Biochemical	B-experimental_method
and	I-experimental_method
structural	I-experimental_method
characterization	I-experimental_method
of	O
a	O
DNA	B-protein_type
N6	I-protein_type
-	I-protein_type
adenine	I-protein_type
methyltransferase	I-protein_type
from	O
Helicobacter	B-species
pylori	I-species

DNA	B-ptm
N6	I-ptm
-	I-ptm
methyladenine	I-ptm
modification	O
plays	O
an	O
important	O
role	O
in	O
regulating	O
a	O
variety	O
of	O
biological	O
functions	O
in	O
bacteria	B-taxonomy_domain
.	O

However	O
,	O
the	O
mechanism	O
of	O
sequence	O
-	O
specific	O
recognition	O
in	O
N6	B-ptm
-	I-ptm
methyladenine	I-ptm
modification	O
remains	O
elusive	O
.	O

M1	B-protein
.	I-protein
HpyAVI	I-protein
,	O
a	O
DNA	B-protein_type
N6	I-protein_type
-	I-protein_type
adenine	I-protein_type
methyltransferase	I-protein_type
from	O
Helicobacter	B-species
pylori	I-species
,	O
shows	O
more	O
promiscuous	O
substrate	O
specificity	O
than	O
other	O
enzymes	O
.	O

Here	O
,	O
we	O
present	O
the	O
crystal	B-evidence
structures	I-evidence
of	O
cofactor	B-protein_state
-	I-protein_state
free	I-protein_state
and	O
AdoMet	B-protein_state
-	I-protein_state
bound	I-protein_state
structures	B-evidence
of	O
this	O
enzyme	O
,	O
which	O
were	O
determined	O
at	O
resolutions	O
of	O
3	O
.	O
0	O
Å	O
and	O
3	O
.	O
1	O
Å	O
,	O
respectively	O
.	O

The	O
core	O
structure	O
of	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
resembles	O
the	O
canonical	O
AdoMet	B-protein_type
-	I-protein_type
dependent	I-protein_type
MTase	I-protein_type
fold	O
,	O
while	O
the	O
putative	O
DNA	B-site
binding	I-site
regions	I-site
considerably	O
differ	O
from	O
those	O
of	O
the	O
other	O
MTases	B-protein_type
,	O
which	O
may	O
account	O
for	O
the	O
substrate	O
promiscuity	O
of	O
this	O
enzyme	O
.	O

Site	B-experimental_method
-	I-experimental_method
directed	I-experimental_method
mutagenesis	I-experimental_method
experiments	O
identified	O
residues	O
D29	B-residue_name_number
and	O
E216	B-residue_name_number
as	O
crucial	O
amino	O
acids	O
for	O
cofactor	O
binding	O
and	O
the	O
methyl	B-chemical
transfer	O
activity	O
of	O
the	O
enzyme	O
,	O
while	O
P41	B-residue_name_number
,	O
located	O
in	O
a	O
highly	B-protein_state
flexible	I-protein_state
loop	B-structure_element
,	O
playing	O
a	O
determinant	O
role	O
for	O
substrate	O
specificity	O
.	O

Taken	O
together	O
,	O
our	O
data	O
revealed	O
the	O
structural	O
basis	O
underlying	O
DNA	B-protein_type
N6	I-protein_type
-	I-protein_type
adenine	I-protein_type
methyltransferase	I-protein_type
substrate	O
promiscuity	O
.	O

DNA	B-ptm
methylation	I-ptm
is	O
a	O
common	O
form	O
of	O
modification	O
on	O
nucleic	O
acids	O
occurring	O
in	O
both	O
prokaryotes	B-taxonomy_domain
and	O
eukaryotes	B-taxonomy_domain
.	O

Such	O
a	O
modification	O
creates	O
a	O
signature	O
motif	O
recognized	O
by	O
DNA	B-chemical
-	O
interacting	O
proteins	O
and	O
functions	O
as	O
a	O
mechanism	O
to	O
regulate	O
gene	O
expression	O
.	O

DNA	B-ptm
methylation	I-ptm
is	O
mediated	O
by	O
DNA	B-protein_type
methyltransferases	I-protein_type
(	O
MTases	B-protein_type
),	O
which	O
catalyze	O
the	O
transfer	O
of	O
a	O
methyl	B-chemical
group	O
from	O
S	B-chemical
-	I-chemical
adenosyl	I-chemical
-	I-chemical
L	I-chemical
-	I-chemical
methionine	I-chemical
(	O
AdoMet	B-chemical
)	O
to	O
a	O
given	O
position	O
of	O
a	O
particular	O
DNA	B-chemical
base	O
within	O
a	O
specific	O
DNA	B-chemical
sequence	O
.	O

Three	O
classes	O
of	O
DNA	B-protein_type
MTases	I-protein_type
have	O
been	O
identified	O
to	O
transfer	O
a	O
methyl	B-chemical
group	O
to	O
different	O
positions	O
of	O
DNA	B-chemical
bases	O
.	O

C5	B-protein_type
-	I-protein_type
cytosine	I-protein_type
MTases	I-protein_type
,	O
for	O
example	O
,	O
methylate	O
C5	O
of	O
cytosine	B-residue_name
(	O
m5C	B-ptm
).	O

In	O
eukaryotes	B-taxonomy_domain
,	O
m5C	B-ptm
plays	O
an	O
important	O
role	O
in	O
gene	O
expression	O
,	O
chromatin	O
organization	O
,	O
genome	O
maintenance	O
and	O
parental	O
imprinting	O
,	O
and	O
is	O
involved	O
in	O
a	O
variety	O
of	O
human	B-species
diseases	O
including	O
cancer	O
.	O

By	O
contrast	O
,	O
the	O
functions	O
of	O
the	O
prokaryotic	B-taxonomy_domain
DNA	B-protein_type
cytosine	I-protein_type
MTase	I-protein_type
remain	O
unknown	O
.	O

N4	B-protein_type
-	I-protein_type
cytosine	I-protein_type
MTases	I-protein_type
,	O
which	O
are	O
frequently	O
present	O
in	O
thermophilic	B-taxonomy_domain
or	O
mesophilic	B-taxonomy_domain
bacteria	B-taxonomy_domain
,	O
transfer	O
a	O
methyl	B-chemical
group	O
to	O
the	O
exocyclic	O
amino	O
group	O
of	O
cytosine	B-residue_name
(	O
4mC	B-ptm
).	O

N4	B-ptm
methylation	I-ptm
seems	O
to	O
be	O
primarily	O
a	O
component	O
of	O
bacterial	B-taxonomy_domain
immune	O
system	O
against	O
invasion	O
by	O
foreign	O
DNA	B-chemical
,	O
such	O
as	O
conjugative	O
plasmids	O
and	O
bacteriophages	B-taxonomy_domain
.	O

The	O
third	O
group	O
,	O
N6	B-protein_type
-	I-protein_type
adenine	I-protein_type
MTases	I-protein_type
methylate	O
the	O
exocyclic	O
amino	O
groups	O
of	O
adenine	B-residue_name
(	O
6mA	B-ptm
),	O
which	O
exists	O
in	O
prokaryotes	B-taxonomy_domain
as	O
a	O
signal	O
for	O
genome	O
defense	O
,	O
DNA	B-chemical
replication	O
and	O
repair	O
,	O
regulation	O
of	O
gene	O
expression	O
,	O
control	O
of	O
transposition	O
and	O
host	O
-	O
pathogen	O
interactions	O
.	O

Recent	O
studies	O
utilizing	O
new	O
sequencing	O
approaches	O
have	O
showed	O
the	O
existence	O
of	O
6mA	B-ptm
in	O
several	O
eukaryotic	B-taxonomy_domain
species	O
.	O

DNA	B-chemical
6mA	B-ptm
modification	O
is	O
associated	O
with	O
important	O
biological	O
processes	O
including	O
nucleosome	O
distribution	O
close	O
to	O
the	O
transcription	O
start	O
sites	O
in	O
Chlamydomonas	B-taxonomy_domain
,	O
carrying	O
heritable	O
epigenetic	O
information	O
in	O
C	B-species
.	I-species
elegans	I-species
or	O
controlling	O
development	O
of	O
Drosophila	B-taxonomy_domain
.	O

All	O
the	O
three	O
types	O
of	O
methylation	B-ptm
exist	O
in	O
prokaryotes	B-taxonomy_domain
,	O
and	O
most	O
DNA	B-protein_type
MTases	I-protein_type
are	O
components	O
of	O
the	O
restriction	O
-	O
modification	O
(	O
R	O
-	O
M	O
)	O
systems	O
.	O

“	O
R	O
”	O
stands	O
for	O
a	O
restriction	B-protein_type
endonuclease	I-protein_type
cleaving	O
specific	O
DNA	B-chemical
sequences	O
,	O
while	O
“	O
M	O
”	O
symbolizes	O
a	O
modification	B-protein_type
methyltransferase	I-protein_type
rendering	O
these	O
sequences	O
resistant	O
to	O
cleavage	O
.	O

The	O
cooperation	O
of	O
these	O
two	O
enzymes	O
provides	O
a	O
defensive	O
mechanism	O
to	O
protect	O
bacteria	B-taxonomy_domain
from	O
infection	O
by	O
bacteriophages	B-taxonomy_domain
.	O

The	O
R	O
-	O
M	O
systems	O
are	O
classified	O
into	O
three	O
types	O
based	O
on	O
specific	O
structural	O
features	O
,	O
position	O
of	O
DNA	B-chemical
cleavage	O
and	O
cofactor	O
requirements	O
.	O

In	O
types	O
I	O
and	O
III	O
,	O
the	O
DNA	B-protein_type
adenine	I-protein_type
or	I-protein_type
cytosine	I-protein_type
methyltransferase	I-protein_type
is	O
part	O
of	O
a	O
multi	O
-	O
subunit	O
enzyme	O
that	O
catalyzes	O
both	O
restriction	O
and	O
modification	O
.	O

By	O
contrast	O
,	O
two	O
separate	O
enzymes	O
exist	O
in	O
type	O
II	O
systems	O
,	O
where	O
a	O
restriction	B-protein_type
endonuclease	I-protein_type
and	O
a	O
DNA	B-protein_type
adenine	I-protein_type
or	I-protein_type
cytosine	I-protein_type
methyltransferase	I-protein_type
recognize	O
the	O
same	O
targets	O
.	O

To	O
date	O
,	O
a	O
number	O
of	O
bacterial	B-taxonomy_domain
DNA	B-protein_type
MTases	I-protein_type
have	O
been	O
structurally	B-experimental_method
characterized	I-experimental_method
,	O
covering	O
enzymes	O
from	O
all	O
the	O
three	O
classes	O
.	O

All	O
these	O
MTases	B-protein_type
exhibit	O
high	O
similarity	O
in	O
their	O
overall	O
architectures	O
,	O
which	O
are	O
generally	O
folded	O
into	O
two	O
domains	O
:	O
a	O
conserved	B-protein_state
larger	O
catalytic	B-structure_element
domain	I-structure_element
comprising	O
an	O
active	B-site
site	I-site
for	O
methyl	B-chemical
transfer	O
and	O
a	O
site	O
for	O
AdoMet	B-chemical
-	O
binding	O
,	O
and	O
a	O
smaller	O
target	B-structure_element
(	I-structure_element
DNA	I-structure_element
)-	I-structure_element
recognition	I-structure_element
domain	I-structure_element
(	O
TRD	B-structure_element
)	O
containing	O
variable	O
regions	O
implicated	O
in	O
sequence	O
-	O
specific	O
DNA	B-chemical
recognition	O
and	O
the	O
infiltration	O
of	O
the	O
DNA	B-chemical
to	O
flip	O
the	O
target	O
base	O
.	O

Conserved	B-protein_state
amino	O
acid	O
motifs	O
have	O
been	O
identified	O
from	O
reported	O
structures	B-evidence
,	O
including	O
ten	O
motifs	O
(	O
I	B-structure_element
-	I-structure_element
X	I-structure_element
)	O
in	O
cytosine	B-protein_type
MTases	I-protein_type
and	O
nine	O
motifs	O
(	O
I	B-structure_element
-	I-structure_element
VIII	I-structure_element
and	O
X	B-structure_element
)	O
in	O
adenine	B-protein_type
MTases	I-protein_type
,	O
all	O
of	O
which	O
are	O
arranged	O
in	O
an	O
almost	O
constant	O
order	O
.	O

According	O
to	O
the	O
linear	O
arrangement	O
of	O
three	O
conserved	B-protein_state
domains	O
,	O
exocyclic	B-protein_type
amino	I-protein_type
MTases	I-protein_type
are	O
subdivided	O
into	O
six	O
groups	O
(	O
namely	O
α	B-protein_type
,	O
β	B-protein_type
,	O
γ	B-protein_type
,	O
ζ	B-protein_type
,	O
δ	B-protein_type
and	O
ε	B-protein_type
).	O

N6	B-protein_type
-	I-protein_type
adenine	I-protein_type
and	I-protein_type
N4	I-protein_type
-	I-protein_type
cytosine	I-protein_type
MTases	I-protein_type
,	O
in	O
particular	O
,	O
are	O
closely	O
related	O
by	O
sharing	O
common	O
structural	O
features	O
.	O

Despite	O
the	O
considerable	O
similarity	O
among	O
bacterial	B-taxonomy_domain
MTases	B-protein_type
,	O
some	O
differences	O
were	O
observed	O
among	O
the	O
enzymes	O
from	O
various	O
species	O
.	O

For	O
example	O
,	O
the	O
structural	O
regions	O
of	O
MTases	B-protein_type
beyond	O
the	O
catalytic	B-structure_element
domain	I-structure_element
are	O
rather	O
variable	O
,	O
such	O
as	O
the	O
C	B-structure_element
-	I-structure_element
terminal	I-structure_element
domain	I-structure_element
of	O
M	B-protein
.	I-protein
TaqI	I-protein
,	O
the	O
extended	O
arm	O
of	O
M	B-protein
.	I-protein
MboIIA	I-protein
and	O
M	B-protein
.	I-protein
RsrI	I-protein
,	O
the	O
helix	B-structure_element
bundle	I-structure_element
of	O
EcoDam	B-protein
,	O
and	O
so	O
on	O
.	O

DNA	B-ptm
methylation	I-ptm
is	O
thought	O
to	O
influence	O
bacterial	B-taxonomy_domain
virulence	O
.	O

DNA	B-protein_type
adenine	I-protein_type
methyltransferase	I-protein_type
has	O
been	O
shown	O
to	O
play	O
a	O
crucial	O
role	O
in	O
colonization	O
of	O
deep	O
tissue	O
sites	O
in	O
Salmonella	B-species
typhimurium	I-species
and	O
Aeromonas	B-species
hydrophila	I-species
.	O

Importantly	O
,	O
DNA	B-ptm
adenine	I-ptm
methylation	I-ptm
is	O
a	O
global	O
regulator	O
of	O
genes	O
expressed	O
during	O
infection	O
and	O
inhibitors	O
of	O
DNA	B-ptm
adenine	I-ptm
methylation	I-ptm
are	O
likely	O
to	O
have	O
a	O
broad	O
antimicrobial	O
action	O
.	O

Dam	B-protein_type
was	O
considered	O
a	O
promising	O
target	O
for	O
antimicrobial	O
drug	O
development	O
.	O

Helicobacter	B-species
pylori	I-species
is	O
a	O
Gram	B-taxonomy_domain
-	I-taxonomy_domain
negative	I-taxonomy_domain
bacterium	I-taxonomy_domain
that	O
persistently	O
colonizes	O
in	O
human	B-species
stomach	O
worldwide	O
.	O

H	B-species
.	I-species
pylori	I-species
is	O
involved	O
in	O
90	O
%	O
of	O
all	O
gastric	O
malignancies	O
,	O
infecting	O
nearly	O
50	O
%	O
of	O
the	O
world	O
'	O
s	O
population	O
and	O
is	O
the	O
most	O
crucial	O
etiologic	O
agent	O
for	O
gastric	O
adenocarcinoma	O
.	O

H	B-species
.	I-species
pylori	I-species
strains	O
possess	O
a	O
few	O
R	O
-	O
M	O
systems	O
like	O
other	O
bacteria	B-taxonomy_domain
to	O
function	O
as	O
defensive	O
systems	O
.	O

H	B-species
.	I-species
pylori	I-species
26695	I-species
,	O
for	O
example	O
,	O
has	O
23	O
R	O
-	O
M	O
systems	O
.	O

Methyltransferases	B-protein_type
were	O
suggested	O
to	O
be	O
involved	O
in	O
H	B-species
.	I-species
pylori	I-species
pathogenicity	O
.	O

M1	B-protein
.	I-protein
HpyAVI	I-protein
is	O
a	O
DNA	B-protein_type
adenine	I-protein_type
MTase	I-protein_type
that	O
belongs	O
to	O
the	O
type	O
II	O
R	O
-	O
M	O
system	O
.	O

This	O
system	O
contains	O
two	O
DNA	B-protein_type
MTases	I-protein_type
named	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
and	O
M2	B-protein
.	I-protein
HpyAVI	I-protein
,	O
and	O
a	O
putative	O
restriction	B-protein_type
enzyme	I-protein_type
.	O

M1	B-protein
.	I-protein
HpyAVI	I-protein
encoded	O
by	O
ORF	O
hp0050	B-gene
is	O
an	O
N6	B-protein_type
-	I-protein_type
adenine	I-protein_type
methyltransferase	I-protein_type
belonging	O
to	O
the	O
β	B-protein_type
-	I-protein_type
class	I-protein_type
MTase	I-protein_type
.	O

It	O
has	O
been	O
reported	O
recently	O
that	O
this	O
enzyme	O
recognizes	O
the	O
sequence	O
of	O
5	B-chemical
′-	I-chemical
GAGG	I-chemical
-	I-chemical
3	I-chemical
′,	I-chemical
5	B-chemical
′-	I-chemical
GGAG	I-chemical
-	I-chemical
3	I-chemical
′	I-chemical
or	O
5	B-chemical
′-	I-chemical
GAAG	I-chemical
-	I-chemical
3	I-chemical
′	I-chemical
and	O
methylates	O
adenines	B-residue_name
in	O
these	O
sequences	O
.	O

Given	O
that	O
methylation	B-ptm
of	O
two	O
adjacent	O
adenines	B-residue_name
on	O
the	O
same	O
strand	O
have	O
never	O
been	O
observed	O
for	O
other	O
N6	B-protein_type
-	I-protein_type
adenine	I-protein_type
MTases	I-protein_type
,	O
the	O
methylation	B-ptm
activity	O
on	O
5	B-chemical
′-	I-chemical
GAAG	I-chemical
-	I-chemical
3	I-chemical
′	I-chemical
seems	O
to	O
be	O
a	O
unique	O
feature	O
of	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
,	O
compared	O
with	O
the	O
homologs	O
from	O
other	O
strains	O
of	O
H	B-species
.	I-species
pylori	I-species
which	O
is	O
able	O
to	O
methylate	O
only	O
5	B-chemical
′-	I-chemical
GAGG	I-chemical
-	I-chemical
3	I-chemical
′.	I-chemical
The	O
structural	O
basis	O
and	O
the	O
catalytic	O
mechanism	O
underlying	O
such	O
a	O
distinct	O
activity	O
are	O
not	O
well	O
understood	O
due	O
to	O
the	O
lack	O
of	O
an	O
available	O
3D	O
structure	B-evidence
of	O
this	O
enzyme	O
.	O

Here	O
,	O
we	O
report	O
the	O
crystal	B-evidence
structure	I-evidence
of	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
from	O
H	B-species
.	I-species
pylori	I-species
26695	I-species
,	O
which	O
is	O
the	O
first	O
determined	O
N6	B-protein_type
-	I-protein_type
adenine	I-protein_type
MTase	I-protein_type
structure	B-evidence
in	O
H	B-species
.	I-species
pylori	I-species
.	O

The	O
structure	B-evidence
reveals	O
a	O
similar	O
architecture	O
as	O
the	O
canonical	O
fold	O
of	O
homologous	O
proteins	O
,	O
but	O
displays	O
several	O
differences	O
in	O
the	O
loop	B-structure_element
regions	O
and	O
TRD	B-structure_element
.	O

Based	O
on	O
structural	B-experimental_method
and	I-experimental_method
biochemical	I-experimental_method
analyses	I-experimental_method
,	O
we	O
then	O
identified	O
two	O
conserved	B-protein_state
amino	O
acids	O
,	O
D29	B-residue_name_number
at	O
the	O
catalytic	B-site
site	I-site
and	O
E216	B-residue_name_number
close	O
to	O
the	O
C	O
-	O
terminus	O
,	O
as	O
crucial	O
residues	O
for	O
cofactor	O
binding	O
and	O
methyltransferase	B-protein_type
activity	O
of	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
.	O

In	O
addition	O
,	O
a	O
non	B-protein_state
-	I-protein_state
conserved	I-protein_state
amino	O
acid	O
,	O
P41	B-residue_name_number
,	O
seems	O
to	O
play	O
a	O
key	O
role	O
in	O
substrate	O
recognition	O
.	O

Overall	O
structure	B-evidence

Recombinant	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
M1	B-protein
.	I-protein
HpyAVI	I-protein
was	O
produced	O
as	O
a	O
soluble	O
protein	O
in	O
Escherichia	B-species
coli	I-species
,	O
but	O
was	O
quite	O
unstable	O
and	O
tended	O
to	O
aggregate	O
in	O
low	O
salt	O
environment	O
.	O

The	O
protein	O
,	O
however	O
,	O
remained	O
fully	O
soluble	O
in	O
a	O
buffer	O
containing	O
higher	O
concentration	O
of	O
sodium	B-chemical
chloride	I-chemical
(>	O
300	O
mM	O
),	O
which	O
prompted	O
that	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
is	O
likely	O
a	O
halophilic	B-protein_state
protein	O
.	O

The	O
cofactor	B-protein_state
-	I-protein_state
free	I-protein_state
and	O
AdoMet	B-protein_state
-	I-protein_state
bound	I-protein_state
proteins	O
were	O
crystallized	B-experimental_method
at	O
different	O
conditions	O
.	O

Both	O
structures	B-evidence
were	O
determined	O
by	O
means	O
of	O
molecular	B-experimental_method
replacement	I-experimental_method
,	O
and	O
refined	O
to	O
3	O
.	O
0	O
Å	O
and	O
3	O
.	O
1	O
Å	O
,	O
respectively	O
.	O

Statistics	O
of	O
X	B-experimental_method
-	I-experimental_method
ray	I-experimental_method
data	I-experimental_method
collection	I-experimental_method
and	O
structure	B-experimental_method
refinement	I-experimental_method
were	O
summarized	O
in	O
Table	O
1	O
.	O

Data	O
collection	O
and	O
structure	B-evidence
refinement	I-evidence
statistics	I-evidence
of	O
M1	B-protein
.	I-protein
HpyAVI	I-protein

M1	B-protein
.	I-protein
HpyAVI	I-protein
M1	B-complex_assembly
.	I-complex_assembly
HpyAVI	I-complex_assembly
-	I-complex_assembly
AdoMet	I-complex_assembly
complex	O
Data	O
collection	O
Wavelength	O
(	O
Å	O
)	O
1	O
.	O
0000	O
0	O
.	O
97772	O
Space	O
group	O
P43212	O
P65	O
Unit	O
-	O
cell	O
parameters	O
(	O
Å	O
,	O
˚)	O
a	O
=	O
b	O
=	O
69	O
.	O
73	O
,	O
c	O
=	O
532	O
.	O
75α	O
=	O
β	O
=	O
γ	O
=	O
90	O
a	O
=	O
b	O
=	O
135	O
.	O
60	O
,	O
c	O
=	O
265	O
.	O
15α	O
=	O
β	O
=	O
90	O
,	O
γ	O
=	O
120	O
Resolution	O
range	O
(	O
Å	O
)	O
a	O
49	O
.	O
09	O
-	O
3	O
.	O
00	O
(	O
3	O
.	O
09	O
-	O
3	O
.	O
00	O
)	O
48	O
.	O
91	O
-	O
3	O
.	O
10	O
(	O
3	O
.	O
18	O
-	O
3	O
.	O
10	O
)	O
Unique	O
reflections	O
a	O
27243	O
49833	O
Multiplicity	O
a	O
3	O
.	O
7	O
(	O
3	O
.	O
8	O
)	O
5	O
.	O
6	O
(	O
4	O
.	O
0	O
)	O
Completeness	O
(%)	O
a	O
98	O
.	O
7	O
(	O
98	O
.	O
9	O
)	O
99	O
.	O
7	O
(	O
97	O
.	O
8	O
)	O
Mean	O
I	O
/	O
δ	O
(	O
I	O
)	O
a	O
12	O
.	O
1	O
(	O
3	O
.	O
4	O
)	O
14	O
.	O
0	O
(	O
1	O
.	O
9	O
)	O
Solvent	O
content	O
(%)	O
58	O
.	O
67	O
61	O
.	O
96	O
Rmergea	O
0	O
.	O
073	O
(	O
0	O
.	O
378	O
)	O
0	O
.	O
106	O
(	O
0	O
.	O
769	O
)	O
Structure	O
refinement	O
Rwork	O
0	O
.	O
251	O
0	O
.	O
221	O
Rfree	O
0	O
.	O
308	O
0	O
.	O
276	O
R	B-evidence
.	I-evidence
m	I-evidence
.	I-evidence
s	I-evidence
.	I-evidence
d	I-evidence
.,	O
bond	O
lengths	O
(	O
Å	O
)	O
0	O
.	O
007	O
0	O
.	O
007	O
R	B-evidence
.	I-evidence
m	I-evidence
.	I-evidence
s	I-evidence
.	I-evidence
d	I-evidence
.,	O
bond	O
angles	O
(˚)	O
1	O
.	O
408	O
1	O
.	O
651	O
Ramachandran	O
plot	O
Favoured	O
region	O
(%)	O
89	O
.	O
44	O
91	O
.	O
44	O
Allowed	O
region	O
(%)	O
9	O
.	O
58	O
7	O
.	O
11	O
Outliers	O
(%)	O
0	O
.	O
99	O
1	O
.	O
45	O

Four	O
and	O
eight	O
protein	O
monomers	B-oligomeric_state
resided	O
in	O
the	O
asymmetric	O
units	O
of	O
the	O
two	O
crystal	B-evidence
structures	I-evidence
.	O

Some	O
amino	O
acids	O
,	O
particularly	O
those	O
within	O
two	O
loops	B-structure_element
(	O
residues	O
32	B-residue_range
-	I-residue_range
61	I-residue_range
and	O
152	B-residue_range
-	I-residue_range
172	I-residue_range
)	O
in	O
both	O
structures	B-evidence
,	O
were	O
poorly	O
defined	O
in	O
electron	B-evidence
density	I-evidence
and	O
had	O
to	O
be	O
omitted	O
from	O
the	O
refined	O
models	O
.	O

The	O
two	O
structures	B-evidence
are	O
very	O
similar	O
to	O
each	O
other	O
(	O
Figure	O
1	O
)	O
and	O
could	O
be	O
well	O
overlaid	O
with	O
an	O
RMSD	B-evidence
of	O
0	O
.	O
76	O
Å	O
on	O
191	O
Cα	O
atoms	O
.	O

The	O
overall	O
architecture	O
of	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
revealed	O
in	O
these	O
structures	B-evidence
resembles	O
the	O
AdoMet	B-protein_type
-	I-protein_type
dependent	I-protein_type
MTase	I-protein_type
fold	O
in	O
which	O
a	O
twisted	O
seven	O
-	O
stranded	O
β	B-structure_element
-	I-structure_element
sheet	I-structure_element
flanked	O
by	O
six	O
α	B-structure_element
-	I-structure_element
helices	I-structure_element
forms	O
the	O
structural	O
core	O
.	O

Like	O
the	O
reported	O
structures	B-evidence
of	O
the	O
larger	O
domain	O
of	O
MTases	B-protein_type
,	O
three	O
helices	B-structure_element
(	O
αA	B-structure_element
,	O
αB	B-structure_element
and	O
αZ	B-structure_element
)	O
are	O
located	O
at	O
one	O
face	O
of	O
the	O
central	O
β	B-structure_element
-	I-structure_element
sheet	I-structure_element
,	O
while	O
the	O
other	O
three	O
αD	B-structure_element
,	O
αE	B-structure_element
and	O
αC	B-structure_element
sit	O
at	O
the	O
other	O
side	O
.	O

All	O
these	O
conserved	B-protein_state
structural	O
motifs	O
form	O
a	O
typical	O
α	B-structure_element
/	I-structure_element
β	I-structure_element
Rossmann	I-structure_element
fold	I-structure_element
.	O

The	O
catalytic	B-structure_element
motif	I-structure_element
DPPY	B-structure_element
lies	O
in	O
a	O
loop	B-structure_element
connecting	O
αD	B-structure_element
and	O
β4	B-structure_element
,	O
and	O
the	O
cofactor	O
AdoMet	B-chemical
binds	O
in	O
a	O
neighboring	O
cavity	B-site
.	O

The	O
loop	B-structure_element
(	O
residues	O
136	B-residue_range
-	I-residue_range
166	I-residue_range
)	O
located	O
between	O
β7	B-structure_element
and	O
αZ	B-structure_element
corresponds	O
to	O
a	O
highly	B-protein_state
diverse	I-protein_state
region	O
in	O
other	O
MTases	B-protein_type
that	O
is	O
involved	O
in	O
target	O
DNA	B-chemical
recognition	O
.	O

The	O
hairpin	B-structure_element
loop	I-structure_element
(	O
residues	O
101	B-residue_range
-	I-residue_range
133	I-residue_range
)	O
bridging	O
β6	B-structure_element
and	O
β7	B-structure_element
,	O
which	O
is	O
proposed	O
to	O
bind	O
DNA	B-chemical
in	O
the	O
minor	B-structure_element
groove	I-structure_element
,	O
displays	O
a	O
similar	O
conformation	O
as	O
those	O
observed	O
in	O
M	B-protein
.	I-protein
MboIIA	I-protein
,	O
M	B-protein
.	I-protein
RsrI	I-protein
and	O
M	B-protein
.	I-protein
pvuII	I-protein
.	O

The	O
missing	B-protein_state
loop	B-structure_element
(	O
residues	O
33	B-residue_range
-	I-residue_range
58	I-residue_range
)	O
in	O
the	O
structure	B-evidence
of	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
corresponds	O
to	O
loop	B-structure_element
I	I-structure_element
in	O
M	B-protein
.	I-protein
TaqI	I-protein
,	O
which	O
was	O
also	O
invisible	O
in	O
a	O
structure	B-evidence
without	B-protein_state
DNA	I-protein_state
.	O

This	O
loop	B-structure_element
,	O
however	O
,	O
was	O
well	B-protein_state
ordered	I-protein_state
in	O
an	O
M	B-evidence
.	I-evidence
TaqI	I-evidence
-	I-evidence
DNA	I-evidence
complex	I-evidence
structure	I-evidence
and	O
was	O
shown	O
to	O
play	O
a	O
crucial	O
role	O
in	O
DNA	B-ptm
methylation	I-ptm
by	O
contacting	O
the	O
flipping	O
adenine	B-residue_name
and	O
recognizing	O
specific	O
DNA	B-chemical
sequence	O
.	O

Overall	O
structure	B-evidence
of	O
M1	B-protein
.	I-protein
HpyAVI	I-protein

A	O
.	O
Free	B-protein_state
form	O
B	O
.	O
AdoMet	B-protein_state
-	I-protein_state
bound	I-protein_state
form	O
.	O

Ribbon	O
diagram	O
of	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
resembles	O
an	O
“	O
AdoMet	B-protein_type
-	I-protein_type
dependent	I-protein_type
MTase	I-protein_type
fold	O
”,	O
a	O
mixed	O
seven	O
-	O
stranded	O
β	B-structure_element
-	I-structure_element
sheet	I-structure_element
flanked	O
by	O
six	O
α	B-structure_element
-	I-structure_element
helices	I-structure_element
,	O
αA	B-structure_element
,	O
αB	B-structure_element
,	O
αZ	B-structure_element
on	O
one	O
side	O
and	O
αD	B-structure_element
,	O
αE	B-structure_element
,	O
αC	B-structure_element
on	O
the	O
other	O
side	O
,	O
the	O
cofactor	O
AdoMet	B-chemical
is	O
bound	B-protein_state
in	I-protein_state
a	O
cavity	B-site
near	O
the	O
conserved	B-protein_state
enzyme	O
activity	O
motif	O
DPPY	B-structure_element
.	O

The	O
α	B-structure_element
-	I-structure_element
helices	I-structure_element
and	O
β	B-structure_element
-	I-structure_element
strands	I-structure_element
are	O
labelled	O
and	O
numbered	O
according	O
to	O
the	O
commonly	O
numbering	O
rule	O
for	O
the	O
known	O
MTases	B-protein_type
.	O

The	O
AdoMet	B-chemical
molecule	O
is	O
shown	O
in	O
green	O
.	O

Dimeric	B-oligomeric_state
state	O
of	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
in	O
crystal	B-evidence
and	O
solution	B-experimental_method

Previous	O
studies	O
showed	O
that	O
some	O
DNA	B-protein_type
MTases	I-protein_type
,	O
e	O
.	O
g	O
.	O
M	B-protein
.	I-protein
BamHI	I-protein
and	O
M	B-protein
.	I-protein
EcoRI	I-protein
,	O
exist	O
as	O
monomer	B-oligomeric_state
in	O
solution	O
,	O
in	O
agreement	O
with	O
the	O
fact	O
that	O
a	O
DNA	B-chemical
substrate	O
for	O
a	O
typical	O
MTase	B-protein_type
is	O
hemimethylated	B-protein_state
and	O
therefore	O
needs	O
only	O
a	O
single	O
methylation	B-ptm
event	O
to	O
convert	O
it	O
into	O
a	O
fully	B-protein_state
methylated	I-protein_state
state	O
.	O

Increasing	O
number	O
of	O
dimeric	B-oligomeric_state
DNA	B-protein_type
MTases	I-protein_type
,	O
however	O
,	O
has	O
been	O
identified	O
from	O
later	O
studies	O
.	O

For	O
instance	O
,	O
M	B-protein
.	I-protein
DpnII	I-protein
,	O
M	B-protein
.	I-protein
RsrI	I-protein
,	O
M	B-protein
.	I-protein
KpnI	I-protein
,	O
and	O
M	B-protein
.	I-protein
MboIIA	I-protein
have	O
been	O
found	O
as	O
dimers	B-oligomeric_state
in	O
solution	O
.	O

In	O
addition	O
,	O
several	O
MTases	B-protein_type
including	O
M	B-protein
.	I-protein
MboIIA	I-protein
,	O
M	B-protein
.	I-protein
RsrI	I-protein
and	O
TTH0409	B-protein
form	O
tightly	O
associated	O
dimers	B-oligomeric_state
in	O
crystal	B-evidence
structures	I-evidence
.	O

Nonetheless	O
,	O
some	O
DNA	B-protein_type
MTases	I-protein_type
such	O
as	O
M	B-protein
.	I-protein
CcrMI	I-protein
and	O
the	O
Bacillus	B-species
amyloliquefaciens	I-species
MTase	B-protein_type
dissociate	O
from	O
dimer	B-oligomeric_state
into	O
monomer	B-oligomeric_state
upon	O
DNA	B-chemical
-	O
binding	O
.	O

According	O
to	O
the	O
arrangement	O
of	O
the	O
three	O
conserved	B-protein_state
domains	O
,	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
belongs	O
to	O
the	O
β	B-protein_type
-	I-protein_type
subgroup	I-protein_type
,	O
in	O
which	O
a	O
conserved	B-protein_state
motif	O
NXXTX9	B-structure_element
−	I-structure_element
11AXRXFSXXHX4WX6	I-structure_element
−	I-structure_element
9	I-structure_element
YXFXLX3RX9	I-structure_element
−	I-structure_element
26NPX1	I-structure_element
−	I-structure_element
6NVWX29	I-structure_element
−	I-structure_element
34A	I-structure_element
has	O
been	O
identified	O
at	O
the	O
dimerization	B-site
interface	I-site
in	O
crystal	B-evidence
structures	I-evidence
.	O

Most	O
of	O
conserved	B-protein_state
amino	O
acids	O
within	O
that	O
motif	O
are	O
present	O
in	O
the	O
sequence	O
of	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
(	O
Figure	O
2A	O
),	O
implying	O
dimerization	B-oligomeric_state
of	O
this	O
protein	O
.	O

In	O
agreement	O
,	O
a	O
dimer	B-oligomeric_state
of	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
was	O
observed	O
in	O
our	O
crystal	B-evidence
structures	I-evidence
with	O
the	O
two	O
monomers	B-oligomeric_state
related	O
by	O
a	O
two	O
-	O
fold	O
axis	O
(	O
Figure	O
2B	O
and	O
2C	O
).	O

An	O
area	O
of	O
~	O
1900	O
Å2	O
was	O
buried	O
at	O
the	O
dimeric	B-site
interface	I-site
,	O
taking	O
up	O
ca	O
17	O
%	O
of	O
the	O
total	O
area	O
.	O

The	O
dimeric	B-oligomeric_state
architecture	O
was	O
greatly	O
stabilized	O
by	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
and	O
salt	B-bond_interaction
bridges	I-bond_interaction
formed	O
among	O
residues	O
R86	B-residue_name_number
,	O
D93	B-residue_name_number
and	O
E96	B-residue_name_number
.	O

In	O
addition	O
,	O
comparison	O
of	O
the	O
dimer	B-oligomeric_state
structure	B-evidence
of	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
with	O
some	O
other	O
β	B-protein_type
-	I-protein_type
class	I-protein_type
MTases	I-protein_type
(	O
M1	B-protein
.	I-protein
MboIIA	I-protein
,	O
M	B-protein
.	I-protein
RsrI	I-protein
and	O
TTHA0409	B-protein
)	O
suggested	O
that	O
the	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
dimer	B-oligomeric_state
organized	O
in	O
a	O
similar	O
form	O
as	O
others	O
(	O
Figure	O
S3	O
).	O

M1	B-protein
.	I-protein
HpyAVI	I-protein
exists	O
as	O
dimer	B-oligomeric_state
in	O
crystal	B-evidence
and	O
solution	O

A	O
.	O
A	O
conserved	B-protein_state
interface	B-site
area	I-site
of	O
β	B-protein_type
-	I-protein_type
class	I-protein_type
MTases	I-protein_type
is	O
defined	O
in	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
.	O

Residues	O
that	O
involved	O
are	O
signed	O
in	O
red	O
color	O
;	O
Dimerization	B-oligomeric_state
of	O
free	B-protein_state
-	O
form	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
B	O
.	O
and	O
cofactor	B-protein_state
-	I-protein_state
bound	I-protein_state
M1	B-protein
.	I-protein
HpyAVI	I-protein
C	O
.	O
The	O
two	O
monomers	B-oligomeric_state
are	O
marked	O
in	O
green	O
and	O
blue	O
,	O
AdoMet	B-chemical
molecules	O
are	O
marked	O
in	O
magenta	O
.	O

D	O
.	O
Gel	B-experimental_method
-	I-experimental_method
filtration	I-experimental_method
analysis	I-experimental_method
revealed	O
that	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
exist	O
as	O
a	O
dimer	B-oligomeric_state
in	O
solution	O
.	O

FPLC	B-experimental_method
system	O
coupled	O
to	O
a	O
Superdex	O
75	O
10	O
/	O
300	O
column	O
.	O

Elution	B-evidence
profiles	I-evidence
at	O
280	O
nm	O
(	O
blue	O
)	O
and	O
260	O
nm	O
(	O
red	O
)	O
are	O
:	O
different	O
concentration	O
(	O
0	O
.	O
05	O
,	O
0	O
.	O
1	O
,	O
0	O
.	O
2	O
,	O
0	O
.	O
5	O
mg	O
/	O
ml	O
)	O
of	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
protein	O
.	O

To	O
probe	O
the	O
oligomeric	O
form	O
of	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
in	O
solution	O
,	O
different	O
concentrations	O
of	O
purified	O
enzyme	O
was	O
loaded	O
onto	O
a	O
Superdex	O
75	O
10	O
/	O
300	O
column	O
.	O

The	O
protein	O
was	O
eluted	O
at	O
~	O
10	O
ml	O
regardless	O
of	O
the	O
protein	O
concentrations	O
,	O
corresponding	O
to	O
a	O
dimeric	B-oligomeric_state
molecular	B-evidence
mass	I-evidence
of	O
54	O
kDa	O
(	O
Figure	O
2D	O
).	O

Our	O
results	O
clearly	O
showed	O
that	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
forms	O
a	O
dimer	B-oligomeric_state
in	O
both	O
crystal	B-evidence
and	O
solution	O
as	O
other	O
β	B-protein_type
-	I-protein_type
class	I-protein_type
MTases	I-protein_type
,	O
which	O
however	O
disagrees	O
with	O
a	O
previous	O
investigation	O
using	O
dynamic	B-experimental_method
light	I-experimental_method
scattering	I-experimental_method
(	O
DLS	B-experimental_method
)	O
measurement	O
and	O
gel	B-experimental_method
-	I-experimental_method
filtration	I-experimental_method
chromatography	I-experimental_method
,	O
suggesting	O
that	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
is	O
taking	O
a	O
monomeric	B-oligomeric_state
state	O
in	O
solution	O
.	O

This	O
variance	O
might	O
be	O
caused	O
by	O
an	O
addition	O
of	O
100	O
mM	O
arginine	B-chemical
before	O
cell	O
lysis	O
to	O
keep	O
protein	O
solubility	O
and	O
also	O
by	O
later	O
replacement	O
of	O
arginine	B-chemical
with	O
30	O
%	O
glycerol	B-chemical
by	O
dialysis	O
.	O

Structure	B-experimental_method
comparisons	I-experimental_method

As	O
a	O
β	B-protein_type
-	I-protein_type
class	I-protein_type
N6	I-protein_type
adenine	I-protein_type
MTase	I-protein_type
,	O
the	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
structure	B-evidence
displayed	O
a	O
good	O
similarity	O
with	O
M	B-protein
.	I-protein
MboIIA	I-protein
(	O
PDB	O
ID	O
1G60	O
)	O
and	O
M	B-protein
.	I-protein
RsrI	I-protein
(	O
PDB	O
ID	O
1NW7	O
),	O
which	O
are	O
falling	O
into	O
the	O
same	O
subgroup	O
.	O

Superimposition	B-experimental_method
of	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
onto	O
them	O
gave	O
RMSDs	B-evidence
of	O
1	O
.	O
63	O
Å	O
and	O
1	O
.	O
9	O
Å	O
on	O
168	O
and	O
190	O
Cα	O
atoms	O
,	O
respectively	O
.	O

The	O
most	O
striking	O
structural	O
difference	O
was	O
found	O
to	O
locate	O
on	O
the	O
TRD	B-structure_element
region	O
(	O
residues	O
133	B-residue_range
-	I-residue_range
163	I-residue_range
in	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
)	O
(	O
Figure	O
3A	O
–	O
3C	O
),	O
where	O
the	O
secondary	O
structures	O
vary	O
among	O
these	O
structures	O
.	O

By	O
comparison	O
with	O
the	O
other	O
two	O
enzymes	O
that	O
possess	O
protruding	O
arms	O
containing	O
several	O
α	B-structure_element
-	I-structure_element
helices	I-structure_element
and	O
/	O
or	O
β	B-structure_element
-	I-structure_element
strands	I-structure_element
,	O
the	O
TRD	B-structure_element
of	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
is	O
much	O
shorter	O
in	O
length	O
(	O
Figure	O
S1	O
),	O
wrapping	O
more	O
closely	O
around	O
the	O
structural	O
core	O
and	O
lacking	B-protein_state
apparent	O
secondary	O
structures	O
.	O

Given	O
the	O
proposed	O
role	O
of	O
the	O
TRD	B-structure_element
for	O
DNA	B-chemical
interaction	O
at	O
the	O
major	B-structure_element
groove	I-structure_element
,	O
some	O
differences	O
of	O
DNA	B-chemical
recognition	O
mode	O
can	O
be	O
expected	O
.	O

Another	O
difference	O
locates	O
at	O
the	O
highly	B-protein_state
flexible	I-protein_state
loop	B-structure_element
between	O
β4	B-structure_element
and	O
αD	B-structure_element
(	O
residues	O
33	B-residue_range
-	I-residue_range
58	I-residue_range
)	O
of	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
,	O
which	O
was	O
invisible	O
in	O
our	O
structures	B-evidence
but	O
present	O
in	O
the	O
structures	B-evidence
of	O
M	B-protein
.	I-protein
MboIIA	I-protein
and	O
M	B-protein
.	I-protein
RsrI	I-protein
.	O
Sequence	B-experimental_method
alignment	I-experimental_method
revealed	O
that	O
this	O
region	O
of	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
was	O
longer	O
than	O
its	O
counterparts	O
by	O
13	O
and	O
16	O
amino	O
acids	O
respectively	O
,	O
which	O
likely	O
renders	O
the	O
H	B-species
.	I-species
pylori	I-species
enzyme	O
more	O
flexible	B-protein_state
.	O

Structural	B-experimental_method
comparisons	I-experimental_method
between	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
and	O
other	O
DNA	B-protein_type
MTases	I-protein_type

A	O
.	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
;	O
B	O
.	O
M	B-protein
.	I-protein
MboIIA	I-protein
;	O
C	O
.	O
M	B-protein
.	I-protein
RsrI	I-protein
;	O
D	O
.	O
TTHA0409	B-protein
;	O
E	O
.	O
DpnM	B-protein
;	O
F	O
.	O
M	B-protein
.	I-protein
TaqI	I-protein
.	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
possesses	O
only	O
a	O
long	B-protein_state
disorder	I-protein_state
TRD	B-structure_element
region	O
,	O
compared	O
with	O
the	O
structure	B-protein_state
-	I-protein_state
rich	I-protein_state
TRD	B-structure_element
of	O
M	B-protein
.	I-protein
MboIIA	I-protein
,	O
M	B-protein
.	I-protein
RsrI	I-protein
and	O
TTHA0409	B-protein
,	O
or	O
the	O
extra	O
DNA	B-structure_element
-	I-structure_element
binding	I-structure_element
domain	I-structure_element
of	O
DpnM	B-protein
and	O
M	B-protein
.	I-protein
TaqI	I-protein
.	O
The	O
core	O
structure	O
is	O
in	O
cyan	O
;	O
TRD	B-structure_element
of	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
,	O
M	B-protein
.	I-protein
MboIIA	I-protein
,	O
M	B-protein
.	I-protein
RsrI	I-protein
and	O
TTHA0409	B-protein
is	O
in	O
red	O
;	O
The	O
region	O
between	O
β4	B-structure_element
and	O
αD	B-structure_element
of	O
M	B-protein
.	I-protein
MboIIA	I-protein
and	O
M	B-protein
.	I-protein
RsrI	I-protein
is	O
in	O
green	O
;	O
DNA	B-structure_element
-	I-structure_element
binding	I-structure_element
domain	I-structure_element
of	O
DpnM	B-protein
is	O
in	O
magenta	O
;	O
The	O
C	B-structure_element
-	I-structure_element
terminal	I-structure_element
domain	I-structure_element
of	O
M	B-protein
.	I-protein
TaqI	I-protein
is	O
in	O
orange	O
.	O

Structural	B-experimental_method
comparison	I-experimental_method
between	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
and	O
a	O
putative	O
β	B-protein_type
-	I-protein_type
class	I-protein_type
N4	I-protein_type
cytosine	I-protein_type
MTase	I-protein_type
named	O
TTHA0409	B-protein
(	O
PDB	O
ID	O
2ZIF	O
)	O
showed	O
a	O
good	O
similarity	O
as	O
well	O
,	O
giving	O
an	O
RMSD	B-evidence
of	O
1	O
.	O
73	O
Å	O
on	O
164	O
Cα	O
atoms	O
(	O
Figure	O
3D	O
).	O

Exactly	O
like	O
the	O
above	O
comparison	O
,	O
the	O
most	O
significant	O
difference	O
exists	O
in	O
the	O
TRD	B-structure_element
,	O
where	O
the	O
structures	B-evidence
vary	O
in	O
terms	O
of	O
length	O
and	O
presence	O
of	O
α	B-structure_element
-	I-structure_element
helices	I-structure_element
(	O
Figure	O
S1	O
).	O

M1	B-protein
.	I-protein
HpyAVI	I-protein
displayed	O
a	O
considerable	O
structural	O
dissimilarity	O
in	O
comparison	O
with	O
N6	B-protein_type
-	I-protein_type
adenine	I-protein_type
MTases	I-protein_type
from	O
other	O
subgroups	O
including	O
the	O
α	B-protein_type
-	I-protein_type
class	I-protein_type
DpnM	B-protein
(	O
PDB	O
ID	O
2DPM	O
)	O
and	O
the	O
γ	B-protein_type
-	I-protein_type
class	I-protein_type
M	B-protein
.	I-protein
TaqI	I-protein
(	O
PDB	O
ID	O
2ADM	O
).	O

Both	O
comparisons	O
gave	O
RMSDs	B-evidence
above	O
3	O
.	O
0	O
Å	O
(	O
Figure	O
3E	O
and	O
3F	O
).	O

These	O
two	O
enzymes	O
lack	B-protein_state
a	O
counterpart	B-structure_element
loop	I-structure_element
present	O
in	O
the	O
TRD	B-structure_element
of	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
,	O
but	O
instead	O
rely	O
on	O
an	O
extra	O
domain	O
for	O
DNA	B-chemical
binding	O
and	O
sequence	O
recognition	O
.	O

Collectively	O
,	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
possesses	O
a	O
long	B-protein_state
disordered	I-protein_state
TRD	B-structure_element
,	O
which	O
is	O
in	O
sharp	O
contrast	O
to	O
the	O
secondary	B-protein_state
structure	I-protein_state
-	I-protein_state
rich	I-protein_state
TRD	B-structure_element
in	O
other	O
β	B-protein_type
-	I-protein_type
class	I-protein_type
N6	I-protein_type
adenine	I-protein_type
or	I-protein_type
N4	I-protein_type
cytosine	I-protein_type
MTases	I-protein_type
or	O
the	O
extra	O
DNA	O
binding	O
domain	O
present	O
in	O
DNA	B-protein_type
MTases	I-protein_type
from	O
other	O
subgroups	O
.	O

This	O
striking	O
difference	O
may	O
be	O
a	O
significant	O
determinant	O
of	O
the	O
wider	O
substrate	O
spectrum	O
of	O
this	O
H	B-species
.	I-species
pylori	I-species
enzyme	O
.	O

AdoMet	B-site
-	I-site
binding	I-site
pocket	I-site

The	O
cofactor	B-site
binding	I-site
pocket	I-site
of	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
is	O
surrounded	O
by	O
residues	O
7	B-residue_range
-	I-residue_range
9	I-residue_range
,	O
29	B-residue_range
-	I-residue_range
31	I-residue_range
,	O
165	B-residue_range
-	I-residue_range
167	I-residue_range
,	O
216	B-residue_range
-	I-residue_range
218	I-residue_range
and	O
221	B-residue_number
(	O
Figure	O
4A	O
),	O
which	O
are	O
conserved	B-protein_state
among	O
most	O
of	O
DNA	B-protein_type
MTases	I-protein_type
.	O

A	O
hydrogen	B-bond_interaction
bond	I-bond_interaction
between	O
D29	B-residue_name_number
in	O
the	O
catalytic	B-structure_element
motif	I-structure_element
DPPY	B-structure_element
and	O
the	O
amino	O
group	O
of	O
bound	B-protein_state
AdoMet	B-chemical
is	O
preserved	O
as	O
other	O
MTase	B-protein_type
structures	B-evidence
.	O

Residues	O
D8	B-residue_name_number
and	O
A9	B-residue_name_number
from	O
hydrogen	B-bond_interaction
-	I-bond_interaction
bonds	I-bond_interaction
with	O
N6	O
and	O
N1	O
of	O
the	O
purine	B-chemical
ring	O
,	O
respectively	O
,	O
and	O
E216	B-residue_name_number
also	O
locates	O
at	O
hydrogen	B-bond_interaction
bonding	I-bond_interaction
distance	O
with	O
O2	O
′	O
and	O
O3	O
′	O
of	O
the	O
ribose	B-chemical
.	O

In	O
addition	O
,	O
H168	B-residue_name_number
,	O
T200	B-residue_name_number
and	O
S198	B-residue_name_number
contact	O
the	O
terminal	O
carboxyl	O
of	O
AdoMet	B-chemical
.	O

Superposition	B-experimental_method
of	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
with	O
the	O
five	O
structures	B-evidence
shown	O
in	O
Figure	O
3	O
reveals	O
that	O
the	O
orientation	O
of	O
cofactor	O
is	O
rather	B-protein_state
conserved	I-protein_state
except	O
for	O
M	B-protein
.	I-protein
TaqI	I-protein
(	O
Figure	O
4B	O
).	O

The	O
different	O
conformation	O
of	O
the	O
bound	B-protein_state
cofactor	O
observed	O
in	O
M	B-protein
.	I-protein
TaqI	I-protein
might	O
be	O
attributable	O
to	O
the	O
absence	B-protein_state
of	I-protein_state
corresponding	O
residues	O
of	O
the	O
conserved	B-protein_state
AdoMet	B-chemical
-	O
binding	O
motif	O
FXGXG	B-structure_element
in	O
that	O
structure	B-evidence
.	O

Structural	B-experimental_method
and	I-experimental_method
biochemical	I-experimental_method
analyses	I-experimental_method
define	O
two	O
conserved	B-protein_state
residues	O
D29	B-residue_name_number
and	O
E216	B-residue_name_number
to	O
be	O
the	O
key	O
sites	O
for	O
AdoMet	B-chemical
binding	O

A	O
.	O
The	O
cofactor	B-site
-	I-site
binding	I-site
cavity	I-site
of	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
.	O

Residues	O
(	O
yellow	O
)	O
that	O
form	O
direct	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
with	O
AdoMet	B-chemical
(	O
green	O
)	O
are	O
indicated	O
,	O
distance	O
of	O
the	O
hydrogen	B-bond_interaction
bond	I-bond_interaction
is	O
marked	O
.	O

B	O
.	O
Superposition	B-experimental_method
of	O
AdoMet	B-chemical
in	O
the	O
structures	B-evidence
of	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
(	O
green	O
),	O
DpnM	B-protein
(	O
yellow	O
)	O
and	O
M	B-protein
.	I-protein
TaqI	I-protein
(	O
orange	O
).	O

The	O
AdoMet	B-chemical
terminal	O
carboxyl	O
of	O
M	B-protein
.	I-protein
TaqI	I-protein
reveals	O
different	O
orientations	O
.	O

C	O
.	O
Cofactor	B-evidence
binding	I-evidence
affinity	I-evidence
of	O
wt	B-protein_state
-/	O
mutants	B-protein_state
M1	B-protein
.	I-protein
HpyAVI	I-protein
proteins	O
analyzed	O
by	O
microscale	B-experimental_method
thermophoresis	I-experimental_method
(	O
MST	B-experimental_method
).	O

The	O
binding	B-evidence
affinity	I-evidence
was	O
determined	O
between	O
fluorescently	O
labelled	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
protein	O
and	O
unlabeled	B-protein_state
AdoMet	B-chemical
.	O

AdoMet	B-chemical
(	O
15	O
nM	O
to	O
1	O
mM	O
)	O
was	O
titrated	B-experimental_method
into	O
a	O
fixed	O
concentration	O
of	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
wt	B-protein_state
/	O
mutant	B-protein_state
proteins	O
(	O
800	O
nM	O
).	O

The	O
dissociation	B-evidence
constant	I-evidence
(	O
KD	B-evidence
)	O
is	O
yielded	O
according	O
to	O
the	O
law	O
of	O
mass	O
action	O
from	O
the	O
isotherm	B-evidence
derived	O
of	O
the	O
raw	O
data	O
:	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
-	O
wt	B-protein_state
:	O
41	O
±	O
6	O
μM	O
;	O
M1	B-mutant
.	I-mutant
HpyAVI	I-mutant
-	I-mutant
D8A	I-mutant
:	O
212	O
±	O
11	O
μM	O
;	O
M1	B-mutant
.	I-mutant
HpyAVI	I-mutant
-	I-mutant
D29A	I-mutant
:	O
0	O
μM	O
;	O
M1	B-mutant
.	I-mutant
HpyAVI	I-mutant
-	I-mutant
H168A	I-mutant
:	O
471	O
±	O
51	O
μM	O
;	O
M1	B-mutant
.	I-mutant
HpyAVI	I-mutant
-	I-mutant
S198A	I-mutant
:	O
242	O
±	O
32	O
μM	O
;	O
M1	B-mutant
.	I-mutant
HpyAVI	I-mutant
-	I-mutant
T200A	I-mutant
:	O
252	O
±	O
28	O
μM	O
;	O
M1	B-mutant
.	I-mutant
HpyAVI	I-mutant
-	I-mutant
E216A	I-mutant
:	O
0	O
μM	O
.	O
Standard	O
for	O
three	O
replicates	O
is	O
indicated	O
.	O

D	O
.	O
DNA	B-protein_type
methyltransferase	I-protein_type
activity	O
of	O
wide	B-protein_state
type	I-protein_state
protein	O
and	O
the	O
mutants	B-protein_state
is	O
quantified	O
using	O
radioactive	B-experimental_method
assay	I-experimental_method
.	O

[	B-chemical
3H	I-chemical
]-	I-chemical
methyl	I-chemical
transferred	O
to	O
duplex	O
DNA	B-chemical
containing	O
5	B-chemical
′-	I-chemical
GAGG	I-chemical
-	I-chemical
3	I-chemical
′	I-chemical
was	O
quantified	O
by	O
Beckman	O
LS6500	O
for	O
10	O
min	O
,	O
experiments	O
were	O
repeated	O
for	O
three	O
times	O
and	O
data	O
were	O
corrected	O
by	O
subtraction	O
of	O
the	O
background	O
.	O

E	O
.	O
Superposition	B-experimental_method
of	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
(	O
green	O
)	O
with	O
M	B-protein
.	I-protein
MboIIA	I-protein
(	O
cyan	O
)	O
and	O
M	B-protein
.	I-protein
RsrI	I-protein
(	O
magenta	O
).	O

Residues	O
D29	B-residue_name_number
and	O
E216	B-residue_name_number
are	O
conserved	B-protein_state
through	O
all	O
the	O
DNA	B-protein_type
MTases	I-protein_type
mentioned	O
in	O
Figure	O
3	O
(	O
not	O
shown	O
in	O
Figure	O
4	O
).	O

To	O
confirm	O
the	O
key	O
residues	O
for	O
ligand	O
binding	O
,	O
we	O
prepared	O
a	O
series	O
of	O
single	B-experimental_method
mutants	I-experimental_method
by	O
replacing	B-experimental_method
D8	B-residue_name_number
,	O
D29	B-residue_name_number
,	O
H168	B-residue_name_number
,	O
S198	B-residue_name_number
,	O
T200	B-residue_name_number
,	O
E216	B-residue_name_number
with	O
alanine	B-residue_name
and	O
investigated	O
their	O
ligand	B-evidence
binding	I-evidence
affinity	I-evidence
using	O
microscale	B-experimental_method
thermophoresis	I-experimental_method
(	O
MST	B-experimental_method
)	O
assay	O
.	O

As	O
shown	O
in	O
Figure	O
4C	O
,	O
by	O
contrast	O
to	O
the	O
wild	B-protein_state
type	I-protein_state
enzyme	O
,	O
most	O
mutants	B-protein_state
displayed	O
variable	O
reduction	O
of	O
KD	B-evidence
value	O
,	O
among	O
them	O
the	O
D29A	B-mutant
and	O
E216A	B-mutant
mutants	B-protein_state
displayed	O
no	O
protein	B-evidence
-	I-evidence
AdoMet	I-evidence
affinity	I-evidence
at	O
all	O
.	O

The	O
results	O
suggested	O
that	O
the	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
formed	O
by	O
D29	B-residue_name_number
and	O
E216	B-residue_name_number
with	O
AdoMet	B-chemical
were	O
most	O
crucial	O
interactions	O
for	O
cofactor	O
binding	O
.	O

Mutation	B-experimental_method
of	O
the	O
two	O
residues	O
may	O
directly	O
prevent	O
the	O
methyl	B-chemical
transfer	O
reaction	O
of	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
.	O

The	O
importance	O
of	O
D29	B-residue_name_number
is	O
preserved	O
because	O
it	O
belongs	O
to	O
the	O
catalytic	B-site
active	I-site
site	I-site
DPPY	B-structure_element
,	O
but	O
the	O
residue	O
E216	B-residue_name_number
has	O
not	O
been	O
fully	O
investigated	O
even	O
being	O
a	O
conserved	B-protein_state
amino	B-chemical
acid	I-chemical
throughout	O
MTases	B-protein_type
(	O
Figure	O
4E	O
).	O

E216	B-residue_name_number
is	O
the	O
last	O
residue	O
of	O
β2	B-structure_element
,	O
which	O
contacts	O
the	O
two	O
hydroxyls	O
of	O
the	O
ribose	B-chemical
of	O
AdoMet	B-chemical
.	O

Replacement	B-experimental_method
of	O
this	O
residue	O
by	O
alanine	B-residue_name
completely	O
abolishes	O
the	O
key	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
for	O
AdoMet	B-chemical
-	O
binding	O
,	O
and	O
very	O
likely	O
blocks	O
the	O
methyl	B-chemical
transfer	O
reaction	O
.	O

To	O
confirm	O
this	O
notion	O
,	O
[	B-experimental_method
3H	I-experimental_method
]	I-experimental_method
AdoMet	I-experimental_method
radiological	I-experimental_method
assay	I-experimental_method
was	O
applied	O
to	O
quantify	O
the	O
methyl	B-chemical
transfer	O
activity	O
of	O
the	O
mutants	B-protein_state
.	O

As	O
shown	O
in	O
Figure	O
4D	O
,	O
the	O
result	O
of	O
radiological	B-experimental_method
assay	I-experimental_method
agreed	O
well	O
with	O
the	O
MST	B-experimental_method
measurement	O
.	O

The	O
D29A	B-mutant
and	O
E216A	B-mutant
mutants	B-protein_state
showed	O
little	O
or	O
no	O
methyl	B-chemical
transfer	O
activity	O
,	O
while	O
other	O
mutants	B-protein_state
exhibited	O
reduced	O
methyltransferase	B-protein_type
activity	O
.	O

As	O
mentioned	O
previously	O
,	O
FXGXG	B-structure_element
is	O
a	O
conserved	B-protein_state
AdoMet	B-chemical
-	O
binding	O
motif	O
of	O
DNA	B-protein_type
MTases	I-protein_type
.	O

We	O
also	O
made	O
mutants	B-protein_state
of	O
“	O
FMGSG	B-structure_element
”	O
to	O
alanine	B-residue_name
for	O
every	O
amino	B-chemical
acid	I-chemical
,	O
and	O
found	O
that	O
the	O
F195A	B-mutant
mutant	B-protein_state
was	O
insoluble	O
probably	O
due	O
to	O
decreasing	O
the	O
local	O
hydrophobicity	O
upon	O
this	O
mutation	O
.	O

We	O
subsequently	O
investigated	O
the	O
ligand	B-evidence
binding	I-evidence
affinity	I-evidence
and	O
methyl	B-chemical
transfer	O
reaction	O
of	O
the	O
other	O
mutants	B-protein_state
using	O
MST	B-experimental_method
and	O
a	O
radiological	B-experimental_method
assay	I-experimental_method
.	O

We	O
found	O
that	O
G197	B-residue_name_number
played	O
a	O
crucial	O
role	O
in	O
AdoMet	B-chemical
-	O
binding	O
,	O
while	O
mutagenesis	B-experimental_method
of	O
M196	B-residue_name_number
and	O
G199	B-residue_name_number
did	O
not	O
influence	O
cofactor	O
binding	O
and	O
catalytic	O
activity	O
(	O
Figure	O
S2A	O
and	O
B	O
).	O

G197	B-residue_name_number
is	O
a	O
conserved	B-protein_state
residue	O
throughout	O
the	O
DNA	B-protein_type
MTases	I-protein_type
,	O
and	O
replacing	B-experimental_method
by	O
alanine	B-residue_name
at	O
this	O
site	O
likely	O
change	O
the	O
local	O
conformation	O
of	O
cofactor	B-site
-	I-site
binding	I-site
pocket	I-site
.	O

Mutagenesis	B-experimental_method
on	O
this	O
glycine	B-residue_name
residue	O
in	O
M	B-protein
.	I-protein
EcoKI	I-protein
or	O
M	B-protein
.	I-protein
EcoP15I	I-protein
also	O
abolished	O
the	O
AdoMet	B-chemical
-	O
binding	O
activity	O
.	O

Although	O
mutational	B-experimental_method
study	I-experimental_method
could	O
not	O
tell	O
the	O
role	O
of	O
F195	B-residue_name_number
in	O
ligand	O
binding	O
due	O
to	O
the	O
insolubility	O
of	O
the	O
F195A	B-mutant
mutant	B-protein_state
,	O
structural	B-experimental_method
analysis	I-experimental_method
suggested	O
the	O
importance	O
of	O
this	O
residue	O
in	O
AdoMet	B-chemical
-	O
binding	O
.	O

The	O
phenyl	O
ring	O
of	O
F195	B-residue_name_number
forms	O
a	O
perpendicular	O
π	B-bond_interaction
-	I-bond_interaction
stacking	I-bond_interaction
interaction	I-bond_interaction
with	O
the	O
purine	O
ring	O
of	O
AdoMet	B-chemical
,	O
which	O
stabilizes	O
the	O
orientation	O
of	O
AdoMet	B-chemical
bound	B-protein_state
in	I-protein_state
the	O
pocket	B-site
of	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
(	O
Figure	O
S2C	O
).	O

In	O
a	O
separate	O
scenario	O
,	O
mutagenesis	B-experimental_method
of	O
this	O
residue	O
in	O
M	B-protein
.	I-protein
EcoRV	I-protein
has	O
been	O
proven	O
to	O
play	O
an	O
important	O
role	O
in	O
AdoMet	B-chemical
binding	O
.	O

Potential	O
DNA	B-site
-	I-site
binding	I-site
sites	I-site

The	O
putative	O
DNA	B-site
binding	I-site
region	I-site
of	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
involves	O
the	O
hairpin	B-structure_element
loop	I-structure_element
(	O
residue	O
101	B-residue_range
-	I-residue_range
133	I-residue_range
),	O
the	O
TRD	B-structure_element
(	O
residues	O
136	B-residue_range
-	I-residue_range
166	I-residue_range
),	O
and	O
a	O
highly	B-protein_state
flexible	I-protein_state
loop	B-structure_element
(	O
residues	O
33	B-residue_range
-	I-residue_range
58	I-residue_range
).	O

The	O
hairpin	B-structure_element
loop	I-structure_element
between	O
β6	B-structure_element
and	O
β7	B-structure_element
strands	O
that	O
carries	O
a	O
conserved	B-protein_state
HRRY	B-structure_element
sequence	O
signature	O
in	O
the	O
middle	O
is	O
proposed	O
to	O
insert	O
into	O
the	O
minor	B-structure_element
groove	I-structure_element
of	O
the	O
bound	B-protein_state
DNA	B-chemical
.	O

As	O
aforementioned	O
,	O
the	O
TRD	B-structure_element
of	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
shows	O
striking	O
difference	O
from	O
the	O
other	O
DNA	B-protein_type
MTases	I-protein_type
,	O
and	O
the	O
relaxed	O
specificity	O
of	O
substrate	O
recognition	O
may	O
be	O
at	O
least	O
partially	O
attributable	O
to	O
the	O
disordered	B-protein_state
TRD	B-structure_element
.	O

In	O
addition	O
,	O
the	O
highly	B-protein_state
flexible	I-protein_state
loop	B-structure_element
immediately	O
following	O
the	O
DPPY	B-structure_element
motif	O
in	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
was	O
poorly	O
defined	O
in	O
electron	B-evidence
density	I-evidence
,	O
exactly	O
like	O
the	O
corresponding	O
loops	B-structure_element
in	O
the	O
AdoMet	B-protein_state
-	I-protein_state
bound	I-protein_state
structures	B-evidence
of	O
M	B-protein
.	I-protein
PvuII	I-protein
,	O
DpnM	B-protein
or	O
M	B-protein
.	I-protein
TaqI	I-protein
that	O
were	O
invisible	O
either	O
.	O

This	O
loop	B-structure_element
,	O
however	O
,	O
was	O
largely	O
stabilized	O
upon	O
DNA	B-chemical
binding	O
,	O
as	O
observed	O
in	O
the	O
protein	B-evidence
-	I-evidence
DNA	I-evidence
complex	I-evidence
structures	I-evidence
of	O
M	B-protein
.	I-protein
TaqI	I-protein
(	O
PDB	O
ID	O
2IBS	O
),	O
M	B-protein
.	I-protein
HhaI	I-protein
(	O
PDB	O
ID	O
1MHT	O
)	O
and	O
M	B-protein
.	I-protein
HaeIII	I-protein
(	O
PDB	O
ID	O
1DCT	O
).	O

The	O
well	B-protein_state
-	I-protein_state
ordered	I-protein_state
loop	B-structure_element
in	O
those	O
structures	B-evidence
directly	O
contacts	O
the	O
flipping	O
adenine	B-residue_name
and	O
forms	O
hydrogen	B-bond_interaction
bond	I-bond_interaction
with	O
neighboring	O
bases	O
.	O

These	O
observations	O
implied	O
that	O
the	O
corresponding	O
loop	B-structure_element
in	O
other	O
MTases	B-protein_type
,	O
e	O
.	O
g	O
.	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
,	O
is	O
likely	O
responsible	O
for	O
reducing	O
sequence	O
recognition	O
specificity	O
and	O
thus	O
plays	O
crucial	O
roles	O
in	O
catalysis	O
.	O

Previous	O
research	O
suggested	O
that	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
from	O
strain	O
26695	O
was	O
the	O
first	O
N6	B-protein_type
adenine	I-protein_type
MTase	I-protein_type
that	O
can	O
methylate	O
the	O
adenine	B-residue_name
of	O
5	B-chemical
′-	I-chemical
GAGG	I-chemical
-	I-chemical
3	I-chemical
′/	I-chemical
5	B-chemical
′-	I-chemical
GGAG	I-chemical
-	I-chemical
3	I-chemical
′	I-chemical
or	O
both	O
two	O
adenines	B-residue_name
of	O
5	B-chemical
′-	I-chemical
GAAG	I-chemical
-	I-chemical
3	I-chemical
′,	I-chemical
compared	O
with	O
the	O
homologs	O
from	O
other	O
strains	O
that	O
can	O
methylate	O
only	O
one	O
adenine	B-residue_name
of	O
5	B-chemical
′-	I-chemical
GAGG	I-chemical
-	I-chemical
3	I-chemical
′.	I-chemical
To	O
answer	O
why	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
displayed	O
a	O
wider	O
specificity	O
for	O
DNA	B-chemical
recognition	O
,	O
we	O
randomly	O
choose	O
fifty	O
of	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
sequences	O
from	O
hundreds	O
of	O
H	B-species
.	I-species
pylori	I-species
strains	O
for	O
multiple	B-experimental_method
sequence	I-experimental_method
alignment	I-experimental_method
.	O

Based	O
on	O
sequence	B-experimental_method
comparison	I-experimental_method
and	O
structural	B-experimental_method
analysis	I-experimental_method
,	O
four	O
residues	O
including	O
P41	B-residue_name_number
,	O
N111	B-residue_name_number
,	O
K165	B-residue_name_number
and	O
T166	B-residue_name_number
were	O
selected	O
and	O
replaced	B-experimental_method
by	O
serine	B-residue_name
,	O
threonine	B-residue_name
,	O
threonine	B-residue_name
and	O
valine	B-residue_name
,	O
respectively	O
(	O
Figure	O
5A	O
).	O

Then	O
,	O
a	O
[	B-experimental_method
3H	I-experimental_method
]	I-experimental_method
AdoMet	I-experimental_method
radiological	I-experimental_method
assay	I-experimental_method
was	O
applied	O
to	O
quantify	O
the	O
methyl	B-chemical
transfer	O
activity	O
of	O
the	O
wide	B-protein_state
type	I-protein_state
protein	O
and	O
the	O
mutants	B-protein_state
.	O

As	O
shown	O
in	O
Figure	O
5	O
,	O
when	O
the	O
substrate	O
DNA	B-chemical
contains	O
5	B-chemical
′-	I-chemical
GAGG	I-chemical
-	I-chemical
3	I-chemical
′	I-chemical
or	O
5	B-chemical
′-	I-chemical
GAAG	I-chemical
-	I-chemical
3	I-chemical
′,	I-chemical
all	O
the	O
mutants	B-protein_state
showed	O
no	O
apparent	O
difference	O
of	O
methyl	B-chemical
transfer	O
activity	O
compared	O
to	O
the	O
wt	B-protein_state
-	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
;	O
but	O
when	O
the	O
recognition	O
sequence	O
was	O
5	B-chemical
′-	I-chemical
GGAG	I-chemical
-	I-chemical
3	I-chemical
′,	I-chemical
the	O
methyl	B-chemical
transfer	O
activity	O
of	O
the	O
P41S	B-mutant
mutant	B-protein_state
was	O
significantly	O
reduced	O
compared	O
to	O
the	O
wild	B-protein_state
type	I-protein_state
M1	B-protein
.	I-protein
HpyAVI	I-protein
.	O

Sequence	B-experimental_method
alignment	I-experimental_method
,	O
structural	B-experimental_method
analysis	I-experimental_method
and	O
radioactive	B-experimental_method
methyl	I-experimental_method
transfer	I-experimental_method
activity	I-experimental_method
define	O
the	O
key	O
residue	O
for	O
wider	O
substrate	O
specificity	O
of	O
M1	B-protein
.	I-protein
HpyAVI	I-protein

A	O
.	O
Sequence	B-experimental_method
alignment	I-experimental_method
of	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
from	O
50	O
H	B-species
.	I-species
pylori	I-species
strains	O
including	O
26695	O
revealed	O
several	O
variant	O
residues	O
.	O

Residues	O
P41	B-residue_name_number
,	O
N111	B-residue_name_number
,	O
K165	B-residue_name_number
and	O
T166	B-residue_name_number
of	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
from	O
strain	O
26695	B-species
were	O
chosen	O
based	O
on	O
structural	B-experimental_method
analysis	I-experimental_method
and	O
sequence	B-experimental_method
alignment	I-experimental_method
(	O
shown	O
in	O
red	O
arrow	O
).	O

Amino	O
-	O
acid	O
conservation	O
is	O
depicted	O
using	O
WebLogo	B-experimental_method
(	O
Crooks	O
et	O
al	O
,	O
2004	O
).	O

B	O
.,	O
C	O
.,	O
D	O
.	O
Methyl	B-chemical
transfer	O
reactions	O
were	O
performed	O
using	O
wt	B-protein_state
-	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
,	O
M1	B-mutant
.	I-mutant
HpyAVI	I-mutant
-	I-mutant
P41S	I-mutant
,	O
M1	B-mutant
.	I-mutant
HpyAVI	I-mutant
-	I-mutant
N111T	I-mutant
,	O
and	O
M1	B-mutant
.	I-mutant
HpyAVI	I-mutant
-	I-mutant
K165R	I-mutant
T166V	I-mutant
,	O
respectively	O
.	O

Radioactivity	O
incorporated	O
into	O
the	O
duplex	O
DNA	B-chemical
containing	O
5	B-chemical
′-	I-chemical
GAGG	I-chemical
-	I-chemical
3	I-chemical
′,	I-chemical
5	B-chemical
′-	I-chemical
GAAG	I-chemical
-	I-chemical
3	I-chemical
′	I-chemical
or	O
5	B-chemical
′-	I-chemical
GGAG	I-chemical
-	I-chemical
3	I-chemical
′	I-chemical
was	O
quantified	O
by	O
Beckman	O
LS6500	O
for	O
10	O
min	O
.	O

Our	O
experimental	O
data	O
identified	O
P41	B-residue_name_number
as	O
a	O
key	O
residue	O
determining	O
the	O
recognition	O
of	O
GGAG	B-structure_element
of	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
.	O

This	O
amino	O
acid	O
locates	O
in	O
the	O
highly	B-protein_state
flexible	I-protein_state
loop	B-structure_element
between	O
residues	O
33	B-residue_range
and	I-residue_range
58	I-residue_range
,	O
which	O
is	O
involved	O
in	O
DNA	B-chemical
binding	O
and	O
substrate	O
recognition	O
as	O
shown	O
above	O
.	O

Replacement	B-experimental_method
by	O
serine	B-residue_name
at	O
this	O
position	O
definitely	O
changes	O
the	O
local	O
conformation	O
and	O
hydrophobicity	O
,	O
and	O
probably	O
some	O
structural	O
properties	O
of	O
the	O
whole	O
loop	B-structure_element
,	O
which	O
may	O
in	O
turn	O
result	O
in	O
reduced	O
specificity	O
for	O
sequence	O
recognition	O
of	O
the	O
enzyme	O
from	O
strain	O
26695	B-species
.	O

Although	O
the	O
DNA	B-protein_state
-	I-protein_state
bound	I-protein_state
structure	B-evidence
of	O
previous	O
investigation	O
on	O
a	O
γ	B-protein_type
-	I-protein_type
class	I-protein_type
N6	I-protein_type
-	I-protein_type
adenine	I-protein_type
MTase	I-protein_type
revealed	O
that	O
the	O
target	O
adenine	B-residue_name
was	O
rotated	O
out	O
of	O
DNA	B-chemical
helix	O
,	O
details	O
of	O
the	O
methyl	B-chemical
transfer	O
process	O
were	O
still	O
unclear	O
.	O

Additionally	O
,	O
recent	O
studies	O
reported	O
the	O
importance	O
of	O
N6	B-ptm
-	I-ptm
methyladenine	I-ptm
in	O
some	O
eukaryotic	B-taxonomy_domain
species	O
,	O
but	O
until	O
now	O
there	O
has	O
not	O
been	O
any	O
N6	B-protein_type
-	I-protein_type
adenine	I-protein_type
MTases	I-protein_type
being	O
identified	O
in	O
eukaryotes	B-taxonomy_domain
.	O

Biochemical	B-experimental_method
and	I-experimental_method
structural	I-experimental_method
characterization	I-experimental_method
of	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
provides	O
a	O
new	O
model	O
for	O
uncovering	O
the	O
methyl	B-chemical
transfer	O
mechanism	O
and	O
for	O
investigating	O
the	O
N6	B-ptm
-	I-ptm
methyladenine	I-ptm
in	O
eukaryotes	B-taxonomy_domain
.	O

Oligomeric	O
state	O
of	O
DNA	B-protein_type
MTases	I-protein_type
was	O
long	O
accepted	O
as	O
monomer	B-oligomeric_state
,	O
but	O
our	O
study	O
indicated	O
here	O
that	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
exists	O
as	O
a	O
dimer	B-oligomeric_state
both	O
in	O
crystal	B-evidence
and	O
solution	O
.	O

Interestingly	O
,	O
some	O
other	O
β	B-protein_type
-	I-protein_type
class	I-protein_type
DNA	I-protein_type
exocyclic	I-protein_type
MTases	I-protein_type
showed	O
similar	O
oligomeric	O
state	O
in	O
crystal	B-evidence
and	O
in	O
solution	O
,	O
indicating	O
that	O
dimer	B-oligomeric_state
may	O
be	O
the	O
functional	O
state	O
shared	O
by	O
a	O
subgroup	O
of	O
DNA	B-protein_type
MTases	I-protein_type
.	O

The	O
highly	B-protein_state
flexible	I-protein_state
region	O
(	O
residues	O
33	B-residue_range
-	I-residue_range
58	I-residue_range
)	O
and	O
TRD	B-structure_element
(	O
residues	O
133	B-residue_range
-	I-residue_range
163	I-residue_range
)	O
of	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
are	O
supposed	O
to	O
interact	O
with	O
DNA	B-chemical
at	O
minor	B-structure_element
and	I-structure_element
major	I-structure_element
grooves	I-structure_element
,	O
respectively	O
.	O

And	O
residue	O
P41	B-residue_name_number
might	O
be	O
a	O
key	O
residue	O
partially	O
determining	O
the	O
substrate	O
spectrum	O
of	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
.	O

The	O
missing	B-protein_state
loop	B-structure_element
between	O
residues	O
33	B-residue_range
and	I-residue_range
58	I-residue_range
may	O
need	O
DNA	B-chemical
binding	O
so	O
as	O
to	O
form	O
a	O
stable	B-protein_state
conformation	O
,	O
which	O
is	O
similar	O
to	O
the	O
condition	O
of	O
M	B-protein
.	I-protein
TaqI	I-protein
.	O
Crystallization	B-experimental_method
of	O
M1	B-complex_assembly
.	I-complex_assembly
HpyAVI	I-complex_assembly
-	I-complex_assembly
DNA	I-complex_assembly
complex	O
warrants	O
future	O
investigations	O
,	O
with	O
the	O
purpose	O
of	O
revealing	O
the	O
mechanism	O
behind	O
the	O
wider	O
substrate	O
specificity	O
of	O
this	O
enzyme	O
.	O

DNA	B-ptm
methylation	I-ptm
plays	O
an	O
important	O
role	O
in	O
bacterial	B-taxonomy_domain
pathogenicity	O
.	O

DNA	B-ptm
adenine	I-ptm
methylation	I-ptm
was	O
known	O
to	O
regulate	O
the	O
expression	O
of	O
some	O
virulence	O
genes	O
in	O
bacteria	B-taxonomy_domain
including	O
H	B-species
.	I-species
pylori	I-species
.	O

Inhibitors	O
of	O
DNA	B-ptm
adenine	I-ptm
methylation	I-ptm
may	O
have	O
a	O
broad	O
antimicrobial	O
action	O
by	O
targeting	O
DNA	B-protein_type
adenine	I-protein_type
methyltransferase	I-protein_type
.	O

As	O
an	O
important	O
biological	O
modification	O
,	O
DNA	B-ptm
methylation	I-ptm
directly	O
influences	O
bacterial	B-taxonomy_domain
survival	O
.	O

Knockout	B-experimental_method
of	I-experimental_method
M1	B-protein
.	I-protein
HpyAVI	I-protein
largely	O
prevents	O
the	O
growth	O
of	O
H	B-species
.	I-species
pylori	I-species
.	O

Importantly	O
,	O
H	B-species
.	I-species
pylori	I-species
is	O
involved	O
in	O
90	O
%	O
of	O
all	O
gastric	O
malignancies	O
.	O

Appropriate	O
antibiotic	O
regimens	O
could	O
successfully	O
cure	O
gastric	O
diseases	O
caused	O
by	O
H	B-species
.	I-species
pylori	I-species
infection	O
.	O

However	O
,	O
eradication	O
of	O
H	B-species
.	I-species
pylori	I-species
infection	O
remains	O
a	O
big	O
challenge	O
for	O
the	O
significantly	O
increasing	O
prevalence	O
of	O
its	O
resistance	O
to	O
antibiotics	O
.	O

The	O
development	O
of	O
new	O
drugs	O
targeting	O
adenine	B-protein_type
MTases	I-protein_type
such	O
as	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
offers	O
a	O
new	O
opportunity	O
for	O
inhibition	O
of	O
H	B-species
.	I-species
pylori	I-species
infection	O
.	O

Residues	O
that	O
play	O
crucial	O
roles	O
for	O
catalytic	O
activity	O
like	O
D29	B-residue_name_number
or	O
E216	B-residue_name_number
may	O
influence	O
the	O
H	B-species
.	I-species
pylori	I-species
survival	O
.	O

Small	O
molecules	O
targeting	O
these	O
highly	B-protein_state
conserved	I-protein_state
residues	O
are	O
likely	O
to	O
emerge	O
less	O
drug	O
resistance	O
.	O

In	O
summary	O
,	O
the	O
structure	B-evidence
of	O
M1	B-protein
.	I-protein
HpyAVI	I-protein
is	O
featured	O
with	O
a	O
disordered	B-protein_state
TRD	B-structure_element
and	O
a	O
key	O
residue	O
P41that	B-residue_name_number
located	O
in	O
the	O
putative	O
DNA	B-site
binding	I-site
region	I-site
that	O
may	O
associate	O
with	O
the	O
wider	O
substrate	O
specificity	O
.	O

Residues	O
D29	B-residue_name_number
and	O
E216	B-residue_name_number
were	O
identified	O
to	O
play	O
a	O
crucial	O
role	O
in	O
cofactor	O
binding	O
.	O

As	O
the	O
first	O
crystal	B-evidence
structure	I-evidence
of	O
N6	B-protein_type
-	I-protein_type
adenine	I-protein_type
MTase	I-protein_type
in	O
H	B-species
.	I-species
pylori	I-species
,	O
this	O
model	O
may	O
shed	O
light	O
on	O
design	O
of	O
new	O
antibiotics	O
to	O
interfere	O
the	O
growth	O
and	O
pathogenesis	O
of	O
H	B-species
.	I-species
pylori	I-species
in	O
human	B-species
.	O