Biochemical	B-experimental_method
analysis	I-experimental_method
reveals	O
that	O
these	O
outer	B-protein_type
membrane	I-protein_type
-	I-protein_type
anchored	I-protein_type
proteins	I-protein_type
are	O
in	O
fact	O
exquisitely	O
specific	O
for	O
the	O
highly	O
branched	O
xyloglucan	B-chemical
(	O
XyG	B-chemical
)	O
polysaccharide	B-chemical
.	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

This	O
microbial	B-taxonomy_domain
community	O
is	O
largely	O
bacterial	B-taxonomy_domain
,	O
with	O
the	O
Bacteroidetes	B-taxonomy_domain
,	O
Firmicutes	B-taxonomy_domain
,	O
and	O
Actinobacteria	B-taxonomy_domain
comprising	O
the	O
dominant	O
phyla	O
.	O

In	O
the	O
archetypal	O
starch	B-complex_assembly
utilization	I-complex_assembly
system	I-complex_assembly
of	O
B	B-species
.	I-species
thetaiotaomicron	I-species
,	O
starch	O
binding	O
to	O
the	O
cell	O
surface	O
is	O
mediated	O
at	O
eight	O
distinct	O
starch	B-site
-	I-site
binding	I-site
sites	I-site
distributed	O
among	O
four	O
surface	B-protein_type
glycan	I-protein_type
-	I-protein_type
binding	I-protein_type
proteins	I-protein_type
(	O
SGBPs	B-protein_type
):	O
two	O
within	O
the	O
amylase	B-protein_type
SusG	B-protein
,	O
one	O
within	O
SusD	B-protein
,	O
two	O
within	O
SusE	B-protein
,	O
and	O
three	O
within	O
SusF	B-protein
.	O
The	O
functional	O
redundancy	O
of	O
many	O
of	O
these	O
sites	O
is	O
high	O
:	O
whereas	O
SusD	B-protein
is	O
essential	O
for	O
growth	O
on	O
starch	B-chemical
,	O
combined	O
mutations	O
of	O
the	O
SusE	B-protein
,	O
SusF	B-protein
,	O
and	O
SusG	B-protein
binding	B-site
sites	I-site
are	O
required	O
to	O
impair	O
growth	O
on	O
the	O
polysaccharide	B-chemical
.	O

Combined	O
biochemical	B-experimental_method
,	I-experimental_method
structural	I-experimental_method
,	I-experimental_method
and	I-experimental_method
reverse	I-experimental_method
-	I-experimental_method
genetic	I-experimental_method
approaches	I-experimental_method
clearly	O
illuminate	O
the	O
distinct	O
,	O
yet	O
complementary	O
,	O
functions	O
that	O
these	O
two	O
proteins	O
play	O
in	O
XyG	B-chemical
recognition	O
as	O
it	O
impacts	O
the	O
physiology	O
of	O
B	B-species
.	I-species
ovatus	I-species
.	O

Hence	O
,	O
there	O
is	O
a	O
critical	O
need	O
for	O
the	O
elucidation	O
of	O
detailed	O
structure	O
-	O
function	O
relationships	O
among	O
PUL	B-gene
SGBPs	B-protein_type
,	O
in	O
light	O
of	O
the	O
manifold	O
glycan	B-chemical
structures	O
in	O
nature	O
.	O

Formalin	O
-	O
fixed	O
,	O
nonpermeabilized	O
B	B-species
.	I-species
ovatus	I-species
cells	O
were	O
grown	O
in	O
minimal	O
medium	O
plus	O
XyG	B-chemical
,	O
probed	O
with	O
custom	O
rabbit	O
antibodies	O
to	O
SGBP	B-protein
-	I-protein
A	I-protein
or	O
SGBP	B-protein
-	I-protein
B	I-protein
,	O
and	O
then	O
stained	O
with	O
Alexa	O
Fluor	O
488	O
goat	O
anti	O
-	O
rabbit	O
IgG	O
.	O
(	O
A	O
)	O
Overlay	B-experimental_method
of	O
bright	B-evidence
-	I-evidence
field	I-evidence
and	I-evidence
FITC	I-evidence
images	I-evidence
of	O
B	B-species
.	I-species
ovatus	I-species
cells	O
labeled	O
with	O
anti	O
-	O
SGBP	O
-	O
A	O
.	O
(	O
B	O
)	O
Overlay	B-experimental_method
of	O
bright	B-evidence
-	I-evidence
field	I-evidence
and	I-evidence
FITC	I-evidence
images	I-evidence
of	O
B	B-species
.	I-species
ovatus	I-species
cells	O
labeled	O
with	O
anti	O
-	O
SGBP	O
-	O
B	O
.	O
(	O
C	O
)	O
Bright	B-evidence
-	I-evidence
field	I-evidence
image	I-evidence
of	O
ΔSGBP	B-mutant
-	I-mutant
B	I-mutant
cells	O
labeled	O
with	O
anti	O
-	O
SGBP	O
-	O
B	O
antibodies	O
.	O

In	O
our	O
initial	O
study	O
focused	O
on	O
the	O
functional	O
characterization	O
of	O
the	O
glycoside	B-protein_type
hydrolases	I-protein_type
of	O
the	O
XyGUL	B-gene
,	O
we	O
reported	O
preliminary	O
affinity	B-experimental_method
PAGE	I-experimental_method
and	O
isothermal	B-experimental_method
titration	I-experimental_method
calorimetry	I-experimental_method
(	O
ITC	B-experimental_method
)	O
data	O
indicating	O
that	O
both	O
SGBP	B-protein
-	I-protein
A	I-protein
and	O
SGBP	B-protein
-	I-protein
B	I-protein
are	O
competent	O
xyloglucan	B-protein_type
-	I-protein_type
binding	I-protein_type
proteins	I-protein_type
(	O
affinity	B-evidence
constant	I-evidence
[	O
Ka	B-evidence
]	O
values	O
of	O
3	O
.	O
74	O
×	O
105	O
M	O
−	O
1	O
and	O
4	O
.	O
98	O
×	O
104	O
M	O
−	O
1	O
,	O
respectively	O
[	O
23	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

Molecular	O
structure	B-evidence
of	O
SGBP	B-protein
-	I-protein
A	I-protein
(	O
Bacova_02651	B-gene
).	O
(	O
A	O
)	O
Overlay	B-experimental_method
of	O
SGBP	B-protein
-	I-protein
A	I-protein
from	O
the	O
apo	B-protein_state
(	O
rainbow	O
)	O
and	O
XyGO2	B-chemical
(	O
gray	O
)	O
structures	B-evidence
.	O

The	O
backbone	O
glucose	B-chemical
residues	O
are	O
numbered	O
from	O
the	O
nonreducing	O
end	O
;	O
xylose	B-chemical
residues	O
are	O
labeled	O
X1	B-residue_name_number
and	O
X2	B-residue_name_number
.	O

Most	O
surprising	O
in	O
light	O
of	O
the	O
saccharide	B-evidence
-	I-evidence
binding	I-evidence
data	I-evidence
,	O
however	O
,	O
was	O
a	O
lack	O
of	O
extensive	O
recognition	O
of	O
the	O
XyG	B-chemical
side	O
chains	O
;	O
only	O
Y84	B-residue_name_number
appeared	O
to	O
provide	O
a	O
hydrophobic	B-site
interface	I-site
for	O
a	O
xylosyl	B-chemical
residue	O
(	O
Xyl1	B-residue_name_number
).	O

Protein	O
name	O
Ka	B-evidence
ΔG	O
(	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

Weak	O
binding	O
represents	O
a	O
Ka	B-evidence
of	O
<	O
500	O
M	O
−	O
1	O
.	O

SGBP	B-protein
-	I-protein
B	I-protein
has	O
a	O
multimodular	O
structure	O
with	O
a	O
single	O
,	O
C	O
-	O
terminal	O
glycan	B-structure_element
-	I-structure_element
binding	I-structure_element
domain	I-structure_element
.	O

The	O
crystal	B-evidence
structure	I-evidence
of	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
SGBP	B-protein
-	I-protein
B	I-protein
in	B-protein_state
complex	I-protein_state
with	I-protein_state
XyGO2	B-chemical
(	O
2	O
.	O
37	O
Å	O
,	O
Rwork	B-evidence
=	O
19	O
.	O
9	O
%,	O
Rfree	B-evidence
=	O
23	O
.	O
9	O
%,	O
residues	O
34	B-residue_range
to	I-residue_range
489	I-residue_range
)	O
(	O
Table	O
2	O
)	O
revealed	O
an	O
extended	O
structure	B-evidence
composed	O
of	O
three	O
tandem	B-structure_element
immunoglobulin	I-structure_element
(	I-structure_element
Ig	I-structure_element
)-	I-structure_element
like	I-structure_element
domains	I-structure_element
(	O
domains	O
A	B-structure_element
,	O
B	B-structure_element
,	O
and	O
C	B-structure_element
)	O
followed	O
at	O
the	O
C	O
terminus	O
by	O
a	O
novel	O
xyloglucan	B-structure_element
-	I-structure_element
binding	I-structure_element
domain	I-structure_element
(	O
domain	O
D	B-structure_element
)	O
(	O
Fig	O
.	O
5A	O
).	O

Analogously	O
,	O
the	O
outer	B-protein_state
membrane	I-protein_state
-	I-protein_state
anchored	I-protein_state
endo	B-protein_type
-	I-protein_type
xyloglucanase	I-protein_type
BoGH5	B-protein
of	O
the	O
XyGUL	B-gene
contains	O
a	O
100	B-structure_element
-	I-structure_element
amino	I-structure_element
-	I-structure_element
acid	I-structure_element
,	I-structure_element
all	I-structure_element
-	I-structure_element
β	I-structure_element
-	I-structure_element
strand	I-structure_element
,	O
N	B-structure_element
-	I-structure_element
terminal	I-structure_element
module	I-structure_element
and	O
flexible	B-structure_element
linker	I-structure_element
that	O
imparts	O
conformational	O
flexibility	O
and	O
distances	O
the	O
catalytic	B-structure_element
module	I-structure_element
from	O
the	O
cell	O
surface	O
.	O

The	O
Y363A	B-mutant
site	B-experimental_method
-	I-experimental_method
directed	I-experimental_method
mutant	I-experimental_method
of	O
SGBP	B-protein
-	I-protein
B	I-protein
displays	O
a	O
20	O
-	O
fold	O
decrease	O
in	O
the	O
Ka	B-evidence
for	O
XyG	B-chemical
,	O
while	O
the	O
W364A	B-mutant
mutant	B-protein_state
lacks	B-protein_state
XyG	I-protein_state
binding	I-protein_state
(	O
Table	O
3	O
;	O
see	O
Fig	O
.	O
S6	O
in	O
the	O
supplemental	O
material	O
).	O

Hoping	O
to	O
achieve	O
a	O
higher	O
-	O
resolution	O
view	O
of	O
the	O
SGBP	B-protein
-	I-protein
B	I-protein
–	O
xyloglucan	B-chemical
interaction	O
,	O
we	O
solved	B-experimental_method
the	O
crystal	B-evidence
structure	I-evidence
of	O
the	O
fused	B-mutant
CD	I-mutant
domains	I-mutant
in	B-protein_state
complex	I-protein_state
with	I-protein_state
XyGO2	B-chemical
(	O
1	O
.	O
57	O
Å	O
,	O
Rwork	B-evidence
=	O
15	O
.	O
6	O
%,	O
Rfree	B-evidence
=	O
17	O
.	O
1	O
%,	O
residues	O
230	B-residue_range
to	I-residue_range
489	I-residue_range
)	O
(	O
Table	O
2	O
).	O

Growth	O
on	O
glucose	B-chemical
displayed	O
the	O
shortest	O
lag	B-evidence
time	I-evidence
for	O
each	O
strain	O
,	O
and	O
so	O
lag	B-evidence
times	I-evidence
were	O
normalized	O
for	O
each	O
carbohydrate	B-chemical
by	O
subtracting	O
the	O
lag	B-evidence
time	I-evidence
of	O
that	O
strain	O
in	O
glucose	B-chemical
(	O
Fig	O
.	O
6	O
;	O
see	O
Fig	O
.	O
S8	O
in	O
the	O
supplemental	O
material	O
).	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

Fig	O
.	O
1B	O
)	O
was	O
completely	O
incapable	O
of	O
growth	O
on	O
XyG	B-chemical
,	O
XyGO1	B-chemical
,	O
and	O
XyGO2	B-chemical
,	O
indicating	O
that	O
SGBP	B-protein
-	I-protein
A	I-protein
is	O
essential	O
for	O
XyG	B-chemical
utilization	O
(	O
Fig	O
.	O
6	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

In	O
the	O
BtSus	B-gene
,	O
SusD	B-protein
and	O
the	O
TBDT	B-protein_type
SusC	B-protein
interact	O
,	O
and	O
we	O
speculate	O
that	O
this	O
interaction	O
is	O
necessary	O
for	O
glycan	B-chemical
uptake	O
,	O
as	O
suggested	O
by	O
the	O
fact	O
that	O
a	O
ΔsusD	B-mutant
mutant	B-protein_state
cannot	O
grow	O
on	O
starch	B-chemical
,	O
but	O
a	O
ΔsusD	B-mutant
::	O
SusD	B-mutant
*	I-mutant
strain	O
regains	O
this	O
ability	O
if	O
a	O
transcriptional	B-protein_type
activator	I-protein_type
of	O
the	O
sus	B-gene
operon	I-gene
is	O
supplied	O
.	O

Recent	O
work	O
has	O
elucidated	O
that	O
Bacteroidetes	B-taxonomy_domain
cross	O
-	O
feed	O
during	O
growth	O
on	O
many	O
glycans	B-chemical
;	O
the	O
glycoside	B-protein_type
hydrolases	I-protein_type
expressed	O
by	O
one	O
species	O
liberate	O
oligosaccharides	B-chemical
for	O
consumption	O
by	O
other	O
members	O
of	O
the	O
community	O
.	O

In	O
this	O
instance	O
,	O
coexpression	O
of	O
the	O
susD	B-gene
-	O
like	O
gene	O
nanU	B-gene
was	O
not	O
required	O
,	O
nor	O
did	O
the	O
expression	O
of	O
the	O
nanU	B-gene
gene	O
enhance	O
growth	O
kinetics	O
.	O

However	O
,	O
the	O
natural	O
diversity	O
of	O
these	O
proteins	O
represents	O
a	O
rich	O
source	O
for	O
the	O
discovery	O
of	O
unique	O
carbohydrate	B-structure_element
-	I-structure_element
binding	I-structure_element
motifs	I-structure_element
to	O
both	O
inform	O
gut	O
microbiology	O
and	O
generate	O
new	O
,	O
specific	O
carbohydrate	B-chemical
analytical	O
reagents	O
.	O

The	O
ability	O
of	O
our	O
resident	O
gut	O
bacteria	B-taxonomy_domain
to	O
recognize	O
polysaccharides	B-chemical
is	O
the	O
first	O
committed	O
step	O
of	O
glycan	B-chemical
consumption	O
by	O
these	O
organisms	O
,	O
a	O
critical	O
process	O
that	O
influences	O
the	O
community	O
structure	O
and	O
thus	O
the	O
metabolic	O
output	O
(	O
i	O
.	O
e	O
.,	O
short	O
-	O
chain	O
fatty	O
acid	O
and	O
metabolite	O
profile	O
)	O
of	O
these	O
organisms	O
.	O

Mucosal	O
glycan	B-chemical
foraging	O
enhances	O
fitness	O
and	O
transmission	O
of	O
a	O
saccharolytic	O
human	O
gut	O
bacterial	O
symbiont	O

Neisseria	B-protein
adhesin	I-protein
A	I-protein
(	O
NadA	B-protein
)	O
present	O
on	O
the	O
meningococcal	B-taxonomy_domain
surface	O
can	O
mediate	O
binding	O
to	O
human	B-species
cells	O
and	O
is	O
one	O
of	O
the	O
three	O
MenB	B-species
vaccine	O
protein	O
antigens	O
.	O

MarR	B-protein_type
family	O
proteins	O
can	O
promote	O
bacterial	B-taxonomy_domain
survival	O
in	O
the	O
presence	O
of	O
antibiotics	O
,	O
toxic	O
chemicals	O
,	O
organic	O
solvents	O
or	O
reactive	O
oxygen	O
species	O
and	O
can	O
regulate	O
virulence	O
factor	O
expression	O
.	O

MarR	B-protein_type
homologues	O
can	O
act	O
either	O
as	O
transcriptional	O
repressors	O
or	O
as	O
activators	O
.	O

A	O
potentially	O
interesting	O
exception	O
comes	O
from	O
the	O
ligand	B-protein_state
-	I-protein_state
free	I-protein_state
and	O
salicylate	B-protein_state
-	I-protein_state
bound	I-protein_state
forms	O
of	O
the	O
Methanobacterium	B-species
thermoautotrophicum	I-species
protein	O
MTH313	B-protein
which	O
revealed	O
that	O
two	O
salicylate	B-chemical
molecules	O
bind	O
to	O
one	O
MTH313	B-protein
dimer	B-oligomeric_state
and	O
induce	O
large	O
conformational	O
changes	O
,	O
apparently	O
sufficient	O
to	O
prevent	O
DNA	O
binding	O
.	O

We	O
obtained	O
detailed	O
new	O
insights	O
into	O
ligand	O
specificity	O
,	O
how	O
the	O
ligand	O
allosterically	O
influences	O
the	O
DNA	O
-	O
binding	O
ability	O
of	O
NadR	B-protein
,	O
and	O
the	O
regulation	O
of	O
nadA	B-gene
expression	O
,	O
thus	O
also	O
providing	O
a	O
deeper	O
structural	O
understanding	O
of	O
the	O
ligand	O
-	O
responsive	O
MarR	B-protein_type
super	O
-	O
family	O
.	O

Since	O
ligand	O
-	O
binding	O
often	O
increases	O
protein	O
stability	O
,	O
we	O
also	O
investigated	O
the	O
effect	O
of	O
various	O
HPAs	B-chemical
(	O
Fig	O
1A	O
)	O
on	O
the	O
melting	B-evidence
temperature	I-evidence
(	O
Tm	B-evidence
)	O
of	O
NadR	B-protein
.	O
As	O
a	O
control	O
of	O
specificity	O
,	O
we	O
also	O
tested	O
salicylate	B-chemical
,	O
a	O
known	O
ligand	O
of	O
some	O
MarR	B-protein_type
proteins	O
previously	O
reported	O
to	O
increase	O
the	O
Tm	B-evidence
of	O
ST1710	B-protein
and	O
MTH313	B-protein
.	O

However	O
,	O
an	O
increased	O
thermal	O
stability	O
was	O
induced	O
by	O
4	B-chemical
-	I-chemical
HPA	I-chemical
and	O
,	O
to	O
a	O
lesser	O
extent	O
,	O
by	O
3	B-chemical
-	I-chemical
HPA	I-chemical
.	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

NadR	B-protein
displays	O
distinct	O
binding	B-evidence
affinities	I-evidence
for	O
hydroxyphenylacetate	B-chemical
ligands	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

First	O
,	O
we	O
crystallized	B-experimental_method
NadR	B-protein
(	O
a	O
selenomethionine	B-experimental_method
-	I-experimental_method
labelled	I-experimental_method
derivative	I-experimental_method
)	O
in	O
the	O
presence	O
of	O
a	O
200	O
-	O
fold	O
molar	O
excess	O
of	O
4	B-chemical
-	I-chemical
HPA	I-chemical
.	O

A	O
single	O
conserved	B-protein_state
leucine	B-residue_name
residue	O
(	O
L130	B-residue_name_number
)	O
is	O
crucial	O
for	O
dimerization	O

The	O
NadR	B-protein
dimer	B-site
interface	I-site
is	O
formed	O
by	O
at	O
least	O
32	O
residues	O
,	O
which	O
establish	O
numerous	O
inter	O
-	O
chain	O
salt	O
bridges	O
or	O
hydrogen	O
bonds	O
,	O
and	O
many	O
hydrophobic	O
packing	O
interactions	O
(	O
Fig	O
3A	O
and	O
3B	O
).	O

Only	O
the	O
L130K	B-mutant
mutation	O
induced	O
a	O
notable	O
change	O
in	O
the	O
oligomeric	O
state	O
of	O
NadR	B-protein
(	O
Fig	O
3C	O
).	O

Chain	B-structure_element
B	I-structure_element
,	O
grey	O
surface	O
,	O
is	O
marked	O
blue	O
to	O
highlight	O
residues	O
probed	O
by	O
site	B-experimental_method
-	I-experimental_method
directed	I-experimental_method
mutagenesis	I-experimental_method
(	O
E136	B-residue_name_number
only	O
makes	O
a	O
salt	O
bridge	O
with	O
K126	B-residue_name_number
,	O
therefore	O
it	O
was	O
sufficient	O
to	O
make	O
the	O
K126A	B-mutant
mutation	O
to	O
assess	O
the	O
importance	O
of	O
this	O
ionic	O
interaction	O
;	O
the	O
H7	B-residue_name_number
position	O
is	O
labelled	O
for	O
monomer	B-oligomeric_state
A	B-structure_element
,	O
since	O
electron	B-evidence
density	I-evidence
was	O
lacking	O
for	O
monomer	B-oligomeric_state
B	B-structure_element
).	O
(	O
B	O
)	O
A	O
zoom	O
into	O
the	O
environment	O
of	O
helix	B-structure_element
α6	B-structure_element
to	O
show	O
how	O
residue	O
L130	B-residue_name_number
chain	B-structure_element
B	I-structure_element
(	O
blue	O
side	O
chain	O
)	O
is	O
a	O
focus	O
of	O
hydrophobic	O
packing	O
interactions	O
with	O
L130	B-residue_name_number
,	O
L133	B-residue_name_number
,	O
L134	B-residue_name_number
and	O
L137	B-residue_name_number
of	O
chain	B-structure_element
A	I-structure_element
(	O
red	O
side	O
chains	O
).	O

*	O
Bond	O
distance	O
between	O
the	O
ligand	O
carboxylate	O
group	O
and	O
the	O
water	B-chemical
molecule	O
,	O
which	O
in	O
turn	O
makes	O
H	O
-	O
bond	O
to	O
the	O
SerA9	B-residue_name_number
and	O
AsnA11	B-residue_name_number
side	O
chains	O
.	O

Consequently	O
,	O
residues	O
in	O
the	O
4	B-site
-	I-site
HPA	I-site
binding	I-site
pocket	I-site
are	O
mostly	O
contributed	O
by	O
NadR	B-protein
chain	B-structure_element
B	I-structure_element
,	O
and	O
effectively	O
created	O
a	O
polar	O
‘	O
floor	O
’	O
and	O
a	O
hydrophobic	O
‘	O
ceiling	O
’,	O
which	O
house	O
the	O
ligand	O
.	O

Collectively	O
,	O
this	O
mixed	O
network	O
of	O
polar	O
and	O
hydrophobic	O
interactions	O
endows	O
NadR	B-protein
with	O
a	O
strong	O
recognition	O
pattern	O
for	O
HPAs	B-chemical
,	O
with	O
additional	O
medium	O
-	O
range	O
interactions	O
potentially	O
established	O
with	O
the	O
hydroxyl	O
group	O
at	O
the	O
4	O
-	O
position	O
.	O

Structure	O
-	O
activity	O
relationships	O
:	O
molecular	O
basis	O
of	O
enhanced	O
stabilization	O
by	O
3Cl	B-chemical
,	I-chemical
4	I-chemical
-	I-chemical
HPA	I-chemical

We	O
modelled	B-experimental_method
the	O
binding	O
of	O
other	O
HPAs	B-chemical
by	O
in	B-experimental_method
silico	I-experimental_method
superposition	I-experimental_method
onto	O
4	B-chemical
-	I-chemical
HPA	I-chemical
in	O
the	O
holo	B-protein_state
-	O
NadR	B-protein
structure	B-evidence
,	O
and	O
thereby	O
obtained	O
molecular	O
explanations	O
for	O
the	O
binding	O
specificities	O
of	O
diverse	O
ligands	O
.	O

For	O
example	O
,	O
similar	O
to	O
4	B-chemical
-	I-chemical
HPA	I-chemical
,	O
the	O
binding	O
of	O
3Cl	B-chemical
,	I-chemical
4	I-chemical
-	I-chemical
HPA	I-chemical
could	O
involve	O
multiple	O
bonds	O
towards	O
the	O
carboxylate	O
group	O
of	O
the	O
ligand	O
and	O
some	O
to	O
the	O
4	O
-	O
hydroxyl	O
group	O
.	O

Finally	O
,	O
salicylate	B-chemical
is	O
presumably	O
unable	O
to	O
specifically	O
bind	O
NadR	B-protein
due	O
to	O
the	O
2	O
-	O
hydroxyl	O
substitution	O
and	O
the	O
shorter	O
aliphatic	O
chain	O
connecting	O
its	O
carboxylate	O
group	O
(	O
Fig	O
1A	O
):	O
the	O
compound	O
simply	O
seems	O
too	O
small	O
to	O
simultaneously	O
establish	O
the	O
network	O
of	O
beneficial	O
bonds	O
observed	O
in	O
the	O
NadR	B-protein
/	O
HPA	B-chemical
interactions	O
.	O

The	O
stoichiometry	O
of	O
the	O
NadR	B-complex_assembly
-	I-complex_assembly
HPA	I-complex_assembly
interactions	O
was	O
determined	O
using	O
Eq	O
1	O
(	O
see	O
Materials	O
and	O
Methods	O
),	O
and	O
revealed	O
stoichiometries	B-evidence
of	O
1	O
.	O
13	O
for	O
4	B-chemical
-	I-chemical
HPA	I-chemical
,	O
1	O
.	O
02	O
for	O
3	B-chemical
-	I-chemical
HPA	I-chemical
,	O
and	O
1	O
.	O
21	O
for	O
3Cl	B-chemical
,	I-chemical
4	I-chemical
-	I-chemical
HPA	I-chemical
,	O
strongly	O
suggesting	O
that	O
one	O
NadR	B-protein
dimer	B-oligomeric_state
bound	B-protein_state
to	I-protein_state
1	O
HPA	B-chemical
analyte	O
molecule	O
.	O

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

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

The	O
broad	O
spectral	O
dispersion	O
and	O
the	O
number	O
of	O
peaks	O
observed	O
,	O
which	O
is	O
close	O
to	O
the	O
number	O
of	O
expected	O
backbone	O
amide	O
N	O
-	O
H	O
groups	O
for	O
this	O
polypeptide	O
,	O
confirmed	O
that	O
apo	B-protein_state
-	O
NadR	B-protein
is	O
well	B-protein_state
-	I-protein_state
folded	I-protein_state
under	O
these	O
conditions	O
and	O
exhibits	O
one	O
conformation	O
appreciable	O
on	O
the	O
NMR	B-experimental_method
timescale	O
,	O
i	O
.	O
e	O
.	O
in	O
the	O
NMR	B-experimental_method
experiments	O
at	O
25	O
°	O
C	O
,	O
two	O
or	O
more	O
distinct	O
conformations	O
of	O
apo	B-protein_state
-	O
NadR	B-protein
monomers	B-oligomeric_state
were	O
not	O
readily	O
apparent	O
.	O

(	O
A	O
)	O
Superposition	B-experimental_method
of	O
two	O
1H	B-experimental_method
-	I-experimental_method
15N	I-experimental_method
TROSY	I-experimental_method
-	I-experimental_method
HSQC	I-experimental_method
spectra	B-evidence
recorded	O
at	O
25	O
°	O
C	O
on	O
apo	B-protein_state
-	O
NadR	B-protein
(	O
cyan	O
)	O
and	O
on	O
NadR	B-protein
in	O
the	O
presence	B-protein_state
of	I-protein_state
4	B-chemical
-	I-chemical
HPA	I-chemical
(	O
red	O
).	O

Considering	O
the	O
small	O
size	O
,	O
fast	O
diffusion	O
and	O
relatively	O
low	O
binding	B-evidence
affinity	I-evidence
of	O
4	B-chemical
-	I-chemical
HPA	I-chemical
,	O
it	O
would	O
not	O
be	O
surprising	O
if	O
the	O
ligand	O
associates	O
and	O
dissociates	O
rapidly	O
on	O
the	O
NMR	B-experimental_method
time	O
scale	O
,	O
resulting	O
in	O
only	O
one	O
set	O
of	O
peaks	O
whose	O
chemical	O
shifts	O
represent	O
the	O
average	O
environment	O
of	O
the	O
bound	B-protein_state
and	O
unbound	B-protein_state
states	O
.	O

Overall	O
apo	B-protein_state
-	O
and	O
holo	B-protein_state
-	O
NadR	B-protein
structures	B-evidence
are	O
similar	O
.	O

To	O
further	O
investigate	O
the	O
conformational	O
rearrangements	O
of	O
NadR	B-protein
,	O
we	O
performed	O
local	B-experimental_method
structural	I-experimental_method
alignments	I-experimental_method
using	O
only	O
a	O
subset	O
of	O
residues	O
in	O
the	O
DNA	B-structure_element
-	I-structure_element
binding	I-structure_element
helix	I-structure_element
(	O
α4	B-structure_element
).	O

By	O
selecting	B-experimental_method
and	O
aligning	B-experimental_method
residues	O
Arg64	B-residue_range
-	I-residue_range
Ala77	I-residue_range
of	O
one	O
α4	B-structure_element
helix	I-structure_element
per	O
dimer	B-oligomeric_state
,	O
superposition	B-experimental_method
of	O
the	O
holo	B-protein_state
-	O
homodimer	B-oligomeric_state
onto	O
the	O
two	O
apo	B-protein_state
-	O
homodimers	B-oligomeric_state
revealed	O
differences	O
in	O
the	O
monomer	B-oligomeric_state
conformations	O
of	O
each	O
structure	B-evidence
.	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

However	O
,	O
structural	B-experimental_method
comparisons	I-experimental_method
revealed	O
that	O
the	O
shift	O
of	O
holo	B-protein_state
-	O
NadR	B-protein
helix	B-structure_element
α4	B-structure_element
induced	O
by	O
the	O
presence	B-protein_state
of	I-protein_state
4	B-chemical
-	I-chemical
HPA	I-chemical
was	O
also	O
accompanied	O
by	O
several	O
changes	O
at	O
the	O
holo	B-protein_state
dimer	B-site
interface	I-site
,	O
while	O
such	O
extensive	O
structural	O
differences	O
were	O
not	O
observed	O
in	O
the	O
apo	B-protein_state
dimer	B-site
interfaces	I-site
,	O
particularly	O
notable	O
when	O
comparing	O
the	O
α6	B-structure_element
helices	I-structure_element
(	O
S3	O
Fig	O
).	O

Pairwise	B-experimental_method
superpositions	I-experimental_method
showed	O
that	O
the	O
NadR	B-protein
apo	B-protein_state
-	O
homodimer	B-oligomeric_state
AB	B-structure_element
was	O
the	O
most	O
similar	O
to	O
OhrR	B-protein
(	O
rmsd	B-evidence
2	O
.	O
6	O
Å	O
),	O
while	O
the	O
holo	B-protein_state
-	O
homodimer	B-oligomeric_state
was	O
the	O
most	O
divergent	O
(	O
rmsd	B-evidence
3	O
.	O
3	O
Å	O
)	O
(	O
Fig	O
8C	O
).	O

Interestingly	O
,	O
and	O
on	O
the	O
contrary	O
,	O
the	O
nadR	B-gene
N11A	B-mutant
complemented	O
strain	O
showed	O
hypo	O
-	O
repression	O
(	O
i	O
.	O
e	O
.	O
exhibited	O
high	O
expression	O
of	O
nadA	B-gene
both	O
in	O
absence	O
and	O
presence	O
of	O
4	B-chemical
-	I-chemical
HPA	I-chemical
).	O

Western	B-experimental_method
blot	I-experimental_method
analyses	O
of	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
(	O
WT	B-protein_state
)	O
strain	O
(	O
lanes	O
1	O
–	O
2	O
)	O
or	O
isogenic	O
nadR	B-gene
knockout	O
strains	O
(	O
ΔNadR	B-mutant
)	O
complemented	O
to	O
express	O
the	O
indicated	O
NadR	B-protein
WT	B-protein_state
or	O
mutant	B-protein_state
proteins	O
(	O
lanes	O
3	O
–	O
12	O
)	O
or	O
not	O
complemented	O
(	O
lanes	O
13	O
–	O
14	O
),	O
grown	O
in	O
the	O
presence	O
(	O
even	O
lanes	O
)	O
or	O
absence	O
(	O
odd	O
lanes	O
)	O
of	O
5mM	O
4	B-chemical
-	I-chemical
HPA	I-chemical
,	O
showing	O
NadA	B-protein
and	O
NadR	B-protein
expression	O
.	O

Complementation	O
of	O
ΔNadR	B-mutant
with	O
WT	B-protein_state
NadR	B-protein
enables	O
induction	O
of	O
nadA	B-gene
expression	O
by	O
4	B-chemical
-	I-chemical
HPA	I-chemical
.	O

Here	O
,	O
we	O
determined	O
the	O
first	O
crystal	B-evidence
structures	I-evidence
of	O
apo	B-protein_state
-	O
NadR	B-protein
and	O
holo	B-protein_state
-	O
NadR	B-protein
.	O
These	O
experimentally	O
-	O
determined	O
structures	B-evidence
enabled	O
a	O
new	O
detailed	O
characterization	O
of	O
the	O
ligand	B-site
-	I-site
binding	I-site
pocket	I-site
.	O

Subsequently	O
,	O
we	O
established	O
the	O
functional	O
importance	O
of	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
in	O
the	O
in	O
vitro	O
response	O
of	O
meningococcus	B-taxonomy_domain
to	O
4	B-chemical
-	I-chemical
HPA	I-chemical
,	O
via	O
site	B-experimental_method
-	I-experimental_method
directed	I-experimental_method
mutagenesis	I-experimental_method
.	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

In	O
an	O
alternative	O
and	O
less	O
extensive	O
manner	O
,	O
the	O
binding	O
of	O
two	O
salicylate	B-chemical
molecules	O
to	O
the	O
M	B-species
.	I-species
thermoautotrophicum	I-species
protein	O
MTH313	B-protein
appeared	O
to	O
induce	O
large	O
changes	O
in	O
the	O
wHTH	B-structure_element
domain	I-structure_element
,	O
which	O
was	O
associated	O
with	O
reduced	O
DNA	O
-	O
binding	O
activity	O
.	O

Here	O
,	O
we	O
report	O
two	O
high	O
-	O
resolution	O
PduL	B-protein_type
crystal	B-evidence
structures	I-evidence
with	B-protein_state
bound	I-protein_state
substrates	I-protein_state
.	O

This	O
reaction	O
directly	O
links	O
an	O
acyl	B-chemical
-	I-chemical
CoA	I-chemical
with	O
ATP	B-chemical
generation	O
via	O
substrate	O
-	O
level	O
phosphorylation	O
,	O
producing	O
short	B-chemical
-	I-chemical
chain	I-chemical
fatty	I-chemical
acids	I-chemical
(	O
e	O
.	O
g	O
.,	O
acetate	B-chemical
),	O
and	O
also	O
provides	O
a	O
path	O
for	O
short	B-chemical
-	I-chemical
chain	I-chemical
fatty	I-chemical
acids	I-chemical
to	O
enter	O
central	O
metabolism	O
.	O

Not	O
only	O
does	O
PduL	B-protein_type
facilitate	O
substrate	O
level	O
phosphorylation	O
,	O
but	O
it	O
also	O
is	O
critical	O
for	O
cofactor	O
recycling	O
within	O
,	O
and	O
product	O
efflux	O
from	O
,	O
the	O
organelle	O
.	O

More	O
recently	O
,	O
bioinformatic	B-experimental_method
studies	I-experimental_method
have	O
demonstrated	O
the	O
widespread	O
distribution	O
of	O
BMCs	B-complex_assembly
among	O
diverse	O
bacterial	B-taxonomy_domain
phyla	I-taxonomy_domain
and	O
grouped	O
them	O
into	O
23	O
different	O
functional	O
types	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

Another	O
distinctive	O
feature	O
of	O
BMC	B-protein_state
-	I-protein_state
associated	I-protein_state
PduL	B-protein_type
homologs	O
is	O
an	O
N	O
-	O
terminal	O
encapsulation	B-structure_element
peptide	I-structure_element
(	O
EP	B-structure_element
)	O
that	O
is	O
thought	O
to	O
“	O
target	O
”	O
proteins	O
for	O
encapsulation	O
by	O
the	O
BMC	B-complex_assembly
shell	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

While	O
purifying	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
sPduL	B-protein
,	O
we	O
observed	O
a	O
tendency	O
to	O
aggregation	O
as	O
described	O
previously	O
,	O
with	O
a	O
large	O
fraction	O
of	O
the	O
expressed	O
protein	O
found	O
in	O
the	O
insoluble	O
fraction	O
in	O
a	O
white	O
,	O
cake	O
-	O
like	O
pellet	O
.	O

(	O
a	O
)	O
Primary	O
and	O
secondary	O
structure	O
of	O
rPduL	B-protein
(	O
tubes	O
represent	O
α	B-structure_element
-	I-structure_element
helices	I-structure_element
,	O
arrows	O
β	B-structure_element
-	I-structure_element
sheets	I-structure_element
and	O
dashed	O
line	O
residues	O
disordered	O
in	O
the	O
structure	B-evidence
.	O

The	O
first	B-residue_range
33	I-residue_range
amino	I-residue_range
acids	I-residue_range
are	O
present	O
only	O
in	O
the	O
wildtype	O
construct	O
and	O
contains	O
the	O
predicted	O
EP	B-structure_element
alpha	B-structure_element
helix	I-structure_element
,	O
α0	B-structure_element
);	O
the	O
truncated	B-protein_state
rPduLΔEP	B-mutant
that	O
was	O
crystallized	B-experimental_method
begins	O
with	O
M	B-residue_name
-	O
G	B-residue_name
-	O
V	B-residue_name
.	O
Coloring	O
is	O
according	O
to	O
structural	O
domains	O
(	O
domain	B-structure_element
1	I-structure_element
D36	B-residue_range
-	I-residue_range
N46	I-residue_range
/	O
Q155	B-residue_range
-	I-residue_range
C224	I-residue_range
,	O
blue	O
;	O
loop	B-structure_element
insertion	I-structure_element
G61	B-residue_range
-	I-residue_range
E81	I-residue_range
,	O
grey	O
;	O
domain	B-structure_element
2	I-structure_element
R47	B-residue_range
-	I-residue_range
F60	I-residue_range
/	O
E82	B-residue_range
-	I-residue_range
A154	I-residue_range
,	O
red	O
).	O

Using	O
a	O
mercury	B-experimental_method
-	I-experimental_method
derivative	I-experimental_method
crystal	I-experimental_method
form	O
diffracting	O
to	O
1	O
.	O
99	O
Å	O
(	O
Table	O
2	O
),	O
we	O
obtained	O
high	O
quality	O
electron	B-evidence
density	I-evidence
for	O
model	O
building	O
and	O
used	O
the	O
initial	O
model	O
to	O
refine	O
against	O
the	O
native	O
data	O
to	O
Rwork	B-evidence
/	O
Rfree	B-evidence
values	O
of	O
18	O
.	O
9	O
/	O
22	O
.	O
1	O
%.	O

There	O
are	O
two	O
PduL	B-protein_type
molecules	O
in	O
the	O
asymmetric	O
unit	O
of	O
the	O
P212121	O
unit	O
cell	O
.	O

Structurally	O
,	O
PduL	B-protein_type
consists	O
of	O
two	O
domains	B-structure_element
(	O
Fig	O
2	O
,	O
blue	O
/	O
red	O
),	O
each	O
a	O
beta	B-structure_element
-	I-structure_element
barrel	I-structure_element
that	O
is	O
capped	O
on	O
both	O
ends	O
by	O
short	O
α	B-structure_element
-	I-structure_element
helices	I-structure_element
.	O

Consistent	O
with	O
this	O
,	O
results	O
from	O
size	B-experimental_method
exclusion	I-experimental_method
chromatography	I-experimental_method
of	O
rPduLΔEP	B-mutant
suggest	O
that	O
it	O
is	O
a	O
dimer	B-oligomeric_state
in	O
solution	O
(	O
Fig	O
5e	O
).	O

The	O
asterisk	O
and	O
double	O
arrow	O
marks	O
the	O
location	O
of	O
the	O
π	O
–	O
π	O
interaction	O
between	O
F116	B-residue_name_number
and	O
the	O
CoA	B-chemical
base	O
of	O
the	O
other	O
dimer	B-oligomeric_state
chain	O
.	O

CoA	B-chemical
and	O
the	O
metal	O
ions	O
bind	O
between	O
the	O
two	O
domains	O
,	O
presumably	O
in	O
the	O
active	B-site
site	I-site
(	O
Figs	O
2b	O
and	O
4a	O
).	O

The	O
large	O
differences	O
between	O
the	O
anomalous	O
signals	O
confirm	O
the	O
presence	O
of	O
zinc	B-chemical
at	O
both	O
metal	O
sites	O
(	O
S3	O
Fig	O
).	O

The	O
first	O
zinc	B-chemical
ion	O
(	O
Zn1	B-chemical
)	O
is	O
in	O
a	O
tetrahedral	O
coordination	O
state	O
with	O
His48	B-residue_name_number
,	O
His50	B-residue_name_number
,	O
Glu109	B-residue_name_number
,	O
and	O
the	O
CoA	B-chemical
sulfur	B-chemical
(	O
Fig	O
4a	O
).	O

Oligomeric	O
States	O
of	O
PduL	B-protein_type
Orthologs	O
Are	O
Influenced	O
by	O
the	O
EP	B-structure_element

In	O
contrast	O
,	O
both	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
rPduL	B-protein
and	O
pPduL	B-protein
appeared	O
to	O
exist	O
in	O
two	O
distinct	O
oligomeric	O
states	O
(	O
Fig	O
5b	O
and	O
5c	O
respectively	O
,	O
orange	O
curves	O
),	O
one	O
form	O
of	O
the	O
approximate	O
size	O
of	O
a	O
dimer	B-oligomeric_state
and	O
the	O
second	O
,	O
a	O
higher	O
molecular	O
weight	O
oligomer	B-oligomeric_state
(~	O
150	O
kDa	O
).	O

In	O
contrast	O
,	O
rPduLΔEP	B-mutant
eluted	O
as	O
one	O
smaller	O
oligomer	O
,	O
possibly	O
a	O
dimer	B-oligomeric_state
.	O

Homologs	O
of	O
the	O
predominant	O
cofactor	O
utilizer	O
(	O
aldehyde	B-protein_type
dehydrogenase	I-protein_type
)	O
and	O
NAD	B-chemical
+	I-chemical
regenerator	O
(	O
alcohol	B-protein_type
dehydrogenase	I-protein_type
)	O
have	O
been	O
structurally	O
characterized	O
,	O
but	O
until	O
now	O
structural	O
information	O
was	O
lacking	O
for	O
PduL	B-protein_type
,	O
which	O
recycles	O
CoA	B-chemical
in	O
the	O
organelle	O
lumen	O
.	O

The	O
PduL	B-protein_type
signature	O
primary	O
structure	O
,	O
two	O
PF06130	B-structure_element
domains	O
,	O
occurs	O
in	O
some	O
multidomain	O
proteins	O
,	O
most	O
of	O
them	O
annotated	O
as	O
Acks	B-protein_type
,	O
suggesting	O
that	O
PduL	B-protein_type
may	O
also	O
replace	O
Pta	B-protein_type
in	O
variants	O
of	O
the	O
phosphotransacetylase	B-protein_type
-	O
Ack	B-protein_type
pathway	O
.	O

For	O
BMC	B-complex_assembly
-	O
encapsulated	O
proteins	O
to	O
properly	O
function	O
together	O
,	O
they	O
must	O
be	O
targeted	O
to	O
the	O
lumen	O
and	O
assemble	O
into	O
an	O
organization	O
that	O
facilitates	O
substrate	O
/	O
product	O
channeling	O
among	O
the	O
different	O
catalytic	B-site
sites	I-site
of	O
the	O
signature	O
and	O
core	O
enzymes	O
.	O

Structured	O
aggregation	O
of	O
the	O
core	O
enzymes	O
has	O
been	O
proposed	O
to	O
be	O
the	O
initial	O
step	O
in	O
metabolosome	B-complex_assembly
assembly	O
and	O
is	O
known	O
to	O
be	O
the	O
first	O
step	O
of	O
β	O
-	O
carboxysome	O
biogenesis	O
,	O
where	O
the	O
core	O
enzyme	O
Ribulose	B-protein_type
Bisphosphate	I-protein_type
Carboxylase	I-protein_type
/	I-protein_type
Oxygenase	I-protein_type
(	O
RuBisCO	B-protein_type
)	O
is	O
aggregated	O
by	O
the	O
CcmM	B-protein_type
protein	O
.	O

The	O
close	O
resemblance	O
between	O
the	O
structures	O
binding	O
CoA	B-chemical
and	O
phosphate	B-chemical
likely	O
indicates	O
that	O
no	O
large	O
changes	O
in	O
protein	O
conformation	O
are	O
involved	O
in	O
catalysis	O
,	O
and	O
that	O
our	O
crystal	B-evidence
structures	I-evidence
are	O
representative	O
of	O
the	O
active	B-protein_state
form	O
.	O

This	O
hypothesis	O
is	O
strengthened	O
by	O
the	O
fact	O
that	O
the	O
CoA	B-protein_state
-	I-protein_state
bound	I-protein_state
crystals	B-evidence
were	O
obtained	O
without	O
added	O
CoA	B-chemical
,	O
indicating	O
that	O
the	O
protein	O
bound	B-protein_state
CoA	B-chemical
from	O
the	O
E	B-species
.	I-species
coli	I-species
expression	O
strain	O
and	O
retained	O
it	O
throughout	O
purification	O
and	O
crystallization	O
.	O

PduL	B-protein_type
and	O
Pta	B-protein_type
are	O
mechanistically	O
and	O
structurally	O
distinct	O
enzymes	O
that	O
catalyze	O
the	O
same	O
reaction	O
,	O
a	O
prime	O
example	O
of	O
evolutionary	O
convergence	O
upon	O
a	O
function	O
.	O

This	O
is	O
not	O
surprising	O
,	O
as	O
β	B-protein_type
-	I-protein_type
lactamases	I-protein_type
are	O
not	O
so	O
widespread	O
among	O
bacteria	B-taxonomy_domain
and	O
therefore	O
would	O
be	O
expected	O
to	O
have	O
evolved	O
independently	O
several	O
times	O
as	O
a	O
defense	O
mechanism	O
against	O
β	O
-	O
lactam	O
antibiotics	O
.	O

These	O
results	O
suggest	O
that	O
Regnase	B-protein
-	I-protein
1	I-protein
RNase	B-protein_type
activity	O
is	O
tightly	O
controlled	O
by	O
both	O
intramolecular	O
(	O
NTD	B-structure_element
-	O
PIN	B-structure_element
)	O
and	O
intermolecular	O
(	O
PIN	B-structure_element
-	O
PIN	B-structure_element
)	O
interactions	O
.	O

The	O
initial	O
sensing	O
of	O
infection	O
is	O
mediated	O
by	O
a	O
set	O
of	O
pattern	B-protein_type
-	I-protein_type
recognition	I-protein_type
receptors	I-protein_type
(	O
PRRs	B-protein_type
)	O
such	O
Toll	B-protein_type
-	I-protein_type
like	I-protein_type
receptors	I-protein_type
(	O
TLRs	B-protein_type
)	O
and	O
the	O
intracellular	O
signaling	O
cascades	O
triggered	O
by	O
TLRs	B-protein_type
evoke	O
transcriptional	O
expression	O
of	O
inflammatory	O
mediators	O
that	O
coordinate	O
the	O
elimination	O
of	O
pathogens	O
and	O
infected	O
cells	O
.	O

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

X	B-experimental_method
-	I-experimental_method
ray	I-experimental_method
crystallography	I-experimental_method
was	O
attempted	O
for	O
the	O
fragment	O
containing	O
both	O
the	O
PIN	B-structure_element
and	O
ZF	B-structure_element
domains	O
,	O
however	O
,	O
electron	B-evidence
density	I-evidence
was	O
observed	O
only	O
for	O
the	O
PIN	B-structure_element
domain	O
(	O
Fig	O
.	O
1c	O
),	O
consistent	O
with	O
a	O
previous	O
report	O
on	O
Regnase	B-protein
-	I-protein
1	I-protein
derived	O
from	O
Homo	B-species
sapiens	I-species
.	O

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

Although	O
the	O
PIN	B-structure_element
domain	O
is	O
responsible	O
for	O
the	O
catalytic	O
activity	O
of	O
Regnase	B-protein
-	I-protein
1	I-protein
,	O
the	O
roles	O
of	O
the	O
other	O
domains	O
are	O
largely	O
unknown	O
.	O

Upon	O
addition	O
of	O
a	O
larger	O
amount	O
of	O
Regnase	B-protein
-	I-protein
1	I-protein
,	O
the	O
fluorescence	B-evidence
of	O
free	B-protein_state
RNA	B-chemical
decreased	O
,	O
indicating	O
that	O
Regnase	B-protein
-	I-protein
1	I-protein
bound	B-protein_state
to	I-protein_state
the	O
RNA	B-chemical
.	O

Direct	O
binding	O
of	O
the	O
ZF	B-structure_element
domain	O
and	O
RNA	B-chemical
were	O
confirmed	O
by	O
NMR	B-experimental_method
spectral	B-evidence
changes	I-evidence
.	O

Dimer	B-oligomeric_state
formation	O
of	O
the	O
PIN	B-structure_element
domains	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

Based	O
on	O
the	O
titration	B-evidence
curve	I-evidence
for	O
the	O
chemical	B-evidence
shift	I-evidence
changes	I-evidence
of	O
L58	B-residue_name_number
,	O
the	O
apparent	O
Kd	B-evidence
between	O
the	O
isolated	O
NTD	B-structure_element
and	O
PIN	B-structure_element
was	O
estimated	O
to	O
be	O
110	O
±	O
5	O
.	O
8	O
μM	O
.	O
Considering	O
the	O
fact	O
that	O
the	O
NTD	B-structure_element
and	O
PIN	B-structure_element
domains	O
are	O
attached	O
by	O
a	O
linker	B-structure_element
,	O
the	O
actual	O
binding	B-evidence
affinity	I-evidence
is	O
expected	O
much	O
higher	O
in	O
the	O
native	B-protein_state
protein	O
.	O

An	O
in	B-experimental_method
silico	I-experimental_method
docking	I-experimental_method
of	O
the	O
NTD	B-structure_element
and	O
PIN	B-structure_element
domains	O
using	O
chemical	B-evidence
shift	I-evidence
restraints	I-evidence
provided	O
a	O
model	O
consistent	O
with	O
the	O
NMR	B-experimental_method
experiments	O
(	O
Fig	O
.	O
3c	O
).	O

When	O
any	O
members	O
of	O
the	O
two	O
groups	O
are	O
mixed	O
,	O
two	O
kinds	O
of	O
heterodimers	B-oligomeric_state
can	O
be	O
formed	O
:	O
one	O
is	O
composed	O
of	O
a	O
DDNN	B-mutant
primary	B-protein_state
PIN	B-structure_element
and	O
a	O
basic	O
residue	O
mutant	B-protein_state
secondary	B-protein_state
PIN	B-structure_element
and	O
is	O
expected	O
to	O
exhibit	O
no	O
RNase	B-protein_type
activity	O
;	O
the	O
other	O
is	O
composed	O
of	O
a	O
basic	O
residue	O
mutant	B-protein_state
primary	B-protein_state
PIN	B-structure_element
and	O
a	O
DDNN	B-mutant
secondary	B-protein_state
PIN	B-structure_element
and	O
is	O
predicted	O
to	O
rescue	O
RNase	B-protein_type
activity	O
(	O
Fig	O
.	O
5a	O
).	O

When	O
we	O
compared	O
the	O
fluorescence	B-evidence
intensity	I-evidence
of	O
uncleaved	B-protein_state
IL	B-protein_type
-	I-protein_type
6	I-protein_type
mRNA	B-chemical
,	O
basic	O
residue	O
mutants	B-protein_state
W182A	B-mutant
,	O
K184A	B-mutant
,	O
R214A	B-mutant
,	O
and	O
R220A	B-mutant
were	O
rescued	O
upon	O
addition	O
of	O
the	O
DDNN	B-mutant
mutant	B-protein_state
(	O
Fig	O
.	O
5b	O
).	O

Rescue	O
of	O
K184A	B-mutant
and	O
R214A	B-mutant
by	O
the	O
DDNN	B-mutant
mutant	B-protein_state
was	O
also	O
confirmed	O
by	O
a	O
significant	O
increase	O
in	O
the	O
cleaved	O
products	O
.	O

R214	B-residue_name_number
is	O
an	O
important	O
residue	O
for	O
dimer	B-oligomeric_state
formation	O
as	O
shown	O
in	O
Fig	O
.	O
2	O
,	O
therefore	O
,	O
R214A	B-mutant
in	O
the	O
secondary	B-protein_state
PIN	B-structure_element
cannot	O
dimerize	O
.	O

Due	O
to	O
this	O
limitation	O
,	O
it	O
is	O
difficult	O
to	O
perform	O
further	O
structural	B-experimental_method
analyses	I-experimental_method
of	O
mRNA	B-complex_assembly
-	I-complex_assembly
Regnase	I-complex_assembly
-	I-complex_assembly
1	I-complex_assembly
complexes	O
by	O
X	B-experimental_method
-	I-experimental_method
ray	I-experimental_method
crystallography	I-experimental_method
or	O
NMR	B-experimental_method
.	O

Moreover	O
,	O
our	O
structure	B-experimental_method
-	I-experimental_method
based	I-experimental_method
mutational	I-experimental_method
analyses	I-experimental_method
showed	O
these	O
two	O
Regnase	B-protein
-	I-protein
1	I-protein
specific	O
basic	O
regions	O
were	O
essential	O
for	O
target	O
mRNA	B-chemical
cleavage	O
in	O
vitro	O
.	O

The	O
affinity	B-evidence
of	O
the	O
domain	O
-	O
domain	O
interaction	O
between	O
two	O
PIN	B-structure_element
domains	O
(	O
Kd	B-evidence
=	O
~	O
10	O
−	O
4	O
M	O
)	O
is	O
similar	O
to	O
that	O
of	O
the	O
NTD	B-structure_element
-	O
PIN	B-structure_element
(	O
Kd	B-evidence
=	O
110	O
±	O
5	O
.	O
8	O
μM	O
)	O
interactions	O
;	O
however	O
,	O
the	O
covalent	O
connection	O
corresponding	O
to	O
residues	O
90	B-residue_range
–	I-residue_range
133	I-residue_range
between	O
the	O
NTD	B-structure_element
and	O
the	O
primary	B-protein_state
PIN	B-structure_element
will	O
greatly	O
enhance	O
the	O
intramolecular	O
domain	O
interaction	O
in	O
the	O
case	O
of	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
Regnase	B-protein
-	I-protein
1	I-protein
.	O

In	O
this	O
context	O
,	O
it	O
is	O
interesting	O
that	O
,	O
in	O
response	O
to	O
TCR	O
stimulation	O
,	O
Malt1	B-protein
cleaves	O
Regnase	B-protein
-	I-protein
1	I-protein
at	O
R111	B-residue_name_number
to	O
control	O
immune	O
responses	O
in	O
vivo	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

Eukaryotic	B-taxonomy_domain
ribosome	O
biogenesis	O
is	O
highly	O
complex	O
and	O
requires	O
a	O
large	O
number	O
of	O
non	O
-	O
ribosomal	O
proteins	O
and	O
small	B-chemical
non	I-chemical
-	I-chemical
coding	I-chemical
RNAs	I-chemical
in	O
addition	O
to	O
ribosomal	B-chemical
RNAs	I-chemical
(	O
rRNAs	B-chemical
)	O
and	O
proteins	O
.	O

In	O
addition	O
,	O
18S	B-chemical
and	O
25S	B-chemical
(	O
yeast	B-taxonomy_domain
)/	O
28S	B-chemical
(	O
humans	B-species
)	O
rRNAs	B-chemical
contain	O
several	O
base	O
modifications	O
catalyzed	O
by	O
site	O
-	O
specific	O
and	O
snoRNA	B-chemical
-	O
independent	O
enzymes	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

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

Wild	B-protein_state
type	I-protein_state
(	O
WT	B-protein_state
)	O
and	O
plasmid	O
encoded	O
18S	B-chemical
rRNA	I-chemical
(	O
U1191U	B-mutant
)	O
show	O
the	O
14C	B-chemical
-	I-chemical
acp	I-chemical
signal	O
,	O
whereas	O
the	O
14C	B-chemical
-	I-chemical
acp	I-chemical
signal	O
is	O
missing	O
in	O
the	O
U1191A	B-mutant
mutant	B-protein_state
plasmid	O
encoded	O
18S	B-chemical
rRNA	I-chemical
(	O
U1191A	B-mutant
)	O
and	O
Δtsr3	B-mutant
mutants	O
(	O
Δtsr3	B-mutant
).	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

For	O
the	O
Δtsr3	B-mutant
deletion	O
strain	O
the	O
HPLC	B-evidence
elution	I-evidence
profile	I-evidence
of	O
18S	B-chemical
rRNA	I-chemical
nucleosides	B-chemical
(	O
Figure	O
1B	O
)	O
was	O
very	O
similar	O
to	O
that	O
of	O
the	O
pseudouridine	B-protein_type
-	I-protein_type
N1	I-protein_type
methyltransferase	I-protein_type
mutant	B-protein_state
Δnep1	B-mutant
,	O
where	O
a	O
shoulder	O
at	O
∼	O
7	O
.	O
4	O
min	O
elution	O
time	O
was	O
missing	O
in	O
the	O
elution	O
profile	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

Similar	O
to	O
yeast	B-taxonomy_domain
,	O
siRNA	B-experimental_method
-	I-experimental_method
mediated	I-experimental_method
depletion	I-experimental_method
of	O
the	O
Ψ1248	B-protein_type
N1	I-protein_type
-	I-protein_type
methyltransferase	I-protein_type
Nep1	B-protein
/	O
Emg1	B-protein
had	O
no	O
influence	O
on	O
the	O
primer	B-evidence
extension	I-evidence
arrest	I-evidence
(	O
Figure	O
1E	O
).	O

However	O
,	O
the	O
Δtsr3	B-mutant
deletion	O
was	O
synthetic	O
sick	O
with	O
a	O
Δsnr35	B-mutant
deletion	O
preventing	O
pseudouridylation	B-ptm
and	O
Nep1	B-protein
-	O
catalyzed	O
methylation	O
of	O
nucleotide	O
1191	B-residue_number
(	O
Figure	O
2A	O
).	O

Phenotypic	O
characterization	O
of	O
yeast	B-taxonomy_domain
TSR3	B-protein
deletion	O
(	O
Δtrs3	B-mutant
)	O
and	O
human	B-species
TSR3	B-protein
depletion	O
(	O
siRNAs	B-chemical
544	O
and	O
545	O
)	O
and	O
cellular	O
localization	O
of	O
yeast	B-taxonomy_domain
Tsr3	B-protein
.	O
(	O
A	O
)	O
Growth	O
of	O
yeast	B-taxonomy_domain
wild	B-protein_state
type	I-protein_state
,	O
Δtsr3	B-mutant
,	O
Δsnr35	B-mutant
and	O
Δtsr3	B-mutant
Δsnr35	I-mutant
segregants	O
after	O
meiosis	O
and	O
tetrad	O
dissection	O
of	O
Δtsr3	B-mutant
/	O
TSR3	B-protein
Δsnr35	B-mutant
/	O
SNR35	B-protein
heterozygous	O
diploids	O
.	O

Consistent	O
with	O
its	O
role	O
in	O
late	O
18S	B-chemical
rRNA	I-chemical
processing	O
,	O
TSR3	B-protein
deletion	O
leads	O
to	O
a	O
ribosomal	O
subunit	O
imbalance	O
with	O
a	O
reduced	O
40S	B-complex_assembly
to	O
60S	B-complex_assembly
ratio	O
of	O
0	O
.	O
81	O
(	O
σ	O
=	O
0	O
.	O
024	O
)	O
which	O
was	O
further	O
increased	O
in	O
a	O
Δtsr3	B-mutant
Δsnr35	I-mutant
recombinant	O
to	O
0	O
.	O
73	O
(	O
σ	O
=	O
0	O
.	O
023	O
)	O
(	O
Supplementary	O
Figure	O
S2F	O
).	O

N	O
-	O
terminal	O
deletions	B-experimental_method
of	O
36	B-residue_range
or	O
45	B-residue_range
amino	O
acids	O
and	O
C	O
-	O
terminal	O
deletions	B-experimental_method
of	O
43	B-residue_range
or	O
76	B-residue_range
residues	O
show	O
a	O
primer	B-evidence
extension	I-evidence
stop	I-evidence
comparable	O
to	O
the	O
wild	B-protein_state
type	I-protein_state
.	O

Strong	O
20S	O
rRNA	O
accumulation	O
similar	O
to	O
that	O
of	O
the	O
Δtsr3	B-mutant
deletion	B-experimental_method
is	O
observed	O
for	O
Tsr3	B-protein
fragments	O
37	B-residue_range
–	I-residue_range
223	I-residue_range
or	O
46	B-residue_range
–	I-residue_range
223	I-residue_range
.	O

Well	O
diffracting	O
crystals	B-evidence
were	O
obtained	O
for	O
Tsr3	B-protein
homologs	O
from	O
the	O
two	O
crenarchaeal	B-taxonomy_domain
species	O
Vulcanisaeta	B-species
distributa	I-species
(	O
VdTsr3	B-protein
)	O
and	O
Sulfolobus	B-species
solfataricus	I-species
(	O
SsTsr3	B-protein
)	O
which	O
share	O
36	O
%	O
(	O
VdTsr3	B-protein
)	O
and	O
38	O
%	O
(	O
SsTsr3	B-protein
)	O
identity	O
with	O
the	O
ScTsr3	B-protein
core	B-structure_element
region	I-structure_element
(	O
ScTsr3	B-protein
aa	O
46	B-residue_range
–	I-residue_range
223	I-residue_range
).	O

Crystals	B-evidence
of	O
VdTsr3	B-protein
diffracted	O
to	O
a	O
resolution	O
of	O
1	O
.	O
6	O
Å	O
whereas	O
crystals	B-evidence
of	O
SsTsr3	B-protein
diffracted	O
to	O
2	O
.	O
25	O
Å	O
.	O
Serendipitously	O
,	O
VdTsr3	B-protein
was	O
purified	O
and	O
crystallized	B-experimental_method
in	B-protein_state
complex	I-protein_state
with	I-protein_state
endogenous	B-protein_state
(	O
E	B-species
.	I-species
coli	I-species
)	O
SAM	B-chemical
(	O
Supplementary	O
Figure	O
S4	O
)	O
while	O
SsTsr3	B-protein
crystals	B-evidence
contained	O
the	O
protein	O
in	O
the	O
apo	B-protein_state
state	O
.	O

The	O
structure	B-evidence
of	O
VdTsr3	B-protein
can	O
be	O
divided	O
into	O
two	O
domains	O
(	O
Figure	O
4A	O
).	O

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

Gel	B-experimental_method
filtration	I-experimental_method
experiments	O
with	O
both	O
VdTsr3	B-protein
and	O
SsTsr3	B-protein
(	O
Figure	O
4E	O
)	O
showed	O
that	O
both	O
proteins	O
are	O
monomeric	B-oligomeric_state
in	O
solution	O
thereby	O
extending	O
the	O
structural	O
similarities	O
to	O
Trm10	B-protein
.	O

This	O
enzyme	O
,	O
Tyw2	B-protein
,	O
is	O
part	O
of	O
the	O
biosynthesis	O
pathway	O
of	O
wybutosine	B-chemical
nucleotides	I-chemical
in	O
tRNAs	B-chemical
.	O

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

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

Accordingly	O
,	O
a	O
W66A	B-mutant
-	O
mutation	B-experimental_method
(	O
W73	B-residue_name_number
in	O
VdTsr3	B-protein
)	O
of	O
SsTsr3	B-protein
significantly	O
diminished	O
SAM	B-evidence
-	I-evidence
binding	I-evidence
in	O
a	O
filter	B-experimental_method
binding	I-experimental_method
assay	I-experimental_method
compared	O
to	O
the	O
wild	B-protein_state
type	I-protein_state
(	O
Figure	O
5E	O
).	O

Furthermore	O
,	O
a	O
W	B-experimental_method
to	I-experimental_method
A	I-experimental_method
mutation	I-experimental_method
at	O
the	O
equivalent	O
position	O
W114	B-residue_name_number
in	O
ScTsr3	B-protein
strongly	O
reduced	O
the	O
in	O
vivo	O
acp	B-protein_type
transferase	I-protein_type
activity	O
(	O
Figure	O
5F	O
).	O

Furthermore	O
,	O
a	O
negatively	O
charged	O
MES	B-chemical
-	O
ion	O
is	O
found	O
in	O
the	O
crystal	B-evidence
structure	I-evidence
of	O
VdTsr3	B-protein
complexed	B-protein_state
to	I-protein_state
the	O
side	O
chain	O
of	O
K22	B-residue_name_number
in	O
helix	B-structure_element
α1	B-structure_element
.	O

In	O
the	O
C	B-structure_element
-	I-structure_element
terminal	I-structure_element
domain	I-structure_element
,	O
the	O
surface	O
exposed	O
α	B-structure_element
-	I-structure_element
helices	I-structure_element
α5	B-structure_element
and	O
α7	B-structure_element
carry	O
a	O
significant	O
amount	O
of	O
positively	O
charged	O
amino	O
acids	O
.	O

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

5	O
′-	O
fluoresceine	B-chemical
labeled	O
RNA	B-chemical
oligonucleotides	O
corresponding	O
either	O
to	O
the	O
native	B-protein_state
(	O
20mer	B-oligomeric_state
–	O
see	O
inset	O
)	O
or	O
a	O
stabilized	B-protein_state
(	O
20mer_GC	B-oligomeric_state
-	O
inset	O
)	O
helix	B-structure_element
31	I-structure_element
of	O
the	O
small	O
ribosomal	O
subunit	O
rRNA	B-chemical
from	O
S	B-species
.	I-species
solfataricus	I-species
were	O
titrated	B-experimental_method
with	I-experimental_method
increasing	I-experimental_method
amounts	I-experimental_method
of	O
SsTsr3	B-protein
and	O
the	O
changes	O
in	O
the	O
fluoresceine	B-chemical
fluorescence	B-evidence
anisotropy	I-evidence
were	O
measured	O
and	O
fitted	O
to	O
a	O
binding	B-evidence
curve	I-evidence
(	O
20mer	B-oligomeric_state
–	O
red	O
,	O
20mer_GC	B-oligomeric_state
–	O
blue	O
).	O

This	O
suggests	O
that	O
Tsr3	B-protein
is	O
not	O
stably	O
incorporated	O
into	O
pre	B-complex_assembly
-	I-complex_assembly
ribosomal	I-complex_assembly
particles	I-complex_assembly
and	O
that	O
its	O
binding	O
to	O
the	O
nascent	O
ribosomal	B-complex_assembly
subunit	I-complex_assembly
possibly	O
requires	O
additional	O
interactions	O
with	O
other	O
pre	O
-	O
ribosomal	O
components	O
.	O

The	O
cleavage	O
step	O
most	O
likely	O
acts	O
as	O
a	O
quality	O
control	O
check	O
that	O
ensures	O
the	O
proper	O
40S	B-complex_assembly
subunit	I-complex_assembly
assembly	O
with	O
only	O
completely	O
processed	O
precursors	O
.	O

Finally	O
,	O
termination	B-protein_type
factor	I-protein_type
Rli1	B-protein
,	O
an	O
ATPase	B-protein_type
,	O
promotes	O
the	O
dissociation	O
of	O
assembly	O
factors	O
and	O
the	O
80S	B-complex_assembly
-	I-complex_assembly
like	I-complex_assembly
complex	I-complex_assembly
dissociates	O
and	O
releases	O
the	O
mature	B-protein_state
40S	B-complex_assembly
subunit	I-complex_assembly
.	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

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

PmC11	B-protein
has	O
an	O
acidic	B-site
binding	I-site
pocket	I-site
and	O
a	O
preference	O
for	O
basic	O
substrates	O
,	O
and	O
accepts	O
substrates	O
with	O
Arg	B-residue_name
and	O
Lys	B-residue_name
in	O
P1	B-residue_number
and	O
does	O
not	O
require	O
Ca2	B-chemical
+	I-chemical
for	O
activity	O
.	O

Clan	B-protein_type
CD	I-protein_type
families	I-protein_type
are	O
typically	O
described	O
using	O
the	O
name	O
of	O
their	O
archetypal	O
,	O
or	O
founding	O
,	O
member	O
and	O
also	O
given	O
an	O
identification	O
number	O
preceded	O
by	O
a	O
“	O
C	O
,”	O
to	O
denote	O
cysteine	B-protein_type
peptidase	I-protein_type
.	O

Although	O
seven	O
families	O
(	O
C14	O
is	O
additionally	O
split	O
into	O
three	O
subfamilies	O
)	O
have	O
been	O
described	O
for	O
this	O
clan	O
,	O
crystal	B-evidence
structures	I-evidence
have	O
only	O
been	O
determined	O
from	O
four	O
:	O
legumain	B-protein
(	O
C13	B-protein_type
),	O
caspase	B-protein
(	O
C14a	B-protein_type
),	O
paracaspase	B-protein
(	O
C14b	B-protein_type
(	I-protein_type
P	I-protein_type
),	O
metacaspase	B-protein
(	O
C14b	B-protein_type
(	I-protein_type
M	I-protein_type
),	O
gingipain	B-protein
(	O
C25	B-protein_type
),	O
and	O
the	O
cysteine	B-structure_element
peptidase	I-structure_element
domain	I-structure_element
(	O
CPD	B-structure_element
)	O
of	O
various	O
toxins	O
(	O
C80	B-protein_type
).	O

Clan	B-protein_type
CD	I-protein_type
enzymes	I-protein_type
have	O
a	O
highly	B-protein_state
conserved	I-protein_state
His	B-site
/	I-site
Cys	I-site
catalytic	I-site
dyad	I-site
and	O
exhibit	O
strict	O
specificity	O
for	O
the	O
P1	B-residue_number
residue	O
of	O
their	O
substrates	O
.	O

Structure	B-evidence
of	O
PmC11	B-protein

The	O
position	O
of	O
the	O
catalytic	B-site
dyad	I-site
(	O
H	B-residue_name
,	O
C	B-residue_name
)	O
and	O
the	O
processing	B-site
site	I-site
(	O
Lys147	B-residue_name_number
)	O
are	O
highlighted	O
.	O

Helices	O
(	O
1	O
–	O
14	O
)	O
and	O
β	B-structure_element
-	I-structure_element
strands	I-structure_element
(	O
1	O
–	O
9	O
and	O
A	O
-	O
F	O
)	O
are	O
numbered	O
from	O
the	O
N	O
terminus	O
.	O

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

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

This	B-structure_element
helix	I-structure_element
makes	O
a	O
total	O
of	O
eight	O
hydrogen	O
bonds	O
with	O
the	O
CTD	B-structure_element
,	O
including	O
one	O
salt	O
bridge	O
(	O
Arg191	B-residue_name_number
-	O
Asp255	B-residue_name_number
)	O
and	O
is	O
surrounded	O
by	O
the	O
CTD	B-structure_element
on	O
one	O
side	O
and	O
the	O
main	B-structure_element
core	I-structure_element
of	O
the	O
enzyme	O
on	O
the	O
other	O
,	O
acting	O
like	O
a	O
linchpin	O
holding	O
both	O
components	O
together	O
(	O
Fig	O
.	O
1C	O
).	O

The	O
two	O
ends	O
of	O
the	O
autolytic	B-site
cleavage	I-site
site	I-site
(	O
Lys147	B-residue_name_number
and	O
Ala148	B-residue_name_number
,	O
green	O
)	O
are	O
displaced	O
by	O
19	O
.	O
5	O
Å	O
(	O
thin	O
black	O
line	O
)	O
from	O
one	O
another	O
and	O
residues	O
in	O
the	O
potential	O
substrate	B-site
binding	I-site
pocket	I-site
are	O
highlighted	O
in	O
blue	O
.	O

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

Elution	O
fractions	O
across	O
the	O
major	O
peak	O
(	O
1	O
–	O
6	O
)	O
were	O
analyzed	O
by	O
SDS	B-experimental_method
-	I-experimental_method
PAGE	I-experimental_method
on	O
a	O
4	O
–	O
12	O
%	O
gel	O
in	O
MES	O
buffer	O
.	O

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

PmC11C179A	O
(	O
20	O
μg	O
)	O
was	O
incubated	O
overnight	O
at	O
37	O
°	O
C	O
with	O
increasing	O
amounts	O
of	O
processed	O
PmC11	B-protein
and	O
analyzed	O
on	O
a	O
10	O
%	O
SDS	B-experimental_method
-	I-experimental_method
PAGE	I-experimental_method
gel	O
.	O

A	O
single	O
lane	O
of	O
20	O
μg	O
of	O
active	B-protein_state
PmC11	B-protein
(	O
labeled	O
20	O
)	O
is	O
shown	O
for	O
comparison	O
.	O

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

To	O
investigate	O
this	O
possibility	O
,	O
two	O
mutant	O
forms	O
of	O
the	O
enzyme	O
were	O
created	O
:	O
PmC11C179A	B-mutant
(	O
a	O
catalytically	B-protein_state
inactive	I-protein_state
mutant	I-protein_state
)	O
and	O
PmC11K147A	B-mutant
(	O
a	O
cleavage	B-protein_state
-	I-protein_state
site	I-protein_state
mutant	I-protein_state
).	O

The	O
PmC11K147A	B-mutant
mutant	B-protein_state
enzyme	O
had	O
a	O
markedly	O
different	O
reaction	B-evidence
rate	I-evidence
(	O
Vmax	B-evidence
)	O
compared	O
with	O
WT	B-protein_state
,	O
where	O
the	O
reaction	B-evidence
velocity	I-evidence
of	O
PmC11	B-protein
was	O
10	O
times	O
greater	O
than	O
that	O
of	O
PmC11K147A	B-mutant
(	O
Fig	O
.	O
2D	O
).	O

Thus	O
,	O
Asn50	B-residue_name_number
,	O
Asp177	B-residue_name_number
,	O
and	O
Asp207	B-residue_name_number
are	O
most	O
likely	O
responsible	O
for	O
the	O
substrate	O
specificity	O
of	O
PmC11	B-protein
.	O

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

The	O
structural	O
similarity	O
of	O
PmC11	B-protein
with	O
its	O
nearest	O
structural	O
neighbors	O
in	O
the	O
PDB	O
is	O
decidedly	O
low	O
,	O
overlaying	O
better	O
with	O
six	O
-	O
stranded	O
caspase	B-protein
-	I-protein
7	I-protein
than	O
any	O
of	O
the	O
other	O
larger	O
members	O
of	O
the	O
clan	O
(	O
Table	O
2	O
).	O

PmC11	B-protein
differs	O
from	O
clostripain	B-protein
in	O
that	O
is	O
does	O
not	O
appear	O
to	O
require	O
divalent	O
cations	O
for	O
activation	O
.	O

In	O
addition	O
,	O
several	O
members	O
of	O
clan	B-protein_type
CD	I-protein_type
exhibit	O
self	O
-	O
inhibition	O
,	O
whereby	O
regions	B-structure_element
of	O
the	O
enzyme	O
block	O
access	O
to	O
the	O
active	B-site
site	I-site
.	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

In	O
the	O
present	O
study	O
,	O
we	O
solved	O
the	O
crystal	B-evidence
structures	I-evidence
of	O
an	O
N	O
-	O
terminal	O
truncated	B-protein_state
form	O
of	O
YfiB	B-protein
(	O
34	B-residue_range
–	I-residue_range
168	I-residue_range
)	O
and	O
YfiR	B-protein
in	B-protein_state
complex	I-protein_state
with	I-protein_state
an	O
active	B-protein_state
mutant	B-protein_state
YfiBL43P	B-mutant
.	O

Compared	O
with	O
the	O
reported	O
complex	O
structure	O
,	O
YfiBL43P	B-mutant
in	O
our	O
YfiB	B-complex_assembly
-	I-complex_assembly
YfiR	I-complex_assembly
complex	O
structure	B-evidence
has	O
additional	O
visible	O
N	O
-	O
terminal	O
residues	O
44	B-residue_range
–	I-residue_range
58	I-residue_range
that	O
are	O
shown	O
to	O
play	O
essential	O
roles	O
in	O
YfiB	B-protein
activation	O
and	O
biofilm	O
formation	O
.	O

In	O
addition	O
,	O
there	O
is	O
a	O
short	O
helix	B-structure_element
turn	I-structure_element
connecting	O
the	O
β4	B-structure_element
strand	I-structure_element
and	O
α4	B-structure_element
helix	I-structure_element
(	O
Fig	O
.	O
1A	O
and	O
1B	O
).	O

The	O
YfiR	B-protein
molecules	O
are	O
shown	O
in	O
green	O
and	O
magenta	O
.	O

YfiBL43P	B-mutant
and	O
YfiR	B-protein
are	O
shown	O
in	O
cyan	O
and	O
green	O
,	O
respectively	O
.	O
(	O
E	O
and	O
F	O
)	O
The	O
conserved	B-site
surface	I-site
in	O
YfiR	B-protein
contributes	O
to	O
the	O
interaction	O
with	O
YfiB	B-protein
.	O
(	O
G	O
)	O
The	O
residues	B-structure_element
of	O
YfiR	B-protein
responsible	O
for	O
interacting	O
with	O
YfiB	B-protein
are	O
shown	O
in	O
green	O
sticks	O
,	O
and	O
the	O
proposed	O
YfiN	B-site
-	I-site
interacting	I-site
residues	I-site
are	O
shown	O
in	O
yellow	O
sticks	O
.	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

The	O
observed	O
changes	O
in	O
conformation	O
of	O
YfiB	B-protein
and	O
the	O
results	O
of	O
mutagenesis	B-experimental_method
suggest	O
a	O
mechanism	O
by	O
which	O
YfiB	B-protein
sequesters	O
YfiR	B-protein
.	O

Region	B-structure_element
I	I-structure_element
is	O
formed	O
by	O
numerous	O
main	O
-	O
chain	O
and	O
side	O
-	O
chain	O
hydrophilic	O
interactions	O
between	O
residues	O
E45	B-residue_name_number
,	O
G47	B-residue_name_number
and	O
E53	B-residue_name_number
from	O
the	O
N	O
-	O
terminal	O
extended	O
loop	B-structure_element
of	O
YfiB	B-protein
and	O
residues	O
S57	B-residue_name_number
,	O
R60	B-residue_name_number
,	O
A89	B-residue_name_number
and	O
H177	B-residue_name_number
from	O
YfiR	B-protein
(	O
Fig	O
.	O
3D	O
-	O
I	O
(	O
i	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

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	O
=	O
1	O
.	O
1	O
mmol	O
/	O
L	O
)	O
(	O
Fig	O
.	O
4E	O
–	O
F	O
).	O

As	O
the	O
experiment	O
is	O
performed	O
in	B-protein_state
the	I-protein_state
absence	I-protein_state
of	I-protein_state
YfiR	B-protein
,	O
it	O
suggests	O
that	O
an	O
increase	O
in	O
the	O
PG	B-evidence
-	I-evidence
binding	I-evidence
affinity	I-evidence
of	O
YfiB	B-protein
is	O
not	O
a	O
result	O
of	O
YfiB	B-complex_assembly
-	I-complex_assembly
YfiR	I-complex_assembly
interaction	O
and	O
is	O
highly	O
coupled	O
to	O
the	O
activation	O
of	O
YfiB	B-protein
characterized	O
by	O
a	O
stretched	B-protein_state
N	I-protein_state
-	I-protein_state
terminal	I-protein_state
conformation	I-protein_state
.	O

Calculation	O
using	O
the	O
ConSurf	B-experimental_method
Server	I-experimental_method
(	O
http	O
://	O
consurf	O
.	O
tau	O
.	O
ac	O
.	O
il	O
/),	O
which	O
estimates	O
the	O
evolutionary	B-evidence
conservation	I-evidence
of	O
amino	O
acid	O
positions	O
and	O
visualizes	O
information	O
on	O
the	O
structure	B-site
surface	I-site
,	O
revealed	O
a	O
conserved	B-site
surface	I-site
on	O
YfiR	B-protein
that	O
contributes	O
to	O
the	O
interaction	O
with	O
YfiB	B-protein
(	O
Fig	O
.	O
3E	O
and	O
3F	O
).	O

F151	B-residue_name_number
,	O
E163	B-residue_name_number
and	O
I169	B-residue_name_number
form	O
a	O
hydrophobic	B-site
core	I-site
while	O
,	O
Q187	B-residue_name_number
is	O
located	O
at	O
the	O
end	O
of	O
the	O
α6	B-structure_element
helix	I-structure_element
.	O

YfiR	B-protein
binds	O
small	O
molecules	O

The	O
results	O
showed	O
Kd	B-evidence
values	O
of	O
1	O
.	O
4	O
×	O
10	O
−	O
7	O
mol	O
/	O
L	O
and	O
5	O
.	O
3	O
×	O
10	O
−	O
7	O
mol	O
/	O
L	O
for	O
YfiBL43P	B-mutant
and	O
YfiBL43P	B-mutant
/	O
F57A	B-mutant
,	O
respectively	O
,	O
revealing	O
that	O
the	O
YfiBL43P	B-mutant
/	O
F57A	B-mutant
mutant	B-protein_state
caused	O
a	O
3	O
.	O
8	O
-	O
fold	O
reduction	O
in	O
the	O
binding	B-evidence
affinity	I-evidence
compared	O
with	O
the	O
YfiBL43P	B-mutant
mutant	B-protein_state
(	O
Fig	O
.	O
6F	O
and	O
6G	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

Thus	O
,	O
YfiB	B-protein
alone	B-protein_state
represents	O
an	O
inactive	B-protein_state
form	O
that	O
may	O
only	O
partially	O
insert	O
into	O
the	O
PG	O
matrix	O
.	O

The	O
periplasmic	B-structure_element
domain	I-structure_element
of	O
YfiB	B-protein
and	O
the	O
YfiB	B-complex_assembly
-	I-complex_assembly
YfiR	I-complex_assembly
complex	O
are	O
depicted	O
according	O
to	O
the	O
crystal	B-evidence
structures	I-evidence
.	O

The	O
lipid	O
acceptor	O
Cys26	B-residue_name_number
is	O
indicated	O
as	O
blue	O
ball	O
.	O

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

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

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

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

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

Summary	O
of	O
ligand	B-experimental_method
screening	I-experimental_method
assays	I-experimental_method
used	O
to	O
measure	O
ER	O
‐	O
mediated	O
activities	O
.	O

This	O
wide	O
variance	O
enabled	O
us	O
to	O
probe	O
specific	O
features	O
of	O
ERα	B-protein
signaling	O
using	O
ligand	B-experimental_method
class	I-experimental_method
analyses	I-experimental_method
,	O
and	O
identify	O
signaling	O
patterns	O
shared	O
by	O
specific	O
ligand	O
series	O
or	O
scaffolds	O
.	O

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

Ligand	O
‐	O
specific	O
signaling	O
underlies	O
ERα	B-protein
‐	O
mediated	O
cell	O
proliferation	O

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

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

For	O
example	O
,	O
3	B-chemical
,	I-chemical
4	I-chemical
‐	I-chemical
DTP	I-chemical
,	O
furan	B-chemical
,	O
and	O
S	B-chemical
‐	I-chemical
OBHS	I-chemical
‐	I-chemical
2	I-chemical
drove	O
positively	O
correlated	O
GREB1	B-protein
levels	O
and	O
E	B-experimental_method
‐	I-experimental_method
Luc	I-experimental_method
but	O
not	O
L	B-experimental_method
‐	I-experimental_method
Luc	I-experimental_method
ERα	B-protein
‐	O
WT	B-protein_state
activity	O
(	O
Fig	O
3C	O
lanes	O
5	O
–	O
7	O
).	O

This	O
is	O
demonstrated	O
by	O
directly	O
comparing	O
the	O
signaling	O
specificities	O
of	O
matched	O
OBHS	B-chemical
(	O
indirect	O
modulator	O
,	O
cluster	O
1	O
)	O
and	O
OBHS	B-chemical
‐	I-chemical
BSC	I-chemical
analogs	O
(	O
direct	O
modulator	O
,	O
cluster	O
3	O
),	O
which	O
differ	O
only	O
in	O
the	O
basic	O
side	O
chain	O
(	O
Fig	O
2E	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

Similarly	O
,	O
deletion	B-experimental_method
of	I-experimental_method
the	O
F	B-structure_element
domain	O
did	O
not	O
abolish	O
correlations	O
between	O
the	O
L	B-experimental_method
‐	I-experimental_method
Luc	I-experimental_method
and	O
E	B-experimental_method
‐	I-experimental_method
Luc	I-experimental_method
or	O
GREB1	B-protein
levels	O
induced	O
by	O
OBHS	B-chemical
analogs	O
(	O
Fig	O
EV3F	O
).	O

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

Direct	O
modulators	O
showed	O
low	O
NCOA1	B-protein
/	I-protein
2	I-protein
/	I-protein
3	I-protein
recruitment	O
(	O
Fig	O
EV2F	O
–	O
H	O
),	O
but	O
only	O
OBHS	B-chemical
‐	I-chemical
ASC	I-chemical
analogs	O
had	O
NCOA2	B-protein
recruitment	O
profiles	O
that	O
predicted	O
a	O
full	O
range	O
of	O
effects	O
on	O
GREB1	B-protein
levels	O
(	O
Figs	O
3E	O
lanes	O
9	O
,	O
11	O
,	O
18	O
–	O
19	O
,	O
and	O
EV2A	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

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

Therefore	O
,	O
we	O
first	O
performed	O
a	O
time	B-experimental_method
‐	I-experimental_method
course	I-experimental_method
study	I-experimental_method
,	O
and	O
found	O
that	O
E2	B-chemical
and	O
the	O
WAY	B-chemical
‐	I-chemical
C	I-chemical
analog	O
,	O
AAPII	B-chemical
‐	I-chemical
151	I-chemical
‐	I-chemical
4	I-chemical
,	O
induced	O
recruitment	O
of	O
NCOA3	B-protein
to	O
the	O
GREB1	B-protein
promoter	O
in	O
a	O
temporal	O
cycle	O
that	O
peaked	O
after	O
45	O
min	O
in	O
MCF	O
‐	O
7	O
cells	O
(	O
Fig	O
4A	O
).	O

Kinetic	B-experimental_method
ChIP	I-experimental_method
assay	I-experimental_method
examining	O
recruitment	O
of	O
NCOA3	B-protein
to	O
the	O
GREB1	B-protein
gene	O
in	O
MCF	O
‐	O
7	O
cells	O
stimulated	O
with	O
E2	B-chemical
or	O
the	O
indicated	O
WAY	B-chemical
‐	I-chemical
C	I-chemical
analog	O
.	O

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

However	O
,	O
the	O
ChIP	B-experimental_method
assays	I-experimental_method
for	O
WAY	B-chemical
‐	I-chemical
C	I-chemical
‐	O
induced	O
recruitment	O
of	O
NCOA3	B-protein
to	O
the	O
GREB1	B-protein
promoter	O
did	O
not	O
correlate	O
with	O
any	O
of	O
the	O
other	O
WAY	B-chemical
‐	I-chemical
C	I-chemical
activity	O
profiles	O
(	O
Fig	O
4D	O
),	O
although	O
the	O
positive	O
correlation	O
between	O
ChIP	B-experimental_method
assays	I-experimental_method
and	O
NCOA3	B-protein
recruitment	O
via	O
M2H	B-experimental_method
assay	I-experimental_method
showed	O
a	O
trend	O
toward	O
significance	O
with	O
r	B-evidence
2	I-evidence
=	O
0	O
.	O
36	O
and	O
P	B-evidence
=	O
0	O
.	O
09	O
(	O
F	B-experimental_method
‐	I-experimental_method
test	I-experimental_method
for	O
nonzero	O
slope	O
).	O

Nevertheless	O
,	O
the	O
E	B-experimental_method
‐	I-experimental_method
Luc	I-experimental_method
activities	O
of	O
both	O
2	B-chemical
,	I-chemical
5	I-chemical
‐	I-chemical
DTP	I-chemical
and	O
cyclofenil	B-chemical
analogs	O
were	O
better	O
predicted	O
by	O
their	O
L	B-experimental_method
‐	I-experimental_method
Luc	I-experimental_method
ERα	B-protein
‐	O
WT	B-protein_state
than	O
L	B-experimental_method
‐	I-experimental_method
Luc	I-experimental_method
ERβ	B-protein
activities	O
(	O
Fig	O
EV4A	O
and	O
B	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

Based	O
on	O
our	O
original	O
OBHS	B-chemical
structure	B-evidence
,	O
the	O
OBHS	B-chemical
,	O
OBHS	B-chemical
‐	I-chemical
N	I-chemical
,	O
and	O
triaryl	B-chemical
‐	I-chemical
ethylene	I-chemical
compounds	O
were	O
modified	O
with	O
h11	B-structure_element
‐	O
directed	O
pendant	O
groups	O
(	O
Zheng	O
et	O
al	O
,	O
2012	O
;	O
Zhu	O
et	O
al	O
,	O
2012	O
;	O
Liao	O
et	O
al	O
,	O
2014	O
).	O

For	O
the	O
triaryl	B-chemical
‐	I-chemical
ethylene	I-chemical
analogs	O
,	O
the	O
displacement	O
of	O
h11	B-structure_element
was	O
in	O
a	O
perpendicular	O
direction	O
,	O
away	O
from	O
Ile424	B-residue_name_number
in	O
h8	B-structure_element
and	O
toward	O
h12	B-structure_element
.	O

Structure	B-experimental_method
‐	I-experimental_method
class	I-experimental_method
analysis	I-experimental_method
of	O
triaryl	B-chemical
‐	I-chemical
ethylene	I-chemical
analogs	O
.	O

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

Triaryl	B-chemical
‐	I-chemical
ethylene	I-chemical
analogs	O
induce	O
variance	O
of	O
ERα	B-protein
conformations	O
at	O
the	O
C	O
‐	O
terminal	O
region	O
of	O
h11	B-structure_element
.	O

WAY	B-chemical
‐	I-chemical
C	I-chemical
side	O
groups	O
subtly	O
nudge	O
h12	B-structure_element
Leu540	B-residue_name_number
.	O

Structure	B-experimental_method
‐	I-experimental_method
class	I-experimental_method
analysis	I-experimental_method
of	O
indirect	O
modulators	O
in	O
cluster	O
1	O
.	O

Crystal	B-evidence
structures	I-evidence
of	O
the	O
ERα	B-protein
LBD	B-structure_element
bound	B-protein_state
to	I-protein_state
OBHS	B-chemical
and	O
OBHS	B-chemical
‐	I-chemical
N	I-chemical
analogs	O
were	O
superposed	B-experimental_method
.	O

As	O
visualized	O
in	O
four	O
LBD	B-structure_element
structures	B-evidence
(	O
Srinivasan	O
et	O
al	O
,	O
2013	O
),	O
WAY	B-chemical
‐	I-chemical
C	I-chemical
analogs	O
were	O
designed	O
with	O
small	O
substitutions	O
that	O
slightly	O
nudge	O
h12	B-structure_element
Leu540	B-residue_name_number
,	O
without	O
exiting	O
the	O
ligand	B-site
‐	I-site
binding	I-site
pocket	I-site
(	O
Fig	O
5G	O
and	O
H	O
).	O

This	O
difference	O
in	O
ligand	O
positioning	O
altered	O
the	O
AF	B-site
‐	I-site
2	I-site
surface	I-site
via	O
a	O
shift	O
in	O
the	O
N	O
‐	O
terminus	O
of	O
h12	B-structure_element
,	O
which	O
directly	O
contacts	O
the	O
coactivator	O
.	O

Crystal	B-evidence
structures	I-evidence
show	O
that	O
a	O
3	B-chemical
,	I-chemical
4	I-chemical
‐	I-chemical
DTPD	I-chemical
analog	O
shifts	O
h3	B-structure_element
(	O
F	B-structure_element
)	O
and	O
the	O
NCOA2	B-protein
(	O
G	O
)	O
peptide	O
compared	O
to	O
an	O
A	B-chemical
‐	I-chemical
CD	I-chemical
‐	O
ring	O
estrogen	B-chemical
(	O
PDB	O
4PPS	O
,	O
5DTV	O
).	O

The	O
2	B-chemical
,	I-chemical
5	I-chemical
‐	I-chemical
DTP	I-chemical
analogs	O
showed	O
perturbation	O
of	O
h11	B-structure_element
,	O
as	O
well	O
as	O
h3	B-structure_element
,	O
which	O
forms	O
part	O
of	O
the	O
AF	B-site
‐	I-site
2	I-site
surface	I-site
.	O

We	O
observed	O
a	O
difference	O
of	O
0	O
.	O
4	O
Å	O
that	O
was	O
significant	O
(	O
two	O
‐	O
tailed	O
Student	B-experimental_method
'	I-experimental_method
s	I-experimental_method
t	I-experimental_method
‐	I-experimental_method
test	I-experimental_method
,	O
P	B-evidence
=	O
0	O
.	O
002	O
)	O
due	O
to	O
the	O
very	O
tight	O
clustering	O
of	O
the	O
2	B-chemical
,	I-chemical
5	I-chemical
‐	I-chemical
DTP	I-chemical
‐	O
induced	O
LBD	B-structure_element
conformation	O
.	O

The	O
3	B-chemical
,	I-chemical
4	I-chemical
‐	I-chemical
DTPD	I-chemical
analogs	O
also	O
induced	O
a	O
shift	O
in	O
h3	B-structure_element
positioning	O
,	O
which	O
translated	O
again	O
into	O
a	O
shift	O
in	O
the	O
bound	O
coactivator	O
peptide	O
(	O
Fig	O
6F	O
).	O

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

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

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

Secondly	O
,	O
our	O
finding	O
that	O
WAY	B-chemical
‐	I-chemical
C	I-chemical
compounds	O
do	O
not	O
rely	O
of	O
AF	B-structure_element
‐	I-structure_element
1	I-structure_element
for	O
signaling	O
efficacy	O
may	O
derive	O
from	O
the	O
slight	O
contacts	O
with	O
h12	B-structure_element
observed	O
in	O
crystal	B-evidence
structures	I-evidence
(	O
Figs	O
3B	O
and	O
5H	O
),	O
unlike	O
other	O
compounds	O
in	O
cluster	O
1	O
that	O
dislocate	O
h11	B-structure_element
and	O
rely	O
on	O
AF	B-structure_element
‐	I-structure_element
1	I-structure_element
for	O
signaling	O
efficacy	O
(	O
Figs	O
3B	O
and	O
5C	O
,	O
and	O
EV5B	O
).	O

We	O
have	O
also	O
investigated	O
the	O
binding	O
of	O
the	O
TOCA	B-protein
HR1	B-structure_element
domain	O
to	O
Cdc42	B-protein
and	O
the	O
potential	O
ternary	O
complex	O
between	O
Cdc42	B-protein
and	O
the	O
G	B-site
protein	I-site
-	I-site
binding	I-site
regions	I-site
of	O
TOCA1	B-protein
and	O
a	O
member	O
of	O
the	O
Wiskott	B-protein_type
-	I-protein_type
Aldrich	I-protein_type
syndrome	I-protein_type
protein	I-protein_type
family	I-protein_type
,	O
N	B-protein
-	I-protein
WASP	I-protein
.	O

The	O
guanine	B-protein_type
nucleotide	I-protein_type
exchange	I-protein_type
factors	I-protein_type
mediate	O
formation	O
of	O
the	O
active	B-protein_state
state	O
by	O
promoting	O
the	O
dissociation	O
of	O
GDP	B-chemical
,	O
allowing	O
GTP	B-chemical
to	O
bind	O
.	O

The	O
Rho	B-protein_type
family	I-protein_type
comprises	O
20	O
members	O
,	O
of	O
which	O
three	O
,	O
RhoA	B-protein
,	O
Rac1	B-protein
,	O
and	O
Cdc42	B-protein
,	O
have	O
been	O
relatively	O
well	O
studied	O
.	O

Following	O
their	O
release	O
,	O
the	O
C	B-structure_element
-	I-structure_element
terminal	I-structure_element
regions	I-structure_element
of	O
N	B-protein
-	I-protein
WASP	I-protein
are	O
free	O
to	O
interact	O
with	O
G	B-protein_type
-	I-protein_type
actin	I-protein_type
and	O
a	O
known	O
nucleator	O
of	O
actin	O
assembly	O
,	O
the	O
Arp2	B-complex_assembly
/	I-complex_assembly
3	I-complex_assembly
complex	O
.	O

The	O
structures	B-evidence
of	O
the	O
PRK1	B-protein
HR1a	B-structure_element
domain	O
in	O
complex	B-protein_state
with	I-protein_state
RhoA	B-protein
and	O
the	O
HR1b	B-structure_element
domain	O
in	O
complex	B-protein_state
with	I-protein_state
Rac1	B-protein
show	O
that	O
the	O
HR1	B-structure_element
domain	O
comprises	O
an	O
anti	B-structure_element
-	I-structure_element
parallel	I-structure_element
coiled	I-structure_element
-	I-structure_element
coil	I-structure_element
that	O
interacts	O
with	O
its	O
G	B-protein_type
protein	I-protein_type
binding	O
partner	O
via	O
both	O
helices	B-structure_element
.	O

Both	O
of	O
the	O
G	B-site
protein	I-site
switch	I-site
regions	I-site
are	O
involved	O
in	O
the	O
interaction	O
.	O

The	O
coiled	B-structure_element
-	I-structure_element
coil	I-structure_element
fold	I-structure_element
is	O
shared	O
by	O
the	O
HR1	B-structure_element
domain	O
of	O
the	O
TOCA	B-protein_type
family	I-protein_type
protein	I-protein_type
,	O
CIP4	B-protein
,	O
and	O
,	O
based	O
on	O
sequence	O
homology	O
,	O
by	O
TOCA1	B-protein
itself	O
.	O

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

The	O
interactions	O
of	O
TOCA1	B-protein
and	O
N	B-protein
-	I-protein
WASP	I-protein
with	O
Cdc42	B-protein
as	O
well	O
as	O
with	O
each	O
other	O
have	O
raised	O
questions	O
as	O
to	O
whether	O
the	O
two	O
Cdc42	B-protein
effectors	O
can	O
interact	O
with	O
a	O
single	O
molecule	O
of	O
Cdc42	B-protein
simultaneously	O
.	O

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

Finally	O
,	O
we	O
investigate	O
the	O
potential	O
ternary	O
complex	O
between	O
Cdc42	B-protein
and	O
the	O
G	B-site
protein	I-site
-	I-site
binding	I-site
regions	I-site
of	O
TOCA1	B-protein
and	O
N	B-protein
-	I-protein
WASP	I-protein
,	O
contributing	O
to	O
our	O
understanding	O
of	O
G	B-protein_type
protein	I-protein_type
-	O
effector	O
interactions	O
as	O
well	O
as	O
the	O
roles	O
of	O
Cdc42	B-protein
,	O
N	B-protein
-	I-protein
WASP	I-protein
,	O
and	O
TOCA1	B-protein
in	O
the	O
pathways	O
that	O
govern	O
actin	O
dynamics	O
.	O

TOCA1	B-protein
was	O
identified	O
in	O
Xenopus	B-taxonomy_domain
extracts	O
as	O
a	O
protein	O
necessary	O
for	O
Cdc42	B-protein
-	O
dependent	O
actin	O
assembly	O
and	O
was	O
shown	O
to	O
bind	O
to	O
Cdc42	B-complex_assembly
·	I-complex_assembly
GTPγS	I-complex_assembly
but	O
not	O
to	O
Cdc42	B-complex_assembly
·	I-complex_assembly
GDP	I-complex_assembly
or	O
to	O
Rac1	B-protein
and	O
RhoA	B-protein
.	O
Given	O
its	O
homology	O
to	O
other	O
Rho	B-site
family	I-site
binding	I-site
modules	I-site
,	O
it	O
is	O
likely	O
that	O
the	O
HR1	B-structure_element
domain	O
of	O
TOCA1	B-protein
is	O
sufficient	O
to	O
bind	O
Cdc42	B-protein
.	O

The	O
binding	O
of	O
TOCA1	B-protein
HR1	B-structure_element
to	O
Cdc42	B-protein
was	O
unexpectedly	O
weak	O
,	O
with	O
a	O
Kd	B-evidence
of	O
>	O
1	O
μm	O
.	O

A	O
,	O
curves	O
derived	O
from	O
direct	B-experimental_method
binding	I-experimental_method
assays	I-experimental_method
in	O
which	O
the	O
indicated	O
concentrations	O
of	O
Cdc42Δ7Q61L	B-complex_assembly
·[	I-complex_assembly
3H	I-complex_assembly
]	I-complex_assembly
GTP	I-complex_assembly
were	O
incubated	B-experimental_method
with	O
30	O
nm	O
GST	B-mutant
-	I-mutant
PAK	I-mutant
or	O
HR1	B-mutant
-	I-mutant
His6	I-mutant
in	O
SPAs	B-experimental_method
.	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

Isothermal	B-experimental_method
titration	I-experimental_method
calorimetry	I-experimental_method
was	O
carried	O
out	O
,	O
but	O
no	O
heat	O
changes	O
were	O
observed	O
at	O
a	O
range	O
of	O
concentrations	O
and	O
temperatures	O
(	O
data	O
not	O
shown	O
),	O
suggesting	O
that	O
the	O
interaction	O
is	O
predominantly	O
entropically	O
driven	O
.	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

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
<	O
SA	O
>,	O
the	O
average	B-evidence
root	I-evidence
mean	I-evidence
square	I-evidence
deviations	I-evidence
for	O
the	O
ensemble	O
±	O
S	O
.	O
D	O
.	O

B	O
,	O
a	O
sequence	B-experimental_method
alignment	I-experimental_method
of	O
the	O
HR1	B-structure_element
domains	O
from	O
TOCA1	B-protein
,	O
CIP4	B-protein
,	O
and	O
PRK1	B-protein
.	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

Residues	O
with	O
significantly	O
affected	O
backbone	O
or	O
side	O
chain	O
chemical	O
shifts	O
when	O
Cdc42	B-protein_state
bound	I-protein_state
and	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
yellow	O
.	O

Therefore	O
,	O
13C	B-experimental_method
HSQC	I-experimental_method
and	O
methyl	B-experimental_method
-	I-experimental_method
selective	I-experimental_method
SOFAST	I-experimental_method
-	I-experimental_method
HMQC	I-experimental_method
experiments	O
were	O
also	O
recorded	O
on	O
15N	B-chemical
,	O
13C	B-chemical
-	O
labeled	B-protein_state
TOCA1	B-protein
HR1	B-structure_element
to	O
yield	O
more	O
information	O
on	O
side	O
chain	O
involvement	O
.	O

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

B	O
,	O
CSPs	B-experimental_method
are	O
shown	O
for	O
backbone	O
NH	O
groups	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

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

HADDOCK	B-experimental_method
was	O
therefore	O
used	O
to	O
perform	O
rigid	O
body	B-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

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

Residues	O
of	O
Cdc42	B-protein
that	O
are	O
affected	O
in	O
the	O
presence	B-protein_state
of	I-protein_state
the	O
HR1	B-structure_element
domain	O
but	O
are	O
not	O
in	O
close	O
proximity	O
to	O
it	O
are	O
colored	O
in	O
red	O
and	O
labeled	O
.	O

B	O
,	O
structure	B-evidence
of	O
Rac1	B-protein
in	B-protein_state
complex	I-protein_state
with	I-protein_state
the	O
HR1b	B-structure_element
domain	O
of	O
PRK1	B-protein
(	O
PDB	O
code	O
2RMK	O
).	O

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

D	O
,	O
selected	O
regions	O
of	O
the	O
15N	B-experimental_method
HSQC	I-experimental_method
of	O
600	O
μm	O
TOCA1	B-protein
HR1	B-structure_element
domain	O
in	B-protein_state
complex	I-protein_state
with	I-protein_state
Cdc42	B-protein
in	O
the	O
absence	B-protein_state
and	O
presence	B-protein_state
of	I-protein_state
the	O
N	B-protein
-	I-protein
WASP	I-protein
GBD	B-structure_element
,	O
showing	O
displacement	O
of	O
Cdc42	B-protein
from	O
the	O
HR1	B-structure_element
domain	O
by	O
N	B-protein
-	I-protein
WASP	I-protein
.	O

The	O
Kd	B-evidence
that	O
was	O
determined	O
(	O
37	O
nm	O
)	O
is	O
consistent	O
with	O
the	O
previously	O
reported	O
affinity	B-evidence
.	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

These	O
assays	O
,	O
described	O
in	O
detail	O
elsewhere	O
,	O
were	O
carried	O
out	O
using	O
pyrene	B-chemical
actin	I-chemical
-	O
supplemented	O
Xenopus	B-taxonomy_domain
extracts	O
into	O
which	O
exogenous	O
TOCA1	B-protein
HR1	B-structure_element
domain	O
or	O
N	B-protein
-	I-protein
WASP	I-protein
GBD	B-structure_element
was	O
added	O
,	O
to	O
assess	O
their	O
effects	O
on	O
actin	B-protein_type
polymerization	O
.	O

The	O
corresponding	O
sequence	O
in	O
CIP4	B-protein
also	O
includes	O
a	O
series	O
of	O
turns	O
but	O
is	O
flexible	O
,	O
whereas	O
in	O
the	O
HR1a	B-structure_element
domain	O
of	O
PRK1	B-protein
,	O
the	O
equivalent	O
region	O
adopts	O
an	O
α	B-structure_element
-	I-structure_element
helical	I-structure_element
structure	I-structure_element
that	O
packs	O
against	O
the	O
coiled	B-structure_element
-	I-structure_element
coil	I-structure_element
.	O

The	O
lowest	O
energy	O
model	B-evidence
produced	O
by	O
HADDOCK	B-experimental_method
using	O
ambiguous	O
interaction	O
restraints	O
from	O
the	O
titration	B-evidence
data	O
resembled	O
the	O
NMR	B-experimental_method
structures	B-evidence
of	O
RhoA	B-protein
and	O
Rac1	B-protein
in	B-protein_state
complex	I-protein_state
with	I-protein_state
their	O
HR1	B-structure_element
domain	O
partners	O
.	O

For	O
example	O
,	O
Phe	B-residue_name_number
-	I-residue_name_number
56Cdc42	I-residue_name_number
,	O
which	O
is	O
not	O
visible	O
in	O
free	B-protein_state
Cdc42	B-protein
or	O
Cdc42	B-complex_assembly
·	I-complex_assembly
HR1TOCA1	I-complex_assembly
,	O
is	O
close	O
to	O
the	O
TOCA1	B-protein
HR1	B-structure_element
(	O
Fig	O
.	O
6A	O
).	O

Phe	B-residue_name_number
-	I-residue_name_number
56Cdc42	I-residue_name_number
is	O
therefore	O
likely	O
to	O
be	O
involved	O
in	O
the	O
Cdc42	B-protein
-	O
TOCA1	B-protein
interaction	O
,	O
probably	O
by	O
stabilizing	O
the	O
position	O
of	O
switch	B-site
I	I-site
.	O

Thr	B-residue_name_number
-	I-residue_name_number
52Cdc42	I-residue_name_number
,	O
which	O
has	O
also	O
been	O
identified	O
as	O
making	O
minor	O
contacts	O
with	O
ACK	B-protein
,	O
falls	O
near	O
the	O
side	O
chains	O
of	O
HR1TOCA1	B-structure_element
helix	B-structure_element
1	I-structure_element
,	O
particularly	O
Lys	B-residue_name_number
-	I-residue_name_number
372TOCA1	I-residue_name_number
,	O
whereas	O
the	O
equivalent	O
position	O
in	O
Rac1	B-protein
is	O
Asn	B-residue_name_number
-	I-residue_name_number
52Rac1	I-residue_name_number
.	O

In	O
contrast	O
,	O
the	O
best	O
estimate	O
of	O
the	O
affinity	B-evidence
of	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
WASP	B-protein_type
for	O
Cdc42	B-protein
is	O
low	O
micromolar	O
.	O

WIP	B-protein
inhibits	O
the	O
activation	O
of	O
N	B-protein
-	I-protein
WASP	I-protein
by	O
Cdc42	B-protein
,	O
an	O
effect	O
that	O
is	O
reversed	O
by	O
TOCA1	B-protein
.	O

It	O
has	O
been	O
postulated	O
that	O
the	O
initial	O
interactions	O
between	O
this	O
basic	O
region	O
and	O
Cdc42	B-protein
could	O
stabilize	O
the	O
active	B-protein_state
conformation	O
of	O
WASP	B-protein
,	O
leading	O
to	O
high	O
affinity	O
binding	O
between	O
the	O
core	O
CRIB	B-structure_element
and	O
Cdc42	B-protein
.	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

It	O
is	O
clear	O
from	O
the	O
data	O
presented	O
here	O
that	O
TOCA1	B-protein
and	O
N	B-protein
-	I-protein
WASP	I-protein
do	O
not	O
bind	O
Cdc42	B-protein
simultaneously	O
and	O
that	O
N	B-protein
-	I-protein
WASP	I-protein
is	O
likely	O
to	O
outcompete	O
TOCA1	B-protein
for	O
Cdc42	B-protein
binding	O
.	O

Furthermore	O
,	O
elevated	O
ACC	B-protein_type
activity	O
is	O
observed	O
in	O
malignant	O
tumours	O
.	O

The	O
principal	O
functional	O
protein	O
components	O
of	O
ACCs	B-protein_type
have	O
been	O
described	O
already	O
in	O
the	O
late	O
1960s	O
for	O
Escherichia	B-species
coli	I-species
(	O
E	B-species
.	I-species
coli	I-species
)	O
ACC	B-protein_type
:	O
Biotin	B-protein_type
carboxylase	I-protein_type
(	O
BC	B-protein_type
)	O
catalyses	O
the	O
ATP	B-chemical
-	O
dependent	O
carboxylation	O
of	O
a	O
biotin	B-chemical
moiety	O
,	O
which	O
is	O
covalently	O
linked	O
to	O
the	O
biotin	B-protein_type
carboxyl	I-protein_type
carrier	I-protein_type
protein	I-protein_type
(	O
BCCP	B-protein_type
).	O

Human	B-species
ACC1	B-protein
is	O
further	O
regulated	O
by	O
specific	O
phosphorylation	B-ptm
-	O
dependent	O
binding	O
of	O
BRCA1	B-protein
to	O
Ser1263	B-residue_name_number
in	O
the	O
CD	B-structure_element
.	O

Furthermore	O
,	O
phosphorylation	B-ptm
by	O
AMP	B-protein
-	I-protein
activated	I-protein
protein	I-protein
kinase	I-protein
(	O
AMPK	B-protein
)	O
and	O
cAMP	B-protein
-	I-protein
dependent	I-protein
protein	I-protein
kinase	I-protein
(	O
PKA	B-protein
)	O
leads	O
to	O
a	O
decrease	O
in	O
ACC1	B-protein
activity	O
.	O

The	O
regulatory	O
Ser1201	B-residue_name_number
shows	O
only	O
moderate	B-protein_state
conservation	I-protein_state
across	O
higher	B-taxonomy_domain
eukaryotes	I-taxonomy_domain
,	O
while	O
the	O
phosphorylated	B-protein_state
Ser1216	B-residue_name_number
is	O
highly	B-protein_state
conserved	I-protein_state
across	O
all	O
eukaryotes	B-taxonomy_domain
.	O

In	O
yeast	B-taxonomy_domain
ACC	B-protein_type
,	O
phosphorylation	B-site
sites	I-site
have	O
been	O
identified	O
at	O
Ser2	B-residue_name_number
,	O
Ser735	B-residue_name_number
,	O
Ser1148	B-residue_name_number
,	O
Ser1157	B-residue_name_number
and	O
Ser1162	B-residue_name_number
(	O
ref	O
.).	O

SceCD	B-species
comprises	O
four	O
distinct	O
domains	O
,	O
an	O
N	O
-	O
terminal	O
α	B-structure_element
-	I-structure_element
helical	I-structure_element
domain	I-structure_element
(	O
CDN	B-structure_element
),	O
and	O
a	O
central	O
four	B-structure_element
-	I-structure_element
helix	I-structure_element
bundle	I-structure_element
linker	I-structure_element
domain	I-structure_element
(	O
CDL	B-structure_element
),	O
followed	O
by	O
two	O
α	B-structure_element
–	I-structure_element
β	I-structure_element
-	I-structure_element
fold	I-structure_element
C	I-structure_element
-	I-structure_element
terminal	I-structure_element
domains	I-structure_element
(	O
CDC1	B-structure_element
/	O
CDC2	B-structure_element
).	O

In	O
insect	B-experimental_method
-	I-experimental_method
cell	I-experimental_method
-	I-experimental_method
expressed	I-experimental_method
full	B-protein_state
-	I-protein_state
length	I-protein_state
SceACC	B-protein
,	O
the	O
highly	B-protein_state
conserved	I-protein_state
Ser1157	B-residue_name_number
is	O
the	O
only	O
fully	B-protein_state
occupied	I-protein_state
phosphorylation	B-site
site	I-site
with	O
functional	O
relevance	O
in	O
S	B-species
.	I-species
cerevisiae	I-species
.	O

Additional	O
phosphorylation	B-ptm
was	O
detected	O
for	O
Ser2101	B-residue_name_number
and	O
Tyr2179	B-residue_name_number
;	O
however	O
,	O
these	O
sites	O
are	O
neither	B-protein_state
conserved	I-protein_state
across	O
fungal	B-taxonomy_domain
ACC	B-protein_type
nor	B-protein_state
natively	I-protein_state
phosphorylated	I-protein_state
in	O
yeast	B-taxonomy_domain
.	O

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

The	O
variable	O
CD	B-structure_element
is	O
conserved	B-protein_state
between	O
yeast	B-taxonomy_domain
and	O
human	B-species

To	O
compare	O
the	O
organization	O
of	O
fungal	B-taxonomy_domain
and	O
human	B-species
ACC	B-protein_type
CD	B-structure_element
,	O
we	O
determined	B-experimental_method
the	I-experimental_method
structure	I-experimental_method
of	O
a	O
human	B-species
ACC1	B-mutant
fragment	I-mutant
that	O
comprises	O
the	O
BT	B-structure_element
and	O
CD	B-structure_element
domains	O
(	O
HsaBT	B-mutant
-	I-mutant
CD	I-mutant
),	O
but	O
lacks	B-protein_state
the	O
mobile	O
BCCP	B-structure_element
in	O
between	O
(	O
Fig	O
.	O
1a	O
).	O

Besides	O
the	O
regulatory	B-structure_element
loop	I-structure_element
,	O
also	O
the	O
phosphopeptide	B-site
target	I-site
region	I-site
for	O
BRCA1	B-protein
interaction	O
is	O
not	O
resolved	O
presumably	O
because	O
of	O
pronounced	O
flexibility	O
.	O

The	O
neighbouring	O
loop	B-structure_element
on	O
the	O
CT	B-structure_element
side	O
(	O
between	O
CT	B-structure_element
β1	B-structure_element
/	O
β2	B-structure_element
)	O
is	O
displaced	O
by	O
2	O
.	O
5	O
Å	O
compared	O
to	O
isolated	B-protein_state
CT	B-structure_element
structures	B-evidence
(	O
Supplementary	O
Fig	O
.	O
3c	O
).	O

The	O
interface	B-site
between	O
CDC2	B-structure_element
and	O
CDL	B-structure_element
/	O
CDC1	B-structure_element
,	O
which	O
is	O
mediated	O
by	O
the	O
phosphorylated	B-protein_state
regulatory	B-structure_element
loop	I-structure_element
in	O
the	O
SceCD	B-species
structure	B-evidence
,	O
is	O
less	O
variable	O
than	O
the	O
CD	B-structure_element
–	I-structure_element
CT	I-structure_element
junction	I-structure_element
,	O
and	O
permits	O
only	O
limited	O
rotation	O
and	O
tilting	O
(	O
Fig	O
.	O
3b	O
).	O

The	O
BC	B-structure_element
domain	O
is	O
not	O
completely	O
disordered	O
,	O
but	O
laterally	O
attached	O
to	O
BT	B-structure_element
/	O
CDN	B-structure_element
in	O
a	O
generally	B-protein_state
conserved	I-protein_state
position	I-protein_state
,	O
albeit	O
with	O
increased	O
flexibility	O
.	O

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

In	O
flACC	B-mutant
,	O
the	O
ACC	B-protein_type
dimer	B-oligomeric_state
obeys	O
twofold	O
symmetry	O
and	O
assembles	O
in	O
a	O
triangular	B-protein_state
architecture	I-protein_state
with	O
dimeric	B-oligomeric_state
BC	B-structure_element
domains	O
(	O
Supplementary	O
Fig	O
.	O
5a	O
).	O

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

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

The	O
only	O
bona	O
fide	O
regulatory	B-protein_state
phophorylation	B-site
site	I-site
of	O
fungal	B-taxonomy_domain
ACC	B-protein_type
in	O
the	O
regulatory	B-structure_element
loop	I-structure_element
is	O
directly	O
participating	O
in	O
CDC1	B-structure_element
/	O
CDC2	B-structure_element
domain	O
interactions	O
and	O
thus	O
stabilizes	O
the	O
hinge	B-structure_element
conformation	I-structure_element
.	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

However	O
,	O
the	O
example	O
of	O
ACC	B-protein_type
now	O
demonstrates	O
the	O
possibility	O
of	O
regulating	O
activity	O
by	O
controlled	O
dynamics	O
of	O
non	B-structure_element
-	I-structure_element
enzymatic	I-structure_element
linker	I-structure_element
regions	I-structure_element
also	O
in	O
other	O
families	O
of	O
carrier	B-protein_type
-	I-protein_type
dependent	I-protein_type
multienzymes	I-protein_type
.	O

CDN	B-structure_element
is	O
linked	O
by	O
a	O
four	B-structure_element
-	I-structure_element
helix	I-structure_element
bundle	I-structure_element
(	O
CDL	B-structure_element
)	O
to	O
two	B-structure_element
α	I-structure_element
–	I-structure_element
β	I-structure_element
-	I-structure_element
fold	I-structure_element
domains	I-structure_element
(	O
CDC1	B-structure_element
and	O
CDC2	B-structure_element
).	O

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

(	O
a	O
–	O
c	O
)	O
Large	O
-	O
scale	O
conformational	O
variability	O
of	O
the	O
CDN	B-structure_element
domain	O
relative	O
to	O
the	O
CDL	B-structure_element
/	O
CDC1	B-structure_element
domain	O
.	O

Domains	O
other	O
than	O
CDN	B-structure_element
and	O
CDL	B-structure_element
/	O
CDC1	B-structure_element
are	O
omitted	O
for	O
clarity	O
.	O

Structural	O
insights	O
into	O
the	O
Escherichia	B-species
coli	I-species
lysine	B-protein_type
decarboxylases	I-protein_type
and	O
molecular	O
determinants	O
of	O
interaction	O
with	O
the	O
AAA	B-protein_type
+	I-protein_type
ATPase	I-protein_type
RavA	B-protein

Previously	O
,	O
we	O
proposed	O
a	O
pseudoatomic	B-evidence
model	I-evidence
of	O
the	O
LdcI	B-complex_assembly
-	I-complex_assembly
RavA	I-complex_assembly
cage	O
based	O
on	O
its	O
cryo	B-experimental_method
-	I-experimental_method
electron	I-experimental_method
microscopy	I-experimental_method
map	B-evidence
and	O
crystal	B-evidence
structures	I-evidence
of	O
an	O
inactive	B-protein_state
LdcI	B-protein
decamer	B-oligomeric_state
and	O
a	O
RavA	B-protein
monomer	B-oligomeric_state
.	O

Enterobacterial	B-taxonomy_domain
inducible	B-protein_state
decarboxylases	B-protein_type
of	O
basic	B-protein_state
amino	B-chemical
acids	I-chemical
lysine	B-residue_name
,	O
arginine	B-residue_name
and	O
ornithine	B-residue_name
have	O
a	O
common	O
evolutionary	O
origin	O
and	O
belong	O
to	O
the	O
α	B-protein_type
-	I-protein_type
family	I-protein_type
of	O
pyridoxal	B-chemical
-	I-chemical
5	I-chemical
′-	I-chemical
phosphate	I-chemical
(	O
PLP	B-chemical
)-	O
dependent	O
enzymes	O
.	O

Inducible	B-protein_state
enterobacterial	B-taxonomy_domain
amino	B-protein_type
acid	I-protein_type
decarboxylases	I-protein_type
have	O
been	O
intensively	O
studied	O
since	O
the	O
early	O
1940	O
because	O
the	O
ability	O
of	O
bacteria	B-taxonomy_domain
to	O
withstand	O
acid	O
stress	O
can	O
be	O
linked	O
to	O
their	O
pathogenicity	O
in	O
humans	B-species
.	O

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

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

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

To	O
solve	O
this	O
discrepancy	O
,	O
in	O
the	O
present	O
work	O
we	O
provided	O
a	O
three	O
-	O
dimensional	O
(	O
3D	O
)	O
cryoEM	B-experimental_method
reconstruction	B-evidence
of	O
LdcC	B-protein
and	O
compared	O
it	O
with	O
the	O
available	O
LdcI	B-protein
and	O
LdcI	B-complex_assembly
-	I-complex_assembly
RavA	I-complex_assembly
structures	B-evidence
.	O

Given	O
that	O
the	O
LdcI	B-protein
crystal	B-evidence
structures	I-evidence
were	O
obtained	O
at	O
high	B-protein_state
pH	I-protein_state
where	O
the	O
enzyme	O
is	O
inactive	B-protein_state
(	O
LdcIi	B-protein
,	O
pH	B-protein_state
8	I-protein_state
.	I-protein_state
5	I-protein_state
),	O
whereas	O
the	O
cryoEM	B-experimental_method
reconstructions	B-evidence
of	O
LdcI	B-complex_assembly
-	I-complex_assembly
RavA	I-complex_assembly
and	O
LdcI	B-complex_assembly
-	I-complex_assembly
LARA	I-complex_assembly
were	O
done	O
at	O
acidic	B-protein_state
pH	I-protein_state
optimal	I-protein_state
for	O
the	O
enzymatic	O
activity	O
,	O
for	O
a	O
meaningful	O
comparison	O
,	O
we	O
also	O
produced	O
a	O
3D	B-evidence
reconstruction	I-evidence
of	O
the	O
LdcI	B-protein
at	O
active	B-protein_state
pH	I-protein_state
(	O
LdcIa	B-protein
,	O
pH	B-protein_state
6	I-protein_state
.	I-protein_state
2	I-protein_state
).	O

As	O
common	O
for	O
the	O
α	B-protein_type
family	I-protein_type
of	O
the	O
PLP	B-protein_type
-	I-protein_type
dependent	I-protein_type
decarboxylases	I-protein_type
,	O
dimerization	O
is	O
required	O
for	O
the	O
enzymatic	O
activity	O
because	O
the	O
active	B-site
site	I-site
is	O
buried	O
in	O
the	O
dimer	B-site
interface	I-site
(	O
Fig	O
.	O
3A	O
,	O
B	O
).	O

In	O
addition	O
,	O
our	O
earlier	O
biochemical	B-experimental_method
observation	I-experimental_method
that	O
the	O
enzymatic	O
activity	O
of	O
LdcIa	B-protein
is	O
unaffected	O
by	O
RavA	B-protein
binding	O
is	O
consistent	O
with	O
the	O
relatively	O
small	O
changes	O
undergone	O
by	O
the	O
active	B-site
site	I-site
upon	O
transition	O
from	O
LdcIa	B-protein
to	O
LdcI	B-complex_assembly
-	I-complex_assembly
LARA	I-complex_assembly
.	O

Second	O
,	O
the	O
phylogenetic	B-experimental_method
analysis	I-experimental_method
clearly	O
split	O
the	O
lysine	B-protein_type
decarboxylases	I-protein_type
into	O
two	O
groups	O
(	O
Fig	O
.	O
6A	O
).	O

Inspection	O
of	O
these	O
consensus	B-evidence
sequences	I-evidence
revealed	O
important	O
differences	O
between	O
the	O
groups	O
regarding	O
charge	O
,	O
size	O
and	O
hydrophobicity	O
of	O
several	O
residues	O
precisely	O
at	O
the	O
level	O
of	O
the	O
C	O
-	O
terminal	O
β	B-structure_element
-	I-structure_element
sheet	I-structure_element
that	O
is	O
responsible	O
for	O
the	O
interaction	O
with	O
RavA	B-protein
(	O
Fig	O
.	O
6B	O
–	O
D	O
).	O

For	O
example	O
,	O
in	O
our	O
previous	O
study	O
,	O
site	B-experimental_method
-	I-experimental_method
directed	I-experimental_method
mutations	I-experimental_method
identified	O
Y697	B-residue_name_number
as	O
critically	O
required	O
for	O
the	O
RavA	B-protein
binding	O
.	O

Our	O
current	O
analysis	O
shows	O
that	O
Y697	B-residue_name_number
is	O
strictly	B-protein_state
conserved	I-protein_state
in	O
the	O
“	O
LdcI	B-protein_type
-	I-protein_type
like	I-protein_type
”	O
group	O
whereas	O
the	O
“	O
LdcC	B-protein_type
-	I-protein_type
like	I-protein_type
”	O
enzymes	O
always	B-protein_state
have	I-protein_state
a	O
lysine	B-residue_name
in	O
this	O
position	O
;	O
it	O
also	O
uncovers	O
several	O
other	O
residues	O
potentially	O
essential	O
for	O
the	O
interaction	O
with	O
RavA	B-protein
which	O
can	O
now	O
be	O
addressed	O
by	O
site	B-experimental_method
-	I-experimental_method
directed	I-experimental_method
mutagenesis	I-experimental_method
.	O

Superposition	B-experimental_method
of	O
the	O
pseudoatomic	B-evidence
models	I-evidence
of	O
LdcC	B-protein
,	O
LdcI	B-protein
from	O
LdcI	B-complex_assembly
-	I-complex_assembly
LARA	I-complex_assembly
and	O
LdcIa	B-protein
colored	O
as	O
in	O
Fig	O
.	O
1	O
,	O
and	O
the	O
crystal	B-evidence
structure	I-evidence
of	O
LdcIi	B-protein
in	O
shades	O
of	O
yellow	O
.	O

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

(	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

Sequence	B-experimental_method
alignment	I-experimental_method
suggests	O
that	O
both	O
kinases	B-protein_type
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

In	O
addition	O
,	O
our	O
enzymatic	B-experimental_method
assays	I-experimental_method
suggested	O
that	O
SePSK	B-protein
has	O
the	O
capability	O
to	O
phosphorylate	O
D	B-chemical
-	I-chemical
ribulose	I-chemical
.	O

These	O
kinases	B-protein_type
exhibit	O
considerable	O
differences	O
in	O
their	O
folding	O
pattern	O
and	O
substrate	O
specificity	O
.	O

Domain	B-structure_element
I	I-structure_element
exhibits	O
a	O
ribonuclease	B-structure_element
H	I-structure_element
-	I-structure_element
like	I-structure_element
folding	I-structure_element
pattern	I-structure_element
,	O
and	O
is	O
responsible	O
for	O
the	O
substrate	O
binding	O
,	O
while	O
domain	B-structure_element
II	I-structure_element
possesses	O
an	O
actin	B-structure_element
-	I-structure_element
like	I-structure_element
ATPase	I-structure_element
domain	I-structure_element
that	O
binds	O
cofactor	O
ATP	B-chemical
.	O

2	B-residue_range
–	I-residue_range
228	I-residue_range
and	O
aa	O
.	O

Domain	B-structure_element
II	I-structure_element
is	O
comprised	O
of	O
aa	O
.	O

229	B-residue_range
–	I-residue_range
401	I-residue_range
and	O
classified	O
into	O
B2	B-structure_element
(	O
β31	B-structure_element
/	O
β29	B-structure_element
/	O
β22	B-structure_element
/	O
β23	B-structure_element
/	O
β25	B-structure_element
/	O
β24	B-structure_element
)	O
and	O
A3	B-structure_element
(	O
α26	B-structure_element
/	O
α27	B-structure_element
/	O
α28	B-structure_element
/	O
α30	B-structure_element
)	O
(	O
Fig	O
1A	O
and	O
S1	O
Fig	O
).	O

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

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

In	O
order	O
to	O
understand	O
the	O
function	O
of	O
these	O
two	O
kinases	O
,	O
we	O
performed	O
structural	B-experimental_method
comparison	I-experimental_method
using	O
Dali	B-experimental_method
server	I-experimental_method
.	O

Both	O
SePSK	B-protein
and	O
AtXK	B-protein
-	I-protein
1	I-protein
showed	O
ATP	B-chemical
hydrolysis	O
activity	O
in	O
the	O
absence	B-protein_state
of	I-protein_state
substrate	O
.	O

This	O
result	O
was	O
consistent	O
with	O
our	O
enzymatic	B-experimental_method
activity	I-experimental_method
assays	I-experimental_method
where	O
SePSK	B-protein
and	O
AtXK	B-protein
-	I-protein
1	I-protein
showed	O
ATP	B-chemical
hydrolysis	O
activity	O
without	O
adding	O
any	O
substrates	O
(	O
Fig	O
2A	O
and	O
2C	O
).	O

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

To	O
better	O
understand	O
the	O
interaction	O
pattern	O
between	O
SePSK	B-protein
and	O
D	B-chemical
-	I-chemical
ribulose	I-chemical
,	O
the	O
apo	B-protein_state
-	O
SePSK	B-protein
crystals	B-experimental_method
were	I-experimental_method
soaked	I-experimental_method
into	I-experimental_method
the	O
reservoir	B-experimental_method
with	O
10	O
mM	O
D	B-chemical
-	I-chemical
ribulose	I-chemical
(	O
RBL	B-chemical
)	O
and	O
the	O
RBL	B-complex_assembly
-	I-complex_assembly
SePSK	I-complex_assembly
structure	B-evidence
was	O
solved	B-experimental_method
.	O

As	O
shown	O
in	O
Fig	O
4A	O
,	O
the	O
nearest	O
distance	O
between	O
the	O
carbon	O
skeleton	O
of	O
two	O
D	B-chemical
-	I-chemical
ribulose	I-chemical
molecules	O
are	O
approx	O
.	O

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

(	O
A	O
)	O
The	O
electrostatic	B-evidence
potential	I-evidence
surface	I-evidence
map	I-evidence
of	O
RBL	B-complex_assembly
-	I-complex_assembly
SePSK	I-complex_assembly
and	O
a	O
zoom	O
-	O
in	O
view	O
of	O
RBL	B-site
binding	I-site
site	I-site
.	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

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

To	O
further	O
verified	O
this	O
result	O
,	O
we	O
measured	O
the	O
binding	B-evidence
affinity	I-evidence
for	O
D	B-chemical
-	I-chemical
ribulose	I-chemical
of	O
both	O
wild	B-protein_state
type	I-protein_state
(	O
WT	B-protein_state
)	O
and	O
D8A	B-mutant
mutant	B-protein_state
of	O
SePSK	B-protein
using	O
a	O
surface	B-experimental_method
plasmon	I-experimental_method
resonance	I-experimental_method
method	I-experimental_method
.	O

Dissociation	B-evidence
rate	I-evidence
constant	I-evidence
(	O
Kd	B-evidence
)	O
of	O
wild	B-protein_state
type	I-protein_state
and	O
D8A	B-mutant
-	O
SePSK	B-protein
are	O
3	O
ms	O
-	O
1	O
and	O
9	O
ms	O
-	O
1	O
,	O
respectively	O
.	O

Simulated	O
conformational	O
change	O
of	O
SePSK	B-protein
during	O
the	O
catalytic	O
process	O
.	O

The	O
crystal	B-evidence
structure	I-evidence
of	O
phosphorylation	B-protein_state
-	I-protein_state
mimicking	I-protein_state
Mep2	B-mutant
variants	I-mutant
from	O
C	B-species
.	I-species
albicans	I-species
show	O
large	O
conformational	O
changes	O
in	O
a	O
conserved	B-protein_state
and	O
functionally	O
important	O
region	O
of	O
the	O
CTR	B-structure_element
.	O

Mep2	B-protein_type
proteins	I-protein_type
are	O
tightly	O
regulated	O
fungal	B-taxonomy_domain
ammonium	B-protein_type
transporters	I-protein_type
.	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

With	O
the	O
exception	O
of	O
the	O
human	B-species
RhCG	B-protein
structure	B-evidence
,	O
no	O
structural	O
information	O
is	O
available	O
for	O
eukaryotic	B-taxonomy_domain
ammonium	B-protein_type
transporters	I-protein_type
.	O

Ammonium	B-chemical
transport	O
is	O
tightly	O
regulated	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

In	O
plants	B-taxonomy_domain
,	O
transporter	B-protein_type
phosphorylation	B-ptm
and	O
dephosphorylation	B-ptm
are	O
known	O
to	O
regulate	O
activity	O
.	O

In	O
S	B-species
.	I-species
cerevisiae	I-species
,	O
phosphorylation	B-ptm
of	O
Ser457	B-residue_name_number
within	O
the	O
C	B-structure_element
-	I-structure_element
terminal	I-structure_element
region	I-structure_element
(	O
CTR	B-structure_element
)	O
in	O
the	O
cytoplasm	O
was	O
recently	O
proposed	O
to	O
cause	O
Mep2	B-protein_type
opening	O
,	O
possibly	O
via	O
inducing	O
a	O
conformational	O
change	O
.	O

The	O
most	O
striking	O
difference	O
is	O
the	O
fact	O
that	O
the	O
Mep2	B-protein_type
proteins	I-protein_type
have	O
closed	B-protein_state
conformations	O
.	O

Electron	B-evidence
density	I-evidence
is	O
visible	O
for	O
the	O
entire	O
polypeptide	O
chains	O
,	O
with	O
the	O
exception	O
of	O
the	O
C	O
-	O
terminal	O
43	B-residue_range
(	O
ScMep2	B-protein
)	O
and	O
25	B-residue_range
residues	O
(	O
CaMep2	B-protein
),	O
which	O
are	O
poorly	B-protein_state
conserved	I-protein_state
and	O
presumably	O
disordered	B-protein_state
.	O

Together	O
with	O
additional	O
,	O
smaller	O
differences	O
in	O
other	O
extracellular	B-structure_element
loops	I-structure_element
,	O
these	O
changes	O
generate	O
a	O
distinct	O
vestibule	B-structure_element
leading	O
to	O
the	O
ammonium	B-site
binding	I-site
site	I-site
that	O
is	O
much	O
more	O
pronounced	O
than	O
in	O
the	O
bacterial	B-taxonomy_domain
proteins	O
.	O

The	O
largest	O
backbone	O
movements	O
of	O
equivalent	O
residues	O
within	O
ICL1	B-structure_element
are	O
∼	O
10	O
Å	O
,	O
markedly	O
affecting	O
the	O
conserved	B-protein_state
basic	B-protein_state
RxK	B-structure_element
motif	I-structure_element
(	O
Fig	O
.	O
4	O
).	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

Compared	O
with	O
ICL1	B-structure_element
,	O
the	O
backbone	O
conformational	O
changes	O
observed	O
for	O
the	O
neighbouring	O
ICL2	B-structure_element
are	O
smaller	O
,	O
but	O
large	O
shifts	O
are	O
nevertheless	O
observed	O
for	O
the	O
conserved	B-protein_state
residues	O
Glu140	B-residue_name_number
and	O
Arg141	B-residue_name_number
(	O
Fig	O
.	O
4	O
).	O

The	O
closed	B-protein_state
state	O
of	O
the	O
channel	B-site
might	O
also	O
explain	O
why	O
no	B-evidence
density	I-evidence
,	O
which	O
could	O
correspond	O
to	O
ammonium	B-chemical
(	O
or	O
water	B-chemical
),	O
is	O
observed	O
in	O
the	O
hydrophobic	O
part	O
of	O
the	O
Mep2	B-protein
channel	B-site
close	O
to	O
the	O
twin	B-structure_element
-	I-structure_element
His	I-structure_element
motif	I-structure_element
.	O

The	O
final	O
region	O
in	O
Mep2	B-protein
that	O
shows	O
large	O
differences	O
compared	O
with	O
the	O
bacterial	B-taxonomy_domain
transporters	B-protein_type
is	O
the	O
CTR	B-structure_element
.	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

This	O
is	O
illustrated	O
by	O
the	O
positions	O
of	O
the	O
five	O
universally	B-protein_state
conserved	I-protein_state
residues	O
within	O
the	O
CTR	B-structure_element
,	O
that	O
is	O
,	O
Arg415	B-residue_name_number
(	O
370	B-residue_number
),	O
Glu421	B-residue_name_number
(	O
376	B-residue_number
),	O
Gly424	B-residue_name_number
(	O
379	B-residue_number
),	O
Asp426	B-residue_name_number
(	O
381	B-residue_number
)	O
and	O
Tyr	B-residue_name_number
435	I-residue_name_number
(	O
390	B-residue_number
)	O
in	O
CaMep2	B-protein
(	O
Amt	B-protein
-	I-protein
1	I-protein
)	O
(	O
Fig	O
.	O
2	O
).	O

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

In	O
the	O
Mep2	B-protein
structures	B-evidence
,	O
none	O
of	O
the	O
interactions	O
mentioned	O
above	O
are	O
present	O
.	O

In	O
the	O
absence	B-protein_state
of	I-protein_state
Npr1	B-protein
,	O
plasmid	B-experimental_method
-	I-experimental_method
encoded	I-experimental_method
WT	B-protein_state
Mep2	B-protein
in	O
a	O
S	B-species
.	I-species
cerevisiae	I-species
mep1	B-mutant
-	I-mutant
3Δ	I-mutant
strain	O
(	O
triple	B-mutant
mepΔ	I-mutant
)	O
does	O
not	O
allow	O
growth	O
on	O
low	O
concentrations	O
of	O
ammonium	B-chemical
,	O
suggesting	O
that	O
the	O
transporter	B-protein_type
is	O
inactive	B-protein_state
(	O
Fig	O
.	O
3	O
and	O
Supplementary	O
Fig	O
.	O
1	O
).	O

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

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

In	O
CaMep2	B-protein
,	O
the	O
visible	O
part	O
of	O
the	O
sequence	O
extends	O
for	O
two	O
residues	O
beyond	O
Ser453	B-residue_name_number
(	O
Fig	O
.	O
6	O
).	O

Boeckstaens	O
et	O
al	O
.	O
proposed	O
that	O
phosphorylation	B-ptm
does	O
not	O
affect	O
channel	O
activity	O
directly	O
,	O
but	O
instead	O
relieves	O
inhibition	O
by	O
the	O
AI	B-structure_element
region	I-structure_element
.	O

The	O
first	O
one	O
is	O
that	O
the	O
open	B-protein_state
state	O
is	O
disfavoured	O
by	O
crystallization	B-experimental_method
because	O
of	O
lower	O
stability	O
or	O
due	O
to	O
crystal	O
packing	O
constraints	O
.	O

The	O
side	O
-	O
chain	O
hydroxyl	O
of	O
Ser457	B-residue_name_number
/	O
453	B-residue_number
is	O
located	O
in	O
a	O
well	O
-	O
defined	O
electronegative	B-site
pocket	I-site
that	O
is	O
solvent	B-protein_state
accessible	I-protein_state
(	O
Fig	O
.	O
6	O
).	O

In	O
the	O
WT	B-protein_state
structure	B-evidence
,	O
the	O
acidic	O
residues	O
Asp419	B-residue_name_number
,	O
Glu420	B-residue_name_number
and	O
Glu421	B-residue_name_number
are	O
within	O
hydrogen	O
bonding	O
distance	O
of	O
Ser453	B-residue_name_number
.	O

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

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

These	O
efforts	O
have	O
advanced	O
our	O
knowledge	O
considerably	O
but	O
have	O
not	O
yet	O
yielded	O
atomic	O
-	O
level	O
answers	O
to	O
several	O
important	O
mechanistic	O
questions	O
,	O
including	O
how	O
ammonium	B-chemical
transport	O
is	O
regulated	O
in	O
eukaryotes	B-taxonomy_domain
and	O
the	O
mechanism	O
of	O
ammonium	B-chemical
signalling	O
.	O

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

Interestingly	O
,	O
phosphomimetic	B-mutant
mutations	I-mutant
introduced	O
into	O
one	O
monomer	B-oligomeric_state
inactivate	O
the	O
entire	O
trimer	B-oligomeric_state
,	O
indicating	O
that	O
(	O
i	O
)	O
heterotrimerization	O
occurs	O
and	O
(	O
ii	O
)	O
the	O
CTR	B-structure_element
mediates	O
allosteric	O
regulation	O
of	O
ammonium	B-chemical
transport	O
activity	O
via	O
phosphorylation	B-ptm
.	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

How	O
exactly	O
the	O
channel	B-site
opens	O
and	O
whether	O
opening	O
is	O
intra	O
-	O
monomeric	O
are	O
still	O
open	B-protein_state
questions	O
;	O
it	O
is	O
possible	O
that	O
the	O
change	O
in	O
the	O
CTR	B-structure_element
may	O
disrupt	O
its	O
interactions	O
with	O
ICL3	B-structure_element
of	O
the	O
neighbouring	O
monomer	B-oligomeric_state
(	O
Fig	O
.	O
9b	O
),	O
which	O
could	O
result	O
in	O
opening	O
of	O
the	O
neighbouring	O
channel	B-site
via	O
inward	O
movement	O
of	O
its	O
ICL3	B-structure_element
.	O

Is	O
our	O
model	O
for	O
opening	O
and	O
closing	O
of	O
Mep2	B-protein
channels	B-site
valid	O
for	O
other	O
eukaryotic	B-taxonomy_domain
ammonium	B-protein_type
transporters	I-protein_type
?	O
Our	O
structural	B-evidence
data	I-evidence
support	O
previous	O
studies	O
and	O
clarify	O
the	O
central	O
role	O
of	O
the	O
CTR	B-structure_element
and	O
cytoplasmic	B-structure_element
loops	I-structure_element
in	O
the	O
transition	O
between	O
closed	B-protein_state
and	O
open	B-protein_state
states	O
.	O

There	O
is	O
generally	O
no	O
equivalent	O
for	O
CaMep2	B-protein
Tyr49	B-residue_name_number
in	O
plant	B-taxonomy_domain
AMTs	B-protein_type
,	O
indicating	O
that	O
a	O
Tyr	B-site
–	I-site
His2	I-site
hydrogen	I-site
bond	I-site
as	O
observed	O
in	O
Mep2	B-protein
may	O
not	O
contribute	O
to	O
the	O
closed	B-protein_state
state	O
in	O
plant	B-taxonomy_domain
transporters	B-protein_type
.	O

The	O
need	O
to	O
regulate	O
in	O
opposite	O
ways	O
may	O
be	O
the	O
reason	O
why	O
the	O
phosphorylation	B-site
sites	I-site
are	O
in	O
different	O
parts	O
of	O
the	O
CTR	B-structure_element
,	O
that	O
is	O
,	O
centrally	O
located	O
close	O
to	O
the	O
ExxGxD	B-structure_element
motif	I-structure_element
in	O
AMTs	B-protein_type
and	O
peripherally	O
in	O
Mep2	B-protein
.	O

With	O
respect	O
to	O
ammonium	B-chemical
transport	O
,	O
phosphorylation	B-ptm
has	O
thus	O
far	O
only	O
been	O
shown	O
for	O
A	B-species
.	I-species
thaliana	I-species
AMTs	B-protein_type
and	O
for	O
S	B-species
.	I-species
cerevisiae	I-species
Mep2	B-protein
(	O
refs	O
).	O

In	O
one	O
model	O
,	O
signalling	O
is	O
proposed	O
to	O
depend	O
on	O
the	O
nature	O
of	O
the	O
transported	O
substrate	O
,	O
which	O
might	O
be	O
different	O
in	O
certain	O
subfamilies	O
of	O
ammonium	B-protein_type
transporters	I-protein_type
(	O
for	O
example	O
,	O
Mep1	B-protein
/	O
Mep3	B-protein
versus	O
Mep2	B-protein
).	O

In	O
the	O
other	O
model	O
,	O
signalling	O
is	O
thought	O
to	O
require	O
a	O
distinct	O
conformation	O
of	O
the	O
Mep2	B-protein
transporter	B-protein_type
occurring	O
during	O
the	O
transport	O
cycle	O
.	O

The	O
region	O
showing	O
ICL1	B-structure_element
(	O
blue	O
),	O
ICL3	B-structure_element
(	O
green	O
)	O
and	O
the	O
CTR	B-structure_element
(	O
red	O
)	O
is	O
boxed	O
for	O
comparison	O
.	O

(	O
a	O
)	O
ICL1	B-structure_element
in	O
AfAmt	B-protein
-	I-protein
1	I-protein
(	O
light	O
blue	O
)	O
and	O
CaMep2	B-protein
(	O
dark	O
blue	O
),	O
showing	O
unwinding	O
and	O
inward	O
movement	O
in	O
the	O
fungal	B-taxonomy_domain
protein	O
.	O
(	O
b	O
)	O
Stereo	O
diagram	O
viewed	O
from	O
the	O
cytosol	O
of	O
ICL1	B-structure_element
,	O
ICL3	B-structure_element
(	O
green	O
)	O
and	O
the	O
CTR	B-structure_element
(	O
red	O
)	O
in	O
AfAmt	B-protein
-	I-protein
1	I-protein
(	O
light	O
colours	O
)	O
and	O
CaMep2	B-protein
(	O
dark	O
colours	O
).	O

The	O
labelled	O
residues	O
are	O
analogous	O
within	O
both	O
structures	B-evidence
.	O

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

The	O
Npr1	B-protein
kinase	B-protein_type
target	O
Ser453	B-residue_name_number
is	O
dephosphorylated	B-protein_state
and	O
located	O
in	O
an	O
electronegative	B-site
pocket	I-site
.	O

(	O
a	O
)	O
Stereoviews	O
of	O
CaMep2	B-protein
showing	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
CTR	B-structure_element
residues	O
Asp419	B-residue_range
-	I-residue_range
Met422	I-residue_range
and	O
for	O
Tyr446	B-residue_range
-	I-residue_range
Thr455	I-residue_range
of	O
the	O
AI	B-structure_element
region	I-structure_element
.	O

Phosphorylation	B-ptm
causes	O
conformational	O
changes	O
in	O
the	O
CTR	B-structure_element
.	O

(	O
a	O
)	O
In	O
the	O
closed	B-protein_state
,	O
non	B-protein_state
-	I-protein_state
phosphorylated	I-protein_state
state	O
(	O
i	O
),	O
the	O
CTR	B-structure_element
(	O
magenta	O
)	O
and	O
ICL3	B-structure_element
(	O
green	O
)	O
are	O
far	O
apart	O
with	O
the	O
latter	O
blocking	O
the	O
intracellular	O
channel	B-site
exit	I-site
(	O
indicated	O
with	O
a	O
hatched	O
circle	O
).	O

Visualizing	O
chaperone	B-protein_type
-	O
assisted	O
protein	O
folding	O

READ	B-experimental_method
enabled	O
us	O
to	O
visualize	O
even	O
sparsely	O
populated	O
conformations	O
of	O
the	O
substrate	O
protein	O
immunity	B-protein
protein	I-protein
7	I-protein
(	O
Im7	B-protein
)	O
in	B-protein_state
complex	I-protein_state
with	I-protein_state
the	O
E	B-species
.	I-species
coli	I-species
chaperone	B-protein_type
Spy	B-protein
.	O

It	O
is	O
clear	O
that	O
molecular	O
chaperones	B-protein_type
aid	O
in	O
protein	O
folding	O
.	O

Structural	B-evidence
models	I-evidence
of	O
chaperone	B-protein_type
-	O
substrate	O
complexes	O
have	O
recently	O
begun	O
to	O
provide	O
information	O
as	O
to	O
how	O
a	O
chaperone	B-protein_type
can	O
recognize	O
its	O
substrate	O
.	O

For	O
most	O
chaperones	B-protein_type
,	O
it	O
is	O
still	O
unclear	O
whether	O
the	O
chaperone	B-protein_type
actively	O
participates	O
in	O
and	O
affects	O
the	O
folding	O
of	O
the	O
substrate	O
proteins	O
,	O
or	O
merely	O
provides	O
a	O
suitable	O
microenvironment	O
enabling	O
the	O
substrate	O
to	O
fold	O
on	O
its	O
own	O
.	O

We	O
therefore	O
screened	B-experimental_method
crystallization	B-experimental_method
conditions	I-experimental_method
for	O
Spy	B-protein
with	O
four	O
different	O
substrate	O
proteins	O
:	O
a	O
fragment	O
of	O
the	O
largely	O
unfolded	B-protein_state
bovine	B-taxonomy_domain
α	B-chemical
-	I-chemical
casein	I-chemical
protein	O
,	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
(	O
WT	B-protein_state
)	O
E	B-species
.	I-species
coli	I-species
Im7	B-protein
,	O
an	O
unfolded	B-protein_state
variant	O
of	O
Im7	B-protein
(	O
L18A	B-mutant
L19A	B-mutant
L37A	B-mutant
),	O
and	O
the	O
N	B-structure_element
-	I-structure_element
terminal	I-structure_element
half	I-structure_element
of	O
Im7	B-protein
(	O
Im76	B-mutant
-	I-mutant
45	I-mutant
),	O
which	O
encompasses	O
the	O
entire	O
Spy	B-structure_element
-	I-structure_element
binding	I-structure_element
portion	I-structure_element
of	O
Im7	B-protein
.	O

Subsequent	O
crystal	B-experimental_method
washing	I-experimental_method
and	I-experimental_method
dissolution	I-experimental_method
experiments	O
confirmed	O
the	O
presence	O
of	O
the	O
substrates	O
in	O
the	O
co	B-experimental_method
-	I-experimental_method
crystals	I-experimental_method
(	O
Supplementary	O
Fig	O
.	O
2	O
).	O

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

The	O
READ	B-experimental_method
sample	B-experimental_method
-	I-experimental_method
and	I-experimental_method
-	I-experimental_method
select	I-experimental_method
algorithm	I-experimental_method
is	O
diagrammed	O
in	O
Fig	O
.	O
2	O
.	O

To	O
make	O
the	O
electron	B-experimental_method
density	I-experimental_method
selection	I-experimental_method
practical	O
,	O
we	O
needed	O
to	O
develop	O
a	O
method	O
to	O
rapidly	O
evaluate	O
the	O
agreement	O
between	O
the	O
selected	O
sub	O
-	O
ensembles	O
and	O
the	O
experimental	O
electron	B-evidence
density	I-evidence
on	O
-	O
the	O
-	O
fly	O
during	O
the	O
selection	O
procedure	O
.	O

To	O
reduce	O
the	O
extent	O
of	O
3D	O
space	O
to	O
be	O
explored	O
,	O
this	O
compressed	O
map	B-evidence
was	O
created	O
by	O
only	O
using	O
density	B-evidence
from	O
regions	O
of	O
space	O
significantly	O
sampled	O
by	O
Im76	B-mutant
-	I-mutant
45	I-mutant
in	O
the	O
Spy	B-complex_assembly
:	I-complex_assembly
Im76	I-complex_assembly
-	I-complex_assembly
45	I-complex_assembly
MD	B-experimental_method
simulations	B-experimental_method
.	O

We	O
were	O
particularly	O
interested	O
in	O
finding	O
answers	O
to	O
one	O
of	O
the	O
most	O
fundamental	O
questions	O
in	O
chaperone	B-protein_type
biology	O
—	O
how	O
does	O
chaperone	B-protein_type
binding	O
affect	O
substrate	O
structure	O
and	O
vice	O
versa	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

This	O
twist	O
yields	O
asymmetry	O
and	O
results	O
in	O
substantially	O
different	O
interaction	O
patterns	O
in	O
the	O
two	O
Spy	B-protein
monomers	B-oligomeric_state
(	O
Fig	O
.	O
4b	O
).	O

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

Importantly	O
,	O
we	O
observed	O
the	O
same	O
structural	O
changes	O
in	O
Spy	B-protein
regardless	O
of	O
which	O
of	O
the	O
four	O
substrates	O
was	O
bound	O
(	O
Fig	O
.	O
5b	O
,	O
Table	O
1	O
).	O

We	O
recently	O
showed	O
that	O
Im7	B-protein
can	O
fold	O
while	O
remaining	O
continuously	B-protein_state
bound	I-protein_state
to	I-protein_state
Spy	B-protein
.	O

This	O
model	O
is	O
consistent	O
with	O
previous	O
studies	O
postulating	O
that	O
the	O
flexible	O
binding	O
of	O
chaperones	B-protein_type
allows	O
for	O
substrate	O
protein	O
folding	O
.	O

The	O
amphipathic	O
concave	B-site
surface	I-site
of	O
Spy	B-protein
likely	O
facilitates	O
this	O
flexible	O
binding	O
and	O
may	O
be	O
a	O
crucial	O
feature	O
for	O
Spy	B-protein
and	O
potentially	O
other	O
chaperones	B-protein_type
,	O
allowing	O
them	O
to	O
bind	O
multiple	O
conformations	O
of	O
many	O
different	O
substrates	O
.	O

The	O
negatively	O
charged	O
Im7	B-protein
residues	O
Glu21	B-residue_name_number
,	O
Asp32	B-residue_name_number
,	O
and	O
Asp35	B-residue_name_number
reside	O
on	O
the	O
surface	O
of	O
Im7	B-protein
and	O
form	O
interactions	O
with	O
Spy	B-protein
’	O
s	O
positively	O
charged	O
cradle	B-site
in	O
both	O
the	O
unfolded	B-protein_state
and	O
native	B-protein_state
-	I-protein_state
like	I-protein_state
states	O
.	O

This	O
selection	O
resulted	O
in	O
“	O
Super	O
Spy	B-protein
”	O
variants	B-protein_state
that	O
were	O
more	O
effective	O
at	O
both	O
preventing	O
aggregation	O
and	O
promoting	O
protein	O
folding	O
.	O

By	O
sampling	O
multiple	O
conformations	O
,	O
this	O
linker	B-structure_element
region	I-structure_element
may	O
allow	O
diverse	O
substrate	O
conformations	O
to	O
be	O
accommodated	O
.	O

Overall	O
,	O
comparison	O
of	O
our	O
ensemble	B-evidence
to	O
the	O
Super	O
Spy	B-protein
variants	B-protein_state
provides	O
specific	O
examples	O
to	O
corroborate	O
the	O
importance	O
of	O
conformational	O
flexibility	O
in	O
chaperone	B-protein_type
-	O
substrate	O
interactions	O
.	O

ATP	B-chemical
and	O
co	O
-	O
chaperone	B-protein_type
dependencies	O
may	O
have	O
emerged	O
later	O
through	O
evolution	O
to	O
better	O
modulate	O
and	O
control	O
chaperone	B-protein_type
action	O
.	O

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

The	O
Super	O
Spy	B-protein
mutants	O
F115L	B-mutant
,	O
F115I	B-mutant
,	O
and	O
L32P	B-mutant
are	O
proposed	O
to	O
gain	O
activity	O
by	O
increasing	O
the	O
flexibility	O
or	O
size	O
of	O
this	O
linker	B-structure_element
region	I-structure_element
.	O

To	O
support	O
antibody	B-protein_type
therapeutic	O
development	O
,	O
the	O
crystal	B-evidence
structures	I-evidence
of	O
a	O
set	O
of	O
16	O
germline	O
variants	O
composed	O
of	O
4	O
different	O
kappa	B-structure_element
light	I-structure_element
chains	I-structure_element
paired	O
with	O
4	O
different	O
heavy	B-structure_element
chains	I-structure_element
have	O
been	O
determined	O
.	O

The	O
longer	B-protein_state
CDRs	B-structure_element
with	O
tandem	O
glycines	B-residue_name
or	O
serines	B-residue_name
have	O
more	O
conformational	O
diversity	O
than	O
the	O
others	O
.	O

Two	O
of	O
16	O
structures	B-evidence
showed	O
particularly	O
large	O
variations	O
in	O
the	O
tilt	B-evidence
angles	I-evidence
when	O
compared	O
with	O
the	O
other	O
pairings	O
.	O

These	O
domains	O
have	O
a	O
common	O
folding	O
pattern	O
often	O
referred	O
to	O
as	O
the	O
“	O
immunoglobulin	B-structure_element
fold	I-structure_element
,”	O
formed	O
by	O
the	O
packing	O
together	O
of	O
2	O
anti	B-structure_element
-	I-structure_element
parallel	I-structure_element
β	I-structure_element
-	I-structure_element
sheets	I-structure_element
.	O

All	O
immunoglobulin	B-protein_type
chains	I-protein_type
have	O
an	O
N	O
-	O
terminal	O
V	B-structure_element
domain	I-structure_element
followed	O
by	O
1	O
to	O
4	O
C	B-structure_element
domains	I-structure_element
,	O
depending	O
upon	O
the	O
chain	O
type	O
.	O

The	O
cataloging	O
and	O
development	O
of	O
the	O
rules	O
for	O
predicting	O
the	O
conformation	O
of	O
the	O
anchor	B-structure_element
region	I-structure_element
of	O
CDR	B-structure_element
H3	B-structure_element
continue	O
to	O
be	O
refined	O
,	O
producing	O
new	O
insight	O
into	O
the	O
CDR	B-structure_element
H3	B-structure_element
conformations	O
and	O
new	O
tools	O
for	O
antibody	B-protein_type
engineering	O
.	O

One	O
important	O
finding	O
of	O
the	O
antibody	B-experimental_method
modeling	I-experimental_method
assessments	I-experimental_method
was	O
that	O
errors	O
in	O
the	O
structural	O
templates	O
that	O
are	O
used	O
as	O
the	O
basis	O
for	O
homology	B-experimental_method
models	I-experimental_method
can	O
propagate	O
into	O
the	O
final	O
models	O
,	O
producing	O
inaccuracies	O
that	O
may	O
negatively	O
influence	O
the	O
predictive	O
nature	O
of	O
the	O
V	B-structure_element
region	I-structure_element
model	O
.	O

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

The	O
similarity	O
in	O
the	O
crystal	B-evidence
forms	I-evidence
is	O
attributed	O
in	O
part	O
to	O
cross	O
-	O
seeding	O
using	O
the	O
microseed	B-experimental_method
matrix	I-experimental_method
screening	I-experimental_method
for	O
groups	O
2	O
and	O
3	O
.	O

The	O
number	O
of	O
Fab	B-structure_element
molecules	O
in	O
the	O
crystallographic	O
asymmetric	O
unit	O
varies	O
from	O
1	O
(	O
for	O
12	O
Fabs	B-structure_element
)	O
to	O
2	O
(	O
for	O
4	O
Fabs	B-structure_element
).	O

For	O
the	O
LC	B-structure_element
,	O
the	O
disorder	B-protein_state
is	O
observed	O
at	O
2	O
of	O
the	O
C	O
-	O
terminal	O
residues	O
with	O
few	O
exceptions	O
.	O

CDR	B-structure_element
H1	B-structure_element

The	O
canonical	O
structures	O
of	O
CDR	B-structure_element
H2	B-structure_element
have	O
fairly	O
consistent	O
conformations	O
(	O
Table	O
2	O
,	O
Fig	O
.	O
2	O
).	O

In	O
one	O
case	O
,	O
in	O
the	O
second	O
Fab	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
,	O
CDR	B-structure_element
H2	B-structure_element
is	O
partially	B-protein_state
disordered	I-protein_state
(	O
Δ55	B-mutant
-	I-mutant
60	I-mutant
).	O

Despite	O
this	O
,	O
the	O
conformations	O
are	O
tightly	O
clustered	O
(	O
rmsd	B-evidence
is	O
0	O
.	O
20	O
Å	O
).	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
CDR	B-structure_element
L2	B-structure_element
conformations	O
for	O
each	O
of	O
the	O
LCs	B-structure_element
paired	O
with	O
the	O
4	O
HCs	B-structure_element
are	O
clustered	O
more	O
tightly	O
than	O
any	O
of	O
the	O
other	O
CDRs	B-structure_element
(	O
rmsd	B-evidence
values	O
are	O
in	O
the	O
range	O
0	O
.	O
09	O
-	O
0	O
.	O
16	O
Å	O
),	O
and	O
all	O
4	O
sets	O
have	O
virtually	O
the	O
same	O
conformation	O
despite	O
the	O
sequence	O
diversity	O
of	O
the	O
loop	B-structure_element
.	O

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

An	O
interesting	O
feature	O
of	O
these	O
CDR	B-structure_element
H3	B-structure_element
structures	B-evidence
is	O
the	O
presence	O
of	O
a	O
water	B-chemical
molecule	O
that	O
interacts	O
with	O
the	O
peptide	O
nitrogens	O
and	O
carbonyl	O
oxygens	O
near	O
the	O
bridging	O
loop	B-structure_element
connecting	O
the	O
2	O
β	B-structure_element
-	I-structure_element
strands	I-structure_element
.	O

A	O
representative	O
CDR	B-structure_element
H3	B-structure_element
structure	B-evidence
for	O
H1	B-complex_assembly
-	I-complex_assembly
69	I-complex_assembly
:	I-complex_assembly
L1	I-complex_assembly
-	I-complex_assembly
39	I-complex_assembly
illustrating	O
this	O
is	O
shown	O
in	O
Fig	O
.	O
7A	O
.	O

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

The	O
stem	B-structure_element
regions	I-structure_element
of	O
CDR	B-structure_element
H3	B-structure_element
for	O
the	O
H5	B-complex_assembly
-	I-complex_assembly
51	I-complex_assembly
:	I-complex_assembly
L4	I-complex_assembly
-	I-complex_assembly
1	I-complex_assembly
Fabs	B-structure_element
are	O
in	O
the	O
‘	O
kinked	B-protein_state
’	O
conformation	O
while	O
,	O
surprisingly	O
,	O
those	O
of	O
the	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
pair	O
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
are	O
in	O
the	O
‘	O
extended	B-protein_state
’	O
conformation	O
(	O
Fig	O
.	O
7B	O
).	O

The	O
two	O
domains	O
pack	O
together	O
such	O
that	O
the	O
5	B-structure_element
-	I-structure_element
stranded	I-structure_element
β	I-structure_element
-	I-structure_element
sheets	I-structure_element
,	O
which	O
have	O
hydrophobic	O
surfaces	O
,	O
interact	O
with	O
each	O
other	O
bringing	O
the	O
CDRs	B-structure_element
from	O
both	O
the	O
VH	B-structure_element
and	O
VL	B-structure_element
domains	O
into	O
close	O
proximity	O
.	O

VH	B-site
:	I-site
VL	I-site
interface	I-site
amino	O
acid	O
residue	O
interactions	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	O
interaction	O
with	O
H	B-structure_element
-	O
Tyr91	B-residue_name_number
is	O
preserved	O
.	O

These	O
core	O
interactions	O
provide	O
enough	O
stability	O
to	O
the	O
VH	B-complex_assembly
:	I-complex_assembly
VL	I-complex_assembly
dimer	B-oligomeric_state
so	O
that	O
additional	O
VH	B-site
-	I-site
VL	I-site
contacts	I-site
can	O
tolerate	O
amino	O
acid	O
sequence	O
variations	O
in	O
CDRs	B-structure_element
H3	B-structure_element
and	O
L3	B-structure_element
that	O
form	O
part	O
of	O
the	O
VH	B-site
:	I-site
VL	I-site
interface	I-site
.	O

One	O
notable	O
exception	O
is	O
H	B-structure_element
-	O
Trp47	B-residue_name_number
,	O
which	O
exhibits	O
2	O
conformations	O
of	O
the	O
indole	O
ring	O
.	O

Apparently	O
,	O
residues	O
flanking	O
CDR	B-structure_element
H3	B-structure_element
in	O
the	O
2	O
VH	B-complex_assembly
:	I-complex_assembly
VL	I-complex_assembly
pairings	O
are	O
inconsistent	O
with	O
any	O
stable	B-protein_state
conformation	O
of	O
CDR	B-structure_element
H3	B-structure_element
,	O
which	O
translates	O
into	O
a	O
less	O
restricted	O
conformational	O
space	O
for	O
some	O
of	O
them	O
,	O
including	O
H	B-structure_element
-	O
Trp47	B-residue_name_number
.	O

Differences	O
in	O
VH	B-complex_assembly
:	I-complex_assembly
VL	I-complex_assembly
tilt	B-evidence
angles	I-evidence
.	O

The	O
differences	B-evidence
in	O
the	O
tilt	B-evidence
angle	I-evidence
are	O
shown	O
for	O
all	O
pairs	O
of	O
V	B-structure_element
regions	I-structure_element
in	O
Table	O
3	O
.	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

Interestingly	O
,	O
the	O
2	O
structures	B-evidence
that	O
have	O
the	O
largest	O
tilt	B-evidence
angle	I-evidence
differences	I-evidence
with	O
the	O
other	O
variants	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
and	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
have	O
the	O
smallest	O
VH	B-site
:	I-site
VL	I-site
interfaces	I-site
,	O
684	O
and	O
725	O
Å2	O
,	O
respectively	O
.	O

It	O
appears	O
that	O
for	O
each	O
given	O
LC	B-structure_element
,	O
the	O
Fabs	B-structure_element
with	O
germlines	O
H1	B-mutant
-	I-mutant
69	I-mutant
and	O
H3	B-mutant
-	I-mutant
23	I-mutant
are	O
substantially	O
more	O
stable	B-protein_state
than	O
those	O
with	O
germlines	O
H3	B-mutant
-	I-mutant
53	I-mutant
and	O
H5	B-mutant
-	I-mutant
51	I-mutant
.	O

Parts	O
of	O
CDR	B-structure_element
H3	B-structure_element
main	O
chain	O
are	O
completely	O
disordered	B-protein_state
,	O
and	O
were	O
not	O
modeled	O
in	O
Fabs	B-structure_element
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
and	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
that	O
have	O
the	O
lowest	O
Tms	B-evidence
in	O
the	O
set	O
.	O

All	O
those	O
molecules	O
are	O
relatively	O
unstable	O
,	O
as	O
is	O
reflected	O
in	O
their	O
low	O
Tms	B-evidence
.	O

Of	O
the	O
4	O
HCs	B-structure_element
,	O
H1	B-mutant
-	I-mutant
69	I-mutant
has	O
the	O
greatest	O
number	O
of	O
canonical	O
structure	O
assignments	O
(	O
Table	O
2	O
).	O

The	O
remaining	O
8	O
structures	B-evidence
exhibit	O
“	O
non	O
-	O
parental	O
”	O
conformations	O
,	O
indicating	O
that	O
the	O
VH	B-structure_element
and	O
VL	B-structure_element
context	O
can	O
also	O
be	O
a	O
dominating	O
factor	O
influencing	O
CDR	B-structure_element
H3	B-structure_element
.	O

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

One	O
of	O
the	O
variants	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
the	O
CDR	B-structure_element
H3	B-structure_element
conformation	O
similar	O
to	O
the	O
parent	O
,	O
but	O
the	O
other	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
is	O
different	O
.	O

These	O
differences	O
undoubtedly	O
influence	O
the	O
conformation	O
of	O
the	O
CDRs	B-structure_element
,	O
in	O
particular	O
CDR	B-structure_element
H1	B-structure_element
(	O
Fig	O
.	O
1A	O
)	O
and	O
CDR	B-structure_element
L1	B-structure_element
(	O
Fig	O
.	O
3C	O
),	O
especially	O
with	O
the	O
tandem	O
glycines	B-residue_name
and	O
multiple	O
serines	B-residue_name
present	O
,	O
respectively	O
.	O

Pairing	O
of	O
different	O
germlines	O
yields	O
antibodies	B-protein_type
with	O
various	O
degrees	O
of	O
stability	O
.	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

The	O
set	O
of	O
16	O
germline	O
Fab	B-structure_element
structures	B-evidence
offers	O
a	O
unique	O
dataset	O
to	O
facilitate	O
software	O
development	O
for	O
antibody	B-protein_type
modeling	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

The	O
U2AF65	B-protein
linker	B-structure_element
residues	O
between	O
the	O
dual	O
RNA	B-structure_element
recognition	I-structure_element
motifs	I-structure_element
(	O
RRMs	B-structure_element
)	O
recognize	O
the	O
central	O
nucleotide	B-chemical
,	O
whereas	O
the	O
N	O
-	O
and	O
C	O
-	O
terminal	O
RRM	B-structure_element
extensions	I-structure_element
recognize	O
the	O
3	B-site
′	I-site
terminus	I-site
and	O
third	B-residue_number
nucleotide	B-chemical
.	O

The	O
splice	B-site
sites	I-site
are	O
marked	O
by	O
relatively	O
short	B-structure_element
consensus	I-structure_element
sequences	I-structure_element
and	O
are	O
regulated	O
by	O
additional	O
pre	B-structure_element
-	I-structure_element
mRNA	I-structure_element
motifs	I-structure_element
(	O
reviewed	O
in	O
ref	O
.).	O

The	O
early	O
-	O
stage	O
pre	B-protein_type
-	I-protein_type
mRNA	I-protein_type
splicing	I-protein_type
factor	I-protein_type
U2AF65	B-protein
is	O
essential	O
for	O
viability	O
in	O
vertebrates	B-taxonomy_domain
and	O
other	O
model	O
organisms	O
(	O
for	O
example	O
,	O
ref	O
.).	O

A	O
tightly	O
controlled	O
assembly	B-complex_assembly
among	O
U2AF65	B-protein
,	O
the	O
pre	B-chemical
-	I-chemical
mRNA	I-chemical
,	O
and	O
partner	O
proteins	O
sequentially	O
identifies	O
the	O
3	B-site
′	I-site
splice	I-site
site	I-site
and	O
promotes	O
association	O
of	O
the	O
spliceosome	B-complex_assembly
,	O
which	O
ultimately	O
accomplishes	O
the	O
task	O
of	O
splicing	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

Likewise	O
,	O
both	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
and	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
U2AF65	B-protein
showed	O
similar	O
sequence	B-evidence
specificity	I-evidence
for	O
U	B-structure_element
-	I-structure_element
rich	I-structure_element
stretches	I-structure_element
in	O
the	O
5	B-site
′-	I-site
region	I-site
of	O
the	O
Py	B-chemical
tract	I-chemical
and	O
promiscuity	O
for	O
C	B-structure_element
-	I-structure_element
rich	I-structure_element
regions	I-structure_element
in	O
the	O
3	B-site
′-	I-site
region	I-site
(	O
Fig	O
.	O
1c	O
,	O
Supplementary	O
Fig	O
.	O
1e	O
–	O
h	O
).	O

We	O
compare	O
the	O
global	O
conformation	O
of	O
the	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
structures	B-evidence
with	O
the	O
prior	O
dU2AF651	B-mutant
,	I-mutant
2	I-mutant
crystal	B-evidence
structure	I-evidence
and	O
U2AF651	B-mutant
,	I-mutant
2	I-mutant
NMR	B-experimental_method
structure	B-evidence
in	O
the	O
Supplementary	O
Discussion	O
and	O
Supplementary	O
Fig	O
.	O
2	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
rU6	B-residue_name_number
base	O
edge	O
is	O
relatively	O
solvent	B-protein_state
exposed	I-protein_state
;	O
accordingly	O
,	O
the	O
rU6	B-residue_name_number
hydrogen	O
bonds	O
with	O
U2AF65	B-protein
are	O
water	B-chemical
mediated	O
apart	O
from	O
a	O
single	O
direct	O
interaction	O
by	O
the	O
RRM1	B-structure_element
-	O
N196	B-residue_name_number
side	O
chain	O
.	O

Consistent	O
with	O
loss	O
of	O
a	O
hydrogen	O
bond	O
with	O
the	O
ninth	B-residue_number
pyrimidine	B-chemical
-	O
O2	O
(	O
ΔΔG	B-evidence
1	O
.	O
0	O
kcal	O
mol	O
−	O
1	O
),	O
mutation	B-experimental_method
of	O
the	O
Q147	B-residue_name_number
to	O
an	O
alanine	B-residue_name
reduced	O
U2AF651	B-evidence
,	I-evidence
2L	I-evidence
affinity	I-evidence
for	O
the	O
AdML	B-gene
Py	B-chemical
tract	I-chemical
by	O
five	O
-	O
fold	O
(	O
Fig	O
.	O
3i	O
;	O
Supplementary	O
Fig	O
.	O
4c	O
).	O

Despite	O
12	B-experimental_method
concurrent	I-experimental_method
mutations	I-experimental_method
,	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
12Gly	I-mutant
variant	B-protein_state
was	O
reduced	O
by	O
only	O
three	O
-	O
fold	O
relative	O
to	O
the	O
unmodified	B-protein_state
protein	B-protein
(	O
Fig	O
.	O
4b	O
),	O
which	O
is	O
less	O
than	O
the	O
penalty	O
of	O
the	O
V254P	B-mutant
mutation	O
that	O
disrupts	O
the	O
rU5	B-residue_name_number
hydrogen	O
bond	O
(	O
Fig	O
.	O
3d	O
,	O
i	O
).	O

This	O
difference	O
indicates	O
that	O
the	O
linearly	B-protein_state
distant	I-protein_state
regions	B-structure_element
of	O
the	O
U2AF65	B-protein
primary	O
sequence	O
,	O
including	O
Q147	B-residue_name_number
in	O
the	O
N	O
-	O
terminal	O
RRM1	B-structure_element
extension	I-structure_element
and	O
R227	B-residue_name_number
/	O
V254	B-residue_name_number
in	O
the	O
N	O
-/	O
C	O
-	O
terminal	O
linker	B-structure_element
regions	I-structure_element
at	O
the	O
fifth	B-site
nucleotide	I-site
site	I-site
,	O
cooperatively	O
recognize	O
the	O
Py	B-chemical
tract	I-chemical
.	O

When	O
transfected	B-experimental_method
into	O
HEK293T	O
cells	O
containing	O
only	O
endogenous	B-protein_state
U2AF65	B-protein
,	O
the	O
PY	B-site
splice	I-site
site	I-site
is	O
used	O
and	O
the	O
remaining	O
transcript	O
remains	O
unspliced	O
.	O

Co	B-experimental_method
-	I-experimental_method
transfection	I-experimental_method
of	O
the	O
U2AF65	B-mutant
-	I-mutant
3Mut	I-mutant
with	O
the	O
pyPY	B-chemical
splicing	O
substrate	O
significantly	O
reduced	O
splicing	O
of	O
the	O
weak	O
‘	B-site
py	I-site
'	I-site
splice	I-site
site	I-site
relative	O
to	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
U2AF65	B-protein
(	O
Fig	O
.	O
5b	O
,	O
c	O
).	O

Paramagnetic	B-experimental_method
resonance	I-experimental_method
enhancement	I-experimental_method
(	O
PRE	B-experimental_method
)	O
measurements	O
previously	O
had	O
suggested	O
a	O
predominant	O
back	B-protein_state
-	I-protein_state
to	I-protein_state
-	I-protein_state
back	I-protein_state
,	O
or	O
‘	O
closed	B-protein_state
'	O
conformation	O
of	O
the	O
apo	B-protein_state
-	O
U2AF651	B-mutant
,	I-mutant
2	I-mutant
RRM1	B-structure_element
and	O
RRM2	B-structure_element
in	O
equilibrium	O
with	O
a	O
minor	O
‘	O
open	B-protein_state
'	O
conformation	O
resembling	O
the	O
RNA	B-protein_state
-	I-protein_state
bound	I-protein_state
inter	B-structure_element
-	I-structure_element
RRM	I-structure_element
arrangement	O
.	O

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

The	O
positions	O
of	O
single	O
cysteine	B-residue_name
mutations	B-experimental_method
for	O
fluorophore	B-chemical
attachment	O
(	O
A181C	B-mutant
in	O
RRM1	B-structure_element
and	O
Q324C	B-mutant
in	O
RRM2	B-structure_element
)	O
were	O
chosen	O
based	O
on	O
inspection	O
of	O
the	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
structures	B-evidence
and	O
the	O
‘	O
closed	B-protein_state
'	O
model	O
of	O
apo	B-protein_state
-	O
U2AF651	B-mutant
,	I-mutant
2	I-mutant
.	O

Approximately	O
70	O
%	O
of	O
observed	O
fluctuations	O
were	O
interchanges	O
between	O
the	O
∼	O
0	O
.	O
65	O
and	O
∼	O
0	O
.	O
45	O
FRET	B-evidence
values	I-evidence
(	O
Supplementary	O
Fig	O
.	O
7b	O
).	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

U2AF65	B-protein
conformational	O
selection	O
and	O
induced	O
fit	O
by	O
bound	B-protein_state
RNA	B-chemical

Insertion	B-experimental_method
of	O
adenine	B-chemical
nucleotides	I-chemical
decreased	O
binding	B-evidence
affinity	I-evidence
of	O
U2AF65	B-protein
to	O
RNA	B-chemical
by	O
approximately	O
five	O
-	O
fold	O
.	O

Further	O
research	O
will	O
be	O
needed	O
to	O
understand	O
the	O
roles	O
of	O
SF1	B-protein
and	O
U2AF35	B-protein
subunits	O
in	O
the	O
conformational	O
equilibria	O
underlying	O
U2AF65	B-protein
association	O
with	O
Py	B-chemical
tracts	I-chemical
.	O

Residues	O
V249	B-residue_name_number
,	O
V250	B-residue_name_number
,	O
V254	B-residue_name_number
(	O
yellow	O
)	O
are	O
mutated	B-experimental_method
to	O
V249G	B-mutant
/	O
V250G	B-mutant
/	O
V254G	B-mutant
in	O
the	O
3Gly	B-mutant
mutant	I-mutant
;	O
residues	O
S251	B-residue_name_number
,	O
T252	B-residue_name_number
,	O
V253	B-residue_name_number
,	O
P255	B-residue_name_number
(	O
red	O
)	O
along	O
with	O
V254	B-residue_name_number
are	O
mutated	B-experimental_method
to	O
S251G	B-mutant
/	O
T252G	B-mutant
/	O
V253G	B-mutant
/	O
V254G	B-mutant
/	O
P255G	B-mutant
in	O
the	O
5Gly	B-mutant
mutant	I-mutant
or	O
to	O
S251N	B-mutant
/	O
T252L	B-mutant
/	O
V253A	B-mutant
/	O
V254L	B-mutant
/	O
P255A	B-mutant
in	O
the	O
NLALA	B-mutant
mutant	I-mutant
;	O
residues	O
M144	B-residue_name_number
,	O
L235	B-residue_name_number
,	O
M238	B-residue_name_number
,	O
V244	B-residue_name_number
,	O
V246	B-residue_name_number
(	O
orange	O
)	O
along	O
with	O
V249	B-residue_name_number
,	O
V250	B-residue_name_number
,	O
S251	B-residue_name_number
,	O
T252	B-residue_name_number
,	O
V253	B-residue_name_number
,	O
V254	B-residue_name_number
,	O
P255	B-residue_name_number
are	O
mutated	B-experimental_method
to	O
M144G	B-mutant
/	O
L235G	B-mutant
/	O
M238G	B-mutant
/	O
V244G	B-mutant
/	O
V246G	B-mutant
/	O
V249G	B-mutant
/	O
V250G	B-mutant
/	O
S251G	B-mutant
/	O
T252G	B-mutant
/	O
V253G	B-mutant
/	O
V254G	B-mutant
/	O
P255G	B-mutant
in	O
the	O
12Gly	B-mutant
mutant	I-mutant
.	O

Other	O
linker	B-structure_element
residues	O
are	O
coloured	O
either	O
dark	O
blue	O
for	O
new	O
residues	O
in	O
the	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
structure	O
or	O
light	O
blue	O
for	O
the	O
remaining	O
inter	B-structure_element
-	I-structure_element
RRM	I-structure_element
residues	O
.	O

The	O
central	O
panel	O
shows	O
an	O
overall	O
view	O
with	O
stick	O
diagrams	O
for	O
mutated	O
residues	O
;	O
boxed	O
regions	O
are	O
expanded	O
to	O
show	O
the	O
C	O
-	O
terminal	O
(	O
bottom	O
left	O
)	O
and	O
central	B-structure_element
linker	I-structure_element
regions	I-structure_element
(	O
top	O
)	O
at	O
the	O
inter	B-structure_element
-	I-structure_element
RRM	I-structure_element
interfaces	I-structure_element
,	O
and	O
N	O
-	O
terminal	O
linker	O
region	O
contacts	O
with	O
RRM1	B-structure_element
(	O
bottom	O
right	O
).	O

The	O
fitted	O
fluorescence	O
anisotropy	O
RNA	B-evidence
-	I-evidence
binding	I-evidence
curves	I-evidence
are	O
shown	O
in	O
Supplementary	O
Fig	O
.	O
4d	O
–	O
j	O
.	O
(	O
c	O
)	O
Close	O
view	O
of	O
the	O
U2AF65	B-protein
RRM1	B-site
/	I-site
RRM2	I-site
interface	I-site
following	O
a	O
two	O
-	O
fold	O
rotation	O
about	O
the	O
x	O
-	O
axis	O
relative	O
to	O
a	O
.	O

(	O
a	O
,	O
b	O
)	O
Views	O
of	O
FRET	B-experimental_method
pairs	O
chosen	O
to	O
follow	O
the	O
relative	O
movement	O
of	O
RRM1	B-structure_element
and	O
RRM2	B-structure_element
on	O
the	O
crystal	B-evidence
structure	I-evidence
of	O
‘	O
side	B-protein_state
-	I-protein_state
by	I-protein_state
-	I-protein_state
side	I-protein_state
'	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
RRMs	B-structure_element
bound	B-protein_state
to	I-protein_state
a	O
Py	B-chemical
-	I-chemical
tract	I-chemical
oligonucleotide	I-chemical
(	O
a	O
,	O
representative	O
structure	O
iv	O
)	O
or	O
‘	O
closed	B-protein_state
'	O
NMR	B-experimental_method
/	O
PRE	B-experimental_method
-	O
based	O
model	O
of	O
U2AF651	B-mutant
,	I-mutant
2	I-mutant
(	O
b	O
,	O
PDB	O
ID	O
2YH0	O
)	O
in	O
identical	O
orientations	O
of	O
RRM2	B-structure_element
.	O

(	O
c	O
–	O
f	O
,	O
i	O
,	O
j	O
)	O
The	O
U2AF651	B-mutant
,	I-mutant
2LFRET	I-mutant
(	O
Cy3	B-chemical
/	O
Cy5	B-chemical
)	O
protein	O
was	O
immobilized	O
on	O
the	O
microscope	O
slide	O
via	O
biotin	B-chemical
-	I-chemical
NTA	I-chemical
/	I-chemical
Ni	I-chemical
+	I-chemical
2	I-chemical
(	O
orange	O
line	O
)	O
on	O
a	O
neutravidin	O
(	O
black	O
X	O
)-	O
biotin	O
-	O
PEG	O
(	O
orange	O
triangle	O
)-	O
treated	O
surface	O
and	O
imaged	O
either	O
in	O
the	O
absence	B-protein_state
of	I-protein_state
ligands	B-chemical
(	O
c	O
,	O
d	O
),	O
in	O
the	O
presence	O
of	O
5	O
μM	O
AdML	B-gene
Py	B-chemical
-	I-chemical
tract	I-chemical
RNA	I-chemical
(	O
5	B-chemical
′-	I-chemical
CCUUUUUUUUCC	I-chemical
-	I-chemical
3	I-chemical
′)	I-chemical
(	O
e	O
,	O
f	O
),	O
or	O
in	O
the	O
presence	O
of	O
10	O
μM	O
adenosine	B-residue_name
-	O
interrupted	O
variant	O
RNA	B-chemical
(	O
5	B-chemical
′-	I-chemical
CUUUUUAAUUUCCA	I-chemical
-	I-chemical
3	I-chemical
′)	I-chemical
(	O
i	O
,	O
j	O
).	O

The	O
untethered	B-protein_state
U2AF651	B-mutant
,	I-mutant
2LFRET	I-mutant
(	O
Cy3	B-chemical
/	O
Cy5	B-chemical
)	O
protein	O
(	O
1	O
nM	O
)	O
was	O
added	O
to	O
AdML	B-gene
RNA	B-chemical
–	I-chemical
polyethylene	I-chemical
-	I-chemical
glycol	I-chemical
-	I-chemical
linker	I-chemical
–	I-chemical
DNA	I-chemical
oligonucleotide	I-chemical
(	O
10	O
nM	O
),	O
which	O
was	O
immobilized	O
on	O
the	O
microscope	O
slide	O
by	O
annealing	O
with	O
a	O
complementary	O
biotinyl	B-chemical
-	I-chemical
DNA	I-chemical
oligonucleotide	I-chemical
(	O
black	O
vertical	O
line	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

RRM1	B-structure_element
,	O
green	O
;	O
RRM2	B-structure_element
,	O
pale	O
blue	O
;	O
RRM	B-structure_element
extensions	I-structure_element
/	O
linker	B-structure_element
,	O
blue	O
.	O

Systematic	O
analysis	O
of	O
radiation	O
damage	O
within	O
a	O
protein	B-complex_assembly
–	I-complex_assembly
RNA	I-complex_assembly
complex	O
over	O
a	O
large	O
dose	O
range	O
(	O
1	O
.	O
3	O
–	O
25	O
MGy	O
)	O
reveals	O
significant	O
differential	O
susceptibility	O
of	O
RNA	B-chemical
and	O
protein	O
.	O

With	O
the	O
wide	O
use	O
of	O
high	O
-	O
flux	O
third	O
-	O
generation	O
synchrotron	O
sources	O
,	O
radiation	O
damage	O
(	O
RD	O
)	O
has	O
once	O
again	O
become	O
a	O
dominant	O
reason	O
for	O
the	O
failure	O
of	O
structure	B-experimental_method
determination	I-experimental_method
using	O
macromolecular	B-experimental_method
crystallography	I-experimental_method
(	O
MX	B-experimental_method
)	O
in	O
experiments	O
conducted	O
both	O
at	O
room	O
temperature	O
and	O
under	O
cryocooled	O
conditions	O
(	O
100	O
K	O
).	O

Significant	O
progress	O
has	O
been	O
made	O
in	O
recent	O
years	O
in	O
understanding	O
the	O
inevitable	O
manifestations	O
of	O
X	O
-	O
ray	O
-	O
induced	O
RD	O
within	O
protein	O
crystals	B-evidence
,	O
and	O
there	O
is	O
now	O
a	O
body	O
of	O
literature	O
on	O
possible	O
strategies	O
to	O
mitigate	O
the	O
effects	O
of	O
RD	O
(	O
e	O
.	O
g	O
.	O
Zeldin	O
,	O
Brockhauser	O
et	O
al	O
.,	O
2013	O
;	O
Bourenkov	O
&	O
Popov	O
,	O
2010	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

Understanding	O
RD	O
to	O
such	O
complexes	O
is	O
crucial	O
,	O
since	O
DNA	B-chemical
is	O
rarely	O
naked	O
within	O
a	O
cell	O
,	O
instead	O
dynamically	O
interacting	O
with	O
proteins	O
,	O
facilitating	O
replication	O
,	O
transcription	O
,	O
modification	O
and	O
DNA	B-chemical
repair	O
.	O

Nucleoproteins	B-complex_assembly
also	O
represent	O
one	O
of	O
the	O
main	O
targets	O
of	O
radiotherapy	O
,	O
and	O
an	O
insight	O
into	O
the	O
damage	O
mechanisms	O
induced	O
by	O
X	O
-	O
ray	O
irradiation	O
could	O
inform	O
innovative	O
treatments	O
.	O

Investigations	O
on	O
sub	O
-	O
ionization	O
-	O
level	O
LEEs	O
(	O
0	O
–	O
15	O
eV	O
)	O
interacting	O
with	O
both	O
dried	O
and	O
aqueous	O
oligonucleotides	O
(	O
Alizadeh	O
&	O
Sanche	O
,	O
2014	O
;	O
Simons	O
,	O
2006	O
)	O
concluded	O
that	O
resonant	O
electron	O
attachment	O
to	O
DNA	B-chemical
bases	O
and	O
the	O
sugar	O
-	O
phosphate	O
backbone	O
could	O
lead	O
to	O
the	O
preferential	O
cleavage	O
of	O
strong	O
(∼	O
4	O
eV	O
,	O
385	O
kJ	O
mol	O
−	O
1	O
)	O
sugar	O
-	O
phosphate	O
C	O
—	O
O	O
covalent	O
bonds	O
within	O
the	O
DNA	B-chemical
backbone	O
and	O
then	O
base	O
-	O
sugar	O
N1	O
—	O
C	O
bonds	O
,	O
eventually	O
leading	O
to	O
single	O
-	O
strand	O
breakages	O
(	O
SSBs	O
;	O
Ptasińska	O
&	O
Sanche	O
,	O
2007	O
).	O

To	O
avoid	O
the	O
previous	O
necessity	O
for	O
visual	O
inspection	O
of	O
electron	B-evidence
-	I-evidence
density	I-evidence
maps	I-evidence
to	O
detect	O
SRD	B-site
sites	I-site
,	O
a	O
computational	O
approach	O
was	O
designed	O
to	O
quantify	O
the	O
electron	B-evidence
-	I-evidence
density	I-evidence
change	I-evidence
for	O
each	O
refined	O
atom	O
with	O
increasing	O
dose	O
,	O
thus	O
providing	O
a	O
rapid	O
systematic	O
method	O
for	O
SRD	O
study	O
on	O
such	O
large	O
multimeric	O
complexes	O
.	O

To	O
quantify	O
the	O
exact	O
effects	O
of	O
nucleic	O
acid	O
binding	O
to	O
a	O
protein	O
on	O
SRD	O
susceptibility	O
,	O
a	O
high	O
-	O
throughput	O
and	O
automated	O
pipeline	O
was	O
created	O
to	O
systematically	O
calculate	O
the	O
electron	B-evidence
-	I-evidence
density	I-evidence
change	I-evidence
for	O
every	O
refined	O
atom	O
within	O
the	O
TRAP	B-complex_assembly
–	I-complex_assembly
RNA	I-complex_assembly
structure	B-evidence
as	O
a	O
function	O
of	O
dose	O
.	O

This	O
provides	O
an	O
atom	O
-	O
specific	O
quantification	O
of	O
density	B-evidence
–	I-evidence
dose	I-evidence
dynamics	I-evidence
,	O
which	O
was	O
previously	O
lacking	O
within	O
the	O
field	O
.	O

However	O
,	O
these	O
σ	B-evidence
levels	O
depend	O
on	O
the	O
standard	B-evidence
deviation	I-evidence
values	O
of	O
the	O
map	B-evidence
,	O
which	O
can	O
deviate	O
between	O
data	O
sets	O
,	O
and	O
are	O
thus	O
unsuitable	O
for	O
quantitative	O
comparison	O
of	O
density	B-evidence
between	O
different	O
dose	O
data	O
sets	O
.	O

The	O
rate	O
of	O
D	B-evidence
loss	I-evidence
(	O
attributed	O
to	O
side	O
-	O
chain	O
decarboxylation	O
)	O
was	O
consistently	O
larger	O
for	O
Glu	B-residue_name
compared	O
with	O
Asp	B-residue_name
residues	O
over	O
the	O
large	O
dose	O
range	O
(	O
Fig	O
.	O
2	O
▸	O
b	O
and	O
Supplementary	O
Fig	O
.	O
S3	O
);	O
this	O
observation	O
is	O
consistent	O
with	O
our	O
calculations	O
on	O
model	O
systems	O
(	O
see	O
above	O
)	O
that	O
suggest	O
that	O
,	O
without	O
considering	O
differential	O
hydrogen	O
-	O
bonding	O
environments	O
,	O
CO2	B-chemical
loss	O
is	O
more	O
exothermic	O
by	O
around	O
8	O
kJ	O
mol	O
−	O
1	O
from	O
oxidized	B-protein_state
Glu	B-residue_name
residues	O
than	O
from	O
their	O
Asp	B-residue_name
counterparts	O
.	O

In	O
our	O
analysis	O
,	O
Asp39	B-residue_name_number
in	O
the	O
TRAP	B-complex_assembly
–(	I-complex_assembly
GAGUU	I-complex_assembly
)	I-complex_assembly
10GAG	I-complex_assembly
structure	B-evidence
appears	O
to	O
exhibit	O
two	O
distinct	O
hydrogen	O
bonds	O
to	O
the	O
G1	B-residue_name_number
base	O
within	O
each	O
of	O
the	O
11	O
TRAP	B-site
–	I-site
RNA	I-site
interfaces	I-site
,	O
as	O
does	O
Glu36	B-residue_name_number
to	O
G3	B-residue_name_number
;	O
however	O
,	O
the	O
reduction	O
in	O
density	B-evidence
disordering	O
upon	O
RNA	B-chemical
binding	O
is	O
far	O
less	O
significant	O
for	O
Asp39	B-residue_name_number
than	O
for	O
Glu36	B-residue_name_number
(	O
Fig	O
.	O
5	O
▸	O
b	O
,	O
p	O
=	O
0	O
.	O
0925	O
).	O

One	O
oxygen	O
(	O
O	O
∊	O
1	O
)	O
of	O
Glu42	B-residue_name_number
appears	O
to	O
form	O
a	O
hydrogen	O
bond	O
to	O
a	O
nearby	O
water	B-chemical
within	O
each	O
TRAP	B-site
RNA	I-site
-	I-site
binding	I-site
pocket	I-site
,	O
with	O
the	O
other	O
(	O
O	O
∊	O
2	O
)	O
being	O
involved	O
in	O
a	O
salt	O
-	O
bridge	O
interaction	O
with	O
Arg58	B-residue_name_number
(	O
Hopcroft	O
et	O
al	O
.,	O
2002	O
;	O
Antson	O
et	O
al	O
.,	O
1999	O
).	O

The	O
density	B-evidence
-	I-evidence
change	I-evidence
dynamics	I-evidence
were	O
statistically	O
indistinguishable	O
between	O
bound	B-protein_state
and	O
nonbound	B-protein_state
TRAP	B-complex_assembly
for	O
each	O
Glu42	B-residue_name_number
carboxyl	O
group	O
Cδ	O
atom	O
(	O
p	O
=	O
0	O
.	O
435	O
),	O
indicating	O
that	O
upon	O
RNA	B-chemical
binding	O
the	O
conserved	O
salt	O
-	O
bridge	O
interaction	O
ultimately	O
dictated	O
the	O
overall	O
Glu42	B-residue_name_number
decarboxylation	O
rate	O
.	O

The	O
RNA	B-chemical
-	O
stabilizing	O
effect	O
was	O
not	O
restricted	O
to	O
radiation	O
-	O
sensitive	O
acidic	O
residues	O
.	O

Here	O
,	O
MX	B-experimental_method
radiation	O
-	O
induced	O
specific	O
structural	O
changes	O
within	O
the	O
large	O
TRAP	B-complex_assembly
–	I-complex_assembly
RNA	I-complex_assembly
assembly	O
over	O
a	O
large	O
dose	O
range	O
(	O
1	O
.	O
3	O
–	O
25	O
.	O
0	O
MGy	O
)	O
have	O
been	O
analysed	O
using	O
a	O
high	O
-	O
throughput	O
quantitative	O
approach	O
,	O
providing	O
a	O
measure	O
of	O
the	O
electron	B-evidence
-	I-evidence
density	I-evidence
distribution	I-evidence
for	O
each	O
refined	O
atom	O
with	O
increasing	O
dose	O
,	O
D	B-evidence
loss	I-evidence
.	O

The	O
RNA	B-chemical
was	O
found	O
to	O
be	O
substantially	O
more	O
radiation	B-protein_state
-	I-protein_state
resistant	I-protein_state
than	O
the	O
protein	O
,	O
even	O
at	O
the	O
highest	O
doses	O
investigated	O
(∼	O
25	O
.	O
0	O
MGy	O
),	O
which	O
is	O
in	O
strong	O
concurrence	O
with	O
our	O
previous	O
SRD	B-experimental_method
investigation	I-experimental_method
of	O
the	O
C	B-complex_assembly
.	I-complex_assembly
Esp1396I	I-complex_assembly
protein	O
–	O
DNA	B-chemical
complex	O
(	O
Bury	O
et	O
al	O
.,	O
2015	O
).	O

For	O
example	O
,	O
Asp17	B-residue_name_number
is	O
located	O
∼	O
6	O
.	O
8	O
Å	O
from	O
the	O
G1	B-residue_name_number
base	O
,	O
outside	O
the	O
RNA	B-site
-	I-site
binding	I-site
interfaces	I-site
,	O
and	O
has	O
indistinguishable	O
Cγ	O
atom	O
D	O
loss	B-evidence
dose	I-evidence
-	I-evidence
dynamics	I-evidence
between	O
RNA	B-protein_state
-	I-protein_state
bound	I-protein_state
and	O
nonbound	B-protein_state
TRAP	B-complex_assembly
(	O
p	O
>	O
0	O
.	O
9	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

The	O
results	O
presented	O
here	O
suggest	O
that	O
biologically	O
relevant	O
nucleoprotein	B-complex_assembly
complexes	O
also	O
exhibit	O
prolonged	O
life	O
-	O
doses	O
under	O
the	O
effect	O
of	O
LEE	O
-	O
induced	O
structural	O
changes	O
,	O
involving	O
direct	O
physical	O
protection	O
of	O
key	O
RNA	B-site
-	I-site
binding	I-site
residues	I-site
.	O

Such	O
reduced	O
radiation	O
-	O
sensitivity	O
in	O
this	O
case	O
ensures	O
that	O
the	O
interacting	O
protein	O
remains	O
bound	B-protein_state
long	O
enough	O
to	O
the	O
RNA	B-chemical
to	O
complete	O
its	O
function	O
,	O
even	O
whilst	O
exposed	O
to	O
ionizing	O
radiation	O
.	O

RNA	B-chemical
is	O
shown	O
is	O
yellow	O
.	O

Only	O
a	O
subset	O
of	O
key	O
TRAP	B-complex_assembly
residue	O
types	O
are	O
included	O
.	O

D	O
loss	O
calculated	O
for	O
all	O
side	O
-	O
chain	O
carboxyl	O
group	O
Glu	B-residue_name
Cδ	O
and	O
Asp	B-residue_name
Cγ	O
atoms	O
within	O
the	O
TRAP	B-complex_assembly
–	I-complex_assembly
RNA	I-complex_assembly
complex	O
for	O
a	O
dose	O
of	O
19	O
.	O
3	O
MGy	O
(	O
d	O
8	O
).	O

Another	O
challenge	O
will	O
be	O
to	O
find	O
out	O
where	O
IDA	B-protein
is	O
produced	O
in	O
the	O
plant	B-taxonomy_domain
and	O
what	O
causes	O
it	O
to	O
accumulate	O
in	O
specific	O
places	O
in	O
preparation	O
for	O
organ	O
shedding	O
.	O

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

(	O
A	O
)	O
Details	O
of	O
the	O
IDA	B-site
binding	I-site
pocket	I-site
.	O

The	O
IDA	B-complex_assembly
-	I-complex_assembly
HAESA	I-complex_assembly
and	O
SERK1	B-complex_assembly
-	I-complex_assembly
HAESA	I-complex_assembly
complex	O
interfaces	B-site
are	O
conserved	B-protein_state
among	O
HAESA	B-protein
and	O
HAESA	B-protein_type
-	I-protein_type
like	I-protein_type
proteins	I-protein_type
from	O
different	O
plant	B-taxonomy_domain
species	O
.	O

The	O
peptide	B-site
binding	I-site
pocket	I-site
covers	O
HAESA	B-protein
LRRs	B-structure_element
2	I-structure_element
–	I-structure_element
14	I-structure_element
.	O
(	O
D	O
)	O
Close	O
-	O
up	O
view	O
of	O
the	O
entire	O
IDA	B-protein
(	O
in	O
yellow	O
)	O
peptide	B-site
binding	I-site
site	I-site
in	O
HAESA	B-protein
(	O
in	O
blue	O
).	O

Hydrogren	O
bonds	O
are	O
depicted	O
as	O
dotted	O
lines	O
(	O
in	O
magenta	O
),	O
a	O
water	B-chemical
molecule	O
is	O
shown	O
as	O
a	O
red	O
sphere	O
.	O

This	B-structure_element
sequence	I-structure_element
motif	I-structure_element
is	O
highly	B-protein_state
conserved	I-protein_state
among	O
IDA	B-protein_type
family	I-protein_type
members	I-protein_type
(	O
IDA	B-protein_type
-	I-protein_type
LIKE	I-protein_type
PROTEINS	I-protein_type
,	O
IDLs	B-protein_type
)	O
and	O
contains	O
a	O
central	O
Pro	B-residue_name
residue	O
,	O
presumed	O
to	O
be	O
post	B-protein_state
-	I-protein_state
translationally	I-protein_state
modified	I-protein_state
to	O
hydroxyproline	B-residue_name
(	O
Hyp	B-residue_name
;	O
Figure	O
1A	O
).	O

Active	B-protein_state
IDA	B-protein_type
-	I-protein_type
family	I-protein_type
peptide	I-protein_type
hormones	I-protein_type
are	O
hydroxyprolinated	B-protein_state
dodecamers	B-structure_element
.	O

no	O
detectable	O
binding	O
).	O
(	O
E	O
)	O
Structural	B-experimental_method
superposition	I-experimental_method
of	O
the	O
active	B-protein_state
IDA	B-protein
(	O
in	O
bonds	O
representation	O
,	O
in	O
gray	O
)	O
and	O
IDL1	B-chemical
peptide	I-chemical
(	O
in	O
yellow	O
)	O
hormones	O
bound	B-protein_state
to	I-protein_state
the	O
HAESA	B-protein
ectodomain	B-structure_element
.	O

Petal	O
break	O
-	O
strength	O
was	O
found	O
significantly	O
increased	O
in	O
almost	O
all	O
positions	O
(	O
indicated	O
with	O
a	O
*)	O
for	O
haesa	B-gene
/	O
hsl2	B-gene
and	O
serk1	B-gene
-	I-gene
1	I-gene
mutant	B-protein_state
plants	B-taxonomy_domain
with	O
respect	O
to	O
the	O
Col	O
-	O
0	O
control	O
.	O

(	O
B	O
)	O
Analytical	B-experimental_method
size	I-experimental_method
-	I-experimental_method
exclusion	I-experimental_method
chromatography	I-experimental_method
.	O

The	O
central	O
Hyp64IDA	B-residue_name_number
is	O
buried	O
in	O
a	O
specific	O
pocket	B-site
formed	O
by	O
HAESA	B-protein
LRRs	B-structure_element
8	I-structure_element
–	I-structure_element
10	I-structure_element
,	O
with	O
its	O
hydroxyl	O
group	O
establishing	O
hydrogen	O
bonds	O
with	O
the	O
strictly	B-protein_state
conserved	I-protein_state
Glu266HAESA	B-residue_name_number
and	O
with	O
a	O
water	B-chemical
molecule	O
,	O
which	O
in	O
turn	O
is	O
coordinated	O
by	O
the	O
main	O
chain	O
oxygens	O
of	O
Phe289HAESA	B-residue_name_number
and	O
Ser311HAESA	B-residue_name_number
(	O
Figure	O
1E	O
;	O
Figure	O
1	O
—	O
figure	O
supplement	O
3	O
).	O

In	O
this	O
structure	B-evidence
,	O
no	O
additional	O
electron	B-evidence
density	I-evidence
accounts	O
for	O
the	O
PKGV	B-structure_element
motif	I-structure_element
at	O
the	O
IDA	B-protein
N	O
-	O
terminus	O
(	O
Figure	O
2A	O
,	O
B	O
).	O

We	O
do	O
not	O
detect	O
interaction	O
between	O
HAESA	B-protein
and	O
a	O
synthetic	B-protein_state
peptide	B-chemical
missing	B-protein_state
the	I-protein_state
C	I-protein_state
-	I-protein_state
terminal	I-protein_state
Asn69IDA	B-residue_name_number
(	O
ΔN69	B-mutant
),	O
highlighting	O
the	O
importance	O
of	O
the	O
polar	O
interactions	O
between	O
the	O
IDA	B-protein
carboxy	O
-	O
terminus	O
and	O
Arg407HAESA	B-residue_name_number
/	O
Arg409HAESA	B-residue_name_number
(	O
Figures	O
1F	O
,	O
2D	O
).	O

The	O
co	B-protein_type
-	I-protein_type
receptor	I-protein_type
kinase	I-protein_type
SERK1	B-protein
allows	O
for	O
high	O
-	O
affinity	O
IDA	O
sensing	O

We	O
found	O
that	O
the	O
force	O
required	O
to	O
remove	O
the	O
petals	O
of	O
serk1	B-gene
-	I-gene
1	I-gene
mutants	B-protein_state
is	O
significantly	O
higher	O
than	O
that	O
needed	O
for	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
plants	B-taxonomy_domain
,	O
as	O
previously	O
observed	O
for	O
haesa	B-gene
/	O
hsl2	B-gene
mutants	B-protein_state
,	O
and	O
that	O
floral	O
abscission	O
is	O
delayed	O
in	O
serk1	B-gene
-	I-gene
1	I-gene
(	O
Figure	O
3A	O
).	O

In	O
vitro	O
,	O
the	O
LRR	B-structure_element
ectodomain	I-structure_element
of	O
SERK1	B-protein
(	O
residues	O
24	B-residue_range
–	I-residue_range
213	I-residue_range
)	O
forms	O
stable	B-protein_state
,	O
IDA	B-protein_state
-	I-protein_state
dependent	I-protein_state
heterodimeric	B-oligomeric_state
complexes	B-protein_state
with	I-protein_state
HAESA	B-protein
in	O
size	B-experimental_method
exclusion	I-experimental_method
chromatography	I-experimental_method
experiments	O
(	O
Figure	O
3B	O
).	O

We	O
next	O
titrated	B-experimental_method
SERK1	B-protein
into	O
a	O
solution	O
containing	O
only	O
the	O
HAESA	B-protein
ectodomain	B-structure_element
.	O

In	O
this	O
case	O
,	O
there	O
was	O
no	O
detectable	O
interaction	O
between	O
receptor	O
and	O
co	O
-	O
receptor	O
,	O
while	O
in	O
the	O
presence	B-protein_state
of	I-protein_state
IDA	B-protein
,	O
SERK1	B-protein
strongly	O
binds	O
HAESA	B-protein
with	O
a	O
dissociation	B-evidence
constant	I-evidence
in	O
the	O
mid	O
-	O
nanomolar	O
range	O
(	O
Figure	O
3C	O
).	O

Upon	O
IDA	B-protein
binding	O
at	O
the	O
cell	O
surface	O
,	O
the	O
kinase	B-structure_element
domains	I-structure_element
of	O
HAESA	B-protein
and	O
SERK1	B-protein
,	O
which	O
have	O
been	O
shown	O
to	O
be	O
active	B-protein_state
protein	B-protein_type
kinases	I-protein_type
,	O
may	O
interact	O
in	O
the	O
cytoplasm	O
to	O
activate	O
each	O
other	O
.	O

Consistently	O
,	O
the	O
HAESA	B-protein
kinase	B-structure_element
domain	I-structure_element
can	O
transphosphorylate	O
SERK1	B-protein
and	O
vice	O
versa	O
in	O
in	O
vitro	O
transphosphorylation	B-experimental_method
assays	I-experimental_method
(	O
Figure	O
3E	O
).	O

Crystal	B-evidence
structure	I-evidence
of	O
a	O
HAESA	B-complex_assembly
–	I-complex_assembly
IDA	I-complex_assembly
–	I-complex_assembly
SERK1	I-complex_assembly
signaling	O
complex	O
.	O

(	O
A	O
)	O
Overview	O
of	O
the	O
ternary	O
complex	O
with	O
HAESA	B-protein
in	O
blue	O
(	O
surface	O
representation	O
),	O
IDA	B-protein
in	O
yellow	O
(	O
bonds	O
representation	O
)	O
and	O
SERK1	B-protein
in	O
orange	O
(	O
surface	O
view	O
).	O
(	O
B	O
)	O
The	O
HAESA	B-protein
ectodomain	B-structure_element
undergoes	O
a	O
conformational	O
change	O
upon	O
SERK1	B-protein
co	O
-	O
receptor	O
binding	O
.	O

Polar	O
contacts	O
of	O
SERK1	B-protein
with	O
IDA	B-protein
are	O
shown	O
in	O
magenta	O
,	O
with	O
the	O
HAESA	B-protein
LRR	B-structure_element
domain	I-structure_element
in	O
gray	O
.	O
(	O
D	O
)	O
Details	O
of	O
the	O
zipper	B-structure_element
-	I-structure_element
like	I-structure_element
SERK1	B-site
-	I-site
HAESA	I-site
interface	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

We	O
found	O
that	O
over	B-experimental_method
-	I-experimental_method
expression	I-experimental_method
of	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
IDA	B-protein
leads	O
to	O
early	O
floral	O
abscission	O
and	O
an	O
enlargement	O
of	O
the	O
abscission	O
zone	O
(	O
Figure	O
5C	O
–	O
E	O
).	O

In	O
contrast	O
to	O
animal	B-taxonomy_domain
LRR	B-protein_type
receptors	I-protein_type
,	O
plant	B-taxonomy_domain
LRR	B-structure_element
-	I-structure_element
RKs	I-structure_element
harbor	O
spiral	B-protein_state
-	I-protein_state
shaped	I-protein_state
ectodomains	B-structure_element
and	O
thus	O
they	O
require	O
shape	B-protein_state
-	I-protein_state
complementary	I-protein_state
co	B-protein_type
-	I-protein_type
receptor	I-protein_type
proteins	I-protein_type
for	O
receptor	O
activation	O
.	O

However	O
our	O
results	O
show	O
that	O
SERK1	B-protein
also	O
can	O
activate	O
this	O
process	O
upon	O
IDA	B-protein
sensing	O
,	O
indicating	O
that	O
SERKs	B-protein_type
may	O
fulfill	O
several	O
different	O
functions	O
in	O
the	O
course	O
of	O
the	O
abscission	O
process	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

A	O
ribbon	O
diagram	O
of	O
SERK1	B-protein
in	O
the	O
same	O
orientation	O
is	O
shown	O
alongside	O
.	O

These	O
residues	O
are	O
not	O
involved	O
in	O
the	O
sensing	O
of	O
the	O
steroid	B-chemical
hormone	I-chemical
brassinolide	B-chemical
.	O

Different	O
plant	B-taxonomy_domain
peptide	B-protein_type
hormone	I-protein_type
families	I-protein_type
contain	O
a	O
C	O
-	O
terminal	O
(	B-structure_element
Arg	I-structure_element
)-	I-structure_element
His	I-structure_element
-	I-structure_element
Asn	I-structure_element
motif	I-structure_element
,	O
which	O
in	O
IDA	B-protein
represents	O
the	O
co	B-site
-	I-site
receptor	I-site
recognition	I-site
site	I-site
.	O

Our	O
experiments	O
reveal	O
that	O
SERK1	B-protein
recognizes	O
a	O
C	O
-	O
terminal	O
Arg	B-structure_element
-	I-structure_element
His	I-structure_element
-	I-structure_element
Asn	I-structure_element
motif	I-structure_element
in	O
IDA	B-protein
.	O

Among	O
these	O
are	O
the	O
CLE	B-chemical
peptides	I-chemical
regulating	O
stem	O
cell	O
maintenance	O
in	O
the	O
shoot	O
and	O
the	O
root	O
.	O

The	O
crotonyllysine	B-residue_name
mark	O
on	O
histone	B-protein_type
H3K18	B-protein_type
is	O
produced	O
by	O
p300	B-protein
,	O
a	O
histone	B-protein_type
acetyltransferase	I-protein_type
also	O
responsible	O
for	O
acetylation	B-ptm
of	O
histones	O
.	O

The	O
family	O
of	O
acetyllysine	B-residue_name
readers	O
has	O
been	O
expanded	O
with	O
the	O
discovery	O
that	O
the	O
YEATS	B-structure_element
(	O
Yaf9	B-protein
,	O
ENL	B-protein
,	O
AF9	B-protein
,	O
Taf14	B-protein
,	O
Sas5	B-protein
)	O
domains	O
of	O
human	B-species
AF9	B-protein
and	O
yeast	B-taxonomy_domain
Taf14	B-protein
are	O
capable	O
of	O
recognizing	O
the	O
histone	B-protein_type
mark	O
H3K9ac	B-protein_type
.	O

Similarly	O
,	O
activation	O
of	O
a	O
subset	O
of	O
genes	O
and	O
DNA	O
damage	O
repair	O
in	O
yeast	B-taxonomy_domain
require	O
the	O
acetyllysine	B-residue_name
binding	O
activity	O
of	O
the	O
Taf14	B-protein
YEATS	B-structure_element
domain	I-structure_element
.	O

However	O
,	O
Taf14	B-protein
is	O
also	O
found	O
in	O
a	O
number	O
of	O
chromatin	O
-	O
remodeling	O
complexes	O
(	O
i	O
.	O
e	O
.,	O
INO80	B-complex_assembly
,	O
SWI	B-complex_assembly
/	I-complex_assembly
SNF	I-complex_assembly
and	O
RSC	B-complex_assembly
)	O
and	O
the	O
histone	B-protein_type
acetyltransferase	I-protein_type
complex	O
NuA3	B-complex_assembly
,	O
indicating	O
a	O
multifaceted	O
role	O
of	O
Taf14	B-protein
in	O
transcriptional	O
regulation	O
and	O
chromatin	O
biology	O
.	O

We	O
found	O
that	O
H3K9cr	B-protein_type
is	O
present	O
in	O
yeast	B-taxonomy_domain
and	O
is	O
dynamically	O
regulated	O
.	O

This	O
distinctive	O
mechanism	O
was	O
corroborated	O
through	O
mapping	O
the	O
Taf14	B-protein
YEATS	B-site
-	I-site
H3K9cr	I-site
binding	I-site
interface	I-site
in	O
solution	O
using	O
NMR	B-experimental_method
chemical	I-experimental_method
shift	I-experimental_method
perturbation	I-experimental_method
analysis	I-experimental_method
(	O
Supplementary	O
Fig	O
.	O
2a	O
,	O
b	O
).	O

Binding	O
of	O
the	O
Taf14	B-protein
YEATS	B-structure_element
domain	I-structure_element
to	O
H3K9cr	B-protein_type
is	O
robust	O
.	O

We	O
concluded	O
that	O
H3K9cr	B-protein_type
is	O
the	O
preferred	O
target	O
of	O
this	O
domain	O
.	O

However	O
,	O
bromodomains	B-structure_element
did	O
not	O
interact	O
(	O
or	O
associated	O
very	O
weakly	O
)	O
with	O
longer	O
acyl	O
modifications	O
,	O
including	O
crotonyllysine	B-residue_name
,	O
as	O
in	O
the	O
case	O
of	O
BDs	B-structure_element
of	O
TAF1	B-protein
and	O
BRD2	B-protein
,	O
supporting	O
recent	O
reports	O
.	O

As	O
we	O
previously	O
showed	O
the	O
importance	O
of	O
acyllysine	B-residue_name
binding	O
by	O
the	O
Taf14	B-protein
YEATS	B-structure_element
domain	I-structure_element
for	O
the	O
DNA	O
damage	O
response	O
and	O
gene	O
transcription	O
,	O
it	O
will	O
be	O
essential	O
in	O
the	O
future	O
to	O
define	O
the	O
physiological	O
role	O
of	O
crotonyllysine	B-residue_name
recognition	O
and	O
to	O
differentiate	O
the	O
activities	O
of	O
Taf14	B-protein
that	O
are	O
due	O
to	O
binding	O
to	O
crotonyllysine	B-residue_name
and	O
acetyllysine	B-residue_name
modifications	O
.	O

Furthermore	O
,	O
the	O
functional	O
significance	O
of	O
crotonyllysine	B-residue_name
recognition	O
by	O
other	O
YEATS	B-protein_type
proteins	O
will	O
be	O
of	O
great	O
importance	O
to	O
elucidate	O
and	O
compare	O
.	O

The	O
structural	O
mechanism	O
for	O
the	O
recognition	O
of	O
H3K9cr	B-protein_type

Total	O
H3	B-protein_type
was	O
used	O
as	O
a	O
loading	O
control	O
.	O

(	O
c	O
)	O
Superimposed	O
1H	B-experimental_method
,	I-experimental_method
15N	I-experimental_method
HSQC	I-experimental_method
spectra	B-evidence
of	O
Taf14	B-protein
YEATS	B-structure_element
recorded	O
as	O
H3K9cr5	B-chemical
-	I-chemical
13	I-chemical
and	O
H3K9ac5	B-chemical
-	I-chemical
13	I-chemical
peptides	O
were	O
titrated	B-experimental_method
in	O
.	O

Although	O
a	O
Thr1Ser	B-mutant
mutant	B-protein_state
is	O
active	B-protein_state
,	O
it	O
is	O
less	O
efficient	O
compared	O
with	O
wild	B-protein_state
type	I-protein_state
because	O
of	O
the	O
unfavourable	O
orientation	O
of	O
Ser1	B-residue_name_number
towards	O
incoming	O
substrates	O
.	O

The	O
proteasome	B-complex_assembly
,	O
an	O
essential	O
molecular	O
machine	O
,	O
is	O
a	O
threonine	B-protein_type
protease	I-protein_type
,	O
but	O
the	O
evolution	O
and	O
the	O
components	O
of	O
its	O
proteolytic	O
centre	O
are	O
unclear	O
.	O

In	O
the	O
last	O
stage	O
of	O
CP	B-complex_assembly
biogenesis	O
,	O
the	O
prosegments	B-structure_element
are	O
autocatalytically	B-ptm
removed	I-ptm
through	O
nucleophilic	O
attack	O
by	O
the	O
active	B-site
site	I-site
residue	I-site
Thr1	B-residue_name_number
on	O
the	O
preceding	O
peptide	O
bond	O
involving	O
Gly	B-residue_name_number
(-	I-residue_name_number
1	I-residue_name_number
).	I-residue_name_number

These	O
results	O
indicate	O
that	O
the	O
β1	B-protein
and	O
β2	B-protein
proteolytic	O
activities	O
are	O
not	O
essential	O
for	O
cell	O
survival	O
.	O

Our	O
present	O
crystallographic	B-experimental_method
analysis	I-experimental_method
of	O
the	O
β5	B-mutant
-	I-mutant
T1A	I-mutant
pp	B-chemical
trans	B-protein_state
mutant	B-protein_state
demonstrates	O
that	O
the	O
mutation	B-experimental_method
per	O
se	O
does	O
not	O
structurally	O
alter	O
the	O
catalytic	B-site
active	I-site
site	I-site
and	O
that	O
the	O
trans	B-experimental_method
-	I-experimental_method
expressed	I-experimental_method
β5	B-protein
propeptide	B-structure_element
is	O
not	B-protein_state
bound	I-protein_state
in	O
the	O
β5	B-protein
substrate	B-site
-	I-site
binding	I-site
channel	I-site
(	O
Supplementary	O
Fig	O
.	O
1a	O
).	O

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

In	O
subunit	O
β1	B-protein
,	O
we	O
found	O
that	O
Gly	B-residue_name_number
(-	I-residue_name_number
1	I-residue_name_number
)	I-residue_name_number
indeed	O
forms	O
a	O
sharp	B-structure_element
turn	I-structure_element
,	O
which	O
relaxes	O
on	O
prosegment	B-ptm
cleavage	I-ptm
(	O
Fig	O
.	O
1a	O
and	O
Supplementary	O
Fig	O
.	O
2a	O
).	O

Regarding	O
the	O
β2	B-protein
propeptide	B-structure_element
,	O
Thr	B-residue_name_number
(-	I-residue_name_number
2	I-residue_name_number
)	I-residue_name_number
occupies	O
the	O
S1	B-site
pocket	I-site
but	O
is	O
less	O
deeply	O
anchored	O
compared	O
with	O
Leu	B-residue_name_number
(-	I-residue_name_number
2	I-residue_name_number
)	I-residue_name_number
in	O
β1	B-protein
,	O
which	O
might	O
be	O
due	O
to	O
the	O
rather	O
large	O
β2	B-protein
-	O
S1	B-site
pocket	I-site
created	O
by	O
Gly45	B-residue_name_number
.	O

Nevertheless	O
,	O
both	O
Leu	B-residue_name_number
(-	I-residue_name_number
2	I-residue_name_number
)	I-residue_name_number
and	O
Thr	B-residue_name_number
(-	I-residue_name_number
2	I-residue_name_number
)	I-residue_name_number
were	O
found	O
to	O
occupy	O
the	O
S1	B-site
specificity	I-site
pocket	I-site
formed	O
by	O
Met45	B-residue_name_number
(	O
Fig	O
.	O
2a	O
,	O
b	O
and	O
Supplementary	O
Fig	O
.	O
4f	O
–	O
h	O
).	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

Notably	O
,	O
the	O
2FO	B-evidence
–	I-evidence
FC	I-evidence
electron	I-evidence
-	I-evidence
density	I-evidence
map	I-evidence
displays	O
a	O
different	O
orientation	O
for	O
the	O
β2	B-protein
propeptide	B-structure_element
than	O
has	O
been	O
observed	O
for	O
the	O
β2	B-mutant
-	I-mutant
T1A	I-mutant
proteasome	B-complex_assembly
.	O

The	O
active	B-site
site	I-site
of	O
the	O
proteasome	B-complex_assembly

Instead	O
,	O
Lys33NH2	B-residue_name_number
,	O
which	O
is	O
in	O
hydrogen	O
-	O
bonding	O
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

In	O
agreement	O
,	O
an	O
E17A	B-mutant
mutant	B-protein_state
in	O
the	O
proteasomal	O
β	B-protein
-	I-protein
subunit	I-protein
of	O
the	O
archaeon	B-taxonomy_domain
Thermoplasma	B-species
acidophilum	I-species
prevents	O
autolysis	B-ptm
and	O
catalysis	O
.	O

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

Whereas	O
Asn	B-residue_name
can	O
to	O
some	O
degree	O
replace	O
Asp166	B-residue_name_number
due	O
to	O
its	O
carbonyl	O
group	O
in	O
the	O
side	O
chain	O
,	O
Ala	B-residue_name
at	O
this	O
position	O
was	O
found	O
to	O
prevent	O
both	O
autolysis	B-ptm
and	O
catalysis	O
.	O

His	B-residue_name_number
(-	I-residue_name_number
2	I-residue_name_number
)	I-residue_name_number
occupies	O
the	O
S2	B-site
pocket	I-site
like	O
observed	O
for	O
the	O
β5	B-mutant
-	I-mutant
T1A	I-mutant
-	I-mutant
K81R	I-mutant
mutant	B-protein_state
,	O
but	O
in	O
contrast	O
to	O
the	O
latter	O
,	O
the	O
propeptide	B-structure_element
in	O
the	O
T1C	B-mutant
mutant	B-protein_state
adopts	O
an	O
antiparallel	B-structure_element
β	I-structure_element
-	I-structure_element
sheet	I-structure_element
conformation	O
as	O
known	O
from	O
inhibitors	O
like	O
MG132	B-chemical
(	O
Fig	O
.	O
4c	O
–	O
e	O
and	O
Supplementary	O
Fig	O
.	O
9b	O
).	O

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

In	O
vitro	O
,	O
the	O
mutant	B-protein_state
proteasome	B-complex_assembly
is	O
less	O
susceptible	O
to	O
proteasome	B-complex_assembly
inhibition	O
by	O
bortezomib	B-chemical
(	O
3	O
.	O
7	O
-	O
fold	O
)	O
and	O
carfilzomib	B-chemical
(	O
1	O
.	O
8	O
-	O
fold	O
;	O
Fig	O
.	O
5	O
).	O

The	O
20S	B-complex_assembly
proteasome	I-complex_assembly
CP	B-complex_assembly
is	O
the	O
major	O
non	B-protein_type
-	I-protein_type
lysosomal	I-protein_type
protease	I-protein_type
in	O
eukaryotic	B-taxonomy_domain
cells	O
,	O
and	O
its	O
assembly	O
is	O
highly	O
organized	O
.	O

The	O
β	B-protein
-	I-protein
subunit	I-protein
propeptides	B-structure_element
,	O
particularly	O
that	O
of	O
β5	B-protein
,	O
are	O
key	O
factors	O
that	O
help	O
drive	O
proper	O
assembly	O
of	O
the	O
CP	B-complex_assembly
complex	O
.	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

The	O
resulting	O
uncharged	O
Thr1NH2	B-residue_name_number
is	O
hydrogen	O
-	O
bridged	O
to	O
the	O
C3	O
-	O
OH	O
group	O
.	O

In	O
agreement	O
,	O
acetylation	B-ptm
of	O
the	O
Thr1	B-residue_name_number
N	O
terminus	O
irreversibly	O
blocks	O
hydrolytic	O
activity	O
,	O
and	O
binding	O
of	O
substrates	O
is	O
prevented	O
for	O
steric	O
reasons	O
.	O

This	O
interpretation	O
agrees	O
with	O
the	O
strongly	O
reduced	O
catalytic	O
activity	O
of	O
the	O
β5	B-mutant
-	I-mutant
D166N	I-mutant
mutant	B-protein_state
on	O
the	O
one	O
hand	O
,	O
and	O
the	O
ability	O
to	O
react	O
readily	O
with	O
carfilzomib	B-chemical
on	O
the	O
other	O
.	O

We	O
also	O
observed	O
slightly	O
lower	O
affinity	O
of	O
the	O
β5	B-mutant
-	I-mutant
T1S	I-mutant
mutant	B-protein_state
yCP	B-complex_assembly
for	O
the	O
Food	O
and	O
Drug	O
Administration	O
-	O
approved	O
proteasome	B-complex_assembly
inhibitors	O
bortezomib	B-chemical
and	O
carfilzomib	B-chemical
.	O

In	O
contrast	O
to	O
Thr1	B-residue_name_number
,	O
the	O
hydroxyl	O
group	O
of	O
Ser1	B-residue_name_number
occupies	O
the	O
position	O
of	O
the	O
Thr1	B-residue_name_number
methyl	O
side	O
chain	O
in	O
the	O
WT	B-protein_state
enzyme	B-complex_assembly
,	O
which	O
requires	O
its	O
reorientation	O
relative	O
to	O
the	O
substrate	O
to	O
allow	O
cleavage	O
(	O
Fig	O
.	O
4g	O
,	O
h	O
).	O

Architecture	O
and	O
proposed	O
reaction	O
mechanism	O
of	O
the	O
proteasomal	O
active	B-site
site	I-site
.	O

Thr1OH	B-residue_name_number
is	O
hydrogen	O
-	O
bonded	O
to	O
Lys33NH2	B-residue_name_number
(	O
2	O
.	O
7	O
Å	O
),	O
which	O
in	O
turn	O
interacts	O
with	O
Asp17Oδ	B-residue_name_number
.	O

Autolysis	B-ptm
(	O
left	O
set	O
of	O
structures	O
)	O
is	O
initiated	O
by	O
deprotonation	O
of	O
Thr1OH	B-residue_name_number
via	O
Lys33NH2	B-residue_name_number
and	O
the	O
formation	O
of	O
a	O
tetrahedral	O
transition	O
state	O
.	O

Next	O
,	O
Thr1NH2	B-residue_name_number
polarizes	O
a	O
water	B-chemical
molecule	O
for	O
the	O
nucleophilic	O
attack	O
of	O
the	O
acyl	O
-	O
enzyme	O
intermediate	O
.	O

(	O
c	O
)	O
Illustration	O
of	O
the	O
2FO	B-evidence
–	I-evidence
FC	I-evidence
electron	I-evidence
-	I-evidence
density	I-evidence
map	I-evidence
(	O
blue	O
mesh	O
contoured	O
at	O
1σ	O
)	O
for	O
the	O
β5	B-mutant
-	I-mutant
T1C	I-mutant
propeptide	B-structure_element
fragment	O
.	O

(	O
h	O
)	O
The	O
methyl	O
group	O
of	O
Thr1	B-residue_name_number
is	O
anchored	O
by	O
hydrophobic	O
interactions	O
with	O
Ala46Cβ	B-residue_name_number
and	O
Thr3Cγ	B-residue_name_number
.	O