", "ner": [ [ 36, 53, "ancillary modules", "structure_element" ], [ 58, 65, "BlCel5B", "protein" ], [ 76, 92, "enzymatic assays", "experimental_method" ], [ 128, 135, "mutants", "protein_state" ], [ 139, 144, "CBM46", "structure_element" ], [ 145, 153, "deletion", "experimental_method" ], [ 155, 161, "\u0394CBM46", "mutant" ], [ 170, 177, "Ig-like", "structure_element" ], [ 180, 185, "CBM46", "structure_element" ], [ 186, 194, "deletion", "experimental_method" ], [ 196, 205, "\u0394Ig-CBM46", "mutant" ], [ 218, 233, "point mutations", "experimental_method" ], [ 241, 246, "CBM46", "structure_element" ], [ 270, 275, "W479A", "mutant" ], [ 280, 285, "W481A", "mutant" ] ] }, { "sid": 60, "sent": "These mutants were expressed and purified as described for the wild-type enzyme.", "section": "RESULTS", "ner": [ [ 6, 13, "mutants", "protein_state" ], [ 19, 41, "expressed and purified", "experimental_method" ], [ 63, 72, "wild-type", "protein_state" ] ] }, { "sid": 61, "sent": "Strikingly, neither of the deletion variants exhibited detectable activity toward any of the substrates tested using full-length BlCel5B (Table 1), demonstrating that the Ig-like module and the CBM46 are essential for BlCel5B activity.", "section": "RESULTS", "ner": [ [ 27, 44, "deletion variants", "protein_state" ], [ 117, 128, "full-length", "protein_state" ], [ 129, 136, "BlCel5B", "protein" ], [ 171, 185, "Ig-like module", "structure_element" ], [ 194, 199, "CBM46", "structure_element" ], [ 218, 225, "BlCel5B", "protein" ] ] }, { "sid": 62, "sent": "Thermal shift assays were conducted to confirm structural stability of the mutants (Supplementary Fig. 1).", "section": "RESULTS", "ner": [ [ 0, 20, "Thermal shift assays", "experimental_method" ], [ 75, 82, "mutants", "protein_state" ] ] }, { "sid": 63, "sent": "All of the constructs showed similar melting temperatures: 62\u2009\u00b0C for BlCel5B, 58\u2009\u00b0C for BlCel5B\u0394CBM46, 56\u2009\u00b0C for BlCel5B\u0394Ig-CBM46, 65\u2009\u00b0C for BlCel5BW479A and 59\u2009\u00b0C for BlCel5BW479A, thus confirming their proper overall fold.", "section": "RESULTS", "ner": [ [ 37, 57, "melting temperatures", "evidence" ], [ 69, 76, "BlCel5B", "protein" ], [ 88, 101, "BlCel5B\u0394CBM46", "mutant" ], [ 113, 129, "BlCel5B\u0394Ig-CBM46", "mutant" ], [ 141, 153, "BlCel5BW479A", "mutant" ], [ 168, 180, "BlCel5BW479A", "mutant" ] ] }, { "sid": 64, "sent": "We also examined the function of the CBM46 inner surface residues W479 and W481 (Fig. 1A) in BlCel5B activity by performing enzymatic assays with W479A and W481A mutants.", "section": "RESULTS", "ner": [ [ 37, 42, "CBM46", "structure_element" ], [ 49, 56, "surface", "site" ], [ 66, 70, "W479", "residue_name_number" ], [ 75, 79, "W481", "residue_name_number" ], [ 93, 100, "BlCel5B", "protein" ], [ 124, 140, "enzymatic assays", "experimental_method" ], [ 146, 151, "W479A", "mutant" ], [ 156, 161, "W481A", "mutant" ], [ 162, 169, "mutants", "protein_state" ] ] }, { "sid": 65, "sent": "Both mutations reduced enzymatic activity toward all tested substrates (Table 1), with W481A having a stronger effect than W479A (~64% vs. 79% activity relative to wt BlCel5B using \u03b2-glucan and ~10% vs. 50% using CMC).", "section": "RESULTS", "ner": [ [ 5, 14, "mutations", "experimental_method" ], [ 87, 92, "W481A", "mutant" ], [ 123, 128, "W479A", "mutant" ], [ 164, 166, "wt", "protein_state" ], [ 167, 174, "BlCel5B", "protein" ], [ 181, 189, "\u03b2-glucan", "chemical" ], [ 213, 216, "CMC", "chemical" ] ] }, { "sid": 66, "sent": "This indicates that CBM46 must interact with the substrate via residues W479 and W481.", "section": "RESULTS", "ner": [ [ 20, 25, "CBM46", "structure_element" ], [ 72, 76, "W479", "residue_name_number" ], [ 81, 85, "W481", "residue_name_number" ] ] }, { "sid": 67, "sent": "However, since the BlCel5B crystal structure exhibits no close contact between these residues and the substrate, these results suggest the existence of large-amplitude interdomain motions that may enable direct interactions between CBM46 and the carbohydrate.", "section": "RESULTS", "ner": [ [ 19, 26, "BlCel5B", "protein" ], [ 27, 44, "crystal structure", "evidence" ], [ 57, 62, "close", "protein_state" ], [ 232, 237, "CBM46", "structure_element" ], [ 246, 258, "carbohydrate", "chemical" ] ] }, { "sid": 68, "sent": "BlCelB5 dynamics and binding-site architecture", "section": "RESULTS", "ner": [ [ 0, 7, "BlCelB5", "protein" ], [ 21, 33, "binding-site", "site" ] ] }, { "sid": 69, "sent": "Molecular dynamics (MD) simulations were performed to investigate the conformational mobility of BlCel5B.", "section": "RESULTS", "ner": [ [ 0, 18, "Molecular dynamics", "experimental_method" ], [ 20, 22, "MD", "experimental_method" ], [ 24, 35, "simulations", "experimental_method" ], [ 97, 104, "BlCel5B", "protein" ] ] }, { "sid": 70, "sent": "In the simulations of the crystal structure for BlCel5B bound to C4, the substrate dissociates from the protein within the first 100\u2009ns of the simulation time (Supplementary Fig. 2A).", "section": "RESULTS", "ner": [ [ 7, 18, "simulations", "experimental_method" ], [ 26, 43, "crystal structure", "evidence" ], [ 48, 55, "BlCel5B", "protein" ], [ 56, 64, "bound to", "protein_state" ], [ 65, 67, "C4", "chemical" ], [ 143, 153, "simulation", "experimental_method" ] ] }, { "sid": 71, "sent": "This observation suggests that cellotetraose does not exhibit detectable affinity for this specific BlCel5B conformation in solution, as one might otherwise expect for a reaction product.", "section": "RESULTS", "ner": [ [ 31, 44, "cellotetraose", "chemical" ], [ 100, 107, "BlCel5B", "protein" ] ] }, { "sid": 72, "sent": "No changes beyond local fluctuations were observed in any of the three BlCel5B domains within the time scale of these runs (400\u2009ns; Supplementary Fig. 2B).", "section": "RESULTS", "ner": [ [ 71, 78, "BlCel5B", "protein" ] ] }, { "sid": 73, "sent": "However, the CBM46 and Ig-like domains did exhibit rigid body-like motions relative to the CD, with rmsd values around 2.3\u2009\u00c5 and 1.8\u2009\u00c5, respectively, suggesting that BlCel5B may execute large-amplitude interdomain motions over longer time scales (Supplementary Fig. 2B,C).", "section": "RESULTS", "ner": [ [ 13, 18, "CBM46", "structure_element" ], [ 23, 38, "Ig-like domains", "structure_element" ], [ 91, 93, "CD", "structure_element" ], [ 100, 104, "rmsd", "evidence" ], [ 166, 173, "BlCel5B", "protein" ] ] }, { "sid": 74, "sent": "Accordingly, simulations were then performed using accelerated molecular dynamics (aMD) techniques to probe BlCel5B interdomain motions.", "section": "RESULTS", "ner": [ [ 13, 24, "simulations", "experimental_method" ], [ 51, 81, "accelerated molecular dynamics", "experimental_method" ], [ 83, 86, "aMD", "experimental_method" ], [ 108, 115, "BlCel5B", "protein" ] ] }, { "sid": 75, "sent": "aMD enhances conformational sampling by raising the basins of the dihedral potential energy surface without affecting the general form of the atomistic potential, thereby increasing transition rates between different local minima.", "section": "RESULTS", "ner": [ [ 0, 3, "aMD", "experimental_method" ], [ 66, 99, "dihedral potential energy surface", "evidence" ] ] }, { "sid": 76, "sent": "aMD trajectories corresponding to more than 1.0\u2009\u03bcs of conventional MD runs were generated.", "section": "RESULTS", "ner": [ [ 0, 3, "aMD", "experimental_method" ], [ 4, 16, "trajectories", "evidence" ], [ 67, 69, "MD", "experimental_method" ] ] }, { "sid": 77, "sent": "During these simulations, we observed occlusive conformations between CBM46 and CD that resulted in a rearrangement of the enzyme\u2019s architecture around the active site (Video S1).", "section": "RESULTS", "ner": [ [ 13, 24, "simulations", "experimental_method" ], [ 70, 75, "CBM46", "structure_element" ], [ 80, 82, "CD", "structure_element" ], [ 156, 167, "active site", "site" ] ] }, { "sid": 78, "sent": "Figure 3A shows BlCel5B in the crystallographic conformation (red) and in a selected configuration obtained with aMD (blue) in the absence of the substrate.", "section": "RESULTS", "ner": [ [ 16, 23, "BlCel5B", "protein" ], [ 31, 47, "crystallographic", "experimental_method" ], [ 113, 116, "aMD", "experimental_method" ], [ 131, 141, "absence of", "protein_state" ] ] }, { "sid": 79, "sent": "Interdomain motions were gauged by the time evolution of the distance between the \u03b1 carbons of residues I120 and E477 (represented as spheres in Fig. 3A), belonging to the CD and CBM46, respectively.", "section": "RESULTS", "ner": [ [ 61, 69, "distance", "evidence" ], [ 104, 108, "I120", "residue_name_number" ], [ 113, 117, "E477", "residue_name_number" ], [ 172, 174, "CD", "structure_element" ], [ 179, 184, "CBM46", "structure_element" ] ] }, { "sid": 80, "sent": "Figure 3C shows that the I120-E477 distance (red curve) gradually decreases from ~35\u2009\u00c5 to ~7\u2009\u00c5 within the first half of the 1.0\u2009\u03bcs aMD trajectory, indicating a transition between the semi-open (crystallographic) and occluded (aMD sampled) configurations.", "section": "RESULTS", "ner": [ [ 25, 29, "I120", "residue_name_number" ], [ 30, 34, "E477", "residue_name_number" ], [ 35, 43, "distance", "evidence" ], [ 131, 134, "aMD", "experimental_method" ], [ 135, 145, "trajectory", "evidence" ], [ 183, 192, "semi-open", "protein_state" ], [ 194, 210, "crystallographic", "experimental_method" ], [ 216, 224, "occluded", "protein_state" ], [ 226, 229, "aMD", "experimental_method" ] ] }, { "sid": 81, "sent": "During the second half of the aMD simulation, the full-length enzyme remained in the closed conformation, with the CBM46 covering the carbohydrate-binding site.", "section": "RESULTS", "ner": [ [ 30, 44, "aMD simulation", "experimental_method" ], [ 50, 61, "full-length", "protein_state" ], [ 85, 91, "closed", "protein_state" ], [ 115, 120, "CBM46", "structure_element" ], [ 134, 159, "carbohydrate-binding site", "site" ] ] }, { "sid": 82, "sent": "These results suggest that BlCel5B undergoes large-scale interdomain movements that enable interactions between CBM46 and the substrate bound to the CD.", "section": "RESULTS", "ner": [ [ 27, 34, "BlCel5B", "protein" ], [ 112, 117, "CBM46", "structure_element" ], [ 136, 144, "bound to", "protein_state" ], [ 149, 151, "CD", "structure_element" ] ] }, { "sid": 83, "sent": "To study the interactions of BlCel5B with a non-hydrolyzed glucan chain, we built a model structure with a cellooctaose (C8) chain spanning the entire positive (+1 to +4) and negative (\u22124 to \u22121) subsites of the enzyme.", "section": "RESULTS", "ner": [ [ 29, 36, "BlCel5B", "protein" ], [ 59, 65, "glucan", "chemical" ], [ 90, 99, "structure", "evidence" ], [ 107, 119, "cellooctaose", "chemical" ], [ 121, 123, "C8", "chemical" ], [ 151, 170, "positive (+1 to +4)", "site" ], [ 175, 194, "negative (\u22124 to \u22121)", "site" ], [ 195, 203, "subsites", "site" ] ] }, { "sid": 84, "sent": "Starting from the crystallographic BlCel5B conformation, the C8 molecule deviated significantly from the active site and assumed a non-productive binding mode (Supplementary Fig. 2D).", "section": "RESULTS", "ner": [ [ 35, 42, "BlCel5B", "protein" ], [ 61, 63, "C8", "chemical" ], [ 105, 116, "active site", "site" ] ] }, { "sid": 85, "sent": "This observation suggests that the open conformation of BlCel5B is not able to hold the substrate in a position suitable for hydrolysis (Supplementary Fig. 2E).", "section": "RESULTS", "ner": [ [ 35, 39, "open", "protein_state" ], [ 56, 63, "BlCel5B", "protein" ] ] }, { "sid": 86, "sent": "However, after subjecting the BlCel5B-C8 complex to a 0.5\u2009\u03bcs aMD simulation with harmonic restraints on the C8 chain to prevent it from deviating from the productive binding mode, the CBM46 readily closed over the CD and trapped the C8 chain in position for hydrolysis (Fig. 3B).", "section": "RESULTS", "ner": [ [ 30, 40, "BlCel5B-C8", "complex_assembly" ], [ 61, 75, "aMD simulation", "experimental_method" ], [ 108, 110, "C8", "chemical" ], [ 184, 189, "CBM46", "structure_element" ], [ 198, 204, "closed", "protein_state" ], [ 214, 216, "CD", "structure_element" ], [ 233, 235, "C8", "chemical" ] ] }, { "sid": 87, "sent": "In the presence of the substrate, CBM46 adopts a final conformation intermediate between the crystallographic structure and that observed in the substrate-free BlCel5B aMD simulations; this is illustrated by the I120-E477 distance, which stabilizes near 20\u2009\u00c5 in the closed configuration that traps the C8 molecule (in contrast to ~7\u2009\u00c5 for substrate-free BlCel5B) (Fig. 3C).", "section": "RESULTS", "ner": [ [ 7, 18, "presence of", "protein_state" ], [ 34, 39, "CBM46", "structure_element" ], [ 93, 119, "crystallographic structure", "evidence" ], [ 145, 159, "substrate-free", "protein_state" ], [ 160, 167, "BlCel5B", "protein" ], [ 168, 183, "aMD simulations", "experimental_method" ], [ 212, 216, "I120", "residue_name_number" ], [ 217, 221, "E477", "residue_name_number" ], [ 222, 230, "distance", "evidence" ], [ 266, 272, "closed", "protein_state" ], [ 302, 304, "C8", "chemical" ], [ 339, 353, "substrate-free", "protein_state" ], [ 354, 361, "BlCel5B", "protein" ] ] }, { "sid": 88, "sent": "This BlCel5B-C8 configuration remains stable over an additional 500 ns of conventional MD simulation with no restraints (Fig. 3C cyan line, Supplementary Fig. 2E,F).", "section": "RESULTS", "ner": [ [ 5, 15, "BlCel5B-C8", "complex_assembly" ], [ 87, 100, "MD simulation", "experimental_method" ] ] }, { "sid": 89, "sent": "A closer inspection of the productive binding mode obtained from these extensive simulations reveals that the CBM46 tryptophan residues W479 and W481 (along with CD tryptophan residues) play important roles in carbohydrate recognition and orientation by creating a tunnel-like topology along the BlCel5B binding cleft, as depicted in Fig. 3D.", "section": "RESULTS", "ner": [ [ 81, 92, "simulations", "experimental_method" ], [ 110, 115, "CBM46", "structure_element" ], [ 116, 126, "tryptophan", "residue_name" ], [ 136, 140, "W479", "residue_name_number" ], [ 145, 149, "W481", "residue_name_number" ], [ 162, 164, "CD", "structure_element" ], [ 165, 175, "tryptophan", "residue_name" ], [ 210, 222, "carbohydrate", "chemical" ], [ 265, 271, "tunnel", "site" ], [ 296, 303, "BlCel5B", "protein" ], [ 304, 317, "binding cleft", "site" ] ] }, { "sid": 90, "sent": "Together, these results indicate that CBM46 is a key component of the catalytic active complex, providing an explanation as to why CBM46 is essential for the enzymatic activity of BlCel5B.", "section": "RESULTS", "ner": [ [ 38, 43, "CBM46", "structure_element" ], [ 70, 86, "catalytic active", "protein_state" ], [ 131, 136, "CBM46", "structure_element" ], [ 180, 187, "BlCel5B", "protein" ] ] }, { "sid": 91, "sent": "To enable substantially longer time scales compared to atomistic simulations, we further explored the dynamics of BlCel5B using coarse-grained MD (CG-MD) simulations.", "section": "RESULTS", "ner": [ [ 55, 76, "atomistic simulations", "experimental_method" ], [ 114, 121, "BlCel5B", "protein" ], [ 128, 145, "coarse-grained MD", "experimental_method" ], [ 147, 152, "CG-MD", "experimental_method" ], [ 154, 165, "simulations", "experimental_method" ] ] }, { "sid": 92, "sent": "We performed three independent ~120\u2009\u03bcs CG-MD simulations, for a total of approximately 360\u2009\u03bcs of sampling.", "section": "RESULTS", "ner": [ [ 39, 56, "CG-MD simulations", "experimental_method" ] ] }, { "sid": 93, "sent": "The distance between the \u03b1 carbons of two residues centrally positioned in the CD and CBM46 (Fig. 4A) was monitored, and the results shown in Fig. 4B indicate that the wide-amplitude events described above frequently appear in this time scale.", "section": "RESULTS", "ner": [ [ 4, 12, "distance", "evidence" ], [ 79, 81, "CD", "structure_element" ], [ 86, 91, "CBM46", "structure_element" ] ] }, { "sid": 94, "sent": "The computed distance distribution depicted in Fig. 4C indicates three main conformational states ranging from (I) closed conformations similar to those encountered in the substrate-free aMD simulations, in which CBM46 interacts with the CD to shape the substrate binding site, to (II) semi-open conformations similar to the crystallographic structure, and (III) extended BlCel5B conformations in which the CD and CBM46 are even further apart than in the crystal structure.", "section": "RESULTS", "ner": [ [ 4, 34, "computed distance distribution", "evidence" ], [ 115, 121, "closed", "protein_state" ], [ 172, 186, "substrate-free", "protein_state" ], [ 187, 202, "aMD simulations", "experimental_method" ], [ 213, 218, "CBM46", "structure_element" ], [ 238, 240, "CD", "structure_element" ], [ 254, 276, "substrate binding site", "site" ], [ 286, 295, "semi-open", "protein_state" ], [ 325, 351, "crystallographic structure", "evidence" ], [ 363, 371, "extended", "protein_state" ], [ 372, 379, "BlCel5B", "protein" ], [ 407, 409, "CD", "structure_element" ], [ 414, 419, "CBM46", "structure_element" ], [ 455, 472, "crystal structure", "evidence" ] ] }, { "sid": 95, "sent": "BlCel5B conformers fit the SAXS envelope", "section": "RESULTS", "ner": [ [ 0, 7, "BlCel5B", "protein" ], [ 27, 31, "SAXS", "experimental_method" ], [ 32, 40, "envelope", "evidence" ] ] }, { "sid": 96, "sent": "SAXS experiments were conducted to assess BlCel5B conformational states in solution, and the results revealed the enzyme in its monomeric form, with average values of Rg\u2009=\u200927.17\u2009\u00c5 and Dmax\u2009=\u200987.59\u2009\u00c5 (Supplementary Table 2).", "section": "RESULTS", "ner": [ [ 0, 4, "SAXS", "experimental_method" ], [ 42, 49, "BlCel5B", "protein" ], [ 128, 137, "monomeric", "oligomeric_state" ], [ 167, 169, "Rg", "evidence" ], [ 184, 188, "Dmax", "evidence" ] ] }, { "sid": 97, "sent": "The ab initio dummy atom model (DAM) demonstrated that the SAXS-derived BlCel5B molecular envelope could not be single-handedly filled by any of the main conformational states encountered in the simulations (Fig. 4D).", "section": "RESULTS", "ner": [ [ 4, 30, "ab initio dummy atom model", "experimental_method" ], [ 32, 35, "DAM", "experimental_method" ], [ 59, 63, "SAXS", "experimental_method" ], [ 72, 79, "BlCel5B", "protein" ], [ 90, 98, "envelope", "evidence" ], [ 195, 206, "simulations", "experimental_method" ] ] }, { "sid": 98, "sent": "It is known that a Kratky plot exhibits a peak with an elevated baseline at high q for a monodisperse system composed of multi-domain particles with flexible extensions.", "section": "RESULTS", "ner": [ [ 19, 30, "Kratky plot", "evidence" ] ] }, { "sid": 99, "sent": "Indeed, an elevation of the baseline toward a hyperbolic-like curve was observed for BlCel5B, indicating a considerable degree of molecular mobility in solution (Supplementary Fig. 3).", "section": "RESULTS", "ner": [ [ 85, 92, "BlCel5B", "protein" ] ] }, { "sid": 100, "sent": "Thus, the conformational heterogeneity of the enzyme can be decomposed in structural terms as a combination of conformational states identified in our crystallographic and MD studies.", "section": "RESULTS", "ner": [ [ 151, 182, "crystallographic and MD studies", "experimental_method" ] ] }, { "sid": 101, "sent": "We found that the SAXS envelope can be well represented by considering the superimposition of three different representative molecular conformations of BlCel5B (Fig. 4E): a closed or CBM46/CD-occluded conformation extracted from the simulations with a relative weight of 26%, a semi-open conformation represented by the crystal structure corresponding to 40%, and an extended conformation based on simulations that is responsible for 34% of the SAXS envelope.", "section": "RESULTS", "ner": [ [ 18, 22, "SAXS", "experimental_method" ], [ 23, 31, "envelope", "evidence" ], [ 75, 90, "superimposition", "experimental_method" ], [ 152, 159, "BlCel5B", "protein" ], [ 173, 179, "closed", "protein_state" ], [ 183, 188, "CBM46", "structure_element" ], [ 189, 191, "CD", "structure_element" ], [ 192, 200, "occluded", "protein_state" ], [ 233, 244, "simulations", "experimental_method" ], [ 278, 287, "semi-open", "protein_state" ], [ 320, 337, "crystal structure", "evidence" ], [ 367, 375, "extended", "protein_state" ], [ 398, 409, "simulations", "experimental_method" ], [ 445, 449, "SAXS", "experimental_method" ], [ 450, 458, "envelope", "evidence" ] ] }, { "sid": 102, "sent": "The resulting average scattering curve from this model fits the experimental protein scattering intensity, with \u03c7\u2009=\u20091.89 (Supplementary Fig. 3).", "section": "RESULTS", "ner": [ [ 14, 38, "average scattering curve", "evidence" ], [ 85, 105, "scattering intensity", "evidence" ], [ 112, 113, "\u03c7", "evidence" ] ] }, { "sid": 103, "sent": "GH5_4 phylogenetic analysis", "section": "RESULTS", "ner": [ [ 0, 5, "GH5_4", "protein_type" ], [ 6, 27, "phylogenetic analysis", "experimental_method" ] ] }, { "sid": 104, "sent": "After the exclusion of partial sequences and the suppression of highly identical members (higher than 90% identity), 144 sequences containing between 277 and 400 residues were aligned and used to construct a phylogenetic tree (Supplementary Fig. 4A).", "section": "RESULTS", "ner": [ [ 150, 161, "277 and 400", "residue_range" ], [ 176, 183, "aligned", "experimental_method" ], [ 208, 225, "phylogenetic tree", "evidence" ] ] }, { "sid": 105, "sent": "According to PFAM database conserved domain classification, 128 GH5 enzymes have an architecture consisting of an N-terminal catalytic module, a CBM_X2 module and an unknown module of approximately 100 residues at the C-terminus (Supplementary Fig. 4B).", "section": "RESULTS", "ner": [ [ 64, 67, "GH5", "protein_type" ], [ 125, 141, "catalytic module", "structure_element" ], [ 145, 151, "CBM_X2", "structure_element" ] ] }, { "sid": 106, "sent": "Of these, 12 enzymes have an additional CBM1, and 5 have a CBM2 at the N-terminal region.", "section": "RESULTS", "ner": [ [ 40, 44, "CBM1", "structure_element" ], [ 59, 63, "CBM2", "structure_element" ] ] }, { "sid": 107, "sent": "Based on this PFAM architecture and CAZy subfamily classification, all the 144 enzymes (including BlCel5B) belong to the GH5_4 subfamily and group together in the same branch of the phylogenetic tree, evidencing a common ancestor.", "section": "RESULTS", "ner": [ [ 98, 105, "BlCel5B", "protein" ], [ 121, 126, "GH5_4", "protein_type" ], [ 182, 199, "phylogenetic tree", "evidence" ] ] }, { "sid": 108, "sent": "These results support the hypothesis that the enzymes may employ the same mechanism by which ligand binding is mediated by an extensive conformational breathing of the enzyme that involves the large-scale movement of CBM46 around the Ig-like module (CBM_X2) as a structural hinge.", "section": "RESULTS", "ner": [ [ 217, 222, "CBM46", "structure_element" ], [ 234, 248, "Ig-like module", "structure_element" ], [ 250, 256, "CBM_X2", "structure_element" ], [ 263, 279, "structural hinge", "structure_element" ] ] }, { "sid": 109, "sent": "Here, we elucidate the trimodular molecular architecture of the full-length BlCel5B, a member of the GH5_4 subfamily, for which large-scale conformational dynamics appears to play a central role in its enzymatic activity.", "section": "DISCUSS", "ner": [ [ 23, 33, "trimodular", "protein_state" ], [ 64, 75, "full-length", "protein_state" ], [ 76, 83, "BlCel5B", "protein" ], [ 101, 106, "GH5_4", "protein_type" ] ] }, { "sid": 110, "sent": "Full-length BlCel5B is active on both cellulosic and hemicellulosic substrates and auxiliary modules are crucial for its activity.", "section": "DISCUSS", "ner": [ [ 0, 11, "Full-length", "protein_state" ], [ 12, 19, "BlCel5B", "protein" ], [ 23, 29, "active", "protein_state" ], [ 38, 48, "cellulosic", "chemical" ], [ 53, 67, "hemicellulosic", "chemical" ] ] }, { "sid": 111, "sent": "Most carbohydrate-active enzymes are modular and consist of a catalytic domain appended to one or more separate AMs.", "section": "DISCUSS", "ner": [ [ 5, 32, "carbohydrate-active enzymes", "protein_type" ], [ 62, 78, "catalytic domain", "structure_element" ], [ 112, 115, "AMs", "structure_element" ] ] }, { "sid": 112, "sent": "AMs, such as CBMs, typically recognize carbohydrates and target their cognate catalytic domains toward the substrate.", "section": "DISCUSS", "ner": [ [ 0, 3, "AMs", "structure_element" ], [ 13, 17, "CBMs", "structure_element" ], [ 39, 52, "carbohydrates", "chemical" ], [ 78, 95, "catalytic domains", "structure_element" ] ] }, { "sid": 113, "sent": "Because the structural analysis of the protein is challenging if the linkers connecting the structural subunits of the enzyme are long and flexible, the standard approach is to study the domains separately.", "section": "DISCUSS", "ner": [ [ 12, 31, "structural analysis", "experimental_method" ], [ 69, 76, "linkers", "structure_element" ] ] }, { "sid": 114, "sent": "In this work, a combination of protein crystallography, computational molecular dynamics, and SAXS analyses enabled the identification of a new conformational selection-based molecular mechanism that involves GH5 catalytic domain and two AMs in full-length BlCel5B.", "section": "DISCUSS", "ner": [ [ 31, 54, "protein crystallography", "experimental_method" ], [ 56, 88, "computational molecular dynamics", "experimental_method" ], [ 94, 98, "SAXS", "experimental_method" ], [ 209, 212, "GH5", "protein_type" ], [ 213, 229, "catalytic domain", "structure_element" ], [ 238, 241, "AMs", "structure_element" ], [ 245, 256, "full-length", "protein_state" ], [ 257, 264, "BlCel5B", "protein" ] ] }, { "sid": 115, "sent": "We observed that the BlCel5B distal CBM46 is directly involved in shaping the local architecture of the substrate-binding site.", "section": "DISCUSS", "ner": [ [ 21, 28, "BlCel5B", "protein" ], [ 36, 41, "CBM46", "structure_element" ], [ 104, 126, "substrate-binding site", "site" ] ] }, { "sid": 116, "sent": "Although the CD alone appears unable to bind the substrate for catalysis, the AMs exhibit open-close motions that allow the substrate to be captured in a suitable position for hydrolysis.", "section": "DISCUSS", "ner": [ [ 13, 15, "CD", "structure_element" ], [ 16, 21, "alone", "protein_state" ], [ 78, 81, "AMs", "structure_element" ], [ 90, 94, "open", "protein_state" ], [ 95, 100, "close", "protein_state" ] ] }, { "sid": 117, "sent": "Here, we advocate that large-amplitude motions of AMs are crucial for assembling the enzyme into its active conformation, highlighting a new function of CBMs.", "section": "DISCUSS", "ner": [ [ 50, 53, "AMs", "structure_element" ], [ 101, 107, "active", "protein_state" ], [ 153, 157, "CBMs", "structure_element" ] ] }, { "sid": 118, "sent": "This mechanism of substrate binding closely resembles the extended conformational selection model, with the induced-fit mechanism of reaction as its limiting case.", "section": "DISCUSS", "ner": [ [ 58, 66, "extended", "protein_state" ] ] }, { "sid": 119, "sent": "To the best of our knowledge, this enzymatic mechanism has not been proposed previously for any GH.", "section": "DISCUSS", "ner": [ [ 96, 98, "GH", "protein_type" ] ] }, { "sid": 120, "sent": "The CD binding site of BlCel5B is open and relatively flat and is thus barely able to properly hold the substrate in position for catalysis without assistance from the CBM46.", "section": "DISCUSS", "ner": [ [ 4, 19, "CD binding site", "site" ], [ 23, 30, "BlCel5B", "protein" ], [ 168, 173, "CBM46", "structure_element" ] ] }, { "sid": 121, "sent": "In contrast, other GH5s belonging to subfamily 4 listed in the Protein Data Bank exhibit a deep binding cleft or tunnel that can effectively entrap the substrate for catalysis (Fig. 5).", "section": "DISCUSS", "ner": [ [ 19, 23, "GH5s", "protein_type" ], [ 96, 109, "binding cleft", "site" ], [ 113, 119, "tunnel", "site" ] ] }, { "sid": 122, "sent": "Due to the marked interdomain conformational rearrangement observed in our simulations, the CBM46 generates a confined binding site in BlCel5B that resembles the binding site architecture of the other GH5 enzymes that lack AMs.", "section": "DISCUSS", "ner": [ [ 75, 86, "simulations", "experimental_method" ], [ 92, 97, "CBM46", "structure_element" ], [ 119, 131, "binding site", "site" ], [ 135, 142, "BlCel5B", "protein" ], [ 162, 174, "binding site", "site" ], [ 201, 204, "GH5", "protein_type" ], [ 218, 222, "lack", "protein_state" ], [ 223, 226, "AMs", "structure_element" ] ] }, { "sid": 123, "sent": "Thus, BlCel5B appears to have adopted a strategy of CBM46-mediated interactions for proper functioning.", "section": "DISCUSS", "ner": [ [ 6, 13, "BlCel5B", "protein" ], [ 52, 57, "CBM46", "structure_element" ] ] }, { "sid": 124, "sent": "Although the homologous BhCel5B has the same domain architecture of BlCel5B and belongs to the same subfamily (a comparison of the sequence and structure of BlCel5B and BhCel5B is presented in Supplementary Fig. 5), its binding site exhibits important differences that may impact the catalytic mechanism.", "section": "DISCUSS", "ner": [ [ 24, 31, "BhCel5B", "protein" ], [ 68, 75, "BlCel5B", "protein" ], [ 144, 153, "structure", "evidence" ], [ 157, 164, "BlCel5B", "protein" ], [ 169, 176, "BhCel5B", "protein" ], [ 220, 232, "binding site", "site" ] ] }, { "sid": 125, "sent": "The BhCel5B binding site is V-shaped and deeper than the BlCel5B binding site (Figs 5 and 6).", "section": "DISCUSS", "ner": [ [ 4, 11, "BhCel5B", "protein" ], [ 12, 24, "binding site", "site" ], [ 28, 36, "V-shaped", "protein_state" ], [ 57, 64, "BlCel5B", "protein" ], [ 65, 77, "binding site", "site" ] ] }, { "sid": 126, "sent": "This is due to the loop between residues F177 and R185 from BhCel5B (absent in the BlCel5B), which contains residue W181 that forms part of the binding cleft (Fig. 6).", "section": "DISCUSS", "ner": [ [ 19, 23, "loop", "structure_element" ], [ 41, 45, "F177", "residue_name_number" ], [ 50, 54, "R185", "residue_name_number" ], [ 60, 67, "BhCel5B", "protein" ], [ 69, 75, "absent", "protein_state" ], [ 83, 90, "BlCel5B", "protein" ], [ 116, 120, "W181", "residue_name_number" ], [ 144, 157, "binding cleft", "site" ] ] }, { "sid": 127, "sent": "Consistently, although BhCel5B CBM46 is important for \u03b2-1,3-1,4-glucan hydrolysis (BhCel5B is about 60-fold less active without CBM46), the truncated enzyme is completely active against xyloglucan, suggesting that the CBM46, in this case, is necessary for the binding to specific substrates.", "section": "DISCUSS", "ner": [ [ 23, 30, "BhCel5B", "protein" ], [ 31, 36, "CBM46", "structure_element" ], [ 54, 70, "\u03b2-1,3-1,4-glucan", "chemical" ], [ 83, 90, "BhCel5B", "protein" ], [ 113, 119, "active", "protein_state" ], [ 120, 127, "without", "protein_state" ], [ 128, 133, "CBM46", "structure_element" ], [ 140, 149, "truncated", "protein_state" ], [ 171, 177, "active", "protein_state" ], [ 186, 196, "xyloglucan", "chemical" ], [ 218, 223, "CBM46", "structure_element" ] ] }, { "sid": 128, "sent": "A closer inspection of results of the phylogenetic analysis, more specifically of the clade composed by GH5_4 enzymes with trimodular architecture (Supplementary Fig. 4C), reveals subclades whose main characteristic is the varying length of the loop located between residues 161 and 163 (BlCel5B residue numbering).", "section": "DISCUSS", "ner": [ [ 38, 59, "phylogenetic analysis", "experimental_method" ], [ 104, 109, "GH5_4", "protein_type" ], [ 123, 133, "trimodular", "protein_state" ], [ 245, 249, "loop", "structure_element" ], [ 275, 286, "161 and 163", "residue_range" ], [ 288, 295, "BlCel5B", "protein" ] ] }, { "sid": 129, "sent": "Therefore, our results show that BlCel5B represents a smaller group of enzymes that are completely dependent on its AMs for hydrolysis of plant cell wall polysaccharides, and that the underlying mechanism may rely on large-scale interdomain motions.", "section": "DISCUSS", "ner": [ [ 33, 40, "BlCel5B", "protein" ], [ 116, 119, "AMs", "structure_element" ], [ 138, 143, "plant", "taxonomy_domain" ], [ 154, 169, "polysaccharides", "chemical" ] ] }, { "sid": 130, "sent": "The amino acid sequence of the BlCel5B Ig-like module is recognized by BLASTP as belonging to CBM_X2, a poorly described group that has been compared with CBM-like accessory modules without a defined function.", "section": "DISCUSS", "ner": [ [ 31, 38, "BlCel5B", "protein" ], [ 39, 53, "Ig-like module", "structure_element" ], [ 71, 77, "BLASTP", "experimental_method" ], [ 94, 100, "CBM_X2", "structure_element" ], [ 155, 181, "CBM-like accessory modules", "structure_element" ] ] }, { "sid": 131, "sent": "Despite the similarity of BlCel5B Ig-like module to CBMs, it lacks an identifiable aromatic residue-rich carbohydrate-binding site.", "section": "DISCUSS", "ner": [ [ 26, 33, "BlCel5B", "protein" ], [ 34, 48, "Ig-like module", "structure_element" ], [ 52, 56, "CBMs", "structure_element" ], [ 105, 130, "carbohydrate-binding site", "site" ] ] }, { "sid": 132, "sent": "Nonetheless, according to our results, the Ig-like module seems to play an important function as a structural hinge, dynamically holding the CBM46 and CD in positions that are appropriate for enzymatic activity.", "section": "DISCUSS", "ner": [ [ 43, 57, "Ig-like module", "structure_element" ], [ 99, 115, "structural hinge", "structure_element" ], [ 141, 146, "CBM46", "structure_element" ], [ 151, 153, "CD", "structure_element" ] ] }, { "sid": 133, "sent": "Based on the results of our crystallographic, computer simulation, and SAXS structural analyses, as well as site-directed mutagenesis and activity assays, we propose a molecular mechanism for BlCel5B substrate binding, which might apply to other GH5_4 subfamily enzymes that share this tri-modular architecture.", "section": "DISCUSS", "ner": [ [ 28, 65, "crystallographic, computer simulation", "experimental_method" ], [ 71, 95, "SAXS structural analyses", "experimental_method" ], [ 108, 133, "site-directed mutagenesis", "experimental_method" ], [ 138, 153, "activity assays", "experimental_method" ], [ 192, 199, "BlCel5B", "protein" ], [ 246, 251, "GH5_4", "protein_type" ], [ 286, 297, "tri-modular", "structure_element" ] ] }, { "sid": 134, "sent": "BlCel5B can be found in several different conformational states ranging from CBM46/CD closed (or occluded) to extended conformations (Fig. 7).", "section": "DISCUSS", "ner": [ [ 0, 7, "BlCel5B", "protein" ], [ 77, 82, "CBM46", "structure_element" ], [ 83, 85, "CD", "structure_element" ], [ 86, 92, "closed", "protein_state" ], [ 97, 105, "occluded", "protein_state" ], [ 110, 118, "extended", "protein_state" ] ] }, { "sid": 135, "sent": "In extended configurations, the substrate may dock at the shallow substrate binding site of CD in one of the semi-closed conformations of the enzyme; however, its binding is properly stabilized for hydrolysis only with the aid of induced-fit repositioning mediated by CBM46.", "section": "DISCUSS", "ner": [ [ 3, 11, "extended", "protein_state" ], [ 66, 88, "substrate binding site", "site" ], [ 92, 94, "CD", "structure_element" ], [ 109, 120, "semi-closed", "protein_state" ], [ 268, 273, "CBM46", "structure_element" ] ] }, { "sid": 136, "sent": "After cleavage, the intrinsic dynamics of BlCel5B would eventually allow the opening of the active site for product release.", "section": "DISCUSS", "ner": [ [ 42, 49, "BlCel5B", "protein" ], [ 92, 103, "active site", "site" ] ] }, { "sid": 137, "sent": "The proposed mechanism is consistent with our mutagenesis and enzymatic activity assays, which show that the Ig-like module and CBM46 are indispensable for BlCel5B catalytic activity and, together with the CD, form the unique catalytic domain of the enzyme.", "section": "DISCUSS", "ner": [ [ 46, 87, "mutagenesis and enzymatic activity assays", "experimental_method" ], [ 109, 123, "Ig-like module", "structure_element" ], [ 128, 133, "CBM46", "structure_element" ], [ 156, 163, "BlCel5B", "protein" ], [ 206, 208, "CD", "structure_element" ], [ 219, 225, "unique", "protein_state" ], [ 226, 242, "catalytic domain", "structure_element" ] ] }, { "sid": 138, "sent": "These experiments reveal a novel function for CBMs in which they are intimately involved in the assembly of the active site and catalytic process.", "section": "DISCUSS", "ner": [ [ 46, 50, "CBMs", "structure_element" ], [ 112, 123, "active site", "site" ] ] }, { "sid": 139, "sent": "Computer simulations suggest that large-scale motions of the CBM46 and Ig-like domains mediate conformational selection and final induced-fit adjustments to trap the substrate at the active site and promote hydrolysis.", "section": "DISCUSS", "ner": [ [ 0, 20, "Computer simulations", "experimental_method" ], [ 61, 66, "CBM46", "structure_element" ], [ 71, 86, "Ig-like domains", "structure_element" ], [ 183, 194, "active site", "site" ] ] }, { "sid": 140, "sent": "SAXS data support the modeling results, providing compelling evidence for highly mobile domains in solution.", "section": "DISCUSS", "ner": [ [ 0, 4, "SAXS", "experimental_method" ], [ 22, 30, "modeling", "experimental_method" ], [ 74, 87, "highly mobile", "protein_state" ] ] }, { "sid": 141, "sent": "A single spectrum was obtained by averaging four independent spectra generated by 300 laser shots at 60% potency.", "section": "METHODS", "ner": [ [ 9, 17, "spectrum", "evidence" ], [ 61, 68, "spectra", "evidence" ] ] }, { "sid": 142, "sent": "The missing residues were taken from the apo BlCel5B structure after structural alignment using the LovoAlign server.", "section": "METHODS", "ner": [ [ 41, 44, "apo", "protein_state" ] ] }, { "sid": 143, "sent": "BlCel5B-cellooctaose", "section": "METHODS", "ner": [ [ 0, 20, "BlCel5B-cellooctaose", "complex_assembly" ] ] }, { "sid": 144, "sent": "To get a model of the BlCel5B-cellooctaose complex in the closed conformation, we took the configuration after 80\u2009ns of the restrained 200-ns MD simulation as the starting point for a 500-ns-long restrained aMD simulation, in which the CBM46 moved towards the CD in the presence of the harmonically-restrained cellooctaose chain.", "section": "METHODS", "ner": [ [ 22, 42, "BlCel5B-cellooctaose", "complex_assembly" ] ] }, { "sid": 145, "sent": "After this procedure, we released the restraints and propagated the closed BlCel5B-cellooctaose complex for additional 500\u2009ns of conventional, restraint-free MD simulation.", "section": "METHODS", "ner": [ [ 75, 95, "BlCel5B-cellooctaose", "complex_assembly" ] ] }, { "sid": 146, "sent": "Crystal models of BlCel5B.", "section": "FIG", "ner": [ [ 0, 14, "Crystal models", "evidence" ], [ 18, 25, "BlCel5B", "protein" ] ] }, { "sid": 147, "sent": "Complete structure is shown as a cartoon illustration in (a) and a van der Waals surface in (b).", "section": "FIG", "ner": [ [ 9, 18, "structure", "evidence" ] ] }, { "sid": 148, "sent": "The CD module (red) has a typical TIM-barrel fold, and its substrate-binding site is adjacent to CBM46 (blue).", "section": "FIG", "ner": [ [ 4, 6, "CD", "structure_element" ], [ 34, 49, "TIM-barrel fold", "structure_element" ], [ 59, 81, "substrate-binding site", "site" ], [ 97, 102, "CBM46", "structure_element" ] ] }, { "sid": 149, "sent": "Despite the proximity of the binding site in the crystallographic model, the CBM46 residues W479 and W481 are distant from the substrate cellotetraose (yellow).", "section": "FIG", "ner": [ [ 29, 41, "binding site", "site" ], [ 77, 82, "CBM46", "structure_element" ], [ 92, 96, "W479", "residue_name_number" ], [ 101, 105, "W481", "residue_name_number" ], [ 137, 150, "cellotetraose", "chemical" ] ] }, { "sid": 150, "sent": "The Ig-like domain (green) has a lateral position, serving as a connector between the CD and CBM46. (c) A superposition of the Ig-like domain and CBM46 illustrates their structural similarity, with most of the structural differences present in the loop highlighted by a red circle. (d) Cellotetraose occupies subsites -1 to -3 and is primarily coordinated by the residues represented in gray.", "section": "FIG", "ner": [ [ 4, 18, "Ig-like domain", "structure_element" ], [ 86, 88, "CD", "structure_element" ], [ 93, 98, "CBM46", "structure_element" ], [ 106, 119, "superposition", "experimental_method" ], [ 127, 141, "Ig-like domain", "structure_element" ], [ 146, 151, "CBM46", "structure_element" ], [ 248, 252, "loop", "structure_element" ], [ 286, 299, "Cellotetraose", "chemical" ], [ 309, 326, "subsites -1 to -3", "site" ], [ 344, 355, "coordinated", "bond_interaction" ] ] }, { "sid": 151, "sent": "BlCel5B enzymatic activity characterization.", "section": "FIG", "ner": [ [ 0, 7, "BlCel5B", "protein" ], [ 8, 43, "enzymatic activity characterization", "experimental_method" ] ] }, { "sid": 152, "sent": "(a) MALDI/TOF-MS spectra of the products released after incubation of BlCel5B and its two deletion constructs (\u0394CBM46 and \u0394Ig-CBM46) with the substrate cellopentaose (C5).", "section": "FIG", "ner": [ [ 4, 16, "MALDI/TOF-MS", "experimental_method" ], [ 17, 24, "spectra", "evidence" ], [ 70, 77, "BlCel5B", "protein" ], [ 90, 109, "deletion constructs", "experimental_method" ], [ 111, 117, "\u0394CBM46", "mutant" ], [ 122, 131, "\u0394Ig-CBM46", "mutant" ], [ 152, 165, "cellopentaose", "chemical" ], [ 167, 169, "C5", "chemical" ] ] }, { "sid": 153, "sent": "The first three spectra show the substrate, enzyme and buffer controls.", "section": "FIG", "ner": [ [ 16, 23, "spectra", "evidence" ] ] }, { "sid": 154, "sent": "The forth spectrum reveals that full length BlCel5B is capable of enzymatic hydrolysis of C5 into smaller oligosaccharides such as C4, C3 and C2.", "section": "FIG", "ner": [ [ 10, 18, "spectrum", "evidence" ], [ 32, 43, "full length", "protein_state" ], [ 44, 51, "BlCel5B", "protein" ], [ 90, 92, "C5", "chemical" ], [ 106, 122, "oligosaccharides", "chemical" ], [ 131, 133, "C4", "chemical" ], [ 135, 137, "C3", "chemical" ], [ 142, 144, "C2", "chemical" ] ] }, { "sid": 155, "sent": "The last two spectra show that the C-terminal deletions eliminate the enzyme activity.", "section": "FIG", "ner": [ [ 13, 20, "spectra", "evidence" ], [ 56, 85, "eliminate the enzyme activity", "protein_state" ] ] }, { "sid": 156, "sent": "BlCel5B activities on CMC as functions of pH and temperature are shown in (b) and (c), respectively.", "section": "FIG", "ner": [ [ 0, 7, "BlCel5B", "protein" ], [ 22, 25, "CMC", "chemical" ] ] }, { "sid": 157, "sent": "(d) Michaelis-Menten curve using CMC as a substrate.", "section": "FIG", "ner": [ [ 4, 26, "Michaelis-Menten curve", "evidence" ], [ 33, 36, "CMC", "chemical" ] ] }, { "sid": 158, "sent": "Open-close transitions of BlCel5B.", "section": "FIG", "ner": [ [ 0, 4, "Open", "protein_state" ], [ 5, 10, "close", "protein_state" ], [ 26, 33, "BlCel5B", "protein" ] ] }, { "sid": 159, "sent": "(a) BlCel5B in the absence of substrate and (b) in the presence of cellooctaose, as observed in our aMD simulations.", "section": "FIG", "ner": [ [ 4, 11, "BlCel5B", "protein" ], [ 19, 29, "absence of", "protein_state" ], [ 55, 66, "presence of", "protein_state" ], [ 67, 79, "cellooctaose", "chemical" ], [ 100, 115, "aMD simulations", "experimental_method" ] ] }, { "sid": 160, "sent": "The distance between the \u03b1 carbon of residues I120 (CD) and E477 (CBM46), illustrated as spheres in (a), is plotted in (c), revealing a transition by the decrease in the distance from 40\u2009\u00c5 to 7\u2009\u00c5 (substrate-free) or 20\u2009\u00c5 (in presence of cellooctaose).", "section": "FIG", "ner": [ [ 4, 12, "distance", "evidence" ], [ 46, 50, "I120", "residue_name_number" ], [ 52, 54, "CD", "structure_element" ], [ 60, 64, "E477", "residue_name_number" ], [ 66, 71, "CBM46", "structure_element" ], [ 170, 178, "distance", "evidence" ], [ 197, 211, "substrate-free", "protein_state" ], [ 225, 236, "presence of", "protein_state" ], [ 237, 249, "cellooctaose", "chemical" ] ] }, { "sid": 161, "sent": "For the substrate-free enzyme, the red line refers to a 1\u2009\u03bcs-long aMD; for the BlCel5B-cellooctaose complex, the first 500\u2009ns refers to aMD (in blue) and the second 500\u2009ns to conventional MD (in turquoise).", "section": "FIG", "ner": [ [ 8, 22, "substrate-free", "protein_state" ], [ 66, 69, "aMD", "experimental_method" ], [ 79, 99, "BlCel5B-cellooctaose", "complex_assembly" ], [ 136, 139, "aMD", "experimental_method" ], [ 188, 190, "MD", "experimental_method" ] ] }, { "sid": 162, "sent": "(d) A snapshot of the BlCel5B-cellooctaose complex, highlighting the tryptophan residues that interact with the glucan chain in subsites \u22124 to +4.", "section": "FIG", "ner": [ [ 22, 42, "BlCel5B-cellooctaose", "complex_assembly" ], [ 69, 79, "tryptophan", "residue_name" ], [ 112, 118, "glucan", "chemical" ], [ 128, 145, "subsites \u22124 to +4", "site" ] ] }, { "sid": 163, "sent": "Residues W479 and W481 belong to CBM46 and only become available for substrate interactions in the closed configuration of BlCel5B.", "section": "FIG", "ner": [ [ 9, 13, "W479", "residue_name_number" ], [ 18, 22, "W481", "residue_name_number" ], [ 33, 38, "CBM46", "structure_element" ], [ 99, 105, "closed", "protein_state" ], [ 123, 130, "BlCel5B", "protein" ] ] }, { "sid": 164, "sent": "Large-scale movements of BlCel5B modules and superposition of their representative conformations with the SAXS envelope.", "section": "FIG", "ner": [ [ 25, 32, "BlCel5B", "protein" ], [ 45, 58, "superposition", "experimental_method" ], [ 106, 110, "SAXS", "experimental_method" ], [ 111, 119, "envelope", "evidence" ] ] }, { "sid": 165, "sent": "(a) BlCel5B structure showing the distance between the backbone beads of residues I120 and E477, which are centrally located in CD and CBM46, respectively, as a metric for the relative disposition between the two domains. (b) Time history of the I120-E477 distance computed using CG-MD simulations.", "section": "FIG", "ner": [ [ 4, 11, "BlCel5B", "protein" ], [ 12, 21, "structure", "evidence" ], [ 34, 42, "distance", "evidence" ], [ 82, 86, "I120", "residue_name_number" ], [ 91, 95, "E477", "residue_name_number" ], [ 128, 130, "CD", "structure_element" ], [ 135, 140, "CBM46", "structure_element" ], [ 246, 250, "I120", "residue_name_number" ], [ 251, 255, "E477", "residue_name_number" ], [ 256, 264, "distance", "evidence" ], [ 280, 297, "CG-MD simulations", "experimental_method" ] ] }, { "sid": 166, "sent": "Different colors separated by vertical lines correspond to independent simulations of approximately 120\u2009\u03bcs. (c) The distance distribution indicates three major peaks: closed or occluded CBM46/CD conformations (I); semi-open (II), which is similar to the crystallographic structure; and extended conformers (III).", "section": "FIG", "ner": [ [ 71, 82, "simulations", "experimental_method" ], [ 116, 137, "distance distribution", "evidence" ], [ 167, 173, "closed", "protein_state" ], [ 177, 185, "occluded", "protein_state" ], [ 186, 191, "CBM46", "structure_element" ], [ 192, 194, "CD", "structure_element" ], [ 214, 223, "semi-open", "protein_state" ], [ 254, 280, "crystallographic structure", "evidence" ], [ 286, 294, "extended", "protein_state" ] ] }, { "sid": 167, "sent": "(d) Superimposition of the three representative molecular conformations of BlCel5B with the SAXS model. (e) Average structures obtained from the simulation segments corresponding to population groups I-III, which are individually superposed on the SAXS envelope.", "section": "FIG", "ner": [ [ 4, 19, "Superimposition", "experimental_method" ], [ 75, 82, "BlCel5B", "protein" ], [ 92, 96, "SAXS", "experimental_method" ], [ 97, 102, "model", "evidence" ], [ 116, 126, "structures", "evidence" ], [ 145, 155, "simulation", "experimental_method" ], [ 230, 240, "superposed", "experimental_method" ], [ 248, 252, "SAXS", "experimental_method" ], [ 253, 261, "envelope", "evidence" ] ] }, { "sid": 168, "sent": "Comparison of the binding site shape of GH5_4 enzymes available on the Protein Data Bank.", "section": "FIG", "ner": [ [ 0, 10, "Comparison", "experimental_method" ], [ 18, 30, "binding site", "site" ], [ 40, 45, "GH5_4", "protein_type" ] ] }, { "sid": 169, "sent": "(a) BlCel5B in the crystallographic and closed configuration; (b) Bacillus halodurans Cel5B (BhCel5B) (PDB id: 4V2X) (c) Piromyces rhizinflata GH5 endoglucanase (PDB id: 3AYR); (d) Clostridium cellulolyticum GH5 endoglucanase (PDB id: 1EDG); (e) Clostridium cellulovorans GH5 endoglucanase (PDB id: 3NDY); (f) Bacteroides ovatus GH5 xyloglucanase (PDB id: 3ZMR); (g) Paenibacillus pabuli GH5 xyloglucanase (PDB id: 2JEP); (h) Prevotella bryantii GH5 endoglucanase (PDB id: 3VDH); (i) Ruminiclostridium thermocellum multifunctional GH5 cellulase, xylanase and mannase (PDB id: 4IM4); (j) Bacteroidetes bacterium AC2a endocellulase (PDB id: 4YHE).", "section": "FIG", "ner": [ [ 4, 11, "BlCel5B", "protein" ], [ 19, 35, "crystallographic", "experimental_method" ], [ 40, 46, "closed", "protein_state" ], [ 66, 85, "Bacillus halodurans", "species" ], [ 86, 91, "Cel5B", "protein" ], [ 93, 100, "BhCel5B", "protein" ], [ 121, 142, "Piromyces rhizinflata", "species" ], [ 143, 146, "GH5", "protein_type" ], [ 147, 160, "endoglucanase", "protein_type" ], [ 181, 207, "Clostridium cellulolyticum", "species" ], [ 208, 211, "GH5", "protein_type" ], [ 212, 225, "endoglucanase", "protein_type" ], [ 246, 271, "Clostridium cellulovorans", "species" ], [ 272, 275, "GH5", "protein_type" ], [ 276, 289, "endoglucanase", "protein_type" ], [ 310, 328, "Bacteroides ovatus", "species" ], [ 329, 332, "GH5", "protein_type" ], [ 333, 346, "xyloglucanase", "protein_type" ], [ 367, 387, "Paenibacillus pabuli", "species" ], [ 388, 391, "GH5", "protein_type" ], [ 392, 405, "xyloglucanase", "protein_type" ], [ 426, 445, "Prevotella bryantii", "species" ], [ 446, 449, "GH5", "protein_type" ], [ 450, 463, "endoglucanase", "protein_type" ], [ 484, 514, "Ruminiclostridium thermocellum", "species" ], [ 531, 534, "GH5", "protein_type" ], [ 535, 544, "cellulase", "protein_type" ], [ 546, 554, "xylanase", "protein_type" ], [ 559, 566, "mannase", "protein_type" ], [ 587, 610, "Bacteroidetes bacterium", "taxonomy_domain" ], [ 611, 615, "AC2a", "protein_type" ], [ 616, 629, "endocellulase", "protein_type" ] ] }, { "sid": 170, "sent": "Comparison of the binding cleft of the BlCel5B and BhCel5B.", "section": "FIG", "ner": [ [ 0, 10, "Comparison", "experimental_method" ], [ 18, 31, "binding cleft", "site" ], [ 39, 46, "BlCel5B", "protein" ], [ 51, 58, "BhCel5B", "protein" ] ] }, { "sid": 171, "sent": "The main difference between BlCel5B and BhCel5B is that the latter exhibits a deeper cleft due to the presence of residue W181 in the loop between F177 and R185.", "section": "FIG", "ner": [ [ 28, 35, "BlCel5B", "protein" ], [ 40, 47, "BhCel5B", "protein" ], [ 85, 90, "cleft", "site" ], [ 102, 113, "presence of", "protein_state" ], [ 122, 126, "W181", "residue_name_number" ], [ 134, 138, "loop", "structure_element" ], [ 147, 151, "F177", "residue_name_number" ], [ 156, 160, "R185", "residue_name_number" ] ] }, { "sid": 172, "sent": "We conjecture that this difference in the binding site architecture relates to the importance that the CBM46 plays in the BlCel5B enzymatic mechanism.", "section": "FIG", "ner": [ [ 42, 54, "binding site", "site" ], [ 103, 108, "CBM46", "structure_element" ], [ 122, 129, "BlCel5B", "protein" ] ] }, { "sid": 173, "sent": "Proposed molecular mechanism of BlCel5B conformational selection.", "section": "FIG", "ner": [ [ 32, 39, "BlCel5B", "protein" ] ] }, { "sid": 174, "sent": "As suggested by the simulations and SAXS data, BlCel5B spans multiple conformations ranging from closed to extended CBM46/CD states.", "section": "FIG", "ner": [ [ 20, 31, "simulations", "experimental_method" ], [ 36, 40, "SAXS", "experimental_method" ], [ 47, 54, "BlCel5B", "protein" ], [ 97, 103, "closed", "protein_state" ], [ 107, 115, "extended", "protein_state" ], [ 116, 121, "CBM46", "structure_element" ], [ 122, 124, "CD", "structure_element" ] ] }, { "sid": 175, "sent": "In a given open state, the substrate may reach the active site and become entrapped by the capping of CBM46 onto CD and induced-fit conformational adjustments.", "section": "FIG", "ner": [ [ 11, 15, "open", "protein_state" ], [ 51, 62, "active site", "site" ], [ 102, 107, "CBM46", "structure_element" ], [ 113, 115, "CD", "structure_element" ] ] }, { "sid": 176, "sent": "After hydrolysis, the reaction product is released to yield apo-BlCel5B, which becomes ready for a new cycle.", "section": "FIG", "ner": [ [ 60, 63, "apo", "protein_state" ], [ 64, 71, "BlCel5B", "protein" ] ] }, { "sid": 177, "sent": "Activity of BlCel5B constructs against tested substrates.", "section": "TABLE", "ner": [ [ 12, 19, "BlCel5B", "protein" ] ] }, { "sid": 178, "sent": "Substrate (1%)\tRelative Activity (%)\t \tWT*\tW479A\tW481A\t\u0394CBM46\t\u0394Ig-CBM46\t \t\u03b2-glucan\t100\t79.1\t63.6\tnd\tnd\t \tCMC\t25.5\t12.2\t2.4\tnd\tnd\t \tLichenan\t52.4\t41\t28.6\tnd\tnd\t \tXyloglucan\t45.2\t41.2\t30.8\tnd\tnd\t \tAzo-Avicel\tnd**\tnd\tnd\tnd\tnd\t \tArabinoxylan\tnd\tnd\tnd\tnd\tnd\t \tGalactomannan\tnd\tnd\tnd\tnd\tnd\t \t1,4-\u03b2-mannan\tnd\tnd\tnd\tnd\tnd\t \t", "section": "TABLE", "ner": [ [ 39, 41, "WT", "protein_state" ], [ 43, 48, "W479A", "mutant" ], [ 49, 54, "W481A", "mutant" ], [ 55, 61, "\u0394CBM46", "mutant" ], [ 62, 71, "\u0394Ig-CBM46", "mutant" ], [ 74, 82, "\u03b2-glucan", "chemical" ], [ 105, 108, "CMC", "chemical" ], [ 131, 139, "Lichenan", "chemical" ], [ 161, 171, "Xyloglucan", "chemical" ], [ 195, 205, "Azo-Avicel", "chemical" ], [ 225, 237, "Arabinoxylan", "chemical" ], [ 255, 268, "Galactomannan", "chemical" ], [ 286, 298, "1,4-\u03b2-mannan", "chemical" ] ] }, { "sid": 179, "sent": "*WT\u2009=\u2009wild type.", "section": "TABLE", "ner": [ [ 1, 3, "WT", "protein_state" ], [ 6, 15, "wild type", "protein_state" ] ] } ] }, "PMC5012862": { "annotations": [ { "sid": 0, "sent": "Structural characterization of encapsulated ferritin provides insight into iron storage in bacterial nanocompartments", "section": "TITLE", "ner": [ [ 0, 27, "Structural characterization", "experimental_method" ], [ 31, 43, "encapsulated", "protein_state" ], [ 44, 52, "ferritin", "protein_type" ], [ 75, 79, "iron", "chemical" ], [ 91, 100, "bacterial", "taxonomy_domain" ], [ 101, 117, "nanocompartments", "complex_assembly" ] ] }, { "sid": 1, "sent": "Ferritins are ubiquitous proteins that oxidise and store iron within a protein shell to protect cells from oxidative damage.", "section": "ABSTRACT", "ner": [ [ 0, 9, "Ferritins", "protein_type" ], [ 57, 61, "iron", "chemical" ], [ 79, 84, "shell", "structure_element" ] ] }, { "sid": 2, "sent": "We have characterized the structure and function of a new member of the ferritin superfamily that is sequestered within an encapsulin capsid.", "section": "ABSTRACT", "ner": [ [ 26, 35, "structure", "evidence" ], [ 72, 80, "ferritin", "protein_type" ], [ 123, 133, "encapsulin", "protein" ] ] }, { "sid": 3, "sent": "We show that this encapsulated ferritin (EncFtn) has two main alpha helices, which assemble in a metal dependent manner to form a ferroxidase center at a dimer interface.", "section": "ABSTRACT", "ner": [ [ 18, 30, "encapsulated", "protein_state" ], [ 31, 39, "ferritin", "protein_type" ], [ 41, 47, "EncFtn", "protein" ], [ 57, 75, "main alpha helices", "structure_element" ], [ 97, 112, "metal dependent", "protein_state" ], [ 130, 148, "ferroxidase center", "site" ], [ 154, 169, "dimer interface", "site" ] ] }, { "sid": 4, "sent": "EncFtn adopts an open decameric structure that is topologically distinct from other ferritins.", "section": "ABSTRACT", "ner": [ [ 0, 6, "EncFtn", "protein" ], [ 17, 21, "open", "protein_state" ], [ 22, 31, "decameric", "oligomeric_state" ], [ 32, 41, "structure", "evidence" ], [ 84, 93, "ferritins", "protein_type" ] ] }, { "sid": 5, "sent": "While EncFtn acts as a ferroxidase, it cannot mineralize iron.", "section": "ABSTRACT", "ner": [ [ 6, 12, "EncFtn", "protein" ], [ 23, 34, "ferroxidase", "protein_type" ], [ 57, 61, "iron", "chemical" ] ] }, { "sid": 6, "sent": "Conversely, the encapsulin shell associates with iron, but is not enzymatically active, and we demonstrate that EncFtn must be housed within the encapsulin for iron storage.", "section": "ABSTRACT", "ner": [ [ 16, 26, "encapsulin", "protein" ], [ 27, 32, "shell", "structure_element" ], [ 49, 53, "iron", "chemical" ], [ 62, 86, "not enzymatically active", "protein_state" ], [ 112, 118, "EncFtn", "protein" ], [ 145, 155, "encapsulin", "protein" ], [ 160, 164, "iron", "chemical" ] ] }, { "sid": 7, "sent": "This encapsulin nanocompartment is widely distributed in bacteria and archaea and represents a distinct class of iron storage system, where the oxidation and mineralization of iron are distributed between two proteins.", "section": "ABSTRACT", "ner": [ [ 5, 15, "encapsulin", "protein" ], [ 16, 31, "nanocompartment", "complex_assembly" ], [ 57, 65, "bacteria", "taxonomy_domain" ], [ 70, 77, "archaea", "taxonomy_domain" ], [ 113, 117, "iron", "chemical" ], [ 176, 180, "iron", "chemical" ] ] }, { "sid": 8, "sent": "Iron is essential for life as it is a key component of many different enzymes that participate in processes such as energy production and metabolism.", "section": "ABSTRACT", "ner": [ [ 0, 4, "Iron", "chemical" ] ] }, { "sid": 9, "sent": "However, iron can also be highly toxic to cells because it readily reacts with oxygen.", "section": "ABSTRACT", "ner": [ [ 9, 13, "iron", "chemical" ], [ 79, 85, "oxygen", "chemical" ] ] }, { "sid": 10, "sent": "To balance the cell\u2019s need for iron against its potential damaging effects, organisms have evolved iron storage proteins known as ferritins that form cage-like structures.", "section": "ABSTRACT", "ner": [ [ 31, 35, "iron", "chemical" ], [ 99, 120, "iron storage proteins", "protein_type" ], [ 130, 139, "ferritins", "protein_type" ], [ 150, 170, "cage-like structures", "structure_element" ] ] }, { "sid": 11, "sent": "The ferritins convert iron into a less reactive form that is mineralised and safely stored in the central cavity of the ferritin cage and is available for cells when they need it.", "section": "ABSTRACT", "ner": [ [ 4, 13, "ferritins", "protein_type" ], [ 22, 26, "iron", "chemical" ], [ 98, 112, "central cavity", "site" ], [ 120, 128, "ferritin", "protein_type" ] ] }, { "sid": 12, "sent": "Recently, a new family of ferritins known as encapsulated ferritins have been found in some microorganisms.", "section": "ABSTRACT", "ner": [ [ 26, 35, "ferritins", "protein_type" ], [ 45, 57, "encapsulated", "protein_state" ], [ 58, 67, "ferritins", "protein_type" ], [ 92, 106, "microorganisms", "taxonomy_domain" ] ] }, { "sid": 13, "sent": "These ferritins are found in bacterial genomes with a gene that codes for a protein cage called an encapsulin.", "section": "ABSTRACT", "ner": [ [ 6, 15, "ferritins", "protein_type" ], [ 29, 38, "bacterial", "taxonomy_domain" ], [ 99, 109, "encapsulin", "protein" ] ] }, { "sid": 14, "sent": "Although the structure of the encapsulin cage is known to look like the shell of a virus, the structure that the encapsulated ferritin itself forms is not known.", "section": "ABSTRACT", "ner": [ [ 13, 22, "structure", "evidence" ], [ 30, 40, "encapsulin", "protein" ], [ 72, 77, "shell", "structure_element" ], [ 83, 88, "virus", "taxonomy_domain" ], [ 94, 103, "structure", "evidence" ], [ 113, 125, "encapsulated", "protein_state" ], [ 126, 134, "ferritin", "protein_type" ] ] }, { "sid": 15, "sent": "It is also not clear how encapsulin and the encapsulated ferritin work together to store iron.", "section": "ABSTRACT", "ner": [ [ 25, 35, "encapsulin", "protein" ], [ 44, 56, "encapsulated", "protein_state" ], [ 57, 65, "ferritin", "protein_type" ], [ 89, 93, "iron", "chemical" ] ] }, { "sid": 16, "sent": "He et al. have now used the techniques of X-ray crystallography and mass spectrometry to determine the structure of the encapsulated ferritin found in some bacteria.", "section": "ABSTRACT", "ner": [ [ 42, 63, "X-ray crystallography", "experimental_method" ], [ 68, 85, "mass spectrometry", "experimental_method" ], [ 103, 112, "structure", "evidence" ], [ 120, 132, "encapsulated", "protein_state" ], [ 133, 141, "ferritin", "protein_type" ], [ 156, 164, "bacteria", "taxonomy_domain" ] ] }, { "sid": 17, "sent": "The encapsulated ferritin forms a ring-shaped doughnut in which ten subunits of ferritin are arranged in a ring; this is totally different from the enclosed cages that other ferritins form.", "section": "ABSTRACT", "ner": [ [ 4, 16, "encapsulated", "protein_state" ], [ 17, 25, "ferritin", "protein_type" ], [ 34, 45, "ring-shaped", "structure_element" ], [ 46, 54, "doughnut", "structure_element" ], [ 68, 76, "subunits", "structure_element" ], [ 80, 88, "ferritin", "protein_type" ], [ 107, 111, "ring", "structure_element" ], [ 157, 162, "cages", "structure_element" ], [ 174, 183, "ferritins", "protein_type" ] ] }, { "sid": 18, "sent": "Biochemical studies revealed that the encapsulated ferritin is able to convert iron into a less reactive form, but it cannot store iron on its own since it does not form a cage.", "section": "ABSTRACT", "ner": [ [ 0, 19, "Biochemical studies", "experimental_method" ], [ 38, 50, "encapsulated", "protein_state" ], [ 51, 59, "ferritin", "protein_type" ], [ 79, 83, "iron", "chemical" ], [ 131, 135, "iron", "chemical" ] ] }, { "sid": 19, "sent": "Thus, the encapsulated ferritin needs to be housed within the encapsulin cage to store iron.", "section": "ABSTRACT", "ner": [ [ 10, 22, "encapsulated", "protein_state" ], [ 23, 31, "ferritin", "protein_type" ], [ 62, 72, "encapsulin", "protein" ], [ 87, 91, "iron", "chemical" ] ] }, { "sid": 20, "sent": "Further work is needed to investigate how iron moves into the encapsulin cage to reach the ferritin proteins.", "section": "ABSTRACT", "ner": [ [ 42, 46, "iron", "chemical" ], [ 62, 72, "encapsulin", "protein" ], [ 91, 99, "ferritin", "protein_type" ] ] }, { "sid": 21, "sent": "Some organisms have both standard ferritin cages and encapsulated ferritins; why this is the case also remains to be discovered.", "section": "ABSTRACT", "ner": [ [ 34, 42, "ferritin", "protein_type" ], [ 53, 65, "encapsulated", "protein_state" ], [ 66, 75, "ferritins", "protein_type" ] ] }, { "sid": 22, "sent": "Encapsulin nanocompartments are a family of proteinaceous metabolic compartments that are widely distributed in bacteria and archaea.", "section": "INTRO", "ner": [ [ 0, 10, "Encapsulin", "protein_type" ], [ 11, 27, "nanocompartments", "complex_assembly" ], [ 112, 120, "bacteria", "taxonomy_domain" ], [ 125, 132, "archaea", "taxonomy_domain" ] ] }, { "sid": 23, "sent": "They share a common architecture, comprising an icosahedral shell formed by the oligomeric assembly of a protein, encapsulin, that is structurally related to the HK97 bacteriophage capsid protein gp5.", "section": "INTRO", "ner": [ [ 48, 59, "icosahedral", "protein_state" ], [ 60, 65, "shell", "structure_element" ], [ 114, 124, "encapsulin", "protein_type" ], [ 162, 180, "HK97 bacteriophage", "taxonomy_domain" ], [ 196, 199, "gp5", "protein" ] ] }, { "sid": 24, "sent": "Gp5 is known to assemble as a 66 nm diameter icosahedral shell of 420 subunits.", "section": "INTRO", "ner": [ [ 0, 3, "Gp5", "protein" ], [ 45, 56, "icosahedral", "protein_state" ], [ 57, 62, "shell", "structure_element" ], [ 70, 78, "subunits", "structure_element" ] ] }, { "sid": 25, "sent": "In contrast, both the Pyrococcus furiosus and\u00a0Myxococcus xanthus encapsulin shell-proteins form 32 nm icosahedra with 180 subunits; while the Thermotoga maritima encapsulin is smaller still with a 25 nm, 60-subunit icosahedron.", "section": "INTRO", "ner": [ [ 22, 41, "Pyrococcus furiosus", "species" ], [ 46, 64, "Myxococcus xanthus", "species" ], [ 65, 75, "encapsulin", "protein" ], [ 76, 81, "shell", "structure_element" ], [ 102, 112, "icosahedra", "structure_element" ], [ 122, 130, "subunits", "structure_element" ], [ 142, 161, "Thermotoga maritima", "species" ], [ 162, 172, "encapsulin", "protein" ], [ 215, 226, "icosahedron", "structure_element" ] ] }, { "sid": 26, "sent": "The high structural similarity of the encapsulin shell-proteins to gp5 suggests a common evolutionary origin for these proteins.", "section": "INTRO", "ner": [ [ 38, 48, "encapsulin", "protein_type" ], [ 49, 54, "shell", "structure_element" ], [ 67, 70, "gp5", "protein" ] ] }, { "sid": 27, "sent": "The genes encoding encapsulin proteins are found downstream of genes for dye-dependent peroxidase (DyP) family enzymes, or encapsulin-associated ferritins (EncFtn).", "section": "INTRO", "ner": [ [ 19, 29, "encapsulin", "protein_type" ], [ 73, 97, "dye-dependent peroxidase", "protein_type" ], [ 99, 102, "DyP", "protein_type" ], [ 123, 154, "encapsulin-associated ferritins", "protein_type" ], [ 156, 162, "EncFtn", "protein_type" ] ] }, { "sid": 28, "sent": "Enzymes in the DyP family are active against polyphenolic compounds such as azo dyes and lignin breakdown products; although their physiological function and natural substrates are not known.", "section": "INTRO", "ner": [ [ 15, 25, "DyP family", "protein_type" ] ] }, { "sid": 29, "sent": "Ferritin family proteins are found in all kingdoms and have a wide range of activities, including ribonucleotide reductase, protecting DNA from oxidative damage, and iron storage.", "section": "INTRO", "ner": [ [ 0, 8, "Ferritin", "protein_type" ], [ 42, 50, "kingdoms", "taxonomy_domain" ], [ 98, 122, "ribonucleotide reductase", "protein_type" ], [ 166, 170, "iron", "chemical" ] ] }, { "sid": 30, "sent": "The classical iron storage ferritin nanocages are found in all kingdoms and are essential in eukaryotes; they play a central role in iron homeostasis, where they protect the cell from toxic free Fe2+ by oxidizing it and storing the resulting Fe3+ as ferrihydrite minerals within their central cavity.", "section": "INTRO", "ner": [ [ 4, 13, "classical", "protein_state" ], [ 14, 45, "iron storage ferritin nanocages", "complex_assembly" ], [ 63, 71, "kingdoms", "taxonomy_domain" ], [ 93, 103, "eukaryotes", "taxonomy_domain" ], [ 133, 137, "iron", "chemical" ], [ 195, 199, "Fe2+", "chemical" ], [ 242, 246, "Fe3+", "chemical" ], [ 250, 262, "ferrihydrite", "chemical" ], [ 285, 299, "central cavity", "site" ] ] }, { "sid": 31, "sent": "The encapsulin-associated enzymes are sequestered within the icosahedral shell through interactions between the shell\u2019s inner surface and a short localization sequence (Gly-Ser-Leu-Lys) appended to their C-termini.", "section": "INTRO", "ner": [ [ 4, 14, "encapsulin", "protein_type" ], [ 61, 72, "icosahedral", "protein_state" ], [ 73, 78, "shell", "structure_element" ], [ 112, 117, "shell", "structure_element" ], [ 140, 167, "short localization sequence", "structure_element" ], [ 169, 184, "Gly-Ser-Leu-Lys", "structure_element" ] ] }, { "sid": 32, "sent": "This motif is well-conserved, and the\u00a0addition of this sequence to heterologous proteins is sufficient to direct them to the interior of encapsulins.", "section": "INTRO", "ner": [ [ 0, 10, "This motif", "structure_element" ], [ 14, 28, "well-conserved", "protein_state" ], [ 137, 148, "encapsulins", "protein_type" ] ] }, { "sid": 33, "sent": "A recent study of the Myxococcus xanthus encapsulin showed that it sequesters a number of different EncFtn proteins and acts as an \u2018iron-megastore\u2019 to protect these bacteria from oxidative stress.", "section": "INTRO", "ner": [ [ 22, 40, "Myxococcus xanthus", "species" ], [ 41, 51, "encapsulin", "protein" ], [ 100, 106, "EncFtn", "protein_type" ], [ 132, 136, "iron", "chemical" ], [ 165, 173, "bacteria", "taxonomy_domain" ] ] }, { "sid": 34, "sent": "At 32 nm in diameter, it is much larger than other members of the ferritin superfamily, such as the 12 nm 24-subunit classical ferritin nanocage and the 8 nm 12-subunit Dps (DNA-binding protein from starved cells) complex; and is thus capable of sequestering up to ten times more iron than these ferritins.", "section": "INTRO", "ner": [ [ 66, 74, "ferritin", "protein_type" ], [ 117, 126, "classical", "protein_state" ], [ 127, 135, "ferritin", "protein_type" ], [ 136, 144, "nanocage", "complex_assembly" ], [ 169, 172, "Dps", "protein_type" ], [ 174, 193, "DNA-binding protein", "protein_type" ], [ 280, 284, "iron", "chemical" ], [ 296, 305, "ferritins", "protein_type" ] ] }, { "sid": 35, "sent": "The primary sequences of EncFtn proteins have Glu-X-X-His metal coordination sites, which are shared features of the ferritin family proteins.", "section": "INTRO", "ner": [ [ 25, 31, "EncFtn", "protein_type" ], [ 46, 57, "Glu-X-X-His", "structure_element" ], [ 58, 82, "metal coordination sites", "site" ], [ 117, 125, "ferritin", "protein_type" ] ] }, { "sid": 36, "sent": "Secondary structure prediction identifies two major \u03b1-helical regions in these proteins; this is in contrast to other members of the ferritin superfamily, which have four major \u03b1-helices (Supplementary file 1).", "section": "INTRO", "ner": [ [ 0, 30, "Secondary structure prediction", "experimental_method" ], [ 46, 69, "major \u03b1-helical regions", "structure_element" ], [ 133, 141, "ferritin", "protein_type" ], [ 171, 186, "major \u03b1-helices", "structure_element" ] ] }, { "sid": 37, "sent": "The \u2018half-ferritin\u2019 primary sequence of the EncFtn family and their association with encapsulin nanocompartments suggests a distinct biochemical and structural organization to other ferritin family proteins.", "section": "INTRO", "ner": [ [ 10, 18, "ferritin", "protein_type" ], [ 44, 50, "EncFtn", "protein_type" ], [ 85, 95, "encapsulin", "protein" ], [ 96, 112, "nanocompartments", "complex_assembly" ], [ 182, 190, "ferritin", "protein_type" ] ] }, { "sid": 38, "sent": "The Rhodospirillum\u00a0rubrum EncFtn protein (Rru_A0973) shares 33% protein sequence identity with the M. xanthus (MXAN_4464), 53% with the T. maritima (Tmari_0787), and 29% with the P. furiosus (PF1192) homologues.", "section": "INTRO", "ner": [ [ 4, 25, "Rhodospirillum\u00a0rubrum", "species" ], [ 26, 32, "EncFtn", "protein" ], [ 42, 51, "Rru_A0973", "gene" ], [ 99, 109, "M. xanthus", "species" ], [ 111, 120, "MXAN_4464", "gene" ], [ 136, 147, "T. maritima", "species" ], [ 149, 159, "Tmari_0787", "gene" ], [ 179, 190, "P. furiosus", "species" ], [ 192, 198, "PF1192", "gene" ] ] }, { "sid": 39, "sent": "The GXXH motifs are strictly conserved in each of these species (Supplementary file 1).", "section": "INTRO", "ner": [ [ 4, 8, "GXXH", "structure_element" ], [ 20, 38, "strictly conserved", "protein_state" ] ] }, { "sid": 40, "sent": "Here we investigate the structure and biochemistry of EncFtn in order to understand iron storage within the encapsulin nanocompartment.", "section": "INTRO", "ner": [ [ 24, 33, "structure", "evidence" ], [ 54, 60, "EncFtn", "protein" ], [ 84, 88, "iron", "chemical" ], [ 108, 118, "encapsulin", "protein" ], [ 119, 134, "nanocompartment", "complex_assembly" ] ] }, { "sid": 41, "sent": "We have produced recombinant encapsulin (Enc) and EncFtn from the aquatic purple-sulfur bacterium R.\u00a0rubrum, which serves as a model organism for the study of the control of the bacterial nitrogen fixation machinery, in Escherichia coli.", "section": "INTRO", "ner": [ [ 29, 39, "encapsulin", "protein" ], [ 41, 44, "Enc", "protein" ], [ 50, 56, "EncFtn", "protein" ], [ 66, 73, "aquatic", "taxonomy_domain" ], [ 74, 97, "purple-sulfur bacterium", "taxonomy_domain" ], [ 98, 107, "R.\u00a0rubrum", "species" ], [ 178, 187, "bacterial", "taxonomy_domain" ], [ 220, 236, "Escherichia coli", "species" ] ] }, { "sid": 42, "sent": "Analysis by transmission electron microscopy (TEM) indicates that their co-expression leads to the production of an icosahedral nanocompartment with encapsulated EncFtn.", "section": "INTRO", "ner": [ [ 12, 44, "transmission electron microscopy", "experimental_method" ], [ 46, 49, "TEM", "experimental_method" ], [ 72, 85, "co-expression", "experimental_method" ], [ 116, 127, "icosahedral", "protein_state" ], [ 128, 143, "nanocompartment", "complex_assembly" ], [ 149, 161, "encapsulated", "protein_state" ], [ 162, 168, "EncFtn", "protein" ] ] }, { "sid": 43, "sent": "The crystal structure of a truncated hexahistidine-tagged variant of the EncFtn protein (EncFtnsH) shows that it forms a decameric structure with an annular \u2018ring-doughnut\u2019 topology, which is distinct from the four-helical bundles of the 24meric ferritins and dodecahedral DPS proteins.", "section": "INTRO", "ner": [ [ 4, 21, "crystal structure", "evidence" ], [ 27, 36, "truncated", "protein_state" ], [ 37, 57, "hexahistidine-tagged", "protein_state" ], [ 73, 79, "EncFtn", "protein" ], [ 89, 97, "EncFtnsH", "protein" ], [ 121, 130, "decameric", "oligomeric_state" ], [ 131, 140, "structure", "evidence" ], [ 158, 171, "ring-doughnut", "structure_element" ], [ 210, 230, "four-helical bundles", "structure_element" ], [ 238, 245, "24meric", "oligomeric_state" ], [ 246, 255, "ferritins", "protein_type" ], [ 260, 272, "dodecahedral", "oligomeric_state" ], [ 273, 276, "DPS", "protein_type" ] ] }, { "sid": 44, "sent": "We identify a symmetrical iron bound ferroxidase center (FOC) formed between subunits in the decamer and additional metal-binding sites close to the center of the ring and on the outer surface.", "section": "INTRO", "ner": [ [ 26, 36, "iron bound", "protein_state" ], [ 37, 55, "ferroxidase center", "site" ], [ 57, 60, "FOC", "site" ], [ 77, 85, "subunits", "structure_element" ], [ 93, 100, "decamer", "oligomeric_state" ], [ 116, 135, "metal-binding sites", "site" ], [ 163, 167, "ring", "structure_element" ] ] }, { "sid": 45, "sent": "We also demonstrate the metal-dependent assembly of EncFtn decamers using native PAGE, analytical gel-filtration, and native mass spectrometry.", "section": "INTRO", "ner": [ [ 52, 58, "EncFtn", "protein" ], [ 59, 67, "decamers", "oligomeric_state" ], [ 74, 85, "native PAGE", "experimental_method" ], [ 87, 112, "analytical gel-filtration", "experimental_method" ], [ 118, 142, "native mass spectrometry", "experimental_method" ] ] }, { "sid": 46, "sent": "Biochemical assays show that EncFtn is active as a ferroxidase enzyme.", "section": "INTRO", "ner": [ [ 0, 18, "Biochemical assays", "experimental_method" ], [ 29, 35, "EncFtn", "protein" ], [ 39, 45, "active", "protein_state" ], [ 51, 62, "ferroxidase", "protein_type" ] ] }, { "sid": 47, "sent": "Through site-directed mutagenesis we show that the conserved glutamic acid and histidine residues in the FOC influence protein assembly and activity.", "section": "INTRO", "ner": [ [ 8, 33, "site-directed mutagenesis", "experimental_method" ], [ 51, 60, "conserved", "protein_state" ], [ 61, 74, "glutamic acid", "residue_name" ], [ 79, 88, "histidine", "residue_name" ], [ 105, 108, "FOC", "site" ] ] }, { "sid": 48, "sent": "We use our combined structural and biochemical data to propose a model for the EncFtn-catalyzed sequestration of iron within the encapsulin shell.", "section": "INTRO", "ner": [ [ 20, 51, "structural and biochemical data", "evidence" ], [ 79, 85, "EncFtn", "protein" ], [ 113, 117, "iron", "chemical" ], [ 129, 139, "encapsulin", "protein" ], [ 140, 145, "shell", "structure_element" ] ] }, { "sid": 49, "sent": "Assembly of R. rubrum EncFtn encapsulin nanocompartments in E. coli", "section": "RESULTS", "ner": [ [ 12, 21, "R. rubrum", "species" ], [ 22, 28, "EncFtn", "protein" ], [ 29, 39, "encapsulin", "protein" ], [ 40, 56, "nanocompartments", "complex_assembly" ], [ 60, 67, "E. coli", "species" ] ] }, { "sid": 50, "sent": "Full-frame transmission electron micrographs of R. rubrum nanocompartments.", "section": "FIG", "ner": [ [ 0, 44, "Full-frame transmission electron micrographs", "evidence" ], [ 48, 57, "R. rubrum", "species" ], [ 58, 74, "nanocompartments", "complex_assembly" ] ] }, { "sid": 51, "sent": "(A/B) Negative stain TEM image of recombinant R. rubrum encapsulin and EncFtn-Enc nanocompartments.", "section": "FIG", "ner": [ [ 6, 24, "Negative stain TEM", "experimental_method" ], [ 25, 30, "image", "evidence" ], [ 46, 55, "R. rubrum", "species" ], [ 56, 66, "encapsulin", "protein" ], [ 71, 81, "EncFtn-Enc", "complex_assembly" ], [ 82, 98, "nanocompartments", "complex_assembly" ] ] }, { "sid": 52, "sent": "All samples were imaged at 143,000 x magnification; the scale bar length corresponds to 50 nm. (C) Histogram showing the distribution of nanocompartment diameters.", "section": "FIG", "ner": [ [ 99, 108, "Histogram", "evidence" ], [ 137, 152, "nanocompartment", "complex_assembly" ] ] }, { "sid": 53, "sent": "A model Gaussian nonlinear least square function was fitted to the data to obtain a mean diameter of 24.6 nm with a\u00a0standard deviation of 2.0 nm for encapsulin (grey) and a mean value of 23.9 nm with a\u00a0standard deviation of 2.2 nm for co-expressed EncFtn and encapsulin (EncFtn-Enc, black).", "section": "FIG", "ner": [ [ 8, 48, "Gaussian nonlinear least square function", "experimental_method" ], [ 149, 159, "encapsulin", "protein" ], [ 235, 247, "co-expressed", "experimental_method" ], [ 248, 254, "EncFtn", "protein" ], [ 259, 269, "encapsulin", "protein" ], [ 271, 281, "EncFtn-Enc", "complex_assembly" ] ] }, { "sid": 54, "sent": "Purification of recombinant R. rubrum encapsulin nanocompartments.", "section": "FIG", "ner": [ [ 28, 37, "R. rubrum", "species" ], [ 38, 48, "encapsulin", "protein" ], [ 49, 65, "nanocompartments", "complex_assembly" ] ] }, { "sid": 55, "sent": "(A) Recombinantly expressed encapsulin (Enc) and co-expressed EncFtn-Enc were purified by sucrose gradient ultracentrifugation from E. coli B834(DE3) grown in SeMet medium.", "section": "FIG", "ner": [ [ 4, 27, "Recombinantly expressed", "experimental_method" ], [ 28, 38, "encapsulin", "protein" ], [ 40, 43, "Enc", "protein" ], [ 49, 61, "co-expressed", "experimental_method" ], [ 62, 72, "EncFtn-Enc", "complex_assembly" ], [ 90, 126, "sucrose gradient ultracentrifugation", "experimental_method" ], [ 132, 139, "E. coli", "species" ], [ 159, 164, "SeMet", "chemical" ] ] }, { "sid": 56, "sent": "Samples were resolved by 18% acrylamide SDS-PAGE; the position of the proteins found in the complexes as resolved on the gel are shown with arrows.", "section": "FIG", "ner": [ [ 40, 48, "SDS-PAGE", "experimental_method" ] ] }, { "sid": 57, "sent": "(B/C) Negative stain TEM image of recombinant encapsulin and EncFtn-Enc nanocompartments.", "section": "FIG", "ner": [ [ 6, 24, "Negative stain TEM", "experimental_method" ], [ 46, 56, "encapsulin", "protein" ], [ 61, 71, "EncFtn-Enc", "complex_assembly" ], [ 72, 88, "nanocompartments", "complex_assembly" ] ] }, { "sid": 58, "sent": "Representative encapsulin and EncFtn-Enc complexes are indicated with red arrows.", "section": "FIG", "ner": [ [ 15, 25, "encapsulin", "protein" ], [ 30, 40, "EncFtn-Enc", "complex_assembly" ] ] }, { "sid": 59, "sent": "We produced recombinant R. rubrum encapsulin nanocompartments in E. coli by co-expression of the encapsulin (Rru_A0974) and EncFtn (Rru_A0973) proteins, and purified these by sucrose gradient ultra-centrifugation (Figure 1A).", "section": "RESULTS", "ner": [ [ 24, 33, "R. rubrum", "species" ], [ 34, 44, "encapsulin", "protein" ], [ 45, 61, "nanocompartments", "complex_assembly" ], [ 65, 72, "E. coli", "species" ], [ 76, 89, "co-expression", "experimental_method" ], [ 97, 107, "encapsulin", "protein" ], [ 109, 118, "Rru_A0974", "gene" ], [ 124, 130, "EncFtn", "protein" ], [ 132, 141, "Rru_A0973", "gene" ], [ 175, 212, "sucrose gradient ultra-centrifugation", "experimental_method" ] ] }, { "sid": 60, "sent": "TEM imaging of uranyl acetate-stained samples revealed that, when expressed in isolation, the encapsulin protein forms empty compartments with an average diameter of 24 nm (Figure 1B and Figure 1\u2014figure supplement 1A/C), consistent with the appearance and size of the T. maritima encapsulin.", "section": "RESULTS", "ner": [ [ 0, 3, "TEM", "experimental_method" ], [ 66, 88, "expressed in isolation", "experimental_method" ], [ 94, 104, "encapsulin", "protein" ], [ 119, 124, "empty", "protein_state" ], [ 125, 137, "compartments", "complex_assembly" ], [ 268, 279, "T. maritima", "species" ], [ 280, 290, "encapsulin", "protein" ] ] }, { "sid": 61, "sent": "We were not able to resolve any higher-order structures of EncFtn by TEM.", "section": "RESULTS", "ner": [ [ 59, 65, "EncFtn", "protein" ], [ 69, 72, "TEM", "experimental_method" ] ] }, { "sid": 62, "sent": "Protein purified from co-expression of the encapsulin and EncFtn resulted in 24 nm compartments with regions in the center that exclude stain, consistent with the presence of the EncFtn within the encapsulin shell (Figure 1C and Figure 1\u2014figure supplement 1B/C).", "section": "RESULTS", "ner": [ [ 22, 35, "co-expression", "experimental_method" ], [ 43, 53, "encapsulin", "protein" ], [ 58, 64, "EncFtn", "protein" ], [ 163, 174, "presence of", "protein_state" ], [ 179, 185, "EncFtn", "protein" ], [ 197, 207, "encapsulin", "protein" ], [ 208, 213, "shell", "structure_element" ] ] }, { "sid": 63, "sent": "R.\u00a0rubrum EncFtn forms a metal-ion stabilized decamer in solution", "section": "RESULTS", "ner": [ [ 0, 9, "R.\u00a0rubrum", "species" ], [ 10, 16, "EncFtn", "protein" ], [ 46, 53, "decamer", "oligomeric_state" ] ] }, { "sid": 64, "sent": "Purification of recombinant R. rubrum EncFtnsH.", "section": "FIG", "ner": [ [ 0, 27, "Purification of recombinant", "experimental_method" ], [ 28, 37, "R. rubrum", "species" ], [ 38, 46, "EncFtnsH", "protein" ] ] }, { "sid": 65, "sent": "(A) Recombinant SeMet-labeled EncFtnsH produced with 1 mM Fe(NH4)2(SO4)2 in the growth medium was purified by nickel affinity chromatography and size-exclusion chromatography using a\u00a0Superdex\u00a0200 16/60 column (GE Healthcare).", "section": "FIG", "ner": [ [ 16, 29, "SeMet-labeled", "protein_state" ], [ 30, 38, "EncFtnsH", "protein" ], [ 58, 72, "Fe(NH4)2(SO4)2", "chemical" ], [ 110, 140, "nickel affinity chromatography", "experimental_method" ], [ 145, 174, "size-exclusion chromatography", "experimental_method" ] ] }, { "sid": 66, "sent": "Chromatogram traces measured at 280 nm and 315 nm are shown with the results from ICP-MS analysis of the iron content of the fractions collected during the experiment.", "section": "FIG", "ner": [ [ 0, 12, "Chromatogram", "evidence" ], [ 82, 88, "ICP-MS", "experimental_method" ], [ 105, 109, "iron", "chemical" ] ] }, { "sid": 67, "sent": "The peak around 73 ml corresponds to a molecular weight of around 130 kDa when compared to calibration standards; this is consistent with a decamer of EncFtnsH. The small peak at 85 ml corresponds to the 13 kDa monomer compared to the standards.", "section": "FIG", "ner": [ [ 39, 55, "molecular weight", "evidence" ], [ 140, 147, "decamer", "oligomeric_state" ], [ 151, 159, "EncFtnsH", "protein" ], [ 211, 218, "monomer", "oligomeric_state" ] ] }, { "sid": 68, "sent": "Only the decamer peak contains significant amounts of iron as indicated by the ICP-MS analysis.", "section": "FIG", "ner": [ [ 9, 16, "decamer", "oligomeric_state" ], [ 54, 58, "iron", "chemical" ], [ 79, 85, "ICP-MS", "experimental_method" ] ] }, { "sid": 69, "sent": "(B) Peak fractions from the gel filtration run were resolved by 15% acrylamide SDS-PAGE and stained with Coomassie blue stain.", "section": "FIG", "ner": [ [ 28, 42, "gel filtration", "experimental_method" ], [ 79, 87, "SDS-PAGE", "experimental_method" ] ] }, { "sid": 70, "sent": "The bands around 13 kDa and 26 kDa correspond to EncFtnsH, as\u00a0identified by MALDI peptide mass fingerprinting.", "section": "FIG", "ner": [ [ 49, 57, "EncFtnsH", "protein" ], [ 76, 109, "MALDI peptide mass fingerprinting", "experimental_method" ] ] }, { "sid": 71, "sent": "The band at 13 kDa is consistent with the monomer mass, while the band at 26 kDa is consistent with a dimer of EncFtnsH. The dimer species only appears in the decamer fractions.", "section": "FIG", "ner": [ [ 42, 49, "monomer", "oligomeric_state" ], [ 102, 107, "dimer", "oligomeric_state" ], [ 111, 119, "EncFtnsH", "protein" ], [ 125, 130, "dimer", "oligomeric_state" ], [ 159, 166, "decamer", "oligomeric_state" ] ] }, { "sid": 72, "sent": "(C) SEC-MALLS analysis of EncFtnsH from decamer fractions and monomer fractions allows assignment of an average mass of 132 kDa to decamer fractions and 13 kDa to monomer fractions, consistent with decamer and monomer species (Table 2).", "section": "FIG", "ner": [ [ 4, 13, "SEC-MALLS", "experimental_method" ], [ 26, 34, "EncFtnsH", "protein" ], [ 40, 47, "decamer", "oligomeric_state" ], [ 62, 69, "monomer", "oligomeric_state" ], [ 131, 138, "decamer", "oligomeric_state" ], [ 163, 170, "monomer", "oligomeric_state" ], [ 198, 205, "decamer", "oligomeric_state" ], [ 210, 217, "monomer", "oligomeric_state" ] ] }, { "sid": 73, "sent": "Determination of the Fe/EncFtnsH protein ratio by ICP-MS.", "section": "TABLE", "ner": [ [ 21, 23, "Fe", "chemical" ], [ 24, 32, "EncFtnsH", "protein" ], [ 50, 56, "ICP-MS", "experimental_method" ] ] }, { "sid": 74, "sent": "EncFtnsH was purified as a SeMet derivative from E. coli B834(DE3) cells grown in SeMet medium with 1 mM Fe(NH4)2(SO4)2.", "section": "TABLE", "ner": [ [ 0, 8, "EncFtnsH", "protein" ], [ 27, 32, "SeMet", "chemical" ], [ 49, 66, "E. coli B834(DE3)", "species" ], [ 82, 87, "SeMet", "chemical" ], [ 105, 119, "Fe(NH4)2(SO4)2", "chemical" ] ] }, { "sid": 75, "sent": "Fractions from SEC were collected, acidified and analysed by ICP-MS.", "section": "TABLE", "ner": [ [ 15, 18, "SEC", "experimental_method" ], [ 61, 67, "ICP-MS", "experimental_method" ] ] }, { "sid": 76, "sent": "EncFtnsH concentration was calculated based on the presence of two SeMet per mature monomer.", "section": "TABLE", "ner": [ [ 0, 8, "EncFtnsH", "protein" ], [ 51, 62, "presence of", "protein_state" ], [ 67, 72, "SeMet", "chemical" ], [ 77, 83, "mature", "protein_state" ], [ 84, 91, "monomer", "oligomeric_state" ] ] }, { "sid": 77, "sent": "These data were collected from EncFtnsH fractions from a single gel-filtration run.", "section": "TABLE", "ner": [ [ 31, 39, "EncFtnsH", "protein" ], [ 64, 78, "gel-filtration", "experimental_method" ] ] }, { "sid": 78, "sent": "Peak\tEncFtnsHretention volume (ml)\tElement concentration (\u00b5M)\tDerived EncFtnsHconcentration (\u00b5M)\tDerived Fe/ EncFtnsH monomer\t \tCa\tFe\tZn\tSe\t \tDecamer\t66.5\tn.d.", "section": "TABLE", "ner": [ [ 5, 13, "EncFtnsH", "protein" ], [ 70, 78, "EncFtnsH", "protein" ], [ 105, 107, "Fe", "chemical" ], [ 109, 117, "EncFtnsH", "protein" ], [ 118, 125, "monomer", "oligomeric_state" ], [ 128, 130, "Ca", "chemical" ], [ 131, 133, "Fe", "chemical" ], [ 134, 136, "Zn", "chemical" ], [ 137, 139, "Se", "chemical" ], [ 142, 149, "Decamer", "oligomeric_state" ] ] }, { "sid": 79, "sent": "Estimates of EncFtnsH molecular weight from SEC-MALLS analysis.", "section": "TABLE", "ner": [ [ 13, 21, "EncFtnsH", "protein" ], [ 22, 38, "molecular weight", "evidence" ], [ 44, 53, "SEC-MALLS", "experimental_method" ] ] }, { "sid": 80, "sent": "EncFtnsH was purified from E. coli BL21(DE3) grown in minimal medium\u00a0(MM) by nickel affinity chromatography\u00a0and size-exclusion chromatography.", "section": "TABLE", "ner": [ [ 0, 8, "EncFtnsH", "protein" ], [ 27, 44, "E. coli BL21(DE3)", "species" ], [ 54, 68, "minimal medium", "experimental_method" ], [ 70, 72, "MM", "experimental_method" ], [ 77, 107, "nickel affinity chromatography", "experimental_method" ], [ 112, 141, "size-exclusion chromatography", "experimental_method" ] ] }, { "sid": 81, "sent": "Fractions from two peaks (decamer and monomer) were pooled separately (Figure 1C) and analysed by SEC-MALLS using a Superdex\u00a0200 10/300 GL column\u00a0(GE\u00a0Healthcare) and Viscotek SEC-MALLS instruments\u00a0(Malvern\u00a0Instruments) (Figure 2C).", "section": "TABLE", "ner": [ [ 19, 24, "peaks", "evidence" ], [ 26, 33, "decamer", "oligomeric_state" ], [ 38, 45, "monomer", "oligomeric_state" ], [ 98, 107, "SEC-MALLS", "experimental_method" ], [ 175, 184, "SEC-MALLS", "experimental_method" ] ] }, { "sid": 82, "sent": "The decamer and monomer peaks were both symmetric and monodisperse, allowing the estimation of the molecular weight of the species in these fractions.", "section": "TABLE", "ner": [ [ 4, 11, "decamer", "oligomeric_state" ], [ 16, 23, "monomer", "oligomeric_state" ], [ 24, 29, "peaks", "evidence" ], [ 99, 115, "molecular weight", "evidence" ] ] }, { "sid": 83, "sent": "The proteins analyzed by SEC-MALLS came from single protein preparation.", "section": "TABLE", "ner": [ [ 25, 34, "SEC-MALLS", "experimental_method" ] ] }, { "sid": 84, "sent": "Molecular Weight (kDa)\tDecamer peak\tMonomer peak\t \tTheoretical\t133\t13\t \tEncFtnsH-decamer fractions\t132\t15\t \tEncFtnsH-monomer fractions\t126\t13\t \t", "section": "TABLE", "ner": [ [ 0, 16, "Molecular Weight", "evidence" ], [ 23, 30, "Decamer", "oligomeric_state" ], [ 36, 43, "Monomer", "oligomeric_state" ], [ 72, 80, "EncFtnsH", "protein" ], [ 81, 88, "decamer", "oligomeric_state" ], [ 108, 116, "EncFtnsH", "protein" ], [ 117, 124, "monomer", "oligomeric_state" ] ] }, { "sid": 85, "sent": "We purified recombinant R. rubrum EncFtn as both the full-length sequence (140 amino acids) and a truncated C-terminal hexahistidine-tagged variant (amino acids 1\u201396 plus the tag; herein EncFtnsH).", "section": "RESULTS", "ner": [ [ 24, 33, "R. rubrum", "species" ], [ 34, 40, "EncFtn", "protein" ], [ 53, 64, "full-length", "protein_state" ], [ 75, 90, "140 amino acids", "residue_range" ], [ 98, 107, "truncated", "protein_state" ], [ 119, 139, "hexahistidine-tagged", "protein_state" ], [ 161, 165, "1\u201396", "residue_range" ], [ 187, 195, "EncFtnsH", "protein" ] ] }, { "sid": 86, "sent": "In both cases the elution profile from size-exclusion chromatography (SEC) displayed two peaks (Figure 2A).", "section": "RESULTS", "ner": [ [ 18, 33, "elution profile", "evidence" ], [ 39, 68, "size-exclusion chromatography", "experimental_method" ], [ 70, 73, "SEC", "experimental_method" ], [ 89, 94, "peaks", "evidence" ] ] }, { "sid": 87, "sent": "SDS-PAGE analysis of fractions from these peaks showed that the high molecular weight peak was partially resistant to SDS and heat-induced denaturation; in contrast, the low molecular weight peak was consistent with monomeric mass of 13 kDa (Figure 2B).", "section": "RESULTS", "ner": [ [ 0, 8, "SDS-PAGE", "experimental_method" ], [ 42, 47, "peaks", "evidence" ], [ 69, 85, "molecular weight", "evidence" ], [ 174, 190, "molecular weight", "evidence" ], [ 216, 225, "monomeric", "oligomeric_state" ] ] }, { "sid": 88, "sent": "MALDI peptide mass fingerprinting of these bands confirmed the identity of both as EncFtn.", "section": "RESULTS", "ner": [ [ 0, 33, "MALDI peptide mass fingerprinting", "experimental_method" ], [ 83, 89, "EncFtn", "protein" ] ] }, { "sid": 89, "sent": "Inductively coupled plasma mass spectrometry (ICP-MS) analysis of the SEC fractions showed 100 times more iron in the oligomeric fraction than the monomer (Figure 2A, blue scatter points; Table 1), suggesting that EncFtn oligomerization is associated with iron binding.", "section": "RESULTS", "ner": [ [ 0, 44, "Inductively coupled plasma mass spectrometry", "experimental_method" ], [ 46, 52, "ICP-MS", "experimental_method" ], [ 70, 73, "SEC", "experimental_method" ], [ 106, 110, "iron", "chemical" ], [ 147, 154, "monomer", "oligomeric_state" ], [ 214, 220, "EncFtn", "protein" ], [ 256, 260, "iron", "chemical" ] ] }, { "sid": 90, "sent": "In order to determine the iron-loading stoichiometry in the EncFtn complex, further ICP-MS experiments were performed using selenomethionine\u00a0(SeMet)-labelled protein EncFtn (Table 1).", "section": "RESULTS", "ner": [ [ 26, 30, "iron", "chemical" ], [ 60, 66, "EncFtn", "protein" ], [ 84, 90, "ICP-MS", "experimental_method" ], [ 124, 140, "selenomethionine", "chemical" ], [ 142, 147, "SeMet", "chemical" ], [ 166, 172, "EncFtn", "protein" ] ] }, { "sid": 91, "sent": "In these experiments, we observed sub-stoichiometric metal binding, which is in contrast to the classical ferritins.", "section": "RESULTS", "ner": [ [ 96, 105, "classical", "protein_state" ], [ 106, 115, "ferritins", "protein_type" ] ] }, { "sid": 92, "sent": "Size-exclusion chromatography with multi-angle laser light scattering (SEC-MALLS) analysis of samples taken from each peak gave calculated molecular weights consistent with a decamer for the high molecular weight peak and a monomer for the low molecular weight peak (Figure 2C, Table 2).", "section": "RESULTS", "ner": [ [ 0, 29, "Size-exclusion chromatography", "experimental_method" ], [ 35, 69, "multi-angle laser light scattering", "experimental_method" ], [ 71, 80, "SEC-MALLS", "experimental_method" ], [ 175, 182, "decamer", "oligomeric_state" ], [ 196, 212, "molecular weight", "evidence" ], [ 224, 231, "monomer", "oligomeric_state" ], [ 244, 260, "molecular weight", "evidence" ] ] }, { "sid": 93, "sent": "Effect of metal ions on the oligomeric state of EncFtnsH\u00a0in solution.", "section": "FIG", "ner": [ [ 48, 56, "EncFtnsH", "protein" ] ] }, { "sid": 94, "sent": "(A/B) EncFtnsH-monomer was incubated with one mole equivalent of various metal salts for two hours prior to analytical gel-filtration using a Superdex 200 PC 3.2/30 column.", "section": "FIG", "ner": [ [ 6, 14, "EncFtnsH", "protein" ], [ 15, 22, "monomer", "oligomeric_state" ], [ 27, 36, "incubated", "experimental_method" ], [ 108, 133, "analytical gel-filtration", "experimental_method" ] ] }, { "sid": 95, "sent": "Co2+ and Zn2+ induced the formation of the decameric form of EncFtnsH; while Mn2+, Mg2+ and Fe3+ did not significantly alter the oligomeric state of EncFtnsH.", "section": "FIG", "ner": [ [ 0, 4, "Co2+", "chemical" ], [ 9, 13, "Zn2+", "chemical" ], [ 43, 52, "decameric", "oligomeric_state" ], [ 61, 69, "EncFtnsH", "protein" ], [ 77, 81, "Mn2+", "chemical" ], [ 83, 87, "Mg2+", "chemical" ], [ 92, 96, "Fe3+", "chemical" ], [ 149, 157, "EncFtnsH", "protein" ] ] }, { "sid": 96, "sent": "PAGE analysis of the effect of metal ions on the oligomeric state of EncFtnsH.", "section": "FIG", "ner": [ [ 0, 4, "PAGE", "experimental_method" ], [ 69, 77, "EncFtnsH", "protein" ] ] }, { "sid": 97, "sent": "50 \u00b5M EncFtnsH monomer or decamer samples were mixed with equal molar metal ions including Fe2+, Co2+, Zn2+, Mn2+, Ca2+, Mg2+ and Fe3+, which were analyzed by Native PAGE alongside SDS-PAGE.", "section": "FIG", "ner": [ [ 6, 14, "EncFtnsH", "protein" ], [ 15, 22, "monomer", "oligomeric_state" ], [ 26, 33, "decamer", "oligomeric_state" ], [ 91, 96, "Fe2+,", "chemical" ], [ 97, 102, "Co2+,", "chemical" ], [ 103, 108, "Zn2+,", "chemical" ], [ 109, 114, "Mn2+,", "chemical" ], [ 115, 120, "Ca2+,", "chemical" ], [ 121, 125, "Mg2+", "chemical" ], [ 130, 135, "Fe3+,", "chemical" ], [ 159, 170, "Native PAGE", "experimental_method" ], [ 181, 189, "SDS-PAGE", "experimental_method" ] ] }, { "sid": 98, "sent": "\u00a0(A) 10% Native PAGE analysis of EncFtnsH monomer fractions mixed with various metal solutions; (B) 10% Native PAGE analysis of EncFtnsH decamer fractions mixed with various metal solutions; (C) 15% SDS-PAGE analysis on the mixtures of EncFtnsH monomer fractions and metal solutions; (D) 15% SDS-PAGE analysis on the mixtures of EncFtnsH decamer fractions and metal solutions.", "section": "FIG", "ner": [ [ 9, 20, "Native PAGE", "experimental_method" ], [ 33, 41, "EncFtnsH", "protein" ], [ 42, 49, "monomer", "oligomeric_state" ], [ 104, 115, "Native PAGE", "experimental_method" ], [ 128, 136, "EncFtnsH", "protein" ], [ 137, 144, "decamer", "oligomeric_state" ], [ 199, 207, "SDS-PAGE", "experimental_method" ], [ 236, 244, "EncFtnsH", "protein" ], [ 245, 252, "monomer", "oligomeric_state" ], [ 292, 300, "SDS-PAGE", "experimental_method" ], [ 329, 337, "EncFtnsH", "protein" ], [ 338, 345, "decamer", "oligomeric_state" ] ] }, { "sid": 99, "sent": "Effect of Fe2+ and protein concentration on the oligomeric state of EncFtnsH in solution.", "section": "FIG", "ner": [ [ 10, 14, "Fe2+", "chemical" ], [ 68, 76, "EncFtnsH", "protein" ] ] }, { "sid": 100, "sent": "(A) Recombinant EncFtnsH was purified by Gel filtration Superdex\u00a0200 chromatography from E. coli BL21(DE3) grown in MM or in MM supplemented with 1 mM Fe(NH4)2(SO4)2 (MM+Fe2+).", "section": "FIG", "ner": [ [ 16, 24, "EncFtnsH", "protein" ], [ 41, 55, "Gel filtration", "experimental_method" ], [ 89, 106, "E. coli BL21(DE3)", "species" ], [ 116, 118, "MM", "experimental_method" ], [ 125, 127, "MM", "experimental_method" ], [ 151, 165, "Fe(NH4)2(SO4)2", "chemical" ], [ 167, 169, "MM", "experimental_method" ], [ 170, 174, "Fe2+", "chemical" ] ] }, { "sid": 101, "sent": "A higher proportion of decamer (peak between 65 and 75 ml) is seen in the sample purified from MM+Fe2+ compared to EncFtnsH-MM, indicating that Fe2+ facilitates the multimerization of EncFtnsH in vivo. (B) EncFtnsH-monomer was incubated with one molar equivalent of Fe2+ salts for two hours prior to analytical gel-filtration using a Superdex 200 PC 3.2/30 column\u00a0(GE\u00a0Healthcare).", "section": "FIG", "ner": [ [ 23, 30, "decamer", "oligomeric_state" ], [ 95, 97, "MM", "experimental_method" ], [ 98, 102, "Fe2+", "chemical" ], [ 115, 123, "EncFtnsH", "protein" ], [ 124, 126, "MM", "experimental_method" ], [ 144, 148, "Fe2+", "chemical" ], [ 184, 192, "EncFtnsH", "protein" ], [ 206, 214, "EncFtnsH", "protein" ], [ 215, 222, "monomer", "oligomeric_state" ], [ 266, 270, "Fe2+", "chemical" ], [ 300, 325, "analytical gel-filtration", "experimental_method" ] ] }, { "sid": 102, "sent": "Both Fe2+ salts tested induced the formation of decamer indicated by the peak between 1.2 and 1.6 ml.", "section": "FIG", "ner": [ [ 5, 9, "Fe2+", "chemical" ], [ 48, 55, "decamer", "oligomeric_state" ] ] }, { "sid": 103, "sent": "Monomeric and decameric samples of EncFtnsH\u00a0are shown as controls.", "section": "FIG", "ner": [ [ 0, 9, "Monomeric", "oligomeric_state" ], [ 14, 23, "decameric", "oligomeric_state" ], [ 35, 43, "EncFtnsH", "protein" ] ] }, { "sid": 104, "sent": "Peaks around 0.8 ml were seen as protein aggregation.", "section": "FIG", "ner": [ [ 0, 5, "Peaks", "evidence" ] ] }, { "sid": 105, "sent": "(C) Analytical gel filtration of EncFtn monomer at different concentrations to illustrate the effect of protein concentration on multimerization.", "section": "FIG", "ner": [ [ 4, 29, "Analytical gel filtration", "experimental_method" ], [ 33, 39, "EncFtn", "protein" ], [ 40, 47, "monomer", "oligomeric_state" ] ] }, { "sid": 106, "sent": "The major peak shows a shift towards a dimer species at high concentration of protein, but the ratio of this peak (1.5\u20131.8 ml) to the decamer peak (1.2\u20131.5 ml) does not change when compared to the low concentration sample.", "section": "FIG", "ner": [ [ 39, 44, "dimer", "oligomeric_state" ], [ 134, 141, "decamer", "oligomeric_state" ] ] }, { "sid": 107, "sent": "Gel-filtration peak area ratios for EncFtnsH decamer and monomer on addition of different metal ions.", "section": "TABLE", "ner": [ [ 0, 14, "Gel-filtration", "experimental_method" ], [ 15, 31, "peak area ratios", "evidence" ], [ 36, 44, "EncFtnsH", "protein" ], [ 45, 52, "decamer", "oligomeric_state" ], [ 57, 64, "monomer", "oligomeric_state" ] ] }, { "sid": 108, "sent": "EncFtnsH was produced in E. coli BL21(DE3) cultured in MM and MM with 1 mM Fe(NH4)2(SO4)2 (MM+Fe2+) and purified by gel-filtration chromatography using an Superdex\u00a0200 16/60\u00a0column (GE Healthcare).", "section": "TABLE", "ner": [ [ 0, 8, "EncFtnsH", "protein" ], [ 25, 42, "E. coli BL21(DE3)", "species" ], [ 55, 57, "MM", "experimental_method" ], [ 62, 64, "MM", "experimental_method" ], [ 75, 89, "Fe(NH4)2(SO4)2", "chemical" ], [ 91, 93, "MM", "experimental_method" ], [ 94, 98, "Fe2+", "chemical" ], [ 116, 145, "gel-filtration chromatography", "experimental_method" ] ] }, { "sid": 109, "sent": "Monomer fractions of EncFtnsH purified from MM were pooled and run in subsequent analytical gel-filtration runs over the course of three days.", "section": "TABLE", "ner": [ [ 0, 7, "Monomer", "oligomeric_state" ], [ 21, 29, "EncFtnsH", "protein" ], [ 44, 46, "MM", "experimental_method" ], [ 81, 106, "analytical gel-filtration", "experimental_method" ] ] }, { "sid": 110, "sent": "Samples of EncFtnsH monomer were incubated with one molar equivalent of metal ion salts at room temperature for two hours before analysis by analytical gel\u00a0filtration chromatography (AGF) using a Superdex 200\u00a010/300 GL column.", "section": "TABLE", "ner": [ [ 11, 19, "EncFtnsH", "protein" ], [ 20, 27, "monomer", "oligomeric_state" ], [ 141, 181, "analytical gel\u00a0filtration chromatography", "experimental_method" ], [ 183, 186, "AGF", "experimental_method" ] ] }, { "sid": 111, "sent": "The area for resulting protein peaks were calculated using the Unicorn software (GE Healthcare); peak ratios were calculated to quantify the propensity of EncFtnsH to multimerize in the presence of the different metal ions.", "section": "TABLE", "ner": [ [ 31, 36, "peaks", "evidence" ], [ 97, 108, "peak ratios", "evidence" ], [ 155, 163, "EncFtnsH", "protein" ], [ 186, 197, "presence of", "protein_state" ] ] }, { "sid": 112, "sent": "The change in the\u00a0ratios of monomer to decamer over the three days of experiments may be a consequence of experimental variability, or the propensity of this protein to equilibrate towards decamer over time.", "section": "TABLE", "ner": [ [ 28, 35, "monomer", "oligomeric_state" ], [ 39, 46, "decamer", "oligomeric_state" ], [ 189, 196, "decamer", "oligomeric_state" ] ] }, { "sid": 113, "sent": "The increased decamer: monomer ratio seen in the presence of Fe2+, Co2+, and Zn2+ indicates that these metal ions facilitate multimerization of the EncFtnsH protein, while the other metal ions tested do not appear to induce multimerization.", "section": "TABLE", "ner": [ [ 14, 21, "decamer", "oligomeric_state" ], [ 23, 30, "monomer", "oligomeric_state" ], [ 49, 60, "presence of", "protein_state" ], [ 61, 65, "Fe2+", "chemical" ], [ 67, 71, "Co2+", "chemical" ], [ 77, 81, "Zn2+", "chemical" ], [ 148, 156, "EncFtnsH", "protein" ] ] }, { "sid": 114, "sent": "The analytical gel filtration experiment was repeated twice using two independent preparations of protein, of which values calculated from one sample are presented here.", "section": "TABLE", "ner": [ [ 4, 29, "analytical gel filtration", "experimental_method" ] ] }, { "sid": 115, "sent": "Method\tSample\tMonomer area\tDecamer area\tDecamer/Monomer\t \tGel filtration Superdex\u00a0200 chromatography\tEncFtnsH-MM\t64.3\t583.6\t0.1\t \tEncFtnsH-MM+Fe2+\t1938.4\t426.4\t4.5\t \tAnalytical Gel filtration Day1\tEncFtnsH-decamer fractions\t20.2\t1.8\t11.2\t \tEncFtnsH-monomer fractions\t2.9\t21.9\t0.1\t \tFe(NH4)2(SO4)2/EncFtnsH-monomer\t11.0\t13.0\t0.8\t \tFeSO4-HCl/EncFtnsH-monomer\t11.3\t11.4\t1.0\t \tAnalytical Gel filtration Day2\tEncFtnsH-monomer fractions\t8.3\t22.8\t0.4\t \tCoCl2/EncFtnsH-monomer\t17.7\t14.5\t1.2\t \tMnCl2/EncFtnsH-monomer\t3.1\t30.5\t0.1\t \tZnSO4/EncFtnsH-monomer\t20.4\t9.0\t2.3\t \tFeCl3/EncFtnsH-monomer\t3.9\t28.6\t0.1\t \tAnalytical Gel filtration Day3\tEncFtnsH-monomer fractions\t6.3\t23.4\t0.3\t \tMgSO4/EncFtnsH-monomer\t5.8\t30.2\t0.2\t \tCa acetate/EncFtnsH-monomer\t5.6\t25.2\t0.2\t \t", "section": "TABLE", "ner": [ [ 14, 21, "Monomer", "oligomeric_state" ], [ 27, 34, "Decamer", "oligomeric_state" ], [ 40, 47, "Decamer", "oligomeric_state" ], [ 48, 55, "Monomer", "oligomeric_state" ], [ 58, 72, "Gel filtration", "experimental_method" ], [ 101, 109, "EncFtnsH", "protein" ], [ 110, 112, "MM", "experimental_method" ], [ 130, 138, "EncFtnsH", "protein" ], [ 139, 141, "MM", "experimental_method" ], [ 142, 146, "Fe2+", "chemical" ], [ 166, 191, "Analytical Gel filtration", "experimental_method" ], [ 197, 205, "EncFtnsH", "protein" ], [ 206, 213, "decamer", "oligomeric_state" ], [ 240, 248, "EncFtnsH", "protein" ], [ 249, 256, "monomer", "oligomeric_state" ], [ 282, 296, "Fe(NH4)2(SO4)2", "chemical" ], [ 297, 305, "EncFtnsH", "protein" ], [ 306, 313, "monomer", "oligomeric_state" ], [ 330, 339, "FeSO4-HCl", "chemical" ], [ 340, 348, "EncFtnsH", "protein" ], [ 349, 356, "monomer", "oligomeric_state" ], [ 373, 398, "Analytical Gel filtration", "experimental_method" ], [ 404, 412, "EncFtnsH", "protein" ], [ 413, 420, "monomer", "oligomeric_state" ], [ 446, 451, "CoCl2", "chemical" ], [ 452, 460, "EncFtnsH", "protein" ], [ 461, 468, "monomer", "oligomeric_state" ], [ 485, 490, "MnCl2", "chemical" ], [ 491, 499, "EncFtnsH", "protein" ], [ 500, 507, "monomer", "oligomeric_state" ], [ 523, 528, "ZnSO4", "chemical" ], [ 529, 537, "EncFtnsH", "protein" ], [ 538, 545, "monomer", "oligomeric_state" ], [ 561, 566, "FeCl3", "chemical" ], [ 567, 575, "EncFtnsH", "protein" ], [ 576, 583, "monomer", "oligomeric_state" ], [ 599, 624, "Analytical Gel filtration", "experimental_method" ], [ 630, 638, "EncFtnsH", "protein" ], [ 639, 646, "monomer", "oligomeric_state" ], [ 672, 677, "MgSO4", "chemical" ], [ 678, 686, "EncFtnsH", "protein" ], [ 687, 694, "monomer", "oligomeric_state" ], [ 710, 720, "Ca acetate", "chemical" ], [ 721, 729, "EncFtnsH", "protein" ], [ 730, 737, "monomer", "oligomeric_state" ] ] }, { "sid": 116, "sent": "We purified EncFtnsH from E. coli grown in MM with or without the addition of 1 mM Fe(NH4)2(SO4)2.", "section": "RESULTS", "ner": [ [ 12, 20, "EncFtnsH", "protein" ], [ 26, 33, "E. coli", "species" ], [ 43, 45, "MM", "experimental_method" ], [ 83, 97, "Fe(NH4)2(SO4)2", "chemical" ] ] }, { "sid": 117, "sent": "The decamer to monomer ratio in the sample purified from cells grown in iron-supplemented media was 4.5, while that from the iron-free media was 0.11, suggesting that iron induces the oligomerization of EncFtnsH in vivo\u00a0(Figure 3A, Table 3).", "section": "RESULTS", "ner": [ [ 4, 11, "decamer", "oligomeric_state" ], [ 15, 22, "monomer", "oligomeric_state" ], [ 72, 76, "iron", "chemical" ], [ 125, 134, "iron-free", "protein_state" ], [ 167, 171, "iron", "chemical" ], [ 203, 211, "EncFtnsH", "protein" ] ] }, { "sid": 118, "sent": "To test the metal-dependent oligomerization of EncFtnsH in vitro, we incubated the protein with various metal cations and subjected samples to analytical SEC and non-denaturing PAGE.", "section": "RESULTS", "ner": [ [ 47, 55, "EncFtnsH", "protein" ], [ 69, 78, "incubated", "experimental_method" ], [ 143, 157, "analytical SEC", "experimental_method" ], [ 162, 181, "non-denaturing PAGE", "experimental_method" ] ] }, { "sid": 119, "sent": "Of the metals tested, only Fe2+, Zn2+ and Co2+ induced the formation of significant amounts of the decamer (Figure 3B, Figure 3\u2014figure supplement 1/2).", "section": "RESULTS", "ner": [ [ 27, 32, "Fe2+,", "chemical" ], [ 33, 37, "Zn2+", "chemical" ], [ 42, 46, "Co2+", "chemical" ], [ 99, 106, "decamer", "oligomeric_state" ] ] }, { "sid": 120, "sent": "While Fe2+ induces the multimerization of EncFtnsH, Fe3+ in the form of FeCl3 does not have this effect on the protein, highlighting the apparent preference this protein has for the ferrous form of iron.", "section": "RESULTS", "ner": [ [ 6, 10, "Fe2+", "chemical" ], [ 42, 50, "EncFtnsH", "protein" ], [ 52, 56, "Fe3+", "chemical" ], [ 72, 77, "FeCl3", "chemical" ], [ 182, 202, "ferrous form of iron", "chemical" ] ] }, { "sid": 121, "sent": "To determine if the oligomerization of EncFtnsH was concentration dependent we performed analytical SEC at 90 and 700 \u00b5M protein concentration (Figure 3C).", "section": "RESULTS", "ner": [ [ 39, 47, "EncFtnsH", "protein" ], [ 89, 103, "analytical SEC", "experimental_method" ] ] }, { "sid": 122, "sent": "At the higher concentration, no increase in the decameric form of EncFtn was observed; however, the shift in the major peak from the position of the monomer species indicated a tendency to dimerize at high concentration.", "section": "RESULTS", "ner": [ [ 48, 57, "decameric", "oligomeric_state" ], [ 66, 72, "EncFtn", "protein" ], [ 149, 156, "monomer", "oligomeric_state" ], [ 189, 197, "dimerize", "oligomeric_state" ] ] }, { "sid": 123, "sent": "Crystal structure of EncFtnsH", "section": "RESULTS", "ner": [ [ 0, 17, "Crystal structure", "evidence" ], [ 21, 29, "EncFtnsH", "protein" ] ] }, { "sid": 124, "sent": "Electrostatic surface of EncFtnsH.", "section": "FIG", "ner": [ [ 25, 33, "EncFtnsH", "protein" ] ] }, { "sid": 125, "sent": "The solvent accessible surface of EncFtnsH is shown, colored by electrostatic potential as calculated using the APBS plugin in PyMOL.", "section": "FIG", "ner": [ [ 34, 42, "EncFtnsH", "protein" ] ] }, { "sid": 126, "sent": "Negatively charged regions are colored red and positive regions in blue, neutral regions in grey. (A) View of the surface of the EncFtnsH decamer looking down the central axis.", "section": "FIG", "ner": [ [ 129, 137, "EncFtnsH", "protein" ], [ 138, 145, "decamer", "oligomeric_state" ] ] }, { "sid": 127, "sent": "(B) Orthogonal view of (A). (C) Cutaway view of (B) showing the charge distribution within the central cavity.", "section": "FIG", "ner": [ [ 95, 109, "central cavity", "site" ] ] }, { "sid": 128, "sent": "Crystal structure of EncFtnsH.", "section": "FIG", "ner": [ [ 0, 17, "Crystal structure", "evidence" ], [ 21, 29, "EncFtnsH", "protein" ] ] }, { "sid": 129, "sent": "(A) Overall architecture of EncFtnsH. Transparent solvent accessible surface view with \u03b1-helices shown as tubes and bound metal ions as spheres.", "section": "FIG", "ner": [ [ 28, 36, "EncFtnsH", "protein" ], [ 87, 96, "\u03b1-helices", "structure_element" ] ] }, { "sid": 130, "sent": "Alternating subunits are colored blue and green for clarity.", "section": "FIG", "ner": [ [ 12, 20, "subunits", "structure_element" ] ] }, { "sid": 131, "sent": "The doughnut-like decamer is 7 nm in diameter and 4.5 nm thick. (B) Monomer of EncFtnsH shown as a secondary structure cartoon. (C/D) Dimer interfaces formed in the decameric ring of EncFtnsH. Subunits are shown as secondary structure cartoons and colored blue and green for clarity.", "section": "FIG", "ner": [ [ 4, 17, "doughnut-like", "structure_element" ], [ 18, 25, "decamer", "oligomeric_state" ], [ 68, 75, "Monomer", "oligomeric_state" ], [ 79, 87, "EncFtnsH", "protein" ], [ 134, 150, "Dimer interfaces", "site" ], [ 165, 174, "decameric", "oligomeric_state" ], [ 175, 179, "ring", "structure_element" ], [ 183, 191, "EncFtnsH", "protein" ], [ 193, 201, "Subunits", "structure_element" ] ] }, { "sid": 132, "sent": "Bound metal ions are shown as orange spheres for Fe3+ and grey and white spheres for Ca2+.", "section": "FIG", "ner": [ [ 49, 53, "Fe3+", "chemical" ], [ 85, 89, "Ca2+", "chemical" ] ] }, { "sid": 133, "sent": "We determined the crystal structure of EncFtnsH by molecular replacement to 2.0 \u00c5 resolution (see Table 1 for X-ray data collection and refinement statistics).", "section": "RESULTS", "ner": [ [ 18, 35, "crystal structure", "evidence" ], [ 39, 47, "EncFtnsH", "protein" ], [ 51, 72, "molecular replacement", "experimental_method" ], [ 110, 157, "X-ray data collection and refinement statistics", "evidence" ] ] }, { "sid": 134, "sent": "The crystallographic asymmetric unit contained thirty monomers of EncFtn with visible electron density for residues 7 \u2013 96 in each chain.", "section": "RESULTS", "ner": [ [ 54, 62, "monomers", "oligomeric_state" ], [ 66, 72, "EncFtn", "protein" ], [ 86, 102, "electron density", "evidence" ], [ 116, 122, "7 \u2013 96", "residue_range" ] ] }, { "sid": 135, "sent": "The protein chains were arranged as three identical annular decamers, each with D5 symmetry.", "section": "RESULTS", "ner": [ [ 52, 59, "annular", "structure_element" ], [ 60, 68, "decamers", "oligomeric_state" ] ] }, { "sid": 136, "sent": "The decamer has a diameter of 7 nm and thickness of 4 nm (Figure 4A).", "section": "RESULTS", "ner": [ [ 4, 11, "decamer", "oligomeric_state" ] ] }, { "sid": 137, "sent": "The monomer of EncFtn has an N-terminal 310-helix that precedes two 4 nm long antiparallel \u03b1-helices arranged with their long axes at 25\u00b0 to each other; these helices are followed by a shorter 1.4 nm helix projecting at 70\u00b0 from \u03b12 (Figure 4B).", "section": "RESULTS", "ner": [ [ 4, 11, "monomer", "oligomeric_state" ], [ 15, 21, "EncFtn", "protein" ], [ 40, 49, "310-helix", "structure_element" ], [ 78, 100, "antiparallel \u03b1-helices", "structure_element" ], [ 159, 166, "helices", "structure_element" ], [ 200, 205, "helix", "structure_element" ], [ 229, 231, "\u03b12", "structure_element" ] ] }, { "sid": 138, "sent": "The C-terminal region of the crystallized construct extends from the outer circumference of the ring, indicating that the encapsulin localization sequence in the full-length protein is on the exterior of the ring and is thus free to interact with its binding site on the encapsulin shell protein.", "section": "RESULTS", "ner": [ [ 4, 21, "C-terminal region", "structure_element" ], [ 96, 100, "ring", "structure_element" ], [ 122, 154, "encapsulin localization sequence", "site" ], [ 162, 173, "full-length", "protein_state" ], [ 208, 212, "ring", "structure_element" ], [ 251, 263, "binding site", "site" ], [ 271, 281, "encapsulin", "protein" ], [ 282, 287, "shell", "structure_element" ] ] }, { "sid": 139, "sent": "The monomer of EncFtnsH forms two distinct dimer interfaces within the decamer (Figure 4 C/D).", "section": "RESULTS", "ner": [ [ 4, 11, "monomer", "oligomeric_state" ], [ 15, 23, "EncFtnsH", "protein" ], [ 43, 59, "dimer interfaces", "site" ], [ 71, 78, "decamer", "oligomeric_state" ] ] }, { "sid": 140, "sent": "The first dimer is formed from two monomers arranged antiparallel to each other, with \u03b11 from each monomer interacting along their lengths and \u03b13 interdigitating with \u03b12 and \u03b13 of the partner chain.", "section": "RESULTS", "ner": [ [ 10, 15, "dimer", "oligomeric_state" ], [ 35, 43, "monomers", "oligomeric_state" ], [ 86, 88, "\u03b11", "structure_element" ], [ 99, 106, "monomer", "oligomeric_state" ], [ 143, 145, "\u03b13", "structure_element" ], [ 167, 169, "\u03b12", "structure_element" ], [ 174, 176, "\u03b13", "structure_element" ] ] }, { "sid": 141, "sent": "This interface buries one third of the surface area from each partner and is stabilized by thirty hydrogen bonds and fourteen salt bridges (Figure 4C).", "section": "RESULTS", "ner": [ [ 5, 14, "interface", "site" ], [ 98, 112, "hydrogen bonds", "bond_interaction" ], [ 126, 138, "salt bridges", "bond_interaction" ] ] }, { "sid": 142, "sent": "The second dimer interface forms an antiparallel four-helix bundle between helices 1 and 2 from each monomer (Figure 4D).", "section": "RESULTS", "ner": [ [ 11, 26, "dimer interface", "site" ], [ 36, 66, "antiparallel four-helix bundle", "structure_element" ], [ 75, 90, "helices 1 and 2", "structure_element" ], [ 101, 108, "monomer", "oligomeric_state" ] ] }, { "sid": 143, "sent": "This interface is less extensive than the first and is stabilized by twenty-one hydrogen bonds, six salt bridges, and a number of metal ions.", "section": "RESULTS", "ner": [ [ 5, 14, "interface", "site" ], [ 80, 94, "hydrogen bonds", "bond_interaction" ], [ 100, 112, "salt bridges", "bond_interaction" ] ] }, { "sid": 144, "sent": "The arrangement of ten monomers in alternating orientation forms the decamer of EncFtn, which assembles as a pentamer of dimers (Figure 4A).", "section": "RESULTS", "ner": [ [ 23, 31, "monomers", "oligomeric_state" ], [ 69, 76, "decamer", "oligomeric_state" ], [ 80, 86, "EncFtn", "protein" ], [ 109, 117, "pentamer", "oligomeric_state" ], [ 121, 127, "dimers", "oligomeric_state" ] ] }, { "sid": 145, "sent": "Each monomer lies at 45\u00b0 relative to the vertical central-axis of the ring, with the N-termini of alternating subunits capping the center of the ring at each end, while the C-termini are arranged around the circumference.", "section": "RESULTS", "ner": [ [ 5, 12, "monomer", "oligomeric_state" ], [ 70, 74, "ring", "structure_element" ], [ 110, 118, "subunits", "structure_element" ], [ 145, 149, "ring", "structure_element" ] ] }, { "sid": 146, "sent": "The central hole in the ring is 2.5 nm at its widest in the center of the complex, and 1.5 nm at its narrowest point near the outer surface, although it should be noted that a number of residues at the N-terminus are not visible in the crystallographic electron density and these may occupy the central channel.", "section": "RESULTS", "ner": [ [ 4, 16, "central hole", "site" ], [ 24, 28, "ring", "structure_element" ], [ 236, 269, "crystallographic electron density", "evidence" ], [ 295, 310, "central channel", "site" ] ] }, { "sid": 147, "sent": "The surface of the decamer has distinct negatively charged patches, both within the central hole and on the outer circumference, which form spokes through the radius of the complex (Figure 4\u2014figure supplement 1).", "section": "RESULTS", "ner": [ [ 19, 26, "decamer", "oligomeric_state" ], [ 40, 66, "negatively charged patches", "site" ], [ 84, 96, "central hole", "site" ], [ 140, 146, "spokes", "structure_element" ] ] }, { "sid": 148, "sent": "EncFtn ferroxidase center", "section": "RESULTS", "ner": [ [ 0, 6, "EncFtn", "protein" ], [ 7, 25, "ferroxidase center", "site" ] ] }, { "sid": 149, "sent": "Putative ligand-binding site in EncFtnsH.", "section": "FIG", "ner": [ [ 9, 28, "ligand-binding site", "site" ], [ 32, 40, "EncFtnsH", "protein" ] ] }, { "sid": 150, "sent": "(A) Wall-eyed stereo view of the dimer interface of EncFtn.", "section": "FIG", "ner": [ [ 33, 48, "dimer interface", "site" ], [ 52, 58, "EncFtn", "protein" ] ] }, { "sid": 151, "sent": "Protein chains are shown as sticks, with 2mFo-DFc electron density shown in blue mesh and contoured at 1.5 \u03c3 and mFo-DFc shown in green mesh and contoured at 3 \u03c3. (B) Wall-eyed stereo view of putative metal binding site at the external surface of EncFtnsH. Protein chains and electron density maps are shown as in (A).", "section": "FIG", "ner": [ [ 41, 66, "2mFo-DFc electron density", "evidence" ], [ 113, 120, "mFo-DFc", "evidence" ], [ 201, 219, "metal binding site", "site" ], [ 247, 255, "EncFtnsH", "protein" ], [ 276, 297, "electron density maps", "evidence" ] ] }, { "sid": 152, "sent": "EncFtnsH metal binding sites.", "section": "FIG", "ner": [ [ 0, 8, "EncFtnsH", "protein" ], [ 9, 28, "metal binding sites", "site" ] ] }, { "sid": 153, "sent": "(A) Wall-eyed stereo view of the metal-binding dimerization interface of EncFtnsH. Protein residues are shown as sticks with blue and green carbons for the different subunits, iron ions are shown as orange spheres and calcium as grey spheres, and the glycolic acid ligand is shown with yellow carbon atoms coordinated above the di-iron center.", "section": "FIG", "ner": [ [ 33, 69, "metal-binding dimerization interface", "site" ], [ 73, 81, "EncFtnsH", "protein" ], [ 166, 174, "subunits", "structure_element" ], [ 176, 180, "iron", "chemical" ], [ 218, 225, "calcium", "chemical" ], [ 251, 264, "glycolic acid", "chemical" ], [ 328, 342, "di-iron center", "site" ] ] }, { "sid": 154, "sent": "The 2mFo-DFc electron density map is shown as a blue mesh contoured at 1.5 \u03c3 and the NCS-averaged\u00a0anomalous difference map is shown as an orange mesh and contoured at 10 \u03c3. (B) Iron coordination within the FOC including residues Glu32, Glu62, His65 and Tyr39 from two chains.", "section": "FIG", "ner": [ [ 4, 33, "2mFo-DFc electron density map", "evidence" ], [ 85, 122, "NCS-averaged\u00a0anomalous difference map", "evidence" ], [ 177, 181, "Iron", "chemical" ], [ 182, 194, "coordination", "bond_interaction" ], [ 206, 209, "FOC", "site" ], [ 229, 234, "Glu32", "residue_name_number" ], [ 236, 241, "Glu62", "residue_name_number" ], [ 243, 248, "His65", "residue_name_number" ], [ 253, 258, "Tyr39", "residue_name_number" ] ] }, { "sid": 155, "sent": "Protein and metal ions are shown as in A. Coordination between the protein and iron ions is shown as yellow dashed lines with distances indicated. (C) Coordination of calcium within the dimer interface by four glutamic acid residues (E31 and E34 from two chains).", "section": "FIG", "ner": [ [ 42, 54, "Coordination", "bond_interaction" ], [ 79, 83, "iron", "chemical" ], [ 151, 163, "Coordination", "bond_interaction" ], [ 167, 174, "calcium", "chemical" ], [ 186, 201, "dimer interface", "site" ], [ 210, 223, "glutamic acid", "residue_name" ], [ 234, 237, "E31", "residue_name_number" ], [ 242, 245, "E34", "residue_name_number" ] ] }, { "sid": 156, "sent": "The calcium ion is shown as a grey sphere and water molecules involved in the coordination of the calcium ion are shown as crosses. (D) Metal coordination site on the outer surface of EncFtnsH. The two calcium ions are coordinated by residues His57, Glu61 and Glu64 from the two chains of the FOC dimer, and are located at the outer surface of the complex, positioned 10 \u00c5 away from the FOC iron.", "section": "FIG", "ner": [ [ 4, 11, "calcium", "chemical" ], [ 46, 51, "water", "chemical" ], [ 78, 90, "coordination", "bond_interaction" ], [ 98, 105, "calcium", "chemical" ], [ 136, 159, "Metal coordination site", "site" ], [ 184, 192, "EncFtnsH", "protein" ], [ 202, 209, "calcium", "chemical" ], [ 219, 233, "coordinated by", "bond_interaction" ], [ 243, 248, "His57", "residue_name_number" ], [ 250, 255, "Glu61", "residue_name_number" ], [ 260, 265, "Glu64", "residue_name_number" ], [ 293, 296, "FOC", "site" ], [ 297, 302, "dimer", "oligomeric_state" ], [ 387, 390, "FOC", "site" ], [ 391, 395, "iron", "chemical" ] ] }, { "sid": 157, "sent": "The electron density maps of the initial EncFtnsH model displayed significant positive peaks in the mFo-DFc map at the center of the 4-helix bundle dimer (Figure 5\u2014figure supplement 1).", "section": "RESULTS", "ner": [ [ 4, 25, "electron density maps", "evidence" ], [ 41, 49, "EncFtnsH", "protein" ], [ 100, 111, "mFo-DFc map", "evidence" ], [ 133, 147, "4-helix bundle", "structure_element" ], [ 148, 153, "dimer", "oligomeric_state" ] ] }, { "sid": 158, "sent": "Informed by the ICP-MS data indicating the presence of iron in the protein we collected diffraction data at the experimentally determined iron absorption edge (1.74 \u00c5) and calculated an anomalous difference Fourier map using this data.", "section": "RESULTS", "ner": [ [ 16, 22, "ICP-MS", "experimental_method" ], [ 43, 54, "presence of", "protein_state" ], [ 55, 59, "iron", "chemical" ], [ 88, 104, "diffraction data", "evidence" ], [ 138, 142, "iron", "chemical" ], [ 186, 218, "anomalous difference Fourier map", "evidence" ] ] }, { "sid": 159, "sent": "Inspection of this map showed two 10-sigma peaks between residues Glu32, Glu62 and His65 of two adjacent chains, and a statistically smaller 5-sigma peak between residues Glu31 and Glu34 of the two chains.", "section": "RESULTS", "ner": [ [ 19, 22, "map", "evidence" ], [ 43, 48, "peaks", "evidence" ], [ 66, 71, "Glu32", "residue_name_number" ], [ 73, 78, "Glu62", "residue_name_number" ], [ 83, 88, "His65", "residue_name_number" ], [ 171, 176, "Glu31", "residue_name_number" ], [ 181, 186, "Glu34", "residue_name_number" ] ] }, { "sid": 160, "sent": "Modeling metal ions into these peaks and refinement of the anomalous scattering parameters allowed us to identify these as two iron ions and a calcium ion respectively (Figure 5A).", "section": "RESULTS", "ner": [ [ 41, 51, "refinement", "experimental_method" ], [ 59, 90, "anomalous scattering parameters", "evidence" ], [ 127, 131, "iron", "chemical" ], [ 143, 150, "calcium", "chemical" ] ] }, { "sid": 161, "sent": "An additional region of asymmetric electron density near the di-iron binding site in the mFo-DFc map was modeled as glycolic acid, presumably a breakdown product of the PEG 3350 used for crystallization.", "section": "RESULTS", "ner": [ [ 35, 51, "electron density", "evidence" ], [ 61, 81, "di-iron binding site", "site" ], [ 89, 100, "mFo-DFc map", "evidence" ], [ 116, 129, "glycolic acid", "chemical" ], [ 169, 177, "PEG 3350", "chemical" ] ] }, { "sid": 162, "sent": "This di-iron center has an Fe-Fe distance of 3.5 \u00c5, Fe-Glu-O distances between 2.3 and 2.5 \u00c5, and Fe-His-N distances of 2.5 \u00c5 (Figure 5B).", "section": "RESULTS", "ner": [ [ 5, 19, "di-iron center", "site" ], [ 27, 41, "Fe-Fe distance", "evidence" ], [ 52, 70, "Fe-Glu-O distances", "evidence" ], [ 98, 116, "Fe-His-N distances", "evidence" ] ] }, { "sid": 163, "sent": "This coordination geometry is consistent with the di-nuclear ferroxidase center (FOC) found in ferritin.", "section": "RESULTS", "ner": [ [ 5, 17, "coordination", "bond_interaction" ], [ 50, 79, "di-nuclear ferroxidase center", "site" ], [ 81, 84, "FOC", "site" ], [ 95, 103, "ferritin", "protein_type" ] ] }, { "sid": 164, "sent": "It is interesting to note that although we did not add any additional iron to the crystallization trials, the FOC was fully occupied with iron in the final structure, implying that this site has a very high affinity for iron.", "section": "RESULTS", "ner": [ [ 70, 74, "iron", "chemical" ], [ 82, 104, "crystallization trials", "experimental_method" ], [ 110, 113, "FOC", "site" ], [ 138, 142, "iron", "chemical" ], [ 156, 165, "structure", "evidence" ], [ 207, 215, "affinity", "evidence" ], [ 220, 224, "iron", "chemical" ] ] }, { "sid": 165, "sent": "The calcium ion coordinated by Glu31 and Glu34 adopts heptacoordinate geometry, with coordination distances of 2.5 \u00c5 between the metal ion and carboxylate oxygens of Glu31 and Glu34 (E31/34-site).", "section": "RESULTS", "ner": [ [ 4, 11, "calcium", "chemical" ], [ 16, 30, "coordinated by", "bond_interaction" ], [ 31, 36, "Glu31", "residue_name_number" ], [ 41, 46, "Glu34", "residue_name_number" ], [ 54, 69, "heptacoordinate", "protein_state" ], [ 85, 97, "coordination", "bond_interaction" ], [ 166, 171, "Glu31", "residue_name_number" ], [ 176, 181, "Glu34", "residue_name_number" ], [ 183, 194, "E31/34-site", "site" ] ] }, { "sid": 166, "sent": "A number of ordered solvent molecules are also coordinated to this metal ion at a distance of 2.5 \u00c5. This heptacoordinate geometry is common in crystal structures with calcium ions (Figure 5C).", "section": "RESULTS", "ner": [ [ 47, 58, "coordinated", "bond_interaction" ], [ 106, 121, "heptacoordinate", "protein_state" ], [ 144, 162, "crystal structures", "evidence" ], [ 168, 175, "calcium", "chemical" ] ] }, { "sid": 167, "sent": "While ICP-MS indicated that there were negligible amounts of calcium in the purified protein, the presence of 140 mM calcium acetate in the crystallization mother liquor favors the coordination of calcium at this site.", "section": "RESULTS", "ner": [ [ 6, 12, "ICP-MS", "experimental_method" ], [ 61, 68, "calcium", "chemical" ], [ 98, 109, "presence of", "protein_state" ], [ 117, 132, "calcium acetate", "chemical" ], [ 181, 193, "coordination", "bond_interaction" ], [ 197, 204, "calcium", "chemical" ] ] }, { "sid": 168, "sent": "The fact that the protein does not multimerize in solution in the presence of Fe3+ may indicate that these metal binding sites have a lower affinity for the ferric form of iron, which is the product of the ferroxidase reaction.", "section": "RESULTS", "ner": [ [ 66, 77, "presence of", "protein_state" ], [ 78, 82, "Fe3+", "chemical" ], [ 107, 126, "metal binding sites", "site" ], [ 172, 176, "iron", "chemical" ], [ 206, 217, "ferroxidase", "protein_type" ] ] }, { "sid": 169, "sent": "A number of additional metal-ions were present at the outer circumference of at least one decamer in the asymmetric unit (Figure 5D).", "section": "RESULTS", "ner": [ [ 90, 97, "decamer", "oligomeric_state" ] ] }, { "sid": 170, "sent": "These ions are coordinated by His57, Glu61 and Glu64 from both chains in the FOC dimer and are 4.5 \u00c5 apart; Fe-Glu-O distances are between 2.5 and 3.5 \u00c5 and the Fe-His-N distances are 4 and 4.5 \u00c5.", "section": "RESULTS", "ner": [ [ 15, 29, "coordinated by", "bond_interaction" ], [ 30, 35, "His57", "residue_name_number" ], [ 37, 42, "Glu61", "residue_name_number" ], [ 47, 52, "Glu64", "residue_name_number" ], [ 77, 80, "FOC", "site" ], [ 81, 86, "dimer", "oligomeric_state" ], [ 108, 116, "Fe-Glu-O", "evidence" ], [ 161, 179, "Fe-His-N distances", "evidence" ] ] }, { "sid": 171, "sent": "Comparison of quaternary structure of EncFtnsH and ferritin.", "section": "FIG", "ner": [ [ 38, 46, "EncFtnsH", "protein" ], [ 51, 59, "ferritin", "protein_type" ] ] }, { "sid": 172, "sent": "(A) Aligned FOC of EncFtnsH and Pseudo-nitzschia multiseries ferritin (PmFtn).", "section": "FIG", "ner": [ [ 4, 11, "Aligned", "experimental_method" ], [ 12, 15, "FOC", "site" ], [ 19, 27, "EncFtnsH", "protein" ], [ 32, 60, "Pseudo-nitzschia multiseries", "species" ], [ 61, 69, "ferritin", "protein" ], [ 71, 76, "PmFtn", "protein" ] ] }, { "sid": 173, "sent": "The metal binding site residues from two EncFtnsH chains are shown in green and blue, while the PmFtn is shown in orange.", "section": "FIG", "ner": [ [ 4, 22, "metal binding site", "site" ], [ 41, 49, "EncFtnsH", "protein" ], [ 96, 101, "PmFtn", "protein" ] ] }, { "sid": 174, "sent": "Fe2+ in the FOC is shown as orange spheres and Ca2+ in EncFtnsH is shown as a grey sphere.", "section": "FIG", "ner": [ [ 0, 4, "Fe2+", "chemical" ], [ 12, 15, "FOC", "site" ], [ 47, 51, "Ca2+", "chemical" ], [ 55, 63, "EncFtnsH", "protein" ] ] }, { "sid": 175, "sent": "The two-fold symmetry axis of the EncFtn FOC is shown with a grey arrow (B) Cross-section surface view of quaternary structure of EncFtnsH and PmFtn as aligned in (A) (dashed black box).", "section": "FIG", "ner": [ [ 34, 40, "EncFtn", "protein" ], [ 41, 44, "FOC", "site" ], [ 130, 138, "EncFtnsH", "protein" ], [ 143, 148, "PmFtn", "protein" ] ] }, { "sid": 176, "sent": "The central channel of EncFtnsH is spatially equivalent to the outer surface of ferritin and its outer surface corresponds to the mineralization surface within ferritin.", "section": "FIG", "ner": [ [ 4, 19, "central channel", "site" ], [ 23, 31, "EncFtnsH", "protein" ], [ 80, 88, "ferritin", "protein_type" ], [ 130, 152, "mineralization surface", "site" ], [ 160, 168, "ferritin", "protein_type" ] ] }, { "sid": 177, "sent": "Comparison of the symmetric metal ion binding site of EncFtnsH and the ferritin FOC.", "section": "FIG", "ner": [ [ 0, 10, "Comparison", "experimental_method" ], [ 28, 50, "metal ion binding site", "site" ], [ 54, 62, "EncFtnsH", "protein" ], [ 71, 79, "ferritin", "protein_type" ], [ 80, 83, "FOC", "site" ] ] }, { "sid": 178, "sent": "(A) Structural alignment of the FOC residues in a dimer of EncFtnsH (green/blue) with a monomer of Pseudo-nitzschia multiseries ferritin (PmFtn) (PDBID: 4ITW) (orange).", "section": "FIG", "ner": [ [ 4, 24, "Structural alignment", "experimental_method" ], [ 32, 35, "FOC", "site" ], [ 50, 55, "dimer", "oligomeric_state" ], [ 59, 67, "EncFtnsH", "protein" ], [ 88, 95, "monomer", "oligomeric_state" ], [ 99, 127, "Pseudo-nitzschia multiseries", "species" ], [ 128, 136, "ferritin", "protein" ], [ 138, 143, "PmFtn", "protein" ] ] }, { "sid": 179, "sent": "Iron ions are shown as orange spheres and a single calcium ion as a grey sphere.", "section": "FIG", "ner": [ [ 0, 4, "Iron", "chemical" ], [ 51, 58, "calcium", "chemical" ] ] }, { "sid": 180, "sent": "Residues within the FOC are conserved between EncFtn and ferritin PmFtn, with the exception of residues in the position equivalent to H65\u2019 in the second subunit in the dimer (blue).", "section": "FIG", "ner": [ [ 20, 23, "FOC", "site" ], [ 28, 37, "conserved", "protein_state" ], [ 46, 52, "EncFtn", "protein" ], [ 57, 65, "ferritin", "protein_type" ], [ 66, 71, "PmFtn", "protein" ], [ 134, 137, "H65", "residue_name_number" ], [ 153, 160, "subunit", "oligomeric_state" ], [ 168, 173, "dimer", "oligomeric_state" ] ] }, { "sid": 181, "sent": "The site in EncFtn with bound calcium is not present in other family members.", "section": "FIG", "ner": [ [ 12, 18, "EncFtn", "protein" ], [ 24, 29, "bound", "protein_state" ], [ 30, 37, "calcium", "chemical" ] ] }, { "sid": 182, "sent": "(B) Secondary structure of aligned dimeric EncFtnsH and monomeric ferritin highlighting the conserved four-helix bundle.", "section": "FIG", "ner": [ [ 27, 34, "aligned", "experimental_method" ], [ 35, 42, "dimeric", "oligomeric_state" ], [ 43, 51, "EncFtnsH", "protein" ], [ 56, 65, "monomeric", "oligomeric_state" ], [ 66, 74, "ferritin", "protein_type" ], [ 92, 101, "conserved", "protein_state" ], [ 102, 119, "four-helix bundle", "structure_element" ] ] }, { "sid": 183, "sent": "EncFtnsH monomers are shown in green and blue and aligned PmFtn monomer in orange as in A. (C) Cartoon of secondary structure elements in EncFtn dimer and ferritin.", "section": "FIG", "ner": [ [ 0, 8, "EncFtnsH", "protein" ], [ 9, 17, "monomers", "oligomeric_state" ], [ 50, 57, "aligned", "experimental_method" ], [ 58, 63, "PmFtn", "protein" ], [ 64, 71, "monomer", "oligomeric_state" ], [ 138, 144, "EncFtn", "protein" ], [ 145, 150, "dimer", "oligomeric_state" ], [ 155, 163, "ferritin", "protein_type" ] ] }, { "sid": 184, "sent": "In the dimer of EncFtn that forms the FOC, the C-terminus of the first monomer (green) and N-terminus of the second monomer (blue) correspond to the position of the long linker between \u03b12 and \u03b13 in ferritin PmFtn.", "section": "FIG", "ner": [ [ 7, 12, "dimer", "oligomeric_state" ], [ 16, 22, "EncFtn", "protein" ], [ 38, 41, "FOC", "site" ], [ 71, 78, "monomer", "oligomeric_state" ], [ 116, 123, "monomer", "oligomeric_state" ], [ 165, 176, "long linker", "structure_element" ], [ 185, 187, "\u03b12", "structure_element" ], [ 192, 194, "\u03b13", "structure_element" ], [ 198, 206, "ferritin", "protein_type" ], [ 207, 212, "PmFtn", "protein" ] ] }, { "sid": 185, "sent": "Structural alignment of the di-iron binding site of EncFtnsH to the FOC of Pseudo-nitzschia multiseries ferritin (PmFtn, PDB ID: 4ITW) reveals a striking similarity between the metal binding sites of EncFtnsH and the classical ferritins\u00a0\u00a0(Figure 6A).", "section": "RESULTS", "ner": [ [ 0, 20, "Structural alignment", "experimental_method" ], [ 28, 48, "di-iron binding site", "site" ], [ 52, 60, "EncFtnsH", "protein" ], [ 68, 71, "FOC", "site" ], [ 75, 103, "Pseudo-nitzschia multiseries", "species" ], [ 104, 112, "ferritin", "protein_type" ], [ 114, 119, "PmFtn", "protein" ], [ 177, 196, "metal binding sites", "site" ], [ 200, 208, "EncFtnsH", "protein" ], [ 217, 226, "classical", "protein_state" ], [ 227, 236, "ferritins", "protein_type" ] ] }, { "sid": 186, "sent": "The di-iron site of EncFtnsH is by necessity symmetrical, as it is formed through a dimer interface, while the FOC of ferritin does not have these constraints and varies in different species at a position equivalent to His65 of the second EncFtn monomer in the FOC interface (His65\u2019) (Figure 6A).", "section": "RESULTS", "ner": [ [ 4, 16, "di-iron site", "site" ], [ 20, 28, "EncFtnsH", "protein" ], [ 84, 99, "dimer interface", "site" ], [ 111, 114, "FOC", "site" ], [ 118, 126, "ferritin", "protein_type" ], [ 219, 224, "His65", "residue_name_number" ], [ 239, 245, "EncFtn", "protein" ], [ 246, 253, "monomer", "oligomeric_state" ], [ 261, 274, "FOC interface", "site" ], [ 276, 281, "His65", "residue_name_number" ] ] }, { "sid": 187, "sent": "Structural superimposition of the FOCs of ferritin and EncFtn brings the four-helix bundle of the ferritin fold into close alignment with the EncFtn dimer, showing that the two families of proteins have essentially the same architecture around the di-iron center (Figure 6B).", "section": "RESULTS", "ner": [ [ 0, 26, "Structural superimposition", "experimental_method" ], [ 34, 38, "FOCs", "site" ], [ 42, 50, "ferritin", "protein_type" ], [ 55, 61, "EncFtn", "protein" ], [ 73, 90, "four-helix bundle", "structure_element" ], [ 98, 106, "ferritin", "protein_type" ], [ 142, 148, "EncFtn", "protein" ], [ 149, 154, "dimer", "oligomeric_state" ], [ 248, 262, "di-iron center", "site" ] ] }, { "sid": 188, "sent": "The linker connecting helices 2 and 3 of ferritin is congruent with the start of the C-terminal helix of one EncFtn monomer and the N-terminal 310 helix of the second monomer (Figure 6C).", "section": "RESULTS", "ner": [ [ 4, 10, "linker", "structure_element" ], [ 22, 37, "helices 2 and 3", "structure_element" ], [ 41, 49, "ferritin", "protein_type" ], [ 96, 101, "helix", "structure_element" ], [ 109, 115, "EncFtn", "protein" ], [ 116, 123, "monomer", "oligomeric_state" ], [ 143, 152, "310 helix", "structure_element" ], [ 167, 174, "monomer", "oligomeric_state" ] ] }, { "sid": 189, "sent": "Mass spectrometry of the EncFtn assembly", "section": "RESULTS", "ner": [ [ 0, 17, "Mass spectrometry", "experimental_method" ], [ 25, 31, "EncFtn", "protein" ] ] }, { "sid": 190, "sent": "Native IM-MS analysis of the apo-EncFtnsH monomer.", "section": "FIG", "ner": [ [ 0, 12, "Native IM-MS", "experimental_method" ], [ 29, 32, "apo", "protein_state" ], [ 33, 41, "EncFtnsH", "protein" ], [ 42, 49, "monomer", "oligomeric_state" ] ] }, { "sid": 191, "sent": "(A) Mass spectrum of apo-EncFtnsH acquired from 100 mM ammonium acetate pH 8.0 under native MS conditions.", "section": "FIG", "ner": [ [ 4, 17, "Mass spectrum", "evidence" ], [ 21, 24, "apo", "protein_state" ], [ 25, 33, "EncFtnsH", "protein" ], [ 85, 94, "native MS", "experimental_method" ] ] }, { "sid": 192, "sent": "The charge state distribution observed is bimodal, with peaks corresponding to the 6+ to 15+ charge states of apo-monomer EncFtnsH (neutral average mass 13,194.3 Da). (B) The arrival time distributions (ion mobility data) of all ions in the apo-EncFtnsH charge state distribution displayed as a greyscale heat map (linear intensity scale). (B) Right, the arrival time distribution of the 6+ (orange) and 7+ (green) charge state (dashed colored\u2010box) has been extracted and plotted; The arrival time distributions for these ion is shown (ms), along with the calibrated collision cross section, \u03a9 (nm2). (C) The collision cross section of a single monomer unit from the crystal structure of the Fe-loaded EncFtnsH decamer was calculated to be 15.8 nm2\u00a0using IMPACT v. 0.9.1.", "section": "FIG", "ner": [ [ 4, 16, "charge state", "evidence" ], [ 56, 61, "peaks", "evidence" ], [ 93, 106, "charge states", "evidence" ], [ 110, 113, "apo", "protein_state" ], [ 114, 121, "monomer", "oligomeric_state" ], [ 122, 130, "EncFtnsH", "protein" ], [ 175, 201, "arrival time distributions", "evidence" ], [ 203, 220, "ion mobility data", "evidence" ], [ 241, 244, "apo", "protein_state" ], [ 245, 253, "EncFtnsH", "protein" ], [ 254, 266, "charge state", "evidence" ], [ 355, 380, "arrival time distribution", "evidence" ], [ 415, 427, "charge state", "evidence" ], [ 485, 511, "arrival time distributions", "evidence" ], [ 567, 590, "collision cross section", "evidence" ], [ 592, 593, "\u03a9", "evidence" ], [ 609, 632, "collision cross section", "evidence" ], [ 645, 652, "monomer", "oligomeric_state" ], [ 667, 684, "crystal structure", "evidence" ], [ 692, 701, "Fe-loaded", "protein_state" ], [ 702, 710, "EncFtnsH", "protein" ], [ 711, 718, "decamer", "oligomeric_state" ] ] }, { "sid": 193, "sent": "The +8 to +15 protein charge states have observed CCS between 20\u201326 nm2, which is significantly higher than the calculated CCS for an EncFtnsH monomer taken from the decameric assembly crystal structure (15.8 nm2).", "section": "FIG", "ner": [ [ 22, 35, "charge states", "evidence" ], [ 50, 53, "CCS", "evidence" ], [ 123, 126, "CCS", "evidence" ], [ 134, 142, "EncFtnsH", "protein" ], [ 143, 150, "monomer", "oligomeric_state" ], [ 166, 175, "decameric", "oligomeric_state" ], [ 185, 202, "crystal structure", "evidence" ] ] }, { "sid": 194, "sent": "The mobility of the +7 charge state displays broad drift-time distribution with maxima consistent with CCS of 15.9 and 17.9 nm2.", "section": "FIG", "ner": [ [ 4, 12, "mobility", "evidence" ], [ 23, 35, "charge state", "evidence" ], [ 51, 74, "drift-time distribution", "evidence" ], [ 103, 106, "CCS", "evidence" ] ] }, { "sid": 195, "sent": "Finally, the 6+ charge state of EncFtnsH has mobility consistent with a CCS of 12.3 nm2, indicating a more compact/collapsed structure.", "section": "FIG", "ner": [ [ 16, 28, "charge state", "evidence" ], [ 32, 40, "EncFtnsH", "protein" ], [ 45, 53, "mobility", "evidence" ], [ 72, 75, "CCS", "evidence" ], [ 107, 114, "compact", "protein_state" ], [ 115, 124, "collapsed", "protein_state" ] ] }, { "sid": 196, "sent": "It is clear from this data that apo-EncFtnsH exists in several gas phase conformations.", "section": "FIG", "ner": [ [ 32, 35, "apo", "protein_state" ], [ 36, 44, "EncFtnsH", "protein" ] ] }, { "sid": 197, "sent": "The range of charge states occupied by the protein (6+ to 15+) and the range of CCS in which the protein is observed (12.3 nm2 \u2013 26 nm2) are both large.", "section": "FIG", "ner": [ [ 13, 26, "charge states", "evidence" ], [ 80, 83, "CCS", "evidence" ] ] }, { "sid": 198, "sent": "In addition, many of the charge states observed have higher charge than the theoretical maximal charge on spherical globular protein, as determined by the De La Mora relationship (ZR = 0.0778m; for the EncFtnsH monomer ZR = 8.9) Fernandez.", "section": "FIG", "ner": [ [ 25, 38, "charge states", "evidence" ], [ 116, 124, "globular", "protein_state" ], [ 155, 178, "De La Mora relationship", "experimental_method" ], [ 180, 182, "ZR", "evidence" ], [ 202, 210, "EncFtnsH", "protein" ], [ 211, 218, "monomer", "oligomeric_state" ], [ 219, 221, "ZR", "evidence" ] ] }, { "sid": 199, "sent": "As described by Beveridge et al., all these factors are indicative of a disordered protein.", "section": "FIG", "ner": [ [ 72, 82, "disordered", "protein_state" ] ] }, { "sid": 200, "sent": "Gas-phase disassembly of the holo-EncFtnsH decameric assembly.", "section": "FIG", "ner": [ [ 29, 33, "holo", "protein_state" ], [ 34, 42, "EncFtnsH", "protein" ], [ 43, 52, "decameric", "oligomeric_state" ] ] }, { "sid": 201, "sent": "The entire charge state distribution of the Fe-loaded holo- EncFtnsH assembly (green circles) was subject to collisional-induced dissociation (CID) by increasing the source cone voltage to 200 V and the trap voltage to 50 V. The resulting CID mass spectrum (A) revealed that dissociation of the holo- EncFtnsH decamer primarily occurred via ejection of a highly charged monomer (blue circles), leaving the \u2018stripped\u2019 complex (a 9mer; 118.7 kDa; yellow circles).", "section": "FIG", "ner": [ [ 11, 23, "charge state", "evidence" ], [ 44, 53, "Fe-loaded", "protein_state" ], [ 54, 58, "holo", "protein_state" ], [ 60, 68, "EncFtnsH", "protein" ], [ 109, 141, "collisional-induced dissociation", "experimental_method" ], [ 143, 146, "CID", "experimental_method" ], [ 239, 242, "CID", "experimental_method" ], [ 243, 256, "mass spectrum", "evidence" ], [ 295, 299, "holo", "protein_state" ], [ 301, 309, "EncFtnsH", "protein" ], [ 310, 317, "decamer", "oligomeric_state" ], [ 370, 377, "monomer", "oligomeric_state" ], [ 407, 415, "stripped", "protein_state" ], [ 428, 432, "9mer", "oligomeric_state" ] ] }, { "sid": 202, "sent": "The mass of the ejected-monomer is consistent with apo- EncFtnsH (13.2 kDa), suggesting unfolding of the monomer (and loss of Fe) occurs during ejection from the complex.", "section": "FIG", "ner": [ [ 24, 31, "monomer", "oligomeric_state" ], [ 51, 54, "apo", "protein_state" ], [ 56, 64, "EncFtnsH", "protein" ], [ 105, 112, "monomer", "oligomeric_state" ], [ 118, 125, "loss of", "protein_state" ], [ 126, 128, "Fe", "chemical" ] ] }, { "sid": 203, "sent": "This observation of asymmetric charge partitioning of the sub-complexes with respect to the mass of the complex is consistent with the 'typical' pathway of dissociation of protein assemblies by CID, as described by.", "section": "FIG", "ner": [ [ 194, 197, "CID", "experimental_method" ] ] }, { "sid": 204, "sent": "In addition, a third, lower abundance, charge state distribution is observed which overlaps the EncFtn ejected monomer charge state distribution; this region of the spectrum is highlighted in (B).", "section": "FIG", "ner": [ [ 39, 51, "charge state", "evidence" ], [ 96, 102, "EncFtn", "protein" ], [ 111, 118, "monomer", "oligomeric_state" ], [ 119, 131, "charge state", "evidence" ] ] }, { "sid": 205, "sent": "This distribution is consistent with an ejected EncFtnsH dimer (orange circles).", "section": "FIG", "ner": [ [ 48, 56, "EncFtnsH", "protein" ], [ 57, 62, "dimer", "oligomeric_state" ] ] }, { "sid": 206, "sent": "Interestingly, closer analysis of the individual charge state of this dimeric CID product shows that this sub-complex exists in three forms \u2013 displaying mass consistent with an EncFtnsH dimer binding 0, 1, and 2 Fe ions.", "section": "FIG", "ner": [ [ 49, 61, "charge state", "evidence" ], [ 70, 77, "dimeric", "oligomeric_state" ], [ 78, 81, "CID", "experimental_method" ], [ 177, 185, "EncFtnsH", "protein" ], [ 186, 191, "dimer", "oligomeric_state" ], [ 212, 214, "Fe", "chemical" ] ] }, { "sid": 207, "sent": "This is highlighted in (C), where the 15+ charge state of the EncFtnsH dimer is shown; 3 peaks are observed with m/z 1760.5, 1763.8, and 1767.0 Th \u2013 the lowest peak corresponds to neutral masses of 26392.5 Da [predicted EncFtnsH dimer, (C572H884N172O185S2)2;\u00a026388.6 Da].", "section": "FIG", "ner": [ [ 42, 54, "charge state", "evidence" ], [ 62, 70, "EncFtnsH", "protein" ], [ 71, 76, "dimer", "oligomeric_state" ], [ 89, 94, "peaks", "evidence" ], [ 220, 228, "EncFtnsH", "protein" ], [ 229, 234, "dimer", "oligomeric_state" ] ] }, { "sid": 208, "sent": "The two further peaks have a delta-mass of ~+50 Da, consistent with Fe binding.", "section": "FIG", "ner": [ [ 16, 21, "peaks", "evidence" ], [ 68, 70, "Fe", "chemical" ] ] }, { "sid": 209, "sent": "We interpret these observations as partial \u2018atypical\u2019 CID fragmentation of the decameric complex \u2013 i.e. fragmentation of the initial complex with retention of subunit and ligand interactions.", "section": "FIG", "ner": [ [ 54, 57, "CID", "experimental_method" ], [ 79, 88, "decameric", "oligomeric_state" ] ] }, { "sid": 210, "sent": "We postulate the high stability of this iron-bound dimer sub-complex is due to the metal coordination at the dimer interface, increasing the strength of the dimer interface.", "section": "FIG", "ner": [ [ 40, 50, "iron-bound", "protein_state" ], [ 51, 56, "dimer", "oligomeric_state" ], [ 83, 88, "metal", "chemical" ], [ 89, 101, "coordination", "bond_interaction" ], [ 109, 124, "dimer interface", "site" ], [ 157, 172, "dimer interface", "site" ] ] }, { "sid": 211, "sent": "Taken together, these observations support our findings that the topology of the decameric EncFtnsH assembly is arranged as a pentamer of dimers, with two Fe ions at each dimer interface.", "section": "FIG", "ner": [ [ 81, 90, "decameric", "oligomeric_state" ], [ 91, 99, "EncFtnsH", "protein" ], [ 126, 134, "pentamer", "oligomeric_state" ], [ 138, 144, "dimers", "oligomeric_state" ], [ 155, 157, "Fe", "chemical" ], [ 171, 186, "dimer interface", "site" ] ] }, { "sid": 212, "sent": "Native mass spectrometry and ion mobility analysis of iron loading in EncFtnsH.", "section": "FIG", "ner": [ [ 0, 24, "Native mass spectrometry", "experimental_method" ], [ 29, 50, "ion mobility analysis", "experimental_method" ], [ 54, 58, "iron", "chemical" ], [ 70, 78, "EncFtnsH", "protein" ] ] }, { "sid": 213, "sent": "All spectra were acquired in 100 mM ammonium acetate, pH 8.0 with a protein concentration of 5 \u00b5M. (A) Native nanoelectrospray ionization (nESI) mass spectrometry of EncFtnsH at varying iron concentrations.", "section": "FIG", "ner": [ [ 4, 11, "spectra", "evidence" ], [ 45, 52, "acetate", "chemical" ], [ 103, 137, "Native nanoelectrospray ionization", "experimental_method" ], [ 139, 143, "nESI", "experimental_method" ], [ 145, 162, "mass spectrometry", "experimental_method" ], [ 166, 174, "EncFtnsH", "protein" ], [ 186, 190, "iron", "chemical" ] ] }, { "sid": 214, "sent": "A1, nESI spectrum of iron-free EncFtnsH displays a charge state distribution consistent with EncFtnsH monomer (blue circles, 13,194 Da).", "section": "FIG", "ner": [ [ 4, 8, "nESI", "experimental_method" ], [ 9, 17, "spectrum", "evidence" ], [ 21, 30, "iron-free", "protein_state" ], [ 31, 39, "EncFtnsH", "protein" ], [ 51, 63, "charge state", "evidence" ], [ 93, 101, "EncFtnsH", "protein" ], [ 102, 109, "monomer", "oligomeric_state" ] ] }, { "sid": 215, "sent": "Addition of 100 \u00b5M (A2) and 300 \u00b5M (A3) Fe2+ results in the appearance of a second higher molecular weight charge state distribution consistent with a decameric assembly of EncFtnsH (green circles, 132.6 kDa).", "section": "FIG", "ner": [ [ 40, 44, "Fe2+", "chemical" ], [ 90, 106, "molecular weight", "evidence" ], [ 107, 119, "charge state", "evidence" ], [ 151, 160, "decameric", "oligomeric_state" ], [ 173, 181, "EncFtnsH", "protein" ] ] }, { "sid": 216, "sent": "(B) Ion mobility (IM)-MS of the iron-bound holo-EncFtnsH decamer.", "section": "FIG", "ner": [ [ 4, 24, "Ion mobility (IM)-MS", "experimental_method" ], [ 32, 42, "iron-bound", "protein_state" ], [ 43, 47, "holo", "protein_state" ], [ 48, 56, "EncFtnsH", "protein" ], [ 57, 64, "decamer", "oligomeric_state" ] ] }, { "sid": 217, "sent": "Top, Peaks corresponding to the 22+ to 26+ charge states of a homo-decameric assembly of EncFtnsH are observed (132.6 kDa).", "section": "FIG", "ner": [ [ 5, 10, "Peaks", "evidence" ], [ 43, 56, "charge states", "evidence" ], [ 62, 76, "homo-decameric", "oligomeric_state" ], [ 89, 97, "EncFtnsH", "protein" ] ] }, { "sid": 218, "sent": "Top Insert, Analysis of the 24+ charge state of the assembly at m/z 5528.2 Th.", "section": "FIG", "ner": [ [ 32, 44, "charge state", "evidence" ] ] }, { "sid": 219, "sent": "The theoretical average m/z of the 24+ charge state with no additional metals bound is marked by a red line (5498.7 Th); the observed m/z of the 24+ charge state indicates that the EncFtnsH assembly binds between 10 (green line, 5521.1 Th) and 15 Fe ions (blue line, 5532.4 Th) per decamer.", "section": "FIG", "ner": [ [ 39, 51, "charge state", "evidence" ], [ 149, 161, "charge state", "evidence" ], [ 181, 189, "EncFtnsH", "protein" ], [ 247, 249, "Fe", "chemical" ], [ 282, 289, "decamer", "oligomeric_state" ] ] }, { "sid": 220, "sent": "Bottom, The arrival time distributions (ion mobility data) of all ions in the EncFtnsH charge state distribution displayed as a greyscale heat map (linear intensity scale).", "section": "FIG", "ner": [ [ 12, 38, "arrival time distributions", "evidence" ], [ 40, 57, "ion mobility data", "evidence" ], [ 78, 86, "EncFtnsH", "protein" ], [ 87, 99, "charge state", "evidence" ] ] }, { "sid": 221, "sent": "Bottom right, The arrival time distribution of the 24+ charge state (dashed blue box) has been extracted and plotted.", "section": "FIG", "ner": [ [ 18, 43, "arrival time distribution", "evidence" ], [ 55, 67, "charge state", "evidence" ] ] }, { "sid": 222, "sent": "The drift time for this ion is shown (ms), along with the calibrated collision cross section (CCS), \u03a9 (nm2).", "section": "FIG", "ner": [ [ 4, 14, "drift time", "evidence" ], [ 69, 92, "collision cross section", "evidence" ], [ 94, 97, "CCS", "evidence" ], [ 100, 101, "\u03a9", "evidence" ] ] }, { "sid": 223, "sent": "In order to confirm the assignment of the oligomeric state of EncFtnsH and investigate further the Fe2+-dependent assembly, we used native nano-electrospray ionization (nESI) and ion-mobility mass spectrometry (IM-MS).", "section": "RESULTS", "ner": [ [ 62, 70, "EncFtnsH", "protein" ], [ 99, 103, "Fe2+", "chemical" ], [ 132, 167, "native nano-electrospray ionization", "experimental_method" ], [ 169, 173, "nESI", "experimental_method" ], [ 179, 209, "ion-mobility mass spectrometry", "experimental_method" ], [ 211, 216, "IM-MS", "experimental_method" ] ] }, { "sid": 224, "sent": "As described above, by recombinant production of EncFtnsH in minimal media we were able to limit the bioavailability of iron.", "section": "RESULTS", "ner": [ [ 23, 45, "recombinant production", "experimental_method" ], [ 49, 57, "EncFtnsH", "protein" ], [ 120, 124, "iron", "chemical" ] ] }, { "sid": 225, "sent": "Native MS analysis of EncFtnsH produced in this way displayed a charge state distribution consistent with an EncFtnsH monomer (blue circles, Figure 7A1) with an average neutral mass of 13,194 Da, in agreement with the predicted mass of the EncFtnsH protein (13,194.53 Da).", "section": "RESULTS", "ner": [ [ 0, 9, "Native MS", "experimental_method" ], [ 22, 30, "EncFtnsH", "protein" ], [ 64, 76, "charge state", "evidence" ], [ 109, 117, "EncFtnsH", "protein" ], [ 118, 125, "monomer", "oligomeric_state" ], [ 240, 248, "EncFtnsH", "protein" ] ] }, { "sid": 226, "sent": "Titration with Fe2+ directly before native MS analysis resulted in the appearance of a new charge state distribution, consistent with an EncFtnsH decameric assembly (+22 to +26; 132.65 kDa) (Figure 7A2/3).", "section": "RESULTS", "ner": [ [ 0, 9, "Titration", "experimental_method" ], [ 15, 19, "Fe2+", "chemical" ], [ 36, 45, "native MS", "experimental_method" ], [ 91, 103, "charge state", "evidence" ], [ 137, 145, "EncFtnsH", "protein" ], [ 146, 155, "decameric", "oligomeric_state" ] ] }, { "sid": 227, "sent": "After instrument optimization, the mass resolving power achieved was sufficient to assign iron-loading in the complex to between 10 and 15 Fe ions per decamer (Figure 7B, inset top right), consistent with the presence of 10 irons in the FOC and the coordination of iron in the Glu31/34-site occupied by calcium in the crystal structure (\u0394mass observed ~0.67 kDa).", "section": "RESULTS", "ner": [ [ 90, 94, "iron", "chemical" ], [ 139, 141, "Fe", "chemical" ], [ 151, 158, "decamer", "oligomeric_state" ], [ 209, 220, "presence of", "protein_state" ], [ 224, 229, "irons", "chemical" ], [ 237, 240, "FOC", "site" ], [ 249, 261, "coordination", "bond_interaction" ], [ 265, 269, "iron", "chemical" ], [ 277, 290, "Glu31/34-site", "site" ], [ 303, 310, "calcium", "chemical" ], [ 318, 335, "crystal structure", "evidence" ], [ 337, 342, "\u0394mass", "evidence" ] ] }, { "sid": 228, "sent": "MS analysis of EncFtnsH after addition of further Fe2+ did not result in iron loading above this stoichiometry.", "section": "RESULTS", "ner": [ [ 0, 2, "MS", "experimental_method" ], [ 15, 23, "EncFtnsH", "protein" ], [ 50, 54, "Fe2+", "chemical" ], [ 73, 77, "iron", "chemical" ] ] }, { "sid": 229, "sent": "Therefore, the extent of iron binding seen is limited to the FOC and Glu31/34 secondary metal binding site.", "section": "RESULTS", "ner": [ [ 25, 29, "iron", "chemical" ], [ 61, 64, "FOC", "site" ], [ 69, 106, "Glu31/34 secondary metal binding site", "site" ] ] }, { "sid": 230, "sent": "These data suggest that the decameric assembly of EncFtnsH does not accrue iron in the same manner as classical ferritin, which is able to sequester around 4500 iron ions within its nanocage.", "section": "RESULTS", "ner": [ [ 28, 37, "decameric", "oligomeric_state" ], [ 50, 58, "EncFtnsH", "protein" ], [ 75, 79, "iron", "chemical" ], [ 102, 111, "classical", "protein_state" ], [ 112, 120, "ferritin", "protein_type" ], [ 161, 165, "iron", "chemical" ], [ 182, 190, "nanocage", "complex_assembly" ] ] }, { "sid": 231, "sent": "Ion mobility analysis of the EncFtnsH decameric assembly, collected with minimal collisional activation, suggested that it consists of a single conformation with a collision cross section (CCS) of 58.2 nm2\u00a0(Figure 7B).", "section": "RESULTS", "ner": [ [ 0, 21, "Ion mobility analysis", "experimental_method" ], [ 29, 37, "EncFtnsH", "protein" ], [ 38, 47, "decameric", "oligomeric_state" ], [ 164, 187, "collision cross section", "evidence" ], [ 189, 192, "CCS", "evidence" ] ] }, { "sid": 232, "sent": "This observation is in agreement with the calculated CCS of 58.7 nm2derived from our crystal structure of the EncFtnsH decamer.", "section": "RESULTS", "ner": [ [ 53, 56, "CCS", "evidence" ], [ 85, 102, "crystal structure", "evidence" ], [ 110, 118, "EncFtnsH", "protein" ], [ 119, 126, "decamer", "oligomeric_state" ] ] }, { "sid": 233, "sent": "By contrast, IM-MS measurements of the monomeric EncFtnsH at pH 8.0 under the same instrumental conditions revealed that the metal-free protein monomer exists in a wide range of charge states (+6 to +16) and adopts many conformations in the gas phase with collision cross sections ranging from 12 nm2\u00a0to 26 nm2 (Figure 7\u2014figure supplement 1).", "section": "RESULTS", "ner": [ [ 13, 18, "IM-MS", "experimental_method" ], [ 39, 48, "monomeric", "oligomeric_state" ], [ 49, 57, "EncFtnsH", "protein" ], [ 61, 67, "pH 8.0", "protein_state" ], [ 125, 135, "metal-free", "protein_state" ], [ 136, 143, "protein", "protein" ], [ 144, 151, "monomer", "oligomeric_state" ], [ 178, 191, "charge states", "evidence" ] ] }, { "sid": 234, "sent": "Thus, IM-MS studies highlight that higher order structure in EncFtnsH is mediated/stabilized by metal binding, an observation that is in agreement with our solution studies.", "section": "RESULTS", "ner": [ [ 6, 11, "IM-MS", "experimental_method" ], [ 61, 69, "EncFtnsH", "protein" ] ] }, { "sid": 235, "sent": "Taken together, these results suggest that di-iron binding, forming the FOC in EncFtnsH, is required to stabilize the 4-helix bundle dimer interface, essentially reconstructing the classical ferritin-like fold; once stabilized, these dimers readily associate as pentamers, and the overall assembly adopts the decameric ring arrangement observed in the crystal structure.", "section": "RESULTS", "ner": [ [ 46, 50, "iron", "chemical" ], [ 72, 75, "FOC", "site" ], [ 79, 87, "EncFtnsH", "protein" ], [ 118, 132, "4-helix bundle", "structure_element" ], [ 133, 148, "dimer interface", "site" ], [ 181, 190, "classical", "protein_state" ], [ 191, 199, "ferritin", "protein_type" ], [ 234, 240, "dimers", "oligomeric_state" ], [ 309, 318, "decameric", "oligomeric_state" ], [ 352, 369, "crystal structure", "evidence" ] ] }, { "sid": 236, "sent": "We subsequently performed gas phase disassembly of the decameric EncFtnsH using collision-induced dissociation (CID) tandem mass spectrometry.", "section": "RESULTS", "ner": [ [ 55, 64, "decameric", "oligomeric_state" ], [ 65, 73, "EncFtnsH", "protein" ], [ 80, 110, "collision-induced dissociation", "experimental_method" ], [ 112, 115, "CID", "experimental_method" ], [ 117, 141, "tandem mass spectrometry", "experimental_method" ] ] }, { "sid": 237, "sent": "Under the correct CID conditions, protein assemblies can dissociate with retention of subunit and ligand interactions, and thus provide structurally-informative evidence as to the topology of the original assembly; this has been termed \u2018atypical\u2019 dissociation.", "section": "RESULTS", "ner": [ [ 18, 21, "CID", "experimental_method" ] ] }, { "sid": 238, "sent": "For EncFtnsH, this atypical dissociation pathway was clearly evident; CID of the EncFtnsH decamer resulted in the appearance of a dimeric EncFtnsH subcomplex containing 0, 1, or 2 iron ions (Figure 7\u2014figure supplement 2).", "section": "RESULTS", "ner": [ [ 4, 12, "EncFtnsH", "protein" ], [ 70, 73, "CID", "experimental_method" ], [ 81, 89, "EncFtnsH", "protein" ], [ 90, 97, "decamer", "oligomeric_state" ], [ 130, 137, "dimeric", "oligomeric_state" ], [ 138, 146, "EncFtnsH", "protein" ], [ 180, 184, "iron", "chemical" ] ] }, { "sid": 239, "sent": "In light of the crystal structure, this observation can be rationalized as dissociation of the EncFtnsH decamer by disruption of the non-FOC interface with at least partial retention of the FOC interface and the FOC-Fe.", "section": "RESULTS", "ner": [ [ 16, 33, "crystal structure", "evidence" ], [ 95, 103, "EncFtnsH", "protein" ], [ 104, 111, "decamer", "oligomeric_state" ], [ 133, 150, "non-FOC interface", "site" ], [ 190, 203, "FOC interface", "site" ], [ 212, 215, "FOC", "site" ], [ 216, 218, "Fe", "chemical" ] ] }, { "sid": 240, "sent": "Thus, this observation supports our crystallographic assignment of the overall topology of the EncFtnsH assembly as a pentameric assembly of dimers with two iron ions located at the FOC dimer interface.", "section": "RESULTS", "ner": [ [ 95, 103, "EncFtnsH", "protein" ], [ 118, 128, "pentameric", "oligomeric_state" ], [ 141, 147, "dimers", "oligomeric_state" ], [ 157, 161, "iron", "chemical" ], [ 182, 201, "FOC dimer interface", "site" ] ] }, { "sid": 241, "sent": "In addition, this analysis provides evidence that the overall architecture of the complex is consistent in the crystal, solution and gas phases.", "section": "RESULTS", "ner": [ [ 111, 118, "crystal", "evidence" ] ] }, { "sid": 242, "sent": "Ferroxidase activity", "section": "RESULTS", "ner": [ [ 0, 11, "Ferroxidase", "protein_type" ] ] }, { "sid": 243, "sent": "TEM visualization of iron-loaded bacterial nanocompartments and ferritin.", "section": "FIG", "ner": [ [ 0, 3, "TEM", "experimental_method" ], [ 21, 32, "iron-loaded", "protein_state" ], [ 33, 42, "bacterial", "taxonomy_domain" ], [ 43, 59, "nanocompartments", "complex_assembly" ], [ 64, 72, "ferritin", "protein_type" ] ] }, { "sid": 244, "sent": "Decameric EncFtnsH, encapsulin, EncFtn-Enc and apoferritin, at 8.5 \u00b5M, were mixed with 147 \u00b5M, 1 mM, 1 mM and 215 \u00b5M acidic Fe(NH4)2(SO4)2, respectively.", "section": "FIG", "ner": [ [ 0, 9, "Decameric", "oligomeric_state" ], [ 10, 18, "EncFtnsH", "protein" ], [ 20, 30, "encapsulin", "protein" ], [ 32, 42, "EncFtn-Enc", "complex_assembly" ], [ 47, 58, "apoferritin", "protein_state" ], [ 124, 138, "Fe(NH4)2(SO4)2", "chemical" ] ] }, { "sid": 245, "sent": "Protein mixtures were incubated at room temperature for 1 hr prior to TEM analysis with or without uranyl acetate stain.", "section": "FIG", "ner": [ [ 70, 73, "TEM", "experimental_method" ], [ 99, 113, "uranyl acetate", "chemical" ] ] }, { "sid": 246, "sent": "(A\u2013D) Unstained EncFtnsH, encapsulin, EncFtn-Enc, apoferritin loaded with Fe2+, respectively, with 35,000 x magnification and scale bars indicate 100 nm. (E) Protein-free sample as a control. (F\u2013I) Stained EncFtnsH, encapsulin, EncFtn-Enc, apoferritin loaded with Fe2+, respectively, with 140,000 x magnification and scale bars indicate 25 nm.", "section": "FIG", "ner": [ [ 16, 24, "EncFtnsH", "protein" ], [ 26, 36, "encapsulin", "protein" ], [ 38, 48, "EncFtn-Enc", "complex_assembly" ], [ 50, 61, "apoferritin", "protein_state" ], [ 62, 73, "loaded with", "protein_state" ], [ 74, 78, "Fe2+", "chemical" ], [ 198, 205, "Stained", "experimental_method" ], [ 206, 214, "EncFtnsH", "protein" ], [ 216, 226, "encapsulin", "protein" ], [ 228, 238, "EncFtn-Enc", "complex_assembly" ], [ 240, 251, "apoferritin", "protein_state" ], [ 252, 263, "loaded with", "protein_state" ], [ 264, 268, "Fe2+", "chemical" ] ] }, { "sid": 247, "sent": "Spectroscopic evidence for the ferroxidase activity and comparison of iron loading capacity of apoferritin, EncFtnsH, encapsulin, and EncFtn-Enc.", "section": "FIG", "ner": [ [ 31, 42, "ferroxidase", "protein_type" ], [ 70, 74, "iron", "chemical" ], [ 95, 106, "apoferritin", "protein_state" ], [ 108, 116, "EncFtnsH", "protein" ], [ 118, 128, "encapsulin", "protein" ], [ 134, 144, "EncFtn-Enc", "complex_assembly" ] ] }, { "sid": 248, "sent": "(A) Apoferritin (10 \u03bcM monomer concentration) and EncFtnsH decamer fractions (20 \u03bcM monomer concentration, 10 \u03bcM FOC concentration) were incubated with 20 and 100 \u03bcM iron (2 and 10 times molar equivalent Fe2+ per FOC) and progress curves of the oxidation of Fe2+ to Fe3+ at 315 nm were recorded in a spectrophotometer.", "section": "FIG", "ner": [ [ 4, 15, "Apoferritin", "protein_state" ], [ 23, 30, "monomer", "oligomeric_state" ], [ 50, 58, "EncFtnsH", "protein" ], [ 59, 66, "decamer", "oligomeric_state" ], [ 84, 91, "monomer", "oligomeric_state" ], [ 113, 116, "FOC", "site" ], [ 166, 170, "iron", "chemical" ], [ 204, 208, "Fe2+", "chemical" ], [ 213, 216, "FOC", "site" ], [ 222, 237, "progress curves", "evidence" ], [ 258, 262, "Fe2+", "chemical" ], [ 266, 270, "Fe3+", "chemical" ] ] }, { "sid": 249, "sent": "The background oxidation of iron at 20 and 100 \u03bcM in enzyme-free controls are shown for reference. (B) Encapsulin and EncFtn-Enc complexes at 10 \u03bcM asymmetric unit concentration were incubated with Fe2+ at 20 and 100 \u03bcM and progress curves for iron oxidation at A315 were measured in a UV/visible spectrophotometer.", "section": "FIG", "ner": [ [ 28, 32, "iron", "chemical" ], [ 103, 113, "Encapsulin", "protein" ], [ 118, 128, "EncFtn-Enc", "complex_assembly" ], [ 183, 192, "incubated", "experimental_method" ], [ 198, 202, "Fe2+", "chemical" ], [ 224, 239, "progress curves", "evidence" ], [ 244, 248, "iron", "chemical" ], [ 286, 314, "UV/visible spectrophotometer", "experimental_method" ] ] }, { "sid": 250, "sent": "Enzyme free controls for background oxidation of Fe2+ are shown for reference. (C) Histogram of the iron loading capacity per biological assembly of EncFtnsH, encapsulin, EncFtn-Enc and apoferritin.", "section": "FIG", "ner": [ [ 49, 53, "Fe2+", "chemical" ], [ 100, 104, "iron", "chemical" ], [ 149, 157, "EncFtnsH", "protein" ], [ 159, 169, "encapsulin", "protein" ], [ 171, 181, "EncFtn-Enc", "complex_assembly" ], [ 186, 197, "apoferritin", "protein_state" ] ] }, { "sid": 251, "sent": "The results shown are for three technical replicates and represent the optimal iron loading by the complexes after three hours when incubated with Fe2+.", "section": "FIG", "ner": [ [ 79, 83, "iron", "chemical" ], [ 147, 151, "Fe2+", "chemical" ] ] }, { "sid": 252, "sent": "In light of the identification of an iron-loaded FOC in the crystal structure of EncFtn and our native mass spectrometry data, we performed ferroxidase and peroxidase assays to demonstrate the catalytic activity of this protein.", "section": "RESULTS", "ner": [ [ 37, 48, "iron-loaded", "protein_state" ], [ 49, 52, "FOC", "site" ], [ 60, 77, "crystal structure", "evidence" ], [ 81, 87, "EncFtn", "protein" ], [ 96, 120, "native mass spectrometry", "experimental_method" ], [ 140, 173, "ferroxidase and peroxidase assays", "experimental_method" ] ] }, { "sid": 253, "sent": "In addition, we also assayed equine apoferritin, an example of a classical ferritin enzyme, as a positive control.", "section": "RESULTS", "ner": [ [ 29, 35, "equine", "taxonomy_domain" ], [ 36, 47, "apoferritin", "protein_state" ], [ 65, 74, "classical", "protein_state" ], [ 75, 83, "ferritin", "protein_type" ] ] }, { "sid": 254, "sent": "Unlike the Dps family of ferritin-like proteins, EncFtn showed no peroxidase activity when assayed with the substrate ortho-phenylenediamine.", "section": "RESULTS", "ner": [ [ 11, 21, "Dps family", "protein_type" ], [ 25, 47, "ferritin-like proteins", "protein_type" ], [ 49, 55, "EncFtn", "protein" ], [ 118, 140, "ortho-phenylenediamine", "chemical" ] ] }, { "sid": 255, "sent": "The ferroxidase activity of EncFtnsH was measured by recording the progress curve of Fe2+ oxidation to Fe3+ at 315 nm after addition of 20 and 100 \u00b5M Fe2+ (2 and 10 times molar ratio Fe2+/FOC).", "section": "RESULTS", "ner": [ [ 4, 15, "ferroxidase", "protein_type" ], [ 28, 36, "EncFtnsH", "protein" ], [ 67, 81, "progress curve", "evidence" ], [ 85, 89, "Fe2+", "chemical" ], [ 103, 107, "Fe3+", "chemical" ], [ 150, 154, "Fe2+", "chemical" ], [ 183, 187, "Fe2+", "chemical" ], [ 188, 191, "FOC", "site" ] ] }, { "sid": 256, "sent": "In both experiments the rate of oxidation was faster than background oxidation of Fe2+ by molecular oxygen, and was highest for 100 \u00b5M Fe2+ (Figure 8A).", "section": "RESULTS", "ner": [ [ 82, 86, "Fe2+", "chemical" ], [ 100, 106, "oxygen", "chemical" ], [ 135, 139, "Fe2+", "chemical" ] ] }, { "sid": 257, "sent": "These data show that recombinant EncFtnsH acts as an active ferroxidase enzyme.", "section": "RESULTS", "ner": [ [ 33, 41, "EncFtnsH", "protein" ], [ 53, 59, "active", "protein_state" ], [ 60, 71, "ferroxidase", "protein_type" ] ] }, { "sid": 258, "sent": "When compared to apoferritin, EncFtnsH oxidized Fe2+ at a slower rate and the reaction did not run to completion over the 1800 s of the experiment.", "section": "RESULTS", "ner": [ [ 17, 28, "apoferritin", "protein_state" ], [ 30, 38, "EncFtnsH", "protein" ], [ 48, 52, "Fe2+", "chemical" ] ] }, { "sid": 259, "sent": "Addition of higher quantities of iron resulted in the formation of a yellow/red precipitate at the end of the reaction.", "section": "RESULTS", "ner": [ [ 33, 37, "iron", "chemical" ] ] }, { "sid": 260, "sent": "We also performed these assays on purified recombinant encapsulin; which, when assayed alone, did not display ferroxidase activity above background Fe2+ oxidation (Figure 8B).", "section": "RESULTS", "ner": [ [ 55, 65, "encapsulin", "protein" ], [ 110, 121, "ferroxidase", "protein_type" ], [ 148, 152, "Fe2+", "chemical" ] ] }, { "sid": 261, "sent": "In contrast, complexes of the full EncFtn encapsulin nanocompartment (i.e. the EncFtn-Enc protein complex) displayed ferroxidase activity comparable to apoferritin without the formation of precipitates (Figure 8B).", "section": "RESULTS", "ner": [ [ 30, 34, "full", "protein_state" ], [ 35, 41, "EncFtn", "protein" ], [ 42, 52, "encapsulin", "protein" ], [ 53, 68, "nanocompartment", "complex_assembly" ], [ 79, 89, "EncFtn-Enc", "complex_assembly" ], [ 117, 128, "ferroxidase", "protein_type" ], [ 152, 163, "apoferritin", "protein_state" ] ] }, { "sid": 262, "sent": "We attributed the precipitates observed in the EncFtnsH ferroxidase assay to the production of insoluble Fe3+ complexes, which led us to propose that EncFtn does not directly store Fe3+ in a mineral form.", "section": "RESULTS", "ner": [ [ 47, 55, "EncFtnsH", "protein" ], [ 56, 73, "ferroxidase assay", "experimental_method" ], [ 105, 109, "Fe3+", "chemical" ], [ 150, 156, "EncFtn", "protein" ], [ 181, 185, "Fe3+", "chemical" ] ] }, { "sid": 263, "sent": "This observation agrees with native MS results, which indicates a maximum iron loading of 10\u201315 iron ions per decameric EncFtn; and the structure, which does not possess the enclosed iron-storage cavity characteristic of classical ferritins and Dps family proteins that can directly accrue mineralized Fe3+ within their nanocompartment structures.", "section": "RESULTS", "ner": [ [ 29, 38, "native MS", "experimental_method" ], [ 74, 78, "iron", "chemical" ], [ 96, 100, "iron", "chemical" ], [ 110, 119, "decameric", "oligomeric_state" ], [ 120, 126, "EncFtn", "protein" ], [ 136, 145, "structure", "evidence" ], [ 183, 202, "iron-storage cavity", "site" ], [ 221, 230, "classical", "protein_state" ], [ 231, 240, "ferritins", "protein_type" ], [ 245, 264, "Dps family proteins", "protein_type" ], [ 302, 306, "Fe3+", "chemical" ], [ 320, 335, "nanocompartment", "complex_assembly" ], [ 336, 346, "structures", "evidence" ] ] }, { "sid": 264, "sent": "To analyze the products of these reactions and determine whether the EncFtn and encapsulin were able to store iron in a mineral form, we performed TEM on the reaction mixtures from the ferroxidase assay.", "section": "RESULTS", "ner": [ [ 69, 75, "EncFtn", "protein" ], [ 80, 90, "encapsulin", "protein" ], [ 110, 114, "iron", "chemical" ], [ 147, 150, "TEM", "experimental_method" ], [ 185, 202, "ferroxidase assay", "experimental_method" ] ] }, { "sid": 265, "sent": "The EncFtnsH reaction mixture showed the formation of large, irregular electron-dense precipitates (Figure 8\u2014figure supplement 1A).", "section": "RESULTS", "ner": [ [ 4, 12, "EncFtnsH", "protein" ] ] }, { "sid": 266, "sent": "A similar distribution of particles was observed after addition of Fe2+ to the encapsulin protein (Figure 8\u2014figure supplement 1B).", "section": "RESULTS", "ner": [ [ 67, 71, "Fe2+", "chemical" ], [ 79, 89, "encapsulin", "protein" ] ] }, { "sid": 267, "sent": "In contrast, addition of Fe2+ to the EncFtn-Enc nanocompartment resulted in small, highly regular, electron dense particles of approximately 5 nm in diameter (Figure 8\u2014figure supplement 1C); we interpret these observations as controlled mineralization of iron within the nanocompartment.", "section": "RESULTS", "ner": [ [ 25, 29, "Fe2+", "chemical" ], [ 37, 47, "EncFtn-Enc", "complex_assembly" ], [ 48, 63, "nanocompartment", "complex_assembly" ], [ 255, 259, "iron", "chemical" ], [ 271, 286, "nanocompartment", "complex_assembly" ] ] }, { "sid": 268, "sent": "Addition of Fe2+ to apoferritin resulted in a mixture of large particles and small (~2 nm) particles consistent with partial mineralization by the ferritin and some background oxidation of the iron (Figure 8\u2014figure supplement 1D).", "section": "RESULTS", "ner": [ [ 12, 16, "Fe2+", "chemical" ], [ 20, 31, "apoferritin", "protein_state" ], [ 147, 155, "ferritin", "protein_type" ], [ 193, 197, "iron", "chemical" ] ] }, { "sid": 269, "sent": "Negative stain TEM of these samples revealed that upon addition of iron, the EncFtnsH\u00a0protein showed significant aggregation (Figure 8\u2014figure supplement 1F); while the encapsulin, EncFtn-Enc system, and apoferritin are present as distinct nanocompartments without significant protein aggregation (Figure\u00a08\u2014figure supplement 1G\u2013I).", "section": "RESULTS", "ner": [ [ 0, 18, "Negative stain TEM", "experimental_method" ], [ 67, 71, "iron", "chemical" ], [ 77, 85, "EncFtnsH", "protein" ], [ 168, 178, "encapsulin", "protein" ], [ 180, 190, "EncFtn-Enc", "complex_assembly" ], [ 203, 214, "apoferritin", "protein_state" ], [ 239, 255, "nanocompartments", "complex_assembly" ] ] }, { "sid": 270, "sent": "Iron storage in encapsulin nanocompartments", "section": "RESULTS", "ner": [ [ 0, 4, "Iron", "chemical" ], [ 16, 26, "encapsulin", "protein" ], [ 27, 43, "nanocompartments", "complex_assembly" ] ] }, { "sid": 271, "sent": "The results of the ferroxidase assay and micrographs of the reaction products suggest that the oxidation and mineralization function of the classical ferritins are split between the EncFtn and encapsulin proteins, with the EncFtn acting as a ferroxidase and the encapsulin shell providing an environment and template for iron mineralization and storage.", "section": "RESULTS", "ner": [ [ 19, 36, "ferroxidase assay", "experimental_method" ], [ 41, 52, "micrographs", "evidence" ], [ 140, 149, "classical", "protein_state" ], [ 150, 159, "ferritins", "protein_type" ], [ 182, 188, "EncFtn", "protein" ], [ 193, 203, "encapsulin", "protein" ], [ 223, 229, "EncFtn", "protein" ], [ 242, 253, "ferroxidase", "protein_type" ], [ 262, 272, "encapsulin", "protein" ], [ 273, 278, "shell", "structure_element" ], [ 321, 325, "iron", "chemical" ] ] }, { "sid": 272, "sent": "To investigate this further, we added Fe2+ at various concentrations to samples of apo-ferritin, EncFtn, isolated encapsulin, and the EncFtn-Enc protein complex, and subjected these samples to a ferrozine assay to quantify the amount of iron associated with the proteins after three hours of incubation.", "section": "RESULTS", "ner": [ [ 38, 42, "Fe2+", "chemical" ], [ 83, 86, "apo", "protein_state" ], [ 87, 95, "ferritin", "protein_type" ], [ 97, 103, "EncFtn", "protein" ], [ 114, 124, "encapsulin", "protein" ], [ 134, 144, "EncFtn-Enc", "complex_assembly" ], [ 195, 210, "ferrozine assay", "experimental_method" ], [ 237, 241, "iron", "chemical" ] ] }, { "sid": 273, "sent": "The maximum iron loading capacity of these systems was calculated as the quantity of iron per biological assembly (Figure 8C).", "section": "RESULTS", "ner": [ [ 12, 16, "iron", "chemical" ], [ 85, 89, "iron", "chemical" ] ] }, { "sid": 274, "sent": "In this assay, the EncFtnsH\u00a0decamer binds a maximum of around 48 iron ions before excess iron induces protein precipitation.", "section": "RESULTS", "ner": [ [ 19, 27, "EncFtnsH", "protein" ], [ 28, 35, "decamer", "oligomeric_state" ], [ 65, 69, "iron", "chemical" ], [ 89, 93, "iron", "chemical" ] ] }, { "sid": 275, "sent": "The encapsulin shell protein can sequester about 2200 iron ions before significant protein loss occurs, and the reconstituted EncFtn-Enc nanocompartment sequestered about 4150 iron ions.", "section": "RESULTS", "ner": [ [ 4, 14, "encapsulin", "protein" ], [ 15, 20, "shell", "structure_element" ], [ 54, 58, "iron", "chemical" ], [ 126, 136, "EncFtn-Enc", "complex_assembly" ], [ 137, 152, "nanocompartment", "complex_assembly" ], [ 176, 180, "iron", "chemical" ] ] }, { "sid": 276, "sent": "This latter result is significantly more than the apoferritin used in our assay, which sequesters approximately 570 iron ions in this assay (Figure 8C, Table 5).", "section": "RESULTS", "ner": [ [ 50, 61, "apoferritin", "protein_state" ], [ 116, 120, "iron", "chemical" ] ] }, { "sid": 277, "sent": "Consideration of the functional oligomeric states of these proteins, where EncFtn is a decamer and encapsulin forms an icosahedral cage, and estimation of the iron loading capacity of these complexes gives insight into the role of the two proteins in iron storage and mineralization.", "section": "RESULTS", "ner": [ [ 75, 81, "EncFtn", "protein" ], [ 87, 94, "decamer", "oligomeric_state" ], [ 99, 109, "encapsulin", "protein" ], [ 119, 130, "icosahedral", "protein_state" ], [ 131, 135, "cage", "complex_assembly" ], [ 159, 163, "iron", "chemical" ], [ 251, 255, "iron", "chemical" ] ] }, { "sid": 278, "sent": "EncFtn decamers bind up to 48 iron ions (Figure 8C), which is significantly higher than the stoichiometry of fifteen metal ions visible in the FOC and E31/34-site of the crystal structure of the EncFtnsH decamer and our MS analysis.", "section": "RESULTS", "ner": [ [ 0, 6, "EncFtn", "protein" ], [ 7, 15, "decamers", "oligomeric_state" ], [ 30, 34, "iron", "chemical" ], [ 143, 146, "FOC", "site" ], [ 151, 162, "E31/34-site", "site" ], [ 170, 187, "crystal structure", "evidence" ], [ 195, 203, "EncFtnsH", "protein" ], [ 204, 211, "decamer", "oligomeric_state" ], [ 220, 222, "MS", "experimental_method" ] ] }, { "sid": 279, "sent": "The discrepancy between these solution measurements and our MS analysis may indicate that there are additional metal-binding sites on the interior channel and exterior faces of the protein; this is consistent with our identification of a number of weak metal-binding sites at the surface of the protein in the crystal structure (Figure 5D).", "section": "RESULTS", "ner": [ [ 30, 51, "solution measurements", "experimental_method" ], [ 60, 62, "MS", "experimental_method" ], [ 111, 130, "metal-binding sites", "site" ], [ 147, 154, "channel", "site" ], [ 253, 272, "metal-binding sites", "site" ], [ 310, 327, "crystal structure", "evidence" ] ] }, { "sid": 280, "sent": "These observations are consistent with hydrated Fe2+ ions being channeled to the active site from the E31/34-site and the subsequent exit of Fe3+ products on the outer surface, as is seen in other ferritin family proteins.", "section": "RESULTS", "ner": [ [ 48, 52, "Fe2+", "chemical" ], [ 81, 92, "active site", "site" ], [ 102, 113, "E31/34-site", "site" ], [ 141, 145, "Fe3+", "chemical" ], [ 197, 205, "ferritin", "protein_type" ] ] }, { "sid": 281, "sent": "While the isolated encapsulin shell does not display any ferroxidase activity, it binds around 2200 iron ions in our assay (Table 5).", "section": "RESULTS", "ner": [ [ 19, 29, "encapsulin", "protein" ], [ 30, 35, "shell", "structure_element" ], [ 57, 68, "ferroxidase", "protein_type" ], [ 100, 104, "iron", "chemical" ] ] }, { "sid": 282, "sent": "This implies that the shell can bind a significant amount of iron on its outer and inner surfaces.", "section": "RESULTS", "ner": [ [ 22, 27, "shell", "structure_element" ], [ 61, 65, "iron", "chemical" ] ] }, { "sid": 283, "sent": "While the maximum reported loading capacity of classical ferritins is approximately 4500 iron ions, in our assay system we were only able to load apoferritin with around 570 iron ions.", "section": "RESULTS", "ner": [ [ 47, 56, "classical", "protein_state" ], [ 57, 66, "ferritins", "protein_type" ], [ 89, 93, "iron", "chemical" ], [ 146, 157, "apoferritin", "protein_state" ], [ 174, 178, "iron", "chemical" ] ] }, { "sid": 284, "sent": "However, the recombinant EncFtn-Enc nanocompartment was able to bind over 4100 iron ions in the same time period, over seven times the amount seen for the apoferritin.", "section": "RESULTS", "ner": [ [ 25, 35, "EncFtn-Enc", "complex_assembly" ], [ 36, 51, "nanocompartment", "complex_assembly" ], [ 79, 83, "iron", "chemical" ], [ 155, 166, "apoferritin", "protein_state" ] ] }, { "sid": 285, "sent": "We note we do not reach the experimental maximum iron\u00a0loading\u00a0for apoferritin and therefore the total iron-loading capacity of our system may be significantly higher than in this experimental system.", "section": "RESULTS", "ner": [ [ 49, 53, "iron", "chemical" ], [ 66, 77, "apoferritin", "protein_state" ], [ 102, 106, "iron", "chemical" ] ] }, { "sid": 286, "sent": "Taken together, our data show that EncFtn can catalytically oxidize Fe2+ to Fe3+; however, iron binding in EncFtn is limited to the FOC and several surface metal binding sites.", "section": "RESULTS", "ner": [ [ 35, 41, "EncFtn", "protein" ], [ 68, 72, "Fe2+", "chemical" ], [ 76, 80, "Fe3+", "chemical" ], [ 91, 95, "iron", "chemical" ], [ 107, 113, "EncFtn", "protein" ], [ 132, 135, "FOC", "site" ], [ 156, 175, "metal binding sites", "site" ] ] }, { "sid": 287, "sent": "In contrast, the encapsulin protein displays no catalytic activity, but has the ability to bind a considerable amount of iron.", "section": "RESULTS", "ner": [ [ 17, 27, "encapsulin", "protein" ], [ 121, 125, "iron", "chemical" ] ] }, { "sid": 288, "sent": "Finally, the EncFtn-Enc nanocompartment complex retains the catalytic activity of EncFtn, and sequesters iron within the encapsulin shell at a higher level than the isolated components of the system, and at a significantly higher level than the classical ferritins.", "section": "RESULTS", "ner": [ [ 13, 23, "EncFtn-Enc", "complex_assembly" ], [ 24, 39, "nanocompartment", "complex_assembly" ], [ 82, 88, "EncFtn", "protein" ], [ 105, 109, "iron", "chemical" ], [ 121, 131, "encapsulin", "protein" ], [ 132, 137, "shell", "structure_element" ], [ 245, 254, "classical", "protein_state" ], [ 255, 264, "ferritins", "protein_type" ] ] }, { "sid": 289, "sent": "\u00a0Furthermore, our recombinant nanocompartments may not have the physiological subunit stoichiometry, and the iron-loading capacity of native nanocompartments is potentially much higher than the level we have observed.", "section": "RESULTS", "ner": [ [ 30, 46, "nanocompartments", "complex_assembly" ], [ 109, 113, "iron", "chemical" ], [ 134, 140, "native", "protein_state" ], [ 141, 157, "nanocompartments", "complex_assembly" ] ] }, { "sid": 290, "sent": "Mutagenesis of the EncFtnsHferroxidase center", "section": "RESULTS", "ner": [ [ 0, 11, "Mutagenesis", "experimental_method" ], [ 19, 27, "EncFtnsH", "protein" ], [ 27, 45, "ferroxidase center", "site" ] ] }, { "sid": 291, "sent": "Purification of recombinant R. rubrum EncFtnsH FOC mutants.", "section": "FIG", "ner": [ [ 28, 37, "R. rubrum", "species" ], [ 38, 46, "EncFtnsH", "protein" ], [ 47, 50, "FOC", "site" ], [ 51, 58, "mutants", "protein_state" ] ] }, { "sid": 292, "sent": "Single mutants E32A, E62A, and H65A of EncFtnsH produced from E. coli BL21(DE3) cells grown in MM and MM supplemented with iron were subjected to Superdex\u00a0200 size-exclusion\u00a0chromatography.", "section": "FIG", "ner": [ [ 7, 14, "mutants", "protein_state" ], [ 15, 19, "E32A", "mutant" ], [ 21, 25, "E62A", "mutant" ], [ 31, 35, "H65A", "mutant" ], [ 39, 47, "EncFtnsH", "protein" ], [ 62, 79, "E. coli BL21(DE3)", "species" ], [ 95, 97, "MM", "experimental_method" ], [ 102, 104, "MM", "experimental_method" ], [ 123, 127, "iron", "chemical" ], [ 159, 188, "size-exclusion\u00a0chromatography", "experimental_method" ] ] }, { "sid": 293, "sent": "(A) Gel-filtration chromatogram of the E32A mutant form of EncFtnsH\u00a0resulted in an elution profile with a majority of the protein eluting as the decameric form of the protein and a small proportion of monomer. (B) Gel-filtration chromatograhy\u00a0of the E62A mutant form of EncFtnsH\u00a0resulted in an elution profile with a single major decameric peak. (C) Gel-filtration chromatography\u00a0of the H65A mutant form of EncFtnsH\u00a0resulted in a single peak corresponding to the protein monomer.", "section": "FIG", "ner": [ [ 4, 31, "Gel-filtration chromatogram", "evidence" ], [ 39, 43, "E32A", "mutant" ], [ 44, 50, "mutant", "protein_state" ], [ 59, 67, "EncFtnsH", "protein" ], [ 83, 98, "elution profile", "evidence" ], [ 145, 154, "decameric", "oligomeric_state" ], [ 201, 208, "monomer", "oligomeric_state" ], [ 214, 242, "Gel-filtration chromatograhy", "experimental_method" ], [ 250, 254, "E62A", "mutant" ], [ 255, 261, "mutant", "protein_state" ], [ 270, 278, "EncFtnsH", "protein" ], [ 294, 309, "elution profile", "evidence" ], [ 330, 339, "decameric", "oligomeric_state" ], [ 350, 379, "Gel-filtration chromatography", "experimental_method" ], [ 387, 391, "H65A", "mutant" ], [ 392, 398, "mutant", "protein_state" ], [ 407, 415, "EncFtnsH", "protein" ], [ 471, 478, "monomer", "oligomeric_state" ] ] }, { "sid": 294, "sent": "To investigate the structural and biochemical role played by the metal binding residues in the di-iron FOC of EncFtnsH we produced alanine mutations in each of these residues: Glu32, Glu62, and His65.", "section": "RESULTS", "ner": [ [ 65, 87, "metal binding residues", "site" ], [ 95, 106, "di-iron FOC", "site" ], [ 110, 118, "EncFtnsH", "protein" ], [ 131, 148, "alanine mutations", "experimental_method" ], [ 176, 181, "Glu32", "residue_name_number" ], [ 183, 188, "Glu62", "residue_name_number" ], [ 194, 199, "His65", "residue_name_number" ] ] }, { "sid": 295, "sent": "These EncFtnsH mutants were produced in E. coli cells grown in MM, both in the absence and presence of additional iron.", "section": "RESULTS", "ner": [ [ 6, 14, "EncFtnsH", "protein" ], [ 15, 22, "mutants", "protein_state" ], [ 40, 47, "E. coli", "species" ], [ 63, 65, "MM", "experimental_method" ], [ 79, 86, "absence", "protein_state" ], [ 91, 102, "presence of", "protein_state" ], [ 114, 118, "iron", "chemical" ] ] }, { "sid": 296, "sent": "The E32A and E62A mutants eluted from SEC at a volume consistent with the decameric form of EncFtnsH, with a small proportion of monomer; the H65A mutant eluted at a volume consistent with the monomeric form of EncFtnsH (Figure 9).", "section": "RESULTS", "ner": [ [ 4, 8, "E32A", "mutant" ], [ 13, 17, "E62A", "mutant" ], [ 18, 25, "mutants", "protein_state" ], [ 38, 41, "SEC", "experimental_method" ], [ 74, 83, "decameric", "oligomeric_state" ], [ 92, 100, "EncFtnsH", "protein" ], [ 129, 136, "monomer", "oligomeric_state" ], [ 142, 146, "H65A", "mutant" ], [ 147, 153, "mutant", "protein_state" ], [ 193, 202, "monomeric", "oligomeric_state" ], [ 211, 219, "EncFtnsH", "protein" ] ] }, { "sid": 297, "sent": "For all of the mutants studied, no change in oligomerization state was apparent upon addition of Fe2+\u00a0in vitro.", "section": "RESULTS", "ner": [ [ 15, 22, "mutants", "protein_state" ], [ 97, 101, "Fe2+", "chemical" ] ] }, { "sid": 298, "sent": "Native mass spectrometry of EncFtnsH mutants.", "section": "FIG", "ner": [ [ 0, 24, "Native mass spectrometry", "experimental_method" ], [ 28, 36, "EncFtnsH", "protein" ], [ 37, 44, "mutants", "protein_state" ] ] }, { "sid": 299, "sent": "All spectra were acquired in 100 mM ammonium acetate, pH 8.0 with a protein concentration of 5 \u00b5M. (A) Wild-type EncFtnsH in the absence of iron displays a charge state distribution consistent with a monomer (see also Figure 8). (B) E32A EncFtnsH displays a charge states consistent with a decamer (green circles); a minor species, consistent with the monomer of E32A mutant\u00a0is also observed (blue circles).", "section": "FIG", "ner": [ [ 4, 11, "spectra", "evidence" ], [ 45, 52, "acetate", "chemical" ], [ 103, 112, "Wild-type", "protein_state" ], [ 113, 121, "EncFtnsH", "protein" ], [ 129, 139, "absence of", "protein_state" ], [ 140, 144, "iron", "chemical" ], [ 156, 181, "charge state distribution", "evidence" ], [ 200, 207, "monomer", "oligomeric_state" ], [ 233, 237, "E32A", "mutant" ], [ 238, 246, "EncFtnsH", "protein" ], [ 258, 271, "charge states", "evidence" ], [ 290, 297, "decamer", "oligomeric_state" ], [ 352, 359, "monomer", "oligomeric_state" ], [ 363, 367, "E32A", "mutant" ], [ 368, 374, "mutant", "protein_state" ] ] }, { "sid": 300, "sent": "(C) E62A EncFtnsH displays charge states consistent with a decamer (green circles). (D) H65A EncFtnsH displays charge states consistent with both monomer (blue circles) and dimer (purple circles).", "section": "FIG", "ner": [ [ 4, 8, "E62A", "mutant" ], [ 9, 17, "EncFtnsH", "protein" ], [ 27, 40, "charge states", "evidence" ], [ 59, 66, "decamer", "oligomeric_state" ], [ 88, 92, "H65A", "mutant" ], [ 93, 101, "EncFtnsH", "protein" ], [ 111, 124, "charge states", "evidence" ], [ 146, 153, "monomer", "oligomeric_state" ], [ 173, 178, "dimer", "oligomeric_state" ] ] }, { "sid": 301, "sent": "In addition to SEC studies, native mass spectrometry of the apo-EncFtnsH mutants was performed and compared with the wild-type apo-EncFtnsH protein (Figure 10).", "section": "RESULTS", "ner": [ [ 15, 18, "SEC", "experimental_method" ], [ 28, 52, "native mass spectrometry", "experimental_method" ], [ 60, 63, "apo", "protein_state" ], [ 64, 72, "EncFtnsH", "protein" ], [ 73, 80, "mutants", "protein_state" ], [ 117, 126, "wild-type", "protein_state" ], [ 127, 130, "apo", "protein_state" ], [ 131, 139, "EncFtnsH", "protein" ] ] }, { "sid": 302, "sent": "As described above, the apo-EncFtnsH\u00a0has a charge state distribution consistent with an unstructured monomer, and decamer formation is only initiated upon addition of ferrous iron.", "section": "RESULTS", "ner": [ [ 24, 27, "apo", "protein_state" ], [ 28, 36, "EncFtnsH", "protein" ], [ 43, 55, "charge state", "evidence" ], [ 88, 100, "unstructured", "protein_state" ], [ 101, 108, "monomer", "oligomeric_state" ], [ 114, 121, "decamer", "oligomeric_state" ], [ 175, 179, "iron", "chemical" ] ] }, { "sid": 303, "sent": "Both the E32A mutant and E62A mutant displayed charge state distributions consistent with decamers, even in the absence of Fe2+.", "section": "RESULTS", "ner": [ [ 9, 13, "E32A", "mutant" ], [ 14, 20, "mutant", "protein_state" ], [ 25, 29, "E62A", "mutant" ], [ 30, 36, "mutant", "protein_state" ], [ 47, 59, "charge state", "evidence" ], [ 90, 98, "decamers", "oligomeric_state" ], [ 112, 122, "absence of", "protein_state" ], [ 123, 127, "Fe2+", "chemical" ] ] }, { "sid": 304, "sent": "This gas-phase observation is consistent with SEC measurements, which indicate both of these variants were also decamers in solution.", "section": "RESULTS", "ner": [ [ 46, 49, "SEC", "experimental_method" ], [ 112, 120, "decamers", "oligomeric_state" ] ] }, { "sid": 305, "sent": "Thus it seems that these mutations allow the decamer to form in the absence of iron in the FOC.", "section": "RESULTS", "ner": [ [ 45, 52, "decamer", "oligomeric_state" ], [ 68, 78, "absence of", "protein_state" ], [ 79, 83, "iron", "chemical" ], [ 91, 94, "FOC", "site" ] ] }, { "sid": 306, "sent": "In contrast to the glutamic acid mutants, MS analysis of the H65A mutant is similar to wild-type apo-EncFtnsH and is present as a monomer; interestingly a minor population of dimeric H65A was also observed.", "section": "RESULTS", "ner": [ [ 19, 32, "glutamic acid", "residue_name" ], [ 33, 40, "mutants", "protein_state" ], [ 42, 44, "MS", "experimental_method" ], [ 61, 65, "H65A", "mutant" ], [ 66, 72, "mutant", "protein_state" ], [ 87, 96, "wild-type", "protein_state" ], [ 97, 100, "apo", "protein_state" ], [ 101, 109, "EncFtnsH", "protein" ], [ 130, 137, "monomer", "oligomeric_state" ], [ 175, 182, "dimeric", "oligomeric_state" ], [ 183, 187, "H65A", "mutant" ] ] }, { "sid": 307, "sent": "We propose that the observed differences in the oligomerization state of the E32A and E62A mutants compared to wild-type are due to the changes in the electrostatic environment within the FOC.", "section": "RESULTS", "ner": [ [ 77, 81, "E32A", "mutant" ], [ 86, 90, "E62A", "mutant" ], [ 91, 98, "mutants", "protein_state" ], [ 111, 120, "wild-type", "protein_state" ], [ 188, 191, "FOC", "site" ] ] }, { "sid": 308, "sent": "At neutral pH the glutamic acid residues are negatively charged, while the histidine residues are predominantly in their uncharged state.", "section": "RESULTS", "ner": [ [ 3, 13, "neutral pH", "protein_state" ], [ 18, 31, "glutamic acid", "residue_name" ], [ 75, 84, "histidine", "residue_name" ] ] }, { "sid": 309, "sent": "In the wild-type\u00a0(WT) EncFtnsH this leads to electrostatic repulsion between subunits in the absence of iron.", "section": "RESULTS", "ner": [ [ 7, 16, "wild-type", "protein_state" ], [ 18, 20, "WT", "protein_state" ], [ 22, 30, "EncFtnsH", "protein" ], [ 77, 85, "subunits", "structure_element" ], [ 93, 103, "absence of", "protein_state" ], [ 104, 108, "iron", "chemical" ] ] }, { "sid": 310, "sent": "Coordination of Fe2+ in this site stabilizes the dimer and reconstitutes the active FOC.", "section": "RESULTS", "ner": [ [ 0, 12, "Coordination", "bond_interaction" ], [ 16, 20, "Fe2+", "chemical" ], [ 49, 54, "dimer", "oligomeric_state" ], [ 77, 83, "active", "protein_state" ], [ 84, 87, "FOC", "site" ] ] }, { "sid": 311, "sent": "The geometric arrangement of Glu32 and Glu62 in the FOC explains their behavior in solution and the gas phase, where they both favor the formation of decamers due to the loss of a repulsive negative charge.", "section": "RESULTS", "ner": [ [ 29, 34, "Glu32", "residue_name_number" ], [ 39, 44, "Glu62", "residue_name_number" ], [ 52, 55, "FOC", "site" ], [ 150, 158, "decamers", "oligomeric_state" ] ] }, { "sid": 312, "sent": "The FOC in the H65A mutant is destabilized through the loss of this metal coordinating residue and potential positive charge carrier, thus favoring the monomer in solution and the gas phase.", "section": "RESULTS", "ner": [ [ 4, 7, "FOC", "site" ], [ 15, 19, "H65A", "mutant" ], [ 20, 26, "mutant", "protein_state" ], [ 55, 62, "loss of", "protein_state" ], [ 68, 94, "metal coordinating residue", "site" ], [ 152, 159, "monomer", "oligomeric_state" ] ] }, { "sid": 313, "sent": "Data collection and refinement statistics.", "section": "TABLE", "ner": [ [ 0, 41, "Data collection and refinement statistics", "evidence" ] ] }, { "sid": 314, "sent": "\tWT\tE32A\tE62A\tH65A\t \tData collection\t\t\t\t\t \tWavelength (\u00c5)\t1.74\t1.73\t1.73\t1.74\t \tResolution range (\u00c5)\t49.63 - 2.06 (2.10 - 2.06)\t48.84 - 2.59 (2.683 - 2.59)\t48.87 - 2.21 (2.29 - 2.21)\t48.86 - 2.97 (3.08 - 2.97)\t \tSpace group\tP 1 21 1\tP 1 21 1\tP 1 21 1\tP 1 21 1\t \tUnit cell (\u00c5) a\u00a0b \u00a0c\u00a0\u03b2 (\u00b0)\t98.18 120.53 140.30 95.36\t97.78 120.28 140.53 95.41\t98.09 120.23 140.36 95.50\t98.03 120.29 140.43 95.39\t \tTotal reflections\t1,264,922 (41,360)\t405,488 (36,186)\t1,069,345 (95,716)\t323,853 (32,120)\t \tUnique reflections\t197,873 (8,766)\t100,067 (9,735)\t162,379 (15,817)\t66,658 (6,553)\t \tMultiplicity\t6.4 (4.7)\t4.1 (3.7)\t6.6 (6.1)\t4.9 (4.9)\t \tAnomalous multiplicity\t3.2 (2.6)\tN/A\tN/A\tN/A\t \tCompleteness (%)\t99.2 (88.6)\t99.0 (97.0)\t100 (97.0)\t100 (99.0)\t \tAnomalous completeness (%)\t96.7 (77.2)\tN/A\tN/A\tN/A\t \tMean I/sigma(I)\t10.6 (1.60)\t8.46 (1.79)\t13.74 (1.80)\t8.09 (1.74)\t \tWilson B-factor\t26.98\t40.10\t33.97\t52.20\t \tRmerge\t0.123 (0.790)\t0.171 (0.792)\t0.0979 (1.009)\t0.177 (0.863)\t \tRmeas\t0.147 (0.973)\t0.196 (0.923)\t0.1064 (1.107)\t0.199 (0.966)\t \tCC1/2\t0.995 (0.469)\t0.985 (0.557)\t0.998 (0.642)\t0.989 (0.627)\t \tCC*\t0.999 (0.846)\t0.996 (0.846)\t0.999 (0.884)\t0.997 (0.878)\t \tImage DOI\t10.7488/ds/1342\t10.7488/ds/1419\t10.7488/ds/1420\t10.7488/ds/1421\t \tRefinement\t\t\t\t\t \tRwork\t0.171 (0.318)\t0.183 (0.288)\t0.165 (0.299)\t0.186 (0.273)\t \tRfree\t0.206 (0.345)\t0.225 (0351)\t0.216 (0.364)\t0.237 (0.325)\t \tNumber of non-hydrogen atoms\t23,222\t22,366\t22,691\t22,145\t \tmacromolecules\t22,276\t22,019\t21,965\t22,066\t \tligands\t138\t8\t24\t74\t \twater\t808\t339\t702\t5\t \tProtein residues\t2,703\t2,686\t2,675\t2,700\t \tRMS(bonds) (\u00c5)\t0.012\t0.005\t0.011\t0.002\t \tRMS(angles) (\u00b0)\t1.26\t0.58\t1.02\t0.40\t \tRamachandran favored (%)\t100\t99\t100\t99\t \tRamachandran allowed (%)\t0\t1\t0\t1\t \tRamachandran outliers (%)\t0\t0\t0\t0\t \tClash score\t1.42\t1.42\t1.79\t0.97\t \tAverage B-factor (\u00c52)\t33.90\t42.31\t41.34\t47.68\t \tmacromolecules\t33.80\t42.35\t41.31\t47.60\t \tligands\t40.40\t72.80\t65.55\t72.34\t \tsolvent\t36.20\t38.95\t41.46\t33.85\t \tPDB ID\t5DA5\t5L89\t5L8B\t5L8G\t \t", "section": "TABLE", "ner": [ [ 1, 3, "WT", "protein_state" ], [ 4, 8, "E32A", "mutant" ], [ 9, 13, "E62A", "mutant" ], [ 14, 18, "H65A", "mutant" ], [ 1504, 1509, "water", "chemical" ] ] }, { "sid": 315, "sent": "Iron loading capacity of EncFtn, encapsulin and ferritin.", "section": "TABLE", "ner": [ [ 0, 4, "Iron", "chemical" ], [ 25, 31, "EncFtn", "protein" ], [ 33, 43, "encapsulin", "protein" ], [ 48, 56, "ferritin", "protein_type" ] ] }, { "sid": 316, "sent": "Protein samples (at 8.5 \u00b5M) including decameric EncFtnsH, encapsulin, EncFtn-Enc and apoferritin were mixed with Fe(NH4)2(SO4) (in 0.1%\u00a0(v/v) HCl) of different concentrations in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl buffer at room temperature for 3 hrs in the air.", "section": "TABLE", "ner": [ [ 38, 47, "decameric", "oligomeric_state" ], [ 48, 56, "EncFtnsH", "protein" ], [ 58, 68, "encapsulin", "protein" ], [ 70, 80, "EncFtn-Enc", "complex_assembly" ], [ 85, 96, "apoferritin", "protein_state" ], [ 113, 126, "Fe(NH4)2(SO4)", "chemical" ], [ 142, 145, "HCl", "chemical" ], [ 210, 214, "NaCl", "chemical" ] ] }, { "sid": 317, "sent": "Protein-Fe mixtures were centrifuged at 13,000 x g to remove precipitated material and desalted prior to the Fe and protein content analysis by ferrozine assay and BCA microplate assay, respectively.", "section": "TABLE", "ner": [ [ 8, 10, "Fe", "chemical" ], [ 109, 111, "Fe", "chemical" ], [ 144, 159, "ferrozine assay", "experimental_method" ], [ 164, 184, "BCA microplate assay", "experimental_method" ] ] }, { "sid": 318, "sent": "Fe to protein ratio was calculated to indicate the Fe binding capacity of the protein.", "section": "TABLE", "ner": [ [ 0, 2, "Fe", "chemical" ], [ 51, 53, "Fe", "chemical" ] ] }, { "sid": 319, "sent": "Protein stability was compromised at high iron concentrations; therefore, the highest iron loading with the least protein precipitation was used to derive the maximum iron loading capacity per biological assembly (underlined and highlighted in bold).", "section": "TABLE", "ner": [ [ 42, 46, "iron", "chemical" ], [ 86, 90, "iron", "chemical" ], [ 167, 171, "iron", "chemical" ] ] }, { "sid": 320, "sent": "The biological unit assemblies are a decamer for EncFtnsH, a 60mer for encapsulin, a 60mer of encapsulin loaded with 12 copies of decameric EncFtn in the complex, and 24mer for horse spleen apoferritin.", "section": "TABLE", "ner": [ [ 37, 44, "decamer", "oligomeric_state" ], [ 49, 57, "EncFtnsH", "protein" ], [ 61, 66, "60mer", "oligomeric_state" ], [ 71, 81, "encapsulin", "protein" ], [ 85, 90, "60mer", "oligomeric_state" ], [ 94, 104, "encapsulin", "protein" ], [ 105, 116, "loaded with", "protein_state" ], [ 130, 139, "decameric", "oligomeric_state" ], [ 140, 146, "EncFtn", "protein" ], [ 167, 172, "24mer", "oligomeric_state" ], [ 177, 182, "horse", "taxonomy_domain" ], [ 190, 201, "apoferritin", "protein_state" ] ] }, { "sid": 321, "sent": "Errors are quoted as the standard deviation of three technical repeats in both the ferrozine and BCA microplate assays.", "section": "TABLE", "ner": [ [ 83, 118, "ferrozine and BCA microplate assays", "experimental_method" ] ] }, { "sid": 322, "sent": "The proteins used in Fe loading experiment came from a single preparation.", "section": "TABLE", "ner": [ [ 21, 23, "Fe", "chemical" ] ] }, { "sid": 323, "sent": "Protein sample\tFe(NH4)2(SO4)2 loading (\u00b5M)\tFe detected by ferrozine assay (\u00b5M)\tProtein detected by BCA microplate assay (\u00b5M)\tFe / monomeric protein\tMaximum Fe loading per biological assembly unit\t \t8.46 \u00b5M EncFtnsH-10mer\t0\t4.73 \u00b1 2.32\t5.26 \u00b1 0.64\t0.90 \u00b1 0.44\t\t \t39.9\t9.93 \u00b1 1.20\t5.36 \u00b1 0.69\t1.85 \u00b1 0.22\t\t \t84\t17.99 \u00b1 2.01\t4.96 \u00b1 0.04\t3.63 \u00b1 0.41\t\t \t147\t21.09 \u00b1 1.94\t4.44 \u00b1 0.21\t4.75 \u00b1 0.44\t48 \u00b1 4\t \t224\t28.68 \u00b1 0.30\t3.73 \u00b1 0.53\t7.68 \u00b1 0.08\t\t \t301\t11.27 \u00b1 1.10\t2.50 \u00b1 0.05\t4.51 \u00b1 0.44\t\t \t8.50 \u00b5M Encapsulin\t0\t-1.02 \u00b1 0.54\t8.63 \u00b1 0.17\t-0.12 \u00b1 0.06\t\t \t224\t62.24 \u00b1 2.49\t10.01 \u00b1 0.58\t6.22 \u00b1 0.35\t\t \t301\t67.94 \u00b1 3.15\t8.69 \u00b1 0.42\t7.81 \u00b1 0.36\t\t \t450\t107.96 \u00b1 8.88\t8.50 \u00b1 0.69\t12.71 \u00b1 1.05\t\t \t700\t97.51 \u00b1 3.19\t7.26 \u00b1 0.20\t13.44 \u00b1 0.44\t\t \t1000\t308.63 \u00b1 2.06\t8.42 \u00b1 0.34\t36.66 \u00b1 0.24\t2199 \u00b1 15\t \t1500\t57.09 \u00b1 0.90\t1.44 \u00b1 0.21\t39.77 \u00b1 0.62\t\t \t2000\t9.2 \u00b1 1.16\t0.21 \u00b1 0.14\t44.73 \u00b1 5.63\t\t \t8.70 \u00b5M EncFtn-Enc\t0\t3.31 \u00b1 1.57\t6.85 \u00b1 0.07\t0.48 \u00b1 0.23\t\t \t224\t116.27 \u00b1 3.74\t7.63 \u00b1 0.12\t15.25 \u00b1 0.49\t\t \t301\t132.86 \u00b1 4.03\t6.66 \u00b1 0.31\t19.96 \u00b1 0.61\t\t \t450\t220.57 \u00b1 27.33\t6.12 \u00b1 1.07\t36.06 \u00b1 4.47\t\t \t700\t344.03 \u00b1 40.38\t6.94 \u00b1 0.17\t49.58 \u00b1 5.82\t\t \t1000\t496.00 \u00b1 38.48\t7.19 \u00b1 0.08\t68.94 \u00b1 5.35\t4137 \u00b1 321\t \t1500\t569.98 \u00b1 73.63\t5.73 \u00b1 0.03\t99.44 \u00b1 12.84\t\t \t2000\t584.30 \u00b1 28.33\t4.88 \u00b1 0.22\t119.62 \u00b1 5.80\t\t \t8.50 \u00b5M Apoferritin\t0\t3.95 \u00b1 2.26\t9.37 \u00b1 0.24\t0.42 \u00b1 0.25\t\t \t42.5\t10.27 \u00b1 1.12\t8.27 \u00b1 0.30\t1.24 \u00b1 0.18\t\t \t212.5\t44.48 \u00b1 2.76\t7.85 \u00b1 0.77\t5.67 \u00b1 0.83\t\t \t637.5\t160.93 \u00b1 4.27\t6.76 \u00b1 0.81\t23.79 \u00b1 3.12\t571 \u00b1 75\t \t1275\t114.92 \u00b1 3.17\t3.84 \u00b1 0.30\t29.91 \u00b1 2.95\t\t \t1700\t91.40 \u00b1 3.37\t3.14 \u00b1 0.35\t29.13 \u00b1 3.86\t\t \t", "section": "TABLE", "ner": [ [ 15, 29, "Fe(NH4)2(SO4)2", "chemical" ], [ 43, 45, "Fe", "chemical" ], [ 58, 73, "ferrozine assay", "experimental_method" ], [ 99, 119, "BCA microplate assay", "experimental_method" ], [ 125, 127, "Fe", "chemical" ], [ 156, 158, "Fe", "chemical" ], [ 206, 214, "EncFtnsH", "protein" ], [ 215, 220, "10mer", "oligomeric_state" ], [ 495, 505, "Encapsulin", "protein" ], [ 883, 893, "EncFtn-Enc", "complex_assembly" ], [ 1285, 1296, "Apoferritin", "protein_state" ] ] }, { "sid": 324, "sent": "To understand the impact of the mutants on the organization and metal binding of the FOC, we determined the X-ray crystal structures of each of the EncFtnsH\u00a0mutants (See Table 4 for data collection and refinement statistics).", "section": "RESULTS", "ner": [ [ 32, 39, "mutants", "protein_state" ], [ 85, 88, "FOC", "site" ], [ 108, 132, "X-ray crystal structures", "evidence" ], [ 148, 156, "EncFtnsH", "protein" ], [ 157, 164, "mutants", "protein_state" ] ] }, { "sid": 325, "sent": "The crystal packing of all of the mutants in this study is essentially isomorphous to the EncFtnsH structure.", "section": "RESULTS", "ner": [ [ 34, 41, "mutants", "protein_state" ], [ 90, 98, "EncFtnsH", "protein" ], [ 99, 108, "structure", "evidence" ] ] }, { "sid": 326, "sent": "All of the mutants display the same decameric arrangement in the crystals as the EncFtnsH structure, and the monomers superimpose with an average RMSDC\u03b1 of less than 0.2 \u00c5.", "section": "RESULTS", "ner": [ [ 11, 18, "mutants", "protein_state" ], [ 36, 45, "decameric", "oligomeric_state" ], [ 65, 73, "crystals", "evidence" ], [ 81, 89, "EncFtnsH", "protein" ], [ 90, 99, "structure", "evidence" ], [ 109, 117, "monomers", "oligomeric_state" ], [ 118, 129, "superimpose", "experimental_method" ], [ 146, 152, "RMSDC\u03b1", "evidence" ] ] }, { "sid": 327, "sent": "FOC dimer interface of EncFtnsH-E32A mutant.", "section": "FIG", "ner": [ [ 0, 3, "FOC", "site" ], [ 4, 19, "dimer interface", "site" ], [ 23, 36, "EncFtnsH-E32A", "mutant" ], [ 37, 43, "mutant", "protein_state" ] ] }, { "sid": 328, "sent": "(A) Wall-eyed stereo view of the metal-binding dimerization interface of EncFtnsH-E32A.", "section": "FIG", "ner": [ [ 33, 69, "metal-binding dimerization interface", "site" ], [ 73, 86, "EncFtnsH-E32A", "mutant" ] ] }, { "sid": 329, "sent": "Protein residues are shown as sticks with blue and green carbons for the different subunits.", "section": "FIG", "ner": [ [ 83, 91, "subunits", "structure_element" ], [ 83, 91, "subunits", "structure_element" ], [ 83, 91, "subunits", "structure_element" ] ] }, { "sid": 330, "sent": "The 2mFo-DFc electron density map is shown as a blue mesh contoured at 1.5 \u03c3.", "section": "FIG", "ner": [ [ 4, 33, "2mFo-DFc electron density map", "evidence" ], [ 4, 33, "2mFo-DFc electron density map", "evidence" ], [ 4, 33, "2mFo-DFc electron density map", "evidence" ] ] }, { "sid": 331, "sent": "(B) Views of the FOC of the EncFtnsH-E32Amutant.", "section": "FIG", "ner": [ [ 17, 20, "FOC", "site" ], [ 28, 41, "EncFtnsH-E32A", "mutant" ], [ 41, 47, "mutant", "protein_state" ] ] }, { "sid": 332, "sent": "FOC dimer interface of EncFtnsH-E62A mutant.", "section": "FIG", "ner": [ [ 0, 19, "FOC dimer interface", "site" ], [ 23, 36, "EncFtnsH-E62A", "mutant" ], [ 37, 43, "mutant", "protein_state" ] ] }, { "sid": 333, "sent": "(A) Wall-eyed stereo view of the metal-binding dimerization interface of EncFtnsH-E62A.", "section": "FIG", "ner": [ [ 33, 69, "metal-binding dimerization interface", "site" ], [ 73, 86, "EncFtnsH-E62A", "mutant" ] ] }, { "sid": 334, "sent": "The single coordinated calcium ion is shown as a grey sphere. (B) Views of the FOC of the EncFtnsH-E62A mutant.", "section": "FIG", "ner": [ [ 23, 30, "calcium", "chemical" ], [ 79, 82, "FOC", "site" ], [ 90, 103, "EncFtnsH-E62A", "mutant" ], [ 104, 110, "mutant", "protein_state" ] ] }, { "sid": 335, "sent": "FOC dimer interface of EncFtnsH-H65A mutant.", "section": "FIG", "ner": [ [ 0, 19, "FOC dimer interface", "site" ], [ 23, 36, "EncFtnsH-H65A", "mutant" ], [ 37, 43, "mutant", "protein_state" ] ] }, { "sid": 336, "sent": "(A) Wall-eyed stereo view of the metal-binding dimerization interface of EncFtnsH-H65A.", "section": "FIG", "ner": [ [ 33, 69, "metal-binding dimerization interface", "site" ], [ 73, 86, "EncFtnsH-H65A", "mutant" ] ] }, { "sid": 337, "sent": "The coordinated calcium ions are shown as a grey spheres with coordination distances in the FOC highlighted with yellow dashed lines.", "section": "FIG", "ner": [ [ 16, 23, "calcium", "chemical" ], [ 62, 74, "coordination", "bond_interaction" ], [ 92, 95, "FOC", "site" ] ] }, { "sid": 338, "sent": "(B) Views of the FOC of the EncFtnsH-H65A mutant.", "section": "FIG", "ner": [ [ 17, 20, "FOC", "site" ], [ 28, 41, "EncFtnsH-H65A", "mutant" ], [ 42, 48, "mutant", "protein_state" ] ] }, { "sid": 339, "sent": "Comparison of the EncFtnsH FOC mutants vs wild type.", "section": "FIG", "ner": [ [ 18, 26, "EncFtnsH", "protein" ], [ 27, 30, "FOC", "site" ], [ 31, 38, "mutants", "protein_state" ], [ 42, 51, "wild type", "protein_state" ] ] }, { "sid": 340, "sent": "The structures of the three EncFtnsH\u00a0mutants were all determined by X-ray crystallography.", "section": "FIG", "ner": [ [ 4, 14, "structures", "evidence" ], [ 28, 36, "EncFtnsH", "protein" ], [ 37, 44, "mutants", "protein_state" ], [ 68, 89, "X-ray crystallography", "experimental_method" ] ] }, { "sid": 341, "sent": "The E32A, E62A and H65A mutants were crystallized in identical conditions to the wild type.", "section": "FIG", "ner": [ [ 4, 8, "E32A", "mutant" ], [ 10, 14, "E62A", "mutant" ], [ 19, 23, "H65A", "mutant" ], [ 24, 31, "mutants", "protein_state" ], [ 37, 49, "crystallized", "experimental_method" ], [ 81, 90, "wild type", "protein_state" ] ] }, { "sid": 342, "sent": "EncFtnsH structure and were essentially isomorphous in terms of their unit cell dimensions.", "section": "FIG", "ner": [ [ 0, 8, "EncFtnsH", "protein" ], [ 9, 18, "structure", "evidence" ] ] }, { "sid": 343, "sent": "The FOC residues of the mutants and native EncFtnsH structures are shown as sticks with coordinated Fe2+ as orange and Ca2+ as grey spheres and are colored as follows: wild type, grey; E32A, pink; E62A, green; H65A, blue.", "section": "FIG", "ner": [ [ 4, 7, "FOC", "site" ], [ 24, 31, "mutants", "protein_state" ], [ 36, 42, "native", "protein_state" ], [ 43, 51, "EncFtnsH", "protein" ], [ 52, 62, "structures", "evidence" ], [ 88, 99, "coordinated", "bond_interaction" ], [ 100, 104, "Fe2+", "chemical" ], [ 119, 123, "Ca2+", "chemical" ], [ 168, 177, "wild type", "protein_state" ], [ 185, 189, "E32A", "mutant" ], [ 197, 201, "E62A", "mutant" ], [ 210, 214, "H65A", "mutant" ] ] }, { "sid": 344, "sent": "Of the mutants, only H65A has any coordinated metal ions, which appear to be calcium ions from the crystallization condition.", "section": "FIG", "ner": [ [ 7, 14, "mutants", "protein_state" ], [ 21, 25, "H65A", "mutant" ], [ 34, 45, "coordinated", "bond_interaction" ], [ 77, 84, "calcium", "chemical" ] ] }, { "sid": 345, "sent": "The overall organization of FOC residues is retained in the mutants, with almost no backbone movements.", "section": "FIG", "ner": [ [ 28, 31, "FOC", "site" ], [ 60, 67, "mutants", "protein_state" ] ] }, { "sid": 346, "sent": "Significant differences center around Tyr39, which moves to coordinate the bound calcium ions in the H65A mutant; and Glu32, which moves away from the metal ions in this structure.", "section": "FIG", "ner": [ [ 38, 43, "Tyr39", "residue_name_number" ], [ 60, 70, "coordinate", "bond_interaction" ], [ 75, 80, "bound", "protein_state" ], [ 81, 88, "calcium", "chemical" ], [ 101, 105, "H65A", "mutant" ], [ 106, 112, "mutant", "protein_state" ], [ 118, 123, "Glu32", "residue_name_number" ], [ 170, 179, "structure", "evidence" ] ] }, { "sid": 347, "sent": "Close inspection of the region of the protein around the FOC in each of the mutants highlights their effect on metal binding (Figure 11 and Figure 11\u2014figure supplement 1\u20133).", "section": "RESULTS", "ner": [ [ 57, 60, "FOC", "site" ], [ 76, 83, "mutants", "protein_state" ] ] }, { "sid": 348, "sent": "In the E32A mutant the position of the side chains of the remaining iron coordinating residues in the FOC is essentially unchanged, but the absence of the axial-metal coordinating ligand provided by the Glu32 side chain abrogates metal binding in this site.", "section": "RESULTS", "ner": [ [ 7, 11, "E32A", "mutant" ], [ 12, 18, "mutant", "protein_state" ], [ 68, 94, "iron coordinating residues", "site" ], [ 102, 105, "FOC", "site" ], [ 140, 150, "absence of", "protein_state" ], [ 167, 179, "coordinating", "bond_interaction" ], [ 203, 208, "Glu32", "residue_name_number" ], [ 220, 243, "abrogates metal binding", "protein_state" ] ] }, { "sid": 349, "sent": "The Glu31/34-site also lacks metal, with the side chain of Glu31 rotated by 180\u00b0\u00a0at the C\u03b2 in the absence of metal (Figure 11\u2014figure supplement 1).", "section": "RESULTS", "ner": [ [ 4, 17, "Glu31/34-site", "site" ], [ 23, 28, "lacks", "protein_state" ], [ 29, 34, "metal", "chemical" ], [ 59, 64, "Glu31", "residue_name_number" ], [ 98, 108, "absence of", "protein_state" ], [ 109, 114, "metal", "chemical" ] ] }, { "sid": 350, "sent": "The E62A mutant has a similar effect on the FOC to the E32A mutant, however the entry site still has a calcium ion coordinated between residues Glu31 and Glu34 (Figure 11\u2014figure supplement 2).", "section": "RESULTS", "ner": [ [ 4, 8, "E62A", "mutant" ], [ 9, 15, "mutant", "protein_state" ], [ 44, 47, "FOC", "site" ], [ 55, 59, "E32A", "mutant" ], [ 60, 66, "mutant", "protein_state" ], [ 80, 90, "entry site", "site" ], [ 103, 110, "calcium", "chemical" ], [ 115, 126, "coordinated", "bond_interaction" ], [ 144, 149, "Glu31", "residue_name_number" ], [ 154, 159, "Glu34", "residue_name_number" ] ] }, { "sid": 351, "sent": "The H65A mutant diverges significantly from the wild type in the position of the residues Glu32 and Tyr39 in the FOC.", "section": "RESULTS", "ner": [ [ 4, 8, "H65A", "mutant" ], [ 9, 15, "mutant", "protein_state" ], [ 48, 57, "wild type", "protein_state" ], [ 90, 95, "Glu32", "residue_name_number" ], [ 100, 105, "Tyr39", "residue_name_number" ], [ 113, 116, "FOC", "site" ] ] }, { "sid": 352, "sent": "E32 appears in either the original orientation as the wild type and coordinates Ca2+ in this position, or it is flipped by 180\u00b0 at the C\u03b2, moving away from the coordinated calcium ion in the FOC.", "section": "RESULTS", "ner": [ [ 0, 3, "E32", "residue_name_number" ], [ 54, 63, "wild type", "protein_state" ], [ 68, 79, "coordinates", "bond_interaction" ], [ 80, 84, "Ca2+", "chemical" ], [ 160, 171, "coordinated", "bond_interaction" ], [ 172, 179, "calcium", "chemical" ], [ 191, 194, "FOC", "site" ] ] }, { "sid": 353, "sent": "Tyr39 moves closer to Ca2+ compared to the wild-type and coordinates the calcium ion (Figure 11\u2014figure supplement 3).", "section": "RESULTS", "ner": [ [ 0, 5, "Tyr39", "residue_name_number" ], [ 22, 26, "Ca2+", "chemical" ], [ 43, 52, "wild-type", "protein_state" ], [ 57, 68, "coordinates", "bond_interaction" ], [ 73, 80, "calcium", "chemical" ] ] }, { "sid": 354, "sent": "A single calcium ion is present in the entry site of this mutant; however, Glu31 of one chain is rotated away from the metal ion and is not involved in coordination.", "section": "RESULTS", "ner": [ [ 9, 16, "calcium", "chemical" ], [ 39, 49, "entry site", "site" ], [ 58, 64, "mutant", "protein_state" ], [ 75, 80, "Glu31", "residue_name_number" ], [ 152, 164, "coordination", "bond_interaction" ] ] }, { "sid": 355, "sent": "Taken together the results of our data show that these changes to the FOC of EncFtn still permit the formation of the decameric form of the protein.", "section": "RESULTS", "ner": [ [ 70, 73, "FOC", "site" ], [ 77, 83, "EncFtn", "protein" ], [ 118, 127, "decameric", "oligomeric_state" ] ] }, { "sid": 356, "sent": "While the proteins all appear decameric in crystals, their solution and gas-phase behavior differs considerably and the mutants no longer show metal-dependent oligomerization.", "section": "RESULTS", "ner": [ [ 30, 39, "decameric", "oligomeric_state" ], [ 43, 51, "crystals", "evidence" ], [ 120, 127, "mutants", "protein_state" ] ] }, { "sid": 357, "sent": "These results highlight the importance of metal coordination in the FOC for the stability and assembly of the EncFtn protein.", "section": "RESULTS", "ner": [ [ 42, 47, "metal", "chemical" ], [ 48, 60, "coordination", "bond_interaction" ], [ 68, 71, "FOC", "site" ], [ 110, 116, "EncFtn", "protein" ] ] }, { "sid": 358, "sent": "Progress curves recording ferroxidase activity of EncFtnsH mutants.", "section": "FIG", "ner": [ [ 0, 15, "Progress curves", "evidence" ], [ 26, 37, "ferroxidase", "protein_type" ], [ 50, 58, "EncFtnsH", "protein" ], [ 59, 66, "mutants", "protein_state" ] ] }, { "sid": 359, "sent": "20 \u00b5M wild-type\u00a0EncFtnsH, E32A, E62A and H65A mutants\u00a0were mixed with 20 \u00b5M or 100 \u00b5M acidic Fe(NH4)2(SO4)2, respectively.", "section": "FIG", "ner": [ [ 6, 15, "wild-type", "protein_state" ], [ 16, 24, "EncFtnsH", "protein" ], [ 26, 30, "E32A", "mutant" ], [ 32, 36, "E62A", "mutant" ], [ 41, 45, "H65A", "mutant" ], [ 46, 53, "mutants", "protein_state" ], [ 93, 107, "Fe(NH4)2(SO4)2", "chemical" ] ] }, { "sid": 360, "sent": "Absorbance at 315 nm was recorded for 1800 s at 25\u00b0C as an indication of Fe3+ formation.", "section": "FIG", "ner": [ [ 73, 77, "Fe3+", "chemical" ] ] }, { "sid": 361, "sent": "Protein free samples (dashed and dotted lines) were measured for Fe2+ background oxidation as controls.", "section": "FIG", "ner": [ [ 65, 69, "Fe2+", "chemical" ] ] }, { "sid": 362, "sent": "Relative ferroxidase activity of EncFtnsH mutants.", "section": "FIG", "ner": [ [ 9, 20, "ferroxidase", "protein_type" ], [ 33, 41, "EncFtnsH", "protein" ], [ 42, 49, "mutants", "protein_state" ] ] }, { "sid": 363, "sent": "EncFtnsH, and the mutant forms E32A, E62A and H65A, each at 20 \u00b5M, were mixed with 100 \u00b5M acidic Fe(NH4)2(SO4)2.", "section": "FIG", "ner": [ [ 0, 8, "EncFtnsH", "protein" ], [ 18, 24, "mutant", "protein_state" ], [ 31, 35, "E32A", "mutant" ], [ 37, 41, "E62A", "mutant" ], [ 46, 50, "H65A", "mutant" ], [ 97, 111, "Fe(NH4)2(SO4)2", "chemical" ] ] }, { "sid": 364, "sent": "Ferroxidase activity of the mutant forms is determined by measuring the absorbance at 315 nm for 1800 s at 25\u00a0\u00b0C as an indication of Fe3+ formation.", "section": "FIG", "ner": [ [ 0, 11, "Ferroxidase", "protein_type" ], [ 28, 34, "mutant", "protein_state" ], [ 58, 92, "measuring the absorbance at 315 nm", "experimental_method" ], [ 133, 137, "Fe3+", "chemical" ] ] }, { "sid": 365, "sent": "The relative ferroxidase activity of mutants is plotted as a proportion of the activity of the wild-type protein using the endpoint measurement of A315.", "section": "FIG", "ner": [ [ 13, 24, "ferroxidase", "protein_type" ], [ 37, 44, "mutants", "protein_state" ], [ 95, 104, "wild-type", "protein_state" ], [ 132, 151, "measurement of A315", "experimental_method" ] ] }, { "sid": 366, "sent": "The FOC mutants showed reduced ferroxidase activity to varied extents, among which E62A significantly abrogated the ferroxidase activity.", "section": "FIG", "ner": [ [ 4, 7, "FOC", "site" ], [ 8, 15, "mutants", "protein_state" ], [ 31, 42, "ferroxidase", "protein_type" ], [ 83, 87, "E62A", "mutant" ], [ 116, 127, "ferroxidase", "protein_type" ] ] }, { "sid": 367, "sent": "To address the question of how mutagenesis of the iron coordinating residues affects the enzymatic activity of the EncFtnsH protein we recorded progress curves for the oxidation of Fe2+ to Fe3+ by the different mutants as before.", "section": "RESULTS", "ner": [ [ 31, 42, "mutagenesis", "experimental_method" ], [ 50, 76, "iron coordinating residues", "site" ], [ 115, 123, "EncFtnsH", "protein" ], [ 144, 159, "progress curves", "evidence" ], [ 181, 185, "Fe2+", "chemical" ], [ 189, 193, "Fe3+", "chemical" ], [ 211, 218, "mutants", "protein_state" ] ] }, { "sid": 368, "sent": "Mutagenesis of E32A and H65A reduces the activity of EncFtnsH by about 40%-55%;\u00a0the E62A mutant completely abrogates activity, presumably through the loss of the bridging coordination for the formation of the di-nuclear iron center of the FOC (Figure 12).", "section": "RESULTS", "ner": [ [ 0, 11, "Mutagenesis", "experimental_method" ], [ 15, 19, "E32A", "mutant" ], [ 24, 28, "H65A", "mutant" ], [ 53, 61, "EncFtnsH", "protein" ], [ 84, 88, "E62A", "mutant" ], [ 89, 95, "mutant", "protein_state" ], [ 150, 157, "loss of", "protein_state" ], [ 171, 183, "coordination", "bond_interaction" ], [ 209, 231, "di-nuclear iron center", "site" ], [ 239, 242, "FOC", "site" ] ] }, { "sid": 369, "sent": "Collectively, the effect of mutating these residues in the FOC confirms the importance of the iron coordinating residues for the ferroxidase activity of the EncFtnsH protein.", "section": "RESULTS", "ner": [ [ 28, 36, "mutating", "experimental_method" ], [ 59, 62, "FOC", "site" ], [ 94, 120, "iron coordinating residues", "site" ], [ 129, 140, "ferroxidase", "protein_type" ], [ 157, 165, "EncFtnsH", "protein" ] ] }, { "sid": 370, "sent": "Phylogenetic tree of ferritin family proteins.", "section": "FIG", "ner": [ [ 0, 17, "Phylogenetic tree", "evidence" ], [ 21, 29, "ferritin", "protein_type" ] ] }, { "sid": 371, "sent": "The tree was built using the Neighbor-Joining method based on step-wise amino acid sequence alignment of the four-helical bundle portions of ferritin family proteins (Supplementary file 1).", "section": "FIG", "ner": [ [ 29, 52, "Neighbor-Joining method", "experimental_method" ], [ 62, 101, "step-wise amino acid sequence alignment", "experimental_method" ], [ 109, 128, "four-helical bundle", "structure_element" ], [ 141, 149, "ferritin", "protein_type" ] ] }, { "sid": 372, "sent": "The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site.", "section": "FIG", "ner": [ [ 4, 26, "evolutionary distances", "evidence" ], [ 51, 68, "p-distance method", "experimental_method" ] ] }, { "sid": 373, "sent": "Our study reports on a new class of ferritin-like proteins (EncFtn), which are associated with bacterial encapsulin nanocompartments (Enc).", "section": "DISCUSS", "ner": [ [ 36, 44, "ferritin", "protein_type" ], [ 60, 66, "EncFtn", "protein" ], [ 95, 104, "bacterial", "taxonomy_domain" ], [ 105, 115, "encapsulin", "protein" ], [ 116, 132, "nanocompartments", "complex_assembly" ], [ 134, 137, "Enc", "protein" ] ] }, { "sid": 374, "sent": "By studying the EncFtn from R. rubrum we demonstrate that iron binding results in assembly of EncFtn decamers, which display a unique annular architecture.", "section": "DISCUSS", "ner": [ [ 16, 22, "EncFtn", "protein" ], [ 28, 37, "R. rubrum", "species" ], [ 58, 62, "iron", "chemical" ], [ 94, 100, "EncFtn", "protein" ], [ 101, 109, "decamers", "oligomeric_state" ] ] }, { "sid": 375, "sent": "Despite a radically different quaternary structure to the classical ferritins, the four-helical bundle scaffold and FOC of EncFtnsH are strikingly similar to ferritin (Figure 6A).", "section": "DISCUSS", "ner": [ [ 58, 67, "classical", "protein_state" ], [ 68, 77, "ferritins", "protein_type" ], [ 83, 111, "four-helical bundle scaffold", "structure_element" ], [ 116, 119, "FOC", "site" ], [ 123, 131, "EncFtnsH", "protein" ], [ 158, 166, "ferritin", "protein_type" ] ] }, { "sid": 376, "sent": "A sequence-based phylogenetic tree for proteins in the ferritin family was constructed; in addition to the classical ferritins, bacterioferritins and Dps proteins, our analysis included the encapsulin-associated ferritin-like proteins (EncFtns) and a group related to these, but lacking the encapsulin sequence (Non-EncFtn).", "section": "DISCUSS", "ner": [ [ 2, 34, "sequence-based phylogenetic tree", "experimental_method" ], [ 55, 63, "ferritin", "protein_type" ], [ 107, 116, "classical", "protein_state" ], [ 117, 126, "ferritins", "protein_type" ], [ 128, 145, "bacterioferritins", "protein_type" ], [ 150, 153, "Dps", "protein_type" ], [ 190, 234, "encapsulin-associated ferritin-like proteins", "protein_type" ], [ 236, 243, "EncFtns", "protein_type" ], [ 291, 301, "encapsulin", "protein" ], [ 312, 322, "Non-EncFtn", "protein_type" ] ] }, { "sid": 377, "sent": "The analysis revealed that the EncFtn and Non-EncFtn proteins form groups distinct from the other clearly delineated groups of ferritins, and represent outliers in the tree (Figure 13).", "section": "DISCUSS", "ner": [ [ 31, 37, "EncFtn", "protein" ], [ 42, 52, "Non-EncFtn", "protein_type" ], [ 127, 136, "ferritins", "protein_type" ] ] }, { "sid": 378, "sent": "While it is difficult to infer ancestral lineages in protein families, the similarity seen in the active site scaffold of these proteins highlights a shared evolutionary relationship between EncFtn proteins and other members of the ferritin superfamily that has been noted in previous studies (;\u00a0).", "section": "DISCUSS", "ner": [ [ 98, 118, "active site scaffold", "site" ], [ 191, 197, "EncFtn", "protein_type" ], [ 232, 240, "ferritin", "protein_type" ] ] }, { "sid": 379, "sent": "From this analysis, we propose that the four-helical fold of the classical ferritins may have arisen through gene duplication of an ancestor of EncFtn.", "section": "DISCUSS", "ner": [ [ 40, 57, "four-helical fold", "structure_element" ], [ 65, 74, "classical", "protein_state" ], [ 75, 84, "ferritins", "protein_type" ], [ 144, 150, "EncFtn", "protein" ] ] }, { "sid": 380, "sent": "This gene duplication would result in the C-terminal region of one EncFtn monomer being linked to the N-terminus of another and thus stabilizing the four-helix bundle fold within a single polypeptide chain (Figure 6B).", "section": "DISCUSS", "ner": [ [ 42, 59, "C-terminal region", "structure_element" ], [ 67, 73, "EncFtn", "protein" ], [ 74, 81, "monomer", "oligomeric_state" ], [ 149, 171, "four-helix bundle fold", "structure_element" ] ] }, { "sid": 381, "sent": "Linking the protein together in this way relaxes the requirement for the maintenance of a symmetrical FOC and thus provides a path to the diversity in active-site residues seen across the ferritin family (Figure 6A, residues Glu95, Gln128 and Glu131 in PmFtn, Supplementary file 1).", "section": "DISCUSS", "ner": [ [ 102, 105, "FOC", "site" ], [ 151, 171, "active-site residues", "site" ], [ 188, 196, "ferritin", "protein_type" ], [ 225, 230, "Glu95", "residue_name_number" ], [ 232, 238, "Gln128", "residue_name_number" ], [ 243, 249, "Glu131", "residue_name_number" ], [ 253, 258, "PmFtn", "protein" ] ] }, { "sid": 382, "sent": "Relationship between ferritin structure and activity", "section": "DISCUSS", "ner": [ [ 21, 29, "ferritin", "protein_type" ], [ 30, 39, "structure", "evidence" ] ] }, { "sid": 383, "sent": "The quaternary arrangement of classical ferritins into an octahedral nanocage and Dps into a dodecamer is absolutely required for their function as iron storage compartments.", "section": "DISCUSS", "ner": [ [ 30, 39, "classical", "protein_state" ], [ 40, 49, "ferritins", "protein_type" ], [ 58, 68, "octahedral", "protein_state" ], [ 69, 77, "nanocage", "complex_assembly" ], [ 82, 85, "Dps", "protein" ], [ 93, 102, "dodecamer", "oligomeric_state" ], [ 148, 152, "iron", "chemical" ] ] }, { "sid": 384, "sent": "The oxidation and mineralization of iron must be spatially separated from the host cytosol to prevent the formation of damaging hydroxyl radicals in the Fenton and Haber-Weiss reactions.", "section": "DISCUSS", "ner": [ [ 36, 40, "iron", "chemical" ] ] }, { "sid": 385, "sent": "\u00a0This is achieved in all ferritins by confining the oxidation of iron to the interior of the protein complex, thus achieving sequestration of the Fe3+ mineralization product.", "section": "DISCUSS", "ner": [ [ 25, 34, "ferritins", "protein_type" ], [ 65, 69, "iron", "chemical" ], [ 146, 150, "Fe3+", "chemical" ] ] }, { "sid": 386, "sent": "A structural alignment of the FOC of EncFtn with the classical ferritin PmFtn shows that the central ring of EncFtn corresponds to the external surface of ferritin, while the outer circumference of EncFtn is congruent with the inner mineralization surface of ferritin (Figure 6\u2014figure supplement 1A).", "section": "DISCUSS", "ner": [ [ 2, 22, "structural alignment", "experimental_method" ], [ 30, 33, "FOC", "site" ], [ 37, 43, "EncFtn", "protein" ], [ 53, 62, "classical", "protein_state" ], [ 63, 71, "ferritin", "protein_type" ], [ 72, 77, "PmFtn", "protein" ], [ 93, 105, "central ring", "structure_element" ], [ 109, 115, "EncFtn", "protein" ], [ 155, 163, "ferritin", "protein_type" ], [ 198, 204, "EncFtn", "protein" ], [ 233, 255, "mineralization surface", "site" ], [ 259, 267, "ferritin", "protein_type" ] ] }, { "sid": 387, "sent": "This overlay highlights the fact that the ferroxidase center of EncFtn faces in the opposite direction relative to the classical ferritins and is essentially inside out regarding iron storage space (Figure 6\u2014figure supplement 1B, boxed region).", "section": "DISCUSS", "ner": [ [ 5, 12, "overlay", "experimental_method" ], [ 42, 60, "ferroxidase center", "site" ], [ 64, 70, "EncFtn", "protein" ], [ 119, 128, "classical", "protein_state" ], [ 129, 138, "ferritins", "protein_type" ], [ 179, 183, "iron", "chemical" ] ] }, { "sid": 388, "sent": "Analysis of each of the single mutations (E32A, E62A and H65A) made in the FOC highlights the importance of the iron-coordinating residues in the catalytic activity of EncFtn.", "section": "DISCUSS", "ner": [ [ 31, 40, "mutations", "experimental_method" ], [ 42, 46, "E32A", "mutant" ], [ 48, 52, "E62A", "mutant" ], [ 57, 61, "H65A", "mutant" ], [ 75, 78, "FOC", "site" ], [ 112, 138, "iron-coordinating residues", "site" ], [ 168, 174, "EncFtn", "protein" ] ] }, { "sid": 389, "sent": "Furthermore, the position of the calcium ion coordinated by Glu31 and Glu34 seen in the EncFtnsH structure suggests an entry site to channel metal ions into the FOC; we propose that this site binds hydrated iron ions in vivo and acts as a selectivity filter and gate for the FOC.", "section": "DISCUSS", "ner": [ [ 33, 40, "calcium", "chemical" ], [ 45, 59, "coordinated by", "bond_interaction" ], [ 60, 65, "Glu31", "residue_name_number" ], [ 70, 75, "Glu34", "residue_name_number" ], [ 88, 96, "EncFtnsH", "protein" ], [ 97, 106, "structure", "evidence" ], [ 119, 129, "entry site", "site" ], [ 161, 164, "FOC", "site" ], [ 207, 211, "iron", "chemical" ], [ 275, 278, "FOC", "site" ] ] }, { "sid": 390, "sent": "The constellation of charged residues on the outer circumference of EncFtn (His57, Glu61 and Glu64) could function in the same way as the residues lining the mineralization surface within the classical ferritin nanocage, and given their proximity to the FOC these sites may be the exit portal and mineralization site.", "section": "DISCUSS", "ner": [ [ 68, 74, "EncFtn", "protein" ], [ 76, 81, "His57", "residue_name_number" ], [ 83, 88, "Glu61", "residue_name_number" ], [ 93, 98, "Glu64", "residue_name_number" ], [ 158, 180, "mineralization surface", "site" ], [ 192, 201, "classical", "protein_state" ], [ 202, 210, "ferritin", "protein_type" ], [ 211, 219, "nanocage", "complex_assembly" ], [ 254, 257, "FOC", "site" ], [ 281, 292, "exit portal", "site" ], [ 297, 316, "mineralization site", "site" ] ] }, { "sid": 391, "sent": "The absolute requirement for the spatial separation of oxidation and mineralization in ferritins suggests that the EncFtn family proteins are not capable of storing iron minerals due to the absence of an enclosed compartment in their structure (Figure 6\u2014figure supplement 1B).", "section": "DISCUSS", "ner": [ [ 87, 96, "ferritins", "protein_type" ], [ 115, 121, "EncFtn", "protein_type" ], [ 165, 169, "iron", "chemical" ], [ 190, 200, "absence of", "protein_state" ] ] }, { "sid": 392, "sent": "Our biochemical characterization of EncFtn supports this hypothesis, indicating that while this protein is capable of oxidizing iron, it does not accrue mineralized iron in an analogous manner to classical ferritins.", "section": "DISCUSS", "ner": [ [ 4, 32, "biochemical characterization", "experimental_method" ], [ 36, 42, "EncFtn", "protein" ], [ 128, 132, "iron", "chemical" ], [ 165, 169, "iron", "chemical" ], [ 196, 205, "classical", "protein_state" ], [ 206, 215, "ferritins", "protein_type" ] ] }, { "sid": 393, "sent": "While EncFtn does not store iron itself, its association with the encapsulin nanocage suggests that mineralization occurs within the cavity of the encapsulin shell.", "section": "DISCUSS", "ner": [ [ 6, 12, "EncFtn", "protein" ], [ 28, 32, "iron", "chemical" ], [ 66, 76, "encapsulin", "protein" ], [ 77, 85, "nanocage", "complex_assembly" ], [ 133, 139, "cavity", "site" ], [ 147, 157, "encapsulin", "protein" ], [ 158, 163, "shell", "structure_element" ] ] }, { "sid": 394, "sent": "Our ferroxidase assay data on the recombinant EncFtn-Enc nanocompartments, which accrue over 4100 iron ions per complex and form regular nanoparticles, are consistent with the encapsulin protein acting as the store for iron oxidized by the EncFtn enzyme.", "section": "DISCUSS", "ner": [ [ 4, 21, "ferroxidase assay", "experimental_method" ], [ 46, 56, "EncFtn-Enc", "complex_assembly" ], [ 57, 73, "nanocompartments", "complex_assembly" ], [ 98, 102, "iron", "chemical" ], [ 137, 150, "nanoparticles", "complex_assembly" ], [ 176, 186, "encapsulin", "protein" ], [ 219, 223, "iron", "chemical" ], [ 240, 246, "EncFtn", "protein" ] ] }, { "sid": 395, "sent": "TEM analysis of the reaction products shows the production of homogeneous iron nanoparticles only in the EncFtn-Enc nanocompartment (Figure 8\u2014figure supplement 1).", "section": "DISCUSS", "ner": [ [ 0, 3, "TEM", "experimental_method" ], [ 74, 78, "iron", "chemical" ], [ 105, 115, "EncFtn-Enc", "complex_assembly" ], [ 116, 131, "nanocompartment", "complex_assembly" ] ] }, { "sid": 396, "sent": "Model of iron oxidation in encapsulin nanocompartments.", "section": "FIG", "ner": [ [ 9, 13, "iron", "chemical" ], [ 27, 37, "encapsulin", "protein" ], [ 38, 54, "nanocompartments", "complex_assembly" ] ] }, { "sid": 397, "sent": "(A) Model of EncFtnsH docking to the encapsulin shell.", "section": "FIG", "ner": [ [ 13, 21, "EncFtnsH", "protein" ], [ 22, 29, "docking", "experimental_method" ], [ 37, 47, "encapsulin", "protein" ], [ 48, 53, "shell", "structure_element" ] ] }, { "sid": 398, "sent": "A single pentamer of the icosahedral T. maritima encapsulin structure (PDBID: 3DKT) is shown as a blue surface with the encapsulin localization sequence of EncFtn shown as a purple surface.", "section": "FIG", "ner": [ [ 9, 17, "pentamer", "oligomeric_state" ], [ 25, 36, "icosahedral", "protein_state" ], [ 37, 48, "T. maritima", "species" ], [ 49, 59, "encapsulin", "protein" ], [ 60, 69, "structure", "evidence" ], [ 120, 130, "encapsulin", "protein" ], [ 131, 152, "localization sequence", "structure_element" ], [ 156, 162, "EncFtn", "protein" ] ] }, { "sid": 399, "sent": "The C-terminal regions of the EncFtn subunits correspond to the position of the localization sequences seen in 3DKT.", "section": "FIG", "ner": [ [ 30, 36, "EncFtn", "protein" ], [ 37, 45, "subunits", "structure_element" ], [ 80, 102, "localization sequences", "structure_element" ] ] }, { "sid": 400, "sent": "Alignment of EncFtnsH with 3DKT positions the central channel directly above the pore in the 3DKT pentamer axis (shown as a grey pentagon). (B) Surface view of EncFtn within the encapsulin nanocompartment (grey and blue respectively).", "section": "FIG", "ner": [ [ 0, 9, "Alignment", "experimental_method" ], [ 13, 21, "EncFtnsH", "protein" ], [ 46, 61, "central channel", "site" ], [ 81, 85, "pore", "site" ], [ 98, 106, "pentamer", "oligomeric_state" ], [ 160, 166, "EncFtn", "protein" ], [ 178, 188, "encapsulin", "protein" ], [ 189, 204, "nanocompartment", "complex_assembly" ] ] }, { "sid": 401, "sent": "The lumen of the encapsulin nanocompartment is considerably larger than the interior of ferritin (shown in orange behind the encapsulin for reference) and thus allows the storage of significantly more iron.", "section": "FIG", "ner": [ [ 17, 27, "encapsulin", "protein" ], [ 28, 43, "nanocompartment", "complex_assembly" ], [ 88, 96, "ferritin", "protein_type" ], [ 125, 135, "encapsulin", "protein" ], [ 201, 205, "iron", "chemical" ] ] }, { "sid": 402, "sent": "The proposed pathway for iron movement through the encapsulin shell and EncFtn FOC is shown with arrows. (C) Model ofiron oxidation within an encapsulin nanocompartment.", "section": "FIG", "ner": [ [ 25, 29, "iron", "chemical" ], [ 51, 61, "encapsulin", "protein" ], [ 62, 67, "shell", "structure_element" ], [ 72, 78, "EncFtn", "protein" ], [ 79, 82, "FOC", "site" ], [ 142, 152, "encapsulin", "protein" ], [ 153, 168, "nanocompartment", "complex_assembly" ] ] }, { "sid": 403, "sent": "As EncFtn is unable to mineralize iron on its surface directly, Fe2+ must pass through the encapsulin shell to access the first metal binding site within the central channel of EncFtnsH (entry site) prior to oxidation within the FOC and release as Fe3+ to the outer surface of the protein where it can be mineralized within the lumen of the encapsulin cage.", "section": "FIG", "ner": [ [ 3, 9, "EncFtn", "protein" ], [ 34, 38, "iron", "chemical" ], [ 64, 68, "Fe2+", "chemical" ], [ 91, 101, "encapsulin", "protein" ], [ 102, 107, "shell", "structure_element" ], [ 128, 146, "metal binding site", "site" ], [ 158, 173, "central channel", "site" ], [ 177, 185, "EncFtnsH", "protein" ], [ 187, 197, "entry site", "site" ], [ 229, 232, "FOC", "site" ], [ 248, 252, "Fe3+", "chemical" ], [ 341, 351, "encapsulin", "protein" ] ] }, { "sid": 404, "sent": "Docking the decamer structure of EncFtnsH into the pentamer of the T. maritima encapsulin Tmari_0786 (PDB ID: 3DKT) \u00a0shows that the position of the C-terminal extensions of our EncFtnsH structure are consistent with the localization sequences seen bound to the encapsulin protein (Figure 14A).", "section": "DISCUSS", "ner": [ [ 0, 7, "Docking", "experimental_method" ], [ 12, 19, "decamer", "oligomeric_state" ], [ 20, 29, "structure", "evidence" ], [ 33, 41, "EncFtnsH", "protein" ], [ 51, 59, "pentamer", "oligomeric_state" ], [ 67, 78, "T. maritima", "species" ], [ 79, 89, "encapsulin", "protein" ], [ 90, 100, "Tmari_0786", "gene" ], [ 148, 169, "C-terminal extensions", "structure_element" ], [ 177, 185, "EncFtnsH", "protein" ], [ 186, 195, "structure", "evidence" ], [ 220, 242, "localization sequences", "structure_element" ], [ 248, 256, "bound to", "protein_state" ], [ 261, 271, "encapsulin", "protein" ] ] }, { "sid": 405, "sent": "Thus, it appears that the EncFtn decamer is the physiological state of this protein.", "section": "DISCUSS", "ner": [ [ 26, 32, "EncFtn", "protein" ], [ 33, 40, "decamer", "oligomeric_state" ] ] }, { "sid": 406, "sent": "This arrangement positions the central ring of EncFtn directly above the pore at the five-fold symmetry axis of the encapsulin shell and highlights a potential route for the entry of iron into the encapsulin and towards the active site of EncFtn.", "section": "DISCUSS", "ner": [ [ 31, 43, "central ring", "structure_element" ], [ 47, 53, "EncFtn", "protein" ], [ 73, 77, "pore", "site" ], [ 116, 126, "encapsulin", "protein" ], [ 127, 132, "shell", "structure_element" ], [ 183, 187, "iron", "chemical" ], [ 197, 207, "encapsulin", "protein" ], [ 224, 235, "active site", "site" ], [ 239, 245, "EncFtn", "protein" ] ] }, { "sid": 407, "sent": "A comparison of the encapsulin nanocompartment and the ferritin nanocage highlights the size differential between the two complexes (Figure 14B) that allows the encapsulin to store significantly more iron.", "section": "DISCUSS", "ner": [ [ 20, 30, "encapsulin", "protein" ], [ 31, 46, "nanocompartment", "complex_assembly" ], [ 55, 63, "ferritin", "protein_type" ], [ 64, 72, "nanocage", "complex_assembly" ], [ 161, 171, "encapsulin", "protein" ], [ 200, 204, "iron", "chemical" ] ] }, { "sid": 408, "sent": "The presence of five FOCs per EncFtnsH decamer and the fact that the icosahedral encapsulin nanocage can hold up to twelve of decameric EncFtn between each of the internal five-fold vertices means that they can achieve a high rate of iron mineralization across the entire nanocompartment.", "section": "DISCUSS", "ner": [ [ 4, 15, "presence of", "protein_state" ], [ 21, 25, "FOCs", "site" ], [ 30, 38, "EncFtnsH", "protein" ], [ 39, 46, "decamer", "oligomeric_state" ], [ 69, 80, "icosahedral", "protein_state" ], [ 81, 91, "encapsulin", "protein" ], [ 92, 100, "nanocage", "complex_assembly" ], [ 126, 135, "decameric", "oligomeric_state" ], [ 136, 142, "EncFtn", "protein" ], [ 234, 238, "iron", "chemical" ], [ 272, 287, "nanocompartment", "complex_assembly" ] ] }, { "sid": 409, "sent": "This arrangement of multiple reaction centers in a single protein assembly is reminiscent of classical ferritins, which has 24 FOCs distributed around the nanocage.", "section": "DISCUSS", "ner": [ [ 93, 102, "classical", "protein_state" ], [ 103, 112, "ferritins", "protein_type" ], [ 127, 131, "FOCs", "site" ], [ 155, 163, "nanocage", "complex_assembly" ] ] }, { "sid": 410, "sent": "Our structural data, coupled with biochemical and ICP-MS analysis, suggest a model for the activity of the encapsulin iron-megastore (Figure 14C).", "section": "DISCUSS", "ner": [ [ 4, 19, "structural data", "evidence" ], [ 34, 56, "biochemical and ICP-MS", "experimental_method" ], [ 107, 117, "encapsulin", "protein" ], [ 118, 132, "iron-megastore", "complex_assembly" ] ] }, { "sid": 411, "sent": "The crystal structure of the T. maritima encapsulin shell protein has a negatively charged pore positioned to allow the passage of Fe2+ into the encapsulin and directs the metal towards the central, negatively charged hole of the EncFtn ring (Figure 4\u2014figure supplement 1).", "section": "DISCUSS", "ner": [ [ 4, 21, "crystal structure", "evidence" ], [ 29, 40, "T. maritima", "species" ], [ 41, 51, "encapsulin", "protein" ], [ 52, 57, "shell", "structure_element" ], [ 72, 95, "negatively charged pore", "site" ], [ 131, 135, "Fe2+", "chemical" ], [ 145, 155, "encapsulin", "protein" ], [ 199, 222, "negatively charged hole", "site" ], [ 230, 236, "EncFtn", "protein" ], [ 237, 241, "ring", "structure_element" ] ] }, { "sid": 412, "sent": "The five metal-binding sites on the interior of the ring (Glu31/34-sites) may select for the Fe2+ ion and direct it towards their cognate FOCs.", "section": "DISCUSS", "ner": [ [ 9, 28, "metal-binding sites", "site" ], [ 52, 56, "ring", "structure_element" ], [ 58, 72, "Glu31/34-sites", "site" ], [ 93, 97, "Fe2+", "chemical" ], [ 138, 142, "FOCs", "site" ] ] }, { "sid": 413, "sent": "We propose that the oxidation of Fe2+ to Fe3+ occurs within the FOC according to the model postulated by \u00a0in which the FOC acts as a substrate site through which iron passes and is released on to weakly coordinating sites at the outer circumference of the protein (His57, Glu61 and Glu64), where it is able to form ferrihydrite minerals which can be safely deposited within the lumen of the encapsulin nanocompartment (Figure 14).", "section": "DISCUSS", "ner": [ [ 33, 37, "Fe2+", "chemical" ], [ 41, 45, "Fe3+", "chemical" ], [ 64, 67, "FOC", "site" ], [ 119, 122, "FOC", "site" ], [ 133, 147, "substrate site", "site" ], [ 162, 166, "iron", "chemical" ], [ 196, 221, "weakly coordinating sites", "site" ], [ 265, 270, "His57", "residue_name_number" ], [ 272, 277, "Glu61", "residue_name_number" ], [ 282, 287, "Glu64", "residue_name_number" ], [ 315, 327, "ferrihydrite", "chemical" ], [ 391, 401, "encapsulin", "protein" ], [ 402, 417, "nanocompartment", "complex_assembly" ] ] }, { "sid": 414, "sent": "Here we describe for the first time the structure and biochemistry of a new class of encapsulin-associated ferritin-like protein and demonstrate that it has an absolute requirement for compartmentalization within an encapsulin nanocage to act as an iron store.", "section": "DISCUSS", "ner": [ [ 40, 49, "structure", "evidence" ], [ 85, 128, "encapsulin-associated ferritin-like protein", "protein_type" ], [ 216, 226, "encapsulin", "protein" ], [ 227, 235, "nanocage", "complex_assembly" ], [ 249, 253, "iron", "chemical" ] ] }, { "sid": 415, "sent": "Further work on the EncFtn-Enc nanocompartment will establish the structural basis for the movement of iron through the encapsulin shell, the mechanism of iron oxidation by the EncFtn FOC and its subsequent storage in the lumen of the encapsulin nanocompartment.", "section": "DISCUSS", "ner": [ [ 20, 30, "EncFtn-Enc", "complex_assembly" ], [ 31, 46, "nanocompartment", "complex_assembly" ], [ 103, 107, "iron", "chemical" ], [ 120, 130, "encapsulin", "protein" ], [ 131, 136, "shell", "structure_element" ], [ 155, 159, "iron", "chemical" ], [ 177, 183, "EncFtn", "protein" ], [ 184, 187, "FOC", "site" ], [ 235, 245, "encapsulin", "protein" ], [ 246, 261, "nanocompartment", "complex_assembly" ] ] }, { "sid": 416, "sent": "TEM imaging was performed on purified encapsulin, EncFtn, and EncFtn-Enc and apoferritin.", "section": "METHODS", "ner": [ [ 77, 88, "apoferritin", "protein_state" ] ] }, { "sid": 417, "sent": "To observe iron mineral formation by TEM, protein samples at 8.5 \u00b5M concentration including EncFtnsH, encapsulin, EncFtn-Enc and apoferritin were supplemented with acidic Fe(NH4)2(SO4)2 at their maximum iron loading ratio in room temperature for 1 hr.", "section": "METHODS", "ner": [ [ 129, 140, "apoferritin", "protein_state" ] ] }, { "sid": 418, "sent": "Horse spleen apoferritin preparation", "section": "METHODS", "ner": [ [ 13, 24, "apoferritin", "protein_state" ] ] }, { "sid": 419, "sent": "Horse spleen apoferritin purchased from Sigma Aldrich (UK)\u00a0was dissolved in deaerated MOPS buffer (100 mM MOPS, 100 mM NaCl, 3 g/100 ml Na2S2O4 and 0.5 M EDTA, pH 6.5).", "section": "METHODS", "ner": [ [ 13, 24, "apoferritin", "protein_state" ] ] }, { "sid": 420, "sent": "Fe content of apoferritin was detected using ferrozine assay.", "section": "METHODS", "ner": [ [ 14, 25, "apoferritin", "protein_state" ] ] }, { "sid": 421, "sent": "Apoferritin containing less than 0.5 Fe per 24-mer was used in the ferroxidase assay.", "section": "METHODS", "ner": [ [ 0, 11, "Apoferritin", "protein_state" ] ] }, { "sid": 422, "sent": "Apoferritin used in the Fe loading capacity experiment was prepared in the same way with 5\u201315 Fe per 24-mer.", "section": "METHODS", "ner": [ [ 0, 11, "Apoferritin", "protein_state" ] ] }, { "sid": 423, "sent": "In order to determine the maximum iron loading capacity, around 8.5 \u00b5M proteins including decameric EncFtnsH, Encapsulin, EncFtn-Enc and apoferritin were loaded with various amount of acidic Fe(NH4)2(SO4)2 ranging from 0 to 1700 \u00b5M. Protein mixtures were incubated in room temperature for 3 hrs before desalting in Zebra spin desalting columns (7 kDa cut-off, Thermo Fisher Scientific,\u00a0UK) to remove free iron ions.", "section": "METHODS", "ner": [ [ 137, 148, "apoferritin", "protein_state" ] ] }, { "sid": 424, "sent": "Both monomer and decamer fractions of EncFtnsH left at room temperature for 2 hrs, or overnight, were also analysed as controls to show the stability of the protein samples in the absence of additional metal ions.", "section": "METHODS", "ner": [ [ 180, 190, "absence of", "protein_state" ] ] }, { "sid": 425, "sent": "Characterization of a Mycobacterium tuberculosis nanocompartment and its potential cargo proteins", "section": "REF", "ner": [ [ 49, 64, "nanocompartment", "complex_assembly" ] ] }, { "sid": 426, "sent": "A virus capsid-like nanocompartment that stores iron and protects bacteria from oxidative stress", "section": "REF", "ner": [ [ 20, 35, "nanocompartment", "complex_assembly" ] ] }, { "sid": 427, "sent": "Self-sorting of foreign proteins in a bacterial nanocompartment", "section": "REF", "ner": [ [ 48, 63, "nanocompartment", "complex_assembly" ] ] }, { "sid": 428, "sent": "Structural basis of enzyme encapsulation into a bacterial nanocompartment", "section": "REF", "ner": [ [ 58, 73, "nanocompartment", "complex_assembly" ] ] }, { "sid": 429, "sent": "1) Methods: What procedures and analyses did the author use to assess whether the iron added to the various ferritin derivatives was protein coated or was simply balls of rust attached to protein fragments? If the latter, it could easily generate reactive oxygen species in air under physiological conditions.", "section": "REVIEW_INFO", "ner": [ [ 256, 262, "oxygen", "chemical" ] ] }, { "sid": 430, "sent": "Even an experimental situation: 24 subunit (monomer) ferritin with a biomineral prepared experimentally from apoferritin and containing, on average, only 1000 iron atoms/24 subunit cage, the equivalent parameter appears to be 1000/24 = 42.", "section": "REVIEW_INFO", "ner": [ [ 109, 120, "apoferritin", "protein_state" ], [ 109, 120, "apoferritin", "protein_state" ] ] }, { "sid": 431, "sent": "Missing are data for the starting material, 24 subunit ferritin or apoferritin (ferritin with the iron removed, by reduction and chelation, as a control.)", "section": "REVIEW_INFO", "ner": [ [ 67, 78, "apoferritin", "protein_state" ], [ 67, 78, "apoferritin", "protein_state" ] ] }, { "sid": 432, "sent": "4) I would have liked to see some mutagenesis experiments to test the models of assembly, iron binding and ferroxidase activity.", "section": "REVIEW_INFO", "ner": [ [ 34, 45, "mutagenesis", "experimental_method" ], [ 107, 118, "ferroxidase", "protein_type" ] ] }, { "sid": 433, "sent": "These results show the production of ROS by apoferritin, which is consistent with the published data on the reaction mechanism of certain ferritins; however, no significant ROS were detected for the EncFtn or encapsulin proteins.", "section": "REVIEW_INFO", "ner": [ [ 44, 55, "apoferritin", "protein_state" ] ] }, { "sid": 434, "sent": "We have clarified this key difference in the discussion of the iron storage function of the encapsulin nanocompartment (subsection \u201cIron storage in encapsulin nanocompartments\u201d, second paragraph).", "section": "REVIEW_INFO", "ner": [ [ 103, 118, "nanocompartment", "complex_assembly" ] ] }, { "sid": 435, "sent": "The key conclusion of the paper is that the iron storage and iron oxidation functions that are combined in classical ferritins are split between the encapsulin nanocompartment and the EncFtn protein.", "section": "REVIEW_INFO", "ner": [ [ 160, 175, "nanocompartment", "complex_assembly" ] ] }, { "sid": 436, "sent": "Control data for apoferritin have been added to this table and are illustrated in Figure 8.", "section": "REVIEW_INFO", "ner": [ [ 17, 28, "apoferritin", "protein_state" ] ] }, { "sid": 437, "sent": "We note that we do not reach the experimental maximum loading capacity for apoferritin; however, we also note that the EncFtn-encapsulin nanocompartment sequesters five times more iron than the ferritin under the same reaction conditions, supporting the published observations that these nanocompartments can store more iron than classical ferritin nanocages.", "section": "REVIEW_INFO", "ner": [ [ 75, 86, "apoferritin", "protein_state" ], [ 137, 152, "nanocompartment", "complex_assembly" ] ] } ] }, "PMC4981400": { "annotations": [ { "sid": 0, "sent": "Crystal Structure of the SPOC Domain of the Arabidopsis Flowering Regulator FPA", "section": "TITLE", "ner": [ [ 0, 17, "Crystal Structure", "evidence" ], [ 25, 29, "SPOC", "structure_element" ], [ 44, 55, "Arabidopsis", "taxonomy_domain" ], [ 56, 75, "Flowering Regulator", "protein_type" ], [ 76, 79, "FPA", "protein" ] ] }, { "sid": 1, "sent": "The Arabidopsis protein FPA controls flowering time by regulating the alternative 3\u2032-end processing of the FLOWERING LOCUS (FLC) antisense RNA.", "section": "ABSTRACT", "ner": [ [ 4, 15, "Arabidopsis", "taxonomy_domain" ], [ 24, 27, "FPA", "protein" ], [ 107, 122, "FLOWERING LOCUS", "gene" ], [ 124, 127, "FLC", "gene" ], [ 129, 142, "antisense RNA", "chemical" ] ] }, { "sid": 2, "sent": "FPA belongs to the split ends (SPEN) family of proteins, which contain N-terminal RNA recognition motifs (RRMs) and a SPEN paralog and ortholog C-terminal (SPOC) domain.", "section": "ABSTRACT", "ner": [ [ 0, 3, "FPA", "protein" ], [ 19, 29, "split ends", "protein_type" ], [ 31, 35, "SPEN", "protein_type" ], [ 82, 104, "RNA recognition motifs", "structure_element" ], [ 106, 110, "RRMs", "structure_element" ], [ 118, 154, "SPEN paralog and ortholog C-terminal", "structure_element" ], [ 156, 160, "SPOC", "structure_element" ] ] }, { "sid": 3, "sent": "The SPOC domain is highly conserved among FPA homologs in plants, but the conservation with the domain in other SPEN proteins is much lower.", "section": "ABSTRACT", "ner": [ [ 4, 8, "SPOC", "structure_element" ], [ 19, 35, "highly conserved", "protein_state" ], [ 42, 45, "FPA", "protein" ], [ 58, 64, "plants", "taxonomy_domain" ], [ 112, 116, "SPEN", "protein_type" ] ] }, { "sid": 4, "sent": "We have determined the crystal structure of Arabidopsis thaliana FPA SPOC domain at 2.7 \u00c5 resolution.", "section": "ABSTRACT", "ner": [ [ 23, 40, "crystal structure", "evidence" ], [ 44, 64, "Arabidopsis thaliana", "species" ], [ 65, 68, "FPA", "protein" ], [ 69, 73, "SPOC", "structure_element" ] ] }, { "sid": 5, "sent": "The overall structure is similar to that of the SPOC domain in human SMRT/HDAC1 Associated Repressor Protein (SHARP), although there are also substantial conformational differences between them.", "section": "ABSTRACT", "ner": [ [ 12, 21, "structure", "evidence" ], [ 48, 52, "SPOC", "structure_element" ], [ 63, 68, "human", "species" ], [ 69, 108, "SMRT/HDAC1 Associated Repressor Protein", "protein" ], [ 110, 115, "SHARP", "protein" ] ] }, { "sid": 6, "sent": "Structural and sequence analyses identify a surface patch that is conserved among plant FPA homologs.", "section": "ABSTRACT", "ner": [ [ 0, 32, "Structural and sequence analyses", "experimental_method" ], [ 44, 57, "surface patch", "site" ], [ 66, 75, "conserved", "protein_state" ], [ 82, 87, "plant", "taxonomy_domain" ], [ 88, 91, "FPA", "protein" ] ] }, { "sid": 7, "sent": "Mutations of two residues in this surface patch did not disrupt FPA functions, suggesting that either the SPOC domain is not required for the role of FPA in regulating RNA 3\u2032-end formation or the functions of the FPA SPOC domain cannot be disrupted by the combination of mutations, in contrast to observations with the SHARP SPOC domain.", "section": "ABSTRACT", "ner": [ [ 0, 9, "Mutations", "experimental_method" ], [ 34, 47, "surface patch", "site" ], [ 64, 67, "FPA", "protein" ], [ 106, 110, "SPOC", "structure_element" ], [ 150, 153, "FPA", "protein" ], [ 168, 171, "RNA", "chemical" ], [ 213, 216, "FPA", "protein" ], [ 217, 221, "SPOC", "structure_element" ], [ 319, 324, "SHARP", "protein" ], [ 325, 329, "SPOC", "structure_element" ] ] }, { "sid": 8, "sent": "Eukaryotic messenger RNAs (mRNAs) are made as precursors through transcription by RNA polymerase II (Pol II), and these primary transcripts undergo extensive processing, including 3\u2032-end cleavage and polyadenylation.", "section": "INTRO", "ner": [ [ 0, 10, "Eukaryotic", "taxonomy_domain" ], [ 11, 25, "messenger RNAs", "chemical" ], [ 27, 32, "mRNAs", "chemical" ], [ 82, 99, "RNA polymerase II", "complex_assembly" ], [ 101, 107, "Pol II", "complex_assembly" ] ] }, { "sid": 9, "sent": "In addition, alternative 3\u2032-end cleavage and polyadenylation is an essential and ubiquitous process in eukaryotes.", "section": "INTRO", "ner": [ [ 103, 113, "eukaryotes", "taxonomy_domain" ] ] }, { "sid": 10, "sent": "Recently, the split ends (SPEN) family of proteins was identified as RNA binding proteins that regulate alternative 3\u2032-end cleavage and polyadenylation.", "section": "INTRO", "ner": [ [ 14, 24, "split ends", "protein_type" ], [ 26, 30, "SPEN", "protein_type" ], [ 69, 89, "RNA binding proteins", "protein_type" ] ] }, { "sid": 11, "sent": "They are characterized by possessing N-terminal RNA recognition motifs (RRMs) and a conserved SPEN paralog and ortholog C-terminal (SPOC) domain (Fig 1A).", "section": "INTRO", "ner": [ [ 48, 70, "RNA recognition motifs", "structure_element" ], [ 72, 76, "RRMs", "structure_element" ], [ 84, 93, "conserved", "protein_state" ], [ 94, 130, "SPEN paralog and ortholog C-terminal", "structure_element" ], [ 132, 136, "SPOC", "structure_element" ] ] }, { "sid": 12, "sent": "The SPOC domain is believed to mediate protein-protein interactions and has diverse functions among SPEN family proteins, but the molecular mechanism of these functions is not well understood.", "section": "INTRO", "ner": [ [ 4, 8, "SPOC", "structure_element" ], [ 100, 104, "SPEN", "protein_type" ] ] }, { "sid": 13, "sent": "Sequence conservation of SPOC domains.", "section": "FIG", "ner": [ [ 0, 21, "Sequence conservation", "evidence" ], [ 25, 29, "SPOC", "structure_element" ] ] }, { "sid": 14, "sent": "Domain organization of A. thaliana FPA. (B).", "section": "FIG", "ner": [ [ 23, 34, "A. thaliana", "species" ], [ 35, 38, "FPA", "protein" ] ] }, { "sid": 15, "sent": "Sequence alignment of the SPOC domains of Arabidopsis thaliana FPA, human RBM15, Drosophila SPEN, mouse MINT, and human SHARP.", "section": "FIG", "ner": [ [ 0, 18, "Sequence alignment", "experimental_method" ], [ 26, 30, "SPOC", "structure_element" ], [ 42, 62, "Arabidopsis thaliana", "species" ], [ 63, 66, "FPA", "protein" ], [ 68, 73, "human", "species" ], [ 74, 79, "RBM15", "protein" ], [ 81, 91, "Drosophila", "taxonomy_domain" ], [ 92, 96, "SPEN", "protein_type" ], [ 98, 103, "mouse", "taxonomy_domain" ], [ 104, 108, "MINT", "protein" ], [ 114, 119, "human", "species" ], [ 120, 125, "SHARP", "protein" ] ] }, { "sid": 16, "sent": "Residues in surface patch 1 are indicated with the orange dots, and those in surface patch 2 with the green dots.", "section": "FIG", "ner": [ [ 12, 27, "surface patch 1", "site" ], [ 77, 92, "surface patch 2", "site" ] ] }, { "sid": 17, "sent": "The secondary structure elements in the structure of FPA SPOC are labeled.", "section": "FIG", "ner": [ [ 40, 49, "structure", "evidence" ], [ 53, 56, "FPA", "protein" ], [ 57, 61, "SPOC", "structure_element" ] ] }, { "sid": 18, "sent": "Residues that are strictly conserved among the five proteins are shown in white with a red background, and those that are mostly conserved in red.", "section": "FIG", "ner": [ [ 18, 36, "strictly conserved", "protein_state" ], [ 122, 138, "mostly conserved", "protein_state" ] ] }, { "sid": 19, "sent": "FPA, a SPEN family protein in Arabidopsis thaliana and other plants, was found to regulate the 3\u2032-end alternative cleavage and polyadenylation of the antisense RNAs of FLOWERING LOCUS (FLC), a flowering repressor gene.", "section": "INTRO", "ner": [ [ 0, 3, "FPA", "protein" ], [ 7, 11, "SPEN", "protein_type" ], [ 30, 50, "Arabidopsis thaliana", "species" ], [ 61, 67, "plants", "taxonomy_domain" ], [ 150, 164, "antisense RNAs", "chemical" ], [ 168, 183, "FLOWERING LOCUS", "gene" ], [ 185, 188, "FLC", "gene" ] ] }, { "sid": 20, "sent": "FPA promotes the 3\u2032-end processing of class I FLC antisense RNAs, which includes the proximal polyadenylation site.", "section": "INTRO", "ner": [ [ 0, 3, "FPA", "protein" ], [ 46, 49, "FLC", "gene" ], [ 50, 64, "antisense RNAs", "chemical" ], [ 94, 114, "polyadenylation site", "site" ] ] }, { "sid": 21, "sent": "This is associated with histone demethylase activity and down-regulation of FLC transcription.", "section": "INTRO", "ner": [ [ 24, 43, "histone demethylase", "protein_type" ], [ 76, 79, "FLC", "gene" ] ] }, { "sid": 22, "sent": "Although a SPOC domain is found in all the SPEN family proteins, its sequence conservation is rather low.", "section": "INTRO", "ner": [ [ 11, 15, "SPOC", "structure_element" ], [ 43, 47, "SPEN", "protein_type" ] ] }, { "sid": 23, "sent": "For example, the sequence identity between the SPOC domains of A. thaliana FPA and human SMRT/HDAC1 Associated Repressor Protein (SHARP) is only 19% (Fig 1B).", "section": "INTRO", "ner": [ [ 47, 51, "SPOC", "structure_element" ], [ 63, 74, "A. thaliana", "species" ], [ 75, 78, "FPA", "protein" ], [ 83, 88, "human", "species" ], [ 89, 128, "SMRT/HDAC1 Associated Repressor Protein", "protein" ], [ 130, 135, "SHARP", "protein" ] ] }, { "sid": 24, "sent": "Currently, the SHARP SPOC domain is the only one with structural information.", "section": "INTRO", "ner": [ [ 15, 20, "SHARP", "protein" ], [ 21, 25, "SPOC", "structure_element" ] ] }, { "sid": 25, "sent": "As a first step toward understanding the molecular basis for the regulation of alternative 3\u2032-end processing and flowering by FPA, we have determined the crystal structure of the SPOC domain of A. thaliana FPA at 2.7 \u00c5 resolution.", "section": "INTRO", "ner": [ [ 126, 129, "FPA", "protein" ], [ 154, 171, "crystal structure", "evidence" ], [ 179, 183, "SPOC", "structure_element" ], [ 194, 205, "A. thaliana", "species" ], [ 206, 209, "FPA", "protein" ] ] }, { "sid": 26, "sent": "The overall structure is similar to that of the SHARP SPOC domain, although there are also substantial conformational differences between them.", "section": "INTRO", "ner": [ [ 12, 21, "structure", "evidence" ], [ 48, 53, "SHARP", "protein" ], [ 54, 58, "SPOC", "structure_element" ] ] }, { "sid": 27, "sent": "The structure reveals a surface patch that is conserved among FPA homologs.", "section": "INTRO", "ner": [ [ 4, 13, "structure", "evidence" ], [ 24, 37, "surface patch", "site" ], [ 46, 55, "conserved", "protein_state" ], [ 62, 65, "FPA", "protein" ] ] }, { "sid": 28, "sent": "Structure of FPA SPOC domain", "section": "RESULTS", "ner": [ [ 0, 9, "Structure", "evidence" ], [ 13, 16, "FPA", "protein" ], [ 17, 21, "SPOC", "structure_element" ] ] }, { "sid": 29, "sent": "The crystal structure of the SPOC domain of A. thaliana FPA has been determined at 2.7 \u00c5 resolution using the selenomethionyl single-wavelength anomalous dispersion method.", "section": "RESULTS", "ner": [ [ 4, 21, "crystal structure", "evidence" ], [ 29, 33, "SPOC", "structure_element" ], [ 44, 55, "A. thaliana", "species" ], [ 56, 59, "FPA", "protein" ], [ 110, 171, "selenomethionyl single-wavelength anomalous dispersion method", "experimental_method" ] ] }, { "sid": 30, "sent": "The expression construct contained residues 433\u2013565 of FPA, but only residues 439\u2013460 and 465\u2013565 are ordered in the crystal.", "section": "RESULTS", "ner": [ [ 44, 51, "433\u2013565", "residue_range" ], [ 55, 58, "FPA", "protein" ], [ 78, 85, "439\u2013460", "residue_range" ], [ 90, 97, "465\u2013565", "residue_range" ], [ 117, 124, "crystal", "evidence" ] ] }, { "sid": 31, "sent": "The atomic model has good agreement with the X-ray diffraction data and the expected bond lengths, bond angles and other geometric parameters (Table 1).", "section": "RESULTS", "ner": [ [ 4, 16, "atomic model", "evidence" ], [ 45, 67, "X-ray diffraction data", "evidence" ] ] }, { "sid": 32, "sent": "All the residues are located in the favored regions of the Ramachandran plot (data not shown).", "section": "RESULTS", "ner": [ [ 59, 76, "Ramachandran plot", "evidence" ] ] }, { "sid": 33, "sent": "The structure has been deposited in the Protein Data Bank, with accession code 5KXF.", "section": "RESULTS", "ner": [ [ 4, 13, "structure", "evidence" ] ] }, { "sid": 34, "sent": "Resolution range (\u00c5)1\t50\u20132.7 (2.8\u20132.7)\t \tNumber of observations\t78,008\t \tRmerge (%)\t10.5 (45.3)\t \tI/\u03c3I\t24.1 (6.3)\t \tRedundancy\t\t \tCompleteness (%)\t100 (100)\t \tR factor (%)\t19.2 (25.0)\t \tFree R factor (%)\t25.4 (35.4)\t \tRms deviation in bond lengths (\u00c5)\t0.017\t \tRms deviation in bond angles (\u00b0)\t1.9\t \t", "section": "TABLE", "ner": [ [ 159, 167, "R factor", "evidence" ], [ 186, 199, "Free R factor", "evidence" ] ] }, { "sid": 35, "sent": "The crystal structure of the FPA SPOC domain contains a seven-stranded, mostly anti-parallel \u03b2-barrel (\u03b21-\u03b27) and three helices (\u03b1A-\u03b1C) (Fig 2A).", "section": "RESULTS", "ner": [ [ 4, 21, "crystal structure", "evidence" ], [ 29, 32, "FPA", "protein" ], [ 33, 37, "SPOC", "structure_element" ], [ 56, 101, "seven-stranded, mostly anti-parallel \u03b2-barrel", "structure_element" ], [ 103, 108, "\u03b21-\u03b27", "structure_element" ], [ 120, 127, "helices", "structure_element" ], [ 129, 134, "\u03b1A-\u03b1C", "structure_element" ] ] }, { "sid": 36, "sent": "Only two of the neighboring strands, \u03b21 and \u03b23, are parallel to each other.", "section": "RESULTS", "ner": [ [ 28, 35, "strands", "structure_element" ], [ 37, 39, "\u03b21", "structure_element" ], [ 44, 46, "\u03b23", "structure_element" ] ] }, { "sid": 37, "sent": "Helix \u03b1B covers one end of the barrel, while helices \u03b1A and \u03b1C are located next to each other at one side of the barrel (Fig 2B).", "section": "RESULTS", "ner": [ [ 0, 5, "Helix", "structure_element" ], [ 6, 8, "\u03b1B", "structure_element" ], [ 31, 37, "barrel", "structure_element" ], [ 45, 52, "helices", "structure_element" ], [ 53, 55, "\u03b1A", "structure_element" ], [ 60, 62, "\u03b1C", "structure_element" ], [ 113, 119, "barrel", "structure_element" ] ] }, { "sid": 38, "sent": "The other end of the \u03b2-barrel is covered by the loop connecting strands \u03b22 and \u03b23, which contains the disordered 461\u2013464 segment.", "section": "RESULTS", "ner": [ [ 21, 29, "\u03b2-barrel", "structure_element" ], [ 48, 52, "loop", "structure_element" ], [ 64, 71, "strands", "structure_element" ], [ 72, 74, "\u03b22", "structure_element" ], [ 79, 81, "\u03b23", "structure_element" ], [ 102, 112, "disordered", "protein_state" ], [ 113, 120, "461\u2013464", "residue_range" ] ] }, { "sid": 39, "sent": "The center of the barrel is filled with hydrophobic side chains and is not accessible to the solvent.", "section": "RESULTS", "ner": [ [ 18, 24, "barrel", "structure_element" ] ] }, { "sid": 40, "sent": "Crystal structure of the SPOC domain of A. thaliana FPA.", "section": "FIG", "ner": [ [ 0, 17, "Crystal structure", "evidence" ], [ 25, 29, "SPOC", "structure_element" ], [ 40, 51, "A. thaliana", "species" ], [ 52, 55, "FPA", "protein" ] ] }, { "sid": 41, "sent": "Schematic drawing of the structure of FPA SPOC domain, colored from blue at the N terminus to red at the C terminus.", "section": "FIG", "ner": [ [ 25, 34, "structure", "evidence" ], [ 38, 41, "FPA", "protein" ], [ 42, 46, "SPOC", "structure_element" ] ] }, { "sid": 42, "sent": "The view is from the side of the \u03b2-barrel.", "section": "FIG", "ner": [ [ 33, 41, "\u03b2-barrel", "structure_element" ] ] }, { "sid": 43, "sent": "The disordered segment (residues 460\u2013465) is indicated with the dotted line.", "section": "FIG", "ner": [ [ 4, 14, "disordered", "protein_state" ], [ 33, 40, "460\u2013465", "residue_range" ] ] }, { "sid": 44, "sent": "Structure of the FPA SPOC domain, viewed from the end of the \u03b2-barrel, after 90\u00b0 rotation around the horizontal axis from panel A. All structure figures were produced with PyMOL (www.pymol.org).", "section": "FIG", "ner": [ [ 0, 9, "Structure", "evidence" ], [ 17, 20, "FPA", "protein" ], [ 21, 25, "SPOC", "structure_element" ], [ 61, 69, "\u03b2-barrel", "structure_element" ] ] }, { "sid": 45, "sent": "Comparisons to structural homologs of the SPOC domain", "section": "RESULTS", "ner": [ [ 0, 34, "Comparisons to structural homologs", "experimental_method" ], [ 42, 46, "SPOC", "structure_element" ] ] }, { "sid": 46, "sent": "Only five structural homologs of the FPA SPOC domain were found in the Protein Data Bank with the DaliLite server, suggesting that the SPOC domain structure is relatively unique.", "section": "RESULTS", "ner": [ [ 37, 40, "FPA", "protein" ], [ 41, 45, "SPOC", "structure_element" ], [ 98, 113, "DaliLite server", "experimental_method" ], [ 135, 139, "SPOC", "structure_element" ], [ 147, 156, "structure", "evidence" ] ] }, { "sid": 47, "sent": "The top hit is the SPOC domain of human SHARP (Fig 3A), with a Z score of 12.3.", "section": "RESULTS", "ner": [ [ 19, 23, "SPOC", "structure_element" ], [ 34, 39, "human", "species" ], [ 40, 45, "SHARP", "protein" ], [ 63, 70, "Z score", "evidence" ] ] }, { "sid": 48, "sent": "The other four structural homologs include the \u03b2-barrel domain of the proteins Ku70 and Ku80 (Z score 11.4) (Fig 3B), a domain in the chromodomain protein Chp1 (Z score 10.8) (Fig 3C), and the activator interacting domain (ACID) of the Med25 subunit of the Mediator complex (Z score 8.5) (Fig 3D).", "section": "RESULTS", "ner": [ [ 47, 55, "\u03b2-barrel", "structure_element" ], [ 79, 83, "Ku70", "protein" ], [ 88, 92, "Ku80", "protein" ], [ 94, 101, "Z score", "evidence" ], [ 134, 154, "chromodomain protein", "protein_type" ], [ 155, 159, "Chp1", "protein" ], [ 161, 168, "Z score", "evidence" ], [ 193, 221, "activator interacting domain", "structure_element" ], [ 223, 227, "ACID", "structure_element" ], [ 236, 241, "Med25", "protein" ], [ 275, 282, "Z score", "evidence" ] ] }, { "sid": 49, "sent": "The next structural homolog has a Z score of 3.0.", "section": "RESULTS", "ner": [ [ 34, 41, "Z score", "evidence" ] ] }, { "sid": 50, "sent": "Structural homologs of the FPA SPOC domain.", "section": "FIG", "ner": [ [ 27, 30, "FPA", "protein" ], [ 31, 35, "SPOC", "structure_element" ] ] }, { "sid": 51, "sent": "Overlay of the structures of the FPA SPOC domain (cyan) and the SHARP SPOC domain (gray).", "section": "FIG", "ner": [ [ 0, 7, "Overlay", "experimental_method" ], [ 15, 25, "structures", "evidence" ], [ 33, 36, "FPA", "protein" ], [ 37, 41, "SPOC", "structure_element" ], [ 64, 69, "SHARP", "protein" ], [ 70, 74, "SPOC", "structure_element" ] ] }, { "sid": 52, "sent": "The bound position of a doubly-phosphorylated peptide from SMRT is shown in magenta.", "section": "FIG", "ner": [ [ 24, 45, "doubly-phosphorylated", "protein_state" ], [ 46, 53, "peptide", "chemical" ], [ 59, 63, "SMRT", "protein" ] ] }, { "sid": 53, "sent": "Overlay of the structures of the FPA SPOC domain (cyan) and the Ku70 \u03b2-barrel domain (gray).", "section": "FIG", "ner": [ [ 0, 7, "Overlay", "experimental_method" ], [ 15, 25, "structures", "evidence" ], [ 33, 36, "FPA", "protein" ], [ 37, 41, "SPOC", "structure_element" ], [ 64, 68, "Ku70", "protein" ], [ 69, 77, "\u03b2-barrel", "structure_element" ] ] }, { "sid": 54, "sent": "Ku80 contains a homologous domain (green), which forms a hetero-dimer with that in Ku70.", "section": "FIG", "ner": [ [ 0, 4, "Ku80", "protein" ], [ 57, 69, "hetero-dimer", "oligomeric_state" ], [ 83, 87, "Ku70", "protein" ] ] }, { "sid": 55, "sent": "The two domains, and inserted segments on them, mediate the binding of dsDNA (orange).", "section": "FIG", "ner": [ [ 71, 76, "dsDNA", "chemical" ] ] }, { "sid": 56, "sent": "The red rectangle highlights the region of contact between the two \u03b2-barrel domains.", "section": "FIG", "ner": [ [ 67, 75, "\u03b2-barrel", "structure_element" ] ] }, { "sid": 57, "sent": "Overlay of the structures of the FPA SPOC domain (cyan) and the homologous domain in Chp1 (gray).", "section": "FIG", "ner": [ [ 0, 7, "Overlay", "experimental_method" ], [ 15, 25, "structures", "evidence" ], [ 33, 36, "FPA", "protein" ], [ 37, 41, "SPOC", "structure_element" ], [ 85, 89, "Chp1", "protein" ] ] }, { "sid": 58, "sent": "The binding partner of Chp1, Tas3, is shown in green.", "section": "FIG", "ner": [ [ 23, 27, "Chp1", "protein" ], [ 29, 33, "Tas3", "protein" ] ] }, { "sid": 59, "sent": "The red rectangle indicates the region equivalent to the binding site of the SMART phosphopeptide in SHARP SPOC domain, where a loop of Tas3 is also located. (D).", "section": "FIG", "ner": [ [ 57, 69, "binding site", "site" ], [ 77, 82, "SMART", "protein" ], [ 83, 97, "phosphopeptide", "ptm" ], [ 101, 106, "SHARP", "protein" ], [ 107, 111, "SPOC", "structure_element" ], [ 128, 132, "loop", "structure_element" ], [ 136, 140, "Tas3", "protein" ] ] }, { "sid": 60, "sent": "Overlay of the structures of the FPA SPOC domain (cyan) and the Med25 ACID (gray).", "section": "FIG", "ner": [ [ 0, 7, "Overlay", "experimental_method" ], [ 15, 25, "structures", "evidence" ], [ 33, 36, "FPA", "protein" ], [ 37, 41, "SPOC", "structure_element" ], [ 64, 69, "Med25", "protein" ], [ 70, 74, "ACID", "structure_element" ] ] }, { "sid": 61, "sent": "SHARP is a transcriptional co-repressor in the nuclear receptor and Notch/RBP-J\u03ba signaling pathways.", "section": "RESULTS", "ner": [ [ 0, 5, "SHARP", "protein" ], [ 11, 39, "transcriptional co-repressor", "protein_type" ], [ 47, 63, "nuclear receptor", "protein_type" ], [ 68, 73, "Notch", "protein" ], [ 74, 80, "RBP-J\u03ba", "protein" ] ] }, { "sid": 62, "sent": "The SPOC domain of SHARP interacts directly with silencing mediator for retinoid and thyroid receptor (SMRT), nuclear receptor co-repressor (N-CoR), HDAC, and other components to represses transcription.", "section": "RESULTS", "ner": [ [ 4, 8, "SPOC", "structure_element" ], [ 19, 24, "SHARP", "protein" ], [ 49, 101, "silencing mediator for retinoid and thyroid receptor", "protein" ], [ 103, 107, "SMRT", "protein" ], [ 110, 139, "nuclear receptor co-repressor", "protein_type" ], [ 141, 146, "N-CoR", "protein_type" ], [ 149, 153, "HDAC", "protein" ] ] }, { "sid": 63, "sent": "While the overall structure of the FPA SPOC domain is similar to that of the SHARP SPOC domain, there are noticeable differences in the positioning of the \u03b2-strands and the helices, and most of the loops have substantially different conformations as well (Fig 3A).", "section": "RESULTS", "ner": [ [ 18, 27, "structure", "evidence" ], [ 35, 38, "FPA", "protein" ], [ 39, 43, "SPOC", "structure_element" ], [ 77, 82, "SHARP", "protein" ], [ 83, 87, "SPOC", "structure_element" ], [ 155, 164, "\u03b2-strands", "structure_element" ], [ 173, 180, "helices", "structure_element" ], [ 198, 203, "loops", "structure_element" ] ] }, { "sid": 64, "sent": "In addition, the SHARP SPOC domain has three extra helices.", "section": "RESULTS", "ner": [ [ 17, 22, "SHARP", "protein" ], [ 23, 27, "SPOC", "structure_element" ], [ 51, 58, "helices", "structure_element" ] ] }, { "sid": 65, "sent": "One of them covers the other end of the \u03b2-barrel, and the other two shield an additional surface of the side of the \u03b2-barrel from solvent.", "section": "RESULTS", "ner": [ [ 40, 48, "\u03b2-barrel", "structure_element" ], [ 116, 124, "\u03b2-barrel", "structure_element" ] ] }, { "sid": 66, "sent": "A doubly-phosphorylated peptide from SMRT is bound to the side of the barrel, near strands \u03b21 and \u03b23 (Fig 3A).", "section": "RESULTS", "ner": [ [ 2, 23, "doubly-phosphorylated", "protein_state" ], [ 24, 31, "peptide", "chemical" ], [ 37, 41, "SMRT", "protein" ], [ 45, 53, "bound to", "protein_state" ], [ 70, 76, "barrel", "structure_element" ], [ 83, 90, "strands", "structure_element" ], [ 91, 93, "\u03b21", "structure_element" ], [ 98, 100, "\u03b23", "structure_element" ] ] }, { "sid": 67, "sent": "Such a binding mode probably would not be possible in FPA, as the peptide would clash with the \u03b21-\u03b22 loop.", "section": "RESULTS", "ner": [ [ 54, 57, "FPA", "protein" ], [ 66, 73, "peptide", "chemical" ], [ 95, 105, "\u03b21-\u03b22 loop", "structure_element" ] ] }, { "sid": 68, "sent": "The Ku70-Ku80 hetero-dimer is involved in DNA double-strand break repair and the \u03b2-barrel domain contributes to DNA binding.", "section": "RESULTS", "ner": [ [ 4, 13, "Ku70-Ku80", "complex_assembly" ], [ 14, 26, "hetero-dimer", "oligomeric_state" ], [ 81, 89, "\u03b2-barrel", "structure_element" ], [ 112, 115, "DNA", "chemical" ] ] }, { "sid": 69, "sent": "In fact, the \u03b2-barrel domains of Ku70 and Ku80 form a hetero-dimer, primarily through interactions between the loops connecting the third and fourth strands of the barrel (Fig 3B).", "section": "RESULTS", "ner": [ [ 13, 21, "\u03b2-barrel", "structure_element" ], [ 33, 37, "Ku70", "protein" ], [ 42, 46, "Ku80", "protein" ], [ 54, 66, "hetero-dimer", "oligomeric_state" ], [ 111, 116, "loops", "structure_element" ], [ 132, 156, "third and fourth strands", "structure_element" ], [ 164, 170, "barrel", "structure_element" ] ] }, { "sid": 70, "sent": "The open ends of the two \u03b2-barrels face the DNA binding sites, and contact the phosphodiester backbone of the dsDNA.", "section": "RESULTS", "ner": [ [ 25, 34, "\u03b2-barrels", "structure_element" ], [ 44, 61, "DNA binding sites", "site" ], [ 110, 115, "dsDNA", "chemical" ] ] }, { "sid": 71, "sent": "In addition, a long insert connecting strands \u03b22 and \u03b23 in the two domains form an arch-like structure, encircling the dsDNA.", "section": "RESULTS", "ner": [ [ 15, 26, "long insert", "structure_element" ], [ 38, 45, "strands", "structure_element" ], [ 46, 48, "\u03b22", "structure_element" ], [ 53, 55, "\u03b23", "structure_element" ], [ 83, 102, "arch-like structure", "structure_element" ], [ 119, 124, "dsDNA", "chemical" ] ] }, { "sid": 72, "sent": "Chp1 is a subunit of the RNA-induced initiation of transcriptional gene silencing (RITS) complex.", "section": "RESULTS", "ner": [ [ 0, 4, "Chp1", "protein" ], [ 25, 81, "RNA-induced initiation of transcriptional gene silencing", "complex_assembly" ], [ 83, 87, "RITS", "complex_assembly" ] ] }, { "sid": 73, "sent": "The partner of Chp1, Tas3, is bound between the barrel domain and the second domain of Chp1, and the linker between the two domains is also crucial for this interaction (Fig 3C).", "section": "RESULTS", "ner": [ [ 15, 19, "Chp1", "protein" ], [ 21, 25, "Tas3", "protein" ], [ 48, 61, "barrel domain", "structure_element" ], [ 70, 83, "second domain", "structure_element" ], [ 87, 91, "Chp1", "protein" ], [ 101, 107, "linker", "structure_element" ] ] }, { "sid": 74, "sent": "It is probably unlikely that the \u03b2-barrel itself is sufficient to bind Tas3.", "section": "RESULTS", "ner": [ [ 33, 41, "\u03b2-barrel", "structure_element" ], [ 71, 75, "Tas3", "protein" ] ] }, { "sid": 75, "sent": "Interestingly, a loop in Tas3 contacts strand \u03b23 of the barrel domain, at a location somewhat similar to that of the N-terminal segment of the SMRT peptide in complex with SHARP SPOC domain (Fig 3A).", "section": "RESULTS", "ner": [ [ 17, 21, "loop", "structure_element" ], [ 25, 29, "Tas3", "protein" ], [ 39, 45, "strand", "structure_element" ], [ 46, 48, "\u03b23", "structure_element" ], [ 56, 69, "barrel domain", "structure_element" ], [ 143, 147, "SMRT", "protein" ], [ 148, 155, "peptide", "chemical" ], [ 156, 171, "in complex with", "protein_state" ], [ 172, 177, "SHARP", "protein" ], [ 178, 182, "SPOC", "structure_element" ] ] }, { "sid": 76, "sent": "Mediator is a coactivator complex that promotes transcription by Pol II.", "section": "RESULTS", "ner": [ [ 0, 8, "Mediator", "protein_type" ], [ 65, 71, "Pol II", "complex_assembly" ] ] }, { "sid": 77, "sent": "The Med25 subunit ACID is the target of the potent activator VP16 of the herpes simplex virus.", "section": "RESULTS", "ner": [ [ 4, 9, "Med25", "protein" ], [ 18, 22, "ACID", "structure_element" ], [ 61, 65, "VP16", "protein" ], [ 73, 93, "herpes simplex virus", "species" ] ] }, { "sid": 78, "sent": "The structure of ACID contains a helix at the C-terminus as well as an extended \u03b21-\u03b22 loop.", "section": "RESULTS", "ner": [ [ 4, 13, "structure", "evidence" ], [ 17, 21, "ACID", "structure_element" ], [ 33, 38, "helix", "structure_element" ], [ 80, 90, "\u03b21-\u03b22 loop", "structure_element" ] ] }, { "sid": 79, "sent": "Nonetheless, the binding site for VP16 has been mapped to roughly the same surface patch, near strands \u03b21 and \u03b23, that is used by the SHARP and Tas3 SPOC domains for binding their partners.", "section": "RESULTS", "ner": [ [ 17, 29, "binding site", "site" ], [ 34, 38, "VP16", "protein" ], [ 75, 88, "surface patch", "site" ], [ 95, 102, "strands", "structure_element" ], [ 103, 105, "\u03b21", "structure_element" ], [ 110, 112, "\u03b23", "structure_element" ], [ 134, 139, "SHARP", "protein" ], [ 144, 148, "Tas3", "protein" ], [ 149, 153, "SPOC", "structure_element" ] ] }, { "sid": 80, "sent": "A conserved surface patch in the FPA SPOC domain", "section": "RESULTS", "ner": [ [ 2, 11, "conserved", "protein_state" ], [ 12, 25, "surface patch", "site" ], [ 33, 36, "FPA", "protein" ], [ 37, 41, "SPOC", "structure_element" ] ] }, { "sid": 81, "sent": "An analysis of the SPOC domain indicates a large surface patch near strands \u03b21, \u03b23, \u03b25 and \u03b26 that is conserved among plant FPA homologs (Fig 4A).", "section": "RESULTS", "ner": [ [ 19, 23, "SPOC", "structure_element" ], [ 49, 62, "surface patch", "site" ], [ 68, 75, "strands", "structure_element" ], [ 76, 78, "\u03b21", "structure_element" ], [ 80, 82, "\u03b23", "structure_element" ], [ 84, 86, "\u03b25", "structure_element" ], [ 91, 93, "\u03b26", "structure_element" ], [ 102, 111, "conserved", "protein_state" ], [ 118, 123, "plant", "taxonomy_domain" ], [ 124, 127, "FPA", "protein" ] ] }, { "sid": 82, "sent": "This surface patch can be broken into two sub-patches, with residues Lys447 (in strand \u03b21), Arg477 (\u03b23), Tyr515 (\u03b1B) and Arg521 (\u03b25) in one sub-patch, and residues His486 (\u03b1A), Thr478 (\u03b23), Val524 (\u03b25) and Phe534 (\u03b26) in the other sub-patch (Fig 4B).", "section": "RESULTS", "ner": [ [ 5, 18, "surface patch", "site" ], [ 42, 53, "sub-patches", "site" ], [ 69, 75, "Lys447", "residue_name_number" ], [ 80, 86, "strand", "structure_element" ], [ 87, 89, "\u03b21", "structure_element" ], [ 92, 98, "Arg477", "residue_name_number" ], [ 100, 102, "\u03b23", "structure_element" ], [ 105, 111, "Tyr515", "residue_name_number" ], [ 113, 115, "\u03b1B", "structure_element" ], [ 121, 127, "Arg521", "residue_name_number" ], [ 129, 131, "\u03b25", "structure_element" ], [ 140, 149, "sub-patch", "site" ], [ 164, 170, "His486", "residue_name_number" ], [ 172, 174, "\u03b1A", "structure_element" ], [ 177, 183, "Thr478", "residue_name_number" ], [ 185, 187, "\u03b23", "structure_element" ], [ 190, 196, "Val524", "residue_name_number" ], [ 198, 200, "\u03b25", "structure_element" ], [ 206, 212, "Phe534", "residue_name_number" ], [ 214, 216, "\u03b26", "structure_element" ], [ 231, 240, "sub-patch", "site" ] ] }, { "sid": 83, "sent": "The first surface patch is electropositive in nature (Fig 4C), and residues Arg477 and Tyr515 are also conserved in the SHARP SPOC domain (Fig 1B).", "section": "RESULTS", "ner": [ [ 4, 23, "first surface patch", "site" ], [ 27, 42, "electropositive", "protein_state" ], [ 76, 82, "Arg477", "residue_name_number" ], [ 87, 93, "Tyr515", "residue_name_number" ], [ 103, 112, "conserved", "protein_state" ], [ 120, 125, "SHARP", "protein" ], [ 126, 130, "SPOC", "structure_element" ] ] }, { "sid": 84, "sent": "In fact, one of the phosphorylated residues of the SMRT peptide interacts with this surface patch (Fig 3A), suggesting that the FPA SPOC domain might also interact with a phosphorylated segment here.", "section": "RESULTS", "ner": [ [ 20, 34, "phosphorylated", "protein_state" ], [ 51, 55, "SMRT", "protein" ], [ 56, 63, "peptide", "chemical" ], [ 84, 97, "surface patch", "site" ], [ 128, 131, "FPA", "protein" ], [ 132, 136, "SPOC", "structure_element" ], [ 171, 185, "phosphorylated", "protein_state" ] ] }, { "sid": 85, "sent": "In comparison, the second surface patch is more hydrophobic in nature (Fig 4C).", "section": "RESULTS", "ner": [ [ 19, 39, "second surface patch", "site" ], [ 48, 59, "hydrophobic", "protein_state" ] ] }, { "sid": 86, "sent": "A conserved surface patch of FPA SPOC domain.", "section": "FIG", "ner": [ [ 2, 11, "conserved", "protein_state" ], [ 12, 25, "surface patch", "site" ], [ 29, 32, "FPA", "protein" ], [ 33, 37, "SPOC", "structure_element" ] ] }, { "sid": 87, "sent": "Two views of the molecular surface of FPA SPOC domain colored based on sequence conservation among plant FPA homologs.", "section": "FIG", "ner": [ [ 38, 41, "FPA", "protein" ], [ 42, 46, "SPOC", "structure_element" ], [ 99, 104, "plant", "taxonomy_domain" ], [ 105, 108, "FPA", "protein" ] ] }, { "sid": 88, "sent": "Residues in the conserved surface patch of FPA SPOC domain.", "section": "FIG", "ner": [ [ 16, 25, "conserved", "protein_state" ], [ 26, 39, "surface patch", "site" ], [ 43, 46, "FPA", "protein" ], [ 47, 51, "SPOC", "structure_element" ] ] }, { "sid": 89, "sent": "The side chains of the residues are shown in stick models, colored orange in the first sub-patch and green in the second. (C).", "section": "FIG", "ner": [ [ 81, 96, "first sub-patch", "site" ] ] }, { "sid": 90, "sent": "Molecular surface of FPA SPOC domain colored based on electrostatic potential.", "section": "FIG", "ner": [ [ 21, 24, "FPA", "protein" ], [ 25, 29, "SPOC", "structure_element" ] ] }, { "sid": 91, "sent": "Testing the requirement of specific conserved amino acids for FPA functions", "section": "RESULTS", "ner": [ [ 62, 65, "FPA", "protein" ] ] }, { "sid": 92, "sent": "We next examined the potential impact of the conserved surface patch on FPA function in vivo.", "section": "RESULTS", "ner": [ [ 45, 54, "conserved", "protein_state" ], [ 55, 68, "surface patch", "site" ], [ 72, 75, "FPA", "protein" ] ] }, { "sid": 93, "sent": "We mutated two residues, Arg477 and Tyr515, of the surface patch, which are also conserved in the SHARP SPOC domain (Fig 1B) and were found to be functionally important.", "section": "RESULTS", "ner": [ [ 3, 10, "mutated", "experimental_method" ], [ 25, 31, "Arg477", "residue_name_number" ], [ 36, 42, "Tyr515", "residue_name_number" ], [ 51, 64, "surface patch", "site" ], [ 81, 90, "conserved", "protein_state" ], [ 98, 103, "SHARP", "protein" ], [ 104, 108, "SPOC", "structure_element" ] ] }, { "sid": 94, "sent": "The mutations were introduced into a transgene designed to express FPA from its native control elements (promoter, introns and 3\u2032 UTR).", "section": "RESULTS", "ner": [ [ 4, 13, "mutations", "experimental_method" ], [ 19, 29, "introduced", "experimental_method" ], [ 67, 70, "FPA", "protein" ] ] }, { "sid": 95, "sent": "The resulting transgenes were then stably transformed into an fpa-8 mutant background so that the impact of the mutations on FPA function could be assessed.", "section": "RESULTS", "ner": [ [ 35, 53, "stably transformed", "experimental_method" ], [ 62, 67, "fpa-8", "gene" ], [ 68, 74, "mutant", "protein_state" ], [ 112, 121, "mutations", "experimental_method" ], [ 125, 128, "FPA", "protein" ] ] }, { "sid": 96, "sent": "Control transformation of the same expression constructs into fpa-8 designed to express wild-type FPA protein restored FPA protein expression levels to near wild-type levels (panel A in S1 Fig) and rescued the function of FPA in controlling RNA 3\u2032-end formation, for example in FPA pre-mRNA (panel B in S1 Fig).", "section": "RESULTS", "ner": [ [ 35, 56, "expression constructs", "experimental_method" ], [ 62, 67, "fpa-8", "gene" ], [ 88, 97, "wild-type", "protein_state" ], [ 98, 101, "FPA", "protein" ], [ 119, 122, "FPA", "protein" ], [ 131, 148, "expression levels", "evidence" ], [ 157, 166, "wild-type", "protein_state" ], [ 222, 225, "FPA", "protein" ], [ 241, 244, "RNA", "chemical" ], [ 278, 281, "FPA", "protein" ], [ 282, 290, "pre-mRNA", "chemical" ] ] }, { "sid": 97, "sent": "We examined independent transgenic lines expressing each R477A and Y515A mutation.", "section": "RESULTS", "ner": [ [ 57, 62, "R477A", "mutant" ], [ 67, 72, "Y515A", "mutant" ], [ 73, 81, "mutation", "experimental_method" ] ] }, { "sid": 98, "sent": "In each case, we confirmed that detectable levels of FPA protein expression were restored close to wild-type levels in protein blot analyses using antibodies that specifically recognize FPA (S2 Fig).", "section": "RESULTS", "ner": [ [ 53, 56, "FPA", "protein" ], [ 99, 108, "wild-type", "protein_state" ], [ 119, 131, "protein blot", "experimental_method" ], [ 186, 189, "FPA", "protein" ] ] }, { "sid": 99, "sent": "We then examined the impact of the surface patch mutations on FPA\u2019s function in controlling RNA 3\u2032-end formation by determining whether the mutant proteins functioned in FPA autoregulation and the repression of FLC expression.", "section": "RESULTS", "ner": [ [ 35, 48, "surface patch", "site" ], [ 49, 58, "mutations", "experimental_method" ], [ 62, 65, "FPA", "protein" ], [ 140, 146, "mutant", "protein_state" ], [ 170, 173, "FPA", "protein" ], [ 211, 214, "FLC", "gene" ] ] }, { "sid": 100, "sent": "FPA autoregulates its expression by promoting cleavage and polyadenylation within intron 1 of its own pre-mRNA, resulting in a truncated transcript that does not encode functional protein.", "section": "RESULTS", "ner": [ [ 0, 3, "FPA", "protein" ], [ 102, 110, "pre-mRNA", "chemical" ] ] }, { "sid": 101, "sent": "We used RNA gel blot analyses to reveal that in each of three independent transgenic lines for each single mutant, rescue of proximally polyadenylated FPA pre-mRNA can be detected (Fig 5A and 5B).", "section": "RESULTS", "ner": [ [ 8, 29, "RNA gel blot analyses", "experimental_method" ], [ 107, 113, "mutant", "protein_state" ], [ 151, 154, "FPA", "protein" ], [ 155, 163, "pre-mRNA", "chemical" ] ] }, { "sid": 102, "sent": "We therefore conclude that neither of these mutations disrupted the ability of FPA to promote RNA 3\u2032-end formation in its own transcript.", "section": "RESULTS", "ner": [ [ 79, 82, "FPA", "protein" ] ] }, { "sid": 103, "sent": "Impact of individual FPA SPOC domain mutations on alternative polyadenylation of FPA pre-mRNA.", "section": "FIG", "ner": [ [ 21, 24, "FPA", "protein" ], [ 25, 29, "SPOC", "structure_element" ], [ 37, 46, "mutations", "experimental_method" ], [ 81, 84, "FPA", "protein" ], [ 85, 93, "pre-mRNA", "chemical" ] ] }, { "sid": 104, "sent": "RNA gel blot analysis of WT A. thaliana accession Columbia (Col-0) plants fpa-8 and fpa-8 mutants expressing either FPA::FPA R477A (A), or FPA::FPA Y515A (B) using poly(A)+ purified mRNAs.", "section": "FIG", "ner": [ [ 0, 12, "RNA gel blot", "experimental_method" ], [ 25, 27, "WT", "protein_state" ], [ 28, 39, "A. thaliana", "species" ], [ 67, 73, "plants", "taxonomy_domain" ], [ 74, 79, "fpa-8", "gene" ], [ 84, 89, "fpa-8", "gene" ], [ 90, 97, "mutants", "protein_state" ], [ 116, 119, "FPA", "protein" ], [ 121, 130, "FPA R477A", "mutant" ], [ 139, 142, "FPA", "protein" ], [ 144, 153, "FPA Y515A", "mutant" ], [ 182, 187, "mRNAs", "chemical" ] ] }, { "sid": 105, "sent": "A probe corresponding to the 5\u2019UTR region of FPA mRNA was used to detect FPA specific mRNAs.", "section": "FIG", "ner": [ [ 45, 48, "FPA", "protein" ], [ 49, 53, "mRNA", "chemical" ], [ 73, 76, "FPA", "protein" ], [ 86, 91, "mRNAs", "chemical" ], [ 45, 48, "FPA", "protein" ], [ 49, 53, "mRNA", "chemical" ], [ 73, 76, "FPA", "protein" ], [ 86, 91, "mRNAs", "chemical" ] ] }, { "sid": 106, "sent": "Proximally and distally polyadenylated FPA transcripts are marked with arrows.", "section": "FIG", "ner": [ [ 39, 42, "FPA", "protein" ] ] }, { "sid": 107, "sent": "The ratio of distal:proximal polyadenylated forms is given under each lane. (C,D) Impact of individual FPA SPOC domain mutations on FLC transcript levels.", "section": "FIG", "ner": [ [ 103, 106, "FPA", "protein" ], [ 107, 111, "SPOC", "structure_element" ], [ 119, 128, "mutations", "experimental_method" ], [ 132, 135, "FLC", "gene" ] ] }, { "sid": 108, "sent": "qRT-PCR analysis was performed with total RNA purified from Col-0, fpa-8, 35S::FPA:YFP and FPA::FPA R477A (C), FPA::FPA Y515A (D) plants.", "section": "FIG", "ner": [ [ 0, 7, "qRT-PCR", "experimental_method" ], [ 42, 45, "RNA", "chemical" ], [ 67, 72, "fpa-8", "gene" ], [ 79, 82, "FPA", "protein" ], [ 83, 86, "YFP", "experimental_method" ], [ 91, 94, "FPA", "protein" ], [ 96, 105, "FPA R477A", "mutant" ], [ 111, 114, "FPA", "protein" ], [ 116, 125, "FPA Y515A", "mutant" ], [ 130, 136, "plants", "taxonomy_domain" ] ] }, { "sid": 109, "sent": "Histograms show mean values \u00b1SE for three independent PCR amplifications of three biological replicates.", "section": "FIG", "ner": [ [ 0, 10, "Histograms", "evidence" ], [ 54, 57, "PCR", "experimental_method" ], [ 0, 10, "Histograms", "evidence" ], [ 54, 57, "PCR", "experimental_method" ] ] }, { "sid": 110, "sent": "We next examined whether the corresponding mutations disrupted the ability of FPA to control FLC expression.", "section": "RESULTS", "ner": [ [ 78, 81, "FPA", "protein" ], [ 93, 96, "FLC", "gene" ] ] }, { "sid": 111, "sent": "We used RT-qPCR to measure the expression of FLC mRNA and found that in each independent transgenic line encoding each mutated FPA protein, the elevated levels of FLC detected in fpa-8 mutants were restored to near wild-type levels by expression of the FPA SPOC conserved patch mutant proteins (Fig 5C and 5D).", "section": "RESULTS", "ner": [ [ 8, 15, "RT-qPCR", "experimental_method" ], [ 45, 48, "FLC", "gene" ], [ 49, 53, "mRNA", "chemical" ], [ 119, 126, "mutated", "protein_state" ], [ 127, 130, "FPA", "protein" ], [ 163, 166, "FLC", "gene" ], [ 179, 184, "fpa-8", "gene" ], [ 185, 192, "mutants", "protein_state" ], [ 215, 224, "wild-type", "protein_state" ], [ 253, 256, "FPA", "protein" ], [ 257, 261, "SPOC", "structure_element" ], [ 262, 271, "conserved", "protein_state" ], [ 272, 277, "patch", "site" ], [ 278, 284, "mutant", "protein_state" ] ] }, { "sid": 112, "sent": "Since each surface patch mutation appeared to be insufficient to disrupt FPA functions on its own, we combined both mutations into the same transgene.", "section": "RESULTS", "ner": [ [ 11, 24, "surface patch", "site" ], [ 25, 33, "mutation", "experimental_method" ], [ 73, 76, "FPA", "protein" ] ] }, { "sid": 113, "sent": "We could again confirm that near wild-type levels of FPA protein were expressed from three independent transgenic lines expressing the FPA R477A;Y515A doubly mutated protein in an fpa-8 mutant background (S3 Fig).", "section": "RESULTS", "ner": [ [ 33, 42, "wild-type", "protein_state" ], [ 53, 56, "FPA", "protein" ], [ 135, 150, "FPA R477A;Y515A", "mutant" ], [ 151, 165, "doubly mutated", "protein_state" ], [ 180, 185, "fpa-8", "gene" ], [ 186, 192, "mutant", "protein_state" ] ] }, { "sid": 114, "sent": "We found that FPA R477A;Y515A protein functioned like wild-type FPA to restore FPA pre-mRNA proximal polyadenylation (Fig 6A) and FLC expression to wild-type levels (Fig 6B).", "section": "RESULTS", "ner": [ [ 14, 29, "FPA R477A;Y515A", "mutant" ], [ 54, 63, "wild-type", "protein_state" ], [ 64, 67, "FPA", "protein" ], [ 79, 82, "FPA", "protein" ], [ 83, 91, "pre-mRNA", "chemical" ], [ 130, 133, "FLC", "gene" ], [ 148, 157, "wild-type", "protein_state" ] ] }, { "sid": 115, "sent": "Impact of double FPA SPOC domain mutations on alternative polyadenylation of FPA pre-mRNA and FLC expression.", "section": "FIG", "ner": [ [ 17, 20, "FPA", "protein" ], [ 21, 25, "SPOC", "structure_element" ], [ 33, 42, "mutations", "experimental_method" ], [ 77, 80, "FPA", "protein" ], [ 81, 89, "pre-mRNA", "chemical" ], [ 94, 97, "FLC", "gene" ] ] }, { "sid": 116, "sent": "(A) RNA gel blot analysis of WT A. thaliana accession Columbia (Col-0) plants fpa-8 and fpa-8 mutants expressing FPA::FPA R477A;Y515A using poly(A)+ purified mRNAs.", "section": "FIG", "ner": [ [ 4, 16, "RNA gel blot", "experimental_method" ], [ 29, 31, "WT", "protein_state" ], [ 32, 43, "A. thaliana", "species" ], [ 71, 77, "plants", "taxonomy_domain" ], [ 78, 83, "fpa-8", "gene" ], [ 88, 93, "fpa-8", "gene" ], [ 94, 101, "mutants", "protein_state" ], [ 113, 116, "FPA", "protein" ], [ 118, 133, "FPA R477A;Y515A", "mutant" ], [ 158, 163, "mRNAs", "chemical" ] ] }, { "sid": 117, "sent": "Black arrows indicate the proximally and distally polyadenylated FPA mRNAs.", "section": "FIG", "ner": [ [ 65, 68, "FPA", "protein" ], [ 69, 74, "mRNAs", "chemical" ] ] }, { "sid": 118, "sent": "qRT-PCR analysis was performed with total RNA purified from Col-0, fpa-8, and FPA::FPA R477A;Y515A plants.", "section": "FIG", "ner": [ [ 0, 7, "qRT-PCR", "experimental_method" ], [ 42, 45, "RNA", "chemical" ], [ 67, 72, "fpa-8", "gene" ], [ 78, 81, "FPA", "protein" ], [ 83, 98, "FPA R477A;Y515A", "mutant" ], [ 99, 105, "plants", "taxonomy_domain" ] ] }, { "sid": 119, "sent": "Together our findings suggest that either the SPOC domain is not required for the role of FPA in regulating RNA 3\u2032-end formation, or that this combination of mutations is not sufficient to critically disrupt the function of the FPA SPOC domain.", "section": "RESULTS", "ner": [ [ 46, 50, "SPOC", "structure_element" ], [ 90, 93, "FPA", "protein" ], [ 108, 111, "RNA", "chemical" ], [ 158, 167, "mutations", "experimental_method" ], [ 228, 231, "FPA", "protein" ], [ 232, 236, "SPOC", "structure_element" ] ] }, { "sid": 120, "sent": "Since the corresponding mutations in the SHARP SPOC domain do disrupt its recognition of unphosphorylated SMRT peptides, these observations may reinforce the idea that the features and functions of the FPA SPOC domain differ from those of the only other well-characterized SPOC domain.", "section": "RESULTS", "ner": [ [ 24, 33, "mutations", "experimental_method" ], [ 41, 46, "SHARP", "protein" ], [ 47, 51, "SPOC", "structure_element" ], [ 89, 105, "unphosphorylated", "protein_state" ], [ 106, 110, "SMRT", "protein" ], [ 111, 119, "peptides", "chemical" ], [ 202, 205, "FPA", "protein" ], [ 206, 210, "SPOC", "structure_element" ], [ 273, 277, "SPOC", "structure_element" ] ] } ] }, "PMC4806292": { "annotations": [ { "sid": 0, "sent": "Structural insights and in vitro reconstitution of membrane targeting and activation of human PI4KB by the ACBD3 protein", "section": "TITLE", "ner": [ [ 24, 47, "in vitro reconstitution", "experimental_method" ], [ 88, 93, "human", "species" ], [ 94, 99, "PI4KB", "protein" ], [ 107, 112, "ACBD3", "protein" ] ] }, { "sid": 1, "sent": "Phosphatidylinositol 4-kinase beta (PI4KB) is one of four human PI4K enzymes that generate phosphatidylinositol 4-phosphate (PI4P), a minor but essential regulatory lipid found in all eukaryotic cells.", "section": "ABSTRACT", "ner": [ [ 0, 34, "Phosphatidylinositol 4-kinase beta", "protein" ], [ 36, 41, "PI4KB", "protein" ], [ 58, 63, "human", "species" ], [ 64, 68, "PI4K", "protein_type" ], [ 91, 123, "phosphatidylinositol 4-phosphate", "chemical" ], [ 125, 129, "PI4P", "chemical" ], [ 184, 194, "eukaryotic", "taxonomy_domain" ] ] }, { "sid": 2, "sent": "To convert their lipid substrates, PI4Ks must be recruited to the correct membrane compartment.", "section": "ABSTRACT", "ner": [ [ 35, 40, "PI4Ks", "protein_type" ] ] }, { "sid": 3, "sent": "PI4KB is critical for the maintenance of the Golgi and trans Golgi network (TGN) PI4P pools, however, the actual targeting mechanism of PI4KB to the Golgi and TGN membranes is unknown.", "section": "ABSTRACT", "ner": [ [ 0, 5, "PI4KB", "protein" ], [ 81, 85, "PI4P", "chemical" ], [ 136, 141, "PI4KB", "protein" ] ] }, { "sid": 4, "sent": "Here, we present an NMR structure of the complex of PI4KB and its interacting partner, Golgi adaptor protein acyl-coenzyme A binding domain containing protein 3 (ACBD3).", "section": "ABSTRACT", "ner": [ [ 20, 23, "NMR", "experimental_method" ], [ 24, 33, "structure", "evidence" ], [ 52, 57, "PI4KB", "protein" ], [ 87, 108, "Golgi adaptor protein", "protein_type" ], [ 109, 160, "acyl-coenzyme A binding domain containing protein 3", "protein" ], [ 162, 167, "ACBD3", "protein" ] ] }, { "sid": 5, "sent": "We show that ACBD3 is capable of recruiting PI4KB to membranes both in vitro and in vivo, and that membrane recruitment of PI4KB by ACBD3 increases its enzymatic activity and that the ACBD3:PI4KB complex formation is essential for proper function of the Golgi.", "section": "ABSTRACT", "ner": [ [ 13, 18, "ACBD3", "protein" ], [ 44, 49, "PI4KB", "protein" ], [ 123, 128, "PI4KB", "protein" ], [ 132, 137, "ACBD3", "protein" ], [ 152, 170, "enzymatic activity", "evidence" ], [ 184, 195, "ACBD3:PI4KB", "complex_assembly" ] ] }, { "sid": 6, "sent": "Phosphatidylinositol 4-kinase beta (PI4KB, also known as PI4K III\u03b2) is a soluble cytosolic protein yet its function is to phosphorylate membrane lipids.", "section": "INTRO", "ner": [ [ 0, 34, "Phosphatidylinositol 4-kinase beta", "protein" ], [ 36, 41, "PI4KB", "protein" ], [ 57, 66, "PI4K III\u03b2", "protein" ] ] }, { "sid": 7, "sent": "It is one of four human PI4K enzymes that phosphorylate phosphatidylinositol (PI) to generate phosphatidylinositol 4-phosphate (PI4P).", "section": "INTRO", "ner": [ [ 18, 23, "human", "species" ], [ 24, 28, "PI4K", "protein_type" ], [ 56, 76, "phosphatidylinositol", "chemical" ], [ 78, 80, "PI", "chemical" ], [ 94, 126, "phosphatidylinositol 4-phosphate", "chemical" ], [ 128, 132, "PI4P", "chemical" ] ] }, { "sid": 8, "sent": "PI4P is an essential lipid found in various membrane compartments including the Golgi and trans-Golgi network (TGN), the plasma membrane and the endocytic compartments.", "section": "INTRO", "ner": [ [ 0, 4, "PI4P", "chemical" ] ] }, { "sid": 9, "sent": "In these locations, PI4P plays an important role in cell signaling and lipid transport, and serves as a precursor for higher phosphoinositides or as a docking site for clathrin adaptor or lipid transfer proteins.", "section": "INTRO", "ner": [ [ 20, 24, "PI4P", "chemical" ], [ 125, 142, "phosphoinositides", "chemical" ], [ 168, 176, "clathrin", "protein_type" ] ] }, { "sid": 10, "sent": "A wide range of positive-sense single-stranded RNA viruses (+RNA viruses), including many that are important human pathogens, hijack human PI4KA or PI4KB enzymes to generate specific PI4P-enriched organelles called membranous webs or replication factories.", "section": "INTRO", "ner": [ [ 16, 58, "positive-sense single-stranded RNA viruses", "taxonomy_domain" ], [ 60, 72, "+RNA viruses", "taxonomy_domain" ], [ 109, 114, "human", "species" ], [ 133, 138, "human", "species" ], [ 139, 144, "PI4KA", "protein" ], [ 148, 153, "PI4KB", "protein" ], [ 183, 187, "PI4P", "chemical" ] ] }, { "sid": 11, "sent": "These structures are essential for effective viral replication.", "section": "INTRO", "ner": [ [ 6, 16, "structures", "evidence" ], [ 45, 50, "viral", "taxonomy_domain" ] ] }, { "sid": 12, "sent": "Recently, highly specific PI4KB inhibitors were developed as potential antivirals.", "section": "INTRO", "ner": [ [ 26, 31, "PI4KB", "protein" ] ] }, { "sid": 13, "sent": "PI4K kinases must be recruited to the correct membrane type to fulfill their enzymatic functions.", "section": "INTRO", "ner": [ [ 0, 4, "PI4K", "protein_type" ], [ 5, 12, "kinases", "protein_type" ] ] }, { "sid": 14, "sent": "Type II PI4Ks (PI4K2A and PI4K2B) are heavily palmitoylated and thus behave as membrane proteins.", "section": "INTRO", "ner": [ [ 0, 13, "Type II PI4Ks", "protein_type" ], [ 15, 21, "PI4K2A", "protein" ], [ 26, 32, "PI4K2B", "protein" ], [ 38, 59, "heavily palmitoylated", "protein_state" ], [ 79, 96, "membrane proteins", "protein" ] ] }, { "sid": 15, "sent": "In contrast, type III PI4Ks (PI4KA and PI4KB) are soluble cytosolic proteins that are recruited to appropriate membranes indirectly via protein-protein interactions.", "section": "INTRO", "ner": [ [ 13, 27, "type III PI4Ks", "protein_type" ], [ 29, 34, "PI4KA", "protein" ], [ 39, 44, "PI4KB", "protein" ] ] }, { "sid": 16, "sent": "The recruitment of PI4KA to the plasma membrane by EFR3 and TTC7 is relatively well understood even at the structural level, but, the actual molecular mechanism of PI4KB recruitment to the Golgi is still poorly understood.", "section": "INTRO", "ner": [ [ 19, 24, "PI4KA", "protein" ], [ 51, 55, "EFR3", "protein" ], [ 60, 64, "TTC7", "protein" ], [ 164, 169, "PI4KB", "protein" ] ] }, { "sid": 17, "sent": "Acyl-coenzyme A binding domain containing protein 3 (ACBD3, also known as GCP60 and PAP7) is a Golgi resident protein.", "section": "INTRO", "ner": [ [ 0, 51, "Acyl-coenzyme A binding domain containing protein 3", "protein" ], [ 53, 58, "ACBD3", "protein" ], [ 74, 79, "GCP60", "protein" ], [ 84, 88, "PAP7", "protein" ] ] }, { "sid": 18, "sent": "Its membrane localization is mediated by the interaction with the Golgi integral protein golgin B1/giantin.", "section": "INTRO", "ner": [ [ 89, 98, "golgin B1", "protein" ], [ 99, 106, "giantin", "protein" ] ] }, { "sid": 19, "sent": "ACBD3 functions as an adaptor protein and signaling hub across cellular signaling pathways.", "section": "INTRO", "ner": [ [ 0, 5, "ACBD3", "protein" ] ] }, { "sid": 20, "sent": "ACBD3 can interact with a number of proteins including golgin A3/golgin-160 to regulate apoptosis, Numb proteins to control asymmetric cell division and neuronal differentiation, metal transporter DMT1 and monomeric G protein Dexras1 to maintain iron homeostasis, and the lipid kinase PI4KB to regulate lipid homeostasis.", "section": "INTRO", "ner": [ [ 0, 5, "ACBD3", "protein" ], [ 55, 64, "golgin A3", "protein" ], [ 65, 75, "golgin-160", "protein" ], [ 99, 112, "Numb proteins", "protein_type" ], [ 179, 196, "metal transporter", "protein_type" ], [ 197, 201, "DMT1", "protein" ], [ 206, 215, "monomeric", "oligomeric_state" ], [ 216, 225, "G protein", "protein_type" ], [ 226, 233, "Dexras1", "protein" ], [ 246, 250, "iron", "chemical" ], [ 272, 284, "lipid kinase", "protein_type" ], [ 285, 290, "PI4KB", "protein" ] ] }, { "sid": 21, "sent": "ACBD3 has been also implicated in the pathology of neurodegenerative diseases such as Huntington\u2019s disease due to its interactions with a polyglutamine repeat-containing mutant huntingtin and the striatal-selective monomeric G protein Rhes/Dexras2.", "section": "INTRO", "ner": [ [ 0, 5, "ACBD3", "protein" ], [ 138, 158, "polyglutamine repeat", "structure_element" ], [ 170, 176, "mutant", "protein_state" ], [ 177, 187, "huntingtin", "protein" ], [ 215, 224, "monomeric", "oligomeric_state" ], [ 225, 234, "G protein", "protein_type" ], [ 235, 239, "Rhes", "protein" ], [ 240, 247, "Dexras2", "protein" ] ] }, { "sid": 22, "sent": "ACBD3 is a binding partner of viral non-structural 3A proteins and a host factor of several picornaviruses including poliovirus, coxsackievirus B3, and Aichi virus.", "section": "INTRO", "ner": [ [ 0, 5, "ACBD3", "protein" ], [ 30, 35, "viral", "taxonomy_domain" ], [ 36, 62, "non-structural 3A proteins", "protein_type" ], [ 92, 106, "picornaviruses", "taxonomy_domain" ], [ 117, 127, "poliovirus", "taxonomy_domain" ], [ 129, 146, "coxsackievirus B3", "taxonomy_domain" ], [ 152, 163, "Aichi virus", "taxonomy_domain" ] ] }, { "sid": 23, "sent": "We present a biochemical and structural characterization of the molecular complex composed of the ACBD3 protein and the PI4KB enzyme.", "section": "INTRO", "ner": [ [ 13, 56, "biochemical and structural characterization", "experimental_method" ], [ 98, 103, "ACBD3", "protein" ], [ 120, 125, "PI4KB", "protein" ] ] }, { "sid": 24, "sent": "We show that ACBD3 can recruit PI4KB to model membranes as well as redirect PI4KB to cellular membranes where it is not naturally found.", "section": "INTRO", "ner": [ [ 13, 18, "ACBD3", "protein" ], [ 31, 36, "PI4KB", "protein" ], [ 76, 81, "PI4KB", "protein" ] ] }, { "sid": 25, "sent": "Our data also show that ACBD3 regulates the enzymatic activity of PI4KB kinase through membrane recruitment rather than allostery.", "section": "INTRO", "ner": [ [ 24, 29, "ACBD3", "protein" ], [ 44, 62, "enzymatic activity", "evidence" ], [ 66, 71, "PI4KB", "protein" ], [ 72, 78, "kinase", "protein_type" ] ] }, { "sid": 26, "sent": "ACBD3 and PI4KB interact with 1:1 stoichiometry with submicromolar affinity", "section": "RESULTS", "ner": [ [ 0, 5, "ACBD3", "protein" ], [ 10, 15, "PI4KB", "protein" ] ] }, { "sid": 27, "sent": "In order to verify the interactions between ACBD3 and PI4KB we expressed and purified both proteins.", "section": "RESULTS", "ner": [ [ 44, 49, "ACBD3", "protein" ], [ 54, 59, "PI4KB", "protein" ], [ 63, 85, "expressed and purified", "experimental_method" ] ] }, { "sid": 28, "sent": "To increase yields of bacterial expression the intrinsically disordered region of PI4KB (residues 423\u2013522) was removed (Fig. 1A).", "section": "RESULTS", "ner": [ [ 22, 42, "bacterial expression", "experimental_method" ], [ 47, 78, "intrinsically disordered region", "structure_element" ], [ 82, 87, "PI4KB", "protein" ], [ 98, 105, "423\u2013522", "residue_range" ], [ 111, 118, "removed", "experimental_method" ] ] }, { "sid": 29, "sent": "This internal deletion does not significantly affect the kinase activity(SI Fig. 1A) or interaction with ACBD3 (SI Fig. 1B,C).", "section": "RESULTS", "ner": [ [ 14, 22, "deletion", "experimental_method" ], [ 57, 63, "kinase", "protein_type" ], [ 105, 110, "ACBD3", "protein" ] ] }, { "sid": 30, "sent": "In an in vitro binding assay, ACBD3 co-purified with the NiNTA-immobilized N-terminal His6GB1-tagged PI4KB (Fig. 1B, left panel), suggesting a direct interaction.", "section": "RESULTS", "ner": [ [ 6, 28, "in vitro binding assay", "experimental_method" ], [ 30, 35, "ACBD3", "protein" ], [ 36, 74, "co-purified with the NiNTA-immobilized", "experimental_method" ], [ 86, 100, "His6GB1-tagged", "protein_state" ], [ 101, 106, "PI4KB", "protein" ] ] }, { "sid": 31, "sent": "Using a mammalian two-hybrid assay Greninger and colleagues localized this interaction to the Q domain of ACBD3 (named according to its high content of glutamine residues) and the N-terminal region of PI4KB preceding its helical domain.", "section": "RESULTS", "ner": [ [ 8, 34, "mammalian two-hybrid assay", "experimental_method" ], [ 60, 69, "localized", "evidence" ], [ 94, 102, "Q domain", "structure_element" ], [ 106, 111, "ACBD3", "protein" ], [ 152, 161, "glutamine", "residue_name" ], [ 180, 197, "N-terminal region", "structure_element" ], [ 201, 206, "PI4KB", "protein" ], [ 221, 235, "helical domain", "structure_element" ] ] }, { "sid": 32, "sent": "We expressed the Q domain of ACBD3 (residues 241\u2013308) and the N-terminal region of PI4KB (residues 1\u201368) in E. coli and using purified recombinant proteins, we confirmed that these two domains are sufficient to maintain the interaction (Fig. 1B, middle and right panel).", "section": "RESULTS", "ner": [ [ 3, 12, "expressed", "experimental_method" ], [ 17, 25, "Q domain", "structure_element" ], [ 29, 34, "ACBD3", "protein" ], [ 45, 52, "241\u2013308", "residue_range" ], [ 62, 79, "N-terminal region", "structure_element" ], [ 83, 88, "PI4KB", "protein" ], [ 99, 103, "1\u201368", "residue_range" ], [ 108, 115, "E. coli", "species" ] ] }, { "sid": 33, "sent": "Because it has been reported that ACBD3 can dimerize in a mammalian two-hybrid assay, we were interested in determining the stoichiometry of the ACBD3:PI4KB protein complex.", "section": "RESULTS", "ner": [ [ 34, 39, "ACBD3", "protein" ], [ 44, 52, "dimerize", "oligomeric_state" ], [ 58, 84, "mammalian two-hybrid assay", "experimental_method" ], [ 145, 156, "ACBD3:PI4KB", "complex_assembly" ] ] }, { "sid": 34, "sent": "The sedimentation coefficients of ACBD3 and PI4KB alone, or ACBD3:PI4KB complex were determined by analytical ultracentrifugation and found to be 3.1 S, 4.1 S, and 5.1 S. These values correspond to molecular weights of approximately 55\u2009kDa, 80\u2009kDa, and 130\u2009kDa, respectively.", "section": "RESULTS", "ner": [ [ 4, 30, "sedimentation coefficients", "evidence" ], [ 34, 39, "ACBD3", "protein" ], [ 44, 49, "PI4KB", "protein" ], [ 50, 55, "alone", "protein_state" ], [ 60, 71, "ACBD3:PI4KB", "complex_assembly" ], [ 99, 129, "analytical ultracentrifugation", "experimental_method" ], [ 198, 215, "molecular weights", "evidence" ] ] }, { "sid": 35, "sent": "This result suggests that both proteins are monomeric and the stoichiometry of the ACBD3: PI4KB protein complex is 1:1 (Fig. 1C, left panel).", "section": "RESULTS", "ner": [ [ 44, 53, "monomeric", "oligomeric_state" ], [ 83, 95, "ACBD3: PI4KB", "complex_assembly" ] ] }, { "sid": 36, "sent": "Similar results were obtained for the complex of the Q domain of ACBD3 and the N-terminal region of PI4KB (Fig. 1C, right panel).", "section": "RESULTS", "ner": [ [ 53, 61, "Q domain", "structure_element" ], [ 65, 70, "ACBD3", "protein" ], [ 79, 96, "N-terminal region", "structure_element" ], [ 100, 105, "PI4KB", "protein" ] ] }, { "sid": 37, "sent": "We also determined the strength of the interaction between recombinant full length ACBD3 and PI4KB using surface plasmon resonance (SPR).", "section": "RESULTS", "ner": [ [ 71, 82, "full length", "protein_state" ], [ 83, 88, "ACBD3", "protein" ], [ 93, 98, "PI4KB", "protein" ], [ 105, 130, "surface plasmon resonance", "experimental_method" ], [ 132, 135, "SPR", "experimental_method" ] ] }, { "sid": 38, "sent": "SPR measurements revealed a strong interaction with a Kd value of 320\u2009+/\u2212130\u2009nM (Fig. 1D, SI Fig. 1D).", "section": "RESULTS", "ner": [ [ 0, 3, "SPR", "experimental_method" ], [ 54, 56, "Kd", "evidence" ] ] }, { "sid": 39, "sent": "We concluded that ACBD3 and PI4KB interact directly through the Q domain of ACBD3 and the N-terminal region of PI4KB forming a 1:1 complex with a dissociation constant in the submicromolar range.", "section": "RESULTS", "ner": [ [ 18, 23, "ACBD3", "protein" ], [ 28, 33, "PI4KB", "protein" ], [ 64, 72, "Q domain", "structure_element" ], [ 76, 81, "ACBD3", "protein" ], [ 90, 107, "N-terminal region", "structure_element" ], [ 111, 116, "PI4KB", "protein" ], [ 146, 167, "dissociation constant", "evidence" ] ] }, { "sid": 40, "sent": "Structural analysis of the ACBD3:PI4KB complex", "section": "RESULTS", "ner": [ [ 0, 19, "Structural analysis", "experimental_method" ], [ 27, 38, "ACBD3:PI4KB", "complex_assembly" ] ] }, { "sid": 41, "sent": "Full length ACBD3 and PI4KB both contain large intrinsically disordered regions that impede crystallization.", "section": "RESULTS", "ner": [ [ 0, 11, "Full length", "protein_state" ], [ 12, 17, "ACBD3", "protein" ], [ 22, 27, "PI4KB", "protein" ], [ 47, 79, "intrinsically disordered regions", "structure_element" ] ] }, { "sid": 42, "sent": "We used hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis of the complex to determine which parts of the complex are well folded (SI Fig. 2).", "section": "RESULTS", "ner": [ [ 8, 53, "hydrogen-deuterium exchange mass spectrometry", "experimental_method" ], [ 55, 61, "HDX-MS", "experimental_method" ], [ 131, 142, "well folded", "protein_state" ] ] }, { "sid": 43, "sent": "However, we were unable to obtain crystals even when using significantly truncated constructs that included only the ACBD3 Q domain and the N-terminal region of PI4KB.", "section": "RESULTS", "ner": [ [ 34, 42, "crystals", "evidence" ], [ 73, 82, "truncated", "protein_state" ], [ 117, 122, "ACBD3", "protein" ], [ 123, 131, "Q domain", "structure_element" ], [ 140, 157, "N-terminal region", "structure_element" ], [ 161, 166, "PI4KB", "protein" ] ] }, { "sid": 44, "sent": "For this reason, we produced an isotopically labeled ACBD3 Q domain and isotopically labeled ACBD3 Q domain:PI4KB N-terminal region protein complex and used NMR spectroscopy for structural characterization.", "section": "RESULTS", "ner": [ [ 32, 52, "isotopically labeled", "protein_state" ], [ 53, 58, "ACBD3", "protein" ], [ 59, 67, "Q domain", "structure_element" ], [ 72, 92, "isotopically labeled", "protein_state" ], [ 93, 98, "ACBD3", "protein" ], [ 99, 107, "Q domain", "structure_element" ], [ 108, 113, "PI4KB", "protein" ], [ 114, 131, "N-terminal region", "structure_element" ], [ 157, 173, "NMR spectroscopy", "experimental_method" ] ] }, { "sid": 45, "sent": "As the N-terminal region protein complex was prepared by co-expression of both proteins, the samples consisted of an equimolar mixture of two uniformly 15N/13C labelled molecules.", "section": "RESULTS", "ner": [ [ 7, 24, "N-terminal region", "structure_element" ], [ 57, 70, "co-expression", "experimental_method" ], [ 152, 155, "15N", "chemical" ], [ 156, 159, "13C", "chemical" ], [ 160, 168, "labelled", "protein_state" ] ] }, { "sid": 46, "sent": "Comprehensive backbone and side-chain resonance assignments for the free ACBD3 Q domain and the complex, as illustrated by the 2D 15N/1H HSQC spectra (SI Figs 3 and 4), were obtained using a standard combination of triple-resonance experiments, as described previously.", "section": "RESULTS", "ner": [ [ 68, 72, "free", "protein_state" ], [ 73, 78, "ACBD3", "protein" ], [ 79, 87, "Q domain", "structure_element" ], [ 127, 141, "2D 15N/1H HSQC", "experimental_method" ], [ 142, 149, "spectra", "evidence" ], [ 215, 243, "triple-resonance experiments", "experimental_method" ] ] }, { "sid": 47, "sent": "Backbone amide signals (15N and 1H) for the free ACBD3 Q domain were nearly completely assigned apart from the first four N-terminal residues (Met1-Lys4) and Gln44.", "section": "RESULTS", "ner": [ [ 24, 27, "15N", "chemical" ], [ 32, 34, "1H", "chemical" ], [ 44, 48, "free", "protein_state" ], [ 49, 54, "ACBD3", "protein" ], [ 55, 63, "Q domain", "structure_element" ], [ 143, 152, "Met1-Lys4", "residue_range" ], [ 158, 163, "Gln44", "residue_name_number" ] ] }, { "sid": 48, "sent": "Over 93% of non-exchangeable side-chain signals were assigned for the free ACBD3 Q domain.", "section": "RESULTS", "ner": [ [ 70, 74, "free", "protein_state" ], [ 75, 80, "ACBD3", "protein" ], [ 81, 89, "Q domain", "structure_element" ] ] }, { "sid": 49, "sent": "Apart from the four N-terminal residues, the side-chain assignments were missing for Gln (Hg3), Gln (Ha/Hb/Hg), Gln44 (Ha/Hb/Hg) and Gln48 (Hg) mainly due to extensive overlaps within the spectral regions populated by highly abundant glutamine side-chain resonances.", "section": "RESULTS", "ner": [ [ 85, 88, "Gln", "residue_name" ], [ 96, 99, "Gln", "residue_name" ], [ 112, 117, "Gln44", "residue_name_number" ], [ 133, 138, "Gln48", "residue_name_number" ], [ 234, 243, "glutamine", "residue_name" ] ] }, { "sid": 50, "sent": "The protein complex yielded relatively well resolved spectra (SI Fig. 4) that resulted in assignment of backbone amide signals for all residues apart from Gln (ACBD3) and Ala2 (PI4KB).", "section": "RESULTS", "ner": [ [ 53, 60, "spectra", "evidence" ], [ 155, 158, "Gln", "residue_name" ], [ 160, 165, "ACBD3", "protein" ], [ 171, 175, "Ala2", "residue_name_number" ], [ 177, 182, "PI4KB", "protein" ] ] }, { "sid": 51, "sent": "The essentially complete 15N, 13C and 1H resonance assignments allowed automated assignment of the NOEs identified in the 3D 15N/1H NOESY-HSQC and 13C/1H HMQC-NOESY spectra that were subsequently used in structural calculation.", "section": "RESULTS", "ner": [ [ 25, 28, "15N", "chemical" ], [ 30, 33, "13C", "chemical" ], [ 38, 40, "1H", "chemical" ], [ 99, 103, "NOEs", "evidence" ], [ 122, 142, "3D 15N/1H NOESY-HSQC", "experimental_method" ], [ 147, 164, "13C/1H HMQC-NOESY", "experimental_method" ], [ 165, 172, "spectra", "evidence" ], [ 204, 226, "structural calculation", "experimental_method" ] ] }, { "sid": 52, "sent": "Structural statistics for the final water-refined sets of structures are shown in SI Table 1.", "section": "RESULTS", "ner": [ [ 0, 21, "Structural statistics", "evidence" ], [ 58, 68, "structures", "evidence" ] ] }, { "sid": 53, "sent": "This structure revealed that the Q domain forms a two helix hairpin.", "section": "RESULTS", "ner": [ [ 5, 14, "structure", "evidence" ], [ 33, 41, "Q domain", "structure_element" ], [ 50, 67, "two helix hairpin", "structure_element" ] ] }, { "sid": 54, "sent": "The first helix bends sharply over the second helix and creates a fold resembling a three helix bundle that serves as a nest for one helix of the PI4KB N-terminus (residues 44\u201364, from this point on referred to as the kinase helix) (Fig. 2A).", "section": "RESULTS", "ner": [ [ 10, 15, "helix", "structure_element" ], [ 46, 51, "helix", "structure_element" ], [ 84, 102, "three helix bundle", "structure_element" ], [ 133, 138, "helix", "structure_element" ], [ 146, 151, "PI4KB", "protein" ], [ 173, 178, "44\u201364", "residue_range" ], [ 218, 230, "kinase helix", "structure_element" ] ] }, { "sid": 55, "sent": "Preceding the kinase helix are three ordered residues (Val42, Ile43, and Asp44) that also contribute to the interaction (Fig. 2B).", "section": "RESULTS", "ner": [ [ 14, 26, "kinase helix", "structure_element" ], [ 55, 60, "Val42", "residue_name_number" ], [ 62, 67, "Ile43", "residue_name_number" ], [ 73, 78, "Asp44", "residue_name_number" ] ] }, { "sid": 56, "sent": "The remaining part of the PI4KB N-termini, however, is disordered (SI Fig. 5).", "section": "RESULTS", "ner": [ [ 26, 31, "PI4KB", "protein" ] ] }, { "sid": 57, "sent": "Almost all of the PI4KB:ACBD3 interactions are hydrophobic with the exception of hydrogen bonds between the side chains of ACBD3 Tyr261 and PI4KB His63, and between the sidechain of ACBD3 Tyr288 and the PI4KB backbone (Asp44) (Fig. 2B).", "section": "RESULTS", "ner": [ [ 18, 29, "PI4KB:ACBD3", "complex_assembly" ], [ 30, 58, "interactions are hydrophobic", "bond_interaction" ], [ 81, 95, "hydrogen bonds", "bond_interaction" ], [ 123, 128, "ACBD3", "protein" ], [ 129, 135, "Tyr261", "residue_name_number" ], [ 140, 145, "PI4KB", "protein" ], [ 146, 151, "His63", "residue_name_number" ], [ 182, 187, "ACBD3", "protein" ], [ 188, 194, "Tyr288", "residue_name_number" ], [ 203, 208, "PI4KB", "protein" ], [ 219, 224, "Asp44", "residue_name_number" ] ] }, { "sid": 58, "sent": "Interestingly, we noted that the PI4KB helix is amphipathic and its hydrophobic surface leans on the Q domain (Fig. 2C).", "section": "RESULTS", "ner": [ [ 33, 38, "PI4KB", "protein" ], [ 39, 44, "helix", "structure_element" ], [ 48, 59, "amphipathic", "protein_state" ], [ 68, 87, "hydrophobic surface", "site" ], [ 101, 109, "Q domain", "structure_element" ] ] }, { "sid": 59, "sent": "To corroborate the structural data, we introduced a number of point mutations and validated their effect on complex formation using an in vitro pull-down assay (Fig. 2D).", "section": "RESULTS", "ner": [ [ 19, 34, "structural data", "evidence" ], [ 39, 49, "introduced", "experimental_method" ], [ 62, 77, "point mutations", "experimental_method" ], [ 135, 159, "in vitro pull-down assay", "experimental_method" ] ] }, { "sid": 60, "sent": "Wild type ACBD3 protein co-purified together with the NiNTA-immobilized His6-tagged wild type PI4KB as well as with the PI4KB V42A and V47A mutants, but not with mutants within the imminent binding interface (I43A, V55A, L56A).", "section": "RESULTS", "ner": [ [ 0, 9, "Wild type", "protein_state" ], [ 10, 15, "ACBD3", "protein" ], [ 24, 35, "co-purified", "experimental_method" ], [ 72, 83, "His6-tagged", "protein_state" ], [ 84, 93, "wild type", "protein_state" ], [ 94, 99, "PI4KB", "protein" ], [ 120, 125, "PI4KB", "protein" ], [ 126, 130, "V42A", "mutant" ], [ 135, 139, "V47A", "mutant" ], [ 140, 147, "mutants", "protein_state" ], [ 162, 169, "mutants", "protein_state" ], [ 190, 207, "binding interface", "site" ], [ 209, 213, "I43A", "mutant" ], [ 215, 219, "V55A", "mutant" ], [ 221, 225, "L56A", "mutant" ] ] }, { "sid": 61, "sent": "As predicted, wild type PI4KB interacted with the ACBD3 Y266A mutant and slightly with the Y285A mutant, but not with the F258A, H284A, and Y288A mutants (Fig. 2D).", "section": "RESULTS", "ner": [ [ 14, 23, "wild type", "protein_state" ], [ 24, 29, "PI4KB", "protein" ], [ 50, 55, "ACBD3", "protein" ], [ 56, 61, "Y266A", "mutant" ], [ 62, 68, "mutant", "protein_state" ], [ 91, 96, "Y285A", "mutant" ], [ 97, 103, "mutant", "protein_state" ], [ 122, 127, "F258A", "mutant" ], [ 129, 134, "H284A", "mutant" ], [ 140, 145, "Y288A", "mutant" ], [ 146, 153, "mutants", "protein_state" ] ] }, { "sid": 62, "sent": "ACBD3 efficiently recruits the PI4KB enzyme to membranes", "section": "RESULTS", "ner": [ [ 0, 5, "ACBD3", "protein" ], [ 31, 36, "PI4KB", "protein" ] ] }, { "sid": 63, "sent": "We next sought to determine if the ACBD3:PI4KB interaction drives membrane localization of the PI4KB enzyme.", "section": "RESULTS", "ner": [ [ 35, 46, "ACBD3:PI4KB", "complex_assembly" ], [ 95, 100, "PI4KB", "protein" ] ] }, { "sid": 64, "sent": "To do this, we first established an in vitro membrane recruitment system using Giant Unilamellar Vesicles (GUVs) containing the PI4KB substrate \u2013 the PI lipid.", "section": "RESULTS", "ner": [ [ 36, 72, "in vitro membrane recruitment system", "experimental_method" ], [ 79, 105, "Giant Unilamellar Vesicles", "experimental_method" ], [ 107, 111, "GUVs", "experimental_method" ], [ 128, 133, "PI4KB", "protein" ], [ 150, 152, "PI", "chemical" ] ] }, { "sid": 65, "sent": "We observed that PI4KB kinase was not membrane localized when added to the GUVs at 600\u2009nM concentration, whereas non-covalent tethering of ACBD3 to the surface of the GUVs, using the His6 tag on ACBD3 and the DGS-NTA (Ni) lipid, led to efficient PI4KB membrane localization (Fig. 3A).", "section": "RESULTS", "ner": [ [ 17, 22, "PI4KB", "protein" ], [ 23, 29, "kinase", "protein_type" ], [ 47, 56, "localized", "evidence" ], [ 75, 79, "GUVs", "experimental_method" ], [ 139, 144, "ACBD3", "protein" ], [ 167, 171, "GUVs", "experimental_method" ], [ 195, 200, "ACBD3", "protein" ], [ 209, 227, "DGS-NTA (Ni) lipid", "chemical" ], [ 246, 251, "PI4KB", "protein" ] ] }, { "sid": 66, "sent": "We hypothesized that if ACBD3 is one of the main Golgi localization signals for PI4KB, overexpression of the Q domain should decrease the amount of the endogenous kinase on the Golgi. Indeed, we observed loss for endogenous PI4KB signal on the Golgi in cells overexpressing the GFP \u2013 Q domain construct (Fig. 3B upper panel).", "section": "RESULTS", "ner": [ [ 24, 29, "ACBD3", "protein" ], [ 55, 75, "localization signals", "evidence" ], [ 80, 85, "PI4KB", "protein" ], [ 87, 101, "overexpression", "experimental_method" ], [ 109, 117, "Q domain", "structure_element" ], [ 163, 169, "kinase", "protein_type" ], [ 224, 229, "PI4KB", "protein" ], [ 259, 273, "overexpressing", "experimental_method" ], [ 278, 281, "GFP", "experimental_method" ], [ 284, 292, "Q domain", "structure_element" ] ] }, { "sid": 67, "sent": "We attribute the loss of signal to the immunostaining protocol-the kinase that is not bound to Golgi is lost during the permeabilization step and hence the \u201cdisappearance\u201d of the signal because overexpression of GFP alone or a non-binding Q domain mutant has no effect on the localization of the endogenous PI4KB (Fig. 3B).", "section": "RESULTS", "ner": [ [ 25, 31, "signal", "evidence" ], [ 67, 73, "kinase", "protein_type" ], [ 179, 185, "signal", "evidence" ], [ 194, 208, "overexpression", "experimental_method" ], [ 212, 215, "GFP", "experimental_method" ], [ 227, 238, "non-binding", "protein_state" ], [ 239, 247, "Q domain", "structure_element" ], [ 248, 254, "mutant", "protein_state" ], [ 276, 288, "localization", "evidence" ], [ 307, 312, "PI4KB", "protein" ] ] }, { "sid": 68, "sent": "Given this result, overexpression of the Q domain should also interfere with the PI4KB dependent Golgi functions.", "section": "RESULTS", "ner": [ [ 19, 33, "overexpression", "experimental_method" ], [ 41, 49, "Q domain", "structure_element" ], [ 81, 86, "PI4KB", "protein" ] ] }, { "sid": 69, "sent": "Ceramide transport and accumulation in Golgi is a well-known PI4KB dependent process.", "section": "RESULTS", "ner": [ [ 0, 8, "Ceramide", "chemical" ], [ 61, 66, "PI4KB", "protein" ] ] }, { "sid": 70, "sent": "We have used fluorescently labeled ceramide and analyzed its trafficking in non-transfected cells and cell overexpressing the Q domain.", "section": "RESULTS", "ner": [ [ 13, 34, "fluorescently labeled", "protein_state" ], [ 35, 43, "ceramide", "chemical" ], [ 107, 121, "overexpressing", "experimental_method" ], [ 126, 134, "Q domain", "structure_element" ] ] }, { "sid": 71, "sent": "As expected, the Golgi accumulation of ceramide was not observed in cells expressing the wt Q domain while cells expressing RFP or the mutant Q domain accumulated ceramide normally (Fig. 3C) suggesting that ACBD3:PI4KB complex formation is crucial for the normal function of Golgi.", "section": "RESULTS", "ner": [ [ 39, 47, "ceramide", "chemical" ], [ 74, 84, "expressing", "experimental_method" ], [ 89, 91, "wt", "protein_state" ], [ 92, 100, "Q domain", "structure_element" ], [ 124, 127, "RFP", "experimental_method" ], [ 135, 141, "mutant", "protein_state" ], [ 142, 150, "Q domain", "structure_element" ], [ 163, 171, "ceramide", "chemical" ], [ 207, 218, "ACBD3:PI4KB", "complex_assembly" ] ] }, { "sid": 72, "sent": "We further analyzed the function of ACBD3:PI4KB interaction in membrane recruitment of PI4KB in living cells using fluorescently tagged proteins.", "section": "RESULTS", "ner": [ [ 36, 47, "ACBD3:PI4KB", "complex_assembly" ], [ 87, 92, "PI4KB", "protein" ], [ 115, 135, "fluorescently tagged", "protein_state" ] ] }, { "sid": 73, "sent": "We used the rapamycin-inducible heteromerization of FKBP12 (FK506 binding protein 12) and FRB (fragment of mTOR that binds rapamycin) system.", "section": "RESULTS", "ner": [ [ 12, 21, "rapamycin", "chemical" ], [ 52, 58, "FKBP12", "protein" ], [ 60, 84, "FK506 binding protein 12", "protein" ], [ 90, 93, "FRB", "structure_element" ], [ 95, 103, "fragment", "structure_element" ], [ 107, 111, "mTOR", "protein" ], [ 123, 132, "rapamycin", "chemical" ] ] }, { "sid": 74, "sent": "We fused the FRB to residues 34\u201363 of the mitochondrial localization signal from mitochondrial A-kinase anchor protein 1 (AKAP1) and CFP.", "section": "RESULTS", "ner": [ [ 3, 8, "fused", "experimental_method" ], [ 13, 16, "FRB", "structure_element" ], [ 29, 34, "34\u201363", "residue_range" ], [ 42, 75, "mitochondrial localization signal", "structure_element" ], [ 81, 120, "mitochondrial A-kinase anchor protein 1", "protein" ], [ 122, 127, "AKAP1", "protein" ], [ 133, 136, "CFP", "experimental_method" ] ] }, { "sid": 75, "sent": "The ACBD3 Q domain was then fused to FKBP12 and mRFP (Fig. 3D).", "section": "RESULTS", "ner": [ [ 4, 9, "ACBD3", "protein" ], [ 10, 18, "Q domain", "structure_element" ], [ 28, 36, "fused to", "experimental_method" ], [ 37, 43, "FKBP12", "protein" ], [ 48, 52, "mRFP", "experimental_method" ] ] }, { "sid": 76, "sent": "We analyzed localization of the ACBD3 Q domain and GFP \u2013 PI4KB before and after the addition of rapamycin.", "section": "RESULTS", "ner": [ [ 12, 24, "localization", "evidence" ], [ 32, 37, "ACBD3", "protein" ], [ 38, 46, "Q domain", "structure_element" ], [ 51, 54, "GFP", "experimental_method" ], [ 57, 62, "PI4KB", "protein" ], [ 96, 105, "rapamycin", "chemical" ] ] }, { "sid": 77, "sent": "As a control we used H284A mutant of the ACBD3 Q domain that does not significantly bind PI4KB kinase.", "section": "RESULTS", "ner": [ [ 21, 26, "H284A", "mutant" ], [ 27, 33, "mutant", "protein_state" ], [ 41, 46, "ACBD3", "protein" ], [ 47, 55, "Q domain", "structure_element" ], [ 89, 94, "PI4KB", "protein" ], [ 95, 101, "kinase", "protein_type" ] ] }, { "sid": 78, "sent": "In every case the ACDB3 Q domain was rapidly (within 5\u2009minutes) recruited to the mitochondrial membrane upon addition of rapamycin, but only the wild-type protein effectively directed the kinase to the mitochondria (Fig. 3E, Movie 1 and 2).", "section": "RESULTS", "ner": [ [ 18, 23, "ACDB3", "protein" ], [ 24, 32, "Q domain", "structure_element" ], [ 121, 130, "rapamycin", "chemical" ], [ 145, 154, "wild-type", "protein_state" ], [ 188, 194, "kinase", "protein_type" ] ] }, { "sid": 79, "sent": "Notably, we observed that when the GFP-PI4KB kinase is co-expressed with the wild-type ACDB3 Q domain it loses its typical Golgi localization (Fig. 3E upper panel).", "section": "RESULTS", "ner": [ [ 35, 38, "GFP", "experimental_method" ], [ 39, 44, "PI4KB", "protein" ], [ 45, 51, "kinase", "protein_type" ], [ 55, 67, "co-expressed", "experimental_method" ], [ 77, 86, "wild-type", "protein_state" ], [ 87, 92, "ACDB3", "protein" ], [ 93, 101, "Q domain", "structure_element" ], [ 129, 141, "localization", "evidence" ] ] }, { "sid": 80, "sent": "However, PI4KB retains it Golgi localization when co-expressed with the non-interacting Q domain mutant (Fig. 3E lower panel).", "section": "RESULTS", "ner": [ [ 9, 14, "PI4KB", "protein" ], [ 32, 44, "localization", "evidence" ], [ 50, 62, "co-expressed", "experimental_method" ], [ 72, 87, "non-interacting", "protein_state" ], [ 88, 96, "Q domain", "structure_element" ], [ 97, 103, "mutant", "protein_state" ] ] }, { "sid": 81, "sent": "ACBD3 increases PI4KB enzymatic activity by recruiting PI4KB to close vicinity of its substrate", "section": "RESULTS", "ner": [ [ 0, 5, "ACBD3", "protein" ], [ 16, 21, "PI4KB", "protein" ], [ 22, 40, "enzymatic activity", "evidence" ], [ 55, 60, "PI4KB", "protein" ] ] }, { "sid": 82, "sent": "To test whether ACBD3 can stimulate PI4KB kinase enzymatic activity we performed a standard luminescent kinase assay using PI-containing micelles as the substrate.", "section": "RESULTS", "ner": [ [ 16, 21, "ACBD3", "protein" ], [ 36, 41, "PI4KB", "protein" ], [ 42, 48, "kinase", "protein_type" ], [ 49, 67, "enzymatic activity", "evidence" ], [ 92, 116, "luminescent kinase assay", "experimental_method" ], [ 123, 125, "PI", "chemical" ] ] }, { "sid": 83, "sent": "We observed no effect on the kinase activity of PI4KB (Fig. 4A) suggesting that ACBD3 does not directly affect the enzyme (e.g. induction of a conformation change).", "section": "RESULTS", "ner": [ [ 29, 35, "kinase", "protein_type" ], [ 48, 53, "PI4KB", "protein" ], [ 80, 85, "ACBD3", "protein" ] ] }, { "sid": 84, "sent": "However, in vivo ACBD3 is located at the Golgi membranes, whereas in this experiment, ACBD3 was located in the solution and PI is provided as micelles.", "section": "RESULTS", "ner": [ [ 17, 22, "ACBD3", "protein" ], [ 86, 91, "ACBD3", "protein" ], [ 124, 126, "PI", "chemical" ] ] }, { "sid": 85, "sent": "For this, we again turned to the GUV system with ACBD3 localized to the GUV membrane.", "section": "RESULTS", "ner": [ [ 33, 36, "GUV", "experimental_method" ], [ 49, 54, "ACBD3", "protein" ], [ 55, 64, "localized", "evidence" ], [ 72, 75, "GUV", "experimental_method" ] ] }, { "sid": 86, "sent": "The GUVs contained 10% PI to serve as a substrate for PI4KB kinase.", "section": "RESULTS", "ner": [ [ 4, 8, "GUVs", "experimental_method" ], [ 23, 25, "PI", "chemical" ], [ 54, 59, "PI4KB", "protein" ], [ 60, 66, "kinase", "protein_type" ] ] }, { "sid": 87, "sent": "The buffer also contained CFP-SidC, which binds to PI4P with nanomolar affinity.", "section": "RESULTS", "ner": [ [ 26, 29, "CFP", "experimental_method" ], [ 30, 34, "SidC", "protein" ], [ 51, 55, "PI4P", "chemical" ] ] }, { "sid": 88, "sent": "This enabled visualization of the kinase reaction using a confocal microscope.", "section": "RESULTS", "ner": [ [ 34, 40, "kinase", "protein_type" ], [ 58, 77, "confocal microscope", "experimental_method" ] ] }, { "sid": 89, "sent": "We compared the efficiency of the phosphorylation reaction of the kinase alone with that of kinase recruited to the surface of the GUVs by ACBD3.", "section": "RESULTS", "ner": [ [ 34, 49, "phosphorylation", "ptm" ], [ 66, 72, "kinase", "protein_type" ], [ 73, 78, "alone", "protein_state" ], [ 92, 98, "kinase", "protein_type" ], [ 131, 135, "GUVs", "experimental_method" ], [ 139, 144, "ACBD3", "protein" ] ] }, { "sid": 90, "sent": "Reaction was also performed in the absence of ATP as a negative control (Fig. 4B).", "section": "RESULTS", "ner": [ [ 35, 45, "absence of", "protein_state" ], [ 46, 49, "ATP", "chemical" ] ] }, { "sid": 91, "sent": "These experiments showed that PI4KB enzymatic activity increases when ACBD3 is membrane localized (Fig. 4C, SI Fig. 6).", "section": "RESULTS", "ner": [ [ 30, 35, "PI4KB", "protein" ], [ 36, 54, "enzymatic activity", "evidence" ], [ 70, 75, "ACBD3", "protein" ] ] }, { "sid": 92, "sent": "Membrane recruitment of PI4KB enzyme is crucial to ensure its proper function at the Golgi and TGN.", "section": "DISCUSS", "ner": [ [ 24, 29, "PI4KB", "protein" ] ] }, { "sid": 93, "sent": "However, the molecular mechanism and structural basis for PI4KB interaction with the membrane is poorly understood.", "section": "DISCUSS", "ner": [ [ 58, 63, "PI4KB", "protein" ] ] }, { "sid": 94, "sent": "In principle, any of the binding partners of PI4KB could play a role in membrane recruitment.", "section": "DISCUSS", "ner": [ [ 45, 50, "PI4KB", "protein" ] ] }, { "sid": 95, "sent": "To date, several PI4KB interacting proteins have been reported, including the small GTPases Rab11 and Arf1, the Golgi resident acyl-CoA binding domain containing 3 (ACBD3) protein, neuronal calcium sensor-1 (NCS-1 also known as frequenin in yeast) and the 14-3-3 proteins.", "section": "DISCUSS", "ner": [ [ 17, 22, "PI4KB", "protein" ], [ 78, 91, "small GTPases", "protein_type" ], [ 92, 97, "Rab11", "protein" ], [ 102, 106, "Arf1", "protein" ], [ 127, 163, "acyl-CoA binding domain containing 3", "protein" ], [ 165, 170, "ACBD3", "protein" ], [ 181, 206, "neuronal calcium sensor-1", "protein" ], [ 208, 213, "NCS-1", "protein" ], [ 228, 237, "frequenin", "protein" ], [ 241, 246, "yeast", "taxonomy_domain" ], [ 256, 271, "14-3-3 proteins", "protein_type" ] ] }, { "sid": 96, "sent": "The monomeric G protein Rab11 binds mammalian PI4KB through the helical domain of the kinase.", "section": "DISCUSS", "ner": [ [ 4, 13, "monomeric", "oligomeric_state" ], [ 14, 23, "G protein", "protein_type" ], [ 24, 29, "Rab11", "protein" ], [ 36, 45, "mammalian", "taxonomy_domain" ], [ 46, 51, "PI4KB", "protein" ], [ 64, 78, "helical domain", "structure_element" ], [ 86, 92, "kinase", "protein_type" ] ] }, { "sid": 97, "sent": "Although Rab11 does not appear to be required for recruitment of PI4KB to the Golgi, PI4KB is required for Golgi recruitment of Rab11.", "section": "DISCUSS", "ner": [ [ 9, 14, "Rab11", "protein" ], [ 65, 70, "PI4KB", "protein" ], [ 85, 90, "PI4KB", "protein" ], [ 128, 133, "Rab11", "protein" ] ] }, { "sid": 98, "sent": "Arf1, the other small GTP binding protein, is known to influence the activity and localization of PI4KB, but it does not appear to interact directly with PI4KB (our unpublished data).", "section": "DISCUSS", "ner": [ [ 0, 4, "Arf1", "protein" ], [ 16, 41, "small GTP binding protein", "protein_type" ], [ 98, 103, "PI4KB", "protein" ], [ 154, 159, "PI4KB", "protein" ] ] }, { "sid": 99, "sent": "The yeast homologue of NCS1 called frequenin has been shown to interact with Pik1p, the yeast orthologue of PI4KB and regulate its activity and perhaps its membrane association, but the role of NCS-1 in PI4KB recruitment in mammalian cells is unclear.", "section": "DISCUSS", "ner": [ [ 4, 9, "yeast", "taxonomy_domain" ], [ 23, 27, "NCS1", "protein" ], [ 35, 44, "frequenin", "protein" ], [ 77, 82, "Pik1p", "protein" ], [ 88, 93, "yeast", "taxonomy_domain" ], [ 108, 113, "PI4KB", "protein" ], [ 194, 199, "NCS-1", "protein" ], [ 203, 208, "PI4KB", "protein" ], [ 224, 233, "mammalian", "taxonomy_domain" ] ] }, { "sid": 100, "sent": "NCS-1 is an N-terminally myristoylated protein that participates in exocytosis.", "section": "DISCUSS", "ner": [ [ 0, 5, "NCS-1", "protein" ], [ 25, 38, "myristoylated", "protein_state" ] ] }, { "sid": 101, "sent": "It is expressed only in certain cell types, suggesting that if it contributes to PI4KB membrane recruitment, it does so in a tissues specific manner.", "section": "DISCUSS", "ner": [ [ 81, 86, "PI4KB", "protein" ] ] }, { "sid": 102, "sent": "The interaction of PI4KB with 14-3-3 proteins, promoted by phosphorylation of PI4KB by protein kinase D, influences the activity of PI4KB by stabilizing its active conformation.", "section": "DISCUSS", "ner": [ [ 19, 24, "PI4KB", "protein" ], [ 30, 45, "14-3-3 proteins", "protein_type" ], [ 59, 74, "phosphorylation", "ptm" ], [ 78, 83, "PI4KB", "protein" ], [ 87, 103, "protein kinase D", "protein" ], [ 132, 137, "PI4KB", "protein" ], [ 157, 163, "active", "protein_state" ] ] }, { "sid": 103, "sent": "However, 14-3-3 proteins do not appear to interfere with membrane recruitment of this kinase.", "section": "DISCUSS", "ner": [ [ 9, 24, "14-3-3 proteins", "protein_type" ], [ 86, 92, "kinase", "protein_type" ] ] }, { "sid": 104, "sent": "ACBD3 is a Golgi resident protein, conserved among vertebrates (SI Fig. 7), that interacts directly with PI4KB (see also SI Fig. 8 and SI Discussion), and whose genetic inactivation interferes with the Golgi localization of the kinase.", "section": "DISCUSS", "ner": [ [ 0, 5, "ACBD3", "protein" ], [ 35, 44, "conserved", "protein_state" ], [ 51, 62, "vertebrates", "taxonomy_domain" ], [ 105, 110, "PI4KB", "protein" ], [ 228, 234, "kinase", "protein_type" ] ] }, { "sid": 105, "sent": "For these reasons we focused on the interaction of the PI4KB enzyme with the Golgi resident ACBD3 protein in this study.", "section": "DISCUSS", "ner": [ [ 55, 60, "PI4KB", "protein" ], [ 92, 97, "ACBD3", "protein" ] ] }, { "sid": 106, "sent": "Here we present the mechanism for membrane recruitment of PI4KB by the Golgi resident ACBD3 protein.", "section": "DISCUSS", "ner": [ [ 58, 63, "PI4KB", "protein" ], [ 86, 91, "ACBD3", "protein" ] ] }, { "sid": 107, "sent": "We show that these proteins interact directly with a Kd value in the submicromolar range.", "section": "DISCUSS", "ner": [ [ 53, 55, "Kd", "evidence" ] ] }, { "sid": 108, "sent": "The interaction is sufficient to recruit PI4KB to model membranes in vitro as well as to the mitochondria where PI4KB is never naturally found.", "section": "DISCUSS", "ner": [ [ 41, 46, "PI4KB", "protein" ], [ 112, 117, "PI4KB", "protein" ] ] }, { "sid": 109, "sent": "To understand this process at the atomic level we solved the solution structure of ACBD3:PI4KB sub complex (Fig. 1A) and found that the PI4KB N-terminal region contains a short amphipatic helix (residues 44\u201364) that binds the ACBD3 Q domain.", "section": "DISCUSS", "ner": [ [ 50, 56, "solved", "experimental_method" ], [ 61, 79, "solution structure", "evidence" ], [ 83, 94, "ACBD3:PI4KB", "complex_assembly" ], [ 136, 141, "PI4KB", "protein" ], [ 142, 159, "N-terminal region", "structure_element" ], [ 171, 193, "short amphipatic helix", "structure_element" ], [ 204, 209, "44\u201364", "residue_range" ], [ 226, 231, "ACBD3", "protein" ], [ 232, 240, "Q domain", "structure_element" ] ] }, { "sid": 110, "sent": "The Q domain adopts a helical hairpin fold that is further stabilized upon binding the kinase helix (Fig. 2A).", "section": "DISCUSS", "ner": [ [ 4, 12, "Q domain", "structure_element" ], [ 22, 42, "helical hairpin fold", "structure_element" ], [ 87, 99, "kinase helix", "structure_element" ] ] }, { "sid": 111, "sent": "Our data strongly suggest that formation of the complex does not directly influence the catalytic abilities of the kinase but experiments with model membranes revealed that ACBD3 enhances catalytic activity of the kinase by a recruitment based mechanism; it recruits the kinase to the membrane and thus increases the local concentration of the substrate in the vicinity of the kinase.", "section": "DISCUSS", "ner": [ [ 115, 121, "kinase", "protein_type" ], [ 173, 178, "ACBD3", "protein" ], [ 214, 220, "kinase", "protein_type" ], [ 271, 277, "kinase", "protein_type" ], [ 377, 383, "kinase", "protein_type" ] ] }, { "sid": 112, "sent": "Based on our and previously published structures we built a pseudoatomic model of PI4KB multi-protein assembly on the membrane (Fig. 5) that illustrates how the enzyme is recruited and positioned towards its lipidic substrate and how it in turn recruits Rab11.", "section": "DISCUSS", "ner": [ [ 38, 48, "structures", "evidence" ], [ 60, 78, "pseudoatomic model", "evidence" ], [ 82, 87, "PI4KB", "protein" ], [ 254, 259, "Rab11", "protein" ] ] }, { "sid": 113, "sent": "+RNA viruses replicate at specific PI4P-enriched membranous compartments.", "section": "DISCUSS", "ner": [ [ 0, 12, "+RNA viruses", "taxonomy_domain" ], [ 35, 39, "PI4P", "chemical" ] ] }, { "sid": 114, "sent": "These are called replication factories (because they enhance viral replication) or membranous webs (because of their appearance under the electron microscope).", "section": "DISCUSS", "ner": [ [ 61, 66, "viral", "taxonomy_domain" ] ] }, { "sid": 115, "sent": "To generate replication factories, viruses hijack several host factors including the PI4K kinases to secure high content of the PI4P lipid.", "section": "DISCUSS", "ner": [ [ 35, 42, "viruses", "taxonomy_domain" ], [ 85, 89, "PI4K", "protein_type" ], [ 90, 97, "kinases", "protein_type" ], [ 128, 132, "PI4P", "chemical" ], [ 133, 138, "lipid", "chemical" ] ] }, { "sid": 116, "sent": "Non-structural 3A proteins from many picornaviruses from the Enterovirus (e.g. poliovirus, coxsackievirus-B3, rhinovirus-14) and Kobuvirus (e.g. Aichi virus-1) genera directly interact with ACBD3.", "section": "DISCUSS", "ner": [ [ 0, 26, "Non-structural 3A proteins", "protein_type" ], [ 37, 51, "picornaviruses", "taxonomy_domain" ], [ 61, 72, "Enterovirus", "taxonomy_domain" ], [ 79, 89, "poliovirus", "species" ], [ 91, 108, "coxsackievirus-B3", "species" ], [ 110, 123, "rhinovirus-14", "species" ], [ 129, 138, "Kobuvirus", "taxonomy_domain" ], [ 145, 158, "Aichi virus-1", "species" ], [ 190, 195, "ACBD3", "protein" ] ] }, { "sid": 117, "sent": "Our data suggest that they could do this via 3A:ACBD3:PI4KB complex formation.", "section": "DISCUSS", "ner": [ [ 45, 59, "3A:ACBD3:PI4KB", "complex_assembly" ] ] }, { "sid": 118, "sent": "The structure of the ACBD3 Q domain and the kinase helix described here provides a novel opportunity for further research on the role of ACBD3, PI4KB, and the ACBD3:PI4KB interaction in picornaviral replication.", "section": "DISCUSS", "ner": [ [ 4, 13, "structure", "evidence" ], [ 21, 26, "ACBD3", "protein" ], [ 27, 35, "Q domain", "structure_element" ], [ 44, 56, "kinase helix", "structure_element" ], [ 137, 142, "ACBD3", "protein" ], [ 144, 149, "PI4KB", "protein" ], [ 159, 170, "ACBD3:PI4KB", "complex_assembly" ], [ 186, 198, "picornaviral", "taxonomy_domain" ] ] }, { "sid": 119, "sent": "This could eventually have implications for therapeutic intervention to combat picornaviruses-mediated diseases ranging from polio to the common cold.", "section": "DISCUSS", "ner": [ [ 79, 93, "picornaviruses", "taxonomy_domain" ] ] }, { "sid": 120, "sent": "Biochemical characterization of the ACBD3:PI4KB complex.", "section": "FIG", "ner": [ [ 0, 28, "Biochemical characterization", "experimental_method" ], [ 36, 47, "ACBD3:PI4KB", "complex_assembly" ] ] }, { "sid": 121, "sent": "(A) Schematic representation of the ACBD3 and PI4KB constructs used for the experiments.", "section": "FIG", "ner": [ [ 36, 41, "ACBD3", "protein" ], [ 46, 51, "PI4KB", "protein" ] ] }, { "sid": 122, "sent": "ACBD3 contains the acyl-CoA binding domain (ACBD), charged amino acids region (CAR), glutamine rich region (Q), and Golgi dynamics domain (GOLD).", "section": "FIG", "ner": [ [ 0, 5, "ACBD3", "protein" ], [ 19, 42, "acyl-CoA binding domain", "structure_element" ], [ 44, 48, "ACBD", "structure_element" ], [ 51, 77, "charged amino acids region", "structure_element" ], [ 79, 82, "CAR", "structure_element" ], [ 85, 106, "glutamine rich region", "structure_element" ], [ 108, 109, "Q", "structure_element" ], [ 116, 137, "Golgi dynamics domain", "structure_element" ], [ 139, 143, "GOLD", "structure_element" ] ] }, { "sid": 123, "sent": "PI4KB is composed of the N-terminal region, helical domain, and kinase domain which can be divided into N- and C-terminal lobes.", "section": "FIG", "ner": [ [ 0, 5, "PI4KB", "protein" ], [ 25, 42, "N-terminal region", "structure_element" ], [ 44, 58, "helical domain", "structure_element" ], [ 64, 77, "kinase domain", "structure_element" ], [ 104, 127, "N- and C-terminal lobes", "structure_element" ] ] }, { "sid": 124, "sent": "(B) In vitro pull-down assay.", "section": "FIG", "ner": [ [ 4, 28, "In vitro pull-down assay", "experimental_method" ] ] }, { "sid": 125, "sent": "Pull-down assays were performed using NiNTA-immobilized N-terminal His6GB1-tagged proteins as indicated and untagged full-length PI4KB or ACBD3.", "section": "FIG", "ner": [ [ 0, 16, "Pull-down assays", "experimental_method" ], [ 67, 81, "His6GB1-tagged", "protein_state" ], [ 108, 116, "untagged", "protein_state" ], [ 117, 128, "full-length", "protein_state" ], [ 129, 134, "PI4KB", "protein" ], [ 138, 143, "ACBD3", "protein" ] ] }, { "sid": 126, "sent": "The inputs and bound proteins were analyzed on SDS gels stained with Coomassie Blue.", "section": "FIG", "ner": [ [ 47, 55, "SDS gels", "experimental_method" ] ] }, { "sid": 127, "sent": "Please, see SI Fig. 9 for original full-length gels. (C) Analytical Ultracentrifugation.", "section": "FIG", "ner": [ [ 35, 46, "full-length", "protein_state" ], [ 57, 87, "Analytical Ultracentrifugation", "experimental_method" ] ] }, { "sid": 128, "sent": "AUC analysis of the ACBD3:PI4KB full-length complex at the concentration of 5\u2009\u03bcM (both proteins, left panel) and ACBD3 Q domain: PI4KB N terminal region complex at the concentration of 35\u2009\u03bcM (both proteins, right panel). (D) Surface plasmon resonance.", "section": "FIG", "ner": [ [ 0, 3, "AUC", "experimental_method" ], [ 20, 31, "ACBD3:PI4KB", "complex_assembly" ], [ 32, 43, "full-length", "protein_state" ], [ 113, 152, "ACBD3 Q domain: PI4KB N terminal region", "complex_assembly" ], [ 225, 250, "Surface plasmon resonance", "experimental_method" ] ] }, { "sid": 129, "sent": "SPR analysis of the PI4KB binding to immobilized ACBD3.", "section": "FIG", "ner": [ [ 0, 3, "SPR", "experimental_method" ], [ 20, 25, "PI4KB", "protein" ], [ 49, 54, "ACBD3", "protein" ] ] }, { "sid": 130, "sent": "Sensorgrams for four concentrations of PI4KB are shown.", "section": "FIG", "ner": [ [ 0, 11, "Sensorgrams", "evidence" ], [ 39, 44, "PI4KB", "protein" ] ] }, { "sid": 131, "sent": "Structural analysis of the ACBD3:PI4KB complex.", "section": "FIG", "ner": [ [ 0, 19, "Structural analysis", "experimental_method" ], [ 27, 38, "ACBD3:PI4KB", "complex_assembly" ] ] }, { "sid": 132, "sent": "(A) Overall structure of the ACBD3 Q domain by itself and in complex with the PI4KB N-terminal region.", "section": "FIG", "ner": [ [ 12, 21, "structure", "evidence" ], [ 29, 34, "ACBD3", "protein" ], [ 35, 43, "Q domain", "structure_element" ], [ 58, 73, "in complex with", "protein_state" ], [ 78, 83, "PI4KB", "protein" ], [ 84, 101, "N-terminal region", "structure_element" ] ] }, { "sid": 133, "sent": "Superposition of the 30 converged structures obtained for the Q domain (top) and the 45 converged structures obtained for the complex (bottom), with only the folded part of PI4KB shown (see SI Fig. 2 for the complete view). (B) Detailed view of the complex.", "section": "FIG", "ner": [ [ 0, 13, "Superposition", "experimental_method" ], [ 34, 44, "structures", "evidence" ], [ 62, 70, "Q domain", "structure_element" ], [ 98, 108, "structures", "evidence" ], [ 158, 164, "folded", "protein_state" ], [ 173, 178, "PI4KB", "protein" ] ] }, { "sid": 134, "sent": "The interaction is facilitated by only two hydrogen bonds (ACBD3 Tyr261: PI4KB His63 and ACBD3 Tyr288: PI4KB Asp44), while the hydrophobic surface of the kinase helix nests in the ACBD3 Q domain.", "section": "FIG", "ner": [ [ 43, 57, "hydrogen bonds", "bond_interaction" ], [ 59, 64, "ACBD3", "protein" ], [ 65, 71, "Tyr261", "residue_name_number" ], [ 73, 78, "PI4KB", "protein" ], [ 79, 84, "His63", "residue_name_number" ], [ 89, 94, "ACBD3", "protein" ], [ 95, 101, "Tyr288", "residue_name_number" ], [ 103, 108, "PI4KB", "protein" ], [ 109, 114, "Asp44", "residue_name_number" ], [ 127, 146, "hydrophobic surface", "site" ], [ 154, 166, "kinase helix", "structure_element" ], [ 180, 185, "ACBD3", "protein" ], [ 186, 194, "Q domain", "structure_element" ] ] }, { "sid": 135, "sent": "ACBD3 is shown in magenta and PI4KB in orange.", "section": "FIG", "ner": [ [ 0, 5, "ACBD3", "protein" ], [ 30, 35, "PI4KB", "protein" ] ] }, { "sid": 136, "sent": "(C) Top view of the kinase helix.", "section": "FIG", "ner": [ [ 20, 32, "kinase helix", "structure_element" ] ] }, { "sid": 137, "sent": "The kinase helix is amphipathic and its hydrophobic surface overlaps with the ACBD3 binding surface (shown in magenta).", "section": "FIG", "ner": [ [ 4, 16, "kinase helix", "structure_element" ], [ 20, 31, "amphipathic", "protein_state" ], [ 40, 59, "hydrophobic surface", "site" ], [ 78, 83, "ACBD3", "protein" ], [ 84, 99, "binding surface", "site" ] ] }, { "sid": 138, "sent": "Strong and weak hydrophobes are in green and cyan respectively, basic residues in blue, acidic residues in red and nonpolar hydrophilic residues in orange. (D) Pull-down assay with a NiNTA-immobilized N-terminally His6GB1-tagged PI4KB kinase and untagged ACBD3 protein.", "section": "FIG", "ner": [ [ 160, 175, "Pull-down assay", "experimental_method" ], [ 214, 228, "His6GB1-tagged", "protein_state" ], [ 229, 234, "PI4KB", "protein" ], [ 235, 241, "kinase", "protein_type" ], [ 246, 254, "untagged", "protein_state" ], [ 255, 260, "ACBD3", "protein" ] ] }, { "sid": 139, "sent": "Wild type proteins and selected point mutants of both PI4KB and ACBD3 were used.", "section": "FIG", "ner": [ [ 0, 9, "Wild type", "protein_state" ], [ 38, 45, "mutants", "protein_state" ], [ 54, 59, "PI4KB", "protein" ], [ 64, 69, "ACBD3", "protein" ] ] }, { "sid": 140, "sent": "Please, see SI Fig. 9 for original full-length gels.", "section": "FIG", "ner": [ [ 35, 46, "full-length", "protein_state" ] ] }, { "sid": 141, "sent": "ACBD3 is sufficient to recruit the PI4KB kinase to membranes.", "section": "FIG", "ner": [ [ 0, 5, "ACBD3", "protein" ], [ 35, 40, "PI4KB", "protein" ], [ 41, 47, "kinase", "protein_type" ] ] }, { "sid": 142, "sent": "(A) GUVs recruitment assay.", "section": "FIG", "ner": [ [ 4, 26, "GUVs recruitment assay", "experimental_method" ] ] }, { "sid": 143, "sent": "Top \u2013 Virtually no membrane bound kinase was observed when 600\u2009nM PI4KB was added to the GUVs.", "section": "FIG", "ner": [ [ 34, 40, "kinase", "protein_type" ], [ 66, 71, "PI4KB", "protein" ], [ 89, 93, "GUVs", "experimental_method" ] ] }, { "sid": 144, "sent": "Bottom \u2013 in the presence of 600\u2009nM GUV tethered ACBD3 a significant signal of the kinase is detected on the surface of GUVs.", "section": "FIG", "ner": [ [ 16, 27, "presence of", "protein_state" ], [ 35, 47, "GUV tethered", "protein_state" ], [ 48, 53, "ACBD3", "protein" ], [ 82, 88, "kinase", "protein_type" ], [ 119, 123, "GUVs", "experimental_method" ] ] }, { "sid": 145, "sent": "(B) Golgi displacement experiment.", "section": "FIG", "ner": [ [ 4, 33, "Golgi displacement experiment", "experimental_method" ] ] }, { "sid": 146, "sent": "Upper panel: ACBD3 Q domain fused to GFP was overexpressed and the endogenous PI4KB was immunostained.", "section": "FIG", "ner": [ [ 13, 18, "ACBD3", "protein" ], [ 19, 27, "Q domain", "structure_element" ], [ 37, 40, "GFP", "experimental_method" ], [ 45, 58, "overexpressed", "experimental_method" ], [ 78, 83, "PI4KB", "protein" ], [ 88, 101, "immunostained", "experimental_method" ] ] }, { "sid": 147, "sent": "Middle panel: The same experiment performed with GFP alone.", "section": "FIG", "ner": [ [ 49, 52, "GFP", "experimental_method" ] ] }, { "sid": 148, "sent": "Lower panel: The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (C) ACBD3 Q domain overexpression inhibits ceramide transport to Golgi \u2013 COS-7 cells transfected with wild-type ACBD3 Q domain-FKBP-mRFP were loaded with 0.05\u2009\u03bcM Bodipy FL-Ceramide for 20\u2009min, then washed and depicted after 20\u2009min.", "section": "FIG", "ner": [ [ 48, 54, "mutant", "protein_state" ], [ 55, 63, "Q domain", "structure_element" ], [ 65, 70, "F258A", "mutant" ], [ 72, 77, "H284A", "mutant" ], [ 79, 84, "Y288A", "mutant" ], [ 109, 114, "PI4KB", "protein" ], [ 120, 125, "ACBD3", "protein" ], [ 126, 134, "Q domain", "structure_element" ], [ 135, 149, "overexpression", "experimental_method" ], [ 159, 167, "ceramide", "chemical" ], [ 218, 227, "wild-type", "protein_state" ], [ 228, 233, "ACBD3", "protein" ], [ 234, 242, "Q domain", "structure_element" ], [ 243, 247, "FKBP", "protein" ], [ 248, 252, "mRFP", "experimental_method" ], [ 278, 296, "Bodipy FL-Ceramide", "chemical" ] ] }, { "sid": 149, "sent": "Middle panel \u2013 The same experiment performed with mRFP-FKBP alone.", "section": "FIG", "ner": [ [ 50, 54, "mRFP", "experimental_method" ], [ 55, 59, "FKBP", "protein" ] ] }, { "sid": 150, "sent": "Lower panel \u2013 The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (D) Scheme of the mitochondria recruitment experiment.", "section": "FIG", "ner": [ [ 49, 55, "mutant", "protein_state" ], [ 56, 64, "Q domain", "structure_element" ], [ 66, 71, "F258A", "mutant" ], [ 73, 78, "H284A", "mutant" ], [ 80, 85, "Y288A", "mutant" ], [ 110, 115, "PI4KB", "protein" ], [ 135, 170, "mitochondria recruitment experiment", "experimental_method" ] ] }, { "sid": 151, "sent": "\u2013 The AKAP1-FRB-CFP construct is localized at the outer mitochondrial membrane, while the GFP-PI4KB and Q domain-FKBP-mRFP constructs are localized in the cytoplasm where they can form a complex.", "section": "FIG", "ner": [ [ 6, 11, "AKAP1", "protein" ], [ 12, 15, "FRB", "structure_element" ], [ 16, 19, "CFP", "experimental_method" ], [ 33, 42, "localized", "evidence" ], [ 90, 93, "GFP", "experimental_method" ], [ 94, 99, "PI4KB", "protein" ], [ 104, 112, "Q domain", "structure_element" ], [ 113, 117, "FKBP", "protein" ], [ 118, 122, "mRFP", "experimental_method" ], [ 138, 147, "localized", "evidence" ] ] }, { "sid": 152, "sent": "Upon addition of rapamycin the Q domain-FKBP-mRFP construct translocates to the mitochondria and takes GFP-PI4KB with it. (E) Mitochondria recruitment experiment.", "section": "FIG", "ner": [ [ 17, 26, "rapamycin", "chemical" ], [ 31, 39, "Q domain", "structure_element" ], [ 40, 44, "FKBP", "protein" ], [ 45, 49, "mRFP", "experimental_method" ], [ 103, 106, "GFP", "experimental_method" ], [ 107, 112, "PI4KB", "protein" ], [ 126, 161, "Mitochondria recruitment experiment", "experimental_method" ] ] }, { "sid": 153, "sent": "Left \u2013 cells transfected with AKAP1-FRB-CFP, GFP-PI4KB and wild-type Q domain-FKBP-mRFP constructs before and five minutes after addition of rapamycin.", "section": "FIG", "ner": [ [ 30, 35, "AKAP1", "protein" ], [ 36, 39, "FRB", "structure_element" ], [ 40, 43, "CFP", "experimental_method" ], [ 45, 48, "GFP", "experimental_method" ], [ 49, 54, "PI4KB", "protein" ], [ 59, 68, "wild-type", "protein_state" ], [ 69, 77, "Q domain", "structure_element" ], [ 78, 82, "FKBP", "protein" ], [ 83, 87, "mRFP", "experimental_method" ], [ 141, 150, "rapamycin", "chemical" ] ] }, { "sid": 154, "sent": "Right \u2013 The same experiment performed using the H264A Q domain mutant.", "section": "FIG", "ner": [ [ 48, 53, "H264A", "mutant" ], [ 54, 62, "Q domain", "structure_element" ], [ 63, 69, "mutant", "protein_state" ] ] }, { "sid": 155, "sent": "ACBD3 indirectly increases the activity of PI4KB.", "section": "FIG", "ner": [ [ 0, 5, "ACBD3", "protein" ], [ 43, 48, "PI4KB", "protein" ] ] }, { "sid": 156, "sent": "(A) Micelles-based kinase assay \u2013 PI in TX100 micelles was used in a luminescent kinase assay and the production of PI4P was measured.", "section": "FIG", "ner": [ [ 4, 31, "Micelles-based kinase assay", "experimental_method" ], [ 34, 36, "PI", "chemical" ], [ 69, 93, "luminescent kinase assay", "experimental_method" ], [ 116, 120, "PI4P", "chemical" ] ] }, { "sid": 157, "sent": "Bar graph presents the mean values of PI4P generated in the presence of the proteins as indicated, normalized to the amount of PI4P generated by PI4KB alone.", "section": "FIG", "ner": [ [ 38, 42, "PI4P", "chemical" ], [ 60, 71, "presence of", "protein_state" ], [ 127, 131, "PI4P", "chemical" ], [ 145, 150, "PI4KB", "protein" ] ] }, { "sid": 158, "sent": "Error bars are standard errors of the mean (SEM) based on three independent experiments. (B) GUV-based phosphorylation assay \u2013 GUVs containing 10% PI were used as a substrate and the production of PI4P was measured using the CFP-SidC biosensor.", "section": "FIG", "ner": [ [ 15, 42, "standard errors of the mean", "evidence" ], [ 44, 47, "SEM", "evidence" ], [ 93, 124, "GUV-based phosphorylation assay", "experimental_method" ], [ 127, 131, "GUVs", "experimental_method" ], [ 147, 149, "PI", "chemical" ], [ 197, 201, "PI4P", "chemical" ], [ 225, 243, "CFP-SidC biosensor", "experimental_method" ] ] }, { "sid": 159, "sent": "(C)\u2013Quantification of the GUV phosphorylation assay \u2013 Mean membrane fluorescence intensity of the PI4P reporter (SidC-label) under different protein/ATP conditions.", "section": "FIG", "ner": [ [ 26, 51, "GUV phosphorylation assay", "experimental_method" ], [ 54, 90, "Mean membrane fluorescence intensity", "evidence" ], [ 98, 102, "PI4P", "chemical" ], [ 113, 117, "SidC", "protein" ], [ 149, 152, "ATP", "chemical" ] ] }, { "sid": 160, "sent": "The mean membrane intensity value is relative to the background signal and the difference between the membrane and background signal in the reference system lacking ATP.", "section": "FIG", "ner": [ [ 4, 27, "mean membrane intensity", "evidence" ], [ 165, 168, "ATP", "chemical" ] ] }, { "sid": 161, "sent": "The error bars stand for SEM based on three independent experiments (also SI Fig. 6).", "section": "FIG", "ner": [ [ 25, 28, "SEM", "evidence" ] ] }, { "sid": 162, "sent": "Pseudoatomic model of the PI4KB multiprotein complex assembly.", "section": "FIG", "ner": [ [ 0, 18, "Pseudoatomic model", "evidence" ], [ 26, 31, "PI4KB", "protein" ] ] }, { "sid": 163, "sent": "PI4KB in orange, Rab11 in purple, ACBD3 in blue.", "section": "FIG", "ner": [ [ 0, 5, "PI4KB", "protein" ], [ 17, 22, "Rab11", "protein" ], [ 34, 39, "ACBD3", "protein" ] ] }, { "sid": 164, "sent": "The model is based on our NMR structure and a previously published crystal structure of PI4KB:Rab11 complex (PDB code 4D0L), ACBD and GOLD domain were homology modeled based on high sequence identity structures produced by the Phyre2 web server.", "section": "FIG", "ner": [ [ 26, 29, "NMR", "experimental_method" ], [ 30, 39, "structure", "evidence" ], [ 67, 84, "crystal structure", "evidence" ], [ 88, 99, "PI4KB:Rab11", "complex_assembly" ], [ 125, 129, "ACBD", "structure_element" ], [ 134, 138, "GOLD", "structure_element" ], [ 151, 167, "homology modeled", "experimental_method" ], [ 200, 210, "structures", "evidence" ], [ 227, 233, "Phyre2", "experimental_method" ] ] }, { "sid": 165, "sent": "The GOLD domain is tethered to the membrane by GolginB1 (also known as Giantin) which is not shown for clarity.", "section": "FIG", "ner": [ [ 4, 8, "GOLD", "structure_element" ], [ 47, 55, "GolginB1", "protein" ], [ 71, 78, "Giantin", "protein" ] ] }, { "sid": 166, "sent": "Intrinsically disordered linkers are modeled in an arbitrary but physically plausible conformation.", "section": "FIG", "ner": [ [ 0, 32, "Intrinsically disordered linkers", "structure_element" ] ] } ] }, "PMC5603727": { "annotations": [ { "sid": 0, "sent": "Roquin recognizes a non-canonical hexaloop structure in the 3\u2032-UTR of Ox40", "section": "TITLE", "ner": [ [ 0, 6, "Roquin", "protein" ], [ 34, 42, "hexaloop", "structure_element" ], [ 60, 66, "3\u2032-UTR", "structure_element" ], [ 70, 74, "Ox40", "protein" ] ] }, { "sid": 1, "sent": "The RNA-binding protein Roquin is required to prevent autoimmunity.", "section": "ABSTRACT", "ner": [ [ 4, 23, "RNA-binding protein", "protein_type" ], [ 24, 30, "Roquin", "protein" ] ] }, { "sid": 2, "sent": "Roquin controls T-helper cell activation and differentiation by limiting the induced expression of costimulatory receptors such as tumor necrosis factor receptor superfamily 4 (Tnfrs4 or Ox40).", "section": "ABSTRACT", "ner": [ [ 0, 6, "Roquin", "protein" ], [ 99, 122, "costimulatory receptors", "protein_type" ], [ 131, 175, "tumor necrosis factor receptor superfamily 4", "protein" ], [ 177, 183, "Tnfrs4", "protein" ], [ 187, 191, "Ox40", "protein" ] ] }, { "sid": 3, "sent": "A constitutive decay element (CDE) with a characteristic triloop hairpin was previously shown to be recognized by Roquin.", "section": "ABSTRACT", "ner": [ [ 2, 28, "constitutive decay element", "structure_element" ], [ 30, 33, "CDE", "structure_element" ], [ 57, 72, "triloop hairpin", "structure_element" ], [ 114, 120, "Roquin", "protein" ] ] }, { "sid": 4, "sent": "Here we use SELEX assays to identify a novel U-rich hexaloop motif, representing an alternative decay element (ADE).", "section": "ABSTRACT", "ner": [ [ 12, 24, "SELEX assays", "experimental_method" ], [ 45, 66, "U-rich hexaloop motif", "structure_element" ], [ 84, 109, "alternative decay element", "structure_element" ], [ 111, 114, "ADE", "structure_element" ] ] }, { "sid": 5, "sent": "Crystal structures and NMR data show that the Roquin-1 ROQ domain recognizes hexaloops in the SELEX-derived ADE and in an ADE-like variant present in the Ox40 3\u2032-UTR with identical binding modes.", "section": "ABSTRACT", "ner": [ [ 0, 18, "Crystal structures", "evidence" ], [ 23, 26, "NMR", "experimental_method" ], [ 46, 54, "Roquin-1", "protein" ], [ 55, 58, "ROQ", "structure_element" ], [ 77, 86, "hexaloops", "structure_element" ], [ 94, 99, "SELEX", "experimental_method" ], [ 108, 111, "ADE", "structure_element" ], [ 122, 125, "ADE", "structure_element" ], [ 154, 158, "Ox40", "protein" ], [ 159, 165, "3\u2032-UTR", "structure_element" ] ] }, { "sid": 6, "sent": "In cells, ADE-like and CDE-like motifs cooperate in the repression of Ox40 by Roquin.", "section": "ABSTRACT", "ner": [ [ 10, 13, "ADE", "structure_element" ], [ 23, 26, "CDE", "structure_element" ], [ 70, 74, "Ox40", "protein" ], [ 78, 84, "Roquin", "protein" ] ] }, { "sid": 7, "sent": "Our data reveal an unexpected recognition of hexaloop cis elements for the posttranscriptional regulation of target messenger RNAs by Roquin.", "section": "ABSTRACT", "ner": [ [ 45, 66, "hexaloop cis elements", "structure_element" ], [ 116, 130, "messenger RNAs", "chemical" ], [ 134, 140, "Roquin", "protein" ] ] }, { "sid": 8, "sent": " Roquin is an RNA-binding protein that prevents autoimmunity by limiting expression of receptors such as Ox40.", "section": "ABSTRACT", "ner": [ [ 1, 7, "Roquin", "protein" ], [ 14, 33, "RNA-binding protein", "protein_type" ], [ 105, 109, "Ox40", "protein" ] ] }, { "sid": 9, "sent": "Here, the authors identify an RNA structure that they describe as an alternative decay element, and they characterise its interaction with Roquin using structural and biochemical techniques.", "section": "ABSTRACT", "ner": [ [ 30, 33, "RNA", "chemical" ], [ 34, 43, "structure", "evidence" ], [ 69, 94, "alternative decay element", "structure_element" ], [ 139, 145, "Roquin", "protein" ], [ 152, 189, "structural and biochemical techniques", "experimental_method" ] ] }, { "sid": 10, "sent": "The Roquin protein is essential in T cells for the prevention of autoimmune disease.", "section": "INTRO", "ner": [ [ 4, 10, "Roquin", "protein" ] ] }, { "sid": 11, "sent": "This is evident from the so-called sanroque mutation in Roquin-1, a single amino acid exchange from Met199 to Arg that causes the development of systemic lupus erythematosus-like symptoms in homozygous mice.", "section": "INTRO", "ner": [ [ 56, 64, "Roquin-1", "protein" ], [ 100, 106, "Met199", "residue_name_number" ], [ 110, 113, "Arg", "residue_name" ], [ 202, 206, "mice", "taxonomy_domain" ] ] }, { "sid": 12, "sent": "The Rc3h1 and Rc3h2 genes, encoding for Roquin-1 and Roquin-2 proteins in vertebrates, respectively, have both been shown to be essential for the survival of mice, but apparently serve redundant functions in T cells.", "section": "INTRO", "ner": [ [ 4, 9, "Rc3h1", "gene" ], [ 14, 19, "Rc3h2", "gene" ], [ 40, 48, "Roquin-1", "protein" ], [ 53, 61, "Roquin-2", "protein" ], [ 74, 85, "vertebrates", "taxonomy_domain" ], [ 158, 162, "mice", "taxonomy_domain" ] ] }, { "sid": 13, "sent": "Consistently, CD4+ and CD8+ T cells with the combined deletion of Roquin-encoding genes are spontaneously activated and CD4+ T-helper cells preferentially differentiate into the Th1, Tfh or Th17 subsets.", "section": "INTRO", "ner": [ [ 54, 65, "deletion of", "experimental_method" ], [ 66, 72, "Roquin", "protein" ] ] }, { "sid": 14, "sent": "Roquin-1 was shown to negatively regulate expression of transcripts encoding for co-stimulatory receptors such as Icos, Ox40 and CTLA-4, for cytokines such as interleukin (IL)-6 and tumour necrosis factor or for transcription factors such as IRF4, I\u03baBNS and I\u03baB\u03b6 (refs).", "section": "INTRO", "ner": [ [ 0, 8, "Roquin-1", "protein" ], [ 81, 105, "co-stimulatory receptors", "protein_type" ], [ 114, 118, "Icos", "protein" ], [ 120, 124, "Ox40", "protein" ], [ 129, 135, "CTLA-4", "protein" ], [ 141, 150, "cytokines", "protein_type" ], [ 159, 177, "interleukin (IL)-6", "protein" ], [ 182, 204, "tumour necrosis factor", "protein" ], [ 212, 233, "transcription factors", "protein_type" ], [ 242, 246, "IRF4", "protein" ], [ 248, 253, "I\u03baBNS", "protein" ], [ 258, 262, "I\u03baB\u03b6", "protein" ] ] }, { "sid": 15, "sent": "We have recently reported structural and functional data of the Roquin-1 ROQ domain bound to a canonical constitutive decay element (CDE), a short stem loop (SL) that acts as a cis-regulatory RNA element in the 3\u2032-untranslated regions (3\u2032-UTRs) of target genes such as Tnf (ref).", "section": "INTRO", "ner": [ [ 26, 56, "structural and functional data", "evidence" ], [ 64, 72, "Roquin-1", "protein" ], [ 73, 76, "ROQ", "structure_element" ], [ 84, 92, "bound to", "protein_state" ], [ 105, 131, "constitutive decay element", "structure_element" ], [ 133, 136, "CDE", "structure_element" ], [ 141, 156, "short stem loop", "structure_element" ], [ 158, 160, "SL", "structure_element" ], [ 192, 195, "RNA", "chemical" ], [ 211, 234, "3\u2032-untranslated regions", "structure_element" ], [ 236, 243, "3\u2032-UTRs", "structure_element" ], [ 269, 272, "Tnf", "protein" ] ] }, { "sid": 16, "sent": "The ROQ domain adopts an extended winged helix fold that engages predominantly non-sequence-specific protein\u2013RNA contacts and mainly recognizes the shape of the canonical Tnf CDE RNA.", "section": "INTRO", "ner": [ [ 4, 7, "ROQ", "structure_element" ], [ 25, 51, "extended winged helix fold", "structure_element" ], [ 109, 112, "RNA", "chemical" ], [ 171, 174, "Tnf", "protein" ], [ 175, 178, "CDE", "structure_element" ], [ 179, 182, "RNA", "chemical" ] ] }, { "sid": 17, "sent": "The structural data and mutational analysis indicated that a broader, extended range of sequence variations in both the loop and stem of the CDE element is recognized and regulated by Roquin.", "section": "INTRO", "ner": [ [ 4, 19, "structural data", "evidence" ], [ 24, 43, "mutational analysis", "experimental_method" ], [ 120, 124, "loop", "structure_element" ], [ 129, 133, "stem", "structure_element" ], [ 141, 144, "CDE", "structure_element" ], [ 184, 190, "Roquin", "protein" ] ] }, { "sid": 18, "sent": "At the same time, Tan et al. described the crystal structure and supporting functional data of a similar interaction with a CDE-like SL, and reported a second binding site for a double-stranded RNA (dsRNA) within an extended ROQ domain.", "section": "INTRO", "ner": [ [ 43, 60, "crystal structure", "evidence" ], [ 124, 127, "CDE", "structure_element" ], [ 133, 135, "SL", "structure_element" ], [ 152, 171, "second binding site", "site" ], [ 178, 197, "double-stranded RNA", "chemical" ], [ 199, 204, "dsRNA", "chemical" ], [ 216, 224, "extended", "protein_state" ], [ 225, 228, "ROQ", "structure_element" ] ] }, { "sid": 19, "sent": "The structural basis for CDE recognition by the Roquin-2 ROQ domain has also been recently reported.", "section": "INTRO", "ner": [ [ 25, 28, "CDE", "structure_element" ], [ 48, 56, "Roquin-2", "protein" ], [ 57, 60, "ROQ", "structure_element" ] ] }, { "sid": 20, "sent": "We found that the posttranscriptional activity of Roquin-1 and Roquin-2 is regulated through cleavage by the paracaspase MALT1 (refs).", "section": "INTRO", "ner": [ [ 50, 58, "Roquin-1", "protein" ], [ 63, 71, "Roquin-2", "protein" ], [ 109, 120, "paracaspase", "protein_type" ], [ 121, 126, "MALT1", "protein" ] ] }, { "sid": 21, "sent": "Enhanced MALT1-dependent cleavage and inactivation of Roquin, and thus less effective repression of target genes, result from increased strength of antigen recognition in T cells.", "section": "INTRO", "ner": [ [ 9, 14, "MALT1", "protein" ], [ 54, 60, "Roquin", "protein" ] ] }, { "sid": 22, "sent": "These findings suggest that dependent on the strength of cognate antigen recognition differential gene expression and cell fate decisions can be established in naive T cells by a graded cleavage and inactivation of Roquin.", "section": "INTRO", "ner": [ [ 215, 221, "Roquin", "protein" ] ] }, { "sid": 23, "sent": "In addition to this mechanism, the composition and binding affinity of cis-regulatory SL elements in the 3\u2032-UTRs of target mRNAs may determine the sensitivity to repression by the trans-acting factor Roquin. Defining the SL RNA structures that are recognized by Roquin is therefore essential for our understanding of posttranscriptional gene regulation by Roquin and its involvement in T-cell biology and T-cell-driven pathology.", "section": "INTRO", "ner": [ [ 51, 67, "binding affinity", "evidence" ], [ 86, 88, "SL", "structure_element" ], [ 105, 112, "3\u2032-UTRs", "structure_element" ], [ 123, 128, "mRNAs", "chemical" ], [ 200, 206, "Roquin", "protein" ], [ 221, 223, "SL", "structure_element" ], [ 224, 227, "RNA", "chemical" ], [ 262, 268, "Roquin", "protein" ], [ 356, 362, "Roquin", "protein" ] ] }, { "sid": 24, "sent": "Here we present structural and functional evidence for a greatly expanded repertoire of RNA elements that are regulated by Roquin as demonstrated with a novel U-rich hexaloop SL in the 3\u2032-UTR of Ox40 bound to the Roquin-1 ROQ domain.", "section": "INTRO", "ner": [ [ 88, 91, "RNA", "chemical" ], [ 123, 129, "Roquin", "protein" ], [ 159, 174, "U-rich hexaloop", "structure_element" ], [ 175, 177, "SL", "structure_element" ], [ 185, 191, "3\u2032-UTR", "structure_element" ], [ 195, 199, "Ox40", "protein" ], [ 200, 208, "bound to", "protein_state" ], [ 213, 221, "Roquin-1", "protein" ], [ 222, 225, "ROQ", "structure_element" ] ] }, { "sid": 25, "sent": "We find an additive regulation of Ox40 gene expression based on both its CDE-like and hexaloop SL RNAs that we identified using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) experiments.", "section": "INTRO", "ner": [ [ 34, 38, "Ox40", "protein" ], [ 73, 76, "CDE", "structure_element" ], [ 86, 94, "hexaloop", "structure_element" ], [ 95, 97, "SL", "structure_element" ], [ 98, 102, "RNAs", "chemical" ], [ 128, 185, "Systematic Evolution of Ligands by Exponential Enrichment", "experimental_method" ], [ 187, 192, "SELEX", "experimental_method" ] ] }, { "sid": 26, "sent": "Our X-ray crystallographic, NMR, biochemical and functional data combined with mutational analysis demonstrate that both triloop and hexaloop SL RNAs contribute to the functional activity of Roquin in T cells.", "section": "INTRO", "ner": [ [ 4, 26, "X-ray crystallographic", "experimental_method" ], [ 28, 31, "NMR", "experimental_method" ], [ 33, 64, "biochemical and functional data", "evidence" ], [ 79, 98, "mutational analysis", "experimental_method" ], [ 121, 128, "triloop", "structure_element" ], [ 133, 141, "hexaloop", "structure_element" ], [ 142, 144, "SL", "structure_element" ], [ 145, 149, "RNAs", "chemical" ], [ 191, 197, "Roquin", "protein" ] ] }, { "sid": 27, "sent": "SELEX identifies novel RNA ligands of Roquin-1", "section": "RESULTS", "ner": [ [ 0, 5, "SELEX", "experimental_method" ], [ 23, 26, "RNA", "chemical" ], [ 38, 46, "Roquin-1", "protein" ] ] }, { "sid": 28, "sent": "We set out to identify Roquin-bound RNA motifs in an unbiased manner by performing SELEX experiments.", "section": "RESULTS", "ner": [ [ 23, 35, "Roquin-bound", "protein_state" ], [ 36, 39, "RNA", "chemical" ], [ 83, 88, "SELEX", "experimental_method" ] ] }, { "sid": 29, "sent": "A biotinylated amino-terminal protein fragment of Roquin-1 (residues 2\u2013440) was used to enrich RNAs from a library containing 47 random nucleotides over three sequential selection rounds.", "section": "RESULTS", "ner": [ [ 2, 14, "biotinylated", "protein_state" ], [ 50, 58, "Roquin-1", "protein" ], [ 69, 74, "2\u2013440", "residue_range" ], [ 95, 99, "RNAs", "chemical" ] ] }, { "sid": 30, "sent": "Next-generation sequencing (NGS) of the RNA before and after each selection round revealed that the starting pool represented about 99.6% unique reads in \u223c4.2 \u00d7 106 sequences.", "section": "RESULTS", "ner": [ [ 0, 26, "Next-generation sequencing", "experimental_method" ], [ 28, 31, "NGS", "experimental_method" ], [ 40, 43, "RNA", "chemical" ] ] }, { "sid": 31, "sent": "Bioinformatic analysis of NGS data sets derived from the starting pool and enriched selection rounds revealed that the complexity was reduced to 78.6% unique reads in 3.7 \u00d7 106 sequences that were analysed after 3 rounds of selection and enrichment.", "section": "RESULTS", "ner": [ [ 0, 22, "Bioinformatic analysis", "experimental_method" ], [ 26, 29, "NGS", "experimental_method" ] ] }, { "sid": 32, "sent": "For NGS data analysis, the COMPAS software (AptaIT, Munich, Germany) was applied.", "section": "RESULTS", "ner": [ [ 4, 7, "NGS", "experimental_method" ] ] }, { "sid": 33, "sent": "Enriched sequences were clustered into so-called patterns with highly homologous sequences.", "section": "RESULTS", "ner": [ [ 9, 33, "sequences were clustered", "experimental_method" ] ] }, { "sid": 34, "sent": "Based on this so-called co-occurrence approach, patterns on the basis of frequent motifs were generated and were searched for prominent hexamer sequences (Supplementary Fig. 1a).", "section": "RESULTS", "ner": [ [ 24, 46, "co-occurrence approach", "experimental_method" ] ] }, { "sid": 35, "sent": "We identified 5\u2032-CGTTTT-3\u2032, 5\u2032-GCGTTT-3\u2032, 5\u2032-TGCGTT-3\u2032 and 5\u2032-GTTTTA-3\u2032 motifs that were also reconfirmed in an independent experiment (Supplementary Fig. 1a) and are located within highly similar sequences (Fig. 1a and Supplementary Fig. 1b).", "section": "RESULTS", "ner": [ [ 14, 27, "5\u2032-CGTTTT-3\u2032,", "chemical" ], [ 28, 40, "5\u2032-GCGTTT-3\u2032", "chemical" ], [ 42, 54, "5\u2032-TGCGTT-3\u2032", "chemical" ], [ 59, 71, "5\u2032-GTTTTA-3\u2032", "chemical" ] ] }, { "sid": 36, "sent": "Consistent with previous findings showing that the sanroque mutation does not impair RNA binding of Roquin, we found similarly enriched sequences in SELEX approaches using a corresponding Roquin-1 fragment harbouring the M199R mutation (Fig. 1a and Supplementary Fig. 1b).", "section": "RESULTS", "ner": [ [ 51, 68, "sanroque mutation", "mutant" ], [ 85, 88, "RNA", "chemical" ], [ 100, 106, "Roquin", "protein" ], [ 149, 154, "SELEX", "experimental_method" ], [ 188, 196, "Roquin-1", "protein" ], [ 221, 226, "M199R", "mutant" ] ] }, { "sid": 37, "sent": "Notably, our SELEX approach did not reveal the previously identified CDE sequence.", "section": "RESULTS", "ner": [ [ 13, 18, "SELEX", "experimental_method" ], [ 69, 72, "CDE", "structure_element" ] ] }, { "sid": 38, "sent": "We assume that the region of sequence identity in the CDE is too short for our sequence clustering algorithm.", "section": "RESULTS", "ner": [ [ 54, 57, "CDE", "structure_element" ], [ 79, 108, "sequence clustering algorithm", "experimental_method" ] ] }, { "sid": 39, "sent": "Evaluation of the structural context for the SELEX-derived motif suggested a putative SL formation with six unpaired nucleotides in a loop followed by a 5\u20138\u2009nt stem, with one base in the stem not being paired (Supplementary Fig. 1c).", "section": "RESULTS", "ner": [ [ 45, 50, "SELEX", "experimental_method" ], [ 86, 88, "SL", "structure_element" ], [ 134, 138, "loop", "structure_element" ], [ 160, 164, "stem", "structure_element" ], [ 187, 191, "stem", "structure_element" ] ] }, { "sid": 40, "sent": "Searching the 3\u2032-UTRs of known Roquin targets with the consensus 5\u2032-TGCGTTTTAGGA-3\u2032, obtained by Motif-based sequence analysis (MEME), revealed a homologous sequence with the potential to form a hexaloop structure in the 3\u2032-UTR of Ox40 (Fig. 1b).", "section": "RESULTS", "ner": [ [ 14, 21, "3\u2032-UTRs", "structure_element" ], [ 31, 37, "Roquin", "protein" ], [ 65, 83, "5\u2032-TGCGTTTTAGGA-3\u2032", "chemical" ], [ 97, 126, "Motif-based sequence analysis", "experimental_method" ], [ 128, 132, "MEME", "experimental_method" ], [ 195, 203, "hexaloop", "structure_element" ], [ 221, 227, "3\u2032-UTR", "structure_element" ], [ 231, 235, "Ox40", "protein" ] ] }, { "sid": 41, "sent": "Importantly, this motif is present across species in the 3\u2032-UTRs of respective mRNAs and showed highest conservation in the loop and the upper stem sequences with a drop of conservation towards the boundaries of the motif (Fig. 1c,d).", "section": "RESULTS", "ner": [ [ 57, 64, "3\u2032-UTRs", "structure_element" ], [ 79, 84, "mRNAs", "chemical" ], [ 124, 128, "loop", "structure_element" ], [ 143, 147, "stem", "structure_element" ] ] }, { "sid": 42, "sent": "The predicted SL for the consensus SELEX-derived motif (from here on referred to as alternative decay element SL, ADE SL), the ADE-like SL, is positioned 5\u2032 to another CDE-like SL in the 3\u2032-UTR of Ox40 mRNA.", "section": "RESULTS", "ner": [ [ 14, 16, "SL", "structure_element" ], [ 35, 40, "SELEX", "experimental_method" ], [ 84, 109, "alternative decay element", "structure_element" ], [ 110, 112, "SL", "structure_element" ], [ 114, 117, "ADE", "structure_element" ], [ 118, 120, "SL", "structure_element" ], [ 127, 130, "ADE", "structure_element" ], [ 136, 138, "SL", "structure_element" ], [ 168, 171, "CDE", "structure_element" ], [ 177, 179, "SL", "structure_element" ], [ 187, 193, "3\u2032-UTR", "structure_element" ], [ 197, 201, "Ox40", "protein" ], [ 202, 206, "mRNA", "chemical" ] ] }, { "sid": 43, "sent": "This CDE-like SL differs in the sequence of the upper stem from the canonical CDE from the 3\u2032-UTR of Tnf mRNA (CDE SL) (Fig. 1d).", "section": "RESULTS", "ner": [ [ 5, 8, "CDE", "structure_element" ], [ 14, 16, "SL", "structure_element" ], [ 78, 81, "CDE", "structure_element" ], [ 91, 97, "3\u2032-UTR", "structure_element" ], [ 101, 104, "Tnf", "protein" ], [ 105, 109, "mRNA", "chemical" ], [ 111, 114, "CDE", "structure_element" ], [ 115, 117, "SL", "structure_element" ] ] }, { "sid": 44, "sent": "NMR analysis of Roquin-bound SL RNAs", "section": "RESULTS", "ner": [ [ 0, 3, "NMR", "experimental_method" ], [ 16, 28, "Roquin-bound", "protein_state" ], [ 29, 31, "SL", "structure_element" ], [ 32, 36, "RNAs", "chemical" ] ] }, { "sid": 45, "sent": "We used NMR to analyse the secondary structure of Roquin-1-binding motifs derived from SELEX.", "section": "RESULTS", "ner": [ [ 8, 11, "NMR", "experimental_method" ], [ 50, 73, "Roquin-1-binding motifs", "structure_element" ], [ 87, 92, "SELEX", "experimental_method" ] ] }, { "sid": 46, "sent": "Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3\u2032-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2).", "section": "RESULTS", "ner": [ [ 0, 74, "Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy", "experimental_method" ], [ 76, 81, "NOESY", "experimental_method" ], [ 83, 86, "NMR", "experimental_method" ], [ 87, 94, "spectra", "evidence" ], [ 102, 106, "free", "protein_state" ], [ 107, 110, "RNA", "chemical" ], [ 120, 128, "bound to", "protein_state" ], [ 133, 141, "Roquin-1", "protein" ], [ 142, 145, "ROQ", "structure_element" ], [ 175, 178, "ADE", "structure_element" ], [ 179, 181, "SL", "structure_element" ], [ 187, 190, "ADE", "structure_element" ], [ 196, 198, "SL", "structure_element" ], [ 206, 212, "3\u2032-UTR", "structure_element" ], [ 216, 220, "Ox40", "protein" ], [ 251, 255, "Ox40", "protein" ], [ 256, 259, "CDE", "structure_element" ], [ 265, 267, "SL", "structure_element" ] ] }, { "sid": 47, "sent": "The NMR data of the free RNAs show that almost all predicted base pairs in the stem regions of the hexa- and triloop SL including the closing base pairs are formed in all three RNAs.", "section": "RESULTS", "ner": [ [ 4, 7, "NMR", "experimental_method" ], [ 20, 24, "free", "protein_state" ], [ 25, 29, "RNAs", "chemical" ], [ 79, 91, "stem regions", "structure_element" ], [ 99, 116, "hexa- and triloop", "structure_element" ], [ 117, 119, "SL", "structure_element" ], [ 177, 181, "RNAs", "chemical" ] ] }, { "sid": 48, "sent": "Notably, we also found an unambiguous imino proton signal for G15, but not G6, in the ADE SL, indicating a non-Watson\u2013Crick G\u2013G base pair at this position (Fig. 2a).", "section": "RESULTS", "ner": [ [ 62, 65, "G15", "residue_name_number" ], [ 75, 77, "G6", "residue_name_number" ], [ 86, 89, "ADE", "structure_element" ], [ 90, 92, "SL", "structure_element" ], [ 107, 137, "non-Watson\u2013Crick G\u2013G base pair", "bond_interaction" ] ] }, { "sid": 49, "sent": "Significant chemical shift perturbations (CSPs) are observed for imino proton signals on binding to the ROQ domain, demonstrating that formation of protein\u2013RNA complexes involves contacts of the ROQ domain to the stem region of the RNA ligands (Fig. 2, bases coloured red).", "section": "RESULTS", "ner": [ [ 12, 40, "chemical shift perturbations", "evidence" ], [ 42, 46, "CSPs", "evidence" ], [ 104, 107, "ROQ", "structure_element" ], [ 156, 159, "RNA", "chemical" ], [ 195, 198, "ROQ", "structure_element" ], [ 213, 224, "stem region", "structure_element" ], [ 232, 235, "RNA", "chemical" ] ] }, { "sid": 50, "sent": "No imino correlations are observed for the predicted Watson\u2013Crick base pairs at the bottom of the ADE SL and the Ox40 ADE-like SL RNAs, as well as for the A\u2013U base pair flanking the bulge in the Ox40 ADE-like SL RNA (Fig. 2a,b), suggesting that these base pairs are dynamic.", "section": "RESULTS", "ner": [ [ 53, 76, "Watson\u2013Crick base pairs", "bond_interaction" ], [ 98, 101, "ADE", "structure_element" ], [ 102, 104, "SL", "structure_element" ], [ 113, 117, "Ox40", "protein" ], [ 118, 121, "ADE", "structure_element" ], [ 127, 129, "SL", "structure_element" ], [ 130, 134, "RNAs", "chemical" ], [ 155, 156, "A", "residue_name" ], [ 157, 158, "U", "residue_name" ], [ 182, 187, "bulge", "structure_element" ], [ 195, 199, "Ox40", "protein" ], [ 200, 203, "ADE", "structure_element" ], [ 209, 211, "SL", "structure_element" ], [ 212, 215, "RNA", "chemical" ] ] }, { "sid": 51, "sent": "In contrast, all expected base pairs are observed for the Ox40 CDE-like SL RNA (Fig. 2c; see also Supplementary Notes).", "section": "RESULTS", "ner": [ [ 58, 62, "Ox40", "protein" ], [ 63, 66, "CDE", "structure_element" ], [ 72, 74, "SL", "structure_element" ], [ 75, 78, "RNA", "chemical" ] ] }, { "sid": 52, "sent": "Structures of ROQ bound to ADE SL RNAs", "section": "RESULTS", "ner": [ [ 0, 10, "Structures", "evidence" ], [ 14, 17, "ROQ", "structure_element" ], [ 18, 26, "bound to", "protein_state" ], [ 27, 30, "ADE", "structure_element" ], [ 31, 33, "SL", "structure_element" ], [ 34, 38, "RNAs", "chemical" ] ] }, { "sid": 53, "sent": "To elucidate how Roquin can recognize the novel SL elements identified in the SELEX approach, we solved crystal structures of the Roquin-1 ROQ domain bound to these non-canonical RNA elements.", "section": "RESULTS", "ner": [ [ 17, 23, "Roquin", "protein" ], [ 48, 50, "SL", "structure_element" ], [ 78, 83, "SELEX", "experimental_method" ], [ 97, 103, "solved", "experimental_method" ], [ 104, 122, "crystal structures", "evidence" ], [ 130, 138, "Roquin-1", "protein" ], [ 139, 142, "ROQ", "structure_element" ], [ 150, 158, "bound to", "protein_state" ], [ 179, 182, "RNA", "chemical" ] ] }, { "sid": 54, "sent": "The structures of ROQ bound to the 20-mer ADE SL (Supplementary Fig. 2a) and to the 22-mer Ox40 ADE-like SL RNAs (Fig. 3a) were refined to a resolution of 3.0 and 2.2\u2009\u00c5, respectively.", "section": "RESULTS", "ner": [ [ 4, 14, "structures", "evidence" ], [ 18, 21, "ROQ", "structure_element" ], [ 22, 30, "bound to", "protein_state" ], [ 42, 45, "ADE", "structure_element" ], [ 46, 48, "SL", "structure_element" ], [ 91, 95, "Ox40", "protein" ], [ 96, 99, "ADE", "structure_element" ], [ 105, 107, "SL", "structure_element" ], [ 108, 112, "RNAs", "chemical" ] ] }, { "sid": 55, "sent": "In both structures the RNA adopts an SL fold, where the hexaloop is located in the vicinity of the carboxy-terminal end of ROQ helix \u03b14 and the N-terminal part of \u03b23 (Fig. 3a,b and Supplementary Fig. 2a,b).", "section": "RESULTS", "ner": [ [ 8, 18, "structures", "evidence" ], [ 23, 26, "RNA", "chemical" ], [ 37, 39, "SL", "structure_element" ], [ 56, 64, "hexaloop", "structure_element" ], [ 123, 126, "ROQ", "structure_element" ], [ 127, 132, "helix", "structure_element" ], [ 133, 135, "\u03b14", "structure_element" ], [ 163, 165, "\u03b23", "structure_element" ] ] }, { "sid": 56, "sent": "The dsRNA stem is recognized in the same way as previously reported for the Tnf CDE SL RNA (Supplementary Fig. 2c\u2013e).", "section": "RESULTS", "ner": [ [ 4, 9, "dsRNA", "chemical" ], [ 10, 14, "stem", "structure_element" ], [ 76, 79, "Tnf", "protein" ], [ 80, 83, "CDE", "structure_element" ], [ 84, 86, "SL", "structure_element" ], [ 87, 90, "RNA", "chemical" ] ] }, { "sid": 57, "sent": "As may be expected, the recognition of the hexaloop is significantly different from the triloop in the CDE RNA (Fig. 3b,c and Supplementary Fig. 2b).", "section": "RESULTS", "ner": [ [ 43, 51, "hexaloop", "structure_element" ], [ 88, 95, "triloop", "structure_element" ], [ 103, 106, "CDE", "structure_element" ], [ 107, 110, "RNA", "chemical" ] ] }, { "sid": 58, "sent": "Interestingly, although the sequences of the ADE SL and ADE-like SL RNAs are different, the overall structures and protein\u2013RNA contacts are virtually identical (Supplementary Fig. 2a,d,e).", "section": "RESULTS", "ner": [ [ 45, 48, "ADE", "structure_element" ], [ 49, 51, "SL", "structure_element" ], [ 56, 59, "ADE", "structure_element" ], [ 65, 67, "SL", "structure_element" ], [ 68, 72, "RNAs", "chemical" ], [ 100, 110, "structures", "evidence" ], [ 123, 126, "RNA", "chemical" ] ] }, { "sid": 59, "sent": "The only differences are a C19 bulge, the non-Watson\u2013Crick G6\u2013G15 base pair and the interaction of U1 with Trp184 and Phe194 in the ADE-like SL RNA (Supplementary Fig. 2a,e\u2013g).", "section": "RESULTS", "ner": [ [ 27, 30, "C19", "residue_name_number" ], [ 31, 36, "bulge", "structure_element" ], [ 42, 58, "non-Watson\u2013Crick", "bond_interaction" ], [ 59, 61, "G6", "residue_name_number" ], [ 62, 65, "G15", "residue_name_number" ], [ 66, 75, "base pair", "bond_interaction" ], [ 99, 101, "U1", "residue_name_number" ], [ 107, 113, "Trp184", "residue_name_number" ], [ 118, 124, "Phe194", "residue_name_number" ], [ 132, 135, "ADE", "structure_element" ], [ 141, 143, "SL", "structure_element" ], [ 144, 147, "RNA", "chemical" ] ] }, { "sid": 60, "sent": "Given their highly similar binding modes we focus the following discussion on the structure of the Ox40 ADE-like SL RNA, as it naturally exists in the Ox40 3\u2032-UTR and was solved at higher resolution.", "section": "RESULTS", "ner": [ [ 82, 91, "structure", "evidence" ], [ 99, 103, "Ox40", "protein" ], [ 104, 107, "ADE", "structure_element" ], [ 113, 115, "SL", "structure_element" ], [ 116, 119, "RNA", "chemical" ], [ 151, 155, "Ox40", "protein" ], [ 156, 162, "3\u2032-UTR", "structure_element" ] ] }, { "sid": 61, "sent": "The overall orientation and recognition of the double-stranded stem in the Ox40 ADE-like SL is similar to the CDE triloop.", "section": "RESULTS", "ner": [ [ 47, 67, "double-stranded stem", "structure_element" ], [ 75, 79, "Ox40", "protein" ], [ 80, 83, "ADE", "structure_element" ], [ 89, 91, "SL", "structure_element" ], [ 110, 113, "CDE", "structure_element" ], [ 114, 121, "triloop", "structure_element" ] ] }, { "sid": 62, "sent": "Notably, the U-rich hexaloop in the Ox40 ADE-like SL RNA binds to an extended surface on the ROQ domain that cannot be accessed by the CDE triloop (Fig. 3b,c) and includes a few pyrimidine-specific contacts.", "section": "RESULTS", "ner": [ [ 13, 28, "U-rich hexaloop", "structure_element" ], [ 36, 40, "Ox40", "protein" ], [ 41, 44, "ADE", "structure_element" ], [ 50, 52, "SL", "structure_element" ], [ 53, 56, "RNA", "chemical" ], [ 78, 85, "surface", "site" ], [ 93, 96, "ROQ", "structure_element" ], [ 135, 138, "CDE", "structure_element" ], [ 139, 146, "triloop", "structure_element" ] ] }, { "sid": 63, "sent": "For example, the main chain atoms of Phe255 form two hydrogen bonds with the Watson\u2013Crick face of the U11 base (Fig. 3d).", "section": "RESULTS", "ner": [ [ 37, 43, "Phe255", "residue_name_number" ], [ 53, 67, "hydrogen bonds", "bond_interaction" ], [ 102, 105, "U11", "residue_name_number" ] ] }, { "sid": 64, "sent": "Although in the structure of the Tnf CDE triloop the Tyr250 side chain engages only one hydrogen bond to the phosphate group of G12 (ref.), a number of contacts are observed with the hexaloop (Fig. 3d\u2013f): the side chain hydroxyl of Tyr250 contacts the phosphate group of U11, while the aromatic ring is positioned by parallel and orthogonal stacking interactions with the U10 and U11 bases, on either side, respectively (Fig. 3e).", "section": "RESULTS", "ner": [ [ 16, 25, "structure", "evidence" ], [ 33, 36, "Tnf", "protein" ], [ 37, 40, "CDE", "structure_element" ], [ 41, 48, "triloop", "structure_element" ], [ 53, 59, "Tyr250", "residue_name_number" ], [ 88, 101, "hydrogen bond", "bond_interaction" ], [ 128, 131, "G12", "residue_name_number" ], [ 183, 191, "hexaloop", "structure_element" ], [ 232, 238, "Tyr250", "residue_name_number" ], [ 271, 274, "U11", "residue_name_number" ], [ 341, 362, "stacking interactions", "bond_interaction" ], [ 372, 375, "U10", "residue_name_number" ], [ 380, 383, "U11", "residue_name_number" ] ] }, { "sid": 65, "sent": "In addition, the Tyr250 main-chain carbonyl interacts with U13 imino proton (Fig. 3d,e).", "section": "RESULTS", "ner": [ [ 17, 23, "Tyr250", "residue_name_number" ], [ 59, 62, "U13", "residue_name_number" ] ] }, { "sid": 66, "sent": "Val257 and Lys259 in strand \u03b23 are too far to contact the UGU triloop in the Tnf CDE RNA, but mediate a number of contacts with the longer hexaloop.", "section": "RESULTS", "ner": [ [ 0, 6, "Val257", "residue_name_number" ], [ 11, 17, "Lys259", "residue_name_number" ], [ 21, 27, "strand", "structure_element" ], [ 28, 30, "\u03b23", "structure_element" ], [ 58, 61, "UGU", "structure_element" ], [ 62, 69, "triloop", "structure_element" ], [ 77, 80, "Tnf", "protein" ], [ 81, 84, "CDE", "structure_element" ], [ 85, 88, "RNA", "chemical" ], [ 139, 147, "hexaloop", "structure_element" ] ] }, { "sid": 67, "sent": "The side chain of Lys259 forms hydrogen bonds with the phosphate groups of U10 and U11 (Fig. 3e,f) and the hydrophobic side chain of Val257 stacks with the U11 base (Fig. 3d,f).", "section": "RESULTS", "ner": [ [ 18, 24, "Lys259", "residue_name_number" ], [ 31, 45, "hydrogen bonds", "bond_interaction" ], [ 75, 78, "U10", "residue_name_number" ], [ 83, 86, "U11", "residue_name_number" ], [ 133, 139, "Val257", "residue_name_number" ], [ 140, 146, "stacks", "bond_interaction" ], [ 156, 159, "U11", "residue_name_number" ] ] }, { "sid": 68, "sent": "The RNA stem is closed by a Watson\u2013Crick base pair (C8\u2013G15 in the hexaloop SL RNA).", "section": "RESULTS", "ner": [ [ 4, 7, "RNA", "chemical" ], [ 8, 12, "stem", "structure_element" ], [ 28, 50, "Watson\u2013Crick base pair", "bond_interaction" ], [ 52, 54, "C8", "residue_name_number" ], [ 55, 58, "G15", "residue_name_number" ], [ 66, 74, "hexaloop", "structure_element" ], [ 75, 77, "SL", "structure_element" ], [ 78, 81, "RNA", "chemical" ] ] }, { "sid": 69, "sent": "Interestingly, the G9 base stacks on top of this closing base pair and takes a position that is very similar to the purine base of G12 in the CDE triloop (Fig. 3b,c and Supplementary Fig. 2b).", "section": "RESULTS", "ner": [ [ 19, 21, "G9", "residue_name_number" ], [ 27, 33, "stacks", "bond_interaction" ], [ 131, 134, "G12", "residue_name_number" ], [ 142, 145, "CDE", "structure_element" ], [ 146, 153, "triloop", "structure_element" ] ] }, { "sid": 70, "sent": "The G9 base does not form a base pair with A14 but rather the A14 base packs into the minor groove of the RNA duplex.", "section": "RESULTS", "ner": [ [ 4, 6, "G9", "residue_name_number" ], [ 43, 46, "A14", "residue_name_number" ], [ 62, 65, "A14", "residue_name_number" ], [ 86, 98, "minor groove", "site" ], [ 106, 109, "RNA", "chemical" ] ] }, { "sid": 71, "sent": "This arrangement provides an extended stacking interaction of G9, U10 and Tyr250 in the ROQ domain at the 5\u2032-side of the RNA stem (Fig. 3e).", "section": "RESULTS", "ner": [ [ 38, 58, "stacking interaction", "bond_interaction" ], [ 62, 64, "G9", "residue_name_number" ], [ 66, 69, "U10", "residue_name_number" ], [ 74, 80, "Tyr250", "residue_name_number" ], [ 88, 91, "ROQ", "structure_element" ], [ 121, 124, "RNA", "chemical" ], [ 125, 129, "stem", "structure_element" ] ] }, { "sid": 72, "sent": "The U11 and U13 bases stack with each other in the vicinity of the ROQ domain wing (Fig. 3b,d,f).", "section": "RESULTS", "ner": [ [ 4, 7, "U11", "residue_name_number" ], [ 12, 15, "U13", "residue_name_number" ], [ 22, 27, "stack", "bond_interaction" ], [ 67, 70, "ROQ", "structure_element" ], [ 78, 82, "wing", "structure_element" ] ] }, { "sid": 73, "sent": "This is possible by exposing the base C12 of the Ox-40 ADE-like SL towards the solvent, which accordingly does not show any contacts to the protein.", "section": "RESULTS", "ner": [ [ 38, 41, "C12", "residue_name_number" ], [ 49, 54, "Ox-40", "protein" ], [ 55, 58, "ADE", "structure_element" ], [ 64, 66, "SL", "structure_element" ] ] }, { "sid": 74, "sent": "In summary, similar to the CDE SL, both the ADE SL and ADE-like SL RNAs are recognized mainly by non-sequence-specific contacts.", "section": "RESULTS", "ner": [ [ 27, 30, "CDE", "structure_element" ], [ 31, 33, "SL", "structure_element" ], [ 44, 47, "ADE", "structure_element" ], [ 48, 50, "SL", "structure_element" ], [ 55, 58, "ADE", "structure_element" ], [ 64, 66, "SL", "structure_element" ], [ 67, 71, "RNAs", "chemical" ] ] }, { "sid": 75, "sent": "However, these involve an extended binding surface on the ROQ domain with a number of additional residues compared with the triloop RNA.", "section": "RESULTS", "ner": [ [ 58, 61, "ROQ", "structure_element" ], [ 132, 135, "RNA", "chemical" ] ] }, { "sid": 76, "sent": "NMR analysis of ROQ interactions with ADE SLs", "section": "RESULTS", "ner": [ [ 0, 3, "NMR", "experimental_method" ], [ 16, 19, "ROQ", "structure_element" ], [ 38, 41, "ADE", "structure_element" ], [ 42, 45, "SLs", "structure_element" ] ] }, { "sid": 77, "sent": "We next used NMR spectroscopy to compare the ROQ domain interaction of ADE-like and CDE-like SL RNAs in solution.", "section": "RESULTS", "ner": [ [ 13, 29, "NMR spectroscopy", "experimental_method" ], [ 45, 48, "ROQ", "structure_element" ], [ 71, 74, "ADE", "structure_element" ], [ 84, 87, "CDE", "structure_element" ], [ 93, 95, "SL", "structure_element" ], [ 96, 100, "RNAs", "chemical" ] ] }, { "sid": 78, "sent": "CSPs observed for amides in the ROQ domain on binding to the Ox40 ADE-like SL RNA (Fig. 4a,b) map to residues that also mediate key interactions with CDE SLs, such as Lys220, Lys239/Thr240 and Lys259/Arg260 (Fig. 4b).", "section": "RESULTS", "ner": [ [ 0, 4, "CSPs", "evidence" ], [ 32, 35, "ROQ", "structure_element" ], [ 61, 65, "Ox40", "protein" ], [ 66, 69, "ADE", "structure_element" ], [ 75, 77, "SL", "structure_element" ], [ 78, 81, "RNA", "chemical" ], [ 150, 153, "CDE", "structure_element" ], [ 154, 157, "SLs", "structure_element" ], [ 167, 173, "Lys220", "residue_name_number" ], [ 175, 181, "Lys239", "residue_name_number" ], [ 182, 188, "Thr240", "residue_name_number" ], [ 193, 199, "Lys259", "residue_name_number" ], [ 200, 206, "Arg260", "residue_name_number" ] ] }, { "sid": 79, "sent": "This is fully consistent with the interactions observed in the crystal structure (Supplementary Fig. 2c\u2013e) and indicates a similar binding surface.", "section": "RESULTS", "ner": [ [ 63, 80, "crystal structure", "evidence" ], [ 131, 146, "binding surface", "site" ] ] }, { "sid": 80, "sent": "However, there are also notable CSP differences when comparing binding of the ROQ domain to Ox40 ADE-like SL RNAs and to the CDE-like SL RNA in the Ox40 3\u2032-UTR (Fig. 4c), or to the Tnf CDE SL RNA (Supplementary Fig. 3 and Supplementary Notes).", "section": "RESULTS", "ner": [ [ 32, 47, "CSP differences", "evidence" ], [ 78, 81, "ROQ", "structure_element" ], [ 92, 96, "Ox40", "protein" ], [ 97, 100, "ADE", "structure_element" ], [ 106, 108, "SL", "structure_element" ], [ 109, 113, "RNAs", "chemical" ], [ 125, 128, "CDE", "structure_element" ], [ 134, 136, "SL", "structure_element" ], [ 137, 140, "RNA", "chemical" ], [ 148, 152, "Ox40", "protein" ], [ 153, 159, "3\u2032-UTR", "structure_element" ], [ 181, 184, "Tnf", "protein" ], [ 185, 188, "CDE", "structure_element" ], [ 189, 191, "SL", "structure_element" ], [ 192, 195, "RNA", "chemical" ] ] }, { "sid": 81, "sent": "For example, Ser253 is strongly affected only on binding to the Ox40 ADE-like SL (Fig. 4a,b) in line with tight interactions with the hexaloop (Fig. 3d).", "section": "RESULTS", "ner": [ [ 13, 19, "Ser253", "residue_name_number" ], [ 64, 68, "Ox40", "protein" ], [ 69, 72, "ADE", "structure_element" ], [ 78, 80, "SL", "structure_element" ], [ 134, 142, "hexaloop", "structure_element" ] ] }, { "sid": 82, "sent": "On the other hand, comparison of ROQ domain binding with the ADE and with the ADE-like SL RNAs indicates almost identical NMR spectra and CSPs.", "section": "RESULTS", "ner": [ [ 33, 36, "ROQ", "structure_element" ], [ 61, 64, "ADE", "structure_element" ], [ 78, 81, "ADE", "structure_element" ], [ 87, 89, "SL", "structure_element" ], [ 90, 94, "RNAs", "chemical" ], [ 122, 125, "NMR", "experimental_method" ], [ 126, 133, "spectra", "evidence" ], [ 138, 142, "CSPs", "evidence" ] ] }, { "sid": 83, "sent": "This is consistent with the very similar structural features and mode of RNA recognition of the ROQ domain with these RNAs (Supplementary Fig. 2a,d,e).", "section": "RESULTS", "ner": [ [ 73, 76, "RNA", "chemical" ], [ 96, 99, "ROQ", "structure_element" ], [ 118, 122, "RNAs", "chemical" ] ] }, { "sid": 84, "sent": "Mutational analysis of the ROQ-ADE interaction", "section": "RESULTS", "ner": [ [ 0, 19, "Mutational analysis", "experimental_method" ], [ 27, 30, "ROQ", "structure_element" ], [ 31, 34, "ADE", "structure_element" ] ] }, { "sid": 85, "sent": "To examine the individual contributions of ROQ\u2013hexaloop interactions for complex formation, we performed electrophoretic mobility shift assays (EMSAs) with variants of the ROQ domain and the Ox40 ADE-like RNA (Fig. 5a and Supplementary Fig. 4).", "section": "RESULTS", "ner": [ [ 43, 46, "ROQ", "structure_element" ], [ 105, 142, "electrophoretic mobility shift assays", "experimental_method" ], [ 144, 149, "EMSAs", "experimental_method" ], [ 172, 175, "ROQ", "structure_element" ], [ 191, 195, "Ox40", "protein" ], [ 196, 199, "ADE", "structure_element" ], [ 205, 208, "RNA", "chemical" ] ] }, { "sid": 86, "sent": "Analysis of the interaction with wild-type ROQ revealed an apparent affinity in a similar range as for the Tnf CDE (Fig. 5a and ) Table 2).", "section": "RESULTS", "ner": [ [ 33, 42, "wild-type", "protein_state" ], [ 43, 46, "ROQ", "structure_element" ], [ 68, 76, "affinity", "evidence" ], [ 107, 110, "Tnf", "protein" ], [ 111, 114, "CDE", "structure_element" ] ] }, { "sid": 87, "sent": "We next tested a set of mutants (Supplementary Fig. 4), which were designed based on contacts observed in the crystal structure (Fig. 3) and the NMR CSPs (Fig. 4a,b).", "section": "RESULTS", "ner": [ [ 110, 127, "crystal structure", "evidence" ], [ 145, 148, "NMR", "experimental_method" ], [ 149, 153, "CSPs", "evidence" ] ] }, { "sid": 88, "sent": "In line with expectations from ROQ-Tnf CDE binding (see comparison in Supplementary Fig. 4) and based on our structural analysis, the key residues Lys220, Lys239, Lys259 and Arg260 strongly reduce or abolish binding after replacement by alanine.", "section": "RESULTS", "ner": [ [ 31, 42, "ROQ-Tnf CDE", "complex_assembly" ], [ 109, 128, "structural analysis", "experimental_method" ], [ 147, 153, "Lys220", "residue_name_number" ], [ 155, 161, "Lys239", "residue_name_number" ], [ 163, 169, "Lys259", "residue_name_number" ], [ 174, 180, "Arg260", "residue_name_number" ], [ 222, 233, "replacement", "experimental_method" ], [ 237, 244, "alanine", "residue_name" ] ] }, { "sid": 89, "sent": "We also observe an almost complete loss of binding in the Y250A mutant to the hexaloop SL RNA, which had not been seen for the Tnf CDE previously (Fig. 5a).", "section": "RESULTS", "ner": [ [ 58, 63, "Y250A", "mutant" ], [ 64, 70, "mutant", "protein_state" ], [ 78, 86, "hexaloop", "structure_element" ], [ 87, 89, "SL", "structure_element" ], [ 90, 93, "RNA", "chemical" ], [ 127, 130, "Tnf", "protein" ], [ 131, 134, "CDE", "structure_element" ] ] }, { "sid": 90, "sent": "This underlines the central role of Tyr250 for stabilization of the hexaloop structure and recognition by stacking interactions (Fig. 3b,e).", "section": "RESULTS", "ner": [ [ 36, 42, "Tyr250", "residue_name_number" ], [ 68, 76, "hexaloop", "structure_element" ], [ 106, 127, "stacking interactions", "bond_interaction" ] ] }, { "sid": 91, "sent": "Mutation of Ser253, which shows large CSPs in the NMR titrations (Fig. 4a,b), does not significantly impair complex formation (Supplementary Fig. 4).", "section": "RESULTS", "ner": [ [ 0, 8, "Mutation", "experimental_method" ], [ 12, 18, "Ser253", "residue_name_number" ], [ 38, 42, "CSPs", "evidence" ], [ 50, 64, "NMR titrations", "experimental_method" ] ] }, { "sid": 92, "sent": "The large chemical shift change is probably caused by ring current effects induced by the close proximity of the U11 and U13 bases.", "section": "RESULTS", "ner": [ [ 10, 31, "chemical shift change", "evidence" ], [ 113, 116, "U11", "residue_name_number" ], [ 121, 124, "U13", "residue_name_number" ] ] }, { "sid": 93, "sent": "Finally, a mutant in the wing of the ROQ domain (S265Y) does only slightly impair binding, as has been previously observed for the interaction with the Tnf CDE (Supplementary Fig. 4).", "section": "RESULTS", "ner": [ [ 11, 17, "mutant", "protein_state" ], [ 25, 29, "wing", "structure_element" ], [ 37, 40, "ROQ", "structure_element" ], [ 49, 54, "S265Y", "mutant" ], [ 152, 155, "Tnf", "protein" ], [ 156, 159, "CDE", "structure_element" ] ] }, { "sid": 94, "sent": "This indicates that replacement by Tyr does not strongly affect the RNA interaction, and that some conformational variations are tolerated.", "section": "RESULTS", "ner": [ [ 20, 31, "replacement", "experimental_method" ], [ 35, 38, "Tyr", "residue_name" ], [ 68, 71, "RNA", "chemical" ] ] }, { "sid": 95, "sent": "Thus, the mutational analysis is fully consistent with the recognition of the hexaloop observed in our crystal structures.", "section": "RESULTS", "ner": [ [ 10, 29, "mutational analysis", "experimental_method" ], [ 78, 86, "hexaloop", "structure_element" ], [ 103, 121, "crystal structures", "evidence" ] ] }, { "sid": 96, "sent": "To prove the contribution of the key residue Tyr250 in Roquin-1 to Ox40 mRNA recognition and regulation, we set up a retroviral reconstitution system in Roquin-deficient CD4+ T cells.", "section": "RESULTS", "ner": [ [ 45, 51, "Tyr250", "residue_name_number" ], [ 55, 63, "Roquin-1", "protein" ], [ 67, 71, "Ox40", "protein" ], [ 72, 76, "mRNA", "chemical" ], [ 117, 149, "retroviral reconstitution system", "experimental_method" ], [ 153, 159, "Roquin", "protein" ] ] }, { "sid": 97, "sent": "Isolated CD4+ T cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice harbouring floxed Roquin-1/2 encoding alleles, a tamoxifen-inducible Cre recombinase and the reverse tetracycline-controlled transactivator rtTA were treated in vitro with 4-hydroxy tamoxifen, to induce deletion.", "section": "RESULTS", "ner": [ [ 27, 32, "Rc3h1", "gene" ], [ 33, 36, "2fl", "gene" ], [ 37, 39, "fl", "gene" ], [ 60, 64, "mice", "taxonomy_domain" ], [ 83, 91, "Roquin-1", "protein" ], [ 92, 93, "2", "protein" ], [ 114, 123, "tamoxifen", "chemical" ], [ 158, 204, "reverse tetracycline-controlled transactivator", "protein_type" ], [ 205, 209, "rtTA", "protein" ], [ 237, 256, "4-hydroxy tamoxifen", "chemical" ] ] }, { "sid": 98, "sent": "The cells were then transduced with doxycycline-inducible retroviral vectors to reconstitute Roquin-1 expression (Fig. 5b).", "section": "RESULTS", "ner": [ [ 36, 47, "doxycycline", "chemical" ], [ 93, 101, "Roquin-1", "protein" ] ] }, { "sid": 99, "sent": "Depletion of Roquin proteins on tamoxifen treatment (Supplementary Fig. 5a) strongly increased surface expression of Ox40 and Icos (Fig. 5c).", "section": "RESULTS", "ner": [ [ 13, 19, "Roquin", "protein" ], [ 32, 41, "tamoxifen", "chemical" ], [ 117, 121, "Ox40", "protein" ], [ 126, 130, "Icos", "protein" ] ] }, { "sid": 100, "sent": "This increase in surface expression of both costimulatory receptors was partially corrected by the doxycycline-induced reconstitution with Roquin-1 WT protein (Fig. 5c left panels).", "section": "RESULTS", "ner": [ [ 44, 67, "costimulatory receptors", "protein_type" ], [ 99, 110, "doxycycline", "chemical" ], [ 139, 147, "Roquin-1", "protein" ], [ 148, 150, "WT", "protein_state" ] ] }, { "sid": 101, "sent": "Importantly, no effect was observed on expression of the Y250A mutant of Roquin-1 or the K220A, K239A and R260 mutant, which is strongly impaired in CDE SL interactions (Fig. 5c middle and right panels).", "section": "RESULTS", "ner": [ [ 57, 62, "Y250A", "mutant" ], [ 63, 69, "mutant", "protein_state" ], [ 73, 81, "Roquin-1", "protein" ], [ 89, 94, "K220A", "mutant" ], [ 96, 101, "K239A", "mutant" ], [ 106, 110, "R260", "mutant" ], [ 111, 117, "mutant", "protein_state" ], [ 149, 152, "CDE", "structure_element" ], [ 153, 155, "SL", "structure_element" ] ] }, { "sid": 102, "sent": "However, it is also possible that continuous overexpression of targets following Roquin deletion induces a hyperactivated state in the T cells.", "section": "RESULTS", "ner": [ [ 45, 59, "overexpression", "experimental_method" ], [ 81, 87, "Roquin", "protein" ] ] }, { "sid": 103, "sent": "This hyperactivation, compared with the actual posttranscriptional derepression, may contribute even stronger to the increased Icos and Ox40 expression levels.", "section": "RESULTS", "ner": [ [ 127, 131, "Icos", "protein" ], [ 136, 140, "Ox40", "protein" ] ] }, { "sid": 104, "sent": "Hence, our structure\u2013function analyses conclusively show that the Y250 residue is essential for Roquin interaction and regulation of Ox40, and potentially also for other Roquin targets such as Icos.", "section": "RESULTS", "ner": [ [ 11, 38, "structure\u2013function analyses", "experimental_method" ], [ 66, 70, "Y250", "residue_name_number" ], [ 96, 102, "Roquin", "protein" ], [ 133, 137, "Ox40", "protein" ], [ 170, 176, "Roquin", "protein" ], [ 193, 197, "Icos", "protein" ] ] }, { "sid": 105, "sent": "We also investigated the role of individual nucleotides in the Ox40 ADE-like SL for complex formation with the ROQ domain.", "section": "RESULTS", "ner": [ [ 63, 67, "Ox40", "protein" ], [ 68, 71, "ADE", "structure_element" ], [ 77, 79, "SL", "structure_element" ], [ 111, 114, "ROQ", "structure_element" ] ] }, { "sid": 106, "sent": "We designed four mutants (Mut1\u20134, see Supplementary Fig. 6) that were expected to disrupt key interactions with the protein according to our co-crystal structure (Fig. 3d\u2013f and Supplementary Fig. 2).", "section": "RESULTS", "ner": [ [ 141, 161, "co-crystal structure", "evidence" ] ] }, { "sid": 107, "sent": "NMR analysis confirmed that all mutant RNAs formed the same base pairs in the stem region, identical to the wild-type ADE-like SL (Fig. 2b and Supplementary Fig. 6).", "section": "RESULTS", "ner": [ [ 0, 3, "NMR", "experimental_method" ], [ 32, 38, "mutant", "protein_state" ], [ 39, 43, "RNAs", "chemical" ], [ 78, 89, "stem region", "structure_element" ], [ 108, 117, "wild-type", "protein_state" ], [ 118, 121, "ADE", "structure_element" ], [ 127, 129, "SL", "structure_element" ] ] }, { "sid": 108, "sent": "We next used surface plasmon resonance experiments to determine dissociation constants for the ROQ-RNA interaction (Table 2 and Supplementary Fig. 7).", "section": "RESULTS", "ner": [ [ 13, 38, "surface plasmon resonance", "experimental_method" ], [ 64, 86, "dissociation constants", "evidence" ], [ 95, 98, "ROQ", "structure_element" ], [ 99, 102, "RNA", "chemical" ] ] }, { "sid": 109, "sent": "Although the replacement of a C8\u2013G15 closing base pair by A-U (Mut 4) only reduces the affinity threefold, reduction of loop size in the A14C mutant (Mut 1, see Table 2) reduces the affinity and binding is not detected by surface plasmon resonance.", "section": "RESULTS", "ner": [ [ 13, 24, "replacement", "experimental_method" ], [ 30, 32, "C8", "residue_name_number" ], [ 33, 36, "G15", "residue_name_number" ], [ 58, 59, "A", "residue_name" ], [ 60, 61, "U", "residue_name" ], [ 63, 68, "Mut 4", "mutant" ], [ 87, 95, "affinity", "evidence" ], [ 120, 124, "loop", "structure_element" ], [ 137, 141, "A14C", "mutant" ], [ 142, 148, "mutant", "protein_state" ], [ 150, 155, "Mut 1", "mutant" ], [ 182, 190, "affinity", "evidence" ], [ 222, 247, "surface plasmon resonance", "experimental_method" ] ] }, { "sid": 110, "sent": "As intended, the mutation Mut 1 allows the formation of an additional base pair and thus leads to the formation of a tetraloop with a new G-C closing base pair (Supplementary Fig. 6a).", "section": "RESULTS", "ner": [ [ 26, 31, "Mut 1", "mutant" ], [ 117, 126, "tetraloop", "structure_element" ], [ 138, 139, "G", "residue_name" ], [ 140, 141, "C", "residue_name" ] ] }, { "sid": 111, "sent": "Consistent with the structural analysis, we assume that this variant alters the hexaloop conformation and thus reduces the interaction with ROQ.", "section": "RESULTS", "ner": [ [ 20, 39, "structural analysis", "experimental_method" ], [ 80, 88, "hexaloop", "structure_element" ], [ 140, 143, "ROQ", "structure_element" ] ] }, { "sid": 112, "sent": "Disruption of stacking interactions between G15, G9 and Y250 in the G9C mutant (Mut 2) completely abolished binding of ROQ to the SL RNA (Table 2 and Supplementary Fig. 7).", "section": "RESULTS", "ner": [ [ 14, 35, "stacking interactions", "bond_interaction" ], [ 44, 47, "G15", "residue_name_number" ], [ 49, 51, "G9", "residue_name_number" ], [ 56, 60, "Y250", "residue_name_number" ], [ 68, 71, "G9C", "mutant" ], [ 72, 78, "mutant", "protein_state" ], [ 80, 85, "Mut 2", "mutant" ], [ 119, 122, "ROQ", "structure_element" ], [ 130, 132, "SL", "structure_element" ], [ 133, 136, "RNA", "chemical" ] ] }, { "sid": 113, "sent": "No binding is also observed for the U11AU13G double mutant (Mut 3) (Table 2 and Supplementary Fig. 7), which abolishes specific interactions mediated by U11 and U13 in the hexaloop with ROQ (Fig. 3d).", "section": "RESULTS", "ner": [ [ 36, 44, "U11AU13G", "mutant" ], [ 45, 58, "double mutant", "protein_state" ], [ 60, 65, "Mut 3", "mutant" ], [ 153, 156, "U11", "residue_name_number" ], [ 161, 164, "U13", "residue_name_number" ], [ 172, 180, "hexaloop", "structure_element" ], [ 186, 189, "ROQ", "structure_element" ] ] }, { "sid": 114, "sent": "Consistent with the SELEX consensus (Fig. 1b), all of the tested mutations of conserved nucleotides in the loop reduce or abolish the interaction with ROQ.", "section": "RESULTS", "ner": [ [ 20, 25, "SELEX", "experimental_method" ], [ 65, 74, "mutations", "experimental_method" ], [ 78, 87, "conserved", "protein_state" ], [ 88, 99, "nucleotides", "chemical" ], [ 107, 111, "loop", "structure_element" ], [ 151, 154, "ROQ", "structure_element" ] ] }, { "sid": 115, "sent": "Interestingly, the affinity of the wild-type Tnf CDE and the Ox40 ADE-like SLs to ROQ are very similar (42 and 81\u2009nM, respectively, Table 2 and Supplementary Fig. 7).", "section": "RESULTS", "ner": [ [ 19, 27, "affinity", "evidence" ], [ 35, 44, "wild-type", "protein_state" ], [ 45, 48, "Tnf", "protein" ], [ 49, 52, "CDE", "structure_element" ], [ 61, 65, "Ox40", "protein" ], [ 66, 69, "ADE", "structure_element" ], [ 75, 78, "SLs", "structure_element" ], [ 82, 85, "ROQ", "structure_element" ] ] }, { "sid": 116, "sent": "Roquin binding to different SLs in the Ox40 3\u2032-UTR", "section": "RESULTS", "ner": [ [ 0, 6, "Roquin", "protein" ], [ 28, 31, "SLs", "structure_element" ], [ 39, 43, "Ox40", "protein" ], [ 44, 50, "3\u2032-UTR", "structure_element" ] ] }, { "sid": 117, "sent": "We have recently shown that Roquin-1 binds to a CDE-like motif in the 3\u2032-UTR of Ox40 mRNA (Figs 1d and 4c).", "section": "RESULTS", "ner": [ [ 28, 36, "Roquin-1", "protein" ], [ 48, 51, "CDE", "structure_element" ], [ 70, 76, "3\u2032-UTR", "structure_element" ], [ 80, 84, "Ox40", "protein" ], [ 85, 89, "mRNA", "chemical" ] ] }, { "sid": 118, "sent": "We therefore investigated whether the interactions with the CDE-like and the ADE-like SL RNAs both contribute to Roquin-1 binding in the context of the full-length Ox40 3\u2032-UTR.", "section": "RESULTS", "ner": [ [ 60, 63, "CDE", "structure_element" ], [ 77, 80, "ADE", "structure_element" ], [ 86, 88, "SL", "structure_element" ], [ 89, 93, "RNAs", "chemical" ], [ 113, 121, "Roquin-1", "protein" ], [ 152, 163, "full-length", "protein_state" ], [ 164, 168, "Ox40", "protein" ], [ 169, 175, "3\u2032-UTR", "structure_element" ] ] }, { "sid": 119, "sent": "The binding affinities of either motif for the N-terminal domain of Roquin-1 (residues 2\u2013440) (Supplementary Fig. 8a,b) or the ROQ domain alone are in a similar range (Table 2).", "section": "RESULTS", "ner": [ [ 4, 22, "binding affinities", "evidence" ], [ 47, 64, "N-terminal domain", "structure_element" ], [ 68, 76, "Roquin-1", "protein" ], [ 87, 92, "2\u2013440", "residue_range" ], [ 127, 130, "ROQ", "structure_element" ], [ 138, 143, "alone", "protein_state" ] ] }, { "sid": 120, "sent": "The dissociation constants for the ROQ interaction with the Ox40 CDE-like SL and the ADE-like SL RNAs are 1,460 and 81\u2009nM, respectively (Table 2).", "section": "RESULTS", "ner": [ [ 4, 26, "dissociation constants", "evidence" ], [ 35, 38, "ROQ", "structure_element" ], [ 60, 64, "Ox40", "protein" ], [ 65, 68, "CDE", "structure_element" ], [ 74, 76, "SL", "structure_element" ], [ 85, 88, "ADE", "structure_element" ], [ 94, 96, "SL", "structure_element" ], [ 97, 101, "RNAs", "chemical" ] ] }, { "sid": 121, "sent": "This is consistent with the extended binding interface and additional interactions observed with the hexaloop, and suggests a preferential binding to the hexaloop SL RNA in the Ox40 3\u2032-UTR.", "section": "RESULTS", "ner": [ [ 37, 54, "binding interface", "site" ], [ 101, 109, "hexaloop", "structure_element" ], [ 154, 162, "hexaloop", "structure_element" ], [ 163, 165, "SL", "structure_element" ], [ 166, 169, "RNA", "chemical" ], [ 177, 181, "Ox40", "protein" ], [ 182, 188, "3\u2032-UTR", "structure_element" ] ] }, { "sid": 122, "sent": "We designed different variants of the 3\u2032-UTR by point mutagenesis abrogating base pairing in the stem region, where none, individual, or both SL RNA motifs were mutated to impair Roquin-1 binding (Fig. 6a).", "section": "RESULTS", "ner": [ [ 38, 44, "3\u2032-UTR", "structure_element" ], [ 48, 65, "point mutagenesis", "experimental_method" ], [ 97, 108, "stem region", "structure_element" ], [ 142, 144, "SL", "structure_element" ], [ 145, 148, "RNA", "chemical" ], [ 161, 168, "mutated", "experimental_method" ], [ 179, 187, "Roquin-1", "protein" ] ] }, { "sid": 123, "sent": "These RNAs were then tested in EMSAs with the Roquin-1 N terminus (residues 2\u2013440) (Fig. 6b).", "section": "RESULTS", "ner": [ [ 6, 10, "RNAs", "chemical" ], [ 31, 36, "EMSAs", "experimental_method" ], [ 46, 54, "Roquin-1", "protein" ], [ 76, 81, "2\u2013440", "residue_range" ] ] }, { "sid": 124, "sent": "Gel shift assays show that binding to the wild-type 3\u2032-UTR construct leads to two distinct bands during the titrations, which should reflect binding to one and both RNA motifs, respectively.", "section": "RESULTS", "ner": [ [ 0, 16, "Gel shift assays", "experimental_method" ], [ 42, 51, "wild-type", "protein_state" ], [ 52, 58, "3\u2032-UTR", "structure_element" ], [ 108, 118, "titrations", "experimental_method" ], [ 165, 168, "RNA", "chemical" ] ] }, { "sid": 125, "sent": "Consistent with this, both bands are strongly reduced when mutations are introduced that interfere with the formation of both SLs.", "section": "RESULTS", "ner": [ [ 126, 129, "SLs", "structure_element" ] ] }, { "sid": 126, "sent": "Notably, among these, the slower migrating band disappears when either of the two SL RNA motifs is altered to impair Roquin binding, indicating an interaction with the remaining wild-type SL.", "section": "RESULTS", "ner": [ [ 82, 84, "SL", "structure_element" ], [ 85, 88, "RNA", "chemical" ], [ 117, 123, "Roquin", "protein" ], [ 178, 187, "wild-type", "protein_state" ], [ 188, 190, "SL", "structure_element" ] ] }, { "sid": 127, "sent": "We thus conclude that Roquin is able to bind to both SL RNA motifs in the context of the full-length Ox40 3\u2032-UTR.", "section": "RESULTS", "ner": [ [ 22, 28, "Roquin", "protein" ], [ 53, 55, "SL", "structure_element" ], [ 56, 59, "RNA", "chemical" ], [ 89, 100, "full-length", "protein_state" ], [ 101, 105, "Ox40", "protein" ], [ 106, 112, "3\u2032-UTR", "structure_element" ] ] }, { "sid": 128, "sent": "Regulation of Ox40 expression via two motifs in its 3\u2032-UTR", "section": "RESULTS", "ner": [ [ 14, 18, "Ox40", "protein" ], [ 52, 58, "3\u2032-UTR", "structure_element" ] ] }, { "sid": 129, "sent": "To investigate the role of the new ADE-like motif in target mRNA regulation, we introduced Ox40 mRNA variants harbouring altered 3\u2032-UTRs in cells.", "section": "RESULTS", "ner": [ [ 35, 38, "ADE", "structure_element" ], [ 60, 64, "mRNA", "chemical" ], [ 80, 90, "introduced", "experimental_method" ], [ 91, 95, "Ox40", "protein" ], [ 96, 100, "mRNA", "chemical" ], [ 121, 128, "altered", "protein_state" ], [ 129, 136, "3\u2032-UTRs", "structure_element" ] ] }, { "sid": 130, "sent": "Considering the close proximity of the ADE-like and CDE-like SL RNAs in the 3\u2032-UTR (Fig. 6a), which is essential for Roquin-mediated posttranscriptional regulation of Ox40 (ref.) we tested individual contributions and the functional cooperation of the two RNA elements by deletion and point mutagenesis abrogating base pairing in the stem region (Fig. 6a,c and Supplementary Fig. 8c).", "section": "RESULTS", "ner": [ [ 39, 42, "ADE", "structure_element" ], [ 52, 55, "CDE", "structure_element" ], [ 61, 63, "SL", "structure_element" ], [ 64, 68, "RNAs", "chemical" ], [ 76, 82, "3\u2032-UTR", "structure_element" ], [ 117, 123, "Roquin", "protein" ], [ 167, 171, "Ox40", "protein" ], [ 256, 259, "RNA", "chemical" ], [ 272, 302, "deletion and point mutagenesis", "experimental_method" ], [ 303, 313, "abrogating", "protein_state" ], [ 314, 326, "base pairing", "bond_interaction" ], [ 334, 345, "stem region", "structure_element" ] ] }, { "sid": 131, "sent": "Specifically, using retroviruses we introduced Ox40 expression constructs placed under the control of different 3\u2032-UTRs into Roquin-1/2-deficient mouse embryonic fibroblasts.", "section": "RESULTS", "ner": [ [ 20, 32, "retroviruses", "taxonomy_domain" ], [ 47, 51, "Ox40", "protein" ], [ 112, 119, "3\u2032-UTRs", "structure_element" ], [ 125, 133, "Roquin-1", "protein" ], [ 134, 135, "2", "protein" ], [ 146, 151, "mouse", "taxonomy_domain" ] ] }, { "sid": 132, "sent": "Doxycycline treatment of cells from this cell line enabled ectopic Roquin-1 and co-translational mCherry expression due to the stable integration of an inducible lentiviral vector (Supplementary Fig. 8c).", "section": "RESULTS", "ner": [ [ 0, 11, "Doxycycline", "chemical" ], [ 67, 75, "Roquin-1", "protein" ], [ 162, 172, "lentiviral", "taxonomy_domain" ] ] }, { "sid": 133, "sent": "The expression of Ox40 in cells with and without doxycycline treatment was then quantified by flow cytometry (Supplementary Fig. 8c).", "section": "RESULTS", "ner": [ [ 18, 22, "Ox40", "protein" ], [ 49, 60, "doxycycline", "chemical" ], [ 94, 108, "flow cytometry", "experimental_method" ] ] }, { "sid": 134, "sent": "Comparing the ratio of Ox40 mean fluorescence intensities in cells with and without doxycycline treatment normalized to the values from cells that expressed Ox40 constructs without 3\u2032-UTR revealed a comparable importance of both structural elements (Fig. 6c).", "section": "RESULTS", "ner": [ [ 23, 27, "Ox40", "protein" ], [ 28, 57, "mean fluorescence intensities", "evidence" ], [ 84, 95, "doxycycline", "chemical" ], [ 157, 161, "Ox40", "protein" ], [ 173, 180, "without", "protein_state" ], [ 181, 187, "3\u2032-UTR", "structure_element" ] ] }, { "sid": 135, "sent": "In fact, only deletion or point mutagenesis of the sequences encoding both structures at the same time (3\u2032-UTR 1\u201380 and double mut) neutralized Roquin-dependent repression of Ox40.", "section": "RESULTS", "ner": [ [ 14, 43, "deletion or point mutagenesis", "experimental_method" ], [ 104, 110, "3\u2032-UTR", "structure_element" ], [ 111, 115, "1\u201380", "residue_range" ], [ 120, 130, "double mut", "protein_state" ], [ 144, 150, "Roquin", "protein" ], [ 175, 179, "Ox40", "protein" ] ] }, { "sid": 136, "sent": "In contrast, individual mutations that left the hexaloop (3\u2032-UTR 1\u2013120 or CDE mut) or the CDE-like triloop intact still enabled Roquin-dependent repression, which occurred in an attenuated manner compared with the full-length 3\u2032-UTR (Fig. 6c).", "section": "RESULTS", "ner": [ [ 24, 33, "mutations", "experimental_method" ], [ 48, 56, "hexaloop", "structure_element" ], [ 58, 64, "3\u2032-UTR", "structure_element" ], [ 65, 70, "1\u2013120", "residue_range" ], [ 74, 81, "CDE mut", "mutant" ], [ 90, 93, "CDE", "structure_element" ], [ 99, 106, "triloop", "structure_element" ], [ 107, 113, "intact", "protein_state" ], [ 128, 134, "Roquin", "protein" ], [ 214, 225, "full-length", "protein_state" ], [ 226, 232, "3\u2032-UTR", "structure_element" ] ] }, { "sid": 137, "sent": "To further analyse the functional consequences of Roquin binding to the 3\u2032-UTR, we also measured mRNA decay rates after introducing the different Ox40 constructs into HeLa tet-off cells that allow to turn off transcription from the tetracycline-repressed vectors by addition of doxycycline (Fig. 6d).", "section": "RESULTS", "ner": [ [ 50, 56, "Roquin", "protein" ], [ 72, 78, "3\u2032-UTR", "structure_element" ], [ 97, 113, "mRNA decay rates", "evidence" ], [ 146, 150, "Ox40", "protein" ], [ 278, 289, "doxycycline", "chemical" ] ] }, { "sid": 138, "sent": "Quantitative reverse transcriptase\u2013PCR revealed a strong stabilization of the Ox40 mRNA by deletion of the 3\u2032-UTR (CDS t1/2=311\u2009min vs full-length t1/2=96\u2009min).", "section": "RESULTS", "ner": [ [ 0, 38, "Quantitative reverse transcriptase\u2013PCR", "experimental_method" ], [ 78, 82, "Ox40", "protein" ], [ 83, 87, "mRNA", "chemical" ], [ 91, 102, "deletion of", "experimental_method" ], [ 107, 113, "3\u2032-UTR", "structure_element" ], [ 115, 118, "CDS", "structure_element" ], [ 119, 123, "t1/2", "evidence" ], [ 135, 146, "full-length", "protein_state" ], [ 147, 151, "t1/2", "evidence" ] ] }, { "sid": 139, "sent": "A comparable stabilization was achieved by combined mutation of the CDE-like and the ADE-like SLs (ADE/CDE-like mut t1/2=255\u2009min).", "section": "RESULTS", "ner": [ [ 43, 60, "combined mutation", "experimental_method" ], [ 68, 71, "CDE", "structure_element" ], [ 85, 88, "ADE", "structure_element" ], [ 94, 97, "SLs", "structure_element" ], [ 99, 102, "ADE", "structure_element" ], [ 103, 106, "CDE", "structure_element" ], [ 112, 115, "mut", "protein_state" ], [ 116, 120, "t1/2", "evidence" ] ] }, { "sid": 140, "sent": "Individual mutations of either the ADE-like or the CDE-like SLs showed intermediate effects (ADE-like mut t1/2=170\u2009min, CDE-like mut t1/2=167\u2009min), respectively.", "section": "RESULTS", "ner": [ [ 11, 20, "mutations", "experimental_method" ], [ 35, 38, "ADE", "structure_element" ], [ 51, 54, "CDE", "structure_element" ], [ 60, 63, "SLs", "structure_element" ], [ 93, 96, "ADE", "structure_element" ], [ 102, 105, "mut", "protein_state" ], [ 106, 110, "t1/2", "evidence" ], [ 120, 123, "CDE", "structure_element" ], [ 129, 132, "mut", "protein_state" ], [ 133, 137, "t1/2", "evidence" ] ] }, { "sid": 141, "sent": "These findings underscore the importance of both structural motifs and reveal that they have an additive effect on the regulation of Ox40 mRNA expression in cells.", "section": "RESULTS", "ner": [ [ 133, 137, "Ox40", "protein" ], [ 138, 142, "mRNA", "chemical" ] ] }, { "sid": 142, "sent": "Recent structural and functional studies have provided first insight into the RNA binding of Roquin.", "section": "DISCUSS", "ner": [ [ 7, 40, "structural and functional studies", "experimental_method" ], [ 78, 81, "RNA", "chemical" ], [ 93, 99, "Roquin", "protein" ] ] }, { "sid": 143, "sent": "Structures of Roquin bound to CDE SL RNAs indicated mainly shape recognition of the SL RNA in the so-called A-site of the N-terminal region of the Roquin protein with no sequence specificity, except the requirement for a pyrimidine\u2013purine\u2013pyrimidine triloop.", "section": "DISCUSS", "ner": [ [ 0, 10, "Structures", "evidence" ], [ 14, 20, "Roquin", "protein" ], [ 21, 29, "bound to", "protein_state" ], [ 30, 33, "CDE", "structure_element" ], [ 34, 36, "SL", "structure_element" ], [ 37, 41, "RNAs", "chemical" ], [ 84, 86, "SL", "structure_element" ], [ 87, 90, "RNA", "chemical" ], [ 108, 114, "A-site", "site" ], [ 122, 139, "N-terminal region", "structure_element" ], [ 147, 153, "Roquin", "protein" ], [ 221, 257, "pyrimidine\u2013purine\u2013pyrimidine triloop", "structure_element" ] ] }, { "sid": 144, "sent": "Considering that the CDE RNA recognition is mostly structure specific and not sequence dependent, a wide spectrum of target mRNA might be recognized by Roquin.", "section": "DISCUSS", "ner": [ [ 21, 24, "CDE", "structure_element" ], [ 25, 28, "RNA", "chemical" ], [ 124, 128, "mRNA", "chemical" ], [ 152, 158, "Roquin", "protein" ] ] }, { "sid": 145, "sent": "Here we have used SELEX assays to identify a novel RNA recognition motif of Roquin-1, which is present in the Ox40 3\u2032-UTR and variations of which may be found in the 3\u2032-UTRs of many other genes.", "section": "DISCUSS", "ner": [ [ 18, 30, "SELEX assays", "experimental_method" ], [ 51, 72, "RNA recognition motif", "structure_element" ], [ 76, 84, "Roquin-1", "protein" ], [ 110, 114, "Ox40", "protein" ], [ 115, 121, "3\u2032-UTR", "structure_element" ], [ 166, 173, "3\u2032-UTRs", "structure_element" ] ] }, { "sid": 146, "sent": "Our experiments show that this SELEX-derived ADE shows functional activity comparable to the previously established CDE motif.", "section": "DISCUSS", "ner": [ [ 31, 36, "SELEX", "experimental_method" ], [ 45, 48, "ADE", "structure_element" ], [ 116, 119, "CDE", "structure_element" ] ] }, { "sid": 147, "sent": "The ADE and Ox40 ADE-like SL RNAs adopt SL folds with a hexaloop instead of a triloop.", "section": "DISCUSS", "ner": [ [ 4, 7, "ADE", "structure_element" ], [ 12, 16, "Ox40", "protein" ], [ 17, 20, "ADE", "structure_element" ], [ 26, 28, "SL", "structure_element" ], [ 29, 33, "RNAs", "chemical" ], [ 40, 42, "SL", "structure_element" ], [ 56, 64, "hexaloop", "structure_element" ], [ 78, 85, "triloop", "structure_element" ] ] }, { "sid": 148, "sent": "Notably, the recognition of the respective RNA-helical stem regions by the ROQ domain is identical for the triloop and hexaloop motifs.", "section": "DISCUSS", "ner": [ [ 43, 67, "RNA-helical stem regions", "structure_element" ], [ 75, 78, "ROQ", "structure_element" ], [ 107, 114, "triloop", "structure_element" ], [ 119, 127, "hexaloop", "structure_element" ] ] }, { "sid": 149, "sent": "However, the U-rich hexaloops in the ADE and ADE-like SL RNAs mediate a number of additional contacts with the helix \u03b14 and strand \u03b23 in the ROQ domain that are absent in the triloop CDE (Fig. 3b\u2013f).", "section": "DISCUSS", "ner": [ [ 13, 29, "U-rich hexaloops", "structure_element" ], [ 37, 40, "ADE", "structure_element" ], [ 45, 48, "ADE", "structure_element" ], [ 54, 56, "SL", "structure_element" ], [ 57, 61, "RNAs", "chemical" ], [ 111, 116, "helix", "structure_element" ], [ 117, 119, "\u03b14", "structure_element" ], [ 124, 130, "strand", "structure_element" ], [ 131, 133, "\u03b23", "structure_element" ], [ 141, 144, "ROQ", "structure_element" ], [ 175, 182, "triloop", "structure_element" ], [ 183, 186, "CDE", "structure_element" ] ] }, { "sid": 150, "sent": "Of particular importance for the hexaloop recognition is Tyr250, which acts as a stabilizing element for the integrity of a defined loop conformation.", "section": "DISCUSS", "ner": [ [ 33, 41, "hexaloop", "structure_element" ], [ 57, 63, "Tyr250", "residue_name_number" ], [ 132, 136, "loop", "structure_element" ] ] }, { "sid": 151, "sent": "It stacks with nucleotides in the hexaloop but not the CDE triloop (Fig. 3b,c).", "section": "DISCUSS", "ner": [ [ 3, 9, "stacks", "bond_interaction" ], [ 34, 42, "hexaloop", "structure_element" ], [ 55, 58, "CDE", "structure_element" ], [ 59, 66, "triloop", "structure_element" ] ] }, { "sid": 152, "sent": "The functional role of Tyr250 for ADE-mediated mRNA regulation by Roquin-1 is thus explained by our experiments (Fig. 5b,c).", "section": "DISCUSS", "ner": [ [ 23, 29, "Tyr250", "residue_name_number" ], [ 34, 37, "ADE", "structure_element" ], [ 47, 51, "mRNA", "chemical" ], [ 66, 74, "Roquin-1", "protein" ] ] }, { "sid": 153, "sent": "The preference for U-rich hexaloops depends on nucleotide-specific interactions of ROQ with U10, U11 and U13 in the Ox40 ADE-like SL.", "section": "DISCUSS", "ner": [ [ 19, 35, "U-rich hexaloops", "structure_element" ], [ 83, 86, "ROQ", "structure_element" ], [ 92, 95, "U10", "residue_name_number" ], [ 97, 100, "U11", "residue_name_number" ], [ 105, 108, "U13", "residue_name_number" ], [ 116, 120, "Ox40", "protein" ], [ 121, 124, "ADE", "structure_element" ], [ 130, 132, "SL", "structure_element" ] ] }, { "sid": 154, "sent": "Consistent with this, loss of ROQ binding is observed on replacement of U11 and U13 by other bases (Table 2).", "section": "DISCUSS", "ner": [ [ 30, 33, "ROQ", "structure_element" ], [ 57, 68, "replacement", "experimental_method" ], [ 72, 75, "U11", "residue_name_number" ], [ 80, 83, "U13", "residue_name_number" ] ] }, { "sid": 155, "sent": "In spite of these differences in some aspects of the RNA recognition, overall features of Roquin targets are conserved in ADE and CDE-like RNAs, namely, a crucial role of non-sequence-specific contacts to the RNA stem and mainly shape recognition of the hexa- and triloops, respectively.", "section": "DISCUSS", "ner": [ [ 53, 56, "RNA", "chemical" ], [ 90, 96, "Roquin", "protein" ], [ 122, 125, "ADE", "structure_element" ], [ 130, 133, "CDE", "structure_element" ], [ 139, 143, "RNAs", "chemical" ], [ 209, 212, "RNA", "chemical" ], [ 213, 217, "stem", "structure_element" ], [ 254, 272, "hexa- and triloops", "structure_element" ] ] }, { "sid": 156, "sent": "A unique feature of the bound RNA structure, common to both tri- and hexaloops, is the stacking of a purine base onto the closing base pair (Fig. 3b,c).", "section": "DISCUSS", "ner": [ [ 24, 29, "bound", "protein_state" ], [ 30, 33, "RNA", "chemical" ], [ 34, 43, "structure", "evidence" ], [ 60, 78, "tri- and hexaloops", "structure_element" ], [ 87, 95, "stacking", "bond_interaction" ] ] }, { "sid": 157, "sent": "Previous structural data and the results presented here therefore suggest that Roquin may recognize additional SL RNA motifs, potentially with larger loops.", "section": "DISCUSS", "ner": [ [ 9, 24, "structural data", "evidence" ], [ 79, 85, "Roquin", "protein" ], [ 111, 113, "SL", "structure_element" ], [ 114, 117, "RNA", "chemical" ], [ 150, 155, "loops", "structure_element" ] ] }, { "sid": 158, "sent": "Interestingly, the SELEX-derived motif resembles the U-rich motifs that were identified recently by Murakawa et al.. In their study, several U-rich loops of various sizes were identified by crosslinking and immunoprecipitation of Roquin-1 using PAR-CLIP and the data also included sequences comprising the U-rich hexaloop identified in our present work.", "section": "DISCUSS", "ner": [ [ 19, 24, "SELEX", "experimental_method" ], [ 53, 66, "U-rich motifs", "structure_element" ], [ 141, 153, "U-rich loops", "structure_element" ], [ 190, 226, "crosslinking and immunoprecipitation", "experimental_method" ], [ 230, 238, "Roquin-1", "protein" ], [ 245, 253, "PAR-CLIP", "experimental_method" ], [ 306, 321, "U-rich hexaloop", "structure_element" ] ] }, { "sid": 159, "sent": "Most probably, the experimental setup of Murakawa et al. revealed both high- and low-affinity target motifs for Roquin, whereas our structural study reports on a high-affinity binding motif.", "section": "DISCUSS", "ner": [ [ 112, 118, "Roquin", "protein" ], [ 132, 148, "structural study", "experimental_method" ] ] }, { "sid": 160, "sent": "Notably, Murakawa et al. neither found the Roquin-regulated Ox40 nor the Tnf 3\u2032-UTRs, as both genes are not expressed in HEK 293 cells.", "section": "DISCUSS", "ner": [ [ 43, 49, "Roquin", "protein" ], [ 60, 64, "Ox40", "protein" ], [ 73, 76, "Tnf", "protein" ], [ 77, 84, "3\u2032-UTRs", "structure_element" ] ] }, { "sid": 161, "sent": "However, their newly identified U-rich target SL within the 3\u2032-UTR of A20 mRNA supports our conclusion that Roquin can accept alternative target motifs apart from the classical CDE triloop arrangement.", "section": "DISCUSS", "ner": [ [ 46, 48, "SL", "structure_element" ], [ 60, 66, "3\u2032-UTR", "structure_element" ], [ 70, 73, "A20", "protein" ], [ 74, 78, "mRNA", "chemical" ], [ 108, 114, "Roquin", "protein" ], [ 177, 180, "CDE", "structure_element" ], [ 181, 188, "triloop", "structure_element" ] ] }, { "sid": 162, "sent": "It remains to be seen which exact features govern the recognition of the A20 SL by Roquin.", "section": "DISCUSS", "ner": [ [ 73, 76, "A20", "protein" ], [ 77, 79, "SL", "structure_element" ], [ 83, 89, "Roquin", "protein" ] ] }, { "sid": 163, "sent": "The regulatory cis RNA elements in 3\u2032-UTRs may also be targeted by additional trans-acting factors.", "section": "DISCUSS", "ner": [ [ 15, 31, "cis RNA elements", "structure_element" ], [ 35, 42, "3\u2032-UTRs", "structure_element" ] ] }, { "sid": 164, "sent": "We have recently identified the endonuclease Regnase-1 as a cofactor of Roquin function that shares an overlapping set of target mRNAs.", "section": "DISCUSS", "ner": [ [ 32, 44, "endonuclease", "protein_type" ], [ 45, 54, "Regnase-1", "protein" ], [ 72, 78, "Roquin", "protein" ], [ 129, 134, "mRNAs", "chemical" ] ] }, { "sid": 165, "sent": "In another study, the overlap in targets was confirmed, but a mutually exclusive regulation was proposed based on studies in lipopolysaccharide (LPS)-stimulated myeloid cells.", "section": "DISCUSS", "ner": [ [ 125, 143, "lipopolysaccharide", "chemical" ], [ 145, 148, "LPS", "chemical" ] ] }, { "sid": 166, "sent": "In these cells, Roquin induced mRNA decay only for translationally inactive mRNAs, while Regnase-1-induced mRNA decay depended on active translation of the target.", "section": "DISCUSS", "ner": [ [ 16, 22, "Roquin", "protein" ], [ 31, 35, "mRNA", "chemical" ], [ 67, 75, "inactive", "protein_state" ], [ 76, 81, "mRNAs", "chemical" ], [ 89, 98, "Regnase-1", "protein" ], [ 107, 111, "mRNA", "chemical" ] ] }, { "sid": 167, "sent": "In CD4+ T cells, Ox40 does not show derepression in individual knockouts of Roquin-1 or Roquin-2 encoding genes, but is strongly induced upon combined deficiency of both genes.", "section": "DISCUSS", "ner": [ [ 17, 21, "Ox40", "protein" ], [ 76, 84, "Roquin-1", "protein" ], [ 88, 96, "Roquin-2", "protein" ], [ 151, 161, "deficiency", "experimental_method" ] ] }, { "sid": 168, "sent": "In addition, conditional deletion of the Regnase-1-encoding gene induced Ox40 expression in these cells.", "section": "DISCUSS", "ner": [ [ 25, 36, "deletion of", "experimental_method" ], [ 41, 50, "Regnase-1", "protein" ], [ 73, 77, "Ox40", "protein" ] ] }, { "sid": 169, "sent": "Whether induced decay of Ox40 mRNA by Roquin or Regnase proteins occurs in a mutually exclusive manner at different points during T-cell activation or shows cooperative regulation will have to await a direct comparison of T cells with single, double and triple knockouts of these genes.", "section": "DISCUSS", "ner": [ [ 25, 29, "Ox40", "protein" ], [ 30, 34, "mRNA", "chemical" ], [ 38, 44, "Roquin", "protein" ], [ 48, 55, "Regnase", "protein_type" ], [ 243, 270, "double and triple knockouts", "experimental_method" ] ] }, { "sid": 170, "sent": "However, in cultures of CD4+ T cells, Ox40 is translated on day 4\u20135 and is expressed much higher in T cells with combined deficiency of Roquin-1 and Roquin-2.", "section": "DISCUSS", "ner": [ [ 38, 42, "Ox40", "protein" ], [ 136, 144, "Roquin-1", "protein" ], [ 149, 157, "Roquin-2", "protein" ] ] }, { "sid": 171, "sent": "At this time point, the short-term inducible reconstitution with WT Roquin-1 was effective to reduced Ox40 expression, demonstrating the regulation of a translationally active mRNA by Roquin-1 in T cells (Fig. 5c).", "section": "DISCUSS", "ner": [ [ 45, 59, "reconstitution", "experimental_method" ], [ 65, 67, "WT", "protein_state" ], [ 68, 76, "Roquin-1", "protein" ], [ 102, 106, "Ox40", "protein" ], [ 169, 175, "active", "protein_state" ], [ 176, 180, "mRNA", "chemical" ], [ 184, 192, "Roquin-1", "protein" ] ] }, { "sid": 172, "sent": "Recombinant N-terminal protein fragments of Roquin-1 or Roquin-2 bind with comparable affinity to Ox40 mRNA in EMSAs and the 3\u2032-UTR of Ox40 is similarly retained by the two recombinant proteins in filter binding assays.", "section": "DISCUSS", "ner": [ [ 44, 52, "Roquin-1", "protein" ], [ 56, 64, "Roquin-2", "protein" ], [ 98, 102, "Ox40", "protein" ], [ 103, 107, "mRNA", "chemical" ], [ 111, 116, "EMSAs", "experimental_method" ], [ 125, 131, "3\u2032-UTR", "structure_element" ], [ 135, 139, "Ox40", "protein" ], [ 197, 218, "filter binding assays", "experimental_method" ] ] }, { "sid": 173, "sent": "Given the almost identical RNA contacts in both paralogues, we assume a similar recognition of ADE and CDE motifs in the Ox40 3\u2032-UTR by both proteins.", "section": "DISCUSS", "ner": [ [ 27, 30, "RNA", "chemical" ], [ 95, 98, "ADE", "structure_element" ], [ 103, 106, "CDE", "structure_element" ], [ 121, 125, "Ox40", "protein" ], [ 126, 132, "3\u2032-UTR", "structure_element" ] ] }, { "sid": 174, "sent": "In contrast, structural details on how Regnase-1 can interact with these SL RNAs are currently missing.", "section": "DISCUSS", "ner": [ [ 39, 48, "Regnase-1", "protein" ], [ 73, 75, "SL", "structure_element" ], [ 76, 80, "RNAs", "chemical" ] ] }, { "sid": 175, "sent": "Surprisingly, transcriptome-wide mapping of Regnase-1-binding sites in crosslinking and immunoprecipitation experiments identified specific triloop structures with pyrimidine\u2013purine\u2013pyrimidine loops in 3- to 7-nt-long stems, as well as a novel hexaloop structure in the Ptgs2 gene.", "section": "DISCUSS", "ner": [ [ 44, 67, "Regnase-1-binding sites", "site" ], [ 71, 119, "crosslinking and immunoprecipitation experiments", "experimental_method" ], [ 140, 147, "triloop", "structure_element" ], [ 164, 198, "pyrimidine\u2013purine\u2013pyrimidine loops", "structure_element" ], [ 218, 223, "stems", "structure_element" ], [ 244, 252, "hexaloop", "structure_element" ], [ 270, 275, "Ptgs2", "gene" ] ] }, { "sid": 176, "sent": "Both were required for Regnase-1-mediated repression.", "section": "DISCUSS", "ner": [ [ 23, 32, "Regnase-1", "protein" ] ] }, { "sid": 177, "sent": "These findings therefore raise the possibility that Regnase-1 interacts with ADE-like hexaloop structures either in a direct or indirect manner.", "section": "DISCUSS", "ner": [ [ 52, 61, "Regnase-1", "protein" ], [ 77, 80, "ADE", "structure_element" ], [ 86, 94, "hexaloop", "structure_element" ] ] }, { "sid": 178, "sent": "Nevertheless, it becomes clear that composite cis-elements, that is, the presence of several SLs as in Ox40 or Icos, could attract multiple trans-acting factors that may potentially co-regulate or even act cooperatively to control mRNA expression through posttranscriptional pathways of gene regulation.", "section": "DISCUSS", "ner": [ [ 46, 58, "cis-elements", "structure_element" ], [ 93, 96, "SLs", "structure_element" ], [ 103, 107, "Ox40", "protein" ], [ 111, 115, "Icos", "protein" ], [ 231, 235, "mRNA", "chemical" ] ] }, { "sid": 179, "sent": "The novel 3\u2032-UTR loop motif that we have identified as a bona fide target of Roquin now expands this multilayer mode of co-regulation.", "section": "DISCUSS", "ner": [ [ 10, 16, "3\u2032-UTR", "structure_element" ], [ 17, 27, "loop motif", "structure_element" ], [ 77, 83, "Roquin", "protein" ] ] }, { "sid": 180, "sent": "We suggest that differential regulation of mRNA expression is not only achieved through multiple regulators with individual preferences for a given motif or variants thereof, but that regulators may also identify and use distinct motifs, as long as they exhibit some basic features regarding shape, size and sequence.", "section": "DISCUSS", "ner": [ [ 43, 47, "mRNA", "chemical" ] ] }, { "sid": 181, "sent": "The presence of distinct motifs in 3\u2032-UTRs offers a broader variability for gene regulation by RNA cis elements.", "section": "DISCUSS", "ner": [ [ 35, 42, "3\u2032-UTRs", "structure_element" ], [ 95, 98, "RNA", "chemical" ], [ 99, 111, "cis elements", "structure_element" ] ] }, { "sid": 182, "sent": "Their accessibility can be modulated by trans-acting factors that may bind regulatory motifs, unfold higher-order structures in the RNA or maintain a preference for duplex structures as was shown recently for mRNAs that are recognized by Staufen-1 (ref.).", "section": "DISCUSS", "ner": [ [ 132, 135, "RNA", "chemical" ], [ 209, 214, "mRNAs", "chemical" ], [ 238, 247, "Staufen-1", "protein" ] ] }, { "sid": 183, "sent": "In the 3\u2032-UTR of the Ox40 mRNA, we find one ADE-like and one CDE-like SL, with similar binding to the ROQ domain.", "section": "DISCUSS", "ner": [ [ 7, 13, "3\u2032-UTR", "structure_element" ], [ 21, 25, "Ox40", "protein" ], [ 26, 30, "mRNA", "chemical" ], [ 44, 47, "ADE", "structure_element" ], [ 61, 64, "CDE", "structure_element" ], [ 70, 72, "SL", "structure_element" ], [ 102, 105, "ROQ", "structure_element" ] ] }, { "sid": 184, "sent": "The exact stoichiometry of Roquin bound to the Ox40 3\u2032-UTR is unknown.", "section": "DISCUSS", "ner": [ [ 27, 33, "Roquin", "protein" ], [ 34, 42, "bound to", "protein_state" ], [ 47, 51, "Ox40", "protein" ], [ 52, 58, "3\u2032-UTR", "structure_element" ] ] }, { "sid": 185, "sent": "The recently identified secondary binding site for dsRNA in Roquin (B-site) could potentially allow for simultaneous binding of dsRNA and thereby promote engagement of Roquin and target RNAs before recognition of high-affinity SLs.", "section": "DISCUSS", "ner": [ [ 24, 46, "secondary binding site", "site" ], [ 51, 56, "dsRNA", "chemical" ], [ 60, 66, "Roquin", "protein" ], [ 68, 74, "B-site", "site" ], [ 128, 133, "dsRNA", "chemical" ], [ 168, 174, "Roquin", "protein" ], [ 186, 190, "RNAs", "chemical" ], [ 218, 226, "affinity", "evidence" ], [ 227, 230, "SLs", "structure_element" ] ] }, { "sid": 186, "sent": "In this respect, it is interesting to note that symmetry-related RNA molecules of both Tnf CDE and ADE SL RNAs are found in the respective crystal lattice in a position that corresponds to the recognition of dsRNA in the B site.", "section": "DISCUSS", "ner": [ [ 65, 68, "RNA", "chemical" ], [ 87, 90, "Tnf", "protein" ], [ 91, 94, "CDE", "structure_element" ], [ 99, 102, "ADE", "structure_element" ], [ 103, 105, "SL", "structure_element" ], [ 106, 110, "RNAs", "chemical" ], [ 139, 154, "crystal lattice", "evidence" ], [ 208, 213, "dsRNA", "chemical" ], [ 221, 227, "B site", "site" ] ] }, { "sid": 187, "sent": "This opens the possibility that one Roquin molecule may cluster two motifs in a given 3\u2032-UTR and/or cluster motifs from distinct 3\u2032-UTRs to enhance downstream processing.", "section": "DISCUSS", "ner": [ [ 36, 42, "Roquin", "protein" ], [ 86, 92, "3\u2032-UTR", "structure_element" ], [ 129, 136, "3\u2032-UTRs", "structure_element" ] ] }, { "sid": 188, "sent": "Interestingly, two SL RNA elements that resemble bona fide ligands of Roquin have also been identified in the 3\u2032-UTR of the Nfkbid mRNA.", "section": "DISCUSS", "ner": [ [ 19, 21, "SL", "structure_element" ], [ 22, 25, "RNA", "chemical" ], [ 70, 76, "Roquin", "protein" ], [ 110, 116, "3\u2032-UTR", "structure_element" ], [ 124, 130, "Nfkbid", "protein" ], [ 131, 135, "mRNA", "chemical" ] ] }, { "sid": 189, "sent": "We therefore hypothesize that the combination of multiple binding sites may be more commonly used to enhance the functional activity of Roquin.", "section": "DISCUSS", "ner": [ [ 58, 71, "binding sites", "site" ], [ 136, 142, "Roquin", "protein" ] ] }, { "sid": 190, "sent": "At the same time, the combination of cis elements may be important for differential gene regulation, as composite cis elements with lower affinity may be less sensitive to Roquin.", "section": "DISCUSS", "ner": [ [ 37, 49, "cis elements", "structure_element" ], [ 114, 126, "cis elements", "structure_element" ], [ 138, 146, "affinity", "evidence" ], [ 172, 178, "Roquin", "protein" ] ] }, { "sid": 191, "sent": "This will lead to less effective repression in T cells when antigen recognition is of moderate signal strength and only incomplete cleavage of Roquin by MALT1 occurs.", "section": "DISCUSS", "ner": [ [ 143, 149, "Roquin", "protein" ], [ 153, 158, "MALT1", "protein" ] ] }, { "sid": 192, "sent": "For understanding the intricate complexity of 3\u2032-UTR regulation, future work will be necessary by combining large-scale approaches, such as cross-linking and immunoprecipitation experiments to identify RNA-binding sites, and structural biology to dissect the underlying molecular mechanisms.", "section": "DISCUSS", "ner": [ [ 46, 52, "3\u2032-UTR", "structure_element" ], [ 140, 189, "cross-linking and immunoprecipitation experiments", "experimental_method" ], [ 202, 219, "RNA-binding sites", "site" ], [ 225, 243, "structural biology", "experimental_method" ] ] }, { "sid": 193, "sent": "SELEX identifies a novel SL RNA ligand of Roquin-1.", "section": "FIG", "ner": [ [ 0, 5, "SELEX", "experimental_method" ], [ 25, 27, "SL", "structure_element" ], [ 28, 31, "RNA", "chemical" ], [ 42, 50, "Roquin-1", "protein" ] ] }, { "sid": 194, "sent": "(a) Enriched hexamers that were found by Roquin-1 N terminus (residues 2\u2013440) or Roquin-1 M199R N terminus (residues 2\u2013440) (see also Supplementary Fig. 1). (b) An ADE sequence motif in the Ox40 3\u2032-UTR closely resembles the MEME motif found in SELEX-enriched RNA sequences.", "section": "FIG", "ner": [ [ 41, 49, "Roquin-1", "protein" ], [ 71, 76, "2\u2013440", "residue_range" ], [ 81, 95, "Roquin-1 M199R", "mutant" ], [ 117, 122, "2\u2013440", "residue_range" ], [ 164, 167, "ADE", "structure_element" ], [ 190, 194, "Ox40", "protein" ], [ 195, 201, "3\u2032-UTR", "structure_element" ], [ 224, 228, "MEME", "experimental_method" ], [ 244, 249, "SELEX", "experimental_method" ], [ 259, 262, "RNA", "chemical" ] ] }, { "sid": 195, "sent": "(c) Conservation of the motif found in Ox40 3\u2032-UTRs for various species as indicated.", "section": "FIG", "ner": [ [ 39, 43, "Ox40", "protein" ], [ 44, 51, "3\u2032-UTRs", "structure_element" ] ] }, { "sid": 196, "sent": "rn5 is the fifth assembly version of the rat (Rattus novegicus). (d) Schematic representation of the predicted SELEX-derived consensus SL, ADE and the Ox40 ADE-like hexaloop SL.", "section": "FIG", "ner": [ [ 0, 3, "rn5", "gene" ], [ 41, 44, "rat", "taxonomy_domain" ], [ 46, 62, "Rattus novegicus", "species" ], [ 111, 116, "SELEX", "experimental_method" ], [ 135, 137, "SL", "structure_element" ], [ 139, 142, "ADE", "structure_element" ], [ 151, 155, "Ox40", "protein" ], [ 156, 159, "ADE", "structure_element" ], [ 165, 173, "hexaloop", "structure_element" ], [ 174, 176, "SL", "structure_element" ] ] }, { "sid": 197, "sent": "The broken line between the G\u2013G base pair in the ADE SL indicates a putative non-Watson\u2013Crick pairing.", "section": "FIG", "ner": [ [ 49, 52, "ADE", "structure_element" ], [ 53, 55, "SL", "structure_element" ], [ 77, 101, "non-Watson\u2013Crick pairing", "bond_interaction" ] ] }, { "sid": 198, "sent": "The Ox40 CDE-like SL and the Tnf CDE SL are shown for comparison.", "section": "FIG", "ner": [ [ 4, 8, "Ox40", "protein" ], [ 9, 12, "CDE", "structure_element" ], [ 18, 20, "SL", "structure_element" ], [ 29, 32, "Tnf", "protein" ], [ 33, 36, "CDE", "structure_element" ], [ 37, 39, "SL", "structure_element" ] ] }, { "sid": 199, "sent": "NMR analysis of the SL RNAs used in this study.", "section": "FIG", "ner": [ [ 0, 3, "NMR", "experimental_method" ], [ 20, 22, "SL", "structure_element" ], [ 23, 27, "RNAs", "chemical" ] ] }, { "sid": 200, "sent": "Imino proton regions of one-dimensional 1H NMR spectra of (a) the ADE SL (b), the Ox40 ADE-like SL and (c) the Ox40 CDE-like SL are shown for free RNAs (black) and in complex with the Roquin-1 ROQ domain (red).", "section": "FIG", "ner": [ [ 40, 46, "1H NMR", "experimental_method" ], [ 47, 54, "spectra", "evidence" ], [ 66, 69, "ADE", "structure_element" ], [ 70, 72, "SL", "structure_element" ], [ 82, 86, "Ox40", "protein" ], [ 87, 90, "ADE", "structure_element" ], [ 96, 98, "SL", "structure_element" ], [ 111, 115, "Ox40", "protein" ], [ 116, 119, "CDE", "structure_element" ], [ 125, 127, "SL", "structure_element" ], [ 142, 146, "free", "protein_state" ], [ 147, 151, "RNAs", "chemical" ], [ 164, 179, "in complex with", "protein_state" ], [ 184, 192, "Roquin-1", "protein" ], [ 193, 196, "ROQ", "structure_element" ] ] }, { "sid": 201, "sent": "The respective SL RNAs and their base pairs are indicated.", "section": "FIG", "ner": [ [ 15, 17, "SL", "structure_element" ], [ 18, 22, "RNAs", "chemical" ] ] }, { "sid": 202, "sent": "Red asterisks indicate NMR signals of the protein.", "section": "FIG", "ner": [ [ 23, 26, "NMR", "experimental_method" ] ] }, { "sid": 203, "sent": "Green lines in the secondary structure schemes on the left refer to visible imino NMR signals and thus experimental confirmation of the base pairs indicated.", "section": "FIG", "ner": [ [ 82, 85, "NMR", "experimental_method" ], [ 86, 93, "signals", "evidence" ] ] }, { "sid": 204, "sent": "The dotted green line between G6 and G15 in a highlights a G\u2013G base pair.", "section": "FIG", "ner": [ [ 30, 32, "G6", "residue_name_number" ], [ 37, 40, "G15", "residue_name_number" ], [ 59, 60, "G", "residue_name" ], [ 61, 62, "G", "residue_name" ] ] }, { "sid": 205, "sent": "Structure of the Roquin-1 ROQ domain bound to Ox40 ADE-like RNA.", "section": "FIG", "ner": [ [ 0, 9, "Structure", "evidence" ], [ 17, 25, "Roquin-1", "protein" ], [ 26, 29, "ROQ", "structure_element" ], [ 37, 45, "bound to", "protein_state" ], [ 46, 50, "Ox40", "protein" ], [ 51, 54, "ADE", "structure_element" ], [ 60, 63, "RNA", "chemical" ] ] }, { "sid": 206, "sent": "(a) Cartoon presentation of the crystal structure of the ROQ domain (residues 174\u2013325; blue) and the Ox40 ADE-like SL RNA (magenta).", "section": "FIG", "ner": [ [ 32, 49, "crystal structure", "evidence" ], [ 57, 60, "ROQ", "structure_element" ], [ 78, 85, "174\u2013325", "residue_range" ], [ 101, 105, "Ox40", "protein" ], [ 106, 109, "ADE", "structure_element" ], [ 115, 117, "SL", "structure_element" ], [ 118, 121, "RNA", "chemical" ] ] }, { "sid": 207, "sent": "Selected RNA bases and protein secondary structure elements are labelled.", "section": "FIG", "ner": [ [ 9, 12, "RNA", "chemical" ] ] }, { "sid": 208, "sent": "(b) Close-up view of the Ox40 ADE-like SL (bases in the RNA hexaloop are shown in magenta) and (c) the previously reported structure of the ROQ-Tnf CDE complex (bases of the triloop RNA are shown in green).", "section": "FIG", "ner": [ [ 25, 29, "Ox40", "protein" ], [ 30, 33, "ADE", "structure_element" ], [ 39, 41, "SL", "structure_element" ], [ 56, 59, "RNA", "chemical" ], [ 60, 68, "hexaloop", "structure_element" ], [ 123, 132, "structure", "evidence" ], [ 140, 151, "ROQ-Tnf CDE", "complex_assembly" ], [ 182, 185, "RNA", "chemical" ] ] }, { "sid": 209, "sent": "Only RNA-interacting residues that are different in both structures are shown.", "section": "FIG", "ner": [ [ 5, 29, "RNA-interacting residues", "site" ], [ 57, 67, "structures", "evidence" ] ] }, { "sid": 210, "sent": "Both protein chains and remaining parts of both RNAs are shown in grey and protein residue side chains are shown in turquoise. (d) Close-up view of the contacts between the ROQ domain and nucleotides U11 and U13 of the Ox40 ADE-like SL RNA.", "section": "FIG", "ner": [ [ 48, 52, "RNAs", "chemical" ], [ 173, 176, "ROQ", "structure_element" ], [ 200, 203, "U11", "residue_name_number" ], [ 208, 211, "U13", "residue_name_number" ], [ 219, 223, "Ox40", "protein" ], [ 224, 227, "ADE", "structure_element" ], [ 233, 235, "SL", "structure_element" ], [ 236, 239, "RNA", "chemical" ] ] }, { "sid": 211, "sent": "The nucleotides interact with the C-terminal end of helix \u03b14 (Tyr250 and Ser253) and the N-terminal part of strand \u03b23 (Phe255 and Val257).", "section": "FIG", "ner": [ [ 52, 57, "helix", "structure_element" ], [ 58, 60, "\u03b14", "structure_element" ], [ 62, 68, "Tyr250", "residue_name_number" ], [ 73, 79, "Ser253", "residue_name_number" ], [ 108, 114, "strand", "structure_element" ], [ 115, 117, "\u03b23", "structure_element" ], [ 119, 125, "Phe255", "residue_name_number" ], [ 130, 136, "Val257", "residue_name_number" ] ] }, { "sid": 212, "sent": "The protein chain is shown in turquoise and the RNA is shown in grey.", "section": "FIG", "ner": [ [ 48, 51, "RNA", "chemical" ] ] }, { "sid": 213, "sent": "(e) Close-up view of the contacts between the ROQ domain and nucleotides U10, U11 and U13 in the RNA hexaloop.", "section": "FIG", "ner": [ [ 46, 49, "ROQ", "structure_element" ], [ 73, 76, "U10", "residue_name_number" ], [ 78, 81, "U11", "residue_name_number" ], [ 86, 89, "U13", "residue_name_number" ], [ 97, 100, "RNA", "chemical" ], [ 101, 109, "hexaloop", "structure_element" ] ] }, { "sid": 214, "sent": "U11 and U13 contact the C-terminal end of helix \u03b14: residues Tyr250 and Gln247.", "section": "FIG", "ner": [ [ 0, 3, "U11", "residue_name_number" ], [ 8, 11, "U13", "residue_name_number" ], [ 42, 47, "helix", "structure_element" ], [ 48, 50, "\u03b14", "structure_element" ], [ 61, 67, "Tyr250", "residue_name_number" ], [ 72, 78, "Gln247", "residue_name_number" ] ] }, { "sid": 215, "sent": "The side chain of Tyr250 makes hydrophobic interactions with the pyrimidine side chain of U10 on one side and U11 on the other side.", "section": "FIG", "ner": [ [ 18, 24, "Tyr250", "residue_name_number" ], [ 31, 55, "hydrophobic interactions", "bond_interaction" ], [ 90, 93, "U10", "residue_name_number" ], [ 110, 113, "U11", "residue_name_number" ] ] }, { "sid": 216, "sent": "Lys259 interacts with the phosphate groups of U10 and U11.", "section": "FIG", "ner": [ [ 0, 6, "Lys259", "residue_name_number" ], [ 46, 49, "U10", "residue_name_number" ], [ 54, 57, "U11", "residue_name_number" ] ] }, { "sid": 217, "sent": "(f) Close-up view of the hydrophobic interaction between Val257 and U11, as well as the double hydrogen bond of Lys259 with phosphate groups of U10 and U11.", "section": "FIG", "ner": [ [ 25, 48, "hydrophobic interaction", "bond_interaction" ], [ 57, 63, "Val257", "residue_name_number" ], [ 68, 71, "U11", "residue_name_number" ], [ 95, 108, "hydrogen bond", "bond_interaction" ], [ 112, 118, "Lys259", "residue_name_number" ], [ 144, 147, "U10", "residue_name_number" ], [ 152, 155, "U11", "residue_name_number" ] ] }, { "sid": 218, "sent": "NMR analysis of ROQ domain interactions with the Ox40 ADE-like hexaloop RNA.", "section": "FIG", "ner": [ [ 0, 3, "NMR", "experimental_method" ], [ 16, 19, "ROQ", "structure_element" ], [ 49, 53, "Ox40", "protein" ], [ 54, 57, "ADE", "structure_element" ], [ 63, 71, "hexaloop", "structure_element" ], [ 72, 75, "RNA", "chemical" ] ] }, { "sid": 219, "sent": "(a) Overlay of 1H,15N HSQC spectra of either the free ROQ domain (171\u2013326, black) or in complex with stoichiometric amounts of the Ox40 ADE-like SL (red).", "section": "FIG", "ner": [ [ 4, 11, "Overlay", "experimental_method" ], [ 15, 26, "1H,15N HSQC", "experimental_method" ], [ 27, 34, "spectra", "evidence" ], [ 49, 53, "free", "protein_state" ], [ 54, 57, "ROQ", "structure_element" ], [ 66, 73, "171\u2013326", "residue_range" ], [ 85, 100, "in complex with", "protein_state" ], [ 131, 135, "Ox40", "protein" ], [ 136, 139, "ADE", "structure_element" ], [ 145, 147, "SL", "structure_element" ] ] }, { "sid": 220, "sent": "(b) Plot of chemical shift change versus residue number in the ROQ domain (residues 171\u2013326) from a. Grey negative bars indicate missing assignments in one of the spectra.", "section": "FIG", "ner": [ [ 12, 33, "chemical shift change", "evidence" ], [ 63, 66, "ROQ", "structure_element" ], [ 84, 91, "171\u2013326", "residue_range" ], [ 163, 170, "spectra", "evidence" ] ] }, { "sid": 221, "sent": "Gaps indicate prolines.", "section": "FIG", "ner": [ [ 14, 22, "prolines", "residue_name" ] ] }, { "sid": 222, "sent": "(c) Overlay of the ROQ domain alone (black) or in complex with the Ox40 ADE-like SL (red) or the Ox40 CDE-like SL (green).", "section": "FIG", "ner": [ [ 4, 11, "Overlay", "experimental_method" ], [ 19, 22, "ROQ", "structure_element" ], [ 30, 35, "alone", "protein_state" ], [ 47, 62, "in complex with", "protein_state" ], [ 67, 71, "Ox40", "protein" ], [ 72, 75, "ADE", "structure_element" ], [ 81, 83, "SL", "structure_element" ], [ 97, 101, "Ox40", "protein" ], [ 102, 105, "CDE", "structure_element" ], [ 111, 113, "SL", "structure_element" ] ] }, { "sid": 223, "sent": "Mutational analysis of Roquin-1-interactions with Ox40 ADE-like SL and Ox40 3\u2032-UTR.", "section": "FIG", "ner": [ [ 0, 19, "Mutational analysis", "experimental_method" ], [ 23, 31, "Roquin-1", "protein" ], [ 50, 54, "Ox40", "protein" ], [ 55, 58, "ADE", "structure_element" ], [ 64, 66, "SL", "structure_element" ], [ 71, 75, "Ox40", "protein" ], [ 76, 82, "3\u2032-UTR", "structure_element" ] ] }, { "sid": 224, "sent": "(a) EMSA assay comparing binding of the wild-type and of the Y250A mutant ROQ domain for binding to the Ox40 ADE-like SL (left) or the previously described Tnf CDE SL (right).", "section": "FIG", "ner": [ [ 4, 14, "EMSA assay", "experimental_method" ], [ 40, 49, "wild-type", "protein_state" ], [ 61, 66, "Y250A", "mutant" ], [ 67, 73, "mutant", "protein_state" ], [ 74, 77, "ROQ", "structure_element" ], [ 104, 108, "Ox40", "protein" ], [ 109, 112, "ADE", "structure_element" ], [ 118, 120, "SL", "structure_element" ], [ 156, 159, "Tnf", "protein" ], [ 160, 163, "CDE", "structure_element" ], [ 164, 166, "SL", "structure_element" ] ] }, { "sid": 225, "sent": "A comparison of further mutants is shown in Supplementary Fig. 4. (b) Schematic overview of the timeline used for the reconstitution experiment shown in c. (c) Flow cytometry of Ox40 and Icos surface expression on CD4+ Th1 cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice treated with tamoxifen (+tam) to induce Rc3h1/2fl/fl deletion or left untreated (\u2212 tam).", "section": "FIG", "ner": [ [ 160, 174, "Flow cytometry", "experimental_method" ], [ 178, 182, "Ox40", "protein" ], [ 187, 191, "Icos", "protein" ], [ 234, 239, "Rc3h1", "gene" ], [ 240, 243, "2fl", "gene" ], [ 244, 246, "fl", "gene" ], [ 267, 271, "mice", "taxonomy_domain" ], [ 285, 294, "tamoxifen", "chemical" ], [ 312, 317, "Rc3h1", "gene" ], [ 318, 321, "2fl", "gene" ], [ 322, 324, "fl", "gene" ], [ 325, 333, "deletion", "experimental_method" ] ] }, { "sid": 226, "sent": "The cells were then either left untransduced (UT) or were transduced with retrovirus containing a doxycycline-inducible cassette, to express Roquin-1 WT, Roquin-1 Y250A or Roquin-1 K220A, K239A and R260A mutants (see also Supplementary Fig. 5).", "section": "FIG", "ner": [ [ 74, 84, "retrovirus", "taxonomy_domain" ], [ 98, 109, "doxycycline", "chemical" ], [ 141, 149, "Roquin-1", "protein" ], [ 150, 152, "WT", "protein_state" ], [ 154, 162, "Roquin-1", "protein" ], [ 163, 168, "Y250A", "mutant" ], [ 172, 180, "Roquin-1", "protein" ], [ 181, 186, "K220A", "mutant" ], [ 188, 193, "K239A", "mutant" ], [ 198, 203, "R260A", "mutant" ], [ 204, 211, "mutants", "protein_state" ] ] }, { "sid": 227, "sent": "Functional importance of Roquin-1 target motifs in cells.", "section": "FIG", "ner": [ [ 25, 33, "Roquin-1", "protein" ] ] }, { "sid": 228, "sent": "(a) Overview of the Ox40 3\u2032-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2\u2013440) and either the complete wild-type Ox40 3\u2032-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a).", "section": "FIG", "ner": [ [ 20, 24, "Ox40", "protein" ], [ 25, 31, "3\u2032-UTR", "structure_element" ], [ 36, 45, "truncated", "protein_state" ], [ 46, 53, "mutated", "protein_state" ], [ 83, 87, "EMSA", "experimental_method" ], [ 134, 138, "Ox40", "protein" ], [ 155, 159, "EMSA", "experimental_method" ], [ 208, 216, "Roquin-1", "protein" ], [ 245, 250, "2\u2013440", "residue_range" ], [ 276, 285, "wild-type", "protein_state" ], [ 286, 290, "Ox40", "protein" ], [ 291, 297, "3\u2032-UTR", "structure_element" ], [ 315, 324, "mutations", "experimental_method" ], [ 332, 335, "CDE", "structure_element" ], [ 341, 343, "SL", "structure_element" ], [ 349, 352, "ADE", "structure_element" ], [ 358, 360, "SL", "structure_element" ], [ 369, 372, "SLs", "structure_element" ] ] }, { "sid": 229, "sent": "It is noteworthy that the higher bands observed at large protein concentrations are probably additional nonspecific, lower-affinity interactions of Roquin-1 with the 3\u2032-UTR or protein aggregates.", "section": "FIG", "ner": [ [ 148, 156, "Roquin-1", "protein" ], [ 166, 172, "3\u2032-UTR", "structure_element" ] ] }, { "sid": 230, "sent": "(c) Relative Ox40 MFI normalized to expression levels from the Ox40 CDS construct.", "section": "FIG", "ner": [ [ 13, 17, "Ox40", "protein" ], [ 18, 53, "MFI normalized to expression levels", "evidence" ], [ 63, 67, "Ox40", "protein" ], [ 68, 71, "CDS", "structure_element" ] ] }, { "sid": 231, "sent": "Error bars show s.d. of seven (CDS, 1\u201340, 1\u201380, 1\u2013120 and full-length), six (ADE-like mut and CDE mut) or three (double mut) independent experiments.", "section": "FIG", "ner": [ [ 31, 34, "CDS", "structure_element" ], [ 36, 40, "1\u201340", "residue_range" ], [ 42, 46, "1\u201380", "residue_range" ], [ 48, 53, "1\u2013120", "residue_range" ], [ 58, 69, "full-length", "protein_state" ], [ 77, 80, "ADE", "structure_element" ], [ 86, 89, "mut", "protein_state" ], [ 94, 97, "CDE", "structure_element" ], [ 98, 101, "mut", "protein_state" ], [ 113, 123, "double mut", "protein_state" ] ] }, { "sid": 232, "sent": "Statistical significance was calculated by one-way analysis of variance (ANOVA) Kruskal\u2013Wallis test followed by Dunn\u2019s multiple comparison test (**P<0.01).", "section": "FIG", "ner": [ [ 43, 71, "one-way analysis of variance", "experimental_method" ], [ 73, 78, "ANOVA", "experimental_method" ], [ 80, 99, "Kruskal\u2013Wallis test", "experimental_method" ], [ 112, 143, "Dunn\u2019s multiple comparison test", "experimental_method" ] ] }, { "sid": 233, "sent": "(d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3\u2032-UTR (CDS, red line), Ox40 CDS with its wild-type 3\u2032-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line).", "section": "FIG", "ner": [ [ 4, 21, "mRNA decay curves", "evidence" ], [ 67, 79, "retroviruses", "taxonomy_domain" ], [ 91, 95, "Ox40", "protein" ], [ 96, 99, "CDS", "structure_element" ], [ 108, 114, "3\u2032-UTR", "structure_element" ], [ 116, 119, "CDS", "structure_element" ], [ 132, 136, "Ox40", "protein" ], [ 137, 140, "CDS", "structure_element" ], [ 150, 159, "wild-type", "protein_state" ], [ 160, 166, "3\u2032-UTR", "structure_element" ], [ 168, 179, "full length", "protein_state" ], [ 194, 198, "Ox40", "protein" ], [ 199, 210, "full length", "protein_state" ], [ 216, 223, "mutated", "protein_state" ], [ 224, 227, "ADE", "structure_element" ], [ 240, 243, "ADE", "structure_element" ], [ 249, 252, "mut", "protein_state" ], [ 266, 270, "Ox40", "protein" ], [ 271, 282, "full length", "protein_state" ], [ 288, 295, "mutated", "protein_state" ], [ 296, 299, "CDE", "structure_element" ], [ 312, 315, "CDE", "structure_element" ], [ 321, 324, "mut", "protein_state" ], [ 341, 345, "Ox40", "protein" ], [ 346, 357, "full length", "protein_state" ], [ 363, 370, "mutated", "protein_state" ], [ 371, 374, "ADE", "structure_element" ], [ 379, 382, "CDE", "structure_element" ], [ 391, 401, "Double mut", "protein_state" ] ] }, { "sid": 234, "sent": "mRNA half-life times were calculated with Graph Pad Prism.", "section": "FIG", "ner": [ [ 0, 20, "mRNA half-life times", "evidence" ] ] }, { "sid": 235, "sent": "Data collection and refinement statistics.", "section": "TABLE", "ner": [ [ 0, 41, "Data collection and refinement statistics", "evidence" ] ] }, { "sid": 236, "sent": "\u00a0\tROQ-Ox40ADE-like SL\tROQ-ADE SL\t \tData collection\t \t\u2003space group\tP21212\tP212121\t \t\u00a0\t\u00a0\t\u00a0\t \t\u2003Cell dimensions\t \t\u2003a, b, c (\u00c5)\t89.66, 115.79, 42.61\t72.90, 89.30, 144.70\t \t\u2003\u03b1, \u03b2, \u03b3 (\u00b0)\t90, 90, 90\t90, 90, 90\t \t\u2003Resolution (\u00c5)\t50\u20132.23 (2.29\u20132.23)\t50\u20133.0 (3.08\u20133.00)\t \t\u2003Rmerge\t5.9 (68.3)\t14.8 (93.8)\t \t\u2003I/\u03c3I\t14.9 (2.1)\t16.7 (3.1)\t \t\u2003Completeness (%)\t98.7 (97.7)\t99.9 (99.9)\t \t\u2003Redundancy\t3.9 (3.7)\t13.2 (12.7)\t \t\u00a0\t\u00a0\t\u00a0\t \tRefinement\t \t\u2003Resolution (\u00c5)\t2.23\t3.00\t \t\u2003No. reflections\t21,018\t18,598\t \t\u2003Rwork/Rfree\t21.8/25.7\t18.6/23.4\t \t\u00a0\t\u00a0\t\u00a0\t \t\u2003No. atoms\t \t\u2003Protein\t2,404\t4,820\t \t\u2003Ligand/ion\t894\t1,708\t \t\u2003Water\t99\t49\t \t\u2003B-factor overall\t47.2\t60.4\t \t\u00a0\t\u00a0\t\u00a0\t \tRoot mean squared deviations\t \t\u2003Bond lengths (\u00c5)\t0.006\t0.014\t \t\u2003Bond angles (\u00b0)\t1.07\t1.77\t \t\u00a0\t\u00a0\t\u00a0\t \tRamachandran plot\t \t\u2003Most favoured (%)\t98.6\t99.8\t \t\u2003Additional allowed (%)\t1.4\t0.2\t \t", "section": "TABLE", "ner": [ [ 2, 5, "ROQ", "structure_element" ], [ 6, 10, "Ox40", "protein" ], [ 10, 13, "ADE", "structure_element" ], [ 19, 21, "SL", "structure_element" ], [ 22, 25, "ROQ", "structure_element" ], [ 26, 29, "ADE", "structure_element" ], [ 30, 32, "SL", "structure_element" ], [ 642, 670, "Root mean squared deviations", "evidence" ] ] }, { "sid": 237, "sent": "ADE, alternative decay element; CDE, constitutive decay element; SL, stem loop.", "section": "TABLE", "ner": [ [ 0, 3, "ADE", "structure_element" ], [ 5, 30, "alternative decay element", "structure_element" ], [ 32, 35, "CDE", "structure_element" ], [ 37, 63, "constitutive decay element", "structure_element" ], [ 65, 67, "SL", "structure_element" ], [ 69, 78, "stem loop", "structure_element" ] ] }, { "sid": 238, "sent": "For each data set, only one crystal has been used.", "section": "TABLE", "ner": [ [ 28, 35, "crystal", "evidence" ] ] }, { "sid": 239, "sent": "KD for selected RNAs obtained from SPR measurements with immobilized ROQ domain of Roquin-1.", "section": "TABLE", "ner": [ [ 0, 2, "KD", "evidence" ], [ 16, 20, "RNAs", "chemical" ], [ 35, 51, "SPR measurements", "experimental_method" ], [ 69, 72, "ROQ", "structure_element" ], [ 83, 91, "Roquin-1", "protein" ] ] } ] } }