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+<collection><source>PMC</source><date>20201216</date><key>pmc.key</key><document><id>4746701</id><infon key="license">CC BY</infon><passage><infon key="article-id_doi">10.1038/srep20261</infon><infon key="article-id_pii">srep20261</infon><infon key="article-id_pmc">4746701</infon><infon key="article-id_pmid">27064360</infon><infon key="elocation-id">20261</infon><infon key="license">This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/</infon><infon key="name_0">surname:Jeong;given-names:Hanbin</infon><infon key="name_1">surname:Sim;given-names:Hyo Jung</infon><infon key="name_2">surname:Song;given-names:Eun Kyung</infon><infon key="name_3">surname:Lee;given-names:Hakbong</infon><infon key="name_4">surname:Ha;given-names:Sung Chul</infon><infon key="name_5">surname:Jun;given-names:Youngsoo</infon><infon key="name_6">surname:Park;given-names:Tae Joo</infon><infon key="name_7">surname:Lee;given-names:Changwook</infon><infon key="section_type">TITLE</infon><infon key="type">front</infon><infon key="volume">6</infon><infon key="year">2016</infon><offset>0</offset><text>Crystal structure of SEL1L: Insight into the roles of SLR motifs in ERAD pathway</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>81</offset><text>Terminally misfolded proteins are selectively recognized and cleared by the endoplasmic reticulum-associated degradation (ERAD) pathway. SEL1L, a component of the ERAD machinery, plays an important role in selecting and transporting ERAD substrates for degradation. We have determined the crystal structure of the mouse SEL1L central domain comprising five Sel1-Like Repeats (SLR motifs 5 to 9; hereafter called SEL1Lcent). Strikingly, SEL1Lcent forms a homodimer with two-fold symmetry in a head-to-tail manner. Particularly, the SLR motif 9 plays an important role in dimer formation by adopting a domain-swapped structure and providing an extensive dimeric interface. We identified that the full-length SEL1L forms a self-oligomer through the SEL1Lcent domain in mammalian cells. Furthermore, we discovered that the SLR-C, comprising SLR motifs 10 and 11, of SEL1L directly interacts with the N-terminus luminal loops of HRD1. Therefore, we propose that certain SLR motifs of SEL1L play a unique role in membrane bound ERAD machinery.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1119</offset><text>Protein quality control in the endoplasmic reticulum (ER) is essential for maintenance of cellular homeostasis in eukaryotes and is implicated in many severe diseases. Terminally misfolded proteins in the lumen or membrane of the ER are retrotranslocated into the cytosol, polyubiquitinated, and degraded by the proteasome. The process is called ER-associated protein degradation (ERAD) and is conserved in all eukaryotes. Accumulating studies have identified key components for ERAD, including HRD1, SEL1L (Hrd3p), Derlin-1, -2, -3 (Der1p), HERP-1, -2 (Usa1p), OS9 (Yos9), XTP-B, and Grp94, all of which are involved in the recognition and translocation of the ERAD substrates in yeast and metazoans. The components are differentially localized from the lumen and membrane of the ER to the cytosol, and have different functions in the ERAD process. Yeast ERAD components, which have been extensively characterized through genetic and biochemical studies, are comparable with mammalian ERAD components, sharing similar molecular functions and structural composition.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2186</offset><text>The HRD1 E3 ubiquitin ligase, which is embedded in the ER membrane, is involved in translocating ERAD substrates across the ER membrane and catalyzing substrate ubiquitination via its cytosolic RING finger domain. SEL1L, the mammalian homolog of Hrd3p, associates with HRD1, mediates HRD1 interactions with the ER luminal lectin OS9, and recognizes substrates to be degraded. In particular, SEL1L is crucial for translocation of Class I major histocompatibility complex (MHC) heavy chains (HCs). Recent research based on the inducible Sel1l knockout mouse model highlights the physiological functions of SEL1L. SEL1L is required for ER homeostasis, which is essential for protein translation, pancreatic function, and cellular and organismal survival. However, despite the functional importance of SEL1L, the molecular structure of SEL1L has not been solved. Previous biochemical studies reveal that SEL1L is a type I transmembrane protein and has a large luminal domain comprising sets of repeated Sel1-like (SLR) motifs. The SLR motif is a structural motif that closely resembles the tetratricopeptide-repeat (TPR) motif, which is a protein-protein interaction module. Although there is evidence that the luminal domain of SEL1L is involved in substrate recognition or in forming complexes with chaperones, it is not known how the unique structure of the repeated SLR motifs contributes to the molecular function of the HRD1-SEL1L E3 ligase complex and affects ERAD at the molecular level. Furthermore, while repeated SLR motifs are commonly found in tandem arrays, the SLR motifs in SEL1L are, according to the primary structure prediction of SEL1L, interspersed among other sequences in the luminal domain and form three SLR domain clusters. Therefore, the way in which these unique structural features of SEL1L are related to its critical function in ERAD remains to be elucidated.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>4073</offset><text>To clearly understand the biochemical role of the SLR domains of SEL1L in ERAD, we determined the crystal structure of the central SLR domain of SEL1L. We found that the central domain of SEL1L, comprising SLR motifs 5 through 9 (SEL1Lcent), forms a tight dimer with two-fold symmetry due to domain swapping of the SLR motif 9. We also found that SLR-C, consisting of SLR motifs 10 and 11, directly interacts with the N-terminus luminal loop of HRD1. Based on these observations, we propose a model wherein the SLR domains of SEL1L contribute to the formation of stable oligomers of the ERAD translocation machinery, which is indispensable for ERAD.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>4723</offset><text>Results</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>4731</offset><text>Structure Determination of SEL1Lcent</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>4768</offset><text>The Mus musculus SEL1L protein contains 790 amino acids and has 17% sequence identity to its yeast homolog, Hrd3p. Mouse SEL1L contains a fibronectin type II domain at the N-terminus, followed by 11 SLR motifs and a single transmembrane domain at the C-terminus (Fig. 1A). The 11 SLR motifs are located in the ER lumen and account for more than two thirds of the mass of full-length SEL1L. The SLR motifs can be grouped into three regions due to the presence of linker sequences among the groups of SLR motifs: SLR-N (SLR motifs 1 to 4), SLR-M (SLR motifs 5 to 9), and SLR-C (SLR motifs 10 to 11) (Fig. 1A). Sequence alignment of the SLR motifs revealed that there is a short linker sequence (residues 336–345) between SLR-N and SLR-M and a long linker sequence (residues 528–635) between SLR-M and SLR-C (Fig. 1A). We first tried to prepare the full-length mouse SEL1L protein, excluding the transmembrane domain at the C-terminus (residues 735–755), by expression in bacteria. However, the full-length SEL1L protein aggregated in solution and produced no soluble protein. To identify a soluble form of SEL1L, we generated serial truncation constructs of SEL1L based on the predicted SLR motifs and highly conserved regions across several different species. Both SLR-N (residues 194–343) and SLR-C (residues 639–719) alone could be solubilized with an MBP tag at the N-terminus, but appeared to be polydisperse when analyzed by size-exclusion chromatography. However, the central region of SEL1L, comprising residues 337–554, was soluble and homogenous in size, as determined by size-exclusion chromatography. To define compact domain boundaries for the central region of SEL1L, we digested the protein with trypsin and analyzed the proteolysis products by SDS-PAGE and N-terminal sequencing. The results of this preliminary biochemical analysis suggested that SEL1L residues 348–533 (SEL1Lcent) would be amenable to structural analysis (Fig. 1A).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>6731</offset><text>Crystals of SEL1Lcent grew in space group P21 with four copies of SEL1Lcent (a total of 82 kDa) in the asymmetric unit. The structure was determined by the single-wavelength anomalous diffraction (SAD) method using selenium as the anomalous scatterer (Table 1 and Methods). The assignment of residues during model building was aided by the selenium atom positions, and the structure was refined with native data to 2.6 Å resolution with Rwork/Rfree values of 20.7/27.7%. Statistics for data collection and refinement are presented in Table 1.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>7279</offset><text>Overall Structure of SEL1Lcent</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>7310</offset><text>The mouse SEL1Lcent crystallized as a homodimer, and there were two homodimers in the crystal asymmetric unit (Fig. 1B,C, Supplementary Fig. 1). The two SEL1Lcent molecules dimerize in a head-to-tail manner through a two-fold symmetry interface resulting in a cosmos-like shaped structure (Fig. 1B). The resulting structure resembles the yin-yang symbol with overall dimensions of 60 × 60 × 25 Å, where a SEL1Lcent monomer corresponds to half the symbol. The dimer formation buries a surface area of 1670 Å2 for each monomer, and no significant differences between the protomers were displayed (final root mean square deviation (RMSD) of 0.6 Å for all Cα atoms). Each protomer is composed of ten α-helices, which form the five SLRs, resulting in an elongated curved structure, confirming the primary structure prediction (Fig. 1D).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>8163</offset><text>The α-helices subdivide the structure into five pairs (A and B) as shown in a number of TPRs and SLRs. Helices A and B are 14 and 13 residues long, respectively, and the two helices are connected by a short turn and loop (Fig. 1D). In addition, a longer loop, consisting of approximately eight amino acids, is inserted between helix B of one SLR and helix A of the next SLR. This arrangement is a unique feature for SLRs among the major classes of repeats containing an α-solenoid. Starting from its N-terminus, the α-solenoid of SEL1L extends across a semi-circle in a right-handed superhelix fashion along the rotation axis of the yin-yang circle. However, the last helix, 9B, at the C-terminus adopts a different conformation, lying parallel to the long axis of helix 9A instead of forming an antiparallel SLR. This unique conformation of helix 9B most likely contributes to formation of the dimer structure of SEL1Lcent, as detailed below. With the exception of the last SLR, the four α-helix pairs possess similar conformations, with RMSD values of 0.7 Å for all Cα atoms. Although the sequence similarity for the pairwise alignments varies between 25% and 35%, all the residues present in the SLR motifs are conserved among the five pairs. The SLR domain of SLR-M ends at residue 524, and C-terminal amino acids 525–533 of the protein are not visible in the electron density map, suggesting that this region is highly flexible.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9617</offset><text>Since no information regarding dimer formation by SEL1L through its SLR motifs is available, we tested whether the SEL1Lcent dimer shown in our crystal structure is a biological unit. First, we cross-linked SEL1Lcent or a longer construct of SEL1L (SEL1Llong, residues 337–554) using various concentrations of glutaraldehyde (GA) or dimethyl suberimidate (DMS) and analyzed the products by SDS-PAGE. We detected bands at the mass of a dimer for both SEL1Lcent and SEL1Llong when cross-linked with low concentrations of GA (0.005%) or DMS (0.3 mM) (Supplementary Fig. 2A,B). Next, we conducted analytical ultracentrifugation of SEL1Lcent. Consistent with the cross-linking data, analytical ultracentrifugation revealed that the molecular weight of SEL1Lcent corresponds to a dimer (Supplementary Fig. 2C). Taken together, these data indicate that some kind of dimer is formed in solution.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>10509</offset><text>Dimer Interface of SEL1Lcent</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10538</offset><text>In contrast to a previously described SLR motif containing proteins that exist as monomers in solution, SEL1Lcent forms an intimate two-fold homotypic dimer interface (Figs 1B and 2A). The concave surface of each SEL1L domain comprising helix 5A to 9A encircles its dimer counterpart in an interlocking clasp-like arrangement. However, no interactions were seen between the two-fold-related protomers through the concave inner surfaces themselves. Rather, the unique structure of SLR motif 9, consisting of two parallel helices (9A and 9B), is located in the space generated by the concave surface and provides an extensive dimerization interface between the two-fold-related molecules (Fig. 2A). Helix 9B from one protomer inserts into the empty space surrounded by the concave region in the other monomer, forming a domain-swapped conformation.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>11385</offset><text>Three major contact interfaces are involved in the interactions, and all interfaces are symmetrically related between the dimer subunits (Fig. 2A). Structure-based sequence alignment of 135 SEL1L phylogenetic sequences using a ConSurf server revealed that the surface residues in the dimer interfaces were highly conserved among the SEL1L orthologs (Fig. 1E). First, helix 9B of each SEL1Lcent subunit interacts with residues lining the inner groove from the SLR α-helices (5B, 6B, 7B, and 8B) from its counterpart. In this interface, Leu 516 and Tyr 519 on helix 9B are located in the center, making hydrophobic interactions with Trp 478 on helix 8B, Val 444 on helix 7B, Phe 411 on helix 6B, and Leu 380 on helix 5B from the SEL1Lcent counterpart (Fig. 2A, Interface 1 detail). In addition to hydrophobic interactions, the side chain hydroxyl group of Tyr 519 and the main-chain oxygen of Ile 515 form H-bonds to the side chain of the conserved Gln 377 and His 381 on helix 5B of the two-fold-related protomer. The side chain of Gln 523 forms an H-bond to the side chain of Asp 480 on the two-fold-related protomer (Fig. 2A, Interface 1 detail). Second, the residues from helix 9A interact with the residues from helix 5A of its counterpart in a head-to-tail orientation. In this interface, the interacting residues on helix 9A, including Leu 503, Tyr 499, and the aliphatic side chain of Lys 500, form an extensive network of van der Waals contacts with the hydrophobic residues of the counterpart helix 5A, including Tyr 360, Leu 356, Tyr 359, and Leu 363. In addition to hydrophobic interactions, the side chains of Asn 507 and Ser 510 on helix 9A make H-bonds with highly conserved Arg 384 in the loop between helix 5B and 6A from the two-fold-related protomer (Fig. 2A, Interface 2 detail). Third, the helix 9B from each protomer is involved in the dimer interaction by forming a two-fold antiparallel symmetry. In particular, the side chains of hydrophobic residues, including Phe 518, Leu 521, and Met 524, are directed toward each other, where they make both inter- and intramolecular contacts (Fig. 2A, Interface 3 detail).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>13524</offset><text>To further investigate the interactions observed in our crystal structure, we generated a C-terminal deletion mutant (SEL1L348–497) lacking SLR motif 9 (helix 9A and 9B) from SEL1Lcent for comparative analysis. The deletion mutant and the wild-type SEL1Lcent showed no difference in spectra by CD spectroscopy, indicating that the deletion of the SLR motif 9 did not affect the secondary structure of SEL1Lcent (Supplementary Fig. 3). However, the mutant behaved as a monomer in size-exclusion chromatography and analytical ultracentrifugation experiments (Fig. 2B, Supplementary Fig. 2C). Additionally, to further validate the key residues involved in dimer formation, we generated a triple point mutant (Interface 1, I515A, L516A, and Y519A) of the hydrophobic residues that are involved in dimerization. The triple point mutant eluted at the monomer position upon size-exclusion chromatography, while the negative control point mutant (Q460A) eluted at the same position as the wild-type. Notably, a single-residue mutation (L521A in interface 3) abolished the dimerization of SEL1Lcent (Fig. 2B). Leu 521 is located in the dimerization center of the antiparallel 9B helices in the SEL1Lcent dimer.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>14728</offset><text>Taken together, these structural and biochemical data demonstrate that SEL1Lcent exists as a dimer in solution and that SLR motif 9 in SEL1Lcent plays an important role in generating a two-fold dimerization interface.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>14946</offset><text>The Two Glycine Residues (G512 and G513) Create a Hinge for Domain Swapping of SLR Motif 9</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15037</offset><text>SLRs of mouse SEL1L were predicted using the TPRpred server. Based on the prediction, full-length SEL1L contains a total of 11 SLR motifs, and our construct corresponds to SLR motifs 5 through 9. Although amino acid sequences from helix 9A and 9B correctly aligned with the regular SLR repeats and corresponded to SLR motif 9 (Fig. 3A), the structural arrangement of the two helices deviated from the common structure for the SLR motif. According to our crystal structure, the central axis of helix 9B is almost parallel to that of helix 9A (Fig. 3B). However, this unusual conformation of SLR motif 9 seems to be essential for dimer formation, as described earlier. For this structural geometry, two adjacent residues, Gly 512 and Gly 513, in SEL1L confer flexibility at this position by adopting main-chain dihedral angles that are disallowed for non-glycine residues. The phi and psi dihedrals are 100° and 20° for Gly 512, and 110° and −20° for Gly 513, respectively (Fig. 3C). Gly 513 is conserved among other SLR motifs in the SEL1Lcent, but Gly 512 is present only in the SLR motif 9 of SEL1Lcent (Fig. 3A). Thus, the Gly-Gly residues generate an unusual sharp bend at the C-terminal SLR motif 9. The involvement of a glycine residue in forming a hinge for domain swapping has been reported previously. The significance of Gly 513 is further highlighted by its absolute conservation among different species, including the budding yeast homolog Hrd3p.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>16501</offset><text>To further investigate the importance of Gly 512 and Gly 513 in the unusual SLR motif geometry, we generated a point mutation (Gly to Ala), which restricts the flexibility. Although the Gly 512 and Gly 513 residues are closely surrounded by helix 9B from the counter protomer, there is enough space for the side chain of alanine, suggesting that no steric hindrance would be caused by the mutation (Fig. 3C). This means that the effect of the mutation is mainly to generate a more restricted geometry at the hinge region. G512A or G513A alone showed no differences from wild-type in terms of the size-exclusion chromatography elution profile (Fig. 3D), suggesting that the restriction for single glycine flexibility would not be enough to break the swapped structure of helix 9B. However, the double mutant (G512A/G513A) eluted over a broad range and much earlier than the wild-type, suggesting that mutation of the residues involved in the hinge linking helix 9A and 9B significantly affected the geometry of helix 9B in generating domain swapping, and eventually altered the overall oligomeric state of SEL1Lcent into a polydisperse pattern (Fig. 3D, Supplementary Fig. 6). When the residues were mutated to lysine (G512K/G513K), the mutant not only restricted the geometry of residues at the hinge but also generated steric hindrance during interaction with the counter protomer of SEL1Lcent, thereby inhibiting self-association of SEL1Lcent completely. The G512K/G513K double mutant eluted at the monomer position in size-exclusion chromatography (Fig. 3D). A previous study shows that induction of steric hindrance by mutation destabilizes the dimerization interface of a different protein, ClC transporter.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>18214</offset><text>Collectively, these data suggest that the Gly 512 and Gly 513 at the connection between helix 9A and 9B play a crucial role in forming the domain-swapped conformation that enables dimer formation.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>18411</offset><text>SEL1L Forms Self-oligomers through SEL1Lcent domain in vivo</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>18471</offset><text>Next, we examined if SEL1L also forms self-oligomers in vivo using HEK293T cells. We generated full-length SEL1L-HA and SEL1L-FLAG fusion constructs and co-transfected the constructs into HEK293T cells. A co-immunoprecipitation assay using an anti-FLAG antibody followed by Western blot analysis using an anti-HA antibody showed that full-length SEL1L forms self-oligomers in vivo (Fig. 4A). To further examine whether the SEL1Lcent domain is sufficient to physically interact with full-length SEL1L, we generated SEL1Lcent and SLR motif 9 deletion (SEL1L348–497) construct, which were fused to the C-terminus of SEL1L signal peptides. Co-immunoprecipitation analysis showed that the SEL1Lcent was sufficient to physically interact with the full-length SEL1L, while SEL1L348–497 failed to do so (Fig. 4A). Interestingly, however, the expression level of SEL1L348–497 was consistently lower than that of SEL1Lcent (Fig. 4A,B). Semi-quantitative RT-PCR revealed no significant difference in transcriptional levels of the two constructs (data not shown). We speculated that SEL1L348–497 could be secreted while the SEL1Lcent is retained in the ER by association with the endogenous ERAD complex. Indeed, immunoprecipitation followed by western blot analysis using the culture medium detected secreted SEL1L348–497 fragment, but not SEL1Lcent (Fig. 4B). We next examined if the reason why SEL1L348–497 failed to bind to the full-length SEL1L may be because of the lower level of SEL1L348–497 in the ER lumen compared to SEL1Lcent fragment. In order to retain two SEL1L fragments in the ER lumen, we added KDEL ER retention sequence to the C-terminus of both fragments. Indeed, the addition of KDEL peptide increased the level of SEL1L348–497 in the ER lumen (Fig. 4D,E) and the immunostaining analysis showed both constructs were well localized to the ER (Fig. 4C). We further analyzed whether SEL1Lcent may competitively inhibit the self-oligomerization of SEL1L in vivo. To this end, we co-transfected the differentially tagged full-length SEL1L (SEL1L-HA and SEL1L-FLAG) and increasing doses of SEL1Lcent-KDEL, SEL1L348–497-KDEL or SEL1Lcent (L521A)-KDEL, respectively. Co-immunoprecipitation assay revealed that wild-type SEL1Lcent-KDEL, indeed, competitively disrupted the self-association of the full-length SEL1L (Fig. 4E). In contrast, SEL1L348–497-KDEL and the single-residue mutation L521A in SEL1Lcent did not competitively inhibit the self-association of full-length SEL1L (Fig. 4E,F). These data suggest that the SEL1L forms self-oligomers and the oligomerization is mediated by the SEL1Lcent domain in vivo.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>21109</offset><text>Structural Comparison of SEL1L SLRs with TPRs or SLRs of Other Proteins</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>21181</offset><text>Previous studies reveal that TPRs and SLRs have similar consensus sequences, suggesting that their three-dimensional structures are also similar. The superposition of isolated TPRs from Cdc23 (S. pombe, cell division cycle 23 homolog, PDB code 3ZN3) and SLRs from HcpC (Helicobacter Cysteine-rich Protein C, PDB code 1OUV) yields RMSDs below 1 Å, confirming that the isolated repeats are indeed similar. This is relevant to SLR motifs in SEL1L, as isolated SLR motifs from SEL1Lcent showed good structural alignment with isolated TPRs (RMSD 1.6 Å for all Cα chains) from Cdc23N-term and SLRs (RMSD 0.6 Å for all Cα chains) from HcpC (Fig. 5A). However, superimposing the structure of SLR motifs 5 to 9 from SEL1Lcent onto the overall Cdc23N-term or full-length HcpC structures revealed that SLR motifs 5 to 9 in SEL1Lcent have a different superhelical structure than either Cdc23 or HcpC (RMSD values of &gt;2.5 Å for Cα atoms) (Fig. 5B). The differences may result from the differing numbers of residues in the loops and differences in antiparallel helix packing. Moreover, there are conserved disulfide bonds in the SLR motifs of HcpC and HcpB, but no such bonds are observed in SEL1Lcent. These factors contribute to the differences in the overall conformation of the SLR motifs in SEL1L and other SLR or TPR motif-containing proteins.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>22532</offset><text>Another major difference in the structure of SLR motifs between SEL1L and HcpC is the oligomeric state of proteins. The TPR motif is involved in the dimerization of proteins such as Cdc23, Cdc16, and Cdc27. In particular, the N-terminal domain of Cdc23 (Cdc23N-term) has a TPR-motif organization similar to that of the SLR motif in SEL1Lcent. The seven TPR motifs of Cdc23N-term are assembled into a superhelical structure, generating a hollow surface and encircling its dimer counterpart in an interlocking clasp-like arrangement (Fig. 5C). The TPR motif 1 (TPR1) of each Cdc23N-term subunit is located in the hollow surface of the counter subunit and interacts with residues lining the inner groove TPR α-helices, generating two-fold symmetry homotype interactions. However, in this structure, a conformational change in the TPR motif itself is not observed.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>23397</offset><text>Self-association of HcpC has not been reported, and there is no domain-swapped structure in the SLR motifs of HcpC, in contrast to that observed in SEL1Lcent. Although SEL1L contains a number of SLR motifs comparable to HcpC, the SLR motifs in SEL1L are interrupted by other sequences, making three SLR motif clusters (Fig. 1A). The interrupted SLR motifs may be required for dimerization of SEL1Lcent, as five SLR motifs are more than enough to form the semicircle of the yin-yang symbol (Fig. 1B). Helix 5A from SLR motif 5 meets helix 9A from SLR motif 9 of the counterpart SEL1L. If the SLR motifs 5 to 9 were not isolated from other SLR motifs, steric hindrance could interfere with dimerization of SEL1L. This is one of the biggest differences from TPRs in Cdc23 and from the SLRs in HcpC, where the motifs exist in tandem. TPR and SLR motifs are generally involved in protein-protein interaction modules, and the sequences between the SLR motifs of SEL1L might actually facilitate the self-association of this protein.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>24423</offset><text>SLR-C of SEL1L Binds HRD1 N-terminus Luminal Loop</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>24473</offset><text>Based on the structural data presented herein, a possible arrangement of membrane-associated ERAD components in mammals, highlighting the molecular functions of SLR domains in SEL1L, is shown in Fig. 6C. We suggest that the middle SLR domains are involved in the dimerization of SEL1L based on the crystal structure and biochemical data. SLR-C, which contains SLR motifs 10 to 11, might be involved in the interaction with HRD1. Indirect evidence from a previous yeast study shows that the circumscribed region of C-terminal Hrd3p, specifically residues 664–695, forms contacts with the Hrd1 luminal loops. The Hrd3p residues 664–695 correspond to mouse SEL1L residues 696–727, which include the entire helix 11B (residue 697–709) of SLR motif 11 and a well-conserved adjacent region (Supplementary Fig. 4). This observation is supported by the following: (1) the meticulous range of SLR motif 10 to 11 is newly established from a structure-guided SLR motif alignment, based on the present structure study, and (2) the relatively high sequence conservation between mammalian SEL1L and yeast Hrd3p around SLR motifs 10 to 11, which contain contact regions with HRD1 (Hrd1p) (Supplementary Figs. 4 and 5). To address this hypothesis, we prepared constructs encoding mouse HRD1 luminal fragments fused to GST as shown in Fig. 6A, and tested their ability to bind certain SLR motifs in SEL1L. The fusion proteins were immobilized on glutathione-Sepharose beads and probed for binding to SLR-N, SLR-M, SLR-C, and monomer form of SLR-M (SLR-ML521A). Figure 6B shows that the SLR-C, consisting of SLR motifs 10 and 11, exclusively interacts with N-terminal luminal loop (residues 21–42) of HRD1.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>26172</offset><text>The molecular functions of SLR-N are unclear. One possibility is that SLR-N contributes to substrate recognition of proteins to be degraded because there are a couple of putative glycosylation sites within the SLR-N domain (Fig. 1A). SEL1Lcent contains a putative N-glycosylation site, Asn 427, which is highly conserved among different species and structurally exposed to the surface of the SEL1L dimer according to the crystal structure (Fig. 6C).</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_1</infon><offset>26622</offset><text>Discussion</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>26633</offset><text>Many reports demonstrate that membrane-bound ERAD machinery proteins in yeast, such as Hrd1p, Der1p, and Usa1p, are involved in oligomerization of ERAD components. The Hrd1p complex forms dimers upon sucrose gradient sedimentation and size-exclusion chromatography. Previous data show that HA-epitope-tagged Hrd3p or Hrd1p efficiently co-precipitate with unmodified Hrd3p and Hrd1p, respectively, suggesting that both Hrd1p and Hrd3p homodimers are involved in self-association of the Hrd complex. Considering that the functional and structural composition of ERAD components are conserved in both yeast and mammals, we propose that the mammalian ERAD components also form self-associating oligomers. This hypothesis is supported by cross-linking data suggesting that human HRD1 forms a homodimer. Consistent with the previous data, our crystal structure and biochemical data demonstrate that mouse SEL1Lcent exists as a homodimer in the ER lumen via domain swapping of SLR motif 9. We need to further test whether there are contacts involved in dimer formation in SEL1L in addition to those in the SLR-M region.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>27746</offset><text>In yeast, Usa1p acts as a scaffold for Hrd1p and Der1p, in which the N-terminus of Usa1p interacts with the C-terminal 34 amino acids of Hrd1p in the cytosol to induce oligomerization of Hrd1p, which is essential for its activity. However, metazoans lack a clear Usa1p homolog. Although mammalian HERP has sequences and domains that are conserved in Usa1p, the molecular function of HERP is not clearly related to that of Usa1p. Rather, recent research shows that a transiently expressed HRD1-SEL1L complex alone associates with the ERAD lectins OS9 or XTP-B and is sufficient to facilitate the retrotranslocation and degradation of the model ERAD substrate α-antitrypsin null Hong-Kong (NHK) and its variant, NHK-QQQ, which lacks the N-glycosylation sites. Assuming that the correct oligomerization of ERAD components may be critical for their function, we hypothesize that homodimer formation of SEL1L in the ER lumen may stabilize oligomerization of the HRD complex, given that SEL1L forms a stoichiometric complex with HRD1. This is further supported by our data showing that the SLR-C of SEL1L directly interacts with the luminal fragment of HRD1 in the ER lumen.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>28919</offset><text>Although the organization of membrane-bound HRD complex components may be very similar between metazoans and yeast, the molecular details of interactions between the components may not necessarily be conserved. In yeast, it is unclear whether self-association of Hrd3p is due to SLR motifs because the sequence of Hrd3p does not align precisely with the SLR motifs in SEL1L. Furthermore, we are uncertain whether self-association of Hrd3p contributes to formation of the active form of the Hrd1p complex. Recently, a truncated version of Yos9 was shown to form a dimer in the ER lumen and to contribute to the dimeric state of the Hrd1p complex. This interaction seems to be weak because direct Yos9-Yos9 interactions were not detected in immunoprecipitation experiments from yeast cell extracts containing different epitope-tagged variants of Yos9. However, the dimerization of Yos9 could provide a higher stability for the Hrd1p complex oligomer. Likewise, the dimerization of SEL1L might provide stability for the mammalian HRD oligomer complex. Further cell biological studies are required to clarify whether SEL1L (Hrd3p) dimerization could be cooperative with the oligomerization of the HRD complex.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>30125</offset><text>Considering that it is very important for the function of the HRD complex that the components assemble as oligomers, we believe that the self-association of SEL1L strongly contributes to generating active forms of the HRD complex, even in the absence of Usa1p, in metazoans. These findings should provide a foundation for molecular-level studies to understand the membrane-associated HRD complex assembly in ERAD.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>30539</offset><text>Methods</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>30547</offset><text>Protein Production</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>30566</offset><text>The expression and purification of SEL1L was performed as described previously.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>30646</offset><text>Crystallization and SAD Structure Determination of SEL1Lcent</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>30707</offset><text>Crystals were grown using the hanging-drop vapor diffusion method at 4 °C. For crystallization of the M. musculus SEL1Lcent, 1 μl of protein solution (in 25 mM Tris-HCl, 150 mM NaCl, and 5 mM DTT, pH 7.5) was equilibrated with 1 μl of well solution (30% isopropanol, 100 mM NaCl, 100 mM Tris, 5 mM DTT, and 20 mM phenol, pH 8.5). The crystals, which appeared after 4 days, contain two SEL1Lcent dimers in the asymmetric unit (space group P21, a = 29.13, b = 110.52, c = 109.81 Å, α = 90.00, β = 90.61, γ = 90.00, 44% solvent). For X-ray diffraction experiments, crystals were transferred to well solution plus paraffin-oil, then flash frozen in liquid nitrogen.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>31422</offset><text>SAD data were collected with a Se-Met crystal at beamline 7A of the Pohang Accelerator Laboratory (PAL) and processed using HKL2000 software. Native data (2.6 Å resolution) were collected from a single frozen crystal at the same beamline of PAL and were integrated and scaled as described above. The SAD data analysis was performed using Phenix software using data between 50 and 2.9 Å resolution. Phenix identified 31 of the 32 selenium sites and refined these to give a mean f.o.m. = 0.472. Electron density modification, including non-crystallographic symmetry (NCS) averaging, using the RESOLVE software yielded an initial electron density map of excellent quality. Model building and refinement were carried out with the Coot and Phenix programs, respectively. The final model was refined to an R factor of 20.7% (Rfree = 27.7%) for native data between 30 and 2.6 Å resolution (Table 1). The final model consisted of 5402 protein atoms and 47 water molecules. There were no outliers in a Ramachandran plot of the final model. The model contained four copies of SEL1Lcent (residues 348–533) in the asymmetric unit. Of these, the following residues were not modeled due to weak electron densities: SEL1Lcent residues 348–351, 420, 421, and 525–533 in the first copy; residues 348–351 and 525–533 in the second and third copies; and residues 348–352 and 525–533 in the fourth copy. The X-ray data and refinement statistics are summarized in Table 1.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>32905</offset><text>Cell Culture and Plasmids Construction</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>32944</offset><text>HEK293T cells were cultured in DMEM (Gibco) supplemented with 10% FBS. The mouse Sel1L gene was cloned into pCS108 and the 3 × HA or 3 × FLAG tag was fused to the C-terminus of SEL1L. The signal peptide from Xenopus Sel1L was cloned into pCS108 and the mouse SEL1Lcent domain, SEL1L (348-497) fragments, and SEL1Lcent (L521A) were fused to the C-terminus of the signal peptide. Then, a 3 × HA or a 3 × FLAG tag was fused to the C-terminus of the constructs. For the ER retention signal, the KDEL sequences were added to the C-terminus of the fragments. The plasmids were transfected using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s manual.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>33634</offset><text>Western Blot Analysis and Immunostaining</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>33675</offset><text>For western blot analysis, HEK293T cells were transfected with the indicated construct and harvested after washing in PBS. The cells were homogenized in lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Triton X-100, 5% glycerol), supplemented with protease and phosphatase inhibitor cocktails. Homogenates were cleared by centrifugation at 13,200 rpm for 15 minutes at 4 °C. The lysates were subsequently used for either co-immunoprecipitation experiment or western blot analysis. For the western blot analysis, the samples were run onto 6–12% polyacrylamide gel. Blots were blocked in 5% TBS + 0.05% Tween 20 and incubated with anti-DDDD-K (Abcam) or anti-HA (Roche) antibodies. Proteins were visualized using HRP-conjugated secondary antibodies (1:4000) and SuperSignal West Pico Chemiluminescent Substrate or SuperSignal West Dura Extended Duration Substrate (Thermo) and exposed to ChemiDoc MP (Bio-Rad).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>34604</offset><text>For immunostaining, the cells were fixed in 4% formaldehyde and incubated with the indicated antibodies. The coverslips were incubated in blocking solution (10% FBS + 2% DMSO in TBS + 0.1% Triton X-100) at room temperature for 30 minutes to block non-specific binding. Fluorescent labeling was performed using Alexa Fluor 555 or 488-conjugated secondary antibodies and nuclei were stained with DAPI. The samples were mounted and confocal images were obtained using a Zeiss LSM700.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>35095</offset><text>GST Pull-down Assay</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>35115</offset><text>For pull-down experiments, 400 μg of HRD1 luminal fragment GST-fusion proteins were incubated with 5 μl of a 50% (v/v) slurry of glutathione sepharose 4B beads (GE Healthcare) for 50 min at 4 °C. Beads were washed twice with buffer A (150 mM NaCl, 25 mM sodium phosphate pH 7.5, 5 mM DTT), and then mixed with 100 μg of MBP-SEL1L protein (SLR-N, SLR-M, SLR-C, and SLR-ML521A) in buffer A, in a total assay volume of 500 μl. The assay mix was incubated at 4 °C for 15 minutes, and beads were washed twice with 500 μl buffer A. Proteins were eluted with SDS sample buffer, and analyzed by SDS-PAGE.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>35743</offset><text>Additional Information</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>35766</offset><text>Accession Numbers: The coordinates and structure factors have been deposited in the Protein Data Bank with the accession code of 5B26.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>35901</offset><text>How to cite this article: Jeong, H. et al. Crystal structure of SEL1L: Insight into the roles of SLR motifs in ERAD pathway. Sci. 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G.</infon><infon key="name_3">surname:Cowtan;given-names:K.</infon><infon key="pub-id_pmid">20383002</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr D</infon><infon key="type">ref</infon><infon key="volume">66</infon><infon key="year">2010</infon><offset>39759</offset><text>Features and development of Coot</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>39792</offset><text>Author Contributions H.J. and H.L. performed cloning, expression, and protein purification. H.J., S.H. and C.L. crystallized, collected X-ray data, and solved the protein structure. H.J., H.S., E.S. and Y.J performed biochemical experiments. H.J., T.P. and C.L. wrote the manuscript. T.P. and C.L. designed and supervised the project. All the authors discussed the results, commented on the manuscript, and approved the manuscript.</text></passage><passage><infon key="file">srep20261-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>40224</offset><text>Crystal Structure of SEL1Lcent.</text></passage><passage><infon key="file">srep20261-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>40256</offset><text>(A) The diagram shows the domain structure of Mus musculus SEL1L, as defined by proteolytic mapping and sequence/structure analysis. The 11 SLR motifs were divided into three groups (SLR-N, SLR-M, and SLR-C) due to the presence of linker sequences that are not predicted SLR motifs. Putative N-glycosylation sites are indicated by black triangles. We determined the crystal structure of the SLR-M, residues 348–533. (B) Ribbon diagram of the biological unit of the SEL1Lcent, viewed along the two-fold NCS axis. The crystal structure was determined by SAD phasing using selenium as the anomalous scatterer and refined to 2.6 Å resolution (Table 1). (C) SEL1Lcent ribbon diagram rotated 90° around a horizontal axis relative to (B). (D) One protomer of the SEL1Lcent dimer. This view is rotated about 90° anticlockwise from the bottom copy in (B), along the two-fold NCS axis. Starting from the N-terminus, SEL1Lcent has five SLR motifs comprising ten α helices. Each SLR motif (from 5 to 9) is indicated in a different color. (E) Evolutionary conservation of surface residues in SEL1Lcent, calculated using ConSurf, from a structure-based alignment of 135 SEL1L sequences. The surface is colored from red (high) to white (poor) according to the degree of conservation in the SEL1L phylogenetic orthologs. The ribbon diagram of the counterpart protomer is drawn to show the orientation of the SEL1Lcent dimer.</text></passage><passage><infon key="file">srep20261-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>41671</offset><text>Dimer Interface of SEL1Lcent.</text></passage><passage><infon key="file">srep20261-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>41701</offset><text>(A) The diagram on the left shows the SEL1Lcent dimer viewed along the two-fold symmetry axis. Three distinct contact regions are indicated with labeled boxes. The close-up view on the right shows the residues of SEL1Lcent that contribute to dimer formation via the three contact interfaces. Oxygen and nitrogen atoms are shown as red and blue, respectively. The yellow dotted lines indicate intermolecular hydrogen bonds between two protomers of SEL1Lcent. (B) Size-exclusion chromatography (SEC) analysis of the wild-type and dimeric interface SEL1Lcent mutants to compare the oligomeric states of the proteins. The standard molecular masses for the SEC experiments (top) were obtained from the following proteins: aldolase, 158 kDa; cobalbumin, 75 kDa; ovalbumin, 44 kDa; and carbonic anhydrase, 29 kDa. Chromatography was performed on a Superdex 200 column with a buffer containing 25 mM Tris, 150 mM NaCl, and 5 mM DTT (pH 7.5). The elution fractions, indicated by the gray shading, were run on SDS-PAGE and are shown below the gel-filtration elution profile. The schematic diagrams representing the protein constructs used in the SEC are shown on the left of each SDS-PAGE profile.</text></passage><passage><infon key="file">srep20261-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>42903</offset><text>Domain Swapping for Dimerization of SEL1Lcent.</text></passage><passage><infon key="file">srep20261-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>42950</offset><text>(A) Sequence alignment of the SLR motifs in SEL1L. The 11 SLR motifs were aligned based on the present crystal structure of SEL1Lcent. The sequences of SEL1Lcent included in the crystal structure are highlighted by the blue box. The secondary structure elements are indicated above the sequences, with helices depicted as cylinders. Residues that are conserved in at least 7 out of 11 sequences are red. The GG sequence in SLR motif 9, which creates the hinge for domain swapping (see text), is shaded yellow. Stars below the sequences indicate the specific residues that commonly appear in SLRs. (B) Structure alignment of five SLR motifs in SEL1Lcent is shown to highlight the unusual geometry of SLR motif 9. Each SLR motif is shown in a different color. The arrow indicates the direction of the helical axes. In SLR motif 9, the axes for the two helices are almost parallel, while the other SLR motifs adopt an α-hairpin structure. (C) Stereo view shows that the Gly 512 and Gly 513 residues are surrounded by neighboring residues from helix 9B from the counterpart dimer. Oxygen and nitrogen atoms are colored red and blue, respectively. The Gly 512 and Gly 513 residues are colored green and red, respectively. (D) The following point mutations were generated to check the effect of the Gly 512 and Gly 513 residues in terms of generating the hinge of SLR motif 9: G512A, G513A, G512A/G513A, and G512K/G513K. Size-exclusion chromatography was conducted as described in Fig. 2B. The standard molecular masses are shown at the top as in Fig. 2B.</text></passage><passage><infon key="file">srep20261-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>44504</offset><text>SEL1L forms self-oligomer mediated by the SEL1Lcent domain in vivo.</text></passage><passage><infon key="file">srep20261-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>44572</offset><text>(A) HEK293T cells were transfected with the indicated plasmid constructs and the lysates were immunoprecipitated with an anti-FLAG antibody followed by western blot analysis using an anti-HA antibody. The full-length SEL1L-FLAG was co-immunoprecipitated with the full-length SEL1L-HA. Also, SEL1Lcent was co-immunoprecipitated with the full-length SEL1L while the SLR motif 9 deletion failed to do so. (B) The HEK293T cells were transfected with the indicated plasmid constructs and the cell lysate and culture media were analyzed by western blot analysis and immunoprecipitation respectively. The SEL1L348–497 fragment was secreted to the culture media but the SEL1Lcent was retained in the ER. (C) SEL1Lcent-FLAG-KDEL and SEL1L348–497-FLAG-KDEL localized to the ER. The nuclei were stained with DAPI in blue. The ER was visualized with the anti-calnexin antibody in green. The SEL1L fragments were stained in red. (D) HEK293T cells were transfected with the indicated plasmid constructs and the lysates were immunoprecipitated with an anti-HA antibody followed by Western blot analysis using an anti-FLAG antibody. The full-length SEL1L forms self-oligomers and the SEL1Lcent-FLAG-KDEL was co-immunoprecipitated with full-length SEL1L-HA. The red asterisk indicates the expected signal for SEL1L348–497-FLAG-KDEL. SEL1L348–497-FLAG-KDEL did not co-immunoprecipitate with full-length SEL1L-HA. The white asterisks indicate non-specific bands. (E) SEL1Lcent-HA-KDEL competitively inhibited self-oligomerization of full-length SEL1L. The indicated plasmid constructs were transfected and immunoprecipitation assay was performed using an anti-FLAG antibody followed by western blot analysis using an anti-HA antibody. The red rectangle indicates competitively inhibited SEL1L self-oligomer formation by the increasing doses of SEL1Lcent-HA-KDEL. (F) L521A point mutant in SEL1Lcent did not inhibit the self-association of SEL1L.</text></passage><passage><infon key="file">srep20261-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>46507</offset><text>Comparison of SLR in SEL1L with TPR or Other SLR-Containing Proteins.</text></passage><passage><infon key="file">srep20261-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>46577</offset><text>(A) Ribbon diagram showing superimposition of an isolated TPR motif from Cdc23 and an SLR motif from SEL1Lcent (left), and SLR motifs in HcpC and SEL1Lcent (right). The SEL1L, Cdc23, and HcpC are colored magenta, green and cyan, respectively. Black arrows indicate the helical axes. The red arrow indicates disulfide bonds in the HcpC, and Cys residues involved in disulfide bonding are shown by a yellow line. (B) Ribbon representation showing superimposition of Cdc23 and SEL1Lcent (left) or HcpC and SEL1Lcent (right) to compare the overall organization of the α-solenoid domain. Both SEL1Lcent schematics are identically oriented for comparison. The Cα atoms of the residues in each α-solenoid domain are superimposed with a root-mean-squared deviation of 3.3 Å for Cdc23 and SEL1Lcent (left), and 2.5 Å for HcpC and SEL1Lcent (right). SEL1Lcent, Cdc23, and HcpC are colored as in (A). (C) Ribbon diagram showing the overall structure of Cdc23N-term (left) and SEL1Lcent (right) to compare their similarities regarding dimer formation through domain swapping. The view is along the two-fold axis.</text></passage><passage><infon key="file">srep20261-f6.jpg</infon><infon key="id">f6</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>47690</offset><text>The Role of SLR-C in ERAD machinery and Model for the Organization of Proteins in Membrane-Associated ERAD Components.</text></passage><passage><infon key="file">srep20261-f6.jpg</infon><infon key="id">f6</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>47809</offset><text>(A) Schematic diagram shows three HRD1 fragment constructs used in the GST pull-down experiment. (B) Pull-down experiments to examine the interactions between HRD luminal loops and certain SLR motifs of SEL1L. Fragments of the luminal loop of HRD1 fused to GST were immobilized on glutathione sepharose beads and incubated with purified three clusters of SLR motifs and monomer form of SLR-M (SLR-ML521A, right panel) in SEL1L. Proteins were analyzed by 12% SDS-PAGE and Coomassie blue staining. (C) Schematic representation of the organization of metazoan ERAD components in the ER membrane. The 11 SLR motifs of SEL1L were expressed with red cylinders and grouped into three parts (SLR-N, SLR-M, and SLR-C) based on the sequence alignment across the motifs and the crystal structure presented herein. We hypothesized that the interrupted SLR motifs of SEL1L have distinct functions such that the SLR-M is important for dimer formation of the protein, and SLR-C is involved in the interaction with HRD1 in the ER lumen. The surface representation of SEL1Lcent is placed in the same orientation as that shown in the schematic model to show that the putative N-glycosylation site, residue N427 (indicated in yellow), is exposed on the surface of the protein. The yellow arrow indicates self-association among the respective components.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_title_caption</infon><offset>49144</offset><text>Data Collection and Refinement Statistics.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot; border=&quot;1&quot;&gt;&lt;colgroup&gt;&lt;col align=&quot;left&quot;/&gt;&lt;col align=&quot;center&quot;/&gt;&lt;col align=&quot;center&quot;/&gt;&lt;/colgroup&gt;&lt;tbody valign=&quot;top&quot;&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;SEL1L&lt;sup&gt;cent&lt;/sup&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Data set:&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Native&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Se-SAD&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;PDB accession #:&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;5B26&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;X-ray source&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Beamline 7A, PAL&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Beamline 7A, PAL&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Temperature (K)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;100&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;100&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Space group:&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;P2&lt;sub&gt;1&lt;/sub&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;P2&lt;sub&gt;1&lt;/sub&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Cell parameters a, b, c (Å)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;29.13, 110.52, 109.81&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;29.51, 110.49, 109.81&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;90.00, 90.61, 90.00&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;90.00, 90.74, 90.00&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;3&quot; align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;Data processing&lt;/bold&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Wavelength (Å)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;1.00000&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;0.97923&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Resolution (Å)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;50-2.60&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;50–2.90&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;R&lt;sub&gt;merge&lt;/sub&gt; (%)&lt;xref ref-type=&quot;fn&quot; rid=&quot;t1-fn1&quot;&gt;a&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;6.1 (38.7)&lt;xref ref-type=&quot;fn&quot; rid=&quot;t1-fn1&quot;&gt;*&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;9.4 (40.6)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;I/σ&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;29.4 (4.6)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;21.0 (3.3)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Completeness (%)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;99.5 (99.3)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;99.9 (100.0)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Redundancy&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;4.1 (4.1)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;3.8 (3.8)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Measured reflections&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;88070&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;116951&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Unique reflections&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;21479&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;30823&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;3&quot; align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;Refinement statistics&lt;/bold&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Data range (Å)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;30-2.60&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Reflections&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;21446&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Nonhydrogen atoms&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;5402&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Water molecules&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;47&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;R.m.s. ∆ bonds (Å)&lt;xref ref-type=&quot;fn&quot; rid=&quot;t1-fn2&quot;&gt;b&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;0.010&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;R.m.s. ∆ angles (°)&lt;xref ref-type=&quot;fn&quot; rid=&quot;t1-fn2&quot;&gt;b&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;1.365&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;R-factor (%)&lt;xref ref-type=&quot;fn&quot; rid=&quot;t1-fn3&quot;&gt;c&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;20.7&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;R&lt;sub&gt;free&lt;/sub&gt; (%)&lt;xref ref-type=&quot;fn&quot; rid=&quot;t1-fn3&quot;&gt;c&lt;/xref&gt;&lt;sup&gt;,&lt;xref ref-type=&quot;fn&quot; rid=&quot;t1-fn4&quot;&gt;d&lt;/xref&gt;&lt;/sup&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;27.7&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Ramachandran plot, residues in&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Most favored regions (%)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;92.8&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Additional allowed regions (%)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;6.5&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Generously allowed regions (%)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;0.7&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Disallowed regions (%)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;0.0&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>49187</offset><text> 	SEL1Lcent	 	 	Data set:	Native	Se-SAD	 	PDB accession #:	5B26	 	 	X-ray source	Beamline 7A, PAL	Beamline 7A, PAL	 	Temperature (K)	100	100	 	Space group:	P21	P21	 	Cell parameters a, b, c (Å)	29.13, 110.52, 109.81	29.51, 110.49, 109.81	 	 	90.00, 90.61, 90.00	90.00, 90.74, 90.00	 	Data processing	 	Wavelength (Å)	1.00000	0.97923	 	Resolution (Å)	50-2.60	50–2.90	 	Rmerge (%)a	6.1 (38.7)*	9.4 (40.6)	 	I/σ	29.4 (4.6)	21.0 (3.3)	 	Completeness (%)	99.5 (99.3)	99.9 (100.0)	 	Redundancy	4.1 (4.1)	3.8 (3.8)	 	Measured reflections	88070	116951	 	Unique reflections	21479	30823	 	Refinement statistics	 	Data range (Å)	30-2.60	 	 	Reflections	21446	 	 	Nonhydrogen atoms	5402	 	 	Water molecules	47	 	 	R.m.s. ∆ bonds (Å)b	0.010	 	 	R.m.s. ∆ angles (°)b	1.365	 	 	R-factor (%)c	20.7	 	 	Rfree (%)c,d	27.7	 	 	Ramachandran plot, residues in	 	 	 	Most favored regions (%)	92.8	 	 	Additional allowed regions (%)	6.5	 	 	Generously allowed regions (%)	0.7	 	 	Disallowed regions (%)	0.0	 	 	</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>50207</offset><text>*Highest resolution shell is shown in parenthesis.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>50258</offset><text>aRmerge = 100 × ∑h∑i | Ii(h) − &lt;I(h) &gt;|/∑h &lt;I(h)&gt;, where Ii(h) is the ith measurement and &lt;I(h)&gt; is the weighted mean of all measurement of I(h) for Miller indices h.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>50446</offset><text>bRoot-mean-squared deviation (r.m.s. ∆) from target geometries.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>50512</offset><text>cR-factor = 100 × ∑|FP – FP(calc)|/∑ FP.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>50572</offset><text>dRfree was calculated with 5% of the data.</text></passage></document></collection>

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+<collection><source>PMC</source><date>20201220</date><key>pmc.key</key><document><id>4772114</id><infon key="license">CC BY</infon><passage><infon key="article-id_doi">10.1038/srep22324</infon><infon key="article-id_pii">srep22324</infon><infon key="article-id_pmc">4772114</infon><infon key="article-id_pmid">26927947</infon><infon key="elocation-id">22324</infon><infon key="license">This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/</infon><infon key="name_0">surname:Yokogawa;given-names:Mariko</infon><infon key="name_1">surname:Tsushima;given-names:Takashi</infon><infon key="name_2">surname:Noda;given-names:Nobuo N.</infon><infon key="name_3">surname:Kumeta;given-names:Hiroyuki</infon><infon key="name_4">surname:Enokizono;given-names:Yoshiaki</infon><infon key="name_5">surname:Yamashita;given-names:Kazuo</infon><infon key="name_6">surname:Standley;given-names:Daron M.</infon><infon key="name_7">surname:Takeuchi;given-names:Osamu</infon><infon key="name_8">surname:Akira;given-names:Shizuo</infon><infon key="name_9">surname:Inagaki;given-names:Fuyuhiko</infon><infon key="section_type">TITLE</infon><infon key="type">front</infon><infon key="volume">6</infon><infon key="year">2016</infon><offset>0</offset><text>Structural basis for the regulation of enzymatic activity of Regnase-1 by domain-domain interactions</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>101</offset><text>Regnase-1 is an RNase that directly cleaves mRNAs of inflammatory genes such as IL-6 and IL-12p40, and negatively regulates cellular inflammatory responses. Here, we report the structures of four domains of Regnase-1 from Mus musculus—the N-terminal domain (NTD), PilT N-terminus like (PIN) domain, zinc finger (ZF) domain and C-terminal domain (CTD). The PIN domain harbors the RNase catalytic center; however, it is insufficient for enzymatic activity. We found that the NTD associates with the PIN domain and significantly enhances its RNase activity. The PIN domain forms a head-to-tail oligomer and the dimer interface overlaps with the NTD binding site. Interestingly, mutations blocking PIN oligomerization had no RNase activity, indicating that both oligomerization and NTD binding are crucial for RNase activity in vitro. These results suggest that Regnase-1 RNase activity is tightly controlled by both intramolecular (NTD-PIN) and intermolecular (PIN-PIN) interactions.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1084</offset><text>The initial sensing of infection is mediated by a set of pattern-recognition receptors (PRRs) such Toll-like receptors (TLRs) and the intracellular signaling cascades triggered by TLRs evoke transcriptional expression of inflammatory mediators that coordinate the elimination of pathogens and infected cells. Since aberrant activation of this system leads to auto immune disorders, it must be tightly regulated. Regnase-1 (also known as Zc3h12a and MCPIP1) is an RNase whose expression level is stimulated by lipopolysaccharides and prevents autoimmune diseases by directly controlling the stability of mRNAs of inflammatory genes such as interleukin (IL)-6, IL-1β, IL-2, and IL-12p40. Regnase-1 accelerates target mRNA degradation via their 3′-terminal untranslated region (3′UTR), and also degrades its own mRNA.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1904</offset><text>Regnase-1 is a member of Regnase family and is composed of a PilT N-terminus like (PIN) domain followed by a CCCH-type zinc–finger (ZF) domain, which are conserved among Regnase family members. Recently, the crystal structure of the Regnase-1 PIN domain derived from Homo sapiens was reported. The structure combined with functional analyses revealed that four catalytically important Asp residues form the catalytic center and stabilize Mg2+ binding that is crucial for RNase activity. Several CCCH-type ZF motifs in RNA-binding proteins have been reported to directly bind RNA. In addition, Regnase-1 has been predicted to possess other domains in the N- and C- terminal regions. However, the structure and function of the ZF domain, N-terminal domain (NTD) and C-terminal domain (CTD) of Regnase-1 have not been solved.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2729</offset><text>Here, we performed structural and functional analyses of individual domains of Regnase-1 derived from Mus musculus in order to understand the catalytic activity in vitro. Our data revealed that the catalytic activity of Regnase-1 is regulated through both intra and intermolecular domain interactions in vitro. The NTD plays a crucial role in efficient cleavage of target mRNA, through intramolecular NTD-PIN interactions. Moreover, Regnase-1 functions as a dimer through intermolecular PIN-PIN interactions during cleavage of target mRNA. Our findings suggest that Regnase-1 cleaves its target mRNA by an NTD-activated functional PIN dimer, while the ZF increases RNA affinity in the vicinity of the PIN dimer.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>3441</offset><text>Results</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>3449</offset><text>Domain structures of Regnase-1</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>3480</offset><text>We analyzed Rengase-1 derived from Mus musculus and solved the structures of the four domains; NTD, PIN, ZF, and CTD individually by X-ray crystallography or NMR (Fig. 1a–e). X-ray crystallography was attempted for the fragment containing both the PIN and ZF domains, however, electron density was observed only for the PIN domain (Fig. 1c), consistent with a previous report on Regnase-1 derived from Homo sapiens. This suggests that the PIN and ZF domains exist independently without interacting with each other. The domain structures of NTD, ZF, and CTD were determined by NMR (Fig. 1b,d,e). The NTD and CTD are both composed of three α helices, and structurally resemble ubiquitin conjugating enzyme E2 K (PDB ID: 3K9O) and ubiquitin associated protein 1 (PDB ID: 4AE4), respectively, according to the Dali server.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>4303</offset><text>Contribution of each domain of Regnase-1 to the mRNA binding activity</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>4373</offset><text>Although the PIN domain is responsible for the catalytic activity of Regnase-1, the roles of the other domains are largely unknown. First, we evaluated a role of the NTD and ZF domains for mRNA binding by an in vitro gel shift assay (Fig. 1f). Fluorescently 5′-labeled RNA corresponding to nucleotides 82–106 of the IL-6 mRNA 3′UTR and the catalytically inactive mutant (D226N and D244N) of Regnase-1—hereafter referred to as the DDNN mutant—were utilized. Upon addition of a larger amount of Regnase-1, the fluorescence of free RNA decreased, indicating that Regnase-1 bound to the RNA. Based on the decrease in the free RNA fluorescence band, we evaluated the contribution of each domain of Regnase-1 to RNA binding. While the RNA binding ability was not significantly changed in the presence of NTD, it increased in the presence of the ZF domain (Fig. 1f,g and Supplementary Fig. 1). Direct binding of the ZF domain and RNA were confirmed by NMR spectral changes. The fitting of the titration curve of Y314 resulted in an apparent dissociation constant (Kd) of 10 ± 1.1 μM (Supplementary Fig. 2). These results indicate that not only the PIN but also the ZF domain contribute to RNA binding, while the NTD is not likely to be involved in direct interaction with RNA.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>5661</offset><text>Contribution of each domain of Regnase-1 to RNase activity</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>5720</offset><text>In order to characterize the role of each domain in the RNase activity of Regnase-1, we performed an in vitro cleavage assay using fluorescently 5′-labeled RNA corresponding to nucleotides 82–106 of the IL-6 mRNA 3′UTR (Fig. 1g). Regnase-1 constructs consisting of NTD-PIN-ZF completely cleaved the target mRNA and generated the cleaved products. The apparent half-life (T1/2) of the RNase activity was about 20 minutes. Regnase-1 lacking the ZF domain generated a smaller but appreciable amount of cleaved product (T1/2 ~ 70 minutes), while those lacking the NTD did not generate cleaved products (T1/2 &gt; 90 minutes). It should be noted that NTD-PIN(DDNN)-ZF, which possesses the NTD but lacks the catalytic residues in PIN, completely lost all RNase activity (Fig. 1g, right panel), as expected, confirming that the RNase catalytic center is located in the PIN domain. Taken together with the results in the previous section, we conclude that the NTD is crucial for the RNase activity of Regnase-1 in vitro, although it does not contribute to the direct mRNA binding.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>6810</offset><text>Dimer formation of the PIN domains</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>6845</offset><text>During purification by gel filtration, the PIN domain exhibited extremely asymmetric elution peaks in a concentration dependent manner (Fig. 2a). By comparison with the elution volume of standard marker proteins, the PIN domain was assumed to be in equilibrium between a monomer and a dimer in solution at concentrations in the 20–200 μM range. The crystal structure of the PIN domain has been determined in three distinct crystal forms with a space group of P3121 (form I in this study and PDB ID 3V33), P3221 (form II in this study), and P41 (PDB ID 3V32 and 3V34), respectively. We found that the PIN domain formed a head-to-tail oligomer that was commonly observed in all three crystal forms in spite of the different crystallization conditions (Supplementary Fig. 3). Mutation of Arg215, whose side chain faces to the opposite side of the oligomeric surface, to Glu preserved the monomer/dimer equilibrium, similar to the wild type. On the other hand, single mutations of side chains involved in the PIN–PIN oligomeric interaction resulted in monomer formation, judging from gel filtration (Fig. 2a,b). Wild type and monomeric PIN mutants (P212A and D278R) were also analyzed by NMR. The spectra indicate that the dimer interface of the wild type PIN domain were significantly broadened compared to the monomeric mutants (Supplementary Fig. 4). These results indicate that the PIN domain forms a head-to-tail oligomer in solution similar to the crystal structure. Interestingly, the monomeric PIN mutants P212A, R214A, and D278R had no significant RNase activity for IL-6 mRNA in vitro (Fig. 2c). The side chains of these residues point away from the catalytic center on the same molecule (Fig. 2b). Therefore, we concluded that head-to-tail PIN dimerization, together with the NTD, are required for Regnase-1 RNase activity in vitro.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>8692</offset><text>Domain-domain interaction between the NTD and the PIN domain</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>8753</offset><text>While the NTD does not contribute to RNA binding (Fig. 1f,g, and Supplementary Fig. 1), it increases the RNase activity of Regnase-1 (Fig. 1h). In order to gain insight into the molecular mechanism of the NTD-mediated enhancement of Regnase-1 RNase activity, we further investigated the domain-domain interaction between the NTD and the PIN domain using NMR. We used the catalytically inactive monomeric PIN mutant possessing both the DDNN and D278R mutations to avoid dimer formation of the PIN domain. The NMR signals from the PIN domain (residues V177, F210-T211, R214, F228-L232, and F234-S236) exhibited significant chemical shift changes upon addition of the NTD (Fig. 3a). Likewise, upon addition of the PIN domain, NMR signals derived from R56, L58-G59, and V86-H88 in the NTD exhibited large chemical shift changes and residues D53, F55, K57, Y60-S61, V68, T80-G83, L85, and G89 of the NTD as well as side chain amide signals of N79 exhibited small but appreciable chemical shift changes (Fig. 3b and Supplementary Fig. 5). These results clearly indicate a direct interaction between the PIN domain and the NTD. Based on the titration curve for the chemical shift changes of L58, the apparent Kd between the isolated NTD and PIN was estimated to be 110 ± 5.8 μM. Considering the fact that the NTD and PIN domains are attached by a linker, the actual binding affinity is expected much higher in the native protein. Mapping the residues with chemical shift changes reveals the putative PIN/NTD interface, which includes a helix that harbors catalytic residues D225 and D226 on the PIN domain (Fig. 3a). Interestingly, the putative binding site for the NTD overlaps with the PIN-PIN dimer interface, implying that NTD binding can “terminate” PIN-PIN oligomerization (Fig. 2b). An in silico docking of the NTD and PIN domains using chemical shift restraints provided a model consistent with the NMR experiments (Fig. 3c).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>10692</offset><text>Residues critical for Regnase-1 RNase activity</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10739</offset><text>To gain insight into the residues critical for Regnase-1 RNase activity, each basic or aromatic residue located around the catalytic site of the PIN oligomer was mutated to alanine, and the oligomerization and RNase activity were investigated (Fig. 4). From the gel filtration assays, all mutants except R214A formed dimers, suggesting that any lack of RNase activity in the mutants, except R214A, was directly due to mutational effects of the specific residues and not to abrogation of dimer formation. The W182A, R183A, and R214A mutants markedly lost cleavage activity for IL-6 mRNA as well as for Regnase-1 mRNA. The K184A, R215A, and R220A mutants moderately but significantly decreased the cleavage activity for both target mRNAs. The importance of K219 and R247 was slightly different for IL-6 and Regnase-1 mRNA; both K219 and R247 were more important in the cleavage of IL-6 mRNA than for Regnase-1 mRNA. The other mutated residues—K152, R158, R188, R200, K204, K206, K257, and R258—were not critical for RNase activity. The importance of residues W182 and R183 can readily be understood in terms of the monomeric PIN structure as they are located near to the RNase catalytic site; however, the importance of residue K184, which points away from the active site is more easily rationalized in terms of the oligomeric structure, in which the “secondary” chain’s residue K184 is positioned near the “primary” chain’s catalytic site (Fig. 4). In contrast, R214 is important for oligomerization of the PIN domain and the “secondary” chain’s residue R214 is also positioned near the “primary” chain’s active site within the dimer interface. It should be noted that the putative-RNA binding residues K184 and R214 are unique to Regnase-1 among PIN domains.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>12527</offset><text>Molecular mechanism of target mRNA cleavage by the PIN dimer</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>12588</offset><text>Our mutational experiments indicated that the observed dimer is functional and that the role of the secondary PIN domain is to position Regnase-1-unique RNA binding residues near the active site of the primary PIN domain. If this model is correct, then we reasoned that a catalytically inactive PIN and a PIN lacking the putative RNA-binding residues ought to be inactive in isolation but become active when mixed together. In order to test this hypothesis, we performed in vitro cleavage assays using combinations of Regnase-1 mutants that had no or decreased RNase activities by themselves (Fig. 5). One group consisted of catalytically active PIN domains with mutation of basic residues found in the previous section to confer decreased RNase activity (Fig. 4). These were paired with a DDNN mutant that had no RNase activity by itself. When any members of the two groups are mixed, two kinds of heterodimers can be formed: one is composed of a DDNN primary PIN and a basic residue mutant secondary PIN and is expected to exhibit no RNase activity; the other is composed of a basic residue mutant primary PIN and a DDNN secondary PIN and is predicted to rescue RNase activity (Fig. 5a). When we compared the fluorescence intensity of uncleaved IL-6 mRNA, basic residue mutants W182A, K184A, R214A, and R220A were rescued upon addition of the DDNN mutant (Fig. 5b). Consistently, when we compared the fluorescence intensity of the uncleaved Regnase-1 mRNA, basic residue mutants K184A and R214A were rescued upon addition of the DDNN mutant (Fig. 5c). Rescue of K184A and R214A by the DDNN mutant was also confirmed by a significant increase in the cleaved products. This is particularly significant because the side chains of K184 and R214 in the primary PIN are oriented away from their own catalytic center, while those in the secondary PIN face toward the catalytic center of the primary PIN. R214 is an important residue for dimer formation as shown in Fig. 2, therefore, R214A in the secondary PIN cannot dimerize. According to the proposed model, an R214A PIN domain can only form a dimer when the DDNN PIN acts as the secondary PIN. Taken together, the rescue experiments above support the proposed model in which the head-to-tail dimer is functional in vitro.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_1</infon><offset>14859</offset><text>Discussion</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>14870</offset><text>We determined the individual domain structures of Regnase-1 by NMR and X-ray crystallography. Although the function of the CTD remains elusive, we revealed the functions of the NTD, PIN, and ZF domains. A Regnase-1 construct consisting of PIN and ZF domains derived from Mus musculus was crystallized; however, the electron density of the ZF domain was low, indicating that the ZF domain is highly mobile in the absence of target mRNA or possibly other protein-protein interactions. Our NMR experiments confirmed direct binding of the ZF domain to IL-6 mRNA with a Kd of 10 ± 1.1 μM. Furthermore, an in vitro gel shift assay indicated that Regnase-1 containing the ZF domain enhanced target mRNA-binding, but the protein-RNA complex remained in the bottom of the well without entering into the polyacrylamide gel. These results indicate that Regnase-1 directly binds to RNA and precipitates under such experimental conditions. Due to this limitation, it is difficult to perform further structural analyses of mRNA-Regnase-1 complexes by X-ray crystallography or NMR.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>15945</offset><text>The previously reported crystal structure of the Regnase-1 PIN domain derived from Homo sapiens is nearly identical to the one derived from Mus musculus in this study, with a backbone RMSD of 0.2 Å. The amino acid sequences corresponding to PIN (residues 134–295) are the two non-identical residues are substituted with similar amino acids. Both the mouse and human PIN domains form head-to-tail oligomers in three distinct crystal forms. Rao and co-workers previously argued that PIN dimerization is likely to be a crystallographic artifact with no physiological significance, since monomers were dominant in their analytical ultra-centrifugation experiments. In contrast, our gel filtration data, mutational analyses, and NMR spectra all indicate that the PIN domain forms a head-to-tail dimer in solution in a manner similar to the crystal structure. This inconsistency might be due to difference in the analytical methods and/or protein concentrations used in each experiment, since the oligomer formation of PIN was dependent on the protein concentration in our study.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>17024</offset><text>Single mutations to residues involved in the putative oligomeric interaction of PIN monomerized as expected and these mutants lost their RNase activity as well. Since the NMR spectra of monomeric mutants overlaps with those of the oligomeric forms, it is unlikely that the tertiary structure of the monomeric mutants were affected by the mutations. (Supplementary Fig. 4b,c). Based on these observations, we concluded that PIN-PIN dimer formation is critical for Regnase-1 RNase activity in vitro. Within the crystal structure of the PIN dimer, the Regnase-1 specific basic regions in both the “primary” and “secondary” PINs are located around the catalytic site of the primary PIN (Supplementary Fig. 6). Moreover, our structure-based mutational analyses showed these two Regnase-1 specific basic regions were essential for target mRNA cleavage in vitro.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>17888</offset><text>The cleavage assay also showed that the NTD is crucial for efficient mRNA cleavage. Moreover, we found that the NTD associates with the oligomeric surface of the primary PIN, docking to a helix that harbors its catalytic residues (Figs 2b and 3a). Taken together, this suggests that the NTD and the PIN domain compete for a common binding site. The affinity of the domain-domain interaction between two PIN domains (Kd = ~10−4 M) is similar to that of the NTD-PIN (Kd = 110 ± 5.8 μM) interactions; however, the covalent connection corresponding to residues 90–133 between the NTD and the primary PIN will greatly enhance the intramolecular domain interaction in the case of full-length Regnase-1. While further analyses are necessary to prove this point, our preliminary docking and molecular dynamics simulations indicate that NTD-binding rearranges the catalytic residues of the PIN domain toward an active conformation suitable for binding Mg2+. In this context, it is interesting that, in response to TCR stimulation, Malt1 cleaves Regnase-1 at R111 to control immune responses in vivo. This result is consistent with a model in which the NTD acts as an enhancer, and cleavage of the linker lowers enzymatic activity dramatically.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>19145</offset><text>Based on these structural and functional analyses of Regnase-1 domain-domain interactions, we performed docking simulations of the NTD, PIN dimer, and IL-6 mRNA. We incorporated information from the cleavage site of IL-6 mRNA in vitro is indicated by denaturing polyacrylamide gel electrophoresis (Supplementary Fig. 7a,b). The docking result revealed multiple RNA binding modes that satisfied the experimental results in vitro (Supplementary Fig. 7c,d), however, it should be noted that, in vivo, there would likely be many other RNA-binding proteins that would protect loop regions from cleavage by Regnase-1.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>19757</offset><text>The overall model of regulation of Regnase-1 RNase activity through domain-domain interactions in vitro is summarized in Fig. 6. In the absence of target mRNA, the PIN domain forms head-to-tail oligomers at high concentration. A fully active catalytic center can be formed only when the NTD associates with the oligomer surface of the PIN domain, which terminates the head-to-tail oligomer formation in one direction (primary PIN), and forms a functional dimer together with the neighboring PIN (secondary PIN). While further investigations on the domain-domain interactions of Regnase-1 in vivo are necessary, these intramolecular and intermolecular domain interactions of Regnase-1 appear to structurally constrain Regnase-1activity, which, in turn, enables tight regulation of immune responses.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>20555</offset><text>Methods</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>20563</offset><text>Protein expression and purification</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>20599</offset><text>The DNA fragment encoding Regnase-1 derived from Mus musculus was cloned into pGEX6p vector (GE Healthcare). All the mutants were generated by PCR-mediated site-directed mutagenesis and confirmed by the DNA sequence analyses. As a catalytically deficient mutant, both Asp226 and Asp244 at the catalytic center of PIN were mutated to Asn, which is referred to as DDNN mutant. Regnase-1 was expressed at 16 °C using the Escherichia coli RosettaTM(DE3)pLysS strain. After purification with a GST-affinity resin, an N-terminal GST tag was digested by HRV-3 C protease. NTD was further purified by gel filtration chromatography using a HiLoad 16/60 Superdex 75 pg (GE Healthcare). The other domains were further purified by cation exchange chromatography using Resource S (GE Healthcare), followed by gel filtration chromatography using a HiLoad 16/60 Superdex 75 pg (GE Healthcare). Uniformly 15N or 13C, 15N-double labeled proteins for NMR experiments were prepared by growing E. coli host in M9 minimal medium containing 15NH4Cl, unlabeled glucose and 15N CELTONE® Base Powder (CIL) or 15NH4Cl, 13C6-glucose, and13C, 15N CELTONE® Base Powder (CIL), respectively.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>21771</offset><text>X-ray crystallography</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>21793</offset><text>Crystallization was performed using the sitting drop vapor diffusion method at 20 °C and two crystal forms (I and II) were obtained. In the case of form I crystals, drops (0.5 μl) of 6 mg/ml selenomethionine-labeled Regnase-1 PIN-ZF (residues 134–339 derived from Mus musculus) in 20 mM HEPES-NaOH (pH 6.8), 200 mM NaCl and 5 mM DTT were mixed with reservoir solution consisting of 1 M (NH4)2HPO4, 200 mM NaCl and 100 mM sodium citrate (pH 5.5) whereas in the case of form II crystals, drops (0.5 μl) of 6 mg/ml native Regnase-1 PIN-ZF (residues 134–339) in 20 mM HEPES-NaOH (pH 6.8), 200 mM NaCl and 5 mM DTT were mixed with reservoir solution consisting of 1.7 M NaCl and 100 mM HEPES-NaOH (pH 7.0). Diffraction data were collected at a Photon Factory Advanced Ring beamline NE3A (form I) or at a SPring-8 beamline BL41XU (form II), and were processed with HKL2000. The structure of the form I crystal was determined by the multiple anomalous dispersion (MAD) method. Nine Se sites were found using the program SOLVE; however, the electron density obtained by MAD phases calculated using SOLVE was not good enough to build a model even after density modification using the program RESOLVE. Then the program CNS was used to find additional three Se sites and calculate MAD phases using 12 Se sites. The electron density after density modification using CNS was good enough to build a model. Structure of the form II crystal was determined by the molecular replacement method using CNS and using the structure of the form I crystal as a search model. For all structures, further model building was performed manually with COOT, and TLS and restrained refinement using isotropic individual B factors was performed with REFMAC5 in the CCP4 program suite. Crystallographic parameters are summarized in Supplementary Table 1.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>23654</offset><text>NMR measurements</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>23671</offset><text>All NMR experiments were carried out at 298 K on Inova 500-MHz, 600-MHz, and 800-MHz spectrometer (Agilent). The NMR data were processed using the NMRPipe, the Olivia (fermi.pharm.hokudai.ac.jp/olivia/), and the Sparky program (Sparky3, University of California, San Francisco).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>23952</offset><text>For structure calculation, NOE distance restraints were obtained from 3D 15N-NOESY-HSQC (100 ms mixing time for the NTD, 75 ms mixing time for the ZF domain and the CTD) and 13C-NOESY-HSQC spectra (100 ms mixing time for the NTD, 75 ms mixing time for the ZF domain and the CTD). The NMR structures were determined using the CANDID/CYANA2.1. Dihedral restraints were derived from backbone chemical shifts using TALOS.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>24378</offset><text>For the domain-domain interaction analyses between the NTD and the PIN domain, 1H-15N HSQC spectra of uniformly 15N-labeled proteins in the concentration of 100 μM were obtained in the presence of 3 or 6 molar equivalents of unlabeled proteins.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>24626</offset><text>Preparation of RNAs</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>24646</offset><text>The fluorescently labeled RNAs at the 5′-end by 6-FAM were purchased from SIGMA-ALDORICH. The RNA sequences used in this study were shown below.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>24793</offset><text>IL-6 mRNA 3′UTR (82–106): 5′-UGUUGUUCUCUACGAAGAACUGACA-3′ (25 nts)</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>24868</offset><text>Regnase-1 mRNA 3′UTR (191–211): 5′- CUGUUGAUACACAUUGUAUCU-3′ (21 nts)</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>24946</offset><text>Electrophoretic mobility shift assay</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>24983</offset><text>Catalytically deficient Regnase-1 proteins, containing DDNN mutations, and 5′-terminally 6-FAM labeled RNAs were incubated in the RNA-binding buffer (20 mM HEPES-NaOH (pH 6.8), 150 mM NaCl, 1 mM DTT, 10% glycerol (v/v), and 0.1% NP-40 (v/v)) at 4 °C for 30 minutes, then analyzed by non-denaturing polyacrylamide gel electrophoresis. The electrophoreses were performed at 4 °C using the 7.5% polyacrylamide (w/v) gel (monomer : bis = 29 : 1) in the electrophoresis buffer (25 mM Tris-HCl (pH 7.5) and 200 mM glycine). The fluorescence of 6-FAM labeled RNA was directly detected at the excitation wavelength of 460 nm with a fluorescence filter (Y515-Di) using a fluoroimaging analyzer (LAS-4000 (FUJIFILM)). The fluorescence intensity of each sample was quantified using ImageJ software.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>25797</offset><text>In vitro RNA cleavage assay</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>25825</offset><text>Regnase-1 (2 μM) and 5′-terminally 6-FAM labeled RNA (1 μM) were incubated in the RNA-cleavage buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM MgCl2, and 1 mM DTT) at 37 °C. For the assay using combinations of Regnase-1 mutants, equimolar amounts of Regnase-1 mutants (2 μM each) were mixed with fluorescently labeled RNA (1 μM). After incubation for 30–120 minutes, the reaction was stopped by the addition of 1.5-fold volume of denaturing buffer containing 8 M urea and 100 mM EDTA, and samples were boiled. The electrophoreses were performed at room temperature using the 8 M urea containing denaturing gel with 20% polyacrylamide (w/v) (monomer : bis = 19 : 1) in 0.5 × TBE as the electrophoresis buffer.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>26581</offset><text>Docking calculations</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>26602</offset><text>For docking NTD to PIN, OSCAR-star was first used to rebuild sidechains in the head-to-tail PIN dimer. Docking was carried out by surFit (http://sysimm.ifrec.osaka-u.ac.jp/docking/main/) with restraints obtained from NMR data (Fig. 3a,b) as follows. NTD: R56, L58, G59, V86, K87, H88; PIN: V177, F210, T211, R214, F228, I229, V230, K231, L232, F234, D235, S236. Top-scoring model was selected.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>26996</offset><text>For docking IL-6 mRNA 3′UTR to the PIN dimer, each domain of the PIN dimer structure was superimposed onto the PIN dimer of the human X-ray structure (PDB ID: 3V34) in order to graft both water molecules and Mg2+ ions to the mouse model. Each IL-6 representative structure was submitted to the HADDOCK 2.0 server, for total of 10 independent jobs. In order to be consistent with the cleavage assay, active residues consisted of all nucleotides in RNA, Mg2+ and W182, R183, K184, R188, R214, R215, K219, R220, and R247 in the protein. Docked models were selected based on the following criteria: one heavy atom within 7, 8, or 9th nucleotide from the 5′ end was &lt;5 Å from the Mg2+ ion on the primary PIN. Further classification was done manually in order to divide the selected models into two clusters.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>27806</offset><text>Additional Information</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>27829</offset><text>Accession codes: The crystal structure of the Regnase-1 PIN domain has been deposited in the Protein Data Bank (accession codes: 5H9V (Form I) and 5H9W (Form II)). The chemical shift assignments of the NTD, the ZF domain, and the CTD have been deposited at Biological Magnetic Resonance Bank (accession codes: 25718, 25719, and 25720, respectively), and the coordinates for the ensemble have been deposited in the Protein Data Bank (accession codes: 2N5J, 2N5K, and 2N5L, respectively). </text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>28317</offset><text>How to cite this article: Yokogawa, M. et al. Structural basis for the regulation of enzymatic activity of Regnase-1 by domain-domain interactions. Sci. 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All authors reviewed the manuscript.</text></passage><passage><infon key="file">srep22324-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>31249</offset><text>Structural and functional analyses of Regnase-1.</text></passage><passage><infon key="file">srep22324-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>31298</offset><text>(a) Domain architecture of Regnase-1. (b) Solution structure of the NTD. (c) Crystal structure of the PIN domain. Catalytic Asp residues were shown in sticks. (d) Solution structure of the ZF domain. Three Cys residues and one His residue responsible for Zn2+-binding were shown in sticks. (e) Solution structure of the CTD. All the structures were colored in rainbow from N-terminus (blue) to C-terminus (red). (f) In vitro gel shift binding assay between Regnase-1 and IL-6 mRNA. Fluorescence intensity of the free IL-6 in each sample was indicated as the percentage against that in the absence of Regnase-1. (g) Binding of Regnase-1 and IL-6 mRNA was plotted. The percentage of the bound IL-6 was calculated based on the fluorescence intensities of the free IL-6 quantified in (f). (h) In vitro cleavage assay of Regnase-1 to IL-6 mRNA. Fluorescence intensity of the uncleaved IL-6 mRNA was indicated as the percentage against that in the absence of Regnase-1.</text></passage><passage><infon key="file">srep22324-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>32262</offset><text>Head-to-tail oligomer formation of the PIN domain is crucial for the RNase activity of Regnase-1.</text></passage><passage><infon key="file">srep22324-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>32360</offset><text>(a) Gel filtration analyses of the PIN domain. Elution volumes of the standard marker proteins were indicated by arrows at the upper part. (b) Dimer structure of the PIN domain. Two PIN molecules in the crystal were colored white and green, respectively. Catalytic residues and mutated residues were shown in sticks. Residues important for the oligomeric interaction were colored red, while R215 that was dispensable for the oligomeric interaction was colored blue. (c) RNase activity of monomeric mutants for IL-6 mRNA was analyzed.</text></passage><passage><infon key="file">srep22324-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>32894</offset><text>Domain-domain interaction between the NTD and the PIN domain.</text></passage><passage><infon key="file">srep22324-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>32956</offset><text>(a) NMR analyses of the NTD-binding to the PIN domain. The residues with significant chemical shift changes were labeled in the overlaid spectra (left) and colored red on the surface and ribbon structure of the PIN domain (right). Pro and the residues without analysis were colored black and gray, respectively. (b) NMR analyses of the PIN-binding to the NTD. The residues with significant chemical shift changes were labeled in the overlaid spectra (left) and colored red, yellow, or green on the surface and ribbon structure of the NTD. S62 was colored gray and excluded from the analysis, due to low signal intensity. (c) Docking model of the NTD and the PIN domain. The NTD and the PIN domain are shown in cyan and white, respectively. Residues in close proximity (&lt;5 Å) to each other in the docking structure were colored yellow. Catalytic residues of the PIN domain are shown in sticks, and the residues that exhibited significant chemical shift changes in (a,b) were labeled.</text></passage><passage><infon key="file">srep22324-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>33942</offset><text>Critical residues in the PIN domain for the RNase activity of Regnase-1.</text></passage><passage><infon key="file">srep22324-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>34015</offset><text>(a) In vitro cleavage assay of basic residue mutants for IL-6 mRNA. The results indicate mean ± SD of four independent experiments. (b)
+In vitro cleavage assay of basic residue mutants for Regnase-1 mRNA. The results indicate mean ± SD of three independent experiments. The fluorescence intensity of the uncleaved mRNA was quantified and the results were mapped on the PIN dimer structure. Mutated basic residues were shown in sticks and those with significantly reduced RNase activities were colored red or yellow.</text></passage><passage><infon key="file">srep22324-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>34541</offset><text>Heterodimer formation by combination of the Regnase-1 basic residue mutants and the DDNN mutant restored the RNase activity.</text></passage><passage><infon key="file">srep22324-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>34666</offset><text>(a) Cartoon representation of the concept of the experiment. (b) In vitro cleavage assay of Regnase-1 for IL-6 mRNA. (c) In vitro cleavage assay of Regnase-1 for Regnase-1 mRNA. The results indicate mean ± SD of three independent experiments. The fluorescence intensity of the uncleaved mRNA was quantified and the results were mapped on the PIN dimer. The mutations whose RNase activities were not increased in the presence of DDNN mutant were colored in blue on the primary PIN. The mutations whose RNase activities were restored in the presence of DDNN mutant were colored in red or yellow on the primary PIN.</text></passage><passage><infon key="file">srep22324-f6.jpg</infon><infon key="id">f6</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>35284</offset><text>Schematic representation of regulation of the Regnase-1 catalytic activity through the domain-domain interactions.</text></passage></document></collection>

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+<collection><source>PMC</source><date>20201215</date><key>pmc.key</key><document><id>4774019</id><infon key="license">CC BY</infon><passage><infon key="article-id_doi">10.1186/s12915-016-0236-7</infon><infon key="article-id_pmc">4774019</infon><infon key="article-id_pmid">26934976</infon><infon key="article-id_publisher-id">236</infon><infon key="elocation-id">14</infon><infon key="kwd">Innate immunity IRG proteins GTPase Dynamin superfamily Dimerization Oligomerization</infon><infon key="license">
+Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.</infon><infon key="name_0">surname:Schulte;given-names:Kathrin</infon><infon key="name_1">surname:Pawlowski;given-names:Nikolaus</infon><infon key="name_2">surname:Faelber;given-names:Katja</infon><infon key="name_3">surname:Fröhlich;given-names:Chris</infon><infon key="name_4">surname:Howard;given-names:Jonathan</infon><infon key="name_5">surname:Daumke;given-names:Oliver</infon><infon key="name_6">surname:Howard;given-names:Jonathan</infon><infon key="name_7">surname:Daumke;given-names:Oliver</infon><infon key="section_type">TITLE</infon><infon key="title">Keywords</infon><infon key="type">front</infon><infon key="volume">14</infon><infon key="year">2016</infon><offset>0</offset><text>The immunity-related GTPase Irga6 dimerizes in a parallel head-to-head fashion</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract_title_1</infon><offset>79</offset><text>Background</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>90</offset><text>The immunity-related GTPases (IRGs) constitute a powerful cell-autonomous resistance system against several intracellular pathogens. Irga6 is a dynamin-like protein that oligomerizes at the parasitophorous vacuolar membrane (PVM) of Toxoplasma gondii leading to its vesiculation. Based on a previous biochemical analysis, it has been proposed that the GTPase domains of Irga6 dimerize in an antiparallel fashion during oligomerization.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract_title_1</infon><offset>526</offset><text>Results</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>534</offset><text>We determined the crystal structure of an oligomerization-impaired Irga6 mutant bound to a non-hydrolyzable GTP analog. Contrary to the previous model, the structure shows that the GTPase domains dimerize in a parallel fashion. The nucleotides in the center of the interface participate in dimerization by forming symmetric contacts with each other and with the switch I region of the opposing Irga6 molecule. The latter contact appears to activate GTP hydrolysis by stabilizing the position of the catalytic glutamate 106 in switch I close to the active site. Further dimerization contacts involve switch II, the G4 helix and the trans stabilizing loop.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract_title_1</infon><offset>1189</offset><text>Conclusions</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>1201</offset><text>The Irga6 structure features a parallel GTPase domain dimer, which appears to be a unifying feature of all dynamin and septin superfamily members. This study contributes important insights into the assembly and catalytic mechanisms of IRG proteins as prerequisite to understand their anti-microbial action.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract_title_1</infon><offset>1508</offset><text>Electronic supplementary material</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>1542</offset><text>The online version of this article (doi:10.1186/s12915-016-0236-7) contains supplementary material, which is available to authorized users.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">title_1</infon><offset>1682</offset><text>Background</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1693</offset><text>Immunity-related GTPases (IRGs) comprise a family of dynamin-related cell-autonomous resistance proteins targeting intracellular pathogens, such as Mycobacterium tuberculosis, Mycobacterium avium, Listeria monocytogenes, Trypanosoma cruzi, and Toxoplasma gondii. In mice, the 23 IRG members are induced by interferons, whereas the single human homologue is constitutively expressed in some tissues, especially in testis. In non-infected cells, most IRGs are largely cytosolic. However, members of a small sub-family with regulatory function associate with specific intracellular membranes, with one member favoring the endoplasmic reticulum and others the Golgi membrane and the endolysosomal system. Infection by certain intracellular pathogens initiates the redistribution of several effector members to the parasitophorous vacuole, followed by its disruption. In this way, IRGs contribute to the release of the pathogen into the cytoplasm and its subsequent destruction.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2667</offset><text>Irga6, one of the effector IRG proteins, localizes to the intact parasitophorous vacuole membrane (PVM) and, after disruption of the PVM, is found associated with vesicular accumulations, presumably derived from the PVM. A myristoylation site at Gly2 is necessary for the recruitment to the PVM but not for the weak constitutive binding to the ER membrane. An internally oriented antibody epitope on helix A between positions 20 and 24 was demonstrated to be accessible in the GTP-, but not in the GDP-bound state. This indicates large-scale structural changes upon GTP binding that probably include exposure of the myristoyl group, enhancing binding to the PVM. Biochemical studies indicated that Irga6 hydrolyses GTP in a cooperative manner and forms GTP-dependent oligomers in vitro and in vivo.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3466</offset><text>Crystal structures of Irga6 in various nucleotide-loaded states revealed the basic architecture of IRG proteins, including a GTPase domain and a composite helical domain. These studies additionally showed a dimerization interface in the nucleotide-free protein as well as in all nucleotide-bound states. It involves a GTPase domain surface, which is located at the opposite side of the nucleotide, and an interface in the helical domain, with a water-filled gap between the two contact surfaces. Mutagenesis of the contact surfaces suggests that this &quot;backside&quot; interface is not required for GTP-dependent oligomerization or cooperative hydrolysis, despite an earlier suggestion to the contrary.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>4162</offset><text>Extensive biochemical studies suggested that GTP-induced oligomerization of Irga6 requires an interface in the GTPase domain across the nucleotide-binding site. Recent structural studies indicated that a 'G interface' is typical of dynamin superfamily members, such as dynamin, MxA, the guanylate binding protein-1 (GBP-1), atlastin and the bacterial dynamin-like proteins (BDLP). For several of these proteins, formation of the G interface was shown to trigger GTP hydrolysis by inducing rearrangements of catalytic residues in cis. In dynamin, the G interface includes residues in the phosphate binding loop, the two switch regions, the 'trans stabilizing loop' and the 'G4 loop'. For Irga6, it was demonstrated that besides residues in the switch I and switch II regions, the 3'-OH group of the ribose participates in this interface. Since the signal recognition particle GTPase and its homologous receptor (called FfH and FtsY in bacteria) also employ the 3'-OH ribose group to dimerize in an anti-parallel orientation therefore activating its GTPase, an analogous dimerization model was proposed for Irga6. However, the crystal structure of Irga6 in the presence of the non-hydrolyzable GTP analogue 5'-guanylyl imidodiphosphate (GMPPNP) showed only subtle differences relative to the apo or GDP-bound protein and did not reveal a new dimer interface associated with the GTPase domain. This structure was obtained by soaking GMPPNP in nucleotide-free crystals of Irga6, an approach which may have interfered with nucleotide-induced domain rearrangements.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>5722</offset><text>To clarify the dimerization mode via the G interface, we determined the GMPPNP-bound crystal structure of a non-oligomerizing Irga6 variant. The structure revealed that Irga6 can dimerize via the G interface in a parallel head-to-head fashion. This dimerization mode explains previously published biochemical data, and shows in particular how the 3'-OH group of the ribose participates in the assembly. Our data suggest that a parallel dimerization mode may be a unifying feature in all dynamin and septin superfamily proteins.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>6250</offset><text>Results</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>6258</offset><text>Previous results indicated that Irga6 mutations in a loosely defined surface region (the &quot;secondary patch&quot;), which is distant from the G-interface and only slightly overlapping with the backside interface (see below), individually reduced GTP-dependent oligomerization. A combination of four of these mutations (R31E, K32E, K176E, and K246E) essentially eliminated GTP-dependent assembly (Additional file 1: Figure S1) and allowed crystallization of Irga6 in the presence of GMPPNP. Crystals diffracted to 3.2 Å resolution and displayed one exceptionally long unit cell axis of 1289 Å (Additional file 1: Table S1). The structure was solved by molecular replacement and refined to Rwork/Rfree of 29.7 %/31.7 % (Additional file 1: Table S2). The asymmetric unit contained seven Irga6 molecules that were arranged in a helical pattern along the long cell axis (Additional file 1: Figure S2).</text></passage><passage><infon key="file">12915_2016_236_Fig1_HTML.jpg</infon><infon key="id">Fig1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>7150</offset><text>Structure of the Irga6 dimer. a Schematic view of the domain architecture of mouse Irga6. The first and last amino acids of each domain are indicated. b Ribbon-type representation of the Irga6 dimer. In the left molecule, domains are colored according to the domain architecture, the right molecule is colored in grey. The nucleotide and Mg2+ ion (green) are shown in sphere representation. The GTPase domain dimer is boxed. The dotted line indicates a 2-fold axis. Secondary structure was numbered according to ref.. c
+Top view on the GTPase domain dimer. d Magnification of the contact sites. Dotted lines indicate interactions. e Superposition of different switch I conformations in the asymmetric unit; the same colors as in Additional file 1: Figure S2 are used for the switch I regions of the individual subunits. Switch I residues of subunit A (yellow) involved in ribose binding are labelled and shown in stick representation. Irga6 immunity-related GTPase 6</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>8117</offset><text>Like other dynamin superfamily members, the GTPase domain of Irga6 comprises a canonical GTPase domain fold, with a central β-sheet surrounded by helices on both sides (Fig. 1a-c). The helical domain is a bipartite structure composed of helices αA-C at the N-terminus and helix αF-L at the C-terminus of the GTPase domain. Overall, the seven molecules in the asymmetric unit are very similar to each other, with root mean square deviations (rmsd) ranging from 0.32 – 0.45 Å over all Cα atoms. The structures of the seven molecules also agree well with the previously determined structure of native GMPPNP-bound Irga6 (PDB: 1TQ6; rmsd of 1.00-1.13 Å over all Cα atoms).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>8805</offset><text>The seven Irga6 molecules in the asymmetric unit form various higher order contacts in the crystals. Within the asymmetric unit, six molecules dimerize via the symmetric backside dimer interface (buried surface area 930 Å2), and the remaining seventh molecule forms the same type of interaction with its symmetry mate of the adjacent asymmetric unit (Additional file 1: Figure S2a, b, Figure S3). This indicates that the introduced mutations in the secondary patch, from which only Lys176 is part of the backside interface, do, in fact, not prevent this interaction.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9373</offset><text>Another assembly interface with a buried surface area of 450 Å2, which we call the “tertiary patch”, was formed via two interaction sites in the helical domains (Additional file 1: Figure S2c, d, S3). In this interface, helices αK from two adjacent molecules form a hydrogen bonding network involving residues 373-376. Furthermore, two adjacent helices αA form hydrophobic contacts. It was previously shown that the double mutation L372R/A373R did not prevent GTP-induced assembly, so there is currently no evidence supporting an involvement of this interface in higher-order oligomerization.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9974</offset><text>Strikingly, molecule A of one asymmetric unit assembled with an equivalent molecule of the adjacent asymmetric unit via the G-interface in a symmetric parallel fashion via a 470 Å2 interface. This assembly results in a butterfly-shaped Irga6 dimer in which the helical domains protrude in parallel orientations (Fig. 1b, Additional file 1: Figure S3). In contrast, the other six molecules in the asymmetric unit do not assemble via the G interface.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10425</offset><text>The G interface in molecule A can be subdivided into three distinct contact sites (Fig. 1c, d). Contact site I is formed between R159 and K161 in the trans stabilizing loops, and S132 in the switch II regions of the opposing molecules. Contact site II features polar and hydrophobic interactions formed by switch I (V104, V107) with a helix following the guanine specificity motif (G4 helix, K184 and S187) and the trans stabilizing loop (T158) of the opposing GTPase domain. In contact site III, G103 of switch I interacts via its main chain nitrogen with the exocyclic 2’-OH and 3’-OH groups of the opposing ribose in trans, whereas the two opposing exocyclic 3’-OH group of the ribose form hydrogen bonds with each other. Via the ribose contact, switch I is pulled towards the opposing nucleotide (Fig. 1e). In turn, E106 of switch I reorients towards the nucleotide and now participates in the coordination of the Mg2+ ion (Fig. 1e, Additional file 1: Figure S4). E106 was previously shown to be essential for catalysis, and the observed interactions in contact site III explain how dimerization via the ribose is directly coupled to the activation of GTP hydrolysis.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>11606</offset><text>The G interface is in full agreement with previously published biochemical data that indicate crucial roles of E77, G103, E106, S132, R159, K161, K162, D164, N191, and K196 for oligomerization and oligomerization-induced GTP hydrolysis. All of these residues directly participate in contacts (G103, S132, R159, and K161) or are in direct vicinity to the interface (E77, E106, K162, D164, and N191). Residues E77, K162, and D164 appear to orient the trans stabilizing loop which is involved in interface formation in contact site II. In the earlier model of an anti-parallel G interface, it was not possible to position the side chain of R159 to avoid steric conflict. In the present structure, the side-chain of R159 projects laterally along the G interface and, therefore, does not cause a steric conflict.</text></passage><passage><infon key="file">12915_2016_236_Fig2_HTML.jpg</infon><infon key="id">Fig2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>12414</offset><text>A conserved dimerization mode via the G interface in dynamin and septin GTPases. The overall architecture of the parallel GTPase domain dimer of Irga6 is related to that of other dynamin and septin superfamily proteins. The following structures are shown in cylinder representations, in similar orientations of their GTPase domains: a the GMPPNP-bound Irga6 dimer, b the GDP-AlF4
+--bound dynamin 1 GTPase-minimal BSE construct [pdb 2X2E], c the GDP-bound atlastin 1 dimer [pdb 3Q5E], d the GDP-AlF3- bound GBP1 GTPase domain dimer [pdb 2B92], e the BDLP dimer bound to GDP [pdb 2J68] and f the GTP-bound GIMAP2 dimer [pdb 2XTN]. The GTPase domains of the left molecules are shown in orange, helical domains or extensions in blue. Nucleotide, Mg2+ (green) and AlF4
+- are shown in sphere representation, the buried interface sizes per molecule are indicated on the right. Irga6 immunity-related GTPase 6, GMPPNP 5'-guanylyl imidodiphosphate, GTP guanosine-triphosphate, BDLP bacterial dynamin like protein, GIMAP2, GTPase of immunity associated protein 2</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>13467</offset><text>The buried surface area per molecule (BSA) of the G interface in Irga6 is relatively small (470 Å2) compared to that of other dynamin superfamily members, such as dynamin (BSA: 1400 Å2), atlastin (BSA: 820 Å2), GBP-1 (BSA: 2060 Å2), BDLP (BSA: 2300 Å2) or the septin-related GTPase of immunity associated protein 2 (GIMAP2) (BSA: 590 Å2) (Fig. 2). However, the relative orientations of the GTPase domains in these dimers are strikingly similar, and the same elements, such as switch I, switch II, the trans activating and G4 loops are involved in the parallel dimerization mode in all of these GTPase families.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_1</infon><offset>14085</offset><text>Discussion</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>14096</offset><text>IRG proteins are crucial mediators of the innate immune response in mice against a specific subset of intracellular pathogens, all of which enter the cell to form a membrane-bounded vacuole without engagement of the phagocytic machinery. As members of the dynamin superfamily, IRGs oligomerize at cellular membranes in response to GTP binding. Oligomerization and oligomerization-induced GTP hydrolysis are thought to induce membrane remodeling events ultimately leading to disruption of the PVM. Recent structural and mechanistic analyses have begun to unravel the molecular basis for the membrane-remodeling activity and mechano-chemical function of some members (reviewed in). For example, for dynamin and atlastin, it was shown that GTP binding and/or hydrolysis leads to dimerization of the GTPase domains and to the reorientation of the adjacent helical domains. The resulting domain movement was suggested to act as a “power stroke” during membrane remodeling events. However, for other dynamin superfamily members such as IRGs, the molecular basis for GTP hydrolysis and the exact role of the mechano-chemical function are still unclear.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>15246</offset><text>Our structural analysis of an oligomerization- and GTPase-defective Irga6 mutant indicates that Irga6 dimerizes via the G interface in a parallel orientation. Only one of the seven Irga6 molecules in the asymmetric unit formed this contact pointing to a low affinity interaction via the G interface, which is in agreement with its small size. In the crystals, dimerization via the G interface is promoted by the high protein concentrations which may mimic a situation when Irga6 oligomerizes on a membrane surface. Such a low affinity interaction mode may allow reversibility of oligomerization following GTP hydrolysis. Similar low affinity G interface interactions were reported for dynamin and MxA.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>15948</offset><text>The dimerization mode is strikingly different from the previously proposed anti-parallel model that was based on the crystal structure of the signal recognition particle GTPase, SRP54 and its homologous receptor. However, the G dimer interface is reminiscent of the GTPase domain dimers observed for several other dynamin superfamily members, such as dynamin, GBP1, atlastin, and BDLP. It was recently shown that septin and septin-related GTPases, such as the Tocs GTPases or GTPases of immunity related proteins (GIMAPs), also employ a GTP-dependent parallel dimerization mode. Based on phylogenetic and structural analysis, these observations suggest that dynamin and septin superfamilies are derived from a common ancestral membrane-associated GTPase that featured a GTP-dependent parallel dimerization mode. Importantly, our analysis indicates that IRGs are not outliers, but bona-fide representatives of the dynamin superfamily.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>16882</offset><text>Whereas the overall dimerization mode is similar in septin and dynamin GTPases, family-specific differences in the G interface and the oligomerization interfaces exist. For example, the involvement of the 2’ and 3’-OH groups of the ribose in the dimerization interface of Irga6 has not been observed for other dynamin and septin superfamily members. The surface-exposed location of the ribose in the Irga6 structure, with a wide-open nucleotide-binding pocket, facilitates its engagement in the dimerization interface. This contact, in turn, appears to activate GTP hydrolysis by inducing rearrangements in switch I and the positioning of the catalytic E106. During dimerization of GBP1, an arginine finger from the P loop reorients towards the nucleotide in cis to trigger GTP hydrolysis. In dynamin, the corresponding serine residue coordinates a sodium ion that is crucial for GTP hydrolysis. Irga6 bears Gly79 at this position, which in the dimerizing molecule A appears to approach the bridging imido group of GMPPNP via a main chain hydrogen bond. Higher resolution structures of the Irga6 dimer in the presence of a transition state analogue are required to show whether Gly79 directly participates in GTP hydrolysis or whether it may also position a catalytic cation.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>18162</offset><text>In dynamin, further assembly sites are provided by the helical domains which assemble in a criss-cross fashion to form a helical filament. In dynamin-related Eps15 homology domain containing proteins (EHDs), a second assembly interface is present in the GTPase domain. For Irga6, additional interfaces in the helical domain are presumably involved in oligomerization, such as the secondary patch residues whose mutation prevented oligomerization in the crystallized mutant. Further structural studies, especially electron microscopy analysis of the Irga6 oligomers, are required to clarify the assembly mode via the helical domains and to show how these interfaces cooperate with the G interface to mediate the regulated assembly on a membrane surface. Notably, we did not observe major rearrangements of the helical domain versus the GTPase domain in the Irga6 molecules that dimerized via the G interface. In a manner similar to BDLP, such large-scale conformational changes may be induced by membrane binding. Our structural analysis and the identification of the G-interface paves the way for determining the specific assembly of Irga6 into a membrane-associated scaffold as the prerequisite to understand its action as an anti-parasitic machine.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>19413</offset><text>Methods</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>19421</offset><text>Protein expression and purification</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>19457</offset><text>Selenomethionine-substituted Mus musculus Irga6R31E, K32E, K176E, K246E was expressed as a GST-fusion from the vector pGEX-4T-2 in BL21 Rosetta2(DE3) cells according to reference. Protein was purified as previously described and the protein stored in small aliquots at a concentration of 118 mg/mL in 50 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 2 mM DTT.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>19804</offset><text>Biochemical analyses</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>19825</offset><text>Oligomerization and GTPase assays for the Irga6R31E, K32E, K176E, K246E mutant were carried out as described in.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>19938</offset><text>Protein crystallization</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>19962</offset><text>The protein was gently thawed on ice and diluted to a final concentration of 10 mg/mL with buffer containing 20 mM Tris-HCl, pH 7.5, 8 mM MgCl2, 3 mM DTT. GMPPNP was added to a final concentration of 2 mM. Crystallization was carried out in a 96 well format using the sitting drop vapor diffusion method. The reservoir contained 100 mM HEPES-NaOH pH 7.0, 9 % PEG4000, 6 % isopropanol. The sitting drop was set up using an Art Robbins Gryphon system and consisted of 200 nL protein solution and 200 nL reservoir solution.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>20483</offset><text>For cryo-protection, crystals were transferred into a cryo solution containing 33 % PEG4000, 3 % isopropanol, 50 mM HEPES pH 7.0, 4 mM MgCl2, 2 mM DTT, and 2 mM GMPPNP at 4 °C for at least 5 sec. Crystals were screened for diffraction at beamline BL 14.1 at BESSY II, Berlin, Germany.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>20769</offset><text>Data collection</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>20785</offset><text>All data were recorded at beamline P11 at PETRA III, DESY Hamburg, Germany using a PILATUS 6 M detector. To achieve spot separation along the long cell axis, three data sets were collected with a φ increment of 0.05/0.1° at a temperature of 100 K using detector distances between 1300 and 598.5 mm (Additional file 1: Table S1). The wavelength was 0.972/0.979 Å. Calculation of an optimal data collection strategy was done with the Mosflm software. The high- and low-resolution datasets were processed and merged using the XDS program suite.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>21330</offset><text>Structure solution and refinement</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>21364</offset><text>Structure solution was done by molecular replacement with Phaser employing the structure of Irga6 without nucleotide [PDB: 1TQ2] as search model. Atomic model building was done by Coot. Iterative refinement was done using Phenix at a maximum resolution of 3.2 Å. For the refinement strategy, a seven-fold non-crystallographic symmetry as well as one molecule of Irga6 [PDB: 1TQ4] as high resolution reference structure was chosen. Five percent of the measured X-ray intensities were set aside from the refinement as cross-validation. Methionine sites in the protein were confirmed by the anomalous signal of the selenium atoms. Protein superposition was done with lsqkab and the PyMol Molecular Graphics System, Version 1.3 Schrödinger, LLC. Figures were prepared using the PyMOL Molecular Graphics System, Version 1.7.4 Schrödinger, LLC. Evaluation of atom contacts and geometry of the atomic model was done by the Molprobity server. Interface sizes were calculated by the PISA server.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>22354</offset><text>Accession numbers</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>22372</offset><text>The Irga6 coordinates were submitted to the Protein Data Bank (pdb) database with accession code 5fph. http://www.rcsb.org/pdb/explore/explore.do?structureId=5fph.</text></passage><passage><infon key="section_type">CONCL</infon><infon key="type">title_1</infon><offset>22536</offset><text>Conclusions</text></passage><passage><infon key="section_type">CONCL</infon><infon key="type">paragraph</infon><offset>22548</offset><text>Our study indicates that Irg proteins dimerize via the G interface in a parallel head-to-head fashion thereby facilitating GTPase activation. These findings contribute to a molecular understanding of the anti-parasitic action of the Irg protein family and suggest that Irgs are bona-fide members of the dynamin superfamily.</text></passage><passage><infon key="section_type">CONCL</infon><infon key="type">title_1</infon><offset>22872</offset><text>Additional file</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">title</infon><offset>22888</offset><text>Abbreviations</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>22902</offset><text>BDLP</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>22907</offset><text>Bacterial dynamin like protein</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>22938</offset><text>EHD2</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>22943</offset><text>Eps15 homology domain containing protein 2</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>22986</offset><text>GBP</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>22990</offset><text>Guanylate-binding protein</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>23016</offset><text>GDP</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>23020</offset><text>Guanosine-diphosphate</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>23042</offset><text>GIMAP2</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>23049</offset><text>GTPase of immunity associated protein 2</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>23089</offset><text>GMPPNP</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>23096</offset><text>5'-guanylyl imidodiphosphate</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>23125</offset><text>GTP</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>23129</offset><text>Guanosine-triphosphate</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>23152</offset><text>IRG</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>23156</offset><text>Immunity-related GTPase</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>23180</offset><text>Irga6</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>23186</offset><text>Immunity-related GTPase 6</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>23212</offset><text>MxA</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>23216</offset><text>Myxovirus resistance protein A</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>23247</offset><text>PVM</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>23251</offset><text>Parasitophorous vacuolar membrane</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">footnote</infon><offset>23285</offset><text>Competing interests</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">footnote</infon><offset>23305</offset><text>The authors declare that they have no competing interests.</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">footnote</infon><offset>23364</offset><text>Authors’ contributions</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">footnote</infon><offset>23389</offset><text>All authors planned the experimental design. NP cloned, characterized, and purified the Irga6 construct and found initial crystallization conditions. KS and CF optimized the crystallization condition and found suitable cryo conditions. KS and KF collected data, KF solved, and KS and KF refined the structure. KS, NP, CF, JCH, and OD wrote the manuscript. 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raw_BioC_XML/PMC4781976_raw.xml

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+<!DOCTYPE collection SYSTEM "BioC.dtd">
+<collection><source>PMC</source><date>20201222</date><key>pmc.key</key><document><id>4781976</id><infon key="license">CC BY</infon><passage><infon key="article-id_doi">10.1016/j.dib.2016.02.042</infon><infon key="article-id_pmc">4781976</infon><infon key="article-id_pmid">26977434</infon><infon key="article-id_publisher-id">S2352-3409(16)30064-6</infon><infon key="fpage">344</infon><infon key="kwd">Tom1, GAT domain, Tollip, Ubiquitin, nuclear magnetic resonance</infon><infon key="license">This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).</infon><infon key="lpage">348</infon><infon key="name_0">surname:Xiao;given-names:Shuyan</infon><infon key="name_1">surname:Ellena;given-names:Jeffrey F.</infon><infon key="name_2">surname:Armstrong;given-names:Geoffrey S.</infon><infon key="name_3">surname:Capelluto;given-names:Daniel G.S.</infon><infon key="section_type">TITLE</infon><infon key="title">Keywords</infon><infon key="type">front</infon><infon key="volume">7</infon><infon key="year">2016</infon><offset>0</offset><text>Structure of the GAT domain of the endosomal adapter protein Tom1</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>66</offset><text>Cellular homeostasis requires correct delivery of cell-surface receptor proteins (cargo) to their target subcellular compartments. The adapter proteins Tom1 and Tollip are involved in sorting of ubiquitinated cargo in endosomal compartments. Recruitment of Tom1 to the endosomal compartments is mediated by its GAT domain’s association to Tollip’s Tom1-binding domain (TBD). In this data article, we report the solution NMR-derived structure of the Tom1 GAT domain. The estimated protein structure exhibits a bundle of three helical elements. We compare the Tom1 GAT structure with those structures corresponding to the Tollip TBD- and ubiquitin-bound states.</text></passage><passage><infon key="section_type">TABLE</infon><infon key="type">title_1</infon><offset>730</offset><text>Specifications table</text></passage><passage><infon key="file">t0010.xml</infon><infon key="id">t0010</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td&gt;Subject area&lt;/td&gt;&lt;td&gt;&lt;italic&gt;Biology&lt;/italic&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;More specific subject area&lt;/td&gt;&lt;td&gt;&lt;italic&gt;Structural biology&lt;/italic&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Type of data&lt;/td&gt;&lt;td&gt;&lt;italic&gt;Table, text file, graph, figures&lt;/italic&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;How data was acquired&lt;/td&gt;&lt;td&gt;&lt;italic&gt;Circular dichroism and NMR. NMR data was recorded using a Bruker 800 MHz&lt;/italic&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Data format&lt;/td&gt;&lt;td&gt;&lt;italic&gt;PDB format text file. Analyzed by CS-Rosetta, Protein Structure Validation Server (PSVS), NMRPipe, NMRDraw, and PyMol&lt;/italic&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Experimental factors&lt;/td&gt;&lt;td&gt;&lt;italic&gt;Recombinant human Tom1 GAT domain was purified to homogeneity before use&lt;/italic&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Experimental features&lt;/td&gt;&lt;td&gt;&lt;italic&gt;Solution structure of Tom1 GAT was determined from NMR chemical shift data&lt;/italic&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Data source location&lt;/td&gt;&lt;td&gt;&lt;italic&gt;Virginia and Colorado, United States.&lt;/italic&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Data accessibility&lt;/td&gt;&lt;td&gt;&lt;italic&gt;Data is available within this article. Tom1 GAT structural data is publicly available in the RCSB Protein Data Bank (http://www.rscb.org/) under the accession number PDB: 2n9d&lt;/italic&gt;&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>751</offset><text>Table	 	</text></passage><passage><infon key="file">t0010.xml</infon><infon key="id">t0010</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td&gt;Subject area&lt;/td&gt;&lt;td&gt;&lt;italic&gt;Biology&lt;/italic&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;More specific subject area&lt;/td&gt;&lt;td&gt;&lt;italic&gt;Structural biology&lt;/italic&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Type of data&lt;/td&gt;&lt;td&gt;&lt;italic&gt;Table, text file, graph, figures&lt;/italic&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;How data was acquired&lt;/td&gt;&lt;td&gt;&lt;italic&gt;Circular dichroism and NMR. NMR data was recorded using a Bruker 800 MHz&lt;/italic&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Data format&lt;/td&gt;&lt;td&gt;&lt;italic&gt;PDB format text file. Analyzed by CS-Rosetta, Protein Structure Validation Server (PSVS), NMRPipe, NMRDraw, and PyMol&lt;/italic&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Experimental factors&lt;/td&gt;&lt;td&gt;&lt;italic&gt;Recombinant human Tom1 GAT domain was purified to homogeneity before use&lt;/italic&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Experimental features&lt;/td&gt;&lt;td&gt;&lt;italic&gt;Solution structure of Tom1 GAT was determined from NMR chemical shift data&lt;/italic&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Data source location&lt;/td&gt;&lt;td&gt;&lt;italic&gt;Virginia and Colorado, United States.&lt;/italic&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Data accessibility&lt;/td&gt;&lt;td&gt;&lt;italic&gt;Data is available within this article. Tom1 GAT structural data is publicly available in the RCSB Protein Data Bank (http://www.rscb.org/) under the accession number PDB: 2n9d&lt;/italic&gt;&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>760</offset><text>Subject area	Biology	 	More specific subject area	Structural biology	 	Type of data	Table, text file, graph, figures	 	How data was acquired	Circular dichroism and NMR. NMR data was recorded using a Bruker 800 MHz	 	Data format	PDB format text file. Analyzed by CS-Rosetta, Protein Structure Validation Server (PSVS), NMRPipe, NMRDraw, and PyMol	 	Experimental factors	Recombinant human Tom1 GAT domain was purified to homogeneity before use	 	Experimental features	Solution structure of Tom1 GAT was determined from NMR chemical shift data	 	Data source location	Virginia and Colorado, United States.	 	Data accessibility	Data is available within this article. Tom1 GAT structural data is publicly available in the RCSB Protein Data Bank (http://www.rscb.org/) under the accession number PDB: 2n9d	 	</text></passage><passage><infon key="section_type">TABLE</infon><infon key="type">title_1</infon><offset>1563</offset><text>Value of the data</text></passage><passage><infon key="section_type">TABLE</infon><infon key="type">paragraph</infon><offset>1581</offset><text>The Tom1 GAT domain solution structure will provide additional tools for modulating its biological function.</text></passage><passage><infon key="section_type">TABLE</infon><infon key="type">paragraph</infon><offset>1690</offset><text>Tom1 GAT can adopt distinct conformations upon ligand binding.</text></passage><passage><infon key="section_type">TABLE</infon><infon key="type">paragraph</infon><offset>1753</offset><text>A conformational response of the Tom1 GAT domain upon Tollip TBD binding can serve as an example to explain mutually exclusive ligand binding events.</text></passage><passage><infon key="section_type">TABLE</infon><infon key="type">title_1</infon><offset>1903</offset><text>Data</text></passage><passage><infon key="section_type">TABLE</infon><infon key="type">paragraph</infon><offset>1908</offset><text>Analysis of the far-UV circular dichroism (CD) spectrum of the Tom 1 GAT domain (Fig. 1) predicts 58.7% α-helix, 3% β-strand, 15.5% turn, and 22.8% disordered regions. The Tom1 GAT structural restraints yielded ten helical structures (Fig. 2A,B) with a root mean square deviation (RMSD) of 0.9 Å for backbone and 1.3 Å for all heavy atoms (Table 1) and estimated the presence of three helices spanning residues Q216-E240 (α-helix 1), P248-Q274 (α-helix 2), and E278-T306 (α-helix 3). Unlike ubiquitin binding, data suggest that conformational changes of the Tom1 GAT α-helices 1 and 2 occur upon Tollip TBD binding (Fig. 3A,B).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>2559</offset><text>Experimental design, materials, and methods</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>2603</offset><text>Protein expression and purification</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>2639</offset><text>Human Tom1 GAT (residues 215–309) cDNA was cloned into both pGEX6P1 and pET28a vectors, and expressed as GST-tagged and His-tagged fusion proteins, respectively, using Escherichia coli [Rosetta (DE3) strain]. The 13C, 15N-labeled Tom1 GAT domain was expressed and purified as described previously.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>2939</offset><text>Circular dichroism</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>2958</offset><text>Far-UV CD spectra of the His-Tom1 GAT domain were collected on a Jasco J-815 spectropolarimeter using a 1 mm path length quartz cell at room temperature. The protein (10 μM) was solubilized in 5 mM Tris–HCl (pH 7) and 100 mM KF. Spectra were obtained from five accumulated scans from 190 to 260 nm using a bandwidth of 1 nm and a response time of 1 s at a scan speed of 20 nm/min. Buffer backgrounds were employed to subtract the protein spectra. Data was processed using the Dichroweb server and the CONTIN algorithm (http://dichroweb.cryst.bbk.ac.uk/html/home.shtml).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>3539</offset><text>NMR structure determination</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>3567</offset><text>NMR experiments were performed using 1 mM 13C, 15N-labeled Tom1 GAT domain in a buffer containing 20 mM d11-TrisHCl (pH 7), 50 mM KCl, 1 mM d18-DTT, and 1 mM NaN3. NMR spectra were recorded at 25 °C on a Bruker 800-MHz spectrometer (University of Virginia). The individual structure of Tom1 GAT was generated using CS-Rosetta (https://csrosetta.bmrb.wisc.edu/csrosetta). Chemical shift information (BMRB #26574) was used to obtain the structure calculation. The Rosetta calculations yielded 3000 structures of Tom1 GAT. From these, ten structures were selected based on their score and RMSDs, and converted to Protein Data Bank (PDB) format. NMR structural statistics for the ten lowest energy conformers of Tom1 GAT was generated using the Protein Structure Validation Suite. By using MolProbity, the Ramachandran analysis of the ten superimposed Tom1 GAT structures identified that 100% of the residues were in the most favored regions and there were no Ramachandran outliers in the allowed and disallowed regions. Protein structure images were obtained using PyMol (http://www.pymol.org). The structures of the ubiquitin- and Tollip TBD-bound states of the Tom1 GAT domain were obtained from data reported in Refs. and.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">title</infon><offset>4797</offset><text>References</text></passage><passage><infon key="fpage">1910</infon><infon key="lpage">1920</infon><infon key="name_0">surname:Xiao;given-names:S.</infon><infon key="name_1">surname:Brannon;given-names:M.K.</infon><infon key="name_2">surname:Zhao;given-names:X.</infon><infon key="name_3">surname:Fread;given-names:K.I.</infon><infon key="name_4">surname:Ellena;given-names:J.F.</infon><infon key="name_5">surname:Bushweller;given-names:J.H.</infon><infon key="name_6">surname:Finkielstein;given-names:C.V.</infon><infon key="name_7">surname:Armstrong;given-names:G.S.</infon><infon key="name_8">surname:Capelluto;given-names:D.G.</infon><infon key="section_type">REF</infon><infon key="source">Structure</infon><infon key="type">ref</infon><infon key="volume">23</infon><infon key="year">2015</infon><offset>4808</offset><text>Tom1 modulates binding of Tollip to phosphatidylinositol 3-phosphate via a coupled folding and binding mechanism</text></passage><passage><infon key="fpage">5385</infon><infon key="lpage">5391</infon><infon key="name_0">surname:Akutsu;given-names:M.</infon><infon key="name_1">surname:Kawasaki;given-names:M.</infon><infon key="name_2">surname:Katoh;given-names:Y.</infon><infon key="name_3">surname:Shiba;given-names:T.</infon><infon key="name_4">surname:Yamaguchi;given-names:Y.</infon><infon key="name_5">surname:Kato;given-names:R.</infon><infon key="name_6">surname:Kato;given-names:K.</infon><infon key="name_7">surname:Nakayama;given-names:K.</infon><infon key="name_8">surname:Wakatsuki;given-names:S.</infon><infon key="pub-id_pmid">16199040</infon><infon key="section_type">REF</infon><infon key="source">FEBS Lett.</infon><infon key="type">ref</infon><infon key="volume">579</infon><infon key="year">2005</infon><offset>4921</offset><text>Structural basis for recognition of ubiquitinated cargo by Tom1-GAT domain</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">title_1</infon><offset>4996</offset><text>Supplementary material</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>5019</offset><text>Supplementary data associated with this article can be found in the online version at doi:10.1016/j.dib.2016.02.042.</text></passage><passage><infon key="file">gr1.jpg</infon><infon key="id">f0005</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>5136</offset><text>Representative far-UV CD spectrum of the His-Tom1 GAT domain.</text></passage><passage><infon key="file">gr1.jpg</infon><infon key="id">f0005</infon><infon key="section_type">FIG</infon><infon key="type">fig</infon><offset>5198</offset><text>Fig. 1.</text></passage><passage><infon key="file">gr2.jpg</infon><infon key="id">f0010</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>5206</offset><text>(A) Stereo view displaying the best-fit backbone superposition of the refined structures for the Tom1 GAT domain. Helices are shown in orange, whereas loops are colored in green. (B) Ribbon illustration of the Tom1 GAT domain.</text></passage><passage><infon key="file">gr2.jpg</infon><infon key="id">f0010</infon><infon key="section_type">FIG</infon><infon key="type">fig</infon><offset>5433</offset><text>Fig. 2.</text></passage><passage><infon key="file">gr3.jpg</infon><infon key="id">f0015</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>5441</offset><text>(A) Two views of the superimposed structures of the Tom1 GAT domain in the free state (gray) with that in the Tollip TBD-bound state (red). (B) Two views of the superimposed structures of the Tom1 GAT domain (gray) with that in the Ub-bound state (green).</text></passage><passage><infon key="file">gr3.jpg</infon><infon key="id">f0015</infon><infon key="section_type">FIG</infon><infon key="type">fig</infon><offset>5697</offset><text>Fig. 3.</text></passage><passage><infon key="file">t0005.xml</infon><infon key="id">t0005</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>5705</offset><text>NMR and refinement statistics for the Tom1 GAT domain. NMR structural statistics for lowest energy conformers of Tom1 GAT using PSVS.</text></passage><passage><infon key="file">t0005.xml</infon><infon key="id">t0005</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;thead&gt;&lt;tr&gt;&lt;th/&gt;&lt;th&gt;&lt;bold&gt;Tom1 GAT&lt;/bold&gt;&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td&gt;&lt;bold&gt;NMR distance and dihedral constraints&lt;/bold&gt;&lt;/td&gt;&lt;td/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt; Dihedral angle restraints total&lt;/td&gt;&lt;td&gt;178&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;&lt;italic&gt; ϕ&lt;/italic&gt;&lt;/td&gt;&lt;td&gt;89&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;&lt;italic&gt; ψ&lt;/italic&gt;&lt;/td&gt;&lt;td&gt;89&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;&lt;bold&gt;Structure statistics&lt;/bold&gt;&lt;/td&gt;&lt;td/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt; Dihedral angle constraints (deg)&lt;/td&gt;&lt;td&gt;8.8±0.2&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt; Max. dihedral angle violation (deg)&lt;/td&gt;&lt;td&gt;111±3&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Deviations from idealized geometry&lt;/td&gt;&lt;td/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt; Bond lengths (Å)&lt;/td&gt;&lt;td&gt;0.011&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt; Bond angles (deg)&lt;/td&gt;&lt;td&gt;0.7&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Average pairwise r.m.s. deviation (Å)&lt;xref rid=&quot;tbl1fna&quot; ref-type=&quot;table-fn&quot;&gt;a&lt;/xref&gt;&lt;/td&gt;&lt;td/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt; Protein&lt;/td&gt;&lt;td/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt; Heavy&lt;/td&gt;&lt;td&gt;1.3&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt; Backbone&lt;/td&gt;&lt;td&gt;0.9&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>5839</offset><text>Table 1.	 	</text></passage><passage><infon key="file">t0005.xml</infon><infon key="id">t0005</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;thead&gt;&lt;tr&gt;&lt;th/&gt;&lt;th&gt;&lt;bold&gt;Tom1 GAT&lt;/bold&gt;&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td&gt;&lt;bold&gt;NMR distance and dihedral constraints&lt;/bold&gt;&lt;/td&gt;&lt;td/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt; Dihedral angle restraints total&lt;/td&gt;&lt;td&gt;178&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;&lt;italic&gt; ϕ&lt;/italic&gt;&lt;/td&gt;&lt;td&gt;89&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;&lt;italic&gt; ψ&lt;/italic&gt;&lt;/td&gt;&lt;td&gt;89&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;&lt;bold&gt;Structure statistics&lt;/bold&gt;&lt;/td&gt;&lt;td/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt; Dihedral angle constraints (deg)&lt;/td&gt;&lt;td&gt;8.8±0.2&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt; Max. dihedral angle violation (deg)&lt;/td&gt;&lt;td&gt;111±3&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Deviations from idealized geometry&lt;/td&gt;&lt;td/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt; Bond lengths (Å)&lt;/td&gt;&lt;td&gt;0.011&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt; Bond angles (deg)&lt;/td&gt;&lt;td&gt;0.7&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Average pairwise r.m.s. deviation (Å)&lt;xref rid=&quot;tbl1fna&quot; ref-type=&quot;table-fn&quot;&gt;a&lt;/xref&gt;&lt;/td&gt;&lt;td/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt; Protein&lt;/td&gt;&lt;td/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt; Heavy&lt;/td&gt;&lt;td&gt;1.3&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt; Backbone&lt;/td&gt;&lt;td&gt;0.9&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>5851</offset><text>	Tom1 GAT	 	NMR distance and dihedral constraints		 	 Dihedral angle restraints total	178	 	 ϕ	89	 	 ψ	89	 	Structure statistics		 	 Dihedral angle constraints (deg)	8.8±0.2	 	 Max. dihedral angle violation (deg)	111±3	 	Deviations from idealized geometry		 	 Bond lengths (Å)	0.011	 	 Bond angles (deg)	0.7	 	Average pairwise r.m.s. deviation (Å)a		 	 Protein		 	 Heavy	1.3	 	 Backbone	0.9	 	</text></passage><passage><infon key="file">t0005.xml</infon><infon key="id">t0005</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>6261</offset><text>Pairwise backbone and heavy-atom r.m.s. deviations were obtained by superimposing residues 215–309 of Tom1 GAT among 10 lowest energy refined structures.</text></passage></document></collection>

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+<collection><source>PMC</source><date>20201223</date><key>pmc.key</key><document><id>4820378</id><infon key="license">CC BY-NC</infon><passage><infon key="article-id_doi">10.1126/sciadv.1501397</infon><infon key="article-id_pmc">4820378</infon><infon key="article-id_pmid">27051866</infon><infon key="article-id_publisher-id">1501397</infon><infon key="elocation-id">e1501397</infon><infon key="issue">3</infon><infon key="kwd">biomolecules nucleotides tRNA 3′-5′ addition reverse polymerization TLP crystal structure</infon><infon key="license">This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.</infon><infon key="name_0">surname:Kimura;given-names:Shoko</infon><infon key="name_1">surname:Suzuki;given-names:Tateki</infon><infon key="name_2">surname:Chen;given-names:Meirong</infon><infon key="name_3">surname:Kato;given-names:Koji</infon><infon key="name_4">surname:Yu;given-names:Jian</infon><infon key="name_5">surname:Nakamura;given-names:Akiyoshi</infon><infon key="name_6">surname:Tanaka;given-names:Isao</infon><infon key="name_7">surname:Yao;given-names:Min</infon><infon key="name_8">surname:Tanaka;given-names:Isao</infon><infon key="section_type">TITLE</infon><infon key="title">Keywords</infon><infon key="type">front</infon><infon key="volume">2</infon><infon key="year">2016</infon><offset>0</offset><text>Template-dependent nucleotide addition in the reverse (3′-5′) direction by Thg1-like protein</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>97</offset><text>Structures of Thg1-like proteins provide insight into the template-dependent nucleotide addition in the reverse (3′-5′) direction.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>232</offset><text>Thg1-like protein (TLP) catalyzes the addition of a nucleotide to the 5′-end of truncated transfer RNA (tRNA) species in a Watson-Crick template–dependent manner. The reaction proceeds in two steps: the activation of the 5′-end by adenosine 5′-triphosphate (ATP)/guanosine 5′-triphosphate (GTP), followed by nucleotide addition. Structural analyses of the TLP and its reaction intermediates have revealed the atomic detail of the template-dependent elongation reaction in the 3′-5′ direction. The enzyme creates two substrate binding sites for the first- and second-step reactions in the vicinity of one reaction center consisting of two Mg2+ ions, and the two reactions are executed at the same reaction center in a stepwise fashion. When the incoming nucleotide is bound to the second binding site with Watson-Crick hydrogen bonds, the 3′-OH of the incoming nucleotide and the 5′-triphosphate of the tRNA are moved to the reaction center where the first reaction has occurred. That the 3′-5′ elongation enzyme performs this elaborate two-step reaction in one catalytic center suggests that these two reactions have been inseparable throughout the process of protein evolution. Although TLP and Thg1 have similar tetrameric organization, the tRNA binding mode of TLP is different from that of Thg1, a tRNAHis-specific G−1 addition enzyme. Each tRNAHis binds to three of the four Thg1 tetramer subunits, whereas in TLP, tRNA only binds to a dimer interface and the elongation reaction is terminated by measuring the accepter stem length through the flexible β-hairpin. Furthermore, mutational analyses show that tRNAHis is bound to TLP in a similar manner as Thg1, thus indicating that TLP has a dual binding mode.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">title_1</infon><offset>1972</offset><text>INTRODUCTION</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1985</offset><text>All polynucleotide chain elongation reactions, whether with DNA or RNA, proceed in the 5′-3′ direction. This reaction involves the nucleophilic attack of a 3′-OH of the terminal nucleotide in the elongating chain on the α-phosphate of an incoming nucleotide. The energy in the high-energy bond of the incoming nucleotide is used for its addition [termed tail polymerization ]. This elongation reaction of DNA/RNA chains is in clear contrast to the elongation of protein chains in which the high energy of the incoming aminoacyl-tRNA is not used for its own addition but for the addition of the next monomer (termed head polymerization). However, recent studies have shown that the Thg1/Thg1-like protein (TLP) family of proteins extends tRNA nucleotide chains in the reverse (3′-5′) direction. In this case, the 5′-end of tRNA is first activated using adenosine 5′-triphosphate (ATP)/guanosine 5′-triphosphate (GTP), followed by nucleophilic attack of a 3′-OH on the incoming nucleotide [nucleoside 5′-triphosphate (NTP)] to yield pppN-tRNA. Thus, the energy in the triphosphate bond of the incoming nucleotide is not used for its own addition but is reserved for subsequent polymerization (that is, head polymerization) (Fig. 1).</text></passage><passage><infon key="file">1501397-F1.jpg</infon><infon key="id">F1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>3237</offset><text>Reaction schemes of 3′-5′ and 5′-3′ elongation.</text></passage><passage><infon key="file">1501397-F1.jpg</infon><infon key="id">F1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>3293</offset><text>Top: Reaction scheme of 3′-5′ elongation by Thg1/TLP family proteins. Bottom: Reaction scheme of 5′-3′ elongation by DNA/RNA polymerases. In 3′-5′ elongation by Thg1/TLP family proteins, the 5′-monophosphate of the tRNA is first activated by ATP/GTP, followed by the actual elongation reaction. The energy of the incoming nucleotide is not used for its own addition but is reserved for the subsequent addition (head polymerization). In 5′-3′ elongation by DNA/RNA polymerases, the energy of the incoming nucleotide is used for its own addition (tail polymerization).</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3878</offset><text>The best-characterized member of this family of proteins is eukaryotic Thg1 (tRNAHis guanylyltransferase), which catalyzes the nontemplated addition of a guanylate to the 5′-end of immature tRNAHis. This guanosine at position −1 (G−1) of tRNAHis is a critical identity element for recognition by the histidyl-tRNA synthase. Therefore, Thg1 is essential to the fidelity of protein synthesis in eukaryotes. However, Thg1 homologs or TLPs are found in organisms in which G−1 is genetically encoded, and thus, posttranscriptional modification is not required. This finding suggests that TLPs may have potential functions other than tRNAHis maturation. TLPs have been shown to catalyze 5′-end nucleotide addition to truncated tRNA species in vitro in a Watson-Crick template–dependent manner. This function of TLPs is not limited to tRNAHis but occurs efficiently with other tRNAs. Furthermore, the yeast homolog (Thg1p) has been shown to interact with the replication origin recognition complex for DNA replication, and the plant homolog (ICA1) was identified as a protein affecting the capacity to repair DNA damage. These observations suggest that TLPs may have more general functions in DNA/RNA repair.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>5092</offset><text>The 3′-5′ addition reaction catalyzed by Thg1 occurs through three reaction steps. In the first step, the 5′-monophosphorylated tRNAHis, which is cleaved by ribonuclease P from pre-tRNAHis, is activated by ATP, creating a 5′-adenylylated tRNAHis intermediate. In the second step, the 3′-OH of the incoming GTP attacks the activated intermediate, yielding pppG−1-tRNAHis. Finally, the pyrophosphate is removed, and mature pG−1-tRNAHis is created. The crystal structure of human Thg1 (HsThg1) shows that its catalytic core shares structural homology with canonical 5′-3′ nucleotide polymerases, such as T7 DNA/RNA polymerases. This finding suggests that 3′-5′ elongation enzymes are related to 5′-3′ polymerases and raises important questions on why 5′-3′ polymerases predominate in nature. The crystal structure of TLP from Bacillus thuringiensis shows that it shares a similar tetrameric assembly and active-site architecture with HsThg1. Furthermore, the structure of Candida albicans Thg1 (CaThg1) complexed with tRNAHis reveals that the tRNA substrate accesses the reaction center from a direction opposite to that of canonical DNA/RNA polymerase. However, in this structural analysis, the 5′-end of tRNAHis was not activated and the second substrate (GTP) was not bound. Thus, a detailed reaction mechanism remains unknown.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>6452</offset><text>Here, we successfully solved the structure of TLP from the methanogenic archaeon Methanosarcina acetivorans (MaTLP) in complex with ppptRNAPheΔ1, which mimics the activated intermediate of the repair substrate. Although TLP and Thg1 have similar tetrameric organization, the mode of tRNA binding is different in TLP. Furthermore, we obtained the structure in which the GTP analog (GDPNP) was inserted into this complex to form a Watson-Crick base pair with C72 at the 3′-end region of the tRNA. On the basis of these structures, we discuss the reaction mechanism of template-dependent reverse (3′-5′) polymerization in comparison with canonical 5′-3′ polymerization.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>7130</offset><text>RESULTS</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>7138</offset><text>Anticodon-independent binding of ppptRNAPheΔ1 to MaTLP</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>7197</offset><text>Previous biochemical experiments have suggested that ppptRNAPheΔ1, in which the 5′-end of tRNAPhe was triphosphorylated and G1 was deleted, can be an efficient substrate for the repair reaction (guanylyl transfer) of Thg1/TLP. Therefore, we prepared a crystal of MaTLP complexed with ppptRNAPheΔ1 and solved its structure to study the template-directed 3′-5′ elongation reaction by TLP (fig. S1). The crystal contained a dimer of TLP (A and B molecules) and one tRNA in an asymmetric unit. Two dimers in the crystal further assembled as a dimer of dimers by the crystallographic twofold axis (Fig. 2). This tetrameric structure and 4:2 stoichiometry of the TLP-tRNA complex are the same as those of the CaThg1-tRNA complex. However, whereas the AB and CD dimers of tetrameric CaThg1 play different roles, respectively recognizing the accepter stem and anticodon of tRNAHis, the AB dimer and its symmetry mate (CD dimer) on tetrameric MaTLP independently bind one molecule of tRNA (fig. S2), recognizing the tRNA accepter stem and elbow region. Thus, consistent with the notion that MaTLP is an anticodon-independent repair enzyme, the anticodon was not recognized in the MaTLP-tRNA complex, whereas the binding mode of CaThg1 is for the G−1 addition reaction, therefore the His anticodon has to be recognized (see “Dual binding mode for tRNA repair”).</text></passage><passage><infon key="file">1501397-F2.jpg</infon><infon key="id">F2</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>8563</offset><text>Structure of the MaTLP complex with ppptRNAPheΔ1.</text></passage><passage><infon key="file">1501397-F2.jpg</infon><infon key="id">F2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>8617</offset><text>Left: One molecule of the tRNA substrate (ppptRNAPheΔ1) is bound to the MaTLP dimer. The AB and CD dimers are further dimerized by the crystallographic twofold axis to form a tetrameric structure (dimer of dimers). Right: Left figure rotated by 90o. The CD dimer is omitted for clarity. The accepter stem of the tRNA is recognized by molecule A (yellow), and the elbow region by molecule B (blue). Residues important for binding are depicted in stick form. The β-hairpin region of molecule B is shown in red.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9133</offset><text>The elbow region of the tRNA substrate was recognized by the β-hairpin of molecule B of MaTLP. The N atoms in the side chain of R215 in the β-hairpin region of MaTLP were hydrogen-bonded to the phosphate groups of U55 and G57. The O atom on the S213 side chain was also hydrogen-bonded to the phosphate moiety of G57 of the tRNA (Fig. 2). This β-hairpin region was disordered in the crystal structure of the CaThg1-tRNA complex.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9571</offset><text>The accepter stem of the tRNA substrate was recognized by molecule A of MaTLP. The N7 atom of G2 at the 5′-end was hydrogen-bonded to the N atom of the R136 side chain, whereas the α-phosphate was bonded to the N137 side chain (Fig. 2). R136 was also hydrogen-bonded to the base of C72 (the Watson-Crick bond partner of ΔG1). The triphosphate moiety at the 5′-end of the tRNA was bonded to the D21-K26 region. These phosphates were also coordinated to two metal ions, presumably Mg2+ (Mg2+A and Mg2+B) because they were observed at the same positions (figs. S3 and S4) previously identified by CaThg1 and HsThg1 structures. These ions were in turn coordinated by the O atoms of the side chains of D21 and D69 and the main-chain O of G22 (fig. S3A). Mutation of D29 and D76 in HsThg1 (corresponding to D21 and D69 of MaTLP) has been shown to markedly decrease G−1 addition activity.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>10461</offset><text>Template-dependent binding of the GTP analog to the MaTLP-ppptRNAPheΔ1 complex</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10544</offset><text>Here, we successfully obtained the structure of the ternary complex of MaTLP, 5′-activated tRNA (ppptRNAPheΔ1), and the GTP analog (GDPNP) (Fig. 3 and fig. S4) by soaking the MaTLP-ppptRNAPheΔ1 complex crystal in a solution containing GDPNP. The obtained structure showed that the guanine base of the incoming GDPNP formed Watson-Crick hydrogen bonds with C72 and accompanied base-stacking interactions with G2 of the tRNA (Fig. 3B), whereas no interaction was observed between the guanine base and the enzyme. These features are consistent with the fact that this elongation reaction is template-dependent. The 5′-end (position 2) of the tRNA moved significantly (Fig. 3C) due to the insertion of GDPNP. Surprisingly, the 5′-triphosphate moiety after movement occupied the GTP/ATP triphosphate position during the activation step (Fig. 3D). Together with the observation that the 3′-OH of the incoming GTP analog was within coordination distance (2.8 Å) to Mg2+A (fig. S3B) and was able to execute a nucleophilic attack on the α-phosphate of the 5′-end, this structure indicates that the elongation reaction (second reaction) takes place at the same reaction center where the activation reaction (first reaction) occurs.</text></passage><passage><infon key="file">1501397-F3.jpg</infon><infon key="id">F3</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>11780</offset><text>Structural change of the tRNA (ppptRNAPheΔ1).</text></passage><passage><infon key="file">1501397-F3.jpg</infon><infon key="id">F3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>11830</offset><text>Structural change of the tRNA (ppptRNAPheΔ1) accepter stem in MaTLP caused by insertion of GDPNP. (A) Structure before GDPNP binding. (B) Structure after GDPNP binding. (C) Superposition of the two structures showing movement of the 5′-end of the tRNA before (blue) and after (red) insertion of GDPNP. (D) Superposition of the 5′-end of the tRNA after GDPNP insertion (red) with GTP at the activation step (green), showing that both triphosphate moieties superpose well.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>12306</offset><text>The triphosphate moiety of GDPNP was at the interface between molecules A and B and was recognized by the side chains of both molecules, including R19 (molecule A), R83 (molecule B), K86 (molecule B), and R114 (molecule A) (Fig. 3B). All of these residues are well conserved (fig. S5), and mutation of corresponding residues in ScThg1 (R27, R93, K96, and R133) decreased the catalytic efficiency of G−1 addition. The triphosphate of the GDPNP was also bonded to the third Mg2+ (Mg2+C), which, unlike Mg2+A and Mg2+B, is not coordinated by the TLP molecule (fig. S3B). This triphosphate binding mode is the same as that for the second nucleotide binding site in Thg1. However, in previous analyses, the base moiety at the second site was either invisible or far beyond the reaction distance of the phosphate, and therefore, flipping of the base was expected to occur.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>13175</offset><text>tRNA binding and repair experiments of the β-hairpin mutants</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>13239</offset><text>To confirm tRNA recognition by the β-hairpin, we created mutation variants with altered residues in the β-hairpin region. Then, tRNA binding and enzymatic activities were measured. β-Hairpin deletion variant delR198-R215 almost completely abolished the binding of tRNAPheΔ1 (fig. S6). Furthermore, the enzymatic activities of delR198-R215 and delG202-E210 were very weak (5.2 and 13.5%, respectively) compared with wild type, whereas mutations (N179A and F174A/N179A/R188A) on the anticodon recognition site [deduced from the Thg1-tRNAHis complex structure ] had no effect on the catalytic activity (Fig. 4A). Experiments using the tRNAHisΔ1 substrate gave similar results (Fig. 4A). All these results are consistent with the crystal structure and suggest that the β-hairpin plays an important role in anticodon-independent binding of the tRNA substrate. Residues in the β-hairpin are not well conserved, except for R215 (fig. S5). Mutants R215A and R215A/S213A, in which the completely conserved R215 was changed to alanine, showed a moderate effect on the activity (27.3 and 16.3%, respectively). Thus, specific interactions with the conserved R215 and van der Waals contacts to residues in the β-hairpin would be important for tRNA recognition.</text></passage><passage><infon key="file">1501397-F4.jpg</infon><infon key="id">F4</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>14513</offset><text>Mutational analysis of the β-hairpin and anticodon binding region.</text></passage><passage><infon key="file">1501397-F4.jpg</infon><infon key="id">F4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>14583</offset><text>The rates of guanylylation by various mutants were measured. Error bars represent the SD of three independent experiments. (A) Guanylylation of ppptRNAPheΔ1 and ppptRNAHisΔ1 by various TLP mutants. The activity using [α-32P]GTP, wild-type MaTLP, and ppptRNAPheΔ1 is denoted as 100. (B) Guanylylation of tRNAPheΔ1, tRNAPhe, and tRNAHisΔ−1 by various TLP mutants. The activity to tRNAPheΔ1 is about 10% of ppptRNAPheΔ1.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>15033</offset><text>Termination of the elongation reaction by measuring the accepter stem</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15103</offset><text>TLPs catalyze the Watson-Crick template–dependent elongation or repair reaction for 5′-end truncated tRNAPhe substrates lacking G1 only (tRNAPheΔ1), or lacking both G1 and G2 (tRNAPheΔ1,2), whereas they do not show any activity with intact tRNAPhe (thus, repair is unnecessary). How TLP distinguishes between tRNAs that need 5′-end repair from ones that do not, or in other words, how the elongation reaction is properly terminated, remains unknown. The present structure of the MaTLP-ppptRNAPheΔ1 complex shows that, unlike Thg1, the TLP dimer binds one molecule of tRNA by recognizing the elbow region by the β-hairpin of molecule B and the 5′-end by molecule A. Therefore, we speculated that the flexible nature of the β-hairpin enables the recognition of tRNA substrates with different accepter stem lengths. To confirm this speculation, we used computer graphics to examine whether the β-hairpin region was able to bind tRNA substrates with different accepter stem lengths when the 5′-end was properly placed in the reaction site. When the 5′-end was placed in the reaction site, the body of the tRNA molecule shifted in a manner dependent on the accepter stem length. The tRNA body also rotated because of the helical nature of the accepter stem (fig. S7). This model structure showed that the accepter stem of intact tRNAPhe was too long for the β-hairpin to recognize its elbow region, whereas tRNAPheΔ1 and tRNAPheΔ1,2 were recognized by the β-hairpin region (fig. S7), which is consistent with previous experiments. On the basis of these model structures, we concluded that the TLP molecule can properly terminate elongation by measuring the accepter stem length of tRNA substrates.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>16831</offset><text>Dual binding mode for tRNA repair</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>16865</offset><text>The present structural analysis revealed that although TLP and Thg1 have a similar tetrameric architecture, they have different binding modes for tRNAs: Thg1 is bound to tRNAHis as a tetramer, whereas TLP is bound to tRNAPhe as a dimer. This difference in the tRNA binding modes is closely related to their enzymatic functions. The tRNAHis-specific G−1 addition enzyme Thg1 needs to recognize both the accepter stem and anticodon of tRNAHis. The tetrameric architecture of the Thg1 molecule allows it to access both regions located at the opposite side of the tRNA molecule [the AB dimer recognizes the accepter stem and CD dimer anticodon ]. In contrast, the binding mode of TLP corresponds to the anticodon-independent repair reactions of 5′-truncated general tRNAs. This binding mode is also suitable for the correct termination of the elongation or repair reaction by measuring the length of the accepter stem by the flexible β-hairpin.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>17811</offset><text>Because tRNAHis requires an extra guanosine (G−1) at the 5′-end, the repair enzyme has to extend the 5′-end by one more nucleotide than other tRNAs. TLP has been shown to confer such catalytic activity on tRNAHisΔ−1 (Fig. 4B). Here, we showed that the TLP mutants, wherein the β-hairpin is truncated and tRNAPheΔ1 binding ability is lost, can still bind to tRNAPhe (GUG) whose anticodon is changed to that for His (fig. S6, C, H, and I). Also, the intact tRNAPhe, which is not recognized by TLP (Fig. 4B and fig. S6E), can be recognized when its anticodon is changed to that for His (fig. S6D). Furthermore, the TLP variant (F174A/N179A/R188A) whose anticodon recognition site [deduced from the Thg1-tRNAHis complex structure ] is disrupted has been shown to have a reduced catalytic activity to tRNAHisΔ−1 (Fig. 4B). All these experimental results indicate that TLP recognizes and binds tRNAs carrying the His anticodon in the same way that Thg1 recognizes tRNAHis. Thus, we concluded that TLP has two tRNA binding modes that are selectively used, depending on both the length of the accepter stem and the anticodon. The elongation or repair reaction normally terminates when the 5′-end reaches position 1, but when the His anticodon is present, TLP binds the tRNA in the second mode by recognizing the anticodon to execute the G−1 addition reaction. By having two different binding modes, TLP can manage this special feature of tRNAHis.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_1</infon><offset>19268</offset><text>DISCUSSION</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>19279</offset><text>The Thg1/TLP family of proteins extends tRNA chains in the 3′-5′ direction. The reaction involves two steps. First, the 5′-phosphate is activated by GTP/ATP. Then, the activated phosphate is attacked by the incoming nucleotide, resulting in an extension by one nucleotide at the 5′-end. Here, we successfully solved for the first time the intermediate structures of the template-dependent 3′-5′ elongation complex of MaTLP. On the basis of these structures, we will discuss the 3′-5′ addition reaction compared with canonical 5′-3′ elongation by DNA/RNA polymerases.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>19866</offset><text>Figure 5 is a schematic diagram of the 3′-5′ addition reaction of TLP. This enzyme has two triphosphate binding sites and one reaction center at the position overlapping these two binding sites (Fig. 5A). In the first activation step, when GTP/ATP is bound to site 1 (Fig. 5B), the 5′-phosphate of the tRNA is deprotonated by Mg2+A and attacks the α-phosphate of the GTP/ATP, resulting in an activated intermediate (Fig. 5C). The structure of the MaTLP-ppptRNAPheΔ1 complex, wherein β- and γ-phosphates coordinate with Mg2+A and Mg2+B, respectively (Figs. 3A and 5C′), may represent this activated intermediate. Subsequent binding of an incoming nucleotide to site 2 followed by formation of the Watson-Crick base pair with a nucleotide in the template strand conveys the 3′-OH of the incoming nucleotide to the position of deprotonation by Mg2+A and the 5′-triphosphate of the tRNA to the reaction center (Figs. 3B and 5D). Then, the elongation reaction of step 2 occurs (Fig. 5E). Thus, the present structure shows that this 3′-5′ elongation enzyme utilizes a reaction center homologous to that of 5′-3′ elongation enzymes for both activation and elongation in a stepwise fashion. Although these two reactions are similar in chemistry, their substrate characteristics are very different. It should be noted that TLP has evolved to allow the occurrence of these two elaborate reaction steps within one reaction center.</text></passage><passage><infon key="file">1501397-F5.jpg</infon><infon key="id">F5</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>21310</offset><text>Schematic representation of the 3′-5′ elongation mechanism.</text></passage><passage><infon key="file">1501397-F5.jpg</infon><infon key="id">F5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>21374</offset><text>(A) The reaction center overlapped with two triphosphate binding sites. A, B, and C (in green) represent binding sites for Mg2+A, Mg2+B, and Mg2+C. P (in blue) represents the phosphate binding sites; O− (in red) is the binding site for the deprotonated OH group. Important TLP residues for tRNA and Mg2+ binding are also shown. (B) Structure of the activation complex (corresponding to fig. S8). GTP/ATP binds to triphosphate binding site 1; the deprotonated OH group of the 5′-phosphate attacks the α-phosphate of GTP/ATP, and PPi (inorganic pyrophosphate) is released. (C) Possible structure after the activation step as suggested from the structure of (C′). (C′) Structure before the elongation reaction (corresponding to Fig. 3A). The 5′-triphosphate of the tRNA binds to the same site as for activation of the 5′-terminus of the tRNA in (B). (D) Structure of initiation of the elongation reaction (corresponding to Fig. 3B). The base of the incoming GTP forms a Watson-Crick hydrogen bond with the nucleotide at position 72 in the template chain and a base-stacking interaction with a neighboring base (G2). Movement of the 5′-terminal chain leaves the 5′-triphosphate of the tRNA in the same site as the activation step in (B). The 3′-OH of the incoming GTP is deprotonated by Mg2+A and attacks the α-phosphate to form a covalent bond. (E) After the elongation reaction, the triphosphate of the new nucleotide is placed on site 1, as in (C′), and is ready for the next reaction.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>22881</offset><text>Figure 6 compares the 3′-5′ and 5′-3′ elongation mechanisms, showing the symmetrical nature of both elongation reactions using a similar reaction center composed of Mg2+A and Mg2+B in the conserved catalytic core. In TLP, which carries out 3′-5′ elongation, the 3′-OH of the incoming nucleotide attacks the 5′-activated phosphate of the tRNA to form a phosphodiester bond, whereas in the T7 RNA polymerase, a representative 5′-3′ DNA/RNA polymerase, the 3′-OH of the 3′-terminal nucleotide of the RNA attacks the activated phosphate of the incoming nucleotide to form a phosphodiester bond. In these reactions, the roles of the two Mg ions are identical. Mg2+A activates the 3′-OH of the incoming nucleotide in TLP and the 3′-OH of the 3′-end of the RNA chain in T7 RNA polymerase. The role of Mg2+B is to position the 5′-triphosphate of the tRNA in TLP and the incoming nucleotide in T7 RNA polymerase. These two Mg2+ ions are coordinated by a conserved Asp (D21 and D69 in TLP) in the conserved catalytic core.</text></passage><passage><infon key="file">1501397-F6.jpg</infon><infon key="id">F6</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>23926</offset><text>Structures of template-dependent nucleotide elongation in the 3′-5′ and 5′-3′ directions.</text></passage><passage><infon key="file">1501397-F6.jpg</infon><infon key="id">F6</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>24024</offset><text>Symmetrical relationship between 3′-5′ elongation by TLP (this study) (left) and 5′-3′ elongation by T7 RNA polymerase [Protein Data Bank (PDB) ID: 1S76] (right). Red arrows represent elongation directions. In the 3′-5′ elongation reaction, the 3′-OH of the incoming nucleotide attacks the 5′-activated phosphate of the tRNA to form a phosphodiester bond, whereas in the 5′-3′ elongation reaction, the 3′-OH of the 3′-terminal nucleotide of the RNA attacks the activated phosphate of the incoming nucleotide to form a phosphodiester bond. Green spheres represent Mg2+ ions.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>24622</offset><text>Because the chemical roles of tRNA and the incoming nucleotide are reversed in these two reactions, these two substrates are inserted into a similar reaction center from opposite directions (Fig. 6). In spite of this difference, their fundamental reaction scheme is conserved. However, from an energetic viewpoint, these two reactions are clearly different: Whereas the high energy of the incoming nucleotide is used for its own addition in DNA/RNA polymerases, the high energy of the incoming nucleotide is used for subsequent addition in TLP. For this reason, TLP requires a mechanism that activates the 5′-terminus of the tRNA during the initial step of the reaction. Our analysis showed that the initial activation and subsequent elongation reactions occur sequentially at one reaction center. In this case, the enzyme needs to create two substrate binding sites for two different reactions in the vicinities of one reaction center. TLP has successfully created such sites by utilizing a conformational change in the tRNA through Watson-Crick base pairing (Fig. 3). These structural features of the TLP molecule suggest that development of an activation reaction site is a prerequisite for developing the 3′-5′ elongation enzyme. This is clearly more difficult than developing the 5′-3′ elongation enzyme, wherein the activation reaction site is not necessary, and which may be the primary reason why the 5′-3′ elongation enzyme has been exclusively developed.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>26101</offset><text>Here, we established a structural basis for 3′-5′ nucleotide elongation and showed that TLP has evolved to acquire a two-step Watson-Crick template–dependent 3′-5′ elongation reaction using the catalytic center homologous to 5′-3′ elongation enzymes. The active site of this enzyme is created at the dimerization interface. The dimerization also endows this protein with the ability to measure the length of the accepter stem of the tRNA substrate, so that the enzyme can properly terminate the elongation reaction. Furthermore, the dual binding mode of this protein suggests that it has further evolved to cover G−1 addition of tRNAHis by additional dimerization (dimer of dimers). Thus, the present structural analysis is consistent with the scenario in which TLP began as a 5′-end repair enzyme and evolved into a tRNAHis-specific G−1 addition enzyme. The detailed molecular mechanism of the Thg1/TLP family established by our analysis will open up new perspectives in our understanding of 3′-5′ versus 5′-3′ polymerization and the molecular evolution of template-dependent polymerases.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>27219</offset><text>MATERIALS AND METHODS</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>27241</offset><text>Plasmid construction</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>27262</offset><text>Genomic DNA from M. acetivorans NBRC100939 was obtained from the NITE Biological Resource Center. The MaTLP gene was amplified by polymerase chain reaction from genomic DNA. The DNA fragment encoding MaTLP was then cloned between the Nde I and Xho I restriction sites in a pET26b vector with a C-terminal His tag. In the MaTLP gene, the amber stop codon (UAG) at position 142 was translated as Pyl. To express the full-length MaTLP in Escherichia coli, the TAG codon was altered to TGG (encoding Trp) with the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) as previously described. The inserted sequence was verified by DNA sequencing.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>27915</offset><text>Preparation of MaTLP and mutants</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>27948</offset><text>Plasmids were transformed into E. coli strain BL21 (DE3) pLysSRARE by electroporation, and cells were grown in LB medium containing kanamycin (25 μg/ml) and chloramphenicol (34 μg/ml) at 37°C until reaching an optical density at 600 nm (OD600) of 0.45. The cells were then induced by the addition of isopropyl-β-d-thiogalactopyranoside to a final concentration of 250 μM and shifted to 18°C for approximately 20 hours before harvest. The cells were harvested and resuspended in buffer A [50 mM Hepes-NaOH (pH 7.5), 1 M NaCl, 4 mM MgCl2, 10% glycerol, 0.5 mM β-mercaptoethanol, lysozyme (0.5 mg/ml), and deoxyibonuclease (0.1 mg/ml)]. After sonication and centrifugation, the His6-tagged protein was purified by immobilized metal-ion affinity chromatography using a HisTrap HP column (GE Healthcare). The sample was washed with 75 mM imidazole and eluted with a 75 to 400 mM imidazole gradient in buffer B [50 mM tris-HCl (pH 7.5), 500 mM NaCl, 4 mM MgCl2, 20% glycerol, and 0.5 mM β-mercaptoethanol]. Then, the collected fractions were diluted in 300 mM NaCl with buffer C [25 mM tris-HCl (pH 7.5), 10% glycerol, 5 mM MgCl2, and 1 mM dithiothreitol (DTT)] and further purified on a HiTrap Heparin HP column (GE Healthcare) by elution with a 300 to 1000 mM NaCl gradient in buffer C. Finally, the protein was loaded onto a HiLoad 16/60 Superdex 200 prep grade column (GE Healthcare) equilibrated with buffer D [20 mM Hepes-NaOH (pH 7.5), 500 mM NaCl, 5 mM MgCl2, 10% glycerol, and 1 mM DTT]. The protein was concentrated to 3.9 mg/ml by ultrafiltration. All MaTLP mutants were constructed with the QuikChange Site-Directed Mutagenesis Kit. MaTLP mutants were purified by a HisTrap HP column for RNA binding assay and further purified by a HiLoad 16/60 Superdex 200 prep grade column for 3′-5′ nucleotide addition assay.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>29779</offset><text>Preparation of tRNA and its mutants</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>29815</offset><text>tRNA transcripts derived from yeast tRNAPhe and tRNAHis were prepared using T7 RNA polymerase as previously described. ppptRNA transcripts were prepared by excluding guanosine 5′-monophosphate (GMP) from the reaction mixture. Transcribed tRNAs were purified by a HiTrap DEAE FF column (GE Healthcare) as previously described. Pooled tRNAs were precipitated with isopropanol and dissolved in buffer E [20 mM Hepes-NaOH (pH 7.5), 100 mM NaCl, and 10 mM MgCl2].</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>30276</offset><text>Preparation of the MaTLP-ppptRNAPheΔ1 complex</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>30326</offset><text>MaTLP and ppptRNAPheΔ1 (tRNAPhe with a triphosphorylated 5′-end and deleted G1) were mixed in a molar ratio of 1.7:1 and incubated for 30 min at room temperature. The mixture was then loaded onto a HiLoad 16/60 Superdex 200 prep grade column equilibrated with buffer F [20 mM Hepes-NaOH (pH 7.5), 400 mM NaCl, 5 mM MgCl2, 10% glycerol, and 1 mM DTT]. Fractions containing the MaTLP-ppptRNAPheΔ1 complex were mixed with 1 mM spermine and concentrated to an OD280 of 16 by ultrafiltration.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>30822</offset><text>Crystallization and data collection</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>30858</offset><text>All crystallization experiments were performed with the sitting-drop vapor diffusion method at 293 K. Initial crystals of MaTLP were obtained by mixing 1 μl of protein solution (3.9 mg/ml) with 1 μl of a reservoir solution containing 0.1 M Hepes-NaOH buffer (pH 7.5), 0.2 M magnesium chloride, and 30% polyethylene glycol 400 (PEG 400). MaTLP-GTP complex crystals were obtained by soaking the MaTLP crystals in the above reservoir solution supplemented with 1 mM GTP overnight. High-resolution crystals of MaTLP in apo form (MaTLP-apo) were obtained unexpectedly by mixing MaTLP with tRNAHis in 0.1 M sodium/potassium phosphate (pH 6.2) containing 2.5 M NaCl. Crystals of the MaTLP-ppptRNAPheΔ1 complex were obtained from a solution containing 0.2 M tripotassium citrate, 0.1 M tris (pH 8.0), 37% PEG3350, and 10 mM praseodymium (III) acetate. Crystals of the MaTLP-ppptRNAPheΔ1-GDPNP complex were obtained by soaking MaTLP-ppptRNAPheΔ1 complex crystals in a reservoir solution containing 0.2 M tripotassium citrate, 0.1 M tris (pH 8.0), 30% PEG3350, 5% glycerol, and 15 mM GDPNP overnight. Crystals of MaTLP-apo and MaTLP-GTP were cryoprotected with a reservoir solution containing 50% PEG400 before flash-cooling, whereas crystals of the MaTLP-ppptRNAPheΔ1-GDPNP and MaTLP-ppptRNAPheΔ1 complexes were flash-cooled without any cryoprotectant under a stream of liquid nitrogen at 100 K. X-ray diffraction data were collected from beamline BL41XU at SPring-8 (Hyogo, Japan) and beamlines BL5A and BL17A at Photon Factory (Ibaraki, Japan). All diffraction data were indexed, integrated, scaled, and merged using XDS.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>32495</offset><text>Structure determination and refinement</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>32534</offset><text>The crystal structure of MaTLP-apo was determined by the molecular replacement (MR) method with Molrep, using the protomer structure of CaThg1 (PDB ID: 3WBZ) as a search model. The protomer structure of MaTLP-apo was then used as a search model to solve the structures of MaTLP-GTP. The crystal structure of the MaTLP-ppptRNAPheΔ1 complex was determined by the MR method with PHASER, using the protomer structures of MaTLP-apo and tRNAPhe from Saccharomyces cerevisiae (PDB ID: 1EHZ) as search models. The structure of the MaTLP-ppptRNAPheΔ1 complex was then used as a search model to solve the MaTLP-ppptRNAPheΔ1-GDPNP complex structure. Initial protein models were fitted manually using Coot, and tRNA models were automatically rebuilt by LAFIRE_NAFIT; these models were then refined using phenix.refine. The data collection and refinement statistics are summarized in Table 1. All structure figures were generated by PyMol.</text></passage><passage><infon key="file">T1.xml</infon><infon key="id">T1</infon><infon key="section_type">TABLE</infon><infon key="type">table_title_caption</infon><offset>33473</offset><text>Summary of data collection and refinement statistics.</text></passage><passage><infon key="file">T1.xml</infon><infon key="id">T1</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>33527</offset><text>Values in parentheses are for the highest-resolution shell. PF, Photon Factory; Rmsd, root-mean-square deviation.</text></passage><passage><infon key="file">T1.xml</infon><infon key="id">T1</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;col width=&quot;%&quot; span=&quot;1&quot;/&gt;&lt;col width=&quot;%&quot; span=&quot;1&quot;/&gt;&lt;col width=&quot;%&quot; span=&quot;1&quot;/&gt;&lt;col width=&quot;%&quot; span=&quot;1&quot;/&gt;&lt;col width=&quot;%&quot; span=&quot;1&quot;/&gt;&lt;thead&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; scope=&quot;col&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; scope=&quot;col&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;bold&gt;&lt;italic&gt;Ma&lt;/italic&gt;TLP-apo&lt;/bold&gt;&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; scope=&quot;col&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;bold&gt;&lt;italic&gt;Ma&lt;/italic&gt;TLP-GTP&lt;/bold&gt;&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; scope=&quot;col&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;bold&gt;&lt;italic&gt;Ma&lt;/italic&gt;TLP-ppptRNA&lt;sup&gt;Phe&lt;/sup&gt;Δ&lt;sub&gt;1&lt;/sub&gt;&lt;/bold&gt;&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; scope=&quot;col&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;bold&gt;&lt;italic&gt;Ma&lt;/italic&gt;TLP-ppptRNA&lt;sup&gt;Phe&lt;/sup&gt;Δ&lt;sub&gt;1&lt;/sub&gt;-GDPNP&lt;/bold&gt;&lt;/td&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; scope=&quot;row&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;PDB ID&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5AXK&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5AXL&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5AXM&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5AXN&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;5&quot; valign=&quot;top&quot; align=&quot;left&quot; scope=&quot;row&quot; rowspan=&quot;1&quot;&gt;&lt;bold&gt;Data collection&lt;/bold&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; scope=&quot;row&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;  Beamline&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;SPring-8 BL41XU&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;SPring-8 BL41XU&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;PF BL17A&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;PF BL5A&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; scope=&quot;row&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;  Space group&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;C&lt;/italic&gt;222&lt;sub&gt;1&lt;/sub&gt;&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;C&lt;/italic&gt;222&lt;sub&gt;1&lt;/sub&gt;&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;I&lt;/italic&gt;222&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;I&lt;/italic&gt;222&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; scope=&quot;row&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;  Unit cell parameters &lt;italic&gt;a&lt;/italic&gt;, &lt;italic&gt;b&lt;/italic&gt;, &lt;italic&gt;c&lt;/italic&gt; (Å)&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;98.3, 120.5, 157.4&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;103.1, 115.7, 144.9&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;75.3, 127.6, 143.8&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;82.3, 134.1, 147.4&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; scope=&quot;row&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;  Wavelength (Å)&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.9780&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.0000&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.97319&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.0000&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; scope=&quot;row&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;  Resolution range (Å)&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;50.0–2.29 (2.43–2.29)&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;50.0–2.99 (3.17–2.99)&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;50.0–2.21 (2.34–2.21)&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;50.0–2.70 (2.87–2.70)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; scope=&quot;row&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;  &lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;meas&lt;/sub&gt; (%)*&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;8.9 (76.3)&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;15.2 (90.0)&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;9.7 (74.4)&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;11.0 (87.2)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; scope=&quot;row&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;  CC&lt;sub&gt;1/2&lt;/sub&gt; (%)&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;99.8 (80.4)&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;99.5 (81.2)&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;99.9 (83.6)&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;99.9 (83.5)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; scope=&quot;row&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;  〈&lt;italic&gt;I&lt;/italic&gt;/σ(&lt;italic&gt;I&lt;/italic&gt;)〉&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;14.7 (2.8)&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;12.0 (2.6)&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;19.4 (3.2)&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;16.9 (2.5)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; scope=&quot;row&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;  Completeness (%)&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;98.3 (93.8)&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;98.8 (93.4)&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;99.7 (98.6)&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;99.7 (99.3)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; scope=&quot;row&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;  Redundancy&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;6.7 (6.6)&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;7.2 (7.2)&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;7.4 (7.3)&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;8.1 (8.2)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;5&quot; valign=&quot;top&quot; align=&quot;left&quot; scope=&quot;row&quot; rowspan=&quot;1&quot;&gt;&lt;bold&gt;Refinement&lt;/bold&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; scope=&quot;row&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;  No. of reflections&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;41,650&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;17,581&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;35,102&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;22,669&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; scope=&quot;row&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;  &lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;work&lt;/sub&gt;/&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;free&lt;/sub&gt; (%)&lt;sup&gt;†&lt;/sup&gt;&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;20.6/24.0&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;21.5/25.3&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;21.6/24.3&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;22.5/26.7&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;5&quot; valign=&quot;top&quot; align=&quot;left&quot; scope=&quot;row&quot; rowspan=&quot;1&quot;&gt;  No. of atoms&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; scope=&quot;row&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    Macromolecules&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3760&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3622&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5247&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5142&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; scope=&quot;row&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    Ligand/ion&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;30&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;68&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;36&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;101&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; scope=&quot;row&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    Water&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;89&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;8&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;102&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;16&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;5&quot; valign=&quot;top&quot; align=&quot;left&quot; scope=&quot;row&quot; rowspan=&quot;1&quot;&gt;  &lt;italic&gt;B&lt;/italic&gt;-factors (Å&lt;sup&gt;2&lt;/sup&gt;)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; scope=&quot;row&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    Macromolecules&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; scope=&quot;col&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;57.0&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;68.4&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;45.3&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;57.3&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; scope=&quot;row&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    Ligand/ion&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;60.5&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;86.2&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;46.6&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;59.9&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; scope=&quot;row&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    Water&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;49.0&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;59.5&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;33.0&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;38.1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; scope=&quot;row&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;  Estimated coordinate error (Å)&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.32&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.48&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.25&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.41&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;5&quot; valign=&quot;top&quot; align=&quot;left&quot; scope=&quot;row&quot; rowspan=&quot;1&quot;&gt;  Rmsd from ideal&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; scope=&quot;row&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    Bond lengths (Å)&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.009&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.003&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.003&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.003&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; scope=&quot;row&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    Bond angles (°)&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.11&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.92&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.72&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.80&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>33641</offset><text>	MaTLP-apo	MaTLP-GTP	MaTLP-ppptRNAPheΔ1	MaTLP-ppptRNAPheΔ1-GDPNP	 	PDB ID	5AXK	5AXL	5AXM	5AXN	 	Data collection	 	  Beamline	SPring-8 BL41XU	SPring-8 BL41XU	PF BL17A	PF BL5A	 	  Space group	C2221	C2221	I222	I222	 	  Unit cell parameters a, b, c (Å)	98.3, 120.5, 157.4	103.1, 115.7, 144.9	75.3, 127.6, 143.8	82.3, 134.1, 147.4	 	  Wavelength (Å)	0.9780	1.0000	0.97319	1.0000	 	  Resolution range (Å)	50.0–2.29 (2.43–2.29)	50.0–2.99 (3.17–2.99)	50.0–2.21 (2.34–2.21)	50.0–2.70 (2.87–2.70)	 	  Rmeas (%)*	8.9 (76.3)	15.2 (90.0)	9.7 (74.4)	11.0 (87.2)	 	  CC1/2 (%)	99.8 (80.4)	99.5 (81.2)	99.9 (83.6)	99.9 (83.5)	 	  〈I/σ(I)〉	14.7 (2.8)	12.0 (2.6)	19.4 (3.2)	16.9 (2.5)	 	  Completeness (%)	98.3 (93.8)	98.8 (93.4)	99.7 (98.6)	99.7 (99.3)	 	  Redundancy	6.7 (6.6)	7.2 (7.2)	7.4 (7.3)	8.1 (8.2)	 	Refinement	 	  No. of reflections	41,650	17,581	35,102	22,669	 	  Rwork/Rfree (%)†	20.6/24.0	21.5/25.3	21.6/24.3	22.5/26.7	 	  No. of atoms	 	    Macromolecules	3760	3622	5247	5142	 	    Ligand/ion	30	68	36	101	 	    Water	89	8	102	16	 	  B-factors (Å2)	 	    Macromolecules	57.0	68.4	45.3	57.3	 	    Ligand/ion	60.5	86.2	46.6	59.9	 	    Water	49.0	59.5	33.0	38.1	 	  Estimated coordinate error (Å)	0.32	0.48	0.25	0.41	 	  Rmsd from ideal	 	    Bond lengths (Å)	0.009	0.003	0.003	0.003	 	    Bond angles (°)	1.11	0.92	0.72	0.80	 	</text></passage><passage><infon key="file">T1.xml</infon><infon key="id">T1</infon><infon key="section_type">TABLE</infon><infon key="type">table_foot</infon><offset>35061</offset><text>*Rmeas = Σhkl {N(hkl)/[N(hkl) − 1]}1/2 Σi | Ii(hkl) − 〈I(hkl)〉 |/Σhkl Σi
+Ii(hkl), where 〈I(hkl)〉 and N(hkl) are the mean intensity of a set of equivalent reflections and the multiplicity, respectively.</text></passage><passage><infon key="file">T1.xml</infon><infon key="id">T1</infon><infon key="section_type">TABLE</infon><infon key="type">table_foot</infon><offset>35279</offset><text>†Rwork
+= Σhkl ||Fobs| − |Fcalc||/Σhkl |Fobs|; Rfree was calculated for 5% randomly selected test sets that were not used in the refinement.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>35427</offset><text>Nucleotide addition assay</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>35453</offset><text>Nucleotide addition assays were performed as previously described. A reaction mixture containing 25 mM Hepes-NaOH (pH 7.5), 400 mM NaCl, 10 mM MgCl2, 3 mM DTT, 5% glycerol, 0.1 μM [α-32P]GTP, 100 μM GTP, 1 μM MaTLP variants, and 10 μM tRNA transcript was incubated at 30°C for 2 hours. Then, the reaction was quenched with phenol/chloroform, and the supernatant was resolved on a 10% polyacrylamide gel containing 8 M urea. The radioactivity was visualized with a BAS-1800 II bioimaging analyzer (Fujifilm).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>35969</offset><text>tRNA binding assay</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>35988</offset><text>A reaction mixture containing 34 μM MaTLP variants and 20 μM tRNA transcript was incubated in buffer F at room temperature for 30 min. Then, the mixture was loaded onto a Superdex 200 10/300 GL column (GE Healthcare) equilibrated with the same buffer.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">title_1</infon><offset>36242</offset><text>Supplementary Material</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">title</infon><offset>36265</offset><text>SUPPLEMENTARY MATERIALS</text></passage><passage><infon key="section_type">REF</infon><infon key="type">title</infon><offset>36289</offset><text>REFERENCES AND NOTES</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>36310</offset><text>B. Alberts, A. Johnson, J. Lewis, M. Raff, K. Roberts, P. Walter, Molecular Biology of the Cell (Garland Science, New York, ed. 5, 2008).</text></passage><passage><infon key="fpage">333</infon><infon key="lpage">344</infon><infon key="name_0">surname:Heinemann;given-names:I. 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+<collection><source>PMC</source><date>20201216</date><key>pmc.key</key><document><id>4822561</id><infon key="license">CC BY</infon><passage><infon key="alt-title">Quinolone-stabilized cleavage complex of topoisomerase IV</infon><infon key="article-id_coden">ACSDAD</infon><infon key="article-id_doi">10.1107/S2059798316001212</infon><infon key="article-id_pii">S2059798316001212</infon><infon key="article-id_pmc">4822561</infon><infon key="article-id_pmid">27050128</infon><infon key="article-id_publisher-id">mn5108</infon><infon key="fpage">488</infon><infon key="issue">Pt 4</infon><infon key="kwd">Klebsiella pneumoniae cleavage complex quinolone levofloxacin topoisomerase IV DNA binding isomerase isomerase–DNA complex topoisomerases Gram-negative complexes X-ray crystallography protein–DNA–drug complexes</infon><infon key="license">This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits
+unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.</infon><infon key="lpage">496</infon><infon key="name_0">surname:Veselkov;given-names:Dennis A.</infon><infon key="name_1">surname:Laponogov;given-names:Ivan</infon><infon key="name_2">surname:Pan;given-names:Xiao-Su</infon><infon key="name_3">surname:Selvarajah;given-names:Jogitha</infon><infon key="name_4">surname:Skamrova;given-names:Galyna B.</infon><infon key="name_5">surname:Branstrom;given-names:Arthur</infon><infon key="name_6">surname:Narasimhan;given-names:Jana</infon><infon key="name_7">surname:Prasad;given-names:Josyula V. N. Vara</infon><infon key="name_8">surname:Fisher;given-names:L. Mark</infon><infon key="name_9">surname:Sanderson;given-names:Mark R.</infon><infon key="section_type">TITLE</infon><infon key="type">front</infon><infon key="volume">72</infon><infon key="year">2016</infon><offset>0</offset><text>Structure of a quinolone-stabilized cleavage complex of topoisomerase IV from Klebsiella pneumoniae and comparison with a related Streptococcus pneumoniae complex</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>163</offset><text>Crystal structures of the cleavage complexes of topoisomerase IV from Gram-negative (K. pneumoniae) and Gram-positive (S. pneumoniae) bacterial pathogens stabilized by the clinically important antibacterial drug levofloxacin are presented, analysed and compared. For K. pneumoniae, this is the first high-resolution cleavage complex structure to be reported.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>522</offset><text> Klebsiella pneumoniae is a Gram-negative bacterium that is responsible for a range of common infections, including pulmonary pneumonia, bloodstream infections and meningitis. Certain strains of Klebsiella have become highly resistant to antibiotics. Despite the vast amount of research carried out on this class of bacteria, the molecular structure of its topoisomerase IV, a type II topoisomerase essential for catalysing chromosomal segregation, had remained unknown. In this paper, the structure of its DNA-cleavage complex is reported at 3.35 Å resolution. The complex is comprised of ParC breakage-reunion and ParE TOPRIM domains of K. pneumoniae topoisomerase IV with DNA stabilized by levofloxacin, a broad-spectrum fluoroquinolone antimicrobial agent. This complex is compared with a similar complex from Streptococcus pneumoniae, which has recently been solved.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">title_1</infon><offset>1397</offset><text>Introduction   </text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1414</offset><text> Klebsiella is a genus belonging to the Enterobacteriaceae family of Gram-negative bacilli, which is divided into seven species with demonstrated similarities in DNA homology: K. pneumoniae, K. ozaenae, K. rhinoscleromatis, K. oxytoca, K. planticola, K. terrigena and K. ornithinolytica. K. pneumoniae is the most medically important species of the genus owing to its high resistance to antibiotics. Significant morbidity and mortality has been associated with an emerging, highly drug-resistant strain of K. pneumoniae characterized as producing the carbapenemase enzyme (KPC-producing bacteria; Nordmann et al., 2009). The best therapeutic approach to KPC-producing organisms has yet to be defined. However, common treatments (based on in vitro susceptibility testing) are the polymyxins, tigecycline and, less frequently, aminoglycoside antibiotics (Arnold et al., 2011). Another effective strategy involves the limited use of certain antimicrobials, specifically fluoroquinolones and cephalo­sporins (Gasink et al., 2009). Several new antibiotics are under development for KPC producers. These include combinations of existing β-lactam antibiotics with new β-lactamase inhibitors able to circumvent KPC resistance. Neoglycosides are novel aminoglycosides that have activity against KPC-producing bacteria that are also being developed (Chen et al., 2012).</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2779</offset><text>Type II topoisomerase enzymes play important roles in prokaryotic and eukaryotic DNA replication, recombination and transcription (Drlica et al., 2008; Laponogov et al., 2013; Lee et al., 2013; Nitiss, 2009a ,b ; Schoeffler &amp; Berger, 2008; Sissi &amp; Palumbo, 2009; Vos et al., 2011; Wendorff et al., 2012; Wu et al., 2011, 2013). In bacteria, topoisomerase IV, a tetramer of two ParC and two ParE subunits, unlinks daughter chromosomes prior to cell division, whereas the related enzyme gyrase, a GyrA2GyrB2 tetramer, supercoils DNA and helps unwind DNA at replication forks. Both enzymes act via a double-strand DNA break involving a cleavage complex and are targets for quinolone antimicrobials that act by trapping these enzymes at the DNA-cleavage stage and preventing strand re-joining (Drlica et al., 2008).</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3591</offset><text>Levofloxacin is a broad-spectrum third-generation fluoro­quinolone antibiotic. It is active against Gram-positive and Gram-negative bacteria and functions by inhibiting gyrase and topoisomerase IV (Drlica &amp; Zhao, 1997; Laponogov et al., 2010). Acquiring a deep structural and functional understanding of the mode of action of fluoroquinolones (Tomašić &amp; Mašič, 2014) and the development of new drugs targeted against topoisomerase IV and gyrase from a wide range of Gram-positive and Gram-negative pathogenic bacteria are highly active areas of current research directed at overcoming the vexed problem of drug resistance (Bax et al., 2010; Chan et al., 2015; Drlica et al., 2014; Mutsaev et al., 2014; Pommier, 2013; Srikannathasan et al., 2015).</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>4344</offset><text>Here, we report the first three-dimensional X-ray structure of a K. pneumoniae topoisomerase IV ParC/ParE cleavage complex with DNA stabilized by levofloxacin. The crystal structure provides structural information on topoisomerase IV from K. pneumoniae, a pathogen for which drug resistance is a serious concern. The structure of the ParC/ParE–DNA–levofloxacin binding site highlights the details of the cleavage-complex assembly that are essential for the rational design of Klebsiella topoisomerase inhibitors.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>4861</offset><text>Materials and methods   </text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>4887</offset><text>Cloning, expression and purification of K. pneumoniae and Streptococcus pneumoniae ParC55/ParE30   </text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>4988</offset><text>Cloning, expression and purification protocols are described in detail in the Supporting Information. Table 1 ▸ contains the sequence information for all of the components of the complexes. Fig. 1 ▸ provides information about the protein and DNA constructs used in the experimental work.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>5280</offset><text>Preparation of the DNA oligomer   </text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>5316</offset><text>For the K. pneumoniae cleavage complex, two DNA oligomers (5′-CGTATTACGTTGTAT-3′ and 5′-GATCATACAACGTAATACG-3′) were synthesized by solid-phase phosphoramidite chemistry and doubly HPLC purified by Metabion, Munich, Germany. The DNA sequence was designed to make a complementary DNA 34-mer that contained the ‘pre-cut’ binding-site fragment: 5′-CGTATTACGTTGTAT↓GATCATACAACGTAATACG-3′ and 3′-GCATAATGCA­ACATACTAG↓TATGTTGCATTATGC-5′ (the cuts are shown by arrows; see Fig. 1 ▸ b).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>5822</offset><text>For the S. pneumoniae cleavage complex, two DNA oligomers (5′-CATGAATGACTATGCACG-3′ and 5′-CGTGCATAGTCATTCATG-3′) were synthesized by solid-phase phosphoramidite chemistry and doubly HPLC purified by Metabion, Munich, Germany. The DNA sequence corresponds to the E-site 18-mer, which was found to be a better DNA length for crystallization of the S. pneumoniae topo­isomerase IV cleavage complexes in order to give stable reproducible crystals (see Fig. 1 ▸ b).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>6295</offset><text>DNA stock solutions were made by mixing the required oligomers (at 1 mM in 20 mM Tris pH 7.5, 200 mM NaCl, 1 mM β-mercaptothanol, 0.05% NaN3) in equal volumes. For DNA annealing, the mixtures of complementary oligomers were heated to 98°C and then slowly cooled to 4°C over a 48 h period.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>6597</offset><text>Crystallization and data collection   </text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>6637</offset><text>Crystallization information for both the S. pneumoniae and the K. pneumoniae topoisomerase IV cleavage complexes is summarized in Table 2 ▸. Data-collection statistics and details are provided in Table 3 ▸. Structure-solution and refinement details are provided in Table 4 ▸.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_3</infon><offset>6919</offset><text> S. pneumoniae topoisomerase IV   </text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>6955</offset><text>Protein was mixed with DNA in a 1:1:1.2 molar ratio (ParC55:ParE30:18-mer E-site DNA) with an overall concentration of 4 mg ml−1. Levofloxacin and magnesium chloride were added to final concentrations of 2 and 10 mM, respectively. The mixture was pre-incubated at room temperature overnight. Initial crystallization screening was performed by sitting-drop vapour diffusion in a 96-well MRC crystallization plate (600 nl protein mixture + 400 nl reservoir solution) using a Mosquito robot (TTP Labtech; http://www.ttplabtech.com). The best crystals were obtained using capillary counter-diffusion against 50 mM sodium cacodylate pH 6.5, 2.5% Tacsimate (Hampton Research; McPherson &amp; Cudney, 2006), 7% 2-propanol, 62.5 mM KCl, 7.5 mM MgCl2 at 304 K. The crystals were flash-cooled at 100 K in cryoprotectant buffer C [50 mM sodium cacodyl­ate pH 6.5, 2.5% Tacsimate, 62.5 mM KCl, 7.5 mM MgCl2, 1 mM β-mercapto­ethanol, 30%(v/v) MPD]. The best data set was collected on beamline I03 at Diamond Light Source at a wavelength of 0.9763 Å using an ADSC Quantum 315 detector. The data extended to 2.6 Å resolution anisotropically and were used in refinement with a maximum-likelihood target in the initial refinement cycles; they were deposited in the PDB without introducing a resolution cutoff. However, owing to the high R merge values in the outer shells, the final resolution is given as 2.9 Å and the statistics are reported according to this ‘trimmed’ resolution. The resolution cutoff was based on the rejection criteria R merge &lt; 50% and I/σ(I) &gt; 1.5 in the highest resolution shell. The data were integrated using HKL-2000 (Otwinowski &amp; Minor, 1997). The space group was determined to be P3121, with unit-cell parameters a = b = 157.83, c = 211.15 Å.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>8761</offset><text>The structure was solved by molecular replacement using Phaser (McCoy et al., 2007) as implemented within the CCP4 suite (Winn et al., 2011) and our previously published topo­isomerase IV–levofloxacin structure (PDB entry 3k9f; Laponogov et al., 2010). Refinement was performed in PHENIX (Adams et al., 2002, 2010) with manual inspection and corrections performed in WinCoot (Emsley &amp; Cowtan, 2004; Emsley et al., 2010).The structure was verified using WinCoot and PROCHECK (Laskowski et al., 1993).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_3</infon><offset>9264</offset><text> K. pneumoniae topoisomerase IV   </text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>9300</offset><text>ParC55/ParE30 protein stock in incubation buffer (at 4.5 mg ml−1) was mixed with the ‘pre-cut’ 34-mer DNA stock in a 1:1.2 protein:DNA molar ratio. High-concentration stocks of levofloxacin and MgCl2 were added to give final concentrations of 2 and 10 mM, respectively. The mixture was incubated overnight at room temperature. Initial crystallization screening was performed by sitting-drop vapour diffusion in 96-well MRC crystallization plates (600 nl protein mixture + 300 nl reservoir solution) using a Mosquito robot. When the optimal crystallization conditions had been established, conventional hanging-drop vapour diffusion in 24-well Linbro plates (4 µl protein mixture + 2 µl reservoir solution) was used to increase the crystal size.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>10067</offset><text>Crystals formed after ∼7–10 d at room temperature. The crystallization conditions varied slightly from batch to batch in the range 0.1 M Tris pH 7.5–8.0, 0–50 mM NaCl, 4–8% PEG 4000, 12–15% glycerol.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>10285</offset><text>It should be mentioned that several other DNA oligomers with the same binding-site sequence were tried for crystallization (i.e. 20-mer, ‘pre-cut’ 20-mer and 34-mer DNA sequences). However, these protein–DNA–drug complexes did not produce good-quality crystals for data collection.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>10575</offset><text>Crystals were tested in-house for diffraction quality using an Oxford Xcalibur Nova CCD diffractometer and were then transported for high-resolution data collection at Diamond Light Source (Harwell Science and Innovation Campus, Oxfordshire, England). The data were collected on beamline I03 (wavelength 0.9762 Å) using a Pilatus 6M-F detector (0.2° oscillation per image, 100 K nitrogen stream). The best crystals diffracted to ∼3.2 Å resolution.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>11034</offset><text>All data sets were integrated with MOSFLM (Leslie &amp; Powell, 2007) and merged with SCALA (Evans, 2006) as implemented in CCP4 (Winn et al., 2011). The ParC55/ParE30–DNA–levofloxacin crystals belonged to space group P21, with unit-cell parameters a = 102.07, b = 161.53, c = 138.60 Å, α = 90.00, β = 94.22, γ = 90.00°. They contained two ParC/ParE–DNA heterodimers in the asymmetric unit.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>11438</offset><text>Several data sets were collected, some of which contained visible diffraction to 3.2 Å resolution, but owing to potential internal twinning and space-group ambiguity (most data sets could be integrated in space groups P21 and P212121) and the fact that the structure solution could be obtained in both space groups, careful selection of the integration ranges as well as appropriate data truncation were necessary. The best region of data was integrated to 3.35 Å (see Table 3 ▸ for statistics). The resolution cutoff was based on the rejection criteria R merge &lt; 50% and I/σ(I) &gt; 1.5 in the highest resolution shell.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>12065</offset><text>The structure was solved by the molecular-replacement method in Phaser (McCoy et al., 2007) using the levofloxacin–DNA cleavage complex of topoisomerase IV from S. pneumoniae as a search model (PDB entry 3rae; ∼41.8% sequence identity). Refinement was performed in PHENIX (Adams et al., 2002, 2010) using secondary-structure restraints derived by superposition of the K. pneumoniae ParC/ParE model with the previously solved complex of S. pneumoniae ParC/ParE. Rigid-body, positional and TLS refinements were performed. Levofloxacin molecules and magnesium ions were placed during the final stages of refinement based on missing electron density in the σA-weighted 2F obs − F calc and F obs − F calc maps. WinCoot (Emsley &amp; Cowtan, 2004) was used for interactive model fitting. The structure was verified using WinCoot and PROCHECK (Laskowski et al., 1993). The resulting model had good geometry, with 87.8, 9.9 and 1.3% of residues in the favoured, allowed and generously allowed regions of the Ramachandran plot, respectively, and no more than 1% of residues in disallowed regions. The data-collection and final refinement statistics are given in Tables 3 ▸ and 4 ▸. Sequence alignment was performed in ClustalW (Larkin et al., 2007, McWilliam et al., 2013). Figures were prepared using PyMOL (DeLano, 2008), CHEMDRAW (Evans, 2014) and CorelDRAW (http://www.coreldraw.com).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>13454</offset><text>Results and discussion   </text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>13481</offset><text>We have co-crystallized the K. pneumoniae topoisomerase IV ParC/ParE breakage-reunion domain (ParC55; residues 1–490) and ParE TOPRIM domain (ParE30; residues 390–631) with a precut 34 bp DNA duplex (the E-site), stabilized by levofloxacin. The X-ray crystal structure of the complex was determined to 3.35 Å resolution, revealing a closed ParC55 dimer flanked by two ParE30 monomers (Figs. 1 ▸, 2 ▸ and 3 ▸). The overall architecture of this complex is similar to that found for S. pneumoniae topoisomerase–DNA–drug complexes (Laponogov et al., 2009, 2010). Residues 6–30 of the N-terminal α-helix α1 of the ParC subunit again embrace the ParE subunit, ‘hugging’ the ParE subunits close to either side of the ParC dimer (Laponogov et al., 2010). Deletion of this ‘arm’ α1 results in loss of DNA-cleavage activity (Laponogov et al., 2007) and is clearly very important in complex stability (Fig. 3 ▸). This structural feature was absent in our original ParC55 structure (Laponogov et al., 2007; Sohi et al., 2008). The upper region of the topoisomerase complex consists of the E-subunit TOPRIM metal-binding domain formed of four parallel β-sheets and the surrounding α-helices. The C-subunit provides the WHD (winged-helix domain; a CAP-like structure; McKay &amp; Steitz, 1981) and the ‘tower’ which form the U groove-shaped protein region into which the G-gate DNA binds with an induced U-shaped bend. The lower C-gate region (Fig. 3 ▸) consists of the same disposition of pairs of two long α-helices terminated by a spanning short α-helix forming a 30 Å wide DNA-accommodating cavity through which the T-gate DNA passes as found in the S. pneumoniae complex. Owing to the structural similarity, it appears that the topo­isomerases IV from K. pneumoniae and S. pneumoniae are likely to follow a similar overall topoisomerase catalytic cycle as shown in Fig. 4 ▸; we have confirmation of one intermediate from our recent structure of the full complex (the holoenzyme less the CTD β-pinwheel domain) with the ATPase domain in the open conformation (Laponogov et al., 2013).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15603</offset><text>The G-gate DNA for the S. pneumoniae complex consists of an 18-base-pair E-site sequence (our designation for a DNA site which we first found from DNA-mapping studies; Leo et al., 2005; Arnoldi et al., 2013; Fig. 1 ▸). The crystallized complex was formed by turning over the topoisomerase tetramer in the presence of DNA and levofloxacin and crystallizing the product. In contrast, the K. pneumoniae complex was formed by co-crystallizing the topoisomerase tetramer complex in the presence of a 34-base-pair pre-cleaved DNA in the presence of levofloxacin. In both cases the DNA is bent into a U-form and bound snugly against the protein of the G-gate. We have been able to unambiguously read off the DNA sequences in the electron-density maps.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>16350</offset><text>There is 41.6% sequence identity and 54.4% sequence homology between the ParE subunit of K. pneumoniae and that of S. pneumoniae. For the ParC subunits, the figures are 40.8 identity and 55.6% homology between the two organisms. The sequence alignment is given in Supplementary Fig. S1, with the key metal-binding residues and those which give rise to quinolone resistance highlighted. The binding of levofloxacin in the K. pneumoniae complex is shown in Figs. 2 ▸, 3 ▸ and 5 ▸ and is hemi-intercalated into the DNA and stacked against the DNA bases at the cleavage site (positions −1 and +1 of the four-base-pair staggered cut in the 34-mer DNA) which is similar to that found for the S. pneumoniae complex. Fig. 5 ▸ presents side-by-side views of the K. pneumoniae and S. pneumoniae active sites which shows that levofloxacin binds in a very similar manner in these two complexes with extensive π–π stacking interaction between the bases and the drug. The methylpiperazine at C7 (using the conventional quinolone numbering; C9 in the IUPAC numbering) on the drug extends towards residues Glu474 and Glu475 for S. pneumoniae and towards Gln460 and Glu461 for K. pneumoniae, where the glutamate at 474 is substituted by a glutamine at 460 in the Klebsiella strain. Interestingly, for S. pneumoniae we observe only one possible orientation of the C7 groups in both sub­units, while for K. pneumoniae we can see two: one with the same orientation as in S. pneumoniae and other rotated 180° away. They both exist within the same crystal in the two different dimers in the asymmetric unit. The side chains surrounding them in ParE are quite disordered and are more defined in K. pneumoniae (even though this complex is at lower resolution) than in S. pneumoniae. There are no direct hydrogen bonds from the drug to these residues (although it is possible that some are formed through water, which cannot be observed at this resolution). Obviously, the drug–ParE interaction in this region is less strong compared with PD 0305970 binding to the S. pneumoniae DNA complex, where PD 0305970 forms a hydrogen bond to ParE residue Asp475 and can form one to Asp474 if the bond rotates (Laponogov et al., 2010). This may explain why drug-resistance mutations for levofloxacin are more likely to form in the ParC subunits rather than in the ParE subunits.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>18713</offset><text>For both complexes there is a Mg2+ ion bound to levofloxacin between the carbonyl group at position 4 of the quinolone and the carboxyl at position 6 (Figs. 2 ▸ and 5 ▸ and Supplementary Fig. 2 ▸). For S. pneumoniae topoisomerase IV, one of the O atoms of the carboxyl of Asp83 points towards the Mg2+ ion and is within hydrogen-bonding distance (5.04 Å) through an Mg2+-coordinated water. For K. pneumoniae both of the carboxyl O atoms are pointing towards the Mg2+ ion at distances of 4.86 and 4.23 Å. These residues are ordered in only one of the two dimers in the K. pneumoniae crystal (the one in which the C7 group is pointing towards the DNA away from ParE, although the conformations of these two groups on the drug are probably not correlated).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>19479</offset><text>The topoisomerase IV ParE27-ParC55 fusion protein from K. pneumoniae was fully active in promoting levofloxacin-mediated cleavage of DNA (Fig. 6 ▸). In the absence of the drug and ATP, the protein converted supercoiled pBR322 into a ladder of bands corresponding to relaxed DNA. The inclusion of levofloxacin produced linear DNA in a dose-dependent and ATP-independent fashion. Similar behaviour was observed for the S. pneumoniae topo­isomerase IV ParE30-ParC55 fusion protein. The CC25 (the drug concentration that converted 25% of the supercoiled DNA substrate to a linear form) was 0.5 µM for the Klebsiella enzyme and 1 µM for the pneumococcal enzyme. Interestingly, K. pneumoniae strains are much more susceptible to levofloxacin than S. pneumoniae, with typical MIC values of 0.016 and 1 mg l−1, respectively (Odenholt &amp; Cars, 2006), reflecting differences in multiple factors (in addition to binding affinity) that influence drug responses, including membrane, peptidoglycan structure, drug-uptake and efflux mechanisms. Moreover, although topoisomerase IV is primarily the target of levofloxacin in S. pneumoniae, it is likely to be gyrase in the Gram-negative K. pneumoniae.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>20679</offset><text>In summary, we have determined the first structure of a quinolone–DNA cleavage complex involving a type II topo­isomerase from K. pneumoniae. Given the current concerns about drug-resistant strains of Klebsiella, the structure reported here provides key information in understanding the action of currently used quinolones and should aid in the development of other topoisomerase-targeting therapeutics active against this major human pathogen.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">title_1</infon><offset>21127</offset><text>Supplementary Material</text></passage><passage><infon key="section_type">REF</infon><infon key="type">title</infon><offset>21150</offset><text>References</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>21161</offset><text>Adams, P. D. et al. (2010). Acta Cryst. D66, 213–221.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>21217</offset><text>Adams, P. D., Grosse-Kunstleve, R. W., Hung, L.-W., Ioerger, T. R., McCoy, A. J., Moriarty, N. W., Read, R. 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(a) Coloured diagram of the protein constructs used in crystallization. (b) DNA sequences used in crystallization. The 4 bp overhang is shown in red. Cleavage points are indicated by arrows.</text></passage><passage><infon key="file">d-72-00488-fig2.jpg</infon><infon key="id">fig2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>26265</offset><text>Chemical structure of levofloxacin (a) and its conformations observed within the active sites of S. pneumoniae topoisomerase IV (b) and K. pneumoniae topoisomerase IV (c, d). Electron-density maps (2F
+obs − F
+calc) are shown as meshes for the drug molecules contoured at 1.5σ and are limited to a distance of 2.3 Å from the drug atoms.</text></passage><passage><infon key="file">d-72-00488-fig3.jpg</infon><infon key="id">fig3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>26608</offset><text>Overall orthogonal views of the cleavage complex of topoisomerase IV from K. pneumoniae in surface (left) and cartoon (right) representations. The ParC subunit is in blue, ParE is in yellow and DNA is in cyan. The bound quinolone molecules (levofloxacin) are in red and are shown using van der Waals representation.</text></passage><passage><infon key="file">d-72-00488-fig4.jpg</infon><infon key="id">fig4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>26924</offset><text>Schematic representation of the catalytic cycle of type II topoisomerases. The ParC N-terminal domain (ParC55) is in grey, the ParC C-terminal β-­pinwheel domain is in silver, the ParE N-terminal ATPase domain is in red, the ParE C-terminal domain (ParE30) is in yellow, the G-gate DNA is in green and the T-segment DNA is in purple. Bound ATP is indicated by pink circles in the ATPase domains (reproduced with permission from Fig. 1 of Lapanogov et al., 2013).</text></passage><passage><infon key="file">d-72-00488-fig5.jpg</infon><infon key="id">fig5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>27389</offset><text>Detailed views of the active sites of topoisomerase IV from S. pneumoniae and K. pneumoniae with quinolone molecules bound. The magnesium ions and their coordinating amino acids are shown in purple. The drug molecules and residues known to lead to drug resistance upon mutation are in red. The active-site tyrosine and arginine are in orange. The DNA is shown in silver/cyan. The ParC and ParE backbones are shown in blue and yellow, respectively.</text></passage><passage><infon key="file">d-72-00488-fig6.jpg</infon><infon key="id">fig6</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>27837</offset><text>Comparison of DNA cleavage by topoisomerase IV core ParE-ParC fusion proteins from K. pneumoniae (KP) and S. pneumoniae (SP) promoted by levofloxacin. Supercoiled plasmid pBR322 (400 ng) was incubated with topoisomerase IV proteins (400 ng) in the absence or presence of levofloxacin at the indicated concentrations. After 60 min incubation, samples were treated with SDS and proteinase K to remove proteins covalent bound to DNA, and the DNA products were examined by gel electrophoresis in 1% agarose. Lane A, supercoiled pBR322 DNA; N, L and S, nicked, linear and supercoiled pBR322, respectively.</text></passage><passage><infon key="file"></infon><infon key="id">table1</infon><infon key="section_type">TABLE</infon><infon key="type">table_title_caption</infon><offset>28445</offset><text>Macromolecule-production information</text></passage><passage><infon key="file">d36e1283.xml</infon><infon key="id">table1</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>28482</offset><text>
+K. pneumoniae topoisomerase IV.</text></passage><passage><infon key="file">d36e1283.xml</infon><infon key="id">table1</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;tbody valign=&quot;top&quot;&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;Source organism&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;
+&lt;italic&gt;K. pneumoniae&lt;/italic&gt; (strain ATCC 35657)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;Expression vector&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;pET-29a&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;Expression host&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;
+&lt;italic&gt;E. coli&lt;/italic&gt; BL21(λDE3) pLysS&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;2&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;Complete amino-acid sequence of the construct produced&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt; Topoisomerase IV (ParE CTD 390–631 and ParC NTD 1–490 fused) &lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;
+&lt;underline&gt;M&lt;/underline&gt;KKLTSGPALPGKLADCTAQDLNRTELFLVEGDSAGGSAKQARDREYQAIMPLKGKILNTWEVSSDEVLASQEVHDISVAIGIDPDSDDLSQLRYGKICILADADSDGLHIATLLCALFVRHFRTLVKEGHVYVALPPLYRIDLGKEVYYALTEEEKTGVLEQLKRKKGKPNVQRFKGLGEMNPMQLRETTLDPNTRRLVQLVISDEDEQQTTAIMDMLLAKKRSEDRRNWLQEKGDMADLEV&lt;underline&gt;EF&lt;/underline&gt;MSDMAERLALHEFTENAYLNYSMYVIMDRALPFIGDGLKPVQRRIVYAMSELGLNASAKFKKSARTVGDVLGKYHPHGDSACYEAMVLMAQPFSYRYPLGDGQGNWGAPDDPKSFAAMRYTESRLSKYAELLLSELGQGTVDWVPNFDGTLQEPKMLPARLPNILLNGTTGIAVGMATDIPPHNLREVAKAAITLIEQPKTTLDELLDIVQGPDFPTEAEIITSRAEIRKIYQNGRGSVRMRAVWSKEDGAVVITALPHQVSGAKVLEQIAAQMRNKKLPMVDDLRDESDHENPTRLVIVPRSNRVDMEQVMNHLFATTDLEKSYRINLNMIGLDGRPAVKNLLEILSEWLVFRRDTVRRRLNHRLEKVLKRLHILEGLLVAFLNIDEVIEIIRTEDEPKPALMSRFGISETQAEAILELKLRHLAKLEEMKIRGEQSELEKERDQLQAILASERKMNNLLKKELQADADAFGDDRRSPLHEREEAKAMS&lt;underline&gt;HHHHHH&lt;/underline&gt;
+&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt; Symmetrized E-site (pre-cut) DNA1&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;5′-CGTATTACGTTGTAT-3′&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt; Symmetrized E-site (pre-cut) DNA2&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;5′-GATCATACAACGTAATACG-3′&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>28515</offset><text>Source organism	K. pneumoniae (strain ATCC 35657)	 	Expression vector	pET-29a	 	Expression host	E. coli BL21(λDE3) pLysS	 	Complete amino-acid sequence of the construct produced	 	 Topoisomerase IV (ParE CTD 390–631 and ParC NTD 1–490 fused) 	MKKLTSGPALPGKLADCTAQDLNRTELFLVEGDSAGGSAKQARDREYQAIMPLKGKILNTWEVSSDEVLASQEVHDISVAIGIDPDSDDLSQLRYGKICILADADSDGLHIATLLCALFVRHFRTLVKEGHVYVALPPLYRIDLGKEVYYALTEEEKTGVLEQLKRKKGKPNVQRFKGLGEMNPMQLRETTLDPNTRRLVQLVISDEDEQQTTAIMDMLLAKKRSEDRRNWLQEKGDMADLEVEFMSDMAERLALHEFTENAYLNYSMYVIMDRALPFIGDGLKPVQRRIVYAMSELGLNASAKFKKSARTVGDVLGKYHPHGDSACYEAMVLMAQPFSYRYPLGDGQGNWGAPDDPKSFAAMRYTESRLSKYAELLLSELGQGTVDWVPNFDGTLQEPKMLPARLPNILLNGTTGIAVGMATDIPPHNLREVAKAAITLIEQPKTTLDELLDIVQGPDFPTEAEIITSRAEIRKIYQNGRGSVRMRAVWSKEDGAVVITALPHQVSGAKVLEQIAAQMRNKKLPMVDDLRDESDHENPTRLVIVPRSNRVDMEQVMNHLFATTDLEKSYRINLNMIGLDGRPAVKNLLEILSEWLVFRRDTVRRRLNHRLEKVLKRLHILEGLLVAFLNIDEVIEIIRTEDEPKPALMSRFGISETQAEAILELKLRHLAKLEEMKIRGEQSELEKERDQLQAILASERKMNNLLKKELQADADAFGDDRRSPLHEREEAKAMSHHHHHH	 	 Symmetrized E-site (pre-cut) DNA1	5′-CGTATTACGTTGTAT-3′	 	 Symmetrized E-site (pre-cut) DNA2	5′-GATCATACAACGTAATACG-3′	 	</text></passage><passage><infon key="file">d36e1342.xml</infon><infon key="id">table1</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>29644</offset><text>
+S. pneumoniae topoisomerase IV.</text></passage><passage><infon key="file">d36e1342.xml</infon><infon key="id">table1</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;tbody valign=&quot;top&quot;&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;Source organism&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;
+&lt;italic&gt;S. pneumoniae&lt;/italic&gt; (isolate 7785 St George’s Hospital; Pan &amp;amp; Fisher, 1996&lt;xref ref-type=&quot;bibr&quot; rid=&quot;bb50&quot;&gt; ▸&lt;/xref&gt;)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;Expression vector&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;pET-19b (N-terminal His&lt;sub&gt;10&lt;/sub&gt;), pET-29a (C-terminal His&lt;sub&gt;6&lt;/sub&gt;)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;Expression host&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;
+&lt;italic&gt;E. coli&lt;/italic&gt; BL21(λDE3) pLysS&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;2&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;Complete amino-acid sequence of the construct produced&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt; ParC55&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;MSNIQNMSLEDIMGERFGRYSKYIIQDRALPDIRDGLKPVQRRILYSMNKDSNTFDKSYRKSAKSVGNIMGNFHPHGDSSIYDAMVRMSQNWKNREILVEMHGNNGSMDGDPPAAMRYTEARLSEIAGYLLQDIEKKTVPFAWNFDDTEKEPTVLPAAFPNLLVNGSTGISAGYATDIPPHNLAEVIDAAVYMIDHPTAKIDKLMEFLPGPDFPTGAIIQGRDEIKKAYETGKGRVVVRSKTEIEKLKGGKEQIVITEIPYEINKANLVKKIDDVRVNNKVAGIAEVRDESDRDGLRIAIELKKDANTELVLNYLFKYTDLQINYNFNMVAIDNFTPRQVGIVPILSSYIAHRREVILARSRFDKEKAEKRLHIVEGLIRVISILDEVIALIRASENKADAKENLKVSYDFTEEQAEAIVTLQLYRLTNTDVVVLQEEEAELREKIAMLAAIIGDERTMYNLMKKELREVKKKFATPRLSSLEDTAKA&lt;underline&gt;L&lt;/underline&gt;E&lt;underline&gt;HHHHHH&lt;/underline&gt;
+&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt; ParE30&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;
+&lt;underline&gt;MGHHHHHHHHHHSSGHIDDDDKHM&lt;/underline&gt;KNKKDKGLLSGKLTPAQSKNPAKNELYLVEGDSAGGSAKQGRDRKFQAILPLRGKVINTAKAKMADILKNEEINTMIYTIGAGVGADFSIEDANYDKIIIMTDADTDGAHIQTLLLTFFYRYMRPLVEAGHVYIALPPLYKMSKGKGKKEEVAYAWTDGELEELRKQFGKGATLQRYKGLGEMNADQLWETTMNPETRTLIRVTIEDLARAERRVNVLMGDKVEPRRKWIEDNVKFTLEEATVF &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt; E-site DNA1 &lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;5′-CATGAATGACTATGCACG-3′&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt; E-site DNA2 &lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;5′-CGTGCATAGTCATTCATG-3′&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>29677</offset><text>Source organism	S. pneumoniae (isolate 7785 St George’s Hospital; Pan &amp; Fisher, 1996)	 	Expression vector	pET-19b (N-terminal His10), pET-29a (C-terminal His6)	 	Expression host	E. coli BL21(λDE3) pLysS	 	Complete amino-acid sequence of the construct produced	 	 ParC55	MSNIQNMSLEDIMGERFGRYSKYIIQDRALPDIRDGLKPVQRRILYSMNKDSNTFDKSYRKSAKSVGNIMGNFHPHGDSSIYDAMVRMSQNWKNREILVEMHGNNGSMDGDPPAAMRYTEARLSEIAGYLLQDIEKKTVPFAWNFDDTEKEPTVLPAAFPNLLVNGSTGISAGYATDIPPHNLAEVIDAAVYMIDHPTAKIDKLMEFLPGPDFPTGAIIQGRDEIKKAYETGKGRVVVRSKTEIEKLKGGKEQIVITEIPYEINKANLVKKIDDVRVNNKVAGIAEVRDESDRDGLRIAIELKKDANTELVLNYLFKYTDLQINYNFNMVAIDNFTPRQVGIVPILSSYIAHRREVILARSRFDKEKAEKRLHIVEGLIRVISILDEVIALIRASENKADAKENLKVSYDFTEEQAEAIVTLQLYRLTNTDVVVLQEEEAELREKIAMLAAIIGDERTMYNLMKKELREVKKKFATPRLSSLEDTAKALEHHHHHH	 	 ParE30	MGHHHHHHHHHHSSGHIDDDDKHMKNKKDKGLLSGKLTPAQSKNPAKNELYLVEGDSAGGSAKQGRDRKFQAILPLRGKVINTAKAKMADILKNEEINTMIYTIGAGVGADFSIEDANYDKIIIMTDADTDGAHIQTLLLTFFYRYMRPLVEAGHVYIALPPLYKMSKGKGKKEEVAYAWTDGELEELRKQFGKGATLQRYKGLGEMNADQLWETTMNPETRTLIRVTIEDLARAERRVNVLMGDKVEPRRKWIEDNVKFTLEEATVF 	 	 E-site DNA1 	5′-CATGAATGACTATGCACG-3′	 	 E-site DNA2 	5′-CGTGCATAGTCATTCATG-3′	 	</text></passage><passage><infon key="file">table2.xml</infon><infon key="id">table2</infon><infon key="section_type">TABLE</infon><infon key="type">table_title_caption</infon><offset>30828</offset><text>Crystallization</text></passage><passage><infon key="file">table2.xml</infon><infon key="id">table2</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;thead valign=&quot;top&quot;&gt;&lt;tr&gt;&lt;th style=&quot;border-bottom:1px solid black;&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;bottom&quot;&gt; &lt;/th&gt;&lt;th style=&quot;border-bottom:1px solid black;&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;bottom&quot;&gt;
+&lt;italic&gt;K. pneumoniae&lt;/italic&gt; topoisomerase IV&lt;/th&gt;&lt;th style=&quot;border-bottom:1px solid black;&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;bottom&quot;&gt;
+&lt;italic&gt;S. pneumoniae&lt;/italic&gt; topoisomerase IV&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody valign=&quot;top&quot;&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;Method&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;Vapour diffusion&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;Capillary counter-diffusion&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;Plate type&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;24-well Limbro&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;N/A&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;Temperature (K)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;298&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;304&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;Protein concentration (mg ml&lt;sup&gt;−1&lt;/sup&gt;)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;4.5 &lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;4&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;Buffer composition of protein solution&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;2&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;20 m&lt;italic&gt;M&lt;/italic&gt; Tris pH 7.5, 100 m&lt;italic&gt;M&lt;/italic&gt; NaCl, 1 m&lt;italic&gt;M&lt;/italic&gt; β-mercaptoethanol, 0.05% NaN&lt;sub&gt;3&lt;/sub&gt;
+&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;Composition of reservoir solution&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;0.1 &lt;italic&gt;M&lt;/italic&gt; Tris pH 7.5–8.0, 0–50 m&lt;italic&gt;M&lt;/italic&gt; NaCl, 4–8% PEG 4000, 12–15% glycerol&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;50 m&lt;italic&gt;M&lt;/italic&gt; sodium cacodylate pH 6.5, 2.5% Tacsimate, 7% 2-propanol, 62.5 m&lt;italic&gt;M&lt;/italic&gt; KCl, 7.5 m&lt;italic&gt;M&lt;/italic&gt; MgCl&lt;sub&gt;2&lt;/sub&gt;
+&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;Volume and ratio of drop&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;4 + 2 µl&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;N/A&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;Volume of reservoir (ml)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;0.5&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;N/A&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>30844</offset><text> 	K. pneumoniae topoisomerase IV	S. pneumoniae topoisomerase IV	 	Method	Vapour diffusion	Capillary counter-diffusion	 	Plate type	24-well Limbro	N/A	 	Temperature (K)	298	304	 	Protein concentration (mg ml−1)	4.5 	4	 	Buffer composition of protein solution	20 mM Tris pH 7.5, 100 mM NaCl, 1 mM β-mercaptoethanol, 0.05% NaN3	 	Composition of reservoir solution	0.1 M Tris pH 7.5–8.0, 0–50 mM NaCl, 4–8% PEG 4000, 12–15% glycerol	50 mM sodium cacodylate pH 6.5, 2.5% Tacsimate, 7% 2-propanol, 62.5 mM KCl, 7.5 mM MgCl2	 	Volume and ratio of drop	4 + 2 µl	N/A	 	Volume of reservoir (ml)	0.5	N/A	 	</text></passage><passage><infon key="file">table3.xml</infon><infon key="id">table3</infon><infon key="section_type">TABLE</infon><infon key="type">table_title_caption</infon><offset>31473</offset><text>Data collection and processing</text></passage><passage><infon key="file">table3.xml</infon><infon key="id">table3</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>31504</offset><text>Values in parentheses are for the outer shell.</text></passage><passage><infon key="file">table3.xml</infon><infon key="id">table3</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;thead valign=&quot;bottom&quot;&gt;&lt;tr&gt;&lt;th style=&quot;border-bottom:1px solid black;&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;bottom&quot;&gt; &lt;/th&gt;&lt;th style=&quot;border-bottom:1px solid black;&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;bottom&quot;&gt;
+&lt;italic&gt;K. pneumoniae&lt;/italic&gt; topoisomerase IV&lt;/th&gt;&lt;th style=&quot;border-bottom:1px solid black;&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;bottom&quot;&gt;
+&lt;italic&gt;S. pneumoniae&lt;/italic&gt; topoisomerase IV&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody valign=&quot;top&quot;&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;Diffraction source&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;2&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;Beamline I03, Diamond Light Source&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;Wavelength (Å)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;0.97620&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;0.97630&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;Temperature (K)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;100.0&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;100.0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;Detector&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;Pilatus 6M-F&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;ADSC Quantum 315&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;Crystal-to-detector distance (mm)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;502.22&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;377.629&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;Rotation range per image (°)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;0.2&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;0.25&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;Total rotation range (°)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;180&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;75&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;Exposure time per image (s)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;0.2&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;1.0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;Space group&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;
+&lt;italic&gt;P&lt;/italic&gt;2&lt;sub&gt;1&lt;/sub&gt;
+&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;
+&lt;italic&gt;P&lt;/italic&gt;3&lt;sub&gt;1&lt;/sub&gt;21&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;
+&lt;italic&gt;a&lt;/italic&gt;, &lt;italic&gt;b&lt;/italic&gt;, &lt;italic&gt;c&lt;/italic&gt; (Å)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;102.07, 161.53, 138.60&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;157.83, 157.83, 211.15&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;α, β, γ (°)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;90, 94.22, 90&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;Mosaicity (°)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;0.237&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;0.466&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;Resolution range (Å)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;86.12–3.35 (3.53–3.35)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;50–2.90 (3.00–2.90)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;Total No. of reflections&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;160764&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;311576&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;No. of unique reflections&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;63406&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;67471&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;Completeness (%)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;98.5 (98.4)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;99.4 (99.9)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;Multiplicity&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;2.53 (2.59)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;4.6 (4.7)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;〈&lt;italic&gt;I&lt;/italic&gt;/σ(&lt;italic&gt;I&lt;/italic&gt;)〉&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;3.48 (1.95)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;16.14 (3.48)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;
+&lt;italic&gt;R&lt;/italic&gt;
+&lt;sub&gt;r.i.m.&lt;/sub&gt;
+&lt;xref ref-type=&quot;table-fn&quot; rid=&quot;tfn1&quot;&gt;†&lt;/xref&gt;
+&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;0.116 (0.434)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;0.08 (0.515)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;Overall &lt;italic&gt;B&lt;/italic&gt; factor from Wilson plot (Å&lt;sup&gt;2&lt;/sup&gt;)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;53.29&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;73.37&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>31551</offset><text> 	K. pneumoniae topoisomerase IV	S. pneumoniae topoisomerase IV	 	Diffraction source	Beamline I03, Diamond Light Source	 	Wavelength (Å)	0.97620	0.97630	 	Temperature (K)	100.0	100.0	 	Detector	Pilatus 6M-F	ADSC Quantum 315	 	Crystal-to-detector distance (mm)	502.22	377.629	 	Rotation range per image (°)	0.2	0.25	 	Total rotation range (°)	180	75	 	Exposure time per image (s)	0.2	1.0	 	Space group	P21	P3121	 	a, b, c (Å)	102.07, 161.53, 138.60	157.83, 157.83, 211.15	 	α, β, γ (°)	90, 94.22, 90	 	 	Mosaicity (°)	0.237	0.466	 	Resolution range (Å)	86.12–3.35 (3.53–3.35)	50–2.90 (3.00–2.90)	 	Total No. of reflections	160764	311576	 	No. of unique reflections	63406	67471	 	Completeness (%)	98.5 (98.4)	99.4 (99.9)	 	Multiplicity	2.53 (2.59)	4.6 (4.7)	 	〈I/σ(I)〉	3.48 (1.95)	16.14 (3.48)	 	Rr.i.m.†	0.116 (0.434)	0.08 (0.515)	 	Overall B factor from Wilson plot (Å2)	53.29	73.37	 	</text></passage><passage><infon key="file">table3.xml</infon><infon key="id">table3</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>32465</offset><text>Estimated R
+r.i.m. = R
+merge[N/(N − 1)]1/2, where N is the data multiplicity.</text></passage><passage><infon key="file">table4.xml</infon><infon key="id">table4</infon><infon key="section_type">TABLE</infon><infon key="type">table_title_caption</infon><offset>32545</offset><text>Structure solution and refinement</text></passage><passage><infon key="file">table4.xml</infon><infon key="id">table4</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>32579</offset><text>Values in parentheses are for the outer shell.</text></passage><passage><infon key="file">table4.xml</infon><infon key="id">table4</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;thead valign=&quot;bottom&quot;&gt;&lt;tr&gt;&lt;th style=&quot;border-bottom:1px solid black;&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;bottom&quot;&gt; &lt;/th&gt;&lt;th style=&quot;border-bottom:1px solid black;&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;bottom&quot;&gt;
+&lt;italic&gt;K. pneumoniae&lt;/italic&gt; topoisomerase IV&lt;/th&gt;&lt;th style=&quot;border-bottom:1px solid black;&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;bottom&quot;&gt;
+&lt;italic&gt;S. pneumoniae&lt;/italic&gt; topoisomerase IV&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody valign=&quot;top&quot;&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;Resolution range (Å)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;85.01–3.35 (3.40–3.35)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;41.83–2.90 (2.93–2.90)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;Completeness (%)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;98.3&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;99.5&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;σ Cutoff&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;
+&lt;italic&gt;F&lt;/italic&gt; &amp;gt; 1.350σ(&lt;italic&gt;F&lt;/italic&gt;)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;
+&lt;italic&gt;F&lt;/italic&gt; &amp;gt; 1.34σ(&lt;italic&gt;F&lt;/italic&gt;)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;No. of reflections, working set&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;60158 (2615)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;67471 (1992)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;No. of reflections, test set&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;3208 (142)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;6838 (218)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;Final &lt;italic&gt;R&lt;/italic&gt;
+&lt;sub&gt;cryst&lt;/sub&gt;
+&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;0.224 (0.2990)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;0.186 (0.2806)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;Final &lt;italic&gt;R&lt;/italic&gt;
+&lt;sub&gt;free&lt;/sub&gt;
+&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;0.259 (0.3537)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;0.226 (0.3562)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;3&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;No. of non-H atoms&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt; Protein&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;18741&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;10338&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt; Nucleic acid&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;1608&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;730&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt; Ligand&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;104&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;52&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt; Ion&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;8&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;6&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt; Water&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;—&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;54&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt; Total&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;20461&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;11180&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;3&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;R.m.s. deviations&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt; Bonds (Å)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;0.002&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;0.008&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt; Angles (°)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;0.611&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;1.221&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;3&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;Average &lt;italic&gt;B&lt;/italic&gt; factors (Å&lt;sup&gt;2&lt;/sup&gt;)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt; Protein&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;58.05&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;76.7&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt; Nucleic acid&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;64.85&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;90.7&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt; Ligand&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;60.14&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;95.7&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt; Ion&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;42.62&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;84.5&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt; Water&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;—&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;64.2&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;3&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;Ramachandran plot&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt; Most favoured (%)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;93&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;94&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt; Allowed (%)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;6&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot; align=&quot;left&quot; valign=&quot;top&quot;&gt;6&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>32626</offset><text> 	K. pneumoniae topoisomerase IV	S. pneumoniae topoisomerase IV	 	Resolution range (Å)	85.01–3.35 (3.40–3.35)	41.83–2.90 (2.93–2.90)	 	Completeness (%)	98.3	99.5	 	σ Cutoff	F &gt; 1.350σ(F)	F &gt; 1.34σ(F)	 	No. of reflections, working set	60158 (2615)	67471 (1992)	 	No. of reflections, test set	3208 (142)	6838 (218)	 	Final Rcryst	0.224 (0.2990)	0.186 (0.2806)	 	Final Rfree	0.259 (0.3537)	0.226 (0.3562)	 	No. of non-H atoms	 	 Protein	18741	10338	 	 Nucleic acid	1608	730	 	 Ligand	104	52	 	 Ion	8	6	 	 Water	—	54	 	 Total	20461	11180	 	R.m.s. deviations	 	 Bonds (Å)	0.002	0.008	 	 Angles (°)	0.611	1.221	 	Average B factors (Å2)	 	 Protein	58.05	76.7	 	 Nucleic acid	64.85	90.7	 	 Ligand	60.14	95.7	 	 Ion	42.62	84.5	 	 Water	—	64.2	 	Ramachandran plot	 	 Most favoured (%)	93	94	 	 Allowed (%)	6	6	 	</text></passage></document></collection>

raw_BioC_XML/PMC4831588_raw.xml

@@ -0,0 +1,73 @@
+<?xml version="1.0" encoding="UTF-8"?>
+<!DOCTYPE collection SYSTEM "BioC.dtd">
+<collection><source>PMC</source><date>20201216</date><key>pmc.key</key><document><id>4831588</id><infon key="license">NO-CC CODE</infon><passage><infon key="article-id_doi">10.1021/jacs.6b01332</infon><infon key="article-id_pmc">4831588</infon><infon key="article-id_pmid">26967810</infon><infon key="fpage">4634</infon><infon key="issue">13</infon><infon key="license">This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.</infon><infon key="lpage">4642</infon><infon key="name_0">surname:Kreutzer;given-names:Adam G.</infon><infon key="name_1">surname:Hamza;given-names:Imane L.</infon><infon key="name_2">surname:Spencer;given-names:Ryan K.</infon><infon key="name_3">surname:Nowick;given-names:James S.</infon><infon key="section_type">TITLE</infon><infon key="type">front</infon><infon key="volume">138</infon><infon key="year">2017</infon><offset>0</offset><text>X-ray Crystallographic Structures of a Trimer, Dodecamer, and Annular Pore Formed by an Aβ17–36 β-Hairpin</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>112</offset><text>High-resolution structures of oligomers formed by the β-amyloid peptide Aβ are needed to understand the molecular basis of Alzheimer’s disease and develop therapies. This paper presents the X-ray crystallographic structures of oligomers formed by a 20-residue peptide segment derived from Aβ. The development of a peptide in which Aβ17–36 is stabilized as a β-hairpin is described, and the X-ray crystallographic structures of oligomers it forms are reported. Two covalent constraints act in tandem to stabilize the Aβ17–36 peptide in a hairpin conformation: a δ-linked ornithine turn connecting positions 17 and 36 to create a macrocycle and an intramolecular disulfide linkage between positions 24 and 29. An N-methyl group at position 33 blocks uncontrolled aggregation. The peptide readily crystallizes as a folded β-hairpin, which assembles hierarchically in the crystal lattice. Three β-hairpin monomers assemble to form a triangular trimer, four trimers assemble in a tetrahedral arrangement to form a dodecamer, and five dodecamers pack together to form an annular pore. This hierarchical assembly provides a model, in which full-length Aβ transitions from an unfolded monomer to a folded β-hairpin, which assembles to form oligomers that further pack to form an annular pore. This model may provide a better understanding of the molecular basis of Alzheimer’s disease at atomic resolution.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">title_1</infon><offset>1545</offset><text>Introduction</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1558</offset><text>High-resolution structures of oligomers formed by the β-amyloid peptide Aβ are desperately needed to understand the molecular basis of Alzheimer’s disease and ultimately develop preventions or treatments. In Alzheimer’s disease, monomeric Aβ aggregates to form soluble low molecular weight oligomers, such as dimers, trimers, tetramers, hexamers, nonamers, and dodecamers, as well as high molecular weight aggregates, such as annular protofibrils. Over the last two decades the role of Aβ oligomers in the pathophysiology of Alzheimer’s disease has begun to unfold.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2136</offset><text>Mouse models for Alzheimer’s disease have helped shape our current understanding about the Aβ oligomerization that precedes neurodegeneration. Aβ isolated from the brains of young plaque-free Tg2576 mice forms a mixture of low molecular weight oligomers. A 56 kDa soluble oligomer identified by SDS-PAGE was found to be especially important within this mixture. This oligomer was termed Aβ*56 and appears to be a dodecamer of Aβ. Purified Aβ*56 injected intercranially into healthy rats was found to impair memory, providing evidence that this Aβ oligomer may cause memory loss in Alzheimer’s disease. Smaller oligomers with molecular weights consistent with trimers, hexamers, and nonamers were also identified within the mixture of low molecular weight oligomers. Treatment of the mixture of low molecular weight oligomers with hexafluoroisopropanol resulted in the dissociation of the putative dodecamers, nonamers, and hexamers into trimers and monomers, suggesting that trimers may be the building block of the dodecamers, nonamers, and hexamers. Recently, Aβ trimers and Aβ*56 were identified in the brains of cognitively normal humans and were found to increase with age.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3339</offset><text>A type of large oligomers called annular protofibrils (APFs) have also been observed in the brains of transgenic mice and isolated from the brains of Alzheimer’s patients. APFs were first discovered in vitro using chemically synthesized Aβ that aggregated into porelike structures that could be observed by atomic force microscopy (AFM) and transmission electron microscopy (TEM). The sizes of APFs prepared in vitro vary among different studies. Lashuel et al. observed APFs with an outer diameter that ranged from 7–10 nm and an inner diameter that ranged from 1.5–2 nm, consistent with molecular weights of 150–250 kDa. Quist et al. observed APFs with an outer diameter of 16 nm embedded in a lipid bilayer. Kayed et al. observed APFs with an outer diameter that ranged from 8–25 nm, which were composed of small spherical Aβ oligomers, 3–5 nm in diameter. Although the APFs in these studies differ in size, they share a similar annular morphology and appear to be composed of smaller oligomers.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>4352</offset><text>APFs have also been observed in the brains of APP23 transgenic mice by immunofluorescence with an anti-APF antibody and were found to accumulate in neuronal processes and synapses. In a subsequent study, APFs were isolated from the brains of Alzheimer’s patients by immunoprecipitation with an anti-APF antibody. These APFs had an outer diameter that ranged from 11–14 nm and an inner diameter that ranged from 2.5–4 nm.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>4779</offset><text>Dimers of Aβ have also been isolated from the brains of Alzheimer’s patients.− Aβ dimers inhibit long-term potentiation in mice and promote hyperphosphorylation of the microtubule-associated protein tau, leading to neuritic damage. Aβ dimers have only been isolated from human or transgenic mouse brains that contain the pathognomonic fibrillar Aβ plaques associated with Alzheimer’s disease. Furthermore, the endogenous rise of Aβ dimers in the brains of Tg2576 and J20 transgenic mice coincides with the deposition of Aβ plaques. These observations suggest that the Aβ trimers, hexamers, dodecamers, and related assemblies may be associated with presymptomatic neurodegeneration, while Aβ dimers are more closely associated with fibril formation and plaque deposition during symptomatic Alzheimer’s disease.−</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>5614</offset><text>The approach of isolating and characterizing Aβ oligomers has not provided any high-resolution structures of Aβ oligomers. Techniques such as SDS-PAGE, TEM, and AFM have only provided information about the molecular weights, sizes, morphologies, and stoichiometry of Aβ oligomers. High-resolution structural studies of Aβ have primarily focused on Aβ fibrils and Aβ monomers. Solid-state NMR spectroscopy studies of Aβ fibrils revealed that Aβ fibrils are generally composed of extended networks of in-register parallel β-sheets.− X-ray crystallographic studies using fragments of Aβ have provided additional information about how Aβ fibrils pack. Solution-phase NMR and solid-state NMR have been used to study the structures of the Aβ monomers within oligomeric assemblies.− A major finding from these studies is that oligomeric assemblies of Aβ are primarily composed of antiparallel β-sheets. Many of these studies have reported the monomer subunit as adopting a β-hairpin conformation, in which the hydrophobic central and C-terminal regions form an antiparallel β-sheet.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>6738</offset><text>In 2008, Hoyer et al. reported the NMR structure of an Aβ monomer bound to an artificial binding protein called an affibody (PDB 2OTK). The structure revealed that monomeric Aβ forms a β-hairpin when bound to the affibody. This Aβ β-hairpin encompasses residues 17–37 and contains two β-strands comprising Aβ17–24 and Aβ30–37 connected by an Aβ25–29 loop. Sequestering Aβ within the affibody prevents its fibrilization and reduces its neurotoxicity, providing evidence that the β-hairpin structure may contribute to the ability of Aβ to form neurotoxic oligomers. In a related study, Sandberg et al. constrained Aβ in a β-hairpin conformation by mutating residues A21 and A30 to cysteine and forming an intramolecular disulfide bond. Locking Aβ into a β-hairpin structure resulted in the formation Aβ oligomers, which were observed by size exclusion chromatography (SEC) and SDS-PAGE. The oligomers with a molecular weight of ∼100 kDa that were isolated by SEC were toxic toward neuronally derived SH-SY5Y cells. This study provides evidence for the role of β-hairpin structure in Aβ oligomerization and neurotoxicity.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>7914</offset><text>Inspired by these β-hairpin structures, our laboratory developed a macrocyclic β-sheet peptide derived from Aβ17–36 designed to mimic an Aβ β-hairpin and reported its X-ray crystallographic structure. This peptide (peptide 1) consists of two β-strands comprising Aβ17–23 and Aβ30–36 covalently linked by two δ-linked ornithine (δOrn) β-turn mimics. The δOrn that connects residues D23 and A30 replaces the Aβ24–29 loop. The δOrn that connects residues L17 and V36 enforces β-hairpin structure. We incorporated an N-methyl group at position G33 to prevent uncontrolled aggregation and precipitation of the peptide. To improve the solubility of the peptide we replaced M35 with the hydrophilic isostere of methionine, ornithine (α-linked) (Figure 1B). The X-ray crystallographic structure of peptide 1 reveals that it folds to form a β-hairpin that assembles to form trimers and that the trimers further assemble to form hexamers and dodecamers.</text></passage><passage><infon key="file">ja-2016-013325_0002.jpg</infon><infon key="id">fig1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>8914</offset><text>(A) Cartoon
+illustrating the design of peptides 1 and 2 and their relationship to an Aβ17–36 β-hairpin.
+(B) Chemical structure of peptide 1 illustrating Aβ17–23 and Aβ30–36, M35Orn, the N-methyl group, and the δ-linked
+ornithine turns. (C) Chemical structure of peptide 2 illustrating
+Aβ17–36, the N-methyl group,
+the disulfide bond across positions 24 and 29, and the δ-linked
+ornithine turn.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>9331</offset><text>Our design of peptide 1 omitted the Aβ24–29 loop. To visualize the Aβ24–29 loop, we performed replica-exchange molecular dynamics (REMD) simulations on Aβ17–36 using the X-ray crystallographic coordinates of Aβ17–23 and Aβ30–36 from peptide 1. These studies provided a working model for a trimer of Aβ17–36 β-hairpins and demonstrated that the trimer should be capable of accommodating the Aβ24–29 loop.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>9759</offset><text>In the current study we set out to restore the Aβ24–29 loop, reintroduce the methionine residue at position 35, and determine the X-ray crystallographic structures of oligomers that form. We designed peptide 2 as a homologue of peptide 1 that embodies these ideas. Peptide 2 contains a methionine residue at position 35 and an Aβ24–29 loop with residues 24 and 29 (Val and Gly) mutated to cysteine and linked by a disulfide bond (Figure 1C). Here, we describe the development of peptide 2 and report the X-ray crystallographic structures of the trimer, dodecamer, and annular pore observed within the crystal structure.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>10385</offset><text>Results</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>10393</offset><text>Development of Peptide 2</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10418</offset><text>We developed peptide 2 from peptide 1 by an iterative process, in which we first attempted to restore the Aβ24–29 loop without a disulfide linkage. We envisioned peptide 3 as a homologue of peptide 1 with the Aβ24–29 loop in place of the δOrn that connects D23 and A30 and p-iodophenylalanine (FI) in place of F19. We routinely use p-iodophenylalanine to determine the X-ray crystallographic phases. After determining the X-ray crystallographic structure of the p-iodophenylalanine variant we attempt to determine the structure of the native phenylalanine compound by isomorphous replacement. Upon synthesizing peptide 3, we found that it formed an amorphous precipitate in most crystallization conditions screened and failed to afford crystals in any condition.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>11191</offset><text>We postulate that the loss of the δOrn constraint leads to conformational heterogeneity that prevents peptide 3 from crystallizing. To address this issue, we next incorporated a disulfide bond between residues 24 and 29 as a conformational constraint that serves as a surrogate for δOrn. We designed peptide 4 to embody this idea, mutating Val24 and Gly29 to cysteine and forming an interstrand disulfide linkage. We mutated these residues because they occupy the same position as the δOrn that connects D23 and A30 in peptide 1. Residues V24 and G29 form a non-hydrogen-bonded pair, which can readily accommodate disulfide linkages in antiparallel β-sheets. Disulfide bonds across non-hydrogen-bonded pairs stabilize β-hairpins, while disulfide bonds across hydrogen-bonded pairs do not. Although the disulfide bond between positions 24 and 29 helps stabilize the β-hairpin, it does not alter the charge or substantially change the hydrophobicity of the Aβ17–36 β-hairpin. We were gratified to find that peptide 4 afforded crystals suitable for X-ray crystallography. As the next step in the iterative process, we determined the X-ray crystallographic structure of this peptide (PDB 5HOW).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>12409</offset><text>After determining the X-ray crystallographic structure of peptide 4 we reintroduced the native phenylalanine at position 19 and the methionine at position 35 to afford peptide 2. We completed the iterative process—from 1 to 3 to 4 to 2—by successfully determining the X-ray crystallographic structure of peptide 2 (PDB 5HOX and 5HOY). The following sections describe the synthesis of peptides 2–4 and the X-ray crystallographic structure of peptide 2.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>12867</offset><text>Synthesis of Peptides 2–4</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>12895</offset><text>We synthesized peptides 2–4 by similar procedures to those we have developed for other macrocyclic peptides. Our laboratory routinely prepares macrocyclic peptides by solid-phase synthesis of the corresponding linear peptide on 2-chlorotrityl resin, followed by cleavage of the protected linear peptide from the resin, solution-phase macrolactamization, and deprotection of the resulting macrocyclic peptide. In synthesizing peptides 2 and 4 we formed the disulfide linkage after macrolactamization and deprotection of the acid-labile side chain protecting groups. We used acid-stable Acm-protected cysteine residues at positions 24 and 29 and removed the Acm groups by oxidation with I2 in aqueous acetic acid to afford the disulfide linkage. Peptides 2–4 were purified by RP-HPLC.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>13682</offset><text>Crystallization, X-ray Crystallographic Data Collection, Data Processing, and Structure Determination of Peptides 2 and 4</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>13804</offset><text>We screened crystallization conditions for peptide 4 in a 96-well-plate format using three different Hampton Research crystallization kits (Crystal Screen, Index, and PEG/Ion) with three ratios of peptide and mother liquor per condition (864 experiments). Peptide 4 afforded crystals in a single set of conditions containing HEPES buffer and Jeffamine M-600—the same crystallization conditions that afforded crystals of peptide 1. Peptide 2 also afforded crystals in these conditions. We further optimized these conditions to rapidly (∼72 h) yield crystals suitable for X-ray crystallography. The optimized conditions consist of 0.1 M HEPES at pH 6.4 with 31% Jeffamine M-600 for peptide 4 and 0.1 M HEPES pH 7.1 with 29% Jeffamine M-600 for peptide 2.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>14561</offset><text>Crystal diffraction data for peptides 4 and 2 were collected in-house with a Rigaku MicroMax 007HF X-ray diffractometer at 1.54 Å wavelength. Crystal diffraction data for peptide 2 were also collected at the Advanced Light Source at Lawrence Berkeley National Laboratory with a synchrotron source at 1.00 Å wavelength to achieve higher resolution. Data from peptides 4 and 2 suitable for refinement at 2.30 Å were obtained from the diffractometer; data from peptide 2 suitable for refinement at 1.90 Å were obtained from the synchrotron.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15103</offset><text>Data for peptides 4 and 2 were scaled and merged using XDS. Phases for peptide 4 were determined by single-wavelength anomalous diffraction (SAD) phasing by using the coordinates of the iodine anomalous signal from p-iodophenylalanine. Phases for peptide 2 were determined by isomorphous replacement of peptide 4. The structures of peptides 2 and 4 were solved and refined in the P6122 space group. Coordinates for hydrogens were generated by phenix.refine during refinement. The asymmetric unit of each peptide consists of six monomers, arranged as two trimers. Peptides 2 and 4 form morphologically identical structures and assemblies in the crystal lattice.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>15764</offset><text>X-ray Crystallographic Structure of Peptide 2 and the Oligomers It Forms</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15837</offset><text>The X-ray crystallographic structure of peptide 2 reveals that it folds to form a twisted β-hairpin comprising two β-strands connected by a loop (Figure 2A). Eight residues make up each surface of the β-hairpin: L17, F19, A21, D23, A30, I32, L34, and V36 make up one surface; V18, F20, E22, C24, C29, I31, G33, and M35 make up the other surface. The β-strands of the monomers in the asymmetric unit are virtually identical, differing primarily in rotamers of F20, E22, C24, C29, I31, and M35 (Figure S1). The disulfide linkages suffered radiation damage under synchrotron radiation. We refined three of the β-hairpins with intact disulfide linkages and three with thiols to represent cleaved disulfide linkages in the synchrotron data set (PDB 5HOX). No evidence for cleavage of the disulfides was observed in the refinement of the data set collected on the X-ray diffractometer, and we refined all disulfide linkages as intact (PDB 5HOY).</text></passage><passage><infon key="file">ja-2016-013325_0003.jpg</infon><infon key="id">fig2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>16792</offset><text>X-ray crystallographic
+structure of peptide 2 (PDB 5HOX, synchrotron data
+set). (A) X-ray crystallographic structure of a representative β-hairpin
+monomer formed by peptide 2. (B) Overlay of the six β-hairpin
+monomers in the asymmetric unit. The β-hairpins are shown as
+cartoons to illustrate the differences in the Aβ25–28 loops.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>17135</offset><text>The Aβ25–28 loops of the six monomers within the asymmetric unit vary substantially in backbone geometry and side chain rotamers (Figures 2B and S1). The electron density for the loops is weak and diffuse compared to the electron density for the β-strands. The B values for the loops are large, indicating that the loops are dynamic and not well ordered. Thus, the differences in backbone geometry and side chain rotamers among the loops are likely of little significance and should be interpreted with caution.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>17653</offset><text>Peptide 2 assembles into oligomers similar in morphology to those formed by peptide 1. Like peptide 1, peptide 2 forms a triangular trimer, and four trimers assemble to form a dodecamer. In the higher-order assembly of the dodecamers formed by peptide 2 a new structure emerges, not seen in peptide 1, an annular pore consisting of five dodecamers.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_3</infon><offset>18002</offset><text>Trimer</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>18009</offset><text>Peptide 2 forms a trimer, much like that which we observed previously for peptide 1, in which three β-hairpins assemble to form an equilateral triangle (Figure 3A). The trimer maintains all of the same stabilizing contacts as those of peptide 1. Hydrogen bonding and hydrophobic interactions between residues on the β-strands comprising Aβ17–23 and Aβ30–36 stabilize the core of the trimer. The disulfide bonds between residues 24 and 29 are adjacent to the structural core of the trimer and do not make any substantial intermolecular contacts. Two crystallographically distinct trimers comprise the peptide portion of the asymmetric unit. The two trimers are almost identical in structure, differing slightly among side chain rotamers and loop conformations.</text></passage><passage><infon key="file">ja-2016-013325_0004.jpg</infon><infon key="id">fig3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>18781</offset><text>X-ray crystallographic structure of the trimer formed
+by peptide 2. (A) Triangular trimer. The three water
+molecules in the
+center hole of the trimer are shown as spheres. (B) Detailed view
+of the intermolecular hydrogen bonds between the main chains of V18 and E22 and δOrn and C24, at the three corners of the triangular trimer. (C) The F19 face of the trimer, with key side chains shown as spheres. (D) The
+F20 face of the trimer, with key side chains as spheres.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>19252</offset><text>A network of 18 intermolecular hydrogen bonds helps stabilize the trimer. At the corners of the trimer, the pairs of β-hairpin monomers form four hydrogen bonds: two between the main chains of V18 and E22 and two between δOrn and the main chain of C24 (Figure 3B). Three ordered water molecules fill the hole in the center of the trimer, hydrogen bonding to each other and to the main chain of F20 (Figure 3A).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>19670</offset><text>Hydrophobic contacts between residues at the three corners of the trimer, where the β-hairpins meet, further stabilize the trimer. At each corner, the side chains of residues L17, F19, and V36 of one β-hairpin pack against the side chains of residues A21, I32, L34, and also D23 of the adjacent β-hairpin to create a hydrophobic cluster (Figure 3C). The three hydrophobic clusters create a large hydrophobic surface on one face of the trimer. The other face of the trimer displays a smaller hydrophobic surface, which includes the side chains of residues V18, F20, and I31 of the three β-hairpins (Figure 3D). In subsequent discussion, we designate the former surface the “F19 face” and the latter surface the “F20 face”.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_3</infon><offset>20404</offset><text>Dodecamer</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>20414</offset><text>Four trimers assemble to form a dodecamer. The four trimers arrange in a tetrahedral fashion, creating a central cavity inside the dodecamer. Because each trimer is triangular, the resulting arrangement resembles an octahedron. Each of the 12 β-hairpins constitutes an edge of the octahedron, and the triangular trimers occupy four of the eight faces of the octahedron. Figure 4A illustrates the octahedral shape of the dodecamer. Figure 4B illustrates the tetrahedral arrangement of the four trimers.</text></passage><passage><infon key="file">ja-2016-013325_0005.jpg</infon><infon key="id">fig4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>20919</offset><text>X-ray
+crystallographic structure of the dodecamer formed by peptide 2. (A) View of the dodecamer that illustrates the octahedral
+shape. (B) View of the dodecamer that illustrates the tetrahedral
+arrangement of the four trimers that comprise the dodecamer. (C) View
+of two trimer subunits from inside the cavity of the dodecamer. Residues
+L17, L34, and V36 are shown as spheres,
+illustrating the hydrophobic packing that occurs at the six vertices
+of the dodecamer. (D) Detailed view of one of the six vertices of
+the dodecamer.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>21447</offset><text>The F19 faces of the trimers line the interior of the dodecamer. At the six vertices, hydrophobic packing between the side chains of L17, L34, and V36 helps stabilize the dodecamer (Figures 4C and D). Salt bridges between the side chains of D23 and δOrn at the vertices further stabilize the dodecamer. Each of the six vertices includes two Aβ25–28 loops that extend past the core of the dodecamer without making any substantial intermolecular contacts. The exterior of the dodecamer displays four F20 faces (Figure S3). In the crystal lattice, each F20 face of one dodecamer packs against an F20 face of another dodecamer. Although the asymmetric unit comprises half a dodecamer, the crystal lattice may be thought of as being built of dodecamers.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>22203</offset><text>The electron density map for the X-ray crystallographic structure of peptide 2 has long tubes of electron density inside the central cavity of the dodecamer. The shape and length of the electron density is consistent with the structure of Jeffamine M-600, which is an essential component of the crystallization conditions. Jeffamine M-600 is a polypropylene glycol derivative with a 2-methoxyethoxy unit at one end and a 2-aminopropyl unit at the other end. Its average molecular weight is about 600 Da, which corresponds to nine propylene glycol units. Although Jeffamine M-600 is a heterogeneous mixture with varying chain lengths and stereochemistry, we modeled a single stereoisomer with nine propylene glycol units (n = 9) to fit the electron density. The Jeffamine M-600 appears to stabilize the dodecamer by occupying the central cavity and making hydrophobic contacts with residues lining the cavity (Figure S3). In a dodecamer formed by full-length Aβ, the hydrophobic C-terminal residues (Aβ37–40 or Aβ37–42) might play a similar role in filling the dodecamer and thus create a packed hydrophobic core within the central cavity of the dodecamer.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_3</infon><offset>23368</offset><text>Annular Pore</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>23381</offset><text>Five dodecamers assemble to form an annular porelike structure (Figure 5A). Hydrophobic packing between the F20 faces of trimers displayed on the outer surface of each dodecamer stabilizes the porelike assembly. Two morphologically distinct interactions between trimers occur at the interfaces of the five dodecamers: one in which the trimers are eclipsed (Figure 5B), and one in which the trimers are staggered (Figure 5C). Hydrophobic packing between the side chains of F20, I31, and E22 stabilizes these interfaces (Figure 5D and E). The annular pore contains three eclipsed interfaces and two staggered interfaces. The eclipsed interfaces occur between dodecamers 1 and 2, 1 and 5, and 3 and 4, as shown in Figure 5A. The staggered interfaces occur between dodecamers 2 and 3 and 4 and 5. The annular pore is not completely flat, instead, adopting a slightly puckered shape, which accommodates the eclipsed and staggered interfaces. Ten Aβ25–28 loops from the vertices of the five dodecamers line the hole in the center of the pore. The hydrophilic side chains of S26, N27, and K28 decorate the hole.</text></passage><passage><infon key="file">ja-2016-013325_0006.jpg</infon><infon key="id">fig5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>24489</offset><text>X-ray crystallographic structure of the annular pore formed by
+peptide 2. (A) Annular porelike structure illustrating
+the relationship of the five dodecamers that form the pore (top view).
+(B) Eclipsed interface between dodecamers 1 and 2 (side view). The
+same eclipsed interface also occurs between dodecamers 1 and 5 and
+3 and 4. (C) Staggered interface between dodecamers 2 and 3 (side
+view). The same staggered interface also occurs between dodecamers
+4 and 5. (D) Eclipsed interface between dodecamers 1 and 5 (top view).
+Residues F20, I31, and E22 are shown
+as spheres to detail the hydrophobic packing. (E) Staggered interface
+between dodecamers 2 and 3 (top view). Residues F20, I31, and E22 are shown as spheres to detail the hydrophobic
+packing.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>25245</offset><text>The annular pore is comparable in size to other large protein assemblies. The outer diameter is ∼11–12 nm. The diameter of the hole in the center of the pore is ∼2 nm. The thickness of the pore is ∼5 nm, which is comparable to that of a lipid bilayer membrane. It is important to note that the annular pore formed by peptide 2 is not a discrete unit in the crystal lattice. Rather, the crystal lattice is composed of conjoined annular pores in which all four F20 faces on the surface of each dodecamer contact F20 faces on other dodecamers (Figure S4). The crystal lattice shows how the dodecamers can further assemble to form larger structures. Each dodecamer may be thought of as a tetravalent building block with the potential to assemble on all four faces to form higher-order supramolecular assemblies.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_1</infon><offset>26061</offset><text>Discussion</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>26072</offset><text>The X-ray crystallographic study of peptide 2 described here provides high-resolution structures of oligomers formed by an Aβ17–36 β-hairpin. The crystallographic assembly of peptide 2 into a trimer, dodecamer, and annular pore provides a model for the assembly of the full-length Aβ peptide to form oligomers. In this model Aβ folds to form a β-hairpin comprising the hydrophobic central and C-terminal regions. Three β-hairpins assemble to form a trimer, and four trimers assemble to form a dodecamer. The dodecamers further assemble to form an annular pore (Figure 6).</text></passage><passage><infon key="file">ja-2016-013325_0007.jpg</infon><infon key="id">fig6</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>26662</offset><text>Model for the hierarchical assembly of an Aβ
+β-hairpin
+into a trimer, dodecamer, and annular pore based on the crystallographic
+assembly of peptide 2. Monomeric Aβ folds to form
+a β-hairpin in which the hydrophobic central and C-terminal regions form an antiparallel β-sheet. Three β-hairpin
+monomers assemble to form a triangular trimer. Four triangular trimers
+assemble to form a dodecamer. Five dodecamers assemble to form an
+annular pore. The molecular weights shown correspond to an Aβ42 monomer (∼4.5 kDa), an Aβ42 trimer
+(∼13.5 kDa), an Aβ42 dodecamer (∼54
+kDa), and an Aβ42 annular pore composed of five dodecamers
+(∼270 kDa).</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>27327</offset><text>The model put forth in Figure 6 is consistent with the current understanding of endogenous Aβ oligomerization and explains at atomic resolution many key observations about Aβ oligomers. Two general types of endogenous Aβ oligomers have been observed: Aβ oligomers that occur on a pathway to fibrils, or “fibrillar oligomers”, and Aβ oligomers that evade a fibrillar fate, or “nonfibrillar oligomers”.− Fibrillar oligomers accumulate in Alzheimer’s disease later than nonfibrillar oligomers and coincide with the deposition of plaques. Nonfibrillar oligomers accumulate early in Alzheimer’s disease before plaque deposition.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>27970</offset><text>Fibrillar and nonfibrillar oligomers have structurally distinct characteristics, which are reflected in their reactivity with the fibril-specific OC antibody and the oligomer-specific A11 antibody. Fibrillar oligomers are recognized by the OC antibody but not the A11 antibody, whereas nonfibrillar oligomers are recognized by the A11 antibody but not the OC antibody. These criteria have been used to classify the Aβ oligomers that accumulate in vivo. Aβ dimers have been classified as fibrillar oligomers, whereas Aβ trimers, Aβ*56, and APFs have been classified as nonfibrillar oligomers.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>28574</offset><text>Larson and Lesné proposed a model for the endogenous production of nonfibrillar oligomers that explains these observations. In this model, folded Aβ monomer assembles into a trimer, the trimer further assembles into hexamers and dodecamers, and the dodecamers further assemble to form annular protofibrils. The hierarchical assembly of peptide 2 is consistent with this model; and the trimer, dodecamer, and annular pore formed by peptide 2 may share similarities to the trimers, Aβ*56, and APFs observed in vivo. At this point, we can only speculate whether the trimer and dodecamer formed by peptide 2 share structural similarities to Aβ trimers and Aβ*56, as little is known about the structure of Aβ trimers and Aβ*56.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>29315</offset><text>The crystallographically observed annular pore formed by peptide 2 is morphologically similar to the APFs formed by full-length Aβ. The annular pore formed by peptide 2 is comparable in size to the APFs prepared in vitro or isolated from Alzheimer’s brains (Figure 7 and Table 1). The varying sizes of APFs formed by full-length Aβ might result from differences in the number of oligomer subunits comprising each APF. Although the annular pore formed by peptide 2 contains five dodecamer subunits, pores containing fewer or more subunits can easily be envisioned. The dodecamers that comprise the annular pore exhibit two modes of assembly—eclipsed interactions and staggered interactions between the F20 faces of trimers within dodecamers. These two modes of assembly might reflect a dynamic interaction between dodecamers, which could permit assemblies of more dodecamers into larger annular pores.</text></passage><passage><infon key="file">ja-2016-013325_0008.jpg</infon><infon key="id">fig7</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>30222</offset><text>Surface views of the annular pore formed
+by peptide 2. (A) Top view. (B) Side view.</text></passage><passage><infon key="file">tbl1.xml</infon><infon key="id">tbl1</infon><infon key="section_type">TABLE</infon><infon key="type">table_title_caption</infon><offset>30306</offset><text>Annular Pores Formed by Aβ and
+Peptide 2</text></passage><passage><infon key="file">tbl1.xml</infon><infon key="id">tbl1</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot; border=&quot;0&quot;&gt;&lt;colgroup&gt;&lt;col align=&quot;left&quot;/&gt;&lt;col align=&quot;center&quot;/&gt;&lt;col align=&quot;center&quot;/&gt;&lt;col align=&quot;left&quot;/&gt;&lt;/colgroup&gt;&lt;thead&gt;&lt;tr&gt;&lt;th style=&quot;border:none;&quot; align=&quot;center&quot;&gt;annular pore
+source&lt;/th&gt;&lt;th style=&quot;border:none;&quot; align=&quot;center&quot;&gt;outer diameter&lt;/th&gt;&lt;th style=&quot;border:none;&quot; align=&quot;center&quot;&gt;inner diameter&lt;/th&gt;&lt;th style=&quot;border:none;&quot; align=&quot;center&quot;&gt;observation
+method&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td style=&quot;border:none;&quot; align=&quot;left&quot;&gt;peptide &lt;bold&gt;2&lt;/bold&gt;&lt;/td&gt;&lt;td style=&quot;border:none;&quot; align=&quot;center&quot;&gt; ∼11–12 nm&lt;/td&gt;&lt;td style=&quot;border:none;&quot; align=&quot;center&quot;&gt;∼2 nm&lt;/td&gt;&lt;td style=&quot;border:none;&quot; align=&quot;left&quot;&gt;X-ray crystallography&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td style=&quot;border:none;&quot; align=&quot;left&quot;&gt;synthetic Aβ&lt;sup&gt;&lt;xref ref-type=&quot;bibr&quot; rid=&quot;ref6&quot;&gt;6&lt;/xref&gt;&lt;/sup&gt;&lt;/td&gt;&lt;td style=&quot;border:none;&quot; align=&quot;center&quot;&gt;7–10 nm&lt;/td&gt;&lt;td style=&quot;border:none;&quot; align=&quot;center&quot;&gt;1.5–2 nm&lt;/td&gt;&lt;td style=&quot;border:none;&quot; align=&quot;left&quot;&gt;TEM&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td style=&quot;border:none;&quot; align=&quot;left&quot;&gt;synthetic Aβ&lt;sup&gt;&lt;xref ref-type=&quot;bibr&quot; rid=&quot;ref7&quot;&gt;7&lt;/xref&gt;&lt;/sup&gt;&lt;/td&gt;&lt;td style=&quot;border:none;&quot; align=&quot;center&quot;&gt;16 nm&lt;/td&gt;&lt;td style=&quot;border:none;&quot; align=&quot;center&quot;&gt;not reported&lt;/td&gt;&lt;td style=&quot;border:none;&quot; align=&quot;left&quot;&gt;AFM&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td style=&quot;border:none;&quot; align=&quot;left&quot;&gt;synthetic Aβ&lt;sup&gt;&lt;xref ref-type=&quot;bibr&quot; rid=&quot;ref8&quot;&gt;8&lt;/xref&gt;&lt;/sup&gt;&lt;/td&gt;&lt;td style=&quot;border:none;&quot; align=&quot;center&quot;&gt;8–25 nm&lt;/td&gt;&lt;td style=&quot;border:none;&quot; align=&quot;center&quot;&gt;not reported&lt;/td&gt;&lt;td style=&quot;border:none;&quot; align=&quot;left&quot;&gt;TEM&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td style=&quot;border:none;&quot; align=&quot;left&quot;&gt;Alzheimer’s brain&lt;sup&gt;&lt;xref ref-type=&quot;bibr&quot; rid=&quot;ref10&quot;&gt;10&lt;/xref&gt;&lt;/sup&gt;&lt;/td&gt;&lt;td style=&quot;border:none;&quot; align=&quot;center&quot;&gt;11–14 nm&lt;/td&gt;&lt;td style=&quot;border:none;&quot; align=&quot;center&quot;&gt;2.5–4 nm&lt;/td&gt;&lt;td style=&quot;border:none;&quot; align=&quot;left&quot;&gt;TEM&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>30350</offset><text>annular pore	 	source	outer diameter	inner diameter	observation	 	method	 	peptide 2	 ∼11–12 nm	∼2 nm	X-ray crystallography	 	synthetic Aβ	7–10 nm	1.5–2 nm	TEM	 	synthetic Aβ	16 nm	not reported	AFM	 	synthetic Aβ	8–25 nm	not reported	TEM	 	Alzheimer’s brain	11–14 nm	2.5–4 nm	TEM	 	</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>30663</offset><text>Dot blot analysis shows that peptide 2 is reactive toward the A11 antibody (Figure S5). This reactivity suggests that peptide 2 forms oligomers in solution that share structural similarities to the nonfibrillar oligomers formed by full-length Aβ. Further studies are needed to elucidate the species that peptide 2 forms in solution and to study their biological properties. This is an active area of research in our laboratory. Preliminary attempts to study these species by SEC and SDS-PAGE have not provided a clear measure of the structures formed in solution. The difficulty in studying the oligomers formed in solution may reflect the propensity of the dodecamer to assemble on all four F20 faces.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>31369</offset><text>The X-ray crystallographic structure and A11 reactivity of peptide 2 support the model proposed by Larsen and Lesné and suggest that β-hairpins constitute a fundamental building block for nonfibrillar oligomers. What makes β-hairpins special is that three β-hairpins can nestle together to form trimers, stabilized by a network of hydrogen bonds and hydrophobic interactions. This mode of assembly is not unique to Aβ. The foldon domain of bacteriophage T4 fibritin is composed of three β-hairpins that assemble into a triangular trimer similar to the triangular trimer formed by peptide 2. Additionally, our research group has observed a similar assembly of a β-hairpin peptide derived from β2-microglobulin.</text></passage><passage><infon key="section_type">CONCL</infon><infon key="type">title_1</infon><offset>32100</offset><text>Conclusion</text></passage><passage><infon key="section_type">CONCL</infon><infon key="type">paragraph</infon><offset>32111</offset><text>Although we began these studies with a relatively simple hypothesis—that the trimers and dodecamers formed by peptide 1 could accommodate the Aβ24–29 loop—an even more exciting finding has emerged—that the dodecamers can assemble to form annular pores. This finding could not have been anticipated from the X-ray crystallographic structure of peptide 1 and reveals a new level of hierarchical assembly that recapitulates micrographic observations of annular protofibrils. The crystallographically observed dodecamer, in turn, recapitulates the observation of Aβ*56, which appears to be a dodecamer of Aβ. The crystallographically observed trimer recapitulates the Aβ trimers that are observed even before the onset of symptoms in Alzheimer’s disease.</text></passage><passage><infon key="section_type">CONCL</infon><infon key="type">paragraph</infon><offset>32876</offset><text>Our approach of constraining Aβ17–36 into a β-hairpin conformation and blocking aggregation with an N-methyl group has allowed us to crystallize a large fragment of what is generally considered to be an uncrystallizable peptide. We believe this iterative, “bottom up” approach of identifying the minimal modification required to crystallize Aβ peptides will ultimately allow larger fragments of Aβ to be crystallized, thus providing greater insights into the structures of Aβ oligomers.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">title</infon><offset>33378</offset><text>Supporting Information Available</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">paragraph</infon><offset>33411</offset><text>Procedures for the synthesis and crystallization of peptides 2–4; details of X-ray crystallographic data collection, processing, and refinement; procedure and data for dot blot analysis (PDF)</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">paragraph</infon><offset>33605</offset><text>Crystallographic coordinates of peptide 2 deposited into the Protein Data Bank (PDB) with code 5HOX (data collected on a synchrotron at 1.00 Å wavelength) (PDB)</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">paragraph</infon><offset>33767</offset><text>Crystallographic coordinates of peptide 2 deposited into the Protein Data Bank (PDB) with code 5HOY (data collected on an X-ray diffractometer at 1.54 Å wavelength) (PDB)</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">paragraph</infon><offset>33939</offset><text>Crystallographic coordinates of peptide 4 deposited into the Protein Data Bank (PDB) with code 5HOW (PDB)</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">paragraph</infon><offset>34045</offset><text>Crystallographic coordinates of the dodecamer formed by peptide 2 (PDB)</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">paragraph</infon><offset>34117</offset><text>Crystallographic coordinates of the annular pore formed by peptide 2 (PDB)</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">paragraph</infon><offset>34192</offset><text>Crystallographic data for 5HOW (CIF)</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">paragraph</infon><offset>34229</offset><text>Crystallographic data for 5HOX (CIF)</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">paragraph</infon><offset>34266</offset><text>Crystallographic data for 5HOY (CIF)</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">paragraph</infon><offset>34303</offset><text>The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.6b01332.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">title_1</infon><offset>34420</offset><text>Supplementary Material</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">paragraph</infon><offset>34443</offset><text>The authors declare no competing financial interest.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">title</infon><offset>34496</offset><text>References</text></passage><passage><infon key="fpage">349</infon><infon key="lpage">357</infon><infon key="name_0">surname:Benilova;given-names:I.</infon><infon key="name_1">surname:Karran;given-names:E.</infon><infon key="name_2">surname:De Strooper;given-names:B.</infon><infon key="pub-id_doi">10.1038/nn.3028</infon><infon key="pub-id_pmid">22286176</infon><infon key="section_type">REF</infon><infon key="source">Nat. 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raw_BioC_XML/PMC4832331_raw.xml

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+<collection><source>PMC</source><date>20201215</date><key>pmc.key</key><document><id>4832331</id><infon key="license">CC BY</infon><passage><infon key="article-id_doi">10.1038/srep24601</infon><infon key="article-id_pii">srep24601</infon><infon key="article-id_pmc">4832331</infon><infon key="article-id_pmid">27080013</infon><infon key="elocation-id">24601</infon><infon key="license">This work is licensed under a Creative Commons Attribution 4.0
+International License. The images or other third party material in this article are
+included in the article’s Creative Commons license, unless indicated
+otherwise in the credit line; if the material is not included under the Creative
+Commons license, users will need to obtain permission from the license holder to
+reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/</infon><infon key="name_0">surname:Kandiah;given-names:Eaazhisai</infon><infon key="name_1">surname:Carriel;given-names:Diego</infon><infon key="name_10">surname:Gutsche;given-names:Irina</infon><infon key="name_2">surname:Perard;given-names:Julien</infon><infon key="name_3">surname:Malet;given-names:Hélène</infon><infon key="name_4">surname:Bacia;given-names:Maria</infon><infon key="name_5">surname:Liu;given-names:Kaiyin</infon><infon key="name_6">surname:Chan;given-names:Sze W. S.</infon><infon key="name_7">surname:Houry;given-names:Walid A.</infon><infon key="name_8">surname:Ollagnier de Choudens;given-names:Sandrine</infon><infon key="name_9">surname:Elsen;given-names:Sylvie</infon><infon key="section_type">TITLE</infon><infon key="type">front</infon><infon key="volume">6</infon><infon key="year">2016</infon><offset>0</offset><text>Structural insights into the Escherichia coli lysine decarboxylases and molecular determinants of interaction with the AAA+ ATPase RavA</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>136</offset><text>The inducible lysine decarboxylase LdcI is an important enterobacterial acid stress response enzyme whereas LdcC is its close paralogue thought to play mainly a metabolic role. A unique macromolecular cage formed by two decamers of the Escherichia coli LdcI and five hexamers of the AAA+ ATPase RavA was shown to counteract acid stress under starvation. Previously, we proposed a pseudoatomic model of the LdcI-RavA cage based on its cryo-electron microscopy map and crystal structures of an inactive LdcI decamer and a RavA monomer. We now present cryo-electron microscopy 3D reconstructions of the E. coli LdcI and LdcC, and an improved map of the LdcI bound to the LARA domain of RavA, at pH optimal for their enzymatic activity. Comparison with each other and with available structures uncovers differences between LdcI and LdcC explaining why only the acid stress response enzyme is capable of binding RavA. We identify interdomain movements associated with the pH-dependent enzyme activation and with the RavA binding. Multiple sequence alignment coupled to a phylogenetic analysis reveals that certain enterobacteria exert evolutionary pressure on the lysine decarboxylase towards the cage-like assembly with RavA, implying that this complex may have an important function under particular stress conditions.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1452</offset><text>Enterobacterial inducible decarboxylases of basic amino acids lysine, arginine and ornithine have a common evolutionary origin and belong to the α-family of pyridoxal-5′-phosphate (PLP)-dependent enzymes. They counteract acid stress experienced by the bacterium in the host digestive and urinary tract, and in particular in the extremely acidic stomach. Each decarboxylase is induced by an excess of the target amino acid and a specific range of extracellular pH, and works in conjunction with a cognate inner membrane antiporter. Decarboxylation of the amino acid into a polyamine is catalysed by a PLP cofactor in a multistep reaction that consumes a cytoplasmic proton and produces a CO2 molecule passively diffusing out of the cell, while the polyamine is excreted by the antiporter in exchange for a new amino acid substrate. Consequently, these enzymes buffer both the bacterial cytoplasm and the local extracellular environment. These amino acid decarboxylases are therefore called acid stress inducible or biodegradative to distinguish them from their biosynthetic lysine and ornithine decarboxylase paralogs catalysing the same reaction but responsible for the polyamine production at neutral pH.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2662</offset><text>Inducible enterobacterial amino acid decarboxylases have been intensively studied since the early 1940 because the ability of bacteria to withstand acid stress can be linked to their pathogenicity in humans. In particular, the inducible lysine decarboxylase LdcI (or CadA) attracts attention due to its broad pH range of activity and its capacity to promote survival and growth of pathogenic enterobacteria such as Salmonella enterica serovar Typhimurium, Vibrio cholerae and Vibrio vulnificus under acidic conditions. Furthermore, both LdcI and the biosynthetic lysine decarboxylase LdcC of uropathogenic Escherichia coli (UPEC) appear to play an important role in increased resistance of this pathogen to nitrosative stress produced by nitric oxide and other damaging reactive nitrogen intermediates accumulating during the course of urinary tract infections (UTI). This effect is attributed to cadaverine, the diamine produced by decarboxylation of lysine by LdcI and LdcC, that was shown to enhance UPEC colonisation of the bladder. In addition, the biosynthetic E. coli lysine decarboxylase LdcC, long thought to be constitutively expressed in low amounts, was demonstrated to be strongly upregulated by fluoroquinolones via their induction of RpoS. A direct correlation between the level of cadaverine and the resistance of E. coli to these antibiotics commonly used as a first-line treatment of UTI could be established. Both acid pH and cadaverine induce closure of outer membrane porins thereby contributing to bacterial protection from acid stress, but also from certain antibiotics, by reduction in membrane permeability.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>4295</offset><text>The crystal structure of the E. coli LdcI as well as its low resolution characterisation by electron microscopy (EM) showed that it is a decamer made of two pentameric rings. Each monomer is composed of three domains – an N-terminal wing domain (residues 1–129), a PLP-binding core domain (residues 130–563), and a C-terminal domain (CTD, residues 564–715). Monomers tightly associate via their core domains into 2-fold symmetrical dimers with two complete active sites, and further build a toroidal D5-symmetrical structure held by the wing and core domain interactions around the central pore, with the CTDs at the periphery.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>4931</offset><text>Ten years ago we showed that the E. coli AAA+ ATPase RavA, involved in multiple stress response pathways, tightly interacted with LdcI but was not capable of binding to LdcC. We described how two double pentameric rings of the LdcI tightly associate with five hexameric rings of RavA to form a unique cage-like architecture that enables the bacterium to withstand acid stress even under conditions of nutrient deprivation eliciting stringent response. Furthermore, we recently solved the structure of the E. coli LdcI-RavA complex by cryo-electron microscopy (cryoEM) and combined it with the crystal structures of the individual proteins. This allowed us to make a pseudoatomic model of the whole assembly, underpinned by a cryoEM map of the LdcI-LARA complex (with LARA standing for LdcI associating domain of RavA), and to identify conformational rearrangements and specific elements essential for complex formation. The main determinants of the LdcI-RavA cage assembly appeared to be the N-terminal loop of the LARA domain of RavA and the C-terminal β-sheet of LdcI.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>6005</offset><text>In spite of this wealth of structural information, the fact that LdcC does not interact with RavA, although the two lysine decarboxylases are 69% identical and 84% similar, and the physiological significance of the absence of this interaction remained unexplored. To solve this discrepancy, in the present work we provided a three-dimensional (3D) cryoEM reconstruction of LdcC and compared it with the available LdcI and LdcI-RavA structures. Given that the LdcI crystal structures were obtained at high pH where the enzyme is inactive (LdcIi, pH 8.5), whereas the cryoEM reconstructions of LdcI-RavA and LdcI-LARA were done at acidic pH optimal for the enzymatic activity, for a meaningful comparison, we also produced a 3D reconstruction of the LdcI at active pH (LdcIa, pH 6.2). This comparison pinpointed differences between the biodegradative and the biosynthetic lysine decarboxylases and brought to light interdomain movements associated to pH-dependent enzyme activation and RavA binding, notably at the predicted RavA binding site at the level of the C-terminal β-sheet of LdcI. Consequently, we tested the capacity of cage formation by LdcI-LdcC chimeras where we interchanged the C-terminal β-sheets in question. Finally, we performed multiple sequence alignment of 22 lysine decarboxylases from Enterobacteriaceae containing the ravA-viaA operon in their genome. Remarkably, this analysis revealed that several specific residues in the above-mentioned β-sheet, independently of the rest of the protein sequence, are sufficient to define if a particular lysine decarboxylase should be classified as an “LdcC-like” or an “LdcI-like”. Moreover, this classification perfectly agrees with the genetic environment of the lysine decarboxylase genes. This fascinating parallelism between the propensity for RavA binding and the genetic environment of an enterobacterial lysine decarboxylase, as well as the high degree of conservation of this small structural motif, emphasize the functional importance of the interaction between biodegradative enterobacterial lysine decarboxylases and the AAA+ ATPase RavA.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>8130</offset><text>Results and Discussion</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>8153</offset><text>CryoEM 3D reconstructions of LdcC, LdcIa and LdcI-LARA</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>8208</offset><text>In the frame of this work, we produced two novel subnanometer resolution cryoEM reconstructions of the E. coli lysine decarboxylases at pH optimal for their enzymatic activity – a 5.5 Å resolution cryoEM map of the LdcC (pH 7.5) for which no 3D structural information has been previously available (Figs 1A,B and S1), and a 6.1 Å resolution cryoEM map of the LdcIa, (pH 6.2) (Figs 1C,D and S2). In addition, we improved our earlier cryoEM map of the LdcI-LARA complex from 7.5 Å to 6.2 Å resolution (Figs 1E,F and S3). Based on these reconstructions, reliable pseudoatomic models of the three assemblies were obtained by flexible fitting of either the crystal structure of LdcIi or a derived structural homology model of LdcC (Table S1). Significant differences between these pseudoatomic models can be interpreted as movements between specific biological states of the proteins as described below.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>9121</offset><text>The wing domains as a stable anchor at the center of the double-ring</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9190</offset><text>As a first step of a comparative analysis, we superimposed the three cryoEM reconstructions (LdcIa, LdcI-LARA and LdcC) and the crystal structure of the LdcIi decamer (Fig. 2 and Movie S1). This superposition reveals that the densities lining the central hole of the toroid are roughly at the same location, while the rest of the structure exhibits noticeable changes. Specifically, at the center of the double-ring the wing domains of the subunits provide the conserved basis for the assembly with the lowest root mean square deviation (RMSD) (between 1.4 and 2 Å for the Cα atoms only), whereas the peripheral CTDs containing the RavA binding interface manifest the highest RMSD (up to 4.2 Å) (Table S2). In addition, the wing domains of all structures are very similar, with the RMSD after optimal rigid body alignment (RMSDmin) less than 1.1 Å. Thus, taking the limited resolution of the cryoEM maps into account, we consider that the wing domains of all the four structures are essentially identical and that in the present study the RMSD of less than 2 Å can serve as a baseline below which differences may be assumed as insignificant. This preservation of the central part of the double-ring assembly may help the enzymes to maintain their decameric state upon activation and incorporation into the LdcI-RavA cage.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>10525</offset><text>The core domain and the active site rearrangements upon pH-dependent enzyme activation and LARA binding</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10629</offset><text>Both visual inspection (Fig. 2) and RMSD calculations (Table S2) show that globally the three structures at active pH (LdcIa, LdcI-LARA and LdcC) are more similar to each other than to the structure determined at high pH conditions (LdcIi). The decameric enzyme is built of five dimers associating into a 5-fold symmetrical double-ring (two monomers making a dimer are delineated in Fig. 1). As common for the α family of the PLP-dependent decarboxylases, dimerization is required for the enzymatic activity because the active site is buried in the dimer interface (Fig. 3A,B). This interface is formed essentially by the core domains with some contribution of the CTDs. The core domain is built by the PLP-binding subdomain (PLP-SD, residues 184–417) flanked by two smaller subdomains rich in partly disordered loops – the linker region (residues 130–183) and the subdomain 4 (residues 418–563). Zooming in the variations in the PLP-SD shows that most of the structural changes concern displacements in the active site (Fig. 3C–F). The most conspicuous differences between the PLP-SDs can be linked to the pH-dependent activation of the enzymes. The resolution of the cryoEM maps does not allow modeling the position of the PLP moiety and calls for caution in detailed mechanistic interpretations in terms of individual amino acids. Therefore we restrict our analysis to secondary structure elements. In particular, transition from LdcIi to LdcI-LARA involves ~3.5 Å and ~4.5 Å shifts away from the 5-fold axis in the active site α-helices spanning residues 218–232 and 246–254 respectively (Fig. 3C–E). Between these two extremes, the PLP-SDs of LdcIa and LdcC are similar both in the context of the decamer (Fig. 3F) and in terms of RMSDmin = 0.9 Å, which probably reflects the fact that, at the optimal pH, these lysine decarboxylases have a similar enzymatic activity. In addition, our earlier biochemical observation that the enzymatic activity of LdcIa is unaffected by RavA binding is consistent with the relatively small changes undergone by the active site upon transition from LdcIa to LdcI-LARA. Worthy of note, our previous comparison of the crystal structure of LdcIi with that of the inducible arginine decarboxylase AdiA revealed high conservation of the PLP-coordinating residues and identified a patch of negatively charged residues lining the active site channel as a potential binding site for the target amino acid substrate (Figs S3 and S4 in ref.).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>13132</offset><text>Rearrangements of the ppGpp binding pocket upon pH-dependent enzyme activation and LARA binding</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>13228</offset><text>An inhibitor of the LdcI and LdcC activity, the stringent response alarmone ppGpp, is known to bind at the interface between neighboring monomers within each ring (Fig. S4). The ppGpp binding pocket is made up by residues from all domains and is located approximately 30 Å away from the PLP moiety. Whereas the crystal structure of the ppGpp-LdcIi was solved to 2 Å resolution, only a 4.1 Å resolution structure of the ppGpp-free LdcIi could be obtained. At this resolution, the apo-LdcIi and ppGpp-LdcIi structures (both solved at pH 8.5) appeared indistinguishable except for the presence of ppGpp (Fig. S11 in ref. ). Thus, we speculated that inhibition of LdcI by ppGpp would be accompanied by a transduction of subtle structural changes at the level of individual amino acid side chains between the ppGpp binding pocket and the active site of the enzyme. All our current cryoEM reconstructions of the lysine decarboxylases were obtained in the absence of ppGpp in order to be closer to the active state of the enzymes under study. While differences in the ppGpp binding site could indeed be visualized (Fig. S4), the level of resolution warns against speculations about their significance. The fact that interaction with RavA reduces the ppGpp affinity for LdcI despite the long distance of ~30 Å between the LARA domain binding site and the closest ppGpp binding pocket (Fig. S5) seems to favor an allosteric regulation mechanism. Interestingly, although a number of ppGpp binding residues are strictly conserved between LdcI and AdiA that also forms decamers at low pH optimal for its arginine decarboxylase activity, no ppGpp regulation of AdiA could be demonstrated.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>14916</offset><text>Swinging and stretching of the CTDs upon pH-dependent LdcI activation and LARA binding</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15003</offset><text>Inspection of the superimposed decameric structures (Figs 2 and S6) suggests a depiction of the wing domains as an anchor around which the peripheral CTDs swing. This swinging movement seems to be mediated by the core domains and is accompanied by a stretching of the whole LdcI subunits attracted by the RavA magnets. Indeed, all CTDs have very similar structures (RMSDmin &lt;1 Å). Yet the superposition of the decamers lays bare a progressive movement of the CTD as a whole upon enzyme activation by pH and the binding of LARA. The LdcIi monomer is the most compact, whereas LdcIa and especially LdcI-LARA gradually extend their CTDs towards the LARA domain of RavA (Figs 2 and 4). These small but noticeable swinging and stretching (up to ~4 Å) may be related to the incorporation of the LdcI decamer into the LdcI-RavA cage.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>15836</offset><text>The C-terminal β-sheet of a lysine decarboxylase as a major determinant of the interaction with RavA</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15940</offset><text>In our previous contribution, based on the fit of the LdcIi and the LARA crystal structures into the LdcI-LARA cryoEM density, we predicted that the LdcI-RavA interaction should involve the C-terminal two-stranded β-sheet of the LdcI. Our present cryoEM maps and pseudoatomic models provide first structure-based insights into the differences between the inducible and the constitutive lysine decarboxylases. However, at the level of this structural element the two proteins are actually surpisingly similar. Therefore, we wanted to check the influence of the primary sequence of the two proteins in this region on their ability to interact with RavA. To this end, we swapped the relevant β-sheets of the two proteins and produced their chimeras, namely LdcIC (i.e. LdcI with the C-terminal β-sheet of LdcC) and LdcCI (i.e. LdcC with the C-terminal β-sheet of LdcI) (Fig. 5A–C). Both constructs could be purified and could form decamers visually indistinguishable from the wild-type proteins. As expected, binding of LdcI to RavA was completely abolished by this procedure and no LdcIC-RavA complex could be detected. On the contrary, introduction of the C-terminal β-sheet of LdcI into LdcC led to an assembly of the LdcCI-RavA complex. On the negative stain EM grid, the chimeric cages appeared less rigid than the native LdcI-RavA, which probably means that the environment of the β-sheet contributes to the efficiency of the interaction and the stability of the entire architecture (Fig. 5D–F).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>17457</offset><text>The C-terminal β-sheet of a lysine decarboxylase is a highly conserved signature allowing to distinguish between LdcI and LdcC</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>17587</offset><text>Alignment of the primary sequences of the E. coli LdcI and LdcC shows that some amino acid residues of the C-terminal β-sheet are the same in the two proteins, whereas others are notably different in chemical nature. Importantly, most of the amino acid differences between the two enzymes are located in this very region. Thus, to advance beyond our experimental confirmation of the C-terminal β-sheet as a major determinant of the capacity of a particular lysine decarboxylase to form a cage with RavA, we set out to investigate whether certain residues in this β-sheet are conserved in lysine decarboxylases of different enterobacteria that have the ravA-viaA operon in their genome. We inspected the genetic environment of lysine decarboxylases from 22 enterobacterial species referenced in the NCBI database, corrected the gene annotation where necessary (Tables S3 and S4), and performed multiple sequence alignment coupled to a phylogenetic analysis (see Methods). This procedure yielded several unexpected and exciting results. First of all, consensus sequence for the entire lysine decarboxylase family was derived. Second, the phylogenetic analysis clearly split the lysine decarboxylases into two groups (Fig. 6A). All lysine decarboxylases predicted to be “LdcI-like” or biodegradable based on their genetic environment, as for example their organization in an operon with a gene encoding the CadB antiporter (see Methods), were found in one group, whereas all enzymes predicted as “LdcC-like” or biosynthetic partitioned into another group. Thus, consensus sequences could also be determined for each of the two groups (Figs 6B,C and S7). Inspection of these consensus sequences revealed important differences between the groups regarding charge, size and hydrophobicity of several residues precisely at the level of the C-terminal β-sheet that is responsible for the interaction with RavA (Fig. 6B–D). For example, in our previous study, site-directed mutations identified Y697 as critically required for the RavA binding. Our current analysis shows that Y697 is strictly conserved in the “LdcI-like” group whereas the “LdcC-like” enzymes always have a lysine in this position; it also uncovers several other residues potentially essential for the interaction with RavA which can now be addressed by site-directed mutagenesis. The third and most remarkable finding was that exactly the same separation into “LdcI-like” and “LdcC”-like groups can be obtained based on a comparison of the C-terminal β-sheets only, without taking the rest of the primary sequence into account. Therefore the C-terminal β-sheet emerges as being a highly conserved signature sequence, sufficient to unambiguously discriminate between the “LdcI-like” and “LdcC-like” enterobacterial lysine decarboxylases independently of any other information (Figs 6 and S7). Our structures show that this motif is not involved in the enzymatic activity or the oligomeric state of the proteins. Thus, enterobacteria identified here (Fig. 6, Table S4) appear to exert evolutionary pressure on the biodegradative lysine decarboxylase towards the RavA binding. One of the elucidated roles of the LdcI-RavA cage is to maintain LdcI activity under conditions of enterobacterial starvation by preventing LdcI inhibition by the stringent response alarmone ppGpp. Furthermore, the recently documented interaction of both LdcI and RavA with specific subunits of the respiratory complex I, together with the unanticipated link between RavA and maturation of numerous iron-sulfur proteins, tend to suggest an additional intriguing function for this 3.5 MDa assembly. The conformational rearrangements of LdcI upon enzyme activation and RavA binding revealed in this work, and our amazing finding that the molecular determinant of the LdcI-RavA interaction is the one that straightforwardly determines if a particular enterobacterial lysine decarboxylase belongs to “LdcI-like” or “LdcC-like” proteins, should give a new impetus to functional studies of the unique LdcI-RavA cage. Besides, the structures and the pseudoatomic models of the active ppGpp-free states of both the biodegradative and the biosynthetic E. coli lysine decarboxylases offer an additional tool for analysis of their role in UPEC infectivity. Together with the apo-LdcI and ppGpp-LdcIi crystal structures, our cryoEM reconstructions provide a structural framework for future studies of structure-function relationships of lysine decarboxylases from other enterobacteria and even of their homologues outside Enterobacteriaceae. For example, the lysine decarboxylase of Eikenella corrodens is thought to play a major role in the periodontal disease and its inhibitors were shown to retard gingivitis development. Finally, cadaverine being an important platform chemical for the production of industrial polymers such as nylon, structural information is valuable for optimisation of bacterial lysine decarboxylases used for its production in biotechnology.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>22628</offset><text>Methods</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>22636</offset><text>Protein expression and purification</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>22672</offset><text>LdcI and LdcC were expressed and purified as described from an E. coli strain that cannot produce ppGpp (MG1655 ΔrelA ΔspoT strain). LdcI was stored in 20 mM Tris-HCl, 100 mM NaCl, 1 mM DTT, 0.1 mM PLP, pH 6.8 (buffer A) and LdcC in 20 mM Tris-HCl, 100 mM NaCl, 1 mM DTT, 0.1 mM PLP, pH 7.5 (buffer B).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>22993</offset><text>Chimeric LdcIC and LdcCI were constructed, expressed and purified as follows. The chimeras were designed by exchange, between LdcI and LdcC, of residues from 631 to 640 and from 697 to the C-terminus, corresponding to the regions around the two strands of the C-terminal β-sheet (Fig. 5B,C), while leaving the rest of the sequence unaltered. The synthetic ldcIC and ldcCI genes (2148 bp and 2154 bp respectively), provided within a pUC57 vector (GenScript) were subcloned into pET-TEV vector based on pET-28a (Invitrogen) containing an N-terminal TEV-cleavable 6x-His-Tag. Proteins were expressed in Rosetta 2 (DE3) cells (Novagen) in LB medium supplemented with kanamycin and chloramphenicol at 37 °C, upon induction with 0.5 mM IPTG at 18 °C. Cells were harvested by centrifugation, the pellet resuspended in a 50 mM Tris-HCl, 150 mM NaCl, pH 8 buffer supplemented with Complete EDTA free (Roche) and 0.1 mM PMSF (Sigma), and disrupted by sonication at 4 °C. After centrifugation at 75000 g at 4 °C for 20 min, the supernatant was loaded on a Ni-NTA column. The eluted protein-containing fractions were pooled and the His-Tag removed by incubation with the TEV protease at 1/100 ratio and an extensive dialysis in a 50 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, 5 mM EDTA, pH 8 buffer. After a second dialysis in a 50 mM Tris-HCl, 150 mM NaCl, pH 8 buffer supplemented with 10 mM imidazole, the sample was loaded on a Ni-NTA column in the same buffer, which allowed to separate the TEV protease and LdcCI/LdcIC. Finally, the pure proteins were obtained by size exclusion chromatography on a Superdex-S200 column in buffer A.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>24652</offset><text>LdcIa -cryoEM data collection and 3D reconstruction</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>24704</offset><text>LdcI was prepared at 2 mg/ml in a buffer containing 25 mM MES, 100 mM NaCl, 0.2 mM PLP, 1 mM DTT, pH 6.2. 3 μl of sample were applied to glow-discharged quantifoil grids 300 mesh 2/1 (Quantifoil Micro Tools GmbH, Germany), excess solution was blotted during 2.5 s with a Vitrobot (FEI) and the grid frozen in liquid ethane. Data collection was performed on a FEI Polara microscope operated at 300 kV under low dose conditions. Micrographs were recorded on Kodak SO-163 film at 59,000 magnification, with defocus ranging from 0.6 to 4.9 μm. Films were digitized on a Zeiss scanner (Photoscan) at a step size of 7 μm giving a pixel size of 1.186 Å. The contrast transfer function (CTF) for each micrograph was determined with CTFFIND3.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>25462</offset><text>Initially ~2500 particles of 256 × 256 pixels were extracted manually, binned 4 times and subjected to one round of multivariate statistical analysis and classification using IMAGIC. Representative class averages corresponding to projections in different orientations were used as input for an ab-initio 3D reconstruction by RICOserver (rico.ibs.fr/). The resulting 3D reconstruction was refined using RELION, which yielded an 18 Å resolution map. Using projections of this model as a template, particles of size 256 × 256 pixels were semi-automatically selected from all the micrographs using the Fast Projection Matching (FPM) algorithm. The resulting dataset of ~46000 particles was processed in RELION with the previous map as an initial model and with a full CTF correction after the first peak. The final map comprised 44207 particles with a resolution of 7.4 Å as per the gold-standard FSC = 0.143 criterion. It was sharpened with EMBfactor using calculated B-factor of −350 Å2 and masked with a soft mask to obtain a final map with a resolution of 6.1 Å (Fig. S3, Table S1).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>26571</offset><text>LdcC - cryoEM data collection and 3D reconstruction</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>26623</offset><text>LdcC was prepared at 2 mg/ml in a buffer containing 25 mM HEPES, 100 mM NaCl, 0.2 mM PLP, 1 mM DTT, pH 7.2. Grids were prepared and sample imaged as LdcIa. Data were processed essentially as LdcIa described above. Briefly, an initial ~20 Å resolution model was generated by angular reconstitution after manual picking of circa 3000 particles from the first micrographs, filtered to 60 Å resolution, refined with RELION and used for a semi-automatic selection with FPM. The dataset was processed in RELION with a full CTF correction to yield a final reconstruction comprising 61000 particles. The map was sharpened with Relion postprocessing, using a soft mask and automated B-factor estimation (−690 Å2), yielding a map at 5.5 Å resolution (Fig. S1, Table S1).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>27400</offset><text>LdcI-LARA - 3D reconstruction</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>27430</offset><text>In our first study, the dataset was processed in SPIDER and the CTF correction involved a simple phase-flipping. Therefore, for consistency with the present work, we revisited the dataset and processed it in RELION with a full CTF correction after the first peak. It was sharpened with EMBfactor using calculated B-factor of −350 Å2 and masked with a soft mask to obtain a final map with a resolution of 6.2 Å (Fig. S2). This reconstruction is of a slightly better quality in terms of the continuity of the internal density. Therefore we used this improved map for fitting of the atomic model and further analysis (Fig. S2, Table S1).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>28073</offset><text>Additional image processing</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>28101</offset><text>As a crosscheck, each data set was also refined either from other initial models: a “featureless donut” with approximate dimensions of the decamer, and low pass-filtered reconstructions from the two other data sets (i.e. the LdcC reconstruction was used as a model for the LdcIa and LdcI-LARA data sets, etc). All refinements converged to the same solutions independently of the starting model. Local resolution of all maps was determined with ResMap.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>28557</offset><text>LdcCI and LdcIC chimeras —negative stain EM and 2D image analysis</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>28625</offset><text>0.4 mg/ml of RavA (in a 20 mM Tris-HCl, 500 mM NaCl, 10 mM MgCl2, 1 mM DTT, 5% glycerol, pH 6.8 buffer) was mixed with 0.3 mg/ml of either LdcI, LdcC, LdcCI or LdcIC in the presence of 2 mM ADP and 10 mM MgCl2 in a buffer containing 20 mM Hepes and 150 mM NaCl at pH 7.4. After 10 minutes incubation at room temperature, 3 μl of mixture were applied to the clear side of the carbon on a carbon-mica interface and negatively stained with 2% uranyl acetate. Images were recorded with a JEOL 1200 EX II microscope at 100 kV at a nominal magnification of 15000 on a CCD camera yielding a pixel size of 4.667 Å. No complexes between RavA and LdcC or LdcIC could be observed, whereas the LdcCI-RavA preparation manifested cage-like particles similar to the previously published LdcI-RavA, but also unbound RavA and LdcCI, which implies that the affinity of RavA to the LdcCI chimera is lower than its affinity to the native LdcI. 1260 particles of 96 × 96 pixels were extracted interactively from several micrographs. 2D centering, multivariate statistical analysis and classification were performed using IMAGIC. Class-averages similar to the cage-like LdcI-RavA complex were used as references for multi-reference alignment followed by multivariate statistical analysis and classification.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>29946</offset><text>Fitting of atomic models into cryoEM maps</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>29988</offset><text>A homology model of LdcC was obtained using the atomic coordinates of the LdcI monomer (3N75) as the template in SWISS-MODEL server. The RMSD between the template and the resulting model was 0.26 Å. The LdcC model was then fitted as a rigid body into the LdcC cryoEM map using the fit-in-map module of UCSF Chimera. This rigid fit indicated movements of several parts of the protein. Therefore, the density corresponding to one LdcC monomer was extracted and flexible fitting was performed using IMODFIT at 8 Å resolution. This monomeric model was then docked into the decameric cryoEM map with URO and its graphical version VEDA that use symmetry information for fitting in Fourier space. The Cα RMSDmin between the initial model of the LdcC monomer and the final IMODFIT LdcC model is 1.2 Å. In the case of LdcIa, the density corresponding to an individual monomer was extracted and the fit performed similarly to the one described above, with the final model of the decameric particle obtained with URO and VEDA. The Cα RMSDmin between the LdcIi monomer and the final IMODFIT model is 1.4 Å. For LdcI-LARA, the density accounting for one LdcI monomer bound to a LARA domain was extracted and further separated into individual densities corresponding to LdcI and to LARA. The fit of LdcI was performed as for LdcC and LdcIa, while the crystal structure of LARA was docked into the monomeric LdcI-LARA map as a rigid body using SITUS. The resulting pseudoatomic models were used to create the final model of the LdcI-LARA decamer with URO and VEDA. The Cα RMSDmin between the LdcIi monomer and the final IMODFIT model is 1.4 Å. A brief summary of relevant parameters is provided in Table S1.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>31699</offset><text>Sequence analysis</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>31717</offset><text>Out of a non-exhaustive list of 50 species of Enterobacteriaceae (Table S3), 22 were found to contain genes annotated as ldcI or ldcC and containing the ravA-viaA operon (Table S4). An analysis using MUSCLE with default parameters showed that these genes share more than 65% identity. To verify annotation of these genes, we compared their genetic environment with that of E. coli ldcI and ldcC. Indeed, in E. coli the ldcI gene is in operon with the cadB gene encoding a lysine-cadaverine antiporter, whereas the ldcC gene is present between the accA gene (encoding an acetyl-CoA carboxylase alpha subunit carboxyltransferase) and the yaeR gene (coding for an unknown protein belonging to the family of Glyoxalase/Dioxygenase/Bleomycin resistance proteins). Compared with this genetic environment, the annotation of several ldcI and ldcC genes in enterobacteria was found to be inconsistent (Table S4). For example, several strains contain genes annotated as ldcC in the genetic background of ldcI and vice versa, as in the case of Salmonella enterica and Trabulsiella guamensi. Furthermore, the gene with an “ldcC-like” environment was found to be annotated as cadA in particular strains of Citrobacter freundii, Cronobacter sakazakii, Enterobacter cloacae subsp. Cloaca, Erwinia amylovora, Pantoea agglomerans, Rahnella aquatilis, Shigella dysenteriae, and Yersinia enterocolitica subsp. enterocolitica, whereas in Hafnia alvei, Kluyvera ascorbata, and Serratia marcescens subsp. marcescens, the gene with an “ldcI-like” environment was found to be annotated as ldcC. In addition, as far as the genetic environment is concerned, Plesiomonas appears to have two ldc genes with the organization of the E. coli ldcI (operon cadA-cadB). Consequently, we corrected for gene annotation consistent with the genetic environment and made multiple sequence alignments using version 8.0.1 of the CLC Genomics Workbench software. A phylogenetic tree was generated based on Maximum Likelihood and following the Neighbor-Joining method with the WAG protein substitution model. The reliability of the generated phylogenetic tree was assessed by bootstrap analysis. The presented unrooted phylogenetic tree shows the nodes that are reliable over 95% (Fig. 6A). Remarkably, the multiple sequence alignment and the resulting phylogenetic tree clearly grouped together all sequences annotated as ldcI on the one side, and all sequences annotated as ldcC on the other side. Thus, we conclude that all modifications in gene annotations that we introduced for the sake of consistency with the genetic environment are perfectly corroborated by the multiple sequence alignment and the phylogenetic analysis. Since the regulation of the lysine decarboxylase gene (i.e. inducible or constitutive) cannot be assessed by this analysis, we call the resulting groups “ldcI-like” and “ldcC-like” as referred to the E. coli enzymes, and make a parallel between the membership in a given group and the ability of the protein to form a cage complex with RavA.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>34762</offset><text>Additional Information</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>34785</offset><text>Accession codes: CryoEM maps and corresponding fitted atomic structures (main chain atoms) have been deposited in the Electron Microscopy Data Bank and Protein Data Bank, respectively, with accession codes EMD-3205 and 5FKZ for LdcC, EMD-3204 and 5FKX for LdcIa and EMD-3206 and 5FL2 for LdcI-LARA.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>35084</offset><text>How to cite this article: Kandiah, E. et al. 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+Evol.</infon><infon key="type">ref</infon><infon key="volume">18</infon><infon key="year">2001</infon><offset>39873</offset><text>A general empirical model of protein evolution derived from multiple protein families using a maximum-likelihood approach</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>39995</offset><text>Author Contributions E.K., H.M. and I.G. carried out EM data collection with assistance of M.B. and analyzed the data. D.C. performed cloning, multiple sequence alignment and phylogenetic analysis under the direction of S.E. and I.G., J.P. cloned and purified chimeric proteins under the direction of S.O.C., K.L. and S.W.S.C. purified LdcI, LdcC and LARA under the direction of W.A.H., I.G. conceived and directed the studies and wrote the manuscript with input from E.K.</text></passage><passage><infon key="file">srep24601-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>40468</offset><text>3D cryoEM reconstructions of LdcC, LdcI-LARA and LdcIa.</text></passage><passage><infon key="file">srep24601-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>40524</offset><text>(A,C,E) cryoEM map of the LdcC (A), LdcIa
+(C) and LdcI-LARA (E) decamers with one protomer in light
+grey. In the rest of the protomers, the wing, core and C-terminal domains
+are colored from light to dark in shades of green for LdcC (A), pink
+for LdcIa (C) and blue for LdcI in LdcI-LARA (E).
+In (E), the LARA domain density is shown in dark grey. Two monomers
+making a dimer are delineated. Scale bar 50 Å.
+(B,D,F) One protomer from the cryoEM map of the LdcC (B),
+LdcIa (D) and LdcI-LARA (F) in light grey with
+the pseudoatomic model represented as cartoons and colored as the densities
+in (A,C,E). Each domain is indicated for clarity. Scale bar
+50 Å. See also Figs S1 and S3.</text></passage><passage><infon key="file">srep24601-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>41210</offset><text>Analysis of conformational rearrangements.</text></passage><passage><infon key="file">srep24601-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>41253</offset><text>Superposition of the pseudoatomic models of LdcC, LdcI from LdcI-LARA and
+LdcIa colored as in Fig. 1, and the
+crystal structure of LdcIi in shades of yellow. Only one of the
+two rings of the double toroid is shown for clarity. The dashed circle
+indicates the central region that remains virtually unchanged between all
+the structures, while the periphery undergoes visible movements. Scale bar
+50 Å.</text></passage><passage><infon key="file">srep24601-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>41656</offset><text>Conformational rearrangements in the enzyme active site.</text></passage><passage><infon key="file">srep24601-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>41713</offset><text>(A) LdcIi crystal structure, with one ring represented as a
+grey surface and the second as a cartoon. A monomer with its PLP cofactor is
+delineated. The PLP moieties of the cartoon ring are shown in red.
+(B) The LdcIi dimer extracted from the crystal structure
+of the decamer. One monomer is colored in shades of yellow as in Figs 1 and 2, while the monomer
+related by C2 symmetry is grey. The PLP is red. The active site is boxed.
+(C–F) Close-up views of the active site. The PLP
+moiety in red is from the LdcIi crystal structure. We did not
+attempt to model it in the cryoEM maps. The dimer interface is shown as a
+dashed line and the active site α-helices mentioned in the text
+are highlighted. (C) Compares LdcIi (yellow) and
+LdcIa (pink), (D) compares LdcIa (pink) and
+LdcI-LARA (blue), and (E) compares LdcIi (yellow),
+LdcIa (pink) and LdcI-LARA (blue) simultaneously in order to
+show the progressive shift described in the text. (F) Shows the
+similarity between LdcIa and LdcC at the level of the secondary
+structure elements composing the active site. Colors are as in the other
+figures.</text></passage><passage><infon key="file">srep24601-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>42813</offset><text>Stretching of the LdcI monomer upon pH-dependent enzyme activation and LARA
+binding.</text></passage><passage><infon key="file">srep24601-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>42898</offset><text>(A–C) A slice through the pseudoatomic models of the LdcI
+monomers extracted from the superimposed decamers (Fig.
+2) The rectangle indicates the regions enlarged in
+(D–F). (A) compares LdcIi (yellow)
+and LdcIa (pink), (B) compares LdcIa (pink) and
+LdcI-LARA (blue), and (C) compares LdcIi (yellow),
+LdcIa (pink) and LdcI-LARA (blue) simultaneously in order to
+show the progressive stretching described in the text. The cryoEM density of
+the LARA domain is represented as a grey surface to show the position of the
+binding site and the direction of the movement. (D–F)
+Inserts zooming at the CTD part in proximity of the LARA binding site. Loop
+regions are removed for a clearer visual comparison. An arrow indicates a
+swinging movement.</text></passage><passage><infon key="file">srep24601-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>43641</offset><text>Analysis of the LdcIC and LdcCI chimeras.</text></passage><passage><infon key="file">srep24601-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>43683</offset><text>(A) A slice through the pseudoatomic models of the LdcIa
+(purple) and LdcC (green) monomers extracted from the superimposed decamers
+(Fig. 2). (B) The C-terminal
+β-sheet in LdcIa and LdcC enlarged from
+(A,C) Exchanged primary sequences (capital letters) and
+their immediate vicinity (lower case letters) colored as in
+(A,B), with the corresponding secondary structure elements
+and the amino acid numbering shown. (D,E) A gallery of negative stain
+EM images of (D) the wild type LdcI-RavA cage and (E) the
+LdcCI-RavA cage-like particles. (F) Some representative class
+averages of the LdcCI-RavA cage-like particles. Scale bar
+20 nm.</text></passage><passage><infon key="file">srep24601-f6.jpg</infon><infon key="id">f6</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>44318</offset><text>Sequence analysis of enterobacterial lysine decarboxylases.</text></passage><passage><infon key="file">srep24601-f6.jpg</infon><infon key="id">f6</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>44378</offset><text>(A) Maximum likelihood tree with the
+“LdcC-like” and the
+“LdcI-like” groups highlighted in green and pink,
+respectively. Only nodes with higher values than 95% are shown (500
+replicates of the original dataset, see Methods for details). Scale bar
+indicates the average number of substitutions per site. (B) Analysis
+of consensus “LdcI-like” and
+“LdcC-like” sequences around the first and second
+C-terminal β-strands. The height of the bars and the letters
+representing the amino acids reflects the degree of conservation of each
+particular position is in the alignment. Amino acids are colored according
+to a polarity color scheme with hydrophobic residues in black, hydrophilic
+in green, acidic in red and basic in blue. Numbering as in E. coli.
+(C) Signature sequences of LdcI and LdcC in the C-terminal
+β-sheet. Polarity differences are highlighted. (D)
+Position and nature of these differences at the surface of the respective
+cryoEM maps with the color code as in B. See also Fig. S7 and Tables S3 and S4.</text></passage></document></collection>

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+<collection><source>PMC</source><date>20201216</date><key>pmc.key</key><document><id>4848761</id><infon key="license">CC BY</infon><passage><infon key="alt-title">Jerome C Nwachukwu et al</infon><infon key="article-id_doi">10.15252/msb.20156701</infon><infon key="article-id_pmc">4848761</infon><infon key="article-id_pmid">27107013</infon><infon key="article-id_publisher-id">MSB156701</infon><infon key="elocation-id">864</infon><infon key="issue">4</infon><infon key="kwd">Breast cancer Chemical biology Crystal structure Nuclear receptor Signal transduction Chemical Biology Structural Biology Transcription</infon><infon key="license">This is an open access article under the terms of the Creative Commons Attribution 4.0 License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.</infon><infon key="name_0">surname:Nwachukwu;given-names:Jerome C</infon><infon key="name_1">surname:Srinivasan;given-names:Sathish</infon><infon key="name_10">surname:Carlson;given-names:Kathryn E</infon><infon key="name_11">surname:Josan;given-names:Jatinder S</infon><infon key="name_12">surname:Elemento;given-names:Olivier</infon><infon key="name_13">surname:Katzenellenbogen;given-names:John A</infon><infon key="name_14">surname:Zhou;given-names:Hai‐Bing</infon><infon key="name_15">surname:Nettles;given-names:Kendall W</infon><infon key="name_2">surname:Zheng;given-names:Yangfan</infon><infon key="name_3">surname:Wang;given-names:Song</infon><infon key="name_4">surname:Min;given-names:Jian</infon><infon key="name_5">surname:Dong;given-names:Chune</infon><infon key="name_6">surname:Liao;given-names:Zongquan</infon><infon key="name_7">surname:Nowak;given-names:Jason</infon><infon key="name_8">surname:Wright;given-names:Nicholas J</infon><infon key="name_9">surname:Houtman;given-names:René</infon><infon key="notes">
+
+. () : 864
+</infon><infon key="section_type">TITLE</infon><infon key="source">Mol Syst Biol</infon><infon key="title">Subject Categories</infon><infon key="type">front</infon><infon key="volume">12</infon><infon key="year">2016</infon><offset>0</offset><text>Predictive features of ligand‐specific signaling through the estrogen receptor</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract_title_1</infon><offset>81</offset><text>Abstract</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>90</offset><text>Some estrogen receptor‐α (ERα)‐targeted breast cancer therapies such as tamoxifen have tissue‐selective or cell‐specific activities, while others have similar activities in different cell types. To identify biophysical determinants of cell‐specific signaling and breast cancer cell proliferation, we synthesized 241 ERα ligands based on 19 chemical scaffolds, and compared ligand response using quantitative bioassays for canonical ERα activities and X‐ray crystallography. Ligands that regulate the dynamics and stability of the coactivator‐binding site in the C‐terminal ligand‐binding domain, called activation function‐2 (AF‐2), showed similar activity profiles in different cell types. Such ligands induced breast cancer cell proliferation in a manner that was predicted by the canonical recruitment of the coactivators NCOA1/2/3 and induction of the GREB1 proliferative gene. For some ligand series, a single inter‐atomic distance in the ligand‐binding domain predicted their proliferative effects. In contrast, the N‐terminal coactivator‐binding site, activation function‐1 (AF‐1), determined cell‐specific signaling induced by ligands that used alternate mechanisms to control cell proliferation. Thus, incorporating systems structural analyses with quantitative chemical biology reveals how ligands can achieve distinct allosteric signaling outcomes through ERα.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">title_1</infon><offset>1503</offset><text>Introduction</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1516</offset><text>Many drugs are small‐molecule ligands of allosteric signaling proteins, including G protein‐coupled receptors (GPCRs) and nuclear receptors such as ERα. These receptors regulate distinct phenotypic outcomes (i.e., observable characteristics of cells and tissues, such as cell proliferation or the inflammatory response) in a ligand‐dependent manner. Small‐molecule ligands control receptor activity by modulating recruitment of effector enzymes to distal regions of the receptor, relative to the ligand‐binding site. Some of these ligands achieve selectivity for a subset of tissue‐ or pathway‐specific signaling outcomes, which is called selective modulation, functional selectivity, or biased signaling, through structural mechanisms that are poorly understood (Frolik et al, 1996; Nettles &amp; Greene, 2005; Overington et al, 2006; Katritch et al, 2012; Wisler et al, 2014). For example, selective estrogen receptor modulators (SERMs) such as tamoxifen (Nolvadex®; AstraZeneca) or raloxifene (Evista®; Eli Lilly) (Fig 1A) block the ERα‐mediated proliferative effects of the native estrogen, 17β‐estradiol (E2), on breast cancer cells, but promote beneficial estrogenic effects on bone mineral density and adverse estrogenic effects such as uterine proliferation, fatty liver, or stroke (Frolik et al, 1996; Fisher et al, 1998; McDonnell et al, 2002; Jordan, 2003).</text></passage><passage><infon key="file">MSB-12-864-g002.jpg</infon><infon key="id">msb156701-fig-0001</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>2912</offset><text>Allosteric control of ERα activity</text></passage><passage><infon key="file">MSB-12-864-g002.jpg</infon><infon key="id">msb156701-fig-0001</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>2951</offset><text>Chemical structures of some common ERα ligands. BSC, basic side chain. E2‐rings are numbered A‐D. The E‐ring is the common site of attachment for BSC found in many SERMS.</text></passage><passage><infon key="file">MSB-12-864-g002.jpg</infon><infon key="id">msb156701-fig-0001</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>3129</offset><text>ERα domain organization lettered, A‐F. DBD, DNA‐binding domain; LBD, ligand‐binding domain; AF, activation function</text></passage><passage><infon key="file">MSB-12-864-g002.jpg</infon><infon key="id">msb156701-fig-0001</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>3252</offset><text>Schematic illustration of the canonical ERα signaling pathway.</text></passage><passage><infon key="file">MSB-12-864-g002.jpg</infon><infon key="id">msb156701-fig-0001</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>3319</offset><text>Linear causality model for ERα‐mediated cell proliferation.</text></passage><passage><infon key="file">MSB-12-864-g002.jpg</infon><infon key="id">msb156701-fig-0001</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>3383</offset><text>Branched causality model for ERα‐mediated cell proliferation.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3449</offset><text>ERα contains structurally conserved globular domains of the nuclear receptor superfamily, including a DNA‐binding domain (DBD) that is connected by a flexible hinge region to the ligand‐binding domain (LBD), as well as unstructured AB and F domains at its amino and carboxyl termini, respectively (Fig 1B). The LBD contains a ligand‐dependent coactivator‐binding site called activation function‐2 (AF‐2). However, the agonist activity of SERMs derives from activation function‐1 (AF‐1)—a coactivator recruitment site located in the AB domain (Berry et al, 1990; Shang &amp; Brown, 2002; Abot et al, 2013).</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>4073</offset><text>AF‐1 and AF‐2 bind distinct but overlapping sets of coregulators (Webb et al, 1998; Endoh et al, 1999; Delage‐Mourroux et al, 2000; Yi et al, 2015). AF‐2 binds the signature LxxLL motif peptides of coactivators such as NCOA1/2/3 (also known as SRC‐1/2/3). AF‐1 binds a separate surface on these coactivators (Webb et al, 1998; Yi et al, 2015). Yet, it is unknown how different ERα ligands control AF‐1 through the LBD, and whether this inter‐domain communication is required for cell‐specific signaling or anti‐proliferative responses.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>4636</offset><text>In the canonical model of the ERα signaling pathway (Fig 1C), E2‐bound ERα forms a homodimer that binds DNA at estrogen‐response elements (EREs), recruits NCOA1/2/3 (Metivier et al, 2003; Johnson &amp; O'Malley, 2012), and activates the GREB1 gene, which is required for proliferation of ERα‐positive breast cancer cells (Ghosh et al, 2000; Rae et al, 2005; Deschenes et al, 2007; Liu et al, 2012; Srinivasan et al, 2013). However, ERα‐mediated proliferative responses vary in a ligand‐dependent manner (Srinivasan et al, 2013); thus, it is not known whether this canonical model is widely applicable across diverse ERα ligands.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>5284</offset><text>Our long‐term goal is to be able to predict proliferative or anti‐proliferative activity of a ligand in different tissues from its crystal structure by identifying different structural perturbations that lead to specific signaling outcomes. The simplest response model for ligand‐specific proliferative effects is a linear causality model, where the degree of NCOA1/2/3 recruitment determines GREB1 expression, which in turn drives ligand‐specific cell proliferation (Fig 1D). Alternatively, a more complicated branched causality model could explain ligand‐specific proliferative responses (Fig 1E). In this signaling model, multiple coregulator binding events and target genes (Won Jeong et al, 2012; Nwachukwu et al, 2014), LBD conformation, nucleocytoplasmic shuttling, the occupancy and dynamics of DNA binding, and other biophysical features could contribute independently to cell proliferation (Lickwar et al, 2012).</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>6222</offset><text>To test these signaling models, we profiled a diverse library of ERα ligands using systems biology approaches to X‐ray crystallography and chemical biology (Srinivasan et al, 2013), including a series of quantitative bioassays for ERα function that were statistically robust and reproducible, based on the Z’‐statistic (Fig EV1A and B; see Materials and Methods). We also determined the structures of 76 distinct ERα LBD complexes bound to different ligand types, which allowed us to understand how diverse ligand scaffolds distort the active conformation of the ERα LBD. Our findings here indicate that specific structural perturbations can be tied to ligand‐selective domain usage and signaling patterns, thus providing a framework for structure‐based design of improved breast cancer therapeutics, and understanding the different phenotypic effects of environmental estrogens.</text></passage><passage><infon key="file">MSB-12-864-g003.jpg</infon><infon key="id">msb156701-fig-0001ev</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>7118</offset><text>High‐throughput screens for ERα ligand profiling</text></passage><passage><infon key="file">MSB-12-864-g003.jpg</infon><infon key="id">msb156701-fig-0001ev</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>7171</offset><text>Summary of ligand screening assays used to measure ER‐mediated activities. ERE, estrogen‐response element; Luc, luciferase reporter gene; M2H, mammalian 2‐hybrid; UAS, upstream‐activating sequence.</text></passage><passage><infon key="file">MSB-12-864-g003.jpg</infon><infon key="id">msb156701-fig-0001ev</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>7377</offset><text>Controls for screening assays described in panel (A), above. Error bars indicate mean ± SEM, n = 3.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>7478</offset><text>Results</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>7486</offset><text>Strength of AF‐1 signaling does not determine cell‐specific signaling</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>7560</offset><text>To compare ERα signaling induced by diverse ligand types, we synthesized and assayed a library of 241 ERα ligands containing 19 distinct molecular scaffolds. These include 15 indirect modulator series, which lack a SERM‐like side chain and modulate coactivator binding indirectly from the ligand‐binding pocket (Fig 2A–E; Dataset EV1) (Zheng et al, 2012) (Zhu et al, 2012) (Muthyala et al, 2003; Seo et al, 2006) (Srinivasan et al, 2013) (Wang et al, 2012) (Liao et al, 2014) (Min et al, 2013). We also generated four direct modulator series with side chains designed to directly dislocate h12 and thereby completely occlude the AF‐2 surface (Fig 2C and E; Dataset EV1) (Kieser et al, 2010). Ligand profiling using our quantitative bioassays revealed a wide range of ligand‐induced GREB1 expression, reporter gene activities, ERα‐coactivator interactions, and proliferative effects on MCF‐7 breast cancer cells (Figs EV1 and EV2A–J). This wide variance enabled us to probe specific features of ERα signaling using ligand class analyses, and identify signaling patterns shared by specific ligand series or scaffolds.</text></passage><passage><infon key="file">MSB-12-864-g004.jpg</infon><infon key="id">msb156701-fig-0002</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>8708</offset><text>Classes of compounds in the ERα ligand library</text></passage><passage><infon key="file">MSB-12-864-g004.jpg</infon><infon key="id">msb156701-fig-0002</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>8759</offset><text>Structure of the E2‐bound ERα LBD in complex with an NCOA2 peptide of (PDB 1GWR).</text></passage><passage><infon key="file">MSB-12-864-g004.jpg</infon><infon key="id">msb156701-fig-0002</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>8845</offset><text>Structural details of the ERα LBD bound to the indicated ligands. Unlike E2 (PDB 1GWR), TAM is a direct modulator with a BSC that dislocates h12 to block the NCOA2‐binding site (PDB 3ERT). OBHS is an indirect modulator that dislocates the h11 C‐terminus to destabilize the h11–h12 interface (PDB 4ZN9).</text></passage><passage><infon key="file">MSB-12-864-g004.jpg</infon><infon key="id">msb156701-fig-0002</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>9155</offset><text>The ERα ligand library contains 241 ligands representing 15 indirect modulator scaffolds, plus 4 direct modulator scaffolds. The number of compounds per scaffold is shown in parentheses (see Dataset EV1 for individual compound information and Appendix Supplementary Methods for synthetic protocols).</text></passage><passage><infon key="file">MSB-12-864-g005.jpg</infon><infon key="id">msb156701-fig-0002ev</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>9459</offset><text>ERα ligands induced a range of agonist activity profiles</text></passage><passage><infon key="file">MSB-12-864-g005.jpg</infon><infon key="id">msb156701-fig-0002ev</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>9520</offset><text>Screening data from individual ligands are shown, grouped by scaffold. Each data point represents the activity of a distinct compound. Error bars indicate the class average (mean) ± range. *Direct modulator.</text></passage><passage><infon key="file">MSB-12-864-g005.jpg</infon><infon key="id">msb156701-fig-0002ev</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>9731</offset><text>
+Source data are available online for this figure.
+</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9783</offset><text>We first asked whether direct modulation of the receptor with an extended side chain is required for cell‐specific signaling. To this end, we compared the average ligand‐induced GREB1 mRNA levels in MCF‐7 cells and 3×ERE‐Luc reporter gene activity in Ishikawa endometrial cancer cells (E‐Luc) or in HepG2 cells transfected with wild‐type ERα (L‐Luc ERα‐WT) (Figs 3A and EV2A–C). Direct modulators showed significant differences in average activity between cell types except OBHS‐ASC analogs, which had similar low agonist activities in the three cell types. The other direct modulators had low agonist activity in Ishikawa cells, no or inverse agonist activity in MCF‐7 cells, and more variable activity in HepG2 liver cells. While it was known that direct modulators such as tamoxifen drive cell‐specific signaling, these experiments reveal that indirect modulators also drive cell‐specific signaling, since eight of fourteen classes showed significant differences in average activity (Figs 3A and EV2A–C).</text></passage><passage><infon key="file">MSB-12-864-g006.jpg</infon><infon key="id">msb156701-fig-0003</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>10824</offset><text>Ligand‐specific signaling underlies ERα‐mediated cell proliferation</text></passage><passage><infon key="file">MSB-12-864-g006.jpg</infon><infon key="id">msb156701-fig-0003</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>10897</offset><text>(A) Ligand‐specific ERα activities in HepG2, Ishikawa and MCF‐7 cells. The ligand‐induced L‐Luc ERα‐WT and E‐Luc activities and GREB1 mRNA levels are shown by scaffold (mean + SD). (B) Ligand class analysis of the L‐Luc ERα‐WT and ERα‐ΔAB activities in HepG2 cells. Significant sensitivity to AB domain deletion was determined by Student's t‐test (n = number of ligands per scaffold in Fig 2). The average activities of ligands classes are shown (mean + SEM).</text></passage><passage><infon key="file">MSB-12-864-g006.jpg</infon><infon key="id">msb156701-fig-0003</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>11389</offset><text>Correlation and regression analyses in a large test set. The r
+2 values are plotted as a heat map. In cluster 1, the first three comparisons (rows) showed significant positive correlations (F‐test for nonzero slope, P ≤ 0.05). In cluster 2, only one of these comparisons revealed a significant positive correlation, while none was significant in cluster 3. +, statistically significant correlations gained by deletion of the AB or F domains. −, significant correlations lost upon deletion of AB or F domains.</text></passage><passage><infon key="file">MSB-12-864-g006.jpg</infon><infon key="id">msb156701-fig-0003</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>11906</offset><text>
+Source data are available online for this figure.
+</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>11958</offset><text>Tamoxifen depends on AF‐1 for its cell‐specific activity (Sakamoto et al, 2002); therefore, we asked whether cell‐specific signaling observed here is due to a similar dependence on AF‐1 for activity (Fig EV1). To test this idea, we compared the average L‐Luc activities of each scaffold in HepG2 cells co‐transfected with wild‐type ERα or with ERα lacking the AB domain (Figs 1B and EV1). While E2 showed similar L‐Luc ERα‐WT and ERα‐ΔAB activities, tamoxifen showed complete loss of activity without the AB domain (Fig EV1B). Deletion of the AB domain significantly reduced the average L‐Luc activities of 14 scaffolds (Student's t‐test, P ≤ 0.05) (Fig 3B). These “AF‐1‐sensitive” activities were exhibited by both direct and indirect modulators, and were not limited to scaffolds that showed cell‐specific signaling (Fig 3A and B). Thus, the strength of AF‐1 signaling does not determine cell‐specific signaling.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>12926</offset><text>Identifying cell‐specific signaling clusters in ERα ligand classes</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>12997</offset><text>As another approach to identifying cell‐specific signaling, we determined the degree of correlation between ligand‐induced activities in the different cell types. Here, we compared ligands within each class (Fig 3C), instead of comparing average activities (Fig 3A and B). For each ligand class or scaffold, we calculated the Pearson's correlation coefficient, r, for pairwise comparison of activity profiles in breast (GREB1), liver (L‐Luc), and endometrial cells (E‐Luc). The value of r ranges from −1 to 1, and it defines the extent to which the data fit a straight line when compounds show similar agonist/antagonist activity profiles between cell types (Fig EV3A). We also calculated the coefficient of determination, r 2, which describes the percentage of variance in a dependent variable such as proliferation that can be predicted by an independent variable such as GREB1 expression. We present both calculations as r 2 to readily compare signaling specificities using a heat map on which the red–yellow palette indicates significant positive correlations (P ≤ 0.05, F‐test for nonzero slope), while the blue palette denotes negative correlations (Fig 3C–F).</text></passage><passage><infon key="file">MSB-12-864-g007.jpg</infon><infon key="id">msb156701-fig-0003ev</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>14188</offset><text>The side chain of OBHS‐BSC analogs induces cell‐specific signaling</text></passage><passage><infon key="file">MSB-12-864-g007.jpg</infon><infon key="id">msb156701-fig-0003ev</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>14259</offset><text>Correlation analysis of OBHS versus OBHS‐BSC activity across cell types.</text></passage><passage><infon key="file">MSB-12-864-g007.jpg</infon><infon key="id">msb156701-fig-0003ev</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>14334</offset><text>Correlation analysis of L‐Luc ERα‐ΔAB activity versus endogenous ERα activity of OBHS analogs. In panel (D), L‐Luc ERα‐WT activity from panel (B) is shown for comparison.</text></passage><passage><infon key="file">MSB-12-864-g007.jpg</infon><infon key="id">msb156701-fig-0003ev</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>14521</offset><text>Correlation analysis of L‐Luc ERα‐ΔF activity versus endogenous ERα activities of OBHS analogs.</text></passage><passage><infon key="file">MSB-12-864-g007.jpg</infon><infon key="id">msb156701-fig-0003ev</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>14629</offset><text>Correlation analysis of MCF‐7 cell proliferation versus NCOA2/3 recruitment or GREB1 levels observed in response to (G) OBHS‐N and (H) OBHS‐BSC analogs.</text></passage><passage><infon key="file">MSB-12-864-g007.jpg</infon><infon key="id">msb156701-fig-0003ev</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>14788</offset><text>
+Data information: In each panel, a data point indicates the activity of a distinct compound.Source data are available online for this figure.
+</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>14932</offset><text>This analysis revealed diverse signaling specificities that we grouped into three clusters. Scaffolds in cluster 1 exhibited strongly correlated GREB1 levels, E‐Luc and L‐Luc activity profiles across the three cell types (Fig 3C lanes 1–4), suggesting these ligands use similar ERα signaling pathways in the breast, endometrial, and liver cell types. This cluster includes WAY‐C, OBHS, OBHS‐N, and triaryl‐ethylene analogs, all of which are indirect modulators. Cluster 2 contains scaffolds with activities that were positively correlated in only two of the three cell types, indicating cell‐specific signaling (Fig 3C lanes 5–12). This cluster includes two classes of direct modulators (cyclofenil‐ASC and WAY dimer), and six classes of indirect modulators (2,5‐DTP, 3,4‐DTP, S‐OBHS‐2 and S‐OBHS‐3, furan, and WAY‐D). In this cluster, the correlated activities varied by scaffold. For example, 3,4‐DTP, furan, and S‐OBHS‐2 drove positively correlated GREB1 levels and E‐Luc but not L‐Luc ERα‐WT activity (Fig 3C lanes 5–7). In contrast, WAY dimer and WAY‐D analogs drove positively correlated GREB1 levels and L‐Luc ERα‐WT but not E‐Luc activity (Fig 3C lanes 8 and 9). The last set of scaffolds, cluster 3, displayed cell‐specific activities that were not correlated in any of the three cell types (Fig 3C lanes 13–19). This cluster includes two direct modulator scaffolds (OBHS‐ASC and OBHS‐BSC), and five indirect modulator scaffolds (A‐CD, cyclofenil, 3,4‐DTPD, imine, and imidazopyridine).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>16503</offset><text>These results suggest that addition of an extended side chain to an ERα ligand scaffold is sufficient to induce cell‐specific signaling, where the relative activity profiles of the individual ligands change between cell types. This is demonstrated by directly comparing the signaling specificities of matched OBHS (indirect modulator, cluster 1) and OBHS‐BSC analogs (direct modulator, cluster 3), which differ only in the basic side chain (Fig 2E). The activities of OBHS analogs were positively correlated across the three cell types, but the side chain of OBHS‐BSC analogs was sufficient to abolish these correlations (Figs 3C lanes 1 and 19, and EV3A–C).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>17173</offset><text>The indirect modulator scaffolds in clusters 2 and 3 showed cell‐specific signaling patterns without the extended side chain typically viewed as the primary chemical and structural mechanism driving cell‐specific activity. Many of these scaffolds drove similar average activities of the ligand class in the different cell types (Fig 3A), but the individual ligands in each class had different cell‐specific activities (Fig EV2A–C). Thus, examining the correlated patterns of ERα activity within each scaffold demonstrates that an extended side chain is not required for cell‐specific signaling.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>17781</offset><text>Modulation of signaling specificity by AF‐1</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>17827</offset><text>To evaluate the role of AF‐1 and the F domain in ERα signaling specificity, we compared activity of truncated ERα constructs in HepG2 liver cells with endogenous ERα activity in the other cell types. The positive correlation between the L‐Luc and E‐Luc activities or GREB1 levels induced by scaffolds in cluster 1 was generally retained without the AB domain, or the F domain (Fig 3D lanes 1–4). This demonstrates that the signaling specificities underlying these positive correlations are not modified by AF‐1. OBHS analogs showed an average L‐Luc ERα‐ΔAB activity of 3.2% ± 3 (mean + SEM) relative to E2. Despite this nearly complete lack of activity, the pattern of L‐Luc ERα‐ΔAB activity was still highly correlated with the E‐Luc activity and GREB1 expression (Fig EV3D and E), demonstrating that very small AF‐2 activities can be amplified by AF‐1 to produce robust signals. Similarly, deletion of the F domain did not abolish correlations between the L‐Luc and E‐Luc or GREB1 levels induced by OBHS analogs (Fig EV3F). These similar patterns of ligand activity in the wild‐type and deletion mutants suggest that AF‐1 and the F domain purely amplify the AF‐2 activities of ligands in cluster 1.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>19077</offset><text>In contrast, AF‐1 was a determinant of signaling specificity for scaffolds in cluster 2. Deletion of the AB or F domain altered correlations for six of the eight scaffolds in this cluster (2,5‐DTP, 3,4‐DTP, S‐OBHS‐3, WAY‐D, WAY dimer, and cyclofenil‐ASC) (Fig 3D lanes 5–12). Comparing Fig 3C and D, the + and − signs indicate where the deletion mutant assays led to a gain or loss of statically significant correlation, respectively. Thus, in cluster 2, AF‐1 substantially modulated the specificity of ligands with cell‐specific activity (Fig 3D lanes 5–12). For ligands in cluster 3, we could not eliminate a role for AF‐1 in determining signaling specificity, since this cluster lacked positively correlated activity profiles (Fig 3C), and deletion of the AB or F domain rarely induced such correlations (Fig 3D), except for A‐CD and OBHS‐ASC analogs, where deletion of the AB domain or F domain led to positive correlations with E‐Luc activity and/or GREB1 levels (Fig 3D lanes 13 and 18). Thus, ligands in cluster 2 rely on AF‐1 for both activity (Fig 3B) and signaling specificity (Fig 3D). As discussed below, this cell specificity derives from alternate coactivator preferences.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>20306</offset><text>Ligand‐specific control of GREB1 expression</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>20352</offset><text>To determine whether ligand classes control expression of native ERα target genes through the canonical linear signaling pathway, we performed pairwise linear regression analyses using ERα–NCOA1/2/3 interactions in M2H assay as independent predictors of GREB1 expression (the dependent variable) (Figs EV1 and EV2A, F–H). In cluster 1, the recruitment of NCOA1 and NCOA2 was highest for WAY‐C, followed by triaryl‐ethylene, OBHS‐N, and OBHS series, while for NCOA3, OBHS‐N compounds induced the most recruitment and OBHS ligands were inverse agonists (Fig EV2F–H). The average induction of GREB1 by cluster 1 ligands showed greater variance, with a range between ~25 and ~75% for OBHS and a range from full agonist to inverse agonist for the others in cluster 1 (Fig EV2A). GREB1 levels induced by OBHS analogs were determined by recruitment of NCOA1 but not NCOA2/3 (Fig 3E lane 1), suggesting that there may be alternate or preferential use of these coactivators by different classes. However, in cluster 1, NCOA1/2/3 recruitment generally predicted GREB1 levels (Fig 3E lanes 1–4), consistent with the canonical signaling model (Fig 1D).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>21517</offset><text>For clusters 2 and 3, GREB1 activity was generally not predicted by NCOA1/2/3 recruitment. Direct modulators showed low NCOA1/2/3 recruitment (Fig EV2F–H), but only OBHS‐ASC analogs had NCOA2 recruitment profiles that predicted a full range of effects on GREB1 levels (Figs 3E lanes 9, 11, 18–19, and EV2A). The indirect modulators in clusters 2 and 3 stimulated NCOA1/2/3 recruitment and GREB1 expression with substantial variance (Figs 3A and EV2F–H). However, ligand‐induced GREB1 levels were generally not determined by NCOA1/2/3 recruitment (Fig 3E lanes 5–19), consistent with an alternate causality model (Fig 1E). Out of 11 indirect modulator series in cluster 2 or 3, only the S‐OBHS‐3 class had NCOA1/2/3 recruitment profiles that predicted GREB1 levels (Fig 3E lane 12). These results suggest that compounds that show cell‐specific signaling do not activate GREB1, or use coactivators other than NCOA1/2/3 to control GREB1 expression (Fig 1E).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>22497</offset><text>Ligand‐specific control of cell proliferation</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>22545</offset><text>To determine mechanisms for ligand‐dependent control of breast cancer cell proliferation, we performed linear regression analyses across the 19 scaffolds using MCF‐7 cell proliferation as the dependent variable, and the other activities as independent variables (Fig 3F). In cluster 1, E‐Luc and L‐Luc activities, NCOA1/2/3 recruitment, and GREB1 levels generally predicted the proliferative response (Fig 3F lanes 2–4). With the OBHS‐N compounds, NCOA3 and GREB1 showed near perfect prediction of proliferation (Fig EV3G), with unexplained variance similar to the noise in the assays. The lack of significant predictors for OBHS analogs (Fig 3F lane 1) reflects their small range of proliferative effects on MCF‐7 cells (Fig EV2I). The significant correlations with GREB1 expression and NCOA1/2/3 recruitment observed in this cluster are consistent with the canonical signaling model (Fig 1D), where NCOA1/2/3 recruitment determines GREB1 expression, which then drives proliferation.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>23550</offset><text>Ligands in cluster 2 and cluster 3 showed a wide range of proliferative effects on MCF‐7 cells (Fig EV2I). Despite this phenotypic variance, proliferation was not generally predicted by correlated NCOA1/2/3 recruitment and GREB1 induction (Figs 3F lanes 5–19, and EV3H). Out of 15 ligand series in these clusters, only 2,5‐DTP analogs induced a proliferative response that was predicted by GREB1 levels, which were not determined by NCOA1/2/3 recruitment (Fig 3E and F lane 10). 3,4‐DTP, cyclofenil, 3,4‐DTPD, and imidazopyridine analogs had NCOA1/3 recruitment profiles that predicted their proliferative effects, without determining GREB1 levels (Fig 3E and F, lanes 5 and 14–16). Similarly, S‐OBHS‐3, cyclofenil‐ASC, and OBHS‐ASC had positively correlated NCOA1/2/3 recruitment and GREB1 levels, but none of these activities determined their proliferative effects (Fig 3E and F lanes 11–12 and 18). For ligands that show cell‐specific signaling, ERα‐mediated recruitment of other coregulators and activation of other target genes likely determine their proliferative effects on MCF‐7 cells.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>24678</offset><text>NCOA3 occupancy at GREB1 did not predict the proliferative response</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>24746</offset><text>We also questioned whether promoter occupancy by coactivators is statistically robust and reproducible for ligand class analysis using a chromatin immunoprecipitation (ChIP)‐based quantitative assay, and whether it has a better predictive power than the M2H assay. ERα and NCOA3 cycle on and off the GREB1 promoter (Nwachukwu et al, 2014). Therefore, we first performed a time‐course study, and found that E2 and the WAY‐C analog, AAPII‐151‐4, induced recruitment of NCOA3 to the GREB1 promoter in a temporal cycle that peaked after 45 min in MCF‐7 cells (Fig 4A). At this time point, other WAY‐C analogs also induced recruitment of NCOA3 at this site to varying degrees (Fig 4B). The Z’ for this assay was 0.6, showing statistical robustness (see Materials and Methods). We prepared biological replicates with different cell passage numbers and separately prepared samples, which showed r 2 of 0.81, demonstrating high reproducibility (Fig 4C).</text></passage><passage><infon key="file">MSB-12-864-g008.jpg</infon><infon key="id">msb156701-fig-0004</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>25714</offset><text>
+NCOA3 occupancy at GREB1 is statistically robust but does not predict transcriptional activity</text></passage><passage><infon key="file">MSB-12-864-g008.jpg</infon><infon key="id">msb156701-fig-0004</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>25810</offset><text>Kinetic ChIP assay examining recruitment of NCOA3 to the GREB1 gene in MCF‐7 cells stimulated with E2 or the indicated WAY‐C analog. The average of duplicate experiments (mean ± SEM) is shown.</text></passage><passage><infon key="file">MSB-12-864-g008.jpg</infon><infon key="id">msb156701-fig-0004</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>26010</offset><text>NCOA3 occupancy at GREB1 was compared by ChIP assay 45 min after stimulation with vehicle, E2, or the WAY‐C analogs. In panel (B), the average recruitment of two biological replicates are shown as mean + SEM, and the Z‐score is indicated. In panel (C), correlation analysis was performed for two biological replicates.</text></passage><passage><infon key="file">MSB-12-864-g008.jpg</infon><infon key="id">msb156701-fig-0004</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>26336</offset><text>Linear regression analyses comparing the ability of NCOA3 recruitment, measured by ChIP or M2H, to predict other agonist activities of WAY‐C analogs. *Significant positive correlation (F‐test for nonzero slope, P‐value).</text></passage><passage><infon key="file">MSB-12-864-g008.jpg</infon><infon key="id">msb156701-fig-0004</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>26563</offset><text>
+Source data are available online for this figure.
+</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>26615</offset><text>The M2H assay for NCOA3 recruitment broadly correlated with the other assays, and was predictive for GREB1 expression and cell proliferation (Fig 3E). However, the ChIP assays for WAY‐C‐induced recruitment of NCOA3 to the GREB1 promoter did not correlate with any of the other WAY‐C activity profiles (Fig 4D), although the positive correlation between ChIP assays and NCOA3 recruitment via M2H assay showed a trend toward significance with r 2 = 0.36 and P = 0.09 (F‐test for nonzero slope). Thus, the simplified coactivator‐binding assay showed much greater predictive power than the ChIP assay for ligand‐specific effects on GREB1 expression and cell proliferation.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>27302</offset><text>ERβ activity is not an independent predictor of cell‐specific activity</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>27376</offset><text>One difference between MCF‐7 breast cancer cells and Ishikawa endometrial cancer cells is the contribution of ERβ to estrogenic response, as Ishikawa cells may express ERβ (Bhat &amp; Pezzuto, 2001). When overexpressed in MCF‐7 cells, ERβ alters E2‐induced expression of only a subset of ERα‐target genes (Wu et al, 2011), raising the possibility that ligand‐induced ERβ activity may contribute to E‐Luc activities, and thus underlie the lack of correlation between the E‐Luc and L‐Luc ERα‐WT activities or GREB1 levels induced by cell‐specific modulators in cluster 2 and cluster 3 (Fig 3C).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>27993</offset><text>To test this idea, we determined the L‐Luc ERβ activity profiles of the ligands (Fig EV1). All direct modulator and two indirect modulator scaffolds (OBHS and S‐OBHS‐3) lacked ERβ agonist activity. However, the other ligands showed a range of ERβ activities (Fig EV2J). For most scaffolds, L‐Luc ERβ and E‐Luc activities were not correlated, except for 2,5‐DTP and cyclofenil analogs, which showed moderate but significant correlations (Fig EV4A). Nevertheless, the E‐Luc activities of both 2,5‐DTP and cyclofenil analogs were better predicted by their L‐Luc ERα‐WT than L‐Luc ERβ activities (Fig EV4A and B). Thus, ERβ activity was not an independent determinant of the observed activity profiles.</text></passage><passage><infon key="file">MSB-12-864-g009.jpg</infon><infon key="id">msb156701-fig-0004ev</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>28725</offset><text>ERβ activity is not an independent predictor of E‐Luc activity</text></passage><passage><infon key="file">MSB-12-864-g009.jpg</infon><infon key="id">msb156701-fig-0004ev</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>28791</offset><text>ERβ activity in HepG2 cells rarely correlates with E‐Luc activity.</text></passage><passage><infon key="file">MSB-12-864-g009.jpg</infon><infon key="id">msb156701-fig-0004ev</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>28861</offset><text>ERα activity of 2,5‐DTP and cyclofenil analogs correlates with E‐Luc activity.</text></passage><passage><infon key="file">MSB-12-864-g009.jpg</infon><infon key="id">msb156701-fig-0004ev</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>28945</offset><text>
+Data information: The r
+2 and P values for the indicated correlations are shown in both panels. *Significant positive correlation (F‐test for nonzero slope, P‐value)</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>29116</offset><text>Structural features of consistent signaling across cell types</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>29178</offset><text>To overcome barriers to crystallization of ERα LBD complexes, we developed a conformation‐trapping X‐ray crystallography approach using the ERα‐Y537S mutation (Nettles et al, 2008; Bruning et al, 2010; Srinivasan et al, 2013). To further validate this approach, we solved the structure of the ERα‐Y537S LBD in complex with diethylstilbestrol (DES), which bound identically in the wild‐type and ERα‐Y537S LBDs, demonstrating again that this surface mutation stabilizes h12 dynamics to facilitate crystallization without changing ligand binding (Appendix Fig S1A and B) (Nettles et al, 2008; Bruning et al, 2010; Delfosse et al, 2012). Using this approach, we solved 76 ERα LBD structures in the active conformation and bound to ligands studied here (Appendix Fig S1C). Eleven of these structures have been published, while 65 are new, including the DES‐bound ERα‐Y537S LBD. We present 57 of these new structures here (Dataset EV2), while the remaining eight new structures bound to OBHS‐N analogs will be published elsewhere (S. Srinivasan et al, in preparation). Examining many closely related structures allows us to visualize subtle structural differences, in effect using X‐ray crystallography as a systems biology tool.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>30436</offset><text>The indirect modulator scaffolds in cluster 1 did not show cell‐specific signaling (Fig 3C), but shared common structural perturbations that we designed to modulate h12 dynamics. Based on our original OBHS structure, the OBHS, OBHS‐N, and triaryl‐ethylene compounds were modified with h11‐directed pendant groups (Zheng et al, 2012; Zhu et al, 2012; Liao et al, 2014). Superposing the LBDs based on the class of bound ligands provides an ensemble view of the structural variance and clarifies what part of the ligand‐binding pocket is differentially perturbed or targeted.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>31023</offset><text>The 24 structures containing OBHS, OBHS‐N, or triaryl‐ethylene analogs showed structural diversity in the same part of the scaffolds (Figs 5A and EV5A), and the same region of the LBD—the C‐terminal end of h11 (Figs 5B and C, and EV5B), which in turn nudges h12 (Fig 5C and D). We observed that the OBHS‐N analogs displaced h11 along a vector away from Leu354 in a region of h3 that is unaffected by the ligands, and toward the dimer interface. For the triaryl‐ethylene analogs, the displacement of h11 was in a perpendicular direction, away from Ile424 in h8 and toward h12. Remarkably, these individual inter‐atomic distances showed a ligand class‐specific ability to significantly predict proliferative effects (Fig 5E and F), demonstrating the feasibility of developing a minimal set of activity predictors from crystal structures.</text></passage><passage><infon key="file">MSB-12-864-g010.jpg</infon><infon key="id">msb156701-fig-0005</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>31879</offset><text>Structural determinants of consistent signaling</text></passage><passage><infon key="file">MSB-12-864-g010.jpg</infon><infon key="id">msb156701-fig-0005</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>31927</offset><text>Structure‐class analysis of triaryl‐ethylene analogs. Triaryl‐ethylene analogs bound to the superposed crystal structures of the ERα LBD are shown. Arrows indicate chemical variance in the orientation of the different h11‐directed ligand side groups (PDB 5DK9, 5DKB, 5DKE, 5DKG, 5DKS, 5DL4, 5DLR, 5DMC, 5DMF and 5DP0).</text></passage><passage><infon key="file">MSB-12-864-g010.jpg</infon><infon key="id">msb156701-fig-0005</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>32255</offset><text>Triaryl‐ethylene analogs induce variance of ERα conformations at the C‐terminal region of h11. Panel (B) shows the crystal structure of a triaryl‐ethylene analog‐bound ERα LBD (PDB 5DLR). The h11–h12 interface (circled) includes the C‐terminal part of h11. This region was expanded in panel (C), where the 10 triaryl‐ethylene analog‐bound ERα LBD structures (see Datasets EV1 and EV2) were superposed to show variations in the h11 C‐terminus (PDB 5DK9, 5DKB, 5DKE, 5DKG, 5DKS, 5DL4, 5DLR, 5DMC, 5DMF, and 5DP0).</text></passage><passage><infon key="file">MSB-12-864-g010.jpg</infon><infon key="id">msb156701-fig-0005</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>32789</offset><text>ERα LBDs in complex with diethylstilbestrol (DES) or a triaryl‐ethylene analog were superposed to show that the ligand‐induced difference in h11 conformation is transmitted to the C‐terminus of h12 (PDB 4ZN7, 5DMC).</text></passage><passage><infon key="file">MSB-12-864-g010.jpg</infon><infon key="id">msb156701-fig-0005</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>33012</offset><text>Inter‐atomic distances predict the proliferative effects of specific ligand series. Ile424–His524 distance measured in the crystal structures correlates with the proliferative effect of triaryl‐ethylene analogs in MCF‐7 cells. In contrast, the Leu354–Leu525 distance correlates with the proliferative effects of OBHS‐N analogs in MCF‐7 cells.</text></passage><passage><infon key="file">MSB-12-864-g010.jpg</infon><infon key="id">msb156701-fig-0005</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>33369</offset><text>Structure‐class analysis of WAY‐C analogs. WAY‐C side groups subtly nudge h12 Leu540. ERα LBD structures bound to 4 distinct WAY‐C analogs were superposed (PDB 4 IU7, 4IV4, 4IVW, 4IW6) (see Datasets EV1 and EV2).</text></passage><passage><infon key="file">MSB-12-864-g010.jpg</infon><infon key="id">msb156701-fig-0005</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>33592</offset><text>
+Source data are available online for this figure.
+</text></passage><passage><infon key="file">MSB-12-864-g011.jpg</infon><infon key="id">msb156701-fig-0005ev</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>33644</offset><text>Structure‐class analysis of indirect modulators</text></passage><passage><infon key="file">MSB-12-864-g011.jpg</infon><infon key="id">msb156701-fig-0005ev</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>33694</offset><text>Structure‐class analysis of indirect modulators in cluster 1. Crystal structures of the ERα LBD bound to OBHS and OBHS‐N analogs were superposed. The bound ligands are shown in panel (A). Arrows indicate chemical variance in the orientation of the different h11‐directed ligand side groups. Panel (B) shows the ligand‐induced conformational variation at the C‐terminal region of h11 (OBHS: PDB 4ZN9, 4ZNH, 4ZNS, 4ZNT, 4ZNU, 4ZNV, and 4ZNW; OBHS‐N: PDB 4ZUB, 4ZUC, 4ZWH, 4ZWK, 5BNU, 5BP6, 5BPR, and 5BQ4).</text></passage><passage><infon key="file">MSB-12-864-g011.jpg</infon><infon key="id">msb156701-fig-0005ev</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>34213</offset><text>Structure‐class analysis of indirect modulators in clusters 2 and 3. Crystal structures of the ERα LBD bound to ligands with cell‐specific activities were superposed. The bound ligands are shown, and arrows indicate considerable variation in the orientation of the different h3‐, h8‐, h11‐, or h12‐directed ligand side groups.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>34553</offset><text>As visualized in four LBD structures (Srinivasan et al, 2013), WAY‐C analogs were designed with small substitutions that slightly nudge h12 Leu540, without exiting the ligand‐binding pocket (Fig 5G and H). Therefore, changing h12 dynamics maintains the canonical signaling pathway defined by E2 (Fig 1D) to support AF‐2‐driven signaling and recruit NCOA1/2/3 for GREB1‐stimulated proliferation.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>34961</offset><text>Ligands with cell‐specific activity alter the shape of the AF‐2 surface</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>35037</offset><text>Direct modulators like tamoxifen drive AF‐1‐dependent cell‐specific activity by completely occluding AF‐2, but it is not known how indirect modulators produce cell‐specific ERα activity. Therefore, we examined another 50 LBD structures containing ligands in clusters 2 and 3. These structures demonstrated that cell‐specific activity derived from altering the shape of the AF‐2 surface without an extended side chain.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>35470</offset><text>Ligands in cluster 2 and cluster 3 showed conformational heterogeneity in parts of the scaffold that were directed toward multiple regions of the receptor including h3, h8, h11, h12, and/or the β‐sheets (Fig EV5C–G). For instance, S‐OBHS‐2 and S‐OBHS‐3 analogs (Fig 2) had similar ERα activity profiles in the different cell types (Fig EV2A–C), but the 2‐ versus 3‐methyl substituted phenol rings altered the correlated signaling patterns in different cell types (Fig 3B lanes 7 and 12). Structurally, the 2‐ versus 3‐methyl substitutions changed the binding position of the A‐ and E‐ring phenols by 1.0 Å and 2.2 Å, respectively (Fig EV5C). This difference in ligand positioning altered the AF‐2 surface via a shift in the N‐terminus of h12, which directly contacts the coactivator. This effect is evident in a single structure due to its 1 Å magnitude (Fig 6A and B). The shifts in h12 residues Asp538 and Leu539 led to rotation of the coactivator peptide (Fig 6C). Thus, cell‐specific activity can stem from perturbation of the AF‐2 surface without an extended side chain, which presumably alters the receptor–coregulator interaction profile.</text></passage><passage><infon key="file">MSB-12-864-g012.jpg</infon><infon key="id">msb156701-fig-0006</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>36665</offset><text>Structural correlates of cell‐specific signaling</text></passage><passage><infon key="file">MSB-12-864-g012.jpg</infon><infon key="id">msb156701-fig-0006</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>36716</offset><text>S‐OBHS‐2/3 analogs subtly distort the AF‐2 surface. Panel (A) shows the crystal structure of an S‐OBHS‐3‐bound ERα LBD (PDB 5DUH). The h3–h12 interface (circled) at AF‐2 (pink) was expanded in panels (B, C). The S‐OBHS‐2/3‐bound ERα LBDs were superposed to show shifts in h3 (panel B) and the NCOA2 peptide docked at the AF‐2 surface (panel C).</text></passage><passage><infon key="file">MSB-12-864-g012.jpg</infon><infon key="id">msb156701-fig-0006</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>37088</offset><text>Crystal structures show that 2,5‐DTP analogs shift h3 and h11 further apart compared to an A‐CD‐ring estrogen (PDB 4PPS, 5DRM, 5DRJ). The 2F
+o‐F
+c electron density map and F
+o‐F
+c difference map of a 2,5‐DTP‐bound structure (PDB 5DRJ) were contoured at 1.0 sigma and ± 3.0 sigma, respectively.</text></passage><passage><infon key="file">MSB-12-864-g012.jpg</infon><infon key="id">msb156701-fig-0006</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>37398</offset><text>Average (mean + SEM) α‐carbon distance measured from h3 Thr347 to h11 Leu525 of A‐CD‐, 2,5‐DTP‐, and 3,4‐DTPD‐bound ERα LBDs. *Two‐tailed Student's t‐test, P = 0.002 (PDB A‐CD: 5DI7, 5DID, 5DIE, 5DIG, and 4PPS; 2,5‐DTP: 4IWC, 5DRM, and 5DRJ; 3,4‐DTPD: 5DTV and 5DU5).</text></passage><passage><infon key="file">MSB-12-864-g012.jpg</infon><infon key="id">msb156701-fig-0006</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>37696</offset><text>Crystal structures show that a 3,4‐DTPD analog shifts h3 (F) and the NCOA2 (G) peptide compared to an A‐CD‐ring estrogen (PDB 4PPS, 5DTV).</text></passage><passage><infon key="file">MSB-12-864-g012.jpg</infon><infon key="id">msb156701-fig-0006</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>37841</offset><text>Hierarchical clustering of ligand‐specific binding of 154 interacting peptides to the ERα LBD was performed in triplicate by MARCoNI analysis.</text></passage><passage><infon key="file">MSB-12-864-g012.jpg</infon><infon key="id">msb156701-fig-0006</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>37988</offset><text>
+Source data are available online for this figure.
+</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>38040</offset><text>The 2,5‐DTP analogs showed perturbation of h11, as well as h3, which forms part of the AF‐2 surface. These compounds bind the LBD in an unusual fashion because they have a phenol‐to‐phenol length of ~12 Å, which is longer than steroids and other prototypical ERα agonists that are ~10 Å in length. One phenol pushed further toward h3 (Fig 6D), while the other phenol pushed toward the C‐terminus of h11 to a greater extent than A‐CD‐ring estrogens (Nwachukwu et al, 2014), which are close structural analogs of E2 that lack a B‐ring (Fig 2). To quantify this difference, we compared the distance between α‐carbons at h3 Thr347 and h11 Leu525 in the set of structures containing 2,5‐DTP analogs (n = 3) or A‐CD‐ring analogs (n = 5) (Fig 6E). We observed a difference of 0.4 Å that was significant (two‐tailed Student's t‐test, P = 0.002) due to the very tight clustering of the 2,5‐DTP‐induced LBD conformation. The shifts in h3 suggest these compounds are positioned to alter coregulator preferences.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>39088</offset><text>The 2,5‐DTP and 3,4‐DTP scaffolds are isomeric, but with aryl groups at obtuse and acute angles, respectively (Fig 2). The crystal structure of ERα in complex with a 3,4‐DTP is unknown; however, we solved two crystal structures of ERα bound to 3,4‐DTPD analogs and one structure containing a furan ligand—all of which have a 3,4‐diaryl configuration (Fig 2; Datasets EV1 and EV2). In these structures, the A‐ring mimetic of the 3,4‐DTPD scaffold bound h3 Glu353 as expected, but the other phenol wrapped around h3 to form a hydrogen bond with Thr347, indicating a change in binding epitopes in the ERα ligand‐binding pocket (Fig 6F). The 3,4‐DTPD analogs also induced a shift in h3 positioning, which translated again into a shift in the bound coactivator peptide (Fig 6F). Therefore, these indirect modulators, including S‐OBHS‐2, S‐OBHS‐3, 2,5‐DTP, and 3,4‐DTPD analogs—all of which show cell‐specific activity profiles—induced shifts in h3 and h12 that were transmitted to the coactivator peptide via an altered AF‐2 surface.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>40164</offset><text>To test whether the AF‐2 surface shows changes in shape in solution, we used the microarray assay for real‐time coregulator–nuclear receptor interaction (MARCoNI) analysis (Aarts et al, 2013). Here, the ligand‐dependent interactions of the ERα LBD with over 150 distinct LxxLL motif peptides were assayed to define structural fingerprints for the AF‐2 surface, in a manner similar to the use of phage display peptides as structural probes (Connor et al, 2001). Despite the similar average activities of these ligand classes (Fig 3A and B), 2,5‐DTP and 3,4‐DTP analogs displayed remarkably different peptide recruitment patterns (Fig 6H), consistent with the structural analyses.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>40862</offset><text>Hierarchical clustering revealed that many of the 2,5‐DTP analogs recapitulated most of the peptide recruitment and dismissal patterns observed with E2 (Fig 6H). However, there was a unique cluster of peptides that were recruited by E2 but not the 2,5‐DTP analogs. In contrast, 3,4‐DTP analogs dismissed most of the peptides from the AF‐2 surface (Fig 6H). Thus, the isomeric attachment of diaryl groups to the thiophene core changed the AF‐2 surface from inside the ligand‐binding pocket, as predicted by the crystal structures. Together, these findings suggest that without an extended side chain, cell‐specific activity stems from different coregulator recruitment profiles, due to unique ligand‐induced conformations of the AF‐2 surface, in addition to differential usage of AF‐1. Indirect modulators in cluster 1 avoid this by perturbing the h11–h12 interface, and modulating the dynamics of h12 without changing the shape of AF‐2 when stabilized.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_1</infon><offset>41841</offset><text>Discussion</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>41852</offset><text>Our goal was to identify a minimal set of predictors that would link specific structural perturbations to ERα signaling pathways that control cell‐specific signaling and proliferation. We found a very strong set of predictors, where ligands in cluster 1, defined by similar signaling across cell types, showed indirect modulation of h12 dynamics via the h11–12 interface or slight contact with h12. This perturbation determined proliferation that correlated strongly with AF‐2 activity, recruitment of NCOA1/2/3 family members, and induction of the GREB1 gene, consistent with the canonical ERα signaling pathway (Fig 1D). For ligands in cluster 1, deletion of AF‐1 reduced activity to varying degrees, but did not change the underlying signaling patterns established through AF‐2. In contrast, an extended side chain designed to directly reposition h12 and completely disrupt the AF‐2 surface results in cell‐specific signaling. This was demonstrated with direct modulators in clusters 2 and 3. Cluster 2 was defined by ligand classes that showed correlated activities in two of the three cell types tested, while ligand classes in cluster 3 did not show correlated activities among any of the three cell types. Compared to cluster 1, the structural rules are less clear in clusters 2 and 3, but a number of indirect modulator classes perturbed the LBD conformation at the intersection of h3, the h12 N‐terminus, and the AF‐2 surface. Ligands in these classes altered the shape of AF‐2 to affect coregulator preferences. For direct and indirect modulators in cluster 2 or 3, the canonical ERα signaling pathway involving recruitment of NCOA1/2/3 and induction of GREB1 did not generally predict their proliferative effects, indicating an alternate causal model (Fig 1E).</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>43650</offset><text>These principles outlined above provide a structural basis for how the ligand–receptor interface leads to different signaling specificities through AF‐1 and AF‐2. It is noteworthy that regulation of h12 dynamics indirectly through h11 can virtually abolish AF‐2 activity, and yet still drive robust transcriptional activity through AF‐1, as demonstrated with the OBHS series. This finding can be explained by the fact that NCOA1/2/3 contain distinct binding sites for interaction with AF‐1 and AF‐2 (McInerney et al, 1996; Webb et al, 1998), which allows ligands to nucleate ERα–NCOA1/2/3 interaction through AF‐2, and reinforce this interaction with additional binding to AF‐1. Completely blocking AF‐2 with an extended side chain or altering the shape of AF‐2 changes the preference away from NCOA1/2/3 for determining GREB1 levels and proliferation of breast cancer cells. AF‐2 blockade also allows AF‐1 to function independently, which is important since AF‐1 drives tissue‐selective effects in vivo. This was demonstrated with AF‐1 knockout mice that show E2‐dependent vascular protection, but not uterine proliferation, thus highlighting the role of AF‐1 in tissue‐selective or cell‐specific signaling (Billon‐Gales et al, 2009; Abot et al, 2013).</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>44954</offset><text>One current limitation to our approach is the identification of statistical variables that predict ligand‐specific activity. Here, we examined many LBD structures and tested several variables that were not predictive, including ERβ activity, the strength of AF‐1 signaling, and NCOA3 occupancy at the GREB1 gene. Similarly, we visualized structures to identify patterns. There are many systems biology approaches that could contribute to the unbiased identification of predictive variables for statistical modeling. For example, phage display was used to identify the androgen receptor interactome, which was cloned into an M2H library and used to identify clusters of ligand‐selective interactions (Norris et al, 2009). Also, we have used siRNA screening to identify a number of coregulators required for ERα‐mediated repression of the IL‐6 gene (Nwachukwu et al, 2014). However, the use of larger datasets to identify such predictor variables has its own limitations, one of the major ones being the probability of false positives from multiple hypothesis testing. If we calculated inter‐atomic distance matrices containing 4,000 atoms per structure × 76 ligand–receptor complexes, we would have 3 × 105 predictions. One way to address this issue is to use the cross‐validation concept, where hypotheses are generated on training sets of ligands and tested with another set of ligands.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>46367</offset><text>Based on this work, we propose several testable hypotheses for drug discovery. We have identified atomic vectors for the OBHS‐N and triaryl‐ethylene classes that predict ligand response (Fig 5E and F). These ligands in cluster 1 drive consistent, canonical signaling across cell types, which is desirable for generating full antagonists. Indeed, the most anti‐proliferative compound in the OBHS‐N series had a fulvestrant‐like profile across a battery of assays (S. Srinivasan et al, in preparation). Secondly, our finding that WAY‐C compounds do not rely of AF‐1 for signaling efficacy may derive from the slight contacts with h12 observed in crystal structures (Figs 3B and 5H), unlike other compounds in cluster 1 that dislocate h11 and rely on AF‐1 for signaling efficacy (Figs 3B and 5C, and EV5B). Thirdly, we found ligands that achieved cell‐specific activity without a prototypical extended side chain. Some of these ligands altered the shape of the AF‐2 surface by perturbing the h3–h12 interface, thus providing a route to new SERM‐like activity profiles by combining indirect and direct modulation of receptor structure. Incorporation of statistical approaches to understand relationships between structure and signaling variables moves us toward predictive models for complex ERα‐mediated responses such as in vivo uterine proliferation or tumor growth, and more generally toward structure‐based design for other allosteric drug targets including GPCRs and other nuclear receptors.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>47895</offset><text>Materials and Methods</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>47917</offset><text>Statistical analysis</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>47938</offset><text>Correlation and linear regression analyses were performed using GraphPad Prism software. For correlation analysis, the degree to which two datasets vary together was calculated with the Pearson correlation coefficient (r). However, we reported r 2 rather than r, to facilitate comparison with the linear regression results for which we calculated and reported r 2 (Fig 3C–F). Significance for r 2 was determined using the F‐test for nonzero slope. High‐throughput assays were considered statistically robust if they show Z’ &gt; 0.5, where Z’ = 1 − (3(σp+σn)/|μp−μn|), for the mean (σ) and standard deviations (μ) of the positive and negative controls (Fig EV1A and B).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>48631</offset><text>ERα ligand library</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>48654</offset><text>The library of compounds examined includes both previously reported (Srinivasan et al, 2013) and newly synthesized compound series (see Dataset EV1 for individual compound information, and Appendix Supplementary Methods for synthetic protocols).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>48902</offset><text>Luciferase reporter assays</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>48929</offset><text>Cells were transfected with FugeneHD reagent (Roche Applied Sciences, Indianapolis, IN) in 384‐well plates. After 24 h, cells were stimulated with 10 μM compounds dispensed using a 100‐nl pintool Biomeck NXP workstation (Beckman Coulter Inc.). Luciferase activity was measured 24 h later (see Appendix Supplementary Methods for more details).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>49281</offset><text>Mammalian 2‐hybrid (M2H) assays</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>49315</offset><text>HEK293T cells were transfected with 5× UAS‐luciferase reporter, and wild‐type ERα‐VP16 activation domain plus full‐length NCOA1/2/3‐GAL4 DBD fusion protein expression plasmids, using the TransIT‐LT1 transfection reagent (Mirus Bio LLC, Madison, WI). The next day, cells were stimulated with 10 μM compounds using a 100‐nl pintool Biomeck NXP workstation (Beckman Coulter Inc.). Luciferase activity was measured after 24 h (see Appendix Supplementary Methods for more details).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>49813</offset><text>Cell proliferation assay</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>49838</offset><text>MCF‐7 cells were plated on 384‐well plates in phenol red‐free media plus 10% FBS and stimulated with 10 μM compounds using 100‐nl pintool Biomeck NXP workstation (Beckman Coulter Inc.). Cell numbers determined 1 week later (see Appendix Supplementary Methods for more details).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>50128</offset><text>Quantitative RT–PCR</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>50150</offset><text>MCF‐7 cells were steroid‐deprived and stimulated with compounds for 24 h. Total RNA was extracted and reverse‐transcribed. The cDNA was analyzed using TaqMan Gene Expression Master Mix (Life Technologies, Grand Island, NY), GREB1 and GAPDH (control) primers, and hybridization probes (see Appendix Supplementary Methods for more details).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>50497</offset><text>MARCoNI coregulator‐interaction profiling</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>50541</offset><text>This assay was performed as previously described with the ERα LBD, 10 μM compounds, and a PamChiP peptide microarray (PamGene International) containing 154 unique coregulator peptides (Aarts et al, 2013) (see Appendix Supplementary Methods for more details).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>50805</offset><text>Protein production and X‐ray crystallography</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>50852</offset><text>ERα protein was produced as previously described (Bruning et al, 2010). New ERα LBD structures (see Dataset EV2 for data collection and refinement statistics) were solved by molecular replacement using PHENIX (Adams et al, 2010), refined using ExCoR as previously described (Nwachukwu et al, 2013), and COOT (Emsley &amp; Cowtan, 2004) for ligand‐docking and rebuilding.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>51227</offset><text>Data availability</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>51245</offset><text>Crystal structures analyzed in this study include the following: 1GWR (Warnmark et al, 2002), 3ERD and 3ERT (Shiau et al, 1998), 4ZN9 (Zheng et al, 2012), 4IWC, 4 IU7, 4IV4, 4IVW, 4IW6, 4IUI, 4IV2, 4IVY and 4IW8 (Srinivasan et al, 2013), and 4PPS (Nwachukwu et al, 2014). New crystal structures analyzed in this study were deposited in the RCSB protein data bank (http://www.pdb.org): 4ZN7, 4ZNH, 4ZNS, 4ZNT, 4ZNU, 4ZNV, 4ZNW, 5DI7, 5DID, 5DIE, 5DIG, 5DK9, 5DKB, 5DKE, 5DKG, 5DKS, 5DL4, 5DLR, 5DMC, 5DMF, 5DP0, 5DRM, 5DRJ, 5DTV, 5DU5, 5DUE, 5DUG, 5DUH, 5DXK, 5DXM, 5DXP, 5DXQ, 5DXR, 5EHJ, 5DY8, 5DYB, 5DYD, 5DZ0, 5DZ1, 5DZ3, 5DZH, 5DZI, 5E0W, 5E0X, 5E14, 5E15, 5E19, 5E1C, 5DVS, 5DVV, 5DWE, 5DWG, 5DWI, 5DWJ, 5EGV, 5EI1, 5EIT.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">title_1</infon><offset>51978</offset><text>Author contributions</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">paragraph</infon><offset>51999</offset><text>JCN and SS contributed equally to this work. JCN and SS designed and performed experiments and wrote the manuscript; YZ, KEC, SW, JM, CD, ZL, VC, JN, NJW, JSJ, and RH performed experiments; HBZ designed experiments; and JAK and KWN designed experiments and wrote the manuscript.</text></passage><passage><infon key="section_type">COMP_INT</infon><infon key="type">title_1</infon><offset>52278</offset><text>Conflict of Interest</text></passage><passage><infon key="section_type">COMP_INT</infon><infon key="type">paragraph</infon><offset>52299</offset><text>The authors declare that they have no conflict of interest.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">title_1</infon><offset>52359</offset><text>Supporting information</text></passage><passage><infon key="section_type">REF</infon><infon key="type">title</infon><offset>52382</offset><text>References</text></passage><passage><infon key="fpage">336</infon><infon key="lpage">346</infon><infon key="pub-id_pmid">23383871</infon><infon 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+<!DOCTYPE collection SYSTEM "BioC.dtd">
+<collection><source>PMC</source><date>20201218</date><key>pmc.key</key><document><id>4852598</id><infon key="license">CC BY</infon><passage><infon key="article-id_doi">10.1038/ncomms11337</infon><infon key="article-id_pii">ncomms11337</infon><infon key="article-id_pmc">4852598</infon><infon key="article-id_pmid">27088325</infon><infon key="elocation-id">11337</infon><infon key="license">This work is licensed under a Creative Commons Attribution 4.0
+International License. The images or other third party material in this article are
+included in the article's Creative Commons license, unless indicated otherwise
+in the credit line; if the material is not included under the Creative Commons
+license, users will need to obtain permission from the license holder to reproduce
+the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/</infon><infon key="name_0">surname:van den Berg;given-names:Bert</infon><infon key="name_1">surname:Chembath;given-names:Anupama</infon><infon key="name_2">surname:Jefferies;given-names:Damien</infon><infon key="name_3">surname:Basle;given-names:Arnaud</infon><infon key="name_4">surname:Khalid;given-names:Syma</infon><infon key="name_5">surname:Rutherford;given-names:Julian C.</infon><infon key="section_type">TITLE</infon><infon key="type">front</infon><infon key="volume">7</infon><infon key="year">2016</infon><offset>0</offset><text>Structural basis for Mep2 ammonium transceptor activation by phosphorylation</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>77</offset><text>Mep2 proteins are fungal transceptors that play an important role as ammonium sensors in fungal development. Mep2 activity is tightly regulated by phosphorylation, but how this is achieved at the molecular level is not clear. Here we report X-ray crystal structures of the Mep2 orthologues from Saccharomyces cerevisiae and Candida albicans and show that under nitrogen-sufficient conditions the transporters are not phosphorylated and present in closed, inactive conformations. Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif. The phosphorylation site in the CTR is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. The crystal structure of phosphorylation-mimicking Mep2 variants from C. albicans show large conformational changes in a conserved and functionally important region of the CTR. The results allow us to propose a model for regulation of eukaryotic ammonium transport by phosphorylation.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>1224</offset><text> Mep2 proteins are tightly regulated fungal ammonium transporters. Here, the authors report the crystal structures of closed states of Mep2 proteins and propose a model for their regulation by comparing them with the open ammonium transporters of bacteria.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1481</offset><text>Transceptors are membrane proteins that function not only as transporters but also as receptors/sensors during nutrient sensing to activate downstream signalling pathways. A common feature of transceptors is that they are induced when cells are starved for their substrate. While most studies have focused on the Saccharomyces cerevisiae transceptors for phosphate (Pho84), amino acids (Gap1) and ammonium (Mep2), transceptors are found in higher eukaryotes as well (for example, the mammalian SNAT2 amino-acid transporter and the GLUT2 glucose transporter). One of the most important unresolved questions in the field is how the transceptors couple to downstream signalling pathways. One hypothesis is that downstream signalling is dependent on a specific conformation of the transporter.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2271</offset><text>Mep2 (methylammonium (MA) permease) proteins are ammonium transceptors that are ubiquitous in fungi. They belong to the Amt/Mep/Rh family of transporters that are present in all kingdoms of life and they take up ammonium from the extracellular environment. Fungi typically have more than one Mep paralogue, for example, Mep1-3 in S. cerevisiae. Of these, only Mep2 proteins function as ammonium receptors/sensors in fungal development. Under conditions of nitrogen limitation, Mep2 initiates a signalling cascade that results in a switch from the yeast form to filamentous (pseudohyphal) growth that may be required for fungal pathogenicity. As is the case for other transceptors, it is not clear how Mep2 interacts with downstream signalling partners, but the protein kinase A and mitogen-activated protein kinase pathways have been proposed as downstream effectors of Mep2 (refs). Compared with Mep1 and Mep3, Mep2 is highly expressed and functions as a low-capacity, high-affinity transporter in the uptake of MA. In addition, Mep2 is also important for uptake of ammonium produced by growth on other nitrogen sources.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3393</offset><text>With the exception of the human RhCG structure, no structural information is available for eukaryotic ammonium transporters. By contrast, several bacterial Amt orthologues have been characterized in detail via high-resolution crystal structures and a number of molecular dynamics (MD) studies. All the solved structures including that of RhCG are very similar, establishing the basic architecture of ammonium transporters. The proteins form stable trimers, with each monomer having 11 transmembrane (TM) helices and a central channel for the transport of ammonium. All structures show the transporters in open conformations. Intriguingly, fundamental questions such as the nature of the transported substrate and the transport mechanism are still controversial. Where earlier studies favoured the transport of ammonia gas, recent data and theoretical considerations suggest that Amt/Mep proteins are instead active, electrogenic transporters of either NH4+ (uniport) or NH3/H+ (symport). A highly conserved pair of channel-lining histidine residues dubbed the twin-His motif may serve as a proton relay system while NH3 moves through the channel during NH3/H+ symport.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>4562</offset><text>Ammonium transport is tightly regulated. In animals, this is due to toxicity of elevated intracellular ammonium levels, whereas for microorganisms ammonium is a preferred nitrogen source. In bacteria, amt genes are present in an operon with glnK, encoding a PII-like signal transduction class protein. By binding tightly to Amt proteins without inducing a conformational change in the transporter, GlnK sterically blocks ammonium conductance when nitrogen levels are sufficient. Under conditions of nitrogen limitation, GlnK becomes uridylated, blocking its ability to bind and inhibit Amt proteins. Importantly, eukaryotes do not have GlnK orthologues and have a different mechanism for regulation of ammonium transport activity. In plants, transporter phosphorylation and dephosphorylation are known to regulate activity. In S. cerevisiae, phosphorylation of Ser457 within the C-terminal region (CTR) in the cytoplasm was recently proposed to cause Mep2 opening, possibly via inducing a conformational change.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>5574</offset><text>To elucidate the mechanism of Mep2 transport regulation, we present here X-ray crystal structures of the Mep2 transceptors from S. cerevisiae and C. albicans. The structures are similar to each other but show considerable differences to all other ammonium transporter structures. The most striking difference is the fact that the Mep2 proteins have closed conformations. The putative phosphorylation site is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. The channels of phosphorylation-mimicking mutants of C. albicans Mep2 are still closed but show large conformational changes within a conserved part of the CTR. Together with a structure of a C-terminal Mep2 variant lacking the segment containing the phosphorylation site, the results allow us to propose a structural model for phosphorylation-based regulation of eukaryotic ammonium transport.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>6478</offset><text>Results</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>6486</offset><text>General architecture of Mep2 ammonium transceptors</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>6537</offset><text>The Mep2 protein of S. cerevisiae (ScMep2) was overexpressed in S. cerevisiae in high yields, enabling structure determination by X-ray crystallography using data to 3.2 Å resolution by molecular replacement (MR) with the archaebacterial Amt-1 structure (see Methods section). Given that the modest resolution of the structure and the limited detergent stability of ScMep2 would likely complicate structure–function studies, several other fungal Mep2 orthologues were subsequently overexpressed and screened for diffraction-quality crystals. Of these, Mep2 from C. albicans (CaMep2) showed superior stability in relatively harsh detergents such as nonyl-glucoside, allowing structure determination in two different crystal forms to high resolution (up to 1.5 Å). Despite different crystal packing (Supplementary Table 1), the two CaMep2 structures are identical to each other and very similar to ScMep2 (Cα r.m.s.d. (root mean square deviation)=0.7 Å for 434 residues), with the main differences confined to the N terminus and the CTR (Fig. 1). Electron density is visible for the entire polypeptide chains, with the exception of the C-terminal 43 (ScMep2) and 25 residues (CaMep2), which are poorly conserved and presumably disordered. Both Mep2 proteins show the archetypal trimeric assemblies in which each monomer consists of 11 TM helices surrounding a central pore. Important functional features such as the extracellular ammonium binding site, the Phe gate and the twin-His motif within the hydrophobic channel are all very similar to those present in the bacterial transporters and RhCG. In the remainder of the manuscript, we will specifically discuss CaMep2 due to the superior resolution of the structure. Unless specifically stated, the drawn conclusions also apply to ScMep2.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>8338</offset><text>While the overall architecture of Mep2 is similar to that of the prokaryotic transporters (Cα r.m.s.d. with Amt-1=1.4 Å for 361 residues), there are large differences within the N terminus, intracellular loops (ICLs) ICL1 and ICL3, and the CTR. The N termini of the Mep2 proteins are ∼20–25 residues longer compared with their bacterial counterparts (Figs 1 and 2), substantially increasing the size of the extracellular domain. Moreover, the N terminus of one monomer interacts with the extended extracellular loop ECL5 of a neighbouring monomer. Together with additional, smaller differences in other extracellular loops, these changes generate a distinct vestibule leading to the ammonium binding site that is much more pronounced than in the bacterial proteins. The N-terminal vestibule and the resulting inter-monomer interactions likely increase the stability of the Mep2 trimer, in support of data for plant AMT proteins. However, given that an N-terminal deletion mutant (2-27Δ) grows as well as wild-type (WT) Mep2 on minimal ammonium medium (Fig. 3 and Supplementary Fig. 1), the importance of the N terminus for Mep2 activity is not clear.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>9498</offset><text>Mep2 channels are closed by a two-tier channel block</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9551</offset><text>The largest differences between the Mep2 structures and the other known ammonium transporter structures are located on the intracellular side of the membrane. In the vicinity of the Mep2 channel exit, the cytoplasmic end of TM2 has unwound, generating a longer ICL1 even though there are no insertions in this region compared to the bacterial proteins (Figs 2 and 4). ICL1 has also moved inwards relative to its position in the bacterial Amts. The largest backbone movements of equivalent residues within ICL1 are ∼10 Å, markedly affecting the conserved basic RxK motif (Fig. 4). The head group of Arg54 has moved ∼11 Å relative to that in Amt-1, whereas the shift of the head group of the variable Lys55 residue is almost 20 Å. The side chain of Lys56 in the basic motif points in an opposite direction in the Mep2 structures compared with that of, for example, Amt-1 (Fig. 4). In addition to changing the RxK motif, the movement of ICL1 has another, crucial functional consequence. At the C-terminal end of TM1, the side-chain hydroxyl group of the relatively conserved Tyr49 (Tyr53 in ScMep2) makes a strong hydrogen bond with the ɛ2 nitrogen atom of the absolutely conserved His342 of the twin-His motif (His348 in ScMep2), closing the channel (Figs 4 and 5). In bacterial Amt proteins, this Tyr side chain is rotated ∼4 Å away as a result of the different conformation of TM1, leaving the channel open and the histidine available for its putative role in substrate transport (Supplementary Fig. 2).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>11074</offset><text>Compared with ICL1, the backbone conformational changes observed for the neighbouring ICL2 are smaller, but large shifts are nevertheless observed for the conserved residues Glu140 and Arg141 (Fig. 4). Finally, the important ICL3 linking the pseudo-symmetrical halves (TM1-5 and TM6-10) of the transporter is also shifted up to ∼10 Å and forms an additional barrier that closes the channel on the cytoplasmic side (Fig. 5). This two-tier channel block likely ensures that very little ammonium transport will take place under nitrogen-sufficient conditions. The closed state of the channel might also explain why no density, which could correspond to ammonium (or water), is observed in the hydrophobic part of the Mep2 channel close to the twin-His motif. Significantly, this is also true for ScMep2, which was crystallized in the presence of 0.2 M ammonium ions (see Methods section).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>11967</offset><text>The final region in Mep2 that shows large differences compared with the bacterial transporters is the CTR. In Mep2, the CTR has moved away and makes relatively few contacts with the main body of the transporter, generating a more elongated protein (Figs 1 and 4). By contrast, in the structures of bacterial proteins, the CTR is docked tightly onto the N-terminal half of the transporters (corresponding to TM1-5), resulting in a more compact structure. This is illustrated by the positions of the five universally conserved residues within the CTR, that is, Arg415(370), Glu421(376), Gly424(379), Asp426(381) and Tyr 435(390) in CaMep2(Amt-1) (Fig. 2). These residues include those of the ‘ExxGxD' motif, which when mutated generate inactive transporters. In Amt-1 and other bacterial ammonium transporters, these CTR residues interact with residues within the N-terminal half of the protein. On one side, the Tyr390 hydroxyl in Amt-1 is hydrogen bonded with the side chain of the conserved His185 at the C-terminal end of loop ICL3. At the other end of ICL3, the backbone carbonyl groups of Gly172 and Lys173 are hydrogen bonded to the side chain of Arg370. Similar interactions were also modelled in the active, non-phosphorylated plant AtAmt-1;1 structure (for example, Y467-H239 and D458-K71). The result of these interactions is that the CTR ‘hugs' the N-terminal half of the transporters (Fig. 4). Also noteworthy is Asp381, the side chain of which interacts strongly with the positive dipole on the N-terminal end of TM2. This interaction in the centre of the protein may be particularly important to stabilize the open conformations of ammonium transporters. In the Mep2 structures, none of the interactions mentioned above are present.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>13717</offset><text>Phosphorylation target site is at the periphery of Mep2</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>13773</offset><text>Recently Boeckstaens et al. provided evidence that Ser457 in ScMep2 (corresponding to Ser453 in CaMep2) is phosphorylated by the TORC1 effector kinase Npr1 under nitrogen-limiting conditions. In the absence of Npr1, plasmid-encoded WT Mep2 in a S. cerevisiae mep1-3Δ strain (triple mepΔ) does not allow growth on low concentrations of ammonium, suggesting that the transporter is inactive (Fig. 3 and Supplementary Fig. 1). Conversely, the phosphorylation-mimicking S457D variant is active both in the triple mepΔ background and in a triple mepΔ npr1Δ strain (Fig. 3). Mutation of other potential phosphorylation sites in the CTR did not support growth in the npr1Δ background. Collectively, these data suggest that phosphorylation of Ser457 opens the Mep2 channel to allow ammonium uptake. Ser457 is located in a part of the CTR that is conserved in a subgroup of Mep2 proteins, but which is not present in bacterial proteins (Fig. 2). This segment (residues 450–457 in ScMep2 and 446–453 in CaMep2) was dubbed an autoinhibitory (AI) region based on the fact that its removal generates an active transporter in the absence of Npr1 (Fig. 3).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>14939</offset><text>Where is the AI region and the Npr1 phosphorylation site located? Our structures reveal that surprisingly, the AI region is folded back onto the CTR and is not located near the centre of the trimer as expected from the bacterial structures (Fig. 4). The AI region packs against the cytoplasmic ends of TM2 and TM4, physically linking the main body of the transporter with the CTR via main chain interactions and side-chain interactions of Val447, Asp449, Pro450 and Arg452 (Fig. 6). The AI regions have very similar conformations in CaMep2 and ScMep2, despite considerable differences in the rest of the CTR (Fig. 6). Strikingly, the Npr1 target serine residue is located at the periphery of the trimer, far away (∼30 Å) from any channel exit (Fig. 6). Despite its location at the periphery of the trimer, the electron density for the serine is well defined in both Mep2 structures and corresponds to the non-phosphorylated state (Fig. 6). This makes sense since the proteins were expressed in rich medium and confirms the recent suggestion by Boeckstaens et al. that the non-phosphorylated form of Mep2 corresponds to the inactive state. For ScMep2, Ser457 is the most C-terminal residue for which electron density is visible, indicating that the region beyond Ser457 is disordered. In CaMep2, the visible part of the sequence extends for two residues beyond Ser453 (Fig. 6). The peripheral location and disorder of the CTR beyond the kinase target site should facilitate the phosphorylation by Npr1. The disordered part of the CTR is not conserved in ammonium transporters (Fig. 2), suggesting that it is not important for transport. Interestingly, a ScMep2 457Δ truncation mutant in which a His-tag directly follows Ser457 is highly expressed but has low activity (Fig. 3 and Supplementary Fig. 1b), suggesting that the His-tag interferes with phosphorylation by Npr1. The same mutant lacking the His-tag has WT properties (Supplementary Fig. 1b), confirming that the region following the phosphorylation site is dispensable for function.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>16987</offset><text>Mep2 lacking the AI region is conformationally heterogeneous</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>17048</offset><text>Given that Ser457/453 is far from any channel exit (Fig. 6), the crucial question is how phosphorylation opens the Mep2 channel to generate an active transporter. Boeckstaens et al. proposed that phosphorylation does not affect channel activity directly, but instead relieves inhibition by the AI region. The data behind this hypothesis is the observation that a ScMep2 449-485Δ deletion mutant lacking the AI region is highly active in MA uptake both in the triple mepΔ and triple mepΔ npr1Δ backgrounds, implying that this Mep2 variant has a constitutively open channel. We obtained a similar result for ammonium uptake by the 446Δ mutant (Fig. 3), supporting the data from Marini et al. We then constructed and purified the analogous CaMep2 442Δ truncation mutant and determined the crystal structure using data to 3.4 Å resolution. The structure shows that removal of the AI region markedly increases the dynamics of the cytoplasmic parts of the transporter. This is not unexpected given the fact that the AI region bridges the CTR and the main body of Mep2 (Fig. 6). Density for ICL3 and the CTR beyond residue Arg415 is missing in the 442Δ mutant, and the density for the other ICLs including ICL1 is generally poor with visible parts of the structure having high B-factors (Fig. 7). Interestingly, however, the Tyr49-His342 hydrogen bond that closes the channel in the WT protein is still present (Fig. 7 and Supplementary Fig. 2). Why then does this mutant appear to be constitutively active? We propose two possibilities. The first one is that the open state is disfavoured by crystallization because of lower stability or due to crystal packing constraints. The second possibility is that the Tyr–His hydrogen bond has to be disrupted by the incoming substrate to open the channel. The latter model would fit well with the NH3/H+ symport model in which the proton is relayed by the twin-His motif. The importance of the Tyr–His hydrogen bond is underscored by the fact that its removal in the ScMep2 Y53A mutant results in a constitutively active transporter (Fig. 3).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>19155</offset><text>Phosphorylation causes a conformational change in the CTR</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>19213</offset><text>Do the Mep2 structures provide any clues regarding the potential effect of phosphorylation? The side-chain hydroxyl of Ser457/453 is located in a well-defined electronegative pocket that is solvent accessible (Fig. 6). The closest atoms to the serine hydroxyl group are the backbone carbonyl atoms of Asp419, Glu420 and Glu421, which are 3–4 Å away. We therefore predict that phosphorylation of Ser453 will result in steric clashes as well as electrostatic repulsion, which in turn might cause substantial conformational changes within the CTR. To test this hypothesis, we determined the structure of the phosphorylation-mimicking R452D/S453D protein (hereafter termed ‘DD mutant'), using data to a resolution of 2.4 Å. The additional mutation of the arginine preceding the phosphorylation site was introduced (i) to increase the negative charge density and make it more comparable to a phosphate at neutral pH, and (ii) to further destabilize the interactions of the AI region with the main body of the transporter (Fig. 6). The ammonium uptake activity of the S. cerevisiae version of the DD mutant is the same as that of WT Mep2 and the S453D mutant, indicating that the mutations do not affect transporter functionality in the triple mepΔ background (Fig. 3). Unexpectedly, the AI segment containing the mutated residues has only undergone a slight shift compared with the WT protein (Fig. 8 and Supplementary Fig. 3). By contrast, the conserved part of the CTR has undergone a large conformational change involving formation of a 12-residue-long α-helix from Leu427 to Asp438. In addition, residues Glu420-Leu423 including Glu421 of the ExxGxD motif are now disordered (Fig. 8 and Supplementary Fig. 3). Overall, ∼20 residues are affected by the introduced mutations. This is the first time a large conformational change has been observed in an ammonium transporter as a result of a mutation, and confirms previous hypotheses that phosphorylation causes structural changes in the CTR. To exclude the possibility that the additional R452D mutation is responsible for the observed changes, we also determined the structure of the ‘single D' S453D mutant. As shown in Supplementary Fig. 4, the consequence of the single D mutation is very similar to that of the DD substitution, with conformational changes and increased dynamics confined to the conserved part of the CTR (Supplementary Fig. 4). To supplement the crystal structures, we also performed modelling and MD studies of WT CaMep2, the DD mutant and phosphorylated protein (S453J). In the WT structure, the acidic residues Asp419, Glu420 and Glu421 are within hydrogen bonding distance of Ser453. After 200 ns of MD simulation, the interactions between these residues and Ser453 remain intact. The protein backbone has an average r.m.s.d. of only ∼3 Å during the 200-ns simulation, indicating that the protein is stable. There is flexibility in the side chains of the acidic residues so that they are able to form stable hydrogen bonds with Ser453. In particular, persistent hydrogen bonds are observed between the Ser453 hydroxyl group and the acidic group of Glu420, and also between the amine group of Ser453 and the backbone carbonyl of Glu420 (Supplementary Fig. 5). The DD mutant is also stable during the simulations, but the average backbone r.m.s.d of ∼3.6 Å suggests slightly more conformational flexibility than WT. As the simulation proceeds, the side chains of the acidic residues move away from Asp452 and Asp453, presumably to avoid electrostatic repulsion. For example, the distance between the Asp453 acidic oxygens and the Glu420 acidic oxygens increases from ∼7 to &gt;22 Å after 200 ns simulations, and thus these residues are not interacting. The protein is structurally stable throughout the simulation with little deviation in the other parts of the protein. Finally, the S453J mutant is also stable throughout the 200-ns simulation and has an average backbone deviation of ∼3.8 Å, which is similar to the DD mutant. The movement of the acidic residues away from Arg452 and Sep453 is more pronounced in this simulation in comparison with the movement away from Asp452 and Asp453 in the DD mutant. The distance between the phosphate of Sep453 and the acidic oxygen atoms of Glu420 is initially ∼11 Å, but increases to &gt;30 Å after 200 ns. The short helix formed by residues Leu427 to Asp438 unravels during the simulations to a disordered state. The remainder of the protein is not affected (Supplementary Fig. 5). Thus, the MD simulations support the notion from the crystal structures that phosphorylation generates conformational changes in the conserved part of the CTR. However, the conformational changes for the phosphomimetic mutants in the crystals are confined to the CTR (Fig. 8), and the channels are still closed (Supplementary Fig. 2). One possible explanation is that the mutants do not accurately mimic a phosphoserine, but the observation that the S453D and DD mutants are fully active in the absence of Npr1 suggests that the mutations do mimic the effect of phosphorylation (Fig. 3). The fact that the S453D structure was obtained in the presence of 10 mM ammonium ions suggests that the crystallization process favours closed states of the Mep2 channels.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_1</infon><offset>24519</offset><text>Discussion</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>24530</offset><text>Knowledge about ammonium transporter structure has been obtained from experimental and theoretical studies on bacterial family members. In addition, a number of biochemical and genetic studies are available for bacterial, fungal and plant proteins. These efforts have advanced our knowledge considerably but have not yet yielded atomic-level answers to several important mechanistic questions, including how ammonium transport is regulated in eukaryotes and the mechanism of ammonium signalling. In Arabidopsis thaliana Amt-1;1, phosphorylation of the CTR residue T460 under conditions of high ammonium inhibits transport activity, that is, the default (non-phosphorylated) state of the plant transporter is open. Interestingly, phosphomimetic mutations introduced into one monomer inactivate the entire trimer, indicating that (i) heterotrimerization occurs and (ii) the CTR mediates allosteric regulation of ammonium transport activity via phosphorylation. Owing to the lack of structural information for plant AMTs, the details of channel closure and inter-monomer crosstalk are not yet clear. Contrasting with the plant transporters, the inactive states of Mep2 proteins under conditions of high ammonium are non-phosphorylated, with channels that are closed on the cytoplasmic side. The reason why similar transporters such as A. thaliana Amt-1;1 and Mep2 are regulated in opposite ways by phosphorylation (inactivation in plants and activation in fungi) is not known. In fungi, preventing ammonium entry via channel closure in ammonium transporters would be one way to alleviate ammonium toxicity, in addition to ammonium excretion via Ato transporters and amino-acid secretion.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>26215</offset><text>By determining the first structures of closed ammonium transporters and comparing those structures with the permanently open bacterial proteins, we demonstrate that Mep2 channel closure is likely due to movements of the CTR and ICL1 and ICL3. More specifically, the close interactions between the CTR and ICL1/ICL3 present in open transporters are disrupted, causing ICL3 to move outwards and block the channel (Figs 4 and 9a). In addition, ICL1 has shifted inwards to contribute to the channel closure by engaging His2 from the twin-His motif via hydrogen bonding with a highly conserved tyrosine hydroxyl group. Upon phosphorylation by the Npr1 kinase in response to nitrogen limitation, the region around the conserved ExxGxD motif undergoes a conformational change that opens the channel (Fig. 9). Importantly, the structural similarities in the TM parts of Mep2 and AfAmt-1 (Fig. 5a) suggest that channel opening/closure does not require substantial changes in the residues lining the channel. How exactly the channel opens and whether opening is intra-monomeric are still open questions; it is possible that the change in the CTR may disrupt its interactions with ICL3 of the neighbouring monomer (Fig. 9b), which could result in opening of the neighbouring channel via inward movement of its ICL3. Owing to the crosstalk between monomers, a single phosphorylation event might lead to opening of the entire trimer, although this has not yet been tested (Fig. 9b). Whether or not Mep2 channel opening requires, in addition to phosphorylation, disruption of the Tyr–His2 interaction by the ammonium substrate is not yet clear.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>27848</offset><text>Is our model for opening and closing of Mep2 channels valid for other eukaryotic ammonium transporters? Our structural data support previous studies and clarify the central role of the CTR and cytoplasmic loops in the transition between closed and open states. However, even the otherwise highly similar Mep2 proteins of S. cerevisiae and C. albicans have different structures for their CTRs (Fig. 1 and Supplementary Fig. 6). In addition, the AI region of the CTR containing the Npr1 kinase site is conserved in only a subset of fungal transporters, suggesting that the details of the structural changes underpinning regulation vary. Nevertheless, given the central role of absolutely conserved residues within the ICL1-ICL3-CTR interaction network (Fig. 4), we propose that the structural basics of fungal ammonium transporter activation are conserved. The fact that Mep2 orthologues of distantly related fungi are fully functional in ammonium transport and signalling in S. cerevisiae supports this notion. It should also be noted that the tyrosine residue interacting with His2 is highly conserved in fungal Mep2 orthologues, suggesting that the Tyr–His2 hydrogen bond might be a general way to close Mep2 proteins.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>29070</offset><text>With regards to plant AMTs, it has been proposed that phosphorylation at T460 generates conformational changes that would close the neighbouring pore via the C terminus. This assumption was based partly on a homology model for Amt-1;1 based on the (open) archaebacterial AfAmt-1 structure, which suggested that the C terminus of Amt-1;1 would extend further to the neighbouring monomer. Our Mep2 structures show that this assumption may not be correct (Fig. 4 and Supplementary Fig. 6). In addition, the considerable differences between structurally resolved CTR domains means that the exact environment of T460 in Amt-1;1 is also not known (Supplementary Fig. 6). Based on the available structural information, we consider it more likely that phosphorylation-mediated pore closure in Amt-1;1 is intra-monomeric, via disruption of the interactions between the CTR and ICL1/ICL3 (for example, Y467-H239 and D458-K71). There is generally no equivalent for CaMep2 Tyr49 in plant AMTs, indicating that a Tyr–His2 hydrogen bond as observed in Mep2 may not contribute to the closed state in plant transporters.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>30177</offset><text>We propose that intra-monomeric CTR-ICL1/ICL3 interactions lie at the basis of regulation of both fungal and plant ammonium transporters; close interactions generate open channels, whereas the lack of ‘intra-' interactions leads to inactive states. The need to regulate in opposite ways may be the reason why the phosphorylation sites are in different parts of the CTR, that is, centrally located close to the ExxGxD motif in AMTs and peripherally in Mep2. In this way, phosphorylation can either lead to channel closing (in the case of AMTs) or channel opening in the case of Mep2. Our model also provides an explanation for the observation that certain mutations within the CTR completely abolish transport activity. An example of an inactivating residue is the glycine of the ExxGxD motif of the CTR. Mutation of this residue (G393 in EcAmtB; G456 in AtAmt-1;1) inactivates transporters as diverse as Escherichia coli AmtB and A. thaliana Amt-1;1 (refs). Such mutations likely cause structural changes in the CTR that prevent close contacts between the CTR and ICL1/ICL3, thereby stabilizing a closed state that may be similar to that observed in Mep2.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>31335</offset><text>Regulation and modulation of membrane transport by phosphorylation is known to occur in, for example, aquaporins and urea transporters, and is likely to be a common theme for eukaryotic channels and transporters. Recently, phosphorylation was also shown to modulate substrate affinity in nitrate transporters. With respect to ammonium transport, phosphorylation has thus far only been shown for A. thaliana AMTs and for S. cerevisiae Mep2 (refs). However, the absence of GlnK proteins in eukaryotes suggests that phosphorylation-based regulation of ammonium transport may be widespread. Nevertheless, as discussed above, considerable differences may exist between different species.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>32018</offset><text>With respect to Mep2-mediated signalling to induce pseudohyphal growth, two models have been put forward as to how this occurs and why it is specific to Mep2 proteins. In one model, signalling is proposed to depend on the nature of the transported substrate, which might be different in certain subfamilies of ammonium transporters (for example, Mep1/Mep3 versus Mep2). For example, NH3 uniport or symport of NH3/H+ might result in changes in local pH, but NH4+ uniport might not, and this difference might determine signalling. In the other model, signalling is thought to require a distinct conformation of the Mep2 transporter occurring during the transport cycle. While the current study does not specifically address the mechanism of signalling underlying pseudohyphal growth, our structures do show that Mep2 proteins can assume different conformations.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>32878</offset><text>It is clear that ammonium transport across biomembranes remains a fascinating and challenging field in large part due to the unique properties of the substrate. Our Mep2 structural work now provides a foundation for future studies to uncover the details of the structural changes that occur during eukaryotic ammonium transport and signaling, and to assess the possibility to utilize small molecules to shut down ammonium sensing and downstream signalling pathways in pathogenic fungi.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>33364</offset><text>Methods</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>33372</offset><text>Mep2 overexpression and purification</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>33409</offset><text>Ammonium transporter genes were amplified from genomic DNA or cDNA by PCR (Phusion, New England Biolabs). In both ScMEP2 and CaMEP2, Asn4 was replaced by a glutamine to prevent glycosylation. In order to allow transformation of yeast by recombination, the following primer extensions were used: forward 5′-GAAAAAACCCCGGATTCTAGAACTAGTGGATCCTCC-3′ and reverse 5′-TGACTCGAGTTATGCACCGTGGTGGTGATGGTGATG-3′. These primers result in a construct that lacks the cleavable N- and C-terminal tags present in the original vector, and replaces these with a C-terminal hexa-histidine tag. Recombination in yeast strain W303 pep4Δ was carried out using ∼50–100 ng of SmaI-digested vector 83νΔ (ref.) and at least a fourfold molar excess of PCR product via the lithium acetate method. Transformants were selected on SCD -His plates incubated at 30 °C. Construction of mutant CaMEP2 genes was done using the Q5 site-directed mutagenesis kit (NEB) per manufacturer's instructions. Three CaMep2 mutants were made for crystallization: the first mutant is a C-terminal truncation mutant 442Δ, lacking residues 443–480 including the AI domain. The second mutant, R452D/S453D, mimics the protein phosphorylated at Ser453. Given that phosphate is predominantly charged −2 at physiological pH, we introduced the second aspartate residue for Arg452. However, we also constructed the ‘single D', S453D CaMep2 variant.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>34827</offset><text>For expression, cells were grown in shaker flasks at 30 °C for ∼24 h in synthetic minimal medium lacking histidine and with 1% (w/v) glucose to a typical OD600 of 6–8. Cells were subsequently spun down for 15 min at 4,000g and resuspended in YP medium containing 1.5% (w/v) galactose, followed by another 16–20 h growth at 30 °C/160 r.p.m. and harvesting by centrifugation. Final OD600 values typically reached 18–20. Cells were lysed by bead beating (Biospec) for 5 × 1 min with 1 min intervals on ice, or by 1–2 passes through a cell disrupter operated at 35,000 p.s.i. (TS-Series 0.75 kW; Constant Systems). Membranes were collected from the suspension by centrifugation at 200,000g for 90 min (45Ti rotor; Beckmann Coulter). Membrane protein extraction was performed by homogenization in a 1:1 (w/w) mixture of dodecyl-β-D-maltoside and decyl-β-D-maltoside (DDM/DM) followed by stirring at 4 °C overnight. Typically, 1 g (1% w/v) of total detergent was used for membranes from 2 l of cells. The membrane extract was centrifuged for 35 min at 200,000g and the supernatant was loaded onto a 10-ml Nickel column (Chelating Sepharose; GE Healthcare) equilibrated in 20 mM Tris/300 mM NaCl/0.2% DDM, pH 8. The column was washed with 15 column volumes buffer containing 30 mM imidazole and eluted in 3 column volumes with 250 mM imidazole. Proteins were purified to homogeneity by gel filtration chromatography in 10 mM HEPES/100 mM NaCl/0.05% DDM, pH 7–7.5. For polishing and detergent exchange, a second gel filtration column was performed using various detergents. In the case of ScMep2, diffracting crystals were obtained only with 0.05% decyl-maltose neopentyl glycol. For the more stable CaMep2 protein, we obtained crystals in, for example, nonyl-glucoside, decyl-maltoside and octyl-glucose neopentyl glycol. Proteins were concentrated to 7–15 mg ml−1 using 100 kDa cutoff centrifugal devices (Millipore), flash-frozen and stored at −80 °C before use.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>36860</offset><text>Crystallization and structure determination</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>36904</offset><text>Crystallization screening trials by sitting drop vapour diffusion were set up at 4 and 20 °C using in-house screens and the MemGold 1 and 2 screens (Molecular Dimensions) with a Mosquito crystallization robot. Crystals were harvested directly from the initial trials or optimized by sitting or hanging drop vapour diffusion using larger drops (typically 2–3 μl total volume). Bar-shaped crystals for ScMep2 diffracting to 3.2 Å resolution were obtained from 50 mM 2-(N-morpholino)ethanesulfonic acid (MES)/0.2 M di-ammonium hydrogen phosphate/30% PEG 400, pH 6. They belong to space group P212121 and have nine molecules (three trimers) in the asymmetric unit (AU). Well-diffracting crystals for CaMep2 were obtained in space group P3 from 0.1 M MES/0.2 M lithium sulphate/20% PEG400, pH 6 (two molecules per AU). An additional crystal form in space group R3 was grown in 0.04 M Tris/0.04 M NaCl/27% PEG350 MME, pH 8 (one molecule per AU). Diffracting crystals for the phosporylation-mimicking CaMep2 DD mutant were obtained in space group P6322 from 0.1 M sodium acetate/15–20% PEG400, pH 5 (using decyl-maltoside as detergent; one molecule per AU), while S453D mutant crystals grew in 24% PEG400/0.05 M sodium acetate, pH 5.4/0.05 M magnesium acetate tetrahydrate/10 mM NH4Cl (space group R32; one molecule per AU). Finally, the 442Δ truncation mutant gave crystals under many different conditions, but most of these diffracted poorly or not at all. A reasonable low-resolution data set (3.4 Å resolution) was eventually obtained from a crystal grown in 24% PEG400/0.05 M sodium acetate/0.05 M magnesium acetate, pH 6.1 (space group R32). Diffraction data were collected at the Diamond Light Source and processed with XDS or HKL2000 (ref. ).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>38689</offset><text>For MR, a search model was constructed with Sculptor within Phenix, using a sequence alignment of ScMep2 with Archaeoglobus fulgidus Amt-1 (PDB ID 2B2H; ∼40% sequence identity to ScMep2). A clear solution with nine molecules (three trimers) in the AU was obtained using Phaser. The model was subsequently completed by iterative rounds of manual building within Coot followed by refinement within Phenix. The structures for WT CaMep2 were solved using the best-defined monomer of ScMep2 (60% sequence identity with CaMep2) in MR with Phaser, followed by automated model building within Phenix. Finally, the structures of the three mutant CaMep2 proteins were solved using WT CaMep2 as the search model. The data collection and refinement statistics for all six solved structures have been summarized in Supplementary Tables 1 and 2.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>39523</offset><text>Growth assays</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>39537</offset><text>The S. cerevisiae haploid triple mepΔ strain (Σ1278b MATα mep1::LEU2 mep2::LEU2 mep3::G418 ura3-52) and triple mepΔ npr1Δ strain (Σ1278b MATα mep1::LEU2 mep2::LEU2 mep3::G418 npr1::NAT1 ura3-52) were generated by integrating the NAT1 resistance gene at one NPR1 locus in the diploid strain MLY131 (ref.), followed by isolation of individual haploid strains. Cells were grown in synthetic minimal medium with glucose (2%) as the carbon source and ammonium sulphate (1 mM) or glutamate (0.1%) as the nitrogen source. Yeast cells were transformed as described. All DNA sequences encoding epitope-tagged ScMep2 and its mutant derivatives were generated by PCR and homologous recombination using the vector pRS316 (ref. ). In each case, the ScMEP2 sequences included the ScMEP2 promoter (1 kb), the ScMEP2 terminator and sequences coding for a His-6 epitope at the C-terminal end of the protein. All Mep2-His fusions contain the N4Q mutation to prevent glycosylation of Mep2 (ref.). All newly generated plasmid inserts were verified by DNA sequencing. For growth assays, S. cerevisiae cells containing plasmids expressing ScMep2 or mutant derivatives were grown overnight in synthetic minimal glutamate medium, washed, spotted by robot onto solid agar plates and culture growth followed by time course photography. Images were then processed to quantify the growth of each strain over 3 days as described.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>40966</offset><text>Protein modelling</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>40984</offset><text>The MODELLER (version 9.15) software package was used to build protein structures for MD simulations. This method was required to construct two complete protein models, the double mutant R452D/S453D (with the four missing residues from the X-ray structure added) and also the construct in which the mutation at position 452 is reverted to R, and D453 is replaced with a phosphoserine. The quality of these models was assessed using normalized Discrete Optimized Protein Energy (DOPE) values and the molpdf assessment function within the MODELLER package. The model R452D/S453D mutant has a molpdf assessment score of 1854.05, and a DOPE assessment score of -60920.55. The model of the S453J mutant has a molpdf assessment score of 1857.01 and a DOPE assessment score of −61032.15.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>41767</offset><text>MD simulations</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>41782</offset><text>WT and model structures were embedded into a pre-equilibrated lipid bilayer composed of 512 dipalmitoylphosphatidylcholine lipids using the InflateGRO2 computer programme. The bilayers were then solvated with the SPC water model and counterions were added to achieve a charge neutral state. All simulations were performed with the GROMACS package (version 4.5.5), and the GROMOS96 43a1p force field. During simulation time, the temperature was maintained at 310 K using the Nosé-Hoover thermostat with a coupling constant of 0.5 ps. Pressure was maintained at 1 bar using semi-isotropic coupling with the Parrinello-Rahman barostat and a time constant of 5 ps. Electrostatic interactions were treated using the smooth particle mesh Ewald algorithm with a short-range cutoff of 0.9 nm. Van der Waals interactions were truncated at 1.4 nm with a long-range dispersion correction applied to energy and pressure. The neighbour list was updated every five steps. All bonds were constrained with the LINCS algorithm, so that a 2-fs time step could be applied throughout. The phospholipid parameters for the dipalmitoylphosphatidylcholine lipids were based on the work of Berger. The embedded proteins were simulated for 200 ns each; a repeat simulation was performed for each system with different initial velocities to ensure reproducibility. To keep the c.p.u. times within reasonable limits, all simulations were performed on Mep2 monomers. This is also consistent with previous simulations for E. coli AmtB.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>43303</offset><text>Additional information</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>43326</offset><text>Accession codes: The atomic coordinates and the associated structure factors have been deposited in the Protein Data Bank (http:// www.pdbe.org) with accession codes 5AEX (ScMep2), 5AEZ(CaMep2; R3), 5AF1(CaMep2; P3), 5AID(CaMep2; 442D), 5AH3 (CaMep2; R452D/S453D) and 5FUF (CaMep2; S453D).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>43616</offset><text>How to cite this article: van den Berg, B. et al. Structural basis for Mep2 ammonium transceptor activation by phosphorylation. Nat. 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+Comput. Chem.</infon><infon key="type">ref</infon><infon key="volume">18</infon><infon key="year">1997</infon><offset>49120</offset><text>LINCS: a linear constraint solver for molecular simulations</text></passage><passage><infon key="fpage">2002</infon><infon key="lpage">2013</infon><infon key="name_0">surname:Berger;given-names:O.</infon><infon key="name_1">surname:Edholm;given-names:O.</infon><infon key="name_2">surname:Jähnig;given-names:F.</infon><infon key="pub-id_pmid">9129804</infon><infon key="section_type">REF</infon><infon key="source">Biophys. J.</infon><infon key="type">ref</infon><infon key="volume">72</infon><infon key="year">1997</infon><offset>49180</offset><text>Molecular dynamics simulations of a fluid bilayer of dipalmitoylphosphatidylcholine at full hydration, constant pressure, and constant temperature</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>49327</offset><text> The PyMOL Molecular Graphics System. version 1.7.4 (Schrödinger, LLC).</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>49400</offset><text>Author contributions B.v.d.B. performed the experiments related to Mep2 structure determination, designed research and wrote the paper. A.C. performed ammonium growth experiments of Mep variants. D.J. and S.K. performed modelling studies and MD simulations. A.B. collected the X-ray synchrotron data and maintained the Newcastle Structural Biology Laboratory. J.C.R. designed research related to the S. cerevisiae growth assays.</text></passage><passage><infon key="file">ncomms11337-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>49829</offset><text>X-ray crystal structures of Mep2 transceptors.</text></passage><passage><infon key="file">ncomms11337-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>49876</offset><text>(a) Monomer cartoon models viewed from the side for (left) A.
+fulgidus Amt-1 (PDB ID 2B2H), S. cerevisiae Mep2 (middle) and
+C. albicans Mep2 (right). The cartoons are in rainbow
+representation. The region showing ICL1 (blue), ICL3 (green) and the CTR
+(red) is boxed for comparison. (b) CaMep2 trimer viewed from the
+intracellular side (right). One monomer is coloured as in a and one
+monomer is coloured by B-factor (blue, low; red; high). The CTR is boxed.
+(c) Overlay of ScMep2 (grey) and CaMep2 (rainbow), illustrating
+the differences in the CTRs. All structure figures were generated with
+Pymol.</text></passage><passage><infon key="file">ncomms11337-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>50476</offset><text>Sequence conservation in ammonium transporters.</text></passage><passage><infon key="file">ncomms11337-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>50524</offset><text>ClustalW alignment of CaMep2, ScMep2, A. fulgidus Amt-1, E.
+coli AmtB and A. thaliana Amt-1;1. The secondary structure
+elements observed for CaMep2 are indicated, with the numbers corresponding
+to the centre of the TM segment. Important regions are labelled. The
+conserved RxK motif in ICL1 is boxed in blue, the ER motif in ICL2 in cyan,
+the conserved ExxGxD motif of the CTR in red and the AI region in yellow.
+Coloured residues are functionally important and correspond to those of the
+Phe gate (blue), the binding site Trp residue (magenta) and the twin-His
+motif (red). The Npr1 kinase site in the AI region is highlighted pink. The
+grey sequences at the C termini of CaMep2 and ScMep2 are not visible in the
+structures and are likely disordered.</text></passage><passage><infon key="file">ncomms11337-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>51276</offset><text>Growth of ScMep2 variants on low ammonium medium.</text></passage><passage><infon key="file">ncomms11337-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>51326</offset><text>(a) The triple mepΔ strain (black) and triple
+mepΔ npr1Δ strain (grey) containing plasmids
+expressing WT and variant ScMep2 were grown on minimal medium containing
+1 mM ammonium sulphate. The quantified cell density reflects
+logarithmic growth after 24 h. Error bars are the s.d. for three
+replicates of each strain (b) The strains used in a were also
+serially diluted and spotted onto minimal agar plates containing glutamate
+(0.1%) or ammonium sulphate (1 mM), and grown for 3 days at
+30 °C.</text></passage><passage><infon key="file">ncomms11337-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>51832</offset><text>Structural differences between Mep2 and bacterial ammonium
+transporters.</text></passage><passage><infon key="file">ncomms11337-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>51905</offset><text>(a) ICL1 in AfAmt-1 (light blue) and CaMep2 (dark blue), showing
+unwinding and inward movement in the fungal protein. (b) Stereo
+diagram viewed from the cytosol of ICL1, ICL3 (green) and the CTR (red) in
+AfAmt-1 (light colours) and CaMep2 (dark colours). The side chains of
+residues in the RxK motif as well as those of Tyr49 and His342 are labelled.
+The numbering is for CaMep2. (c) Conserved residues in ICL1-3 and the
+CTR. Views from the cytosol for CaMep2 (left) and AfAmt-1, highlighting the
+large differences in conformation of the conserved residues in ICL1 (RxK
+motif; blue), ICL2 (ER motif; cyan), ICL3 (green) and the CTR (red). The
+labelled residues are analogous within both structures. In b and
+c, the centre of the trimer is at top.</text></passage><passage><infon key="file">ncomms11337-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>52652</offset><text>Channel closures in Mep2.</text></passage><passage><infon key="file">ncomms11337-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>52678</offset><text>(a) Stereo superposition of AfAmt-1 and CaMep2 showing the residues of
+the Phe gate, His2 of the twin-His motif and the tyrosine residue Y49 in TM1
+that forms a hydrogen bond with His2 in CaMep2. (b) Surface views
+from the side in rainbow colouring, showing the two-tier channel block
+(indicated by the arrows) in CaMep2.</text></passage><passage><infon key="file">ncomms11337-f6.jpg</infon><infon key="id">f6</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>53000</offset><text>The Npr1 kinase target Ser453 is dephosphorylated and located in an
+electronegative pocket.</text></passage><passage><infon key="file">ncomms11337-f6.jpg</infon><infon key="id">f6</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>53092</offset><text>(a) Stereoviews of CaMep2 showing 2Fo–Fc
+electron density (contoured at 1.0 σ) for CTR residues
+Asp419-Met422 and for Tyr446-Thr455 of the AI region. For clarity, the
+residues shown are coloured white, with oxygen atoms in red and nitrogen
+atoms in blue. The phosphorylation target residue Ser453 is labelled in
+bold. (b) Overlay of the CTRs of ScMep2 (grey) and CaMep2 (green),
+showing the similar electronegative environment surrounding the
+phosphorylation site (P). The AI regions are coloured magenta. (c)
+Cytoplasmic view of the Mep2 trimer indicating the large distance between
+Ser453 and the channel exits (circles; Ile52 lining the channel exit is
+shown).</text></passage><passage><infon key="file">ncomms11337-f7.jpg</infon><infon key="id">f7</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>53761</offset><text>Effect of removal of the AI region on Mep2 structure.</text></passage><passage><infon key="file">ncomms11337-f7.jpg</infon><infon key="id">f7</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>53815</offset><text>(a) Side views for WT CaMep2 (left) and the truncation mutant
+442Δ (right). The latter is shown as a putty model according to
+B-factors to illustrate the disorder in the protein on the cytoplasmic side.
+Missing regions are labelled. (b) Stereo superpositions of WT CaMep2
+and the truncation mutant. 2Fo–Fc electron
+density (contoured at 1.0 σ) for residues Tyr49 and His342 is
+shown for the truncation mutant.</text></passage><passage><infon key="file">ncomms11337-f8.jpg</infon><infon key="id">f8</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>54233</offset><text>Phosphorylation causes conformational changes in the CTR.</text></passage><passage><infon key="file">ncomms11337-f8.jpg</infon><infon key="id">f8</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>54291</offset><text>(a) Cytoplasmic view of the DD mutant trimer, with WT CaMep2
+superposed in grey for one of the monomers. The arrow indicates the
+phosphorylation site. The AI region is coloured magenta. (b) Monomer
+side-view superposition of WT CaMep2 and the DD mutant, showing the
+conformational change and disorder around the ExxGxD motif. Side chains for
+residues 452 and 453 are shown as stick models.</text></passage><passage><infon key="file">ncomms11337-f9.jpg</infon><infon key="id">f9</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>54681</offset><text>Schematic model for phosphorylation-based regulation of Mep2 ammonium
+transporters.</text></passage><passage><infon key="file">ncomms11337-f9.jpg</infon><infon key="id">f9</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>54765</offset><text>(a) In the closed, non-phosphorylated state (i), the CTR (magenta) and
+ICL3 (green) are far apart with the latter blocking the intracellular
+channel exit (indicated with a hatched circle). Upon phosphorylation and
+mimicked by the CaMep2 S453D and DD mutants (ii), the region around the
+ExxGxD motif undergoes a conformational change that results in the CTR
+interacting with the inward-moving ICL3, opening the channel (full circle)
+(iii). The arrows depict the movements of important structural elements. The
+open-channel Mep2 structure is represented by archaebacterial Amt-1 and
+shown in lighter colours consistent with Fig. 4. As
+discussed in the text, similar structural arrangements may occur in plant
+AMTs. In this case however, the open channel corresponds to the
+non-phosphorylated state; phosphorylation breaks the CTR–ICL3
+interactions leading to channel closure. (b) Model based on AMT
+transporter analogy showing how phosphorylation of a
+Mep2 monomer might allosterically open channels in the entire trimer via
+disruption of the interactions between the CTR and ICL3 of a neighbouring
+monomer (arrow).</text></passage></document></collection>

raw_BioC_XML/PMC4854314_raw.xml

@@ -0,0 +1,28 @@
+<?xml version="1.0" encoding="UTF-8"?>
+<!DOCTYPE collection SYSTEM "BioC.dtd">
+<collection><source>PMC</source><date>20201222</date><key>pmc.key</key><document><id>4854314</id><infon key="license">CC BY</infon><passage><infon key="alt-title">RNA protects a nucleoprotein complex against radiation damage</infon><infon key="article-id_coden">ACSDAD</infon><infon key="article-id_doi">10.1107/S2059798316003351</infon><infon key="article-id_pii">S2059798316003351</infon><infon key="article-id_pmc">4854314</infon><infon key="article-id_pmid">27139628</infon><infon key="article-id_publisher-id">rr5121</infon><infon key="fpage">648</infon><infon key="issue">Pt 5</infon><infon key="kwd">radiation damage protein–RNA complex electron difference density specific damage decarboxylation</infon><infon key="license">This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits
+unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.</infon><infon key="lpage">657</infon><infon key="name_0">surname:Bury;given-names:Charles S.</infon><infon key="name_1">surname:McGeehan;given-names:John E.</infon><infon key="name_2">surname:Antson;given-names:Alfred A.</infon><infon key="name_3">surname:Carmichael;given-names:Ian</infon><infon key="name_4">surname:Gerstel;given-names:Markus</infon><infon key="name_5">surname:Shevtsov;given-names:Mikhail B.</infon><infon key="name_6">surname:Garman;given-names:Elspeth F.</infon><infon key="section_type">TITLE</infon><infon key="type">front</infon><infon key="volume">72</infon><infon key="year">2016</infon><offset>0</offset><text>RNA protects a nucleoprotein complex against radiation damage</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>62</offset><text>Systematic analysis of radiation damage within a protein–RNA complex over a large dose range (1.3–25 MGy) reveals significant differential susceptibility of RNA and protein. A new method of difference electron-density quantification is presented.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>315</offset><text>Radiation damage during macromolecular X-ray crystallographic data collection is still the main impediment for many macromolecular structure determinations. Even when an eventual model results from the crystallographic pipeline, the manifestations of radiation-induced structural and conformation changes, the so-called specific damage, within crystalline macromolecules can lead to false interpretations of biological mechanisms. Although this has been well characterized within protein crystals, far less is known about specific damage effects within the larger class of nucleoprotein complexes. Here, a methodology has been developed whereby per-atom density changes could be quantified with increasing dose over a wide (1.3–25.0 MGy) range and at higher resolution (1.98 Å) than the previous systematic specific damage study on a protein–DNA complex. Specific damage manifestations were determined within the large trp RNA-binding attenuation protein (TRAP) bound to a single-stranded RNA that forms a belt around the protein. Over a large dose range, the RNA was found to be far less susceptible to radiation-induced chemical changes than the protein. The availability of two TRAP molecules in the asymmetric unit, of which only one contained bound RNA, allowed a controlled investigation into the exact role of RNA binding in protein specific damage susceptibility. The 11-fold symmetry within each TRAP ring permitted statistically significant analysis of the Glu and Asp damage patterns, with RNA binding unexpectedly being observed to protect these otherwise highly sensitive residues within the 11 RNA-binding pockets distributed around the outside of the protein molecule. Additionally, the method enabled a quantification of the reduction in radiation-induced Lys and Phe disordering upon RNA binding directly from the electron density.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">title_1</infon><offset>2173</offset><text>Introduction   </text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2190</offset><text>With the wide use of high-flux third-generation synchrotron sources, radiation damage (RD) has once again become a dominant reason for the failure of structure determination using macromolecular crystallography (MX) in experiments conducted both at room temperature and under cryocooled conditions (100 K). Significant progress has been made in recent years in understanding the inevitable manifestations of X-ray-induced RD within protein crystals, and there is now a body of literature on possible strategies to mitigate the effects of RD (e.g. Zeldin, Brockhauser et al., 2013; Bourenkov &amp; Popov, 2010). However, there is still no general consensus within the field on how to minimize RD during MX data collection, and debates on the dependence of RD progression on incident X-ray energy (Shimizu et al., 2007; Liebschner et al., 2015) and the efficacy of radical scavengers (Allan et al., 2013) have yet to be resolved.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3116</offset><text>RD manifests in two forms. Global radiation damage is observed within reciprocal space as the overall decay of the summed intensity of reflections detected within the diffraction pattern as dose increases (Garman, 2010; Murray &amp; Garman, 2002). Dose is defined as the absorbed energy per unit mass of crystal in grays (Gy; 1 Gy = 1 J kg−1), and is the metric against which damage progression should be monitored during MX data collection, as opposed to time. At 100 K, an experimental dose limit of 30 MGy has been recommended as an upper limit beyond which the biological information derived from any macromolecular crystal may be compromised (Owen et al., 2006).</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3792</offset><text> Specific radiation damage (SRD) is observed in the real-space electron density, and has been detected at much lower doses than any observable decay in the intensity of reflections. Indeed, the C—Se bond in selenomethionine, the stability of which is key for the success of experimental phasing methods, can be cleaved at a dose as low as 2 MGy for a crystal maintained at 100 K (Holton, 2007). SRD has been well characterized in a large range of proteins, and is seen to follow a reproducible order: metallo-centre reduction, disulfide-bond cleavage, acidic residue decarboxylation and methionine methylthio cleavage (Ravelli &amp; McSweeney, 2000; Burmeister, 2000; Weik et al., 2000; Yano et al., 2005). Furthermore, damage susceptibility within each residue type follows a preferential ordering influenced by a combination of local environment factors (solvent accessibility, conformational strain, proximity to active sites/high X-ray cross-section atoms; Holton, 2009). Deconvoluting the individual roles of these parameters has been surprisingly challenging, with factors such as solvent accessibility currently under active investigation (Weik et al., 2000; Fioravanti et al., 2007; Gerstel et al., 2015).</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>5008</offset><text>There are a number of cases where SRD manifestations have compromised the biological information extracted from MX-determined structures at much lower doses than the recommended 30 MGy limit, leading to false structural interpretations of protein mechanisms. Active-site residues appear to be particularly susceptible, particularly for photosensitive proteins and in instances where chemical strain is an intrinsic feature of the reaction mechanism. For instance, structure determination of the purple membrane protein bacterio­rhodopsin required careful corrections for radiation-induced structural changes before the correct photosensitive intermediate states could be isolated (Matsui et al., 2002). The significant chemical strain required for catalysis within the active site of phosphoserine aminotransferase has been observed to diminish during X-ray exposure (Dubnovitsky et al., 2005).</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>5906</offset><text>Since the majority of SRD studies to date have focused on proteins, much less is known about the effects of X-ray irradiation on the wider class of crystalline nucleoprotein complexes or how to correct for such radiation-induced structural changes. Understanding RD to such complexes is crucial, since DNA is rarely naked within a cell, instead dynamically interacting with proteins, facilitating replication, transcription, modification and DNA repair. As of early 2016, &gt;5400 nucleoprotein complex structures have been deposited within the PDB, with 91% solved by MX. It is essential to understand how these increasingly complex macromolecular structures are affected by the radiation used to solve them. Nucleoproteins also represent one of the main targets of radiotherapy, and an insight into the damage mechanisms induced by X-ray irradiation could inform innovative treatments.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>6791</offset><text>When a typical macromolecular crystal is irradiated with ionizing X-rays, each photoelectron produced via interactions with both the macromolecule (direct damage) and solvent (indirect damage) can induce cascades of up to 500 secondary low-energy electrons (LEEs) that are capable of inducing further ionizations. Investigations on sub-ionization-level LEEs (0–15 eV) interacting with both dried and aqueous oligonucleotides (Alizadeh &amp; Sanche, 2014; Simons, 2006) concluded that resonant electron attachment to DNA bases and the sugar-phosphate backbone could lead to the preferential cleavage of strong (∼4 eV, 385 kJ mol−1) sugar-phosphate C—O covalent bonds within the DNA backbone and then base-sugar N1—C bonds, eventually leading to single-strand breakages (SSBs; Ptasińska &amp; Sanche, 2007). Electrons have been shown to be mobile at 77 K by electron spin resonance spectroscopy studies (Symons, 1997; Jones et al., 1987), with rapid electron quantum tunnelling and positive hole migration along the protein backbone and through stacked DNA bases indicated as a dominant mechanism by which oxidative and reductive damage localizes at distances from initial ionization sites at 100 K (O’Neill et al., 2002).</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>8029</offset><text>The investigation of naturally forming nucleoprotein complexes circumvents the inherent challenges in making controlled comparisons of damage mechanisms between protein and nucleic acids crystallized separately. Recently, for a well characterized bacterial protein–DNA complex (C.Esp1396I; PDB entry 3clc; resolution 2.8 Å; McGeehan et al., 2008) it was concluded that over a wide dose range (2.1–44.6 MGy) the protein was far more susceptible to SRD than the DNA within the crystal (Bury et al., 2015). Only at doses above 20 MGy were precursors of phosphodiester-bond cleavage observed within AT-rich regions of the 35-mer DNA.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>8670</offset><text>For crystalline complexes such as C.Esp1396I, whether the protein is intrinsically more susceptible to X-ray-induced damage or whether the protein scavenges electrons to protect the DNA remains unclear in the absence of a non-nucleic acid-bound protein control obtained under exactly the same crystallization and data-collection conditions. To monitor the effects of nucleic acid binding on protein damage susceptibility, a crystal containing two protein molecules per asymmetric unit, only one of which was bound to RNA, is reported here (Fig. 1 ▸). Using newly developed methodology, we present a controlled SRD investigation at 1.98 Å resolution using a large (∼91 kDa) crystalline protein–RNA complex: trp RNA-binding attenuation protein (TRAP) bound to a 53 bp RNA sequence (GAGUU)10GAG (PDB entry 1gtf; Hopcroft et al., 2002). TRAP consists of 11 identical subunits assembled into a ring with 11-fold rotational symmetry. It binds with high affinity (K d ≃ 1.0 nM) to RNA segments containing 11 GAG/UAG triplets separated by two or three spacer nucleotides (Elliott et al., 2001) to regulate the transcription of tryptophan biosynthetic genes in Bacillus subtilis (Antson et al., 1999). In this structure, the bases of the G1-A2-G3 nucleotides form direct hydrogen bonds to the protein, unlike the U4-U5 nucleotides, which appear to be more flexible.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>10044</offset><text>Ten successive 1.98 Å resolution MX data sets were collected from the same TRAP–RNA crystal to analyse X-ray-induced structural changes over a large dose range (d 1 = 1.3 MGy to d 10 = 25.0 MGy). To avoid the previous necessity for visual inspection of electron-density maps to detect SRD sites, a computational approach was designed to quantify the electron-density change for each refined atom with increasing dose, thus providing a rapid systematic method for SRD study on such large multimeric complexes. By employing the high 11-fold structural symmetry within each TRAP macromolecule, this approach permitted a thorough statistical quantification of the RD effects of RNA binding to TRAP.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>10748</offset><text>Materials and methods   </text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>10774</offset><text>RNA synthesis and protein preparation   </text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>10816</offset><text>As previously described (Hopcroft et al., 2002), the 53-base RNA (GAGUU)10GAG was synthesized by in vitro transcription with T7 RNA polymerase and gel-purified. TRAP from B. stearothermophilus was overexpressed in Escherichia coli and purified.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>11061</offset><text>Crystallization   </text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>11081</offset><text>TRAP–RNA crystals were prepared using a previously established hanging-drop crystallization protocol (Antson et al., 1999). By using a 2:1 molar ratio of TRAP to RNA, crystals successfully formed from the protein–RNA complex (∼15 mg ml−1) in a solution containing 70 mM potassium phosphate pH 7.8 and 10 mM l-tryptophan. The reservoir consisted of 0.2 M potassium glutamate, 50 mM triethanol­amine pH 8.0, 10 mM MgCl2, 8–11% monomethyl ether PEG 2000. In order to accelerate crystallization, a further gradient was induced by adding 0.4 M KCl to the reservoir after 1.5 µl protein solution had been mixed with an equal volume of the reservoir solution. Wedge-shaped crystals of approximate length 70 µm (longest dimension) grew within 3 d and were vitrified and stored in liquid nitrogen immediately after growth. The cryosolution consisted of 12% monomethyl ether PEG 2000, 30 mM triethanolamine pH 8.0, 6 mM l-tryptophan, 0.1 M potassium glutamate, 35 mM potassium phosphate pH 7.8, 5 mM MgCl2 with 25% 2-methyl-2,4-pentanediol (MPD) included as a cryoprotectant.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>12192</offset><text>X-ray data collection   </text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>12218</offset><text>Data were collected at 100 K from a wedge-shaped TRAP–RNA crystal of approximate dimensions 70 × 20 × 40 µm (see Supplementary Fig. S2) on beamline ID14-4 at the ESRF using an incident wavelength of 0.940 Å (13.2 keV) and an ADSC Q315R mosaic CCD detector at 304.5 mm from the crystal throughout the data collection. The beam size was slitted to 0.100 mm (vertical) × 0.160 mm (horizontal), with a uniformly distributed profile, such that the crystal was completely bathed within the beam throughout data collection. Ten successive (1.98 Å resolution) 180° data sets (with Δφ = 1°) were collected over the same angular range from a TRAP–RNA crystal at 28.9% beam transmission. The TRAP–RNA macromolecule crystallized in space group C2, with unit-cell parameters a = 140.9, b = 110.9, c = 137.8 Å, α = γ = 90, β = 137.8° (the values quoted are for the first data set; see Supplementary Table S1 for subsequent values). For the first nine data sets the attenuated flux was recorded to be ∼5 × 1011 photons s−1. A beam refill took place immediately before data set 10, requiring a flux-scale factor increase of 1.42 to be applied, based on the ratio of observed relative intensity I D/I 1 at data set 10 to that extrapolated from data set 9.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>13507</offset><text>Dose calculation   </text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>13528</offset><text> RADDOSE-3D (Zeldin, Gerstel et al., 2013) was used to calculate the absorbed dose distribution during each data set (see input file; Supplementary Figs. S1 and S2). The crystal composition was calculated from the deposited TRAP–RNA structure (PDB entry 1gtf; Hopcroft et al., 2002). Crystal absorption coefficients were calculated in RADDOSE-3D using the concentration (mmol l−1) of solvent heavy elements from the crystallization conditions. The beam-intensity profile was modelled as a uniform (‘top-hat’) distribution. The diffraction-weighted dose (DWD) values (Zeldin, Brock­hauser et al., 2013) are given in Supplementary Table S1.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>14178</offset><text>Data processing and model refinement   </text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>14219</offset><text>Each data set was integrated using iMosflm (Leslie &amp; Powell, 2007) and was scaled using AIMLESS (Evans &amp; Murshudov, 2013; Winn et al., 2011) using the same 5% R free set of test reflections for each data set. To phase the structure obtained from the first data set, molecular replacement was carried out with Phaser (McCoy et al., 2007), using an identical TRAP–RNA structure (PDB entry 1gtf; resolution 1.75 Å; Hopcroft et al., 2002) as a search model. The resulting TRAP–RNA structure (TR1) was refined using REFMAC5 (Murshudov et al., 2011), initially using rigid-body refinement, followed by repeated cycles of restrained, TLS and isotropic B-factor refinement, coupled with visual inspection in Coot (Emsley et al., 2010). TR1 was refined to 1.98 Å resolution, with a dimeric assembly of non-RNA-bound and RNA-bound TRAP rings within the asymmetric unit. Consistent with previous structures of the TRAP–RNA complex, the RNA sequence termini were not observed within the 2F o − F c map; the first spacer (U4) was then modelled at all 11 repeats around the TRAP ring and the second spacer (U5) was omitted from the final refined structure. For the later data sets, the observed structure-factor amplitudes from each separately scaled data set (output from AIMLESS) were combined with the phases of TR1 and the resulting higher-dose model was refined with phenix.refine (Adams et al., 2010) using only rigid-body and isotropic B-factor refinement. During this refinement, the TRAP–RNA complex and nonbound TRAP ring were treated as two separate rigid bodies within the asymmetric unit. Supplementary Table S1 shows the relevant summary statistics.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>15885</offset><text> D loss metric calculation   </text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>15916</offset><text>The CCP4 program CAD was used to create a series of nine merged .mtz files combining observed structure-factor amplitudes for the first data set F obs(d 1) with each later data set F obs(d n) (for n = 2, …, 10). All later data sets were scaled against the initial low-dose data set in SCALEIT. For each data set an atom-tagged .map file was generated using the ATMMAP mode in SFALL (Winn et al., 2011). A full set of nine Fourier difference maps F obs(d n) − F obs(d 1) were calculated using FFT (Ten Eyck, 1973) over the full TRAP–RNA unit-cell dimensions, with the same grid-sampling dimensions as the atom-tagged .map file. All maps were cropped to the TRAP asymmetric unit in MAPMASK. Comparing the atom-tagged .map file and F obs(d n) − F obs(d 1) difference map at each dose, each refined atom was assigned a set of density-change values X. The maximum density-loss metric, D loss (units of e Å−3), was calculated to quantify the per-atom electron-density decay at each dose, assigned as the absolute magnitude of the most negative Fourier difference map voxel value in a local volume around each atom as defined by the set X.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>17062</offset><text>Model system calculation   </text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>17091</offset><text>Model calculations were run for the simple amino acids glutamate and aspartate. In order to avoid decarboxylation at the C-terminus instead of the side chain on the Cα atom, the C-terminus of each amino acid was methylated. While the structures of the closed shell acids are well known, the same is not true of those in the oxidized state. The quantum-chemical calculations employed were chosen to provide a satisfactory description of the structure of such radical species and also provide a reliable estimation of the relative C—C(O2) bond strengths, which are otherwise not available.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>17683</offset><text>Structures of methyl-terminated (at the N- and C-termini) carboxylates were determined using analytic energy gradients with density functional theory (B3LYP functional; Becke, 1993) and a flexible basis set of polarized valence triple-zeta size with diffuse functions on the non-H atoms [6-311+G(d,p)] in the Gaussian 09 computational chemistry package (Frisch et al., 2009). The stationary points obtained were characterized as at least local minima by examination of the associated analytic Hessian. Effects of the medium were modelled using a dielectric cavity approach (Tomasi et al., 1999) parameterized for water.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>18303</offset><text>Results   </text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>18315</offset><text>Per-atom quantification of electron density   </text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>18363</offset><text>To quantify the exact effects of nucleic acid binding to a protein on SRD susceptibility, a high-throughput and automated pipeline was created to systematically calculate the electron-density change for every refined atom within the TRAP–RNA structure as a function of dose. This provides an atom-specific quantification of density–dose dynamics, which was previously lacking within the field. Previous studies have characterized SRD sites by reporting magnitudes of F obs(d n) − F obs(d 1) Fourier difference map peaks in terms of the sigma (σ) contour level (the number of standard deviations from the mean map electron-density value) at which peaks become visible. However, these σ levels depend on the standard deviation values of the map, which can deviate between data sets, and are thus unsuitable for quantitative comparison of density between different dose data sets. Instead, we use here a maximum density-loss metric (D loss), which is the per-atom equivalent of the magnitude of these negative Fourier difference map peaks in units of e Å−3. Large positive D loss values indicate radiation-induced atomic disordering reproducibly throughout the unit cells with respect to the initial low-dose data set.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>19592</offset><text>For each TRAP–RNA data set, the D loss metric successfully identified the recognized forms of protein SRD (Fig. 2 ▸ a), with clear Glu and Asp side-chain decarboxylation even in the first difference map calculated (3.9 MGy; Fig. 3 ▸ a). The main sequence of TRAP does not contain any Trp and Cys residues (and thus contains no disulfide bonds). The substrate Trp amino-acid ligands also exhibited disordering of the free terminal carboxyl groups at higher doses (Fig. 2 ▸ a); however, no clear Fourier difference peaks could be observed visually. Even for radiation-insensitive residues (e.g. Gly) the average D loss increases with dose: this is the effect of global radiation damage, since as dose increases the electron density associated with each refined atom becomes weaker as the atomic occupancy decreases (Fig. 2 ▸ b). Only Glu and Asp residues exhibit a rate of D loss increase that consistently exceeds the average decay (Fig. 2 ▸ b, dashed line) at each dose. Additionally, the density surrounding ordered solvent molecules was determined to significantly diminish with increasing dose (Fig. 2 ▸ b). The rate of D loss (attributed to side-chain decarboxylation) was consistently larger for Glu compared with Asp residues over the large dose range (Fig. 2 ▸ b and Supplementary Fig. S3); this observation is consistent with our calculations on model systems (see above) that suggest that, without considering differential hydrogen-bonding environments, CO2 loss is more exothermic by around 8 kJ mol−1 from oxidized Glu residues than from their Asp counterparts.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>21189</offset><text>RNA is less susceptible to electron-density loss than protein within the TRAP–RNA complex   </text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>21285</offset><text>Visual inspection of Fourier difference maps illustrated the clear lack of RNA electron-density degradation with increasing dose compared with the obvious protein damage manifestations (Figs. 3 ▸ b and 3 ▸ c). Only at the highest doses investigated (&gt;20 MGy) was density loss observed at the RNA phosphate and C—O bonds of the phosphodiester backbone. However, the median D loss was lower by a factor of &gt;2 for RNA P atoms than for Glu and Asp side-chain groups at 25.0 MGy (Supplementary Fig. S4), and furthermore could not be numerically distinguished from Gly Cα atoms within TRAP, which are not radiation-sensitive at the doses tested here (Supplementary Fig. S3).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>21965</offset><text>RNA binding protects radiation-sensitive residues   </text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>22019</offset><text>For the large number of acidic residues per TRAP ring (four Asp and six Glu residues per protein monomer), a strong dependence of decarboxylation susceptibility on local environment was observed (Fig. 4 ▸). For each Glu Cδ or Asp Cγ atom, D loss provided a direct measure of the rate of side-chain carboxyl-group disordering and subsequent decarboxylation. For acidic residues with no differing interactions between nonbound and bound TRAP (Fig. 4 ▸ a), similar damage was apparent between the two rings within the asymmetric unit, as expected. However, TRAP residues directly on the RNA-binding interfaces exhibited greater damage accumulation in nonbound TRAP (Fig. 4 ▸ b), and for residues at the ring–ring interfaces (where crystal contacts were detected) bound TRAP exhibited enhanced SRD accumulation (Fig. 4 ▸ c).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>22856</offset><text>Three acidic residues (Glu36, Asp39 and Glu42) are involved in RNA interactions within each of the 11 TRAP ring subunits, and Fig. 5 ▸ shows their density changes with increasing dose. Hotelling’s T-squared test (the multivariate counterpart of Student’s t-test) was used to reject the null hypothesis that the means of the D loss metric were equal for the bound and nonbound groups in Fig. 5 ▸.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>23260</offset><text>A significant reduction in D loss is seen for Glu36 in RNA-bound compared with nonbound TRAP, indicative of a lower rate of side-chain decarboxylation (Fig. 5 ▸ a; p = 6.06 × 10−5). For each TRAP ring subunit, the Glu36 side-chain carboxyl group accepts a pair of hydrogen bonds from the two N atoms of the G3 RNA base. In our analysis, Asp39 in the TRAP–(GAGUU)10GAG structure appears to exhibit two distinct hydrogen bonds to the G1 base within each of the 11 TRAP–RNA interfaces, as does Glu36 to G3; however, the reduction in density disordering upon RNA binding is far less significant for Asp39 than for Glu36 (Fig. 5 ▸ b, p = 0.0925).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>23913</offset><text>RNA binding reduces radiation-induced disorder on the atomic scale   </text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>23984</offset><text>One oxygen (O∊1) of Glu42 appears to form a hydrogen bond to a nearby water within each TRAP RNA-binding pocket, with the other (O∊2) being involved in a salt-bridge interaction with Arg58 (Hopcroft et al., 2002; Antson et al., 1999). Salt-bridge interactions have previously been suggested to reduce the glutamate decarboxylation rate within the large (∼62.4 kDa) myrosinase protein structure (Burmeister, 2000). A significant difference was observed between the D loss dynamics for the nonbound/bound Glu42 O∊1 atoms (Fig. 5 ▸ c; p = 0.007) but not for the Glu42 O∊2 atoms (Fig. 5 ▸ d; p = 0.239), indicating that the stabilizing strength of this salt-bridge interaction was conserved upon RNA binding and that the water-mediated hydrogen bond had a greater relative susceptibility to atomic disordering in the absence of RNA. The density-change dynamics were statistically indistinguishable between bound and nonbound TRAP for each Glu42 carboxyl group Cδ atom (p = 0.435), indicating that upon RNA binding the conserved salt-bridge interaction ultimately dictated the overall Glu42 decarboxylation rate.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>25115</offset><text>The RNA-stabilizing effect was not restricted to radiation-sensitive acidic residues. The side chain of Phe32 stacks against the G3 base within the 11 TRAP RNA-binding interfaces (Antson et al., 1999). With increasing dose, the D loss associated with the Phe32 side chain was significantly reduced upon RNA binding (Fig. 5 ▸ e; Phe32 Cζ; p = 0.0014), an indication that radiation-induced conformation disordering of Phe32 had been reduced. The extended aliphatic Lys37 side chain stacks against the nearby G1 base, making a series of nonpolar contacts within each RNA-binding interface. The D loss for Lys37 side-chain atoms was also reduced when stacked against the G1 base (Fig. 5 ▸ f; p = 0.0243 for Lys37 C∊ atoms). Representative Phe32 and Lys37 atoms were selected to illustrate these trends.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_1</infon><offset>25925</offset><text>Discussion   </text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>25940</offset><text>Here, MX radiation-induced specific structural changes within the large TRAP–RNA assembly over a large dose range (1.3–25.0 MGy) have been analysed using a high-throughput quantitative approach, providing a measure of the electron-density distribution for each refined atom with increasing dose, D loss. Compared with previous studies, the results provide a further step in the detailed characterization of SRD effects in MX. Our method­ology, which eliminated tedious and error-prone visual inspection, permitted the determination on a per-atom basis of the most damaged sites, as characterized by F obs(d n) − F obs(d 1) Fourier difference map peaks between successive data sets collected from the same crystal. Here, it provided the precision required to quantify the role of RNA in the damage susceptibilities of equivalent atoms between RNA-bound and nonbound TRAP, but it is applicable to any MX SRD study.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>26862</offset><text>The RNA was found to be substantially more radiation-resistant than the protein, even at the highest doses investigated (∼25.0 MGy), which is in strong concurrence with our previous SRD investigation of the C.Esp1396I protein–DNA complex (Bury et al., 2015). Consistent with that study, at high doses of above ∼20 MGy, F obs(d n) − F obs(d 1) map density was detected around P, O3′ and O5′ atoms of the RNA backbone, with no significant difference density localized to RNA ribose and basic subunits. RNA backbone disordering thus appears to be the main radiation-induced effect in RNA, with the protein–base interactions maintained even at high doses (&gt;20 MGy). The U4 phosphate exhibited marginally larger D loss values above 20 MGy than G1, A2 and G3 (Supplementary Fig. S4). Since U4 is the only refined nucleotide not to exhibit significant base–protein interactions around TRAP (with a water-mediated hydrogen bond detected in only three of the 11 subunits and a single Arg58 hydrogen bond suggested in a further four subunits), this increased U4 D loss can be explained owing to its greater flexibility. At 25.0 MGy, the magnitude of the RNA backbone D loss was of the same order as for the radiation-insensitive Gly Cα atoms and on average less than half that of the acidic residues of the protein (Supplementary Fig. S3). Consequently, no clear single-strand breaks could be located, and since RNA-binding within the current TRAP–(GAGUU)10GAG complex is mediated predominantly through base–protein interactions, the biological integrity of the RNA complex was dictated by the rate at which protein decarboxylation occurred.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>28527</offset><text>RNA interacting with TRAP was shown to offer significant protection against radiation-induced structural changes. Both Glu36 and Asp39 bind directly to RNA, each through two hydrogen bonds to guanine bases (G3 and G1, respectively). However, compared with Asp39, Glu36 is strikingly less decarboxylated when bound to RNA (Fig. 4 ▸). This is in good agreement with previous mutagenesis and nucleoside analogue studies (Elliott et al., 2001), which indicated that the G1 nucleotide does not bind to TRAP as strongly as do A2 and G3, and plays little role in the high RNA-binding affinity of TRAP (K d ≃ 1.1 ± 0.4 nM). For Glu36 and Asp39, no direct quantitative correlation could be established between hydrogen-bond length and D loss (linear R 2 of &lt;0.23 for all doses; Supplementary Fig. S5). Thus, another factor must be responsible for this clear reduction in Glu36 CO2 decarboxyl­ation in RNA-bound TRAP. The Glu36 carboxyl side chain also potentially forms hydrogen bonds to His34 and Lys56, but since these interactions are conserved irrespective of G3 nucleotide binding, this cannot directly account for the stabilization effect on Glu36 in RNA-bound TRAP. Radiation-induced decarboxylation has been proposed to be mediated by preferential positive-hole migration to the side-chain carboxyl group, with rapid proton transfer trapping the hole at the carboxyl group (Burmeister, 2000; Symons, 1997):where the forward rate is K 1 and the backward rate is K −1, where the forward rate is K 2.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>30034</offset><text>When bound to RNA, the average solvent-accessible area of the Glu36 side-chain O atoms is reduced from ∼15 to 0 Å2. We propose that with no solvent accessibility Glu36 decarboxylation is inhibited, since the CO2-formation rate K 2 is greatly reduced, and suggest that steric hindrance prevents each radicalized Glu36 CO2 group from achieving the planar conformation required for complete dissociation from TRAP. The electron-recombination rate K −1 remains high, however, owing to rapid electron migration through the protein–RNA complex to refill the Glu36 positive hole (the precursor for Glu decarboxylation). Upon RNA binding, the Asp39 side-chain carboxyl group solvent-accessible area changes from ∼75 to 35 Å2, still allowing a high CO2-formation rate K 2.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>30812</offset><text>Previous studies have reported inconsistent results concerning the dependence of the acidic residue decarboxylation rate on solvent accessibility (Weik et al., 2000; Fioravanti et al., 2007; Gerstel et al., 2015). The prevalence of radical attack from solvent channels surrounding the protein in the crystal is a questionable cause, considering previous observations indicating that the strongly oxidizing hydroxyl radical is immobile at 100 K (Allan et al., 2013; Owen et al., 2012). Furthermore, the suggested electron hole-trapping mechanism which induces decarboxylation within proteins at 100 K has no clear mechanistic dependence on the solvent-accessible area of each carboxyl group. By comparing equivalent acidic residues with and without RNA, we have now deconvoluted the role of solvent accessibility from other local protein environment factors, and thus propose a suitable mechanism by which exceptionally low solvent accessibility can reduce the rate of decarboxylation. Overall, no direct correlation between solvent accessibility and decarboxylation susceptibility was observed, but it is very clear that inaccessible residues are protected.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>31974</offset><text>Apart from these RNA-binding interfaces, RNA binding was seen to enhance decarboxylation for residues Glu50, Glu71 and Glu73, all of which are involved in crystal contacts between TRAP rings (Fig. 4 ▸ c). However, for each of these residues the exact crystal contacts are not preserved between bound and nonbound TRAP or even between monomers within one TRAP ring. For example, in bound TRAP, Glu73 hydrogen-bonds to a nearby lysine on each of the 11 subunits, whereas in nonbound TRAP no such interaction exists and Glu73 interacts with a variable number of refined waters in each subunit. Thus, the dependence of decarboxylation rates on these interactions could not be established.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>32661</offset><text>Radiation-induced side-chain conformational changes have been poorly characterized in previous SRD investigations owing to their strong dependence on packing density and geometric strain. Such structural changes are known to have significant roles within enzymatic pathways, and experimenters must be aware of these possible confounding factors when assigning true functional mechanisms using MX. Our results show that RNA binding to TRAP physically stabilizes non-acidic residues within the TRAP macromolecule, most notably Lys37 and Phe32, which stack against the G1 and G3 bases, respectively. It has been suggested (Burmeister, 2000) that Tyr residues can lose their aromatic –OH group owing to radiation-induced effects; however, no energetically favourable pathway for –OH cleavage exists and this has not been detected in aqueous radiation-chemistry studies. In TRAP, D loss increased at a similar rate for both the Tyr O atoms and aromatic ring atoms, suggesting that full ring conformational disordering is more likely. Indeed, no convincing reproducible Fourier difference peaks above the background map noise were observed around any Tyr terminal –OH groups.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>33837</offset><text>The RNA-stabilization effects on protein are observed at short ranges and are restricted to within the RNA-binding interfaces around the TRAP ring. For example, Asp17 is located ∼6.8 Å from the G1 base, outside the RNA-binding interfaces, and has indistinguishable Cγ atom D loss dose-dynamics between RNA-bound and nonbound TRAP (p &gt; 0.9). An increase in the dose at which functionally important residues remain intact has biological ramifications for understanding the mechanisms at which ionizing radiation damage is mitigated within naturally forming DNA–protein and RNA–protein complexes. Observations of lower protein radiation-sensitivity in DNA-bound forms have been recorded in solution at RT at much lower doses (∼1 kGy) than those used for typical MX experiments [e.g. an oestrogen response element–receptor complex (Stísová et al., 2006) and a DNA glycosylase and its abasic DNA target site (Gillard et al., 2004)]. In these studies, the main damaging species is predicted to be the oxidizing hydroxyl radical produced through solvent irradiation, which is known to add to double covalent bonds within both DNA and RNA bases to induce strand breaks and base modification (Spotheim-Maurizot &amp; Davídková, 2011; Chance et al., 1997). It was suggested that physical screening of DNA by protein shielded the DNA–protein interaction sites from radical damage, yielding an extended life-dose for the nucleoprotein complex compared with separate protein and DNA constituents at RT.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>35345</offset><text>However, in the current MX study at 100 K, the main damaging species are believed to be migrating LEEs and holes produced directly within the protein–RNA components or in closely associated solvent. The results presented here suggest that biologically relevant nucleoprotein complexes also exhibit prolonged life-doses under the effect of LEE-induced structural changes, involving direct physical protection of key RNA-binding residues. Such reduced radiation-sensitivity in this case ensures that the interacting protein remains bound long enough to the RNA to complete its function, even whilst exposed to ionizing radiation. Within the nonbound TRAP macromolecule, the acidic residues within the unoccupied RNA-binding interfaces (Asp39, Glu36, Glu42) are notably amongst the most susceptible residues within the asymmetric unit (Fig. 4 ▸). When exposed to X-rays, these residues will be preferentially damaged by X-rays and subsequently reduce the affinity with which TRAP binds to RNA. Within the cellular environment, this mechanism could reduce the risk that radiation-damaged proteins might bind to RNA, thus avoiding the detrimental introduction of incorrect DNA-repair, transcriptional and base-modification pathways.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>36579</offset><text>The Python scripts written to calculate the per atom D loss metric are available from the authors on request.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">title_1</infon><offset>36689</offset><text>Related literature   </text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>36712</offset><text>The following references are cited in the Supporting Information for this article: Chen et al. (2010).</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">title_1</infon><offset>36815</offset><text>Supplementary Material</text></passage><passage><infon key="section_type">REF</infon><infon key="type">title</infon><offset>36838</offset><text>References</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>36849</offset><text>Adams, P. D. et al. (2010). Acta Cryst. D66, 213–221.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>36905</offset><text>Alizadeh, E. &amp; Sanche, L. (2014). Eur. Phys. J. D, 68, 97.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>36964</offset><text>Allan, E. G., Kander, M. C., Carmichael, I. &amp; Garman, E. F. (2013). J. Synchrotron Rad. 20, 23–36.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>37065</offset><text>Antson, A. 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USA, 97, 623–628.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>40994</offset><text>Winn, M. D. et al. (2011). Acta Cryst. D67, 235–242.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>41049</offset><text>Yano, J., Kern, J., Irrgang, K. D., Latimer, M. J., Bergmann, U., Glatzel, P., Pushkar, Y., Biesiadka, J., Loll, B., Sauer, K., Messinger, J., Zouni, A. &amp; Yachandra, V. K. (2005). Proc. Natl Acad. Sci. USA, 102, 12047–12052.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>41276</offset><text>Zeldin, O. B., Brockhauser, S., Bremridge, J., Holton, J. M. &amp; Garman, E. F. (2013). Proc. Natl Acad. Sci. USA, 110, 20551–20556.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>41408</offset><text>Zeldin, O. B., Gerstel, M. &amp; Garman, E. F. (2013). J. Appl. Cryst. 46, 1225–1230.</text></passage><passage><infon key="file">d-72-00648-fig1.jpg</infon><infon key="id">fig1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>41492</offset><text>The TRAP–(GAGUU)10GAG complex asymmetric unit (PDB entry 1gtf; Hopcroft et al., 2002). Bound tryptophan ligands are represented as coloured spheres. RNA is shown is yellow.</text></passage><passage><infon key="file">d-72-00648-fig2.jpg</infon><infon key="id">fig2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>41667</offset><text>(a) Electron-density loss sites as indicated by D
+loss in the TRAP–RNA complex crystal by residue/nucleotide type for five doses [sites determined above the 4× average D
+loss threshold, calculated over the TRAP–RNA structure for the first difference map: F
+obs(d
+2) − F
+obs(d
+1)]. Cumulative frequencies are normalized to both the total number of non-H atoms per residue/nucleotide and the total number of that residue/nucleotide type present. (b) Average D
+loss for each residue/nucleotide type with respect to the DWD (diffraction-weighted dose; Zeldin, Brock­hauser et al., 2013). 95% confidence intervals (CI) are shown. Only a subset of key TRAP residue types are included. The average D
+loss (calculated over the whole TRAP asymmetric unit) is shown at each dose (dashed line).</text></passage><passage><infon key="file">d-72-00648-fig3.jpg</infon><infon key="id">fig3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>42459</offset><text>
+F
+obs(d
+n) − F
+obs(d
+1) Fourier difference maps for (a) n = 2 (3.9 MGy), (b) n = 3 (6.5 MGy) and (c) n = 7 (16.7 MGy) contoured at ±4σ (a) and ±3.5σ (b, c). In (a) clear difference density is observed around the Glu42 carboxyl side chain in chain H, within the lowest dose difference map at d
+2 = 3.9 MGy. Radiation-induced protein disordering is evident across the large dose range (b, c); in comparison, no clear deterioration of the RNA density was observed.</text></passage><passage><infon key="file">d-72-00648-fig4.jpg</infon><infon key="id">fig4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>42938</offset><text>
+D
+loss calculated for all side-chain carboxyl group Glu Cδ and Asp Cγ atoms within the TRAP–RNA complex for a dose of 19.3 MGy (d
+8). Residues have been grouped by amino-acid number, and split into bound and nonbound groupings, with each bar representing the mean calculated over 11 equivalent atoms around a TRAP ring. Whiskers indicate 95% CI. The D
+loss behaviour shown here was consistently exhibited across the entire investigated dose range.</text></passage><passage><infon key="file">d-72-00648-fig5.jpg</infon><infon key="id">fig5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>43397</offset><text>
+D
+loss against dose for (a) Glu36 Cδ, (b) Asp39 Cγ, (c) Glu42 O∊1, (d) Glu42 O∊2, (e) Phe32 Cζ and (f) Lys37 C∊ atoms. 95% CI are included for each set of 11 equivalent atoms grouped as bound/nonbound. RNA-binding interface interactions are shown for TRAP chain N, with the F
+obs(d
+7) − F
+obs(d
+1) Fourier difference map (dose 16.7 MGy) overlaid and contoured at a ±4σ level.</text></passage></document></collection>

raw_BioC_XML/PMC4855620_raw.xml

@@ -0,0 +1,104 @@
+<?xml version="1.0" encoding="UTF-8"?>
+<!DOCTYPE collection SYSTEM "BioC.dtd">
+<collection><source>PMC</source><date>20201222</date><key>pmc.key</key><document><id>4855620</id><infon key="license">CC BY</infon><passage><infon key="alt-title">D. E. Scott et al.</infon><infon key="article-id_doi">10.1002/1873-3468.12139</infon><infon key="article-id_pmc">4855620</infon><infon key="article-id_pmid">26992456</infon><infon key="article-id_publisher-id">FEB212139</infon><infon key="fn">Edited by Alfonso Valencia</infon><infon key="fpage">1094</infon><infon key="issue">8</infon><infon key="kwd">alanine scanning biophysics/ITC peptides protein–protein interaction RAD51 X‐ray crystallography</infon><infon key="license">This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.</infon><infon key="lpage">1102</infon><infon key="name_0">surname:Scott;given-names:Duncan E.</infon><infon key="name_1">surname:Marsh;given-names:May</infon><infon key="name_2">surname:Blundell;given-names:Tom L.</infon><infon key="name_3">surname:Abell;given-names:Chris</infon><infon key="name_4">surname:Hyvönen;given-names:Marko</infon><infon key="section_type">TITLE</infon><infon key="type">front</infon><infon key="volume">590</infon><infon key="year">2016</infon><offset>0</offset><text>Structure‐activity relationship of the peptide binding‐motif mediating the BRCA2:RAD51 protein–protein interaction</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>121</offset><text> RAD51 is a recombinase involved in the homologous recombination of double‐strand breaks in DNA. RAD51 forms oligomers by binding to another molecule of RAD51 via an ‘FxxA’ motif, and the same recognition sequence is similarly utilised to bind BRCA2. We have tabulated the effects of mutation of this sequence, across a variety of experimental methods and from relevant mutations observed in the clinic. We use mutants of a tetrapeptide sequence to probe the binding interaction, using both isothermal titration calorimetry and X‐ray crystallography. Where possible, comparison between our tetrapeptide mutational study and the previously reported mutations is made, discrepancies are discussed and the importance of secondary structure in interpreting alanine scanning and mutational data of this nature is considered.</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">title_1</infon><offset>949</offset><text>Abbreviations</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>963</offset><text> BRCA2, breast cancer type‐2 susceptibility protein</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>1017</offset><text> HR, homologous recombination</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>1047</offset><text> ITC, isothermal titration calorimetry</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>1086</offset><text> PPI, protein–protein interaction</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>1122</offset><text> SAR, structure activity relationship</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>1160</offset><text>Eukaryotic RAD51, archeal RadA and prokaryotic RecA are a family of ATP‐dependent recombinases involved in homologous recombination (HR) of double‐strand breaks in DNA 1. RAD51 interacts with BRCA2, and is thought to localise RAD51 to sites of DNA damage 2, 3. Both BRCA2 and RAD51 together are vital for helping to repair and maintain a high fidelity in DNA replication. BRCA2 especially has garnered much attention in a clinical context, as many mutations have been identified that drive an increased risk of cancer in individuals 4, 5. Although the inactivation of the BRCA2:RAD51 DNA repair pathway can cause genomic instability and eventual tumour development, an inability to repair breaks in DNA may also engender a sensitivity to ionising radiation 6, 7. For this reason it is hypothesised that in tumour cells with an intact BRCA2:RAD51 repair pathway, small molecules which prevent the interaction between RAD51 and BRCA2 may confer radiosensitivity by disabling the HR pathway 8. The interaction between the two proteins is mediated by eight BRC repeats, which are characterised by a conserved ‘FxxA’ motif 9. RAD51 and RadA proteins also contain an ‘FxxA’ sequence (FTTA for human RAD51) through which it can bind to other RAD51 and RadA molecules, and oligomerise to form higher order filament structures on DNA. The common FxxA motifs of both the BRC repeats and RAD51 oligomerisation sequence are recognised by the same FxxA‐binding site of RAD51.</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>2637</offset><text>In general, the dominant contribution of certain residues to the overall binding energy of a protein–protein interaction are known as ‘hot‐spot’ residues. Interestingly, small molecule inhibitors of PPIs are often found to occupy the same pockets which are otherwise occupied by hot‐spot residues in the native complex. It is therefore of great interest to identify hot‐spots in an effort to guide drug discovery efforts against a PPI. Further, a correlation between residues that are strongly conserved and hot‐spot residues has been reported 10. Purely based on the amino acid consensus sequence reported by Pellegrini et al., 11 phenylalanine and alanine would both be expected to be hot‐spots and to a lesser extent, threonine. However, whilst the identification of highly conserved residues may be a good starting point for identifying hot‐spots, experimental validation by mutation of these sequences is vital.</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>3572</offset><text>The importance of residues in the FxxA motif has been probed by a variety of techniques, collated in Table 1. Briefly, mutating phenylalanine to glutamic acid inactivated the BRC4 peptide and prevented RAD51 oligomerisation 11, 12. A phenylalanine‐truncated BRC4 is also found to be inactive 13, however, introducing a tryptophan for phenylalanine was found to have no significant effect on BRC4 affinity 12. A glutamine replacing the histidine in BRC4 maintains BRC4 activity 13. The ability of BRC3 to interact with RAD51 nucleoprotein filaments is disrupted when threonine is mutated to an alanine 3. Similarly, mutating alanine to glutamic acid in the RAD51 oligomerisation sequence 11 or to serine in BRC4 13 leads to loss of interaction in both cases. The BRC5 repeat in humans has serine in the place of alanine, and is thought to be a nonbinding repeat 12. Mutations identified in the clinic, in the FxxA region of the BRC repeats of BRCA2 are collated in Table 1 14. It is difficult to state the clinical relevance of these mutations as they are annotated as ‘unvalidated’, that is, it is not known whether they contribute to the disease phenotype or are neutral variants. For completeness, we present them here with this caveat, and to make the comment that these clinical mutations are consistent with abrogating the interaction with RAD51.</text></passage><passage><infon key="file">feb212139-tbl-0001.xml</infon><infon key="id">feb212139-tbl-0001</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>4930</offset><text>Summary of FxxA‐relevant mutations previously reported and degree of characterisation. The mutation, relevant peptide context, resulting FxxA motif sequence and experimental technique for each entry is given. For clarity, mutated residues are shown in bold</text></passage><passage><infon key="file">feb212139-tbl-0001.xml</infon><infon key="id">feb212139-tbl-0001</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;col style=&quot;border-right:solid 1px #000000&quot; span=&quot;1&quot;/&gt;&lt;col style=&quot;border-right:solid 1px #000000&quot; span=&quot;1&quot;/&gt;&lt;col style=&quot;border-right:solid 1px #000000&quot; span=&quot;1&quot;/&gt;&lt;col style=&quot;border-right:solid 1px #000000&quot; span=&quot;1&quot;/&gt;&lt;col style=&quot;border-right:solid 1px #000000&quot; span=&quot;1&quot;/&gt;&lt;thead valign=&quot;top&quot;&gt;&lt;tr style=&quot;border-bottom:solid 1px #000000&quot;&gt;&lt;th align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Mutation context&lt;xref ref-type=&quot;fn&quot; rid=&quot;feb212139-note-0002&quot;&gt;a&lt;/xref&gt;
+&lt;/th&gt;&lt;th align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Mutation&lt;/th&gt;&lt;th align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;FxxA motif&lt;/th&gt;&lt;th align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Technique used&lt;/th&gt;&lt;th align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Effect&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;RAD51 (FTTA)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;F86E&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;monospace&gt;&lt;bold&gt;E&lt;/bold&gt;TTA&lt;/monospace&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Immunoprecipitation &lt;xref rid=&quot;feb212139-bib-0011&quot; ref-type=&quot;ref&quot;&gt;11&lt;/xref&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;No binding&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;BRC4 (FHTA)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;F1524E&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;monospace&gt;&lt;bold&gt;E&lt;/bold&gt;HTA&lt;/monospace&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Competitive ELISA &lt;xref rid=&quot;feb212139-bib-0012&quot; ref-type=&quot;ref&quot;&gt;12&lt;/xref&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Peptide inactive&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;BRC4 (FHTA)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;F1524W&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;monospace&gt;&lt;bold&gt;W&lt;/bold&gt;HTA&lt;/monospace&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Competitive ELISA &lt;xref rid=&quot;feb212139-bib-0012&quot; ref-type=&quot;ref&quot;&gt;12&lt;/xref&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Comparable activity to WT&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;BRC4 (FHTA)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;F1524V&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;monospace&gt;&lt;bold&gt;V&lt;/bold&gt;HTA&lt;/monospace&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;BRCA2 mutations database &lt;xref rid=&quot;feb212139-bib-0014&quot; ref-type=&quot;ref&quot;&gt;14&lt;/xref&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;–&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;BRC4 (FHTA)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;ΔF1524&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;monospace&gt;&lt;bold&gt;‐&lt;/bold&gt;HTA&lt;/monospace&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Dissociation of RAD51‐DNA complex &lt;xref rid=&quot;feb212139-bib-0013&quot; ref-type=&quot;ref&quot;&gt;13&lt;/xref&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Peptide inactive&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;BRC4 (FHTA)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;H1525Q&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;monospace&gt;F&lt;bold&gt;Q&lt;/bold&gt;TA&lt;/monospace&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Dissociation of RAD51‐DNA complex &lt;xref rid=&quot;feb212139-bib-0013&quot; ref-type=&quot;ref&quot;&gt;13&lt;/xref&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Comparable activity&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;BRC7 (FSTA)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;S1979R&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;monospace&gt;F&lt;bold&gt;R&lt;/bold&gt;TA&lt;/monospace&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;BRCA2 mutations database &lt;xref rid=&quot;feb212139-bib-0014&quot; ref-type=&quot;ref&quot;&gt;14&lt;/xref&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;–&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;BRC3 (FQTA)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;T1430A&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;monospace&gt;FQ&lt;bold&gt;A&lt;/bold&gt;A&lt;/monospace&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;RAD51:DNA bandshift assay &lt;xref rid=&quot;feb212139-bib-0003&quot; ref-type=&quot;ref&quot;&gt;3&lt;/xref&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Peptide inactive&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;BRC3 (FQTA)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;T1430A&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;monospace&gt;FQ&lt;bold&gt;A&lt;/bold&gt;A&lt;/monospace&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Electron microscopic visualisation of nucleoprotein filaments &lt;xref rid=&quot;feb212139-bib-0003&quot; ref-type=&quot;ref&quot;&gt;3&lt;/xref&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Peptide inactive&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;BRC1 (FRTA)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;T1011R&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;monospace&gt;FR&lt;bold&gt;R&lt;/bold&gt;A&lt;/monospace&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;BRCA2 mutations database &lt;xref rid=&quot;feb212139-bib-0014&quot; ref-type=&quot;ref&quot;&gt;14&lt;/xref&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;–&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;BRC2 (FYSA)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;S1221P&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;monospace&gt;FY&lt;bold&gt;P&lt;/bold&gt;A&lt;/monospace&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;BRCA2 mutations database &lt;xref rid=&quot;feb212139-bib-0014&quot; ref-type=&quot;ref&quot;&gt;14&lt;/xref&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;–&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;BRC2 (FYSA)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;S1221Y&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;monospace&gt;FY&lt;bold&gt;Y&lt;/bold&gt;A&lt;/monospace&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;BRCA2 mutations database &lt;xref rid=&quot;feb212139-bib-0014&quot; ref-type=&quot;ref&quot;&gt;14&lt;/xref&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;–&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;RAD51 (FTTA)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;A89E&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;monospace&gt;FTT&lt;bold&gt;E&lt;/bold&gt;&lt;/monospace&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Immunoprecipitation &lt;xref rid=&quot;feb212139-bib-0011&quot; ref-type=&quot;ref&quot;&gt;11&lt;/xref&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;No binding&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;BRC4 (FHTA)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;A1527S&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;monospace&gt;FHT&lt;bold&gt;S&lt;/bold&gt;&lt;/monospace&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Dissociation of RAD51‐DNA complex &lt;xref rid=&quot;feb212139-bib-0013&quot; ref-type=&quot;ref&quot;&gt;13&lt;/xref&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Peptide inactive&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>5189</offset><text>Mutation contexta	Mutation	FxxA motif	Technique used	Effect	 	RAD51 (FTTA)	F86E	ETTA	Immunoprecipitation 11	No binding	 	BRC4 (FHTA)	F1524E	EHTA	Competitive ELISA 12	Peptide inactive	 	BRC4 (FHTA)	F1524W	WHTA	Competitive ELISA 12	Comparable activity to WT	 	BRC4 (FHTA)	F1524V	VHTA	BRCA2 mutations database 14	–	 	BRC4 (FHTA)	ΔF1524	‐HTA	Dissociation of RAD51‐DNA complex 13	Peptide inactive	 	BRC4 (FHTA)	H1525Q	FQTA	Dissociation of RAD51‐DNA complex 13	Comparable activity	 	BRC7 (FSTA)	S1979R	FRTA	BRCA2 mutations database 14	–	 	BRC3 (FQTA)	T1430A	FQAA	RAD51:DNA bandshift assay 3	Peptide inactive	 	BRC3 (FQTA)	T1430A	FQAA	Electron microscopic visualisation of nucleoprotein filaments 3	Peptide inactive	 	BRC1 (FRTA)	T1011R	FRRA	BRCA2 mutations database 14	–	 	BRC2 (FYSA)	S1221P	FYPA	BRCA2 mutations database 14	–	 	BRC2 (FYSA)	S1221Y	FYYA	BRCA2 mutations database 14	–	 	RAD51 (FTTA)	A89E	FTTE	Immunoprecipitation 11	No binding	 	BRC4 (FHTA)	A1527S	FHTS	Dissociation of RAD51‐DNA complex 13	Peptide inactive	 	</text></passage><passage><infon key="file">feb212139-tbl-0001.xml</infon><infon key="id">feb212139-tbl-0001</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>6227</offset><text>The wild‐type FxxA sequence is indicated in parenthesis.</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>6286</offset><text>In this work, we report the most detailed study of systematic mutations of peptides to probe the FxxA‐binding motif to date. We have chosen to focus on tetrapeptides, which allows us to examine the effect of mutation on the fundamental unit of binding, FxxA, rather than in the context of either the BRC repeat or self‐oligomerisation sequence. Affinities of peptides were measured directly using Isothermal Titration Calorimetry (ITC) and the structures of many of the peptides bound to humanised RadA were determined by X‐ray crystallography. The use of ITC is generally perceived as a gold‐standard in protein–ligand characterisation, rather than a competitive assay which may be prone to aggregation artefacts. Wild‐type human RAD51, however, is a heterogeneous mixture of oligomers and when monomerised by mutation, is highly unstable. In this context, we have previously reported the use of stable monomeric forms of RAD51, humanised from Pyrococcus furiosus homologue RadA, for ITC experiments and X‐ray crystallography 8, 15.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>7334</offset><text>Materials and methods</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>7356</offset><text>Peptide synthesis</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>7374</offset><text>Peptides were synthesised using solid‐phase FMOC chemistry by Alta Biosciences (Birmingham, UK) or the Protein and Nucleic Acid Service at the Department of Biochemistry (University of Cambridge). All peptides prepared and used in the study were N‐acetylated and C‐amide terminated.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>7663</offset><text>Protein preparation</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>7683</offset><text>Protein expression and purification was performed as described previously 15. In brief, monomeric HumRadA2 was expressed in E. coli using T7‐based expression vector at 37 °C for 3 h. Soluble cell lysate was heat treated to precipitate most of the cellular proteins and the soluble fraction containing HumRadA2 was purified using a combination of cation exchange chromatography at pH 6.0 and size‐exclusion chromatography in 10 mm MES, 100 mm NaCl pH 6.0 buffer. Protein concentration was determined using the calculated extinction coefficient at 280 nm, and stored at −80 °C in small aliquots after flash freezing.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>8306</offset><text>Isothermal titration calorimetry</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>8339</offset><text>Isothermal titration calorimetry experiments were performed at 25 °C on a MicroCal iTC200. HumRadA2 (600 μm in 20 mm MES pH 6.0 with 100 mm NaCl and 0.5 mm EDTA) was diluted with Tris buffer (200 mm, pH 7.5 with 100 mm NaCl) to 64–83 μm. Peptides were dissolved in MilliQ water (50 mm) and an aliquot taken and diluted with 200 mm Tris, pH 7.5, 100 mm NaCl to give a ligand solution of 2.5–5 mm. The peptide solution was titrated into the protein solution; 16 injections (2.4 μL) of 4.8 s duration were made at 80‐s intervals. The initial injection of ligand (0.4 μL) was discarded during data analysis. Control experiments of peptides to buffer showed insignificant heats. The data were processed and thermodynamic parameters obtained by fitting the data to a single‐site‐binding model using Origin software and fixing the stoichiometry as 1.0 for weak‐binding ligands 16. All data from ITC measurements are shown in the Figs S1 and S2.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>9294</offset><text>X‐ray crystallography</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>9318</offset><text>Monomerised RadA proteins were crystallised in the same conditions as described previously 15. Peptides were soaked into the crystals at 2–5 mm concentration overnight in the presence of 10% glycerol as a cryoprotectant. Crystals were cryo‐cooled in liquid nitrogen and data collected at synchrotron light sources and processed using XDS: details of this are found in crystallographic table (Table S1 in Supporting Information). Structures were solved by molecular replacement using unliganded, monomeric RadA coordinates (PDB: 4b3b, after removal of FHTA peptide) as a search model and refined with an automated procedure using Refmac5 17. After inspection of the resulting electron density, the bound peptides were modelled into the density and structures were further refined using Refmac5 18 and phenix.refine 19, and manually rebuilt using Coot 20. Coordinates and structure factors have been deposited in the PDB under accession codes as listed in Table 2 and in the crystallographic data table in the Supporting Information. With the exception of FATA peptide complex, which was crystallised with wild‐type RadA, the structures are determined using HumRadA1 mutant.</text></passage><passage><infon key="file">feb212139-tbl-0002.xml</infon><infon key="id">feb212139-tbl-0002</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>10497</offset><text>Summary of peptide‐binding data determined by ITC against HumRadA2. Mutated residues are highlighted in bold. All peptides were N‐acetylated and C‐amide terminated</text></passage><passage><infon key="file">feb212139-tbl-0002.xml</infon><infon key="id">feb212139-tbl-0002</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table xmlns:xlink=&quot;http://www.w3.org/1999/xlink&quot; frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;col style=&quot;border-right:solid 1px #000000&quot; span=&quot;1&quot;/&gt;&lt;col style=&quot;border-right:solid 1px #000000&quot; span=&quot;1&quot;/&gt;&lt;col style=&quot;border-right:solid 1px #000000&quot; span=&quot;1&quot;/&gt;&lt;col style=&quot;border-right:solid 1px #000000&quot; span=&quot;1&quot;/&gt;&lt;col style=&quot;border-right:solid 1px #000000&quot; span=&quot;1&quot;/&gt;&lt;col style=&quot;border-right:solid 1px #000000&quot; span=&quot;1&quot;/&gt;&lt;thead valign=&quot;top&quot;&gt;&lt;tr style=&quot;border-bottom:solid 1px #000000&quot;&gt;&lt;th align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Table entry&lt;/th&gt;&lt;th align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Peptide&lt;/th&gt;&lt;th align=&quot;char&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;italic&gt;K&lt;/italic&gt;
+&lt;sub&gt;D&lt;/sub&gt;/μ&lt;sc&gt;m&lt;/sc&gt;
+&lt;/th&gt;&lt;th align=&quot;char&quot; char=&quot; &quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Δ&lt;italic&gt;H&lt;/italic&gt;/cal·mol&lt;sup&gt;−1&lt;/sup&gt;
+&lt;/th&gt;&lt;th align=&quot;char&quot; char=&quot; &quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;italic&gt;T&lt;/italic&gt;Δ&lt;italic&gt;S&lt;/italic&gt;/cal·mol&lt;sup&gt;−1&lt;/sup&gt;
+&lt;/th&gt;&lt;th align=&quot;left&quot; valign=&quot;top&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;PDB code&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; colspan=&quot;6&quot; rowspan=&quot;1&quot;&gt;First position variation&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; style=&quot;padding-left:10%&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;monospace&gt;FHTA&lt;/monospace&gt;
+&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;±&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;280 ± 20&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;±&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;−2388 ± 94&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2453&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;ext-link ext-link-type=&quot;uri&quot; xlink:href=&quot;http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4b3b&quot;&gt;4b3b&lt;/ext-link&gt;
+&lt;xref rid=&quot;feb212139-bib-0015&quot; ref-type=&quot;ref&quot;&gt;15&lt;/xref&gt;
+&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; style=&quot;padding-left:10%&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;monospace&gt;&lt;bold&gt;W&lt;/bold&gt;HTA&lt;/monospace&gt;
+&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;±&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;93 ± 3&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;±&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;−2768 ± 34&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2727&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;ext-link ext-link-type=&quot;uri&quot; xlink:href=&quot;http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5fow&quot;&gt;5fow&lt;/ext-link&gt;
+&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; colspan=&quot;6&quot; rowspan=&quot;1&quot;&gt;Second position variation&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; style=&quot;padding-left:10%&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;monospace&gt;F&lt;bold&gt;A&lt;/bold&gt;TA&lt;/monospace&gt;
+&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;±&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;280 ± 29&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;±&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;−1820 ± 109&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3010&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;ext-link ext-link-type=&quot;uri&quot; xlink:href=&quot;http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5fpk&quot;&gt;5fpk&lt;/ext-link&gt;
+&lt;xref ref-type=&quot;fn&quot; rid=&quot;feb212139-note-0003&quot;&gt;a&lt;/xref&gt;
+&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; style=&quot;padding-left:10%&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;4&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;monospace&gt;F&lt;bold&gt;N&lt;/bold&gt;TA&lt;/monospace&gt;
+&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;±&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;613 ± 44&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;±&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;−4036 ± 177&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;346&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;–&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; style=&quot;padding-left:10%&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;monospace&gt;F&lt;bold&gt;P&lt;/bold&gt;TA&lt;/monospace&gt;
+&lt;/td&gt;&lt;td align=&quot;char&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;No detectable binding&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;–&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; colspan=&quot;6&quot; rowspan=&quot;1&quot;&gt;Third position variation&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; style=&quot;padding-left:10%&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;6&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;monospace&gt;FH&lt;bold&gt;P&lt;/bold&gt;A&lt;/monospace&gt;
+&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;±&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;113 ± 3&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;±&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;−2155 ± 26&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3218&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;ext-link ext-link-type=&quot;uri&quot; xlink:href=&quot;http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5fou&quot;&gt;5fou&lt;/ext-link&gt;
+&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; style=&quot;padding-left:10%&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;7&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;monospace&gt;FH&lt;bold&gt;A&lt;/bold&gt;A&lt;/monospace&gt;
+&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;±&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;675 ± 60&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;±&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;−7948 ± 466&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;−3636&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;ext-link ext-link-type=&quot;uri&quot; xlink:href=&quot;http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5fox&quot;&gt;5fox&lt;/ext-link&gt;
+&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; colspan=&quot;6&quot; rowspan=&quot;1&quot;&gt;Fourth position variation&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; style=&quot;padding-left:10%&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;8&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;monospace&gt;FHT&lt;bold&gt;G&lt;/bold&gt;&lt;/monospace&gt;
+&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;±&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1590 ± 300&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;±&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;−5518 ± 924&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;−1702&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;ext-link ext-link-type=&quot;uri&quot; xlink:href=&quot;http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5fov&quot;&gt;5fov&lt;/ext-link&gt;
+&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; style=&quot;padding-left:10%&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;9&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;monospace&gt;FHT&lt;bold&gt;U&lt;/bold&gt;&lt;/monospace&gt;
+&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;±&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;680 ± 51&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;±&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;−14 600 ± 771&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;−10 281&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;ext-link ext-link-type=&quot;uri&quot; xlink:href=&quot;http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5fot&quot;&gt;5fot&lt;/ext-link&gt;
+&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; colspan=&quot;6&quot; rowspan=&quot;1&quot;&gt;Combination&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; style=&quot;padding-left:10%&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;10&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;monospace&gt;&lt;bold&gt;W&lt;/bold&gt;H&lt;bold&gt;P&lt;/bold&gt;A&lt;/monospace&gt;
+&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;±&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;330 ± 25&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;±&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;−6801 ± 318&lt;/td&gt;&lt;td align=&quot;char&quot; char=&quot;.&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;−2044&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;–&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; colspan=&quot;6&quot; rowspan=&quot;1&quot;&gt;Peptide truncations&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; style=&quot;padding-left:10%&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;11&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;monospace&gt;FH&lt;/monospace&gt;
+&lt;/td&gt;&lt;td align=&quot;char&quot; rowspan=&quot;3&quot; colspan=&quot;1&quot;&gt;
+No binding detected&lt;break/&gt;
+No binding detected&lt;break/&gt;
+No binding detected
+&lt;/td&gt;&lt;td align=&quot;char&quot; rowspan=&quot;3&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;char&quot; rowspan=&quot;3&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;–&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; style=&quot;padding-left:10%&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;12&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;monospace&gt;FHT&lt;/monospace&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;–&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; style=&quot;padding-left:10%&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;13&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;
+&lt;monospace&gt;HTA&lt;/monospace&gt;
+&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;–&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>10667</offset><text>Table entry	Peptide	KD/μm	ΔH/cal·mol−1	TΔS/cal·mol−1	PDB code	 	First position variation	 	1	FHTA	280 ± 20	−2388 ± 94	2453	4b3b15	 	2	WHTA	93 ± 3	−2768 ± 34	2727	5fow	 	Second position variation	 	3	FATA	280 ± 29	−1820 ± 109	3010	5fpka	 	4	FNTA	613 ± 44	−4036 ± 177	346	–	 	5	FPTA	No detectable binding			–	 	Third position variation	 	6	FHPA	113 ± 3	−2155 ± 26	3218	5fou	 	7	FHAA	675 ± 60	−7948 ± 466	−3636	5fox	 	Fourth position variation	 	8	FHTG	1590 ± 300	−5518 ± 924	−1702	5fov	 	9	FHTU	680 ± 51	−14 600 ± 771	−10 281	5fot	 	Combination	 	10	WHPA	330 ± 25	−6801 ± 318	−2044	–	 	Peptide truncations	 	11	FH		 	No binding detected	 	No binding detected	 	No binding detected	 				–	 	12	FHT	–	 	13	HTA	–	 	</text></passage><passage><infon key="file">feb212139-tbl-0002.xml</infon><infon key="id">feb212139-tbl-0002</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>11443</offset><text>Structure solved with wild‐type RadA.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>11483</offset><text>Sequence analysis</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>11501</offset><text>Sequences of mammalian RAD51 proteins and archeal RadA orthologues were obtained from Ensembl (www.ensembl.org) and Uniprot (www.uniprot.org) databases. Sequences were aligned using ClustalX2, and aligned sequences for the FxxA motifs were used in WebLogo (weblogo.berkely.edu/logo.cgi) server 21 to derive the consensus diagrams shown in Figs 1 and 4. All the sequences used in these analyses are shown in Figs S4, S5 and S6.</text></passage><passage><infon key="file">FEB2-590-1094-g001.jpg</infon><infon key="id">feb212139-fig-0001</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>11928</offset><text>Conservation of FxxA motif (A) BRC4 peptide (green cartoon) bound to truncated human RAD51 (grey surface) (PDB: 1n0w, 11). The blue dashed box highlights the FxxA interaction pocket. (B) Two interacting protein molecules of RAD51 from Saccharomyces cerevisiae are shown. One RAD51 (green cartoon) interacts with another molecule of RAD51 (grey and pink surface) via the FxxA pocket indicated by the dashed blue box. The N‐terminal domain of one RAD51 protomer is highlighted in pink for clarity and the green arrow indicates the location of this protomer's FxxA oligomerisation sequence (PDB: 1szp, 29). (C) Conservation of FxxA motif across the human BRC repeats and (D) across 21 eukaryotic RAD51s and 24 RadAs, with the size of the letters proportional to the degree of conservation. Sequence figures generated using Weblogo 3.0 21, sequence details are found in the Supporting Information.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>12824</offset><text>Results</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>12832</offset><text>We have mutated and truncated the tetrapeptide epitope FHTA, and examined the effects both structurally and on the binding affinity with humanised RadA. As a comparative reference, we are using the FHTA sequence derived from the most tightly binding BRC repeat, BRC4 22. The peptides used are N‐acetylated and C‐amide terminated in order to provide the most relevant peptide in the context of a longer peptide chain. A summary of the peptide sequence, PDB codes and K D data measured by ITC with the corresponding ΔH and TΔS values are collated in Table 2.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>13397</offset><text>Phe1524 of BRC4 binds in a small surface pocket of human RAD51, defined by the hydrophobic side chains of residues Met158, Ile160, Ala192, Leu203 and Met210. The residue is highly conserved across BRC repeats and oligomerisation sequences. Consistent with this, the truncated HTA tripeptide could not be detected to bind to humanised, monomeric RadA, HumRadA2 (Table 2, entry 13). As previously discussed, there is some evidence that substituting a tryptophan for the phenylalanine at this position was tolerated in the context of BRC4 12. Therefore, the WHTA peptide was tested and found to not only be tolerated, but to increase the binding affinity of the peptide approximately threefold.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>14089</offset><text>The second position of the tetrapeptide was found to be largely invariant to changes in the side chains that were investigated. The residue makes no interactions with the RAD51 protein, but may make an internal hydrogen bond with Thr1520 in the context of BRC4, Fig. 3A. Replacing the histidine with an asparagine, chosen to potentially mimic the hydrogen bond donor–acceptor nature of histidine, resulted in a moderate, twofold decrease in potency (Table 2, entry 4). Mutating to an alanine, recapitulated the potency of FHTA, implying that the interactions made by histidine do not contribute overall to binding affinity (Table 2, entry 3). FPTA was also tested, but was found to have no affinity for the protein (Table 2, entry 5). Modelling suggests that a proline in the second position would be expected to clash sterically with the surface of the protein, and provides a rationale for the lack of binding observed.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15013</offset><text>Threonine was mutated to an alanine, resulting in only a moderately weaker K D (twofold, Table 2, entry 7). In the context of a tetrapeptide at least, this result implies a lack of importance of a threonine at this position. Interestingly, it was found that a proline at this position improved the affinity almost threefold, to 113 μm (Table 2, entry 6). This beneficial mutation was incorporated with another previously identified variant to produce the peptide WHPA. Disappointingly, the combined effect of the mutations was not additive and the potency was weakened to 690 μm.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15595</offset><text>While the importance of the phenylalanine may be possible to predict from examination of the crystal structure, the alanine appears to be of much less importance in this regard. It is, however, a highly conserved residue and clearly of interest for systematic mutation. Removing the alanine residue entirely produced the truncated tripeptide FHT, which did not bind (Table 2, entry 12). The unnatural amino acid, α‐amino butyric acid (U), was introduced at the fourth position, positioning an ethyl group into the alanine pocket (Table 2, entry 9). Perhaps surprisingly, it was accommodated and the affinity dropped only by twofold as compared to FHTA. The effect of simply removing the β‐carbon of alanine, by mutation to glycine (FHTG), produced an approximately sixfold drop in binding affinity (Table 2, entry 8). This is in line with the observation that alanine is not 100% conserved and some archeal RadA proteins contain a glycine in the place of alanine 23.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>16569</offset><text>Structural characterisation of peptide complexes</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>16618</offset><text>Structures of the key tetrapeptides were solved by soaking into crystals of a humanised form of RAD51, HumRadA1, which we have previously reported as a convenient surrogate system for RAD51 crystallography 15. The corresponding PDB codes are indicated in Table 2 and crystallographic data are found in the Supporting Information. All structures are of high resolution (1.2–1.7 Å) and the electron density for the peptide was clearly visible after the first refinement using unliganded RadA coordinates (Fig. S1).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>17134</offset><text>Some of the SAR observed in the binding analysis can be interpreted in terms of these X‐ray crystal structures. For example, an overlay of the bound poses of the ligands FHTA and FHPA (Fig. 2B) reveals a high similarity in the binding modes, indicating that the conformational rigidity conferred by the proline is compatible with the FHTA‐binding mode, and a reduction in an entropic penalty of binding may be the source of the improvement in affinity. WHTA peptide shows a relative dislocation when compared to FHTA (Fig 2A), with the entire ligand backbone of WHTA shifted to accommodate the change in the position of the main chain carbon of the first residue, as the larger indole side chain fills the Phe pocket. This shift is translated all the way to the alanine side chain. It is possible that this mutation is beneficial in the tetrapeptide context and neutral in the full‐length BRC4 context because the smaller peptide is less constrained and allowed to explore more conformations. An attempt to combine both the tryptophan and proline mutations, however, led to no improvement for WHPA peptide compared to FHTA. One possible explanation is that the ‘shifted’ binding mode observed in WHTA was not compatible with the conformational restriction that the proline of WHPA introduced.</text></passage><passage><infon key="file">FEB2-590-1094-g002.jpg</infon><infon key="id">feb212139-fig-0002</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>18437</offset><text>Comparison of different peptide complexes (A) Overlay with FHTA (grey) and WHTA (purple) showing a small relative displacement of the peptide backbone. (B) Superposition of FHTA (grey) and FHPA (yellow), showing conservation of backbone orientation (C) Overlay of FHTU (green), FHTA (grey) and FHTG (cyan).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>18744</offset><text>The thermodynamic data of peptide binding are also shown in Table 2. Although we have both thermodynamic data and high‐quality X‐ray structural information for some of the mutant peptides, we do not attempt to interpret differences in thermodynamic profiles between ligands, that is, to analyse ΔΔH and ΔΔS. Although ΔH and ΔS are tabulated, the K Ds measured are relatively weak and necessarily performed under low c‐value conditions. In this experimental regime, nonsigmoidal curves are generated and therefore errors in ΔH are expected to be much higher than the errors from model fitting given in Table 2 16. As ΔS is derived from ΔG by subtracting ΔH, errors in ΔH will be correlated with errors in ΔS, giving rise to a ‘phantom’ enthalpy–entropy compensation. Such effects have been discussed by Klebe 24 and Chodera and Mobley 25 and will frustrate attempts to interpret the measured ΔΔH and ΔΔS.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>19712</offset><text>Understanding mutations, residue conservation and epitope secondary structure</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>19790</offset><text>The conserved phenylalanine and alanine residues of the FHTA sequence were both found to be essential for binding by ITC. Conversely the second position histidine residue, corresponding to the unconserved His1525 in the BRC4 sequence, could be mutated without significant effect on the peptide affinity. The more general correlation between hot‐spot residues in protein–protein interactions and the high conservation of such residues has been previously reported 10, 26. Interestingly, however, the highly conserved threonine residue could be mutated without affecting the peptide affinity. This unexpected result, in the light of its very high conservation in the BRC and oligomerisation sequences, begs the question of what the role of Thr1526 is and highlights a potential pitfall and need for caution in the experimental design of alanine mutation studies.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>20655</offset><text>As the FHTA peptide is potentially a surrogate peptide for both the BRC repeat peptides and the RAD51 self‐oligomerisation peptide, it is useful to examine the role of Thr1526 (BRC4) and the analogous Thr87 (RAD51) in both binding contexts in more detail. Structural information for these two interactions is limited. Only one structure of BRC4 is published in complex with human RAD51 (PDB: 1n0w). Figure 3A shows the binding pose of BRC4 when bound to RAD51 and the intrapeptide hydrogen bonds that are made by BRC4. While Phe1524 and Ala1527 are buried in hydrophobic pockets on the surface, His1525 is close enough to form a hydrogen bond with the carbonyl of Thr1520, but the rotamer of His1525, supported by clearly positioned water molecules, is not compatible with hydrogen bonding. Also, Thr1520 is constrained by crystal contacts in this structure. Lack of conservation of this residue supports the idea that this interaction is not crucial for RAD51:BRC repeat binding.</text></passage><passage><infon key="file">FEB2-590-1094-g003.jpg</infon><infon key="id">feb212139-fig-0003</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>21638</offset><text>(A) Highlight of intra‐BRC4 interactions when bound to RAD51 (omitted for clarity) (PDB: 1n0w), with key residues shown in colour. (B) Intrapeptide interactions from oligomerisation epitope of S. cerevisiae 
+RAD51 when bound to next RAD51 in the filament (PDB: 1szp). Colouring as in (A). Residue numbering relates to the S. cerevisiae 
+RAD51 protein, the corresponding human residues are in parentheses.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>22045</offset><text>Either a threonine or serine is most commonly found in the third position of the FxxA motif. Thr1526 makes no direct interactions with the RAD51 protein, but instead forms a hydrogen bond network with the highly conserved S1528 and K1530 (Fig. 1C). The high degree of conservation of these three residues suggests an important possible role in facilitating a turn and stabilising the conformation of the peptide as it continues its way to a second interaction site on the side of RAD51. With respect to understanding the RAD51:RAD51 interaction, no human crystal structure has been published, however, several oligomeric structures of archaeal RadA as well that of Saccharomyces cerevisiae RAD51 have been reported 27, 28, 29. Figure 3B shows a highlight of the FxxA portion of oligomerisation peptide from the S. cerevisiae RAD51 structure, with residues in parentheses corresponding to the human RAD51 protein. The conserved threonine residue at the third position forms a hydrogen bond with the peptide backbone amide, which forms the base of an α‐helix.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>23107</offset><text>In both structural contexts, the role of the third position threonine in FxxA seems to be in stabilising secondary structure; a β‐turn in the case of BRC binding and an α‐helix in the case of RAD51 oligomerisation. In the tetrapeptide context these secondary interactions are not present and mutation of threonine to alanine would be expected to have little effect on affinity. In line with this, although we observe a slight twofold weakening of peptide affinity, the effect is far from being as drastic or inactivating as reported in longer peptide backgrounds 3. It would be interesting to investigate the importance of this residue in the context of the BRC4 peptide, and the oligomerisation peptide. Rather than indifference to alanine mutation, a significant effect, via lack of secondary structure stabilisation, would be predicted, as indeed has been reported for BRC3 3.</text></passage><passage><infon key="section_type">CONCL</infon><infon key="type">title_1</infon><offset>23994</offset><text>Conclusions</text></passage><passage><infon key="section_type">CONCL</infon><infon key="type">paragraph</infon><offset>24006</offset><text>The key observations from this work are shown in Fig 4. Two residues in the FxxA motif, phenylalanine and alanine, are highly conserved (Fig 4a). Phenylalanine mutated to tryptophan, in the context of the tetrapeptide improved potency, contrary to the reported result of comparable activity in the context of BRC4 12. Proline at the third position similarly improved potency. Activity was lost by mutating the terminal alanine to glycine, but recovered somewhat with the novel α‐amino butyric acid (U). Threonine was found to be relatively unimportant in the tetrapeptides but has been previously reported to be crucial in the context of BRC3. The reason for this disconnection is suggested to be that threonine plays a role in stabilising the β‐turn in the BRC repeats, which is absent in the tetrapeptides studied. This may lead to a more general caution, that hot‐spot data should be interpreted by considering the bound interaction with the protein, as well as the potential role in stabilising the bound peptide secondary structure. In either case, the requirement for structural data in correctly interpreting alanine‐scanning experiments is reinforced.</text></passage><passage><infon key="file">FEB2-590-1094-g004.jpg</infon><infon key="id">feb212139-fig-0004</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>25176</offset><text>Summary of key observations (A) FxxA motif sequence conservation of Rad51 oligomerisation sequences and BRC repeats. (B) Highlight of SAR identified for the tetrapeptide. The differences in ΔG for different peptide variants relative to FHTA are shown in the bar chart with colouring matching with the structural overlay below. (C) Overlay of tetrapeptide structures, with wild‐type FHTA peptide across the figure for reference and truncated segments of mutated residues shown in each panel. Purple carbon is WHTA, light blue is FATA, yellow is FHPA, cyan is FHTG and grey carbon is FHTA. Note the C‐terminal amide changes position in FHTG without the anchoring methyl group.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">title_1</infon><offset>25856</offset><text>Supporting information</text></passage><passage><infon key="section_type">REF</infon><infon key="type">title</infon><offset>25879</offset><text>References</text></passage><passage><infon key="fpage">457</infon><infon key="lpage">470</infon><infon key="pub-id_pmid">1581961</infon><infon key="section_type">REF</infon><infon key="source">Cell</infon><infon key="type">ref</infon><infon key="volume">69</infon><infon key="year">1992</infon><offset>25890</offset><text>RAD51 protein involved in repair and recombination in S. cerevisiae is a RecA‐like protein</text></passage><passage><infon key="fpage">171</infon><infon key="lpage">182</infon><infon key="pub-id_pmid">11832208</infon><infon key="section_type">REF</infon><infon key="source">Cell</infon><infon key="type">ref</infon><infon 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+<collection><source>PMC</source><date>20201221</date><key>pmc.key</key><document><id>4871749</id><infon key="license">NO-CC CODE</infon><passage><infon key="article-id_doi">10.1038/nchembio.2065</infon><infon key="article-id_manuscript">NIHMS769551</infon><infon key="article-id_pmc">4871749</infon><infon key="article-id_pmid">27089029</infon><infon key="fpage">396</infon><infon key="issue">6</infon><infon key="kwd">YEATS domain crotonylated lysine chromatin Taf14 histone PTM</infon><infon key="license">Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
+</infon><infon key="lpage">398</infon><infon key="name_0">surname:Andrews;given-names:Forest H.</infon><infon key="name_1">surname:Shinsky;given-names:Stephen A.</infon><infon key="name_2">surname:Shanle;given-names:Erin K.</infon><infon key="name_3">surname:Bridgers;given-names:Joseph B.</infon><infon key="name_4">surname:Gest;given-names:Anneliese</infon><infon key="name_5">surname:Tsun;given-names:Ian K.</infon><infon key="name_6">surname:Krajewski;given-names:Krzysztof</infon><infon key="name_7">surname:Shi;given-names:Xiaobing</infon><infon key="name_8">surname:Strahl;given-names:Brian D.</infon><infon key="name_9">surname:Kutateladze;given-names:Tatiana G.</infon><infon key="section_type">TITLE</infon><infon key="type">front</infon><infon key="volume">12</infon><infon key="year">2016</infon><offset>0</offset><text>The Taf14 YEATS domain is a reader of histone crotonylation</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>60</offset><text>The discovery of new histone modifications is unfolding at startling rates, however, the identification of effectors capable of interpreting these modifications has lagged behind. Here we report the YEATS domain as an effective reader of histone lysine crotonylation – an epigenetic signature associated with active transcription. We show that the Taf14 YEATS domain engages crotonyllysine via a unique π-π-π-stacking mechanism and that other YEATS domains have crotonyllysine binding activity.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>560</offset><text>Crotonylation of lysine residues (crotonyllysine, Kcr) has emerged as one of the fundamental histone post-translational modifications (PTMs) found in mammalian chromatin. This epigenetic PTM is widespread and enriched at active gene promoters and potentially enhancers. The crotonyllysine mark on histone H3K18 is produced by p300, a histone acetyltransferase also responsible for acetylation of histones. Owing to some differences in their genomic distribution, the crotonyllysine and acetyllysine (Kac) modifications have been linked to distinct functional outcomes. p300-catalyzed histone crotonylation, which is likely metabolically regulated, stimulates transcription to a greater degree than p300-catalyzed acetylation. The discovery of individual biological roles for the crotonyllysine and acetyllysine marks suggests that these PTMs can be read by distinct readers. While a number of acetyllysine readers have been identified and characterized, a specific reader of the crotonyllysine mark remains unknown (reviewed in). A recent survey of bromodomains (BDs) demonstrates that only one BD associates very weakly with a crotonylated peptide, however it binds more tightly to acetylated peptides, inferring that bromodomains do not possess physiologically relevant crotonyllysine binding activity.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1865</offset><text>The family of acetyllysine readers has been expanded with the discovery that the YEATS (Yaf9, ENL, AF9, Taf14, Sas5) domains of human AF9 and yeast Taf14 are capable of recognizing the histone mark H3K9ac. The acetyllysine binding function of the AF9 YEATS domain is essential for the recruitment of the histone methyltransferase DOT1L to H3K9ac-containing chromatin and for DOT1L-mediated H3K79 methylation and transcription. Similarly, activation of a subset of genes and DNA damage repair in yeast require the acetyllysine binding activity of the Taf14 YEATS domain. Consistent with its role in gene regulation, Taf14 was identified as a core component of the transcription factor complexes TFIID and TFIIF. However, Taf14 is also found in a number of chromatin-remodeling complexes (i.e., INO80, SWI/SNF and RSC) and the histone acetyltransferase complex NuA3, indicating a multifaceted role of Taf14 in transcriptional regulation and chromatin biology. In this study, we identified the Taf14 YEATS domain as a reader of crotonyllysine that binds to histone H3 crotonylated at lysine 9 (H3K9cr) via a distinctive binding mechanism. We found that H3K9cr is present in yeast and is dynamically regulated.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3072</offset><text>To elucidate the molecular basis for recognition of the H3K9cr mark, we obtained a crystal structure of the Taf14 YEATS domain in complex with H3K9cr5-13 (residues 5–13 of H3) peptide (Fig. 1, Supplementary Results, Supplementary Fig. 1 and Supplementary Table 1). The Taf14 YEATS domain adopts an immunoglobin-like β sandwich fold containing eight anti-parallel β strands linked by short loops that form a binding site for H3K9cr (Fig. 1b). The H3K9cr peptide lays in an extended conformation in an orientation orthogonal to the β strands and is stabilized through an extensive network of direct and water-mediated hydrogen bonds and a salt bridge (Fig. 1c).</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3741</offset><text>The most striking feature of the crotonyllysine recognition mechanism is the unique coordination of crotonylated lysine residue. The fully extended side chain of K9cr transverses the narrow tunnel, crossing the β sandwich at right angle in a corkscrew-like manner (Fig. 1b and Supplementary Figure 1b). The planar crotonyl group is inserted between Trp81 and Phe62 of the protein, the aromatic rings of which are positioned strictly parallel to each other and at equal distance from the crotonyl group, yielding a novel aromatic-amide/aliphatic-aromatic π-π-π-stacking system that, to our knowledge, has not been reported previously for any protein-protein interaction (Fig. 1d and Supplementary Fig. 1c). The side chain of Trp81 appears to adopt two conformations, one of which provides maximum π-stacking with the alkene functional group while the other rotamer affords maximum π-stacking with the amide π electrons (Supplementary Fig. 1c). The dual conformation of Trp81 is likely due to the conjugated nature of the C=C and C=O π-orbitals within the crotonyl functional group.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>4832</offset><text>In addition to π-π-π stacking, the crotonyl group is stabilized by a set of hydrogen bonds and electrostatic interactions. The π bond conjugation of the crotonyl group gives rise to a dipole moment of the alkene moiety, resulting in a partial positive charge on the β-carbon (Cβ) and a partial negative charge on the α-carbon (Cα). This provides the capability for the alkene moiety to form electrostatic contacts, as Cα and Cβ lay within electrostatic interaction distances of the carbonyl oxygen of Gln79 and of the hydroxyl group of Thr61, respectively. The hydroxyl group of Thr61 also participates in a hydrogen bond with the amide nitrogen of the K9cr side chain (Fig. 1d). The fixed position of the Thr61 hydroxyl group, which facilitates interactions with both the amide and Cα of K9cr, is achieved through a hydrogen bond with imidazole ring of His59. Extra stabilization of K9cr is attained by a hydrogen bond formed between its carbonyl oxygen and the backbone nitrogen of Trp81, as well as a water-mediated hydrogen bond with the backbone carbonyl group of Gly82 (Fig 1d). This distinctive mechanism was corroborated through mapping the Taf14 YEATS-H3K9cr binding interface in solution using NMR chemical shift perturbation analysis (Supplementary Fig. 2a, b).</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>6134</offset><text>Binding of the Taf14 YEATS domain to H3K9cr is robust. The dissociation constant (Kd) for the Taf14 YEATS-H3K9cr5-13 complex was found to be 9.5 μM, as measured by fluorescence spectroscopy (Supplementary Fig. 2c). This value is in the range of binding affinities exhibited by the majority of histone readers, thus attesting to the physiological relevance of the H3K9cr recognition by Taf14.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>6527</offset><text>To determine whether H3K9cr is present in yeast, we generated whole cell extracts from logarithmically growing yeast cells and subjected them to Western blot analysis using antibodies directed towards H3K9cr, H3K9ac and H3 (Fig. 2a, b, Supplementary Fig. 3 and Supplementary Table 2). Both H3K9cr and H3K9ac were detected in yeast histones; to our knowledge, this is the first report of H3K9cr occurring in yeast. We next asked if H3K9cr is regulated by the actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Towards this end, we probed extracts derived from yeast cells in which major yeast HATs (HAT1, Gcn5, and Rtt109) or HDACs (Rpd3, Hos1, and Hos2) were deleted. As shown in Figure 2a, b and Supplementary Fig. 3e, H3K9cr levels were abolished or reduced considerably in the HAT deletion strains, whereas they were dramatically increased in the HDAC deletion strains. Furthermore, fluctuations in the H3K9cr levels were more substantial than fluctuations in the corresponding H3K9ac levels. Together, these results reveal that H3K9cr is a dynamic mark of chromatin in yeast and suggest an important role for this modification in transcription as it is regulated by HATs and HDACs.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>7741</offset><text>We have previously shown that among acetylated histone marks, the Taf14 YEATS domain prefers acetylated H3K9 (also see Supplementary Fig. 3b), however it binds to H3K9cr tighter. The selectivity of Taf14 towards crotonyllysine was substantiated by 1H,15N HSQC experiments, in which either H3K9cr5-13 or H3K9ac5-13 peptide was titrated into the 15N-labeled Taf14 YEATS domain (Fig. 2c and Supplementary Fig. 4a, b). Binding of H3K9cr induced resonance changes in slow exchange regime on the NMR time scale, indicative of strong interaction. In contrast, binding of H3K9ac resulted in an intermediate exchange, which is characteristic of a weaker association. Furthermore, crosspeaks of Gly80 and Trp81 of the YEATS domain were uniquely perturbed by H3K9cr and H3K9ac, indicating a different chemical environment in the respective crotonyllysine and acetyllysine binding pockets (Supplementary Fig. 4a). These differences support our model that Trp81 adopts two conformations upon complex formation with the H3K9cr mark as compared to H3K9ac (Supplementary Figs. 1c, d and 4c). One of the conformations, characterized by the π stacking involving two aromatic residues and the alkene group, is observed only in the YEATS-H3K9cr complex.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>8976</offset><text>To establish whether the Taf14 YEATS domain is able to recognize other recently identified acyllysine marks, we performed solution pull-down assays using H3 peptides acetylated, propionylated, butyrylated, and crotonylated at lysine 9 (residues 1–20 of H3). As shown in Figure 2d and Supplementary Fig. 5a, the Taf14 YEATS domain binds more strongly to H3K9cr1-20, as compared to other acylated histone peptides. The preference for H3K9cr over H3K9ac, H3K9pr and H3K9bu was supported by 1H,15N HSQC titration experiments. Addition of H3K9ac1-20, H3K9pr1-20, and H3K9bu1-20 peptides caused chemical shift perturbations in the Taf14 YEATS domain in intermediate exchange regime, implying that these interactions are weaker compared to the interaction with the H3K9cr1-20 peptide (Supplementary Fig. 5b). We concluded that H3K9cr is the preferred target of this domain. From comparative structural analysis of the YEATS complexes, Gly80 emerged as candidate residue potentially responsible for the preference for crotonyllysine. In attempt to generate a mutant capable of accommodating a short acetyl moiety but discriminating against a longer, planar crotonyl moiety, we mutated Gly80 to more bulky residues, however all mutants of Gly80 lost their binding activities towards either acylated peptide, suggesting that Gly80 is absolutely required for the interaction. In contrast, mutation of Val24, a residue located on another side of Trp81, had no effect on binding (Fig. 2d and Supplementary Fig. 5a, c).</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>10484</offset><text>To determine if the binding to crotonyllysine is conserved, we tested human YEATS domains by pull-down experiments using singly and multiply acetylated, propionylated, butyrylated, and crotonylated histone peptides (Supplementary Fig. 6). We found that all YEATS domains tested are capable of binding to crotonyllysine peptides, though they display variable preferences for the acyl moieties. While YEATS2 and ENL showed selectivity for the crotonylated peptides, GAS41 and AF9 bound acylated peptides almost equally well.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>11007</offset><text>Unlike the YEATS domain, a known acetyllysine reader, bromodomain, does not recognize crotonyllysine. We assayed a large set of BDs in pull-down experiments and found that this module is highly specific for acetyllysine and propionyllysine containing peptides (Supplementary Fig. 7). However, bromodomains did not interact (or associated very weakly) with longer acyl modifications, including crotonyllysine, as in the case of BDs of TAF1 and BRD2, supporting recent reports. These results demonstrate that the YEATS domain is currently the sole reader of crotonyllysine.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>11579</offset><text>In conclusion, we have identified the YEATS domain of Taf14 as the first reader of histone crotonylation. The unique and previously unobserved aromatic-amide/aliphatic-aromatic π-π-π-stacking mechanism facilitates the specific recognition of the crotonyl moiety. We further demonstrate that H3K9cr exists in yeast and is dynamically regulated by HATs and HDACs. As we previously showed the importance of acyllysine binding by the Taf14 YEATS domain for the DNA damage response and gene transcription, it will be essential in the future to define the physiological role of crotonyllysine recognition and to differentiate the activities of Taf14 that are due to binding to crotonyllysine and acetyllysine modifications. Furthermore, the functional significance of crotonyllysine recognition by other YEATS proteins will be of great importance to elucidate and compare.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>12449</offset><text>ONLINE METHODS</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>12464</offset><text>Protein expression and purification</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>12500</offset><text>The Taf14 YEATS constructs (residues 1–132 or 1–137) were expressed in E. coli BL21 (DE3) RIL in either Luria Broth or M19 minimal media supplemented with 15NH4Cl and purified as N-terminal GST fusion proteins. Cells were harvested by centrifugation and resuspended in 50 mM HEPES (pH 7.5) supplemented with 150 mM NaCl and 1 mM TCEP. Cells are lysed by freeze-thaw followed by sonication. Proteins were purified on glutathione Sepharose 4B beads and the GST tag was cleaved with PreScission protease.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>13006</offset><text>X-ray data collection and structure determination</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>13056</offset><text>Taf14 YEATS (residues 1–137) was concentrated to 9 mg/mL in 25 mM MES (pH 6.5) and incubated with 2 molar equivalence of the H3K9cr5-13 at RT for 30 mins prior to crystallization. Crystals were obtain via sitting drop diffusion method at 18°C by mixing 800 nL of protein/peptide solution with 800 nL of well solution composed of 44% PEG600 (v/v) and 0.2 M citric acid (pH 6.0). X-ray diffraction data was collected at a wavelength of 1.54 Å at 100 K from a single crystal on the UC Denver Biophysical Core home source composed of a Rigaku Micromax 007 high frequency microfocus X-ray generator with a Pilatus 200K 2D area detector. HKL3000 was used for indexing, scaling, and data reduction. Solution was solved via molecular replacement with Phaser using the Taf14 YEATS domain (PDB 5D7E) as search model with waters, ligands, and peptide removed. Phenix was used for refinement of structure and waters were manually placed by inception of difference maps in Coot. Ramachandran plot indicates good stereochemistry of the three-dimensional structure with 100% of all residues falling within the favored (98%) and allowed (2%) regions. The crystallographic statistics are shown in Supplementary Table 1.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>14263</offset><text>NMR spectroscopy</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>14280</offset><text>NMR spectroscopy was carried out on a Varian INOVA 600 MHz spectrometer outfitted with a cryogenic probe. Chemical shift perturbation (CSP) analysis was performed using uniformly 15N-labeled Taf14 (1–132). 1H,15N heteronuclear single quantum coherence (HSQC) spectra of the Taf14 YEATS domain were collected in the presence of increasing concentrations of either H3K9cr5-13, H3K9ac5-13, H3K9cr1-20, H3K9ac1-20 H3K9pr1-20, H3K9bu1-20 or free Kcr in PBS buffer pH 6.8, 8% D2O.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>14757</offset><text>Fluorescence binding assays</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>14785</offset><text>Tryptophan fluorescence measurements were performed on a Fluorolog spectrofluorometer at room temperature as described. The samples containing 2 μM of Taf14 YEATS in PBS (pH 7.4) and increasing concentrations of H3K9cr5-13 were excited at 295 nm. Emission spectra were recorded from 310 to 340 nm with a 1 nm step size and a 0.5 sec integration time. The Kd value was determined using a nonlinear least-squares analysis and the equation:   where [L] is the concentration of the peptide, [P] is the concentration of the protein, ΔI is the observed change of signal intensity, and ΔImax is the difference in signal intensity of the free and bound states. The Kd values were averaged over 3 separate experiments, with error calculated as the standard deviation (SD).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>15558</offset><text>Peptide pull-downs</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>15577</offset><text>YEATS domains in pGEX vectors were expressed in SoluBL21 cells (Amsbio) by induction with 1 mM IPTG at 16–18°C overnight with shaking. Cells were lysed by freeze-thaw and sonication then purified over glutathione agarose (Pierce) in a buffer containing 50 mM Tris pH 8.0, 500 mM NaCl, 20% glycerol (v/v) and 1 mM dithiothreitol (DTT). Peptide pull-downs were performed essentially as described except that the assay buffer contained 50 mM Tris pH 8.0, 500 mM NaCl, and 0.1% NP-40, and 500 pmols of biotinylated histone peptides were loaded onto streptavidin coated magnetic beads before incubation with 40 pmols of protein. Bound proteins were detected with rabbit GST antibody (Sigma, G7781). Point mutants were generated by site-directed mutagenesis and purified/assayed as described above. The YEATS domains of Taf14, AF9, ENL, and GAS41 were previously described.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>16448</offset><text>Western blotting</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>16465</offset><text>Yeast cultures were grown in YPD media at 30°C to mid-log phase and extracts were prepared as previously described. Proteins from cell lysates were separated by SDS-PAGE and transferred to a PVDF membrane. Anti-H3K9ac (Millipore, 07-352) and anti-H3K9cr (PTM Biolabs, PTM-516) were diluted to 1:2000 and 1:1000, respectively, in 1x Superblock (ThermoScientific). An HRP-conjugated anti-rabbit (GE Healthcare) was used for detection. Bands were quantified using the ImageJ program.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>16947</offset><text>Dot blotting</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>16960</offset><text>Increasing concentrations of biotinylated histone peptides (0.06–1.5 μg) were spotted onto a PVDF membrane then probed with the anti-H3K9ac (Millipore, 07-352) or H3K9cr (PTM Biolabs, PTM-516) at 1:2000 in a 5% non-fat milk solution and detected with an HRP-conjugated anti-rabbit by enhanced chemiluminesence (ECL).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>17280</offset><text>Bromodomains pull-downs</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>17304</offset><text>cDNAs of GST-fused bromodomains were obtained either from EpiCypher Inc. or as a kind gift from Katrin Chua (Stanford University). GST fusions were expressed as described above except that the preparation buffer contained 50 mM Tris (pH 7.5), 150 mM NaCl, 10% glycerol (v/v), and 1 mM DTT. Pull-down assays were preformed as described above except that the assay buffer contained 50 mM Tris (pH 8.0), 300 mM NaCl, and 0.1% NP-40.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">title_1</infon><offset>17734</offset><text>Supplementary Material</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>17757</offset><text>Accession codes. Coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 5IOK.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>17881</offset><text>Author contributions</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>17902</offset><text>F.H.A., S.A.S., E.K.S., J.B.B., A.G., I.K.T and K.K. performed experiments and together with X.S., B.D.S and T.G.K. analyzed the data. F.H.A., S.A.S., B.D.S. and T.G.K. wrote the manuscript with input from all authors.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>18121</offset><text>Competing Financial Interest</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>18150</offset><text>The authors declare no competing financial interests.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>18204</offset><text>Additional information</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>18227</offset><text>Any supplementary information is available in the online version of this paper.</text></passage><passage><infon key="fpage">1016</infon><infon key="lpage">28</infon><infon key="name_0">surname:Tan;given-names:M</infon><infon key="pub-id_pmid">21925322</infon><infon key="section_type">REF</infon><infon 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key="name_0">surname:Keogh;given-names:MC</infon><infon key="pub-id_pmid">16299494</infon><infon key="section_type">REF</infon><infon key="source">Nature</infon><infon key="type">ref</infon><infon key="volume">439</infon><infon key="year">2006</infon><offset>20415</offset><text>A phosphatase complex that dephosphorylates gammaH2AX regulates DNA damage checkpoint recovery</text></passage><passage><infon key="file">nihms769551f1.jpg</infon><infon key="id">F1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>20510</offset><text>The structural mechanism for the recognition of H3K9cr</text></passage><passage><infon key="file">nihms769551f1.jpg</infon><infon key="id">F1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>20565</offset><text>(a) Chemical structure of crotonyllysine. (b) The crystal structure of the Taf14 YEATS domain (wheat) in complex with the H3K9cr5-13 peptide (green). (c) H3K9cr is stabilized via an extensive network of intermolecular electrostatic and polar interactions with the Taf14 YEATS domain. (d) The π-π-π stacking mechanism involving the alkene moiety of crotonyllysine.</text></passage><passage><infon key="file">nihms769551f2.jpg</infon><infon key="id">F2</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>20932</offset><text>H3K9cr is a selective target of the Taf14 YEATS domain</text></passage><passage><infon key="file">nihms769551f2.jpg</infon><infon key="id">F2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>20987</offset><text>(a, b) Western blot analysis comparing the levels of H3K9cr and H3K9ac in wild type (WT), HAT deletion, or HDAC deletion yeast strains. Total H3 was used as a loading control. (c) Superimposed 1H,15N HSQC spectra of Taf14 YEATS recorded as H3K9cr5-13 and H3K9ac5-13 peptides were titrated in. Spectra are color coded according to the protein:peptide molar ratio. (d) Western blot analyses of peptide pull-down assays using wild-type and mutated Taf14 YEATS domains and indicated peptides.</text></passage></document></collection>

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+<collection><source>PMC</source><date>20201215</date><key>pmc.key</key><document><id>4880283</id><infon key="license">CC BY</infon><passage><infon key="alt-title">Crystal Structures of Putative Sugar Kinases</infon><infon key="article-id_doi">10.1371/journal.pone.0156067</infon><infon key="article-id_pmc">4880283</infon><infon key="article-id_pmid">27223615</infon><infon key="article-id_publisher-id">PONE-D-16-05184</infon><infon key="elocation-id">e0156067</infon><infon key="issue">5</infon><infon key="license">This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</infon><infon key="name_0">surname:Xie;given-names:Yuan</infon><infon key="name_1">surname:Li;given-names:Mei</infon><infon key="name_2">surname:Chang;given-names:Wenrui</infon><infon key="name_3">surname:Zeth;given-names:Kornelius</infon><infon key="name_4">surname:Chang;given-names:Wenrui</infon><infon key="name_5">surname:Li;given-names:Mei</infon><infon key="name_6">surname:Chang;given-names:Wenrui</infon><infon key="name_7">surname:Li;given-names:Mei</infon><infon key="notes">All structural files are available from the Protein Data Bank (accession numbers 5HTN, 5HTP, 5HUX, 5HV7, 5HTJ, 5HU2, 5HTY, 5HTR, 5HTV and 5HTX).</infon><infon key="section_type">TITLE</infon><infon key="title">Data Availability</infon><infon key="type">front</infon><infon key="volume">11</infon><infon key="year">2016</infon><offset>0</offset><text>Crystal Structures of Putative Sugar Kinases from Synechococcus Elongatus PCC 7942 and Arabidopsis Thaliana</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>108</offset><text>The genome of the Synechococcus elongatus strain PCC 7942 encodes a putative sugar kinase (SePSK), which shares 44.9% sequence identity with the xylulose kinase-1 (AtXK-1) from Arabidopsis thaliana. Sequence alignment suggests that both kinases belong to the ribulokinase-like carbohydrate kinases, a sub-family of FGGY family carbohydrate kinases. However, their exact physiological function and real substrates remain unknown. Here we solved the structures of SePSK and AtXK-1 in both their apo forms and in complex with nucleotide substrates. The two kinases exhibit nearly identical overall architecture, with both kinases possessing ATP hydrolysis activity in the absence of substrates. In addition, our enzymatic assays suggested that SePSK has the capability to phosphorylate D-ribulose. In order to understand the catalytic mechanism of SePSK, we solved the structure of SePSK in complex with D-ribulose and found two potential substrate binding pockets in SePSK. Using mutation and activity analysis, we further verified the key residues important for its catalytic activity. Moreover, our structural comparison with other family members suggests that there are major conformational changes in SePSK upon substrate binding, facilitating the catalytic process. Together, these results provide important information for a more detailed understanding of the cofactor and substrate binding mode as well as the catalytic mechanism of SePSK, and possible similarities with its plant homologue AtXK-1.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">title_1</infon><offset>1612</offset><text>Introduction</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1625</offset><text>Carbohydrates are essential cellular compounds involved in the metabolic processes present in all organisms. Phosphorylation is one of the various pivotal modifications of carbohydrates, and is catalyzed by specific sugar kinases. These kinases exhibit considerable differences in their folding pattern and substrate specificity. Based on sequence analysis, they can be divided into four families, namely HSP 70_NBD family, FGGY family, Mer_B like family and Parm_like family. The FGGY family carbohydrate kinases contain different types of sugar kinases, all of which possess different catalytic substrates with preferences for short-chained sugar substrates, ranging from triose to heptose. These sugar substrates include L-ribulose, erythritol, L-fuculose, D-glycerol, D-gluconate, L-xylulose, D-ribulose, L-rhamnulose and D-xylulose. Structures reported in the Protein Data Bank of the FGGY family carbohydrate kinases exhibit a similar overall architecture containing two protein domains, one of which is responsible for the binding of substrate, while the second is used for binding cofactor ATP. While the binding pockets for substrates are at the same position, each FGGY family carbohydrate kinases uses different substrate-binding residues, resulting in high substrate specificity.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2917</offset><text>Synpcc7942_2462 from the cyanobacteria Synechococcus elongatus PCC 7942 encodes a putative sugar kinase (SePSK), and this kinase contains 426 amino acids. The At2g21370 gene product from Arabidopsis thaliana, xylulose kinase-1 (AtXK-1), whose mature form contains 436 amino acids, is located in the chloroplast (ChloroP 1.1 Server). SePSK and AtXK-1 display a sequence identity of 44.9%, and belong to the ribulokinase-like carbohydrate kinases, a sub-family of FGGY family carbohydrate kinases. Members of this sub-family are responsible for the phosphorylation of sugars similar to L-ribulose and D-ribulose. The sequence and the substrate specificity of ribulokinase-like carbohydrate kinases are different, but they share the common folding feature with two domains. Domain I exhibits a ribonuclease H-like folding pattern, and is responsible for the substrate binding, while domain II possesses an actin-like ATPase domain that binds cofactor ATP.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3870</offset><text>Two possible xylulose kinases (xylulose kinase-1: XK-1 and xylulose kinase-2: XK-2) from Arabidopsis thaliana were previously proposed. It was shown that XK-2 (At5g49650) located in the cytosol is indeed xylulose kinase. However, the function of XK-1 (At2g21370) inside the chloroplast stroma has remained unknown. SePSK from Synechococcus elongatus strain PCC 7942 is the homolog of AtXK-1, though its physiological function and substrates remain unclear. In order to obtain functional and structural information about these two proteins, here we reported the crystal structures of SePSK and AtXK-1. Our findings provide new details of the catalytic mechanism of SePSK and lay the foundation for future studies into its homologs in eukaryotes.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>4615</offset><text>Results and Discussion</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>4638</offset><text>Overall structures of apo-SePSK and apo-AtXK-1</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>4685</offset><text>The attempt to solve the SePSK structure by molecular replacement method failed with ribulokinase from Bacillus halodurans (PDB code: 3QDK, 15.7% sequence identity) as an initial model. We therefore used single isomorphous replacement anomalous scattering method (SIRAS) for successful solution of the apo-SePSK structure at a resolution of 2.3 Å. Subsequently, the apo-SePSK structure was used as molecular replacement model to solve all other structures identified in this study.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>5168</offset><text>Our structural analysis showed that apo-SePSK consists of one SePSK protein molecule in an asymmetric unit. The amino-acid residues were traced from Val2 to His419, except for the Met1 residue and the seven residues at the C-termini. Apo-SePSK contains two domains referred to further on as domain I and domain II (Fig 1A). Domain I consists of non-contiguous portions of the polypeptide chains (aa. 2–228 and aa. 402–419), exhibiting 11 α-helices and 11 β-sheets. Among all these structural elements, α4/α5/α11/α18, β3/β2/β1/β6/β19/β20/β17 and α21/α32 form three patches, referred to as A1, B1 and A2, exhibiting the core region. In addition, four β-sheets (β7, β10, β12 and β16) and five α-helices (α8, α9, α13, α14 and α15) flank the left side of the core region. Domain II is comprised of aa. 229–401 and classified into B2 (β31/β29/β22/β23/β25/β24) and A3 (α26/α27/α28/α30) (Fig 1A and S1 Fig). In the SePSK structure, B1 and B2 are sandwiched by A1, A2 and A3, and the whole structure shows the A1/B1/A2/B2/A3 (α/β/α/β/α) folding pattern, which is in common with other members of FGGY family carbohydrate kinases (S2 Fig). The overall folding of SePSK resembles a clip, with A2 of domain I acting as a hinge region. As a consequence, a deep cleft is formed between the two domains.</text></passage><passage><infon key="file">pone.0156067.g001.jpg</infon><infon key="id">pone.0156067.g001</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>6599</offset><text>Overall structures of SePSK and AtXK-1.</text></passage><passage><infon key="file">pone.0156067.g001.jpg</infon><infon key="id">pone.0156067.g001</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>6639</offset><text>(A) Three-dimensional structure of apo-SePSK. The secondary structural elements are indicated (α-helix: cyan, β-sheet: yellow). (B) Three-dimensional structure of apo-AtXK-1. The secondary structural elements are indicated (α-helix: green, β-sheet: wheat).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>6910</offset><text>Apo-AtXK-1 exhibits a folding pattern similar to that of SePSK in line with their high sequence identity (Fig 1B and S1 Fig). However, superposition of structures of AtXK-1 and SePSK shows some differences, especially at the loop regions. A considerable difference is found in the loop3 linking β3 and α4, which is stretched out in the AtXK-1 structure, while in the SePSK structure, it is bent back towards the inner part. The corresponding residues between these two structures (SePSK-Lys35 and AtXK-1-Lys48) have a distance of 15.4 Å (S3 Fig).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>7464</offset><text>Activity assays of SePSK and AtXK-1</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>7500</offset><text>In order to understand the function of these two kinases, we performed structural comparison using Dali server. The structures most closely related to SePSK are xylulose kinase, glycerol kinase and ribulose kinase, implying that SePSK and AtXK-1 might function similarly to these kinases. We first tested whether both enzymes possessed ATP hydrolysis activity in the absence of substrates. As shown in Fig 2A, both SePSK and AtXK-1 exhibited ATP hydrolysis activity. This finding is in agreement with a previous result showing that xylulose kinase (PDB code: 2ITM) possessed ATP hydrolysis activity without adding substrate. To further identify the actual substrate of SePSK and AtXK-1, five different sugar molecules, including D-ribulose, L-ribulose, D-xylulose, L-xylulose and Glycerol, were used in enzymatic activity assays. As shown in Fig 2B, the ATP hydrolysis activity of SePSK greatly increased upon adding D-ribulose than adding other potential substrates, suggesting that it has D-ribulose kinase activity. In contrary, limited increasing of ATP hydrolysis activity was detected for AtXK-1 upon addition of D-ribulose (Fig 2C), despite its structural similarity with SePSK.</text></passage><passage><infon key="file">pone.0156067.g002.jpg</infon><infon key="id">pone.0156067.g002</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>8686</offset><text>The enzymatic activity assays of SePSK and AtXK-1.</text></passage><passage><infon key="file">pone.0156067.g002.jpg</infon><infon key="id">pone.0156067.g002</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>8737</offset><text>(A) The ATP hydrolysis activity of SePSK and AtXK-1. Both SePSK and AtXK-1 showed ATP hydrolysis activity in the absence of substrate. While the ATP hydrolysis activity of SePSK greatly increases upon addition of D-ribulose (DR). (B) The ATP hydrolysis activity of SePSK with addition of five different substrates. The substrates are DR (D-ribulose), LR (L-ribulose), DX (D-xylulose), LX (L-xylulose) and GLY (Glycerol). (C) The ATP hydrolysis activity of SePSK and AtXK-1 with or without D-ribulose. (D) The ATP hydrolysis activity of wild-type (WT) and single-site mutants of SePSK. Three single-site mutants of SePSK are D8A-SePSK, T11A-SePSK and D221A-SePSK. The ATP hydrolysis activity measured via luminescent ADP-Glo assay (Promega).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9478</offset><text>To understand the catalytic mechanism of SePSK, we performed structural comparisons among xylulose kinase, glycerol kinase, ribulose kinase and SePSK. Our results suggested that three conserved residues (D8, T11 and D221 of SePSK) play an important role in SePSK function. Mutations of the corresponding residue in xylulose kinase and glycerol kinase from Escherichia coli greatly reduced their activity. To identify the function of these three residues of SePSK, we constructed D8A, T11A and D221A mutants. Using enzymatic activity assays, we found that all of these mutants exhibit much lower activity of ATP hydrolysis after adding D-ribulose than that of wild type, indicating the possibility that these three residues are involved in the catalytic process of phosphorylation D-ribulose and are vital for the function of SePSK (Fig 2D).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>10319</offset><text>SePSK and AtXK-1 possess a similar ATP binding site</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10371</offset><text>To obtain more detailed information of SePSK and AtXK-1 in complex with ATP, we soaked the apo-crystals in the reservoir adding cofactor ATP, and obtained the structures of SePSK and AtXK-1 bound with ATP at the resolution of 2.3 Å and 1.8 Å, respectively. In both structures, a strong electron density was found in the conserved ATP binding pocket, but can only be fitted with an ADP molecule (S4 Fig). Thus the two structures were named ADP-SePSK and ADP-AtXK-1, respectively. The extremely weak electron densities of ATP γ-phosphate in both structures suggest that the γ-phosphate group of ATP is either flexible or hydrolyzed by SePSK and AtXK-1. This result was consistent with our enzymatic activity assays where SePSK and AtXK-1 showed ATP hydrolysis activity without adding any substrates (Fig 2A and 2C).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>11193</offset><text>To avoid hydrolysis of ATP, we soaked the crystals of apo-SePSK and apo-AtXK-1 into the reservoir adding AMP-PNP. However, we found that the electron densities of γ-phosphate group of AMP-PNP (AMP-PNP γ-phosphate) are still weak in the AMP-PNP-SePSK and AMP-PNP-AtXK-1 structures, suggesting high flexibility of ATP-γ-phosphate. The γ-phosphate group of ATP is transferred to the sugar substrate during the reaction process, so this flexibility might be important for the ability of these kinases. The overall structures as well as the coordination modes of ADP and AMP-PNP in the AMP-PNP-AtXK-1, ADP-AtXK-1, ADP-SePSK and AMP-PNP-SePSK structures are nearly identical (S5 Fig), therefore the structure of AMP-PNP-SePSK is used here to describe the structural details and to compare with those of other family members. As shown in Fig 3A, one SePSK protein molecule is in an asymmetric unit with one AMP-PNP molecule. The AMP-PNP is bound at the domain II, where it fits well inside a positively charged groove. The AMP-PNP binding pocket consists of four α-helices (α26, α28, α27 and α30) and forms a shape resembling a half-fist (Fig 3A and 3B). The head group of the AMP-PNP is embedded in a pocket surrounded by Trp383, Asn380, Gly376 and Gly377. The purine ring of AMP-PNP is positioned in parallel to the indole ring of Trp383. In addition, it is hydrogen-bonded with the side chain amide of Asn380 (Fig 3B). The tail of AMP-PNP points to the hinge region of SePSK, and its α-phosphate and β-phosphate groups are stabilized by Gly376 and Ser243, respectively. Together, this structure clearly shows that the AMP-PNP-β-phosphate is sticking out of the ATP binding pocket, thus the γ-phosphate group is at the empty space between domain I and domain II and is unconstrained in its movement by the protein.</text></passage><passage><infon key="file">pone.0156067.g003.jpg</infon><infon key="id">pone.0156067.g003</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>13053</offset><text>Structure of SePSK in complex with AMP-PNP.</text></passage><passage><infon key="file">pone.0156067.g003.jpg</infon><infon key="id">pone.0156067.g003</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>13097</offset><text>(A) The electron density of AMP-PNP. The SePSK structure is shown in the electrostatic potential surface mode. The AMP-PNP is depicted as sticks with its ǀFoǀ-ǀFcǀ map contoured at 3 σ shown as cyan mesh. (B) The AMP-PNP binding pocket. The head of AMP-PNP is sandwiched by four residues (Leu293, Gly376, Gly377 and Trp383). The protein skeleton is shown as cartoon (cyan). The four α-helices (α26, α28, α27 and α30) are labeled in red. The AMP-PNP and coordinated residues are shown as sticks. The interactions between them are represented as black dashed lines. The numerical note near the black dashed line indicates the distance (Å).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>13759</offset><text>The potential substrate binding site in SePSK</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>13805</offset><text>The results from our activity assays suggested that SePSK has D-ribulose kinase activity. To better understand the interaction pattern between SePSK and D-ribulose, the apo-SePSK crystals were soaked into the reservoir with 10 mM D-ribulose (RBL) and the RBL-SePSK structure was solved. As shown in S6 Fig, two residual electron densities are visible in domain I, which can be interpreted as two D-ribulose molecules with reasonable fit.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>14243</offset><text>As shown in Fig 4A, the nearest distance between the carbon skeleton of two D-ribulose molecules are approx. 7.1 Å (RBL1-C4 and RBL2-C1). RBL1 is located in the pocket consisting of α21 and the loop between β6 and β7. The O4 and O5 of RBL1 are coordinated with the side chain carboxyl group of Asp221. Furthermore, the O2 of RBL1 interacts with the main chain amide nitrogen of Ser72 (Fig 4B). This pocket is at a similar position of substrate binding site of other sugar kinase, such as L-ribulokinase (PDB code: 3QDK) (S7 Fig). However, structural comparison shows that the substrate ligating residues between the two structures are not strictly conserved. Based on the structures, the ligating residues of RBL1 in RBL-SePSK structure are Ser72, Asp221 and Ser222, and the interacting residues of L-ribulose with L-ribulokinase are Ala96, Lys208, Asp274 and Glu329 (S7 Fig). Glu329 in 3QDK has no counterpart in RBL-SePSK structure. In addition, although Lys208 of L-ribulokinase has the corresponding residue (Lys163) in RBL-SePSK structure, the hydrogen bond of Lys163 is broken because of the conformational change of two α-helices (α9 and α13) of SePSK. These differences might account for their different substrate specificity.</text></passage><passage><infon key="file">pone.0156067.g004.jpg</infon><infon key="id">pone.0156067.g004</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>15500</offset><text>The binding of D-ribulose (RBL) with SePSK.</text></passage><passage><infon key="file">pone.0156067.g004.jpg</infon><infon key="id">pone.0156067.g004</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>15544</offset><text>(A) The electrostatic potential surface map of RBL-SePSK and a zoom-in view of RBL binding site. The RBL1 and RBL2 are depicted as sticks. (B) Interaction of two D-ribulose molecules (RBL1 and RBL2) with SePSK. The RBL molecules (carbon atoms colored yellow) and amino acid residues of SePSK (carbon atoms colored green) involved in RBL interaction are shown as sticks. The hydrogen bonds are indicated by the black dashed lines and the numbers near the dashed lines are the distances (Å). (C) The binding affinity assays of SePSK with D-ribulose. Single-cycle kinetic data are reflecting the interaction of SePSK and D8A-SePSK with D-ribulose. It shows two experimental sensorgrams after minus the empty sensorgrams. The original data is shown as black curve, and the fitted data is shown as different color (wild type SePSK: red curve, D8A-SePSK: green curve). Dissociation rate constant of wild type and D8A-SePSK are 3 ms-1 and 9 ms-1, respectively.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>16499</offset><text>The binding pocket of RBL2 with relatively weak electron density is near the N-terminal region of SePSK and is negatively charged. The side chain of Asp8 interacts strongly with O3 and O4 of RBL2. The hydroxyl group of Ser12 coordinates with O2 of RBL2. The backbone amide nitrogens of Gly13 and Arg15 also keep hydrogen bonds with RBL2 (Fig 4B). Structural comparison of SePSK and AtXK-1 showed that while the RBL1 binding pocket is conserved, the RBL2 pocket is disrupted in AtXK-1 structure, despite the fact that the residues interacting with RBL2 are highly conserved between the two proteins. In the RBL-SePSK structure, a 2.6 Å hydrogen bond is present between RBL2 and Ser12 (Fig 4B), while in the AtXK-1 structure this hydrogen bond with the corresponding residue (Ser22) is broken. This break is probably induced by the conformational change of the two β-sheets (β1 and β2), with the result that the linking loop (loop 1) is located further away from the RBL2 binding site. This change might be the reason that AtXK-1 only shows limited increasing in its ATP hydrolysis ability upon adding D-ribulose as a substrate after comparing with SePSK (Fig 2C).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>17671</offset><text>Our SePSK structure shows that the Asp8 residue forms strong hydrogen bond with RBL2 (Fig 4B). In addition, our enzymatic assays indicated that Asp8 is important for the activity of SePSK (Fig 2D). To further verified this result, we measured the binding affinity for D-ribulose of both wild type (WT) and D8A mutant of SePSK using a surface plasmon resonance method. The results showed that the affinity of D8A-SePSK with D-ribulose is weaker than that of WT with a reduction of approx. two third (Fig 4C). Dissociation rate constant (Kd) of wild type and D8A-SePSK are 3 ms-1 and 9 ms-1, respectively. The results implied that the second RBL binding site plays a role in the D-ribulose kinase function of SePSK. However, considering the high concentration of D-ribulose used for crystal soaking, as well as the relatively weak electron density of RBL2, it is also possible that the second binding site of D-ribulose in SePSK is an artifact.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>18614</offset><text>Simulated conformational change of SePSK during the catalytic process</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>18684</offset><text>It was reported earlier that the crossing angle between the domain I and domain II in FGGY family carbohydrate kinases is different. In addition, this difference may be caused by the binding of substrates and/or ATP. As reported previously, members of the sugar kinase family undergo a conformational change to narrow the crossing angle between two domains and reduce the distance between substrate and ATP in order to facilitate the catalytic reaction of phosphorylation of sugar substrates. After comparing structures of apo-SePSK, RBL-SePSK and AMP-PNP-SePSK, we noticed that these structures presented here are similar. Superposing the structures of RBL-SePSK and AMP-PNP-SePSK, the results show that the nearest distance between AMP-PNP γ-phosphate and RBL1/RBL2 is 7.5 Å (RBL1-O5)/6.7 Å (RBL2-O1) (S8 Fig). This distance is too long to transfer the γ-phosphate group from ATP to the substrate. Since the two domains of SePSK are widely separated in this structure, we hypothesize that our structures of SePSK represent its open form, and that a conformational rearrangement must occur to switch to the closed state in order to facilitate the catalytic process of phosphorylation of sugar substrates.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>19898</offset><text>For studying such potential conformational change, a simulation on the Hingeprot Server was performed to predict the movement of different SePSK domains. The results showed that domain I and domain II are closer to each other with Ala228 and Thr401 in A2 as Hinge-residues. Based on the above results, SePSK is divided into two rigid parts. The domain I of RBL-SePSK (aa. 1–228, aa. 402–421) and the domain II of AMP-PNP-SePSK (aa. 229–401) were superposed with structures, including apo-AtXK-1, apo-SePSK, xylulose kinase from Lactobacillus acidophilus (PDB code: 3LL3) and the S58W mutant form of glycerol kinase from Escherichia coli (PDB code: 1GLJ). The results of superposition displayed different crossing angle between these two domains. After superposition, the distances of AMP-PNP γ-phosphate and the fifth hydroxyl group of RBL1 are 7.9 Å (superposed with AtXK-1), 7.4 Å (superposed with SePSK), 6.6 Å (superposed with 3LL3) and 6.1 Å (superposed with 1GLJ). Meanwhile, the distances of AMP-PNP γ-phosphate and the first hydroxyl group of RBL2 are 7.2 Å (superposed with AtXK-1), 6.7 Å (superposed with SePSK), 3.7 Å (superposed with 3LL3), until AMP-PNP γ-phosphate fully contacts RBL2 after superposition with 1GLJ (Fig 5). This distance between RBL2 and AMP-PNP-γ-phosphate is close enough to facilitate phosphate transferring. Together, our superposition results provided snapshots of the conformational changes at different catalytic stages of SePSK and potentially revealed the closed form of SePSK.</text></passage><passage><infon key="file">pone.0156067.g005.jpg</infon><infon key="id">pone.0156067.g005</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>21433</offset><text>Simulated conformational change of SePSK during the catalytic process.</text></passage><passage><infon key="file">pone.0156067.g005.jpg</infon><infon key="id">pone.0156067.g005</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>21504</offset><text>The structures are shown as cartoon and the ligands are shown as sticks. Domain I from D-ribulose-SePSK (green) and Domain II from AMP-PNP-SePSK (cyan) are superposed with apo-AtXK-1 (1st), apo-SePSK (2nd), 3LL3 (3rd) and 1GLJ (4th), respectively. The numbers near the black dashed lines show the distances (Å) between two nearest atoms of RBL and AMP-PNP.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>21862</offset><text>In summary, our structural and enzymatic analyses provide evidence that SePSK shows D-ribulose kinase activity, and exhibits the conserved features of FGGY family carbohydrate kinases. Three conserved residues in SePSK were identified to be essential for this function. Our results provide the detailed information about the interaction of SePSK with ATP and substrates. Moreover, structural superposition results enable us to visualize the conformational change of SePSK during the catalytic process. In conclusion, our results provide important information for a more detailed understanding of the mechanisms of SePSK and other members of FGGY family carbohydrate kinases.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>22537</offset><text>Materials and Methods</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>22559</offset><text>Cloning, expression and purification of SePSK</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>22605</offset><text>The gene encoding SePSK was amplified by polymerase chain reaction (PCR) with forward primer 5' CATGCCATGGGCATGGTCGTTGCACTTGGCCTCG 3' containing an internal Nco I restriction site (underlined) and reverse primer 5' CCGCTCGAGGGTTCTCTTTAACCCCGCCG 3' including an internal Xho I restriction site (underlined) from Synechococcus elongatus PCC 7942 genomic DNA. The amplified PCR product was digested with Nco I and Xho I (Takara) and ligated into linearized pET28-a vector (Novagen) between Nco I and Xho I restriction sites with a C-terminal his6 tag. The recombinant plasmids were transformed into competent Escherichia coli Trans10 cells for DNA production and purification, and the final constructs were verified by sequencing. The recombinant vectors were transformed into Escherichia coli BL21 (DE3) to express the protein. After induction with the 1 mM IPTG at 289 K in Luria-Bertani medium until the cell density reached an OD 600 nm of 0.6–0.8, the cells were harvested by centrifugation at 6,000 g at 4°C for 15 min, re-suspended in buffer A (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM imidazole) and disrupted by sonication. After centrifuge 40,000 g for 30 min, the protein was purified by passage through a Ni2+ affinity column in buffer A, and then washed the unbound protein with buffer B (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 60 mM imidazole), and eluted the fraction with the buffer C (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 500 mM imidazole). After that, the protein was further purified by size exclusion chromatography with Superdex 200 10/300 GL (GE Healthcare) equilibrated with the buffer D (20 mM Tris-HCl, pH 8.0, 300 mM NaCl). The eluted major peak fraction was concentrated to 20 mg/mL protein using 10,000 MCWO centrifugal filter units (Millipore) and stored at -80°C for crystallization trials. The purified product was analyzed by SDS-PAGE with a single band visible only.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>24505</offset><text>Cloning, expression and purification of AtXK-1</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>24552</offset><text>The gene encoding AtXK-1 was amplified by PCR using a forward primer 5' TACTTCCAATCCAATGCTGTTATGAGTGGCAATAAAGGAACGA 3' and reverse primer 5' TTATCCACTTCCAATGTTACAAACCACTGTTCTGTTTTGCGCCC 3' from cDNA library of Arabidopsis thaliana. The underlined nucleotides were used for the ligation-independent cloning. The PCR product was treated by T4 DNA polymerase (LIC-qualified, Novagen) and then cloned into linearized pMCSG7 vector treated by T4 DNA polymerase (LIC-qualified, Novagen) with an N-terminal his6 tag though ligation-independent cloning method. The final construct was confirmed by DNA sequencing after amplified in competent Escherichia coli Trans10 cells. The recombinant vectors were transformed into Escherichia coli BL21 (DE3) for protein expression. After induction with 1 mM IPTG at 289 K in Luria-Bertani medium, cells were grown until the cell density reached an OD 600 nm of 0.6–0.8. Subsequent purification was identical to that used for SePSK, except that there was one additional step, during which tobacco etch virus protease was used to digest the crude AtXK-1 protein for removal of the N-terminal his6 tag following Ni2+ affinity purification. Ni2+ affinity column buffer contained extra 20% glycerol. The protein was further purified by size exclusion chromatography with Superdex 200 10/300 GL (GE Healthcare) in elution buffer consisting of 20 mM HEPES, pH 7.5, 100 mM NaCl. Finally, AtXK-1 protein was concentrated to 40 mg/mL protein using 10,000 MCWO centrifugal filter units (Millipore) and stored at -80°C prior to crystallization trials. Purity was verified by SDS-PAGE with a single band visible only.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>26192</offset><text>Site-directed mutagenesis of SePSK</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>26227</offset><text>The gene of D8A and T11A mutations were amplified by PCR with the forward primer 5' CATGCCATGGGCATGGTCGTTGCACTTGGCCTCGCCTTCGGCAC 3' and forward primer 5' CATGCCATGGGCATGGTCGTTGCACTTGGCCTCGACTTCGGCGCCTCTGGAGCCC 3' (mismatched base pairs are underlined). The reverse primers of D8A and T11A mutants, the further constructions and purification procedures were identical with those used for wild type SePSK.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>26631</offset><text>The N-terminal sequence of D221A was amplified with forward primer (T7 promoter primer) 5' TAATACGACTCACTATA 3' and reverse primer 5' AGCAGCAATGCTAGCCGTTGTACCG 3’, and the C-terminal sequence of D221A was amplified with forward primer 5' TGCCGGTACAACGGCTAGCATTGCT 3' and reverse primer (T7 terminator primer) CGATCAATAACGAGTCGCC (mismatched base pairs are underlined). The second cycle PCR used the above PCR products as templates, and the construction and purification procedures were identical to those used for wild type SePSK.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>27164</offset><text>Crystallization and data collection</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>27200</offset><text>Crystallization trials of SePSK and AtXK-1 were carried out at 281 K by mixing equal volume of 20 mg/ml protein and reservoir solution with the sitting-drop vapor diffusion method. The reservoir solution was PEG Rx I-35 (0.1 M BIS-TRIS pH 6.5, 20% w/v Polyethylene glycol monomethyl ether 5,000) (Hampton research). After 2 or 3 days, the rod-like crystals could be observed. For phasing, the high-quality apo-SePSK crystals were soaked in mother liquor containing 1 mM ethylmercuricthiosalicylic acid, sodium salt (Hampton research, heavy atom kit) overnight at 281 K. In order to get the complexes with ATP and AMP-PNP, the crystals of apo-SePSK and apo-AtXK-1 were incubated with the reservoir including 10 mM ATP and 20 mM MgCl2 as well as 10 mM AMP-PNP and 20 mM MgCl2, respectively. The apo-SePSK crystals were incubated with the reservoir including 10 mM D-ribulose in order to obtain the complex D-ribulose-bound SePSK (RBL-SePSK). The crystals of three mutants (D8A, T11A and D221A) grew in the same condition as that of the wild type SePSK. The crystals were dipped into reservoir solution supplemented with 15% glycerol and then flash frozen in a nitrogen gas stream at 100 K. All data sets were collected at Shanghai Synchrotron Radiation Facility, Photo Factory in Japan and Institute of Biophysics, Chinese Academy of Sciences. Diffraction data were processed using the HKL2000 package.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>28601</offset><text>Structure determination and refinement</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>28640</offset><text>The initial phases of SePSK were obtained from the Hg-derivative crystals by single isomorphous replacement anomalous scattering (SIRAS) using AutoSol from the PHENIX suite. AutoBuild from the PHENIX suite was used to build 75% of the main chain of apo-SePSK, and the remaining residues were built manually by Coot. All other structures were solved by molecular replacement method using apo-SePSK as an initial model. The model was refined using phenix.refine and REFMAC5. The final model was checked with PROCHECK. All structural figures were prepared by PyMOL. The summary of the data-collection and structure-refinement statistics is shown in Table 1 and S1 Table. Atomic coordinates and structure factors in this article have been deposited in the Protein Data Bank. The deposited codes of all structures listed in the Table 1 and S1 Table.</text></passage><passage><infon key="file">pone.0156067.t001.xml</infon><infon key="id">pone.0156067.t001</infon><infon key="section_type">TABLE</infon><infon key="type">table_title_caption</infon><offset>29485</offset><text>Data collection and refinement statistics.</text></passage><passage><infon key="file">pone.0156067.t001.xml</infon><infon key="id">pone.0156067.t001</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;colgroup span=&quot;1&quot;&gt;&lt;col align=&quot;left&quot; valign=&quot;middle&quot; span=&quot;1&quot;/&gt;&lt;col align=&quot;left&quot; valign=&quot;middle&quot; span=&quot;1&quot;/&gt;&lt;col align=&quot;left&quot; valign=&quot;middle&quot; span=&quot;1&quot;/&gt;&lt;col align=&quot;left&quot; valign=&quot;middle&quot; span=&quot;1&quot;/&gt;&lt;col align=&quot;left&quot; valign=&quot;middle&quot; span=&quot;1&quot;/&gt;&lt;col align=&quot;left&quot; valign=&quot;middle&quot; span=&quot;1&quot;/&gt;&lt;/colgroup&gt;&lt;thead&gt;&lt;tr&gt;&lt;th align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Data set&lt;/th&gt;&lt;th align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Hg-SePSK&lt;/th&gt;&lt;th align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;apo-SePSK&lt;/th&gt;&lt;th align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;AMP-PNP-SePSK&lt;/th&gt;&lt;th align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;RBL-SePSK&lt;/th&gt;&lt;th align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;apo-AtXK-1&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Data collection&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Space group&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;C 1 2 1&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;C 1 2 1&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;C 1 2 1&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;C 1 2 1&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;P21&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Wavelength (Å)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.54178&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.54178&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.54178&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.54178&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.54178&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Cell parameters&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;a/b/c(Å)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;103.1, 46.6, 88.3&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;110.2, 49.0, 86.9&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;103.5, 46.6, 88.0&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;102.6, 47.0, 88.7&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;49.7, 87.9, 53.6&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;α/β/γ(°)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;90.0, 91.9, 90.0&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;90.0, 110.3, 90.0&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;90.0, 91.0, 90.0&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;90.0, 91.4, 90.0&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;90.0, 97.0, 90.0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Resolution (Å)&lt;xref ref-type=&quot;table-fn&quot; rid=&quot;t001fn001&quot;&gt;&lt;sup&gt;a&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;50.00–2.20(2.28–2.20)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;50.00–2.30(2.38–2.30)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;50.00–2.30(2.38–2.30)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;50.00–2.35(2.43–2.35)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;50.00–2.00(2.07–2.00)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;R merge&lt;xref ref-type=&quot;table-fn&quot; rid=&quot;t001fn002&quot;&gt;&lt;sup&gt;b&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.105(0.514)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.149(0.501)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.082(0.503)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.095(0.507)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.106(0.454)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;〈 I/σ(I)〉&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;28.89(4.07)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;13.85(4.10)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;10.18(1.79)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;19.4(4.6)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;12.91(4.08)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Completeness (%)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;92.3(99.2)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;96.1(94.2)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;98.9(99.8)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;99.8(100.0)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;97.1(94.5)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Redundancy&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;6.7(5.1)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;7.4(7.5)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2.4(2.4)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;6.9(6.7)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;7.2(6.9)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Refinement statistics&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Resolution (Å)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;32.501–2.301&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;24.707–2.300&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;24.475–2.344&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;23.771–1.998&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;R&lt;sub&gt;work&lt;/sub&gt;/ R&lt;sub&gt;free&lt;/sub&gt;&lt;xref ref-type=&quot;table-fn&quot; rid=&quot;t001fn003&quot;&gt;&lt;sup&gt;c&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.1834/0.2276&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.1975/0.2327&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.2336/0.2687&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.1893/0.2161&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;No. atoms&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Protein&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3503&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3196&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3209&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3256&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;ligand/ion&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;-&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;31&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;20&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;-&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Water&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;313&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;146&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;143&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;486&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;RMSD Bond lengths (Å)&lt;xref ref-type=&quot;table-fn&quot; rid=&quot;t001fn004&quot;&gt;&lt;sup&gt;d&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.003&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.005&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.003&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.003&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;RMSD Bond angles (°)&lt;xref ref-type=&quot;table-fn&quot; rid=&quot;t001fn004&quot;&gt;&lt;sup&gt;d&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.674&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.886&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.649&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.838&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Ramachandran plot (%)&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;favoured&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;98.1&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;97.8&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;96.7&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;99.1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;allowed&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.9&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2.2&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3.3&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.9&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;disallowed&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.0&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.0&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.0&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;PDB code&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5HTN&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5HTP&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5HV7&lt;/td&gt;&lt;td align=&quot;justify&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5HTR&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>29528</offset><text>Data set	Hg-SePSK	apo-SePSK	AMP-PNP-SePSK	RBL-SePSK	apo-AtXK-1	 	Data collection						 	Space group	C 1 2 1	C 1 2 1	C 1 2 1	C 1 2 1	P21	 	Wavelength (Å)	1.54178	1.54178	1.54178	1.54178	1.54178	 	Cell parameters						 	a/b/c(Å)	103.1, 46.6, 88.3	110.2, 49.0, 86.9	103.5, 46.6, 88.0	102.6, 47.0, 88.7	49.7, 87.9, 53.6	 	α/β/γ(°)	90.0, 91.9, 90.0	90.0, 110.3, 90.0	90.0, 91.0, 90.0	90.0, 91.4, 90.0	90.0, 97.0, 90.0	 	Resolution (Å)a	50.00–2.20(2.28–2.20)	50.00–2.30(2.38–2.30)	50.00–2.30(2.38–2.30)	50.00–2.35(2.43–2.35)	50.00–2.00(2.07–2.00)	 	R mergeb	0.105(0.514)	0.149(0.501)	0.082(0.503)	0.095(0.507)	0.106(0.454)	 	〈 I/σ(I)〉	28.89(4.07)	13.85(4.10)	10.18(1.79)	19.4(4.6)	12.91(4.08)	 	Completeness (%)	92.3(99.2)	96.1(94.2)	98.9(99.8)	99.8(100.0)	97.1(94.5)	 	Redundancy	6.7(5.1)	7.4(7.5)	2.4(2.4)	6.9(6.7)	7.2(6.9)	 	Refinement statistics						 	Resolution (Å)		32.501–2.301	24.707–2.300	24.475–2.344	23.771–1.998	 	Rwork/ Rfreec		0.1834/0.2276	0.1975/0.2327	0.2336/0.2687	0.1893/0.2161	 	No. atoms						 	Protein		3503	3196	3209	3256	 	ligand/ion		-	31	20	-	 	Water		313	146	143	486	 	RMSD Bond lengths (Å)d		0.003	0.005	0.003	0.003	 	RMSD Bond angles (°)d		0.674	0.886	0.649	0.838	 	Ramachandran plot (%)						 	favoured		98.1	97.8	96.7	99.1	 	allowed		1.9	2.2	3.3	0.9	 	disallowed		0.0	0.0	0.0	0.0	 	PDB code		5HTN	5HTP	5HV7	5HTR	 	</text></passage><passage><infon key="file">pone.0156067.t001.xml</infon><infon key="id">pone.0156067.t001</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>30913</offset><text>a The values in parentheses correspond to the highest resolution shell.</text></passage><passage><infon key="file">pone.0156067.t001.xml</infon><infon key="id">pone.0156067.t001</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>30985</offset><text>b Rmerge = ∑j∑h|Ij,h-&lt;Ih&gt;|/∑j∑h&lt;Ih&gt; where h are unique reflection indices and Ij,h are intensities of symmetry-related reflections and &lt;Ih&gt; is the mean intensity.</text></passage><passage><infon key="file">pone.0156067.t001.xml</infon><infon key="id">pone.0156067.t001</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>31156</offset><text>c R-work and R-free were calculated as follows: R = Σ (|Fobs-Fcalc|)/Σ |Fobs| ×100, where Fobs and Fcalc are the observed and calculated structure factor amplitudes, respectively.</text></passage><passage><infon key="file">pone.0156067.t001.xml</infon><infon key="id">pone.0156067.t001</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>31344</offset><text>d Root mean square deviations (r.m.s.d.) from standard values.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>31407</offset><text>ADP-Glo kinase assay</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>31428</offset><text>ADP-Glo kinase assay was used according to the manufacturer’s instructions (Promega). Each reaction mixture system consisted of 8 uM enzyme, 100 uM ATP, 1 mM MgCl2, 20 mM HEPES (pH 7.4), 5 mM substrate. The reaction was initiated by adding the purified enzyme into the reaction system. After incubation at 298 K for different time, equal volume ADP-Glo™ reagent was added to terminate the kinase reaction and to deplete any remaining ATP. Subsequently, kinase detection reagent with double volume of reaction system was added to convert ADP to ATP and allowed the newly synthesized ATP to be measured using a luciferase/luciferin reaction which produced luminescence signal and could be recorded. After incubation at room temperature for another 60 min, luminescence was detected by Varioskan Flash Multimode Reader (Thermo). The reference experiment was carried out in the same reaction system without the enzyme. For each assay, at least three repeats were performed for the calculation of mean values and standard deviations (SDs). The purity of five substrates in the activity assays was ≥98% (D-ribulose, Santa cruz), 99.7% (L-ribulose, Carbosynth), 99.3% (D-xylulose, Carbosynth), 99.5% (L-xylulose, Carbosynth) and 99.0% (Glycerol, AMRESCO).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>32683</offset><text>Surface plasmon resonance</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>32709</offset><text>Surface plasmon resonance (SPR) was used to analyze the interaction of SePSK and D-ribulose. The SPR experiments were performed on a Biacore T100 system using series S CM5 sensor chips (GE Healthcare). All sensorgrams were recorded at 298 K. The proteins in buffer containing 20 mM HEPES, pH 7.5, 100 mM NaCl, was diluted to 40 ug/ml by 10 mM sodium acetate buffer at pH 4.5. All flow cells on a CM5 sensor chip were activated with a freshly prepared solution of 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and 0.05 M N-hydroxysuccinimide (NHS) in a ratio of 1:1 at a constant flow rate of 10 ul/min for 420 s. Deactivation of the surface was performed with an injection of a 1 M solution of ethanolamine-HCl (pH 8.5) using the same flow rate and duration. Kinetic parameters were derived from data sets acquired in single-cycle mode. Each run consisted of five consecutive analytic injections at 125, 250, 500, 1000 and 2000 uM. Analytic injections lasted for 60 s, separated by 30 s dissociation periods. Each cycle was completed with an extended dissociation period of 300 s. The specific binding to a blank flow cell was subtracted to obtain corrected sensorgrams. Biacore data were analyzed using BiaEvaluation software (GE Healthcare) by fitting to a 1:1 Langmuir binding fitting model.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>34017</offset><text>Accession Codes</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>34033</offset><text>Coordinates and structure factors for all the structures in this article have been deposited in the Protein Data Bank. These accession codes are 5HTN, 5HTP, 5HUX, 5HV7, 5HTJ, 5HU2, 5HTY, 5HTR, 5HTV and 5HTX. The corresponding-structures are apo-SePSK, AMP-PNP-SePSK, ADP-SePSK, RBL-SePSK, D8A-SePSK, T11A-SePSK, D221A-SePSK, apo-AtXK1, AMP-PNP-AtXK1 and ADP-AtXK1, respectively.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">title_1</infon><offset>34412</offset><text>Supporting Information</text></passage><passage><infon key="section_type">REF</infon><infon key="type">title</infon><offset>34435</offset><text>References</text></passage><passage><infon key="fpage">e1002318</infon><infon key="issue">12</infon><infon key="name_0">surname:Zhang;given-names:Y</infon><infon key="name_1">surname:Zagnitko;given-names:O</infon><infon key="name_2">surname:Rodionova;given-names:I</infon><infon key="name_3">surname:Osterman;given-names:A</infon><infon key="name_4">surname:Godzik;given-names:A</infon><infon key="pub-id_doi">10.1371/journal.pcbi.1002318</infon><infon key="pub-id_pmid">22215998</infon><infon key="section_type">REF</infon><infon key="source">PLoS 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+<collection><source>PMC</source><date>20201220</date><key>pmc.key</key><document><id>4885502</id><infon key="license">NO-CC CODE</infon><passage><infon key="alt-title">Cryo-EM Studies with Glutamate Dehydrogenase</infon><infon key="article-id_doi">10.1124/mol.116.103382</infon><infon key="article-id_pmc">4885502</infon><infon key="article-id_pmid">27036132</infon><infon key="article-id_publisher-id">MOL_103382</infon><infon key="fpage">645</infon><infon key="issue">6</infon><infon key="lpage">651</infon><infon key="name_0">surname:Borgnia;given-names:Mario J.</infon><infon key="name_1">surname:Banerjee;given-names:Soojay</infon><infon key="name_10">surname:Milne;given-names:Jacqueline L. S.</infon><infon key="name_2">surname:Merk;given-names:Alan</infon><infon key="name_3">surname:Matthies;given-names:Doreen</infon><infon key="name_4">surname:Bartesaghi;given-names:Alberto</infon><infon key="name_5">surname:Rao;given-names:Prashant</infon><infon key="name_6">surname:Pierson;given-names:Jason</infon><infon key="name_7">surname:Earl;given-names:Lesley A.</infon><infon key="name_8">surname:Falconieri;given-names:Veronica</infon><infon key="name_9">surname:Subramaniam;given-names:Sriram</infon><infon key="section_type">TITLE</infon><infon key="type">front</infon><infon key="volume">89</infon><infon key="year">2016</infon><offset>0</offset><text>Using Cryo-EM to Map Small Ligands on Dynamic Metabolic Enzymes: Studies with Glutamate Dehydrogenase</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>102</offset><text>Cryo-electron microscopy (cryo-EM) methods are now being used to determine structures at near-atomic resolution and have great promise in molecular pharmacology, especially in the context of mapping the binding of small-molecule ligands to protein complexes that display conformational flexibility. We illustrate this here using glutamate dehydrogenase (GDH), a 336-kDa metabolic enzyme that catalyzes the oxidative deamination of glutamate. Dysregulation of GDH leads to a variety of metabolic and neurologic disorders. Here, we report near-atomic resolution cryo-EM structures, at resolutions ranging from 3.2 Å to 3.6 Å for GDH complexes, including complexes for which crystal structures are not available. We show that the binding of the coenzyme NADH alone or in concert with GTP results in a binary mixture in which the enzyme is in either an “open” or “closed” state. Whereas the structure of NADH in the active site is similar between the open and closed states, it is unexpectedly different at the regulatory site. Our studies thus demonstrate that even in instances when there is considerable structural information available from X-ray crystallography, cryo-EM methods can provide useful complementary insights into regulatory mechanisms for dynamic protein complexes.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">title_1</infon><offset>1392</offset><text>Introduction</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1405</offset><text>Recent advances in cryo-electron microscopy (cryo-EM) allow determination of structures of small protein complexes and membrane proteins at near-atomic resolution, marking a critical shift in the structural biology field. One specific area of broad general interest in drug discovery is the localization of bound ligands and cofactors under conditions in which efforts at crystallization have not been successful because of structural heterogeneity. Recent cryo-EM analyses have already demonstrated that it is now possible to use single-particle cryo-EM methods to localize small bound ligands or inhibitors on target proteins. Whether ligand binding can be visualized at high resolution is an important question, even in the more general case when multiple conformations are present simultaneously. Here, we address this question using mammalian glutamate dehydrogenase as an example.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2292</offset><text>Glutamate dehydrogenase (GDH) is a highly conserved enzyme expressed in most organisms. GDH plays a central role in glutamate metabolism by catalyzing the reversible oxidative deamination of glutamate to generate α-ketoglutarate and ammonia, with the concomitant transfer of a pair of electrons to either NAD+ or NADP+. Regulation of GDH is tightly controlled through multiple allosteric mechanisms. Extensive biochemical and crystallographic studies have characterized the enzymatic activity of GDH and its modulation by a chemically diverse group of compounds such as nucleotides, amino acids, steroid hormones, antipsychotic drugs, and natural products. X-ray crystallographic studies have shown that the functional unit of GDH is a homohexamer composed of a trimer of dimers, with a 3-fold axis and an equatorial plane that define its D3 symmetry (Fig. 1A). Each 56-kDa protomer consists of three domains. The first is located near the dimer interface and forms the core of the hexamer. The second, a nucleotide-binding domain (NBD) with a Rossmann fold, defines one face of the catalytic cleft bounded by the core domain. During the catalytic cycle, the NBD executes a large movement, hinged around a “pivot” helix, that closes the catalytic cleft, and drives a large conformational change in the hexamer from open to closed states (Fig. 1B). The third domain, dubbed the “antenna,” is an evolutionary acquisition in protista and animals. Antennae of adjacent protomers in each trimer intercalate to form a bundle, perpendicular to the pivot helices, that protrudes along the distal extremes of the 3-fold axis. When a protomer undergoes a conformational change, the rotation of its pivot helix is transferred through the antenna to the adjacent subunit. The influence of the antenna, present only in protozoan and metazoan enzymes, has been proposed to explain its cooperative behavior, which is absent in bacterial homologs. Deletion of this domain leads to loss of cooperativity.</text></passage><passage><infon key="file">mol.116.103382f1.jpg</infon><infon key="id">F1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>4289</offset><text>Structure and quaternary conformational changes in GDH. (A) Views of open (PDB ID 1NR7) and closed (PDB 3MW9) states of the GDH hexamer, shown in ribbon representation perpendicular to the 2-fold symmetry axis (side view, top) and 3-fold symmetry axis (top view, bottom). Only three protomers are shown in the top view for purposes of visual clarity. The dashed lines and arrows, respectively, highlight the slight extension in length, and twist in shape that occurs with transition from open to the closed state. The open state shown is for unliganded GDH, whereas the closed state has NADH, GTP, and glutamate bound. (B) Superposition of structures for closed and open conformations, along with a series of possible intermediate conformations along the trajectory that serve to illustrate the extent of change in structure across different regions of the protein.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>5155</offset><text>The transition between “closed” and “open” states of GDH is modulated by two allosteric sites in each protomer (Fig. 1A), which are differentially bound by GTP (an inhibitor) and ADP (an activator). These allosteric modulators tightly control GDH function in vivo. In the first site, which sits next to the pivot helix at the base of the antenna (the “GTP binding site”), GTP binding is known to act as an inhibitor, preventing release of the reaction product from the catalytic site by stabilizing the closed conformation of the catalytic cleft. In the second “regulatory site”, which is situated near the pivot helix between adjacent protomers, ADP acts as an activator of enzymatic activity, presumably by hastening the opening of the catalytic cleft that leads to the release of the reaction product. Interestingly, it has also been shown that the coenzyme NADH can bind to the regulatory site (also bound by the activator ADP), exerting a converse, inhibitory effect on GDH product release, although the role this may play in vivo is not entirely clear.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>6230</offset><text>Although there are numerous crystal structures available for GDH in complex with cofactors and nucleotides, they are limited to the combinations that have been amenable to crystallization. Nearly all X-ray structures of mammalian GDH are in the closed conformation, and the few structures that are in the open conformation are at lower resolution (Table 1). Of those structures in the closed conformation, most include NAD[P]H, GTP, and glutamate (or, alternately, NAD+, GTP, and α-ketoglutarate). However, the effects of coenzyme and GTP, bound alone or in concert in the absence of glutamate, have not been analyzed by crystallographic methods. Here, we report single-particle cryo-electron microscopy (cryo-EM) studies that show that under these conditions enzyme complexes coexist in both closed and open conformations. We show that the structures in both states can be resolved at near-atomic resolution, suggesting a molecular mechanism for synergistic inhibition of GDH by NADH and GTP (see Table 2 for detailed information on all cryo-EM-derived structures that we report in this work).</text></passage><passage><infon key="file">T1.xml</infon><infon key="id">T1</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>7329</offset><text>X-ray structures of mammalian GDH reported in both the open and closed conformations</text></passage><passage><infon key="file">T1.xml</infon><infon key="id">T1</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;col width=&quot;17.06%&quot; span=&quot;1&quot;/&gt;&lt;col width=&quot;33.34%&quot; span=&quot;1&quot;/&gt;&lt;col width=&quot;13.13%&quot; span=&quot;1&quot;/&gt;&lt;col width=&quot;20.31%&quot; span=&quot;1&quot;/&gt;&lt;col width=&quot;16.16%&quot; span=&quot;1&quot;/&gt;&lt;thead&gt;&lt;tr&gt;&lt;th valign=&quot;top&quot; align=&quot;center&quot; scope=&quot;col&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;GDH&lt;/th&gt;&lt;th valign=&quot;top&quot; align=&quot;center&quot; scope=&quot;col&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Ligands&lt;/th&gt;&lt;th valign=&quot;top&quot; align=&quot;center&quot; scope=&quot;col&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;PDB ID&lt;/th&gt;&lt;th valign=&quot;top&quot; align=&quot;center&quot; scope=&quot;col&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Conformation&lt;/th&gt;&lt;th valign=&quot;top&quot; align=&quot;center&quot; scope=&quot;col&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Resolution&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; scope=&quot;row&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;WT&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NADH + GLU + GTP&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3MW9&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Closed&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2.4&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; scope=&quot;row&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;WT&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Glu, GTP, NADPH, and Bithionol&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3ETD&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Closed&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2.5&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; scope=&quot;row&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;WT&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Glu, NADPH, GTP + GW5074&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3ETG&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Closed&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2.5&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; scope=&quot;row&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;WT&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;apo&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1L1F&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Open&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2.7&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; scope=&quot;row&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;WT&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NADPH, glutamate, and GTP&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1HWZ&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Closed&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2.8&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; scope=&quot;row&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;WT&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NADPH + GLU + GTP + Zinc&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3MVQ&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Closed&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2.94&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; scope=&quot;row&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;WT&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NADPH, Glu, GTP, Hexachlorophene&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3ETE&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Closed&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; scope=&quot;row&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;WT&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NAD, PO4, and 2-oxoglutarate&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1HWY&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Closed&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3.2&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; scope=&quot;row&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;WT&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NADPH + GLU + Eu&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3MVO&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Closed&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3.23&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; scope=&quot;row&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;R463A mutant&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;apo&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1NR1&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Open&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3.3&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; scope=&quot;row&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;WT&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;apo&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1NR7&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Open&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3.3&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; scope=&quot;row&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;WT&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;ADP&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1NQT&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Open&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3.5&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; scope=&quot;row&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;WT&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NADPH and Epicatechin-3-gallate (Ecg)&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3QMU&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Open&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3.62&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>7414</offset><text>GDH	Ligands	PDB ID	Conformation	Resolution	 	WT	NADH + GLU + GTP	3MW9	Closed	2.4	 	WT	Glu, GTP, NADPH, and Bithionol	3ETD	Closed	2.5	 	WT	Glu, NADPH, GTP + GW5074	3ETG	Closed	2.5	 	WT	apo	1L1F	Open	2.7	 	WT	NADPH, glutamate, and GTP	1HWZ	Closed	2.8	 	WT	NADPH + GLU + GTP + Zinc	3MVQ	Closed	2.94	 	WT	NADPH, Glu, GTP, Hexachlorophene	3ETE	Closed	3	 	WT	NAD, PO4, and 2-oxoglutarate	1HWY	Closed	3.2	 	WT	NADPH + GLU + Eu	3MVO	Closed	3.23	 	R463A mutant	apo	1NR1	Open	3.3	 	WT	apo	1NR7	Open	3.3	 	WT	ADP	1NQT	Open	3.5	 	WT	NADPH and Epicatechin-3-gallate (Ecg)	3QMU	Open	3.62	 	</text></passage><passage><infon key="file">T2.xml</infon><infon key="id">T2</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>7991</offset><text>Cryo-EM structures of mammalian GDH determined for this study</text></passage><passage><infon key="file">T2.xml</infon><infon key="id">T2</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;col width=&quot;7.86%&quot; span=&quot;1&quot;/&gt;&lt;col width=&quot;15.31%&quot; span=&quot;1&quot;/&gt;&lt;col width=&quot;18.73%&quot; span=&quot;1&quot;/&gt;&lt;col width=&quot;14.12%&quot; span=&quot;1&quot;/&gt;&lt;col width=&quot;16.09%&quot; span=&quot;1&quot;/&gt;&lt;col width=&quot;12.62%&quot; span=&quot;1&quot;/&gt;&lt;col width=&quot;15.27%&quot; span=&quot;1&quot;/&gt;&lt;thead&gt;&lt;tr&gt;&lt;th valign=&quot;top&quot; align=&quot;center&quot; scope=&quot;col&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;GDH&lt;/th&gt;&lt;th valign=&quot;top&quot; align=&quot;center&quot; scope=&quot;col&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Ligands&lt;/th&gt;&lt;th valign=&quot;top&quot; align=&quot;center&quot; scope=&quot;col&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;EMDB ID&lt;/th&gt;&lt;th valign=&quot;top&quot; align=&quot;center&quot; scope=&quot;col&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;PDB ID&lt;/th&gt;&lt;th valign=&quot;top&quot; align=&quot;center&quot; scope=&quot;col&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Conformation&lt;/th&gt;&lt;th valign=&quot;top&quot; align=&quot;center&quot; scope=&quot;col&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Resolution&lt;/th&gt;&lt;th valign=&quot;top&quot; align=&quot;center&quot; scope=&quot;col&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Particles&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; scope=&quot;row&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;WT&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;apo&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;EMD-6630&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3JCZ&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Open&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3.26&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;22462&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; scope=&quot;row&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;WT&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;GTP&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;EMD-6631&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3JD0&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Open&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3.47&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;39439&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; scope=&quot;row&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;WT&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NADH&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;EMD-6635&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3JD2&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Open&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3.27&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;34716&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; scope=&quot;row&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;WT&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NADH&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;EMD-6634&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3JD1&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Closed&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3.27&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;34926&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; scope=&quot;row&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;WT&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NADH + GTP&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;EMD-6632&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3JD3&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Open&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3.55&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;14793&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; scope=&quot;row&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;WT&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NADH + GTP&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;EMD-6633&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3JD4&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Closed&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3.40&lt;/td&gt;&lt;td valign=&quot;top&quot; align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;20429&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>8053</offset><text>GDH	Ligands	EMDB ID	PDB ID	Conformation	Resolution	Particles	 	WT	apo	EMD-6630	3JCZ	Open	3.26	22462	 	WT	GTP	EMD-6631	3JD0	Open	3.47	39439	 	WT	NADH	EMD-6635	3JD2	Open	3.27	34716	 	WT	NADH	EMD-6634	3JD1	Closed	3.27	34926	 	WT	NADH + GTP	EMD-6632	3JD3	Open	3.55	14793	 	WT	NADH + GTP	EMD-6633	3JD4	Closed	3.40	20429	 	</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>8371</offset><text>Materials and Methods</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_3</infon><offset>8393</offset><text>Specimen Preparation.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>8415</offset><text>Bovine glutamate dehydrogenase (Enzyme Commission 1.4.1.3; Sigma-Aldrich/MilliporeSigma, St. Louis, MO) was dialyzed overnight against fractionation buffer (100 mM potassium phosphate, pH 6.8) prior to fractionation by size-exclusion chromatography using a Superdex 200 10/30 column connected to an ÄKTA FPLC apparatus (GE Healthcare Bio-Sciences, Piscataway, NJ ). The concentration of GDH was adjusted to ∼2 mg/ml by rapid mixing with potassium phosphate buffer containing the concentration of ligand as necessary and with n-octyl glucopyranoside at a final concentration of 0.1%. The final concentration of each ligand was 20 mM. Small volumes of sample, typically 3 µl, were deposited on 200 mesh Quantifoil R2/2 grids (Quantifoil Micro Tools, Großlöbichaum, Germany), blotted, and plunge-frozen in liquid ethane using an FEI Vitrobot Mark IV (FEI Company, Hillsboro, OR). Frozen grids were mounted into autoloader cartridges and transferred to the microscope.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_3</infon><offset>9386</offset><text>Cryo-Electron Microscopy.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>9412</offset><text>Specimens were imaged on an FEI Titan Krios microscope (FEI Company) aligned for parallel illumination and operated at 300 kV. The instrument was furnished with a Gatan K2 Summit camera placed at the end of a GIF Quantum energy filter (Gatan Inc., Pleasanton, CA), operated in zero-energy-loss mode with a slit width of 20 eV. Images were collected manually at a dose rate of ∼5 e– pixel−1 s−1, i.e., in the linear range of the detector. The physical pixel size at the plane of the specimen was 1.275 Å, corresponding to a super-resolution pixel size of 0.6375 Å. The total exposure time was 15.2 s, and intermediate frames were recorded every 0.4 s, giving an accumulated dose of ∼45 e–/Å2 and a total of 38 frames per image. The majority of images were collected at under-focus values between 1 μm and 3 μm.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_3</infon><offset>10239</offset><text>Data Processing.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>10256</offset><text>Drift and beam-induced motion were compensated by whole-frame alignment of movies, and CTF was estimated as described in. Integrated frames were manually examined and selected on the basis of the quality of the CTF estimation, astigmatism, drift, and particle distribution. Molecular images were automatically identified from selected integrated micrographs by detecting the local maxima of correlation of each image with a Gaussian disk of 150 Å in radius. Individual particle projections were extracted from integrated super-resolution images using a binning factor of 4 and a box size of 96 × 96 pixels and assigned into 20–100 groups by iterative reference free two-dimensional classification as implemented in EMAN2. Following 8 iterations of classification, a subset of classes depicting intact particles were used to build symmetric (D3) density maps using the program e2initialmodel.py from the EMAN2 suite. One or more maps were selected as reference for further processing on the basis of consistency between projections and the original classes. These “initial models” were refined to ∼15–20 Å using e2refine_easy.py. Unbinned particles were then re-extracted from the original super-resolution images using a binning of 2 and a box size of 384 × 384, and subject to classification in three dimensions using the maximum likelihood method implemented in RELION (; MRC Laboratory, Cambridge, UK) (regularization parameter of T=4). Unless noted otherwise, D3 symmetry was imposed for three-dimensional classification runs, the number of classes was initially determined on the basis of the number of particles included in the analysis and later adjusted on the basis of the number of conformations detected in the sample (see Supplemental Table 1 for details). Iteration over classification in three dimensions was continued until convergence as judged by resolution and distribution of particles among the classes. Particles belonging to “good” three-dimensional classes were pooled into one or more classes, depending on the conformational landscape of the complex, and refined using the “gold standard” method in RELION. The refined maps were corrected for the MTF of the camera and for B-factor in the framework of RELION. Figures were generated using UCSF Chimera and Maxon Cinema4D (Maxon Computer Inc., Newbury Park, CA), and two-dimensionally composited in Adobe Photoshop and Illustrator.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_3</infon><offset>12684</offset><text>Building of Atomic Models.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>12711</offset><text>The deposited models for unliganded GDH (1NR7), the binary complex with ADP (1NQT), and the quaternary complex with NADH, GTP, and Glu (3MW9) were used to derive models from the six structures reported here. Conflicts in sequence between the deposited models were solved by conforming to the primary sequence as reported in 3MW9. The models were placed in the corresponding map (open or closed) by rigid body fitting as implemented in Chimera. In the closed state, in which all relative orientations of the ligands are known, only the ligands known to be present in each structure were retained, all other nonstandard residues were deleted. For the open state, ligands were initially placed on the basis of their orientation relative to the corresponding binding site. The models were refined against a map derived from one-half of the dataset using Rosetta as described (https://faculty.washington.edu/dimaio/files/density_tutorial.pdf), followed by real space refinement in PHENIX (; PHENIX, Berkeley, CA) and evaluated by calculating the Fourier shell correlation between the model and the map derived from the second half of the dataset. For each complex, ten best scoring instances on the basis of the Fourier shell correlation were selected among one hundred runs, visually examined, and the one deemed best interactively corrected in Coot. Each corrected model was subject to a final instance of real space refinement using PHENIX.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>14150</offset><text>Results and Discussion</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>14173</offset><text>To explore the conformational landscape of apo-GDH, we first determined its structure in the absence of any added ligands (Supplemental Fig. 1, Fig. 2, A–C). The density map, refined to an average resolution of ∼3.0 Å (Supplemental Fig. 2), is in the open conformation and closely matches the model of unliganded GDH derived by X-ray crystallography at 3.3 Å resolution (PDB ID 1NR7). The variation in local resolution from the core to the periphery, as reported by ResMap (Supplemental Fig. 3D), is consistent with the B-factor gradient observed in the crystal structure (Supplemental Fig. 3A). Extensive classification without imposing symmetry yielded only open structures and failed to detect any closed catalytic cleft in the unliganded enzyme, suggesting that all six protomers are in the open conformation. Consistent with this conclusion, the loops connecting the β-strands of the Rossmann fold are well-defined (Fig. 2B), implying that there is little movement at the NBD, as the transition between closed and open states is associated with NBD movement (Fig. 1B).</text></passage><passage><infon key="file">mol.116.103382f2.jpg</infon><infon key="id">F2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>15254</offset><text>Cryo-EM structures of GDH in unliganded and NADH-bound states. (A) Refined cryo-EM map of unliganded GDH at ∼3 Å resolution. (B, C) Illustration of density map in the regions that contain the Rossmann nucleotide binding fold (B), pivot and antenna helices (C) in the unliganded GDH map. (D) Cryo-EM-derived density maps for two coexisting conformations that are present when GDH is bound to the cofactor NADH. Each protomer is shown in a different color and densities for NADH bound in both regulatory (red) and catalytic (purple) sites on one protomer are indicated. The overall quaternary structures of the two conformations are essentially the same as that of the open and closed states observed by X-ray crystallography.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15982</offset><text>When GDH is bound to NADH, GTP, and glutamate, the enzyme adopts a closed conformation; this “abortive complex” has been determined to 2.4-Å resolution by X-ray crystallography (PDB 3MW9). However, crystal structures of GDH bound only to NADH or to GTP have not yet been reported. To test the effect of NADH binding on GDH conformation in solution, we determined the structure of this binary complex using cryo-EM methods combined with three-dimensional classification. Two dominant conformational states, in an all open or all closed conformation were detected, segregated (Fig. 2D), and further refined to near-atomic resolution (∼3.3 Å; Supplemental Fig. 2). Densities for 12 molecules of bound NADH were identified in maps of both open and closed states (Supplemental Fig. 4). The NADH-bound closed conformation matches the structure of the quaternary complex observed by X-ray crystallography, with the exception that density corresponding to GTP and glutamate was absent in the cryo-EM-derived map.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>16995</offset><text>Comparison of the NADH-bound closed conformation to the NADH-bound open conformation shows that, as expected, the catalytic cleft is closed and the NBDs are displaced toward the equatorial plane, accompanied by a rotation of the pivot helix by ∼7°, concomitant with a large conformational change in the antennae domains (Figs. 1 and 2D). A comparison between NADH-bound open and closed conformations also involves a displacement of helix 5 (residues 171–186), as well as a tilt of the core β-sheets relative to the equatorial plane of the enzyme (residues 57–97, 122–130) and α-helix 2 (residues 36–54), and a bending of the N-terminal helix. Thus, closure of the catalytic cleft is accompanied by a quaternary structural change that can be described as a global bending of the structure about an axis that runs parallel to the pivot helix, accompanied by an expansion of the core (Figs. 1A and 2D).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>17908</offset><text>Detailed analysis of the GDH/NADH structures shows that both the adenosine and nicotinamide moieties of NADH bind to the catalytic site within the NBD in nearly the same orientation in both the open and the closed states, and display closely comparable interactions with the Rossmann fold (Fig. 3, A and B). At the regulatory site, where either ADP can bind as an activator or NADH can bind as an inhibitor, the binding of the adenine moiety of NADH is nearly identical between the two conformers. However, there is a significant difference in the orientation of the nicotinamide and phosphate moieties in the two conformational states (Fig. 3, C and D). In the closed state, the nicotinamide group is oriented toward the center of the hexamer, inserted into a narrow cavity between two adjacent subunits in the trimer. There are extensive interactions between NADH and the residues lining this cavity, which may explain the well-defined density of this portion of NADH in the closed state. In contrast, in the open conformation, the cavity present in the closed state becomes too narrow for the nicotinamide group; instead, the group is oriented in the opposite direction, parallel to the pivot helix with the amido group extending toward the C-terminal end of the helix.</text></passage><passage><infon key="file">mol.116.103382f3.jpg</infon><infon key="id">F3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>19181</offset><text>Detailed view of NADH conformation in catalytic and regulatory sites. (A, B) NADH density (purple) and interactions in the catalytic sites of closed (A) and open (B) states. (C, D) NADH density (red) and interactions in the regulatory sites of closed (C) and open (D) states.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>19457</offset><text>Although there is a difference in orientation of the nicotinamide moiety between the closed and open states in the regulatory site, in both structures the adenine portion of NADH has a similar binding pocket and is located in almost exactly the same position as ADP, a potent activator of GDH function (Supplemental Fig. 5). In the open state, the binding of ADP or NADH is further stabilized by His209, a residue that undergoes a large movement during the transition from open to closed conformation (Fig. 3, C and D). In the open conformation, the distance between His209 and the α-phosphate of NADH is ∼4.4 Å, which is comparable with the corresponding distance in the ADP-bound conformation. In the closed conformation, however, this key histidine residue is &gt;10.5 Å away from the nearest phosphate group on NADH, altering a critical stabilization point within the regulatory site. This suggests that although the conformation of NADH in the open state regulatory site more closely mimics the binding of ADP, the conformation of NADH in the closed state regulatory site is significantly different; these differences may contribute to the opposite effects of NADH and ADP on GDH enzymatic activity.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>20664</offset><text>In the absence of NADH, GTP binds weakly to GDH with a dissociation constant of ∼20 μM. Cryo-EM analysis of GDH incubated with GTP resulted in a structure at an overall resolution of 3.5 Å, showing that it is in an open conformation (Supplemental Fig. 6), with all NBDs in the open state. The density for GTP is not very well defined, suggesting considerable wobble in the binding site. Subtraction of the GTP-bound map with that of the apo state shows that GTP binding can nevertheless be visualized specifically in the GTP binding site (Supplemental Fig. 6). Importantly, the binding of GTP alone does not appear to drive the transition from the open to the closed state of GDH.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>21349</offset><text>To further dissect the roles of NADH and GTP in the transition from the open to closed conformations, we next determined structures of GDH in complex with both NADH and GTP, but without glutamate. When NADH and GTP are both present, classification reveals the presence of both closed and open GDH conformations, similar to the condition when only NADH is present (Fig. 4, A and B). Reconstruction without classification, however, yields a structure clearly in the closed conformation, suggesting that, in coordination with NADH, GTP may further stabilize the closed conformation. The location of GTP in the open and closed states of the GDH/NADH/GTP complex is similar to that in the crystal structure observed in the presence of NADH, GTP, and glutamate. Likewise, the position of NADH in the open and closed states closely resembles the position of NADH in the GDH/NADH open and closed structures. One key difference between the open and closed states of these structures is the position of the His209 residue: As mentioned above, His209 swings away from the adenine moiety of NADH in the closed state. When GTP is present in the GTP binding site, His209 instead interacts with GTP, probably stabilizing the closed conformation (Fig. 4, C and D). Thus, GTP binding to GDH appears synergistic with NADH and displaces the conformational landscape toward the closed state.</text></passage><passage><infon key="file">mol.116.103382f4.jpg</infon><infon key="id">F4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>22721</offset><text>Cryo-EM structure of GDH bound to both NADH and GTP. (A, B) Observation of co-existing open (A) and closed (B) conformations in the GDH-NADH-GTP ternary complex. Densities for GTP (yellow) as well as NADH bound to both catalytic (purple) and regulatory (red) sites in each protomer are shown. (C, D) Detailed inspection of the interactions near the regulatory site show that the orientation of His209 switches between the two states, which may allow interactions with bound GTP in the closed (D), but not open (C) conformation.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>23249</offset><text>Our structural studies thus establish that whether or not GTP is bound, NADH binding is detectable at catalytic and regulatory sites, in both the open and closed conformational states. Whereas the orientation in which NADH binds at the catalytic site is similar for both conformations, the orientation of the nicotinamide portion of NADH in the regulatory site is different between the open and closed conformations (Figs. 3 and 4). In the closed state, the nicotinamide moiety is inserted into a well-defined cavity at the interface between two adjacent protomers in the trimer. As mentioned above, this cavity is much narrower in the open state, suggesting that this cavity may be unavailable to the NADH nicotinamide moiety when the enzyme is in the open conformation. These structural features provide a potential explanation of the weaker density for the nicotinamide moiety of NADH in the open state, and may account for the higher reported affinity of NADH for the closed state. The role of the nicotinamide moiety in acting as a wedge that prevents the transition to the open conformation also suggests a structural explanation of the mechanism by which NADH binding inhibits the activity of the enzyme by stabilizing the closed conformation state.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>24506</offset><text>The rapid emergence of cryo-EM as a tool for near-atomic resolution structure determination provides new opportunities for complementing atomic resolution information from X-ray crystallography, as illustrated here with GDH. Perhaps the most important contribution of these methods is the prospect that when there are discrete subpopulations present, the structure of each state can be determined at near-atomic resolution. What we demonstrate here with GDH is that by employing three-dimensional image classification approaches, we not only can isolate distinct, coexisting conformations, but we can also localize small molecule ligands in each of these conformations. These kinds of approaches will probably become increasingly important in molecular pharmacology, especially in the context of better understanding drug-target interactions in dynamic protein complexes.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">title_1</infon><offset>25378</offset><text>Supplementary Material</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>25401</offset><text>This research was supported by funds from the  National Cancer Institute Center for Cancer Research, the IATAP program at NIH, and the NIH-FEI Living Laboratory for Structural Biology (S.S., J.L.S.M.). This work was supported by the Intramural Research Program of the National Institutes of Health National Cancer Institute.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>25726</offset><text>dx.doi.org/10.1124/mol.116.103382.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>25761</offset><text>This article has supplemental material available at molpharm.aspetjournals.org.</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">title</infon><offset>25841</offset><text>Abbreviations</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>25855</offset><text>cryo-EM</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>25863</offset><text>cryo-electron microscopy</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>25888</offset><text>GDH</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>25892</offset><text>glutamate dehydrogenase</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>25916</offset><text>NBD</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>25920</offset><text>nucleotide binding domain</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">title</infon><offset>25946</offset><text>Authorship Contributions</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">paragraph</infon><offset>25971</offset><text>Participated in research design: Borgnia, Banerjee, Merk, Subramaniam, Milne.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">paragraph</infon><offset>26049</offset><text>Conducted experiments: Borgnia, Banerjee, Merk, Rao, Pierson.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">paragraph</infon><offset>26111</offset><text>Performed data analysis: Borgnia, Banerjee, Merk, Matthies, Bartesaghi, Earl, Falconieri, Subramaniam, Milne.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">paragraph</infon><offset>26221</offset><text>Wrote or contributed to the writing of the manuscript: Borgnia, Banerjee, Earl, Falconieri, Subramaniam, Milne.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">title</infon><offset>26333</offset><text>References</text></passage><passage><infon key="fpage">213</infon><infon key="lpage">221</infon><infon key="name_0">surname:Adams;given-names:PD</infon><infon key="name_1">surname:Afonine;given-names:PV</infon><infon key="name_2">surname:Bunkóczi;given-names:G</infon><infon key="name_3">surname:Chen;given-names:VB</infon><infon key="name_4">surname:Davis;given-names:IW</infon><infon key="name_5">surname:Echols;given-names:N</infon><infon key="name_6">surname:Headd;given-names:JJ</infon><infon key="name_7">surname:Hung;given-names:LW</infon><infon key="name_8">surname:Kapral;given-names:GJ</infon><infon key="name_9">surname:Grosse-Kunstleve;given-names:RW</infon><infon key="pub-id_pmid">20124702</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr D Biol Crystallogr</infon><infon key="type">ref</infon><infon key="volume">66</infon><infon key="year">2010</infon><offset>26344</offset><text>PHENIX: a comprehensive Python-based system for macromolecular structure solution</text></passage><passage><infon key="fpage">14431</infon><infon key="lpage">14443</infon><infon key="name_0">surname:Allen;given-names:A</infon><infon key="name_1">surname:Kwagh;given-names:J</infon><infon key="name_2">surname:Fang;given-names:J</infon><infon key="name_3">surname:Stanley;given-names:CA</infon><infon key="name_4">surname:Smith;given-names:TJ</infon><infon key="pub-id_pmid">15533048</infon><infon key="section_type">REF</infon><infon key="source">Biochemistry</infon><infon key="type">ref</infon><infon key="volume">43</infon><infon key="year">2004</infon><offset>26426</offset><text>Evolution of glutamate dehydrogenase regulation of insulin homeostasis is an example of molecular exaptation</text></passage><passage><infon key="fpage">5579</infon><infon key="lpage">5583</infon><infon key="name_0">surname:Bailey;given-names:J</infon><infon key="name_1">surname:Bell;given-names:ET</infon><infon key="name_2">surname:Bell;given-names:JE</infon><infon key="pub-id_pmid">7068608</infon><infon key="section_type">REF</infon><infon key="source">J Biol Chem</infon><infon key="type">ref</infon><infon key="volume">257</infon><infon key="year">1982</infon><offset>26535</offset><text>Regulation of bovine glutamate dehydrogenase. 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+<collection><source>PMC</source><date>20201221</date><key>pmc.key</key><document><id>4888278</id><infon key="license">CC BY</infon><passage><infon key="article-id_doi">10.1186/s12900-016-0059-3</infon><infon key="article-id_pmc">4888278</infon><infon key="article-id_pmid">27246200</infon><infon key="article-id_publisher-id">59</infon><infon key="elocation-id">7</infon><infon key="kwd">RORγ Agonist Inverse Agonist Activation Function 2 Helix (AF2) TH17cells IL-17 Autoimmune Disease</infon><infon key="license">
+Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.</infon><infon key="name_0">surname:Marcotte;given-names:Douglas J.</infon><infon key="name_1">surname:Liu;given-names:YuTing</infon><infon key="name_2">surname:Little;given-names:Kevin</infon><infon key="name_3">surname:Jones;given-names:John H.</infon><infon key="name_4">surname:Powell;given-names:Noel A.</infon><infon key="name_5">surname:Wildes;given-names:Craig P.</infon><infon key="name_6">surname:Silvian;given-names:Laura F.</infon><infon key="name_7">surname:Chodaparambil;given-names:Jayanth V.</infon><infon key="section_type">TITLE</infon><infon key="title">Keywords</infon><infon key="type">front</infon><infon key="volume">16</infon><infon key="year">2016</infon><offset>0</offset><text>Structural determinant for inducing RORgamma specific inverse agonism triggered by a synthetic benzoxazinone ligand</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract_title_1</infon><offset>116</offset><text>Background</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>127</offset><text>The nuclear hormone receptor RORγ regulates transcriptional genes involved in the production of the pro-inflammatory interleukin IL-17 which has been linked to autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease. This transcriptional activity of RORγ is modulated through a protein-protein interaction involving the activation function 2 (AF2) helix on the ligand binding domain of RORγ and a conserved LXXLL helix motif on coactivator proteins. Our goal was to develop a RORγ specific inverse agonist that would help down regulate pro-inflammatory gene transcription by disrupting the protein protein interaction with coactivator proteins as a therapeutic agent.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract_title_1</infon><offset>855</offset><text>Results</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>863</offset><text>We identified a novel series of synthetic benzoxazinone ligands having an agonist (BIO592) and inverse agonist (BIO399) mode of action in a FRET based assay. We show that the AF2 helix of RORγ is proteolytically sensitive when inverse agonist BIO399 binds. Using x-ray crystallography we show how small modifications on the benzoxazinone agonist BIO592 trigger inverse agonism of RORγ. Using an in vivo reporter assay, we show that the inverse agonist BIO399 displayed specificity for RORγ over ROR sub-family members α and β.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract_title_1</infon><offset>1409</offset><text>Conclusion</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>1420</offset><text>The synthetic benzoxazinone ligands identified in our FRET assay have an agonist (BIO592) or inverse agonist (BIO399) effect by stabilizing or destabilizing the agonist conformation of RORγ. The proteolytic sensitivity of the AF2 helix of RORγ demonstrates that it destabilizes upon BIO399 inverse agonist binding perturbing the coactivator protein binding site. Our structural investigation of the BIO592 agonist and BIO399 inverse agonist structures identified residue Met358 on RORγ as the trigger for RORγ specific inverse agonism.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract_title_1</infon><offset>1972</offset><text>Electronic supplementary material</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>2006</offset><text>The online version of this article (doi:10.1186/s12900-016-0059-3) contains supplementary material, which is available to authorized users.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">title_1</infon><offset>2146</offset><text>Background</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2157</offset><text>Retinoid-related orphan receptor gamma (RORγ) is a transcription factor belonging to a sub-family of nuclear receptors that includes two closely related members RORα and RORβ. Even though a high degree of sequence similarity exists between the RORs, their functional roles in regulation for physiological processes involved in development and immunity are distinct. During development, RORγ regulates the transcriptional genes involved in the functioning of multiple pro-inflammatory lymphocyte lineages including T helper cells (TH17cells) which are necessary for IL-17 production. IL-17 is a pro-inflammatory interleukin linked to autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease; making its transcriptional regulation through RORγ an attractive therapeutic target.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2995</offset><text>RORγ consists of an N-terminal DNA binding domain (DBD) connected to a C-terminal ligand binding domain (LBD) via a flexible hinge region. The DBD is composed of two zinc fingers that allow it to interact with specifically encoded regions on the DNA called the nuclear receptor response elements. The LBD consists of a coactivator protein binding pocket and a hydrophobic ligand binding site (LBS) which are responsible for regulating transcription. The coactivator binding pocket of RORγ recognizes a conserved helix motif LXXLL (where X can be any amino acid) on transcriptional coactivator complexes and recruits it to activate transcription. Like other nuclear hormone receptors, RORγ’s helix12 which makes up the C-termini of the LBD is an essential part of the coactivator binding pocket and is commonly referred to as the activation function helix 2 (AF2). In RORγ, the conformation of the AF2 helix required to form the coactivator binding pocket is mediated by a salt bridge between His479 and Tyr502 in addition to π- π interactions between Tyr502 and Phe506. The conformation of the AF2 helix can be modulated through targeted ligands which bind the LBS and increase the binding of the coactivator protein (agonists) or disrupt binding (inverse agonists) thereby enhancing or inhibiting transcription. Since RORγ has been demonstrated to play an important role in pro-inflammatory gene expression patterns implicated in several major autoimmune diseases, our aim was to develop RORγ inverse agonists that would help down regulate pro-inflammatory gene transcription.</text></passage><passage><infon key="file">12900_2016_59_Fig1_HTML.jpg</infon><infon key="id">Fig1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>4599</offset><text>FRET results for agonist BIO592 (a) and Inverse Agonist BIO399 (b)</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>4666</offset><text>Here we present the identification of two synthetic benzoxazinone RORγ ligands, a weak agonist BIO592 (Fig. 1a) and an inverse agonist BIO399 (Fig. 1b) which were identified using a Fluorescence Resonance Energy transfer (FRET) based assay that monitored coactivator peptide recruitment. Using partial proteolysis in combination with mass spectrometry analysis we demonstrate that the AF2 helix of RORγ destabilizes upon BIO399 (inverse agonist) binding. Finally, comparing binding modes of our benzoxazinone RORγ crystal structures to other ROR structures, we hypothesize a new mode of action for achieving inverse agonism and selectivity.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>5319</offset><text>Methods</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>5327</offset><text>Cloning, protein expression and purification of RORγ518</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>5387</offset><text>GST-RORγ518 was constructed by sub-cloning residues 259 to 518 of a human RORγ cDNA into a pGEX-6P vector with a cleavable N-terminal GST fusion tag. BL21 (DE3) Escherichia coli cells were transformed with the plasmid encoding the GST-PreScission-hRORgamma 259–518 protein (GST-RORγ518) and were grown at 37 °C in LB media supplemented with ampicillin to an OD of 1. The temperature was reduced to 18 °C and protein expression was induced by adding 1 mM IPTG and was shaking for an additional 16 h. The cells were harvested and resuspended in lysis buffer (25 mM TRIS pH 8.0, 250 mM NaCl, 10 % Glycerol, 5 mM DTT and Roche EDTA-free protease inhibitor cocktail) and were lysed using a microfluidizer. The lysate was clarified by centrifugation at 20,000 × g for 1 h at 4 °C and GST-RORγ518 was captured by batch binding to Glutathione Sepharose resin overnight at 4 °C. The resin was washed with buffer A (25 mM TRIS pH 8.0, 250 mM NaCl, 10 % glycerol, 5 mM DTT) and loaded onto a XK column and washed until no non-specific unbound protein was detected. GST- RORγ518 was eluted from the column using buffer A supplemented with 10 mM Glutathione pH 8.0 and analyzed by SDS-PAGE. The eluate was then treated with PreScission Protease (10units/mg of protein) and further purified on a Superdex 75 column equilibrated in buffer B (25 mM TRIS pH 8.0, 250 mM NaCl, 5 % glycerol and 2 mM DTT). RORγ518 eluted as a monomer and was approximately 95 % pure as observed by SDS-PAGE.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>6901</offset><text>Additional constructs including c-terminal truncations, surface entropy reduction and cysteine scrubbed mutations were also expressed and purified in the same manner as RORγ518 if an expression level of &gt;1 mg/L was achieved.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>7130</offset><text>RORγ FRET based assay and GAL4 reporter assay</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>7180</offset><text>FRET-based (Fluorescence Resonance Energy Transfer) assay and the GAL4 Reporter assay were performed as described previously. BIO592 and BIO399 were synthesized (Additional file 1) and belonged to a proprietary library where they were identified as RORγ activity modulators using the FRET-based assay.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>7486</offset><text>Partial proteolysis of RORγ518</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>7521</offset><text>RORγ518 at 8 mg/ml or in complex with 1 mM BIO399 or 1 mM BIO592 and 0.5 mM coactivator peptide EBI96 EFPYLLSLLGEVSPQ (New England Peptide) were treated with Actinase E (Hampton Research) added at a ratio of 1.25ugs of protease/1 mg of RORγ518 for 6 h at 4 °C. The reactions were quenched using 1X Protease inhibitor cocktail (Roche) + 1 mM EDTA and subjected to mass spectrometry analysis.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>7927</offset><text>Mass spectrometry of partially proteolyzed RORγ518</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>7982</offset><text>Proteolyzed RORγ518 samples were reduced with 50 mM dithiothreitol in 50 mM Tris pH 8.0, 150 mM NaCl containing 4 M urea and 5 mM EDTA. The sample was then analyzed on a LC-MS system comprised of a UPLC (ACQUITY, Waters Corp.), a TUV dual-wavelength UV detector (Waters Corp.), and a ZQ mass spectrometer (Waters Corp.). A Vydac C4 cartridge was used for desalting. Molecular masses for the Actinase E treated RORγ518 samples were obtained by deconvoluting the raw mass spectra using MaxLynx 4.1 software (Waters Corp.).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>8511</offset><text>Crystallization of RORγ518 with agonist BIO592 and inverse agonist BIO399</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>8589</offset><text>RORγ518 was concentrated to 8 mg/ml and EBI96 was added to a final concentration of 0.5 mM and agonist BIO592 to 1 mM and incubated on ice for 1 h. The coactivator peptide EBI96 which was identified by phage display was chosen for crystallization because of its strong interaction with RORγ in a mammalian two-hybrid analysis system that assessed the transactivation of RORγ. Diffraction quality crystals were grown through vapor diffusion in a buffer containing 0.1 M HEPES pH 8.0, 25 % PEG3350 and 0.2 M NaCl at 18 °C. Crystals were cryoprotected in the mother liquor containing 20 % glycerol as cryoprotectant prior to being frozen in liquid nitrogen for data collection.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>9278</offset><text>Actinase E proteolyzed RORγ518 BIO399 concentrated to 8 mg/ml was crystallized using vapor diffusion in a buffer containing 0.1 M BisTRIS pH 5.5, 0.2 M ammonium acetate and 15 % PEG3350 at 18 °C. Crystals were cryoprotected for data collection by transferring them to a mother liquor containing 15 % PEG400 prior to being frozen in liquid nitrogen.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>9636</offset><text>Data collection and structure determination for RORγ518 BIO592 and BIO399 complexes</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>9724</offset><text>X-ray diffraction data for all the crystals were measured at beam line ID31 at the Argonne Photon Source. The data were processed with Mosflm in case of the RORγ518-BIO592-EBI96 ternary complex and with HKL2000 in the case of the Actinase E treated aeRORγ518/BIO399 complex. For both datasets, PDB ID: 3LOL was used as the search model, and the molecular replacement solutions were determined using MOLREP. The refinement was carried out using Refmac5 and model building was carried out in Coot. The data processing and refinement statistics are provided in Additional file 2.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>10309</offset><text>RORγ518-BIO592-EBI96 ternary complex:</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>10351</offset><text>The data for the ternary complex were measured to 2.63 Å. It crystallized in a P21 space group with four molecules of the ternary complex in the asymmetric unit. The final model was refined to a Rcryst of 19.9 % and Rfree of 25.5 %.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>10588</offset><text>aeRORγ518/BIO399 complex:</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>10618</offset><text>Diffraction data for the aeRORγ518-BIO399 complex were measured to 2.35 Å. It crystallized in C2 space group with two molecules in the asymmetric unit. The final model was refined to a Rcryst of 21.1 % and Rfree of 26.3 %.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>10846</offset><text>Results and discussion</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>10869</offset><text>Identification of BIO592 and BIO399 as ligands that modulate RORγ coactivator peptide recruitment</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10971</offset><text>Using a FRET based assay we discovered agonist BIO592 (Fig. 1a) which increased the coactivator peptide TRAP220 recruitment to RORγ (EC50 0f 58nM and Emax of 130 %) and a potent inverse agonist BIO399 (Fig. 1b) which inhibited coactivator recruitment (IC50: 4.7nM). Interestingly, the structural difference between the agonist BIO592 and inverse agonist BIO399 was minor; with the 2,3-dihydrobenzo[1,4]oxazepin-4-one ring system of BIO399 being 3 atoms larger than the benzo[1,4]oxazine-3-one ring system of BIO592. In order to understand how small changes in the core ring system leads to inverse agonism, we wanted to structurally determine the binding mode of both BIO592 and BIO399 in the LBS of RORγ using x-ray crystallography.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>11713</offset><text>Structure of the RORγ518-BIO592-EBI96 ternary complex is in a transcriptionally active conformation</text></passage><passage><infon key="file">12900_2016_59_Fig2_HTML.jpg</infon><infon key="id">Fig2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>11817</offset><text>
+a The ternary structure of RORγ518 BIO592 and EBI96. b RORγ AF2 helix in the agonist conformation. c EBI96 coactivator peptide bound in the coactivator pocket of RORγ</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>11997</offset><text>RORγ518 bound to agonist BIO592 was crystallized with a truncated form of the coactivator peptide EBI96 to a resolution of 2.6 Å (Fig. 2a). The structure of the ternary complex had features similar to other ROR agonist coactivator structures in a transcriptionally active canonical three layer helix fold with the AF2 helix in the agonist conformation. The agonist conformation is stabilized by a hydrogen bond between His479 and Tyr502, in addition to π-π interactions between His479, Tyr502 and Phe506 (Fig. 2b). The hydrogen bond between His479 and Tyr502 has been reported to be critical for RORγ agonist activity. Disrupting this interaction through mutagenesis reduced transcriptional activity of RORγ. This reduced transcriptional activity has been attributed to the inability of the AF2 helix to complete the formation of the coactivator binding pocket necessary for coactivator proteins to bind.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>12916</offset><text>Electron density for the coactivator peptide EBI96 was observed for residues EFPYLLSLLG which formed a α-helix stabilized through hydrophobic interactions with the coactivator binding pocket on RORγ (Fig. 2c). This interaction is further stabilized through a conserved charged clamp wherein the backbone amide of Tyr7 and carbonyl of Leu11 of EBI96 form hydrogen bonds with Glu504 (helix12) and Lys336 (helix3) of RORγ. Formation of this charged clamp is essential for RORγ’s function for playing a role in transcriptional activation and this has been corroborated through mutagenic studies in this region.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>13539</offset><text>BIO592 binds in a collapsed conformation stabilizing the agonist conformation of RORγ</text></passage><passage><infon key="file">12900_2016_59_Fig3_HTML.jpg</infon><infon key="id">Fig3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>13629</offset><text>
+a Collapsed binding mode of agonist BIO592 in the hydrophobic LBS of RORγ. b Benzoxazinone ring system of agonist BIO592 packing against His479 of RORγ stabilizing agonist conformation of the AF2 helix</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>13840</offset><text>BIO592 bound in a collapsed conformational state in the LBS of RORγ with the xylene ring positioned at the bottom of the pocket making hydrophobic interactions with Val376, Phe378, Phe388 and Phe401, with the ethyl-benzoxazinone ring making several hydrophobic interactions with Trp317, Leu324, Met358, Leu391, Ile 400 and His479 (Fig. 3a, Additional file 3). The sulfonyl group faces the entrance of the pocket, while the CF3 makes a hydrophobic contact with Ala327. Hydrophobic interaction between the ethyl group of the benzoxazinone and His479 reinforce the His479 sidechain position for making the hydrogen bond with Tyr502 thereby stabilizing the agonist conformation (Fig. 3b).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>14529</offset><text>RORγ AF2 helix is sensitive to proteolysis in the presence of Inverse Agonist BIO399</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>14618</offset><text>Next, we attempted co-crystallization with the inverse agonist BIO399. However, extensive crystallization efforts with BIO399 and RORγ518 or other AF2 intact constructs did not produce crystals. We hypothesized that the RORγ518 coactivator peptide interaction in the FRET assay was disrupted upon BIO399 binding and that a conformational rearrangement of the AF2 helix could have occurred, hindering crystallization.</text></passage><passage><infon key="file">12900_2016_59_Fig4_HTML.jpg</infon><infon key="id">Fig4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>15043</offset><text>Specific proteolytic positions on RORγ518 when treated with Actinase E alone (Green) or in the presence of BIO399 (Red) and shared proteolytic sites (Yellow)</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15205</offset><text>The unfolding of the AF2 helix has been observed for other nuclear hormone receptors when bound to an inverse agonist or antagonist. We used partial proteolysis in combination with mass spectrometry to determine if BIO399 was causing the AF2 helix to unfold. Results of the Actinase E proteolysis experiments on RORγ518, the ternary complex of RORγ518 with agonist BIO592 and coactivator EBI96, or in the presence of inverse agonist BIO399 supported our hypothesis. Analysis of the fragmentation pattern showed minimal proteolytic removal of the AF2 helix by Actinase E on RORγ518 alone (ending at 504 to 506) and the ternary complex remained primarily intact (ending at 515/518) (Additional file 4). However, in the presence of inverse agonist BIO399, the proteolytic pattern showed significantly less protection, albeit the products were more heterogeneous (majority ending at 494/495), indicating the destabilization of the AF2 helix compared to either the APO or ternary agonist complex (Fig. 4, Additional file 5).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>16237</offset><text>Several rounds of cocrystallization attempts with RORγ518 or other RORγ AF2 helix containing constructs complexed with BIO399 had not produced crystals. We attributed the inability to form crystals to the unfolding of the AF2 helix induced by BIO399. We reasoned that if we could remove the unfolded AF2 helix using proteolysis we could produce a binary complex more amenable to crystallization.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>16641</offset><text>AF2 truncated RORγ BIO399 complex is more amenable to crystallization</text></passage><passage><infon key="file">12900_2016_59_Fig5_HTML.jpg</infon><infon key="id">Fig5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>16715</offset><text>
+a The binary structure of AF2-truncated RORγ and BIO399. b The superposition of inverse agonist BIO399 (Cyan) and agonist BIO592 (Green). c Movement of Met358 and His479 in the BIO399 (Cyan) and BIO592 (Green) structures</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>16941</offset><text>The Actinase E treated RORγ518 BIO399 ternary complex (aeRORγ493/4) co-crystallized readily in several PEG based conditions. The structure of aeRORγ493/4 BIO399 complex was solved to 2.3 Å and adopted a similar core fold to the BIO592 agonist crystal structure (Fig. 5a, Additional file 3). The aeRORγ493/4 BIO399 structure diverged at the c-terminal end of Helix 11 from the RORγ518 BIO592 EBI96 structure, where helix 11 unwinds into a random coil after residue L475.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>17431</offset><text>Inverse agonist BIO399 uses Met358 as a trigger for inverse agonism</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>17499</offset><text>BIO399 binds to the ligand binding site of RORγ adopting a collapsed conformation as seen with BIO592 where the two compounds superimpose with an RMSD of 0.72 Å (Fig. 5b). The majority of the side chains within 4 Å of BIO399 and BIO592 adopt similar rotomer conformations with the exceptions of Met358 and His479 (Fig. 5c). The difference density map showed clear positive density for Met358 in an alternate rotomer conformation compared to the one observed in the molecular replacement model or the other agonist containing models (Additional file 6). We tried to refine Met358 in the same conformation as the molecular replacement model or the other agonist containing models, but the results clearly indicated that this was not possible, thus confirming the new rotamer conformation for the Met358 sidechain in the inverse agonist bound structure. The change in rotomer conformation of Met358 between the agonist and inverse agonist structures is attributed to the gem-dimethyl group on the larger 7 membered benzoxazinone ring system of BIO399. The comparison of the two structures shows that the agonist conformation observed in the BIO592 structure would be perturbed by BIO399 pushing Met358 into Phe506 of the AF2 helix indicating that Met358 is a trigger for inducing inverse agonism in RORγ (Fig. 5c).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>18820</offset><text>BIO399 and Inverse agonist T0901317 bind in a collapsed conformation distinct from other RORγ Inverse Agonists Cocrystal structures</text></passage><passage><infon key="file">12900_2016_59_Fig6_HTML.jpg</infon><infon key="id">Fig6</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>18956</offset><text>
+a Overlay of RORγ structures bound to BIO596 (Green), BIO399 (Cyan) and T0901317 (Pink). b Overlay of M358 in RORγ structure BIO596 (Green), BIO399 (Cyan), Digoxin (Yellow), Compound 2 (Grey), Compound 48 (Salmon) and Compound 4j (Orange)</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>19204</offset><text>The co-crystal structure of RORγ with T0901317 (PDB code: 4NB6), an inverse agonist of RORγ (IC50 of 54nM in an SRC1 displacement FRET assay and an IC50 of 59nM in our FRET assay (Additional file 7)) shows that it adopts a collapsed conformation similar to the structure of BIO399 described here. The two compounds superimpose with an RMSD of 0.81 Å (Fig. 6a). The CF3 group on the hexafluoropropanol group of T0901317 was reported to fit the electron density in two conformations one of which pushes Met358 into the vicinity of Phe506 in the RORγ BIO592 agonist structure. We hypothesize that since the Met358 sidechain conformation in the T0901317 RORγ structure is not in the BIO399 conformation, this difference could account for the 10-fold reduction in the inverse agonism for T0901317 compared to BIO399 in the FRET assay.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>20050</offset><text>Co-crystal structures of RORγ have been generated with several potent inverse agonists adopting a linear conformation distinct from the collapsed conformations seen for BIO399 and T090131718. The inverse agonist activity for these compounds has been attributed to orientating Trp317 to clash with Tyr502 or a direct inverse agonist hydrogen bonding event with His479, both of which would perturb the agonist conformation of RORγ. BIO399 neither orients the sidechain of Trp317 toward Tyr502 nor forms a hydrogen bond with His479 suggesting its mode of action is distinct from linear inverse agonists (Additional file 8). In the linear inverse agonist crystal structures the side chain of Met358 resides in a similar position as the rotomer observed in RORγ agonist structures with BIO592 described here or as observed in the hydroxycholesterol derivatives and therefore would not trigger inverse agonism with these ligands (Fig. 6b).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>20996</offset><text>BIO399 shows selectivity for RORγ over RORα and RORβ in a GAL4 Cellular Reporter Assay</text></passage><passage><infon key="file">Tab1.xml</infon><infon key="id">Tab1</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>21094</offset><text>GAL4 cell assay selectivity profile for BIO399 toward RORα and RORβ in GAL4</text></passage><passage><infon key="file">Tab1.xml</infon><infon key="id">Tab1</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;thead&gt;&lt;tr&gt;&lt;th&gt;ROR&lt;/th&gt;&lt;th&gt;γ&lt;/th&gt;&lt;th&gt;α&lt;/th&gt;&lt;th&gt;β&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td&gt;IC50 (uM)&lt;/td&gt;&lt;td&gt;0.043 (+/− 0.01uM; N = 6)&lt;/td&gt;&lt;td&gt;&amp;gt;10 (N = 2)&lt;/td&gt;&lt;td&gt;&amp;gt;1.2 (N = 2)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Selectivity (X)&lt;/td&gt;&lt;td&gt;-&lt;/td&gt;&lt;td&gt;&amp;gt;235&lt;/td&gt;&lt;td&gt;&amp;gt;28.2&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>21177</offset><text>ROR	γ	α	β	 	IC50 (uM)	0.043 (+/− 0.01uM; N = 6)	&gt;10 (N = 2)	&gt;1.2 (N = 2)	 	Selectivity (X)	-	&gt;235	&gt;28.2	 	</text></passage><passage><infon key="file">12900_2016_59_Fig7_HTML.jpg</infon><infon key="id">Fig7</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>21301</offset><text>
+a Overlay of RORα (yellow), β (pink) and γ (cyan) showing side chain differences at Met358 inverse agonism trigger position and (b) around the benzoxazinone ring system of BIO399</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>21492</offset><text>In order to assess the in vivo selectivity profile of BIO399 a cellular reporter assay was implemented where the ligand binding domains of ROR α, β and γ were fused to the DNA binding domain of the transcriptional factor GAL4. The ROR-GAL4 fusion proteins were expressed in cells with the luciferase reporter gene under the control of a GAL4 promoter. BIO399 inhibited the luciferase activity when added to the cells expressing the RORγ-GAL4 fusion with an in vivo IC50 of 42.5nM while showing &gt;235 and 28 fold selectivity over cells expressing GAL4 fused to the LBD of ROR α or β, respectively (Table 1). The LBS of RORs share a high degree of similarity. However, the inverse agonism trigger of BIO399, residue Met358, is a leucine in both RORα and β. This selectivity profile for BIO399 is attributed to the shorter leucine side chain in RORα and β which would not reach the phenylalanine on the AF2 helix further underscoring the role of Met358 as a trigger for RORγ specific inverse agonism (Fig. 7a). Furthermore, RORα contains two phenylalanine residues in its LBS whereas RORβ and γ have a leucine in the same position (Fig. 6b). We hypothesize that the two phenylalanine residues in the LBS of RORα occlude the dihydrobenzoxazepinone ring system of BIO399 from binding it and responsible for the increase in selectivity for RORα over β.</text></passage><passage><infon key="section_type">CONCL</infon><infon key="type">title_1</infon><offset>22900</offset><text>Conclusions</text></passage><passage><infon key="section_type">CONCL</infon><infon key="type">paragraph</infon><offset>22912</offset><text>We have identified a novel series of synthetic benzoxazinone ligands which modulate the transcriptional activity of RORγ in a FRET based assay. Using partial proteolysis we show a conformational change which destabilizes the AF2 helix of RORγ when the inverse agonist BIO399 binds. The two RORγ co-crystal structures reported here show how a small change to the core ring system can modulate the mode of action from agonist (BIO592) to inverse agonism (BIO399). Finally, we are reporting a newly identified trigger for achieving RORγ specific inverse agonism in an in vivo setting through Met358 which perturbs the agonist conformation of the AF2 helix and prevents coactivator protein binding.</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">title_1</infon><offset>23623</offset><text>Abbreviations</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">paragraph</infon><offset>23637</offset><text>AF2, activation function 2; BisTRIS, 2-[Bis(2-hydroxyethyl)amino]-a-(hydroxymethyl)propane-1,3-diol; DND, DNA binding domain; DTT, 1,4-Dithiothreitol; EDTA, 2-({2-[Bis(carboxymethyl)amino]ethyl}(carboxymethyl)amino)acetic acid; FRET, fluorescence resonance energy transfer; GST, Glutathione-S-Transferase; HEPES, 2-[4(2-hydroxyethyl)-1-piperazineethanesulfonic acid; IC50, half maximal inhibitory concentration; IL-17, Interleukin-17; IPTG, isopropyl β-D-1-thiogalactopyranoside; LBD, Ligand Binding Domain; LBS, ligand binding site; LC-MS, liquid chromatography/mass spectrometry; PDB, Protein Data Bank; ROR, retinoid orphan receptor; SRC-1, steroid receptor coactivator-1; TH17 Cells, T helper cells; TRIS, 2-amino-2-hydroxymethyl-propane-1,3,diol.</text></passage><passage><infon key="section_type">ABBR</infon><infon key="type">title_1</infon><offset>24392</offset><text>Additional files</text></passage><passage><infon key="section_type">COMP_INT</infon><infon key="type">title_1</infon><offset>24409</offset><text>Competing interests</text></passage><passage><infon key="section_type">COMP_INT</infon><infon key="type">paragraph</infon><offset>24429</offset><text>The authors declare that they have no competing interests.</text></passage><passage><infon key="section_type">COMP_INT</infon><infon key="type">title_1</infon><offset>24488</offset><text>Consent to publish</text></passage><passage><infon key="section_type">COMP_INT</infon><infon key="type">paragraph</infon><offset>24507</offset><text>Not applicable.</text></passage><passage><infon key="section_type">COMP_INT</infon><infon key="type">title_1</infon><offset>24523</offset><text>Ethics</text></passage><passage><infon key="section_type">COMP_INT</infon><infon key="type">paragraph</infon><offset>24530</offset><text>Not applicable.</text></passage><passage><infon key="section_type">REF</infon><infon key="type">title</infon><offset>24546</offset><text>References</text></passage><passage><infon key="section_type">REF</infon><infon key="type">ref</infon><offset>24557</offset><text>Jetten AM. 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