The	O
Bacteroidetes	B-taxonomy_domain
are	O
dominant	O
bacteria	B-taxonomy_domain
in	O
the	O
human	B-species
gut	O
that	O
are	O
responsible	O
for	O
the	O
digestion	O
of	O
the	O
complex	B-chemical
polysaccharides	I-chemical
that	O
constitute	O
“	O
dietary	O
fiber	O
.”	O
Although	O
this	O
symbiotic	O
relationship	O
has	O
been	O
appreciated	O
for	O
decades	O
,	O
little	O
is	O
currently	O
known	O
about	O
how	O
Bacteroidetes	B-taxonomy_domain
seek	O
out	O
and	O
bind	O
plant	B-taxonomy_domain
cell	O
wall	O
polysaccharides	B-chemical
as	O
a	O
necessary	O
first	O
step	O
in	O
their	O
metabolism	O
.	O

Here	O
,	O
we	O
provide	O
the	O
first	O
biochemical	B-experimental_method
,	I-experimental_method
crystallographic	I-experimental_method
,	I-experimental_method
and	I-experimental_method
genetic	I-experimental_method
insight	I-experimental_method
into	O
how	O
two	O
surface	B-protein_type
glycan	I-protein_type
-	I-protein_type
binding	I-protein_type
proteins	I-protein_type
from	O
the	O
complex	O
Bacteroides	B-species
ovatus	I-species
xyloglucan	B-gene
utilization	I-gene
locus	I-gene
(	O
XyGUL	B-gene
)	O
enable	O
recognition	O
and	O
uptake	O
of	O
this	O
ubiquitous	O
vegetable	B-taxonomy_domain
polysaccharide	B-chemical
.	O

More	O
importantly	O
,	O
this	O
makes	O
diet	O
a	O
tractable	O
way	O
to	O
manipulate	O
the	O
abundance	O
and	O
metabolic	O
output	O
of	O
the	O
microbiota	B-taxonomy_domain
toward	O
improved	O
human	B-species
health	O
.	O

The	O
archetypal	O
PUL	B-gene
-	O
encoded	O
system	O
is	O
the	O
starch	B-complex_assembly
utilization	I-complex_assembly
system	I-complex_assembly
(	O
Sus	B-complex_assembly
)	O
(	O
Fig	O
.	O
1B	O
)	O
of	O
Bacteroides	B-species
thetaiotaomicron	I-species
.	O

The	O
location	O
of	O
SGBP	B-protein
-	I-protein
A	I-protein
/	O
B	B-protein
is	O
presented	O
in	O
this	O
work	O
;	O
the	O
location	O
of	O
GH5	B-protein
has	O
been	O
empirically	O
determined	O
,	O
and	O
the	O
enzymes	O
have	O
been	O
placed	O
based	O
upon	O
their	O
predicted	O
cellular	O
location	O
.	O

We	O
recently	O
reported	O
the	O
detailed	O
molecular	O
characterization	O
of	O
a	O
PUL	B-gene
that	O
confers	O
the	O
ability	O
of	O
the	O
human	B-species
gut	O
commensal	O
B	B-species
.	I-species
ovatus	I-species
ATCC	I-species
8483	I-species
to	O
grow	O
on	O
a	O
prominent	O
family	O
of	O
plant	B-taxonomy_domain
cell	O
wall	O
glycans	B-chemical
,	O
the	O
xyloglucans	B-chemical
(	O
XyG	B-chemical
).	O

As	O
the	O
Sus	B-complex_assembly
SGBPs	B-protein_type
remain	O
the	O
only	O
structurally	O
characterized	O
cohort	O
to	O
date	O
,	O
we	O
therefore	O
wondered	O
whether	O
such	O
glycan	B-chemical
binding	O
and	O
function	O
are	O
extended	O
to	O
other	O
PUL	B-gene
that	O
target	O
more	O
complex	O
and	O
heterogeneous	O
polysaccharides	B-chemical
,	O
such	O
as	O
XyG	B-chemical
.	O

These	O
data	O
extend	O
our	O
current	O
understanding	O
of	O
the	O
Sus	O
-	O
like	O
glycan	B-chemical
uptake	O
paradigm	O
within	O
the	O
Bacteroidetes	B-taxonomy_domain
and	O
reveals	O
how	O
the	O
complex	O
dietary	O
polysaccharide	B-chemical
xyloglucan	B-chemical
is	O
recognized	O
at	O
the	O
cell	O
surface	O
.	O

Similarly	O
,	O
SGBP	B-protein
-	I-protein
B	I-protein
also	O
bound	B-protein_state
to	I-protein_state
XyG	B-chemical
and	O
XyGO2	B-chemical
with	O
approximately	O
equal	O
affinities	B-evidence
,	O
although	O
in	O
both	O
cases	O
,	O
Ka	B-evidence
values	O
were	O
nearly	O
10	O
-	O
fold	O
lower	O
than	O
those	O
for	O
SGBP	B-protein
-	I-protein
A	I-protein
.	O
Also	O
in	O
contrast	O
to	O
SGBP	B-protein
-	I-protein
A	I-protein
,	O
SGBP	B-protein
-	I-protein
B	I-protein
also	O
bound	B-protein_state
to	I-protein_state
XyGO1	B-chemical
,	O
yet	O
the	O
affinity	B-evidence
for	O
this	O
minimal	B-structure_element
repeating	I-structure_element
unit	I-structure_element
was	O
poor	O
,	O
with	O
a	O
Ka	B-evidence
value	O
of	O
ca	O
.	O
1	O
order	O
of	O
magnitude	O
lower	O
than	O
for	O
XyG	B-chemical
and	O
XyGO2	B-chemical
.	O

As	O
anticipated	O
by	O
sequence	O
similarity	O
,	O
the	O
high	O
-	O
resolution	O
tertiary	O
structure	B-evidence
of	O
apo	B-protein_state
-	O
SGBP	B-protein
-	I-protein
A	I-protein
(	O
1	O
.	O
36	O
Å	O
,	O
Rwork	B-evidence
=	O
14	O
.	O
7	O
%,	O
Rfree	B-evidence
=	O
17	O
.	O
4	O
%,	O
residues	O
28	B-residue_range
to	I-residue_range
546	I-residue_range
)	O
(	O
Table	O
2	O
)	O
displays	O
the	O
canonical	O
“	B-structure_element
SusD	I-structure_element
-	I-structure_element
like	I-structure_element
”	I-structure_element
protein	I-structure_element
fold	I-structure_element
dominated	O
by	O
four	O
tetratrico	B-structure_element
-	I-structure_element
peptide	I-structure_element
repeat	I-structure_element
(	O
TPR	B-structure_element
)	O
motifs	O
that	O
cradle	O
the	O
rest	O
of	O
the	O
structure	B-evidence
(	O
Fig	O
.	O
4A	O
).	O

Cocrystallization	B-experimental_method
of	O
SGBP	B-protein
-	I-protein
A	I-protein
with	O
XyGO2	B-chemical
generated	O
a	O
substrate	B-complex_assembly
complex	I-complex_assembly
structure	B-evidence
(	O
2	O
.	O
3	O
Å	O
,	O
Rwork	B-evidence
=	O
21	O
.	O
8	O
%,	O
Rfree	B-evidence
=	O
24	O
.	O
8	O
%,	O
residues	O
36	B-residue_range
to	I-residue_range
546	I-residue_range
)	O
(	O
Fig	O
.	O
4A	O
and	O
B	O
;	O
Table	O
2	O
)	O
that	O
revealed	O
the	O
distinct	O
binding	B-site
-	I-site
site	I-site
architecture	O
of	O
the	O
XyG	B-protein_type
binding	I-protein_type
protein	I-protein_type
.	O

Seven	O
of	O
the	O
eight	O
backbone	O
glucosyl	B-chemical
residues	O
of	O
XyGO2	B-chemical
could	O
be	O
convincingly	O
modeled	O
in	O
the	O
ligand	B-evidence
electron	I-evidence
density	I-evidence
,	O
and	O
only	O
two	O
α	B-chemical
(	I-chemical
1	I-chemical
→	I-chemical
6	I-chemical
)-	I-chemical
linked	I-chemical
xylosyl	I-chemical
residues	O
were	O
observed	O
(	O
Fig	O
.	O
4B	O
;	O
cf	O
.	O

The	O
functional	O
importance	O
of	O
this	O
platform	B-site
is	O
underscored	O
by	O
the	O
observation	O
that	O
the	O
W82A	B-mutant
W283A	B-mutant
W306A	B-mutant
mutant	B-protein_state
of	O
SGBP	B-protein
-	I-protein
A	I-protein
,	O
designated	O
SGBP	B-mutant
-	I-mutant
A	I-mutant
*,	I-mutant
is	O
completely	B-protein_state
devoid	I-protein_state
of	I-protein_state
XyG	I-protein_state
affinity	I-protein_state
(	O
Table	O
3	O
;	O
see	O
Fig	O
.	O
S4	O
in	O
the	O
supplemental	O
material	O
).	O

Protein	O
name	O
Ka	B-evidence
ΔG	B-evidence
(	O
kcal	O
⋅	O
mol	O
−	O
1	O
)	O
ΔH	B-evidence
(	O
kcal	O
⋅	O
mol	O
−	O
1	O
)	O
TΔS	B-evidence
(	O
kcal	O
⋅	O
mol	O
−	O
1	O
)	O
Fold	O
changeb	O
M	O
−	O
1	O
SGBP	B-protein
-	I-protein
A	I-protein
(	O
W82A	B-mutant
W283A	B-mutant
W306A	B-mutant
)	O
ND	O
NB	O
NB	O
NB	O
NB	O
SGBP	B-protein
-	I-protein
A	I-protein
(	O
W82A	B-mutant
)	O
c	O
4	O
.	O
9	O
9	O
.	O
1	O
×	O
104	O
−	O
6	O
.	O
8	O
−	O
6	O
.	O
3	O
0	O
.	O
5	O
SGBP	B-protein
-	I-protein
A	I-protein
(	O
W306	B-residue_name_number
)	O
ND	O
NB	O
NB	O
NB	O
NB	O
SGBP	B-protein
-	I-protein
B	I-protein
(	O
230	B-residue_range
–	I-residue_range
489	I-residue_range
)	O
0	O
.	O
7	O
(	O
8	O
.	O
6	O
±	O
0	O
.	O
20	O
)	O
×	O
104	O
−	O
6	O
.	O
7	O
−	O
14	O
.	O
9	O
±	O
0	O
.	O
1	O
−	O
8	O
.	O
2	O
SGBP	B-protein
-	I-protein
B	I-protein
(	O
Y363A	B-mutant
)	O
19	O
.	O
7	O
(	O
2	O
.	O
9	O
±	O
0	O
.	O
10	O
)	O
×	O
103	O
−	O
4	O
.	O
7	O
−	O
18	O
.	O
1	O
±	O
0	O
.	O
1	O
−	O
13	O
.	O
3	O
SGBP	B-protein
-	I-protein
B	I-protein
(	O
W364A	B-mutant
)	O
ND	O
Weak	O
Weak	O
Weak	O
Weak	O
SGBP	B-protein
-	I-protein
B	I-protein
(	O
F414A	B-mutant
)	O
3	O
.	O
2	O
(	O
1	O
.	O
80	O
±	O
0	O
.	O
03	O
)	O
×	O
104	O
−	O
5	O
.	O
8	O
−	O
11	O
.	O
4	O
±	O
0	O
.	O
1	O
−	O
5	O
.	O
6	O

Binding	O
thermodynamics	O
are	O
based	O
on	O
the	O
concentration	O
of	O
the	O
binding	O
unit	O
,	O
XyGO2	B-chemical
.	O

Domains	O
A	B-structure_element
,	O
B	B-structure_element
,	O
and	O
C	B-structure_element
display	O
similar	O
β	B-structure_element
-	I-structure_element
sandwich	I-structure_element
folds	I-structure_element
;	O
domains	O
B	B-structure_element
(	O
residues	O
134	B-residue_range
to	I-residue_range
230	I-residue_range
)	O
and	O
C	B-structure_element
(	O
residues	O
231	B-residue_range
to	I-residue_range
313	I-residue_range
)	O
can	O
be	O
superimposed	B-experimental_method
onto	O
domain	O
A	B-structure_element
(	O
residues	O
34	B-residue_range
to	I-residue_range
133	I-residue_range
)	O
with	O
RMSDs	B-evidence
of	O
1	O
.	O
1	O
and	O
1	O
.	O
2	O
Å	O
,	O
respectively	O
,	O
for	O
47	O
atom	O
pairs	O
(	O
23	O
%	O
and	O
16	O
%	O
sequence	O
identity	O
,	O
respectively	O
).	O

While	O
there	O
is	O
no	O
substrate	O
-	O
complexed	O
structure	O
of	O
Bacova_04391	B-protein
available	O
,	O
the	O
binding	B-site
site	I-site
is	O
predicted	O
to	O
include	O
W241	B-residue_name_number
and	O
Y404	B-residue_name_number
,	O
which	O
are	O
proximal	O
to	O
the	O
XyGO	B-site
binding	I-site
site	I-site
in	O
SGBP	B-protein
-	I-protein
B	I-protein
.	O
However	O
,	O
the	O
opposing	B-protein_state
,	I-protein_state
clamp	I-protein_state
-	I-protein_state
like	I-protein_state
arrangement	I-protein_state
of	O
these	B-structure_element
residues	I-structure_element
in	O
Bacova_04391	B-protein
is	O
clearly	O
distinct	O
from	O
the	O
planar	B-site
surface	I-site
arrangement	I-site
of	O
the	O
residues	B-structure_element
that	O
interact	O
with	O
XyG	B-chemical
in	O
SGBP	B-protein
-	I-protein
B	I-protein
(	O
described	O
below	O
).	O

Inspection	O
of	O
the	O
tertiary	O
structure	B-evidence
indicates	O
that	O
domains	O
C	B-structure_element
and	O
D	B-structure_element
are	O
effectively	O
inseparable	O
,	O
with	O
a	O
contact	O
interface	O
of	O
396	O
Å2	O
.	O

Despite	O
the	O
lack	O
of	O
sequence	O
and	O
structural	O
conservation	O
,	O
a	O
similarly	O
positioned	O
proline	B-residue_name
joins	O
the	O
Ig	B-structure_element
-	I-structure_element
like	I-structure_element
domains	I-structure_element
of	O
the	O
xylan	O
-	O
binding	O
Bacova_04391	B-protein
and	O
the	O
starch	B-protein_type
-	I-protein_type
binding	I-protein_type
proteins	I-protein_type
SusE	B-protein
and	O
SusF	B-protein
.	O
We	O
speculate	O
that	O
this	O
is	O
a	O
biologically	O
important	O
adaptation	O
that	O
serves	O
to	O
project	O
the	O
glycan	B-site
binding	I-site
site	I-site
of	O
these	O
proteins	O
far	O
from	O
the	O
membrane	O
surface	O
.	O

In	O
these	O
growth	B-experimental_method
experiments	I-experimental_method
,	O
overnight	O
cultures	O
of	O
strains	O
grown	O
on	O
minimal	O
medium	O
plus	O
glucose	B-chemical
were	O
back	O
-	O
diluted	O
1	O
:	O
100	O
-	O
fold	O
into	O
minimal	O
medium	O
containing	O
5	O
mg	O
/	O
ml	O
of	O
the	O
reported	O
carbohydrate	B-chemical
.	O

Complementation	B-experimental_method
of	O
the	O
ΔSGBP	B-mutant
-	I-mutant
A	I-mutant
strain	O
(	O
ΔSGBP	B-mutant
-	I-mutant
A	I-mutant
::	O
SGBP	B-protein
-	I-protein
A	I-protein
)	O
restores	O
growth	O
to	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
rates	O
on	O
xyloglucan	B-chemical
and	O
XyGO1	B-chemical
,	O
yet	O
the	O
calculated	O
rate	O
of	O
the	O
complemented	O
strain	O
is	O
~	O
72	O
%	O
that	O
of	O
the	O
WT	B-protein_state
Δtdk	B-mutant
strain	O
on	O
XyGO2	B-chemical
;	O
similar	O
results	O
were	O
obtained	O
for	O
the	O
SGBP	B-protein
-	I-protein
B	I-protein
complemented	O
strain	O
despite	O
the	O
fact	O
that	O
the	O
growth	O
curves	O
do	O
not	O
appear	O
much	O
different	O
(	O
see	O
Fig	O
.	O
S8C	O
and	O
F	O
).	O

Growth	O
was	O
measured	O
over	O
time	O
in	O
minimal	O
medium	O
containing	O
(	O
A	O
)	O
XyG	B-chemical
,	O
(	O
B	O
)	O
XyGO2	B-chemical
,	O
(	O
C	O
)	O
XyGO1	B-chemical
,	O
(	O
D	O
)	O
glucose	B-chemical
,	O
and	O
(	O
E	O
)	O
xylose	B-chemical
.	O

In	O
panel	O
F	O
,	O
the	O
growth	O
rate	O
of	O
each	O
strain	O
on	O
the	O
five	O
carbon	O
sources	O
is	O
displayed	O
,	O
and	O
in	O
panel	O
G	O
,	O
the	O
normalized	O
lag	B-evidence
time	I-evidence
of	O
each	O
culture	O
,	O
relative	O
to	O
its	O
growth	O
on	O
glucose	B-chemical
,	O
is	O
displayed	O
.	O

Intriguingly	O
,	O
the	O
ΔSGBP	B-mutant
-	I-mutant
B	I-mutant
strain	O
(	O
ΔBacova_02650	B-mutant
)	O
(	O
cf	O
.	O

Fig	O
.	O
1B	O
)	O
exhibited	O
a	O
minor	O
growth	O
defect	O
on	O
both	O
XyG	B-chemical
and	O
XyGO2	B-chemical
,	O
with	O
rates	O
84	O
.	O
6	O
%	O
and	O
93	O
.	O
9	O
%	O
that	O
of	O
the	O
WT	B-protein_state
Δtdk	B-mutant
strain	O
.	O

However	O
,	O
growth	O
of	O
the	O
ΔSGBP	B-mutant
-	I-mutant
B	I-mutant
strain	O
on	O
XyGO1	B-chemical
was	O
54	O
.	O
2	O
%	O
the	O
rate	O
of	O
the	O
parental	O
strain	O
,	O
despite	O
the	O
fact	O
that	O
SGBP	B-protein
-	I-protein
B	I-protein
binds	O
this	O
substrate	O
ca	O
.	O

Taken	O
together	O
,	O
the	O
data	O
indicate	O
that	O
SGBP	B-protein
-	I-protein
A	I-protein
and	O
SGBP	B-protein
-	I-protein
B	I-protein
functionally	O
complement	O
each	O
other	O
in	O
the	O
capture	O
of	O
XyG	B-chemical
polysaccharide	B-chemical
,	O
while	O
SGBP	B-protein
-	I-protein
B	I-protein
may	O
allow	O
B	B-species
.	I-species
ovatus	I-species
to	O
scavenge	O
smaller	O
XyGOs	B-chemical
liberated	O
by	O
other	O
gut	O
commensals	O
.	O

It	O
may	O
then	O
be	O
that	O
only	O
after	O
a	O
sufficient	O
amount	O
of	O
glycan	B-chemical
is	O
processed	O
and	O
imported	O
by	O
the	O
cell	O
is	O
XyGUL	B-gene
upregulated	O
and	O
exponential	O
growth	O
on	O
the	O
glycan	B-chemical
can	O
begin	O
.	O

Likewise	O
,	O
such	O
cognate	O
interactions	O
between	O
homologous	O
protein	O
pairs	O
such	O
as	O
SGBP	B-protein
-	I-protein
A	I-protein
and	O
its	O
TBDT	B-protein_type
may	O
underlie	O
our	O
observation	O
that	O
a	O
ΔSGBP	B-mutant
-	I-mutant
A	I-mutant
mutant	B-protein_state
cannot	O
grow	O
on	O
xyloglucan	B-chemical
.	O

Thus	O
,	O
understanding	O
glycan	B-chemical
capture	O
at	O
the	O
cell	O
surface	O
is	O
fundamental	O
to	O
explaining	O
,	O
and	O
eventually	O
predicting	O
,	O
how	O
the	O
carbohydrate	O
content	O
of	O
the	O
diet	O
shapes	O
the	O
gut	O
community	O
structure	O
as	O
well	O
as	O
its	O
causative	O
health	O
effects	O
.	O

PUL	B-gene
-	O
encoded	O
TBDTs	B-protein_type
in	O
Bacteroidetes	B-taxonomy_domain
are	O
larger	O
than	O
the	O
well	O
-	O
characterized	O
iron	B-protein_type
-	I-protein_type
targeting	I-protein_type
TBDTs	I-protein_type
from	O
many	O
Proteobacteria	B-taxonomy_domain
and	O
are	O
further	O
distinguished	O
as	O
the	O
only	O
known	O
glycan	B-protein_type
-	I-protein_type
importing	I-protein_type
TBDTs	I-protein_type
coexpressed	O
with	O
an	O
SGBP	B-protein_type
.	O

Our	O
observation	O
here	O
that	O
the	O
physical	O
presence	O
of	O
the	O
SusD	B-protein
homolog	O
SGBP	B-protein
-	I-protein
A	I-protein
,	O
independent	O
of	O
XyG	B-chemical
-	O
binding	O
ability	O
,	O
is	O
both	O
necessary	O
and	O
sufficient	O
for	O
XyG	B-chemical
utilization	O
further	O
supports	O
a	O
model	O
of	O
glycan	B-chemical
import	O
whereby	O
the	O
SusC	B-protein_type
-	I-protein_type
like	I-protein_type
TBDTs	I-protein_type
and	O
the	O
SusD	B-protein_type
-	I-protein_type
like	I-protein_type
SGBPs	I-protein_type
must	O
be	O
intimately	O
associated	O
to	O
support	O
glycan	B-chemical
uptake	O
(	O
Fig	O
.	O
1C	O
).	O

A	O
molecular	O
understanding	O
of	O
glycan	B-chemical
uptake	O
by	O
human	B-species
gut	O
bacteria	B-taxonomy_domain
is	O
therefore	O
central	O
to	O
the	O
development	O
of	O
strategies	O
to	O
improve	O
human	B-species
health	O
through	O
manipulation	O
of	O
the	O
microbiota	B-taxonomy_domain
.	O

A	O
high	B-chemical
affinity	I-chemical
IL	I-chemical
-	I-chemical
17A	I-chemical
peptide	I-chemical
antagonist	I-chemical
(	O
HAP	B-chemical
)	O
of	O
15	B-residue_range
residues	I-residue_range
was	O
identified	O
through	O
phage	B-experimental_method
-	I-experimental_method
display	I-experimental_method
screening	I-experimental_method
followed	O
by	O
saturation	B-experimental_method
mutagenesis	I-experimental_method
optimization	I-experimental_method
and	O
amino	B-experimental_method
acid	I-experimental_method
substitutions	I-experimental_method
.	O

The	O
family	O
of	O
IL	B-protein_type
-	I-protein_type
17	I-protein_type
cytokines	I-protein_type
and	O
receptors	O
consists	O
of	O
six	O
polypeptides	O
,	O
IL	B-protein
-	I-protein
17A	I-protein
-	I-protein
F	I-protein
,	O
and	O
five	O
receptors	O
,	O
IL	B-protein
-	I-protein
17RA	I-protein
-	I-protein
E	I-protein
.	O
IL	B-protein
-	I-protein
17A	I-protein
is	O
secreted	O
from	O
activated	O
Th17	O
cells	O
,	O
and	O
several	O
innate	O
immune	O
T	O
cell	O
types	O
including	O
macrophages	O
,	O
neutrophils	O
,	O
natural	O
killer	O
cells	O
,	O
and	O
dendritic	O
cells	O
.	O

There	O
has	O
been	O
active	O
research	O
in	O
identifying	O
orally	O
available	O
chemical	O
entities	O
that	O
would	O
functionally	O
antagonize	O
IL	B-protein
-	I-protein
17A	I-protein
-	O
mediated	O
signaling	O
.	O

Since	O
IL	B-protein
-	I-protein
17RA	I-protein
is	O
a	O
shared	O
receptor	B-protein_type
for	O
at	O
least	O
IL	B-protein
-	I-protein
17A	I-protein
,	O
IL	B-protein
-	I-protein
17F	I-protein
,	O
IL	B-complex_assembly
-	I-complex_assembly
17A	I-complex_assembly
/	I-complex_assembly
IL	I-complex_assembly
-	I-complex_assembly
17F	I-complex_assembly
and	O
IL	B-protein
-	I-protein
17E	I-protein
,	O
we	O
chose	O
to	O
seek	O
IL	B-protein
-	I-protein
17A	I-protein
-	O
specific	O
inhibitors	O
that	O
may	O
have	O
more	O
defined	O
pharmacological	O
responses	O
than	O
IL	B-protein
-	I-protein
17RA	I-protein
inhibitors	O
.	O

Positive	B-experimental_method
phage	I-experimental_method
pools	I-experimental_method
were	O
then	O
sub	B-experimental_method
-	I-experimental_method
cloned	I-experimental_method
into	O
a	O
maltose	B-experimental_method
-	I-experimental_method
binding	I-experimental_method
protein	I-experimental_method
(	I-experimental_method
MBP	I-experimental_method
)	I-experimental_method
fusion	I-experimental_method
system	I-experimental_method
.	O

Sequences	O
identified	O
from	O
phage	B-experimental_method
clones	I-experimental_method
were	O
chemically	B-experimental_method
synthesized	I-experimental_method
(	O
Supplementary	O
Table	O
1	O
)	O
and	O
tested	O
for	O
inhibition	O
of	O
IL	B-protein
-	I-protein
17A	I-protein
binding	O
to	O
IL	B-protein
-	I-protein
17RA	I-protein
(	O
Table	O
1	O
).	O

In	O
particular	O
,	O
at	O
position	O
5	B-residue_number
(	O
13	B-chemical
),	O
substitution	B-experimental_method
of	O
methionine	B-residue_name
with	O
alanine	B-residue_name
resulted	O
in	O
a	O
seven	O
fold	O
improvement	O
in	O
potency	O
(	O
80	O
nM	O
versus	O
11	O
nM	O
respectively	O
).	O

Since	O
the	O
replacement	B-experimental_method
of	O
methionine	B-residue_name
at	O
position	O
5	B-residue_number
with	O
alanine	B-residue_name
was	O
beneficial	O
,	O
the	O
additional	O
hydrophobic	O
amino	O
acids	O
isoleucine	B-residue_name
(	O
24	B-chemical
),	O
leucine	B-residue_name
(	O
25	B-chemical
)	O
and	O
valine	B-residue_name
(	O
26	B-chemical
)	O
were	O
evaluated	O
and	O
an	O
additional	O
two	O
-	O
three	O
fold	O
improvement	O
in	O
binding	O
was	O
observed	O
for	O
the	O
valine	B-residue_name
and	O
isoleucine	B-residue_name
replacements	B-experimental_method
in	O
comparison	O
with	O
alanine	B-residue_name
.	O

Dimerization	O
of	O
HAP	B-chemical
can	O
further	O
increase	O
its	O
potency	O

Orthogonal	O
assays	O
to	O
confirm	O
HAP	B-chemical
antagonism	O

The	O
relatively	O
high	O
IC50	B-evidence
values	O
in	O
this	O
assay	O
(	O
Table	O
3	O
)	O
are	O
probably	O
due	O
to	O
the	O
high	O
IL	B-protein
-	I-protein
17A	I-protein
concentration	O
(	O
100	O
ng	O
/	O
ml	O
)	O
needed	O
for	O
detection	O
of	O
IL	B-protein_type
-	I-protein_type
6	I-protein_type
.	O

Crystallization	B-experimental_method
and	I-experimental_method
structure	I-experimental_method
determination	I-experimental_method

Crystals	B-evidence
of	O
the	O
Fab	B-complex_assembly
/	I-complex_assembly
truncated	I-complex_assembly
IL	I-complex_assembly
-	I-complex_assembly
17A	I-complex_assembly
/	I-complex_assembly
HAP	I-complex_assembly
complex	O
diffracted	O
to	O
2	O
.	O
2	O
Å	O
,	O
and	O
the	O
Fab	B-complex_assembly
/	I-complex_assembly
full	I-complex_assembly
length	I-complex_assembly
IL	I-complex_assembly
-	I-complex_assembly
17A	I-complex_assembly
/	I-complex_assembly
HAP	I-complex_assembly
complex	O
diffracted	O
to	O
3	O
.	O
0	O
Å	O
(	O
Supplementary	O
Table	O
S3	O
).	O

Two	O
copies	O
of	O
HAP	B-chemical
bind	O
to	O
the	O
N	O
-	O
terminal	O
of	O
the	O
cytokine	B-protein_type
dimer	B-oligomeric_state
,	O
also	O
symmetrically	O
,	O
and	O
each	O
HAP	B-chemical
molecule	O
also	O
interacts	O
with	O
both	O
IL	B-protein
-	I-protein
17A	I-protein
monomers	B-oligomeric_state
(	O
Fig	O
.	O
2	O
).	O

Inhibition	O
mechanism	O
of	O
IL	B-protein
-	I-protein
17A	I-protein
signaling	O
by	O
HAP	B-chemical

Structure	O
basis	O
for	O
the	O
observed	O
SAR	B-experimental_method
of	O
peptides	O

The	O
C	O
-	O
terminal	O
Asn14	B-residue_name_number
and	O
Lys15	B-residue_name_number
of	O
HAP	B-chemical
are	O
not	O
directly	O
involved	O
in	O
interactions	O
with	O
IL	B-protein
-	I-protein
17A	I-protein
,	O
and	O
this	O
is	O
reflected	O
in	O
the	O
gradual	O
reduction	O
in	O
activity	O
caused	O
by	O
C	O
-	O
terminal	O
truncations	B-experimental_method
(	O
35	B-chemical
and	O
36	B-chemical
,	O
Table	O
2	O
).	O

For	O
example	O
,	O
inspection	O
of	O
the	O
published	O
IL	B-protein
-	I-protein
17F	I-protein
crystal	B-evidence
structure	I-evidence
(	O
PDB	O
code	O
1JPY	O
)	O
revealed	O
a	O
pocket	B-site
of	O
IL	B-protein
-	I-protein
17F	I-protein
similar	O
to	O
that	O
of	O
IL	B-protein
-	I-protein
17A	I-protein
for	O
W12	B-residue_name_number
of	O
HAP	B-chemical
binding	O
,	O
but	O
it	O
is	O
occupied	O
by	O
a	O
Phe	B-structure_element
-	I-structure_element
Phe	I-structure_element
motif	I-structure_element
at	O
the	O
N	O
-	O
terminal	O
peptide	O
of	O
IL	B-protein
-	I-protein
17F	I-protein
.	O

We	O
have	O
also	O
determined	B-experimental_method
the	O
complex	B-evidence
structure	I-evidence
of	O
IL	B-complex_assembly
-	I-complex_assembly
17A	I-complex_assembly
/	I-complex_assembly
HAP	I-complex_assembly
,	O
which	O
provides	O
the	O
structural	O
basis	O
for	O
HAP	B-chemical
’	O
s	O
antagonism	O
to	O
IL	B-protein
-	I-protein
17A	I-protein
signaling	O
.	O

Since	O
apo	B-protein_state
IL	B-protein
-	I-protein
17A	I-protein
is	O
a	O
homodimer	B-oligomeric_state
with	O
2	O
fold	O
symmetry	O
,	O
IL	B-protein
-	I-protein
17RA	I-protein
potentially	O
can	O
bind	O
to	O
either	O
face	O
of	O
the	O
IL	B-protein
-	I-protein
17A	I-protein
dimer	B-oligomeric_state
.	O

The	O
interaction	O
of	O
IL	B-protein
-	I-protein
17A	I-protein
with	O
IL	B-protein
-	I-protein
17RA	I-protein
has	O
an	O
extensive	O
interface	B-site
,	O
covering	O
~	O
2	O
,	O
200	O
Å2	O
surface	O
area	O
of	O
IL	B-protein
-	I-protein
17A	I-protein
.	O

One	O
way	O
of	O
further	O
improving	O
HAP	B-chemical
’	O
s	O
potency	O
is	O
by	O
dimerization	O
.	O

KD	B-evidence
determined	O
by	O
the	O
standard	O
equation	O
,	O
KD	B-evidence
=	O
kd	B-evidence
/	O
ka	B-evidence
.	O
(	O
B	O
)	O
HAP	B-chemical
inhibits	O
SPR	B-experimental_method
signaling	O
of	O
IL	B-protein
-	I-protein
17A	I-protein
binding	O
to	O
immobilized	B-protein_state
IL	B-protein
-	I-protein
17RA	I-protein
.	O

Overall	O
structure	B-evidence
of	O
the	O
Fab	B-complex_assembly
/	I-complex_assembly
IL	I-complex_assembly
-	I-complex_assembly
17A	I-complex_assembly
/	I-complex_assembly
HAP	I-complex_assembly
complex	O
in	O
ribbon	O
presentation	O
.	O

(	O
C	O
)	O
As	O
a	O
comparison	O
,	O
the	O
IL	B-complex_assembly
-	I-complex_assembly
17A	I-complex_assembly
/	I-complex_assembly
IL	I-complex_assembly
-	I-complex_assembly
17RA	I-complex_assembly
complex	O
was	O
shown	O
with	O
IL	B-protein
-	I-protein
17A	I-protein
in	O
the	O
same	O
orientation	O
.	O

ELISA	B-experimental_method
competition	I-experimental_method
activity	I-experimental_method
of	O
peptide	O
analogues	O
of	O
1	O
.	O

The	O
amount	O
of	O
NadA	B-protein
on	O
the	O
bacterial	B-taxonomy_domain
surface	O
is	O
of	O
direct	O
relevance	O
in	O
the	O
constant	O
battle	O
of	O
host	O
-	O
pathogen	O
interactions	O
:	O
it	O
influences	O
the	O
ability	O
of	O
the	O
pathogen	O
to	O
engage	O
human	B-species
cell	O
surface	O
-	O
exposed	O
receptors	O
and	O
,	O
conversely	O
,	O
the	O
bacterial	B-taxonomy_domain
susceptibility	O
to	O
the	O
antibody	O
-	O
mediated	O
immune	O
response	O
.	O

NadR	B-protein
also	O
mediates	O
ligand	O
-	O
dependent	O
regulation	O
of	O
many	O
other	O
meningococcal	B-taxonomy_domain
genes	O
,	O
for	O
example	O
the	O
highly	O
-	O
conserved	O
multiple	O
adhesin	O
family	O
(	O
maf	O
)	O
genes	O
,	O
which	O
encode	O
proteins	O
emerging	O
with	O
important	O
roles	O
in	O
host	O
-	O
pathogen	O
interactions	O
,	O
immune	O
evasion	O
and	O
niche	O
adaptation	O
.	O

The	O
abundance	O
of	O
surface	O
-	O
exposed	O
NadA	B-protein
is	O
regulated	O
by	O
the	O
ligand	B-protein_type
-	I-protein_type
responsive	I-protein_type
transcriptional	I-protein_type
repressor	I-protein_type
NadR	B-protein
.	O
Here	O
,	O
we	O
present	O
functional	B-evidence
,	I-evidence
biochemical	I-evidence
and	I-evidence
high	I-evidence
-	I-evidence
resolution	I-evidence
structural	I-evidence
data	I-evidence
on	O
NadR	B-protein
.	O
Our	O
studies	O
provide	O
detailed	O
insights	O
into	O
how	O
small	O
molecule	O
ligands	O
,	O
such	O
as	O
hydroxyphenylacetate	B-chemical
derivatives	O
,	O
found	O
in	O
relevant	O
host	O
niches	O
,	O
modulate	O
the	O
structure	O
and	O
activity	O
of	O
NadR	B-protein
,	O
by	O
‘	O
conformational	O
selection	O
’	O
of	O
inactive	B-protein_state
forms	O
.	O

The	O
DNA	O
-	O
binding	O
activity	O
of	O
NadR	B-protein
is	O
attenuated	O
in	O
vitro	O
upon	O
addition	O
of	O
various	O
hydroxyphenylacetate	B-chemical
(	O
HPA	B-chemical
)	O
derivatives	O
,	O
including	O
4	B-chemical
-	I-chemical
HPA	I-chemical
.	O

Moreover	O
,	O
these	O
findings	O
are	O
important	O
because	O
the	O
activity	O
of	O
NadR	B-protein
impacts	O
the	O
potential	O
coverage	O
provided	O
by	O
anti	O
-	O
NadA	B-protein
antibodies	O
elicited	O
by	O
the	O
Bexsero	O
vaccine	O
and	O
influences	O
host	O
-	O
bacteria	B-taxonomy_domain
interactions	O
that	O
contribute	O
to	O
meningococcal	B-taxonomy_domain
pathogenesis	O
.	O

In	O
analytical	B-experimental_method
size	I-experimental_method
-	I-experimental_method
exclusion	I-experimental_method
high	I-experimental_method
-	I-experimental_method
performance	I-experimental_method
liquid	I-experimental_method
chromatography	I-experimental_method
(	O
SE	B-experimental_method
-	I-experimental_method
HPLC	I-experimental_method
)	O
experiments	O
coupled	O
with	O
multi	B-experimental_method
-	I-experimental_method
angle	I-experimental_method
laser	I-experimental_method
light	I-experimental_method
scattering	I-experimental_method
(	O
MALLS	B-experimental_method
),	O
NadR	B-protein
presented	O
a	O
single	O
species	O
with	O
an	O
absolute	O
molecular	O
mass	O
of	O
35	O
kDa	O
(	O
S1	O
Fig	O
).	O

(	O
A	O
)	O
Molecular	O
structures	O
of	O
3	B-chemical
-	I-chemical
HPA	I-chemical
(	O
MW	O
152	O
.	O
2	O
),	O
4	B-chemical
-	I-chemical
HPA	I-chemical
(	O
MW	O
152	O
.	O
2	O
),	O
3Cl	B-chemical
,	I-chemical
4	I-chemical
-	I-chemical
HPA	I-chemical
(	O
MW	O
186	O
.	O
6	O
)	O
and	O
salicylic	B-chemical
acid	I-chemical
(	O
MW	O
160	O
.	O
1	O
).	O
(	O
B	O
)	O
DSC	B-experimental_method
profiles	B-evidence
,	O
colored	O
as	O
follows	O
:	O
apo	B-protein_state
-	O
NadR	B-protein
(	O
violet	O
),	O
NadR	B-complex_assembly
+	I-complex_assembly
salicylate	I-complex_assembly
(	O
red	O
),	O
NadR	B-complex_assembly
+	I-complex_assembly
3	I-complex_assembly
-	I-complex_assembly
HPA	I-complex_assembly
(	O
green	O
),	O
NadR	B-complex_assembly
+	I-complex_assembly
4	I-complex_assembly
-	I-complex_assembly
HPA	I-complex_assembly
(	O
blue	O
),	O
NadR	B-complex_assembly
+	I-complex_assembly
3Cl	I-complex_assembly
,	I-complex_assembly
4	I-complex_assembly
-	I-complex_assembly
HPA	I-complex_assembly
(	O
pink	O
).	O

All	O
DSC	B-experimental_method
profiles	B-evidence
are	O
representative	O
of	O
triplicate	O
experiments	O
.	O

However	O
,	O
steady	B-experimental_method
-	I-experimental_method
state	I-experimental_method
SPR	I-experimental_method
analyses	O
of	O
the	O
NadR	B-complex_assembly
-	I-complex_assembly
HPA	I-complex_assembly
interactions	O
allowed	O
determination	O
of	O
the	O
equilibrium	B-evidence
dissociation	I-evidence
constants	I-evidence
(	O
KD	B-evidence
)	O
(	O
Table	O
1	O
and	O
S2	O
Fig	O
).	O

The	O
interactions	O
of	O
4	B-chemical
-	I-chemical
HPA	I-chemical
and	O
3Cl	B-chemical
,	I-chemical
4	I-chemical
-	I-chemical
HPA	I-chemical
with	O
NadR	B-protein
exhibited	O
KD	B-evidence
values	O
of	O
1	O
.	O
5	O
mM	O
and	O
1	O
.	O
1	O
mM	O
,	O
respectively	O
.	O

To	O
fully	O
characterize	O
the	O
NadR	B-protein
/	O
HPA	B-chemical
interactions	O
,	O
we	O
sought	O
to	O
determine	O
crystal	B-evidence
structures	I-evidence
of	O
NadR	B-protein
in	O
ligand	B-protein_state
-	I-protein_state
bound	I-protein_state
(	O
holo	B-protein_state
)	O
and	O
ligand	B-protein_state
-	I-protein_state
free	I-protein_state
(	O
apo	B-protein_state
)	O
forms	O
.	O

The	O
map	B-evidence
is	O
contoured	O
at	O
1σ	O
and	O
the	O
figure	O
was	O
prepared	O
with	O
a	O
density	B-evidence
mesh	I-evidence
carve	O
factor	O
of	O
1	O
.	O
7	O
,	O
using	O
Pymol	O
(	O
www	O
.	O
pymol	O
.	O
org	O
).	O

Only	O
the	O
mutation	O
L130K	B-mutant
has	O
a	O
noteworthy	O
effect	O
on	O
the	O
oligomeric	O
state	O
,	O
inducing	O
a	O
second	O
peak	O
with	O
a	O
longer	O
retention	O
time	O
and	O
a	O
second	O
peak	O
maximum	O
at	O
18	O
.	O
6	O
min	O
.	O

To	O
a	O
much	O
lesser	O
extent	O
,	O
the	O
L133K	B-mutant
mutation	O
also	O
appears	O
to	O
induce	O
a	O
‘	O
shoulder	O
’	O
to	O
the	O
main	O
peak	O
,	O
suggesting	O
very	O
weak	O
ability	O
to	O
disrupt	O
the	O
dimer	B-oligomeric_state
.	O
(	O
D	O
)	O
SE	B-experimental_method
-	I-experimental_method
HPLC	I-experimental_method
/	I-experimental_method
MALLS	I-experimental_method
analyses	O
of	O
the	O
L130K	B-mutant
mutant	B-protein_state
,	O
shows	O
20	O
%	O
dimer	B-oligomeric_state
and	O
80	O
%	O
monomer	B-oligomeric_state
.	O

The	O
ligand	O
showed	O
a	O
different	O
position	O
and	O
orientation	O
compared	O
to	O
salicylate	B-chemical
complexed	B-protein_state
with	I-protein_state
MTH313	B-protein
and	O
ST1710	B-protein
(	O
see	O
Discussion	O
).	O

At	O
the	O
other	O
‘	O
end	O
’	O
of	O
the	O
ligand	O
,	O
the	O
4	O
-	O
hydroxyl	O
group	O
was	O
proximal	O
to	O
AspB36	B-residue_name_number
,	O
with	O
which	O
it	O
may	O
establish	O
an	O
H	B-bond_interaction
-	I-bond_interaction
bond	I-bond_interaction
(	O
see	O
bond	O
distances	O
in	O
Table	O
3	O
).	O

The	O
water	B-chemical
molecule	O
observed	O
in	O
the	O
pocket	O
was	O
bound	O
by	O
the	O
carboxylate	O
group	O
and	O
the	O
side	O
chains	O
of	O
SerA9	B-residue_name_number
and	O
AsnA11	B-residue_name_number
.	O

In	O
addition	O
to	O
the	O
H	B-bond_interaction
-	I-bond_interaction
bonds	I-bond_interaction
involving	O
the	O
carboxylate	O
and	O
hydroxyl	O
groups	O
of	O
4	B-chemical
-	I-chemical
HPA	I-chemical
,	O
binding	O
of	O
the	O
phenyl	O
moiety	O
appeared	O
to	O
be	O
stabilized	O
by	O
several	O
van	B-bond_interaction
der	I-bond_interaction
Waals	I-bond_interaction
’	I-bond_interaction
contacts	I-bond_interaction
,	O
particularly	O
those	O
involving	O
the	O
hydrophobic	O
side	O
chain	O
atoms	O
of	O
LeuB21	B-residue_name_number
,	O
MetB22	B-residue_name_number
,	O
PheB25	B-residue_name_number
,	O
LeuB29	B-residue_name_number
and	O
ValB111	B-residue_name_number
(	O
Fig	O
4A	O
).	O

The	O
presence	O
of	O
a	O
single	O
hydroxyl	O
group	O
at	O
position	O
2	O
,	O
as	O
in	O
2	B-chemical
-	I-chemical
HPA	I-chemical
,	O
rather	O
than	O
at	O
position	O
4	O
,	O
would	O
eliminate	O
the	O
possibility	O
of	O
favorable	O
interactions	O
with	O
AspB36	B-residue_name_number
,	O
resulting	O
in	O
the	O
lack	O
of	O
NadR	B-protein
regulation	O
by	O
2	B-chemical
-	I-chemical
HPA	I-chemical
described	O
previously	O
.	O

Firstly	O
,	O
NadR	B-protein
is	O
expected	O
to	O
be	O
covalently	O
immobilized	O
on	O
the	O
sensor	O
chip	O
as	O
a	O
dimer	B-oligomeric_state
in	O
random	O
orientations	O
,	O
since	O
it	O
is	O
a	O
stable	B-protein_state
dimer	B-oligomeric_state
in	O
solution	O
and	O
has	O
sixteen	O
lysines	B-residue_name
well	O
-	O
distributed	O
around	O
its	O
surface	O
,	O
all	O
able	O
to	O
act	O
as	O
potential	O
sites	O
for	O
amine	O
coupling	O
to	O
the	O
chip	O
,	O
and	O
none	O
of	O
which	O
are	O
close	O
to	O
the	O
ligand	B-site
-	I-site
binding	I-site
pocket	I-site
.	O

Secondly	O
,	O
the	O
HPA	B-chemical
analytes	O
are	O
all	O
very	O
small	O
(	O
MW	O
150	O
–	O
170	O
,	O
Fig	O
1A	O
)	O
and	O
therefore	O
are	O
expected	O
to	O
be	O
able	O
to	O
diffuse	O
readily	O
into	O
all	O
potential	O
binding	B-site
sites	I-site
,	O
irrespective	O
of	O
the	O
random	O
orientations	O
of	O
the	O
immobilized	O
NadR	B-protein
dimers	B-oligomeric_state
on	O
the	O
chip	O
.	O

The	O
crystallographic	B-evidence
data	I-evidence
,	O
supported	O
by	O
the	O
SPR	B-experimental_method
studies	O
of	O
binding	B-evidence
stoichiometry	I-evidence
,	O
revealed	O
the	O
lack	O
of	O
a	O
second	O
4	B-chemical
-	I-chemical
HPA	I-chemical
molecule	O
in	O
the	O
homodimer	B-oligomeric_state
,	O
suggesting	O
negative	O
co	O
-	O
operativity	O
,	O
a	O
phenomenon	O
previously	O
described	O
for	O
the	O
MTH313	B-protein
/	O
salicylate	B-chemical
interaction	O
and	O
for	O
other	O
MarR	B-protein_type
family	O
proteins	O
.	O

However	O
,	O
since	O
residues	O
of	O
helix	B-structure_element
α6	B-structure_element
were	O
not	O
directly	O
involved	O
in	O
ligand	O
binding	O
,	O
an	O
explanation	O
for	O
the	O
lack	O
of	O
4	B-chemical
-	I-chemical
HPA	I-chemical
in	O
monomer	B-oligomeric_state
A	B-structure_element
did	O
not	O
emerge	O
by	O
analyzing	O
only	O
these	O
backbone	O
atom	O
positions	O
,	O
suggesting	O
that	O
a	O
more	O
complex	O
series	O
of	O
allosteric	O
events	O
may	O
occur	O
.	O

Specifically	O
,	O
upon	O
analysis	O
with	O
the	O
CASTp	B-experimental_method
software	O
,	O
the	O
pocket	B-site
in	O
chain	B-structure_element
B	I-structure_element
containing	O
the	O
4	B-chemical
-	I-chemical
HPA	I-chemical
exhibited	O
a	O
total	O
volume	O
of	O
approximately	O
370	O
Å3	O
,	O
while	O
the	O
pocket	B-site
in	O
chain	B-structure_element
A	I-structure_element
was	O
occupied	O
by	O
these	O
three	O
side	O
chains	O
that	O
adopted	O
‘	O
inward	B-protein_state
’	O
positions	O
and	O
thereby	O
divided	O
the	O
space	O
into	O
a	O
few	O
much	O
smaller	O
pockets	O
,	O
each	O
with	O
volume	O
<	O
50	O
Å3	O
,	O
evidently	O
rendering	O
chain	B-structure_element
A	I-structure_element
unfavorable	O
for	O
ligand	O
binding	O
.	O

Although	O
more	O
comprehensive	O
NMR	B-experimental_method
experiments	O
and	O
full	O
chemical	O
shift	O
assignment	O
of	O
the	O
spectra	B-evidence
would	O
be	O
required	O
to	O
precisely	O
define	O
this	O
multi	O
-	O
state	O
behavior	O
,	O
the	O
NMR	B-experimental_method
data	O
clearly	O
demonstrate	O
that	O
NadR	B-protein
exhibits	O
conformational	O
flexibility	O
which	O
is	O
modulated	O
by	O
4	B-chemical
-	I-chemical
HPA	I-chemical
in	O
solution	O
.	O

(	O
A	O
)	O
The	O
holo	B-protein_state
-	O
homodimer	B-oligomeric_state
structure	B-evidence
is	O
shown	O
as	O
green	O
and	O
blue	O
cartoons	O
,	O
for	O
chain	B-structure_element
A	I-structure_element
and	I-structure_element
B	I-structure_element
,	O
respectively	O
,	O
while	O
the	O
two	O
homodimers	B-oligomeric_state
of	O
apo	B-protein_state
-	O
NadR	B-protein
are	O
both	O
cyan	O
and	O
pale	O
blue	O
for	O
chains	O
A	B-structure_element
/	I-structure_element
C	I-structure_element
and	O
B	B-structure_element
/	I-structure_element
D	I-structure_element
,	O
respectively	O
.	O

The	O
three	O
homodimers	B-oligomeric_state
(	O
chains	O
AB	B-structure_element
holo	B-protein_state
,	O
AB	B-structure_element
apo	B-protein_state
,	O
and	O
CD	B-structure_element
apo	B-protein_state
)	O
were	O
overlaid	B-experimental_method
by	O
structural	B-experimental_method
alignment	I-experimental_method
exclusively	O
of	O
all	O
heavy	O
atoms	O
in	O
residues	O
R64	B-residue_range
-	I-residue_range
A77	I-residue_range
(	O
shown	O
in	O
red	O
,	O
with	O
side	O
chain	O
sticks	O
)	O
of	O
chains	O
A	B-structure_element
holo	B-protein_state
,	O
A	B-structure_element
apo	B-protein_state
,	O
and	O
C	B-structure_element
apo	B-protein_state
,	O
belonging	O
to	O
helix	B-structure_element
α4	B-structure_element
(	O
left	O
).	O

Thus	O
,	O
the	O
apo	B-protein_state
-	O
homodimer	B-oligomeric_state
AB	B-structure_element
presented	O
the	O
DNA	B-structure_element
-	I-structure_element
binding	I-structure_element
helices	I-structure_element
in	O
a	O
conformation	O
similar	O
to	O
that	O
observed	O
in	O
the	O
protein	O
:	O
DNA	O
complex	O
of	O
OhrR	B-complex_assembly
:	I-complex_assembly
ohrA	I-complex_assembly
from	O
Bacillus	B-species
subtilis	I-species
(	O
Fig	O
8C	O
).	O

This	O
mutagenesis	B-experimental_method
data	O
revealed	O
that	O
NadR	B-protein
residues	O
His7	B-residue_name_number
,	O
Ser9	B-residue_name_number
,	O
Asn11	B-residue_name_number
and	O
Phe25	B-residue_name_number
play	O
key	O
roles	O
in	O
the	O
ligand	O
-	O
mediated	O
regulation	O
of	O
NadR	B-protein
;	O
they	O
are	O
each	O
involved	O
in	O
the	O
controlled	O
de	O
-	O
repression	O
of	O
the	O
nadA	B-gene
promoter	O
and	O
synthesis	O
of	O
NadA	B-protein
in	O
response	O
to	O
4	B-chemical
-	I-chemical
HPA	I-chemical
in	O
vivo	O
.	O

Given	O
the	O
importance	O
of	O
NadR	B-protein
-	O
mediated	O
regulation	O
of	O
NadA	B-protein
levels	O
in	O
the	O
contexts	O
of	O
meningococcal	B-taxonomy_domain
pathogenesis	O
,	O
we	O
sought	O
to	O
characterize	O
NadR	B-protein
,	O
and	O
its	O
interaction	O
with	O
ligands	O
,	O
at	O
atomic	O
resolution	O
.	O

While	O
some	O
flexibility	O
of	O
helix	B-structure_element
α4	B-structure_element
was	O
also	O
observed	O
in	O
the	O
two	O
apo	B-protein_state
-	O
structures	B-evidence
,	O
concomitant	O
changes	O
in	O
the	O
dimer	B-site
interfaces	I-site
were	O
not	O
observed	O
,	O
possibly	O
due	O
to	O
the	O
absence	B-protein_state
of	I-protein_state
ligand	I-protein_state
.	O

The	O
latter	O
may	O
influence	O
the	O
surface	O
abundance	O
or	O
secretion	O
of	O
maf	O
proteins	O
,	O
an	O
emerging	O
class	O
of	O
highly	B-protein_state
conserved	I-protein_state
meningococcal	B-taxonomy_domain
putative	O
adhesins	O
and	O
toxins	O
with	O
many	O
important	O
roles	O
.	O

Further	O
work	O
is	O
required	O
to	O
investigate	O
how	O
the	O
two	O
different	O
promoter	O
types	O
influence	O
the	O
ligand	O
-	O
responsiveness	O
of	O
NadR	B-protein
during	O
bacterial	B-taxonomy_domain
infection	O
and	O
may	O
provide	O
insights	O
into	O
the	O
regulatory	O
mechanisms	O
occurring	O
during	O
these	O
host	O
-	O
pathogen	O
interactions	O
.	O

Structure	O
of	O
an	O
OhrR	O
-	O
ohrA	B-gene
operator	O
complex	O
reveals	O
the	O
DNA	O
binding	O
mechanism	O
of	O
the	O
MarR	O
family	O

Structural	O
determinant	O
for	O
inducing	O
RORgamma	B-protein
specific	O
inverse	O
agonism	O
triggered	O
by	O
a	O
synthetic	O
benzoxazinone	B-chemical
ligand	O

Our	O
goal	O
was	O
to	O
develop	O
a	O
RORγ	B-protein
specific	O
inverse	B-protein_state
agonist	I-protein_state
that	O
would	O
help	O
down	O
regulate	O
pro	O
-	O
inflammatory	O
gene	O
transcription	O
by	O
disrupting	O
the	O
protein	O
protein	O
interaction	O
with	O
coactivator	O
proteins	O
as	O
a	O
therapeutic	O
agent	O
.	O

Using	O
an	O
in	B-experimental_method
vivo	I-experimental_method
reporter	I-experimental_method
assay	I-experimental_method
,	O
we	O
show	O
that	O
the	O
inverse	B-protein_state
agonist	I-protein_state
BIO399	B-chemical
displayed	O
specificity	O
for	O
RORγ	B-protein
over	O
ROR	B-protein_type
sub	O
-	O
family	O
members	O
α	B-protein
and	O
β	B-protein
.	O

The	O
synthetic	O
benzoxazinone	B-chemical
ligands	O
identified	O
in	O
our	O
FRET	B-experimental_method
assay	I-experimental_method
have	O
an	O
agonist	B-protein_state
(	O
BIO592	B-chemical
)	O
or	O
inverse	B-protein_state
agonist	I-protein_state
(	O
BIO399	B-chemical
)	O
effect	O
by	O
stabilizing	O
or	O
destabilizing	O
the	O
agonist	B-protein_state
conformation	O
of	O
RORγ	B-protein
.	O

Our	O
structural	B-experimental_method
investigation	I-experimental_method
of	O
the	O
BIO592	B-chemical
agonist	B-protein_state
and	O
BIO399	B-chemical
inverse	B-protein_state
agonist	I-protein_state
structures	B-evidence
identified	O
residue	O
Met358	B-residue_name_number
on	O
RORγ	B-protein
as	O
the	O
trigger	O
for	O
RORγ	B-protein
specific	O
inverse	O
agonism	O
.	O

Retinoid	B-protein
-	I-protein
related	I-protein
orphan	I-protein
receptor	I-protein
gamma	I-protein
(	O
RORγ	B-protein
)	O
is	O
a	O
transcription	B-protein_type
factor	I-protein_type
belonging	O
to	O
a	O
sub	O
-	O
family	O
of	O
nuclear	B-protein_type
receptors	I-protein_type
that	O
includes	O
two	O
closely	O
related	O
members	O
RORα	B-protein
and	O
RORβ	B-protein
.	O

Here	O
we	O
present	O
the	O
identification	O
of	O
two	O
synthetic	O
benzoxazinone	B-chemical
RORγ	B-protein
ligands	O
,	O
a	O
weak	O
agonist	B-protein_state
BIO592	B-chemical
(	O
Fig	O
.	O
1a	O
)	O
and	O
an	O
inverse	B-protein_state
agonist	I-protein_state
BIO399	B-chemical
(	O
Fig	O
.	O
1b	O
)	O
which	O
were	O
identified	O
using	O
a	O
Fluorescence	B-experimental_method
Resonance	I-experimental_method
Energy	I-experimental_method
transfer	I-experimental_method
(	I-experimental_method
FRET	I-experimental_method
)	I-experimental_method
based	I-experimental_method
assay	I-experimental_method
that	O
monitored	O
coactivator	O
peptide	O
recruitment	O
.	O

Using	O
partial	B-experimental_method
proteolysis	I-experimental_method
in	O
combination	O
with	O
mass	B-experimental_method
spectrometry	I-experimental_method
analysis	O
we	O
demonstrate	O
that	O
the	O
AF2	B-structure_element
helix	I-structure_element
of	O
RORγ	B-protein
destabilizes	O
upon	O
BIO399	B-chemical
(	O
inverse	B-protein_state
agonist	I-protein_state
)	O
binding	O
.	O

Using	O
a	O
FRET	B-experimental_method
based	I-experimental_method
assay	I-experimental_method
we	O
discovered	O
agonist	B-protein_state
BIO592	B-chemical
(	O
Fig	O
.	O
1a	O
)	O
which	O
increased	O
the	O
coactivator	O
peptide	O
TRAP220	B-chemical
recruitment	O
to	O
RORγ	B-protein
(	O
EC50	B-evidence
0f	O
58nM	O
and	O
Emax	B-evidence
of	O
130	O
%)	O
and	O
a	O
potent	O
inverse	B-protein_state
agonist	I-protein_state
BIO399	B-chemical
(	O
Fig	O
.	O
1b	O
)	O
which	O
inhibited	O
coactivator	O
recruitment	O
(	O
IC50	B-evidence
:	O
4	O
.	O
7nM	O
).	O

c	O
EBI96	B-chemical
coactivator	O
peptide	O
bound	B-protein_state
in	I-protein_state
the	O
coactivator	B-site
pocket	I-site
of	O
RORγ	B-protein

Specific	O
proteolytic	O
positions	O
on	O
RORγ518	B-protein
when	O
treated	B-experimental_method
with	I-experimental_method
Actinase	B-protein
E	I-protein
alone	O
(	O
Green	O
)	O
or	O
in	O
the	O
presence	B-protein_state
of	I-protein_state
BIO399	B-chemical
(	O
Red	O
)	O
and	O
shared	O
proteolytic	B-site
sites	I-site
(	O
Yellow	O
)	O

Several	O
rounds	O
of	O
cocrystallization	B-experimental_method
attempts	O
with	O
RORγ518	B-protein
or	O
other	O
RORγ	B-protein
AF2	B-structure_element
helix	I-structure_element
containing	O
constructs	O
complexed	B-protein_state
with	I-protein_state
BIO399	B-chemical
had	O
not	O
produced	O
crystals	B-evidence
.	O

We	O
reasoned	O
that	O
if	O
we	O
could	O
remove	O
the	O
unfolded	B-protein_state
AF2	B-structure_element
helix	I-structure_element
using	O
proteolysis	B-experimental_method
we	O
could	O
produce	O
a	O
binary	O
complex	O
more	O
amenable	O
to	O
crystallization	B-experimental_method
.	O

The	O
aeRORγ493	B-complex_assembly
/	I-complex_assembly
4	I-complex_assembly
BIO399	I-complex_assembly
structure	B-evidence
diverged	O
at	O
the	O
c	O
-	O
terminal	O
end	O
of	O
Helix	B-structure_element
11	I-structure_element
from	O
the	O
RORγ518	B-complex_assembly
BIO592	I-complex_assembly
EBI96	I-complex_assembly
structure	B-evidence
,	O
where	O
helix	B-structure_element
11	I-structure_element
unwinds	O
into	O
a	O
random	O
coil	O
after	O
residue	O
L475	B-residue_name_number
.	O

BIO399	B-chemical
and	O
Inverse	B-protein_state
agonist	I-protein_state
T0901317	B-chemical
bind	O
in	O
a	O
collapsed	B-protein_state
conformation	O
distinct	O
from	O
other	O
RORγ	B-protein
Inverse	O
Agonists	O
Cocrystal	B-evidence
structures	I-evidence

However	O
,	O
the	O
inverse	O
agonism	O
trigger	O
of	O
BIO399	B-chemical
,	O
residue	O
Met358	B-residue_name_number
,	O
is	O
a	O
leucine	B-residue_name
in	O
both	O
RORα	B-protein
and	O
β	B-protein
.	O

The	O
Structural	O
Basis	O
of	O
Coenzyme	B-chemical
A	I-chemical
Recycling	O
in	O
a	O
Bacterial	B-taxonomy_domain
Organelle	O

The	O
majority	O
of	O
catabolic	B-protein_state
BMCs	B-complex_assembly
(	O
metabolosomes	B-complex_assembly
)	O
compartmentalize	O
a	O
common	O
core	O
of	O
enzymes	O
to	O
metabolize	O
compounds	O
via	O
a	O
toxic	O
and	O
/	O
or	O
volatile	O
aldehyde	B-chemical
intermediate	O
.	O

Accordingly	O
,	O
PduL	B-protein_type
and	O
Pta	B-protein_type
exemplify	O
functional	O
,	O
but	O
not	O
structural	O
,	O
convergent	O
evolution	O
.	O

This	O
enzyme	O
,	O
PduL	B-protein_type
,	O
is	O
exclusively	B-protein_state
associated	O
with	O
organelles	O
called	O
bacterial	B-taxonomy_domain
microcompartments	B-complex_assembly
,	O
which	O
are	O
used	O
to	O
catabolize	O
various	O
compounds	O
.	O

The	O
aldehyde	B-chemical
is	O
subsequently	O
converted	O
into	O
an	O
acyl	B-chemical
-	I-chemical
CoA	I-chemical
by	O
aldehyde	B-protein_type
dehydrogenase	I-protein_type
,	O
which	O
uses	O
NAD	B-chemical
+	I-chemical
and	O
CoA	B-chemical
as	O
cofactors	O
.	O

NAD	B-chemical
+	I-chemical
is	O
recycled	O
via	O
alcohol	B-protein_type
dehydrogenase	I-protein_type
,	O
and	O
CoA	B-chemical
is	O
recycled	O
via	O
phosphotransacetylase	B-protein_type
(	O
PTAC	B-protein_type
)	O
(	O
Fig	O
1	O
).	O

They	O
can	O
also	O
work	O
in	O
the	O
reverse	O
direction	O
to	O
activate	O
acetate	B-chemical
to	O
the	O
CoA	B-chemical
-	I-chemical
thioester	I-chemical
.	O

The	O
canonical	O
PTAC	B-protein_type
,	O
Pta	B-protein_type
,	O
is	O
an	O
ancient	O
enzyme	O
found	O
in	O
some	O
eukaryotes	B-taxonomy_domain
and	O
archaea	B-taxonomy_domain
,	O
and	O
widespread	O
among	O
the	O
bacteria	B-taxonomy_domain
;	O
90	O
%	O
of	O
the	O
bacterial	B-taxonomy_domain
genomes	O
in	O
the	O
Integrated	O
Microbial	O
Genomes	O
database	O
contain	O
a	O
gene	O
encoding	O
the	O
PTA_PTB	B-protein_type
phosphotransacylase	I-protein_type
(	O
Pfam	O
domain	O
PF01515	B-structure_element
).	O

The	O
primary	O
structure	O
of	O
PduL	B-protein_type
homologs	O
is	O
subdivided	O
into	O
two	O
PF06130	B-structure_element
domains	O
,	O
each	O
roughly	O
80	B-residue_range
residues	I-residue_range
in	I-residue_range
length	I-residue_range
.	O

Structure	B-experimental_method
Determination	I-experimental_method
of	O
PduL	B-protein_type

Remarkably	O
,	O
after	O
removing	B-experimental_method
the	O
N	O
-	O
terminal	O
putative	O
EP	B-structure_element
(	O
27	B-residue_range
amino	I-residue_range
acids	I-residue_range
),	O
most	O
of	O
the	O
sPduLΔEP	B-mutant
protein	O
was	O
in	O
the	O
soluble	O
fraction	O
upon	O
cell	O
lysis	O
.	O

A	O
CoA	B-chemical
cofactor	O
as	O
well	O
as	O
two	O
metal	O
ions	O
are	O
clearly	O
resolved	O
in	O
the	O
density	B-evidence
(	O
for	O
omit	B-evidence
maps	I-evidence
of	O
CoA	B-chemical
see	O
S2	O
Fig	O
).	O

Distances	O
between	O
atom	O
centers	O
are	O
indicated	O
in	O
Å	O
.	O
(	O
a	O
)	O
Coenzyme	B-chemical
A	I-chemical
containing	O
,	O
(	O
b	O
)	O
phosphate	B-protein_state
-	I-protein_state
bound	I-protein_state
structure	B-evidence
.	O

(	O
d	O
)–(	O
f	O
):	O
Chromatograms	B-evidence
of	O
sPduL	B-protein
(	O
d	O
),	O
rPduL	B-protein
(	O
e	O
),	O
and	O
pPduL	B-protein
(	O
f	O
)	O
post	O
-	O
preparative	O
size	B-experimental_method
exclusion	I-experimental_method
chromatography	I-experimental_method
with	O
different	O
size	O
fractions	O
separated	O
,	O
applied	O
over	O
an	O
analytical	O
size	O
exclusion	O
column	O
(	O
see	O
Materials	O
and	O
Methods	O
).	O

The	O
phosphate	B-chemical
contacts	O
both	O
zinc	B-chemical
atoms	O
(	O
Fig	O
4b	O
)	O
and	O
replaces	O
the	O
coordination	O
by	O
CoA	B-chemical
at	O
Zn1	B-chemical
;	O
the	O
coordination	O
for	O
Zn2	B-chemical
changes	O
from	O
octahedral	O
with	O
three	O
bound	O
waters	B-chemical
to	O
tetrahedral	O
with	O
a	O
phosphate	B-chemical
ion	O
as	O
one	O
of	O
the	O
ligands	O
(	O
Fig	O
4b	O
).	O

The	O
two	O
zinc	B-chemical
atoms	O
are	O
slightly	O
closer	O
together	O
in	O
the	O
phosphate	B-protein_state
-	I-protein_state
bound	I-protein_state
form	O
(	O
5	O
.	O
8	O
Å	O
vs	O
6	O
.	O
3	O
Å	O
),	O
possibly	O
due	O
to	O
the	O
bridging	O
effect	O
of	O
the	O
phosphate	B-chemical
.	O

rPduL	B-protein
full	B-protein_state
length	I-protein_state
runs	O
as	O
Mw	B-evidence
=	O
140	O
.	O
3	O
kDa	O
+/−	O
1	O
.	O
2	O
%	O
and	O
Mn	B-evidence
=	O
140	O
.	O
5	O
kDa	O
+/−	O
1	O
.	O
2	O
%.	O

Moreover	O
,	O
the	O
PduL	B-protein_type
crystal	B-evidence
structures	I-evidence
offer	O
a	O
clue	O
as	O
to	O
how	O
required	O
cofactors	O
enter	O
the	O
BMC	B-complex_assembly
lumen	O
during	O
assembly	O
.	O

The	O
native	O
substrate	O
for	O
the	O
forward	O
reaction	O
of	O
rPduL	B-protein
and	O
pPduL	B-protein
,	O
propionyl	B-chemical
-	I-chemical
CoA	I-chemical
,	O
most	O
likely	O
binds	O
to	O
the	O
enzyme	O
in	O
the	O
same	O
way	O
at	O
the	O
observed	O
nucleotide	B-chemical
and	O
pantothenic	B-chemical
acid	I-chemical
moiety	O
,	O
but	O
the	O
propionyl	O
group	O
in	O
the	O
CoA	B-chemical
-	I-chemical
thioester	I-chemical
might	O
point	O
in	O
a	O
different	O
direction	O
.	O

Indeed	O
,	O
in	O
the	O
majority	O
of	O
PduLs	B-protein_type
encoded	O
in	O
pvm	B-gene
loci	I-gene
,	O
Gln77	B-residue_name_number
is	O
substituted	O
by	O
either	O
a	O
Tyr	B-residue_name
or	O
Phe	B-residue_name
,	O
whereas	O
it	O
is	O
typically	O
a	O
Gln	B-residue_name
or	O
Glu	B-residue_name
in	O
PduLs	B-protein_type
in	O
all	O
other	O
BMC	B-complex_assembly
types	O
that	O
degrade	O
acetyl	B-chemical
-	I-chemical
or	O
propionyl	B-chemical
-	I-chemical
CoA	I-chemical
.	O
A	O
comparison	B-experimental_method
of	O
the	O
PduL	B-protein_type
active	B-site
site	I-site
to	O
that	O
of	O
the	O
functionally	O
identical	O
Pta	B-protein_type
suggests	O
that	O
the	O
two	O
enzymes	O
have	O
distinctly	O
different	O
mechanisms	O
.	O

The	O
two	O
high	O
-	O
resolution	O
crystal	B-evidence
structures	I-evidence
presented	O
here	O
will	O
serve	O
as	O
the	O
foundation	O
for	O
mechanistic	O
studies	O
on	O
this	O
noncanonical	O
PTAC	B-protein_type
enzyme	O
to	O
determine	O
how	O
the	O
dimetal	B-site
active	I-site
site	I-site
functions	O
to	O
catalyze	O
both	O
forward	O
and	O
reverse	O
reactions	O
.	O

A	O
detailed	O
understanding	O
of	O
the	O
underlying	O
principles	O
governing	O
the	O
assembly	O
and	O
internal	O
structural	O
organization	O
of	O
BMCs	B-complex_assembly
is	O
a	O
requisite	O
for	O
synthetic	O
biologists	O
to	O
design	O
custom	O
nanoreactors	O
that	O
use	O
BMC	B-complex_assembly
architectures	O
as	O
a	O
template	O
.	O

Furthermore	O
,	O
given	O
the	O
growing	O
number	O
of	O
metabolosomes	B-complex_assembly
implicated	O
in	O
pathogenesis	O
,	O
the	O
PduL	B-protein_type
structure	B-evidence
will	O
be	O
useful	O
in	O
the	O
development	O
of	O
therapeutics	O
.	O

EctC	B-protein
forms	O
a	O
dimer	B-oligomeric_state
with	O
a	O
head	B-protein_state
-	I-protein_state
to	I-protein_state
-	I-protein_state
tail	I-protein_state
arrangement	O
,	O
both	O
in	O
solution	O
and	O
in	O
the	O
crystal	B-evidence
structure	I-evidence
.	O

We	O
show	O
for	O
the	O
first	O
time	O
that	O
ectoine	B-protein_type
synthase	I-protein_type
harbors	O
a	O
catalytically	O
important	O
metal	B-chemical
co	O
-	O
factor	O
;	O
metal	B-experimental_method
depletion	I-experimental_method
and	I-experimental_method
reconstitution	I-experimental_method
experiments	I-experimental_method
suggest	O
that	O
EctC	B-protein
is	O
probably	O
an	O
iron	B-protein_state
-	I-protein_state
dependent	I-protein_state
enzyme	O
.	O

Structure	B-experimental_method
-	I-experimental_method
guided	I-experimental_method
site	I-experimental_method
-	I-experimental_method
directed	I-experimental_method
mutagenesis	I-experimental_method
experiments	O
targeting	O
amino	O
acid	O
residues	O
that	O
are	O
evolutionarily	B-protein_state
highly	I-protein_state
conserved	I-protein_state
among	O
the	O
extended	O
EctC	B-protein_type
protein	I-protein_type
family	I-protein_type
,	O
including	O
those	O
forming	O
the	O
presumptive	O
iron	B-site
-	I-site
binding	I-site
site	I-site
,	O
were	O
conducted	O
to	O
functionally	O
analyze	O
the	O
properties	O
of	O
the	O
resulting	O
EctC	B-protein
variants	O
.	O

This	O
stereospecific	O
chemical	O
modification	O
of	O
ectoine	B-chemical
(	O
Fig	O
1	O
)	O
is	O
catalyzed	O
by	O
the	O
ectoine	B-protein_type
hydroxylase	I-protein_type
(	O
EctD	B-protein_type
)	O
(	O
EC	O
1	O
.	O
14	O
.	O
11	O
),	O
a	O
member	O
of	O
the	O
non	B-protein_type
-	I-protein_type
heme	I-protein_type
containing	I-protein_type
iron	I-protein_type
(	I-protein_type
II	I-protein_type
)	I-protein_type
and	I-protein_type
2	I-protein_type
-	I-protein_type
oxoglutarate	I-protein_type
-	I-protein_type
dependent	I-protein_type
dioxygenase	I-protein_type
superfamily	I-protein_type
.	O

Scheme	O
of	O
the	O
ectoine	B-chemical
and	O
5	B-chemical
-	I-chemical
hydroxyectoine	I-chemical
biosynthetic	O
pathway	O
.	O

The	O
EctC	B-protein
protein	O
forms	O
a	O
dimer	B-oligomeric_state
in	O
solution	O
and	O
our	O
structural	B-experimental_method
analysis	I-experimental_method
identifies	O
it	O
as	O
a	O
member	O
of	O
the	O
cupin	B-protein_type
superfamily	I-protein_type
.	O

(	O
Sa	B-species
)	O
EctC	B-protein
is	O
a	O
highly	O
salt	O
-	O
tolerant	O
enzyme	O
since	O
it	O
exhibited	O
substantial	O
enzyme	O
activity	O
even	O
at	O
NaCl	B-chemical
and	O
KCl	B-chemical
concentrations	O
of	O
1	O
M	O
in	O
the	O
assay	O
buffer	O
(	O
S3c	O
and	O
S3d	O
Fig	O
).	O

The	O
ectoine	B-protein_type
synthase	I-protein_type
is	O
a	O
metal	B-protein_type
-	I-protein_type
containing	I-protein_type
protein	I-protein_type

The	O
amino	O
acid	O
sequences	O
of	O
20	O
selected	O
EctC	B-protein_type
-	I-protein_type
type	I-protein_type
proteins	I-protein_type
are	O
compared	O
.	O

A	O
metal	B-chemical
cofactor	O
is	O
important	O
for	O
the	O
catalytic	O
activity	O
of	O
EctC	B-protein

To	O
address	O
these	O
questions	O
,	O
we	O
incubated	B-experimental_method
the	O
(	O
Sa	B-species
)	O
EctC	B-protein
enzyme	O
with	B-experimental_method
increasing	I-experimental_method
concentrations	I-experimental_method
of	O
the	O
metal	B-chemical
chelator	O
ethylene	B-chemical
-	I-chemical
diamine	I-chemical
-	I-chemical
tetraacetic	I-chemical
-	I-chemical
acid	I-chemical
(	O
EDTA	B-chemical
)	O
and	O
subsequently	O
assayed	O
ectoine	B-protein_type
synthase	I-protein_type
activity	O
.	O

The	O
EctC	B-protein
-	O
catalyzed	O
ring	O
-	O
closure	O
of	O
N	B-chemical
-	I-chemical
γ	I-chemical
-	I-chemical
ADABA	I-chemical
to	O
form	O
ectoine	B-chemical
exhibited	O
Michaelis	B-experimental_method
-	I-experimental_method
Menten	I-experimental_method
-	I-experimental_method
kinetics	I-experimental_method
with	O
an	O
apparent	O
Km	B-evidence
of	O
4	O
.	O
9	O
±	O
0	O
.	O
5	O
mM	O
,	O
a	O
vmax	B-evidence
of	O
25	O
.	O
0	O
±	O
0	O
.	O
8	O
U	O
/	O
mg	O
and	O
a	O
kcat	B-evidence
of	O
7	O
.	O
2	O
s	O
-	O
1	O
(	O
S4a	O
Fig	O
).	O

(	O
Sa	B-species
)	O
EctC	B-protein
catalyzed	O
this	O
reaction	O
with	O
Michaelis	B-experimental_method
-	I-experimental_method
Menten	I-experimental_method
-	I-experimental_method
kinetics	I-experimental_method
exhibiting	O
an	O
apparent	O
Km	B-evidence
of	O
25	O
.	O
4	O
±	O
2	O
.	O
9	O
mM	O
,	O
a	O
vmax	B-evidence
of	O
24	O
.	O
6	O
±	O
1	O
.	O
0	O
U	O
/	O
mg	O
and	O
a	O
kcat	B-evidence
0	O
.	O
6	O
s	O
-	O
1	O
(	O
S4b	O
Fig	O
).	O

However	O
,	O
two	O
crystal	B-evidence
forms	I-evidence
of	O
the	O
(	O
Sa	B-species
)	O
EctC	B-protein
protein	O
in	O
the	O
absence	B-protein_state
of	I-protein_state
the	O
substrate	O
were	O
obtained	O
.	O

Overall	O
fold	O
of	O
the	O
(	O
Sa	B-species
)	O
EctC	B-protein
protein	O

The	O
β	B-structure_element
-	I-structure_element
strands	I-structure_element
are	O
numbered	O
β1	B-structure_element
-	I-structure_element
β11	I-structure_element
and	O
the	O
helices	B-structure_element
α	B-structure_element
-	I-structure_element
I	I-structure_element
to	I-structure_element
α	I-structure_element
-	I-structure_element
II	I-structure_element
.	O

The	O
entrance	O
to	O
the	O
active	B-site
site	I-site
of	O
the	O
ectoine	B-protein_type
synthase	I-protein_type
is	O
marked	O
.	O

(	O
c	O
)	O
Overlay	B-experimental_method
of	O
the	O
“	O
semi	B-protein_state
-	I-protein_state
closed	I-protein_state
”	O
and	O
“	O
open	B-protein_state
”	O
(	O
Sa	B-species
)	O
EctC	B-protein
structures	B-evidence
.	O

Hence	O
,	O
(	O
Sa	B-species
)	O
EctC	B-protein
adopts	O
an	O
overall	O
bowl	O
shape	O
in	O
which	O
one	O
side	O
is	O
opened	O
towards	O
the	O
solvent	O
(	O
Fig	O
4a	O
to	O
4c	O
).	O

The	O
formation	O
of	O
this	O
α	B-structure_element
-	I-structure_element
II	I-structure_element
helix	I-structure_element
induces	O
a	O
reorientation	O
and	O
shift	O
of	O
a	O
long	O
unstructured	B-protein_state
loop	B-structure_element
(	O
as	O
observed	O
in	O
the	O
“	O
open	B-protein_state
”	O
structure	B-evidence
)	O
connecting	O
β4	B-structure_element
and	O
β6	B-structure_element
,	O
resulting	O
in	O
the	O
formation	O
of	O
the	O
stable	B-protein_state
β	B-structure_element
-	I-structure_element
strand	I-structure_element
β5	B-structure_element
as	O
observed	O
in	O
the	O
“	O
semi	B-protein_state
-	I-protein_state
closed	I-protein_state
”	O
state	O
of	O
the	O
(	O
Sa	B-species
)	O
EctC	B-protein
protein	O
(	O
Fig	O
4a	O
).	O

Both	O
the	O
SEC	B-experimental_method
analysis	O
and	O
the	O
HPLC	B-experimental_method
-	I-experimental_method
MALS	I-experimental_method
experiments	O
(	O
S2b	O
Fig	O
)	O
have	O
shown	O
that	O
the	O
ectoine	B-protein_type
synthase	I-protein_type
from	O
S	B-species
.	I-species
alaskensis	I-species
is	O
a	O
dimer	B-oligomeric_state
in	O
solution	O
.	O

The	O
crystal	B-evidence
structure	I-evidence
of	O
this	O
protein	O
reflects	O
this	O
quaternary	O
arrangement	O
.	O

As	O
calculated	O
with	O
PDBePISA	B-experimental_method
,	O
the	O
surface	O
area	O
buried	O
upon	O
dimer	B-oligomeric_state
formation	O
is	O
1462	O
Å2	O
,	O
which	O
is	O
20	O
.	O
5	O
%	O
of	O
the	O
total	O
accessible	O
surface	O
of	O
a	O
monomer	B-oligomeric_state
of	O
this	O
protein	O
.	O

In	O
the	O
“	O
open	B-protein_state
”	O
(	O
Sa	B-species
)	O
EctC	B-protein
structure	B-evidence
,	O
one	O
monomer	B-oligomeric_state
is	O
present	O
in	O
the	O
asymmetric	O
unit	O
.	O

We	O
therefore	O
inspected	O
the	O
crystal	O
packing	B-experimental_method
and	O
analyzed	O
the	O
monomer	B-oligomeric_state
-	O
monomer	B-oligomeric_state
interactions	O
with	O
symmetry	O
related	O
molecules	O
to	O
elucidate	O
whether	O
a	O
physiologically	O
relevant	O
dimer	B-oligomeric_state
could	O
be	O
deduced	O
from	O
this	O
crystal	B-evidence
form	I-evidence
as	O
well	O
.	O

These	O
additional	O
amino	O
acids	O
fold	O
into	O
a	O
small	B-structure_element
helix	I-structure_element
,	O
which	O
seals	O
the	O
open	B-protein_state
cavity	B-site
of	O
the	O
cupin	B-structure_element
-	I-structure_element
fold	I-structure_element
of	O
the	O
(	O
Sa	B-species
)	O
EctC	B-protein
protein	O
(	O
Fig	O
4a	O
).	O

As	O
a	O
result	O
,	O
the	O
newly	O
formed	O
β	B-structure_element
-	I-structure_element
strand	I-structure_element
β5	B-structure_element
is	O
reoriented	O
and	O
moved	O
by	O
2	O
.	O
4	O
Å	O
within	O
the	O
“	O
semi	B-protein_state
-	I-protein_state
closed	I-protein_state
”	O
(	O
Sa	B-species
)	O
EctC	B-protein
structure	B-evidence
(	O
Fig	O
4a	O
to	O
4c	O
).	O

Therefore	O
the	O
sealing	O
of	O
the	O
cupin	B-structure_element
fold	I-structure_element
,	O
as	O
described	O
above	O
,	O
seem	O
to	O
have	O
an	O
indirect	O
influence	O
on	O
the	O
architecture	O
of	O
the	O
postulated	O
iron	B-site
-	I-site
binding	I-site
site	I-site
.	O

In	O
the	O
“	O
open	B-protein_state
”	O
structure	B-evidence
of	O
the	O
(	O
Sa	B-species
)	O
EctC	B-protein
protein	O
,	O
this	O
interaction	O
does	O
not	O
occur	O
since	O
Glu	B-residue_name_number
-	I-residue_name_number
115	I-residue_name_number
is	O
rotated	O
outwards	O
(	O
Fig	O
6a	O
and	O
6b	O
).	O

Hence	O
,	O
one	O
might	O
speculate	O
that	O
this	O
missing	O
interaction	O
might	O
be	O
responsible	O
for	O
the	O
flexibility	O
of	O
the	O
carboxy	B-structure_element
-	I-structure_element
terminus	I-structure_element
in	O
the	O
“	O
open	B-protein_state
”	O
(	O
Sa	B-species
)	O
EctC	B-protein
structure	B-evidence
and	O
consequently	O
results	O
in	O
less	O
well	O
defined	O
electron	B-evidence
density	I-evidence
in	O
this	O
region	O
.	O

These	O
distances	O
are	O
to	O
long	O
when	O
compared	O
to	O
other	O
iron	B-site
binding	I-site
sites	I-site
,	O
a	O
fact	O
that	O
might	O
be	O
caused	O
by	O
the	O
absence	B-protein_state
of	I-protein_state
the	O
proper	O
substrate	O
in	O
the	O
(	O
Sa	B-species
)	O
EctC	B-protein
crystal	B-evidence
structure	I-evidence
.	O

Since	O
both	O
the	O
refinement	O
and	O
the	O
distance	O
did	O
not	O
clearly	O
identify	O
an	O
iron	B-chemical
molecule	O
,	O
we	O
decided	O
to	O
conservatively	O
place	O
a	O
water	B-chemical
molecule	O
at	O
this	O
position	O
.	O

Only	O
His	B-residue_name_number
-	I-residue_name_number
93	I-residue_name_number
is	O
slightly	O
rotated	O
inwards	O
in	O
the	O
“	O
semi	B-protein_state
-	I-protein_state
closed	I-protein_state
”	O
structure	B-evidence
,	O
most	O
likely	O
due	O
to	O
formation	O
of	O
β	B-structure_element
-	I-structure_element
strand	I-structure_element
β5	B-structure_element
as	O
described	O
above	O
.	O

Taken	O
together	O
,	O
this	O
observations	O
indicate	O
,	O
that	O
the	O
architecture	O
of	O
the	O
presumptive	O
iron	B-site
-	I-site
binding	I-site
site	I-site
is	O
pre	O
-	O
set	O
for	O
the	O
binding	O
of	O
the	O
catalytically	O
important	O
metal	B-chemical
by	O
the	O
ectoine	B-protein_type
synthase	I-protein_type
.	O

This	O
is	O
in	O
contrast	O
to	O
the	O
high	O
-	O
resolution	O
“	O
open	B-protein_state
”	O
structure	B-evidence
of	O
the	O
(	O
Sa	B-species
)	O
EctC	B-protein
protein	O
where	O
no	O
additional	O
electron	B-evidence
density	I-evidence
was	O
observed	O
after	O
refinement	O
.	O

When	O
analyzing	O
the	O
interactions	O
of	O
this	O
compound	O
within	O
the	O
(	O
Sa	B-species
)	O
EctC	B-protein
protein	O
,	O
we	O
found	O
that	O
it	O
is	O
bound	B-protein_state
via	O
interactions	O
with	O
Trp	B-residue_name_number
-	I-residue_name_number
21	I-residue_name_number
and	O
Ser	B-residue_name_number
-	I-residue_name_number
23	I-residue_name_number
of	O
β	B-structure_element
-	I-structure_element
sheet	I-structure_element
β3	B-structure_element
,	O
Thr	B-residue_name_number
-	I-residue_name_number
40	I-residue_name_number
located	O
in	O
β	B-structure_element
-	I-structure_element
sheet	I-structure_element
β4	B-structure_element
,	O
and	O
Cys	B-residue_name_number
-	I-residue_name_number
105	I-residue_name_number
and	O
Phe	B-residue_name_number
-	I-residue_name_number
107	I-residue_name_number
,	O
which	O
are	O
both	O
part	O
of	O
β	B-structure_element
-	I-structure_element
sheet	I-structure_element
β11	B-structure_element
.	O

As	O
described	O
above	O
,	O
the	O
side	O
chains	O
of	O
Glu	B-residue_name_number
-	I-residue_name_number
57	I-residue_name_number
,	O
Tyr	B-residue_name_number
-	I-residue_name_number
85	I-residue_name_number
,	O
and	O
His	B-residue_name_number
-	I-residue_name_number
93	I-residue_name_number
are	O
probably	O
involved	O
in	O
iron	B-chemical
binding	O
(	O
Table	O
1	O
and	O
Fig	O
6a	O
).	O

However	O
,	O
the	O
Cys	B-mutant
-	I-mutant
105	I-mutant
/	I-mutant
Ala	I-mutant
variant	B-protein_state
was	O
practically	O
catalytically	B-protein_state
inactive	I-protein_state
while	O
largely	O
maintaining	O
its	O
iron	B-chemical
content	O
(	O
Table	O
1	O
).	O

We	O
observed	O
two	O
amino	B-experimental_method
acid	I-experimental_method
substitutions	I-experimental_method
that	O
simultaneously	O
strongly	O
affected	O
enzyme	O
activity	O
and	O
iron	B-chemical
content	O
;	O
these	O
were	O
the	O
Tyr	B-mutant
-	I-mutant
52	I-mutant
/	I-mutant
Ala	I-mutant
and	O
the	O
His	B-mutant
-	I-mutant
55	I-mutant
/	I-mutant
Ala	I-mutant
(	O
Sa	B-species
)	O
EctC	B-protein
protein	O
variants	O
(	O
Table	O
1	O
).	O

The	O
carboxy	B-structure_element
-	I-structure_element
terminal	I-structure_element
region	I-structure_element
of	O
the	O
(	O
Sa	B-species
)	O
EctC	B-protein
protein	O
is	O
held	O
in	O
its	O
position	O
via	O
an	O
interaction	O
of	O
Glu	B-residue_name_number
-	I-residue_name_number
115	I-residue_name_number
with	O
His	B-residue_name_number
-	I-residue_name_number
55	I-residue_name_number
,	O
where	O
His	B-residue_name_number
-	I-residue_name_number
55	I-residue_name_number
in	O
turn	O
interacts	O
with	O
Pro	B-residue_name_number
-	I-residue_name_number
110	I-residue_name_number
(	O
Fig	O
6a	O
and	O
6b	O
).	O

The	O
Glu	B-mutant
-	I-mutant
115	I-mutant
/	I-mutant
Ala	I-mutant
mutant	B-protein_state
possessed	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
levels	O
of	O
iron	B-chemical
,	O
whereas	O
the	O
iron	B-chemical
content	O
of	O
the	O
His	B-mutant
-	I-mutant
55	I-mutant
/	I-mutant
Ala	I-mutant
substitutions	O
dropped	O
to	O
15	O
%	O
of	O
the	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
level	O
(	O
Table	O
1	O
).	O

As	O
a	O
consequence	O
of	O
the	O
structural	O
relatedness	O
of	O
EctC	B-protein
and	O
RemF	B-protein
and	O
the	O
type	O
of	O
chemical	O
reaction	O
these	O
two	O
enzymes	O
catalyze	O
,	O
is	O
now	O
understandable	O
why	O
bona	O
fide	O
EctC	B-protein_type
-	I-protein_type
type	I-protein_type
proteins	I-protein_type
are	O
frequently	O
(	O
mis	O
)-	O
annotated	O
in	O
microbial	B-taxonomy_domain
genome	O
sequences	O
as	O
“	O
RemF	B-protein_type
-	I-protein_type
like	I-protein_type
”	O
proteins	O
.	O

Except	O
for	O
some	O
cupin	B-protein_type
-	I-protein_type
related	I-protein_type
proteins	I-protein_type
that	O
seem	O
to	O
function	O
as	O
metallo	B-protein_type
-	I-protein_type
chaperones	I-protein_type
,	O
the	O
bound	B-protein_state
metal	B-chemical
is	O
typically	O
an	O
essential	O
part	O
of	O
the	O
active	B-site
sites	I-site
.	O

The	O
architecture	O
of	O
the	O
metal	B-site
center	I-site
of	O
ectoine	B-protein_type
synthase	I-protein_type
seems	O
to	O
be	O
subjected	O
to	O
considerable	O
evolutionary	O
constraints	O
.	O

This	O
set	O
of	O
data	O
and	O
the	O
fact	O
that	O
the	O
targeted	O
residues	O
are	O
strongly	B-protein_state
conserved	I-protein_state
among	O
EctC	B-protein_type
-	I-protein_type
type	I-protein_type
proteins	I-protein_type
(	O
Fig	O
2	O
)	O
is	O
consistent	O
with	O
their	O
potential	O
role	O
in	O
N	B-chemical
-	I-chemical
γ	I-chemical
-	I-chemical
ADABA	I-chemical
binding	O
or	O
enzyme	O
catalysis	O
.	O

Because	O
microbial	B-taxonomy_domain
ectoine	B-chemical
producers	O
can	O
colonize	O
ecological	O
niches	O
with	O
rather	O
different	O
physicochemical	O
attributes	O
,	O
it	O
seems	O
promising	O
to	O
exploit	O
this	O
considerable	O
biodiversity	O
to	O
identify	O
EctC	B-protein_type
proteins	I-protein_type
with	O
enhanced	O
protein	O
stability	O
.	O

Structures	B-evidence
of	O
human	B-species
ADAR2	B-protein
bound	B-protein_state
to	I-protein_state
dsRNA	B-chemical
reveal	O
base	O
-	O
flipping	O
mechanism	O
and	O
basis	O
for	O
site	O
selectivity	O

We	O
then	O
evaluated	O
the	O
importance	O
of	O
protein	O
-	O
RNA	B-chemical
contacts	O
using	O
structure	B-experimental_method
-	I-experimental_method
guided	I-experimental_method
mutagenesis	I-experimental_method
and	O
RNA	B-experimental_method
-	I-experimental_method
modification	I-experimental_method
experiments	I-experimental_method
coupled	O
with	O
adenosine	B-experimental_method
deamination	I-experimental_method
kinetics	I-experimental_method
.	O

For	O
trapping	O
hADAR2d	B-mutant
bound	B-protein_state
to	I-protein_state
RNA	B-chemical
for	O
crystallography	B-experimental_method
,	O
we	O
incorporated	O
8	B-chemical
-	I-chemical
azanebularine	I-chemical
into	O
duplex	B-structure_element
RNAs	I-structure_element
shown	O
recently	O
to	O
be	O
excellent	O
substrates	O
for	O
deamination	O
by	O
hADAR2d	B-mutant
(	O
for	O
duplex	O
sequence	O
see	O
Fig	O
.	O
1c	O
)	O
(	O
for	O
characterization	O
of	O
protein	O
–	O
RNA	B-chemical
complex	O
see	O
Supplementary	O
Fig	O
.	O
1	O
).	O

In	O
each	O
of	O
these	O
complexes	O
,	O
the	O
protein	O
binds	O
the	O
RNA	B-chemical
on	O
one	O
face	O
of	O
the	O
duplex	O
over	O
~	O
20	O
bp	O
using	O
a	O
positively	O
charged	O
surface	O
near	O
the	O
zinc	B-site
-	I-site
containing	I-site
active	I-site
site	I-site
(	O
Fig	O
.	O
2	O
,	O
Supplementary	O
Fig	O
.	O
2a	O
).	O

The	O
side	O
chain	O
of	O
E396	B-residue_name_number
H	B-bond_interaction
-	I-bond_interaction
bonds	I-bond_interaction
to	O
purine	B-chemical
N1	O
and	O
O6	O
.	O

Lastly	O
,	O
the	O
side	O
chain	O
of	O
V351	B-residue_name_number
provides	O
a	O
hydrophobic	B-site
surface	I-site
for	O
interaction	O
with	O
the	O
nucleobase	O
of	O
the	O
edited	B-protein_state
nucleotide	B-chemical
.	O

The	O
side	O
chain	O
of	O
this	O
amino	O
acid	O
penetrates	O
the	O
helix	O
where	O
it	O
occupies	O
the	O
space	O
vacated	O
by	O
the	O
flipped	B-protein_state
out	I-protein_state
base	B-chemical
and	O
H	B-bond_interaction
-	I-bond_interaction
bonds	I-bond_interaction
to	O
the	O
complementary	O
strand	O
orphaned	B-protein_state
base	B-chemical
and	O
to	O
the	O
2	O
’	O
hydroxyl	O
of	O
the	O
nucleotide	O
immediately	O
5	O
’	O
to	O
the	O
editing	B-site
site	I-site
(	O
Figs	O
.	O
3b	O
,	O
3c	O
).	O

In	O
the	O
four	O
structures	B-evidence
reported	O
here	O
,	O
three	O
different	O
combinations	O
of	O
helix	O
-	O
penetrating	O
residue	O
and	O
orphan	B-protein_state
base	B-chemical
are	O
observed	O
(	O
i	O
.	O
e	O
.	O
E488	B-residue_name_number
+	O
U	B-residue_name
,	O
E488	B-residue_name_number
+	O
C	B-residue_name
and	O
Q488	B-residue_name_number
+	O
C	B-residue_name
)	O
and	O
all	O
three	O
combinations	O
show	O
the	O
same	O
side	O
chain	O
and	O
base	O
positions	O
(	O
Figs	O
.	O
3b	O
,	O
3c	O
,	O
Supplementary	O
Fig	O
.	O
4a	O
for	O
overlay	B-experimental_method
of	O
all	O
three	O
).	O

In	O
the	O
complex	B-protein_state
with	I-protein_state
hADAR2d	B-mutant
WT	B-protein_state
and	O
the	O
Bdf2	B-chemical
-	I-chemical
U	I-chemical
duplex	I-chemical
,	O
a	O
similar	O
interaction	O
is	O
observed	O
with	O
the	O
E488	B-residue_name_number
backbone	O
NH	O
hydrogen	B-bond_interaction
bonded	I-bond_interaction
to	O
the	O
uracil	B-residue_name
O2	O
and	O
the	O
E488	B-residue_name_number
side	O
chain	O
H	B-bond_interaction
-	I-bond_interaction
bonded	I-bond_interaction
to	O
the	O
uracil	B-residue_name
N3H	O
(	O
Fig	O
.	O
3c	O
).	O

The	O
flipping	B-structure_element
loop	I-structure_element
in	O
ADAR2	B-protein
(	O
i	O
.	O
e	O
.	O
aa487	O
–	B-residue_range
489	I-residue_range
)	O
also	O
has	O
the	O
helix	O
-	O
penetrating	O
residue	O
flanked	O
by	O
glycines	B-residue_name
.	O

The	O
ADAR	B-protein_type
-	O
induced	O
distortion	O
in	O
RNA	B-chemical
conformation	O
results	O
in	O
a	O
kink	B-structure_element
in	O
the	O
RNA	B-chemical
strand	O
opposite	O
the	O
editing	B-site
site	I-site
(	O
Fig	O
.	O
4b	O
).	O

As	O
described	O
above	O
,	O
the	O
base	O
pair	O
including	O
the	O
5	O
’	O
nearest	O
neighbor	O
U	B-residue_name
(	O
U11	B-residue_name_number
-	O
A13	B-residue_name_number
’	O
in	O
the	O
Bdf2	B-chemical
duplex	O
)	O
is	O
shifted	O
from	O
the	O
position	O
it	O
would	O
occupy	O
in	O
a	O
typical	O
A	B-structure_element
-	I-structure_element
form	I-structure_element
helix	I-structure_element
to	O
accommodate	O
the	O
loop	B-structure_element
(	O
Fig	O
.	O
4a	O
).	O

Modeling	O
a	O
G	B-structure_element
-	I-structure_element
C	I-structure_element
or	I-structure_element
C	I-structure_element
-	I-structure_element
G	I-structure_element
pair	I-structure_element
at	O
this	O
position	O
(	O
i	O
.	O
e	O
.	O
5	O
’	O
G	B-residue_name
or	O
5	O
’	O
C	B-residue_name
)	O
suggests	O
a	O
2	O
-	O
amino	O
group	O
in	O
the	O
minor	B-site
groove	I-site
would	O
clash	O
with	O
the	O
protein	O
at	O
G489	B-residue_name_number
(	O
Fig	O
.	O
5a	O
,	O
Supplementary	O
Fig	O
.	O
7c	O
).	O

Indeed	O
,	O
replacing	O
the	O
U	B-structure_element
-	I-structure_element
A	I-structure_element
pair	I-structure_element
adjacent	O
to	O
the	O
editing	B-site
site	I-site
with	O
a	O
C	B-structure_element
-	I-structure_element
G	I-structure_element
pair	I-structure_element
in	O
the	O
Gli1	B-protein
duplex	O
substrate	O
resulted	O
in	O
an	O
80	O
%	O
reduction	O
in	O
the	O
rate	O
of	O
hADAR2d	B-mutant
-	O
catalyzed	O
deamination	O
(	O
Figs	O
.	O
5b	O
,	O
5c	O
).	O

To	O
determine	O
whether	O
this	O
effect	O
arises	O
from	O
an	O
increase	O
in	O
local	O
duplex	O
stability	O
from	O
the	O
C	O
-	O
G	O
for	O
U	O
-	O
A	O
substitution	O
or	O
from	O
the	O
presence	O
of	O
the	O
2	O
-	O
amino	O
group	O
,	O
we	O
replaced	O
the	O
U	B-structure_element
-	I-structure_element
A	I-structure_element
pair	I-structure_element
with	O
a	O
U	B-structure_element
-	I-structure_element
2	I-structure_element
-	I-structure_element
aminopurine	I-structure_element
(	I-structure_element
2AP	I-structure_element
)	I-structure_element
pair	I-structure_element
.	O

RNA	B-structure_element
-	I-structure_element
binding	I-structure_element
loops	I-structure_element
of	O
the	O
ADAR	B-protein_type
catalytic	B-structure_element
domain	I-structure_element

In	O
addition	O
,	O
mutation	B-experimental_method
of	O
G593	B-residue_name_number
to	O
glutamic	B-residue_name
acid	I-residue_name
(	O
G593E	B-mutant
)	O
resulted	O
in	O
a	O
nearly	O
two	O
orders	O
of	O
magnitude	O
reduction	O
in	O
rate	O
,	O
consistent	O
with	O
proximity	O
of	O
this	O
residue	O
to	O
the	O
negatively	O
charged	O
phosphodiester	O
backbone	O
of	O
the	O
RNA	B-chemical
(	O
Fig	O
.	O
6c	O
).	O

This	O
loop	B-structure_element
binds	O
the	O
RNA	B-structure_element
duplex	I-structure_element
contacting	O
the	O
minor	B-site
groove	I-site
near	O
the	O
editing	B-site
site	I-site
and	O
inserting	O
into	O
the	O
adjacent	O
major	B-site
groove	I-site
(	O
Fig	O
.	O
6e	O
).	O

Thus	O
,	O
this	O
system	O
is	O
not	O
illustrative	O
of	O
base	O
flipping	O
from	O
a	O
normal	B-protein_state
duplex	O
and	O
does	O
not	O
involve	O
an	O
enzyme	O
that	O
must	O
carryout	O
a	O
chemical	O
reaction	O
on	O
the	O
flipped	B-protein_state
out	I-protein_state
nucleotide	B-chemical
.	O

Thus	O
,	O
the	O
E488	B-residue_name_number
side	O
chain	O
directly	O
contacts	O
each	O
orphan	B-protein_state
base	B-chemical
,	O
likely	O
by	O
accepting	O
an	O
H	B-bond_interaction
-	I-bond_interaction
bond	I-bond_interaction
from	O
uracil	B-residue_name
N3H	O
or	O
by	O
donating	O
an	O
H	B-bond_interaction
-	I-bond_interaction
bond	I-bond_interaction
to	O
cytidine	B-residue_name
N3	O
.	O

Aicardi	O
-	O
Goutieres	O
Syndrome	O
(	O
AGS	O
)	O
and	O
Dyschromatosis	O
Symmetrica	O
Hereditaria	O
(	O
DSH	O
)	O
are	O
human	B-species
diseases	O
caused	O
by	O
mutations	O
in	O
the	O
human	B-species
ADAR1	B-protein
gene	O
and	O
several	O
of	O
the	O
disease	O
-	O
associated	O
mutations	O
are	O
found	O
in	O
the	O
deaminase	B-structure_element
domain	I-structure_element
.	O

An	O
arginine	B-residue_name
at	O
this	O
position	O
would	O
preclude	O
close	O
approach	O
of	O
the	O
flipping	B-structure_element
loop	I-structure_element
to	O
the	O
RNA	B-chemical
,	O
preventing	O
E1008	B-residue_name_number
insertion	O
and	O
base	O
flipping	O
into	O
the	O
active	B-site
site	I-site
(	O
Supplementary	O
Fig	O
.	O
8b	O
).	O

This	O
is	O
consistent	O
with	O
the	O
observation	O
that	O
the	O
G1007R	B-mutant
mutation	O
in	O
hADAR1	B-protein
inhibits	O
RNA	B-chemical
editing	O
activity	O
.	O

In	O
addition	O
,	O
this	O
work	O
provides	O
a	O
basis	O
for	O
understanding	O
the	O
role	O
of	O
the	O
ADAR	B-protein_type
catalytic	B-structure_element
domain	I-structure_element
in	O
determining	O
editing	B-site
site	I-site
selectivity	O
and	O
additional	O
structural	O
context	O
to	O
evaluate	O
the	O
impact	O
of	O
ADAR	B-protein_type
mutations	O
associated	O
with	O
human	B-species
disease	O
.	O

Human	B-species
ADAR2	B-protein
and	O
modified	O
RNAs	B-chemical
for	O
crystallography	B-experimental_method

A	O
transparent	O
surface	O
is	O
shown	O
for	O
the	O
hADAR2d	B-mutant
protein	O
.	O

c	O
,	O
Summary	O
of	O
the	O
contacts	O
between	O
hADAR2d	B-mutant
E488Q	B-mutant
and	O
the	O
Bdf2	B-chemical
-	I-chemical
C	I-chemical
RNA	I-chemical
duplex	I-chemical
.	O

b	O
,	O
Orphan	B-protein_state
nucleotide	B-chemical
recognition	O
in	O
the	O
hADAR2d	B-complex_assembly
E488Q	I-complex_assembly
–	I-complex_assembly
Bdf2	I-complex_assembly
-	I-complex_assembly
C	I-complex_assembly
complex	O
.	O

a	O
,	O
The	O
minor	B-site
groove	I-site
edge	O
of	O
the	O
U11	B-residue_name_number
-	O
A13	B-residue_name_number
’	O
base	O
pair	O
from	O
the	O
Bdf2	B-chemical
duplex	I-chemical
approaches	O
G489	B-residue_name_number
;	O
model	O
with	O
a	O
C	B-structure_element
-	I-structure_element
G	I-structure_element
pair	I-structure_element
at	O
this	O
position	O
suggests	O
a	O
clash	O
with	O
the	O
G	B-residue_name
2	O
-	O
amino	O
group	O
b	O
,	O
RNA	B-structure_element
duplex	I-structure_element
substrates	O
prepared	O
with	O
different	O
5	O
’	O
nearest	O
neighbor	O
nucleotides	O
adjacent	O
to	O
editing	B-site
site	I-site
indicated	O
in	O
red	O
(	O
2AP	B-structure_element
=	O
2	B-structure_element
-	I-structure_element
aminopurine	I-structure_element
).	O

Regnase	B-protein
-	I-protein
1	I-protein
is	O
an	O
RNase	B-protein_type
that	O
directly	O
cleaves	O
mRNAs	B-chemical
of	O
inflammatory	O
genes	O
such	O
as	O
IL	B-protein_type
-	I-protein_type
6	I-protein_type
and	O
IL	B-protein_type
-	I-protein_type
12p40	I-protein_type
,	O
and	O
negatively	O
regulates	O
cellular	O
inflammatory	O
responses	O
.	O

Regnase	B-protein
-	I-protein
1	I-protein
is	O
a	O
member	O
of	O
Regnase	B-protein_type
family	I-protein_type
and	O
is	O
composed	O
of	O
a	O
PilT	B-structure_element
N	I-structure_element
-	I-structure_element
terminus	I-structure_element
like	I-structure_element
(	O
PIN	B-structure_element
)	O
domain	O
followed	O
by	O
a	O
CCCH	B-structure_element
-	I-structure_element
type	I-structure_element
zinc	I-structure_element
–	I-structure_element
finger	I-structure_element
(	O
ZF	B-structure_element
)	O
domain	O
,	O
which	O
are	O
conserved	B-protein_state
among	O
Regnase	B-protein_type
family	I-protein_type
members	I-protein_type
.	O

The	O
structure	B-evidence
combined	O
with	O
functional	O
analyses	O
revealed	O
that	O
four	O
catalytically	O
important	O
Asp	B-residue_name
residues	O
form	O
the	O
catalytic	B-site
center	I-site
and	O
stabilize	O
Mg2	B-chemical
+	I-chemical
binding	O
that	O
is	O
crucial	O
for	O
RNase	B-protein_type
activity	O
.	O

Our	O
data	O
revealed	O
that	O
the	O
catalytic	O
activity	O
of	O
Regnase	B-protein
-	I-protein
1	I-protein
is	O
regulated	O
through	O
both	O
intra	O
and	O
intermolecular	O
domain	O
interactions	O
in	O
vitro	O
.	O

In	O
order	O
to	O
characterize	O
the	O
role	O
of	O
each	O
domain	O
in	O
the	O
RNase	B-protein_type
activity	O
of	O
Regnase	B-protein
-	I-protein
1	I-protein
,	O
we	O
performed	O
an	O
in	B-experimental_method
vitro	I-experimental_method
cleavage	I-experimental_method
assay	I-experimental_method
using	O
fluorescently	B-protein_state
5	I-protein_state
′-	I-protein_state
labeled	I-protein_state
RNA	B-chemical
corresponding	O
to	O
nucleotides	O
82	O
–	O
106	O
of	O
the	O
IL	B-protein_type
-	I-protein_type
6	I-protein_type
mRNA	B-chemical
3	B-structure_element
′	I-structure_element
UTR	I-structure_element
(	O
Fig	O
.	O
1g	O
).	O

Regnase	B-protein
-	I-protein
1	I-protein
constructs	O
consisting	O
of	O
NTD	B-mutant
-	I-mutant
PIN	I-mutant
-	I-mutant
ZF	I-mutant
completely	O
cleaved	O
the	O
target	O
mRNA	B-chemical
and	O
generated	O
the	O
cleaved	O
products	O
.	O

During	O
purification	B-experimental_method
by	O
gel	B-experimental_method
filtration	I-experimental_method
,	O
the	O
PIN	B-structure_element
domain	O
exhibited	O
extremely	O
asymmetric	O
elution	O
peaks	O
in	O
a	O
concentration	O
dependent	O
manner	O
(	O
Fig	O
.	O
2a	O
).	O

Likewise	O
,	O
upon	O
addition	B-experimental_method
of	I-experimental_method
the	O
PIN	B-structure_element
domain	O
,	O
NMR	B-experimental_method
signals	O
derived	O
from	O
R56	B-residue_name_number
,	O
L58	B-residue_range
-	I-residue_range
G59	I-residue_range
,	O
and	O
V86	B-residue_range
-	I-residue_range
H88	I-residue_range
in	O
the	O
NTD	B-structure_element
exhibited	O
large	O
chemical	O
shift	O
changes	O
and	O
residues	O
D53	B-residue_name_number
,	O
F55	B-residue_name_number
,	O
K57	B-residue_name_number
,	O
Y60	B-residue_range
-	I-residue_range
S61	I-residue_range
,	O
V68	B-residue_name_number
,	O
T80	B-residue_range
-	I-residue_range
G83	I-residue_range
,	O
L85	B-residue_name_number
,	O
and	O
G89	B-residue_name_number
of	O
the	O
NTD	B-structure_element
as	O
well	O
as	O
side	O
chain	O
amide	O
signals	O
of	O
N79	B-residue_name_number
exhibited	O
small	O
but	O
appreciable	O
chemical	O
shift	O
changes	O
(	O
Fig	O
.	O
3b	O
and	O
Supplementary	O
Fig	O
.	O
5	O
).	O

Our	O
mutational	B-experimental_method
experiments	I-experimental_method
indicated	O
that	O
the	O
observed	O
dimer	B-oligomeric_state
is	O
functional	O
and	O
that	O
the	O
role	O
of	O
the	O
secondary	B-protein_state
PIN	B-structure_element
domain	O
is	O
to	O
position	O
Regnase	B-protein
-	I-protein
1	I-protein
-	O
unique	O
RNA	B-site
binding	I-site
residues	I-site
near	O
the	O
active	B-site
site	I-site
of	O
the	O
primary	B-protein_state
PIN	B-structure_element
domain	O
.	O

If	O
this	O
model	O
is	O
correct	O
,	O
then	O
we	O
reasoned	O
that	O
a	O
catalytically	B-protein_state
inactive	I-protein_state
PIN	B-structure_element
and	O
a	O
PIN	B-structure_element
lacking	B-protein_state
the	O
putative	O
RNA	B-site
-	I-site
binding	I-site
residues	I-site
ought	O
to	O
be	O
inactive	B-protein_state
in	O
isolation	O
but	O
become	O
active	B-protein_state
when	O
mixed	O
together	O
.	O

In	O
order	O
to	O
test	O
this	O
hypothesis	O
,	O
we	O
performed	O
in	B-experimental_method
vitro	I-experimental_method
cleavage	I-experimental_method
assays	I-experimental_method
using	O
combinations	O
of	O
Regnase	B-protein
-	I-protein
1	I-protein
mutants	B-protein_state
that	O
had	O
no	O
or	O
decreased	O
RNase	B-protein_type
activities	O
by	O
themselves	O
(	O
Fig	O
.	O
5	O
).	O

Consistently	O
,	O
when	O
we	O
compared	O
the	O
fluorescence	B-evidence
intensity	I-evidence
of	O
the	O
uncleaved	B-protein_state
Regnase	B-protein
-	I-protein
1	I-protein
mRNA	B-chemical
,	O
basic	O
residue	O
mutants	B-protein_state
K184A	B-mutant
and	O
R214A	B-mutant
were	O
rescued	O
upon	O
addition	O
of	O
the	O
DDNN	B-mutant
mutant	B-protein_state
(	O
Fig	O
.	O
5c	O
).	O

According	O
to	O
the	O
proposed	O
model	O
,	O
an	O
R214A	B-mutant
PIN	B-structure_element
domain	O
can	O
only	O
form	O
a	O
dimer	B-oligomeric_state
when	O
the	O
DDNN	B-mutant
PIN	B-structure_element
acts	O
as	O
the	O
secondary	B-protein_state
PIN	B-structure_element
.	O

This	O
inconsistency	O
might	O
be	O
due	O
to	O
difference	O
in	O
the	O
analytical	O
methods	O
and	O
/	O
or	O
protein	O
concentrations	O
used	O
in	O
each	O
experiment	O
,	O
since	O
the	O
oligomer	B-oligomeric_state
formation	O
of	O
PIN	B-structure_element
was	O
dependent	O
on	O
the	O
protein	O
concentration	O
in	O
our	O
study	O
.	O

Since	O
the	O
NMR	B-experimental_method
spectra	B-evidence
of	O
monomeric	B-oligomeric_state
mutants	B-protein_state
overlaps	O
with	O
those	O
of	O
the	O
oligomeric	O
forms	O
,	O
it	O
is	O
unlikely	O
that	O
the	O
tertiary	O
structure	O
of	O
the	O
monomeric	B-oligomeric_state
mutants	B-protein_state
were	O
affected	O
by	O
the	O
mutations	O
.	O
(	O
Supplementary	O
Fig	O
.	O
4b	O
,	O
c	O
).	O

Based	O
on	O
these	O
observations	O
,	O
we	O
concluded	O
that	O
PIN	B-structure_element
-	O
PIN	B-structure_element
dimer	B-oligomeric_state
formation	O
is	O
critical	O
for	O
Regnase	B-protein
-	I-protein
1	I-protein
RNase	B-protein_type
activity	O
in	O
vitro	O
.	O

Within	O
the	O
crystal	B-evidence
structure	I-evidence
of	O
the	O
PIN	B-structure_element
dimer	B-oligomeric_state
,	O
the	O
Regnase	B-protein
-	I-protein
1	I-protein
specific	O
basic	O
regions	O
in	O
both	O
the	O
“	O
primary	B-protein_state
”	O
and	O
“	O
secondary	B-protein_state
”	O
PINs	B-structure_element
are	O
located	O
around	O
the	O
catalytic	B-site
site	I-site
of	O
the	O
primary	O
PIN	B-structure_element
(	O
Supplementary	O
Fig	O
.	O
6	O
).	O

(	O
d	O
)	O
Solution	B-evidence
structure	I-evidence
of	O
the	O
ZF	B-structure_element
domain	O
.	O

(	O
f	O
)	O
In	B-experimental_method
vitro	I-experimental_method
gel	I-experimental_method
shift	I-experimental_method
binding	I-experimental_method
assay	I-experimental_method
between	O
Regnase	B-protein
-	I-protein
1	I-protein
and	O
IL	B-protein_type
-	I-protein_type
6	I-protein_type
mRNA	B-chemical
.	O

Fluorescence	B-evidence
intensity	I-evidence
of	O
the	O
uncleaved	B-protein_state
IL	B-protein_type
-	I-protein_type
6	I-protein_type
mRNA	B-chemical
was	O
indicated	O
as	O
the	O
percentage	O
against	O
that	O
in	O
the	O
absence	B-protein_state
of	I-protein_state
Regnase	B-protein
-	I-protein
1	I-protein
.	O

(	O
a	O
)	O
Gel	B-experimental_method
filtration	I-experimental_method
analyses	I-experimental_method
of	O
the	O
PIN	B-structure_element
domain	O
.	O

(	O
b	O
)	O
Dimer	B-oligomeric_state
structure	B-evidence
of	O
the	O
PIN	B-structure_element
domain	O
.	O

S62	B-residue_name_number
was	O
colored	O
gray	O
and	O
excluded	O
from	O
the	O
analysis	O
,	O
due	O
to	O
low	O
signal	O
intensity	O
.	O

(	O
c	O
)	O
Docking	O
model	O
of	O
the	O
NTD	B-structure_element
and	O
the	O
PIN	B-structure_element
domain	O
.	O

Residues	O
in	O
close	O
proximity	O
(<	O
5	O
Å	O
)	O
to	O
each	O
other	O
in	O
the	O
docking	B-evidence
structure	I-evidence
were	O
colored	O
yellow	O
.	O

Critical	O
residues	O
in	O
the	O
PIN	B-structure_element
domain	O
for	O
the	O
RNase	B-protein_type
activity	O
of	O
Regnase	B-protein
-	I-protein
1	I-protein
.	O

The	O
mutations	O
whose	O
RNase	B-protein_type
activities	O
were	O
restored	O
in	O
the	O
presence	B-protein_state
of	I-protein_state
DDNN	B-mutant
mutant	B-protein_state
were	O
colored	O
in	O
red	O
or	O
yellow	O
on	O
the	O
primary	O
PIN	B-structure_element
.	O

RAD51	B-protein
is	O
a	O
recombinase	B-protein_type
involved	O
in	O
the	O
homologous	O
recombination	O
of	O
double	O
‐	O
strand	O
breaks	O
in	O
DNA	O
.	O

RAD51	B-protein
interacts	O
with	O
BRCA2	B-protein
,	O
and	O
is	O
thought	O
to	O
localise	O
RAD51	B-protein
to	O
sites	O
of	O
DNA	O
damage	O
2	O
,	O
3	O
.	O

The	O
ability	O
of	O
BRC3	B-chemical
to	O
interact	O
with	O
RAD51	B-protein
nucleoprotein	O
filaments	O
is	O
disrupted	O
when	O
threonine	B-residue_name
is	O
mutated	B-experimental_method
to	O
an	O
alanine	B-residue_name
3	O
.	O

The	O
BRC5	B-chemical
repeat	O
in	O
humans	B-species
has	O
serine	B-residue_name
in	O
the	O
place	O
of	O
alanine	B-residue_name
,	O
and	O
is	O
thought	O
to	O
be	O
a	O
nonbinding	B-structure_element
repeat	I-structure_element
12	O
.	O

Affinities	B-evidence
of	O
peptides	O
were	O
measured	O
directly	O
using	O
Isothermal	B-experimental_method
Titration	I-experimental_method
Calorimetry	I-experimental_method
(	O
ITC	B-experimental_method
)	O
and	O
the	O
structures	B-evidence
of	O
many	O
of	O
the	O
peptides	O
bound	B-protein_state
to	I-protein_state
humanised	B-protein_state
RadA	B-protein
were	O
determined	O
by	O
X	B-experimental_method
‐	I-experimental_method
ray	I-experimental_method
crystallography	I-experimental_method
.	O

In	O
this	O
context	O
,	O
we	O
have	O
previously	O
reported	O
the	O
use	O
of	O
stable	B-protein_state
monomeric	B-oligomeric_state
forms	O
of	O
RAD51	B-protein
,	O
humanised	B-protein_state
from	O
Pyrococcus	B-species
furiosus	I-species
homologue	O
RadA	B-protein
,	O
for	O
ITC	B-experimental_method
experiments	O
and	O
X	B-experimental_method
‐	I-experimental_method
ray	I-experimental_method
crystallography	I-experimental_method
8	O
,	O
15	O
.	O

The	O
residue	O
makes	O
no	O
interactions	O
with	O
the	O
RAD51	B-protein
protein	O
,	O
but	O
may	O
make	O
an	O
internal	O
hydrogen	B-bond_interaction
bond	I-bond_interaction
with	O
Thr1520	B-residue_name_number
in	O
the	O
context	O
of	O
BRC4	B-chemical
,	O
Fig	O
.	O
3A	O
.	O

FPTA	B-structure_element
was	O
also	O
tested	O
,	O
but	O
was	O
found	O
to	O
have	O
no	O
affinity	B-evidence
for	O
the	O
protein	O
(	O
Table	O
2	O
,	O
entry	O
5	O
).	O

For	O
example	O
,	O
an	O
overlay	B-experimental_method
of	O
the	O
bound	O
poses	O
of	O
the	O
ligands	O
FHTA	B-structure_element
and	O
FHPA	B-structure_element
(	O
Fig	O
.	O
2B	O
)	O
reveals	O
a	O
high	O
similarity	O
in	O
the	O
binding	O
modes	O
,	O
indicating	O
that	O
the	O
conformational	O
rigidity	O
conferred	O
by	O
the	O
proline	B-residue_name
is	O
compatible	O
with	O
the	O
FHTA	B-structure_element
‐	O
binding	O
mode	O
,	O
and	O
a	O
reduction	O
in	O
an	O
entropic	B-evidence
penalty	I-evidence
of	O
binding	O
may	O
be	O
the	O
source	O
of	O
the	O
improvement	O
in	O
affinity	B-evidence
.	O

Only	O
one	O
structure	B-evidence
of	O
BRC4	B-chemical
is	O
published	O
in	B-protein_state
complex	I-protein_state
with	I-protein_state
human	B-species
RAD51	B-protein
(	O
PDB	O
:	O
1n0w	O
).	O

Either	O
a	O
threonine	B-residue_name
or	O
serine	B-residue_name
is	O
most	O
commonly	O
found	O
in	O
the	O
third	O
position	O
of	O
the	O
FxxA	B-structure_element
motif	O
.	O

The	O
high	B-protein_state
degree	I-protein_state
of	I-protein_state
conservation	I-protein_state
of	O
these	O
three	O
residues	O
suggests	O
an	O
important	O
possible	O
role	O
in	O
facilitating	O
a	O
turn	O
and	O
stabilising	O
the	O
conformation	O
of	O
the	O
peptide	O
as	O
it	O
continues	O
its	O
way	O
to	O
a	O
second	O
interaction	B-site
site	I-site
on	O
the	O
side	O
of	O
RAD51	B-protein
.	O

Two	O
residues	O
in	O
the	O
FxxA	B-structure_element
motif	O
,	O
phenylalanine	B-residue_name
and	O
alanine	B-residue_name
,	O
are	O
highly	B-protein_state
conserved	I-protein_state
(	O
Fig	O
4a	O
).	O

Phenylalanine	B-residue_name
mutated	B-experimental_method
to	I-experimental_method
tryptophan	B-residue_name
,	O
in	O
the	O
context	O
of	O
the	O
tetrapeptide	B-chemical
improved	O
potency	O
,	O
contrary	O
to	O
the	O
reported	O
result	O
of	O
comparable	O
activity	O
in	O
the	O
context	O
of	O
BRC4	B-chemical
12	O
.	O

These	O
studies	O
also	O
revealed	O
a	O
well	O
ordered	O
break	O
in	O
the	O
polypeptide	O
chain	O
at	O
Lys147	B-residue_name_number
,	O
resulting	O
in	O
a	O
large	O
conformational	O
rearrangement	O
close	O
to	O
the	O
active	B-site
site	I-site
.	O

Cysteine	B-protein_type
peptidases	I-protein_type
play	O
crucial	O
roles	O
in	O
the	O
virulence	O
of	O
bacterial	B-taxonomy_domain
and	O
other	O
eukaryotic	B-taxonomy_domain
pathogens	O
.	O

However	O
,	O
despite	O
these	O
similarities	O
,	O
clan	B-protein_type
CD	I-protein_type
forms	O
a	O
functionally	O
diverse	O
group	O
of	O
enzymes	O
:	O
the	O
overall	O
structural	O
diversity	O
between	O
(	O
and	O
at	O
times	O
within	O
)	O
the	O
various	O
families	O
provides	O
these	O
peptidases	B-protein_type
with	O
a	O
wide	O
variety	O
of	O
substrate	O
specificities	O
and	O
activation	O
mechanisms	O
.	O

The	O
archetypal	O
and	O
arguably	O
most	O
notable	O
family	O
in	O
the	O
clan	O
is	O
that	O
of	O
the	O
mammalian	B-taxonomy_domain
caspases	B-protein_type
(	O
C14a	B-protein_type
),	O
although	O
clan	B-protein_type
CD	I-protein_type
members	O
are	O
distributed	O
throughout	O
the	O
entire	O
phylogenetic	O
kingdom	O
and	O
are	O
often	O
required	O
in	O
fundamental	O
biological	O
processes	O
.	O

The	O
structure	B-evidence
also	O
includes	O
two	O
short	O
β	B-structure_element
-	I-structure_element
hairpins	I-structure_element
(	O
βA	B-structure_element
–	I-structure_element
βB	I-structure_element
and	O
βD	B-structure_element
–	I-structure_element
βE	I-structure_element
)	O
and	O
a	O
small	B-structure_element
β	I-structure_element
-	I-structure_element
sheet	I-structure_element
(	O
βC	B-structure_element
–	I-structure_element
βF	I-structure_element
),	O
which	O
is	O
formed	O
from	O
two	O
distinct	O
regions	O
of	O
the	O
sequence	O
(	O
βC	B-structure_element
precedes	O
α11	B-structure_element
,	O
α12	B-structure_element
and	O
β9	B-structure_element
,	O
whereas	O
βF	B-structure_element
follows	O
the	O
βD	B-structure_element
-	I-structure_element
βE	I-structure_element
hairpin	B-structure_element
)	O
in	O
the	O
middle	O
of	O
the	O
CTD	B-structure_element
(	O
Fig	O
.	O
1B	O
).	O

Crystal	B-evidence
structure	I-evidence
of	O
a	O
C11	B-protein_type
peptidase	I-protein_type
from	O
P	B-species
.	I-species
merdae	I-species
.	O

The	O
N	O
and	O
C	O
termini	O
(	O
N	O
and	O
C	O
)	O
of	O
PmC11	B-protein
along	O
with	O
the	O
central	O
β	B-structure_element
-	I-structure_element
sheet	I-structure_element
(	O
1	O
–	O
9	O
),	O
helix	B-structure_element
α5	B-structure_element
,	O
and	O
helices	B-structure_element
α8	B-structure_element
,	O
α11	B-structure_element
,	O
and	O
α13	B-structure_element
from	O
the	O
C	B-structure_element
-	I-structure_element
terminal	I-structure_element
domain	I-structure_element
,	O
are	O
all	O
labeled	O
.	O

Of	O
the	O
interacting	O
secondary	O
structure	O
elements	O
,	O
α5	B-structure_element
is	O
perhaps	O
the	O
most	O
interesting	O
.	O

PmC11	B-protein
is	O
,	O
as	O
expected	O
,	O
most	O
structurally	O
similar	O
to	O
other	O
members	O
of	O
clan	B-protein_type
CD	I-protein_type
with	O
the	O
top	O
hits	O
in	O
a	O
search	O
of	O
known	O
structures	B-evidence
being	O
caspase	B-protein
-	I-protein
7	I-protein
,	O
gingipain	B-protein
-	I-protein
K	I-protein
,	O
and	O
legumain	B-protein
(	O
PBD	O
codes	O
4hq0	O
,	O
4tkx	O
,	O
and	O
4aw9	O
,	O
respectively	O
)	O
(	O
Table	O
2	O
).	O

E	O
,	O
intermolecular	B-ptm
processing	I-ptm
of	O
PmC11C179A	B-mutant
by	O
PmC11	B-protein
.	O

Inactive	O
PmC11C179A	B-mutant
was	O
not	O
processed	O
to	O
a	O
major	O
extent	O
by	O
active	B-protein_state
PmC11	B-protein
until	O
around	O
a	O
ratio	O
of	O
1	O
:	O
4	O
(	O
5	O
μg	O
of	O
active	B-protein_state
PmC11	B-protein
).	O

The	O
position	O
of	O
the	O
catalytic	B-site
dyad	I-site
,	O
one	O
potential	O
key	B-site
substrate	I-site
binding	I-site
residue	I-site
Asp177	B-residue_name_number
,	O
and	O
the	O
ends	O
of	O
the	O
cleavage	B-site
site	I-site
Lys147	B-residue_name_number
and	O
Ala148	B-residue_name_number
are	O
indicated	O
.	O

Other	O
than	O
its	O
more	O
extended	B-structure_element
β	I-structure_element
-	I-structure_element
sheet	I-structure_element
,	O
PmC11	B-protein
differs	O
most	O
significantly	O
from	O
other	O
clan	B-protein_type
CD	I-protein_type
members	O
at	O
its	O
C	O
terminus	O
,	O
where	O
the	O
CTD	B-structure_element
contains	O
a	O
further	O
seven	O
α	B-structure_element
-	I-structure_element
helices	I-structure_element
and	O
four	O
β	B-structure_element
-	I-structure_element
strands	I-structure_element
after	O
β8	B-structure_element
.	O

To	O
investigate	O
whether	O
processing	O
is	O
a	O
result	O
of	O
intra	O
-	O
or	O
intermolecular	O
cleavage	O
,	O
the	O
PmC11C179A	B-mutant
mutant	B-protein_state
was	O
incubated	B-experimental_method
with	I-experimental_method
increasing	I-experimental_method
concentrations	I-experimental_method
of	O
processed	B-protein_state
and	O
activated	B-protein_state
PmC11	B-protein
.	O

Collectively	O
,	O
these	O
data	O
suggest	O
that	O
the	O
pro	B-protein_state
-	I-protein_state
form	I-protein_state
of	O
PmC11	B-protein
is	O
autoinhibited	B-protein_state
by	O
a	O
section	O
of	O
L5	B-structure_element
blocking	O
access	O
to	O
the	O
active	B-site
site	I-site
,	O
prior	O
to	O
intramolecular	B-ptm
cleavage	I-ptm
at	O
Lys147	B-residue_name_number
.	O

These	O
results	O
confirm	O
that	O
PmC11	B-protein
accepts	O
substrates	O
containing	O
Arg	B-residue_name
or	O
Lys	B-residue_name
in	O
P1	B-residue_number
with	O
a	O
possible	O
preference	O
for	O
Arg	B-residue_name
.	O

Because	O
PmC11	B-protein
recognizes	O
basic	O
substrates	O
,	O
the	O
tetrapeptide	O
inhibitor	O
Z	B-chemical
-	I-chemical
VRPR	I-chemical
-	I-chemical
FMK	I-chemical
was	O
tested	O
as	O
an	O
enzyme	O
inhibitor	O
and	O
was	O
found	O
to	O
inhibit	B-protein_state
both	O
the	O
autoprocessing	B-ptm
and	O
activity	O
of	O
PmC11	B-protein
(	O
Fig	O
.	O
3A	O
).	O

Z	B-chemical
-	I-chemical
VRPR	I-chemical
-	I-chemical
FMK	I-chemical
was	O
also	O
shown	O
to	O
bind	O
to	O
the	O
enzyme	O
:	O
a	O
size	B-evidence
-	I-evidence
shift	I-evidence
was	O
observed	O
,	O
by	O
SDS	B-experimental_method
-	I-experimental_method
PAGE	I-experimental_method
analysis	O
,	O
in	O
the	O
larger	O
processed	O
product	O
of	O
PmC11	B-protein
suggesting	O
that	O
the	O
inhibitor	B-protein_state
bound	I-protein_state
to	O
the	O
active	B-site
site	I-site
(	O
Fig	O
.	O
3B	O
).	O

Asp177	B-residue_name_number
is	O
highly	B-protein_state
conserved	I-protein_state
throughout	O
the	O
clan	B-protein_type
CD	I-protein_type
C11	I-protein_type
peptidases	I-protein_type
and	O
is	O
thought	O
to	O
be	O
primarily	O
responsible	O
for	O
substrate	O
specificity	O
of	O
the	O
clan	B-protein_type
CD	I-protein_type
enzymes	I-protein_type
,	O
as	O
also	O
illustrated	O
from	O
the	O
proximity	O
of	O
these	O
residues	O
relative	O
to	O
the	O
inhibitor	O
Z	B-chemical
-	I-chemical
VRPR	I-chemical
-	I-chemical
FMK	I-chemical
when	O
PmC11	B-protein
is	O
overlaid	B-experimental_method
on	O
the	O
MALT1	B-protein
-	I-protein
P	I-protein
structure	B-evidence
(	O
Fig	O
.	O
3C	O
).	O

Cleavage	O
of	O
Bz	B-chemical
-	I-chemical
R	I-chemical
-	I-chemical
AMC	I-chemical
by	O
PmC11	B-protein
was	O
measured	O
in	O
a	O
fluorometric	B-experimental_method
activity	I-experimental_method
assay	I-experimental_method
with	O
(+,	O
purple	O
)	O
and	O
without	O
(−,	O
red	O
)	O
Z	B-chemical
-	I-chemical
VRPR	I-chemical
-	I-chemical
FMK	I-chemical
.	O

B	O
,	O
gel	B-experimental_method
-	I-experimental_method
shift	I-experimental_method
assay	I-experimental_method
reveals	O
that	O
Z	B-chemical
-	I-chemical
VRPR	I-chemical
-	I-chemical
FMK	I-chemical
binds	O
to	O
PmC11	B-protein
.	O

Furthermore	O
,	O
Cu2	B-chemical
+,	I-chemical
Fe2	B-chemical
+,	I-chemical
and	O
Zn2	B-chemical
+	I-chemical
appear	O
to	O
inhibit	B-protein_state
PmC11	B-protein
.	O

Comparison	O
with	O
Clostripain	B-protein

In	O
addition	O
,	O
the	O
predicted	O
primary	O
S1	B-site
-	I-site
binding	I-site
residue	I-site
in	O
PmC11	B-protein
Asp177	B-residue_name_number
also	O
overlays	B-experimental_method
with	O
the	O
residue	O
predicted	O
to	O
be	O
the	O
P1	B-site
specificity	I-site
determining	I-site
residue	I-site
in	O
clostripain	B-protein
(	O
Asp229	B-residue_name_number
,	O
Fig	O
.	O
1A	O
).	O

This	O
is	O
also	O
the	O
case	O
in	O
PmC11	B-protein
,	O
although	O
the	O
cleavage	B-ptm
loop	B-structure_element
is	O
structurally	O
different	O
to	O
that	O
found	O
in	O
the	O
caspases	B-protein_type
and	O
follows	O
the	O
catalytic	B-protein_state
His	B-residue_name
(	O
Fig	O
.	O
1A	O
),	O
as	O
opposed	O
to	O
the	O
Cys	B-residue_name
in	O
the	O
caspases	B-protein_type
.	O

The	O
PmC11	B-protein
structure	B-evidence
should	O
provide	O
a	O
good	O
basis	O
for	O
structural	B-experimental_method
modeling	I-experimental_method
and	O
,	O
given	O
the	O
importance	O
of	O
other	O
clan	B-protein_type
CD	I-protein_type
enzymes	I-protein_type
,	O
this	O
work	O
should	O
also	O
advance	O
the	O
exploration	O
of	O
these	O
peptidases	B-protein_type
and	O
potentially	O
identify	O
new	O
biologically	O
important	O
substrates	O
.	O

Ribosome	B-protein_type
biogenesis	I-protein_type
factor	I-protein_type
Tsr3	B-protein
is	O
the	O
aminocarboxypropyl	B-protein_type
transferase	I-protein_type
responsible	O
for	O
18S	B-chemical
rRNA	I-chemical
hypermodification	O
in	O
yeast	B-taxonomy_domain
and	O
humans	B-species

Here	O
we	O
identify	O
the	O
cytoplasmic	O
ribosome	O
biogenesis	O
protein	O
Tsr3	B-protein
as	O
the	O
responsible	O
enzyme	O
in	O
yeast	B-taxonomy_domain
and	O
human	B-species
cells	O
.	O

This	O
unique	O
SAM	B-site
binding	I-site
mode	I-site
explains	O
why	O
Tsr3	B-protein
transfers	O
the	O
acp	B-chemical
and	O
not	O
the	O
methyl	O
group	O
of	O
SAM	B-chemical
to	O
its	O
substrate	O
.	O

Defects	O
of	O
rRNA	B-chemical
modification	O
enzymes	O
often	O
lead	O
to	O
disturbed	O
ribosome	O
biogenesis	O
or	O
functionally	O
impaired	O
ribosomes	O
,	O
although	O
the	O
lack	O
of	O
individual	O
rRNA	B-chemical
modifications	O
often	O
has	O
no	O
or	O
only	O
a	O
slight	O
influence	O
on	O
the	O
cell	O
.	O

Methylation	B-ptm
can	O
only	O
occur	O
once	O
pseudouridylation	B-ptm
has	O
taken	O
place	O
,	O
as	O
the	O
latter	O
reaction	O
generates	O
the	O
substrate	O
for	O
the	O
former	O
.	O

Hypermodified	B-protein_state
m1acp3Ψ	B-chemical
elutes	O
at	O
7	O
.	O
4	O
min	O
(	O
wild	B-protein_state
type	I-protein_state
,	O
left	O
profile	O
)	O
and	O
is	O
missing	O
in	O
Δtsr3	B-mutant
(	O
middle	O
profile	O
)	O
and	O
Δnep1	B-mutant
Δnop6	I-mutant
mutants	O
(	O
right	O
profile	O
).	O

Upper	O
lanes	O
show	O
the	O
ethidium	B-chemical
bromide	I-chemical
staining	O
of	O
the	O
18S	B-chemical
rRNAs	I-chemical
for	O
quantification	O
.	O

Archaeal	B-taxonomy_domain
Tyw2	B-protein
has	O
a	O
structure	B-evidence
very	O
similar	O
to	O
Rossmann	B-protein_type
-	I-protein_type
fold	I-protein_type
(	I-protein_type
class	I-protein_type
I	I-protein_type
)	I-protein_type
RNA	I-protein_type
-	I-protein_type
methyltransferases	I-protein_type
,	O
but	O
its	O
distinctive	O
SAM	B-site
-	I-site
binding	I-site
mode	I-site
enables	O
the	O
transfer	O
of	O
the	O
acp	B-chemical
group	O
instead	O
of	O
the	O
methyl	O
group	O
of	O
the	O
cofactor	O
.	O

On	O
this	O
basis	O
,	O
YOR006C	B-gene
was	O
renamed	O
‘	O
Twenty	B-protein
S	I-protein
rRNA	I-protein
accumulation	I-protein
3	I-protein
′	O
(	O
TSR3	B-protein
).	O

In	O
contrast	O
,	O
the	O
only	O
other	O
structurally	O
characterized	O
acp	B-protein_type
transferase	I-protein_type
enzyme	O
Tyw2	B-protein
belongs	O
to	O
the	O
Rossmann	B-protein_type
-	I-protein_type
fold	I-protein_type
class	I-protein_type
of	I-protein_type
methyltransferase	I-protein_type
proteins	I-protein_type
.	O

By	O
comparison	O
,	O
treating	O
cells	O
with	O
siRNA	B-chemical
545	O
,	O
which	O
only	O
reduced	O
the	O
TSR3	B-protein
mRNA	O
to	O
20	O
%,	O
did	O
not	O
markedly	O
reduced	O
the	O
acp	B-chemical
signal	O
.	O

This	O
suggests	O
that	O
low	O
residual	O
levels	O
of	O
HsTsr3	B-protein
are	O
sufficient	O
to	O
modify	O
the	O
RNA	B-chemical
.	O

Similar	O
to	O
a	O
temperature	O
-	O
sensitive	O
nep1	B-gene
mutant	B-protein_state
,	O
the	O
Δtsr3	B-mutant
deletion	O
caused	O
hypersensitivity	O
to	O
paromomycin	B-chemical
and	O
,	O
to	O
a	O
lesser	O
extent	O
,	O
to	O
hygromycin	B-chemical
B	I-chemical
(	O
Figure	O
2B	O
),	O
but	O
not	O
to	O
G418	B-chemical
or	O
cycloheximide	B-chemical
(	O
data	O
not	O
shown	O
).	O

In	O
accordance	O
with	O
the	O
synthetic	O
sick	O
growth	O
phenotype	O
the	O
paromomycin	B-chemical
and	O
hygromycin	B-chemical
B	I-chemical
hypersensitivity	O
further	O
increased	O
in	O
a	O
Δtsr3	B-mutant
Δsnr35	I-mutant
recombination	O
strain	O
(	O
Figure	O
2B	O
).	O

In	O
a	O
yeast	B-taxonomy_domain
Δtsr3	B-mutant
strain	O
as	O
well	O
as	O
in	O
the	O
Δtsr3	B-mutant
Δsnr35	I-mutant
recombinant	O
20S	B-chemical
pre	I-chemical
-	I-chemical
rRNA	I-chemical
accumulated	O
significantly	O
and	O
the	O
level	O
of	O
mature	O
18S	B-chemical
rRNA	I-chemical
was	O
reduced	O
(	O
Supplementary	O
Figures	O
S2C	O
and	O
S3D	O
),	O
as	O
reported	O
previously	O
.	O

However	O
,	O
these	O
archaeal	B-taxonomy_domain
homologs	O
are	O
significantly	O
smaller	O
than	O
ScTsr3	B-protein
(∼	O
190	O
aa	O
in	O
archaea	B-taxonomy_domain
vs	O
.	O
313	O
aa	O
in	O
yeast	B-taxonomy_domain
)	O
due	O
to	O
shortened	O
N	O
-	O
and	O
C	O
-	O
termini	O
(	O
Supplementary	O
Figure	O
S1A	O
).	O

Even	O
a	O
Tsr3	B-protein
fragment	O
with	O
a	O
90	B-residue_range
aa	I-residue_range
C	O
-	O
terminal	O
truncation	O
showed	O
a	O
residual	O
primer	O
extension	O
stop	O
,	O
whereas	O
N	O
-	O
terminal	O
truncations	O
exceeding	O
46	B-residue_range
aa	I-residue_range
almost	O
completely	O
abolished	O
the	O
primer	O
extension	O
arrest	O
(	O
Figure	O
3B	O
).	O

(	O
B	O
)	O
Primer	B-experimental_method
extension	I-experimental_method
analysis	I-experimental_method
of	O
18S	B-chemical
rRNA	I-chemical
acp	B-chemical
modification	O
in	O
yeast	B-taxonomy_domain
cells	O
expressing	O
the	O
indicated	O
TSR3	B-protein
fragments	O
.	O

While	O
for	O
S	B-species
.	I-species
solfataricus	I-species
the	O
existence	O
of	O
a	O
modified	O
nucleotide	B-chemical
of	O
unknown	O
chemical	O
composition	O
in	O
the	O
loop	B-structure_element
capping	I-structure_element
helix	I-structure_element
31	I-structure_element
of	O
its	O
16S	B-chemical
rRNA	I-chemical
has	O
been	O
demonstrated	O
,	O
no	O
information	O
regarding	O
rRNA	O
modifications	O
is	O
yet	O
available	O
for	O
V	B-species
.	I-species
distributa	I-species
.	O

The	O
structure	B-evidence
of	O
VdTsr3	B-protein
was	O
solved	O
ab	O
initio	O
,	O
by	O
single	B-experimental_method
-	I-experimental_method
wavelength	I-experimental_method
anomalous	I-experimental_method
diffraction	I-experimental_method
phasing	I-experimental_method
(	O
Se	B-experimental_method
-	I-experimental_method
SAD	I-experimental_method
)	O
with	O
Se	B-chemical
containing	O
derivatives	O
(	O
selenomethionine	B-chemical
and	O
seleno	B-chemical
-	I-chemical
substituted	I-chemical
SAM	I-chemical
).	O

Thus	O
,	O
the	O
VdTsr3	B-protein
structure	B-evidence
contains	O
a	O
deep	B-structure_element
trefoil	I-structure_element
knot	I-structure_element
.	O

β	B-structure_element
-	I-structure_element
strands	I-structure_element
are	O
colored	O
in	O
crimson	O
whereas	O
α	B-structure_element
-	I-structure_element
helices	I-structure_element
in	O
the	O
N	B-structure_element
-	I-structure_element
terminal	I-structure_element
domain	I-structure_element
are	O
colored	O
light	O
blue	O
and	O
α	B-structure_element
-	I-structure_element
helices	I-structure_element
in	O
the	O
C	B-structure_element
-	I-structure_element
terminal	I-structure_element
domain	I-structure_element
are	O
colored	O
dark	O
blue	O
.	O

A	O
red	O
arrow	O
marks	O
the	O
location	O
of	O
the	O
topological	B-structure_element
knot	I-structure_element
in	O
the	O
structure	B-evidence
.	O
(	O
B	O
)	O
Secondary	O
structure	O
representation	O
of	O
the	O
VdTsr3	B-protein
structure	B-evidence
.	O

However	O
,	O
there	O
are	O
no	O
structural	O
similarities	O
between	O
Tsr3	B-protein
and	O
Tyw2	B-protein
,	O
which	O
contains	O
an	O
all	B-structure_element
-	I-structure_element
parallel	I-structure_element
β	I-structure_element
-	I-structure_element
sheet	I-structure_element
of	O
a	O
different	O
topology	O
and	O
no	O
knot	B-structure_element
structure	I-structure_element
.	O

SAM	B-chemical
-	O
binding	O
by	O
Tsr3	B-protein
.	O

Bound	B-protein_state
SAM	B-chemical
was	O
modelled	O
based	O
on	O
the	O
X	B-evidence
-	I-evidence
ray	I-evidence
structure	I-evidence
of	O
the	O
Trm10	B-complex_assembly
/	I-complex_assembly
SAH	I-complex_assembly
-	O
complex	O
(	O
pdb4jwf	O
).	O

3xHA	B-protein_state
tagged	I-protein_state
Tsr3	B-protein
mutants	B-protein_state
are	O
expressed	O
comparable	O
to	O
the	O
wild	B-protein_state
type	I-protein_state
as	O
shown	O
by	O
western	B-experimental_method
blot	I-experimental_method
(	O
lower	O
left	O
).	O

VdTsr3	B-protein
could	O
not	O
be	O
used	O
in	O
these	O
experiments	O
since	O
we	O
could	O
not	O
purify	O
it	O
in	O
a	O
stable	B-protein_state
SAM	B-protein_state
-	I-protein_state
free	I-protein_state
form	O
.	O

The	O
side	O
chain	O
of	O
D70	B-residue_name_number
(	O
VdTsr3	B-protein
)	O
located	O
in	O
β4	B-structure_element
is	O
∼	O
5	O
Å	O
away	O
from	O
the	O
SAM	B-chemical
sulfur	O
atom	O
.	O

A	O
second	O
cluster	O
of	O
positively	O
charged	O
residues	O
is	O
found	O
in	O
or	O
near	O
helix	B-structure_element
α3	B-structure_element
(	O
K74	B-residue_name_number
,	O
R75	B-residue_name_number
,	O
K82	B-residue_name_number
,	O
R85	B-residue_name_number
and	O
K87	B-residue_name_number
).	O

A	O
triple	B-experimental_method
mutation	I-experimental_method
of	O
the	O
conserved	B-protein_state
positively	O
charged	O
residues	O
R60	B-residue_name_number
,	O
K65	B-residue_name_number
and	O
R131	B-residue_name_number
to	O
A	B-residue_name
in	O
ScTsr3	B-protein
resulted	O
in	O
a	O
protein	O
with	O
a	O
significantly	O
impaired	O
acp	B-protein_type
transferase	I-protein_type
activity	O
in	O
vivo	O
(	O
Figure	O
6D	O
)	O
in	O
line	O
with	O
an	O
important	O
functional	O
role	O
for	O
these	O
positively	O
charged	O
residues	O
.	O

For	O
S	B-species
.	I-species
solfataricus	I-species
the	O
chemical	O
identity	O
of	O
the	O
hypermodified	B-protein_state
nucleotide	B-chemical
is	O
not	O
known	O
but	O
the	O
existence	O
of	O
NEP1	B-protein
and	O
TSR3	B-protein
homologs	O
suggest	O
that	O
it	O
is	O
indeed	O
N1	B-chemical
-	I-chemical
methyl	I-chemical
-	I-chemical
N3	I-chemical
-	I-chemical
acp	I-chemical
-	I-chemical
pseudouridine	I-chemical
.	O

The	O
formation	O
of	O
1	B-chemical
-	I-chemical
methyl	I-chemical
-	I-chemical
3	I-chemical
-(	I-chemical
3	I-chemical
-	I-chemical
amino	I-chemical
-	I-chemical
3	I-chemical
-	I-chemical
carboxypropyl	I-chemical
)-	I-chemical
pseudouridine	I-chemical
(	O
m1acp3Ψ	B-chemical
)	O
is	O
very	O
complex	O
requiring	O
three	O
successive	O
modification	O
reactions	O
involving	O
one	O
H	B-structure_element
/	I-structure_element
ACA	I-structure_element
snoRNP	B-complex_assembly
(	O
snR35	B-protein
)	O
and	O
two	O
protein	O
enzymes	O
(	O
Nep1	B-protein
/	O
Emg1	B-protein
and	O
Tsr3	B-protein
).	O

This	O
makes	O
it	O
unique	O
in	O
eukaryotic	B-taxonomy_domain
rRNA	B-chemical
modification	O
.	O

A	O
similar	O
modification	O
(	O
acp3U	B-chemical
)	O
was	O
identified	O
in	O
Haloferax	B-species
volcanii	I-species
and	O
corresponding	O
modified	O
nucleotides	B-chemical
were	O
also	O
shown	O
to	O
occur	O
in	O
other	O
archaea	B-taxonomy_domain
.	O

As	O
shown	O
here	O
TSR3	B-protein
encodes	O
the	O
transferase	O
catalyzing	O
the	O
acp	B-chemical
modification	O
as	O
the	O
last	O
step	O
in	O
the	O
biosynthesis	O
of	O
m1acp3Ψ	B-chemical
in	O
yeast	B-taxonomy_domain
and	O
human	B-species
cells	O
.	O

In	O
contrast	O
,	O
in	O
the	O
structurally	O
closely	O
related	O
RNA	B-protein_type
methyltransferase	I-protein_type
Trm10	B-protein
the	O
methyl	O
group	O
of	O
the	O
cofactor	O
SAM	B-chemical
is	O
accessible	O
whereas	O
its	O
acp	B-chemical
side	O
chain	O
is	O
buried	O
inside	O
the	O
protein	O
.	O

In	O
response	O
to	O
cell	O
stress	O
,	O
YfiB	B-protein
in	O
the	O
outer	O
membrane	O
can	O
sequester	O
the	O
periplasmic	O
protein	O
YfiR	B-protein
,	O
releasing	O
its	O
inhibition	O
of	O
YfiN	B-protein
on	O
the	O
inner	O
membrane	O
and	O
thus	O
provoking	O
the	O
diguanylate	O
cyclase	O
activity	O
of	O
YfiN	B-protein
to	O
induce	O
c	B-chemical
-	I-chemical
di	I-chemical
-	I-chemical
GMP	I-chemical
production	O
.	O

Based	O
on	O
the	O
structural	B-evidence
and	I-evidence
biochemical	I-evidence
data	I-evidence
,	O
we	O
propose	O
an	O
updated	O
regulatory	O
model	O
of	O
the	O
YfiBNR	B-complex_assembly
system	O
.	O

The	O
functional	O
role	O
of	O
a	O
number	O
of	O
downstream	O
effectors	O
of	O
c	B-chemical
-	I-chemical
di	I-chemical
-	I-chemical
GMP	I-chemical
has	O
been	O
characterized	O
as	O
affecting	O
exopolysaccharide	B-chemical
(	O
EPS	B-chemical
)	O
production	O
,	O
transcription	O
,	O
motility	O
,	O
and	O
surface	O
attachment	O
(	O
Caly	O
et	O
al	O
.,;	O
Camilli	O
and	O
Bassler	O
,;	O
Ha	O
and	O
O	O
’	O
Toole	O
,;	O
Pesavento	O
and	O
Hengge	O
,).	O

Recently	O
,	O
Malone	O
and	O
coworkers	O
identified	O
the	O
tripartite	B-protein_state
c	B-chemical
-	I-chemical
di	I-chemical
-	I-chemical
GMP	I-chemical
signaling	O
module	O
system	O
YfiBNR	B-complex_assembly
(	O
also	O
known	O
as	O
AwsXRO	B-complex_assembly
(	O
Beaumont	O
et	O
al	O
.,;	O
Giddens	O
et	O
al	O
.,)	O
or	O
Tbp	B-complex_assembly
(	O
Ueda	O
and	O
Wood	O
,))	O
by	O
genetic	B-experimental_method
screening	I-experimental_method
for	O
mutants	O
that	O
displayed	O
SCV	O
phenotypes	O
in	O
P	B-species
.	I-species
aeruginosa	I-species
PAO1	I-species
(	O
Malone	O
et	O
al	O
.,;	O
Malone	O
et	O
al	O
.,).	O

Together	O
with	O
functional	O
data	O
,	O
these	O
results	O
provide	O
new	O
mechanistic	O
insights	O
into	O
how	O
activated	B-protein_state
YfiB	B-protein
sequesters	O
YfiR	B-protein
and	O
releases	O
the	O
suppression	O
of	O
YfiN	B-protein
.	O
These	O
findings	O
may	O
facilitate	O
the	O
development	O
and	O
optimization	O
of	O
anti	O
-	O
biofilm	O
drugs	O
for	O
the	O
treatment	O
of	O
chronic	O
infections	O
.	O

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

Each	O
crystal	O
form	O
contains	O
three	O
different	O
dimeric	B-oligomeric_state
types	O
of	O
YfiB	B-protein
,	O
two	O
of	O
which	O
are	O
present	O
in	O
both	O
,	O
suggesting	O
that	O
the	O
rest	O
of	O
the	O
dimeric	B-oligomeric_state
types	O
may	O
result	O
from	O
crystal	O
packing	O
.	O

The	O
dimerization	O
occurs	O
mainly	O
via	O
hydrophobic	B-bond_interaction
interactions	I-bond_interaction
formed	O
by	O
A37	B-residue_name_number
and	O
I40	B-residue_name_number
on	O
the	O
α1	B-structure_element
helices	I-structure_element
,	O
L50	B-residue_name_number
on	O
the	O
β1	B-structure_element
strands	I-structure_element
,	O
and	O
W55	B-residue_name_number
on	O
the	O
β2	B-structure_element
strands	I-structure_element
of	O
both	O
molecules	O
,	O
making	O
a	O
hydrophobic	B-site
interacting	I-site
core	I-site
(	O
Fig	O
.	O
2A	O
–	O
C	O
).	O

Two	O
interacting	O
regions	O
are	O
highlighted	O
by	O
red	O
rectangles	O
.	O
(	O
B	O
)	O
Structural	B-experimental_method
superposition	I-experimental_method
of	O
apo	B-protein_state
YfiB	B-protein
and	O
YfiR	B-protein_state
-	I-protein_state
bound	I-protein_state
YfiBL43P	B-mutant
.	O

To	O
gain	O
structural	O
insights	O
into	O
the	O
YfiB	B-complex_assembly
-	I-complex_assembly
YfiR	I-complex_assembly
interaction	O
,	O
we	O
co	B-experimental_method
-	I-experimental_method
expressed	I-experimental_method
YfiB	B-protein
(	O
residues	O
34	B-residue_range
–	I-residue_range
168	I-residue_range
)	O
and	O
YfiR	B-protein
(	O
residues	O
35	B-residue_range
–	I-residue_range
190	I-residue_range
,	O
lacking	B-protein_state
the	O
signal	B-structure_element
peptide	I-structure_element
),	O
but	O
failed	O
to	O
obtain	O
the	O
complex	O
,	O
in	O
accordance	O
with	O
a	O
previous	O
report	O
in	O
which	O
no	B-protein_state
stable	I-protein_state
complex	O
of	O
YfiB	B-complex_assembly
-	I-complex_assembly
YfiR	I-complex_assembly
was	O
observed	O
(	O
Malone	O
et	O
al	O
.,).	O

The	O
N	O
-	O
terminal	O
structural	O
conformation	O
of	O
YfiBL43P	B-mutant
,	O
from	O
the	O
foremost	O
N	O
-	O
terminus	O
to	O
residue	O
D70	B-residue_name_number
,	O
is	O
significantly	O
altered	O
compared	O
with	O
that	O
of	O
the	O
apo	B-protein_state
YfiB	B-protein
.	O
The	O
majority	O
of	O
the	O
α1	B-structure_element
helix	I-structure_element
(	O
residues	O
34	B-residue_range
–	I-residue_range
43	I-residue_range
)	O
is	O
invisible	O
on	O
the	O
electron	B-evidence
density	I-evidence
map	I-evidence
,	O
and	O
the	O
α2	B-structure_element
helix	I-structure_element
and	O
β1	B-structure_element
and	O
β2	B-structure_element
strands	I-structure_element
are	O
rearranged	O
to	O
form	O
a	O
long	O
loop	B-structure_element
containing	O
two	O
short	O
α	B-structure_element
-	I-structure_element
helix	I-structure_element
turns	I-structure_element
(	O
Fig	O
.	O
3B	O
and	O
3C	O
),	O
thus	O
embracing	O
the	O
YfiR	B-protein
dimer	B-oligomeric_state
.	O

Therefore	O
,	O
it	O
is	O
possible	O
that	O
both	O
dimeric	B-oligomeric_state
types	O
might	O
exist	O
in	O
solution	O
.	O

(	O
C	O
)	O
Close	O
-	O
up	O
view	O
showing	O
the	O
key	O
residues	O
of	O
YfiR	B-protein_state
-	I-protein_state
bound	I-protein_state
YfiBL43P	B-mutant
interacting	O
with	O
a	O
sulfate	B-chemical
ion	O
.	O

YfiR	B-protein_state
-	I-protein_state
bound	I-protein_state
YfiBL43P	B-mutant
is	O
shown	O
in	O
cyan	O
;	O
the	O
sulfate	B-chemical
ion	O
,	O
in	O
green	O
;	O
and	O
the	O
water	B-chemical
molecule	O
,	O
in	O
yellow	O
.	O
(	O
D	O
)	O
Structural	B-experimental_method
superposition	I-experimental_method
of	O
the	O
PG	B-site
-	I-site
binding	I-site
sites	I-site
of	O
apo	B-protein_state
YfiB	B-protein
and	O
YfiR	B-protein_state
-	I-protein_state
bound	I-protein_state
YfiBL43P	B-mutant
,	O
the	O
key	O
residues	O
are	O
shown	O
in	O
stick	O
.	O

In	O
the	O
Pal	B-complex_assembly
/	I-complex_assembly
PG	I-complex_assembly
-	I-complex_assembly
P	I-complex_assembly
complex	O
structure	B-evidence
,	O
the	O
m	B-chemical
-	I-chemical
Dap5	I-chemical
ϵ	I-chemical
-	I-chemical
carboxylate	I-chemical
group	O
interacts	O
with	O
the	O
side	O
-	O
chain	O
atoms	O
of	O
D71	B-residue_name_number
and	O
the	O
main	O
-	O
chain	O
amide	O
of	O
D37	B-residue_name_number
(	O
Fig	O
.	O
4B	O
).	O

In	O
addition	O
,	O
sequence	B-experimental_method
alignment	I-experimental_method
of	O
YfiB	B-protein
with	O
Pal	B-protein_type
and	O
the	O
periplasmic	B-structure_element
domain	I-structure_element
of	O
OmpA	B-protein_type
(	O
proteins	O
containing	O
PG	B-site
-	I-site
binding	I-site
site	I-site
)	O
showed	O
that	O
N68	B-residue_name_number
and	O
D102	B-residue_name_number
are	O
highly	B-protein_state
conserved	I-protein_state
(	O
Fig	O
.	O
4G	O
,	O
blue	O
stars	O
),	O
suggesting	O
that	O
these	O
residues	O
contribute	O
to	O
the	O
PG	O
-	O
binding	O
ability	O
of	O
YfiB	B-protein
.	O

Therefore	O
,	O
we	O
proposed	O
that	O
the	O
PG	B-chemical
-	O
binding	O
ability	O
of	O
inactive	B-protein_state
YfiB	B-protein
might	O
be	O
weaker	O
than	O
that	O
of	O
active	B-protein_state
YfiB	B-protein
.	O
To	O
validate	O
this	O
,	O
we	O
performed	O
a	O
microscale	B-experimental_method
thermophoresis	I-experimental_method
(	O
MST	B-experimental_method
)	O
assay	O
to	O
measure	O
the	O
binding	B-evidence
affinities	I-evidence
of	O
PG	B-chemical
to	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
YfiB	B-protein
and	O
YfiBL43P	B-mutant
,	O
respectively	O
.	O

Interestingly	O
,	O
these	O
residues	O
are	O
part	O
of	O
the	O
conserved	B-site
surface	I-site
of	O
YfiR	B-protein
(	O
Fig	O
.	O
3G	O
).	O

Collectively	O
,	O
a	O
part	O
of	O
the	O
YfiB	B-site
-	I-site
YfiR	I-site
interface	I-site
overlaps	O
with	O
the	O
proposed	O
YfiR	B-site
-	I-site
YfiN	I-site
interface	I-site
,	O
suggesting	O
alteration	O
in	O
the	O
association	O
-	O
disassociation	O
equilibrium	O
of	O
YfiR	B-protein
-	O
YfiN	B-protein
and	O
hence	O
the	O
ability	O
of	O
YfiB	B-protein
to	O
sequester	O
YfiR	B-protein
.	O

However	O
,	O
whether	O
YfiR	B-protein
is	O
involved	O
in	O
other	O
regulatory	O
mechanisms	O
is	O
still	O
an	O
open	O
question	O
.	O

Overall	O
Structures	B-evidence
of	O
VB6	B-protein_state
-	I-protein_state
bound	I-protein_state
and	O
Trp	B-protein_state
-	I-protein_state
bound	I-protein_state
YfiR	B-protein
.	O
(	O
A	O
)	O
Superposition	B-experimental_method
of	O
the	O
overall	O
structures	B-evidence
of	O
VB6	B-protein_state
-	I-protein_state
bound	I-protein_state
and	O
Trp	B-protein_state
-	I-protein_state
bound	I-protein_state
YfiR	B-protein
.	O
(	O
B	O
)	O
Close	O
-	O
up	O
views	O
showing	O
the	O
key	O
residues	O
of	O
YfiR	B-protein
that	O
bind	O
VB6	B-chemical
and	O
L	B-chemical
-	I-chemical
Trp	I-chemical
.	O

In	O
parallel	O
,	O
to	O
better	O
understand	O
the	O
putative	O
functional	O
role	O
of	O
VB6	B-chemical
and	O
L	B-chemical
-	I-chemical
Trp	I-chemical
,	O
yfiB	B-gene
was	O
deleted	B-experimental_method
in	O
a	O
PAO1	B-species
wild	B-protein_state
-	I-protein_state
type	I-protein_state
strain	O
,	O
and	O
a	O
construct	B-experimental_method
expressing	I-experimental_method
the	O
YfiBL43P	B-mutant
mutant	B-protein_state
was	O
transformed	B-experimental_method
into	I-experimental_method
the	O
PAO1	B-species
ΔyfiB	B-mutant
strain	O
to	O
trigger	O
YfiBL43P	B-mutant
-	O
induced	O
biofilm	O
formation	O
.	O

By	O
contrast	O
,	O
YfiR	B-protein_state
-	I-protein_state
bound	I-protein_state
YfiBL43P	B-mutant
(	O
residues	O
44	B-residue_range
–	I-residue_range
168	I-residue_range
)	O
has	O
a	O
stretched	B-protein_state
conformation	I-protein_state
of	O
approximately	O
55	O
Å	O
in	O
length	O
.	O

Provided	O
that	O
the	O
diameter	O
of	O
the	O
widest	O
part	O
of	O
the	O
YfiB	B-protein
dimer	B-oligomeric_state
is	O
approximately	O
64	O
Å	O
,	O
which	O
is	O
slightly	O
smaller	O
than	O
the	O
smallest	O
diameter	O
of	O
the	O
PG	O
pore	O
(	O
70	O
Å	O
)	O
(	O
Meroueh	O
et	O
al	O
.,),	O
the	O
YfiB	B-protein
dimer	B-oligomeric_state
should	O
be	O
able	O
to	O
penetrate	O
the	O
PG	O
layer	O
.	O

Regulatory	O
model	O
of	O
the	O
YfiBNR	B-complex_assembly
tripartite	B-protein_state
system	O
.	O

Once	O
activated	B-protein_state
by	O
certain	O
cell	O
stress	O
,	O
the	O
dimeric	B-oligomeric_state
YfiB	B-protein
transforms	O
from	O
a	O
compact	B-protein_state
conformation	I-protein_state
to	O
a	O
stretched	B-protein_state
conformation	I-protein_state
,	O
allowing	O
the	O
periplasmic	B-structure_element
domain	I-structure_element
of	O
the	O
membrane	B-protein_state
-	I-protein_state
anchored	I-protein_state
YfiB	B-protein
to	O
penetrate	O
the	O
cell	O
wall	O
and	O
sequester	O
the	O
YfiR	B-protein
dimer	B-oligomeric_state
,	O
thus	O
relieving	O
the	O
repression	O
of	O
YfiN	B-protein

These	O
results	O
,	O
together	O
with	O
our	O
observation	O
that	O
activated	B-protein_state
YfiB	B-protein
has	O
a	O
much	O
higher	O
cell	B-evidence
wall	I-evidence
binding	I-evidence
affinity	I-evidence
,	O
and	O
previous	O
mutagenesis	O
data	O
showing	O
that	O
(	O
1	O
)	O
both	O
PG	B-chemical
binding	O
and	O
membrane	O
anchoring	O
are	O
required	O
for	O
YfiB	B-protein
activity	O
and	O
(	O
2	O
)	O
activating	O
mutations	O
possessing	O
an	O
altered	O
N	O
-	O
terminal	O
loop	B-structure_element
length	O
are	O
dominant	O
over	O
the	O
loss	O
of	O
PG	B-chemical
binding	O
(	O
Malone	O
et	O
al	O
.,),	O
suggest	O
an	O
updated	O
regulatory	O
model	O
of	O
the	O
YfiBNR	B-complex_assembly
system	O
(	O
Fig	O
.	O
7	O
).	O

The	O
mechanism	O
by	O
which	O
activated	B-protein_state
YfiB	B-protein
relieves	O
the	O
repression	O
of	O
YfiN	B-protein
may	O
be	O
applicable	O
to	O
the	O
YfiBNR	B-complex_assembly
system	O
in	O
other	O
bacteria	B-taxonomy_domain
and	O
to	O
analogous	O
outside	O
-	O
in	O
signaling	O
for	O
c	B-chemical
-	I-chemical
di	I-chemical
-	I-chemical
GMP	I-chemical
production	O
,	O
which	O
in	O
turn	O
may	O
be	O
relevant	O
to	O
the	O
development	O
of	O
drugs	O
that	O
can	O
circumvent	O
complicated	O
antibiotic	O
resistance	O
.	O

UHRF1	B-protein
recognizes	O
hemi	B-chemical
-	I-chemical
methylated	I-chemical
DNA	I-chemical
(	O
hm	B-chemical
-	I-chemical
DNA	I-chemical
)	O
and	O
trimethylation	B-ptm
of	O
histone	B-protein_type
H3K9	B-protein_type
(	O
H3K9me3	B-protein_type
),	O
but	O
the	O
regulatory	O
mechanism	O
remains	O
unknown	O
.	O

UHRF1	B-protein
also	O
plays	O
an	O
important	O
role	O
in	O
promoting	O
proliferation	O
and	O
is	O
shown	O
to	O
be	O
upregulated	O
in	O
a	O
number	O
of	O
cancers	O
,	O
suggesting	O
that	O
UHRF1	B-protein
may	O
serve	O
as	O
a	O
potential	O
drug	O
target	O
for	O
therapeutic	O
applications	O
.	O

Here	O
we	O
report	O
that	O
UHRF1	B-protein
adopts	O
a	O
closed	B-protein_state
conformation	O
,	O
in	O
which	O
the	O
C	O
-	O
terminal	O
Spacer	B-structure_element
binds	B-protein_state
to	I-protein_state
the	O
TTD	B-structure_element
and	O
inhibits	O
its	O
recognition	O
of	O
H3K9me3	B-protein_type
,	O
whereas	O
the	O
SRA	B-structure_element
binds	B-protein_state
to	I-protein_state
the	O
PHD	B-structure_element
and	O
inhibits	O
its	O
recognition	O
of	O
H3R2	B-site
(	O
unmethylated	B-protein_state
histone	B-protein_type
H3	B-protein_type
at	O
residue	O
R2	B-residue_name_number
).	O

As	O
a	O
result	O
,	O
UHRF1	B-protein
is	O
locked	O
in	O
the	O
open	B-protein_state
conformation	O
by	O
the	O
association	O
of	O
H3K9me3	B-protein_type
by	O
TTD	B-structure_element
–	I-structure_element
PHD	I-structure_element
,	O
and	O
thus	O
SRA	B-structure_element
-	I-structure_element
Spacer	I-structure_element
either	O
recognizes	O
hm	B-chemical
-	I-chemical
DNA	I-chemical
or	O
recruits	O
DNMT1	B-protein
for	O
DNA	B-chemical
methylation	B-ptm
.	O

To	O
investigate	O
how	O
UHRF1	B-protein
coordinates	O
the	O
recognition	O
of	O
H3K9me3	B-protein_type
and	O
hm	B-chemical
-	I-chemical
DNA	I-chemical
,	O
we	O
purified	O
recombinant	O
UHRF1	B-protein
(	O
truncations	O
and	O
mutations	O
)	O
proteins	O
from	O
bacteria	O
.	O

These	O
results	O
suggest	O
that	O
hm	B-chemical
-	I-chemical
DNA	I-chemical
facilitates	O
histone	B-protein_type
recognition	O
by	O
UHRF1	B-protein
.	O

The	O
gel	B-experimental_method
filtration	I-experimental_method
analysis	I-experimental_method
showed	O
that	O
UHRF1	B-protein
is	O
a	O
monomer	B-oligomeric_state
in	O
solution	O
(	O
Supplementary	O
Fig	O
.	O
1b	O
),	O
indicating	O
that	O
the	O
intramolecular	O
(	O
not	O
intermolecular	O
)	O
interaction	O
of	O
UHRF1	B-protein
regulates	O
histone	B-protein_type
recognition	O
.	O

These	O
results	O
suggest	O
that	O
UHRF1	B-protein
adopts	O
an	O
unfavourable	O
conformation	O
for	O
histone	B-protein_type
H3	B-protein_type
tails	O
recognition	O
,	O
in	O
which	O
TTD	B-structure_element
–	I-structure_element
PHD	I-structure_element
might	O
be	O
blocked	O
by	O
other	O
regions	O
of	O
UHRF1	B-protein
,	O
and	O
hm	B-chemical
-	I-chemical
DNA	I-chemical
impairs	O
this	O
intramolecular	O
interaction	O
to	O
facilitate	O
its	O
recognition	O
of	O
histone	B-protein_type
H3	B-protein_type
tails	O
.	O

The	O
isothermal	B-experimental_method
titration	I-experimental_method
calorimetry	I-experimental_method
(	O
ITC	B-experimental_method
)	O
measurements	O
show	O
that	O
the	O
TTD	B-structure_element
bound	B-protein_state
to	I-protein_state
the	O
Spacer	B-structure_element
(	O
but	O
not	O
the	O
SRA	B-structure_element
)	O
in	O
a	O
1	O
:	O
1	O
stoichiometry	O
with	O
a	O
binding	B-evidence
affinity	I-evidence
(	O
KD	B-evidence
)	O
of	O
1	O
.	O
59	O
μM	O
(	O
Fig	O
.	O
2b	O
).	O

Compared	O
with	O
the	O
PHD	B-structure_element
alone	B-protein_state
,	O
PHD	B-structure_element
-	I-structure_element
SRA	I-structure_element
showed	O
decreased	O
binding	B-evidence
affinity	I-evidence
to	O
H3K9me0	B-protein_type
peptide	O
by	O
a	O
factor	O
of	O
eight	O
(	O
Fig	O
.	O
2e	O
).	O

These	O
results	O
indicate	O
that	O
the	O
SRA	B-structure_element
directly	O
binds	B-protein_state
to	I-protein_state
the	O
PHD	B-structure_element
and	O
inhibits	O
its	O
binding	B-evidence
affinity	I-evidence
to	O
H3K9me0	B-protein_type
.	O

The	O
Spacer	B-structure_element
(	O
residues	O
643	B-residue_range
–	I-residue_range
655	I-residue_range
were	O
built	O
in	O
the	O
model	O
)	O
adopts	O
an	O
extended	B-protein_state
conformation	I-protein_state
and	O
binds	B-protein_state
to	I-protein_state
an	O
acidic	B-site
groove	I-site
on	O
the	O
TTD	B-structure_element
(	O
Fig	O
.	O
3c	O
).	O

Comparison	B-experimental_method
of	O
TTD	B-structure_element
–	I-structure_element
Spacer	I-structure_element
and	O
TTD	B-complex_assembly
–	I-complex_assembly
PHD	I-complex_assembly
–	I-complex_assembly
H3K9me3	I-complex_assembly
(	O
PDB	O
:	O
4GY5	O
)	O
structures	B-evidence
indicates	O
that	O
the	O
Spacer	B-structure_element
and	O
the	O
Linker	B-structure_element
bind	O
to	O
the	O
TTD	B-structure_element
in	O
a	O
similar	O
manner	O
in	O
the	O
two	O
complexes	O
(	O
Fig	O
.	O
3b	O
).	O

The	O
ITC	B-experimental_method
experiment	O
shows	O
that	O
the	O
Linker	B-structure_element
peptide	O
(	O
289	B-residue_range
–	I-residue_range
306	I-residue_range
)	O
bound	B-protein_state
to	I-protein_state
the	O
TTD	B-structure_element
with	O
a	O
binding	B-evidence
affinity	I-evidence
of	O
24	O
.	O
04	O
μM	O
(	O
Supplementary	O
Fig	O
.	O
4b	O
),	O
∼	O
15	O
-	O
fold	O
lower	O
than	O
that	O
of	O
the	O
Spacer	B-structure_element
peptide	O
(	O
KD	B-evidence
=	O
1	O
.	O
59	O
μM	O
,	O
Fig	O
.	O
3e	O
).	O

As	O
shown	O
in	O
Fig	O
.	O
4d	O
,	O
hm	B-chemical
-	I-chemical
DNA	I-chemical
largely	O
enhanced	O
the	O
H3K9me3	B-evidence
-	I-evidence
binding	I-evidence
affinities	I-evidence
of	O
both	O
mutants	B-protein_state
in	O
the	O
presence	B-protein_state
of	I-protein_state
DTT	B-chemical
,	O
but	O
not	O
in	O
the	O
absence	B-protein_state
of	I-protein_state
DTT	B-chemical
,	O
indicating	O
that	O
the	O
disulphide	B-ptm
bond	I-ptm
formation	O
(	O
in	O
the	O
absence	B-protein_state
of	I-protein_state
DTT	B-chemical
)	O
disallows	O
hm	B-chemical
-	I-chemical
DNA	I-chemical
to	O
disrupt	O
TTD	B-structure_element
–	I-structure_element
Spacer	I-structure_element
interaction	O
for	O
H3K9me3	B-protein_type
recognition	O
.	O

The	O
above	O
results	O
collectively	O
demonstrate	O
that	O
(	O
i	O
)	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
UHRF1	B-protein
adopts	O
a	O
closed	B-protein_state
form	O
,	O
in	O
which	O
the	O
Spacer	B-structure_element
binds	B-protein_state
to	I-protein_state
the	O
TTD	B-structure_element
and	O
H3K9me3	B-protein_type
recognition	O
is	O
inhibited	O
;	O
(	O
ii	O
)	O
hm	B-chemical
-	I-chemical
DNA	I-chemical
displaces	O
the	O
Spacer	B-structure_element
from	O
the	O
TTD	B-structure_element
in	O
the	O
context	O
of	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
UHRF1	B-protein
and	O
therefore	O
largely	O
enhances	O
its	O
histone	B-protein_type
H3K9me3	B-protein_type
-	O
binding	O
activity	O
in	O
a	O
manner	O
independent	O
on	O
the	O
PHD	B-structure_element
(	O
SRA	B-structure_element
is	O
required	O
).	O

The	O
results	O
suggest	O
that	O
UHRF1ΔSRA	B-mutant
adopts	O
closed	B-protein_state
conformation	O
so	O
that	O
H3K9me3	B-protein_type
recognition	O
by	O
TTD	B-structure_element
–	I-structure_element
PHD	I-structure_element
is	O
blocked	O
by	O
the	O
intramolecular	O
interaction	O
,	O
and	O
support	O
the	O
regulatory	O
role	O
of	O
the	O
Spacer	B-structure_element
in	O
PCH	O
localization	O
of	O
UHRF1	B-protein
in	O
vivo	O
.	O

Getting	O
these	O
structures	B-evidence
would	O
greatly	O
help	O
for	O
understanding	O
the	O
hm	B-chemical
-	I-chemical
DNA	I-chemical
-	O
mediated	O
regulation	O
of	O
UHRF1	B-protein
.	O

These	O
findings	O
together	O
indicate	O
that	O
the	O
Spacer	B-structure_element
plays	O
a	O
very	O
important	O
role	O
in	O
the	O
dynamic	O
regulation	O
of	O
UHRF1	B-protein
.	O

When	O
our	O
manuscript	O
was	O
in	O
preparation	O
,	O
Gelato	O
et	O
al	O
.	O
reported	O
that	O
binding	O
of	O
PI5P	B-chemical
to	O
the	O
Spacer	B-structure_element
opens	O
the	O
closed	B-protein_state
conformation	O
of	O
UHRF1	B-protein
and	O
increases	O
H3K9me3	B-evidence
-	I-evidence
binding	I-evidence
affinity	I-evidence
of	O
the	O
TTD	B-structure_element
.	O

Interestingly	O
,	O
variant	B-protein
in	I-protein
methylation	I-protein
1	I-protein
(	O
VIM1	B-protein
,	O
a	O
UHRF1	B-protein
homologue	O
in	O
Arabidopsis	B-taxonomy_domain
)	O
contains	O
an	O
equivalent	O
spacer	B-structure_element
region	O
,	O
which	O
was	O
shown	O
to	O
be	O
required	O
for	O
hm	B-chemical
-	I-chemical
DNA	I-chemical
recognition	O
by	O
its	O
SRA	B-structure_element
domain	O
,	O
suggesting	O
a	O
conserved	O
regulatory	O
mechanism	O
in	O
SRA	B-structure_element
domain	O
-	O
containing	O
proteins	O
.	O

As	O
shown	O
in	O
the	O
proposed	O
model	O
,	O
recognition	O
of	O
H3K9me3	B-protein_type
by	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
UHRF1	B-protein
is	O
blocked	O
to	O
avoid	O
its	O
miss	O
-	O
localization	O
to	O
unmethylated	B-protein_state
genomic	O
region	O
,	O
in	O
which	O
chromatin	O
contains	O
H3K9me3	B-protein_type
(	O
KD	B-evidence
=	O
4	O
.	O
61	O
μM	O
)	O
or	O
H3K9me0	B-protein_type
(	O
KD	B-evidence
=	O
25	O
.	O
99	O
μM	O
).	O

This	O
function	O
is	O
probably	O
induced	O
by	O
a	O
direct	O
interaction	O
between	O
the	O
SRA	B-structure_element
and	O
RFTSDNMT1	B-protein
(	O
refs	O
)	O
or	O
interaction	O
between	O
DNMT1	B-protein
and	O
ubiquitylation	B-ptm
of	O
histione	B-protein_type
tail	O
.	O

In	O
our	O
in	B-experimental_method
vitro	I-experimental_method
assays	I-experimental_method
,	O
we	O
could	O
detect	O
interaction	O
between	O
SRA	B-structure_element
–	I-structure_element
Spacer	I-structure_element
and	O
RFTSDNMT1	B-protein
,	O
but	O
not	O
the	O
interaction	O
between	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
UHRF1	B-protein
and	O
RFTSDNMT1	B-protein
(	O
Supplementary	O
Fig	O
.	O
8a	O
,	O
b	O
and	O
Fig	O
.	O
5e	O
).	O

The	O
results	O
suggest	O
that	O
UHRF1	B-protein
adopts	O
multiple	O
conformations	O
.	O

SRA	B-structure_element
–	I-structure_element
Spacer	I-structure_element
was	O
incubated	B-experimental_method
with	O
GST	B-protein_state
-	I-protein_state
tagged	I-protein_state
TTD	B-structure_element
–	I-structure_element
PHD	I-structure_element
or	O
TTD	B-structure_element
in	O
the	O
presence	B-protein_state
of	I-protein_state
increasing	O
concentrations	O
of	O
hm	B-chemical
-	I-chemical
DNA	I-chemical
and	O
analysed	O
in	O
pull	B-experimental_method
-	I-experimental_method
down	I-experimental_method
experiment	I-experimental_method
as	O
described	O
in	O
a	O
.	O
The	O
quantified	O
band	B-experimental_method
densitometries	I-experimental_method
are	O
indicated	O
below	O
the	O
Coomassie	B-experimental_method
blue	I-experimental_method
staining	I-experimental_method
.	O

This	O
hierarchical	O
assembly	O
provides	O
a	O
model	O
,	O
in	O
which	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
Aβ	B-protein
transitions	O
from	O
an	O
unfolded	B-protein_state
monomer	B-oligomeric_state
to	O
a	O
folded	B-protein_state
β	B-structure_element
-	I-structure_element
hairpin	I-structure_element
,	O
which	O
assembles	O
to	O
form	O
oligomers	B-oligomeric_state
that	O
further	O
pack	O
to	O
form	O
an	O
annular	B-site
pore	I-site
.	O

In	O
Alzheimer	O
’	O
s	O
disease	O
,	O
monomeric	B-oligomeric_state
Aβ	B-protein
aggregates	O
to	O
form	O
soluble	O
low	O
molecular	O
weight	O
oligomers	B-oligomeric_state
,	O
such	O
as	O
dimers	B-oligomeric_state
,	O
trimers	B-oligomeric_state
,	O
tetramers	B-oligomeric_state
,	O
hexamers	B-oligomeric_state
,	O
nonamers	B-oligomeric_state
,	O
and	O
dodecamers	B-oligomeric_state
,	O
as	O
well	O
as	O
high	O
molecular	O
weight	O
aggregates	O
,	O
such	O
as	O
annular	B-complex_assembly
protofibrils	I-complex_assembly
.	O

Mouse	B-taxonomy_domain
models	O
for	O
Alzheimer	O
’	O
s	O
disease	O
have	O
helped	O
shape	O
our	O
current	O
understanding	O
about	O
the	O
Aβ	B-protein
oligomerization	O
that	O
precedes	O
neurodegeneration	O
.	O

Purified	O
Aβ	B-complex_assembly
*	I-complex_assembly
56	I-complex_assembly
injected	B-experimental_method
intercranially	I-experimental_method
into	O
healthy	O
rats	B-taxonomy_domain
was	O
found	O
to	O
impair	O
memory	O
,	O
providing	O
evidence	O
that	O
this	O
Aβ	B-protein
oligomer	B-oligomeric_state
may	O
cause	O
memory	O
loss	O
in	O
Alzheimer	O
’	O
s	O
disease	O
.	O

The	O
approach	O
of	O
isolating	O
and	O
characterizing	O
Aβ	B-protein
oligomers	B-oligomeric_state
has	O
not	O
provided	O
any	O
high	O
-	O
resolution	O
structures	B-evidence
of	O
Aβ	B-protein
oligomers	B-oligomeric_state
.	O

The	O
structure	B-evidence
revealed	O
that	O
monomeric	B-oligomeric_state
Aβ	B-protein
forms	O
a	O
β	B-structure_element
-	I-structure_element
hairpin	I-structure_element
when	O
bound	B-protein_state
to	I-protein_state
the	O
affibody	B-chemical
.	O

In	O
the	O
current	O
study	O
we	O
set	O
out	O
to	O
restore	B-experimental_method
the	O
Aβ24	B-protein
–	B-residue_range
29	I-residue_range
loop	B-structure_element
,	O
reintroduce	B-experimental_method
the	O
methionine	B-residue_name
residue	O
at	O
position	O
35	B-residue_number
,	O
and	O
determine	O
the	O
X	B-evidence
-	I-evidence
ray	I-evidence
crystallographic	I-evidence
structures	I-evidence
of	O
oligomers	B-oligomeric_state
that	O
form	O
.	O

We	O
routinely	O
use	O
p	B-chemical
-	I-chemical
iodophenylalanine	I-chemical
to	O
determine	O
the	O
X	B-evidence
-	I-evidence
ray	I-evidence
crystallographic	I-evidence
phases	I-evidence
.	O

Upon	O
synthesizing	O
peptide	B-mutant
3	I-mutant
,	O
we	O
found	O
that	O
it	O
formed	O
an	O
amorphous	O
precipitate	O
in	O
most	O
crystallization	O
conditions	O
screened	O
and	O
failed	O
to	O
afford	O
crystals	B-evidence
in	O
any	O
condition	O
.	O

To	O
address	O
this	O
issue	O
,	O
we	O
next	O
incorporated	O
a	O
disulfide	B-ptm
bond	I-ptm
between	O
residues	O
24	B-residue_number
and	O
29	B-residue_number
as	O
a	O
conformational	O
constraint	O
that	O
serves	O
as	O
a	O
surrogate	O
for	O
δOrn	B-structure_element
.	O

We	O
used	O
acid	B-protein_state
-	I-protein_state
stable	I-protein_state
Acm	B-protein_state
-	I-protein_state
protected	I-protein_state
cysteine	B-residue_name
residues	O
at	O
positions	O
24	B-residue_number
and	O
29	B-residue_number
and	O
removed	O
the	O
Acm	O
groups	O
by	O
oxidation	O
with	O
I2	O
in	O
aqueous	O
acetic	B-chemical
acid	I-chemical
to	O
afford	O
the	O
disulfide	B-ptm
linkage	I-ptm
.	O

Peptide	B-mutant
2	I-mutant
also	O
afforded	O
crystals	B-evidence
in	O
these	O
conditions	O
.	O

Crystal	B-evidence
diffraction	I-evidence
data	I-evidence
for	O
peptide	B-mutant
2	I-mutant
were	O
also	O
collected	O
at	O
the	O
Advanced	O
Light	O
Source	O
at	O
Lawrence	O
Berkeley	O
National	O
Laboratory	O
with	O
a	O
synchrotron	O
source	O
at	O
1	O
.	O
00	O
Å	O
wavelength	O
to	O
achieve	O
higher	O
resolution	O
.	O

Data	O
for	O
peptides	B-mutant
4	I-mutant
and	I-mutant
2	I-mutant
were	O
scaled	O
and	O
merged	O
using	O
XDS	O
.	O

No	O
evidence	O
for	O
cleavage	O
of	O
the	O
disulfides	B-ptm
was	O
observed	O
in	O
the	O
refinement	B-experimental_method
of	O
the	O
data	O
set	O
collected	O
on	O
the	O
X	O
-	O
ray	O
diffractometer	O
,	O
and	O
we	O
refined	B-experimental_method
all	O
disulfide	B-ptm
linkages	I-ptm
as	O
intact	B-protein_state
(	O
PDB	O
5HOY	O
).	O

The	O
trimer	B-oligomeric_state
maintains	O
all	O
of	O
the	O
same	O
stabilizing	O
contacts	O
as	O
those	O
of	O
peptide	B-mutant
1	I-mutant
.	O

Hydrogen	B-bond_interaction
bonding	I-bond_interaction
and	O
hydrophobic	B-bond_interaction
interactions	I-bond_interaction
between	O
residues	O
on	O
the	O
β	B-structure_element
-	I-structure_element
strands	I-structure_element
comprising	O
Aβ17	B-protein
–	B-residue_range
23	I-residue_range
and	O
Aβ30	B-protein
–	B-residue_range
36	I-residue_range
stabilize	O
the	O
core	B-structure_element
of	O
the	O
trimer	B-oligomeric_state
.	O

The	O
disulfide	B-ptm
bonds	I-ptm
between	O
residues	O
24	B-residue_number
and	O
29	B-residue_number
are	O
adjacent	O
to	O
the	O
structural	B-structure_element
core	I-structure_element
of	O
the	O
trimer	B-oligomeric_state
and	O
do	O
not	O
make	O
any	O
substantial	O
intermolecular	O
contacts	O
.	O

Three	O
ordered	O
water	B-chemical
molecules	O
fill	O
the	O
hole	O
in	O
the	O
center	O
of	O
the	O
trimer	B-oligomeric_state
,	O
hydrogen	B-bond_interaction
bonding	I-bond_interaction
to	O
each	O
other	O
and	O
to	O
the	O
main	O
chain	O
of	O
F20	B-residue_name_number
(	O
Figure	O
3A	O
).	O

Figure	O
4A	O
illustrates	O
the	O
octahedral	B-protein_state
shape	O
of	O
the	O
dodecamer	B-oligomeric_state
.	O

The	O
electron	B-evidence
density	I-evidence
map	I-evidence
for	O
the	O
X	B-evidence
-	I-evidence
ray	I-evidence
crystallographic	I-evidence
structure	I-evidence
of	O
peptide	B-mutant
2	I-mutant
has	O
long	O
tubes	O
of	O
electron	B-evidence
density	I-evidence
inside	O
the	O
central	B-site
cavity	I-site
of	O
the	O
dodecamer	B-oligomeric_state
.	O

Jeffamine	B-chemical
M	I-chemical
-	I-chemical
600	I-chemical
is	O
a	O
polypropylene	O
glycol	O
derivative	O
with	O
a	O
2	O
-	O
methoxyethoxy	O
unit	O
at	O
one	O
end	O
and	O
a	O
2	O
-	O
aminopropyl	O
unit	O
at	O
the	O
other	O
end	O
.	O

Annular	B-site
Pore	I-site

The	O
staggered	B-protein_state
interfaces	B-site
occur	O
between	O
dodecamers	B-structure_element
2	I-structure_element
and	I-structure_element
3	I-structure_element
and	O
4	B-structure_element
and	I-structure_element
5	I-structure_element
.	O

The	O
same	O
eclipsed	B-site
interface	I-site
also	O
occurs	O
between	O
dodecamers	B-structure_element
1	I-structure_element
and	I-structure_element
5	I-structure_element
and	O
3	B-structure_element
and	I-structure_element
4	I-structure_element
.	O
(	O
C	O
)	O
Staggered	B-site
interface	I-site
between	O
dodecamers	B-structure_element
2	I-structure_element
and	I-structure_element
3	I-structure_element
(	O
side	O
view	O
).	O

The	O
annular	B-site
pore	I-site
is	O
comparable	O
in	O
size	O
to	O
other	O
large	O
protein	O
assemblies	O
.	O

At	O
this	O
point	O
,	O
we	O
can	O
only	O
speculate	O
whether	O
the	O
trimer	B-oligomeric_state
and	O
dodecamer	B-oligomeric_state
formed	O
by	O
peptide	B-mutant
2	I-mutant
share	O
structural	O
similarities	O
to	O
Aβ	B-protein
trimers	B-oligomeric_state
and	O
Aβ	B-complex_assembly
*	I-complex_assembly
56	I-complex_assembly
,	O
as	O
little	O
is	O
known	O
about	O
the	O
structure	B-evidence
of	O
Aβ	B-protein
trimers	B-oligomeric_state
and	O
Aβ	B-complex_assembly
*	I-complex_assembly
56	I-complex_assembly
.	O

The	O
difficulty	O
in	O
studying	O
the	O
oligomers	B-oligomeric_state
formed	O
in	O
solution	O
may	O
reflect	O
the	O
propensity	O
of	O
the	O
dodecamer	B-oligomeric_state
to	O
assemble	O
on	O
all	O
four	O
F20	B-residue_name_number
faces	O
.	O

The	O
crystallographically	B-evidence
observed	I-evidence
trimer	B-oligomeric_state
recapitulates	O
the	O
Aβ	B-protein
trimers	B-oligomeric_state
that	O
are	O
observed	O
even	O
before	O
the	O
onset	O
of	O
symptoms	O
in	O
Alzheimer	O
’	O
s	O
disease	O
.	O

Our	O
approach	O
of	O
constraining	O
Aβ17	B-protein
–	B-residue_range
36	I-residue_range
into	O
a	O
β	B-structure_element
-	I-structure_element
hairpin	I-structure_element
conformation	O
and	O
blocking	O
aggregation	O
with	O
an	O
N	O
-	O
methyl	O
group	O
has	O
allowed	O
us	O
to	O
crystallize	B-experimental_method
a	O
large	O
fragment	O
of	O
what	O
is	O
generally	O
considered	O
to	O
be	O
an	O
uncrystallizable	O
peptide	O
.	O

Predictive	O
features	O
of	O
ligand	O
‐	O
specific	O
signaling	O
through	O
the	O
estrogen	B-protein_type
receptor	I-protein_type

For	O
some	O
ligand	O
series	O
,	O
a	O
single	O
inter	B-evidence
‐	I-evidence
atomic	I-evidence
distance	I-evidence
in	O
the	O
ligand	B-structure_element
‐	I-structure_element
binding	I-structure_element
domain	I-structure_element
predicted	O
their	O
proliferative	O
effects	O
.	O

In	O
contrast	O
,	O
the	O
N	O
‐	O
terminal	O
coactivator	B-site
‐	I-site
binding	I-site
site	I-site
,	O
activation	B-structure_element
function	I-structure_element
‐	I-structure_element
1	I-structure_element
(	O
AF	B-structure_element
‐	I-structure_element
1	I-structure_element
),	O
determined	O
cell	O
‐	O
specific	O
signaling	O
induced	O
by	O
ligands	O
that	O
used	O
alternate	O
mechanisms	O
to	O
control	O
cell	O
proliferation	O
.	O

Thus	O
,	O
incorporating	O
systems	B-experimental_method
structural	I-experimental_method
analyses	I-experimental_method
with	O
quantitative	B-experimental_method
chemical	I-experimental_method
biology	I-experimental_method
reveals	O
how	O
ligands	O
can	O
achieve	O
distinct	O
allosteric	O
signaling	O
outcomes	O
through	O
ERα	B-protein
.	O

ERα	B-protein
domain	O
organization	O
lettered	O
,	O
A	O
‐	O
F	O
.	O
DBD	B-structure_element
,	O
DNA	B-structure_element
‐	I-structure_element
binding	I-structure_element
domain	I-structure_element
;	O
LBD	B-structure_element
,	O
ligand	B-structure_element
‐	I-structure_element
binding	I-structure_element
domain	I-structure_element
;	O
AF	B-structure_element
,	O
activation	B-structure_element
function	I-structure_element

Linear	O
causality	O
model	O
for	O
ERα	B-protein
‐	O
mediated	O
cell	O
proliferation	O
.	O

Yet	O
,	O
it	O
is	O
unknown	O
how	O
different	O
ERα	B-protein
ligands	O
control	O
AF	B-structure_element
‐	I-structure_element
1	I-structure_element
through	O
the	O
LBD	B-structure_element
,	O
and	O
whether	O
this	O
inter	O
‐	O
domain	O
communication	O
is	O
required	O
for	O
cell	O
‐	O
specific	O
signaling	O
or	O
anti	O
‐	O
proliferative	O
responses	O
.	O

Our	O
long	O
‐	O
term	O
goal	O
is	O
to	O
be	O
able	O
to	O
predict	O
proliferative	O
or	O
anti	O
‐	O
proliferative	O
activity	O
of	O
a	O
ligand	O
in	O
different	O
tissues	O
from	O
its	O
crystal	B-evidence
structure	I-evidence
by	O
identifying	O
different	O
structural	O
perturbations	O
that	O
lead	O
to	O
specific	O
signaling	O
outcomes	O
.	O

In	O
this	O
signaling	O
model	O
,	O
multiple	O
coregulator	O
binding	O
events	O
and	O
target	O
genes	O
(	O
Won	O
Jeong	O
et	O
al	O
,	O
2012	O
;	O
Nwachukwu	O
et	O
al	O
,	O
2014	O
),	O
LBD	B-structure_element
conformation	O
,	O
nucleocytoplasmic	O
shuttling	O
,	O
the	O
occupancy	O
and	O
dynamics	O
of	O
DNA	O
binding	O
,	O
and	O
other	O
biophysical	O
features	O
could	O
contribute	O
independently	O
to	O
cell	O
proliferation	O
(	O
Lickwar	O
et	O
al	O
,	O
2012	O
).	O

We	O
also	O
determined	B-experimental_method
the	O
structures	B-evidence
of	O
76	O
distinct	O
ERα	B-protein
LBD	B-structure_element
complexes	O
bound	B-protein_state
to	I-protein_state
different	O
ligand	O
types	O
,	O
which	O
allowed	O
us	O
to	O
understand	O
how	O
diverse	O
ligand	O
scaffolds	O
distort	O
the	O
active	B-protein_state
conformation	O
of	O
the	O
ERα	B-protein
LBD	B-structure_element
.	O

To	O
compare	O
ERα	B-protein
signaling	O
induced	O
by	O
diverse	O
ligand	O
types	O
,	O
we	O
synthesized	B-experimental_method
and	I-experimental_method
assayed	I-experimental_method
a	O
library	O
of	O
241	O
ERα	B-protein
ligands	O
containing	O
19	O
distinct	O
molecular	O
scaffolds	O
.	O

Structure	B-evidence
of	O
the	O
E2	B-protein_state
‐	I-protein_state
bound	I-protein_state
ERα	B-protein
LBD	B-structure_element
in	B-protein_state
complex	I-protein_state
with	I-protein_state
an	O
NCOA2	B-protein
peptide	O
of	O
(	O
PDB	O
1GWR	O
).	O

The	O
ERα	B-protein
ligand	O
library	O
contains	O
241	O
ligands	O
representing	O
15	O
indirect	O
modulator	O
scaffolds	O
,	O
plus	O
4	O
direct	O
modulator	O
scaffolds	O
.	O

To	O
test	O
this	O
idea	O
,	O
we	O
compared	O
the	O
average	B-evidence
L	I-evidence
‐	I-evidence
Luc	I-evidence
activities	I-evidence
of	O
each	O
scaffold	O
in	O
HepG2	O
cells	O
co	B-experimental_method
‐	I-experimental_method
transfected	I-experimental_method
with	O
wild	B-protein_state
‐	I-protein_state
type	I-protein_state
ERα	B-protein
or	O
with	O
ERα	B-protein
lacking	B-protein_state
the	I-protein_state
AB	B-structure_element
domain	O
(	O
Figs	O
1B	O
and	O
EV1	O
).	O

Deletion	B-experimental_method
of	I-experimental_method
the	O
AB	B-structure_element
domain	O
significantly	O
reduced	O
the	O
average	B-evidence
L	I-evidence
‐	I-evidence
Luc	I-evidence
activities	I-evidence
of	O
14	O
scaffolds	O
(	O
Student	B-experimental_method
'	I-experimental_method
s	I-experimental_method
t	I-experimental_method
‐	I-experimental_method
test	I-experimental_method
,	O
P	B-evidence
≤	O
0	O
.	O
05	O
)	O
(	O
Fig	O
3B	O
).	O

This	O
cluster	O
includes	O
two	O
direct	O
modulator	O
scaffolds	O
(	O
OBHS	B-chemical
‐	I-chemical
ASC	I-chemical
and	O
OBHS	B-chemical
‐	I-chemical
BSC	I-chemical
),	O
and	O
five	O
indirect	O
modulator	O
scaffolds	O
(	O
A	B-chemical
‐	I-chemical
CD	I-chemical
,	O
cyclofenil	B-chemical
,	O
3	B-chemical
,	I-chemical
4	I-chemical
‐	I-chemical
DTPD	I-chemical
,	O
imine	B-chemical
,	O
and	O
imidazopyridine	B-chemical
).	O

Thus	O
,	O
examining	O
the	O
correlated	O
patterns	O
of	O
ERα	B-protein
activity	O
within	O
each	O
scaffold	O
demonstrates	O
that	O
an	O
extended	O
side	O
chain	O
is	O
not	O
required	O
for	O
cell	O
‐	O
specific	O
signaling	O
.	O

To	O
evaluate	O
the	O
role	O
of	O
AF	B-structure_element
‐	I-structure_element
1	I-structure_element
and	O
the	O
F	B-structure_element
domain	O
in	O
ERα	B-protein
signaling	O
specificity	O
,	O
we	O
compared	O
activity	O
of	O
truncated	O
ERα	B-protein
constructs	O
in	O
HepG2	O
liver	O
cells	O
with	O
endogenous	O
ERα	B-protein
activity	O
in	O
the	O
other	O
cell	O
types	O
.	O

Thus	O
,	O
in	O
cluster	O
2	O
,	O
AF	B-structure_element
‐	I-structure_element
1	I-structure_element
substantially	O
modulated	O
the	O
specificity	O
of	O
ligands	O
with	O
cell	O
‐	O
specific	O
activity	O
(	O
Fig	O
3D	O
lanes	O
5	O
–	O
12	O
).	O

Thus	O
,	O
ligands	O
in	O
cluster	O
2	O
rely	O
on	O
AF	B-structure_element
‐	I-structure_element
1	I-structure_element
for	O
both	O
activity	O
(	O
Fig	O
3B	O
)	O
and	O
signaling	O
specificity	O
(	O
Fig	O
3D	O
).	O

The	O
average	O
induction	O
of	O
GREB1	B-protein
by	O
cluster	O
1	O
ligands	O
showed	O
greater	O
variance	O
,	O
with	O
a	O
range	O
between	O
~	O
25	O
and	O
~	O
75	O
%	O
for	O
OBHS	B-chemical
and	O
a	O
range	O
from	O
full	O
agonist	O
to	O
inverse	O
agonist	O
for	O
the	O
others	O
in	O
cluster	O
1	O
(	O
Fig	O
EV2A	O
).	O

The	O
significant	O
correlations	O
with	O
GREB1	B-protein
expression	O
and	O
NCOA1	B-protein
/	I-protein
2	I-protein
/	I-protein
3	I-protein
recruitment	O
observed	O
in	O
this	O
cluster	O
are	O
consistent	O
with	O
the	O
canonical	O
signaling	O
model	O
(	O
Fig	O
1D	O
),	O
where	O
NCOA1	B-protein
/	I-protein
2	I-protein
/	I-protein
3	I-protein
recruitment	O
determines	O
GREB1	B-protein
expression	O
,	O
which	O
then	O
drives	O
proliferation	O
.	O

Despite	O
this	O
phenotypic	O
variance	O
,	O
proliferation	O
was	O
not	O
generally	O
predicted	O
by	O
correlated	O
NCOA1	B-protein
/	I-protein
2	I-protein
/	I-protein
3	I-protein
recruitment	O
and	O
GREB1	B-protein
induction	O
(	O
Figs	O
3F	O
lanes	O
5	O
–	O
19	O
,	O
and	O
EV3H	O
).	O

NCOA3	B-protein
occupancy	O
at	O
GREB1	B-protein
did	O
not	O
predict	O
the	O
proliferative	O
response	O

NCOA3	B-protein
occupancy	O
at	O
GREB1	B-protein
is	O
statistically	O
robust	O
but	O
does	O
not	O
predict	O
transcriptional	O
activity	O

In	O
panel	O
(	O
C	O
),	O
correlation	B-experimental_method
analysis	I-experimental_method
was	O
performed	O
for	O
two	O
biological	O
replicates	O
.	O

The	O
M2H	B-experimental_method
assay	I-experimental_method
for	O
NCOA3	B-protein
recruitment	O
broadly	O
correlated	O
with	O
the	O
other	O
assays	O
,	O
and	O
was	O
predictive	O
for	O
GREB1	B-protein
expression	O
and	O
cell	O
proliferation	O
(	O
Fig	O
3E	O
).	O

ERβ	B-protein
activity	O
is	O
not	O
an	O
independent	O
predictor	O
of	O
cell	O
‐	O
specific	O
activity	O

ERα	B-protein
activity	O
of	O
2	B-chemical
,	I-chemical
5	I-chemical
‐	I-chemical
DTP	I-chemical
and	O
cyclofenil	B-chemical
analogs	O
correlates	O
with	O
E	B-experimental_method
‐	I-experimental_method
Luc	I-experimental_method
activity	O
.	O

Examining	O
many	O
closely	O
related	O
structures	B-evidence
allows	O
us	O
to	O
visualize	O
subtle	O
structural	O
differences	O
,	O
in	O
effect	O
using	O
X	B-experimental_method
‐	I-experimental_method
ray	I-experimental_method
crystallography	I-experimental_method
as	O
a	O
systems	O
biology	O
tool	O
.	O

The	O
24	O
structures	B-evidence
containing	O
OBHS	B-chemical
,	O
OBHS	B-chemical
‐	I-chemical
N	I-chemical
,	O
or	O
triaryl	B-chemical
‐	I-chemical
ethylene	I-chemical
analogs	O
showed	O
structural	O
diversity	O
in	O
the	O
same	O
part	O
of	O
the	O
scaffolds	O
(	O
Figs	O
5A	O
and	O
EV5A	O
),	O
and	O
the	O
same	O
region	O
of	O
the	O
LBD	B-structure_element
—	O
the	O
C	O
‐	O
terminal	O
end	O
of	O
h11	B-structure_element
(	O
Figs	O
5B	O
and	O
C	O
,	O
and	O
EV5B	O
),	O
which	O
in	O
turn	O
nudges	O
h12	B-structure_element
(	O
Fig	O
5C	O
and	O
D	O
).	O

Triaryl	B-chemical
‐	I-chemical
ethylene	I-chemical
analogs	O
bound	B-protein_state
to	I-protein_state
the	O
superposed	B-experimental_method
crystal	B-evidence
structures	I-evidence
of	O
the	O
ERα	B-protein
LBD	B-structure_element
are	O
shown	O
.	O

Panel	O
(	O
A	O
)	O
shows	O
the	O
crystal	B-evidence
structure	I-evidence
of	O
an	O
S	B-protein_state
‐	I-protein_state
OBHS	I-protein_state
‐	I-protein_state
3	I-protein_state
‐	I-protein_state
bound	I-protein_state
ERα	B-protein
LBD	B-structure_element
(	O
PDB	O
5DUH	O
).	O

Hierarchical	B-experimental_method
clustering	I-experimental_method
of	O
ligand	O
‐	O
specific	O
binding	O
of	O
154	O
interacting	O
peptides	O
to	O
the	O
ERα	B-protein
LBD	B-structure_element
was	O
performed	O
in	O
triplicate	O
by	O
MARCoNI	B-experimental_method
analysis	I-experimental_method
.	O

One	O
phenol	O
pushed	O
further	O
toward	O
h3	B-structure_element
(	O
Fig	O
6D	O
),	O
while	O
the	O
other	O
phenol	O
pushed	O
toward	O
the	O
C	O
‐	O
terminus	O
of	O
h11	B-structure_element
to	O
a	O
greater	O
extent	O
than	O
A	B-chemical
‐	I-chemical
CD	I-chemical
‐	O
ring	O
estrogens	B-chemical
(	O
Nwachukwu	O
et	O
al	O
,	O
2014	O
),	O
which	O
are	O
close	O
structural	O
analogs	O
of	O
E2	B-chemical
that	O
lack	O
a	O
B	O
‐	O
ring	O
(	O
Fig	O
2	O
).	O

Despite	O
the	O
similar	O
average	O
activities	O
of	O
these	O
ligand	O
classes	O
(	O
Fig	O
3A	O
and	O
B	O
),	O
2	B-chemical
,	I-chemical
5	I-chemical
‐	I-chemical
DTP	I-chemical
and	O
3	B-chemical
,	I-chemical
4	I-chemical
‐	I-chemical
DTP	I-chemical
analogs	O
displayed	O
remarkably	O
different	O
peptide	O
recruitment	O
patterns	O
(	O
Fig	O
6H	O
),	O
consistent	O
with	O
the	O
structural	B-experimental_method
analyses	I-experimental_method
.	O

Thus	O
,	O
the	O
isomeric	O
attachment	O
of	O
diaryl	O
groups	O
to	O
the	O
thiophene	B-chemical
core	O
changed	O
the	O
AF	B-site
‐	I-site
2	I-site
surface	I-site
from	O
inside	O
the	O
ligand	B-site
‐	I-site
binding	I-site
pocket	I-site
,	O
as	O
predicted	O
by	O
the	O
crystal	B-evidence
structures	I-evidence
.	O

This	O
perturbation	O
determined	O
proliferation	O
that	O
correlated	O
strongly	O
with	O
AF	B-structure_element
‐	I-structure_element
2	I-structure_element
activity	O
,	O
recruitment	O
of	O
NCOA1	B-protein
/	I-protein
2	I-protein
/	I-protein
3	I-protein
family	O
members	O
,	O
and	O
induction	O
of	O
the	O
GREB1	B-protein
gene	O
,	O
consistent	O
with	O
the	O
canonical	O
ERα	B-protein
signaling	O
pathway	O
(	O
Fig	O
1D	O
).	O

This	O
finding	O
can	O
be	O
explained	O
by	O
the	O
fact	O
that	O
NCOA1	B-protein
/	I-protein
2	I-protein
/	I-protein
3	I-protein
contain	O
distinct	O
binding	B-site
sites	I-site
for	O
interaction	O
with	O
AF	B-structure_element
‐	I-structure_element
1	I-structure_element
and	O
AF	B-structure_element
‐	I-structure_element
2	I-structure_element
(	O
McInerney	O
et	O
al	O
,	O
1996	O
;	O
Webb	O
et	O
al	O
,	O
1998	O
),	O
which	O
allows	O
ligands	O
to	O
nucleate	O
ERα	B-complex_assembly
–	I-complex_assembly
NCOA1	I-complex_assembly
/	I-complex_assembly
2	I-complex_assembly
/	I-complex_assembly
3	I-complex_assembly
interaction	O
through	O
AF	B-structure_element
‐	I-structure_element
2	I-structure_element
,	O
and	O
reinforce	O
this	O
interaction	O
with	O
additional	O
binding	O
to	O
AF	B-structure_element
‐	I-structure_element
1	I-structure_element
.	O

For	O
K	B-species
.	I-species
pneumoniae	I-species
,	O
this	O
is	O
the	O
first	O
high	O
-	O
resolution	O
cleavage	O
complex	O
structure	B-evidence
to	O
be	O
reported	O
.	O

This	O
complex	O
is	O
compared	O
with	O
a	O
similar	O
complex	O
from	O
Streptococcus	B-species
pneumoniae	I-species
,	O
which	O
has	O
recently	O
been	O
solved	O
.	O

Acquiring	O
a	O
deep	O
structural	O
and	O
functional	O
understanding	O
of	O
the	O
mode	O
of	O
action	O
of	O
fluoroquinolones	B-chemical
(	O
Tomašić	O
&	O
Mašič	O
,	O
2014	O
)	O
and	O
the	O
development	O
of	O
new	O
drugs	O
targeted	O
against	O
topoisomerase	B-complex_assembly
IV	I-complex_assembly
and	O
gyrase	B-protein_type
from	O
a	O
wide	O
range	O
of	O
Gram	B-taxonomy_domain
-	I-taxonomy_domain
positive	I-taxonomy_domain
and	O
Gram	B-taxonomy_domain
-	I-taxonomy_domain
negative	I-taxonomy_domain
pathogenic	O
bacteria	B-taxonomy_domain
are	O
highly	O
active	O
areas	O
of	O
current	O
research	O
directed	O
at	O
overcoming	O
the	O
vexed	O
problem	O
of	O
drug	O
resistance	O
(	O
Bax	O
et	O
al	O
.,	O
2010	O
;	O
Chan	O
et	O
al	O
.,	O
2015	O
;	O
Drlica	O
et	O
al	O
.,	O
2014	O
;	O
Mutsaev	O
et	O
al	O
.,	O
2014	O
;	O
Pommier	O
,	O
2013	O
;	O
Srikannathasan	O
et	O
al	O
.,	O
2015	O
).	O

In	O
both	O
cases	O
the	O
DNA	B-chemical
is	O
bent	O
into	O
a	O
U	B-protein_state
-	I-protein_state
form	I-protein_state
and	O
bound	B-protein_state
snugly	O
against	O
the	O
protein	O
of	O
the	O
G	B-structure_element
-	I-structure_element
gate	I-structure_element
.	O

The	O
sequence	B-experimental_method
alignment	I-experimental_method
is	O
given	O
in	O
Supplementary	O
Fig	O
.	O
S1	O
,	O
with	O
the	O
key	O
metal	B-site
-	I-site
binding	I-site
residues	I-site
and	O
those	O
which	O
give	O
rise	O
to	O
quinolone	O
resistance	O
highlighted	O
.	O

The	O
side	O
chains	O
surrounding	O
them	O
in	O
ParE	B-protein
are	O
quite	O
disordered	O
and	O
are	O
more	O
defined	O
in	O
K	B-species
.	I-species
pneumoniae	I-species
(	O
even	O
though	O
this	O
complex	O
is	O
at	O
lower	O
resolution	O
)	O
than	O
in	O
S	B-species
.	I-species
pneumoniae	I-species
.	O

There	O
are	O
no	O
direct	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
from	O
the	O
drug	O
to	O
these	O
residues	O
(	O
although	O
it	O
is	O
possible	O
that	O
some	O
are	O
formed	O
through	O
water	B-chemical
,	O
which	O
cannot	O
be	O
observed	O
at	O
this	O
resolution	O
).	O

The	O
CC25	B-evidence
(	O
the	O
drug	O
concentration	O
that	O
converted	O
25	O
%	O
of	O
the	O
supercoiled	O
DNA	B-chemical
substrate	O
to	O
a	O
linear	O
form	O
)	O
was	O
0	O
.	O
5	O
µM	O
for	O
the	O
Klebsiella	B-taxonomy_domain
enzyme	O
and	O
1	O
µM	O
for	O
the	O
pneumococcal	B-taxonomy_domain
enzyme	O
.	O

Moreover	O
,	O
although	O
topoisomerase	B-complex_assembly
IV	I-complex_assembly
is	O
primarily	O
the	O
target	O
of	O
levofloxacin	B-chemical
in	O
S	B-species
.	I-species
pneumoniae	I-species
,	O
it	O
is	O
likely	O
to	O
be	O
gyrase	B-protein_type
in	O
the	O
Gram	B-taxonomy_domain
-	I-taxonomy_domain
negative	I-taxonomy_domain
K	B-species
.	I-species
pneumoniae	I-species
.	O

The	O
magnesium	B-chemical
ions	O
and	O
their	O
coordinating	O
amino	O
acids	O
are	O
shown	O
in	O
purple	O
.	O

The	O
active	B-site
-	I-site
site	I-site
tyrosine	B-residue_name
and	O
arginine	B-residue_name
are	O
in	O
orange	O
.	O

The	O
ParC	B-protein
and	O
ParE	B-protein
backbones	O
are	O
shown	O
in	O
blue	O
and	O
yellow	O
,	O
respectively	O
.	O

Comparison	O
of	O
DNA	B-chemical
cleavage	O
by	O
topoisomerase	B-complex_assembly
IV	I-complex_assembly
core	O
ParE	B-complex_assembly
-	I-complex_assembly
ParC	I-complex_assembly
fusion	O
proteins	O
from	O
K	B-species
.	I-species
pneumoniae	I-species
(	O
KP	B-species
)	O
and	O
S	B-species
.	I-species
pneumoniae	I-species
(	O
SP	B-species
)	O
promoted	O
by	O
levofloxacin	B-chemical
.	O

Lane	O
A	O
,	O
supercoiled	O
pBR322	O
DNA	B-chemical
;	O
N	O
,	O
L	O
and	O
S	O
,	O
nicked	O
,	O
linear	O
and	O
supercoiled	O
pBR322	O
,	O
respectively	O
.	O

Using	O
Cryo	B-experimental_method
-	I-experimental_method
EM	I-experimental_method
to	O
Map	O
Small	O
Ligands	O
on	O
Dynamic	O
Metabolic	O
Enzymes	O
:	O
Studies	O
with	O
Glutamate	B-protein_type
Dehydrogenase	I-protein_type

X	B-experimental_method
-	I-experimental_method
ray	I-experimental_method
crystallographic	I-experimental_method
studies	I-experimental_method
have	O
shown	O
that	O
the	O
functional	O
unit	O
of	O
GDH	B-protein_type
is	O
a	O
homohexamer	B-oligomeric_state
composed	O
of	O
a	O
trimer	B-oligomeric_state
of	O
dimers	B-oligomeric_state
,	O
with	O
a	O
3	O
-	O
fold	O
axis	O
and	O
an	O
equatorial	O
plane	O
that	O
define	O
its	O
D3	O
symmetry	O
(	O
Fig	O
.	O
1A	O
).	O

Each	O
56	O
-	O
kDa	O
protomer	B-oligomeric_state
consists	O
of	O
three	O
domains	O
.	O

Structure	O
and	O
quaternary	O
conformational	O
changes	O
in	O
GDH	B-protein_type
.	O
(	O
A	O
)	O
Views	O
of	O
open	B-protein_state
(	O
PDB	O
ID	O
1NR7	O
)	O
and	O
closed	B-protein_state
(	O
PDB	O
3MW9	O
)	O
states	O
of	O
the	O
GDH	B-protein_type
hexamer	B-oligomeric_state
,	O
shown	O
in	O
ribbon	O
representation	O
perpendicular	O
to	O
the	O
2	O
-	O
fold	O
symmetry	O
axis	O
(	O
side	O
view	O
,	O
top	O
)	O
and	O
3	O
-	O
fold	O
symmetry	O
axis	O
(	O
top	O
view	O
,	O
bottom	O
).	O

The	O
dashed	O
lines	O
and	O
arrows	O
,	O
respectively	O
,	O
highlight	O
the	O
slight	O
extension	O
in	O
length	O
,	O
and	O
twist	O
in	O
shape	O
that	O
occurs	O
with	O
transition	O
from	O
open	B-protein_state
to	O
the	O
closed	B-protein_state
state	O
.	O

The	O
open	B-protein_state
state	O
shown	O
is	O
for	O
unliganded	B-protein_state
GDH	B-protein_type
,	O
whereas	O
the	O
closed	B-protein_state
state	O
has	O
NADH	B-chemical
,	O
GTP	B-chemical
,	O
and	O
glutamate	B-chemical
bound	B-protein_state
.	O
(	O
B	O
)	O
Superposition	B-experimental_method
of	O
structures	B-evidence
for	O
closed	B-protein_state
and	O
open	B-protein_state
conformations	O
,	O
along	O
with	O
a	O
series	O
of	O
possible	O
intermediate	O
conformations	O
along	O
the	O
trajectory	O
that	O
serve	O
to	O
illustrate	O
the	O
extent	O
of	O
change	O
in	O
structure	O
across	O
different	O
regions	O
of	O
the	O
protein	O
.	O

These	O
allosteric	O
modulators	O
tightly	O
control	O
GDH	B-protein_type
function	O
in	O
vivo	O
.	O

Although	O
there	O
are	O
numerous	O
crystal	B-evidence
structures	I-evidence
available	O
for	O
GDH	B-protein_type
in	B-protein_state
complex	I-protein_state
with	I-protein_state
cofactors	O
and	O
nucleotides	O
,	O
they	O
are	O
limited	O
to	O
the	O
combinations	O
that	O
have	O
been	O
amenable	O
to	O
crystallization	B-experimental_method
.	O

When	O
GDH	B-protein
is	O
bound	B-protein_state
to	I-protein_state
NADH	B-chemical
,	O
GTP	B-chemical
,	O
and	O
glutamate	B-chemical
,	O
the	O
enzyme	O
adopts	O
a	O
closed	B-protein_state
conformation	O
;	O
this	O
“	O
abortive	O
complex	O
”	O
has	O
been	O
determined	O
to	O
2	O
.	O
4	O
-	O
Å	O
resolution	O
by	O
X	B-experimental_method
-	I-experimental_method
ray	I-experimental_method
crystallography	I-experimental_method
(	O
PDB	O
3MW9	O
).	O

However	O
,	O
crystal	B-evidence
structures	I-evidence
of	O
GDH	B-protein
bound	B-protein_state
only	I-protein_state
to	I-protein_state
NADH	B-chemical
or	O
to	B-protein_state
GTP	B-chemical
have	O
not	O
yet	O
been	O
reported	O
.	O

Comparison	O
of	O
the	O
NADH	B-protein_state
-	I-protein_state
bound	I-protein_state
closed	B-protein_state
conformation	O
to	O
the	O
NADH	B-protein_state
-	I-protein_state
bound	I-protein_state
open	B-protein_state
conformation	O
shows	O
that	O
,	O
as	O
expected	O
,	O
the	O
catalytic	B-site
cleft	I-site
is	O
closed	B-protein_state
and	O
the	O
NBDs	B-structure_element
are	O
displaced	O
toward	O
the	O
equatorial	O
plane	O
,	O
accompanied	O
by	O
a	O
rotation	O
of	O
the	O
pivot	B-structure_element
helix	I-structure_element
by	O
∼	O
7	O
°,	O
concomitant	O
with	O
a	O
large	O
conformational	O
change	O
in	O
the	O
antennae	B-structure_element
domains	O
(	O
Figs	O
.	O
1	O
and	O
2D	O
).	O

A	O
comparison	O
between	O
NADH	B-protein_state
-	I-protein_state
bound	I-protein_state
open	B-protein_state
and	O
closed	B-protein_state
conformations	O
also	O
involves	O
a	O
displacement	O
of	O
helix	B-structure_element
5	I-structure_element
(	O
residues	O
171	B-residue_range
–	I-residue_range
186	I-residue_range
),	O
as	O
well	O
as	O
a	O
tilt	O
of	O
the	O
core	O
β	B-structure_element
-	I-structure_element
sheets	I-structure_element
relative	O
to	O
the	O
equatorial	O
plane	O
of	O
the	O
enzyme	O
(	O
residues	O
57	B-residue_range
–	I-residue_range
97	I-residue_range
,	O
122	B-residue_range
–	I-residue_range
130	I-residue_range
)	O
and	O
α	B-structure_element
-	I-structure_element
helix	I-structure_element
2	I-structure_element
(	O
residues	O
36	B-residue_range
–	I-residue_range
54	I-residue_range
),	O
and	O
a	O
bending	O
of	O
the	O
N	O
-	O
terminal	O
helix	B-structure_element
.	O

Although	O
there	O
is	O
a	O
difference	O
in	O
orientation	O
of	O
the	O
nicotinamide	O
moiety	O
between	O
the	O
closed	B-protein_state
and	O
open	B-protein_state
states	O
in	O
the	O
regulatory	B-site
site	I-site
,	O
in	O
both	O
structures	B-evidence
the	O
adenine	O
portion	O
of	O
NADH	B-chemical
has	O
a	O
similar	O
binding	B-site
pocket	I-site
and	O
is	O
located	O
in	O
almost	O
exactly	O
the	O
same	O
position	O
as	O
ADP	B-chemical
,	O
a	O
potent	O
activator	O
of	O
GDH	B-protein
function	O
(	O
Supplemental	O
Fig	O
.	O
5	O
).	O

This	O
suggests	O
that	O
although	O
the	O
conformation	O
of	O
NADH	B-chemical
in	O
the	O
open	B-protein_state
state	O
regulatory	B-site
site	I-site
more	O
closely	O
mimics	O
the	O
binding	O
of	O
ADP	B-chemical
,	O
the	O
conformation	O
of	O
NADH	B-chemical
in	O
the	O
closed	B-protein_state
state	O
regulatory	B-site
site	I-site
is	O
significantly	O
different	O
;	O
these	O
differences	O
may	O
contribute	O
to	O
the	O
opposite	O
effects	O
of	O
NADH	B-chemical
and	O
ADP	B-chemical
on	O
GDH	B-protein
enzymatic	O
activity	O
.	O

Cryo	B-experimental_method
-	I-experimental_method
EM	I-experimental_method
structure	B-evidence
of	O
GDH	B-protein
bound	B-protein_state
to	I-protein_state
both	O
NADH	B-chemical
and	O
GTP	B-chemical
.	O

(	O
C	O
,	O
D	O
)	O
Detailed	O
inspection	O
of	O
the	O
interactions	O
near	O
the	O
regulatory	B-site
site	I-site
show	O
that	O
the	O
orientation	O
of	O
His209	B-residue_name_number
switches	O
between	O
the	O
two	O
states	O
,	O
which	O
may	O
allow	O
interactions	O
with	O
bound	B-protein_state
GTP	B-chemical
in	O
the	O
closed	B-protein_state
(	O
D	O
),	O
but	O
not	O
open	B-protein_state
(	O
C	O
)	O
conformation	O
.	O

Our	O
structural	B-experimental_method
studies	I-experimental_method
thus	O
establish	O
that	O
whether	O
or	O
not	O
GTP	B-chemical
is	O
bound	B-protein_state
,	O
NADH	B-chemical
binding	O
is	O
detectable	O
at	O
catalytic	B-site
and	I-site
regulatory	I-site
sites	I-site
,	O
in	O
both	O
the	O
open	B-protein_state
and	O
closed	B-protein_state
conformational	O
states	O
.	O

The	O
role	O
of	O
the	O
nicotinamide	O
moiety	O
in	O
acting	O
as	O
a	O
wedge	O
that	O
prevents	O
the	O
transition	O
to	O
the	O
open	B-protein_state
conformation	O
also	O
suggests	O
a	O
structural	O
explanation	O
of	O
the	O
mechanism	O
by	O
which	O
NADH	B-chemical
binding	O
inhibits	O
the	O
activity	O
of	O
the	O
enzyme	O
by	O
stabilizing	O
the	O
closed	B-protein_state
conformation	O
state	O
.	O

We	O
have	O
solved	B-experimental_method
the	O
structure	B-evidence
of	O
the	O
HR1	B-structure_element
domain	O
of	O
TOCA1	B-protein
,	O
providing	O
the	O
first	O
structural	B-evidence
data	I-evidence
for	O
this	O
protein	O
.	O

All	O
members	O
share	O
a	O
well	O
defined	O
core	O
structure	O
of	O
∼	O
20	O
kDa	O
known	O
as	O
the	O
G	B-structure_element
domain	I-structure_element
,	O
which	O
is	O
responsible	O
for	O
guanine	B-chemical
nucleotide	I-chemical
binding	O
.	O

The	O
overall	O
conformation	O
of	O
small	B-protein_type
G	I-protein_type
proteins	I-protein_type
in	O
the	O
active	B-protein_state
and	O
inactive	B-protein_state
states	O
is	O
similar	O
,	O
but	O
they	O
differ	O
significantly	O
in	O
two	O
main	O
regions	O
known	O
as	O
switch	B-site
I	I-site
and	O
switch	B-site
II	I-site
.	O

However	O
,	O
because	O
each	O
of	O
the	O
150	O
members	O
of	O
the	O
superfamily	O
interacts	O
with	O
multiple	O
effectors	O
,	O
there	O
are	O
still	O
a	O
huge	O
number	O
of	O
known	O
G	B-protein_type
protein	I-protein_type
-	O
effector	O
interactions	O
that	O
have	O
not	O
yet	O
been	O
studied	O
structurally	O
.	O

The	O
most	O
widely	O
studied	O
role	O
of	O
TOCA1	B-protein
is	O
in	O
membrane	O
invagination	O
and	O
endocytosis	O
,	O
although	O
it	O
has	O
also	O
been	O
implicated	O
in	O
filopodia	O
formation	O
,	O
neurite	O
elongation	O
,	O
transcriptional	O
reprogramming	O
via	O
nuclear	O
actin	B-protein_type
,	O
and	O
interaction	O
with	O
ZO	B-protein
-	I-protein
1	I-protein
at	O
tight	O
junctions	O
.	O

How	O
different	O
HR1	B-structure_element
domain	O
proteins	O
distinguish	O
their	O
specific	O
G	B-protein_type
protein	I-protein_type
partners	O
remains	O
only	O
partially	O
understood	O
,	O
and	O
structural	O
characterization	O
of	O
a	O
novel	O
G	B-protein_type
protein	I-protein_type
-	O
HR1	B-structure_element
domain	O
interaction	O
would	O
add	O
to	O
the	O
growing	O
body	O
of	O
information	O
pertaining	O
to	O
these	O
protein	O
complexes	O
.	O

We	O
also	O
present	O
data	O
pertaining	O
to	O
binding	O
of	O
the	O
TOCA	B-protein_type
HR1	B-structure_element
domain	O
to	O
Cdc42	B-protein
,	O
which	O
is	O
the	O
first	O
biophysical	O
description	O
of	O
an	O
HR1	B-structure_element
domain	O
binding	O
this	O
particular	O
Rho	B-protein_type
family	I-protein_type
small	I-protein_type
G	I-protein_type
protein	I-protein_type
.	O

The	O
data	O
were	O
fitted	O
to	O
a	O
binding	B-evidence
isotherm	I-evidence
describing	O
competition	O
.	O

As	O
the	O
observed	O
affinity	B-evidence
between	O
TOCA1	B-protein
HR1	B-structure_element
and	O
Cdc42	B-protein
was	O
much	O
lower	O
than	O
expected	O
,	O
we	O
reasoned	O
that	O
the	O
C	O
terminus	O
of	O
Cdc42	B-protein
might	O
be	O
necessary	O
for	O
a	O
high	O
affinity	B-evidence
interaction	O
.	O

Thus	O
,	O
the	O
C	B-structure_element
-	I-structure_element
terminal	I-structure_element
region	I-structure_element
of	O
Cdc42	B-protein
is	O
not	O
required	O
for	O
maximal	O
binding	O
of	O
TOCA1	B-protein
HR1	B-structure_element
.	O

The	O
SPA	B-experimental_method
signal	O
was	O
corrected	O
by	O
subtraction	O
of	O
control	O
data	O
with	O
no	O
fusion	O
protein	O
.	O

The	O
low	O
affinity	O
of	O
the	O
TOCA1	B-protein
HR1	B-structure_element
-	O
Cdc42	B-protein
interaction	O
raised	O
the	O
question	O
of	O
whether	O
the	O
other	O
known	O
Cdc42	B-protein
-	O
binding	O
TOCA	B-protein_type
family	I-protein_type
proteins	I-protein_type
,	O
FBP17	B-protein
and	O
CIP4	B-protein
,	O
also	O
bind	O
weakly	O
.	O

Initial	O
experiments	O
were	O
performed	O
with	O
TOCA1	B-protein
residues	O
324	B-residue_range
–	I-residue_range
426	I-residue_range
,	O
but	O
we	O
observed	O
that	O
the	O
N	O
terminus	O
was	O
cleaved	O
during	O
purification	O
to	O
yield	O
a	O
new	O
N	O
terminus	O
at	O
residue	O
330	B-residue_number
(	O
data	O
not	O
shown	O
).	O

We	O
therefore	O
engineered	O
a	O
construct	O
comprising	O
residues	O
330	B-residue_range
–	I-residue_range
426	I-residue_range
to	O
produce	O
the	O
minimal	B-protein_state
,	O
stable	B-protein_state
HR1	B-structure_element
domain	O
.	O

A	O
,	O
the	O
backbone	O
trace	B-evidence
of	O
the	O
35	O
lowest	O
energy	O
structures	B-evidence
of	O
the	O
HR1	B-structure_element
domain	O
overlaid	O
with	O
the	O
structure	B-evidence
closest	O
to	O
the	O
mean	O
is	O
shown	O
alongside	O
a	O
schematic	O
representation	O
of	O
the	O
structure	B-evidence
closest	O
to	O
the	O
mean	O
.	O

D	O
,	O
a	O
close	O
-	O
up	O
of	O
the	O
interhelix	B-structure_element
loop	I-structure_element
region	O
showing	O
some	O
of	O
the	O
contacts	O
between	O
the	O
loop	B-structure_element
and	O
helix	B-structure_element
1	I-structure_element
.	O

Overall	O
chemical	B-experimental_method
shift	I-experimental_method
perturbations	I-experimental_method
(	O
CSPs	B-experimental_method
)	O
were	O
calculated	O
for	O
each	O
residue	O
,	O
whereas	O
those	O
that	O
had	O
disappeared	O
were	O
assigned	O
a	O
shift	O
change	O
of	O
0	O
.	O
2	O
(	O
Fig	O
.	O
4B	O
).	O

Mapping	O
the	O
binding	B-site
surface	I-site
of	O
Cdc42	B-protein
onto	O
the	O
TOCA1	B-protein
HR1	B-structure_element
domain	O
.	O

TOCA1	B-protein
residues	O
whose	O
signals	O
were	O
affected	O
by	O
Cdc42	B-protein
binding	O
were	O
mapped	O
onto	O
the	O
structure	B-evidence
of	O
TOCA1	B-protein
HR1	B-structure_element
(	O
Fig	O
.	O
4C	O
).	O

As	O
was	O
the	O
case	O
when	O
labeled	B-protein_state
HR1	B-structure_element
was	O
observed	O
,	O
several	O
peaks	O
were	O
shifted	O
in	O
the	O
complex	O
,	O
but	O
many	O
disappeared	O
,	O
indicating	O
exchange	O
on	O
an	O
unfavorable	O
,	O
millisecond	O
time	O
scale	O
(	O
Fig	O
.	O
5A	O
).	O

A	O
,	O
the	O
15N	B-experimental_method
HSQC	I-experimental_method
of	O
Cdc42Δ7Q61L	B-complex_assembly
·	I-complex_assembly
GMPPNP	I-complex_assembly
is	O
shown	O
in	O
its	O
free	B-protein_state
form	I-protein_state
(	O
black	O
)	O
and	O
in	O
the	O
presence	B-protein_state
of	I-protein_state
excess	O
TOCA1	B-protein
HR1	B-structure_element
domain	O
(	O
1	O
:	O
2	O
.	O
2	O
,	O
red	O
).	O

The	O
flexible	B-protein_state
switch	B-site
regions	I-site
are	O
circled	O
.	O

Although	O
the	O
binding	B-site
interface	I-site
may	O
be	O
overestimated	O
,	O
this	O
suggests	O
that	O
the	O
switch	B-site
regions	I-site
are	O
involved	O
in	O
binding	O
to	O
TOCA1	B-protein
.	O

The	O
Cdc42	B-complex_assembly
·	I-complex_assembly
HR1TOCA1	I-complex_assembly
complex	O
was	O
not	O
amenable	O
to	O
full	O
structural	O
analysis	O
due	O
to	O
the	O
weak	O
interaction	O
and	O
the	O
extensive	O
exchange	O
broadening	O
seen	O
in	O
the	O
NMR	B-experimental_method
experiments	O
.	O

The	O
cluster	O
with	O
the	O
lowest	O
root	B-evidence
mean	I-evidence
square	I-evidence
deviation	I-evidence
from	O
the	O
lowest	O
energy	O
structure	B-evidence
is	O
assumed	O
to	O
be	O
the	O
best	O
model	O
.	O

By	O
these	O
criteria	O
,	O
in	O
the	O
best	O
model	O
,	O
the	O
HR1	B-structure_element
domain	O
is	O
in	O
a	O
similar	O
orientation	O
to	O
the	O
HR1a	B-structure_element
domain	O
of	O
PRK1	B-protein
bound	B-protein_state
to	I-protein_state
RhoA	B-protein
and	O
the	O
HR1b	B-structure_element
domain	O
bound	B-protein_state
to	I-protein_state
Rac1	B-protein
.	O

Model	O
of	O
Cdc42	B-complex_assembly
·	I-complex_assembly
HR1	I-complex_assembly
complex	O
.	O

C	O
,	O
sequence	B-experimental_method
alignment	I-experimental_method
of	O
RhoA	B-protein
,	O
Cdc42	B-protein
and	O
Rac1	B-protein
.	O

Some	O
of	O
these	O
can	O
be	O
rationalized	O
;	O
for	O
example	O
,	O
Thr	B-residue_name_number
-	I-residue_name_number
24Cdc42	I-residue_name_number
,	O
Leu	B-residue_name_number
-	I-residue_name_number
160Cdc42	I-residue_name_number
,	O
and	O
Lys	B-residue_name_number
-	I-residue_name_number
163Cdc42	I-residue_name_number
all	O
pack	O
behind	O
switch	B-site
I	I-site
and	O
are	O
likely	O
to	O
be	O
affected	O
by	O
conformational	O
changes	O
within	O
the	O
switch	B-site
,	O
while	O
Glu	B-residue_name_number
-	I-residue_name_number
95Cdc42	I-residue_name_number
and	O
Lys	B-residue_name_number
-	I-residue_name_number
96Cdc42	I-residue_name_number
are	O
in	O
the	O
helix	B-structure_element
behind	O
switch	B-site
II	I-site
.	O

Lys	B-residue_name_number
-	I-residue_name_number
16Cdc42	I-residue_name_number
is	O
unlikely	O
to	O
be	O
a	O
contact	O
residue	O
because	O
it	O
is	O
involved	O
in	O
nucleotide	O
binding	O
,	O
but	O
the	O
others	O
may	O
represent	O
specific	O
Cdc42	B-complex_assembly
-	I-complex_assembly
TOCA1	I-complex_assembly
contacts	O
.	O

Studies	O
in	O
CHO	O
cells	O
indicated	O
that	O
a	O
Cdc42	B-complex_assembly
·	I-complex_assembly
N	I-complex_assembly
-	I-complex_assembly
WASP	I-complex_assembly
·	I-complex_assembly
TOCA1	I-complex_assembly
complex	O
existed	O
because	O
FRET	B-evidence
was	O
observed	O
between	O
RFP	B-chemical
-	O
TOCA1	B-protein
and	O
GFP	B-chemical
-	O
N	B-protein
-	I-protein
WASP	I-protein
,	O
and	O
the	O
efficiency	O
was	O
decreased	O
when	O
an	O
N	B-protein
-	I-protein
WASP	I-protein
mutant	B-protein_state
was	O
used	O
that	O
no	O
longer	O
binds	O
Cdc42	B-protein
.	O

A	O
basic	O
region	O
in	O
WASP	B-protein
including	O
three	O
lysines	B-residue_name
(	O
residues	O
230	B-residue_range
–	I-residue_range
232	I-residue_range
),	O
N	O
-	O
terminal	O
to	O
the	O
core	O
CRIB	B-structure_element
,	O
has	O
been	O
implicated	O
in	O
an	O
electrostatic	O
steering	O
mechanism	O
,	O
and	O
these	O
residues	O
would	O
be	O
free	O
to	O
bind	O
in	O
the	O
presence	B-protein_state
of	I-protein_state
TOCA1	B-protein
HR1	B-structure_element
(	O
Fig	O
.	O
7A	O
).	O

The	O
core	O
CRIB	B-structure_element
region	O
of	O
WASP	B-protein
is	O
shown	O
in	O
red	O
,	O
whereas	O
its	O
basic	O
region	O
is	O
shown	O
in	O
orange	O
and	O
the	O
C	O
-	O
terminal	O
region	O
required	O
for	O
maximal	O
affinity	O
is	O
shown	O
in	O
cyan	O
.	O

Unlabeled	B-protein_state
N	B-protein
-	I-protein
WASP	I-protein
GBD	B-structure_element
was	O
titrated	B-experimental_method
into	O
15N	B-chemical
-	O
Cdc42Δ7Q61L	B-complex_assembly
·	I-complex_assembly
GMPPNP	I-complex_assembly
,	O
and	O
the	O
backbone	O
NH	O
groups	O
were	O
monitored	O
using	O
HSQCs	B-experimental_method
(	O
Fig	O
.	O
7C	O
).	O

These	O
experiments	O
were	O
recorded	O
at	O
sufficiently	O
high	O
protein	O
concentrations	O
(	O
145	O
μm	O
Cdc42	B-protein
,	O
145	O
μm	O
N	B-protein
-	I-protein
WASP	I-protein
GBD	B-structure_element
,	O
725	O
μm	O
TOCA1	B-protein
HR1	B-structure_element
domain	O
)	O
to	O
be	O
far	O
in	O
excess	O
of	O
the	O
Kd	B-evidence
values	O
of	O
the	O
individual	O
interactions	O
(	O
TOCA1	B-protein
Kd	B-evidence
≈	O
5	O
μm	O
,	O
N	B-protein
-	I-protein
WASP	I-protein
Kd	B-evidence
=	O
37	O
nm	O
).	O

The	O
spectrum	B-evidence
when	O
N	B-protein
-	I-protein
WASP	I-protein
and	O
TOCA1	B-protein
were	O
equimolar	O
was	O
identical	O
to	O
that	O
of	O
the	O
free	B-protein_state
HR1	B-structure_element
domain	O
,	O
whereas	O
the	O
spectrum	B-evidence
in	O
the	O
presence	B-protein_state
of	I-protein_state
0	O
.	O
25	O
eq	O
of	O
N	B-protein
-	I-protein
WASP	I-protein
was	O
intermediate	O
between	O
the	O
TOCA1	B-protein
HR1	B-structure_element
free	B-protein_state
and	O
complex	B-protein_state
spectra	B-evidence
(	O
Fig	O
.	O
7D	O
).	O

Actin	B-protein_type
polymerization	O
in	O
all	O
cases	O
was	O
initiated	O
by	O
the	O
addition	O
of	O
PI	B-chemical
(	I-chemical
4	I-chemical
,	I-chemical
5	I-chemical
)	I-chemical
P2	I-chemical
-	O
containing	O
liposomes	O
.	O

The	O
GBD	B-structure_element
presumably	O
acts	O
as	O
a	O
dominant	O
negative	O
,	O
sequestering	O
endogenous	O
Cdc42	B-protein
and	O
preventing	O
endogenous	B-protein_state
full	B-protein_state
-	I-protein_state
length	I-protein_state
N	B-protein
-	I-protein
WASP	I-protein
from	O
binding	O
and	O
becoming	O
activated	O
.	O

This	O
is	O
over	O
100	O
times	O
lower	O
than	O
that	O
of	O
the	O
N	B-protein
-	I-protein
WASP	I-protein
GBD	B-structure_element
(	O
Kd	B-evidence
=	O
37	O
nm	O
)	O
and	O
considerably	O
lower	O
than	O
other	O
known	O
G	B-protein_type
protein	I-protein_type
-	O
HR1	B-structure_element
domain	O
interactions	O
.	O

The	O
polybasic	O
tract	O
within	O
the	O
C	B-structure_element
-	I-structure_element
terminal	I-structure_element
region	I-structure_element
of	O
Cdc42	B-protein
does	O
not	O
appear	O
to	O
be	O
required	O
for	O
binding	O
to	O
TOCA1	B-protein
,	O
which	O
is	O
in	O
contrast	O
to	O
the	O
interaction	O
between	O
Rac1	B-protein
and	O
the	O
HR1b	B-structure_element
domain	O
of	O
PRK1	B-protein
but	O
more	O
similar	O
to	O
the	O
PRK1	B-protein
HR1a	B-structure_element
-	O
RhoA	B-protein
interaction	O
.	O

The	O
TOCA1	B-protein
HR1	B-structure_element
domain	O
is	O
a	O
left	O
-	O
handed	O
coiled	B-structure_element
-	I-structure_element
coil	I-structure_element
comparable	O
with	O
other	O
known	O
HR1	B-structure_element
domains	O
.	O

This	O
region	O
is	O
distant	O
from	O
the	O
G	B-site
protein	I-site
-	I-site
binding	I-site
interface	I-site
of	O
the	O
HR1	B-structure_element
domains	O
,	O
so	O
the	O
structural	O
differences	O
may	O
relate	O
to	O
the	O
structure	O
and	O
regulation	O
of	O
these	O
domains	O
rather	O
than	O
their	O
G	B-protein_type
protein	I-protein_type
interactions	O
.	O

N52T	B-mutant
is	O
one	O
of	O
a	O
combination	O
of	O
seven	O
residues	O
found	O
to	O
confer	O
ACK	B-protein
binding	O
on	O
Rac1	B-protein
and	O
so	O
may	O
represent	O
a	O
specific	O
Cdc42	B-protein
-	O
effector	O
contact	O
residue	O
.	O

Nonetheless	O
,	O
structural	B-experimental_method
studies	I-experimental_method
of	O
the	O
TOCA1	B-protein
HR1	B-structure_element
domain	O
,	O
combined	O
with	O
chemical	B-experimental_method
shift	I-experimental_method
mapping	I-experimental_method
,	O
have	O
highlighted	O
some	O
potentially	O
interesting	O
differences	O
between	O
Cdc42	B-protein
-	O
HR1TOCA1	B-structure_element
and	O
RhoA	B-protein
/	O
Rac1	B-protein
-	O
HR1PRK1	B-structure_element
binding	O
.	O

Evidence	O
suggests	O
that	O
the	O
TOCA	B-protein_type
family	I-protein_type
of	O
proteins	O
are	O
recruited	O
to	O
the	O
membrane	O
via	O
an	O
interaction	O
between	O
their	O
F	B-structure_element
-	I-structure_element
BAR	I-structure_element
domain	O
and	O
specific	O
signaling	O
lipids	O
.	O

A	O
simplified	O
model	O
of	O
the	O
early	O
stages	O
of	O
Cdc42	B-complex_assembly
·	I-complex_assembly
N	I-complex_assembly
-	I-complex_assembly
WASP	I-complex_assembly
·	I-complex_assembly
TOCA1	I-complex_assembly
-	O
dependent	O
actin	O
polymerization	O
.	O

The	O
HR1TOCA1	B-structure_element
-	O
Cdc42	O
and	O
SH3TOCA1	B-structure_element
-	O
N	O
-	O
WASP	O
interactions	O
position	O
Cdc42	B-protein
and	O
N	B-protein
-	I-protein
WASP	I-protein
for	O
binding	O
.	O

In	O
conclusion	O
,	O
the	O
data	O
presented	O
here	O
show	O
that	O
the	O
TOCA1	B-protein
HR1	B-structure_element
domain	O
is	O
sufficient	O
for	O
Cdc42	B-protein
binding	O
in	O
vitro	O
and	O
that	O
the	O
interaction	O
is	O
of	O
micromolar	O
affinity	O
,	O
lower	O
than	O
that	O
of	O
other	O
G	B-protein_type
protein	I-protein_type
-	O
HR1	B-structure_element
domain	O
interactions	O
.	O

Eukaryotic	B-taxonomy_domain
ACCs	B-protein_type
are	O
single	B-protein_type
-	I-protein_type
chain	I-protein_type
multienzymes	I-protein_type
characterized	O
by	O
a	O
large	O
,	O
non	B-protein_state
-	I-protein_state
catalytic	I-protein_state
central	B-structure_element
domain	I-structure_element
(	O
CD	B-structure_element
),	O
whose	O
role	O
in	O
ACC	B-protein_type
regulation	O
remains	O
poorly	O
characterized	O
.	O

Combining	O
the	O
yeast	B-taxonomy_domain
CD	B-structure_element
structure	B-evidence
with	O
intermediate	O
and	O
low	O
-	O
resolution	O
data	O
of	O
larger	B-mutant
fragments	I-mutant
up	O
to	O
intact	B-protein_state
ACCs	B-protein_type
provides	O
a	O
comprehensive	O
characterization	O
of	O
the	O
dynamic	B-protein_state
fungal	B-taxonomy_domain
ACC	B-protein_type
architecture	O
.	O

ACC	B-experimental_method
inhibition	I-experimental_method
and	I-experimental_method
knock	I-experimental_method
-	I-experimental_method
out	I-experimental_method
studies	I-experimental_method
show	O
the	O
potential	O
of	O
targeting	O
ACC	B-protein_type
for	O
treatment	O
of	O
the	O
metabolic	O
syndrome	O
.	O

BRCA1	B-protein
binds	O
only	O
to	O
the	O
phosphorylated	B-protein_state
form	O
of	O
ACC1	B-protein
and	O
prevents	O
ACC	B-protein_type
activation	O
by	O
phosphatase	B-protein_type
-	O
mediated	O
dephosphorylation	O
.	O

The	O
crystal	B-evidence
structure	I-evidence
of	O
the	O
CD	B-structure_element
of	O
SceACC	B-protein
(	O
SceCD	B-species
)	O
was	O
determined	O
at	O
3	O
.	O
0	O
Å	O
resolution	O
by	O
experimental	B-experimental_method
phasing	I-experimental_method
and	O
refined	B-experimental_method
to	O
Rwork	B-evidence
/	O
Rfree	B-evidence
=	O
0	O
.	O
20	O
/	O
0	O
.	O
24	O
(	O
Table	O
1	O
).	O

A	O
regulatory	B-structure_element
loop	I-structure_element
mediates	O
interdomain	O
interactions	O

To	O
define	O
the	O
functional	O
state	O
of	O
insect	B-experimental_method
-	I-experimental_method
cell	I-experimental_method
-	I-experimental_method
expressed	I-experimental_method
ACC	B-protein_type
variants	O
,	O
we	O
employed	O
mass	B-experimental_method
spectrometry	I-experimental_method
(	O
MS	B-experimental_method
)	O
for	O
phosphorylation	B-experimental_method
site	I-experimental_method
detection	I-experimental_method
.	O

The	O
SceCD	B-species
structure	B-evidence
thus	O
authentically	O
represents	O
the	O
state	O
of	O
SceACC	B-protein
,	O
where	O
the	O
enzyme	B-protein
is	O
inhibited	B-protein_state
by	O
SNF1	B-ptm
-	I-ptm
dependent	I-ptm
phosphorylation	I-ptm
.	O

Already	O
the	O
binding	O
of	O
phosphorylated	B-protein_state
Ser1157	B-residue_name_number
apparently	O
stabilizes	O
the	O
regulatory	B-structure_element
loop	I-structure_element
conformation	O
;	O
the	O
accessory	O
phosphorylation	B-site
sites	I-site
Ser1148	B-residue_name_number
and	O
Ser1162	B-residue_name_number
in	O
the	O
same	B-structure_element
loop	I-structure_element
may	O
further	O
modulate	O
the	O
strength	O
of	O
interaction	O
between	O
the	O
regulatory	B-structure_element
loop	I-structure_element
and	O
the	O
CDC1	B-structure_element
and	O
CDC2	B-structure_element
domains	O
.	O

However	O
,	O
residual	O
phosphorylation	B-ptm
levels	O
were	O
detected	O
for	O
Ser1204	B-residue_name_number
(	O
7	O
%)	O
and	O
Ser1218	B-residue_name_number
(	O
7	O
%)	O
in	O
the	O
same	B-structure_element
loop	I-structure_element
.	O

To	O
further	O
obtain	O
insights	O
into	O
the	O
functional	O
architecture	O
of	O
fungal	B-taxonomy_domain
ACC	B-protein_type
,	O
we	O
characterized	O
larger	B-mutant
multidomain	I-mutant
fragments	I-mutant
up	O
to	O
the	O
intact	B-protein_state
enzymes	B-protein
.	O

Using	O
molecular	B-experimental_method
replacement	I-experimental_method
based	O
on	O
fungal	B-taxonomy_domain
ACC	B-protein_type
CD	B-structure_element
and	O
CT	B-structure_element
models	O
,	O
we	O
obtained	O
structures	B-evidence
of	O
a	O
variant	B-mutant
comprising	O
CthCT	B-species
and	O
CDC1	B-structure_element
/	O
CDC2	B-structure_element
in	O
two	B-evidence
crystal	I-evidence
forms	I-evidence
at	O
resolutions	O
of	O
3	O
.	O
6	O
and	O
4	O
.	O
5	O
Å	O
(	O
CthCD	B-mutant
-	I-mutant
CTCter1	I-mutant
/	I-mutant
2	I-mutant
),	O
respectively	O
,	O
as	O
well	O
as	O
of	O
a	O
CthCT	B-species
linked	O
to	O
the	O
entire	O
CD	B-structure_element
at	O
7	O
.	O
2	O
Å	O
resolution	O
(	O
CthCD	B-mutant
-	I-mutant
CT	I-mutant
;	O
Figs	O
1a	O
and	O
2	O
,	O
Table	O
1	O
).	O

Owing	O
to	O
the	O
limited	O
resolution	O
the	O
discussion	O
of	O
structures	B-evidence
of	O
CthCD	B-mutant
-	I-mutant
CT	I-mutant
and	O
CthΔBCCP	B-mutant
is	O
restricted	O
to	O
the	O
analysis	O
of	O
domain	O
localization	O
.	O

However	O
,	O
MS	B-experimental_method
analysis	O
of	O
CthCD	B-mutant
-	I-mutant
CT	I-mutant
and	O
CthΔBCCP	B-mutant
constructs	O
revealed	O
between	O
60	O
and	O
70	O
%	O
phosphorylation	B-ptm
of	O
Ser1170	B-residue_name_number
(	O
corresponding	O
to	O
SceACC	B-protein
Ser1157	B-residue_name_number
).	O

Class	B-evidence
averages	I-evidence
,	O
obtained	O
by	O
maximum	B-experimental_method
-	I-experimental_method
likelihood	I-experimental_method
-	I-experimental_method
based	I-experimental_method
two	I-experimental_method
-	I-experimental_method
dimensional	I-experimental_method
(	I-experimental_method
2D	I-experimental_method
)	I-experimental_method
classification	I-experimental_method
,	O
are	O
focused	O
on	O
the	O
dimeric	B-oligomeric_state
CT	B-structure_element
domain	O
and	O
the	O
full	B-protein_state
BC	B-mutant
–	I-mutant
BCCP	I-mutant
–	I-mutant
CD	I-mutant
domain	O
of	O
only	O
one	O
protomer	B-oligomeric_state
,	O
due	O
to	O
the	O
non	O
-	O
coordinated	O
motions	O
of	O
the	O
lateral	O
BC	B-structure_element
/	O
CD	B-structure_element
regions	O
relative	O
to	O
the	O
CT	B-structure_element
dimer	B-oligomeric_state
.	O

They	O
identify	O
the	O
connections	O
between	O
CDN	B-structure_element
/	O
CDL	B-structure_element
and	O
between	O
CDC2	B-structure_element
/	O
CT	B-structure_element
as	O
major	O
contributors	O
to	O
conformational	O
heterogeneity	O
(	O
Supplementary	O
Fig	O
.	O
4a	O
,	O
b	O
).	O

The	O
most	O
relevant	O
candidate	O
site	O
for	O
mediating	O
such	O
additional	O
flexibility	O
and	O
permitting	O
an	O
extended	O
set	O
of	O
conformations	O
is	O
the	O
CDC1	B-site
/	I-site
CDC2	I-site
interface	I-site
,	O
which	O
is	O
rigidified	O
by	O
the	O
Ser1157	B-residue_name_number
-	O
phosphorylated	B-protein_state
regulatory	B-structure_element
loop	I-structure_element
,	O
as	O
depicted	O
in	O
the	O
SceCD	B-species
crystal	B-evidence
structure	I-evidence
.	O

The	O
CD	B-structure_element
consists	O
of	O
four	O
distinct	O
subdomains	B-structure_element
and	O
acts	O
as	O
a	O
tether	O
from	O
the	O
CT	B-structure_element
to	O
the	O
mobile	B-protein_state
BCCP	B-structure_element
and	O
an	O
oriented	B-protein_state
BC	B-structure_element
domain	O
.	O

In	O
higher	B-taxonomy_domain
eukaryotic	I-taxonomy_domain
ACCs	B-protein_type
,	O
regulation	O
via	O
phosphorylation	B-ptm
is	O
achieved	O
by	O
combining	O
the	O
effects	O
of	O
phosphorylation	B-ptm
at	O
Ser80	B-residue_name_number
,	O
Ser1201	B-residue_name_number
and	O
Ser1263	B-residue_name_number
.	O

A	O
comparison	O
between	O
fungal	B-taxonomy_domain
and	O
human	B-species
ACC	B-protein_type
will	O
help	O
to	O
further	O
discriminate	O
mechanistic	O
differences	O
that	O
contribute	O
to	O
the	O
extended	O
control	O
and	O
polymerization	O
of	O
human	B-species
ACC	B-protein_type
.	O

In	O
flACC	B-protein
,	O
CDC2	B-structure_element
rotates	O
∼	O
120	O
°	O
with	O
respect	O
to	O
the	O
CT	B-structure_element
domain	O
.	O

On	O
the	O
basis	O
of	O
a	O
superposition	B-experimental_method
of	O
CDC2	B-structure_element
,	O
CDC1	B-structure_element
of	O
the	O
phosphorylated	B-protein_state
SceCD	B-species
is	O
rotated	O
by	O
30	O
°	O
relative	O
to	O
CDC1	B-structure_element
of	O
the	O
non	B-protein_state
-	I-protein_state
phosphorylated	I-protein_state
flACC	B-protein
(	O
Supplementary	O
Fig	O
.	O
5d	O
),	O
similar	O
to	O
what	O
we	O
have	O
observed	O
for	O
the	O
non	B-protein_state
-	I-protein_state
phosphorylated	I-protein_state
HsaBT	B-mutant
-	I-mutant
CD	I-mutant
(	O
Supplementary	O
Fig	O
.	O
1d	O
).	O

When	O
inspecting	B-experimental_method
all	O
individual	O
protomer	B-oligomeric_state
and	O
fragment	B-mutant
structures	B-evidence
in	O
their	O
study	O
,	O
Wei	O
and	O
Tong	O
also	O
identify	O
the	O
CDN	B-structure_element
/	I-structure_element
CDC1	I-structure_element
connection	I-structure_element
as	O
a	O
highly	B-protein_state
flexible	I-protein_state
hinge	B-structure_element
,	O
in	O
agreement	O
with	O
our	O
observations	O
.	O

It	O
disfavours	O
the	O
adoption	O
of	O
a	O
rare	B-protein_state
,	I-protein_state
compact	I-protein_state
conformation	I-protein_state
,	O
in	O
which	O
intramolecular	O
dimerization	O
of	O
the	O
BC	B-structure_element
domains	O
results	O
in	O
catalytic	O
turnover	O
.	O

(	O
e	O
)	O
Structural	O
overview	O
of	O
HsaBT	B-mutant
-	I-mutant
CD	I-mutant
.	O

Cartoon	O
representation	O
of	O
crystal	B-evidence
structures	I-evidence
of	O
multidomain	B-mutant
constructs	I-mutant
of	O
CthACC	B-protein
.	O

(	O
b	O
)	O
The	O
interdomain	B-site
interface	I-site
of	O
CDC1	B-structure_element
and	O
CDC2	B-structure_element
exhibits	O
only	O
limited	O
plasticity	O
.	O

We	O
identified	O
that	O
the	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
SEL1L	B-protein
forms	O
a	O
self	B-oligomeric_state
-	I-oligomeric_state
oligomer	I-oligomeric_state
through	O
the	O
SEL1Lcent	B-structure_element
domain	O
in	O
mammalian	B-taxonomy_domain
cells	O
.	O

The	O
process	O
is	O
called	O
ER	O
-	O
associated	O
protein	O
degradation	O
(	O
ERAD	O
)	O
and	O
is	O
conserved	B-protein_state
in	O
all	O
eukaryotes	B-taxonomy_domain
.	O

SEL1L	B-protein
is	O
required	O
for	O
ER	O
homeostasis	O
,	O
which	O
is	O
essential	O
for	O
protein	O
translation	O
,	O
pancreatic	O
function	O
,	O
and	O
cellular	O
and	O
organismal	O
survival	O
.	O

Based	O
on	O
these	O
observations	O
,	O
we	O
propose	O
a	O
model	O
wherein	O
the	O
SLR	B-structure_element
domains	O
of	O
SEL1L	B-protein
contribute	O
to	O
the	O
formation	O
of	O
stable	B-protein_state
oligomers	B-oligomeric_state
of	O
the	O
ERAD	O
translocation	O
machinery	O
,	O
which	O
is	O
indispensable	O
for	O
ERAD	O
.	O

The	O
SLR	B-structure_element
motifs	O
can	O
be	O
grouped	O
into	O
three	O
regions	O
due	O
to	O
the	O
presence	O
of	O
linker	B-structure_element
sequences	I-structure_element
among	O
the	O
groups	O
of	O
SLR	B-structure_element
motifs	O
:	O
SLR	B-structure_element
-	I-structure_element
N	I-structure_element
(	O
SLR	B-structure_element
motifs	I-structure_element
1	I-structure_element
to	I-structure_element
4	I-structure_element
),	O
SLR	B-structure_element
-	I-structure_element
M	I-structure_element
(	O
SLR	B-structure_element
motifs	I-structure_element
5	I-structure_element
to	I-structure_element
9	I-structure_element
),	O
and	O
SLR	B-structure_element
-	I-structure_element
C	I-structure_element
(	O
SLR	B-structure_element
motifs	I-structure_element
10	I-structure_element
to	I-structure_element
11	I-structure_element
)	O
(	O
Fig	O
.	O
1A	O
).	O

We	O
first	O
tried	O
to	O
prepare	O
the	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
mouse	B-taxonomy_domain
SEL1L	B-protein
protein	O
,	O
excluding	O
the	O
transmembrane	B-structure_element
domain	I-structure_element
at	O
the	O
C	O
-	O
terminus	O
(	O
residues	O
735	B-residue_range
–	I-residue_range
755	I-residue_range
),	O
by	O
expression	B-experimental_method
in	I-experimental_method
bacteria	I-experimental_method
.	O

Both	O
SLR	B-structure_element
-	I-structure_element
N	I-structure_element
(	O
residues	O
194	B-residue_range
–	I-residue_range
343	I-residue_range
)	O
and	O
SLR	B-structure_element
-	I-structure_element
C	I-structure_element
(	O
residues	O
639	B-residue_range
–	I-residue_range
719	I-residue_range
)	O
alone	O
could	O
be	O
solubilized	O
with	O
an	O
MBP	B-experimental_method
tag	I-experimental_method
at	I-experimental_method
the	I-experimental_method
N	I-experimental_method
-	I-experimental_method
terminus	I-experimental_method
,	O
but	O
appeared	O
to	O
be	O
polydisperse	O
when	O
analyzed	O
by	O
size	B-experimental_method
-	I-experimental_method
exclusion	I-experimental_method
chromatography	I-experimental_method
.	O

Starting	O
from	O
its	O
N	O
-	O
terminus	O
,	O
the	O
α	B-structure_element
-	I-structure_element
solenoid	I-structure_element
of	O
SEL1L	B-protein
extends	O
across	O
a	O
semi	O
-	O
circle	O
in	O
a	O
right	O
-	O
handed	O
superhelix	O
fashion	O
along	O
the	O
rotation	O
axis	O
of	O
the	O
yin	B-structure_element
-	I-structure_element
yang	I-structure_element
circle	I-structure_element
.	O

However	O
,	O
the	O
last	O
helix	O
,	O
9B	B-structure_element
,	O
at	O
the	O
C	O
-	O
terminus	O
adopts	O
a	O
different	O
conformation	O
,	O
lying	O
parallel	O
to	O
the	O
long	O
axis	O
of	O
helix	B-structure_element
9A	I-structure_element
instead	O
of	O
forming	O
an	O
antiparallel	O
SLR	B-structure_element
.	O

Helix	B-structure_element
9B	I-structure_element
from	O
one	O
protomer	B-oligomeric_state
inserts	O
into	O
the	O
empty	O
space	O
surrounded	O
by	O
the	O
concave	B-site
region	I-site
in	O
the	O
other	O
monomer	B-oligomeric_state
,	O
forming	O
a	O
domain	B-protein_state
-	I-protein_state
swapped	I-protein_state
conformation	O
.	O

In	O
this	O
interface	B-site
,	O
Leu	B-residue_name_number
516	I-residue_name_number
and	O
Tyr	B-residue_name_number
519	I-residue_name_number
on	O
helix	B-structure_element
9B	I-structure_element
are	O
located	O
in	O
the	O
center	O
,	O
making	O
hydrophobic	B-bond_interaction
interactions	I-bond_interaction
with	O
Trp	B-residue_name_number
478	I-residue_name_number
on	O
helix	B-structure_element
8B	I-structure_element
,	O
Val	B-residue_name_number
444	I-residue_name_number
on	O
helix	B-structure_element
7B	I-structure_element
,	O
Phe	B-residue_name_number
411	I-residue_name_number
on	O
helix	B-structure_element
6B	I-structure_element
,	O
and	O
Leu	B-residue_name_number
380	I-residue_name_number
on	O
helix	B-structure_element
5B	I-structure_element
from	O
the	O
SEL1Lcent	B-structure_element
counterpart	O
(	O
Fig	O
.	O
2A	O
,	O
Interface	B-site
1	I-site
detail	O
).	O

Second	O
,	O
the	O
residues	O
from	O
helix	B-structure_element
9A	I-structure_element
interact	O
with	O
the	O
residues	O
from	O
helix	B-structure_element
5A	I-structure_element
of	O
its	O
counterpart	O
in	O
a	O
head	B-protein_state
-	I-protein_state
to	I-protein_state
-	I-protein_state
tail	I-protein_state
orientation	O
.	O

However	O
,	O
the	O
mutant	B-protein_state
behaved	O
as	O
a	O
monomer	B-oligomeric_state
in	O
size	B-experimental_method
-	I-experimental_method
exclusion	I-experimental_method
chromatography	I-experimental_method
and	O
analytical	B-experimental_method
ultracentrifugation	I-experimental_method
experiments	O
(	O
Fig	O
.	O
2B	O
,	O
Supplementary	O
Fig	O
.	O
2C	O
).	O

SLRs	B-structure_element
of	O
mouse	B-taxonomy_domain
SEL1L	B-protein
were	O
predicted	O
using	O
the	O
TPRpred	B-experimental_method
server	I-experimental_method
.	O

Although	O
amino	O
acid	O
sequences	O
from	O
helix	B-structure_element
9A	I-structure_element
and	O
9B	B-structure_element
correctly	O
aligned	O
with	O
the	O
regular	O
SLR	B-structure_element
repeats	I-structure_element
and	O
corresponded	O
to	O
SLR	B-structure_element
motif	I-structure_element
9	I-structure_element
(	O
Fig	O
.	O
3A	O
),	O
the	O
structural	O
arrangement	O
of	O
the	O
two	O
helices	B-structure_element
deviated	O
from	O
the	O
common	O
structure	O
for	O
the	O
SLR	B-structure_element
motif	O
.	O

Thus	O
,	O
the	O
Gly	B-structure_element
-	I-structure_element
Gly	I-structure_element
residues	O
generate	O
an	O
unusual	O
sharp	O
bend	O
at	O
the	O
C	O
-	O
terminal	O
SLR	B-structure_element
motif	I-structure_element
9	I-structure_element
.	O

G512A	B-mutant
or	O
G513A	B-mutant
alone	O
showed	O
no	O
differences	O
from	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
in	O
terms	O
of	O
the	O
size	B-experimental_method
-	I-experimental_method
exclusion	I-experimental_method
chromatography	I-experimental_method
elution	O
profile	O
(	O
Fig	O
.	O
3D	O
),	O
suggesting	O
that	O
the	O
restriction	O
for	O
single	O
glycine	B-residue_name
flexibility	O
would	O
not	O
be	O
enough	O
to	O
break	O
the	O
swapped	O
structure	O
of	O
helix	B-structure_element
9B	I-structure_element
.	O

We	O
generated	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
SEL1L	B-protein
-	O
HA	B-experimental_method
and	O
SEL1L	B-protein
-	O
FLAG	B-experimental_method
fusion	B-experimental_method
constructs	I-experimental_method
and	O
co	B-experimental_method
-	I-experimental_method
transfected	I-experimental_method
the	O
constructs	O
into	O
HEK293T	O
cells	O
.	O

Indeed	O
,	O
immunoprecipitation	B-experimental_method
followed	O
by	O
western	B-experimental_method
blot	I-experimental_method
analysis	O
using	O
the	O
culture	O
medium	O
detected	O
secreted	O
SEL1L348	B-mutant
–	I-mutant
497	I-mutant
fragment	O
,	O
but	O
not	O
SEL1Lcent	B-structure_element
(	O
Fig	O
.	O
4B	O
).	O

To	O
this	O
end	O
,	O
we	O
co	B-experimental_method
-	I-experimental_method
transfected	I-experimental_method
the	O
differentially	O
tagged	B-protein_state
full	B-protein_state
-	I-protein_state
length	I-protein_state
SEL1L	B-protein
(	O
SEL1L	B-protein
-	O
HA	B-experimental_method
and	O
SEL1L	B-protein
-	O
FLAG	B-experimental_method
)	O
and	O
increasing	B-experimental_method
doses	I-experimental_method
of	O
SEL1Lcent	B-mutant
-	I-mutant
KDEL	I-mutant
,	O
SEL1L348	B-mutant
–	I-mutant
497	I-mutant
-	I-mutant
KDEL	I-mutant
or	O
SEL1Lcent	B-mutant
(	I-mutant
L521A	I-mutant
)-	I-mutant
KDEL	I-mutant
,	O
respectively	O
.	O

Co	B-experimental_method
-	I-experimental_method
immunoprecipitation	I-experimental_method
assay	I-experimental_method
revealed	O
that	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
SEL1Lcent	B-mutant
-	I-mutant
KDEL	I-mutant
,	O
indeed	O
,	O
competitively	O
disrupted	O
the	O
self	O
-	O
association	O
of	O
the	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
SEL1L	O
(	O
Fig	O
.	O
4E	O
).	O

This	O
is	O
one	O
of	O
the	O
biggest	O
differences	O
from	O
TPRs	B-structure_element
in	O
Cdc23	B-protein
and	O
from	O
the	O
SLRs	B-structure_element
in	O
HcpC	B-protein
,	O
where	O
the	O
motifs	O
exist	O
in	O
tandem	O
.	O

Indirect	O
evidence	O
from	O
a	O
previous	O
yeast	B-taxonomy_domain
study	O
shows	O
that	O
the	O
circumscribed	O
region	O
of	O
C	O
-	O
terminal	O
Hrd3p	B-protein
,	O
specifically	O
residues	O
664	B-residue_range
–	I-residue_range
695	I-residue_range
,	O
forms	O
contacts	O
with	O
the	O
Hrd1	B-protein
luminal	B-structure_element
loops	I-structure_element
.	O

This	O
hypothesis	O
is	O
supported	O
by	O
cross	B-experimental_method
-	I-experimental_method
linking	I-experimental_method
data	I-experimental_method
suggesting	O
that	O
human	B-species
HRD1	B-protein
forms	O
a	O
homodimer	B-oligomeric_state
.	O

However	O
,	O
metazoans	B-taxonomy_domain
lack	O
a	O
clear	O
Usa1p	B-protein
homolog	O
.	O

Assuming	O
that	O
the	O
correct	O
oligomerization	O
of	O
ERAD	O
components	O
may	O
be	O
critical	O
for	O
their	O
function	O
,	O
we	O
hypothesize	O
that	O
homodimer	B-oligomeric_state
formation	O
of	O
SEL1L	B-protein
in	O
the	O
ER	O
lumen	O
may	O
stabilize	O
oligomerization	O
of	O
the	O
HRD	B-complex_assembly
complex	O
,	O
given	O
that	O
SEL1L	B-protein
forms	O
a	O
stoichiometric	O
complex	B-protein_state
with	I-protein_state
HRD1	B-protein
.	O

Recently	O
,	O
a	O
truncated	B-protein_state
version	O
of	O
Yos9	B-protein
was	O
shown	O
to	O
form	O
a	O
dimer	B-oligomeric_state
in	O
the	O
ER	O
lumen	O
and	O
to	O
contribute	O
to	O
the	O
dimeric	B-oligomeric_state
state	O
of	O
the	O
Hrd1p	B-protein
complex	O
.	O

Putative	O
N	B-site
-	I-site
glycosylation	I-site
sites	I-site
are	O
indicated	O
by	O
black	O
triangles	O
.	O

The	O
schematic	O
diagrams	O
representing	O
the	O
protein	O
constructs	O
used	O
in	O
the	O
SEC	B-experimental_method
are	O
shown	O
on	O
the	O
left	O
of	O
each	O
SDS	B-experimental_method
-	I-experimental_method
PAGE	I-experimental_method
profile	O
.	O

(	O
A	O
)	O
Sequence	B-experimental_method
alignment	I-experimental_method
of	O
the	O
SLR	B-structure_element
motifs	O
in	O
SEL1L	B-protein
.	O

The	O
11	O
SLR	B-structure_element
motifs	O
of	O
SEL1L	B-protein
were	O
expressed	O
with	O
red	O
cylinders	O
and	O
grouped	O
into	O
three	O
parts	O
(	O
SLR	B-structure_element
-	I-structure_element
N	I-structure_element
,	O
SLR	B-structure_element
-	I-structure_element
M	I-structure_element
,	O
and	O
SLR	B-structure_element
-	I-structure_element
C	I-structure_element
)	O
based	O
on	O
the	O
sequence	B-experimental_method
alignment	I-experimental_method
across	O
the	O
motifs	O
and	O
the	O
crystal	B-evidence
structure	I-evidence
presented	O
herein	O
.	O

Crystal	B-evidence
Structures	I-evidence
of	O
Putative	O
Sugar	B-protein_type
Kinases	I-protein_type
from	O
Synechococcus	B-species
Elongatus	I-species
PCC	I-species
7942	I-species
and	O
Arabidopsis	B-species
Thaliana	I-species

The	O
genome	O
of	O
the	O
Synechococcus	B-species
elongatus	I-species
strain	I-species
PCC	I-species
7942	I-species
encodes	O
a	O
putative	O
sugar	B-protein_type
kinase	I-protein_type
(	O
SePSK	B-protein
),	O
which	O
shares	O
44	O
.	O
9	O
%	O
sequence	O
identity	O
with	O
the	O
xylulose	B-protein
kinase	I-protein
-	I-protein
1	I-protein
(	O
AtXK	B-protein
-	I-protein
1	I-protein
)	O
from	O
Arabidopsis	B-species
thaliana	I-species
.	O

The	O
At2g21370	B-gene
gene	O
product	O
from	O
Arabidopsis	B-species
thaliana	I-species
,	O
xylulose	B-protein
kinase	I-protein
-	I-protein
1	I-protein
(	O
AtXK	B-protein
-	I-protein
1	I-protein
),	O
whose	O
mature	B-protein_state
form	I-protein_state
contains	O
436	B-residue_range
amino	O
acids	O
,	O
is	O
located	O
in	O
the	O
chloroplast	O
(	O
ChloroP	O
1	O
.	O
1	O
Server	O
).	O

Two	O
possible	O
xylulose	B-protein_type
kinases	I-protein_type
(	O
xylulose	B-protein
kinase	I-protein
-	I-protein
1	I-protein
:	O
XK	B-protein
-	I-protein
1	I-protein
and	O
xylulose	B-protein
kinase	I-protein
-	I-protein
2	I-protein
:	O
XK	B-protein
-	I-protein
2	I-protein
)	O
from	O
Arabidopsis	B-species
thaliana	I-species
were	O
previously	O
proposed	O
.	O

The	O
attempt	O
to	O
solve	O
the	O
SePSK	B-protein
structure	B-evidence
by	O
molecular	B-experimental_method
replacement	I-experimental_method
method	I-experimental_method
failed	O
with	O
ribulokinase	B-protein
from	O
Bacillus	B-species
halodurans	I-species
(	O
PDB	O
code	O
:	O
3QDK	O
,	O
15	O
.	O
7	O
%	O
sequence	O
identity	O
)	O
as	O
an	O
initial	O
model	O
.	O

Among	O
all	O
these	O
structural	O
elements	O
,	O
α4	B-structure_element
/	O
α5	B-structure_element
/	O
α11	B-structure_element
/	O
α18	B-structure_element
,	O
β3	B-structure_element
/	O
β2	B-structure_element
/	O
β1	B-structure_element
/	O
β6	B-structure_element
/	O
β19	B-structure_element
/	O
β20	B-structure_element
/	O
β17	B-structure_element
and	O
α21	B-structure_element
/	O
α32	B-structure_element
form	O
three	O
patches	O
,	O
referred	O
to	O
as	O
A1	B-structure_element
,	O
B1	B-structure_element
and	O
A2	B-structure_element
,	O
exhibiting	O
the	O
core	B-structure_element
region	I-structure_element
.	O

(	O
A	O
)	O
Three	O
-	O
dimensional	O
structure	B-evidence
of	O
apo	B-protein_state
-	O
SePSK	B-protein
.	O

(	O
B	O
)	O
Three	O
-	O
dimensional	O
structure	B-evidence
of	O
apo	B-protein_state
-	O
AtXK	B-protein
-	I-protein
1	I-protein
.	O

In	O
contrary	O
,	O
limited	O
increasing	O
of	O
ATP	B-chemical
hydrolysis	O
activity	O
was	O
detected	O
for	O
AtXK	B-protein
-	I-protein
1	I-protein
upon	O
addition	O
of	O
D	B-chemical
-	I-chemical
ribulose	I-chemical
(	O
Fig	O
2C	O
),	O
despite	O
its	O
structural	O
similarity	O
with	O
SePSK	B-protein
.	O

While	O
the	O
ATP	B-chemical
hydrolysis	O
activity	O
of	O
SePSK	B-protein
greatly	O
increases	O
upon	O
addition	O
of	O
D	B-chemical
-	I-chemical
ribulose	I-chemical
(	O
DR	B-chemical
).	O

The	O
ATP	B-chemical
hydrolysis	O
activity	O
measured	O
via	O
luminescent	B-experimental_method
ADP	I-experimental_method
-	I-experimental_method
Glo	I-experimental_method
assay	I-experimental_method
(	O
Promega	O
).	O

Our	O
results	O
suggested	O
that	O
three	O
conserved	O
residues	O
(	O
D8	B-residue_name_number
,	O
T11	B-residue_name_number
and	O
D221	B-residue_name_number
of	O
SePSK	B-protein
)	O
play	O
an	O
important	O
role	O
in	O
SePSK	B-protein
function	O
.	O

Using	O
enzymatic	B-experimental_method
activity	I-experimental_method
assays	I-experimental_method
,	O
we	O
found	O
that	O
all	O
of	O
these	O
mutants	O
exhibit	O
much	O
lower	O
activity	O
of	O
ATP	B-chemical
hydrolysis	O
after	O
adding	O
D	B-chemical
-	I-chemical
ribulose	I-chemical
than	O
that	O
of	O
wild	B-protein_state
type	I-protein_state
,	O
indicating	O
the	O
possibility	O
that	O
these	O
three	O
residues	O
are	O
involved	O
in	O
the	O
catalytic	O
process	O
of	O
phosphorylation	B-ptm
D	B-chemical
-	I-chemical
ribulose	I-chemical
and	O
are	O
vital	O
for	O
the	O
function	O
of	O
SePSK	B-protein
(	O
Fig	O
2D	O
).	O

Thus	O
the	O
two	O
structures	B-evidence
were	O
named	O
ADP	B-complex_assembly
-	I-complex_assembly
SePSK	I-complex_assembly
and	O
ADP	B-complex_assembly
-	I-complex_assembly
AtXK	I-complex_assembly
-	I-complex_assembly
1	I-complex_assembly
,	O
respectively	O
.	O

As	O
shown	O
in	O
Fig	O
3A	O
,	O
one	O
SePSK	B-protein
protein	O
molecule	O
is	O
in	O
an	O
asymmetric	O
unit	O
with	O
one	O
AMP	B-chemical
-	I-chemical
PNP	I-chemical
molecule	O
.	O

The	O
AMP	B-chemical
-	I-chemical
PNP	I-chemical
is	O
bound	O
at	O
the	O
domain	B-structure_element
II	I-structure_element
,	O
where	O
it	O
fits	O
well	O
inside	O
a	O
positively	B-site
charged	I-site
groove	I-site
.	O

(	O
B	O
)	O
The	O
AMP	B-site
-	I-site
PNP	I-site
binding	I-site
pocket	I-site
.	O

7	O
.	O
1	O
Å	O
(	O
RBL1	B-residue_name_number
-	O
C4	O
and	O
RBL2	B-residue_name_number
-	O
C1	O
).	O

Furthermore	O
,	O
the	O
O2	O
of	O
RBL1	B-residue_name_number
interacts	B-bond_interaction
with	I-bond_interaction
the	O
main	O
chain	O
amide	O
nitrogen	O
of	O
Ser72	B-residue_name_number
(	O
Fig	O
4B	O
).	O

The	O
original	O
data	O
is	O
shown	O
as	O
black	O
curve	O
,	O
and	O
the	O
fitted	O
data	O
is	O
shown	O
as	O
different	O
color	O
(	O
wild	B-protein_state
type	I-protein_state
SePSK	B-protein
:	O
red	O
curve	O
,	O
D8A	B-mutant
-	O
SePSK	B-protein
:	O
green	O
curve	O
).	O

The	O
side	O
chain	O
of	O
Asp8	B-residue_name_number
interacts	B-bond_interaction
strongly	I-bond_interaction
with	I-bond_interaction
O3	O
and	O
O4	O
of	O
RBL2	B-residue_name_number
.	O

Structural	B-experimental_method
comparison	I-experimental_method
of	O
SePSK	B-protein
and	O
AtXK	B-protein
-	I-protein
1	I-protein
showed	O
that	O
while	O
the	O
RBL1	B-site
binding	I-site
pocket	I-site
is	O
conserved	B-protein_state
,	O
the	O
RBL2	B-site
pocket	I-site
is	O
disrupted	O
in	O
AtXK	B-protein
-	I-protein
1	I-protein
structure	B-evidence
,	O
despite	O
the	O
fact	O
that	O
the	O
residues	O
interacting	O
with	O
RBL2	B-residue_name_number
are	O
highly	B-protein_state
conserved	I-protein_state
between	O
the	O
two	O
proteins	O
.	O

Our	O
SePSK	B-protein
structure	B-evidence
shows	O
that	O
the	O
Asp8	B-residue_name_number
residue	O
forms	O
strong	O
hydrogen	B-bond_interaction
bond	I-bond_interaction
with	O
RBL2	B-residue_name_number
(	O
Fig	O
4B	O
).	O

In	O
addition	O
,	O
this	O
difference	O
may	O
be	O
caused	O
by	O
the	O
binding	O
of	O
substrates	O
and	O
/	O
or	O
ATP	B-chemical
.	O

The	O
results	O
of	O
superposition	B-experimental_method
displayed	O
different	O
crossing	O
angle	O
between	O
these	O
two	O
domains	O
.	O

Meanwhile	O
,	O
the	O
distances	O
of	O
AMP	B-chemical
-	I-chemical
PNP	I-chemical
γ	O
-	O
phosphate	B-chemical
and	O
the	O
first	O
hydroxyl	O
group	O
of	O
RBL2	B-residue_name_number
are	O
7	O
.	O
2	O
Å	O
(	O
superposed	B-experimental_method
with	O
AtXK	B-protein
-	I-protein
1	I-protein
),	O
6	O
.	O
7	O
Å	O
(	O
superposed	B-experimental_method
with	O
SePSK	B-protein
),	O
3	O
.	O
7	O
Å	O
(	O
superposed	B-experimental_method
with	O
3LL3	O
),	O
until	O
AMP	B-chemical
-	I-chemical
PNP	I-chemical
γ	O
-	O
phosphate	B-chemical
fully	O
contacts	O
RBL2	B-residue_name_number
after	O
superposition	B-experimental_method
with	O
1GLJ	O
(	O
Fig	O
5	O
).	O

In	O
summary	O
,	O
our	O
structural	B-experimental_method
and	I-experimental_method
enzymatic	I-experimental_method
analyses	I-experimental_method
provide	O
evidence	O
that	O
SePSK	B-protein
shows	O
D	B-protein_type
-	I-protein_type
ribulose	I-protein_type
kinase	I-protein_type
activity	O
,	O
and	O
exhibits	O
the	O
conserved	O
features	O
of	O
FGGY	B-protein_type
family	I-protein_type
carbohydrate	I-protein_type
kinases	I-protein_type
.	O

Three	O
conserved	B-site
residues	O
in	O
SePSK	B-protein
were	O
identified	O
to	O
be	O
essential	O
for	O
this	O
function	O
.	O

The	O
inducible	B-protein_state
lysine	B-protein_type
decarboxylase	I-protein_type
LdcI	B-protein
is	O
an	O
important	O
enterobacterial	B-taxonomy_domain
acid	B-protein_type
stress	I-protein_type
response	I-protein_type
enzyme	I-protein_type
whereas	O
LdcC	B-protein
is	O
its	O
close	O
paralogue	O
thought	O
to	O
play	O
mainly	O
a	O
metabolic	O
role	O
.	O

Decarboxylation	O
of	O
the	O
amino	B-chemical
acid	I-chemical
into	O
a	O
polyamine	B-chemical
is	O
catalysed	O
by	O
a	O
PLP	B-chemical
cofactor	O
in	O
a	O
multistep	O
reaction	O
that	O
consumes	O
a	O
cytoplasmic	O
proton	B-chemical
and	O
produces	O
a	O
CO2	B-chemical
molecule	O
passively	O
diffusing	O
out	O
of	O
the	O
cell	O
,	O
while	O
the	O
polyamine	B-chemical
is	O
excreted	O
by	O
the	O
antiporter	B-protein_type
in	O
exchange	O
for	O
a	O
new	O
amino	B-chemical
acid	I-chemical
substrate	O
.	O

Each	O
monomer	B-oligomeric_state
is	O
composed	O
of	O
three	O
domains	O
–	O
an	O
N	O
-	O
terminal	O
wing	B-structure_element
domain	I-structure_element
(	O
residues	O
1	B-residue_range
–	I-residue_range
129	I-residue_range
),	O
a	O
PLP	B-structure_element
-	I-structure_element
binding	I-structure_element
core	I-structure_element
domain	I-structure_element
(	O
residues	O
130	B-residue_range
–	I-residue_range
563	I-residue_range
),	O
and	O
a	O
C	B-structure_element
-	I-structure_element
terminal	I-structure_element
domain	I-structure_element
(	O
CTD	B-structure_element
,	O
residues	O
564	B-residue_range
–	I-residue_range
715	I-residue_range
).	O

Furthermore	O
,	O
we	O
recently	O
solved	B-experimental_method
the	I-experimental_method
structure	I-experimental_method
of	O
the	O
E	B-species
.	I-species
coli	I-species
LdcI	B-complex_assembly
-	I-complex_assembly
RavA	I-complex_assembly
complex	O
by	O
cryo	B-experimental_method
-	I-experimental_method
electron	I-experimental_method
microscopy	I-experimental_method
(	O
cryoEM	B-experimental_method
)	O
and	O
combined	O
it	O
with	O
the	O
crystal	B-evidence
structures	I-evidence
of	O
the	O
individual	O
proteins	O
.	O

In	O
addition	O
,	O
the	O
wing	B-structure_element
domains	I-structure_element
of	O
all	O
structures	B-evidence
are	O
very	O
similar	O
,	O
with	O
the	O
RMSD	B-evidence
after	O
optimal	O
rigid	O
body	O
alignment	O
(	O
RMSDmin	B-evidence
)	O
less	O
than	O
1	O
.	O
1	O
Å	O
.	O
Thus	O
,	O
taking	O
the	O
limited	O
resolution	O
of	O
the	O
cryoEM	B-experimental_method
maps	B-evidence
into	O
account	O
,	O
we	O
consider	O
that	O
the	O
wing	B-structure_element
domains	I-structure_element
of	O
all	O
the	O
four	O
structures	B-evidence
are	O
essentially	O
identical	O
and	O
that	O
in	O
the	O
present	O
study	O
the	O
RMSD	B-evidence
of	O
less	O
than	O
2	O
Å	O
can	O
serve	O
as	O
a	O
baseline	O
below	O
which	O
differences	O
may	O
be	O
assumed	O
as	O
insignificant	O
.	O

This	O
interface	B-site
is	O
formed	O
essentially	O
by	O
the	O
core	B-structure_element
domains	I-structure_element
with	O
some	O
contribution	O
of	O
the	O
CTDs	B-structure_element
.	O

Zooming	O
in	O
the	O
variations	O
in	O
the	O
PLP	B-structure_element
-	I-structure_element
SD	I-structure_element
shows	O
that	O
most	O
of	O
the	O
structural	O
changes	O
concern	O
displacements	O
in	O
the	O
active	B-site
site	I-site
(	O
Fig	O
.	O
3C	O
–	O
F	O
).	O

The	O
most	O
conspicuous	O
differences	O
between	O
the	O
PLP	B-structure_element
-	I-structure_element
SDs	I-structure_element
can	O
be	O
linked	O
to	O
the	O
pH	B-protein_state
-	I-protein_state
dependent	I-protein_state
activation	O
of	O
the	O
enzymes	O
.	O

In	O
particular	O
,	O
transition	O
from	O
LdcIi	B-protein
to	O
LdcI	B-complex_assembly
-	I-complex_assembly
LARA	I-complex_assembly
involves	O
~	O
3	O
.	O
5	O
Å	O
and	O
~	O
4	O
.	O
5	O
Å	O
shifts	O
away	O
from	O
the	O
5	O
-	O
fold	O
axis	O
in	O
the	O
active	B-site
site	I-site
α	B-structure_element
-	I-structure_element
helices	I-structure_element
spanning	O
residues	O
218	B-residue_range
–	I-residue_range
232	I-residue_range
and	O
246	B-residue_range
–	I-residue_range
254	I-residue_range
respectively	O
(	O
Fig	O
.	O
3C	O
–	O
E	O
).	O

An	O
inhibitor	O
of	O
the	O
LdcI	B-protein
and	O
LdcC	B-protein
activity	O
,	O
the	O
stringent	B-chemical
response	I-chemical
alarmone	I-chemical
ppGpp	B-chemical
,	O
is	O
known	O
to	O
bind	O
at	O
the	O
interface	B-site
between	O
neighboring	O
monomers	B-oligomeric_state
within	O
each	O
ring	B-structure_element
(	O
Fig	O
.	O
S4	O
).	O

Interestingly	O
,	O
although	O
a	O
number	O
of	O
ppGpp	B-site
binding	I-site
residues	I-site
are	O
strictly	B-protein_state
conserved	I-protein_state
between	O
LdcI	B-protein
and	O
AdiA	B-protein
that	O
also	O
forms	O
decamers	B-oligomeric_state
at	O
low	B-protein_state
pH	I-protein_state
optimal	I-protein_state
for	O
its	O
arginine	B-protein_type
decarboxylase	I-protein_type
activity	O
,	O
no	O
ppGpp	B-chemical
regulation	O
of	O
AdiA	B-protein
could	O
be	O
demonstrated	O
.	O

All	O
lysine	B-protein_type
decarboxylases	I-protein_type
predicted	O
to	O
be	O
“	O
LdcI	B-protein_type
-	I-protein_type
like	I-protein_type
”	O
or	O
biodegradable	B-protein_state
based	O
on	O
their	O
genetic	O
environment	O
,	O
as	O
for	O
example	O
their	O
organization	O
in	O
an	O
operon	O
with	O
a	O
gene	O
encoding	O
the	O
CadB	B-protein
antiporter	B-protein_type
(	O
see	O
Methods	O
),	O
were	O
found	O
in	O
one	O
group	O
,	O
whereas	O
all	O
enzymes	B-protein_type
predicted	O
as	O
“	O
LdcC	B-protein_type
-	I-protein_type
like	I-protein_type
”	O
or	O
biosynthetic	B-protein_state
partitioned	O
into	O
another	O
group	O
.	O

Our	O
structures	B-evidence
show	O
that	O
this	B-structure_element
motif	I-structure_element
is	O
not	O
involved	O
in	O
the	O
enzymatic	O
activity	O
or	O
the	O
oligomeric	O
state	O
of	O
the	O
proteins	O
.	O

Only	O
one	O
of	O
the	O
two	O
rings	B-structure_element
of	O
the	O
double	B-structure_element
toroid	I-structure_element
is	O
shown	O
for	O
clarity	O
.	O

(	O
B	O
)	O
The	O
LdcIi	B-protein
dimer	B-oligomeric_state
extracted	O
from	O
the	O
crystal	B-evidence
structure	I-evidence
of	O
the	O
decamer	B-oligomeric_state
.	O

The	O
PLP	B-chemical
is	O
red	O
.	O

(	O
A	O
)	O
Maximum	B-evidence
likelihood	I-evidence
tree	I-evidence
with	O
the	O
“	O
LdcC	B-protein_type
-	I-protein_type
like	I-protein_type
”	O
and	O
the	O
“	O
LdcI	B-protein_type
-	I-protein_type
like	I-protein_type
”	O
groups	O
highlighted	O
in	O
green	O
and	O
pink	O
,	O
respectively	O
.	O

(	O
B	O
)	O
Analysis	O
of	O
consensus	O
“	O
LdcI	B-protein_type
-	I-protein_type
like	I-protein_type
”	O
and	O
“	O
LdcC	B-protein_type
-	I-protein_type
like	I-protein_type
”	O
sequences	O
around	O
the	O
first	O
and	O
second	O
C	O
-	O
terminal	O
β	B-structure_element
-	I-structure_element
strands	I-structure_element
.	O

(	O
C	O
)	O
Signature	O
sequences	O
of	O
LdcI	B-protein
and	O
LdcC	B-protein
in	O
the	O
C	O
-	O
terminal	O
β	B-structure_element
-	I-structure_element
sheet	I-structure_element
.	O

Mep2	B-protein_type
proteins	I-protein_type
are	O
fungal	B-taxonomy_domain
transceptors	B-protein_type
that	O
play	O
an	O
important	O
role	O
as	O
ammonium	B-chemical
sensors	O
in	O
fungal	B-taxonomy_domain
development	O
.	O

Mep2	B-protein_type
activity	O
is	O
tightly	O
regulated	O
by	O
phosphorylation	B-ptm
,	O
but	O
how	O
this	O
is	O
achieved	O
at	O
the	O
molecular	O
level	O
is	O
not	O
clear	O
.	O

Relative	O
to	O
the	O
open	B-protein_state
bacterial	B-taxonomy_domain
ammonium	B-protein_type
transporters	I-protein_type
,	O
non	B-protein_state
-	I-protein_state
phosphorylated	I-protein_state
Mep2	B-protein_type
exhibits	O
shifts	O
in	O
cytoplasmic	B-structure_element
loops	I-structure_element
and	O
the	O
C	B-structure_element
-	I-structure_element
terminal	I-structure_element
region	I-structure_element
(	O
CTR	B-structure_element
)	O
to	O
occlude	O
the	O
cytoplasmic	O
exit	B-site
of	O
the	O
channel	B-site
and	O
to	O
interact	O
with	O
His2	B-residue_name_number
of	O
the	O
twin	B-structure_element
-	I-structure_element
His	I-structure_element
motif	I-structure_element
.	O

One	O
of	O
the	O
most	O
important	O
unresolved	O
questions	O
in	O
the	O
field	O
is	O
how	O
the	O
transceptors	B-protein_type
couple	O
to	O
downstream	O
signalling	O
pathways	O
.	O

Mep2	B-protein_type
(	B-protein_type
methylammonium	I-protein_type
(	I-protein_type
MA	I-protein_type
)	I-protein_type
permease	I-protein_type
)	I-protein_type
proteins	I-protein_type
are	O
ammonium	B-protein_type
transceptors	I-protein_type
that	O
are	O
ubiquitous	O
in	O
fungi	B-taxonomy_domain
.	O

Compared	O
with	O
Mep1	B-protein
and	O
Mep3	B-protein
,	O
Mep2	B-protein
is	O
highly	B-protein_state
expressed	I-protein_state
and	O
functions	O
as	O
a	O
low	O
-	O
capacity	O
,	O
high	O
-	O
affinity	O
transporter	O
in	O
the	O
uptake	O
of	O
MA	B-chemical
.	O

All	O
the	O
solved	O
structures	B-evidence
including	O
that	O
of	O
RhCG	B-protein
are	O
very	O
similar	O
,	O
establishing	O
the	O
basic	O
architecture	O
of	O
ammonium	B-protein_type
transporters	I-protein_type
.	O

Where	O
earlier	O
studies	O
favoured	O
the	O
transport	O
of	O
ammonia	B-chemical
gas	O
,	O
recent	O
data	O
and	O
theoretical	O
considerations	O
suggest	O
that	O
Amt	B-protein_type
/	I-protein_type
Mep	I-protein_type
proteins	I-protein_type
are	O
instead	O
active	B-protein_state
,	O
electrogenic	B-protein_type
transporters	I-protein_type
of	O
either	O
NH4	B-chemical
+	I-chemical
(	O
uniport	O
)	O
or	O
NH3	B-chemical
/	O
H	B-chemical
+	I-chemical
(	O
symport	O
).	O

Ammonium	B-chemical
transport	O
is	O
tightly	O
regulated	O
.	O

The	O
structures	B-evidence
are	O
similar	O
to	O
each	O
other	O
but	O
show	O
considerable	O
differences	O
to	O
all	O
other	O
ammonium	B-protein_type
transporter	I-protein_type
structures	B-evidence
.	O

The	O
Mep2	B-protein
protein	O
of	O
S	B-species
.	I-species
cerevisiae	I-species
(	O
ScMep2	B-protein
)	O
was	O
overexpressed	B-experimental_method
in	O
S	B-species
.	I-species
cerevisiae	I-species
in	O
high	O
yields	O
,	O
enabling	O
structure	B-experimental_method
determination	I-experimental_method
by	O
X	B-experimental_method
-	I-experimental_method
ray	I-experimental_method
crystallography	I-experimental_method
using	O
data	O
to	O
3	O
.	O
2	O
Å	O
resolution	O
by	O
molecular	B-experimental_method
replacement	I-experimental_method
(	O
MR	B-experimental_method
)	O
with	O
the	O
archaebacterial	B-taxonomy_domain
Amt	B-protein
-	I-protein
1	I-protein
structure	B-evidence
(	O
see	O
Methods	O
section	O
).	O

Given	O
that	O
the	O
modest	O
resolution	O
of	O
the	O
structure	B-evidence
and	O
the	O
limited	O
detergent	O
stability	O
of	O
ScMep2	B-protein
would	O
likely	O
complicate	O
structure	B-experimental_method
–	I-experimental_method
function	I-experimental_method
studies	I-experimental_method
,	O
several	O
other	O
fungal	B-taxonomy_domain
Mep2	B-protein_type
orthologues	O
were	O
subsequently	O
overexpressed	B-experimental_method
and	I-experimental_method
screened	I-experimental_method
for	I-experimental_method
diffraction	O
-	O
quality	O
crystals	B-evidence
.	O

Mep2	B-protein
channels	B-site
are	O
closed	B-protein_state
by	O
a	O
two	O
-	O
tier	O
channel	B-structure_element
block	I-structure_element

In	O
Mep2	B-protein
,	O
the	O
CTR	B-structure_element
has	O
moved	O
away	O
and	O
makes	O
relatively	O
few	O
contacts	O
with	O
the	O
main	B-structure_element
body	I-structure_element
of	O
the	O
transporter	B-protein_type
,	O
generating	O
a	O
more	O
elongated	B-protein_state
protein	O
(	O
Figs	O
1	O
and	O
4	O
).	O

On	O
one	O
side	O
,	O
the	O
Tyr390	B-residue_name_number
hydroxyl	O
in	O
Amt	B-protein
-	I-protein
1	I-protein
is	O
hydrogen	B-bond_interaction
bonded	I-bond_interaction
with	O
the	O
side	O
chain	O
of	O
the	O
conserved	B-protein_state
His185	B-residue_name_number
at	O
the	O
C	O
-	O
terminal	O
end	O
of	O
loop	B-structure_element
ICL3	B-structure_element
.	O

At	O
the	O
other	O
end	O
of	O
ICL3	B-structure_element
,	O
the	O
backbone	O
carbonyl	O
groups	O
of	O
Gly172	B-residue_name_number
and	O
Lys173	B-residue_name_number
are	O
hydrogen	B-bond_interaction
bonded	I-bond_interaction
to	O
the	O
side	O
chain	O
of	O
Arg370	B-residue_name_number
.	O

The	O
result	O
of	O
these	O
interactions	O
is	O
that	O
the	O
CTR	B-structure_element
‘	O
hugs	O
'	O
the	O
N	B-structure_element
-	I-structure_element
terminal	I-structure_element
half	I-structure_element
of	O
the	O
transporters	B-protein_type
(	O
Fig	O
.	O
4	O
).	O

Conversely	O
,	O
the	O
phosphorylation	B-protein_state
-	I-protein_state
mimicking	I-protein_state
S457D	B-mutant
variant	O
is	O
active	B-protein_state
both	O
in	O
the	O
triple	B-mutant
mepΔ	I-mutant
background	O
and	O
in	O
a	O
triple	B-mutant
mepΔ	I-mutant
npr1Δ	I-mutant
strain	O
(	O
Fig	O
.	O
3	O
).	O

Mutation	B-experimental_method
of	O
other	O
potential	O
phosphorylation	B-site
sites	I-site
in	O
the	O
CTR	B-structure_element
did	O
not	O
support	O
growth	O
in	O
the	O
npr1Δ	B-mutant
background	O
.	O

This	O
segment	B-structure_element
(	O
residues	O
450	B-residue_range
–	I-residue_range
457	I-residue_range
in	O
ScMep2	B-protein
and	O
446	B-residue_range
–	I-residue_range
453	I-residue_range
in	O
CaMep2	B-protein
)	O
was	O
dubbed	O
an	O
autoinhibitory	B-structure_element
(	I-structure_element
AI	I-structure_element
)	I-structure_element
region	I-structure_element
based	O
on	O
the	O
fact	O
that	O
its	O
removal	B-experimental_method
generates	O
an	O
active	B-protein_state
transporter	B-protein_type
in	O
the	O
absence	B-protein_state
of	I-protein_state
Npr1	B-protein
(	O
Fig	O
.	O
3	O
).	O

The	O
AI	B-structure_element
region	I-structure_element
packs	O
against	O
the	O
cytoplasmic	O
ends	O
of	O
TM2	B-structure_element
and	O
TM4	B-structure_element
,	O
physically	O
linking	O
the	O
main	B-structure_element
body	I-structure_element
of	O
the	O
transporter	B-protein_type
with	O
the	O
CTR	B-structure_element
via	O
main	O
chain	O
interactions	O
and	O
side	O
-	O
chain	O
interactions	O
of	O
Val447	B-residue_name_number
,	O
Asp449	B-residue_name_number
,	O
Pro450	B-residue_name_number
and	O
Arg452	B-residue_name_number
(	O
Fig	O
.	O
6	O
).	O

The	O
peripheral	O
location	O
and	O
disorder	B-protein_state
of	O
the	O
CTR	B-structure_element
beyond	O
the	O
kinase	B-site
target	I-site
site	I-site
should	O
facilitate	O
the	O
phosphorylation	B-ptm
by	O
Npr1	B-protein
.	O

The	O
disordered	B-protein_state
part	O
of	O
the	O
CTR	B-structure_element
is	O
not	B-protein_state
conserved	I-protein_state
in	O
ammonium	B-protein_type
transporters	I-protein_type
(	O
Fig	O
.	O
2	O
),	O
suggesting	O
that	O
it	O
is	O
not	O
important	O
for	O
transport	O
.	O

The	O
data	O
behind	O
this	O
hypothesis	O
is	O
the	O
observation	O
that	O
a	O
ScMep2	B-protein
449	B-mutant
-	I-mutant
485Δ	I-mutant
deletion	B-protein_state
mutant	I-protein_state
lacking	B-protein_state
the	O
AI	B-structure_element
region	I-structure_element
is	O
highly	B-protein_state
active	I-protein_state
in	O
MA	B-chemical
uptake	O
both	O
in	O
the	O
triple	B-mutant
mepΔ	I-mutant
and	O
triple	B-mutant
mepΔ	I-mutant
npr1Δ	I-mutant
backgrounds	O
,	O
implying	O
that	O
this	O
Mep2	B-mutant
variant	I-mutant
has	O
a	O
constitutively	B-protein_state
open	I-protein_state
channel	B-site
.	O

Interestingly	O
,	O
however	O
,	O
the	O
Tyr49	B-residue_name_number
-	O
His342	B-residue_name_number
hydrogen	B-bond_interaction
bond	I-bond_interaction
that	O
closes	O
the	O
channel	O
in	O
the	O
WT	B-protein_state
protein	O
is	O
still	O
present	O
(	O
Fig	O
.	O
7	O
and	O
Supplementary	O
Fig	O
.	O
2	O
).	O

We	O
therefore	O
predict	O
that	O
phosphorylation	B-ptm
of	O
Ser453	B-residue_name_number
will	O
result	O
in	O
steric	O
clashes	O
as	O
well	O
as	O
electrostatic	O
repulsion	O
,	O
which	O
in	O
turn	O
might	O
cause	O
substantial	O
conformational	O
changes	O
within	O
the	O
CTR	B-structure_element
.	O

By	O
contrast	O
,	O
the	O
conserved	B-protein_state
part	O
of	O
the	O
CTR	B-structure_element
has	O
undergone	O
a	O
large	O
conformational	O
change	O
involving	O
formation	O
of	O
a	O
12	B-structure_element
-	I-structure_element
residue	I-structure_element
-	I-structure_element
long	I-structure_element
α	I-structure_element
-	I-structure_element
helix	I-structure_element
from	O
Leu427	B-residue_range
to	I-residue_range
Asp438	I-residue_range
.	O

This	O
is	O
the	O
first	O
time	O
a	O
large	O
conformational	O
change	O
has	O
been	O
observed	O
in	O
an	O
ammonium	B-protein_type
transporter	I-protein_type
as	O
a	O
result	O
of	O
a	O
mutation	B-experimental_method
,	O
and	O
confirms	O
previous	O
hypotheses	O
that	O
phosphorylation	B-ptm
causes	O
structural	O
changes	O
in	O
the	O
CTR	B-structure_element
.	O

After	O
200	O
ns	O
of	O
MD	B-experimental_method
simulation	B-experimental_method
,	O
the	O
interactions	O
between	O
these	O
residues	O
and	O
Ser453	B-residue_name_number
remain	O
intact	O
.	O

Finally	O
,	O
the	O
S453J	B-mutant
mutant	B-protein_state
is	O
also	O
stable	B-protein_state
throughout	O
the	O
200	O
-	O
ns	O
simulation	B-experimental_method
and	O
has	O
an	O
average	O
backbone	O
deviation	O
of	O
∼	O
3	O
.	O
8	O
Å	O
,	O
which	O
is	O
similar	O
to	O
the	O
DD	B-mutant
mutant	I-mutant
.	O

The	O
distance	B-evidence
between	O
the	O
phosphate	B-chemical
of	O
Sep453	B-residue_name_number
and	O
the	O
acidic	O
oxygen	O
atoms	O
of	O
Glu420	B-residue_name_number
is	O
initially	O
∼	O
11	O
Å	O
,	O
but	O
increases	O
to	O
>	O
30	O
Å	O
after	O
200	O
ns	O
.	O

Thus	O
,	O
the	O
MD	B-experimental_method
simulations	B-experimental_method
support	O
the	O
notion	O
from	O
the	O
crystal	B-evidence
structures	I-evidence
that	O
phosphorylation	B-ptm
generates	O
conformational	O
changes	O
in	O
the	O
conserved	B-protein_state
part	O
of	O
the	O
CTR	B-structure_element
.	O

In	O
Arabidopsis	B-species
thaliana	I-species
Amt	B-protein
-	I-protein
1	I-protein
;	I-protein
1	I-protein
,	O
phosphorylation	B-ptm
of	O
the	O
CTR	B-structure_element
residue	O
T460	B-residue_name_number
under	O
conditions	O
of	O
high	O
ammonium	B-chemical
inhibits	O
transport	O
activity	O
,	O
that	O
is	O
,	O
the	O
default	O
(	O
non	B-protein_state
-	I-protein_state
phosphorylated	I-protein_state
)	O
state	O
of	O
the	O
plant	B-taxonomy_domain
transporter	B-protein_type
is	O
open	B-protein_state
.	O

Owing	O
to	O
the	O
lack	O
of	O
structural	O
information	O
for	O
plant	B-taxonomy_domain
AMTs	B-protein_type
,	O
the	O
details	O
of	O
channel	B-site
closure	O
and	O
inter	O
-	O
monomer	O
crosstalk	O
are	O
not	O
yet	O
clear	O
.	O

Upon	O
phosphorylation	B-ptm
by	O
the	O
Npr1	B-protein
kinase	B-protein_type
in	O
response	O
to	O
nitrogen	B-chemical
limitation	O
,	O
the	O
region	O
around	O
the	O
conserved	B-protein_state
ExxGxD	B-structure_element
motif	I-structure_element
undergoes	O
a	O
conformational	O
change	O
that	O
opens	O
the	O
channel	B-site
(	O
Fig	O
.	O
9	O
).	O

Our	O
Mep2	B-protein
structures	B-evidence
show	O
that	O
this	O
assumption	O
may	O
not	O
be	O
correct	O
(	O
Fig	O
.	O
4	O
and	O
Supplementary	O
Fig	O
.	O
6	O
).	O

In	O
this	O
way	O
,	O
phosphorylation	B-ptm
can	O
either	O
lead	O
to	O
channel	B-site
closing	O
(	O
in	O
the	O
case	O
of	O
AMTs	B-protein_type
)	O
or	O
channel	B-site
opening	O
in	O
the	O
case	O
of	O
Mep2	B-protein
.	O

Such	O
mutations	O
likely	O
cause	O
structural	O
changes	O
in	O
the	O
CTR	B-structure_element
that	O
prevent	O
close	O
contacts	O
between	O
the	O
CTR	B-structure_element
and	O
ICL1	B-structure_element
/	O
ICL3	B-structure_element
,	O
thereby	O
stabilizing	O
a	O
closed	B-protein_state
state	O
that	O
may	O
be	O
similar	O
to	O
that	O
observed	O
in	O
Mep2	B-protein
.	O

Recently	O
,	O
phosphorylation	B-ptm
was	O
also	O
shown	O
to	O
modulate	O
substrate	O
affinity	O
in	O
nitrate	B-protein_type
transporters	I-protein_type
.	O

X	B-evidence
-	I-evidence
ray	I-evidence
crystal	I-evidence
structures	I-evidence
of	O
Mep2	B-protein
transceptors	B-protein_type
.	O

ClustalW	B-experimental_method
alignment	I-experimental_method
of	O
CaMep2	B-protein
,	O
ScMep2	B-protein
,	O
A	B-species
.	I-species
fulgidus	I-species
Amt	B-protein
-	I-protein
1	I-protein
,	O
E	O
.	O
coli	O
AmtB	B-protein
and	O
A	B-species
.	I-species
thaliana	I-species
Amt	B-protein
-	I-protein
1	I-protein
;	I-protein
1	I-protein
.	O

The	O
grey	O
sequences	O
at	O
the	O
C	O
termini	O
of	O
CaMep2	B-protein
and	O
ScMep2	B-protein
are	O
not	O
visible	O
in	O
the	O
structures	B-evidence
and	O
are	O
likely	B-protein_state
disordered	I-protein_state
.	O

In	O
b	O
and	O
c	O
,	O
the	O
centre	O
of	O
the	O
trimer	B-oligomeric_state
is	O
at	O
top	O
.	O

That	O
the	O
3	B-protein_type
′-	I-protein_type
5	I-protein_type
′	I-protein_type
elongation	I-protein_type
enzyme	I-protein_type
performs	O
this	O
elaborate	O
two	O
-	O
step	O
reaction	O
in	O
one	O
catalytic	B-site
center	I-site
suggests	O
that	O
these	O
two	O
reactions	O
have	O
been	O
inseparable	O
throughout	O
the	O
process	O
of	O
protein	O
evolution	O
.	O

However	O
,	O
recent	O
studies	O
have	O
shown	O
that	O
the	O
Thg1	B-protein
/	O
Thg1	B-protein_type
-	I-protein_type
like	I-protein_type
protein	I-protein_type
(	O
TLP	B-protein_type
)	O
family	O
of	O
proteins	O
extends	O
tRNA	B-chemical
nucleotide	O
chains	O
in	O
the	O
reverse	O
(	O
3	O
′-	O
5	O
′)	O
direction	O
.	O

In	O
5	O
′-	O
3	O
′	O
elongation	O
by	O
DNA	B-protein_type
/	I-protein_type
RNA	I-protein_type
polymerases	I-protein_type
,	O
the	O
energy	O
of	O
the	O
incoming	O
nucleotide	O
is	O
used	O
for	O
its	O
own	O
addition	O
(	O
tail	O
polymerization	O
).	O

This	O
guanosine	B-chemical
at	O
position	O
−	B-residue_number
1	I-residue_number
(	O
G	B-residue_name_number
−	I-residue_name_number
1	I-residue_name_number
)	O
of	O
tRNAHis	B-chemical
is	O
a	O
critical	O
identity	O
element	O
for	O
recognition	O
by	O
the	O
histidyl	B-protein_type
-	I-protein_type
tRNA	I-protein_type
synthase	I-protein_type
.	O

This	O
finding	O
suggests	O
that	O
TLPs	B-protein_type
may	O
have	O
potential	O
functions	O
other	O
than	O
tRNAHis	B-chemical
maturation	O
.	O

This	O
finding	O
suggests	O
that	O
3	B-protein_type
′-	I-protein_type
5	I-protein_type
′	I-protein_type
elongation	I-protein_type
enzymes	I-protein_type
are	O
related	O
to	O
5	B-protein_type
′-	I-protein_type
3	I-protein_type
′	I-protein_type
polymerases	I-protein_type
and	O
raises	O
important	O
questions	O
on	O
why	O
5	B-protein_type
′-	I-protein_type
3	I-protein_type
′	I-protein_type
polymerases	I-protein_type
predominate	O
in	O
nature	O
.	O

The	O
O	O
atom	O
on	O
the	O
S213	B-residue_name_number
side	O
chain	O
was	O
also	O
hydrogen	B-bond_interaction
-	I-bond_interaction
bonded	I-bond_interaction
to	O
the	O
phosphate	B-chemical
moiety	O
of	O
G57	B-residue_name_number
of	O
the	O
tRNA	B-chemical
(	O
Fig	O
.	O
2	O
).	O

The	O
N7	O
atom	O
of	O
G2	B-residue_name_number
at	O
the	O
5	O
′-	O
end	O
was	O
hydrogen	B-bond_interaction
-	I-bond_interaction
bonded	I-bond_interaction
to	O
the	O
N	O
atom	O
of	O
the	O
R136	B-residue_name_number
side	O
chain	O
,	O
whereas	O
the	O
α	O
-	O
phosphate	B-chemical
was	O
bonded	O
to	O
the	O
N137	B-residue_name_number
side	O
chain	O
(	O
Fig	O
.	O
2	O
).	O

The	O
obtained	O
structure	B-evidence
showed	O
that	O
the	O
guanine	B-chemical
base	O
of	O
the	O
incoming	O
GDPNP	B-chemical
formed	O
Watson	B-bond_interaction
-	I-bond_interaction
Crick	I-bond_interaction
hydrogen	I-bond_interaction
bonds	I-bond_interaction
with	O
C72	B-residue_name_number
and	O
accompanied	O
base	B-bond_interaction
-	I-bond_interaction
stacking	I-bond_interaction
interactions	I-bond_interaction
with	O
G2	B-residue_name_number
of	O
the	O
tRNA	B-chemical
(	O
Fig	O
.	O
3B	O
),	O
whereas	O
no	O
interaction	O
was	O
observed	O
between	O
the	O
guanine	B-chemical
base	O
and	O
the	O
enzyme	O
.	O

Surprisingly	O
,	O
the	O
5	B-chemical
′-	I-chemical
triphosphate	I-chemical
moiety	O
after	O
movement	O
occupied	O
the	O
GTP	B-chemical
/	O
ATP	B-chemical
triphosphate	B-chemical
position	O
during	O
the	O
activation	O
step	O
(	O
Fig	O
.	O
3D	O
).	O

All	O
of	O
these	O
residues	O
are	O
well	B-protein_state
conserved	I-protein_state
(	O
fig	O
.	O
S5	O
),	O
and	O
mutation	B-experimental_method
of	O
corresponding	O
residues	O
in	O
ScThg1	B-protein
(	O
R27	B-residue_name_number
,	O
R93	B-residue_name_number
,	O
K96	B-residue_name_number
,	O
and	O
R133	B-residue_name_number
)	O
decreased	O
the	O
catalytic	O
efficiency	O
of	O
G	B-residue_name_number
−	I-residue_name_number
1	I-residue_name_number
addition	O
.	O

(	O
A	O
)	O
Guanylylation	O
of	O
ppptRNAPheΔ1	B-chemical
and	O
ppptRNAHisΔ1	B-chemical
by	O
various	O
TLP	B-protein_type
mutants	B-protein_state
.	O

The	O
activity	O
using	O
[	B-chemical
α	I-chemical
-	I-chemical
32P	I-chemical
]	I-chemical
GTP	I-chemical
,	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
MaTLP	B-protein
,	O
and	O
ppptRNAPheΔ1	B-chemical
is	O
denoted	O
as	O
100	O
.	O
(	O
B	O
)	O
Guanylylation	O
of	O
tRNAPheΔ1	B-chemical
,	O
tRNAPhe	B-chemical
,	O
and	O
tRNAHisΔ	B-chemical
−	I-chemical
1	I-chemical
by	O
various	O
TLP	B-protein_type
mutants	B-protein_state
.	O

The	O
tRNAHis	B-protein_type
-	I-protein_type
specific	I-protein_type
G	I-protein_type
−	I-protein_type
1	I-protein_type
addition	I-protein_type
enzyme	I-protein_type
Thg1	B-protein
needs	O
to	O
recognize	O
both	O
the	O
accepter	B-structure_element
stem	I-structure_element
and	O
anticodon	B-structure_element
of	O
tRNAHis	B-chemical
.	O

TLP	B-protein_type
has	O
been	O
shown	O
to	O
confer	O
such	O
catalytic	O
activity	O
on	O
tRNAHisΔ	B-chemical
−	I-chemical
1	I-chemical
(	O
Fig	O
.	O
4B	O
).	O

Thus	O
,	O
we	O
concluded	O
that	O
TLP	B-protein_type
has	O
two	O
tRNA	B-chemical
binding	O
modes	O
that	O
are	O
selectively	O
used	O
,	O
depending	O
on	O
both	O
the	O
length	O
of	O
the	O
accepter	B-structure_element
stem	I-structure_element
and	O
the	O
anticodon	B-structure_element
.	O

This	O
enzyme	O
has	O
two	O
triphosphate	B-site
binding	I-site
sites	I-site
and	O
one	O
reaction	B-site
center	I-site
at	O
the	O
position	O
overlapping	O
these	O
two	O
binding	B-site
sites	I-site
(	O
Fig	O
.	O
5A	O
).	O

(	O
A	O
)	O
The	O
reaction	B-site
center	I-site
overlapped	O
with	O
two	O
triphosphate	B-site
binding	I-site
sites	I-site
.	O

Movement	O
of	O
the	O
5	O
′-	O
terminal	O
chain	O
leaves	O
the	O
5	B-chemical
′-	I-chemical
triphosphate	I-chemical
of	O
the	O
tRNA	B-chemical
in	O
the	O
same	O
site	O
as	O
the	O
activation	O
step	O
in	O
(	O
B	O
).	O

These	O
two	O
Mg2	B-chemical
+	I-chemical
ions	O
are	O
coordinated	B-bond_interaction
by	I-bond_interaction
a	O
conserved	B-protein_state
Asp	B-residue_name
(	O
D21	B-residue_name_number
and	O
D69	B-residue_name_number
in	O
TLP	B-protein_type
)	O
in	O
the	O
conserved	B-protein_state
catalytic	B-site
core	I-site
.	O

However	O
,	O
from	O
an	O
energetic	O
viewpoint	O
,	O
these	O
two	O
reactions	O
are	O
clearly	O
different	O
:	O
Whereas	O
the	O
high	O
energy	O
of	O
the	O
incoming	O
nucleotide	O
is	O
used	O
for	O
its	O
own	O
addition	O
in	O
DNA	B-protein_type
/	I-protein_type
RNA	I-protein_type
polymerases	I-protein_type
,	O
the	O
high	O
energy	O
of	O
the	O
incoming	O
nucleotide	O
is	O
used	O
for	O
subsequent	O
addition	O
in	O
TLP	B-protein_type
.	O

TLP	B-protein_type
has	O
successfully	O
created	O
such	O
sites	O
by	O
utilizing	O
a	O
conformational	O
change	O
in	O
the	O
tRNA	B-chemical
through	O
Watson	B-bond_interaction
-	I-bond_interaction
Crick	I-bond_interaction
base	I-bond_interaction
pairing	I-bond_interaction
(	O
Fig	O
.	O
3	O
).	O

Structural	O
diversity	O
in	O
a	O
human	B-species
antibody	B-protein_type
germline	O
library	O

The	O
structures	B-evidence
and	O
their	O
analyses	O
provide	O
a	O
rich	O
foundation	O
for	O
future	O
antibody	B-protein_type
modeling	O
and	O
engineering	O
efforts	O
.	O

Our	O
current	O
structural	O
knowledge	O
of	O
antibodies	B-protein_type
is	O
based	O
on	O
a	O
multitude	O
of	O
studies	O
that	O
used	O
many	O
techniques	O
to	O
gain	O
insight	O
into	O
the	O
functional	O
and	O
structural	O
properties	O
of	O
this	O
class	O
of	O
macromolecule	O
.	O

IgG	B-protein
,	O
IgD	B-protein
and	O
IgE	B-protein
isotypes	O
are	O
composed	O
of	O
2	O
heavy	B-structure_element
chains	I-structure_element
(	O
HCs	B-structure_element
)	O
and	O
2	O
light	B-structure_element
chains	I-structure_element
(	O
LCs	B-structure_element
)	O
linked	O
through	O
disulfide	B-ptm
bonds	I-ptm
,	O
while	O
IgA	B-protein
and	O
IgM	B-protein
are	O
double	O
and	O
quintuple	O
versions	O
of	O
antibodies	B-protein_type
,	O
respectively	O
.	O

This	O
site	O
,	O
which	O
interacts	O
with	O
the	O
antigen	O
(	O
or	O
target	O
),	O
is	O
the	O
focus	O
of	O
current	O
antibody	B-protein_type
modeling	O
efforts	O
.	O

This	O
interaction	B-site
site	I-site
is	O
composed	O
of	O
6	O
complementarity	B-structure_element
-	I-structure_element
determining	I-structure_element
regions	I-structure_element
(	O
CDRs	B-structure_element
)	O
that	O
were	O
identified	O
in	O
early	O
antibody	B-experimental_method
amino	I-experimental_method
acid	I-experimental_method
sequence	I-experimental_method
analyses	I-experimental_method
to	O
be	O
hypervariable	B-protein_state
in	O
nature	O
,	O
and	O
thus	O
are	O
responsible	O
for	O
the	O
sequence	O
and	O
structural	O
diversity	O
of	O
our	O
antibody	B-protein_type
repertoire	O
.	O

A	O
CDR	B-structure_element
canonical	O
structure	O
is	O
defined	O
by	O
its	O
length	O
and	O
conserved	O
residues	O
located	O
in	O
the	O
hypervariable	B-structure_element
loop	I-structure_element
and	O
framework	B-structure_element
residues	I-structure_element
(	O
V	B-structure_element
-	I-structure_element
region	I-structure_element
residues	O
that	O
are	O
not	O
part	O
of	O
the	O
CDRs	B-structure_element
).	O

Classification	O
schemes	O
for	O
the	O
canonical	O
structures	O
of	O
these	O
5	O
CDRs	B-structure_element
have	O
emerged	O
and	O
evolved	O
as	O
the	O
number	O
of	O
depositions	O
in	O
the	O
Protein	O
Data	O
Bank	O
of	O
Fab	B-structure_element
fragments	O
of	O
antibodies	B-protein_type
grow	O
.	O

Some	O
clustering	O
of	O
conformations	O
was	O
observed	O
for	O
the	O
shortest	O
lengths	O
;	O
however	O
,	O
for	O
the	O
longer	O
loops	B-structure_element
,	O
only	O
the	O
portions	O
nearest	O
the	O
framework	B-structure_element
(	O
torso	B-structure_element
,	O
stem	B-structure_element
or	O
anchor	B-structure_element
region	I-structure_element
)	O
were	O
found	O
to	O
have	O
defined	O
conformations	O
.	O

Current	O
antibody	B-protein_type
modeling	O
approaches	O
take	O
advantage	O
of	O
the	O
most	O
recent	O
advances	O
in	O
homology	B-experimental_method
modeling	I-experimental_method
,	O
the	O
evolving	O
understanding	O
of	O
the	O
CDR	B-structure_element
canonical	O
structures	B-evidence
,	O
the	O
emerging	O
rules	O
for	O
CDR	B-structure_element
H3	B-structure_element
modeling	O
and	O
the	O
growing	O
body	O
of	O
antibody	B-protein_type
structural	O
data	O
available	O
from	O
the	O
PDB	O
.	O

All	O
16	O
HCs	B-structure_element
of	O
the	O
Fabs	B-structure_element
have	O
the	O
same	O
CDR	B-structure_element
H3	B-structure_element
that	O
was	O
reported	O
in	O
an	O
earlier	O
Fab	B-structure_element
structure	B-evidence
.	O

This	O
is	O
the	O
first	O
systematic	O
study	O
of	O
the	O
same	O
VH	B-structure_element
and	O
VL	B-structure_element
structures	B-evidence
in	O
the	O
context	O
of	O
different	O
pairings	O
.	O

Crystallization	B-experimental_method
of	O
the	O
16	O
Fabs	B-structure_element
was	O
previously	O
reported	O
.	O

Three	O
sets	O
of	O
the	O
crystals	B-evidence
were	O
isomorphous	O
with	O
nearly	O
identical	O
unit	O
cells	O
(	O
Table	O
1	O
).	O

These	O
include	O
(	O
1	O
)	O
H3	B-complex_assembly
-	I-complex_assembly
23	I-complex_assembly
:	I-complex_assembly
L3	I-complex_assembly
-	I-complex_assembly
11	I-complex_assembly
and	O
H3	B-complex_assembly
-	I-complex_assembly
23	I-complex_assembly
:	I-complex_assembly
L4	I-complex_assembly
-	I-complex_assembly
1	I-complex_assembly
in	O
P212121	O
,	O
(	O
2	O
)	O
H3	B-complex_assembly
-	I-complex_assembly
53	I-complex_assembly
:	I-complex_assembly
L1	I-complex_assembly
-	I-complex_assembly
39	I-complex_assembly
,	O
H3	B-complex_assembly
-	I-complex_assembly
53	I-complex_assembly
:	I-complex_assembly
L3	I-complex_assembly
-	I-complex_assembly
11	I-complex_assembly
and	O
H3	B-complex_assembly
-	I-complex_assembly
53	I-complex_assembly
:	I-complex_assembly
L3	I-complex_assembly
-	I-complex_assembly
20	I-complex_assembly
in	O
P6522	O
,	O
and	O
(	O
3	O
)	O
H5	B-complex_assembly
-	I-complex_assembly
51	I-complex_assembly
:	I-complex_assembly
L1	I-complex_assembly
-	I-complex_assembly
39	I-complex_assembly
,	O
H5	B-complex_assembly
-	I-complex_assembly
51	I-complex_assembly
:	I-complex_assembly
L3	I-complex_assembly
-	I-complex_assembly
11	I-complex_assembly
and	O
H5	B-complex_assembly
-	I-complex_assembly
51	I-complex_assembly
:	I-complex_assembly
L3	I-complex_assembly
-	I-complex_assembly
20	I-complex_assembly
in	O
P212121	O
.	O

No	O
assignment	O
(	O
NA	O
)	O
for	O
CDRs	B-structure_element
with	O
missing	O
residues	O
.	O

A	O
major	O
difference	O
of	O
H1	B-mutant
-	I-mutant
69	I-mutant
from	O
the	O
other	O
germlines	O
in	O
the	O
experimental	O
data	O
set	O
is	O
the	O
presence	O
of	O
Gly	B-residue_name
instead	O
of	O
Phe	B-residue_name
or	O
Tyr	B-residue_name
at	O
position	O
27	B-residue_number
(	O
residue	O
5	O
of	O
13	O
in	O
CDR	B-structure_element
H1	B-structure_element
).	O

The	O
superposition	B-experimental_method
of	O
CDR	B-structure_element
L1	B-structure_element
backbones	O
for	O
all	O
HC	B-complex_assembly
:	I-complex_assembly
LC	I-complex_assembly
pairs	O
with	O
light	B-structure_element
chains	I-structure_element
:	O
(	O
A	O
)	O
L1	B-mutant
-	I-mutant
39	I-mutant
,	O
(	O
B	O
)	O
L3	B-mutant
-	I-mutant
11	I-mutant
,	O
(	O
C	O
)	O
L3	B-mutant
-	I-mutant
20	I-mutant
and	O
(	O
D	O
)	O
L4	B-mutant
-	I-mutant
1	I-mutant
.	O

For	O
the	O
remaining	O
2	O
,	O
L3	B-mutant
-	I-mutant
20	I-mutant
has	O
2	O
different	O
assignments	O
,	O
L1	B-mutant
-	I-mutant
12	I-mutant
-	I-mutant
1	I-mutant
and	O
L1	B-mutant
-	I-mutant
12	I-mutant
-	I-mutant
2	I-mutant
,	O
while	O
L4	B-mutant
-	I-mutant
1	I-mutant
has	O
a	O
single	O
assignment	O
,	O
L1	B-mutant
-	I-mutant
17	I-mutant
-	I-mutant
1	I-mutant
.	O

The	O
third	O
structure	O
,	O
H3	B-complex_assembly
-	I-complex_assembly
23	I-complex_assembly
:	I-complex_assembly
L3	I-complex_assembly
-	I-complex_assembly
20	I-complex_assembly
,	O
has	O
CDR	B-structure_element
L1	B-structure_element
as	O
L1	B-mutant
-	I-mutant
12	I-mutant
-	I-mutant
2	I-mutant
,	O
which	O
deviates	O
from	O
L1	B-mutant
-	I-mutant
12	I-mutant
-	I-mutant
1	I-mutant
at	O
residues	O
29	B-residue_range
-	I-residue_range
32	I-residue_range
,	O
i	O
.	O
e	O
.,	O
at	O
the	O
site	O
of	O
insertion	O
with	O
respect	O
to	O
the	O
11	B-residue_range
-	I-residue_range
residue	I-residue_range
CDR	B-structure_element
.	O

The	O
stem	B-structure_element
region	I-structure_element
of	O
CDR	B-structure_element
H3	B-structure_element
in	O
the	O
parental	O
Fab	B-structure_element
is	O
in	O
a	O
‘	O
kinked	B-protein_state
’	O
conformation	O
,	O
in	O
which	O
the	O
indole	O
nitrogen	O
of	O
Trp103	B-residue_name_number
forms	O
a	O
hydrogen	B-bond_interaction
bond	I-bond_interaction
with	O
the	O
carbonyl	O
oxygen	O
of	O
Leu100b	B-residue_name_number
.	O

The	O
structure	B-evidence
of	O
each	O
CDR	B-structure_element
H3	B-structure_element
is	O
represented	O
with	O
a	O
different	O
color	O
.	O

Despite	O
having	O
the	O
same	O
amino	O
acid	O
sequence	O
in	O
all	O
variants	O
,	O
CDR	B-structure_element
H3	B-structure_element
has	O
the	O
highest	O
degree	O
of	O
structural	O
diversity	O
and	O
disorder	O
of	O
all	O
of	O
the	O
CDRs	B-structure_element
in	O
the	O
experimental	O
set	O
.	O

In	O
10	O
of	O
the	O
18	O
Fab	B-structure_element
structures	B-evidence
,	O
H1	B-complex_assembly
-	I-complex_assembly
69	I-complex_assembly
:	I-complex_assembly
L1	I-complex_assembly
-	I-complex_assembly
39	I-complex_assembly
,	O
H1	B-complex_assembly
-	I-complex_assembly
69	I-complex_assembly
:	I-complex_assembly
L3	I-complex_assembly
-	I-complex_assembly
11	I-complex_assembly
(	O
2	O
Fabs	B-structure_element
),	O
H1	B-complex_assembly
-	I-complex_assembly
69	I-complex_assembly
:	I-complex_assembly
L4	I-complex_assembly
-	I-complex_assembly
1	I-complex_assembly
,	O
H3	B-complex_assembly
-	I-complex_assembly
23	I-complex_assembly
:	I-complex_assembly
L3	I-complex_assembly
-	I-complex_assembly
11	I-complex_assembly
(	O
2	O
Fabs	B-structure_element
),	O
H3	B-complex_assembly
-	I-complex_assembly
23	I-complex_assembly
:	I-complex_assembly
L3	I-complex_assembly
-	I-complex_assembly
20	I-complex_assembly
,	O
H3	B-complex_assembly
-	I-complex_assembly
53	I-complex_assembly
:	I-complex_assembly
L3	I-complex_assembly
-	I-complex_assembly
11	I-complex_assembly
,	O
H3	B-complex_assembly
-	I-complex_assembly
53	I-complex_assembly
:	I-complex_assembly
L3	I-complex_assembly
-	I-complex_assembly
20	I-complex_assembly
and	O
H5	B-complex_assembly
-	I-complex_assembly
51	I-complex_assembly
:	I-complex_assembly
L1	I-complex_assembly
-	I-complex_assembly
39	I-complex_assembly
,	O
the	O
CDRs	B-structure_element
have	O
similar	O
conformations	O
to	O
that	O
found	O
in	O
4DN3	O
.	O

The	O
stem	B-structure_element
regions	I-structure_element
in	O
these	O
3	O
cases	O
are	O
in	O
the	O
‘	O
kinked	B-protein_state
’	O
conformation	O
consistent	O
with	O
that	O
observed	O
for	O
4DN3	O
.	O

The	O
domain	O
packing	O
of	O
the	O
variants	O
was	O
assessed	O
by	O
computing	O
the	O
domain	B-site
interface	I-site
interactions	O
,	O
the	O
VH	B-complex_assembly
:	I-complex_assembly
VL	I-complex_assembly
tilt	B-evidence
angles	I-evidence
,	O
the	O
buried	O
surface	O
area	O
and	O
surface	O
complementarity	O
.	O

The	O
VH	B-structure_element
residues	O
are	O
in	O
blue	O
,	O
the	O
VL	B-structure_element
residues	O
are	O
in	O
orange	O
.	O

The	O
VH	B-site
:	I-site
VL	I-site
interface	I-site
is	O
pseudosymmetric	B-protein_state
,	O
and	O
involves	O
2	O
stretches	O
of	O
the	O
polypeptide	O
chain	O
from	O
each	O
domain	O
,	O
namely	O
CDR3	B-structure_element
and	O
the	O
framework	B-structure_element
region	I-structure_element
between	O
CDRs	B-structure_element
1	I-structure_element
and	I-structure_element
2	I-structure_element
.	O

These	O
stretches	O
form	O
antiparallel	B-structure_element
β	I-structure_element
-	I-structure_element
hairpins	I-structure_element
within	O
the	O
internal	O
5	B-structure_element
-	I-structure_element
stranded	I-structure_element
β	I-structure_element
-	I-structure_element
sheet	I-structure_element
.	O

The	O
second	O
approach	O
used	O
for	O
comparing	O
tilt	B-evidence
angles	I-evidence
involved	O
computing	O
the	O
difference	B-evidence
in	O
the	O
tilt	B-evidence
angles	I-evidence
between	O
all	O
pairs	O
of	O
structures	B-evidence
.	O

Indeed	O
,	O
this	O
Fab	B-structure_element
has	O
the	O
largest	O
twist	B-evidence
angle	I-evidence
HC2	B-structure_element
within	O
the	O
experimental	O
set	O
that	O
exceeds	O
the	O
mean	O
value	O
by	O
2	O
.	O
5	O
standard	O
deviations	O
(	O
Table	O
S2	O
).	O

One	O
of	O
the	O
2	O
structures	B-evidence
,	O
H1	B-complex_assembly
-	I-complex_assembly
69	I-complex_assembly
:	I-complex_assembly
L3	I-complex_assembly
-	I-complex_assembly
20	I-complex_assembly
,	O
has	O
its	O
CDR	B-structure_element
H3	B-structure_element
in	O
the	O
‘	O
extended	B-protein_state
’	O
conformation	O
;	O
the	O
other	O
structure	O
has	O
it	O
in	O
the	O
‘	O
kinked	B-protein_state
’	O
conformation	O
.	O

Some	O
side	O
chain	O
atoms	O
in	O
CDR	B-structure_element
H3	B-structure_element
are	O
missing	O
.	O

Having	O
all	O
16	O
VH	B-complex_assembly
:	I-complex_assembly
VL	I-complex_assembly
pairs	O
with	O
the	O
same	O
CDR	B-structure_element
H3	B-structure_element
provides	O
some	O
insights	O
into	O
why	O
molecular	O
modeling	O
efforts	O
of	O
CDR	B-structure_element
H3	B-structure_element
have	O
proven	O
so	O
difficult	O
.	O

This	O
subset	O
also	O
has	O
2	O
structures	B-evidence
with	O
2	O
Fab	B-structure_element
copies	O
in	O
the	O
asymmetric	O
unit	O
.	O

Thus	O
,	O
no	O
patterns	O
of	O
conformational	O
preference	O
for	O
a	O
particular	O
HC	B-structure_element
or	O
LC	B-structure_element
emerge	O
to	O
shed	O
any	O
direct	O
light	O
on	O
what	O
drives	O
the	O
conformational	O
differences	O
.	O

At	O
the	O
other	O
end	O
of	O
the	O
stability	O
range	O
is	O
LC	B-structure_element
germline	O
L3	B-mutant
-	I-mutant
20	I-mutant
,	O
which	O
yields	O
antibodies	B-protein_type
with	O
the	O
lowest	O
Tms	B-evidence
.	O

Yet	O
,	O
for	O
the	O
2	O
antibodies	B-protein_type
,	O
the	O
total	O
gain	O
in	O
stability	O
merits	O
the	O
domain	O
repacking	O
.	O

Quite	O
unexpectedly	O
,	O
2	O
of	O
the	O
variants	O
,	O
H1	B-complex_assembly
-	I-complex_assembly
69	I-complex_assembly
:	I-complex_assembly
L3	I-complex_assembly
-	I-complex_assembly
20	I-complex_assembly
and	O
H3	B-complex_assembly
-	I-complex_assembly
53	I-complex_assembly
:	I-complex_assembly
L4	I-complex_assembly
-	I-complex_assembly
1	I-complex_assembly
,	O
have	O
the	O
‘	O
extended	B-protein_state
’	O
stem	B-structure_element
region	I-structure_element
differing	O
from	O
the	O
other	O
14	O
that	O
have	O
a	O
‘	O
kinked	B-protein_state
’	O
stem	B-structure_element
region	I-structure_element
.	O

For	O
those	O
applications	O
where	O
accurate	O
CDR	B-structure_element
structures	B-evidence
are	O
essential	O
,	O
such	O
as	O
docking	O
,	O
the	O
results	O
in	O
this	O
work	O
demonstrate	O
the	O
importance	O
of	O
experimental	O
structures	B-evidence
.	O

Interestingly	O
,	O
several	O
clinically	O
relevant	O
and	O
human	B-species
pathogenic	O
strains	O
are	O
inherently	O
resistant	O
towards	O
lantibiotics	B-chemical
.	O

The	O
C	B-structure_element
-	I-structure_element
terminal	I-structure_element
domain	I-structure_element
exhibits	O
a	O
fold	O
that	O
classifies	O
NsrR	B-protein
as	O
a	O
member	O
of	O
the	O
OmpR	B-protein_type
/	I-protein_type
PhoB	I-protein_type
subfamily	I-protein_type
of	O
regulators	O
.	O

They	O
are	O
post	O
-	O
translationally	O
modified	O
and	O
contain	O
specific	O
lanthionine	B-chemical
/	O
methyl	B-chemical
-	I-chemical
lanthionine	I-chemical
rings	O
,	O
which	O
are	O
crucial	O
for	O
their	O
high	O
antimicrobial	O
activity	O
.	O

Examples	O
for	O
LanFEG	B-protein_type
are	O
NisI	B-protein
and	O
NisFEG	B-protein
of	O
the	O
nisin	B-chemical
system	O
,	O
SpaI	B-protein
and	O
SpaFEG	B-protein
conferring	O
immunity	O
towards	O
subtilin	B-chemical
,	O
and	O
PepI	B-protein
constituting	O
the	O
immunity	O
system	O
of	O
Pep5	B-chemical
producing	O
strains	O
.	O

Furthermore	O
,	O
the	O
upregulation	O
of	O
these	O
genes	O
is	O
mediated	O
by	O
a	O
specific	O
two	B-complex_assembly
-	I-complex_assembly
component	I-complex_assembly
system	I-complex_assembly
(	O
TCS	B-complex_assembly
)	O
similar	O
to	O
the	O
one	O
found	O
in	O
lantibiotic	B-chemical
producing	O
strains	O
,	O
consisting	O
of	O
a	O
sensor	O
histidine	B-protein_type
kinase	I-protein_type
(	O
HK	B-protein_type
)	O
and	O
a	O
response	B-protein_type
regulator	I-protein_type
(	O
RR	B-protein_type
),	O
apparently	O
mediate	O
the	O
expression	O
of	O
the	O
resistance	O
proteins	O
:	O
HK	B-protein_type
senses	O
the	O
external	O
lantibiotic	B-chemical
and	O
,	O
upon	O
receiving	O
the	O
stimuli	O
,	O
auto	B-ptm
-	I-ptm
phosphorylates	I-ptm
at	O
a	O
conserved	B-protein_state
histidine	B-residue_name
residue	O
within	O
the	O
cytosol	O
;	O
this	O
high	O
-	O
energetic	O
phosphoryl	O
group	O
is	O
then	O
transferred	O
to	O
the	O
associated	O
RR	B-protein_type
inducing	O
a	O
conformational	O
change	O
there	O
,	O
which	O
activates	O
the	O
RR	B-protein_type
to	O
evoke	O
the	O
cellular	O
response	O
.	O

The	O
recently	O
discovered	O
nsr	B-gene
gene	O
cluster	O
of	O
the	O
human	B-species
pathogen	O
S	B-species
.	I-species
agalactiae	I-species
encodes	O
for	O
the	O
resistance	B-protein_type
protein	I-protein_type
NSR	B-protein
and	O
the	O
ABC	B-protein_type
transporter	I-protein_type
NsrFP	B-protein
,	O
both	O
conferring	O
resistance	O
against	O
nisin	B-chemical
.	O

The	O
ED	B-structure_element
is	O
thereby	O
activated	O
and	O
binds	O
to	O
the	O
designated	O
promoters	O
,	O
thus	O
initiating	O
transcription	O
of	O
the	O
target	O
genes	O
.	O

Although	O
numerous	O
structures	B-evidence
of	O
the	O
single	O
domains	O
are	O
known	O
,	O
only	O
a	O
few	O
structures	B-evidence
of	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
OmpR	B-protein_type
/	I-protein_type
PhoB	I-protein_type
-	I-protein_type
type	I-protein_type
RRs	I-protein_type
have	O
been	O
determined	O
:	O
RegX3	B-protein
(	O
PDB	O
code	O
:	O
2OQR	O
),	O
MtrA	B-protein
(	O
PDB	O
code	O
:	O
2GWR	O
),	O
PrrA	B-protein
(	O
PDB	O
code	O
:	O
1YS6	O
)	O
and	O
PhoP	B-protein
(	O
PDB	O
code	O
:	O
3R0J	O
)	O
from	O
Mycobacterium	B-species
tuberculosis	I-species
;	O
DrrB	B-protein
(	O
PDB	O
code	O
:	O
1P2F	O
)	O
and	O
DrrD	B-protein
(	O
PDB	O
code	O
:	O
1KGS	O
)	O
from	O
Thermotoga	B-species
maritima	I-species
;	O
and	O
KdpE	B-protein
from	O
Escherichia	B-species
coli	I-species
(	O
PDB	O
code	O
:	O
4KNY	O
).	O

By	O
calibrating	O
the	O
column	O
with	O
proteins	O
of	O
known	O
molecular	O
weight	O
the	O
NsrR	B-protein
full	B-protein_state
length	I-protein_state
protein	O
elutes	O
as	O
a	O
dimer	B-oligomeric_state
.	O

Surprisingly	O
,	O
over	O
time	O
NsrR	B-protein
degraded	O
into	O
two	O
distinct	O
fragments	O
as	O
visible	O
on	O
SDS	B-experimental_method
-	I-experimental_method
PAGE	I-experimental_method
analysis	O
using	O
the	O
same	O
purified	O
protein	O
sample	O
after	O
one	O
week	O
(	O
Fig	O
1C	O
,	O
indicated	O
by	O
**	O
and	O
***,	O
respectively	O
).	O

Such	O
a	O
cleavage	O
of	O
the	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
RR	B-protein_type
into	O
two	O
specific	O
domains	O
is	O
not	O
unusual	O
and	O
has	O
been	O
previously	O
reported	O
for	O
other	O
RRs	B-protein_type
as	O
well	O
.	O

The	O
bold	O
line	O
represents	O
the	O
chromatogram	B-evidence
of	O
freshly	O
purified	O
NsrR	B-protein
while	O
the	O
dashed	O
line	O
shows	O
the	O
chromatogram	B-evidence
of	O
the	O
same	O
NsrR	B-protein
protein	O
after	O
one	O
week	O
.	O

We	O
also	O
tried	O
to	O
solve	O
the	O
structure	B-evidence
of	O
the	O
thin	O
plate	O
-	O
shaped	O
crystals	B-evidence
with	O
this	O
template	O
,	O
but	O
the	O
resulting	O
model	O
generated	O
was	O
not	O
sufficient	O
.	O

We	O
determined	O
the	O
crystal	B-evidence
structures	I-evidence
of	O
NsrR	B-protein
-	O
RD	B-structure_element
and	O
NsrR	B-protein
-	O
ED	B-structure_element
separately	O
.	O

Ramachandran	B-evidence
validation	I-evidence
revealed	O
that	O
all	O
residues	O
(	O
100	O
%,	O
236	O
amino	O
acids	O
)	O
were	O
in	O
the	O
preferred	O
or	O
allowed	O
regions	O
.	O

Cartoon	O
representation	O
of	O
the	O
helices	B-structure_element
(	O
α1	B-structure_element
–	I-structure_element
α5	I-structure_element
)	O
and	O
β	B-structure_element
-	I-structure_element
sheets	I-structure_element
(	O
β1	B-structure_element
-	I-structure_element
β5	I-structure_element
).	O

Structural	O
areas	O
with	O
the	O
highest	O
variations	O
to	O
the	O
receiver	B-structure_element
domains	I-structure_element
of	O
DrrB	B-protein
(	O
pink	O
,	O
1P2F	O
),	O
MtrA	B-protein
(	O
grey	O
,	O
2GWR	O
),	O
and	O
PhoB	B-protein
(	O
blue	O
,	O
1B00	O
)	O
are	O
marked	O
in	O
separate	O
boxes	O
.	O

Furthermore	O
,	O
NsrR	B-protein
is	O
crystallized	B-experimental_method
as	O
a	O
monomer	B-oligomeric_state
,	O
and	O
investigation	O
of	O
the	O
symmetry	O
-	O
related	O
molecules	O
did	O
not	O
reveal	O
a	O
functional	O
dimer	B-oligomeric_state
within	O
the	O
crystal	B-evidence
.	O

The	O
rmsd	B-evidence
values	O
of	O
the	O
superimpositions	B-experimental_method
of	O
the	O
structures	B-evidence
of	O
NsrR	B-protein
-	O
RD	B-structure_element
and	O
NsrR	B-protein
-	O
ED	B-structure_element
with	O
the	O
available	O
structures	B-evidence
of	O
members	O
of	O
the	O
OmpR	B-protein_type
/	I-protein_type
PhoB	I-protein_type
subfamily	I-protein_type
are	O
highlighted	O
.	O
*	O
Seq	O
ID	O
(%)	O
corresponds	O
to	O
the	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
protein	O
sequence	O
.	O

This	O
site	O
of	O
phosphorylation	B-ptm
is	O
conserved	B-protein_state
throughout	O
the	O
family	O
of	O
response	B-protein_type
regulators	I-protein_type
,	O
including	O
the	O
lantibiotic	B-protein_type
resistance	I-protein_type
-	I-protein_type
associated	I-protein_type
RRs	I-protein_type
such	O
as	O
BraR	B-protein
from	O
L	B-species
.	I-species
monocytogenes	I-species
,	O
BceR	B-protein
from	O
Bacillus	B-species
subtilis	I-species
,	O
CprR	B-protein
from	O
C	B-species
.	I-species
difficile	I-species
,	O
GraR	B-protein
from	O
S	B-species
.	I-species
aureus	I-species
,	O
LcrR	B-protein
from	O
S	B-species
.	I-species
mutans	I-species
,	O
LisR	B-protein
,	O
and	O
VirR	B-protein
from	O
L	B-species
.	I-species
monocytogenes	I-species
(	O
Fig	O
3	O
).	O

The	O
linker	B-structure_element
region	I-structure_element
of	O
the	O
known	O
structures	B-evidence
is	O
underlined	O
within	O
the	O
sequence	O
.	O

This	O
pocket	B-site
is	O
similar	O
to	O
the	O
acidic	B-protein_state
active	B-site
site	I-site
observed	O
within	O
most	O
structures	B-evidence
of	O
RRs	B-protein_type
such	O
as	O
PhoB	B-protein
from	O
E	B-species
.	I-species
coli	I-species
,	O
PhoP	B-protein
from	O
M	B-species
.	I-species
tuberculosis	I-species
,	O
and	O
DivK	B-protein
from	O
Caulobacter	B-species
crescentus	I-species
.	O

The	O
inactive	B-protein_state
conformation	O
of	O
NsrR	B-protein
differs	O
from	O
the	O
active	B-protein_state
state	O
structure	B-evidence
of	O
PhoB	B-protein
(	O
light	O
blue	O
,	O
PDB	O
code	O
1ZES	O
)	O
(	O
b	O
)	O
in	O
the	O
orientation	O
of	O
the	O
corresponding	O
switch	B-site
residues	I-site
,	O
Ser82	B-residue_name_number
and	O
Phe101	B-residue_name_number
,	O
which	O
adopt	O
a	O
conformation	O
pointing	O
away	O
from	O
the	O
active	B-site
site	I-site
(	O
Asp55	B-residue_name_number
in	O
NsrR	B-protein
).	O

In	O
some	O
RRs	B-protein_type
like	O
CheY	B-protein
,	O
Mg2	B-chemical
+	I-chemical
is	O
observed	O
in	O
the	O
structure	B-evidence
,	O
bound	B-protein_state
near	O
the	O
phosphorylation	B-site
site	I-site
.	O

In	O
the	O
KdpE	B-protein
regulator	B-protein_type
from	O
E	B-species
.	I-species
coli	I-species
that	O
is	O
involved	O
in	O
osmoregulation	O
,	O
a	O
divalent	O
calcium	B-chemical
ion	O
is	O
present	O
.	O

Within	O
the	O
β4	B-structure_element
-	I-structure_element
α4	I-structure_element
loop	I-structure_element
and	O
in	O
β5	B-structure_element
of	O
the	O
RD	B-structure_element
of	O
RRs	B-protein_type
,	O
specific	O
amino	O
acids	O
are	O
crucial	O
for	O
signal	O
transduction	O
from	O
the	O
RD	B-structure_element
to	O
the	O
ED	B-structure_element
via	O
conformational	O
changes	O
that	O
are	O
a	O
consequence	O
of	O
phosphorylation	B-ptm
of	O
the	O
RD	B-structure_element
.	O

These	O
amino	O
acids	O
are	O
Ser	B-residue_name
/	O
Thr	B-residue_name
and	O
Phe	B-residue_name
/	O
Tyr	B-residue_name
located	O
at	O
the	O
end	O
of	O
β4	B-structure_element
and	O
before	O
β5	B-structure_element
,	O
respectively	O
,	O
and	O
designated	O
as	O
“	O
signature	B-site
switch	I-site
residues	I-site
”.	O

Although	O
some	O
RRs	B-protein_type
such	O
as	O
KdpE	B-protein
,	O
BraR	B-protein
,	O
BceR	B-protein
,	O
GraR	B-protein
,	O
and	O
VirR	B-protein
contain	O
a	O
serine	B-residue_name
residue	O
as	O
the	O
first	B-site
switch	I-site
residue	I-site
,	O
the	O
others	O
possess	O
a	O
threonine	B-residue_name
instead	O
.	O

The	O
RD	B-structure_element
domain	O
of	O
NsrR	B-protein
was	O
crystallized	B-experimental_method
with	O
two	O
separate	O
monomers	B-oligomeric_state
in	O
the	O
asymmetric	O
unit	O
.	O

Afterwards	O
,	O
helix	B-structure_element
α4	B-structure_element
and	O
the	O
adjacent	O
loops	B-structure_element
were	O
energy	B-experimental_method
minimized	I-experimental_method
with	I-experimental_method
the	I-experimental_method
MAB	I-experimental_method
force	I-experimental_method
field	I-experimental_method
as	O
implemented	O
in	O
the	O
program	O
Moloc	O
;	O
all	O
other	O
atoms	O
of	O
NsrR	B-protein
-	O
RD	B-structure_element
were	O
kept	O
fixed	O
.	O

The	O
result	O
is	O
highlighted	O
in	O
S2B	O
Fig	O
.	O
The	O
energy	B-protein_state
minimized	I-protein_state
structure	B-evidence
of	O
NsrR	B-protein
-	O
RD	B-structure_element
was	O
then	O
superimposed	B-experimental_method
on	O
the	O
dimeric	B-oligomeric_state
structure	B-evidence
of	O
KdpE	B-protein
.	O

In	O
addition	O
,	O
the	O
dimeric	B-site
interface	I-site
of	O
KdpE	B-protein
is	O
characterized	O
by	O
hydrophobic	B-site
patch	I-site
formed	O
by	O
residues	O
Ile88	B-residue_name_number
(	O
α4	B-structure_element
),	O
Leu91	B-residue_name_number
(	O
α4	B-structure_element
),	O
Ala110	B-residue_name_number
(	O
α5	B-structure_element
),	O
and	O
Val114	B-residue_name_number
(	O
α5	B-structure_element
).	O

Dimeric	B-oligomeric_state
structure	B-evidence
of	O
the	O
RD	B-structure_element
of	O
NsrR	B-protein
aligned	O
to	O
the	O
structure	B-evidence
of	O
KdpE	B-protein
(	O
PDB	O
code	O
1ZH2	O
,	O
not	O
shown	O
).	O

(	O
a	O
)	O
The	O
two	O
monomers	B-oligomeric_state
of	O
NsrR	B-protein
as	O
functional	O
dimers	B-oligomeric_state
are	O
represented	O
in	O
a	O
cartoon	O
representation	O
displayed	O
in	O
cyan	O
and	O
yellow	O
colors	O
.	O

Overall	O
Structure	B-evidence
of	O
C	O
-	O
terminal	O
DNA	B-structure_element
-	I-structure_element
binding	I-structure_element
effector	I-structure_element
domain	I-structure_element
of	O
NsrR	B-protein

The	O
asymmetric	O
unit	O
contains	O
two	O
copies	O
of	O
NsrR	B-protein
-	O
ED	B-structure_element
related	O
by	O
two	O
-	O
fold	O
rotational	O
symmetry	O
.	O

The	O
structure	B-evidence
of	O
NsrR	B-protein
-	O
ED	B-structure_element
also	O
contains	O
such	O
a	O
wHTH	B-structure_element
motif	O
built	O
up	O
by	O
helices	B-structure_element
α7	B-structure_element
and	O
α8	B-structure_element
(	O
Fig	O
6	O
).	O

In	O
the	O
structure	B-evidence
of	O
NsrR	B-protein
-	O
ED	B-structure_element
,	O
helix	B-structure_element
α8	B-structure_element
is	O
identified	O
as	O
the	O
recognition	B-structure_element
helix	I-structure_element
,	O
α7	B-structure_element
as	O
the	O
positioning	B-structure_element
helix	I-structure_element
,	O
and	O
the	O
loop	B-structure_element
region	I-structure_element
between	O
helices	O
α7	B-structure_element
-	I-structure_element
α8	I-structure_element
as	O
transactivation	B-structure_element
loop	I-structure_element
as	O
observed	O
in	O
other	O
RRs	B-protein_type
(	O
Fig	O
6	O
).	O

The	O
rmsd	B-evidence
between	O
the	O
three	O
helices	O
of	O
the	O
effector	B-structure_element
domain	I-structure_element
(	O
including	O
the	O
two	O
helices	B-structure_element
forming	O
the	O
wHTH	B-structure_element
motif	O
)	O
of	O
PhoB	B-protein
and	O
NsrR	B-protein
-	O
ED	B-structure_element
is	O
1	O
.	O
6	O
Å	O
over	O
47	O
Cα	O
atoms	O
,	O
clearly	O
indicating	O
that	O
NsrR	B-protein
belongs	O
to	O
the	O
OmpR	B-protein_type
/	I-protein_type
PhoB	I-protein_type
family	I-protein_type
of	I-protein_type
RRs	I-protein_type
.	O

The	O
exact	O
boundaries	O
of	O
these	O
linkers	B-structure_element
are	O
difficult	O
to	O
predict	O
from	O
sequence	B-experimental_method
alignments	I-experimental_method
in	O
the	O
absence	B-protein_state
of	I-protein_state
structural	O
information	O
of	O
the	O
distinct	O
RR	B-protein_type
.	O

Linker	B-structure_element
lengths	O
in	O
OmpR	B-protein_type
/	I-protein_type
PhoB	I-protein_type
proteins	I-protein_type
of	O
unknown	O
structure	O
have	O
been	O
estimated	O
by	O
comparing	O
the	O
number	O
of	O
residues	O
between	O
conserved	O
landmark	O
residues	O
in	O
the	O
regulatory	B-structure_element
and	I-structure_element
effector	I-structure_element
domains	I-structure_element
to	O
those	O
from	O
structurally	O
characterized	O
family	O
members	O
.	O

As	O
seen	O
in	O
the	O
structures	B-evidence
of	O
MtrA	B-protein
and	O
KdpE	B-protein
,	O
this	O
arginine	B-residue_name
residue	O
residing	O
at	O
the	O
end	O
of	O
α5	B-structure_element
participates	O
in	O
the	O
active	B-protein_state
state	O
dimer	B-site
interface	I-site
of	O
the	O
RD	B-structure_element
through	O
a	O
salt	B-bond_interaction
bridge	I-bond_interaction
interaction	O
with	O
an	O
aspartate	B-residue_name
residue	O
.	O

Although	O
we	O
aimed	O
at	O
crystallizing	B-experimental_method
full	B-protein_state
-	I-protein_state
length	I-protein_state
NsrR	B-protein
,	O
this	O
endeavor	O
failed	O
due	O
to	O
proteolytic	O
cleavage	O
within	O
the	O
linker	B-structure_element
region	I-structure_element
during	O
the	O
time	O
period	O
of	O
crystallization	B-experimental_method
.	O

DNA	B-chemical
-	O
binding	O
mode	O
of	O
NsrR	B-protein
using	O
a	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
model	O

Since	O
the	O
structures	B-evidence
of	O
both	O
domains	O
of	O
NsrR	B-protein
were	O
determined	O
,	O
we	O
used	O
this	O
structural	B-evidence
information	I-evidence
together	O
with	O
the	O
available	O
crystal	B-evidence
structures	I-evidence
of	O
related	O
proteins	O
to	O
create	O
a	O
model	O
of	O
the	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
NsrR	B-protein
in	O
its	O
active	B-protein_state
and	O
inactive	B-protein_state
state	O
.	O

Model	O
of	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
NsrR	B-protein
in	O
its	O
inactive	B-protein_state
state	O
and	O
active	B-protein_state
state	O
.	O

In	O
numerous	O
pathogenic	O
bacteria	B-taxonomy_domain
such	O
as	O
S	B-species
.	I-species
agalactiae	I-species
,	O
S	B-species
.	I-species
aureus	I-species
,	O
and	O
C	B-species
.	I-species
difficile	I-species
that	O
apparently	O
do	O
not	O
produce	O
a	O
lantibiotic	B-chemical
,	O
a	O
gene	O
cluster	O
is	O
present	O
to	O
provide	O
resistance	O
against	O
lantibiotics	B-chemical
such	O
as	O
nisin	B-chemical
,	O
nukacin	B-chemical
ISK	I-chemical
-	I-chemical
1	I-chemical
,	O
lacticin	B-chemical
481	I-chemical
gallidermin	B-chemical
,	O
actagardine	B-chemical
,	O
or	O
mersacidin	B-chemical
.	O

NMR	B-experimental_method
can	O
theoretically	O
be	O
used	O
to	O
determine	O
heterogeneous	O
ensembles	O
,	O
but	O
in	O
practice	O
,	O
this	O
proves	O
to	O
be	O
very	O
challenging	O
.	O

However	O
,	O
the	O
impact	O
that	O
chaperones	B-protein_type
have	O
on	O
their	O
substrates	O
,	O
and	O
how	O
these	O
interactions	O
affect	O
the	O
folding	O
process	O
remain	O
largely	O
unknown	O
.	O

Spy	B-protein
prevents	O
protein	O
aggregation	O
and	O
aids	O
in	O
protein	O
folding	O
under	O
various	O
stress	O
conditions	O
,	O
including	O
treatment	O
with	O
tannin	B-chemical
and	O
butanol	B-chemical
.	O

We	O
originally	O
discovered	O
Spy	B-protein
by	O
its	O
ability	O
to	O
stabilize	O
the	O
protein	O
-	O
folding	O
model	O
Im7	B-protein
in	O
vivo	O
and	O
recently	O
demonstrated	O
that	O
Im7	B-protein
folds	O
while	O
associated	O
with	O
Spy	B-protein
.	O

Such	O
residual	O
density	O
is	O
typically	O
not	O
considered	O
usable	O
by	O
traditional	O
X	B-experimental_method
-	I-experimental_method
ray	I-experimental_method
crystallography	I-experimental_method
methods	O
.	O

To	O
determine	O
the	O
structure	B-evidence
of	O
the	O
substrate	O
portion	O
of	O
these	O
Spy	B-protein
:	O
substrate	O
complexes	O
,	O
we	O
conceived	O
of	O
an	O
approach	O
that	O
we	O
term	O
READ	B-experimental_method
,	O
for	O
Residual	B-experimental_method
Electron	I-experimental_method
and	I-experimental_method
Anomalous	I-experimental_method
Density	I-experimental_method
.	O

We	O
split	O
this	O
approach	O
into	O
five	O
steps	O
:	O
(	O
1	O
)	O
By	O
using	O
a	O
well	O
-	O
diffracting	O
Spy	B-protein
:	O
substrate	O
co	B-evidence
-	I-evidence
crystal	I-evidence
,	O
we	O
first	O
determined	O
the	O
structure	B-evidence
of	O
the	O
folded	B-protein_state
domain	B-structure_element
of	O
Spy	B-protein
and	O
obtained	O
high	O
quality	O
residual	B-evidence
electron	I-evidence
density	I-evidence
within	O
the	O
dynamic	B-protein_state
regions	O
of	O
the	O
substrate	O
.	O

We	O
then	O
co	B-experimental_method
-	I-experimental_method
crystallized	I-experimental_method
Spy	B-protein
and	O
the	O
eight	O
Im76	B-mutant
-	I-mutant
45	I-mutant
peptides	O
,	O
each	O
of	O
which	O
harbored	O
an	O
individual	O
pI	B-chemical
-	I-chemical
Phe	I-chemical
substitution	B-experimental_method
at	O
one	O
distinct	O
position	O
,	O
and	O
collected	B-experimental_method
anomalous	B-evidence
data	I-evidence
for	O
all	O
eight	O
Spy	B-complex_assembly
:	I-complex_assembly
Im76	I-complex_assembly
-	I-complex_assembly
45	I-complex_assembly
complexes	O
(	O
Fig	O
.	O
1B	O
,	O
Supplementary	O
Table	O
1	O
Supplementary	O
Dataset	O
1	O
,	O
and	O
Supplementary	O
Table	O
2	O
).	O

During	O
each	O
round	O
of	O
the	O
selection	O
,	O
a	O
genetic	B-experimental_method
algorithm	I-experimental_method
alters	O
the	O
ensemble	O
and	O
its	O
agreement	O
to	O
the	O
experimental	O
data	O
is	O
re	O
-	O
evaluated	O
.	O

This	O
strategy	O
allows	O
a	O
wide	O
range	O
of	O
substrate	O
conformations	O
to	O
interact	O
with	O
the	O
chaperone	B-protein_type
.	O

The	O
ensemble	O
primarily	O
encompasses	O
Im76	B-mutant
-	I-mutant
45	I-mutant
laying	O
diagonally	O
within	O
the	O
Spy	B-protein
cradle	B-site
in	O
several	O
different	O
orientations	O
,	O
but	O
some	O
conformations	O
traverse	O
as	O
far	O
as	O
the	O
tips	O
or	O
even	O
extend	O
over	O
the	O
side	O
of	O
the	O
cradle	B-site
(	O
Figs	O
.	O
3	O
,	O
4a	O
).	O

The	O
Spy	B-site
-	I-site
contacting	I-site
residues	I-site
comprise	O
a	O
mixture	O
of	O
charged	O
,	O
polar	O
,	O
and	O
hydrophobic	O
residues	O
.	O

For	O
example	O
,	O
the	O
N	B-structure_element
-	I-structure_element
terminal	I-structure_element
half	I-structure_element
of	O
Im76	B-mutant
-	I-mutant
45	I-mutant
binds	O
more	O
consistently	O
in	O
the	O
Spy	B-protein
cradle	B-site
,	O
whereas	O
the	O
C	B-structure_element
-	I-structure_element
terminal	I-structure_element
half	I-structure_element
predominantly	O
binds	O
to	O
the	O
outer	O
edges	O
of	O
Spy	B-protein
’	O
s	O
concave	B-site
surface	I-site
.	O

Although	O
we	O
do	O
not	O
yet	O
have	O
time	O
resolution	O
data	O
of	O
these	O
various	O
snapshots	O
of	O
Im76	B-mutant
-	I-mutant
45	I-mutant
,	O
this	O
ensemble	O
illustrates	O
how	O
a	O
substrate	O
samples	O
its	O
folding	O
landscape	O
while	O
bound	B-protein_state
to	I-protein_state
a	O
chaperone	B-protein_type
.	O

Additionally	O
,	O
we	O
observed	O
that	O
the	O
linker	B-structure_element
region	I-structure_element
(	O
residues	O
47	B-residue_range
–	I-residue_range
57	I-residue_range
)	O
of	O
Spy	B-protein
,	O
which	O
participates	O
in	O
substrate	O
interaction	O
,	O
becomes	O
mostly	O
disordered	B-protein_state
upon	O
binding	O
the	O
substrate	O
.	O

In	O
the	O
chaperone	B-protein_state
-	I-protein_state
bound	I-protein_state
ensemble	O
,	O
Im76	B-mutant
-	I-mutant
45	I-mutant
samples	O
unfolded	B-protein_state
,	O
partially	O
folded	B-protein_state
,	O
and	O
native	B-protein_state
-	O
like	O
states	O
.	O

The	O
ensemble	O
provides	O
an	O
unprecedented	O
description	O
of	O
the	O
conformations	O
that	O
a	O
substrate	O
assumes	O
while	O
exploring	O
its	O
chaperone	B-protein_type
-	O
associated	O
folding	O
landscape	O
.	O

The	O
high	O
-	O
resolution	O
ensemble	B-evidence
obtained	O
here	O
now	O
provides	O
insight	O
into	O
exactly	O
how	O
this	O
occurs	O
.	O

The	O
ensemble	B-evidence
suggests	O
a	O
model	O
in	O
which	O
Spy	B-protein
provides	O
an	O
amphipathic	B-site
surface	I-site
that	O
allows	O
substrate	O
proteins	O
to	O
assume	O
different	O
conformations	O
while	O
bound	B-protein_state
to	I-protein_state
the	O
chaperone	B-protein_type
.	O

Residues	O
Asp32	B-residue_name_number
and	O
Asp35	B-residue_name_number
are	O
close	O
to	O
each	O
other	O
in	O
the	O
folded	B-protein_state
state	O
of	O
Im7	B-protein
.	O

Our	O
study	O
indicates	O
that	O
the	O
chaperone	B-protein_type
Spy	B-protein
employs	O
a	O
simple	O
surface	O
binding	O
approach	O
that	O
allows	O
the	O
substrate	O
to	O
explore	O
various	O
conformations	O
and	O
form	O
transiently	O
favorable	O
interactions	O
while	O
being	O
protected	O
from	O
aggregation	O
.	O

Crystallographic	O
data	O
and	O
ensemble	O
selection	O
.	O
(	O
a	O
)	O
2mFo	B-evidence
−	I-evidence
DFc	I-evidence
omit	I-evidence
map	I-evidence
of	O
residual	O
Im76	B-mutant
-	I-mutant
45	I-mutant
and	O
flexible	B-structure_element
linker	I-structure_element
electron	B-evidence
density	I-evidence
contoured	O
at	O
0	O
.	O
5	O
σ	O
.	O

Spy	B-complex_assembly
:	I-complex_assembly
Im76	I-complex_assembly
-	I-complex_assembly
45	I-complex_assembly
ensemble	O
,	O
arranged	O
by	O
RMSD	B-evidence
to	O
native	B-protein_state
state	O
of	O
Im76	B-mutant
-	I-mutant
45	I-mutant
.	O
Although	O
the	O
six	O
-	O
membered	O
ensemble	O
from	O
the	O
READ	B-experimental_method
selection	O
should	O
be	O
considered	O
only	O
as	O
an	O
ensemble	O
,	O
for	O
clarity	O
,	O
the	O
individual	O
conformers	O
are	O
shown	O
separately	O
here	O
.	O

Dashed	O
lines	O
connect	O
non	O
-	O
contiguous	O
segments	O
of	O
the	O
Im76	B-mutant
-	I-mutant
45	I-mutant
substrate	O
.	O

(	O
a	O
)	O
Spy	B-complex_assembly
:	I-complex_assembly
Im76	I-complex_assembly
-	I-complex_assembly
45	I-complex_assembly
contact	B-evidence
map	I-evidence
projected	O
onto	O
the	O
bound	B-protein_state
Spy	B-protein
dimer	B-oligomeric_state
(	O
above	O
)	O
and	O
Im76	B-mutant
-	I-mutant
45	I-mutant
(	O
below	O
)	O
structures	B-evidence
.	O

For	O
clarity	O
,	O
Im76	B-mutant
-	I-mutant
45	I-mutant
is	O
represented	O
with	O
a	O
single	O
conformation	O
.	O

As	O
the	O
residues	O
involved	O
in	O
contacts	O
are	O
more	O
evenly	O
distributed	O
in	O
Im76	B-mutant
-	I-mutant
45	I-mutant
compared	O
to	O
Spy	B-protein
,	O
its	O
contact	B-evidence
map	I-evidence
was	O
amplified	O
.	O
(	O
b	O
)	O
Detailed	O
contact	B-evidence
maps	I-evidence
of	O
Spy	B-complex_assembly
:	I-complex_assembly
Im76	I-complex_assembly
-	I-complex_assembly
45	I-complex_assembly
.	O

(	O
a	O
)	O
Overlay	B-experimental_method
of	O
apo	B-protein_state
Spy	B-protein
(	O
PDB	O
ID	O
:	O
3O39	O
,	O
gray	O
)	O
and	O
bound	B-protein_state
Spy	B-protein
(	O
green	O
).	O
(	O
b	O
)	O
Overlay	B-experimental_method
of	O
WT	B-protein_state
Spy	B-protein
bound	B-protein_state
to	I-protein_state
Im76	B-mutant
-	I-mutant
45	I-mutant
(	O
green	O
),	O
H96L	B-mutant
Spy	B-protein
bound	B-protein_state
to	I-protein_state
Im7	B-protein
L18A	B-mutant
L19	B-mutant
AL13A	I-mutant
(	O
blue	O
),	O
H96L	B-mutant
Spy	B-protein
bound	B-protein_state
to	I-protein_state
WT	B-protein_state
Im7	B-protein
(	O
yellow	O
),	O
and	O
WT	B-protein_state
Spy	B-protein
bound	B-protein_state
to	I-protein_state
casein	B-chemical
(	O
salmon	O
).	O
(	O
c	O
)	O
Competition	B-experimental_method
assay	I-experimental_method
showing	O
Im76	B-mutant
-	I-mutant
45	I-mutant
competes	O
with	O
Im7	B-protein
L18A	B-mutant
L19A	B-mutant
L37A	B-mutant
H40W	B-mutant
for	O
the	O
same	O
binding	B-site
site	I-site
on	O
Spy	B-protein
(	O
further	O
substrate	B-experimental_method
competition	I-experimental_method
assays	I-experimental_method
are	O
shown	O
in	O
Supplementary	O
Fig	O
.	O
8	O
).	O

Reversal	O
of	O
DNA	B-chemical
damage	O
induced	O
Topoisomerase	B-protein_type
2	I-protein_type
DNA	B-chemical
–	O
protein	O
crosslinks	O
by	O
Tdp2	B-protein

Mammalian	B-taxonomy_domain
Tyrosyl	B-protein
-	I-protein
DNA	I-protein
phosphodiesterase	I-protein
2	I-protein
(	O
Tdp2	B-protein
)	O
reverses	O
Topoisomerase	B-protein_type
2	I-protein_type
(	O
Top2	B-protein_type
)	O
DNA	B-chemical
–	O
protein	O
crosslinks	O
triggered	O
by	O
Top2	B-protein_type
engagement	O
of	O
DNA	B-chemical
damage	O
or	O
poisoning	O
by	O
anticancer	O
drugs	O
.	O

(	O
B	O
)	O
DNA	B-chemical
oligonucleotide	O
substrates	O
synthesized	O
by	O
EDC	O
-	O
imidazole	O
coupling	O
and	O
used	O
in	O
Tdp2	B-experimental_method
enzyme	I-experimental_method
assays	I-experimental_method
contain	O
deoxyadenine	B-chemical
(	O
dA	B-chemical
),	O
Ethenoadenine	B-chemical
(	O
ϵA	B-chemical
)	O
or	O
an	O
abasic	B-site
site	I-site
(	O
THF	B-chemical
)	O
and	O
a	O
5	O
′–	O
nitrophenol	O
moiety	O
.	O

P	B-evidence
-	I-evidence
values	I-evidence
calculated	O
using	O
two	O
-	O
tailed	O
t	B-experimental_method
-	I-experimental_method
test	I-experimental_method
;	O
error	O
bars	O
,	O
s	O
.	O
d	O
.	O
n	O
=	O
4	O
,	O
n	O
.	O
s	O
.	O
=	O
not	O
statistically	O
significant	O
.	O
(	O
D	O
)	O
Structure	B-evidence
of	O
mTdp2cat	B-structure_element
bound	B-protein_state
to	I-protein_state
5	B-chemical
′-	I-chemical
phosphate	I-chemical
DNA	I-chemical
(	O
product	O
complex	O
)	O
containing	O
ϵA	B-chemical
(	O
yellow	O
).	O

DNA	B-site
binding	I-site
β2Hβ	I-site
–	I-site
grasp	I-site
(	O
tan	O
)	O
and	O
cap	O
elements	O
engage	O
the	O
5	O
′-	O
nucleotide	O
as	O
well	O
as	O
the	O
+	O
2	O
and	O
+	O
3	O
nucleotides	O
(	O
blue	O
)	O
of	O
substrate	O
DNA	B-chemical
.	O

Our	O
integrated	O
results	O
from	O
structural	B-experimental_method
analysis	I-experimental_method
,	O
mutagenesis	B-experimental_method
,	O
functional	B-experimental_method
assays	I-experimental_method
and	O
quanyum	B-experimental_method
mechanics	I-experimental_method
/	I-experimental_method
molecular	I-experimental_method
mechanics	I-experimental_method
(	O
QM	B-experimental_method
/	I-experimental_method
MM	I-experimental_method
)	O
modeling	B-experimental_method
of	O
the	O
Tdp2	B-protein
reaction	O
coordinate	O
describe	O
in	O
detail	O
how	O
Tdp2	B-protein
mediates	O
a	O
single	O
-	O
metal	O
ion	O
tyrosyl	B-protein_type
DNA	I-protein_type
phosphodiesterase	I-protein_type
reaction	O
capable	O
of	O
acting	O
on	O
diverse	O
DNA	B-chemical
end	O
damage	O
.	O

To	O
understand	O
the	O
molecular	O
basis	O
for	O
Tdp2	B-protein
processing	O
of	O
Top2cc	B-complex_assembly
in	O
the	O
context	O
of	O
DNA	B-chemical
damage	O
,	O
we	O
crystallized	B-experimental_method
and	I-experimental_method
determined	I-experimental_method
X	B-experimental_method
-	I-experimental_method
ray	I-experimental_method
crystal	B-evidence
structures	I-evidence
of	O
mTdp2cat	B-structure_element
bound	B-protein_state
to	I-protein_state
5	B-chemical
′-	I-chemical
phosphate	I-chemical
DNA	I-chemical
(	O
product	O
complex	O
)	O
with	O
a	O
5	B-chemical
′-	I-chemical
ϵA	I-chemical
at	O
1	O
.	O
43	O
Å	O
resolution	O
(	O
PDB	O
entry	O
5HT2	O
)	O
and	O
the	O
abasic	O
DNA	B-chemical
damage	O
mimic	O
5	B-chemical
′-	I-chemical
THF	I-chemical
at	O
2	O
.	O
15	O
Å	O
resolution	O
(	O
PDB	O
entry	O
5INK	O
;	O
Figure	O
1D	O
and	O
E	O
,	O
Table	O
1	O
).	O

(	O
A	O
)	O
Structure	B-evidence
of	O
mTdp2cat	B-structure_element
bound	B-protein_state
to	I-protein_state
5	B-chemical
′-	I-chemical
phosphate	I-chemical
DNA	I-chemical
(	O
product	O
complex	O
)	O
containing	O
ϵA	B-chemical
(	O
yellow	O
),	O
Mg2	B-chemical
+	I-chemical
(	O
magenta	O
)	O
and	O
its	O
inner	O
-	O
sphere	O
waters	B-chemical
(	O
gray	O
).	O

The	O
THF	B-chemical
is	O
shown	O
in	O
yellow	O
and	O
a	O
hydrogen	B-bond_interaction
bond	I-bond_interaction
from	O
the	O
THF	B-chemical
O4	O
′	O
to	O
inner	O
-	O
sphere	O
water	B-chemical
is	O
shown	O
as	O
gray	O
dashes	O
.	O

Structural	O
plasticity	O
in	O
the	O
Tdp2	B-protein
DNA	B-site
binding	I-site
trench	I-site

The	O
paucity	O
of	O
hydrophobic	B-bond_interaction
interactions	I-bond_interaction
stabilizing	O
the	O
β2Hβ	B-structure_element
DNA	B-protein_state
-	I-protein_state
bound	I-protein_state
conformation	O
suggests	O
that	O
conformational	O
plasticity	O
in	O
the	O
β2Hβ	B-structure_element
might	O
be	O
a	O
feature	O
of	O
DNA	B-chemical
damage	O
and	O
metal	O
cofactor	O
engagement	O
.	O

Conformational	O
plasticity	O
in	O
the	O
Tdp2	B-protein
active	B-site
site	I-site
.	O

Wall	O
-	O
eyed	O
stereo	O
view	O
is	O
displayed	O
.	O
(	O
B	O
)	O
The	O
closed	B-protein_state
β2Hβ	B-structure_element
conformation	O
in	O
the	O
mTdp2cat	B-complex_assembly
–	I-complex_assembly
DNA	I-complex_assembly
product	O
structure	B-evidence
containing	O
5	B-chemical
′-	I-chemical
ϵA	I-chemical
(	O
yellow	O
,	O
PDB	O
entry	O
5HT2	O
).	O
T309	B-residue_name_number
(	O
green	O
)	O
is	O
an	O
integral	O
part	O
of	O
the	O
β2Hβ	B-site
DNA	I-site
-	I-site
binding	I-site
grasp	I-site
(	O
tan	O
)	O
and	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
to	O
the	O
backbone	O
of	O
Y321	B-residue_name_number
,	O
while	O
N314	B-residue_name_number
(	O
orange	O
)	O
occupies	O
the	O
β2Hβ	B-site
docking	I-site
pocket	I-site
.	O

Experiments	O
performed	O
as	O
in	O
panel	O
D	O
for	O
mTdp2cat	B-structure_element
WT	B-protein_state
(	O
lanes	O
27	O
–	O
39	O
)	O
or	O
mTdp2cat	B-structure_element
D358N	B-mutant
(	O
lanes	O
40	O
–	O
52	O
),	O
but	O
with	O
chymotrypsin	B-experimental_method
instead	O
of	O
trypsin	B-experimental_method
.	O

Thus	O
,	O
X	B-experimental_method
-	I-experimental_method
ray	I-experimental_method
structures	B-evidence
and	O
limited	B-experimental_method
proteolysis	I-experimental_method
analysis	I-experimental_method
indicate	O
that	O
DNA	B-chemical
-	O
and	O
metal	O
-	O
induced	O
conformational	O
changes	O
are	O
a	O
conserved	B-protein_state
feature	O
of	O
the	O
vertebrate	B-taxonomy_domain
Tdp2	B-protein
-	O
substrate	O
interaction	O
.	O

However	O
,	O
previous	O
biochemical	B-experimental_method
analysis	I-experimental_method
has	O
suggested	O
an	O
alternative	O
two	O
-	O
metal	O
ion	O
mechanism	O
for	O
the	O
Tdp2	B-protein
-	O
phosphotyrosyl	B-protein_type
phosphodiesterase	I-protein_type
reaction	O
.	O

Either	O
Mg2	B-chemical
+	I-chemical
or	O
Ca2	B-chemical
+	I-chemical
were	O
titrated	B-experimental_method
in	O
the	O
presence	B-protein_state
or	O
absence	B-protein_state
of	I-protein_state
5	B-chemical
′-	I-chemical
P	I-chemical
DNA	I-chemical
,	O
and	O
the	O
tryptophan	B-evidence
fluorescence	I-evidence
was	O
monitored	O
with	O
an	O
excitation	O
wavelength	O
of	O
280	O
nm	O
and	O
emission	O
wavelength	O
of	O
350	O
nm	O
using	O
10	O
nm	O
band	O
pass	O
filters	O
.	O

PNP	B-chemical
release	O
(	O
monitored	O
by	O
absorbance	O
at	O
415	O
nm	O
)	O
as	O
a	O
function	O
of	O
Mg2	B-chemical
+	I-chemical
concentration	O
and	O
in	O
the	O
absence	B-protein_state
or	O
presence	B-protein_state
of	I-protein_state
1	O
or	O
10	O
mM	O
Ca2	B-chemical
+	I-chemical
is	O
shown	O
;	O
error	O
bars	O
,	O
s	O
.	O
d	O
.	O
n	O
=	O
4	O
.	O
(	O
C	O
)	O
σ	B-evidence
-	I-evidence
A	I-evidence
weighted	I-evidence
2Fo	I-evidence
-	I-evidence
Fc	I-evidence
electron	I-evidence
density	I-evidence
map	I-evidence
(	O
blue	O
)	O
and	O
model	B-evidence
-	I-evidence
phased	I-evidence
anomalous	I-evidence
difference	I-evidence
Fourier	I-evidence
(	O
magenta	O
)	O
maps	B-evidence
for	O
the	O
mTdp2cat	B-complex_assembly
–	I-complex_assembly
DNA	I-complex_assembly
–	I-complex_assembly
Mn2	I-complex_assembly
+	I-complex_assembly
complex	O
(	O
PDB	O
entry	O
5INP	O
)	O
show	O
a	O
single	O
Mn2	B-chemical
+	I-chemical
(	O
cyan	O
)	O
is	O
bound	O
with	O
expected	O
octahedral	O
coordination	O
geometry	O
.	O

These	O
data	O
were	O
an	O
excellent	O
fit	O
to	O
a	O
single	O
-	O
site	O
binding	O
model	O
both	O
in	O
the	O
presence	B-protein_state
and	O
absence	B-protein_state
of	I-protein_state
DNA	B-chemical
(	O
Figure	O
4A	O
).	O

Overall	O
,	O
these	O
metal	B-experimental_method
binding	I-experimental_method
analyses	I-experimental_method
are	O
consistent	O
with	O
a	O
single	O
metal	O
ion	O
mediated	O
reaction	O
.	O

Altogether	O
,	O
QM	B-experimental_method
/	I-experimental_method
MM	I-experimental_method
modeling	I-experimental_method
identifies	O
new	O
determinants	O
of	O
the	O
Tdp2	B-protein
reaction	O
,	O
and	O
demonstrates	O
our	O
proposed	O
single	O
Mg2	B-chemical
+	I-chemical
catalyzed	O
reaction	O
model	O
is	O
a	O
viable	O
mechanism	O
for	O
Tdp2	B-protein
-	O
catalyzed	O
5	B-residue_name
′-	I-residue_name
phosphotyrosine	I-residue_name
bond	O
hydrolysis	O
.	O

To	O
test	O
the	O
aspects	O
of	O
the	O
Tdp2	B-protein
reaction	O
mechanism	O
described	O
here	O
derived	O
from	O
high	O
-	O
resolution	O
mouse	B-taxonomy_domain
Tdp2	B-protein
crystal	B-evidence
structures	I-evidence
(	O
denoted	O
with	O
superscript	O
numbering	O
‘	O
m	O
’	O
for	O
numbering	O
of	O
the	O
mouse	B-taxonomy_domain
protein	O
),	O
we	O
engineered	B-experimental_method
and	I-experimental_method
purified	I-experimental_method
thirteen	O
human	B-species
MBP	B-experimental_method
-	O
hTdp2cat	B-structure_element
mutant	B-protein_state
proteins	O
(	O
denoted	O
with	O
superscript	O
numbering	O
and	O
‘	O
h	O
’	O
for	O
the	O
human	B-species
protein	O
)	O
and	O
assayed	O
the	O
impacts	O
of	O
mutations	B-experimental_method
on	O
Tdp2	B-protein
catalytic	O
activity	O
using	O
three	O
in	O
vitro	O
reporter	O
substrates	O
including	O
a	O
tyrosylated	B-protein_state
DNA	B-chemical
substrate	O
(	O
5	B-ptm
′-	I-ptm
Y	I-ptm
),	O
p	B-chemical
-	I-chemical
nitrophenyl	I-chemical
phosphate	I-chemical
(	O
PNPP	B-chemical
)	O
and	O
thymidine	B-chemical
5	I-chemical
′-	I-chemical
monophosphate	I-chemical
p	I-chemical
-	I-chemical
nitrophenyl	I-chemical
ester	I-chemical
(	O
T5PNP	B-chemical
)	O
(	O
Figure	O
5E	O
,	O
Supplementary	O
Figures	O
S5B	O
and	O
S5C	O
).	O

We	O
found	O
that	O
mutations	B-experimental_method
that	O
removed	B-experimental_method
the	O
charge	O
yet	O
retained	O
the	O
ability	O
to	O
hydrogen	B-bond_interaction
bond	I-bond_interaction
(	O
hH351Q	B-mutant
)	O
or	O
should	O
abrogate	O
the	O
elevated	O
pKa	B-evidence
of	O
the	O
Histidine	B-residue_name
(	O
hD316N	B-mutant
)	O
had	O
severe	O
impacts	O
on	O
catalysis	O
.	O

Interestingly	O
,	O
the	O
Tdp2	B-protein
single	O
Mg2	B-chemical
+	I-chemical
ion	O
octahedral	B-bond_interaction
coordination	I-bond_interaction
shell	I-bond_interaction
also	O
involves	O
an	O
extended	O
hydrogen	B-bond_interaction
-	I-bond_interaction
bonding	I-bond_interaction
network	I-bond_interaction
mediated	O
by	O
hAsp350	B-residue_name_number
(	O
mAsp358	B-residue_name_number
)	O
that	O
stabilizes	O
the	O
DNA	B-protein_state
-	I-protein_state
bound	I-protein_state
conformation	O
of	O
the	O
β2Hβ	B-structure_element
substrate	I-structure_element
-	I-structure_element
binding	I-structure_element
loop	I-structure_element
through	O
hydrogen	B-bond_interaction
bonding	I-bond_interaction
to	O
mTrp307	B-residue_name_number
.	O

Samples	O
were	O
withdrawn	O
from	O
reactions	O
,	O
neutralized	O
with	O
TBE	O
-	O
urea	O
loading	O
dye	O
at	O
the	O
indicated	O
timepoints	O
,	O
and	O
electrophoresed	O
on	O
a	O
20	O
%	O
TBE	B-experimental_method
-	I-experimental_method
urea	I-experimental_method
PAGE	I-experimental_method
.	O

(	O
D	O
)	O
Relative	O
activity	O
of	O
WT	B-protein_state
and	O
indicated	O
mutant	B-protein_state
human	B-species
MBP	B-experimental_method
-	O
hTdp2cat	B-structure_element
fusion	O
proteins	O
on	O
three	O
model	O
Tdp2	B-protein
substrates	O
.	O

Although	O
Mg2	B-chemical
+	I-chemical
is	O
present	O
at	O
the	O
same	O
concentration	O
as	O
the	O
WT	B-protein_state
-	O
mTdpcat	B-protein
crystals	B-evidence
(	O
10	O
mM	O
),	O
we	O
find	O
the	O
metal	B-site
site	I-site
is	O
unoccupied	B-protein_state
in	O
the	O
mD358N	B-mutant
crystals	B-evidence
.	O

Therefore	O
,	O
metal	O
-	O
regulated	O
opening	O
/	O
closure	O
of	O
the	O
active	B-site
site	I-site
may	O
modulate	O
Tdp2	B-protein
activity	O
,	O
and	O
D350N	B-mutant
is	O
sufficient	O
to	O
block	O
both	O
metal	O
binding	O
and	O
conformational	O
change	O
.	O

In	O
support	O
of	O
this	O
,	O
we	O
also	O
find	O
that	O
hD350N	B-mutant
(	O
mD358N	B-mutant
)	O
impairs	O
Mg2	B-chemical
+	I-chemical
binding	O
as	O
measured	O
by	O
intrinsic	B-evidence
tryptophan	I-evidence
fluorescence	I-evidence
(	O
Figure	O
4A	O
),	O
and	O
abrogates	O
Mg2	B-chemical
+-	I-chemical
stimulated	O
active	B-site
site	I-site
conformational	O
changes	O
detected	O
by	O
trypsin	O
and	O
chymotrypsin	O
sensitivity	O
of	O
the	O
Tdp2	B-protein
metamorphic	O
loop	B-structure_element
(	O
Figure	O
3D	O
).	O

Error	O
bars	O
,	O
s	O
.	O
d	O
,	O
n	O
=	O
3	O
.	O
(	O
D	O
)	O
Junctions	O
recovered	O
from	O
cellular	B-experimental_method
end	I-experimental_method
-	I-experimental_method
joining	I-experimental_method
assays	I-experimental_method
in	O
the	O
noted	O
cell	O
types	O
were	O
characterized	O
by	O
sequencing	B-experimental_method
to	O
assess	O
the	O
end	B-evidence
-	I-evidence
joining	I-evidence
error	I-evidence
rate	I-evidence
.	O

By	O
comparison	O
the	O
more	O
frequent	O
I307V	B-mutant
has	O
only	O
mild	O
effects	O
on	O
in	O
vitro	O
activity	O
,	O
and	O
no	O
detectable	O
impact	O
on	O
cellular	O
assays	O
.	O

Tdp2	B-protein
was	O
originally	O
identified	O
as	O
a	O
protein	O
conferring	O
resistance	O
to	O
both	O
Top1	B-protein_type
and	O
Top2	B-protein_type
anti	O
-	O
cancer	O
drugs	O
,	O
however	O
it	O
is	O
hypothesized	O
that	O
the	O
predominant	O
natural	O
source	O
of	O
substrates	O
for	O
Tdp2	B-protein
are	O
likely	O
the	O
potent	O
DNA	B-chemical
damage	O
triggers	O
of	O
Top2	B-protein_type
poisoning	O
and	O
Top2	B-protein_type
DNA	B-chemical
protein	O
crosslinks	O
encountered	O
during	O
transcription	O
.	O

The	O
mechanism	O
of	O
NCX	B-protein_type
proteins	O
is	O
therefore	O
highly	O
likely	O
to	O
be	O
consistent	O
with	O
the	O
alternating	O
-	O
access	O
model	O
of	O
secondary	O
-	O
active	O
transport	O
.	O

Our	O
recent	O
atomic	O
-	O
resolution	O
structure	B-evidence
of	O
NCX_Mj	B-protein
from	O
Methanococcus	B-species
jannaschii	I-species
provided	O
the	O
first	O
view	O
of	O
the	O
basic	O
functional	O
unit	O
of	O
an	O
NCX	B-protein_type
protein	O
.	O

These	O
structures	B-evidence
reveal	O
the	O
mode	O
of	O
recognition	O
of	O
these	O
ions	O
,	O
their	O
relative	O
affinities	O
,	O
and	O
the	O
mechanism	O
of	O
extracellular	O
ion	O
exchange	O
,	O
for	O
a	O
well	O
-	O
defined	O
,	O
functional	O
conformation	O
in	O
a	O
membrane	O
-	O
like	O
environment	O
.	O

The	O
water	B-chemical
molecule	O
at	O
Smid	B-site
forms	O
hydrogen	B-bond_interaction
-	I-bond_interaction
bonds	I-bond_interaction
with	O
the	O
highly	B-protein_state
conserved	I-protein_state
Glu54	B-residue_name_number
and	O
Glu213	B-residue_name_number
(	O
Supplementary	O
Fig	O
.	O
1d	O
),	O
stabilizing	O
their	O
orientation	O
to	O
properly	O
coordinate	B-bond_interaction
multiple	O
Na	B-chemical
+	I-chemical
ions	O
at	O
Sext	B-site
,	O
SCa	B-site
and	O
Sint	B-site
.	O

When	O
Na	B-chemical
+	I-chemical
binds	O
to	O
Sext	B-site
at	O
high	B-protein_state
concentrations	O
,	O
the	O
N	B-structure_element
-	I-structure_element
terminal	I-structure_element
half	I-structure_element
of	O
TM7	B-structure_element
is	O
bent	O
into	O
two	O
short	B-structure_element
helices	I-structure_element
,	O
TM7a	B-structure_element
and	O
TM7b	B-structure_element
(	O
Fig	O
.	O
2a	O
).	O

These	O
contacts	O
are	O
absent	O
in	O
the	O
structure	B-evidence
with	O
Na	B-chemical
+	I-chemical
at	O
Sext	B-site
,	O
in	O
which	O
there	O
is	O
an	O
open	O
gap	O
between	O
the	O
two	O
helices	B-structure_element
(	O
Fig	O
.	O
2b	O
).	O

This	O
difference	O
is	O
noteworthy	O
because	O
TM6	B-structure_element
and	O
TM1	B-structure_element
are	O
believed	O
to	O
undergo	O
a	O
sliding	O
motion	O
,	O
relative	O
to	O
the	O
rest	O
of	O
the	O
protein	O
,	O
when	O
the	O
transporter	B-protein_type
switches	O
to	O
the	O
inward	B-protein_state
-	I-protein_state
facing	I-protein_state
conformation	O
.	O

Thus	O
,	O
in	O
100	O
mM	O
Na	B-chemical
+	I-chemical
and	O
10	O
mM	O
Sr2	B-chemical
+,	I-chemical
Na	B-chemical
+	I-chemical
completely	O
replaced	O
Sr2	B-chemical
+	I-chemical
(	O
Fig	O
.	O
3a	O
)	O
and	O
reverted	O
NCX_Mj	B-protein
to	O
the	O
Na	B-protein_state
+-	I-protein_state
loaded	I-protein_state
,	O
fully	B-protein_state
occluded	I-protein_state
state	O
.	O

Similar	O
titration	B-experimental_method
experiments	I-experimental_method
showed	O
that	O
Ca2	B-chemical
+	I-chemical
and	O
Sr2	B-chemical
+	I-chemical
binding	O
to	O
NCX_Mj	B-protein
are	O
not	O
exactly	O
alike	O
The	O
electron	B-evidence
density	I-evidence
distribution	I-evidence
from	O
crystals	B-experimental_method
soaked	I-experimental_method
in	I-experimental_method
high	B-protein_state
Ca2	B-chemical
+	I-chemical
and	O
low	B-protein_state
Na	B-chemical
+,	I-chemical
indicates	O
that	O
Ca2	B-chemical
+	I-chemical
can	O
bind	O
to	O
Smid	B-site
as	O
well	O
as	O
SCa	B-site
,	O
with	O
a	O
preference	O
for	O
SCa	B-site
(	O
Fig	O
.	O
3b	O
).	O

An	O
apo	B-protein_state
state	O
of	O
outward	B-protein_state
-	I-protein_state
facing	I-protein_state
NCX_Mj	B-protein
is	O
likely	O
to	O
exist	O
transiently	O
in	O
physiological	O
conditions	O
,	O
despite	O
the	O
high	O
amounts	O
of	O
extracellular	O
Na	B-chemical
+	I-chemical
(~	O
150	O
mM	O
)	O
and	O
Ca2	B-chemical
+	I-chemical
(~	O
2	O
mM	O
).	O

That	O
secondary	B-protein_type
-	I-protein_type
active	I-protein_type
transporters	I-protein_type
are	O
able	O
to	O
harness	O
an	O
electrochemical	O
gradient	O
of	O
one	O
substrate	O
to	O
power	O
the	O
uphill	O
transport	O
of	O
another	O
relies	O
on	O
a	O
seemingly	O
simple	O
principle	O
:	O
they	O
must	O
not	O
transition	O
between	O
outward	B-protein_state
-	I-protein_state
and	O
inward	B-protein_state
-	I-protein_state
open	I-protein_state
conformations	O
unless	O
in	O
two	O
precise	O
substrate	O
occupancy	O
states	O
.	O

To	O
examine	O
this	O
central	O
question	O
,	O
we	O
sought	O
to	O
characterize	O
the	O
conformational	B-evidence
free	I-evidence
-	I-evidence
energy	I-evidence
landscape	I-evidence
of	O
NCX_Mj	B-protein
and	O
to	O
examine	O
its	O
dependence	O
on	O
the	O
ion	O
-	O
occupancy	O
state	O
,	O
using	O
molecular	B-experimental_method
dynamics	I-experimental_method
(	O
MD	B-experimental_method
)	O
simulations	B-experimental_method
.	O

With	O
Sext	B-site
empty	B-protein_state
,	O
however	O
,	O
TM7ab	B-structure_element
forms	O
a	O
canonical	O
α	B-structure_element
-	I-structure_element
helix	I-structure_element
(	O
Fig	O
.	O
4a	O
-	O
b	O
,	O
4g	O
),	O
thereby	O
creating	O
an	O
opening	O
between	O
TM3	B-structure_element
and	O
TM7	B-structure_element
,	O
which	O
in	O
turn	O
allows	O
water	B-chemical
molecules	O
from	O
the	O
external	O
solution	O
to	O
reach	O
into	O
Sext	B-site
(	O
Fig	O
.	O
4e	O
,	O
4h	O
-	O
i	O
),	O
i	O
.	O
e	O
.	O
the	O
transporter	B-protein_type
is	O
no	B-protein_state
longer	I-protein_state
occluded	I-protein_state
.	O

Displacement	O
of	O
Na	B-chemical
+	I-chemical
from	O
SCa	B-site
and	O
Sint	B-site
induces	O
further	O
changes	O
(	O
Fig	O
.	O
4c	O
).	O

These	O
calculations	B-experimental_method
demonstrate	O
that	O
the	O
Na	B-chemical
+	I-chemical
occupancy	O
state	O
of	O
the	O
transporter	B-protein_type
has	O
a	O
profound	O
effect	O
on	O
its	O
conformational	B-evidence
free	I-evidence
-	I-evidence
energy	I-evidence
landscape	I-evidence
.	O

At	O
a	O
small	O
energetic	O
cost	O
,	O
however	O
,	O
the	O
transporter	B-protein_type
can	O
adopt	O
a	O
metastable	B-protein_state
‘	O
half	B-protein_state
-	I-protein_state
open	I-protein_state
’	O
conformation	O
in	O
which	O
TM7ab	B-structure_element
is	O
completely	O
straight	O
and	O
Sext	B-site
is	O
open	B-protein_state
to	O
the	O
exterior	O
(	O
Fig	O
.	O
5a	O
,	O
5b	O
).	O

Similarly	O
puzzling	O
is	O
that	O
a	O
given	O
antiporter	B-protein_type
will	O
undergo	O
this	O
transition	O
upon	O
recognition	O
of	O
substrates	O
of	O
different	O
charge	O
,	O
size	O
and	O
number	O
.	O

Yet	O
,	O
when	O
multiple	O
species	O
are	O
to	O
be	O
co	O
-	O
translocated	O
,	O
by	O
either	O
an	O
antiporter	B-protein_type
or	O
a	O
symporter	B-protein_type
,	O
partial	O
occupancies	O
must	O
not	O
be	O
conducive	O
to	O
the	O
alternating	B-site
-	I-site
access	I-site
switch	I-site
.	O

Nonetheless	O
,	O
the	O
calculated	B-evidence
free	I-evidence
-	I-evidence
energy	I-evidence
landscapes	I-evidence
,	O
derived	O
without	O
knowledge	O
of	O
the	O
experimental	O
data	O
,	O
reassuringly	O
confirm	O
that	O
the	O
crystallized	B-evidence
structures	I-evidence
correspond	O
to	O
mechanistically	O
relevant	O
,	O
interconverting	O
states	O
.	O

Specifically	O
,	O
our	O
crystal	B-experimental_method
titrations	I-experimental_method
suggest	O
that	O
,	O
during	O
forward	O
Na	B-chemical
+/	I-chemical
Ca2	B-chemical
+	I-chemical
exchange	O
,	O
sites	O
Sint	B-site
and	O
SCa	B-site
,	O
which	O
Ca2	B-chemical
+	I-chemical
and	O
Na	B-chemical
+	I-chemical
compete	O
for	O
,	O
can	O
be	O
grouped	O
into	O
one	O
;	O
Na	B-chemical
+	I-chemical
binding	O
to	O
these	O
sites	O
does	O
not	O
require	O
high	O
Na	B-chemical
+	I-chemical
concentrations	O
,	O
and	O
two	O
Na	B-chemical
+	I-chemical
ions	O
along	O
with	O
a	O
water	B-chemical
molecule	O
(	O
at	O
Smid	B-site
)	O
are	O
sufficient	O
to	O
displace	O
Ca2	B-chemical
+,	I-chemical
explaining	O
the	O
Hill	B-evidence
coefficient	I-evidence
of	O
~	O
2	O
for	O
Na	B-chemical
+-	I-chemical
dependent	O
inhibition	O
of	O
Ca2	B-chemical
+	I-chemical
fluxes	O
.	O

Interestingly	O
,	O
binding	O
of	O
Ca2	B-chemical
+	I-chemical
to	O
Smid	B-site
appears	O
to	O
be	O
also	O
possible	O
,	O
but	O
available	O
evidence	O
indicates	O
that	O
this	O
event	O
transiently	O
blocks	O
the	O
exchange	O
cycle	O
.	O

The	O
electron	B-evidence
density	I-evidence
(	O
grey	O
mesh	O
,	O
1	O
.	O
9	O
Å	O
Fo	B-evidence
-	I-evidence
Fc	I-evidence
ion	I-evidence
omit	I-evidence
map	I-evidence
contoured	O
at	O
4σ	O
)	O
at	O
Smid	B-site
was	O
modeled	O
as	O
water	B-chemical
(	O
red	O
sphere	O
)	O
and	O
those	O
at	O
Sext	B-site
,	O
SCa	B-site
and	O
Sint	B-site
as	O
Na	B-chemical
+	I-chemical
ions	O
(	O
green	O
spheres	O
).	O

The	O
displacement	O
of	O
A206	B-residue_name_number
reflects	O
the	O
[	O
Na	B-chemical
+]-	I-chemical
dependent	O
conformational	O
change	O
from	O
the	O
partially	B-protein_state
open	I-protein_state
to	O
the	O
occluded	B-protein_state
state	O
(	O
observed	O
at	O
low	O
and	O
high	O
Na	B-chemical
+	I-chemical
concentrations	O
,	O
respectively	O
).	O

Residues	O
forming	O
van	O
-	O
der	O
-	O
Waals	O
contacts	O
in	O
the	O
structure	B-evidence
at	O
low	B-protein_state
Na	B-chemical
+	I-chemical
concentration	O
are	O
shown	O
in	O
detail	O
.	O

The	O
vacant	O
Sext	B-site
site	O
in	O
the	O
structure	B-evidence
at	O
low	B-protein_state
Na	B-chemical
+	I-chemical
concentration	O
is	O
indicated	O
with	O
a	O
white	O
sphere	O
.	O

Residues	O
involved	O
in	O
Sr2	B-chemical
+	I-chemical
coordination	O
are	O
labeled	O
.	O

(	O
d	O
)	O
Close	O
-	O
up	O
of	O
the	O
ion	B-site
-	I-site
binding	I-site
region	I-site
in	O
the	O
fully	B-protein_state
Na	I-protein_state
+-	I-protein_state
occupied	I-protein_state
state	O
.	O

These	O
descriptors	O
were	O
employed	O
as	O
collective	O
variables	O
in	O
the	O
Bias	B-experimental_method
-	I-experimental_method
Exchange	I-experimental_method
Metadynamics	I-experimental_method
simulations	I-experimental_method
(	O
Methods	O
).	O

Thermodynamic	O
basis	O
for	O
the	O
proposed	O
mechanism	O
of	O
substrate	O
control	O
of	O
the	O
alternating	O
-	O
access	O
transition	O
of	O
NCX	B-protein_type
.	O
(	O
a	O
)	O
Calculated	B-evidence
conformational	I-evidence
free	I-evidence
-	I-evidence
energy	I-evidence
landscapes	I-evidence
for	O
outward	B-protein_state
-	I-protein_state
facing	I-protein_state
NCX_Mj	B-protein
,	O
for	O
two	O
different	O
Na	B-chemical
+-	I-chemical
occupancy	O
states	O
,	O
and	O
for	O
a	O
state	O
with	O
no	B-protein_state
ions	I-protein_state
bound	I-protein_state
.	O

The	O
uncorrected	O
map	B-evidence
overstabilizes	O
the	O
open	B-protein_state
state	O
relative	O
to	O
the	O
semi	B-protein_state
-	I-protein_state
open	I-protein_state
and	O
occluded	B-protein_state
because	O
it	O
also	O
overestimates	O
the	O
cost	O
of	O
dehydration	O
of	O
the	O
ion	O
,	O
once	O
it	O
is	O
bound	B-protein_state
to	I-protein_state
the	O
protein	O
(	O
this	O
effect	O
is	O
negligible	O
for	O
Na	B-chemical
+).	I-chemical

How	O
the	O
essential	O
pre	B-protein_type
-	I-protein_type
mRNA	I-protein_type
splicing	I-protein_type
factor	I-protein_type
U2AF65	B-protein
recognizes	O
the	O
polypyrimidine	B-chemical
(	O
Py	B-chemical
)	O
signals	O
of	O
the	O
major	O
class	O
of	O
3	B-site
′	I-site
splice	I-site
sites	I-site
in	O
human	B-species
gene	O
transcripts	O
remains	O
incompletely	O
understood	O
.	O

Single	B-experimental_method
-	I-experimental_method
molecule	I-experimental_method
FRET	I-experimental_method
experiments	O
suggest	O
that	O
conformational	O
selection	O
and	O
induced	O
fit	O
of	O
the	O
U2AF65	B-protein
RRMs	B-structure_element
are	O
complementary	O
mechanisms	O
for	O
Py	B-chemical
-	I-chemical
tract	I-chemical
association	O
.	O

The	O
pre	B-protein_type
-	I-protein_type
mRNA	I-protein_type
splicing	I-protein_type
factor	I-protein_type
U2AF65	B-protein
recognizes	O
3	B-site
′	I-site
splice	I-site
sites	I-site
in	O
human	B-species
gene	O
transcripts	O
,	O
but	O
the	O
details	O
are	O
not	O
fully	O
understood	O
.	O

Milestone	O
crystal	B-evidence
structures	I-evidence
of	O
the	O
core	B-protein_state
U2AF65	B-protein
RRM1	B-structure_element
and	O
RRM2	B-structure_element
connected	O
by	O
a	O
shortened	B-protein_state
inter	B-structure_element
-	I-structure_element
RRM	I-structure_element
linker	I-structure_element
(	O
dU2AF651	B-mutant
,	I-mutant
2	I-mutant
)	O
detailed	O
a	O
subset	O
of	O
nucleotide	O
interactions	O
with	O
the	O
individual	O
U2AF65	B-protein
RRMs	B-structure_element
.	O

In	O
a	O
fluorescence	B-experimental_method
anisotropy	I-experimental_method
assay	I-experimental_method
for	O
binding	O
a	O
representative	O
Py	B-chemical
tract	I-chemical
derived	O
from	O
the	O
well	O
-	O
characterized	O
splice	B-site
site	I-site
of	O
the	O
adenovirus	B-gene
major	I-gene
late	I-gene
promoter	I-gene
(	O
AdML	B-gene
),	O
the	O
RNA	B-evidence
affinity	I-evidence
of	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
increased	O
by	O
100	O
-	O
fold	O
relative	O
to	O
U2AF651	B-mutant
,	I-mutant
2	I-mutant
to	O
comparable	O
levels	O
as	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
U2AF65	B-protein
(	O
Fig	O
.	O
1b	O
;	O
Supplementary	O
Fig	O
.	O
1a	O
–	O
d	O
).	O

The	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
structures	B-evidence
characterize	O
ribose	B-chemical
(	O
r	B-chemical
)	O
nucleotides	B-chemical
at	O
all	O
of	O
the	O
binding	B-site
sites	I-site
except	O
the	O
seventh	B-residue_number
and	O
eighth	B-residue_number
deoxy	B-chemical
-(	I-chemical
d	I-chemical
)	I-chemical
U	I-chemical
,	O
which	O
are	O
likely	O
to	O
lack	O
2	O
′-	O
hydroxyl	O
contacts	O
based	O
on	O
the	O
RNA	B-protein_state
-	I-protein_state
bound	I-protein_state
dU2AF651	B-mutant
,	I-mutant
2	I-mutant
structure	B-evidence
.	O

U2AF65	B-protein
inter	B-structure_element
-	I-structure_element
RRM	I-structure_element
linker	I-structure_element
interacts	O
with	O
the	O
Py	B-chemical
tract	I-chemical

Otherwise	O
,	O
the	O
rU4	B-residue_name_number
nucleotide	B-chemical
packs	O
against	O
F304	B-residue_name_number
in	O
the	O
signature	O
ribonucleoprotein	B-structure_element
consensus	I-structure_element
motif	I-structure_element
(	I-structure_element
RNP	I-structure_element
)-	I-structure_element
2	I-structure_element
of	O
RRM2	B-structure_element
.	O

At	O
the	O
opposite	O
side	O
of	O
the	O
central	O
fifth	B-residue_number
nucleotide	B-chemical
,	O
the	O
sixth	B-residue_number
rU6	B-residue_name_number
nucleotide	B-chemical
is	O
located	O
at	O
the	O
inter	B-site
-	I-site
RRM1	I-site
/	I-site
RRM2	I-site
interface	I-site
(	O
Fig	O
.	O
3e	O
;	O
Supplementary	O
Movie	O
1	O
).	O

Versatile	O
primary	O
sequence	O
of	O
the	O
U2AF65	B-protein
inter	B-structure_element
-	I-structure_element
RRM	I-structure_element
linker	I-structure_element

However	O
,	O
stretching	O
of	O
the	O
truncated	B-protein_state
dU2AF651	B-mutant
,	I-mutant
2L	I-mutant
linker	B-structure_element
to	O
connect	O
the	O
RRM	B-structure_element
termini	I-structure_element
is	O
expected	O
to	O
disrupt	O
its	O
nucleotide	O
interactions	O
.	O

Likewise	O
,	O
deletion	B-experimental_method
of	O
the	O
N	O
-	O
terminal	O
RRM1	B-structure_element
extension	I-structure_element
in	O
the	O
shortened	B-protein_state
constructs	O
would	O
remove	O
packing	O
interactions	O
that	O
position	O
the	O
linker	B-structure_element
in	O
a	O
kinked	B-structure_element
turn	I-structure_element
following	O
P229	B-residue_name_number
(	O
Fig	O
.	O
4a	O
),	O
consistent	O
with	O
the	O
lower	O
RNA	B-evidence
affinities	I-evidence
of	O
dU2AF651	B-mutant
,	I-mutant
2L	I-mutant
,	O
dU2AF651	B-mutant
,	I-mutant
2	I-mutant
and	O
U2AF651	B-mutant
,	I-mutant
2	I-mutant
compared	O
with	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
.	O

Notably	O
,	O
the	O
Q147A	B-mutant
/	O
V254P	B-mutant
/	O
R227A	B-mutant
mutation	B-experimental_method
reduced	O
the	O
RNA	B-evidence
affinity	I-evidence
of	O
the	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
-	I-mutant
3Mut	I-mutant
protein	O
by	O
30	O
-	O
fold	O
more	O
than	O
would	O
be	O
expected	O
based	O
on	O
simple	O
addition	O
of	O
the	O
ΔΔG	B-evidence
'	O
s	O
for	O
the	O
single	O
mutations	O
.	O

As	O
a	O
representative	O
splicing	O
substrate	O
,	O
we	O
utilized	O
a	O
well	O
-	O
characterized	O
minigene	B-chemical
splicing	I-chemical
reporter	I-chemical
(	O
called	O
pyPY	B-chemical
)	O
comprising	O
a	O
weak	O
(	O
that	O
is	O
,	O
degenerate	O
,	O
py	B-chemical
)	O
and	O
strong	O
(	O
that	O
is	O
,	O
U	B-structure_element
-	I-structure_element
rich	I-structure_element
,	O
PY	B-chemical
)	O
polypyrimidine	B-chemical
tracts	I-chemical
preceding	O
two	O
alternative	O
splice	B-site
sites	I-site
(	O
Fig	O
.	O
5a	O
).	O

Paramagnetic	B-experimental_method
resonance	I-experimental_method
enhancement	I-experimental_method
(	O
PRE	B-experimental_method
)	O
measurements	O
previously	O
had	O
suggested	O
a	O
predominant	O
back	B-protein_state
-	I-protein_state
to	I-protein_state
-	I-protein_state
back	I-protein_state
,	O
or	O
‘	O
closed	B-protein_state
'	O
conformation	O
of	O
the	O
apo	B-protein_state
-	O
U2AF651	B-mutant
,	I-mutant
2	I-mutant
RRM1	B-structure_element
and	O
RRM2	B-structure_element
in	O
equilibrium	O
with	O
a	O
minor	O
‘	O
open	B-protein_state
'	O
conformation	O
resembling	O
the	O
RNA	B-protein_state
-	I-protein_state
bound	I-protein_state
inter	B-structure_element
-	I-structure_element
RRM	I-structure_element
arrangement	O
.	O

Approximately	O
40	O
%	O
of	O
the	O
smFRET	B-experimental_method
traces	B-evidence
showed	O
apparent	O
transitions	O
between	O
multiple	O
FRET	B-evidence
values	I-evidence
(	O
for	O
example	O
,	O
Fig	O
.	O
6c	O
).	O

Nevertheless	O
,	O
the	O
predominant	O
0	O
.	O
45	O
FRET	B-evidence
state	I-evidence
in	O
the	O
presence	O
of	O
RNA	B-chemical
agrees	O
with	O
the	O
Py	B-protein_state
-	I-protein_state
tract	I-protein_state
-	I-protein_state
bound	I-protein_state
crystal	B-evidence
structure	I-evidence
of	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
.	O

The	O
majority	O
of	O
traces	B-evidence
that	O
show	O
fluctuations	O
began	O
at	O
high	O
(	O
0	O
.	O
65	O
–	O
0	O
.	O
8	O
)	O
FRET	B-evidence
value	I-evidence
and	O
transitioned	O
to	O
a	O
∼	O
0	O
.	O
45	O
FRET	B-evidence
value	I-evidence
(	O
Supplementary	O
Fig	O
.	O
7c	O
–	O
g	O
).	O

Altogether	O
,	O
these	O
data	O
indicate	O
that	O
interactions	O
among	O
the	O
U2AF65	B-protein
RRM1	B-structure_element
/	O
RRM2	B-structure_element
,	O
inter	B-structure_element
-	I-structure_element
RRM	I-structure_element
linker	I-structure_element
,	O
N	B-structure_element
-	I-structure_element
and	I-structure_element
C	I-structure_element
-	I-structure_element
terminal	I-structure_element
extensions	I-structure_element
are	O
mutually	O
inter	O
-	O
dependent	O
for	O
cognate	O
Py	B-chemical
-	I-chemical
tract	I-chemical
recognition	O
.	O

Our	O
smFRET	B-experimental_method
results	O
agree	O
with	O
prior	O
NMR	B-experimental_method
/	O
PRE	B-experimental_method
evidence	O
for	O
multi	O
-	O
domain	O
conformational	O
selection	O
as	O
one	O
mechanistic	O
basis	O
for	O
U2AF65	B-protein
–	O
RNA	B-chemical
association	O
(	O
Fig	O
.	O
7b	O
).	O

An	O
∼	O
0	O
.	O
45	O
FRET	B-evidence
value	I-evidence
is	O
likely	O
to	O
correspond	O
to	O
the	O
U2AF65	B-protein
conformation	O
visualized	O
in	O
our	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
crystal	B-evidence
structures	I-evidence
,	O
in	O
which	O
the	O
RRM1	B-structure_element
and	O
RRM2	B-structure_element
bind	O
side	B-protein_state
-	I-protein_state
by	I-protein_state
-	I-protein_state
side	I-protein_state
to	O
the	O
Py	B-chemical
-	I-chemical
tract	I-chemical
oligonucleotide	I-chemical
.	O

As	O
such	O
,	O
the	O
smFRET	B-experimental_method
approach	O
reconciles	O
prior	O
inconsistencies	O
between	O
two	O
major	O
conformations	O
that	O
were	O
detected	O
by	O
NMR	B-experimental_method
/	O
PRE	B-experimental_method
experiments	O
and	O
a	O
broad	O
ensemble	O
of	O
diverse	O
inter	B-structure_element
-	I-structure_element
RRM	I-structure_element
arrangements	O
that	O
fit	O
the	O
SAXS	B-experimental_method
data	O
for	O
the	O
apo	B-protein_state
-	O
protein	B-protein
.	O

Similar	O
interdisciplinary	O
structural	O
approaches	O
are	O
likely	O
to	O
illuminate	O
whether	O
similar	O
mechanistic	O
bases	O
for	O
RNA	O
binding	O
are	O
widespread	O
among	O
other	O
members	O
of	O
the	O
vast	O
multi	O
-	O
RRM	B-structure_element
family	O
.	O

The	O
prior	O
dU2AF651	B-mutant
,	I-mutant
2	I-mutant
nucleotide	B-site
-	I-site
binding	I-site
sites	I-site
are	O
given	O
in	O
parentheses	O
(	O
site	O
4	O
'	O
interacts	O
with	O
dU2AF65	B-mutant
RRM1	B-structure_element
and	O
RRM2	B-structure_element
by	O
crystallographic	O
symmetry	O
).	O

(	O
i	O
)	O
Bar	O
graph	O
of	O
apparent	O
equilibrium	B-evidence
affinities	I-evidence
(	O
KA	B-evidence
)	O
of	O
the	O
wild	B-protein_state
type	I-protein_state
(	O
blue	O
)	O
and	O
the	O
indicated	O
mutant	B-protein_state
(	O
yellow	O
)	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
proteins	O
binding	O
the	O
AdML	B-gene
Py	B-chemical
tract	I-chemical
(	O
5	B-chemical
′-	I-chemical
CCCUUUUUUUUCC	I-chemical
-	I-chemical
3	I-chemical
′).	I-chemical

The	O
average	O
fitted	O
fluorescence	O
anisotropy	O
RNA	B-evidence
-	I-evidence
binding	I-evidence
curves	I-evidence
are	O
shown	O
in	O
Supplementary	O
Fig	O
.	O
4a	O
–	O
c	O
.	O

Protein	B-experimental_method
overexpression	I-experimental_method
and	O
qRT	B-experimental_method
-	I-experimental_method
PCR	I-experimental_method
results	O
are	O
shown	O
in	O
Supplementary	O
Fig	O
.	O
5	O
.	O

(	O
c	O
–	O
f	O
,	O
i	O
,	O
j	O
)	O
The	O
U2AF651	B-mutant
,	I-mutant
2LFRET	I-mutant
(	O
Cy3	B-chemical
/	O
Cy5	B-chemical
)	O
protein	O
was	O
immobilized	O
on	O
the	O
microscope	O
slide	O
via	O
biotin	B-chemical
-	I-chemical
NTA	I-chemical
/	I-chemical
Ni	I-chemical
+	I-chemical
2	I-chemical
(	O
orange	O
line	O
)	O
on	O
a	O
neutravidin	O
(	O
black	O
X	O
)-	O
biotin	O
-	O
PEG	O
(	O
orange	O
triangle	O
)-	O
treated	O
surface	O
and	O
imaged	O
either	O
in	O
the	O
absence	B-protein_state
of	I-protein_state
ligands	B-chemical
(	O
c	O
,	O
d	O
),	O
in	O
the	O
presence	O
of	O
5	O
μM	O
AdML	B-gene
Py	B-chemical
-	I-chemical
tract	I-chemical
RNA	I-chemical
(	O
5	B-chemical
′-	I-chemical
CCUUUUUUUUCC	I-chemical
-	I-chemical
3	I-chemical
′)	I-chemical
(	O
e	O
,	O
f	O
),	O
or	O
in	O
the	O
presence	O
of	O
10	O
μM	O
adenosine	B-residue_name
-	O
interrupted	O
variant	O
RNA	B-chemical
(	O
5	B-chemical
′-	I-chemical
CUUUUUAAUUUCCA	I-chemical
-	I-chemical
3	I-chemical
′)	I-chemical
(	O
i	O
,	O
j	O
).	O

N	O
is	O
the	O
number	O
of	O
single	O
-	O
molecule	O
traces	B-evidence
compiled	O
for	O
each	O
histogram	B-evidence
.	O

Schematic	O
models	O
of	O
U2AF65	B-protein
recognizing	O
the	O
Py	B-chemical
tract	I-chemical
.	O

Alternatively	O
,	O
a	O
conformation	O
of	O
U2AF65	B-protein
corresponding	O
to	O
∼	O
0	O
.	O
45	O
FRET	B-evidence
value	I-evidence
can	O
directly	O
bind	O
to	O
RNA	B-chemical
;	O
RNA	B-chemical
binding	O
stabilizes	O
the	O
‘	O
open	B-protein_state
',	O
side	B-protein_state
-	I-protein_state
by	I-protein_state
-	I-protein_state
side	I-protein_state
conformation	O
and	O
thus	O
shifts	O
the	O
U2AF65	B-protein
population	O
towards	O
the	O
∼	O
0	O
.	O
45	O
FRET	B-evidence
value	I-evidence
.	O

Specific	O
damage	O
manifestations	O
were	O
determined	O
within	O
the	O
large	O
trp	B-protein_type
RNA	I-protein_type
-	I-protein_type
binding	I-protein_type
attenuation	I-protein_type
protein	I-protein_type
(	O
TRAP	B-complex_assembly
)	O
bound	B-protein_state
to	I-protein_state
a	O
single	O
-	O
stranded	O
RNA	B-chemical
that	O
forms	O
a	O
belt	O
around	O
the	O
protein	O
.	O

Over	O
a	O
large	O
dose	O
range	O
,	O
the	O
RNA	B-chemical
was	O
found	O
to	O
be	O
far	O
less	O
susceptible	O
to	O
radiation	O
-	O
induced	O
chemical	O
changes	O
than	O
the	O
protein	O
.	O

The	O
11	O
-	O
fold	O
symmetry	O
within	O
each	O
TRAP	B-complex_assembly
ring	B-structure_element
permitted	O
statistically	O
significant	O
analysis	O
of	O
the	O
Glu	B-residue_name
and	O
Asp	B-residue_name
damage	O
patterns	O
,	O
with	O
RNA	B-chemical
binding	O
unexpectedly	O
being	O
observed	O
to	O
protect	O
these	O
otherwise	O
highly	O
sensitive	O
residues	O
within	O
the	O
11	O
RNA	B-site
-	I-site
binding	I-site
pockets	I-site
distributed	O
around	O
the	O
outside	O
of	O
the	O
protein	O
molecule	O
.	O

Global	O
radiation	O
damage	O
is	O
observed	O
within	O
reciprocal	O
space	O
as	O
the	O
overall	O
decay	O
of	O
the	O
summed	O
intensity	O
of	O
reflections	O
detected	O
within	O
the	O
diffraction	B-evidence
pattern	I-evidence
as	O
dose	O
increases	O
(	O
Garman	O
,	O
2010	O
;	O
Murray	O
&	O
Garman	O
,	O
2002	O
).	O

For	O
instance	O
,	O
structure	B-experimental_method
determination	I-experimental_method
of	O
the	O
purple	O
membrane	O
protein	O
bacterio	B-protein_type
­	I-protein_type
rhodopsin	I-protein_type
required	O
careful	O
corrections	O
for	O
radiation	O
-	O
induced	O
structural	O
changes	O
before	O
the	O
correct	O
photosensitive	O
intermediate	O
states	O
could	O
be	O
isolated	O
(	O
Matsui	O
et	O
al	O
.,	O
2002	O
).	O

As	O
of	O
early	O
2016	O
,	O
>	O
5400	O
nucleoprotein	B-complex_assembly
complex	O
structures	B-evidence
have	O
been	O
deposited	O
within	O
the	O
PDB	O
,	O
with	O
91	O
%	O
solved	O
by	O
MX	B-experimental_method
.	O

TRAP	B-complex_assembly
consists	O
of	O
11	O
identical	O
subunits	B-structure_element
assembled	O
into	O
a	O
ring	B-structure_element
with	O
11	O
-	O
fold	O
rotational	O
symmetry	O
.	O

The	O
substrate	O
Trp	B-chemical
amino	O
-	O
acid	O
ligands	O
also	O
exhibited	O
disordering	O
of	O
the	O
free	O
terminal	O
carboxyl	O
groups	O
at	O
higher	O
doses	O
(	O
Fig	O
.	O
2	O
▸	O
a	O
);	O
however	O
,	O
no	O
clear	O
Fourier	B-evidence
difference	I-evidence
peaks	I-evidence
could	O
be	O
observed	O
visually	O
.	O

The	O
rate	O
of	O
D	B-evidence
loss	I-evidence
(	O
attributed	O
to	O
side	O
-	O
chain	O
decarboxylation	O
)	O
was	O
consistently	O
larger	O
for	O
Glu	B-residue_name
compared	O
with	O
Asp	B-residue_name
residues	O
over	O
the	O
large	O
dose	O
range	O
(	O
Fig	O
.	O
2	O
▸	O
b	O
and	O
Supplementary	O
Fig	O
.	O
S3	O
);	O
this	O
observation	O
is	O
consistent	O
with	O
our	O
calculations	O
on	O
model	O
systems	O
(	O
see	O
above	O
)	O
that	O
suggest	O
that	O
,	O
without	O
considering	O
differential	O
hydrogen	B-bond_interaction
-	I-bond_interaction
bonding	I-bond_interaction
environments	O
,	O
CO2	B-chemical
loss	O
is	O
more	O
exothermic	O
by	O
around	O
8	O
kJ	O
mol	O
−	O
1	O
from	O
oxidized	B-protein_state
Glu	B-residue_name
residues	O
than	O
from	O
their	O
Asp	B-residue_name
counterparts	O
.	O

For	O
the	O
large	O
number	O
of	O
acidic	O
residues	O
per	O
TRAP	B-complex_assembly
ring	B-structure_element
(	O
four	O
Asp	B-residue_name
and	O
six	O
Glu	B-residue_name
residues	O
per	O
protein	O
monomer	B-oligomeric_state
),	O
a	O
strong	O
dependence	O
of	O
decarboxylation	O
susceptibility	O
on	O
local	O
environment	O
was	O
observed	O
(	O
Fig	O
.	O
4	O
▸).	O

For	O
each	O
Glu	B-residue_name
Cδ	O
or	O
Asp	B-residue_name
Cγ	O
atom	O
,	O
D	B-evidence
loss	I-evidence
provided	O
a	O
direct	O
measure	O
of	O
the	O
rate	O
of	O
side	O
-	O
chain	O
carboxyl	O
-	O
group	O
disordering	O
and	O
subsequent	O
decarboxylation	O
.	O

For	O
acidic	O
residues	O
with	O
no	O
differing	O
interactions	O
between	O
nonbound	B-protein_state
and	O
bound	B-protein_state
TRAP	B-complex_assembly
(	O
Fig	O
.	O
4	O
▸	O
a	O
),	O
similar	O
damage	O
was	O
apparent	O
between	O
the	O
two	O
rings	O
within	O
the	O
asymmetric	O
unit	O
,	O
as	O
expected	O
.	O

However	O
,	O
TRAP	B-complex_assembly
residues	O
directly	O
on	O
the	O
RNA	B-site
-	I-site
binding	I-site
interfaces	I-site
exhibited	O
greater	O
damage	O
accumulation	O
in	O
nonbound	B-protein_state
TRAP	B-complex_assembly
(	O
Fig	O
.	O
4	O
▸	O
b	O
),	O
and	O
for	O
residues	O
at	O
the	O
ring	B-site
–	I-site
ring	I-site
interfaces	I-site
(	O
where	O
crystal	O
contacts	O
were	O
detected	O
)	O
bound	B-protein_state
TRAP	B-complex_assembly
exhibited	O
enhanced	O
SRD	O
accumulation	O
(	O
Fig	O
.	O
4	O
▸	O
c	O
).	O

Three	O
acidic	O
residues	O
(	O
Glu36	B-residue_name_number
,	O
Asp39	B-residue_name_number
and	O
Glu42	B-residue_name_number
)	O
are	O
involved	O
in	O
RNA	B-chemical
interactions	O
within	O
each	O
of	O
the	O
11	O
TRAP	B-complex_assembly
ring	B-structure_element
subunits	B-structure_element
,	O
and	O
Fig	O
.	O
5	O
▸	O
shows	O
their	O
density	B-evidence
changes	I-evidence
with	O
increasing	O
dose	O
.	O

Here	O
,	O
MX	B-experimental_method
radiation	O
-	O
induced	O
specific	O
structural	O
changes	O
within	O
the	O
large	O
TRAP	B-complex_assembly
–	I-complex_assembly
RNA	I-complex_assembly
assembly	O
over	O
a	O
large	O
dose	O
range	O
(	O
1	O
.	O
3	O
–	O
25	O
.	O
0	O
MGy	O
)	O
have	O
been	O
analysed	O
using	O
a	O
high	O
-	O
throughput	O
quantitative	O
approach	O
,	O
providing	O
a	O
measure	O
of	O
the	O
electron	B-evidence
-	I-evidence
density	I-evidence
distribution	I-evidence
for	O
each	O
refined	O
atom	O
with	O
increasing	O
dose	O
,	O
D	B-evidence
loss	I-evidence
.	O

Here	O
,	O
it	O
provided	O
the	O
precision	O
required	O
to	O
quantify	O
the	O
role	O
of	O
RNA	B-chemical
in	O
the	O
damage	O
susceptibilities	O
of	O
equivalent	O
atoms	O
between	O
RNA	B-protein_state
-	I-protein_state
bound	I-protein_state
and	O
nonbound	B-protein_state
TRAP	B-complex_assembly
,	O
but	O
it	O
is	O
applicable	O
to	O
any	O
MX	B-experimental_method
SRD	O
study	O
.	O

Since	O
U4	B-residue_name_number
is	O
the	O
only	O
refined	O
nucleotide	O
not	O
to	O
exhibit	O
significant	O
base	O
–	O
protein	O
interactions	O
around	O
TRAP	B-complex_assembly
(	O
with	O
a	O
water	B-chemical
-	O
mediated	O
hydrogen	B-bond_interaction
bond	I-bond_interaction
detected	O
in	O
only	O
three	O
of	O
the	O
11	O
subunits	B-structure_element
and	O
a	O
single	O
Arg58	B-residue_name_number
hydrogen	B-bond_interaction
bond	I-bond_interaction
suggested	O
in	O
a	O
further	O
four	O
subunits	B-structure_element
),	O
this	O
increased	O
U4	B-residue_name_number
D	B-evidence
loss	I-evidence
can	O
be	O
explained	O
owing	O
to	O
its	O
greater	O
flexibility	O
.	O

For	O
Glu36	B-residue_name_number
and	O
Asp39	B-residue_name_number
,	O
no	O
direct	O
quantitative	O
correlation	O
could	O
be	O
established	O
between	O
hydrogen	B-bond_interaction
-	I-bond_interaction
bond	I-bond_interaction
length	O
and	O
D	B-evidence
loss	I-evidence
(	O
linear	B-evidence
R	I-evidence
2	I-evidence
of	O
<	O
0	O
.	O
23	O
for	O
all	O
doses	O
;	O
Supplementary	O
Fig	O
.	O
S5	O
).	O

The	O
Glu36	B-residue_name_number
carboxyl	O
side	O
chain	O
also	O
potentially	O
forms	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
to	O
His34	B-residue_name_number
and	O
Lys56	B-residue_name_number
,	O
but	O
since	O
these	O
interactions	O
are	O
conserved	B-protein_state
irrespective	O
of	O
G3	B-residue_name_number
nucleotide	O
binding	O
,	O
this	O
cannot	O
directly	O
account	O
for	O
the	O
stabilization	O
effect	O
on	O
Glu36	B-residue_name_number
in	O
RNA	B-protein_state
-	I-protein_state
bound	I-protein_state
TRAP	B-complex_assembly
.	O

We	O
propose	O
that	O
with	O
no	O
solvent	O
accessibility	O
Glu36	B-residue_name_number
decarboxylation	O
is	O
inhibited	O
,	O
since	O
the	O
CO2	B-evidence
-	I-evidence
formation	I-evidence
rate	I-evidence
K	I-evidence
2	I-evidence
is	O
greatly	O
reduced	O
,	O
and	O
suggest	O
that	O
steric	O
hindrance	O
prevents	O
each	O
radicalized	O
Glu36	B-residue_name_number
CO2	O
group	O
from	O
achieving	O
the	O
planar	O
conformation	O
required	O
for	O
complete	O
dissociation	O
from	O
TRAP	B-complex_assembly
.	O

Such	O
reduced	O
radiation	O
-	O
sensitivity	O
in	O
this	O
case	O
ensures	O
that	O
the	O
interacting	O
protein	O
remains	O
bound	B-protein_state
long	O
enough	O
to	O
the	O
RNA	B-chemical
to	O
complete	O
its	O
function	O
,	O
even	O
whilst	O
exposed	O
to	O
ionizing	O
radiation	O
.	O

RNA	B-chemical
is	O
shown	O
is	O
yellow	O
.	O

(	O
b	O
)	O
Average	O
D	O
loss	O
for	O
each	O
residue	O
/	O
nucleotide	O
type	O
with	O
respect	O
to	O
the	O
DWD	B-evidence
(	O
diffraction	B-evidence
-	I-evidence
weighted	I-evidence
dose	I-evidence
;	O
Zeldin	O
,	O
Brock	O
­	O
hauser	O
et	O
al	O
.,	O
2013	O
).	O

Only	O
a	O
subset	O
of	O
key	O
TRAP	B-complex_assembly
residue	O
types	O
are	O
included	O
.	O

Mitogen	B-protein_type
-	I-protein_type
activated	I-protein_type
protein	I-protein_type
kinases	I-protein_type
(	O
MAPKs	B-protein_type
),	O
important	O
in	O
a	O
large	O
array	O
of	O
signalling	O
pathways	O
,	O
are	O
tightly	O
controlled	O
by	O
a	O
cascade	O
of	O
protein	B-protein_type
kinases	I-protein_type
and	O
by	O
MAPK	B-protein_type
phosphatases	I-protein_type
(	O
MKPs	B-protein_type
).	O

Two	O
types	O
of	O
docking	O
interactions	O
have	O
been	O
identified	O
:	O
D	B-structure_element
-	I-structure_element
motif	I-structure_element
-	O
mediated	O
interaction	O
and	O
FXF	B-site
-	I-site
docking	I-site
interaction	I-site
.	O

The	O
285FNFL288	B-structure_element
segment	I-structure_element
in	O
MKP7	B-protein
directly	O
binds	O
to	O
a	O
hydrophobic	B-site
site	I-site
on	O
JNK1	B-protein
that	O
is	O
near	O
the	O
MAPK	B-protein_type
insertion	O
and	O
helix	B-structure_element
αG	B-structure_element
.	O
Biochemical	B-experimental_method
studies	I-experimental_method
further	O
reveal	O
that	O
this	O
highly	B-protein_state
conserved	I-protein_state
structural	B-structure_element
motif	I-structure_element
is	O
present	O
in	O
all	O
members	O
of	O
the	O
MKP	B-protein_type
family	I-protein_type
,	O
and	O
the	O
interaction	O
mode	O
is	O
universal	O
and	O
critical	O
for	O
the	O
MKP	B-protein_type
-	O
MAPK	B-protein_type
recognition	O
and	O
biological	O
function	O
.	O

The	O
MAPKs	B-protein_type
are	O
activated	O
by	O
MAPK	B-protein_type
kinases	I-protein_type
that	O
phosphorylate	O
the	O
MAPKs	B-protein_type
at	O
conserved	B-protein_state
threonine	B-residue_name
and	O
tyrosine	B-residue_name
residues	O
within	O
their	O
activation	B-structure_element
loop	I-structure_element
.	O

MAPKs	B-protein_type
lie	O
at	O
the	O
bottom	O
of	O
conserved	O
three	O
-	O
component	O
phosphorylation	O
cascades	O
and	O
utilize	O
docking	O
interactions	O
to	O
link	O
module	O
components	O
and	O
bind	O
substrates	O
.	O

Downregulation	O
of	O
MAPK	B-protein_type
activity	O
can	O
be	O
achieved	O
through	O
direct	O
dephosphorylation	O
of	O
the	O
phospho	B-residue_name
-	I-residue_name
threonine	I-residue_name
and	I-residue_name
/	I-residue_name
or	I-residue_name
tyrosine	I-residue_name
residues	O
by	O
various	O
serine	B-protein_type
/	I-protein_type
threonine	I-protein_type
phosphatases	I-protein_type
,	O
tyrosine	B-protein_type
phosphatases	I-protein_type
and	O
dual	B-protein_type
-	I-protein_type
specificity	I-protein_type
phosphatases	I-protein_type
(	O
DUSPs	B-protein_type
)	O
termed	O
MKPs	B-protein_type
.	O

DUSPs	B-protein_type
belong	O
to	O
the	O
protein	B-protein_type
-	I-protein_type
tyrosine	I-protein_type
phosphatases	I-protein_type
(	O
PTPase	B-protein_type
)	O
superfamily	O
,	O
which	O
is	O
defined	O
by	O
the	O
PTPase	B-protein_type
-	O
signature	O
motif	O
CXXGXXR	B-structure_element
.	O

The	O
KBD	B-structure_element
is	O
homologous	O
to	O
the	O
rhodanese	B-protein_type
family	I-protein_type
and	O
contains	O
an	O
intervening	O
cluster	O
of	O
basic	O
amino	O
acids	O
,	O
which	O
has	O
been	O
suggested	O
to	O
be	O
important	O
for	O
interacting	O
with	O
the	O
target	O
MAPKs	B-protein_type
.	O

As	O
shown	O
in	O
Fig	O
.	O
2d	O
,	O
the	O
CD	B-structure_element
of	O
MKP7	B-protein
can	O
be	O
pulled	O
down	O
by	O
JNK1	B-protein
,	O
while	O
the	O
KBD	B-structure_element
failed	O
to	O
bind	O
to	O
the	O
counterpart	O
protein	O
.	O

In	O
the	O
complex	O
,	O
JNK1	B-protein
has	O
its	O
characteristic	O
bilobal	O
structure	O
comprising	O
an	O
N	B-structure_element
-	I-structure_element
terminal	I-structure_element
lobe	I-structure_element
rich	O
in	O
β	B-structure_element
-	I-structure_element
sheet	I-structure_element
and	O
a	O
C	B-structure_element
-	I-structure_element
terminal	I-structure_element
lobe	I-structure_element
that	O
is	O
mostly	O
α	B-structure_element
-	I-structure_element
helical	I-structure_element
.	O

In	O
an	O
alignment	B-experimental_method
of	O
the	O
structure	B-evidence
of	O
MKP7	B-protein
-	O
CD	B-structure_element
with	O
that	O
of	O
VHR	B-protein
,	O
an	O
atypical	O
‘	O
MKP	B-protein_type
'	O
consisting	O
of	O
only	O
a	O
catalytic	B-structure_element
domain	I-structure_element
,	O
119	O
of	O
147	O
MKP7	B-protein
-	O
CD	B-structure_element
residues	O
could	O
be	O
superimposed	B-experimental_method
with	O
a	O
r	B-evidence
.	I-evidence
m	I-evidence
.	I-evidence
s	I-evidence
.	I-evidence
d	I-evidence
.	I-evidence
(	O
root	B-evidence
mean	I-evidence
squared	I-evidence
deviation	I-evidence
)	O
of	O
1	O
.	O
05	O
Å	O
(	O
Fig	O
.	O
3c	O
).	O

Since	O
helix	B-structure_element
α0	B-structure_element
and	O
the	O
following	O
loop	B-structure_element
α0	B-structure_element
–	I-structure_element
β1	I-structure_element
are	O
known	O
for	O
a	O
substrate	B-site
-	I-site
recognition	I-site
motif	I-site
of	O
VHR	B-protein
and	O
other	O
phosphatases	B-protein_type
,	O
the	O
absence	O
of	O
these	O
moieties	O
implicates	O
a	O
different	O
substrate	O
-	O
binding	O
mode	O
of	O
MKP7	B-protein
.	O

We	O
also	O
observed	O
the	O
binding	O
of	O
a	O
chloride	B-chemical
ion	O
in	O
the	O
active	B-site
site	I-site
of	O
MKP7	B-protein
-	O
CD	B-structure_element
.	O

Thus	O
this	O
chloride	B-chemical
ion	O
is	O
a	O
mimic	O
for	O
the	O
phosphate	B-chemical
group	O
of	O
the	O
substrate	O
,	O
as	O
revealed	O
by	O
a	O
comparison	O
with	O
the	O
structure	B-evidence
of	O
PTP1B	B-protein
in	B-protein_state
complex	I-protein_state
with	I-protein_state
phosphotyrosine	B-residue_name
(	O
Supplementary	O
Fig	O
.	O
1d	O
).	O

The	O
aromatic	O
ring	O
of	O
Phe285	B-residue_name_number
on	O
MKP7	B-protein
α5	B-structure_element
-	I-structure_element
helix	I-structure_element
is	O
nestled	O
in	O
a	O
hydrophobic	B-site
pocket	I-site
on	O
JNK1	B-protein
,	O
formed	O
by	O
side	O
chains	O
of	O
Ile197	B-residue_name_number
,	O
Leu198	B-residue_name_number
,	O
Ile231	B-residue_name_number
,	O
Trp234	B-residue_name_number
,	O
Val256	B-residue_name_number
,	O
Tyr259	B-residue_name_number
,	O
Val260	B-residue_name_number
and	O
the	O
aliphatic	O
portion	O
of	O
His230	B-residue_name_number
(	O
Fig	O
.	O
3d	O
,	O
f	O
and	O
Supplementary	O
Fig	O
.	O
1g	O
).	O

Interestingly	O
,	O
mutation	B-experimental_method
of	O
Phe287	B-residue_name_number
results	O
in	O
a	O
considerable	O
loss	O
of	O
activity	O
against	O
pJNK1	B-protein_state
without	O
altering	O
the	O
affinity	B-evidence
of	O
MKP7	B-protein
-	O
CD	B-structure_element
for	O
JNK1	B-protein
(	O
Supplementary	O
Fig	O
.	O
2a	O
).	O

Incubation	B-experimental_method
of	O
MKP7	B-protein
with	O
JNK1	B-protein
did	O
not	O
markedly	O
stimulate	O
the	O
phosphatase	B-protein_type
activity	O
,	O
which	O
is	O
consistent	O
with	O
previous	O
results	O
that	O
MKP7	B-protein
solely	O
possesses	O
the	O
intrinsic	O
activity	O
(	O
Supplementary	O
Fig	O
.	O
2b	O
).	O

Biochemical	O
results	O
suggested	O
that	O
the	O
affinity	O
and	O
specificity	O
between	O
KAP	B-protein
and	O
CDK2	B-protein
results	O
from	O
the	O
recognition	B-site
site	I-site
comprising	O
CDK2	B-protein
residues	O
from	O
the	O
αG	B-structure_element
helix	I-structure_element
and	O
L14	B-structure_element
loop	I-structure_element
and	O
the	O
N	B-structure_element
-	I-structure_element
terminal	I-structure_element
helical	I-structure_element
region	I-structure_element
of	O
KAP	B-protein
(	O
Fig	O
.	O
5b	O
).	O

There	O
is	O
a	O
hydrogen	B-bond_interaction
bond	I-bond_interaction
between	O
the	O
main	O
-	O
chain	O
nitrogen	O
of	O
Ile183	B-residue_name_number
(	O
KAP	B-protein
)	O
and	O
side	O
chain	O
oxygen	O
of	O
Glu208	B-residue_name_number
(	O
CDK2	B-protein
),	O
and	O
salt	O
bridges	O
between	O
Lys184	B-residue_name_number
of	O
KAP	B-protein
and	O
Asp235	B-residue_name_number
of	O
CDK2	B-protein
.	O

JNK	B-protein_type
is	O
activated	O
following	O
cellular	O
exposure	O
to	O
a	O
number	O
of	O
acute	O
stimuli	O
such	O
as	O
anisomycin	B-chemical
,	O
H2O2	B-chemical
,	O
ultraviolet	O
light	O
,	O
sorbitol	B-chemical
,	O
DNA	O
-	O
damaging	O
agents	O
and	O
several	O
strong	O
apoptosis	O
inducers	O
(	O
etoposide	B-chemical
,	O
cisplatin	B-chemical
and	O
taxol	B-chemical
).	O

Expressions	B-experimental_method
of	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
MKP7	B-protein
,	O
MKP7ΔC304	B-mutant
and	O
MKP7	B-protein
-	O
CD	B-structure_element
significantly	O
decreased	O
the	O
proportion	O
of	O
apoptotic	O
cells	O
after	O
ultraviolet	O
treatment	O
.	O

As	O
in	O
the	O
case	O
of	O
MKP7	B-protein
,	O
all	O
the	O
mutants	B-protein_state
,	O
except	O
F451D	B-mutant
/	I-mutant
A	I-mutant
,	O
showed	O
no	O
pNPPase	B-protein_type
activity	O
changes	O
compared	O
with	O
the	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
MKP5	B-protein
-	O
CD	B-structure_element
(	O
Fig	O
.	O
7g	O
),	O
and	O
the	O
point	B-experimental_method
mutations	I-experimental_method
in	O
JNK1	B-protein
also	O
reduced	O
the	O
binding	B-evidence
affinity	I-evidence
of	O
MKP5	B-protein
-	O
CD	B-structure_element
for	O
JNK1	B-protein
(	O
Fig	O
.	O
7h	O
).	O

In	O
this	O
model	O
,	O
the	O
MKP5	B-protein
-	O
CD	B-structure_element
adopts	O
a	O
conformation	O
nearly	O
identical	O
to	O
that	O
in	O
its	O
unbound	B-protein_state
form	O
,	O
suggesting	O
that	O
the	O
conformation	O
of	O
the	O
catalytic	B-structure_element
domain	I-structure_element
undergoes	O
little	O
change	O
,	O
if	O
any	O
at	O
all	O
,	O
upon	O
JNK1	B-protein
binding	O
.	O

The	O
JNK1	B-complex_assembly
–	I-complex_assembly
MKP7	I-complex_assembly
-	I-complex_assembly
CD	I-complex_assembly
interaction	O
is	O
better	O
and	O
more	O
extensive	O
.	O

These	O
structures	B-evidence
revealed	O
that	O
linear	B-structure_element
docking	I-structure_element
motifs	I-structure_element
in	O
interacting	O
proteins	O
bind	O
to	O
a	O
common	O
docking	B-site
site	I-site
on	O
MAPKs	B-protein_type
outside	O
the	O
kinase	B-protein_type
active	B-site
site	I-site
.	O

In	O
contrast	O
to	O
the	O
canonical	O
D	B-site
-	I-site
motif	I-site
-	I-site
binding	I-site
mode	I-site
,	O
separate	O
helices	B-structure_element
,	O
α2	B-structure_element
and	O
α3	B-structure_element
′,	I-structure_element
in	O
the	O
KBD	B-structure_element
of	O
MKP5	B-protein
engage	O
the	O
p38α	B-site
-	I-site
docking	I-site
site	I-site
.	O

Sequence	B-experimental_method
alignment	I-experimental_method
of	O
all	O
MKPs	B-protein_type
reveals	O
a	O
high	O
degree	O
of	O
conservation	O
of	O
residues	O
surrounding	O
the	O
interacting	B-site
region	I-site
observed	O
in	O
JNK1	B-complex_assembly
–	I-complex_assembly
MKP7	I-complex_assembly
-	I-complex_assembly
CD	I-complex_assembly
complex	O
(	O
Supplementary	O
Fig	O
.	O
5	O
).	O

In	O
summary	O
,	O
we	O
have	O
resolved	O
the	O
structure	B-evidence
of	O
JNK1	B-protein
in	B-protein_state
complex	I-protein_state
with	I-protein_state
the	O
catalytic	B-structure_element
domain	I-structure_element
of	O
MKP7	B-protein
.	O

In	O
addition	O
,	O
JIP	B-protein
-	I-protein
1	I-protein
can	O
also	O
associate	O
with	O
MKP7	B-protein
via	O
the	O
C	B-structure_element
-	I-structure_element
terminal	I-structure_element
region	I-structure_element
of	O
MKP7	B-protein
(	O
ref	O
.).	O

When	O
MKP7	B-protein
is	O
bound	B-protein_state
to	I-protein_state
JIP	B-protein
-	I-protein
1	I-protein
,	O
it	O
reduces	O
JNK	B-protein_type
activation	O
,	O
leading	O
to	O
reduced	O
phosphorylation	O
of	O
the	O
JNK	B-protein_type
target	O
c	B-protein_type
-	I-protein_type
Jun	I-protein_type
.	O

Thus	O
,	O
our	O
biochemical	B-evidence
and	I-evidence
structural	I-evidence
data	I-evidence
allow	O
us	O
to	O
present	O
a	O
model	O
for	O
the	O
JNK1	B-complex_assembly
–	I-complex_assembly
JIP	I-complex_assembly
-	I-complex_assembly
1	I-complex_assembly
–	I-complex_assembly
MKP7	I-complex_assembly
ternary	O
complex	O
and	O
provide	O
an	O
important	O
insight	O
into	O
the	O
assembly	O
and	O
function	O
of	O
JNK	B-protein_type
signalling	O
modules	O
(	O
Supplementary	O
Fig	O
.	O
6	O
).	O

The	O
colour	O
scheme	O
is	O
the	O
same	O
in	O
the	O
following	O
figures	O
unless	O
indicated	O
otherwise	O
.	O
(	O
b	O
)	O
Plots	B-evidence
of	I-evidence
initial	I-evidence
velocity	I-evidence
of	O
the	O
MKP7	B-protein
-	O
catalysed	O
reaction	O
versus	O
phospho	B-ptm
-	O
JNK1	B-protein
concentration	O
.	O

(	O
d	O
)	O
GST	B-experimental_method
-	I-experimental_method
mediated	I-experimental_method
pull	I-experimental_method
-	I-experimental_method
down	I-experimental_method
assay	I-experimental_method
for	O
interaction	O
of	O
JNK1	B-protein
with	O
MKP7	B-protein
-	O
CD	B-structure_element
and	O
MKP7	B-protein
-	O
KBD	B-structure_element
.	O

Structure	B-evidence
of	O
JNK1	B-protein
in	B-protein_state
complex	I-protein_state
with	I-protein_state
MKP7	B-protein
-	O
CD	B-structure_element
.	O

(	O
a	O
)	O
Ribbon	O
diagram	O
of	O
JNK1	B-complex_assembly
–	I-complex_assembly
MKP7	I-complex_assembly
-	I-complex_assembly
CD	I-complex_assembly
complex	O
in	O
two	O
views	O
related	O
by	O
a	O
45	O
°	O
rotation	O
around	O
a	O
vertical	O
axis	O
.	O
(	O
b	O
)	O
Structure	B-evidence
of	O
MKP7	B-protein
-	O
CD	B-structure_element
with	O
its	O
active	B-site
site	I-site
highlight	O
in	O
cyan	O
.	O

The	O
2Fo	B-evidence
−	I-evidence
Fc	I-evidence
omit	I-evidence
map	I-evidence
(	O
contoured	O
at	O
1	O
.	O
5σ	O
)	O
for	O
the	O
P	B-structure_element
-	I-structure_element
loop	I-structure_element
of	O
MKP7	B-protein
-	O
CD	B-structure_element
is	O
shown	O
at	O
inset	O
of	O
b	O
.	O
(	O
c	O
)	O
Structure	B-evidence
of	O
VHR	B-protein
with	O
its	O
active	B-site
site	I-site
highlighted	O
in	O
marine	O
blue	O
.	O
(	O
d	O
)	O
Close	O
-	O
up	O
view	O
of	O
the	O
JNK1	B-site
–	I-site
MKP7	I-site
interface	I-site
showing	O
interacting	O
amino	O
acids	O
of	O
JNK1	B-protein
(	O
orange	O
)	O
and	O
MKP7	B-protein
-	O
CD	B-structure_element
(	O
cyan	O
).	O

(	O
a	O
)	O
Effects	O
of	O
mutations	O
in	O
MKP7	B-protein
-	O
CD	B-structure_element
on	O
the	O
JNK1	B-protein
dephosphorylation	B-ptm
(	O
mean	O
±	O
s	O
.	O
e	O
.	O
m	O
.,	O
n	O
=	O
3	O
).	O

However	O
,	O
in	O
contrast	O
to	O
the	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
MKP7	B-protein
-	O
CD	B-structure_element
,	O
mutant	B-protein_state
F285D	B-mutant
did	O
not	O
co	O
-	O
migrate	O
with	O
JNK1	B-protein
.	O

(	O
f	O
)	O
Effects	O
of	O
mutations	B-experimental_method
in	O
MKP7	B-protein
-	O
CD	B-structure_element
on	O
the	O
pNPP	B-chemical
hydrolysis	O
reaction	O
(	O
mean	O
±	O
s	O
.	O
e	O
.	O
m	O
.,	O
n	O
=	O
3	O
).	O

FXF	B-structure_element
-	I-structure_element
motif	I-structure_element
is	O
critical	O
for	O
controlling	O
the	O
phosphorylation	B-ptm
of	O
JNK	B-protein_type
and	O
ultraviolet	O
-	O
induced	O
apoptosis	O
.	O

After	O
36	O
h	O
infection	O
,	O
cells	O
were	O
untreated	O
in	O
a	O
,	O
stimulated	O
with	O
30	O
μM	O
etoposide	B-chemical
for	O
3	O
h	O
in	O
b	O
or	O
irradiated	O
with	O
25	O
J	O
m	O
−	O
2	O
ultraviolet	O
light	O
at	O
30	O
min	O
before	O
lysis	O
in	O
c	O
.	O
Whole	O
-	O
cell	O
extracts	O
were	O
then	O
immunoblotted	O
with	O
antibody	O
indicated	O
.	O

Apoptotic	O
cells	O
were	O
determined	O
by	O
Annexin	B-chemical
-	I-chemical
V	I-chemical
-	I-chemical
APC	I-chemical
/	O
PI	B-chemical
staining	O
.	O

Floral	O
abscission	O
is	O
controlled	O
by	O
the	O
leucine	B-protein_type
-	I-protein_type
rich	I-protein_type
repeat	I-protein_type
receptor	I-protein_type
kinase	I-protein_type
(	O
LRR	B-protein_type
-	I-protein_type
RK	I-protein_type
)	O
HAESA	B-protein
and	O
the	O
peptide	B-protein_type
hormone	I-protein_type
IDA	B-protein
.	O

Crystal	B-evidence
structures	I-evidence
of	O
HAESA	B-protein
in	B-protein_state
complex	I-protein_state
with	I-protein_state
IDA	B-protein
reveal	O
a	O
hormone	B-site
binding	I-site
pocket	I-site
that	O
accommodates	O
an	O
active	B-protein_state
dodecamer	B-structure_element
peptide	B-chemical
.	O

The	O
HAESA	B-protein
co	B-protein_type
-	I-protein_type
receptor	I-protein_type
SERK1	B-protein
,	O
a	O
positive	O
regulator	O
of	O
the	O
floral	O
abscission	O
pathway	O
,	O
allows	O
for	O
high	O
-	O
affinity	O
sensing	O
of	O
the	O
peptide	B-protein_type
hormone	I-protein_type
by	O
binding	O
to	O
an	O
Arg	B-structure_element
-	I-structure_element
His	I-structure_element
-	I-structure_element
Asn	I-structure_element
motif	I-structure_element
in	O
IDA	B-protein
.	O

This	O
sequence	O
pattern	O
is	O
conserved	B-protein_state
among	O
diverse	O
plant	B-taxonomy_domain
peptides	B-chemical
,	O
suggesting	O
that	O
plant	B-taxonomy_domain
peptide	B-protein_type
hormone	I-protein_type
receptors	I-protein_type
may	O
share	O
a	O
common	O
ligand	O
binding	O
mode	O
and	O
activation	O
mechanism	O
.	O

Another	O
challenge	O
will	O
be	O
to	O
find	O
out	O
where	O
IDA	B-protein
is	O
produced	O
in	O
the	O
plant	B-taxonomy_domain
and	O
what	O
causes	O
it	O
to	O
accumulate	O
in	O
specific	O
places	O
in	O
preparation	O
for	O
organ	O
shedding	O
.	O

(	O
A	O
)	O
SDS	B-experimental_method
PAGE	I-experimental_method
analysis	O
of	O
the	O
purified	O
Arabidopsis	B-species
thaliana	I-species
HAESA	B-protein
ectodomain	B-structure_element
(	O
residues	O
20	B-residue_range
–	I-residue_range
620	I-residue_range
)	O
obtained	O
by	O
secreted	B-experimental_method
expression	I-experimental_method
in	I-experimental_method
insect	I-experimental_method
cells	I-experimental_method
.	O

Residues	O
mediating	O
hydrophobic	B-bond_interaction
interactions	I-bond_interaction
with	O
the	O
IDA	B-chemical
peptide	I-chemical
are	O
highlighted	O
in	O
blue	O
,	O
residues	O
contributing	O
to	O
hydrogen	B-bond_interaction
bond	I-bond_interaction
interactions	I-bond_interaction
and	O
/	O
or	O
salt	B-bond_interaction
bridges	I-bond_interaction
are	O
shown	O
in	O
red	O
.	O

The	O
alignment	O
includes	O
a	O
secondary	O
structure	O
assignment	O
calculated	O
with	O
the	O
program	O
DSSP	O
and	O
colored	O
according	O
to	O
Figure	O
1	O
,	O
with	O
the	O
N	O
-	O
and	O
C	O
-	O
terminal	O
caps	B-structure_element
and	O
the	O
21	O
LRR	B-structure_element
motifs	I-structure_element
indicated	O
in	O
orange	O
and	O
blue	O
,	O
respectively	O
.	O

Cysteine	B-residue_name
residues	O
engaged	O
in	O
disulphide	B-ptm
bonds	I-ptm
are	O
depicted	O
in	O
green	O
.	O

HAESA	B-protein
residues	O
interacting	O
with	O
the	O
IDA	B-chemical
peptide	I-chemical
and	O
/	O
or	O
the	O
SERK1	B-protein
co	B-protein_type
-	I-protein_type
receptor	I-protein_type
kinase	I-protein_type
ectodomain	B-structure_element
are	O
highlighted	O
in	O
blue	O
and	O
orange	O
,	O
respectively	O
.	O

The	O
PKGV	B-structure_element
motif	I-structure_element
present	O
in	O
our	O
N	B-protein_state
-	I-protein_state
terminally	I-protein_state
extended	I-protein_state
IDA	B-chemical
peptide	I-chemical
is	O
highlighted	O
in	O
red	O
.	O
(	O
B	O
)	O
Isothermal	B-experimental_method
titration	I-experimental_method
calorimetry	I-experimental_method
of	O
the	O
HAESA	B-protein
ectodomain	B-structure_element
vs	O
.	O
IDA	B-protein
and	O
including	O
the	O
synthetic	B-protein_state
peptide	B-chemical
sequence	O
.	O

(	O
C	O
)	O
Structure	O
of	O
the	O
HAESA	B-complex_assembly
–	I-complex_assembly
IDA	I-complex_assembly
complex	O
with	O
HAESA	B-protein
shown	O
in	O
blue	O
(	O
ribbon	O
diagram	O
).	O

We	O
purified	B-experimental_method
the	O
HAESA	B-protein
ectodomain	B-structure_element
(	O
residues	O
20	B-residue_range
–	I-residue_range
620	I-residue_range
)	O
from	O
baculovirus	B-experimental_method
-	I-experimental_method
infected	I-experimental_method
insect	I-experimental_method
cells	I-experimental_method
(	O
Figure	O
1	O
—	O
figure	O
supplement	O
1A	O
,	O
see	O
Materials	O
and	O
methods	O
)	O
and	O
quantified	O
the	O
interaction	O
of	O
the	O
~	O
75	O
kDa	O
glycoprotein	B-protein_type
with	O
synthetic	B-protein_state
IDA	B-chemical
peptides	I-chemical
using	O
isothermal	B-experimental_method
titration	I-experimental_method
calorimetry	I-experimental_method
(	O
ITC	B-experimental_method
).	O

We	O
obtained	O
a	O
structure	B-evidence
of	O
HAESA	B-protein
in	B-protein_state
complex	I-protein_state
with	I-protein_state
a	O
PKGV	B-mutant
-	I-mutant
IDA	I-mutant
peptide	B-chemical
at	O
1	O
.	O
94	O
Å	O
resolution	O
(	O
Table	O
2	O
).	O

We	O
next	O
tested	O
if	O
HAESA	B-protein
binds	O
other	O
IDA	B-chemical
peptide	I-chemical
family	I-chemical
members	I-chemical
.	O

We	O
do	O
not	O
detect	O
interaction	O
between	O
HAESA	B-protein
and	O
a	O
synthetic	B-protein_state
peptide	B-chemical
missing	B-protein_state
the	I-protein_state
C	I-protein_state
-	I-protein_state
terminal	I-protein_state
Asn69IDA	B-residue_name_number
(	O
ΔN69	B-mutant
),	O
highlighting	O
the	O
importance	O
of	O
the	O
polar	B-bond_interaction
interactions	I-bond_interaction
between	O
the	O
IDA	B-protein
carboxy	O
-	O
terminus	O
and	O
Arg407HAESA	B-residue_name_number
/	O
Arg409HAESA	B-residue_name_number
(	O
Figures	O
1F	O
,	O
2D	O
).	O

We	O
found	O
that	O
the	O
force	O
required	O
to	O
remove	O
the	O
petals	O
of	O
serk1	B-gene
-	I-gene
1	I-gene
mutants	B-protein_state
is	O
significantly	O
higher	O
than	O
that	O
needed	O
for	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
plants	B-taxonomy_domain
,	O
as	O
previously	O
observed	O
for	O
haesa	B-gene
/	O
hsl2	B-gene
mutants	B-protein_state
,	O
and	O
that	O
floral	O
abscission	O
is	O
delayed	O
in	O
serk1	B-gene
-	I-gene
1	I-gene
(	O
Figure	O
3A	O
).	O

The	O
serk2	B-gene
-	I-gene
2	I-gene
,	O
serk3	B-gene
-	I-gene
1	I-gene
,	O
serk4	B-gene
-	I-gene
1	I-gene
and	O
serk5	B-gene
-	I-gene
1	I-gene
mutant	B-protein_state
lines	O
showed	O
a	O
petal	O
break	O
-	O
strength	O
profile	O
not	O
significantly	O
different	O
from	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
plants	B-taxonomy_domain
.	O

In	O
this	O
case	O
,	O
there	O
was	O
no	O
detectable	O
interaction	O
between	O
receptor	O
and	O
co	O
-	O
receptor	O
,	O
while	O
in	O
the	O
presence	B-protein_state
of	I-protein_state
IDA	B-protein
,	O
SERK1	B-protein
strongly	O
binds	O
HAESA	B-protein
with	O
a	O
dissociation	B-evidence
constant	I-evidence
in	O
the	O
mid	O
-	O
nanomolar	O
range	O
(	O
Figure	O
3C	O
).	O

SERK1	B-protein
senses	O
a	O
conserved	B-protein_state
motif	B-structure_element
in	O
IDA	B-chemical
family	I-chemical
peptides	I-chemical

The	O
conformational	O
change	O
in	O
the	O
C	O
-	O
terminal	O
LRRs	B-structure_element
and	O
capping	B-structure_element
domain	I-structure_element
is	O
indicated	O
by	O
an	O
arrow	O
.	O
(	O
C	O
)	O
SERK1	B-protein
forms	O
an	O
integral	O
part	O
of	O
the	O
receptor	O
'	O
s	O
peptide	B-site
binding	I-site
pocket	I-site
.	O

Ribbon	O
diagrams	O
of	O
HAESA	B-protein
(	O
in	O
blue	O
)	O
and	O
SERK1	B-protein
(	O
in	O
orange	O
)	O
are	O
shown	O
with	O
selected	O
interface	B-site
residues	I-site
(	O
in	O
bonds	O
representation	O
).	O

To	O
understand	O
in	O
molecular	O
terms	O
how	O
SERK1	B-protein
contributes	O
to	O
high	O
-	O
affinity	O
IDA	B-protein
recognition	O
,	O
we	O
solved	O
a	O
2	O
.	O
43	O
Å	O
crystal	B-evidence
structure	I-evidence
of	O
the	O
ternary	O
HAESA	B-complex_assembly
–	I-complex_assembly
IDA	I-complex_assembly
–	I-complex_assembly
SERK1	I-complex_assembly
complex	O
(	O
Figure	O
4A	O
,	O
Table	O
2	O
).	O

SERK1	B-protein
LRRs	B-structure_element
1	I-structure_element
–	I-structure_element
5	I-structure_element
and	O
its	O
C	O
-	O
terminal	O
capping	B-structure_element
domain	I-structure_element
form	O
an	O
additional	O
zipper	B-structure_element
-	I-structure_element
like	I-structure_element
interface	B-site
with	O
residues	O
originating	O
from	O
HAESA	B-protein
LRRs	B-structure_element
15	I-structure_element
–	I-structure_element
21	I-structure_element
and	O
from	O
the	O
HAESA	B-protein
C	O
-	O
terminal	O
cap	B-structure_element
(	O
Figure	O
4D	O
).	O

Deletion	B-experimental_method
of	O
the	O
buried	O
Asn69IDA	B-residue_name_number
completely	B-protein_state
inhibits	I-protein_state
receptor	O
–	O
co	O
-	O
receptor	O
complex	O
formation	O
and	O
HSL2	O
activation	O
(	O
Figure	O
5A	O
,	O
B	O
).	O

Comparison	O
of	O
35S	B-gene
::	O
IDA	B-protein
wild	B-protein_state
-	I-protein_state
type	I-protein_state
and	O
mutant	B-protein_state
plants	B-taxonomy_domain
further	O
indicates	O
that	O
mutation	B-experimental_method
of	O
Lys66IDA	B-mutant
/	I-mutant
Arg67IDA	I-mutant
→	I-mutant
Ala	I-mutant
may	O
cause	O
a	O
weak	O
dominant	O
negative	O
effect	O
(	O
Figure	O
5C	O
–	O
E	O
).	O

In	O
contrast	O
to	O
animal	B-taxonomy_domain
LRR	B-protein_type
receptors	I-protein_type
,	O
plant	B-taxonomy_domain
LRR	B-structure_element
-	I-structure_element
RKs	I-structure_element
harbor	O
spiral	B-protein_state
-	I-protein_state
shaped	I-protein_state
ectodomains	B-structure_element
and	O
thus	O
they	O
require	O
shape	B-protein_state
-	I-protein_state
complementary	I-protein_state
co	B-protein_type
-	I-protein_type
receptor	I-protein_type
proteins	I-protein_type
for	O
receptor	O
activation	O
.	O

As	O
serk1	B-gene
-	I-gene
1	I-gene
mutant	B-protein_state
plants	B-taxonomy_domain
show	O
intermediate	O
abscission	O
phenotypes	O
when	O
compared	O
to	O
haesa	B-gene
/	O
hsl2	O
mutants	B-protein_state
,	O
SERK1	B-protein
likely	O
acts	O
redundantly	O
with	O
other	O
SERKs	B-protein_type
in	O
the	O
abscission	O
zone	O
(	O
Figure	O
3A	O
).	O

Our	O
comparative	B-experimental_method
structural	I-experimental_method
and	I-experimental_method
biochemical	I-experimental_method
analysis	I-experimental_method
further	O
suggests	O
that	O
IDLs	B-protein_type
share	O
a	O
common	O
receptor	O
binding	O
mode	O
,	O
but	O
may	O
preferably	O
bind	O
to	O
HAESA	B-protein
,	O
HSL1	B-protein
or	O
HSL2	B-protein
in	O
different	O
plant	B-taxonomy_domain
tissues	O
and	O
organs	O
.	O

Several	O
residues	O
in	O
the	O
SERK1	B-protein
N	O
-	O
terminal	O
capping	B-structure_element
domain	I-structure_element
(	O
Thr59SERK1	B-residue_name_number
,	O
Phe61SERK1	B-residue_name_number
)	O
and	O
the	O
LRR	B-site
inner	I-site
surface	I-site
(	O
Asp75SERK1	B-residue_name_number
,	O
Tyr101SERK1	B-residue_name_number
,	O
SER121SERK1	B-residue_name_number
,	O
Phe145SERK1	B-residue_name_number
)	O
contribute	O
to	O
the	O
formation	O
of	O
both	O
complexes	O
(	O
Figures	O
4C	O
,	O
D	O
,	O
6B	O
).	O

This	O
fact	O
together	O
with	O
the	O
largely	O
overlapping	O
SERK1	B-site
binding	I-site
surfaces	I-site
in	O
HAESA	B-protein
and	O
BRI1	B-protein
allows	O
us	O
to	O
speculate	O
that	O
SERK1	B-protein
may	O
promote	O
high	O
-	O
affinity	O
peptide	B-protein_type
hormone	I-protein_type
and	O
brassinosteroid	O
sensing	O
by	O
simply	O
slowing	O
down	O
dissociation	O
of	O
the	O
ligand	O
from	O
its	O
cognate	O
receptor	O
.	O

Ensemble	O
cryo	B-experimental_method
-	I-experimental_method
EM	I-experimental_method
uncovers	O
inchworm	B-protein_state
-	O
like	O
translocation	O
of	O
a	O
viral	B-taxonomy_domain
IRES	B-site
through	O
the	O
ribosome	B-complex_assembly

This	O
unlocks	O
40S	B-complex_assembly
domains	O
,	O
facilitating	O
head	B-structure_element
swivel	O
and	O
biasing	O
IRES	B-site
translocation	O
via	O
hitherto	O
-	O
elusive	O
intermediates	O
with	O
PKI	B-structure_element
captured	O
between	O
the	O
A	B-site
and	I-site
P	I-site
sites	I-site
.	O

Virus	B-taxonomy_domain
propagation	O
relies	O
on	O
the	O
host	O
translational	O
apparatus	O
.	O

A	O
recent	O
demonstration	O
of	O
bacterial	B-taxonomy_domain
translation	O
initiation	B-protein_state
by	O
an	O
IGR	B-structure_element
IRES	B-site
indicates	O
that	O
the	O
IRESs	B-site
take	O
advantage	O
of	O
conserved	O
structural	O
and	O
dynamic	O
properties	O
of	O
the	O
ribosome	B-complex_assembly
.	O

Nucleotides	O
C1274	B-residue_name_number
,	O
U1191	B-residue_name_number
of	O
the	O
40S	B-complex_assembly
head	B-structure_element
and	O
G904	B-residue_name_number
of	O
the	O
platform	B-structure_element
(	O
corresponding	O
to	O
C1054	B-residue_name_number
,	O
G966	B-residue_name_number
and	O
G693	B-residue_name_number
in	O
E	B-species
.	I-species
coli	I-species
16S	B-chemical
rRNA	I-chemical
)	O
are	O
shown	O
in	O
black	O
to	O
denote	O
the	O
A	B-site
,	I-site
P	I-site
and	I-site
E	I-site
sites	I-site
,	O
respectively	O
.	O

The	O
extents	O
of	O
the	O
40S	B-complex_assembly
subunit	B-structure_element
rotation	O
and	O
head	B-structure_element
swivel	O
relative	O
to	O
their	O
positions	O
in	O
the	O
post	B-protein_state
-	I-protein_state
translocation	I-protein_state
structure	B-evidence
are	O
shown	O
with	O
arrows	O
.	O

In	O
panels	O
(	O
a	O
-	O
e	O
),	O
the	O
maps	B-evidence
are	O
segmented	O
and	O
colored	O
as	O
in	O
Figure	O
1	O
.	O

Cryo	B-experimental_method
-	I-experimental_method
EM	I-experimental_method
structures	B-evidence
of	O
the	O
80S	B-complex_assembly
•	I-complex_assembly
TSV	I-complex_assembly
IRES	I-complex_assembly
bound	B-protein_state
with	I-protein_state
eEF2	B-complex_assembly
•	I-complex_assembly
GDP	I-complex_assembly
•	I-complex_assembly
sordarin	I-complex_assembly
.	O

Unsupervised	B-experimental_method
cryo	I-experimental_method
-	I-experimental_method
EM	I-experimental_method
data	I-experimental_method
classification	I-experimental_method
was	O
combined	O
with	O
the	O
use	O
of	O
three	B-experimental_method
-	I-experimental_method
dimensional	I-experimental_method
and	I-experimental_method
two	I-experimental_method
-	I-experimental_method
dimensional	I-experimental_method
masking	I-experimental_method
around	O
the	O
ribosomal	O
A	B-site
site	I-site
(	O
Figure	O
1	O
—	O
figure	O
supplement	O
2	O
).	O

The	O
views	O
were	O
obtained	O
by	O
structural	B-experimental_method
alignment	I-experimental_method
of	O
the	O
25S	B-chemical
rRNAs	I-chemical
;	O
the	O
sarcin	B-structure_element
-	I-structure_element
ricin	I-structure_element
loop	I-structure_element
(	O
SRL	B-structure_element
)	O
of	O
25S	B-chemical
rRNA	I-chemical
is	O
shown	O
in	O
gray	O
for	O
reference	O
.	O

Conformation	O
of	O
the	O
non	B-protein_state
-	I-protein_state
swiveled	I-protein_state
40S	B-complex_assembly
subunit	B-structure_element
in	O
the	O
S	B-species
.	I-species
cerevisiae	I-species
80S	B-complex_assembly
ribosome	I-complex_assembly
bound	B-protein_state
with	I-protein_state
two	O
tRNAs	B-chemical
is	O
shown	O
for	O
reference	O
(	O
blue	O
).	O

Structure	B-evidence
I	I-evidence
comprises	O
the	O
most	B-protein_state
rotated	I-protein_state
ribosome	B-complex_assembly
conformation	O
(~	O
10	O
°),	O
characteristic	O
of	O
pre	B-protein_state
-	I-protein_state
translocation	I-protein_state
hybrid	B-protein_state
-	I-protein_state
tRNA	I-protein_state
states	O
.	O

Structure	B-evidence
IV	I-evidence
adopts	O
a	O
slightly	B-protein_state
rotated	I-protein_state
conformation	O
(~	O
1	O
°).	O

Thus	O
,	O
intersubunit	O
rotation	O
of	O
~	O
9	O
°	O
from	O
Structure	B-evidence
I	I-evidence
to	I-evidence
V	I-evidence
covers	O
a	O
nearly	O
complete	O
range	O
of	O
relative	O
subunit	B-structure_element
positions	O
,	O
similar	O
to	O
what	O
was	O
reported	O
for	O
tRNA	B-protein_state
-	I-protein_state
bound	I-protein_state
yeast	B-taxonomy_domain
,	O
bacterial	B-taxonomy_domain
and	O
mammalian	B-taxonomy_domain
ribosomes	B-complex_assembly
.	O

40S	B-complex_assembly
head	B-structure_element
swivel	O

As	O
with	O
the	O
intersubunit	O
rotation	O
,	O
the	O
small	O
head	B-structure_element
swivel	O
(~	O
1	O
°)	O
in	O
the	O
non	B-protein_state
-	I-protein_state
rotated	I-protein_state
Structure	B-evidence
V	I-evidence
is	O
closest	O
to	O
that	O
in	O
the	O
80S	B-complex_assembly
•	I-complex_assembly
2tRNA	I-complex_assembly
•	I-complex_assembly
mRNA	I-complex_assembly
post	B-protein_state
-	I-protein_state
translocation	I-protein_state
ribosome	B-complex_assembly
.	O

The	O
head	B-structure_element
samples	O
a	O
mid	B-protein_state
-	I-protein_state
swiveled	I-protein_state
position	O
in	O
Structure	B-evidence
I	I-evidence
(	O
12	O
°),	O
then	O
a	O
highly	B-protein_state
-	I-protein_state
swiveled	I-protein_state
position	O
in	O
Structures	B-evidence
II	I-evidence
and	I-evidence
III	I-evidence
(	O
17	O
°)	O
and	O
a	O
less	B-protein_state
swiveled	I-protein_state
position	O
in	O
Structure	B-evidence
IV	I-evidence
(	O
14	O
°).	O

Superpositions	O
were	O
obtained	O
by	O
structural	B-experimental_method
alignments	I-experimental_method
of	O
the	O
18S	B-chemical
rRNAs	I-chemical
excluding	O
the	O
head	B-structure_element
domains	O
(	O
nt	O
1150	B-residue_range
–	I-residue_range
1620	I-residue_range
).	O

Interactions	O
of	O
the	O
stem	B-structure_element
loops	I-structure_element
4	I-structure_element
and	I-structure_element
5	I-structure_element
of	O
the	O
TSV	B-species
with	O
proteins	O
uS7	B-protein
and	O
eS25	B-protein
.	O

Position	O
and	O
interactions	O
of	O
loop	B-structure_element
3	I-structure_element
(	O
variable	B-structure_element
loop	I-structure_element
region	I-structure_element
)	O
of	O
the	O
PKI	B-structure_element
domain	O
in	O
Structure	B-evidence
V	I-evidence
(	O
this	O
work	O
)	O
resembles	O
those	O
of	O
the	O
anticodon	B-structure_element
stem	I-structure_element
loop	I-structure_element
of	O
the	O
E	B-site
-	I-site
site	I-site
tRNA	B-chemical
(	O
blue	O
)	O
in	O
the	O
80S	B-complex_assembly
•	I-complex_assembly
2tRNA	I-complex_assembly
•	I-complex_assembly
mRNA	I-complex_assembly
complex	O
.	O

Positions	O
of	O
tRNAs	B-chemical
and	O
the	O
TSV	B-species
IRES	B-site
relative	O
to	O
the	O
A	B-structure_element
-	I-structure_element
site	I-structure_element
finger	I-structure_element
(	O
blue	O
,	O
nt	O
1008	B-residue_range
–	I-residue_range
1043	I-residue_range
of	O
25S	B-chemical
rRNA	I-chemical
)	O
and	O
the	O
P	B-site
site	I-site
of	O
the	O
large	B-structure_element
subunit	I-structure_element
,	O
comprising	O
helix	B-structure_element
84	I-structure_element
of	O
25S	B-chemical
rRNA	I-chemical
(	O
nt	O
.	O

Interactions	O
of	O
the	O
TSV	B-species
IRES	B-site
with	O
uL5	B-protein
and	O
eL42	B-protein
.	O

Structures	B-evidence
of	O
80S	B-complex_assembly
•	I-complex_assembly
IRES	I-complex_assembly
complexes	O
in	O
the	O
absence	B-protein_state
of	I-protein_state
eEF2	B-protein
(	O
INIT	B-complex_assembly
;	O
PDB	O
3J6Y	O
,)	O
and	O
in	O
the	O
presence	B-protein_state
of	I-protein_state
eEF2	B-protein
(	O
this	O
work	O
)	O
are	O
shown	O
in	O
the	O
upper	O
row	O
and	O
labeled	O
.	O

6758	B-residue_range
–	I-residue_range
6888	I-residue_range
)	O
binds	O
near	O
the	O
E	B-site
site	I-site
,	O
contacting	O
the	O
ribosome	B-complex_assembly
mostly	O
by	O
means	O
of	O
three	O
protruding	O
structural	O
elements	O
:	O
the	O
L1	B-structure_element
.	I-structure_element
1	I-structure_element
region	I-structure_element
and	O
stem	B-structure_element
loops	I-structure_element
4	I-structure_element
and	I-structure_element
5	I-structure_element
(	O
SL4	B-structure_element
and	O
SL5	B-structure_element
).	O

The	O
minor	B-site
groove	I-site
of	O
SL5	B-structure_element
(	O
at	O
nt	O
6862	B-residue_range
–	I-residue_range
6868	I-residue_range
)	O
contacts	O
the	O
positively	O
charged	O
region	O
of	O
eS25	B-protein
(	O
R49	B-residue_name_number
,	O
R58	B-residue_name_number
and	O
R68	B-residue_name_number
)	O
(	O
Figure	O
3	O
—	O
figure	O
supplement	O
4	O
).	O

Conformations	O
and	O
positions	O
of	O
the	O
IRES	B-site
in	O
the	O
initiation	B-protein_state
state	O
and	O
in	O
Structures	B-evidence
I	I-evidence
-	I-evidence
V	I-evidence
are	O
shown	O
relative	O
to	O
those	O
of	O
the	O
A	B-site
-,	I-site
P	I-site
-	I-site
and	I-site
E	I-site
-	I-site
site	I-site
tRNAs	B-chemical
.	O

In	O
Structure	B-evidence
I	I-evidence
,	O
SL3	B-structure_element
of	O
the	O
PKI	B-structure_element
domain	O
is	O
positioned	O
between	O
the	O
A	B-structure_element
-	I-structure_element
site	I-structure_element
finger	I-structure_element
(	O
nt	O
1008	B-residue_range
–	I-residue_range
1043	I-residue_range
of	O
25S	B-chemical
rRNA	I-chemical
)	O
and	O
the	O
P	B-site
site	I-site
of	O
the	O
60S	B-complex_assembly
subunit	B-structure_element
,	O
comprising	O
helix	B-structure_element
84	I-structure_element
of	O
25S	B-chemical
rRNA	I-chemical
(	O
nt	O
.	O

In	O
Structure	B-evidence
V	I-evidence
,	O
loop	B-structure_element
3	I-structure_element
is	O
bound	B-protein_state
in	I-protein_state
the	O
40S	B-complex_assembly
E	B-site
site	I-site
and	O
the	O
backbone	O
of	O
loop	B-structure_element
3	I-structure_element
near	O
the	O
codon	B-structure_element
-	I-structure_element
like	I-structure_element
part	I-structure_element
of	O
PKI	B-structure_element
(	O
at	O
nt	O
.	O

The	O
interaction	O
of	O
loop	B-structure_element
3	I-structure_element
backbone	O
with	O
uS7	B-protein
resembles	O
that	O
of	O
the	O
anticodon	B-structure_element
-	I-structure_element
stem	I-structure_element
loop	I-structure_element
of	O
E	B-site
-	I-site
site	I-site
tRNA	B-chemical
in	O
the	O
post	B-protein_state
-	I-protein_state
translocation	I-protein_state
80S	B-complex_assembly
•	I-complex_assembly
2tRNA	I-complex_assembly
•	I-complex_assembly
mRNA	I-complex_assembly
structure	B-evidence
(	O
Figure	O
3	O
—	O
figure	O
supplement	O
5	O
).	O

Ordering	O
of	O
loop	B-structure_element
3	I-structure_element
suggests	O
that	O
this	O
flexible	O
region	O
contributes	O
to	O
the	O
stabilization	O
of	O
the	O
PKI	B-structure_element
domain	O
in	O
the	O
post	B-protein_state
-	I-protein_state
translocation	I-protein_state
state	O
.	O

Colors	O
for	O
the	O
ribosome	B-complex_assembly
and	O
eEF2	B-protein
are	O
as	O
in	O
Figure	O
1	O
.	O

Switch	B-structure_element
loop	I-structure_element
I	I-structure_element
(	O
SWI	B-structure_element
)	O
in	O
Structure	B-evidence
I	I-evidence
is	O
in	O
blue	O
;	O
dashed	O
line	O
shows	O
the	O
putative	O
location	O
of	O
unresolved	O
switch	B-structure_element
loop	I-structure_element
I	I-structure_element
in	O
Structure	B-evidence
II	I-evidence
.	O

Elongation	B-protein_type
factor	I-protein_type
eEF2	B-protein
in	O
all	O
five	O
structures	B-evidence
is	O
bound	B-protein_state
with	I-protein_state
GDP	B-chemical
and	O
sordarin	B-chemical
(	O
Figure	O
5	O
).	O

GDP	B-chemical
in	O
our	O
structures	B-evidence
is	O
bound	B-protein_state
in	I-protein_state
the	O
GTPase	B-site
center	I-site
(	O
Figures	O
5d	O
,	O
e	O
and	O
f	O
)	O
and	O
sordarin	B-chemical
is	O
sandwiched	O
between	O
the	O
β	B-structure_element
-	I-structure_element
platforms	I-structure_element
of	O
domains	O
III	B-structure_element
and	O
V	B-structure_element
(	O
Figures	O
5g	O
and	O
h	O
),	O
as	O
in	O
the	O
structure	B-evidence
of	O
free	B-protein_state
eEF2	B-complex_assembly
•	I-complex_assembly
sordarin	I-complex_assembly
complex	O
.	O

From	O
Structure	B-evidence
I	I-evidence
to	I-evidence
V	I-evidence
,	O
eEF2	B-protein
is	O
rigidly	O
attached	O
to	O
the	O
GTPase	B-site
-	I-site
associated	I-site
center	I-site
of	O
the	O
60S	B-complex_assembly
subunit	B-structure_element
.	O

The	O
tips	O
of	O
25S	B-chemical
rRNA	I-chemical
helices	B-structure_element
43	I-structure_element
and	I-structure_element
44	I-structure_element
of	O
the	O
P	B-structure_element
stalk	I-structure_element
(	O
nucleotides	O
G1242	B-residue_name_number
and	O
A1270	B-residue_name_number
,	O
respectively	O
)	O
stack	B-bond_interaction
on	O
V754	B-residue_name_number
and	O
Y744	B-residue_name_number
of	O
domain	O
V	B-structure_element
.	O
An	O
αββ	B-structure_element
motif	I-structure_element
of	O
the	O
eukaryote	B-taxonomy_domain
-	O
specific	O
protein	O
P0	B-protein
(	O
aa	O
126	B-residue_range
–	I-residue_range
154	I-residue_range
)	O
packs	O
in	O
the	O
crevice	O
between	O
the	O
long	B-structure_element
α	I-structure_element
-	I-structure_element
helix	I-structure_element
D	I-structure_element
(	O
aa	O
172	B-residue_range
–	I-residue_range
188	I-residue_range
)	O
of	O
the	O
GTPase	B-structure_element
domain	I-structure_element
and	O
the	O
β	B-structure_element
-	I-structure_element
sheet	I-structure_element
region	I-structure_element
(	O
aa	O
246	B-residue_range
–	I-residue_range
263	I-residue_range
)	O
of	O
the	O
GTPase	B-structure_element
domain	I-structure_element
insert	I-structure_element
(	O
or	O
G	B-structure_element
’	I-structure_element
insert	I-structure_element
)	O
of	O
eEF2	B-protein
(	O
secondary	O
-	O
structure	O
nomenclatures	O
for	O
eEF2	B-protein
and	O
EF	B-protein
-	I-protein
G	I-protein
are	O
the	O
same	O
).	O

While	O
the	O
overall	O
mode	O
of	O
this	O
interaction	O
is	O
similar	O
to	O
that	O
seen	O
in	O
70S	B-complex_assembly
•	I-complex_assembly
EF	I-complex_assembly
-	I-complex_assembly
G	I-complex_assembly
crystal	B-evidence
structures	I-evidence
,	O
there	O
is	O
an	O
important	O
local	O
difference	O
between	O
Structure	B-evidence
I	I-evidence
and	O
Structures	B-evidence
II	I-evidence
-	I-evidence
V	I-evidence
in	O
switch	B-structure_element
loop	I-structure_element
I	I-structure_element
,	O
as	O
discussed	O
below	O
.	O

Structures	B-evidence
I	I-evidence
through	I-evidence
V	I-evidence
are	O
shown	O
.	O

The	O
view	O
was	O
obtained	O
by	O
superpositions	B-experimental_method
of	O
the	O
body	B-structure_element
domains	O
of	O
18S	B-chemical
rRNAs	I-chemical
.	O

At	O
the	O
head	B-structure_element
,	O
C1274	B-residue_name_number
of	O
the	O
18S	B-chemical
rRNA	I-chemical
(	O
C1054	B-residue_name_number
in	O
E	B-species
.	I-species
coli	I-species
)	O
base	O
pairs	O
with	O
the	O
first	O
nucleotide	O
of	O
the	O
ORF	B-structure_element
immediately	O
downstream	O
of	O
PKI	B-structure_element
.	O

Key	O
elements	O
of	O
the	O
decoding	B-site
center	I-site
of	O
the	O
'	O
locked	B-protein_state
'	O
initiation	B-protein_state
structure	B-evidence
,	O
'	O
unlocked	B-protein_state
'	O
Structure	B-evidence
I	I-evidence
,	O
and	O
post	B-protein_state
-	I-protein_state
translocation	I-protein_state
Structure	B-evidence
V	I-evidence
(	O
this	O
work	O
)	O
are	O
shown	O
.	O

The	O
codon	B-structure_element
-	I-structure_element
anticodon	I-structure_element
-	I-structure_element
like	I-structure_element
helix	I-structure_element
of	O
PKI	B-structure_element
is	O
shown	O
in	O
red	O
,	O
the	O
downstream	O
first	O
codon	O
of	O
the	O
ORF	B-structure_element
in	O
magenta	O
.	O

Nucleotides	O
of	O
the	O
18S	B-chemical
rRNA	I-chemical
body	B-structure_element
are	O
in	O
orange	O
and	O
head	B-structure_element
in	O
yellow	O
;	O
25S	B-chemical
rRNA	I-chemical
nucleotide	O
A2256	B-residue_name_number
is	O
blue	O
.	O

In	O
the	O
latter	O
,	O
PKI	B-structure_element
is	O
stabilized	O
by	O
interactions	O
with	O
the	O
universally	B-protein_state
conserved	I-protein_state
decoding	B-site
-	I-site
center	I-site
nucleotides	O
G577	B-residue_name_number
,	O
A1755	B-residue_name_number
and	O
A1756	B-residue_name_number
('	O
body	B-structure_element
A	B-site
site	I-site
'),	O
as	O
in	O
the	O
A	B-site
-	I-site
site	I-site
tRNA	B-protein_state
bound	I-protein_state
complexes	O
.	O

Histidines	B-residue_name_number
583	I-residue_name_number
and	I-residue_name_number
694	I-residue_name_number
interact	O
with	O
the	O
phosphate	O
backbone	O
of	O
the	O
anticodon	B-structure_element
-	I-structure_element
like	I-structure_element
strand	I-structure_element
(	O
at	O
G6907	B-residue_name_number
and	O
C6908	B-residue_name_number
).	O

The	O
N	O
-	O
terminal	O
part	O
of	O
the	O
loop	B-structure_element
(	O
aa	O
50	B-residue_range
–	I-residue_range
60	I-residue_range
)	O
is	O
sandwiched	O
between	O
the	O
tip	O
of	O
helix	B-structure_element
14	I-structure_element
(	O
415CAAA418	B-structure_element
)	O
of	O
the	O
18S	B-chemical
rRNA	I-chemical
of	O
the	O
40S	B-complex_assembly
subunit	B-structure_element
and	O
helix	B-structure_element
A	I-structure_element
(	O
aa	O
32	B-residue_range
–	I-residue_range
42	I-residue_range
)	O
of	O
eEF2	B-protein
(	O
Figure	O
5d	O
).	O

Among	O
the	O
five	O
structures	B-evidence
,	O
the	O
PKI	B-structure_element
domain	O
is	O
least	O
ordered	O
in	O
Structure	B-evidence
III	I-evidence
and	O
lacks	O
density	B-evidence
for	O
SL3	B-structure_element
.	O

Thus	O
,	O
in	O
Structure	B-evidence
III	I-evidence
,	O
PKI	B-structure_element
has	O
translocated	O
along	O
the	O
40S	B-complex_assembly
body	B-structure_element
,	O
but	O
the	O
head	B-structure_element
remains	O
fully	B-protein_state
swiveled	I-protein_state
so	O
that	O
PKI	B-structure_element
is	O
between	O
the	O
head	B-structure_element
A	B-site
and	I-site
P	I-site
sites	I-site
.	O

The	O
position	O
of	O
eEF2	B-protein
is	O
similar	O
to	O
that	O
in	O
Structure	B-evidence
II	I-evidence
.	O

To	O
this	O
end	O
,	O
the	O
head	B-site
-	I-site
interacting	I-site
interface	I-site
of	O
domain	O
IV	B-structure_element
slides	O
along	O
the	O
surface	O
of	O
the	O
head	B-structure_element
by	O
5	O
Å	O
.	O
Helix	B-structure_element
A	I-structure_element
of	O
domain	O
IV	B-structure_element
is	O
positioned	O
next	O
to	O
the	O
backbone	O
of	O
h34	B-structure_element
,	O
with	O
positively	O
charged	O
residues	O
K613	B-residue_name_number
,	O
R617	B-residue_name_number
and	O
R631	B-residue_name_number
rearranged	O
from	O
the	O
backbone	O
of	O
h33	B-structure_element
(	O
Figure	O
6c	O
;	O
see	O
also	O
Figure	O
6	O
—	O
figure	O
supplement	O
1	O
).	O

In	O
the	O
nearly	B-protein_state
non	I-protein_state
-	I-protein_state
rotated	I-protein_state
and	O
non	B-protein_state
-	I-protein_state
swiveled	I-protein_state
ribosome	B-complex_assembly
conformation	O
in	O
Structure	B-evidence
V	I-evidence
closely	O
resembling	O
that	O
of	O
the	O
post	B-protein_state
-	I-protein_state
translocation	I-protein_state
80S	B-complex_assembly
•	I-complex_assembly
2tRNA	I-complex_assembly
•	I-complex_assembly
mRNA	I-complex_assembly
complex	O
,	O
PKI	B-structure_element
is	O
fully	O
accommodated	O
in	O
the	O
P	B-site
site	I-site
.	O

In	O
this	O
position	O
,	O
uS12	B-protein
forms	O
extensive	O
interactions	O
with	O
eEF2	B-protein
domains	O
II	B-structure_element
and	O
III	B-structure_element
.	O

Domain	O
IV	B-structure_element
of	O
eEF2	B-protein
is	O
fully	O
accommodated	O
in	O
the	O
A	B-site
site	I-site
.	O

The	O
first	O
codon	O
of	O
the	O
open	B-structure_element
reading	I-structure_element
frame	I-structure_element
is	O
also	O
positioned	O
in	O
the	O
A	B-site
site	I-site
,	O
with	O
bases	O
exposed	O
toward	O
eEF2	B-protein
(	O
Figure	O
7	O
),	O
resembling	O
the	O
conformations	O
of	O
the	O
A	B-site
-	I-site
site	I-site
codons	O
in	O
EF	B-protein_state
-	I-protein_state
G	I-protein_state
-	I-protein_state
bound	I-protein_state
70S	B-complex_assembly
complexes	O
.	O

Diph699	B-ptm
slightly	O
rearranges	O
,	O
relative	O
to	O
that	O
in	O
Structure	B-evidence
I	I-evidence
(	O
Figure	O
7	O
),	O
and	O
interacts	O
with	O
four	O
out	O
of	O
six	O
codon	O
-	O
anticodon	O
nucleotides	O
.	O

The	O
imidazole	O
moiety	O
stacks	O
on	O
G6907	B-residue_name_number
(	O
corresponding	O
to	O
nt	O
36	O
in	O
the	O
tRNA	B-chemical
anticodon	O
)	O
and	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
with	O
O2	O
’	O
of	O
G6906	B-residue_name_number
(	O
nt	O
35	O
of	O
tRNA	B-chemical
).	O

In	O
scenes	O
1	O
,	O
2	O
and	O
3	O
,	O
nucleotides	O
C1274	B-residue_name_number
,	O
U1191	B-residue_name_number
of	O
the	O
40S	B-complex_assembly
head	B-structure_element
and	O
G904	B-residue_name_number
of	O
the	O
40S	B-site
platform	I-site
are	O
shown	O
in	O
black	O
to	O
denote	O
the	O
A	B-site
,	I-site
P	I-site
and	I-site
E	I-site
sites	I-site
,	O
respectively	O
.	O

Translocation	O
of	O
the	O
TSV	B-species
IRES	B-site
on	O
the	O
40S	B-complex_assembly
subunit	B-structure_element
globally	O
resembles	O
a	O
step	O
of	O
an	O
inchworm	B-protein_state
(	O
Figure	O
4	O
;	O
see	O
also	O
Figure	O
3	O
—	O
figure	O
supplement	O
2	O
).	O

Finally	O
(	O
Structures	B-evidence
IV	I-evidence
to	I-evidence
V	I-evidence
),	O
as	O
the	O
hind	B-structure_element
end	I-structure_element
is	O
accommodated	O
in	O
the	O
P	B-site
site	I-site
,	O
the	O
front	B-structure_element
'	I-structure_element
legs	I-structure_element
'	I-structure_element
advance	O
by	O
departing	O
from	O
their	O
initial	B-site
binding	I-site
sites	I-site
.	O

This	O
converts	O
the	O
IRES	B-site
into	O
an	O
extended	B-protein_state
conformation	O
,	O
rendering	O
the	O
inchworm	B-protein_state
prepared	O
for	O
the	O
next	O
translocation	O
step	O
.	O

Recent	O
studies	O
have	O
shown	O
that	O
in	O
some	O
cases	O
a	O
fraction	O
of	O
IGR	B-structure_element
IRES	B-site
-	O
driven	O
translation	O
results	O
from	O
an	O
alternative	O
reading	O
frame	O
,	O
which	O
is	O
shifted	O
by	O
one	O
nucleotide	O
relative	O
to	O
the	O
normal	O
ORF	B-structure_element
.	O

Specifically	O
,	O
biochemical	B-experimental_method
toe	I-experimental_method
-	I-experimental_method
printing	I-experimental_method
studies	I-experimental_method
in	O
the	O
presence	B-protein_state
of	I-protein_state
eEF2	B-complex_assembly
•	I-complex_assembly
GTP	I-complex_assembly
identified	O
IRES	B-site
in	O
a	O
non	B-protein_state
-	I-protein_state
translocated	I-protein_state
position	O
unless	O
eEF1a	B-complex_assembly
•	I-complex_assembly
aa	I-complex_assembly
-	I-complex_assembly
tRNA	I-complex_assembly
is	O
also	O
present	O
.	O

Thus	O
,	O
the	O
meta	O
-	O
stability	O
of	O
the	O
post	B-protein_state
-	I-protein_state
translocation	I-protein_state
IRES	B-site
is	O
likely	O
due	O
to	O
the	O
absence	B-protein_state
of	I-protein_state
stabilizing	O
structural	O
features	O
present	O
in	O
the	O
2tRNA	B-complex_assembly
•	I-complex_assembly
mRNA	I-complex_assembly
complex	O
.	O

Reverse	O
intersubunit	O
rotation	O
from	O
Structure	B-evidence
I	I-evidence
to	I-evidence
V	I-evidence
shifts	O
the	O
translocation	B-site
tunnel	I-site
(	O
the	O
tunnel	B-site
between	O
the	O
A	B-site
,	I-site
P	I-site
and	I-site
E	I-site
sites	I-site
)	O
toward	O
eEF2	B-protein
,	O
which	O
is	O
rigidly	O
attached	O
to	O
the	O
60S	B-complex_assembly
subunit	B-structure_element
.	O

The	O
head	B-structure_element
swivel	O
allows	O
gradual	O
translocation	O
of	O
PKI	B-structure_element
to	O
the	O
P	B-site
site	I-site
,	O
first	O
with	O
respect	O
to	O
the	O
body	B-structure_element
and	O
then	O
to	O
the	O
head	B-structure_element
.	O

The	O
bulky	O
ADP	B-chemical
-	O
ribosyl	O
moiety	O
at	O
this	O
position	O
would	O
disrupt	O
the	O
interaction	O
,	O
rendering	O
eEF2	B-protein
unable	O
to	O
bind	O
to	O
the	O
A	B-site
site	I-site
and	O
/	O
or	O
stalled	O
on	O
ribosomes	B-complex_assembly
in	O
a	O
non	O
-	O
productive	O
conformation	O
.	O

However	O
,	O
the	O
structural	O
and	O
mechanistic	O
definitions	O
of	O
the	O
locked	B-protein_state
and	O
unlocked	B-protein_state
states	O
have	O
remained	O
unclear	O
,	O
ranging	O
from	O
the	O
globally	O
distinct	O
ribosome	B-complex_assembly
conformations	O
to	O
unknown	O
local	O
rearrangements	O
,	O
e	O
.	O
g	O
.	O
those	O
in	O
the	O
decoding	B-site
center	I-site
.	O

Structure	B-evidence
I	I-evidence
demonstrates	O
that	O
at	O
an	O
early	O
pre	B-protein_state
-	I-protein_state
translocation	I-protein_state
step	O
,	O
the	O
histidine	B-site
-	I-site
diphthamide	I-site
tip	I-site
of	O
eEF2	B-protein
is	O
wedged	O
between	O
A1755	B-residue_name_number
and	O
A1756	B-residue_name_number
and	O
PKI	B-structure_element
.	O

This	O
destabilization	O
allows	O
PKI	B-structure_element
to	O
detach	O
from	O
the	O
body	B-structure_element
A	B-site
site	I-site
upon	O
spontaneous	O
reverse	O
40S	B-complex_assembly
body	B-structure_element
rotation	O
,	O
while	O
maintaining	O
interactions	O
with	O
the	O
head	B-structure_element
A	B-site
site	I-site
.	O

Destabilization	O
of	O
the	O
head	B-protein_state
-	I-protein_state
bound	I-protein_state
PKI	B-structure_element
at	O
the	O
body	B-structure_element
A	B-site
site	I-site
thus	O
allows	O
mobility	O
of	O
the	O
head	B-structure_element
relative	O
to	O
the	O
body	B-structure_element
.	O

Here	O
,	O
switch	B-structure_element
loop	I-structure_element
I	I-structure_element
interacts	O
with	O
helix	B-structure_element
14	I-structure_element
(	O
415CAAA418	B-structure_element
)	O
of	O
the	O
18S	B-chemical
rRNA	I-chemical
.	O

This	O
stabilization	O
renders	O
the	O
GTPase	B-site
center	I-site
to	O
adopt	O
a	O
GTP	B-protein_state
-	I-protein_state
bound	I-protein_state
conformation	O
,	O
similar	O
to	O
those	O
observed	O
in	O
other	O
translational	B-protein_type
GTPases	I-protein_type
in	O
the	O
presence	B-protein_state
of	I-protein_state
GTP	B-chemical
analogs	O
and	O
in	O
the	O
80S	B-complex_assembly
•	I-complex_assembly
eEF2	I-complex_assembly
complex	O
bound	B-protein_state
with	I-protein_state
a	O
transition	O
-	O
state	O
mimic	O
GDP	B-complex_assembly
•	I-complex_assembly
AlF4	I-complex_assembly
–.	I-complex_assembly
The	O
switch	B-structure_element
loop	I-structure_element
contacts	O
the	O
base	O
of	O
A416	B-residue_name_number
(	O
invariable	B-protein_state
A344	B-residue_name_number
in	O
E	B-species
.	I-species
coli	I-species
and	O
A463	B-residue_name_number
in	O
H	B-species
.	I-species
sapiens	I-species
).	O

Mutations	B-experimental_method
of	O
residues	O
flanking	O
A344	B-residue_name_number
in	O
E	B-species
.	I-species
coli	I-species
16S	B-chemical
rRNA	I-chemical
modestly	O
inhibit	O
translation	O
but	O
do	O
not	O
specifically	O
affect	O
EF	B-protein
-	I-protein
G	I-protein
-	O
mediated	O
translocation	O
.	O

However	O
,	O
the	O
effect	O
of	O
A344	B-residue_name_number
mutation	B-experimental_method
on	O
translation	O
was	O
not	O
addressed	O
in	O
that	O
study	O
,	O
leaving	O
the	O
question	O
open	O
whether	O
this	O
residue	O
is	O
critical	O
for	O
eEF2	B-protein
/	O
EF	B-protein
-	I-protein
G	I-protein
function	O
.	O

Based	O
on	O
biochemical	B-experimental_method
experiments	I-experimental_method
,	O
two	O
alternative	O
mechanisms	O
of	O
action	O
were	O
proposed	O
:	O
sordarin	B-chemical
either	O
prevents	O
eEF2	B-protein
departure	O
by	O
inhibiting	O
GTP	B-chemical
hydrolysis	O
or	O
acts	O
after	O
GTP	B-chemical
hydrolysis	O
.	O

The	O
mechanism	O
of	O
stalling	O
is	O
suggested	O
by	O
comparison	O
of	O
pre	B-protein_state
-	I-protein_state
translocation	I-protein_state
and	O
post	B-protein_state
-	I-protein_state
translocation	I-protein_state
structures	B-evidence
in	O
our	O
ensemble	O
.	O

We	O
note	O
that	O
Structure	B-evidence
V	I-evidence
is	O
slightly	O
more	O
rotated	O
than	O
the	O
80S	B-complex_assembly
•	I-complex_assembly
2tRNA	I-complex_assembly
•	I-complex_assembly
mRNA	I-complex_assembly
complex	O
in	O
the	O
absence	B-protein_state
of	I-protein_state
eEF2	B-complex_assembly
•	I-complex_assembly
sordarin	I-complex_assembly
,	O
implying	O
that	O
sordarin	B-chemical
interferes	O
with	O
the	O
final	O
stages	O
of	O
reverse	O
rotation	O
of	O
the	O
post	B-protein_state
-	I-protein_state
translocation	I-protein_state
ribosome	B-complex_assembly
.	O

The	O
structural	O
understanding	O
of	O
ribosome	B-complex_assembly
and	O
tRNA	B-chemical
dynamics	O
has	O
been	O
greatly	O
aided	O
by	O
a	O
wealth	O
of	O
X	B-experimental_method
-	I-experimental_method
ray	I-experimental_method
and	O
cryo	B-experimental_method
-	I-experimental_method
EM	I-experimental_method
structures	B-evidence
(	O
reviewed	O
in	O
).	O

Our	O
study	O
provides	O
new	O
insights	O
into	O
the	O
structural	O
understanding	O
of	O
tRNA	B-chemical
translocation	O
.	O

These	O
interactions	O
are	O
maintained	O
for	O
the	O
classical	B-protein_state
-	O
and	O
hybrid	B-protein_state
-	O
state	O
tRNAs	B-chemical
in	O
the	O
spontaneously	O
sampled	O
non	B-protein_state
-	I-protein_state
rotated	I-protein_state
and	O
rotated	B-protein_state
ribosomes	B-complex_assembly
,	O
respectively	O
.	O

This	O
unlatches	O
the	O
head	B-structure_element
,	O
allowing	O
creation	O
of	O
hitherto	O
elusive	O
intermediate	O
tRNA	B-chemical
positions	O
during	O
spontaneous	O
reverse	O
body	B-structure_element
rotation	O
.	O

For	O
example	O
,	O
Förster	B-experimental_method
resonance	I-experimental_method
energy	I-experimental_method
transfer	I-experimental_method
can	O
provide	O
insight	O
into	O
the	O
macromolecular	O
dynamics	O
of	O
an	O
assembly	O
at	O
the	O
single	O
-	O
molecule	O
level	O
but	O
is	O
limited	O
to	O
specifically	O
labeled	O
locations	O
within	O
the	O
assembly	O
.	O

A	O
unified	O
mechanism	O
for	O
proteolysis	O
and	O
autocatalytic	B-ptm
activation	I-ptm
in	O
the	O
20S	B-complex_assembly
proteasome	I-complex_assembly

Biogenesis	O
of	O
the	O
20S	B-complex_assembly
proteasome	I-complex_assembly
is	O
tightly	O
regulated	O
.	O

This	O
work	O
provides	O
insights	O
into	O
the	O
basic	O
mechanism	O
of	O
proteolysis	O
and	O
propeptide	B-ptm
autolysis	I-ptm
,	O
as	O
well	O
as	O
the	O
evolutionary	O
pressures	O
that	O
drove	O
the	O
proteasome	B-complex_assembly
to	O
become	O
a	O
threonine	B-protein_type
protease	I-protein_type
.	O

This	O
mechanism	O
,	O
however	O
,	O
cannot	O
explain	O
autocatalytic	B-ptm
precursor	I-ptm
processing	I-ptm
because	O
in	O
the	O
immature	B-protein_state
active	B-site
sites	I-site
,	O
Thr1N	B-residue_name_number
is	O
part	O
of	O
the	O
peptide	O
bond	O
with	O
Gly	B-residue_name_number
(-	I-residue_name_number
1	I-residue_name_number
),	I-residue_name_number
the	O
bond	O
that	O
needs	O
to	O
be	O
hydrolysed	O
.	O

Inactivation	O
of	O
proteasome	B-complex_assembly
subunits	B-protein
by	O
T1A	B-mutant
mutations	B-experimental_method

Yeast	B-taxonomy_domain
strains	O
carrying	O
the	O
single	O
mutations	O
β1	B-mutant
-	I-mutant
T1A	I-mutant
or	O
β2	B-mutant
-	I-mutant
T1A	I-mutant
,	O
or	O
both	O
,	O
are	O
viable	O
,	O
even	O
though	O
one	O
or	O
two	O
of	O
the	O
three	O
distinct	O
catalytic	B-protein_state
β	B-protein
subunits	I-protein
are	O
disabled	B-protein_state
and	O
carry	B-protein_state
remnants	I-protein_state
of	I-protein_state
their	O
N	O
-	O
terminal	O
propeptides	B-structure_element
(	O
Table	O
1	O
).	O

Thr	B-residue_name_number
(-	I-residue_name_number
2	I-residue_name_number
)	I-residue_name_number
positions	O
Gly	B-residue_name_number
(-	I-residue_name_number
1	I-residue_name_number
)	I-residue_name_number
O	O
via	O
hydrogen	B-bond_interaction
bonding	I-bond_interaction
(∼	O
2	O
.	O
8	O
Å	O
)	O
in	O
a	O
perfect	O
trajectory	O
for	O
the	O
nucleophilic	O
attack	O
by	O
Thr1Oγ	B-residue_name_number
(	O
Fig	O
.	O
1b	O
and	O
Supplementary	O
Fig	O
.	O
2b	O
).	O

Next	O
,	O
we	O
examined	O
the	O
position	O
of	O
the	O
β5	B-protein
propeptide	B-structure_element
in	O
the	O
β5	B-mutant
-	I-mutant
T1A	I-mutant
-	I-mutant
K81R	I-mutant
mutant	B-protein_state
.	O

Processing	O
of	O
β	O
-	O
subunit	O
precursors	O
requires	O
deprotonation	O
of	O
Thr1OH	B-residue_name_number
;	O
however	O
,	O
the	O
general	O
base	O
initiating	O
autolysis	B-ptm
is	O
unknown	O
.	O

Structural	B-experimental_method
analyses	I-experimental_method
revealed	O
that	O
the	O
propeptides	B-structure_element
of	O
all	O
mutant	B-protein_state
yCPs	B-complex_assembly
shared	O
residual	O
2FO	B-evidence
–	I-evidence
FC	I-evidence
electron	I-evidence
densities	I-evidence
.	O

Although	O
we	O
observed	O
fragments	O
of	O
2FO	B-evidence
–	I-evidence
FC	I-evidence
electron	I-evidence
density	I-evidence
in	O
the	O
β1	B-protein
active	B-site
site	I-site
,	O
the	O
data	O
were	O
not	O
interpretable	O
.	O

Notably	O
,	O
the	O
2FO	B-evidence
–	I-evidence
FC	I-evidence
electron	I-evidence
-	I-evidence
density	I-evidence
map	I-evidence
displays	O
a	O
different	O
orientation	O
for	O
the	O
β2	B-protein
propeptide	B-structure_element
than	O
has	O
been	O
observed	O
for	O
the	O
β2	B-mutant
-	I-mutant
T1A	I-mutant
proteasome	B-complex_assembly
.	O

The	O
active	B-site
site	I-site
of	O
the	O
proteasome	B-complex_assembly

Mutation	B-experimental_method
of	O
β5	B-protein
-	O
Lys33	B-residue_name_number
to	O
Ala	B-residue_name
causes	O
a	O
strongly	O
deleterious	O
phenotype	O
,	O
and	O
previous	O
structural	B-experimental_method
and	I-experimental_method
biochemical	I-experimental_method
analyses	I-experimental_method
confirmed	O
that	O
this	O
is	O
caused	O
by	O
failure	O
of	O
propeptide	B-ptm
cleavage	I-ptm
,	O
and	O
consequently	O
,	O
lack	O
of	O
ChT	O
-	O
L	O
activity	O
(	O
Fig	O
.	O
4a	O
,	O
Supplementary	O
Fig	O
.	O
3b	O
and	O
Table	O
1	O
;	O
for	O
details	O
see	O
Supplementary	O
Note	O
1	O
).	O

The	O
phenotype	O
of	O
the	O
β5	B-mutant
-	I-mutant
K33A	I-mutant
mutant	B-protein_state
was	O
however	O
less	O
pronounced	O
than	O
for	O
the	O
β5	B-mutant
-	I-mutant
T1A	I-mutant
-	I-mutant
K81R	I-mutant
yeast	B-taxonomy_domain
(	O
Fig	O
.	O
4a	O
).	O

Structural	B-experimental_method
comparison	I-experimental_method
of	O
the	O
β5	B-mutant
-	I-mutant
L	I-mutant
(-	I-mutant
49	I-mutant
)	I-mutant
S	I-mutant
-	I-mutant
K33A	I-mutant
and	O
β5	B-mutant
-	I-mutant
T1A	I-mutant
-	I-mutant
K81R	I-mutant
active	B-site
sites	I-site
revealed	O
that	O
mutation	B-experimental_method
of	O
Lys33	B-residue_name_number
to	O
Ala	B-residue_name
creates	O
a	O
cavity	O
that	O
is	O
filled	O
with	O
Thr1	B-residue_name_number
and	O
the	O
remnant	O
propeptide	B-structure_element
.	O

Furthermore	O
,	O
the	O
crystal	B-evidence
structure	I-evidence
of	O
the	O
β5	B-mutant
-	I-mutant
K33A	I-mutant
pp	B-chemical
trans	B-protein_state
mutant	B-protein_state
without	B-protein_state
inhibitor	I-protein_state
revealed	O
that	O
Thr1Oγ	B-residue_name_number
strongly	O
coordinates	B-bond_interaction
a	O
well	O
-	O
defined	O
water	B-chemical
molecule	O
(∼	O
2	O
Å	O
;	O
Fig	O
.	O
3c	O
and	O
Supplementary	O
Fig	O
.	O
5c	O
,	O
d	O
).	O

This	O
water	B-chemical
hydrogen	B-bond_interaction
bonds	I-bond_interaction
also	O
to	O
Arg19O	B-residue_name_number
(∼	O
3	O
.	O
0	O
Å	O
)	O
and	O
Asp17Oδ	B-residue_name_number
(∼	O
3	O
.	O
0	O
Å	O
),	O
and	O
thereby	O
presumably	O
enables	O
residual	O
activity	O
of	O
the	O
mutant	B-protein_state
.	O

Even	O
though	O
the	O
β5	B-mutant
-	I-mutant
D17N	I-mutant
pp	B-chemical
trans	B-protein_state
yCP	B-complex_assembly
crystal	B-evidence
structure	I-evidence
appeared	O
identical	O
to	O
the	O
WT	B-protein_state
yCP	B-complex_assembly
(	O
Supplementary	O
Fig	O
.	O
7b	O
),	O
the	O
co	B-evidence
-	I-evidence
crystal	I-evidence
structure	I-evidence
with	O
the	O
α	B-chemical
′,	I-chemical
β	I-chemical
′	I-chemical
epoxyketone	I-chemical
inhibitor	O
carfilzomib	B-chemical
visualized	O
only	O
partial	O
occupancy	O
of	O
the	O
ligand	O
in	O
the	O
β5	B-protein
active	B-site
site	I-site
(	O
Supplementary	O
Fig	O
.	O
7a	O
).	O

In	O
agreement	O
,	O
an	O
E17A	B-mutant
mutant	B-protein_state
in	O
the	O
proteasomal	O
β	B-protein
-	I-protein
subunit	I-protein
of	O
the	O
archaeon	B-taxonomy_domain
Thermoplasma	B-species
acidophilum	I-species
prevents	O
autolysis	B-ptm
and	O
catalysis	O
.	O

The	O
β5	B-mutant
-	I-mutant
D166N	I-mutant
pp	B-chemical
cis	B-protein_state
yeast	B-taxonomy_domain
mutant	B-protein_state
is	O
significantly	O
impaired	O
in	O
growth	O
and	O
its	O
ChT	O
-	O
L	O
activity	O
is	O
drastically	O
reduced	O
(	O
Supplementary	O
Fig	O
.	O
6a	O
,	O
b	O
and	O
Table	O
1	O
).	O

X	B-evidence
-	I-evidence
ray	I-evidence
data	I-evidence
on	O
the	O
β5	B-mutant
-	I-mutant
D166N	I-mutant
mutant	B-protein_state
indicate	O
that	O
the	O
β5	B-protein
propeptide	B-structure_element
is	O
hydrolysed	O
,	O
but	O
due	O
to	O
reorientation	O
of	O
Ser129OH	B-residue_name_number
,	O
the	O
interaction	O
with	O
Asn166Oδ	B-residue_name_number
is	O
disrupted	O
(	O
Supplementary	O
Fig	O
.	O
8a	O
).	O

His	B-residue_name_number
(-	I-residue_name_number
2	I-residue_name_number
)	I-residue_name_number
occupies	O
the	O
S2	B-site
pocket	I-site
like	O
observed	O
for	O
the	O
β5	B-mutant
-	I-mutant
T1A	I-mutant
-	I-mutant
K81R	I-mutant
mutant	B-protein_state
,	O
but	O
in	O
contrast	O
to	O
the	O
latter	O
,	O
the	O
propeptide	B-structure_element
in	O
the	O
T1C	B-mutant
mutant	B-protein_state
adopts	O
an	O
antiparallel	B-structure_element
β	I-structure_element
-	I-structure_element
sheet	I-structure_element
conformation	O
as	O
known	O
from	O
inhibitors	O
like	O
MG132	B-chemical
(	O
Fig	O
.	O
4c	O
–	O
e	O
and	O
Supplementary	O
Fig	O
.	O
9b	O
).	O

Owing	O
to	O
the	O
unequal	O
positions	O
of	O
the	O
two	O
β5	B-protein
subunits	O
within	O
the	O
CP	B-complex_assembly
in	O
the	O
crystal	O
lattice	O
,	O
maturation	O
and	O
propeptide	B-structure_element
displacement	O
may	O
occur	O
at	O
different	O
timescales	O
in	O
the	O
two	O
subunits	O
.	O

Notably	O
,	O
the	O
2FO	B-evidence
–	I-evidence
FC	I-evidence
electron	I-evidence
-	I-evidence
density	I-evidence
map	I-evidence
of	O
the	O
T1C	B-mutant
mutant	B-protein_state
also	O
indicates	O
that	O
Lys33NH2	B-residue_name_number
is	O
disordered	B-protein_state
.	O

However	O
,	O
the	O
apo	B-protein_state
crystal	B-evidence
structure	I-evidence
revealed	O
that	O
Ser1Oγ	B-residue_name_number
is	O
turned	O
away	O
from	O
the	O
substrate	B-site
-	I-site
binding	I-site
channel	I-site
(	O
Fig	O
.	O
4g	O
).	O

The	O
20S	B-complex_assembly
proteasome	I-complex_assembly
CP	B-complex_assembly
is	O
the	O
major	O
non	B-protein_type
-	I-protein_type
lysosomal	I-protein_type
protease	I-protein_type
in	O
eukaryotic	B-taxonomy_domain
cells	O
,	O
and	O
its	O
assembly	O
is	O
highly	O
organized	O
.	O

By	O
contrast	O
,	O
the	O
prosegments	B-structure_element
of	O
β	B-protein
subunits	I-protein
are	O
dispensable	O
for	O
archaeal	B-taxonomy_domain
proteasome	B-complex_assembly
assembly	O
,	O
at	O
least	O
when	O
heterologously	B-experimental_method
expressed	I-experimental_method
in	O
Escherichia	B-species
coli	I-species
.	O

Here	O
we	O
have	O
described	O
the	O
atomic	B-evidence
structures	I-evidence
of	O
various	O
β5	B-mutant
-	I-mutant
T1A	I-mutant
mutants	O
,	O
which	O
allowed	O
for	O
the	O
first	O
time	O
visualization	O
of	O
the	O
residual	O
β5	B-protein
propeptide	B-structure_element
.	O

From	O
these	O
data	O
we	O
conclude	O
that	O
only	O
the	O
positioning	O
of	O
Gly	B-residue_name_number
(-	I-residue_name_number
1	I-residue_name_number
)	I-residue_name_number
and	O
Thr1	B-residue_name_number
as	O
well	O
as	O
the	O
integrity	O
of	O
the	O
proteasomal	O
active	B-site
site	I-site
are	O
required	O
for	O
autolysis	B-ptm
.	O

The	O
propeptide	B-structure_element
needs	O
some	O
anchoring	O
in	O
the	O
substrate	B-site
-	I-site
binding	I-site
channel	I-site
to	O
properly	O
position	O
Gly	B-residue_name_number
(-	I-residue_name_number
1	I-residue_name_number
),	I-residue_name_number
but	O
this	O
seems	O
to	O
be	O
independent	O
of	O
the	O
orientation	O
of	O
residue	O
(-	B-residue_number
2	I-residue_number
).	I-residue_number

Lys33NH2	B-residue_name_number
is	O
expected	O
to	O
act	O
as	O
the	O
proton	O
acceptor	O
during	O
autocatalytic	B-ptm
removal	I-ptm
of	O
the	O
propeptides	B-structure_element
,	O
as	O
well	O
as	O
during	O
substrate	O
proteolysis	O
,	O
while	O
Asp17Oδ	B-residue_name_number
orients	O
Lys33NH2	B-residue_name_number
and	O
makes	O
it	O
more	O
prone	O
to	O
protonation	O
by	O
raising	O
its	O
pKa	O
(	O
hydrogen	B-bond_interaction
bond	I-bond_interaction
distance	O
:	O
Lys33NH3	B-residue_name_number
+–	O
Asp17Oδ	B-residue_name_number
:	O
2	O
.	O
9	O
Å	O
).	O

Furthermore	O
,	O
opening	O
of	O
the	O
β	O
-	O
lactone	O
compound	O
omuralide	B-chemical
by	O
Thr1	B-residue_name_number
creates	O
a	O
C3	O
-	O
hydroxyl	O
group	O
,	O
whose	O
proton	O
originates	O
from	O
Thr1NH3	B-residue_name_number
+.	O

By	O
acting	O
as	O
a	O
proton	O
donor	O
during	O
catalysis	O
,	O
the	O
Thr1	B-residue_name_number
N	O
terminus	O
may	O
also	O
favour	O
cleavage	O
of	O
substrate	O
peptide	O
bonds	O
(	O
Fig	O
.	O
3d	O
).	O

During	O
autolysis	B-ptm
the	O
Thr1	B-residue_name_number
N	O
terminus	O
is	O
engaged	O
in	O
a	O
hydroxyoxazolidine	O
ring	O
intermediate	O
(	O
Fig	O
.	O
3d	O
),	O
which	O
is	O
unstable	O
and	O
short	O
-	O
lived	O
.	O

The	O
mutation	B-experimental_method
D166N	B-mutant
lowers	O
the	O
pKa	O
of	O
Thr1N	B-residue_name_number
,	O
which	O
is	O
thus	O
more	O
likely	O
to	O
exist	O
in	O
the	O
uncharged	O
deprotonated	O
state	O
(	O
Thr1NH2	B-residue_name_number
).	O

We	O
also	O
observed	O
slightly	O
lower	O
affinity	O
of	O
the	O
β5	B-mutant
-	I-mutant
T1S	I-mutant
mutant	B-protein_state
yCP	B-complex_assembly
for	O
the	O
Food	O
and	O
Drug	O
Administration	O
-	O
approved	O
proteasome	B-complex_assembly
inhibitors	O
bortezomib	B-chemical
and	O
carfilzomib	B-chemical
.	O

Thr1	B-residue_name_number
is	O
well	O
anchored	O
in	O
the	O
active	B-site
site	I-site
by	O
hydrophobic	B-bond_interaction
interactions	I-bond_interaction
of	O
its	O
Cγ	O
methyl	O
group	O
with	O
Ala46	B-residue_name_number
(	O
Cβ	O
),	O
Lys33	B-residue_name_number
(	O
carbon	O
side	O
chain	O
)	O
and	O
Thr3	B-residue_name_number
(	O
Cγ	O
).	O

In	O
contrast	O
to	O
Thr1	B-residue_name_number
,	O
the	O
hydroxyl	O
group	O
of	O
Ser1	B-residue_name_number
occupies	O
the	O
position	O
of	O
the	O
Thr1	B-residue_name_number
methyl	O
side	O
chain	O
in	O
the	O
WT	B-protein_state
enzyme	B-complex_assembly
,	O
which	O
requires	O
its	O
reorientation	O
relative	O
to	O
the	O
substrate	O
to	O
allow	O
cleavage	O
(	O
Fig	O
.	O
4g	O
,	O
h	O
).	O

Notably	O
,	O
in	O
the	O
threonine	B-protein_type
aspartase	I-protein_type
Taspase1	B-protein
,	O
mutation	B-experimental_method
of	O
the	O
active	B-site
-	I-site
site	I-site
Thr234	B-residue_name_number
to	O
Ser	B-residue_name
also	O
places	O
the	O
side	O
chain	O
in	O
the	O
position	O
of	O
the	O
methyl	O
group	O
of	O
Thr234	B-residue_name_number
in	O
the	O
WT	B-protein_state
,	O
thereby	O
reducing	O
catalytic	O
activity	O
.	O

Only	O
the	O
residues	O
(-	B-residue_range
5	I-residue_range
)	I-residue_range
to	I-residue_range
(-	I-residue_range
1	I-residue_range
)	I-residue_range
of	O
the	O
β1	B-mutant
-	I-mutant
T1A	I-mutant
propeptide	B-structure_element
are	O
displayed	O
.	O

Thr	B-residue_name_number
(-	I-residue_name_number
2	I-residue_name_number
)	I-residue_name_number
OH	O
is	O
hydrogen	B-bond_interaction
-	I-bond_interaction
bonded	I-bond_interaction
to	O
Gly	B-residue_name_number
(-	I-residue_name_number
1	I-residue_name_number
)	I-residue_name_number
O	O
(∼	O
2	O
.	O
8	O
Å	O
;	O
black	O
dashed	O
line	O
).	O

Mutations	B-experimental_method
of	O
residue	O
(-	B-residue_number
2	I-residue_number
)	I-residue_number
and	O
their	O
influence	O
on	O
propeptide	B-structure_element
conformation	O
and	O
autolysis	B-ptm
.	O

(	O
a	O
)	O
Hydrogen	B-site
-	I-site
bonding	I-site
network	I-site
at	O
the	O
mature	B-protein_state
WT	B-protein_state
β5	B-protein
proteasomal	O
active	B-site
site	I-site
(	O
dotted	O
lines	O
).	O

(	O
c	O
)	O
Structural	B-experimental_method
superposition	I-experimental_method
of	O
the	O
WT	B-protein_state
β5	B-protein
and	O
the	O
β5	B-mutant
-	I-mutant
K33A	I-mutant
pp	B-chemical
trans	B-protein_state
mutant	B-protein_state
active	B-site
site	I-site
.	O

Note	O
,	O
the	O
strong	O
interaction	O
with	O
the	O
water	B-chemical
molecule	O
causes	O
a	O
minor	O
shift	O
of	O
Thr1	B-residue_name_number
,	O
while	O
all	O
other	O
active	B-site
-	I-site
site	I-site
residues	I-site
remain	O
in	O
place	O
.	O

Notably	O
,	O
His	B-residue_name_number
(-	I-residue_name_number
2	I-residue_name_number
)	I-residue_name_number
does	O
not	O
occupy	O
the	O
S1	B-site
pocket	I-site
formed	O
by	O
Met45	B-residue_name_number
,	O
similar	O
to	O
what	O
was	O
observed	O
for	O
the	O
β5	B-mutant
-	I-mutant
T1A	I-mutant
-	I-mutant
K81R	I-mutant
mutant	B-protein_state
.	O

(	O
f	O
)	O
Structural	B-experimental_method
superposition	I-experimental_method
of	O
the	O
WT	B-protein_state
β5	B-protein
and	O
β5	B-mutant
-	I-mutant
T1C	I-mutant
mutant	B-protein_state
active	B-site
sites	I-site
illustrates	O
the	O
different	O
orientations	O
of	O
the	O
hydroxyl	O
group	O
of	O
Thr1	B-residue_name_number
and	O
the	O
thiol	O
side	O
chain	O
of	O
Cys1	B-residue_name_number
.	O

(	O
g	O
)	O
Structural	B-experimental_method
superposition	I-experimental_method
of	O
the	O
WT	B-protein_state
β5	B-protein
and	O
β5	B-mutant
-	I-mutant
T1S	I-mutant
mutant	B-protein_state
active	B-site
sites	I-site
reveals	O
different	O
orientations	O
of	O
the	O
hydroxyl	O
groups	O
of	O
Thr1	B-residue_name_number
and	O
Ser1	B-residue_name_number
,	O
respectively	O
.	O

A	O
recent	O
survey	O
of	O
bromodomains	B-structure_element
(	O
BDs	B-structure_element
)	O
demonstrates	O
that	O
only	O
one	O
BD	B-structure_element
associates	O
very	O
weakly	O
with	O
a	O
crotonylated	B-protein_state
peptide	O
,	O
however	O
it	O
binds	O
more	O
tightly	O
to	O
acetylated	B-protein_state
peptides	O
,	O
inferring	O
that	O
bromodomains	B-structure_element
do	O
not	O
possess	O
physiologically	O
relevant	O
crotonyllysine	B-residue_name
binding	O
activity	O
.	O

To	O
elucidate	O
the	O
molecular	O
basis	O
for	O
recognition	O
of	O
the	O
H3K9cr	B-protein_type
mark	O
,	O
we	O
obtained	O
a	O
crystal	B-evidence
structure	I-evidence
of	O
the	O
Taf14	B-protein
YEATS	B-structure_element
domain	I-structure_element
in	B-protein_state
complex	I-protein_state
with	I-protein_state
H3K9cr5	B-chemical
-	I-chemical
13	I-chemical
(	O
residues	O
5	B-residue_range
–	I-residue_range
13	I-residue_range
of	O
H3	B-protein_type
)	O
peptide	O
(	O
Fig	O
.	O
1	O
,	O
Supplementary	O
Results	O
,	O
Supplementary	O
Fig	O
.	O
1	O
and	O
Supplementary	O
Table	O
1	O
).	O

The	O
H3K9cr	B-protein_type
peptide	O
lays	O
in	O
an	O
extended	B-protein_state
conformation	I-protein_state
in	O
an	O
orientation	O
orthogonal	O
to	O
the	O
β	B-structure_element
strands	I-structure_element
and	O
is	O
stabilized	O
through	O
an	O
extensive	O
network	O
of	O
direct	O
and	O
water	B-chemical
-	O
mediated	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
and	O
a	O
salt	B-bond_interaction
bridge	I-bond_interaction
(	O
Fig	O
.	O
1c	O
).	O

The	O
hydroxyl	O
group	O
of	O
Thr61	B-residue_name_number
also	O
participates	O
in	O
a	O
hydrogen	B-bond_interaction
bond	I-bond_interaction
with	O
the	O
amide	O
nitrogen	O
of	O
the	O
K9cr	B-ptm
side	O
chain	O
(	O
Fig	O
.	O
1d	O
).	O

To	O
determine	O
whether	O
H3K9cr	B-protein_type
is	O
present	O
in	O
yeast	B-taxonomy_domain
,	O
we	O
generated	O
whole	B-experimental_method
cell	I-experimental_method
extracts	I-experimental_method
from	O
logarithmically	O
growing	O
yeast	B-taxonomy_domain
cells	O
and	O
subjected	O
them	O
to	O
Western	B-experimental_method
blot	I-experimental_method
analysis	I-experimental_method
using	O
antibodies	O
directed	O
towards	O
H3K9cr	B-protein_type
,	O
H3K9ac	B-protein_type
and	O
H3	B-protein_type
(	O
Fig	O
.	O
2a	O
,	O
b	O
,	O
Supplementary	O
Fig	O
.	O
3	O
and	O
Supplementary	O
Table	O
2	O
).	O

We	O
next	O
asked	O
if	O
H3K9cr	B-protein_type
is	O
regulated	O
by	O
the	O
actions	O
of	O
histone	B-protein_type
acetyltransferases	I-protein_type
(	O
HATs	B-protein_type
)	O
and	O
histone	B-protein_type
deacetylases	I-protein_type
(	O
HDACs	B-protein_type
).	O

Unlike	O
the	O
YEATS	B-structure_element
domain	I-structure_element
,	O
a	O
known	O
acetyllysine	B-protein_type
reader	I-protein_type
,	O
bromodomain	B-structure_element
,	O
does	O
not	O
recognize	O
crotonyllysine	B-residue_name
.	O

The	O
unique	O
and	O
previously	O
unobserved	O
aromatic	O
-	O
amide	O
/	O
aliphatic	O
-	O
aromatic	O
π	B-bond_interaction
-	I-bond_interaction
π	I-bond_interaction
-	I-bond_interaction
π	I-bond_interaction
-	I-bond_interaction
stacking	I-bond_interaction
mechanism	O
facilitates	O
the	O
specific	O
recognition	O
of	O
the	O
crotonyl	B-chemical
moiety	O
.	O

(	O
d	O
)	O
The	O
π	B-bond_interaction
-	I-bond_interaction
π	I-bond_interaction
-	I-bond_interaction
π	I-bond_interaction
stacking	I-bond_interaction
mechanism	O
involving	O
the	O
alkene	O
moiety	O
of	O
crotonyllysine	B-residue_name
.	O

H3K9cr	B-protein_type
is	O
a	O
selective	O
target	O
of	O
the	O
Taf14	B-protein
YEATS	B-structure_element
domain	I-structure_element

(	O
a	O
,	O
b	O
)	O
Western	B-experimental_method
blot	I-experimental_method
analysis	O
comparing	O
the	O
levels	O
of	O
H3K9cr	B-protein_type
and	O
H3K9ac	B-protein_type
in	O
wild	B-protein_state
type	I-protein_state
(	O
WT	B-protein_state
),	O
HAT	B-protein_type
deletion	O
,	O
or	O
HDAC	B-protein_type
deletion	B-experimental_method
yeast	B-taxonomy_domain
strains	O
.	O

Structure	B-evidence
of	O
the	O
GAT	B-structure_element
domain	O
of	O
the	O
endosomal	O
adapter	B-protein_type
protein	I-protein_type
Tom1	B-protein

Analyzed	O
by	O
CS	B-experimental_method
-	I-experimental_method
Rosetta	I-experimental_method
,	O
Protein	B-experimental_method
Structure	I-experimental_method
Validation	I-experimental_method
Server	I-experimental_method
(	O
PSVS	B-experimental_method
),	O
NMRPipe	B-experimental_method
,	O
NMRDraw	B-experimental_method
,	O
and	O
PyMol	O
Experimental	O
factors	O
Recombinant	O
human	B-species
Tom1	B-protein
GAT	B-structure_element
domain	O
was	O
purified	O
to	O
homogeneity	O
before	O
use	O
Experimental	O
features	O
Solution	B-evidence
structure	I-evidence
of	O
Tom1	B-protein
GAT	B-structure_element
was	O
determined	O
from	O
NMR	B-experimental_method
chemical	B-evidence
shift	I-evidence
data	O
Data	O
source	O
location	O
Virginia	O
and	O
Colorado	O
,	O
United	O
States	O
.	O

The	O
Tom1	B-protein
GAT	B-structure_element
structural	B-evidence
restraints	I-evidence
yielded	O
ten	O
helical	O
structures	B-evidence
(	O
Fig	O
.	O
2A	O
,	O
B	O
)	O
with	O
a	O
root	B-evidence
mean	I-evidence
square	I-evidence
deviation	I-evidence
(	O
RMSD	B-evidence
)	O
of	O
0	O
.	O
9	O
Å	O
for	O
backbone	O
and	O
1	O
.	O
3	O
Å	O
for	O
all	O
heavy	O
atoms	O
(	O
Table	O
1	O
)	O
and	O
estimated	O
the	O
presence	O
of	O
three	O
helices	O
spanning	O
residues	O
Q216	B-residue_range
-	I-residue_range
E240	I-residue_range
(	O
α	B-structure_element
-	I-structure_element
helix	I-structure_element
1	I-structure_element
),	O
P248	B-residue_range
-	I-residue_range
Q274	I-residue_range
(	O
α	B-structure_element
-	I-structure_element
helix	I-structure_element
2	I-structure_element
),	O
and	O
E278	B-residue_range
-	I-residue_range
T306	I-residue_range
(	O
α	B-structure_element
-	I-structure_element
helix	I-structure_element
3	I-structure_element
).	O

Unlike	O
ubiquitin	B-chemical
binding	O
,	O
data	O
suggest	O
that	O
conformational	O
changes	O
of	O
the	O
Tom1	B-protein
GAT	B-structure_element
α	B-structure_element
-	I-structure_element
helices	I-structure_element
1	I-structure_element
and	I-structure_element
2	I-structure_element
occur	O
upon	O
Tollip	B-protein
TBD	B-structure_element
binding	O
(	O
Fig	O
.	O
3A	O
,	O
B	O
).	O

NMR	B-experimental_method
structural	B-evidence
statistics	I-evidence
for	O
lowest	O
energy	O
conformers	O
of	O
Tom1	B-protein
GAT	B-structure_element
using	O
PSVS	B-experimental_method
.	O

The	O
dimer	B-oligomeric_state
is	O
dissociated	O
to	O
monomers	B-oligomeric_state
by	O
physiological	O
levels	O
of	O
CO	B-chemical
,	O
suggesting	O
that	O
PGRMC1	B-protein
serves	O
as	O
a	O
CO	B-chemical
-	O
sensitive	O
molecular	O
switch	O
regulating	O
cancer	O
cell	O
proliferation	O
.	O

While	O
the	O
overall	O
fold	O
of	O
PGRMC1	B-protein
is	O
similar	O
to	O
that	O
of	O
canonical	O
cytochrome	B-protein_type
b5	I-protein_type
,	O
their	O
haem	B-chemical
irons	O
are	O
coordinated	O
differently	O
.	O

Our	O
structural	B-evidence
data	I-evidence
revealed	O
that	O
Tyr164	B-residue_name_number
and	O
a	O
few	O
other	O
residues	O
such	O
as	O
Tyr107	B-residue_name_number
and	O
Lys163	B-residue_name_number
are	O
in	O
fact	O
hydrogen	B-bond_interaction
-	I-bond_interaction
bonded	I-bond_interaction
to	O
haem	B-chemical
propionates	O
.	O

When	O
chemical	B-evidence
shifts	I-evidence
of	O
apo	B-protein_state
-	O
and	O
haem	B-protein_state
-	I-protein_state
bound	I-protein_state
forms	O
of	O
PGMRC1	B-protein
were	O
compared	O
,	O
some	O
amino	O
acid	O
residues	O
close	O
to	O
those	O
which	O
disappeared	O
because	O
of	O
the	O
paramagnetic	O
relaxation	O
effect	O
of	O
haem	B-chemical
exhibit	O
notable	O
chemical	O
shifts	O
(	O
Supplementary	O
Fig	O
.	O
6a	O
,	O
b	O
;	O
dark	O
yellow	O
).	O

Similarly	O
,	O
the	O
C129S	B-mutant
mutant	B-protein_state
of	O
PGRMC1	B-protein
converted	O
from	O
monomer	B-oligomeric_state
to	O
dimer	B-oligomeric_state
by	O
binding	O
to	O
haem	B-chemical
(	O
Fig	O
.	O
2b	O
).	O

SV	B-experimental_method
-	I-experimental_method
AUC	I-experimental_method
analyses	O
also	O
allowed	O
us	O
to	O
examine	O
the	O
stability	O
of	O
haem	B-chemical
/	O
PGRMC1	B-protein
dimer	B-oligomeric_state
.	O

To	O
this	O
end	O
,	O
we	O
used	O
different	O
concentrations	O
(	O
3	O
.	O
5	O
–	O
147	O
μmol	O
l	O
−	O
1	O
)	O
of	O
haem	B-protein_state
-	I-protein_state
bound	I-protein_state
PGRMC1	B-protein
protein	O
(	O
a	O
.	O
a	O
.	O
72	B-residue_range
–	I-residue_range
195	I-residue_range
),	O
which	O
were	O
identical	O
to	O
that	O
used	O
in	O
the	O
crystallographic	B-experimental_method
analysis	I-experimental_method
.	O

We	O
also	O
showed	O
by	O
haem	B-experimental_method
titration	I-experimental_method
experiments	I-experimental_method
that	O
haem	B-chemical
binding	O
to	O
PGRMC1	B-protein
was	O
of	O
low	O
affinity	O
with	O
a	O
Kd	B-evidence
value	O
of	O
50	O
nmol	O
l	O
−	O
1	O
;	O
this	O
is	O
comparable	O
with	O
that	O
of	O
iron	B-protein
regulatory	I-protein
protein	I-protein
2	I-protein
,	O
which	O
is	O
known	O
to	O
be	O
regulated	O
by	O
intracellular	O
levels	O
of	O
haem	B-chemical
(	O
Fig	O
.	O
2c	O
and	O
Supplementary	O
Table	O
1	O
).	O

Under	O
these	O
circumstances	O
,	O
CO	B-chemical
application	O
induced	O
dissociation	O
of	O
the	O
haem	B-chemical
-	O
mediated	O
dimers	B-oligomeric_state
of	O
PGRMC1	B-protein
to	O
generate	O
a	O
peak	O
of	O
monomers	B-oligomeric_state
(	O
Supplementary	O
Fig	O
.	O
15	O
,	O
lower	O
panel	O
).	O

This	O
interaction	O
was	O
disrupted	O
by	O
the	O
ruthenium	B-chemical
-	O
based	O
CO	B-chemical
-	O
releasing	O
molecule	O
,	O
CORM3	B-chemical
,	O
but	O
not	O
by	O
RuCl3	B-chemical
as	O
a	O
control	O
reagent	O
(	O
Fig	O
.	O
4b	O
).	O

To	O
further	O
investigate	O
the	O
role	O
of	O
the	O
dimerized	B-protein_state
form	O
of	O
PGRMC1	B-protein
in	O
cancer	O
proliferation	O
,	O
we	O
performed	O
PGRMC1	B-protein
knockdown	B-experimental_method
-	I-experimental_method
rescue	I-experimental_method
experiments	I-experimental_method
using	O
FLAG	B-protein_state
-	I-protein_state
tagged	I-protein_state
wild	B-protein_state
-	I-protein_state
type	I-protein_state
and	O
Y113F	B-mutant
PGRMC1	B-protein
expression	B-experimental_method
vectors	I-experimental_method
,	O
in	O
which	O
silent	B-experimental_method
mutations	I-experimental_method
were	O
introduced	B-experimental_method
into	O
the	O
nucleotide	O
sequence	O
targeted	O
by	O
shRNA	B-chemical
(	O
Fig	O
.	O
5a	O
).	O

While	O
proliferation	O
of	O
HCT116	O
cells	O
was	O
not	O
affected	O
by	O
knocking	B-experimental_method
down	I-experimental_method
PGRMC1	B-protein
,	O
PGRMC1	B-mutant
-	I-mutant
KD	I-mutant
cells	O
were	O
more	O
sensitive	O
to	O
the	O
EGFR	B-protein_type
inhibitor	O
erlotinib	B-chemical
than	O
control	O
HCT116	O
cells	O
,	O
and	O
the	O
knockdown	O
effect	O
was	O
reversed	O
by	O
co	B-experimental_method
-	I-experimental_method
expression	I-experimental_method
of	O
shRNA	B-protein_state
-	I-protein_state
resistant	I-protein_state
wild	B-protein_state
-	I-protein_state
type	I-protein_state
PGRMC1	B-protein
but	O
not	O
of	O
the	O
Y113F	B-mutant
mutant	B-protein_state
(	O
Fig	O
.	O
5b	O
).	O

Interaction	O
of	O
PGRMC1	B-protein
dimer	B-oligomeric_state
with	O
cytochromes	B-protein_type
P450	I-protein_type

We	O
showed	O
that	O
the	O
haem	B-chemical
-	O
mediated	O
dimer	B-oligomeric_state
of	O
PGRMC1	B-protein
enables	O
interaction	O
with	O
different	O
subclasses	O
of	O
cytochromes	B-protein_type
P450	I-protein_type
(	O
CYP	B-protein_type
)	O
(	O
Fig	O
.	O
6	O
).	O

Thus	O
,	O
HO	B-protein
-	I-protein
1	I-protein
induction	O
in	O
cancer	O
cells	O
may	O
inhibit	O
the	O
haem	B-chemical
-	O
mediated	O
dimerization	B-oligomeric_state
of	O
PGRMC1	B-protein
through	O
the	O
production	O
of	O
CO	B-chemical
and	O
thereby	O
suppress	O
tumour	O
progression	O
.	O

(	O
b	O
)	O
Haem	B-chemical
coordination	B-bond_interaction
of	O
PGRMC1	B-protein
with	O
Tyr113	B-residue_name_number
.	O

(	O
a	O
)	O
UV	B-evidence
-	I-evidence
visible	I-evidence
absorption	I-evidence
spectra	I-evidence
of	O
PGRMC1	B-protein
(	O
a	O
.	O
a	O
.	O
44	B-residue_range
–	I-residue_range
195	I-residue_range
).	O

Measurements	O
were	O
performed	O
in	O
the	O
presence	B-protein_state
of	I-protein_state
the	O
oxidized	B-protein_state
form	O
of	O
haem	B-chemical
(	O
ferric	B-protein_state
),	O
the	O
reduced	B-protein_state
form	O
of	O
haem	B-chemical
(	O
ferrous	B-protein_state
)	O
and	O
the	O
reduced	B-protein_state
form	O
of	O
haem	B-chemical
plus	O
CO	B-chemical
gas	O
(	O
ferrous	B-protein_state
+	O
CO	B-chemical
).	O

(	O
c	O
)	O
Gel	B-experimental_method
-	I-experimental_method
filtration	I-experimental_method
chromatography	I-experimental_method
analyses	O
of	O
PGRMC1	B-protein
(	O
a	O
.	O
a	O
.	O
44	B-residue_range
–	I-residue_range
195	I-residue_range
)	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
(	O
wt	B-protein_state
)	O
and	O
the	O
Y113F	B-mutant
or	O
C129S	B-mutant
mutant	B-protein_state
in	O
the	O
presence	B-protein_state
or	O
absence	B-protein_state
of	I-protein_state
haem	B-chemical
,	O
dithionite	B-chemical
and	O
/	O
or	O
CO	B-chemical
.	O
(	O
d	O
)	O
Transition	O
model	O
for	O
structural	O
regulation	O
of	O
PGRMC1	B-protein
in	O
response	O
to	O
haem	B-chemical
and	O
CO	B-chemical
.	O

Input	O
and	O
bound	O
proteins	O
were	O
detected	O
by	O
Western	B-experimental_method
blotting	I-experimental_method
.	O

(	O
b	O
)	O
In	B-experimental_method
vitro	I-experimental_method
binding	I-experimental_method
assay	I-experimental_method
was	O
performed	O
as	O
in	O
(	O
a	O
)	O
using	O
haem	B-protein_state
-	I-protein_state
bound	I-protein_state
FLAG	O
-	O
PGRMC1	B-protein
wt	B-protein_state
(	O
a	O
.	O
a	O
.	O
44	B-residue_range
–	I-residue_range
195	I-residue_range
)	O
and	O
purified	O
EGFR	B-protein_type
with	O
or	O
without	O
treatment	O
of	O
RuCl3	B-chemical
and	O
CORM3	B-chemical
.	O

Co	B-experimental_method
-	I-experimental_method
immunoprecipitated	I-experimental_method
proteins	O
(	O
FLAG	O
-	O
PGRMC1	B-protein
,	O
endogenous	B-protein_state
PGRMC1	B-protein
and	O
EGFR	B-protein_type
)	O
were	O
detected	O
with	O
Western	B-experimental_method
blotting	I-experimental_method
by	O
using	O
anti	O
-	O
PGRMC1	B-protein
or	O
anti	O
-	O
EGFR	B-protein_type
antibody	O
.	O

(	O
f	O
)	O
HCT116	O
cells	O
expressing	O
control	O
shRNA	B-chemical
or	O
those	O
knocking	B-experimental_method
down	I-experimental_method
PGRMC1	B-protein
(	O
PGRMC1	B-mutant
-	I-mutant
KD	I-mutant
)	O
were	O
treated	O
with	O
EGF	B-protein_type
or	O
left	O
untreated	O
,	O
and	O
components	O
of	O
the	O
EGFR	B-protein_type
signaling	O
pathway	O
were	O
detected	O
by	O
Western	B-experimental_method
blotting	I-experimental_method
.	O

Haem	B-chemical
-	O
dependent	O
PGRMC1	B-protein
dimerization	B-oligomeric_state
enhances	O
tumour	O
chemoresistance	O
through	O
interaction	O
with	O
cytochromes	B-protein_type
P450	I-protein_type
.	O

Doxorubicin	B-chemical
was	O
incubated	B-experimental_method
with	O
HCT116	O
cells	O
expressing	O
control	O
shRNA	B-chemical
or	O
shPGRMC1	B-chemical
(	O
PGRMC1	B-mutant
-	I-mutant
KD	I-mutant
),	O
and	O
the	O
doxorubicinol	B-chemical
/	O
doxorubicin	B-chemical
ratios	O
in	O
cell	O
pellets	O
were	O
determined	O
using	O
LC	B-experimental_method
-	I-experimental_method
MS	I-experimental_method
.	O

of	O
four	O
separate	O
experiments	O
.	O
**	O
P	B-evidence
<	O
0	O
.	O
01	O
versus	O
control	O
using	O
unpaired	O
Student	B-experimental_method
'	I-experimental_method
s	I-experimental_method
t	I-experimental_method
-	I-experimental_method
test	I-experimental_method
.	O
(	O
e	O
)	O
Indicated	O
amounts	O
of	O
doxorubicin	B-chemical
were	O
added	O
to	O
HCT116	O
(	O
control	O
)	O
cells	O
,	O
PGRMC1	B-mutant
-	I-mutant
KD	I-mutant
cells	O
,	O
or	O
PGRMC1	B-mutant
-	I-mutant
KD	I-mutant
cells	O
expressing	O
shRNA	B-protein_state
-	I-protein_state
resistant	I-protein_state
full	B-protein_state
-	I-protein_state
length	I-protein_state
PGRMC1	B-protein
wt	B-protein_state
or	O
Y113F	B-mutant
,	O
and	O
cell	O
viability	O
was	O
examined	O
by	O
MTT	B-experimental_method
assay	I-experimental_method
.	O

PGRMC1	O
proteins	O
exhibit	O
haem	O
-	O
dependent	O
dimerization	B-oligomeric_state
in	O
solution	O
.	O

T	O
cells	O
perform	O
an	O
essential	O
role	O
in	O
adaptive	O
immunity	O
by	O
interrogating	O
the	O
host	O
proteome	O
for	O
anomalies	O
,	O
classically	O
by	O
recognizing	O
peptides	O
bound	O
in	O
major	B-complex_assembly
histocompatibility	I-complex_assembly
(	O
MHC	B-complex_assembly
)	O
molecules	O
at	O
the	O
cell	O
surface	O
.	O

Structures	B-evidence
of	O
unligated	B-protein_state
and	O
ligated	B-protein_state
TCRs	B-complex_assembly
have	O
shown	O
that	O
the	O
TCR	B-complex_assembly
complementarity	B-structure_element
determining	I-structure_element
region	I-structure_element
(	O
CDR	B-structure_element
)	O
loops	B-structure_element
can	O
be	O
flexible	O
,	O
perhaps	O
enabling	O
peptide	O
binding	O
using	O
different	O
loop	B-structure_element
conformations	O
.	O

Here	O
,	O
the	O
A2	B-chemical
-	I-chemical
RQFGPDFPTI	I-chemical
tetramer	B-oligomeric_state
stained	O
1E6	O
with	O
the	O
greatest	O
MFI	O
,	O
gradually	O
decreasing	O
to	O
the	O
weakest	O
tetramers	B-oligomeric_state
:	O
A2	B-chemical
-	I-chemical
MVWGPDPLYV	I-chemical
and	O
-	O
YLGGPDFPTI	B-chemical
.	O

The	O
range	O
of	O
Tm	B-evidence
was	O
between	O
49	O
.	O
4	O
°	O
C	O
(	O
RQFGPDWIVA	B-chemical
)	O
and	O
60	O
.	O
3	O
°	O
C	O
(	O
YQFGPDFPIA	B-chemical
),	O
with	O
an	O
average	O
approximately	O
55	O
°	O
C	O
,	O
similar	O
to	O
our	O
previous	O
findings	O
.	O

In	O
accordance	O
with	O
this	O
trend	O
,	O
the	O
1E6	B-complex_assembly
TCR	I-complex_assembly
bound	B-protein_state
the	O
natural	O
preproinsulin	B-protein
peptide	O
,	O
ALWGPDPAAA	B-chemical
,	O
with	O
the	O
weakest	O
affinity	B-evidence
currently	O
published	O
for	O
a	O
human	B-species
CD8	O
+	O
T	O
cell	O
–	O
derived	O
TCR	B-complex_assembly
with	O
a	O
biologically	O
relevant	O
ligand	O
(	O
KD	B-evidence
>	O
200	O
μM	O
;	O
KD	B-evidence
,	O
equilibrium	B-evidence
binding	I-evidence
constant	I-evidence
).	O

Surface	B-experimental_method
plasmon	I-experimental_method
resonance	I-experimental_method
(	O
SPR	B-experimental_method
)	O
analysis	O
of	O
the	O
1E6	B-complex_assembly
TCR	I-complex_assembly
–	O
pMHC	B-complex_assembly
interaction	O
for	O
all	O
7	O
APLs	B-chemical
(	O
Figure	O
2	O
,	O
A	O
–	O
H	O
)	O
demonstrated	O
that	O
stronger	O
binding	B-evidence
affinity	I-evidence
(	O
represented	O
as	O
ΔG	B-evidence
°,	I-evidence
kcal	O
/	O
mol	O
)	O
correlated	O
well	O
with	O
the	O
EC50	B-evidence
values	O
(	O
peptide	O
concentration	O
required	O
to	O
reach	O
half	O
-	O
maximal	O
1E6	O
T	O
cell	O
killing	O
)	O
for	O
each	O
ligand	O
,	O
demonstrated	O
by	O
a	O
Pearson	B-experimental_method
’	I-experimental_method
s	I-experimental_method
correlation	I-experimental_method
analysis	I-experimental_method
value	O
of	O
0	O
.	O
8	O
(	O
P	O
=	O
0	O
.	O
01	O
)	O
(	O
Figure	O
2I	O
).	O

It	O
should	O
be	O
noted	O
that	O
this	O
correlation	O
,	O
although	O
consistent	O
with	O
the	O
T	O
cell	O
killing	O
experiments	O
,	O
uses	O
only	O
approximate	O
affinities	B-evidence
calculated	O
for	O
the	O
2	O
weakest	O
ligands	O
.	O

Finally	O
,	O
these	O
data	O
demonstrate	O
the	O
largest	O
range	O
of	O
binding	B-evidence
affinities	I-evidence
reported	O
for	O
a	O
natural	O
,	O
endogenous	B-protein_state
human	B-species
TCR	B-complex_assembly
of	O
more	O
than	O
3	O
logs	O
of	O
magnitude	O
(	O
A2	B-chemical
-	I-chemical
MVWGPDPLYV	I-chemical
vs	O
.	O
A2	B-chemical
-	I-chemical
RQFGPDFPTI	I-chemical
).	O

The	O
1E6	B-complex_assembly
TCR	I-complex_assembly
uses	O
a	O
consensus	O
binding	O
mode	O
to	O
engage	O
multiple	O
APLs	B-chemical
.	O

Our	O
previous	O
structure	B-evidence
of	O
the	O
1E6	B-complex_assembly
-	I-complex_assembly
A2	I-complex_assembly
-	I-complex_assembly
ALWGPDPAAA	I-complex_assembly
complex	O
demonstrated	O
a	O
limited	O
binding	B-site
footprint	I-site
between	O
the	O
TCR	B-complex_assembly
and	O
pMHC	B-complex_assembly
.	O

The	O
surface	B-evidence
complementarity	I-evidence
values	I-evidence
(	O
0	O
.	O
52	O
–	O
0	O
.	O
7	O
)	O
correlated	O
slightly	O
with	O
affinity	B-evidence
(	O
Pearson	B-evidence
’	I-evidence
s	I-evidence
correlation	I-evidence
=	O
0	O
.	O
7	O
,	O
P	B-evidence
=	O
0	O
.	O
05	O
)	O
but	O
could	O
not	O
explain	O
all	O
differences	O
in	O
binding	O
(	O
Figure	O
3A	O
and	O
Table	O
2	O
).	O

The	O
TCR	B-complex_assembly
CDR	B-structure_element
loops	I-structure_element
were	O
in	O
a	O
very	O
similar	O
position	O
in	O
all	O
complexes	O
,	O
apart	O
from	O
some	O
slight	O
deviations	O
in	O
the	O
TCR	B-complex_assembly
β	B-structure_element
-	I-structure_element
chain	I-structure_element
(	O
Figure	O
3B	O
);	O
the	O
peptides	O
were	O
all	O
presented	O
in	O
a	O
similar	O
conformation	O
(	O
Figure	O
3C	O
);	O
and	O
there	O
was	O
minimal	O
variation	O
in	O
crossing	O
angles	O
of	O
the	O
TCR	B-complex_assembly
(	O
42	O
.	O
3	O
°–	O
45	O
.	O
6	O
°)	O
(	O
Figure	O
3D	O
).	O

Focused	O
hotspot	O
binding	O
around	O
a	O
conserved	B-protein_state
GPD	B-structure_element
motif	I-structure_element
enables	O
the	O
1E6	B-complex_assembly
TCR	I-complex_assembly
to	O
tolerate	O
peptide	O
degeneracy	O
.	O

In	O
addition	O
to	O
changes	O
between	O
the	O
TCR	B-complex_assembly
and	O
peptide	O
component	O
,	O
we	O
also	O
observed	O
that	O
different	O
APLs	B-chemical
had	O
different	O
knock	O
-	O
on	O
effects	O
between	O
the	O
TCR	B-complex_assembly
and	O
MHC	B-complex_assembly
.	O

Generally	O
,	O
the	O
weaker	O
-	O
affinity	B-evidence
APLs	B-chemical
made	O
fewer	O
contacts	O
with	O
the	O
MHC	B-complex_assembly
surface	O
(	O
27	O
–	O
29	O
interactions	O
)	O
compared	O
with	O
the	O
stronger	O
-	O
affinity	B-evidence
APLs	B-chemical
(	O
29	O
–	O
35	O
contacts	O
),	O
consistent	O
with	O
a	O
better	O
Pearson	B-evidence
’	I-evidence
s	I-evidence
correlation	I-evidence
value	I-evidence
(	O
0	O
.	O
55	O
)	O
compared	O
with	O
TCR	B-complex_assembly
-	O
peptide	O
interactions	O
versus	O
affinity	B-evidence
(	O
0	O
.	O
045	O
).	O

For	O
example	O
,	O
the	O
1E6	B-complex_assembly
TCR	I-complex_assembly
bound	B-protein_state
to	I-protein_state
A2	B-chemical
-	I-chemical
RQWGPDPAAV	I-chemical
with	O
the	O
third	O
strongest	O
affinity	B-evidence
(	O
KD	B-evidence
=	O
7	O
.	O
8	O
μM	O
)	O
but	O
made	O
fewer	O
contacts	O
than	O
with	O
A2	B-chemical
-	I-chemical
ALWGPDPAAA	I-chemical
(	O
KD	B-evidence
=	O
~	O
208	O
μM	O
)	O
(	O
Table	O
2	O
).	O

Thus	O
,	O
we	O
performed	O
an	O
in	O
-	O
depth	O
thermodynamic	B-experimental_method
analysis	I-experimental_method
of	O
6	O
of	O
the	O
ligands	O
under	O
investigation	O
(	O
Figure	O
8	O
and	O
Supplemental	O
Table	O
3	O
).	O

However	O
,	O
there	O
was	O
a	O
clear	O
switch	O
in	O
entropy	B-evidence
between	O
the	O
weaker	O
-	O
affinity	B-evidence
and	O
stronger	O
-	O
affinity	B-evidence
ligands	O
,	O
indicated	O
by	O
a	O
strong	O
Pearson	B-evidence
’	I-evidence
s	I-evidence
correlation	I-evidence
value	I-evidence
between	O
entropy	B-evidence
and	O
affinity	B-evidence
(	O
Pearson	B-evidence
’	I-evidence
s	I-evidence
correlation	I-evidence
value	I-evidence
0	O
.	O
93	O
,	O
P	B-evidence
=	O
0	O
.	O
007	O
).	O

Conversely	O
,	O
the	O
stronger	O
-	O
affinity	B-evidence
ligands	O
A2	B-chemical
-	I-chemical
RQWGPDPAAV	I-chemical
(	O
KD	B-evidence
=	O
7	O
.	O
8	O
μM	O
),	O
A2	B-chemical
-	I-chemical
YQFGPDFPIA	I-chemical
(	O
KD	B-evidence
=	O
7	O
.	O
4	O
μM	O
),	O
and	O
A2	B-chemical
-	I-chemical
RQFGPDFPTI	I-chemical
(	O
KD	B-evidence
=	O
0	O
.	O
5	O
μM	O
)	O
exhibited	O
favorable	O
entropy	B-evidence
(	O
TΔS	B-evidence
°	I-evidence
=	O
2	O
.	O
2	O
to	O
14	O
.	O
9	O
kcal	O
/	O
mol	O
),	O
indicating	O
an	O
order	O
-	O
to	O
-	O
disorder	O
change	O
during	O
binding	O
,	O
possibly	O
through	O
the	O
expulsion	O
of	O
ordered	O
water	O
molecules	O
.	O

The	O
binding	B-evidence
affinity	I-evidence
of	O
the	O
1E6	B-complex_assembly
TCR	I-complex_assembly
interaction	O
with	O
A2	B-chemical
-	I-chemical
RQFGPDWIVA	I-chemical
is	O
considerably	O
higher	O
than	O
with	O
the	O
disease	O
-	O
implicated	O
A2	B-chemical
-	I-chemical
ALWGPDPAAA	I-chemical
sequence	O
(	O
KD	B-evidence
=	O
44	O
.	O
4	O
μM	O
and	O
KD	B-evidence
>	O
200	O
μM	O
,	O
respectively	O
),	O
highlighting	O
how	O
a	O
pathogen	O
-	O
derived	O
sequence	O
might	O
be	O
capable	O
of	O
priming	O
a	O
1E6	O
-	O
like	O
T	O
cell	O
.	O

Focused	O
binding	O
around	O
a	O
minimal	O
peptide	O
motif	O
has	O
also	O
been	O
implicated	O
as	O
an	O
alternative	O
mechanism	O
enabling	O
TCR	B-complex_assembly
cross	O
-	O
reactivity	O
.	O

Ligand	O
engagement	O
is	O
dominated	O
by	O
peptide	O
interactions	O
,	O
but	O
hotspot	O
-	O
like	O
interactions	O
with	O
the	O
central	O
GPD	B-structure_element
motif	I-structure_element
enable	O
the	O
1E6	B-complex_assembly
TCR	I-complex_assembly
to	O
tolerate	O
peptide	O
residues	O
that	O
vary	O
outside	O
of	O
this	O
region	O
,	O
explaining	O
how	O
T	O
cells	O
expressing	O
this	O
TCR	B-complex_assembly
may	O
cross	O
-	O
react	O
with	O
a	O
large	O
number	O
of	O
different	O
peptides	O
.	O

These	O
data	O
also	O
explain	O
our	O
previous	O
findings	O
that	O
alteration	O
of	O
the	O
anchor	B-structure_element
residue	I-structure_element
at	O
peptide	O
position	O
2	B-residue_number
(	O
Leu	B-mutant
-	I-mutant
Gln	I-mutant
)	O
has	O
a	O
direct	O
effect	O
on	O
1E6	B-evidence
TCR	I-evidence
binding	I-evidence
affinity	I-evidence
because	O
our	O
structural	B-experimental_method
analysis	I-experimental_method
demonstrated	O
that	O
1E6	O
made	O
3	O
additional	O
bonds	O
with	O
A2	B-chemical
-	I-chemical
AQWGPDPAAA	I-chemical
compared	O
with	O
A2	B-chemical
-	I-chemical
ALWGPDPAAA	I-chemical
,	O
consistent	O
with	O
the	O
>	O
3	O
-	O
fold	O
stronger	O
binding	B-evidence
affinity	I-evidence
.	O

Further	O
experiments	O
will	O
be	O
required	O
to	O
determine	O
whether	O
any	O
naturally	O
presented	O
,	O
human	B-species
pathogen	O
–	O
derived	O
peptides	O
act	O
as	O
active	O
ligands	O
for	O
1E6	O
,	O
but	O
our	O
work	O
presented	O
here	O
demonstrates	O
that	O
it	O
is	O
at	O
least	O
feasible	O
for	O
an	O
autoimmune	O
TCR	B-complex_assembly
to	O
bind	O
to	O
a	O
different	O
peptide	O
sequence	O
that	O
could	O
be	O
present	O
in	O
a	O
pathogen	O
proteome	O
with	O
substantially	O
higher	O
affinity	B-evidence
and	O
potency	O
than	O
the	O
interaction	O
it	O
might	O
use	O
to	O
attack	O
self	O
-	O
tissue	O
.	O

3D	B-experimental_method
and	I-experimental_method
2D	I-experimental_method
binding	I-experimental_method
analysis	I-experimental_method
of	O
the	O
1E6	B-complex_assembly
TCR	I-complex_assembly
with	O
A2	B-chemical
-	I-chemical
ALW	I-chemical
and	O
the	O
APLs	B-chemical
.	O

The	O
equilibrium	B-evidence
binding	I-evidence
constant	I-evidence
(	O
KD	B-evidence
)	O
values	O
were	O
calculated	O
using	O
a	O
nonlinear	B-experimental_method
curve	I-experimental_method
fit	I-experimental_method
(	O
y	O
=	O
[	O
P1x	O
]/[	O
P2	O
+	O
X	O
]).	O

All	O
unligated	B-protein_state
pMHCs	B-complex_assembly
are	O
shown	O
as	O
light	O
green	O
illustrations	O
.	O

Boxes	O
show	O
total	O
contacts	O
between	O
the	O
1E6	B-complex_assembly
TCR	I-complex_assembly
and	O
these	O
key	O
residues	O
(	O
green	O
boxes	O
are	O
MHC	B-complex_assembly
residues	O
;	O
white	O
boxes	O
are	O
TCR	B-complex_assembly
residues	O
).	O

We	O
determined	B-experimental_method
the	O
crystal	B-evidence
structure	I-evidence
of	O
an	O
oligomerization	B-protein_state
-	I-protein_state
impaired	I-protein_state
Irga6	B-protein
mutant	B-protein_state
bound	B-protein_state
to	I-protein_state
a	O
non	O
-	O
hydrolyzable	O
GTP	B-chemical
analog	O
.	O

This	O
study	O
contributes	O
important	O
insights	O
into	O
the	O
assembly	O
and	O
catalytic	O
mechanisms	O
of	O
IRG	B-protein_type
proteins	O
as	O
prerequisite	O
to	O
understand	O
their	O
anti	O
-	O
microbial	O
action	O
.	O

Mutagenesis	B-experimental_method
of	O
the	O
contact	B-site
surfaces	I-site
suggests	O
that	O
this	O
""""	O
backside	B-site
""""	O
interface	B-site
is	O
not	O
required	O
for	O
GTP	B-chemical
-	O
dependent	O
oligomerization	O
or	O
cooperative	O
hydrolysis	O
,	O
despite	O
an	O
earlier	O
suggestion	O
to	O
the	O
contrary	O
.	O

For	O
several	O
of	O
these	O
proteins	O
,	O
formation	O
of	O
the	O
G	B-site
interface	I-site
was	O
shown	O
to	O
trigger	O
GTP	B-chemical
hydrolysis	O
by	O
inducing	O
rearrangements	O
of	O
catalytic	O
residues	O
in	O
cis	O
.	O

However	O
,	O
the	O
crystal	B-evidence
structure	I-evidence
of	O
Irga6	B-protein
in	O
the	O
presence	B-protein_state
of	I-protein_state
the	O
non	O
-	O
hydrolyzable	O
GTP	B-chemical
analogue	O
5	B-chemical
'-	I-chemical
guanylyl	I-chemical
imidodiphosphate	I-chemical
(	O
GMPPNP	B-chemical
)	O
showed	O
only	O
subtle	O
differences	O
relative	O
to	O
the	O
apo	B-protein_state
or	O
GDP	B-protein_state
-	I-protein_state
bound	I-protein_state
protein	O
and	O
did	O
not	O
reveal	O
a	O
new	O
dimer	B-site
interface	I-site
associated	O
with	O
the	O
GTPase	B-structure_element
domain	I-structure_element
.	O

Structure	B-evidence
of	O
the	O
Irga6	B-protein
dimer	B-oligomeric_state
.	O

b	O
Ribbon	O
-	O
type	O
representation	O
of	O
the	O
Irga6	B-protein
dimer	B-oligomeric_state
.	O

Irga6	B-protein
immunity	B-protein
-	I-protein
related	I-protein
GTPase	I-protein
6	I-protein

Within	O
the	O
asymmetric	O
unit	O
,	O
six	O
molecules	O
dimerize	B-oligomeric_state
via	O
the	O
symmetric	O
backside	B-site
dimer	I-site
interface	I-site
(	O
buried	O
surface	O
area	O
930	O
Å2	O
),	O
and	O
the	O
remaining	O
seventh	O
molecule	O
forms	O
the	O
same	O
type	O
of	O
interaction	O
with	O
its	O
symmetry	O
mate	O
of	O
the	O
adjacent	O
asymmetric	O
unit	O
(	O
Additional	O
file	O
1	O
:	O
Figure	O
S2a	O
,	O
b	O
,	O
Figure	O
S3	O
).	O

In	O
turn	O
,	O
E106	B-residue_name_number
of	O
switch	B-site
I	I-site
reorients	O
towards	O
the	O
nucleotide	B-chemical
and	O
now	O
participates	O
in	O
the	O
coordination	B-bond_interaction
of	I-bond_interaction
the	O
Mg2	B-chemical
+	I-chemical
ion	O
(	O
Fig	O
.	O
1e	O
,	O
Additional	O
file	O
1	O
:	O
Figure	O
S4	O
).	O

The	O
following	O
structures	B-evidence
are	O
shown	O
in	O
cylinder	O
representations	O
,	O
in	O
similar	O
orientations	O
of	O
their	O
GTPase	B-structure_element
domains	I-structure_element
:	O
a	O
the	O
GMPPNP	B-protein_state
-	I-protein_state
bound	I-protein_state
Irga6	B-protein
dimer	B-oligomeric_state
,	O
b	O
the	O
GDP	O
-	O
AlF4	O
--	O
bound	O
dynamin	B-protein
1	I-protein
GTPase	B-structure_element
-	I-structure_element
minimal	I-structure_element
BSE	O
construct	O
[	O
pdb	O
2X2E	O
],	O
c	O
the	O
GDP	B-protein_state
-	I-protein_state
bound	I-protein_state
atlastin	B-protein
1	I-protein
dimer	B-oligomeric_state
[	O
pdb	O
3Q5E	O
],	O
d	O
the	O
GDP	B-protein_state
-	I-protein_state
AlF3	I-protein_state
-	I-protein_state
bound	I-protein_state
GBP1	B-protein
GTPase	B-structure_element
domain	I-structure_element
dimer	B-oligomeric_state
[	O
pdb	O
2B92	O
],	O
e	O
the	O
BDLP	B-protein_type
dimer	B-oligomeric_state
bound	B-protein_state
to	I-protein_state
GDP	B-chemical
[	O
pdb	O
2J68	O
]	O
and	O
f	O
the	O
GTP	B-protein_state
-	I-protein_state
bound	I-protein_state
GIMAP2	B-protein
dimer	B-oligomeric_state
[	O
pdb	O
2XTN	O
].	O

Nucleotide	O
,	O
Mg2	B-chemical
+	I-chemical
(	O
green	O
)	O
and	O
AlF4	O
-	O
are	O
shown	O
in	O
sphere	O
representation	O
,	O
the	O
buried	O
interface	B-site
sizes	O
per	O
molecule	O
are	O
indicated	O
on	O
the	O
right	O
.	O

For	O
example	O
,	O
for	O
dynamin	B-protein_type
and	O
atlastin	B-protein_type
,	O
it	O
was	O
shown	O
that	O
GTP	B-chemical
binding	O
and	O
/	O
or	O
hydrolysis	O
leads	O
to	O
dimerization	O
of	O
the	O
GTPase	B-structure_element
domains	I-structure_element
and	O
to	O
the	O
reorientation	O
of	O
the	O
adjacent	O
helical	B-structure_element
domains	I-structure_element
.	O

Only	O
one	O
of	O
the	O
seven	O
Irga6	B-protein
molecules	O
in	O
the	O
asymmetric	O
unit	O
formed	O
this	O
contact	O
pointing	O
to	O
a	O
low	O
affinity	O
interaction	O
via	O
the	O
G	B-site
interface	I-site
,	O
which	O
is	O
in	O
agreement	O
with	O
its	O
small	O
size	O
.	O

Based	O
on	O
phylogenetic	B-experimental_method
and	I-experimental_method
structural	I-experimental_method
analysis	I-experimental_method
,	O
these	O
observations	O
suggest	O
that	O
dynamin	B-protein_type
and	O
septin	B-protein_type
superfamilies	O
are	O
derived	O
from	O
a	O
common	O
ancestral	O
membrane	B-protein_type
-	I-protein_type
associated	I-protein_type
GTPase	I-protein_type
that	O
featured	O
a	O
GTP	B-chemical
-	O
dependent	O
parallel	B-protein_state
dimerization	O
mode	O
.	O

During	O
dimerization	O
of	O
GBP1	B-protein
,	O
an	O
arginine	B-structure_element
finger	I-structure_element
from	O
the	O
P	B-structure_element
loop	I-structure_element
reorients	O
towards	O
the	O
nucleotide	B-chemical
in	O
cis	O
to	O
trigger	O
GTP	B-chemical
hydrolysis	O
.	O