The	O
human	B-species
gut	O
microbiota	B-taxonomy_domain
influences	O
the	O
course	O
of	O
human	B-species
development	O
and	O
health	O
,	O
playing	O
key	O
roles	O
in	O
immune	O
stimulation	O
,	O
intestinal	O
cell	O
proliferation	O
,	O
and	O
metabolic	O
balance	O
.	O

The	O
ability	O
to	O
acquire	O
energy	O
from	O
carbohydrates	B-chemical
of	O
dietary	O
or	O
host	O
origin	O
is	O
central	O
to	O
the	O
adaptation	O
of	O
human	B-species
gut	O
bacterial	B-taxonomy_domain
species	O
to	O
their	O
niche	O
.	O

Xyloglucan	B-chemical
and	O
the	O
Bacteroides	B-species
ovatus	I-species
xyloglucan	B-gene
utilization	I-gene
locus	I-gene
(	O
XyGUL	B-gene
).	O
(	O
A	O
)	O
Representative	O
structures	B-evidence
of	O
common	O
xyloglucans	B-chemical
using	O
the	O
Consortium	O
for	O
Functional	O
Glycomics	O
Symbol	O
Nomenclature	O
(	O
http	O
://	O
www	O
.	O
functionalglycomics	O
.	O
org	O
/	O
static	O
/	O
consortium	O
/	O
Nomenclature	O
.	O
shtml	O
).	O

Whereas	O
our	O
previous	O
study	O
focused	O
on	O
the	O
characterization	O
of	O
the	O
linkage	O
specificity	O
of	O
these	O
GHs	B-protein_type
,	O
a	O
key	O
outstanding	O
question	O
regarding	O
this	O
locus	O
is	O
how	O
XyG	B-chemical
recognition	O
is	O
mediated	O
at	O
the	O
cell	O
surface	O
.	O

Here	O
,	O
the	O
SGBPs	B-protein_type
very	O
likely	O
work	O
in	O
concert	O
with	O
the	O
cell	B-protein_type
-	I-protein_type
surface	I-protein_type
-	I-protein_type
localized	I-protein_type
endo	I-protein_type
-	I-protein_type
xyloglucanase	I-protein_type
B	B-species
.	I-species
ovatus	I-species
GH5	B-protein
(	O
BoGH5	B-protein
)	O
to	O
recruit	O
and	O
cleave	O
XyG	B-chemical
for	O
subsequent	O
periplasmic	O
import	O
via	O
the	O
SusC	B-protein_type
-	I-protein_type
like	I-protein_type
TBDT	I-protein_type
of	O
the	O
XyGUL	B-gene
(	O
Fig	O
.	O
1B	O
and	O
C	O
).	O

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

Together	O
,	O
these	O
results	O
highlight	O
the	O
high	O
specificities	O
of	O
SGBP	B-protein
-	I-protein
A	I-protein
and	O
SGBP	B-protein
-	I-protein
B	I-protein
for	O
XyG	B-chemical
,	O
which	O
is	O
concordant	O
with	O
their	O
association	O
with	O
XyG	B-protein_type
-	I-protein_type
specific	I-protein_type
GHs	I-protein_type
in	O
the	O
XyGUL	B-gene
,	O
as	O
well	O
as	O
transcriptomic	O
analysis	O
indicating	O
that	O
B	B-species
.	I-species
ovatus	I-species
has	O
discrete	O
PUL	B-gene
for	O
MLG	B-chemical
,	O
GM	B-chemical
,	O
and	O
GGM	B-chemical
(	O
11	O
).	O

The	O
apo	B-protein_state
structure	B-evidence
is	O
color	O
ramped	O
from	O
blue	O
to	O
red	O
.	O

The	O
approximate	O
length	O
of	O
each	O
glycan	B-site
-	I-site
binding	I-site
site	I-site
is	O
displayed	O
,	O
colored	O
to	O
match	O
the	O
protein	B-evidence
structures	I-evidence
.	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
A	I-protein
,	O
displaying	O
all	O
residues	O
within	O
4	O
Å	O
of	O
the	O
ligand	O
.	O

Potential	O
hydrogen	B-bond_interaction
-	I-bond_interaction
bonding	I-bond_interaction
interactions	I-bond_interaction
are	O
shown	O
as	O
dashed	O
lines	O
,	O
and	O
the	O
distance	O
is	O
shown	O
in	O
angstroms	O
.	O

Dissection	O
of	O
the	O
individual	O
contribution	O
of	O
these	O
residues	O
reveals	O
that	O
the	O
W82A	B-mutant
mutant	B-protein_state
displays	O
a	O
significant	O
4	O
.	O
9	O
-	O
fold	O
decrease	O
in	O
the	O
Ka	B-evidence
value	O
for	O
XyG	B-chemical
,	O
while	O
the	O
W306A	B-mutant
substitution	B-experimental_method
completely	O
abolishes	B-protein_state
XyG	I-protein_state
binding	I-protein_state
.	O

Prolines	B-residue_name
between	O
domains	O
are	O
indicated	O
as	O
spheres	O
.	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

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

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

The	O
similarity	O
of	O
the	O
glycan	B-chemical
specificity	O
of	O
SGBP	B-protein
-	I-protein
A	I-protein
and	O
SGBP	B-protein
-	I-protein
B	I-protein
presents	O
an	O
intriguing	O
conundrum	O
regarding	O
their	O
individual	O
roles	O
in	O
XyG	B-chemical
utilization	O
by	O
B	B-species
.	I-species
ovatus	I-species
.	O

The	O
ΔSGBP	B-mutant
-	I-mutant
A	I-mutant
(	O
ΔBacova_02651	B-mutant
)	O
strain	O
(	O
cf	O
.	O

The	O
specific	O
glycan	B-chemical
signal	O
that	O
upregulates	O
BoXyGUL	B-gene
is	O
currently	O
unknown	O
.	O

From	O
our	O
present	O
data	O
,	O
we	O
cannot	O
eliminate	O
the	O
possibility	O
that	O
the	O
glycan	B-chemical
binding	O
by	O
SGBP	B-protein
-	I-protein
A	I-protein
enhances	O
transcriptional	O
activation	O
of	O
the	O
XyGUL	B-gene
.	O

Beyond	O
SGBP	B-protein
-	I-protein
A	I-protein
and	O
SGBP	B-protein
-	I-protein
B	I-protein
,	O
we	O
speculated	O
that	O
the	O
catalytically	B-protein_state
feeble	I-protein_state
endo	B-protein_type
-	I-protein_type
xyloglucanase	I-protein_type
GH9	B-protein
,	O
which	O
is	O
expendable	O
for	O
growth	O
in	O
the	O
presence	O
of	O
GH5	B-protein
,	O
might	O
also	O
play	O
a	O
role	O
in	O
glycan	B-chemical
binding	O
to	O
the	O
cell	O
surface	O
.	O

We	O
hypothesize	O
that	O
during	O
exponential	O
growth	O
the	O
essential	O
role	O
of	O
SGBP	B-protein
-	I-protein
A	I-protein
extends	O
beyond	O
glycan	B-chemical
recognition	O
,	O
perhaps	O
due	O
to	O
a	O
critical	O
interaction	O
with	O
the	O
TBDT	B-protein_type
.	O

A	O
particularly	O
understudied	O
aspect	O
of	O
glycan	B-chemical
utilization	O
is	O
the	O
mechanism	O
of	O
import	O
via	O
TBDTs	B-protein_type
(	O
SusC	B-protein
homologs	O
)	O
(	O
Fig	O
.	O
1	O
),	O
which	O
are	O
ubiquitous	O
and	O
defining	O
components	O
of	O
all	O
PUL	B-gene
.	O

Similarly	O
,	O
the	O
deletion	O
of	O
BT1762	B-gene
encoding	O
a	O
fructan	B-protein_type
-	I-protein_type
targeting	I-protein_type
SusD	I-protein_type
-	I-protein_type
like	I-protein_type
protein	I-protein_type
in	O
B	B-species
.	I-species
thetaiotaomicron	I-species
did	O
not	O
result	O
in	O
a	O
dramatic	O
loss	O
of	O
growth	O
on	O
fructans	B-chemical
.	O

Furthermore	O
,	O
considering	O
the	O
broader	O
distribution	O
of	O
TBDTs	B-protein_type
in	O
PUL	B-gene
lacking	O
SGBPs	B-protein_type
(	O
sometimes	O
known	O
as	O
carbohydrate	B-gene
utilization	I-gene
containing	I-gene
TBDT	I-gene
[	I-gene
CUT	I-gene
]	I-gene
loci	I-gene
;	O
see	O
reference	O
and	O
reviewed	O
in	O
reference	O
)	O
across	O
bacterial	B-taxonomy_domain
phyla	O
,	O
it	O
appears	O
that	O
the	O
intimate	O
biophysical	O
association	O
of	O
these	O
substrate	O
-	O
transport	O
and	O
-	O
binding	O
proteins	O
is	O
the	O
result	O
of	O
specific	O
evolution	O
within	O
the	O
Bacteroidetes	B-taxonomy_domain
.	O

Equally	O
intriguing	O
is	O
the	O
observation	O
that	O
while	O
SusD	B-protein_type
-	I-protein_type
like	I-protein_type
proteins	I-protein_type
such	O
as	O
SGBP	B-protein
-	I-protein
A	I-protein
share	O
moderate	O
primary	O
and	O
high	O
tertiary	O
structural	O
conservation	O
,	O
the	O
genes	O
for	O
the	O
SGBPs	B-protein_type
encoded	O
immediately	O
downstream	O
(	O
Fig	O
.	O
1B	O
[	O
sometimes	O
referred	O
to	O
as	O
“	O
susE	O
positioned	O
”])	O
encode	O
glycan	B-protein_type
-	I-protein_type
binding	I-protein_type
lipoproteins	I-protein_type
with	O
little	O
or	O
no	O
sequence	O
or	O
structural	O
conservation	O
,	O
even	O
among	O
syntenic	O
PUL	B-gene
that	O
target	O
the	O
same	O
polysaccharide	B-chemical
.	O

Because	O
the	O
intestinal	O
ecosystem	O
is	O
a	O
dense	O
consortium	O
of	O
bacteria	B-taxonomy_domain
that	O
must	O
compete	O
for	O
their	O
nutrients	O
,	O
these	O
multimodular	O
SGBPs	B-protein_type
may	O
reflect	O
ongoing	O
evolutionary	O
experiments	O
to	O
enhance	O
glycan	B-chemical
uptake	O
efficiency	O
.	O

Whether	O
organisms	O
that	O
express	O
longer	O
SGBPs	B-protein_type
,	O
extending	O
further	O
above	O
the	O
cell	O
surface	O
toward	O
the	O
extracellular	O
environment	O
,	O
are	O
better	O
equipped	O
to	O
compete	O
for	O
available	O
carbohydrates	B-chemical
is	O
presently	O
unknown	O
.	O

Monoclonal	O
antibodies	B-protein_type
inhibiting	O
IL	B-protein
-	I-protein
17A	I-protein
signaling	O
have	O
demonstrated	O
remarkable	O
efficacy	O
,	O
but	O
an	O
oral	O
therapy	O
is	O
still	O
lacking	O
.	O

Tested	O
in	O
primary	O
human	B-species
cells	O
,	O
HAP	B-chemical
blocked	O
the	O
production	O
of	O
multiple	O
inflammatory	O
cytokines	B-protein_type
.	O

These	O
polypeptides	O
form	O
covalent	B-protein_state
homodimers	B-oligomeric_state
,	O
and	O
IL	B-protein
-	I-protein
17A	I-protein
and	O
IL	B-protein
-	I-protein
17F	I-protein
also	O
form	O
an	O
IL	B-complex_assembly
-	I-complex_assembly
17A	I-complex_assembly
/	I-complex_assembly
IL	I-complex_assembly
-	I-complex_assembly
17F	I-complex_assembly
hetereodimer	B-oligomeric_state
.	O

In	O
these	O
structures	B-evidence
,	O
both	O
IL	B-protein
-	I-protein
17A	I-protein
and	O
IL	B-protein
-	I-protein
17F	I-protein
adopt	O
a	O
cysteine	B-structure_element
-	I-structure_element
knot	I-structure_element
fold	O
with	O
two	O
intramolecular	O
disulfides	B-ptm
and	O
two	O
interchain	O
disulfide	B-ptm
bonds	I-ptm
that	O
covalently	O
link	O
two	O
monomers	B-oligomeric_state
.	O

Identification	O
of	O
IL	B-protein
-	I-protein
17A	I-protein
peptide	O
inhibitors	O

Peptides	O
specifically	O
binding	O
to	O
human	B-species
IL	B-protein
-	I-protein
17A	I-protein
were	O
identified	O
from	O
phage	B-experimental_method
panning	I-experimental_method
using	O
cyclic	B-experimental_method
and	I-experimental_method
linear	I-experimental_method
peptide	I-experimental_method
libraries	I-experimental_method
(	O
Supplementary	O
Figure	O
S1	O
).	O

The	O
positive	O
binding	O
supernatants	O
were	O
tested	O
for	O
the	O
ability	O
to	O
block	O
biotinylated	B-protein_state
IL	B-protein
-	I-protein
17A	I-protein
signaling	O
through	O
IL	B-protein
-	I-protein
17RA	I-protein
in	O
an	O
IL	B-complex_assembly
-	I-complex_assembly
17A	I-complex_assembly
/	I-complex_assembly
IL	I-complex_assembly
-	I-complex_assembly
17RA	I-complex_assembly
competition	B-experimental_method
ELISA	I-experimental_method
assay	I-experimental_method
where	O
unlabeled	O
IL	B-protein
-	I-protein
17A	I-protein
was	O
used	O
as	O
positive	O
control	O
to	O
inhibit	O
biotinylated	B-protein_state
IL	B-protein
-	I-protein
17A	I-protein
binding	O
.	O

An	O
alanine	B-experimental_method
scan	I-experimental_method
of	O
peptide	B-chemical
2	I-chemical
,	O
an	O
analogue	O
of	O
1	B-chemical
with	O
a	O
lysine	B-residue_name
to	O
arginine	B-residue_name
substitution	B-experimental_method
at	O
position	O
14	B-residue_number
,	O
was	O
initiated	O
.	O

Modifications	O
at	O
positions	O
2	B-residue_number
and	O
14	B-residue_number
were	O
shown	O
to	O
display	O
improvement	O
in	O
binding	B-evidence
affinity	I-evidence
(	O
data	O
not	O
shown	O
).	O

In	O
this	O
work	O
,	O
32	B-chemical
–	I-chemical
34	I-chemical
are	O
capped	B-protein_state
by	O
protective	O
acetyl	O
group	O
and	O
reflect	O
the	O
same	O
inactivity	O
as	O
reported	O
.	O

Peptide	B-chemical
45	I-chemical
,	O
dimerized	B-oligomeric_state
via	O
attachment	O
of	O
a	O
PEG21	B-chemical
spacer	O
at	O
position	O
14	B-residue_number
(	O
Supplementary	O
Scheme	O
S1	O
and	O
Figure	O
S3	O
),	O
was	O
the	O
most	O
potent	O
with	O
cellular	O
IC50	B-evidence
of	O
0	O
.	O
1	O
nM	O
.	O
This	O
significant	O
improvement	O
in	O
antagonism	O
was	O
not	O
seen	O
in	O
the	O
peptide	O
monomer	B-oligomeric_state
functionalized	O
with	O
a	O
PEG21	B-chemical
group	O
at	O
position	O
14	B-residue_number
as	O
peptide	B-chemical
48	I-chemical
had	O
an	O
IC50	B-evidence
of	O
21	O
nM	O
(	O
Supplementary	O
Scheme	O
S2	O
).	O

To	O
further	O
characterize	O
the	O
interaction	O
of	O
HAP	B-chemical
with	O
IL	B-protein
-	I-protein
17A	I-protein
,	O
we	O
set	O
out	O
to	O
determine	O
its	O
in	O
vitro	O
binding	B-evidence
affinity	I-evidence
,	O
specificity	O
and	O
kinetic	B-evidence
profile	I-evidence
using	O
Surface	B-experimental_method
Plasmon	I-experimental_method
Resonance	I-experimental_method
(	O
SPR	B-experimental_method
)	O
methods	O
(	O
Fig	O
.	O
1A	O
).	O

HAP	B-chemical
blocks	O
IL	B-protein
-	I-protein
17A	I-protein
signaling	O
in	O
a	O
human	B-species
primary	O
cell	O
assay	O

In	O
patients	O
,	O
the	O
concentration	O
of	O
IL	B-protein
-	I-protein
17A	I-protein
in	O
psoriatic	O
lesions	O
is	O
reported	O
to	O
be	O
0	O
.	O
01	O
ng	O
/	O
ml	O
,	O
well	O
below	O
the	O
EC50	O
(	O
5	O
–	O
10ng	O
/	O
ml	O
)	O
of	O
IL	B-protein
-	I-protein
17A	I-protein
induced	O
IL	B-protein_type
-	I-protein_type
8	I-protein_type
production	O
in	O
vitro	O
.	O

It	O
is	O
known	O
that	O
an	O
antibody	B-protein_type
antigen	B-structure_element
-	I-structure_element
binding	I-structure_element
fragment	I-structure_element
(	O
Fab	B-structure_element
)	O
can	O
be	O
used	O
as	O
crystallization	O
chaperones	O
in	O
crystallizing	O
difficult	O
targets	O
.	O

Furthermore	O
,	O
since	O
it	O
binds	O
to	O
an	O
area	O
far	O
away	O
from	O
that	O
of	O
HAP	B-chemical
(	O
see	O
below	O
),	O
this	O
Fab	B-structure_element
should	O
have	O
minimum	O
effects	O
on	O
HAP	B-chemical
binding	O
conformation	O
.	O

Crystals	B-evidence
of	O
Fab	B-complex_assembly
/	I-complex_assembly
IL	I-complex_assembly
-	I-complex_assembly
17A	I-complex_assembly
/	I-complex_assembly
HAP	I-complex_assembly
ternary	O
complex	O
were	O
obtained	O
readily	O
in	O
crystallization	B-experimental_method
screens	I-experimental_method
.	O

Crystallization	B-experimental_method
of	O
IL	B-protein
-	I-protein
17A	I-protein
and	O
its	O
binding	O
partners	O
was	O
accomplished	O
using	O
two	O
forms	O
of	O
IL	B-protein
-	I-protein
17A	I-protein
.	O

Both	O
structures	B-evidence
were	O
solved	O
by	O
molecular	B-experimental_method
replacement	I-experimental_method
.	O

The	O
C	O
-	O
terminal	O
8	B-residue_range
residues	I-residue_range
of	O
the	O
HAP	B-chemical
that	O
are	O
ordered	O
in	O
the	O
structure	B-evidence
,	O
7ADLWDWIN	B-chemical
,	O
form	O
an	O
amphipathic	B-protein_state
α	B-structure_element
-	I-structure_element
helix	I-structure_element
interacting	O
with	O
the	O
second	O
IL	B-protein
-	I-protein
17A	I-protein
monomer	B-oligomeric_state
.	O

Pro6	B-residue_name_number
of	O
HAP	B-chemical
makes	O
a	O
transition	O
between	O
the	O
N	O
-	O
terminal	O
β	B-structure_element
-	I-structure_element
strand	I-structure_element
and	O
the	O
C	O
-	O
terminal	O
α	B-structure_element
-	I-structure_element
helix	I-structure_element
of	O
HAP	B-chemical
.	O

Conformational	O
changes	O
in	O
region	B-structure_element
I	I-structure_element
induced	O
by	O
HAP	B-chemical
binding	O
alone	O
may	O
allosterically	O
affect	O
IL	B-protein
-	I-protein
17RA	I-protein
binding	O
,	O
but	O
more	O
importantly	O
,	O
the	O
α	B-structure_element
-	I-structure_element
helix	I-structure_element
of	O
HAP	B-chemical
directly	O
competes	O
with	O
IL	B-protein
-	I-protein
17RA	I-protein
for	O
binding	O
to	O
IL	B-protein
-	I-protein
17A	I-protein
(	O
Fig	O
.	O
3	O
).	O

However	O
,	O
it	O
mimics	O
the	O
β	B-structure_element
-	I-structure_element
strand	I-structure_element
0	I-structure_element
of	O
IL	B-protein
-	I-protein
17A	I-protein
.	O

Conformational	O
changes	O
of	O
IL	B-protein
-	I-protein
17A	I-protein
are	O
needed	O
for	O
both	O
HAP	B-chemical
and	O
IL	B-protein
-	I-protein
17RA	I-protein
to	O
bind	O
to	O
that	O
region	O
.	O

During	O
IL	B-protein
-	I-protein
17A	I-protein
signaling	O
,	O
IL	B-protein
-	I-protein
17A	I-protein
binds	O
to	O
one	O
copy	O
of	O
IL	B-protein
-	I-protein
17RA	I-protein
and	O
one	O
copy	O
of	O
IL	B-protein
-	I-protein
17RC	I-protein
.	O

HAP	B-chemical
,	O
with	O
only	O
15	B-residue_range
residues	I-residue_range
,	O
can	O
achieve	O
almost	O
the	O
same	O
binding	B-evidence
affinity	I-evidence
as	O
the	O
much	O
larger	O
IL	B-protein
-	I-protein
17RA	I-protein
molecule	O
,	O
indicating	O
a	O
more	O
efficient	O
way	O
of	O
binding	O
to	O
IL	B-protein
-	I-protein
17A	I-protein
.	O

As	O
demonstrated	O
by	O
the	O
crystal	B-evidence
structure	I-evidence
,	O
binding	O
of	O
the	O
α	B-structure_element
-	I-structure_element
helix	I-structure_element
of	O
HAP	B-chemical
should	O
be	O
sufficient	O
for	O
preventing	O
IL	B-protein
-	I-protein
17RA	I-protein
binding	O
to	O
IL	B-protein
-	I-protein
17A	I-protein
.	O

Theoretically	O
,	O
it	O
is	O
possible	O
to	O
design	O
chemicals	O
such	O
as	O
stapled	O
α	O
-	O
helical	O
peptides	O
to	O
block	O
α	B-structure_element
-	I-structure_element
helix	I-structure_element
-	O
mediated	O
IL	B-complex_assembly
-	I-complex_assembly
17A	I-complex_assembly
/	I-complex_assembly
IL	I-complex_assembly
-	I-complex_assembly
17RA	I-complex_assembly
interactions	O
.	O

(	O
A	O
)	O
HAP	B-chemical
binds	O
at	O
region	B-structure_element
I	I-structure_element
of	O
IL	B-protein
-	I-protein
17A	I-protein
.	O

Polar	B-bond_interaction
interactions	I-bond_interaction
are	O
shown	O
in	O
dashes	O
.	O

Notice	O
that	O
the	O
Trp	B-site
binding	I-site
pocket	I-site
for	O
W12	B-residue_name_number
of	O
HAP	B-chemical
or	O
W31	B-residue_name_number
of	O
IL	B-protein
-	I-protein
17RA	I-protein
is	O
missing	O
in	O
the	O
apo	B-protein_state
structure	B-evidence
.	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

NadR	B-protein
binds	O
the	O
nadA	B-gene
promoter	O
and	O
represses	O
gene	O
transcription	O
.	O

Serogroup	B-taxonomy_domain
B	I-taxonomy_domain
meningococcus	I-taxonomy_domain
(	O
MenB	B-species
)	O
causes	O
fatal	O
sepsis	O
and	O
invasive	O
meningococcal	B-taxonomy_domain
disease	O
,	O
particularly	O
in	O
young	O
children	O
and	O
adolescents	O
,	O
as	O
highlighted	O
by	O
recent	O
MenB	B-species
outbreaks	O
in	O
universities	O
of	O
the	O
United	O
States	O
and	O
Canada	O
.	O

The	O
amount	O
of	O
NadA	B-protein
exposed	O
on	O
the	O
meningococcal	B-taxonomy_domain
surface	O
also	O
influences	O
the	O
antibody	O
-	O
mediated	O
serum	O
bactericidal	O
response	O
measured	O
in	O
vitro	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

However	O
,	O
the	O
homologous	O
archeal	B-taxonomy_domain
Sulfolobus	B-species
tokodaii	I-species
protein	O
ST1710	B-protein
presented	O
essentially	O
the	O
same	O
structure	B-evidence
in	O
ligand	B-protein_state
-	I-protein_state
free	I-protein_state
and	O
salicylate	B-protein_state
-	I-protein_state
bound	I-protein_state
forms	O
,	O
apparently	O
contrasting	O
the	O
mechanism	O
proposed	O
for	O
MTH313	B-protein
.	O

Despite	O
these	O
apparent	O
differences	O
,	O
MTH313	B-protein
and	O
ST1710	B-protein
bind	O
salicylate	B-chemical
in	O
approximately	O
the	O
same	O
site	O
,	O
between	O
their	O
dimerization	B-structure_element
and	I-structure_element
DNA	I-structure_element
-	I-structure_element
binding	I-structure_element
domains	I-structure_element
.	O

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

NadR	B-protein
is	O
dimeric	B-oligomeric_state
and	O
is	O
stabilized	O
by	O
specific	O
hydroxyphenylacetate	B-chemical
ligands	O

Recombinant	O
NadR	B-protein
was	O
produced	O
in	O
E	B-species
.	I-species
coli	I-species
using	O
an	O
expression	B-experimental_method
construct	I-experimental_method
prepared	O
from	O
N	B-species
.	I-species
meningitidis	I-species
serogroup	I-species
B	I-species
strain	I-species
MC58	I-species
.	O

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

3	B-chemical
-	I-chemical
HPA	I-chemical
70	O
.	O
0	O
±	O
0	O
.	O
1	O
2	O
.	O
7	O
2	O
.	O
7	O
±	O
0	O
.	O
1	O
4	B-chemical
-	I-chemical
HPA	I-chemical
70	O
.	O
7	O
±	O
0	O
.	O
1	O
3	O
.	O
3	O
1	O
.	O
5	O
±	O
0	O
.	O
1	O
3Cl	B-chemical
,	I-chemical
4	I-chemical
-	I-chemical
HPA	I-chemical
71	O
.	O
3	O
±	O
0	O
.	O
2	O
3	O
.	O
9	O
1	O
.	O
1	O
±	O
0	O
.	O
1	O

To	O
further	O
investigate	O
the	O
binding	O
of	O
HPAs	B-chemical
to	O
NadR	B-protein
,	O
we	O
used	O
surface	B-experimental_method
plasmon	I-experimental_method
resonance	I-experimental_method
(	O
SPR	B-experimental_method
).	O

Data	O
collection	O
and	O
refinement	O
statistics	O
for	O
NadR	B-protein
structures	B-evidence
.	O

In	O
the	O
apo	B-protein_state
-	O
NadR	B-protein
crystals	B-evidence
,	O
the	O
two	O
homodimers	B-oligomeric_state
were	O
related	O
by	O
a	O
rotation	O
of	O
~	O
90	O
°;	O
the	O
observed	O
association	O
of	O
the	O
two	O
dimers	B-oligomeric_state
was	O
presumably	O
merely	O
an	O
effect	O
of	O
crystal	O
packing	O
,	O
since	O
the	O
interface	B-site
between	O
the	O
two	O
homodimers	B-oligomeric_state
is	O
small	O
(<	O
550	O
Å2	O
of	O
buried	O
surface	O
area	O
),	O
and	O
is	O
not	O
predicted	O
to	O
be	O
physiologically	O
relevant	O
by	O
the	O
PISA	O
software	O
.	O

Helices	B-structure_element
α3	B-structure_element
and	O
α4	B-structure_element
form	O
a	O
helix	B-structure_element
-	I-structure_element
turn	I-structure_element
-	I-structure_element
helix	I-structure_element
motif	I-structure_element
,	O
followed	O
by	O
the	O
“	O
wing	B-structure_element
motif	I-structure_element
”	O
comprised	O
of	O
two	O
short	B-structure_element
antiparallel	I-structure_element
β	I-structure_element
-	I-structure_element
strands	I-structure_element
(	O
β1	B-structure_element
-	I-structure_element
β2	I-structure_element
)	O
linked	O
by	O
a	O
relatively	O
long	O
and	O
flexible	O
loop	B-structure_element
.	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

Using	O
site	B-experimental_method
-	I-experimental_method
directed	I-experimental_method
mutagenesis	I-experimental_method
,	O
a	O
panel	O
of	O
eight	O
mutant	B-protein_state
NadR	B-protein
proteins	O
was	O
prepared	O
(	O
including	O
mutations	O
H7A	B-mutant
,	O
S9A	B-mutant
,	O
N11A	B-mutant
,	O
D112A	B-mutant
,	O
R114A	B-mutant
,	O
Y115A	B-mutant
,	O
K126A	B-mutant
,	O
L130K	B-mutant
and	O
L133K	B-mutant
),	O
sufficient	O
to	O
explore	O
the	O
entire	O
dimer	B-site
interface	I-site
.	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

The	O
NadR	B-complex_assembly
/	I-complex_assembly
4	I-complex_assembly
-	I-complex_assembly
HPA	I-complex_assembly
structure	B-evidence
revealed	O
the	O
ligand	B-site
-	I-site
binding	I-site
site	I-site
nestled	O
between	O
the	O
dimerization	B-structure_element
and	I-structure_element
DNA	I-structure_element
-	I-structure_element
binding	I-structure_element
domains	I-structure_element
(	O
Fig	O
2	O
).	O

The	O
binding	B-site
pocket	I-site
was	O
almost	O
entirely	O
filled	O
by	O
4	B-chemical
-	I-chemical
HPA	I-chemical
and	O
one	O
water	B-chemical
molecule	O
,	O
although	O
there	O
also	O
remained	O
a	O
small	O
tunnel	B-site
2	O
-	O
4Å	O
in	O
diameter	O
and	O
5	O
-	O
6Å	O
long	O
leading	O
from	O
the	O
pocket	B-site
(	O
proximal	O
to	O
the	O
4	O
-	O
hydroxyl	O
position	O
)	O
to	O
the	O
protein	O
surface	O
.	O

Green	O
and	O
blue	O
ribbons	O
depict	O
NadR	B-protein
chains	B-structure_element
A	I-structure_element
and	I-structure_element
B	I-structure_element
,	O
respectively	O
.	O

Residues	O
AsnA11	B-residue_name_number
and	O
ArgB18	B-residue_name_number
likely	O
make	O
indirect	O
yet	O
local	O
contributions	O
to	O
ligand	O
binding	O
,	O
mainly	O
by	O
stabilizing	O
the	O
position	O
of	O
AspB36	B-residue_name_number
.	O

List	O
of	O
4	B-chemical
-	I-chemical
HPA	I-chemical
atoms	O
bound	O
to	O
NadR	B-protein
via	O
ionic	B-bond_interaction
interactions	I-bond_interaction
and	O
/	O
or	O
H	B-bond_interaction
-	I-bond_interaction
bonds	I-bond_interaction
.	O

4	B-chemical
-	I-chemical
HPA	I-chemical
atom	O
NadR	B-protein
residue	O
/	O
atom	O
Distance	O
(	O
Å	O
)	O
O2	O
TrpB39	B-residue_name_number
/	O
NE1	O
2	O
.	O
83	O
O2	O
ArgB43	B-residue_name_number
/	O
NH1	O
2	O
.	O
76	O
O1	O
ArgB43	B-residue_name_number
/	O
NH1	O
3	O
.	O
84	O
O1	O
SerA9	B-residue_name_number
/	O
OG	O
2	O
.	O
75	O
O1	O
TyrB115	B-residue_name_number
/	O
OH	O
2	O
.	O
50	O
O2	O
Water	B-chemical
(*	O
Ser9	B-residue_name_number
/	O
Asn11	B-residue_name_number
)	O
2	O
.	O
88	O
OH	O
AspB36	B-residue_name_number
/	O
OD1	O
/	O
OD2	O
3	O
.	O
6	O
/	O
3	O
.	O
7	O

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

In	O
SPR	B-experimental_method
,	O
the	O
signal	O
measured	O
is	O
proportional	O
to	O
the	O
total	O
molecular	O
mass	O
proximal	O
to	O
the	O
sensor	O
surface	O
;	O
consequently	O
,	O
if	O
the	O
molecular	O
weights	O
of	O
the	O
interactors	O
are	O
known	O
,	O
then	O
the	O
stoichiometry	O
of	O
the	O
resulting	O
complex	O
can	O
be	O
determined	O
.	O

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

Indeed	O
,	O
we	O
noted	O
interesting	O
differences	O
in	O
the	O
side	O
chains	O
of	O
Met22	B-residue_name_number
,	O
Phe25	B-residue_name_number
and	O
Arg43	B-residue_name_number
,	O
which	O
in	O
monomer	B-oligomeric_state
B	B-structure_element
are	O
used	O
to	O
contact	O
the	O
ligand	O
while	O
in	O
monomer	B-oligomeric_state
A	B-structure_element
they	O
partially	O
occupied	O
the	O
pocket	B-site
and	O
collectively	O
reduced	O
its	O
volume	O
significantly	O
.	O

In	O
contrast	O
,	O
the	O
apo	B-protein_state
-	O
form	O
Met22	B-residue_name_number
and	O
Phe25	B-residue_name_number
residues	O
were	O
still	O
encroaching	O
the	O
spaces	O
of	O
the	O
4	O
-	O
hydroxyl	O
group	O
and	O
the	O
phenyl	O
ring	O
of	O
the	O
ligand	O
,	O
respectively	O
(	O
Fig	O
5C	O
).	O

The	O
‘	O
outward	B-protein_state
’	O
position	O
of	O
Arg43	B-residue_name_number
generated	O
an	O
open	B-protein_state
apo	B-protein_state
-	O
form	O
pocket	B-site
with	O
volume	O
approximately	O
380Å3	O
.	O

Taken	O
together	O
,	O
these	O
observations	O
suggest	O
that	O
Arg43	B-residue_name_number
is	O
a	O
major	O
determinant	O
of	O
ligand	O
binding	O
,	O
and	O
that	O
its	O
‘	O
inward	B-protein_state
’	O
position	O
inhibits	O
the	O
binding	O
of	O
4	B-chemical
-	I-chemical
HPA	I-chemical
to	O
the	O
empty	O
pocket	B-site
of	O
holo	B-protein_state
-	O
NadR	B-protein
.	O

The	O
inner	O
conformer	O
is	O
the	O
one	O
that	O
would	O
display	O
major	O
clashes	O
if	O
4	B-chemical
-	I-chemical
HPA	I-chemical
were	O
present	O
.	O
(	O
C	O
)	O
Comparison	O
of	O
the	O
empty	O
pocket	B-site
from	O
holo	B-protein_state
-	O
NadR	B-protein
(	O
green	O
residues	O
)	O
with	O
the	O
four	O
empty	O
pockets	B-site
of	O
apo	B-protein_state
-	O
NadR	B-protein
(	O
grey	O
residues	O
),	O
shows	O
that	O
in	O
the	O
absence	B-protein_state
of	I-protein_state
4	B-chemical
-	I-chemical
HPA	I-chemical
the	O
Arg43	B-residue_name_number
side	O
chain	O
is	O
always	O
observed	O
in	O
the	O
‘	O
outward	B-protein_state
’	O
conformation	O
.	O

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

(	O
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

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

Interestingly	O
,	O
by	O
cooling	O
the	O
samples	O
to	O
10	O
°	O
C	O
,	O
we	O
observed	O
that	O
a	O
number	O
of	O
those	O
peaks	O
strongly	O
affected	O
by	O
4	B-chemical
-	I-chemical
HPA	I-chemical
(	O
and	O
therefore	O
likely	O
to	O
be	O
in	O
the	O
ligand	B-site
-	I-site
binding	I-site
site	I-site
)	O
demonstrated	O
evidence	O
of	O
peak	O
splitting	O
,	O
i	O
.	O
e	O
.	O
a	O
tendency	O
to	O
become	O
two	O
distinct	O
peaks	O
rather	O
than	O
one	O
single	O
peak	O
(	O
Fig	O
6B	O
and	O
6C	O
).	O

Similarly	O
,	O
the	O
entire	O
holo	B-protein_state
-	O
homodimer	B-oligomeric_state
could	O
be	O
closely	B-experimental_method
superposed	I-experimental_method
onto	O
each	O
of	O
the	O
apo	B-protein_state
-	O
homodimers	B-oligomeric_state
,	O
showing	O
rmsd	B-evidence
values	O
of	O
1	O
.	O
29Å	O
and	O
1	O
.	O
31Å	O
,	O
and	O
with	O
more	O
notable	O
differences	O
in	O
the	O
α6	B-structure_element
helix	I-structure_element
positions	O
(	O
Fig	O
7B	O
).	O

Structural	B-experimental_method
comparisons	I-experimental_method
of	O
NadR	B-protein
and	O
modelling	O
of	O
interactions	O
with	O
DNA	B-chemical
.	O

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

Interestingly	O
,	O
OhrR	B-protein
contacts	O
ohrA	B-gene
across	O
22	O
base	O
pairs	O
(	O
bp	O
),	O
and	O
similarly	O
the	O
main	O
NadR	B-protein
target	B-site
sites	I-site
identified	O
in	O
the	O
nadA	B-gene
promoter	O
(	O
the	O
operators	O
Op	O
I	O
and	O
Op	O
II	O
)	O
both	O
span	O
22	O
bp	O
.	O

When	O
aligned	B-experimental_method
with	O
OhrR	B-protein
,	O
the	O
apo	B-protein_state
-	O
homodimer	B-oligomeric_state
CD	B-structure_element
presented	O
yet	O
another	O
different	O
intermediate	O
conformation	O
(	O
rmsd	B-evidence
2	O
.	O
9Å	O
),	O
apparently	O
not	O
ideally	O
pre	O
-	O
configured	O
for	O
DNA	B-chemical
binding	O
,	O
but	O
which	O
in	O
solution	O
can	O
presumably	O
readily	O
adopt	O
the	O
AB	B-structure_element
conformation	O
due	O
to	O
the	O
intrinsic	O
flexibility	O
described	O
above	O
.	O

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

The	O
H7A	B-mutant
,	O
S9A	B-mutant
and	O
F25A	B-mutant
mutants	O
efficiently	O
repress	O
nadA	B-gene
expression	O
but	O
are	O
less	O
ligand	O
-	O
responsive	O
than	O
WT	B-protein_state
NadR	B-protein
.	O
The	O
N11A	B-mutant
mutant	B-protein_state
does	O
not	O
efficiently	O
repress	O
nadA	B-gene
expression	O
either	O
in	O
presence	O
or	O
absence	O
of	O
4	B-chemical
-	I-chemical
HPA	I-chemical
.	O
(	O
The	O
protein	O
abundance	O
levels	O
of	O
the	O
meningococcal	B-taxonomy_domain
factor	B-protein
H	I-protein
binding	I-protein
protein	I-protein
(	O
fHbp	B-protein
)	O
were	O
used	O
as	O
a	O
gel	O
loading	O
control	O
).	O

NadA	B-protein
is	O
a	O
surface	O
-	O
exposed	O
meningococcal	B-taxonomy_domain
protein	O
contributing	O
to	O
pathogenesis	O
,	O
and	O
is	O
one	O
of	O
three	O
main	O
antigens	O
present	O
in	O
the	O
vaccine	O
Bexsero	O
.	O

We	O
confirmed	O
this	O
stoichiometry	O
in	O
solution	O
using	O
SPR	B-experimental_method
methods	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

In	O
the	O
S	B-species
.	I-species
tokodaii	I-species
protein	O
ST1710	B-protein
,	O
salicylate	B-chemical
binds	O
to	O
the	O
same	O
position	O
in	O
each	O
monomer	B-oligomeric_state
of	O
the	O
dimer	B-oligomeric_state
,	O
in	O
a	O
site	O
equivalent	O
to	O
the	O
putative	O
biologically	O
relevant	O
site	O
of	O
MTH313	B-protein
(	O
Fig	O
10B	O
).	O

Unlike	O
other	O
MarR	B-protein_type
family	O
proteins	O
which	O
revealed	O
multiple	O
ligand	O
binding	O
interactions	O
,	O
we	O
observed	O
only	O
1	O
molecule	O
of	O
4	B-chemical
-	I-chemical
HPA	I-chemical
bound	B-protein_state
to	I-protein_state
NadR	B-protein
,	O
suggesting	O
a	O
more	O
specific	O
and	O
less	O
promiscuous	O
interaction	O
.	O

NadR	B-protein
shows	O
a	O
ligand	B-site
binding	I-site
site	I-site
distinct	O
from	O
other	O
MarR	B-protein_type
homologues	O
.	O

Alternatively	O
,	O
it	O
is	O
possible	O
that	O
other	O
MarR	B-protein_type
homologues	O
(	O
e	O
.	O
g	O
.	O
NMB1585	B-protein
)	O
may	O
have	O
no	O
extant	O
functional	O
binding	B-site
pocket	I-site
and	O
thus	O
may	O
have	O
lost	O
the	O
ability	O
to	O
respond	O
to	O
a	O
ligand	O
,	O
acting	O
instead	O
as	O
constitutive	O
DNA	B-chemical
-	O
binding	O
regulatory	O
proteins	O
.	O

The	O
noted	O
flexibility	O
may	O
also	O
explain	O
how	O
NadR	B-protein
can	O
adapt	O
to	O
bind	O
various	O
DNA	B-chemical
target	O
sequences	O
with	O
slightly	O
different	O
structural	O
features	O
.	O

Like	O
other	O
nuclear	B-protein_type
hormone	I-protein_type
receptors	I-protein_type
,	O
RORγ	B-protein
’	O
s	O
helix12	B-structure_element
which	O
makes	O
up	O
the	O
C	O
-	O
termini	O
of	O
the	O
LBD	B-structure_element
is	O
an	O
essential	O
part	O
of	O
the	O
coactivator	B-site
binding	I-site
pocket	I-site
and	O
is	O
commonly	O
referred	O
to	O
as	O
the	O
activation	B-structure_element
function	I-structure_element
helix	I-structure_element
2	I-structure_element
(	O
AF2	B-structure_element
).	O

FRET	B-evidence
results	I-evidence
for	O
agonist	B-protein_state
BIO592	B-chemical
(	O
a	O
)	O
and	O
Inverse	B-protein_state
Agonist	I-protein_state
BIO399	B-chemical
(	O
b	O
)	O

a	O
The	O
ternary	B-evidence
structure	I-evidence
of	O
RORγ518	B-protein
BIO592	B-chemical
and	O
EBI96	B-chemical
.	O

b	O
RORγ	B-protein
AF2	B-structure_element
helix	I-structure_element
in	O
the	O
agonist	B-protein_state
conformation	O
.	O

The	O
structure	B-evidence
of	O
the	O
ternary	O
complex	O
had	O
features	O
similar	O
to	O
other	O
ROR	B-protein_type
agonist	B-protein_state
coactivator	O
structures	B-evidence
in	O
a	O
transcriptionally	B-protein_state
active	I-protein_state
canonical	B-protein_state
three	I-protein_state
layer	I-protein_state
helix	I-protein_state
fold	I-protein_state
with	O
the	O
AF2	B-structure_element
helix	I-structure_element
in	O
the	O
agonist	B-protein_state
conformation	O
.	O

The	O
agonist	B-protein_state
conformation	O
is	O
stabilized	O
by	O
a	O
hydrogen	B-bond_interaction
bond	I-bond_interaction
between	O
His479	B-residue_name_number
and	O
Tyr502	B-residue_name_number
,	O
in	O
addition	O
to	O
π	B-bond_interaction
-	I-bond_interaction
π	I-bond_interaction
interactions	I-bond_interaction
between	O
His479	B-residue_name_number
,	O
Tyr502	B-residue_name_number
and	O
Phe506	B-residue_name_number
(	O
Fig	O
.	O
2b	O
).	O

Electron	B-evidence
density	I-evidence
for	O
the	O
coactivator	O
peptide	O
EBI96	B-chemical
was	O
observed	O
for	O
residues	O
EFPYLLSLLG	B-structure_element
which	O
formed	O
a	O
α	B-structure_element
-	I-structure_element
helix	I-structure_element
stabilized	O
through	O
hydrophobic	B-bond_interaction
interactions	I-bond_interaction
with	O
the	O
coactivator	B-site
binding	I-site
pocket	I-site
on	O
RORγ	B-protein
(	O
Fig	O
.	O
2c	O
).	O

b	O
Benzoxazinone	B-chemical
ring	O
system	O
of	O
agonist	B-protein_state
BIO592	B-chemical
packing	O
against	O
His479	B-residue_name_number
of	O
RORγ	B-protein
stabilizing	O
agonist	B-protein_state
conformation	O
of	O
the	O
AF2	B-structure_element
helix	I-structure_element

BIO592	B-chemical
bound	B-protein_state
in	I-protein_state
a	O
collapsed	B-protein_state
conformational	O
state	O
in	O
the	O
LBS	B-site
of	O
RORγ	B-protein
with	O
the	O
xylene	B-chemical
ring	O
positioned	O
at	O
the	O
bottom	O
of	O
the	O
pocket	B-site
making	O
hydrophobic	B-bond_interaction
interactions	I-bond_interaction
with	O
Val376	B-residue_name_number
,	O
Phe378	B-residue_name_number
,	O
Phe388	B-residue_name_number
and	O
Phe401	B-residue_name_number
,	O
with	O
the	O
ethyl	B-chemical
-	I-chemical
benzoxazinone	I-chemical
ring	O
making	O
several	O
hydrophobic	B-bond_interaction
interactions	I-bond_interaction
with	O
Trp317	B-residue_name_number
,	O
Leu324	B-residue_name_number
,	O
Met358	B-residue_name_number
,	O
Leu391	B-residue_name_number
,	O
Ile	B-residue_name_number
400	I-residue_name_number
and	O
His479	B-residue_name_number
(	O
Fig	O
.	O
3a	O
,	O
Additional	O
file	O
3	O
).	O

Hydrophobic	B-bond_interaction
interaction	I-bond_interaction
between	O
the	O
ethyl	O
group	O
of	O
the	O
benzoxazinone	B-chemical
and	O
His479	B-residue_name_number
reinforce	O
the	O
His479	B-residue_name_number
sidechain	O
position	O
for	O
making	O
the	O
hydrogen	B-bond_interaction
bond	I-bond_interaction
with	O
Tyr502	B-residue_name_number
thereby	O
stabilizing	O
the	O
agonist	B-protein_state
conformation	O
(	O
Fig	O
.	O
3b	O
).	O

However	O
,	O
in	O
the	O
presence	B-protein_state
of	I-protein_state
inverse	B-protein_state
agonist	I-protein_state
BIO399	B-chemical
,	O
the	O
proteolytic	B-evidence
pattern	I-evidence
showed	O
significantly	O
less	O
protection	O
,	O
albeit	O
the	O
products	O
were	O
more	O
heterogeneous	O
(	O
majority	O
ending	O
at	O
494	B-residue_number
/	O
495	B-residue_number
),	O
indicating	O
the	O
destabilization	O
of	O
the	O
AF2	B-structure_element
helix	I-structure_element
compared	O
to	O
either	O
the	O
APO	B-protein_state
or	O
ternary	B-protein_state
agonist	I-protein_state
complex	I-protein_state
(	O
Fig	O
.	O
4	O
,	O
Additional	O
file	O
5	O
).	O

a	O
Overlay	B-experimental_method
of	O
RORγ	B-protein
structures	B-evidence
bound	B-protein_state
to	I-protein_state
BIO596	B-chemical
(	O
Green	O
),	O
BIO399	B-chemical
(	O
Cyan	O
)	O
and	O
T0901317	B-chemical
(	O
Pink	O
).	O

We	O
hypothesize	O
that	O
since	O
the	O
Met358	B-residue_name_number
sidechain	O
conformation	O
in	O
the	O
T0901317	B-chemical
RORγ	B-protein
structure	B-evidence
is	O
not	O
in	O
the	O
BIO399	B-chemical
conformation	O
,	O
this	O
difference	O
could	O
account	O
for	O
the	O
10	O
-	O
fold	O
reduction	O
in	O
the	O
inverse	O
agonism	O
for	O
T0901317	B-chemical
compared	O
to	O
BIO399	B-chemical
in	O
the	O
FRET	B-experimental_method
assay	I-experimental_method
.	O

The	O
inverse	B-protein_state
agonist	I-protein_state
activity	O
for	O
these	O
compounds	O
has	O
been	O
attributed	O
to	O
orientating	O
Trp317	B-residue_name_number
to	O
clash	O
with	O
Tyr502	B-residue_name_number
or	O
a	O
direct	O
inverse	B-protein_state
agonist	I-protein_state
hydrogen	B-bond_interaction
bonding	I-bond_interaction
event	O
with	O
His479	B-residue_name_number
,	O
both	O
of	O
which	O
would	O
perturb	O
the	O
agonist	B-protein_state
conformation	O
of	O
RORγ	B-protein
.	O

GAL4	B-experimental_method
cell	I-experimental_method
assay	I-experimental_method
selectivity	O
profile	O
for	O
BIO399	B-chemical
toward	O
RORα	B-protein
and	O
RORβ	B-protein
in	O
GAL4	B-protein

Furthermore	O
,	O
RORα	B-protein
contains	O
two	O
phenylalanine	B-residue_name
residues	O
in	O
its	O
LBS	B-site
whereas	O
RORβ	B-protein
and	O
γ	B-protein
have	O
a	O
leucine	B-residue_name
in	O
the	O
same	O
position	O
(	O
Fig	O
.	O
6b	O
).	O

In	O
metabolism	O
,	O
molecules	O
with	O
“	O
high	O
-	O
energy	O
”	O
bonds	O
(	O
e	O
.	O
g	O
.,	O
ATP	B-chemical
and	O
Acetyl	B-chemical
~	I-chemical
CoA	I-chemical
)	O
are	O
critical	O
for	O
both	O
catabolic	O
and	O
anabolic	O
processes	O
.	O

The	O
facets	O
of	O
the	O
shell	B-structure_element
are	O
composed	O
primarily	O
of	O
hexamers	B-oligomeric_state
that	O
are	O
typically	O
perforated	O
by	O
pores	B-site
lined	O
with	O
highly	B-protein_state
conserved	I-protein_state
,	O
polar	B-protein_state
residues	B-structure_element
that	O
presumably	O
function	O
as	O
the	O
conduits	O
for	O
metabolites	O
into	O
and	O
out	O
of	O
the	O
shell	B-structure_element
.	O

Substrates	O
and	O
cofactors	O
involving	O
the	O
PTAC	B-protein_type
reaction	O
are	O
shown	O
in	O
red	O
;	O
other	O
substrates	O
and	O
enzymes	O
are	O
shown	O
in	O
black	O
,	O
and	O
other	O
cofactors	O
are	O
shown	O
in	O
gray	O
.	O

The	O
activities	O
of	O
core	O
enzymes	O
are	O
not	O
confined	O
to	O
BMC	B-complex_assembly
-	O
associated	O
functions	O
:	O
aldehyde	B-protein_type
and	I-protein_type
alcohol	I-protein_type
dehydrogenases	I-protein_type
are	O
utilized	O
in	O
diverse	O
metabolic	O
reactions	O
,	O
and	O
PTAC	B-protein_type
catalyzes	O
a	O
key	O
biochemical	O
reaction	O
in	O
the	O
process	O
of	O
obtaining	O
energy	O
during	O
fermentation	O
.	O

This	O
occurs	O
,	O
for	O
example	O
,	O
during	O
acetoclastic	O
methanogenesis	O
in	O
the	O
archaeal	B-taxonomy_domain
Methanosarcina	B-taxonomy_domain
species	I-taxonomy_domain
.	O

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

EPs	B-structure_element
are	O
frequently	O
found	O
on	O
BMC	B-protein_type
-	I-protein_type
associated	I-protein_type
proteins	I-protein_type
and	O
have	O
been	O
shown	O
to	O
interact	O
with	O
shell	O
proteins	O
.	O

Here	O
we	O
report	O
high	O
-	O
resolution	O
crystal	B-evidence
structures	I-evidence
of	O
a	O
PduL	B-protein_type
-	I-protein_type
type	I-protein_type
PTAC	I-protein_type
in	O
both	O
CoA	B-protein_state
-	I-protein_state
and	O
phosphate	B-protein_state
-	I-protein_state
bound	I-protein_state
forms	O
,	O
completing	O
our	O
understanding	O
of	O
the	O
structural	O
basis	O
of	O
catalysis	O
by	O
the	O
metabolosome	B-complex_assembly
common	O
core	O
enzymes	O
.	O

β	B-structure_element
-	I-structure_element
Barrel	I-structure_element
1	I-structure_element
consists	O
of	O
the	O
N	O
-	O
terminal	O
β	B-structure_element
strand	I-structure_element
and	O
β	B-structure_element
strands	I-structure_element
from	O
the	O
C	B-structure_element
-	I-structure_element
terminal	I-structure_element
half	I-structure_element
of	O
the	O
polypeptide	O
chain	O
(	O
β1	B-structure_element
,	O
β10	B-structure_element
-	I-structure_element
β14	I-structure_element
;	O
residues	O
37	B-residue_range
–	I-residue_range
46	I-residue_range
and	O
155	B-residue_range
–	I-residue_range
224	I-residue_range
).	O

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

Sequence	O
logo	O
calculated	O
from	O
the	O
multiple	B-experimental_method
sequence	I-experimental_method
alignment	I-experimental_method
of	O
PduL	B-protein_type
homologs	O
(	O
see	O
Materials	O
and	O
Methods	O
),	O
but	O
not	B-protein_state
including	I-protein_state
putative	O
EP	B-structure_element
sequences	O
.	O

The	O
position	O
numbers	O
shown	O
correspond	O
to	O
the	O
residue	O
numbering	O
of	O
rPduL	B-protein
;	O
note	O
that	O
some	O
positions	O
in	O
the	O
logo	O
represent	O
gaps	O
in	O
the	O
rPduL	B-protein
sequence	O
.	O

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

Size	B-experimental_method
exclusion	I-experimental_method
chromatography	I-experimental_method
of	O
PduL	B-protein_type
homologs	O
.	O

(	O
a	O
)–(	O
c	O
):	O
Chromatograms	B-evidence
of	O
sPduL	B-protein
(	O
a	O
),	O
rPduL	B-protein
(	O
b	O
),	O
and	O
pPduL	B-protein
(	O
c	O
)	O
with	O
(	O
orange	O
)	O
or	O
without	O
(	O
blue	O
)	O
the	O
predicted	O
EP	B-structure_element
,	O
post	O
-	O
nickel	B-experimental_method
affinity	I-experimental_method
purification	I-experimental_method
,	O
applied	O
over	O
a	O
preparative	O
size	O
exclusion	O
column	O
(	O
see	O
Materials	O
and	O
Methods	O
).	O

The	O
second	O
(	O
Zn2	B-chemical
)	O
is	O
in	O
octahedral	O
coordination	O
by	O
three	O
conserved	B-protein_state
histidine	B-residue_name
residues	O
(	O
His157	B-residue_name_number
,	O
His159	B-residue_name_number
and	O
His204	B-residue_name_number
)	O
as	O
well	O
as	O
three	O
water	B-chemical
molecules	O
(	O
Fig	O
4a	O
).	O

When	O
the	O
crystals	B-experimental_method
were	I-experimental_method
soaked	I-experimental_method
in	O
a	O
sodium	B-chemical
phosphate	I-chemical
solution	O
for	O
2	O
d	O
prior	O
to	O
data	O
collection	O
,	O
the	O
CoA	B-chemical
dissociates	O
,	O
and	O
density	B-evidence
for	O
a	O
phosphate	B-chemical
molecule	O
is	O
visible	O
at	O
the	O
active	B-site
site	I-site
(	O
Table	O
2	O
,	O
Fig	O
4b	O
).	O

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

Given	O
the	O
diversity	O
of	O
signature	O
enzymes	O
(	O
Table	O
1	O
),	O
it	O
is	O
plausible	O
that	O
PduL	B-protein_type
orthologs	O
may	O
adopt	O
different	O
oligomeric	O
states	O
that	O
reflect	O
the	O
differences	O
in	O
the	O
proteins	O
being	O
packaged	O
with	O
them	O
in	O
the	O
organelle	O
lumen	O
.	O

pPduLΔEP	B-mutant
eluted	O
as	O
two	O
smaller	O
forms	O
,	O
possibly	O
corresponding	O
to	O
a	O
trimer	B-oligomeric_state
and	O
a	O
monomer	B-oligomeric_state
.	O

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

Refined	O
domain	O
assignment	O
based	O
on	O
our	O
structure	B-evidence
should	O
be	O
able	O
to	O
predict	O
domains	O
of	O
PF06130	B-structure_element
homologs	O
much	O
more	O
accurately	O
.	O

Implications	O
for	O
Metabolosome	B-complex_assembly
Core	O
Assembly	O

Free	O
CoA	B-chemical
and	O
NAD	B-chemical
+/	I-chemical
H	B-chemical
could	O
potentially	O
be	O
bound	O
to	O
the	O
enzymes	O
as	O
the	O
core	O
assembles	O
and	O
is	O
encapsulated	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

The	O
phosphate	B-protein_state
-	I-protein_state
bound	I-protein_state
structure	B-evidence
indicates	O
that	O
in	O
the	O
opposite	O
reaction	O
direction	O
phosphate	B-chemical
is	O
bound	O
first	O
,	O
and	O
then	O
an	O
acyl	B-chemical
-	I-chemical
CoA	I-chemical
enters	O
.	O

The	O
two	O
crystal	B-evidence
structures	I-evidence
that	O
we	O
report	O
here	O
for	O
the	O
(	O
Sa	B-species
)	O
EctC	B-protein
protein	O
(	O
with	O
resolutions	O
of	O
1	O
.	O
2	O
Å	O
and	O
2	O
.	O
0	O
Å	O
,	O
respectively	O
),	O
and	O
data	O
derived	O
from	O
extensive	O
site	B-experimental_method
-	I-experimental_method
directed	I-experimental_method
mutagenesis	I-experimental_method
experiments	O
targeting	O
evolutionarily	B-protein_state
highly	I-protein_state
conserved	I-protein_state
residues	O
within	O
the	O
extended	O
EctC	B-protein_type
protein	I-protein_type
family	O
,	O
provide	O
a	O
first	O
view	O
into	O
the	O
architecture	O
of	O
the	O
catalytic	B-site
core	I-site
of	O
the	O
ectoine	B-protein_type
synthase	I-protein_type
.	O

The	O
(	O
Sa	B-species
)	O
EctC	B-protein
protein	O
was	O
overproduced	O
and	O
isolated	O
with	O
good	O
yields	O
(	O
30	O
–	O
40	O
mg	O
L	O
-	O
1	O
of	O
culture	O
)	O
and	O
purity	O
(	O
S2a	O
Fig	O
).	O

Biochemical	O
properties	O
of	O
the	O
ectoine	B-protein_type
synthase	I-protein_type

N	B-chemical
-	I-chemical
α	I-chemical
-	I-chemical
ADABA	I-chemical
has	O
so	O
far	O
not	O
been	O
considered	O
as	O
a	O
substrate	O
for	O
EctC	B-protein
,	O
but	O
microorganisms	B-taxonomy_domain
that	O
use	O
ectoine	B-chemical
as	O
a	O
nutrient	O
produce	O
it	O
as	O
an	O
intermediate	O
during	O
catabolism	O
.	O

The	O
stimulation	O
of	O
EctC	B-protein
enzyme	O
activity	O
by	O
salts	O
has	O
previously	O
also	O
been	O
observed	O
for	O
other	O
ectoine	B-protein_type
synthases	I-protein_type
.	O

Since	O
variations	O
of	O
the	O
above	O
-	O
described	O
metal	B-structure_element
-	I-structure_element
binding	I-structure_element
motif	I-structure_element
occur	O
frequently	O
,	O
we	O
experimentally	O
investigated	O
the	O
presence	O
and	O
nature	O
of	O
the	O
metal	B-chemical
that	O
might	O
be	O
contained	O
in	O
the	O
(	O
Sa	B-species
)	O
EctC	B-protein
protein	O
by	O
inductive	B-experimental_method
-	I-experimental_method
coupled	I-experimental_method
plasma	I-experimental_method
mass	I-experimental_method
spectrometry	I-experimental_method
(	O
ICP	B-experimental_method
-	I-experimental_method
MS	I-experimental_method
).	O

We	O
note	O
in	O
this	O
context	O
,	O
that	O
the	O
values	O
obtained	O
for	O
the	O
iron	B-chemical
content	O
of	O
the	O
(	O
Sa	B-species
)	O
EctC	B-protein
proteins	O
varied	O
by	O
approximately	O
10	O
to	O
20	O
%	O
between	O
the	O
two	O
methods	O
.	O

Dependency	O
of	O
the	O
ectoine	B-protein_type
synthase	I-protein_type
activity	O
on	O
metals	O
.	O

We	O
then	O
took	O
such	O
an	O
inactivated	B-protein_state
enzyme	O
preparation	O
,	O
removed	O
the	O
EDTA	B-chemical
by	O
dialysis	B-experimental_method
,	O
and	O
added	O
stoichiometric	O
amounts	O
(	O
10	O
μM	O
)	O
of	O
various	O
metals	O
to	O
the	O
(	O
Sa	B-species
)	O
EctC	B-protein
enzyme	O
.	O

Hence	O
,	O
N	B-chemical
-	I-chemical
α	I-chemical
-	I-chemical
ADABA	I-chemical
is	O
a	O
newly	O
recognized	O
substrate	O
for	O
ectoine	B-protein_type
synthase	I-protein_type
.	O

The	O
Km	B-evidence
dropped	O
fife	O
-	O
fold	O
from	O
4	O
.	O
9	O
±	O
0	O
.	O
5	O
mM	O
to	O
25	O
.	O
4	O
±	O
2	O
.	O
9	O
mM	O
,	O
and	O
the	O
catalytic	B-evidence
efficiency	I-evidence
was	O
reduced	O
from	O
1	O
.	O
47	O
mM	O
-	O
1	O
s	O
-	O
1	O
to	O
0	O
.	O
02	O
mM	O
-	O
1	O
s	O
-	O
1	O
,	O
a	O
73	O
-	O
fold	O
decrease	O
.	O

Finally	O
,	O
a	O
monomer	B-oligomeric_state
of	O
this	O
structure	B-evidence
was	O
used	O
as	O
a	O
template	O
for	O
molecular	B-experimental_method
replacement	I-experimental_method
to	O
phase	O
the	O
high	O
-	O
resolution	O
(	O
1	O
.	O
2	O
Å	O
)	O
dataset	O
of	O
crystal	O
form	O
A	O
,	O
which	O
was	O
subsequently	O
refined	O
to	O
a	O
final	O
Rcryst	B-evidence
of	O
12	O
.	O
4	O
%	O
and	O
an	O
Rfree	B-evidence
of	O
14	O
.	O
9	O
%	O
(	O
S1	O
Table	O
).	O

This	O
structure	B-evidence
adopts	O
an	O
open	B-protein_state
conformation	O
with	O
respect	O
to	O
the	O
typical	O
fold	O
of	O
cupin	B-structure_element
barrels	I-structure_element
and	O
is	O
therefore	O
termed	O
in	O
the	O
following	O
the	O
“	O
open	B-protein_state
”	O
(	O
Sa	B-species
)	O
EctC	B-protein
structure	B-evidence
(	O
Fig	O
4b	O
).	O

Interestingly	O
,	O
the	O
three	O
other	O
monomers	B-oligomeric_state
present	O
in	O
the	O
asymmetric	O
unit	O
all	O
range	O
from	O
Met	B-residue_range
-	I-residue_range
1	I-residue_range
to	I-residue_range
Glu	I-residue_range
-	I-residue_range
115	I-residue_range
and	O
adopt	O
a	O
conformation	O
similar	O
to	O
the	O
“	O
open	B-protein_state
”	O
EctC	B-protein
structure	B-evidence
.	O

The	O
structure	B-evidence
of	O
the	O
“	O
semi	B-protein_state
-	I-protein_state
closed	I-protein_state
”	O
(	O
Sa	B-species
)	O
EctC	B-protein
protein	O
consists	O
of	O
11	O
β	B-structure_element
-	I-structure_element
strands	I-structure_element
(	O
β1	B-structure_element
-	I-structure_element
β11	I-structure_element
)	O
and	O
two	O
α	B-structure_element
-	I-structure_element
helices	I-structure_element
(	O
α	B-structure_element
-	I-structure_element
I	I-structure_element
and	O
α	B-structure_element
-	I-structure_element
II	I-structure_element
)	O
(	O
Fig	O
4a	O
).	O

Our	O
data	O
classify	O
EctC	B-protein
,	O
in	O
addition	O
to	O
the	O
polyketide	B-protein_type
cyclase	I-protein_type
RemF	B-protein
,	O
as	O
the	O
second	O
known	O
cupin	B-protein_type
-	I-protein_type
related	I-protein_type
enzyme	O
that	O
catalyze	O
a	O
cyclocondensation	O
reaction	O
.	O

Analysis	O
of	O
the	O
EctC	B-protein
dimer	B-site
interface	I-site
as	O
observed	O
in	O
the	O
(	O
Sa	B-species
)	O
EctC	B-protein
crystal	B-evidence
structure	I-evidence

It	O
is	O
worth	O
mentioning	O
that	O
β	B-structure_element
-	I-structure_element
strand	I-structure_element
β5	B-structure_element
is	O
located	O
next	O
to	O
His	B-residue_name_number
-	I-residue_name_number
93	I-residue_name_number
,	O
which	O
in	O
all	O
likelihood	O
involved	O
in	O
metal	B-chemical
binding	O
(	O
see	O
below	O
).	O

In	O
the	O
“	O
open	B-protein_state
”	O
(	O
Sa	B-species
)	O
EctC	B-protein
structure	B-evidence
,	O
both	O
proline	B-residue_name
residues	O
are	O
visible	O
in	O
the	O
electron	B-evidence
density	I-evidence
;	O
however	O
,	O
almost	O
directly	O
after	O
Pro	B-residue_name_number
-	I-residue_name_number
110	I-residue_name_number
,	O
the	O
electron	B-evidence
density	I-evidence
is	O
drastically	O
diminished	O
caused	O
by	O
the	O
flexibility	O
of	O
the	O
carboxy	B-structure_element
-	I-structure_element
terminus	I-structure_element
.	O

Since	O
these	O
proline	B-residue_name
residues	O
are	O
followed	O
by	O
the	O
carboxy	B-structure_element
-	I-structure_element
terminal	I-structure_element
region	I-structure_element
of	O
the	O
(	O
Sa	B-species
)	O
EctC	B-protein
protein	O
,	O
the	O
interaction	O
of	O
His	B-residue_name_number
-	I-residue_name_number
55	I-residue_name_number
with	O
Pro	B-residue_name_number
-	I-residue_name_number
109	I-residue_name_number
will	O
likely	O
play	O
a	O
substantial	O
role	O
in	O
spatially	O
orienting	O
this	O
very	O
flexible	O
part	O
of	O
the	O
protein	O
.	O

The	O
interaction	O
between	O
Glu	B-residue_name_number
-	I-residue_name_number
115	I-residue_name_number
and	O
His	B-residue_name_number
-	I-residue_name_number
55	I-residue_name_number
is	O
only	O
visible	O
in	O
the	O
“	O
semi	B-protein_state
-	I-protein_state
closed	I-protein_state
”	O
structure	B-evidence
where	O
the	O
partially	B-protein_state
extended	I-protein_state
carboxy	B-structure_element
-	I-structure_element
terminus	I-structure_element
is	O
resolved	O
in	O
the	O
electron	B-evidence
density	I-evidence
.	O

(	O
b	O
)	O
An	O
overlay	B-experimental_method
of	O
the	O
“	O
open	B-protein_state
”	O
(	O
colored	O
in	O
light	O
blue	O
)	O
and	O
the	O
“	O
semi	B-protein_state
-	I-protein_state
closed	I-protein_state
”	O
(	O
colored	O
in	O
green	O
)	O
structure	B-evidence
of	O
the	O
(	O
Sa	B-species
)	O
EctC	B-protein
protein	O
.	O

The	O
putative	O
iron	B-site
binding	I-site
site	I-site
of	O
(	O
Sa	B-species
)	O
EctC	B-protein

In	O
the	O
“	O
semi	B-protein_state
-	I-protein_state
closed	I-protein_state
”	O
structure	B-evidence
of	O
(	O
Sa	B-species
)	O
EctC	B-protein
,	O
each	O
of	O
the	O
four	O
monomers	B-oligomeric_state
in	O
the	O
asymmetric	O
unit	O
contains	O
a	O
relative	O
strong	O
electron	B-evidence
density	I-evidence
positioned	O
within	O
the	O
cupin	B-structure_element
barrel	I-structure_element
.	O

Of	O
note	O
is	O
the	O
different	O
spatial	O
arrangement	O
of	O
the	O
side	O
-	O
chain	O
of	O
Tyr	B-residue_name_number
-	I-residue_name_number
52	I-residue_name_number
(	O
located	O
in	O
a	O
loop	B-structure_element
after	O
the	O
end	O
of	O
β	B-structure_element
-	I-structure_element
strand	I-structure_element
β5	B-structure_element
)	O
in	O
the	O
“	O
open	B-protein_state
”	O
and	O
“	O
semi	B-protein_state
-	I-protein_state
closed	I-protein_state
”	O
(	O
Sa	B-species
)	O
EctC	B-protein
structures	B-evidence
.	O

It	O
becomes	O
apparent	O
from	O
an	O
overlay	B-experimental_method
of	O
the	O
“	O
open	B-protein_state
”	O
and	O
“	O
semi	B-protein_state
-	I-protein_state
closed	I-protein_state
”	O
(	O
Sa	B-species
)	O
EctC	B-protein
crystal	B-evidence
structures	I-evidence
that	O
the	O
side	O
-	O
chain	O
of	O
Tyr	B-residue_name_number
-	I-residue_name_number
52	I-residue_name_number
rotates	O
away	O
from	O
the	O
position	O
of	O
the	O
presumptive	O
iron	B-chemical
,	O
whereas	O
the	O
side	O
-	O
chains	O
of	O
those	O
residues	O
that	O
probably	O
contacting	O
the	O
metal	B-chemical
directly	O
[	O
Glu	B-residue_name_number
-	I-residue_name_number
57	I-residue_name_number
,	O
Tyr	B-residue_name_number
-	I-residue_name_number
85	I-residue_name_number
,	O
and	O
His	B-residue_name_number
-	I-residue_name_number
93	I-residue_name_number
],	O
remain	O
in	O
place	O
(	O
Fig	O
6a	O
and	O
6b	O
).	O

We	O
also	O
replaced	B-experimental_method
Tyr	B-residue_name_number
-	I-residue_name_number
85	I-residue_name_number
with	O
either	O
a	O
Phe	B-residue_name
or	O
a	O
Trp	B-residue_name
residue	O
and	O
both	O
mutant	B-protein_state
proteins	O
largely	O
lost	O
their	O
catalytic	O
activity	O
and	O
iron	B-chemical
content	O
(	O
Table	O
1	O
)	O
despite	O
the	O
fact	O
that	O
these	O
substitutions	O
were	O
conservative	O
.	O

Since	O
we	O
used	O
PEG	B-chemical
molecules	O
in	O
the	O
crystallization	O
conditions	O
,	O
the	O
observed	O
density	B-evidence
might	O
stem	O
from	O
an	O
ordered	O
part	O
of	O
a	O
PEG	B-chemical
molecule	O
,	O
or	O
low	O
molecular	O
weight	O
PEG	B-chemical
species	O
that	O
might	O
have	O
been	O
present	O
in	O
the	O
PEG	B-chemical
preparation	O
used	O
in	O
our	O
experiments	O
.	O

Despite	O
these	O
notable	O
limitations	O
,	O
we	O
considered	O
the	O
serendipitously	O
trapped	O
compound	O
as	O
a	O
mock	O
ligand	O
that	O
might	O
provide	O
useful	O
insights	O
into	O
the	O
spatial	O
positioning	O
of	O
the	O
true	O
EctC	B-protein
substrate	O
and	O
those	O
residues	O
that	O
coordinate	O
it	O
within	O
the	O
ectoine	B-protein_type
synthase	I-protein_type
active	B-site
site	I-site
.	O

The	O
electron	B-evidence
density	I-evidence
was	O
calculated	O
as	O
an	O
omit	B-evidence
map	I-evidence
and	O
contoured	O
at	O
1	O
.	O
0	O
σ	O
.	O

We	O
also	O
calculated	O
an	O
omit	B-evidence
map	I-evidence
and	O
the	O
electron	B-evidence
density	I-evidence
reappeared	O
(	O
Fig	O
7b	O
).	O

These	O
correspond	O
to	O
amino	O
acids	O
Thr	B-residue_name_number
-	I-residue_name_number
40	I-residue_name_number
,	O
Tyr	B-residue_name_number
-	I-residue_name_number
52	I-residue_name_number
,	O
His	B-residue_name_number
-	I-residue_name_number
55	I-residue_name_number
,	O
Glu	B-residue_name_number
-	I-residue_name_number
57	I-residue_name_number
,	O
Gly	B-residue_name_number
-	I-residue_name_number
64	I-residue_name_number
,	O
Tyr	B-residue_name_number
-	I-residue_name_number
85	I-residue_name_number
-	O
Leu	B-residue_name_number
-	I-residue_name_number
87	I-residue_name_number
,	O
His	B-residue_name_number
-	I-residue_name_number
93	I-residue_name_number
,	O
Phe	B-residue_name_number
-	I-residue_name_number
107	I-residue_name_number
,	O
Pro	B-residue_name_number
-	I-residue_name_number
109	I-residue_name_number
,	O
Gly	B-residue_name_number
-	I-residue_name_number
113	I-residue_name_number
,	O
Glu	B-residue_name_number
-	I-residue_name_number
115	I-residue_name_number
,	O
and	O
His	B-residue_name_number
-	I-residue_name_number
117	I-residue_name_number
in	O
the	O
(	O
Sa	B-species
)	O
EctC	B-protein
protein	O
(	O
Fig	O
2	O
).	O

Each	O
of	O
these	O
mutant	B-protein_state
(	O
Sa	B-species
)	O
EctC	B-protein
proteins	O
was	O
overproduced	O
in	O
E	B-species
.	I-species
coli	I-species
and	O
purified	O
by	O
affinity	B-experimental_method
chromatography	I-experimental_method
;	O
they	O
all	O
yielded	O
pure	O
and	O
stable	O
protein	O
preparations	O
.	O

We	O
replaced	B-experimental_method
each	O
of	O
these	O
residues	O
with	O
an	O
Ala	B-residue_name
residue	O
and	O
found	O
that	O
none	O
of	O
them	O
had	O
an	O
influence	O
on	O
the	O
iron	B-chemical
content	O
of	O
the	O
mutant	B-protein_state
proteins	O
.	O

Each	O
of	O
these	O
residues	O
is	O
evolutionarily	B-protein_state
highly	I-protein_state
conserved	I-protein_state
.	O

His	B-residue_name_number
-	I-residue_name_number
117	I-residue_name_number
is	O
a	O
strictly	B-protein_state
conserved	I-protein_state
residue	O
and	O
its	O
substitution	B-experimental_method
by	O
an	O
Ala	B-residue_name
residue	O
results	O
in	O
a	O
drop	O
of	O
enzyme	O
activity	O
(	O
down	O
to	O
44	O
%)	O
and	O
an	O
iron	B-chemical
content	O
of	O
83	O
%	O
(	O
Table	O
1	O
).	O

As	O
an	O
internal	O
control	O
for	O
our	O
mutagenesis	B-experimental_method
experiments	I-experimental_method
,	O
we	O
also	O
substituted	B-experimental_method
Thr	B-residue_name_number
-	I-residue_name_number
41	I-residue_name_number
and	O
His	B-residue_name_number
-	I-residue_name_number
51	I-residue_name_number
,	O
two	O
residues	O
that	O
are	O
not	B-protein_state
evolutionarily	I-protein_state
conserved	I-protein_state
in	O
EctC	B-protein_type
-	I-protein_type
type	I-protein_type
proteins	I-protein_type
with	O
Ala	B-residue_name
residues	O
.	O

Hence	O
,	O
the	O
active	B-site
site	I-site
of	O
ectoine	B-protein_type
synthase	I-protein_type
must	O
possess	O
a	O
certain	O
degree	O
of	O
structural	O
plasticity	O
,	O
a	O
notion	O
that	O
is	O
supported	O
by	O
the	O
report	O
on	O
the	O
EctC	B-protein
-	O
catalyzed	O
formation	O
of	O
the	O
synthetic	O
compatible	O
solute	O
ADPC	B-chemical
through	O
the	O
cyclic	O
condensation	O
of	O
two	O
glutamine	B-chemical
molecules	O
.	O

We	O
assumed	O
that	O
its	O
location	O
and	O
mode	O
of	O
binding	O
gives	O
,	O
in	O
all	O
likelihood	O
,	O
clues	O
as	O
to	O
the	O
position	O
of	O
the	O
true	O
substrate	O
N	B-chemical
-	I-chemical
γ	I-chemical
-	I-chemical
ADABA	I-chemical
within	O
the	O
EctC	B-protein
active	B-site
site	I-site
.	O

This	O
probably	O
worked	O
to	O
the	O
detriment	O
of	O
our	O
efforts	O
in	O
solving	O
crystal	B-evidence
structures	I-evidence
of	O
the	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
(	O
Sa	B-species
)	O
EctC	B-protein
protein	O
in	B-protein_state
complex	I-protein_state
with	I-protein_state
either	O
N	B-chemical
-	I-chemical
γ	I-chemical
-	I-chemical
ADABA	I-chemical
or	O
ectoine	B-chemical
.	O

Interestingly	O
,	O
mutations	B-experimental_method
blocking	O
PIN	B-structure_element
oligomerization	O
had	O
no	O
RNase	B-protein_type
activity	O
,	O
indicating	O
that	O
both	O
oligomerization	O
and	O
NTD	B-structure_element
binding	O
are	O
crucial	O
for	O
RNase	B-protein_type
activity	O
in	O
vitro	O
.	O

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

Moreover	O
,	O
Regnase	B-protein
-	I-protein
1	I-protein
functions	O
as	O
a	O
dimer	B-oligomeric_state
through	O
intermolecular	O
PIN	B-structure_element
-	O
PIN	B-structure_element
interactions	O
during	O
cleavage	O
of	O
target	O
mRNA	B-chemical
.	O

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

These	O
results	O
indicate	O
that	O
not	O
only	O
the	O
PIN	B-structure_element
but	O
also	O
the	O
ZF	B-structure_element
domain	O
contribute	O
to	O
RNA	B-chemical
binding	O
,	O
while	O
the	O
NTD	B-structure_element
is	O
not	O
likely	O
to	O
be	O
involved	O
in	O
direct	O
interaction	O
with	O
RNA	B-chemical
.	O

Regnase	B-protein
-	I-protein
1	I-protein
lacking	B-protein_state
the	O
ZF	B-structure_element
domain	O
generated	O
a	O
smaller	O
but	O
appreciable	O
amount	O
of	O
cleaved	O
product	O
(	O
T1	O
/	O
2	O
~	O
70	O
minutes	O
),	O
while	O
those	O
lacking	B-protein_state
the	O
NTD	B-structure_element
did	O
not	O
generate	O
cleaved	O
products	O
(	O
T1	O
/	O
2	O
>	O
90	O
minutes	O
).	O

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

Domain	O
-	O
domain	O
interaction	O
between	O
the	O
NTD	B-structure_element
and	O
the	O
PIN	B-structure_element
domain	O

Residues	O
critical	O
for	O
Regnase	B-protein
-	I-protein
1	I-protein
RNase	B-protein_type
activity	O

The	O
other	O
mutated	O
residues	O
—	O
K152	B-residue_name_number
,	O
R158	B-residue_name_number
,	O
R188	B-residue_name_number
,	O
R200	B-residue_name_number
,	O
K204	B-residue_name_number
,	O
K206	B-residue_name_number
,	O
K257	B-residue_name_number
,	O
and	O
R258	B-residue_name_number
—	O
were	O
not	O
critical	O
for	O
RNase	B-protein_type
activity	O
.	O

One	O
group	O
consisted	O
of	O
catalytically	B-protein_state
active	I-protein_state
PIN	B-structure_element
domains	O
with	O
mutation	B-experimental_method
of	I-experimental_method
basic	O
residues	O
found	O
in	O
the	O
previous	O
section	O
to	O
confer	O
decreased	O
RNase	B-protein_type
activity	O
(	O
Fig	O
.	O
4	O
).	O

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

The	O
previously	O
reported	O
crystal	B-evidence
structure	I-evidence
of	O
the	O
Regnase	B-protein
-	I-protein
1	I-protein
PIN	B-structure_element
domain	O
derived	O
from	O
Homo	B-species
sapiens	I-species
is	O
nearly	O
identical	O
to	O
the	O
one	O
derived	O
from	O
Mus	B-species
musculus	I-species
in	O
this	O
study	O
,	O
with	O
a	O
backbone	O
RMSD	B-evidence
of	O
0	O
.	O
2	O
Å	O
.	O
The	O
amino	O
acid	O
sequences	O
corresponding	O
to	O
PIN	B-structure_element
(	O
residues	O
134	B-residue_range
–	I-residue_range
295	I-residue_range
)	O
are	O
the	O
two	O
non	O
-	O
identical	O
residues	O
are	O
substituted	O
with	O
similar	O
amino	O
acids	O
.	O

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

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

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

Based	O
on	O
these	O
structural	B-experimental_method
and	I-experimental_method
functional	I-experimental_method
analyses	I-experimental_method
of	O
Regnase	B-protein
-	I-protein
1	I-protein
domain	O
-	O
domain	O
interactions	O
,	O
we	O
performed	O
docking	B-experimental_method
simulations	I-experimental_method
of	O
the	O
NTD	B-structure_element
,	O
PIN	B-structure_element
dimer	B-oligomeric_state
,	O
and	O
IL	B-protein_type
-	I-protein_type
6	I-protein_type
mRNA	B-chemical
.	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

Structural	B-experimental_method
and	I-experimental_method
functional	I-experimental_method
analyses	I-experimental_method
of	O
Regnase	B-protein
-	I-protein
1	I-protein
.	O

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

Domain	O
-	O
domain	O
interaction	O
between	O
the	O
NTD	B-structure_element
and	O
the	O
PIN	B-structure_element
domain	O
.	O

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

Mutated	O
basic	O
residues	O
were	O
shown	O
in	O
sticks	O
and	O
those	O
with	O
significantly	O
reduced	O
RNase	B-protein_type
activities	O
were	O
colored	O
red	O
or	O
yellow	O
.	O

(	O
c	O
)	O
In	B-experimental_method
vitro	I-experimental_method
cleavage	I-experimental_method
assay	I-experimental_method
of	O
Regnase	B-protein
-	I-protein
1	I-protein
for	O
Regnase	B-protein
-	I-protein
1	I-protein
mRNA	B-chemical
.	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

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

Structure	B-evidence
of	O
PmC11	B-protein

The	O
central	O
nine	B-structure_element
-	I-structure_element
stranded	I-structure_element
β	I-structure_element
-	I-structure_element
sheet	I-structure_element
(	O
β1	B-structure_element
–	I-structure_element
β9	I-structure_element
)	O
of	O
PmC11	B-protein
consists	O
of	O
six	O
parallel	B-structure_element
and	O
three	O
anti	B-structure_element
-	I-structure_element
parallel	I-structure_element
β	I-structure_element
-	I-structure_element
strands	I-structure_element
with	O
4	O
↑	O
3	O
↓	O
2	O
↑	O
1	O
↑	O
5	O
↑	O
6	O
↑	O
7	O
↓	O
8	O
↓	O
9	O
↑	O
topology	O
(	O
Fig	O
.	O
1A	O
)	O
and	O
the	O
overall	O
structure	B-evidence
includes	O
14	O
α	B-structure_element
-	I-structure_element
helices	I-structure_element
with	O
six	O
(	O
α1	B-structure_element
–	I-structure_element
α2	I-structure_element
and	O
α4	B-structure_element
–	I-structure_element
α7	I-structure_element
)	O
closely	O
surrounding	O
the	O
β	B-structure_element
-	I-structure_element
sheet	I-structure_element
in	O
an	O
approximately	O
parallel	O
orientation	O
.	O

Helices	B-structure_element
α1	B-structure_element
,	O
α7	B-structure_element
,	O
and	O
α6	B-structure_element
are	O
located	O
on	O
one	O
side	O
of	O
the	O
β	B-structure_element
-	I-structure_element
sheet	I-structure_element
with	O
α2	B-structure_element
,	O
α4	B-structure_element
,	O
and	O
α5	B-structure_element
on	O
the	O
opposite	O
side	O
(	O
Fig	O
.	O
1A	O
).	O

The	O
core	B-structure_element
caspase	I-structure_element
-	I-structure_element
fold	I-structure_element
is	O
highlighted	O
in	O
a	O
box	O
.	O

The	O
CTD	B-structure_element
of	O
PmC11	B-protein
is	O
composed	O
of	O
a	O
tight	B-structure_element
helical	I-structure_element
bundle	I-structure_element
formed	O
from	O
helices	B-structure_element
α8	B-structure_element
–	I-structure_element
α14	I-structure_element
and	O
includes	O
strands	B-structure_element
βC	B-structure_element
and	O
βF	B-structure_element
,	O
and	O
β	B-structure_element
-	I-structure_element
hairpin	I-structure_element
βD	B-structure_element
–	I-structure_element
βE	I-structure_element
.	O
The	O
CTD	B-structure_element
sits	O
entirely	O
on	O
one	O
side	O
of	O
the	O
enzyme	O
interacting	O
only	O
with	O
α3	B-structure_element
,	O
α5	B-structure_element
,	O
β9	B-structure_element
,	O
and	O
the	O
loops	B-structure_element
surrounding	O
β8	B-structure_element
.	O

D	O
,	O
cysteine	O
peptidase	O
activity	O
of	O
PmC11	B-protein
.	O

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

F	O
,	O
activity	B-evidence
of	O
PmC11	B-protein
against	O
basic	O
substrates	O
.	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

Asp177	B-residue_name_number
is	O
located	O
near	O
the	O
catalytic	B-protein_state
cysteine	B-residue_name
and	O
is	O
conserved	B-protein_state
throughout	I-protein_state
the	O
C11	B-protein_type
family	I-protein_type
,	O
suggesting	O
it	O
is	O
the	O
primary	O
S1	B-site
binding	I-site
site	I-site
residue	I-site
.	O

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

A	O
,	O
PmC11	O
activity	O
is	O
inhibited	O
by	O
Z	B-chemical
-	I-chemical
VRPR	I-chemical
-	I-chemical
FMK	I-chemical
.	O

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

The	O
primary	B-experimental_method
structural	I-experimental_method
alignment	I-experimental_method
also	O
shows	O
that	O
the	O
catalytic	B-site
dyad	I-site
in	O
PmC11	B-protein
is	O
structurally	B-protein_state
conserved	I-protein_state
in	O
clostripain	B-protein
(	O
Fig	O
.	O
1A	O
).	O

Unlike	O
PmC11	B-protein
,	O
clostripain	B-protein
has	O
two	O
cleavage	B-site
sites	I-site
(	O
Arg181	B-residue_name_number
and	O
Arg190	B-residue_name_number
),	O
which	O
results	O
in	O
the	O
removal	O
of	O
a	O
nonapeptide	B-structure_element
,	O
and	O
is	O
required	O
for	O
full	B-protein_state
activation	I-protein_state
of	O
the	O
enzyme	O
(	O
highlighted	O
in	O
Fig	O
.	O
1A	O
).	O

Interestingly	O
,	O
Arg190	B-residue_name_number
was	O
found	O
to	O
align	O
with	O
Lys147	B-residue_name_number
in	O
PmC11	B-protein
.	O

As	O
studies	O
on	O
clostripain	B-protein
revealed	O
addition	O
of	O
Ca2	B-chemical
+	I-chemical
ions	O
are	O
required	O
for	O
full	B-protein_state
activation	I-protein_state
,	O
the	O
Ca2	B-chemical
+	I-chemical
dependence	O
of	O
PmC11	B-protein
was	O
examined	O
.	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

The	O
enzyme	O
exhibits	O
all	O
of	O
the	O
key	O
structural	O
elements	O
of	O
clan	B-protein_type
CD	I-protein_type
members	I-protein_type
,	O
but	O
is	O
unusual	O
in	O
that	O
it	O
has	O
a	O
nine	O
-	O
stranded	O
central	O
β	B-structure_element
-	I-structure_element
sheet	I-structure_element
with	O
a	O
novel	O
C	B-structure_element
-	I-structure_element
terminal	I-structure_element
domain	I-structure_element
.	O

In	O
addition	O
,	O
the	O
structure	B-evidence
suggested	O
a	O
mechanism	O
of	O
self	O
-	O
inhibition	O
in	O
both	O
PmC11	B-protein
and	O
clostripain	B-protein
and	O
an	O
activation	O
mechanism	O
that	O
requires	O
autoprocessing	B-ptm
.	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

Like	O
PmC11	B-protein
,	O
these	O
structures	O
show	O
preformed	O
catalytic	O
machinery	O
and	O
,	O
for	O
a	O
substrate	O
to	O
gain	O
access	O
,	O
movement	O
and	O
/	O
or	O
cleavage	B-ptm
of	O
the	O
blocking	B-structure_element
region	I-structure_element
is	O
required	O
.	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

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

In	O
a	O
second	O
step	O
,	O
the	O
essential	O
SPOUT	B-protein_type
-	I-protein_type
class	I-protein_type
methyltransferase	I-protein_type
Nep1	B-protein
/	O
Emg1	B-protein
modifies	O
the	O
pseudouridine	B-chemical
to	O
N1	B-chemical
-	I-chemical
methylpseudouridine	I-chemical
.	O

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

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

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

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

Indeed	O
,	O
in	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
yeast	B-taxonomy_domain
a	O
strong	O
primer	B-evidence
extension	I-evidence
stop	I-evidence
signal	I-evidence
occurred	O
at	O
position	O
1192	B-residue_number
.	O

As	O
expected	O
,	O
in	O
a	O
Δsnr35	B-mutant
deletion	B-experimental_method
preventing	O
pseudouridylation	B-ptm
and	O
N1	B-ptm
-	I-ptm
methylation	I-ptm
(	O
resulting	O
in	O
acp3U	B-chemical
)	O
as	O
well	O
as	O
in	O
a	O
Δnep1	B-mutant
deletion	O
strain	O
where	O
pseudouridine	B-chemical
is	O
not	B-protein_state
methylated	I-protein_state
(	O
resulting	O
in	O
acp3Ψ	B-chemical
)	O
a	O
primer	B-evidence
extension	I-evidence
stop	I-evidence
signal	I-evidence
of	O
similar	O
intensity	O
as	O
in	O
the	O
wild	B-protein_state
type	I-protein_state
was	O
observed	O
.	O

The	O
efficiency	O
of	O
siRNA	B-chemical
-	O
mediated	O
depletion	O
was	O
established	O
by	O
RT	B-experimental_method
-	I-experimental_method
qPCR	I-experimental_method
and	O
found	O
to	O
be	O
very	O
high	O
with	O
siRNA	B-chemical
544	O
(	O
Supplementary	O
Figure	O
S2A	O
,	O
remaining	O
TSR3	B-protein
mRNA	O
level	O
of	O
2	O
%).	O

Phenotypic	O
characterization	O
of	O
Δtsr3	B-mutant
mutants	O

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

Surprisingly	O
,	O
early	O
nucleolar	O
processing	O
reactions	O
were	O
also	O
inhibited	O
,	O
and	O
this	O
was	O
observed	O
in	O
both	O
yeast	B-taxonomy_domain
Δtsr3	B-mutant
cells	O
(	O
see	O
accumulation	O
of	O
35S	B-complex_assembly
in	O
Supplementary	O
Figure	O
S2C	O
)	O
and	O
Tsr3	B-protein
depleted	O
human	B-species
cells	O
(	O
see	O
47S	B-complex_assembly
/	O
45S	B-complex_assembly
accumulation	O
in	O
Figure	O
2C	O
and	O
Northern	B-experimental_method
blot	I-experimental_method
quantification	O
in	O
Supplementary	O
Figure	O
S2B	O
).	O

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

After	O
polysome	B-experimental_method
gradient	I-experimental_method
separation	I-experimental_method
C	O
-	O
terminally	O
epitope	O
-	O
labeled	O
Tsr3	B-mutant
-	I-mutant
3xHA	I-mutant
was	O
exclusively	O
detectable	O
in	O
the	O
low	O
-	O
density	O
fraction	O
(	O
Figure	O
2E	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

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

N	O
-	O
terminal	O
truncations	B-experimental_method
of	O
up	O
to	O
45	B-residue_range
aa	I-residue_range
and	O
C	O
-	O
terminal	O
truncations	B-experimental_method
of	O
up	O
to	O
76	B-residue_range
aa	I-residue_range
mediated	O
acp	B-chemical
modification	O
as	O
efficiently	O
as	O
the	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
protein	O
and	O
no	O
significant	O
increased	O
levels	O
of	O
20S	B-chemical
pre	I-chemical
-	I-chemical
RNA	I-chemical
were	O
detected	O
.	O

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

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

We	O
focused	O
on	O
archaeal	B-taxonomy_domain
species	O
containing	O
a	O
putative	O
Nep1	B-protein
homolog	O
suggesting	O
that	O
these	O
species	O
are	O
in	O
principle	O
capable	O
of	O
synthesizing	O
N1	B-chemical
-	I-chemical
methyl	I-chemical
-	I-chemical
N3	I-chemical
-	I-chemical
acp	I-chemical
-	I-chemical
pseudouridine	I-chemical
.	O

The	O
C	B-structure_element
-	I-structure_element
terminal	I-structure_element
domain	I-structure_element
(	O
aa	O
93	B-residue_range
–	I-residue_range
184	I-residue_range
)	O
has	O
a	O
globular	B-structure_element
all	I-structure_element
α	I-structure_element
-	I-structure_element
helical	I-structure_element
structure	I-structure_element
comprising	O
α	B-structure_element
-	I-structure_element
helices	I-structure_element
α4	B-structure_element
to	I-structure_element
α9	I-structure_element
.	O

Remarkably	O
,	O
the	O
entire	O
C	B-structure_element
-	I-structure_element
terminal	I-structure_element
domain	I-structure_element
(	O
92	B-residue_range
aa	I-residue_range
)	O
of	O
the	O
protein	O
is	O
threaded	O
through	O
the	O
loop	B-structure_element
which	O
connects	O
β	B-structure_element
-	I-structure_element
strand	I-structure_element
β3	B-structure_element
and	O
α	B-structure_element
-	I-structure_element
helix	I-structure_element
α2	B-structure_element
of	O
the	O
N	B-structure_element
-	I-structure_element
terminal	I-structure_element
domain	I-structure_element
.	O

Tsr3	B-protein
has	O
a	O
fold	O
similar	O
to	O
SPOUT	B-protein_type
-	I-protein_type
class	I-protein_type
RNA	I-protein_type
methyltransferases	I-protein_type
.	O
(	O
A	O
)	O
Cartoon	O
representation	O
of	O
the	O
X	B-evidence
-	I-evidence
ray	I-evidence
structure	I-evidence
of	O
VdTsr3	B-protein
in	O
two	O
orientations	O
.	O

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

Structure	B-experimental_method
predictions	I-experimental_method
suggested	O
that	O
Tsr3	B-protein
might	O
contain	O
a	O
so	O
-	O
called	O
RLI	B-structure_element
domain	I-structure_element
which	O
contains	O
a	O
‘	O
bacterial	B-structure_element
like	I-structure_element
’	I-structure_element
ferredoxin	I-structure_element
fold	I-structure_element
and	O
binds	O
two	O
iron	O
-	O
sulfur	O
clusters	O
through	O
eight	O
conserved	B-protein_state
cysteine	B-residue_name
residues	O
.	O

A	O
notable	O
exception	O
is	O
Trm10	B-protein
.	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

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	B-bond_interaction
bonded	I-bond_interaction
to	O
the	O
backbone	O
carbonyl	O
group	O
of	O
I69	B-residue_name_number
.	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

Hydrogen	B-bond_interaction
bonds	I-bond_interaction
are	O
indicated	O
by	O
dashed	O
lines	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

This	O
correlates	O
with	O
a	O
20S	B-chemical
pre	I-chemical
-	I-chemical
rRNA	I-chemical
accumulation	O
comparable	O
to	O
the	O
Δtsr3	B-mutant
deletion	O
(	O
right	O
:	O
northern	B-experimental_method
blot	I-experimental_method
).	O

This	O
suggests	O
that	O
the	O
hydrophobic	B-bond_interaction
interaction	I-bond_interaction
between	O
SAM	B-chemical
'	O
s	O
methyl	O
group	O
and	O
the	O
hydrophobic	B-site
pocket	I-site
of	O
Tsr3	B-protein
is	O
thermodynamically	O
important	O
for	O
the	O
interaction	O
.	O

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

The	O
side	O
chain	O
hydroxyl	O
group	O
of	O
T19	B-residue_name_number
seems	O
of	O
minor	O
importance	O
for	O
SAM	B-chemical
binding	O
since	O
mutations	B-experimental_method
of	O
T17	B-residue_name_number
(	O
T19	B-residue_name_number
in	O
VdTsr3	B-protein
)	O
to	O
either	O
A	B-residue_name
or	O
D	B-residue_name
did	O
not	O
significantly	O
influence	O
the	O
SAM	B-evidence
-	I-evidence
binding	I-evidence
affinity	I-evidence
of	O
SsTsr3	B-protein
(	O
KD	B-evidence
'	O
s	O
=	O
3	O
.	O
9	O
or	O
11	O
.	O
2	O
mM	O
,	O
respectively	O
).	O

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

RNA	O
-	O
binding	O
of	O
Tsr3	B-protein
.	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

The	O
presence	O
of	O
saturating	O
amounts	O
of	O
SAM	B-chemical
(	O
2	O
mM	O
)	O
did	O
not	O
have	O
a	O
significant	O
influence	O
on	O
the	O
RNA	B-evidence
-	I-evidence
affinity	I-evidence
of	O
SsTsr3	B-protein
(	O
KD	B-evidence
of	O
1	O
.	O
7	O
μM	O
for	O
the	O
20mer	B-oligomeric_state
-	I-oligomeric_state
GC	I-oligomeric_state
-	O
RNA	B-chemical
)	O
suggesting	O
no	O
cooperativity	O
in	O
substrate	O
binding	O
.	O

This	O
suggests	O
that	O
enzymes	O
with	O
a	O
SAM	B-protein_type
-	I-protein_type
dependent	I-protein_type
acp	I-protein_type
transferase	I-protein_type
activity	O
might	O
have	O
evolved	O
from	O
SAM	B-protein_type
-	I-protein_type
dependent	I-protein_type
methyltransferases	I-protein_type
by	O
slight	O
modifications	O
of	O
the	O
SAM	B-site
-	I-site
binding	I-site
pocket	I-site
.	O

In	O
contrast	O
to	O
Nep1	B-protein
,	O
the	O
enzyme	O
preceding	O
Tsr3	B-protein
in	O
the	O
m1acp3Ψ	B-chemical
biosynthesis	O
pathway	O
,	O
Tsr3	B-protein
binds	O
rather	O
weakly	O
and	O
with	O
little	O
specificity	O
to	O
its	O
isolated	O
substrate	O
RNA	B-chemical
.	O

Recently	O
,	O
structural	B-experimental_method
,	I-experimental_method
functional	I-experimental_method
,	I-experimental_method
and	I-experimental_method
CRAC	I-experimental_method
(	I-experimental_method
cross	I-experimental_method
-	I-experimental_method
linking	I-experimental_method
and	I-experimental_method
cDNA	I-experimental_method
analysis	I-experimental_method
)	I-experimental_method
experiments	I-experimental_method
of	O
late	O
assembly	O
factors	O
involved	O
in	O
cytoplasmic	O
processing	O
of	O
40S	B-complex_assembly
subunits	I-complex_assembly
,	O
along	O
with	O
cryo	B-experimental_method
-	I-experimental_method
EM	I-experimental_method
studies	O
of	O
the	O
late	B-protein_state
pre	B-complex_assembly
-	I-complex_assembly
40S	I-complex_assembly
subunits	I-complex_assembly
have	O
provided	O
important	O
insights	O
into	O
late	O
pre	B-complex_assembly
-	I-complex_assembly
40S	I-complex_assembly
processing	O
.	O

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

The	O
YfiBNR	B-complex_assembly
system	O
contains	O
three	O
protein	O
members	O
and	O
modulates	O
intracellular	O
c	B-chemical
-	I-chemical
di	I-chemical
-	I-chemical
GMP	I-chemical
levels	O
in	O
response	O
to	O
signals	O
received	O
in	O
the	O
periplasm	O
(	O
Malone	O
et	O
al	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

Whether	O
YfiB	B-protein
directly	O
recruits	O
YfiR	B-protein
or	O
recruits	O
YfiR	B-protein
via	O
a	O
third	O
partner	O
is	O
an	O
open	O
question	O
.	O

It	O
has	O
been	O
reported	O
that	O
the	O
activation	O
of	O
YfiN	B-protein
may	O
be	O
induced	O
by	O
redox	O
-	O
driven	O
misfolding	O
of	O
YfiR	B-protein
(	O
Giardina	O
et	O
al	O
.,;	O
Malone	O
et	O
al	O
.,;	O
Malone	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
crystal	B-evidence
structure	I-evidence
of	O
YfiB	B-protein
monomer	B-oligomeric_state
consists	O
of	O
a	O
five	B-structure_element
-	I-structure_element
stranded	I-structure_element
β	I-structure_element
-	I-structure_element
sheet	I-structure_element
(	O
β1	B-structure_element
-	I-structure_element
2	I-structure_element
-	I-structure_element
5	I-structure_element
-	I-structure_element
3	I-structure_element
-	I-structure_element
4	I-structure_element
)	O
flanked	O
by	O
five	B-structure_element
α	I-structure_element
-	I-structure_element
helices	I-structure_element
(	O
α1	B-structure_element
–	I-structure_element
5	I-structure_element
)	O
on	O
one	O
side	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

It	O
has	O
been	O
reported	O
that	O
single	B-experimental_method
mutants	I-experimental_method
of	I-experimental_method
Q39	B-residue_name_number
,	O
L43	B-residue_name_number
,	O
F48	B-residue_name_number
and	O
W55	B-residue_name_number
contribute	O
to	O
YfiB	B-protein
activation	O
leading	O
to	O
the	O
induction	O
of	O
the	O
SCV	O
phenotype	O
in	O
P	B-species
.	I-species
aeruginosa	I-species
PAO1	I-species
(	O
Malone	O
et	O
al	O
.,).	O

These	O
two	O
regions	O
contribute	O
a	O
robust	O
hydrogen	B-site
-	I-site
bonding	I-site
network	I-site
to	O
the	O
YfiB	B-site
-	I-site
YfiR	I-site
interface	I-site
,	O
resulting	O
in	O
a	O
tightly	O
bound	O
complex	O
.	O

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

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

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

E163	B-residue_name_number
and	O
I169	B-residue_name_number
are	O
YfiB	B-site
-	I-site
interacting	I-site
residues	I-site
of	O
YfiR	B-protein
,	O
in	O
which	O
E163	B-residue_name_number
forms	O
a	O
hydrogen	B-bond_interaction
bond	I-bond_interaction
with	O
R96	B-residue_name_number
of	O
YfiB	B-protein
(	O
Fig	O
.	O
3D	O
-	O
II	O
)	O
and	O
I169	B-residue_name_number
is	O
involved	O
in	O
forming	O
the	O
L166	B-residue_name_number
/	O
I169	B-residue_name_number
/	O
V176	B-residue_name_number
/	O
P178	B-residue_name_number
/	O
L181	B-residue_name_number
hydrophobic	B-site
core	I-site
for	O
anchoring	O
F57	B-residue_name_number
of	O
YfiB	B-protein
(	O
Fig	O
.	O
3D	O
-	O
I	O
(	O
ii	O
)).	O

Intriguingly	O
,	O
a	O
Dali	B-experimental_method
search	I-experimental_method
(	O
Holm	O
and	O
Rosenstrom	O
,)	O
indicated	O
that	O
the	O
closest	O
homologs	O
of	O
YfiR	B-protein
shared	O
the	O
characteristic	O
of	O
being	O
able	O
to	O
bind	O
several	O
structurally	O
similar	O
small	O
molecules	O
,	O
such	O
as	O
L	B-chemical
-	I-chemical
Trp	I-chemical
,	O
L	B-chemical
-	I-chemical
Phe	I-chemical
,	O
B	O
-	O
group	O
vitamins	O
and	O
their	O
analogs	O
,	O
encouraging	O
us	O
to	O
test	O
whether	O
YfiR	B-protein
can	O
recognize	O
these	O
molecules	O
.	O

Structural	B-experimental_method
analyses	I-experimental_method
revealed	O
that	O
the	O
VB6	B-chemical
and	O
L	B-chemical
-	I-chemical
Trp	I-chemical
molecules	O
are	O
bound	B-protein_state
at	I-protein_state
the	O
periphery	O
of	O
the	O
YfiR	B-protein
dimer	B-oligomeric_state
,	O
but	O
not	O
at	O
the	O
dimer	B-site
interface	I-site
.	O

To	O
evaluate	O
the	O
importance	O
of	O
F57	B-residue_name_number
in	O
YfiBL43P	B-complex_assembly
-	I-complex_assembly
YfiR	I-complex_assembly
interaction	O
,	O
the	O
binding	B-evidence
affinities	I-evidence
of	O
YfiBL43P	B-mutant
and	O
YfiBL43P	B-mutant
/	O
F57A	B-mutant
for	O
YfiR	B-protein
were	O
measured	O
by	O
isothermal	B-experimental_method
titration	I-experimental_method
calorimetry	I-experimental_method
(	O
ITC	B-experimental_method
).	O

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

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

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

These	O
APFs	B-complex_assembly
had	O
an	O
outer	O
diameter	O
that	O
ranged	O
from	O
11	O
–	O
14	O
nm	O
and	O
an	O
inner	O
diameter	O
that	O
ranged	O
from	O
2	O
.	O
5	O
–	O
4	O
nm	O
.	O

These	O
observations	O
suggest	O
that	O
the	O
Aβ	B-protein
trimers	B-oligomeric_state
,	O
hexamers	B-oligomeric_state
,	O
dodecamers	B-oligomeric_state
,	O
and	O
related	O
assemblies	O
may	O
be	O
associated	O
with	O
presymptomatic	O
neurodegeneration	O
,	O
while	O
Aβ	B-protein
dimers	B-oligomeric_state
are	O
more	O
closely	O
associated	O
with	O
fibril	O
formation	O
and	O
plaque	O
deposition	O
during	O
symptomatic	O
Alzheimer	O
’	O
s	O
disease	O
.−	O

Many	O
of	O
these	O
studies	O
have	O
reported	O
the	O
monomer	B-oligomeric_state
subunit	B-structure_element
as	O
adopting	O
a	O
β	B-structure_element
-	I-structure_element
hairpin	I-structure_element
conformation	O
,	O
in	O
which	O
the	O
hydrophobic	O
central	B-structure_element
and	O
C	B-structure_element
-	I-structure_element
terminal	I-structure_element
regions	I-structure_element
form	O
an	O
antiparallel	B-structure_element
β	I-structure_element
-	I-structure_element
sheet	I-structure_element
.	O

In	O
2008	O
,	O
Hoyer	O
et	O
al	O
.	O
reported	O
the	O
NMR	B-experimental_method
structure	B-evidence
of	O
an	O
Aβ	B-protein
monomer	B-oligomeric_state
bound	B-protein_state
to	I-protein_state
an	O
artificial	B-chemical
binding	I-chemical
protein	I-chemical
called	O
an	O
affibody	B-chemical
(	O
PDB	O
2OTK	O
).	O

This	O
Aβ	B-protein
β	B-structure_element
-	I-structure_element
hairpin	I-structure_element
encompasses	O
residues	O
17	B-residue_range
–	I-residue_range
37	I-residue_range
and	O
contains	O
two	O
β	B-structure_element
-	I-structure_element
strands	I-structure_element
comprising	O
Aβ17	B-protein
–	B-residue_range
24	I-residue_range
and	O
Aβ30	B-protein
–	B-residue_range
37	I-residue_range
connected	O
by	O
an	O
Aβ25	B-protein
–	B-residue_range
29	I-residue_range
loop	B-structure_element
.	O

In	O
a	O
related	O
study	O
,	O
Sandberg	O
et	O
al	O
.	O
constrained	O
Aβ	B-protein
in	O
a	O
β	B-structure_element
-	I-structure_element
hairpin	I-structure_element
conformation	O
by	O
mutating	B-experimental_method
residues	O
A21	B-residue_name_number
and	O
A30	B-residue_name_number
to	O
cysteine	B-residue_name
and	O
forming	O
an	O
intramolecular	O
disulfide	B-ptm
bond	I-ptm
.	O

After	O
determining	O
the	O
X	B-evidence
-	I-evidence
ray	I-evidence
crystallographic	I-evidence
structure	I-evidence
of	O
the	O
p	B-chemical
-	I-chemical
iodophenylalanine	I-chemical
variant	O
we	O
attempt	O
to	O
determine	O
the	O
structure	B-evidence
of	O
the	O
native	O
phenylalanine	B-residue_name
compound	O
by	O
isomorphous	B-experimental_method
replacement	I-experimental_method
.	O

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

Although	O
the	O
disulfide	B-ptm
bond	I-ptm
between	O
positions	O
24	B-residue_number
and	O
29	B-residue_number
helps	O
stabilize	O
the	O
β	B-structure_element
-	I-structure_element
hairpin	I-structure_element
,	O
it	O
does	O
not	O
alter	O
the	O
charge	O
or	O
substantially	O
change	O
the	O
hydrophobicity	O
of	O
the	O
Aβ17	B-protein
–	B-residue_range
36	I-residue_range
β	B-structure_element
-	I-structure_element
hairpin	I-structure_element
.	O

Crystallization	B-experimental_method
,	O
X	B-experimental_method
-	I-experimental_method
ray	I-experimental_method
Crystallographic	I-experimental_method
Data	I-experimental_method
Collection	I-experimental_method
,	O
Data	O
Processing	O
,	O
and	O
Structure	B-experimental_method
Determination	I-experimental_method
of	O
Peptides	B-mutant
2	I-mutant
and	I-mutant
4	I-mutant

Data	O
from	O
peptides	B-mutant
4	I-mutant
and	I-mutant
2	I-mutant
suitable	O
for	O
refinement	O
at	O
2	O
.	O
30	O
Å	O
were	O
obtained	O
from	O
the	O
diffractometer	O
;	O
data	O
from	O
peptide	B-mutant
2	I-mutant
suitable	O
for	O
refinement	O
at	O
1	O
.	O
90	O
Å	O
were	O
obtained	O
from	O
the	O
synchrotron	O
.	O

Peptide	B-mutant
2	I-mutant
assembles	O
into	O
oligomers	B-oligomeric_state
similar	O
in	O
morphology	O
to	O
those	O
formed	O
by	O
peptide	B-mutant
1	I-mutant
.	O

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

At	O
the	O
corners	O
of	O
the	O
trimer	B-oligomeric_state
,	O
the	O
pairs	O
of	O
β	B-structure_element
-	I-structure_element
hairpin	I-structure_element
monomers	B-oligomeric_state
form	O
four	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
:	O
two	O
between	O
the	O
main	O
chains	O
of	O
V18	B-residue_name_number
and	O
E22	B-residue_name_number
and	O
two	O
between	O
δOrn	B-structure_element
and	O
the	O
main	O
chain	O
of	O
C24	B-residue_name_number
(	O
Figure	O
3B	O
).	O

The	O
other	O
face	O
of	O
the	O
trimer	B-oligomeric_state
displays	O
a	O
smaller	O
hydrophobic	B-site
surface	I-site
,	O
which	O
includes	O
the	O
side	O
chains	O
of	O
residues	O
V18	B-residue_name_number
,	O
F20	B-residue_name_number
,	O
and	O
I31	B-residue_name_number
of	O
the	O
three	O
β	B-structure_element
-	I-structure_element
hairpins	I-structure_element
(	O
Figure	O
3D	O
).	O

Dodecamer	B-oligomeric_state

The	O
four	O
trimers	B-oligomeric_state
arrange	O
in	O
a	O
tetrahedral	B-protein_state
fashion	O
,	O
creating	O
a	O
central	B-site
cavity	I-site
inside	O
the	O
dodecamer	B-oligomeric_state
.	O
Because	O
each	O
trimer	B-oligomeric_state
is	O
triangular	B-protein_state
,	O
the	O
resulting	O
arrangement	O
resembles	O
an	O
octahedron	B-protein_state
.	O

Residues	O
L17	B-residue_name_number
,	O
L34	B-residue_name_number
,	O
and	O
V36	B-residue_name_number
are	O
shown	O
as	O
spheres	O
,	O
illustrating	O
the	O
hydrophobic	B-bond_interaction
packing	I-bond_interaction
that	O
occurs	O
at	O
the	O
six	O
vertices	O
of	O
the	O
dodecamer	B-oligomeric_state
.	O
(	O
D	O
)	O
Detailed	O
view	O
of	O
one	O
of	O
the	O
six	O
vertices	O
of	O
the	O
dodecamer	B-oligomeric_state
.	O

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

Although	O
Jeffamine	B-chemical
M	I-chemical
-	I-chemical
600	I-chemical
is	O
a	O
heterogeneous	O
mixture	O
with	O
varying	O
chain	O
lengths	O
and	O
stereochemistry	O
,	O
we	O
modeled	O
a	O
single	O
stereoisomer	O
with	O
nine	O
propylene	O
glycol	O
units	O
(	O
n	O
=	O
9	O
)	O
to	O
fit	O
the	O
electron	B-evidence
density	I-evidence
.	O

Annular	B-site
Pore	I-site

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

It	O
is	O
important	O
to	O
note	O
that	O
the	O
annular	B-site
pore	I-site
formed	O
by	O
peptide	B-mutant
2	I-mutant
is	O
not	O
a	O
discrete	O
unit	O
in	O
the	O
crystal	B-evidence
lattice	I-evidence
.	O

The	O
dodecamers	B-oligomeric_state
further	O
assemble	O
to	O
form	O
an	O
annular	B-site
pore	I-site
(	O
Figure	O
6	O
).	O

Monomeric	B-oligomeric_state
Aβ	B-protein
folds	O
to	O
form	O
a	O
β	B-structure_element
-	I-structure_element
hairpin	I-structure_element
in	O
which	O
the	O
hydrophobic	O
central	B-structure_element
and	O
C	B-structure_element
-	I-structure_element
terminal	I-structure_element
regions	I-structure_element
form	O
an	O
antiparallel	B-structure_element
β	I-structure_element
-	I-structure_element
sheet	I-structure_element
.	O

Four	O
triangular	B-protein_state
trimers	B-oligomeric_state
assemble	O
to	O
form	O
a	O
dodecamer	B-oligomeric_state
.	O

Five	O
dodecamers	B-oligomeric_state
assemble	O
to	O
form	O
an	O
annular	B-site
pore	I-site
.	O

These	O
criteria	O
have	O
been	O
used	O
to	O
classify	O
the	O
Aβ	B-protein
oligomers	B-oligomeric_state
that	O
accumulate	O
in	O
vivo	O
.	O

Aβ	B-protein
dimers	B-oligomeric_state
have	O
been	O
classified	O
as	O
fibrillar	B-protein_state
oligomers	B-oligomeric_state
,	O
whereas	O
Aβ	B-protein
trimers	B-oligomeric_state
,	O
Aβ	B-complex_assembly
*	I-complex_assembly
56	I-complex_assembly
,	O
and	O
APFs	B-complex_assembly
have	O
been	O
classified	O
as	O
nonfibrillar	B-protein_state
oligomers	B-oligomeric_state
.	O

The	O
hierarchical	O
assembly	O
of	O
peptide	B-mutant
2	I-mutant
is	O
consistent	O
with	O
this	O
model	O
;	O
and	O
the	O
trimer	B-oligomeric_state
,	O
dodecamer	B-oligomeric_state
,	O
and	O
annular	B-site
pore	I-site
formed	O
by	O
peptide	B-mutant
2	I-mutant
may	O
share	O
similarities	O
to	O
the	O
trimers	B-oligomeric_state
,	O
Aβ	B-complex_assembly
*	I-complex_assembly
56	I-complex_assembly
,	O
and	O
APFs	B-complex_assembly
observed	O
in	O
vivo	O
.	O

Annular	B-site
Pores	I-site
Formed	O
by	O
Aβ	B-protein
and	O
Peptide	B-mutant
2	I-mutant

This	O
mode	O
of	O
assembly	O
is	O
not	O
unique	O
to	O
Aβ	B-protein
.	O

We	O
believe	O
this	O
iterative	O
,	O
“	O
bottom	O
up	O
”	O
approach	O
of	O
identifying	O
the	O
minimal	O
modification	O
required	O
to	O
crystallize	B-experimental_method
Aβ	B-protein
peptides	O
will	O
ultimately	O
allow	O
larger	O
fragments	O
of	O
Aβ	B-protein
to	O
be	O
crystallized	B-experimental_method
,	O
thus	O
providing	O
greater	O
insights	O
into	O
the	O
structures	B-evidence
of	O
Aβ	B-protein
oligomers	B-oligomeric_state
.	O

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

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

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

However	O
,	O
the	O
agonist	O
activity	O
of	O
SERMs	B-protein_type
derives	O
from	O
activation	B-structure_element
function	I-structure_element
‐	I-structure_element
1	I-structure_element
(	O
AF	B-structure_element
‐	I-structure_element
1	I-structure_element
)—	O
a	O
coactivator	B-site
recruitment	I-site
site	I-site
located	O
in	O
the	O
AB	B-structure_element
domain	O
(	O
Berry	O
et	O
al	O
,	O
1990	O
;	O
Shang	O
&	O
Brown	O
,	O
2002	O
;	O
Abot	O
et	O
al	O
,	O
2013	O
).	O

The	O
simplest	O
response	O
model	O
for	O
ligand	O
‐	O
specific	O
proliferative	O
effects	O
is	O
a	O
linear	O
causality	O
model	O
,	O
where	O
the	O
degree	O
of	O
NCOA1	B-protein
/	I-protein
2	I-protein
/	I-protein
3	I-protein
recruitment	O
determines	O
GREB1	B-protein
expression	O
,	O
which	O
in	O
turn	O
drives	O
ligand	O
‐	O
specific	O
cell	O
proliferation	O
(	O
Fig	O
1D	O
).	O

OBHS	B-chemical
is	O
an	O
indirect	O
modulator	O
that	O
dislocates	O
the	O
h11	B-structure_element
C	O
‐	O
terminus	O
to	O
destabilize	O
the	O
h11	B-site
–	I-site
h12	I-site
interface	I-site
(	O
PDB	O
4ZN9	O
).	O

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

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

(	O
B	O
)	O
Ligand	O
class	B-experimental_method
analysis	I-experimental_method
of	O
the	O
L	B-experimental_method
‐	I-experimental_method
Luc	I-experimental_method
ERα	B-protein
‐	O
WT	B-protein_state
and	O
ERα	B-mutant
‐	I-mutant
ΔAB	I-mutant
activities	O
in	O
HepG2	O
cells	O
.	O

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

The	O
value	O
of	O
r	B-evidence
ranges	O
from	O
−	O
1	O
to	O
1	O
,	O
and	O
it	O
defines	O
the	O
extent	O
to	O
which	O
the	O
data	O
fit	O
a	O
straight	O
line	O
when	O
compounds	O
show	O
similar	O
agonist	O
/	O
antagonist	O
activity	O
profiles	O
between	O
cell	O
types	O
(	O
Fig	O
EV3A	O
).	O

This	O
cluster	O
includes	O
two	O
classes	O
of	O
direct	O
modulators	O
(	O
cyclofenil	B-chemical
‐	I-chemical
ASC	I-chemical
and	O
WAY	B-chemical
dimer	I-chemical
),	O
and	O
six	O
classes	O
of	O
indirect	O
modulators	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
2	I-chemical
and	O
S	B-chemical
‐	I-chemical
OBHS	I-chemical
‐	I-chemical
3	I-chemical
,	O
furan	B-chemical
,	O
and	O
WAY	B-chemical
‐	I-chemical
D	I-chemical
).	O

In	O
contrast	O
,	O
AF	B-structure_element
‐	I-structure_element
1	I-structure_element
was	O
a	O
determinant	O
of	O
signaling	O
specificity	O
for	O
scaffolds	O
in	O
cluster	O
2	O
.	O

For	O
ligands	O
in	O
cluster	O
3	O
,	O
we	O
could	O
not	O
eliminate	O
a	O
role	O
for	O
AF	B-structure_element
‐	I-structure_element
1	I-structure_element
in	O
determining	O
signaling	O
specificity	O
,	O
since	O
this	O
cluster	O
lacked	O
positively	O
correlated	O
activity	O
profiles	O
(	O
Fig	O
3C	O
),	O
and	O
deletion	B-experimental_method
of	I-experimental_method
the	O
AB	B-structure_element
or	O
F	B-structure_element
domain	O
rarely	O
induced	O
such	O
correlations	O
(	O
Fig	O
3D	O
),	O
except	O
for	O
A	B-chemical
‐	I-chemical
CD	I-chemical
and	O
OBHS	B-chemical
‐	I-chemical
ASC	I-chemical
analogs	O
,	O
where	O
deletion	B-experimental_method
of	I-experimental_method
the	O
AB	B-structure_element
domain	O
or	O
F	B-structure_element
domain	O
led	O
to	O
positive	O
correlations	O
with	O
E	B-experimental_method
‐	I-experimental_method
Luc	I-experimental_method
activity	O
and	O
/	O
or	O
GREB1	B-protein
levels	O
(	O
Fig	O
3D	O
lanes	O
13	O
and	O
18	O
).	O

To	O
determine	O
mechanisms	O
for	O
ligand	O
‐	O
dependent	O
control	O
of	O
breast	O
cancer	O
cell	O
proliferation	O
,	O
we	O
performed	O
linear	B-experimental_method
regression	I-experimental_method
analyses	I-experimental_method
across	O
the	O
19	O
scaffolds	O
using	O
MCF	O
‐	O
7	O
cell	O
proliferation	O
as	O
the	O
dependent	O
variable	O
,	O
and	O
the	O
other	O
activities	O
as	O
independent	O
variables	O
(	O
Fig	O
3F	O
).	O

The	O
lack	O
of	O
significant	O
predictors	O
for	O
OBHS	B-chemical
analogs	O
(	O
Fig	O
3F	O
lane	O
1	O
)	O
reflects	O
their	O
small	O
range	O
of	O
proliferative	O
effects	O
on	O
MCF	O
‐	O
7	O
cells	O
(	O
Fig	O
EV2I	O
).	O

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

3	B-chemical
,	I-chemical
4	I-chemical
‐	I-chemical
DTP	I-chemical
,	O
cyclofenil	B-chemical
,	O
3	B-chemical
,	I-chemical
4	I-chemical
‐	I-chemical
DTPD	I-chemical
,	O
and	O
imidazopyridine	B-chemical
analogs	O
had	O
NCOA1	B-protein
/	I-protein
3	I-protein
recruitment	O
profiles	O
that	O
predicted	O
their	O
proliferative	O
effects	O
,	O
without	O
determining	O
GREB1	B-protein
levels	O
(	O
Fig	O
3E	O
and	O
F	O
,	O
lanes	O
5	O
and	O
14	O
–	O
16	O
).	O

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

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

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

To	O
further	O
validate	O
this	O
approach	O
,	O
we	O
solved	B-experimental_method
the	O
structure	B-evidence
of	O
the	O
ERα	B-mutant
‐	I-mutant
Y537S	I-mutant
LBD	B-structure_element
in	B-protein_state
complex	I-protein_state
with	I-protein_state
diethylstilbestrol	B-chemical
(	O
DES	B-chemical
),	O
which	O
bound	O
identically	O
in	O
the	O
wild	B-protein_state
‐	I-protein_state
type	I-protein_state
and	O
ERα	B-mutant
‐	I-mutant
Y537S	I-mutant
LBDs	B-structure_element
,	O
demonstrating	O
again	O
that	O
this	O
surface	O
mutation	O
stabilizes	O
h12	B-structure_element
dynamics	O
to	O
facilitate	O
crystallization	O
without	O
changing	O
ligand	O
binding	O
(	O
Appendix	O
Fig	O
S1A	O
and	O
B	O
)	O
(	O
Nettles	O
et	O
al	O
,	O
2008	O
;	O
Bruning	O
et	O
al	O
,	O
2010	O
;	O
Delfosse	O
et	O
al	O
,	O
2012	O
).	O

Using	O
this	O
approach	O
,	O
we	O
solved	B-experimental_method
76	O
ERα	B-protein
LBD	B-structure_element
structures	B-evidence
in	O
the	O
active	B-protein_state
conformation	I-protein_state
and	O
bound	B-protein_state
to	I-protein_state
ligands	I-protein_state
studied	O
here	O
(	O
Appendix	O
Fig	O
S1C	O
).	O

The	O
indirect	O
modulator	O
scaffolds	O
in	O
cluster	O
1	O
did	O
not	O
show	O
cell	O
‐	O
specific	O
signaling	O
(	O
Fig	O
3C	O
),	O
but	O
shared	O
common	O
structural	O
perturbations	O
that	O
we	O
designed	O
to	O
modulate	O
h12	B-structure_element
dynamics	O
.	O

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

We	O
observed	O
that	O
the	O
OBHS	B-chemical
‐	I-chemical
N	I-chemical
analogs	O
displaced	O
h11	B-structure_element
along	O
a	O
vector	O
away	O
from	O
Leu354	B-residue_name_number
in	O
a	O
region	O
of	O
h3	B-structure_element
that	O
is	O
unaffected	O
by	O
the	O
ligands	O
,	O
and	O
toward	O
the	O
dimer	B-site
interface	I-site
.	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

The	O
h11	B-site
–	I-site
h12	I-site
interface	I-site
(	O
circled	O
)	O
includes	O
the	O
C	O
‐	O
terminal	O
part	O
of	O
h11	B-structure_element
.	O

Direct	O
modulators	O
like	O
tamoxifen	B-chemical
drive	O
AF	B-structure_element
‐	I-structure_element
1	I-structure_element
‐	O
dependent	O
cell	O
‐	O
specific	O
activity	O
by	O
completely	O
occluding	O
AF	B-structure_element
‐	I-structure_element
2	I-structure_element
,	O
but	O
it	O
is	O
not	O
known	O
how	O
indirect	O
modulators	O
produce	O
cell	O
‐	O
specific	O
ERα	B-protein
activity	O
.	O

The	O
2F	O
o	O
‐	O
F	O
c	O
electron	O
density	O
map	O
and	O
F	O
o	O
‐	O
F	O
c	O
difference	O
map	O
of	O
a	O
2	B-protein_state
,	I-protein_state
5	I-protein_state
‐	I-protein_state
DTP	I-protein_state
‐	I-protein_state
bound	I-protein_state
structure	B-evidence
(	O
PDB	O
5DRJ	O
)	O
were	O
contoured	O
at	O
1	O
.	O
0	O
sigma	O
and	O
±	O
3	O
.	O
0	O
sigma	O
,	O
respectively	O
.	O

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

The	O
shifts	O
in	O
h3	B-structure_element
suggest	O
these	O
compounds	O
are	O
positioned	O
to	O
alter	O
coregulator	O
preferences	O
.	O

Therefore	O
,	O
these	O
indirect	O
modulators	O
,	O
including	O
S	B-chemical
‐	I-chemical
OBHS	I-chemical
‐	I-chemical
2	I-chemical
,	O
S	B-chemical
‐	I-chemical
OBHS	I-chemical
‐	I-chemical
3	I-chemical
,	O
2	B-chemical
,	I-chemical
5	I-chemical
‐	I-chemical
DTP	I-chemical
,	O
and	O
3	B-chemical
,	I-chemical
4	I-chemical
‐	I-chemical
DTPD	I-chemical
analogs	O
—	O
all	O
of	O
which	O
show	O
cell	O
‐	O
specific	O
activity	O
profiles	O
—	O
induced	O
shifts	O
in	O
h3	B-structure_element
and	O
h12	B-structure_element
that	O
were	O
transmitted	O
to	O
the	O
coactivator	O
peptide	O
via	O
an	O
altered	O
AF	B-site
‐	I-site
2	I-site
surface	I-site
.	O

In	O
contrast	O
,	O
an	O
extended	O
side	O
chain	O
designed	O
to	O
directly	O
reposition	O
h12	B-structure_element
and	O
completely	O
disrupt	O
the	O
AF	B-site
‐	I-site
2	I-site
surface	I-site
results	O
in	O
cell	O
‐	O
specific	O
signaling	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
found	O
that	O
the	O
TOCA1	B-protein
HR1	B-structure_element
,	O
like	O
the	O
closely	O
related	O
CIP4	B-protein
HR1	B-structure_element
,	O
has	O
interesting	O
structural	O
features	O
that	O
are	O
not	O
observed	O
in	O
other	O
HR1	B-structure_element
domains	O
.	O

NMR	B-experimental_method
experiments	O
show	O
that	O
the	O
Cdc42	B-site
-	I-site
binding	I-site
domain	I-site
from	O
N	B-protein
-	I-protein
WASP	I-protein
is	O
able	O
to	O
displace	O
TOCA1	B-protein
HR1	B-structure_element
from	O
Cdc42	B-protein
,	O
whereas	O
the	O
N	B-protein
-	I-protein
WASP	I-protein
domain	O
but	O
not	O
the	O
TOCA1	B-protein
HR1	B-structure_element
domain	O
inhibits	O
actin	O
polymerization	O
.	O

This	O
suggests	O
that	O
TOCA1	B-protein
binding	O
to	O
Cdc42	B-protein
is	O
an	O
early	O
step	O
in	O
the	O
Cdc42	B-protein
-	O
dependent	O
pathways	O
that	O
govern	O
actin	O
dynamics	O
,	O
and	O
the	O
differential	O
binding	B-evidence
affinities	I-evidence
of	O
the	O
effectors	O
facilitate	O
a	O
handover	O
from	O
TOCA1	B-protein
to	O
N	B-protein
-	I-protein
WASP	I-protein
,	O
which	O
can	O
then	O
drive	O
recruitment	O
of	O
the	O
actin	O
-	O
modifying	O
machinery	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

In	O
the	O
active	B-protein_state
state	O
,	O
G	B-protein_type
proteins	I-protein_type
bind	O
to	O
an	O
array	O
of	O
downstream	O
effectors	O
,	O
through	O
which	O
they	O
exert	O
their	O
extensive	O
roles	O
within	O
the	O
cell	O
.	O

RhoA	B-protein
acts	O
to	O
rearrange	O
existing	O
actin	O
structures	O
to	O
form	O
stress	O
fibers	O
,	O
whereas	O
Rac1	B-protein
and	O
Cdc42	B-protein
promote	O
de	O
novo	O
actin	O
polymerization	O
to	O
form	O
lamellipodia	O
and	O
filopodia	O
,	O
respectively	O
.	O

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

The	O
TOCA1	B-protein
SH3	B-structure_element
domain	O
has	O
many	O
known	O
binding	O
partners	O
,	O
including	O
N	B-protein
-	I-protein
WASP	I-protein
and	O
dynamin	B-protein
.	O

The	O
HR1	B-structure_element
domain	O
has	O
been	O
directly	O
implicated	O
in	O
the	O
interaction	O
between	O
TOCA1	B-protein
and	O
Cdc42	B-protein
,	O
representing	O
the	O
first	O
Cdc42	B-protein
-	O
HR1	B-structure_element
domain	O
interaction	O
to	O
be	O
identified	O
.	O

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

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

Cdc42	B-protein
-	O
TOCA1	B-protein
Binding	O

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

The	O
Kd	B-evidence
values	O
derived	O
for	O
the	O
ACK	B-protein
GBD	B-structure_element
with	O
Cdc42Δ7	B-mutant
and	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
Cdc42	B-protein
were	O
0	O
.	O
032	O
±	O
0	O
.	O
01	O
and	O
0	O
.	O
011	O
±	O
0	O
.	O
01	O
μm	O
,	O
respectively	O
.	O

Other	O
G	B-protein_type
protein	I-protein_type
-	O
HR1	B-structure_element
domain	O
interactions	O
have	O
also	O
failed	O
to	O
show	O
heat	O
changes	O
in	O
our	O
hands	O
.	O
7	O
Infrared	B-experimental_method
interferometry	I-experimental_method
with	O
immobilized	B-protein_state
Cdc42	B-protein
was	O
also	O
attempted	O
but	O
was	O
unsuccessful	O
for	O
both	O
TOCA1	B-protein
HR1	B-structure_element
and	O
for	O
the	O
positive	O
control	O
,	O
ACK	B-protein
.	O

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

Free	B-protein_state
ACK	B-protein
competed	O
with	O
itself	O
with	O
an	O
affinity	B-evidence
of	O
32	O
nm	O
,	O
similar	O
to	O
the	O
value	O
obtained	O
by	O
direct	B-experimental_method
binding	I-experimental_method
of	O
23	O
nm	O
.	O

The	O
TOCA1	B-protein
HR1	B-structure_element
domain	O
also	O
fully	O
competed	O
with	O
the	O
GST	B-mutant
-	I-mutant
ACK	I-mutant
but	O
bound	B-protein_state
with	O
an	O
affinity	B-evidence
of	O
6	O
μm	O
(	O
Fig	O
.	O
1	O
,	O
B	O
and	O
C	O
),	O
in	O
agreement	O
with	O
the	O
low	O
affinity	B-evidence
observed	O
in	O
the	O
direct	B-experimental_method
binding	I-experimental_method
experiments	I-experimental_method
.	O

These	O
residues	O
are	O
not	O
generally	O
required	O
for	O
G	B-protein_type
protein	I-protein_type
-	O
effector	O
interactions	O
,	O
including	O
the	O
interaction	O
between	O
RhoA	B-protein
and	O
the	O
PRK1	B-protein
HR1a	B-structure_element
domain	O
.	O

In	O
contrast	O
,	O
the	O
C	O
terminus	O
of	O
Rac1	B-protein
contains	O
a	O
polybasic	O
sequence	O
,	O
which	O
is	O
crucial	O
for	O
Rac1	B-protein
binding	O
to	O
the	O
HR1b	B-structure_element
domain	O
from	O
PRK1	B-protein
.	O

This	O
construct	O
competed	O
with	O
GST	B-mutant
-	I-mutant
ACK	I-mutant
GBD	B-structure_element
to	O
give	O
a	O
similar	O
affinity	O
to	O
the	O
HR1	B-structure_element
domain	O
alone	B-protein_state
(	O
Kd	B-evidence
=	O
4	O
.	O
6	O
±	O
4	O
μm	O
;	O
Fig	O
.	O
2C	O
).	O

Domain	O
boundaries	O
are	O
derived	O
from	O
secondary	O
structure	O
predictions	O
;	O
B	O
,	O
binding	B-evidence
curves	I-evidence
derived	O
from	O
direct	B-experimental_method
binding	I-experimental_method
assays	I-experimental_method
,	O
in	O
which	O
the	O
indicated	O
concentrations	O
of	O
Cdc42Δ7Q61L	B-complex_assembly
·[	I-complex_assembly
3H	I-complex_assembly
]	I-complex_assembly
GTP	I-complex_assembly
were	O
incubated	B-experimental_method
with	O
30	O
nm	O
GST	B-mutant
-	I-mutant
ACK	I-mutant
or	O
His	B-protein_state
-	I-protein_state
tagged	I-protein_state
TOCA1	B-protein
constructs	O
,	O
as	O
indicated	O
,	O
in	O
SPAs	B-experimental_method
.	O

The	O
data	O
were	O
fitted	O
to	O
a	O
binding	B-evidence
isotherm	I-evidence
to	O
give	O
an	O
apparent	O
Kd	B-evidence
and	O
are	O
expressed	O
as	O
a	O
percentage	O
of	O
the	O
maximum	O
signal	O
.	O

The	O
structure	B-evidence
of	O
the	O
TOCA1	B-protein
HR1	B-structure_element
domain	O
.	O

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

Residues	O
with	O
significantly	O
affected	O
backbone	O
or	O
side	O
chain	O
chemical	O
shifts	O
when	O
Cdc42	B-protein_state
bound	I-protein_state
and	O
that	O
are	O
buried	O
are	O
colored	O
dark	O
blue	O
,	O
whereas	O
those	O
that	O
are	O
solvent	B-protein_state
-	I-protein_state
accessible	I-protein_state
are	O
colored	O
yellow	O
.	O

Side	O
chains	O
whose	O
CH	O
groups	O
disappeared	O
in	O
the	O
presence	B-protein_state
of	I-protein_state
Cdc42	B-protein
are	O
marked	O
on	O
the	O
graph	O
in	O
Fig	O
.	O
4B	O
with	O
green	O
asterisks	O
.	O

The	O
overall	O
CSP	B-experimental_method
was	O
calculated	O
for	O
each	O
residue	O
.	O

The	O
red	O
line	O
indicates	O
the	O
mean	O
CSP	B-experimental_method
,	O
plus	O
one	O
S	O
.	O
D	O
.	O
Residues	O
that	O
disappeared	O
in	O
the	O
presence	B-protein_state
of	I-protein_state
Cdc42	B-protein
were	O
assigned	O
a	O
CSP	B-experimental_method
of	O
0	O
.	O
1	O
and	O
are	O
indicated	O
with	O
open	O
bars	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

Modeling	O
the	O
Cdc42	B-complex_assembly
·	I-complex_assembly
TOCA1	I-complex_assembly
HR1	I-complex_assembly
Complex	O

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

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

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

A	O
,	O
the	O
model	O
of	O
the	O
Cdc42	B-complex_assembly
·	I-complex_assembly
TOCA1	I-complex_assembly
HR1	B-structure_element
domain	O
complex	O
overlaid	O
with	O
the	O
Cdc42	B-complex_assembly
-	I-complex_assembly
WASP	I-complex_assembly
structure	B-evidence
.	O

B	O
,	O
competition	B-experimental_method
SPA	I-experimental_method
experiments	O
carried	O
out	O
with	O
indicated	O
concentrations	O
of	O
the	O
N	B-protein
-	I-protein
WASP	I-protein
GBD	B-structure_element
construct	O
titrated	B-experimental_method
into	O
30	O
nm	O
GST	B-mutant
-	I-mutant
ACK	I-mutant
or	O
GST	B-mutant
-	I-mutant
WASP	I-mutant
GBD	B-structure_element
and	O
30	O
nm	O
Cdc42Δ7Q61L	B-complex_assembly
·[	I-complex_assembly
3H	I-complex_assembly
]	I-complex_assembly
GTP	I-complex_assembly
.	O

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

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

Endogenous	O
N	B-protein
-	I-protein
WASP	I-protein
is	O
present	O
at	O
∼	O
100	O
nm	O
in	O
Xenopus	B-taxonomy_domain
extracts	O
,	O
whereas	O
TOCA1	B-protein
is	O
present	O
at	O
a	O
10	O
-	O
fold	O
lower	O
concentration	O
than	O
N	B-protein
-	I-protein
WASP	I-protein
.	O

This	O
is	O
consistent	O
with	O
endogenous	B-protein_state
N	B-protein
-	I-protein
WASP	I-protein
,	O
activated	O
by	O
other	O
components	O
of	O
the	O
assay	O
,	O
outcompeting	O
the	O
TOCA1	B-protein
HR1	B-structure_element
domain	O
for	O
Cdc42	B-protein
binding	O
.	O

Fluorescence	B-evidence
curves	I-evidence
show	O
actin	O
polymerization	O
in	O
the	O
presence	B-protein_state
of	I-protein_state
increasing	B-experimental_method
concentrations	I-experimental_method
of	O
N	B-protein
-	I-protein
WASP	I-protein
GBD	B-structure_element
or	O
TOCA1	B-protein
HR1	B-structure_element
domain	O
as	O
indicated	O
.	O

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

The	O
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

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

This	O
region	O
lies	O
within	O
the	O
G	B-site
protein	I-site
-	I-site
binding	I-site
surface	I-site
of	O
all	O
of	O
the	O
HR1	B-structure_element
domains	O
(	O
Fig	O
.	O
4D	O
).	O

Many	O
of	O
these	O
residues	O
are	O
significantly	O
affected	O
in	O
the	O
presence	B-protein_state
of	I-protein_state
Cdc42	B-protein
,	O
so	O
it	O
is	O
likely	O
that	O
the	O
conformation	O
of	O
this	O
loop	B-structure_element
is	O
altered	O
in	O
the	O
Cdc42	B-protein
complex	O
.	O

These	O
observations	O
therefore	O
provide	O
a	O
molecular	O
mechanism	O
whereby	O
mutation	B-experimental_method
of	O
Met383	B-residue_name_number
-	O
Gly384	B-residue_name_number
-	O
Asp385	B-residue_name_number
to	O
Ile383	B-residue_name_number
-	O
Ser384	B-residue_name_number
-	O
Thr385	B-residue_name_number
abolishes	O
TOCA1	B-protein
binding	O
to	O
Cdc42	B-protein
.	O

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

Some	O
residues	O
that	O
are	O
affected	O
in	O
the	O
Cdc42	B-complex_assembly
·	I-complex_assembly
HR1TOCA1	I-complex_assembly
complex	O
but	O
do	O
not	O
correspond	O
to	O
contact	O
residues	O
of	O
RhoA	B-protein
or	O
Rac1	B-protein
(	O
Fig	O
.	O
6C	O
)	O
may	O
contact	O
HR1TOCA1	B-structure_element
directly	O
(	O
Fig	O
.	O
6D	O
).	O

The	O
weak	O
binding	O
prevented	O
detailed	O
structural	B-experimental_method
and	I-experimental_method
thermodynamic	I-experimental_method
studies	I-experimental_method
of	O
the	O
complex	O
.	O

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

As	O
such	O
,	O
the	O
ability	O
of	O
the	O
TOCA1	B-protein
HR1	B-structure_element
domain	O
to	O
bind	O
to	O
Cdc42	B-protein
(	O
a	O
close	O
relative	O
of	O
Rac1	B-protein
rather	O
than	O
RhoA	B-protein
)	O
fits	O
this	O
trend	O
.	O

The	O
low	O
affinity	O
of	O
the	O
HR1TOCA1	B-structure_element
-	O
Cdc42	B-protein
interaction	O
in	O
the	O
context	O
of	O
the	O
physiological	O
concentration	O
of	O
TOCA1	B-protein
in	O
Xenopus	B-taxonomy_domain
extracts	O
(∼	O
10	O
nm	O
)	O
suggests	O
that	O
binding	O
between	O
TOCA1	B-protein
and	O
Cdc42	B-protein
is	O
likely	O
to	O
occur	O
in	O
vivo	O
only	O
when	O
TOCA1	B-protein
is	O
at	O
high	O
local	O
concentrations	O
and	O
membrane	O
-	O
localized	O
and	O
therefore	O
in	O
close	O
proximity	O
to	O
activated	B-protein_state
Cdc42	B-protein
.	O

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

A	O
combination	O
of	O
allosteric	O
activation	O
by	O
PI	B-chemical
(	I-chemical
4	I-chemical
,	I-chemical
5	I-chemical
)	I-chemical
P2	I-chemical
,	O
activated	B-protein_state
Cdc42	B-protein
and	O
TOCA1	B-protein
,	O
and	O
oligomeric	O
activation	O
implemented	O
by	O
TOCA1	B-protein
would	O
lead	O
to	O
full	B-protein_state
activation	I-protein_state
of	O
N	B-protein
-	I-protein
WASP	I-protein
and	O
downstream	O
actin	O
polymerization	O
.	O

The	O
commonly	O
used	O
MGD	B-mutant
→	I-mutant
IST	I-mutant
(	O
Cdc42	B-protein_state
-	I-protein_state
binding	I-protein_state
deficient	I-protein_state
)	O
mutant	O
of	O
TOCA1	B-protein
has	O
a	O
reduced	O
ability	O
to	O
activate	O
the	O
N	B-complex_assembly
-	I-complex_assembly
WASP	I-complex_assembly
·	I-complex_assembly
WIP	I-complex_assembly
complex	O
,	O
further	O
indicating	O
the	O
importance	O
of	O
the	O
Cdc42	B-protein
-	O
HR1TOCA1	B-structure_element
interaction	O
prior	O
to	O
downstream	O
activation	O
of	O
N	B-protein
-	I-protein
WASP	I-protein
.	O

Step	O
1	O
,	O
TOCA1	B-protein
is	O
recruited	O
to	O
the	O
membrane	O
via	O
its	O
F	B-structure_element
-	I-structure_element
BAR	I-structure_element
domain	O
and	O
/	O
or	O
Cdc42	B-protein
interactions	O
.	O

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

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

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

Its	O
phosphorylation	B-ptm
by	O
the	O
AMPK	B-protein
homologue	O
SNF1	B-protein
results	O
in	O
strongly	O
reduced	O
ACC	B-protein_type
activity	O
.	O

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

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

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

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

CDC1	B-structure_element
/	O
CDC2	B-structure_element
share	O
a	O
common	O
fold	O
;	O
they	O
are	O
composed	O
of	O
six	B-structure_element
-	I-structure_element
stranded	I-structure_element
β	I-structure_element
-	I-structure_element
sheets	I-structure_element
flanked	O
on	O
one	O
side	O
by	O
two	O
long	B-structure_element
,	I-structure_element
bent	I-structure_element
helices	I-structure_element
inserted	O
between	O
strands	B-structure_element
β3	B-structure_element
/	I-structure_element
β4	I-structure_element
and	O
β4	B-structure_element
/	I-structure_element
β5	I-structure_element
.	O

On	O
the	O
basis	O
of	O
a	O
root	B-evidence
mean	I-evidence
square	I-evidence
deviation	I-evidence
of	O
main	O
chain	O
atom	O
positions	O
of	O
2	O
.	O
2	O
Å	O
,	O
CDC1	B-structure_element
/	O
CDC2	B-structure_element
are	O
structurally	O
more	O
closely	O
related	O
to	O
each	O
other	O
than	O
to	O
any	O
other	O
protein	O
(	O
Fig	O
.	O
1c	O
);	O
they	O
may	O
thus	O
have	O
evolved	O
by	O
duplication	O
.	O

Phosphorylated	B-protein_state
SceACC	B-protein
shows	O
only	O
residual	O
activity	O
(	O
kcat	B-evidence
=	O
0	O
.	O
4	O
±	O
0	O
.	O
2	O
s	O
−	O
1	O
,	O
s	O
.	O
d	O
.	O
based	O
on	O
five	O
replicate	O
measurements	O
),	O
which	O
increases	O
16	O
-	O
fold	O
(	O
kcat	B-evidence
=	O
6	O
.	O
5	O
±	O
0	O
.	O
3	O
s	O
−	O
1	O
)	O
after	O
dephosphorylation	O
with	O
λ	B-protein_type
protein	I-protein_type
phosphatase	I-protein_type
.	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

To	O
improve	B-experimental_method
crystallizability	I-experimental_method
,	O
we	O
generated	B-experimental_method
ΔBCCP	B-mutant
variants	I-mutant
of	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
ACC	B-protein_type
,	O
which	O
,	O
based	O
on	O
SAXS	B-experimental_method
analysis	I-experimental_method
,	O
preserve	O
properties	O
of	O
intact	B-protein_state
ACC	B-protein_type
(	O
Supplementary	O
Table	O
1	O
and	O
Supplementary	O
Fig	O
.	O
2a	O
–	O
c	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

Surprisingly	O
,	O
in	O
both	O
the	O
linear	B-protein_state
and	I-protein_state
U	I-protein_state
-	I-protein_state
shaped	I-protein_state
conformations	I-protein_state
,	O
the	O
approximate	O
distances	O
between	O
the	O
BC	B-structure_element
and	O
CT	B-structure_element
active	B-site
sites	I-site
would	O
remain	O
larger	O
than	O
110	O
Å	O
.	O
These	O
observed	O
distances	O
are	O
considerably	O
larger	O
than	O
in	O
static	B-protein_state
structures	B-evidence
of	O
any	O
other	O
related	O
biotin	B-protein_type
-	I-protein_type
dependent	I-protein_type
carboxylase	I-protein_type
.	O

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

A	O
second	B-structure_element
hinge	I-structure_element
can	O
be	O
identified	O
between	O
CDC1	B-structure_element
/	O
CDC2	B-structure_element
.	O

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

In	O
flACC	B-protein
,	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

Thus	O
,	O
in	O
accordance	O
with	O
the	O
results	O
presented	O
here	O
,	O
phosphorylation	B-ptm
of	O
Ser1157	B-residue_name_number
in	O
SceACC	B-protein
most	O
likely	O
limits	O
flexibility	O
in	O
the	O
CDC1	B-structure_element
/	I-structure_element
CDC2	I-structure_element
hinge	I-structure_element
such	O
that	O
activation	O
through	O
BC	B-structure_element
dimerization	O
is	O
not	O
possible	O
(	O
Fig	O
.	O
4d	O
),	O
which	O
however	O
does	O
not	O
exclude	O
intermolecular	O
dimerization	O
.	O

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

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

The	O
conformational	O
dynamics	O
of	O
fungal	B-taxonomy_domain
ACC	B-protein_type
.	O

CthCD	B-mutant
-	I-mutant
CT1	I-mutant
(	O
in	O
colour	O
)	O
serves	O
as	O
reference	O
,	O
the	O
compared	B-experimental_method
structures	I-experimental_method
(	O
as	O
indicated	O
,	O
numbers	O
after	O
construct	O
name	O
differentiate	O
between	O
individual	O
protomers	B-oligomeric_state
)	O
are	O
shown	O
in	O
grey	O
.	O

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

Here	O
we	O
solved	B-experimental_method
the	O
structures	B-evidence
of	O
SePSK	B-protein
and	O
AtXK	B-protein
-	I-protein
1	I-protein
in	O
both	O
their	O
apo	B-protein_state
forms	O
and	O
in	B-protein_state
complex	I-protein_state
with	I-protein_state
nucleotide	B-chemical
substrates	O
.	O

Phosphorylation	B-ptm
is	O
one	O
of	O
the	O
various	O
pivotal	O
modifications	O
of	O
carbohydrates	B-chemical
,	O
and	O
is	O
catalyzed	O
by	O
specific	O
sugar	B-protein_type
kinases	I-protein_type
.	O

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

Based	O
on	O
sequence	B-experimental_method
analysis	I-experimental_method
,	O
they	O
can	O
be	O
divided	O
into	O
four	O
families	O
,	O
namely	O
HSP	B-protein_type
70_NBD	I-protein_type
family	I-protein_type
,	O
FGGY	B-protein_type
family	I-protein_type
,	O
Mer_B	B-protein_type
like	I-protein_type
family	I-protein_type
and	O
Parm_like	B-protein_type
family	I-protein_type
.	O

Our	O
findings	O
provide	O
new	O
details	O
of	O
the	O
catalytic	O
mechanism	O
of	O
SePSK	B-protein
and	O
lay	O
the	O
foundation	O
for	O
future	O
studies	O
into	O
its	O
homologs	O
in	O
eukaryotes	B-taxonomy_domain
.	O

Apo	B-protein_state
-	O
SePSK	B-protein
contains	O
two	O
domains	O
referred	O
to	O
further	O
on	O
as	O
domain	B-structure_element
I	I-structure_element
and	O
domain	B-structure_element
II	I-structure_element
(	O
Fig	O
1A	O
).	O

2	B-residue_range
–	I-residue_range
228	I-residue_range
and	O
aa	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
green	O
,	O
β	B-structure_element
-	I-structure_element
sheet	I-structure_element
:	O
wheat	O
).	O

As	O
shown	O
in	O
Fig	O
2A	O
,	O
both	O
SePSK	B-protein
and	O
AtXK	B-protein
-	I-protein
1	I-protein
exhibited	O
ATP	B-chemical
hydrolysis	O
activity	O
.	O

(	O
A	O
)	O
The	O
ATP	B-chemical
hydrolysis	O
activity	O
of	O
SePSK	B-protein
and	O
AtXK	B-protein
-	I-protein
1	I-protein
.	O

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

SePSK	B-protein
and	O
AtXK	B-protein
-	I-protein
1	I-protein
possess	O
a	O
similar	O
ATP	B-site
binding	I-site
site	I-site

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

The	O
tail	O
of	O
AMP	B-chemical
-	I-chemical
PNP	I-chemical
points	O
to	O
the	O
hinge	B-structure_element
region	I-structure_element
of	O
SePSK	B-protein
,	O
and	O
its	O
α	O
-	O
phosphate	B-chemical
and	O
β	O
-	O
phosphate	B-chemical
groups	O
are	O
stabilized	O
by	O
Gly376	B-residue_name_number
and	O
Ser243	B-residue_name_number
,	O
respectively	O
.	O

The	O
four	O
α	B-structure_element
-	I-structure_element
helices	I-structure_element
(	O
α26	B-structure_element
,	O
α28	B-structure_element
,	O
α27	B-structure_element
and	O
α30	B-structure_element
)	O
are	O
labeled	O
in	O
red	O
.	O

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

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

The	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
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

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

Multiple	B-experimental_method
sequence	I-experimental_method
alignment	I-experimental_method
coupled	O
to	O
a	O
phylogenetic	B-experimental_method
analysis	I-experimental_method
reveals	O
that	O
certain	O
enterobacteria	B-taxonomy_domain
exert	O
evolutionary	O
pressure	O
on	O
the	O
lysine	B-protein_type
decarboxylase	I-protein_type
towards	O
the	O
cage	O
-	O
like	O
assembly	O
with	O
RavA	B-protein
,	O
implying	O
that	O
this	O
complex	O
may	O
have	O
an	O
important	O
function	O
under	O
particular	O
stress	O
conditions	O
.	O

These	O
amino	B-protein_type
acid	I-protein_type
decarboxylases	I-protein_type
are	O
therefore	O
called	O
acid	O
stress	O
inducible	B-protein_state
or	O
biodegradative	B-protein_state
to	O
distinguish	O
them	O
from	O
their	O
biosynthetic	B-protein_state
lysine	B-protein_type
and	I-protein_type
ornithine	I-protein_type
decarboxylase	I-protein_type
paralogs	O
catalysing	O
the	O
same	O
reaction	O
but	O
responsible	O
for	O
the	O
polyamine	B-chemical
production	O
at	O
neutral	B-protein_state
pH	I-protein_state
.	O

In	O
particular	O
,	O
the	O
inducible	B-protein_state
lysine	B-protein_type
decarboxylase	I-protein_type
LdcI	B-protein
(	O
or	O
CadA	B-protein
)	O
attracts	O
attention	O
due	O
to	O
its	O
broad	B-protein_state
pH	I-protein_state
range	I-protein_state
of	O
activity	O
and	O
its	O
capacity	O
to	O
promote	O
survival	O
and	O
growth	O
of	O
pathogenic	O
enterobacteria	B-taxonomy_domain
such	O
as	O
Salmonella	B-species
enterica	I-species
serovar	I-species
Typhimurium	I-species
,	O
Vibrio	B-species
cholerae	I-species
and	O
Vibrio	B-species
vulnificus	I-species
under	O
acidic	O
conditions	O
.	O

The	O
crystal	B-evidence
structure	I-evidence
of	O
the	O
E	B-species
.	I-species
coli	I-species
LdcI	B-protein
as	O
well	O
as	O
its	O
low	O
resolution	O
characterisation	O
by	O
electron	B-experimental_method
microscopy	I-experimental_method
(	O
EM	B-experimental_method
)	O
showed	O
that	O
it	O
is	O
a	O
decamer	B-oligomeric_state
made	O
of	O
two	O
pentameric	B-oligomeric_state
rings	B-structure_element
.	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

In	O
addition	O
,	O
we	O
improved	O
our	O
earlier	O
cryoEM	B-experimental_method
map	B-evidence
of	O
the	O
LdcI	B-complex_assembly
-	I-complex_assembly
LARA	I-complex_assembly
complex	O
from	O
7	O
.	O
5	O
Å	O
to	O
6	O
.	O
2	O
Å	O
resolution	O
(	O
Figs	O
1E	O
,	O
F	O
and	O
S3	O
).	O

Based	O
on	O
these	O
reconstructions	B-evidence
,	O
reliable	O
pseudoatomic	B-evidence
models	I-evidence
of	O
the	O
three	O
assemblies	O
were	O
obtained	O
by	O
flexible	B-experimental_method
fitting	I-experimental_method
of	I-experimental_method
either	O
the	O
crystal	B-evidence
structure	I-evidence
of	O
LdcIi	B-protein
or	O
a	O
derived	O
structural	B-experimental_method
homology	I-experimental_method
model	I-experimental_method
of	O
LdcC	B-protein
(	O
Table	O
S1	O
).	O

The	O
resolution	O
of	O
the	O
cryoEM	B-experimental_method
maps	B-evidence
does	O
not	O
allow	O
modeling	O
the	O
position	O
of	O
the	O
PLP	B-chemical
moiety	O
and	O
calls	O
for	O
caution	O
in	O
detailed	O
mechanistic	O
interpretations	O
in	O
terms	O
of	O
individual	O
amino	B-chemical
acids	I-chemical
.	O

While	O
differences	O
in	O
the	O
ppGpp	B-site
binding	I-site
site	I-site
could	O
indeed	O
be	O
visualized	O
(	O
Fig	O
.	O
S4	O
),	O
the	O
level	O
of	O
resolution	O
warns	O
against	O
speculations	O
about	O
their	O
significance	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

Inspection	O
of	O
the	O
superimposed	B-experimental_method
decameric	B-oligomeric_state
structures	B-evidence
(	O
Figs	O
2	O
and	O
S6	O
)	O
suggests	O
a	O
depiction	O
of	O
the	O
wing	B-structure_element
domains	I-structure_element
as	O
an	O
anchor	O
around	O
which	O
the	O
peripheral	O
CTDs	B-structure_element
swing	O
.	O

This	O
swinging	O
movement	O
seems	O
to	O
be	O
mediated	O
by	O
the	O
core	B-structure_element
domains	I-structure_element
and	O
is	O
accompanied	O
by	O
a	O
stretching	O
of	O
the	O
whole	O
LdcI	B-protein
subunits	B-structure_element
attracted	O
by	O
the	O
RavA	B-protein
magnets	O
.	O

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

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

Furthermore	O
,	O
the	O
recently	O
documented	O
interaction	O
of	O
both	O
LdcI	B-protein
and	O
RavA	B-protein
with	O
specific	O
subunits	B-structure_element
of	O
the	O
respiratory	B-protein_type
complex	I-protein_type
I	I-protein_type
,	O
together	O
with	O
the	O
unanticipated	O
link	O
between	O
RavA	B-protein
and	O
maturation	O
of	O
numerous	O
iron	B-protein_type
-	I-protein_type
sulfur	I-protein_type
proteins	I-protein_type
,	O
tend	O
to	O
suggest	O
an	O
additional	O
intriguing	O
function	O
for	O
this	O
3	O
.	O
5	O
MDa	O
assembly	O
.	O

Besides	O
,	O
the	O
structures	B-evidence
and	O
the	O
pseudoatomic	B-evidence
models	I-evidence
of	O
the	O
active	B-protein_state
ppGpp	B-protein_state
-	I-protein_state
free	I-protein_state
states	O
of	O
both	O
the	O
biodegradative	B-protein_state
and	O
the	O
biosynthetic	B-protein_state
E	B-species
.	I-species
coli	I-species
lysine	B-protein_type
decarboxylases	I-protein_type
offer	O
an	O
additional	O
tool	O
for	O
analysis	O
of	O
their	O
role	O
in	O
UPEC	B-species
infectivity	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

Numbering	O
as	O
in	O
E	B-species
.	I-species
coli	I-species
.	O

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

Here	O
,	O
the	O
authors	O
report	O
the	O
crystal	B-evidence
structures	I-evidence
of	O
closed	B-protein_state
states	O
of	O
Mep2	B-protein_type
proteins	I-protein_type
and	O
propose	O
a	O
model	O
for	O
their	O
regulation	O
by	B-experimental_method
comparing	I-experimental_method
them	I-experimental_method
with	I-experimental_method
the	O
open	B-protein_state
ammonium	B-protein_type
transporters	I-protein_type
of	O
bacteria	B-taxonomy_domain
.	O

A	O
common	O
feature	O
of	O
transceptors	B-protein_type
is	O
that	O
they	O
are	O
induced	O
when	O
cells	O
are	O
starved	O
for	O
their	O
substrate	O
.	O

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

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

Despite	O
different	O
crystal	O
packing	O
(	O
Supplementary	O
Table	O
1	O
),	O
the	O
two	O
CaMep2	B-protein
structures	B-evidence
are	O
identical	O
to	O
each	O
other	O
and	O
very	O
similar	O
to	O
ScMep2	B-protein
(	O
Cα	O
r	B-evidence
.	I-evidence
m	I-evidence
.	I-evidence
s	I-evidence
.	I-evidence
d	I-evidence
.	I-evidence

Unless	O
specifically	O
stated	O
,	O
the	O
drawn	O
conclusions	O
also	O
apply	O
to	O
ScMep2	B-protein
.	O

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

The	O
largest	O
differences	O
between	O
the	O
Mep2	B-protein
structures	B-evidence
and	O
the	O
other	O
known	O
ammonium	B-protein_type
transporter	I-protein_type
structures	B-evidence
are	O
located	O
on	O
the	O
intracellular	O
side	O
of	O
the	O
membrane	O
.	O

ICL1	B-structure_element
has	O
also	O
moved	O
inwards	O
relative	O
to	O
its	O
position	O
in	O
the	O
bacterial	B-taxonomy_domain
Amts	B-protein_type
.	O

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

Phosphorylation	B-site
target	I-site
site	I-site
is	O
at	O
the	O
periphery	O
of	O
Mep2	B-protein

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

We	O
obtained	O
a	O
similar	O
result	O
for	O
ammonium	O
uptake	O
by	O
the	O
446Δ	B-mutant
mutant	B-protein_state
(	O
Fig	O
.	O
3	O
),	O
supporting	O
the	O
data	O
from	O
Marini	O
et	O
al	O
.	O
We	O
then	O
constructed	B-experimental_method
and	I-experimental_method
purified	I-experimental_method
the	O
analogous	O
CaMep2	B-protein
442Δ	B-mutant
truncation	B-protein_state
mutant	I-protein_state
and	O
determined	B-experimental_method
the	O
crystal	B-evidence
structure	I-evidence
using	O
data	O
to	O
3	O
.	O
4	O
Å	O
resolution	O
.	O

The	O
structure	B-evidence
shows	O
that	O
removal	B-experimental_method
of	I-experimental_method
the	O
AI	B-structure_element
region	I-structure_element
markedly	O
increases	O
the	O
dynamics	O
of	O
the	O
cytoplasmic	B-structure_element
parts	I-structure_element
of	O
the	O
transporter	B-protein_type
.	O

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

The	O
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

The	O
movement	O
of	O
the	O
acidic	O
residues	O
away	O
from	O
Arg452	B-residue_name_number
and	O
Sep453	B-residue_name_number
is	O
more	O
pronounced	O
in	O
this	O
simulation	B-experimental_method
in	O
comparison	O
with	O
the	O
movement	O
away	O
from	O
Asp452	B-residue_name_number
and	O
Asp453	B-residue_name_number
in	O
the	O
DD	B-mutant
mutant	I-mutant
.	O

The	O
reason	O
why	O
similar	O
transporters	B-protein_type
such	O
as	O
A	B-species
.	I-species
thaliana	I-species
Amt	B-protein
-	I-protein
1	I-protein
;	I-protein
1	I-protein
and	O
Mep2	B-protein
are	O
regulated	O
in	O
opposite	O
ways	O
by	O
phosphorylation	B-ptm
(	O
inactivation	B-protein_state
in	O
plants	B-taxonomy_domain
and	O
activation	B-protein_state
in	O
fungi	B-taxonomy_domain
)	O
is	O
not	O
known	O
.	O

By	O
determining	O
the	O
first	O
structures	B-evidence
of	O
closed	B-protein_state
ammonium	B-protein_type
transporters	I-protein_type
and	O
comparing	B-experimental_method
those	O
structures	B-evidence
with	O
the	O
permanently	B-protein_state
open	I-protein_state
bacterial	B-taxonomy_domain
proteins	O
,	O
we	O
demonstrate	O
that	O
Mep2	B-protein_type
channel	B-site
closure	O
is	O
likely	O
due	O
to	O
movements	O
of	O
the	O
CTR	B-structure_element
and	O
ICL1	B-structure_element
and	O
ICL3	B-structure_element
.	O

Owing	O
to	O
the	O
crosstalk	O
between	O
monomers	B-oligomeric_state
,	O
a	O
single	O
phosphorylation	B-ptm
event	O
might	O
lead	O
to	O
opening	O
of	O
the	O
entire	O
trimer	B-oligomeric_state
,	O
although	O
this	O
has	O
not	O
yet	O
been	O
tested	O
(	O
Fig	O
.	O
9b	O
).	O

It	O
should	O
also	O
be	O
noted	O
that	O
the	O
tyrosine	B-residue_name
residue	O
interacting	O
with	O
His2	B-residue_name_number
is	O
highly	B-protein_state
conserved	I-protein_state
in	O
fungal	B-taxonomy_domain
Mep2	B-protein_type
orthologues	O
,	O
suggesting	O
that	O
the	O
Tyr	B-site
–	I-site
His2	I-site
hydrogen	I-site
bond	I-site
might	O
be	O
a	O
general	O
way	O
to	O
close	B-protein_state
Mep2	B-protein_type
proteins	I-protein_type
.	O

For	O
example	O
,	O
NH3	B-chemical
uniport	O
or	O
symport	O
of	O
NH3	B-chemical
/	O
H	B-chemical
+	I-chemical
might	O
result	O
in	O
changes	O
in	O
local	O
pH	O
,	O
but	O
NH4	B-chemical
+	I-chemical
uniport	O
might	O
not	O
,	O
and	O
this	O
difference	O
might	O
determine	O
signalling	O
.	O

(	O
a	O
)	O
Monomer	B-oligomeric_state
cartoon	O
models	O
viewed	O
from	O
the	O
side	O
for	O
(	O
left	O
)	O
A	O
.	O
fulgidus	O
Amt	B-protein
-	I-protein
1	I-protein
(	O
PDB	O
ID	O
2B2H	O
),	O
S	B-species
.	I-species
cerevisiae	I-species
Mep2	B-protein
(	O
middle	O
)	O
and	O
C	B-species
.	I-species
albicans	I-species
Mep2	B-protein
(	O
right	O
).	O

One	O
monomer	B-oligomeric_state
is	O
coloured	O
as	O
in	O
a	O
and	O
one	O
monomer	B-oligomeric_state
is	O
coloured	O
by	O
B	O
-	O
factor	O
(	O
blue	O
,	O
low	O
;	O
red	O
;	O
high	O
).	O

The	O
secondary	O
structure	O
elements	O
observed	O
for	O
CaMep2	B-protein
are	O
indicated	O
,	O
with	O
the	O
numbers	O
corresponding	O
to	O
the	O
centre	O
of	O
the	O
TM	B-structure_element
segment	I-structure_element
.	O

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

(	O
b	O
)	O
Overlay	B-experimental_method
of	O
the	O
CTRs	B-structure_element
of	O
ScMep2	B-protein
(	O
grey	O
)	O
and	O
CaMep2	B-protein
(	O
green	O
),	O
showing	O
the	O
similar	O
electronegative	O
environment	O
surrounding	O
the	O
phosphorylation	B-site
site	I-site
(	O
P	O
).	O

The	O
AI	B-structure_element
regions	I-structure_element
are	O
coloured	O
magenta	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

Schematic	O
model	O
for	O
phosphorylation	O
-	O
based	O
regulation	O
of	O
Mep2	B-protein
ammonium	O
transporters	O
.	O

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

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

About	O
half	O
of	O
the	O
structures	B-evidence
have	O
CDR	B-structure_element
H3	B-structure_element
conformations	O
similar	O
to	O
that	O
of	O
the	O
parent	O
;	O
the	O
others	O
diverge	O
significantly	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

Isotypes	O
IgG	B-protein
,	O
IgD	B-protein
and	O
IgA	B-protein
each	O
have	O
4	O
domains	O
,	O
one	O
variable	B-structure_element
(	O
V	B-structure_element
)	O
and	O
3	O
constant	B-structure_element
(	O
C	B-structure_element
)	O
domains	O
,	O
while	O
IgE	B-protein
and	O
IgM	B-protein
each	O
have	O
the	O
same	O
4	O
domains	O
along	O
with	O
an	O
additional	O
C	B-structure_element
domain	I-structure_element
.	O

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

In	O
antibodies	B-protein_type
,	O
the	O
heavy	B-structure_element
and	I-structure_element
light	I-structure_element
chain	I-structure_element
V	B-structure_element
domains	I-structure_element
pack	O
together	O
forming	O
the	O
antigen	B-site
combining	I-site
site	I-site
.	O

Later	O
studies	O
found	O
that	O
the	O
CDR	B-structure_element
loop	I-structure_element
length	O
is	O
the	O
primary	O
determining	O
factor	O
of	O
antigen	B-site
-	I-site
binding	I-site
site	I-site
topography	O
because	O
it	O
is	O
the	O
primary	O
factor	O
for	O
determining	O
a	O
canonical	O
structure	O
.	O

In	O
the	O
torso	B-structure_element
region	I-structure_element
,	O
2	O
primary	O
groups	O
could	O
be	O
identified	O
,	O
which	O
led	O
to	O
sequence	O
-	O
based	O
rules	O
that	O
can	O
predict	O
with	O
some	O
degree	O
of	O
reliability	O
the	O
conformation	O
of	O
the	O
stem	B-structure_element
region	I-structure_element
.	O

The	O
“	O
kinked	B-protein_state
”	O
or	O
“	O
bulged	B-protein_state
”	O
conformation	O
is	O
the	O
most	O
prevalent	O
,	O
but	O
an	O
“	O
extended	B-protein_state
”	O
or	O
“	O
non	B-protein_state
-	I-protein_state
bulged	I-protein_state
”	O
conformation	O
is	O
also	O
,	O
but	O
less	O
frequently	O
,	O
observed	O
.	O

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

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

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

Overall	O
the	O
structures	B-evidence
are	O
fairly	O
complete	O
,	O
and	O
,	O
as	O
can	O
be	O
expected	O
,	O
the	O
models	O
for	O
the	O
higher	O
resolution	O
structures	B-evidence
are	O
more	O
complete	O
than	O
those	O
for	O
the	O
lower	O
resolution	O
structures	B-evidence
(	O
Table	O
S1	O
).	O

CDRs	B-structure_element
are	O
defined	O
using	O
the	O
Dunbrack	O
convention	O
[	O
12	O
].	O

CDR	B-structure_element
H2	B-structure_element

Most	O
likely	O
this	O
is	O
the	O
result	O
of	O
interaction	O
of	O
CDR	B-structure_element
H2	B-structure_element
with	O
CDR	B-structure_element
H1	B-structure_element
,	O
namely	O
with	O
the	O
residue	O
at	O
position	O
33	B-residue_number
(	O
residue	O
11	O
of	O
13	O
in	O
CDR	B-structure_element
H1	B-structure_element
).	O

The	O
four	O
LC	B-structure_element
CDRs	B-structure_element
L1	B-structure_element
feature	O
3	O
different	O
lengths	O
(	O
11	B-residue_range
,	O
12	B-residue_range
and	O
17	B-residue_range
residues	O
)	O
having	O
a	O
total	O
of	O
4	O
different	O
canonical	O
structure	O
assignments	O
.	O

Two	O
structures	B-evidence
,	O
H3	B-complex_assembly
-	I-complex_assembly
53	I-complex_assembly
:	I-complex_assembly
L3	I-complex_assembly
-	I-complex_assembly
20	I-complex_assembly
and	O
H5	B-complex_assembly
-	I-complex_assembly
51	I-complex_assembly
:	I-complex_assembly
L3	I-complex_assembly
-	I-complex_assembly
20	I-complex_assembly
are	O
assigned	O
to	O
canonical	O
structure	O
L1	B-mutant
-	I-mutant
12	I-mutant
-	I-mutant
1	I-mutant
with	O
virtually	O
identical	O
backbone	O
conformations	O
.	O

As	O
with	O
CDR	B-structure_element
L2	B-structure_element
,	O
all	O
4	O
LCs	B-structure_element
have	O
CDR	B-structure_element
L3	B-structure_element
of	O
the	O
same	O
length	O
and	O
canonical	O
structure	B-evidence
,	O
L3	B-mutant
-	I-mutant
9	I-mutant
-	I-mutant
cis7	I-mutant
-	I-mutant
1	I-mutant
(	O
Table	O
2	O
).	O

CDR	B-structure_element
H3	B-structure_element
conformational	O
diversity	O

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

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

In	O
fact	O
,	O
it	O
is	O
the	O
only	O
Fab	B-structure_element
in	O
the	O
set	O
that	O
has	O
a	O
water	B-chemical
molecule	O
present	O
at	O
this	O
site	O
.	O

Three	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
23	I-complex_assembly
:	I-complex_assembly
L4	I-complex_assembly
-	I-complex_assembly
1	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
have	O
distinctive	O
conformations	O
.	O

VH	B-complex_assembly
:	I-complex_assembly
VL	I-complex_assembly
domain	O
packing	O

The	O
VH	B-structure_element
and	O
VL	B-structure_element
domains	O
have	O
a	O
β	B-structure_element
-	I-structure_element
sandwich	I-structure_element
structure	I-structure_element
(	O
also	O
often	O
referred	O
as	O
a	O
Greek	B-structure_element
key	I-structure_element
motif	I-structure_element
)	O
and	O
each	O
is	O
composed	O
of	O
a	O
4	B-structure_element
-	I-structure_element
stranded	I-structure_element
and	I-structure_element
a	I-structure_element
5	I-structure_element
-	I-structure_element
stranded	I-structure_element
antiparallel	I-structure_element
β	I-structure_element
-	I-structure_element
sheets	I-structure_element
.	O

They	O
include	O
:	O
1	O
)	O
a	O
bidentate	O
hydrogen	B-bond_interaction
bond	I-bond_interaction
between	O
L	B-structure_element
-	O
Gln38	B-residue_name_number
and	O
H	B-structure_element
-	O
Gln39	B-residue_name_number
;	O
2	O
)	O
H	B-structure_element
-	O
Leu45	B-residue_name_number
in	O
a	O
hydrophobic	B-site
pocket	I-site
between	O
L	B-structure_element
-	O
Phe98	B-residue_name_number
,	O
L	B-structure_element
-	O
Tyr87	B-residue_name_number
and	O
L	B-structure_element
-	O
Pro44	B-residue_name_number
;	O
3	O
)	O
L	B-structure_element
-	O
Pro44	B-residue_name_number
stacked	O
against	O
H	B-structure_element
-	O
Trp103	B-residue_name_number
;	O
and	O
4	O
)	O
L	B-structure_element
-	O
Ala43	B-residue_name_number
opposite	O
the	O
face	O
of	O
H	B-structure_element
-	O
Tyr91	B-residue_name_number
(	O
Fig	O
.	O
8	O
).	O

With	O
the	O
exception	O
of	O
L	B-structure_element
-	O
Ala43	B-residue_name_number
,	O
all	O
other	O
residues	O
are	O
conserved	O
in	O
human	B-species
germlines	O
.	O

The	O
first	O
approach	O
uses	O
ABangles	B-experimental_method
,	O
the	O
results	O
of	O
which	O
are	O
shown	O
in	O
Table	O
S2	O
.	O

For	O
structures	B-evidence
with	O
2	O
copies	O
of	O
the	O
Fab	B-structure_element
in	O
the	O
asymmetric	O
unit	O
,	O
only	O
one	O
structure	B-evidence
was	O
used	O
.	O

The	O
largest	O
deviations	O
in	O
the	O
tilt	B-evidence
angle	I-evidence
,	O
up	O
to	O
11	O
.	O
0	O
°,	O
are	O
found	O
for	O
2	O
structures	B-evidence
,	O
H1	B-complex_assembly
-	I-complex_assembly
69	I-complex_assembly
:	I-complex_assembly
L3	I-complex_assembly
-	I-complex_assembly
20	I-complex_assembly
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
that	O
stand	O
out	O
from	O
the	O
other	O
Fabs	B-structure_element
.	O

Two	O
examples	O
illustrating	O
large	O
(	O
10	O
.	O
5	O
°)	O
and	O
small	O
(	O
1	O
.	O
6	O
°)	O
differences	O
in	O
the	O
tilt	B-evidence
angles	I-evidence
are	O
shown	O
in	O
Fig	O
.	O
9	O
.	O

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

Parts	O
of	O
CDR	B-structure_element
H3	B-structure_element
main	O
chain	O
are	O
completely	O
disordered	B-protein_state
,	O
and	O
were	O
not	O
modeled	O
in	O
Fabs	B-structure_element
H5	B-complex_assembly
-	I-complex_assembly
51	I-complex_assembly
:	I-complex_assembly
L3	I-complex_assembly
-	I-complex_assembly
20	I-complex_assembly
and	O
H5	B-complex_assembly
-	I-complex_assembly
51	I-complex_assembly
:	I-complex_assembly
L3	I-complex_assembly
-	I-complex_assembly
11	I-complex_assembly
that	O
have	O
the	O
lowest	O
Tms	B-evidence
in	O
the	O
set	O
.	O

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

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

Note	O
that	O
most	O
of	O
the	O
VH	B-site
:	I-site
VL	I-site
interface	I-site
residues	O
are	O
invariant	O
;	O
therefore	O
,	O
significant	O
change	O
of	O
the	O
tilt	O
angle	O
must	O
come	O
with	O
a	O
penalty	O
in	O
free	O
energy	O
.	O

Comparison	O
of	O
the	O
CDR	B-structure_element
H3s	B-structure_element
reveals	O
a	O
large	O
set	O
of	O
variants	O
with	O
conformations	O
similar	O
to	O
the	O
parent	O
,	O
while	O
a	O
second	O
set	O
has	O
significant	O
conformational	O
variability	O
,	O
indicating	O
that	O
both	O
the	O
sequence	O
and	O
the	O
structural	O
context	O
define	O
the	O
CDR	B-structure_element
H3	B-structure_element
conformation	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

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

Importantly	O
,	O
even	O
though	O
we	O
only	O
labeled	O
a	O
subset	O
of	O
the	O
residues	O
in	O
the	O
flexible	B-protein_state
regions	O
of	O
the	O
substrate	O
with	O
iodine	B-chemical
,	O
the	O
residual	B-evidence
electron	I-evidence
density	I-evidence
can	O
provide	O
spatial	O
information	O
on	O
many	O
of	O
the	O
other	O
flexible	B-protein_state
residues	O
.	O

As	O
described	O
in	O
detail	O
below	O
,	O
we	O
developed	O
the	O
READ	B-experimental_method
method	O
to	O
uncover	O
the	O
ensemble	O
of	O
conformations	O
that	O
the	O
Spy	B-structure_element
-	I-structure_element
binding	I-structure_element
domain	I-structure_element
of	O
Im7	B-protein
(	O
i	O
.	O
e	O
.,	O
Im76	B-mutant
-	I-mutant
45	I-mutant
)	O
adopts	O
while	O
bound	B-protein_state
to	I-protein_state
Spy	B-protein
.	O

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

To	O
determine	O
the	O
structural	O
ensemble	O
that	O
Im76	B-mutant
-	I-mutant
45	I-mutant
adopts	O
while	O
bound	B-protein_state
to	I-protein_state
Spy	B-protein
,	O
we	O
combined	O
the	O
residual	B-evidence
electron	I-evidence
density	I-evidence
and	O
the	O
anomalous	B-evidence
signals	I-evidence
from	O
our	O
pI	B-chemical
-	I-chemical
Phe	I-chemical
substituted	O
Spy	B-complex_assembly
:	I-complex_assembly
Im76	I-complex_assembly
-	I-complex_assembly
45	I-complex_assembly
complexes	O
.	O

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

Prior	O
to	O
performing	O
the	O
selection	O
,	O
we	O
generated	O
a	O
large	O
and	O
diverse	O
pool	O
of	O
chaperone	B-protein_type
-	O
substrate	O
complexes	O
using	O
coarse	B-experimental_method
-	I-experimental_method
grained	I-experimental_method
MD	I-experimental_method
simulations	I-experimental_method
in	O
a	O
pseudo	B-experimental_method
-	I-experimental_method
crystal	I-experimental_method
environment	I-experimental_method
(	O
Fig	O
.	O
2	O
and	O
Supplementary	O
Fig	O
.	O
4	O
).	O

The	O
initial	O
conditions	O
of	O
the	O
binding	B-experimental_method
simulations	I-experimental_method
are	O
not	O
biased	O
toward	O
a	O
particular	O
conformation	O
of	O
the	O
substrate	O
or	O
any	O
specific	O
chaperone	B-protein_type
-	O
substrate	O
interaction	O
(	O
Online	O
Methods	O
).	O

Im76	B-mutant
-	I-mutant
45	I-mutant
binds	O
and	O
unbinds	O
to	O
Spy	B-protein
throughout	O
the	O
simulations	B-experimental_method
.	O

The	O
anomalous	B-evidence
scattering	I-evidence
portion	O
of	O
the	O
selection	O
uses	O
our	O
basic	O
knowledge	O
of	O
pI	B-chemical
-	I-chemical
Phe	I-chemical
geometry	O
:	O
the	O
iodine	B-chemical
is	O
separated	O
from	O
its	O
respective	O
Cα	O
atom	O
in	O
each	O
coarse	O
-	O
grained	O
conformer	O
by	O
6	O
.	O
5	O
Å	O
.	O
The	O
selection	O
then	O
picks	O
ensembles	O
that	O
best	O
reproduce	O
the	O
collection	O
of	O
iodine	B-chemical
anomalous	B-evidence
signals	I-evidence
.	O

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

Folding	O
and	O
interactions	O
of	O
Im7	B-protein
while	O
bound	B-protein_state
to	I-protein_state
Spy	B-protein

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

This	O
mixture	O
suggests	O
the	O
importance	O
of	O
both	O
electrostatic	O
and	O
hydrophobic	O
components	O
in	O
binding	O
the	O
Im76	B-mutant
-	I-mutant
45	I-mutant
ensemble	O
.	O

This	O
shift	O
in	O
contacts	O
is	O
likely	O
due	O
to	O
hydrophobic	O
residues	O
of	O
Im76	B-mutant
-	I-mutant
45	I-mutant
preferentially	O
forming	O
intra	O
-	O
molecular	O
contacts	O
upon	O
folding	O
(	O
i	O
.	O
e	O
.,	O
hydrophobic	O
collapse	O
),	O
effectively	O
removing	O
themselves	O
from	O
the	O
interaction	B-site
sites	I-site
.	O

The	O
diversity	O
of	O
conformations	O
and	O
binding	B-site
sites	I-site
observed	O
here	O
emphasizes	O
the	O
dynamic	O
and	O
heterogeneous	O
nature	O
of	O
the	O
chaperone	B-protein_type
-	O
substrate	O
ensemble	O
.	O

It	O
is	O
possible	O
that	O
this	O
twist	O
serves	O
to	O
increase	O
heterogeneity	O
in	O
Spy	B-protein
by	O
providing	O
more	O
binding	O
poses	O
.	O

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

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

This	O
substrate	O
-	O
chaperone	B-protein_type
ensemble	O
helps	O
accomplish	O
the	O
longstanding	O
goal	O
of	O
obtaining	O
a	O
detailed	O
view	O
of	O
how	O
a	O
chaperone	B-protein_type
aids	O
protein	O
folding	O
.	O

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

Nearly	O
all	O
Im76	B-mutant
-	I-mutant
45	I-mutant
residues	O
come	O
in	O
contact	O
with	O
Spy	B-protein
.	O

Unfolded	B-protein_state
substrate	O
conformers	O
interact	O
with	O
Spy	B-protein
through	O
both	O
hydrophobic	B-bond_interaction
and	I-bond_interaction
hydrophilic	I-bond_interaction
interactions	I-bond_interaction
,	O
whereas	O
the	O
binding	O
of	O
native	B-protein_state
-	I-protein_state
like	I-protein_state
states	O
is	O
mainly	O
hydrophilic	O
.	O

Previous	O
analysis	O
revealed	O
that	O
the	O
Super	O
Spy	B-protein
variants	B-protein_state
either	O
bound	B-protein_state
Im7	B-protein
tighter	O
than	O
WT	B-protein_state
Spy	B-protein
,	O
increased	O
chaperone	B-protein_type
flexibility	O
as	O
measured	O
via	O
H	B-experimental_method
/	I-experimental_method
D	I-experimental_method
exchange	I-experimental_method
,	O
or	O
both	O
.	O

Moreover	O
,	O
our	O
co	B-evidence
-	I-evidence
structure	I-evidence
suggests	O
that	O
the	O
L32P	B-mutant
substitution	O
,	O
which	O
increases	O
Spy	B-protein
’	O
s	O
flexibility	O
,	O
could	O
operate	O
by	O
unhinging	O
the	O
N	B-structure_element
-	I-structure_element
terminal	I-structure_element
helix	I-structure_element
and	O
effectively	O
expanding	O
the	O
size	O
of	O
the	O
disordered	B-protein_state
linker	B-structure_element
.	O

This	O
possibility	O
is	O
supported	O
by	O
the	O
Spy	B-protein
:	O
substrate	O
structures	B-evidence
,	O
in	O
which	O
the	O
linker	B-structure_element
region	I-structure_element
becomes	O
more	O
flexible	O
compared	O
to	O
the	O
apo	B-protein_state
state	O
(	O
Fig	O
.	O
6a	O
).	O

Instead	O
,	O
when	O
Spy	B-protein
is	O
bound	B-protein_state
to	I-protein_state
substrate	O
,	O
F115	B-residue_name_number
engages	O
in	O
close	O
CH	O
⋯	O
π	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
with	O
Tyr104	B-residue_name_number
(	O
Fig	O
.	O
6b	O
).	O

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

Spy	B-protein
is	O
depicted	O
as	O
a	O
gray	O
surface	O
and	O
the	O
Im76	B-mutant
-	I-mutant
45	I-mutant
conformer	O
is	O
shown	O
as	O
orange	O
balls	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

The	O
mechanism	O
of	O
NCX	B-protein_type
proteins	O
is	O
therefore	O
highly	O
likely	O
to	O
be	O
consistent	O
with	O
the	O
alternating	O
-	O
access	O
model	O
of	O
secondary	O
-	O
active	O
transport	O
.	O

With	O
similar	O
ion	O
exchange	O
properties	O
to	O
those	O
of	O
its	O
eukaryotic	B-taxonomy_domain
counterparts	O
,	O
NCX_Mj	B-protein
provides	O
a	O
compelling	O
model	O
system	O
to	O
investigate	O
the	O
structural	O
basis	O
for	O
the	O
specificity	O
,	O
stoichiometry	O
and	O
mechanism	O
of	O
the	O
ion	O
-	O
exchange	O
reaction	O
catalyzed	O
by	O
NCX	B-protein_type
.	O

The	O
assignment	O
of	O
the	O
four	O
central	B-site
binding	I-site
sites	I-site
identified	O
in	O
the	O
previously	O
reported	O
NCX_Mj	B-protein
structure	B-evidence
was	O
hampered	O
by	O
the	O
presence	O
of	O
both	O
Na	B-chemical
+	I-chemical
and	O
Ca2	B-chemical
+	I-chemical
in	O
the	O
protein	O
crystals	B-evidence
.	O

First	O
,	O
the	O
electron	B-evidence
density	I-evidence
at	O
Smid	B-site
does	O
not	O
depend	O
significantly	O
on	O
the	O
Na	B-chemical
+	I-chemical
concentration	O
.	O

This	O
structurally	O
-	O
derived	O
Na	B-evidence
+	I-evidence
affinity	I-evidence
agrees	O
well	O
with	O
the	O
external	O
Na	B-chemical
+	I-chemical
concentration	O
required	O
for	O
NCX	B-protein_type
activation	O
in	O
eukaryotes	B-taxonomy_domain
.	O

Similarly	O
to	O
Sr2	B-chemical
+,	I-chemical
Ca2	B-chemical
+	I-chemical
binds	O
with	O
low	O
affinity	B-evidence
to	O
outward	B-protein_state
-	I-protein_state
facing	I-protein_state
NCX_Mj	B-protein
and	O
can	O
be	O
readily	O
displaced	O
by	O
Na	B-chemical
+	I-chemical
(	O
Supplementary	O
Note	O
1	O
and	O
Supplementary	O
Fig	O
.	O
2c	O
).	O

This	O
finding	O
is	O
consistent	O
with	O
physiological	B-evidence
and	I-evidence
biochemical	I-evidence
data	I-evidence
for	O
both	O
eukaryotic	B-taxonomy_domain
NCX	B-protein_type
and	O
NCX_Mj	B-protein
indicating	O
that	O
the	O
apparent	O
Ca2	B-evidence
+	I-evidence
affinity	I-evidence
is	O
much	O
lower	O
on	O
the	O
extracellular	O
than	O
the	O
cytoplasmic	O
side	O
.	O

We	O
were	O
able	O
to	O
determine	O
an	O
apo	B-protein_state
-	O
state	O
structure	B-evidence
of	O
NCX_Mj	B-protein
,	O
by	O
crystallizing	B-experimental_method
the	O
protein	O
at	O
lower	B-protein_state
pH	I-protein_state
and	O
in	O
the	O
absence	B-protein_state
of	I-protein_state
Na	B-chemical
+	I-chemical
(	O
Methods	O
).	O

To	O
examine	O
this	O
central	O
question	O
,	O
we	O
sought	O
to	O
characterize	O
the	O
conformational	B-evidence
free	I-evidence
-	I-evidence
energy	I-evidence
landscape	I-evidence
of	O
NCX_Mj	B-protein
and	O
to	O
examine	O
its	O
dependence	O
on	O
the	O
ion	O
-	O
occupancy	O
state	O
,	O
using	O
molecular	B-experimental_method
dynamics	I-experimental_method
(	O
MD	B-experimental_method
)	O
simulations	B-experimental_method
.	O

This	O
computational	O
analysis	O
was	O
based	O
solely	O
on	O
the	O
published	O
structure	B-evidence
of	O
NCX_Mj	B-protein
,	O
independently	O
of	O
the	O
crystallographic	B-experimental_method
studies	I-experimental_method
described	O
above	O
.	O

These	O
initial	O
simulations	B-experimental_method
revealed	O
noticeable	O
changes	O
in	O
the	O
transporter	B-protein_type
,	O
consistent	O
with	O
those	O
observed	O
in	O
the	O
new	O
crystal	B-evidence
structures	I-evidence
.	O

The	O
most	O
noticeable	O
is	O
an	O
increased	O
separation	O
between	O
TM7	B-structure_element
and	O
TM2	B-structure_element
(	O
Fig	O
.	O
4f	O
),	O
previously	O
brought	O
together	O
by	O
concurrent	O
backbone	O
interactions	O
with	O
the	O
Na	B-chemical
+	I-chemical
ion	O
at	O
SCa	B-site
(	O
Fig	O
.	O
4d	O
-	O
e	O
).	O

TM1	B-structure_element
and	O
TM6	B-structure_element
also	O
slide	O
further	O
towards	O
the	O
membrane	O
center	O
,	O
relative	O
to	O
the	O
outward	B-protein_state
-	I-protein_state
occluded	I-protein_state
state	O
(	O
Fig	O
.	O
4c	O
).	O

To	O
more	O
rigorously	O
characterize	O
the	O
influence	O
of	O
the	O
ion	O
-	O
occupancy	O
state	O
on	O
the	O
conformational	O
dynamics	O
of	O
the	O
exchanger	B-protein_type
,	O
we	O
carried	O
out	O
a	O
series	O
of	O
enhanced	O
-	O
sampling	O
MD	B-experimental_method
calculations	I-experimental_method
designed	O
to	O
reversibly	O
simulate	O
the	O
transition	O
between	O
the	O
outward	B-protein_state
-	I-protein_state
occluded	I-protein_state
and	O
fully	B-protein_state
outward	I-protein_state
-	I-protein_state
open	I-protein_state
states	O
,	O
and	O
thus	O
quantify	O
the	O
free	B-evidence
-	I-evidence
energy	I-evidence
landscape	I-evidence
encompassing	O
these	O
states	O
(	O
Methods	O
).	O

It	O
is	O
however	O
also	O
non	O
-	O
trivial	O
:	O
antiporters	B-protein_type
,	O
for	O
example	O
,	O
do	O
not	O
undergo	O
the	O
alternating	O
-	O
access	O
transition	O
without	O
a	O
cargo	O
,	O
but	O
this	O
is	O
precisely	O
how	O
membrane	B-protein_type
symporters	I-protein_type
reset	O
their	O
transport	O
cycles	O
.	O

Similarly	O
puzzling	O
is	O
that	O
a	O
given	O
antiporter	B-protein_type
will	O
undergo	O
this	O
transition	O
upon	O
recognition	O
of	O
substrates	O
of	O
different	O
charge	O
,	O
size	O
and	O
number	O
.	O

The	O
internal	O
symmetry	O
of	O
outward	B-protein_state
-	I-protein_state
facing	I-protein_state
NCX_Mj	B-protein
and	O
the	O
inward	B-protein_state
-	I-protein_state
facing	I-protein_state
crystal	B-evidence
structures	I-evidence
of	O
several	O
Ca2	B-protein_type
+/	I-protein_type
H	I-protein_type
+	I-protein_type
exchangers	I-protein_type
indicate	O
that	O
the	O
alternating	O
-	O
access	O
mechanism	O
of	O
NCX	B-protein_type
proteins	O
entails	O
a	O
sliding	O
motion	O
of	O
TM1	B-structure_element
and	O
TM6	B-structure_element
relative	O
to	O
the	O
rest	O
of	O
the	O
transporter	B-protein_type
.	O

Indeed	O
,	O
we	O
show	O
that	O
it	O
is	O
the	O
presence	O
or	O
absence	O
of	O
the	O
occluded	B-protein_state
state	O
in	O
this	O
landscape	O
that	O
explains	O
the	O
antiport	O
function	O
of	O
NCX_Mj	B-protein
and	O
its	O
3Na	O
+:	B-chemical
1Ca2	O
+	B-chemical
stoichiometry	O
.	O

Consistent	O
with	O
that	O
finding	O
,	O
mutations	O
that	O
have	O
been	O
shown	O
to	O
inactivate	O
or	O
diminish	O
the	O
transport	O
activity	O
of	O
NCX_Mj	B-protein
and	O
cardiac	O
NCX	B-protein_type
perfectly	O
map	O
to	O
the	O
first	O
ion	O
-	O
coordination	O
shell	O
in	O
our	O
NCX_Mj	B-protein
structures	B-evidence
(	O
Supplementary	O
Fig	O
.	O
4c	O
-	O
d	O
).	O

The	O
Sext	B-site
site	O
,	O
by	O
contrast	O
,	O
might	O
be	O
thought	O
as	O
an	O
activation	B-site
site	I-site
for	O
inward	O
Na	B-chemical
+	I-chemical
translocation	O
,	O
since	O
this	O
is	O
where	O
the	O
third	O
Na	B-chemical
+	I-chemical
ion	O
binds	O
at	O
high	O
Na	B-chemical
+	I-chemical
concentration	O
,	O
enabling	O
the	O
transition	O
to	O
the	O
occluded	B-protein_state
state	O
.	O

Lastly	O
,	O
our	O
theory	O
that	O
occlusion	O
of	O
NCX_Mj	B-protein
is	O
selectively	O
induced	O
upon	O
Ca2	B-chemical
+	I-chemical
or	O
Na	B-chemical
+	I-chemical
recognition	O
is	O
consonant	O
with	O
a	O
recent	O
analysis	O
of	O
the	O
rate	O
of	O
hydrogen	B-experimental_method
-	I-experimental_method
deuterium	I-experimental_method
exchange	I-experimental_method
(	O
HDX	B-experimental_method
)	O
in	O
NCX_Mj	B-protein
,	O
in	O
the	O
presence	B-protein_state
or	O
absence	B-protein_state
of	I-protein_state
these	O
ions	O
,	O
in	O
conditions	O
that	O
favor	O
outward	B-protein_state
-	I-protein_state
facing	I-protein_state
conformations	O
.	O

Specifically	O
,	O
saturating	O
amounts	O
of	O
Ca2	B-chemical
+	I-chemical
or	O
Na	B-chemical
+	I-chemical
resulted	O
in	O
a	O
noticeable	O
slowdown	O
in	O
the	O
HDX	B-evidence
rate	I-evidence
for	O
extracellular	O
portions	O
of	O
the	O
α	B-structure_element
-	I-structure_element
repeat	I-structure_element
helices	I-structure_element
.	O

We	O
interpret	O
these	O
observations	O
as	O
reflecting	O
that	O
the	O
solvent	O
accessibility	O
of	O
the	O
protein	O
interior	O
is	O
diminished	O
upon	O
ion	O
recognition	O
,	O
consistent	O
with	O
our	O
finding	O
that	O
opening	O
and	O
closing	O
of	O
extracellular	O
aqueous	O
pathways	O
to	O
the	O
ion	B-site
-	I-site
binding	I-site
sites	I-site
depend	O
on	O
ion	O
occupancy	O
state	O
.	O

Our	O
data	O
would	O
also	O
explain	O
the	O
observation	O
that	O
the	O
reduction	O
in	O
the	O
HDX	B-evidence
rate	I-evidence
is	O
comparable	O
for	O
Na	B-chemical
+	I-chemical
and	O
Ca2	B-chemical
+,	I-chemical
as	O
well	O
as	O
the	O
finding	O
that	O
the	O
degree	O
of	O
deuterium	O
incorporation	O
remains	O
non	O
-	O
negligible	O
even	O
under	O
saturating	O
ion	O
concentrations	O
.	O

(	O
c	O
)	O
Close	O
-	O
up	O
view	O
of	O
the	O
Na	B-site
+-	I-site
binding	I-site
sites	I-site
.	O

Residues	O
surrounding	O
this	O
site	O
are	O
also	O
indicated	O
;	O
note	O
A206	B-residue_name_number
(	O
labeled	O
in	O
red	O
)	O
coordinates	B-bond_interaction
Na	B-chemical
+	I-chemical
at	O
Sext	B-site
via	O
its	O
backbone	O
carbonyl	O
oxygen	O
.	O

Divalent	O
cation	O
binding	O
and	O
apo	B-protein_state
structure	B-evidence
of	O
NCX_Mj	B-protein
.	O
(	O
a	O
)	O
A	O
single	O
Sr2	B-chemical
+	I-chemical
(	O
dark	O
blue	O
sphere	O
)	O
binds	O
at	O
SCa	B-site
in	O
crystals	B-experimental_method
titrated	I-experimental_method
with	O
10	O
mM	O
Sr2	B-chemical
+	I-chemical
and	O
2	O
.	O
5	O
mM	O
Na	B-chemical
+	I-chemical
(	O
see	O
also	O
Supplementary	O
Fig	O
.	O
2	O
).	O

There	O
are	O
no	O
significant	O
changes	O
in	O
the	O
side	O
-	O
chains	O
involved	O
in	O
ion	O
coordination	O
,	O
relative	O
to	O
the	O
Na	B-protein_state
+-	I-protein_state
bound	I-protein_state
state	O
.	O

The	O
relative	O
occupancies	O
are	O
55	O
%	O
and	O
45	O
%,	O
respectively	O
.	O
(	O
c	O
)	O
Superimposition	B-experimental_method
of	O
NCX_Mj	B-protein
structures	B-evidence
obtained	O
at	O
low	O
Na	B-chemical
+	I-chemical
concentration	O
(	O
10	O
mM	O
)	O
and	O
pH	O
6	O
.	O
5	O
(	O
brown	O
)	O
and	O
in	O
the	O
absence	B-protein_state
of	I-protein_state
Na	B-chemical
+	I-chemical
and	O
pH	B-protein_state
4	I-protein_state
(	O
light	O
green	O
),	O
referred	O
to	O
as	O
apo	B-protein_state
state	O
.	O
(	O
d	O
)	O
Close	O
-	O
up	O
view	O
of	O
the	O
ion	B-site
-	I-site
binding	I-site
sites	I-site
in	O
the	O
apo	B-protein_state
(	O
or	O
high	B-protein_state
H	I-protein_state
+)	I-protein_state
state	O
.	O

(	O
a	O
)	O
Representative	O
simulation	B-experimental_method
snapshots	O
of	O
NCX_Mj	B-protein
(	O
Methods	O
)	O
with	O
Na	B-chemical
+	I-chemical
bound	B-protein_state
at	I-protein_state
Sext	B-site
,	O
SCa	B-site
and	O
Sint	B-site
(	O
orange	O
cartoons	O
,	O
green	O
spheres	O
)	O
and	O
with	O
Na	B-chemical
+	I-chemical
bound	B-protein_state
only	I-protein_state
at	I-protein_state
SCa	B-site
and	O
Sint	B-site
(	O
marine	O
cartoons	O
,	O
yellow	O
spheres	O
)	O
(	O
b	O
)	O
Close	O
-	O
up	O
of	O
the	O
backbone	O
of	O
the	O
N	B-structure_element
-	I-structure_element
terminal	I-structure_element
half	I-structure_element
of	O
TM7	B-structure_element
(	O
TM7ab	B-structure_element
),	O
in	O
the	O
same	O
Na	B-chemical
+	I-chemical
occupancy	O
states	O
depicted	O
in	O
(	O
a	O
).	O

(	O
g	O
)	O
Probability	B-evidence
distributions	I-evidence
of	O
an	O
analytical	O
descriptor	O
of	O
the	O
backbone	O
hydrogen	B-bond_interaction
-	I-bond_interaction
bonding	I-bond_interaction
pattern	O
in	O
TM7ab	B-structure_element
(	O
Eq	O
.	O
2	O
).	O
(	O
h	O
)	O
Mean	O
value	O
(	O
with	O
standard	O
deviation	O
)	O
of	O
a	O
quantitative	O
descriptor	O
of	O
the	O
solvent	O
accessibility	O
of	O
the	O
Sext	B-site
site	O
(	O
Eq	O
.	O
1	O
).	O
(	O
i	O
)	O
Mean	O
value	O
(	O
with	O
standard	O
deviation	O
)	O
of	O
a	O
quantitative	O
descriptor	O
of	O
the	O
solvent	O
accessibility	O
of	O
the	O
SCa	B-site
site	O
(	O
Eq	O
.	O
1	O
).	O

The	O
free	B-evidence
energy	I-evidence
is	O
plotted	O
as	O
a	O
function	O
of	O
two	O
coordinates	O
,	O
each	O
describing	O
the	O
degree	O
of	O
opening	O
of	O
the	O
aqueous	B-site
channels	I-site
leading	O
to	O
the	O
Sext	B-site
and	O
SCa	B-site
sites	O
,	O
respectively	O
(	O
see	O
Methods	O
).	O

(	O
b	O
)	O
Density	B-evidence
isosurfaces	I-evidence
for	O
water	B-chemical
molecules	O
within	O
12	O
Å	O
of	O
the	O
ion	B-site
-	I-site
binding	I-site
region	I-site
(	O
grey	O
volumes	O
),	O
for	O
each	O
of	O
the	O
major	O
conformational	B-evidence
free	I-evidence
-	I-evidence
energy	I-evidence
minima	I-evidence
in	O
each	O
ion	O
-	O
occupancy	O
state	O
.	O

Na	B-chemical
+	I-chemical
ions	O
are	O
shown	O
as	O
green	O
spheres	O
.	O

Black	O
circles	O
map	O
the	O
crystal	B-evidence
structures	I-evidence
obtained	O
at	O
high	O
Ca2	B-chemical
+	I-chemical
concentration	O
and	O
at	O
low	B-protein_state
pH	I-protein_state
(	O
or	O
high	B-protein_state
H	I-protein_state
+)	I-protein_state
reported	O
in	O
this	O
study	O
.	O

(	O
b	O
)	O
Water	B-evidence
-	I-evidence
density	I-evidence
isosurfaces	I-evidence
analogous	O
to	O
those	O
in	O
Fig	O
.	O
5	O
are	O
shown	O
for	O
each	O
of	O
the	O
major	O
conformational	O
free	B-evidence
-	I-evidence
energy	I-evidence
minima	I-evidence
in	O
the	O
free	B-evidence
-	I-evidence
energy	I-evidence
maps	I-evidence
.	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

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

In	O
turn	O
,	O
the	O
ternary	B-complex_assembly
complex	I-complex_assembly
of	O
U2AF65	B-protein
with	O
SF1	B-protein
and	O
U2AF35	B-protein
identifies	O
the	O
surrounding	O
BPS	B-site
and	O
3	B-site
′	I-site
splice	I-site
site	I-site
junctions	O
.	O

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

By	O
sequential	B-experimental_method
boot	I-experimental_method
strapping	I-experimental_method
(	O
Methods	O
),	O
we	O
optimized	O
the	O
oligonucleotide	B-chemical
length	O
,	O
the	O
position	O
of	O
a	O
Br	B-chemical
-	I-chemical
dU	I-chemical
,	O
and	O
the	O
identity	O
of	O
the	O
terminal	O
nucleotide	B-chemical
(	O
rU	B-residue_name
,	O
dU	B-residue_name
and	O
rC	B-residue_name
)	O
to	O
achieve	O
full	O
views	O
of	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
bound	B-protein_state
to	I-protein_state
contiguous	B-structure_element
Py	B-chemical
tracts	I-chemical
at	O
up	O
to	O
1	O
.	O
5	O
Å	O
resolution	O
.	O

We	O
compare	O
the	O
global	O
conformation	O
of	O
the	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
structures	B-evidence
with	O
the	O
prior	O
dU2AF651	B-mutant
,	I-mutant
2	I-mutant
crystal	B-evidence
structure	I-evidence
and	O
U2AF651	B-mutant
,	I-mutant
2	I-mutant
NMR	B-experimental_method
structure	B-evidence
in	O
the	O
Supplementary	O
Discussion	O
and	O
Supplementary	O
Fig	O
.	O
2	O
.	O

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

In	O
the	O
C	O
-	O
terminal	O
β	B-structure_element
-	I-structure_element
strand	I-structure_element
of	O
RRM1	B-structure_element
,	O
the	O
side	O
chains	O
of	O
K225	B-residue_name_number
and	O
R227	B-residue_name_number
donate	O
additional	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
to	O
the	O
rU5	B-residue_name_number
-	O
O2	O
lone	O
pair	O
electrons	O
.	O

We	O
tested	B-experimental_method
the	I-experimental_method
contribution	I-experimental_method
of	O
the	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
interactions	O
with	O
the	O
new	O
central	O
nucleotide	B-chemical
to	O
Py	B-evidence
-	I-evidence
tract	I-evidence
affinity	I-evidence
(	O
Fig	O
.	O
3i	O
;	O
Supplementary	O
Fig	O
.	O
4a	O
,	O
b	O
).	O

U2AF65	B-protein
RRM	B-structure_element
extensions	I-structure_element
interact	O
with	O
the	O
Py	B-chemical
tract	I-chemical

Indirectly	O
,	O
the	O
additional	O
contacts	O
with	O
the	O
third	B-residue_number
nucleotide	B-chemical
shift	O
the	O
rU2	B-residue_name_number
nucleotide	B-chemical
in	O
the	O
second	B-site
binding	I-site
site	I-site
closer	O
to	O
the	O
C	O
-	O
terminal	O
β	B-structure_element
-	I-structure_element
strand	I-structure_element
of	O
RRM2	B-structure_element
.	O

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

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	B-bond_interaction
bonds	I-bond_interaction
between	O
backbone	O
atoms	O
and	O
the	O
base	O
.	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

We	O
proceeded	O
to	O
test	O
the	O
importance	O
of	O
new	O
U2AF65	B-complex_assembly
–	I-complex_assembly
Py	I-complex_assembly
-	I-complex_assembly
tract	I-complex_assembly
interactions	O
for	O
splicing	O
of	O
a	O
model	O
pre	B-chemical
-	I-chemical
mRNA	I-chemical
substrate	O
in	O
a	O
human	B-species
cell	O
line	O
(	O
Fig	O
.	O
5	O
;	O
Supplementary	O
Fig	O
.	O
5	O
).	O

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

The	O
strong	O
PY	B-site
splice	I-site
site	I-site
is	O
insensitive	O
to	O
added	O
U2AF65	B-protein
,	O
suggesting	O
that	O
endogenous	B-protein_state
U2AF65	B-protein
levels	O
are	O
sufficient	O
to	O
saturate	O
this	O
site	O
(	O
Supplementary	O
Fig	O
.	O
5b	O
).	O

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

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

We	O
examined	O
the	O
effect	O
on	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
conformations	O
of	O
purine	B-experimental_method
interruptions	I-experimental_method
that	O
often	O
occur	O
in	O
relatively	O
degenerate	O
human	B-species
Py	B-chemical
tracts	I-chemical
.	O

Nevertheless	O
,	O
the	O
predominant	O
0	O
.	O
45	O
FRET	B-evidence
state	I-evidence
in	O
the	O
presence	O
of	O
RNA	B-chemical
agrees	O
with	O
the	O
Py	B-protein_state
-	I-protein_state
tract	I-protein_state
-	I-protein_state
bound	I-protein_state
crystal	B-evidence
structure	I-evidence
of	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
.	O

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

The	O
U2AF65	B-protein
structures	B-evidence
and	O
analyses	B-evidence
presented	O
here	O
represent	O
a	O
successful	O
step	O
towards	O
defining	O
a	O
molecular	O
map	O
of	O
the	O
3	B-site
′	I-site
splice	I-site
site	I-site
.	O

The	O
intact	B-protein_state
U2AF65	B-protein
RRM1	B-structure_element
/	O
RRM2	B-structure_element
-	O
containing	O
domain	O
and	O
flanking	O
residues	O
are	O
required	O
for	O
binding	O
contiguous	B-structure_element
Py	B-chemical
tracts	I-chemical
.	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

For	O
clarity	O
,	O
we	O
consistently	O
number	O
the	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
nucleotide	B-site
-	I-site
binding	I-site
sites	I-site
from	O
one	O
to	O
nine	O
,	O
although	O
in	O
some	O
cases	O
the	O
co	B-experimental_method
-	I-experimental_method
crystallized	I-experimental_method
oligonucleotide	B-chemical
comprises	O
eight	O
nucleotides	B-chemical
and	O
as	O
such	O
leaves	O
the	O
first	B-site
binding	I-site
site	I-site
empty	O
.	O

(	O
b	O
)	O
Bar	O
graph	O
of	O
apparent	O
equilibrium	B-evidence
affinities	I-evidence
(	O
KA	B-evidence
)	O
for	O
the	O
AdML	B-gene
Py	B-chemical
tract	I-chemical
(	O
5	B-chemical
′-	I-chemical
CCCUUUUUUUUCC	I-chemical
-	I-chemical
3	I-chemical
′)	I-chemical
of	O
the	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
(	O
blue	O
)	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
protein	O
compared	O
with	O
mutations	O
of	O
the	O
residues	O
shown	O
in	O
a	O
:	O
3Gly	B-mutant
(	O
yellow	O
),	O
5Gly	B-mutant
(	O
red	O
),	O
NLALA	B-mutant
(	O
hatched	O
red	O
),	O
12Gly	B-mutant
(	O
orange	O
)	O
and	O
the	O
linker	B-experimental_method
deletions	I-experimental_method
dU2AF651	B-mutant
,	I-mutant
2	I-mutant
in	O
the	O
minimal	B-protein_state
RRM1	B-structure_element
–	I-structure_element
RRM2	I-structure_element
region	I-structure_element
(	O
residues	O
148	B-residue_range
–	I-residue_range
237	I-residue_range
,	O
258	B-residue_range
–	I-residue_range
336	I-residue_range
)	O
or	O
dU2AF651	B-mutant
,	I-mutant
2L	I-mutant
(	O
residues	O
141	B-residue_range
–	I-residue_range
237	I-residue_range
,	O
258	B-residue_range
–	I-residue_range
342	I-residue_range
).	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
a	O
,	O
b	O
)	O
Views	O
of	O
FRET	B-experimental_method
pairs	O
chosen	O
to	O
follow	O
the	O
relative	O
movement	O
of	O
RRM1	B-structure_element
and	O
RRM2	B-structure_element
on	O
the	O
crystal	B-evidence
structure	I-evidence
of	O
‘	O
side	B-protein_state
-	I-protein_state
by	I-protein_state
-	I-protein_state
side	I-protein_state
'	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
RRMs	B-structure_element
bound	B-protein_state
to	I-protein_state
a	O
Py	B-chemical
-	I-chemical
tract	I-chemical
oligonucleotide	I-chemical
(	O
a	O
,	O
representative	O
structure	O
iv	O
)	O
or	O
‘	O
closed	B-protein_state
'	O
NMR	B-experimental_method
/	O
PRE	B-experimental_method
-	O
based	O
model	O
of	O
U2AF651	B-mutant
,	I-mutant
2	I-mutant
(	O
b	O
,	O
PDB	O
ID	O
2YH0	O
)	O
in	O
identical	O
orientations	O
of	O
RRM2	B-structure_element
.	O

RNA	B-chemical
protects	O
a	O
nucleoprotein	B-complex_assembly
complex	O
against	O
radiation	O
damage	O

The	O
availability	O
of	O
two	O
TRAP	B-complex_assembly
molecules	O
in	O
the	O
asymmetric	O
unit	O
,	O
of	O
which	O
only	O
one	O
contained	O
bound	B-protein_state
RNA	B-chemical
,	O
allowed	O
a	O
controlled	O
investigation	O
into	O
the	O
exact	O
role	O
of	O
RNA	B-chemical
binding	O
in	O
protein	O
specific	O
damage	O
susceptibility	O
.	O

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

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

SRD	O
has	O
been	O
well	O
characterized	O
in	O
a	O
large	O
range	O
of	O
proteins	O
,	O
and	O
is	O
seen	O
to	O
follow	O
a	O
reproducible	O
order	O
:	O
metallo	O
-	O
centre	O
reduction	O
,	O
disulfide	B-ptm
-	I-ptm
bond	I-ptm
cleavage	O
,	O
acidic	O
residue	O
decarboxylation	O
and	O
methionine	O
methylthio	O
cleavage	O
(	O
Ravelli	O
&	O
McSweeney	O
,	O
2000	O
;	O
Burmeister	O
,	O
2000	O
;	O
Weik	O
et	O
al	O
.,	O
2000	O
;	O
Yano	O
et	O
al	O
.,	O
2005	O
).	O

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

As	O
of	O
early	O
2016	O
,	O
>	O
5400	O
nucleoprotein	B-complex_assembly
complex	O
structures	B-evidence
have	O
been	O
deposited	O
within	O
the	O
PDB	O
,	O
with	O
91	O
%	O
solved	O
by	O
MX	B-experimental_method
.	O

Using	O
newly	O
developed	O
methodology	O
,	O
we	O
present	O
a	O
controlled	B-experimental_method
SRD	I-experimental_method
investigation	O
at	O
1	O
.	O
98	O
Å	O
resolution	O
using	O
a	O
large	O
(∼	O
91	O
kDa	O
)	O
crystalline	O
protein	B-complex_assembly
–	I-complex_assembly
RNA	I-complex_assembly
complex	O
:	O
trp	B-protein_type
RNA	I-protein_type
-	I-protein_type
binding	I-protein_type
attenuation	I-protein_type
protein	I-protein_type
(	O
TRAP	B-complex_assembly
)	O
bound	B-protein_state
to	I-protein_state
a	O
53	O
bp	O
RNA	B-chemical
sequence	O
(	B-chemical
GAGUU	I-chemical
)	I-chemical
10GAG	I-chemical
(	O
PDB	O
entry	O
1gtf	O
;	O
Hopcroft	O
et	O
al	O
.,	O
2002	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

Previous	O
studies	O
have	O
characterized	O
SRD	B-site
sites	I-site
by	O
reporting	O
magnitudes	O
of	O
F	B-evidence
obs	I-evidence
(	I-evidence
d	I-evidence
n	I-evidence
)	I-evidence
−	I-evidence
F	I-evidence
obs	I-evidence
(	I-evidence
d	I-evidence
1	I-evidence
)	I-evidence
Fourier	I-evidence
difference	I-evidence
map	I-evidence
peaks	I-evidence
in	O
terms	O
of	O
the	O
sigma	B-evidence
(	O
σ	B-evidence
)	O
contour	O
level	O
(	O
the	O
number	O
of	O
standard	B-evidence
deviations	I-evidence
from	O
the	O
mean	B-evidence
map	I-evidence
electron	I-evidence
-	I-evidence
density	I-evidence
value	I-evidence
)	O
at	O
which	O
peaks	O
become	O
visible	O
.	O

Visual	B-experimental_method
inspection	I-experimental_method
of	I-experimental_method
Fourier	B-evidence
difference	I-evidence
maps	I-evidence
illustrated	O
the	O
clear	O
lack	O
of	O
RNA	B-chemical
electron	B-evidence
-	I-evidence
density	I-evidence
degradation	I-evidence
with	O
increasing	O
dose	O
compared	O
with	O
the	O
obvious	O
protein	O
damage	O
manifestations	O
(	O
Figs	O
.	O
3	O
▸	O
b	O
and	O
3	O
▸	O
c	O
).	O

For	O
each	O
TRAP	B-complex_assembly
ring	B-structure_element
subunit	B-structure_element
,	O
the	O
Glu36	B-residue_name_number
side	O
-	O
chain	O
carboxyl	O
group	O
accepts	O
a	O
pair	O
of	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
from	O
the	O
two	O
N	O
atoms	O
of	O
the	O
G3	B-residue_name_number
RNA	B-chemical
base	O
.	O

With	O
increasing	O
dose	O
,	O
the	O
D	B-evidence
loss	I-evidence
associated	O
with	O
the	O
Phe32	B-residue_name_number
side	O
chain	O
was	O
significantly	O
reduced	O
upon	O
RNA	B-chemical
binding	O
(	O
Fig	O
.	O
5	O
▸	O
e	O
;	O
Phe32	B-residue_name_number
Cζ	O
;	O
p	O
=	O
0	O
.	O
0014	O
),	O
an	O
indication	O
that	O
radiation	O
-	O
induced	O
conformation	O
disordering	O
of	O
Phe32	B-residue_name_number
had	O
been	O
reduced	O
.	O

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

Consistent	O
with	O
that	O
study	O
,	O
at	O
high	O
doses	O
of	O
above	O
∼	O
20	O
MGy	O
,	O
F	B-evidence
obs	I-evidence
(	I-evidence
d	I-evidence
n	I-evidence
)	I-evidence
−	I-evidence
F	I-evidence
obs	I-evidence
(	I-evidence
d	I-evidence
1	I-evidence
)	I-evidence
map	I-evidence
density	I-evidence
was	O
detected	O
around	O
P	O
,	O
O3	O
′	O
and	O
O5	O
′	O
atoms	O
of	O
the	O
RNA	B-chemical
backbone	O
,	O
with	O
no	O
significant	O
difference	B-evidence
density	I-evidence
localized	O
to	O
RNA	B-chemical
ribose	O
and	O
basic	O
subunits	B-structure_element
.	O

This	O
is	O
in	O
good	O
agreement	O
with	O
previous	O
mutagenesis	B-experimental_method
and	I-experimental_method
nucleoside	I-experimental_method
analogue	I-experimental_method
studies	I-experimental_method
(	O
Elliott	O
et	O
al	O
.,	O
2001	O
),	O
which	O
indicated	O
that	O
the	O
G1	B-residue_name_number
nucleotide	O
does	O
not	O
bind	O
to	O
TRAP	B-complex_assembly
as	O
strongly	O
as	O
do	O
A2	B-residue_name_number
and	O
G3	B-residue_name_number
,	O
and	O
plays	O
little	O
role	O
in	O
the	O
high	O
RNA	B-evidence
-	I-evidence
binding	I-evidence
affinity	I-evidence
of	O
TRAP	B-complex_assembly
(	O
K	B-evidence
d	I-evidence
≃	O
1	O
.	O
1	O
±	O
0	O
.	O
4	O
nM	O
).	O

The	O
prevalence	O
of	O
radical	O
attack	O
from	O
solvent	O
channels	O
surrounding	O
the	O
protein	O
in	O
the	O
crystal	B-evidence
is	O
a	O
questionable	O
cause	O
,	O
considering	O
previous	O
observations	O
indicating	O
that	O
the	O
strongly	O
oxidizing	O
hydroxyl	O
radical	O
is	O
immobile	O
at	O
100	O
K	O
(	O
Allan	O
et	O
al	O
.,	O
2013	O
;	O
Owen	O
et	O
al	O
.,	O
2012	O
).	O

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

This	O
structure	B-evidence
reveals	O
the	O
molecular	O
mechanism	O
underlying	O
the	O
docking	O
interaction	O
between	O
MKP7	B-protein
and	O
JNK1	B-protein
.	O

On	O
the	O
basis	O
of	O
sequence	O
similarity	O
,	O
substrate	O
specificity	O
and	O
predominant	O
subcellular	O
localization	O
,	O
the	O
MKP	B-protein_type
family	I-protein_type
can	O
be	O
further	O
divided	O
into	O
three	O
groups	O
(	O
Fig	O
.	O
1	O
).	O

In	O
addition	O
to	O
the	O
CD	B-structure_element
and	O
KBD	B-structure_element
,	O
MKP7	B-protein
has	O
a	O
long	O
C	B-structure_element
-	I-structure_element
terminal	I-structure_element
region	I-structure_element
that	O
contains	O
both	O
nuclear	O
localization	O
and	O
export	O
sequences	O
by	O
which	O
MKP7	B-protein
shuttles	O
between	O
the	O
nucleus	O
and	O
the	O
cytoplasm	O
(	O
Fig	O
.	O
2a	O
).	O

Thus	O
,	O
the	O
kinetic	B-evidence
data	I-evidence
were	O
analysed	O
using	O
the	O
general	O
initial	B-evidence
velocity	I-evidence
equation	I-evidence
,	O
taking	O
substrate	O
depletion	O
into	O
account	O
:	O

The	O
overall	O
folding	O
of	O
MKP7	B-protein
-	O
CD	B-structure_element
is	O
typical	O
of	O
DUSPs	B-protein_type
,	O
with	O
a	O
central	O
twisted	B-structure_element
five	I-structure_element
-	I-structure_element
stranded	I-structure_element
β	I-structure_element
-	I-structure_element
sheet	I-structure_element
surrounded	O
by	O
six	O
α	B-structure_element
-	I-structure_element
helices	I-structure_element
.	O

Since	O
helix	B-structure_element
α0	B-structure_element
and	O
the	O
following	O
loop	B-structure_element
α0	B-structure_element
–	I-structure_element
β1	I-structure_element
are	O
known	O
for	O
a	O
substrate	B-site
-	I-site
recognition	I-site
motif	I-site
of	O
VHR	B-protein
and	O
other	O
phosphatases	B-protein_type
,	O
the	O
absence	O
of	O
these	O
moieties	O
implicates	O
a	O
different	O
substrate	O
-	O
binding	O
mode	O
of	O
MKP7	B-protein
.	O

The	O
MKP7	B-protein
-	O
CD	B-structure_element
structure	B-evidence
near	O
the	O
active	B-site
site	I-site
exhibits	O
a	O
typical	O
active	B-protein_state
conformation	I-protein_state
as	O
found	O
in	O
VHR	B-protein
and	O
other	O
PTPs	B-protein_type
.	O

The	O
catalytic	B-site
residue	I-site
,	O
Cys244	B-residue_name_number
,	O
is	O
located	O
just	O
after	O
strand	B-structure_element
β5	B-structure_element
and	O
optimally	O
positioned	O
for	O
nucleophilic	O
attack	O
.	O

The	O
carboxylate	O
of	O
Asp268	B-residue_name_number
in	O
MKP7	B-protein
forms	O
a	O
salt	B-bond_interaction
bridge	I-bond_interaction
with	O
side	O
chain	O
of	O
Arg263	B-residue_name_number
in	O
JNK1	B-protein
,	O
and	O
Lys275	B-residue_name_number
of	O
MKP7	B-protein
forms	O
a	O
hydrogen	B-bond_interaction
bond	I-bond_interaction
and	O
a	O
salt	B-bond_interaction
bridge	I-bond_interaction
with	O
Thr228	B-residue_name_number
and	O
Asp229	B-residue_name_number
of	O
JNK1	B-protein
,	O
respectively	O
.	O

Gel	B-experimental_method
filtration	I-experimental_method
analysis	I-experimental_method
further	O
confirmed	O
the	O
key	O
role	O
of	O
Phe285	B-residue_name_number
in	O
the	O
MKP7	B-protein
–	O
JNK1	B-protein
interaction	O
:	O
no	O
F285D	B-complex_assembly
–	I-complex_assembly
JNK1	I-complex_assembly
complex	O
was	O
detected	O
when	O
3	O
molar	O
equivalents	O
of	O
MKP7	B-protein
-	O
CD	B-structure_element
(	O
F285D	B-mutant
)	O
were	O
mixed	O
with	O
1	O
molar	O
equivalent	O
of	O
JNK1	B-protein
(	O
Fig	O
.	O
4b	O
).	O

To	O
determine	O
whether	O
the	O
deficiencies	O
in	O
their	O
abilities	O
to	O
bind	O
partner	O
proteins	O
or	O
carry	O
out	O
catalytic	O
function	O
are	O
owing	O
to	O
misfolding	O
of	O
the	O
purified	O
mutant	B-protein_state
proteins	O
,	O
we	O
also	O
examined	O
the	O
folding	O
properties	O
of	O
the	O
JNK1	B-protein
and	O
MKP7	B-protein
mutants	B-protein_state
with	O
circular	B-experimental_method
dichroism	I-experimental_method
.	O

The	O
spectra	B-evidence
of	O
these	O
mutants	B-protein_state
are	O
similar	O
to	O
the	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
proteins	O
,	O
indicating	O
that	O
these	O
mutants	B-protein_state
fold	O
as	O
well	O
as	O
the	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
proteins	O
(	O
Fig	O
.	O
4d	O
,	O
e	O
).	O

It	O
has	O
previously	O
been	O
reported	O
that	O
several	O
cytosolic	O
and	O
inducible	O
nuclear	O
MKPs	B-protein_type
undergo	O
catalytic	O
activation	O
upon	O
interaction	O
with	O
the	O
MAPK	B-protein_type
substrates	O
.	O

Incubation	B-experimental_method
of	O
MKP7	B-protein
with	O
JNK1	B-protein
did	O
not	O
markedly	O
stimulate	O
the	O
phosphatase	B-protein_type
activity	O
,	O
which	O
is	O
consistent	O
with	O
previous	O
results	O
that	O
MKP7	B-protein
solely	O
possesses	O
the	O
intrinsic	O
activity	O
(	O
Supplementary	O
Fig	O
.	O
2b	O
).	O

In	O
addition	O
,	O
His230	B-residue_name_number
and	O
Val256	B-residue_name_number
in	O
JNK1	B-protein
are	O
replaced	O
by	O
the	O
negatively	O
charged	O
residues	O
Glu208	B-residue_name_number
and	O
Asp235	B-residue_name_number
in	O
CDK2	B-protein
(	O
Fig	O
.	O
5d	O
),	O
and	O
the	O
charge	O
distribution	O
on	O
the	O
CDK2	B-protein
interactive	B-site
surface	I-site
is	O
quite	O
different	O
from	O
that	O
of	O
JNK	B-protein_type
.	O

These	O
data	O
indicated	O
that	O
a	O
unique	O
hydrophobic	B-site
pocket	I-site
formed	O
between	O
the	O
MAPK	B-structure_element
insert	I-structure_element
and	O
αG	B-structure_element
helix	I-structure_element
plays	O
a	O
major	O
role	O
in	O
the	O
substrate	O
recognition	O
by	O
MKPs	B-protein_type
.	O

The	O
KBD	B-structure_element
of	O
MKP5	B-protein
interacts	O
with	O
the	O
D	B-site
-	I-site
site	I-site
of	O
p38α	B-protein
to	O
mediate	O
the	O
enzyme	O
–	O
substrate	O
interaction	O
.	O

The	O
substrate	B-evidence
specificity	I-evidence
constant	I-evidence
kcat	B-evidence
/	I-evidence
Km	I-evidence
value	O
for	O
MKP5	B-protein
-	O
CD	B-structure_element
was	O
calculated	O
as	O
1	O
.	O
0	O
×	O
105	O
M	O
−	O
1	O
s	O
−	O
1	O
,	O
which	O
is	O
very	O
close	O
to	O
that	O
of	O
MKP7	B-protein
-	O
CD	B-structure_element
(	O
1	O
.	O
07	O
×	O
105	O
M	O
−	O
1	O
s	O
−	O
1	O
).	O

Comparisons	O
between	O
catalytic	B-structure_element
domains	I-structure_element
structures	B-evidence
of	O
MKP5	B-protein
and	O
MKP7	B-protein
reveal	O
that	O
the	O
overall	O
folds	O
of	O
the	O
two	O
proteins	O
are	O
highly	O
similar	O
,	O
with	O
only	O
a	O
few	O
regions	O
exhibiting	O
small	O
deviations	O
(	O
r	B-evidence
.	I-evidence
m	I-evidence
.	I-evidence
s	I-evidence
.	I-evidence
d	I-evidence
.	I-evidence
of	O
0	O
.	O
79	O
Å	O
;	O
Fig	O
.	O
7c	O
).	O

Given	O
the	O
distinct	O
interaction	O
mode	O
revealed	O
by	O
the	O
crystal	B-evidence
structure	I-evidence
of	O
JNK1	B-complex_assembly
–	I-complex_assembly
MKP7	I-complex_assembly
-	I-complex_assembly
CD	I-complex_assembly
,	O
one	O
obvious	O
question	O
is	O
whether	O
this	O
is	O
a	O
general	O
mechanism	O
used	O
by	O
all	O
members	O
of	O
the	O
JNK	B-protein_type
-	I-protein_type
specific	I-protein_type
MKPs	I-protein_type
.	O

Pull	B-experimental_method
-	I-experimental_method
down	I-experimental_method
assays	I-experimental_method
also	O
confirmed	O
the	O
protein	O
–	O
protein	O
interactions	O
observed	O
above	O
.	O

In	O
addition	O
,	O
there	O
were	O
no	O
significant	O
differences	O
in	O
the	O
CD	B-evidence
spectra	I-evidence
between	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
and	O
mutant	B-protein_state
proteins	O
,	O
indicating	O
that	O
the	O
overall	O
structures	B-evidence
of	O
these	O
mutants	B-protein_state
did	O
not	O
change	O
significantly	O
from	O
that	O
of	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
MKP5	B-protein
protein	O
(	O
Supplementary	O
Fig	O
.	O
4a	O
).	O

In	O
addition	O
,	O
the	O
key	O
interacting	O
residues	O
of	O
MKP7	B-protein
-	O
CD	B-structure_element
,	O
Phe215	B-residue_name_number
,	O
Leu267	B-residue_name_number
and	O
Leu288	B-residue_name_number
,	O
are	O
replaced	O
by	O
less	O
hydrophobic	O
residues	O
,	O
Asn379	B-residue_name_number
,	O
Met431	B-residue_name_number
and	O
Met452	B-residue_name_number
in	O
MKP5	B-protein
-	O
CD	B-structure_element
(	O
Fig	O
.	O
5c	O
),	O
respectively	O
,	O
which	O
may	O
result	O
in	O
weaker	O
hydrophobic	B-bond_interaction
interactions	I-bond_interaction
between	O
MKP5	B-protein
-	O
CD	B-structure_element
and	O
JNK1	B-protein
.	O

The	O
propagation	O
of	O
MAPK	B-protein_type
signals	O
is	O
attenuated	O
through	O
the	O
actions	O
of	O
the	O
MKPs	B-protein_type
.	O

Most	O
studies	O
have	O
focused	O
on	O
the	O
dephosphorylation	O
of	O
MAPKs	B-protein_type
by	O
phosphatases	B-protein_type
containing	O
the	O
‘	O
kinase	B-structure_element
-	I-structure_element
interaction	I-structure_element
motif	I-structure_element
'	O
(	O
D	B-structure_element
-	I-structure_element
motif	I-structure_element
),	O
including	O
a	O
group	O
of	O
DUSPs	B-protein_type
(	O
MKPs	B-protein_type
)	O
and	O
a	O
distinct	O
subfamily	O
of	O
tyrosine	B-protein_type
phosphatases	I-protein_type
(	O
HePTP	B-protein
,	O
STEP	B-protein
and	O
PTP	B-protein
-	I-protein
SL	I-protein
).	O

In	O
contrast	O
to	O
MKP5	B-protein
,	O
removal	B-experimental_method
of	I-experimental_method
the	O
KBD	B-structure_element
domain	O
from	O
MKP7	B-protein
does	O
not	O
drastically	O
affect	O
enzyme	O
catalysis	O
,	O
and	O
the	O
kinetic	O
parameters	O
of	O
MKP7	B-protein
-	O
CD	B-structure_element
for	O
p38α	B-protein
substrate	O
are	O
very	O
similar	O
to	O
those	O
for	O
JNK1	B-protein
substrate	O
.	O

The	O
MKP7	B-protein
-	O
KBD	B-structure_element
docks	O
to	O
the	O
D	B-site
-	I-site
site	I-site
located	O
on	O
the	O
back	O
side	O
of	O
the	O
p38α	B-protein
catalytic	B-site
pocket	I-site
for	O
high	O
-	O
affinity	O
association	O
,	O
whereas	O
the	O
interaction	O
of	O
the	O
MKP7	B-protein
-	O
CD	B-structure_element
with	O
another	O
p38α	B-protein
structural	O
region	O
,	O
which	O
is	O
close	O
to	O
the	O
activation	B-structure_element
loop	I-structure_element
,	O
may	O
not	O
only	O
stabilize	O
binding	O
but	O
also	O
provide	O
contacts	O
crucial	O
for	O
organizing	O
the	O
MKP7	B-protein
active	B-site
site	I-site
with	O
respect	O
to	O
the	O
phosphoreceptor	O
in	O
the	O
p38α	B-protein
substrate	O
for	O
efficient	O
dephosphorylation	O
.	O

This	O
hydrophobic	B-site
site	I-site
was	O
first	O
identified	O
by	O
changes	B-evidence
in	I-evidence
deuterium	I-evidence
exchange	I-evidence
profiles	I-evidence
,	O
and	O
is	O
near	O
the	O
MAPK	B-structure_element
insertion	I-structure_element
and	O
helix	B-structure_element
αG	B-structure_element
.	O
Interestingly	O
,	O
many	O
of	O
the	O
equivalent	O
residues	O
in	O
JNK1	B-protein
,	O
important	O
for	O
MKP7	B-protein
-	O
CD	B-structure_element
recognition	O
,	O
are	O
also	O
used	O
for	O
substrate	O
binding	O
by	O
ERK2	B-protein
(	O
ref	O
.),	O
indicating	O
that	O
this	O
site	O
is	O
overlapped	O
with	O
the	O
DEF	B-site
-	I-site
site	I-site
previously	O
identified	O
in	O
ERK2	B-protein
(	O
Fig	O
.	O
5d	O
).	O

Therefore	O
,	O
it	O
is	O
tempting	O
to	O
speculate	O
that	O
the	O
catalytic	B-structure_element
domain	I-structure_element
of	O
MKP3	B-protein
may	O
bind	O
to	O
ERK2	B-protein
in	O
a	O
manner	O
analogous	O
to	O
the	O
way	O
by	O
which	O
MKP7	B-protein
-	O
CD	B-structure_element
binds	O
to	O
JNK1	B-protein
.	O

The	O
ongoing	O
work	O
demonstrates	O
that	O
although	O
the	O
overall	O
interaction	O
modes	O
are	O
similar	O
between	O
the	O
JNK1	B-complex_assembly
–	I-complex_assembly
MKP7	I-complex_assembly
-	I-complex_assembly
CD	I-complex_assembly
and	O
ERK2	B-complex_assembly
–	I-complex_assembly
MKP3	I-complex_assembly
-	I-complex_assembly
CD	I-complex_assembly
complexes	O
,	O
the	O
ERK2	B-complex_assembly
–	I-complex_assembly
MKP3	I-complex_assembly
-	I-complex_assembly
CD	I-complex_assembly
interaction	O
is	O
less	O
extensive	O
and	O
helix	B-structure_element
α4	B-structure_element
from	O
MKP3	B-protein
-	O
CD	B-structure_element
does	O
not	O
interact	O
directly	O
with	O
ERK2	B-protein
.	O

Phe285	B-residue_name_number
is	O
essential	O
for	O
JNK1	B-protein
substrate	O
binding	O
,	O
whereas	O
Phe287	B-residue_name_number
plays	O
a	O
role	O
for	O
the	O
precise	O
alignment	O
of	O
active	B-site
-	I-site
site	I-site
residues	I-site
,	O
which	O
are	O
important	O
for	O
transition	O
-	O
state	O
stabilization	O
.	O

(	O
a	O
)	O
Domain	O
organization	O
of	O
human	B-species
MKP7	B-protein
and	O
JNK1	B-protein
.	O

The	O
2Fo	B-evidence
−	I-evidence
Fc	I-evidence
omit	I-evidence
map	I-evidence
(	O
contoured	O
at	O
1	O
.	O
5σ	O
)	O
for	O
the	O
P	B-structure_element
-	I-structure_element
loop	I-structure_element
of	O
MKP7	B-protein
-	O
CD	B-structure_element
is	O
shown	O
at	O
inset	O
of	O
b	O
.	O
(	O
c	O
)	O
Structure	B-evidence
of	O
VHR	B-protein
with	O
its	O
active	B-site
site	I-site
highlighted	O
in	O
marine	O
blue	O
.	O
(	O
d	O
)	O
Close	O
-	O
up	O
view	O
of	O
the	O
JNK1	B-site
–	I-site
MKP7	I-site
interface	I-site
showing	O
interacting	O
amino	O
acids	O
of	O
JNK1	B-protein
(	O
orange	O
)	O
and	O
MKP7	B-protein
-	O
CD	B-structure_element
(	O
cyan	O
).	O

MKP7	B-protein
-	O
CD	B-structure_element
is	O
shown	O
in	O
surface	O
representation	O
coloured	O
according	O
to	O
electrostatic	O
potential	O
(	O
positive	O
,	O
blue	O
;	O
negative	O
,	O
red	O
).	O

Mutational	B-experimental_method
analysis	I-experimental_method
on	O
interactions	O
between	O
MKP7	B-protein
-	O
CD	B-structure_element
and	O
JNK1	B-protein
.	O

However	O
,	O
in	O
contrast	O
to	O
the	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
MKP7	B-protein
-	O
CD	B-structure_element
,	O
mutant	B-protein_state
F285D	B-mutant
did	O
not	O
co	O
-	O
migrate	O
with	O
JNK1	B-protein
.	O

(	O
c	O
)	O
Pull	B-experimental_method
-	I-experimental_method
down	I-experimental_method
assays	I-experimental_method
of	O
MKP7	B-protein
-	O
CD	B-structure_element
by	O
GST	B-protein_state
-	I-protein_state
tagged	I-protein_state
JNK1	B-protein
mutants	B-protein_state
.	O

Measurements	O
were	O
averaged	O
for	O
three	O
scans	O
.	O
(	O
e	O
)	O
Circular	B-experimental_method
dichroism	I-experimental_method
spectra	B-evidence
for	O
JNK1	B-protein
wild	B-protein_state
type	I-protein_state
and	O
mutants	B-protein_state
.	O

The	O
N	B-structure_element
-	I-structure_element
lobe	I-structure_element
and	O
C	B-structure_element
-	I-structure_element
lobe	I-structure_element
of	O
CDK2	B-protein
are	O
coloured	O
in	O
grey	O
and	O
pink	O
,	O
respectively	O
,	O
and	O
KAP	B-protein
is	O
coloured	O
in	O
green	O
.	O

Interestingly	O
,	O
the	O
recognition	O
of	O
CDK2	B-protein
by	O
KAP	B-protein
is	O
augmented	O
by	O
a	O
similar	O
interface	B-site
as	O
that	O
observed	O
in	O
the	O
complex	O
of	O
JNK1	B-protein
and	O
MKP7	B-protein
-	O
CD	B-structure_element
(	O
region	B-structure_element
II	I-structure_element
).	O

One	O
remarkable	O
difference	O
between	O
these	O
two	O
kinase	O
-	O
phosphatase	O
complexes	O
is	O
that	O
helix	B-structure_element
α6	B-structure_element
of	O
KAP	B-protein
(	O
corresponding	O
to	O
helix	B-structure_element
α4	B-structure_element
of	O
MKP7	B-protein
-	O
CD	B-structure_element
)	O
plays	O
little	O
,	O
if	O
any	O
,	O
role	O
in	O
the	O
formation	O
of	O
a	O
stable	B-protein_state
heterodimer	B-oligomeric_state
of	O
CDK2	B-protein
and	O
KAP	B-protein
.	O
(	O
c	O
)	O
Sequence	B-experimental_method
alignment	I-experimental_method
of	O
the	O
JNK	B-site
-	I-site
interacting	I-site
regions	I-site
on	O
MKPs	B-protein_type
.	O

(	O
d	O
)	O
F	B-site
-	I-site
site	I-site
is	O
required	O
for	O
JNK1	B-protein
to	O
interact	O
with	O
MKP7	B-protein
.	O

(	O
e	O
)	O
Effect	O
of	O
MKP7	B-protein
(	O
wild	B-protein_state
type	I-protein_state
or	O
mutants	B-protein_state
)	O
expression	O
on	O
ultraviolet	O
-	O
induced	O
apoptosis	O
.	O

(	O
f	O
)	O
Statistical	O
analysis	O
of	O
apoptotic	O
cells	O
(	O
mean	O
±	O
s	O
.	O
e	O
.	O
m	O
.,	O
n	O
=	O
3	O
),	O
*	B-evidence
P	I-evidence
<	O
0	O
.	O
05	O
,	O
***	B-evidence
P	I-evidence
<	O
0	O
.	O
001	O
(	O
ANOVA	B-experimental_method
followed	O
by	O
Tukey	B-experimental_method
'	I-experimental_method
s	I-experimental_method
test	I-experimental_method
).	O

The	O
error	O
bars	O
represent	O
s	O
.	O
e	O
.	O
m	O
.	O
(	O
c	O
)	O
Structural	B-experimental_method
comparison	I-experimental_method
of	O
the	O
JNK	B-site
-	I-site
interacting	I-site
residues	I-site
on	O
MKP5	B-protein
-	O
CD	B-structure_element
(	O
PDB	O
1ZZW	O
)	O
and	O
MKP7	B-protein
-	O
CD	B-structure_element
.	O

Mechanistic	O
insight	O
into	O
a	O
peptide	B-protein_type
hormone	I-protein_type
signaling	O
complex	O
mediating	O
floral	O
organ	O
abscission	O

It	O
is	O
unknown	O
how	O
expression	O
of	O
IDA	B-protein
in	O
the	O
abscission	O
zone	O
leads	O
to	O
HAESA	B-protein
activation	O
.	O

The	O
HAESA	B-protein
co	B-protein_type
-	I-protein_type
receptor	I-protein_type
SERK1	B-protein
,	O
a	O
positive	O
regulator	O
of	O
the	O
floral	O
abscission	O
pathway	O
,	O
allows	O
for	O
high	O
-	O
affinity	O
sensing	O
of	O
the	O
peptide	B-protein_type
hormone	I-protein_type
by	O
binding	O
to	O
an	O
Arg	B-structure_element
-	I-structure_element
His	I-structure_element
-	I-structure_element
Asn	I-structure_element
motif	I-structure_element
in	O
IDA	B-protein
.	O

Plants	B-taxonomy_domain
can	O
shed	O
their	O
leaves	O
,	O
flowers	O
or	O
other	O
organs	O
when	O
they	O
no	O
longer	O
need	O
them	O
.	O
But	O
how	O
does	O
a	O
leaf	O
or	O
a	O
flower	O
know	O
when	O
to	O
let	O
go	O
?	O
A	O
receptor	B-protein_type
protein	I-protein_type
called	O
HAESA	B-protein
is	O
found	O
on	O
the	O
surface	O
of	O
the	O
cells	O
that	O
surround	O
a	O
future	O
break	O
point	O
on	O
the	O
plant	O
.	O
When	O
its	O
time	O
to	O
shed	O
an	O
organ	O
,	O
a	O
hormone	B-chemical
called	O
IDA	B-protein
instructs	O
HAESA	B-protein
to	O
trigger	O
the	O
shedding	O
process	O
.	O

Cysteine	B-residue_name
residues	O
engaged	O
in	O
disulphide	B-ptm
bonds	I-ptm
are	O
depicted	O
in	O
green	O
.	O

IDA	B-protein
(	O
in	O
bonds	O
representation	O
,	O
surface	O
view	O
included	O
)	O
is	O
depicted	O
in	O
yellow	O
.	O

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

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

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

The	O
available	O
genetic	O
and	O
biochemical	O
evidence	O
suggests	O
that	O
IDA	B-protein
and	O
HAESA	B-protein
together	O
control	O
floral	O
abscission	O
,	O
but	O
it	O
is	O
poorly	O
understood	O
if	O
IDA	B-protein
is	O
directly	O
sensed	O
by	O
the	O
receptor	B-protein_type
kinase	I-protein_type
HAESA	B-protein
and	O
how	O
IDA	B-protein
binding	O
at	O
the	O
cell	O
surface	O
would	O
activate	O
the	O
receptor	O
.	O

Close	O
-	O
up	O
views	O
of	O
(	O
A	O
)	O
IDA	B-protein
,	O
(	O
B	O
)	O
the	O
N	B-protein_state
-	I-protein_state
terminally	I-protein_state
extended	I-protein_state
PKGV	B-mutant
-	I-mutant
IDA	I-mutant
and	O
(	O
C	O
)	O
IDL1	B-protein
bound	B-protein_state
to	I-protein_state
the	O
HAESA	B-protein
hormone	B-site
binding	I-site
pocket	I-site
(	O
in	O
bonds	O
representation	O
,	O
in	O
yellow	O
)	O
and	O
including	O
simulated	B-experimental_method
annealing	I-experimental_method
2Fo	B-evidence
–	I-evidence
Fc	I-evidence
omit	I-evidence
electron	I-evidence
density	I-evidence
maps	I-evidence
contoured	O
at	O
1	O
.	O
0	O
σ	O
.	O

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

A	O
SDS	B-experimental_method
PAGE	I-experimental_method
of	O
the	O
peak	O
fractions	O
is	O
shown	O
alongside	O
.	O

Purified	O
HAESA	B-protein
and	O
SERK1	B-protein
are	O
~	O
75	O
and	O
~	O
28	O
kDa	O
,	O
respectively	O
.	O
(	O
C	O
)	O
Isothermal	B-experimental_method
titration	I-experimental_method
calorimetry	I-experimental_method
of	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
and	O
Hyp64	B-ptm
→	I-ptm
Pro	I-ptm
IDA	B-protein
versus	O
the	O
HAESA	B-protein
and	O
SERK1	B-protein
ectodomains	B-structure_element
.	O

The	O
titration	B-experimental_method
of	O
IDA	B-protein
wild	B-protein_state
-	I-protein_state
type	I-protein_state
versus	O
the	O
isolated	O
HAESA	B-protein
ectodomain	B-structure_element
from	O
Figure	O
1B	O
is	O
shown	O
for	O
comparison	O
(	O
red	O
line	O
;	O
n	O
.	O
d	O
.	O

We	O
purified	B-experimental_method
the	O
HAESA	B-protein
ectodomain	B-structure_element
(	O
residues	O
20	B-residue_range
–	I-residue_range
620	I-residue_range
)	O
from	O
baculovirus	B-experimental_method
-	I-experimental_method
infected	I-experimental_method
insect	I-experimental_method
cells	I-experimental_method
(	O
Figure	O
1	O
—	O
figure	O
supplement	O
1A	O
,	O
see	O
Materials	O
and	O
methods	O
)	O
and	O
quantified	O
the	O
interaction	O
of	O
the	O
~	O
75	O
kDa	O
glycoprotein	B-protein_type
with	O
synthetic	B-protein_state
IDA	B-chemical
peptides	I-chemical
using	O
isothermal	B-experimental_method
titration	I-experimental_method
calorimetry	I-experimental_method
(	O
ITC	B-experimental_method
).	O

IDA	B-protein
binds	O
in	O
a	O
completely	B-protein_state
extended	I-protein_state
conformation	I-protein_state
along	O
the	O
inner	O
surface	O
of	O
the	O
HAESA	B-protein
ectodomain	B-structure_element
,	O
covering	O
LRRs	B-structure_element
2	I-structure_element
–	I-structure_element
14	I-structure_element
(	O
Figure	O
1C	O
,	O
D	O
,	O
Figure	O
1	O
—	O
figure	O
supplement	O
2	O
).	O

Other	O
hydrophobic	B-bond_interaction
and	I-bond_interaction
polar	I-bond_interaction
interactions	I-bond_interaction
are	O
mediated	O
by	O
Ser62IDA	B-residue_name_number
,	O
Ser65IDA	B-residue_name_number
and	O
by	O
backbone	O
atoms	O
along	O
the	O
IDA	B-chemical
peptide	I-chemical
(	O
Figure	O
1D	O
,	O
Figure	O
1	O
—	O
figure	O
supplement	O
2A	O
–	O
C	O
).	O

Consistently	O
,	O
PKGV	B-mutant
-	I-mutant
IDA	I-mutant
and	O
IDA	B-protein
have	O
similar	O
binding	B-evidence
affinities	I-evidence
in	O
our	O
ITC	B-experimental_method
assays	I-experimental_method
,	O
further	O
indicating	O
that	O
HAESA	B-protein
senses	O
a	O
dodecamer	B-structure_element
peptide	B-chemical
comprising	O
residues	O
58	B-residue_range
-	I-residue_range
69IDA	I-residue_range
(	O
Figure	O
2D	O
).	O

Replacing	B-experimental_method
Hyp64IDA	B-ptm
,	O
which	O
is	O
common	O
to	O
all	O
IDLs	B-protein_type
,	O
with	O
proline	B-residue_name
impairs	O
the	O
interaction	O
with	O
the	O
receptor	O
,	O
as	O
does	O
the	O
Lys66IDA	B-mutant
/	I-mutant
Arg67IDA	I-mutant
→	I-mutant
Ala	I-mutant
double	B-protein_state
-	I-protein_state
mutant	I-protein_state
discussed	O
below	O
(	O
Figure	O
1A	O
,	O
2D	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

The	O
serk2	B-gene
-	I-gene
2	I-gene
,	O
serk3	B-gene
-	I-gene
1	I-gene
,	O
serk4	B-gene
-	I-gene
1	I-gene
and	O
serk5	B-gene
-	I-gene
1	I-gene
mutant	B-protein_state
lines	O
showed	O
a	O
petal	O
break	O
-	O
strength	O
profile	O
not	O
significantly	O
different	O
from	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
plants	B-taxonomy_domain
.	O

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

We	O
found	O
that	O
HAESA	B-protein
senses	O
IDA	B-protein
with	O
a	O
~	O
60	O
fold	O
higher	O
binding	B-evidence
affinity	I-evidence
in	O
the	O
presence	B-protein_state
of	I-protein_state
SERK1	B-protein
,	O
suggesting	O
that	O
SERK1	B-protein
is	O
involved	O
in	O
the	O
specific	O
recognition	O
of	O
the	O
peptide	B-protein_type
hormone	I-protein_type
(	O
Figure	O
3C	O
).	O

Importantly	O
,	O
hydroxyprolination	B-ptm
of	O
IDA	B-protein
is	O
critical	O
for	O
HAESA	B-complex_assembly
-	I-complex_assembly
IDA	I-complex_assembly
-	I-complex_assembly
SERK1	I-complex_assembly
complex	O
formation	O
(	O
Figure	O
3C	O
,	O
D	O
).	O

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

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

Polar	B-bond_interaction
interactions	I-bond_interaction
are	O
highlighted	O
as	O
dotted	O
lines	O
(	O
in	O
magenta	O
).	O

(	O
A	O
)	O
Size	B-experimental_method
exclusion	I-experimental_method
chromatography	I-experimental_method
experiments	O
similar	O
to	O
Figure	O
3B	O
,	O
D	O
reveal	O
that	O
IDA	B-protein
mutant	B-protein_state
peptides	B-chemical
targeting	O
the	O
C	B-structure_element
-	I-structure_element
terminal	I-structure_element
motif	I-structure_element
do	O
not	O
form	O
biochemically	B-protein_state
stable	I-protein_state
HAESA	B-complex_assembly
-	I-complex_assembly
IDA	I-complex_assembly
-	I-complex_assembly
SERK1	I-complex_assembly
complexes	O
.	O

We	O
over	B-experimental_method
-	I-experimental_method
expressed	I-experimental_method
full	B-protein_state
-	I-protein_state
length	I-protein_state
wild	B-protein_state
-	I-protein_state
type	I-protein_state
IDA	B-protein
or	O
this	O
Lys66IDA	B-mutant
/	I-mutant
Arg67IDA	I-mutant
→	I-mutant
Ala	I-mutant
double	B-protein_state
-	I-protein_state
mutant	I-protein_state
to	O
similar	O
levels	O
in	O
Col	O
-	O
0	O
Arabidopsis	B-taxonomy_domain
plants	B-taxonomy_domain
(	O
Figure	O
5D	O
).	O

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

It	O
will	O
be	O
thus	O
interesting	O
to	O
see	O
if	O
proteolytic	O
processing	O
of	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
IDA	B-protein
in	O
vivo	O
is	O
regulated	O
in	O
a	O
cell	O
-	O
type	O
or	O
tissue	O
-	O
specific	O
manner	O
.	O

This	O
observation	O
is	O
consistent	O
with	O
our	O
complex	O
structure	B-evidence
in	O
which	O
receptor	O
and	O
co	O
-	O
receptor	O
together	O
form	O
the	O
IDA	B-site
binding	I-site
pocket	I-site
.	O

In	O
addition	O
,	O
residues	O
53	B-residue_range
-	I-residue_range
55SERK1	I-residue_range
from	O
the	O
SERK1	B-protein
N	O
-	O
terminal	O
cap	B-structure_element
mediate	O
specific	O
interactions	O
with	O
the	O
IDA	B-chemical
peptide	I-chemical
(	O
Figures	O
4C	O
,	O
6B	O
).	O

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

Importantly	O
,	O
this	B-structure_element
motif	I-structure_element
can	O
also	O
be	O
found	O
in	O
other	O
peptide	B-protein_type
hormone	I-protein_type
families	I-protein_type
(	O
Figure	O
7	O
).	O

Using	O
electron	B-experimental_method
cryo	I-experimental_method
-	I-experimental_method
microscopy	I-experimental_method
of	O
a	O
single	O
specimen	O
,	O
we	O
present	O
five	O
ribosome	B-complex_assembly
structures	B-evidence
formed	O
with	O
the	O
Taura	B-species
syndrome	I-species
virus	I-species
IRES	B-site
and	O
translocase	B-protein_type
eEF2	B-complex_assembly
•	I-complex_assembly
GTP	I-complex_assembly
bound	B-protein_state
with	I-protein_state
sordarin	B-chemical
.	O

The	O
structures	B-evidence
suggest	O
missing	O
links	O
in	O
our	O
understanding	O
of	O
tRNA	B-chemical
translocation	O
.	O

To	O
this	O
end	O
,	O
internal	B-site
ribosome	I-site
entry	I-site
site	I-site
(	O
IRES	B-site
)	O
RNAs	B-chemical
are	O
employed	O
(	O
reviewed	O
in	O
.	O

An	O
unusual	O
strategy	O
of	O
initiation	B-protein_state
is	O
used	O
by	O
intergenic	B-structure_element
-	I-structure_element
region	I-structure_element
(	O
IGR	B-structure_element
)	O
IRESs	B-site
found	O
in	O
Dicistroviridae	B-species
arthropod	I-species
-	O
infecting	O
viruses	B-taxonomy_domain
.	O

These	O
include	O
shrimp	B-taxonomy_domain
-	O
infecting	O
Taura	B-species
syndrome	I-species
virus	I-species
(	O
TSV	B-species
),	O
and	O
insect	B-taxonomy_domain
viruses	O
Plautia	B-species
stali	I-species
intestine	I-species
virus	I-species
(	O
PSIV	B-species
)	O
and	O
Cricket	B-species
paralysis	I-species
virus	I-species
(	O
CrPV	B-species
).	O

A	O
recent	O
demonstration	O
of	O
bacterial	B-taxonomy_domain
translation	O
initiation	B-protein_state
by	O
an	O
IGR	B-structure_element
IRES	B-site
indicates	O
that	O
the	O
IRESs	B-site
take	O
advantage	O
of	O
conserved	O
structural	O
and	O
dynamic	O
properties	O
of	O
the	O
ribosome	B-complex_assembly
.	O

For	O
a	O
cognate	O
aminoacyl	B-chemical
-	I-chemical
tRNA	I-chemical
to	O
bind	O
the	O
first	O
viral	B-taxonomy_domain
mRNA	B-chemical
codon	O
,	O
PKI	B-structure_element
has	O
to	O
be	O
translocated	O
from	O
the	O
A	B-site
site	I-site
,	O
so	O
that	O
the	O
first	O
codon	O
can	O
be	O
presented	O
in	O
the	O
A	B-site
site	I-site
.	O

First	O
,	O
studies	O
of	O
bacterial	B-taxonomy_domain
ribosomes	B-complex_assembly
showed	O
that	O
a	O
~	O
10	O
°	O
rotation	O
of	O
the	O
small	B-structure_element
subunit	I-structure_element
relative	O
to	O
the	O
large	B-structure_element
subunit	I-structure_element
,	O
known	O
as	O
intersubunit	O
rotation	O
,	O
or	O
ratcheting	O
,	O
is	O
required	O
for	O
translocation	O
.	O

Schematic	O
of	O
cryo	B-experimental_method
-	I-experimental_method
EM	I-experimental_method
refinement	O
and	O
classification	O
procedures	O
.	O

All	O
particles	B-experimental_method
were	O
initially	O
aligned	O
to	O
a	O
single	O
model	O
.	O

All	O
measurements	O
are	O
relative	O
to	O
the	O
non	B-protein_state
-	I-protein_state
rotated	I-protein_state
80S	B-complex_assembly
•	I-complex_assembly
2tRNA	I-complex_assembly
•	I-complex_assembly
mRNA	I-complex_assembly
structure	B-evidence
.	O

This	O
approach	O
revealed	O
five	O
80S	B-complex_assembly
•	I-complex_assembly
IRES	I-complex_assembly
•	I-complex_assembly
eEF2	I-complex_assembly
•	I-complex_assembly
GDP	I-complex_assembly
structures	B-evidence
at	O
average	O
resolutions	O
of	O
3	O
.	O
5	O
to	O
4	O
.	O
2	O
Å	O
,	O
sufficient	O
to	O
locate	O
IRES	B-site
domains	O
and	O
to	O
resolve	O
individual	O
residues	O
in	O
the	O
core	O
regions	O
of	O
the	O
ribosome	B-complex_assembly
and	O
eEF2	B-protein
(	O
Figures	O
3c	O
,	O
d	O
,	O
and	O
5f	O
,	O
h	O
;	O
see	O
also	O
Figure	O
1	O
—	O
figure	O
supplement	O
2	O
and	O
Figure	O
5	O
—	O
figure	O
supplement	O
2	O
),	O
including	O
the	O
post	O
-	O
translational	O
modification	O
diphthamide	B-ptm
699	I-ptm
(	O
Figure	O
3c	O
).	O

The	O
views	O
were	O
obtained	O
by	O
structural	B-experimental_method
alignment	I-experimental_method
of	O
the	O
25S	B-chemical
rRNAs	I-chemical
;	O
the	O
sarcin	B-structure_element
-	I-structure_element
ricin	I-structure_element
loop	I-structure_element
(	O
SRL	B-structure_element
)	O
of	O
25S	B-chemical
rRNA	I-chemical
is	O
shown	O
in	O
gray	O
for	O
reference	O
.	O

(	O
c	O
)	O
Comparison	O
of	O
the	O
40S	B-complex_assembly
conformations	O
in	O
Structures	B-evidence
I	I-evidence
through	I-evidence
V	I-evidence
shows	O
distinct	O
positions	O
of	O
the	O
head	B-structure_element
relative	O
to	O
the	O
body	B-structure_element
of	O
the	O
40S	B-complex_assembly
subunit	B-structure_element
(	O
head	B-structure_element
swivel	O
).	O

Conformation	O
of	O
the	O
non	B-protein_state
-	I-protein_state
swiveled	I-protein_state
40S	B-complex_assembly
subunit	B-structure_element
in	O
the	O
S	B-species
.	I-species
cerevisiae	I-species
80S	B-complex_assembly
ribosome	I-complex_assembly
bound	B-protein_state
with	I-protein_state
two	O
tRNAs	B-chemical
is	O
shown	O
for	O
reference	O
(	O
blue	O
).	O

The	O
central	B-structure_element
protuberance	I-structure_element
(	O
CP	B-structure_element
)	O
is	O
labeled	O
.	O

Structures	B-evidence
II	I-evidence
and	I-evidence
III	I-evidence
are	O
in	O
mid	B-protein_state
-	I-protein_state
rotation	I-protein_state
conformations	O
(~	O
5	O
°).	O

IRES	B-site
rearrangements	O

Position	O
and	O
interactions	O
of	O
loop	B-structure_element
3	I-structure_element
(	O
variable	B-structure_element
loop	I-structure_element
region	I-structure_element
)	O
of	O
the	O
PKI	B-structure_element
domain	O
in	O
Structure	B-evidence
V	I-evidence
(	O
this	O
work	O
)	O
resembles	O
those	O
of	O
the	O
anticodon	B-structure_element
stem	I-structure_element
loop	I-structure_element
of	O
the	O
E	B-site
-	I-site
site	I-site
tRNA	B-chemical
(	O
blue	O
)	O
in	O
the	O
80S	B-complex_assembly
•	I-complex_assembly
2tRNA	I-complex_assembly
•	I-complex_assembly
mRNA	I-complex_assembly
complex	O
.	O

Structures	B-evidence
of	O
translocation	O
complexes	O
of	O
the	O
bacterial	B-taxonomy_domain
70S	B-complex_assembly
ribosome	I-complex_assembly
bound	B-protein_state
with	I-protein_state
two	O
tRNAs	B-chemical
and	O
yeast	B-taxonomy_domain
80S	B-complex_assembly
complexes	B-protein_state
with	I-protein_state
tRNAs	B-chemical
are	O
shown	O
in	O
the	O
upper	O
row	O
and	O
labeled	O
.	O

Positions	O
of	O
the	O
IRES	B-site
relative	O
to	O
eEF2	B-protein
and	O
elements	O
of	O
the	O
ribosome	B-complex_assembly
in	O
Structures	B-evidence
I	I-evidence
through	I-evidence
V	I-evidence
.	O

The	O
minor	B-site
groove	I-site
of	O
SL5	B-structure_element
(	O
at	O
nt	O
6862	B-residue_range
–	I-residue_range
6868	I-residue_range
)	O
contacts	O
the	O
positively	O
charged	O
region	O
of	O
eS25	B-protein
(	O
R49	B-residue_name_number
,	O
R58	B-residue_name_number
and	O
R68	B-residue_name_number
)	O
(	O
Figure	O
3	O
—	O
figure	O
supplement	O
4	O
).	O

The	O
view	O
was	O
obtained	O
by	O
structural	B-experimental_method
alignment	I-experimental_method
of	O
the	O
body	B-structure_element
domains	O
of	O
18S	B-chemical
rRNAs	I-chemical
of	O
the	O
corresponding	O
80S	B-complex_assembly
structures	B-evidence
.	O

Rearrangements	O
of	O
the	O
IRES	B-site
involve	O
restructuring	O
of	O
several	O
interactions	O
with	O
the	O
ribosome	B-complex_assembly
.	O

In	O
Structure	B-evidence
I	I-evidence
,	O
SL3	B-structure_element
of	O
the	O
PKI	B-structure_element
domain	O
is	O
positioned	O
between	O
the	O
A	B-structure_element
-	I-structure_element
site	I-structure_element
finger	I-structure_element
(	O
nt	O
1008	B-residue_range
–	I-residue_range
1043	I-residue_range
of	O
25S	B-chemical
rRNA	I-chemical
)	O
and	O
the	O
P	B-site
site	I-site
of	O
the	O
60S	B-complex_assembly
subunit	B-structure_element
,	O
comprising	O
helix	B-structure_element
84	I-structure_element
of	O
25S	B-chemical
rRNA	I-chemical
(	O
nt	O
.	O

The	O
second	O
set	O
of	O
major	O
structural	O
changes	O
involves	O
interaction	O
of	O
the	O
P	B-site
site	I-site
region	I-site
of	O
the	O
large	B-structure_element
subunit	I-structure_element
with	O
the	O
hinge	B-structure_element
point	I-structure_element
of	O
the	O
IRES	B-site
bending	O
between	O
the	O
5	B-structure_element
´	I-structure_element
domain	I-structure_element
and	O
the	O
PKI	B-structure_element
domain	O
(	O
nt	O
.	O
6886	B-residue_range
–	I-residue_range
6890	I-residue_range
).	O

The	O
view	O
and	O
colors	O
are	O
as	O
in	O
Figure	O
5b	O
:	O
eEF2	B-protein
is	O
shown	O
in	O
green	O
,	O
IRES	B-site
RNA	B-chemical
in	O
red	O
,	O
40S	B-complex_assembly
subunit	B-structure_element
elements	O
in	O
orange	O
,	O
60S	B-complex_assembly
in	O
cyan	O
/	O
teal	O
.	O

Cryo	B-experimental_method
-	I-experimental_method
EM	I-experimental_method
density	B-evidence
of	O
the	O
GTPase	B-structure_element
region	I-structure_element
in	O
Structures	B-evidence
I	I-evidence
and	I-evidence
II	I-evidence
.	O

Repositioning	O
(	O
sliding	O
)	O
of	O
the	O
positively	B-site
-	I-site
charged	I-site
cluster	I-site
of	O
domain	O
IV	B-structure_element
of	O
eEF2	B-protein
over	O
the	O
phosphate	O
backbone	O
(	O
red	O
)	O
of	O
the	O
18S	B-structure_element
helices	I-structure_element
33	I-structure_element
and	I-structure_element
34	I-structure_element
.	O

Interactions	O
of	O
eEF2	B-protein
with	O
the	O
40S	B-complex_assembly
subunit	B-structure_element
.	O

From	O
Structure	B-evidence
I	I-evidence
to	I-evidence
V	I-evidence
,	O
these	O
central	O
domains	O
migrate	O
by	O
~	O
10	O
Å	O
along	O
the	O
40S	B-complex_assembly
surface	O
(	O
Figure	O
6c	O
).	O

At	O
the	O
head	B-structure_element
,	O
C1274	B-residue_name_number
of	O
the	O
18S	B-chemical
rRNA	I-chemical
(	O
C1054	B-residue_name_number
in	O
E	B-species
.	I-species
coli	I-species
)	O
base	O
pairs	O
with	O
the	O
first	O
nucleotide	O
of	O
the	O
ORF	B-structure_element
immediately	O
downstream	O
of	O
PKI	B-structure_element
.	O

The	O
codon	B-structure_element
-	I-structure_element
anticodon	I-structure_element
-	I-structure_element
like	I-structure_element
helix	I-structure_element
of	O
PKI	B-structure_element
is	O
shown	O
in	O
red	O
,	O
the	O
downstream	O
first	O
codon	O
of	O
the	O
ORF	B-structure_element
in	O
magenta	O
.	O

Histidines	B-residue_name_number
583	I-residue_name_number
and	I-residue_name_number
694	I-residue_name_number
interact	O
with	O
the	O
phosphate	O
backbone	O
of	O
the	O
anticodon	B-structure_element
-	I-structure_element
like	I-structure_element
strand	I-structure_element
(	O
at	O
G6907	B-residue_name_number
and	O
C6908	B-residue_name_number
).	O

Thus	O
,	O
in	O
comparison	O
with	O
the	O
initiation	B-protein_state
state	O
,	O
the	O
histidine	B-site
-	I-site
diphthamide	I-site
tip	I-site
of	O
eEF2	B-protein
replaces	O
the	O
codon	B-structure_element
-	I-structure_element
anticodon	I-structure_element
–	I-structure_element
like	I-structure_element
helix	I-structure_element
of	O
PKI	B-structure_element
.	O

The	O
histidine	B-residue_name
resides	O
next	O
to	O
the	O
backbone	O
of	O
G3028	B-residue_name_number
of	O
the	O
sarcin	B-structure_element
-	I-structure_element
ricin	I-structure_element
loop	I-structure_element
and	O
near	O
the	O
diphosphate	O
of	O
GDP	B-chemical
(	O
Figure	O
5e	O
).	O

By	O
contrast	O
,	O
switch	B-structure_element
loop	I-structure_element
I	I-structure_element
(	O
aa	O
50	B-residue_range
–	I-residue_range
70	I-residue_range
in	O
S	B-species
.	I-species
cerevisiae	I-species
eEF2	B-protein
)	O
is	O
resolved	O
only	O
in	O
Structure	B-evidence
I	I-evidence
(	O
Figure	O
5	O
—	O
figure	O
supplement	O
2	O
).	O

As	O
such	O
,	O
the	O
conformations	O
of	O
SWI	B-structure_element
and	O
the	O
GTPase	B-site
center	I-site
in	O
general	O
are	O
similar	O
to	O
those	O
observed	O
in	O
ribosome	B-protein_state
-	I-protein_state
bound	I-protein_state
EF	B-protein
-	I-protein
Tu	I-protein
and	O
EF	B-protein
-	I-protein
G	I-protein
in	O
the	O
presence	B-protein_state
of	I-protein_state
GTP	B-chemical
analogs	O
.	O

Structure	B-evidence
II	I-evidence
reveals	O
PKI	B-structure_element
between	O
the	O
body	B-structure_element
A	B-site
and	I-site
P	I-site
sites	I-site
and	O
eEF2	B-protein
partially	O
advanced	O
into	O
the	O
A	B-site
site	I-site

Domain	O
IV	B-structure_element
of	O
eEF2	B-protein
is	O
further	O
entrenched	O
in	O
the	O
A	B-site
site	I-site
by	O
~	O
3	O
Å	O
relative	O
to	O
the	O
body	B-structure_element
and	O
~	O
8	O
Å	O
relative	O
to	O
the	O
head	B-structure_element
,	O
preserving	O
its	O
interactions	O
with	O
PKI	B-structure_element
.	O

In	O
Structure	B-evidence
IV	I-evidence
,	O
the	O
40S	B-complex_assembly
subunit	B-structure_element
is	O
almost	O
non	B-protein_state
-	I-protein_state
rotated	I-protein_state
relative	O
to	O
the	O
60S	B-complex_assembly
subunit	B-structure_element
,	O
and	O
the	O
40S	B-complex_assembly
head	B-structure_element
is	O
mid	B-protein_state
-	I-protein_state
swiveled	I-protein_state
.	O

In	O
Structure	B-evidence
V	I-evidence
,	O
protein	O
uS12	B-protein
is	O
shifted	O
along	O
with	O
the	O
40S	B-complex_assembly
body	B-structure_element
as	O
a	O
result	O
of	O
intersubunit	O
rotation	O
.	O

This	O
shifts	O
the	O
tip	O
of	O
helix	B-structure_element
A	I-structure_element
of	O
domain	O
III	B-structure_element
(	O
at	O
aa	O
500	B-residue_number
)	O
by	O
~	O
5	O
Å	O
(	O
relative	O
to	O
that	O
in	O
Structure	B-evidence
I	I-evidence
)	O
toward	O
domain	O
I	B-structure_element
.	O
Although	O
domain	O
III	B-structure_element
remains	O
in	O
contact	O
with	O
domain	O
V	B-structure_element
,	O
the	O
shift	O
occurs	O
in	O
the	O
direction	O
that	O
could	O
eventually	O
disconnect	O
the	O
β	B-structure_element
-	I-structure_element
platforms	I-structure_element
of	O
these	O
domains	O
.	O

The	O
imidazole	O
moiety	O
stacks	O
on	O
G6907	B-residue_name_number
(	O
corresponding	O
to	O
nt	O
36	O
in	O
the	O
tRNA	B-chemical
anticodon	O
)	O
and	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
with	O
O2	O
’	O
of	O
G6906	B-residue_name_number
(	O
nt	O
35	O
of	O
tRNA	B-chemical
).	O

The	O
front	B-structure_element
'	I-structure_element
legs	I-structure_element
'	O
(	O
SL4	B-structure_element
and	O
SL5	B-structure_element
)	O
of	O
the	O
5	B-structure_element
’-	I-structure_element
domain	I-structure_element
(	O
front	B-structure_element
end	I-structure_element
)	O
are	O
attached	O
to	O
the	O
40S	B-complex_assembly
head	B-structure_element
proteins	O
uS7	B-protein
,	O
uS11	B-protein
and	O
eS25	B-protein
(	O
Figure	O
3	O
—	O
figure	O
supplement	O
2	O
).	O

Notably	O
,	O
at	O
all	O
steps	O
,	O
the	O
head	B-structure_element
of	O
the	O
IRES	B-site
inchworm	B-protein_state
(	O
L1	B-structure_element
.	I-structure_element
1	I-structure_element
region	I-structure_element
)	O
is	O
supported	O
by	O
the	O
mobile	B-protein_state
L1	B-structure_element
stalk	I-structure_element
.	O

Partitioned	O
roles	O
of	O
40S	B-complex_assembly
subunit	B-structure_element
rearrangements	O

Specifically	O
,	O
intersubunit	O
rotation	O
allows	O
eEF2	B-protein
entry	O
into	O
the	O
A	B-site
site	I-site
,	O
while	O
the	O
head	B-structure_element
swivel	O
mediates	O
PKI	B-structure_element
translocation	O
.	O

Because	O
the	O
histidine	B-site
-	I-site
diphthamide	I-site
tip	I-site
of	O
eEF2	B-protein
(	O
H583	B-residue_name_number
,	O
H694	B-residue_name_number
and	O
Diph699	B-ptm
)	O
attaches	O
to	O
the	O
codon	B-structure_element
-	I-structure_element
anticodon	I-structure_element
-	I-structure_element
like	I-structure_element
helix	I-structure_element
of	O
PKI	B-structure_element
,	O
eEF2	B-protein
appears	O
to	O
directly	O
force	O
PKI	B-structure_element
out	O
of	O
the	O
A	B-site
site	I-site
.	O

To	O
our	O
knowledge	O
,	O
our	O
work	O
provides	O
the	O
first	O
high	O
-	O
resolution	O
view	O
of	O
the	O
dynamics	O
of	O
a	O
ribosomal	B-protein_type
translocase	I-protein_type
that	O
is	O
inferred	O
from	O
an	O
ensemble	O
of	O
structures	B-evidence
sampled	O
under	O
uniform	O
conditions	O
.	O

While	O
the	O
ribosome	B-complex_assembly
itself	O
has	O
the	O
capacity	O
to	O
translocate	O
in	O
the	O
absence	B-protein_state
of	I-protein_state
the	O
translocase	B-protein_type
,	O
spontaneous	O
translocation	O
is	O
slow	O
.	O

The	O
80S	B-complex_assembly
•	I-complex_assembly
IRES	I-complex_assembly
•	I-complex_assembly
eEF2	I-complex_assembly
structures	B-evidence
reported	O
here	O
suggest	O
two	O
main	O
roles	O
for	O
eEF2	B-protein
in	O
translocation	O
.	O

In	O
our	O
structures	B-evidence
,	O
the	O
tip	O
of	O
domain	O
IV	B-structure_element
docks	O
next	O
to	O
PKI	B-structure_element
,	O
with	O
diphthamide	B-ptm
699	I-ptm
fit	O
into	O
the	O
minor	B-site
groove	I-site
of	O
the	O
codon	B-structure_element
-	I-structure_element
anticodon	I-structure_element
-	I-structure_element
like	I-structure_element
helix	I-structure_element
of	O
PKI	B-structure_element
(	O
Figure	O
7	O
).	O

This	O
arrangement	O
rationalizes	O
inactivation	O
of	O
eEF2	B-protein
by	O
diphtheria	B-protein_type
toxin	I-protein_type
,	O
which	O
catalyzes	O
ADP	B-ptm
-	I-ptm
ribosylation	I-ptm
of	O
the	O
diphthamide	B-ptm
(	O
reviewed	O
in	O
).	O

The	O
enzyme	O
ADP	B-ptm
-	I-ptm
ribosylates	I-ptm
the	O
NE2	O
atom	O
of	O
the	O
imidazole	O
ring	O
,	O
which	O
in	O
our	O
structures	B-evidence
interacts	O
with	O
the	O
first	O
two	O
residues	O
of	O
the	O
anticodon	B-structure_element
-	I-structure_element
like	I-structure_element
strand	I-structure_element
of	O
PKI	B-structure_element
.	O

However	O
,	O
the	O
structural	O
and	O
mechanistic	O
definitions	O
of	O
the	O
locked	B-protein_state
and	O
unlocked	B-protein_state
states	O
have	O
remained	O
unclear	O
,	O
ranging	O
from	O
the	O
globally	O
distinct	O
ribosome	B-complex_assembly
conformations	O
to	O
unknown	O
local	O
rearrangements	O
,	O
e	O
.	O
g	O
.	O
those	O
in	O
the	O
decoding	B-site
center	I-site
.	O

FRET	B-evidence
data	I-evidence
indicate	O
that	O
translocation	O
of	O
2tRNA	B-complex_assembly
•	I-complex_assembly
mRNA	I-complex_assembly
on	O
the	O
70S	B-complex_assembly
ribosome	I-complex_assembly
requires	O
a	O
forward	O
-	O
and	O
-	O
reverse	O
head	B-structure_element
swivel	O
,	O
which	O
may	O
be	O
related	O
to	O
the	O
unlocking	O
phenomenon	O
.	O

Mutations	B-experimental_method
of	O
residues	O
flanking	O
A344	B-residue_name_number
in	O
E	B-species
.	I-species
coli	I-species
16S	B-chemical
rRNA	I-chemical
modestly	O
inhibit	O
translation	O
but	O
do	O
not	O
specifically	O
affect	O
EF	B-protein
-	I-protein
G	I-protein
-	O
mediated	O
translocation	O
.	O

However	O
,	O
the	O
effect	O
of	O
A344	B-residue_name_number
mutation	B-experimental_method
on	O
translation	O
was	O
not	O
addressed	O
in	O
that	O
study	O
,	O
leaving	O
the	O
question	O
open	O
whether	O
this	O
residue	O
is	O
critical	O
for	O
eEF2	B-protein
/	O
EF	B-protein
-	I-protein
G	I-protein
function	O
.	O

The	O
interaction	O
between	O
h14	B-structure_element
and	O
switch	B-structure_element
loop	I-structure_element
I	I-structure_element
is	O
not	O
resolved	O
in	O
Structures	B-evidence
II	I-evidence
to	I-evidence
V	I-evidence
,	O
in	O
all	O
of	O
which	O
the	O
small	B-structure_element
subunit	I-structure_element
is	O
partially	B-protein_state
rotated	I-protein_state
or	O
non	B-protein_state
-	I-protein_state
rotated	I-protein_state
,	O
so	O
that	O
helix	B-structure_element
14	I-structure_element
is	O
placed	O
at	O
least	O
6	O
Å	O
farther	O
from	O
eEF2	B-protein
(	O
Figure	O
5d	O
).	O

We	O
conclude	O
that	O
unlike	O
other	O
conformations	O
of	O
the	O
ribosome	B-complex_assembly
,	O
the	O
fully	B-protein_state
rotated	I-protein_state
40S	B-complex_assembly
subunit	B-structure_element
of	O
the	O
pre	B-protein_state
-	I-protein_state
translocation	I-protein_state
ribosome	B-complex_assembly
provides	O
an	O
interaction	B-site
surface	I-site
,	O
complementing	O
the	O
P	B-structure_element
stalk	I-structure_element
and	O
SRL	B-structure_element
,	O
for	O
binding	O
of	O
the	O
GTP	B-protein_state
-	I-protein_state
bound	I-protein_state
translocase	B-protein_type
.	O

This	O
displacement	O
is	O
caused	O
by	O
the	O
8	O
Å	O
movement	O
of	O
the	O
40S	B-complex_assembly
body	B-structure_element
protein	O
uS12	B-protein
upon	O
reverse	O
intersubunit	O
rotation	O
from	O
Structure	B-evidence
I	I-evidence
to	I-evidence
V	I-evidence
(	O
Figure	O
6d	O
).	O

As	O
we	O
discuss	O
below	O
,	O
Structure	B-evidence
V	I-evidence
captures	O
a	O
'	O
pre	B-protein_state
-	I-protein_state
unstacking	I-protein_state
'	O
state	O
due	O
to	O
stabilization	O
of	O
the	O
interface	B-site
between	O
domains	O
III	B-structure_element
and	O
V	B-structure_element
by	O
sordarin	B-chemical
.	O

Intersubunit	O
rearrangements	O
and	O
tRNA	B-chemical
hybrid	B-protein_state
states	O
have	O
been	O
proposed	O
to	O
play	O
key	O
roles	O
half	O
a	O
century	O
ago	O
.	O

Despite	O
an	O
impressive	O
body	B-structure_element
of	O
biochemical	B-evidence
,	I-evidence
fluorescence	I-evidence
and	I-evidence
structural	I-evidence
data	I-evidence
accumulated	O
since	O
then	O
,	O
translocation	O
remains	O
the	O
least	O
understood	O
step	O
of	O
elongation	O
.	O

Furthermore	O
,	O
the	O
step	O
-	O
wise	O
coupling	O
of	O
ribosome	B-complex_assembly
dynamics	O
with	O
IRES	B-site
translocation	O
is	O
overall	O
consistent	O
with	O
that	O
observed	O
for	O
2tRNA	B-complex_assembly
•	I-complex_assembly
mRNA	I-complex_assembly
translocation	O
in	O
solution	O
.	O

We	O
deem	O
the	O
pre	B-protein_state
-	I-protein_state
translocation	I-protein_state
complex	O
locked	B-protein_state
,	O
because	O
the	O
A	B-protein_state
-	I-protein_state
site	I-protein_state
bound	I-protein_state
ASL	O
-	O
mRNA	B-chemical
is	O
stabilized	O
by	O
interactions	O
with	O
the	O
decoding	B-site
center	I-site
.	O

This	O
unlatches	O
the	O
head	B-structure_element
,	O
allowing	O
creation	O
of	O
hitherto	O
elusive	O
intermediate	O
tRNA	B-chemical
positions	O
during	O
spontaneous	O
reverse	O
body	B-structure_element
rotation	O
.	O

Finally	O
,	O
the	O
similar	O
populations	O
of	O
particles	B-experimental_method
(	O
within	O
a	O
2X	O
range	O
)	O
in	O
our	O
80S	B-complex_assembly
•	I-complex_assembly
IRES	I-complex_assembly
•	I-complex_assembly
eEF2	I-complex_assembly
reconstructions	B-evidence
(	O
Figure	O
1	O
—	O
figure	O
supplement	O
2	O
)	O
suggest	O
that	O
the	O
intermediate	O
translocation	O
states	O
sample	O
several	O
energetically	O
similar	O
and	O
interconverting	O
conformations	O
.	O

The	O
cryo	B-experimental_method
-	I-experimental_method
EM	I-experimental_method
structures	B-evidence
demonstrate	O
that	O
the	O
TSV	B-species
IRES	B-site
structurally	O
and	O
dynamically	O
represents	O
a	O
chimera	O
of	O
the	O
2tRNA	B-complex_assembly
•	I-complex_assembly
mRNA	I-complex_assembly
translocating	O
complex	O
(	O
A	B-complex_assembly
/	I-complex_assembly
P	I-complex_assembly
-	I-complex_assembly
tRNA	I-complex_assembly
•	I-complex_assembly
P	I-complex_assembly
/	I-complex_assembly
E	I-complex_assembly
-	I-complex_assembly
tRNA	I-complex_assembly
•	I-complex_assembly
mRNA	I-complex_assembly
).	O

Intergenic	O
IRESs	B-site
,	O
in	O
turn	O
,	O
represent	O
a	O
striking	O
example	O
of	O
convergent	O
molecular	O
evolution	O
.	O

This	O
work	O
provides	O
insights	O
into	O
the	O
basic	O
mechanism	O
of	O
proteolysis	O
and	O
propeptide	B-ptm
autolysis	I-ptm
,	O
as	O
well	O
as	O
the	O
evolutionary	O
pressures	O
that	O
drove	O
the	O
proteasome	B-complex_assembly
to	O
become	O
a	O
threonine	B-protein_type
protease	I-protein_type
.	O

Data	O
from	O
biochemical	B-experimental_method
and	I-experimental_method
structural	I-experimental_method
analyses	I-experimental_method
of	O
proteasome	O
variants	O
with	O
mutations	O
in	O
the	O
β5	B-protein
propeptide	B-structure_element
and	O
the	O
active	B-site
site	I-site
strongly	O
support	O
the	O
model	O
and	O
deliver	O
novel	O
insights	O
into	O
the	O
structural	O
constraints	O
required	O
for	O
the	O
autocatalytic	B-ptm
activation	I-ptm
of	O
the	O
proteasome	B-complex_assembly
.	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

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

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

Thr	B-residue_name_number
(-	I-residue_name_number
2	I-residue_name_number
)	I-residue_name_number
positions	O
Gly	B-residue_name_number
(-	I-residue_name_number
1	I-residue_name_number
)	I-residue_name_number
O	O
via	O
hydrogen	B-bond_interaction
bonding	I-bond_interaction
(∼	O
2	O
.	O
8	O
Å	O
)	O
in	O
a	O
perfect	O
trajectory	O
for	O
the	O
nucleophilic	O
attack	O
by	O
Thr1Oγ	B-residue_name_number
(	O
Fig	O
.	O
1b	O
and	O
Supplementary	O
Fig	O
.	O
2b	O
).	O

As	O
histidine	B-residue_name
commonly	O
functions	O
as	O
a	O
proton	O
shuttle	O
in	O
the	O
catalytic	B-site
triads	I-site
of	O
serine	B-protein_type
proteases	I-protein_type
,	O
we	O
investigated	O
the	O
role	O
of	O
His	B-residue_name_number
(-	I-residue_name_number
2	I-residue_name_number
)	I-residue_name_number
in	O
processing	O
of	O
the	O
β5	B-protein
propeptide	B-structure_element
by	O
exchanging	B-experimental_method
it	I-experimental_method
for	I-experimental_method
Asn	B-residue_name
,	O
Lys	B-residue_name
,	O
Phe	B-residue_name
and	O
Ala	B-residue_name
.	O
All	O
yeast	B-taxonomy_domain
mutants	O
were	O
viable	O
at	O
30	O
°	O
C	O
,	O
but	O
suffered	O
from	O
growth	O
defects	O
at	O
37	O
°	O
C	O
with	O
the	O
H	B-mutant
(-	I-mutant
2	I-mutant
)	I-mutant
N	I-mutant
and	O
H	B-mutant
(-	I-mutant
2	I-mutant
)	I-mutant
F	I-mutant
mutants	O
being	O
most	O
affected	O
(	O
Supplementary	O
Fig	O
.	O
3b	O
and	O
Table	O
1	O
).	O

In	O
agreement	O
,	O
the	O
chymotrypsin	O
-	O
like	O
(	O
ChT	O
-	O
L	O
)	O
activity	O
of	O
H	B-mutant
(-	I-mutant
2	I-mutant
)	I-mutant
N	I-mutant
and	O
H	B-mutant
(-	I-mutant
2	I-mutant
)	I-mutant
F	I-mutant
mutant	B-protein_state
yCPs	B-complex_assembly
was	O
impaired	O
in	O
situ	O
and	O
in	O
vitro	O
(	O
Supplementary	O
Fig	O
.	O
3c	O
).	O

Next	O
,	O
we	O
examined	O
the	O
effect	O
of	O
residue	O
(-	B-residue_number
2	I-residue_number
)	I-residue_number
on	O
the	O
orientation	O
of	O
the	O
propeptide	B-structure_element
by	O
creating	B-experimental_method
mutants	I-experimental_method
that	I-experimental_method
combine	I-experimental_method
the	O
T1A	B-mutant
(	O
K81R	B-mutant
)	O
mutation	B-experimental_method
(	I-experimental_method
s	I-experimental_method
)	I-experimental_method
with	O
H	B-mutant
(-	I-mutant
2	I-mutant
)	I-mutant
L	I-mutant
,	O
H	B-mutant
(-	I-mutant
2	I-mutant
)	I-mutant
T	I-mutant
or	O
H	B-mutant
(-	I-mutant
2	I-mutant
)	I-mutant
A	I-mutant
substitutions	B-experimental_method
.	O

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

The	O
phenotype	O
of	O
the	O
β5	B-mutant
-	I-mutant
K33A	I-mutant
mutant	B-protein_state
was	O
however	O
less	O
pronounced	O
than	O
for	O
the	O
β5	B-mutant
-	I-mutant
T1A	I-mutant
-	I-mutant
K81R	I-mutant
yeast	B-taxonomy_domain
(	O
Fig	O
.	O
4a	O
).	O

Structural	B-experimental_method
comparison	I-experimental_method
of	O
the	O
β5	B-mutant
-	I-mutant
L	I-mutant
(-	I-mutant
49	I-mutant
)	I-mutant
S	I-mutant
-	I-mutant
K33A	I-mutant
and	O
β5	B-mutant
-	I-mutant
T1A	I-mutant
-	I-mutant
K81R	I-mutant
active	B-site
sites	I-site
revealed	O
that	O
mutation	B-experimental_method
of	O
Lys33	B-residue_name_number
to	O
Ala	B-residue_name
creates	O
a	O
cavity	O
that	O
is	O
filled	O
with	O
Thr1	B-residue_name_number
and	O
the	O
remnant	O
propeptide	B-structure_element
.	O

In	O
contrast	O
to	O
the	O
cis	B-protein_state
-	O
construct	O
,	O
expression	B-experimental_method
of	O
the	O
β5	B-protein
propeptide	B-structure_element
in	O
trans	B-protein_state
allowed	O
straightforward	O
isolation	B-experimental_method
and	O
crystallization	B-experimental_method
of	O
the	O
D17N	B-mutant
mutant	B-protein_state
proteasome	B-complex_assembly
.	O

This	O
observation	O
is	O
consistent	O
with	O
a	O
strongly	O
reduced	O
reactivity	O
of	O
β5	B-protein
-	O
Thr1	B-residue_name_number
and	O
the	O
crystal	B-evidence
structure	I-evidence
of	O
the	O
β5	B-mutant
-	I-mutant
D17N	I-mutant
pp	B-chemical
cis	B-protein_state
mutant	B-protein_state
in	B-protein_state
complex	I-protein_state
with	I-protein_state
carfilzomib	B-chemical
.	O

Asp166Oδ	B-residue_name_number
is	O
hydrogen	B-bond_interaction
-	I-bond_interaction
bonded	I-bond_interaction
to	O
Thr1NH2	B-residue_name_number
via	O
Ser129OH	B-residue_name_number
and	O
Ser169OH	B-residue_name_number
,	O
and	O
therefore	O
was	O
proposed	O
to	O
be	O
involved	O
in	O
catalysis	O
.	O

Instead	O
,	O
a	O
water	B-chemical
molecule	O
is	O
bound	B-protein_state
to	I-protein_state
Ser129OH	B-residue_name_number
and	O
Thr1NH2	B-residue_name_number
(	O
Supplementary	O
Fig	O
.	O
8b	O
),	O
which	O
may	O
enable	O
precursor	B-ptm
processing	I-ptm
.	O

In	O
the	O
carfilzomib	B-complex_assembly
complex	I-complex_assembly
structure	B-evidence
,	O
Thr1Oγ	B-residue_name_number
and	O
Thr1N	B-residue_name_number
incorporate	O
into	O
a	O
morpholine	O
ring	O
structure	O
and	O
Ser129	B-residue_name_number
adopts	O
its	O
WT	B-protein_state
-	O
like	O
orientation	O
.	O

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

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

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

In	O
agreement	O
,	O
soaking	B-experimental_method
crystals	I-experimental_method
with	O
the	O
CP	B-complex_assembly
inhibitors	O
bortezomib	B-chemical
or	O
carfilzomib	B-chemical
modifies	O
only	O
the	O
β1	B-protein
and	O
β2	B-protein
active	B-site
sites	I-site
,	O
while	O
leaving	O
the	O
β5	B-mutant
-	I-mutant
T1C	I-mutant
proteolytic	B-site
centres	I-site
unmodified	B-protein_state
even	O
though	O
they	O
are	O
only	O
partially	O
occupied	O
by	O
the	O
cleaved	B-protein_state
propeptide	B-structure_element
remnant	O
.	O

In	O
agreement	O
,	O
at	O
an	O
elevated	O
growing	O
temperature	O
of	O
37	O
°	O
C	O
the	O
T1S	B-mutant
mutant	B-protein_state
is	O
unable	O
to	O
grow	O
(	O
Fig	O
.	O
4a	O
).	O

Hence	O
,	O
the	O
mean	B-evidence
residence	I-evidence
time	I-evidence
of	O
carfilzomib	B-chemical
at	O
the	O
active	B-site
site	I-site
is	O
prolonged	O
and	O
the	O
probability	O
to	O
covalently	O
react	O
with	O
Ser1	B-residue_name_number
is	O
increased	O
.	O

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

Depending	O
on	O
the	O
(-	B-residue_number
2	I-residue_number
)	I-residue_number
residue	O
we	O
observed	O
various	O
propeptide	B-structure_element
conformations	O
,	O
but	O
Gly	B-residue_name_number
(-	I-residue_name_number
1	I-residue_name_number
)	I-residue_name_number
is	O
in	O
all	O
structures	B-evidence
perfectly	O
located	O
for	O
the	O
nucleophilic	O
attack	O
by	O
Thr1Oγ	B-residue_name_number
,	O
although	O
it	O
does	O
not	O
adopt	O
the	O
tight	B-structure_element
turn	I-structure_element
observed	O
for	O
the	O
prosegment	B-structure_element
of	O
subunit	O
β1	B-protein
.	O

Lys33NH2	B-residue_name_number
is	O
expected	O
to	O
act	O
as	O
the	O
proton	O
acceptor	O
during	O
autocatalytic	B-ptm
removal	I-ptm
of	O
the	O
propeptides	B-structure_element
,	O
as	O
well	O
as	O
during	O
substrate	O
proteolysis	O
,	O
while	O
Asp17Oδ	B-residue_name_number
orients	O
Lys33NH2	B-residue_name_number
and	O
makes	O
it	O
more	O
prone	O
to	O
protonation	O
by	O
raising	O
its	O
pKa	O
(	O
hydrogen	B-bond_interaction
bond	I-bond_interaction
distance	O
:	O
Lys33NH3	B-residue_name_number
+–	O
Asp17Oδ	B-residue_name_number
:	O
2	O
.	O
9	O
Å	O
).	O

Analogously	O
to	O
the	O
proteasome	B-complex_assembly
,	O
a	O
Thr	B-site
–	I-site
Lys	I-site
–	I-site
Asp	I-site
triad	I-site
is	O
also	O
found	O
in	O
L	B-protein_type
-	I-protein_type
asparaginase	I-protein_type
.	O

In	O
this	O
new	O
view	O
of	O
the	O
proteasomal	O
active	B-site
site	I-site
,	O
the	O
positively	O
charged	O
Thr1NH3	B-residue_name_number
+-	O
terminus	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
to	O
the	O
amide	O
nitrogen	O
of	O
incoming	O
peptide	O
substrates	O
and	O
stabilizes	O
as	O
well	O
as	O
activates	O
them	O
for	O
the	O
endoproteolytic	B-ptm
cleavage	I-ptm
by	O
Thr1Oγ	B-residue_name_number
(	O
Fig	O
.	O
3d	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

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

In	O
accord	O
with	O
the	O
proposed	O
Thr1	B-residue_name_number
–	O
Lys33	B-residue_name_number
–	O
Asp17	B-residue_name_number
catalytic	B-site
triad	I-site
,	O
crystallographic	B-evidence
data	I-evidence
on	O
the	O
proteolytically	B-protein_state
inactive	I-protein_state
β5	B-mutant
-	I-mutant
T1C	I-mutant
mutant	B-protein_state
demonstrate	O
that	O
the	O
interaction	O
of	O
Lys33NH2	B-residue_name_number
and	O
Cys1	B-residue_name_number
is	O
broken	O
.	O

Thr1	B-residue_name_number
is	O
well	O
anchored	O
in	O
the	O
active	B-site
site	I-site
by	O
hydrophobic	B-bond_interaction
interactions	I-bond_interaction
of	O
its	O
Cγ	O
methyl	O
group	O
with	O
Ala46	B-residue_name_number
(	O
Cβ	O
),	O
Lys33	B-residue_name_number
(	O
carbon	O
side	O
chain	O
)	O
and	O
Thr3	B-residue_name_number
(	O
Cγ	O
).	O

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

(	O
b	O
)	O
Structural	B-experimental_method
superposition	I-experimental_method
of	O
the	O
β5	B-protein
propeptides	B-structure_element
in	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
,	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
and	O
β5	B-mutant
-	I-mutant
T1A	I-mutant
-	I-mutant
K81R	I-mutant
mutant	B-protein_state
proteasomes	B-complex_assembly
.	O

While	O
the	O
residues	O
(-	B-residue_range
2	I-residue_range
)	I-residue_range
to	I-residue_range
(-	I-residue_range
4	I-residue_range
)	I-residue_range
vary	O
in	O
their	O
conformation	O
,	O
Gly	B-residue_name_number
(-	I-residue_name_number
1	I-residue_name_number
)	I-residue_name_number
and	O
Ala1	B-residue_name_number
are	O
located	O
in	O
all	O
structures	B-evidence
at	O
the	O
same	O
positions	O
.	O

The	O
strictly	B-protein_state
conserved	I-protein_state
oxyanion	O
hole	O
Gly47NH	B-residue_name_number
stabilizing	O
the	O
negatively	O
charged	O
intermediate	O
is	O
illustrated	O
as	O
a	O
semicircle	O
.	O

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

On	O
hydrolysis	O
of	O
the	O
latter	O
,	O
the	O
active	B-site
-	I-site
site	I-site
Thr1	B-residue_name_number
is	O
ready	O
for	O
catalysis	O
(	O
right	O
set	O
of	O
structures	O
).	O

The	O
proteasome	B-complex_assembly
favours	O
threonine	B-residue_name
as	O
the	O
active	O
-	O
site	O
nucleophile	O
.	O

(	O
a	O
)	O
Growth	B-experimental_method
tests	I-experimental_method
by	I-experimental_method
serial	I-experimental_method
dilution	I-experimental_method
of	O
WT	B-protein_state
and	O
pre2	O
(	O
β5	B-protein
)	O
mutant	B-protein_state
yeast	B-taxonomy_domain
cultures	O
reveal	O
growth	O
defects	O
of	O
the	O
active	B-site
-	I-site
site	I-site
mutants	B-experimental_method
under	O
the	O
indicated	O
conditions	O
after	O
2	O
days	O
(	O
2	O
d	O
)	O
of	O
incubation	O
.	O

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

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

Inhibition	B-experimental_method
assays	I-experimental_method
(	O
left	O
panel	O
).	O

p300	B-protein
-	O
catalyzed	O
histone	B-protein_type
crotonylation	B-ptm
,	O
which	O
is	O
likely	O
metabolically	O
regulated	O
,	O
stimulates	O
transcription	O
to	O
a	O
greater	O
degree	O
than	O
p300	B-protein
-	O
catalyzed	O
acetylation	B-ptm
.	O

The	O
discovery	O
of	O
individual	O
biological	O
roles	O
for	O
the	O
crotonyllysine	B-residue_name
and	O
acetyllysine	B-residue_name
marks	O
suggests	O
that	O
these	O
PTMs	O
can	O
be	O
read	O
by	O
distinct	O
readers	O
.	O

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

In	O
this	O
study	O
,	O
we	O
identified	O
the	O
Taf14	B-protein
YEATS	B-structure_element
domain	I-structure_element
as	O
a	O
reader	O
of	O
crotonyllysine	B-residue_name
that	O
binds	O
to	O
histone	B-protein_type
H3	B-protein_type
crotonylated	B-protein_state
at	O
lysine	B-residue_name_number
9	I-residue_name_number
(	O
H3K9cr	B-protein_type
)	O
via	O
a	O
distinctive	O
binding	O
mechanism	O
.	O

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

To	O
determine	O
whether	O
H3K9cr	B-protein_type
is	O
present	O
in	O
yeast	B-taxonomy_domain
,	O
we	O
generated	O
whole	B-experimental_method
cell	I-experimental_method
extracts	I-experimental_method
from	O
logarithmically	O
growing	O
yeast	B-taxonomy_domain
cells	O
and	O
subjected	O
them	O
to	O
Western	B-experimental_method
blot	I-experimental_method
analysis	I-experimental_method
using	O
antibodies	O
directed	O
towards	O
H3K9cr	B-protein_type
,	O
H3K9ac	B-protein_type
and	O
H3	B-protein_type
(	O
Fig	O
.	O
2a	O
,	O
b	O
,	O
Supplementary	O
Fig	O
.	O
3	O
and	O
Supplementary	O
Table	O
2	O
).	O

We	O
next	O
asked	O
if	O
H3K9cr	B-protein_type
is	O
regulated	O
by	O
the	O
actions	O
of	O
histone	B-protein_type
acetyltransferases	I-protein_type
(	O
HATs	B-protein_type
)	O
and	O
histone	B-protein_type
deacetylases	I-protein_type
(	O
HDACs	B-protein_type
).	O

Furthermore	O
,	O
fluctuations	O
in	O
the	O
H3K9cr	B-protein_type
levels	O
were	O
more	O
substantial	O
than	O
fluctuations	O
in	O
the	O
corresponding	O
H3K9ac	B-protein_type
levels	O
.	O

Together	O
,	O
these	O
results	O
reveal	O
that	O
H3K9cr	B-protein_type
is	O
a	O
dynamic	O
mark	O
of	O
chromatin	O
in	O
yeast	B-taxonomy_domain
and	O
suggest	O
an	O
important	O
role	O
for	O
this	O
modification	O
in	O
transcription	O
as	O
it	O
is	O
regulated	O
by	O
HATs	B-protein_type
and	O
HDACs	B-protein_type
.	O

The	O
selectivity	O
of	O
Taf14	B-protein
towards	O
crotonyllysine	B-residue_name
was	O
substantiated	O
by	O
1H	B-experimental_method
,	I-experimental_method
15N	I-experimental_method
HSQC	I-experimental_method
experiments	O
,	O
in	O
which	O
either	O
H3K9cr5	B-chemical
-	I-chemical
13	I-chemical
or	O
H3K9ac5	B-chemical
-	I-chemical
13	I-chemical
peptide	O
was	O
titrated	B-experimental_method
into	O
the	O
15N	B-protein_state
-	I-protein_state
labeled	I-protein_state
Taf14	B-protein
YEATS	B-structure_element
domain	I-structure_element
(	O
Fig	O
.	O
2c	O
and	O
Supplementary	O
Fig	O
.	O
4a	O
,	O
b	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

These	O
results	O
demonstrate	O
that	O
the	O
YEATS	B-structure_element
domain	I-structure_element
is	O
currently	O
the	O
sole	O
reader	O
of	O
crotonyllysine	B-residue_name
.	O

The	O
unique	O
and	O
previously	O
unobserved	O
aromatic	O
-	O
amide	O
/	O
aliphatic	O
-	O
aromatic	O
π	B-bond_interaction
-	I-bond_interaction
π	I-bond_interaction
-	I-bond_interaction
π	I-bond_interaction
-	I-bond_interaction
stacking	I-bond_interaction
mechanism	O
facilitates	O
the	O
specific	O
recognition	O
of	O
the	O
crotonyl	B-chemical
moiety	O
.	O

We	O
further	O
demonstrate	O
that	O
H3K9cr	B-protein_type
exists	O
in	O
yeast	B-taxonomy_domain
and	O
is	O
dynamically	O
regulated	O
by	O
HATs	B-protein_type
and	O
HDACs	B-protein_type
.	O

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

Structure	B-evidence
of	O
the	O
GAT	B-structure_element
domain	O
of	O
the	O
endosomal	O
adapter	B-protein_type
protein	I-protein_type
Tom1	B-protein

The	O
Tom1	B-protein
GAT	B-structure_element
domain	O
solution	B-evidence
structure	I-evidence
will	O
provide	O
additional	O
tools	O
for	O
modulating	O
its	O
biological	O
function	O
.	O

A	O
conformational	O
response	O
of	O
the	O
Tom1	B-protein
GAT	B-structure_element
domain	O
upon	O
Tollip	B-protein
TBD	B-structure_element
binding	O
can	O
serve	O
as	O
an	O
example	O
to	O
explain	O
mutually	O
exclusive	O
ligand	O
binding	O
events	O
.	O

The	O
Tom1	B-protein
GAT	B-structure_element
structural	B-evidence
restraints	I-evidence
yielded	O
ten	O
helical	O
structures	B-evidence
(	O
Fig	O
.	O
2A	O
,	O
B	O
)	O
with	O
a	O
root	B-evidence
mean	I-evidence
square	I-evidence
deviation	I-evidence
(	O
RMSD	B-evidence
)	O
of	O
0	O
.	O
9	O
Å	O
for	O
backbone	O
and	O
1	O
.	O
3	O
Å	O
for	O
all	O
heavy	O
atoms	O
(	O
Table	O
1	O
)	O
and	O
estimated	O
the	O
presence	O
of	O
three	O
helices	O
spanning	O
residues	O
Q216	B-residue_range
-	I-residue_range
E240	I-residue_range
(	O
α	B-structure_element
-	I-structure_element
helix	I-structure_element
1	I-structure_element
),	O
P248	B-residue_range
-	I-residue_range
Q274	I-residue_range
(	O
α	B-structure_element
-	I-structure_element
helix	I-structure_element
2	I-structure_element
),	O
and	O
E278	B-residue_range
-	I-residue_range
T306	I-residue_range
(	O
α	B-structure_element
-	I-structure_element
helix	I-structure_element
3	I-structure_element
).	O

NMR	B-experimental_method
structural	B-evidence
statistics	I-evidence
for	O
lowest	O
energy	O
conformers	O
of	O
Tom1	B-protein
GAT	B-structure_element
using	O
PSVS	B-experimental_method
.	O

Much	O
attention	O
has	O
been	O
paid	O
to	O
the	O
roles	O
of	O
haem	B-chemical
-	O
iron	B-chemical
in	O
cancer	O
development	O
.	O

Thus	O
,	O
a	O
tenuous	O
balance	O
between	O
free	O
haem	B-chemical
and	O
CO	B-chemical
plays	O
key	O
roles	O
in	O
cancer	O
development	O
and	O
chemoresistance	O
,	O
although	O
the	O
underlying	O
mechanisms	O
are	O
not	O
fully	O
understood	O
.	O

Consequently	O
,	O
the	O
five	O
-	O
coordinated	O
haem	B-chemical
of	O
PGRMC1	B-protein
has	O
an	O
open	O
surface	B-site
that	O
allows	O
its	O
dimerization	B-oligomeric_state
through	O
hydrophobic	B-bond_interaction
haem	I-bond_interaction
–	I-bond_interaction
haem	I-bond_interaction
stacking	I-bond_interaction
.	O

Furthermore	O
,	O
free	B-evidence
energy	I-evidence
of	I-evidence
dissociation	I-evidence
predicted	O
by	O
PISA	B-experimental_method
suggested	O
that	O
the	O
haem	B-chemical
-	O
mediated	O
dimer	B-oligomeric_state
is	O
stable	B-protein_state
in	O
solution	O
while	O
the	O
other	O
potential	O
interactions	O
are	O
not	O
.	O

A	O
value	O
of	O
this	O
kind	O
implies	O
that	O
the	O
PGRMC1	B-protein
dimer	B-oligomeric_state
is	O
more	O
stable	O
than	O
other	O
dimers	B-oligomeric_state
of	O
extracellular	B-structure_element
domain	I-structure_element
of	O
membrane	B-protein_type
proteins	I-protein_type
such	O
as	O
Toll	B-protein
like	I-protein
receptor	I-protein
9	I-protein
(	O
dimerization	B-oligomeric_state
Kd	B-evidence
of	O
20	O
μmol	O
l	O
−	O
1	O
)	O
(	O
ref	O
.)	O
and	O
plexin	B-protein
A2	I-protein
receptor	I-protein
(	O
dimerization	B-oligomeric_state
Kd	B-evidence
higher	O
than	O
300	O
μmol	O
l	O
−	O
1	O
)	O
(	O
ref	O
.).	O

These	O
results	O
suggest	O
that	O
CO	B-chemical
favours	O
the	O
six	O
-	O
coordinate	O
form	O
of	O
haem	B-chemical
and	O
interferes	O
with	O
the	O
haem	B-chemical
-	O
mediated	O
dimerization	B-oligomeric_state
of	O
PGRMC1	B-protein
.	O

Because	O
PGRMC1	B-protein
is	O
known	O
to	O
interact	O
with	O
EGFR	B-protein_type
and	O
to	O
accelerate	O
tumour	O
progression	O
,	O
we	O
examined	O
the	O
effect	O
of	O
haem	B-chemical
-	O
dependent	O
dimerization	B-oligomeric_state
of	O
PGRMC1	B-protein
on	O
its	O
interaction	O
with	O
EGFR	B-protein_type
by	O
using	O
purified	O
proteins	O
.	O

We	O
also	O
examined	O
the	O
effect	O
of	O
succinylacetone	B-chemical
(	O
SA	B-chemical
),	O
an	O
inhibitor	O
of	O
haem	B-chemical
biosynthesis	O
(	O
Fig	O
.	O
4d	O
).	O

Thus	O
,	O
PGRMC1	B-protein
dimerization	B-oligomeric_state
is	O
important	O
for	O
cancer	O
cell	O
proliferation	O
and	O
chemoresistance	O
.	O

We	O
examined	O
the	O
role	O
of	O
PGRMC1	B-protein
in	O
metastatic	O
progression	O
by	O
xenograft	B-experimental_method
transplantation	I-experimental_method
assays	I-experimental_method
using	O
super	O
-	O
immunodeficient	O
NOD	O
/	O
scid	O
/	O
γnull	O
(	O
NOG	O
)	O
mice	O
.	O

Recombinant	O
CYP1A2	B-protein
and	O
CYP3A4	B-protein
including	O
a	O
microsomal	O
formulation	O
containing	O
cytochrome	B-protein_type
b5	I-protein_type
and	O
cytochrome	B-protein
P450	I-protein
reductase	I-protein
,	O
drug	O
-	O
metabolizing	O
cytochromes	B-protein_type
P450	I-protein_type
,	O
interacted	O
with	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
PGRMC1	B-protein
,	O
but	O
not	O
with	O
the	O
Y113F	B-mutant
mutant	B-protein_state
,	O
in	O
a	O
haem	B-chemical
-	O
dependent	O
manner	O
(	O
Fig	O
.	O
6a	O
,	O
b	O
).	O

Enhanced	O
doxorubicin	B-chemical
sensitivity	O
was	O
modestly	O
but	O
significantly	O
induced	O
by	O
PGRMC1	B-mutant
-	I-mutant
KD	I-mutant
.	O

Recently	O
,	O
Lucas	O
et	O
al	O
.	O
reported	O
that	O
translationally	B-protein_type
-	I-protein_type
controlled	I-protein_type
tumour	I-protein_type
protein	I-protein_type
was	O
dimerized	B-protein_state
by	O
binding	O
with	O
haem	B-chemical
,	O
but	O
its	O
structural	O
basis	O
remains	O
unclear	O
.	O

Sequence	B-experimental_method
alignments	I-experimental_method
show	O
that	O
haem	B-site
-	I-site
binding	I-site
residues	I-site
(	O
Tyr113	B-residue_name_number
,	O
Tyr107	B-residue_name_number
,	O
Lys163	B-residue_name_number
and	O
Tyr164	B-residue_name_number
)	O
in	O
PGRMC1	B-protein
are	O
conserved	B-protein_state
among	O
MAPR	B-protein_type
proteins	O
(	O
Supplementary	O
Fig	O
.	O
5	O
).	O

Moreover	O
,	O
exposure	O
of	O
cancer	O
cells	O
to	O
stimuli	O
such	O
as	O
hypoxia	O
,	O
radiation	O
and	O
chemotherapy	O
causes	O
cell	O
damages	O
and	O
leads	O
to	O
protein	O
degradation	O
,	O
resulting	O
in	O
increased	O
levels	O
of	O
TCA	O
cycle	O
intermediates	O
and	O
in	O
an	O
enhanced	O
haem	B-chemical
biosynthesis	O
.	O

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

(	O
g	O
,	O
h	O
)	O
HCT116	O
cells	O
were	O
treated	O
with	O
or	O
without	O
EGF	B-protein_type
,	O
SA	B-chemical
,	O
RuCl3	B-chemical
and	O
CORM3	B-chemical
as	O
indicated	O
,	O
and	O
components	O
of	O
the	O
EGFR	B-protein_type
signaling	O
pathway	O
were	O
detected	O
by	O
Western	B-experimental_method
blotting	I-experimental_method
.	O

(	O
c	O
)	O
Binding	B-experimental_method
assay	I-experimental_method
was	O
performed	O
as	O
in	O
(	O
a	O
)	O
using	O
haem	B-protein_state
-	I-protein_state
bound	I-protein_state
FLAG	O
-	O
PGRMC1	B-protein
wt	B-protein_state
and	O
CYP1A2	B-protein
with	O
or	O
without	O
RuCl3	B-chemical
and	O
CORM3	B-chemical
.	O

We	O
generated	O
high	O
-	O
resolution	O
structures	B-evidence
of	O
the	O
1E6	B-complex_assembly
TCR	I-complex_assembly
bound	B-protein_state
to	I-protein_state
7	O
altered	B-chemical
peptide	I-chemical
ligands	I-chemical
,	O
including	O
a	O
pathogen	O
-	O
derived	O
peptide	O
that	O
was	O
an	O
order	O
of	O
magnitude	O
more	O
potent	O
than	O
the	O
natural	O
self	O
-	O
peptide	O
.	O

Highly	O
potent	O
antigens	O
of	O
the	O
1E6	B-complex_assembly
TCR	I-complex_assembly
engaged	O
with	O
a	O
strong	O
antipathogen	B-evidence
-	I-evidence
like	I-evidence
binding	I-evidence
affinity	I-evidence
;	O
this	O
engagement	O
was	O
governed	O
though	O
an	O
energetic	O
switch	O
from	O
an	O
enthalpically	O
to	O
entropically	O
driven	O
interaction	O
compared	O
with	O
the	O
natural	O
autoimmune	O
ligand	O
.	O

This	O
ability	O
is	O
required	O
to	O
enable	O
the	O
estimated	O
25	O
million	O
distinct	O
TCRs	B-complex_assembly
expressed	O
in	O
humans	B-species
to	O
provide	O
effective	O
immune	O
coverage	O
against	O
all	O
possible	O
foreign	O
peptide	O
antigens	O
.	O

The	O
RQFGPDWIVA	B-chemical
sequence	O
(	O
present	O
in	O
C	B-species
.	I-species
asparagiforme	I-species
)	O
activated	O
the	O
1E6	O
T	O
cell	O
with	O
around	O
1	O
log	O
–	O
greater	O
potency	O
compared	O
with	O
ALWGPDPAAA	B-chemical
.	O

The	O
range	O
of	O
Tm	B-evidence
was	O
between	O
49	O
.	O
4	O
°	O
C	O
(	O
RQFGPDWIVA	B-chemical
)	O
and	O
60	O
.	O
3	O
°	O
C	O
(	O
YQFGPDFPIA	B-chemical
),	O
with	O
an	O
average	O
approximately	O
55	O
°	O
C	O
,	O
similar	O
to	O
our	O
previous	O
findings	O
.	O

First	O
,	O
the	O
1E6	O
T	O
cell	O
could	O
still	O
functionally	O
respond	O
to	O
peptide	O
when	O
the	O
TCR	B-evidence
binding	I-evidence
affinity	I-evidence
was	O
extremely	O
weak	O
,	O
e	O
.	O
g	O
.,	O
the	O
1E6	B-evidence
TCR	I-evidence
binding	I-evidence
affinity	I-evidence
for	O
the	O
A2	B-chemical
-	I-chemical
MVWGPDPLYV	I-chemical
peptide	O
was	O
KD	B-evidence
=	O
~	O
600	O
μM	O
.	O
Second	O
,	O
the	O
1E6	B-complex_assembly
TCR	I-complex_assembly
bound	B-protein_state
to	I-protein_state
A2	B-chemical
-	I-chemical
RQFGPDFPTI	I-chemical
with	O
KD	B-evidence
=	O
0	O
.	O
5	O
μM	O
,	O
equivalent	O
to	O
the	O
binding	B-evidence
affinity	I-evidence
of	O
the	O
very	O
strongest	O
antipathogen	O
TCRs	B-complex_assembly
.	O

To	O
confirm	O
the	O
affinity	B-evidence
spread	O
detected	O
by	O
SPR	B-experimental_method
,	O
and	O
to	O
evaluate	O
whether	O
experiments	O
performed	O
using	O
soluble	O
molecules	O
were	O
biologically	O
relevant	O
to	O
events	O
at	O
the	O
T	O
cell	O
surface	O
,	O
we	O
determined	O
the	O
effective	O
2D	B-evidence
affinity	I-evidence
of	O
each	O
APL	B-chemical
using	O
an	O
adhesion	B-experimental_method
frequency	I-experimental_method
assay	I-experimental_method
in	O
which	O
a	O
human	B-species
rbc	O
coated	O
in	O
pMHC	B-complex_assembly
acted	O
as	O
an	O
adhesion	O
sensor	O
.	O

As	O
with	O
the	O
3D	B-evidence
affinity	I-evidence
measurements	O
,	O
the	O
2D	B-evidence
affinity	I-evidence
measurements	O
correlated	O
well	O
with	O
the	O
EC50	B-evidence
values	O
for	O
each	O
ligand	O
(	O
Figure	O
2K	O
)	O
demonstrating	O
a	O
strong	O
correlation	O
(	O
Pearson	B-evidence
’	I-evidence
s	I-evidence
correlation	I-evidence
=	O
0	O
.	O
8	O
,	O
P	B-evidence
=	O
0	O
.	O
01	O
)	O
between	O
T	O
cell	O
antigen	O
sensitivity	O
and	O
TCR	B-evidence
binding	I-evidence
affinity	I-evidence
.	O

Overall	O
,	O
the	O
1E6	B-complex_assembly
TCR	I-complex_assembly
used	O
a	O
canonical	O
binding	O
mode	O
to	O
engage	O
each	O
APL	B-chemical
with	O
the	O
TCR	B-complex_assembly
α	B-structure_element
-	I-structure_element
chain	I-structure_element
positioned	O
over	O
the	O
MHC	B-complex_assembly
class	I-complex_assembly
I	I-complex_assembly
(	O
MHCI	B-complex_assembly
)	O
α2	B-structure_element
-	I-structure_element
helix	I-structure_element
and	O
the	O
TCR	B-complex_assembly
β	B-structure_element
-	I-structure_element
chain	I-structure_element
over	O
the	O
MHCI	B-complex_assembly
α	B-structure_element
-	I-structure_element
1	I-structure_element
helix	I-structure_element
,	O
straddling	O
the	O
peptide	O
cargo	O
.	O

Focused	O
hotspot	O
binding	O
around	O
a	O
conserved	B-protein_state
GPD	B-structure_element
motif	I-structure_element
enables	O
the	O
1E6	B-complex_assembly
TCR	I-complex_assembly
to	O
tolerate	O
peptide	O
degeneracy	O
.	O

Although	O
the	O
number	O
of	O
peptide	O
contacts	O
was	O
a	O
good	O
predictor	O
of	O
TCR	B-evidence
binding	I-evidence
affinity	I-evidence
for	O
some	O
of	O
the	O
APLs	B-chemical
,	O
for	O
others	O
,	O
the	O
correlation	O
was	O
poor	O
(	O
Pearson	B-evidence
’	I-evidence
s	I-evidence
correlation	I-evidence
=	O
0	O
.	O
045	O
,	O
P	O
=	O
0	O
.	O
92	O
),	O
possibly	O
because	O
of	O
different	O
resolutions	O
for	O
each	O
complex	O
structure	B-evidence
.	O

The	O
unligated	B-protein_state
A2	B-chemical
-	I-chemical
MVWGPDPLYV	I-chemical
(	O
KD	B-evidence
=	O
~	O
600	O
μM	O
)	O
structure	B-evidence
revealed	O
that	O
the	O
side	O
chain	O
Tyr9	B-residue_name_number
swung	O
around	O
8	O
Å	O
in	O
the	O
complex	O
structure	B-evidence
,	O
subsequently	O
making	O
contacts	O
with	O
TCR	B-complex_assembly
residues	O
Asp30β	B-residue_name_number
and	O
Asn51β	B-residue_name_number
(	O
Figure	O
6A	O
and	O
Figure	O
5A	O
,	O
respectively	O
).	O

The	O
overall	O
free	B-evidence
binding	I-evidence
energies	I-evidence
(	O
ΔG	B-evidence
°)	I-evidence
were	O
between	O
–	O
4	O
.	O
4	O
and	O
–	O
8	O
.	O
6	O
kcal	O
/	O
mol	O
,	O
reflecting	O
the	O
wide	O
range	O
of	O
TCR	B-evidence
binding	I-evidence
affinities	I-evidence
we	O
observed	O
for	O
the	O
different	O
APLs	B-chemical
.	O

The	O
enthalpic	O
contribution	O
in	O
each	O
complex	O
did	O
not	O
follow	O
a	O
clear	O
trend	O
with	O
affinity	B-evidence
,	O
with	O
all	O
but	O
the	O
1E6	B-complex_assembly
-	I-complex_assembly
A2	I-complex_assembly
-	I-complex_assembly
RQFGPDFPTI	I-complex_assembly
interaction	O
(	O
ΔH	B-evidence
°	I-evidence
=	O
6	O
.	O
3	O
kcal	O
/	O
mol	O
)	O
generating	O
an	O
energetically	O
favorable	O
enthalpy	B-evidence
value	O
(	O
ΔH	B-evidence
°	I-evidence
=	O
–	O
3	O
.	O
7	O
to	O
–	O
11	O
.	O
4	O
kcal	O
/	O
mol	O
);	O
this	O
indicated	O
a	O
net	O
gain	O
in	O
electrostatic	O
interactions	O
during	O
complex	O
formation	O
.	O

Furthermore	O
,	O
the	O
structures	O
of	O
the	O
unligated	B-protein_state
pMHCs	B-complex_assembly
demonstrated	O
that	O
,	O
for	O
these	O
stronger	O
-	O
affinity	B-evidence
ligands	O
,	O
there	O
was	O
less	O
conformational	O
difference	O
between	O
the	O
TCR	B-complex_assembly
ligated	B-protein_state
pMHCs	B-complex_assembly
compared	O
with	O
the	O
weaker	O
-	O
affinity	B-evidence
ligands	O
(	O
Figure	O
6	O
).	O

Sethi	O
and	O
colleagues	O
recently	O
demonstrated	O
that	O
the	O
MHCII	B-protein_type
-	O
restricted	O
Hy	B-protein
.	I-protein
1B11	I-protein
TCR	B-complex_assembly
,	O
which	O
was	O
isolated	O
from	O
a	O
patient	O
with	O
multiple	O
sclerosis	O
,	O
could	O
anchor	O
into	O
a	O
deep	B-site
pocket	I-site
formed	O
from	O
peptide	O
residues	O
2	B-residue_number
,	O
3	B-residue_number
,	O
and	O
5	B-residue_number
(	O
from	O
MBP85	B-protein
–	I-protein
99	I-protein
bound	B-protein_state
to	I-protein_state
HLA	B-protein
-	I-protein
DQ1	I-protein
).	O

Although	O
the	O
1E6	O
T	O
cell	O
was	O
able	O
to	O
activate	O
weakly	O
with	O
peptides	O
that	O
lacked	B-protein_state
this	O
motif	O
,	O
we	O
were	O
unable	O
to	O
robustly	O
measure	O
binding	B-evidence
affinities	I-evidence
or	O
generate	O
complex	O
structures	B-evidence
with	O
these	O
ligands	O
,	O
highlighting	O
the	O
central	O
role	O
of	O
this	O
interaction	O
during	O
1E6	O
T	O
cell	O
antigen	O
recognition	O
.	O

These	O
findings	O
are	O
also	O
analogous	O
to	O
the	O
observed	O
binding	O
mode	O
of	O
the	O
Hy	B-protein
.	I-protein
1B11	I-protein
TCR	B-complex_assembly
,	O
in	O
which	O
one	O
aromatic	O
residue	O
of	O
the	O
TCR	B-complex_assembly
CDR3α	B-structure_element
loop	I-structure_element
anchored	O
into	O
a	O
pocket	O
created	O
by	O
a	O
conserved	O
peptide	O
motif	O
.	O

Despite	O
some	O
weak	O
statistical	O
correlation	O
between	O
the	O
surface	B-evidence
complementarity	I-evidence
(	O
SC	B-evidence
)	O
and	O
affinity	B-evidence
,	O
closer	O
inspection	O
of	O
the	O
interface	B-site
revealed	O
no	O
obvious	O
structural	O
signature	O
that	O
could	O
definitively	O
explain	O
the	O
differences	O
in	O
antigen	B-evidence
potency	I-evidence
and	O
TCR	B-evidence
binding	I-evidence
strength	I-evidence
between	O
the	O
different	O
ligands	O
.	O

However	O
,	O
similar	O
to	O
our	O
findings	O
in	O
other	O
systems	O
,	O
modifications	O
to	O
residues	O
outside	O
of	O
the	O
canonical	O
central	B-structure_element
peptide	I-structure_element
bulge	I-structure_element
were	O
important	O
for	O
generating	O
new	O
interactions	O
.	O

These	O
data	O
also	O
explain	O
our	O
previous	O
findings	O
that	O
alteration	O
of	O
the	O
anchor	B-structure_element
residue	I-structure_element
at	O
peptide	O
position	O
2	B-residue_number
(	O
Leu	B-mutant
-	I-mutant
Gln	I-mutant
)	O
has	O
a	O
direct	O
effect	O
on	O
1E6	B-evidence
TCR	I-evidence
binding	I-evidence
affinity	I-evidence
because	O
our	O
structural	B-experimental_method
analysis	I-experimental_method
demonstrated	O
that	O
1E6	O
made	O
3	O
additional	O
bonds	O
with	O
A2	B-chemical
-	I-chemical
AQWGPDPAAA	I-chemical
compared	O
with	O
A2	B-chemical
-	I-chemical
ALWGPDPAAA	I-chemical
,	O
consistent	O
with	O
the	O
>	O
3	O
-	O
fold	O
stronger	O
binding	B-evidence
affinity	I-evidence
.	O

Although	O
no	O
energetic	O
signature	O
appears	O
to	O
exist	O
for	O
different	O
TCRs	B-complex_assembly
,	O
we	O
used	O
thermodynamic	B-experimental_method
analysis	I-experimental_method
here	O
to	O
explore	O
whether	O
changes	O
in	O
energetics	O
could	O
help	O
explain	O
ligand	O
discrimination	O
by	O
a	O
single	O
TCR	B-complex_assembly
.	O

This	O
analysis	O
demonstrated	O
a	O
strong	O
relationship	O
(	O
according	O
to	O
the	O
Pearson	B-experimental_method
’	I-experimental_method
s	I-experimental_method
correlation	I-experimental_method
analysis	I-experimental_method
)	O
between	O
the	O
energetic	O
signature	O
used	O
by	O
the	O
1E6	B-complex_assembly
TCR	I-complex_assembly
and	O
the	O
sensitivity	O
of	O
the	O
1E6	O
T	O
cell	O
clone	O
to	O
different	O
APLs	B-chemical
.	O

(	O
C	O
)	O
The	O
1E6	O
T	O
cell	O
clone	O
was	O
stained	O
,	O
in	O
duplicate	O
,	O
with	O
tetramers	B-oligomeric_state
composed	O
of	O
each	O
APL	B-chemical
(	O
colored	O
as	O
above	O
)	O
presented	O
by	O
HLA	B-protein
-	I-protein
A	I-protein
*	I-protein
0201	I-protein
.	O
(	O
D	O
)	O
The	O
stability	O
of	O
each	O
APL	B-chemical
(	O
colored	O
as	O
above	O
)	O
was	O
tested	O
,	O
in	O
duplicate	O
,	O
using	O
CD	B-experimental_method
by	O
recording	O
the	O
peak	O
at	O
218	O
nm	O
absorbance	O
from	O
5	O
°	O
C	O
–	O
90	O
°	O
C	O
.	O

The	O
1E6	B-complex_assembly
TCR	I-complex_assembly
uses	O
a	O
conserved	O
binding	O
mode	O
to	O
engage	O
A2	B-chemical
-	I-chemical
ALWGPDPAAA	I-chemical
and	O
the	O
APLs	B-chemical
.	O

Interaction	O
between	O
1E6	B-complex_assembly
TCR	I-complex_assembly
(	O
gray	O
illustration	O
)	O
residues	O
Tyr97α	B-residue_name_number
and	O
Tyr97β	B-residue_name_number
(	O
the	O
position	O
of	O
these	O
side	O
chains	O
in	O
the	O
TCR	B-complex_assembly
in	B-protein_state
complex	I-protein_state
with	I-protein_state
all	O
7	O
APLs	B-chemical
,	O
and	O
the	O
previously	O
reported	O
A2	B-chemical
-	I-chemical
ALWGPDPAAA	I-chemical
epitope	O
,	O
is	O
shown	O
in	O
multicolored	O
sticks	O
;	O
ref	O
.)	O
and	O
the	O
GPD	B-structure_element
peptide	I-structure_element
motif	I-structure_element
(	O
the	O
position	O
of	O
these	O
side	O
chains	O
in	O
all	O
7	O
APLs	B-chemical
and	O
A2	B-chemical
-	I-chemical
ALWGPDPAAA	I-chemical
in	B-protein_state
complex	I-protein_state
with	I-protein_state
the	O
1E6	B-complex_assembly
TCR	I-complex_assembly
is	O
shown	O
in	O
multicolored	O
sticks	O
).	O

Interactions	O
between	O
the	O
1E6	B-complex_assembly
TCR	I-complex_assembly
and	O
peptide	O
residues	O
outside	O
of	O
the	O
conserved	B-protein_state
GPD	B-structure_element
motif	I-structure_element
.	O

Hydrogen	B-bond_interaction
bonds	I-bond_interaction
are	O
shown	O
as	O
red	O
dotted	O
lines	O
;	O
van	B-bond_interaction
der	I-bond_interaction
Waals	I-bond_interaction
(	I-bond_interaction
vdW	I-bond_interaction
)	I-bond_interaction
contacts	I-bond_interaction
are	O
shown	O
as	O
black	O
dotted	O
lines	O
.	O

(	O
A	O
)	O
Interaction	O
between	O
the	O
1E6	B-complex_assembly
TCR	I-complex_assembly
(	O
black	O
illustration	O
and	O
sticks	O
)	O
and	O
A2	B-chemical
-	I-chemical
MVWGPDPLYV	I-chemical
(	O
black	O
illustration	O
and	O
sticks	O
).	O
(	O
B	O
)	O
Interaction	O
between	O
the	O
1E6	B-complex_assembly
TCR	I-complex_assembly
(	O
red	O
illustration	O
and	O
sticks	O
)	O
and	O
A2	B-chemical
-	I-chemical
YLGGPDFPTI	I-chemical
(	O
red	O
illustration	O
and	O
sticks	O
).	O
(	O
C	O
)	O
Interaction	O
between	O
the	O
1E6	B-complex_assembly
TCR	I-complex_assembly
(	O
blue	O
illustration	O
and	O
sticks	O
)	O
and	O
A2	B-chemical
-	I-chemical
ALWGPDPAAA	I-chemical
(	O
blue	O
illustration	O
and	O
sticks	O
)	O
reproduced	O
from	O
previous	O
published	O
data	O
.	O

The	O
1E6	B-complex_assembly
TCR	I-complex_assembly
makes	O
distinct	O
peptide	O
contacts	O
with	O
the	O
MHC	B-site
surface	I-site
depending	O
on	O
the	O
peptide	O
cargo	O
.	O

Boxes	O
show	O
total	O
contacts	O
between	O
the	O
1E6	B-complex_assembly
TCR	I-complex_assembly
and	O
these	O
key	O
residues	O
(	O
green	O
boxes	O
are	O
MHC	B-complex_assembly
residues	O
;	O
white	O
boxes	O
are	O
TCR	B-complex_assembly
residues	O
).	O