annotation_IOB/PMC4993997.tsv

Structure	O
and	O
function	O
of	O
human	B-species
Naa60	B-protein
(	O
NatF	B-complex_assembly
),	O
a	O
Golgi	O
-	O
localized	O
bi	O
-	O
functional	O
acetyltransferase	B-protein_type

N	B-ptm
-	I-ptm
terminal	I-ptm
acetylation	I-ptm
(	O
Nt	B-ptm
-	I-ptm
acetylation	I-ptm
),	O
carried	O
out	O
by	O
N	B-protein_type
-	I-protein_type
terminal	I-protein_type
acetyltransferases	I-protein_type
(	O
NATs	B-protein_type
),	O
is	O
a	O
conserved	O
and	O
primary	O
modification	O
of	O
nascent	O
peptide	B-chemical
chains	O
.	O

Naa60	B-protein
(	O
also	O
named	O
NatF	B-complex_assembly
)	O
is	O
a	O
recently	O
identified	O
NAT	B-protein_type
found	O
only	O
in	O
multicellular	B-taxonomy_domain
eukaryotes	I-taxonomy_domain
.	O

This	O
protein	O
was	O
shown	O
to	O
locate	O
on	O
the	O
Golgi	O
apparatus	O
and	O
mainly	O
catalyze	O
the	O
Nt	B-ptm
-	I-ptm
acetylation	I-ptm
of	O
transmembrane	O
proteins	O
,	O
and	O
it	O
also	O
harbors	O
lysine	B-protein_type
Nε	I-protein_type
-	I-protein_type
acetyltransferase	I-protein_type
(	O
KAT	B-protein_type
)	O
activity	O
to	O
catalyze	O
the	O
acetylation	B-ptm
of	O
lysine	B-residue_name
ε	O
-	O
amine	O
.	O

Here	O
,	O
we	O
report	O
the	O
crystal	B-evidence
structures	I-evidence
of	O
human	B-species
Naa60	B-protein
(	O
hNaa60	B-protein
)	O
in	B-protein_state
complex	I-protein_state
with	I-protein_state
Acetyl	B-chemical
-	I-chemical
Coenzyme	I-chemical
A	I-chemical
(	O
Ac	B-chemical
-	I-chemical
CoA	I-chemical
)	O
or	O
Coenzyme	B-chemical
A	I-chemical
(	O
CoA	B-chemical
).	O

The	O
hNaa60	B-protein
protein	O
contains	O
an	O
amphipathic	B-structure_element
helix	I-structure_element
following	O
its	O
GNAT	B-structure_element
domain	I-structure_element
that	O
may	O
contribute	O
to	O
Golgi	O
localization	O
of	O
hNaa60	B-protein
,	O
and	O
the	O
β7	B-structure_element
-	I-structure_element
β8	I-structure_element
hairpin	I-structure_element
adopted	O
different	O
conformations	O
in	O
the	O
hNaa60	B-protein
(	O
1	B-residue_range
-	I-residue_range
242	I-residue_range
)	O
and	O
hNaa60	B-mutant
(	I-mutant
1	I-mutant
-	I-mutant
199	I-mutant
)	I-mutant
crystal	B-evidence
structures	I-evidence
.	O

Remarkably	O
,	O
we	O
found	O
that	O
the	O
side	O
-	O
chain	O
of	O
Phe	B-residue_name_number
34	I-residue_name_number
can	O
influence	O
the	O
position	O
of	O
the	O
coenzyme	B-chemical
,	O
indicating	O
a	O
new	O
regulatory	O
mechanism	O
involving	O
enzyme	O
,	O
co	O
-	O
factor	O
and	O
substrates	O
interactions	O
.	O

Moreover	O
,	O
structural	B-experimental_method
comparison	I-experimental_method
and	I-experimental_method
biochemical	I-experimental_method
studies	I-experimental_method
indicated	O
that	O
Tyr	B-residue_name_number
97	I-residue_name_number
and	O
His	B-residue_name_number
138	I-residue_name_number
are	O
key	O
residues	O
for	O
catalytic	O
reaction	O
and	O
that	O
a	O
non	B-protein_state
-	I-protein_state
conserved	I-protein_state
β3	B-structure_element
-	I-structure_element
β4	I-structure_element
long	I-structure_element
loop	I-structure_element
participates	O
in	O
the	O
regulation	O
of	O
hNaa60	B-protein
activity	O
.	O

Acetylation	B-ptm
is	O
one	O
of	O
the	O
most	O
ubiquitous	O
modifications	O
that	O
plays	O
a	O
vital	O
role	O
in	O
many	O
biological	O
processes	O
,	O
such	O
as	O
transcriptional	O
regulation	O
,	O
protein	O
-	O
protein	O
interaction	O
,	O
enzyme	O
activity	O
,	O
protein	O
stability	O
,	O
antibiotic	O
resistance	O
,	O
biological	O
rhythm	O
and	O
so	O
on	O
.	O

Protein	O
acetylation	B-ptm
can	O
be	O
grouped	O
into	O
lysine	B-ptm
Nε	I-ptm
-	I-ptm
acetylation	I-ptm
and	O
peptide	B-chemical
N	B-ptm
-	I-ptm
terminal	I-ptm
acetylation	I-ptm
(	O
Nt	B-ptm
-	I-ptm
acetylation	I-ptm
).	O

Generally	O
,	O
Nε	B-ptm
-	I-ptm
acetylation	I-ptm
refers	O
to	O
the	O
transfer	O
of	O
an	O
acetyl	B-chemical
group	O
from	O
an	O
acetyl	B-chemical
coenzyme	I-chemical
A	I-chemical
(	O
Ac	B-chemical
-	I-chemical
CoA	I-chemical
)	O
to	O
the	O
ε	O
-	O
amino	O
group	O
of	O
lysine	B-residue_name
.	O

This	O
kind	O
of	O
modification	O
is	O
catalyzed	O
by	O
lysine	B-protein_type
acetyltransferases	I-protein_type
(	O
KATs	B-protein_type
),	O
some	O
of	O
which	O
are	O
named	O
histone	B-protein_type
acetyltransferases	I-protein_type
(	O
HATs	B-protein_type
)	O
because	O
early	O
studies	O
focused	O
mostly	O
on	O
the	O
post	O
-	O
transcriptional	O
acetylation	B-ptm
of	O
histones	B-protein_type
.	O

Despite	O
the	O
prominent	O
accomplishments	O
in	O
the	O
field	O
regarding	O
Nε	B-ptm
-	I-ptm
acetylation	I-ptm
by	O
KATs	B-protein_type
for	O
over	O
50	O
years	O
,	O
the	O
significance	O
of	O
the	O
more	O
evolutionarily	O
conserved	O
Nt	B-ptm
-	I-ptm
acetylation	I-ptm
is	O
still	O
inconclusive	O
.	O

Nt	B-ptm
-	I-ptm
acetylation	I-ptm
is	O
an	O
abundant	O
and	O
evolutionarily	O
conserved	O
modification	O
occurring	O
in	O
bacteria	B-taxonomy_domain
,	O
archaea	B-taxonomy_domain
and	O
eukaryotes	B-taxonomy_domain
.	O

It	O
is	O
estimated	O
that	O
about	O
80	O
–	O
90	O
%	O
of	O
soluble	O
human	B-species
proteins	O
and	O
50	O
–	O
70	O
%	O
of	O
yeast	B-taxonomy_domain
proteins	O
are	O
subjected	O
to	O
Nt	B-ptm
-	I-ptm
acetylation	I-ptm
,	O
where	O
an	O
acetyl	B-chemical
moiety	O
is	O
transferred	O
from	O
Ac	B-chemical
-	I-chemical
CoA	I-chemical
to	O
the	O
α	O
-	O
amino	O
group	O
of	O
the	O
first	O
residue	O
.	O

Recently	O
Nt	O
-	O
acetylome	O
expands	O
the	O
Nt	B-ptm
-	I-ptm
acetylation	I-ptm
to	O
transmembrane	O
proteins	O
.	O

Unlike	O
Nε	B-ptm
-	I-ptm
acetylation	I-ptm
that	O
can	O
be	O
eliminated	O
by	O
deacetylases	B-protein_type
,	O
Nt	B-ptm
-	I-ptm
acetylation	I-ptm
is	O
considered	O
irreversible	B-protein_state
since	O
no	O
corresponding	O
deacetylase	B-protein_type
is	O
found	O
to	O
date	O
.	O

Although	O
Nt	B-ptm
-	I-ptm
acetylation	I-ptm
has	O
been	O
regarded	O
as	O
a	O
co	O
-	O
translational	O
modification	O
traditionally	O
,	O
there	O
is	O
evidence	O
that	O
post	O
-	O
translational	O
Nt	B-ptm
-	I-ptm
acetylation	I-ptm
exists	O
.	O

During	O
the	O
past	O
decades	O
,	O
a	O
large	O
number	O
of	O
Nt	O
-	O
acetylome	O
researches	O
have	O
shed	O
light	O
on	O
the	O
functional	O
roles	O
of	O
Nt	B-ptm
-	I-ptm
acetylation	I-ptm
,	O
including	O
protein	O
degradation	O
,	O
subcellular	O
localization	O
,	O
protein	O
-	O
protein	O
interaction	O
,	O
protein	O
-	O
membrane	O
interaction	O
,	O
plant	B-taxonomy_domain
development	O
,	O
stress	O
-	O
response	O
and	O
protein	O
stability	O
.	O

The	O
Nt	B-ptm
-	I-ptm
acetylation	I-ptm
is	O
carried	O
out	O
by	O
N	B-protein_type
-	I-protein_type
terminal	I-protein_type
acetyltransferases	I-protein_type
(	O
NATs	B-protein_type
)	O
that	O
belong	O
to	O
the	O
GNAT	B-protein_type
superfamily	I-protein_type
.	O

To	O
date	O
,	O
six	O
NATs	B-protein_type
(	O
NatA	B-complex_assembly
/	O
B	B-complex_assembly
/	O
C	B-complex_assembly
/	O
D	B-complex_assembly
/	O
E	B-complex_assembly
/	O
F	B-complex_assembly
)	O
have	O
been	O
identified	O
in	O
eukaryotes	B-taxonomy_domain
.	O

About	O
40	O
percent	O
of	O
Nt	B-ptm
-	I-ptm
acetylation	I-ptm
of	O
soluble	O
proteins	O
in	O
cells	O
is	O
catalyzed	O
by	O
NatA	B-complex_assembly
complex	O
which	O
is	O
composed	O
of	O
the	O
catalytic	O
subunit	O
Naa10p	B-protein
and	O
the	O
auxiliary	O
subunit	O
Naa15p	B-protein
.	O

NatE	B-complex_assembly
was	O
found	O
to	O
physically	O
interact	O
with	O
the	O
NatA	B-complex_assembly
complex	O
without	O
any	O
observation	O
of	O
impact	O
on	O
NatA	B-complex_assembly
-	O
activity	O
.	O

Two	O
other	O
multimeric	O
complexes	O
of	O
NATs	B-protein_type
are	O
NatB	B-complex_assembly
and	O
NatC	B-complex_assembly
which	O
contain	O
the	O
catalytic	O
subunits	O
Naa20	B-protein
and	O
Naa30	B-protein
and	O
the	O
auxiliary	O
subunits	O
Naa25	B-protein
and	O
Naa35	B-protein
/	O
Naa38	B-protein
,	O
respectively	O
.	O

Furthermore	O
,	O
only	O
the	O
catalytic	O
subunits	O
Naa40	B-protein
and	O
Naa60	B-protein
were	O
found	O
for	O
NatD	B-complex_assembly
and	O
NatF	B-complex_assembly
,	O
respectively	O
.	O

Besides	O
Nt	B-ptm
-	I-ptm
acetylation	I-ptm
,	O
accumulating	O
reports	O
have	O
proposed	O
Nε	B-ptm
-	I-ptm
acetylation	I-ptm
carried	O
out	O
by	O
NATs	B-protein_type
.	O

There	O
is	O
an	O
evolutionary	O
increasing	O
in	O
the	O
degree	O
of	O
Nt	B-ptm
-	I-ptm
acetylation	I-ptm
between	O
yeast	B-taxonomy_domain
and	O
human	B-species
which	O
could	O
partly	O
be	O
explained	O
by	O
the	O
contribution	O
of	O
NatF	B-complex_assembly
.	O
As	O
the	O
first	O
N	B-protein_type
-	I-protein_type
terminal	I-protein_type
acetyltransferase	I-protein_type
discovered	O
on	O
an	O
organelle	O
,	O
NatF	B-complex_assembly
,	O
encoded	O
by	O
NAA60	B-protein
and	O
also	O
named	O
as	O
Histone	B-protein
acetyltransferase	I-protein
type	I-protein
B	I-protein
protein	I-protein
4	I-protein
(	O
HAT4	B-protein
),	O
Naa60	B-protein
or	O
N	B-protein
-	I-protein
acetyltransferase	I-protein
15	I-protein
(	O
NAT15	B-protein
),	O
is	O
the	O
youngest	O
member	O
of	O
the	O
NAT	B-protein_type
family	O
.	O

Unlike	O
other	O
NATs	B-protein_type
that	O
are	O
highly	B-protein_state
conserved	I-protein_state
among	O
lower	B-taxonomy_domain
and	O
higher	B-taxonomy_domain
eukaryotes	I-taxonomy_domain
,	O
NatF	B-complex_assembly
only	O
exists	O
in	O
higher	B-taxonomy_domain
eukaryotes	I-taxonomy_domain
.	O

Subsequent	O
researches	O
indicated	O
that	O
NatF	B-complex_assembly
displays	O
its	O
catalytic	O
ability	O
with	O
both	O
Nt	B-ptm
-	I-ptm
acetylation	I-ptm
and	O
lysine	B-ptm
Nε	I-ptm
-	I-ptm
acetylation	I-ptm
.	O

As	O
an	O
N	B-protein_type
-	I-protein_type
terminal	I-protein_type
acetyltransferase	I-protein_type
,	O
NatF	B-complex_assembly
can	O
specifically	O
catalyze	O
acetylation	B-ptm
of	O
the	O
N	O
-	O
terminal	O
α	O
-	O
amine	O
of	O
most	O
transmembrane	O
proteins	O
and	O
has	O
substrate	O
preference	O
towards	O
proteins	O
with	O
Met	B-structure_element
-	I-structure_element
Lys	I-structure_element
-,	I-structure_element
Met	B-structure_element
-	I-structure_element
Val	I-structure_element
-,	I-structure_element
Met	B-structure_element
-	I-structure_element
Ala	I-structure_element
-	I-structure_element
and	O
Met	B-structure_element
-	I-structure_element
Met	I-structure_element
-	I-structure_element
N	O
-	O
termini	O
,	O
thus	O
partially	O
overlapping	O
substrate	O
selectivity	O
with	O
NatC	B-complex_assembly
and	O
NatE	B-complex_assembly
.	O
On	O
the	O
other	O
hand	O
,	O
NatF	B-complex_assembly
,	O
with	O
its	O
lysine	B-protein_type
acetyltransferase	I-protein_type
activity	O
,	O
mediates	O
the	O
lysine	B-ptm
acetylation	I-ptm
of	O
free	O
histone	B-protein_type
H4	B-protein_type
,	O
including	O
H4K20	B-protein_type
,	O
H4K79	B-protein_type
and	O
H4K91	B-protein_type
.	O

Another	O
important	O
feature	O
of	O
NatF	B-complex_assembly
is	O
that	O
this	O
protein	O
is	O
anchored	O
on	O
the	O
Golgi	O
apparatus	O
through	O
its	O
C	O
-	O
terminal	O
membrane	B-structure_element
-	I-structure_element
integrating	I-structure_element
region	I-structure_element
and	O
takes	O
part	O
in	O
the	O
maintaining	O
of	O
Golgi	O
integrity	O
.	O

With	O
its	O
unique	O
intracellular	O
organellar	O
localization	O
and	O
substrate	O
selectivity	O
,	O
NatF	B-complex_assembly
appears	O
to	O
provide	O
more	O
evolutionary	O
information	O
among	O
the	O
NAT	B-protein_type
family	O
members	O
.	O

It	O
was	O
recently	O
found	O
that	O
NatF	B-complex_assembly
facilitates	O
nucleosomes	B-complex_assembly
assembly	O
and	O
that	O
NAA60	B-protein
knockdown	O
in	O
MCF7	O
-	O
cell	O
inhibits	O
cell	O
proliferation	O
,	O
sensitizes	O
cells	O
to	O
DNA	O
damage	O
and	O
induces	O
cell	O
apoptosis	O
.	O

In	O
Drosophila	B-taxonomy_domain
cells	O
,	O
NAA60	B-protein
knockdown	O
induces	O
chromosomal	O
segregation	O
defects	O
during	O
anaphase	O
including	O
lagging	O
chromosomes	O
and	O
chromosomal	O
bridges	O
.	O

Much	O
recent	O
attention	O
has	O
also	O
been	O
focused	O
on	O
the	O
requirement	O
of	O
NatF	B-complex_assembly
for	O
regulation	O
of	O
organellar	O
structure	O
.	O

In	O
HeLa	O
cells	O
,	O
NAA60	B-protein
knockdown	O
causes	O
Golgi	O
apparatus	O
fragmentation	O
which	O
can	O
be	O
rescued	O
by	O
overexpression	B-experimental_method
Naa60	B-protein
.	O

The	O
systematic	O
investigation	O
of	O
publicly	O
available	O
microarray	O
data	O
showed	O
that	O
NATs	B-protein_type
share	O
distinct	O
tissue	O
-	O
specific	O
expression	O
patterns	O
in	O
Drosophila	B-taxonomy_domain
and	O
NatF	B-complex_assembly
shows	O
a	O
higher	O
expression	O
level	O
in	O
central	O
nervous	O
system	O
of	O
Drosophila	B-taxonomy_domain
.	O

In	O
this	O
study	O
,	O
we	O
solved	B-experimental_method
the	O
structures	B-evidence
of	O
human	B-species
Naa60	B-protein
(	O
NatF	B-complex_assembly
)	O
in	B-protein_state
complex	I-protein_state
with	I-protein_state
coenzyme	B-chemical
.	O

The	O
hNaa60	B-protein
protein	O
contains	O
a	O
unique	O
amphipathic	B-structure_element
α	I-structure_element
-	I-structure_element
helix	I-structure_element
(	O
α5	B-structure_element
)	O
following	O
its	O
GNAT	B-structure_element
domain	I-structure_element
that	O
might	O
account	O
for	O
the	O
Golgi	O
localization	O
of	O
this	O
protein	O
.	O

Crystal	B-evidence
structures	I-evidence
showed	O
that	O
the	O
β7	B-structure_element
-	I-structure_element
β8	I-structure_element
hairpin	I-structure_element
rotated	O
about	O
50	O
degrees	O
upon	O
removing	O
the	O
C	B-structure_element
-	I-structure_element
terminal	I-structure_element
region	I-structure_element
of	O
the	O
protein	O
and	O
this	O
movement	O
substantially	O
changed	O
the	O
geometry	O
of	O
the	O
substrate	B-site
-	I-site
binding	I-site
pocket	I-site
.	O

Remarkably	O
,	O
we	O
find	O
that	O
Phe	B-residue_name_number
34	I-residue_name_number
may	O
participate	O
in	O
the	O
proper	O
positioning	O
of	O
the	O
coenzyme	B-chemical
for	O
the	O
transfer	O
reaction	O
to	O
occur	O
.	O

Further	O
structure	B-experimental_method
comparison	I-experimental_method
and	O
biochemical	B-experimental_method
studies	I-experimental_method
also	O
identified	O
other	O
key	O
structural	O
elements	O
essential	O
for	O
the	O
enzyme	O
activity	O
of	O
Naa60	B-protein
.	O

Overall	O
structure	B-evidence
of	O
hNaa60	B-protein

In	O
the	O
effort	O
to	O
prepare	O
the	O
protein	O
for	O
structural	O
studies	O
,	O
we	O
tried	O
a	O
large	O
number	O
of	O
hNaa60	B-protein
constructs	O
but	O
all	O
failed	O
due	O
to	O
heavy	O
precipitation	O
or	O
aggregation	O
.	O

Sequence	B-experimental_method
alignment	I-experimental_method
of	O
Naa60	B-protein
from	O
different	O
species	O
revealed	O
a	O
Glu	B-structure_element
-	I-structure_element
Glu	I-structure_element
-	I-structure_element
Arg	I-structure_element
(	O
EER	B-structure_element
)	O
versus	O
Val	B-structure_element
-	I-structure_element
Val	I-structure_element
-	I-structure_element
Pro	I-structure_element
(	O
VVP	B-structure_element
)	O
sequence	O
difference	O
near	O
the	O
N	O
-	O
terminus	O
of	O
the	O
protein	O
in	O
Xenopus	B-species
Laevis	I-species
versus	O
Homo	B-species
sapiens	I-species
(	O
Fig	O
.	O
1A	O
).	O

Considering	O
that	O
terminal	O
residues	O
may	O
lack	O
higher	O
-	O
order	O
structure	O
and	O
hydrophobic	O
residues	O
in	O
this	O
region	O
may	O
expose	O
to	O
solvent	O
and	O
hence	O
cause	O
protein	O
aggregation	O
,	O
we	O
mutated	B-experimental_method
residues	O
4	B-residue_range
–	I-residue_range
6	I-residue_range
from	O
VVP	B-mutant
to	I-mutant
EER	I-mutant
for	O
the	O
purpose	O
of	O
improving	O
solubility	O
of	O
this	O
protein	O
.	O

According	O
to	O
previous	O
studies	O
,	O
this	O
N	O
-	O
terminal	O
region	O
should	O
not	O
interfere	O
with	O
hNaa60	B-protein
’	O
s	O
Golgi	O
localization	O
.	O

We	O
tried	O
many	O
hNaa60	B-protein
constructs	O
with	O
the	O
three	O
-	O
residues	O
mutation	B-experimental_method
but	O
only	O
the	O
truncated	B-protein_state
variant	O
1	B-residue_range
-	I-residue_range
199	I-residue_range
and	O
the	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
protein	O
behaved	O
well	O
.	O

We	O
obtained	O
the	O
crystal	B-evidence
of	O
the	O
truncated	B-protein_state
variant	O
1	B-residue_range
-	I-residue_range
199	I-residue_range
in	B-protein_state
complex	I-protein_state
with	I-protein_state
CoA	B-chemical
first	O
,	O
and	O
after	O
extensive	O
trials	O
we	O
got	O
the	O
crystal	B-evidence
of	O
the	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
protein	O
(	O
spanning	O
residues	O
1	B-residue_range
-	I-residue_range
242	I-residue_range
)	O
in	B-protein_state
complex	I-protein_state
with	I-protein_state
Ac	B-chemical
-	I-chemical
CoA	I-chemical
(	O
Fig	O
.	O
1B	O
,	O
C	O
).	O

Hereafter	O
,	O
all	O
deletions	O
or	O
point	O
mutants	B-protein_state
of	O
hNaa60	B-protein
we	O
describe	O
here	O
are	O
with	O
the	O
EER	B-structure_element
mutation	B-experimental_method
.	O

The	O
crystal	B-evidence
structures	I-evidence
of	O
hNaa60	B-complex_assembly
(	I-complex_assembly
1	I-complex_assembly
-	I-complex_assembly
242	I-complex_assembly
)/	I-complex_assembly
Ac	I-complex_assembly
-	I-complex_assembly
CoA	I-complex_assembly
and	O
hNaa60	B-complex_assembly
(	I-complex_assembly
1	I-complex_assembly
-	I-complex_assembly
199	I-complex_assembly
)/	I-complex_assembly
CoA	I-complex_assembly
were	O
determined	O
by	O
molecular	B-experimental_method
replacement	I-experimental_method
and	O
refined	O
to	O
1	O
.	O
38	O
Å	O
and	O
1	O
.	O
60	O
Å	O
resolution	O
,	O
respectively	O
(	O
Table	O
1	O
).	O

The	O
electron	B-evidence
density	I-evidence
maps	I-evidence
were	O
of	O
sufficient	O
quality	O
to	O
trace	O
residues	O
1	B-residue_range
-	I-residue_range
211	I-residue_range
of	O
hNaa60	B-protein
(	O
1	B-residue_range
-	I-residue_range
242	I-residue_range
)	O
and	O
residues	O
5	B-residue_range
-	I-residue_range
199	I-residue_range
of	O
hNaa60	B-mutant
(	I-mutant
1	I-mutant
-	I-mutant
199	I-mutant
).	I-mutant

The	O
structure	B-evidence
of	O
hNaa60	B-protein
protein	O
contains	O
a	O
central	B-structure_element
domain	I-structure_element
exhibiting	O
a	O
classic	O
GCN5	B-protein_type
-	I-protein_type
related	I-protein_type
N	I-protein_type
-	I-protein_type
acetyltransferase	I-protein_type
(	O
GNAT	B-protein_type
)	O
folding	O
,	O
along	O
with	O
the	O
extended	B-protein_state
N	B-structure_element
-	I-structure_element
and	I-structure_element
C	I-structure_element
-	I-structure_element
terminal	I-structure_element
regions	I-structure_element
(	O
Fig	O
.	O
1B	O
,	O
C	O
).	O

The	O
central	B-structure_element
domain	I-structure_element
includes	O
nine	O
β	B-structure_element
strands	I-structure_element
(	O
β1	B-structure_element
-	I-structure_element
β9	I-structure_element
)	O
and	O
four	O
α	B-structure_element
-	I-structure_element
helixes	I-structure_element
(	O
α1	B-structure_element
-	I-structure_element
α4	I-structure_element
)	O
and	O
is	O
highly	B-protein_state
similar	I-protein_state
to	O
the	O
known	O
hNaa50p	B-protein
and	O
other	O
reported	O
NATs	B-protein_type
(	O
Fig	O
.	O
1D	O
).	O

However	O
,	O
in	O
hNaa60	B-protein
,	O
there	O
is	O
an	O
extra	B-structure_element
20	I-structure_element
-	I-structure_element
residue	I-structure_element
loop	I-structure_element
between	O
β3	B-structure_element
and	O
β4	B-structure_element
that	O
forms	O
a	O
small	B-structure_element
subdomain	I-structure_element
with	O
well	O
-	O
defined	O
3D	O
structure	O
(	O
Fig	O
.	O
1B	O
–	O
D	O
).	O

Furthermore	O
,	O
the	O
β7	B-structure_element
-	I-structure_element
β8	I-structure_element
strands	I-structure_element
form	O
an	O
approximately	B-structure_element
antiparallel	I-structure_element
β	I-structure_element
-	I-structure_element
hairpin	I-structure_element
structure	I-structure_element
remarkably	O
different	O
from	O
that	O
in	O
hNaa50p	B-protein
(	O
Fig	O
.	O
1D	O
).	O

The	O
N	B-structure_element
-	I-structure_element
and	I-structure_element
C	I-structure_element
-	I-structure_element
terminal	I-structure_element
regions	I-structure_element
form	O
helical	B-structure_element
structures	I-structure_element
(	O
α0	B-structure_element
and	O
α5	B-structure_element
)	O
stretching	O
out	O
from	O
the	O
central	O
GCN5	B-structure_element
-	I-structure_element
domain	I-structure_element
(	O
Fig	O
.	O
1C	O
).	O

Interestingly	O
,	O
we	O
found	O
that	O
the	O
catalytic	O
activity	O
of	O
hNaa60	B-protein
(	O
1	B-residue_range
-	I-residue_range
242	I-residue_range
)	O
is	O
much	O
lower	O
than	O
that	O
of	O
hNaa60	B-mutant
(	I-mutant
1	I-mutant
-	I-mutant
199	I-mutant
)	I-mutant
(	O
Figure	O
S1	O
),	O
indicating	O
that	O
residues	O
200	B-residue_range
–	I-residue_range
242	I-residue_range
may	O
have	O
some	O
auto	O
-	O
inhibitory	O
effect	O
on	O
the	O
activity	O
of	O
the	O
enzyme	O
.	O

However	O
,	O
since	O
this	O
region	O
was	O
not	O
visible	O
in	O
the	O
hNaa60	B-protein
(	O
1	B-residue_range
-	I-residue_range
242	I-residue_range
)	O
crystal	B-evidence
structure	I-evidence
,	O
we	O
do	O
not	O
yet	O
understand	O
how	O
this	O
happens	O
.	O

Another	O
possibility	O
is	O
that	O
since	O
hNaa60	B-protein
is	O
localized	O
on	O
Golgi	O
apparatus	O
,	O
the	O
observed	O
low	O
activity	O
of	O
the	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
hNaa60	B-protein
might	O
be	O
related	O
to	O
lack	O
of	O
Golgi	O
localization	O
of	O
the	O
enzyme	O
in	O
our	O
in	O
vitro	O
studies	O
.	O

For	O
the	O
convenience	O
of	O
studying	O
the	O
kinetics	O
of	O
mutants	B-protein_state
,	O
the	O
mutagenesis	B-experimental_method
studies	I-experimental_method
described	O
hereafter	O
were	O
all	O
based	O
on	O
hNaa60	B-mutant
(	I-mutant
1	I-mutant
-	I-mutant
199	I-mutant
).	I-mutant

An	O
amphipathic	B-structure_element
α	I-structure_element
-	I-structure_element
helix	I-structure_element
in	O
the	O
C	B-structure_element
-	I-structure_element
terminal	I-structure_element
region	I-structure_element
may	O
contribute	O
to	O
Golgi	O
localization	O
of	O
hNaa60	B-protein

There	O
is	O
one	O
hNaa60	B-protein
molecule	O
in	O
the	O
asymmetric	O
unit	O
in	O
the	O
hNaa60	B-complex_assembly
(	I-complex_assembly
1	I-complex_assembly
-	I-complex_assembly
242	I-complex_assembly
)/	I-complex_assembly
Ac	I-complex_assembly
-	I-complex_assembly
CoA	I-complex_assembly
structure	B-evidence
.	O

The	O
C	B-structure_element
-	I-structure_element
terminal	I-structure_element
region	I-structure_element
extended	O
from	O
the	O
GCN5	B-structure_element
-	I-structure_element
domain	I-structure_element
forms	O
an	O
amphipathic	B-structure_element
helix	I-structure_element
(	O
α5	B-structure_element
)	O
and	O
interacts	O
with	O
a	O
molecule	O
in	O
a	O
neighbor	O
asymmetric	O
unit	O
through	O
hydrophobic	B-bond_interaction
interactions	I-bond_interaction
between	O
α5	B-structure_element
-	I-structure_element
helix	I-structure_element
and	O
a	O
hydrophobic	B-site
groove	I-site
between	O
the	O
N	O
-	O
terminal	O
β1	B-structure_element
and	O
β3	B-structure_element
strands	I-structure_element
of	O
the	O
neighbor	O
molecule	O
(	O
Fig	O
.	O
2A	O
).	O

The	O
C	B-structure_element
-	I-structure_element
terminal	I-structure_element
extension	I-structure_element
following	O
α5	B-structure_element
-	I-structure_element
helix	I-structure_element
forms	O
a	O
β	B-structure_element
-	I-structure_element
turn	I-structure_element
that	O
wraps	O
around	O
and	O
interacts	O
with	O
the	O
neighbor	O
protein	O
molecule	O
through	O
hydrophobic	B-bond_interaction
interactions	I-bond_interaction
,	O
too	O
.	O

In	O
the	O
hNaa60	B-complex_assembly
(	I-complex_assembly
1	I-complex_assembly
-	I-complex_assembly
199	I-complex_assembly
)/	I-complex_assembly
CoA	I-complex_assembly
structure	B-evidence
,	O
a	O
part	O
of	O
the	O
α5	B-structure_element
-	I-structure_element
helix	I-structure_element
is	O
deleted	O
due	O
to	O
truncation	O
of	O
the	O
C	B-structure_element
-	I-structure_element
terminal	I-structure_element
region	I-structure_element
(	O
Fig	O
.	O
1B	O
).	O

Interestingly	O
,	O
the	O
remaining	O
residues	O
in	O
α5	B-structure_element
-	I-structure_element
helix	I-structure_element
still	O
form	O
an	O
amphipathic	B-structure_element
helix	I-structure_element
although	O
the	O
hydrophobic	B-bond_interaction
interaction	I-bond_interaction
with	O
the	O
N	O
-	O
terminal	O
hydrophobic	B-site
groove	I-site
of	O
a	O
neighbor	O
molecule	O
is	O
abolished	O
and	O
the	O
helix	B-structure_element
is	O
largely	O
exposed	O
in	O
solvent	O
due	O
to	O
different	O
crystal	B-evidence
packing	I-evidence
(	O
Fig	O
.	O
2B	O
).	O

A	O
recent	O
research	O
showed	O
that	O
residues	O
182	B-residue_range
–	I-residue_range
216	I-residue_range
are	O
important	O
for	O
the	O
localization	O
of	O
hNaa60	B-protein
on	O
Golgi	O
.	O
According	O
to	O
our	O
structure	B-evidence
,	O
the	O
solvent	B-protein_state
-	I-protein_state
exposed	I-protein_state
amphipathic	B-structure_element
helix	I-structure_element
(	O
α5	B-structure_element
)	O
formed	O
by	O
residues	O
190	B-residue_range
-	I-residue_range
202	I-residue_range
with	O
an	O
array	O
of	O
hydrophobic	O
residues	O
located	O
on	O
one	O
side	O
(	O
Ile	B-residue_name_number
190	I-residue_name_number
,	O
Leu	B-residue_name_number
191	I-residue_name_number
,	O
Ile	B-residue_name_number
194	I-residue_name_number
,	O
Leu	B-residue_name_number
197	I-residue_name_number
and	O
Leu	B-residue_name_number
201	I-residue_name_number
)	O
and	O
hydrophilic	O
residues	O
on	O
the	O
other	O
side	O
(	O
Fig	O
.	O
S2	O
)	O
might	O
account	O
for	O
interaction	O
between	O
hNaa60	B-protein
and	O
Golgi	O
membrane	O
,	O
as	O
it	O
is	O
a	O
typical	O
structure	O
accounting	O
for	O
membrane	O
association	O
through	O
immersing	O
into	O
the	O
lipid	O
bi	O
-	O
layer	O
with	O
its	O
hydrophobic	O
side	O
as	O
was	O
observed	O
with	O
KalSec14	B-protein
,	O
Atg3	B-protein
,	O
PB1	B-protein
-	I-protein
F2	I-protein
etc	O
.	O

The	O
β7	B-structure_element
-	I-structure_element
β8	I-structure_element
hairpin	I-structure_element
showed	O
alternative	O
conformations	O
in	O
the	O
hNaa60	B-protein
crystal	B-evidence
structures	I-evidence

Superposition	B-experimental_method
of	O
hNaa60	B-complex_assembly
(	I-complex_assembly
1	I-complex_assembly
-	I-complex_assembly
242	I-complex_assembly
)/	I-complex_assembly
Ac	I-complex_assembly
-	I-complex_assembly
CoA	I-complex_assembly
,	O
hNaa60	B-complex_assembly
(	I-complex_assembly
1	I-complex_assembly
-	I-complex_assembly
199	I-complex_assembly
)/	I-complex_assembly
CoA	I-complex_assembly
and	O
hNaa50	B-complex_assembly
/	I-complex_assembly
CoA	I-complex_assembly
/	I-complex_assembly
peptide	I-complex_assembly
(	O
PDB	O
3TFY	O
)	O
revealed	O
considerable	O
difference	O
in	O
the	O
β7	B-structure_element
-	I-structure_element
β8	I-structure_element
hairpin	I-structure_element
region	O
despite	O
the	O
overall	O
stability	O
and	O
similarity	O
of	O
the	O
GNAT	B-structure_element
domain	I-structure_element
(	O
Fig	O
.	O
1D	O
).	O

In	O
hNaa60	B-protein
(	O
1	B-residue_range
-	I-residue_range
242	I-residue_range
),	O
the	O
β7	B-structure_element
-	I-structure_element
β8	I-structure_element
hairpin	I-structure_element
is	O
located	O
in	O
close	O
proximity	O
to	O
the	O
α1	B-structure_element
-	I-structure_element
α2	I-structure_element
loop	I-structure_element
,	O
creating	O
a	O
more	O
compact	O
substrate	B-site
binding	I-site
site	I-site
than	O
that	O
in	O
hNaa50	B-protein
,	O
where	O
this	O
region	O
adopts	O
a	O
more	O
flexible	B-protein_state
loop	B-structure_element
conformation	O
(	O
β6	B-structure_element
-	I-structure_element
β7	I-structure_element
loop	I-structure_element
).	O

Upon	O
removing	B-experimental_method
the	O
C	B-structure_element
-	I-structure_element
terminal	I-structure_element
region	I-structure_element
of	O
hNaa60	B-protein
,	O
we	O
observed	O
that	O
hNaa60	B-mutant
(	I-mutant
1	I-mutant
-	I-mutant
199	I-mutant
)	I-mutant
molecules	O
pack	O
in	O
a	O
different	O
way	O
involving	O
the	O
β7	B-structure_element
-	I-structure_element
β8	I-structure_element
hairpin	I-structure_element
in	O
the	O
crystal	B-evidence
,	O
leading	O
to	O
about	O
50	O
degree	O
rotation	O
of	O
the	O
hairpin	B-structure_element
which	O
moves	O
away	O
from	O
the	O
α1	B-structure_element
-	I-structure_element
α2	I-structure_element
loop	I-structure_element
(	O
Figs	O
1D	O
and	O
2C	O
).	O

This	O
conformational	O
change	O
substantially	O
altered	O
the	O
geometry	O
of	O
the	O
substrate	B-site
binding	I-site
site	I-site
,	O
which	O
could	O
potentially	O
change	O
the	O
way	O
in	O
which	O
the	O
substrate	O
accesses	O
the	O
active	B-site
site	I-site
of	O
the	O
enzyme	O
.	O

In	O
hNaa60	B-protein
(	O
1	B-residue_range
-	I-residue_range
242	I-residue_range
),	O
the	O
β7	B-structure_element
-	I-structure_element
β8	I-structure_element
hairpin	I-structure_element
covers	O
the	O
active	B-site
site	I-site
in	O
a	O
way	O
similar	O
to	O
that	O
observed	O
in	O
hNaa50	B-protein
,	O
presumably	O
leaving	O
only	O
one	O
way	O
for	O
the	O
substrate	O
to	O
access	O
the	O
active	B-site
site	I-site
,	O
i	O
.	O
e	O
.	O
to	O
enter	O
from	O
the	O
opposite	O
end	O
into	O
the	O
same	O
tunnel	B-site
where	O
Ac	B-chemical
-	I-chemical
CoA	I-chemical
/	O
CoA	B-chemical
binds	O
(	O
Fig	O
.	O
2D	O
),	O
which	O
may	O
accommodate	O
access	O
of	O
a	O
NAT	B-protein_type
substrate	O
only	O
.	O

KAT	B-protein_type
activity	O
of	O
hNaa60	B-protein
toward	O
histone	B-protein_type
H4	B-protein_type
has	O
been	O
noted	O
in	O
previous	O
study	O
,	O
and	O
our	O
enzyme	B-evidence
kinetic	I-evidence
data	I-evidence
also	O
indicated	O
that	O
hNaa60	B-protein
can	O
acetylate	O
H3	B-complex_assembly
-	I-complex_assembly
H4	I-complex_assembly
tetramer	B-oligomeric_state
in	O
vitro	O
(	O
Figure	O
S3	O
).	O

Furthermore	O
,	O
we	O
analyzed	O
the	O
acetylation	B-ptm
status	O
of	O
histone	B-protein_type
H3	B-complex_assembly
-	I-complex_assembly
H4	I-complex_assembly
tetramer	B-oligomeric_state
using	O
mass	B-experimental_method
spectrometry	I-experimental_method
and	O
observed	O
that	O
multiple	O
lysine	B-residue_name
residues	O
in	O
the	O
protein	O
showed	O
significantly	O
increased	O
acetylation	B-ptm
level	O
and	O
changed	O
acetylation	B-ptm
profile	O
upon	O
treatment	O
with	O
hNaa60	B-mutant
(	I-mutant
1	I-mutant
-	I-mutant
199	I-mutant
)	I-mutant
(	O
Figure	O
S4	O
).	O

We	O
also	O
conducted	O
liquid	B-experimental_method
chromatography	I-experimental_method
-	I-experimental_method
tandem	I-experimental_method
mass	I-experimental_method
spectrometry	I-experimental_method
(	O
LC	B-experimental_method
/	I-experimental_method
MS	I-experimental_method
/	I-experimental_method
MS	I-experimental_method
)	O
analysis	O
on	O
a	O
synthetic	O
peptide	B-chemical
(	O
NH2	B-chemical
-	I-chemical
MKGKEEKEGGAR	I-chemical
-	I-chemical
COOH	I-chemical
)	O
after	O
treatment	O
with	O
hNaa60	B-mutant
(	I-mutant
1	I-mutant
-	I-mutant
199	I-mutant
),	I-mutant
and	O
the	O
data	O
confirmed	O
that	O
both	O
the	O
N	O
-	O
terminal	O
α	O
-	O
amine	O
and	O
lysine	B-residue_name
side	O
-	O
chain	O
ε	O
-	O
amine	O
were	O
robustly	O
acetylated	B-protein_state
after	O
the	O
treatment	O
(	O
Table	O
S1	O
).	O

Recent	O
structural	B-experimental_method
investigation	I-experimental_method
of	O
other	O
NATs	B-protein_type
proposed	O
that	O
the	O
β6	B-structure_element
-	I-structure_element
β7	I-structure_element
loop	I-structure_element
,	O
corresponding	O
to	O
the	O
β7	B-structure_element
-	I-structure_element
β8	I-structure_element
hairpin	I-structure_element
in	O
hNaa60	B-protein
,	O
and	O
the	O
α1	B-structure_element
-	I-structure_element
α2	I-structure_element
loop	I-structure_element
flanking	O
the	O
substrate	B-site
-	I-site
binding	I-site
site	I-site
of	O
NATs	B-protein_type
,	O
prevent	O
the	O
lysine	B-residue_name
side	O
-	O
chain	O
of	O
the	O
KAT	B-protein_type
substrates	O
from	O
inserting	O
into	O
the	O
active	B-site
site	I-site
.	O

Indeed	O
,	O
superposition	B-experimental_method
of	O
hNaa60	B-protein
(	O
1	B-residue_range
-	I-residue_range
242	I-residue_range
)	O
structure	B-evidence
on	O
that	O
of	O
Hat1p	B-protein
,	O
a	O
typical	O
KAT	B-protein_type
,	O
in	B-protein_state
complex	I-protein_state
with	I-protein_state
a	O
histone	B-protein_type
H4	B-protein_type
peptide	B-chemical
revealed	O
obvious	O
overlapping	O
/	O
clashing	O
of	O
the	O
H4	B-protein_type
peptide	B-chemical
(	O
a	O
KAT	B-protein_type
substrate	O
)	O
with	O
the	O
β7	B-structure_element
-	I-structure_element
β8	I-structure_element
hairpin	I-structure_element
of	O
hNaa60	B-protein
(	O
1	B-residue_range
-	I-residue_range
242	I-residue_range
)	O
(	O
Fig	O
.	O
2D	O
).	O

Interestingly	O
,	O
in	O
the	O
hNaa60	B-mutant
(	I-mutant
1	I-mutant
-	I-mutant
199	I-mutant
)	I-mutant
crystal	B-evidence
structure	I-evidence
,	O
the	O
displaced	O
β7	B-structure_element
-	I-structure_element
β8	I-structure_element
hairpin	I-structure_element
opened	O
a	O
second	O
way	O
for	O
the	O
substrate	O
to	O
access	O
the	O
active	B-site
center	I-site
that	O
would	O
readily	O
accommodate	O
the	O
binding	O
of	O
the	O
H4	B-protein_type
peptide	B-chemical
(	O
Fig	O
.	O
2E	O
),	O
thus	O
implied	O
a	O
potential	O
explanation	O
for	O
KAT	B-protein_type
activity	O
of	O
this	O
enzyme	O
from	O
a	O
structural	O
biological	O
view	O
.	O

However	O
,	O
since	O
hNaa60	B-protein
(	O
1	B-residue_range
-	I-residue_range
242	I-residue_range
)	O
and	O
hNaa60	B-protein
(	O
1	O
-	O
199	O
)	O
were	O
crystallized	B-experimental_method
in	O
different	O
crystal	B-evidence
forms	I-evidence
,	O
the	O
observed	O
conformational	O
change	O
of	O
the	O
β7	B-structure_element
-	I-structure_element
β8	I-structure_element
hairpin	I-structure_element
may	O
simply	O
be	O
an	O
artifact	O
related	O
to	O
the	O
different	O
crystal	B-evidence
packing	I-evidence
.	O

Whether	O
the	O
KAT	B-protein_type
substrates	O
bind	O
to	O
the	O
β7	B-structure_element
-	I-structure_element
β8	I-structure_element
hairpin	I-structure_element
displaced	O
conformation	O
of	O
the	O
enzyme	O
needs	O
to	O
be	O
verified	O
by	O
further	O
structural	B-experimental_method
and	I-experimental_method
functional	I-experimental_method
studies	I-experimental_method
.	O

Phe	B-residue_name_number
34	I-residue_name_number
facilitates	O
proper	O
positioning	O
of	O
the	O
cofactor	O
for	O
acetyl	B-chemical
-	O
transfer	O

The	O
electron	B-evidence
density	I-evidence
of	O
Phe	B-residue_name_number
34	I-residue_name_number
side	O
-	O
chain	O
is	O
well	O
defined	O
in	O
the	O
hNaa60	B-complex_assembly
(	I-complex_assembly
1	I-complex_assembly
-	I-complex_assembly
242	I-complex_assembly
)/	I-complex_assembly
Ac	I-complex_assembly
-	I-complex_assembly
CoA	I-complex_assembly
structure	B-evidence
,	O
but	O
becomes	O
invisible	O
in	O
the	O
hNaa60	B-complex_assembly
(	I-complex_assembly
1	I-complex_assembly
-	I-complex_assembly
199	I-complex_assembly
)/	I-complex_assembly
CoA	I-complex_assembly
structure	B-evidence
,	O
indicating	O
displacement	O
of	O
the	O
Phe	B-residue_name_number
34	I-residue_name_number
side	O
-	O
chain	O
in	O
the	O
latter	O
(	O
Fig	O
.	O
3A	O
,	O
B	O
).	O

A	O
solvent	O
-	O
derived	O
malonate	B-chemical
molecule	O
is	O
found	O
beside	O
Phe	B-residue_name_number
34	I-residue_name_number
and	O
the	O
ethanethioate	B-chemical
moiety	O
of	O
Ac	B-chemical
-	I-chemical
CoA	I-chemical
in	O
the	O
high	O
-	O
resolution	O
hNaa60	B-complex_assembly
(	I-complex_assembly
1	I-complex_assembly
-	I-complex_assembly
242	I-complex_assembly
)/	I-complex_assembly
Ac	I-complex_assembly
-	I-complex_assembly
CoA	I-complex_assembly
structure	B-evidence
(	O
Fig	O
.	O
3A	O
).	O

Superposition	B-experimental_method
of	O
this	O
structure	B-evidence
on	O
that	O
of	O
hNaa50p	B-complex_assembly
/	I-complex_assembly
CoA	I-complex_assembly
/	I-complex_assembly
peptide	I-complex_assembly
shows	O
that	O
the	O
malonate	B-chemical
molecule	O
overlaps	O
well	O
on	O
the	O
N	O
-	O
terminal	O
methionine	B-residue_name
of	O
the	O
substrate	O
peptide	B-chemical
and	O
residue	O
Phe	B-residue_name_number
34	I-residue_name_number
in	O
hNaa60	B-protein
overlaps	O
well	O
on	O
Phe	B-residue_name_number
27	I-residue_name_number
in	O
hNaa50	B-protein
(	O
Fig	O
.	O
4A	O
).	O

Interestingly	O
,	O
in	O
the	O
structure	B-evidence
of	O
hNaa60	B-complex_assembly
(	I-complex_assembly
1	I-complex_assembly
-	I-complex_assembly
199	I-complex_assembly
)/	I-complex_assembly
CoA	I-complex_assembly
,	O
the	O
terminal	O
thiol	O
of	O
CoA	B-chemical
adopts	O
alternative	O
conformations	O
.	O

One	O
is	O
to	O
approach	O
the	O
substrate	O
amine	B-chemical
(	O
as	O
indicated	O
by	O
the	O
superimposed	B-experimental_method
hNaa50	B-complex_assembly
/	I-complex_assembly
CoA	I-complex_assembly
/	I-complex_assembly
peptide	I-complex_assembly
structure	B-evidence
),	O
similar	O
to	O
the	O
terminal	O
ethanethioate	B-chemical
of	O
Ac	B-chemical
-	I-chemical
CoA	I-chemical
in	O
the	O
structure	B-evidence
of	O
hNaa60	B-complex_assembly
(	I-complex_assembly
1	I-complex_assembly
-	I-complex_assembly
242	I-complex_assembly
)/	I-complex_assembly
Ac	I-complex_assembly
-	I-complex_assembly
CoA	I-complex_assembly
;	O
the	O
other	O
is	O
to	O
approach	O
the	O
α1	B-structure_element
-	I-structure_element
α2	I-structure_element
loop	I-structure_element
and	O
away	O
from	O
the	O
substrate	O
amine	O
(	O
Fig	O
.	O
3B	O
).	O

To	O
rule	O
out	O
the	O
possibility	O
that	O
the	O
electron	B-evidence
density	I-evidence
we	O
define	O
as	O
the	O
alternative	O
conformation	O
of	O
the	O
thiol	O
terminus	O
is	O
residual	O
electron	B-evidence
density	I-evidence
of	O
the	O
displaced	O
side	O
-	O
chain	O
of	O
Phe	B-residue_name_number
34	I-residue_name_number
,	O
we	O
solved	B-experimental_method
the	O
crystal	B-evidence
structure	I-evidence
of	O
hNaa60	B-complex_assembly
(	I-complex_assembly
1	I-complex_assembly
-	I-complex_assembly
199	I-complex_assembly
)	I-complex_assembly
F34A	I-complex_assembly
/	I-complex_assembly
CoA	I-complex_assembly
.	O
The	O
structure	B-evidence
of	O
this	O
mutant	B-protein_state
is	O
highly	O
similar	O
to	O
hNaa60	B-complex_assembly
(	I-complex_assembly
1	I-complex_assembly
-	I-complex_assembly
199	I-complex_assembly
)/	I-complex_assembly
CoA	I-complex_assembly
and	O
there	O
is	O
essentially	O
the	O
same	O
electron	B-evidence
density	I-evidence
corresponding	O
to	O
the	O
alternative	O
conformation	O
of	O
the	O
thiol	O
(	O
Fig	O
.	O
3C	O
).	O

Phe	B-residue_name_number
27	I-residue_name_number
in	O
hNaa50p	B-protein
(	O
equivalent	O
to	O
Phe	B-residue_name_number
34	I-residue_name_number
in	O
hNaa60	B-protein
)	O
has	O
been	O
implicated	O
to	O
facilitate	O
the	O
binding	O
of	O
N	O
-	O
terminal	O
methionine	B-residue_name
of	O
the	O
substrate	O
peptide	B-chemical
through	O
hydrophobic	B-bond_interaction
interaction	I-bond_interaction
.	O

However	O
,	O
in	O
the	O
hNaa60	B-complex_assembly
/	I-complex_assembly
Ac	I-complex_assembly
-	I-complex_assembly
CoA	I-complex_assembly
structure	B-evidence
,	O
a	O
hydrophilic	O
malonate	B-chemical
molecule	O
is	O
found	O
at	O
the	O
same	O
location	O
where	O
the	O
N	O
-	O
terminal	O
methionine	B-residue_name
should	O
bind	O
as	O
is	O
indicated	O
by	O
the	O
superposition	B-experimental_method
(	O
Fig	O
.	O
3A	O
),	O
suggesting	O
that	O
Phe	B-residue_name_number
34	I-residue_name_number
may	O
accommodate	O
binding	O
of	O
hydrophilic	O
substrate	O
,	O
too	O
.	O

Moreover	O
,	O
orientation	O
of	O
Phe	B-residue_name_number
34	I-residue_name_number
side	O
-	O
chain	O
seems	O
to	O
be	O
co	O
-	O
related	O
to	O
positioning	O
of	O
the	O
terminus	O
of	O
the	O
co	O
-	O
enzyme	O
and	O
important	O
for	O
placing	O
it	O
at	O
a	O
location	O
in	O
close	O
proximity	O
to	O
the	O
substrate	O
amine	O
.	O

We	O
hypothesize	O
that	O
if	O
Phe	B-residue_name_number
34	I-residue_name_number
only	O
works	O
to	O
facilitate	O
the	O
binding	O
of	O
the	O
hydrophobic	O
N	O
-	O
terminal	O
Met	B-residue_name
residue	O
,	O
to	O
mutate	B-experimental_method
it	O
from	O
Phe	B-residue_name
to	O
Ala	B-residue_name
would	O
not	O
abolish	O
the	O
catalytic	O
activity	O
of	O
this	O
enzyme	O
,	O
while	O
if	O
Phe	B-residue_name_number
34	I-residue_name_number
also	O
plays	O
an	O
essential	O
role	O
to	O
position	O
the	O
ethanethioate	B-chemical
moiety	O
of	O
Ac	B-chemical
-	I-chemical
CoA	I-chemical
,	O
the	O
mutation	B-experimental_method
would	O
be	O
expected	O
to	O
abrogate	O
the	O
activity	O
of	O
the	O
enzyme	O
.	O

Indeed	O
,	O
our	O
enzyme	B-evidence
kinetic	I-evidence
data	I-evidence
showed	O
that	O
hNaa60	B-mutant
(	I-mutant
1	I-mutant
-	I-mutant
199	I-mutant
)	I-mutant
F34A	B-mutant
mutant	B-protein_state
showed	O
no	O
detectable	O
activity	O
(	O
Fig	O
.	O
5A	O
).	O

In	O
order	O
to	O
rule	O
out	O
the	O
possibility	O
that	O
the	O
observed	O
loss	O
of	O
activity	O
may	O
be	O
related	O
to	O
bad	O
folding	O
of	O
the	O
mutant	B-protein_state
protein	O
,	O
we	O
studied	O
the	O
circular	B-experimental_method
dichroism	I-experimental_method
(	O
CD	B-experimental_method
)	O
spectrum	B-evidence
of	O
the	O
protein	O
(	O
Fig	O
.	O
5B	O
)	O
and	O
determined	O
its	O
crystal	B-evidence
structure	I-evidence
(	O
Fig	O
.	O
3C	O
).	O

Both	O
studies	O
proved	O
that	O
the	O
F34A	B-mutant
mutant	B-protein_state
protein	O
is	O
well	B-protein_state
-	I-protein_state
folded	I-protein_state
.	O

Many	O
studies	O
have	O
addressed	O
the	O
crucial	O
effect	O
of	O
α1	B-structure_element
-	I-structure_element
α2	I-structure_element
loop	I-structure_element
on	O
catalysis	O
,	O
showing	O
that	O
some	O
residues	O
located	O
in	O
this	O
area	O
are	O
involved	O
in	O
the	O
binding	O
of	O
substrates	O
.	O

We	O
propose	O
that	O
Phe	B-residue_name_number
34	I-residue_name_number
may	O
play	O
a	O
dual	O
role	O
both	O
in	O
interacting	O
with	O
the	O
peptide	B-chemical
substrate	O
(	O
recognition	O
)	O
and	O
in	O
positioning	O
of	O
the	O
ethanethioate	B-chemical
moiety	O
of	O
Ac	B-chemical
-	I-chemical
CoA	I-chemical
to	O
the	O
right	O
location	O
to	O
facilitate	O
acetyl	B-chemical
-	O
transfer	O
.	O

Structural	O
basis	O
for	O
hNaa60	B-protein
substrate	O
binding	O

Several	O
studies	O
have	O
demonstrated	O
that	O
the	O
substrate	O
specificities	O
of	O
hNaa60	B-protein
and	O
hNaa50	B-protein
are	O
highly	O
overlapped	O
.	O

The	O
structure	B-evidence
of	O
hNaa50p	B-complex_assembly
/	I-complex_assembly
CoA	I-complex_assembly
/	I-complex_assembly
peptide	I-complex_assembly
provides	O
detailed	O
information	O
about	O
the	O
position	O
of	O
substrate	O
N	O
-	O
terminal	O
residues	O
in	O
the	O
active	B-site
site	I-site
of	O
hNaa50	B-protein
.	O

Comparing	O
the	O
active	B-site
site	I-site
of	O
hNaa60	B-complex_assembly
(	I-complex_assembly
1	I-complex_assembly
-	I-complex_assembly
242	I-complex_assembly
)/	I-complex_assembly
Ac	I-complex_assembly
-	I-complex_assembly
CoA	I-complex_assembly
with	O
hNaa50p	B-complex_assembly
/	I-complex_assembly
CoA	I-complex_assembly
/	I-complex_assembly
peptide	I-complex_assembly
revealed	O
that	O
key	O
catalytic	B-site
and	I-site
substrate	I-site
binding	I-site
residues	I-site
are	O
highly	B-protein_state
conserved	I-protein_state
in	O
both	O
proteins	O
(	O
Fig	O
.	O
4A	O
).	O

With	O
respect	O
to	O
catalysis	O
,	O
hNaa50p	B-protein
has	O
been	O
shown	O
to	O
employ	O
residues	O
Tyr	B-residue_name_number
73	I-residue_name_number
and	O
His	B-residue_name_number
112	I-residue_name_number
to	O
abstract	O
proton	O
from	O
the	O
α	O
-	O
amino	O
group	O
from	O
the	O
substrate	O
’	O
s	O
first	O
residue	O
through	O
a	O
well	B-protein_state
-	I-protein_state
ordered	I-protein_state
water	B-chemical
.	O

A	O
well	B-protein_state
-	I-protein_state
ordered	I-protein_state
water	B-chemical
was	O
also	O
found	O
between	O
Tyr	B-residue_name_number
97	I-residue_name_number
and	O
His	B-residue_name_number
138	I-residue_name_number
in	O
hNaa60	B-complex_assembly
(	I-complex_assembly
1	I-complex_assembly
-	I-complex_assembly
199	I-complex_assembly
)/	I-complex_assembly
CoA	I-complex_assembly
and	O
hNaa60	B-complex_assembly
(	I-complex_assembly
1	I-complex_assembly
-	I-complex_assembly
242	I-complex_assembly
)/	I-complex_assembly
Ac	I-complex_assembly
-	I-complex_assembly
CoA	I-complex_assembly
(	O
Fig	O
.	O
4B	O
).	O

To	O
determine	O
the	O
function	O
of	O
Tyr	B-residue_name_number
97	I-residue_name_number
and	O
His	B-residue_name_number
138	I-residue_name_number
in	O
hNaa60	B-protein
catalysis	O
,	O
we	O
mutated	B-experimental_method
these	O
residues	O
to	O
alanine	B-residue_name
and	O
phenylalanine	B-residue_name
,	O
respectively	O
,	O
and	O
confirmed	O
that	O
all	O
these	O
mutants	B-protein_state
used	O
in	O
our	O
kinetic	B-experimental_method
assays	I-experimental_method
are	O
well	B-protein_state
-	I-protein_state
folded	I-protein_state
by	O
CD	B-experimental_method
spectra	B-evidence
(	O
Fig	O
.	O
5B	O
).	O

Purity	O
of	O
all	O
proteins	O
were	O
also	O
analyzed	O
by	O
SDS	B-experimental_method
-	I-experimental_method
PAGE	I-experimental_method
(	O
Figure	O
S5	O
).	O

As	O
show	O
in	O
Fig	O
.	O
5A	O
,	O
the	O
mutants	B-protein_state
Y97A	B-mutant
,	O
Y97F	B-mutant
,	O
H138A	B-mutant
and	O
H138F	B-mutant
abolished	B-protein_state
the	I-protein_state
activity	I-protein_state
of	O
hNaa60	B-protein
.	O

In	O
contrast	O
,	O
to	O
mutate	B-experimental_method
the	O
nearby	O
solvent	B-protein_state
exposed	I-protein_state
residue	O
Glu	B-residue_name_number
37	I-residue_name_number
to	O
Ala	B-residue_name
(	O
E37A	B-mutant
)	O
has	O
little	O
impact	O
on	O
the	O
activity	O
of	O
hNaa60	B-protein
(	O
Figs	O
4B	O
and	O
5A	O
).	O

In	O
conclusion	O
,	O
the	O
structural	B-experimental_method
and	I-experimental_method
functional	I-experimental_method
studies	I-experimental_method
indicate	O
that	O
hNaa60	B-protein
applies	O
the	O
same	O
two	O
base	O
mechanism	O
through	O
Tyr	B-residue_name_number
97	I-residue_name_number
,	O
His	B-residue_name_number
138	I-residue_name_number
and	O
a	O
well	B-protein_state
-	I-protein_state
ordered	I-protein_state
water	B-chemical
as	O
was	O
described	O
for	O
hNaa50	B-protein
.	O

The	O
malonate	B-chemical
molecule	O
observed	O
in	O
the	O
hNaa60	B-complex_assembly
(	I-complex_assembly
1	I-complex_assembly
-	I-complex_assembly
242	I-complex_assembly
)/	I-complex_assembly
Ac	I-complex_assembly
-	I-complex_assembly
CoA	I-complex_assembly
crystal	B-evidence
structure	I-evidence
may	O
be	O
indicative	O
of	O
the	O
substrate	O
binding	O
position	O
of	O
hNaa60	B-protein
since	O
it	O
is	O
located	O
in	O
the	O
active	B-site
site	I-site
and	O
overlaps	O
the	O
N	O
-	O
terminal	O
Met	B-residue_name
of	O
the	O
substrate	O
peptide	B-chemical
in	O
the	O
superposition	B-experimental_method
with	O
the	O
hNaa50p	B-complex_assembly
/	I-complex_assembly
CoA	I-complex_assembly
/	I-complex_assembly
peptide	I-complex_assembly
structure	B-evidence
(	O
Fig	O
.	O
4A	O
).	O

Residues	O
Tyr	B-residue_name_number
38	I-residue_name_number
,	O
Asn	B-residue_name_number
143	I-residue_name_number
and	O
Tyr	B-residue_name_number
165	I-residue_name_number
are	O
located	O
around	O
the	O
malonate	B-chemical
and	O
interact	O
with	O
it	O
through	O
direct	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
or	O
water	B-bond_interaction
bridge	I-bond_interaction
(	O
Fig	O
.	O
4C	O
).	O

Although	O
malonate	B-chemical
is	O
negatively	O
charged	O
,	O
which	O
is	O
different	O
from	O
that	O
of	O
lysine	B-residue_name
ε	O
-	O
amine	O
or	O
peptide	B-chemical
N	O
-	O
terminal	O
amine	O
,	O
similar	O
hydrophilic	B-bond_interaction
interactions	I-bond_interaction
may	O
take	O
place	O
when	O
substrate	O
amine	O
presents	O
in	O
the	O
same	O
position	O
,	O
since	O
Tyr	B-residue_name_number
38	I-residue_name_number
,	O
Asn	B-residue_name_number
143	I-residue_name_number
and	O
Tyr	B-residue_name_number
165	I-residue_name_number
are	O
not	O
positively	O
or	O
negatively	O
charged	O
.	O

In	O
agreement	O
with	O
this	O
hypothesis	O
,	O
it	O
was	O
found	O
that	O
the	O
Y38A	B-mutant
,	O
N143A	B-mutant
and	O
Y165A	B-mutant
mutants	B-protein_state
all	O
showed	O
remarkably	O
reduced	O
activities	O
as	O
compared	O
to	O
WT	B-protein_state
,	O
implying	O
that	O
these	O
residues	O
may	O
be	O
critical	O
for	O
substrate	O
binding	O
(	O
Figs	O
4C	O
and	O
5A	O
).	O

The	O
β3	B-structure_element
-	I-structure_element
β4	I-structure_element
loop	I-structure_element
participates	O
in	O
the	O
regulation	O
of	O
hNaa60	B-protein
-	O
activity	O

Residues	O
between	O
β3	B-structure_element
and	O
β4	B-structure_element
of	O
hNaa60	B-protein
form	O
a	O
unique	O
20	B-structure_element
-	I-structure_element
residue	I-structure_element
long	I-structure_element
loop	I-structure_element
(	O
residues	O
73	B-residue_range
–	I-residue_range
92	I-residue_range
)	O
that	O
is	O
a	O
short	B-structure_element
turn	I-structure_element
in	O
many	O
other	O
NAT	B-protein_type
members	O
(	O
Fig	O
.	O
1D	O
).	O

Previous	O
study	O
indicated	O
that	O
auto	B-ptm
-	I-ptm
acetylation	I-ptm
of	O
hNaa60K79	B-protein
could	O
influence	O
the	O
activity	O
of	O
hNaa60	B-protein
;	O
however	O
,	O
we	O
were	O
not	O
able	O
to	O
determine	O
if	O
Lys	B-residue_name_number
79	I-residue_name_number
is	O
acetylated	B-protein_state
in	O
our	O
crystal	B-evidence
structures	I-evidence
due	O
to	O
poor	O
quality	O
of	O
the	O
electron	B-evidence
density	I-evidence
of	O
Lys	B-residue_name_number
79	I-residue_name_number
side	O
-	O
chain	O
.	O

We	O
therefore	O
used	O
mass	B-experimental_method
spectrometry	I-experimental_method
to	O
analyze	O
if	O
Lys	B-residue_name_number
79	I-residue_name_number
was	O
acetylated	B-protein_state
in	O
our	O
bacterially	O
purified	O
proteins	O
,	O
and	O
observed	O
no	O
modification	O
on	O
this	O
residue	O
(	O
Figure	O
S6	O
).	O

To	O
assess	O
the	O
impact	O
of	O
hNaa60K79	B-protein
auto	B-ptm
-	I-ptm
acetylation	I-ptm
,	O
we	O
studied	O
the	O
kinetics	O
of	O
K79R	B-mutant
and	O
K79Q	B-mutant
mutants	B-protein_state
which	O
mimic	O
the	O
un	B-protein_state
-	I-protein_state
acetylated	I-protein_state
and	O
acetylated	B-protein_state
form	O
of	O
Lys	B-residue_name_number
79	I-residue_name_number
,	O
respectively	O
.	O

Interestingly	O
,	O
both	O
K79R	B-mutant
and	O
K79Q	B-mutant
mutants	B-protein_state
led	O
to	O
an	O
increase	O
in	O
the	O
catalytic	O
activity	O
of	O
hNaa60	B-protein
,	O
while	O
K79A	B-mutant
mutant	B-protein_state
led	O
to	O
modest	O
decrease	O
of	O
the	O
activity	O
(	O
Fig	O
.	O
5A	O
).	O

These	O
data	O
indicate	O
that	O
the	O
acetylation	B-ptm
of	O
Lys	B-residue_name_number
79	I-residue_name_number
is	O
not	O
required	O
for	O
optimal	O
catalytic	O
activity	O
of	O
hNaa60	B-protein
in	O
vitro	O
.	O

It	O
is	O
noted	O
that	O
the	O
β3	B-structure_element
-	I-structure_element
β4	I-structure_element
loop	I-structure_element
of	O
hNaa60	B-protein
acts	O
like	O
a	O
door	O
leaf	O
to	O
partly	O
cover	O
the	O
substrate	B-site
-	I-site
binding	I-site
pathway	I-site
.	O

We	O
hence	O
hypothesize	O
that	O
the	O
β3	B-structure_element
-	I-structure_element
β4	I-structure_element
loop	I-structure_element
may	O
interfere	O
with	O
the	O
access	O
of	O
the	O
peptide	B-chemical
substrates	O
and	O
that	O
the	O
solvent	B-protein_state
-	I-protein_state
exposing	I-protein_state
Lys	B-residue_name_number
79	I-residue_name_number
may	O
play	O
a	O
potential	O
role	O
to	O
remove	O
the	O
door	O
leaf	O
when	O
it	O
hovers	O
in	O
solvent	O
(	O
Fig	O
.	O
4D	O
).	O

Acidic	O
residues	O
Glu	B-residue_name_number
80	I-residue_name_number
,	O
Asp	B-residue_name_number
81	I-residue_name_number
and	O
Asp	B-residue_name_number
83	I-residue_name_number
interact	O
with	O
His	B-residue_name_number
138	I-residue_name_number
,	O
His	B-residue_name_number
159	I-residue_name_number
and	O
His	B-residue_name_number
158	I-residue_name_number
to	O
maintain	O
the	O
conformation	O
of	O
the	O
β3	B-structure_element
-	I-structure_element
β4	I-structure_element
loop	I-structure_element
,	O
thus	O
contribute	O
to	O
control	O
the	O
substrate	O
binding	O
(	O
Fig	O
.	O
4D	O
).	O

To	O
verify	O
this	O
hypothesis	O
,	O
we	O
mutated	B-experimental_method
Glu	B-residue_name_number
80	I-residue_name_number
,	O
Asp	B-residue_name_number
81	I-residue_name_number
and	O
Asp	B-residue_name_number
83	I-residue_name_number
to	O
Ala	B-residue_name
respectively	O
.	O

In	O
line	O
with	O
our	O
hypothesis	O
,	O
E80A	B-mutant
,	O
D81A	B-mutant
and	O
D83A	B-mutant
mutants	B-protein_state
exhibit	O
at	O
least	O
2	O
-	O
fold	O
increase	O
in	O
hNaa60	B-protein
-	O
activity	O
(	O
Fig	O
.	O
5A	O
).	O

Interestingly	O
,	O
the	O
structure	B-evidence
of	O
an	O
ancestral	O
NAT	B-protein_type
from	O
S	B-species
.	I-species
solfataricus	I-species
also	O
exhibits	O
a	O
10	B-structure_element
-	I-structure_element
residue	I-structure_element
long	I-structure_element
extension	I-structure_element
between	O
β3	B-structure_element
and	O
β4	B-structure_element
,	O
and	O
the	O
structure	B-experimental_method
and	I-experimental_method
biochemical	I-experimental_method
studies	I-experimental_method
showed	O
that	O
the	O
extension	B-structure_element
of	O
SsNat	B-protein
has	O
the	O
ability	O
to	O
stabilize	O
structure	O
of	O
the	O
active	B-site
site	I-site
and	O
potentiate	O
SsNat	B-protein
-	O
activity	O
.	O

Nt	B-ptm
-	I-ptm
acetylation	I-ptm
,	O
which	O
is	O
carried	O
out	O
by	O
the	O
NAT	B-protein_type
family	I-protein_type
acetyltransferases	I-protein_type
,	O
is	O
an	O
ancient	O
and	O
essential	O
modification	O
of	O
proteins	O
.	O

Although	O
many	O
NATs	B-protein_type
are	O
highly	B-protein_state
conserved	I-protein_state
from	O
lower	B-taxonomy_domain
to	O
higher	B-taxonomy_domain
eukaryotes	I-taxonomy_domain
and	O
the	O
substrate	O
bias	O
of	O
them	O
appears	O
to	O
be	O
partially	O
overlapped	O
,	O
there	O
is	O
a	O
significant	O
increase	O
in	O
the	O
overall	O
level	O
of	O
N	B-ptm
-	I-ptm
terminal	I-ptm
acetylation	I-ptm
from	O
lower	B-taxonomy_domain
to	O
higher	B-taxonomy_domain
eukaryotes	I-taxonomy_domain
.	O

In	O
this	O
study	O
we	O
provide	O
structural	O
insights	O
into	O
Naa60	B-protein
found	O
only	O
in	O
multicellular	B-taxonomy_domain
eukaryotes	I-taxonomy_domain
.	O

The	O
N	O
-	O
terminus	O
of	O
hNaa60	B-protein
harbors	O
three	O
hydrophobic	O
residues	O
(	O
VVP	B-structure_element
)	O
that	O
makes	O
it	O
very	O
difficult	O
to	O
express	O
and	O
purify	O
the	O
protein	O
.	O

This	O
problem	O
was	O
solved	O
by	O
replacing	B-experimental_method
residues	O
4	B-residue_range
–	I-residue_range
6	I-residue_range
from	O
VVP	B-structure_element
to	O
EER	B-structure_element
that	O
are	O
found	O
in	O
Naa60	B-protein
from	O
Xenopus	B-species
Laevis	I-species
.	O

Since	O
Naa60	B-protein
from	O
human	B-species
and	O
from	O
Xenopus	B-species
Laevis	I-species
are	O
highly	B-protein_state
homologous	I-protein_state
(	O
Fig	O
.	O
1A	O
),	O
we	O
speculate	O
that	O
these	O
two	O
proteins	O
should	O
have	O
the	O
same	O
biological	O
function	O
.	O

Therefore	O
it	O
is	O
deduced	O
that	O
the	O
VVP	B-mutant
to	I-mutant
EER	I-mutant
replacement	B-experimental_method
on	O
the	O
N	O
-	O
terminus	O
of	O
hNaa60	B-protein
may	O
not	O
interfere	O
with	O
its	O
function	O
.	O

However	O
,	O
in	O
the	O
hNaa60	B-protein
(	O
1	B-residue_range
-	I-residue_range
242	I-residue_range
)	O
structure	B-evidence
the	O
N	O
-	O
terminus	O
adopts	O
an	O
α	B-structure_element
-	I-structure_element
helical	I-structure_element
structure	I-structure_element
which	O
will	O
probably	O
be	O
kinked	O
if	O
residue	O
6	B-residue_number
is	O
proline	B-residue_name
(	O
Fig	O
.	O
1C	O
),	O
and	O
in	O
the	O
hNaa60	B-mutant
(	I-mutant
1	I-mutant
-	I-mutant
199	I-mutant
)	I-mutant
structure	B-evidence
the	O
N	O
-	O
terminus	O
adopts	O
a	O
different	O
semi	B-structure_element
-	I-structure_element
helical	I-structure_element
structure	I-structure_element
(	O
Fig	O
.	O
1B	O
)	O
likely	O
due	O
to	O
different	O
crystal	B-evidence
packing	I-evidence
.	O

Hence	O
it	O
is	O
not	O
clear	O
if	O
the	O
N	O
-	O
terminal	O
end	O
of	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
hNaa60	B-protein
is	O
an	O
α	B-structure_element
-	I-structure_element
helix	I-structure_element
,	O
and	O
what	O
roles	O
the	O
hydrophobic	O
residues	O
4	B-residue_range
–	I-residue_range
6	I-residue_range
play	O
in	O
structure	O
and	O
function	O
of	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
hNaa60	B-protein
.	O

In	O
addition	O
to	O
the	O
three	O
-	O
residue	O
mutation	B-experimental_method
(	O
VVP	B-structure_element
to	O
EER	B-structure_element
),	O
we	O
also	O
tried	O
many	O
other	O
hNaa60	B-protein
constructs	O
,	O
but	O
only	O
the	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
protein	O
and	O
the	O
truncated	B-protein_state
variant	O
1	B-residue_range
-	I-residue_range
199	I-residue_range
behaved	O
well	O
.	O

The	O
finding	O
that	O
the	O
catalytic	O
activity	O
of	O
hNaa60	B-protein
(	O
1	B-residue_range
-	I-residue_range
242	I-residue_range
)	O
is	O
much	O
lower	O
than	O
that	O
of	O
hNaa60	B-mutant
(	I-mutant
1	I-mutant
-	I-mutant
199	I-mutant
)	I-mutant
is	O
intriguing	O
.	O

We	O
speculate	O
that	O
low	O
activity	O
of	O
the	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
hNaa60	B-protein
might	O
be	O
related	O
to	O
lack	O
of	O
Golgi	O
localization	O
of	O
the	O
enzyme	O
in	O
our	O
in	O
vitro	O
studies	O
or	O
there	O
remains	O
some	O
undiscovered	O
auto	O
-	O
inhibitory	O
regulation	O
in	O
the	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
protein	O
.	O

The	O
hNaa60	B-protein
protein	O
was	O
proven	O
to	O
be	O
localized	O
on	O
Golgi	O
apparatus	O
.	O

Aksnes	O
and	O
colleagues	O
predicted	O
putative	O
transmembrane	B-structure_element
domains	I-structure_element
and	O
two	O
putative	O
sites	O
of	O
S	B-ptm
-	I-ptm
palmitoylation	I-ptm
,	O
by	O
bioinformatics	O
means	O
,	O
to	O
account	O
for	O
Golgi	O
localization	O
of	O
the	O
protein	O
.	O

They	O
then	O
mutated	B-experimental_method
all	O
five	O
cysteine	B-residue_name
residues	O
of	O
hNaa60	B-protein
’	O
s	O
to	O
serine	B-residue_name
,	O
including	O
the	O
two	O
putative	O
S	B-site
-	I-site
palmitoylation	I-site
sites	I-site
.	O

However	O
,	O
these	O
mutations	B-experimental_method
did	O
not	O
abolish	O
Naa60	B-protein
membrane	O
localization	O
,	O
indicating	O
that	O
S	B-ptm
-	I-ptm
palmitoylation	I-ptm
is	O
unlikely	O
to	O
(	O
solely	O
)	O
account	O
for	O
targeting	O
hNaa60	B-protein
on	O
Golgi	O
.	O

Furthermore	O
,	O
adding	B-experimental_method
residues	O
217	B-residue_range
–	I-residue_range
242	I-residue_range
of	O
hNaa60	B-protein
(	O
containing	O
residues	O
217	B-residue_range
–	I-residue_range
236	I-residue_range
,	O
one	O
of	O
the	O
putative	O
transmembrane	B-structure_element
domains	I-structure_element
)	O
to	O
the	O
C	O
terminus	O
of	O
eGFP	B-experimental_method
were	O
not	O
sufficient	O
to	O
localize	O
the	O
protein	O
on	O
Golgi	O
apparatus	O
,	O
while	O
eGFP	B-experimental_method
-	O
hNaa60182	B-mutant
-	I-mutant
242	I-mutant
was	O
sufficient	O
to	O
,	O
suggesting	O
that	O
residues	O
182	B-residue_range
–	I-residue_range
216	I-residue_range
are	O
important	O
for	O
Golgi	O
localization	O
of	O
hNaa60	B-protein
.	O

We	O
found	O
that	O
residues	O
190	B-residue_range
–	I-residue_range
202	I-residue_range
formed	O
an	O
amphipathic	B-structure_element
helix	I-structure_element
with	O
an	O
array	O
of	O
hydrophobic	O
residues	O
located	O
on	O
one	O
side	O
.	O

This	O
observation	O
is	O
reminiscent	O
of	O
the	O
protein	O
/	O
membrane	O
interaction	O
through	O
amphipathic	B-structure_element
helices	I-structure_element
in	O
the	O
cases	O
of	O
KalSec14	B-protein
,	O
Atg3	B-protein
,	O
PB1	B-protein
-	I-protein
F2	I-protein
etc	O
.	O

In	O
this	O
model	O
an	O
amphipathic	B-structure_element
helix	I-structure_element
can	O
immerse	O
its	O
hydrophobic	O
side	O
into	O
the	O
lipid	O
bilayer	O
through	O
hydrophobic	B-bond_interaction
interactions	I-bond_interaction
.	O

Therefore	O
we	O
propose	O
that	O
the	O
amphipathic	B-structure_element
helix	I-structure_element
α5	B-structure_element
may	O
contribute	O
to	O
Golgi	O
localization	O
of	O
hNaa60	B-protein
.	O

Previous	O
studies	O
indicated	O
that	O
members	O
of	O
NAT	B-protein_type
family	O
are	O
bi	O
-	O
functional	O
NAT	B-protein_type
and	O
KAT	B-protein_type
enzymes	O
.	O

However	O
,	O
known	O
structures	B-evidence
of	O
NATs	B-protein_type
do	O
not	O
well	O
support	O
this	O
hypothesis	O
,	O
since	O
the	O
β6	B-structure_element
-	I-structure_element
β7	I-structure_element
hairpin	I-structure_element
/	O
loop	B-structure_element
of	O
most	O
of	O
NATs	B-protein_type
is	O
involved	O
in	O
the	O
formation	O
of	O
a	O
tunnel	B-site
-	I-site
like	I-site
substrate	I-site
-	I-site
binding	I-site
site	I-site
with	O
the	O
α1	B-structure_element
-	I-structure_element
α2	I-structure_element
loop	I-structure_element
,	O
which	O
would	O
be	O
good	O
for	O
the	O
NAT	B-protein_type
but	O
not	O
KAT	B-protein_type
activity	O
of	O
the	O
enzyme	O
.	O

Kinetic	B-experimental_method
studies	I-experimental_method
have	O
been	O
conducted	O
to	O
compare	O
the	O
NAT	B-protein_type
and	O
KAT	B-protein_type
activity	O
of	O
hNaa50	B-protein
in	O
vitro	O
,	O
and	O
indicate	O
that	O
the	O
NAT	B-protein_type
activity	O
of	O
Naa50	B-protein
is	O
much	O
higher	O
than	O
KAT	B-protein_type
activity	O
.	O

However	O
,	O
the	O
substrate	O
used	O
in	O
this	O
study	O
for	O
assessing	O
KAT	B-protein_type
activity	O
was	O
a	O
small	O
peptide	B-chemical
which	O
could	O
not	O
really	O
mimic	O
the	O
3D	B-evidence
structure	I-evidence
of	O
a	O
folded	B-protein_state
protein	O
substrate	O
in	O
vivo	O
.	O

Our	O
mass	B-experimental_method
spectrometry	I-experimental_method
data	B-evidence
indicated	O
that	O
there	O
were	O
robust	O
acetylation	B-ptm
of	O
histone	B-protein_type
H3	B-complex_assembly
-	I-complex_assembly
H4	I-complex_assembly
tetramer	B-oligomeric_state
lysines	B-residue_name
and	O
both	O
N	B-ptm
-	I-ptm
terminal	I-ptm
acetylation	I-ptm
and	O
lysine	B-ptm
acetylation	I-ptm
of	O
the	O
peptide	B-chemical
used	O
in	O
the	O
activity	B-experimental_method
assay	I-experimental_method
,	O
thus	O
confirmed	O
the	O
KAT	B-protein_type
activity	O
of	O
this	O
enzyme	O
in	O
vitro	O
.	O

Conformational	O
change	O
of	O
the	O
β7	B-structure_element
-	I-structure_element
β8	I-structure_element
hairpin	I-structure_element
(	O
corresponding	O
to	O
the	O
β6	B-structure_element
-	I-structure_element
β7	I-structure_element
loop	I-structure_element
of	O
other	O
NATs	B-protein_type
)	O
is	O
noted	O
in	O
our	O
structures	B-evidence
(	O
Figs	O
1D	O
and	O
2C	O
),	O
which	O
might	O
provide	O
an	O
explanation	O
to	O
the	O
NAT	B-protein_type
/	O
KAT	B-protein_type
dual	O
-	O
activity	O
in	O
a	O
structural	O
biological	O
view	O
,	O
but	O
we	O
were	O
unable	O
to	O
rule	O
out	O
the	O
possibility	O
that	O
the	O
observed	O
conformational	O
change	O
of	O
this	O
hairpin	B-structure_element
might	O
be	O
an	O
artifact	O
related	O
to	O
crystal	B-evidence
packing	I-evidence
or	O
truncation	O
of	O
the	O
C	O
-	O
terminal	O
end	O
of	O
the	O
protein	O
.	O

Further	O
studies	O
are	O
therefore	O
needed	O
to	O
reveal	O
the	O
mechanism	O
for	O
the	O
KAT	B-protein_type
activity	O
of	O
this	O
enzyme	O
.	O

In	O
early	O
years	O
,	O
researchers	O
found	O
adjustment	O
of	O
GCN5	B-protein_type
histone	I-protein_type
acetyltransferase	I-protein_type
structure	B-evidence
when	O
it	O
binds	O
CoA	B-chemical
molecule	O
.	O

The	O
complexed	B-protein_state
form	O
of	O
NatA	B-complex_assembly
is	O
more	O
suitable	O
for	O
catalytic	O
activation	O
,	O
since	O
the	O
α1	B-structure_element
-	I-structure_element
α2	I-structure_element
loop	I-structure_element
undergoes	O
a	O
conformation	O
change	O
to	O
participate	O
in	O
the	O
formation	O
of	O
substrate	B-site
-	I-site
binding	I-site
site	I-site
when	O
the	O
auxiliary	O
subunit	O
Naa15	B-protein
interacts	O
with	O
Naa10	B-protein
(	O
the	O
catalytic	B-protein_state
subunit	B-structure_element
of	O
NatA	B-complex_assembly
).	O

In	O
the	O
structure	B-evidence
of	O
hNaa50	B-complex_assembly
/	I-complex_assembly
CoA	I-complex_assembly
/	I-complex_assembly
peptide	I-complex_assembly
,	O
Phe	B-residue_name_number
27	I-residue_name_number
in	O
the	O
α1	B-structure_element
-	I-structure_element
α2	I-structure_element
loop	I-structure_element
appears	O
to	O
make	O
hydrophobic	B-bond_interaction
interaction	I-bond_interaction
with	O
the	O
N	O
-	O
terminal	O
Met	B-residue_name
of	O
substrate	O
peptide	B-chemical
.	O

However	O
,	O
the	O
hNaa60	B-complex_assembly
(	I-complex_assembly
1	I-complex_assembly
-	I-complex_assembly
242	I-complex_assembly
)/	I-complex_assembly
Ac	I-complex_assembly
-	I-complex_assembly
CoA	I-complex_assembly
crystal	B-evidence
structure	I-evidence
indicated	O
that	O
its	O
counterpart	O
in	O
hNaa60	B-protein
,	O
Phe	B-residue_name_number
34	I-residue_name_number
,	O
could	O
also	O
accommodate	O
the	O
binding	O
of	O
a	O
hydrophilic	O
malonate	B-chemical
that	O
occupied	O
the	O
substrate	B-site
binding	I-site
site	I-site
although	O
it	O
maintained	O
the	O
same	O
conformation	O
as	O
that	O
observed	O
in	O
hNaa50	B-protein
.	O

Interestingly	O
,	O
the	O
terminal	O
thiol	B-chemical
of	O
CoA	B-chemical
adopted	O
alternative	O
conformations	O
in	O
the	O
structure	B-evidence
of	O
hNaa60	B-complex_assembly
(	I-complex_assembly
1	I-complex_assembly
-	I-complex_assembly
199	I-complex_assembly
)/	I-complex_assembly
CoA	I-complex_assembly
.	O
One	O
was	O
to	O
approach	O
the	O
substrate	O
amine	O
;	O
the	O
other	O
was	O
to	O
approach	O
the	O
α1	B-structure_element
-	I-structure_element
α2	I-structure_element
loop	I-structure_element
and	O
away	O
from	O
the	O
substrate	O
amine	O
.	O

Same	O
alternative	O
conformations	O
of	O
CoA	B-chemical
were	O
observed	O
in	O
the	O
hNaa60	B-mutant
(	I-mutant
1	I-mutant
-	I-mutant
199	I-mutant
)(	I-mutant
F34A	I-mutant
)	I-mutant
crystal	B-evidence
structure	I-evidence
,	O
and	O
our	O
kinetic	B-evidence
data	I-evidence
showed	O
that	O
the	O
F34A	B-mutant
mutation	B-experimental_method
abolished	O
the	O
activity	O
of	O
the	O
enzyme	O
.	O

Taken	O
together	O
,	O
our	O
data	O
indicated	O
that	O
Phe	B-residue_name_number
34	I-residue_name_number
in	O
hNaa60	B-protein
may	O
play	O
a	O
role	O
in	O
placing	O
co	O
-	O
enzyme	O
at	O
the	O
right	O
location	O
to	O
facilitate	O
the	O
acetyl	B-chemical
-	O
transfer	O
.	O

However	O
,	O
these	O
data	O
did	O
not	O
rule	O
out	O
that	O
possibility	O
that	O
Phe	B-residue_name_number
34	I-residue_name_number
may	O
coordinate	O
the	O
binding	O
of	O
the	O
N	O
-	O
terminal	O
Met	B-residue_name
through	O
hydrophobic	B-bond_interaction
interaction	I-bond_interaction
as	O
was	O
proposed	O
by	O
previous	O
studies	O
.	O

Furthermore	O
,	O
we	O
showed	O
that	O
hNaa60	B-protein
adopts	O
the	O
classical	O
two	O
base	O
mechanism	O
to	O
catalyze	O
acetyl	B-chemical
-	O
transfer	O
.	O

Although	O
sequence	O
identity	O
between	O
hNaa60	B-protein
and	O
hNaa50	B-protein
is	O
low	O
,	O
key	O
residues	O
in	O
the	O
active	B-site
site	I-site
of	O
both	O
enzymes	O
are	O
highly	B-protein_state
conserved	I-protein_state
.	O

This	O
can	O
reasonably	O
explain	O
the	O
high	O
overlapping	O
substrates	O
specificities	O
between	O
hNaa60	B-protein
and	O
hNaa50	B-protein
.	O

Another	O
structural	O
feature	O
of	O
hNaa60	B-protein
that	O
distinguishes	O
it	O
from	O
other	O
NATs	B-protein_type
is	O
the	O
β3	B-structure_element
-	I-structure_element
β4	I-structure_element
long	I-structure_element
loop	I-structure_element
which	O
appears	O
to	O
inhibit	O
the	O
catalytic	O
activity	O
of	O
hNaa60	B-protein
.	O

However	O
,	O
this	O
loop	B-structure_element
also	O
seems	O
to	O
stabilize	O
the	O
whole	O
hNaa60	B-protein
structure	B-evidence
,	O
because	O
deletion	B-experimental_method
mutations	I-experimental_method
of	O
this	O
region	O
led	O
to	O
protein	O
precipitation	O
and	O
aggregation	O
(	O
Figure	O
S7	O
).	O

A	O
previous	O
study	O
suggested	O
that	O
the	O
auto	B-ptm
-	I-ptm
acetylation	I-ptm
of	O
Lys	B-residue_name_number
79	I-residue_name_number
was	O
important	O
for	O
hNaa60	B-protein
-	O
activity	O
,	O
whereas	O
the	O
point	B-experimental_method
mutation	I-experimental_method
K79R	B-mutant
did	O
not	O
decrease	O
the	O
activity	O
of	O
hNaa60	B-protein
in	O
our	O
study	O
.	O

Meanwhile	O
,	O
no	O
electron	B-evidence
density	I-evidence
of	O
acetyl	B-chemical
group	O
was	O
found	O
on	O
Lys	B-residue_name_number
79	I-residue_name_number
in	O
our	O
structures	B-evidence
and	O
mass	B-experimental_method
spectrometry	I-experimental_method
analysis	O
.	O

Hence	O
,	O
it	O
appears	O
that	O
the	O
auto	B-ptm
-	I-ptm
acetylation	I-ptm
of	O
hNaa60	B-protein
is	O
not	O
an	O
essential	O
modification	O
for	O
its	O
activity	O
for	O
the	O
protein	O
we	O
used	O
here	O
.	O

As	O
for	O
the	O
reason	O
why	O
K79R	B-mutant
in	O
Yang	O
’	O
s	O
previous	O
studies	O
reduced	O
the	O
activity	O
of	O
the	O
enzyme	O
,	O
but	O
in	O
our	O
studies	O
it	O
didn	O
’	O
t	O
,	O
we	O
suspect	O
that	O
the	O
stability	O
of	O
this	O
mutant	B-protein_state
may	O
play	O
some	O
role	O
.	O

K79R	B-mutant
is	O
less	O
stable	B-protein_state
than	O
the	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
enzyme	O
as	O
was	O
judged	O
by	O
its	O
poorer	O
gel	B-experimental_method
-	I-experimental_method
filtration	I-experimental_method
behavior	O
and	O
tendency	O
to	O
precipitate	O
.	O

In	O
our	O
studies	O
we	O
have	O
paid	O
special	O
attention	O
and	O
carefully	O
handled	O
this	O
protein	O
to	O
ensure	O
that	O
we	O
did	O
get	O
enough	O
of	O
the	O
protein	O
in	O
good	O
condition	O
for	O
kinetic	B-experimental_method
assays	I-experimental_method
.	O

The	O
intracellular	O
environment	O
is	O
more	O
complicated	O
than	O
our	O
in	O
vitro	O
assay	O
and	O
the	O
substrate	O
specificity	O
of	O
hNaa60	B-protein
most	O
focuses	O
on	O
transmembrane	O
proteins	O
.	O

The	O
interaction	O
between	O
hNaa60	B-protein
and	O
its	O
substrates	O
may	O
involve	O
the	O
protein	O
-	O
membrane	O
interaction	O
which	O
would	O
further	O
increase	O
the	O
complexity	O
.	O

It	O
is	O
not	O
clear	O
if	O
the	O
structure	B-evidence
of	O
hNaa60	B-protein
is	O
different	O
in	O
vivo	O
or	O
if	O
other	O
potential	O
partner	O
proteins	O
may	O
help	O
to	O
regulate	O
its	O
activity	O
.	O

Nevertheless	O
,	O
our	O
study	O
may	O
be	O
an	O
inspiration	O
for	O
further	O
studies	O
on	O
the	O
functions	O
and	O
regulation	O
of	O
this	O
youngest	O
member	O
of	O
the	O
NAT	B-protein_type
family	O
.	O

Overall	O
structure	B-evidence
of	O
Naa60	B-protein
.	O

(	O
A	O
)	O
Sequence	B-experimental_method
alignment	I-experimental_method
of	O
Naa60	B-protein
(	O
NatF	B-complex_assembly
,	O
HAT4	B-protein
)	O
from	O
different	O
species	O
including	O
Homo	B-species
sapiens	I-species
(	O
Homo	B-species
),	O
Bos	B-species
mutus	I-species
(	O
Bos	B-species
),	O
Salmo	B-species
salar	I-species
(	O
Salmo	B-species
)	O
and	O
Xenopus	B-species
(	O
Silurana	B-species
)	O
tropicalis	B-species
(	O
Xenopus	B-species
).	O

Alignment	B-experimental_method
was	O
generated	O
using	O
NPS	O
@	O
and	O
ESPript	O
.	O
3	O
.	O
0	O
(	O
http	O
://	O
espript	O
.	O
ibcp	O
.	O
fr	O
/	O
ESPript	O
/	O
ESPript	O
/).	O

Residues	O
4	B-residue_range
–	I-residue_range
6	I-residue_range
are	O
highlighted	O
in	O
red	O
box	O
.	O

(	O
B	O
)	O
The	O
structure	B-evidence
of	O
hNaa60	B-complex_assembly
(	I-complex_assembly
1	I-complex_assembly
-	I-complex_assembly
199	I-complex_assembly
)/	I-complex_assembly
CoA	I-complex_assembly
complex	O
is	O
shown	O
as	O
a	O
yellow	O
cartoon	O
model	O
.	O

The	O
CoA	B-chemical
molecule	O
is	O
shown	O
as	O
sticks	O
.	O
(	O
C	O
)	O
The	O
structure	B-evidence
of	O
hNaa60	B-complex_assembly
(	I-complex_assembly
1	I-complex_assembly
-	I-complex_assembly
242	I-complex_assembly
)/	I-complex_assembly
Ac	I-complex_assembly
-	I-complex_assembly
CoA	I-complex_assembly
complex	O
is	O
presented	O
as	O
a	O
cartoon	O
model	O
in	O
cyan	O
.	O

The	O
Ac	B-chemical
-	I-chemical
CoA	I-chemical
and	O
malonate	B-chemical
molecules	O
are	O
shown	O
as	O
cyan	O
and	O
purple	O
sticks	O
,	O
respectively	O
.	O

The	O
secondary	O
structures	O
are	O
labeled	O
starting	O
with	O
α0	B-structure_element
.	O
(	O
D	O
)	O
Superposition	B-experimental_method
of	O
hNaa60	B-protein
(	O
1	B-residue_range
-	I-residue_range
242	I-residue_range
)	O
(	O
cyan	O
),	O
hNaa60	B-mutant
(	I-mutant
1	I-mutant
-	I-mutant
199	I-mutant
)	I-mutant
(	O
yellow	O
)	O
and	O
hNaa50	B-protein
(	O
pink	O
,	O
PDB	O
3TFY	O
).	O

The	O
Ac	B-chemical
-	I-chemical
CoA	I-chemical
of	O
hNaa60	B-complex_assembly
(	I-complex_assembly
1	I-complex_assembly
-	I-complex_assembly
242	I-complex_assembly
)/	I-complex_assembly
Ac	I-complex_assembly
-	I-complex_assembly
CoA	I-complex_assembly
complex	O
is	O
represented	O
as	O
cyan	O
sticks	O
.	O

Amphipathicity	B-protein_state
of	O
the	O
α5	B-structure_element
helix	I-structure_element
and	O
alternative	O
conformations	O
of	O
the	O
β7	B-structure_element
-	I-structure_element
β8	I-structure_element
hairpin	I-structure_element
.	O

(	O
A	O
)	O
The	O
α5	B-structure_element
helix	I-structure_element
of	O
hNaa60	B-protein
(	O
1	B-residue_range
-	I-residue_range
242	I-residue_range
)	O
in	O
one	O
asymmetric	O
unit	O
(	O
slate	O
)	O
interacts	O
with	O
another	O
hNaa60	B-protein
molecule	O
in	O
a	O
neighboring	O
asymmetric	O
unit	O
(	O
cyan	O
).	O

Side	O
-	O
chains	O
of	O
hydrophobic	O
residues	O
on	O
α5	B-structure_element
helix	I-structure_element
and	O
the	O
neighboring	O
molecule	O
participating	O
in	O
the	O
interaction	O
are	O
shown	O
as	O
yellow	O
and	O
green	O
sticks	O
,	O
respectively	O
.	O
(	O
B	O
)	O
The	O
α5	B-structure_element
helix	I-structure_element
of	O
hNaa60	B-mutant
(	I-mutant
1	I-mutant
-	I-mutant
199	I-mutant
)	I-mutant
in	O
one	O
asymmetric	O
unit	O
(	O
yellow	O
)	O
interacts	O
with	O
another	O
hNaa60	B-protein
molecule	O
in	O
the	O
neighboring	O
asymmetric	O
units	O
(	O
green	O
).	O

Side	O
-	O
chains	O
of	O
hydrophobic	O
residues	O
on	O
α5	B-structure_element
helix	I-structure_element
and	O
the	O
neighboring	O
molecule	O
(	O
green	O
)	O
participating	O
in	O
the	O
interaction	O
are	O
shown	O
as	O
yellow	O
and	O
green	O
sticks	O
,	O
respectively	O
.	O

The	O
third	O
molecule	O
(	O
pink	O
)	O
does	O
not	O
directly	O
interact	O
with	O
the	O
α5	B-structure_element
helix	I-structure_element
.	O

(	O
C	O
)	O
Superposition	B-experimental_method
of	O
hNaa60	B-mutant
(	I-mutant
1	I-mutant
-	I-mutant
199	I-mutant
)	I-mutant
(	O
yellow	O
)	O
and	O
hNaa60	B-protein
(	O
1	B-residue_range
-	I-residue_range
242	I-residue_range
)	O
(	O
cyan	O
)	O
showing	O
conformational	O
change	O
of	O
the	O
β7	B-structure_element
-	I-structure_element
β8	I-structure_element
hairpin	I-structure_element
in	O
these	O
two	O
structures	B-evidence
.	O
(	O
D	O
,	O
E	O
)	O
Superposition	B-experimental_method
of	O
Hat1p	B-protein
/	O
H4	B-protein_type
(	O
gray	O
,	O
drawn	O
from	O
PDB	O
4PSW	O
)	O
with	O
hNaa60	B-protein
(	O
1	B-residue_range
-	I-residue_range
242	I-residue_range
)	O
(	O
cyan	O
,	O
D	O
)	O
or	O
hNaa60	B-mutant
(	I-mutant
1	I-mutant
-	I-mutant
199	I-mutant
)	I-mutant
(	O
yellow	O
,	O
E	O
).	O

The	O
histone	B-protein_type
H4	B-protein_type
peptide	B-chemical
(	O
a	O
KAT	B-protein_type
substrate	O
)	O
bound	B-protein_state
to	I-protein_state
Hat1p	B-protein
is	O
shown	O
in	O
purple	O
(	O
D	O
,	O
E	O
),	O
while	O
the	O
peptide	B-chemical
bound	B-protein_state
to	I-protein_state
hNaa50	B-protein
(	O
a	O
NAT	B-protein_type
substrate	O
,	O
drawn	O
from	O
PDB	O
3TFY	O
)	O
is	O
shown	O
in	O
orange	O
(	O
Nt	B-chemical
-	I-chemical
peptide	I-chemical
)	O
after	O
superimposing	B-experimental_method
hNaa50	B-protein
(	O
not	O
shown	O
in	O
figure	O
)	O
on	O
hNaa60	B-protein
(	O
D	O
).	O

The	O
α	O
-	O
amine	O
of	O
the	O
NAT	B-protein_type
substrate	O
and	O
ε	O
-	O
amine	O
of	O
the	O
KAT	B-protein_type
substrate	O
(	O
along	O
with	O
the	O
lysine	B-residue_name
side	O
-	O
chain	O
)	O
subject	O
to	O
acetylation	B-ptm
are	O
shown	O
as	O
sticks	O
.	O

Electron	B-evidence
density	I-evidence
map	I-evidence
of	O
the	O
active	B-site
site	I-site
.	O

The	O
2Fo	B-evidence
-	I-evidence
Fc	I-evidence
maps	I-evidence
contoured	O
at	O
1	O
.	O
0σ	O
are	O
shown	O
for	O
hNaa60	B-complex_assembly
(	I-complex_assembly
1	I-complex_assembly
-	I-complex_assembly
242	I-complex_assembly
)/	I-complex_assembly
Ac	I-complex_assembly
-	I-complex_assembly
CoA	I-complex_assembly
(	O
A	O
),	O
hNaa60	B-complex_assembly
(	I-complex_assembly
1	I-complex_assembly
-	I-complex_assembly
199	I-complex_assembly
)/	I-complex_assembly
CoA	I-complex_assembly
(	O
B	O
)	O
and	O
hNaa60	B-complex_assembly
(	I-complex_assembly
1	I-complex_assembly
-	I-complex_assembly
199	I-complex_assembly
)	I-complex_assembly
F34A	I-complex_assembly
/	I-complex_assembly
CoA	I-complex_assembly
(	O
C	O
).	O

The	O
putative	O
substrate	B-site
peptide	I-site
binding	I-site
site	I-site
is	O
indicated	O
by	O
the	O
peptide	B-chemical
(	O
shown	O
as	O
pink	O
sticks	O
)	O
from	O
the	O
hNaa50	B-complex_assembly
/	I-complex_assembly
CoA	I-complex_assembly
/	I-complex_assembly
peptide	I-complex_assembly
complex	O
structure	B-evidence
after	O
superimposing	B-experimental_method
hNaa50	B-protein
on	O
the	O
hNaa60	B-protein
structures	B-evidence
determined	O
in	O
this	O
study	O
.	O

The	O
black	O
arrow	O
indicates	O
the	O
α	O
-	O
amine	O
of	O
the	O
first	B-residue_name_number
Met	I-residue_name_number
(	O
M1	B-residue_name_number
)	O
(	O
all	O
panels	O
).	O

The	O
purple	O
arrow	O
indicates	O
the	O
acetyl	B-chemical
moiety	O
of	O
Ac	B-chemical
-	I-chemical
CoA	I-chemical
(	O
A	O
).	O

The	O
red	O
arrow	O
indicates	O
the	O
alternative	O
conformation	O
of	O
the	O
thiol	O
moiety	O
of	O
the	O
co	O
-	O
enzyme	O
when	O
Phe	B-residue_name_number
34	I-residue_name_number
side	O
-	O
chain	O
is	O
displaced	O
(	O
B	O
)	O
or	O
mutated	B-experimental_method
to	O
Ala	B-residue_name
(	O
C	O
).	O

Structural	O
basis	O
for	O
hNaa60	B-protein
catalytic	O
activity	O
.	O

(	O
A	O
)	O
Superposition	B-experimental_method
of	O
hNaa60	B-protein
active	B-site
site	I-site
(	O
cyan	O
)	O
on	O
that	O
of	O
hNaa50	B-protein
(	O
pink	O
,	O
PDB	O
3TFY	O
).	O

Side	O
-	O
chains	O
of	O
key	O
catalytic	B-site
and	I-site
substrate	I-site
-	I-site
binding	I-site
residues	I-site
are	O
highlighted	O
as	O
sticks	O
.	O

The	O
malonate	B-chemical
molecule	O
in	O
the	O
hNaa60	B-complex_assembly
(	I-complex_assembly
1	I-complex_assembly
-	I-complex_assembly
242	I-complex_assembly
)/	I-complex_assembly
Ac	I-complex_assembly
-	I-complex_assembly
CoA	I-complex_assembly
structure	B-evidence
and	O
the	O
peptide	B-chemical
in	O
the	O
hNaa50	B-complex_assembly
/	I-complex_assembly
CoA	I-complex_assembly
/	I-complex_assembly
peptide	I-complex_assembly
structure	B-evidence
are	O
shown	O
as	O
purple	O
and	O
yellow	O
sticks	O
respectively	O
.	O
(	O
B	O
)	O
A	O
close	O
view	O
of	O
the	O
active	B-site
site	I-site
of	O
hNaa60	B-protein
.	O

Residues	O
Glu	B-residue_name_number
37	I-residue_name_number
,	O
Tyr	B-residue_name_number
97	I-residue_name_number
and	O
His	B-residue_name_number
138	I-residue_name_number
in	O
hNaa60	B-protein
(	O
cyan	O
)	O
and	O
corresponding	O
residues	O
(	O
Tyr	B-residue_name_number
73	I-residue_name_number
and	O
His	B-residue_name_number
112	I-residue_name_number
)	O
in	O
hNaa50	B-protein
(	O
pink	O
)	O
as	O
well	O
as	O
the	O
side	O
-	O
chain	O
of	O
corresponding	O
residues	O
(	O
Glu	B-residue_name_number
24	I-residue_name_number
,	O
His	B-residue_name_number
72	I-residue_name_number
and	O
His	B-residue_name_number
111	I-residue_name_number
)	O
in	O
complexed	B-protein_state
formed	O
hNaa10p	B-protein
(	O
warmpink	O
)	O
are	O
highlighted	O
as	O
sticks	O
.	O

The	O
water	B-chemical
molecules	O
participating	O
in	O
catalysis	O
in	O
the	O
hNaa60	B-protein
and	O
hNaa50	B-protein
structures	B-evidence
are	O
showed	O
as	O
green	O
and	O
red	O
spheres	O
,	O
separately	O
.	O
(	O
C	O
)	O
The	O
interaction	O
between	O
the	O
malonate	B-chemical
molecule	O
and	O
surrounding	O
residues	O
observed	O
in	O
the	O
hNaa60	B-complex_assembly
(	I-complex_assembly
1	I-complex_assembly
-	I-complex_assembly
242	I-complex_assembly
)/	I-complex_assembly
Ac	I-complex_assembly
-	I-complex_assembly
CoA	I-complex_assembly
structure	B-evidence
.	O

The	O
yellow	O
dotted	O
lines	O
indicate	O
the	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
.	O
(	O
D	O
)	O
A	O
zoomed	O
view	O
of	O
β3	B-structure_element
-	I-structure_element
β4	I-structure_element
loop	I-structure_element
of	O
hNaa60	B-protein
.	O

Key	O
residues	O
discussed	O
in	O
the	O
text	O
(	O
cyan	O
),	O
the	O
malonate	B-chemical
(	O
purple	O
)	O
and	O
Ac	B-chemical
-	I-chemical
CoA	I-chemical
(	O
gray	O
)	O
are	O
shown	O
as	O
sticks	O
.	O

The	O
yellow	O
dotted	O
lines	O
indicate	O
the	O
salt	B-bond_interaction
bridges	I-bond_interaction
.	O

Catalytic	O
activity	O
of	O
hNaa60	B-protein
and	O
mutant	B-protein_state
proteins	O
.	O

(	O
A	O
)	O
Catalytic	B-evidence
efficiency	I-evidence
(	O
shown	O
as	O
kcat	B-evidence
/	O
Km	B-evidence
values	O
)	O
of	O
hNaa60	B-mutant
(	I-mutant
1	I-mutant
-	I-mutant
199	I-mutant
)	I-mutant
WT	B-protein_state
and	O
mutants	B-protein_state
.	O

(	O
B	O
)	O
CD	B-experimental_method
spectra	B-evidence
of	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
and	O
mutant	B-protein_state
proteins	O
from	O
250	O
nm	O
to	O
190	O
nm	O
.	O

The	O
sample	O
concentration	O
was	O
4	O
.	O
5	O
μM	O
in	O
20	O
mM	O
Tris	O
,	O
pH	O
8	O
.	O
0	O
,	O
150	O
mM	O
NaCl	O
,	O
1	O
%	O
glycerol	O
and	O
1	O
mM	O
TCEP	B-chemical
at	O
room	O
temperature	O
.	O

Data	B-evidence
collection	I-evidence
and	I-evidence
refinement	I-evidence
statistics	I-evidence
.	O

Structure	O
and	O
PDB	O
ID	O
hNaa60	B-complex_assembly
(	I-complex_assembly
1	I-complex_assembly
-	I-complex_assembly
242	I-complex_assembly
)/	I-complex_assembly
Ac	I-complex_assembly
-	I-complex_assembly
CoA	I-complex_assembly
5HGZ	O
hNaa60	B-complex_assembly
(	I-complex_assembly
1	I-complex_assembly
-	I-complex_assembly
199	I-complex_assembly
)/	I-complex_assembly
CoA	I-complex_assembly
5HH0	O
hNaa60	B-complex_assembly
(	I-complex_assembly
1	I-complex_assembly
-	I-complex_assembly
199	I-complex_assembly
)	I-complex_assembly
F34A	I-complex_assembly
/	I-complex_assembly
CoA	I-complex_assembly
5HH1	O
Data	O
collection	O
*	O
Space	O
group	O
P212121	O
P21212	O
P21212	O
Cell	O
dimensions	O
a	O
,	O
b	O
,	O
c	O
(	O
Å	O
)	O
53	O
.	O
3	O
,	O
57	O
.	O
4	O
,	O
68	O
.	O
8	O
67	O
.	O
8	O
,	O
73	O
.	O
8	O
,	O
43	O
.	O
2	O
66	O
.	O
7	O
,	O
74	O
.	O
0	O
,	O
43	O
.	O
5	O
α	O
,	O
β	O
,	O
γ	O
(°)	O
90	O
.	O
0	O
,	O
90	O
.	O
0	O
,	O
90	O
.	O
0	O
90	O
.	O
0	O
,	O
90	O
.	O
0	O
,	O
90	O
.	O
0	O
90	O
.	O
0	O
,	O
90	O
.	O
0	O
,	O
90	O
.	O
0	O
Resolution	O
(	O
Å	O
)	O
50	O
–	O
1	O
.	O
38	O
(	O
1	O
.	O
42	O
–	O
1	O
.	O
38	O
)	O
50	O
–	O
1	O
.	O
60	O
(	O
1	O
.	O
66	O
–	O
1	O
.	O
60	O
)	O
50	O
–	O
1	O
.	O
80	O
(	O
1	O
.	O
86	O
–	O
1	O
.	O
80	O
)	O
Rp	O
.	O
i	O
.	O
m	O
.(%)**	O
3	O
.	O
0	O
(	O
34	O
.	O
4	O
)	O
2	O
.	O
1	O
(	O
32	O
.	O
5	O
)	O
2	O
.	O
6	O
(	O
47	O
.	O
8	O
)	O
I	O
/	O
σ	O
21	O
.	O
5	O
(	O
2	O
.	O
0	O
)	O
31	O
.	O
8	O
(	O
2	O
.	O
0	O
)	O
28	O
.	O
0	O
(	O
2	O
.	O
4	O
)	O
Completeness	O
(%)	O
99	O
.	O
8	O
(	O
99	O
.	O
1	O
)	O
99	O
.	O
6	O
(	O
98	O
.	O
5	O
)	O
99	O
.	O
9	O
(	O
99	O
.	O
7	O
)	O
Redundancy	O
6	O
.	O
9	O
(	O
5	O
.	O
0	O
)	O
6	O
.	O
9	O
(	O
6	O
.	O
2	O
)	O
6	O
.	O
3	O
(	O
5	O
.	O
9	O
)	O
Refinement	O
Resolution	O
(	O
Å	O
)	O
25	O
.	O
81	O
–	O
1	O
.	O
38	O
33	O
.	O
55	O
–	O
1	O
.	O
60	O
43	O
.	O
52	O
–	O
1	O
.	O
80	O
No	O
.	O
reflections	O
43660	O
28588	O
20490	O
Rwork	O
/	O
Rfree	O
0	O
.	O
182	O
/	O
0	O
.	O
192	O
0	O
.	O
181	O
/	O
0	O
.	O
184	O
0	O
.	O
189	O
/	O
0	O
.	O
209	O
No	O
.	O
atoms	O
Protein	O
1717	O
1576	O
1566	O
Ligand	O
/	O
ion	O
116	O
96	O
96	O
Water	B-chemical
289	O
258	O
168	O
B	O
-	O
factors	O
Protein	O
23	O
.	O
8	O
32	O
.	O
0	O
37	O
.	O
4	O
Ligand	O
/	O
ion	O
22	O
.	O
2	O
34	O
.	O
6	O
43	O
.	O
7	O
Water	B-chemical
35	O
.	O
1	O
46	O
.	O
4	O
49	O
.	O
1	O
R	O
.	O
m	O
.	O
s	O
.	O

One	O
crystal	B-evidence
was	O
used	O
for	O
each	O
data	O
set	O
.	O

**	O
Rp	O
.	O
i	O
.	O
m	O
.,	O
a	O
redundancy	O
-	O
independent	O
R	B-evidence
factor	I-evidence
was	O
used	O
to	O
evaluate	O
the	O
diffraction	B-evidence
data	I-evidence
quality	O
as	O
was	O
proposed	O
by	O
Evans	O
.	O