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

To	O
explore	O
the	O
molecular	O
basis	O
of	O
asymmetry	O
in	O
holo	B-protein_state
-	O
NadR	B-protein
,	O
we	O
superposed	B-experimental_method
its	O
ligand	B-protein_state
-	I-protein_state
free	I-protein_state
monomer	B-oligomeric_state
(	O
chain	B-structure_element
A	I-structure_element
)	O
onto	O
the	O
ligand	B-protein_state
-	I-protein_state
occupied	I-protein_state
monomer	B-oligomeric_state
(	O
chain	B-structure_element
B	I-structure_element
).	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

(	O
B	O
)	O
A	O
structural	B-experimental_method
alignment	I-experimental_method
of	O
MTH313	B-protein
chain	B-structure_element
A	I-structure_element
and	O
ST1710	B-protein
(	O
pink	O
)	O
(	O
Cα	O
rmsd	B-evidence
2	O
.	O
3Å	O
),	O
shows	O
that	O
they	O
bind	O
salicylate	B-chemical
in	O
equivalent	O
sites	O
(	O
differing	O
by	O
only	O
~	O
3Å	O
)	O
and	O
with	O
the	O
same	O
orientation	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
binds	O
to	O
the	O
ligand	B-site
binding	I-site
site	I-site
of	O
RORγ	B-protein
adopting	O
a	O
collapsed	B-protein_state
conformation	O
as	O
seen	O
with	O
BIO592	B-chemical
where	O
the	O
two	O
compounds	O
superimpose	B-experimental_method
with	O
an	O
RMSD	B-evidence
of	O
0	O
.	O
72	O
Å	O
(	O
Fig	O
.	O
5b	O
).	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

The	O
sequences	O
aligning	O
to	O
the	O
PF06130	B-structure_element
domain	O
(	O
determined	O
by	O
BLAST	O
)	O
are	O
highlighted	O
in	O
red	O
and	O
blue	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

There	O
could	O
be	O
some	O
intrinsic	O
biochemical	O
difference	O
between	O
the	O
two	O
enzymes	O
that	O
renders	O
PduL	B-protein_type
a	O
more	O
attractive	O
candidate	O
for	O
encapsulation	O
in	O
a	O
BMC	B-complex_assembly
—	O
for	O
example	O
,	O
PduL	B-protein_type
might	O
be	O
more	O
amenable	O
to	O
tight	O
packaging	O
,	O
or	O
is	O
better	O
suited	O
for	O
the	O
chemical	O
microenvironment	O
formed	O
within	O
the	O
lumen	O
of	O
the	O
BMC	B-complex_assembly
,	O
which	O
can	O
be	O
quite	O
different	O
from	O
the	O
cytosol	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

Structural	O
basis	O
for	O
the	O
regulation	O
of	O
enzymatic	O
activity	O
of	O
Regnase	B-protein
-	I-protein
1	I-protein
by	O
domain	O
-	O
domain	O
interactions	O

Domain	O
structures	B-evidence
of	O
Regnase	B-protein
-	I-protein
1	I-protein

The	O
domain	O
structures	B-evidence
of	O
NTD	B-structure_element
,	O
ZF	B-structure_element
,	O
and	O
CTD	B-structure_element
were	O
determined	O
by	O
NMR	B-experimental_method
(	O
Fig	O
.	O
1b	O
,	O
d	O
,	O
e	O
).	O

Based	O
on	O
the	O
decrease	O
in	O
the	O
free	O
RNA	B-chemical
fluorescence	O
band	O
,	O
we	O
evaluated	O
the	O
contribution	O
of	O
each	O
domain	O
of	O
Regnase	B-protein
-	I-protein
1	I-protein
to	O
RNA	B-chemical
binding	O
.	O

Contribution	O
of	O
each	O
domain	O
of	O
Regnase	B-protein
-	I-protein
1	I-protein
to	O
RNase	B-protein_type
activity	O

It	O
should	O
be	O
noted	O
that	O
NTD	B-mutant
-	I-mutant
PIN	I-mutant
(	I-mutant
DDNN	I-mutant
)-	I-mutant
ZF	I-mutant
,	O
which	O
possesses	O
the	O
NTD	B-structure_element
but	O
lacks	B-protein_state
the	O
catalytic	B-site
residues	I-site
in	O
PIN	B-structure_element
,	O
completely	O
lost	O
all	O
RNase	B-protein_type
activity	O
(	O
Fig	O
.	O
1g	O
,	O
right	O
panel	O
),	O
as	O
expected	O
,	O
confirming	O
that	O
the	O
RNase	B-protein_type
catalytic	B-site
center	I-site
is	O
located	O
in	O
the	O
PIN	B-structure_element
domain	O
.	O

By	O
comparison	B-experimental_method
with	I-experimental_method
the	I-experimental_method
elution	I-experimental_method
volume	I-experimental_method
of	I-experimental_method
standard	I-experimental_method
marker	I-experimental_method
proteins	I-experimental_method
,	O
the	O
PIN	B-structure_element
domain	O
was	O
assumed	O
to	O
be	O
in	O
equilibrium	O
between	O
a	O
monomer	B-oligomeric_state
and	O
a	O
dimer	B-oligomeric_state
in	O
solution	O
at	O
concentrations	O
in	O
the	O
20	O
–	O
200	O
μM	O
range	O
.	O

The	O
crystal	B-evidence
structure	I-evidence
of	O
the	O
PIN	B-structure_element
domain	O
has	O
been	O
determined	O
in	O
three	O
distinct	O
crystal	B-evidence
forms	I-evidence
with	O
a	O
space	O
group	O
of	O
P3121	O
(	O
form	O
I	O
in	O
this	O
study	O
and	O
PDB	O
ID	O
3V33	O
),	O
P3221	O
(	O
form	O
II	O
in	O
this	O
study	O
),	O
and	O
P41	O
(	O
PDB	O
ID	O
3V32	O
and	O
3V34	O
),	O
respectively	O
.	O

Mutation	B-experimental_method
of	O
Arg215	B-residue_name_number
,	O
whose	O
side	O
chain	O
faces	O
to	O
the	O
opposite	O
side	O
of	O
the	O
oligomeric	B-site
surface	I-site
,	O
to	O
Glu	B-residue_name
preserved	O
the	O
monomer	B-oligomeric_state
/	O
dimer	B-oligomeric_state
equilibrium	O
,	O
similar	O
to	O
the	O
wild	B-protein_state
type	I-protein_state
.	O

Therefore	O
,	O
we	O
concluded	O
that	O
head	B-protein_state
-	I-protein_state
to	I-protein_state
-	I-protein_state
tail	I-protein_state
PIN	B-structure_element
dimerization	O
,	O
together	O
with	O
the	O
NTD	B-structure_element
,	O
are	O
required	O
for	O
Regnase	B-protein
-	I-protein
1	I-protein
RNase	B-protein_type
activity	O
in	O
vitro	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

The	O
importance	O
of	O
residues	O
W182	B-residue_name_number
and	O
R183	B-residue_name_number
can	O
readily	O
be	O
understood	O
in	O
terms	O
of	O
the	O
monomeric	B-oligomeric_state
PIN	B-structure_element
structure	B-evidence
as	O
they	O
are	O
located	O
near	O
to	O
the	O
RNase	B-protein_type
catalytic	B-site
site	I-site
;	O
however	O
,	O
the	O
importance	O
of	O
residue	O
K184	B-residue_name_number
,	O
which	O
points	O
away	O
from	O
the	O
active	B-site
site	I-site
is	O
more	O
easily	O
rationalized	O
in	O
terms	O
of	O
the	O
oligomeric	O
structure	B-evidence
,	O
in	O
which	O
the	O
“	O
secondary	O
”	O
chain	O
’	O
s	O
residue	O
K184	B-residue_name_number
is	O
positioned	O
near	O
the	O
“	O
primary	B-protein_state
”	I-protein_state
chain	O
’	O
s	O
catalytic	B-site
site	I-site
(	O
Fig	O
.	O
4	O
).	O

It	O
should	O
be	O
noted	O
that	O
the	O
putative	B-site
-	I-site
RNA	I-site
binding	I-site
residues	I-site
K184	B-residue_name_number
and	O
R214	B-residue_name_number
are	O
unique	O
to	O
Regnase	B-protein
-	I-protein
1	I-protein
among	O
PIN	B-structure_element
domains	O
.	O

Molecular	O
mechanism	O
of	O
target	O
mRNA	B-chemical
cleavage	O
by	O
the	O
PIN	B-structure_element
dimer	B-oligomeric_state

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

We	O
determined	O
the	O
individual	O
domain	O
structures	B-evidence
of	O
Regnase	B-protein
-	I-protein
1	I-protein
by	O
NMR	B-experimental_method
and	O
X	B-experimental_method
-	I-experimental_method
ray	I-experimental_method
crystallography	I-experimental_method
.	O

Both	O
the	O
mouse	B-taxonomy_domain
and	O
human	B-species
PIN	B-structure_element
domains	O
form	O
head	B-protein_state
-	I-protein_state
to	I-protein_state
-	I-protein_state
tail	I-protein_state
oligomers	B-oligomeric_state
in	O
three	O
distinct	O
crystal	B-evidence
forms	I-evidence
.	O

In	O
contrast	O
,	O
our	O
gel	B-experimental_method
filtration	I-experimental_method
data	O
,	O
mutational	B-experimental_method
analyses	I-experimental_method
,	O
and	O
NMR	B-experimental_method
spectra	B-evidence
all	O
indicate	O
that	O
the	O
PIN	B-structure_element
domain	O
forms	O
a	O
head	B-protein_state
-	I-protein_state
to	I-protein_state
-	I-protein_state
tail	I-protein_state
dimer	B-oligomeric_state
in	O
solution	O
in	O
a	O
manner	O
similar	O
to	O
the	O
crystal	B-evidence
structure	I-evidence
.	O

Taken	O
together	O
,	O
this	O
suggests	O
that	O
the	O
NTD	B-structure_element
and	O
the	O
PIN	B-structure_element
domain	O
compete	O
for	O
a	O
common	B-site
binding	I-site
site	I-site
.	O

While	O
further	O
investigations	O
on	O
the	O
domain	O
-	O
domain	O
interactions	O
of	O
Regnase	B-protein
-	I-protein
1	I-protein
in	O
vivo	O
are	O
necessary	O
,	O
these	O
intramolecular	O
and	O
intermolecular	O
domain	O
interactions	O
of	O
Regnase	B-protein
-	I-protein
1	I-protein
appear	O
to	O
structurally	O
constrain	O
Regnase	B-protein
-	I-protein
1activity	I-protein
,	O
which	O
,	O
in	O
turn	O
,	O
enables	O
tight	O
regulation	O
of	O
immune	O
responses	O
.	O

The	O
percentage	O
of	O
the	O
bound	O
IL	B-protein_type
-	I-protein_type
6	I-protein_type
was	O
calculated	O
based	O
on	O
the	O
fluorescence	B-evidence
intensities	I-evidence
of	O
the	O
free	O
IL	B-protein_type
-	I-protein_type
6	I-protein_type
quantified	O
in	O
(	O
f	O
).	O

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

Two	O
PIN	B-structure_element
molecules	O
in	O
the	O
crystal	B-evidence
were	O
colored	O
white	O
and	O
green	O
,	O
respectively	O
.	O

(	O
a	O
)	O
NMR	B-experimental_method
analyses	I-experimental_method
of	O
the	O
NTD	B-structure_element
-	O
binding	O
to	O
the	O
PIN	B-structure_element
domain	O
.	O

The	O
residues	O
with	O
significant	O
chemical	O
shift	O
changes	O
were	O
labeled	O
in	O
the	O
overlaid	B-experimental_method
spectra	B-evidence
(	O
left	O
)	O
and	O
colored	O
red	O
on	O
the	O
surface	O
and	O
ribbon	O
structure	O
of	O
the	O
PIN	B-structure_element
domain	O
(	O
right	O
).	O

The	O
NTD	B-structure_element
and	O
the	O
PIN	B-structure_element
domain	O
are	O
shown	O
in	O
cyan	O
and	O
white	O
,	O
respectively	O
.	O

Catalytic	B-site
residues	I-site
of	O
the	O
PIN	B-structure_element
domain	O
are	O
shown	O
in	O
sticks	O
,	O
and	O
the	O
residues	O
that	O
exhibited	O
significant	B-evidence
chemical	I-evidence
shift	I-evidence
changes	I-evidence
in	O
(	O
a	O
,	O
b	O
)	O
were	O
labeled	O
.	O

(	O
b	O
)	O
In	B-experimental_method
vitro	I-experimental_method
cleavage	I-experimental_method
assay	I-experimental_method
of	O
basic	O
residue	O
mutants	B-protein_state
for	O
Regnase	B-protein
-	I-protein
1	I-protein
mRNA	B-chemical
.	O

The	O
mutations	O
whose	O
RNase	B-protein_type
activities	O
were	O
not	O
increased	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
blue	O
on	O
the	O
primary	O
PIN	B-structure_element
.	O

In	O
the	O
MEROPS	O
peptidase	O
database	O
,	O
clan	B-protein_type
CD	I-protein_type
contains	O
groups	O
(	O
or	O
families	O
)	O
of	O
cysteine	B-protein_type
peptidases	I-protein_type
that	O
share	O
some	O
highly	B-protein_state
conserved	I-protein_state
structural	O
elements	O
.	O

The	O
structure	B-experimental_method
was	I-experimental_method
analyzed	I-experimental_method
,	O
and	O
the	O
enzyme	O
was	O
biochemically	B-experimental_method
characterized	I-experimental_method
to	O
provide	O
the	O
first	O
structure	O
/	O
function	O
correlation	O
for	O
a	O
C11	B-protein_type
peptidase	I-protein_type
.	O

The	O
crystal	B-evidence
structure	I-evidence
of	O
the	O
catalytically	B-protein_state
active	I-protein_state
form	O
of	O
PmC11	B-protein
revealed	O
an	O
extended	B-structure_element
caspase	I-structure_element
-	I-structure_element
like	I-structure_element
α	I-structure_element
/	I-structure_element
β	I-structure_element
/	I-structure_element
α	I-structure_element
sandwich	I-structure_element
architecture	O
comprised	O
of	O
a	O
central	O
nine	B-structure_element
-	I-structure_element
stranded	I-structure_element
β	I-structure_element
-	I-structure_element
sheet	I-structure_element
,	O
with	O
an	O
unusual	O
C	B-structure_element
-	I-structure_element
terminal	I-structure_element
domain	I-structure_element
(	O
CTD	B-structure_element
),	O
starting	O
at	O
Lys250	B-residue_name_number
.	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

His133	B-residue_name_number
and	O
Cys179	B-residue_name_number
were	O
found	O
at	O
locations	O
structurally	O
homologous	O
to	O
the	O
caspase	B-protein_type
catalytic	B-site
dyad	I-site
,	O
and	O
other	O
clan	B-protein_type
CD	I-protein_type
structures	B-evidence
,	O
at	O
the	O
C	O
termini	O
of	O
strands	B-structure_element
β5	B-structure_element
and	O
β6	B-structure_element
,	O
respectively	O
(	O
Figs	O
.	O
1	O
,	O
A	O
and	O
B	O
,	O
and	O
2A	O
).	O

A	O
multiple	B-experimental_method
sequence	I-experimental_method
alignment	I-experimental_method
of	O
C11	B-protein_type
proteins	O
revealed	O
that	O
these	O
residues	O
are	O
highly	B-protein_state
conserved	I-protein_state
(	O
data	O
not	O
shown	O
).	O

B	O
,	O
size	B-experimental_method
exclusion	I-experimental_method
chromatography	I-experimental_method
of	O
PmC11	B-protein
.	O

Incubation	B-experimental_method
of	O
PmC11	B-protein
at	O
37	O
°	O
C	O
for	O
16	O
h	O
,	O
resulted	O
in	O
a	O
fully	B-protein_state
processed	I-protein_state
enzyme	O
that	O
remained	O
as	O
an	O
intact	B-protein_state
monomer	B-oligomeric_state
when	O
applied	O
to	O
a	O
size	O
-	O
exclusion	O
column	O
(	O
Fig	O
.	O
2B	O
).	O

As	O
expected	O
,	O
PmC11	B-protein
showed	O
no	O
activity	O
against	O
substrates	O
with	O
Pro	B-residue_name
or	O
Asp	B-residue_name
in	O
P1	B-residue_number
but	O
was	O
active	B-protein_state
toward	O
substrates	O
with	O
a	O
basic	O
residue	O
in	O
P1	B-residue_number
such	O
as	O
Bz	B-chemical
-	I-chemical
R	I-chemical
-	I-chemical
AMC	I-chemical
,	O
Z	B-chemical
-	I-chemical
GGR	I-chemical
-	I-chemical
AMC	I-chemical
,	O
and	O
BOC	B-chemical
-	I-chemical
VLK	I-chemical
-	I-chemical
AMC	I-chemical
.	O

The	O
rate	O
of	O
cleavage	O
was	O
∼	O
3	O
-	O
fold	O
greater	O
toward	O
the	O
single	O
Arg	B-residue_name
substrate	O
Bz	B-chemical
-	I-chemical
R	I-chemical
-	I-chemical
AMC	I-chemical
than	O
for	O
the	O
other	O
two	O
(	O
Fig	O
.	O
2F	O
)	O
and	O
,	O
unexpectedly	O
,	O
PmC11	B-protein
showed	O
no	O
activity	O
toward	O
BOC	B-chemical
-	I-chemical
K	I-chemical
-	I-chemical
AMC	I-chemical
.	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

In	O
the	O
structure	B-evidence
of	O
PmC11	B-protein
,	O
Asp207	B-residue_name_number
resides	O
on	O
a	O
flexible	O
loop	B-structure_element
pointing	O
away	O
from	O
the	O
S1	B-site
binding	I-site
pocket	I-site
(	O
Fig	O
.	O
3C	O
).	O

The	O
position	O
and	O
orientation	O
of	O
Z	B-chemical
-	I-chemical
VRPR	I-chemical
-	I-chemical
FMK	I-chemical
was	O
taken	O
from	O
superposition	B-experimental_method
of	O
the	O
PmC11	B-protein
and	O
MALTI_P	B-protein
structures	B-evidence
and	O
indicates	O
the	O
presumed	O
active	B-site
site	I-site
of	O
PmC11	B-protein
.	O

C	O
,	O
divalent	O
cations	O
do	O
not	O
increase	O
the	O
activity	O
of	O
PmC11	B-protein
.	O

The	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
the	O
presence	O
of	O
the	O
cations	O
Ca2	B-chemical
+,	I-chemical
Mn2	B-chemical
+,	I-chemical
Zn2	B-chemical
+,	I-chemical
Co2	B-chemical
+,	I-chemical
Cu2	B-chemical
+,	I-chemical
Mg2	B-chemical
+,	I-chemical
and	O
Fe3	B-chemical
+	I-chemical
with	O
EGTA	B-chemical
as	O
a	O
negative	O
control	O
,	O
and	O
relative	B-experimental_method
fluorescence	I-experimental_method
measured	I-experimental_method
against	I-experimental_method
time	I-experimental_method
(	O
min	O
).	O

The	O
addition	B-experimental_method
of	I-experimental_method
cations	I-experimental_method
produced	O
no	O
improvement	O
in	O
activity	O
of	O
PmC11	B-protein
when	O
compared	O
in	O
the	O
presence	O
of	O
EGTA	B-chemical
,	O
suggesting	O
that	O
PmC11	B-protein
does	O
not	O
require	O
metal	O
ions	O
for	O
proteolytic	O
activity	O
.	O

Several	O
other	O
members	O
of	O
clan	B-protein_type
CD	I-protein_type
require	O
processing	B-ptm
for	O
full	B-protein_state
activation	I-protein_state
including	O
legumain	B-protein
,	O
gingipain	B-protein
-	I-protein
R	I-protein
,	O
MARTX	B-protein
-	I-protein
CPD	I-protein
,	O
and	O
the	O
effector	B-protein_type
caspases	I-protein_type
,	O
e	O
.	O
g	O
.	O
caspase	B-protein
-	I-protein
7	I-protein
.	O

The	O
caspases	B-protein_type
and	O
gingipain	B-protein
-	I-protein
R	I-protein
both	O
undergo	O
intermolecular	B-ptm
(	I-ptm
trans	I-ptm
)	I-ptm
cleavage	I-ptm
and	O
legumain	B-protein
and	O
MARTX	B-protein
-	I-protein
CPD	I-protein
are	O
reported	O
to	O
perform	O
intramolecular	B-ptm
(	I-ptm
cis	I-ptm
)	I-ptm
cleavage	I-ptm
.	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

The	O
chemically	O
most	O
complex	O
modification	O
in	O
eukaryotic	B-taxonomy_domain
rRNA	B-chemical
is	O
the	O
conserved	B-protein_state
hypermodified	B-protein_state
nucleotide	B-chemical
N1	B-chemical
-	I-chemical
methyl	I-chemical
-	I-chemical
N3	I-chemical
-	I-chemical
aminocarboxypropyl	I-chemical
-	I-chemical
pseudouridine	I-chemical
(	O
m1acp3Ψ	B-chemical
)	O
located	O
next	O
to	O
the	O
P	B-site
-	I-site
site	I-site
tRNA	B-chemical
on	O
the	O
small	O
subunit	O
18S	B-chemical
rRNA	I-chemical
.	O

While	O
S	B-chemical
-	I-chemical
adenosylmethionine	I-chemical
was	O
identified	O
as	O
the	O
source	O
of	O
the	O
aminocarboxypropyl	B-chemical
(	O
acp	B-chemical
)	O
group	O
more	O
than	O
40	O
years	O
ago	O
the	O
enzyme	O
catalyzing	O
the	O
acp	B-chemical
transfer	O
remained	O
elusive	O
.	O

In	O
Saccharomyces	B-species
cerevisiae	I-species
18S	B-chemical
rRNA	I-chemical
contains	O
four	O
base	O
methylations	B-ptm
,	O
two	O
acetylations	B-ptm
and	O
a	O
single	O
3	B-chemical
-	I-chemical
amino	I-chemical
-	I-chemical
3	I-chemical
-	I-chemical
carboxypropyl	I-chemical
(	O
acp	B-chemical
)	O
modification	O
,	O
whereas	O
six	O
base	O
methylations	B-ptm
are	O
present	O
in	O
the	O
25S	B-chemical
rRNA	I-chemical
.	O

While	O
in	O
humans	B-species
the	O
18S	B-chemical
rRNA	I-chemical
base	O
modifications	O
are	O
highly	B-protein_state
conserved	I-protein_state
,	O
only	O
three	O
of	O
the	O
yeast	B-taxonomy_domain
base	O
modifications	O
catalyzed	O
by	O
ScRrp8	B-protein
/	O
HsNML	B-protein
,	O
ScRcm1	B-protein
/	O
HsNSUN5	B-protein
and	O
ScNop2	B-protein
/	O
HsNSUN1	B-protein
are	O
preserved	O
in	O
the	O
corresponding	O
human	B-species
28S	B-chemical
rRNA	I-chemical
.	O

They	O
might	O
contribute	O
to	O
increased	O
RNA	B-chemical
stability	O
by	O
providing	O
additional	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
(	O
pseudouridines	B-chemical
),	O
improved	O
base	B-bond_interaction
stacking	I-bond_interaction
(	O
pseudouridines	B-chemical
and	O
base	B-ptm
methylations	I-ptm
)	O
or	O
an	O
increased	O
resistance	O
against	O
hydrolysis	O
(	O
ribose	B-ptm
methylations	I-ptm
).	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

The	O
asterisk	O
indicates	O
the	O
C1	O
-	O
atom	O
labeled	O
in	O
the	O
14C	B-experimental_method
-	I-experimental_method
incorporation	I-experimental_method
assay	I-experimental_method
.	O

(	O
C	O
)	O
14C	B-chemical
-	I-chemical
acp	I-chemical
labeling	O
of	O
18S	B-chemical
rRNAs	I-chemical
.	O

The	O
primer	O
extension	O
arrest	O
is	O
reduced	O
in	O
HTC116	O
cells	O
transfected	O
with	O
siRNAs	B-chemical
544	O
and	O
545	O
.	O

As	O
previously	O
reported	O
this	O
shoulder	O
was	O
identified	O
by	O
ESI	B-experimental_method
-	I-experimental_method
MS	I-experimental_method
as	O
corresponding	O
to	O
m1acp3Ψ	B-chemical
.	O

Whereas	O
the	O
acp	B-chemical
labeling	O
of	O
18S	B-chemical
rRNA	I-chemical
was	O
clearly	O
present	O
in	O
the	O
wild	B-protein_state
type	I-protein_state
strain	O
no	O
radioactive	O
labeling	O
could	O
be	O
observed	O
in	O
a	O
Δtsr3	B-mutant
strain	O
(	O
Figure	O
1C	O
).	O

Human	B-species
18S	B-chemical
rRNA	I-chemical
has	O
also	O
been	O
shown	O
to	O
contain	O
m1acp3Ψ	B-ptm
in	O
the	O
18S	B-chemical
rRNA	I-chemical
at	O
position	O
1248	B-residue_number
.	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

The	O
TSR3	B-protein
gene	O
was	O
genetically	O
modified	O
at	O
its	O
native	O
locus	O
,	O
resulting	O
in	O
a	O
C	O
-	O
terminal	O
fusion	B-protein_state
of	O
Tsr3	B-protein
with	O
a	O
3xHA	B-chemical
epitope	O
expressed	O
by	O
the	O
native	O
promotor	O
in	O
yeast	B-taxonomy_domain
strain	O
CEN	O
.	O
BM258	O
-	O
5B	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

Domain	O
characterization	O
of	O
yeast	B-taxonomy_domain
Tsr3	B-protein
and	O
correlation	O
of	O
acp	B-chemical
modification	O
with	O
late	O
18S	B-chemical
rRNA	I-chemical
processing	O
steps	O
.	O
(	O
A	O
)	O
Scheme	O
of	O
the	O
TSR3	B-protein
gene	O
with	O
truncation	O
positions	O
in	O
the	O
open	O
reading	O
frame	O
.	O

The	O
loop	B-structure_element
connecting	O
β2	B-structure_element
and	O
β3	B-structure_element
contains	O
a	O
single	O
turn	O
of	O
a	O
310	B-structure_element
-	I-structure_element
helix	I-structure_element
.	O
Helices	B-structure_element
α1	B-structure_element
and	O
α2	B-structure_element
are	O
located	O
on	O
one	O
side	O
of	O
the	O
five	B-structure_element
-	I-structure_element
stranded	I-structure_element
β	I-structure_element
-	I-structure_element
sheet	I-structure_element
while	O
α3	B-structure_element
packs	O
against	O
the	O
opposite	O
β	B-structure_element
-	I-structure_element
sheet	I-structure_element
surface	O
.	O

The	O
bound	O
S	B-chemical
-	I-chemical
adenosylmethionine	I-chemical
is	O
shown	O
in	O
a	O
stick	O
representation	O
and	O
colored	O
by	O
atom	O
type	O
.	O

The	O
color	O
coding	O
is	O
the	O
same	O
as	O
in	O
(	O
A	O
).	O
(	O
C	O
)	O
Structural	B-experimental_method
superposition	I-experimental_method
of	O
the	O
X	B-evidence
-	I-evidence
ray	I-evidence
structures	I-evidence
of	O
VdTsr3	B-protein
in	O
the	O
SAM	B-protein_state
-	I-protein_state
bound	I-protein_state
state	O
(	O
red	O
)	O
and	O
SsTsr3	B-protein
(	O
blue	O
)	O
in	O
the	O
apo	B-protein_state
state	O
.	O

In	O
comparison	O
to	O
Tsr3	B-protein
the	O
central	O
β	B-structure_element
-	I-structure_element
sheet	I-structure_element
element	I-structure_element
of	O
Trm10	B-protein
is	O
extended	O
by	O
one	O
additional	O
β	B-structure_element
-	I-structure_element
strand	I-structure_element
pairing	O
to	O
β2	B-structure_element
.	O

Furthermore	O
,	O
the	O
trefoil	B-structure_element
knot	I-structure_element
of	O
Trm10	B-protein
is	O
not	O
as	O
deep	O
as	O
that	O
of	O
Tsr3	B-protein
(	O
Figure	O
4D	O
).	O

W73	B-residue_name_number
is	O
highly	B-protein_state
conserved	I-protein_state
in	O
all	O
known	O
Tsr3	B-protein_type
proteins	I-protein_type
,	O
whereas	O
A76	B-residue_name_number
can	O
be	O
replaced	O
by	O
other	O
hydrophobic	O
amino	B-chemical
acids	I-chemical
.	O

(	O
A	O
)	O
Close	O
-	O
up	O
view	O
of	O
the	O
SAM	B-site
-	I-site
binding	I-site
pocket	I-site
of	O
VdTsr3	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

A	O
red	O
arrow	O
indicates	O
the	O
SAM	B-chemical
methyl	O
group	O
.	O
(	O
D	O
)	O
Binding	O
of	O
SAM	B-chemical
analogs	O
to	O
SsTsr3	B-protein
.	O

SsTsr3	B-protein
bound	B-protein_state
SAM	B-chemical
with	O
a	O
KD	B-evidence
of	O
6	O
.	O
5	O
μM	O
,	O
which	O
is	O
similar	O
to	O
SAM	B-evidence
-	I-evidence
KD	I-evidence
'	I-evidence
s	I-evidence
reported	O
for	O
several	O
SPOUT	B-protein_type
-	I-protein_type
class	I-protein_type
methyltransferases	I-protein_type
.	O

5	B-chemical
′-	I-chemical
methylthioadenosin	I-chemical
—	O
the	O
reaction	O
product	O
after	O
the	O
acp	B-chemical
-	O
transfer	O
—	O
binds	O
only	O
∼	O
2	O
.	O
5	O
-	O
fold	O
weaker	O
(	O
KD	O
=	O
16	O
.	O
7	O
μM	O
)	O
compared	O
to	O
SAM	B-chemical
.	O

Mutations	B-experimental_method
of	O
the	O
corresponding	O
residue	O
in	O
SsTsr3	B-protein
to	O
A	B-residue_name
(	O
D63	B-residue_name_number
)	O
does	O
not	O
significantly	O
alter	O
the	O
SAM	B-evidence
-	I-evidence
binding	I-evidence
affinity	I-evidence
of	O
the	O
protein	O
(	O
KD	B-evidence
=	O
11	O
.	O
0	O
μM	O
).	O

Analysis	B-experimental_method
of	I-experimental_method
the	I-experimental_method
electrostatic	I-experimental_method
surface	I-experimental_method
properties	I-experimental_method
of	O
VdTsr3	B-protein
clearly	O
identified	O
positively	B-site
charged	I-site
surface	I-site
patches	I-site
in	O
the	O
vicinity	O
of	O
the	O
SAM	B-site
-	I-site
binding	I-site
site	I-site
suggesting	O
a	O
putative	O
RNA	B-site
-	I-site
binding	I-site
site	I-site
(	O
Figure	O
6A	O
).	O

Its	O
negatively	O
charged	O
sulfate	B-chemical
group	O
might	O
mimic	O
an	O
RNA	B-chemical
backbone	O
phosphate	O
.	O

In	O
order	O
to	O
explore	O
the	O
RNA	O
-	O
ligand	O
specificity	O
of	O
Tsr3	B-protein
we	O
titrated	B-experimental_method
SsTsr3	B-protein
prepared	O
in	O
RNase	B-protein_state
-	I-protein_state
free	I-protein_state
form	O
with	O
5	O
′-	O
fluoresceine	B-chemical
-	O
labeled	O
RNA	B-chemical
and	O
determined	O
the	O
affinity	B-evidence
by	O
fluorescence	B-experimental_method
anisotropy	I-experimental_method
measurements	I-experimental_method
.	O

A	O
single	O
stranded	O
oligoU	B-chemical
-	I-chemical
RNA	I-chemical
bound	B-protein_state
with	O
a	O
10	O
-	O
fold	O
-	O
reduced	O
affinity	B-evidence
(	O
6	O
.	O
0	O
μM	O
).	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

This	O
demonstrates	O
that	O
,	O
unlike	O
the	O
other	O
small	O
subunit	O
rRNA	B-chemical
base	O
modifications	O
,	O
the	O
acp	B-chemical
modification	O
is	O
required	O
for	O
efficient	O
pre	B-chemical
-	I-chemical
rRNA	I-chemical
processing	O
.	O

After	O
structural	O
changes	O
,	O
possibly	O
driven	O
by	O
GTP	B-chemical
hydrolysis	O
,	O
which	O
go	O
together	O
with	O
the	O
formation	O
of	O
the	O
decoding	B-site
site	I-site
,	O
the	O
20S	B-chemical
pre	I-chemical
-	I-chemical
rRNA	I-chemical
becomes	O
accessible	O
for	O
Nob1	B-protein
cleavage	O
at	O
site	B-site
D	I-site
.	O
This	O
also	O
involves	O
joining	O
of	O
pre	B-complex_assembly
-	I-complex_assembly
40S	I-complex_assembly
and	O
60S	B-complex_assembly
subunits	I-complex_assembly
to	O
80S	B-complex_assembly
-	I-complex_assembly
like	I-complex_assembly
particles	I-complex_assembly
in	O
a	O
translation	O
-	O
like	O
cycle	O
promoted	O
by	O
eIF5B	B-protein
.	O

Thus	O
,	O
the	O
acp	B-chemical
transfer	O
to	O
m1Ψ1191	B-residue_name_number
occurs	O
during	O
the	O
step	O
at	O
which	O
Rio2	B-protein
leaves	O
the	O
pre	B-complex_assembly
-	I-complex_assembly
40S	I-complex_assembly
particle	I-complex_assembly
.	O

The	O
current	O
data	O
together	O
with	O
the	O
finding	O
that	O
acp	B-chemical
modification	O
takes	O
place	O
at	O
the	O
very	O
last	O
step	O
in	O
pre	B-complex_assembly
-	I-complex_assembly
40S	I-complex_assembly
subunit	I-complex_assembly
maturation	O
indicate	O
that	O
the	O
acp	B-chemical
modification	O
probably	O
supports	O
the	O
formation	O
of	O
the	O
decoding	B-site
site	I-site
and	O
efficient	O
20S	B-chemical
pre	I-chemical
-	I-chemical
rRNA	I-chemical
D	B-site
-	I-site
site	I-site
cleavage	O
.	O

Furthermore	O
,	O
our	O
structural	B-evidence
data	I-evidence
unravelled	O
how	O
the	O
regioselectivity	O
of	O
SAM	B-chemical
-	O
dependent	O
group	O
transfer	O
reactions	O
can	O
be	O
tuned	O
by	O
distinct	O
small	O
evolutionary	O
adaptions	O
of	O
the	O
ligand	B-site
binding	I-site
pocket	I-site
of	O
SAM	B-protein_type
-	I-protein_type
binding	I-protein_type
enzymes	I-protein_type
.	O

In	O
addition	O
,	O
our	O
crystallographic	B-experimental_method
analyses	I-experimental_method
revealed	O
that	O
YfiR	B-protein
binds	O
Vitamin	B-chemical
B6	I-chemical
(	O
VB6	B-chemical
)	O
or	O
L	B-chemical
-	I-chemical
Trp	I-chemical
at	O
a	O
YfiB	B-site
-	I-site
binding	I-site
site	I-site
and	O
that	O
both	O
VB6	B-chemical
and	O
L	B-chemical
-	I-chemical
Trp	I-chemical
are	O
able	O
to	O
reduce	O
YfiBL43P	B-mutant
-	O
induced	O
biofilm	O
formation	O
.	O

An	O
increase	O
in	O
c	B-chemical
-	I-chemical
di	I-chemical
-	I-chemical
GMP	I-chemical
promotes	O
biofilm	O
formation	O
,	O
and	O
a	O
decrease	O
results	O
in	O
biofilm	O
degradation	O
(	O
Boehm	O
et	O
al	O
.,;	O
Duerig	O
et	O
al	O
.,;	O
Hickman	O
et	O
al	O
.,;	O
Jenal	O
,;	O
Romling	O
et	O
al	O
.,).	O

The	O
c	B-chemical
-	I-chemical
di	I-chemical
-	I-chemical
GMP	I-chemical
level	O
is	O
regulated	O
by	O
two	O
reciprocal	O
enzyme	O
systems	O
,	O
namely	O
,	O
diguanylate	B-protein_type
cyclases	I-protein_type
(	O
DGCs	B-protein_type
)	O
that	O
synthesize	O
c	B-chemical
-	I-chemical
di	I-chemical
-	I-chemical
GMP	I-chemical
and	O
phosphodiesterases	B-protein_type
(	O
PDEs	B-protein_type
)	O
that	O
hydrolyze	O
c	B-chemical
-	I-chemical
di	I-chemical
-	I-chemical
GMP	I-chemical
(	O
Kulasakara	O
et	O
al	O
.,;	O
Ross	O
et	O
al	O
.,;	O
Ross	O
et	O
al	O
.,).	O
Many	O
of	O
these	O
enzymes	O
are	O
multiple	O
-	O
domain	O
proteins	O
containing	O
a	O
variable	O
N	B-structure_element
-	I-structure_element
terminal	I-structure_element
domain	I-structure_element
that	O
commonly	O
acts	O
as	O
a	O
signal	O
sensor	O
or	O
transduction	O
module	O
,	O
followed	O
by	O
the	O
relatively	B-protein_state
conserved	I-protein_state
GGDEF	B-structure_element
motif	I-structure_element
in	O
DGCs	B-protein_type
or	O
EAL	B-structure_element
/	I-structure_element
HD	I-structure_element
-	I-structure_element
GYP	I-structure_element
domains	I-structure_element
in	O
PDEs	B-protein_type
(	O
Hengge	O
,;	O
Navarro	O
et	O
al	O
.,;	O
Schirmer	O
and	O
Jenal	O
,).	O

YfiN	B-protein
is	O
an	O
integral	O
inner	O
-	O
membrane	O
protein	O
with	O
two	O
potential	O
transmembrane	B-structure_element
helices	I-structure_element
,	O
a	O
periplasmic	O
Per	B-structure_element
-	I-structure_element
Arnt	I-structure_element
-	I-structure_element
Sim	I-structure_element
(	O
PAS	B-structure_element
)	O
domain	O
,	O
and	O
cytosolic	O
domains	O
containing	O
a	O
HAMP	B-structure_element
domain	I-structure_element
(	O
mediate	O
input	O
-	O
output	O
signaling	O
in	O
histidine	B-protein_type
kinases	I-protein_type
,	O
adenylyl	B-protein_type
cyclases	I-protein_type
,	O
methyl	B-protein_type
-	I-protein_type
accepting	I-protein_type
chemotaxis	I-protein_type
proteins	I-protein_type
,	O
and	O
phosphatases	B-protein_type
)	O
and	O
a	O
C	O
-	O
terminal	O
GGDEF	B-structure_element
domain	I-structure_element
indicating	O
a	O
DGC	B-protein_type
’	O
s	O
function	O
(	O
Giardina	O
et	O
al	O
.,;	O
Malone	O
et	O
al	O
.,).	O

YfiN	B-protein
is	O
repressed	B-protein_state
by	I-protein_state
specific	O
interaction	O
between	O
its	O
periplasmic	O
PAS	B-structure_element
domain	I-structure_element
and	O
the	O
periplasmic	O
protein	O
YfiR	B-protein
(	O
Malone	O
et	O
al	O
.,).	O

After	O
the	O
sequestration	O
of	O
YfiR	B-protein
by	O
YfiB	B-protein
,	O
the	O
c	B-chemical
-	I-chemical
di	I-chemical
-	I-chemical
GMP	I-chemical
produced	O
by	O
activated	B-protein_state
YfiN	B-protein
increases	O
the	O
biosynthesis	O
of	O
the	O
Pel	B-chemical
and	O
Psl	B-chemical
EPSs	B-chemical
,	O
resulting	O
in	O
the	O
appearance	O
of	O
the	O
SCV	O
phenotype	O
,	O
which	O
indicates	O
enhanced	O
biofilm	O
formation	O
(	O
Malone	O
et	O
al	O
.,).	O

Recently	O
,	O
we	O
solved	O
the	O
crystal	B-evidence
structure	I-evidence
of	O
YfiR	B-protein
in	O
both	O
the	O
non	B-protein_state
-	I-protein_state
oxidized	I-protein_state
and	O
the	O
oxidized	B-protein_state
states	O
,	O
revealing	O
breakage	O
/	O
formation	O
of	O
one	O
disulfide	B-ptm
bond	I-ptm
(	O
Cys71	B-residue_name_number
-	O
Cys110	B-residue_name_number
)	O
and	O
local	O
conformational	O
change	O
around	O
the	O
other	O
one	O
(	O
Cys145	B-residue_name_number
-	O
Cys152	B-residue_name_number
),	O
indicating	O
that	O
Cys145	B-residue_name_number
-	O
Cys152	B-residue_name_number
plays	O
an	O
important	O
role	O
in	O
maintaining	O
the	O
correct	O
folding	O
of	O
YfiR	B-protein
(	O
Yang	O
et	O
al	O
.,).	O

The	O
“	O
back	B-protein_state
to	I-protein_state
back	I-protein_state
”	O
dimer	B-oligomeric_state
.	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

The	O
“	O
back	B-protein_state
to	I-protein_state
back	I-protein_state
”	O
dimer	B-oligomeric_state
presents	O
a	O
Y	B-protein_state
shape	I-protein_state
.	O

Overall	O
structure	B-evidence
of	O
the	O
YfiB	B-complex_assembly
-	I-complex_assembly
YfiR	I-complex_assembly
complex	O
and	O
the	O
conserved	B-site
surface	I-site
in	O
YfiR	B-protein
.	O
(	O
A	O
)	O
The	O
overall	O
structure	B-evidence
of	O
the	O
YfiB	B-complex_assembly
-	I-complex_assembly
YfiR	I-complex_assembly
complex	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

The	O
YfiB	B-complex_assembly
-	I-complex_assembly
YfiR	I-complex_assembly
complex	O
is	O
a	O
2	O
:	O
2	O
heterotetramer	B-oligomeric_state
(	O
Fig	O
.	O
3A	O
)	O
in	O
which	O
the	O
YfiR	B-protein
dimer	B-oligomeric_state
is	O
clamped	O
by	O
two	O
separated	O
YfiBL43P	B-mutant
molecules	O
with	O
a	O
total	O
buried	O
surface	O
area	O
of	O
3161	O
.	O
2	O
Å2	O
.	O

Additionally	O
,	O
three	O
hydrophobic	B-site
anchoring	I-site
sites	I-site
exist	O
in	O
region	B-structure_element
I	I-structure_element
.	O
The	O
residues	O
F48	B-residue_name_number
and	O
W55	B-residue_name_number
of	O
YfiB	B-protein
are	O
inserted	O
into	O
the	O
hydrophobic	B-site
cores	I-site
mainly	O
formed	O
by	O
the	O
main	O
chain	O
and	O
side	O
chain	O
carbon	O
atoms	O
of	O
residues	O
S57	B-residue_name_number
/	O
Q88	B-residue_name_number
/	O
A89	B-residue_name_number
/	O
N90	B-residue_name_number
and	O
R60	B-residue_name_number
/	O
R175	B-residue_name_number
/	O
H177	B-residue_name_number
of	O
YfiR	B-protein
,	O
respectively	O
;	O
and	O
F57	B-residue_name_number
of	O
YfiB	B-protein
is	O
inserted	O
into	O
the	O
hydrophobic	B-site
pocket	I-site
formed	O
by	O
L166	B-residue_name_number
/	O
I169	B-residue_name_number
/	O
V176	B-residue_name_number
/	O
P178	B-residue_name_number
/	O
L181	B-residue_name_number
of	O
YfiR	B-protein
(	O
Fig	O
.	O
3D	O
-	O
I	O
(	O
ii	O
)).	O

This	O
suggests	O
that	O
the	O
N	O
-	O
terminus	O
of	O
YfiB	B-protein
plays	O
an	O
important	O
role	O
in	O
forming	O
the	O
dimeric	B-oligomeric_state
YfiB	B-protein
in	O
solution	O
and	O
that	O
the	O
conformational	O
change	O
of	O
residue	O
L43	B-residue_name_number
is	O
associated	O
with	O
the	O
stretch	O
of	O
the	O
N	O
-	O
terminus	O
and	O
opening	O
of	O
the	O
dimer	B-oligomeric_state
.	O

The	O
PG	B-site
-	I-site
binding	I-site
site	I-site
of	O
YfiB	B-protein

In	O
the	O
YfiB	B-complex_assembly
-	I-complex_assembly
YfiR	I-complex_assembly
complex	O
,	O
one	O
sulfate	B-chemical
ion	O
is	O
found	O
at	O
the	O
bottom	O
of	O
each	O
YfiBL43P	B-mutant
molecule	O
(	O
Fig	O
.	O
3A	O
)	O
and	O
forms	O
a	O
strong	O
hydrogen	B-bond_interaction
bond	I-bond_interaction
with	O
D102	B-residue_name_number
of	O
YfiBL43P	B-mutant
(	O
Fig	O
.	O
4A	O
and	O
4C	O
).	O

Moreover	O
,	O
a	O
water	B-chemical
molecule	O
was	O
found	O
to	O
bridge	O
the	O
sulfate	B-chemical
ion	O
and	O
the	O
side	O
chains	O
of	O
N67	B-residue_name_number
and	O
D102	B-residue_name_number
,	O
strengthening	O
the	O
hydrogen	B-site
bond	I-site
network	I-site
(	O
Fig	O
.	O
4C	O
).	O

The	O
results	O
indicated	O
that	O
the	O
PG	B-evidence
-	I-evidence
binding	I-evidence
affinity	I-evidence
of	O
YfiBL43P	B-mutant
is	O
65	O
.	O
5	O
μmol	O
/	O
L	O
,	O
which	O
is	O
about	O
16	O
-	O
fold	O
stronger	O
than	O
that	O
of	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
YfiB	B-protein
(	O
Kd	B-evidence
=	O
1	O
.	O
1	O
mmol	O
/	O
L	O
)	O
(	O
Fig	O
.	O
4E	O
–	O
F	O
).	O

The	O
relative	B-evidence
optical	I-evidence
density	I-evidence
is	O
represented	O
as	O
curves	O
.	O

Wild	B-protein_state
-	I-protein_state
type	I-protein_state
YfiB	B-protein
is	O
used	O
as	O
negative	O
control	O
.	O

Previous	O
studies	O
suggested	O
that	O
in	O
response	O
to	O
cell	O
stress	O
,	O
YfiB	B-protein
in	O
the	O
outer	O
membrane	O
sequesters	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
inducing	O
the	O
diguanylate	O
cyclase	O
activity	O
of	O
YfiN	B-protein
to	O
allow	O
c	B-chemical
-	I-chemical
di	I-chemical
-	I-chemical
GMP	I-chemical
production	O
(	O
Giardina	O
et	O
al	O
.,;	O
Malone	O
et	O
al	O
.,;	O
Malone	O
et	O
al	O
.,).	O

Here	O
,	O
we	O
report	O
the	O
crystal	B-evidence
structures	I-evidence
of	O
YfiB	B-protein
alone	B-protein_state
and	O
an	O
active	B-protein_state
mutant	B-protein_state
YfiBL43P	B-mutant
in	B-protein_state
complex	I-protein_state
with	I-protein_state
YfiR	B-protein
,	O
indicating	O
that	O
YfiR	B-protein
forms	O
a	O
2	O
:	O
2	O
complex	B-protein_state
with	I-protein_state
YfiB	B-protein
via	O
a	O
region	O
composed	O
of	O
conserved	O
residues	O
.	O

Our	O
structural	B-experimental_method
data	I-experimental_method
analysis	I-experimental_method
shows	O
that	O
the	O
activated	B-protein_state
YfiB	B-protein
has	O
an	O
N	B-structure_element
-	I-structure_element
terminal	I-structure_element
portion	I-structure_element
that	O
is	O
largely	O
altered	O
,	O
adopting	O
a	O
stretched	B-protein_state
conformation	I-protein_state
compared	O
with	O
the	O
compact	B-protein_state
conformation	I-protein_state
of	O
the	O
apo	B-protein_state
YfiB	B-protein
.	O
The	O
apo	B-protein_state
YfiB	B-protein
structure	B-evidence
constructed	O
beginning	O
at	O
residue	O
34	B-residue_number
has	O
a	O
compact	B-protein_state
conformation	I-protein_state
of	O
approximately	O
45	O
Å	O
in	O
length	O
.	O

In	O
this	O
model	O
,	O
in	O
response	O
to	O
a	O
particular	O
cell	O
stress	O
that	O
is	O
yet	O
to	O
be	O
identified	O
,	O
the	O
dimeric	B-oligomeric_state
YfiB	B-protein
is	O
activated	B-protein_state
from	O
a	O
compact	B-protein_state
,	O
inactive	B-protein_state
conformation	B-protein_state
to	O
a	O
stretched	B-protein_state
conformation	I-protein_state
,	O
which	O
possesses	O
increased	O
PG	B-chemical
binding	O
affinity	O
.	O

Homologs	O
of	O
the	O
YfiBNR	B-complex_assembly
system	O
are	O
functionally	B-protein_state
conserved	I-protein_state
in	O
P	B-species
.	I-species
aeruginosa	I-species
(	O
Malone	O
et	O
al	O
.,;	O
Malone	O
et	O
al	O
.,),	O
E	B-species
.	I-species
coli	I-species
(	O
Hufnagel	O
et	O
al	O
.,;	O
Raterman	O
et	O
al	O
.,;	O
Sanchez	O
-	O
Torres	O
et	O
al	O
.,),	O
K	B-species
.	I-species
pneumonia	I-species
(	O
Huertas	O
et	O
al	O
.,)	O
and	O
Y	B-species
.	I-species
pestis	I-species
(	O
Ren	O
et	O
al	O
.,),	O
where	O
they	O
affect	O
c	B-chemical
-	I-chemical
di	I-chemical
-	I-chemical
GMP	I-chemical
production	O
and	O
biofilm	O
formation	O
.	O

High	O
-	O
resolution	O
structures	B-evidence
of	O
oligomers	B-oligomeric_state
formed	O
by	O
the	O
β	B-protein
-	I-protein
amyloid	I-protein
peptide	I-protein
Aβ	B-protein
are	O
needed	O
to	O
understand	O
the	O
molecular	O
basis	O
of	O
Alzheimer	O
’	O
s	O
disease	O
and	O
develop	O
therapies	O
.	O

Over	O
the	O
last	O
two	O
decades	O
the	O
role	O
of	O
Aβ	B-protein
oligomers	B-oligomeric_state
in	O
the	O
pathophysiology	O
of	O
Alzheimer	O
’	O
s	O
disease	O
has	O
begun	O
to	O
unfold	O
.	O

Aβ	B-protein
isolated	O
from	O
the	O
brains	O
of	O
young	O
plaque	O
-	O
free	O
Tg2576	O
mice	B-taxonomy_domain
forms	O
a	O
mixture	O
of	O
low	O
molecular	O
weight	O
oligomers	B-oligomeric_state
.	O

Smaller	O
oligomers	B-oligomeric_state
with	O
molecular	O
weights	O
consistent	O
with	O
trimers	B-oligomeric_state
,	O
hexamers	B-oligomeric_state
,	O
and	O
nonamers	B-oligomeric_state
were	O
also	O
identified	O
within	O
the	O
mixture	O
of	O
low	O
molecular	O
weight	O
oligomers	B-oligomeric_state
.	O

A	O
type	O
of	O
large	O
oligomers	B-oligomeric_state
called	O
annular	B-complex_assembly
protofibrils	I-complex_assembly
(	O
APFs	B-complex_assembly
)	O
have	O
also	O
been	O
observed	O
in	O
the	O
brains	O
of	O
transgenic	O
mice	B-taxonomy_domain
and	O
isolated	O
from	O
the	O
brains	O
of	O
Alzheimer	O
’	O
s	O
patients	O
.	O

Lashuel	O
et	O
al	O
.	O
observed	O
APFs	B-complex_assembly
with	O
an	O
outer	O
diameter	O
that	O
ranged	O
from	O
7	O
–	O
10	O
nm	O
and	O
an	O
inner	O
diameter	O
that	O
ranged	O
from	O
1	O
.	O
5	O
–	O
2	O
nm	O
,	O
consistent	O
with	O
molecular	O
weights	O
of	O
150	O
–	O
250	O
kDa	O
.	O

Kayed	O
et	O
al	O
.	O
observed	O
APFs	B-complex_assembly
with	O
an	O
outer	O
diameter	O
that	O
ranged	O
from	O
8	O
–	O
25	O
nm	O
,	O
which	O
were	O
composed	O
of	O
small	B-protein_state
spherical	I-protein_state
Aβ	B-protein
oligomers	B-oligomeric_state
,	O
3	O
–	O
5	O
nm	O
in	O
diameter	O
.	O

Sequestering	O
Aβ	B-protein
within	O
the	O
affibody	B-chemical
prevents	O
its	O
fibrilization	O
and	O
reduces	O
its	O
neurotoxicity	O
,	O
providing	O
evidence	O
that	O
the	O
β	B-structure_element
-	I-structure_element
hairpin	I-structure_element
structure	O
may	O
contribute	O
to	O
the	O
ability	O
of	O
Aβ	B-protein
to	O
form	O
neurotoxic	O
oligomers	B-oligomeric_state
.	O

(	O
A	O
)	O
Cartoon	O
illustrating	O
the	O
design	O
of	O
peptides	B-chemical
1	I-chemical
and	I-chemical
2	I-chemical
and	O
their	O
relationship	O
to	O
an	O
Aβ17	B-protein
–	B-residue_range
36	I-residue_range
β	B-structure_element
-	I-structure_element
hairpin	I-structure_element
.	O

Peptide	B-mutant
2	I-mutant
contains	O
a	O
methionine	B-residue_name
residue	O
at	O
position	O
35	B-residue_number
and	O
an	O
Aβ24	B-protein
–	B-residue_range
29	I-residue_range
loop	B-structure_element
with	O
residues	O
24	B-residue_number
and	O
29	B-residue_number
(	O
Val	B-residue_name
and	O
Gly	B-residue_name
)	O
mutated	B-experimental_method
to	O
cysteine	B-residue_name
and	O
linked	O
by	O
a	O
disulfide	B-ptm
bond	I-ptm
(	O
Figure	O
1C	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
mutated	B-experimental_method
these	O
residues	O
because	O
they	O
occupy	O
the	O
same	O
position	O
as	O
the	O
δOrn	B-structure_element
that	O
connects	O
D23	B-residue_name_number
and	O
A30	B-residue_name_number
in	O
peptide	B-mutant
1	I-mutant
.	O

In	O
synthesizing	O
peptides	B-mutant
2	I-mutant
and	I-mutant
4	I-mutant
we	O
formed	O
the	O
disulfide	B-ptm
linkage	I-ptm
after	O
macrolactamization	O
and	O
deprotection	O
of	O
the	O
acid	O
-	O
labile	O
side	O
chain	O
protecting	O
groups	O
.	O

Crystal	B-evidence
diffraction	I-evidence
data	I-evidence
for	O
peptides	B-mutant
4	I-mutant
and	I-mutant
2	I-mutant
were	O
collected	O
in	O
-	O
house	O
with	O
a	O
Rigaku	O
MicroMax	O
007HF	O
X	O
-	O
ray	O
diffractometer	O
at	O
1	O
.	O
54	O
Å	O
wavelength	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

X	B-evidence
-	I-evidence
ray	I-evidence
Crystallographic	I-evidence
Structure	I-evidence
of	O
Peptide	B-mutant
2	I-mutant
and	O
the	O
Oligomers	B-oligomeric_state
It	O
Forms	O

The	O
B	B-evidence
values	I-evidence
for	O
the	O
loops	B-structure_element
are	O
large	O
,	O
indicating	O
that	O
the	O
loops	B-structure_element
are	O
dynamic	O
and	O
not	O
well	O
ordered	O
.	O

Thus	O
,	O
the	O
differences	O
in	O
backbone	O
geometry	O
and	O
side	O
chain	O
rotamers	O
among	O
the	O
loops	B-structure_element
are	O
likely	O
of	O
little	O
significance	O
and	O
should	O
be	O
interpreted	O
with	O
caution	O
.	O

Like	O
peptide	B-mutant
1	I-mutant
,	O
peptide	B-mutant
2	I-mutant
forms	O
a	O
triangular	B-protein_state
trimer	B-oligomeric_state
,	O
and	O
four	O
trimers	B-oligomeric_state
assemble	O
to	O
form	O
a	O
dodecamer	B-oligomeric_state
.	O

In	O
the	O
higher	O
-	O
order	O
assembly	O
of	O
the	O
dodecamers	B-oligomeric_state
formed	O
by	O
peptide	B-mutant
2	I-mutant
a	O
new	O
structure	B-evidence
emerges	O
,	O
not	O
seen	O
in	O
peptide	B-mutant
1	I-mutant
,	O
an	O
annular	B-site
pore	I-site
consisting	O
of	O
five	O
dodecamers	B-oligomeric_state
.	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

In	O
the	O
crystal	B-evidence
lattice	I-evidence
,	O
each	O
F20	B-residue_name_number
face	O
of	O
one	O
dodecamer	B-oligomeric_state
packs	O
against	O
an	O
F20	B-residue_name_number
face	O
of	O
another	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

Hydrophobic	B-bond_interaction
packing	I-bond_interaction
between	O
the	O
F20	B-residue_name_number
faces	O
of	O
trimers	B-oligomeric_state
displayed	O
on	O
the	O
outer	O
surface	O
of	O
each	O
dodecamer	B-oligomeric_state
stabilizes	O
the	O
porelike	O
assembly	O
.	O

The	O
eclipsed	B-protein_state
interfaces	B-site
occur	O
between	O
dodecamers	B-structure_element
1	I-structure_element
and	I-structure_element
2	I-structure_element
,	O
1	B-structure_element
and	I-structure_element
5	I-structure_element
,	O
and	O
3	B-structure_element
and	I-structure_element
4	I-structure_element
,	O
as	O
shown	O
in	O
Figure	O
5A	O
.	O

The	O
crystallographic	B-evidence
assembly	I-evidence
of	O
peptide	B-mutant
2	I-mutant
into	O
a	O
trimer	B-oligomeric_state
,	O
dodecamer	B-oligomeric_state
,	O
and	O
annular	B-site
pore	I-site
provides	O
a	O
model	O
for	O
the	O
assembly	O
of	O
the	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
Aβ	B-protein
peptide	O
to	O
form	O
oligomers	B-oligomeric_state
.	O

In	O
this	O
model	O
Aβ	B-protein
folds	O
to	O
form	O
a	O
β	B-structure_element
-	I-structure_element
hairpin	I-structure_element
comprising	O
the	O
hydrophobic	O
central	B-structure_element
and	I-structure_element
C	I-structure_element
-	I-structure_element
terminal	I-structure_element
regions	I-structure_element
.	O

Three	O
β	B-structure_element
-	I-structure_element
hairpins	I-structure_element
assemble	O
to	O
form	O
a	O
trimer	B-oligomeric_state
,	O
and	O
four	O
trimers	B-oligomeric_state
assemble	O
to	O
form	O
a	O
dodecamer	B-oligomeric_state
.	O

The	O
model	O
put	O
forth	O
in	O
Figure	O
6	O
is	O
consistent	O
with	O
the	O
current	O
understanding	O
of	O
endogenous	O
Aβ	B-protein
oligomerization	O
and	O
explains	O
at	O
atomic	O
resolution	O
many	O
key	O
observations	O
about	O
Aβ	B-protein
oligomers	B-oligomeric_state
.	O

Fibrillar	B-protein_state
and	O
nonfibrillar	B-protein_state
oligomers	B-oligomeric_state
have	O
structurally	O
distinct	O
characteristics	O
,	O
which	O
are	O
reflected	O
in	O
their	O
reactivity	O
with	O
the	O
fibril	O
-	O
specific	O
OC	O
antibody	O
and	O
the	O
oligomer	B-oligomeric_state
-	O
specific	O
A11	O
antibody	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

These	O
two	O
modes	O
of	O
assembly	O
might	O
reflect	O
a	O
dynamic	O
interaction	O
between	O
dodecamers	B-oligomeric_state
,	O
which	O
could	O
permit	O
assemblies	O
of	O
more	O
dodecamers	B-oligomeric_state
into	O
larger	O
annular	B-site
pores	I-site
.	O

Preliminary	O
attempts	O
to	O
study	O
these	O
species	O
by	O
SEC	B-experimental_method
and	O
SDS	B-experimental_method
-	I-experimental_method
PAGE	I-experimental_method
have	O
not	O
provided	O
a	O
clear	O
measure	O
of	O
the	O
structures	B-evidence
formed	O
in	O
solution	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

Ligands	O
that	O
regulate	O
the	O
dynamics	O
and	O
stability	O
of	O
the	O
coactivator	B-site
‐	I-site
binding	I-site
site	I-site
in	O
the	O
C	O
‐	O
terminal	O
ligand	B-structure_element
‐	I-structure_element
binding	I-structure_element
domain	I-structure_element
,	O
called	O
activation	B-structure_element
function	I-structure_element
‐	I-structure_element
2	I-structure_element
(	O
AF	B-structure_element
‐	I-structure_element
2	I-structure_element
),	O
showed	O
similar	O
activity	O
profiles	O
in	O
different	O
cell	O
types	O
.	O

Such	O
ligands	O
induced	O
breast	O
cancer	O
cell	O
proliferation	O
in	O
a	O
manner	O
that	O
was	O
predicted	O
by	O
the	O
canonical	O
recruitment	O
of	O
the	O
coactivators	O
NCOA1	B-protein
/	I-protein
2	I-protein
/	I-protein
3	I-protein
and	O
induction	O
of	O
the	O
GREB1	B-protein
proliferative	O
gene	O
.	O

For	O
example	O
,	O
selective	O
estrogen	B-protein_type
receptor	I-protein_type
modulators	I-protein_type
(	O
SERMs	B-protein_type
)	O
such	O
as	O
tamoxifen	B-chemical
(	O
Nolvadex	B-chemical
®;	I-chemical
AstraZeneca	O
)	O
or	O
raloxifene	B-chemical
(	O
Evista	B-chemical
®;	I-chemical
Eli	O
Lilly	O
)	O
(	O
Fig	O
1A	O
)	O
block	O
the	O
ERα	B-protein
‐	O
mediated	O
proliferative	O
effects	O
of	O
the	O
native	O
estrogen	B-chemical
,	O
17β	B-chemical
‐	I-chemical
estradiol	I-chemical
(	O
E2	B-chemical
),	O
on	O
breast	O
cancer	O
cells	O
,	O
but	O
promote	O
beneficial	O
estrogenic	O
effects	O
on	O
bone	O
mineral	O
density	O
and	O
adverse	O
estrogenic	O
effects	O
such	O
as	O
uterine	O
proliferation	O
,	O
fatty	O
liver	O
,	O
or	O
stroke	O
(	O
Frolik	O
et	O
al	O
,	O
1996	O
;	O
Fisher	O
et	O
al	O
,	O
1998	O
;	O
McDonnell	O
et	O
al	O
,	O
2002	O
;	O
Jordan	O
,	O
2003	O
).	O

E2	B-chemical
‐	O
rings	O
are	O
numbered	O
A	O
‐	O
D	O
.	O
The	O
E	O
‐	O
ring	O
is	O
the	O
common	O
site	O
of	O
attachment	O
for	O
BSC	O
found	O
in	O
many	O
SERMS	B-protein_type
.	O

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

AF	B-structure_element
‐	I-structure_element
1	I-structure_element
binds	O
a	O
separate	O
surface	O
on	O
these	O
coactivators	O
(	O
Webb	O
et	O
al	O
,	O
1998	O
;	O
Yi	O
et	O
al	O
,	O
2015	O
).	O

However	O
,	O
ERα	B-protein
‐	O
mediated	O
proliferative	O
responses	O
vary	O
in	O
a	O
ligand	O
‐	O
dependent	O
manner	O
(	O
Srinivasan	O
et	O
al	O
,	O
2013	O
);	O
thus	O
,	O
it	O
is	O
not	O
known	O
whether	O
this	O
canonical	O
model	O
is	O
widely	O
applicable	O
across	O
diverse	O
ERα	B-protein
ligands	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

To	O
test	O
these	O
signaling	O
models	O
,	O
we	O
profiled	O
a	O
diverse	O
library	O
of	O
ERα	B-protein
ligands	O
using	O
systems	O
biology	O
approaches	O
to	O
X	B-experimental_method
‐	I-experimental_method
ray	I-experimental_method
crystallography	I-experimental_method
and	O
chemical	B-experimental_method
biology	I-experimental_method
(	O
Srinivasan	O
et	O
al	O
,	O
2013	O
),	O
including	O
a	O
series	O
of	O
quantitative	O
bioassays	O
for	O
ERα	B-protein
function	O
that	O
were	O
statistically	O
robust	O
and	O
reproducible	O
,	O
based	O
on	O
the	O
Z	B-evidence
’‐	I-evidence
statistic	I-evidence
(	O
Fig	O
EV1A	O
and	O
B	O
;	O
see	O
Materials	O
and	O
Methods	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

In	O
cluster	O
1	O
,	O
the	O
first	O
three	O
comparisons	O
(	O
rows	O
)	O
showed	O
significant	O
positive	O
correlations	O
(	O
F	B-experimental_method
‐	I-experimental_method
test	I-experimental_method
for	O
nonzero	O
slope	O
,	O
P	B-evidence
≤	O
0	O
.	O
05	O
).	O

−,	O
significant	O
correlations	O
lost	O
upon	O
deletion	O
of	O
AB	B-structure_element
or	O
F	B-structure_element
domains	O
.	O

Tamoxifen	B-chemical
depends	O
on	O
AF	B-structure_element
‐	I-structure_element
1	I-structure_element
for	O
its	O
cell	O
‐	O
specific	O
activity	O
(	O
Sakamoto	O
et	O
al	O
,	O
2002	O
);	O
therefore	O
,	O
we	O
asked	O
whether	O
cell	O
‐	O
specific	O
signaling	O
observed	O
here	O
is	O
due	O
to	O
a	O
similar	O
dependence	O
on	O
AF	B-structure_element
‐	I-structure_element
1	I-structure_element
for	O
activity	O
(	O
Fig	O
EV1	O
).	O

Thus	O
,	O
the	O
strength	O
of	O
AF	B-structure_element
‐	I-structure_element
1	I-structure_element
signaling	O
does	O
not	O
determine	O
cell	O
‐	O
specific	O
signaling	O
.	O

Identifying	O
cell	O
‐	O
specific	O
signaling	O
clusters	O
in	O
ERα	B-protein
ligand	O
classes	O

The	O
side	O
chain	O
of	O
OBHS	B-chemical
‐	I-chemical
BSC	I-chemical
analogs	O
induces	O
cell	O
‐	O
specific	O
signaling	O

In	O
panel	O
(	O
D	O
),	O
L	B-experimental_method
‐	I-experimental_method
Luc	I-experimental_method
ERα	B-protein
‐	O
WT	B-protein_state
activity	O
from	O
panel	O
(	O
B	O
)	O
is	O
shown	O
for	O
comparison	O
.	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

Deletion	B-experimental_method
of	I-experimental_method
the	O
AB	B-structure_element
or	O
F	B-structure_element
domain	O
altered	O
correlations	O
for	O
six	O
of	O
the	O
eight	O
scaffolds	O
in	O
this	O
cluster	O
(	O
2	B-chemical
,	I-chemical
5	I-chemical
‐	I-chemical
DTP	I-chemical
,	O
3	B-chemical
,	I-chemical
4	I-chemical
‐	I-chemical
DTP	I-chemical
,	O
S	B-chemical
‐	I-chemical
OBHS	I-chemical
‐	I-chemical
3	I-chemical
,	O
WAY	B-chemical
‐	I-chemical
D	I-chemical
,	O
WAY	B-chemical
dimer	I-chemical
,	O
and	O
cyclofenil	B-chemical
‐	I-chemical
ASC	I-chemical
)	O
(	O
Fig	O
3D	O
lanes	O
5	O
–	O
12	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

To	O
determine	O
whether	O
ligand	O
classes	O
control	O
expression	O
of	O
native	O
ERα	B-protein
target	O
genes	O
through	O
the	O
canonical	O
linear	O
signaling	O
pathway	O
,	O
we	O
performed	O
pairwise	B-experimental_method
linear	I-experimental_method
regression	I-experimental_method
analyses	I-experimental_method
using	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
interactions	O
in	O
M2H	B-experimental_method
assay	I-experimental_method
as	O
independent	O
predictors	O
of	O
GREB1	B-protein
expression	O
(	O
the	O
dependent	O
variable	O
)	O
(	O
Figs	O
EV1	O
and	O
EV2A	O
,	O
F	O
–	O
H	O
).	O

For	O
clusters	O
2	O
and	O
3	O
,	O
GREB1	B-protein
activity	O
was	O
generally	O
not	O
predicted	O
by	O
NCOA1	B-protein
/	I-protein
2	I-protein
/	I-protein
3	I-protein
recruitment	O
.	O

However	O
,	O
ligand	O
‐	O
induced	O
GREB1	B-protein
levels	O
were	O
generally	O
not	O
determined	O
by	O
NCOA1	B-protein
/	I-protein
2	I-protein
/	I-protein
3	I-protein
recruitment	O
(	O
Fig	O
3E	O
lanes	O
5	O
–	O
19	O
),	O
consistent	O
with	O
an	O
alternate	O
causality	O
model	O
(	O
Fig	O
1E	O
).	O

Out	O
of	O
11	O
indirect	O
modulator	O
series	O
in	O
cluster	O
2	O
or	O
3	O
,	O
only	O
the	O
S	B-chemical
‐	I-chemical
OBHS	I-chemical
‐	I-chemical
3	I-chemical
class	O
had	O
NCOA1	B-protein
/	I-protein
2	I-protein
/	I-protein
3	I-protein
recruitment	O
profiles	O
that	O
predicted	O
GREB1	B-protein
levels	O
(	O
Fig	O
3E	O
lane	O
12	O
).	O

With	O
the	O
OBHS	B-chemical
‐	I-chemical
N	I-chemical
compounds	O
,	O
NCOA3	B-protein
and	O
GREB1	B-protein
showed	O
near	O
perfect	O
prediction	O
of	O
proliferation	O
(	O
Fig	O
EV3G	O
),	O
with	O
unexplained	O
variance	O
similar	O
to	O
the	O
noise	O
in	O
the	O
assays	O
.	O

Out	O
of	O
15	O
ligand	O
series	O
in	O
these	O
clusters	O
,	O
only	O
2	B-chemical
,	I-chemical
5	I-chemical
‐	I-chemical
DTP	I-chemical
analogs	O
induced	O
a	O
proliferative	O
response	O
that	O
was	O
predicted	O
by	O
GREB1	B-protein
levels	O
,	O
which	O
were	O
not	O
determined	O
by	O
NCOA1	B-protein
/	I-protein
2	I-protein
/	I-protein
3	I-protein
recruitment	O
(	O
Fig	O
3E	O
and	O
F	O
lane	O
10	O
).	O

Similarly	O
,	O
S	B-chemical
‐	I-chemical
OBHS	I-chemical
‐	I-chemical
3	I-chemical
,	O
cyclofenil	B-chemical
‐	I-chemical
ASC	I-chemical
,	O
and	O
OBHS	B-chemical
‐	I-chemical
ASC	I-chemical
had	O
positively	O
correlated	O
NCOA1	B-protein
/	I-protein
2	I-protein
/	I-protein
3	I-protein
recruitment	O
and	O
GREB1	B-protein
levels	O
,	O
but	O
none	O
of	O
these	O
activities	O
determined	O
their	O
proliferative	O
effects	O
(	O
Fig	O
3E	O
and	O
F	O
lanes	O
11	O
–	O
12	O
and	O
18	O
).	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

All	O
direct	O
modulator	O
and	O
two	O
indirect	O
modulator	O
scaffolds	O
(	O
OBHS	B-chemical
and	O
S	B-chemical
‐	I-chemical
OBHS	I-chemical
‐	I-chemical
3	I-chemical
)	O
lacked	O
ERβ	O
agonist	O
activity	O
.	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

Therefore	O
,	O
we	O
examined	O
another	O
50	O
LBD	B-structure_element
structures	B-evidence
containing	O
ligands	O
in	O
clusters	O
2	O
and	O
3	O
.	O

Ligands	O
in	O
cluster	O
2	O
and	O
cluster	O
3	O
showed	O
conformational	O
heterogeneity	O
in	O
parts	O
of	O
the	O
scaffold	O
that	O
were	O
directed	O
toward	O
multiple	O
regions	O
of	O
the	O
receptor	O
including	O
h3	B-structure_element
,	O
h8	B-structure_element
,	O
h11	B-structure_element
,	O
h12	B-structure_element
,	O
and	O
/	O
or	O
the	O
β	B-structure_element
‐	I-structure_element
sheets	I-structure_element
(	O
Fig	O
EV5C	O
–	O
G	O
).	O

Hierarchical	B-experimental_method
clustering	I-experimental_method
revealed	O
that	O
many	O
of	O
the	O
2	B-chemical
,	I-chemical
5	I-chemical
‐	I-chemical
DTP	I-chemical
analogs	O
recapitulated	O
most	O
of	O
the	O
peptide	O
recruitment	O
and	O
dismissal	O
patterns	O
observed	O
with	O
E2	B-chemical
(	O
Fig	O
6H	O
).	O

Also	O
,	O
we	O
have	O
used	O
siRNA	B-experimental_method
screening	I-experimental_method
to	O
identify	O
a	O
number	O
of	O
coregulators	O
required	O
for	O
ERα	B-protein
‐	O
mediated	O
repression	O
of	O
the	O
IL	O
‐	O
6	O
gene	O
(	O
Nwachukwu	O
et	O
al	O
,	O
2014	O
).	O

Some	O
of	O
these	O
ligands	O
altered	O
the	O
shape	O
of	O
the	O
AF	B-site
‐	I-site
2	I-site
surface	I-site
by	O
perturbing	O
the	O
h3	B-site
–	I-site
h12	I-site
interface	I-site
,	O
thus	O
providing	O
a	O
route	O
to	O
new	O
SERM	O
‐	O
like	O
activity	O
profiles	O
by	O
combining	O
indirect	O
and	O
direct	O
modulation	O
of	O
receptor	O
structure	O
.	O

Incorporation	O
of	O
statistical	O
approaches	O
to	O
understand	O
relationships	O
between	O
structure	O
and	O
signaling	O
variables	O
moves	O
us	O
toward	O
predictive	O
models	O
for	O
complex	O
ERα	B-protein
‐	O
mediated	O
responses	O
such	O
as	O
in	O
vivo	O
uterine	O
proliferation	O
or	O
tumor	O
growth	O
,	O
and	O
more	O
generally	O
toward	O
structure	O
‐	O
based	O
design	O
for	O
other	O
allosteric	O
drug	O
targets	O
including	O
GPCRs	B-protein_type
and	O
other	O
nuclear	B-protein_type
receptors	I-protein_type
.	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

The	O
superfamily	O
can	O
be	O
divided	O
into	O
five	O
families	O
based	O
on	O
structural	O
and	O
functional	O
similarities	O
:	O
Ras	B-protein_type
,	O
Rho	B-protein_type
,	O
Rab	B-protein_type
,	O
Arf	B-protein_type
,	O
and	O
Ran	B-protein_type
.	O

These	O
regions	O
are	O
responsible	O
for	O
“	O
sensing	O
”	O
the	O
nucleotide	O
state	O
,	O
with	O
the	O
GTP	B-protein_state
-	I-protein_state
bound	I-protein_state
state	O
showing	O
greater	O
rigidity	O
and	O
the	O
GDP	B-protein_state
-	I-protein_state
bound	I-protein_state
state	O
adopting	O
a	O
more	O
relaxed	O
conformation	O
(	O
reviewed	O
in	O
Ref	O
.).	O

A	O
number	O
of	O
RhoA	B-protein
and	O
Rac1	B-protein
effector	O
proteins	O
,	O
including	O
the	O
formins	O
and	O
members	O
of	O
the	O
protein	B-protein_type
kinase	I-protein_type
C	I-protein_type
-	I-protein_type
related	I-protein_type
kinase	I-protein_type
(	O
PRK	B-protein_type
)	O
6	B-protein_type
family	O
,	O
along	O
with	O
Cdc42	B-protein
effectors	O
,	O
including	O
the	O
Wiskott	B-protein_type
-	I-protein_type
Aldrich	I-protein_type
syndrome	I-protein_type
(	O
WASP	B-protein_type
)	O
family	O
and	O
the	O
transducer	O
of	O
Cdc42	B-protein_type
-	I-protein_type
dependent	I-protein_type
actin	I-protein_type
assembly	I-protein_type
(	O
TOCA	B-protein_type
)	O
family	O
,	O
have	O
also	O
been	O
linked	O
to	O
the	O
pathways	O
that	O
govern	O
cytoskeletal	O
dynamics	O
.	O

Cdc42	B-protein
effectors	O
,	O
TOCA1	B-protein
and	O
the	O
ubiquitously	O
expressed	O
member	O
of	O
the	O
WASP	B-protein_type
family	I-protein_type
,	O
N	B-protein
-	I-protein
WASP	I-protein
,	O
have	O
been	O
implicated	O
in	O
the	O
regulation	O
of	O
actin	O
polymerization	O
downstream	O
of	O
Cdc42	B-protein
and	O
phosphatidylinositol	B-chemical
4	I-chemical
,	I-chemical
5	I-chemical
-	I-chemical
bisphosphate	I-chemical
(	O
PI	B-chemical
(	I-chemical
4	I-chemical
,	I-chemical
5	I-chemical
)	I-chemical
P2	I-chemical
).	O

The	O
data	O
were	O
fitted	O
to	O
a	O
binding	B-evidence
isotherm	I-evidence
to	O
give	O
an	O
apparent	O
Kd	B-evidence
and	O
are	O
expressed	O
as	O
a	O
percentage	O
of	O
the	O
maximum	O
signal	O
;	O
B	O
and	O
C	O
,	O
competition	B-experimental_method
SPA	I-experimental_method
experiments	O
were	O
carried	O
out	O
with	O
the	O
indicated	O
concentrations	O
of	O
ACK	B-protein
GBD	B-structure_element
(	O
B	O
)	O
or	O
HR1	B-structure_element
domain	O
(	O
C	O
)	O
titrated	B-experimental_method
into	O
30	O
nm	O
GST	B-mutant
-	I-mutant
ACK	I-mutant
and	O
either	O
30	O
nm	O
Cdc42Δ7Q61L	B-complex_assembly
·[	I-complex_assembly
3H	I-complex_assembly
]	I-complex_assembly
GTP	I-complex_assembly
or	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
Cdc42Q61L	B-complex_assembly
·[	I-complex_assembly
3H	I-complex_assembly
]	I-complex_assembly
GTP	I-complex_assembly
.	O

The	O
binding	B-experimental_method
experiments	I-experimental_method
were	O
repeated	O
with	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
[	B-complex_assembly
3H	I-complex_assembly
]	I-complex_assembly
GTP	I-complex_assembly
·	I-complex_assembly
Cdc42	I-complex_assembly
,	O
but	O
the	O
affinity	B-evidence
of	O
the	O
HR1	B-structure_element
domain	O
for	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
Cdc42	B-protein
was	O
similar	O
to	O
its	O
affinity	B-evidence
for	O
truncated	B-protein_state
Cdc42	B-protein
(	O
Kd	B-evidence
≈	O
5	O
μm	O
;	O
Fig	O
.	O
1C	O
).	O

Another	O
possible	O
explanation	O
for	O
the	O
low	O
affinities	B-evidence
observed	O
was	O
that	O
the	O
HR1	B-structure_element
domain	O
alone	B-protein_state
is	O
not	O
sufficient	O
for	O
maximal	O
binding	O
of	O
the	O
TOCA	B-protein_type
proteins	I-protein_type
to	O
Cdc42	B-protein
and	O
that	O
the	O
other	O
domains	O
are	O
required	O
.	O

Furthermore	O
,	O
both	O
BAR	B-structure_element
and	O
SH3	B-structure_element
domains	O
have	O
been	O
implicated	O
in	O
interactions	O
with	O
small	O
G	B-protein_type
proteins	I-protein_type
(	O
e	O
.	O
g	O
.	O
the	O
BAR	B-structure_element
domain	O
of	O
Arfaptin2	B-protein
binds	O
to	O
Rac1	B-protein
and	O
Arl1	B-protein
),	O
while	O
an	O
SH3	B-structure_element
domain	O
mediates	O
the	O
interaction	O
between	O
Rac1	B-protein
and	O
the	O
guanine	B-protein
nucleotide	I-protein
exchange	I-protein
factor	I-protein
,	O
β	B-protein
-	I-protein
PIX	I-protein
.	O

Full	B-protein_state
-	I-protein_state
length	I-protein_state
TOCA1	B-protein
and	O
ΔSH3	B-mutant
TOCA1	B-protein
bound	B-protein_state
with	O
micromolar	O
affinity	O
(	O
Fig	O
.	O
2B	O
),	O
in	O
a	O
similar	O
manner	O
to	O
the	O
isolated	O
HR1	B-structure_element
domain	O
(	O
Fig	O
.	O
1A	O
).	O

There	O
were	O
1	O
,	O
845	O
unambiguous	O
NOEs	B-evidence
and	O
757	O
ambiguous	O
NOEs	B-evidence
after	O
eight	O
iterations	O
.	O

A	O
sequence	B-experimental_method
alignment	I-experimental_method
illustrating	O
the	O
secondary	O
structure	O
elements	O
of	O
the	O
TOCA1	B-protein
and	O
CIP4	B-protein
HR1	B-structure_element
domains	O
and	O
the	O
HR1a	B-structure_element
and	O
HR1b	B-structure_element
domains	O
from	O
PRK1	B-protein
is	O
shown	O
in	O
Fig	O
.	O
3B	O
.	O

A	O
series	O
of	O
15N	B-experimental_method
HSQC	I-experimental_method
experiments	O
was	O
recorded	O
on	O
15N	B-chemical
-	O
labeled	B-protein_state
TOCA1	B-protein
HR1	B-structure_element
domain	O
in	O
the	O
presence	B-protein_state
of	I-protein_state
increasing	B-experimental_method
concentrations	I-experimental_method
of	O
unlabeled	B-protein_state
Cdc42Δ7Q61L	B-complex_assembly
·	I-complex_assembly
GMPPNP	I-complex_assembly
to	O
map	O
the	O
Cdc42	B-site
-	I-site
binding	I-site
surface	I-site
.	O

B	O
,	O
CSPs	B-experimental_method
were	O
calculated	O
as	O
described	O
under	O
“	O
Experimental	O
Procedures	O
”	O
and	O
are	O
shown	O
for	O
backbone	O
and	O
side	O
chain	O
NH	O
groups	O
.	O

Residues	O
that	O
disappeared	O
in	O
the	O
presence	B-protein_state
of	I-protein_state
Cdc42	B-protein
were	O
assigned	O
a	O
CSP	B-experimental_method
of	O
0	O
.	O
2	O
but	O
were	O
excluded	O
when	O
calculating	O
the	O
mean	O
CSP	B-experimental_method
and	O
are	O
indicated	O
with	O
open	O
bars	O
.	O

Residues	O
with	O
affected	O
side	O
chain	O
CSPs	B-experimental_method
derived	O
from	O
13C	B-experimental_method
HSQCs	I-experimental_method
are	O
marked	O
with	O
green	O
asterisks	O
above	O
the	O
bars	O
.	O

The	O
corresponding	O
15N	B-experimental_method
and	O
13C	B-experimental_method
NMR	I-experimental_method
experiments	O
were	O
also	O
recorded	O
on	O
15N	B-chemical
-	O
Cdc42Δ7Q61L	B-complex_assembly
·	I-complex_assembly
GMPPNP	I-complex_assembly
or	O
15N	B-chemical
/	O
13C	B-chemical
-	O
Cdc42Δ7Q61L	B-complex_assembly
·	I-complex_assembly
GMPPNP	I-complex_assembly
in	O
the	O
presence	B-protein_state
of	I-protein_state
unlabeled	B-protein_state
HR1	B-structure_element
domain	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

C	O
,	O
the	O
residues	O
with	O
significantly	O
affected	O
backbone	O
and	O
side	O
chain	O
groups	O
are	O
highlighted	O
on	O
an	O
NMR	B-experimental_method
structure	B-evidence
of	O
free	B-protein_state
Cdc42Δ7Q61L	B-complex_assembly
·	I-complex_assembly
GMPPNP	I-complex_assembly
;	O
those	O
that	O
are	O
buried	O
are	O
colored	O
dark	O
blue	O
,	O
whereas	O
those	O
that	O
are	O
solvent	B-protein_state
-	I-protein_state
accessible	I-protein_state
are	O
colored	O
red	O
.	O

Residues	O
without	O
information	O
from	O
shift	B-experimental_method
mapping	I-experimental_method
are	O
colored	O
gray	O
.	O

HADDOCK	B-experimental_method
was	O
therefore	O
used	O
to	O
perform	O
rigid	B-experimental_method
body	I-experimental_method
docking	I-experimental_method
based	O
on	O
the	O
structures	B-evidence
of	O
free	B-protein_state
HR1	B-structure_element
domain	O
and	O
Cdc42	B-protein
and	O
ambiguous	O
interaction	O
restraints	O
derived	O
from	O
the	O
titration	B-experimental_method
experiments	I-experimental_method
described	O
above	O
.	O

Residues	O
equivalent	O
to	O
Rac1	B-protein
and	O
RhoA	B-protein
contact	B-site
sites	I-site
but	O
that	O
are	O
invisible	O
in	O
free	B-protein_state
Cdc42	B-protein
are	O
gray	O
.	O

D	O
,	O
regions	O
of	O
interest	O
of	O
the	O
Cdc42	B-complex_assembly
·	I-complex_assembly
HR1	I-complex_assembly
domain	O
model	O
.	O

The	O
four	O
lowest	O
energy	O
structures	B-evidence
in	O
the	O
chosen	O
HADDOCK	B-experimental_method
cluster	O
are	O
shown	O
overlaid	O
,	O
with	O
the	O
residues	O
of	O
interest	O
shown	O
as	O
sticks	O
and	O
labeled	O
.	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

Cdc42	O
is	O
shown	O
in	O
green	O
,	O
and	O
TOCA1	B-protein
is	O
shown	O
in	O
purple	O
.	O

A	O
comparison	O
of	O
the	O
HSQC	B-experimental_method
experiments	O
recorded	O
on	O
15N	B-chemical
-	O
Cdc42	B-protein
alone	B-protein_state
,	O
in	O
the	O
presence	B-protein_state
of	I-protein_state
TOCA1	B-protein
HR1	B-structure_element
,	O
N	B-protein
-	I-protein
WASP	I-protein
GBD	B-structure_element
,	O
or	O
both	O
,	O
shows	O
that	O
the	O
spectra	B-evidence
in	O
the	O
presence	B-protein_state
of	I-protein_state
N	B-protein
-	I-protein
WASP	I-protein
and	O
in	O
the	O
presence	B-protein_state
of	I-protein_state
both	O
N	B-protein
-	I-protein
WASP	I-protein
and	O
TOCA1	B-protein
HR1	B-structure_element
are	O
identical	O
(	O
Fig	O
.	O
7C	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

Taken	O
together	O
,	O
the	O
data	O
in	O
Fig	O
.	O
7	O
,	O
C	O
and	O
D	O
,	O
indicate	O
unidirectional	O
competition	O
for	O
Cdc42	B-protein
binding	O
in	O
which	O
the	O
N	B-protein
-	I-protein
WASP	I-protein
GBD	B-structure_element
displaces	O
TOCA1	B-protein
HR1	B-structure_element
but	O
not	O
vice	O
versa	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

The	O
TOCA1	B-protein
HR1	B-structure_element
domain	O
alone	B-protein_state
is	O
sufficient	O
for	O
Cdc42	B-protein
binding	O
in	O
vitro	O
,	O
yet	O
the	O
affinity	B-evidence
of	O
the	O
TOCA1	B-protein
HR1	B-structure_element
domain	O
for	O
Cdc42	B-protein
is	O
remarkably	O
low	O
(	O
Kd	B-evidence
≈	O
5	O
μm	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
equivalent	O
Arg	B-residue_name
in	O
Rac1	B-protein
and	O
RhoA	B-protein
is	O
pointing	O
away	O
from	O
the	O
HR1	B-structure_element
domains	O
of	O
PRK1	B-protein
.	O

Furthermore	O
,	O
the	O
isolated	B-experimental_method
F	B-structure_element
-	I-structure_element
BAR	I-structure_element
domain	O
of	O
FBP17	B-protein
has	O
been	O
shown	O
to	O
induce	O
membrane	O
tubulation	O
of	O
brain	O
liposomes	O
and	O
BAR	B-structure_element
domain	O
proteins	O
that	O
promote	O
tubulation	O
cluster	O
on	O
membranes	O
at	O
high	O
densities	O
.	O

A	O
substantial	O
body	O
of	O
data	O
has	O
illuminated	O
the	O
complex	O
regulation	O
of	O
WASP	B-protein_type
/	I-protein_type
N	I-protein_type
-	I-protein_type
WASP	I-protein_type
proteins	I-protein_type
,	O
and	O
current	O
evidence	O
suggests	O
that	O
these	O
allosteric	O
activation	O
mechanisms	O
and	O
oligomerization	O
combine	O
to	O
regulate	O
WASP	B-protein_type
activity	O
,	O
allowing	O
the	O
synchronization	O
and	O
integration	O
of	O
multiple	O
potential	O
activation	O
signals	O
(	O
reviewed	O
in	O
Ref	O
.).	O

We	O
envisage	O
that	O
TOCA1	B-protein
is	O
first	O
recruited	O
to	O
the	O
appropriate	O
membrane	O
in	O
response	O
to	O
PI	B-chemical
(	I-chemical
4	I-chemical
,	I-chemical
5	I-chemical
)	I-chemical
P2	I-chemical
via	O
its	O
F	B-structure_element
-	I-structure_element
BAR	I-structure_element
domain	O
,	O
where	O
the	O
local	O
increase	O
in	O
concentration	O
favors	O
F	B-structure_element
-	I-structure_element
BAR	I-structure_element
-	O
mediated	O
dimerization	B-oligomeric_state
of	O
TOCA1	B-protein
.	O

TOCA1	B-protein
can	O
then	O
recruit	O
N	B-protein
-	I-protein
WASP	I-protein
via	O
an	O
interaction	O
between	O
its	O
SH3	B-structure_element
domain	O
and	O
the	O
N	B-protein
-	I-protein
WASP	I-protein
proline	B-structure_element
-	I-structure_element
rich	I-structure_element
region	I-structure_element
.	O

In	O
a	O
cellular	O
context	O
,	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
TOCA1	B-protein
and	O
N	B-protein
-	I-protein
WASP	I-protein
are	O
likely	O
to	O
have	O
similar	O
affinities	B-evidence
for	O
active	B-protein_state
Cdc42	B-protein
,	O
but	O
in	O
the	O
unfolded	B-protein_state
,	O
active	B-protein_state
conformation	O
,	O
the	O
affinity	B-evidence
of	O
N	B-protein
-	I-protein
WASP	I-protein
for	O
Cdc42	B-protein
dramatically	O
increases	O
.	O

Our	O
binding	B-evidence
data	I-evidence
suggest	O
that	O
TOCA1	B-protein
HR1	B-structure_element
binding	O
is	O
not	O
allosterically	O
regulated	O
,	O
and	O
our	O
NMR	B-experimental_method
data	O
,	O
along	O
with	O
the	O
high	O
stability	B-protein_state
of	O
TOCA1	B-protein
HR1	B-structure_element
,	O
suggest	O
that	O
there	O
is	O
no	O
widespread	O
conformational	O
change	O
in	O
the	O
presence	B-protein_state
of	I-protein_state
Cdc42	B-protein
.	O

As	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
TOCA1	B-protein
and	O
the	O
isolated	B-protein_state
HR1	B-structure_element
domain	O
bind	O
Cdc42	B-protein
with	O
similar	O
affinities	O
,	O
the	O
N	B-protein
-	I-protein
WASP	I-protein
-	O
Cdc42	B-protein
interaction	O
will	O
be	O
favored	O
because	O
the	O
N	B-protein
-	I-protein
WASP	I-protein
GBD	B-structure_element
can	O
easily	O
outcompete	O
the	O
TOCA1	B-protein
HR1	B-structure_element
for	O
Cdc42	B-protein
.	O

Potentially	O
,	O
the	O
TOCA1	B-protein
-	O
Cdc42	B-protein
interaction	O
functions	O
to	O
position	O
N	B-protein
-	I-protein
WASP	I-protein
and	O
Cdc42	B-protein
such	O
that	O
they	O
are	O
poised	O
to	O
interact	O
with	O
high	O
affinity	O
.	O

There	O
is	O
an	O
advantage	O
to	O
such	O
an	O
effector	O
handover	O
,	O
in	O
that	O
N	B-protein
-	I-protein
WASP	I-protein
would	O
only	O
be	O
robustly	O
recruited	O
when	O
F	B-structure_element
-	I-structure_element
BAR	I-structure_element
domains	O
are	O
already	O
present	O
.	O

F	O
-	O
BAR	O
oligomerization	O
is	O
expected	O
to	O
occur	O
following	O
membrane	O
binding	O
,	O
but	O
a	O
single	O
monomer	B-oligomeric_state
is	O
shown	O
for	O
clarity	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

Step	O
4	O
,	O
the	O
core	O
CRIB	B-structure_element
binds	O
with	O
high	O
affinity	O
while	O
the	O
region	O
C	O
-	O
terminal	O
to	O
the	O
CRIB	B-structure_element
displaces	O
the	O
TOCA1	B-protein
HR1	B-structure_element
domain	O
and	O
increases	O
the	O
affinity	O
of	O
the	O
N	B-protein
-	I-protein
WASP	I-protein
-	O
Cdc42	O
interaction	O
further	O
.	O

WH1	O
,	O
WASP	B-structure_element
homology	I-structure_element
1	I-structure_element
domain	I-structure_element
;	O
PP	B-structure_element
,	O
proline	B-structure_element
-	I-structure_element
rich	I-structure_element
region	I-structure_element
;	O
VCA	B-structure_element
,	O
verprolin	B-structure_element
homology	I-structure_element
,	I-structure_element
cofilin	I-structure_element
homology	I-structure_element
,	I-structure_element
acidic	I-structure_element
region	I-structure_element
.	O

We	O
envisage	O
a	O
complex	O
interplay	O
of	O
equilibria	O
between	O
free	B-protein_state
and	O
bound	B-protein_state
,	O
active	B-protein_state
and	O
inactive	B-protein_state
Cdc42	B-protein
,	O
TOCA	B-protein_type
family	I-protein_type
,	O
and	O
WASP	B-protein_type
family	O
proteins	O
,	O
facilitating	O
a	O
tightly	O
spatially	O
and	O
temporally	O
regulated	O
pathway	O
requiring	O
numerous	O
simultaneous	O
events	O
in	O
order	O
to	O
achieve	O
appropriate	O
and	O
robust	O
activation	O
of	O
the	O
downstream	O
pathway	O
.	O

Acetyl	B-protein_type
-	I-protein_type
CoA	I-protein_type
carboxylases	I-protein_type
(	O
ACCs	B-protein_type
)	O
catalyse	O
the	O
committed	O
step	O
in	O
fatty	O
-	O
acid	O
biosynthesis	O
:	O
the	O
ATP	B-chemical
-	O
dependent	O
carboxylation	O
of	O
acetyl	B-chemical
-	I-chemical
CoA	I-chemical
to	O
malonyl	B-chemical
-	I-chemical
CoA	I-chemical
.	O
They	O
are	O
important	O
regulatory	O
hubs	O
for	O
metabolic	O
control	O
and	O
relevant	O
drug	O
targets	O
for	O
the	O
treatment	O
of	O
the	O
metabolic	O
syndrome	O
and	O
cancer	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

In	O
addition	O
to	O
the	O
canonical	O
ACC	B-structure_element
components	I-structure_element
,	O
eukaryotic	B-taxonomy_domain
ACCs	B-protein_type
contain	O
two	O
non	B-protein_state
-	I-protein_state
catalytic	I-protein_state
regions	B-structure_element
,	O
the	O
large	O
central	B-structure_element
domain	I-structure_element
(	O
CD	B-structure_element
)	O
and	O
the	O
BC	B-structure_element
–	I-structure_element
CT	I-structure_element
interaction	I-structure_element
domain	I-structure_element
(	O
BT	B-structure_element
).	O

The	O
function	O
of	O
this	O
domain	O
remains	O
poorly	O
characterized	O
,	O
although	O
phosphorylation	B-ptm
of	O
several	O
serine	B-residue_name
residues	O
in	O
the	O
CD	B-structure_element
regulates	O
ACC	B-protein_type
activity	O
.	O

Of	O
these	O
,	O
only	O
Ser1157	B-residue_name_number
is	O
highly	B-protein_state
conserved	I-protein_state
in	O
fungal	B-taxonomy_domain
ACC	B-protein_type
and	O
aligns	B-experimental_method
to	I-experimental_method
Ser1216	B-residue_name_number
in	O
human	B-species
ACC1	B-protein
.	O

Integrating	O
these	O
data	O
with	O
small	B-experimental_method
-	I-experimental_method
angle	I-experimental_method
X	I-experimental_method
-	I-experimental_method
ray	I-experimental_method
scattering	I-experimental_method
(	O
SAXS	B-experimental_method
)	O
and	O
electron	B-experimental_method
microscopy	I-experimental_method
(	O
EM	B-experimental_method
)	O
observations	O
yield	O
a	O
comprehensive	O
representation	O
of	O
the	O
dynamic	O
structure	O
and	O
regulation	O
of	O
fungal	B-taxonomy_domain
ACC	B-protein_type
.	O

First	O
,	O
we	O
focused	O
on	O
structure	B-experimental_method
determination	I-experimental_method
of	O
the	O
82	O
-	O
kDa	O
CD	B-structure_element
.	O

Close	O
structural	O
homologues	O
could	O
not	O
be	O
found	O
for	O
the	O
CDN	B-structure_element
or	O
the	O
CDC	B-structure_element
domains	O
.	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

Each	O
of	O
the	O
four	O
CD	B-structure_element
domains	O
in	O
HsaBT	B-mutant
-	I-mutant
CD	I-mutant
individually	O
resembles	O
the	O
corresponding	O
SceCD	B-species
domain	O
;	O
however	O
,	O
human	B-species
and	O
yeast	B-taxonomy_domain
CDs	B-structure_element
exhibit	O
distinct	O
overall	O
structures	B-evidence
.	O

In	O
agreement	O
with	O
their	O
tight	O
interaction	O
in	O
SceCD	B-species
,	O
the	O
relative	O
spatial	O
arrangement	O
of	O
CDL	B-structure_element
and	O
CDC1	B-structure_element
is	O
preserved	O
in	O
HsaBT	B-mutant
-	I-mutant
CD	I-mutant
,	O
but	O
the	O
human	B-species
CDL	B-structure_element
/	O
CDC1	B-structure_element
didomain	O
is	O
tilted	O
by	O
30	O
°	O
based	O
on	O
a	O
superposition	B-experimental_method
of	O
human	B-species
and	O
yeast	B-taxonomy_domain
CDC2	B-structure_element
(	O
Supplementary	O
Fig	O
.	O
1c	O
).	O

It	O
resembles	O
the	O
BT	B-structure_element
of	O
propionyl	B-protein_type
-	I-protein_type
CoA	I-protein_type
carboxylase	I-protein_type
;	O
only	O
the	O
four	O
C	O
-	O
terminal	O
strands	B-structure_element
of	I-structure_element
the	I-structure_element
β	I-structure_element
-	I-structure_element
barrel	I-structure_element
are	O
slightly	O
tilted	O
.	O

The	O
absence	B-protein_state
of	I-protein_state
the	O
regulatory	B-structure_element
loop	I-structure_element
might	O
be	O
linked	O
to	O
the	O
less	B-protein_state
-	I-protein_state
restrained	I-protein_state
interface	B-site
of	O
CDL	B-structure_element
/	O
CDC1	B-structure_element
and	O
CDC2	B-structure_element
and	O
altered	O
relative	O
orientations	O
of	O
these	O
domains	B-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

No	O
crystals	O
diffracting	O
to	O
sufficient	O
resolution	O
were	O
obtained	O
for	O
larger	B-mutant
BC	I-mutant
-	I-mutant
containing	I-mutant
fragments	I-mutant
,	O
or	O
for	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
Cth	B-species
or	O
SceACC	B-protein
.	O

However	O
,	O
molecular	B-experimental_method
replacement	I-experimental_method
did	O
not	O
reveal	O
a	O
unique	O
positioning	O
of	O
the	O
BC	B-structure_element
domain	O
.	O

Indeed	O
,	O
the	O
comparison	O
of	O
the	O
positioning	O
of	O
eight	O
instances	O
of	O
the	O
C	O
-	O
terminal	O
part	O
of	O
CD	B-structure_element
relative	O
to	O
CT	B-structure_element
in	O
crystal	B-evidence
structures	I-evidence
determined	B-experimental_method
here	O
,	O
reveals	O
flexible	O
interdomain	O
linking	O
(	O
Fig	O
.	O
3a	O
).	O

Conformational	O
variability	O
in	O
the	O
CD	B-structure_element
thus	O
contributes	O
considerably	O
to	O
variations	O
in	O
the	O
spacing	O
between	O
the	O
BC	B-structure_element
and	O
CT	B-structure_element
domains	O
,	O
and	O
may	O
extend	O
to	O
distance	O
variations	O
beyond	O
the	O
mobility	O
range	O
of	O
the	O
flexibly	B-protein_state
tethered	I-protein_state
BCCP	B-structure_element
.	O

SAXS	B-experimental_method
analysis	O
of	O
CthACC	B-protein
agrees	O
with	O
a	O
dimeric	B-oligomeric_state
state	O
and	O
an	O
elongated	B-protein_state
shape	I-protein_state
with	O
a	O
maximum	O
extent	O
of	O
350	O
Å	O
(	O
Supplementary	O
Table	O
1	O
).	O

The	O
flexibility	O
in	O
the	O
CDC2	B-structure_element
/	I-structure_element
CT	I-structure_element
hinge	I-structure_element
appears	O
substantially	O
larger	O
than	O
the	O
variations	O
observed	O
in	O
the	O
set	O
of	O
crystal	B-evidence
structures	I-evidence
.	O

The	O
phosphorylated	B-protein_state
regulatory	B-structure_element
loop	I-structure_element
binds	O
to	O
an	O
allosteric	B-site
site	I-site
at	O
the	O
interface	B-site
of	O
two	O
non	B-protein_state
-	I-protein_state
catalytic	I-protein_state
domains	O
and	O
restricts	O
conformational	O
freedom	O
at	O
several	O
hinges	B-structure_element
in	O
the	O
dynamic	B-protein_state
ACC	B-protein_type
.	O

(	O
b	O
)	O
Cartoon	O
representation	O
of	O
the	O
SceCD	B-species
crystal	B-evidence
structure	I-evidence
.	O

(	O
c	O
)	O
Superposition	B-experimental_method
of	O
CDC1	B-structure_element
and	O
CDC2	B-structure_element
reveals	O
highly	B-protein_state
conserved	I-protein_state
folds	B-structure_element
.	O
(	O
d	O
)	O
The	O
regulatory	B-structure_element
loop	I-structure_element
with	O
the	O
phosphorylated	B-protein_state
Ser1157	B-residue_name_number
is	O
bound	O
into	O
a	O
crevice	O
between	O
CDC1	B-structure_element
and	O
CDC2	B-structure_element
,	O
the	O
conserved	B-protein_state
residues	O
Arg1173	B-residue_name_number
and	O
Arg1260	B-residue_name_number
coordinate	O
the	O
phosphoryl	B-chemical
-	O
group	O
.	O

The	O
range	O
of	O
hinge	O
bending	O
is	O
indicated	O
and	O
the	O
connection	O
points	O
between	O
CDC2	B-structure_element
and	O
CT	B-structure_element
(	O
blue	O
)	O
as	O
well	O
as	O
between	O
CDC1	B-structure_element
and	O
CDC2	B-structure_element
(	O
green	O
and	O
grey	O
)	O
are	O
marked	O
as	O
spheres	O
.	O

The	O
connection	O
points	O
from	O
CDC1	B-structure_element
to	O
CDC2	B-structure_element
and	O
to	O
CDL	B-structure_element
are	O
represented	O
by	O
green	O
spheres	O
.	O

The	O
domains	O
are	O
labelled	O
and	O
the	O
distances	O
between	O
the	O
N	O
termini	O
of	O
CDN	B-structure_element
(	O
spheres	O
)	O
in	O
the	O
compared	O
structures	O
are	O
indicated	O
.	O

The	O
two	O
kinases	O
exhibit	O
nearly	O
identical	O
overall	O
architecture	O
,	O
with	O
both	O
kinases	B-protein_type
possessing	O
ATP	B-chemical
hydrolysis	O
activity	O
in	O
the	O
absence	B-protein_state
of	I-protein_state
substrates	I-protein_state
.	O

SePSK	B-protein
and	O
AtXK	B-protein
-	I-protein
1	I-protein
display	O
a	O
sequence	O
identity	O
of	O
44	O
.	O
9	O
%,	O
and	O
belong	O
to	O
the	O
ribulokinase	B-protein_type
-	I-protein_type
like	I-protein_type
carbohydrate	I-protein_type
kinases	I-protein_type
,	O
a	O
sub	O
-	O
family	O
of	O
FGGY	B-protein_type
family	I-protein_type
carbohydrate	I-protein_type
kinases	I-protein_type
.	O

However	O
,	O
the	O
function	O
of	O
XK	B-protein
-	I-protein
1	I-protein
(	O
At2g21370	B-gene
)	O
inside	O
the	O
chloroplast	O
stroma	O
has	O
remained	O
unknown	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

The	O
structures	B-evidence
most	O
closely	O
related	O
to	O
SePSK	B-protein
are	O
xylulose	B-protein_type
kinase	I-protein_type
,	O
glycerol	B-protein_type
kinase	I-protein_type
and	O
ribulose	B-protein_type
kinase	I-protein_type
,	O
implying	O
that	O
SePSK	B-protein
and	O
AtXK	B-protein
-	I-protein
1	I-protein
might	O
function	O
similarly	O
to	O
these	O
kinases	B-protein_type
.	O

To	O
further	O
identify	O
the	O
actual	O
substrate	O
of	O
SePSK	B-protein
and	O
AtXK	B-protein
-	I-protein
1	I-protein
,	O
five	O
different	O
sugar	O
molecules	O
,	O
including	O
D	B-chemical
-	I-chemical
ribulose	I-chemical
,	O
L	B-chemical
-	I-chemical
ribulose	I-chemical
,	O
D	B-chemical
-	I-chemical
xylulose	I-chemical
,	O
L	B-chemical
-	I-chemical
xylulose	I-chemical
and	O
Glycerol	B-chemical
,	O
were	O
used	O
in	O
enzymatic	B-experimental_method
activity	I-experimental_method
assays	I-experimental_method
.	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

(	O
B	O
)	O
The	O
ATP	B-chemical
hydrolysis	O
activity	O
of	O
SePSK	B-protein
with	O
addition	O
of	O
five	O
different	O
substrates	O
.	O

To	O
obtain	O
more	O
detailed	O
information	O
of	O
SePSK	B-protein
and	O
AtXK	B-protein
-	I-protein
1	I-protein
in	B-protein_state
complex	I-protein_state
with	I-protein_state
ATP	B-chemical
,	O
we	O
soaked	B-experimental_method
the	O
apo	B-protein_state
-	O
crystals	B-evidence
in	O
the	O
reservoir	O
adding	O
cofactor	O
ATP	B-chemical
,	O
and	O
obtained	O
the	O
structures	B-evidence
of	O
SePSK	B-protein
and	O
AtXK	B-protein
-	I-protein
1	I-protein
bound	B-protein_state
with	I-protein_state
ATP	B-chemical
at	O
the	O
resolution	O
of	O
2	O
.	O
3	O
Å	O
and	O
1	O
.	O
8	O
Å	O
,	O
respectively	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

Structure	B-evidence
of	O
SePSK	B-protein
in	B-protein_state
complex	I-protein_state
with	I-protein_state
AMP	B-chemical
-	I-chemical
PNP	I-chemical
.	O

(	O
A	O
)	O
The	O
electron	B-evidence
density	I-evidence
of	O
AMP	B-chemical
-	I-chemical
PNP	I-chemical
.	O

The	O
AMP	B-chemical
-	I-chemical
PNP	I-chemical
is	O
depicted	O
as	O
sticks	O
with	O
its	O
ǀFoǀ	B-evidence
-	I-evidence
ǀFcǀ	I-evidence
map	I-evidence
contoured	O
at	O
3	O
σ	O
shown	O
as	O
cyan	O
mesh	O
.	O

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

The	O
AMP	B-chemical
-	I-chemical
PNP	I-chemical
and	O
coordinated	O
residues	O
are	O
shown	O
as	O
sticks	O
.	O

The	O
potential	O
substrate	B-site
binding	I-site
site	I-site
in	O
SePSK	B-protein

The	O
RBL1	B-residue_name_number
and	O
RBL2	B-residue_name_number
are	O
depicted	O
as	O
sticks	O
.	O
(	O
B	O
)	O
Interaction	O
of	O
two	O
D	B-chemical
-	I-chemical
ribulose	I-chemical
molecules	O
(	O
RBL1	B-residue_name_number
and	O
RBL2	B-residue_name_number
)	O
with	O
SePSK	B-protein
.	O

The	O
RBL	B-chemical
molecules	O
(	O
carbon	O
atoms	O
colored	O
yellow	O
)	O
and	O
amino	O
acid	O
residues	O
of	O
SePSK	B-protein
(	O
carbon	O
atoms	O
colored	O
green	O
)	O
involved	O
in	O
RBL	B-chemical
interaction	O
are	O
shown	O
as	O
sticks	O
.	O

The	O
hydroxyl	O
group	O
of	O
Ser12	B-residue_name_number
coordinates	B-bond_interaction
with	I-bond_interaction
O2	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

In	O
the	O
RBL	B-complex_assembly
-	I-complex_assembly
SePSK	I-complex_assembly
structure	B-evidence
,	O
a	O
2	O
.	O
6	O
Å	O
hydrogen	B-bond_interaction
bond	I-bond_interaction
is	O
present	O
between	O
RBL2	B-residue_name_number
and	O
Ser12	B-residue_name_number
(	O
Fig	O
4B	O
),	O
while	O
in	O
the	O
AtXK	B-protein
-	I-protein
1	I-protein
structure	B-evidence
this	O
hydrogen	B-bond_interaction
bond	I-bond_interaction
with	O
the	O
corresponding	O
residue	O
(	O
Ser22	B-residue_name_number
)	O
is	O
broken	O
.	O

This	O
change	O
might	O
be	O
the	O
reason	O
that	O
AtXK	B-protein
-	I-protein
1	I-protein
only	O
shows	O
limited	O
increasing	O
in	O
its	O
ATP	B-chemical
hydrolysis	O
ability	O
upon	O
adding	O
D	B-chemical
-	I-chemical
ribulose	I-chemical
as	O
a	O
substrate	O
after	O
comparing	O
with	O
SePSK	B-protein
(	O
Fig	O
2C	O
).	O

The	O
results	O
showed	O
that	O
the	O
affinity	B-evidence
of	O
D8A	B-mutant
-	O
SePSK	B-protein
with	O
D	B-chemical
-	I-chemical
ribulose	I-chemical
is	O
weaker	O
than	O
that	O
of	O
WT	B-protein_state
with	O
a	O
reduction	O
of	O
approx	O
.	O

This	O
distance	O
between	O
RBL2	B-residue_name_number
and	O
AMP	B-chemical
-	I-chemical
PNP	I-chemical
-	O
γ	O
-	O
phosphate	B-chemical
is	O
close	O
enough	O
to	O
facilitate	O
phosphate	B-chemical
transferring	O
.	O

Together	O
,	O
our	O
superposition	B-experimental_method
results	O
provided	O
snapshots	O
of	O
the	O
conformational	O
changes	O
at	O
different	O
catalytic	O
stages	O
of	O
SePSK	B-protein
and	O
potentially	O
revealed	O
the	O
closed	B-protein_state
form	O
of	O
SePSK	B-protein
.	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

We	O
now	O
present	O
cryo	B-experimental_method
-	I-experimental_method
electron	I-experimental_method
microscopy	I-experimental_method
3D	B-evidence
reconstructions	I-evidence
of	O
the	O
E	B-species
.	I-species
coli	I-species
LdcI	B-protein
and	O
LdcC	B-protein
,	O
and	O
an	O
improved	B-evidence
map	I-evidence
of	O
the	O
LdcI	B-protein
bound	B-protein_state
to	I-protein_state
the	O
LARA	B-structure_element
domain	I-structure_element
of	O
RavA	B-protein
,	O
at	O
pH	B-protein_state
optimal	I-protein_state
for	O
their	O
enzymatic	O
activity	O
.	O

They	O
counteract	O
acid	O
stress	O
experienced	O
by	O
the	O
bacterium	B-taxonomy_domain
in	O
the	O
host	O
digestive	O
and	O
urinary	O
tract	O
,	O
and	O
in	O
particular	O
in	O
the	O
extremely	O
acidic	O
stomach	O
.	O

Monomers	B-oligomeric_state
tightly	O
associate	O
via	O
their	O
core	B-structure_element
domains	I-structure_element
into	O
2	B-protein_state
-	I-protein_state
fold	I-protein_state
symmetrical	I-protein_state
dimers	B-oligomeric_state
with	O
two	O
complete	O
active	B-site
sites	I-site
,	O
and	O
further	O
build	O
a	O
toroidal	B-structure_element
D5	I-structure_element
-	I-structure_element
symmetrical	I-structure_element
structure	I-structure_element
held	O
by	O
the	O
wing	B-structure_element
and	O
core	B-structure_element
domain	I-structure_element
interactions	O
around	O
the	O
central	B-structure_element
pore	I-structure_element
,	O
with	O
the	O
CTDs	B-structure_element
at	O
the	O
periphery	O
.	O

This	O
allowed	O
us	O
to	O
make	O
a	O
pseudoatomic	B-evidence
model	I-evidence
of	O
the	O
whole	O
assembly	O
,	O
underpinned	O
by	O
a	O
cryoEM	B-experimental_method
map	B-evidence
of	O
the	O
LdcI	B-complex_assembly
-	I-complex_assembly
LARA	I-complex_assembly
complex	O
(	O
with	O
LARA	B-structure_element
standing	O
for	O
LdcI	B-structure_element
associating	I-structure_element
domain	I-structure_element
of	I-structure_element
RavA	I-structure_element
),	O
and	O
to	O
identify	O
conformational	O
rearrangements	O
and	O
specific	O
elements	O
essential	O
for	O
complex	O
formation	O
.	O

The	O
main	O
determinants	O
of	O
the	O
LdcI	B-complex_assembly
-	I-complex_assembly
RavA	I-complex_assembly
cage	O
assembly	O
appeared	O
to	O
be	O
the	O
N	O
-	O
terminal	O
loop	B-structure_element
of	O
the	O
LARA	B-structure_element
domain	I-structure_element
of	O
RavA	B-protein
and	O
the	O
C	O
-	O
terminal	O
β	B-structure_element
-	I-structure_element
sheet	I-structure_element
of	O
LdcI	B-protein
.	O

Finally	O
,	O
we	O
performed	O
multiple	B-experimental_method
sequence	I-experimental_method
alignment	I-experimental_method
of	O
22	O
lysine	B-protein_type
decarboxylases	I-protein_type
from	O
Enterobacteriaceae	B-taxonomy_domain
containing	O
the	O
ravA	B-gene
-	I-gene
viaA	I-gene
operon	I-gene
in	O
their	O
genome	O
.	O

Significant	O
differences	O
between	O
these	O
pseudoatomic	B-evidence
models	I-evidence
can	O
be	O
interpreted	O
as	O
movements	O
between	O
specific	O
biological	O
states	O
of	O
the	O
proteins	O
as	O
described	O
below	O
.	O

Both	O
visual	B-experimental_method
inspection	I-experimental_method
(	O
Fig	O
.	O
2	O
)	O
and	O
RMSD	B-experimental_method
calculations	I-experimental_method
(	O
Table	O
S2	O
)	O
show	O
that	O
globally	O
the	O
three	O
structures	B-evidence
at	O
active	B-protein_state
pH	I-protein_state
(	O
LdcIa	B-protein
,	O
LdcI	B-complex_assembly
-	I-complex_assembly
LARA	I-complex_assembly
and	O
LdcC	B-protein
)	O
are	O
more	O
similar	O
to	O
each	O
other	O
than	O
to	O
the	O
structure	O
determined	O
at	O
high	B-protein_state
pH	I-protein_state
conditions	O
(	O
LdcIi	B-protein
).	O

The	O
core	B-structure_element
domain	I-structure_element
is	O
built	O
by	O
the	O
PLP	B-structure_element
-	I-structure_element
binding	I-structure_element
subdomain	I-structure_element
(	O
PLP	B-structure_element
-	I-structure_element
SD	I-structure_element
,	O
residues	O
184	B-residue_range
–	I-residue_range
417	I-residue_range
)	O
flanked	O
by	O
two	O
smaller	O
subdomains	B-structure_element
rich	O
in	O
partly	B-protein_state
disordered	I-protein_state
loops	B-structure_element
–	O
the	O
linker	B-structure_element
region	I-structure_element
(	O
residues	O
130	B-residue_range
–	I-residue_range
183	I-residue_range
)	O
and	O
the	O
subdomain	B-structure_element
4	I-structure_element
(	O
residues	O
418	B-residue_range
–	I-residue_range
563	I-residue_range
).	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

At	O
this	O
resolution	O
,	O
the	O
apo	B-protein_state
-	O
LdcIi	B-protein
and	O
ppGpp	B-complex_assembly
-	I-complex_assembly
LdcIi	I-complex_assembly
structures	B-evidence
(	O
both	O
solved	O
at	O
pH	B-protein_state
8	I-protein_state
.	I-protein_state
5	I-protein_state
)	O
appeared	O
indistinguishable	O
except	O
for	O
the	O
presence	O
of	O
ppGpp	B-chemical
(	O
Fig	O
.	O
S11	O
in	O
ref	O
.	O
).	O

Yet	O
the	O
superposition	B-experimental_method
of	O
the	O
decamers	B-oligomeric_state
lays	O
bare	O
a	O
progressive	O
movement	O
of	O
the	O
CTD	B-structure_element
as	O
a	O
whole	O
upon	O
enzyme	O
activation	O
by	O
pH	O
and	O
the	O
binding	O
of	O
LARA	B-structure_element
.	O

On	O
the	O
contrary	O
,	O
introduction	B-experimental_method
of	O
the	O
C	O
-	O
terminal	O
β	B-structure_element
-	I-structure_element
sheet	I-structure_element
of	O
LdcI	B-protein
into	O
LdcC	B-protein
led	O
to	O
an	O
assembly	O
of	O
the	O
LdcCI	B-complex_assembly
-	I-complex_assembly
RavA	I-complex_assembly
complex	O
.	O

(	O
A	O
,	O
C	O
,	O
E	O
)	O
cryoEM	B-experimental_method
map	B-evidence
of	O
the	O
LdcC	B-protein
(	O
A	O
),	O
LdcIa	B-protein
(	O
C	O
)	O
and	O
LdcI	B-complex_assembly
-	I-complex_assembly
LARA	I-complex_assembly
(	O
E	O
)	O
decamers	B-oligomeric_state
with	O
one	O
protomer	B-oligomeric_state
in	O
light	O
grey	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

Conformational	O
rearrangements	O
in	O
the	O
enzyme	O
active	B-site
site	I-site
.	O

(	O
A	O
)	O
A	O
slice	O
through	O
the	O
pseudoatomic	B-evidence
models	I-evidence
of	O
the	O
LdcIa	B-protein
(	O
purple	O
)	O
and	O
LdcC	B-protein
(	O
green	O
)	O
monomers	B-oligomeric_state
extracted	O
from	O
the	O
superimposed	B-experimental_method
decamers	B-oligomeric_state
(	O
Fig	O
.	O
2	O
).	O
(	O
B	O
)	O
The	O
C	O
-	O
terminal	O
β	B-structure_element
-	I-structure_element
sheet	I-structure_element
in	O
LdcIa	B-protein
and	O
LdcC	B-protein
enlarged	O
from	O
(	O
A	O
,	O
C	O
)	O
Exchanged	O
primary	O
sequences	O
(	O
capital	O
letters	O
)	O
and	O
their	O
immediate	O
vicinity	O
(	O
lower	O
case	O
letters	O
)	O
colored	O
as	O
in	O
(	O
A	O
,	O
B	O
),	O
with	O
the	O
corresponding	O
secondary	O
structure	O
elements	O
and	O
the	O
amino	O
acid	O
numbering	O
shown	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

Structural	O
basis	O
for	O
Mep2	B-protein_type
ammonium	B-protein_type
transceptor	I-protein_type
activation	O
by	O
phosphorylation	B-ptm

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

While	O
most	O
studies	O
have	O
focused	O
on	O
the	O
Saccharomyces	B-species
cerevisiae	I-species
transceptors	B-protein_type
for	O
phosphate	B-chemical
(	O
Pho84	B-protein
),	O
amino	B-chemical
acids	I-chemical
(	O
Gap1	B-protein
)	O
and	O
ammonium	B-chemical
(	O
Mep2	B-protein
),	O
transceptors	B-protein_type
are	O
found	O
in	O
higher	B-taxonomy_domain
eukaryotes	I-taxonomy_domain
as	O
well	O
(	O
for	O
example	O
,	O
the	O
mammalian	B-taxonomy_domain
SNAT2	B-protein
amino	B-protein_type
-	I-protein_type
acid	I-protein_type
transporter	I-protein_type
and	O
the	O
GLUT2	B-protein
glucose	B-protein_type
transporter	I-protein_type
).	O

Of	O
these	O
,	O
only	O
Mep2	B-protein_type
proteins	I-protein_type
function	O
as	O
ammonium	B-chemical
receptors	O
/	O
sensors	O
in	O
fungal	B-taxonomy_domain
development	O
.	O

In	O
bacteria	B-taxonomy_domain
,	O
amt	B-gene
genes	O
are	O
present	O
in	O
an	O
operon	O
with	O
glnK	B-gene
,	O
encoding	O
a	O
PII	B-protein_type
-	I-protein_type
like	I-protein_type
signal	I-protein_type
transduction	I-protein_type
class	I-protein_type
protein	I-protein_type
.	O

Under	O
conditions	O
of	O
nitrogen	B-chemical
limitation	O
,	O
GlnK	B-protein_type
becomes	O
uridylated	B-protein_state
,	O
blocking	O
its	O
ability	O
to	O
bind	O
and	O
inhibit	O
Amt	B-protein_type
proteins	I-protein_type
.	O

(	O
root	B-evidence
mean	I-evidence
square	I-evidence
deviation	I-evidence
)=	O
0	O
.	O
7	O
Å	O
for	O
434	O
residues	O
),	O
with	O
the	O
main	O
differences	O
confined	O
to	O
the	O
N	O
terminus	O
and	O
the	O
CTR	B-structure_element
(	O
Fig	O
.	O
1	O
).	O

The	O
N	O
termini	O
of	O
the	O
Mep2	B-protein_type
proteins	I-protein_type
are	O
∼	O
20	B-residue_range
–	I-residue_range
25	I-residue_range
residues	O
longer	O
compared	O
with	O
their	O
bacterial	B-taxonomy_domain
counterparts	O
(	O
Figs	O
1	O
and	O
2	O
),	O
substantially	O
increasing	O
the	O
size	O
of	O
the	O
extracellular	B-structure_element
domain	I-structure_element
.	O

The	O
N	O
-	O
terminal	O
vestibule	B-structure_element
and	O
the	O
resulting	O
inter	O
-	O
monomer	B-oligomeric_state
interactions	O
likely	O
increase	O
the	O
stability	O
of	O
the	O
Mep2	B-protein
trimer	B-oligomeric_state
,	O
in	O
support	O
of	O
data	O
for	O
plant	B-taxonomy_domain
AMT	B-protein_type
proteins	I-protein_type
.	O

The	O
head	O
group	O
of	O
Arg54	B-residue_name_number
has	O
moved	O
∼	O
11	O
Å	O
relative	O
to	O
that	O
in	O
Amt	B-protein
-	I-protein
1	I-protein
,	O
whereas	O
the	O
shift	O
of	O
the	O
head	O
group	O
of	O
the	O
variable	O
Lys55	B-residue_name_number
residue	O
is	O
almost	O
20	O
Å	O
.	O
The	O
side	O
chain	O
of	O
Lys56	B-residue_name_number
in	O
the	O
basic	B-protein_state
motif	B-structure_element
points	O
in	O
an	O
opposite	O
direction	O
in	O
the	O
Mep2	B-protein
structures	B-evidence
compared	O
with	O
that	O
of	O
,	O
for	O
example	O
,	O
Amt	B-protein
-	I-protein
1	I-protein
(	O
Fig	O
.	O
4	O
).	O

Significantly	O
,	O
this	O
is	O
also	O
true	O
for	O
ScMep2	B-protein
,	O
which	O
was	O
crystallized	B-experimental_method
in	O
the	O
presence	O
of	O
0	O
.	O
2	O
M	O
ammonium	B-chemical
ions	O
(	O
see	O
Methods	O
section	O
).	O

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

These	O
residues	O
include	O
those	O
of	O
the	O
‘	B-structure_element
ExxGxD	I-structure_element
'	I-structure_element
motif	I-structure_element
,	O
which	O
when	O
mutated	B-experimental_method
generate	O
inactive	B-protein_state
transporters	B-protein_type
.	O

In	O
Amt	B-protein
-	I-protein
1	I-protein
and	O
other	O
bacterial	B-taxonomy_domain
ammonium	B-protein_type
transporters	I-protein_type
,	O
these	O
CTR	B-structure_element
residues	O
interact	O
with	O
residues	O
within	O
the	O
N	B-structure_element
-	I-structure_element
terminal	I-structure_element
half	I-structure_element
of	O
the	O
protein	O
.	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

This	O
interaction	O
in	O
the	O
centre	O
of	O
the	O
protein	O
may	O
be	O
particularly	O
important	O
to	O
stabilize	O
the	O
open	B-protein_state
conformations	O
of	O
ammonium	B-protein_type
transporters	I-protein_type
.	O

Where	O
is	O
the	O
AI	B-structure_element
region	I-structure_element
and	O
the	O
Npr1	B-protein
phosphorylation	B-site
site	I-site
located	O
?	O
Our	O
structures	B-evidence
reveal	O
that	O
surprisingly	O
,	O
the	O
AI	B-structure_element
region	I-structure_element
is	O
folded	O
back	O
onto	O
the	O
CTR	B-structure_element
and	O
is	O
not	O
located	O
near	O
the	O
centre	O
of	O
the	O
trimer	B-oligomeric_state
as	O
expected	O
from	O
the	O
bacterial	B-taxonomy_domain
structures	B-evidence
(	O
Fig	O
.	O
4	O
).	O

The	O
AI	B-structure_element
regions	I-structure_element
have	O
very	O
similar	O
conformations	O
in	O
CaMep2	B-protein
and	O
ScMep2	B-protein
,	O
despite	O
considerable	O
differences	O
in	O
the	O
rest	O
of	O
the	O
CTR	B-structure_element
(	O
Fig	O
.	O
6	O
).	O

This	O
makes	O
sense	O
since	O
the	O
proteins	O
were	O
expressed	O
in	O
rich	O
medium	O
and	O
confirms	O
the	O
recent	O
suggestion	O
by	O
Boeckstaens	O
et	O
al	O
.	O
that	O
the	O
non	B-protein_state
-	I-protein_state
phosphorylated	I-protein_state
form	O
of	O
Mep2	B-protein
corresponds	O
to	O
the	O
inactive	B-protein_state
state	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

Mep2	B-protein
lacking	B-protein_state
the	O
AI	B-structure_element
region	I-structure_element
is	O
conformationally	B-protein_state
heterogeneous	I-protein_state

Density	B-evidence
for	O
ICL3	B-structure_element
and	O
the	O
CTR	B-structure_element
beyond	O
residue	O
Arg415	B-residue_name_number
is	O
missing	O
in	O
the	O
442Δ	B-mutant
mutant	B-protein_state
,	O
and	O
the	O
density	B-evidence
for	O
the	O
other	O
ICLs	B-structure_element
including	O
ICL1	B-structure_element
is	O
generally	O
poor	O
with	O
visible	O
parts	O
of	O
the	O
structure	B-evidence
having	O
high	O
B	O
-	O
factors	O
(	O
Fig	O
.	O
7	O
).	O

Why	O
then	O
does	O
this	O
mutant	O
appear	O
to	O
be	O
constitutively	O
active	B-protein_state
?	O
We	O
propose	O
two	O
possibilities	O
.	O

The	O
latter	O
model	O
would	O
fit	O
well	O
with	O
the	O
NH3	B-chemical
/	O
H	B-chemical
+	I-chemical
symport	O
model	O
in	O
which	O
the	O
proton	O
is	O
relayed	O
by	O
the	O
twin	B-structure_element
-	I-structure_element
His	I-structure_element
motif	I-structure_element
.	O

To	O
test	O
this	O
hypothesis	O
,	O
we	O
determined	B-experimental_method
the	O
structure	B-evidence
of	O
the	O
phosphorylation	B-protein_state
-	I-protein_state
mimicking	I-protein_state
R452D	B-mutant
/	I-mutant
S453D	I-mutant
protein	O
(	O
hereafter	O
termed	O
‘	O
DD	B-mutant
mutant	I-mutant
'),	O
using	O
data	O
to	O
a	O
resolution	O
of	O
2	O
.	O
4	O
Å	O
.	O
The	O
additional	B-experimental_method
mutation	I-experimental_method
of	I-experimental_method
the	O
arginine	B-residue_name
preceding	O
the	O
phosphorylation	B-site
site	I-site
was	O
introduced	O
(	O
i	O
)	O
to	O
increase	O
the	O
negative	O
charge	O
density	O
and	O
make	O
it	O
more	O
comparable	O
to	O
a	O
phosphate	B-chemical
at	O
neutral	O
pH	O
,	O
and	O
(	O
ii	O
)	O
to	O
further	O
destabilize	O
the	O
interactions	O
of	O
the	O
AI	B-structure_element
region	I-structure_element
with	O
the	O
main	B-structure_element
body	I-structure_element
of	O
the	O
transporter	B-protein_type
(	O
Fig	O
.	O
6	O
).	O

In	O
addition	O
,	O
residues	O
Glu420	B-residue_range
-	I-residue_range
Leu423	I-residue_range
including	O
Glu421	B-residue_name_number
of	O
the	O
ExxGxD	B-structure_element
motif	I-structure_element
are	O
now	O
disordered	B-protein_state
(	O
Fig	O
.	O
8	O
and	O
Supplementary	O
Fig	O
.	O
3	O
).	O

The	O
protein	O
backbone	O
has	O
an	O
average	O
r	B-evidence
.	I-evidence
m	I-evidence
.	I-evidence
s	I-evidence
.	I-evidence
d	I-evidence
.	I-evidence
of	O
only	O
∼	O
3	O
Å	O
during	O
the	O
200	O
-	O
ns	O
simulation	B-experimental_method
,	O
indicating	O
that	O
the	O
protein	O
is	O
stable	B-protein_state
.	O

There	O
is	O
flexibility	O
in	O
the	O
side	O
chains	O
of	O
the	O
acidic	O
residues	O
so	O
that	O
they	O
are	O
able	O
to	O
form	O
stable	B-protein_state
hydrogen	B-bond_interaction
bonds	I-bond_interaction
with	O
Ser453	B-residue_name_number
.	O

For	O
example	O
,	O
the	O
distance	B-evidence
between	O
the	O
Asp453	B-residue_name_number
acidic	O
oxygens	O
and	O
the	O
Glu420	B-residue_name_number
acidic	O
oxygens	O
increases	O
from	O
∼	O
7	O
to	O
>	O
22	O
Å	O
after	O
200	O
ns	O
simulations	B-experimental_method
,	O
and	O
thus	O
these	O
residues	O
are	O
not	O
interacting	O
.	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

More	O
specifically	O
,	O
the	O
close	O
interactions	O
between	O
the	O
CTR	B-structure_element
and	O
ICL1	B-structure_element
/	O
ICL3	B-structure_element
present	O
in	O
open	B-protein_state
transporters	B-protein_type
are	O
disrupted	O
,	O
causing	O
ICL3	B-structure_element
to	O
move	O
outwards	O
and	O
block	O
the	O
channel	B-site
(	O
Figs	O
4	O
and	O
9a	O
).	O

However	O
,	O
even	O
the	O
otherwise	O
highly	O
similar	O
Mep2	B-protein_type
proteins	I-protein_type
of	O
S	B-species
.	I-species
cerevisiae	I-species
and	O
C	B-species
.	I-species
albicans	I-species
have	O
different	O
structures	B-evidence
for	O
their	O
CTRs	B-structure_element
(	O
Fig	O
.	O
1	O
and	O
Supplementary	O
Fig	O
.	O
6	O
).	O

In	O
addition	O
,	O
the	O
considerable	O
differences	O
between	O
structurally	O
resolved	O
CTR	B-structure_element
domains	O
means	O
that	O
the	O
exact	O
environment	O
of	O
T460	B-residue_name_number
in	O
Amt	B-protein
-	I-protein
1	I-protein
;	I-protein
1	I-protein
is	O
also	O
not	O
known	O
(	O
Supplementary	O
Fig	O
.	O
6	O
).	O

(	O
a	O
)	O
The	O
triple	B-mutant
mepΔ	I-mutant
strain	O
(	O
black	O
)	O
and	O
triple	O
mepΔ	O
npr1Δ	O
strain	O
(	O
grey	O
)	O
containing	O
plasmids	O
expressing	O
WT	B-protein_state
and	O
variant	B-mutant
ScMep2	I-mutant
were	O
grown	B-experimental_method
on	I-experimental_method
minimal	I-experimental_method
medium	I-experimental_method
containing	O
1	O
mM	O
ammonium	B-chemical
sulphate	I-chemical
.	O

The	O
numbering	O
is	O
for	O
CaMep2	B-protein
.	O

Channel	O
closures	O
in	O
Mep2	B-protein
.	O

2Fo	O
–	O
Fc	O
electron	O
density	O
(	O
contoured	O
at	O
1	O
.	O
0	O
σ	O
)	O
for	O
residues	O
Tyr49	B-residue_name_number
and	O
His342	B-residue_name_number
is	O
shown	O
for	O
the	O
truncation	B-protein_state
mutant	I-protein_state
.	O

The	O
arrow	O
indicates	O
the	O
phosphorylation	B-site
site	I-site
.	O

Upon	O
phosphorylation	B-ptm
and	O
mimicked	B-protein_state
by	O
the	O
CaMep2	B-protein
S453D	B-mutant
and	O
DD	B-mutant
mutants	I-mutant
(	O
ii	O
),	O
the	O
region	O
around	O
the	O
ExxGxD	B-structure_element
motif	I-structure_element
undergoes	O
a	O
conformational	O
change	O
that	O
results	O
in	O
the	O
CTR	B-structure_element
interacting	O
with	O
the	O
inward	O
-	O
moving	O
ICL3	B-structure_element
,	O
opening	O
the	O
channel	B-site
(	O
full	O
circle	O
)	O
(	O
iii	O
).	O

Once	O
a	O
candidate	O
antibody	B-protein_type
is	O
identified	O
,	O
protein	B-experimental_method
engineering	I-experimental_method
is	O
usually	O
required	O
to	O
produce	O
a	O
molecule	O
with	O
the	O
right	O
biophysical	O
and	O
functional	O
properties	O
.	O

The	O
sequence	O
diversity	O
of	O
the	O
CDR	B-structure_element
regions	I-structure_element
presents	O
a	O
substantial	O
challenge	O
to	O
antibody	B-protein_type
modeling	O
.	O

In	O
contrast	O
to	O
CDRs	B-structure_element
L1	B-structure_element
,	O
L2	B-structure_element
,	O
L3	B-structure_element
,	O
H1	B-structure_element
and	O
H2	B-structure_element
,	O
no	O
canonical	O
structures	B-evidence
have	O
been	O
observed	O
for	O
CDR	B-structure_element
H3	B-structure_element
,	O
which	O
is	O
the	O
most	O
variable	O
in	O
length	O
and	O
amino	O
acid	O
sequence	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

To	O
support	O
antibody	B-protein_type
engineering	O
and	O
therapeutic	O
development	O
efforts	O
,	O
a	O
phage	B-experimental_method
library	I-experimental_method
was	O
designed	O
and	O
constructed	O
based	O
on	O
a	O
limited	O
number	O
of	O
scaffolds	O
built	O
with	O
frequently	O
used	O
human	B-species
germ	O
-	O
line	O
IGV	B-structure_element
and	O
IGJ	B-structure_element
gene	O
segments	O
that	O
encode	O
antigen	B-site
combining	I-site
sites	I-site
suitable	O
for	O
recognition	O
of	O
peptides	O
and	O
proteins	O
.	O

Variations	O
occur	O
in	O
the	O
pH	O
(	O
buffer	O
)	O
and	O
the	O
additives	O
,	O
and	O
,	O
in	O
group	O
3	O
,	O
PEG	B-chemical
3350	I-chemical
is	O
the	O
precipitant	O
for	O
one	O
variants	O
while	O
ammonium	B-chemical
sulfate	I-chemical
is	O
the	O
precipitant	O
for	O
the	O
other	O
two	O
.	O

Apart	O
from	O
the	O
C	O
-	O
terminus	O
,	O
only	O
a	O
few	O
surface	O
residues	O
in	O
LC	B-structure_element
are	O
disordered	B-protein_state
.	O

The	O
HCs	B-structure_element
feature	O
the	O
largest	O
number	O
of	O
disordered	B-protein_state
residues	O
,	O
with	O
the	O
lower	O
resolution	O
structures	B-evidence
having	O
the	O
most	O
.	O

CDR	B-structure_element
H1	B-structure_element
and	O
CDR	B-structure_element
H2	B-structure_element
also	O
show	O
some	O
degree	O
of	O
disorder	B-protein_state
,	O
but	O
to	O
a	O
lesser	O
extent	O
.	O

Three	O
of	O
the	O
HCs	B-structure_element
,	O
H3	B-mutant
-	I-mutant
23	I-mutant
,	O
H3	B-mutant
-	I-mutant
53	I-mutant
and	O
H5	B-mutant
-	I-mutant
51	I-mutant
,	O
have	O
the	O
same	O
canonical	O
structure	O
,	O
H1	B-mutant
-	I-mutant
13	I-mutant
-	I-mutant
1	I-mutant
,	O
and	O
the	O
backbone	O
conformations	O
are	O
tightly	O
clustered	O
for	O
each	O
set	O
of	O
Fab	B-structure_element
structures	B-evidence
as	O
reflected	O
in	O
the	O
rmsd	B-evidence
values	I-evidence
(	O
Fig	O
.	O
1B	O
-	O
D	O
).	O

Each	O
of	O
the	O
4	O
HCs	B-structure_element
adopts	O
only	O
one	O
canonical	O
structure	O
regardless	O
of	O
the	O
pairing	O
LC	B-structure_element
.	O

Germlines	O
H1	B-mutant
-	I-mutant
69	I-mutant
and	O
H5	B-mutant
-	I-mutant
51	I-mutant
have	O
the	O
same	O
canonical	O
structure	O
assignment	O
H2	B-mutant
-	I-mutant
10	I-mutant
-	I-mutant
1	I-mutant
,	O
H3	B-mutant
-	I-mutant
23	I-mutant
has	O
H2	B-mutant
-	I-mutant
10	I-mutant
-	I-mutant
2	I-mutant
,	O
and	O
H3	B-mutant
-	I-mutant
53	I-mutant
has	O
H2	B-mutant
-	I-mutant
9	I-mutant
-	I-mutant
3	I-mutant
.	O

Germlines	O
H1	B-mutant
-	I-mutant
69	I-mutant
and	O
H5	B-mutant
-	I-mutant
51	I-mutant
are	O
unique	O
in	O
the	O
human	B-species
repertoire	O
in	O
having	O
an	O
Ala	B-residue_name
at	O
position	O
71	B-residue_number
that	O
leaves	O
enough	O
space	O
for	O
H	B-structure_element
-	O
Pro52a	B-residue_name_number
to	O
pack	O
deeper	O
against	O
CDR	B-structure_element
H4	B-structure_element
so	O
that	O
the	O
following	O
residues	O
53	B-residue_number
and	O
54	B-residue_number
point	O
toward	O
the	O
putative	O
antigen	O
.	O

However	O
,	O
there	O
is	O
a	O
significant	O
shift	O
of	O
the	O
CDR	B-structure_element
as	O
a	O
rigid	O
body	O
when	O
the	O
2	O
sets	O
are	O
superimposed	B-experimental_method
.	O

Germline	O
H1	B-mutant
-	I-mutant
69	I-mutant
has	O
Ala	B-residue_name
at	O
position	O
33	B-residue_number
whereas	O
in	O
H5	B-mutant
-	I-mutant
51	I-mutant
position	O
33	B-residue_number
is	O
occupied	O
by	O
a	O
bulky	O
Trp	B-residue_name
,	O
which	O
stacks	O
against	O
H	B-structure_element
-	O
Tyr52	B-residue_name_number
and	O
drives	O
CDR	B-structure_element
H2	B-structure_element
away	O
from	O
the	O
center	O
.	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

L3	B-mutant
-	I-mutant
20	I-mutant
is	O
the	O
most	O
variable	O
in	O
CDR	B-structure_element
L1	B-structure_element
among	O
the	O
4	O
germlines	O
as	O
indicated	O
by	O
an	O
rmsd	B-evidence
of	O
0	O
.	O
54	O
Å	O
(	O
Fig	O
.	O
3C	O
).	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
fourth	O
member	O
of	O
the	O
set	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
,	O
was	O
crystallized	B-experimental_method
with	O
2	O
Fabs	B-structure_element
in	O
the	O
asymmetric	O
unit	O
.	O

As	O
mentioned	O
earlier	O
,	O
all	O
16	O
Fabs	B-structure_element
have	O
the	O
same	O
CDR	B-structure_element
H3	B-structure_element
,	O
for	O
which	O
the	O
amino	O
acid	O
sequence	O
is	O
derived	O
from	O
the	O
anti	O
-	O
CCL2	O
antibody	B-protein_type
CNTO	B-chemical
888	I-chemical
.	O

The	O
variations	O
in	O
CDR	B-structure_element
H3	B-structure_element
conformation	O
are	O
illustrated	O
in	O
Fig	O
.	O
6	O
for	O
the	O
18	O
Fab	B-structure_element
structures	B-evidence
that	O
have	O
ordered	O
backbone	O
atoms	O
.	O

(	O
B	O
)	O
The	O
“	O
extended	B-protein_state
”	O
CDR	B-structure_element
H3	B-structure_element
of	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
with	O
green	O
carbon	O
atoms	O
and	O
yellow	O
dashed	O
lines	O
connecting	O
the	O
H	O
-	O
bond	O
pairs	O
for	O
Asp101	B-residue_name_number
OD1	O
and	O
OD2	O
and	O
Trp103	B-residue_name_number
NE1	O
.	O

The	O
remaining	O
8	O
Fabs	B-structure_element
can	O
be	O
grouped	O
into	O
5	O
different	O
conformational	O
classes	O
.	O

Position	O
43	B-residue_number
may	O
be	O
alternatively	O
occupied	O
by	O
Ser	B-residue_name
,	O
Val	B-residue_name
or	O
Pro	B-residue_name
(	O
as	O
in	O
L4	B-mutant
-	I-mutant
1	I-mutant
),	O
but	O
the	O
hydrophobic	B-bond_interaction
interaction	I-bond_interaction
with	O
H	B-structure_element
-	O
Tyr91	B-residue_name_number
is	O
preserved	O
.	O

In	O
most	O
of	O
the	O
structures	B-evidence
,	O
it	O
has	O
the	O
χ2	B-evidence
angle	O
of	O
∼	O
80	O
°,	O
while	O
the	O
ring	O
is	O
flipped	O
over	O
(	O
χ2	B-evidence
=	O
−	O
100	O
°)	O
in	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
.	O

In	O
fact	O
,	O
the	O
parameter	O
values	O
for	O
the	O
set	O
of	O
16	O
Fabs	B-structure_element
are	O
in	O
the	O
middle	O
of	O
the	O
distribution	O
observed	O
for	O
351	O
non	O
-	O
redundant	O
antibody	B-protein_type
structures	B-evidence
determined	O
at	O
3	O
.	O
0	O
Å	O
resolution	O
or	O
better	O
.	O

An	O
illustration	O
of	O
the	O
difference	O
in	O
tilt	O
angle	O
for	O
2	O
pairs	O
of	O
variants	O
by	O
the	O
superposition	B-experimental_method
of	O
the	O
VH	B-structure_element
domains	O
of	O
(	O
A	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
on	O
that	O
of	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
VL	B-structure_element
domain	O
is	O
off	O
by	O
a	O
rigid	O
-	O
body	O
roatation	O
of	O
10	O
.	O
5	O
°)	O
and	O
(	O
B	O
)	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
on	O
that	O
of	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
VL	B-structure_element
domain	O
is	O
off	O
by	O
a	O
rigid	O
-	O
body	O
roatation	O
of	O
1	O
.	O
6	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

VH	B-complex_assembly
:	I-complex_assembly
VL	I-complex_assembly
buried	O
surface	O
area	O
and	O
complementarity	O

Residues	O
in	O
CDR	B-structure_element
H3	B-structure_element
are	O
missing	O
:	O
YGE	B-structure_element
in	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
,	O
GIY	B-structure_element
in	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
.	O

This	O
is	O
the	O
first	O
report	O
of	O
a	O
systematic	B-experimental_method
structural	I-experimental_method
investigation	I-experimental_method
of	O
a	O
phage	B-experimental_method
germline	I-experimental_method
library	I-experimental_method
.	O

The	O
16	O
Fab	B-structure_element
structures	B-evidence
offer	O
a	O
unique	O
look	O
at	O
all	O
pairings	O
of	O
4	O
different	O
HCs	B-structure_element
(	O
H1	B-mutant
-	I-mutant
69	I-mutant
,	O
H3	B-mutant
-	I-mutant
23	I-mutant
,	O
H3	B-mutant
-	I-mutant
53	I-mutant
,	O
and	O
H5	B-mutant
-	I-mutant
51	I-mutant
)	O
and	O
4	O
different	O
LCs	B-structure_element
(	O
L1	B-mutant
-	I-mutant
39	I-mutant
,	O
L3	B-mutant
-	I-mutant
11	I-mutant
,	O
L3	B-mutant
-	I-mutant
20	I-mutant
and	O
L4	B-mutant
-	I-mutant
1	I-mutant
),	O
all	O
with	O
the	O
same	O
CDR	B-structure_element
H3	B-structure_element
.	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

Thus	O
,	O
it	O
is	O
likely	O
that	O
the	O
CDR	B-structure_element
H3	B-structure_element
conformation	O
is	O
dependent	O
upon	O
2	O
dominating	O
factors	O
:	O
1	O
)	O
amino	O
acid	O
sequence	O
;	O
and	O
2	O
)	O
VH	B-structure_element
and	O
VL	B-structure_element
context	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

The	O
same	O
variability	O
is	O
observed	O
for	O
the	O
sets	O
of	O
variants	O
composed	O
of	O
one	O
LC	B-structure_element
paired	O
with	O
each	O
of	O
the	O
4	O
HCs	B-structure_element
.	O

As	O
noted	O
in	O
the	O
Results	O
section	O
,	O
the	O
2	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
23	I-complex_assembly
:	I-complex_assembly
L3	I-complex_assembly
-	I-complex_assembly
20	I-complex_assembly
,	O
are	O
outliers	O
in	O
terms	O
of	O
the	O
tilt	B-evidence
angle	I-evidence
;	O
at	O
the	O
same	O
time	O
,	O
both	O
have	O
the	O
smallest	O
VH	B-site
:	I-site
VL	I-site
interface	I-site
.	O

Other	O
germlines	O
have	O
bulky	O
residues	O
,	O
Tyr	B-residue_name
,	O
Arg	B-residue_name
and	O
Trp	B-residue_name
,	O
at	O
these	O
positions	O
,	O
whereas	O
L1	B-mutant
-	I-mutant
39	I-mutant
has	O
Ser	B-residue_name
and	O
Thr	B-residue_name
.	O

A	O
more	O
compact	B-protein_state
CDR	B-structure_element
L3	B-structure_element
may	O
be	O
beneficial	O
in	O
this	O
situation	O
.	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

From	O
this	O
point	O
of	O
view	O
,	O
a	O
novel	O
approach	O
to	O
design	O
combinatorial	O
antibody	B-protein_type
libraries	O
would	O
be	O
to	O
cover	O
the	O
range	O
of	O
CDR	B-structure_element
conformations	O
that	O
may	O
not	O
necessarily	O
coincide	O
with	O
the	O
germline	O
usage	O
in	O
the	O
human	B-species
repertoire	O
.	O

This	O
study	O
resulted	O
in	O
a	O
series	O
of	O
snapshots	O
depicting	O
the	O
various	O
folding	O
states	O
of	O
Im7	B-protein
while	O
bound	B-protein_state
to	I-protein_state
Spy	B-protein
.	O

Recent	O
advances	O
in	O
X	B-experimental_method
-	I-experimental_method
ray	I-experimental_method
crystallography	I-experimental_method
and	O
NMR	B-experimental_method
spectroscopy	I-experimental_method
continue	O
to	O
improve	O
our	O
ability	O
to	O
analyze	O
biomolecules	O
that	O
exist	O
in	O
multiple	O
conformations	O
.	O

X	B-experimental_method
-	I-experimental_method
ray	I-experimental_method
crystallography	I-experimental_method
has	O
historically	O
provided	O
valuable	O
information	O
on	O
small	O
-	O
scale	O
conformational	O
changes	O
,	O
but	O
observing	O
large	O
-	O
amplitude	O
heterogeneous	O
conformational	O
changes	O
often	O
falls	O
beyond	O
the	O
reach	O
of	O
current	O
crystallographic	O
techniques	O
.	O

However	O
,	O
modeling	O
of	O
the	O
substrate	O
in	O
the	O
complex	O
proved	O
to	O
be	O
a	O
substantial	O
challenge	O
,	O
as	O
the	O
electron	B-evidence
density	I-evidence
of	O
the	O
substrate	O
was	O
discontinuous	O
and	O
fragmented	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

Its	O
strong	O
anomalous	B-evidence
scattering	I-evidence
allowed	O
us	O
to	O
track	O
the	O
positions	O
of	O
these	O
individual	O
Im76	B-mutant
-	I-mutant
45	I-mutant
residues	O
one	O
at	O
a	O
time	O
,	O
potentially	O
even	O
if	O
the	O
residue	O
was	O
found	O
in	O
several	O
locations	O
in	O
the	O
same	O
crystal	B-evidence
.	O

Together	O
,	O
these	O
results	O
indicated	O
that	O
the	O
Im7	B-protein
substrate	O
binds	O
Spy	B-protein
in	O
multiple	O
conformations	O
.	O

To	O
generate	O
an	O
accurate	O
depiction	O
of	O
the	O
chaperone	B-protein_type
-	O
substrate	O
interactions	O
,	O
we	O
devised	O
a	O
selection	O
protocol	O
based	O
on	O
a	O
sample	B-experimental_method
-	I-experimental_method
and	I-experimental_method
-	I-experimental_method
select	I-experimental_method
procedure	O
employed	O
in	O
NMR	B-experimental_method
spectroscopy	I-experimental_method
.	O

The	O
coarse	B-experimental_method
-	I-experimental_method
grained	I-experimental_method
simulations	I-experimental_method
are	O
based	O
on	O
a	O
single	O
-	O
residue	O
resolution	O
model	O
for	O
protein	O
folding	O
and	O
were	O
extended	O
here	O
to	O
describe	O
Spy	B-complex_assembly
-	I-complex_assembly
Im76	I-complex_assembly
-	I-complex_assembly
45	I-complex_assembly
binding	O
events	O
(	O
Online	O
Methods	O
).	O

To	O
accomplish	O
this	O
task	O
,	O
we	O
generated	O
a	O
compressed	O
version	O
of	O
the	O
experimental	O
2mFo	B-evidence
−	I-evidence
DFc	I-evidence
electron	I-evidence
density	I-evidence
map	I-evidence
for	O
use	O
in	O
the	O
selection	O
.	O

We	O
constructed	O
a	O
contact	B-evidence
map	I-evidence
of	O
the	O
complex	O
,	O
which	O
shows	O
the	O
frequency	O
of	O
interactions	O
for	O
chaperone	B-protein_type
-	O
substrate	O
residue	O
pairs	O
(	O
Fig	O
.	O
4	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

Once	O
the	O
substrate	O
begins	O
to	O
fold	O
within	O
this	O
protected	O
environment	O
,	O
it	O
progressively	O
buries	O
its	O
own	O
hydrophobic	O
residues	O
,	O
and	O
its	O
interactions	O
with	O
the	O
chaperone	B-protein_type
shift	O
towards	O
becoming	O
more	O
electrostatic	O
.	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

This	O
proximity	O
likely	O
causes	O
electrostatic	O
repulsion	O
that	O
destabilizes	O
Im7	B-protein
’	O
s	O
native	B-protein_state
state	O
.	O

In	O
conjunction	O
with	O
our	O
bound	B-protein_state
Im76	B-mutant
-	I-mutant
45	I-mutant
ensemble	B-evidence
,	O
these	O
mutants	O
now	O
allowed	O
us	O
to	O
investigate	O
structural	O
features	O
important	O
to	O
chaperone	B-protein_type
function	O
.	O

Despite	O
extensive	O
studies	O
,	O
exactly	O
how	O
complex	O
chaperone	B-protein_type
machines	O
help	O
proteins	O
fold	O
remains	O
controversial	O
.	O

Heterogeneous	O
dynamic	O
complexes	O
or	O
disordered	B-protein_state
regions	O
of	O
single	O
proteins	O
,	O
once	O
considered	O
solely	O
approachable	O
by	O
NMR	B-experimental_method
spectroscopy	I-experimental_method
,	O
can	O
now	O
be	O
visualized	O
through	O
X	B-experimental_method
-	I-experimental_method
ray	I-experimental_method
crystallography	I-experimental_method
.	O

Flowchart	O
of	O
the	O
READ	B-experimental_method
sample	B-experimental_method
-	I-experimental_method
and	I-experimental_method
-	I-experimental_method
select	I-experimental_method
process	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

(	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

(	O
b	O
)	O
F115	B-residue_name_number
and	O
L32	B-residue_name_number
tether	O
Spy	B-protein
’	O
s	O
linker	B-structure_element
region	I-structure_element
to	O
its	O
cradle	B-site
,	O
decreasing	O
Spy	B-protein
activity	O
by	O
limiting	O
linker	B-structure_element
region	I-structure_element
flexibility	O
.	O

Despite	O
a	O
long	O
history	O
of	O
physiological	O
and	O
functional	O
studies	O
,	O
the	O
molecular	O
mechanism	O
of	O
NCX	B-protein_type
has	O
been	O
elusive	O
,	O
owing	O
to	O
the	O
lack	O
of	O
structural	O
information	O
.	O

In	O
this	O
study	O
,	O
we	O
set	O
out	O
to	O
determine	O
the	O
structures	B-evidence
of	O
outward	B-protein_state
-	I-protein_state
facing	I-protein_state
wild	B-protein_state
-	I-protein_state
type	I-protein_state
NCX_Mj	B-protein
in	B-protein_state
complex	I-protein_state
with	I-protein_state
Na	B-chemical
+,	I-chemical
Ca2	B-chemical
+	I-chemical
and	O
Sr2	B-chemical
+,	I-chemical
at	O
various	O
concentrations	O
.	O

Extracellular	O
Na	B-chemical
+	I-chemical
binding	O

To	O
conclusively	O
clarify	O
this	O
assignment	O
,	O
we	O
first	O
set	O
out	O
to	O
examine	O
the	O
Na	B-chemical
+	I-chemical
occupancy	O
of	O
these	O
sites	O
without	O
Ca2	B-chemical
+.	I-chemical

X	B-experimental_method
-	I-experimental_method
ray	I-experimental_method
diffraction	I-experimental_method
of	O
these	O
soaked	O
crystals	B-evidence
revealed	O
a	O
Na	B-chemical
+-	I-chemical
dependent	O
variation	O
in	O
the	O
electron	B-evidence
-	I-evidence
density	I-evidence
distribution	I-evidence
at	O
sites	O
Sext	B-site
,	O
SCa	B-site
and	O
Sint	B-site
,	O
indicating	O
a	O
Na	B-chemical
+	I-chemical
occupancy	O
change	O
(	O
Fig	O
.	O
1c	O
).	O

Indeed	O
,	O
two	O
observations	O
indicate	O
that	O
a	O
water	B-chemical
molecule	O
rather	O
than	O
a	O
Na	B-chemical
+	I-chemical
ion	O
occupies	O
Smid	B-site
,	O
as	O
was	O
predicted	O
in	O
a	O
recent	O
simulation	B-experimental_method
study	O
.	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

TM7b	B-structure_element
occludes	O
the	O
four	O
central	B-site
binding	I-site
sites	I-site
from	O
the	O
external	O
solution	O
,	O
with	O
the	O
backbone	O
carbonyl	O
of	O
Ala206	B-residue_name_number
coordinating	B-bond_interaction
the	O
Na	B-chemical
+	I-chemical
ion	O
(	O
Fig	O
.	O
2b	O
-	O
d	O
).	O

Extracellular	O
Ca2	B-chemical
+	I-chemical
and	O
Sr2	B-chemical
+	I-chemical
binding	O
and	O
their	O
competition	O
with	O
Na	B-chemical
+	I-chemical

Binding	O
of	O
Ca2	B-chemical
+	I-chemical
to	O
both	O
sites	O
simultaneously	O
is	O
highly	O
improbable	O
due	O
to	O
their	O
close	O
proximity	O
,	O
and	O
at	O
least	O
one	O
water	B-chemical
molecule	O
can	O
be	O
discerned	O
coordinating	B-bond_interaction
the	O
ion	O
(	O
Fig	O
.	O
3b	O
).	O

Indeed	O
,	O
in	O
most	O
NCX	B-protein_type
proteins	O
Asp240	B-residue_name_number
is	O
substituted	B-experimental_method
by	O
Asn	B-residue_name
,	O
which	O
would	O
likely	O
weaken	O
or	O
abrogate	O
Ca2	B-chemical
+	I-chemical
binding	O
to	O
Smid	B-site
.	O

Although	O
the	O
binding	B-site
sites	I-site
are	O
thus	O
fully	B-protein_state
accessible	I-protein_state
to	O
the	O
external	O
solution	O
(	O
Fig	O
.	O
3e	O
),	O
the	O
lack	O
of	O
electron	B-evidence
density	I-evidence
therein	O
indicates	O
no	O
ions	O
or	O
ordered	O
solvent	O
molecules	O
.	O

Such	O
interpretation	O
would	O
be	O
consistent	O
with	O
the	O
computer	B-experimental_method
simulations	I-experimental_method
reported	O
below	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

As	O
it	O
happens	O
,	O
the	O
results	O
confirm	O
that	O
the	O
structures	B-evidence
now	O
available	O
are	O
representing	O
interconverting	O
states	O
of	O
the	O
functional	O
cycle	O
of	O
NCX_Mj	B-protein
,	O
while	O
revealing	O
how	O
the	O
alternating	O
-	O
access	O
mechanism	O
is	O
controlled	O
by	O
the	O
ion	O
-	O
occupancy	O
state	O
.	O

This	O
distortion	O
occludes	O
Sext	B-site
from	O
the	O
exterior	O
(	O
Fig	O
.	O
4d	O
,	O
4h	O
-	O
i	O
)	O
and	O
appears	O
to	O
be	O
induced	O
by	O
the	O
Na	B-chemical
+	I-chemical
ion	O
itself	O
,	O
which	O
pulls	O
the	O
carbonyl	O
group	O
of	O
A206	B-residue_name_number
into	O
its	O
coordination	O
sphere	O
(	O
Fig	O
.	O
4g	O
).	O

When	O
all	O
Na	B-site
+	I-site
sites	I-site
are	O
occupied	O
,	O
the	O
global	O
free	B-evidence
-	I-evidence
energy	I-evidence
minimum	I-evidence
corresponds	O
to	O
a	O
conformation	O
in	O
which	O
the	O
ions	O
are	O
maximally	O
coordinated	O
by	O
the	O
protein	O
(	O
Fig	O
.	O
5a	O
,	O
5c	O
);	O
TM7ab	B-structure_element
is	O
bent	O
and	O
packs	O
closely	O
with	O
TM2	B-structure_element
and	O
TM3	B-structure_element
,	O
and	O
so	O
the	O
binding	B-site
sites	I-site
are	O
occluded	O
from	O
the	O
solvent	O
(	O
Fig	O
.	O
5b	O
).	O

The	O
Na	B-chemical
+	I-chemical
ion	O
at	O
Sext	B-site
remains	O
fully	B-protein_state
coordinated	I-protein_state
,	O
but	O
an	O
ordered	O
water	B-chemical
molecule	O
now	O
mediates	O
its	O
interaction	O
with	O
A206	B-residue_name_number
:	O
O	O
,	O
relieving	O
the	O
strain	O
on	O
the	O
F202	B-residue_name_number
:	O
O	O
–	O
A206	B-residue_name_number
:	O
N	O
hydrogen	B-bond_interaction
-	I-bond_interaction
bond	I-bond_interaction
(	O
Fig	O
.	O
5c	O
).	O

Interestingly	O
,	O
this	O
doubly	O
occupied	O
state	O
can	O
also	O
access	O
conformations	O
in	O
which	O
the	O
second	O
aqueous	B-site
channel	I-site
mentioned	O
above	O
,	O
i	O
.	O
e	O
.	O
leading	O
to	O
SCa	B-site
between	O
TM7	B-structure_element
and	O
TM2	B-structure_element
and	O
over	O
the	O
gating	B-structure_element
helices	I-structure_element
TM1	B-structure_element
and	O
TM6	B-structure_element
,	O
also	O
becomes	O
open	B-protein_state
(	O
Fig	O
.	O
5b	O
-	O
c	O
).	O

This	O
processivity	O
is	O
logical	O
since	O
three	O
Na	B-chemical
+	I-chemical
ions	O
are	O
involved	O
,	O
but	O
also	O
implies	O
that	O
in	O
the	O
Ca2	B-protein_state
+-	I-protein_state
bound	I-protein_state
state	O
,	O
which	O
includes	O
a	O
single	O
ion	O
,	O
the	O
transporter	B-protein_type
ought	O
to	O
be	O
able	O
to	O
access	O
all	O
three	O
major	O
conformations	O
,	O
i	O
.	O
e	O
.	O
the	O
outward	B-protein_state
-	I-protein_state
open	I-protein_state
state	O
,	O
in	O
order	O
to	O
release	O
(	O
or	O
re	O
-	O
bind	O
)	O
Ca2	B-chemical
+,	I-chemical
but	O
also	O
the	O
occluded	B-protein_state
conformation	O
,	O
and	O
thus	O
the	O
semi	B-protein_state
-	I-protein_state
open	I-protein_state
intermediate	O
,	O
in	O
order	O
to	O
transition	O
to	O
the	O
inward	B-protein_state
-	I-protein_state
open	I-protein_state
state	O
.	O

By	O
contrast	O
,	O
occupancy	O
by	O
H	B-chemical
+,	I-chemical
which	O
as	O
mentioned	O
are	O
not	O
transported	O
,	O
might	O
be	O
compatible	O
with	O
a	O
semi	B-protein_state
-	I-protein_state
open	I-protein_state
state	O
as	O
well	O
as	O
with	O
the	O
fully	B-protein_state
open	I-protein_state
conformation	O
,	O
but	O
should	O
not	O
be	O
conducive	O
to	O
occlusion	O
.	O

This	O
occluded	B-protein_state
conformation	O
,	O
which	O
is	O
a	O
necessary	O
intermediate	O
between	O
the	O
outward	B-protein_state
and	O
inward	B-protein_state
-	I-protein_state
open	I-protein_state
states	O
,	O
and	O
which	O
entails	O
the	O
internal	O
dehydration	B-protein_state
of	O
the	O
protein	O
,	O
is	O
only	O
attainable	O
upon	O
complete	B-protein_state
occupancy	I-protein_state
of	O
the	O
binding	B-site
sites	I-site
.	O

The	O
most	O
apparent	O
of	O
these	O
changes	O
involves	O
the	O
N	B-structure_element
-	I-structure_element
terminal	I-structure_element
half	I-structure_element
of	O
TM7	B-structure_element
(	O
TM7ab	B-structure_element
);	O
together	O
with	O
more	O
subtle	O
displacements	O
in	O
TM2	B-structure_element
and	O
TM3	B-structure_element
,	O
this	O
change	O
in	O
TM7ab	B-structure_element
correlates	O
with	O
the	O
opening	O
and	O
closing	O
of	O
two	O
distinct	O
aqueous	B-site
channels	I-site
leading	O
into	O
the	O
ion	B-site
-	I-site
binding	I-site
sites	I-site
from	O
the	O
extracellular	O
solution	O
.	O

The	O
striking	O
quantitative	O
agreement	O
between	O
the	O
ion	B-evidence
-	I-evidence
binding	I-evidence
affinities	I-evidence
inferred	O
from	O
our	O
crystallographic	B-experimental_method
titrations	I-experimental_method
and	O
the	O
Km	B-evidence
and	O
K1	B-evidence
/	I-evidence
2	I-evidence
values	I-evidence
previously	O
deduced	O
from	O
functional	B-experimental_method
assays	I-experimental_method
has	O
been	O
discussed	O
above	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

No	O
significant	O
changes	O
were	O
observed	O
in	O
the	O
side	O
-	O
chains	O
involved	O
in	O
ion	O
or	O
water	B-chemical
coordination	O
at	O
the	O
SCa	B-site
,	O
Sint	B-site
and	O
Smid	B-site
sites	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

(	O
d	O
)	O
Extracellular	O
solvent	O
accessibility	O
of	O
the	O
ion	B-site
binding	I-site
sites	I-site
in	O
the	O
structures	B-evidence
at	O
high	B-protein_state
and	O
low	B-protein_state
[	O
Na	B-chemical
+].	I-chemical

Putative	O
solvent	B-site
channels	I-site
are	O
represented	O
as	O
light	O
-	O
purple	O
surfaces	O
.	O

Residues	O
involved	O
in	O
Sr2	B-chemical
+	I-chemical
coordination	O
are	O
labeled	O
.	O

(	O
b	O
)	O
Ca2	B-chemical
+	I-chemical
(	O
tanned	O
spheres	O
)	O
binds	O
either	O
to	O
SCa	B-site
or	O
Smid	B-site
in	O
crystals	B-experimental_method
titrated	I-experimental_method
with	O
10	O
mM	O
Ca2	B-chemical
+	I-chemical
and	O
2	O
.	O
5	O
mM	O
Na	B-chemical
+	I-chemical
(	O
see	O
also	O
Supplementary	O
Fig	O
.	O
2	O
).	O

Approximate	O
distances	O
between	O
TM2	B-structure_element
,	O
TM3	B-structure_element
and	O
TM7	B-structure_element
are	O
indicated	O
in	O
Å	O
.	O
(	O
e	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
partially	B-protein_state
Na	I-protein_state
+-	I-protein_state
occupied	I-protein_state
state	O
.	O

The	O
water	B-evidence
-	I-evidence
density	I-evidence
maps	I-evidence
in	O
(	O
b	O
)	O
are	O
shown	O
here	O
as	O
a	O
grey	O
mesh	O
.	O

An	O
extended	B-protein_state
U2AF65	B-structure_element
–	I-structure_element
RNA	I-structure_element
-	I-structure_element
binding	I-structure_element
domain	I-structure_element
recognizes	O
the	O
3	B-site
′	I-site
splice	I-site
site	I-site
signal	O

Initially	O
U2AF65	B-protein
recognizes	O
the	O
Py	B-chemical
-	I-chemical
tract	I-chemical
splice	B-site
site	I-site
signal	O
.	O

As	O
such	O
,	O
the	O
molecular	O
mechanisms	O
for	O
Py	B-chemical
-	I-chemical
tract	I-chemical
recognition	O
by	O
the	O
intact	B-protein_state
U2AF65	B-structure_element
–	I-structure_element
RNA	I-structure_element
-	I-structure_element
binding	I-structure_element
domain	I-structure_element
remained	O
unknown	O
.	O

We	O
use	O
single	B-experimental_method
-	I-experimental_method
molecule	I-experimental_method
Förster	I-experimental_method
resonance	I-experimental_method
energy	I-experimental_method
transfer	I-experimental_method
(	O
smFRET	B-experimental_method
)	O
to	O
characterize	O
the	O
conformational	B-evidence
dynamics	I-evidence
of	O
this	O
extended	B-protein_state
U2AF65	B-structure_element
–	I-structure_element
RNA	I-structure_element
-	I-structure_element
binding	I-structure_element
domain	I-structure_element
during	O
Py	B-chemical
-	I-chemical
tract	I-chemical
recognition	O
.	O

The	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
RRM1	B-structure_element
and	O
RRM2	B-structure_element
associate	O
with	O
the	O
Py	B-chemical
tract	I-chemical
in	O
a	O
parallel	B-protein_state
,	O
side	B-protein_state
-	I-protein_state
by	I-protein_state
-	I-protein_state
side	I-protein_state
arrangement	O
(	O
shown	O
for	O
representative	O
structure	O
iv	O
in	O
Fig	O
.	O
2b	O
,	O
c	O
;	O
Supplementary	O
Movie	O
1	O
).	O

An	O
extended	B-protein_state
conformation	I-protein_state
of	O
the	O
U2AF65	B-protein
inter	B-structure_element
-	I-structure_element
RRM	I-structure_element
linker	I-structure_element
traverses	O
across	O
the	O
α	B-structure_element
-	I-structure_element
helical	I-structure_element
surface	I-structure_element
of	O
RRM1	B-structure_element
and	O
the	O
central	O
β	B-structure_element
-	I-structure_element
strands	I-structure_element
of	O
RRM2	B-structure_element
and	O
is	O
well	O
defined	O
in	O
the	O
electron	B-evidence
density	I-evidence
(	O
Fig	O
.	O
2b	O
).	O

Both	O
RRM1	B-structure_element
/	O
RRM2	B-structure_element
extensions	B-structure_element
and	O
the	O
inter	B-structure_element
-	I-structure_element
RRM	I-structure_element
linker	I-structure_element
of	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
directly	O
recognize	O
the	O
bound	B-protein_state
oligonucleotide	B-chemical
.	O

Based	O
on	O
dU2AF651	B-mutant
,	I-mutant
2	I-mutant
structures	B-evidence
,	O
we	O
originally	O
hypothesized	O
that	O
the	O
U2AF65	B-protein
RRMs	B-structure_element
would	O
bind	O
the	O
minimal	B-protein_state
seven	O
nucleotides	B-chemical
observed	O
in	O
these	O
structures	B-evidence
.	O

Surprisingly	O
,	O
the	O
RRM2	B-structure_element
extension	I-structure_element
/	O
inter	B-structure_element
-	I-structure_element
RRM	I-structure_element
linker	I-structure_element
contribute	O
new	O
central	O
nucleotide	B-site
-	I-site
binding	I-site
sites	I-site
near	O
the	O
RRM1	B-site
/	I-site
RRM2	I-site
junction	I-site
and	O
the	O
RRM1	B-structure_element
extension	I-structure_element
recognizes	O
the	O
3	O
′-	O
terminal	O
nucleotide	B-chemical
(	O
Fig	O
.	O
2c	O
;	O
Supplementary	O
Movie	O
1	O
).	O

Qualitatively	O
,	O
a	O
subset	O
of	O
the	O
U2AF651	B-site
,	I-site
2L	I-site
-	I-site
nucleotide	I-site
-	I-site
binding	I-site
sites	I-site
(	O
sites	B-site
1	I-site
–	I-site
3	I-site
and	O
7	B-site
–	I-site
9	I-site
)	O
share	O
similar	O
locations	O
to	O
those	O
of	O
the	O
dU2AF651	B-mutant
,	I-mutant
2	I-mutant
structures	B-evidence
(	O
Supplementary	O
Figs	O
2c	O
,	O
d	O
and	O
3	O
).	O

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

This	O
nucleotide	B-chemical
twists	O
to	O
face	O
away	O
from	O
the	O
U2AF65	B-protein
linker	B-structure_element
and	O
instead	O
inserts	O
the	O
rU6	B-residue_name_number
-	O
uracil	B-residue_name
into	O
a	O
sandwich	O
between	O
the	O
β2	B-structure_element
/	I-structure_element
β3	I-structure_element
loops	I-structure_element
of	O
RRM1	B-structure_element
and	O
RRM2	B-structure_element
.	O

The	O
N	B-structure_element
-	I-structure_element
and	I-structure_element
C	I-structure_element
-	I-structure_element
terminal	I-structure_element
extensions	I-structure_element
of	O
the	O
U2AF65	B-protein
RRM1	B-structure_element
and	O
RRM2	B-structure_element
directly	O
contact	O
the	O
bound	B-protein_state
Py	B-chemical
tract	I-chemical
.	O

Consequently	O
,	O
the	O
U2AF651	B-protein_state
,	I-protein_state
2L	I-protein_state
-	I-protein_state
bound	I-protein_state
rU2	B-residue_name_number
-	O
O4	O
and	O
-	O
N3H	O
form	O
dual	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
with	O
the	O
K329	B-residue_name_number
backbone	O
atoms	O
(	O
Fig	O
.	O
3a	O
),	O
rather	O
than	O
a	O
single	O
hydrogen	B-bond_interaction
bond	I-bond_interaction
with	O
the	O
K329	B-residue_name_number
side	O
chain	O
as	O
in	O
the	O
prior	O
dU2AF651	B-mutant
,	I-mutant
2	I-mutant
structure	B-evidence
(	O
Supplementary	O
Fig	O
.	O
3b	O
).	O

The	O
adjacent	O
R146	B-residue_name_number
guanidinium	O
group	O
donates	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
to	O
the	O
3	O
′-	O
terminal	O
ribose	B-chemical
-	O
O2	O
′	O
and	O
O3	O
′	O
atoms	O
,	O
where	O
it	O
could	O
form	O
a	O
salt	B-bond_interaction
bridge	I-bond_interaction
with	O
a	O
phospho	O
-	O
diester	O
group	O
in	O
the	O
context	O
of	O
a	O
longer	O
pre	B-chemical
-	I-chemical
mRNA	I-chemical
.	O

We	O
compare	B-experimental_method
U2AF65	B-protein
interactions	O
with	O
uracil	B-residue_name
relative	O
to	O
cytosine	B-residue_name
pyrimidines	B-chemical
at	O
the	O
ninth	B-site
binding	I-site
site	I-site
in	O
Fig	O
.	O
3g	O
,	O
h	O
and	O
the	O
Supplementary	O
Discussion	O
.	O

At	O
the	O
RNA	B-chemical
surface	O
,	O
the	O
key	O
V254	B-residue_name_number
that	O
recognizes	O
the	O
fifth	B-residue_number
uracil	B-residue_name
is	O
secured	O
via	O
hydrophobic	B-bond_interaction
contacts	I-bond_interaction
between	O
its	O
side	O
chain	O
and	O
the	O
β	B-structure_element
-	I-structure_element
sheet	I-structure_element
surface	I-structure_element
of	O
RRM2	B-structure_element
,	O
chiefly	O
the	O
consensus	O
RNP1	B-structure_element
-	O
F304	B-residue_name_number
residue	O
that	O
stacks	B-bond_interaction
with	O
the	O
fourth	B-residue_number
uracil	B-residue_name
(	O
Fig	O
.	O
4a	O
,	O
lower	O
left	O
).	O

However	O
,	O
the	O
resulting	O
decrease	O
in	O
the	O
AdML	B-gene
RNA	B-evidence
affinity	I-evidence
of	O
the	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
-	I-mutant
3Gly	I-mutant
mutant	B-protein_state
relative	O
to	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
protein	B-protein
was	O
not	O
significant	O
(	O
Fig	O
.	O
4b	O
).	O

In	O
parallel	O
,	O
we	O
replaced	B-experimental_method
five	O
linker	B-structure_element
residues	I-structure_element
(	O
S251	B-residue_name_number
,	O
T252	B-residue_name_number
,	O
V253	B-residue_name_number
,	O
V254	B-residue_name_number
and	O
P255	B-residue_name_number
)	O
at	O
the	O
fifth	B-site
nucleotide	I-site
-	I-site
binding	I-site
site	I-site
with	O
glycines	B-residue_name
(	O
5Gly	B-mutant
)	O
and	O
also	O
found	O
that	O
the	O
RNA	B-evidence
affinity	I-evidence
of	O
the	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
-	I-mutant
5Gly	I-mutant
mutant	B-protein_state
likewise	O
decreased	O
only	O
slightly	O
relative	O
to	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
protein	B-protein
.	O

Importance	O
of	O
U2AF65	B-complex_assembly
–	I-complex_assembly
RNA	I-complex_assembly
contacts	O
for	O
pre	B-chemical
-	I-chemical
mRNA	I-chemical
splicing	O

To	O
complement	O
the	O
static	O
portraits	O
of	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
structure	B-evidence
that	O
we	O
had	O
determined	O
by	O
X	B-experimental_method
-	I-experimental_method
ray	I-experimental_method
crystallography	I-experimental_method
,	O
we	O
used	O
smFRET	B-experimental_method
to	O
characterize	O
the	O
probability	B-evidence
distribution	I-evidence
functions	I-evidence
and	O
time	O
dependence	O
of	O
U2AF65	B-protein
inter	B-structure_element
-	I-structure_element
RRM	I-structure_element
conformational	O
dynamics	O
in	O
solution	O
.	O

Double	O
-	O
cysteine	B-residue_name
variant	B-protein_state
of	O
U2AF651	B-mutant
,	I-mutant
2	I-mutant
was	O
modified	B-experimental_method
with	O
equimolar	O
amount	O
of	O
Cy3	B-chemical
and	O
Cy5	B-chemical
.	O

Only	O
traces	B-evidence
that	O
showed	O
single	O
photobleaching	O
events	O
for	O
both	O
donor	O
and	O
acceptor	O
dyes	O
and	O
anti	O
-	O
correlated	O
changes	O
in	O
acceptor	O
and	O
donor	O
fluorescence	O
were	O
included	O
in	O
smFRET	B-experimental_method
data	O
analysis	O
.	O

The	O
double	O
-	O
labelled	O
U2AF651	B-mutant
,	I-mutant
2LFRET	I-mutant
(	O
Cy3	B-chemical
/	O
Cy5	B-chemical
)	O
protein	O
was	O
tethered	B-protein_state
to	O
a	O
slide	O
via	O
biotin	B-chemical
-	I-chemical
NTA	I-chemical
/	I-chemical
Ni	I-chemical
+	I-chemical
2	I-chemical
resin	I-chemical
.	O

However	O
,	O
the	O
presence	O
of	O
repetitive	O
fluctuations	O
between	O
particular	O
FRET	B-evidence
values	I-evidence
supports	O
the	O
hypothesis	O
that	O
RNA	B-protein_state
-	I-protein_state
free	I-protein_state
U2AF65	B-protein
samples	O
several	O
distinct	O
conformations	O
.	O

This	O
result	O
is	O
consistent	O
with	O
the	O
broad	O
ensembles	O
of	O
extended	B-protein_state
solution	O
conformations	O
that	O
best	O
fit	O
the	O
SAXS	B-experimental_method
data	O
collected	O
for	O
U2AF651	B-mutant
,	I-mutant
2	I-mutant
as	O
well	O
as	O
for	O
a	O
longer	O
construct	O
(	O
residues	O
136	B-residue_range
–	I-residue_range
347	I-residue_range
).	O

We	O
next	O
used	O
smFRET	B-experimental_method
to	O
probe	O
the	O
conformational	O
selection	O
of	O
distinct	O
inter	B-structure_element
-	I-structure_element
RRM	I-structure_element
arrangements	O
following	O
association	O
of	O
U2AF65	B-protein
with	O
the	O
AdML	B-gene
Py	B-chemical
-	I-chemical
tract	I-chemical
prototype	O
.	O

To	O
assess	O
the	O
possible	O
contributions	O
of	O
RNA	B-protein_state
-	I-protein_state
free	I-protein_state
conformations	O
of	O
U2AF65	B-protein
and	O
/	O
or	O
structural	O
heterogeneity	O
introduced	O
by	O
tethering	B-experimental_method
of	O
U2AF651	B-mutant
,	I-mutant
2LFRET	I-mutant
(	O
Cy3	B-chemical
/	O
Cy5	B-chemical
)	O
to	O
the	O
slide	O
to	O
the	O
observed	O
distribution	B-evidence
of	I-evidence
FRET	I-evidence
values	I-evidence
,	O
we	O
reversed	B-experimental_method
the	I-experimental_method
immobilization	I-experimental_method
scheme	I-experimental_method
.	O

Therefore	O
,	O
RRM1	B-structure_element
-	O
to	O
-	O
RRM2	B-structure_element
distance	O
remains	O
similar	O
regardless	O
of	O
whether	O
U2AF65	B-protein
is	O
bound	B-protein_state
to	I-protein_state
interrupted	O
or	O
continuous	O
Py	B-chemical
tract	I-chemical
.	O

The	O
inter	B-evidence
-	I-evidence
fluorophore	I-evidence
distances	I-evidence
derived	O
from	O
the	O
observed	O
0	O
.	O
45	O
FRET	B-evidence
state	I-evidence
agree	O
with	O
the	O
distances	O
between	O
the	O
α	O
-	O
carbon	O
atoms	O
of	O
the	O
respective	O
residues	O
in	O
the	O
crystal	B-evidence
structures	I-evidence
of	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
bound	B-protein_state
to	I-protein_state
Py	B-chemical
-	I-chemical
tract	I-chemical
oligonucleotides	I-chemical
.	O

Hidden	B-experimental_method
Markov	I-experimental_method
modelling	I-experimental_method
analysis	I-experimental_method
of	O
smFRET	B-experimental_method
traces	B-evidence
suggests	O
that	O
RNA	B-protein_state
-	I-protein_state
bound	I-protein_state
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
can	O
sample	O
at	O
least	O
two	O
other	O
conformations	O
corresponding	O
to	O
∼	O
0	O
.	O
7	O
–	O
0	O
.	O
8	O
and	O
∼	O
0	O
.	O
3	O
FRET	B-evidence
values	I-evidence
in	O
addition	O
to	O
the	O
predominant	O
conformation	O
corresponding	O
to	O
the	O
0	O
.	O
45	O
FRET	B-evidence
state	I-evidence
.	O

Truncation	B-experimental_method
of	O
U2AF65	B-protein
to	O
the	O
core	B-protein_state
RRM1	B-structure_element
–	I-structure_element
RRM2	I-structure_element
region	I-structure_element
reduces	O
its	O
RNA	B-evidence
affinity	I-evidence
by	O
100	O
-	O
fold	O
.	O

As	O
such	O
,	O
we	O
suggest	O
that	O
the	O
MDS	O
-	O
relevant	O
U2AF65	B-protein
mutations	O
contribute	O
to	O
MDS	O
progression	O
indirectly	O
,	O
by	O
destabilizing	O
a	O
relevant	O
conformation	O
of	O
the	O
conjoined	O
U2AF35	B-protein
subunit	O
rather	O
than	O
affecting	O
U2AF65	B-protein
functions	O
in	O
RNA	B-chemical
binding	O
or	O
spliceosome	B-complex_assembly
recruitment	O
per	O
se	O
.	O

An	O
increased	O
prevalence	O
of	O
the	O
∼	O
0	O
.	O
45	O
FRET	B-evidence
value	I-evidence
following	O
U2AF65	B-protein
–	O
RNA	B-chemical
binding	O
,	O
coupled	O
with	O
the	O
apparent	O
absence	B-protein_state
of	I-protein_state
transitions	O
in	O
many	O
∼	O
0	O
.	O
45	O
-	O
value	O
single	O
molecule	O
traces	B-evidence
(	O
for	O
example	O
,	O
Fig	O
.	O
6e	O
),	O
suggests	O
a	O
population	O
shift	O
in	O
which	O
RNA	B-chemical
binds	O
to	O
(	O
and	O
draws	O
the	O
equilibrium	O
towards	O
)	O
a	O
pre	B-protein_state
-	I-protein_state
configured	I-protein_state
inter	B-structure_element
-	I-structure_element
RRM	I-structure_element
proximity	O
that	O
most	O
often	O
corresponds	O
to	O
the	O
∼	O
0	O
.	O
45	O
FRET	B-evidence
value	I-evidence
.	O

Examples	O
of	O
‘	O
extended	B-protein_state
conformational	O
selection	O
'	O
during	O
ligand	O
binding	O
have	O
been	O
characterized	O
for	O
a	O
growing	O
number	O
of	O
macromolecules	O
(	O
for	O
example	O
,	O
adenylate	B-protein_type
kinase	I-protein_type
,	O
LAO	B-protein_type
-	I-protein_type
binding	I-protein_type
protein	I-protein_type
,	O
poly	B-protein_type
-	I-protein_type
ubiquitin	I-protein_type
,	O
maltose	B-protein_type
-	I-protein_type
binding	I-protein_type
protein	I-protein_type
and	O
the	O
preQ1	B-protein_type
riboswitch	I-protein_type
,	O
among	O
others	O
).	O

These	O
transitions	O
could	O
correspond	O
to	O
rearrangement	O
from	O
the	O
‘	O
closed	B-protein_state
'	O
NMR	B-experimental_method
/	O
PRE	B-experimental_method
-	O
based	O
U2AF65	B-protein
conformation	O
in	O
which	O
the	O
RNA	B-site
-	I-site
binding	I-site
surface	I-site
of	O
only	O
a	O
single	B-protein_state
RRM	B-structure_element
is	O
exposed	O
and	O
available	O
for	O
RNA	O
binding	O
,	O
to	O
the	O
structural	O
state	O
seen	O
in	O
the	O
side	B-protein_state
-	I-protein_state
by	I-protein_state
-	I-protein_state
side	I-protein_state
,	O
RNA	B-protein_state
-	I-protein_state
bound	I-protein_state
crystal	B-evidence
structure	I-evidence
.	O

The	O
finding	O
that	O
U2AF65	B-protein
recognizes	O
a	O
nine	O
base	O
pair	O
Py	B-chemical
tract	I-chemical
contributes	O
to	O
an	O
elusive	O
‘	O
code	O
'	O
for	O
predicting	O
splicing	O
patterns	O
from	O
primary	O
sequences	O
in	O
the	O
post	O
-	O
genomic	O
era	O
(	O
reviewed	O
in	O
ref	O
.).	O

(	O
b	O
)	O
Comparison	O
of	O
the	O
apparent	O
equilibrium	B-evidence
affinities	I-evidence
of	O
various	O
U2AF65	B-protein
constructs	O
for	O
binding	O
the	O
prototypical	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
apparent	O
equilibrium	B-evidence
dissociation	I-evidence
constants	I-evidence
(	O
KD	B-evidence
)	O
for	O
binding	O
the	O
AdML	B-gene
13mer	O
are	O
as	O
follows	O
:	O
flU2AF65	B-protein
,	O
30	O
±	O
3	O
nM	O
;	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
,	O
35	O
±	O
6	O
nM	O
;	O
U2AF651	B-mutant
,	I-mutant
2	I-mutant
,	O
3	O
,	O
600	O
±	O
300	O
nM	O
.	O
(	O
c	O
)	O
Comparison	O
of	O
the	O
RNA	B-evidence
sequence	I-evidence
specificities	I-evidence
of	O
flU2AF65	B-protein
and	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
constructs	O
binding	O
C	B-structure_element
-	I-structure_element
rich	I-structure_element
Py	B-chemical
tracts	I-chemical
with	O
4U	O
'	O
s	O
embedded	O
in	O
either	O
the	O
5	O
′-	O
(	O
light	O
grey	O
fill	O
)	O
or	O
3	O
′-	O
(	O
dark	O
grey	O
fill	O
)	O
regions	O
.	O

The	O
purified	O
protein	O
and	O
average	B-evidence
fitted	I-evidence
fluorescence	I-evidence
anisotropy	I-evidence
RNA	I-evidence
-	I-evidence
binding	I-evidence
curves	I-evidence
are	O
shown	O
in	O
Supplementary	O
Fig	O
.	O
1	O
.	O

(	O
b	O
)	O
Stereo	O
views	O
of	O
a	O
‘	O
kicked	O
'	O
2	B-evidence
|	I-evidence
Fo	I-evidence
|−|	I-evidence
Fc	I-evidence
|	I-evidence
electron	I-evidence
density	I-evidence
map	I-evidence
contoured	O
at	O
1σ	O
for	O
the	O
inter	B-structure_element
-	I-structure_element
RRM	I-structure_element
linker	I-structure_element
,	O
N	O
-	O
and	O
C	O
-	O
terminal	O
residues	O
(	O
blue	O
)	O
or	O
bound	O
oligonucleotide	B-chemical
of	O
a	O
representative	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
structure	O
(	O
structure	O
iv	O
,	O
bound	B-protein_state
to	I-protein_state
5	O
′-(	O
P	O
)	O
rUrUrUdUrUrU	O
(	O
BrdU	O
)	O
dUrC	O
)	O
(	O
magenta	O
).	O

BrdU	B-chemical
,	O
5	B-chemical
-	I-chemical
bromo	I-chemical
-	I-chemical
deoxy	I-chemical
-	I-chemical
uridine	I-chemical
;	O
d	B-chemical
,	O
deoxy	B-chemical
-	I-chemical
ribose	I-chemical
;	O
P	B-chemical
-,	I-chemical
5	B-chemical
′-	I-chemical
phosphorylation	I-chemical
;	O
r	B-chemical
,	O
ribose	B-chemical
.	O

The	O
U2AF65	B-protein
linker	B-structure_element
/	O
RRM	B-structure_element
and	O
inter	O
-	O
RRM	B-structure_element
interactions	O
.	O

RNA	O
binding	O
stabilizes	O
the	O
side	B-protein_state
-	I-protein_state
by	I-protein_state
-	I-protein_state
side	I-protein_state
conformation	O
of	O
U2AF65	B-protein
RRMs	B-structure_element
.	O

Additional	O
traces	B-evidence
for	O
untethered	B-protein_state
,	O
RNA	B-protein_state
-	I-protein_state
bound	I-protein_state
U2AF651	B-mutant
,	I-mutant
2LFRET	I-mutant
(	O
Cy3	B-chemical
/	O
Cy5	B-chemical
)	O
are	O
shown	O
in	O
Supplementary	O
Fig	O
.	O
7c	O
,	O
d	O
.	O
Histograms	B-evidence
(	O
d	O
,	O
f	O
,	O
h	O
,	O
j	O
)	O
show	O
the	O
distribution	B-evidence
of	I-evidence
FRET	I-evidence
values	I-evidence
in	O
RNA	B-protein_state
-	I-protein_state
free	I-protein_state
,	O
slide	B-protein_state
-	I-protein_state
tethered	I-protein_state
U2AF651	B-mutant
,	I-mutant
2LFRET	I-mutant
(	O
Cy3	B-chemical
/	O
Cy5	B-chemical
)	O
(	O
d	O
);	O
AdML	B-gene
RNA	B-protein_state
-	I-protein_state
bound	I-protein_state
,	O
slide	B-protein_state
-	I-protein_state
tethered	I-protein_state
U2AF651	B-mutant
,	I-mutant
2LFRET	I-mutant
(	O
Cy3	B-chemical
/	O
Cy5	B-chemical
)	O
(	O
f	O
);	O
AdML	B-gene
RNA	B-protein_state
-	I-protein_state
bound	I-protein_state
,	O
untethered	B-protein_state
U2AF651	B-mutant
,	I-mutant
2LFRET	I-mutant
(	O
Cy3	B-chemical
/	O
Cy5	B-chemical
)	O
(	O
h	O
)	O
and	O
adenosine	O
-	O
interrupted	O
RNA	B-protein_state
-	I-protein_state
bound	I-protein_state
,	O
slide	B-protein_state
-	I-protein_state
tethered	I-protein_state
U2AF651	B-mutant
,	I-mutant
2LFRET	I-mutant
(	O
Cy3	B-chemical
/	O
Cy5	B-chemical
)	O
(	O
j	O
).	O

A	O
surface	O
representation	O
of	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
is	O
shown	O
bound	B-protein_state
to	I-protein_state
nine	O
nucleotides	B-chemical
(	O
nt	O
);	O
the	O
relative	O
distances	O
and	O
juxtaposition	O
of	O
the	O
branch	B-site
point	I-site
sequence	I-site
(	O
BPS	B-site
)	O
and	O
consensus	O
AG	B-chemical
dinucleotide	I-chemical
at	O
the	O
3	B-site
′	I-site
splice	I-site
site	I-site
are	O
unknown	O
.	O

(	O
b	O
)	O
Following	O
binding	O
to	O
the	O
Py	B-chemical
-	I-chemical
tract	I-chemical
RNA	I-chemical
,	O
a	O
conformation	O
corresponding	O
to	O
high	B-evidence
FRET	I-evidence
and	O
consistent	O
with	O
the	O
‘	O
closed	B-protein_state
',	O
back	B-protein_state
-	I-protein_state
to	I-protein_state
-	I-protein_state
back	I-protein_state
apo	B-protein_state
-	O
U2AF65	B-protein
model	O
resulting	O
from	O
PRE	B-experimental_method
/	O
NMR	B-experimental_method
characterization	O
(	O
PDB	O
ID	O
2YH0	O
)	O
often	O
transitions	O
to	O
a	O
conformation	O
corresponding	O
to	O
∼	O
0	O
.	O
45	O
FRET	B-evidence
value	I-evidence
,	O
which	O
is	O
consistent	O
with	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
RRMs	B-structure_element
such	O
as	O
the	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
crystal	B-evidence
structures	I-evidence
.	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

Dose	O
is	O
defined	O
as	O
the	O
absorbed	O
energy	O
per	O
unit	O
mass	O
of	O
crystal	O
in	O
grays	O
(	O
Gy	O
;	O
1	O
Gy	O
=	O
1	O
J	O
kg	O
−	O
1	O
),	O
and	O
is	O
the	O
metric	O
against	O
which	O
damage	O
progression	O
should	O
be	O
monitored	O
during	O
MX	B-experimental_method
data	O
collection	O
,	O
as	O
opposed	O
to	O
time	O
.	O

There	O
are	O
a	O
number	O
of	O
cases	O
where	O
SRD	O
manifestations	O
have	O
compromised	O
the	O
biological	O
information	O
extracted	O
from	O
MX	B-experimental_method
-	I-experimental_method
determined	I-experimental_method
structures	B-evidence
at	O
much	O
lower	O
doses	O
than	O
the	O
recommended	O
30	O
MGy	O
limit	O
,	O
leading	O
to	O
false	O
structural	O
interpretations	O
of	O
protein	O
mechanisms	O
.	O

The	O
investigation	O
of	O
naturally	O
forming	O
nucleoprotein	O
complexes	O
circumvents	O
the	O
inherent	O
challenges	O
in	O
making	O
controlled	O
comparisons	O
of	O
damage	O
mechanisms	O
between	O
protein	O
and	O
nucleic	O
acids	O
crystallized	B-experimental_method
separately	O
.	O
Recently	O
,	O
for	O
a	O
well	O
characterized	O
bacterial	B-taxonomy_domain
protein	O
–	O
DNA	B-chemical
complex	O
(	O
C	B-complex_assembly
.	I-complex_assembly
Esp1396I	I-complex_assembly
;	O
PDB	O
entry	O
3clc	O
;	O
resolution	O
2	O
.	O
8	O
Å	O
;	O
McGeehan	O
et	O
al	O
.,	O
2008	O
)	O
it	O
was	O
concluded	O
that	O
over	O
a	O
wide	O
dose	O
range	O
(	O
2	O
.	O
1	O
–	O
44	O
.	O
6	O
MGy	O
)	O
the	O
protein	O
was	O
far	O
more	O
susceptible	O
to	O
SRD	O
than	O
the	O
DNA	B-chemical
within	O
the	O
crystal	B-evidence
(	O
Bury	O
et	O
al	O
.,	O
2015	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

Salt	B-bond_interaction
-	I-bond_interaction
bridge	I-bond_interaction
interactions	O
have	O
previously	O
been	O
suggested	O
to	O
reduce	O
the	O
glutamate	B-residue_name
decarboxylation	O
rate	O
within	O
the	O
large	O
(∼	O
62	O
.	O
4	O
kDa	O
)	O
myrosinase	B-protein_type
protein	O
structure	B-evidence
(	O
Burmeister	O
,	O
2000	O
).	O

The	O
extended	O
aliphatic	O
Lys37	B-residue_name_number
side	O
chain	O
stacks	O
against	O
the	O
nearby	O
G1	B-residue_name_number
base	O
,	O
making	O
a	O
series	O
of	O
nonpolar	B-bond_interaction
contacts	I-bond_interaction
within	O
each	O
RNA	B-site
-	I-site
binding	I-site
interface	I-site
.	O

Representative	O
Phe32	B-residue_name_number
and	O
Lys37	B-residue_name_number
atoms	O
were	O
selected	O
to	O
illustrate	O
these	O
trends	O
.	O

Our	O
method	O
­	O
ology	O
,	O
which	O
eliminated	O
tedious	O
and	O
error	O
-	O
prone	O
visual	O
inspection	O
,	O
permitted	O
the	O
determination	O
on	O
a	O
per	O
-	O
atom	O
basis	O
of	O
the	O
most	O
damaged	O
sites	O
,	O
as	O
characterized	O
by	O
F	B-evidence
obs	I-evidence
(	I-evidence
d	I-evidence
n	I-evidence
)	I-evidence
−	I-evidence
F	I-evidence
obs	I-evidence
(	I-evidence
d	I-evidence
1	I-evidence
)	I-evidence
Fourier	I-evidence
difference	I-evidence
map	I-evidence
peaks	I-evidence
between	O
successive	O
data	O
sets	O
collected	O
from	O
the	O
same	O
crystal	B-evidence
.	O

Both	O
Glu36	B-residue_name_number
and	O
Asp39	B-residue_name_number
bind	O
directly	O
to	O
RNA	B-chemical
,	O
each	O
through	O
two	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
to	O
guanine	B-chemical
bases	O
(	O
G3	B-residue_name_number
and	O
G1	B-residue_name_number
,	O
respectively	O
).	O

Observations	O
of	O
lower	O
protein	O
radiation	O
-	O
sensitivity	O
in	O
DNA	B-protein_state
-	I-protein_state
bound	I-protein_state
forms	O
have	O
been	O
recorded	O
in	O
solution	O
at	O
RT	O
at	O
much	O
lower	O
doses	O
(∼	O
1	O
kGy	O
)	O
than	O
those	O
used	O
for	O
typical	O
MX	B-experimental_method
experiments	O
[	O
e	O
.	O
g	O
.	O
an	O
oestrogen	O
response	O
element	O
–	O
receptor	O
complex	O
(	O
Stísová	O
et	O
al	O
.,	O
2006	O
)	O
and	O
a	O
DNA	B-protein_type
glycosylase	I-protein_type
and	O
its	O
abasic	B-site
DNA	I-site
target	I-site
site	I-site
(	O
Gillard	O
et	O
al	O
.,	O
2004	O
)].	O

However	O
,	O
in	O
the	O
current	O
MX	B-experimental_method
study	O
at	O
100	O
K	O
,	O
the	O
main	O
damaging	O
species	O
are	O
believed	O
to	O
be	O
migrating	O
LEEs	O
and	O
holes	O
produced	O
directly	O
within	O
the	O
protein	B-complex_assembly
–	I-complex_assembly
RNA	I-complex_assembly
components	O
or	O
in	O
closely	O
associated	O
solvent	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

Residues	O
have	O
been	O
grouped	O
by	O
amino	O
-	O
acid	O
number	O
,	O
and	O
split	O
into	O
bound	B-protein_state
and	O
nonbound	B-protein_state
groupings	O
,	O
with	O
each	O
bar	O
representing	O
the	O
mean	O
calculated	O
over	O
11	O
equivalent	O
atoms	O
around	O
a	O
TRAP	B-complex_assembly
ring	B-structure_element
.	O

The	O
three	O
best	O
-	O
characterized	O
MAPK	B-protein_type
signalling	O
pathways	O
are	O
mediated	O
by	O
the	O
kinases	B-protein_type
extracellular	B-protein_type
signal	I-protein_type
-	I-protein_type
regulated	I-protein_type
kinase	I-protein_type
(	O
ERK	B-protein_type
),	O
c	B-protein_type
-	I-protein_type
Jun	I-protein_type
N	I-protein_type
-	I-protein_type
terminal	I-protein_type
kinase	I-protein_type
(	O
JNK	B-protein_type
)	O
and	O
p38	B-protein_type
.	O

The	O
ERK	B-protein_type
pathway	O
is	O
activated	O
by	O
various	O
mitogens	O
and	O
phorbol	O
esters	O
,	O
whereas	O
the	O
JNK	B-protein_type
and	O
p38	B-protein_type
pathways	O
are	O
stimulated	O
mainly	O
by	O
environmental	O
stress	O
and	O
inflammatory	O
cytokines	B-protein_type
.	O

MKPs	B-protein_type
constitute	O
a	O
group	O
of	O
DUSPs	B-protein_type
that	O
are	O
characterized	O
by	O
their	O
ability	O
to	O
dephosphorylate	O
both	O
phosphotyrosine	B-residue_name
and	O
phosphoserine	B-residue_name
/	O
phospho	B-residue_name
-	I-residue_name
threonine	I-residue_name
residues	O
within	O
a	O
substrate	O
.	O

Biochemical	B-experimental_method
and	I-experimental_method
modelling	I-experimental_method
studies	I-experimental_method
further	O
demonstrate	O
that	O
the	O
molecular	O
interactions	O
mediate	O
this	O
key	O
element	O
for	O
substrate	O
recognition	O
are	O
highly	O
conserved	O
among	O
all	O
MKP	B-protein_type
-	I-protein_type
family	I-protein_type
members	I-protein_type
.	O

In	O
mammalian	B-taxonomy_domain
cells	O
,	O
the	O
MKP	B-protein_type
subfamily	I-protein_type
includes	O
10	O
distinct	O
catalytically	B-protein_state
active	I-protein_state
MKPs	B-protein_type
.	O

Figure	O
2b	O
shows	O
the	O
variation	B-evidence
of	I-evidence
initial	I-evidence
rates	I-evidence
of	O
the	O
MKP7ΔC304	B-mutant
and	O
MKP7	B-protein
-	O
CD	B-structure_element
-	O
catalysed	O
reaction	O
with	O
the	O
concentration	O
of	O
phospho	B-protein_state
-	O
JNK1	B-protein
.	O
Because	O
the	O
concentrations	O
of	O
MKP7	B-protein
and	O
pJNK1	B-protein_state
were	O
comparable	O
in	O
the	O
reaction	O
,	O
the	O
assumption	O
that	O
the	O
free	O
-	O
substrate	O
concentration	O
is	O
equal	O
to	O
the	O
total	O
substrate	O
concentration	O
is	O
not	O
valid	O
.	O

To	O
further	O
confirm	O
the	O
JNK1	B-complex_assembly
–	I-complex_assembly
MKP7	I-complex_assembly
-	I-complex_assembly
CD	I-complex_assembly
interaction	O
,	O
we	O
performed	O
a	O
pull	B-experimental_method
-	I-experimental_method
down	I-experimental_method
assay	I-experimental_method
using	O
the	O
purified	O
proteins	O
.	O

The	O
catalytic	B-structure_element
domain	I-structure_element
of	O
MKP7	B-protein
interacts	O
with	O
JNK1	B-protein
through	O
a	O
contiguous	O
surface	O
area	O
that	O
is	O
remote	O
from	O
the	O
active	B-site
site	I-site
.	O

The	O
active	B-site
site	I-site
of	O
MKP7	B-protein
consists	O
of	O
the	O
phosphate	B-structure_element
-	I-structure_element
binding	I-structure_element
loop	I-structure_element
(	O
P	B-structure_element
-	I-structure_element
loop	I-structure_element
,	O
Cys244	B-residue_name_number
-	O
Leu245	B-residue_name_number
-	O
Ala246	B-residue_name_number
-	O
Gly247	B-residue_name_number
-	O
Ile248	B-residue_name_number
-	O
Ser249	B-residue_name_number
-	O
Arg250	B-residue_name_number
),	O
and	O
Asp213	B-residue_name_number
in	O
the	O
general	B-structure_element
acid	I-structure_element
loop	I-structure_element
(	O
Fig	O
.	O
3b	O
and	O
Supplementary	O
Fig	O
.	O
1b	O
).	O

The	O
side	O
chain	O
of	O
strictly	B-protein_state
conserved	I-protein_state
Arg250	B-residue_name_number
is	O
oriented	O
towards	O
the	O
negatively	O
charged	O
chloride	B-chemical
,	O
similar	O
to	O
the	O
canonical	O
phosphate	B-structure_element
-	I-structure_element
coordinating	I-structure_element
conformation	I-structure_element
.	O

In	O
the	O
complex	O
,	O
MKP7	B-protein
-	O
CD	B-structure_element
and	O
JNK1	B-protein
form	O
extensive	O
protein	O
–	O
protein	O
interactions	O
involving	O
the	O
C	B-structure_element
-	I-structure_element
terminal	I-structure_element
helices	I-structure_element
of	O
MKP7	B-protein
-	O
CD	B-structure_element
and	O
C	B-structure_element
-	I-structure_element
lobe	I-structure_element
of	O
JNK1	B-protein
(	O
Fig	O
.	O
3d	O
,	O
e	O
).	O

Mutation	B-experimental_method
of	O
Leu288	B-residue_name_number
markedly	O
reduced	O
its	O
solubility	O
when	O
expressed	O
in	O
Escherichia	B-species
coli	I-species
,	O
resulting	O
in	O
the	O
insoluble	O
aggregation	O
of	O
the	O
mutant	B-protein_state
protein	O
.	O

The	O
small	O
pNPP	B-chemical
molecule	O
binds	O
directly	O
at	O
the	O
enzyme	O
active	B-site
site	I-site
and	O
can	O
be	O
used	O
to	O
probe	O
the	O
reaction	O
mechanism	O
of	O
protein	B-protein_type
phosphatases	I-protein_type
.	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

Structural	B-experimental_method
analysis	I-experimental_method
and	O
sequence	B-experimental_method
alignment	I-experimental_method
reveal	O
that	O
one	O
of	O
the	O
few	O
differences	O
between	O
MKP7	B-protein
-	O
CD	B-structure_element
and	O
KAP	B-protein
in	O
the	O
substrate	B-site
-	I-site
binding	I-site
region	I-site
is	O
the	O
presence	O
of	O
the	O
motif	O
FNFL	B-structure_element
in	O
MKP7	B-protein
-	O
CD	B-structure_element
,	O
which	O
corresponds	O
to	O
IKQY	B-structure_element
in	O
KAP	B-protein
(	O
Fig	O
.	O
5c	O
).	O

Parallel	O
experiments	O
showed	O
clearly	O
that	O
the	O
D	B-structure_element
-	I-structure_element
motif	I-structure_element
mutants	B-protein_state
(	O
R56A	B-mutant
/	O
R57A	B-mutant
and	O
V63A	B-mutant
/	O
I65A	B-mutant
)	O
dephosphorylated	B-protein_state
JNK	B-protein_type
as	O
did	O
the	O
wild	B-protein_state
type	I-protein_state
under	O
the	O
same	O
conditions	O
,	O
further	O
confirming	O
that	O
the	O
MKP7	B-protein
-	O
KBD	B-structure_element
is	O
not	O
required	O
for	O
the	O
JNK	B-protein_type
inactivation	O
in	O
vivo	O
.	O

Consistent	O
with	O
the	O
in	O
vitro	O
data	O
,	O
the	O
level	O
of	O
phosphorylated	B-protein_state
JNK	B-protein_type
was	O
not	O
or	O
little	O
altered	O
in	O
MKP7	B-protein
FXF	B-structure_element
-	I-structure_element
motif	I-structure_element
mutants	B-protein_state
(	O
F285D	B-mutant
,	O
F287D	B-mutant
and	O
L288D	B-mutant
)-	O
transfected	O
cells	O
,	O
and	O
the	O
MKP7	B-protein
D268A	B-mutant
and	O
N286A	B-mutant
mutants	B-protein_state
retained	O
the	O
ability	O
to	O
reduce	O
the	O
phosphorylation	O
levels	O
of	O
JNK	B-protein_type
.	O

In	O
agreement	O
with	O
the	O
in	B-experimental_method
vitro	I-experimental_method
pull	I-experimental_method
-	I-experimental_method
down	I-experimental_method
results	O
,	O
the	O
mutants	B-protein_state
D229A	B-mutant
,	O
W234D	B-mutant
and	O
Y259D	B-mutant
were	O
not	O
co	O
-	O
precipitated	O
with	O
MKP7	B-protein
,	O
and	O
the	O
I231D	B-mutant
mutant	B-protein_state
had	O
only	O
little	O
effect	O
on	O
the	O
JNK1	B-complex_assembly
–	I-complex_assembly
MKP7	I-complex_assembly
interaction	O
(	O
Fig	O
.	O
6d	O
and	O
Supplementary	O
Fig	O
.	O
3a	O
).	O

Moreover	O
,	O
treatment	O
of	O
cells	O
expressing	O
MKP7	B-protein
-	O
KBD	B-structure_element
mutants	B-protein_state
(	O
R56A	B-mutant
/	O
R57A	B-mutant
and	O
V63A	B-mutant
/	O
I65A	B-mutant
)	O
decreased	O
the	O
apoptosis	O
rates	O
to	O
a	O
similar	O
extent	O
as	O
MKP7	B-protein
wild	B-protein_state
type	I-protein_state
did	O
.	O

MKP5	B-protein
belongs	O
to	O
the	O
same	O
subfamily	O
as	O
MKP7	B-protein
.	O

In	O
contrast	O
to	O
p38α	B-protein
substrate	O
,	O
deletion	B-experimental_method
of	I-experimental_method
the	O
MKP5	B-protein
-	O
KBD	B-structure_element
had	O
little	O
effects	O
on	O
the	O
kinetic	O
parameters	O
for	O
the	O
JNK1	B-protein
dephosphorylation	O
,	O
indicating	O
that	O
the	O
KBD	B-structure_element
of	O
MKP5	B-protein
is	O
not	O
required	O
for	O
the	O
JNK1	B-protein
dephosphorylation	O
(	O
Fig	O
.	O
7b	O
).	O

As	O
shown	O
in	O
Fig	O
.	O
7f	O
,	O
the	O
T432A	B-mutant
and	O
L449F	B-mutant
MKP5	B-protein
mutant	B-protein_state
showed	O
little	O
or	O
no	O
difference	O
in	O
phosphatase	O
activity	O
,	O
whereas	O
the	O
other	O
mutants	B-protein_state
showed	O
reduced	O
specific	O
activities	O
of	O
MKP5	B-protein
.	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

This	O
is	O
consistent	O
with	O
the	O
experimental	O
observation	O
showing	O
that	O
JNK1	B-protein
binds	O
to	O
MKP7	B-protein
-	O
CD	B-structure_element
much	O
more	O
tightly	O
than	O
MKP5	B-protein
-	O
CD	B-structure_element
(	O
Km	O
value	O
of	O
MKP5	B-protein
-	O
CD	B-structure_element
for	O
pJNK1	B-protein_state
substrate	O
is	O
∼	O
20	O
-	O
fold	O
higher	O
than	O
that	O
of	O
MKP7	B-protein
-	O
CD	B-structure_element
).	O

The	O
MAPKs	B-protein_type
p38	B-protein_type
,	O
ERK	B-protein_type
and	O
JNK	B-protein_type
,	O
are	O
central	O
to	O
evolutionarily	O
conserved	O
signalling	O
pathways	O
that	O
are	O
present	O
in	O
all	O
eukaryotic	B-taxonomy_domain
cells	O
.	O

Each	O
MAPK	B-protein_type
cascade	O
is	O
activated	O
in	O
response	O
to	O
a	O
diverse	O
array	O
of	O
extracellular	O
signals	O
and	O
culminates	O
in	O
the	O
dual	B-ptm
-	I-ptm
phosphorylation	I-ptm
of	O
a	O
threonine	B-residue_name
and	O
a	O
tyrosine	B-residue_name
residue	O
in	O
the	O
MAPK	B-structure_element
-	I-structure_element
activation	I-structure_element
loop	I-structure_element
.	O

This	O
structure	B-evidence
reveals	O
an	O
FXF	B-site
-	I-site
docking	I-site
interaction	I-site
mode	I-site
between	O
MAPK	B-protein_type
and	O
MKP	B-protein_type
.	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

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

The	O
top	O
panel	O
shows	O
the	O
relative	O
affinities	B-evidence
of	O
MKP7	B-protein
-	O
CD	B-structure_element
and	O
MKP7	B-protein
-	O
KBD	B-structure_element
to	O
JNK1	B-protein
,	O
with	O
the	O
affinity	B-evidence
of	O
MKP7	B-protein
-	O
CD	B-structure_element
defined	O
as	O
100	O
%;	O
the	O
middle	O
panel	O
is	O
the	O
electrophoretic	O
pattern	O
of	O
MKP7	B-protein
and	O
JNK1	B-protein
after	O
GST	B-experimental_method
pull	I-experimental_method
-	I-experimental_method
down	I-experimental_method
assays	I-experimental_method
.	O

Blue	O
dashed	O
lines	O
represent	O
polar	B-bond_interaction
interactions	I-bond_interaction
.	O

The	O
CDK2	B-protein
is	O
shown	O
in	O
surface	O
representation	O
coloured	O
according	O
to	O
the	O
electrostatic	O
potential	O
(	O
positive	O
,	O
blue	O
;	O
negative	O
,	O
red	O
).	O

Residues	O
of	O
MKP7	B-protein
-	O
CD	B-structure_element
involved	O
in	O
JNK1	B-protein
recognition	O
are	O
indicated	O
by	O
cyan	O
asterisks	O
,	O
and	O
the	O
conserved	B-protein_state
FXF	B-structure_element
-	I-structure_element
motif	I-structure_element
is	O
highlighted	O
in	O
cyan	O
.	O

The	O
secondary	O
structure	O
assignments	O
of	O
MKP7	B-protein
-	O
CD	B-structure_element
and	O
KAP	B-protein
are	O
shown	O
above	O
and	O
below	O
each	O
sequence	O
.	O

Shown	O
is	O
a	O
typical	O
immunoblot	O
for	O
phosphorylated	B-protein_state
JNK	B-protein_type
from	O
three	O
independent	O
experiments	O
.	O

The	O
results	O
using	O
Annexin	B-chemical
-	I-chemical
V	I-chemical
stain	O
for	O
membrane	O
phosphatidylserine	O
eversion	O
,	O
combined	O
with	O
propidium	B-chemical
iodide	I-chemical
(	O
PI	B-chemical
)	O
uptake	O
to	O
evaluate	O
cells	O
whose	O
membranes	O
had	O
been	O
compromised	O
.	O

The	O
solid	O
lines	O
are	O
best	O
-	O
fitting	O
results	O
according	O
to	O
the	O
Michaelis	O
–	O
Menten	O
equation	O
with	O
Km	B-evidence
and	O
kcat	B-evidence
values	O
indicated	O
.	O

(	O
d	O
)	O
Gel	B-experimental_method
filtration	I-experimental_method
analysis	I-experimental_method
for	O
interaction	O
of	O
JNK1	B-protein
with	O
MKP5	B-protein
-	O
CD	B-structure_element
and	O
MKP5	B-protein
-	O
KBD	B-structure_element
.	O
(	O
e	O
)	O
GST	B-experimental_method
-	I-experimental_method
mediated	I-experimental_method
pull	I-experimental_method
-	I-experimental_method
down	I-experimental_method
assays	I-experimental_method
for	O
interaction	O
of	O
JNK1	B-protein
with	O
MKP5	B-protein
-	O
CD	B-structure_element
and	O
MKP5	B-protein
-	O
KBD	B-structure_element
.	O

The	O
panels	O
are	O
arranged	O
the	O
same	O
as	O
in	O
Fig	O
.	O
2d	O
.	O
(	O
f	O
)	O
Effects	O
of	O
mutations	B-experimental_method
in	O
MKP5	B-protein
-	O
CD	B-structure_element
on	O
the	O
JNK1	B-protein
dephosphorylation	O
(	O
mean	O
±	O
s	O
.	O
e	O
.	O
m	O
.,	O
n	O
=	O
3	O
).	O

(	O
g	O
)	O
Effects	O
of	O
mutations	B-experimental_method
in	O
MKP5	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

The	O
HAESA	B-protein
ectodomain	B-structure_element
folds	O
into	O
a	O
superhelical	B-structure_element
assembly	I-structure_element
of	O
21	O
leucine	B-structure_element
-	I-structure_element
rich	I-structure_element
repeats	I-structure_element
.	O

The	O
HAESA	B-protein
ectodomain	B-structure_element
is	O
shown	O
in	O
blue	O
(	O
in	O
surface	O
representation	O
),	O
the	O
glycan	B-chemical
structures	O
are	O
shown	O
in	O
yellow	O
.	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

Void	O
(	O
V0	O
)	O
volume	O
and	O
total	O
volume	O
(	O
Vt	O
)	O
are	O
shown	O
,	O
together	O
with	O
elution	O
volumes	O
for	O
molecular	O
mass	O
standards	O
(	O
A	O
,	O
Thyroglobulin	B-protein
,	O
669	O
,	O
000	O
Da	O
;	O
B	O
,	O
Ferritin	B-protein
,	O
440	O
,	O
00	O
Da	O
,	O
C	O
,	O
Aldolase	B-protein
,	O
158	O
,	O
000	O
Da	O
;	O
D	O
,	O
Conalbumin	B-protein
,	O
75	O
,	O
000	O
Da	O
;	O
E	O
,	O
Ovalbumin	B-protein
,	O
44	O
,	O
000	O
Da	O
;	O
F	O
,	O
Carbonic	B-protein
anhydrase	I-protein
,	O
29	O
,	O
000	O
Da	O
).	O

Mutant	B-protein_state
(	O
m	O
)	O
versions	O
,	O
which	O
carry	O
point	B-experimental_method
mutations	I-experimental_method
in	O
their	O
active	B-site
sites	I-site
(	O
Asp837HAESA	B-mutant
→	I-mutant
Asn	I-mutant
,	O
Asp447SERK1	B-mutant
→	I-mutant
Asn	I-mutant
)	O
possess	O
no	O
autophosphorylation	O
activity	O
(	O
lanes	O
2	O
+	O
4	O
).	O

We	O
next	O
determined	O
crystal	B-evidence
structures	I-evidence
of	O
the	O
apo	B-protein_state
HAESA	B-protein
ectodomain	B-structure_element
and	O
of	O
a	O
HAESA	B-complex_assembly
-	I-complex_assembly
IDA	I-complex_assembly
complex	O
,	O
at	O
1	O
.	O
74	O
and	O
1	O
.	O
86	O
Å	O
resolution	O
,	O
respectively	O
(	O
Figure	O
1C	O
;	O
Figure	O
1	O
—	O
figure	O
supplement	O
1B	O
–	O
D	O
;	O
Tables	O
1	O
,	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

Notably	O
,	O
HAESA	B-protein
can	O
discriminate	O
between	O
IDLs	B-protein_type
and	O
functionally	B-protein_state
unrelated	I-protein_state
dodecamer	B-structure_element
peptides	B-chemical
with	O
Hyp	B-ptm
modifications	I-ptm
,	O
such	O
as	O
CLV3	B-protein
(	O
Figures	O
2D	O
,	O
7	O
).	O

Our	O
experiments	O
suggest	O
that	O
among	O
the	O
SERK	B-protein_type
family	I-protein_type
members	I-protein_type
,	O
SERK1	B-protein
is	O
a	O
positive	O
regulator	O
of	O
floral	O
abscission	O
.	O

We	O
thus	O
focused	O
on	O
analyzing	O
the	O
contribution	O
of	O
SERK1	B-protein
to	O
HAESA	B-protein
ligand	O
sensing	O
and	O
receptor	O
activation	O
.	O

Our	O
calorimetry	B-experimental_method
experiments	O
now	O
reveal	O
that	O
SERKs	B-protein_type
may	O
render	O
HAESA	B-protein
,	O
and	O
potentially	O
other	O
receptor	B-protein_type
kinases	I-protein_type
,	O
competent	O
for	O
high	O
-	O
affinity	O
sensing	O
of	O
their	O
cognate	O
ligands	O
.	O

Together	O
,	O
our	O
genetic	B-experimental_method
and	I-experimental_method
biochemical	I-experimental_method
experiments	I-experimental_method
implicate	O
SERK1	B-protein
as	O
a	O
HAESA	B-protein_type
co	I-protein_type
-	I-protein_type
receptor	I-protein_type
in	O
the	O
Arabidopsis	B-taxonomy_domain
abscission	O
zone	O
.	O

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

The	O
SERK1	B-protein
ectodomain	B-structure_element
interacts	O
with	O
the	O
IDA	B-site
peptide	I-site
binding	I-site
site	I-site
using	O
a	O
loop	B-structure_element
region	I-structure_element
(	O
residues	O
51	B-residue_range
-	I-residue_range
59SERK1	I-residue_range
)	O
from	O
its	O
N	O
-	O
terminal	O
cap	B-structure_element
(	O
Figure	O
4A	O
,	O
C	O
).	O

Deletion	B-experimental_method
of	O
the	O
C	O
-	O
terminal	O
Asn69IDA	B-residue_name_number
completely	O
inhibits	B-protein_state
complex	O
formation	O
.	O

15	O
out	O
of	O
15	O
35S	B-gene
::	O
IDA	B-protein
plants	B-taxonomy_domain
,	O
0	O
out	O
of	O
15	O
Col	O
-	O
0	O
plants	B-taxonomy_domain
and	O
0	O
out	O
of	O
15	O
35S	B-gene
::	O
IDA	B-mutant
K66A	I-mutant
/	I-mutant
R67A	I-mutant
double	B-protein_state
-	I-protein_state
mutant	I-protein_state
plants	B-taxonomy_domain
,	O
showed	O
an	O
enlarged	O
abscission	O
zone	O
,	O
respectively	O
(	O
3	O
independent	O
lines	O
were	O
analyzed	O
).	O

In	O
contrast	O
,	O
over	B-experimental_method
-	I-experimental_method
expression	I-experimental_method
of	O
the	O
IDA	B-mutant
Lys66IDA	I-mutant
/	I-mutant
Arg67IDA	I-mutant
→	I-mutant
Ala	I-mutant
double	B-protein_state
mutant	I-protein_state
significantly	O
delays	O
floral	O
abscission	O
when	O
compared	O
to	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
control	O
plants	B-taxonomy_domain
,	O
suggesting	O
that	O
the	O
mutant	B-protein_state
IDA	B-chemical
peptide	I-chemical
has	O
reduced	O
activity	O
in	O
planta	B-taxonomy_domain
(	O
Figure	O
5C	O
–	O
E	O
).	O

For	O
a	O
rapidly	O
growing	O
number	O
of	O
plant	B-taxonomy_domain
signaling	O
pathways	O
,	O
SERK	B-protein_type
proteins	I-protein_type
act	O
as	O
these	O
essential	O
co	B-protein_type
-	I-protein_type
receptors	I-protein_type
(;	O
).	O

The	O
central	O
Hyp	B-residue_name
residue	O
in	O
IDA	B-protein
is	O
found	O
buried	O
in	O
the	O
HAESA	B-protein
peptide	B-site
binding	I-site
surface	I-site
and	O
thus	O
this	O
post	O
-	O
translational	O
modification	O
may	O
regulate	O
IDA	B-protein
bioactivity	O
.	O

In	O
our	O
quantitative	B-experimental_method
biochemical	I-experimental_method
assays	I-experimental_method
,	O
the	O
presence	B-protein_state
of	I-protein_state
SERK1	B-protein
dramatically	O
increases	O
the	O
HAESA	B-protein
binding	O
specificity	O
and	O
affinity	O
for	O
IDA	B-protein
.	O

It	O
is	O
of	O
note	O
that	O
our	O
reported	O
binding	B-evidence
affinities	I-evidence
for	O
IDA	B-protein
and	O
SERK1	B-protein
have	O
been	O
measured	O
using	O
synthetic	B-protein_state
peptides	B-chemical
and	O
the	O
isolated	B-experimental_method
HAESA	B-protein
and	O
SERK1	B-protein
ectodomains	B-structure_element
,	O
and	O
thus	O
might	O
differ	O
in	O
the	O
context	O
of	O
the	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
,	O
membrane	B-protein_state
-	I-protein_state
embedded	I-protein_state
signaling	O
complex	O
.	O

(	O
B	O
)	O
View	O
of	O
the	O
inner	O
surface	O
of	O
the	O
SERK1	B-protein
LRR	B-structure_element
domain	I-structure_element
(	O
PDB	O
-	O
ID	O
4lsc	O
,	O
surface	O
representation	O
,	O
in	O
gray	O
).	O

Structure	B-experimental_method
-	I-experimental_method
guided	I-experimental_method
multiple	I-experimental_method
sequence	I-experimental_method
alignment	I-experimental_method
of	O
IDA	B-protein
and	O
IDA	B-chemical
-	I-chemical
like	I-chemical
peptides	I-chemical
with	O
other	O
plant	B-taxonomy_domain
peptide	B-protein_type
hormone	I-protein_type
families	I-protein_type
,	O
including	O
CLAVATA3	B-protein_type
–	I-protein_type
EMBRYO	I-protein_type
SURROUNDING	I-protein_type
REGION	I-protein_type
-	I-protein_type
RELATED	I-protein_type
(	O
CLV3	B-protein_type
/	I-protein_type
CLE	I-protein_type
),	O
ROOT	B-protein_type
GROWTH	I-protein_type
FACTOR	I-protein_type
–	I-protein_type
GOLVEN	I-protein_type
(	O
RGF	B-protein_type
/	I-protein_type
GLV	I-protein_type
),	O
PRECURSOR	B-protein_type
GENE	I-protein_type
PROPEP1	I-protein_type
(	O
PEP1	B-protein_type
)	O
from	O
Arabidopsis	B-species
thaliana	I-species
.	O

It	O
is	O
interesting	O
to	O
note	O
,	O
that	O
CLEs	B-protein_type
in	O
their	O
mature	B-protein_state
form	I-protein_state
are	O
also	O
hydroxyprolinated	B-protein_state
dodecamers	B-structure_element
,	O
which	O
bind	O
to	O
a	O
surface	B-site
area	I-site
in	O
the	O
BARELY	B-protein_type
ANY	I-protein_type
MERISTEM	I-protein_type
1	I-protein_type
receptor	I-protein_type
that	O
would	O
correspond	O
to	O
part	O
of	O
the	O
IDA	B-site
binding	I-site
cleft	I-site
in	O
HAESA	B-protein
.	O

The	O
structures	B-evidence
suggest	O
a	O
trajectory	O
of	O
IRES	B-site
translocation	O
,	O
required	O
for	O
translation	O
initiation	B-protein_state
,	O
and	O
provide	O
an	O
unprecedented	O
view	O
of	O
eEF2	B-protein
dynamics	O
.	O

To	O
initiate	O
translation	O
,	O
a	O
structured	B-protein_state
IRES	B-site
RNA	B-chemical
interacts	O
with	O
the	O
40S	B-complex_assembly
subunit	B-structure_element
or	O
the	O
80S	B-complex_assembly
ribosome	I-complex_assembly
,	O
resulting	O
in	O
precise	O
positioning	O
of	O
the	O
downstream	O
start	O
codon	O
in	O
the	O
small	B-protein_state
40S	B-complex_assembly
subunit	B-structure_element
.	O

The	O
canonical	O
scenario	O
of	O
cap	O
-	O
dependent	O
and	O
IRES	B-site
-	O
dependent	O
initiation	O
involves	O
positioning	O
of	O
the	O
AUG	O
start	O
codon	O
and	O
the	O
initiator	O
tRNAMet	B-chemical
in	O
the	O
ribosomal	O
peptidyl	B-site
-	I-site
tRNA	I-site
(	I-site
P	I-site
)	I-site
site	I-site
,	O
facilitated	O
by	O
interaction	O
with	O
initiation	B-protein_type
factors	I-protein_type
.	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
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
G530	B-residue_name_number
,	O
A1492	B-residue_name_number
and	O
A1493	B-residue_name_number
in	O
E	B-species
.	I-species
coli	I-species
16S	O
ribosomal	O
RNA	B-chemical
,	O
or	O
rRNA	B-chemical
).	O

How	O
this	O
non	O
-	O
canonical	O
initiation	B-protein_state
complex	O
transitions	O
to	O
the	O
elongation	O
step	O
is	O
not	O
fully	O
understood	O
.	O

Translocation	O
of	O
2tRNA	B-complex_assembly
•	I-complex_assembly
mRNA	I-complex_assembly
involves	O
two	O
major	O
large	O
-	O
scale	O
ribosome	B-complex_assembly
rearrangements	O
(	O
Figure	O
1	O
—	O
figure	O
supplement	O
1	O
)	O
(	O
reviewed	O
in	O
).	O

Concurrently	O
,	O
the	O
deacyl	B-chemical
-	I-chemical
tRNA	I-chemical
interacts	O
with	O
the	O
P	B-site
site	I-site
of	O
the	O
small	B-structure_element
subunit	I-structure_element
and	O
the	O
E	B-site
site	I-site
of	O
the	O
large	B-structure_element
subunit	I-structure_element
(	O
P	B-protein_state
/	I-protein_state
E	I-protein_state
hybrid	I-protein_state
state	O
).	O

Binding	O
of	O
EF	B-protein
-	I-protein
G	I-protein
next	O
to	O
the	O
A	B-site
site	I-site
and	O
reverse	O
rotation	O
of	O
the	O
small	B-structure_element
subunit	I-structure_element
results	O
in	O
translocation	O
of	O
both	O
ASLs	B-structure_element
on	O
the	O
small	B-structure_element
subunit	I-structure_element
.	O

Structures	B-evidence
of	O
the	O
70S	B-complex_assembly
•	I-complex_assembly
EF	I-complex_assembly
-	I-complex_assembly
G	I-complex_assembly
complex	O
bound	B-protein_state
with	I-protein_state
two	O
nearly	B-protein_state
translocated	I-protein_state
tRNAs	B-chemical
,	O
exhibit	O
a	O
large	O
18	O
°	O
to	O
21	O
°	O
head	B-structure_element
swivel	O
in	O
a	O
mid	B-protein_state
-	I-protein_state
rotated	I-protein_state
subunit	B-structure_element
,	O
whereas	O
no	O
head	B-structure_element
swivel	O
is	O
observed	O
in	O
the	O
fully	B-protein_state
rotated	I-protein_state
pre	B-protein_state
-	I-protein_state
translocation	I-protein_state
or	O
in	O
the	O
non	B-protein_state
-	I-protein_state
rotated	I-protein_state
post	B-protein_state
-	I-protein_state
translocation	I-protein_state
70S	B-complex_assembly
•	I-complex_assembly
2tRNA	I-complex_assembly
•	I-complex_assembly
EF	I-complex_assembly
-	I-complex_assembly
G	I-complex_assembly
structures	B-evidence
.	O

The	O
head	B-structure_element
swivel	O
was	O
proposed	O
to	O
facilitate	O
transition	O
of	O
the	O
tRNA	B-chemical
from	O
the	O
P	B-site
to	I-site
E	I-site
site	I-site
by	O
widening	O
a	O
constriction	B-site
between	O
these	O
sites	O
on	O
the	O
30S	B-complex_assembly
subunit	B-structure_element
.	O

This	O
widening	O
allows	O
the	O
ASL	B-structure_element
to	O
sample	O
positions	O
between	O
the	O
P	B-site
and	I-site
E	I-site
sites	I-site
.	O

(	O
a	O
)	O
Structures	B-evidence
of	O
bacterial	B-taxonomy_domain
70S	B-complex_assembly
•	I-complex_assembly
2tRNA	I-complex_assembly
•	I-complex_assembly
mRNA	I-complex_assembly
translocation	O
complexes	O
,	O
ordered	O
according	O
to	O
the	O
position	O
of	O
the	O
translocating	O
A	B-site
->	I-site
P	I-site
tRNA	B-chemical
(	O
orange	O
).	O

The	O
large	O
ribosomal	O
subunit	B-structure_element
is	O
shown	O
in	O
cyan	O
;	O
the	O
small	B-structure_element
subunit	I-structure_element
in	O
light	O
yellow	O
(	O
head	B-structure_element
)	O
and	O
wheat	O
-	O
yellow	O
(	O
body	B-structure_element
),	O
elongation	B-protein
factor	I-protein
G	I-protein
(	O
EF	B-protein
-	I-protein
G	I-protein
)	O
is	O
shown	O
in	O
green	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
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

Subsequent	O
3D	B-experimental_method
classification	I-experimental_method
using	O
a	O
2D	B-evidence
mask	I-evidence
comprising	O
PKI	B-structure_element
and	O
domain	O
IV	B-structure_element
of	O
eEF2	B-protein
yielded	O
5	O
'	O
purified	O
'	O
classes	O
representing	O
Structures	B-evidence
I	I-evidence
through	I-evidence
V	I-evidence
.	O
Sub	B-experimental_method
-	I-experimental_method
classification	I-experimental_method
of	O
each	O
class	O
did	O
not	O
yield	O
additional	O
classes	O
,	O
but	O
helped	O
improve	O
density	B-evidence
in	O
the	O
PKI	B-structure_element
region	O
of	O
class	O
III	O
(	O
estimated	O
resolution	O
and	O
percentage	O
of	O
particles	B-experimental_method
in	O
the	O
sub	B-experimental_method
-	I-experimental_method
classified	I-experimental_method
reconstruction	B-evidence
are	O
shown	O
in	O
parentheses	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

(	O
a	O
)	O
Structures	B-evidence
I	I-evidence
through	I-evidence
V	I-evidence
.	O
In	O
all	O
panels	O
,	O
the	O
large	B-structure_element
ribosomal	I-structure_element
subunit	I-structure_element
is	O
shown	O
in	O
cyan	O
;	O
the	O
small	B-structure_element
subunit	I-structure_element
in	O
light	O
yellow	O
(	O
head	B-structure_element
)	O
and	O
wheat	O
-	O
yellow	O
(	O
body	B-structure_element
);	O
the	O
TSV	B-species
IRES	B-site
in	O
red	O
,	O
eEF2	B-protein
in	O
green	O
.	O

We	O
sought	O
to	O
address	O
the	O
following	O
questions	O
by	O
structural	B-experimental_method
visualization	I-experimental_method
of	O
80S	B-complex_assembly
•	I-complex_assembly
IRES	I-complex_assembly
•	I-complex_assembly
eEF2	I-complex_assembly
translocation	O
complexes	O
:	O
(	O
1	O
)	O
How	O
does	O
a	O
large	O
IRES	B-site
RNA	B-chemical
move	O
through	O
the	O
restricted	O
intersubunit	O
space	O
,	O
bringing	O
PKI	B-structure_element
from	O
the	O
A	B-site
to	I-site
P	I-site
site	I-site
of	O
the	O
small	B-structure_element
subunit	I-structure_element
?	O
(	O
2	O
)	O
How	O
does	O
eEF2	B-protein
mediate	O
IRES	B-site
translocation	O
?	O
(	O
3	O
)	O
Does	O
IRES	B-site
translocation	O
involve	O
large	O
rearrangements	O
in	O
the	O
ribosome	B-complex_assembly
,	O
similar	O
to	O
tRNA	B-chemical
translocation	O
?	O
(	O
4	O
)	O
What	O
,	O
if	O
any	O
,	O
is	O
the	O
mechanistic	O
role	O
of	O
40S	B-complex_assembly
head	B-structure_element
rotation	O
in	O
IRES	B-site
translocation	O
?	O

Maximum	B-experimental_method
-	I-experimental_method
likelihood	I-experimental_method
classification	I-experimental_method
using	O
FREALIGN	B-experimental_method
identified	O
five	O
IRES	B-protein_state
-	I-protein_state
eEF2	I-protein_state
-	I-protein_state
bound	I-protein_state
ribosome	B-complex_assembly
structures	B-evidence
within	O
a	O
single	O
sample	O
(	O
Figures	O
1	O
and	O
2	O
).	O

The	O
structures	B-evidence
differ	O
in	O
the	O
positions	O
and	O
conformations	O
of	O
ribosomal	O
subunits	O
(	O
Figures	O
1b	O
and	O
2	O
),	O
IRES	B-site
RNA	B-chemical
(	O
Figures	O
3	O
and	O
4	O
)	O
and	O
eEF2	B-protein
(	O
Figures	O
5	O
and	O
6	O
).	O

18S	B-chemical
ribosomal	I-chemical
RNA	I-chemical
is	O
shown	O
and	O
ribosomal	O
proteins	O
are	O
omitted	O
for	O
clarity	O
.	O

The	O
superpositions	B-experimental_method
of	O
structures	B-evidence
were	O
performed	O
by	O
structural	B-experimental_method
alignments	I-experimental_method
of	O
the	O
18S	B-chemical
ribosomal	I-chemical
RNAs	I-chemical
excluding	O
the	O
head	B-structure_element
region	O
(	O
nt	O
1150	B-residue_range
–	I-residue_range
1620	I-residue_range
).	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

40S	B-complex_assembly
head	B-structure_element
swivel	O

Comparison	O
of	O
the	O
TSV	B-species
IRES	B-site
and	O
eEF2	B-protein
positions	O
in	O
Structures	B-evidence
I	I-evidence
through	I-evidence
V	I-evidence
.	O

In	O
all	O
panels	O
,	O
superpositions	B-experimental_method
were	O
obtained	O
by	O
structural	B-experimental_method
alignments	I-experimental_method
of	O
the	O
18S	B-chemical
rRNAs	I-chemical
.	O

Ribosomal	O
proteins	O
of	O
the	O
initiation	B-protein_state
state	O
are	O
shown	O
in	O
gray	O
for	O
comparison	O
.	O

Loop	B-structure_element
1	I-structure_element
.	I-structure_element
1	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
of	O
the	O
IRES	B-site
are	O
labeled	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

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
lower	O
row	O
and	O
labeled	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

Pseudoknots	O
and	O
stem	B-structure_element
loops	I-structure_element
are	O
labeled	O
and	O
colored	O
as	O
in	O
(	O
a	O
).	O

The	O
L1	B-structure_element
.	I-structure_element
1	I-structure_element
region	I-structure_element
remains	O
in	O
contact	O
with	O
the	O
L1	B-structure_element
stalk	I-structure_element
(	O
Figure	O
3	O
—	O
figure	O
supplement	O
3	O
).	O

As	O
such	O
,	O
the	O
transition	O
from	O
the	O
initiation	B-protein_state
state	O
to	O
Structure	B-evidence
I	I-evidence
involves	O
repositioning	O
of	O
SL3	B-structure_element
around	O
the	O
A	B-structure_element
-	I-structure_element
site	I-structure_element
finger	I-structure_element
,	O
resembling	O
the	O
transition	O
between	O
the	O
pre	B-protein_state
-	I-protein_state
translocation	I-protein_state
A	B-site
/	I-site
P	I-site
and	O
A	B-site
/	I-site
P	I-site
*	I-site
tRNA	B-chemical
.	O

Another	O
local	O
rearrangement	O
concerns	O
loop	B-structure_element
3	I-structure_element
,	O
also	O
known	O
as	O
the	O
variable	B-structure_element
loop	I-structure_element
region	I-structure_element
,	O
which	O
connects	O
the	O
ASL	B-structure_element
-	I-structure_element
and	I-structure_element
mRNA	I-structure_element
-	I-structure_element
like	I-structure_element
parts	I-structure_element
of	O
PKI	B-structure_element
.	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

(	O
c	O
)	O
Comparison	O
of	O
conformations	O
of	O
eEF2	B-complex_assembly
•	I-complex_assembly
sordarin	I-complex_assembly
in	O
Structure	B-evidence
I	I-evidence
(	O
light	O
green	O
)	O
with	O
those	O
of	O
free	B-protein_state
apo	B-protein_state
-	O
eEF2	B-protein
(	O
magenta	O
)	O
and	O
eEF2	B-complex_assembly
•	I-complex_assembly
sordarin	I-complex_assembly
(	O
teal	O
).	O

Superposition	B-experimental_method
was	O
obtained	O
by	O
structural	B-experimental_method
alignment	I-experimental_method
of	O
the	O
25S	B-chemical
rRNAs	I-chemical
.	O

(	O
e	O
)	O
Comparison	O
of	O
the	O
GTP	B-protein_state
-	I-protein_state
like	I-protein_state
conformation	O
of	O
eEF2	B-complex_assembly
•	I-complex_assembly
GDP	I-complex_assembly
in	O
Structure	B-evidence
I	I-evidence
(	O
light	O
green	O
)	O
with	O
those	O
of	O
70S	B-protein_state
-	I-protein_state
bound	I-protein_state
elongation	B-protein_type
factors	I-protein_type
EF	B-complex_assembly
-	I-complex_assembly
Tu	I-complex_assembly
•	I-complex_assembly
GDPCP	I-complex_assembly
(	O
teal	O
)	O
and	O
EF	B-complex_assembly
-	I-complex_assembly
G	I-complex_assembly
•	I-complex_assembly
GDP	I-complex_assembly
•	I-complex_assembly
fusidic	I-complex_assembly
acid	I-complex_assembly
(	O
magenta	O
;	O
fusidic	O
acid	O
not	O
shown	O
).	O
(	O
f	O
)	O
Cryo	B-experimental_method
-	I-experimental_method
EM	I-experimental_method
density	B-evidence
showing	O
guanosine	B-chemical
diphosphate	I-chemical
bound	B-protein_state
in	I-protein_state
the	O
GTPase	B-site
center	I-site
(	O
green	O
)	O
next	O
to	O
the	O
sarcin	B-structure_element
-	I-structure_element
ricin	I-structure_element
loop	I-structure_element
of	O
25S	B-chemical
rRNA	I-chemical
(	O
cyan	O
)	O
of	O
Structure	B-evidence
II	I-evidence
.	O
(	O
g	O
)	O
Comparison	O
of	O
the	O
sordarin	B-site
-	I-site
binding	I-site
sites	I-site
in	O
the	O
ribosome	B-protein_state
-	I-protein_state
bound	I-protein_state
(	O
light	O
green	O
;	O
Structure	B-evidence
II	I-evidence
)	O
and	O
isolated	O
eEF2	B-protein
(	O
teal	O
).	O

The	O
sarcin	B-structure_element
-	I-structure_element
ricin	I-structure_element
loop	I-structure_element
interacts	O
with	O
the	O
GTP	B-site
-	I-site
binding	I-site
site	I-site
of	O
eEF2	B-protein
(	O
Figures	O
5d	O
and	O
f	O
).	O

(	O
a	O
)	O
eEF2	B-protein
(	O
green	O
)	O
interacts	O
only	O
with	O
the	O
body	B-structure_element
in	O
Structure	B-evidence
I	I-evidence
(	O
eEF2	B-protein
domains	O
are	O
labeled	O
with	O
roman	O
numerals	O
in	O
white	O
),	O
and	O
with	O
both	O
the	O
head	B-structure_element
and	O
body	B-structure_element
in	O
Structures	B-evidence
II	I-evidence
through	I-evidence
V	I-evidence
.	O
Colors	O
are	O
as	O
in	O
Figure	O
1	O
,	O
except	O
for	O
the	O
40S	B-complex_assembly
structural	O
elements	O
that	O
contact	O
eEF2	B-protein
,	O
which	O
are	O
labeled	O
and	O
shown	O
in	O
purple	O
.	O
(	O
b	O
)	O
Entry	O
of	O
eEF2	B-protein
into	O
the	O
40S	B-complex_assembly
A	B-site
site	I-site
,	O
from	O
Structure	B-evidence
I	I-evidence
through	I-evidence
V	I-evidence
.	O
Distances	O
to	O
the	O
A	B-site
-	I-site
site	I-site
accommodated	O
eEF2	B-protein
(	O
Structure	B-evidence
V	I-evidence
)	O
are	O
shown	O
.	O

Because	O
eEF2	B-protein
is	O
rigidly	O
attached	O
to	O
the	O
60S	B-complex_assembly
subunit	B-structure_element
and	O
does	O
not	O
undergo	O
large	O
inter	O
-	O
subunit	B-structure_element
rearrangements	O
,	O
gradual	O
entry	O
of	O
domain	O
IV	B-structure_element
into	O
the	O
A	B-site
site	I-site
between	O
Structures	B-evidence
I	I-evidence
and	I-evidence
V	I-evidence
is	O
due	O
to	O
40S	B-complex_assembly
subunit	B-structure_element
rotation	O
and	O
head	B-structure_element
swivel	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

Domain	O
IV	B-structure_element
is	O
partially	O
engaged	O
with	O
the	O
body	B-structure_element
A	B-site
site	I-site
.	O

The	O
trimethylamino	O
end	O
of	O
Diph699	B-ptm
packs	O
over	O
A1756	B-residue_name_number
(	O
Figure	O
7	O
).	O

In	O
translational	B-protein_type
GTPases	I-protein_type
,	O
switch	B-structure_element
loops	I-structure_element
I	I-structure_element
and	I-structure_element
II	I-structure_element
are	O
involved	O
in	O
the	O
GTPase	B-protein_type
activity	O
(	O
reviewed	O
in	O
).	O

Next	O
to	O
GDP	B-chemical
,	O
the	O
C	O
-	O
terminal	O
part	O
of	O
the	O
switch	B-structure_element
loop	I-structure_element
(	O
aa	O
61	B-residue_range
–	I-residue_range
67	I-residue_range
)	O
adopts	O
a	O
helical	B-protein_state
fold	I-protein_state
.	O

The	O
decoding	B-site
center	I-site
residues	O
A1755	B-residue_name_number
and	O
A1756	B-residue_name_number
are	O
rearranged	O
to	O
pack	O
inside	O
helix	B-structure_element
44	I-structure_element
,	O
making	O
room	O
for	O
eEF2	B-protein
.	O

This	O
conformation	O
of	O
decoding	B-site
center	I-site
residues	O
is	O
also	O
observed	O
in	O
the	O
absence	B-protein_state
of	I-protein_state
A	B-site
-	I-site
site	I-site
ligands	O
.	O

Structure	B-evidence
III	I-evidence
represents	O
a	O
highly	B-protein_state
bent	I-protein_state
IRES	B-site
with	O
PKI	B-structure_element
captured	O
between	O
the	O
head	B-structure_element
A	B-site
and	I-site
P	I-site
sites	I-site

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

Unwinding	O
of	O
the	O
40S	B-complex_assembly
head	B-structure_element
also	O
positions	O
the	O
head	B-structure_element
A	B-site
site	I-site
closer	O
to	O
the	O
body	B-structure_element
A	B-site
site	I-site
.	O

Four	O
views	O
(	O
scenes	O
)	O
are	O
shown	O
:	O
(	O
1	O
)	O
A	O
view	O
down	O
the	O
intersubunit	O
space	O
,	O
with	O
the	O
head	B-structure_element
of	O
the	O
40S	B-complex_assembly
subunit	B-structure_element
oriented	O
toward	O
a	O
viewer	O
,	O
as	O
in	O
Figure	O
1a	O
;	O
(	O
2	O
)	O
A	O
view	O
at	O
the	O
solvent	O
side	O
of	O
the	O
40S	B-complex_assembly
subunit	B-structure_element
,	O
with	O
the	O
40S	B-complex_assembly
head	B-structure_element
shown	O
at	O
the	O
top	O
,	O
as	O
in	O
Figure	O
2	O
—	O
figure	O
supplement	O
1	O
;	O
(	O
3	O
)	O
A	O
view	O
down	O
at	O
the	O
subunit	O
interface	O
of	O
the	O
40S	B-complex_assembly
subunit	B-structure_element
;	O
(	O
4	O
)	O
A	O
close	O
-	O
up	O
view	O
of	O
the	O
decoding	B-site
center	I-site
(	O
A	B-site
site	I-site
)	O
and	O
the	O
P	B-site
site	I-site
,	O
as	O
in	O
Figure	O
2g	O
.	O
Each	O
scene	O
is	O
shown	O
twice	O
.	O

Our	O
structures	B-evidence
reveal	O
previously	O
unseen	O
intermediate	O
states	O
of	O
eEF2	B-protein
or	O
EF	B-protein
-	I-protein
G	I-protein
engagement	O
with	O
the	O
A	B-site
site	I-site
,	O
providing	O
the	O
structural	O
basis	O
for	O
the	O
mechanism	O
of	O
translocase	B-protein_type
action	O
.	O

In	O
the	O
first	O
sub	O
-	O
step	O
(	O
Structures	B-evidence
I	I-evidence
to	I-evidence
IV	I-evidence
),	O
the	O
hind	B-structure_element
end	I-structure_element
advances	O
from	O
the	O
A	B-site
to	I-site
the	I-site
P	I-site
site	I-site
and	O
approaches	O
the	O
front	B-structure_element
end	I-structure_element
,	O
which	O
remains	O
attached	O
to	O
the	O
40S	B-complex_assembly
surface	O
.	O

Upon	O
translocation	O
,	O
the	O
GCU	O
start	O
codon	O
is	O
positioned	O
in	O
the	O
A	B-site
site	I-site
(	O
Structure	B-evidence
V	I-evidence
),	O
ready	O
for	O
interaction	O
with	O
Ala	B-chemical
-	I-chemical
tRNAAla	I-chemical
upon	O
eEF2	B-protein
departure	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

In	O
our	O
structures	B-evidence
,	O
the	O
IRES	B-site
presents	O
to	O
the	O
decoding	B-site
center	I-site
a	O
pre	B-protein_state
-	I-protein_state
translocated	I-protein_state
or	O
fully	B-protein_state
translocated	I-protein_state
ORF	B-structure_element
,	O
rather	O
than	O
a	O
+	O
1	O
(	O
more	O
translocated	O
)	O
ORF	B-structure_element
,	O
suggesting	O
that	O
eEF2	B-protein
does	O
not	O
induce	O
a	O
highly	O
populated	O
fraction	O
of	O
+	O
1	O
shifted	O
IRES	B-site
mRNAs	B-chemical
.	O

This	O
is	O
consistent	O
with	O
the	O
observations	O
that	O
the	O
intergenic	O
IRESs	B-site
are	O
prone	O
to	O
reverse	O
translocation	O
.	O

In	O
the	O
initiation	B-protein_state
state	O
,	O
the	O
IRES	B-site
resembles	O
a	O
pre	B-protein_state
-	I-protein_state
translocation	I-protein_state
2tRNA	B-complex_assembly
•	I-complex_assembly
mRNA	I-complex_assembly
complex	O
reduced	O
to	O
the	O
A	B-site
/	I-site
P	I-site
-	O
tRNA	B-chemical
anticodon	B-structure_element
-	I-structure_element
stem	I-structure_element
loop	I-structure_element
and	O
elbow	B-structure_element
in	O
the	O
A	B-site
site	I-site
and	O
the	O
P	B-site
/	I-site
E	I-site
-	O
tRNA	B-chemical
elbow	B-structure_element
contacting	O
the	O
L1	B-structure_element
stalk	I-structure_element
.	O

Because	O
the	O
anticodon	B-structure_element
-	I-structure_element
stem	I-structure_element
loop	I-structure_element
of	O
the	O
A	B-site
-	O
tRNA	B-chemical
is	O
sufficient	O
for	O
translocation	O
completion	O
,	O
we	O
ascribe	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
to	O
the	O
absence	B-protein_state
of	I-protein_state
the	O
P	B-site
/	I-site
E	I-site
-	O
tRNA	B-chemical
elements	O
,	O
either	O
the	O
ASL	B-structure_element
or	O
the	O
acceptor	O
arm	O
,	O
or	O
both	O
.	O

Translocases	B-protein_type
are	O
efficient	O
enzymes	O
.	O

EF	B-protein
-	I-protein
G	I-protein
enhances	O
the	O
translocation	O
rate	O
by	O
several	O
orders	O
of	O
magnitude	O
,	O
aided	O
by	O
an	O
additional	O
2	O
-	O
to	O
50	O
-	O
fold	O
boost	O
from	O
GTP	B-chemical
hydrolysis	O
.	O

Due	O
to	O
the	O
lack	O
of	O
structures	B-evidence
of	O
translocation	O
intermediates	O
,	O
the	O
mechanistic	O
role	O
of	O
eEF2	B-protein
/	O
EF	B-protein
-	I-protein
G	I-protein
is	O
not	O
fully	O
understood	O
.	O

The	O
unlocking	O
model	O
of	O
the	O
ribosome	B-complex_assembly
•	I-complex_assembly
2tRNA	I-complex_assembly
•	I-complex_assembly
mRNA	I-complex_assembly
pre	B-protein_state
-	I-protein_state
translocation	I-protein_state
complex	O
has	O
been	O
proposed	O
decades	O
ago	O
and	O
functional	O
requirement	O
of	O
the	O
translocase	B-protein_type
in	O
this	O
process	O
has	O
been	O
implicated	O
.	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

In	O
the	O
fully	B-protein_state
-	I-protein_state
rotated	I-protein_state
pre	B-protein_state
-	I-protein_state
translocation	I-protein_state
-	O
like	O
Structure	B-evidence
I	I-evidence
,	O
an	O
additional	O
interaction	O
exists	O
.	O

We	O
propose	O
that	O
the	O
shift	O
of	O
domain	O
III	B-structure_element
by	O
uS12	B-protein
initiates	O
intra	O
-	O
domain	O
rearrangements	O
in	O
eEF2	B-protein
,	O
which	O
unstack	O
the	O
β	B-structure_element
-	I-structure_element
platform	I-structure_element
of	O
domain	O
III	B-structure_element
from	O
that	O
of	O
domain	O
V	B-structure_element
.	O
This	O
would	O
result	O
in	O
a	O
conformation	O
characteristic	O
of	O
free	B-protein_state
eEF2	B-protein
and	O
EF	B-protein
-	I-protein
G	I-protein
in	O
which	O
the	O
β	B-structure_element
-	I-structure_element
platforms	I-structure_element
are	O
nearly	O
perpendicular	O
.	O

Sordarin	B-chemical
is	O
a	O
potent	O
antifungal	O
antibiotic	O
that	O
inhibits	O
translation	O
.	O

Although	O
our	O
complex	O
was	O
assembled	O
using	O
eEF2	B-complex_assembly
•	I-complex_assembly
GTP	I-complex_assembly
,	O
density	B-evidence
maps	I-evidence
clearly	O
show	O
GDP	B-chemical
and	O
Mg2	B-chemical
+	I-chemical
in	O
each	O
structure	B-evidence
(	O
Figure	O
5g	O
).	O

In	O
all	O
five	O
structures	B-evidence
,	O
sordarin	B-chemical
is	O
bound	B-protein_state
between	O
domains	O
III	B-structure_element
and	O
V	B-structure_element
of	O
eEF2	B-protein
,	O
stabilized	O
by	O
hydrophobic	B-bond_interaction
interactions	I-bond_interaction
identical	O
to	O
those	O
in	O
the	O
isolated	B-protein_state
eEF2	B-complex_assembly
•	I-complex_assembly
sordarin	I-complex_assembly
complex	O
(	O
Figures	O
5g	O
and	O
h	O
).	O

Implications	O
for	O
tRNA	B-chemical
and	O
mRNA	B-chemical
translocation	O
during	O
translation	O

First	O
,	O
we	O
propose	O
that	O
tRNA	B-chemical
and	O
IRES	B-site
translocations	O
occur	O
via	O
the	O
same	O
general	O
trajectory	O
.	O

This	O
is	O
consistent	O
with	O
the	O
idea	O
of	O
a	O
rather	O
flat	O
energy	O
landscape	O
of	O
translocation	O
,	O
suggested	O
by	O
recent	O
work	O
that	O
measured	O
mechanical	O
work	O
produced	O
by	O
the	O
ribosome	B-complex_assembly
during	O
translocation	O
.	O

We	O
note	O
that	O
four	O
of	O
our	O
near	O
-	O
atomic	O
resolution	O
maps	B-evidence
comprised	O
~	O
30	O
,	O
000	O
particles	B-experimental_method
each	O
,	O
the	O
minimum	O
number	O
required	O
for	O
a	O
near	B-evidence
-	I-evidence
atomic	I-evidence
-	I-evidence
resolution	I-evidence
reconstruction	I-evidence
of	O
the	O
ribosome	B-complex_assembly
.	O

This	O
difference	O
likely	O
accounts	O
for	O
the	O
inefficient	O
translocation	O
of	O
the	O
IRES	B-site
,	O
which	O
is	O
difficult	O
to	O
stabilize	O
in	O
the	O
post	B-protein_state
-	I-protein_state
translocation	I-protein_state
state	O
and	O
therefore	O
is	O
prone	O
to	O
reverse	O
translocation	O
.	O

The	O
uniformity	O
of	O
ribosome	B-complex_assembly
dynamics	O
underscores	O
the	O
idea	O
that	O
translocation	O
is	O
an	O
inherent	O
and	O
structurally	O
-	O
optimized	O
property	O
of	O
the	O
ribosome	B-complex_assembly
,	O
supported	O
also	O
by	O
translocation	O
activity	O
in	O
the	O
absence	B-protein_state
of	I-protein_state
the	O
elongation	B-protein_type
factor	I-protein_type
.	O

Our	O
current	O
understanding	O
of	O
macromolecular	O
machines	O
,	O
such	O
as	O
the	O
ribosome	B-complex_assembly
,	O
is	O
often	O
limited	O
by	O
a	O
gap	O
between	O
biophysical	B-experimental_method
/	I-experimental_method
biochemical	I-experimental_method
studies	I-experimental_method
and	O
structural	B-experimental_method
studies	I-experimental_method
.	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

The	O
classification	O
,	O
which	O
followed	O
an	O
initial	O
alignment	O
of	O
all	O
particles	B-experimental_method
to	O
a	O
single	O
reference	O
,	O
required	O
about	O
130	O
,	O
000	O
CPU	O
hours	O
or	O
about	O
five	O
to	O
six	O
full	O
days	O
on	O
a	O
1000	O
-	O
CPU	O
cluster	O
.	O

The	O
N	O
-	O
terminal	O
propeptides	B-structure_element
protecting	O
the	O
active	B-site
-	I-site
site	I-site
threonines	B-residue_name
are	O
autocatalytically	B-ptm
released	O
only	O
on	O
completion	O
of	O
assembly	O
.	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

Sequencing	B-experimental_method
of	I-experimental_method
the	I-experimental_method
plasmids	I-experimental_method
,	O
testing	O
them	O
in	O
both	O
published	O
yeast	B-taxonomy_domain
strain	O
backgrounds	O
and	O
site	B-experimental_method
-	I-experimental_method
directed	I-experimental_method
mutagenesis	I-experimental_method
revealed	O
that	O
the	O
β5	B-mutant
-	I-mutant
T1A	I-mutant
mutant	B-protein_state
pp	B-chemical
cis	B-protein_state
is	O
viable	O
,	O
but	O
suffers	O
from	O
a	O
marked	O
growth	O
defect	O
that	O
requires	O
extended	O
incubation	O
of	O
4	O
–	O
5	O
days	O
for	O
initial	O
colony	O
formation	O
(	O
Table	O
1	O
and	O
Supplementary	O
Methods	O
).	O

For	O
subunit	O
β1	B-protein
,	O
this	O
process	O
was	O
previously	O
inferred	O
to	O
require	O
that	O
the	O
propeptide	B-structure_element
residue	O
at	O
position	O
(-	B-residue_number
2	I-residue_number
)	I-residue_number
of	O
the	O
subunit	O
precursor	O
occupies	O
the	O
S1	B-site
specificity	I-site
pocket	I-site
of	O
the	O
substrate	B-site
-	I-site
binding	I-site
channel	I-site
formed	O
by	O
amino	O
acid	O
45	B-residue_number
(	O
for	O
details	O
see	O
Supplementary	O
Note	O
2	O
).	O

Here	O
we	O
again	O
analysed	O
the	O
β1	B-mutant
-	I-mutant
T1A	I-mutant
mutant	B-protein_state
crystallographically	B-experimental_method
but	O
in	O
addition	O
determined	O
the	O
structures	B-evidence
of	O
the	O
β2	B-mutant
-	I-mutant
T1A	I-mutant
single	O
and	O
β1	B-mutant
-	I-mutant
T1A	I-mutant
-	I-mutant
β2	I-mutant
-	I-mutant
T1A	I-mutant
double	O
mutants	O
(	O
Protein	O
Data	O
Bank	O
(	O
PDB	O
)	O
entry	O
codes	O
are	O
provided	O
in	O
Supplementary	O
Table	O
1	O
).	O

Instead	O
,	O
the	O
plasticity	O
of	O
the	O
β5	B-protein
S1	B-site
pocket	I-site
caused	O
by	O
the	O
rotational	O
flexibility	O
of	O
Met45	B-residue_name_number
might	O
prevent	O
stable	O
accommodation	O
of	O
His	B-residue_name_number
(-	I-residue_name_number
2	I-residue_name_number
)	I-residue_name_number
in	O
the	O
S1	B-site
site	I-site
and	O
thus	O
also	O
promote	O
its	O
immediate	O
release	O
after	O
autolysis	B-ptm
.	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

By	O
contrast	O
,	O
the	O
prosegments	B-structure_element
of	O
the	O
β5	B-mutant
-	I-mutant
H	I-mutant
(-	I-mutant
2	I-mutant
)	I-mutant
L	I-mutant
-	I-mutant
T1A	I-mutant
and	O
the	O
β5	B-mutant
-	I-mutant
H	I-mutant
(-	I-mutant
2	I-mutant
)	I-mutant
T	I-mutant
-	I-mutant
T1A	I-mutant
mutants	O
were	O
significantly	O
better	O
resolved	O
in	O
the	O
2FO	B-evidence
–	I-evidence
FC	I-evidence
electron	I-evidence
-	I-evidence
density	I-evidence
maps	I-evidence
yet	O
not	O
at	O
full	O
occupancy	O
(	O
Supplementary	O
Fig	O
.	O
4b	O
,	O
c	O
and	O
Supplementary	O
Table	O
1	O
),	O
suggesting	O
that	O
the	O
natural	O
propeptide	B-structure_element
bearing	O
His	B-residue_name_number
(-	I-residue_name_number
2	I-residue_name_number
)	I-residue_name_number
is	O
most	O
favourable	O
.	O

This	O
result	O
proves	O
that	O
the	O
naturally	O
occurring	O
His	B-residue_name_number
(-	I-residue_name_number
2	I-residue_name_number
)	I-residue_name_number
of	O
the	O
β5	B-protein
propeptide	B-structure_element
does	O
not	O
stably	O
fit	O
into	O
the	O
S1	B-site
site	I-site
.	O

Bearing	O
in	O
mind	O
that	O
in	O
contrast	O
to	O
Thr	B-residue_name_number
(-	I-residue_name_number
2	I-residue_name_number
)	I-residue_name_number
in	O
β2	B-protein
,	O
Leu	B-residue_name_number
(-	I-residue_name_number
2	I-residue_name_number
)	I-residue_name_number
in	O
subunit	O
β1	B-protein
is	O
not	B-protein_state
conserved	I-protein_state
among	O
species	O
(	O
Supplementary	O
Fig	O
.	O
3a	O
),	O
we	O
created	B-experimental_method
a	O
β2	B-mutant
-	I-mutant
T	I-mutant
(-	I-mutant
2	I-mutant
)	I-mutant
V	I-mutant
proteasome	B-complex_assembly
mutant	B-protein_state
.	O

However	O
,	O
in	O
the	O
immature	B-protein_state
particle	B-complex_assembly
Thr1NH2	B-residue_name_number
is	O
blocked	O
by	O
the	O
propeptide	B-structure_element
and	O
cannot	O
activate	O
Thr1Oγ	B-residue_name_number
.	O

Instead	O
,	O
Lys33NH2	B-residue_name_number
,	O
which	O
is	O
in	O
hydrogen	B-bond_interaction
-	I-bond_interaction
bonding	I-bond_interaction
distance	O
to	O
Thr1Oγ	B-residue_name_number
(	O
2	O
.	O
7	O
Å	O
)	O
in	O
all	O
catalytically	B-protein_state
active	I-protein_state
β	B-protein
subunits	I-protein
(	O
Fig	O
.	O
3a	O
,	O
b	O
),	O
was	O
proposed	O
to	O
serve	O
as	O
the	O
proton	O
acceptor	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

The	O
ChT	O
-	O
L	O
activity	O
of	O
the	O
β5	B-mutant
-	I-mutant
D17N	I-mutant
pp	B-chemical
in	O
trans	B-protein_state
CP	B-complex_assembly
towards	O
the	O
canonical	O
β5	B-protein
model	O
substrates	O
N	B-chemical
-	I-chemical
succinyl	I-chemical
-	I-chemical
Leu	I-chemical
-	I-chemical
Leu	I-chemical
-	I-chemical
Val	I-chemical
-	I-chemical
Tyr	I-chemical
-	I-chemical
7	I-chemical
-	I-chemical
amino	I-chemical
-	I-chemical
4	I-chemical
-	I-chemical
methylcoumarin	I-chemical
(	O
Suc	B-chemical
-	I-chemical
LLVY	I-chemical
-	I-chemical
AMC	I-chemical
)	O
and	O
carboxybenzyl	B-chemical
-	I-chemical
Gly	I-chemical
-	I-chemical
Gly	I-chemical
-	I-chemical
Leu	I-chemical
-	I-chemical
para	I-chemical
-	I-chemical
nitroanilide	I-chemical
(	O
Z	B-chemical
-	I-chemical
GGL	I-chemical
-	I-chemical
pNA	I-chemical
)	O
was	O
severely	O
reduced	O
(	O
Supplementary	O
Fig	O
.	O
6b	O
),	O
confirming	O
that	O
Asp17	B-residue_name_number
is	O
of	O
fundamental	O
importance	O
for	O
the	O
catalytic	O
activity	O
of	O
the	O
mature	B-protein_state
proteasome	B-complex_assembly
.	O

Strikingly	O
,	O
although	O
the	O
X	B-evidence
-	I-evidence
ray	I-evidence
data	I-evidence
on	O
the	O
β5	B-mutant
-	I-mutant
D17N	I-mutant
mutant	B-protein_state
with	O
the	O
propeptide	B-structure_element
expressed	B-experimental_method
in	O
cis	B-protein_state
and	O
in	O
trans	B-protein_state
looked	O
similar	O
,	O
there	O
was	O
a	O
pronounced	O
difference	O
in	O
their	O
growth	O
phenotypes	O
observed	O
(	O
Supplementary	O
Fig	O
.	O
6a	O
and	O
Supplementary	O
Fig	O
.	O
7b	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

The	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
involving	O
Ser169OH	B-residue_name_number
are	O
intact	O
and	O
may	O
account	O
for	O
residual	O
substrate	O
turnover	O
.	O

Together	O
,	O
these	O
observations	O
suggest	O
that	O
efficient	O
peptide	O
-	O
bond	O
hydrolysis	O
requires	O
that	O
Lys33NH2	B-residue_name_number
hydrogen	B-bond_interaction
bonds	I-bond_interaction
to	O
the	O
active	O
site	O
nucleophile	O
.	O

Activity	B-experimental_method
assays	I-experimental_method
with	O
the	O
β5	B-protein
-	O
specific	O
substrate	O
Suc	B-chemical
-	I-chemical
LLVY	I-chemical
-	I-chemical
AMC	I-chemical
demonstrated	O
that	O
the	O
ChT	O
-	O
L	O
activity	O
of	O
the	O
T1S	B-mutant
mutant	B-protein_state
is	O
reduced	O
by	O
40	O
–	O
45	O
%	O
compared	O
with	O
WT	B-protein_state
proteasomes	B-complex_assembly
depending	O
on	O
the	O
incubation	O
temperature	O
(	O
Fig	O
.	O
4b	O
and	O
Supplementary	O
Fig	O
.	O
9c	O
).	O

Compared	O
with	O
Thr1Oγ	B-residue_name_number
in	O
WT	B-protein_state
CP	B-complex_assembly
structures	B-evidence
,	O
Ser1Oγ	B-residue_name_number
is	O
rotated	O
by	O
60	O
°.	O

In	O
addition	O
,	O
they	O
prevent	O
irreversible	O
inactivation	O
of	O
the	O
Thr1	B-residue_name_number
N	O
terminus	O
by	O
N	B-ptm
-	I-ptm
acetylation	I-ptm
.	O

However	O
,	O
removal	B-experimental_method
of	I-experimental_method
the	O
β5	B-protein
prosegment	B-structure_element
or	O
any	O
interference	O
with	O
its	O
cleavage	O
causes	O
severe	O
phenotypic	O
defects	O
.	O

On	O
the	O
basis	O
of	O
the	O
numerous	O
CP	B-complex_assembly
:	I-complex_assembly
ligand	I-complex_assembly
complexes	O
solved	O
during	O
the	O
past	O
18	O
years	O
and	O
in	O
the	O
current	O
study	O
,	O
we	O
provide	O
a	O
revised	O
interpretation	O
of	O
proteasome	B-complex_assembly
active	B-site
-	I-site
site	I-site
architecture	I-site
.	O

We	O
propose	O
a	O
catalytic	B-site
triad	I-site
for	O
the	O
active	B-site
site	I-site
of	O
the	O
CP	B-complex_assembly
consisting	O
of	O
residues	O
Thr1	B-residue_name_number
,	O
Lys33	B-residue_name_number
and	O
Asp	B-residue_name
/	O
Glu17	B-residue_name_number
,	O
which	O
are	O
conserved	O
among	O
all	O
proteolytically	O
active	O
eukaryotic	B-taxonomy_domain
,	O
bacterial	B-taxonomy_domain
and	O
archaeal	B-taxonomy_domain
proteasome	B-complex_assembly
subunits	O
.	O

Cleavage	O
of	O
the	O
scissile	O
peptide	O
bond	O
requires	O
protonation	O
of	O
the	O
emerging	O
free	O
amine	O
,	O
and	O
in	O
the	O
proteasome	B-complex_assembly
,	O
the	O
Thr1	B-residue_name_number
amine	O
group	O
is	O
likely	O
to	O
assume	O
this	O
function	O
.	O

Analogously	O
,	O
Thr1NH3	B-residue_name_number
+	O
might	O
promote	O
the	O
bivalent	O
reaction	O
mode	O
of	O
epoxyketone	O
inhibitors	O
by	O
protonating	O
the	O
epoxide	O
moiety	O
to	O
create	O
a	O
positively	O
charged	O
trivalent	O
oxygen	O
atom	O
that	O
is	O
subsequently	O
nucleophilically	O
attacked	O
by	O
Thr1NH2	B-residue_name_number
.	O

The	O
residues	O
Ser129	B-residue_name_number
and	O
Asp166	B-residue_name_number
are	O
expected	O
to	O
increase	O
the	O
pKa	O
value	O
of	O
Thr1N	B-residue_name_number
,	O
thereby	O
favouring	O
its	O
charged	O
state	O
.	O

Consistent	O
with	O
playing	O
an	O
essential	O
role	O
in	O
proton	O
shuttling	O
,	O
the	O
mutation	B-experimental_method
D166A	B-mutant
prevents	O
autolysis	B-ptm
of	O
the	O
archaeal	B-taxonomy_domain
CP	B-complex_assembly
and	O
the	O
exchange	B-experimental_method
D166N	B-mutant
impairs	O
catalytic	O
activity	O
of	O
the	O
yeast	B-taxonomy_domain
CP	B-complex_assembly
about	O
60	O
%.	O

While	O
Lys33NH2	B-residue_name_number
and	O
Asp17Oδ	B-residue_name_number
are	O
required	O
to	O
deprotonate	O
the	O
Thr1	B-residue_name_number
hydroxyl	O
side	O
chain	O
,	O
Ser129OH	B-residue_name_number
and	O
Asp166OH	B-residue_name_number
serve	O
to	O
protonate	O
the	O
N	O
-	O
terminal	O
amine	O
group	O
of	O
Thr1	B-residue_name_number
.	O

Structural	B-evidence
analyses	I-evidence
support	O
these	O
findings	O
with	O
the	O
T1S	B-mutant
mutant	B-protein_state
and	O
provide	O
an	O
explanation	O
for	O
the	O
strict	B-protein_state
use	I-protein_state
of	I-protein_state
Thr	B-residue_name
residues	O
in	O
proteasomes	B-complex_assembly
.	O

Notably	O
,	O
proteolytically	B-protein_state
active	I-protein_state
proteasome	B-complex_assembly
subunits	O
from	O
archaea	B-taxonomy_domain
,	O
yeast	B-taxonomy_domain
and	O
mammals	B-taxonomy_domain
,	O
including	O
constitutive	O
,	O
immuno	O
-	O
and	O
thymoproteasome	O
subunits	O
,	O
either	O
encode	O
Thr	B-residue_name
or	O
Ile	B-residue_name
at	O
position	O
3	B-residue_number
,	O
indicating	O
the	O
importance	O
of	O
the	O
Cγ	O
for	O
fixing	O
the	O
position	O
of	O
the	O
nucleophilic	O
Thr1	B-residue_name_number
.	O

The	O
major	O
determinant	O
of	O
the	O
S1	B-site
specificity	I-site
pocket	I-site
,	O
residue	O
45	B-residue_number
,	O
is	O
depicted	O
.	O

Note	O
the	O
tight	O
conformation	O
of	O
Gly	B-residue_name_number
(-	I-residue_name_number
1	I-residue_name_number
)	I-residue_name_number
and	O
Ala1	B-residue_name_number
before	O
propeptide	B-structure_element
removal	O
(	O
G	B-residue_name_number
(-	I-residue_name_number
1	I-residue_name_number
)	I-residue_name_number
turn	O
;	O
cyan	O
double	O
arrow	O
)	O
compared	O
with	O
the	O
relaxed	O
,	O
processed	B-protein_state
WT	B-protein_state
active	B-site
-	I-site
site	I-site
Thr1	B-residue_name_number
(	O
red	O
double	O
arrow	O
).	O

The	O
black	O
arrow	O
indicates	O
the	O
attack	O
of	O
Thr1Oγ	B-residue_name_number
onto	O
the	O
carbonyl	O
carbon	O
atom	O
of	O
Gly	B-residue_name_number
(-	I-residue_name_number
1	I-residue_name_number
).	I-residue_name_number

While	O
residue	O
(-	B-residue_number
2	I-residue_number
)	I-residue_number
of	O
the	O
β1	B-protein
and	O
β2	B-protein
prosegments	B-structure_element
fit	O
the	O
S1	B-site
pocket	I-site
,	O
His	B-residue_name_number
(-	I-residue_name_number
2	I-residue_name_number
)	I-residue_name_number
of	O
the	O
β5	B-protein
propeptide	B-structure_element
occupies	O
the	O
S2	B-site
pocket	I-site
.	O

The	O
(-	B-residue_number
2	I-residue_number
)	I-residue_number
residues	O
of	O
both	O
prosegments	B-structure_element
point	O
into	O
the	O
S1	B-site
pocket	I-site
,	O
but	O
only	O
Thr	B-residue_name_number
(-	I-residue_name_number
2	I-residue_name_number
)	I-residue_name_number
OH	O
of	O
β2	B-protein
forms	O
a	O
hydrogen	B-bond_interaction
bridge	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
black	O
dashed	O
line	O
).	O

(	O
d	O
)	O
Structural	B-experimental_method
superposition	I-experimental_method
of	O
the	O
matured	B-protein_state
β2	B-protein
active	B-site
site	I-site
,	O
the	O
WT	B-protein_state
β2	B-mutant
-	I-mutant
T1A	I-mutant
propeptide	B-structure_element
and	O
the	O
β2	B-mutant
-	I-mutant
T	I-mutant
(-	I-mutant
2	I-mutant
)	I-mutant
V	I-mutant
mutant	B-protein_state
propeptide	B-structure_element
.	O

The	O
Thr1	B-residue_name_number
N	O
terminus	O
is	O
engaged	O
in	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
with	O
Ser129Oγ	B-residue_name_number
,	O
the	O
carbonyl	O
oxygen	O
of	O
residue	O
168	B-residue_number
,	O
Ser169Oγ	B-residue_name_number
and	O
Asp166Oδ	B-residue_name_number
.	O
(	O
b	O
)	O
The	O
orientations	O
of	O
the	O
active	B-site
-	I-site
site	I-site
residues	I-site
involved	O
in	O
hydrogen	B-bond_interaction
bonding	I-bond_interaction
are	O
strictly	B-protein_state
conserved	I-protein_state
in	O
each	O
proteolytic	B-site
centre	I-site
,	O
as	O
shown	O
by	O
superposition	B-experimental_method
of	O
the	O
β	B-protein
subunits	I-protein
.	O

In	O
the	O
latter	O
,	O
a	O
water	B-chemical
molecule	O
(	O
red	O
sphere	O
)	O
is	O
found	O
at	O
the	O
position	O
where	O
in	O
the	O
WT	B-protein_state
structure	O
the	O
side	O
chain	O
amine	O
group	O
of	O
Lys33	B-residue_name_number
is	O
located	O
.	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

The	O
charged	O
Thr1	B-residue_name_number
N	O
terminus	O
may	O
engage	O
in	O
the	O
orientation	O
of	O
the	O
amide	O
moiety	O
and	O
donate	O
a	O
proton	O
to	O
the	O
emerging	O
N	O
terminus	O
of	O
the	O
C	O
-	O
terminal	O
cleavage	O
product	O
.	O

The	O
2FO	B-evidence
–	I-evidence
FC	I-evidence
electron	I-evidence
-	I-evidence
density	I-evidence
maps	I-evidence
(	O
blue	O
mesh	O
)	O
for	O
Ser1	B-residue_name_number
(	O
brown	O
)	O
and	O
the	O
covalently	O
bound	O
ligands	O
(	O
green	O
;	O
only	O
the	O
P1	B-site
site	I-site
(	O
Leu1	B-residue_name_number
)	O
is	O
shown	O
)	O
are	O
contoured	O
at	O
1σ	O
.	O

The	O
Taf14	B-protein
YEATS	B-structure_element
domain	I-structure_element
is	O
a	O
reader	O
of	O
histone	B-protein_type
crotonylation	B-ptm

Owing	O
to	O
some	O
differences	O
in	O
their	O
genomic	O
distribution	O
,	O
the	O
crotonyllysine	B-residue_name
and	O
acetyllysine	B-residue_name
(	O
Kac	B-residue_name
)	O
modifications	O
have	O
been	O
linked	O
to	O
distinct	O
functional	O
outcomes	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

The	O
most	O
striking	O
feature	O
of	O
the	O
crotonyllysine	B-residue_name
recognition	O
mechanism	O
is	O
the	O
unique	O
coordination	O
of	O
crotonylated	B-protein_state
lysine	B-residue_name
residue	O
.	O

The	O
π	B-bond_interaction
bond	I-bond_interaction
conjugation	O
of	O
the	O
crotonyl	B-chemical
group	O
gives	O
rise	O
to	O
a	O
dipole	O
moment	O
of	O
the	O
alkene	O
moiety	O
,	O
resulting	O
in	O
a	O
partial	O
positive	O
charge	O
on	O
the	O
β	O
-	O
carbon	O
(	O
Cβ	O
)	O
and	O
a	O
partial	O
negative	O
charge	O
on	O
the	O
α	O
-	O
carbon	O
(	O
Cα	O
).	O

The	O
dissociation	B-evidence
constant	I-evidence
(	O
Kd	B-evidence
)	O
for	O
the	O
Taf14	B-complex_assembly
YEATS	I-complex_assembly
-	I-complex_assembly
H3K9cr5	I-complex_assembly
-	I-complex_assembly
13	I-complex_assembly
complex	O
was	O
found	O
to	O
be	O
9	O
.	O
5	O
μM	O
,	O
as	O
measured	O
by	O
fluorescence	B-experimental_method
spectroscopy	I-experimental_method
(	O
Supplementary	O
Fig	O
.	O
2c	O
).	O

Towards	O
this	O
end	O
,	O
we	O
probed	O
extracts	O
derived	O
from	O
yeast	B-taxonomy_domain
cells	O
in	O
which	O
major	O
yeast	B-taxonomy_domain
HATs	B-protein_type
(	O
HAT1	B-protein
,	O
Gcn5	B-protein
,	O
and	O
Rtt109	B-protein
)	O
or	O
HDACs	B-protein_type
(	O
Rpd3	B-protein
,	O
Hos1	B-protein
,	O
and	O
Hos2	B-protein
)	O
were	O
deleted	B-experimental_method
.	O

In	O
contrast	O
,	O
binding	O
of	O
H3K9ac	B-protein_type
resulted	O
in	O
an	O
intermediate	O
exchange	O
,	O
which	O
is	O
characteristic	O
of	O
a	O
weaker	O
association	O
.	O

The	O
preference	O
for	O
H3K9cr	B-protein_type
over	O
H3K9ac	B-protein_type
,	O
H3K9pr	B-protein_type
and	O
H3K9bu	B-protein_type
was	O
supported	O
by	O
1H	B-experimental_method
,	I-experimental_method
15N	I-experimental_method
HSQC	I-experimental_method
titration	I-experimental_method
experiments	I-experimental_method
.	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

Cellular	O
homeostasis	O
requires	O
correct	O
delivery	O
of	O
cell	B-protein_type
-	I-protein_type
surface	I-protein_type
receptor	I-protein_type
proteins	O
(	O
cargo	O
)	O
to	O
their	O
target	O
subcellular	O
compartments	O
.	O

The	O
adapter	B-protein_type
proteins	I-protein_type
Tom1	B-protein
and	O
Tollip	B-protein
are	O
involved	O
in	O
sorting	O
of	O
ubiquitinated	B-ptm
cargo	O
in	O
endosomal	O
compartments	O
.	O

Recruitment	O
of	O
Tom1	B-protein
to	O
the	O
endosomal	O
compartments	O
is	O
mediated	O
by	O
its	O
GAT	B-structure_element
domain	O
’	O
s	O
association	O
to	O
Tollip	B-protein
’	O
s	O
Tom1	B-structure_element
-	I-structure_element
binding	I-structure_element
domain	I-structure_element
(	O
TBD	B-structure_element
).	O

Subject	O
area	O
Biology	O
More	O
specific	O
subject	O
area	O
Structural	O
biology	O
Type	O
of	O
data	O
Table	O
,	O
text	O
file	O
,	O
graph	O
,	O
figures	O
How	O
data	O
was	O
acquired	O
Circular	B-experimental_method
dichroism	I-experimental_method
and	O
NMR	B-experimental_method
.	O

Analysis	O
of	O
the	O
far	B-experimental_method
-	I-experimental_method
UV	I-experimental_method
circular	I-experimental_method
dichroism	I-experimental_method
(	O
CD	B-experimental_method
)	O
spectrum	B-evidence
of	O
the	O
Tom	B-protein
1	I-protein
GAT	B-structure_element
domain	O
(	O
Fig	O
.	O
1	O
)	O
predicts	O
58	O
.	O
7	O
%	O
α	B-structure_element
-	I-structure_element
helix	I-structure_element
,	O
3	O
%	O
β	B-structure_element
-	I-structure_element
strand	I-structure_element
,	O
15	O
.	O
5	O
%	O
turn	O
,	O
and	O
22	O
.	O
8	O
%	O
disordered	O
regions	O
.	O

Helices	O
are	O
shown	O
in	O
orange	O
,	O
whereas	O
loops	O
are	O
colored	O
in	O
green	O
.	O
(	O
B	O
)	O
Ribbon	O
illustration	O
of	O
the	O
Tom1	B-protein
GAT	B-structure_element
domain	O
.	O

deviations	O
were	O
obtained	O
by	O
superimposing	B-experimental_method
residues	O
215	B-residue_range
–	I-residue_range
309	I-residue_range
of	O
Tom1	B-protein
GAT	B-structure_element
among	O
10	O
lowest	O
energy	O
refined	O
structures	B-evidence
.	O

PGRMC1	B-protein
is	O
a	O
member	O
of	O
the	O
membrane	B-protein_type
-	I-protein_type
associated	I-protein_type
progesterone	I-protein_type
receptor	I-protein_type
(	O
MAPR	B-protein_type
)	O
family	O
with	O
a	O
cytochrome	B-structure_element
b5	I-structure_element
-	I-structure_element
like	I-structure_element
haem	B-site
-	I-site
binding	I-site
region	I-site
,	O
and	O
is	O
known	O
to	O
be	O
highly	B-protein_state
expressed	I-protein_state
in	O
various	O
types	O
of	O
cancers	O
.	O

These	O
histidines	B-residue_name
are	O
missing	B-protein_state
in	O
PGRMC1	B-protein
,	O
and	O
the	O
haem	B-chemical
iron	B-chemical
is	O
five	B-bond_interaction
-	I-bond_interaction
coordinated	I-bond_interaction
by	I-bond_interaction
Tyr113	B-residue_name_number
(	O
Y113	B-residue_name_number
)	O
alone	B-protein_state
(	O
Fig	O
.	O
1b	O
and	O
Supplementary	O
Fig	O
.	O
3	O
).	O

However	O
,	O
at	O
the	O
interfaces	B-site
of	O
the	O
other	O
possible	O
dimeric	B-oligomeric_state
structures	B-evidence
(	O
Supplementary	O
Fig	O
.	O
6a	O
,	O
chain	O
A	O
–	O
A	O
″;	O
cyan	O
and	O
chain	O
A	O
–	O
B	O
;	O
violet	O
),	O
no	O
significant	O
difference	O
was	O
observed	O
.	O

It	O
should	O
be	O
noted	O
that	O
a	O
disulfide	B-ptm
bond	I-ptm
between	O
two	O
Cys129	B-residue_name_number
residues	O
is	O
observed	O
in	O
the	O
crystal	B-evidence
of	O
PGRMC1	B-protein
(	O
Fig	O
.	O
1a	O
),	O
while	O
Cys129	B-residue_name_number
is	O
not	B-protein_state
conserved	I-protein_state
among	O
the	O
MAPR	B-protein_type
family	O
proteins	O
(	O
Supplementary	O
Fig	O
.	O
5a	O
).	O

The	O
current	O
analytical	O
data	O
confirmed	O
that	O
apo	B-protein_state
-	O
PGRMC1	B-protein
monomer	B-oligomeric_state
converts	O
into	O
dimer	B-oligomeric_state
by	O
binding	O
to	O
haem	B-chemical
in	O
solution	O
(	O
Table	O
2	O
).	O

Furthermore	O
,	O
the	O
UV	B-evidence
-	I-evidence
visible	I-evidence
spectrum	I-evidence
of	O
the	O
wild	B-protein_state
type	I-protein_state
PGRMC1	B-protein
was	O
the	O
same	O
as	O
that	O
of	O
the	O
C129S	B-mutant
mutant	B-protein_state
of	O
PGRMC1	B-protein
,	O
and	O
the	O
R	B-evidence
/	I-evidence
Z	I-evidence
ratio	I-evidence
determined	O
by	O
the	O
intensities	O
between	O
the	O
Soret	O
band	O
(	O
394	O
nm	O
)	O
peak	O
and	O
the	O
274	O
-	O
nm	O
peak	O
showed	O
that	O
these	O
proteins	O
were	O
fully	B-protein_state
loaded	I-protein_state
with	I-protein_state
haem	B-chemical
(	O
Supplementary	O
Fig	O
.	O
12	O
).	O

To	O
examine	O
the	O
inhibitory	O
effects	O
of	O
CO	B-chemical
on	O
haem	B-chemical
-	O
mediated	O
PGRMC1	B-protein
dimerization	B-oligomeric_state
,	O
SV	B-experimental_method
-	I-experimental_method
AUC	I-experimental_method
analysis	O
was	O
carried	O
out	O
.	O

By	O
binding	O
with	O
haem	B-chemical
(	O
binding	O
Kd	B-evidence
=	O
50	O
nmol	O
l	O
−	O
1	O
),	O
PGRMC1	B-protein
forms	O
a	O
stable	B-protein_state
dimer	B-oligomeric_state
(	O
dimerization	B-oligomeric_state
Kd	B-evidence
<<	O
3	O
.	O
5	O
μmol	O
l	O
−	O
1	O
)	O
through	O
stacking	B-bond_interaction
of	O
the	O
two	O
open	O
surfaces	B-site
of	O
the	O
five	O
-	O
coordinated	O
haem	B-chemical
molecules	O
in	O
each	O
monomer	B-oligomeric_state
.	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

Furthermore	O
,	O
PGRMC1	B-mutant
-	I-mutant
KD	I-mutant
inhibited	O
spheroid	O
formation	O
of	O
HCT116	O
cells	O
in	O
culture	O
,	O
and	O
this	O
inhibition	O
was	O
reversed	O
by	O
co	B-experimental_method
-	I-experimental_method
expression	I-experimental_method
of	O
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
5c	O
and	O
Supplementary	O
Fig	O
.	O
18	O
).	O

The	O
Kd	B-evidence
value	O
of	O
PGRMC1	B-protein
binding	O
to	O
CYP51	B-protein
was	O
in	O
a	O
micromolar	O
range	O
and	O
comparable	O
with	O
those	O
of	O
other	O
haem	B-chemical
proteins	O
,	O
such	O
as	O
cytochrome	B-protein
P450	I-protein
reductase	I-protein
and	O
neuroglobin	B-protein
/	O
Gαi1	B-protein
(	O
ref	O
.),	O
suggesting	O
that	O
haem	B-chemical
-	O
dependent	O
PGRMC1	B-protein
interaction	O
with	O
CYP51	B-protein
is	O
biologically	O
relevant	O
.	O

In	O
this	O
study	O
,	O
we	O
showed	O
that	O
PGRMC1	B-protein
dimerizes	B-oligomeric_state
by	O
stacking	B-bond_interaction
interactions	I-bond_interaction
of	O
haem	B-chemical
molecules	O
from	O
each	O
monomer	B-oligomeric_state
.	O

In	O
the	O
current	O
study	O
,	O
the	O
Y113	B-residue_name_number
residue	O
plays	O
a	O
crucial	O
role	O
for	O
the	O
haem	B-chemical
-	O
dependent	O
dimerization	B-oligomeric_state
of	O
PGRMC1	B-protein
and	O
resultant	O
regulation	O
of	O
cancer	O
proliferation	O
and	O
chemoresistance	O
(	O
Figs	O
5c	O
and	O
6e	O
).	O

Since	O
the	O
Y113	B-residue_name_number
residue	O
is	O
involved	O
in	O
the	O
putative	O
consensus	B-structure_element
motif	I-structure_element
of	O
phosphorylation	B-ptm
by	O
tyrosine	B-protein_type
kinases	I-protein_type
such	O
as	O
Abl	B-protein_type
and	O
Lck	B-protein_type
,	O
we	O
investigated	O
whether	O
phosphorylated	B-protein_state
Y113	B-residue_name_number
is	O
present	O
in	O
HCT116	O
cells	O
by	O
ESI	B-experimental_method
-	I-experimental_method
MS	I-experimental_method
analysis	O
.	O

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

On	O
the	O
other	O
hand	O
,	O
Oda	O
et	O
al	O
.	O
reported	O
that	O
PGRMC1	B-protein
had	O
no	O
effect	O
to	O
CYP2E1	B-protein
and	O
CYP3A4	B-protein
activities	O
in	O
HepG2	O
cell	O
.	O

Besides	O
the	O
regulatory	O
roles	O
of	O
PGRMC1	B-protein
/	O
Sigma	B-protein
-	I-protein
2	I-protein
receptor	O
in	O
proliferation	O
and	O
chemoresistance	O
in	O
cancer	O
cells	O
(	O
ref	O
.),	O
recent	O
reports	O
show	O
that	O
PGRMC1	B-protein
is	O
able	O
to	O
bind	O
to	O
amyloid	B-protein
beta	I-protein
oligomer	B-oligomeric_state
to	O
enhance	O
its	O
neurotoxicity	O
.	O

Alzheimer	O
'	O
s	O
therapeutics	O
targeting	O
amyloid	O
beta	O
1	O
-	O
42	O
oligomers	O
II	O
:	O
Sigma	O
-	O
2	O
/	O
PGRMC1	O
receptors	O
mediate	O
Abeta	O
42	O
oligomer	B-oligomeric_state
binding	O
and	O
synaptotoxicity	O

X	B-evidence
-	I-evidence
ray	I-evidence
crystal	I-evidence
structure	I-evidence
of	O
PGRMC1	B-protein
.	O

(	O
a	O
)	O
Structure	O
of	O
the	O
PGRMC1	B-protein
dimer	B-oligomeric_state
formed	O
through	O
stacked	O
haems	B-chemical
.	O

(	O
b	O
)	O
SV	B-experimental_method
-	I-experimental_method
AUC	I-experimental_method
analyses	O
of	O
the	O
wt	B-protein_state
-	O
PGRMC1	B-protein
and	O
the	O
C129S	B-mutant
mutant	B-protein_state
(	O
a	O
.	O
a	O
.	O
44	B-residue_range
–	I-residue_range
195	I-residue_range
)	O
in	O
the	O
presence	B-protein_state
or	O
absence	B-protein_state
of	I-protein_state
haem	B-chemical
.	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

Stable	O
PGRMC1	B-mutant
-	I-mutant
knockdown	I-mutant
(	O
PGRMC1	B-mutant
-	I-mutant
KD	I-mutant
)	O
HCT116	O
cells	O
were	O
transiently	B-experimental_method
transfected	I-experimental_method
with	O
the	O
shRNA	B-protein_state
-	I-protein_state
resistant	I-protein_state
expression	B-experimental_method
vector	I-experimental_method
of	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
PGRMC1	B-protein
(	O
wt	B-protein_state
)	O
or	O
the	O
Y113F	B-mutant
mutant	B-protein_state
(	O
Y113F	B-mutant
).	O

(	O
b	O
)	O
Erlotinib	B-chemical
was	O
added	O
to	O
HCT116	O
(	O
control	O
)	O
cells	O
,	O
PGRMC1	B-mutant
-	I-mutant
KD	I-mutant
cells	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
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

The	O
graph	O
represents	O
mean	O
±	O
s	O
.	O
e	O
.	O
of	O
each	O
spheroid	O
size	O
.	O
*	B-evidence
P	I-evidence
<	O
0	O
.	O
01	O
using	O
ANOVA	B-experimental_method
with	O
Fischer	B-experimental_method
'	I-experimental_method
s	I-experimental_method
LSD	I-experimental_method
test	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

(	O
a	O
,	O
b	O
)	O
FLAG	O
-	O
PGRMC1	B-protein
wild	B-protein_state
-	I-protein_state
type	I-protein_state
(	O
wt	B-protein_state
)	O
and	O
Y113F	B-mutant
mutant	B-protein_state
proteins	O
(	O
a	O
.	O
a	O
.	O
44	B-residue_range
–	I-residue_range
195	I-residue_range
),	O
in	O
either	O
apo	B-protein_state
or	O
haem	B-protein_state
-	I-protein_state
bound	I-protein_state
form	O
,	O
were	O
incubated	B-experimental_method
with	O
CYP1A2	B-protein
(	O
a	O
)	O
or	O
CYP3A4	B-protein
(	O
b	O
)	O
and	O
immunoprecipitated	B-experimental_method
with	O
anti	O
-	O
FLAG	O
antibody	O
-	O
conjugated	O
beads	O
.	O

Schematic	O
diagram	O
for	O
the	O
regulation	O
of	O
PGRMC1	B-protein
functions	O
.	O

Differences	O
in	O
molecular	O
weights	O
of	O
the	O
wild	O
-	O
type	O
(	O
wt	O
;	O
a	O
)	O
and	O
the	O
C129S	B-mutant
mutant	O
(	O
b	O
)	O
PGRMC1	O
proteins	O
in	O
the	O
absence	O
(	O
apo	O
form	O
)	O
or	O
the	O
presence	O
of	O
haem	O
(	O
haem	O
-	O
bound	O
form	O
).	O

Hotspot	O
autoimmune	O
T	B-protein_type
cell	I-protein_type
receptor	I-protein_type
binding	O
underlies	O
pathogen	O
and	O
insulin	B-chemical
peptide	O
cross	O
-	O
reactivity	O

Both	O
MHC	B-complex_assembly
and	O
peptide	B-chemical
have	O
also	O
been	O
shown	O
to	O
undergo	O
structural	O
changes	O
upon	O
TCR	B-complex_assembly
binding	O
,	O
mediating	O
an	O
induced	O
fit	O
between	O
the	O
TCR	B-complex_assembly
and	O
pMHC	B-complex_assembly
.	O

We	O
recently	O
reported	O
that	O
the	O
1E6	O
human	B-species
CD8	O
+	O
T	O
cell	O
clone	O
—	O
which	O
mediates	O
the	O
destruction	O
of	O
β	O
cells	O
through	O
the	O
recognition	O
of	O
a	O
major	O
,	O
HLA	B-protein
-	I-protein
A	I-protein
*	I-protein
0201	I-protein
–	O
restricted	O
,	O
preproinsulin	B-protein
signal	B-structure_element
peptide	I-structure_element
(	O
ALWGPDPAAA15	B-chemical
–	I-chemical
24	I-chemical
)	O
—	O
can	O
recognize	O
upwards	O
of	O
1	O
million	O
different	O
peptides	O
.	O

This	O
first	O
experimental	O
evidence	O
of	O
a	O
high	O
level	O
of	O
CD8	O
+	O
T	O
cell	O
cross	O
-	O
reactivity	O
in	O
a	O
human	B-species
autoimmune	O
disease	O
system	O
hinted	O
toward	O
molecular	O
mimicry	O
by	O
a	O
more	O
potent	O
pathogenic	O
peptide	O
as	O
a	O
potential	O
mechanism	O
leading	O
to	O
β	O
cell	O
destruction	O
.	O

These	O
APLs	B-chemical
differed	O
from	O
the	O
natural	O
preproinsulin	B-protein
peptide	O
by	O
up	O
to	O
7	O
of	O
10	O
residues	O
.	O

Two	O
of	O
these	O
peptides	O
,	O
MVWGPDPLYV	B-chemical
and	O
RQFGPDWIVA	B-chemical
(	O
bold	O
text	O
signifies	O
amino	O
acids	O
that	O
are	O
different	O
from	O
the	O
index	O
preproinsulin	B-protein
–	O
derived	O
sequence	O
),	O
are	O
contained	O
within	O
the	O
proteomes	O
of	O
the	O
human	B-species
pathogens	O
Bacteroides	B-species
fragilis	I-species
/	I-species
thetaiotaomicron	I-species
and	O
Clostridium	B-species
asparagiforme	I-species
,	O
respectively	O
.	O

The	O
low	O
number	O
of	O
contacts	O
between	O
the	O
2	O
molecules	O
most	O
likely	O
contributed	O
to	O
the	O
weak	O
binding	B-evidence
affinity	I-evidence
of	O
the	O
interaction	O
.	O

Although	O
the	O
1E6	B-complex_assembly
TCR	I-complex_assembly
formed	O
a	O
similar	O
overall	O
interaction	O
with	O
each	O
APL	B-chemical
,	O
the	O
stabilization	O
between	O
the	O
TCR	B-complex_assembly
and	O
the	O
GPD	B-structure_element
motif	I-structure_element
enabled	O
fine	O
differences	O
in	O
the	O
contact	B-site
network	I-site
with	O
both	O
the	O
peptide	B-chemical
and	O
MHC	B-site
surface	I-site
that	O
allowed	O
discrimination	O
between	O
each	O
ligand	O
(	O
Figure	O
5	O
).	O

These	O
data	O
demonstrated	O
that	O
the	O
unligated	B-protein_state
structure	B-evidence
of	O
the	O
1E6	B-complex_assembly
TCR	I-complex_assembly
was	O
virtually	O
identical	O
to	O
its	O
ligated	B-protein_state
counterparts	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

We	O
searched	O
a	O
database	O
of	O
over	O
1	O
,	O
924	O
,	O
572	O
unique	O
decamer	O
peptides	B-chemical
from	O
the	O
proteome	O
of	O
viral	B-taxonomy_domain
pathogens	O
that	O
are	O
known	O
,	O
or	O
strongly	O
suspected	O
,	O
to	O
infect	O
humans	B-species
.	O

This	O
notion	O
is	O
attractive	O
because	O
the	O
CDR	B-structure_element
loops	I-structure_element
,	O
which	O
form	O
the	O
TCR	B-site
antigen	I-site
-	I-site
binding	I-site
site	I-site
,	O
are	O
usually	O
the	O
most	O
flexible	O
part	O
of	O
the	O
TCR	B-complex_assembly
and	O
have	O
the	O
ability	O
to	O
mold	O
around	O
differently	O
shaped	O
ligands	O
.	O

This	O
motif	O
was	O
conserved	B-protein_state
in	O
at	O
least	O
2	O
potential	O
foreign	O
peptides	O
,	O
originating	O
from	O
Herpes	B-species
simplex	I-species
virus	I-species
and	O
Pseudomonas	B-species
aeruginosa	I-species
,	O
enabling	O
TCR	B-complex_assembly
recognition	O
of	O
foreign	O
epitopes	O
.	O

We	O
have	O
previously	O
demonstrated	O
the	O
importance	O
of	O
the	O
GPD	B-structure_element
motif	I-structure_element
using	O
a	O
peptide	B-experimental_method
library	I-experimental_method
scan	I-experimental_method
,	O
as	O
well	O
as	O
a	O
CPL	B-experimental_method
scan	I-experimental_method
approach	O
.	O

These	O
results	O
challenge	O
the	O
notion	O
that	O
the	O
most	O
potent	O
peptide	O
antigens	O
exhibit	O
the	O
greatest	O
pMHC	B-complex_assembly
stability	O
and	O
have	O
implications	O
for	O
the	O
design	O
of	O
anchor	O
residue	O
–	O
modified	O
heteroclitic	O
peptides	O
for	O
vaccination	O
.	O

These	O
parameters	O
aligned	O
well	O
with	O
structural	B-evidence
data	I-evidence
,	O
demonstrating	O
that	O
TCRs	B-complex_assembly
engaged	O
pMHC	B-complex_assembly
using	O
an	O
induced	O
fit	O
binding	O
mode	O
.	O

These	O
differences	O
were	O
consistent	O
with	O
a	O
greater	O
degree	O
of	O
movement	O
between	O
the	O
unligated	B-protein_state
and	O
ligated	B-protein_state
pMHCs	B-complex_assembly
for	O
the	O
weaker	O
ligands	O
,	O
suggesting	O
a	O
greater	O
requirement	O
for	O
disorder	O
-	O
to	O
-	O
order	O
changes	O
during	O
TCR	B-complex_assembly
binding	O
.	O

Indeed	O
,	O
we	O
found	O
over	O
50	O
decamer	O
peptides	O
from	O
the	O
proteome	O
of	O
likely	O
,	O
or	O
known	O
,	O
human	B-species
viral	B-taxonomy_domain
pathogens	O
alone	O
that	O
contained	O
both	O
the	O
conserved	B-protein_state
central	O
GPD	B-structure_element
motif	I-structure_element
and	O
anchor	B-structure_element
residues	I-structure_element
at	O
positions	O
2	B-residue_number
and	O
10	B-residue_number
that	O
would	O
enable	O
binding	O
to	O
HLA	B-protein
-	I-protein
A	I-protein
*	I-protein
02	I-protein
:	I-protein
01	I-protein
.	O

(	O
K	O
)	O
Effective	B-evidence
2D	I-evidence
affinity	I-evidence
plotted	O
against	O
1	O
/	O
EC50	B-evidence
showing	O
Pearson	B-experimental_method
’	I-experimental_method
s	I-experimental_method
coefficient	I-experimental_method
analysis	I-experimental_method
(	O
r	B-evidence
)	O
and	O
P	B-evidence
value	I-evidence
.	O

(	O
A	O
)	O
Superposition	B-experimental_method
of	O
the	O
1E6	B-complex_assembly
TCR	I-complex_assembly
(	O
multicolored	O
illustration	O
)	O
in	B-protein_state
complex	I-protein_state
with	I-protein_state
all	O
7	O
APLs	B-chemical
(	O
multicolored	O
sticks	O
)	O
and	O
the	O
A2	B-chemical
-	I-chemical
ALWGPDPAAA	I-chemical
ligand	O
using	O
the	O
HLA	B-protein
-	I-protein
A	I-protein
*	I-protein
0201	I-protein
(	O
gray	O
illustration	O
)	O
molecule	O
to	O
align	B-experimental_method
all	O
of	O
the	O
structures	B-evidence
.	O

The	O
MHCα1	B-complex_assembly
helix	B-structure_element
is	O
shown	O
in	O
gray	O
illustrations	O
.	O

(	O
A	O
)	O
A2	B-chemical
-	I-chemical
MVWGPDPLYV	I-chemical
(	O
black	O
sticks	O
).	O