Despite	O
displaying	O
similar	O
affinities	B-evidence
for	O
XyG	B-chemical
,	O
reverse	B-experimental_method
-	I-experimental_method
genetic	I-experimental_method
analysis	I-experimental_method
reveals	O
that	O
SGBP	B-protein
-	I-protein
B	I-protein
is	O
only	O
required	O
for	O
the	O
efficient	O
capture	O
of	O
smaller	O
oligosaccharides	B-chemical
,	O
whereas	O
the	O
presence	O
of	O
SGBP	B-protein
-	I-protein
A	I-protein
is	O
more	O
critical	O
than	O
its	O
carbohydrate	B-chemical
-	O
binding	O
ability	O
for	O
growth	O
on	O
XyG	B-chemical
.	O
Together	O
,	O
these	O
data	O
demonstrate	O
that	O
SGBP	B-protein
-	I-protein
A	I-protein
and	O
SGBP	B-protein
-	I-protein
B	I-protein
play	O
complementary	O
,	O
specialized	O
roles	O
in	O
carbohydrate	B-chemical
capture	O
by	O
B	B-species
.	I-species
ovatus	I-species
and	O
elaborate	O
a	O
model	O
of	O
how	O
vegetable	B-taxonomy_domain
xyloglucans	B-chemical
are	O
accessed	O
by	O
the	O
Bacteroidetes	B-taxonomy_domain
.	O

The	O
human	B-species
gut	O
bacteria	B-taxonomy_domain
Bacteroidetes	B-taxonomy_domain
share	O
a	O
profound	O
capacity	O
for	O
dietary	O
glycan	B-chemical
degradation	O
,	O
with	O
many	O
species	O
containing	O
>	O
250	O
predicted	O
carbohydrate	O
-	O
active	O
enzymes	O
(	O
CAZymes	O
),	O
compared	O
to	O
50	O
to	O
100	O
within	O
many	O
Firmicutes	B-taxonomy_domain
and	O
only	O
17	O
in	O
the	O
human	B-species
genome	O
devoted	O
toward	O
carbohydrate	O
utilization	O
.	O

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

We	O
describe	O
here	O
the	O
detailed	O
functional	B-experimental_method
and	I-experimental_method
structural	I-experimental_method
characterization	I-experimental_method
of	O
the	O
noncatalytic	B-protein_state
SGBPs	B-protein_type
encoded	O
by	O
Bacova_02651	B-gene
and	O
Bacova_02650	B-gene
of	O
the	O
XyGUL	B-gene
,	O
here	O
referred	O
to	O
as	O
SGBP	B-protein
-	I-protein
A	I-protein
and	O
SGBP	B-protein
-	I-protein
B	I-protein
,	O
to	O
elucidate	O
their	O
molecular	O
roles	O
in	O
carbohydrate	O
acquisition	O
in	O
vivo	O
.	O

Immunofluorescence	B-experimental_method
of	O
formaldehyde	O
-	O
fixed	O
,	O
nonpermeabilized	O
cells	O
grown	O
in	O
minimal	O
medium	O
with	O
XyG	B-chemical
as	O
the	O
sole	O
carbon	O
source	O
to	O
induce	O
XyGUL	B-gene
expression	O
,	O
reveals	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
presented	O
on	O
the	O
cell	O
surface	O
by	O
N	O
-	O
terminal	O
lipidation	B-ptm
,	O
as	O
predicted	O
by	O
signal	O
peptide	O
analysis	O
with	O
SignalP	O
(	O
Fig	O
.	O
2	O
).	O

Neither	O
SGBP	B-protein_type
recognized	O
galactomannan	B-chemical
(	O
GGM	B-chemical
),	O
starch	B-chemical
,	O
carboxymethylcellulose	B-chemical
,	O
or	O
mucin	B-chemical
(	O
see	O
Fig	O
.	O
S1	O
in	O
the	O
supplemental	O
material	O
).	O

Affinity	B-experimental_method
electrophoresis	I-experimental_method
(	O
10	O
%	O
acrylamide	O
)	O
of	O
SGBP	B-protein
-	I-protein
A	I-protein
and	O
SGBP	B-protein
-	I-protein
B	I-protein
with	O
BSA	B-protein
as	O
a	O
control	O
protein	O
.	O

All	O
samples	O
were	O
loaded	O
on	O
the	O
same	O
gel	O
next	O
to	O
the	O
BSA	B-protein
controls	O
;	O
thin	O
black	O
lines	O
indicate	O
where	O
intervening	O
lanes	O
were	O
removed	O
from	O
the	O
final	O
image	O
for	O
both	O
space	O
and	O
clarity	O
.	O

SGBP	B-protein
-	I-protein
A	I-protein
is	O
a	O
SusD	B-protein
homolog	O
with	O
an	O
extensive	O
glycan	B-site
-	I-site
binding	I-site
platform	I-site
.	O

It	O
is	O
particularly	O
notable	O
that	O
although	O
the	O
location	O
of	O
the	O
ligand	B-site
-	I-site
binding	I-site
site	I-site
is	O
conserved	B-protein_state
between	O
SGBP	B-protein
-	I-protein
A	I-protein
and	O
SusD	B-protein
,	O
that	O
of	O
SGBP	B-protein
-	I-protein
A	I-protein
displays	O
an	O
~	O
29	O
-	O
Å	O
-	O
long	O
aromatic	B-site
platform	I-site
to	O
accommodate	O
the	O
extended	O
,	O
linear	O
XyG	B-chemical
chain	O
(	O
see	O
reference	O
for	O
a	O
review	O
of	O
XyG	B-chemical
secondary	O
structure	O
),	O
versus	O
the	O
shorter	O
,	O
~	O
18	O
-	O
Å	O
-	O
long	O
,	O
site	B-site
within	O
SusD	B-protein
that	O
complements	O
the	O
helical	O
conformation	O
of	O
amylose	B-chemical
(	O
Fig	O
.	O
4C	O
and	O
D	O
).	O

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

Three	O
aromatic	O
residues	O
—	O
W82	B-residue_name_number
,	O
W283	B-residue_name_number
,	O
W306	B-residue_name_number
—	O
comprise	O
the	O
flat	B-site
platform	I-site
that	O
stacks	O
along	O
the	O
naturally	O
twisted	O
β	B-chemical
-	I-chemical
glucan	I-chemical
backbone	O
(	O
Fig	O
.	O
4E	O
).	O

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

Multimodular	O
structure	O
of	O
SGBP	B-protein
-	I-protein
B	I-protein
(	O
Bacova_02650	B-gene
).	O
(	O
A	O
)	O
Full	B-protein_state
-	I-protein_state
length	I-protein_state
structure	B-evidence
of	O
SGBP	B-protein
-	I-protein
B	I-protein
,	O
color	O
coded	O
by	O
domain	O
as	O
indicated	O
.	O

An	O
omit	B-evidence
map	I-evidence
(	O
2σ	O
)	O
for	O
XyGO2	B-chemical
is	O
displayed	O
to	O
highlight	O
the	O
location	O
of	O
the	O
glycan	B-site
-	I-site
binding	I-site
site	I-site
.	O

(	O
D	O
)	O
Close	O
-	O
up	O
omit	B-evidence
map	I-evidence
for	O
the	O
XyGO2	B-chemical
ligand	O
,	O
contoured	O
at	O
2σ	O
.	O
(	O
E	O
)	O
Stereo	O
view	O
of	O
the	O
xyloglucan	B-site
-	I-site
binding	I-site
site	I-site
of	O
SGBP	B-protein
-	I-protein
B	I-protein
,	O
displaying	O
all	O
residues	O
within	O
4	O
Å	O
of	O
the	O
ligand	O
.	O

XyG	B-chemical
binds	B-protein_state
to	I-protein_state
domain	O
D	B-structure_element
of	O
SGBP	B-protein
-	I-protein
B	I-protein
at	O
the	O
concave	B-site
interface	I-site
of	O
the	O
top	O
β	B-structure_element
-	I-structure_element
sheet	I-structure_element
,	O
with	O
binding	O
mediated	O
by	O
loops	B-structure_element
connecting	O
the	O
β	B-structure_element
-	I-structure_element
strands	I-structure_element
.	O

The	O
backbone	O
is	O
flat	O
,	O
with	O
less	O
of	O
the	O
“	O
twisted	O
-	O
ribbon	O
”	O
geometry	O
observed	O
in	O
some	O
cello	B-chemical
-	I-chemical
and	I-chemical
xylogluco	I-chemical
-	I-chemical
oligosaccharides	I-chemical
.	O

While	O
this	O
may	O
occur	O
for	O
a	O
number	O
of	O
reasons	O
in	O
crystal	B-evidence
structures	I-evidence
,	O
it	O
is	O
likely	O
that	O
the	O
poor	O
ligand	O
density	O
even	O
at	O
higher	O
resolution	O
is	O
due	O
to	O
movement	O
or	O
multiple	O
orientations	O
of	O
the	O
sugar	B-chemical
averaged	O
throughout	O
the	O
lattice	O
.	O

Solid	O
bars	O
indicate	O
conditions	O
that	O
are	O
not	O
statistically	O
significant	O
from	O
the	O
WT	B-protein_state
Δtdk	B-mutant
cultures	O
grown	O
on	O
the	O
indicated	O
carbohydrate	B-chemical
,	O
while	O
open	O
bars	O
indicate	O
a	O
P	O
value	O
of	O
<	O
0	O
.	O
005	O
compared	O
to	O
the	O
WT	B-protein_state
Δtdk	B-mutant
strain	O
.	O

In	O
previous	O
studies	O
,	O
we	O
observed	O
that	O
carbohydrate	B-chemical
binding	O
by	O
SusD	B-protein
enhanced	O
the	O
sensitivity	O
of	O
the	O
cells	O
to	O
limiting	O
concentrations	O
of	O
malto	O
-	O
oligosaccharides	O
by	O
several	O
orders	O
of	O
magnitude	O
,	O
such	O
that	O
the	O
addition	O
of	O
0	O
.	O
5	O
g	O
/	O
liter	O
maltose	B-chemical
was	O
required	O
to	O
restore	O
growth	O
of	O
the	O
ΔsusD	B-mutant
::	O
SusD	B-mutant
*	I-mutant
strain	O
on	O
starch	B-chemical
,	O
which	O
nonetheless	O
occurred	O
following	O
an	O
extended	O
lag	B-evidence
phase	I-evidence
.	O

As	O
such	O
,	O
the	O
data	O
suggest	O
that	O
SGBP	B-protein
-	I-protein
A	I-protein
can	O
compensate	O
for	O
the	O
loss	O
of	O
function	O
of	O
SGBP	B-protein
-	I-protein
B	I-protein
on	O
longer	O
oligo	B-chemical
-	I-chemical
and	I-chemical
polysaccharides	I-chemical
,	O
while	O
SGBP	B-protein
-	I-protein
B	I-protein
may	O
adapt	O
the	O
cell	O
to	O
recognize	O
smaller	O
oligosaccharides	B-chemical
efficiently	O
.	O

Presumably	O
without	O
glycan	B-chemical
binding	O
by	O
the	O
SGBPs	B-protein_type
,	O
the	O
GH5	B-protein
protein	O
cannot	O
efficiently	O
process	O
xyloglucan	B-chemical
,	O
and	O
/	O
or	O
the	O
lack	O
of	O
SGBP	B-protein_type
function	O
prevents	O
efficient	O
capture	O
and	O
import	O
of	O
the	O
processed	O
oligosaccharides	B-chemical
.	O

However	O
,	O
unlike	O
the	O
Sus	B-complex_assembly
,	O
in	O
which	O
elimination	B-experimental_method
of	I-experimental_method
SusE	B-protein
and	O
SusF	B-protein
does	O
not	O
affect	O
growth	O
on	O
starch	B-chemical
,	O
SGBP	B-protein
-	I-protein
B	I-protein
appears	O
to	O
have	O
a	O
dedicated	O
role	O
in	O
growth	O
on	O
small	O
xylogluco	B-chemical
-	I-chemical
oligosaccharides	I-chemical
.	O

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

Yet	O
,	O
a	O
number	O
of	O
questions	O
remain	O
regarding	O
the	O
molecular	O
interplay	O
of	O
SGBPs	B-protein_type
with	O
their	O
cotranscribed	O
cohort	O
of	O
glycoside	B-protein_type
hydrolases	I-protein_type
and	O
TonB	B-protein_type
-	I-protein_type
dependent	I-protein_type
transporters	I-protein_type
.	O

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

Thus	O
,	O
the	O
strict	O
dependence	O
on	O
a	O
SusD	B-protein_type
-	I-protein_type
like	I-protein_type
SGBP	I-protein_type
for	O
glycan	B-chemical
uptake	O
in	O
the	O
Bacteroidetes	B-taxonomy_domain
may	O
be	O
variable	O
and	O
substrate	O
dependent	O
.	O

Such	O
is	O
the	O
case	O
for	O
XyGUL	B-gene
from	O
related	O
Bacteroides	B-taxonomy_domain
species	O
,	O
which	O
may	O
encode	O
either	O
one	O
or	O
two	O
of	O
these	O
predicted	O
SGBPs	B-protein_type
,	O
and	O
these	O
proteins	O
vary	O
considerably	O
in	O
length	O
.	O

It	O
is	O
therefore	O
important	O
to	O
understand	O
the	O
mechanisms	O
which	O
regulate	O
nadA	B-gene
expression	O
levels	O
,	O
which	O
are	O
predominantly	O
controlled	O
by	O
the	O
transcriptional	B-protein_type
regulator	I-protein_type
NadR	B-protein
(	O
Neisseria	B-protein
adhesin	I-protein
A	I-protein
Regulator	I-protein
)	O
both	O
in	O
vitro	O
and	O
in	O
vivo	O
.	O

These	O
findings	O
shed	O
light	O
on	O
the	O
regulation	O
of	O
NadR	B-protein
,	O
a	O
key	O
MarR	B-protein_type
-	O
family	O
virulence	O
factor	O
of	O
this	O
important	O
human	B-species
pathogen	O
.	O

The	O
‘	O
Reverse	B-experimental_method
Vaccinology	I-experimental_method
’	O
approach	O
was	O
pioneered	O
to	O
identify	O
antigens	O
for	O
a	O
protein	O
-	O
based	O
vaccine	O
against	O
serogroup	B-species
B	I-species
Neisseria	I-species
meningitidis	I-species
(	O
MenB	B-species
),	O
a	O
human	B-species
pathogen	O
causing	O
potentially	O
-	O
fatal	O
sepsis	O
and	O
invasive	O
meningococcal	B-taxonomy_domain
disease	O
.	O

Indeed	O
,	O
Reverse	B-experimental_method
Vaccinology	I-experimental_method
identified	O
Neisseria	B-protein
adhesin	I-protein
A	I-protein
(	O
NadA	B-protein
),	O
a	O
surface	O
-	O
exposed	O
protein	O
involved	O
in	O
epithelial	O
cell	O
invasion	O
and	O
found	O
in	O
~	O
30	O
%	O
of	O
clinical	O
isolates	O
.	O

Although	O
additional	O
factors	O
influence	O
nadA	B-gene
expression	O
,	O
we	O
focused	O
on	O
its	O
regulation	O
by	O
NadR	B-protein
,	O
the	O
major	O
mediator	O
of	O
NadA	B-protein
phase	O
variable	O
expression	O
.	O

Stability	O
of	O
NadR	B-protein
is	O
increased	O
by	O
small	O
molecule	O
ligands	O
.	O

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

The	O
structure	B-evidence
of	O
the	O
NadR	B-complex_assembly
/	I-complex_assembly
4	I-complex_assembly
-	I-complex_assembly
HPA	I-complex_assembly
complex	O
was	O
determined	O
at	O
2	O
.	O
3	O
Å	O
resolution	O
using	O
a	O
combination	O
of	O
the	O
single	B-experimental_method
-	I-experimental_method
wavelength	I-experimental_method
anomalous	I-experimental_method
dispersion	I-experimental_method
(	O
SAD	B-experimental_method
)	O
and	O
molecular	B-experimental_method
replacement	I-experimental_method
(	O
MR	B-experimental_method
)	O
methods	O
,	O
and	O
was	O
refined	O
to	O
R	B-evidence
work	I-evidence
/	I-evidence
R	I-evidence
free	I-evidence
values	O
of	O
20	O
.	O
9	O
/	O
26	O
.	O
0	O
%	O
(	O
Table	O
2	O
).	O

Despite	O
numerous	O
attempts	O
,	O
we	O
were	O
unable	O
to	O
obtain	O
high	O
-	O
quality	O
crystals	B-evidence
of	O
NadR	B-protein
complexed	B-protein_state
with	I-protein_state
3Cl	B-chemical
,	I-chemical
4	I-chemical
-	I-chemical
HPA	I-chemical
,	O
3	B-chemical
,	I-chemical
4	I-chemical
-	I-chemical
HPA	I-chemical
,	O
3	B-chemical
-	I-chemical
HPA	I-chemical
or	O
DNA	O
targets	O
.	O

However	O
,	O
it	O
was	O
eventually	O
possible	O
to	O
crystallize	B-experimental_method
apo	B-protein_state
-	O
NadR	B-protein
,	O
and	O
the	O
structure	B-evidence
was	O
determined	O
at	O
2	O
.	O
7	O
Å	O
resolution	O
by	O
MR	B-experimental_method
methods	O
using	O
the	O
NadR	B-complex_assembly
/	I-complex_assembly
4	I-complex_assembly
-	I-complex_assembly
HPA	I-complex_assembly
complex	O
as	O
the	O
search	O
model	O
.	O

Interestingly	O
,	O
in	O
the	O
α4	B-structure_element
-	I-structure_element
β2	I-structure_element
region	I-structure_element
,	O
the	O
stretch	O
of	O
residues	O
from	O
R64	B-residue_range
-	I-residue_range
R91	I-residue_range
presents	O
seven	O
positively	O
-	O
charged	O
side	O
chains	O
,	O
all	O
available	O
for	O
potential	O
interactions	O
with	O
DNA	B-chemical
.	O

The	O
crystal	B-evidence
structure	I-evidence
of	O
NadR	B-protein
in	B-protein_state
complex	I-protein_state
with	I-protein_state
4	B-chemical
-	I-chemical
HPA	I-chemical
.	O

It	O
is	O
notable	O
that	O
L130	B-residue_name_number
is	O
usually	O
present	O
as	O
Leu	B-residue_name
,	O
or	O
an	O
alternative	O
bulky	O
hydrophobic	O
amino	O
acid	O
(	O
e	O
.	O
g	O
.	O
Phe	B-residue_name
,	O
Val	B-residue_name
),	O
in	O
many	O
MarR	B-protein_type
family	O
proteins	O
,	O
suggesting	O
a	O
conserved	B-protein_state
role	O
in	O
stabilizing	O
the	O
dimer	B-site
interface	I-site
.	O

(	O
C	O
)	O
SE	B-experimental_method
-	I-experimental_method
HPLC	I-experimental_method
analyses	O
of	O
all	O
mutant	B-protein_state
forms	O
of	O
NadR	B-protein
are	O
compared	O
with	O
the	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
(	O
WT	B-protein_state
)	O
protein	O
.	O

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

A	O
water	B-chemical
molecule	O
is	O
shown	O
by	O
the	O
red	O
sphere	O
.	O

The	O
entire	O
set	O
of	O
residues	O
making	O
H	O
-	O
bonds	O
or	O
non	O
-	O
bonded	O
contacts	O
with	O
4	B-chemical
-	I-chemical
HPA	I-chemical
is	O
as	O
follows	O
:	O
SerA9	B-residue_name_number
,	O
AsnA11	B-residue_name_number
,	O
LeuB21	B-residue_name_number
,	O
MetB22	B-residue_name_number
,	O
PheB25	B-residue_name_number
,	O
LeuB29	B-residue_name_number
,	O
AspB36	B-residue_name_number
,	O
TrpB39	B-residue_name_number
,	O
ArgB43	B-residue_name_number
,	O
ValB111	B-residue_name_number
and	O
TyrB115	B-residue_name_number
(	O
automated	O
analysis	O
performed	O
using	O
PDBsum	B-experimental_method
and	O
verified	O
manually	O
).	O

Side	O
chains	O
mediating	O
hydrophobic	O
interactions	O
are	O
shown	O
in	O
orange	O
.	O
(	O
B	O
)	O
A	O
model	O
was	O
prepared	O
to	O
visualize	O
putative	O
interactions	O
of	O
3Cl	B-chemical
,	I-chemical
4	I-chemical
-	I-chemical
HPA	I-chemical
(	O
pink	O
)	O
with	O
NadR	B-protein
,	O
revealing	O
the	O
potential	O
for	O
additional	O
contacts	O
(	O
dashed	O
lines	O
)	O
of	O
the	O
chloro	O
moiety	O
(	O
green	O
stick	O
)	O
with	O
LeuB29	B-residue_name_number
and	O
AspB36	B-residue_name_number
.	O

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

Analysis	O
of	O
the	O
pockets	B-site
reveals	O
the	O
molecular	O
basis	O
for	O
asymmetric	O
binding	O
and	O
stoichiometry	O

In	O
these	O
crystals	B-evidence
,	O
the	O
ArgA43	B-residue_name_number
side	O
chain	O
showed	O
two	O
alternate	O
conformations	O
,	O
modelled	O
with	O
50	O
%	O
occupancy	O
in	O
each	O
state	O
,	O
as	O
indicated	O
by	O
the	O
two	O
‘	O
mirrored	O
’	O
arrows	O
.	O

The	O
1H	B-experimental_method
-	I-experimental_method
15N	I-experimental_method
TROSY	I-experimental_method
-	I-experimental_method
HSQC	I-experimental_method
spectrum	B-evidence
of	O
apo	B-protein_state
-	O
NadR	B-protein
,	O
acquired	O
at	O
25	O
°	O
C	O
,	O
displayed	O
approximately	O
140	O
distinct	O
peaks	O
(	O
Fig	O
6A	O
),	O
most	O
of	O
which	O
correspond	O
to	O
backbone	O
amide	O
N	O
-	O
H	O
groups	O
.	O

(	O
B	O
,	O
C	O
)	O
Overlay	B-experimental_method
of	O
selected	O
regions	O
of	O
the	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
acquired	O
at	O
25	O
°	O
C	O
of	O
apo	B-protein_state
-	O
NadR	B-protein
(	O
cyan	O
)	O
and	O
NadR	B-complex_assembly
/	I-complex_assembly
4	I-complex_assembly
-	I-complex_assembly
HPA	I-complex_assembly
(	O
red	O
)	O
superimposed	B-experimental_method
with	O
the	O
spectra	B-evidence
acquired	O
at	O
10	O
°	O
C	O
of	O
apo	B-protein_state
-	O
NadR	B-protein
(	O
blue	O
)	O
and	O
NadR	B-complex_assembly
/	I-complex_assembly
4	I-complex_assembly
-	I-complex_assembly
HPA	I-complex_assembly
(	O
green	O
).	O

Most	O
notably	O
,	O
the	O
position	O
of	O
the	O
DNA	B-chemical
-	O
binding	O
helix	B-structure_element
α4	B-structure_element
shifted	O
by	O
as	O
much	O
as	O
6	O
Å	O
(	O
Fig	O
8B	O
).	O

For	O
clarity	O
,	O
only	O
the	O
α4	B-structure_element
helices	I-structure_element
are	O
shown	O
in	O
panels	O
(	O
B	O
)	O
and	O
(	O
C	O
).	O
(	O
D	O
)	O
Upon	O
comparison	O
with	O
the	O
experimentally	O
-	O
determined	O
OhrR	B-complex_assembly
:	I-complex_assembly
ohrA	I-complex_assembly
structure	B-evidence
(	O
grey	O
),	O
the	O
α4	B-structure_element
helix	I-structure_element
of	O
holo	B-protein_state
-	O
NadR	B-protein
(	O
blue	O
)	O
is	O
shifted	O
~	O
8Å	O
out	O
of	O
the	O
major	O
groove	O
.	O

Specifically	O
,	O
in	O
addition	O
to	O
the	O
different	O
inter	B-evidence
-	I-evidence
helical	I-evidence
translational	I-evidence
distances	I-evidence
,	O
the	O
α4	B-structure_element
helices	I-structure_element
in	O
the	O
holo	B-protein_state
-	O
NadR	B-protein
homodimer	B-oligomeric_state
were	O
also	O
reoriented	O
,	O
resulting	O
in	O
movement	O
of	O
α4	B-structure_element
out	O
of	O
the	O
major	O
groove	O
,	O
by	O
up	O
to	O
8Å	O
,	O
and	O
presumably	O
preventing	O
efficient	O
DNA	B-chemical
binding	O
in	O
the	O
presence	O
of	O
4	B-chemical
-	I-chemical
HPA	I-chemical
(	O
Fig	O
8D	O
).	O

Firstly	O
,	O
we	O
confirmed	O
that	O
NadR	B-protein
is	O
dimeric	B-oligomeric_state
in	O
solution	O
and	O
demonstrated	O
that	O
it	O
retains	O
its	O
dimeric	B-oligomeric_state
state	O
in	O
the	O
presence	B-protein_state
of	I-protein_state
4	B-chemical
-	I-chemical
HPA	I-chemical
,	O
indicating	O
that	O
induction	O
of	O
a	O
monomeric	B-oligomeric_state
status	O
is	O
not	O
the	O
manner	O
by	O
which	O
4	B-chemical
-	I-chemical
HPA	I-chemical
regulates	O
NadR	B-protein
.	O
These	O
observations	O
were	O
in	O
agreement	O
with	O
(	O
i	O
)	O
a	O
previous	O
study	O
of	O
NadR	B-protein
performed	O
using	O
SEC	B-experimental_method
and	O
mass	B-experimental_method
spectrometry	I-experimental_method
,	O
and	O
(	O
ii	O
)	O
crystallographic	B-experimental_method
studies	I-experimental_method
showing	O
that	O
several	O
MarR	B-protein_type
homologues	O
are	O
dimeric	B-oligomeric_state
.	O

Although	O
these	O
NadR	B-protein
/	O
HPA	O
interactions	O
appeared	O
rather	O
weak	O
,	O
their	O
distinct	O
affinities	O
and	O
specificities	O
matched	O
their	O
in	O
vitro	O
effects	O
and	O
their	O
biological	O
relevance	O
appears	O
similar	O
to	O
previous	O
proposals	O
that	O
certain	O
small	O
molecules	O
,	O
including	O
some	O
antibiotics	O
,	O
in	O
the	O
millimolar	O
concentration	O
range	O
may	O
be	O
broad	O
inhibitors	O
of	O
MarR	B-protein_type
family	O
proteins	O
.	O

Structural	B-experimental_method
analyses	I-experimental_method
suggested	O
that	O
‘	O
inward	B-protein_state
’	O
side	O
chain	O
positions	O
of	O
Met22	B-residue_name_number
,	O
Phe25	B-residue_name_number
and	O
especially	O
Arg43	B-residue_name_number
precluded	O
binding	O
of	O
a	O
second	O
ligand	O
molecule	O
.	O

Such	O
a	O
mechanism	O
indicates	O
negative	O
cooperativity	O
,	O
which	O
may	O
enhance	O
the	O
ligand	O
-	O
responsiveness	O
of	O
NadR	B-protein
.	O

Comparisons	O
of	O
the	O
NadR	B-complex_assembly
/	I-complex_assembly
4	I-complex_assembly
-	I-complex_assembly
HPA	I-complex_assembly
complex	O
with	O
available	O
MarR	B-protein_type
family	O
/	O
salicylate	B-chemical
complexes	O
revealed	O
that	O
4	B-chemical
-	I-chemical
HPA	I-chemical
has	O
a	O
previously	O
unobserved	O
binding	O
mode	O
.	O

Ultimately	O
,	O
knowledge	O
of	O
the	O
ligand	O
-	O
dependent	O
activity	O
of	O
NadR	B-protein
will	O
continue	O
to	O
deepen	O
our	O
understanding	O
of	O
nadA	B-gene
expression	O
levels	O
,	O
which	O
influence	O
meningococcal	B-taxonomy_domain
pathogenesis	O
.	O

The	O
structure	O
of	O
NMB1585	B-protein
,	O
a	O
MarR	O
-	O
family	O
regulator	O
from	O
Neisseria	O
meningitidis	O

The	O
PduL	B-protein_type
structure	B-evidence
,	O
in	O
the	O
context	O
of	O
the	O
catalytic	O
core	O
,	O
completes	O
our	O
understanding	O
of	O
the	O
structural	O
basis	O
of	O
cofactor	O
recycling	O
in	O
the	O
metabolosome	B-complex_assembly
lumen	O
.	O

The	O
phosphotransacylase	B-protein_type
(	O
Pta	B-protein_type
)	O
enzyme	O
catalyzes	O
the	O
conversion	O
between	O
acyl	B-chemical
-	I-chemical
CoA	I-chemical
and	O
acyl	B-chemical
-	I-chemical
phosphate	I-chemical
.	O

We	O
solved	B-experimental_method
the	O
structure	B-evidence
of	O
this	O
convergent	B-protein_state
phosphotransacylase	B-protein_type
and	O
show	O
that	O
it	O
is	O
completely	O
structurally	O
different	O
from	O
Pta	B-protein_type
,	O
including	O
its	O
active	B-site
site	I-site
architecture	O
.	O

Bacterial	B-taxonomy_domain
Microcompartments	B-complex_assembly
(	O
BMCs	B-complex_assembly
)	O
are	O
organelles	O
that	O
encapsulate	O
enzymes	O
for	O
sequential	O
biochemical	O
reactions	O
within	O
a	O
protein	O
shell	B-structure_element
.	O

The	O
vitamin	B-complex_assembly
B12	I-complex_assembly
-	I-complex_assembly
dependent	I-complex_assembly
propanediol	I-complex_assembly
-	I-complex_assembly
utilizing	I-complex_assembly
(	I-complex_assembly
PDU	I-complex_assembly
)	I-complex_assembly
BMC	I-complex_assembly
was	O
one	O
of	O
the	O
first	O
functionally	O
characterized	O
catabolic	B-protein_state
BMCs	B-complex_assembly
;	O
subsequently	O
,	O
other	O
types	O
have	O
been	O
implicated	O
in	O
the	O
degradation	O
of	O
ethanolamine	B-chemical
,	O
choline	B-chemical
,	O
fucose	B-chemical
,	O
rhamnose	B-chemical
,	O
and	O
ethanol	B-chemical
,	O
all	O
of	O
which	O
produce	O
different	O
aldehyde	B-chemical
intermediates	O
(	O
Table	O
1	O
).	O

These	O
two	O
cofactors	O
are	O
relatively	O
large	O
,	O
and	O
their	O
diffusion	O
across	O
the	O
protein	B-structure_element
shell	I-structure_element
is	O
thought	O
to	O
be	O
restricted	O
,	O
necessitating	O
their	O
regeneration	O
within	O
the	O
BMC	B-complex_assembly
lumen	O
.	O

Both	O
enzymes	O
are	O
,	O
however	O
,	O
not	O
restricted	O
to	O
fermentative	B-taxonomy_domain
organisms	I-taxonomy_domain
.	O

Reaction	O
2	O
:	O
acetyl	B-chemical
phosphate	I-chemical
+	O
ADP	B-chemical
←→	O
acetate	B-chemical
+	O
ATP	B-chemical
(	O
Ack	B-protein_type
)	O

As	O
a	O
member	O
of	O
the	O
core	O
biochemical	O
machinery	O
of	O
functionally	O
diverse	O
aldehyde	B-protein_state
-	I-protein_state
oxidizing	I-protein_state
metabolosomes	B-complex_assembly
,	O
PduL	B-protein_type
must	O
have	O
a	O
certain	O
level	O
of	O
substrate	O
plasticity	O
(	O
see	O
Table	O
1	O
)	O
that	O
is	O
not	O
required	O
of	O
Pta	B-protein_type
,	O
which	O
has	O
generally	O
been	O
observed	O
to	O
prefer	O
acetyl	B-chemical
-	I-chemical
CoA	I-chemical
.	O
PduL	B-protein_type
from	O
the	O
PDU	B-complex_assembly
BMC	I-complex_assembly
of	O
Salmonella	B-species
enterica	I-species
favors	O
propionyl	B-chemical
-	I-chemical
CoA	I-chemical
over	O
acetyl	B-chemical
-	I-chemical
CoA	I-chemical
,	O
and	O
it	O
is	O
likely	O
that	O
PduL	B-protein_type
orthologs	O
in	O
functionally	O
diverse	O
BMCs	B-complex_assembly
would	O
have	O
substrate	O
preferences	O
for	O
other	O
CoA	B-chemical
derivatives	O
.	O

Of	O
the	O
three	O
common	O
metabolosome	B-complex_assembly
core	O
enzymes	O
,	O
crystal	B-evidence
structures	I-evidence
are	O
available	O
for	O
both	O
the	O
alcohol	B-protein_type
and	I-protein_type
aldehyde	I-protein_type
dehydrogenases	I-protein_type
.	O

No	O
available	O
protein	O
structures	O
contain	O
the	O
PF06130	B-structure_element
domain	O
,	O
and	O
homology	B-experimental_method
searches	I-experimental_method
using	O
the	O
primary	O
structure	O
of	O
PduL	B-protein_type
do	O
not	O
return	O
any	O
significant	O
results	O
that	O
would	O
allow	O
prediction	O
of	O
the	O
structure	B-evidence
.	O

Metal	B-site
coordination	I-site
residues	I-site
are	O
highlighted	O
in	O
light	O
blue	O
and	O
CoA	B-site
contacting	I-site
residues	I-site
in	O
magenta	O
,	O
residues	O
contacting	O
the	O
CoA	B-chemical
of	O
the	O
other	O
chain	O
are	O
also	O
outlined	O
.	O

We	O
were	O
able	O
to	O
fit	O
all	O
of	O
the	O
primary	O
structure	O
of	O
PduLΔEP	B-mutant
into	O
the	O
electron	B-evidence
density	I-evidence
with	O
the	O
exception	O
of	O
three	O
amino	O
acids	O
at	O
the	O
N	O
-	O
terminus	O
and	O
two	O
amino	O
acids	O
at	O
the	O
C	O
-	O
terminus	O
(	O
Fig	O
2a	O
);	O
the	O
model	O
is	O
of	O
excellent	O
quality	O
(	O
Table	O
2	O
).	O

This	O
β	B-structure_element
-	I-structure_element
sheet	I-structure_element
is	O
involved	O
in	O
contacts	O
between	O
the	O
two	O
domains	O
and	O
forms	O
a	O
lid	O
over	O
the	O
active	B-site
site	I-site
.	O

Primary	O
structure	O
conservation	O
of	O
the	O
PduL	B-protein_type
protein	O
family	O
.	O

The	O
interface	B-site
between	O
the	O
two	O
chains	O
buries	O
882	O
Å2	O
per	O
monomer	B-oligomeric_state
and	O
is	O
mainly	O
formed	O
by	O
α	B-structure_element
-	I-structure_element
helices	I-structure_element
2	I-structure_element
and	I-structure_element
4	I-structure_element
and	O
parts	O
of	O
β	B-structure_element
-	I-structure_element
sheets	I-structure_element
12	I-structure_element
and	I-structure_element
14	I-structure_element
,	O
as	O
well	O
as	O
a	O
π	O
–	O
π	O
stacking	O
of	O
the	O
adenine	B-chemical
moiety	O
of	O
CoA	B-chemical
with	O
Phe116	B-residue_name_number
of	O
the	O
adjacent	O
chain	O
(	O
Fig	O
4c	O
).	O

The	O
peripheral	O
helices	B-structure_element
and	O
the	O
short	B-structure_element
antiparallel	I-structure_element
β3	I-structure_element
–	I-structure_element
4	I-structure_element
sheet	I-structure_element
mediate	O
most	O
of	O
the	O
crystal	O
contacts	O
.	O

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

The	O
BMC	B-complex_assembly
shell	B-structure_element
not	O
only	O
sequesters	O
specific	O
enzymes	O
but	O
also	O
their	O
cofactors	O
,	O
thereby	O
establishing	O
a	O
private	O
cofactor	O
pool	O
dedicated	O
to	O
the	O
encapsulated	O
reactions	O
.	O

In	O
catabolic	B-protein_state
BMCs	B-complex_assembly
,	O
CoA	B-chemical
and	O
NAD	B-chemical
+	I-chemical
must	O
be	O
continually	O
recycled	O
within	O
the	O
organelle	O
(	O
Fig	O
1	O
).	O

The	O
Tertiary	O
Structure	O
of	O
PduL	B-protein_type
Is	O
Formed	O
by	O
Discontinuous	O
Segments	O
of	O
Primary	O
Structure	O

In	O
contrast	O
to	O
PduL	B-protein_type
,	O
there	O
is	O
only	O
one	O
barrel	B-structure_element
present	O
in	O
ethylbenzene	B-protein_type
dehydrogenase	I-protein_type
,	O
and	O
there	O
is	O
no	O
comparable	O
active	B-site
site	I-site
arrangement	O
.	O

EP	B-structure_element
-	O
mediated	O
oligomerization	O
has	O
been	O
observed	O
for	O
the	O
signature	O
and	O
core	O
BMC	B-complex_assembly
enzymes	O
;	O
for	O
example	O
,	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
propanediol	B-protein_type
dehydratase	I-protein_type
and	O
ethanolamine	B-protein_type
ammonia	I-protein_type
-	I-protein_type
lyase	I-protein_type
(	O
signature	O
enzymes	O
for	O
PDU	B-complex_assembly
and	O
EUT	B-complex_assembly
BMCs	I-complex_assembly
)	O
subunits	O
are	O
also	O
insoluble	O
,	O
but	O
become	O
soluble	O
upon	O
removal	O
of	O
the	O
predicted	O
EP	B-structure_element
.	O

This	O
propensity	O
of	O
the	O
EP	B-structure_element
to	O
cause	O
proteins	O
to	O
form	O
complexes	O
(	O
Fig	O
5	O
)	O
might	O
not	O
be	O
a	O
coincidence	O
,	O
but	O
could	O
be	O
a	O
necessary	O
step	O
in	O
the	O
assembly	O
of	O
BMCs	B-complex_assembly
.	O

There	O
is	O
a	O
pocket	B-site
nearby	O
the	O
active	B-site
site	I-site
between	O
the	O
well	B-protein_state
-	I-protein_state
conserved	I-protein_state
residues	O
Ser45	B-residue_name_number
and	O
Ala154	B-residue_name_number
,	O
which	O
could	O
accommodate	O
the	O
propionyl	O
group	O
(	O
S6	O
Fig	O
).	O

The	O
catalytic	O
mechanism	O
of	O
Pta	B-protein_type
involves	O
the	O
abstraction	O
of	O
a	O
thiol	O
hydrogen	O
by	O
an	O
aspartate	B-residue_name
residue	O
,	O
resulting	O
in	O
the	O
nucleophilic	O
attack	O
of	O
thiolate	O
upon	O
the	O
carbonyl	O
carbon	O
of	O
acetyl	B-chemical
-	I-chemical
phosphate	I-chemical
,	O
oriented	O
by	O
an	O
arginine	B-residue_name
and	O
stabilized	O
by	O
a	O
serine	B-residue_name
—	O
there	O
are	O
no	O
metals	O
involved	O
.	O

These	O
observations	O
strongly	O
suggest	O
that	O
an	O
acidic	B-protein_state
residue	B-structure_element
is	O
not	O
directly	O
involved	O
in	O
catalysis	O
by	O
PduL	B-protein_type
.	O
Instead	O
,	O
the	O
dimetal	B-site
active	I-site
site	I-site
of	O
PduL	B-protein_type
may	O
create	O
a	O
nucleophile	O
from	O
one	O
of	O
the	O
hydroxyl	O
groups	O
on	O
free	O
phosphate	B-chemical
to	O
attack	O
the	O
carbonyl	O
carbon	O
of	O
the	O
thioester	O
bond	O
of	O
an	O
acyl	B-chemical
-	I-chemical
CoA	I-chemical
.	O
In	O
the	O
reverse	O
direction	O
,	O
the	O
metal	O
ion	O
(	O
s	O
)	O
could	O
stabilize	O
the	O
thiolate	O
anion	O
that	O
would	O
attack	O
the	O
carbonyl	O
carbon	O
of	O
an	O
acyl	B-chemical
-	I-chemical
phosphate	I-chemical
;	O
a	O
similar	O
mechanism	O
has	O
been	O
described	O
for	O
phosphatases	B-protein_type
where	O
hydroxyl	O
groups	O
or	O
hydroxide	O
ions	O
can	O
act	O
as	O
a	O
base	O
when	O
coordinated	O
by	O
a	O
dimetal	B-site
active	I-site
site	I-site
.	O

Alternatively	O
,	O
Arg103	B-residue_name_number
might	O
act	O
as	O
a	O
base	O
to	O
render	O
the	O
phosphate	B-chemical
more	O
nucleophilic	O
.	O

The	O
free	O
CoA	B-protein_state
-	I-protein_state
bound	I-protein_state
form	O
is	O
presumably	O
poised	O
for	O
attack	O
upon	O
an	O
acyl	B-chemical
-	I-chemical
phosphate	I-chemical
,	O
indicating	O
that	O
the	O
enzyme	O
initially	O
binds	O
CoA	B-chemical
as	O
opposed	O
to	O
acyl	B-chemical
-	I-chemical
phosphate	I-chemical
.	O

We	O
have	O
observed	O
the	O
oligomeric	O
state	O
differences	O
of	O
PduL	B-protein_type
to	O
correlate	O
with	O
the	O
presence	O
of	O
an	O
EP	B-structure_element
,	O
providing	O
new	O
insight	O
into	O
the	O
function	O
of	O
this	O
sequence	O
extension	O
in	O
BMC	B-complex_assembly
assembly	O
.	O

Moreover	O
,	O
our	O
results	O
suggest	O
a	O
means	O
for	O
Coenzyme	B-chemical
A	I-chemical
incorporation	O
during	O
metabolosome	B-complex_assembly
biogenesis	O
.	O

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

We	O
found	O
that	O
the	O
NTD	B-structure_element
associates	O
with	O
the	O
PIN	B-structure_element
domain	O
and	O
significantly	O
enhances	O
its	O
RNase	B-protein_type
activity	O
.	O

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

The	O
NTD	B-structure_element
and	O
CTD	B-structure_element
are	O
both	O
composed	O
of	O
three	O
α	B-structure_element
helices	I-structure_element
,	O
and	O
structurally	O
resemble	O
ubiquitin	B-protein
conjugating	I-protein
enzyme	I-protein
E2	I-protein
K	I-protein
(	O
PDB	O
ID	O
:	O
3K9O	O
)	O
and	O
ubiquitin	B-protein
associated	I-protein
protein	I-protein
1	I-protein
(	O
PDB	O
ID	O
:	O
4AE4	O
),	O
respectively	O
,	O
according	O
to	O
the	O
Dali	B-experimental_method
server	I-experimental_method
.	O

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

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

On	O
the	O
other	O
hand	O
,	O
single	B-experimental_method
mutations	I-experimental_method
of	O
side	O
chains	O
involved	O
in	O
the	O
PIN	B-structure_element
–	O
PIN	B-structure_element
oligomeric	O
interaction	O
resulted	O
in	O
monomer	B-oligomeric_state
formation	O
,	O
judging	O
from	O
gel	B-experimental_method
filtration	I-experimental_method
(	O
Fig	O
.	O
2a	O
,	O
b	O
).	O

The	O
spectra	B-evidence
indicate	O
that	O
the	O
dimer	B-site
interface	I-site
of	O
the	O
wild	B-protein_state
type	I-protein_state
PIN	B-structure_element
domain	O
were	O
significantly	O
broadened	O
compared	O
to	O
the	O
monomeric	B-oligomeric_state
mutants	B-protein_state
(	O
Supplementary	O
Fig	O
.	O
4	O
).	O

These	O
results	O
indicate	O
that	O
the	O
PIN	B-structure_element
domain	O
forms	O
a	O
head	B-protein_state
-	I-protein_state
to	I-protein_state
-	I-protein_state
tail	I-protein_state
oligomer	B-oligomeric_state
in	O
solution	O
similar	O
to	O
the	O
crystal	B-evidence
structure	I-evidence
.	O

These	O
results	O
clearly	O
indicate	O
a	O
direct	O
interaction	O
between	O
the	O
PIN	B-structure_element
domain	O
and	O
the	O
NTD	B-structure_element
.	O

The	O
K184A	B-mutant
,	O
R215A	B-mutant
,	O
and	O
R220A	B-mutant
mutants	B-protein_state
moderately	O
but	O
significantly	O
decreased	O
the	O
cleavage	O
activity	O
for	O
both	O
target	O
mRNAs	B-chemical
.	O

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

Our	O
NMR	B-experimental_method
experiments	O
confirmed	O
direct	O
binding	O
of	O
the	O
ZF	B-structure_element
domain	O
to	O
IL	B-protein_type
-	I-protein_type
6	I-protein_type
mRNA	B-chemical
with	O
a	O
Kd	B-evidence
of	O
10	O
±	O
1	O
.	O
1	O
μM	O
.	O
Furthermore	O
,	O
an	O
in	B-experimental_method
vitro	I-experimental_method
gel	I-experimental_method
shift	I-experimental_method
assay	I-experimental_method
indicated	O
that	O
Regnase	B-protein
-	I-protein
1	I-protein
containing	O
the	O
ZF	B-structure_element
domain	O
enhanced	O
target	O
mRNA	B-chemical
-	O
binding	O
,	O
but	O
the	O
protein	O
-	O
RNA	B-chemical
complex	O
remained	O
in	O
the	O
bottom	O
of	O
the	O
well	O
without	O
entering	O
into	O
the	O
polyacrylamide	O
gel	O
.	O

These	O
results	O
indicate	O
that	O
Regnase	B-protein
-	I-protein
1	I-protein
directly	O
binds	O
to	O
RNA	B-chemical
and	O
precipitates	O
under	O
such	O
experimental	O
conditions	O
.	O

Moreover	O
,	O
we	O
found	O
that	O
the	O
NTD	B-structure_element
associates	O
with	O
the	O
oligomeric	B-site
surface	I-site
of	O
the	O
primary	B-protein_state
PIN	B-structure_element
,	O
docking	O
to	O
a	O
helix	B-structure_element
that	O
harbors	O
its	O
catalytic	B-site
residues	I-site
(	O
Figs	O
2b	O
and	O
3a	O
).	O

While	O
further	O
analyses	O
are	O
necessary	O
to	O
prove	O
this	O
point	O
,	O
our	O
preliminary	O
docking	B-experimental_method
and	I-experimental_method
molecular	I-experimental_method
dynamics	I-experimental_method
simulations	I-experimental_method
indicate	O
that	O
NTD	B-structure_element
-	O
binding	O
rearranges	O
the	O
catalytic	B-site
residues	I-site
of	O
the	O
PIN	B-structure_element
domain	O
toward	O
an	O
active	B-protein_state
conformation	O
suitable	O
for	O
binding	O
Mg2	B-chemical
+.	I-chemical

The	O
docking	B-experimental_method
result	O
revealed	O
multiple	O
RNA	B-chemical
binding	O
modes	O
that	O
satisfied	O
the	O
experimental	O
results	O
in	O
vitro	O
(	O
Supplementary	O
Fig	O
.	O
7c	O
,	O
d	O
),	O
however	O
,	O
it	O
should	O
be	O
noted	O
that	O
,	O
in	O
vivo	O
,	O
there	O
would	O
likely	O
be	O
many	O
other	O
RNA	B-protein_type
-	I-protein_type
binding	I-protein_type
proteins	I-protein_type
that	O
would	O
protect	O
loop	B-structure_element
regions	O
from	O
cleavage	O
by	O
Regnase	B-protein
-	I-protein
1	I-protein
.	O

The	O
overall	O
model	O
of	O
regulation	O
of	O
Regnase	B-protein
-	I-protein
1	I-protein
RNase	B-protein_type
activity	O
through	O
domain	O
-	O
domain	O
interactions	O
in	O
vitro	O
is	O
summarized	O
in	O
Fig	O
.	O
6	O
.	O

In	O
the	O
absence	B-protein_state
of	I-protein_state
target	O
mRNA	B-chemical
,	O
the	O
PIN	B-structure_element
domain	O
forms	O
head	B-protein_state
-	I-protein_state
to	I-protein_state
-	I-protein_state
tail	I-protein_state
oligomers	B-oligomeric_state
at	O
high	O
concentration	O
.	O

A	O
fully	B-protein_state
active	I-protein_state
catalytic	B-site
center	I-site
can	O
be	O
formed	O
only	O
when	O
the	O
NTD	B-structure_element
associates	O
with	O
the	O
oligomer	B-oligomeric_state
surface	O
of	O
the	O
PIN	B-structure_element
domain	O
,	O
which	O
terminates	O
the	O
head	B-protein_state
-	I-protein_state
to	I-protein_state
-	I-protein_state
tail	I-protein_state
oligomer	B-oligomeric_state
formation	O
in	O
one	O
direction	O
(	O
primary	B-protein_state
PIN	B-structure_element
),	O
and	O
forms	O
a	O
functional	B-protein_state
dimer	B-oligomeric_state
together	O
with	O
the	O
neighboring	O
PIN	B-structure_element
(	O
secondary	B-protein_state
PIN	B-structure_element
).	O

Catalytic	B-protein_state
Asp	B-residue_name
residues	O
were	O
shown	O
in	O
sticks	O
.	O

Three	O
Cys	B-residue_name
residues	O
and	O
one	O
His	B-residue_name
residue	O
responsible	O
for	O
Zn2	O
+-	O
binding	O
were	O
shown	O
in	O
sticks	O
.	O

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

Catalytic	B-site
residues	I-site
and	O
mutated	O
residues	O
were	O
shown	O
in	O
sticks	O
.	O

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

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

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

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

Schematic	O
representation	O
of	O
regulation	O
of	O
the	O
Regnase	B-protein
-	I-protein
1	I-protein
catalytic	O
activity	O
through	O
the	O
domain	O
-	O
domain	O
interactions	O
.	O

Ribosomal	B-chemical
RNA	I-chemical
modifications	O
have	O
been	O
suggested	O
to	O
optimize	O
ribosome	O
function	O
,	O
although	O
in	O
most	O
cases	O
this	O
remains	O
to	O
be	O
clearly	O
established	O
.	O

Most	O
modified	O
rRNA	B-chemical
nucleotides	B-chemical
cluster	O
in	O
the	O
vicinity	O
of	O
the	O
decoding	B-site
or	O
the	O
peptidyl	B-site
transferase	I-site
center	I-site
,	O
suggesting	O
an	O
influence	O
on	O
ribosome	O
functionality	O
and	O
stability	O
.	O

Both	O
the	O
methyl	O
and	O
the	O
acp	O
group	O
are	O
derived	O
from	O
S	B-chemical
-	I-chemical
adenosylmethionine	I-chemical
(	O
SAM	B-chemical
),	O
but	O
the	O
enzyme	O
responsible	O
for	O
acp	B-chemical
modification	O
remained	O
elusive	O
for	O
more	O
than	O
40	O
years	O
.	O

Tsr3	B-protein
is	O
necessary	O
for	O
acp	B-chemical
modification	O
of	O
18S	B-chemical
rRNA	I-chemical
in	O
yeast	B-taxonomy_domain
and	O
human	B-species
.	O
(	O
A	O
)	O
Hypermodified	B-protein_state
nucleotide	B-chemical
m1acp3Ψ	B-chemical
is	O
synthesized	O
in	O
three	O
steps	O
:	O
pseudouridylation	B-ptm
catalyzed	O
by	O
snoRNP35	B-complex_assembly
,	O
N1	B-ptm
-	I-ptm
methylation	I-ptm
catalyzed	O
by	O
methyltransferase	B-protein_type
Nep1	B-protein
and	O
N3	O
-	O
acp	B-chemical
modification	O
catalyzed	O
by	O
Tsr3	B-protein
.	O

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

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

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

The	O
primer	O
extension	O
stop	O
at	O
nucleotide	O
1191	B-residue_number
is	O
missing	O
exclusively	O
in	O
Δtsr3	B-mutant
mutants	O
and	O
Δtsr3	B-mutant
Δsnr35	I-mutant
recombinants	O
.	O

The	O
efficiency	O
of	O
siRNA	B-chemical
mediated	O
HsTSR3	B-protein
repression	O
correlates	O
with	O
the	O
primer	B-evidence
extension	I-evidence
signals	I-evidence
(	O
see	O
Supplementary	O
Figure	O
S2A	O
).	O

During	O
the	O
biosynthesis	O
of	O
wybutosine	B-chemical
,	O
a	O
tricyclic	O
nucleoside	B-chemical
present	O
in	O
eukaryotic	B-taxonomy_domain
and	O
archaeal	B-taxonomy_domain
phenylalanine	B-chemical
tRNA	B-chemical
,	O
Tyw2	B-protein
(	O
Trm12	B-protein
in	O
yeast	B-taxonomy_domain
)	O
transfers	O
an	O
acp	B-chemical
group	O
from	O
SAM	B-chemical
to	O
an	O
acidic	O
carbon	O
atom	O
.	O

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

In	O
a	O
recent	O
bioinformatic	O
study	O
,	O
the	O
uncharacterized	O
yeast	B-taxonomy_domain
gene	O
YOR006c	B-gene
was	O
predicted	O
to	O
be	O
involved	O
in	O
ribosome	O
biogenesis	O
.	O

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

However	O
,	O
its	O
function	O
remained	O
unclear	O
although	O
recently	O
a	O
putative	O
nuclease	O
function	O
during	O
18S	B-chemical
rRNA	I-chemical
maturation	O
was	O
predicted	O
.	O

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

No	O
radioactive	O
labeling	O
was	O
detected	O
in	O
the	O
18S	B-mutant
U1191A	I-mutant
mutant	B-protein_state
which	O
served	O
as	O
a	O
control	O
for	O
the	O
specificity	O
of	O
the	O
14C	B-chemical
-	I-chemical
aminocarboxypropyl	I-chemical
incorporation	O
.	O

The	O
Tsr3	B-protein
protein	O
is	O
highly	B-protein_state
conserved	I-protein_state
in	O
yeast	B-taxonomy_domain
and	O
humans	B-species
(	O
50	O
%	O
identity	O
).	O

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

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

(	O
D	O
)	O
Cytoplasmic	O
localization	O
of	O
yeast	B-taxonomy_domain
Tsr3	B-protein
shown	O
by	O
fluorescence	B-experimental_method
microscopy	I-experimental_method
of	O
GFP	B-mutant
-	I-mutant
fused	I-mutant
Tsr3	I-mutant
.	O

From	O
left	O
to	O
right	O
:	O
differential	B-experimental_method
interference	I-experimental_method
contrast	I-experimental_method
(	O
DIC	B-experimental_method
),	O
green	O
fluorescence	O
of	O
GFP	B-mutant
-	I-mutant
Tsr3	I-mutant
,	O
red	O
fluorescence	O
of	O
Nop56	B-mutant
-	I-mutant
mRFP	I-mutant
as	O
nucleolar	O
marker	O
,	O
and	O
merge	O
of	O
GFP	B-mutant
-	I-mutant
Tsr3	I-mutant
/	O
Nop56	B-mutant
-	I-mutant
mRFP	I-mutant
with	O
DIC	B-experimental_method
.	O
(	O
E	O
)	O
Elution	B-evidence
profile	I-evidence
(	O
A254	O
)	O
after	O
sucrose	B-experimental_method
gradient	I-experimental_method
separation	I-experimental_method
of	O
yeast	B-taxonomy_domain
ribosomal	B-complex_assembly
subunits	I-complex_assembly
and	O
polysomes	B-complex_assembly
(	O
upper	O
part	O
)	O
and	O
western	B-experimental_method
blot	I-experimental_method
analysis	O
of	O
3xHA	B-chemical
tagged	O
Tsr3	B-protein
(	O
Tsr3	B-mutant
-	I-mutant
3xHA	I-mutant
)	O
after	O
SDS	B-experimental_method
-	I-experimental_method
PAGE	I-experimental_method
separation	O
of	O
polysome	O
profile	O
fractions	O
taken	O
every	O
20	O
s	O
(	O
lower	O
part	O
).	O

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

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

This	O
agrees	O
with	O
previous	O
biochemical	O
data	O
suggesting	O
that	O
the	O
acp	B-chemical
modification	O
of	O
18S	B-chemical
rRNA	I-chemical
occurs	O
late	O
during	O
40S	B-complex_assembly
subunit	O
biogenesis	O
in	O
the	O
cytoplasm	O
,	O
and	O
makes	O
an	O
additional	O
nuclear	O
localization	O
as	O
reported	O
in	O
a	O
previous	O
large	O
-	O
scale	O
analysis	O
unlikely	O
.	O

Such	O
distribution	B-evidence
on	I-evidence
a	I-evidence
density	I-evidence
gradient	I-evidence
suggests	O
that	O
Tsr3	B-protein
only	O
interacts	O
transiently	O
with	O
pre	B-complex_assembly
-	I-complex_assembly
40S	I-complex_assembly
subunits	I-complex_assembly
,	O
which	O
presumably	O
explains	O
why	O
it	O
was	O
not	O
characterized	O
in	O
pre	B-experimental_method
-	I-experimental_method
ribosome	I-experimental_method
affinity	I-experimental_method
purifications	I-experimental_method
.	O

Structure	B-evidence
of	O
Tsr3	B-protein

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

A	O
weak	O
20S	B-chemical
rRNA	I-chemical
signal	O
,	O
indicating	O
normal	O
processing	O
,	O
is	O
observed	O
for	O
Tsr3	B-protein
fragment	O
46	B-residue_range
–	I-residue_range
270	I-residue_range
(	O
highlighted	O
in	O
a	O
box	O
)	O
showing	O
its	O
functionality	O
.	O

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

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

The	O
closest	O
structural	O
homolog	O
identified	O
in	O
a	O
DALI	B-experimental_method
search	I-experimental_method
is	O
the	O
tRNA	B-protein_type
methyltransferase	I-protein_type
Trm10	B-protein
(	O
DALI	B-evidence
Z	I-evidence
-	I-evidence
score	I-evidence
6	O
.	O
8	O
)	O
which	O
methylates	O
the	O
N1	O
nitrogen	O
of	O
G9	B-residue_name_number
/	O
A9	B-residue_name_number
in	O
many	O
archaeal	B-taxonomy_domain
and	O
eukaryotic	B-taxonomy_domain
tRNAs	B-chemical
by	O
using	O
SAM	B-chemical
as	O
the	O
methyl	O
group	O
donor	O
.	O

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

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

Furthermore	O
,	O
the	O
adenine	B-chemical
base	O
of	O
SAM	B-chemical
is	O
involved	O
in	O
hydrophobic	O
packing	O
interactions	O
with	O
the	O
side	O
chains	O
of	O
L45	B-residue_name_number
(	O
β3	B-structure_element
),	O
P47	B-residue_name_number
and	O
W73	B-residue_name_number
(	O
α3	B-structure_element
)	O
in	O
the	O
N	B-structure_element
-	I-structure_element
terminal	I-structure_element
domain	I-structure_element
as	O
well	O
as	O
with	O
L93	B-residue_name_number
,	O
L110	B-residue_name_number
(	O
both	O
in	O
the	O
loop	B-structure_element
connecting	O
β5	B-structure_element
and	O
α4	B-structure_element
)	O
and	O
A115	B-residue_name_number
(	O
α5	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
ribose	B-chemical
2	O
′	O
and	O
3	O
′	O
hydroxyl	O
groups	O
of	O
SAM	B-chemical
are	O
hydrogen	O
bonded	O
to	O
the	O
backbone	O
carbonyl	O
group	O
of	O
I69	B-residue_name_number
.	O

Most	O
importantly	O
,	O
the	O
methyl	O
group	O
of	O
SAM	B-chemical
is	O
buried	O
in	O
a	O
hydrophobic	B-site
pocket	I-site
formed	O
by	O
the	O
sidechains	O
of	O
W73	B-residue_name_number
and	O
A76	B-residue_name_number
both	O
located	O
in	O
α3	B-structure_element
(	O
Figure	O
5A	O
and	O
B	O
).	O

Consequently	O
,	O
the	O
accessibility	O
of	O
this	O
methyl	O
group	O
for	O
a	O
nucleophilic	O
attack	O
is	O
strongly	O
reduced	O
in	O
comparison	O
with	O
RNA	B-protein_type
-	I-protein_type
methyltransferases	I-protein_type
such	O
as	O
Trm10	B-protein
(	O
Figure	O
5B	O
,	O
C	O
).	O

In	O
contrast	O
,	O
the	O
acp	B-chemical
side	O
chain	O
of	O
SAM	B-chemical
is	O
accessible	O
for	O
reactions	O
in	O
the	O
Tsr3	B-protein_state
-	I-protein_state
bound	I-protein_state
state	O
(	O
Figure	O
5B	O
).	O

(	O
E	O
)	O
Binding	O
of	O
14C	B-chemical
-	I-chemical
labeled	I-chemical
SAM	I-chemical
to	O
SsTsr3	B-protein
.	O

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

On	O
the	O
other	O
hand	O
,	O
the	O
loss	O
of	O
hydrogen	O
bonds	O
between	O
the	O
acp	B-chemical
sidechain	O
carboxylate	O
group	O
and	O
the	O
protein	O
appears	O
to	O
be	O
thermodynamically	O
less	O
important	O
but	O
these	O
hydrogen	O
bonds	O
might	O
play	O
a	O
crucial	O
role	O
for	O
the	O
proper	O
orientation	O
of	O
the	O
cofactor	O
side	O
chain	O
in	O
the	O
substrate	B-site
binding	I-site
pocket	I-site
.	O

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

Helix	B-structure_element
α1	B-structure_element
contains	O
two	O
more	O
positively	O
charged	O
amino	O
acids	O
K17	B-residue_name_number
and	O
R25	B-residue_name_number
as	O
does	O
the	O
loop	B-structure_element
preceding	O
it	O
(	O
R9	B-residue_name_number
).	O

Also	O
shown	O
in	O
stick	O
representation	O
is	O
a	O
negatively	O
charged	O
MES	B-chemical
ion	O
.	O

Conserved	B-protein_state
basic	O
amino	B-chemical
acids	I-chemical
are	O
labeled	O
.	O
(	O
B	O
)	O
Comparison	O
of	O
the	O
secondary	O
structures	O
of	O
helix	B-structure_element
31	I-structure_element
from	O
the	O
small	O
ribosomal	O
subunit	O
rRNAs	B-chemical
in	O
S	B-species
.	I-species
cerevisiae	I-species
and	O
S	B-species
.	I-species
solfataricus	I-species
with	O
the	O
location	O
of	O
the	O
hypermodified	B-protein_state
nucleotide	B-chemical
indicated	O
in	O
red	O
.	O

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

Similar	O
to	O
the	O
structurally	O
unrelated	O
Rossmann	B-protein_type
-	I-protein_type
fold	I-protein_type
Tyw2	I-protein_type
acp	I-protein_type
transferase	I-protein_type
,	O
the	O
SAM	B-chemical
methyl	O
group	O
of	O
Tsr3	B-protein
is	O
bound	O
in	O
an	O
inaccessible	O
hydrophobic	B-site
pocket	I-site
whereas	O
the	O
acp	B-chemical
side	O
chain	O
becomes	O
accessible	O
for	O
a	O
nucleophilic	O
attack	O
by	O
the	O
N3	O
of	O
pseudouridine	B-chemical
.	O

Thus	O
,	O
additional	O
examples	O
for	O
acp	B-protein_type
transferase	I-protein_type
enzymes	O
might	O
be	O
found	O
with	O
similarities	O
to	O
other	O
structural	O
classes	O
of	O
methyltransferases	B-protein_type
.	O

These	O
data	O
and	O
the	O
finding	O
that	O
a	O
missing	O
acp	B-chemical
modification	O
hinders	O
pre	B-chemical
-	I-chemical
20S	I-chemical
rRNA	I-chemical
processing	O
,	O
suggest	O
that	O
the	O
acp	B-chemical
modification	O
together	O
with	O
the	O
release	O
of	O
Rio2	B-protein
promotes	O
the	O
formation	O
of	O
the	O
decoding	B-site
site	I-site
and	O
thus	O
D	B-site
-	I-site
site	I-site
cleavage	O
by	O
Nob1	B-protein
.	O

Therefore	O
,	O
Rio2	B-protein
either	O
blocks	O
the	O
access	O
of	O
Tsr3	B-protein
to	O
helix	B-structure_element
31	I-structure_element
,	O
and	O
acp	B-chemical
modification	O
can	O
only	O
occur	O
after	O
Rio2	B-protein
is	O
released	O
,	O
or	O
the	O
acp	B-chemical
modification	O
of	O
m1Ψ1191	B-residue_name_number
and	O
putative	O
subsequent	O
conformational	O
changes	O
of	O
20S	B-chemical
rRNA	I-chemical
weaken	O
the	O
binding	O
of	O
Rio2	B-protein
to	O
helix	B-structure_element
31	I-structure_element
and	O
support	O
its	O
release	O
from	O
the	O
pre	B-chemical
-	I-chemical
rRNA	I-chemical
.	O

Presence	O
of	O
a	O
hypermodified	B-protein_state
nucleotide	O
in	O
HeLa	O
cell	O
18	O
S	O
and	O
Saccharomyces	O
carlsbergensis	O
17	O
S	O
ribosomal	O
RNAs	O

Crystal	B-evidence
Structure	I-evidence
and	O
Activity	B-experimental_method
Studies	I-experimental_method
of	O
the	O
C11	B-protein_type
Cysteine	B-protein_type
Peptidase	I-protein_type
from	O
Parabacteroides	B-species
merdae	I-species
in	O
the	O
Human	B-species
Gut	O
Microbiome	O
*	O

Collectively	O
,	O
these	O
data	O
provide	O
insights	O
into	O
the	O
mechanism	O
and	O
activity	O
of	O
PmC11	B-protein
and	O
a	O
detailed	O
framework	O
for	O
studies	O
on	O
C11	B-protein_type
peptidases	I-protein_type
from	O
other	O
phylogenetic	O
kingdoms	O
.	O

Interestingly	O
,	O
little	O
is	O
known	O
about	O
the	O
structure	O
or	O
function	O
of	O
the	O
C11	B-protein_type
proteins	O
,	O
despite	O
their	O
widespread	O
distribution	O
and	O
its	O
archetypal	O
member	O
,	O
clostripain	B-protein
from	O
Clostridium	B-species
histolyticum	I-species
,	O
first	O
reported	O
in	O
the	O
literature	O
in	O
1938	O
.	O

As	O
part	O
of	O
an	O
ongoing	O
project	O
to	O
characterize	O
commensal	O
bacteria	B-taxonomy_domain
in	O
the	O
microbiome	O
that	O
inhabit	O
the	O
human	B-species
gut	O
,	O
the	O
structure	B-evidence
of	O
C11	B-protein_type
peptidase	I-protein_type
,	O
PmC11	B-protein
,	O
from	O
Parabacteroides	B-species
merdae	I-species
was	O
determined	O
using	O
the	O
Joint	O
Center	O
for	O
Structural	O
Genomics	O
(	O
JCSG	O
)	O
4	O
HTP	O
structural	O
biology	O
pipeline	O
.	O

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

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

B	O
,	O
topology	O
diagram	O
of	O
PmC11	B-protein
colored	O
as	O
in	O
A	O
except	O
that	O
additional	O
(	O
non	O
-	O
core	O
)	O
β	B-structure_element
-	I-structure_element
strands	I-structure_element
are	O
in	O
yellow	O
.	O

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

PmC11	O
migrates	O
as	O
a	O
monomer	B-oligomeric_state
with	O
a	O
molecular	O
mass	O
around	O
41	O
kDa	O
calculated	O
from	O
protein	O
standards	O
of	O
known	O
molecular	O
weights	O
.	O

Km	O
and	O
Vmax	B-evidence
of	O
PmC11	B-protein
and	O
K147A	B-mutant
mutant	O
were	O
determined	O
by	O
monitoring	O
change	O
in	O
the	O
fluorescence	O
corresponding	O
to	O
AMC	O
release	O
from	O
Bz	B-chemical
-	I-chemical
R	I-chemical
-	I-chemical
AMC	I-chemical
.	O

G	O
,	O
electrostatic	O
surface	O
potential	O
of	O
PmC11	B-protein
shown	O
in	O
a	O
similar	O
orientation	O
,	O
where	O
blue	O
and	O
red	O
denote	O
positively	O
and	O
negatively	O
charged	O
surface	O
potential	O
,	O
respectively	O
,	O
contoured	O
at	O
±	O
5	O
kT	O
/	O
e	O
.	O

Purification	B-experimental_method
of	O
recombinant	O
PmC11	B-protein
(	O
molecular	O
mass	O
=	O
42	O
.	O
6	O
kDa	O
)	O
revealed	O
partial	O
processing	O
into	O
two	O
cleavage	O
products	O
of	O
26	O
.	O
4	O
and	O
16	O
.	O
2	O
kDa	O
,	O
related	O
to	O
the	O
observed	O
cleavage	B-ptm
at	O
Lys147	B-residue_name_number
in	O
the	O
crystal	B-evidence
structure	I-evidence
(	O
Fig	O
.	O
2A	O
).	O

Moreover	O
,	O
the	O
C	O
-	O
terminal	O
side	O
of	O
the	O
cleavage	B-site
site	I-site
resides	O
near	O
the	O
catalytic	B-site
dyad	I-site
with	O
Ala148	B-residue_name_number
being	O
4	O
.	O
5	O
and	O
5	O
.	O
7	O
Å	O
from	O
His133	B-residue_name_number
and	O
Cys179	B-residue_name_number
,	O
respectively	O
.	O

However	O
,	O
this	O
loop	B-structure_element
has	O
been	O
shown	O
to	O
be	O
important	O
for	O
substrate	O
binding	O
in	O
clan	B-protein_type
CD	I-protein_type
and	O
this	O
residue	O
could	O
easily	O
rotate	O
and	O
be	O
involved	O
in	O
substrate	O
binding	O
in	O
PmC11	B-protein
.	O

In	O
support	O
of	O
these	O
findings	O
,	O
EGTA	B-chemical
did	O
not	O
inhibit	O
PmC11	B-protein
suggesting	O
that	O
,	O
unlike	O
clostripain	B-protein
,	O
PmC11	B-protein
does	O
not	O
require	O
Ca2	B-chemical
+	I-chemical
or	O
other	O
divalent	O
cations	O
,	O
for	O
activity	O
.	O

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

All	O
other	O
clan	B-protein_type
CD	I-protein_type
members	I-protein_type
requiring	O
cleavage	B-ptm
for	O
full	B-protein_state
activation	I-protein_state
do	O
so	O
at	O
sites	B-site
external	O
to	O
their	O
central	O
sheets	B-structure_element
.	O

Indeed	O
,	O
insights	O
gained	O
from	O
an	O
analysis	O
of	O
the	O
PmC11	B-protein
structure	B-evidence
revealed	O
the	O
identity	O
of	O
the	O
Trypanosoma	B-species
brucei	I-species
PNT1	B-protein
protein	O
as	O
a	O
C11	B-protein_type
cysteine	I-protein_type
peptidase	I-protein_type
with	O
an	O
essential	O
role	O
in	O
organelle	O
replication	O
.	O

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

More	O
recently	O
,	O
this	O
system	O
was	O
also	O
reported	O
in	O
other	O
Gram	B-taxonomy_domain
-	I-taxonomy_domain
negative	I-taxonomy_domain
bacteria	I-taxonomy_domain
,	O
such	O
as	O
Escherichia	B-species
coli	I-species
(	O
Hufnagel	O
et	O
al	O
.,;	O
Raterman	O
et	O
al	O
.,;	O
Sanchez	O
-	O
Torres	O
et	O
al	O
.,),	O
Klebsiella	B-species
pneumonia	I-species
(	O
Huertas	O
et	O
al	O
.,)	O
and	O
Yersinia	B-species
pestis	I-species
(	O
Ren	O
et	O
al	O
.,).	O

In	O
addition	O
,	O
quorum	O
sensing	O
-	O
related	O
dephosphorylation	O
of	O
the	O
PAS	B-structure_element
domain	I-structure_element
of	O
YfiN	B-protein
may	O
also	O
be	O
involved	O
in	O
the	O
regulation	O
(	O
Ueda	O
and	O
Wood	O
,;	O
Xu	O
et	O
al	O
.,).	O

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

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

The	O
residues	O
proposed	O
to	O
contribute	O
to	O
YfiB	B-protein
activation	O
are	O
illustrated	O
in	O
sticks	O
.	O

The	O
key	O
residues	O
in	O
apo	B-protein_state
YfiB	B-protein
are	O
shown	O
in	O
red	O
and	O
those	O
in	O
YfiBL43P	B-mutant
are	O
shown	O
in	O
blue	O
.	O
(	O
D	O
)	O
Close	O
-	O
up	O
views	O
showing	O
interactions	O
in	O
regions	B-structure_element
I	I-structure_element
and	I-structure_element
II	I-structure_element
.	O

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

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

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

Previous	O
homology	B-experimental_method
modeling	I-experimental_method
studies	O
suggested	O
that	O
YfiB	B-protein
contains	O
a	O
Pal	B-site
-	I-site
like	I-site
PG	I-site
-	I-site
binding	I-site
site	I-site
(	O
Parsons	O
et	O
al	O
.,),	O
and	O
the	O
mutation	B-experimental_method
of	I-experimental_method
two	I-experimental_method
residues	I-experimental_method
at	O
this	O
site	O
,	O
D102	B-residue_name_number
and	O
G105	B-residue_name_number
,	O
reduces	O
the	O
ability	O
for	O
biofilm	O
formation	O
and	O
surface	O
attachment	O
(	O
Malone	O
et	O
al	O
.,).	O

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

Previous	O
studies	O
indicated	O
that	O
YfiR	B-protein
constitutes	O
a	O
YfiB	B-protein
-	O
independent	O
sensing	O
device	O
that	O
can	O
activate	O
YfiN	B-protein
in	O
response	O
to	O
the	O
redox	O
status	O
of	O
the	O
periplasm	O
,	O
and	O
we	O
have	O
reported	O
YfiR	B-protein
structures	B-evidence
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
earlier	O
,	O
revealing	O
that	O
the	O
Cys145	B-residue_name_number
-	O
Cys152	B-residue_name_number
disulfide	B-ptm
bond	I-ptm
plays	O
an	O
essential	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
E	B-species
.	I-species
coli	I-species
,	O
mutants	O
with	O
decreased	O
tryptophan	B-chemical
synthesis	O
show	O
greater	O
biofilm	O
formation	O
,	O
and	O
matured	O
biofilm	O
is	O
degraded	O
by	O
L	B-chemical
-	I-chemical
tryptophan	I-chemical
addition	O
(	O
Shimazaki	O
et	O
al	O
.,).	O

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

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

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

This	O
allows	O
the	O
C	B-structure_element
-	I-structure_element
terminal	I-structure_element
portion	I-structure_element
of	O
the	O
membrane	B-protein_state
-	I-protein_state
anchored	I-protein_state
YfiB	B-protein
to	O
reach	O
,	O
bind	O
and	O
penetrate	O
the	O
cell	O
wall	O
and	O
sequester	O
the	O
YfiR	B-protein
dimer	B-oligomeric_state
.	O

The	O
YfiBNR	B-complex_assembly
system	O
provides	O
a	O
good	O
example	O
of	O
a	O
delicate	O
homeostatic	O
system	O
that	O
integrates	O
multiple	O
signals	O
to	O
regulate	O
the	O
c	B-chemical
-	I-chemical
di	I-chemical
-	I-chemical
GMP	I-chemical
level	O
.	O

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

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

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

In	O
the	O
canonical	O
model	O
of	O
the	O
ERα	B-protein
signaling	O
pathway	O
(	O
Fig	O
1C	O
),	O
E2	B-protein_state
‐	I-protein_state
bound	I-protein_state
ERα	B-protein
forms	O
a	O
homodimer	B-oligomeric_state
that	O
binds	O
DNA	O
at	O
estrogen	B-site
‐	I-site
response	I-site
elements	I-site
(	O
EREs	B-site
),	O
recruits	O
NCOA1	B-protein
/	I-protein
2	I-protein
/	I-protein
3	I-protein
(	O
Metivier	O
et	O
al	O
,	O
2003	O
;	O
Johnson	O
&	O
O	O
'	O
Malley	O
,	O
2012	O
),	O
and	O
activates	O
the	O
GREB1	B-protein
gene	O
,	O
which	O
is	O
required	O
for	O
proliferation	O
of	O
ERα	B-protein
‐	O
positive	O
breast	O
cancer	O
cells	O
(	O
Ghosh	O
et	O
al	O
,	O
2000	O
;	O
Rae	O
et	O
al	O
,	O
2005	O
;	O
Deschenes	O
et	O
al	O
,	O
2007	O
;	O
Liu	O
et	O
al	O
,	O
2012	O
;	O
Srinivasan	O
et	O
al	O
,	O
2013	O
).	O

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

Our	O
findings	O
here	O
indicate	O
that	O
specific	O
structural	O
perturbations	O
can	O
be	O
tied	O
to	O
ligand	O
‐	O
selective	O
domain	O
usage	O
and	O
signaling	O
patterns	O
,	O
thus	O
providing	O
a	O
framework	O
for	O
structure	O
‐	O
based	O
design	O
of	O
improved	O
breast	O
cancer	O
therapeutics	O
,	O
and	O
understanding	O
the	O
different	O
phenotypic	O
effects	O
of	O
environmental	O
estrogens	B-chemical
.	O

Structural	O
details	O
of	O
the	O
ERα	B-protein
LBD	B-structure_element
bound	B-protein_state
to	I-protein_state
the	O
indicated	O
ligands	O
.	O

To	O
this	O
end	O
,	O
we	O
compared	O
the	O
average	O
ligand	O
‐	O
induced	O
GREB1	B-protein
mRNA	O
levels	O
in	O
MCF	O
‐	O
7	O
cells	O
and	O
3	B-experimental_method
×	I-experimental_method
ERE	I-experimental_method
‐	I-experimental_method
Luc	I-experimental_method
reporter	O
gene	O
activity	O
in	O
Ishikawa	O
endometrial	O
cancer	O
cells	O
(	O
E	B-experimental_method
‐	I-experimental_method
Luc	I-experimental_method
)	O
or	O
in	O
HepG2	O
cells	O
transfected	O
with	O
wild	B-protein_state
‐	I-protein_state
type	I-protein_state
ERα	B-protein
(	O
L	B-experimental_method
‐	I-experimental_method
Luc	I-experimental_method
ERα	B-protein
‐	O
WT	B-protein_state
)	O
(	O
Figs	O
3A	O
and	O
EV2A	O
–	O
C	O
).	O

Direct	O
modulators	O
showed	O
significant	O
differences	O
in	O
average	O
activity	O
between	O
cell	O
types	O
except	O
OBHS	B-chemical
‐	I-chemical
ASC	I-chemical
analogs	O
,	O
which	O
had	O
similar	O
low	O
agonist	O
activities	O
in	O
the	O
three	O
cell	O
types	O
.	O

Significant	O
sensitivity	O
to	O
AB	B-structure_element
domain	O
deletion	O
was	O
determined	O
by	O
Student	B-experimental_method
'	I-experimental_method
s	I-experimental_method
t	I-experimental_method
‐	I-experimental_method
test	I-experimental_method
(	O
n	O
=	O
number	O
of	O
ligands	O
per	O
scaffold	O
in	O
Fig	O
2	O
).	O

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

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

OBHS	B-chemical
analogs	O
showed	O
an	O
average	O
L	B-experimental_method
‐	I-experimental_method
Luc	I-experimental_method
ERα	B-mutant
‐	I-mutant
ΔAB	I-mutant
activity	O
of	O
3	O
.	O
2	O
%	O
±	O
3	O
(	O
mean	O
+	O
SEM	O
)	O
relative	O
to	O
E2	B-chemical
.	O

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

These	O
results	O
suggest	O
that	O
compounds	O
that	O
show	O
cell	O
‐	O
specific	O
signaling	O
do	O
not	O
activate	O
GREB1	B-protein
,	O
or	O
use	O
coactivators	O
other	O
than	O
NCOA1	B-protein
/	I-protein
2	I-protein
/	I-protein
3	I-protein
to	O
control	O
GREB1	B-protein
expression	O
(	O
Fig	O
1E	O
).	O

For	O
ligands	O
that	O
show	O
cell	O
‐	O
specific	O
signaling	O
,	O
ERα	B-protein
‐	O
mediated	O
recruitment	O
of	O
other	O
coregulators	O
and	O
activation	O
of	O
other	O
target	O
genes	O
likely	O
determine	O
their	O
proliferative	O
effects	O
on	O
MCF	O
‐	O
7	O
cells	O
.	O

At	O
this	O
time	O
point	O
,	O
other	O
WAY	B-chemical
‐	I-chemical
C	I-chemical
analogs	O
also	O
induced	O
recruitment	O
of	O
NCOA3	B-protein
at	O
this	O
site	O
to	O
varying	O
degrees	O
(	O
Fig	O
4B	O
).	O

The	O
Z	B-evidence
’	I-evidence
for	O
this	O
assay	O
was	O
0	O
.	O
6	O
,	O
showing	O
statistical	O
robustness	O
(	O
see	O
Materials	O
and	O
Methods	O
).	O

For	O
most	O
scaffolds	O
,	O
L	B-experimental_method
‐	I-experimental_method
Luc	I-experimental_method
ERβ	O
and	O
E	B-experimental_method
‐	I-experimental_method
Luc	I-experimental_method
activities	O
were	O
not	O
correlated	O
,	O
except	O
for	O
2	B-chemical
,	I-chemical
5	I-chemical
‐	I-chemical
DTP	I-chemical
and	O
cyclofenil	B-chemical
analogs	O
,	O
which	O
showed	O
moderate	O
but	O
significant	O
correlations	O
(	O
Fig	O
EV4A	O
).	O

ERβ	B-protein
activity	O
is	O
not	O
an	O
independent	O
predictor	O
of	O
E	B-experimental_method
‐	I-experimental_method
Luc	I-experimental_method
activity	O

ERβ	B-protein
activity	O
in	O
HepG2	O
cells	O
rarely	O
correlates	O
with	O
E	B-experimental_method
‐	I-experimental_method
Luc	I-experimental_method
activity	O
.	O

Data	O
information	O
:	O
The	O
r	O
2	O
and	O
P	B-evidence
values	I-evidence
for	O
the	O
indicated	O
correlations	O
are	O
shown	O
in	O
both	O
panels	O
.	O
*	O
Significant	O
positive	O
correlation	O
(	O
F	B-experimental_method
‐	I-experimental_method
test	I-experimental_method
for	O
nonzero	O
slope	O
,	O
P	B-evidence
‐	I-evidence
value	I-evidence
)	O

Remarkably	O
,	O
these	O
individual	O
inter	B-evidence
‐	I-evidence
atomic	I-evidence
distances	I-evidence
showed	O
a	O
ligand	O
class	O
‐	O
specific	O
ability	O
to	O
significantly	O
predict	O
proliferative	O
effects	O
(	O
Fig	O
5E	O
and	O
F	O
),	O
demonstrating	O
the	O
feasibility	O
of	O
developing	O
a	O
minimal	O
set	O
of	O
activity	O
predictors	O
from	O
crystal	B-evidence
structures	I-evidence
.	O

ERα	B-protein
LBD	B-structure_element
structures	B-evidence
bound	B-protein_state
to	I-protein_state
4	O
distinct	O
WAY	B-chemical
‐	I-chemical
C	I-chemical
analogs	O
were	O
superposed	B-experimental_method
(	O
PDB	O
4	O
IU7	O
,	O
4IV4	O
,	O
4IVW	O
,	O
4IW6	O
)	O
(	O
see	O
Datasets	O
EV1	O
and	O
EV2	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
ligands	O
with	O
cell	O
‐	O
specific	O
activities	O
were	O
superposed	B-experimental_method
.	O

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

Thus	O
,	O
cell	O
‐	O
specific	O
activity	O
can	O
stem	O
from	O
perturbation	O
of	O
the	O
AF	B-site
‐	I-site
2	I-site
surface	I-site
without	O
an	O
extended	O
side	O
chain	O
,	O
which	O
presumably	O
alters	O
the	O
receptor	O
–	O
coregulator	O
interaction	O
profile	O
.	O

The	O
h3	B-site
–	I-site
h12	I-site
interface	I-site
(	O
circled	O
)	O
at	O
AF	B-structure_element
‐	I-structure_element
2	I-structure_element
(	O
pink	O
)	O
was	O
expanded	O
in	O
panels	O
(	O
B	O
,	O
C	O
).	O

Average	O
(	O
mean	O
+	O
SEM	O
)	O
α	B-evidence
‐	I-evidence
carbon	I-evidence
distance	I-evidence
measured	O
from	O
h3	B-structure_element
Thr347	B-residue_name_number
to	O
h11	B-structure_element
Leu525	B-residue_name_number
of	O
A	B-protein_state
‐	I-protein_state
CD	I-protein_state
‐,	I-protein_state
2	I-protein_state
,	I-protein_state
5	I-protein_state
‐	I-protein_state
DTP	I-protein_state
‐,	I-protein_state
and	I-protein_state
3	I-protein_state
,	I-protein_state
4	I-protein_state
‐	I-protein_state
DTPD	I-protein_state
‐	I-protein_state
bound	I-protein_state
ERα	B-protein
LBDs	B-structure_element
.	O

Ligands	O
in	O
these	O
classes	O
altered	O
the	O
shape	O
of	O
AF	B-structure_element
‐	I-structure_element
2	I-structure_element
to	O
affect	O
coregulator	O
preferences	O
.	O

It	O
is	O
noteworthy	O
that	O
regulation	O
of	O
h12	B-structure_element
dynamics	O
indirectly	O
through	O
h11	B-structure_element
can	O
virtually	O
abolish	O
AF	B-structure_element
‐	I-structure_element
2	I-structure_element
activity	O
,	O
and	O
yet	O
still	O
drive	O
robust	O
transcriptional	O
activity	O
through	O
AF	B-structure_element
‐	I-structure_element
1	I-structure_element
,	O
as	O
demonstrated	O
with	O
the	O
OBHS	B-chemical
series	O
.	O

If	O
we	O
calculated	O
inter	B-evidence
‐	I-evidence
atomic	I-evidence
distance	I-evidence
matrices	I-evidence
containing	O
4	O
,	O
000	O
atoms	O
per	O
structure	O
×	O
76	O
ligand	O
–	O
receptor	O
complexes	O
,	O
we	O
would	O
have	O
3	O
×	O
105	O
predictions	O
.	O

We	O
have	O
identified	O
atomic	B-evidence
vectors	I-evidence
for	O
the	O
OBHS	B-chemical
‐	I-chemical
N	I-chemical
and	O
triaryl	B-chemical
‐	I-chemical
ethylene	I-chemical
classes	O
that	O
predict	O
ligand	O
response	O
(	O
Fig	O
5E	O
and	O
F	O
).	O

TOCA1	B-protein
binding	O
to	O
Cdc42	B-protein
leads	O
to	O
actin	O
rearrangements	O
,	O
which	O
are	O
thought	O
to	O
be	O
involved	O
in	O
processes	O
such	O
as	O
endocytosis	O
,	O
filopodia	O
formation	O
,	O
and	O
cell	O
migration	O
.	O

These	O
molecular	O
switches	O
cycle	O
between	O
active	B-protein_state
,	O
GTP	B-protein_state
-	I-protein_state
bound	I-protein_state
,	O
and	O
inactive	B-protein_state
,	O
GDP	B-protein_state
-	I-protein_state
bound	I-protein_state
,	O
states	O
with	O
the	O
help	O
of	O
auxiliary	O
proteins	O
.	O

N	B-protein
-	I-protein
WASP	I-protein
exists	O
in	O
an	O
autoinhibited	B-protein_state
conformation	I-protein_state
,	O
which	O
is	O
released	O
upon	O
PI	B-chemical
(	I-chemical
4	I-chemical
,	I-chemical
5	I-chemical
)	I-chemical
P2	I-chemical
and	O
Cdc42	B-protein
binding	O
or	O
by	O
other	O
factors	O
,	O
such	O
as	O
phosphorylation	O
.	O

The	O
F	B-structure_element
-	I-structure_element
BAR	I-structure_element
domain	O
is	O
a	O
known	O
dimerization	O
,	O
membrane	O
-	O
binding	O
,	O
and	O
membrane	O
-	O
deforming	O
module	O
found	O
in	O
a	O
number	O
of	O
cell	O
signaling	O
proteins	O
.	O

These	O
HR1	B-structure_element
domains	O
,	O
however	O
,	O
show	O
specificity	O
for	O
Cdc42	B-protein
,	O
rather	O
than	O
RhoA	B-protein
or	O
Rac1	B-protein
.	O

Here	O
,	O
we	O
present	O
the	O
solution	B-experimental_method
NMR	I-experimental_method
structure	B-evidence
of	O
the	O
HR1	B-structure_element
domain	O
of	O
TOCA1	B-protein
,	O
providing	O
the	O
first	O
structural	B-evidence
data	I-evidence
for	O
this	O
protein	O
.	O

The	O
HR1	B-structure_element
domains	O
from	O
the	O
PRK	B-protein_type
family	I-protein_type
bind	O
their	O
G	B-protein_type
protein	I-protein_type
partners	O
with	O
a	O
high	O
affinity	O
,	O
exhibiting	O
a	O
range	O
of	O
submicromolar	O
dissociation	B-evidence
constants	I-evidence
(	O
Kd	B-evidence
)	O
as	O
low	O
as	O
26	O
nm	O
.	O

This	O
region	O
comprises	O
the	O
complete	O
HR1	B-structure_element
domain	O
based	O
on	O
secondary	O
structure	O
predictions	O
and	O
sequence	B-experimental_method
alignments	I-experimental_method
with	O
another	O
TOCA	B-protein_type
family	I-protein_type
member	O
,	O
CIP4	B-protein
,	O
whose	O
structure	B-evidence
has	O
been	O
determined	O
.	O

The	O
interaction	O
between	O
[	B-complex_assembly
3H	I-complex_assembly
]	I-complex_assembly
GTP	I-complex_assembly
·	I-complex_assembly
Cdc42	I-complex_assembly
and	O
a	O
C	O
-	O
terminally	O
His	B-protein_state
-	I-protein_state
tagged	I-protein_state
TOCA1	B-protein
HR1	B-structure_element
domain	O
construct	O
was	O
investigated	O
using	O
SPA	B-experimental_method
.	O

The	O
affinity	B-evidence
was	O
therefore	O
determined	O
using	O
competition	B-experimental_method
SPAs	I-experimental_method
.	O

Competition	O
of	O
GST	B-mutant
-	I-mutant
ACK	I-mutant
GBD	B-structure_element
bound	B-protein_state
to	I-protein_state
[	B-complex_assembly
3H	I-complex_assembly
]	I-complex_assembly
GTP	I-complex_assembly
·	I-complex_assembly
Cdc42	I-complex_assembly
by	O
free	B-protein_state
ACK	B-protein
GBD	B-structure_element
was	O
used	O
as	O
a	O
control	O
and	O
to	O
establish	O
the	O
value	O
of	O
background	O
counts	O
when	O
Cdc42	B-protein
is	O
fully	O
displaced	O
.	O

Indeed	O
,	O
GST	B-experimental_method
pull	I-experimental_method
-	I-experimental_method
downs	I-experimental_method
performed	O
with	O
in	O
vitro	O
translated	O
human	B-species
TOCA1	B-protein
fragments	O
had	O
suggested	O
that	O
residues	O
N	O
-	O
terminal	O
to	O
the	O
HR1	B-structure_element
domain	O
may	O
be	O
required	O
to	O
stabilize	O
the	O
HR1	B-structure_element
domain	O
structure	O
.	O

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

The	O
structure	B-evidence
closest	O
to	O
the	O
mean	O
is	O
shown	O
in	O
Fig	O
.	O
3A	O
.	O

C	O
,	O
a	O
close	O
-	O
up	O
of	O
the	O
N	O
-	O
terminal	O
region	O
of	O
TOCA1	B-protein
HR1	B-structure_element
,	O
indicating	O
some	O
of	O
the	O
NOEs	B-evidence
defining	O
its	O
position	O
with	O
respect	O
to	O
the	O
two	O
α	B-structure_element
-	I-structure_element
helices	I-structure_element
.	O

In	O
the	O
HR1a	B-structure_element
domain	O
of	O
PRK1	B-protein
,	O
a	O
region	O
N	O
-	O
terminal	O
to	O
helix	B-structure_element
1	I-structure_element
forms	O
a	O
short	B-structure_element
α	I-structure_element
-	I-structure_element
helix	I-structure_element
,	O
which	O
packs	O
against	O
both	O
helices	O
of	O
the	O
HR1	B-structure_element
domain	O
.	O

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

The	O
mean	O
CSP	B-experimental_method
is	O
marked	O
with	O
a	O
red	O
line	O
.	O

Residues	O
with	O
significantly	O
affected	O
backbone	O
and	O
side	O
chain	O
groups	O
that	O
are	O
solvent	B-protein_state
-	I-protein_state
accessible	I-protein_state
are	O
colored	O
red	O
.	O

A	O
close	O
-	O
up	O
of	O
the	O
binding	B-site
region	I-site
is	O
shown	O
,	O
with	O
affected	O
side	O
chain	O
heavy	O
atoms	O
shown	O
as	O
sticks	O
.	O

D	O
,	O
the	O
G	B-site
protein	I-site
-	I-site
binding	I-site
region	I-site
is	O
marked	O
in	O
red	O
onto	O
structures	B-evidence
of	O
the	O
HR1	B-structure_element
domains	O
as	O
indicated	O
.	O

These	O
switch	B-site
regions	I-site
become	O
visible	O
in	O
Cdc42	B-protein
and	O
other	O
small	O
G	B-protein_type
protein	I-protein_type
·	O
effector	O
complexes	O
due	O
to	O
a	O
decrease	O
in	O
conformational	O
freedom	O
upon	O
complex	O
formation	O
.	O

This	O
suggests	O
that	O
the	O
switch	B-site
regions	I-site
are	O
not	O
rigidified	O
in	O
the	O
HR1	B-structure_element
complex	O
and	O
are	O
still	O
in	O
conformational	O
exchange	O
.	O

The	O
orientation	O
of	O
the	O
HR1	B-structure_element
domain	O
with	O
respect	O
to	O
Cdc42	B-protein
cannot	O
be	O
definitively	O
concluded	O
in	O
the	O
absence	O
of	O
unambiguous	O
distance	O
restraints	O
;	O
hence	O
,	O
HADDOCK	B-experimental_method
produced	O
a	O
set	O
of	O
models	O
in	O
which	O
the	O
HR1	B-structure_element
domain	O
contacts	O
the	O
same	O
surface	O
on	O
Cdc42	B-protein
but	O
is	O
in	O
various	O
orientations	O
with	O
respect	O
to	O
Cdc42	B-protein
.	O

A	O
representative	O
model	O
from	O
this	O
cluster	O
is	O
shown	O
in	O
Fig	O
.	O
6A	O
alongside	O
the	O
Rac1	B-complex_assembly
-	I-complex_assembly
HR1b	I-complex_assembly
structure	B-evidence
(	O
PDB	O
code	O
2RMK	O
)	O
in	O
Fig	O
.	O
6B	O
.	O

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

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

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

Competition	O
between	O
N	B-protein
-	I-protein
WASP	I-protein
and	O
TOCA1	B-protein

Interestingly	O
,	O
the	O
presence	B-protein_state
of	I-protein_state
the	O
TOCA1	B-protein
HR1	B-structure_element
would	O
not	O
prevent	O
the	O
core	O
CRIB	B-structure_element
of	O
WASP	B-protein
from	O
binding	O
to	O
Cdc42	B-protein
,	O
although	O
the	O
regions	O
C	O
-	O
terminal	O
to	O
the	O
CRIB	B-structure_element
that	O
are	O
required	O
for	O
high	O
affinity	O
binding	O
of	O
WASP	B-protein
would	O
interfere	O
sterically	O
with	O
the	O
TOCA1	B-protein
HR1	B-structure_element
.	O

These	O
data	O
indicate	O
that	O
the	O
HR1	B-structure_element
domain	O
is	O
displaced	O
from	O
Cdc42	B-protein
by	O
N	B-protein
-	I-protein
WASP	I-protein
and	O
that	O
a	O
ternary	O
complex	O
comprising	O
TOCA1	B-protein
HR1	B-structure_element
,	O
N	B-protein
-	I-protein
WASP	I-protein
GBD	B-structure_element
,	O
and	O
Cdc42	B-protein
is	O
not	O
formed	O
.	O

To	O
extend	O
these	O
studies	O
to	O
a	O
more	O
complex	O
system	O
and	O
to	O
assess	O
the	O
ability	O
of	O
TOCA1	B-protein
HR1	B-structure_element
to	O
compete	O
with	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
N	B-protein
-	I-protein
WASP	I-protein
,	O
pyrene	B-experimental_method
actin	I-experimental_method
assays	I-experimental_method
were	O
employed	O
.	O

Actin	B-protein_type
polymerization	O
triggered	O
by	O
the	O
addition	O
of	O
PI	B-chemical
(	I-chemical
4	I-chemical
,	I-chemical
5	I-chemical
)	I-chemical
P2	I-chemical
-	O
containing	O
liposomes	O
has	O
previously	O
been	O
shown	O
to	O
depend	O
on	O
TOCA1	B-protein
and	O
N	B-protein
-	I-protein
WASP	I-protein
.	O

The	O
Cdc42	B-protein
-	O
TOCA1	B-protein
Interaction	O

The	O
TOCA1	B-protein
HR1	B-structure_element
domain	O
alone	B-protein_state
is	O
sufficient	O
for	O
Cdc42	B-protein
binding	O
in	O
vitro	O
,	O
yet	O
the	O
affinity	B-evidence
of	O
the	O
TOCA1	B-protein
HR1	B-structure_element
domain	O
for	O
Cdc42	B-protein
is	O
remarkably	O
low	O
(	O
Kd	B-evidence
≈	O
5	O
μm	O
).	O

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

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

The	O
interhelical	B-structure_element
loops	I-structure_element
of	O
TOCA1	B-protein
and	O
CIP4	B-protein
differ	O
from	O
the	O
same	O
region	O
in	O
the	O
HR1	B-structure_element
domains	O
of	O
PRK1	B-protein
in	O
that	O
they	O
are	O
longer	O
and	O
contain	O
two	O
short	O
stretches	O
of	O
310	B-structure_element
-	I-structure_element
helix	I-structure_element
.	O

In	O
free	B-protein_state
TOCA1	B-protein
,	O
the	O
side	O
chains	O
of	O
the	O
interhelical	B-structure_element
region	I-structure_element
make	O
extensive	O
contacts	O
with	O
residues	O
in	O
helix	B-structure_element
1	I-structure_element
.	O

Arg	B-residue_name_number
-	I-residue_name_number
68Cdc42	I-residue_name_number
of	O
switch	B-site
II	I-site
is	O
positioned	O
close	O
to	O
Glu	B-residue_name_number
-	I-residue_name_number
395TOCA1	I-residue_name_number
(	O
Fig	O
.	O
6D	O
),	O
suggesting	O
a	O
direct	O
electrostatic	O
contact	O
between	O
switch	B-site
II	I-site
of	O
Cdc42	B-protein
and	O
helix	B-structure_element
2	I-structure_element
of	O
the	O
HR1	B-structure_element
domain	O
.	O

The	O
solution	B-evidence
structure	I-evidence
of	O
the	O
TOCA1	B-protein
HR1	B-structure_element
domain	O
presented	O
here	O
,	O
along	O
with	O
the	O
model	O
of	O
the	O
HR1TOCA1	B-complex_assembly
·	I-complex_assembly
Cdc42	I-complex_assembly
complex	O
is	O
consistent	O
with	O
a	O
conserved	O
mode	O
of	O
binding	O
across	O
the	O
known	O
HR1	B-structure_element
domain	O
-	O
Rho	O
family	O
interactions	O
,	O
despite	O
their	O
differing	O
affinities	O
.	O

We	O
have	O
previously	O
postulated	O
that	O
the	O
inherent	O
flexibility	O
of	O
HR1	B-structure_element
domains	O
contributes	O
to	O
their	O
ability	O
to	O
bind	O
to	O
different	O
Rho	B-protein_type
family	I-protein_type
G	I-protein_type
proteins	I-protein_type
,	O
with	O
Rho	O
-	O
binding	O
HR1	B-structure_element
domains	O
displaying	O
increased	O
flexibility	O
,	O
reflected	O
in	O
their	O
lower	O
melting	B-evidence
temperatures	I-evidence
(	O
Tm	B-evidence
)	O
and	O
Rac	B-protein_type
binders	O
being	O
more	O
rigid	O
.	O

Cdc42	B-protein
-	O
HR1TOCA1	B-structure_element
binding	O
would	O
then	O
be	O
favorable	O
,	O
as	O
long	O
as	O
coincident	O
activation	O
of	O
Cdc42	B-protein
had	O
occurred	O
,	O
leading	O
to	O
stabilization	O
of	O
TOCA1	B-protein
at	O
the	O
membrane	O
and	O
downstream	O
activation	O
of	O
N	B-protein
-	I-protein
WASP	I-protein
.	O

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

It	O
may	O
therefore	O
be	O
envisaged	O
that	O
WIP	B-protein
and	O
TOCA1	B-protein
exert	O
opposing	O
allosteric	O
effects	O
on	O
N	B-protein
-	I-protein
WASP	I-protein
,	O
with	O
TOCA1	B-protein
favoring	O
the	O
unfolded	B-protein_state
,	O
active	B-protein_state
conformation	O
of	O
N	B-protein
-	I-protein
WASP	I-protein
and	O
increasing	O
its	O
affinity	O
for	O
Cdc42	B-protein
.	O

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

Furthermore	O
,	O
TOCA1	B-protein
is	O
required	O
for	O
Cdc42	B-protein
-	O
mediated	O
activation	O
of	O
N	B-complex_assembly
-	I-complex_assembly
WASP	I-complex_assembly
·	I-complex_assembly
WIP	I-complex_assembly
,	O
implying	O
that	O
it	O
may	O
not	O
be	O
possible	O
for	O
Cdc42	B-protein
to	O
bind	O
and	O
activate	O
N	B-protein
-	I-protein
WASP	I-protein
prior	O
to	O
TOCA1	B-protein
-	O
Cdc42	B-protein
binding	O
.	O

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

The	O
lysine	B-residue_name
residues	O
thought	O
to	O
be	O
involved	O
in	O
an	O
electrostatic	O
steering	O
mechanism	O
in	O
WASP	B-protein
-	O
Cdc42	B-protein
binding	O
are	O
conserved	O
in	O
N	B-protein
-	I-protein
WASP	I-protein
and	O
would	O
be	O
able	O
to	O
interact	O
with	O
Cdc42	B-protein
even	O
when	O
the	O
TOCA1	B-protein
HR1	B-structure_element
domain	O
is	O
already	O
bound	B-protein_state
.	O

The	O
dynamic	B-protein_state
organization	O
of	O
fungal	B-taxonomy_domain
acetyl	B-protein_type
-	I-protein_type
CoA	I-protein_type
carboxylase	I-protein_type

In	O
contrast	O
to	O
related	O
carboxylases	B-protein_type
,	O
large	O
-	O
scale	O
conformational	O
changes	O
are	O
required	O
for	O
substrate	O
turnover	O
,	O
and	O
are	O
mediated	O
by	O
the	O
CD	B-structure_element
under	O
phosphorylation	B-ptm
control	O
.	O

Biotin	B-protein_type
-	I-protein_type
dependent	I-protein_type
acetyl	I-protein_type
-	I-protein_type
CoA	I-protein_type
carboxylases	I-protein_type
(	O
ACCs	B-protein_type
)	O
are	O
essential	O
enzymes	O
that	O
catalyse	O
the	O
ATP	B-chemical
-	O
dependent	O
carboxylation	O
of	O
acetyl	B-chemical
-	I-chemical
CoA	I-chemical
to	O
malonyl	B-chemical
-	I-chemical
CoA	I-chemical
.	O
This	O
reaction	O
provides	O
the	O
committed	O
activated	O
substrate	O
for	O
the	O
biosynthesis	O
of	O
fatty	B-chemical
acids	I-chemical
via	O
fatty	B-protein_type
-	I-protein_type
acid	I-protein_type
synthase	I-protein_type
.	O

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

The	O
CD	B-structure_element
comprises	O
one	O
-	O
third	O
of	O
the	O
protein	O
and	O
is	O
a	O
unique	B-protein_state
feature	I-protein_state
of	I-protein_state
eukaryotic	B-taxonomy_domain
ACCs	B-protein_type
without	O
homologues	O
in	O
other	O
proteins	O
.	O

The	O
structure	B-experimental_method
determination	I-experimental_method
of	O
the	O
holoenzymes	B-protein_state
of	O
bacterial	B-taxonomy_domain
biotin	B-protein_type
-	I-protein_type
dependent	I-protein_type
carboxylases	I-protein_type
,	O
which	O
lack	B-protein_state
the	O
characteristic	O
CD	B-structure_element
,	O
such	O
as	O
the	O
pyruvate	B-protein_type
carboxylase	I-protein_type
(	O
PC	B-protein_type
),	O
propionyl	B-protein_type
-	I-protein_type
CoA	I-protein_type
carboxylase	I-protein_type
,	O
3	B-protein_type
-	I-protein_type
methyl	I-protein_type
-	I-protein_type
crotonyl	I-protein_type
-	I-protein_type
CoA	I-protein_type
carboxylase	I-protein_type
and	O
a	O
long	B-protein_type
-	I-protein_type
chain	I-protein_type
acyl	I-protein_type
-	I-protein_type
CoA	I-protein_type
carboxylase	I-protein_type
revealed	O
strikingly	O
divergent	O
architectures	O
despite	O
a	O
general	O
conservation	O
of	O
all	O
functional	O
components	O
.	O

Human	B-species
ACC1	B-protein
is	O
regulated	B-protein_state
allosterically	I-protein_state
,	O
via	O
specific	O
protein	O
–	O
protein	O
interactions	O
,	O
and	O
by	O
reversible	O
phosphorylation	B-ptm
.	O

Dynamic	O
polymerization	O
of	O
human	B-species
ACC1	B-protein
is	O
linked	O
to	O
increased	O
activity	O
and	O
is	O
regulated	B-protein_state
allosterically	I-protein_state
by	O
the	O
activator	O
citrate	B-chemical
and	O
the	O
inhibitor	O
palmitate	B-chemical
,	O
or	O
by	O
binding	O
of	O
the	O
small	O
protein	O
MIG	B-protein
-	I-protein
12	I-protein
(	O
ref	O
.).	O

AMPK	B-protein
phosphorylates	O
ACC1	B-protein
in	O
vitro	O
at	O
Ser80	B-residue_name_number
,	O
Ser1201	B-residue_name_number
and	O
Ser1216	B-residue_name_number
and	O
PKA	B-protein
at	O
Ser78	B-residue_name_number
and	O
Ser1201	B-residue_name_number
.	O

However	O
,	O
regulatory	O
effects	O
on	O
ACC1	B-protein
activity	O
are	O
mainly	O
mediated	O
by	O
phosphorylation	B-ptm
of	O
Ser80	B-residue_name_number
and	O
Ser1201	B-residue_name_number
(	O
refs	O
).	O

The	O
organization	O
of	O
the	O
yeast	B-taxonomy_domain
ACC	B-protein_type
CD	B-structure_element

CDL	B-structure_element
is	O
composed	O
of	O
a	O
small	B-structure_element
,	I-structure_element
irregular	I-structure_element
four	I-structure_element
-	I-structure_element
helix	I-structure_element
bundle	I-structure_element
(	O
Lα1	B-structure_element
–	I-structure_element
4	I-structure_element
)	O
and	O
tightly	O
interacts	O
with	O
the	O
open	O
face	O
of	O
CDC1	B-structure_element
via	O
an	O
interface	B-site
of	O
1	O
,	O
300	O
Å2	O
involving	O
helices	B-structure_element
Lα3	B-structure_element
and	O
Lα4	B-structure_element
.	O

CDL	B-structure_element
does	O
not	O
interact	O
with	O
CDN	B-structure_element
apart	O
from	O
the	O
covalent	O
linkage	O
and	O
forms	O
only	O
a	O
small	O
contact	O
to	O
CDC2	B-structure_element
via	O
a	O
loop	B-structure_element
between	O
Lα2	B-structure_element
/	I-structure_element
α3	I-structure_element
and	O
the	O
N	O
-	O
terminal	O
end	O
of	O
Lα1	B-structure_element
,	O
with	O
an	O
interface	B-site
area	O
of	O
400	O
Å2	O
.	O

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

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

An	O
experimentally	B-evidence
phased	I-evidence
map	I-evidence
was	O
obtained	O
at	O
3	O
.	O
7	O
Å	O
resolution	O
for	O
a	O
cadmium	B-chemical
-	O
derivatized	O
crystal	O
and	O
was	O
interpreted	O
by	O
a	O
poly	O
-	O
alanine	O
model	O
(	O
Fig	O
.	O
1e	O
and	O
Table	O
1	O
).	O

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

As	O
a	O
result	O
,	O
the	O
N	O
terminus	O
of	O
CDL	B-structure_element
at	O
helix	B-structure_element
Lα1	B-structure_element
,	O
which	O
connects	O
to	O
CDN	B-structure_element
,	O
is	O
shifted	O
by	O
12	O
Å	O
.	O
Remarkably	O
,	O
CDN	B-structure_element
of	O
HsaBT	B-mutant
-	I-mutant
CD	I-mutant
adopts	O
a	O
completely	O
different	O
orientation	O
compared	O
with	O
SceCD	B-species
.	O

This	O
rotation	O
displaces	O
the	O
N	O
terminus	O
of	O
CDN	B-structure_element
in	O
HsaBT	B-mutant
-	I-mutant
CD	I-mutant
by	O
51	O
Å	O
compared	O
with	O
SceCD	B-species
,	O
resulting	O
in	O
a	O
separation	O
of	O
the	O
attachment	O
points	O
of	O
the	O
N	O
-	O
terminal	O
linker	B-structure_element
to	O
the	O
BCCP	B-structure_element
domain	I-structure_element
and	O
the	O
C	O
-	O
terminal	O
CT	B-structure_element
domain	O
by	O
67	O
Å	O
(	O
the	O
attachment	O
points	O
are	O
indicated	O
with	O
spheres	O
in	O
Fig	O
.	O
1e	O
).	O

The	O
highly	B-protein_state
conserved	I-protein_state
Ser1216	B-residue_name_number
(	O
corresponding	O
to	O
S	B-species
.	I-species
cerevisiae	I-species
Ser1157	B-residue_name_number
),	O
as	O
well	O
as	O
Ser1201	B-residue_name_number
,	O
both	O
in	O
the	O
regulatory	B-structure_element
loop	I-structure_element
discussed	O
above	O
,	O
are	O
not	B-protein_state
phosphorylated	I-protein_state
.	O

At	O
the	O
level	O
of	O
isolated	B-experimental_method
yeast	B-taxonomy_domain
and	O
human	B-species
CD	B-structure_element
,	O
the	O
structural	B-experimental_method
analysis	I-experimental_method
indicates	O
the	O
presence	O
of	O
at	O
least	O
two	O
hinges	B-structure_element
,	O
one	O
with	O
large	O
-	O
scale	O
flexibility	O
at	O
the	O
CDN	B-structure_element
/	I-structure_element
CDL	I-structure_element
connection	I-structure_element
,	O
and	O
one	O
with	O
tunable	O
plasticity	O
between	O
CDL	B-structure_element
/	O
CDC1	B-structure_element
and	O
CDC2	B-structure_element
,	O
plausibly	O
affected	O
by	O
phosphorylation	B-ptm
in	O
the	O
regulatory	B-structure_element
loop	I-structure_element
region	O
.	O

The	O
integration	O
of	O
CD	B-structure_element
into	O
the	O
fungal	B-taxonomy_domain
ACC	B-protein_type
multienzyme	I-protein_type

In	O
all	O
these	O
crystal	B-evidence
structures	I-evidence
,	O
the	O
CT	B-structure_element
domains	O
build	O
a	O
canonical	O
head	B-protein_state
-	I-protein_state
to	I-protein_state
-	I-protein_state
tail	I-protein_state
dimer	B-oligomeric_state
,	O
with	O
active	B-site
sites	I-site
formed	O
by	O
contributions	O
from	O
both	O
protomers	B-oligomeric_state
(	O
Fig	O
.	O
2	O
and	O
Supplementary	O
Fig	O
.	O
3a	O
).	O

The	O
connecting	B-structure_element
region	I-structure_element
is	O
remarkably	O
similar	O
in	O
isolated	B-protein_state
CD	B-structure_element
and	O
CthCD	B-mutant
-	I-mutant
CTCter	I-mutant
structures	B-evidence
,	O
indicating	O
inherent	O
conformational	O
stability	O
.	O

CD	B-structure_element
/	O
CT	B-structure_element
contacts	O
are	O
only	O
formed	O
in	O
direct	O
vicinity	O
of	O
the	O
covalent	O
linkage	O
and	O
involve	O
the	O
β	B-structure_element
-	I-structure_element
hairpin	I-structure_element
extension	I-structure_element
of	O
CDC2	B-structure_element
as	O
well	O
as	O
the	O
loop	B-structure_element
between	O
strands	B-structure_element
β2	I-structure_element
/	I-structure_element
β3	I-structure_element
of	O
the	O
CT	B-structure_element
N	I-structure_element
-	I-structure_element
lobe	I-structure_element
,	O
which	O
contains	O
a	O
conserved	B-protein_state
RxxGxN	B-structure_element
motif	I-structure_element
.	O

On	O
the	O
basis	O
of	O
an	O
interface	O
area	O
of	O
∼	O
600	O
Å2	O
and	O
its	O
edge	O
-	O
to	O
-	O
edge	O
connection	O
characteristics	O
,	O
the	O
interface	B-site
between	O
CT	B-structure_element
and	O
CD	B-structure_element
might	O
be	O
classified	O
as	O
conformationally	O
variable	O
.	O

In	O
addition	O
,	O
CDN	B-structure_element
can	O
rotate	O
around	O
hinges	B-structure_element
in	O
the	O
connection	O
between	O
CDN	B-structure_element
/	O
CDL	B-structure_element
by	O
70	O
°	O
(	O
Fig	O
.	O
4b	O
,	O
observed	O
in	O
the	O
second	O
protomer	B-oligomeric_state
of	O
CthΔBCCP	B-mutant
,	O
denoted	O
as	O
CthΔBCCP2	B-mutant
)	O
and	O
160	O
°	O
(	O
Fig	O
.	O
4c	O
,	O
observed	O
in	O
SceCD	B-species
)	O
leading	O
to	O
displacement	O
of	O
the	O
anchor	B-site
site	I-site
for	O
the	O
BCCP	B-structure_element
linker	I-structure_element
by	O
up	O
to	O
33	O
and	O
40	O
Å	O
,	O
respectively	O
.	O

The	O
current	O
data	O
thus	O
suggest	O
that	O
regulation	O
of	O
fungal	B-taxonomy_domain
ACC	B-protein_type
is	O
mediated	O
by	O
controlling	O
the	O
dynamics	O
of	O
the	O
unique	B-protein_state
CD	B-structure_element
,	O
rather	O
than	O
directly	O
affecting	O
catalytic	O
turnover	O
at	O
the	O
active	B-site
sites	I-site
of	O
BC	B-structure_element
and	O
CT	B-structure_element
.	O

In	O
their	O
study	O
,	O
mutational	B-experimental_method
data	I-experimental_method
indicate	O
a	O
requirement	O
for	O
BC	O
dimerization	O
for	O
catalytic	O
activity	O
.	O

In	O
flACC	B-mutant
,	O
the	O
regulatory	B-structure_element
loop	I-structure_element
is	O
mostly	B-protein_state
disordered	I-protein_state
,	O
illustrating	O
the	O
increased	O
flexibility	O
due	O
to	O
the	O
absence	O
of	O
the	O
phosphoryl	B-chemical
group	O
.	O

Only	O
in	O
three	O
out	O
of	O
eight	O
observed	O
protomers	B-oligomeric_state
a	O
short	B-structure_element
peptide	I-structure_element
stretch	O
(	O
including	O
Ser1157	B-residue_name_number
)	O
was	O
modelled	B-evidence
.	O

In	O
those	O
instances	O
the	O
Ser1157	B-residue_name_number
residue	O
is	O
located	O
at	O
a	O
distance	O
of	O
14	O
–	O
20	O
Å	O
away	O
from	O
the	O
location	O
of	O
the	O
phosphorylated	B-protein_state
serine	B-residue_name
observed	O
here	O
,	O
based	O
on	O
superposition	B-experimental_method
of	O
either	O
CDC1	B-structure_element
or	O
CDC2	B-structure_element
.	O

This	O
implicates	O
that	O
the	O
triangular	B-protein_state
shape	I-protein_state
with	O
dimeric	B-oligomeric_state
BC	B-structure_element
domains	O
has	O
a	O
low	O
population	O
also	O
in	O
the	O
active	B-protein_state
form	I-protein_state
,	O
even	O
though	O
a	O
biasing	O
influence	O
of	O
grid	O
preparation	O
cannot	O
be	O
excluded	O
completely	O
.	O

Large	O
-	O
scale	O
conformational	O
variability	O
has	O
also	O
been	O
observed	O
in	O
most	O
other	O
carrier	B-protein_type
protein	I-protein_type
-	I-protein_type
based	I-protein_type
multienzymes	I-protein_type
,	O
including	O
polyketide	B-protein_type
and	I-protein_type
fatty	I-protein_type
-	I-protein_type
acid	I-protein_type
synthases	I-protein_type
(	O
with	O
the	O
exception	O
of	O
fungal	B-protein_type
-	I-protein_type
type	I-protein_type
fatty	I-protein_type
-	I-protein_type
acid	I-protein_type
synthases	I-protein_type
),	O
non	B-protein_type
-	I-protein_type
ribosomal	I-protein_type
peptide	I-protein_type
synthetases	I-protein_type
and	O
the	O
pyruvate	B-protein_type
dehydrogenase	I-protein_type
complexes	I-protein_type
,	O
although	O
based	O
on	O
completely	O
different	O
architectures	O
.	O

The	O
regulation	O
of	O
activity	O
thus	O
results	O
from	O
restrained	O
large	O
-	O
scale	O
conformational	O
dynamics	O
rather	O
than	O
a	O
direct	O
or	O
indirect	O
influence	O
on	O
active	B-site
site	I-site
structure	I-site
.	O

The	O
phosphorylated	B-protein_state
central	B-structure_element
domain	I-structure_element
of	O
yeast	B-taxonomy_domain
ACC	B-protein_type
.	O

The	O
attachment	O
points	O
to	O
the	O
N	O
-	O
terminal	O
BCCP	B-structure_element
domain	O
and	O
the	O
C	O
-	O
terminal	O
CT	B-structure_element
domain	O
are	O
indicated	O
with	O
spheres	O
.	O

Variability	O
of	O
the	O
connections	O
of	O
CDC2	B-structure_element
to	O
CT	B-structure_element
and	O
CDC1	B-structure_element
in	O
fungal	B-taxonomy_domain
ACC	B-protein_type
.	O

For	O
clarity	O
,	O
only	O
one	O
protomer	B-oligomeric_state
of	O
CthCD	B-mutant
-	I-mutant
CTCter1	I-mutant
is	O
shown	O
in	O
full	O
colour	O
as	O
reference	O
.	O

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

(	O
d	O
)	O
Schematic	O
model	O
of	O
fungal	B-taxonomy_domain
ACC	B-protein_type
showing	O
the	O
intrinsic	O
,	O
regulated	O
flexibility	O
of	O
CD	B-structure_element
in	O
the	O
phosphorylated	B-protein_state
inhibited	B-protein_state
or	O
the	O
non	B-protein_state
-	I-protein_state
phosphorylated	I-protein_state
activated	B-protein_state
state	O
.	O

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

A	O
unique	O
macromolecular	O
cage	O
formed	O
by	O
two	O
decamers	B-oligomeric_state
of	O
the	O
Escherichia	B-species
coli	I-species
LdcI	B-protein
and	O
five	O
hexamers	B-oligomeric_state
of	O
the	O
AAA	B-protein_type
+	I-protein_type
ATPase	I-protein_type
RavA	B-protein
was	O
shown	O
to	O
counteract	O
acid	O
stress	O
under	O
starvation	O
.	O

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

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

Both	O
acid	B-protein_state
pH	I-protein_state
and	O
cadaverine	B-chemical
induce	O
closure	O
of	O
outer	O
membrane	O
porins	B-protein_type
thereby	O
contributing	O
to	O
bacterial	B-taxonomy_domain
protection	O
from	O
acid	O
stress	O
,	O
but	O
also	O
from	O
certain	O
antibiotics	O
,	O
by	O
reduction	O
in	O
membrane	O
permeability	O
.	O

Ten	O
years	O
ago	O
we	O
showed	O
that	O
the	O
E	B-species
.	I-species
coli	I-species
AAA	B-protein_type
+	I-protein_type
ATPase	I-protein_type
RavA	B-protein
,	O
involved	O
in	O
multiple	O
stress	O
response	O
pathways	O
,	O
tightly	O
interacted	O
with	O
LdcI	B-protein
but	O
was	O
not	O
capable	O
of	O
binding	O
to	O
LdcC	B-protein
.	O
We	O
described	O
how	O
two	O
double	O
pentameric	B-oligomeric_state
rings	B-structure_element
of	O
the	O
LdcI	B-protein
tightly	O
associate	O
with	O
five	O
hexameric	B-oligomeric_state
rings	B-structure_element
of	O
RavA	B-protein
to	O
form	O
a	O
unique	O
cage	O
-	O
like	O
architecture	O
that	O
enables	O
the	O
bacterium	B-taxonomy_domain
to	O
withstand	O
acid	O
stress	O
even	O
under	O
conditions	O
of	O
nutrient	O
deprivation	O
eliciting	O
stringent	O
response	O
.	O

This	O
comparison	O
pinpointed	O
differences	O
between	O
the	O
biodegradative	B-protein_state
and	O
the	O
biosynthetic	B-protein_state
lysine	B-protein_type
decarboxylases	I-protein_type
and	O
brought	O
to	O
light	O
interdomain	O
movements	O
associated	O
to	O
pH	B-protein_state
-	I-protein_state
dependent	I-protein_state
enzyme	O
activation	O
and	O
RavA	B-protein
binding	O
,	O
notably	O
at	O
the	O
predicted	O
RavA	B-site
binding	I-site
site	I-site
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
of	O
LdcI	B-protein
.	O
Consequently	O
,	O
we	O
tested	O
the	O
capacity	O
of	O
cage	O
formation	O
by	O
LdcI	B-mutant
-	I-mutant
LdcC	I-mutant
chimeras	I-mutant
where	O
we	O
interchanged	B-experimental_method
the	O
C	O
-	O
terminal	O
β	B-structure_element
-	I-structure_element
sheets	I-structure_element
in	O
question	O
.	O

CryoEM	B-experimental_method
3D	B-evidence
reconstructions	I-evidence
of	O
LdcC	B-protein
,	O
LdcIa	B-protein
and	O
LdcI	B-complex_assembly
-	I-complex_assembly
LARA	I-complex_assembly

In	O
the	O
frame	O
of	O
this	O
work	O
,	O
we	O
produced	O
two	O
novel	O
subnanometer	O
resolution	O
cryoEM	B-experimental_method
reconstructions	B-evidence
of	O
the	O
E	B-species
.	I-species
coli	I-species
lysine	B-protein_type
decarboxylases	I-protein_type
at	O
pH	B-protein_state
optimal	I-protein_state
for	O
their	O
enzymatic	O
activity	O
–	O
a	O
5	O
.	O
5	O
Å	O
resolution	O
cryoEM	B-experimental_method
map	B-evidence
of	O
the	O
LdcC	B-protein
(	O
pH	B-protein_state
7	I-protein_state
.	I-protein_state
5	I-protein_state
)	O
for	O
which	O
no	O
3D	O
structural	O
information	O
has	O
been	O
previously	O
available	O
(	O
Figs	O
1A	O
,	O
B	O
and	O
S1	O
),	O
and	O
a	O
6	O
.	O
1	O
Å	O
resolution	O
cryoEM	B-experimental_method
map	B-evidence
of	O
the	O
LdcIa	B-protein
,	O
(	O
pH	B-protein_state
6	I-protein_state
.	I-protein_state
2	I-protein_state
)	O
(	O
Figs	O
1C	O
,	O
D	O
and	O
S2	O
).	O

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

The	O
core	B-structure_element
domain	I-structure_element
and	O
the	O
active	B-site
site	I-site
rearrangements	O
upon	O
pH	B-protein_state
-	I-protein_state
dependent	I-protein_state
enzyme	O
activation	O
and	O
LARA	O
binding	O

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

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

The	O
ppGpp	B-site
binding	I-site
pocket	I-site
is	O
made	O
up	O
by	O
residues	O
from	O
all	O
domains	O
and	O
is	O
located	O
approximately	O
30	O
Å	O
away	O
from	O
the	O
PLP	B-chemical
moiety	O
.	O

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

Swinging	O
and	O
stretching	O
of	O
the	O
CTDs	B-structure_element
upon	O
pH	B-protein_state
-	I-protein_state
dependent	I-protein_state
LdcI	B-protein
activation	O
and	O
LARA	B-structure_element
binding	O

Importantly	O
,	O
most	O
of	O
the	O
amino	O
acid	O
differences	O
between	O
the	O
two	O
enzymes	O
are	O
located	O
in	O
this	O
very	B-structure_element
region	I-structure_element
.	O

One	O
of	O
the	O
elucidated	O
roles	O
of	O
the	O
LdcI	B-complex_assembly
-	I-complex_assembly
RavA	I-complex_assembly
cage	O
is	O
to	O
maintain	O
LdcI	B-protein
activity	O
under	O
conditions	O
of	O
enterobacterial	B-taxonomy_domain
starvation	O
by	O
preventing	O
LdcI	B-protein
inhibition	O
by	O
the	O
stringent	B-chemical
response	I-chemical
alarmone	I-chemical
ppGpp	B-chemical
.	O

The	O
dashed	O
circle	O
indicates	O
the	O
central	O
region	B-structure_element
that	O
remains	O
virtually	O
unchanged	O
between	O
all	O
the	O
structures	B-evidence
,	O
while	O
the	O
periphery	O
undergoes	O
visible	O
movements	O
.	O

The	O
PLP	B-chemical
moieties	O
of	O
the	O
cartoon	O
ring	B-structure_element
are	O
shown	O
in	O
red	O
.	O

The	O
active	B-site
site	I-site
is	O
boxed	O
.	O

(	O
D	O
,	O
E	O
)	O
A	O
gallery	O
of	O
negative	O
stain	O
EM	O
images	O
of	O
(	O
D	O
)	O
the	O
wild	B-protein_state
type	I-protein_state
LdcI	B-complex_assembly
-	I-complex_assembly
RavA	I-complex_assembly
cage	O
and	O
(	O
E	O
)	O
the	O
LdcCI	B-mutant
-	I-mutant
RavA	I-mutant
cage	I-mutant
-	I-mutant
like	I-mutant
particles	I-mutant
.	O
(	O
F	O
)	O
Some	O
representative	O
class	O
averages	O
of	O
the	O
LdcCI	B-mutant
-	I-mutant
RavA	I-mutant
cage	I-mutant
-	I-mutant
like	I-mutant
particles	I-mutant
.	O

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

Together	O
,	O
these	O
results	O
provide	O
important	O
information	O
for	O
a	O
more	O
detailed	O
understanding	O
of	O
the	O
cofactor	O
and	O
substrate	O
binding	O
mode	O
as	O
well	O
as	O
the	O
catalytic	O
mechanism	O
of	O
SePSK	B-protein
,	O
and	O
possible	O
similarities	O
with	O
its	O
plant	B-taxonomy_domain
homologue	O
AtXK	B-protein
-	I-protein
1	I-protein
.	O

Structures	B-evidence
reported	O
in	O
the	O
Protein	O
Data	O
Bank	O
of	O
the	O
FGGY	B-protein_type
family	I-protein_type
carbohydrate	I-protein_type
kinases	I-protein_type
exhibit	O
a	O
similar	O
overall	O
architecture	O
containing	O
two	O
protein	O
domains	O
,	O
one	O
of	O
which	O
is	O
responsible	O
for	O
the	O
binding	O
of	O
substrate	O
,	O
while	O
the	O
second	O
is	O
used	O
for	O
binding	O
cofactor	O
ATP	B-chemical
.	O

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

It	O
was	O
shown	O
that	O
XK	B-protein
-	I-protein
2	I-protein
(	O
At5g49650	B-gene
)	O
located	O
in	O
the	O
cytosol	O
is	O
indeed	O
xylulose	B-protein_type
kinase	I-protein_type
.	O

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

The	O
secondary	O
structural	O
elements	O
are	O
indicated	O
(	O
α	B-structure_element
-	I-structure_element
helix	I-structure_element
:	O
cyan	O
,	O
β	B-structure_element
-	I-structure_element
sheet	I-structure_element
:	O
yellow	O
).	O

However	O
,	O
superposition	B-experimental_method
of	O
structures	B-evidence
of	O
AtXK	B-protein
-	I-protein
1	I-protein
and	O
SePSK	B-protein
shows	O
some	O
differences	O
,	O
especially	O
at	O
the	O
loop	B-structure_element
regions	I-structure_element
.	O

The	O
corresponding	O
residues	O
between	O
these	O
two	O
structures	B-evidence
(	O
SePSK	B-protein
-	O
Lys35	B-residue_name_number
and	O
AtXK	B-protein
-	I-protein
1	I-protein
-	O
Lys48	B-residue_name_number
)	O
have	O
a	O
distance	O
of	O
15	O
.	O
4	O
Å	O
(	O
S3	O
Fig	O
).	O

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

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

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

In	O
both	O
structures	B-evidence
,	O
a	O
strong	O
electron	B-evidence
density	I-evidence
was	O
found	O
in	O
the	O
conserved	B-protein_state
ATP	B-site
binding	I-site
pocket	I-site
,	O
but	O
can	O
only	O
be	O
fitted	O
with	O
an	O
ADP	B-chemical
molecule	O
(	O
S4	O
Fig	O
).	O

The	O
purine	O
ring	O
of	O
AMP	B-chemical
-	I-chemical
PNP	I-chemical
is	O
positioned	O
in	O
parallel	O
to	O
the	O
indole	O
ring	O
of	O
Trp383	B-residue_name_number
.	O

In	O
addition	O
,	O
it	O
is	O
hydrogen	O
-	O
bonded	O
with	O
the	O
side	O
chain	O
amide	O
of	O
Asn380	B-residue_name_number
(	O
Fig	O
3B	O
).	O

Glu329	B-residue_name_number
in	O
3QDK	O
has	O
no	O
counterpart	O
in	O
RBL	B-complex_assembly
-	I-complex_assembly
SePSK	I-complex_assembly
structure	B-evidence
.	O

The	O
hydrogen	O
bonds	O
are	O
indicated	O
by	O
the	O
black	O
dashed	O
lines	O
and	O
the	O
numbers	O
near	O
the	O
dashed	O
lines	O
are	O
the	O
distances	O
(	O
Å	O
).	O
(	O
C	O
)	O
The	O
binding	B-experimental_method
affinity	I-experimental_method
assays	I-experimental_method
of	O
SePSK	B-protein
with	O
D	B-chemical
-	I-chemical
ribulose	I-chemical
.	O

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

As	O
reported	O
previously	O
,	O
members	O
of	O
the	O
sugar	B-protein_type
kinase	I-protein_type
family	O
undergo	O
a	O
conformational	O
change	O
to	O
narrow	O
the	O
crossing	O
angle	O
between	O
two	O
domains	O
and	O
reduce	O
the	O
distance	O
between	O
substrate	O
and	O
ATP	B-chemical
in	O
order	O
to	O
facilitate	O
the	O
catalytic	O
reaction	O
of	O
phosphorylation	B-ptm
of	O
sugar	O
substrates	O
.	O

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

After	O
superposition	B-experimental_method
,	O
the	O
distances	O
of	O
AMP	B-chemical
-	I-chemical
PNP	I-chemical
γ	O
-	O
phosphate	B-chemical
and	O
the	O
fifth	O
hydroxyl	O
group	O
of	O
RBL1	B-residue_name_number
are	O
7	O
.	O
9	O
Å	O
(	O
superposed	B-experimental_method
with	O
AtXK	B-protein
-	I-protein
1	I-protein
),	O
7	O
.	O
4	O
Å	O
(	O
superposed	B-experimental_method
with	O
SePSK	B-protein
),	O
6	O
.	O
6	O
Å	O
(	O
superposed	B-experimental_method
with	O
3LL3	O
)	O
and	O
6	O
.	O
1	O
Å	O
(	O
superposed	B-experimental_method
with	O
1GLJ	O
).	O

The	O
structures	B-evidence
are	O
shown	O
as	O
cartoon	O
and	O
the	O
ligands	O
are	O
shown	O
as	O
sticks	O
.	O

Domain	B-structure_element
I	I-structure_element
from	O
D	B-complex_assembly
-	I-complex_assembly
ribulose	I-complex_assembly
-	I-complex_assembly
SePSK	I-complex_assembly
(	O
green	O
)	O
and	O
Domain	B-structure_element
II	I-structure_element
from	O
AMP	B-complex_assembly
-	I-complex_assembly
PNP	I-complex_assembly
-	I-complex_assembly
SePSK	I-complex_assembly
(	O
cyan	O
)	O
are	O
superposed	B-experimental_method
with	O
apo	B-protein_state
-	O
AtXK	B-protein
-	I-protein
1	I-protein
(	O
1st	O
),	O
apo	B-protein_state
-	O
SePSK	B-protein
(	O
2nd	O
),	O
3LL3	O
(	O
3rd	O
)	O
and	O
1GLJ	O
(	O
4th	O
),	O
respectively	O
.	O

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

Here	O
we	O
report	O
X	B-evidence
-	I-evidence
ray	I-evidence
crystal	I-evidence
structures	I-evidence
of	O
the	O
Mep2	B-protein_type
orthologues	O
from	O
Saccharomyces	B-species
cerevisiae	I-species
and	O
Candida	B-species
albicans	I-species
and	O
show	O
that	O
under	O
nitrogen	O
-	O
sufficient	O
conditions	O
the	O
transporters	B-protein_type
are	O
not	B-protein_state
phosphorylated	I-protein_state
and	O
present	O
in	O
closed	B-protein_state
,	O
inactive	B-protein_state
conformations	O
.	O

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

They	O
belong	O
to	O
the	O
Amt	B-protein_type
/	I-protein_type
Mep	I-protein_type
/	I-protein_type
Rh	I-protein_type
family	I-protein_type
of	I-protein_type
transporters	I-protein_type
that	O
are	O
present	O
in	O
all	B-taxonomy_domain
kingdoms	I-taxonomy_domain
of	I-taxonomy_domain
life	I-taxonomy_domain
and	O
they	O
take	O
up	O
ammonium	B-chemical
from	O
the	O
extracellular	O
environment	O
.	O

As	O
is	O
the	O
case	O
for	O
other	O
transceptors	B-protein_type
,	O
it	O
is	O
not	O
clear	O
how	O
Mep2	B-protein
interacts	O
with	O
downstream	O
signalling	O
partners	O
,	O
but	O
the	O
protein	O
kinase	O
A	O
and	O
mitogen	O
-	O
activated	O
protein	O
kinase	O
pathways	O
have	O
been	O
proposed	O
as	O
downstream	O
effectors	O
of	O
Mep2	B-protein
(	O
refs	O
).	O

In	O
addition	O
,	O
Mep2	B-protein
is	O
also	O
important	O
for	O
uptake	O
of	O
ammonium	B-chemical
produced	O
by	O
growth	O
on	O
other	O
nitrogen	B-chemical
sources	O
.	O

All	O
structures	B-evidence
show	O
the	O
transporters	B-protein_type
in	O
open	B-protein_state
conformations	O
.	O

To	O
elucidate	O
the	O
mechanism	O
of	O
Mep2	B-protein_type
transport	O
regulation	O
,	O
we	O
present	O
here	O
X	B-evidence
-	I-evidence
ray	I-evidence
crystal	I-evidence
structures	I-evidence
of	O
the	O
Mep2	B-protein_type
transceptors	I-protein_type
from	O
S	B-species
.	I-species
cerevisiae	I-species
and	O
C	B-species
.	I-species
albicans	I-species
.	O

The	O
channels	B-site
of	O
phosphorylation	B-protein_state
-	I-protein_state
mimicking	I-protein_state
mutants	I-protein_state
of	O
C	B-species
.	I-species
albicans	I-species
Mep2	B-protein
are	O
still	O
closed	B-protein_state
but	O
show	O
large	O
conformational	O
changes	O
within	O
a	O
conserved	B-protein_state
part	O
of	O
the	O
CTR	B-structure_element
.	O

Of	O
these	O
,	O
Mep2	B-protein
from	O
C	B-species
.	I-species
albicans	I-species
(	O
CaMep2	B-protein
)	O
showed	O
superior	O
stability	O
in	O
relatively	O
harsh	O
detergents	O
such	O
as	O
nonyl	O
-	O
glucoside	O
,	O
allowing	O
structure	B-experimental_method
determination	I-experimental_method
in	O
two	O
different	O
crystal	B-evidence
forms	I-evidence
to	O
high	O
resolution	O
(	O
up	O
to	O
1	O
.	O
5	O
Å	O
).	O

Important	O
functional	O
features	O
such	O
as	O
the	O
extracellular	O
ammonium	B-site
binding	I-site
site	I-site
,	O
the	O
Phe	B-site
gate	I-site
and	O
the	O
twin	B-structure_element
-	I-structure_element
His	I-structure_element
motif	I-structure_element
within	O
the	O
hydrophobic	B-site
channel	I-site
are	O
all	O
very	O
similar	O
to	O
those	O
present	O
in	O
the	O
bacterial	B-taxonomy_domain
transporters	B-protein_type
and	O
RhCG	B-protein
.	O

In	O
addition	O
to	O
changing	O
the	O
RxK	B-structure_element
motif	I-structure_element
,	O
the	O
movement	O
of	O
ICL1	B-structure_element
has	O
another	O
,	O
crucial	O
functional	O
consequence	O
.	O

This	O
two	O
-	O
tier	O
channel	B-structure_element
block	I-structure_element
likely	O
ensures	O
that	O
very	O
little	O
ammonium	B-chemical
transport	O
will	O
take	O
place	O
under	O
nitrogen	B-chemical
-	O
sufficient	O
conditions	O
.	O

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

Strikingly	O
,	O
the	O
Npr1	B-site
target	I-site
serine	I-site
residue	O
is	O
located	O
at	O
the	O
periphery	O
of	O
the	O
trimer	B-oligomeric_state
,	O
far	O
away	O
(∼	O
30	O
Å	O
)	O
from	O
any	O
channel	B-site
exit	I-site
(	O
Fig	O
.	O
6	O
).	O

Given	O
that	O
Ser457	B-residue_name_number
/	O
453	B-residue_number
is	O
far	O
from	O
any	O
channel	B-site
exit	I-site
(	O
Fig	O
.	O
6	O
),	O
the	O
crucial	O
question	O
is	O
how	O
phosphorylation	B-ptm
opens	O
the	O
Mep2	B-protein
channel	B-site
to	O
generate	O
an	O
active	B-protein_state
transporter	B-protein_type
.	O

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

The	O
second	O
possibility	O
is	O
that	O
the	O
Tyr	B-site
–	I-site
His	I-site
hydrogen	I-site
bond	I-site
has	O
to	O
be	O
disrupted	O
by	O
the	O
incoming	O
substrate	O
to	O
open	B-protein_state
the	O
channel	O
.	O

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

Phosphorylation	B-ptm
causes	O
a	O
conformational	O
change	O
in	O
the	O
CTR	B-structure_element

Do	O
the	O
Mep2	B-protein
structures	B-evidence
provide	O
any	O
clues	O
regarding	O
the	O
potential	O
effect	O
of	O
phosphorylation	B-ptm
?	O

The	O
ammonium	B-chemical
uptake	O
activity	O
of	O
the	O
S	B-species
.	I-species
cerevisiae	I-species
version	O
of	O
the	O
DD	B-mutant
mutant	I-mutant
is	O
the	O
same	O
as	O
that	O
of	O
WT	B-protein_state
Mep2	B-protein
and	O
the	O
S453D	B-mutant
mutant	B-protein_state
,	O
indicating	O
that	O
the	O
mutations	O
do	O
not	O
affect	O
transporter	O
functionality	O
in	O
the	O
triple	B-mutant
mepΔ	I-mutant
background	O
(	O
Fig	O
.	O
3	O
).	O

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

Our	O
model	O
also	O
provides	O
an	O
explanation	O
for	O
the	O
observation	O
that	O
certain	B-mutant
mutations	I-mutant
within	O
the	O
CTR	B-structure_element
completely	O
abolish	O
transport	O
activity	O
.	O

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

While	O
the	O
current	O
study	O
does	O
not	O
specifically	O
address	O
the	O
mechanism	O
of	O
signalling	O
underlying	O
pseudohyphal	O
growth	O
,	O
our	O
structures	B-evidence
do	O
show	O
that	O
Mep2	B-protein_type
proteins	I-protein_type
can	O
assume	O
different	O
conformations	O
.	O

(	O
b	O
)	O
CaMep2	B-protein
trimer	B-oligomeric_state
viewed	O
from	O
the	O
intracellular	O
side	O
(	O
right	O
).	O

The	O
Npr1	B-site
kinase	I-site
site	I-site
in	O
the	O
AI	B-structure_element
region	I-structure_element
is	O
highlighted	O
pink	O
.	O

Growth	B-experimental_method
of	O
ScMep2	B-mutant
variants	I-mutant
on	O
low	O
ammonium	O
medium	O
.	O

The	O
side	O
chains	O
of	O
residues	O
in	O
the	O
RxK	B-structure_element
motif	I-structure_element
as	O
well	O
as	O
those	O
of	O
Tyr49	B-residue_name_number
and	O
His342	B-residue_name_number
are	O
labelled	O
.	O

(	O
c	O
)	O
Conserved	B-protein_state
residues	O
in	O
ICL1	B-structure_element
-	I-structure_element
3	I-structure_element
and	O
the	O
CTR	B-structure_element
.	O

Views	O
from	O
the	O
cytosol	O
for	O
CaMep2	B-protein
(	O
left	O
)	O
and	O
AfAmt	B-protein
-	I-protein
1	I-protein
,	O
highlighting	O
the	O
large	O
differences	O
in	O
conformation	O
of	O
the	O
conserved	B-protein_state
residues	O
in	O
ICL1	B-structure_element
(	O
RxK	O
motif	O
;	O
blue	O
),	O
ICL2	B-structure_element
(	O
ER	B-structure_element
motif	I-structure_element
;	O
cyan	O
),	O
ICL3	B-structure_element
(	O
green	O
)	O
and	O
the	O
CTR	B-structure_element
(	O
red	O
).	O

Missing	O
regions	O
are	O
labelled	O
.	O
(	O
b	O
)	O
Stereo	O
superpositions	B-experimental_method
of	O
WT	B-protein_state
CaMep2	B-protein
and	O
the	O
truncation	B-protein_state
mutant	I-protein_state
.	O

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

(	O
a	O
)	O
Cytoplasmic	O
view	O
of	O
the	O
DD	B-mutant
mutant	I-mutant
trimer	B-oligomeric_state
,	O
with	O
WT	B-protein_state
CaMep2	B-protein
superposed	B-experimental_method
in	O
grey	O
for	O
one	O
of	O
the	O
monomers	B-oligomeric_state
.	O

High	O
-	O
resolution	O
structural	B-evidence
models	I-evidence
of	O
protein	O
-	O
protein	O
interactions	O
are	O
critical	O
for	O
obtaining	O
mechanistic	O
insights	O
into	O
biological	O
processes	O
.	O

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

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

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

Even	O
the	O
minimal	B-structure_element
binding	I-structure_element
portion	I-structure_element
of	O
Im7	B-protein
(	O
Im76	B-mutant
-	I-mutant
45	I-mutant
)	O
showed	O
highly	O
dispersed	O
electron	B-evidence
density	I-evidence
(	O
Fig	O
.	O
1a	O
).	O

Thus	O
,	O
we	O
developed	O
a	O
new	O
approach	O
to	O
interpret	O
the	O
chaperone	B-protein_state
-	I-protein_state
bound	I-protein_state
substrate	O
in	O
multiple	O
conformations	O
.	O

If	O
successful	O
,	O
the	O
selection	O
identifies	O
the	O
smallest	O
group	O
of	O
specific	O
conformations	O
that	O
best	O
fits	O
the	O
residual	B-evidence
electron	I-evidence
density	I-evidence
and	O
anomalous	B-evidence
signals	I-evidence
.	O

Each	O
complex	O
within	O
this	O
pool	O
comprises	O
one	O
Spy	B-protein
dimer	B-oligomeric_state
bound	B-protein_state
to	I-protein_state
a	O
single	O
Im76	B-mutant
-	I-mutant
45	I-mutant
substrate	O
.	O

This	O
process	O
provided	O
us	O
with	O
a	O
target	O
map	B-evidence
that	O
the	O
ensuing	O
selection	O
tried	O
to	O
recapitulate	O
.	O

This	O
approach	O
allowed	O
us	O
to	O
simultaneously	O
use	O
both	O
the	O
iodine	B-chemical
anomalous	B-evidence
signals	I-evidence
and	O
the	O
residual	B-evidence
electron	I-evidence
density	I-evidence
in	O
the	O
selection	O
procedure	O
.	O

The	O
selection	O
resulted	O
in	O
small	O
ensembles	O
from	O
the	O
MD	B-experimental_method
pool	O
that	O
best	O
fit	O
the	O
READ	B-experimental_method
data	O
(	O
Fig	O
.	O
1c	O
,	O
d	O
).	O

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

Surprisingly	O
,	O
we	O
noted	O
that	O
in	O
the	O
ensemble	O
,	O
Im76	B-mutant
-	I-mutant
45	I-mutant
interacts	O
with	O
only	O
38	O
%	O
of	O
the	O
hydrophobic	O
residues	O
in	O
the	O
Spy	B-protein
cradle	B-site
,	O
but	O
interacts	O
with	O
61	O
%	O
of	O
the	O
hydrophilic	O
residues	O
in	O
the	O
cradle	B-site
.	O

The	O
structures	B-evidence
of	O
our	O
ensemble	B-evidence
agree	O
well	O
with	O
lower	O
-	O
resolution	O
crosslinking	O
data	O
,	O
which	O
indicate	O
that	O
chaperone	B-protein_type
-	O
substrate	O
interactions	O
primarily	O
occur	O
on	O
the	O
concave	B-site
surface	I-site
of	O
Spy	B-protein
.	O

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

As	O
inter	O
-	O
molecular	O
hydrophobic	O
interactions	O
between	O
Spy	B-protein
and	O
the	O
substrate	O
become	O
progressively	O
replaced	O
by	O
intra	O
-	O
molecular	O
interactions	O
within	O
the	O
substrate	O
,	O
the	O
affinity	O
between	O
chaperone	B-protein_type
and	O
substrates	O
could	O
decrease	O
,	O
eventually	O
leading	O
to	O
release	O
of	O
the	O
folded	B-protein_state
client	O
protein	O
.	O

Other	O
Super	O
Spy	B-protein
mutations	B-protein_state
(	O
F115I	B-mutant
and	O
F115L	B-mutant
)	O
caused	O
increased	O
flexibility	O
but	O
not	O
tighter	O
substrate	O
binding	O
.	O

In	O
addition	O
to	O
insights	O
into	O
chaperone	B-protein_type
function	O
,	O
this	O
work	O
presents	O
a	O
new	O
method	O
for	O
determining	O
heterogeneous	O
structural	O
ensembles	O
via	O
a	O
hybrid	O
methodology	O
of	O
X	B-experimental_method
-	I-experimental_method
ray	I-experimental_method
crystallography	I-experimental_method
and	O
computational	B-experimental_method
modeling	I-experimental_method
.	O

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

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

Shown	O
below	O
each	O
ensemble	O
member	O
is	O
the	O
RMSD	B-evidence
of	O
each	O
conformer	O
to	O
the	O
native	B-protein_state
state	O
of	O
Im76	B-mutant
-	I-mutant
45	I-mutant
,	O
as	O
well	O
as	O
the	O
percentage	O
of	O
contacts	O
between	O
Im76	B-mutant
-	I-mutant
45	I-mutant
and	O
Spy	B-protein
that	O
are	O
hydrophobic	O
.	O

The	O
frequency	O
plotted	O
is	O
calculated	O
as	O
the	O
average	O
contact	B-evidence
frequency	I-evidence
from	O
Spy	B-protein
to	O
every	O
residue	O
of	O
Im76	B-mutant
-	I-mutant
45	I-mutant
and	O
vice	O
-	O
versa	O
.	O

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

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

All	O
four	O
heavy	B-structure_element
chains	I-structure_element
of	O
the	O
antigen	B-structure_element
-	I-structure_element
binding	I-structure_element
fragments	I-structure_element
(	O
Fabs	B-structure_element
)	O
have	O
the	O
same	O
complementarity	B-structure_element
-	I-structure_element
determining	I-structure_element
region	I-structure_element
(	O
CDR	B-structure_element
)	O
H3	B-structure_element
that	O
was	O
reported	O
in	O
an	O
earlier	O
Fab	B-structure_element
structure	B-evidence
.	O

CDR	B-structure_element
H3	B-structure_element
,	O
despite	O
having	O
the	O
same	O
amino	O
acid	O
sequence	O
,	O
exhibits	O
the	O
largest	O
conformational	O
diversity	O
.	O

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

At	O
present	O
,	O
therapeutic	O
antibodies	B-protein_type
are	O
the	O
largest	O
class	O
of	O
biotherapeutic	O
proteins	O
that	O
are	O
in	O
clinical	O
trials	O
.	O

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

These	O
multimeric	O
forms	O
are	O
linked	O
with	O
an	O
additional	O
J	B-structure_element
chain	O
.	O

Both	O
κ	B-structure_element
and	O
λ	B-structure_element
polypeptide	O
chains	O
are	O
composed	O
of	O
a	O
single	O
V	B-structure_element
domain	I-structure_element
and	O
a	O
single	O
C	B-structure_element
domain	I-structure_element
.	O

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

Additional	O
efforts	O
have	O
led	O
to	O
our	O
current	O
understanding	O
that	O
the	O
LC	B-structure_element
CDRs	B-structure_element
L1	B-structure_element
,	O
L2	B-structure_element
,	O
and	O
L3	B-structure_element
have	O
preferred	O
sets	O
of	O
canonical	O
structures	O
based	O
on	O
length	O
and	O
amino	O
acid	O
sequence	O
composition	O
.	O

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

Recent	O
antibody	B-experimental_method
modeling	I-experimental_method
assessments	I-experimental_method
show	O
continued	O
improvement	O
in	O
the	O
quality	O
of	O
the	O
models	O
being	O
generated	O
by	O
a	O
variety	O
of	O
modeling	O
methods	O
.	O

(	O
Continued	O
)	O
Crystal	B-evidence
data	I-evidence
,	O
X	B-evidence
-	I-evidence
ray	I-evidence
data	I-evidence
,	O
and	O
refinement	B-evidence
statistics	I-evidence
.	O

The	O
crystal	B-evidence
structures	I-evidence
of	O
the	O
16	O
Fabs	B-structure_element
have	O
been	O
determined	O
at	O
resolutions	O
ranging	O
from	O
3	O
.	O
3	O
Å	O
to	O
1	O
.	O
65	O
Å	O
(	O
Table	O
1	O
).	O

One	O
involves	O
the	O
loop	B-structure_element
connecting	O
the	O
first	O
2	O
β	B-structure_element
-	I-structure_element
strands	I-structure_element
of	O
the	O
constant	B-structure_element
domain	I-structure_element
(	O
in	O
all	O
Fabs	B-structure_element
except	O
H3	B-complex_assembly
-	I-complex_assembly
23	I-complex_assembly
:	I-complex_assembly
L1	I-complex_assembly
-	I-complex_assembly
39	I-complex_assembly
,	O
H3	B-complex_assembly
-	I-complex_assembly
23	I-complex_assembly
:	I-complex_assembly
L3	I-complex_assembly
-	I-complex_assembly
11	I-complex_assembly
and	O
H3	B-complex_assembly
-	I-complex_assembly
53	I-complex_assembly
:	I-complex_assembly
L1	I-complex_assembly
-	I-complex_assembly
39	I-complex_assembly
).	O

The	O
CDR	B-structure_element
H1	B-structure_element
structures	B-evidence
with	O
H1	B-mutant
-	I-mutant
69	I-mutant
shown	O
in	O
Fig	O
.	O
1A	O
are	O
quite	O
variable	O
,	O
both	O
for	O
the	O
structures	B-evidence
with	O
different	O
LCs	B-structure_element
and	O
for	O
the	O
copies	O
of	O
the	O
same	O
Fab	B-structure_element
in	O
the	O
asymmetric	O
unit	O
,	O
H1	B-complex_assembly
-	I-complex_assembly
69	I-complex_assembly
:	I-complex_assembly
L3	I-complex_assembly
-	I-complex_assembly
11	I-complex_assembly
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

In	O
total	O
,	O
6	O
independent	O
Fab	B-structure_element
structures	B-evidence
produce	O
5	O
different	O
canonical	O
structures	B-evidence
,	O
namely	O
H1	B-mutant
-	I-mutant
13	I-mutant
-	I-mutant
1	I-mutant
,	O
H1	B-mutant
-	I-mutant
13	I-mutant
-	I-mutant
3	I-mutant
,	O
H1	B-mutant
-	I-mutant
13	I-mutant
-	I-mutant
4	I-mutant
,	O
H1	B-mutant
-	I-mutant
13	I-mutant
-	I-mutant
6	I-mutant
and	O
H1	B-mutant
-	I-mutant
13	I-mutant
-	I-mutant
10	I-mutant
.	O

Although	O
three	O
of	O
the	O
germlines	O
have	O
CDR	B-structure_element
H2	B-structure_element
of	O
the	O
same	O
length	O
,	O
10	B-residue_range
residues	I-residue_range
,	O
they	O
adopt	O
2	O
distinctively	O
different	O
conformations	O
depending	O
mostly	O
on	O
the	O
residue	O
at	O
position	O
71	B-residue_number
from	O
the	O
so	O
-	O
called	O
CDR	B-structure_element
H4	B-structure_element
.	O

Conformations	O
of	O
CDR	B-structure_element
H2	B-structure_element
in	O
H1	B-mutant
-	I-mutant
69	I-mutant
and	O
H5	B-mutant
-	I-mutant
51	I-mutant
,	O
both	O
of	O
which	O
have	O
canonical	O
structure	O
H2	B-mutant
-	I-mutant
10	I-mutant
-	I-mutant
1	I-mutant
,	O
show	O
little	O
deviation	O
within	O
each	O
set	O
of	O
4	O
structures	B-evidence
.	O

L4	B-mutant
-	I-mutant
1	I-mutant
has	O
the	O
longest	O
CDR	B-structure_element
L1	B-structure_element
,	O
composed	O
of	O
17	B-residue_range
amino	I-residue_range
acid	I-residue_range
residues	I-residue_range
(	O
Fig	O
.	O
3D	O
).	O

This	O
is	O
the	O
tip	O
of	O
the	O
loop	B-structure_element
region	I-structure_element
,	O
which	O
appears	O
to	O
have	O
similar	O
conformations	O
that	O
fan	O
out	O
the	O
structures	B-evidence
because	O
of	O
the	O
slight	O
differences	O
in	O
torsion	O
angles	O
in	O
the	O
backbone	O
near	O
Tyr30a	B-residue_name_number
and	O
Lys30f	B-residue_name_number
.	O

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

The	O
superposition	B-experimental_method
of	O
CDR	B-structure_element
L2	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

The	O
slight	O
conformational	O
variability	O
occurs	O
in	O
the	O
region	O
of	O
amino	O
acid	O
residues	O
90	B-residue_range
-	I-residue_range
92	I-residue_range
,	O
which	O
is	O
in	O
contact	O
with	O
CDR	B-structure_element
H3	B-structure_element
.	O

This	O
water	B-chemical
is	O
present	O
in	O
both	O
the	O
bound	B-protein_state
(	O
4DN4	O
)	O
and	O
unbound	B-protein_state
(	O
4DN3	O
)	O
forms	O
of	O
CNTO	B-chemical
888	I-chemical
.	O

Ribbon	O
representations	O
of	O
(	O
A	O
)	O
the	O
superposition	B-experimental_method
of	O
all	O
CDR	B-structure_element
H3s	B-structure_element
of	O
the	O
structures	B-evidence
with	O
complete	O
backbone	O
traces	O
.	O
(	O
B	O
)	O
The	O
CDR	B-structure_element
H3s	B-structure_element
rotated	O
90	O
°	O
about	O
the	O
y	O
axis	O
of	O
the	O
page	O
.	O

Another	O
four	O
of	O
the	O
Fabs	B-structure_element
,	O
H3	B-complex_assembly
-	I-complex_assembly
23	I-complex_assembly
:	I-complex_assembly
L1	I-complex_assembly
-	I-complex_assembly
39	I-complex_assembly
,	O
H3	B-complex_assembly
-	I-complex_assembly
53	I-complex_assembly
:	I-complex_assembly
L1	I-complex_assembly
-	I-complex_assembly
39	I-complex_assembly
,	O
H3	B-complex_assembly
-	I-complex_assembly
53	I-complex_assembly
:	I-complex_assembly
L3	I-complex_assembly
-	I-complex_assembly
11	I-complex_assembly
and	O
H3	B-complex_assembly
-	I-complex_assembly
53	I-complex_assembly
:	I-complex_assembly
L4	I-complex_assembly
-	I-complex_assembly
1	I-complex_assembly
have	O
missing	O
side	O
-	O
chain	O
atoms	O
.	O

A	O
comparison	O
of	O
representatives	O
of	O
the	O
“	O
kinked	B-protein_state
”	O
and	O
“	O
extended	B-protein_state
”	O
structures	B-evidence
.	O

The	O
largest	O
backbone	O
conformational	O
deviation	O
for	O
the	O
set	O
is	O
at	O
Tyr99	B-residue_name_number
,	O
where	O
the	O
C	O
=	O
O	O
is	O
rotated	O
by	O
90	O
°	O
relative	O
to	O
that	O
observed	O
in	O
4DN3	O
.	O

Also	O
,	O
it	O
is	O
worth	O
noting	O
that	O
only	O
one	O
of	O
these	O
structures	B-evidence
,	O
H1	B-complex_assembly
-	I-complex_assembly
69	I-complex_assembly
:	I-complex_assembly
L4	I-complex_assembly
-	I-complex_assembly
1	I-complex_assembly
,	O
has	O
the	O
conserved	B-protein_state
water	B-chemical
molecule	O
in	O
CDR	B-structure_element
H3	B-structure_element
observed	O
in	O
the	O
4DN3	O
and	O
4DN4	O
structures	B-evidence
.	O

The	O
CDR	B-structure_element
H3	B-structure_element
for	O
this	O
structure	B-evidence
is	O
shown	O
in	O
Fig	O
.	O
S3	O
.	O

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

VH	B-complex_assembly
:	I-complex_assembly
VL	I-complex_assembly
tilt	B-evidence
angles	I-evidence

The	O
relative	O
orientation	O
of	O
VH	B-structure_element
and	O
VL	B-structure_element
has	O
been	O
measured	O
in	O
a	O
number	O
of	O
different	O
ways	O
.	O

The	O
four	O
LCs	B-structure_element
all	O
are	O
classified	O
as	O
Type	O
A	O
because	O
they	O
have	O
a	O
proline	B-residue_name
at	O
position	O
44	B-residue_number
,	O
and	O
the	O
results	O
for	O
each	O
orientation	B-evidence
parameter	I-evidence
are	O
within	O
the	O
range	O
of	O
values	O
of	O
this	O
type	O
reported	O
by	O
Dunbar	O
and	O
co	O
-	O
workers	O
.	O

This	O
kind	O
of	O
disorder	O
may	O
compromise	O
the	O
integrity	O
of	O
the	O
VH	B-structure_element
domain	O
and	O
its	O
interaction	O
with	O
the	O
VL	B-structure_element
.	O

The	O
smallest	O
differences	O
in	O
the	O
tilt	B-evidence
angle	I-evidence
are	O
between	O
the	O
Fabs	B-structure_element
in	O
isomorphous	O
crystal	B-evidence
forms	I-evidence
.	O

Among	O
the	O
complete	B-protein_state
structures	B-evidence
,	O
the	O
interface	B-site
areas	O
range	O
from	O
684	O
to	O
836	O
Å2	O
.	O

These	O
findings	O
correlate	O
well	O
with	O
the	O
degree	O
of	O
conformational	O
disorder	O
observed	O
in	O
the	O
crystal	B-evidence
structures	I-evidence
.	O

This	O
variability	O
is	O
likely	O
a	O
result	O
of	O
2	O
factors	O
,	O
crystal	O
packing	O
interactions	O
and	O
internal	O
instability	O
of	O
the	O
variable	B-structure_element
domain	I-structure_element
.	O

The	O
other	O
2	O
HCs	B-structure_element
,	O
H3	B-mutant
-	I-mutant
23	I-mutant
and	O
H5	B-mutant
-	I-mutant
51	I-mutant
,	O
have	O
canonical	O
structures	O
that	O
are	O
remarkably	B-protein_state
well	I-protein_state
conserved	I-protein_state
(	O
Fig	O
.	O
1	O
).	O

As	O
mentioned	O
in	O
the	O
Results	O
section	O
,	O
this	O
data	O
set	O
is	O
composed	O
of	O
21	O
Fabs	B-structure_element
,	O
since	O
5	O
of	O
the	O
16	O
variants	O
have	O
2	O
Fab	B-structure_element
copies	O
in	O
the	O
asymmetric	O
unit	O
.	O

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

More	O
than	O
half	O
of	O
the	O
variants	O
retain	O
the	O
conformation	O
of	O
the	O
parent	O
despite	O
having	O
differences	O
in	O
the	O
VH	B-complex_assembly
:	I-complex_assembly
VL	I-complex_assembly
pairing	O
.	O

The	O
absolute	O
VH	B-complex_assembly
:	I-complex_assembly
VL	I-complex_assembly
orientation	B-evidence
parameters	I-evidence
for	O
the	O
2	O
Fabs	B-structure_element
(	O
Table	O
S2	O
)	O
show	O
significant	O
deviation	B-evidence
in	O
HL	B-structure_element
,	O
LC1	B-structure_element
and	O
HC2	B-structure_element
values	O
(	O
2	O
-	O
3	O
standard	O
deviations	O
from	O
the	O
mean	O
).	O

Curiously	O
,	O
the	O
2	O
Fabs	B-structure_element
,	O
H1	B-complex_assembly
-	I-complex_assembly
69	I-complex_assembly
:	I-complex_assembly
L3	I-complex_assembly
-	I-complex_assembly
20	I-complex_assembly
and	O
H3	B-complex_assembly
-	I-complex_assembly
23	I-complex_assembly
:	I-complex_assembly
L3	I-complex_assembly
-	I-complex_assembly
20	I-complex_assembly
,	O
deviate	O
markedly	O
in	O
their	O
tilt	B-evidence
angles	I-evidence
from	O
the	O
rest	O
of	O
the	O
panel	O
.	O

It	O
is	O
possible	O
that	O
by	O
adopting	O
extreme	O
tilt	B-evidence
angles	I-evidence
the	O
structure	B-evidence
modulates	O
CDR	B-structure_element
H3	B-structure_element
and	O
its	O
environment	O
,	O
which	O
apparently	O
cannot	O
be	O
achieved	O
solely	O
by	O
conformational	O
rearrangement	O
of	O
the	O
CDR	B-structure_element
.	O

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

These	O
data	O
reveal	O
the	O
difficulty	O
of	O
modeling	O
CDR	B-structure_element
H3	B-structure_element
accurately	O
,	O
as	O
shown	O
again	O
in	O
Antibody	O
Modeling	O
Assessment	O
II	O
.	O

Fortunately	O
,	O
for	O
most	O
applications	O
of	O
antibody	B-protein_type
modeling	O
,	O
such	O
as	O
engineering	O
affinity	O
and	O
biophysical	O
properties	O
,	O
an	O
accurate	O
CDR	B-structure_element
H3	B-structure_element
structure	B-evidence
is	O
not	O
always	O
necessary	O
.	O

The	O
results	O
essentially	O
support	O
the	O
underlying	O
idea	O
of	O
canonical	O
structures	B-evidence
,	O
indicating	O
that	O
most	O
CDRs	B-structure_element
with	O
germline	O
sequences	O
tend	O
to	O
adopt	O
predefined	O
conformations	O
.	O

Here	O
,	O
the	O
authors	O
report	O
U2AF65	B-protein
structures	B-evidence
and	O
single	B-experimental_method
molecule	I-experimental_method
FRET	I-experimental_method
that	O
reveal	O
mechanistic	O
insights	O
into	O
splice	B-site
site	I-site
recognition	O
.	O

A	O
subsequent	O
NMR	B-experimental_method
structure	B-evidence
characterized	O
the	O
side	B-protein_state
-	I-protein_state
by	I-protein_state
-	I-protein_state
side	I-protein_state
arrangement	O
of	O
the	O
minimal	B-protein_state
U2AF65	B-protein
RRM1	B-structure_element
and	O
RRM2	B-structure_element
connected	O
by	O
a	O
linker	B-structure_element
of	O
natural	B-protein_state
length	I-protein_state
(	O
U2AF651	B-mutant
,	I-mutant
2	I-mutant
),	O
yet	O
depended	O
on	O
the	O
dU2AF651	B-mutant
,	I-mutant
2	I-mutant
crystal	B-evidence
structures	I-evidence
for	O
RNA	B-chemical
interactions	O
and	O
an	O
ab	O
initio	O
model	O
for	O
the	O
inter	B-structure_element
-	I-structure_element
RRM	I-structure_element
linker	I-structure_element
conformation	O
.	O

Cognate	O
U2AF65	B-protein
–	O
Py	B-chemical
-	I-chemical
tract	I-chemical
recognition	O
requires	O
RRM	B-structure_element
extensions	I-structure_element

Historically	O
,	O
this	O
difference	O
was	O
attributed	O
to	O
the	O
U2AF65	B-protein
arginine	B-structure_element
–	I-structure_element
serine	I-structure_element
rich	I-structure_element
domain	I-structure_element
,	O
which	O
contacts	O
pre	B-complex_assembly
-	I-complex_assembly
mRNA	I-complex_assembly
–	I-complex_assembly
U2	I-complex_assembly
snRNA	I-complex_assembly
duplexes	I-complex_assembly
outside	O
of	O
the	O
Py	B-chemical
tract	I-chemical
.	O

U2AF65	B-protein_state
-	I-protein_state
bound	I-protein_state
Py	B-chemical
tract	I-chemical
comprises	O
nine	O
contiguous	B-structure_element
nucleotides	B-chemical

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

Yet	O
,	O
only	O
the	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
interactions	O
at	O
sites	B-site
1	I-site
and	I-site
7	I-site
are	O
nearly	O
identical	O
to	O
those	O
of	O
the	O
dU2AF651	B-mutant
,	I-mutant
2	I-mutant
structures	B-evidence
(	O
Supplementary	O
Fig	O
.	O
3a	O
,	O
f	O
).	O

Rather	O
than	O
interacting	O
with	O
a	O
new	O
5	O
′-	O
terminal	O
nucleotide	B-chemical
as	O
we	O
had	O
hypothesized	O
,	O
the	O
C	O
-	O
terminal	O
α	B-structure_element
-	I-structure_element
helix	I-structure_element
of	O
RRM2	B-structure_element
instead	O
folds	O
across	O
one	O
surface	O
of	O
rU3	B-residue_name_number
in	O
the	O
third	B-site
binding	I-site
site	I-site
(	O
Fig	O
.	O
3b	O
).	O

The	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
structures	B-evidence
reveal	O
that	O
the	O
inter	B-structure_element
-	I-structure_element
RRM	I-structure_element
linker	I-structure_element
mediates	O
an	O
extensive	B-site
interface	I-site
with	O
the	O
second	O
α	B-structure_element
-	I-structure_element
helix	I-structure_element
of	O
RRM1	B-structure_element
,	O
the	O
β2	B-structure_element
/	I-structure_element
β3	I-structure_element
strands	I-structure_element
of	O
RRM2	B-structure_element
and	O
the	O
N	O
-	O
terminal	O
α	B-structure_element
-	I-structure_element
helical	I-structure_element
extension	I-structure_element
of	O
RRM1	B-structure_element
.	O

We	O
introduced	O
glycine	B-residue_name
substitutions	B-experimental_method
to	O
maximally	O
reduce	O
the	O
buried	O
surface	O
area	O
without	O
directly	O
interfering	O
with	O
its	O
hydrogen	O
bonds	O
between	O
backbone	O
atoms	O
and	O
the	O
base	O
.	O

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

A	O
more	O
conservative	B-experimental_method
substitution	I-experimental_method
of	O
these	O
five	O
residues	O
(	O
251	B-residue_range
–	I-residue_range
255	I-residue_range
)	O
with	O
an	O
unrelated	O
sequence	O
capable	O
of	O
backbone	O
-	O
mediated	O
hydrogen	O
bonds	O
(	O
STVVP	B-mutant
>	I-mutant
NLALA	I-mutant
)	O
confirmed	O
the	O
subtle	O
impact	O
of	O
this	O
versatile	O
inter	B-structure_element
-	I-structure_element
RRM	I-structure_element
sequence	I-structure_element
on	O
affinity	B-evidence
for	O
the	O
AdML	B-gene
Py	B-chemical
tract	I-chemical
.	O

Finally	O
,	O
to	O
ensure	O
that	O
these	O
selective	O
mutations	O
were	O
sufficient	O
to	O
disrupt	O
the	O
linker	B-structure_element
/	O
RRM	B-structure_element
contacts	O
,	O
we	O
substituted	B-experimental_method
glycine	B-residue_name
for	O
the	O
majority	O
of	O
buried	O
hydrophobic	O
residues	O
in	O
the	O
inter	B-structure_element
-	I-structure_element
RRM	I-structure_element
linker	I-structure_element
(	O
including	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
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
;	O
called	O
12Gly	B-mutant
).	O

Yet	O
,	O
it	O
is	O
well	O
known	O
that	O
the	O
linker	B-experimental_method
deletion	I-experimental_method
in	O
the	O
context	O
of	O
the	O
minimal	B-protein_state
RRM1	B-structure_element
–	O
RRM2	B-structure_element
boundaries	O
has	O
no	O
detectable	O
effect	O
on	O
the	O
RNA	B-evidence
affinities	I-evidence
of	O
dU2AF651	B-mutant
,	I-mutant
2	I-mutant
compared	O
with	O
U2AF651	B-mutant
,	I-mutant
2	I-mutant
(	O
refs	O
;	O
Figs	O
1b	O
and	O
4b	O
;	O
Supplementary	O
Fig	O
.	O
4j	O
).	O

The	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
structures	B-evidence
suggest	O
that	O
an	O
extended	B-protein_state
conformation	I-protein_state
of	O
the	O
truncated	B-protein_state
dU2AF651	B-mutant
,	I-mutant
2	I-mutant
inter	B-structure_element
-	I-structure_element
RRM	I-structure_element
linker	I-structure_element
would	O
suffice	O
to	O
connect	O
the	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
RRM1	B-structure_element
C	O
terminus	O
to	O
the	O
N	O
terminus	O
of	O
RRM2	B-structure_element
(	O
24	O
Å	O
distance	O
between	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
R227	B-residue_name_number
-	O
Cα	O
–	O
H259	B-residue_name_number
-	O
Cα	O
atoms	O
),	O
which	O
agrees	O
with	O
the	O
greater	O
RNA	B-evidence
affinities	I-evidence
of	O
dU2AF651	B-mutant
,	I-mutant
2	I-mutant
and	O
U2AF651	B-mutant
,	I-mutant
2	I-mutant
dual	B-protein_state
RRMs	B-structure_element
compared	O
with	O
the	O
individual	B-protein_state
U2AF65	B-protein
RRMs	B-structure_element
.	O

Likewise	O
,	O
deletion	B-experimental_method
of	O
the	O
N	O
-	O
terminal	O
RRM1	B-structure_element
extension	I-structure_element
in	O
the	O
shortened	B-protein_state
constructs	O
would	O
remove	O
packing	O
interactions	O
that	O
position	O
the	O
linker	B-structure_element
in	O
a	O
kinked	B-structure_element
turn	I-structure_element
following	O
P229	B-residue_name_number
(	O
Fig	O
.	O
4a	O
),	O
consistent	O
with	O
the	O
lower	O
RNA	B-evidence
affinities	I-evidence
of	O
dU2AF651	B-mutant
,	I-mutant
2L	I-mutant
,	O
dU2AF651	B-mutant
,	I-mutant
2	I-mutant
and	O
U2AF651	B-mutant
,	I-mutant
2	I-mutant
compared	O
with	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
.	O

To	O
further	O
test	O
cooperation	O
among	O
the	O
U2AF65	B-protein
RRM	B-structure_element
extensions	I-structure_element
and	O
inter	B-structure_element
-	I-structure_element
RRM	I-structure_element
linker	I-structure_element
for	O
RNA	O
recognition	O
,	O
we	O
tested	O
the	O
impact	O
of	O
a	O
triple	O
Q147A	B-mutant
/	O
V254P	B-mutant
/	O
R227A	B-mutant
mutation	B-experimental_method
(	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
-	I-mutant
3Mut	I-mutant
)	O
for	O
RNA	O
binding	O
(	O
Fig	O
.	O
4b	O
;	O
Supplementary	O
Fig	O
.	O
4d	O
).	O

Notably	O
,	O
the	O
Q147A	B-mutant
/	O
V254P	B-mutant
/	O
R227A	B-mutant
mutation	B-experimental_method
reduced	O
the	O
RNA	B-evidence
affinity	I-evidence
of	O
the	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
-	I-mutant
3Mut	I-mutant
protein	O
by	O
30	O
-	O
fold	O
more	O
than	O
would	O
be	O
expected	O
based	O
on	O
simple	O
addition	O
of	O
the	O
ΔΔG	B-evidence
'	O
s	O
for	O
the	O
single	O
mutations	O
.	O

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

As	O
a	O
representative	O
splicing	O
substrate	O
,	O
we	O
utilized	O
a	O
well	O
-	O
characterized	O
minigene	B-chemical
splicing	I-chemical
reporter	I-chemical
(	O
called	O
pyPY	B-chemical
)	O
comprising	O
a	O
weak	O
(	O
that	O
is	O
,	O
degenerate	O
,	O
py	B-chemical
)	O
and	O
strong	O
(	O
that	O
is	O
,	O
U	B-structure_element
-	I-structure_element
rich	I-structure_element
,	O
PY	B-chemical
)	O
polypyrimidine	B-chemical
tracts	I-chemical
preceding	O
two	O
alternative	O
splice	B-site
sites	I-site
(	O
Fig	O
.	O
5a	O
).	O

Sparse	O
inter	B-structure_element
-	I-structure_element
RRM	I-structure_element
contacts	O
underlie	O
apo	B-protein_state
-	O
U2AF65	B-protein
dynamics	O

The	O
direct	O
interface	B-site
between	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
RRM1	B-structure_element
and	O
RRM2	B-structure_element
is	O
minor	O
,	O
burying	O
265	O
Å2	O
of	O
solvent	O
accessible	O
surface	O
area	O
compared	O
with	O
570	O
Å2	O
on	O
average	O
for	O
a	O
crystal	O
packing	O
interface	O
.	O

Criteria	O
included	O
(	O
i	O
)	O
residue	O
locations	O
that	O
are	O
distant	O
from	O
and	O
hence	O
not	O
expected	O
to	O
interfere	O
with	O
the	O
RRM	B-complex_assembly
/	I-complex_assembly
RNA	I-complex_assembly
or	O
inter	B-site
-	I-site
RRM	I-site
interfaces	I-site
,	O
(	O
ii	O
)	O
inter	O
-	O
dye	O
distances	O
(	O
50	O
Å	O
for	O
U2AF651	B-complex_assembly
,	I-complex_assembly
2L	I-complex_assembly
–	I-complex_assembly
Py	I-complex_assembly
tract	I-complex_assembly
and	O
30	O
Å	O
for	O
the	O
closed	B-protein_state
apo	B-protein_state
-	O
model	O
)	O
that	O
are	O
expected	O
to	O
be	O
near	O
the	O
Förster	B-experimental_method
radius	I-experimental_method
(	I-experimental_method
Ro	I-experimental_method
)	I-experimental_method
for	O
the	O
Cy3	B-chemical
/	O
Cy5	B-chemical
pair	O
(	O
56	O
Å	O
),	O
where	O
changes	O
in	O
the	O
efficiency	O
of	O
energy	O
transfer	O
are	O
most	O
sensitive	O
to	O
distance	O
,	O
and	O
(	O
iii	O
)	O
FRET	B-evidence
efficiencies	I-evidence
that	O
are	O
calculated	O
to	O
be	O
significantly	O
greater	O
for	O
the	O
‘	O
closed	B-protein_state
'	O
apo	B-protein_state
-	O
model	O
as	O
opposed	O
to	O
the	O
‘	O
open	B-protein_state
'	O
RNA	B-protein_state
-	I-protein_state
bound	I-protein_state
structures	B-evidence
(	O
by	O
∼	O
30	O
%).	O

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

Importantly	O
,	O
the	O
majority	O
of	O
traces	B-evidence
(∼	O
70	O
%)	O
of	O
U2AF651	B-mutant
,	I-mutant
2LFRET	I-mutant
(	O
Cy3	B-chemical
/	O
Cy5	B-chemical
)	O
bound	B-protein_state
to	I-protein_state
the	O
slide	O
-	O
tethered	O
RNA	B-chemical
lacked	O
FRET	O
fluctuations	O
and	O
predominately	O
exhibited	O
a	O
∼	O
0	O
.	O
45	O
FRET	B-evidence
value	I-evidence
(	O
for	O
example	O
,	O
Fig	O
.	O
6g	O
).	O

Thus	O
,	O
the	O
sequence	O
of	O
structural	O
rearrangements	O
of	O
U2AF65	B-protein
observed	O
in	O
smFRET	B-experimental_method
traces	B-evidence
(	O
Supplementary	O
Fig	O
.	O
7c	O
–	O
g	O
)	O
suggests	O
that	O
a	O
‘	O
conformational	O
selection	O
'	O
mechanism	O
of	O
Py	B-chemical
-	I-chemical
tract	I-chemical
recognition	O
(	O
that	O
is	O
,	O
RNA	O
ligand	O
stabilization	O
of	O
a	O
pre	B-protein_state
-	I-protein_state
configured	I-protein_state
U2AF65	B-protein
conformation	O
)	O
is	O
complemented	O
by	O
‘	O
induced	O
fit	O
'	O
(	O
that	O
is	O
,	O
RNA	O
-	O
induced	O
rearrangement	O
of	O
the	O
U2AF65	B-protein
RRMs	B-structure_element
to	O
achieve	O
the	O
final	O
‘	O
side	B-protein_state
-	I-protein_state
by	I-protein_state
-	I-protein_state
side	I-protein_state
'	O
conformation	O
),	O
as	O
discussed	O
below	O
.	O

Recently	O
,	O
high	B-experimental_method
-	I-experimental_method
throughput	I-experimental_method
sequencing	I-experimental_method
studies	I-experimental_method
have	O
shown	O
that	O
somatic	O
mutations	O
in	O
pre	B-protein_type
-	I-protein_type
mRNA	I-protein_type
splicing	I-protein_type
factors	I-protein_type
occur	O
in	O
the	O
majority	O
of	O
patients	O
with	O
myelodysplastic	O
syndrome	O
(	O
MDS	O
).	O

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

Notably	O
,	O
our	O
smFRET	B-experimental_method
results	O
reveal	O
that	O
U2AF65	B-protein
–	O
Py	B-chemical
-	I-chemical
tract	I-chemical
recognition	O
can	O
be	O
characterized	O
by	O
an	O
‘	O
extended	O
conformational	O
selection	O
'	O
model	O
(	O
Fig	O
.	O
7b	O
).	O

Here	O
,	O
the	O
majority	O
of	O
changes	O
in	O
smFRET	B-experimental_method
traces	B-evidence
for	O
U2AF651	B-mutant
,	I-mutant
2LFRET	I-mutant
(	O
Cy3	B-chemical
/	O
Cy5	B-chemical
)	O
bound	B-protein_state
to	I-protein_state
slide	O
-	O
tethered	O
RNA	B-chemical
began	O
at	O
high	O
(	O
0	O
.	O
65	O
–	O
0	O
.	O
8	O
)	O
FRET	B-evidence
value	I-evidence
and	O
transition	O
to	O
the	O
predominant	O
0	O
.	O
45	O
FRET	B-evidence
value	I-evidence
(	O
Supplementary	O
Fig	O
.	O
7c	O
–	O
g	O
).	O

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

Moreover	O
,	O
structural	O
differences	O
among	O
U2AF65	B-protein
homologues	O
and	O
paralogues	O
may	O
regulate	O
splice	B-site
site	I-site
selection	O
.	O

Ultimately	O
,	O
these	O
guidelines	O
will	O
assist	O
the	O
identification	O
of	O
3	B-site
′	I-site
splice	I-site
sites	I-site
and	O
the	O
relationship	O
of	O
disease	O
-	O
causing	O
mutations	O
to	O
penalties	O
for	O
U2AF65	B-protein
association	O
.	O

(	O
a	O
)	O
Domain	O
organization	O
of	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
(	O
fl	B-protein_state
)	O
U2AF65	B-protein
and	O
constructs	O
used	O
for	O
RNA	B-chemical
binding	O
and	O
structural	O
experiments	O
.	O

Structures	B-evidence
of	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
recognizing	O
a	O
contiguous	B-structure_element
Py	B-chemical
tract	I-chemical
.	O

The	O
prior	O
dU2AF651	B-mutant
,	I-mutant
2	I-mutant
nucleotide	B-site
-	I-site
binding	I-site
sites	I-site
are	O
given	O
in	O
parentheses	O
(	O
site	O
4	O
'	O
interacts	O
with	O
dU2AF65	B-mutant
RRM1	B-structure_element
and	O
RRM2	B-structure_element
by	O
crystallographic	O
symmetry	O
).	O

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

The	O
nucleotide	B-site
-	I-site
binding	I-site
sites	I-site
of	O
the	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
and	O
prior	O
dU2AF651	B-mutant
,	I-mutant
2	I-mutant
structure	B-evidence
are	O
compared	O
in	O
Supplementary	O
Fig	O
.	O
3a	O
–	O
h	O
.	O
The	O
first	B-site
and	I-site
seventh	I-site
U2AF651	I-site
,	I-site
2L	I-site
-	I-site
binding	I-site
sites	I-site
are	O
unchanged	O
from	O
the	O
prior	O
dU2AF651	B-complex_assembly
,	I-complex_assembly
2	I-complex_assembly
–	I-complex_assembly
RNA	I-complex_assembly
structure	B-evidence
and	O
are	O
portrayed	O
in	O
Supplementary	O
Fig	O
.	O
3a	O
,	O
f	O
.	O
The	O
four	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
structures	B-evidence
are	O
similar	O
with	O
the	O
exception	O
of	O
pH	O
-	O
dependent	O
variations	O
at	O
the	O
ninth	B-site
site	I-site
that	O
are	O
detailed	O
in	O
Supplementary	O
Fig	O
.	O
3i	O
,	O
j	O
.	O
The	O
representative	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
structure	B-evidence
shown	O
has	O
the	O
highest	O
resolution	O
and	O
/	O
or	O
ribose	B-chemical
nucleotide	I-chemical
at	O
the	O
given	O
site	O
:	O
(	O
a	O
)	O
rU2	B-residue_name_number
of	O
structure	O
iv	O
;	O
(	O
b	O
)	O
rU3	B-residue_name_number
of	O
structure	O
iii	O
;	O
(	O
c	O
)	O
rU4	B-residue_name_number
of	O
structure	O
i	O
;	O
(	O
d	O
)	O
rU5	B-residue_name_number
of	O
structure	O
iii	O
;	O
(	O
e	O
)	O
rU6	B-residue_name_number
of	O
structure	O
ii	O
;	O
(	O
f	O
)	O
dU8	B-residue_name_number
of	O
structure	O
iii	O
;	O
(	O
g	O
)	O
dU9	B-residue_name_number
of	O
structure	O
iii	O
;	O
(	O
h	O
)	O
rC9	B-residue_name_number
of	O
structure	O
iv	O
.	O

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

The	O
apparent	O
equilibrium	B-evidence
dissociation	I-evidence
constants	I-evidence
(	O
KD	B-evidence
)	O
of	O
the	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
mutant	B-protein_state
proteins	O
are	O
:	O
wild	B-protein_state
type	I-protein_state
(	O
WT	B-protein_state
),	O
35	O
±	O
6	O
nM	O
;	O
3Gly	B-mutant
,	O
47	O
±	O
4	O
nM	O
;	O
5Gly	B-mutant
,	O
61	O
±	O
3	O
nM	O
;	O
12Gly	B-mutant
,	O
88	O
±	O
21	O
nM	O
;	O
NLALA	B-mutant
,	O
45	O
±	O
3	O
nM	O
;	O
dU2AF651	B-mutant
,	I-mutant
2L	I-mutant
,	O
123	O
±	O
5	O
nM	O
;	O
dU2AF651	B-mutant
,	I-mutant
2	I-mutant
,	O
5000	O
±	O
100	O
nM	O
;	O
3Mut	B-mutant
,	O
5630	O
±	O
70	O
nM	O
.	O
The	O
average	O
KA	B-evidence
and	O
s	O
.	O
e	O
.	O
m	O
.	O
for	O
three	O
independent	O
titrations	O
are	O
plotted	O
.	O

(	O
b	O
)	O
Representative	O
RT	B-experimental_method
-	I-experimental_method
PCR	I-experimental_method
of	O
pyPY	B-chemical
transcripts	O
from	O
HEK293T	O
cells	O
co	B-experimental_method
-	I-experimental_method
transfected	I-experimental_method
with	O
constructs	O
encoding	O
the	O
pyPY	B-chemical
minigene	O
and	O
either	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
(	O
WT	B-protein_state
)	O
U2AF65	B-protein
or	O
a	O
triple	O
U2AF65	B-protein
mutant	B-protein_state
(	O
3Mut	B-mutant
)	O
of	O
Q147A	B-mutant
,	O
R227A	B-mutant
and	O
V254P	B-mutant
residues	O
.	O
(	O
c	O
)	O
A	O
bar	O
graph	O
of	O
the	O
average	O
percentage	O
of	O
the	O
py	B-chemical
-	O
spliced	O
mRNA	B-chemical
relative	O
to	O
total	O
detected	O
pyPY	B-chemical
transcripts	O
(	O
spliced	O
and	O
unspliced	O
)	O
for	O
the	O
corresponding	O
gel	O
lanes	O
(	O
black	O
,	O
no	O
U2AF65	B-protein
added	O
;	O
white	O
,	O
WT	B-protein_state
U2AF65	B-protein
;	O
grey	O
,	O
3Mut	B-mutant
U2AF65	B-protein
).	O

Schematic	O
models	O
of	O
U2AF65	B-protein
recognizing	O
the	O
Py	B-chemical
tract	I-chemical
.	O

Alternatively	O
,	O
a	O
conformation	O
of	O
U2AF65	B-protein
corresponding	O
to	O
∼	O
0	O
.	O
45	O
FRET	B-evidence
value	I-evidence
can	O
directly	O
bind	O
to	O
RNA	B-chemical
;	O
RNA	B-chemical
binding	O
stabilizes	O
the	O
‘	O
open	B-protein_state
',	O
side	B-protein_state
-	I-protein_state
by	I-protein_state
-	I-protein_state
side	I-protein_state
conformation	O
and	O
thus	O
shifts	O
the	O
U2AF65	B-protein
population	O
towards	O
the	O
∼	O
0	O
.	O
45	O
FRET	B-evidence
value	I-evidence
.	O

However	O
,	O
there	O
is	O
still	O
no	O
general	O
consensus	O
within	O
the	O
field	O
on	O
how	O
to	O
minimize	O
RD	O
during	O
MX	B-experimental_method
data	O
collection	O
,	O
and	O
debates	O
on	O
the	O
dependence	O
of	O
RD	O
progression	O
on	O
incident	O
X	O
-	O
ray	O
energy	O
(	O
Shimizu	O
et	O
al	O
.,	O
2007	O
;	O
Liebschner	O
et	O
al	O
.,	O
2015	O
)	O
and	O
the	O
efficacy	O
of	O
radical	O
scavengers	O
(	O
Allan	O
et	O
al	O
.,	O
2013	O
)	O
have	O
yet	O
to	O
be	O
resolved	O
.	O

Specific	B-experimental_method
radiation	I-experimental_method
damage	I-experimental_method
(	O
SRD	B-experimental_method
)	O
is	O
observed	O
in	O
the	O
real	B-evidence
-	I-evidence
space	I-evidence
electron	I-evidence
density	I-evidence
,	O
and	O
has	O
been	O
detected	O
at	O
much	O
lower	O
doses	O
than	O
any	O
observable	O
decay	O
in	O
the	O
intensity	O
of	O
reflections	O
.	O

It	O
binds	O
with	O
high	O
affinity	O
(	O
K	B-evidence
d	I-evidence
≃	O
1	O
.	O
0	O
nM	O
)	O
to	O
RNA	B-chemical
segments	O
containing	O
11	O
GAG	B-structure_element
/	I-structure_element
UAG	I-structure_element
triplets	I-structure_element
separated	O
by	O
two	O
or	O
three	O
spacer	B-structure_element
nucleotides	I-structure_element
(	O
Elliott	O
et	O
al	O
.,	O
2001	O
)	O
to	O
regulate	O
the	O
transcription	O
of	O
tryptophan	B-chemical
biosynthetic	O
genes	O
in	O
Bacillus	B-species
subtilis	I-species
(	O
Antson	O
et	O
al	O
.,	O
1999	O
).	O

Ten	O
successive	O
1	O
.	O
98	O
Å	O
resolution	O
MX	B-experimental_method
data	O
sets	O
were	O
collected	O
from	O
the	O
same	O
TRAP	B-complex_assembly
–	I-complex_assembly
RNA	I-complex_assembly
crystal	B-evidence
to	O
analyse	O
X	O
-	O
ray	O
-	O
induced	O
structural	O
changes	O
over	O
a	O
large	O
dose	O
range	O
(	O
d	O
1	O
=	O
1	O
.	O
3	O
MGy	O
to	O
d	O
10	O
=	O
25	O
.	O
0	O
MGy	O
).	O

The	O
substrate	O
Trp	B-chemical
amino	O
-	O
acid	O
ligands	O
also	O
exhibited	O
disordering	O
of	O
the	O
free	O
terminal	O
carboxyl	O
groups	O
at	O
higher	O
doses	O
(	O
Fig	O
.	O
2	O
▸	O
a	O
);	O
however	O
,	O
no	O
clear	O
Fourier	B-evidence
difference	I-evidence
peaks	I-evidence
could	O
be	O
observed	O
visually	O
.	O

A	O
significant	O
reduction	O
in	O
D	B-evidence
loss	I-evidence
is	O
seen	O
for	O
Glu36	B-residue_name_number
in	O
RNA	B-protein_state
-	I-protein_state
bound	I-protein_state
compared	O
with	O
nonbound	B-protein_state
TRAP	B-complex_assembly
,	O
indicative	O
of	O
a	O
lower	O
rate	O
of	O
side	O
-	O
chain	O
decarboxylation	O
(	O
Fig	O
.	O
5	O
▸	O
a	O
;	O
p	O
=	O
6	O
.	O
06	O
×	O
10	O
−	O
5	O
).	O

RNA	B-chemical
binding	O
reduces	O
radiation	O
-	O
induced	O
disorder	O
on	O
the	O
atomic	O
scale	O

RNA	B-chemical
backbone	O
disordering	O
thus	O
appears	O
to	O
be	O
the	O
main	O
radiation	O
-	O
induced	O
effect	O
in	O
RNA	B-chemical
,	O
with	O
the	O
protein	O
–	O
base	O
interactions	O
maintained	O
even	O
at	O
high	O
doses	O
(>	O
20	O
MGy	O
).	O

The	O
U4	B-residue_name_number
phosphate	B-chemical
exhibited	O
marginally	O
larger	O
D	B-evidence
loss	I-evidence
values	O
above	O
20	O
MGy	O
than	O
G1	B-residue_name_number
,	O
A2	B-residue_name_number
and	O
G3	B-residue_name_number
(	O
Supplementary	O
Fig	O
.	O
S4	O
).	O

The	O
Glu36	B-residue_name_number
carboxyl	O
side	O
chain	O
also	O
potentially	O
forms	O
hydrogen	O
bonds	O
to	O
His34	B-residue_name_number
and	O
Lys56	B-residue_name_number
,	O
but	O
since	O
these	O
interactions	O
are	O
conserved	B-protein_state
irrespective	O
of	O
G3	B-residue_name_number
nucleotide	O
binding	O
,	O
this	O
cannot	O
directly	O
account	O
for	O
the	O
stabilization	O
effect	O
on	O
Glu36	B-residue_name_number
in	O
RNA	B-protein_state
-	I-protein_state
bound	I-protein_state
TRAP	B-complex_assembly
.	O

By	O
comparing	O
equivalent	O
acidic	O
residues	O
with	B-protein_state
and	O
without	B-protein_state
RNA	B-chemical
,	O
we	O
have	O
now	O
deconvoluted	O
the	O
role	O
of	O
solvent	O
accessibility	O
from	O
other	O
local	O
protein	O
environment	O
factors	O
,	O
and	O
thus	O
propose	O
a	O
suitable	O
mechanism	O
by	O
which	O
exceptionally	O
low	O
solvent	O
accessibility	O
can	O
reduce	O
the	O
rate	O
of	O
decarboxylation	O
.	O

Apart	O
from	O
these	O
RNA	B-site
-	I-site
binding	I-site
interfaces	I-site
,	O
RNA	B-chemical
binding	O
was	O
seen	O
to	O
enhance	O
decarboxylation	O
for	O
residues	O
Glu50	B-residue_name_number
,	O
Glu71	B-residue_name_number
and	O
Glu73	B-residue_name_number
,	O
all	O
of	O
which	O
are	O
involved	O
in	O
crystal	O
contacts	O
between	O
TRAP	B-complex_assembly
rings	B-structure_element
(	O
Fig	O
.	O
4	O
▸	O
c	O
).	O

In	O
TRAP	B-complex_assembly
,	O
D	B-evidence
loss	I-evidence
increased	O
at	O
a	O
similar	O
rate	O
for	O
both	O
the	O
Tyr	B-residue_name
O	O
atoms	O
and	O
aromatic	O
ring	B-structure_element
atoms	O
,	O
suggesting	O
that	O
full	O
ring	B-structure_element
conformational	O
disordering	O
is	O
more	O
likely	O
.	O

Within	O
the	O
cellular	O
environment	O
,	O
this	O
mechanism	O
could	O
reduce	O
the	O
risk	O
that	O
radiation	O
-	O
damaged	O
proteins	O
might	O
bind	O
to	O
RNA	B-chemical
,	O
thus	O
avoiding	O
the	O
detrimental	O
introduction	O
of	O
incorrect	O
DNA	B-chemical
-	O
repair	O
,	O
transcriptional	O
and	O
base	O
-	O
modification	O
pathways	O
.	O

RNA	B-site
-	I-site
binding	I-site
interface	I-site
interactions	O
are	O
shown	O
for	O
TRAP	B-complex_assembly
chain	O
N	O
,	O
with	O
the	O
F	O
obs	O
(	O
d	O
7	O
)	O
−	O
F	O
obs	O
(	O
d	O
1	O
)	O
Fourier	O
difference	O
map	O
(	O
dose	O
16	O
.	O
7	O
MGy	O
)	O
overlaid	O
and	O
contoured	O
at	O
a	O
±	O
4σ	O
level	O
.	O

Plants	B-taxonomy_domain
constantly	O
renew	O
during	O
their	O
life	O
cycle	O
and	O
thus	O
require	O
to	O
shed	O
senescent	O
and	O
damaged	O
organs	O
.	O

Here	O
we	O
show	O
that	O
IDA	B-protein
is	O
sensed	O
directly	O
by	O
the	O
HAESA	B-protein
ectodomain	B-structure_element
.	O

This	O
sequence	O
pattern	O
is	O
conserved	B-protein_state
among	O
diverse	O
plant	B-taxonomy_domain
peptides	B-chemical
,	O
suggesting	O
that	O
plant	B-taxonomy_domain
peptide	B-protein_type
hormone	I-protein_type
receptors	I-protein_type
may	O
share	O
a	O
common	O
ligand	O
binding	O
mode	O
and	O
activation	O
mechanism	O
.	O

The	O
experiments	O
show	O
that	O
IDA	B-protein
binds	B-protein_state
directly	I-protein_state
to	I-protein_state
a	O
canyon	B-protein_state
shaped	I-protein_state
pocket	B-site
in	O
HAESA	B-protein
that	O
extends	O
out	O
from	O
the	O
surface	O
of	O
the	O
cell	O
.	O

The	O
next	O
step	O
following	O
on	O
from	O
this	O
work	O
is	O
to	O
understand	O
what	O
signals	O
are	O
produced	O
when	O
IDA	B-protein
activates	O
HAESA	B-protein
.	O

The	O
calculated	O
molecular	O
mass	O
is	O
65	O
.	O
7	O
kDa	O
,	O
the	O
actual	O
molecular	O
mass	O
obtained	O
by	O
mass	B-experimental_method
spectrometry	I-experimental_method
is	O
74	O
,	O
896	O
Da	O
,	O
accounting	O
for	O
the	O
N	B-chemical
-	I-chemical
glycans	I-chemical
.	O
(	O
B	O
)	O
Ribbon	O
diagrams	O
showing	O
front	O
(	O
left	O
panel	O
)	O
and	O
side	O
views	O
(	O
right	O
panel	O
)	O
of	O
the	O
isolated	O
HAESA	B-protein
LRR	B-structure_element
domain	I-structure_element
.	O

The	O
N	O
-	O
(	O
residues	O
20	B-residue_range
–	I-residue_range
88	I-residue_range
)	O
and	O
C	O
-	O
terminal	O
(	O
residues	O
593	B-residue_range
–	I-residue_range
615	I-residue_range
)	O
capping	B-structure_element
domains	I-structure_element
are	O
shown	O
in	O
yellow	O
,	O
the	O
central	O
21	O
LRR	B-structure_element
motifs	I-structure_element
are	O
in	O
blue	O
and	O
disulphide	B-ptm
bonds	I-ptm
are	O
highlighted	O
in	O
green	O
(	O
in	O
bonds	O
representation	O
).	O
(	O
C	O
)	O
Structure	B-experimental_method
based	I-experimental_method
sequence	I-experimental_method
alignment	I-experimental_method
of	O
the	O
21	O
leucine	B-structure_element
-	I-structure_element
rich	I-structure_element
repeats	I-structure_element
in	O
HAESA	B-protein
with	O
the	O
plant	B-taxonomy_domain
LRR	B-structure_element
consensus	O
sequence	O
shown	O
for	O
comparison	O
.	O

During	O
their	O
growth	O
,	O
development	O
and	O
reproduction	O
plants	B-taxonomy_domain
use	O
cell	O
separation	O
processes	O
to	O
detach	O
no	O
-	O
longer	O
required	O
,	O
damaged	O
or	O
senescent	O
organs	O
.	O

Abscission	O
of	O
floral	O
organs	O
in	O
Arabidopsis	B-taxonomy_domain
is	O
a	O
model	O
system	O
to	O
study	O
these	O
cell	O
separation	O
processes	O
in	O
molecular	O
detail	O
.	O

The	O
LRR	B-structure_element
-	I-structure_element
RKs	I-structure_element
HAESA	B-protein
(	O
greek	O
:	O
to	O
adhere	O
to	O
)	O
and	O
HAESA	B-protein
-	I-protein
LIKE	I-protein
2	I-protein
(	O
HSL2	B-protein
)	O
redundantly	O
control	O
floral	O
abscission	O
.	O

Transphosphorylation	O
activity	O
from	O
the	O
active	B-protein_state
kinase	O
to	O
the	O
mutated	B-protein_state
form	O
can	O
be	O
observed	O
in	O
both	O
directions	O
(	O
lanes	O
5	O
+	O
6	O
).	O

IDL1	B-protein
,	O
which	O
can	O
rescue	O
IDA	B-protein
loss	O
-	O
of	O
-	O
function	O
mutants	O
when	O
introduced	O
in	O
abscission	O
zone	O
cells	O
,	O
can	O
also	O
be	O
sensed	O
by	O
HAESA	B-protein
,	O
albeit	O
with	O
lower	O
affinity	B-evidence
(	O
Figure	O
2D	O
).	O

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

Our	O
binding	B-experimental_method
assays	I-experimental_method
reveal	O
that	O
IDA	B-chemical
family	I-chemical
peptides	I-chemical
are	O
sensed	O
by	O
the	O
isolated	B-protein_state
HAESA	B-protein
ectodomain	B-structure_element
with	O
relatively	O
weak	O
binding	B-evidence
affinities	I-evidence
(	O
Figures	O
1B	O
,	O
2A	O
–	O
D	O
).	O

Possibly	O
because	O
SERKs	B-protein_type
have	O
additional	O
roles	O
in	O
plant	O
development	O
such	O
as	O
in	O
pollen	O
formation	O
and	O
brassinosteroid	O
signaling	O
,	O
we	O
found	O
that	O
higher	O
-	O
order	O
SERK	O
mutants	O
exhibit	O
pleiotropic	O
phenotypes	O
in	O
the	O
flower	O
,	O
rendering	O
their	O
analysis	O
and	O
comparison	O
by	O
quantitative	B-experimental_method
petal	I-experimental_method
break	I-experimental_method
-	I-experimental_method
strength	I-experimental_method
assays	I-experimental_method
difficult	O
.	O

Ribbon	O
diagrams	O
of	O
HAESA	B-protein
(	O
in	O
blue	O
)	O
and	O
SERK1	B-protein
(	O
in	O
orange	O
)	O
are	O
shown	O
with	O
selected	O
interface	B-site
residues	I-site
(	O
in	O
bonds	O
representation	O
).	O

HAESA	B-protein
LRRs	B-structure_element
16	I-structure_element
–	I-structure_element
21	I-structure_element
and	O
its	O
C	O
-	O
terminal	O
capping	B-structure_element
domain	I-structure_element
undergo	O
a	O
conformational	O
change	O
upon	O
SERK1	B-protein
binding	O
(	O
Figure	O
4B	O
).	O

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

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

Importantly	O
,	O
our	O
calorimetry	B-experimental_method
assays	I-experimental_method
reveal	O
that	O
the	O
SERK1	B-protein
ectodomain	B-structure_element
binds	B-protein_state
HAESA	B-protein
with	O
nanomolar	O
affinity	O
,	O
but	O
only	O
in	O
the	O
presence	B-protein_state
of	I-protein_state
IDA	B-protein
(	O
Figure	O
3C	O
).	O

SERK1	B-protein
uses	O
partially	O
overlapping	O
surface	O
areas	O
to	O
activate	O
different	O
plant	B-taxonomy_domain
signaling	B-protein_type
receptors	I-protein_type
.	O

Residues	O
interacting	O
with	O
the	O
HAESA	B-protein
or	O
BRI1	B-protein
LRR	B-structure_element
domains	I-structure_element
are	O
shown	O
in	O
orange	O
or	O
magenta	O
,	O
respectively	O
.	O

Comparison	B-experimental_method
of	O
our	O
HAESA	B-complex_assembly
–	I-complex_assembly
IDA	I-complex_assembly
–	I-complex_assembly
SERK1	I-complex_assembly
structure	B-evidence
with	O
the	O
brassinosteroid	O
receptor	O
signaling	O
complex	O
,	O
where	O
SERK1	B-protein
also	O
acts	O
as	O
co	B-protein_type
-	I-protein_type
receptor	I-protein_type
,	O
reveals	O
an	O
overall	O
conserved	B-protein_state
mode	O
of	O
SERK1	B-protein
binding	O
,	O
while	O
the	O
ligand	B-site
binding	I-site
pockets	I-site
map	O
to	O
very	O
different	O
areas	O
in	O
the	O
corresponding	O
receptors	O
(	O
LRRs	B-structure_element
2	I-structure_element
–	I-structure_element
14	I-structure_element
;	O
HAESA	B-protein
;	O
LRRs	B-structure_element
21	I-structure_element
–	I-structure_element
25	I-structure_element
,	O
BRI1	B-protein
)	O
and	O
may	O
involve	O
an	O
island	O
domain	O
(	O
BRI1	B-protein
)	O
or	O
not	O
(	O
HAESA	B-protein
)	O
(	O
Figure	O
6A	O
).	O

Several	O
residues	O
in	O
the	O
SERK1	B-protein
N	O
-	O
terminal	O
capping	B-structure_element
domain	I-structure_element
(	O
Thr59SERK1	B-residue_name_number
,	O
Phe61SERK1	B-residue_name_number
)	O
and	O
the	O
LRR	B-site
inner	I-site
surface	I-site
(	O
Asp75SERK1	B-residue_name_number
,	O
Tyr101SERK1	B-residue_name_number
,	O
SER121SERK1	B-residue_name_number
,	O
Phe145SERK1	B-residue_name_number
)	O
contribute	O
to	O
the	O
formation	O
of	O
both	O
complexes	O
(	O
Figures	O
4C	O
,	O
D	O
,	O
6B	O
).	O

This	O
fact	O
together	O
with	O
the	O
largely	O
overlapping	O
SERK1	B-site
binding	I-site
surfaces	I-site
in	O
HAESA	B-protein
and	O
BRI1	B-protein
allows	O
us	O
to	O
speculate	O
that	O
SERK1	B-protein
may	O
promote	O
high	O
-	O
affinity	O
peptide	B-protein_type
hormone	I-protein_type
and	O
brassinosteroid	O
sensing	O
by	O
simply	O
slowing	O
down	O
dissociation	O
of	O
the	O
ligand	O
from	O
its	O
cognate	O
receptor	O
.	O

The	O
conserved	B-protein_state
(	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
is	O
highlighted	O
in	O
red	O
,	O
the	O
central	O
Hyp	B-residue_name
residue	O
in	O
IDLs	B-protein_type
and	O
CLEs	B-protein_type
is	O
marked	O
in	O
blue	O
.	O

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

The	O
acetyllysine	B-residue_name
binding	O
function	O
of	O
the	O
AF9	B-protein
YEATS	B-structure_element
domain	I-structure_element
is	O
essential	O
for	O
the	O
recruitment	O
of	O
the	O
histone	B-protein_type
methyltransferase	I-protein_type
DOT1L	B-protein
to	O
H3K9ac	B-protein_type
-	O
containing	O
chromatin	O
and	O
for	O
DOT1L	B-protein
-	O
mediated	O
H3K79	B-protein_type
methylation	B-ptm
and	O
transcription	O
.	O

To	O
elucidate	O
the	O
molecular	O
basis	O
for	O
recognition	O
of	O
the	O
H3K9cr	B-protein_type
mark	O
,	O
we	O
obtained	O
a	O
crystal	B-evidence
structure	I-evidence
of	O
the	O
Taf14	B-protein
YEATS	B-structure_element
domain	I-structure_element
in	B-protein_state
complex	I-protein_state
with	I-protein_state
H3K9cr5	B-chemical
-	I-chemical
13	I-chemical
(	O
residues	O
5	B-residue_range
–	I-residue_range
13	I-residue_range
of	O
H3	B-protein_type
)	O
peptide	O
(	O
Fig	O
.	O
1	O
,	O
Supplementary	O
Results	O
,	O
Supplementary	O
Fig	O
.	O
1	O
and	O
Supplementary	O
Table	O
1	O
).	O

The	O
hydroxyl	O
group	O
of	O
Thr61	B-residue_name_number
also	O
participates	O
in	O
a	O
hydrogen	O
bond	O
with	O
the	O
amide	O
nitrogen	O
of	O
the	O
K9cr	B-residue_name_number
side	O
chain	O
(	O
Fig	O
.	O
1d	O
).	O

This	O
value	O
is	O
in	O
the	O
range	O
of	O
binding	B-evidence
affinities	I-evidence
exhibited	O
by	O
the	O
majority	O
of	O
histone	O
readers	O
,	O
thus	O
attesting	O
to	O
the	O
physiological	O
relevance	O
of	O
the	O
H3K9cr	B-protein_type
recognition	O
by	O
Taf14	B-protein
.	O

As	O
shown	O
in	O
Figure	O
2a	O
,	O
b	O
and	O
Supplementary	O
Fig	O
.	O
3e	O
,	O
H3K9cr	B-protein_type
levels	O
were	O
abolished	O
or	O
reduced	O
considerably	O
in	O
the	O
HAT	B-protein_type
deletion	B-experimental_method
strains	O
,	O
whereas	O
they	O
were	O
dramatically	O
increased	O
in	O
the	O
HDAC	B-protein_type
deletion	B-experimental_method
strains	O
.	O

We	O
have	O
previously	O
shown	O
that	O
among	O
acetylated	B-protein_state
histone	B-protein_type
marks	O
,	O
the	O
Taf14	B-protein
YEATS	B-structure_element
domain	I-structure_element
prefers	O
acetylated	B-protein_state
H3K9	B-protein_type
(	O
also	O
see	O
Supplementary	O
Fig	O
.	O
3b	O
),	O
however	O
it	O
binds	O
to	O
H3K9cr	B-protein_type
tighter	O
.	O

To	O
determine	O
if	O
the	O
binding	O
to	O
crotonyllysine	B-residue_name
is	O
conserved	B-protein_state
,	O
we	O
tested	O
human	B-species
YEATS	B-structure_element
domains	I-structure_element
by	O
pull	B-experimental_method
-	I-experimental_method
down	I-experimental_method
experiments	I-experimental_method
using	O
singly	O
and	O
multiply	O
acetylated	B-protein_state
,	O
propionylated	B-protein_state
,	O
butyrylated	B-protein_state
,	O
and	O
crotonylated	B-protein_state
histone	B-protein_type
peptides	O
(	O
Supplementary	O
Fig	O
.	O
6	O
).	O

We	O
found	O
that	O
all	O
YEATS	B-structure_element
domains	I-structure_element
tested	O
are	O
capable	O
of	O
binding	O
to	O
crotonyllysine	B-residue_name
peptides	O
,	O
though	O
they	O
display	O
variable	O
preferences	O
for	O
the	O
acyl	O
moieties	O
.	O

While	O
YEATS2	B-protein
and	O
ENL	B-protein
showed	O
selectivity	O
for	O
the	O
crotonylated	B-protein_state
peptides	O
,	O
GAS41	B-protein
and	O
AF9	B-protein
bound	O
acylated	B-protein_state
peptides	O
almost	O
equally	O
well	O
.	O

Spectra	B-evidence
are	O
color	O
coded	O
according	O
to	O
the	O
protein	O
:	O
peptide	O
molar	O
ratio	O
.	O

Substitution	B-experimental_method
of	O
Thr1	B-residue_name_number
by	O
Cys	B-residue_name
disrupts	O
the	O
interaction	O
with	O
Lys33	B-residue_name_number
and	O
inactivates	B-protein_state
the	O
proteasome	B-complex_assembly
.	O

Here	O
,	O
the	O
authors	O
use	O
structural	O
biology	O
and	O
biochemistry	O
to	O
investigate	O
the	O
role	O
of	O
proteasome	B-complex_assembly
active	B-site
site	I-site
residues	O
on	O
maturation	O
and	O
activity	O
.	O

Its	O
seven	O
different	O
α	B-protein
and	O
seven	O
different	O
β	B-protein
subunits	I-protein
assemble	O
into	O
four	O
heptameric	B-oligomeric_state
rings	B-structure_element
that	O
are	O
stacked	O
on	O
each	O
other	O
to	O
form	O
a	O
hollow	B-structure_element
cylinder	I-structure_element
.	O

Only	O
three	O
out	O
of	O
the	O
seven	O
different	O
β	B-protein
subunits	I-protein
,	O
namely	O
β1	B-protein
,	O
β2	B-protein
and	O
β5	B-protein
,	O
bear	O
N	O
-	O
terminal	O
proteolytic	B-site
active	I-site
centres	I-site
,	O
and	O
before	O
CP	B-complex_assembly
maturation	O
these	O
are	O
protected	O
by	O
propeptides	B-structure_element
.	O

In	O
principle	O
it	O
could	O
function	O
as	O
the	O
general	O
base	O
during	O
both	O
autocatalytic	B-ptm
removal	I-ptm
of	O
the	O
propeptide	B-structure_element
and	O
protein	O
substrate	O
cleavage	O
.	O

Proteasome	B-complex_assembly
-	O
mediated	O
degradation	O
of	O
cell	O
-	O
cycle	O
regulators	O
and	O
potentially	O
toxic	O
misfolded	O
proteins	O
is	O
required	O
for	O
the	O
viability	O
of	O
eukaryotic	B-taxonomy_domain
cells	O
.	O

Inactivation	O
of	O
the	O
active	B-site
site	I-site
Thr1	B-residue_name_number
by	O
mutation	B-experimental_method
to	I-experimental_method
Ala	B-residue_name
has	O
been	O
used	O
to	O
study	O
substrate	O
specificity	O
and	O
the	O
hierarchy	O
of	O
the	O
proteasome	B-complex_assembly
active	B-site
sites	I-site
.	O

Viability	O
is	O
restored	O
if	O
the	O
β5	B-mutant
-	I-mutant
T1A	I-mutant
subunit	O
has	O
its	O
propeptide	B-structure_element
(	O
pp	B-chemical
)	O
deleted	B-experimental_method
but	I-experimental_method
expressed	I-experimental_method
separately	I-experimental_method
in	O
trans	B-protein_state
(	O
β5	B-mutant
-	I-mutant
T1A	I-mutant
pp	B-chemical
trans	B-protein_state
),	O
although	O
substantial	O
phenotypic	O
impairment	O
remains	O
(	O
Table	O
1	O
).	O

Processing	O
of	O
β	O
-	O
subunit	O
precursors	O
requires	O
deprotonation	O
of	O
Thr1OH	B-residue_name_number
;	O
however	O
,	O
the	O
general	O
base	O
initiating	O
autolysis	B-ptm
is	O
unknown	O
.	O

Remarkably	O
,	O
eukaryotic	B-taxonomy_domain
proteasomal	O
β5	B-protein
subunits	O
bear	O
a	O
His	B-residue_name
residue	O
in	O
position	O
(-	B-residue_number
2	I-residue_number
)	I-residue_number
of	O
the	O
propeptide	B-structure_element
(	O
Supplementary	O
Fig	O
.	O
3a	O
).	O

We	O
determined	O
crystal	B-evidence
structures	I-evidence
of	O
the	O
β5	B-mutant
-	I-mutant
H	I-mutant
(-	I-mutant
2	I-mutant
)	I-mutant
L	I-mutant
-	I-mutant
T1A	I-mutant
,	O
β5	B-mutant
-	I-mutant
H	I-mutant
(-	I-mutant
2	I-mutant
)	I-mutant
T	I-mutant
-	I-mutant
T1A	I-mutant
and	O
the	O
β5	B-mutant
-	I-mutant
H	I-mutant
(-	I-mutant
2	I-mutant
)	I-mutant
A	I-mutant
-	I-mutant
T1A	I-mutant
-	I-mutant
K81R	I-mutant
mutants	O
(	O
Supplementary	O
Table	O
1	O
).	O

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

Next	O
,	O
we	O
determined	O
the	O
crystal	B-evidence
structure	I-evidence
of	O
a	O
chimeric	B-protein_state
yCP	B-complex_assembly
having	O
the	O
yeast	B-taxonomy_domain
β1	B-protein
-	O
propeptide	B-structure_element
replaced	B-experimental_method
by	I-experimental_method
its	O
β5	B-protein
counterpart	B-structure_element
.	O

These	O
results	O
suggest	O
that	O
Asp166	B-residue_name_number
and	O
Ser129	B-residue_name_number
function	O
as	O
a	O
proton	O
shuttle	O
and	O
affect	O
the	O
protonation	O
state	O
of	O
Thr1N	B-residue_name_number
during	O
autolysis	B-ptm
and	O
catalysis	O
.	O

On	O
the	O
basis	O
of	O
the	O
phenotype	O
of	O
the	O
T1C	B-mutant
mutant	B-protein_state
and	O
the	O
propeptide	B-structure_element
remnant	O
identified	O
in	O
its	O
active	B-site
site	I-site
,	O
we	O
suppose	O
that	O
autolysis	B-ptm
is	O
retarded	O
and	O
may	O
not	O
have	O
been	O
completed	O
before	O
crystallization	B-experimental_method
.	O

Owing	O
to	O
the	O
unequal	O
positions	O
of	O
the	O
two	O
β5	B-protein
subunits	O
within	O
the	O
CP	B-complex_assembly
in	O
the	O
crystal	O
lattice	O
,	O
maturation	O
and	O
propeptide	B-structure_element
displacement	O
may	O
occur	O
at	O
different	O
timescales	O
in	O
the	O
two	O
subunits	O
.	O

Despite	O
propeptide	B-ptm
hydrolysis	I-ptm
,	O
the	O
β5	B-mutant
-	I-mutant
T1C	I-mutant
active	B-site
site	I-site
is	O
catalytically	B-protein_state
inactive	I-protein_state
(	O
Fig	O
.	O
4b	O
and	O
Supplementary	O
Fig	O
.	O
9a	O
).	O

All	O
proteasomes	B-complex_assembly
strictly	B-protein_state
employ	I-protein_state
threonine	B-residue_name
as	O
the	O
active	B-site
-	I-site
site	I-site
residue	I-site
instead	O
of	O
serine	B-residue_name
.	O

From	O
these	O
data	O
we	O
conclude	O
that	O
only	O
the	O
positioning	O
of	O
Gly	B-residue_name_number
(-	I-residue_name_number
1	I-residue_name_number
)	I-residue_name_number
and	O
Thr1	B-residue_name_number
as	O
well	O
as	O
the	O
integrity	O
of	O
the	O
proteasomal	O
active	B-site
site	I-site
are	O
required	O
for	O
autolysis	B-ptm
.	O

In	O
this	O
regard	O
,	O
inappropriate	O
N	B-ptm
-	I-ptm
acetylation	I-ptm
of	O
the	O
Thr1	B-residue_name_number
N	O
terminus	O
cannot	O
be	O
removed	O
by	O
Thr1Oγ	B-residue_name_number
due	O
to	O
the	O
rotational	O
freedom	O
and	O
flexibility	O
of	O
the	O
acetyl	O
group	O
.	O

Thus	O
,	O
specific	O
protein	O
surroundings	O
can	O
significantly	O
alter	O
the	O
chemical	O
properties	O
of	O
amino	O
acids	O
such	O
as	O
Lys	B-residue_name
to	O
function	O
as	O
an	O
acid	O
–	O
base	O
catalyst	O
.	O

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

Breakdown	O
of	O
this	O
tetrahedral	O
transition	O
state	O
releases	O
the	O
Thr1	B-residue_name_number
N	O
terminus	O
that	O
is	O
protonated	O
by	O
aspartic	B-residue_name_number
acid	I-residue_name_number
166	I-residue_name_number
via	O
Ser129OH	B-residue_name_number
to	O
yield	O
Thr1NH3	B-residue_name_number
+.	O

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

However	O
,	O
owing	O
to	O
Cys	B-residue_name
being	O
a	O
strong	O
nucleophile	O
,	O
the	O
propeptide	B-structure_element
can	O
still	O
be	O
cleaved	B-protein_state
off	O
over	O
time	O
.	O

Notably	O
,	O
in	O
the	O
threonine	B-protein_type
aspartase	I-protein_type
Taspase1	B-protein
,	O
mutation	B-experimental_method
of	O
the	O
active	B-site
-	I-site
site	I-site
Thr234	B-residue_name_number
to	O
Ser	B-residue_name
also	O
places	O
the	O
side	O
chain	O
in	O
the	O
position	O
of	O
the	O
methyl	O
group	O
of	O
Thr234	B-residue_name_number
in	O
the	O
WT	B-protein_state
,	O
thereby	O
reducing	O
catalytic	O
activity	O
.	O

Mutations	B-experimental_method
of	O
residue	O
(-	B-residue_number
2	I-residue_number
)	I-residue_number
and	O
their	O
influence	O
on	O
propeptide	B-structure_element
conformation	O
and	O
autolysis	B-ptm
.	O

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

Collapse	O
of	O
the	O
transition	O
state	O
frees	O
the	O
Thr1	B-residue_name_number
N	O
terminus	O
(	O
by	O
completing	O
an	O
N	O
-	O
to	O
-	O
O	O
acyl	O
shift	O
of	O
the	O
propeptide	B-structure_element
),	O
which	O
is	O
subsequently	O
protonated	O
by	O
Asp166OH	B-residue_name_number
via	O
Ser129OH	B-residue_name_number
.	O

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

(	O
g	O
)	O
Structural	B-experimental_method
superposition	I-experimental_method
of	O
the	O
WT	B-protein_state
β5	B-protein
and	O
β5	B-mutant
-	I-mutant
T1S	I-mutant
mutant	B-protein_state
active	B-site
sites	I-site
reveals	O
different	O
orientations	O
of	O
the	O
hydroxyl	O
groups	O
of	O
Thr1	B-residue_name_number
and	O
Ser1	B-residue_name_number
,	O
respectively	O
.	O

Ser1	B-residue_name_number
lacks	B-protein_state
this	O
stabilization	O
and	O
is	O
therefore	O
rotated	O
by	O
60	O
°.	O