dev.tsv

words	labels
The	O
human	O
gut	O
microbiota	O
influences	O
the	O
course	O
of	O
human	O
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
	O
The	O
ability	O
to	O
acquire	O
energy	O
from	O
carbohydrates	O
of	O
dietary	O
or	O
host	O
origin	O
is	O
central	O
to	O
the	O
adaptation	O
of	O
human	O
gut	O
bacterial	O
species	O
to	O
their	O
niche	O
.	O
	O
Xyloglucan	O
and	O
the	O
Bacteroides	O
ovatus	O
xyloglucan	O
utilization	O
locus	O
(	O
XyGUL	O
).	O
(	O
A	O
)	O
Representative	O
structures	O
of	O
common	O
xyloglucans	O
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
	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	O
,	O
a	O
key	O
outstanding	O
question	O
regarding	O
this	O
locus	O
is	O
how	O
XyG	O
recognition	O
is	O
mediated	O
at	O
the	O
cell	O
surface	O
.	O
	O
Here	O
,	O
the	O
SGBPs	O
very	O
likely	O
work	O
in	O
concert	O
with	O
the	O
cell	O
-	O
surface	O
-	O
localized	O
endo	O
-	O
xyloglucanase	O
B	O
.	O
ovatus	O
GH5	O
(	O
BoGH5	O
)	O
to	O
recruit	O
and	O
cleave	O
XyG	O
for	O
subsequent	O
periplasmic	O
import	O
via	O
the	O
SusC	O
-	O
like	O
TBDT	O
of	O
the	O
XyGUL	O
(	O
Fig	O
.	O
1B	O
and	O
C	O
).	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	O
hydrolases	O
of	O
the	O
XyGUL	O
,	O
we	O
reported	O
preliminary	O
affinity	O
PAGE	O
and	O
isothermal	O
titration	O
calorimetry	O
(	O
ITC	O
)	O
data	O
indicating	O
that	O
both	O
SGBP	O
-	O
A	O
and	O
SGBP	O
-	O
B	O
are	O
competent	O
xyloglucan	O
-	O
binding	O
proteins	O
(	O
affinity	O
constant	O
[	O
Ka	O
]	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
	O
Together	O
,	O
these	O
results	O
highlight	O
the	O
high	O
specificities	O
of	O
SGBP	O
-	O
A	O
and	O
SGBP	O
-	O
B	O
for	O
XyG	O
,	O
which	O
is	O
concordant	O
with	O
their	O
association	O
with	O
XyG	O
-	O
specific	O
GHs	O
in	O
the	O
XyGUL	O
,	O
as	O
well	O
as	O
transcriptomic	O
analysis	O
indicating	O
that	O
B	O
.	O
ovatus	O
has	O
discrete	O
PUL	O
for	O
MLG	O
,	O
GM	O
,	O
and	O
GGM	O
(	O
11	O
).	O
	O
The	O
apo	O
structure	O
is	O
color	O
ramped	O
from	O
blue	O
to	O
red	O
.	O
	O
The	O
approximate	O
length	O
of	O
each	O
glycan	O
-	O
binding	O
site	O
is	O
displayed	O
,	O
colored	O
to	O
match	O
the	O
protein	O
structures	O
.	O
(	O
E	O
)	O
Stereo	O
view	O
of	O
the	O
xyloglucan	O
-	O
binding	O
site	O
of	O
SGBP	O
-	O
A	O
,	O
displaying	O
all	O
residues	O
within	O
4	O
Å	O
of	O
the	O
ligand	O
.	O
	O
Potential	O
hydrogen	O
-	O
bonding	O
interactions	O
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
	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	O
displays	O
a	O
significant	O
4	O
.	O
9	O
-	O
fold	O
decrease	O
in	O
the	O
Ka	O
value	O
for	O
XyG	O
,	O
while	O
the	O
W306A	B-mutant
substitution	O
completely	O
abolishes	O
XyG	O
binding	O
.	O
	O
Prolines	O
between	O
domains	O
are	O
indicated	O
as	O
spheres	O
.	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	O
-	O
and	O
xylogluco	O
-	O
oligosaccharides	O
.	O
	O
Hoping	O
to	O
achieve	O
a	O
higher	O
-	O
resolution	O
view	O
of	O
the	O
SGBP	O
-	O
B	O
–	O
xyloglucan	O
interaction	O
,	O
we	O
solved	O
the	O
crystal	O
structure	O
of	O
the	O
fused	B-mutant
CD	I-mutant
domains	I-mutant
in	O
complex	O
with	O
XyGO2	O
(	O
1	O
.	O
57	O
Å	O
,	O
Rwork	O
=	O
15	O
.	O
6	O
%,	O
Rfree	O
=	O
17	O
.	O
1	O
%,	O
residues	O
230	O
to	O
489	O
)	O
(	O
Table	O
2	O
).	O
	O
While	O
this	O
may	O
occur	O
for	O
a	O
number	O
of	O
reasons	O
in	O
crystal	O
structures	O
,	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	O
averaged	O
throughout	O
the	O
lattice	O
.	O
	O
The	O
similarity	O
of	O
the	O
glycan	O
specificity	O
of	O
SGBP	O
-	O
A	O
and	O
SGBP	O
-	O
B	O
presents	O
an	O
intriguing	O
conundrum	O
regarding	O
their	O
individual	O
roles	O
in	O
XyG	O
utilization	O
by	O
B	O
.	O
ovatus	O
.	O
	O
The	O
ΔSGBP	B-mutant
-	I-mutant
A	I-mutant
(	O
ΔBacova_02651	B-mutant
)	O
strain	O
(	O
cf	O
.	O
	O
The	O
specific	O
glycan	O
signal	O
that	O
upregulates	O
BoXyGUL	O
is	O
currently	O
unknown	O
.	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	O
binding	O
by	O
SGBP	O
-	O
A	O
enhances	O
transcriptional	O
activation	O
of	O
the	O
XyGUL	O
.	O
	O
Beyond	O
SGBP	O
-	O
A	O
and	O
SGBP	O
-	O
B	O
,	O
we	O
speculated	O
that	O
the	O
catalytically	O
feeble	O
endo	O
-	O
xyloglucanase	O
GH9	O
,	O
which	O
is	O
expendable	O
for	O
growth	O
in	O
the	O
presence	O
of	O
GH5	O
,	O
might	O
also	O
play	O
a	O
role	O
in	O
glycan	O
binding	O
to	O
the	O
cell	O
surface	O
.	O
	O
We	O
hypothesize	O
that	O
during	O
exponential	O
growth	O
the	O
essential	O
role	O
of	O
SGBP	O
-	O
A	O
extends	O
beyond	O
glycan	O
recognition	O
,	O
perhaps	O
due	O
to	O
a	O
critical	O
interaction	O
with	O
the	O
TBDT	O
.	O
	O
A	O
particularly	O
understudied	O
aspect	O
of	O
glycan	O
utilization	O
is	O
the	O
mechanism	O
of	O
import	O
via	O
TBDTs	O
(	O
SusC	O
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	O
.	O
	O
Similarly	O
,	O
the	O
deletion	O
of	O
BT1762	O
encoding	O
a	O
fructan	O
-	O
targeting	O
SusD	O
-	O
like	O
protein	O
in	O
B	O
.	O
thetaiotaomicron	O
did	O
not	O
result	O
in	O
a	O
dramatic	O
loss	O
of	O
growth	O
on	O
fructans	O
.	O
	O
Furthermore	O
,	O
considering	O
the	O
broader	O
distribution	O
of	O
TBDTs	O
in	O
PUL	O
lacking	O
SGBPs	O
(	O
sometimes	O
known	O
as	O
carbohydrate	O
utilization	O
containing	O
TBDT	O
[	O
CUT	O
]	O
loci	O
;	O
see	O
reference	O
and	O
reviewed	O
in	O
reference	O
)	O
across	O
bacterial	O
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	O
.	O
	O
Equally	O
intriguing	O
is	O
the	O
observation	O
that	O
while	O
SusD	O
-	O
like	O
proteins	O
such	O
as	O
SGBP	O
-	O
A	O
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	O
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	O
-	O
binding	O
lipoproteins	O
with	O
little	O
or	O
no	O
sequence	O
or	O
structural	O
conservation	O
,	O
even	O
among	O
syntenic	O
PUL	O
that	O
target	O
the	O
same	O
polysaccharide	O
.	O
	O
Because	O
the	O
intestinal	O
ecosystem	O
is	O
a	O
dense	O
consortium	O
of	O
bacteria	O
that	O
must	O
compete	O
for	O
their	O
nutrients	O
,	O
these	O
multimodular	O
SGBPs	O
may	O
reflect	O
ongoing	O
evolutionary	O
experiments	O
to	O
enhance	O
glycan	O
uptake	O
efficiency	O
.	O
	O
Whether	O
organisms	O
that	O
express	O
longer	O
SGBPs	O
,	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	O
is	O
presently	O
unknown	O
.	O
	O
Monoclonal	O
antibodies	O
inhibiting	O
IL	O
-	O
17A	O
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
	O
Tested	O
in	O
primary	O
human	O
cells	O
,	O
HAP	O
blocked	O
the	O
production	O
of	O
multiple	O
inflammatory	O
cytokines	O
.	O
	O
These	O
polypeptides	O
form	O
covalent	O
homodimers	O
,	O
and	O
IL	O
-	O
17A	O
and	O
IL	O
-	O
17F	O
also	O
form	O
an	O
IL	O
-	O
17A	O
/	O
IL	O
-	O
17F	O
hetereodimer	O
.	O
	O
In	O
these	O
structures	O
,	O
both	O
IL	O
-	O
17A	O
and	O
IL	O
-	O
17F	O
adopt	O
a	O
cysteine	O
-	O
knot	O
fold	O
with	O
two	O
intramolecular	O
disulfides	O
and	O
two	O
interchain	O
disulfide	O
bonds	O
that	O
covalently	O
link	O
two	O
monomers	O
.	O
	O
Identification	O
of	O
IL	O
-	O
17A	O
peptide	O
inhibitors	O
	O
Peptides	O
specifically	O
binding	O
to	O
human	O
IL	O
-	O
17A	O
were	O
identified	O
from	O
phage	O
panning	O
using	O
cyclic	O
and	O
linear	O
peptide	O
libraries	O
(	O
Supplementary	O
Figure	O
S1	O
).	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	O
IL	O
-	O
17A	O
signaling	O
through	O
IL	O
-	O
17RA	O
in	O
an	O
IL	O
-	O
17A	O
/	O
IL	O
-	O
17RA	O
competition	O
ELISA	O
assay	O
where	O
unlabeled	O
IL	O
-	O
17A	O
was	O
used	O
as	O
positive	O
control	O
to	O
inhibit	O
biotinylated	O
IL	O
-	O
17A	O
binding	O
.	O
	O
An	O
alanine	O
scan	O
of	O
peptide	O
2	O
,	O
an	O
analogue	O
of	O
1	O
with	O
a	O
lysine	O
to	O
arginine	O
substitution	O
at	O
position	O
14	O
,	O
was	O
initiated	O
.	O
	O
Modifications	O
at	O
positions	O
2	O
and	O
14	O
were	O
shown	O
to	O
display	O
improvement	O
in	O
binding	O
affinity	O
(	O
data	O
not	O
shown	O
).	O
	O
In	O
this	O
work	O
,	O
32	O
–	O
34	O
are	O
capped	O
by	O
protective	O
acetyl	O
group	O
and	O
reflect	O
the	O
same	O
inactivity	O
as	O
reported	O
.	O
	O
Peptide	O
45	O
,	O
dimerized	O
via	O
attachment	O
of	O
a	O
PEG21	O
spacer	O
at	O
position	O
14	O
(	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	O
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	O
functionalized	O
with	O
a	O
PEG21	O
group	O
at	O
position	O
14	O
as	O
peptide	O
48	O
had	O
an	O
IC50	O
of	O
21	O
nM	O
(	O
Supplementary	O
Scheme	O
S2	O
).	O
	O
To	O
further	O
characterize	O
the	O
interaction	O
of	O
HAP	O
with	O
IL	O
-	O
17A	O
,	O
we	O
set	O
out	O
to	O
determine	O
its	O
in	O
vitro	O
binding	O
affinity	O
,	O
specificity	O
and	O
kinetic	O
profile	O
using	O
Surface	O
Plasmon	O
Resonance	O
(	O
SPR	O
)	O
methods	O
(	O
Fig	O
.	O
1A	O
).	O
	O
HAP	O
blocks	O
IL	O
-	O
17A	O
signaling	O
in	O
a	O
human	O
primary	O
cell	O
assay	O
	O
In	O
patients	O
,	O
the	O
concentration	O
of	O
IL	O
-	O
17A	O
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	O
-	O
17A	O
induced	O
IL	O
-	O
8	O
production	O
in	O
vitro	O
.	O
	O
It	O
is	O
known	O
that	O
an	O
antibody	O
antigen	O
-	O
binding	O
fragment	O
(	O
Fab	O
)	O
can	O
be	O
used	O
as	O
crystallization	O
chaperones	O
in	O
crystallizing	O
difficult	O
targets	O
.	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	O
(	O
see	O
below	O
),	O
this	O
Fab	O
should	O
have	O
minimum	O
effects	O
on	O
HAP	O
binding	O
conformation	O
.	O
	O
Crystals	O
of	O
Fab	O
/	O
IL	O
-	O
17A	O
/	O
HAP	O
ternary	O
complex	O
were	O
obtained	O
readily	O
in	O
crystallization	O
screens	O
.	O
	O
Crystallization	O
of	O
IL	O
-	O
17A	O
and	O
its	O
binding	O
partners	O
was	O
accomplished	O
using	O
two	O
forms	O
of	O
IL	O
-	O
17A	O
.	O
	O
Both	O
structures	O
were	O
solved	O
by	O
molecular	O
replacement	O
.	O
	O
The	O
C	O
-	O
terminal	O
8	O
residues	O
of	O
the	O
HAP	O
that	O
are	O
ordered	O
in	O
the	O
structure	O
,	O
7ADLWDWIN	O
,	O
form	O
an	O
amphipathic	O
α	O
-	O
helix	O
interacting	O
with	O
the	O
second	O
IL	O
-	O
17A	O
monomer	O
.	O
	O
Pro6	O
of	O
HAP	O
makes	O
a	O
transition	O
between	O
the	O
N	O
-	O
terminal	O
β	O
-	O
strand	O
and	O
the	O
C	O
-	O
terminal	O
α	O
-	O
helix	O
of	O
HAP	O
.	O
	O
Conformational	O
changes	O
in	O
region	O
I	O
induced	O
by	O
HAP	O
binding	O
alone	O
may	O
allosterically	O
affect	O
IL	O
-	O
17RA	O
binding	O
,	O
but	O
more	O
importantly	O
,	O
the	O
α	O
-	O
helix	O
of	O
HAP	O
directly	O
competes	O
with	O
IL	O
-	O
17RA	O
for	O
binding	O
to	O
IL	O
-	O
17A	O
(	O
Fig	O
.	O
3	O
).	O
	O
However	O
,	O
it	O
mimics	O
the	O
β	O
-	O
strand	O
0	O
of	O
IL	O
-	O
17A	O
.	O
	O
Conformational	O
changes	O
of	O
IL	O
-	O
17A	O
are	O
needed	O
for	O
both	O
HAP	O
and	O
IL	O
-	O
17RA	O
to	O
bind	O
to	O
that	O
region	O
.	O
	O
During	O
IL	O
-	O
17A	O
signaling	O
,	O
IL	O
-	O
17A	O
binds	O
to	O
one	O
copy	O
of	O
IL	O
-	O
17RA	O
and	O
one	O
copy	O
of	O
IL	O
-	O
17RC	O
.	O
	O
HAP	O
,	O
with	O
only	O
15	O
residues	O
,	O
can	O
achieve	O
almost	O
the	O
same	O
binding	O
affinity	O
as	O
the	O
much	O
larger	O
IL	O
-	O
17RA	O
molecule	O
,	O
indicating	O
a	O
more	O
efficient	O
way	O
of	O
binding	O
to	O
IL	O
-	O
17A	O
.	O
	O
As	O
demonstrated	O
by	O
the	O
crystal	O
structure	O
,	O
binding	O
of	O
the	O
α	O
-	O
helix	O
of	O
HAP	O
should	O
be	O
sufficient	O
for	O
preventing	O
IL	O
-	O
17RA	O
binding	O
to	O
IL	O
-	O
17A	O
.	O
	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	O
-	O
helix	O
-	O
mediated	O
IL	O
-	O
17A	O
/	O
IL	O
-	O
17RA	O
interactions	O
.	O
	O
(	O
A	O
)	O
HAP	O
binds	O
at	O
region	O
I	O
of	O
IL	O
-	O
17A	O
.	O
	O
Polar	O
interactions	O
are	O
shown	O
in	O
dashes	O
.	O
	O
Notice	O
that	O
the	O
Trp	O
binding	O
pocket	O
for	O
W12	O
of	O
HAP	O
or	O
W31	O
of	O
IL	O
-	O
17RA	O
is	O
missing	O
in	O
the	O
apo	O
structure	O
.	O
	O
It	O
is	O
therefore	O
important	O
to	O
understand	O
the	O
mechanisms	O
which	O
regulate	O
nadA	O
expression	O
levels	O
,	O
which	O
are	O
predominantly	O
controlled	O
by	O
the	O
transcriptional	O
regulator	O
NadR	O
(	O
Neisseria	O
adhesin	O
A	O
Regulator	O
)	O
both	O
in	O
vitro	O
and	O
in	O
vivo	O
.	O
	O
NadR	O
binds	O
the	O
nadA	O
promoter	O
and	O
represses	O
gene	O
transcription	O
.	O
	O
Serogroup	O
B	O
meningococcus	O
(	O
MenB	O
)	O
causes	O
fatal	O
sepsis	O
and	O
invasive	O
meningococcal	O
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	O
outbreaks	O
in	O
universities	O
of	O
the	O
United	O
States	O
and	O
Canada	O
.	O
	O
The	O
amount	O
of	O
NadA	O
exposed	O
on	O
the	O
meningococcal	O
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
	O
Although	O
additional	O
factors	O
influence	O
nadA	O
expression	O
,	O
we	O
focused	O
on	O
its	O
regulation	O
by	O
NadR	O
,	O
the	O
major	O
mediator	O
of	O
NadA	O
phase	O
variable	O
expression	O
.	O
	O
However	O
,	O
the	O
homologous	O
archeal	O
Sulfolobus	O
tokodaii	O
protein	O
ST1710	O
presented	O
essentially	O
the	O
same	O
structure	O
in	O
ligand	O
-	O
free	O
and	O
salicylate	O
-	O
bound	O
forms	O
,	O
apparently	O
contrasting	O
the	O
mechanism	O
proposed	O
for	O
MTH313	O
.	O
	O
Despite	O
these	O
apparent	O
differences	O
,	O
MTH313	O
and	O
ST1710	O
bind	O
salicylate	O
in	O
approximately	O
the	O
same	O
site	O
,	O
between	O
their	O
dimerization	O
and	O
DNA	O
-	O
binding	O
domains	O
.	O
	O
We	O
obtained	O
detailed	O
new	O
insights	O
into	O
ligand	O
specificity	O
,	O
how	O
the	O
ligand	O
allosterically	O
influences	O
the	O
DNA	O
-	O
binding	O
ability	O
of	O
NadR	O
,	O
and	O
the	O
regulation	O
of	O
nadA	O
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	O
super	O
-	O
family	O
.	O
	O
NadR	O
is	O
dimeric	O
and	O
is	O
stabilized	O
by	O
specific	O
hydroxyphenylacetate	O
ligands	O
	O
Recombinant	O
NadR	O
was	O
produced	O
in	O
E	O
.	O
coli	O
using	O
an	O
expression	O
construct	O
prepared	O
from	O
N	O
.	O
meningitidis	O
serogroup	O
B	O
strain	O
MC58	O
.	O
	O
Since	O
ligand	O
-	O
binding	O
often	O
increases	O
protein	O
stability	O
,	O
we	O
also	O
investigated	O
the	O
effect	O
of	O
various	O
HPAs	O
(	O
Fig	O
1A	O
)	O
on	O
the	O
melting	O
temperature	O
(	O
Tm	O
)	O
of	O
NadR	O
.	O
As	O
a	O
control	O
of	O
specificity	O
,	O
we	O
also	O
tested	O
salicylate	O
,	O
a	O
known	O
ligand	O
of	O
some	O
MarR	O
proteins	O
previously	O
reported	O
to	O
increase	O
the	O
Tm	O
of	O
ST1710	O
and	O
MTH313	O
.	O
	O
3	O
-	O
HPA	O
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	O
-	O
HPA	O
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	O
,	O
4	O
-	O
HPA	O
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
	O
To	O
further	O
investigate	O
the	O
binding	O
of	O
HPAs	O
to	O
NadR	O
,	O
we	O
used	O
surface	O
plasmon	O
resonance	O
(	O
SPR	O
).	O
	O
Data	O
collection	O
and	O
refinement	O
statistics	O
for	O
NadR	O
structures	O
.	O
	O
In	O
the	O
apo	O
-	O
NadR	O
crystals	O
,	O
the	O
two	O
homodimers	O
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	O
was	O
presumably	O
merely	O
an	O
effect	O
of	O
crystal	O
packing	O
,	O
since	O
the	O
interface	O
between	O
the	O
two	O
homodimers	O
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
	O
Helices	O
α3	O
and	O
α4	O
form	O
a	O
helix	O
-	O
turn	O
-	O
helix	O
motif	O
,	O
followed	O
by	O
the	O
“	O
wing	O
motif	O
”	O
comprised	O
of	O
two	O
short	O
antiparallel	O
β	O
-	O
strands	O
(	O
β1	O
-	O
β2	O
)	O
linked	O
by	O
a	O
relatively	O
long	O
and	O
flexible	O
loop	O
.	O
	O
Interestingly	O
,	O
in	O
the	O
α4	O
-	O
β2	O
region	O
,	O
the	O
stretch	O
of	O
residues	O
from	O
R64	O
-	O
R91	O
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	O
.	O
	O
Using	O
site	O
-	O
directed	O
mutagenesis	O
,	O
a	O
panel	O
of	O
eight	O
mutant	O
NadR	O
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	O
interface	O
.	O
	O
It	O
is	O
notable	O
that	O
L130	O
is	O
usually	O
present	O
as	O
Leu	O
,	O
or	O
an	O
alternative	O
bulky	O
hydrophobic	O
amino	O
acid	O
(	O
e	O
.	O
g	O
.	O
Phe	O
,	O
Val	O
),	O
in	O
many	O
MarR	O
family	O
proteins	O
,	O
suggesting	O
a	O
conserved	O
role	O
in	O
stabilizing	O
the	O
dimer	O
interface	O
.	O
	O
The	O
NadR	O
/	O
4	O
-	O
HPA	O
structure	O
revealed	O
the	O
ligand	O
-	O
binding	O
site	O
nestled	O
between	O
the	O
dimerization	O
and	O
DNA	O
-	O
binding	O
domains	O
(	O
Fig	O
2	O
).	O
	O
The	O
binding	O
pocket	O
was	O
almost	O
entirely	O
filled	O
by	O
4	O
-	O
HPA	O
and	O
one	O
water	O
molecule	O
,	O
although	O
there	O
also	O
remained	O
a	O
small	O
tunnel	O
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	O
(	O
proximal	O
to	O
the	O
4	O
-	O
hydroxyl	O
position	O
)	O
to	O
the	O
protein	O
surface	O
.	O
	O
Green	O
and	O
blue	O
ribbons	O
depict	O
NadR	O
chains	O
A	O
and	O
B	O
,	O
respectively	O
.	O
	O
Residues	O
AsnA11	O
and	O
ArgB18	O
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	O
.	O
	O
List	O
of	O
4	O
-	O
HPA	O
atoms	O
bound	O
to	O
NadR	O
via	O
ionic	O
interactions	O
and	O
/	O
or	O
H	O
-	O
bonds	O
.	O
	O
4	O
-	O
HPA	O
atom	O
NadR	O
residue	O
/	O
atom	O
Distance	O
(	O
Å	O
)	O
O2	O
TrpB39	O
/	O
NE1	O
2	O
.	O
83	O
O2	O
ArgB43	O
/	O
NH1	O
2	O
.	O
76	O
O1	O
ArgB43	O
/	O
NH1	O
3	O
.	O
84	O
O1	O
SerA9	O
/	O
OG	O
2	O
.	O
75	O
O1	O
TyrB115	O
/	O
OH	O
2	O
.	O
50	O
O2	O
Water	O
(*	O
Ser9	O
/	O
Asn11	O
)	O
2	O
.	O
88	O
OH	O
AspB36	O
/	O
OD1	O
/	O
OD2	O
3	O
.	O
6	O
/	O
3	O
.	O
7	O
	O
*	O
Bond	O
distance	O
between	O
the	O
ligand	O
carboxylate	O
group	O
and	O
the	O
water	O
molecule	O
,	O
which	O
in	O
turn	O
makes	O
H	O
-	O
bond	O
to	O
the	O
SerA9	O
and	O
AsnA11	O
side	O
chains	O
.	O
	O
In	O
SPR	O
,	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
	O
The	O
stoichiometry	O
of	O
the	O
NadR	O
-	O
HPA	O
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	O
of	O
1	O
.	O
13	O
for	O
4	O
-	O
HPA	O
,	O
1	O
.	O
02	O
for	O
3	O
-	O
HPA	O
,	O
and	O
1	O
.	O
21	O
for	O
3Cl	O
,	O
4	O
-	O
HPA	O
,	O
strongly	O
suggesting	O
that	O
one	O
NadR	O
dimer	O
bound	O
to	O
1	O
HPA	O
analyte	O
molecule	O
.	O
	O
Indeed	O
,	O
we	O
noted	O
interesting	O
differences	O
in	O
the	O
side	O
chains	O
of	O
Met22	O
,	O
Phe25	O
and	O
Arg43	O
,	O
which	O
in	O
monomer	O
B	O
are	O
used	O
to	O
contact	O
the	O
ligand	O
while	O
in	O
monomer	O
A	O
they	O
partially	O
occupied	O
the	O
pocket	O
and	O
collectively	O
reduced	O
its	O
volume	O
significantly	O
.	O
	O
In	O
contrast	O
,	O
the	O
apo	O
-	O
form	O
Met22	O
and	O
Phe25	O
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
	O
The	O
‘	O
outward	O
’	O
position	O
of	O
Arg43	O
generated	O
an	O
open	O
apo	O
-	O
form	O
pocket	O
with	O
volume	O
approximately	O
380Å3	O
.	O
	O
Taken	O
together	O
,	O
these	O
observations	O
suggest	O
that	O
Arg43	O
is	O
a	O
major	O
determinant	O
of	O
ligand	O
binding	O
,	O
and	O
that	O
its	O
‘	O
inward	O
’	O
position	O
inhibits	O
the	O
binding	O
of	O
4	O
-	O
HPA	O
to	O
the	O
empty	O
pocket	O
of	O
holo	O
-	O
NadR	O
.	O
	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	O
-	O
HPA	O
were	O
present	O
.	O
(	O
C	O
)	O
Comparison	O
of	O
the	O
empty	O
pocket	O
from	O
holo	O
-	O
NadR	O
(	O
green	O
residues	O
)	O
with	O
the	O
four	O
empty	O
pockets	O
of	O
apo	O
-	O
NadR	O
(	O
grey	O
residues	O
),	O
shows	O
that	O
in	O
the	O
absence	O
of	O
4	O
-	O
HPA	O
the	O
Arg43	O
side	O
chain	O
is	O
always	O
observed	O
in	O
the	O
‘	O
outward	O
’	O
conformation	O
.	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	O
-	O
NadR	O
is	O
well	O
-	O
folded	O
under	O
these	O
conditions	O
and	O
exhibits	O
one	O
conformation	O
appreciable	O
on	O
the	O
NMR	O
timescale	O
,	O
i	O
.	O
e	O
.	O
in	O
the	O
NMR	O
experiments	O
at	O
25	O
°	O
C	O
,	O
two	O
or	O
more	O
distinct	O
conformations	O
of	O
apo	O
-	O
NadR	O
monomers	O
were	O
not	O
readily	O
apparent	O
.	O
	O
(	O
B	O
,	O
C	O
)	O
Overlay	O
of	O
selected	O
regions	O
of	O
the	O
1H	O
-	O
15N	O
TROSY	O
-	O
HSQC	O
spectra	O
acquired	O
at	O
25	O
°	O
C	O
of	O
apo	O
-	O
NadR	O
(	O
cyan	O
)	O
and	O
NadR	O
/	O
4	O
-	O
HPA	O
(	O
red	O
)	O
superimposed	O
with	O
the	O
spectra	O
acquired	O
at	O
10	O
°	O
C	O
of	O
apo	O
-	O
NadR	O
(	O
blue	O
)	O
and	O
NadR	O
/	O
4	O
-	O
HPA	O
(	O
green	O
).	O
	O
Considering	O
the	O
small	O
size	O
,	O
fast	O
diffusion	O
and	O
relatively	O
low	O
binding	O
affinity	O
of	O
4	O
-	O
HPA	O
,	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	O
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	O
and	O
unbound	O
states	O
.	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	O
-	O
HPA	O
(	O
and	O
therefore	O
likely	O
to	O
be	O
in	O
the	O
ligand	O
-	O
binding	O
site	O
)	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
	O
Similarly	O
,	O
the	O
entire	O
holo	O
-	O
homodimer	O
could	O
be	O
closely	O
superposed	O
onto	O
each	O
of	O
the	O
apo	O
-	O
homodimers	O
,	O
showing	O
rmsd	O
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	O
helix	O
positions	O
(	O
Fig	O
7B	O
).	O
	O
Structural	O
comparisons	O
of	O
NadR	O
and	O
modelling	O
of	O
interactions	O
with	O
DNA	O
.	O
	O
However	O
,	O
structural	O
comparisons	O
revealed	O
that	O
the	O
shift	O
of	O
holo	O
-	O
NadR	O
helix	O
α4	O
induced	O
by	O
the	O
presence	O
of	O
4	O
-	O
HPA	O
was	O
also	O
accompanied	O
by	O
several	O
changes	O
at	O
the	O
holo	O
dimer	O
interface	O
,	O
while	O
such	O
extensive	O
structural	O
differences	O
were	O
not	O
observed	O
in	O
the	O
apo	O
dimer	O
interfaces	O
,	O
particularly	O
notable	O
when	O
comparing	O
the	O
α6	O
helices	O
(	O
S3	O
Fig	O
).	O
	O
Interestingly	O
,	O
OhrR	O
contacts	O
ohrA	O
across	O
22	O
base	O
pairs	O
(	O
bp	O
),	O
and	O
similarly	O
the	O
main	O
NadR	O
target	O
sites	O
identified	O
in	O
the	O
nadA	O
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
	O
When	O
aligned	O
with	O
OhrR	O
,	O
the	O
apo	O
-	O
homodimer	O
CD	O
presented	O
yet	O
another	O
different	O
intermediate	O
conformation	O
(	O
rmsd	O
2	O
.	O
9Å	O
),	O
apparently	O
not	O
ideally	O
pre	O
-	O
configured	O
for	O
DNA	O
binding	O
,	O
but	O
which	O
in	O
solution	O
can	O
presumably	O
readily	O
adopt	O
the	O
AB	O
conformation	O
due	O
to	O
the	O
intrinsic	O
flexibility	O
described	O
above	O
.	O
	O
Western	O
blot	O
analyses	O
of	O
wild	O
-	O
type	O
(	O
WT	O
)	O
strain	O
(	O
lanes	O
1	O
–	O
2	O
)	O
or	O
isogenic	O
nadR	O
knockout	O
strains	O
(	O
ΔNadR	B-mutant
)	O
complemented	O
to	O
express	O
the	O
indicated	O
NadR	O
WT	O
or	O
mutant	O
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	O
-	O
HPA	O
,	O
showing	O
NadA	O
and	O
NadR	O
expression	O
.	O
	O
The	O
H7A	B-mutant
,	O
S9A	B-mutant
and	O
F25A	B-mutant
mutants	O
efficiently	O
repress	O
nadA	O
expression	O
but	O
are	O
less	O
ligand	O
-	O
responsive	O
than	O
WT	O
NadR	O
.	O
The	O
N11A	B-mutant
mutant	O
does	O
not	O
efficiently	O
repress	O
nadA	O
expression	O
either	O
in	O
presence	O
or	O
absence	O
of	O
4	O
-	O
HPA	O
.	O
(	O
The	O
protein	O
abundance	O
levels	O
of	O
the	O
meningococcal	O
factor	O
H	O
binding	O
protein	O
(	O
fHbp	O
)	O
were	O
used	O
as	O
a	O
gel	O
loading	O
control	O
).	O
	O
NadA	O
is	O
a	O
surface	O
-	O
exposed	O
meningococcal	O
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
	O
We	O
confirmed	O
this	O
stoichiometry	O
in	O
solution	O
using	O
SPR	O
methods	O
.	O
	O
Structural	O
analyses	O
suggested	O
that	O
‘	O
inward	O
’	O
side	O
chain	O
positions	O
of	O
Met22	O
,	O
Phe25	O
and	O
especially	O
Arg43	O
precluded	O
binding	O
of	O
a	O
second	O
ligand	O
molecule	O
.	O
	O
In	O
the	O
S	O
.	O
tokodaii	O
protein	O
ST1710	O
,	O
salicylate	O
binds	O
to	O
the	O
same	O
position	O
in	O
each	O
monomer	O
of	O
the	O
dimer	O
,	O
in	O
a	O
site	O
equivalent	O
to	O
the	O
putative	O
biologically	O
relevant	O
site	O
of	O
MTH313	O
(	O
Fig	O
10B	O
).	O
	O
Unlike	O
other	O
MarR	O
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	O
-	O
HPA	O
bound	O
to	O
NadR	O
,	O
suggesting	O
a	O
more	O
specific	O
and	O
less	O
promiscuous	O
interaction	O
.	O
	O
NadR	O
shows	O
a	O
ligand	O
binding	O
site	O
distinct	O
from	O
other	O
MarR	O
homologues	O
.	O
	O
Alternatively	O
,	O
it	O
is	O
possible	O
that	O
other	O
MarR	O
homologues	O
(	O
e	O
.	O
g	O
.	O
NMB1585	O
)	O
may	O
have	O
no	O
extant	O
functional	O
binding	O
pocket	O
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	O
-	O
binding	O
regulatory	O
proteins	O
.	O
	O
The	O
noted	O
flexibility	O
may	O
also	O
explain	O
how	O
NadR	O
can	O
adapt	O
to	O
bind	O
various	O
DNA	O
target	O
sequences	O
with	O
slightly	O
different	O
structural	O
features	O
.	O
	O
Like	O
other	O
nuclear	O
hormone	O
receptors	O
,	O
RORγ	O
’	O
s	O
helix12	O
which	O
makes	O
up	O
the	O
C	O
-	O
termini	O
of	O
the	O
LBD	O
is	O
an	O
essential	O
part	O
of	O
the	O
coactivator	O
binding	O
pocket	O
and	O
is	O
commonly	O
referred	O
to	O
as	O
the	O
activation	O
function	O
helix	O
2	O
(	O
AF2	O
).	O
	O
FRET	O
results	O
for	O
agonist	O
BIO592	O
(	O
a	O
)	O
and	O
Inverse	O
Agonist	O
BIO399	O
(	O
b	O
)	O
	O
a	O
The	O
ternary	O
structure	O
of	O
RORγ518	O
BIO592	O
and	O
EBI96	O
.	O
	O
b	O
RORγ	O
AF2	O
helix	O
in	O
the	O
agonist	O
conformation	O
.	O
	O
The	O
structure	O
of	O
the	O
ternary	O
complex	O
had	O
features	O
similar	O
to	O
other	O
ROR	O
agonist	O
coactivator	O
structures	O
in	O
a	O
transcriptionally	O
active	O
canonical	O
three	O
layer	O
helix	O
fold	O
with	O
the	O
AF2	O
helix	O
in	O
the	O
agonist	O
conformation	O
.	O
	O
The	O
agonist	O
conformation	O
is	O
stabilized	O
by	O
a	O
hydrogen	O
bond	O
between	O
His479	O
and	O
Tyr502	O
,	O
in	O
addition	O
to	O
π	O
-	O
π	O
interactions	O
between	O
His479	O
,	O
Tyr502	O
and	O
Phe506	O
(	O
Fig	O
.	O
2b	O
).	O
	O
Electron	O
density	O
for	O
the	O
coactivator	O
peptide	O
EBI96	O
was	O
observed	O
for	O
residues	O
EFPYLLSLLG	O
which	O
formed	O
a	O
α	O
-	O
helix	O
stabilized	O
through	O
hydrophobic	O
interactions	O
with	O
the	O
coactivator	O
binding	O
pocket	O
on	O
RORγ	O
(	O
Fig	O
.	O
2c	O
).	O
	O
b	O
Benzoxazinone	O
ring	O
system	O
of	O
agonist	O
BIO592	O
packing	O
against	O
His479	O
of	O
RORγ	O
stabilizing	O
agonist	O
conformation	O
of	O
the	O
AF2	O
helix	O
	O
BIO592	O
bound	O
in	O
a	O
collapsed	O
conformational	O
state	O
in	O
the	O
LBS	O
of	O
RORγ	O
with	O
the	O
xylene	O
ring	O
positioned	O
at	O
the	O
bottom	O
of	O
the	O
pocket	O
making	O
hydrophobic	O
interactions	O
with	O
Val376	O
,	O
Phe378	O
,	O
Phe388	O
and	O
Phe401	O
,	O
with	O
the	O
ethyl	O
-	O
benzoxazinone	O
ring	O
making	O
several	O
hydrophobic	O
interactions	O
with	O
Trp317	O
,	O
Leu324	O
,	O
Met358	O
,	O
Leu391	O
,	O
Ile	O
400	O
and	O
His479	O
(	O
Fig	O
.	O
3a	O
,	O
Additional	O
file	O
3	O
).	O
	O
Hydrophobic	O
interaction	O
between	O
the	O
ethyl	O
group	O
of	O
the	O
benzoxazinone	O
and	O
His479	O
reinforce	O
the	O
His479	O
sidechain	O
position	O
for	O
making	O
the	O
hydrogen	O
bond	O
with	O
Tyr502	O
thereby	O
stabilizing	O
the	O
agonist	O
conformation	O
(	O
Fig	O
.	O
3b	O
).	O
	O
However	O
,	O
in	O
the	O
presence	O
of	O
inverse	O
agonist	O
BIO399	O
,	O
the	O
proteolytic	O
pattern	O
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	O
/	O
495	O
),	O
indicating	O
the	O
destabilization	O
of	O
the	O
AF2	O
helix	O
compared	O
to	O
either	O
the	O
APO	O
or	O
ternary	O
agonist	O
complex	O
(	O
Fig	O
.	O
4	O
,	O
Additional	O
file	O
5	O
).	O
	O
a	O
Overlay	O
of	O
RORγ	O
structures	O
bound	O
to	O
BIO596	O
(	O
Green	O
),	O
BIO399	O
(	O
Cyan	O
)	O
and	O
T0901317	O
(	O
Pink	O
).	O
	O
We	O
hypothesize	O
that	O
since	O
the	O
Met358	O
sidechain	O
conformation	O
in	O
the	O
T0901317	O
RORγ	O
structure	O
is	O
not	O
in	O
the	O
BIO399	O
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	O
compared	O
to	O
BIO399	O
in	O
the	O
FRET	O
assay	O
.	O
	O
The	O
inverse	O
agonist	O
activity	O
for	O
these	O
compounds	O
has	O
been	O
attributed	O
to	O
orientating	O
Trp317	O
to	O
clash	O
with	O
Tyr502	O
or	O
a	O
direct	O
inverse	O
agonist	O
hydrogen	O
bonding	O
event	O
with	O
His479	O
,	O
both	O
of	O
which	O
would	O
perturb	O
the	O
agonist	O
conformation	O
of	O
RORγ	O
.	O
	O
GAL4	O
cell	O
assay	O
selectivity	O
profile	O
for	O
BIO399	O
toward	O
RORα	O
and	O
RORβ	O
in	O
GAL4	O
	O
Furthermore	O
,	O
RORα	O
contains	O
two	O
phenylalanine	O
residues	O
in	O
its	O
LBS	O
whereas	O
RORβ	O
and	O
γ	O
have	O
a	O
leucine	O
in	O
the	O
same	O
position	O
(	O
Fig	O
.	O
6b	O
).	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	O
and	O
Acetyl	O
~	O
CoA	O
)	O
are	O
critical	O
for	O
both	O
catabolic	O
and	O
anabolic	O
processes	O
.	O
	O
The	O
facets	O
of	O
the	O
shell	O
are	O
composed	O
primarily	O
of	O
hexamers	O
that	O
are	O
typically	O
perforated	O
by	O
pores	O
lined	O
with	O
highly	O
conserved	O
,	O
polar	O
residues	O
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	O
.	O
	O
Substrates	O
and	O
cofactors	O
involving	O
the	O
PTAC	O
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
	O
The	O
activities	O
of	O
core	O
enzymes	O
are	O
not	O
confined	O
to	O
BMC	O
-	O
associated	O
functions	O
:	O
aldehyde	O
and	O
alcohol	O
dehydrogenases	O
are	O
utilized	O
in	O
diverse	O
metabolic	O
reactions	O
,	O
and	O
PTAC	O
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
	O
This	O
occurs	O
,	O
for	O
example	O
,	O
during	O
acetoclastic	O
methanogenesis	O
in	O
the	O
archaeal	O
Methanosarcina	O
species	O
.	O
	O
Another	O
distinctive	O
feature	O
of	O
BMC	O
-	O
associated	O
PduL	O
homologs	O
is	O
an	O
N	O
-	O
terminal	O
encapsulation	O
peptide	O
(	O
EP	O
)	O
that	O
is	O
thought	O
to	O
“	O
target	O
”	O
proteins	O
for	O
encapsulation	O
by	O
the	O
BMC	O
shell	O
.	O
	O
EPs	O
are	O
frequently	O
found	O
on	O
BMC	O
-	O
associated	O
proteins	O
and	O
have	O
been	O
shown	O
to	O
interact	O
with	O
shell	O
proteins	O
.	O
	O
Here	O
we	O
report	O
high	O
-	O
resolution	O
crystal	O
structures	O
of	O
a	O
PduL	O
-	O
type	O
PTAC	O
in	O
both	O
CoA	O
-	O
and	O
phosphate	O
-	O
bound	O
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	O
common	O
core	O
enzymes	O
.	O
	O
β	O
-	O
Barrel	O
1	O
consists	O
of	O
the	O
N	O
-	O
terminal	O
β	O
strand	O
and	O
β	O
strands	O
from	O
the	O
C	O
-	O
terminal	O
half	O
of	O
the	O
polypeptide	O
chain	O
(	O
β1	O
,	O
β10	O
-	O
β14	O
;	O
residues	O
37	O
–	O
46	O
and	O
155	O
–	O
224	O
).	O
	O
Primary	O
structure	O
conservation	O
of	O
the	O
PduL	O
protein	O
family	O
.	O
	O
Sequence	O
logo	O
calculated	O
from	O
the	O
multiple	O
sequence	O
alignment	O
of	O
PduL	O
homologs	O
(	O
see	O
Materials	O
and	O
Methods	O
),	O
but	O
not	O
including	O
putative	O
EP	O
sequences	O
.	O
	O
The	O
position	O
numbers	O
shown	O
correspond	O
to	O
the	O
residue	O
numbering	O
of	O
rPduL	O
;	O
note	O
that	O
some	O
positions	O
in	O
the	O
logo	O
represent	O
gaps	O
in	O
the	O
rPduL	O
sequence	O
.	O
	O
The	O
asterisk	O
and	O
double	O
arrow	O
marks	O
the	O
location	O
of	O
the	O
π	O
–	O
π	O
interaction	O
between	O
F116	O
and	O
the	O
CoA	O
base	O
of	O
the	O
other	O
dimer	O
chain	O
.	O
	O
Size	O
exclusion	O
chromatography	O
of	O
PduL	O
homologs	O
.	O
	O
(	O
a	O
)–(	O
c	O
):	O
Chromatograms	O
of	O
sPduL	O
(	O
a	O
),	O
rPduL	O
(	O
b	O
),	O
and	O
pPduL	O
(	O
c	O
)	O
with	O
(	O
orange	O
)	O
or	O
without	O
(	O
blue	O
)	O
the	O
predicted	O
EP	O
,	O
post	O
-	O
nickel	O
affinity	O
purification	O
,	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
	O
The	O
second	O
(	O
Zn2	O
)	O
is	O
in	O
octahedral	O
coordination	O
by	O
three	O
conserved	O
histidine	O
residues	O
(	O
His157	O
,	O
His159	O
and	O
His204	O
)	O
as	O
well	O
as	O
three	O
water	O
molecules	O
(	O
Fig	O
4a	O
).	O
	O
When	O
the	O
crystals	O
were	O
soaked	O
in	O
a	O
sodium	O
phosphate	O
solution	O
for	O
2	O
d	O
prior	O
to	O
data	O
collection	O
,	O
the	O
CoA	O
dissociates	O
,	O
and	O
density	O
for	O
a	O
phosphate	O
molecule	O
is	O
visible	O
at	O
the	O
active	O
site	O
(	O
Table	O
2	O
,	O
Fig	O
4b	O
).	O
	O
Oligomeric	O
States	O
of	O
PduL	O
Orthologs	O
Are	O
Influenced	O
by	O
the	O
EP	O
	O
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	O
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
	O
pPduLΔEP	B-mutant
eluted	O
as	O
two	O
smaller	O
forms	O
,	O
possibly	O
corresponding	O
to	O
a	O
trimer	O
and	O
a	O
monomer	O
.	O
	O
Homologs	O
of	O
the	O
predominant	O
cofactor	O
utilizer	O
(	O
aldehyde	O
dehydrogenase	O
)	O
and	O
NAD	O
+	O
regenerator	O
(	O
alcohol	O
dehydrogenase	O
)	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	O
,	O
which	O
recycles	O
CoA	O
in	O
the	O
organelle	O
lumen	O
.	O
	O
Refined	O
domain	O
assignment	O
based	O
on	O
our	O
structure	O
should	O
be	O
able	O
to	O
predict	O
domains	O
of	O
PF06130	O
homologs	O
much	O
more	O
accurately	O
.	O
	O
Implications	O
for	O
Metabolosome	O
Core	O
Assembly	O
	O
Free	O
CoA	O
and	O
NAD	O
+/	O
H	O
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
	O
The	O
free	O
CoA	O
-	O
bound	O
form	O
is	O
presumably	O
poised	O
for	O
attack	O
upon	O
an	O
acyl	O
-	O
phosphate	O
,	O
indicating	O
that	O
the	O
enzyme	O
initially	O
binds	O
CoA	O
as	O
opposed	O
to	O
acyl	O
-	O
phosphate	O
.	O
	O
The	O
phosphate	O
-	O
bound	O
structure	O
indicates	O
that	O
in	O
the	O
opposite	O
reaction	O
direction	O
phosphate	O
is	O
bound	O
first	O
,	O
and	O
then	O
an	O
acyl	O
-	O
CoA	O
enters	O
.	O
	O
The	O
two	O
crystal	O
structures	O
that	O
we	O
report	O
here	O
for	O
the	O
(	O
Sa	O
)	O
EctC	O
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	O
-	O
directed	O
mutagenesis	O
experiments	O
targeting	O
evolutionarily	O
highly	O
conserved	O
residues	O
within	O
the	O
extended	O
EctC	O
protein	O
family	O
,	O
provide	O
a	O
first	O
view	O
into	O
the	O
architecture	O
of	O
the	O
catalytic	O
core	O
of	O
the	O
ectoine	O
synthase	O
.	O
	O
The	O
(	O
Sa	O
)	O
EctC	O
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
	O
Biochemical	O
properties	O
of	O
the	O
ectoine	O
synthase	O
	O
N	O
-	O
α	O
-	O
ADABA	O
has	O
so	O
far	O
not	O
been	O
considered	O
as	O
a	O
substrate	O
for	O
EctC	O
,	O
but	O
microorganisms	O
that	O
use	O
ectoine	O
as	O
a	O
nutrient	O
produce	O
it	O
as	O
an	O
intermediate	O
during	O
catabolism	O
.	O
	O
The	O
stimulation	O
of	O
EctC	O
enzyme	O
activity	O
by	O
salts	O
has	O
previously	O
also	O
been	O
observed	O
for	O
other	O
ectoine	O
synthases	O
.	O
	O
Since	O
variations	O
of	O
the	O
above	O
-	O
described	O
metal	O
-	O
binding	O
motif	O
occur	O
frequently	O
,	O
we	O
experimentally	O
investigated	O
the	O
presence	O
and	O
nature	O
of	O
the	O
metal	O
that	O
might	O
be	O
contained	O
in	O
the	O
(	O
Sa	O
)	O
EctC	O
protein	O
by	O
inductive	O
-	O
coupled	O
plasma	O
mass	O
spectrometry	O
(	O
ICP	O
-	O
MS	O
).	O
	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	O
content	O
of	O
the	O
(	O
Sa	O
)	O
EctC	O
proteins	O
varied	O
by	O
approximately	O
10	O
to	O
20	O
%	O
between	O
the	O
two	O
methods	O
.	O
	O
Dependency	O
of	O
the	O
ectoine	O
synthase	O
activity	O
on	O
metals	O
.	O
	O
We	O
then	O
took	O
such	O
an	O
inactivated	O
enzyme	O
preparation	O
,	O
removed	O
the	O
EDTA	O
by	O
dialysis	O
,	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	O
)	O
EctC	O
enzyme	O
.	O
	O
Hence	O
,	O
N	O
-	O
α	O
-	O
ADABA	O
is	O
a	O
newly	O
recognized	O
substrate	O
for	O
ectoine	O
synthase	O
.	O
	O
The	O
Km	O
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	O
efficiency	O
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
	O
Finally	O
,	O
a	O
monomer	O
of	O
this	O
structure	O
was	O
used	O
as	O
a	O
template	O
for	O
molecular	O
replacement	O
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	O
of	O
12	O
.	O
4	O
%	O
and	O
an	O
Rfree	O
of	O
14	O
.	O
9	O
%	O
(	O
S1	O
Table	O
).	O
	O
This	O
structure	O
adopts	O
an	O
open	O
conformation	O
with	O
respect	O
to	O
the	O
typical	O
fold	O
of	O
cupin	O
barrels	O
and	O
is	O
therefore	O
termed	O
in	O
the	O
following	O
the	O
“	O
open	O
”	O
(	O
Sa	O
)	O
EctC	O
structure	O
(	O
Fig	O
4b	O
).	O
	O
Interestingly	O
,	O
the	O
three	O
other	O
monomers	O
present	O
in	O
the	O
asymmetric	O
unit	O
all	O
range	O
from	O
Met	O
-	O
1	O
to	O
Glu	O
-	O
115	O
and	O
adopt	O
a	O
conformation	O
similar	O
to	O
the	O
“	O
open	O
”	O
EctC	O
structure	O
.	O
	O
The	O
structure	O
of	O
the	O
“	O
semi	O
-	O
closed	O
”	O
(	O
Sa	O
)	O
EctC	O
protein	O
consists	O
of	O
11	O
β	O
-	O
strands	O
(	O
β1	O
-	O
β11	O
)	O
and	O
two	O
α	O
-	O
helices	O
(	O
α	O
-	O
I	O
and	O
α	O
-	O
II	O
)	O
(	O
Fig	O
4a	O
).	O
	O
Our	O
data	O
classify	O
EctC	O
,	O
in	O
addition	O
to	O
the	O
polyketide	O
cyclase	O
RemF	O
,	O
as	O
the	O
second	O
known	O
cupin	O
-	O
related	O
enzyme	O
that	O
catalyze	O
a	O
cyclocondensation	O
reaction	O
.	O
	O
Analysis	O
of	O
the	O
EctC	O
dimer	O
interface	O
as	O
observed	O
in	O
the	O
(	O
Sa	O
)	O
EctC	O
crystal	O
structure	O
	O
It	O
is	O
worth	O
mentioning	O
that	O
β	O
-	O
strand	O
β5	O
is	O
located	O
next	O
to	O
His	O
-	O
93	O
,	O
which	O
in	O
all	O
likelihood	O
involved	O
in	O
metal	O
binding	O
(	O
see	O
below	O
).	O
	O
In	O
the	O
“	O
open	O
”	O
(	O
Sa	O
)	O
EctC	O
structure	O
,	O
both	O
proline	O
residues	O
are	O
visible	O
in	O
the	O
electron	O
density	O
;	O
however	O
,	O
almost	O
directly	O
after	O
Pro	O
-	O
110	O
,	O
the	O
electron	O
density	O
is	O
drastically	O
diminished	O
caused	O
by	O
the	O
flexibility	O
of	O
the	O
carboxy	O
-	O
terminus	O
.	O
	O
Since	O
these	O
proline	O
residues	O
are	O
followed	O
by	O
the	O
carboxy	O
-	O
terminal	O
region	O
of	O
the	O
(	O
Sa	O
)	O
EctC	O
protein	O
,	O
the	O
interaction	O
of	O
His	O
-	O
55	O
with	O
Pro	O
-	O
109	O
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
	O
The	O
interaction	O
between	O
Glu	O
-	O
115	O
and	O
His	O
-	O
55	O
is	O
only	O
visible	O
in	O
the	O
“	O
semi	O
-	O
closed	O
”	O
structure	O
where	O
the	O
partially	O
extended	O
carboxy	O
-	O
terminus	O
is	O
resolved	O
in	O
the	O
electron	O
density	O
.	O
	O
(	O
b	O
)	O
An	O
overlay	O
of	O
the	O
“	O
open	O
”	O
(	O
colored	O
in	O
light	O
blue	O
)	O
and	O
the	O
“	O
semi	O
-	O
closed	O
”	O
(	O
colored	O
in	O
green	O
)	O
structure	O
of	O
the	O
(	O
Sa	O
)	O
EctC	O
protein	O
.	O
	O
The	O
putative	O
iron	O
binding	O
site	O
of	O
(	O
Sa	O
)	O
EctC	O
	O
In	O
the	O
“	O
semi	O
-	O
closed	O
”	O
structure	O
of	O
(	O
Sa	O
)	O
EctC	O
,	O
each	O
of	O
the	O
four	O
monomers	O
in	O
the	O
asymmetric	O
unit	O
contains	O
a	O
relative	O
strong	O
electron	O
density	O
positioned	O
within	O
the	O
cupin	O
barrel	O
.	O
	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	O
-	O
52	O
(	O
located	O
in	O
a	O
loop	O
after	O
the	O
end	O
of	O
β	O
-	O
strand	O
β5	O
)	O
in	O
the	O
“	O
open	O
”	O
and	O
“	O
semi	O
-	O
closed	O
”	O
(	O
Sa	O
)	O
EctC	O
structures	O
.	O
	O
It	O
becomes	O
apparent	O
from	O
an	O
overlay	O
of	O
the	O
“	O
open	O
”	O
and	O
“	O
semi	O
-	O
closed	O
”	O
(	O
Sa	O
)	O
EctC	O
crystal	O
structures	O
that	O
the	O
side	O
-	O
chain	O
of	O
Tyr	O
-	O
52	O
rotates	O
away	O
from	O
the	O
position	O
of	O
the	O
presumptive	O
iron	O
,	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	O
directly	O
[	O
Glu	O
-	O
57	O
,	O
Tyr	O
-	O
85	O
,	O
and	O
His	O
-	O
93	O
],	O
remain	O
in	O
place	O
(	O
Fig	O
6a	O
and	O
6b	O
).	O
	O
We	O
also	O
replaced	O
Tyr	O
-	O
85	O
with	O
either	O
a	O
Phe	O
or	O
a	O
Trp	O
residue	O
and	O
both	O
mutant	O
proteins	O
largely	O
lost	O
their	O
catalytic	O
activity	O
and	O
iron	O
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
	O
Since	O
we	O
used	O
PEG	O
molecules	O
in	O
the	O
crystallization	O
conditions	O
,	O
the	O
observed	O
density	O
might	O
stem	O
from	O
an	O
ordered	O
part	O
of	O
a	O
PEG	O
molecule	O
,	O
or	O
low	O
molecular	O
weight	O
PEG	O
species	O
that	O
might	O
have	O
been	O
present	O
in	O
the	O
PEG	O
preparation	O
used	O
in	O
our	O
experiments	O
.	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	O
substrate	O
and	O
those	O
residues	O
that	O
coordinate	O
it	O
within	O
the	O
ectoine	O
synthase	O
active	O
site	O
.	O
	O
The	O
electron	O
density	O
was	O
calculated	O
as	O
an	O
omit	O
map	O
and	O
contoured	O
at	O
1	O
.	O
0	O
σ	O
.	O
	O
We	O
also	O
calculated	O
an	O
omit	O
map	O
and	O
the	O
electron	O
density	O
reappeared	O
(	O
Fig	O
7b	O
).	O
	O
These	O
correspond	O
to	O
amino	O
acids	O
Thr	O
-	O
40	O
,	O
Tyr	O
-	O
52	O
,	O
His	O
-	O
55	O
,	O
Glu	O
-	O
57	O
,	O
Gly	O
-	O
64	O
,	O
Tyr	O
-	O
85	O
-	O
Leu	O
-	O
87	O
,	O
His	O
-	O
93	O
,	O
Phe	O
-	O
107	O
,	O
Pro	O
-	O
109	O
,	O
Gly	O
-	O
113	O
,	O
Glu	O
-	O
115	O
,	O
and	O
His	O
-	O
117	O
in	O
the	O
(	O
Sa	O
)	O
EctC	O
protein	O
(	O
Fig	O
2	O
).	O
	O
Each	O
of	O
these	O
mutant	O
(	O
Sa	O
)	O
EctC	O
proteins	O
was	O
overproduced	O
in	O
E	O
.	O
coli	O
and	O
purified	O
by	O
affinity	O
chromatography	O
;	O
they	O
all	O
yielded	O
pure	O
and	O
stable	O
protein	O
preparations	O
.	O
	O
We	O
replaced	O
each	O
of	O
these	O
residues	O
with	O
an	O
Ala	O
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	O
content	O
of	O
the	O
mutant	O
proteins	O
.	O
	O
Each	O
of	O
these	O
residues	O
is	O
evolutionarily	O
highly	O
conserved	O
.	O
	O
His	O
-	O
117	O
is	O
a	O
strictly	O
conserved	O
residue	O
and	O
its	O
substitution	O
by	O
an	O
Ala	O
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	O
content	O
of	O
83	O
%	O
(	O
Table	O
1	O
).	O
	O
As	O
an	O
internal	O
control	O
for	O
our	O
mutagenesis	O
experiments	O
,	O
we	O
also	O
substituted	O
Thr	O
-	O
41	O
and	O
His	O
-	O
51	O
,	O
two	O
residues	O
that	O
are	O
not	O
evolutionarily	O
conserved	O
in	O
EctC	O
-	O
type	O
proteins	O
with	O
Ala	O
residues	O
.	O
	O
Hence	O
,	O
the	O
active	O
site	O
of	O
ectoine	O
synthase	O
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	O
-	O
catalyzed	O
formation	O
of	O
the	O
synthetic	O
compatible	O
solute	O
ADPC	O
through	O
the	O
cyclic	O
condensation	O
of	O
two	O
glutamine	O
molecules	O
.	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	O
-	O
γ	O
-	O
ADABA	O
within	O
the	O
EctC	O
active	O
site	O
.	O
	O
This	O
probably	O
worked	O
to	O
the	O
detriment	O
of	O
our	O
efforts	O
in	O
solving	O
crystal	O
structures	O
of	O
the	O
full	O
-	O
length	O
(	O
Sa	O
)	O
EctC	O
protein	O
in	O
complex	O
with	O
either	O
N	O
-	O
γ	O
-	O
ADABA	O
or	O
ectoine	O
.	O
	O
Interestingly	O
,	O
mutations	O
blocking	O
PIN	O
oligomerization	O
had	O
no	O
RNase	O
activity	O
,	O
indicating	O
that	O
both	O
oligomerization	O
and	O
NTD	O
binding	O
are	O
crucial	O
for	O
RNase	O
activity	O
in	O
vitro	O
.	O
	O
Regnase	O
-	O
1	O
is	O
a	O
member	O
of	O
Regnase	O
family	O
and	O
is	O
composed	O
of	O
a	O
PilT	O
N	O
-	O
terminus	O
like	O
(	O
PIN	O
)	O
domain	O
followed	O
by	O
a	O
CCCH	O
-	O
type	O
zinc	O
–	O
finger	O
(	O
ZF	O
)	O
domain	O
,	O
which	O
are	O
conserved	O
among	O
Regnase	O
family	O
members	O
.	O
	O
Moreover	O
,	O
Regnase	O
-	O
1	O
functions	O
as	O
a	O
dimer	O
through	O
intermolecular	O
PIN	O
-	O
PIN	O
interactions	O
during	O
cleavage	O
of	O
target	O
mRNA	O
.	O
	O
Although	O
the	O
PIN	O
domain	O
is	O
responsible	O
for	O
the	O
catalytic	O
activity	O
of	O
Regnase	O
-	O
1	O
,	O
the	O
roles	O
of	O
the	O
other	O
domains	O
are	O
largely	O
unknown	O
.	O
	O
These	O
results	O
indicate	O
that	O
not	O
only	O
the	O
PIN	O
but	O
also	O
the	O
ZF	O
domain	O
contribute	O
to	O
RNA	O
binding	O
,	O
while	O
the	O
NTD	O
is	O
not	O
likely	O
to	O
be	O
involved	O
in	O
direct	O
interaction	O
with	O
RNA	O
.	O
	O
Regnase	O
-	O
1	O
lacking	O
the	O
ZF	O
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	O
the	O
NTD	O
did	O
not	O
generate	O
cleaved	O
products	O
(	O
T1	O
/	O
2	O
>	O
90	O
minutes	O
).	O
	O
Dimer	O
formation	O
of	O
the	O
PIN	O
domains	O
	O
Domain	O
-	O
domain	O
interaction	O
between	O
the	O
NTD	O
and	O
the	O
PIN	O
domain	O
	O
Residues	O
critical	O
for	O
Regnase	O
-	O
1	O
RNase	O
activity	O
	O
The	O
other	O
mutated	O
residues	O
—	O
K152	O
,	O
R158	O
,	O
R188	O
,	O
R200	O
,	O
K204	O
,	O
K206	O
,	O
K257	O
,	O
and	O
R258	O
—	O
were	O
not	O
critical	O
for	O
RNase	O
activity	O
.	O
	O
One	O
group	O
consisted	O
of	O
catalytically	O
active	O
PIN	O
domains	O
with	O
mutation	O
of	O
basic	O
residues	O
found	O
in	O
the	O
previous	O
section	O
to	O
confer	O
decreased	O
RNase	O
activity	O
(	O
Fig	O
.	O
4	O
).	O
	O
According	O
to	O
the	O
proposed	O
model	O
,	O
an	O
R214A	B-mutant
PIN	O
domain	O
can	O
only	O
form	O
a	O
dimer	O
when	O
the	O
DDNN	B-mutant
PIN	O
acts	O
as	O
the	O
secondary	O
PIN	O
.	O
	O
The	O
previously	O
reported	O
crystal	O
structure	O
of	O
the	O
Regnase	O
-	O
1	O
PIN	O
domain	O
derived	O
from	O
Homo	O
sapiens	O
is	O
nearly	O
identical	O
to	O
the	O
one	O
derived	O
from	O
Mus	O
musculus	O
in	O
this	O
study	O
,	O
with	O
a	O
backbone	O
RMSD	O
of	O
0	O
.	O
2	O
Å	O
.	O
The	O
amino	O
acid	O
sequences	O
corresponding	O
to	O
PIN	O
(	O
residues	O
134	O
–	O
295	O
)	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
	O
Since	O
the	O
NMR	O
spectra	O
of	O
monomeric	O
mutants	O
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	O
mutants	O
were	O
affected	O
by	O
the	O
mutations	O
.	O
(	O
Supplementary	O
Fig	O
.	O
4b	O
,	O
c	O
).	O
	O
Moreover	O
,	O
we	O
found	O
that	O
the	O
NTD	O
associates	O
with	O
the	O
oligomeric	O
surface	O
of	O
the	O
primary	O
PIN	O
,	O
docking	O
to	O
a	O
helix	O
that	O
harbors	O
its	O
catalytic	O
residues	O
(	O
Figs	O
2b	O
and	O
3a	O
).	O
	O
The	O
affinity	O
of	O
the	O
domain	O
-	O
domain	O
interaction	O
between	O
two	O
PIN	O
domains	O
(	O
Kd	O
=	O
~	O
10	O
−	O
4	O
M	O
)	O
is	O
similar	O
to	O
that	O
of	O
the	O
NTD	O
-	O
PIN	O
(	O
Kd	O
=	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	O
–	O
133	O
between	O
the	O
NTD	O
and	O
the	O
primary	O
PIN	O
will	O
greatly	O
enhance	O
the	O
intramolecular	O
domain	O
interaction	O
in	O
the	O
case	O
of	O
full	O
-	O
length	O
Regnase	O
-	O
1	O
.	O
	O
Based	O
on	O
these	O
structural	O
and	O
functional	O
analyses	O
of	O
Regnase	O
-	O
1	O
domain	O
-	O
domain	O
interactions	O
,	O
we	O
performed	O
docking	O
simulations	O
of	O
the	O
NTD	O
,	O
PIN	O
dimer	O
,	O
and	O
IL	O
-	O
6	O
mRNA	O
.	O
	O
The	O
overall	O
model	O
of	O
regulation	O
of	O
Regnase	O
-	O
1	O
RNase	O
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
	O
Structural	O
and	O
functional	O
analyses	O
of	O
Regnase	O
-	O
1	O
.	O
	O
Fluorescence	O
intensity	O
of	O
the	O
free	O
IL	O
-	O
6	O
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	O
of	O
Regnase	O
-	O
1	O
.	O
	O
Domain	O
-	O
domain	O
interaction	O
between	O
the	O
NTD	O
and	O
the	O
PIN	O
domain	O
.	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	O
structure	O
were	O
colored	O
yellow	O
.	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	O
activities	O
were	O
colored	O
red	O
or	O
yellow	O
.	O
	O
(	O
c	O
)	O
In	O
vitro	O
cleavage	O
assay	O
of	O
Regnase	O
-	O
1	O
for	O
Regnase	O
-	O
1	O
mRNA	O
.	O
	O
Crystal	O
Structure	O
and	O
Activity	O
Studies	O
of	O
the	O
C11	O
Cysteine	O
Peptidase	O
from	O
Parabacteroides	O
merdae	O
in	O
the	O
Human	O
Gut	O
Microbiome	O
*	O
	O
However	O
,	O
despite	O
these	O
similarities	O
,	O
clan	O
CD	O
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	O
with	O
a	O
wide	O
variety	O
of	O
substrate	O
specificities	O
and	O
activation	O
mechanisms	O
.	O
	O
Structure	O
of	O
PmC11	O
	O
The	O
central	O
nine	O
-	O
stranded	O
β	O
-	O
sheet	O
(	O
β1	O
–	O
β9	O
)	O
of	O
PmC11	O
consists	O
of	O
six	O
parallel	O
and	O
three	O
anti	O
-	O
parallel	O
β	O
-	O
strands	O
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	O
includes	O
14	O
α	O
-	O
helices	O
with	O
six	O
(	O
α1	O
–	O
α2	O
and	O
α4	O
–	O
α7	O
)	O
closely	O
surrounding	O
the	O
β	O
-	O
sheet	O
in	O
an	O
approximately	O
parallel	O
orientation	O
.	O
	O
Helices	O
α1	O
,	O
α7	O
,	O
and	O
α6	O
are	O
located	O
on	O
one	O
side	O
of	O
the	O
β	O
-	O
sheet	O
with	O
α2	O
,	O
α4	O
,	O
and	O
α5	O
on	O
the	O
opposite	O
side	O
(	O
Fig	O
.	O
1A	O
).	O
	O
The	O
core	O
caspase	O
-	O
fold	O
is	O
highlighted	O
in	O
a	O
box	O
.	O
	O
The	O
CTD	O
of	O
PmC11	O
is	O
composed	O
of	O
a	O
tight	O
helical	O
bundle	O
formed	O
from	O
helices	O
α8	O
–	O
α14	O
and	O
includes	O
strands	O
βC	O
and	O
βF	O
,	O
and	O
β	O
-	O
hairpin	O
βD	O
–	O
βE	O
.	O
The	O
CTD	O
sits	O
entirely	O
on	O
one	O
side	O
of	O
the	O
enzyme	O
interacting	O
only	O
with	O
α3	O
,	O
α5	O
,	O
β9	O
,	O
and	O
the	O
loops	O
surrounding	O
β8	O
.	O
	O
D	O
,	O
cysteine	O
peptidase	O
activity	O
of	O
PmC11	O
.	O
	O
Inactive	O
PmC11C179A	B-mutant
was	O
not	O
processed	O
to	O
a	O
major	O
extent	O
by	O
active	O
PmC11	O
until	O
around	O
a	O
ratio	O
of	O
1	O
:	O
4	O
(	O
5	O
μg	O
of	O
active	O
PmC11	O
).	O
	O
F	O
,	O
activity	O
of	O
PmC11	O
against	O
basic	O
substrates	O
.	O
	O
G	O
,	O
electrostatic	O
surface	O
potential	O
of	O
PmC11	O
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
	O
Asp177	O
is	O
located	O
near	O
the	O
catalytic	O
cysteine	O
and	O
is	O
conserved	O
throughout	O
the	O
C11	O
family	O
,	O
suggesting	O
it	O
is	O
the	O
primary	O
S1	O
binding	O
site	O
residue	O
.	O
	O
Asp177	O
is	O
highly	O
conserved	O
throughout	O
the	O
clan	O
CD	O
C11	O
peptidases	O
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	O
CD	O
enzymes	O
,	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	O
-	O
VRPR	O
-	O
FMK	O
when	O
PmC11	O
is	O
overlaid	O
on	O
the	O
MALT1	O
-	O
P	O
structure	O
(	O
Fig	O
.	O
3C	O
).	O
	O
A	O
,	O
PmC11	O
activity	O
is	O
inhibited	O
by	O
Z	O
-	O
VRPR	O
-	O
FMK	O
.	O
	O
B	O
,	O
gel	O
-	O
shift	O
assay	O
reveals	O
that	O
Z	O
-	O
VRPR	O
-	O
FMK	O
binds	O
to	O
PmC11	O
.	O
	O
The	O
primary	O
structural	O
alignment	O
also	O
shows	O
that	O
the	O
catalytic	O
dyad	O
in	O
PmC11	O
is	O
structurally	O
conserved	O
in	O
clostripain	O
(	O
Fig	O
.	O
1A	O
).	O
	O
Unlike	O
PmC11	O
,	O
clostripain	O
has	O
two	O
cleavage	O
sites	O
(	O
Arg181	O
and	O
Arg190	O
),	O
which	O
results	O
in	O
the	O
removal	O
of	O
a	O
nonapeptide	O
,	O
and	O
is	O
required	O
for	O
full	O
activation	O
of	O
the	O
enzyme	O
(	O
highlighted	O
in	O
Fig	O
.	O
1A	O
).	O
	O
Interestingly	O
,	O
Arg190	O
was	O
found	O
to	O
align	O
with	O
Lys147	O
in	O
PmC11	O
.	O
	O
As	O
studies	O
on	O
clostripain	O
revealed	O
addition	O
of	O
Ca2	O
+	O
ions	O
are	O
required	O
for	O
full	O
activation	O
,	O
the	O
Ca2	O
+	O
dependence	O
of	O
PmC11	O
was	O
examined	O
.	O
	O
In	O
support	O
of	O
these	O
findings	O
,	O
EGTA	O
did	O
not	O
inhibit	O
PmC11	O
suggesting	O
that	O
,	O
unlike	O
clostripain	O
,	O
PmC11	O
does	O
not	O
require	O
Ca2	O
+	O
or	O
other	O
divalent	O
cations	O
,	O
for	O
activity	O
.	O
	O
The	O
enzyme	O
exhibits	O
all	O
of	O
the	O
key	O
structural	O
elements	O
of	O
clan	O
CD	O
members	O
,	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	O
-	O
sheet	O
with	O
a	O
novel	O
C	O
-	O
terminal	O
domain	O
.	O
	O
In	O
addition	O
,	O
the	O
structure	O
suggested	O
a	O
mechanism	O
of	O
self	O
-	O
inhibition	O
in	O
both	O
PmC11	O
and	O
clostripain	O
and	O
an	O
activation	O
mechanism	O
that	O
requires	O
autoprocessing	O
.	O
	O
This	O
is	O
also	O
the	O
case	O
in	O
PmC11	O
,	O
although	O
the	O
cleavage	O
loop	O
is	O
structurally	O
different	O
to	O
that	O
found	O
in	O
the	O
caspases	O
and	O
follows	O
the	O
catalytic	O
His	O
(	O
Fig	O
.	O
1A	O
),	O
as	O
opposed	O
to	O
the	O
Cys	O
in	O
the	O
caspases	O
.	O
	O
Like	O
PmC11	O
,	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	O
of	O
the	O
blocking	O
region	O
is	O
required	O
.	O
	O
Indeed	O
,	O
insights	O
gained	O
from	O
an	O
analysis	O
of	O
the	O
PmC11	O
structure	O
revealed	O
the	O
identity	O
of	O
the	O
Trypanosoma	O
brucei	O
PNT1	O
protein	O
as	O
a	O
C11	O
cysteine	O
peptidase	O
with	O
an	O
essential	O
role	O
in	O
organelle	O
replication	O
.	O
	O
In	O
addition	O
,	O
18S	O
and	O
25S	O
(	O
yeast	O
)/	O
28S	O
(	O
humans	O
)	O
rRNAs	O
contain	O
several	O
base	O
modifications	O
catalyzed	O
by	O
site	O
-	O
specific	O
and	O
snoRNA	O
-	O
independent	O
enzymes	O
.	O
	O
In	O
a	O
second	O
step	O
,	O
the	O
essential	O
SPOUT	O
-	O
class	O
methyltransferase	O
Nep1	O
/	O
Emg1	O
modifies	O
the	O
pseudouridine	O
to	O
N1	O
-	O
methylpseudouridine	O
.	O
	O
Hypermodified	O
m1acp3Ψ	O
elutes	O
at	O
7	O
.	O
4	O
min	O
(	O
wild	O
type	O
,	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
	O
Upper	O
lanes	O
show	O
the	O
ethidium	O
bromide	O
staining	O
of	O
the	O
18S	O
rRNAs	O
for	O
quantification	O
.	O
	O
The	O
efficiency	O
of	O
siRNA	O
mediated	O
HsTSR3	O
repression	O
correlates	O
with	O
the	O
primer	O
extension	O
signals	O
(	O
see	O
Supplementary	O
Figure	O
S2A	O
).	O
	O
In	O
contrast	O
,	O
the	O
only	O
other	O
structurally	O
characterized	O
acp	O
transferase	O
enzyme	O
Tyw2	O
belongs	O
to	O
the	O
Rossmann	O
-	O
fold	O
class	O
of	O
methyltransferase	O
proteins	O
.	O
	O
Indeed	O
,	O
in	O
wild	O
-	O
type	O
yeast	O
a	O
strong	O
primer	O
extension	O
stop	O
signal	O
occurred	O
at	O
position	O
1192	O
.	O
	O
As	O
expected	O
,	O
in	O
a	O
Δsnr35	B-mutant
deletion	O
preventing	O
pseudouridylation	O
and	O
N1	O
-	O
methylation	O
(	O
resulting	O
in	O
acp3U	O
)	O
as	O
well	O
as	O
in	O
a	O
Δnep1	B-mutant
deletion	O
strain	O
where	O
pseudouridine	O
is	O
not	O
methylated	O
(	O
resulting	O
in	O
acp3Ψ	O
)	O
a	O
primer	O
extension	O
stop	O
signal	O
of	O
similar	O
intensity	O
as	O
in	O
the	O
wild	O
type	O
was	O
observed	O
.	O
	O
The	O
efficiency	O
of	O
siRNA	O
-	O
mediated	O
depletion	O
was	O
established	O
by	O
RT	O
-	O
qPCR	O
and	O
found	O
to	O
be	O
very	O
high	O
with	O
siRNA	O
544	O
(	O
Supplementary	O
Figure	O
S2A	O
,	O
remaining	O
TSR3	O
mRNA	O
level	O
of	O
2	O
%).	O
	O
Phenotypic	O
characterization	O
of	O
Δtsr3	B-mutant
mutants	O
	O
Phenotypic	O
characterization	O
of	O
yeast	O
TSR3	O
deletion	O
(	O
Δtrs3	B-mutant
)	O
and	O
human	O
TSR3	O
depletion	O
(	O
siRNAs	O
544	O
and	O
545	O
)	O
and	O
cellular	O
localization	O
of	O
yeast	O
Tsr3	O
.	O
(	O
A	O
)	O
Growth	O
of	O
yeast	O
wild	O
type	O
,	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	O
Δsnr35	B-mutant
/	O
SNR35	O
heterozygous	O
diploids	O
.	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	O
Δtsr3	B-mutant
cells	O
(	O
see	O
accumulation	O
of	O
35S	O
in	O
Supplementary	O
Figure	O
S2C	O
)	O
and	O
Tsr3	O
depleted	O
human	O
cells	O
(	O
see	O
47S	O
/	O
45S	O
accumulation	O
in	O
Figure	O
2C	O
and	O
Northern	O
blot	O
quantification	O
in	O
Supplementary	O
Figure	O
S2B	O
).	O
	O
Consistent	O
with	O
its	O
role	O
in	O
late	O
18S	O
rRNA	O
processing	O
,	O
TSR3	O
deletion	O
leads	O
to	O
a	O
ribosomal	O
subunit	O
imbalance	O
with	O
a	O
reduced	O
40S	O
to	O
60S	O
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
	O
After	O
polysome	O
gradient	O
separation	O
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
	O
Such	O
distribution	O
on	O
a	O
density	O
gradient	O
suggests	O
that	O
Tsr3	O
only	O
interacts	O
transiently	O
with	O
pre	O
-	O
40S	O
subunits	O
,	O
which	O
presumably	O
explains	O
why	O
it	O
was	O
not	O
characterized	O
in	O
pre	O
-	O
ribosome	O
affinity	O
purifications	O
.	O
	O
Structure	O
of	O
Tsr3	O
	O
However	O
,	O
these	O
archaeal	O
homologs	O
are	O
significantly	O
smaller	O
than	O
ScTsr3	O
(∼	O
190	O
aa	O
in	O
archaea	O
vs	O
.	O
313	O
aa	O
in	O
yeast	O
)	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
	O
N	O
-	O
terminal	O
truncations	O
of	O
up	O
to	O
45	O
aa	O
and	O
C	O
-	O
terminal	O
truncations	O
of	O
up	O
to	O
76	O
aa	O
mediated	O
acp	O
modification	O
as	O
efficiently	O
as	O
the	O
full	O
-	O
length	O
protein	O
and	O
no	O
significant	O
increased	O
levels	O
of	O
20S	O
pre	O
-	O
RNA	O
were	O
detected	O
.	O
	O
Even	O
a	O
Tsr3	O
fragment	O
with	O
a	O
90	O
aa	O
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	O
aa	O
almost	O
completely	O
abolished	O
the	O
primer	O
extension	O
arrest	O
(	O
Figure	O
3B	O
).	O
	O
Strong	O
20S	O
rRNA	O
accumulation	O
similar	O
to	O
that	O
of	O
the	O
Δtsr3	B-mutant
deletion	O
is	O
observed	O
for	O
Tsr3	O
fragments	O
37	O
–	O
223	O
or	O
46	O
–	O
223	O
.	O
	O
We	O
focused	O
on	O
archaeal	O
species	O
containing	O
a	O
putative	O
Nep1	O
homolog	O
suggesting	O
that	O
these	O
species	O
are	O
in	O
principle	O
capable	O
of	O
synthesizing	O
N1	O
-	O
methyl	O
-	O
N3	O
-	O
acp	O
-	O
pseudouridine	O
.	O
	O
The	O
C	O
-	O
terminal	O
domain	O
(	O
aa	O
93	O
–	O
184	O
)	O
has	O
a	O
globular	O
all	O
α	O
-	O
helical	O
structure	O
comprising	O
α	O
-	O
helices	O
α4	O
to	O
α9	O
.	O
	O
Remarkably	O
,	O
the	O
entire	O
C	O
-	O
terminal	O
domain	O
(	O
92	O
aa	O
)	O
of	O
the	O
protein	O
is	O
threaded	O
through	O
the	O
loop	O
which	O
connects	O
β	O
-	O
strand	O
β3	O
and	O
α	O
-	O
helix	O
α2	O
of	O
the	O
N	O
-	O
terminal	O
domain	O
.	O
	O
Tsr3	O
has	O
a	O
fold	O
similar	O
to	O
SPOUT	O
-	O
class	O
RNA	O
methyltransferases	O
.	O
(	O
A	O
)	O
Cartoon	O
representation	O
of	O
the	O
X	O
-	O
ray	O
structure	O
of	O
VdTsr3	O
in	O
two	O
orientations	O
.	O
	O
A	O
red	O
arrow	O
marks	O
the	O
location	O
of	O
the	O
topological	O
knot	O
in	O
the	O
structure	O
.	O
(	O
B	O
)	O
Secondary	O
structure	O
representation	O
of	O
the	O
VdTsr3	O
structure	O
.	O
	O
Structure	O
predictions	O
suggested	O
that	O
Tsr3	O
might	O
contain	O
a	O
so	O
-	O
called	O
RLI	O
domain	O
which	O
contains	O
a	O
‘	O
bacterial	O
like	O
’	O
ferredoxin	O
fold	O
and	O
binds	O
two	O
iron	O
-	O
sulfur	O
clusters	O
through	O
eight	O
conserved	O
cysteine	O
residues	O
.	O
	O
A	O
notable	O
exception	O
is	O
Trm10	O
.	O
	O
However	O
,	O
there	O
are	O
no	O
structural	O
similarities	O
between	O
Tsr3	O
and	O
Tyw2	O
,	O
which	O
contains	O
an	O
all	O
-	O
parallel	O
β	O
-	O
sheet	O
of	O
a	O
different	O
topology	O
and	O
no	O
knot	O
structure	O
.	O
	O
The	O
ribose	O
2	O
′	O
and	O
3	O
′	O
hydroxyl	O
groups	O
of	O
SAM	O
are	O
hydrogen	O
bonded	O
to	O
the	O
backbone	O
carbonyl	O
group	O
of	O
I69	O
.	O
	O
Consequently	O
,	O
the	O
accessibility	O
of	O
this	O
methyl	O
group	O
for	O
a	O
nucleophilic	O
attack	O
is	O
strongly	O
reduced	O
in	O
comparison	O
with	O
RNA	O
-	O
methyltransferases	O
such	O
as	O
Trm10	O
(	O
Figure	O
5B	O
,	O
C	O
).	O
	O
In	O
contrast	O
,	O
the	O
acp	O
side	O
chain	O
of	O
SAM	O
is	O
accessible	O
for	O
reactions	O
in	O
the	O
Tsr3	O
-	O
bound	O
state	O
(	O
Figure	O
5B	O
).	O
	O
Hydrogen	O
bonds	O
are	O
indicated	O
by	O
dashed	O
lines	O
.	O
	O
(	O
E	O
)	O
Binding	O
of	O
14C	O
-	O
labeled	O
SAM	O
to	O
SsTsr3	O
.	O
	O
This	O
correlates	O
with	O
a	O
20S	O
pre	O
-	O
rRNA	O
accumulation	O
comparable	O
to	O
the	O
Δtsr3	B-mutant
deletion	O
(	O
right	O
:	O
northern	O
blot	O
).	O
	O
This	O
suggests	O
that	O
the	O
hydrophobic	O
interaction	O
between	O
SAM	O
'	O
s	O
methyl	O
group	O
and	O
the	O
hydrophobic	O
pocket	O
of	O
Tsr3	O
is	O
thermodynamically	O
important	O
for	O
the	O
interaction	O
.	O
	O
Furthermore	O
,	O
a	O
W	O
to	O
A	O
mutation	O
at	O
the	O
equivalent	O
position	O
W114	O
in	O
ScTsr3	O
strongly	O
reduced	O
the	O
in	O
vivo	O
acp	O
transferase	O
activity	O
(	O
Figure	O
5F	O
).	O
	O
The	O
side	O
chain	O
hydroxyl	O
group	O
of	O
T19	O
seems	O
of	O
minor	O
importance	O
for	O
SAM	O
binding	O
since	O
mutations	O
of	O
T17	O
(	O
T19	O
in	O
VdTsr3	O
)	O
to	O
either	O
A	O
or	O
D	O
did	O
not	O
significantly	O
influence	O
the	O
SAM	O
-	O
binding	O
affinity	O
of	O
SsTsr3	O
(	O
KD	O
'	O
s	O
=	O
3	O
.	O
9	O
or	O
11	O
.	O
2	O
mM	O
,	O
respectively	O
).	O
	O
In	O
the	O
C	O
-	O
terminal	O
domain	O
,	O
the	O
surface	O
exposed	O
α	O
-	O
helices	O
α5	O
and	O
α7	O
carry	O
a	O
significant	O
amount	O
of	O
positively	O
charged	O
amino	O
acids	O
.	O
	O
RNA	O
-	O
binding	O
of	O
Tsr3	O
.	O
	O
Also	O
shown	O
in	O
stick	O
representation	O
is	O
a	O
negatively	O
charged	O
MES	O
ion	O
.	O
	O
The	O
presence	O
of	O
saturating	O
amounts	O
of	O
SAM	O
(	O
2	O
mM	O
)	O
did	O
not	O
have	O
a	O
significant	O
influence	O
on	O
the	O
RNA	O
-	O
affinity	O
of	O
SsTsr3	O
(	O
KD	O
of	O
1	O
.	O
7	O
μM	O
for	O
the	O
20mer	O
-	O
GC	O
-	O
RNA	O
)	O
suggesting	O
no	O
cooperativity	O
in	O
substrate	O
binding	O
.	O
	O
This	O
suggests	O
that	O
enzymes	O
with	O
a	O
SAM	O
-	O
dependent	O
acp	O
transferase	O
activity	O
might	O
have	O
evolved	O
from	O
SAM	O
-	O
dependent	O
methyltransferases	O
by	O
slight	O
modifications	O
of	O
the	O
SAM	O
-	O
binding	O
pocket	O
.	O
	O
In	O
contrast	O
to	O
Nep1	O
,	O
the	O
enzyme	O
preceding	O
Tsr3	O
in	O
the	O
m1acp3Ψ	O
biosynthesis	O
pathway	O
,	O
Tsr3	O
binds	O
rather	O
weakly	O
and	O
with	O
little	O
specificity	O
to	O
its	O
isolated	O
substrate	O
RNA	O
.	O
	O
Recently	O
,	O
structural	O
,	O
functional	O
,	O
and	O
CRAC	O
(	O
cross	O
-	O
linking	O
and	O
cDNA	O
analysis	O
)	O
experiments	O
of	O
late	O
assembly	O
factors	O
involved	O
in	O
cytoplasmic	O
processing	O
of	O
40S	O
subunits	O
,	O
along	O
with	O
cryo	O
-	O
EM	O
studies	O
of	O
the	O
late	O
pre	O
-	O
40S	O
subunits	O
have	O
provided	O
important	O
insights	O
into	O
late	O
pre	O
-	O
40S	O
processing	O
.	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	O
subunit	O
assembly	O
with	O
only	O
completely	O
processed	O
precursors	O
.	O
	O
The	O
YfiBNR	O
system	O
contains	O
three	O
protein	O
members	O
and	O
modulates	O
intracellular	O
c	O
-	O
di	O
-	O
GMP	O
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
	O
More	O
recently	O
,	O
this	O
system	O
was	O
also	O
reported	O
in	O
other	O
Gram	O
-	O
negative	O
bacteria	O
,	O
such	O
as	O
Escherichia	O
coli	O
(	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	O
pneumonia	O
(	O
Huertas	O
et	O
al	O
.,)	O
and	O
Yersinia	O
pestis	O
(	O
Ren	O
et	O
al	O
.,).	O
	O
Whether	O
YfiB	O
directly	O
recruits	O
YfiR	O
or	O
recruits	O
YfiR	O
via	O
a	O
third	O
partner	O
is	O
an	O
open	O
question	O
.	O
	O
It	O
has	O
been	O
reported	O
that	O
the	O
activation	O
of	O
YfiN	O
may	O
be	O
induced	O
by	O
redox	O
-	O
driven	O
misfolding	O
of	O
YfiR	O
(	O
Giardina	O
et	O
al	O
.,;	O
Malone	O
et	O
al	O
.,;	O
Malone	O
et	O
al	O
.,).	O
	O
In	O
addition	O
,	O
quorum	O
sensing	O
-	O
related	O
dephosphorylation	O
of	O
the	O
PAS	O
domain	O
of	O
YfiN	O
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
	O
The	O
crystal	O
structure	O
of	O
YfiB	O
monomer	O
consists	O
of	O
a	O
five	O
-	O
stranded	O
β	O
-	O
sheet	O
(	O
β1	O
-	O
2	O
-	O
5	O
-	O
3	O
-	O
4	O
)	O
flanked	O
by	O
five	O
α	O
-	O
helices	O
(	O
α1	O
–	O
5	O
)	O
on	O
one	O
side	O
.	O
	O
The	O
residues	O
proposed	O
to	O
contribute	O
to	O
YfiB	O
activation	O
are	O
illustrated	O
in	O
sticks	O
.	O
	O
It	O
has	O
been	O
reported	O
that	O
single	O
mutants	O
of	O
Q39	O
,	O
L43	O
,	O
F48	O
and	O
W55	O
contribute	O
to	O
YfiB	O
activation	O
leading	O
to	O
the	O
induction	O
of	O
the	O
SCV	O
phenotype	O
in	O
P	O
.	O
aeruginosa	O
PAO1	O
(	O
Malone	O
et	O
al	O
.,).	O
	O
These	O
two	O
regions	O
contribute	O
a	O
robust	O
hydrogen	O
-	O
bonding	O
network	O
to	O
the	O
YfiB	O
-	O
YfiR	O
interface	O
,	O
resulting	O
in	O
a	O
tightly	O
bound	O
complex	O
.	O
	O
Therefore	O
,	O
it	O
is	O
possible	O
that	O
both	O
dimeric	O
types	O
might	O
exist	O
in	O
solution	O
.	O
	O
In	O
the	O
Pal	O
/	O
PG	O
-	O
P	O
complex	O
structure	O
,	O
the	O
m	O
-	O
Dap5	O
ϵ	O
-	O
carboxylate	O
group	O
interacts	O
with	O
the	O
side	O
-	O
chain	O
atoms	O
of	O
D71	O
and	O
the	O
main	O
-	O
chain	O
amide	O
of	O
D37	O
(	O
Fig	O
.	O
4B	O
).	O
	O
Calculation	O
using	O
the	O
ConSurf	O
Server	O
(	O
http	O
://	O
consurf	O
.	O
tau	O
.	O
ac	O
.	O
il	O
/),	O
which	O
estimates	O
the	O
evolutionary	O
conservation	O
of	O
amino	O
acid	O
positions	O
and	O
visualizes	O
information	O
on	O
the	O
structure	O
surface	O
,	O
revealed	O
a	O
conserved	O
surface	O
on	O
YfiR	O
that	O
contributes	O
to	O
the	O
interaction	O
with	O
YfiB	O
(	O
Fig	O
.	O
3E	O
and	O
3F	O
).	O
	O
E163	O
and	O
I169	O
are	O
YfiB	O
-	O
interacting	O
residues	O
of	O
YfiR	O
,	O
in	O
which	O
E163	O
forms	O
a	O
hydrogen	O
bond	O
with	O
R96	O
of	O
YfiB	O
(	O
Fig	O
.	O
3D	O
-	O
II	O
)	O
and	O
I169	O
is	O
involved	O
in	O
forming	O
the	O
L166	O
/	O
I169	O
/	O
V176	O
/	O
P178	O
/	O
L181	O
hydrophobic	O
core	O
for	O
anchoring	O
F57	O
of	O
YfiB	O
(	O
Fig	O
.	O
3D	O
-	O
I	O
(	O
ii	O
)).	O
	O
Intriguingly	O
,	O
a	O
Dali	O
search	O
(	O
Holm	O
and	O
Rosenstrom	O
,)	O
indicated	O
that	O
the	O
closest	O
homologs	O
of	O
YfiR	O
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	O
-	O
Trp	O
,	O
L	O
-	O
Phe	O
,	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	O
can	O
recognize	O
these	O
molecules	O
.	O
	O
Structural	O
analyses	O
revealed	O
that	O
the	O
VB6	O
and	O
L	O
-	O
Trp	O
molecules	O
are	O
bound	O
at	O
the	O
periphery	O
of	O
the	O
YfiR	O
dimer	O
,	O
but	O
not	O
at	O
the	O
dimer	O
interface	O
.	O
	O
To	O
evaluate	O
the	O
importance	O
of	O
F57	O
in	O
YfiBL43P	O
-	O
YfiR	O
interaction	O
,	O
the	O
binding	O
affinities	O
of	O
YfiBL43P	B-mutant
and	O
YfiBL43P	B-mutant
/	O
F57A	B-mutant
for	O
YfiR	O
were	O
measured	O
by	O
isothermal	O
titration	O
calorimetry	O
(	O
ITC	O
).	O
	O
Provided	O
that	O
the	O
diameter	O
of	O
the	O
widest	O
part	O
of	O
the	O
YfiB	O
dimer	O
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	O
dimer	O
should	O
be	O
able	O
to	O
penetrate	O
the	O
PG	O
layer	O
.	O
	O
Once	O
activated	O
by	O
certain	O
cell	O
stress	O
,	O
the	O
dimeric	O
YfiB	O
transforms	O
from	O
a	O
compact	O
conformation	O
to	O
a	O
stretched	O
conformation	O
,	O
allowing	O
the	O
periplasmic	O
domain	O
of	O
the	O
membrane	O
-	O
anchored	O
YfiB	O
to	O
penetrate	O
the	O
cell	O
wall	O
and	O
sequester	O
the	O
YfiR	O
dimer	O
,	O
thus	O
relieving	O
the	O
repression	O
of	O
YfiN	O
	O
The	O
YfiBNR	O
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	O
-	O
di	O
-	O
GMP	O
level	O
.	O
	O
These	O
APFs	O
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
	O
These	O
observations	O
suggest	O
that	O
the	O
Aβ	O
trimers	O
,	O
hexamers	O
,	O
dodecamers	O
,	O
and	O
related	O
assemblies	O
may	O
be	O
associated	O
with	O
presymptomatic	O
neurodegeneration	O
,	O
while	O
Aβ	O
dimers	O
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
	O
Many	O
of	O
these	O
studies	O
have	O
reported	O
the	O
monomer	O
subunit	O
as	O
adopting	O
a	O
β	O
-	O
hairpin	O
conformation	O
,	O
in	O
which	O
the	O
hydrophobic	O
central	O
and	O
C	O
-	O
terminal	O
regions	O
form	O
an	O
antiparallel	O
β	O
-	O
sheet	O
.	O
	O
In	O
2008	O
,	O
Hoyer	O
et	O
al	O
.	O
reported	O
the	O
NMR	O
structure	O
of	O
an	O
Aβ	O
monomer	O
bound	O
to	O
an	O
artificial	O
binding	O
protein	O
called	O
an	O
affibody	O
(	O
PDB	O
2OTK	O
).	O
	O
This	O
Aβ	O
β	O
-	O
hairpin	O
encompasses	O
residues	O
17	O
–	O
37	O
and	O
contains	O
two	O
β	O
-	O
strands	O
comprising	O
Aβ17	O
–	O
24	O
and	O
Aβ30	O
–	O
37	O
connected	O
by	O
an	O
Aβ25	O
–	O
29	O
loop	O
.	O
	O
In	O
a	O
related	O
study	O
,	O
Sandberg	O
et	O
al	O
.	O
constrained	O
Aβ	O
in	O
a	O
β	O
-	O
hairpin	O
conformation	O
by	O
mutating	O
residues	O
A21	O
and	O
A30	O
to	O
cysteine	O
and	O
forming	O
an	O
intramolecular	O
disulfide	O
bond	O
.	O
	O
After	O
determining	O
the	O
X	O
-	O
ray	O
crystallographic	O
structure	O
of	O
the	O
p	O
-	O
iodophenylalanine	O
variant	O
we	O
attempt	O
to	O
determine	O
the	O
structure	O
of	O
the	O
native	O
phenylalanine	O
compound	O
by	O
isomorphous	O
replacement	O
.	O
	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	O
in	O
any	O
condition	O
.	O
	O
Although	O
the	O
disulfide	O
bond	O
between	O
positions	O
24	O
and	O
29	O
helps	O
stabilize	O
the	O
β	O
-	O
hairpin	O
,	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	O
–	O
36	O
β	O
-	O
hairpin	O
.	O
	O
Crystallization	O
,	O
X	O
-	O
ray	O
Crystallographic	O
Data	O
Collection	O
,	O
Data	O
Processing	O
,	O
and	O
Structure	O
Determination	O
of	O
Peptides	B-mutant
2	I-mutant
and	I-mutant
4	I-mutant
	O
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
	O
Peptide	B-mutant
2	I-mutant
assembles	O
into	O
oligomers	O
similar	O
in	O
morphology	O
to	O
those	O
formed	O
by	O
peptide	B-mutant
1	I-mutant
.	O
	O
Hydrogen	O
bonding	O
and	O
hydrophobic	O
interactions	O
between	O
residues	O
on	O
the	O
β	O
-	O
strands	O
comprising	O
Aβ17	O
–	O
23	O
and	O
Aβ30	O
–	O
36	O
stabilize	O
the	O
core	O
of	O
the	O
trimer	O
.	O
	O
At	O
the	O
corners	O
of	O
the	O
trimer	O
,	O
the	O
pairs	O
of	O
β	O
-	O
hairpin	O
monomers	O
form	O
four	O
hydrogen	O
bonds	O
:	O
two	O
between	O
the	O
main	O
chains	O
of	O
V18	O
and	O
E22	O
and	O
two	O
between	O
δOrn	O
and	O
the	O
main	O
chain	O
of	O
C24	O
(	O
Figure	O
3B	O
).	O
	O
The	O
other	O
face	O
of	O
the	O
trimer	O
displays	O
a	O
smaller	O
hydrophobic	O
surface	O
,	O
which	O
includes	O
the	O
side	O
chains	O
of	O
residues	O
V18	O
,	O
F20	O
,	O
and	O
I31	O
of	O
the	O
three	O
β	O
-	O
hairpins	O
(	O
Figure	O
3D	O
).	O
	O
Dodecamer	O
	O
The	O
four	O
trimers	O
arrange	O
in	O
a	O
tetrahedral	O
fashion	O
,	O
creating	O
a	O
central	O
cavity	O
inside	O
the	O
dodecamer	O
.	O
Because	O
each	O
trimer	O
is	O
triangular	O
,	O
the	O
resulting	O
arrangement	O
resembles	O
an	O
octahedron	O
.	O
	O
Residues	O
L17	O
,	O
L34	O
,	O
and	O
V36	O
are	O
shown	O
as	O
spheres	O
,	O
illustrating	O
the	O
hydrophobic	O
packing	O
that	O
occurs	O
at	O
the	O
six	O
vertices	O
of	O
the	O
dodecamer	O
.	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	O
.	O
	O
The	O
electron	O
density	O
map	O
for	O
the	O
X	O
-	O
ray	O
crystallographic	O
structure	O
of	O
peptide	B-mutant
2	I-mutant
has	O
long	O
tubes	O
of	O
electron	O
density	O
inside	O
the	O
central	O
cavity	O
of	O
the	O
dodecamer	O
.	O
	O
Although	O
Jeffamine	O
M	O
-	O
600	O
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	O
density	O
.	O
	O
Annular	O
Pore	O
	O
The	O
same	O
eclipsed	O
interface	O
also	O
occurs	O
between	O
dodecamers	O
1	O
and	O
5	O
and	O
3	O
and	O
4	O
.	O
(	O
C	O
)	O
Staggered	O
interface	O
between	O
dodecamers	O
2	O
and	O
3	O
(	O
side	O
view	O
).	O
	O
It	O
is	O
important	O
to	O
note	O
that	O
the	O
annular	O
pore	O
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	O
lattice	O
.	O
	O
The	O
dodecamers	O
further	O
assemble	O
to	O
form	O
an	O
annular	O
pore	O
(	O
Figure	O
6	O
).	O
	O
Monomeric	O
Aβ	O
folds	O
to	O
form	O
a	O
β	O
-	O
hairpin	O
in	O
which	O
the	O
hydrophobic	O
central	O
and	O
C	O
-	O
terminal	O
regions	O
form	O
an	O
antiparallel	O
β	O
-	O
sheet	O
.	O
	O
Four	O
triangular	O
trimers	O
assemble	O
to	O
form	O
a	O
dodecamer	O
.	O
	O
Five	O
dodecamers	O
assemble	O
to	O
form	O
an	O
annular	O
pore	O
.	O
	O
These	O
criteria	O
have	O
been	O
used	O
to	O
classify	O
the	O
Aβ	O
oligomers	O
that	O
accumulate	O
in	O
vivo	O
.	O
	O
Aβ	O
dimers	O
have	O
been	O
classified	O
as	O
fibrillar	O
oligomers	O
,	O
whereas	O
Aβ	O
trimers	O
,	O
Aβ	O
*	O
56	O
,	O
and	O
APFs	O
have	O
been	O
classified	O
as	O
nonfibrillar	O
oligomers	O
.	O
	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	O
,	O
dodecamer	O
,	O
and	O
annular	O
pore	O
formed	O
by	O
peptide	B-mutant
2	I-mutant
may	O
share	O
similarities	O
to	O
the	O
trimers	O
,	O
Aβ	O
*	O
56	O
,	O
and	O
APFs	O
observed	O
in	O
vivo	O
.	O
	O
Annular	O
Pores	O
Formed	O
by	O
Aβ	O
and	O
Peptide	B-mutant
2	I-mutant
	O
This	O
mode	O
of	O
assembly	O
is	O
not	O
unique	O
to	O
Aβ	O
.	O
	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	O
Aβ	O
peptides	O
will	O
ultimately	O
allow	O
larger	O
fragments	O
of	O
Aβ	O
to	O
be	O
crystallized	O
,	O
thus	O
providing	O
greater	O
insights	O
into	O
the	O
structures	O
of	O
Aβ	O
oligomers	O
.	O
	O
In	O
contrast	O
,	O
the	O
N	O
‐	O
terminal	O
coactivator	O
‐	O
binding	O
site	O
,	O
activation	O
function	O
‐	O
1	O
(	O
AF	O
‐	O
1	O
),	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
	O
ERα	O
domain	O
organization	O
lettered	O
,	O
A	O
‐	O
F	O
.	O
DBD	O
,	O
DNA	O
‐	O
binding	O
domain	O
;	O
LBD	O
,	O
ligand	O
‐	O
binding	O
domain	O
;	O
AF	O
,	O
activation	O
function	O
	O
Branched	O
causality	O
model	O
for	O
ERα	O
‐	O
mediated	O
cell	O
proliferation	O
.	O
	O
However	O
,	O
the	O
agonist	O
activity	O
of	O
SERMs	O
derives	O
from	O
activation	O
function	O
‐	O
1	O
(	O
AF	O
‐	O
1	O
)—	O
a	O
coactivator	O
recruitment	O
site	O
located	O
in	O
the	O
AB	O
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
	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	O
/	O
2	O
/	O
3	O
recruitment	O
determines	O
GREB1	O
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
	O
OBHS	O
is	O
an	O
indirect	O
modulator	O
that	O
dislocates	O
the	O
h11	O
C	O
‐	O
terminus	O
to	O
destabilize	O
the	O
h11	O
–	O
h12	O
interface	O
(	O
PDB	O
4ZN9	O
).	O
	O
The	O
ERα	O
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
	O
Ligand	O
‐	O
specific	O
signaling	O
underlies	O
ERα	O
‐	O
mediated	O
cell	O
proliferation	O
	O
(	O
B	O
)	O
Ligand	O
class	O
analysis	O
of	O
the	O
L	O
‐	O
Luc	O
ERα	O
‐	O
WT	O
and	O
ERα	B-mutant
‐	I-mutant
ΔAB	I-mutant
activities	O
in	O
HepG2	O
cells	O
.	O
	O
Deletion	O
of	O
the	O
AB	O
domain	O
significantly	O
reduced	O
the	O
average	O
L	O
‐	O
Luc	O
activities	O
of	O
14	O
scaffolds	O
(	O
Student	O
'	O
s	O
t	O
‐	O
test	O
,	O
P	O
≤	O
0	O
.	O
05	O
)	O
(	O
Fig	O
3B	O
).	O
	O
The	O
value	O
of	O
r	O
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
	O
This	O
cluster	O
includes	O
two	O
classes	O
of	O
direct	O
modulators	O
(	O
cyclofenil	O
‐	O
ASC	O
and	O
WAY	O
dimer	O
),	O
and	O
six	O
classes	O
of	O
indirect	O
modulators	O
(	O
2	O
,	O
5	O
‐	O
DTP	O
,	O
3	O
,	O
4	O
‐	O
DTP	O
,	O
S	O
‐	O
OBHS	O
‐	O
2	O
and	O
S	O
‐	O
OBHS	O
‐	O
3	O
,	O
furan	O
,	O
and	O
WAY	O
‐	O
D	O
).	O
	O
In	O
contrast	O
,	O
AF	O
‐	O
1	O
was	O
a	O
determinant	O
of	O
signaling	O
specificity	O
for	O
scaffolds	O
in	O
cluster	O
2	O
.	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	O
‐	O
1	O
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	O
of	O
the	O
AB	O
or	O
F	O
domain	O
rarely	O
induced	O
such	O
correlations	O
(	O
Fig	O
3D	O
),	O
except	O
for	O
A	O
‐	O
CD	O
and	O
OBHS	O
‐	O
ASC	O
analogs	O
,	O
where	O
deletion	O
of	O
the	O
AB	O
domain	O
or	O
F	O
domain	O
led	O
to	O
positive	O
correlations	O
with	O
E	O
‐	O
Luc	O
activity	O
and	O
/	O
or	O
GREB1	O
levels	O
(	O
Fig	O
3D	O
lanes	O
13	O
and	O
18	O
).	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	O
regression	O
analyses	O
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
	O
The	O
lack	O
of	O
significant	O
predictors	O
for	O
OBHS	O
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
	O
The	O
significant	O
correlations	O
with	O
GREB1	O
expression	O
and	O
NCOA1	O
/	O
2	O
/	O
3	O
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	O
/	O
2	O
/	O
3	O
recruitment	O
determines	O
GREB1	O
expression	O
,	O
which	O
then	O
drives	O
proliferation	O
.	O
	O
3	O
,	O
4	O
‐	O
DTP	O
,	O
cyclofenil	O
,	O
3	O
,	O
4	O
‐	O
DTPD	O
,	O
and	O
imidazopyridine	O
analogs	O
had	O
NCOA1	O
/	O
3	O
recruitment	O
profiles	O
that	O
predicted	O
their	O
proliferative	O
effects	O
,	O
without	O
determining	O
GREB1	O
levels	O
(	O
Fig	O
3E	O
and	O
F	O
,	O
lanes	O
5	O
and	O
14	O
–	O
16	O
).	O
	O
Therefore	O
,	O
we	O
first	O
performed	O
a	O
time	O
‐	O
course	O
study	O
,	O
and	O
found	O
that	O
E2	O
and	O
the	O
WAY	O
‐	O
C	O
analog	O
,	O
AAPII	O
‐	O
151	O
‐	O
4	O
,	O
induced	O
recruitment	O
of	O
NCOA3	O
to	O
the	O
GREB1	O
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
	O
However	O
,	O
the	O
ChIP	O
assays	O
for	O
WAY	O
‐	O
C	O
‐	O
induced	O
recruitment	O
of	O
NCOA3	O
to	O
the	O
GREB1	O
promoter	O
did	O
not	O
correlate	O
with	O
any	O
of	O
the	O
other	O
WAY	O
‐	O
C	O
activity	O
profiles	O
(	O
Fig	O
4D	O
),	O
although	O
the	O
positive	O
correlation	O
between	O
ChIP	O
assays	O
and	O
NCOA3	O
recruitment	O
via	O
M2H	O
assay	O
showed	O
a	O
trend	O
toward	O
significance	O
with	O
r	O
2	O
=	O
0	O
.	O
36	O
and	O
P	O
=	O
0	O
.	O
09	O
(	O
F	O
‐	O
test	O
for	O
nonzero	O
slope	O
).	O
	O
ERβ	O
activity	O
is	O
not	O
an	O
independent	O
predictor	O
of	O
E	O
‐	O
Luc	O
activity	O
	O
To	O
further	O
validate	O
this	O
approach	O
,	O
we	O
solved	O
the	O
structure	O
of	O
the	O
ERα	B-mutant
‐	I-mutant
Y537S	I-mutant
LBD	O
in	O
complex	O
with	O
diethylstilbestrol	O
(	O
DES	O
),	O
which	O
bound	O
identically	O
in	O
the	O
wild	O
‐	O
type	O
and	O
ERα	B-mutant
‐	I-mutant
Y537S	I-mutant
LBDs	O
,	O
demonstrating	O
again	O
that	O
this	O
surface	O
mutation	O
stabilizes	O
h12	O
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
	O
Using	O
this	O
approach	O
,	O
we	O
solved	O
76	O
ERα	O
LBD	O
structures	O
in	O
the	O
active	O
conformation	O
and	O
bound	O
to	O
ligands	O
studied	O
here	O
(	O
Appendix	O
Fig	O
S1C	O
).	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	O
dynamics	O
.	O
	O
The	O
24	O
structures	O
containing	O
OBHS	O
,	O
OBHS	O
‐	O
N	O
,	O
or	O
triaryl	O
‐	O
ethylene	O
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	O
—	O
the	O
C	O
‐	O
terminal	O
end	O
of	O
h11	O
(	O
Figs	O
5B	O
and	O
C	O
,	O
and	O
EV5B	O
),	O
which	O
in	O
turn	O
nudges	O
h12	O
(	O
Fig	O
5C	O
and	O
D	O
).	O
	O
We	O
observed	O
that	O
the	O
OBHS	O
‐	O
N	O
analogs	O
displaced	O
h11	O
along	O
a	O
vector	O
away	O
from	O
Leu354	O
in	O
a	O
region	O
of	O
h3	O
that	O
is	O
unaffected	O
by	O
the	O
ligands	O
,	O
and	O
toward	O
the	O
dimer	O
interface	O
.	O
	O
Remarkably	O
,	O
these	O
individual	O
inter	O
‐	O
atomic	O
distances	O
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	O
structures	O
.	O
	O
The	O
h11	O
–	O
h12	O
interface	O
(	O
circled	O
)	O
includes	O
the	O
C	O
‐	O
terminal	O
part	O
of	O
h11	O
.	O
	O
Direct	O
modulators	O
like	O
tamoxifen	O
drive	O
AF	O
‐	O
1	O
‐	O
dependent	O
cell	O
‐	O
specific	O
activity	O
by	O
completely	O
occluding	O
AF	O
‐	O
2	O
,	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	O
activity	O
.	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	O
,	O
5	O
‐	O
DTP	O
‐	O
bound	O
structure	O
(	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
	O
The	O
2	O
,	O
5	O
‐	O
DTP	O
analogs	O
showed	O
perturbation	O
of	O
h11	O
,	O
as	O
well	O
as	O
h3	O
,	O
which	O
forms	O
part	O
of	O
the	O
AF	O
‐	O
2	O
surface	O
.	O
	O
The	O
shifts	O
in	O
h3	O
suggest	O
these	O
compounds	O
are	O
positioned	O
to	O
alter	O
coregulator	O
preferences	O
.	O
	O
Therefore	O
,	O
these	O
indirect	O
modulators	O
,	O
including	O
S	O
‐	O
OBHS	O
‐	O
2	O
,	O
S	O
‐	O
OBHS	O
‐	O
3	O
,	O
2	O
,	O
5	O
‐	O
DTP	O
,	O
and	O
3	O
,	O
4	O
‐	O
DTPD	O
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	O
and	O
h12	O
that	O
were	O
transmitted	O
to	O
the	O
coactivator	O
peptide	O
via	O
an	O
altered	O
AF	O
‐	O
2	O
surface	O
.	O
	O
In	O
contrast	O
,	O
an	O
extended	O
side	O
chain	O
designed	O
to	O
directly	O
reposition	O
h12	O
and	O
completely	O
disrupt	O
the	O
AF	O
‐	O
2	O
surface	O
results	O
in	O
cell	O
‐	O
specific	O
signaling	O
.	O
	O
If	O
we	O
calculated	O
inter	O
‐	O
atomic	O
distance	O
matrices	O
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
	O
We	O
have	O
found	O
that	O
the	O
TOCA1	O
HR1	O
,	O
like	O
the	O
closely	O
related	O
CIP4	O
HR1	O
,	O
has	O
interesting	O
structural	O
features	O
that	O
are	O
not	O
observed	O
in	O
other	O
HR1	O
domains	O
.	O
	O
NMR	O
experiments	O
show	O
that	O
the	O
Cdc42	O
-	O
binding	O
domain	O
from	O
N	O
-	O
WASP	O
is	O
able	O
to	O
displace	O
TOCA1	O
HR1	O
from	O
Cdc42	O
,	O
whereas	O
the	O
N	O
-	O
WASP	O
domain	O
but	O
not	O
the	O
TOCA1	O
HR1	O
domain	O
inhibits	O
actin	O
polymerization	O
.	O
	O
This	O
suggests	O
that	O
TOCA1	O
binding	O
to	O
Cdc42	O
is	O
an	O
early	O
step	O
in	O
the	O
Cdc42	O
-	O
dependent	O
pathways	O
that	O
govern	O
actin	O
dynamics	O
,	O
and	O
the	O
differential	O
binding	O
affinities	O
of	O
the	O
effectors	O
facilitate	O
a	O
handover	O
from	O
TOCA1	O
to	O
N	O
-	O
WASP	O
,	O
which	O
can	O
then	O
drive	O
recruitment	O
of	O
the	O
actin	O
-	O
modifying	O
machinery	O
.	O
	O
These	O
molecular	O
switches	O
cycle	O
between	O
active	O
,	O
GTP	O
-	O
bound	O
,	O
and	O
inactive	O
,	O
GDP	O
-	O
bound	O
,	O
states	O
with	O
the	O
help	O
of	O
auxiliary	O
proteins	O
.	O
	O
In	O
the	O
active	O
state	O
,	O
G	O
proteins	O
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
	O
RhoA	O
acts	O
to	O
rearrange	O
existing	O
actin	O
structures	O
to	O
form	O
stress	O
fibers	O
,	O
whereas	O
Rac1	O
and	O
Cdc42	O
promote	O
de	O
novo	O
actin	O
polymerization	O
to	O
form	O
lamellipodia	O
and	O
filopodia	O
,	O
respectively	O
.	O
	O
Following	O
their	O
release	O
,	O
the	O
C	O
-	O
terminal	O
regions	O
of	O
N	O
-	O
WASP	O
are	O
free	O
to	O
interact	O
with	O
G	O
-	O
actin	O
and	O
a	O
known	O
nucleator	O
of	O
actin	O
assembly	O
,	O
the	O
Arp2	O
/	O
3	O
complex	O
.	O
	O
The	O
TOCA1	O
SH3	O
domain	O
has	O
many	O
known	O
binding	O
partners	O
,	O
including	O
N	O
-	O
WASP	O
and	O
dynamin	O
.	O
	O
The	O
HR1	O
domain	O
has	O
been	O
directly	O
implicated	O
in	O
the	O
interaction	O
between	O
TOCA1	O
and	O
Cdc42	O
,	O
representing	O
the	O
first	O
Cdc42	O
-	O
HR1	O
domain	O
interaction	O
to	O
be	O
identified	O
.	O
	O
Both	O
of	O
the	O
G	O
protein	O
switch	O
regions	O
are	O
involved	O
in	O
the	O
interaction	O
.	O
	O
The	O
interactions	O
of	O
TOCA1	O
and	O
N	O
-	O
WASP	O
with	O
Cdc42	O
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	O
effectors	O
can	O
interact	O
with	O
a	O
single	O
molecule	O
of	O
Cdc42	O
simultaneously	O
.	O
	O
Cdc42	O
-	O
TOCA1	O
Binding	O
	O
A	O
,	O
curves	O
derived	O
from	O
direct	O
binding	O
assays	O
in	O
which	O
the	O
indicated	O
concentrations	O
of	O
Cdc42Δ7Q61L	O
·[	O
3H	O
]	O
GTP	O
were	O
incubated	O
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	O
.	O
	O
The	O
Kd	O
values	O
derived	O
for	O
the	O
ACK	O
GBD	O
with	O
Cdc42Δ7	B-mutant
and	O
full	O
-	O
length	O
Cdc42	O
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
	O
Other	O
G	O
protein	O
-	O
HR1	O
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	O
interferometry	O
with	O
immobilized	O
Cdc42	O
was	O
also	O
attempted	O
but	O
was	O
unsuccessful	O
for	O
both	O
TOCA1	O
HR1	O
and	O
for	O
the	O
positive	O
control	O
,	O
ACK	O
.	O
	O
The	O
affinity	O
was	O
therefore	O
determined	O
using	O
competition	O
SPAs	O
.	O
	O
Free	O
ACK	O
competed	O
with	O
itself	O
with	O
an	O
affinity	O
of	O
32	O
nm	O
,	O
similar	O
to	O
the	O
value	O
obtained	O
by	O
direct	O
binding	O
of	O
23	O
nm	O
.	O
	O
The	O
TOCA1	O
HR1	O
domain	O
also	O
fully	O
competed	O
with	O
the	O
GST	B-mutant
-	I-mutant
ACK	I-mutant
but	O
bound	O
with	O
an	O
affinity	O
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	O
observed	O
in	O
the	O
direct	O
binding	O
experiments	O
.	O
	O
These	O
residues	O
are	O
not	O
generally	O
required	O
for	O
G	O
protein	O
-	O
effector	O
interactions	O
,	O
including	O
the	O
interaction	O
between	O
RhoA	O
and	O
the	O
PRK1	O
HR1a	O
domain	O
.	O
	O
In	O
contrast	O
,	O
the	O
C	O
terminus	O
of	O
Rac1	O
contains	O
a	O
polybasic	O
sequence	O
,	O
which	O
is	O
crucial	O
for	O
Rac1	O
binding	O
to	O
the	O
HR1b	O
domain	O
from	O
PRK1	O
.	O
	O
This	O
construct	O
competed	O
with	O
GST	B-mutant
-	I-mutant
ACK	I-mutant
GBD	O
to	O
give	O
a	O
similar	O
affinity	O
to	O
the	O
HR1	O
domain	O
alone	O
(	O
Kd	O
=	O
4	O
.	O
6	O
±	O
4	O
μm	O
;	O
Fig	O
.	O
2C	O
).	O
	O
Domain	O
boundaries	O
are	O
derived	O
from	O
secondary	O
structure	O
predictions	O
;	O
B	O
,	O
binding	O
curves	O
derived	O
from	O
direct	O
binding	O
assays	O
,	O
in	O
which	O
the	O
indicated	O
concentrations	O
of	O
Cdc42Δ7Q61L	O
·[	O
3H	O
]	O
GTP	O
were	O
incubated	O
with	O
30	O
nm	O
GST	B-mutant
-	I-mutant
ACK	I-mutant
or	O
His	O
-	O
tagged	O
TOCA1	O
constructs	O
,	O
as	O
indicated	O
,	O
in	O
SPAs	O
.	O
	O
The	O
data	O
were	O
fitted	O
to	O
a	O
binding	O
isotherm	O
to	O
give	O
an	O
apparent	O
Kd	O
and	O
are	O
expressed	O
as	O
a	O
percentage	O
of	O
the	O
maximum	O
signal	O
.	O
	O
The	O
structure	O
of	O
the	O
TOCA1	O
HR1	O
domain	O
.	O
	O
B	O
,	O
a	O
sequence	O
alignment	O
of	O
the	O
HR1	O
domains	O
from	O
TOCA1	O
,	O
CIP4	O
,	O
and	O
PRK1	O
.	O
	O
Residues	O
with	O
significantly	O
affected	O
backbone	O
or	O
side	O
chain	O
chemical	O
shifts	O
when	O
Cdc42	O
bound	O
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	O
-	O
accessible	O
are	O
colored	O
yellow	O
.	O
	O
Side	O
chains	O
whose	O
CH	O
groups	O
disappeared	O
in	O
the	O
presence	O
of	O
Cdc42	O
are	O
marked	O
on	O
the	O
graph	O
in	O
Fig	O
.	O
4B	O
with	O
green	O
asterisks	O
.	O
	O
The	O
overall	O
CSP	O
was	O
calculated	O
for	O
each	O
residue	O
.	O
	O
The	O
red	O
line	O
indicates	O
the	O
mean	O
CSP	O
,	O
plus	O
one	O
S	O
.	O
D	O
.	O
Residues	O
that	O
disappeared	O
in	O
the	O
presence	O
of	O
Cdc42	O
were	O
assigned	O
a	O
CSP	O
of	O
0	O
.	O
1	O
and	O
are	O
indicated	O
with	O
open	O
bars	O
.	O
	O
This	O
suggests	O
that	O
the	O
switch	O
regions	O
are	O
not	O
rigidified	O
in	O
the	O
HR1	O
complex	O
and	O
are	O
still	O
in	O
conformational	O
exchange	O
.	O
	O
Modeling	O
the	O
Cdc42	O
·	O
TOCA1	O
HR1	O
Complex	O
	O
Cdc42	O
is	O
shown	O
in	O
cyan	O
,	O
and	O
TOCA1	O
is	O
shown	O
in	O
purple	O
.	O
	O
Some	O
of	O
these	O
can	O
be	O
rationalized	O
;	O
for	O
example	O
,	O
Thr	O
-	O
24Cdc42	O
,	O
Leu	O
-	O
160Cdc42	O
,	O
and	O
Lys	O
-	O
163Cdc42	O
all	O
pack	O
behind	O
switch	O
I	O
and	O
are	O
likely	O
to	O
be	O
affected	O
by	O
conformational	O
changes	O
within	O
the	O
switch	O
,	O
while	O
Glu	O
-	O
95Cdc42	O
and	O
Lys	O
-	O
96Cdc42	O
are	O
in	O
the	O
helix	O
behind	O
switch	O
II	O
.	O
	O
Competition	O
between	O
N	O
-	O
WASP	O
and	O
TOCA1	O
	O
A	O
,	O
the	O
model	O
of	O
the	O
Cdc42	O
·	O
TOCA1	O
HR1	O
domain	O
complex	O
overlaid	O
with	O
the	O
Cdc42	O
-	O
WASP	O
structure	O
.	O
	O
B	O
,	O
competition	O
SPA	O
experiments	O
carried	O
out	O
with	O
indicated	O
concentrations	O
of	O
the	O
N	O
-	O
WASP	O
GBD	O
construct	O
titrated	O
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	O
and	O
30	O
nm	O
Cdc42Δ7Q61L	O
·[	O
3H	O
]	O
GTP	O
.	O
	O
Unlabeled	O
N	O
-	O
WASP	O
GBD	O
was	O
titrated	O
into	O
15N	O
-	O
Cdc42Δ7Q61L	O
·	O
GMPPNP	O
,	O
and	O
the	O
backbone	O
NH	O
groups	O
were	O
monitored	O
using	O
HSQCs	O
(	O
Fig	O
.	O
7C	O
).	O
	O
Actin	O
polymerization	O
in	O
all	O
cases	O
was	O
initiated	O
by	O
the	O
addition	O
of	O
PI	O
(	O
4	O
,	O
5	O
)	O
P2	O
-	O
containing	O
liposomes	O
.	O
	O
Endogenous	O
N	O
-	O
WASP	O
is	O
present	O
at	O
∼	O
100	O
nm	O
in	O
Xenopus	O
extracts	O
,	O
whereas	O
TOCA1	O
is	O
present	O
at	O
a	O
10	O
-	O
fold	O
lower	O
concentration	O
than	O
N	O
-	O
WASP	O
.	O
	O
This	O
is	O
consistent	O
with	O
endogenous	O
N	O
-	O
WASP	O
,	O
activated	O
by	O
other	O
components	O
of	O
the	O
assay	O
,	O
outcompeting	O
the	O
TOCA1	O
HR1	O
domain	O
for	O
Cdc42	O
binding	O
.	O
	O
Fluorescence	O
curves	O
show	O
actin	O
polymerization	O
in	O
the	O
presence	O
of	O
increasing	O
concentrations	O
of	O
N	O
-	O
WASP	O
GBD	O
or	O
TOCA1	O
HR1	O
domain	O
as	O
indicated	O
.	O
	O
This	O
is	O
over	O
100	O
times	O
lower	O
than	O
that	O
of	O
the	O
N	O
-	O
WASP	O
GBD	O
(	O
Kd	O
=	O
37	O
nm	O
)	O
and	O
considerably	O
lower	O
than	O
other	O
known	O
G	O
protein	O
-	O
HR1	O
domain	O
interactions	O
.	O
	O
The	O
TOCA1	O
HR1	O
domain	O
is	O
a	O
left	O
-	O
handed	O
coiled	O
-	O
coil	O
comparable	O
with	O
other	O
known	O
HR1	O
domains	O
.	O
	O
The	O
interhelical	O
loops	O
of	O
TOCA1	O
and	O
CIP4	O
differ	O
from	O
the	O
same	O
region	O
in	O
the	O
HR1	O
domains	O
of	O
PRK1	O
in	O
that	O
they	O
are	O
longer	O
and	O
contain	O
two	O
short	O
stretches	O
of	O
310	O
-	O
helix	O
.	O
	O
This	O
region	O
lies	O
within	O
the	O
G	O
protein	O
-	O
binding	O
surface	O
of	O
all	O
of	O
the	O
HR1	O
domains	O
(	O
Fig	O
.	O
4D	O
).	O
	O
Many	O
of	O
these	O
residues	O
are	O
significantly	O
affected	O
in	O
the	O
presence	O
of	O
Cdc42	O
,	O
so	O
it	O
is	O
likely	O
that	O
the	O
conformation	O
of	O
this	O
loop	O
is	O
altered	O
in	O
the	O
Cdc42	O
complex	O
.	O
	O
These	O
observations	O
therefore	O
provide	O
a	O
molecular	O
mechanism	O
whereby	O
mutation	O
of	O
Met383	O
-	O
Gly384	O
-	O
Asp385	O
to	O
Ile383	O
-	O
Ser384	O
-	O
Thr385	O
abolishes	O
TOCA1	O
binding	O
to	O
Cdc42	O
.	O
	O
For	O
example	O
,	O
Phe	O
-	O
56Cdc42	O
,	O
which	O
is	O
not	O
visible	O
in	O
free	O
Cdc42	O
or	O
Cdc42	O
·	O
HR1TOCA1	O
,	O
is	O
close	O
to	O
the	O
TOCA1	O
HR1	O
(	O
Fig	O
.	O
6A	O
).	O
	O
Some	O
residues	O
that	O
are	O
affected	O
in	O
the	O
Cdc42	O
·	O
HR1TOCA1	O
complex	O
but	O
do	O
not	O
correspond	O
to	O
contact	O
residues	O
of	O
RhoA	O
or	O
Rac1	O
(	O
Fig	O
.	O
6C	O
)	O
may	O
contact	O
HR1TOCA1	O
directly	O
(	O
Fig	O
.	O
6D	O
).	O
	O
The	O
weak	O
binding	O
prevented	O
detailed	O
structural	O
and	O
thermodynamic	O
studies	O
of	O
the	O
complex	O
.	O
	O
Nonetheless	O
,	O
structural	O
studies	O
of	O
the	O
TOCA1	O
HR1	O
domain	O
,	O
combined	O
with	O
chemical	O
shift	O
mapping	O
,	O
have	O
highlighted	O
some	O
potentially	O
interesting	O
differences	O
between	O
Cdc42	O
-	O
HR1TOCA1	O
and	O
RhoA	O
/	O
Rac1	O
-	O
HR1PRK1	O
binding	O
.	O
	O
As	O
such	O
,	O
the	O
ability	O
of	O
the	O
TOCA1	O
HR1	O
domain	O
to	O
bind	O
to	O
Cdc42	O
(	O
a	O
close	O
relative	O
of	O
Rac1	O
rather	O
than	O
RhoA	O
)	O
fits	O
this	O
trend	O
.	O
	O
The	O
low	O
affinity	O
of	O
the	O
HR1TOCA1	O
-	O
Cdc42	O
interaction	O
in	O
the	O
context	O
of	O
the	O
physiological	O
concentration	O
of	O
TOCA1	O
in	O
Xenopus	O
extracts	O
(∼	O
10	O
nm	O
)	O
suggests	O
that	O
binding	O
between	O
TOCA1	O
and	O
Cdc42	O
is	O
likely	O
to	O
occur	O
in	O
vivo	O
only	O
when	O
TOCA1	O
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	O
Cdc42	O
.	O
	O
WIP	O
inhibits	O
the	O
activation	O
of	O
N	O
-	O
WASP	O
by	O
Cdc42	O
,	O
an	O
effect	O
that	O
is	O
reversed	O
by	O
TOCA1	O
.	O
	O
A	O
combination	O
of	O
allosteric	O
activation	O
by	O
PI	O
(	O
4	O
,	O
5	O
)	O
P2	O
,	O
activated	O
Cdc42	O
and	O
TOCA1	O
,	O
and	O
oligomeric	O
activation	O
implemented	O
by	O
TOCA1	O
would	O
lead	O
to	O
full	O
activation	O
of	O
N	O
-	O
WASP	O
and	O
downstream	O
actin	O
polymerization	O
.	O
	O
The	O
commonly	O
used	O
MGD	B-mutant
→	I-mutant
IST	I-mutant
(	O
Cdc42	O
-	O
binding	O
deficient	O
)	O
mutant	O
of	O
TOCA1	O
has	O
a	O
reduced	O
ability	O
to	O
activate	O
the	O
N	O
-	O
WASP	O
·	O
WIP	O
complex	O
,	O
further	O
indicating	O
the	O
importance	O
of	O
the	O
Cdc42	O
-	O
HR1TOCA1	O
interaction	O
prior	O
to	O
downstream	O
activation	O
of	O
N	O
-	O
WASP	O
.	O
	O
Step	O
1	O
,	O
TOCA1	O
is	O
recruited	O
to	O
the	O
membrane	O
via	O
its	O
F	O
-	O
BAR	O
domain	O
and	O
/	O
or	O
Cdc42	O
interactions	O
.	O
	O
It	O
is	O
clear	O
from	O
the	O
data	O
presented	O
here	O
that	O
TOCA1	O
and	O
N	O
-	O
WASP	O
do	O
not	O
bind	O
Cdc42	O
simultaneously	O
and	O
that	O
N	O
-	O
WASP	O
is	O
likely	O
to	O
outcompete	O
TOCA1	O
for	O
Cdc42	O
binding	O
.	O
	O
In	O
contrast	O
to	O
related	O
carboxylases	O
,	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	O
under	O
phosphorylation	O
control	O
.	O
	O
BRCA1	O
binds	O
only	O
to	O
the	O
phosphorylated	O
form	O
of	O
ACC1	O
and	O
prevents	O
ACC	O
activation	O
by	O
phosphatase	O
-	O
mediated	O
dephosphorylation	O
.	O
	O
Its	O
phosphorylation	O
by	O
the	O
AMPK	O
homologue	O
SNF1	O
results	O
in	O
strongly	O
reduced	O
ACC	O
activity	O
.	O
	O
The	O
organization	O
of	O
the	O
yeast	O
ACC	O
CD	O
	O
The	O
crystal	O
structure	O
of	O
the	O
CD	O
of	O
SceACC	O
(	O
SceCD	O
)	O
was	O
determined	O
at	O
3	O
.	O
0	O
Å	O
resolution	O
by	O
experimental	O
phasing	O
and	O
refined	O
to	O
Rwork	O
/	O
Rfree	O
=	O
0	O
.	O
20	O
/	O
0	O
.	O
24	O
(	O
Table	O
1	O
).	O
	O
SceCD	O
comprises	O
four	O
distinct	O
domains	O
,	O
an	O
N	O
-	O
terminal	O
α	O
-	O
helical	O
domain	O
(	O
CDN	O
),	O
and	O
a	O
central	O
four	O
-	O
helix	O
bundle	O
linker	O
domain	O
(	O
CDL	O
),	O
followed	O
by	O
two	O
α	O
–	O
β	O
-	O
fold	O
C	O
-	O
terminal	O
domains	O
(	O
CDC1	O
/	O
CDC2	O
).	O
	O
CDL	O
does	O
not	O
interact	O
with	O
CDN	O
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	O
via	O
a	O
loop	O
between	O
Lα2	O
/	O
α3	O
and	O
the	O
N	O
-	O
terminal	O
end	O
of	O
Lα1	O
,	O
with	O
an	O
interface	O
area	O
of	O
400	O
Å2	O
.	O
	O
CDC1	O
/	O
CDC2	O
share	O
a	O
common	O
fold	O
;	O
they	O
are	O
composed	O
of	O
six	O
-	O
stranded	O
β	O
-	O
sheets	O
flanked	O
on	O
one	O
side	O
by	O
two	O
long	O
,	O
bent	O
helices	O
inserted	O
between	O
strands	O
β3	O
/	O
β4	O
and	O
β4	O
/	O
β5	O
.	O
	O
On	O
the	O
basis	O
of	O
a	O
root	O
mean	O
square	O
deviation	O
of	O
main	O
chain	O
atom	O
positions	O
of	O
2	O
.	O
2	O
Å	O
,	O
CDC1	O
/	O
CDC2	O
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
	O
Phosphorylated	O
SceACC	O
shows	O
only	O
residual	O
activity	O
(	O
kcat	O
=	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	O
=	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	O
protein	O
phosphatase	O
.	O
	O
As	O
a	O
result	O
,	O
the	O
N	O
terminus	O
of	O
CDL	O
at	O
helix	O
Lα1	O
,	O
which	O
connects	O
to	O
CDN	O
,	O
is	O
shifted	O
by	O
12	O
Å	O
.	O
Remarkably	O
,	O
CDN	O
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	O
.	O
	O
To	O
improve	O
crystallizability	O
,	O
we	O
generated	O
ΔBCCP	B-mutant
variants	I-mutant
of	O
full	O
-	O
length	O
ACC	O
,	O
which	O
,	O
based	O
on	O
SAXS	O
analysis	O
,	O
preserve	O
properties	O
of	O
intact	O
ACC	O
(	O
Supplementary	O
Table	O
1	O
and	O
Supplementary	O
Fig	O
.	O
2a	O
–	O
c	O
).	O
	O
The	O
connecting	O
region	O
is	O
remarkably	O
similar	O
in	O
isolated	O
CD	O
and	O
CthCD	B-mutant
-	I-mutant
CTCter	I-mutant
structures	O
,	O
indicating	O
inherent	O
conformational	O
stability	O
.	O
	O
Surprisingly	O
,	O
in	O
both	O
the	O
linear	O
and	O
U	O
-	O
shaped	O
conformations	O
,	O
the	O
approximate	O
distances	O
between	O
the	O
BC	O
and	O
CT	O
active	O
sites	O
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	O
structures	O
of	O
any	O
other	O
related	O
biotin	O
-	O
dependent	O
carboxylase	O
.	O
	O
The	O
CD	O
consists	O
of	O
four	O
distinct	O
subdomains	O
and	O
acts	O
as	O
a	O
tether	O
from	O
the	O
CT	O
to	O
the	O
mobile	O
BCCP	O
and	O
an	O
oriented	O
BC	O
domain	O
.	O
	O
A	O
second	O
hinge	O
can	O
be	O
identified	O
between	O
CDC1	O
/	O
CDC2	O
.	O
	O
The	O
only	O
bona	O
fide	O
regulatory	O
phophorylation	O
site	O
of	O
fungal	O
ACC	O
in	O
the	O
regulatory	O
loop	O
is	O
directly	O
participating	O
in	O
CDC1	O
/	O
CDC2	O
domain	O
interactions	O
and	O
thus	O
stabilizes	O
the	O
hinge	O
conformation	O
.	O
	O
In	O
flACC	O
,	O
the	O
regulatory	O
loop	O
is	O
mostly	O
disordered	O
,	O
illustrating	O
the	O
increased	O
flexibility	O
due	O
to	O
the	O
absence	O
of	O
the	O
phosphoryl	O
group	O
.	O
	O
Thus	O
,	O
in	O
accordance	O
with	O
the	O
results	O
presented	O
here	O
,	O
phosphorylation	O
of	O
Ser1157	O
in	O
SceACC	O
most	O
likely	O
limits	O
flexibility	O
in	O
the	O
CDC1	O
/	O
CDC2	O
hinge	O
such	O
that	O
activation	O
through	O
BC	O
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
	O
Cartoon	O
representation	O
of	O
crystal	O
structures	O
of	O
multidomain	B-mutant
constructs	I-mutant
of	O
CthACC	O
.	O
	O
(	O
b	O
)	O
The	O
interdomain	O
interface	O
of	O
CDC1	O
and	O
CDC2	O
exhibits	O
only	O
limited	O
plasticity	O
.	O
	O
The	O
conformational	O
dynamics	O
of	O
fungal	O
ACC	O
.	O
	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	O
structures	O
(	O
as	O
indicated	O
,	O
numbers	O
after	O
construct	O
name	O
differentiate	O
between	O
individual	O
protomers	O
)	O
are	O
shown	O
in	O
grey	O
.	O
	O
Crystal	O
Structures	O
of	O
Putative	O
Sugar	O
Kinases	O
from	O
Synechococcus	O
Elongatus	O
PCC	O
7942	O
and	O
Arabidopsis	O
Thaliana	O
	O
Here	O
we	O
solved	O
the	O
structures	O
of	O
SePSK	O
and	O
AtXK	O
-	O
1	O
in	O
both	O
their	O
apo	O
forms	O
and	O
in	O
complex	O
with	O
nucleotide	O
substrates	O
.	O
	O
Phosphorylation	O
is	O
one	O
of	O
the	O
various	O
pivotal	O
modifications	O
of	O
carbohydrates	O
,	O
and	O
is	O
catalyzed	O
by	O
specific	O
sugar	O
kinases	O
.	O
	O
These	O
kinases	O
exhibit	O
considerable	O
differences	O
in	O
their	O
folding	O
pattern	O
and	O
substrate	O
specificity	O
.	O
	O
Based	O
on	O
sequence	O
analysis	O
,	O
they	O
can	O
be	O
divided	O
into	O
four	O
families	O
,	O
namely	O
HSP	O
70_NBD	O
family	O
,	O
FGGY	O
family	O
,	O
Mer_B	O
like	O
family	O
and	O
Parm_like	O
family	O
.	O
	O
Our	O
findings	O
provide	O
new	O
details	O
of	O
the	O
catalytic	O
mechanism	O
of	O
SePSK	O
and	O
lay	O
the	O
foundation	O
for	O
future	O
studies	O
into	O
its	O
homologs	O
in	O
eukaryotes	O
.	O
	O
Apo	O
-	O
SePSK	O
contains	O
two	O
domains	O
referred	O
to	O
further	O
on	O
as	O
domain	O
I	O
and	O
domain	O
II	O
(	O
Fig	O
1A	O
).	O
	O
2	O
–	O
228	O
and	O
aa	O
.	O
	O
The	O
secondary	O
structural	O
elements	O
are	O
indicated	O
(	O
α	O
-	O
helix	O
:	O
green	O
,	O
β	O
-	O
sheet	O
:	O
wheat	O
).	O
	O
As	O
shown	O
in	O
Fig	O
2A	O
,	O
both	O
SePSK	O
and	O
AtXK	O
-	O
1	O
exhibited	O
ATP	O
hydrolysis	O
activity	O
.	O
	O
(	O
A	O
)	O
The	O
ATP	O
hydrolysis	O
activity	O
of	O
SePSK	O
and	O
AtXK	O
-	O
1	O
.	O
	O
Both	O
SePSK	O
and	O
AtXK	O
-	O
1	O
showed	O
ATP	O
hydrolysis	O
activity	O
in	O
the	O
absence	O
of	O
substrate	O
.	O
	O
SePSK	O
and	O
AtXK	O
-	O
1	O
possess	O
a	O
similar	O
ATP	O
binding	O
site	O
	O
This	O
result	O
was	O
consistent	O
with	O
our	O
enzymatic	O
activity	O
assays	O
where	O
SePSK	O
and	O
AtXK	O
-	O
1	O
showed	O
ATP	O
hydrolysis	O
activity	O
without	O
adding	O
any	O
substrates	O
(	O
Fig	O
2A	O
and	O
2C	O
).	O
	O
The	O
tail	O
of	O
AMP	O
-	O
PNP	O
points	O
to	O
the	O
hinge	O
region	O
of	O
SePSK	O
,	O
and	O
its	O
α	O
-	O
phosphate	O
and	O
β	O
-	O
phosphate	O
groups	O
are	O
stabilized	O
by	O
Gly376	O
and	O
Ser243	O
,	O
respectively	O
.	O
	O
The	O
four	O
α	O
-	O
helices	O
(	O
α26	O
,	O
α28	O
,	O
α27	O
and	O
α30	O
)	O
are	O
labeled	O
in	O
red	O
.	O
	O
To	O
better	O
understand	O
the	O
interaction	O
pattern	O
between	O
SePSK	O
and	O
D	O
-	O
ribulose	O
,	O
the	O
apo	O
-	O
SePSK	O
crystals	O
were	O
soaked	O
into	O
the	O
reservoir	O
with	O
10	O
mM	O
D	O
-	O
ribulose	O
(	O
RBL	O
)	O
and	O
the	O
RBL	O
-	O
SePSK	O
structure	O
was	O
solved	O
.	O
	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	O
-	O
ribulose	O
molecules	O
are	O
approx	O
.	O
	O
The	O
hydrogen	O
bonds	O
are	O
indicated	O
by	O
the	O
black	O
dashed	O
lines	O
and	O
the	O
numbers	O
near	O
the	O
dashed	O
lines	O
are	O
the	O
distances	O
(	O
Å	O
).	O
(	O
C	O
)	O
The	O
binding	O
affinity	O
assays	O
of	O
SePSK	O
with	O
D	O
-	O
ribulose	O
.	O
	O
A	O
unique	O
macromolecular	O
cage	O
formed	O
by	O
two	O
decamers	O
of	O
the	O
Escherichia	O
coli	O
LdcI	O
and	O
five	O
hexamers	O
of	O
the	O
AAA	O
+	O
ATPase	O
RavA	O
was	O
shown	O
to	O
counteract	O
acid	O
stress	O
under	O
starvation	O
.	O
	O
Multiple	O
sequence	O
alignment	O
coupled	O
to	O
a	O
phylogenetic	O
analysis	O
reveals	O
that	O
certain	O
enterobacteria	O
exert	O
evolutionary	O
pressure	O
on	O
the	O
lysine	O
decarboxylase	O
towards	O
the	O
cage	O
-	O
like	O
assembly	O
with	O
RavA	O
,	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
	O
These	O
amino	O
acid	O
decarboxylases	O
are	O
therefore	O
called	O
acid	O
stress	O
inducible	O
or	O
biodegradative	O
to	O
distinguish	O
them	O
from	O
their	O
biosynthetic	O
lysine	O
and	O
ornithine	O
decarboxylase	O
paralogs	O
catalysing	O
the	O
same	O
reaction	O
but	O
responsible	O
for	O
the	O
polyamine	O
production	O
at	O
neutral	O
pH	O
.	O
	O
In	O
particular	O
,	O
the	O
inducible	O
lysine	O
decarboxylase	O
LdcI	O
(	O
or	O
CadA	O
)	O
attracts	O
attention	O
due	O
to	O
its	O
broad	O
pH	O
range	O
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	O
such	O
as	O
Salmonella	O
enterica	O
serovar	O
Typhimurium	O
,	O
Vibrio	O
cholerae	O
and	O
Vibrio	O
vulnificus	O
under	O
acidic	O
conditions	O
.	O
	O
The	O
crystal	O
structure	O
of	O
the	O
E	O
.	O
coli	O
LdcI	O
as	O
well	O
as	O
its	O
low	O
resolution	O
characterisation	O
by	O
electron	O
microscopy	O
(	O
EM	O
)	O
showed	O
that	O
it	O
is	O
a	O
decamer	O
made	O
of	O
two	O
pentameric	O
rings	O
.	O
	O
This	O
comparison	O
pinpointed	O
differences	O
between	O
the	O
biodegradative	O
and	O
the	O
biosynthetic	O
lysine	O
decarboxylases	O
and	O
brought	O
to	O
light	O
interdomain	O
movements	O
associated	O
to	O
pH	O
-	O
dependent	O
enzyme	O
activation	O
and	O
RavA	O
binding	O
,	O
notably	O
at	O
the	O
predicted	O
RavA	O
binding	O
site	O
at	O
the	O
level	O
of	O
the	O
C	O
-	O
terminal	O
β	O
-	O
sheet	O
of	O
LdcI	O
.	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	O
the	O
C	O
-	O
terminal	O
β	O
-	O
sheets	O
in	O
question	O
.	O
	O
In	O
addition	O
,	O
we	O
improved	O
our	O
earlier	O
cryoEM	O
map	O
of	O
the	O
LdcI	O
-	O
LARA	O
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
	O
Based	O
on	O
these	O
reconstructions	O
,	O
reliable	O
pseudoatomic	O
models	O
of	O
the	O
three	O
assemblies	O
were	O
obtained	O
by	O
flexible	O
fitting	O
of	O
either	O
the	O
crystal	O
structure	O
of	O
LdcIi	O
or	O
a	O
derived	O
structural	O
homology	O
model	O
of	O
LdcC	O
(	O
Table	O
S1	O
).	O
	O
The	O
resolution	O
of	O
the	O
cryoEM	O
maps	O
does	O
not	O
allow	O
modeling	O
the	O
position	O
of	O
the	O
PLP	O
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	O
acids	O
.	O
	O
While	O
differences	O
in	O
the	O
ppGpp	O
binding	O
site	O
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
	O
Swinging	O
and	O
stretching	O
of	O
the	O
CTDs	O
upon	O
pH	O
-	O
dependent	O
LdcI	O
activation	O
and	O
LARA	O
binding	O
	O
Inspection	O
of	O
the	O
superimposed	O
decameric	O
structures	O
(	O
Figs	O
2	O
and	O
S6	O
)	O
suggests	O
a	O
depiction	O
of	O
the	O
wing	O
domains	O
as	O
an	O
anchor	O
around	O
which	O
the	O
peripheral	O
CTDs	O
swing	O
.	O
	O
This	O
swinging	O
movement	O
seems	O
to	O
be	O
mediated	O
by	O
the	O
core	O
domains	O
and	O
is	O
accompanied	O
by	O
a	O
stretching	O
of	O
the	O
whole	O
LdcI	O
subunits	O
attracted	O
by	O
the	O
RavA	O
magnets	O
.	O
	O
Our	O
structures	O
show	O
that	O
this	O
motif	O
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
	O
One	O
of	O
the	O
elucidated	O
roles	O
of	O
the	O
LdcI	O
-	O
RavA	O
cage	O
is	O
to	O
maintain	O
LdcI	O
activity	O
under	O
conditions	O
of	O
enterobacterial	O
starvation	O
by	O
preventing	O
LdcI	O
inhibition	O
by	O
the	O
stringent	O
response	O
alarmone	O
ppGpp	O
.	O
	O
Furthermore	O
,	O
the	O
recently	O
documented	O
interaction	O
of	O
both	O
LdcI	O
and	O
RavA	O
with	O
specific	O
subunits	O
of	O
the	O
respiratory	O
complex	O
I	O
,	O
together	O
with	O
the	O
unanticipated	O
link	O
between	O
RavA	O
and	O
maturation	O
of	O
numerous	O
iron	O
-	O
sulfur	O
proteins	O
,	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
	O
Besides	O
,	O
the	O
structures	O
and	O
the	O
pseudoatomic	O
models	O
of	O
the	O
active	O
ppGpp	O
-	O
free	O
states	O
of	O
both	O
the	O
biodegradative	O
and	O
the	O
biosynthetic	O
E	O
.	O
coli	O
lysine	O
decarboxylases	O
offer	O
an	O
additional	O
tool	O
for	O
analysis	O
of	O
their	O
role	O
in	O
UPEC	O
infectivity	O
.	O
	O
The	O
active	O
site	O
is	O
boxed	O
.	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	O
type	O
LdcI	O
-	O
RavA	O
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
	O
Numbering	O
as	O
in	O
E	O
.	O
coli	O
.	O
	O
Relative	O
to	O
the	O
open	O
bacterial	O
ammonium	O
transporters	O
,	O
non	O
-	O
phosphorylated	O
Mep2	O
exhibits	O
shifts	O
in	O
cytoplasmic	O
loops	O
and	O
the	O
C	O
-	O
terminal	O
region	O
(	O
CTR	O
)	O
to	O
occlude	O
the	O
cytoplasmic	O
exit	O
of	O
the	O
channel	O
and	O
to	O
interact	O
with	O
His2	O
of	O
the	O
twin	O
-	O
His	O
motif	O
.	O
	O
Here	O
,	O
the	O
authors	O
report	O
the	O
crystal	O
structures	O
of	O
closed	O
states	O
of	O
Mep2	O
proteins	O
and	O
propose	O
a	O
model	O
for	O
their	O
regulation	O
by	O
comparing	O
them	O
with	O
the	O
open	O
ammonium	O
transporters	O
of	O
bacteria	O
.	O
	O
A	O
common	O
feature	O
of	O
transceptors	O
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
	O
With	O
the	O
exception	O
of	O
the	O
human	O
RhCG	O
structure	O
,	O
no	O
structural	O
information	O
is	O
available	O
for	O
eukaryotic	O
ammonium	O
transporters	O
.	O
	O
To	O
elucidate	O
the	O
mechanism	O
of	O
Mep2	O
transport	O
regulation	O
,	O
we	O
present	O
here	O
X	O
-	O
ray	O
crystal	O
structures	O
of	O
the	O
Mep2	O
transceptors	O
from	O
S	O
.	O
cerevisiae	O
and	O
C	O
.	O
albicans	O
.	O
	O
Despite	O
different	O
crystal	O
packing	O
(	O
Supplementary	O
Table	O
1	O
),	O
the	O
two	O
CaMep2	O
structures	O
are	O
identical	O
to	O
each	O
other	O
and	O
very	O
similar	O
to	O
ScMep2	O
(	O
Cα	O
r	O
.	O
m	O
.	O
s	O
.	O
d	O
.	O
	O
Unless	O
specifically	O
stated	O
,	O
the	O
drawn	O
conclusions	O
also	O
apply	O
to	O
ScMep2	O
.	O
	O
Together	O
with	O
additional	O
,	O
smaller	O
differences	O
in	O
other	O
extracellular	O
loops	O
,	O
these	O
changes	O
generate	O
a	O
distinct	O
vestibule	O
leading	O
to	O
the	O
ammonium	O
binding	O
site	O
that	O
is	O
much	O
more	O
pronounced	O
than	O
in	O
the	O
bacterial	O
proteins	O
.	O
	O
The	O
largest	O
differences	O
between	O
the	O
Mep2	O
structures	O
and	O
the	O
other	O
known	O
ammonium	O
transporter	O
structures	O
are	O
located	O
on	O
the	O
intracellular	O
side	O
of	O
the	O
membrane	O
.	O
	O
ICL1	O
has	O
also	O
moved	O
inwards	O
relative	O
to	O
its	O
position	O
in	O
the	O
bacterial	O
Amts	O
.	O
	O
Compared	O
with	O
ICL1	O
,	O
the	O
backbone	O
conformational	O
changes	O
observed	O
for	O
the	O
neighbouring	O
ICL2	O
are	O
smaller	O
,	O
but	O
large	O
shifts	O
are	O
nevertheless	O
observed	O
for	O
the	O
conserved	O
residues	O
Glu140	O
and	O
Arg141	O
(	O
Fig	O
.	O
4	O
).	O
	O
Phosphorylation	O
target	O
site	O
is	O
at	O
the	O
periphery	O
of	O
Mep2	O
	O
In	O
the	O
absence	O
of	O
Npr1	O
,	O
plasmid	O
-	O
encoded	O
WT	O
Mep2	O
in	O
a	O
S	O
.	O
cerevisiae	O
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	O
,	O
suggesting	O
that	O
the	O
transporter	O
is	O
inactive	O
(	O
Fig	O
.	O
3	O
and	O
Supplementary	O
Fig	O
.	O
1	O
).	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	O
(	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	O
and	O
purified	O
the	O
analogous	O
CaMep2	O
442Δ	B-mutant
truncation	O
mutant	O
and	O
determined	O
the	O
crystal	O
structure	O
using	O
data	O
to	O
3	O
.	O
4	O
Å	O
resolution	O
.	O
	O
The	O
structure	O
shows	O
that	O
removal	O
of	O
the	O
AI	O
region	O
markedly	O
increases	O
the	O
dynamics	O
of	O
the	O
cytoplasmic	O
parts	O
of	O
the	O
transporter	O
.	O
	O
The	O
first	O
one	O
is	O
that	O
the	O
open	O
state	O
is	O
disfavoured	O
by	O
crystallization	O
because	O
of	O
lower	O
stability	O
or	O
due	O
to	O
crystal	O
packing	O
constraints	O
.	O
	O
The	O
ammonium	O
uptake	O
activity	O
of	O
the	O
S	O
.	O
cerevisiae	O
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	O
Mep2	O
and	O
the	O
S453D	B-mutant
mutant	O
,	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
	O
The	O
movement	O
of	O
the	O
acidic	O
residues	O
away	O
from	O
Arg452	O
and	O
Sep453	O
is	O
more	O
pronounced	O
in	O
this	O
simulation	O
in	O
comparison	O
with	O
the	O
movement	O
away	O
from	O
Asp452	O
and	O
Asp453	O
in	O
the	O
DD	B-mutant
mutant	I-mutant
.	O
	O
The	O
reason	O
why	O
similar	O
transporters	O
such	O
as	O
A	O
.	O
thaliana	O
Amt	O
-	O
1	O
;	O
1	O
and	O
Mep2	O
are	O
regulated	O
in	O
opposite	O
ways	O
by	O
phosphorylation	O
(	O
inactivation	O
in	O
plants	O
and	O
activation	O
in	O
fungi	O
)	O
is	O
not	O
known	O
.	O
	O
By	O
determining	O
the	O
first	O
structures	O
of	O
closed	O
ammonium	O
transporters	O
and	O
comparing	O
those	O
structures	O
with	O
the	O
permanently	O
open	O
bacterial	O
proteins	O
,	O
we	O
demonstrate	O
that	O
Mep2	O
channel	O
closure	O
is	O
likely	O
due	O
to	O
movements	O
of	O
the	O
CTR	O
and	O
ICL1	O
and	O
ICL3	O
.	O
	O
Owing	O
to	O
the	O
crosstalk	O
between	O
monomers	O
,	O
a	O
single	O
phosphorylation	O
event	O
might	O
lead	O
to	O
opening	O
of	O
the	O
entire	O
trimer	O
,	O
although	O
this	O
has	O
not	O
yet	O
been	O
tested	O
(	O
Fig	O
.	O
9b	O
).	O
	O
It	O
should	O
also	O
be	O
noted	O
that	O
the	O
tyrosine	O
residue	O
interacting	O
with	O
His2	O
is	O
highly	O
conserved	O
in	O
fungal	O
Mep2	O
orthologues	O
,	O
suggesting	O
that	O
the	O
Tyr	O
–	O
His2	O
hydrogen	O
bond	O
might	O
be	O
a	O
general	O
way	O
to	O
close	O
Mep2	O
proteins	O
.	O
	O
For	O
example	O
,	O
NH3	O
uniport	O
or	O
symport	O
of	O
NH3	O
/	O
H	O
+	O
might	O
result	O
in	O
changes	O
in	O
local	O
pH	O
,	O
but	O
NH4	O
+	O
uniport	O
might	O
not	O
,	O
and	O
this	O
difference	O
might	O
determine	O
signalling	O
.	O
	O
(	O
a	O
)	O
Monomer	O
cartoon	O
models	O
viewed	O
from	O
the	O
side	O
for	O
(	O
left	O
)	O
A	O
.	O
fulgidus	O
Amt	O
-	O
1	O
(	O
PDB	O
ID	O
2B2H	O
),	O
S	O
.	O
cerevisiae	O
Mep2	O
(	O
middle	O
)	O
and	O
C	O
.	O
albicans	O
Mep2	O
(	O
right	O
).	O
	O
One	O
monomer	O
is	O
coloured	O
as	O
in	O
a	O
and	O
one	O
monomer	O
is	O
coloured	O
by	O
B	O
-	O
factor	O
(	O
blue	O
,	O
low	O
;	O
red	O
;	O
high	O
).	O
	O
The	O
secondary	O
structure	O
elements	O
observed	O
for	O
CaMep2	O
are	O
indicated	O
,	O
with	O
the	O
numbers	O
corresponding	O
to	O
the	O
centre	O
of	O
the	O
TM	O
segment	O
.	O
	O
Growth	O
of	O
ScMep2	B-mutant
variants	I-mutant
on	O
low	O
ammonium	O
medium	O
.	O
	O
(	O
b	O
)	O
Overlay	O
of	O
the	O
CTRs	O
of	O
ScMep2	O
(	O
grey	O
)	O
and	O
CaMep2	O
(	O
green	O
),	O
showing	O
the	O
similar	O
electronegative	O
environment	O
surrounding	O
the	O
phosphorylation	O
site	O
(	O
P	O
).	O
	O
The	O
AI	O
regions	O
are	O
coloured	O
magenta	O
.	O
	O
Missing	O
regions	O
are	O
labelled	O
.	O
(	O
b	O
)	O
Stereo	O
superpositions	O
of	O
WT	O
CaMep2	O
and	O
the	O
truncation	O
mutant	O
.	O
	O
Schematic	O
model	O
for	O
phosphorylation	O
-	O
based	O
regulation	O
of	O
Mep2	O
ammonium	O
transporters	O
.	O
	O
To	O
support	O
antibody	O
therapeutic	O
development	O
,	O
the	O
crystal	O
structures	O
of	O
a	O
set	O
of	O
16	O
germline	O
variants	O
composed	O
of	O
4	O
different	O
kappa	O
light	O
chains	O
paired	O
with	O
4	O
different	O
heavy	O
chains	O
have	O
been	O
determined	O
.	O
	O
CDR	O
H3	O
,	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
	O
About	O
half	O
of	O
the	O
structures	O
have	O
CDR	O
H3	O
conformations	O
similar	O
to	O
that	O
of	O
the	O
parent	O
;	O
the	O
others	O
diverge	O
significantly	O
.	O
	O
The	O
structures	O
and	O
their	O
analyses	O
provide	O
a	O
rich	O
foundation	O
for	O
future	O
antibody	O
modeling	O
and	O
engineering	O
efforts	O
.	O
	O
Isotypes	O
IgG	O
,	O
IgD	O
and	O
IgA	O
each	O
have	O
4	O
domains	O
,	O
one	O
variable	O
(	O
V	O
)	O
and	O
3	O
constant	O
(	O
C	O
)	O
domains	O
,	O
while	O
IgE	O
and	O
IgM	O
each	O
have	O
the	O
same	O
4	O
domains	O
along	O
with	O
an	O
additional	O
C	O
domain	O
.	O
	O
These	O
domains	O
have	O
a	O
common	O
folding	O
pattern	O
often	O
referred	O
to	O
as	O
the	O
“	O
immunoglobulin	O
fold	O
,”	O
formed	O
by	O
the	O
packing	O
together	O
of	O
2	O
anti	O
-	O
parallel	O
β	O
-	O
sheets	O
.	O
	O
In	O
antibodies	O
,	O
the	O
heavy	O
and	O
light	O
chain	O
V	O
domains	O
pack	O
together	O
forming	O
the	O
antigen	O
combining	O
site	O
.	O
	O
Later	O
studies	O
found	O
that	O
the	O
CDR	O
loop	O
length	O
is	O
the	O
primary	O
determining	O
factor	O
of	O
antigen	O
-	O
binding	O
site	O
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
	O
In	O
the	O
torso	O
region	O
,	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	O
region	O
.	O
	O
The	O
“	O
kinked	O
”	O
or	O
“	O
bulged	O
”	O
conformation	O
is	O
the	O
most	O
prevalent	O
,	O
but	O
an	O
“	O
extended	O
”	O
or	O
“	O
non	O
-	O
bulged	O
”	O
conformation	O
is	O
also	O
,	O
but	O
less	O
frequently	O
,	O
observed	O
.	O
	O
Crystallization	O
of	O
the	O
16	O
Fabs	O
was	O
previously	O
reported	O
.	O
	O
Three	O
sets	O
of	O
the	O
crystals	O
were	O
isomorphous	O
with	O
nearly	O
identical	O
unit	O
cells	O
(	O
Table	O
1	O
).	O
	O
The	O
crystal	O
structures	O
of	O
the	O
16	O
Fabs	O
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
	O
Overall	O
the	O
structures	O
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	O
are	O
more	O
complete	O
than	O
those	O
for	O
the	O
lower	O
resolution	O
structures	O
(	O
Table	O
S1	O
).	O
	O
CDRs	O
are	O
defined	O
using	O
the	O
Dunbrack	O
convention	O
[	O
12	O
].	O
	O
CDR	O
H2	O
	O
Most	O
likely	O
this	O
is	O
the	O
result	O
of	O
interaction	O
of	O
CDR	O
H2	O
with	O
CDR	O
H1	O
,	O
namely	O
with	O
the	O
residue	O
at	O
position	O
33	O
(	O
residue	O
11	O
of	O
13	O
in	O
CDR	O
H1	O
).	O
	O
The	O
four	O
LC	O
CDRs	O
L1	O
feature	O
3	O
different	O
lengths	O
(	O
11	O
,	O
12	O
and	O
17	O
residues	O
)	O
having	O
a	O
total	O
of	O
4	O
different	O
canonical	O
structure	O
assignments	O
.	O
	O
Two	O
structures	O
,	O
H3	O
-	O
53	O
:	O
L3	O
-	O
20	O
and	O
H5	O
-	O
51	O
:	O
L3	O
-	O
20	O
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
	O
As	O
with	O
CDR	O
L2	O
,	O
all	O
4	O
LCs	O
have	O
CDR	O
L3	O
of	O
the	O
same	O
length	O
and	O
canonical	O
structure	O
,	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
	O
CDR	O
H3	O
conformational	O
diversity	O
	O
Despite	O
having	O
the	O
same	O
amino	O
acid	O
sequence	O
in	O
all	O
variants	O
,	O
CDR	O
H3	O
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	O
in	O
the	O
experimental	O
set	O
.	O
	O
Another	O
four	O
of	O
the	O
Fabs	O
,	O
H3	O
-	O
23	O
:	O
L1	O
-	O
39	O
,	O
H3	O
-	O
53	O
:	O
L1	O
-	O
39	O
,	O
H3	O
-	O
53	O
:	O
L3	O
-	O
11	O
and	O
H3	O
-	O
53	O
:	O
L4	O
-	O
1	O
have	O
missing	O
side	O
-	O
chain	O
atoms	O
.	O
	O
A	O
representative	O
CDR	O
H3	O
structure	O
for	O
H1	O
-	O
69	O
:	O
L1	O
-	O
39	O
illustrating	O
this	O
is	O
shown	O
in	O
Fig	O
.	O
7A	O
.	O
	O
In	O
fact	O
,	O
it	O
is	O
the	O
only	O
Fab	O
in	O
the	O
set	O
that	O
has	O
a	O
water	O
molecule	O
present	O
at	O
this	O
site	O
.	O
	O
Three	O
of	O
the	O
Fabs	O
,	O
H3	O
-	O
23	O
:	O
L1	O
-	O
39	O
,	O
H3	O
-	O
23	O
:	O
L4	O
-	O
1	O
and	O
H3	O
-	O
53	O
:	O
L1	O
-	O
39	O
,	O
have	O
distinctive	O
conformations	O
.	O
	O
VH	O
:	O
VL	O
domain	O
packing	O
	O
The	O
VH	O
and	O
VL	O
domains	O
have	O
a	O
β	O
-	O
sandwich	O
structure	O
(	O
also	O
often	O
referred	O
as	O
a	O
Greek	O
key	O
motif	O
)	O
and	O
each	O
is	O
composed	O
of	O
a	O
4	O
-	O
stranded	O
and	O
a	O
5	O
-	O
stranded	O
antiparallel	O
β	O
-	O
sheets	O
.	O
	O
They	O
include	O
:	O
1	O
)	O
a	O
bidentate	O
hydrogen	O
bond	O
between	O
L	O
-	O
Gln38	O
and	O
H	O
-	O
Gln39	O
;	O
2	O
)	O
H	O
-	O
Leu45	O
in	O
a	O
hydrophobic	O
pocket	O
between	O
L	O
-	O
Phe98	O
,	O
L	O
-	O
Tyr87	O
and	O
L	O
-	O
Pro44	O
;	O
3	O
)	O
L	O
-	O
Pro44	O
stacked	O
against	O
H	O
-	O
Trp103	O
;	O
and	O
4	O
)	O
L	O
-	O
Ala43	O
opposite	O
the	O
face	O
of	O
H	O
-	O
Tyr91	O
(	O
Fig	O
.	O
8	O
).	O
	O
With	O
the	O
exception	O
of	O
L	O
-	O
Ala43	O
,	O
all	O
other	O
residues	O
are	O
conserved	O
in	O
human	O
germlines	O
.	O
	O
The	O
first	O
approach	O
uses	O
ABangles	O
,	O
the	O
results	O
of	O
which	O
are	O
shown	O
in	O
Table	O
S2	O
.	O
	O
For	O
structures	O
with	O
2	O
copies	O
of	O
the	O
Fab	O
in	O
the	O
asymmetric	O
unit	O
,	O
only	O
one	O
structure	O
was	O
used	O
.	O
	O
The	O
largest	O
deviations	O
in	O
the	O
tilt	O
angle	O
,	O
up	O
to	O
11	O
.	O
0	O
°,	O
are	O
found	O
for	O
2	O
structures	O
,	O
H1	O
-	O
69	O
:	O
L3	O
-	O
20	O
and	O
H3	O
-	O
23	O
:	O
L3	O
-	O
20	O
,	O
that	O
stand	O
out	O
from	O
the	O
other	O
Fabs	O
.	O
	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	O
angles	O
are	O
shown	O
in	O
Fig	O
.	O
9	O
.	O
	O
Some	O
side	O
chain	O
atoms	O
in	O
CDR	O
H3	O
are	O
missing	O
.	O
	O
Parts	O
of	O
CDR	O
H3	O
main	O
chain	O
are	O
completely	O
disordered	O
,	O
and	O
were	O
not	O
modeled	O
in	O
Fabs	O
H5	O
-	O
51	O
:	O
L3	O
-	O
20	O
and	O
H5	O
-	O
51	O
:	O
L3	O
-	O
11	O
that	O
have	O
the	O
lowest	O
Tms	O
in	O
the	O
set	O
.	O
	O
Pairing	O
of	O
different	O
germlines	O
yields	O
antibodies	O
with	O
various	O
degrees	O
of	O
stability	O
.	O
	O
Curiously	O
,	O
the	O
2	O
Fabs	O
,	O
H1	O
-	O
69	O
:	O
L3	O
-	O
20	O
and	O
H3	O
-	O
23	O
:	O
L3	O
-	O
20	O
,	O
deviate	O
markedly	O
in	O
their	O
tilt	O
angles	O
from	O
the	O
rest	O
of	O
the	O
panel	O
.	O
	O
Note	O
that	O
most	O
of	O
the	O
VH	O
:	O
VL	O
interface	O
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
	O
Comparison	O
of	O
the	O
CDR	O
H3s	O
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	O
H3	O
conformation	O
.	O
	O
Fortunately	O
,	O
for	O
most	O
applications	O
of	O
antibody	O
modeling	O
,	O
such	O
as	O
engineering	O
affinity	O
and	O
biophysical	O
properties	O
,	O
an	O
accurate	O
CDR	O
H3	O
structure	O
is	O
not	O
always	O
necessary	O
.	O
	O
Visualizing	O
chaperone	O
-	O
assisted	O
protein	O
folding	O
	O
NMR	O
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
	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	O
regions	O
of	O
the	O
substrate	O
with	O
iodine	O
,	O
the	O
residual	O
electron	O
density	O
can	O
provide	O
spatial	O
information	O
on	O
many	O
of	O
the	O
other	O
flexible	O
residues	O
.	O
	O
As	O
described	O
in	O
detail	O
below	O
,	O
we	O
developed	O
the	O
READ	O
method	O
to	O
uncover	O
the	O
ensemble	O
of	O
conformations	O
that	O
the	O
Spy	O
-	O
binding	O
domain	O
of	O
Im7	O
(	O
i	O
.	O
e	O
.,	O
Im76	B-mutant
-	I-mutant
45	I-mutant
)	O
adopts	O
while	O
bound	O
to	O
Spy	O
.	O
	O
We	O
then	O
co	O
-	O
crystallized	O
Spy	O
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	O
-	O
Phe	O
substitution	O
at	O
one	O
distinct	O
position	O
,	O
and	O
collected	O
anomalous	O
data	O
for	O
all	O
eight	O
Spy	O
:	O
Im76	O
-	O
45	O
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
	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	O
to	O
Spy	O
,	O
we	O
combined	O
the	O
residual	O
electron	O
density	O
and	O
the	O
anomalous	O
signals	O
from	O
our	O
pI	O
-	O
Phe	O
substituted	O
Spy	O
:	O
Im76	O
-	O
45	O
complexes	O
.	O
	O
The	O
READ	O
sample	O
-	O
and	O
-	O
select	O
algorithm	O
is	O
diagrammed	O
in	O
Fig	O
.	O
2	O
.	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	O
-	O
substrate	O
complexes	O
using	O
coarse	O
-	O
grained	O
MD	O
simulations	O
in	O
a	O
pseudo	O
-	O
crystal	O
environment	O
(	O
Fig	O
.	O
2	O
and	O
Supplementary	O
Fig	O
.	O
4	O
).	O
	O
The	O
initial	O
conditions	O
of	O
the	O
binding	O
simulations	O
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	O
-	O
substrate	O
interaction	O
(	O
Online	O
Methods	O
).	O
	O
Im76	B-mutant
-	I-mutant
45	I-mutant
binds	O
and	O
unbinds	O
to	O
Spy	O
throughout	O
the	O
simulations	O
.	O
	O
The	O
anomalous	O
scattering	O
portion	O
of	O
the	O
selection	O
uses	O
our	O
basic	O
knowledge	O
of	O
pI	O
-	O
Phe	O
geometry	O
:	O
the	O
iodine	O
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	O
anomalous	O
signals	O
.	O
	O
To	O
make	O
the	O
electron	O
density	O
selection	O
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	O
density	O
on	O
-	O
the	O
-	O
fly	O
during	O
the	O
selection	O
procedure	O
.	O
	O
Folding	O
and	O
interactions	O
of	O
Im7	O
while	O
bound	O
to	O
Spy	O
	O
The	O
ensemble	O
primarily	O
encompasses	O
Im76	B-mutant
-	I-mutant
45	I-mutant
laying	O
diagonally	O
within	O
the	O
Spy	O
cradle	O
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	O
(	O
Figs	O
.	O
3	O
,	O
4a	O
).	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
	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	O
sites	O
.	O
	O
The	O
diversity	O
of	O
conformations	O
and	O
binding	O
sites	O
observed	O
here	O
emphasizes	O
the	O
dynamic	O
and	O
heterogeneous	O
nature	O
of	O
the	O
chaperone	O
-	O
substrate	O
ensemble	O
.	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	O
by	O
providing	O
more	O
binding	O
poses	O
.	O
	O
Additionally	O
,	O
we	O
observed	O
that	O
the	O
linker	O
region	O
(	O
residues	O
47	O
–	O
57	O
)	O
of	O
Spy	O
,	O
which	O
participates	O
in	O
substrate	O
interaction	O
,	O
becomes	O
mostly	O
disordered	O
upon	O
binding	O
the	O
substrate	O
.	O
	O
Importantly	O
,	O
we	O
observed	O
the	O
same	O
structural	O
changes	O
in	O
Spy	O
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
	O
This	O
substrate	O
-	O
chaperone	O
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	O
aids	O
protein	O
folding	O
.	O
	O
The	O
high	O
-	O
resolution	O
ensemble	O
obtained	O
here	O
now	O
provides	O
insight	O
into	O
exactly	O
how	O
this	O
occurs	O
.	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	O
.	O
	O
Unfolded	O
substrate	O
conformers	O
interact	O
with	O
Spy	O
through	O
both	O
hydrophobic	O
and	O
hydrophilic	O
interactions	O
,	O
whereas	O
the	O
binding	O
of	O
native	O
-	O
like	O
states	O
is	O
mainly	O
hydrophilic	O
.	O
	O
Previous	O
analysis	O
revealed	O
that	O
the	O
Super	O
Spy	O
variants	O
either	O
bound	O
Im7	O
tighter	O
than	O
WT	O
Spy	O
,	O
increased	O
chaperone	O
flexibility	O
as	O
measured	O
via	O
H	O
/	O
D	O
exchange	O
,	O
or	O
both	O
.	O
	O
Moreover	O
,	O
our	O
co	O
-	O
structure	O
suggests	O
that	O
the	O
L32P	B-mutant
substitution	O
,	O
which	O
increases	O
Spy	O
’	O
s	O
flexibility	O
,	O
could	O
operate	O
by	O
unhinging	O
the	O
N	O
-	O
terminal	O
helix	O
and	O
effectively	O
expanding	O
the	O
size	O
of	O
the	O
disordered	O
linker	O
.	O
	O
This	O
possibility	O
is	O
supported	O
by	O
the	O
Spy	O
:	O
substrate	O
structures	O
,	O
in	O
which	O
the	O
linker	O
region	O
becomes	O
more	O
flexible	O
compared	O
to	O
the	O
apo	O
state	O
(	O
Fig	O
.	O
6a	O
).	O
	O
Instead	O
,	O
when	O
Spy	O
is	O
bound	O
to	O
substrate	O
,	O
F115	O
engages	O
in	O
close	O
CH	O
⋯	O
π	O
hydrogen	O
bonds	O
with	O
Tyr104	O
(	O
Fig	O
.	O
6b	O
).	O
	O
Overall	O
,	O
comparison	O
of	O
our	O
ensemble	O
to	O
the	O
Super	O
Spy	O
variants	O
provides	O
specific	O
examples	O
to	O
corroborate	O
the	O
importance	O
of	O
conformational	O
flexibility	O
in	O
chaperone	O
-	O
substrate	O
interactions	O
.	O
	O
Spy	O
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
	O
The	O
frequency	O
plotted	O
is	O
calculated	O
as	O
the	O
average	O
contact	O
frequency	O
from	O
Spy	O
to	O
every	O
residue	O
of	O
Im76	B-mutant
-	I-mutant
45	I-mutant
and	O
vice	O
-	O
versa	O
.	O
	O
The	O
mechanism	O
of	O
NCX	O
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
	O
With	O
similar	O
ion	O
exchange	O
properties	O
to	O
those	O
of	O
its	O
eukaryotic	O
counterparts	O
,	O
NCX_Mj	O
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	O
.	O
	O
The	O
assignment	O
of	O
the	O
four	O
central	O
binding	O
sites	O
identified	O
in	O
the	O
previously	O
reported	O
NCX_Mj	O
structure	O
was	O
hampered	O
by	O
the	O
presence	O
of	O
both	O
Na	O
+	O
and	O
Ca2	O
+	O
in	O
the	O
protein	O
crystals	O
.	O
	O
First	O
,	O
the	O
electron	O
density	O
at	O
Smid	O
does	O
not	O
depend	O
significantly	O
on	O
the	O
Na	O
+	O
concentration	O
.	O
	O
This	O
structurally	O
-	O
derived	O
Na	O
+	O
affinity	O
agrees	O
well	O
with	O
the	O
external	O
Na	O
+	O
concentration	O
required	O
for	O
NCX	O
activation	O
in	O
eukaryotes	O
.	O
	O
Similarly	O
to	O
Sr2	O
+,	O
Ca2	O
+	O
binds	O
with	O
low	O
affinity	O
to	O
outward	O
-	O
facing	O
NCX_Mj	O
and	O
can	O
be	O
readily	O
displaced	O
by	O
Na	O
+	O
(	O
Supplementary	O
Note	O
1	O
and	O
Supplementary	O
Fig	O
.	O
2c	O
).	O
	O
This	O
finding	O
is	O
consistent	O
with	O
physiological	O
and	O
biochemical	O
data	O
for	O
both	O
eukaryotic	O
NCX	O
and	O
NCX_Mj	O
indicating	O
that	O
the	O
apparent	O
Ca2	O
+	O
affinity	O
is	O
much	O
lower	O
on	O
the	O
extracellular	O
than	O
the	O
cytoplasmic	O
side	O
.	O
	O
We	O
were	O
able	O
to	O
determine	O
an	O
apo	O
-	O
state	O
structure	O
of	O
NCX_Mj	O
,	O
by	O
crystallizing	O
the	O
protein	O
at	O
lower	O
pH	O
and	O
in	O
the	O
absence	O
of	O
Na	O
+	O
(	O
Methods	O
).	O
	O
To	O
examine	O
this	O
central	O
question	O
,	O
we	O
sought	O
to	O
characterize	O
the	O
conformational	O
free	O
-	O
energy	O
landscape	O
of	O
NCX_Mj	O
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	O
dynamics	O
(	O
MD	O
)	O
simulations	O
.	O
	O
This	O
computational	O
analysis	O
was	O
based	O
solely	O
on	O
the	O
published	O
structure	O
of	O
NCX_Mj	O
,	O
independently	O
of	O
the	O
crystallographic	O
studies	O
described	O
above	O
.	O
	O
These	O
initial	O
simulations	O
revealed	O
noticeable	O
changes	O
in	O
the	O
transporter	O
,	O
consistent	O
with	O
those	O
observed	O
in	O
the	O
new	O
crystal	O
structures	O
.	O
	O
The	O
most	O
noticeable	O
is	O
an	O
increased	O
separation	O
between	O
TM7	O
and	O
TM2	O
(	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	O
+	O
ion	O
at	O
SCa	O
(	O
Fig	O
.	O
4d	O
-	O
e	O
).	O
	O
TM1	O
and	O
TM6	O
also	O
slide	O
further	O
towards	O
the	O
membrane	O
center	O
,	O
relative	O
to	O
the	O
outward	O
-	O
occluded	O
state	O
(	O
Fig	O
.	O
4c	O
).	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	O
,	O
we	O
carried	O
out	O
a	O
series	O
of	O
enhanced	O
-	O
sampling	O
MD	O
calculations	O
designed	O
to	O
reversibly	O
simulate	O
the	O
transition	O
between	O
the	O
outward	O
-	O
occluded	O
and	O
fully	O
outward	O
-	O
open	O
states	O
,	O
and	O
thus	O
quantify	O
the	O
free	O
-	O
energy	O
landscape	O
encompassing	O
these	O
states	O
(	O
Methods	O
).	O
	O
It	O
is	O
however	O
also	O
non	O
-	O
trivial	O
:	O
antiporters	O
,	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	O
symporters	O
reset	O
their	O
transport	O
cycles	O
.	O
	O
Similarly	O
puzzling	O
is	O
that	O
a	O
given	O
antiporter	O
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
	O
The	O
internal	O
symmetry	O
of	O
outward	O
-	O
facing	O
NCX_Mj	O
and	O
the	O
inward	O
-	O
facing	O
crystal	O
structures	O
of	O
several	O
Ca2	O
+/	O
H	O
+	O
exchangers	O
indicate	O
that	O
the	O
alternating	O
-	O
access	O
mechanism	O
of	O
NCX	O
proteins	O
entails	O
a	O
sliding	O
motion	O
of	O
TM1	O
and	O
TM6	O
relative	O
to	O
the	O
rest	O
of	O
the	O
transporter	O
.	O
	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	O
state	O
in	O
this	O
landscape	O
that	O
explains	O
the	O
antiport	O
function	O
of	O
NCX_Mj	O
and	O
its	O
3Na	O
+:	O
1Ca2	O
+	O
stoichiometry	O
.	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	O
and	O
cardiac	O
NCX	O
perfectly	O
map	O
to	O
the	O
first	O
ion	O
-	O
coordination	O
shell	O
in	O
our	O
NCX_Mj	O
structures	O
(	O
Supplementary	O
Fig	O
.	O
4c	O
-	O
d	O
).	O
	O
The	O
Sext	O
site	O
,	O
by	O
contrast	O
,	O
might	O
be	O
thought	O
as	O
an	O
activation	O
site	O
for	O
inward	O
Na	O
+	O
translocation	O
,	O
since	O
this	O
is	O
where	O
the	O
third	O
Na	O
+	O
ion	O
binds	O
at	O
high	O
Na	O
+	O
concentration	O
,	O
enabling	O
the	O
transition	O
to	O
the	O
occluded	O
state	O
.	O
	O
Lastly	O
,	O
our	O
theory	O
that	O
occlusion	O
of	O
NCX_Mj	O
is	O
selectively	O
induced	O
upon	O
Ca2	O
+	O
or	O
Na	O
+	O
recognition	O
is	O
consonant	O
with	O
a	O
recent	O
analysis	O
of	O
the	O
rate	O
of	O
hydrogen	O
-	O
deuterium	O
exchange	O
(	O
HDX	O
)	O
in	O
NCX_Mj	O
,	O
in	O
the	O
presence	O
or	O
absence	O
of	O
these	O
ions	O
,	O
in	O
conditions	O
that	O
favor	O
outward	O
-	O
facing	O
conformations	O
.	O
	O
Specifically	O
,	O
saturating	O
amounts	O
of	O
Ca2	O
+	O
or	O
Na	O
+	O
resulted	O
in	O
a	O
noticeable	O
slowdown	O
in	O
the	O
HDX	O
rate	O
for	O
extracellular	O
portions	O
of	O
the	O
α	O
-	O
repeat	O
helices	O
.	O
	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	O
-	O
binding	O
sites	O
depend	O
on	O
ion	O
occupancy	O
state	O
.	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	O
rate	O
is	O
comparable	O
for	O
Na	O
+	O
and	O
Ca2	O
+,	O
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
(	O
c	O
)	O
Close	O
-	O
up	O
view	O
of	O
the	O
Na	O
+-	O
binding	O
sites	O
.	O
	O
Residues	O
surrounding	O
this	O
site	O
are	O
also	O
indicated	O
;	O
note	O
A206	O
(	O
labeled	O
in	O
red	O
)	O
coordinates	O
Na	O
+	O
at	O
Sext	O
via	O
its	O
backbone	O
carbonyl	O
oxygen	O
.	O
	O
Divalent	O
cation	O
binding	O
and	O
apo	O
structure	O
of	O
NCX_Mj	O
.	O
(	O
a	O
)	O
A	O
single	O
Sr2	O
+	O
(	O
dark	O
blue	O
sphere	O
)	O
binds	O
at	O
SCa	O
in	O
crystals	O
titrated	O
with	O
10	O
mM	O
Sr2	O
+	O
and	O
2	O
.	O
5	O
mM	O
Na	O
+	O
(	O
see	O
also	O
Supplementary	O
Fig	O
.	O
2	O
).	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	O
+-	O
bound	O
state	O
.	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	O
of	O
NCX_Mj	O
structures	O
obtained	O
at	O
low	O
Na	O
+	O
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	O
of	O
Na	O
+	O
and	O
pH	O
4	O
(	O
light	O
green	O
),	O
referred	O
to	O
as	O
apo	O
state	O
.	O
(	O
d	O
)	O
Close	O
-	O
up	O
view	O
of	O
the	O
ion	O
-	O
binding	O
sites	O
in	O
the	O
apo	O
(	O
or	O
high	O
H	O
+)	O
state	O
.	O
	O
(	O
a	O
)	O
Representative	O
simulation	O
snapshots	O
of	O
NCX_Mj	O
(	O
Methods	O
)	O
with	O
Na	O
+	O
bound	O
at	O
Sext	O
,	O
SCa	O
and	O
Sint	O
(	O
orange	O
cartoons	O
,	O
green	O
spheres	O
)	O
and	O
with	O
Na	O
+	O
bound	O
only	O
at	O
SCa	O
and	O
Sint	O
(	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	O
-	O
terminal	O
half	O
of	O
TM7	O
(	O
TM7ab	O
),	O
in	O
the	O
same	O
Na	O
+	O
occupancy	O
states	O
depicted	O
in	O
(	O
a	O
).	O
	O
(	O
g	O
)	O
Probability	O
distributions	O
of	O
an	O
analytical	O
descriptor	O
of	O
the	O
backbone	O
hydrogen	O
-	O
bonding	O
pattern	O
in	O
TM7ab	O
(	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	O
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	O
site	O
(	O
Eq	O
.	O
1	O
).	O
	O
The	O
free	O
energy	O
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	O
channels	O
leading	O
to	O
the	O
Sext	O
and	O
SCa	O
sites	O
,	O
respectively	O
(	O
see	O
Methods	O
).	O
	O
(	O
b	O
)	O
Density	O
isosurfaces	O
for	O
water	O
molecules	O
within	O
12	O
Å	O
of	O
the	O
ion	O
-	O
binding	O
region	O
(	O
grey	O
volumes	O
),	O
for	O
each	O
of	O
the	O
major	O
conformational	O
free	O
-	O
energy	O
minima	O
in	O
each	O
ion	O
-	O
occupancy	O
state	O
.	O
	O
Na	O
+	O
ions	O
are	O
shown	O
as	O
green	O
spheres	O
.	O
	O
Black	O
circles	O
map	O
the	O
crystal	O
structures	O
obtained	O
at	O
high	O
Ca2	O
+	O
concentration	O
and	O
at	O
low	O
pH	O
(	O
or	O
high	O
H	O
+)	O
reported	O
in	O
this	O
study	O
.	O
	O
(	O
b	O
)	O
Water	O
-	O
density	O
isosurfaces	O
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	O
-	O
energy	O
minima	O
in	O
the	O
free	O
-	O
energy	O
maps	O
.	O
	O
Here	O
,	O
the	O
authors	O
report	O
U2AF65	O
structures	O
and	O
single	O
molecule	O
FRET	O
that	O
reveal	O
mechanistic	O
insights	O
into	O
splice	O
site	O
recognition	O
.	O
	O
The	O
splice	O
sites	O
are	O
marked	O
by	O
relatively	O
short	O
consensus	O
sequences	O
and	O
are	O
regulated	O
by	O
additional	O
pre	O
-	O
mRNA	O
motifs	O
(	O
reviewed	O
in	O
ref	O
.).	O
	O
In	O
turn	O
,	O
the	O
ternary	O
complex	O
of	O
U2AF65	O
with	O
SF1	O
and	O
U2AF35	O
identifies	O
the	O
surrounding	O
BPS	O
and	O
3	O
′	O
splice	O
site	O
junctions	O
.	O
	O
Likewise	O
,	O
both	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
and	O
full	O
-	O
length	O
U2AF65	O
showed	O
similar	O
sequence	O
specificity	O
for	O
U	O
-	O
rich	O
stretches	O
in	O
the	O
5	O
′-	O
region	O
of	O
the	O
Py	O
tract	O
and	O
promiscuity	O
for	O
C	O
-	O
rich	O
regions	O
in	O
the	O
3	O
′-	O
region	O
(	O
Fig	O
.	O
1c	O
,	O
Supplementary	O
Fig	O
.	O
1e	O
–	O
h	O
).	O
	O
By	O
sequential	O
boot	O
strapping	O
(	O
Methods	O
),	O
we	O
optimized	O
the	O
oligonucleotide	O
length	O
,	O
the	O
position	O
of	O
a	O
Br	O
-	O
dU	O
,	O
and	O
the	O
identity	O
of	O
the	O
terminal	O
nucleotide	O
(	O
rU	O
,	O
dU	O
and	O
rC	O
)	O
to	O
achieve	O
full	O
views	O
of	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
bound	O
to	O
contiguous	O
Py	O
tracts	O
at	O
up	O
to	O
1	O
.	O
5	O
Å	O
resolution	O
.	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	O
with	O
the	O
prior	O
dU2AF651	B-mutant
,	I-mutant
2	I-mutant
crystal	O
structure	O
and	O
U2AF651	B-mutant
,	I-mutant
2	I-mutant
NMR	O
structure	O
in	O
the	O
Supplementary	O
Discussion	O
and	O
Supplementary	O
Fig	O
.	O
2	O
.	O
	O
Yet	O
,	O
only	O
the	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
interactions	O
at	O
sites	O
1	O
and	O
7	O
are	O
nearly	O
identical	O
to	O
those	O
of	O
the	O
dU2AF651	B-mutant
,	I-mutant
2	I-mutant
structures	O
(	O
Supplementary	O
Fig	O
.	O
3a	O
,	O
f	O
).	O
	O
In	O
the	O
C	O
-	O
terminal	O
β	O
-	O
strand	O
of	O
RRM1	O
,	O
the	O
side	O
chains	O
of	O
K225	O
and	O
R227	O
donate	O
additional	O
hydrogen	O
bonds	O
to	O
the	O
rU5	O
-	O
O2	O
lone	O
pair	O
electrons	O
.	O
	O
We	O
tested	O
the	O
contribution	O
of	O
the	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
interactions	O
with	O
the	O
new	O
central	O
nucleotide	O
to	O
Py	O
-	O
tract	O
affinity	O
(	O
Fig	O
.	O
3i	O
;	O
Supplementary	O
Fig	O
.	O
4a	O
,	O
b	O
).	O
	O
U2AF65	O
RRM	O
extensions	O
interact	O
with	O
the	O
Py	O
tract	O
	O
Indirectly	O
,	O
the	O
additional	O
contacts	O
with	O
the	O
third	O
nucleotide	O
shift	O
the	O
rU2	O
nucleotide	O
in	O
the	O
second	O
binding	O
site	O
closer	O
to	O
the	O
C	O
-	O
terminal	O
β	O
-	O
strand	O
of	O
RRM2	O
.	O
	O
Consistent	O
with	O
loss	O
of	O
a	O
hydrogen	O
bond	O
with	O
the	O
ninth	O
pyrimidine	O
-	O
O2	O
(	O
ΔΔG	O
1	O
.	O
0	O
kcal	O
mol	O
−	O
1	O
),	O
mutation	O
of	O
the	O
Q147	O
to	O
an	O
alanine	O
reduced	O
U2AF651	O
,	O
2L	O
affinity	O
for	O
the	O
AdML	O
Py	O
tract	O
by	O
five	O
-	O
fold	O
(	O
Fig	O
.	O
3i	O
;	O
Supplementary	O
Fig	O
.	O
4c	O
).	O
	O
We	O
introduced	O
glycine	O
substitutions	O
to	O
maximally	O
reduce	O
the	O
buried	O
surface	O
area	O
without	O
directly	O
interfering	O
with	O
its	O
hydrogen	O
bonds	O
between	O
backbone	O
atoms	O
and	O
the	O
base	O
.	O
	O
To	O
further	O
test	O
cooperation	O
among	O
the	O
U2AF65	O
RRM	O
extensions	O
and	O
inter	O
-	O
RRM	O
linker	O
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	O
(	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
	O
We	O
proceeded	O
to	O
test	O
the	O
importance	O
of	O
new	O
U2AF65	O
–	O
Py	O
-	O
tract	O
interactions	O
for	O
splicing	O
of	O
a	O
model	O
pre	O
-	O
mRNA	O
substrate	O
in	O
a	O
human	O
cell	O
line	O
(	O
Fig	O
.	O
5	O
;	O
Supplementary	O
Fig	O
.	O
5	O
).	O
	O
When	O
transfected	O
into	O
HEK293T	O
cells	O
containing	O
only	O
endogenous	O
U2AF65	O
,	O
the	O
PY	O
splice	O
site	O
is	O
used	O
and	O
the	O
remaining	O
transcript	O
remains	O
unspliced	O
.	O
	O
The	O
strong	O
PY	O
splice	O
site	O
is	O
insensitive	O
to	O
added	O
U2AF65	O
,	O
suggesting	O
that	O
endogenous	O
U2AF65	O
levels	O
are	O
sufficient	O
to	O
saturate	O
this	O
site	O
(	O
Supplementary	O
Fig	O
.	O
5b	O
).	O
	O
The	O
positions	O
of	O
single	O
cysteine	O
mutations	O
for	O
fluorophore	O
attachment	O
(	O
A181C	B-mutant
in	O
RRM1	O
and	O
Q324C	B-mutant
in	O
RRM2	O
)	O
were	O
chosen	O
based	O
on	O
inspection	O
of	O
the	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
structures	O
and	O
the	O
‘	O
closed	O
'	O
model	O
of	O
apo	O
-	O
U2AF651	B-mutant
,	I-mutant
2	I-mutant
.	O
	O
Criteria	O
included	O
(	O
i	O
)	O
residue	O
locations	O
that	O
are	O
distant	O
from	O
and	O
hence	O
not	O
expected	O
to	O
interfere	O
with	O
the	O
RRM	O
/	O
RNA	O
or	O
inter	O
-	O
RRM	O
interfaces	O
,	O
(	O
ii	O
)	O
inter	O
-	O
dye	O
distances	O
(	O
50	O
Å	O
for	O
U2AF651	O
,	O
2L	O
–	O
Py	O
tract	O
and	O
30	O
Å	O
for	O
the	O
closed	O
apo	O
-	O
model	O
)	O
that	O
are	O
expected	O
to	O
be	O
near	O
the	O
Förster	O
radius	O
(	O
Ro	O
)	O
for	O
the	O
Cy3	O
/	O
Cy5	O
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	O
efficiencies	O
that	O
are	O
calculated	O
to	O
be	O
significantly	O
greater	O
for	O
the	O
‘	O
closed	O
'	O
apo	O
-	O
model	O
as	O
opposed	O
to	O
the	O
‘	O
open	O
'	O
RNA	O
-	O
bound	O
structures	O
(	O
by	O
∼	O
30	O
%).	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	O
interruptions	O
that	O
often	O
occur	O
in	O
relatively	O
degenerate	O
human	O
Py	O
tracts	O
.	O
	O
Nevertheless	O
,	O
the	O
predominant	O
0	O
.	O
45	O
FRET	O
state	O
in	O
the	O
presence	O
of	O
RNA	O
agrees	O
with	O
the	O
Py	O
-	O
tract	O
-	O
bound	O
crystal	O
structure	O
of	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
.	O
	O
Importantly	O
,	O
the	O
majority	O
of	O
traces	O
(∼	O
70	O
%)	O
of	O
U2AF651	B-mutant
,	I-mutant
2LFRET	I-mutant
(	O
Cy3	O
/	O
Cy5	O
)	O
bound	O
to	O
the	O
slide	O
-	O
tethered	O
RNA	O
lacked	O
FRET	O
fluctuations	O
and	O
predominately	O
exhibited	O
a	O
∼	O
0	O
.	O
45	O
FRET	O
value	O
(	O
for	O
example	O
,	O
Fig	O
.	O
6g	O
).	O
	O
The	O
U2AF65	O
structures	O
and	O
analyses	O
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	O
′	O
splice	O
site	O
.	O
	O
The	O
intact	O
U2AF65	O
RRM1	O
/	O
RRM2	O
-	O
containing	O
domain	O
and	O
flanking	O
residues	O
are	O
required	O
for	O
binding	O
contiguous	O
Py	O
tracts	O
.	O
	O
Structures	O
of	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
recognizing	O
a	O
contiguous	O
Py	O
tract	O
.	O
	O
For	O
clarity	O
,	O
we	O
consistently	O
number	O
the	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
nucleotide	O
-	O
binding	O
sites	O
from	O
one	O
to	O
nine	O
,	O
although	O
in	O
some	O
cases	O
the	O
co	O
-	O
crystallized	O
oligonucleotide	O
comprises	O
eight	O
nucleotides	O
and	O
as	O
such	O
leaves	O
the	O
first	O
binding	O
site	O
empty	O
.	O
	O
(	O
b	O
)	O
Bar	O
graph	O
of	O
apparent	O
equilibrium	O
affinities	O
(	O
KA	O
)	O
for	O
the	O
AdML	O
Py	O
tract	O
(	O
5	O
′-	O
CCCUUUUUUUUCC	O
-	O
3	O
′)	O
of	O
the	O
wild	O
-	O
type	O
(	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	O
deletions	O
dU2AF651	B-mutant
,	I-mutant
2	I-mutant
in	O
the	O
minimal	O
RRM1	O
–	O
RRM2	O
region	O
(	O
residues	O
148	O
–	O
237	O
,	O
258	O
–	O
336	O
)	O
or	O
dU2AF651	B-mutant
,	I-mutant
2L	I-mutant
(	O
residues	O
141	O
–	O
237	O
,	O
258	O
–	O
342	O
).	O
	O
The	O
apparent	O
equilibrium	O
dissociation	O
constants	O
(	O
KD	O
)	O
of	O
the	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
mutant	O
proteins	O
are	O
:	O
wild	O
type	O
(	O
WT	O
),	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	O
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
(	O
a	O
,	O
b	O
)	O
Views	O
of	O
FRET	O
pairs	O
chosen	O
to	O
follow	O
the	O
relative	O
movement	O
of	O
RRM1	O
and	O
RRM2	O
on	O
the	O
crystal	O
structure	O
of	O
‘	O
side	O
-	O
by	O
-	O
side	O
'	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
RRMs	O
bound	O
to	O
a	O
Py	O
-	O
tract	O
oligonucleotide	O
(	O
a	O
,	O
representative	O
structure	O
iv	O
)	O
or	O
‘	O
closed	O
'	O
NMR	O
/	O
PRE	O
-	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	O
.	O
	O
RNA	O
protects	O
a	O
nucleoprotein	O
complex	O
against	O
radiation	O
damage	O
	O
The	O
availability	O
of	O
two	O
TRAP	O
molecules	O
in	O
the	O
asymmetric	O
unit	O
,	O
of	O
which	O
only	O
one	O
contained	O
bound	O
RNA	O
,	O
allowed	O
a	O
controlled	O
investigation	O
into	O
the	O
exact	O
role	O
of	O
RNA	O
binding	O
in	O
protein	O
specific	O
damage	O
susceptibility	O
.	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	O
determination	O
using	O
macromolecular	O
crystallography	O
(	O
MX	O
)	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
	O
Specific	O
radiation	O
damage	O
(	O
SRD	O
)	O
is	O
observed	O
in	O
the	O
real	O
-	O
space	O
electron	O
density	O
,	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
	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	O
-	O
bond	O
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
	O
Understanding	O
RD	O
to	O
such	O
complexes	O
is	O
crucial	O
,	O
since	O
DNA	O
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	O
repair	O
.	O
	O
As	O
of	O
early	O
2016	O
,	O
>	O
5400	O
nucleoprotein	O
complex	O
structures	O
have	O
been	O
deposited	O
within	O
the	O
PDB	O
,	O
with	O
91	O
%	O
solved	O
by	O
MX	O
.	O
	O
Using	O
newly	O
developed	O
methodology	O
,	O
we	O
present	O
a	O
controlled	O
SRD	O
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	O
–	O
RNA	O
complex	O
:	O
trp	O
RNA	O
-	O
binding	O
attenuation	O
protein	O
(	O
TRAP	O
)	O
bound	O
to	O
a	O
53	O
bp	O
RNA	O
sequence	O
(	O
GAGUU	O
)	O
10GAG	O
(	O
PDB	O
entry	O
1gtf	O
;	O
Hopcroft	O
et	O
al	O
.,	O
2002	O
).	O
	O
It	O
binds	O
with	O
high	O
affinity	O
(	O
K	O
d	O
≃	O
1	O
.	O
0	O
nM	O
)	O
to	O
RNA	O
segments	O
containing	O
11	O
GAG	O
/	O
UAG	O
triplets	O
separated	O
by	O
two	O
or	O
three	O
spacer	O
nucleotides	O
(	O
Elliott	O
et	O
al	O
.,	O
2001	O
)	O
to	O
regulate	O
the	O
transcription	O
of	O
tryptophan	O
biosynthetic	O
genes	O
in	O
Bacillus	O
subtilis	O
(	O
Antson	O
et	O
al	O
.,	O
1999	O
).	O
	O
Previous	O
studies	O
have	O
characterized	O
SRD	O
sites	O
by	O
reporting	O
magnitudes	O
of	O
F	O
obs	O
(	O
d	O
n	O
)	O
−	O
F	O
obs	O
(	O
d	O
1	O
)	O
Fourier	O
difference	O
map	O
peaks	O
in	O
terms	O
of	O
the	O
sigma	O
(	O
σ	O
)	O
contour	O
level	O
(	O
the	O
number	O
of	O
standard	O
deviations	O
from	O
the	O
mean	O
map	O
electron	O
-	O
density	O
value	O
)	O
at	O
which	O
peaks	O
become	O
visible	O
.	O
	O
Visual	O
inspection	O
of	O
Fourier	O
difference	O
maps	O
illustrated	O
the	O
clear	O
lack	O
of	O
RNA	O
electron	O
-	O
density	O
degradation	O
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
	O
For	O
each	O
TRAP	O
ring	O
subunit	O
,	O
the	O
Glu36	O
side	O
-	O
chain	O
carboxyl	O
group	O
accepts	O
a	O
pair	O
of	O
hydrogen	O
bonds	O
from	O
the	O
two	O
N	O
atoms	O
of	O
the	O
G3	O
RNA	O
base	O
.	O
	O
With	O
increasing	O
dose	O
,	O
the	O
D	O
loss	O
associated	O
with	O
the	O
Phe32	O
side	O
chain	O
was	O
significantly	O
reduced	O
upon	O
RNA	O
binding	O
(	O
Fig	O
.	O
5	O
▸	O
e	O
;	O
Phe32	O
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	O
had	O
been	O
reduced	O
.	O
	O
The	O
RNA	O
was	O
found	O
to	O
be	O
substantially	O
more	O
radiation	O
-	O
resistant	O
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	O
investigation	O
of	O
the	O
C	O
.	O
Esp1396I	O
protein	O
–	O
DNA	O
complex	O
(	O
Bury	O
et	O
al	O
.,	O
2015	O
).	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	O
obs	O
(	O
d	O
n	O
)	O
−	O
F	O
obs	O
(	O
d	O
1	O
)	O
map	O
density	O
was	O
detected	O
around	O
P	O
,	O
O3	O
′	O
and	O
O5	O
′	O
atoms	O
of	O
the	O
RNA	O
backbone	O
,	O
with	O
no	O
significant	O
difference	O
density	O
localized	O
to	O
RNA	O
ribose	O
and	O
basic	O
subunits	O
.	O
	O
This	O
is	O
in	O
good	O
agreement	O
with	O
previous	O
mutagenesis	O
and	O
nucleoside	O
analogue	O
studies	O
(	O
Elliott	O
et	O
al	O
.,	O
2001	O
),	O
which	O
indicated	O
that	O
the	O
G1	O
nucleotide	O
does	O
not	O
bind	O
to	O
TRAP	O
as	O
strongly	O
as	O
do	O
A2	O
and	O
G3	O
,	O
and	O
plays	O
little	O
role	O
in	O
the	O
high	O
RNA	O
-	O
binding	O
affinity	O
of	O
TRAP	O
(	O
K	O
d	O
≃	O
1	O
.	O
1	O
±	O
0	O
.	O
4	O
nM	O
).	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	O
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
	O
For	O
example	O
,	O
Asp17	O
is	O
located	O
∼	O
6	O
.	O
8	O
Å	O
from	O
the	O
G1	O
base	O
,	O
outside	O
the	O
RNA	O
-	O
binding	O
interfaces	O
,	O
and	O
has	O
indistinguishable	O
Cγ	O
atom	O
D	O
loss	O
dose	O
-	O
dynamics	O
between	O
RNA	O
-	O
bound	O
and	O
nonbound	O
TRAP	O
(	O
p	O
>	O
0	O
.	O
9	O
).	O
	O
This	O
structure	O
reveals	O
the	O
molecular	O
mechanism	O
underlying	O
the	O
docking	O
interaction	O
between	O
MKP7	O
and	O
JNK1	O
.	O
	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	O
family	O
can	O
be	O
further	O
divided	O
into	O
three	O
groups	O
(	O
Fig	O
.	O
1	O
).	O
	O
In	O
addition	O
to	O
the	O
CD	O
and	O
KBD	O
,	O
MKP7	O
has	O
a	O
long	O
C	O
-	O
terminal	O
region	O
that	O
contains	O
both	O
nuclear	O
localization	O
and	O
export	O
sequences	O
by	O
which	O
MKP7	O
shuttles	O
between	O
the	O
nucleus	O
and	O
the	O
cytoplasm	O
(	O
Fig	O
.	O
2a	O
).	O
	O
Thus	O
,	O
the	O
kinetic	O
data	O
were	O
analysed	O
using	O
the	O
general	O
initial	O
velocity	O
equation	O
,	O
taking	O
substrate	O
depletion	O
into	O
account	O
:	O
	O
The	O
overall	O
folding	O
of	O
MKP7	O
-	O
CD	O
is	O
typical	O
of	O
DUSPs	O
,	O
with	O
a	O
central	O
twisted	O
five	O
-	O
stranded	O
β	O
-	O
sheet	O
surrounded	O
by	O
six	O
α	O
-	O
helices	O
.	O
	O
Since	O
helix	O
α0	O
and	O
the	O
following	O
loop	O
α0	O
–	O
β1	O
are	O
known	O
for	O
a	O
substrate	O
-	O
recognition	O
motif	O
of	O
VHR	O
and	O
other	O
phosphatases	O
,	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	O
.	O
	O
The	O
MKP7	O
-	O
CD	O
structure	O
near	O
the	O
active	O
site	O
exhibits	O
a	O
typical	O
active	O
conformation	O
as	O
found	O
in	O
VHR	O
and	O
other	O
PTPs	O
.	O
	O
The	O
catalytic	O
residue	O
,	O
Cys244	O
,	O
is	O
located	O
just	O
after	O
strand	O
β5	O
and	O
optimally	O
positioned	O
for	O
nucleophilic	O
attack	O
.	O
	O
The	O
carboxylate	O
of	O
Asp268	O
in	O
MKP7	O
forms	O
a	O
salt	O
bridge	O
with	O
side	O
chain	O
of	O
Arg263	O
in	O
JNK1	O
,	O
and	O
Lys275	O
of	O
MKP7	O
forms	O
a	O
hydrogen	O
bond	O
and	O
a	O
salt	O
bridge	O
with	O
Thr228	O
and	O
Asp229	O
of	O
JNK1	O
,	O
respectively	O
.	O
	O
Gel	O
filtration	O
analysis	O
further	O
confirmed	O
the	O
key	O
role	O
of	O
Phe285	O
in	O
the	O
MKP7	O
–	O
JNK1	O
interaction	O
:	O
no	O
F285D	O
–	O
JNK1	O
complex	O
was	O
detected	O
when	O
3	O
molar	O
equivalents	O
of	O
MKP7	O
-	O
CD	O
(	O
F285D	B-mutant
)	O
were	O
mixed	O
with	O
1	O
molar	O
equivalent	O
of	O
JNK1	O
(	O
Fig	O
.	O
4b	O
).	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	O
proteins	O
,	O
we	O
also	O
examined	O
the	O
folding	O
properties	O
of	O
the	O
JNK1	O
and	O
MKP7	O
mutants	O
with	O
circular	O
dichroism	O
.	O
	O
The	O
spectra	O
of	O
these	O
mutants	O
are	O
similar	O
to	O
the	O
wild	O
-	O
type	O
proteins	O
,	O
indicating	O
that	O
these	O
mutants	O
fold	O
as	O
well	O
as	O
the	O
wild	O
-	O
type	O
proteins	O
(	O
Fig	O
.	O
4d	O
,	O
e	O
).	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	O
undergo	O
catalytic	O
activation	O
upon	O
interaction	O
with	O
the	O
MAPK	O
substrates	O
.	O
	O
Incubation	O
of	O
MKP7	O
with	O
JNK1	O
did	O
not	O
markedly	O
stimulate	O
the	O
phosphatase	O
activity	O
,	O
which	O
is	O
consistent	O
with	O
previous	O
results	O
that	O
MKP7	O
solely	O
possesses	O
the	O
intrinsic	O
activity	O
(	O
Supplementary	O
Fig	O
.	O
2b	O
).	O
	O
In	O
addition	O
,	O
His230	O
and	O
Val256	O
in	O
JNK1	O
are	O
replaced	O
by	O
the	O
negatively	O
charged	O
residues	O
Glu208	O
and	O
Asp235	O
in	O
CDK2	O
(	O
Fig	O
.	O
5d	O
),	O
and	O
the	O
charge	O
distribution	O
on	O
the	O
CDK2	O
interactive	O
surface	O
is	O
quite	O
different	O
from	O
that	O
of	O
JNK	O
.	O
	O
These	O
data	O
indicated	O
that	O
a	O
unique	O
hydrophobic	O
pocket	O
formed	O
between	O
the	O
MAPK	O
insert	O
and	O
αG	O
helix	O
plays	O
a	O
major	O
role	O
in	O
the	O
substrate	O
recognition	O
by	O
MKPs	O
.	O
	O
The	O
KBD	O
of	O
MKP5	O
interacts	O
with	O
the	O
D	O
-	O
site	O
of	O
p38α	O
to	O
mediate	O
the	O
enzyme	O
–	O
substrate	O
interaction	O
.	O
	O
The	O
substrate	O
specificity	O
constant	O
kcat	O
/	O
Km	O
value	O
for	O
MKP5	O
-	O
CD	O
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	O
-	O
CD	O
(	O
1	O
.	O
07	O
×	O
105	O
M	O
−	O
1	O
s	O
−	O
1	O
).	O
	O
Comparisons	O
between	O
catalytic	O
domains	O
structures	O
of	O
MKP5	O
and	O
MKP7	O
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	O
.	O
m	O
.	O
s	O
.	O
d	O
.	O
of	O
0	O
.	O
79	O
Å	O
;	O
Fig	O
.	O
7c	O
).	O
	O
Given	O
the	O
distinct	O
interaction	O
mode	O
revealed	O
by	O
the	O
crystal	O
structure	O
of	O
JNK1	O
–	O
MKP7	O
-	O
CD	O
,	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	O
-	O
specific	O
MKPs	O
.	O
	O
Pull	O
-	O
down	O
assays	O
also	O
confirmed	O
the	O
protein	O
–	O
protein	O
interactions	O
observed	O
above	O
.	O
	O
In	O
addition	O
,	O
there	O
were	O
no	O
significant	O
differences	O
in	O
the	O
CD	O
spectra	O
between	O
wild	O
-	O
type	O
and	O
mutant	O
proteins	O
,	O
indicating	O
that	O
the	O
overall	O
structures	O
of	O
these	O
mutants	O
did	O
not	O
change	O
significantly	O
from	O
that	O
of	O
wild	O
-	O
type	O
MKP5	O
protein	O
(	O
Supplementary	O
Fig	O
.	O
4a	O
).	O
	O
In	O
addition	O
,	O
the	O
key	O
interacting	O
residues	O
of	O
MKP7	O
-	O
CD	O
,	O
Phe215	O
,	O
Leu267	O
and	O
Leu288	O
,	O
are	O
replaced	O
by	O
less	O
hydrophobic	O
residues	O
,	O
Asn379	O
,	O
Met431	O
and	O
Met452	O
in	O
MKP5	O
-	O
CD	O
(	O
Fig	O
.	O
5c	O
),	O
respectively	O
,	O
which	O
may	O
result	O
in	O
weaker	O
hydrophobic	O
interactions	O
between	O
MKP5	O
-	O
CD	O
and	O
JNK1	O
.	O
	O
The	O
propagation	O
of	O
MAPK	O
signals	O
is	O
attenuated	O
through	O
the	O
actions	O
of	O
the	O
MKPs	O
.	O
	O
Most	O
studies	O
have	O
focused	O
on	O
the	O
dephosphorylation	O
of	O
MAPKs	O
by	O
phosphatases	O
containing	O
the	O
‘	O
kinase	O
-	O
interaction	O
motif	O
'	O
(	O
D	O
-	O
motif	O
),	O
including	O
a	O
group	O
of	O
DUSPs	O
(	O
MKPs	O
)	O
and	O
a	O
distinct	O
subfamily	O
of	O
tyrosine	O
phosphatases	O
(	O
HePTP	O
,	O
STEP	O
and	O
PTP	O
-	O
SL	O
).	O
	O
In	O
contrast	O
to	O
MKP5	O
,	O
removal	O
of	O
the	O
KBD	O
domain	O
from	O
MKP7	O
does	O
not	O
drastically	O
affect	O
enzyme	O
catalysis	O
,	O
and	O
the	O
kinetic	O
parameters	O
of	O
MKP7	O
-	O
CD	O
for	O
p38α	O
substrate	O
are	O
very	O
similar	O
to	O
those	O
for	O
JNK1	O
substrate	O
.	O
	O
The	O
MKP7	O
-	O
KBD	O
docks	O
to	O
the	O
D	O
-	O
site	O
located	O
on	O
the	O
back	O
side	O
of	O
the	O
p38α	O
catalytic	O
pocket	O
for	O
high	O
-	O
affinity	O
association	O
,	O
whereas	O
the	O
interaction	O
of	O
the	O
MKP7	O
-	O
CD	O
with	O
another	O
p38α	O
structural	O
region	O
,	O
which	O
is	O
close	O
to	O
the	O
activation	O
loop	O
,	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	O
active	O
site	O
with	O
respect	O
to	O
the	O
phosphoreceptor	O
in	O
the	O
p38α	O
substrate	O
for	O
efficient	O
dephosphorylation	O
.	O
	O
This	O
hydrophobic	O
site	O
was	O
first	O
identified	O
by	O
changes	O
in	O
deuterium	O
exchange	O
profiles	O
,	O
and	O
is	O
near	O
the	O
MAPK	O
insertion	O
and	O
helix	O
αG	O
.	O
Interestingly	O
,	O
many	O
of	O
the	O
equivalent	O
residues	O
in	O
JNK1	O
,	O
important	O
for	O
MKP7	O
-	O
CD	O
recognition	O
,	O
are	O
also	O
used	O
for	O
substrate	O
binding	O
by	O
ERK2	O
(	O
ref	O
.),	O
indicating	O
that	O
this	O
site	O
is	O
overlapped	O
with	O
the	O
DEF	O
-	O
site	O
previously	O
identified	O
in	O
ERK2	O
(	O
Fig	O
.	O
5d	O
).	O
	O
Therefore	O
,	O
it	O
is	O
tempting	O
to	O
speculate	O
that	O
the	O
catalytic	O
domain	O
of	O
MKP3	O
may	O
bind	O
to	O
ERK2	O
in	O
a	O
manner	O
analogous	O
to	O
the	O
way	O
by	O
which	O
MKP7	O
-	O
CD	O
binds	O
to	O
JNK1	O
.	O
	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	O
–	O
MKP7	O
-	O
CD	O
and	O
ERK2	O
–	O
MKP3	O
-	O
CD	O
complexes	O
,	O
the	O
ERK2	O
–	O
MKP3	O
-	O
CD	O
interaction	O
is	O
less	O
extensive	O
and	O
helix	O
α4	O
from	O
MKP3	O
-	O
CD	O
does	O
not	O
interact	O
directly	O
with	O
ERK2	O
.	O
	O
Phe285	O
is	O
essential	O
for	O
JNK1	O
substrate	O
binding	O
,	O
whereas	O
Phe287	O
plays	O
a	O
role	O
for	O
the	O
precise	O
alignment	O
of	O
active	O
-	O
site	O
residues	O
,	O
which	O
are	O
important	O
for	O
transition	O
-	O
state	O
stabilization	O
.	O
	O
(	O
a	O
)	O
Domain	O
organization	O
of	O
human	O
MKP7	O
and	O
JNK1	O
.	O
	O
The	O
2Fo	O
−	O
Fc	O
omit	O
map	O
(	O
contoured	O
at	O
1	O
.	O
5σ	O
)	O
for	O
the	O
P	O
-	O
loop	O
of	O
MKP7	O
-	O
CD	O
is	O
shown	O
at	O
inset	O
of	O
b	O
.	O
(	O
c	O
)	O
Structure	O
of	O
VHR	O
with	O
its	O
active	O
site	O
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	O
–	O
MKP7	O
interface	O
showing	O
interacting	O
amino	O
acids	O
of	O
JNK1	O
(	O
orange	O
)	O
and	O
MKP7	O
-	O
CD	O
(	O
cyan	O
).	O
	O
MKP7	O
-	O
CD	O
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
	O
Mutational	O
analysis	O
on	O
interactions	O
between	O
MKP7	O
-	O
CD	O
and	O
JNK1	O
.	O
	O
However	O
,	O
in	O
contrast	O
to	O
the	O
wild	O
-	O
type	O
MKP7	O
-	O
CD	O
,	O
mutant	O
F285D	B-mutant
did	O
not	O
co	O
-	O
migrate	O
with	O
JNK1	O
.	O
	O
(	O
c	O
)	O
Pull	O
-	O
down	O
assays	O
of	O
MKP7	O
-	O
CD	O
by	O
GST	O
-	O
tagged	O
JNK1	O
mutants	O
.	O
	O
Measurements	O
were	O
averaged	O
for	O
three	O
scans	O
.	O
(	O
e	O
)	O
Circular	O
dichroism	O
spectra	O
for	O
JNK1	O
wild	O
type	O
and	O
mutants	O
.	O
	O
The	O
N	O
-	O
lobe	O
and	O
C	O
-	O
lobe	O
of	O
CDK2	O
are	O
coloured	O
in	O
grey	O
and	O
pink	O
,	O
respectively	O
,	O
and	O
KAP	O
is	O
coloured	O
in	O
green	O
.	O
	O
Interestingly	O
,	O
the	O
recognition	O
of	O
CDK2	O
by	O
KAP	O
is	O
augmented	O
by	O
a	O
similar	O
interface	O
as	O
that	O
observed	O
in	O
the	O
complex	O
of	O
JNK1	O
and	O
MKP7	O
-	O
CD	O
(	O
region	O
II	O
).	O
	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	O
α6	O
of	O
KAP	O
(	O
corresponding	O
to	O
helix	O
α4	O
of	O
MKP7	O
-	O
CD	O
)	O
plays	O
little	O
,	O
if	O
any	O
,	O
role	O
in	O
the	O
formation	O
of	O
a	O
stable	O
heterodimer	O
of	O
CDK2	O
and	O
KAP	O
.	O
(	O
c	O
)	O
Sequence	O
alignment	O
of	O
the	O
JNK	O
-	O
interacting	O
regions	O
on	O
MKPs	O
.	O
	O
(	O
d	O
)	O
F	O
-	O
site	O
is	O
required	O
for	O
JNK1	O
to	O
interact	O
with	O
MKP7	O
.	O
	O
(	O
e	O
)	O
Effect	O
of	O
MKP7	O
(	O
wild	O
type	O
or	O
mutants	O
)	O
expression	O
on	O
ultraviolet	O
-	O
induced	O
apoptosis	O
.	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
*	O
P	O
<	O
0	O
.	O
05	O
,	O
***	O
P	O
<	O
0	O
.	O
001	O
(	O
ANOVA	O
followed	O
by	O
Tukey	O
'	O
s	O
test	O
).	O
	O
The	O
error	O
bars	O
represent	O
s	O
.	O
e	O
.	O
m	O
.	O
(	O
c	O
)	O
Structural	O
comparison	O
of	O
the	O
JNK	O
-	O
interacting	O
residues	O
on	O
MKP5	O
-	O
CD	O
(	O
PDB	O
1ZZW	O
)	O
and	O
MKP7	O
-	O
CD	O
.	O
	O
Mechanistic	O
insight	O
into	O
a	O
peptide	O
hormone	O
signaling	O
complex	O
mediating	O
floral	O
organ	O
abscission	O
	O
It	O
is	O
unknown	O
how	O
expression	O
of	O
IDA	O
in	O
the	O
abscission	O
zone	O
leads	O
to	O
HAESA	O
activation	O
.	O
	O
The	O
HAESA	O
co	O
-	O
receptor	O
SERK1	O
,	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	O
hormone	O
by	O
binding	O
to	O
an	O
Arg	O
-	O
His	O
-	O
Asn	O
motif	O
in	O
IDA	O
.	O
	O
Plants	O
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	O
protein	O
called	O
HAESA	O
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	O
called	O
IDA	O
instructs	O
HAESA	O
to	O
trigger	O
the	O
shedding	O
process	O
.	O
	O
Cysteine	O
residues	O
engaged	O
in	O
disulphide	O
bonds	O
are	O
depicted	O
in	O
green	O
.	O
	O
IDA	O
(	O
in	O
bonds	O
representation	O
,	O
surface	O
view	O
included	O
)	O
is	O
depicted	O
in	O
yellow	O
.	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	O
molecule	O
is	O
shown	O
as	O
a	O
red	O
sphere	O
.	O
	O
Abscission	O
of	O
floral	O
organs	O
in	O
Arabidopsis	O
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
	O
This	O
sequence	O
motif	O
is	O
highly	O
conserved	O
among	O
IDA	O
family	O
members	O
(	O
IDA	O
-	O
LIKE	O
PROTEINS	O
,	O
IDLs	O
)	O
and	O
contains	O
a	O
central	O
Pro	O
residue	O
,	O
presumed	O
to	O
be	O
post	O
-	O
translationally	O
modified	O
to	O
hydroxyproline	O
(	O
Hyp	O
;	O
Figure	O
1A	O
).	O
	O
The	O
available	O
genetic	O
and	O
biochemical	O
evidence	O
suggests	O
that	O
IDA	O
and	O
HAESA	O
together	O
control	O
floral	O
abscission	O
,	O
but	O
it	O
is	O
poorly	O
understood	O
if	O
IDA	O
is	O
directly	O
sensed	O
by	O
the	O
receptor	O
kinase	O
HAESA	O
and	O
how	O
IDA	O
binding	O
at	O
the	O
cell	O
surface	O
would	O
activate	O
the	O
receptor	O
.	O
	O
Close	O
-	O
up	O
views	O
of	O
(	O
A	O
)	O
IDA	O
,	O
(	O
B	O
)	O
the	O
N	O
-	O
terminally	O
extended	O
PKGV	B-mutant
-	I-mutant
IDA	I-mutant
and	O
(	O
C	O
)	O
IDL1	O
bound	O
to	O
the	O
HAESA	O
hormone	O
binding	O
pocket	O
(	O
in	O
bonds	O
representation	O
,	O
in	O
yellow	O
)	O
and	O
including	O
simulated	O
annealing	O
2Fo	O
–	O
Fc	O
omit	O
electron	O
density	O
maps	O
contoured	O
at	O
1	O
.	O
0	O
σ	O
.	O
	O
(	O
B	O
)	O
Analytical	O
size	O
-	O
exclusion	O
chromatography	O
.	O
	O
A	O
SDS	O
PAGE	O
of	O
the	O
peak	O
fractions	O
is	O
shown	O
alongside	O
.	O
	O
Purified	O
HAESA	O
and	O
SERK1	O
are	O
~	O
75	O
and	O
~	O
28	O
kDa	O
,	O
respectively	O
.	O
(	O
C	O
)	O
Isothermal	O
titration	O
calorimetry	O
of	O
wild	O
-	O
type	O
and	O
Hyp64	O
→	O
Pro	O
IDA	O
versus	O
the	O
HAESA	O
and	O
SERK1	O
ectodomains	O
.	O
	O
The	O
titration	O
of	O
IDA	O
wild	O
-	O
type	O
versus	O
the	O
isolated	O
HAESA	O
ectodomain	O
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
	O
We	O
purified	O
the	O
HAESA	O
ectodomain	O
(	O
residues	O
20	O
–	O
620	O
)	O
from	O
baculovirus	O
-	O
infected	O
insect	O
cells	O
(	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	O
with	O
synthetic	O
IDA	O
peptides	O
using	O
isothermal	O
titration	O
calorimetry	O
(	O
ITC	O
).	O
	O
IDA	O
binds	O
in	O
a	O
completely	O
extended	O
conformation	O
along	O
the	O
inner	O
surface	O
of	O
the	O
HAESA	O
ectodomain	O
,	O
covering	O
LRRs	O
2	O
–	O
14	O
(	O
Figure	O
1C	O
,	O
D	O
,	O
Figure	O
1	O
—	O
figure	O
supplement	O
2	O
).	O
	O
Other	O
hydrophobic	O
and	O
polar	O
interactions	O
are	O
mediated	O
by	O
Ser62IDA	O
,	O
Ser65IDA	O
and	O
by	O
backbone	O
atoms	O
along	O
the	O
IDA	O
peptide	O
(	O
Figure	O
1D	O
,	O
Figure	O
1	O
—	O
figure	O
supplement	O
2A	O
–	O
C	O
).	O
	O
Consistently	O
,	O
PKGV	B-mutant
-	I-mutant
IDA	I-mutant
and	O
IDA	O
have	O
similar	O
binding	O
affinities	O
in	O
our	O
ITC	O
assays	O
,	O
further	O
indicating	O
that	O
HAESA	O
senses	O
a	O
dodecamer	O
peptide	O
comprising	O
residues	O
58	O
-	O
69IDA	O
(	O
Figure	O
2D	O
).	O
	O
Replacing	O
Hyp64IDA	O
,	O
which	O
is	O
common	O
to	O
all	O
IDLs	O
,	O
with	O
proline	O
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	O
-	O
mutant	O
discussed	O
below	O
(	O
Figure	O
1A	O
,	O
2D	O
).	O
	O
Our	O
binding	O
assays	O
reveal	O
that	O
IDA	O
family	O
peptides	O
are	O
sensed	O
by	O
the	O
isolated	O
HAESA	O
ectodomain	O
with	O
relatively	O
weak	O
binding	O
affinities	O
(	O
Figures	O
1B	O
,	O
2A	O
–	O
D	O
).	O
	O
The	O
serk2	O
-	O
2	O
,	O
serk3	O
-	O
1	O
,	O
serk4	O
-	O
1	O
and	O
serk5	O
-	O
1	O
mutant	O
lines	O
showed	O
a	O
petal	O
break	O
-	O
strength	O
profile	O
not	O
significantly	O
different	O
from	O
wild	O
-	O
type	O
plants	O
.	O
	O
In	O
vitro	O
,	O
the	O
LRR	O
ectodomain	O
of	O
SERK1	O
(	O
residues	O
24	O
–	O
213	O
)	O
forms	O
stable	O
,	O
IDA	O
-	O
dependent	O
heterodimeric	O
complexes	O
with	O
HAESA	O
in	O
size	O
exclusion	O
chromatography	O
experiments	O
(	O
Figure	O
3B	O
).	O
	O
We	O
found	O
that	O
HAESA	O
senses	O
IDA	O
with	O
a	O
~	O
60	O
fold	O
higher	O
binding	O
affinity	O
in	O
the	O
presence	O
of	O
SERK1	O
,	O
suggesting	O
that	O
SERK1	O
is	O
involved	O
in	O
the	O
specific	O
recognition	O
of	O
the	O
peptide	O
hormone	O
(	O
Figure	O
3C	O
).	O
	O
Importantly	O
,	O
hydroxyprolination	O
of	O
IDA	O
is	O
critical	O
for	O
HAESA	O
-	O
IDA	O
-	O
SERK1	O
complex	O
formation	O
(	O
Figure	O
3C	O
,	O
D	O
).	O
	O
Upon	O
IDA	O
binding	O
at	O
the	O
cell	O
surface	O
,	O
the	O
kinase	O
domains	O
of	O
HAESA	O
and	O
SERK1	O
,	O
which	O
have	O
been	O
shown	O
to	O
be	O
active	O
protein	O
kinases	O
,	O
may	O
interact	O
in	O
the	O
cytoplasm	O
to	O
activate	O
each	O
other	O
.	O
	O
Crystal	O
structure	O
of	O
a	O
HAESA	O
–	O
IDA	O
–	O
SERK1	O
signaling	O
complex	O
.	O
	O
Polar	O
interactions	O
are	O
highlighted	O
as	O
dotted	O
lines	O
(	O
in	O
magenta	O
).	O
	O
(	O
A	O
)	O
Size	O
exclusion	O
chromatography	O
experiments	O
similar	O
to	O
Figure	O
3B	O
,	O
D	O
reveal	O
that	O
IDA	O
mutant	O
peptides	O
targeting	O
the	O
C	O
-	O
terminal	O
motif	O
do	O
not	O
form	O
biochemically	O
stable	O
HAESA	O
-	O
IDA	O
-	O
SERK1	O
complexes	O
.	O
	O
We	O
over	O
-	O
expressed	O
full	O
-	O
length	O
wild	O
-	O
type	O
IDA	O
or	O
this	O
Lys66IDA	B-mutant
/	I-mutant
Arg67IDA	I-mutant
→	I-mutant
Ala	I-mutant
double	O
-	O
mutant	O
to	O
similar	O
levels	O
in	O
Col	O
-	O
0	O
Arabidopsis	O
plants	O
(	O
Figure	O
5D	O
).	O
	O
We	O
found	O
that	O
over	O
-	O
expression	O
of	O
wild	O
-	O
type	O
IDA	O
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
	O
It	O
will	O
be	O
thus	O
interesting	O
to	O
see	O
if	O
proteolytic	O
processing	O
of	O
full	O
-	O
length	O
IDA	O
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
	O
This	O
observation	O
is	O
consistent	O
with	O
our	O
complex	O
structure	O
in	O
which	O
receptor	O
and	O
co	O
-	O
receptor	O
together	O
form	O
the	O
IDA	O
binding	O
pocket	O
.	O
	O
In	O
addition	O
,	O
residues	O
53	O
-	O
55SERK1	O
from	O
the	O
SERK1	O
N	O
-	O
terminal	O
cap	O
mediate	O
specific	O
interactions	O
with	O
the	O
IDA	O
peptide	O
(	O
Figures	O
4C	O
,	O
6B	O
).	O
	O
Different	O
plant	O
peptide	O
hormone	O
families	O
contain	O
a	O
C	O
-	O
terminal	O
(	O
Arg	O
)-	O
His	O
-	O
Asn	O
motif	O
,	O
which	O
in	O
IDA	O
represents	O
the	O
co	O
-	O
receptor	O
recognition	O
site	O
.	O
	O
Importantly	O
,	O
this	O
motif	O
can	O
also	O
be	O
found	O
in	O
other	O
peptide	O
hormone	O
families	O
(	O
Figure	O
7	O
).	O
	O
Using	O
electron	O
cryo	O
-	O
microscopy	O
of	O
a	O
single	O
specimen	O
,	O
we	O
present	O
five	O
ribosome	O
structures	O
formed	O
with	O
the	O
Taura	O
syndrome	O
virus	O
IRES	O
and	O
translocase	O
eEF2	O
•	O
GTP	O
bound	O
with	O
sordarin	O
.	O
	O
The	O
structures	O
suggest	O
missing	O
links	O
in	O
our	O
understanding	O
of	O
tRNA	O
translocation	O
.	O
	O
To	O
this	O
end	O
,	O
internal	O
ribosome	O
entry	O
site	O
(	O
IRES	O
)	O
RNAs	O
are	O
employed	O
(	O
reviewed	O
in	O
.	O
	O
An	O
unusual	O
strategy	O
of	O
initiation	O
is	O
used	O
by	O
intergenic	O
-	O
region	O
(	O
IGR	O
)	O
IRESs	O
found	O
in	O
Dicistroviridae	O
arthropod	O
-	O
infecting	O
viruses	O
.	O
	O
These	O
include	O
shrimp	O
-	O
infecting	O
Taura	O
syndrome	O
virus	O
(	O
TSV	O
),	O
and	O
insect	O
viruses	O
Plautia	O
stali	O
intestine	O
virus	O
(	O
PSIV	O
)	O
and	O
Cricket	O
paralysis	O
virus	O
(	O
CrPV	O
).	O
	O
A	O
recent	O
demonstration	O
of	O
bacterial	O
translation	O
initiation	O
by	O
an	O
IGR	O
IRES	O
indicates	O
that	O
the	O
IRESs	O
take	O
advantage	O
of	O
conserved	O
structural	O
and	O
dynamic	O
properties	O
of	O
the	O
ribosome	O
.	O
	O
For	O
a	O
cognate	O
aminoacyl	O
-	O
tRNA	O
to	O
bind	O
the	O
first	O
viral	O
mRNA	O
codon	O
,	O
PKI	O
has	O
to	O
be	O
translocated	O
from	O
the	O
A	O
site	O
,	O
so	O
that	O
the	O
first	O
codon	O
can	O
be	O
presented	O
in	O
the	O
A	O
site	O
.	O
	O
First	O
,	O
studies	O
of	O
bacterial	O
ribosomes	O
showed	O
that	O
a	O
~	O
10	O
°	O
rotation	O
of	O
the	O
small	O
subunit	O
relative	O
to	O
the	O
large	O
subunit	O
,	O
known	O
as	O
intersubunit	O
rotation	O
,	O
or	O
ratcheting	O
,	O
is	O
required	O
for	O
translocation	O
.	O
	O
Schematic	O
of	O
cryo	O
-	O
EM	O
refinement	O
and	O
classification	O
procedures	O
.	O
	O
All	O
particles	O
were	O
initially	O
aligned	O
to	O
a	O
single	O
model	O
.	O
	O
All	O
measurements	O
are	O
relative	O
to	O
the	O
non	O
-	O
rotated	O
80S	O
•	O
2tRNA	O
•	O
mRNA	O
structure	O
.	O
	O
This	O
approach	O
revealed	O
five	O
80S	O
•	O
IRES	O
•	O
eEF2	O
•	O
GDP	O
structures	O
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	O
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	O
and	O
eEF2	O
(	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	O
699	O
(	O
Figure	O
3c	O
).	O
	O
The	O
views	O
were	O
obtained	O
by	O
structural	O
alignment	O
of	O
the	O
25S	O
rRNAs	O
;	O
the	O
sarcin	O
-	O
ricin	O
loop	O
(	O
SRL	O
)	O
of	O
25S	O
rRNA	O
is	O
shown	O
in	O
gray	O
for	O
reference	O
.	O
	O
(	O
c	O
)	O
Comparison	O
of	O
the	O
40S	O
conformations	O
in	O
Structures	O
I	O
through	O
V	O
shows	O
distinct	O
positions	O
of	O
the	O
head	O
relative	O
to	O
the	O
body	O
of	O
the	O
40S	O
subunit	O
(	O
head	O
swivel	O
).	O
	O
Conformation	O
of	O
the	O
non	O
-	O
swiveled	O
40S	O
subunit	O
in	O
the	O
S	O
.	O
cerevisiae	O
80S	O
ribosome	O
bound	O
with	O
two	O
tRNAs	O
is	O
shown	O
for	O
reference	O
(	O
blue	O
).	O
	O
The	O
central	O
protuberance	O
(	O
CP	O
)	O
is	O
labeled	O
.	O
	O
Structures	O
II	O
and	O
III	O
are	O
in	O
mid	O
-	O
rotation	O
conformations	O
(~	O
5	O
°).	O
	O
IRES	O
rearrangements	O
	O
Position	O
and	O
interactions	O
of	O
loop	O
3	O
(	O
variable	O
loop	O
region	O
)	O
of	O
the	O
PKI	O
domain	O
in	O
Structure	O
V	O
(	O
this	O
work	O
)	O
resembles	O
those	O
of	O
the	O
anticodon	O
stem	O
loop	O
of	O
the	O
E	O
-	O
site	O
tRNA	O
(	O
blue	O
)	O
in	O
the	O
80S	O
•	O
2tRNA	O
•	O
mRNA	O
complex	O
.	O
	O
Structures	O
of	O
translocation	O
complexes	O
of	O
the	O
bacterial	O
70S	O
ribosome	O
bound	O
with	O
two	O
tRNAs	O
and	O
yeast	O
80S	O
complexes	O
with	O
tRNAs	O
are	O
shown	O
in	O
the	O
upper	O
row	O
and	O
labeled	O
.	O
	O
Positions	O
of	O
the	O
IRES	O
relative	O
to	O
eEF2	O
and	O
elements	O
of	O
the	O
ribosome	O
in	O
Structures	O
I	O
through	O
V	O
.	O
	O
The	O
minor	O
groove	O
of	O
SL5	O
(	O
at	O
nt	O
6862	O
–	O
6868	O
)	O
contacts	O
the	O
positively	O
charged	O
region	O
of	O
eS25	O
(	O
R49	O
,	O
R58	O
and	O
R68	O
)	O
(	O
Figure	O
3	O
—	O
figure	O
supplement	O
4	O
).	O
	O
The	O
view	O
was	O
obtained	O
by	O
structural	O
alignment	O
of	O
the	O
body	O
domains	O
of	O
18S	O
rRNAs	O
of	O
the	O
corresponding	O
80S	O
structures	O
.	O
	O
Rearrangements	O
of	O
the	O
IRES	O
involve	O
restructuring	O
of	O
several	O
interactions	O
with	O
the	O
ribosome	O
.	O
	O
In	O
Structure	O
I	O
,	O
SL3	O
of	O
the	O
PKI	O
domain	O
is	O
positioned	O
between	O
the	O
A	O
-	O
site	O
finger	O
(	O
nt	O
1008	O
–	O
1043	O
of	O
25S	O
rRNA	O
)	O
and	O
the	O
P	O
site	O
of	O
the	O
60S	O
subunit	O
,	O
comprising	O
helix	O
84	O
of	O
25S	O
rRNA	O
(	O
nt	O
.	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	O
site	O
region	O
of	O
the	O
large	O
subunit	O
with	O
the	O
hinge	O
point	O
of	O
the	O
IRES	O
bending	O
between	O
the	O
5	O
´	O
domain	O
and	O
the	O
PKI	O
domain	O
(	O
nt	O
.	O
6886	O
–	O
6890	O
).	O
	O
The	O
view	O
and	O
colors	O
are	O
as	O
in	O
Figure	O
5b	O
:	O
eEF2	O
is	O
shown	O
in	O
green	O
,	O
IRES	O
RNA	O
in	O
red	O
,	O
40S	O
subunit	O
elements	O
in	O
orange	O
,	O
60S	O
in	O
cyan	O
/	O
teal	O
.	O
	O
Cryo	O
-	O
EM	O
density	O
of	O
the	O
GTPase	O
region	O
in	O
Structures	O
I	O
and	O
II	O
.	O
	O
Repositioning	O
(	O
sliding	O
)	O
of	O
the	O
positively	O
-	O
charged	O
cluster	O
of	O
domain	O
IV	O
of	O
eEF2	O
over	O
the	O
phosphate	O
backbone	O
(	O
red	O
)	O
of	O
the	O
18S	O
helices	O
33	O
and	O
34	O
.	O
	O
Interactions	O
of	O
eEF2	O
with	O
the	O
40S	O
subunit	O
.	O
	O
From	O
Structure	O
I	O
to	O
V	O
,	O
these	O
central	O
domains	O
migrate	O
by	O
~	O
10	O
Å	O
along	O
the	O
40S	O
surface	O
(	O
Figure	O
6c	O
).	O
	O
At	O
the	O
head	O
,	O
C1274	O
of	O
the	O
18S	O
rRNA	O
(	O
C1054	O
in	O
E	O
.	O
coli	O
)	O
base	O
pairs	O
with	O
the	O
first	O
nucleotide	O
of	O
the	O
ORF	O
immediately	O
downstream	O
of	O
PKI	O
.	O
	O
The	O
codon	O
-	O
anticodon	O
-	O
like	O
helix	O
of	O
PKI	O
is	O
shown	O
in	O
red	O
,	O
the	O
downstream	O
first	O
codon	O
of	O
the	O
ORF	O
in	O
magenta	O
.	O
	O
Histidines	O
583	O
and	O
694	O
interact	O
with	O
the	O
phosphate	O
backbone	O
of	O
the	O
anticodon	O
-	O
like	O
strand	O
(	O
at	O
G6907	O
and	O
C6908	O
).	O
	O
Thus	O
,	O
in	O
comparison	O
with	O
the	O
initiation	O
state	O
,	O
the	O
histidine	O
-	O
diphthamide	O
tip	O
of	O
eEF2	O
replaces	O
the	O
codon	O
-	O
anticodon	O
–	O
like	O
helix	O
of	O
PKI	O
.	O
	O
The	O
histidine	O
resides	O
next	O
to	O
the	O
backbone	O
of	O
G3028	O
of	O
the	O
sarcin	O
-	O
ricin	O
loop	O
and	O
near	O
the	O
diphosphate	O
of	O
GDP	O
(	O
Figure	O
5e	O
).	O
	O
By	O
contrast	O
,	O
switch	O
loop	O
I	O
(	O
aa	O
50	O
–	O
70	O
in	O
S	O
.	O
cerevisiae	O
eEF2	O
)	O
is	O
resolved	O
only	O
in	O
Structure	O
I	O
(	O
Figure	O
5	O
—	O
figure	O
supplement	O
2	O
).	O
	O
As	O
such	O
,	O
the	O
conformations	O
of	O
SWI	O
and	O
the	O
GTPase	O
center	O
in	O
general	O
are	O
similar	O
to	O
those	O
observed	O
in	O
ribosome	O
-	O
bound	O
EF	O
-	O
Tu	O
and	O
EF	O
-	O
G	O
in	O
the	O
presence	O
of	O
GTP	O
analogs	O
.	O
	O
Structure	O
II	O
reveals	O
PKI	O
between	O
the	O
body	O
A	O
and	O
P	O
sites	O
and	O
eEF2	O
partially	O
advanced	O
into	O
the	O
A	O
site	O
	O
Domain	O
IV	O
of	O
eEF2	O
is	O
further	O
entrenched	O
in	O
the	O
A	O
site	O
by	O
~	O
3	O
Å	O
relative	O
to	O
the	O
body	O
and	O
~	O
8	O
Å	O
relative	O
to	O
the	O
head	O
,	O
preserving	O
its	O
interactions	O
with	O
PKI	O
.	O
	O
In	O
Structure	O
IV	O
,	O
the	O
40S	O
subunit	O
is	O
almost	O
non	O
-	O
rotated	O
relative	O
to	O
the	O
60S	O
subunit	O
,	O
and	O
the	O
40S	O
head	O
is	O
mid	O
-	O
swiveled	O
.	O
	O
In	O
Structure	O
V	O
,	O
protein	O
uS12	O
is	O
shifted	O
along	O
with	O
the	O
40S	O
body	O
as	O
a	O
result	O
of	O
intersubunit	O
rotation	O
.	O
	O
This	O
shifts	O
the	O
tip	O
of	O
helix	O
A	O
of	O
domain	O
III	O
(	O
at	O
aa	O
500	O
)	O
by	O
~	O
5	O
Å	O
(	O
relative	O
to	O
that	O
in	O
Structure	O
I	O
)	O
toward	O
domain	O
I	O
.	O
Although	O
domain	O
III	O
remains	O
in	O
contact	O
with	O
domain	O
V	O
,	O
the	O
shift	O
occurs	O
in	O
the	O
direction	O
that	O
could	O
eventually	O
disconnect	O
the	O
β	O
-	O
platforms	O
of	O
these	O
domains	O
.	O
	O
The	O
imidazole	O
moiety	O
stacks	O
on	O
G6907	O
(	O
corresponding	O
to	O
nt	O
36	O
in	O
the	O
tRNA	O
anticodon	O
)	O
and	O
hydrogen	O
bonds	O
with	O
O2	O
’	O
of	O
G6906	O
(	O
nt	O
35	O
of	O
tRNA	O
).	O
	O
The	O
front	O
'	O
legs	O
'	O
(	O
SL4	O
and	O
SL5	O
)	O
of	O
the	O
5	O
’-	O
domain	O
(	O
front	O
end	O
)	O
are	O
attached	O
to	O
the	O
40S	O
head	O
proteins	O
uS7	O
,	O
uS11	O
and	O
eS25	O
(	O
Figure	O
3	O
—	O
figure	O
supplement	O
2	O
).	O
	O
Notably	O
,	O
at	O
all	O
steps	O
,	O
the	O
head	O
of	O
the	O
IRES	O
inchworm	O
(	O
L1	O
.	O
1	O
region	O
)	O
is	O
supported	O
by	O
the	O
mobile	O
L1	O
stalk	O
.	O
	O
Partitioned	O
roles	O
of	O
40S	O
subunit	O
rearrangements	O
	O
Specifically	O
,	O
intersubunit	O
rotation	O
allows	O
eEF2	O
entry	O
into	O
the	O
A	O
site	O
,	O
while	O
the	O
head	O
swivel	O
mediates	O
PKI	O
translocation	O
.	O
	O
Because	O
the	O
histidine	O
-	O
diphthamide	O
tip	O
of	O
eEF2	O
(	O
H583	O
,	O
H694	O
and	O
Diph699	O
)	O
attaches	O
to	O
the	O
codon	O
-	O
anticodon	O
-	O
like	O
helix	O
of	O
PKI	O
,	O
eEF2	O
appears	O
to	O
directly	O
force	O
PKI	O
out	O
of	O
the	O
A	O
site	O
.	O
	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	O
translocase	O
that	O
is	O
inferred	O
from	O
an	O
ensemble	O
of	O
structures	O
sampled	O
under	O
uniform	O
conditions	O
.	O
	O
While	O
the	O
ribosome	O
itself	O
has	O
the	O
capacity	O
to	O
translocate	O
in	O
the	O
absence	O
of	O
the	O
translocase	O
,	O
spontaneous	O
translocation	O
is	O
slow	O
.	O
	O
The	O
80S	O
•	O
IRES	O
•	O
eEF2	O
structures	O
reported	O
here	O
suggest	O
two	O
main	O
roles	O
for	O
eEF2	O
in	O
translocation	O
.	O
	O
In	O
our	O
structures	O
,	O
the	O
tip	O
of	O
domain	O
IV	O
docks	O
next	O
to	O
PKI	O
,	O
with	O
diphthamide	O
699	O
fit	O
into	O
the	O
minor	O
groove	O
of	O
the	O
codon	O
-	O
anticodon	O
-	O
like	O
helix	O
of	O
PKI	O
(	O
Figure	O
7	O
).	O
	O
This	O
arrangement	O
rationalizes	O
inactivation	O
of	O
eEF2	O
by	O
diphtheria	O
toxin	O
,	O
which	O
catalyzes	O
ADP	O
-	O
ribosylation	O
of	O
the	O
diphthamide	O
(	O
reviewed	O
in	O
).	O
	O
The	O
enzyme	O
ADP	O
-	O
ribosylates	O
the	O
NE2	O
atom	O
of	O
the	O
imidazole	O
ring	O
,	O
which	O
in	O
our	O
structures	O
interacts	O
with	O
the	O
first	O
two	O
residues	O
of	O
the	O
anticodon	O
-	O
like	O
strand	O
of	O
PKI	O
.	O
	O
However	O
,	O
the	O
structural	O
and	O
mechanistic	O
definitions	O
of	O
the	O
locked	O
and	O
unlocked	O
states	O
have	O
remained	O
unclear	O
,	O
ranging	O
from	O
the	O
globally	O
distinct	O
ribosome	O
conformations	O
to	O
unknown	O
local	O
rearrangements	O
,	O
e	O
.	O
g	O
.	O
those	O
in	O
the	O
decoding	O
center	O
.	O
	O
FRET	O
data	O
indicate	O
that	O
translocation	O
of	O
2tRNA	O
•	O
mRNA	O
on	O
the	O
70S	O
ribosome	O
requires	O
a	O
forward	O
-	O
and	O
-	O
reverse	O
head	O
swivel	O
,	O
which	O
may	O
be	O
related	O
to	O
the	O
unlocking	O
phenomenon	O
.	O
	O
Mutations	O
of	O
residues	O
flanking	O
A344	O
in	O
E	O
.	O
coli	O
16S	O
rRNA	O
modestly	O
inhibit	O
translation	O
but	O
do	O
not	O
specifically	O
affect	O
EF	O
-	O
G	O
-	O
mediated	O
translocation	O
.	O
	O
However	O
,	O
the	O
effect	O
of	O
A344	O
mutation	O
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	O
/	O
EF	O
-	O
G	O
function	O
.	O
	O
The	O
interaction	O
between	O
h14	O
and	O
switch	O
loop	O
I	O
is	O
not	O
resolved	O
in	O
Structures	O
II	O
to	O
V	O
,	O
in	O
all	O
of	O
which	O
the	O
small	O
subunit	O
is	O
partially	O
rotated	O
or	O
non	O
-	O
rotated	O
,	O
so	O
that	O
helix	O
14	O
is	O
placed	O
at	O
least	O
6	O
Å	O
farther	O
from	O
eEF2	O
(	O
Figure	O
5d	O
).	O
	O
We	O
conclude	O
that	O
unlike	O
other	O
conformations	O
of	O
the	O
ribosome	O
,	O
the	O
fully	O
rotated	O
40S	O
subunit	O
of	O
the	O
pre	O
-	O
translocation	O
ribosome	O
provides	O
an	O
interaction	O
surface	O
,	O
complementing	O
the	O
P	O
stalk	O
and	O
SRL	O
,	O
for	O
binding	O
of	O
the	O
GTP	O
-	O
bound	O
translocase	O
.	O
	O
This	O
displacement	O
is	O
caused	O
by	O
the	O
8	O
Å	O
movement	O
of	O
the	O
40S	O
body	O
protein	O
uS12	O
upon	O
reverse	O
intersubunit	O
rotation	O
from	O
Structure	O
I	O
to	O
V	O
(	O
Figure	O
6d	O
).	O
	O
As	O
we	O
discuss	O
below	O
,	O
Structure	O
V	O
captures	O
a	O
'	O
pre	O
-	O
unstacking	O
'	O
state	O
due	O
to	O
stabilization	O
of	O
the	O
interface	O
between	O
domains	O
III	O
and	O
V	O
by	O
sordarin	O
.	O
	O
Intersubunit	O
rearrangements	O
and	O
tRNA	O
hybrid	O
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
	O
Despite	O
an	O
impressive	O
body	O
of	O
biochemical	O
,	O
fluorescence	O
and	O
structural	O
data	O
accumulated	O
since	O
then	O
,	O
translocation	O
remains	O
the	O
least	O
understood	O
step	O
of	O
elongation	O
.	O
	O
Furthermore	O
,	O
the	O
step	O
-	O
wise	O
coupling	O
of	O
ribosome	O
dynamics	O
with	O
IRES	O
translocation	O
is	O
overall	O
consistent	O
with	O
that	O
observed	O
for	O
2tRNA	O
•	O
mRNA	O
translocation	O
in	O
solution	O
.	O
	O
We	O
deem	O
the	O
pre	O
-	O
translocation	O
complex	O
locked	O
,	O
because	O
the	O
A	O
-	O
site	O
bound	O
ASL	O
-	O
mRNA	O
is	O
stabilized	O
by	O
interactions	O
with	O
the	O
decoding	O
center	O
.	O
	O
This	O
unlatches	O
the	O
head	O
,	O
allowing	O
creation	O
of	O
hitherto	O
elusive	O
intermediate	O
tRNA	O
positions	O
during	O
spontaneous	O
reverse	O
body	O
rotation	O
.	O
	O
Finally	O
,	O
the	O
similar	O
populations	O
of	O
particles	O
(	O
within	O
a	O
2X	O
range	O
)	O
in	O
our	O
80S	O
•	O
IRES	O
•	O
eEF2	O
reconstructions	O
(	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
	O
The	O
cryo	O
-	O
EM	O
structures	O
demonstrate	O
that	O
the	O
TSV	O
IRES	O
structurally	O
and	O
dynamically	O
represents	O
a	O
chimera	O
of	O
the	O
2tRNA	O
•	O
mRNA	O
translocating	O
complex	O
(	O
A	O
/	O
P	O
-	O
tRNA	O
•	O
P	O
/	O
E	O
-	O
tRNA	O
•	O
mRNA	O
).	O
	O
Intergenic	O
IRESs	O
,	O
in	O
turn	O
,	O
represent	O
a	O
striking	O
example	O
of	O
convergent	O
molecular	O
evolution	O
.	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	O
autolysis	O
,	O
as	O
well	O
as	O
the	O
evolutionary	O
pressures	O
that	O
drove	O
the	O
proteasome	O
to	O
become	O
a	O
threonine	O
protease	O
.	O
	O
Data	O
from	O
biochemical	O
and	O
structural	O
analyses	O
of	O
proteasome	O
variants	O
with	O
mutations	O
in	O
the	O
β5	O
propeptide	O
and	O
the	O
active	O
site	O
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	O
activation	O
of	O
the	O
proteasome	O
.	O
	O
Proteasome	O
-	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	O
cells	O
.	O
	O
These	O
results	O
indicate	O
that	O
the	O
β1	O
and	O
β2	O
proteolytic	O
activities	O
are	O
not	O
essential	O
for	O
cell	O
survival	O
.	O
	O
Our	O
present	O
crystallographic	O
analysis	O
of	O
the	O
β5	B-mutant
-	I-mutant
T1A	I-mutant
pp	O
trans	O
mutant	O
demonstrates	O
that	O
the	O
mutation	O
per	O
se	O
does	O
not	O
structurally	O
alter	O
the	O
catalytic	O
active	O
site	O
and	O
that	O
the	O
trans	O
-	O
expressed	O
β5	O
propeptide	O
is	O
not	O
bound	O
in	O
the	O
β5	O
substrate	O
-	O
binding	O
channel	O
(	O
Supplementary	O
Fig	O
.	O
1a	O
).	O
	O
Thr	O
(-	O
2	O
)	O
positions	O
Gly	O
(-	O
1	O
)	O
O	O
via	O
hydrogen	O
bonding	O
(∼	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	O
(	O
Fig	O
.	O
1b	O
and	O
Supplementary	O
Fig	O
.	O
2b	O
).	O
	O
As	O
histidine	O
commonly	O
functions	O
as	O
a	O
proton	O
shuttle	O
in	O
the	O
catalytic	O
triads	O
of	O
serine	O
proteases	O
,	O
we	O
investigated	O
the	O
role	O
of	O
His	O
(-	O
2	O
)	O
in	O
processing	O
of	O
the	O
β5	O
propeptide	O
by	O
exchanging	O
it	O
for	O
Asn	O
,	O
Lys	O
,	O
Phe	O
and	O
Ala	O
.	O
All	O
yeast	O
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
	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	O
yCPs	O
was	O
impaired	O
in	O
situ	O
and	O
in	O
vitro	O
(	O
Supplementary	O
Fig	O
.	O
3c	O
).	O
	O
Next	O
,	O
we	O
examined	O
the	O
effect	O
of	O
residue	O
(-	O
2	O
)	O
on	O
the	O
orientation	O
of	O
the	O
propeptide	O
by	O
creating	O
mutants	O
that	O
combine	O
the	O
T1A	B-mutant
(	O
K81R	B-mutant
)	O
mutation	O
(	O
s	O
)	O
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	O
.	O
	O
Notably	O
,	O
the	O
2FO	O
–	O
FC	O
electron	O
-	O
density	O
map	O
displays	O
a	O
different	O
orientation	O
for	O
the	O
β2	O
propeptide	O
than	O
has	O
been	O
observed	O
for	O
the	O
β2	B-mutant
-	I-mutant
T1A	I-mutant
proteasome	O
.	O
	O
The	O
phenotype	O
of	O
the	O
β5	B-mutant
-	I-mutant
K33A	I-mutant
mutant	O
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	O
(	O
Fig	O
.	O
4a	O
).	O
	O
Structural	O
comparison	O
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	O
sites	O
revealed	O
that	O
mutation	O
of	O
Lys33	O
to	O
Ala	O
creates	O
a	O
cavity	O
that	O
is	O
filled	O
with	O
Thr1	O
and	O
the	O
remnant	O
propeptide	O
.	O
	O
In	O
contrast	O
to	O
the	O
cis	O
-	O
construct	O
,	O
expression	O
of	O
the	O
β5	O
propeptide	O
in	O
trans	O
allowed	O
straightforward	O
isolation	O
and	O
crystallization	O
of	O
the	O
D17N	B-mutant
mutant	O
proteasome	O
.	O
	O
This	O
observation	O
is	O
consistent	O
with	O
a	O
strongly	O
reduced	O
reactivity	O
of	O
β5	O
-	O
Thr1	O
and	O
the	O
crystal	O
structure	O
of	O
the	O
β5	B-mutant
-	I-mutant
D17N	I-mutant
pp	O
cis	O
mutant	O
in	O
complex	O
with	O
carfilzomib	O
.	O
	O
Asp166Oδ	O
is	O
hydrogen	O
-	O
bonded	O
to	O
Thr1NH2	O
via	O
Ser129OH	O
and	O
Ser169OH	O
,	O
and	O
therefore	O
was	O
proposed	O
to	O
be	O
involved	O
in	O
catalysis	O
.	O
	O
Instead	O
,	O
a	O
water	O
molecule	O
is	O
bound	O
to	O
Ser129OH	O
and	O
Thr1NH2	O
(	O
Supplementary	O
Fig	O
.	O
8b	O
),	O
which	O
may	O
enable	O
precursor	O
processing	O
.	O
	O
In	O
the	O
carfilzomib	O
complex	O
structure	O
,	O
Thr1Oγ	O
and	O
Thr1N	O
incorporate	O
into	O
a	O
morpholine	O
ring	O
structure	O
and	O
Ser129	O
adopts	O
its	O
WT	O
-	O
like	O
orientation	O
.	O
	O
Whereas	O
Asn	O
can	O
to	O
some	O
degree	O
replace	O
Asp166	O
due	O
to	O
its	O
carbonyl	O
group	O
in	O
the	O
side	O
chain	O
,	O
Ala	O
at	O
this	O
position	O
was	O
found	O
to	O
prevent	O
both	O
autolysis	O
and	O
catalysis	O
.	O
	O
These	O
results	O
suggest	O
that	O
Asp166	O
and	O
Ser129	O
function	O
as	O
a	O
proton	O
shuttle	O
and	O
affect	O
the	O
protonation	O
state	O
of	O
Thr1N	O
during	O
autolysis	O
and	O
catalysis	O
.	O
	O
Owing	O
to	O
the	O
unequal	O
positions	O
of	O
the	O
two	O
β5	O
subunits	O
within	O
the	O
CP	O
in	O
the	O
crystal	O
lattice	O
,	O
maturation	O
and	O
propeptide	O
displacement	O
may	O
occur	O
at	O
different	O
timescales	O
in	O
the	O
two	O
subunits	O
.	O
	O
In	O
agreement	O
,	O
soaking	O
crystals	O
with	O
the	O
CP	O
inhibitors	O
bortezomib	O
or	O
carfilzomib	O
modifies	O
only	O
the	O
β1	O
and	O
β2	O
active	O
sites	O
,	O
while	O
leaving	O
the	O
β5	B-mutant
-	I-mutant
T1C	I-mutant
proteolytic	O
centres	O
unmodified	O
even	O
though	O
they	O
are	O
only	O
partially	O
occupied	O
by	O
the	O
cleaved	O
propeptide	O
remnant	O
.	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	O
is	O
unable	O
to	O
grow	O
(	O
Fig	O
.	O
4a	O
).	O
	O
Hence	O
,	O
the	O
mean	O
residence	O
time	O
of	O
carfilzomib	O
at	O
the	O
active	O
site	O
is	O
prolonged	O
and	O
the	O
probability	O
to	O
covalently	O
react	O
with	O
Ser1	O
is	O
increased	O
.	O
	O
The	O
20S	O
proteasome	O
CP	O
is	O
the	O
major	O
non	O
-	O
lysosomal	O
protease	O
in	O
eukaryotic	O
cells	O
,	O
and	O
its	O
assembly	O
is	O
highly	O
organized	O
.	O
	O
Depending	O
on	O
the	O
(-	O
2	O
)	O
residue	O
we	O
observed	O
various	O
propeptide	O
conformations	O
,	O
but	O
Gly	O
(-	O
1	O
)	O
is	O
in	O
all	O
structures	O
perfectly	O
located	O
for	O
the	O
nucleophilic	O
attack	O
by	O
Thr1Oγ	O
,	O
although	O
it	O
does	O
not	O
adopt	O
the	O
tight	O
turn	O
observed	O
for	O
the	O
prosegment	O
of	O
subunit	O
β1	O
.	O
	O
Lys33NH2	O
is	O
expected	O
to	O
act	O
as	O
the	O
proton	O
acceptor	O
during	O
autocatalytic	O
removal	O
of	O
the	O
propeptides	O
,	O
as	O
well	O
as	O
during	O
substrate	O
proteolysis	O
,	O
while	O
Asp17Oδ	O
orients	O
Lys33NH2	O
and	O
makes	O
it	O
more	O
prone	O
to	O
protonation	O
by	O
raising	O
its	O
pKa	O
(	O
hydrogen	O
bond	O
distance	O
:	O
Lys33NH3	O
+–	O
Asp17Oδ	O
:	O
2	O
.	O
9	O
Å	O
).	O
	O
Analogously	O
to	O
the	O
proteasome	O
,	O
a	O
Thr	O
–	O
Lys	O
–	O
Asp	O
triad	O
is	O
also	O
found	O
in	O
L	O
-	O
asparaginase	O
.	O
	O
In	O
this	O
new	O
view	O
of	O
the	O
proteasomal	O
active	O
site	O
,	O
the	O
positively	O
charged	O
Thr1NH3	O
+-	O
terminus	O
hydrogen	O
bonds	O
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	O
cleavage	O
by	O
Thr1Oγ	O
(	O
Fig	O
.	O
3d	O
).	O
	O
Breakdown	O
of	O
this	O
tetrahedral	O
transition	O
state	O
releases	O
the	O
Thr1	O
N	O
terminus	O
that	O
is	O
protonated	O
by	O
aspartic	O
acid	O
166	O
via	O
Ser129OH	O
to	O
yield	O
Thr1NH3	O
+.	O
	O
This	O
interpretation	O
agrees	O
with	O
the	O
strongly	O
reduced	O
catalytic	O
activity	O
of	O
the	O
β5	B-mutant
-	I-mutant
D166N	I-mutant
mutant	O
on	O
the	O
one	O
hand	O
,	O
and	O
the	O
ability	O
to	O
react	O
readily	O
with	O
carfilzomib	O
on	O
the	O
other	O
.	O
	O
In	O
accord	O
with	O
the	O
proposed	O
Thr1	O
–	O
Lys33	O
–	O
Asp17	O
catalytic	O
triad	O
,	O
crystallographic	O
data	O
on	O
the	O
proteolytically	O
inactive	O
β5	B-mutant
-	I-mutant
T1C	I-mutant
mutant	O
demonstrate	O
that	O
the	O
interaction	O
of	O
Lys33NH2	O
and	O
Cys1	O
is	O
broken	O
.	O
	O
Thr1	O
is	O
well	O
anchored	O
in	O
the	O
active	O
site	O
by	O
hydrophobic	O
interactions	O
of	O
its	O
Cγ	O
methyl	O
group	O
with	O
Ala46	O
(	O
Cβ	O
),	O
Lys33	O
(	O
carbon	O
side	O
chain	O
)	O
and	O
Thr3	O
(	O
Cγ	O
).	O
	O
Notably	O
,	O
in	O
the	O
threonine	O
aspartase	O
Taspase1	O
,	O
mutation	O
of	O
the	O
active	O
-	O
site	O
Thr234	O
to	O
Ser	O
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	O
in	O
the	O
WT	O
,	O
thereby	O
reducing	O
catalytic	O
activity	O
.	O
	O
(	O
b	O
)	O
Structural	O
superposition	O
of	O
the	O
β5	O
propeptides	O
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	O
proteasomes	O
.	O
	O
While	O
the	O
residues	O
(-	O
2	O
)	O
to	O
(-	O
4	O
)	O
vary	O
in	O
their	O
conformation	O
,	O
Gly	O
(-	O
1	O
)	O
and	O
Ala1	O
are	O
located	O
in	O
all	O
structures	O
at	O
the	O
same	O
positions	O
.	O
	O
The	O
strictly	O
conserved	O
oxyanion	O
hole	O
Gly47NH	O
stabilizing	O
the	O
negatively	O
charged	O
intermediate	O
is	O
illustrated	O
as	O
a	O
semicircle	O
.	O
	O
Next	O
,	O
Thr1NH2	O
polarizes	O
a	O
water	O
molecule	O
for	O
the	O
nucleophilic	O
attack	O
of	O
the	O
acyl	O
-	O
enzyme	O
intermediate	O
.	O
	O
On	O
hydrolysis	O
of	O
the	O
latter	O
,	O
the	O
active	O
-	O
site	O
Thr1	O
is	O
ready	O
for	O
catalysis	O
(	O
right	O
set	O
of	O
structures	O
).	O
	O
The	O
proteasome	O
favours	O
threonine	O
as	O
the	O
active	O
-	O
site	O
nucleophile	O
.	O
	O
(	O
a	O
)	O
Growth	O
tests	O
by	O
serial	O
dilution	O
of	O
WT	O
and	O
pre2	O
(	O
β5	O
)	O
mutant	O
yeast	O
cultures	O
reveal	O
growth	O
defects	O
of	O
the	O
active	O
-	O
site	O
mutants	O
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
(	O
c	O
)	O
Illustration	O
of	O
the	O
2FO	O
–	O
FC	O
electron	O
-	O
density	O
map	O
(	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	O
fragment	O
.	O
	O
Ser1	O
lacks	O
this	O
stabilization	O
and	O
is	O
therefore	O
rotated	O
by	O
60	O
°.	O
	O
Inhibition	O
assays	O
(	O
left	O
panel	O
).	O
	O
p300	O
-	O
catalyzed	O
histone	O
crotonylation	O
,	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	O
-	O
catalyzed	O
acetylation	O
.	O
	O
The	O
discovery	O
of	O
individual	O
biological	O
roles	O
for	O
the	O
crotonyllysine	O
and	O
acetyllysine	O
marks	O
suggests	O
that	O
these	O
PTMs	O
can	O
be	O
read	O
by	O
distinct	O
readers	O
.	O
	O
However	O
,	O
Taf14	O
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	O
,	O
SWI	O
/	O
SNF	O
and	O
RSC	O
)	O
and	O
the	O
histone	O
acetyltransferase	O
complex	O
NuA3	O
,	O
indicating	O
a	O
multifaceted	O
role	O
of	O
Taf14	O
in	O
transcriptional	O
regulation	O
and	O
chromatin	O
biology	O
.	O
	O
In	O
this	O
study	O
,	O
we	O
identified	O
the	O
Taf14	O
YEATS	O
domain	O
as	O
a	O
reader	O
of	O
crotonyllysine	O
that	O
binds	O
to	O
histone	O
H3	O
crotonylated	O
at	O
lysine	O
9	O
(	O
H3K9cr	O
)	O
via	O
a	O
distinctive	O
binding	O
mechanism	O
.	O
	O
This	O
distinctive	O
mechanism	O
was	O
corroborated	O
through	O
mapping	O
the	O
Taf14	O
YEATS	O
-	O
H3K9cr	O
binding	O
interface	O
in	O
solution	O
using	O
NMR	O
chemical	O
shift	O
perturbation	O
analysis	O
(	O
Supplementary	O
Fig	O
.	O
2a	O
,	O
b	O
).	O
	O
To	O
determine	O
whether	O
H3K9cr	O
is	O
present	O
in	O
yeast	O
,	O
we	O
generated	O
whole	O
cell	O
extracts	O
from	O
logarithmically	O
growing	O
yeast	O
cells	O
and	O
subjected	O
them	O
to	O
Western	O
blot	O
analysis	O
using	O
antibodies	O
directed	O
towards	O
H3K9cr	O
,	O
H3K9ac	O
and	O
H3	O
(	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
	O
We	O
next	O
asked	O
if	O
H3K9cr	O
is	O
regulated	O
by	O
the	O
actions	O
of	O
histone	O
acetyltransferases	O
(	O
HATs	O
)	O
and	O
histone	O
deacetylases	O
(	O
HDACs	O
).	O
	O
Furthermore	O
,	O
fluctuations	O
in	O
the	O
H3K9cr	O
levels	O
were	O
more	O
substantial	O
than	O
fluctuations	O
in	O
the	O
corresponding	O
H3K9ac	O
levels	O
.	O
	O
Together	O
,	O
these	O
results	O
reveal	O
that	O
H3K9cr	O
is	O
a	O
dynamic	O
mark	O
of	O
chromatin	O
in	O
yeast	O
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	O
and	O
HDACs	O
.	O
	O
The	O
selectivity	O
of	O
Taf14	O
towards	O
crotonyllysine	O
was	O
substantiated	O
by	O
1H	O
,	O
15N	O
HSQC	O
experiments	O
,	O
in	O
which	O
either	O
H3K9cr5	O
-	O
13	O
or	O
H3K9ac5	O
-	O
13	O
peptide	O
was	O
titrated	O
into	O
the	O
15N	O
-	O
labeled	O
Taf14	O
YEATS	O
domain	O
(	O
Fig	O
.	O
2c	O
and	O
Supplementary	O
Fig	O
.	O
4a	O
,	O
b	O
).	O
	O
To	O
determine	O
if	O
the	O
binding	O
to	O
crotonyllysine	O
is	O
conserved	O
,	O
we	O
tested	O
human	O
YEATS	O
domains	O
by	O
pull	O
-	O
down	O
experiments	O
using	O
singly	O
and	O
multiply	O
acetylated	O
,	O
propionylated	O
,	O
butyrylated	O
,	O
and	O
crotonylated	O
histone	O
peptides	O
(	O
Supplementary	O
Fig	O
.	O
6	O
).	O
	O
These	O
results	O
demonstrate	O
that	O
the	O
YEATS	O
domain	O
is	O
currently	O
the	O
sole	O
reader	O
of	O
crotonyllysine	O
.	O
	O
The	O
unique	O
and	O
previously	O
unobserved	O
aromatic	O
-	O
amide	O
/	O
aliphatic	O
-	O
aromatic	O
π	O
-	O
π	O
-	O
π	O
-	O
stacking	O
mechanism	O
facilitates	O
the	O
specific	O
recognition	O
of	O
the	O
crotonyl	O
moiety	O
.	O
	O
We	O
further	O
demonstrate	O
that	O
H3K9cr	O
exists	O
in	O
yeast	O
and	O
is	O
dynamically	O
regulated	O
by	O
HATs	O
and	O
HDACs	O
.	O
	O
Total	O
H3	O
was	O
used	O
as	O
a	O
loading	O
control	O
.	O
	O
Structure	O
of	O
the	O
GAT	O
domain	O
of	O
the	O
endosomal	O
adapter	O
protein	O
Tom1	O
	O
The	O
Tom1	O
GAT	O
domain	O
solution	O
structure	O
will	O
provide	O
additional	O
tools	O
for	O
modulating	O
its	O
biological	O
function	O
.	O
	O
A	O
conformational	O
response	O
of	O
the	O
Tom1	O
GAT	O
domain	O
upon	O
Tollip	O
TBD	O
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
	O
The	O
Tom1	O
GAT	O
structural	O
restraints	O
yielded	O
ten	O
helical	O
structures	O
(	O
Fig	O
.	O
2A	O
,	O
B	O
)	O
with	O
a	O
root	O
mean	O
square	O
deviation	O
(	O
RMSD	O
)	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	O
-	O
E240	O
(	O
α	O
-	O
helix	O
1	O
),	O
P248	O
-	O
Q274	O
(	O
α	O
-	O
helix	O
2	O
),	O
and	O
E278	O
-	O
T306	O
(	O
α	O
-	O
helix	O
3	O
).	O
	O
NMR	O
structural	O
statistics	O
for	O
lowest	O
energy	O
conformers	O
of	O
Tom1	O
GAT	O
using	O
PSVS	O
.	O
	O
Much	O
attention	O
has	O
been	O
paid	O
to	O
the	O
roles	O
of	O
haem	O
-	O
iron	O
in	O
cancer	O
development	O
.	O
	O
Thus	O
,	O
a	O
tenuous	O
balance	O
between	O
free	O
haem	O
and	O
CO	O
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
	O
Consequently	O
,	O
the	O
five	O
-	O
coordinated	O
haem	O
of	O
PGRMC1	O
has	O
an	O
open	O
surface	O
that	O
allows	O
its	O
dimerization	O
through	O
hydrophobic	O
haem	O
–	O
haem	O
stacking	O
.	O
	O
Furthermore	O
,	O
free	O
energy	O
of	O
dissociation	O
predicted	O
by	O
PISA	O
suggested	O
that	O
the	O
haem	O
-	O
mediated	O
dimer	O
is	O
stable	O
in	O
solution	O
while	O
the	O
other	O
potential	O
interactions	O
are	O
not	O
.	O
	O
A	O
value	O
of	O
this	O
kind	O
implies	O
that	O
the	O
PGRMC1	O
dimer	O
is	O
more	O
stable	O
than	O
other	O
dimers	O
of	O
extracellular	O
domain	O
of	O
membrane	O
proteins	O
such	O
as	O
Toll	O
like	O
receptor	O
9	O
(	O
dimerization	O
Kd	O
of	O
20	O
μmol	O
l	O
−	O
1	O
)	O
(	O
ref	O
.)	O
and	O
plexin	O
A2	O
receptor	O
(	O
dimerization	O
Kd	O
higher	O
than	O
300	O
μmol	O
l	O
−	O
1	O
)	O
(	O
ref	O
.).	O
	O
These	O
results	O
suggest	O
that	O
CO	O
favours	O
the	O
six	O
-	O
coordinate	O
form	O
of	O
haem	O
and	O
interferes	O
with	O
the	O
haem	O
-	O
mediated	O
dimerization	O
of	O
PGRMC1	O
.	O
	O
Because	O
PGRMC1	O
is	O
known	O
to	O
interact	O
with	O
EGFR	O
and	O
to	O
accelerate	O
tumour	O
progression	O
,	O
we	O
examined	O
the	O
effect	O
of	O
haem	O
-	O
dependent	O
dimerization	O
of	O
PGRMC1	O
on	O
its	O
interaction	O
with	O
EGFR	O
by	O
using	O
purified	O
proteins	O
.	O
	O
We	O
also	O
examined	O
the	O
effect	O
of	O
succinylacetone	O
(	O
SA	O
),	O
an	O
inhibitor	O
of	O
haem	O
biosynthesis	O
(	O
Fig	O
.	O
4d	O
).	O
	O
Thus	O
,	O
PGRMC1	O
dimerization	O
is	O
important	O
for	O
cancer	O
cell	O
proliferation	O
and	O
chemoresistance	O
.	O
	O
We	O
examined	O
the	O
role	O
of	O
PGRMC1	O
in	O
metastatic	O
progression	O
by	O
xenograft	O
transplantation	O
assays	O
using	O
super	O
-	O
immunodeficient	O
NOD	O
/	O
scid	O
/	O
γnull	O
(	O
NOG	O
)	O
mice	O
.	O
	O
Recombinant	O
CYP1A2	O
and	O
CYP3A4	O
including	O
a	O
microsomal	O
formulation	O
containing	O
cytochrome	O
b5	O
and	O
cytochrome	O
P450	O
reductase	O
,	O
drug	O
-	O
metabolizing	O
cytochromes	O
P450	O
,	O
interacted	O
with	O
wild	O
-	O
type	O
PGRMC1	O
,	O
but	O
not	O
with	O
the	O
Y113F	B-mutant
mutant	O
,	O
in	O
a	O
haem	O
-	O
dependent	O
manner	O
(	O
Fig	O
.	O
6a	O
,	O
b	O
).	O
	O
Enhanced	O
doxorubicin	O
sensitivity	O
was	O
modestly	O
but	O
significantly	O
induced	O
by	O
PGRMC1	B-mutant
-	I-mutant
KD	I-mutant
.	O
	O
Recently	O
,	O
Lucas	O
et	O
al	O
.	O
reported	O
that	O
translationally	O
-	O
controlled	O
tumour	O
protein	O
was	O
dimerized	O
by	O
binding	O
with	O
haem	O
,	O
but	O
its	O
structural	O
basis	O
remains	O
unclear	O
.	O
	O
Sequence	O
alignments	O
show	O
that	O
haem	O
-	O
binding	O
residues	O
(	O
Tyr113	O
,	O
Tyr107	O
,	O
Lys163	O
and	O
Tyr164	O
)	O
in	O
PGRMC1	O
are	O
conserved	O
among	O
MAPR	O
proteins	O
(	O
Supplementary	O
Fig	O
.	O
5	O
).	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	O
biosynthesis	O
.	O
	O
(	O
a	O
)	O
FLAG	O
-	O
PGRMC1	O
wild	O
-	O
type	O
(	O
wt	O
)	O
and	O
Y113F	B-mutant
mutant	O
proteins	O
(	O
a	O
.	O
a	O
.	O
44	O
–	O
195	O
),	O
in	O
either	O
apo	O
-	O
or	O
haem	O
-	O
bound	O
form	O
,	O
were	O
incubated	O
with	O
purified	O
EGFR	O
and	O
co	O
-	O
immunoprecipitated	O
with	O
anti	O
-	O
FLAG	O
antibody	O
-	O
conjugated	O
beads	O
.	O
	O
(	O
g	O
,	O
h	O
)	O
HCT116	O
cells	O
were	O
treated	O
with	O
or	O
without	O
EGF	O
,	O
SA	O
,	O
RuCl3	O
and	O
CORM3	O
as	O
indicated	O
,	O
and	O
components	O
of	O
the	O
EGFR	O
signaling	O
pathway	O
were	O
detected	O
by	O
Western	O
blotting	O
.	O
	O
(	O
c	O
)	O
Binding	O
assay	O
was	O
performed	O
as	O
in	O
(	O
a	O
)	O
using	O
haem	O
-	O
bound	O
FLAG	O
-	O
PGRMC1	O
wt	O
and	O
CYP1A2	O
with	O
or	O
without	O
RuCl3	O
and	O
CORM3	O
.	O
	O
We	O
generated	O
high	O
-	O
resolution	O
structures	O
of	O
the	O
1E6	O
TCR	O
bound	O
to	O
7	O
altered	O
peptide	O
ligands	O
,	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
	O
Highly	O
potent	O
antigens	O
of	O
the	O
1E6	O
TCR	O
engaged	O
with	O
a	O
strong	O
antipathogen	O
-	O
like	O
binding	O
affinity	O
;	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
	O
This	O
ability	O
is	O
required	O
to	O
enable	O
the	O
estimated	O
25	O
million	O
distinct	O
TCRs	O
expressed	O
in	O
humans	O
to	O
provide	O
effective	O
immune	O
coverage	O
against	O
all	O
possible	O
foreign	O
peptide	O
antigens	O
.	O
	O
The	O
RQFGPDWIVA	O
sequence	O
(	O
present	O
in	O
C	O
.	O
asparagiforme	O
)	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	O
.	O
	O
The	O
range	O
of	O
Tm	O
was	O
between	O
49	O
.	O
4	O
°	O
C	O
(	O
RQFGPDWIVA	O
)	O
and	O
60	O
.	O
3	O
°	O
C	O
(	O
YQFGPDFPIA	O
),	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
	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	O
binding	O
affinity	O
was	O
extremely	O
weak	O
,	O
e	O
.	O
g	O
.,	O
the	O
1E6	O
TCR	O
binding	O
affinity	O
for	O
the	O
A2	O
-	O
MVWGPDPLYV	O
peptide	O
was	O
KD	O
=	O
~	O
600	O
μM	O
.	O
Second	O
,	O
the	O
1E6	O
TCR	O
bound	O
to	O
A2	O
-	O
RQFGPDFPTI	O
with	O
KD	O
=	O
0	O
.	O
5	O
μM	O
,	O
equivalent	O
to	O
the	O
binding	O
affinity	O
of	O
the	O
very	O
strongest	O
antipathogen	O
TCRs	O
.	O
	O
To	O
confirm	O
the	O
affinity	O
spread	O
detected	O
by	O
SPR	O
,	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	O
affinity	O
of	O
each	O
APL	O
using	O
an	O
adhesion	O
frequency	O
assay	O
in	O
which	O
a	O
human	O
rbc	O
coated	O
in	O
pMHC	O
acted	O
as	O
an	O
adhesion	O
sensor	O
.	O
	O
As	O
with	O
the	O
3D	O
affinity	O
measurements	O
,	O
the	O
2D	O
affinity	O
measurements	O
correlated	O
well	O
with	O
the	O
EC50	O
values	O
for	O
each	O
ligand	O
(	O
Figure	O
2K	O
)	O
demonstrating	O
a	O
strong	O
correlation	O
(	O
Pearson	O
’	O
s	O
correlation	O
=	O
0	O
.	O
8	O
,	O
P	O
=	O
0	O
.	O
01	O
)	O
between	O
T	O
cell	O
antigen	O
sensitivity	O
and	O
TCR	O
binding	O
affinity	O
.	O
	O
Overall	O
,	O
the	O
1E6	O
TCR	O
used	O
a	O
canonical	O
binding	O
mode	O
to	O
engage	O
each	O
APL	O
with	O
the	O
TCR	O
α	O
-	O
chain	O
positioned	O
over	O
the	O
MHC	O
class	O
I	O
(	O
MHCI	O
)	O
α2	O
-	O
helix	O
and	O
the	O
TCR	O
β	O
-	O
chain	O
over	O
the	O
MHCI	O
α	O
-	O
1	O
helix	O
,	O
straddling	O
the	O
peptide	O
cargo	O
.	O
	O
Focused	O
hotspot	O
binding	O
around	O
a	O
conserved	O
GPD	O
motif	O
enables	O
the	O
1E6	O
TCR	O
to	O
tolerate	O
peptide	O
degeneracy	O
.	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	O
binding	O
affinity	O
for	O
some	O
of	O
the	O
APLs	O
,	O
for	O
others	O
,	O
the	O
correlation	O
was	O
poor	O
(	O
Pearson	O
’	O
s	O
correlation	O
=	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	O
.	O
	O
The	O
unligated	O
A2	O
-	O
MVWGPDPLYV	O
(	O
KD	O
=	O
~	O
600	O
μM	O
)	O
structure	O
revealed	O
that	O
the	O
side	O
chain	O
Tyr9	O
swung	O
around	O
8	O
Å	O
in	O
the	O
complex	O
structure	O
,	O
subsequently	O
making	O
contacts	O
with	O
TCR	O
residues	O
Asp30β	O
and	O
Asn51β	O
(	O
Figure	O
6A	O
and	O
Figure	O
5A	O
,	O
respectively	O
).	O
	O
The	O
overall	O
free	O
binding	O
energies	O
(	O
ΔG	O
°)	O
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	O
binding	O
affinities	O
we	O
observed	O
for	O
the	O
different	O
APLs	O
.	O
	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	O
,	O
with	O
all	O
but	O
the	O
1E6	O
-	O
A2	O
-	O
RQFGPDFPTI	O
interaction	O
(	O
ΔH	O
°	O
=	O
6	O
.	O
3	O
kcal	O
/	O
mol	O
)	O
generating	O
an	O
energetically	O
favorable	O
enthalpy	O
value	O
(	O
ΔH	O
°	O
=	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
	O
Furthermore	O
,	O
the	O
structures	O
of	O
the	O
unligated	O
pMHCs	O
demonstrated	O
that	O
,	O
for	O
these	O
stronger	O
-	O
affinity	O
ligands	O
,	O
there	O
was	O
less	O
conformational	O
difference	O
between	O
the	O
TCR	O
ligated	O
pMHCs	O
compared	O
with	O
the	O
weaker	O
-	O
affinity	O
ligands	O
(	O
Figure	O
6	O
).	O
	O
Sethi	O
and	O
colleagues	O
recently	O
demonstrated	O
that	O
the	O
MHCII	O
-	O
restricted	O
Hy	O
.	O
1B11	O
TCR	O
,	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	O
pocket	O
formed	O
from	O
peptide	O
residues	O
2	O
,	O
3	O
,	O
and	O
5	O
(	O
from	O
MBP85	O
–	O
99	O
bound	O
to	O
HLA	O
-	O
DQ1	O
).	O
	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	O
this	O
motif	O
,	O
we	O
were	O
unable	O
to	O
robustly	O
measure	O
binding	O
affinities	O
or	O
generate	O
complex	O
structures	O
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
	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	O
.	O
1B11	O
TCR	O
,	O
in	O
which	O
one	O
aromatic	O
residue	O
of	O
the	O
TCR	O
CDR3α	O
loop	O
anchored	O
into	O
a	O
pocket	O
created	O
by	O
a	O
conserved	O
peptide	O
motif	O
.	O
	O
Despite	O
some	O
weak	O
statistical	O
correlation	O
between	O
the	O
surface	O
complementarity	O
(	O
SC	O
)	O
and	O
affinity	O
,	O
closer	O
inspection	O
of	O
the	O
interface	O
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	O
potency	O
and	O
TCR	O
binding	O
strength	O
between	O
the	O
different	O
ligands	O
.	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	O
peptide	O
bulge	O
were	O
important	O
for	O
generating	O
new	O
interactions	O
.	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	O
residue	O
at	O
peptide	O
position	O
2	O
(	O
Leu	B-mutant
-	I-mutant
Gln	I-mutant
)	O
has	O
a	O
direct	O
effect	O
on	O
1E6	O
TCR	O
binding	O
affinity	O
because	O
our	O
structural	O
analysis	O
demonstrated	O
that	O
1E6	O
made	O
3	O
additional	O
bonds	O
with	O
A2	O
-	O
AQWGPDPAAA	O
compared	O
with	O
A2	O
-	O
ALWGPDPAAA	O
,	O
consistent	O
with	O
the	O
>	O
3	O
-	O
fold	O
stronger	O
binding	O
affinity	O
.	O
	O
Although	O
no	O
energetic	O
signature	O
appears	O
to	O
exist	O
for	O
different	O
TCRs	O
,	O
we	O
used	O
thermodynamic	O
analysis	O
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	O
.	O
	O
This	O
analysis	O
demonstrated	O
a	O
strong	O
relationship	O
(	O
according	O
to	O
the	O
Pearson	O
’	O
s	O
correlation	O
analysis	O
)	O
between	O
the	O
energetic	O
signature	O
used	O
by	O
the	O
1E6	O
TCR	O
and	O
the	O
sensitivity	O
of	O
the	O
1E6	O
T	O
cell	O
clone	O
to	O
different	O
APLs	O
.	O
	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	O
composed	O
of	O
each	O
APL	O
(	O
colored	O
as	O
above	O
)	O
presented	O
by	O
HLA	O
-	O
A	O
*	O
0201	O
.	O
(	O
D	O
)	O
The	O
stability	O
of	O
each	O
APL	O
(	O
colored	O
as	O
above	O
)	O
was	O
tested	O
,	O
in	O
duplicate	O
,	O
using	O
CD	O
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
	O
The	O
1E6	O
TCR	O
uses	O
a	O
conserved	O
binding	O
mode	O
to	O
engage	O
A2	O
-	O
ALWGPDPAAA	O
and	O
the	O
APLs	O
.	O
	O
Interaction	O
between	O
1E6	O
TCR	O
(	O
gray	O
illustration	O
)	O
residues	O
Tyr97α	O
and	O
Tyr97β	O
(	O
the	O
position	O
of	O
these	O
side	O
chains	O
in	O
the	O
TCR	O
in	O
complex	O
with	O
all	O
7	O
APLs	O
,	O
and	O
the	O
previously	O
reported	O
A2	O
-	O
ALWGPDPAAA	O
epitope	O
,	O
is	O
shown	O
in	O
multicolored	O
sticks	O
;	O
ref	O
.)	O
and	O
the	O
GPD	O
peptide	O
motif	O
(	O
the	O
position	O
of	O
these	O
side	O
chains	O
in	O
all	O
7	O
APLs	O
and	O
A2	O
-	O
ALWGPDPAAA	O
in	O
complex	O
with	O
the	O
1E6	O
TCR	O
is	O
shown	O
in	O
multicolored	O
sticks	O
).	O
	O
Interactions	O
between	O
the	O
1E6	O
TCR	O
and	O
peptide	O
residues	O
outside	O
of	O
the	O
conserved	O
GPD	O
motif	O
.	O
	O
Hydrogen	O
bonds	O
are	O
shown	O
as	O
red	O
dotted	O
lines	O
;	O
van	O
der	O
Waals	O
(	O
vdW	O
)	O
contacts	O
are	O
shown	O
as	O
black	O
dotted	O
lines	O
.	O
	O
(	O
A	O
)	O
Interaction	O
between	O
the	O
1E6	O
TCR	O
(	O
black	O
illustration	O
and	O
sticks	O
)	O
and	O
A2	O
-	O
MVWGPDPLYV	O
(	O
black	O
illustration	O
and	O
sticks	O
).	O
(	O
B	O
)	O
Interaction	O
between	O
the	O
1E6	O
TCR	O
(	O
red	O
illustration	O
and	O
sticks	O
)	O
and	O
A2	O
-	O
YLGGPDFPTI	O
(	O
red	O
illustration	O
and	O
sticks	O
).	O
(	O
C	O
)	O
Interaction	O
between	O
the	O
1E6	O
TCR	O
(	O
blue	O
illustration	O
and	O
sticks	O
)	O
and	O
A2	O
-	O
ALWGPDPAAA	O
(	O
blue	O
illustration	O
and	O
sticks	O
)	O
reproduced	O
from	O
previous	O
published	O
data	O
.	O
	O
The	O
1E6	O
TCR	O
makes	O
distinct	O
peptide	O
contacts	O
with	O
the	O
MHC	O
surface	O
depending	O
on	O
the	O
peptide	O
cargo	O
.	O
	O
Boxes	O
show	O
total	O
contacts	O
between	O
the	O
1E6	O
TCR	O
and	O
these	O
key	O
residues	O
(	O
green	O
boxes	O
are	O
MHC	O
residues	O
;	O
white	O
boxes	O
are	O
TCR	O
residues	O
).	O
	O