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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

(	O
B	O
,	O
C	O
)	O
Overlay	B-experimental_method
of	O
selected	O
regions	O
of	O
the	O
1H	B-experimental_method
-	I-experimental_method
15N	I-experimental_method
TROSY	I-experimental_method
-	I-experimental_method
HSQC	I-experimental_method
spectra	B-evidence
acquired	O
at	O
25	O
°	O
C	O
of	O
apo	B-protein_state
-	O
NadR	B-protein
(	O
cyan	O
)	O
and	O
NadR	B-complex_assembly
/	I-complex_assembly
4	I-complex_assembly
-	I-complex_assembly
HPA	I-complex_assembly
(	O
red	O
)	O
superimposed	B-experimental_method
with	O
the	O
spectra	B-evidence
acquired	O
at	O
10	O
°	O
C	O
of	O
apo	B-protein_state
-	O
NadR	B-protein
(	O
blue	O
)	O
and	O
NadR	B-complex_assembly
/	I-complex_assembly
4	I-complex_assembly
-	I-complex_assembly
HPA	I-complex_assembly
(	O
green	O
).	O

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

(	O
B	O
)	O
A	O
structural	B-experimental_method
alignment	I-experimental_method
of	O
MTH313	B-protein
chain	B-structure_element
A	I-structure_element
and	O
ST1710	B-protein
(	O
pink	O
)	O
(	O
Cα	O
rmsd	B-evidence
2	O
.	O
3Å	O
),	O
shows	O
that	O
they	O
bind	O
salicylate	B-chemical
in	O
equivalent	O
sites	O
(	O
differing	O
by	O
only	O
~	O
3Å	O
)	O
and	O
with	O
the	O
same	O
orientation	O
.	O

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

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

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

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

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

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

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

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

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

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

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

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

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

BIO399	B-chemical
binds	O
to	O
the	O
ligand	B-site
binding	I-site
site	I-site
of	O
RORγ	B-protein
adopting	O
a	O
collapsed	B-protein_state
conformation	O
as	O
seen	O
with	O
BIO592	B-chemical
where	O
the	O
two	O
compounds	O
superimpose	B-experimental_method
with	O
an	O
RMSD	B-evidence
of	O
0	O
.	O
72	O
Å	O
(	O
Fig	O
.	O
5b	O
).	O

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Finally	O
,	O
our	O
results	O
provide	O
a	O
structural	O
framework	O
for	O
understanding	O
the	O
effects	O
of	O
ADAR	B-protein_type
mutations	O
associated	O
with	O
human	B-species
disease	O
.	O

Two	O
different	O
enzymes	O
carry	O
out	O
A	O
to	O
I	O
editing	O
in	O
humans	B-species
;	O
ADAR1	B-protein
and	O
ADAR2	B-protein
.	O

Furthermore	O
,	O
how	O
an	O
ADAR	B-protein_type
deaminase	B-structure_element
domain	I-structure_element
contributes	O
to	O
editing	B-site
site	I-site
selectivity	O
is	O
also	O
unknown	O
,	O
since	O
no	O
structures	B-evidence
of	O
ADAR	B-complex_assembly
deaminase	I-complex_assembly
domain	I-complex_assembly
-	I-complex_assembly
RNA	I-complex_assembly
complexes	O
have	O
been	O
reported	O
.	O

In	O
addition	O
,	O
an	O
inositol	B-chemical
hexakisphosphate	I-chemical
(	O
IHP	B-chemical
)	O
molecule	O
was	O
found	O
buried	O
in	O
the	O
core	O
of	O
the	O
protein	O
hydrogen	B-bond_interaction
bonded	I-bond_interaction
to	O
numerous	O
conserved	O
polar	O
residues	O
.	O

The	O
8	B-chemical
-	I-chemical
azanebularine	I-chemical
is	O
flipped	B-protein_state
out	I-protein_state
of	O
the	O
helix	B-structure_element
and	O
bound	B-protein_state
into	I-protein_state
the	O
active	B-site
site	I-site
as	O
its	O
covalent	O
hydrate	O
where	O
it	O
interacts	O
with	O
several	O
amino	O
acids	O
including	O
V351	B-residue_name_number
,	O
T375	B-residue_name_number
,	O
K376	B-residue_name_number
,	O
E396	B-residue_name_number
and	O
R455	B-residue_name_number
(	O
Fig	O
.	O
3a	O
,	O
Supplementary	O
Fig	O
.	O
3a	O
).	O

The	O
R455	B-residue_name_number
side	O
chain	O
ion	B-bond_interaction
pairs	I-bond_interaction
with	O
the	O
5	O
’-	O
phosphodiester	O
of	O
8	B-chemical
-	I-chemical
azanebularine	I-chemical
while	O
the	O
K376	B-residue_name_number
side	O
chain	O
contacts	O
its	O
3	O
’-	O
phosphodiester	O
.	O

ADARs	B-protein_type
use	O
a	O
unique	O
mechanism	O
to	O
modify	O
duplex	B-structure_element
RNA	I-structure_element

However	O
,	O
unlike	O
the	O
case	O
of	O
the	O
DNA	B-protein_type
MTase	I-protein_type
that	O
approaches	O
the	O
DNA	B-chemical
from	O
the	O
major	B-site
groove	I-site
,	O
the	O
ADAR2	B-protein
loop	B-structure_element
approaches	O
the	O
duplex	B-structure_element
from	O
the	O
minor	B-site
groove	I-site
side	O
.	O

The	O
DNA	B-chemical
B	B-structure_element
-	I-structure_element
form	I-structure_element
helix	I-structure_element
has	O
groove	O
widths	O
and	O
depths	O
that	O
would	O
prevent	O
productive	O
interactions	O
with	O
ADAR	B-protein_type
.	O

ADARs	B-protein_type
have	O
a	O
preference	O
for	O
editing	O
adenosines	B-residue_name
with	O
5	O
’	O
nearest	O
neighbor	O
U	B-residue_name
(	O
or	O
A	B-residue_name
)	O
and	O
3	O
’	O
nearest	O
neighbor	O
G	B-residue_name
.	O
The	O
ADAR2	B-protein
flipping	B-structure_element
loop	I-structure_element
occupies	O
the	O
minor	B-site
groove	I-site
spanning	O
the	O
three	O
base	O
pairs	O
that	O
include	O
the	O
nearest	O
neighbor	O
nucleotides	O
flanking	O
the	O
edited	O
base	O
(	O
Figs	O
.	O
3b	O
,	O
3c	O
).	O

These	O
observations	O
suggest	O
that	O
hADAR2	B-protein
’	O
s	O
5	O
’	O
nearest	O
neighbor	O
preference	O
for	O
U	B-residue_name
(	O
or	O
A	B-residue_name
)	O
is	O
due	O
to	O
a	O
destabilizing	O
clash	O
with	O
the	O
protein	O
backbone	O
at	O
G489	B-residue_name_number
that	O
results	O
from	O
the	O
presence	O
of	O
an	O
amino	O
group	O
in	O
the	O
minor	B-site
groove	I-site
at	O
this	O
location	O
for	O
sequences	O
with	O
5	O
’	O
nearest	O
neighbor	O
G	B-residue_name
or	O
C	B-residue_name
.	O
However	O
,	O
the	O
observed	O
clash	O
is	O
not	O
severe	O
and	O
the	O
enzyme	O
would	O
be	O
able	O
to	O
accommodate	O
G	B-residue_name
or	O
C	B-residue_name
5	O
’	O
nearest	O
neighbors	O
by	O
slight	O
structural	O
perturbations	O
,	O
explaining	O
why	O
this	O
sequence	O
preference	O
is	O
not	O
an	O
absolute	O
requirement	O
.	O

In	O
addition	O
,	O
the	O
substrate	O
with	O
a	O
3	O
’	O
I	B-residue_name
displayed	O
a	O
reduced	B-evidence
deamination	I-evidence
rate	I-evidence
compared	O
to	O
the	O
substrate	O
with	O
a	O
3	O
’	O
G	B-residue_name
suggesting	O
the	O
observed	O
H	B-bond_interaction
-	I-bond_interaction
bond	I-bond_interaction
to	O
the	O
2	O
-	O
amino	O
group	O
contributes	O
to	O
the	O
3	O
’	O
nearest	O
neighbor	O
selectivity	O
(	O
Fig	O
.	O
5f	O
).	O

Mutation	B-experimental_method
of	O
any	O
of	O
these	O
residues	O
to	O
alanine	B-residue_name
(	O
G593A	B-mutant
,	O
K594A	B-mutant
,	O
R348A	B-mutant
)	O
substantially	O
reduces	O
editing	O
activity	O
(	O
Fig	O
.	O
6c	O
).	O

Base	O
flipping	O
is	O
a	O
well	O
-	O
characterized	O
mechanism	O
by	O
which	O
nucleic	B-protein_type
acid	I-protein_type
modifying	I-protein_type
enzymes	I-protein_type
gain	O
access	O
to	O
sites	O
of	O
reaction	O
that	O
are	O
otherwise	O
buried	O
in	O
base	O
-	O
paired	O
structures	B-evidence
.	O

However	O
,	O
these	O
nucleotides	O
are	O
located	O
in	O
a	O
highly	B-protein_state
distorted	I-protein_state
and	O
dynamic	B-protein_state
duplex	B-structure_element
region	I-structure_element
containing	O
several	O
mismatches	O
and	O
are	O
predisposed	O
to	O
undergo	O
this	O
conformational	O
change	O
.	O

Other	O
RNA	B-protein_type
modification	I-protein_type
enzymes	I-protein_type
are	O
known	O
that	O
flip	O
nucleotides	O
out	O
of	O
loops	O
,	O
even	O
from	O
base	O
pairs	O
in	O
loop	O
regions	O
(	O
pseudoU	B-protein_type
synthetase	I-protein_type
,	O
tRNA	B-protein_type
transglycosylase	I-protein_type
,	O
and	O
restrictocin	B-protein
bound	B-protein_state
to	I-protein_state
sarcin	B-structure_element
/	I-structure_element
ricin	I-structure_element
loop	I-structure_element
of	O
28S	B-chemical
rRNA	I-chemical
)	O
(	O
Supplementary	O
Fig	O
.	O
5b	O
).	O

Our	O
use	O
of	O
8	B-chemical
-	I-chemical
azanebularine	I-chemical
,	O
with	O
its	O
high	O
propensity	O
to	O
form	O
a	O
covalent	O
hydrate	O
,	O
allowed	O
us	O
to	O
capture	O
a	O
true	O
mimic	O
of	O
the	O
tetrahedral	O
intermediate	O
and	O
reveal	O
the	O
interactions	O
between	O
the	O
deaminase	B-protein_type
active	B-site
site	I-site
and	O
the	O
reactive	O
nucleotide	O
.	O

Several	O
other	O
base	B-protein_type
-	I-protein_type
flipping	I-protein_type
enzymes	I-protein_type
stabilize	O
the	O
altered	O
nucleic	O
acid	O
conformation	O
by	O
intercalation	O
of	O
an	O
amino	O
acid	O
side	O
chain	O
into	O
the	O
space	O
vacated	O
by	O
the	O
flipped	B-protein_state
out	I-protein_state
base	B-chemical
.	O

The	O
latter	O
interaction	O
requires	O
E488	B-residue_name_number
to	O
be	O
protonated	B-protein_state
.	O

However	O
,	O
since	O
dsRBDs	B-structure_element
are	O
known	O
to	O
bind	O
promiscuously	O
with	O
duplex	B-structure_element
RNA	I-structure_element
,	O
it	O
is	O
possible	O
that	O
the	O
S258	B-residue_name_number
-	O
3	O
’	O
G	B-residue_name
interaction	O
found	O
in	O
a	O
complex	O
lacking	B-protein_state
the	I-protein_state
deaminase	B-structure_element
domain	I-structure_element
is	O
not	O
relevant	O
to	O
catalysis	O
at	O
the	O
editing	B-site
site	I-site
.	O

In	O
summary	O
,	O
the	O
structures	B-evidence
described	O
here	O
establish	O
human	B-species
ADAR2	B-protein
as	O
a	O
base	O
-	O
flipping	O
enzyme	O
that	O
uses	O
a	O
unique	O
mechanism	O
well	O
suited	O
for	O
modifying	O
duplex	B-structure_element
RNA	I-structure_element
.	O

b	O
,	O
View	O
of	O
structure	O
along	O
the	O
dsRNA	B-chemical
helical	O
axis	O
.	O

Other	O
ADAR	B-protein_type
-	O
induced	O
changes	O
in	O
RNA	B-chemical
conformation	O

c	O
,	O
Unusual	O
“	O
wobble	O
”	O
A13	B-residue_name_number
’-	O
U11	B-residue_name_number
interaction	O
in	O
the	O
hADAR2d	B-complex_assembly
WT	I-complex_assembly
–	I-complex_assembly
Bdf2	I-complex_assembly
-	I-complex_assembly
U	I-complex_assembly
complex	O
shown	O
in	O
stick	O
with	O
H	B-bond_interaction
-	I-bond_interaction
bond	I-bond_interaction
indicated	O
with	O
yellow	O
dashes	O
and	O
distances	O
shown	O
in	O
Å	O
.	O
The	O
position	O
of	O
this	O
base	O
pair	O
in	O
the	O
hADAR2d	B-complex_assembly
E488Q	I-complex_assembly
–	I-complex_assembly
Bdf2	I-complex_assembly
-	I-complex_assembly
C	I-complex_assembly
duplex	O
is	O
shown	O
in	O
wire	O
with	O
H	B-bond_interaction
-	I-bond_interaction
bonds	I-bond_interaction
shown	O
with	O
gray	O
dashes	O
.	O

f	O
,	O
Comparison	O
of	O
deamination	B-evidence
rate	I-evidence
constants	I-evidence
by	O
hADAR2d	B-mutant
at	O
the	O
editing	B-site
site	I-site
adenosine	B-residue_name
(	O
red	O
)	O
for	O
duplexes	O
bearing	O
different	O
3	O
’	O
nearest	O
neighbors	O
.	O

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

Recently	O
,	O
the	O
crystal	B-evidence
structure	I-evidence
of	O
the	O
Regnase	B-protein
-	I-protein
1	I-protein
PIN	B-structure_element
domain	O
derived	O
from	O
Homo	B-species
sapiens	I-species
was	O
reported	O
.	O

In	O
addition	O
,	O
Regnase	B-protein
-	I-protein
1	I-protein
has	O
been	O
predicted	O
to	O
possess	O
other	O
domains	O
in	O
the	O
N	B-structure_element
-	I-structure_element
and	I-structure_element
C	I-structure_element
-	I-structure_element
terminal	I-structure_element
regions	I-structure_element
.	O

However	O
,	O
the	O
structure	B-evidence
and	O
function	O
of	O
the	O
ZF	B-structure_element
domain	O
,	O
N	B-structure_element
-	I-structure_element
terminal	I-structure_element
domain	I-structure_element
(	O
NTD	B-structure_element
)	O
and	O
C	B-structure_element
-	I-structure_element
terminal	I-structure_element
domain	I-structure_element
(	O
CTD	B-structure_element
)	O
of	O
Regnase	B-protein
-	I-protein
1	I-protein
have	O
not	O
been	O
solved	O
.	O

Contribution	O
of	O
each	O
domain	O
of	O
Regnase	B-protein
-	I-protein
1	I-protein
to	O
the	O
mRNA	B-chemical
binding	O
activity	O

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

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

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

The	O
side	O
chains	O
of	O
these	O
residues	O
point	O
away	O
from	O
the	O
catalytic	B-site
center	I-site
on	O
the	O
same	O
molecule	O
(	O
Fig	O
.	O
2b	O
).	O

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

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

Although	O
the	O
function	O
of	O
the	O
CTD	B-structure_element
remains	O
elusive	O
,	O
we	O
revealed	O
the	O
functions	O
of	O
the	O
NTD	B-structure_element
,	O
PIN	B-structure_element
,	O
and	O
ZF	B-structure_element
domains	O
.	O

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

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

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

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

This	O
result	O
is	O
consistent	O
with	O
a	O
model	O
in	O
which	O
the	O
NTD	B-structure_element
acts	O
as	O
an	O
enhancer	O
,	O
and	O
cleavage	O
of	O
the	O
linker	B-structure_element
lowers	O
enzymatic	O
activity	O
dramatically	O
.	O

We	O
incorporated	O
information	O
from	O
the	O
cleavage	B-site
site	I-site
of	O
IL	B-protein_type
-	I-protein_type
6	I-protein_type
mRNA	B-chemical
in	O
vitro	O
is	O
indicated	O
by	O
denaturing	O
polyacrylamide	B-experimental_method
gel	I-experimental_method
electrophoresis	I-experimental_method
(	O
Supplementary	O
Fig	O
.	O
7a	O
,	O
b	O
).	O

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

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

(	O
a	O
)	O
Domain	O
architecture	O
of	O
Regnase	B-protein
-	I-protein
1	I-protein
.	O
(	O
b	O
)	O
Solution	B-evidence
structure	I-evidence
of	O
the	O
NTD	B-structure_element
.	O
(	O
c	O
)	O
Crystal	B-evidence
structure	I-evidence
of	O
the	O
PIN	B-structure_element
domain	O
.	O

(	O
e	O
)	O
Solution	B-evidence
structure	I-evidence
of	O
the	O
CTD	B-structure_element
.	O

(	O
g	O
)	O
Binding	O
of	O
Regnase	B-protein
-	I-protein
1	I-protein
and	O
IL	B-protein_type
-	I-protein_type
6	I-protein_type
mRNA	B-chemical
was	O
plotted	O
.	O

(	O
h	O
)	O
In	B-experimental_method
vitro	I-experimental_method
cleavage	I-experimental_method
assay	I-experimental_method
of	O
Regnase	B-protein
-	I-protein
1	I-protein
to	O
IL	B-protein_type
-	I-protein_type
6	I-protein_type
mRNA	B-chemical
.	O

The	O
residues	O
with	O
significant	B-evidence
chemical	I-evidence
shift	I-evidence
changes	I-evidence
were	O
labeled	O
in	O
the	O
overlaid	B-experimental_method
spectra	B-evidence
(	O
left	O
)	O
and	O
colored	O
red	O
,	O
yellow	O
,	O
or	O
green	O
on	O
the	O
surface	O
and	O
ribbon	O
structure	O
of	O
the	O
NTD	B-structure_element
.	O

Heterodimer	O
formation	O
by	O
combination	O
of	O
the	O
Regnase	B-protein
-	I-protein
1	I-protein
basic	O
residue	O
mutants	B-protein_state
and	O
the	O
DDNN	B-mutant
mutant	B-protein_state
restored	O
the	O
RNase	B-protein_type
activity	O
.	O

Structure	O
‐	O
activity	O
relationship	O
of	O
the	O
peptide	B-structure_element
binding	I-structure_element
‐	I-structure_element
motif	I-structure_element
mediating	O
the	O
BRCA2	B-complex_assembly
:	I-complex_assembly
RAD51	I-complex_assembly
protein	O
–	O
protein	O
interaction	O

We	O
have	O
tabulated	O
the	O
effects	O
of	O
mutation	B-experimental_method
of	O
this	O
sequence	O
,	O
across	O
a	O
variety	O
of	O
experimental	O
methods	O
and	O
from	O
relevant	O
mutations	O
observed	O
in	O
the	O
clinic	O
.	O

Both	O
BRCA2	B-protein
and	O
RAD51	B-protein
together	O
are	O
vital	O
for	O
helping	O
to	O
repair	O
and	O
maintain	O
a	O
high	O
fidelity	O
in	O
DNA	O
replication	O
.	O

However	O
,	O
whilst	O
the	O
identification	O
of	O
highly	B-protein_state
conserved	I-protein_state
residues	O
may	O
be	O
a	O
good	O
starting	O
point	O
for	O
identifying	O
hot	B-site
‐	I-site
spots	I-site
,	O
experimental	O
validation	O
by	O
mutation	B-experimental_method
of	O
these	O
sequences	O
is	O
vital	O
.	O

The	O
mutation	O
,	O
relevant	O
peptide	O
context	O
,	O
resulting	O
FxxA	B-structure_element
motif	O
sequence	O
and	O
experimental	O
technique	O
for	O
each	O
entry	O
is	O
given	O
.	O

The	O
wild	B-protein_state
‐	I-protein_state
type	I-protein_state
FxxA	B-structure_element
sequence	O
is	O
indicated	O
in	O
parenthesis	O
.	O

Wild	B-protein_state
‐	I-protein_state
type	I-protein_state
human	B-species
RAD51	B-protein
,	O
however	O
,	O
is	O
a	O
heterogeneous	O
mixture	O
of	O
oligomers	B-oligomeric_state
and	O
when	O
monomerised	B-oligomeric_state
by	O
mutation	B-experimental_method
,	O
is	O
highly	B-protein_state
unstable	I-protein_state
.	O

One	O
RAD51	B-protein
(	O
green	O
cartoon	O
)	O
interacts	O
with	O
another	O
molecule	O
of	O
RAD51	B-protein
(	O
grey	O
and	O
pink	O
surface	O
)	O
via	O
the	O
FxxA	B-site
pocket	I-site
indicated	O
by	O
the	O
dashed	O
blue	O
box	O
.	O

A	O
summary	O
of	O
the	O
peptide	O
sequence	O
,	O
PDB	O
codes	O
and	O
K	B-evidence
D	I-evidence
data	O
measured	O
by	O
ITC	B-experimental_method
with	O
the	O
corresponding	O
ΔH	B-evidence
and	O
TΔS	B-evidence
values	O
are	O
collated	O
in	O
Table	O
2	O
.	O

Interestingly	O
,	O
it	O
was	O
found	O
that	O
a	O
proline	B-residue_name
at	O
this	O
position	O
improved	O
the	O
affinity	B-evidence
almost	O
threefold	O
,	O
to	O
113	O
μm	O
(	O
Table	O
2	O
,	O
entry	O
6	O
).	O

This	O
beneficial	O
mutation	B-experimental_method
was	O
incorporated	O
with	O
another	O
previously	O
identified	O
variant	O
to	O
produce	O
the	O
peptide	O
WHPA	B-structure_element
.	O

Perhaps	O
surprisingly	O
,	O
it	O
was	O
accommodated	O
and	O
the	O
affinity	B-evidence
dropped	O
only	O
by	O
twofold	O
as	O
compared	O
to	O
FHTA	B-structure_element
.	O

The	O
effect	O
of	O
simply	O
removing	B-experimental_method
the	O
β	O
‐	O
carbon	O
of	O
alanine	B-residue_name
,	O
by	O
mutation	B-experimental_method
to	I-experimental_method
glycine	B-residue_name
(	O
FHTG	B-structure_element
),	O
produced	O
an	O
approximately	O
sixfold	O
drop	O
in	O
binding	B-evidence
affinity	I-evidence
(	O
Table	O
2	O
,	O
entry	O
8	O
).	O

This	O
is	O
in	O
line	O
with	O
the	O
observation	O
that	O
alanine	B-residue_name
is	O
not	B-protein_state
100	I-protein_state
%	I-protein_state
conserved	I-protein_state
and	O
some	O
archeal	B-taxonomy_domain
RadA	B-protein_type
proteins	I-protein_type
contain	O
a	O
glycine	B-residue_name
in	O
the	O
place	O
of	O
alanine	B-residue_name
23	O
.	O

Structures	B-evidence
of	O
the	O
key	O
tetrapeptides	B-chemical
were	O
solved	O
by	O
soaking	B-experimental_method
into	I-experimental_method
crystals	B-evidence
of	O
a	O
humanised	B-protein_state
form	O
of	O
RAD51	B-protein
,	O
HumRadA1	B-mutant
,	O
which	O
we	O
have	O
previously	O
reported	O
as	O
a	O
convenient	O
surrogate	O
system	O
for	O
RAD51	B-protein
crystallography	B-experimental_method
15	O
.	O

All	O
structures	B-evidence
are	O
of	O
high	O
resolution	O
(	O
1	O
.	O
2	O
–	O
1	O
.	O
7	O
Å	O
)	O
and	O
the	O
electron	B-evidence
density	I-evidence
for	O
the	O
peptide	O
was	O
clearly	O
visible	O
after	O
the	O
first	O
refinement	O
using	O
unliganded	B-protein_state
RadA	B-protein
coordinates	O
(	O
Fig	O
.	O
S1	O
).	O

Some	O
of	O
the	O
SAR	O
observed	O
in	O
the	O
binding	B-experimental_method
analysis	I-experimental_method
can	O
be	O
interpreted	O
in	O
terms	O
of	O
these	O
X	B-experimental_method
‐	I-experimental_method
ray	I-experimental_method
crystal	B-evidence
structures	I-evidence
.	O

The	O
thermodynamic	B-evidence
data	I-evidence
of	O
peptide	O
binding	O
are	O
also	O
shown	O
in	O
Table	O
2	O
.	O
Although	O
we	O
have	O
both	O
thermodynamic	B-evidence
data	I-evidence
and	O
high	O
‐	O
quality	O
X	B-experimental_method
‐	I-experimental_method
ray	I-experimental_method
structural	B-evidence
information	I-evidence
for	O
some	O
of	O
the	O
mutant	B-protein_state
peptides	B-chemical
,	O
we	O
do	O
not	O
attempt	O
to	O
interpret	O
differences	O
in	O
thermodynamic	B-evidence
profiles	I-evidence
between	O
ligands	O
,	O
that	O
is	O
,	O
to	O
analyse	O
ΔΔH	B-evidence
and	O
ΔΔS	B-evidence
.	O

As	O
ΔS	B-evidence
is	O
derived	O
from	O
ΔG	B-evidence
by	O
subtracting	O
ΔH	B-evidence
,	O
errors	O
in	O
ΔH	B-evidence
will	O
be	O
correlated	O
with	O
errors	O
in	O
ΔS	B-evidence
,	O
giving	O
rise	O
to	O
a	O
‘	O
phantom	O
’	O
enthalpy	O
–	O
entropy	O
compensation	O
.	O

Figure	O
3A	O
shows	O
the	O
binding	O
pose	O
of	O
BRC4	B-chemical
when	O
bound	B-protein_state
to	I-protein_state
RAD51	B-protein
and	O
the	O
intrapeptide	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
that	O
are	O
made	O
by	O
BRC4	B-chemical
.	O

While	O
Phe1524	B-residue_name_number
and	O
Ala1527	B-residue_name_number
are	O
buried	O
in	O
hydrophobic	B-site
pockets	I-site
on	O
the	O
surface	O
,	O
His1525	B-residue_name_number
is	O
close	O
enough	O
to	O
form	O
a	O
hydrogen	B-bond_interaction
bond	I-bond_interaction
with	O
the	O
carbonyl	O
of	O
Thr1520	B-residue_name_number
,	O
but	O
the	O
rotamer	O
of	O
His1525	B-residue_name_number
,	O
supported	O
by	O
clearly	O
positioned	O
water	B-chemical
molecules	O
,	O
is	O
not	O
compatible	O
with	O
hydrogen	B-bond_interaction
bonding	I-bond_interaction
.	O

(	O
A	O
)	O
Highlight	O
of	O
intra	O
‐	O
BRC4	B-chemical
interactions	O
when	O
bound	B-protein_state
to	I-protein_state
RAD51	B-protein
(	O
omitted	O
for	O
clarity	O
)	O
(	O
PDB	O
:	O
1n0w	O
),	O
with	O
key	O
residues	O
shown	O
in	O
colour	O
.	O
(	O
B	O
)	O
Intrapeptide	O
interactions	O
from	O
oligomerisation	B-structure_element
epitope	I-structure_element
of	O
S	B-species
.	I-species
cerevisiae	I-species
RAD51	B-protein
when	O
bound	B-protein_state
to	I-protein_state
next	O
RAD51	B-protein
in	O
the	O
filament	O
(	O
PDB	O
:	O
1szp	O
).	O

Thr1526	B-residue_name_number
makes	O
no	O
direct	O
interactions	O
with	O
the	O
RAD51	B-protein
protein	O
,	O
but	O
instead	O
forms	O
a	O
hydrogen	B-bond_interaction
bond	I-bond_interaction
network	I-bond_interaction
with	O
the	O
highly	B-protein_state
conserved	I-protein_state
S1528	B-residue_name_number
and	O
K1530	B-residue_name_number
(	O
Fig	O
.	O
1C	O
).	O

The	O
conserved	B-protein_state
threonine	B-residue_name
residue	O
at	O
the	O
third	O
position	O
forms	O
a	O
hydrogen	B-bond_interaction
bond	I-bond_interaction
with	O
the	O
peptide	O
backbone	O
amide	O
,	O
which	O
forms	O
the	O
base	O
of	O
an	O
α	B-structure_element
‐	I-structure_element
helix	I-structure_element
.	O

The	O
reason	O
for	O
this	O
disconnection	O
is	O
suggested	O
to	O
be	O
that	O
threonine	B-residue_name
plays	O
a	O
role	O
in	O
stabilising	O
the	O
β	B-structure_element
‐	I-structure_element
turn	I-structure_element
in	O
the	O
BRC	B-structure_element
repeats	I-structure_element
,	O
which	O
is	O
absent	O
in	O
the	O
tetrapeptides	B-chemical
studied	O
.	O

The	O
differences	O
in	O
ΔG	B-evidence
for	O
different	O
peptide	O
variants	O
relative	O
to	O
FHTA	B-structure_element
are	O
shown	O
in	O
the	O
bar	O
chart	O
with	O
colouring	O
matching	O
with	O
the	O
structural	B-experimental_method
overlay	I-experimental_method
below	O
.	O
(	O
C	O
)	O
Overlay	B-experimental_method
of	O
tetrapeptide	B-chemical
structures	B-evidence
,	O
with	O
wild	B-protein_state
‐	I-protein_state
type	I-protein_state
FHTA	B-structure_element
peptide	O
across	O
the	O
figure	O
for	O
reference	O
and	O
truncated	O
segments	O
of	O
mutated	O
residues	O
shown	O
in	O
each	O
panel	O
.	O

Here	O
,	O
we	O
describe	O
the	O
first	O
crystal	B-evidence
structure	I-evidence
of	O
a	O
C11	B-protein_type
protein	O
from	O
the	O
human	B-species
gut	O
bacterium	B-taxonomy_domain
,	O
Parabacteroides	B-species
merdae	I-species
(	O
PmC11	B-protein
),	O
determined	O
to	O
1	O
.	O
7	O
-	O
Å	O
resolution	O
.	O

Biochemical	B-experimental_method
and	I-experimental_method
kinetic	I-experimental_method
analysis	I-experimental_method
revealed	O
Lys147	B-residue_name_number
to	O
be	O
an	O
intramolecular	B-site
processing	I-site
site	I-site
at	O
which	O
cleavage	B-ptm
is	O
required	O
for	O
full	B-protein_state
activation	I-protein_state
of	O
the	O
enzyme	B-protein
,	O
suggesting	O
an	O
autoinhibitory	O
mechanism	O
for	O
self	O
-	O
preservation	O
.	O

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

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

Clostripain	B-protein
has	O
been	O
described	O
as	O
an	O
arginine	B-protein_type
-	I-protein_type
specific	I-protein_type
peptidase	I-protein_type
with	O
a	O
requirement	O
for	O
Ca2	B-chemical
+	I-chemical
and	O
loss	O
of	O
an	O
internal	B-structure_element
nonapeptide	I-structure_element
for	O
full	B-protein_state
activation	I-protein_state
;	O
lack	O
of	O
structural	O
information	O
on	O
the	O
family	O
appears	O
to	O
have	O
prohibited	O
further	O
investigation	O
.	O

A	O
single	B-ptm
cleavage	I-ptm
was	O
observed	O
in	O
the	O
polypeptide	O
chain	O
at	O
Lys147	B-residue_name_number
(	O
Fig	O
.	O
1	O
,	O
A	O
and	O
B	O
),	O
where	O
both	O
ends	O
of	O
the	O
cleavage	B-site
site	I-site
are	O
fully	O
visible	O
and	O
well	O
ordered	O
in	O
the	O
electron	B-evidence
density	I-evidence
.	O

The	O
secondary	O
structure	O
of	O
PmC11	B-protein
from	O
the	O
crystal	B-evidence
structure	I-evidence
is	O
mapped	O
onto	O
its	O
sequence	O
with	O
the	O
position	O
of	O
the	O
PmC11	B-protein
catalytic	B-site
dyad	I-site
,	O
autocatalytic	B-site
cleavage	I-site
site	I-site
(	O
Lys147	B-residue_name_number
),	O
and	O
S1	B-site
binding	I-site
pocket	I-site
Asp	B-residue_name
(	O
Asp177	B-residue_name_number
)	O
highlighted	O
by	O
a	O
red	O
star	O
,	O
a	O
red	O
downturned	O
triangle	O
,	O
and	O
a	O
red	O
upturned	O
triangle	O
,	O
respectively	O
.	O

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

Helices	O
found	O
on	O
either	O
side	O
of	O
the	O
central	O
β	B-structure_element
-	I-structure_element
sheet	I-structure_element
are	O
shown	O
above	O
and	O
below	O
the	O
sheet	B-structure_element
,	O
respectively	O
.	O

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

Summary	O
of	O
PDBeFOLD	B-experimental_method
superposition	I-experimental_method
of	O
structures	O
found	O
to	O
be	O
most	O
similar	O
to	O
PmC11	B-protein
in	O
the	O
PBD	O
based	O
on	O
DaliLite	B-experimental_method

The	O
overall	O
structure	B-evidence
of	O
PmC11	B-protein
is	O
shown	O
in	O
gray	O
,	O
looking	O
down	O
into	O
the	O
catalytic	B-site
site	I-site
with	O
the	O
catalytic	B-site
dyad	I-site
in	O
red	O
.	O

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

The	O
two	O
ends	O
of	O
the	O
cleavage	B-site
site	I-site
are	O
remarkably	O
well	O
ordered	O
in	O
the	O
crystal	B-evidence
structure	I-evidence
and	O
displaced	O
from	O
one	O
another	O
by	O
19	O
.	O
5	O
Å	O
(	O
Fig	O
.	O
2A	O
).	O

Consequently	O
,	O
it	O
appears	O
feasible	O
that	O
the	O
helix	B-structure_element
attached	O
to	O
Lys147	B-residue_name_number
(	O
α3	B-structure_element
)	O
could	O
be	O
responsible	O
for	O
steric	O
autoinhibition	O
of	O
PmC11	B-protein
when	O
Lys147	B-residue_name_number
is	O
covalently	O
bonded	O
to	O
Ala148	B-residue_name_number
.	O

These	O
studies	O
revealed	O
that	O
there	O
was	O
no	O
apparent	O
cleavage	O
of	O
PmC11C179A	B-mutant
by	O
the	O
active	B-protein_state
enzyme	O
at	B-experimental_method
low	I-experimental_method
concentrations	I-experimental_method
of	O
PmC11	B-protein
and	O
that	O
only	O
limited	O
cleavage	O
was	O
observed	O
when	O
the	O
ratio	O
of	O
active	B-protein_state
enzyme	O
(	O
PmC11	B-protein
:	O
PmC11C179A	B-mutant
)	O
was	O
increased	B-experimental_method
to	I-experimental_method
∼	I-experimental_method
1	I-experimental_method
:	I-experimental_method
10	I-experimental_method
and	I-experimental_method
1	I-experimental_method
:	I-experimental_method
4	I-experimental_method
,	O
with	O
complete	O
cleavage	O
observed	O
at	O
a	O
ratio	B-experimental_method
of	I-experimental_method
1	I-experimental_method
:	I-experimental_method
1	I-experimental_method
(	O
Fig	O
.	O
2E	O
).	O

This	O
cleavage	B-ptm
subsequently	O
allows	O
movement	O
of	O
the	O
region	O
containing	O
Lys147	B-residue_name_number
and	O
the	O
active	B-site
site	I-site
to	O
open	B-protein_state
up	O
for	O
substrate	O
access	O
.	O

The	O
cleavage	O
of	O
Bz	B-chemical
-	I-chemical
R	I-chemical
-	I-chemical
AMC	I-chemical
by	O
PmC11	B-protein
was	O
measured	O
in	O
the	O
presence	O
of	O
the	O
cations	O
Ca2	B-chemical
+,	I-chemical
Mn2	B-chemical
+,	I-chemical
Zn2	B-chemical
+,	I-chemical
Co2	B-chemical
+,	I-chemical
Cu2	B-chemical
+,	I-chemical
Mg2	B-chemical
+,	I-chemical
and	O
Fe3	B-chemical
+	I-chemical
with	O
EGTA	B-chemical
as	O
a	O
negative	O
control	O
,	O
and	O
relative	B-experimental_method
fluorescence	I-experimental_method
measured	I-experimental_method
against	I-experimental_method
time	I-experimental_method
(	O
min	O
).	O

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

A	O
multiple	B-experimental_method
sequence	I-experimental_method
alignment	I-experimental_method
revealed	O
that	O
most	O
of	O
the	O
secondary	B-structure_element
structural	I-structure_element
elements	I-structure_element
are	O
conserved	B-protein_state
between	O
the	O
two	O
enzymes	O
,	O
although	O
they	O
are	O
only	O
∼	O
23	O
%	O
identical	O
(	O
Fig	O
.	O
1A	O
).	O

Nevertheless	O
,	O
PmC11	B-protein
may	O
be	O
a	O
good	O
model	O
for	O
the	O
core	O
structure	O
of	O
clostripain	B-protein
.	O

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

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

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

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

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

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

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

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

The	O
S	B-species
.	I-species
cerevisiae	I-species
18S	B-protein_type
rRNA	I-protein_type
acp	I-protein_type
transferase	I-protein_type
was	O
identified	O
in	O
a	O
systematic	O
genetic	O
screen	O
where	O
numerous	O
deletion	O
mutants	O
from	O
the	O
EUROSCARF	O
strain	O
collection	O
(	O
www	O
.	O
euroscarf	O
.	O
de	O
)	O
were	O
analyzed	O
by	O
HPLC	B-experimental_method
for	O
alterations	O
in	O
18S	B-chemical
rRNA	I-chemical
base	O
modifications	O
.	O

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

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

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

Although	O
the	O
acp	B-chemical
modification	O
of	O
18S	B-chemical
rRNA	I-chemical
is	O
highly	B-protein_state
conserved	I-protein_state
in	O
eukaryotes	B-taxonomy_domain
,	O
yeast	B-taxonomy_domain
Δtsr3	B-mutant
mutants	O
showed	O
only	O
a	O
minor	O
growth	O
defect	O
.	O

In	O
polysome	B-evidence
profiles	I-evidence
,	O
a	O
reduced	O
level	O
of	O
80S	B-complex_assembly
ribosomes	I-complex_assembly
and	O
a	O
strong	O
signal	O
for	O
free	O
60S	B-complex_assembly
subunits	O
was	O
observed	O
in	O
line	O
with	O
the	O
40S	B-complex_assembly
subunit	O
deficiency	O
(	O
Supplementary	O
Figure	O
S2G	O
).	O

Structure	B-evidence
of	O
Tsr3	B-protein

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

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

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

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

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

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

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

Instead	O
,	O
Tyw2	B-protein
has	O
a	O
fold	O
typical	O
for	O
the	O
class	B-protein_type
-	I-protein_type
I	I-protein_type
-	I-protein_type
or	I-protein_type
Rossmann	I-protein_type
-	I-protein_type
fold	I-protein_type
class	I-protein_type
of	I-protein_type
methyltransferases	I-protein_type
(	O
Supplementary	O
Figure	O
S5B	O
).	O

A	O
W66A	B-mutant
-	O
mutant	B-protein_state
of	O
SsTsr3	B-protein
(	O
W73	B-residue_name_number
in	O
VdTsr3	B-protein
)	O
does	O
not	O
bind	O
SAM	B-chemical
.	O

(	O
F	O
)	O
Primer	B-experimental_method
extension	I-experimental_method
(	O
upper	O
left	O
)	O
shows	O
a	O
strongly	O
reduced	O
acp	B-chemical
modification	O
of	O
yeast	B-taxonomy_domain
18S	B-chemical
rRNA	I-chemical
in	O
Δtsr3	B-mutant
cells	O
expressing	O
Tsr3	B-mutant
-	I-mutant
S62D	I-mutant
,	O
-	B-mutant
E111A	I-mutant
or	O
–	B-mutant
W114A	I-mutant
.	O

S	B-chemical
-	I-chemical
adenosylhomocysteine	I-chemical
which	O
lacks	O
the	O
methyl	O
group	O
of	O
SAM	B-chemical
binds	O
with	O
significantly	O
lower	O
affinity	B-evidence
(	O
KD	B-evidence
=	O
55	O
.	O
5	O
μM	O
)	O
(	O
Figure	O
5D	O
).	O

However	O
,	O
the	O
mutation	B-experimental_method
of	O
the	O
corresponding	O
residue	O
of	O
ScTsr3	B-protein
(	O
E111A	B-mutant
)	O
leads	O
to	O
a	O
significant	O
decrease	O
of	O
the	O
acp	B-protein_type
transferase	I-protein_type
activity	O
in	O
vivo	O
(	O
Figure	O
5F	O
).	O

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

The	O
m1acp3Ψ	B-chemical
base	O
is	O
located	O
at	O
the	O
tip	O
of	O
helix	B-structure_element
31	I-structure_element
on	O
the	O
18S	B-chemical
rRNA	I-chemical
(	O
Supplementary	O
Figure	O
S1B	O
)	O
which	O
,	O
together	O
with	O
helices	B-structure_element
18	I-structure_element
,	I-structure_element
24	I-structure_element
,	I-structure_element
34	I-structure_element
and	I-structure_element
44	I-structure_element
,	O
contribute	O
to	O
building	O
the	O
decoding	O
center	O
of	O
the	O
small	O
ribosomal	O
subunit	O
.	O

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

In	O
contrast	O
,	O
for	O
several	O
Tsr3	B-protein
mutants	O
(	O
SAM	B-protein_state
-	I-protein_state
binding	I-protein_state
and	O
cysteine	B-protein_state
mutations	I-protein_state
)	O
we	O
found	O
a	O
systematic	O
correlation	O
between	O
the	O
loss	O
of	O
acp	B-chemical
modification	O
and	O
the	O
efficiency	O
of	O
18S	B-chemical
rRNA	I-chemical
maturation	O
.	O

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

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

Early	O
cytoplasmic	O
pre	B-complex_assembly
-	I-complex_assembly
40S	I-complex_assembly
subunits	I-complex_assembly
still	O
containing	O
the	O
ribosome	B-protein_type
assembly	I-protein_type
factors	I-protein_type
Tsr1	B-protein
,	O
Ltv1	B-protein
,	O
Enp1	B-protein
and	O
Rio2	B-protein
were	O
not	O
or	O
only	O
partially	O
acp	B-protein_state
modified	I-protein_state
.	O

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

Structural	B-experimental_method
analyses	I-experimental_method
revealed	O
that	O
in	O
contrast	O
to	O
the	O
compact	B-protein_state
conformation	I-protein_state
of	O
the	O
dimeric	B-oligomeric_state
YfiB	B-protein
alone	B-protein_state
,	O
YfiBL43P	B-mutant
adopts	O
a	O
stretched	B-protein_state
conformation	I-protein_state
allowing	O
activated	B-protein_state
YfiB	B-protein
to	O
penetrate	O
the	O
peptidoglycan	B-chemical
(	O
PG	B-chemical
)	O
layer	O
and	O
access	O
YfiR	B-protein
.	O
YfiBL43P	B-mutant
shows	O
a	O
more	O
compact	O
PG	B-site
-	I-site
binding	I-site
pocket	I-site
and	O
much	O
higher	O
PG	B-evidence
binding	I-evidence
affinity	I-evidence
than	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
YfiB	B-protein
,	O
suggesting	O
a	O
tight	O
correlation	O
between	O
PG	O
binding	O
and	O
YfiB	B-protein
activation	O
.	O

Bis	B-chemical
-(	I-chemical
3	I-chemical
’-	I-chemical
5	I-chemical
’)-	I-chemical
cyclic	I-chemical
dimeric	I-chemical
GMP	I-chemical
(	O
c	B-chemical
-	I-chemical
di	I-chemical
-	I-chemical
GMP	I-chemical
)	O
is	O
a	O
ubiquitous	O
second	O
messenger	O
that	O
bacteria	B-taxonomy_domain
use	O
to	O
facilitate	O
behavioral	O
adaptations	O
to	O
their	O
ever	O
-	O
changing	O
environment	O
.	O

Intriguingly	O
,	O
studies	O
in	O
diverse	O
species	O
have	O
revealed	O
that	O
a	O
single	O
bacterium	B-taxonomy_domain
can	O
have	O
dozens	O
of	O
DGCs	B-protein_type
and	O
PDEs	B-protein_type
(	O
Hickman	O
et	O
al	O
.,;	O
Kirillina	O
et	O
al	O
.,;	O
Kulasakara	O
et	O
al	O
.,;	O
Tamayo	O
et	O
al	O
.,).	O

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

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

Moreover	O
,	O
we	O
found	O
that	O
Vitamin	B-chemical
B6	I-chemical
(	O
VB6	B-chemical
)	O
or	O
L	B-chemical
-	I-chemical
Trp	I-chemical
can	O
bind	O
YfiR	B-protein
with	O
an	O
affinity	B-evidence
in	O
the	O
ten	O
millimolar	O
range	O
.	O

We	O
obtained	O
two	O
crystal	B-evidence
forms	I-evidence
of	O
YfiB	B-protein
(	O
residues	O
34	B-residue_range
–	I-residue_range
168	I-residue_range
,	O
lacking	B-protein_state
the	O
signal	B-structure_element
peptide	I-structure_element
from	O
residues	O
1	B-residue_range
–	I-residue_range
26	I-residue_range
and	O
periplasmic	O
residues	O
27	B-residue_range
–	I-residue_range
33	I-residue_range
),	O
crystal	O
forms	O
I	O
and	O
II	O
,	O
belonging	O
to	O
space	O
groups	O
P21	O
and	O
P41	O
,	O
respectively	O
.	O

Two	O
dimeric	B-oligomeric_state
types	O
of	O
YfiB	B-protein
dimer	B-oligomeric_state
.	O
(	O
A	O
–	O
C	O
)	O
The	O
“	O
head	B-protein_state
to	I-protein_state
head	I-protein_state
”	O
dimer	B-oligomeric_state
.	O

(	O
A	O
)	O
and	O
(	O
E	O
)	O
indicate	O
the	O
front	O
views	O
of	O
the	O
two	O
dimers	B-oligomeric_state
,	O
(	O
B	O
)	O
and	O
(	O
F	O
)	O
indicate	O
the	O
top	O
views	O
of	O
the	O
two	O
dimers	B-oligomeric_state
,	O
and	O
(	O
C	O
)	O
and	O
(	O
D	O
)	O
indicate	O
the	O
details	O
of	O
the	O
two	O
dimeric	B-site
interfaces	I-site

The	O
crystal	B-evidence
structure	I-evidence
of	O
YfiB	B-protein
monomer	B-oligomeric_state
consists	O
of	O
a	O
five	B-structure_element
-	I-structure_element
stranded	I-structure_element
β	I-structure_element
-	I-structure_element
sheet	I-structure_element
(	O
β1	B-structure_element
-	I-structure_element
2	I-structure_element
-	I-structure_element
5	I-structure_element
-	I-structure_element
3	I-structure_element
-	I-structure_element
4	I-structure_element
)	O
flanked	O
by	O
five	B-structure_element
α	I-structure_element
-	I-structure_element
helices	I-structure_element
(	O
α1	B-structure_element
–	I-structure_element
5	I-structure_element
)	O
on	O
one	O
side	O
.	O

The	O
dimeric	B-oligomeric_state
interaction	B-bond_interaction
is	I-bond_interaction
mainly	I-bond_interaction
hydrophilic	I-bond_interaction
,	O
occurring	O
among	O
the	O
main	O
-	O
chain	O
and	O
side	O
-	O
chain	O
atoms	O
of	O
N68	B-residue_name_number
,	O
L69	B-residue_name_number
,	O
D70	B-residue_name_number
and	O
R71	B-residue_name_number
on	O
the	O
α2	B-structure_element
-	I-structure_element
α3	I-structure_element
loops	I-structure_element
and	O
R116	B-residue_name_number
and	O
S120	B-residue_name_number
on	O
the	O
α4	B-structure_element
helices	I-structure_element
of	O
both	O
molecules	O
,	O
resulting	O
in	O
a	O
complex	O
hydrogen	B-site
bond	I-site
network	I-site
(	O
Fig	O
.	O
2D	O
–	O
F	O
).	O

The	O
YfiB	B-complex_assembly
-	I-complex_assembly
YfiR	I-complex_assembly
interaction	O

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

It	O
is	O
likely	O
that	O
these	O
residues	O
may	O
be	O
involved	O
in	O
the	O
conformational	O
changes	O
of	O
YfiB	B-protein
that	O
are	O
related	O
to	O
YfiR	B-protein
sequestration	O
(	O
Fig	O
.	O
3C	O
).	O

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

For	O
simplicity	O
,	O
we	O
only	O
discuss	O
the	O
“	O
head	B-protein_state
to	I-protein_state
head	I-protein_state
”	O
dimer	B-oligomeric_state
in	O
the	O
following	O
text	O
.	O

PG	B-protein_type
-	I-protein_type
associated	I-protein_type
lipoprotein	I-protein_type
(	O
Pal	B-protein_type
)	O
is	O
highly	B-protein_state
conserved	I-protein_state
in	O
Gram	B-taxonomy_domain
-	I-taxonomy_domain
negative	I-taxonomy_domain
bacteria	I-taxonomy_domain
and	O
anchors	O
to	O
the	O
outer	O
membrane	O
through	O
an	O
N	O
-	O
terminal	O
lipid	O
attachment	O
and	O
to	O
PG	B-chemical
layer	O
through	O
its	O
periplasmic	B-structure_element
domain	I-structure_element
,	O
which	O
is	O
implicated	O
in	O
maintaining	O
outer	O
membrane	O
integrity	O
.	O

Interestingly	O
,	O
superposition	B-experimental_method
of	O
apo	B-protein_state
YfiB	B-protein
with	O
YfiR	B-protein_state
-	I-protein_state
bound	I-protein_state
YfiBL43P	B-mutant
revealed	O
that	O
the	O
PG	B-site
-	I-site
binding	I-site
region	I-site
is	O
largely	O
altered	O
mainly	O
due	O
to	O
different	B-protein_state
conformation	I-protein_state
of	O
the	O
N68	B-residue_name_number
containing	O
loop	B-structure_element
.	O

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

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

Previous	O
studies	O
indicated	O
that	O
YfiR	B-protein
constitutes	O
a	O
YfiB	B-protein
-	O
independent	O
sensing	O
device	O
that	O
can	O
activate	O
YfiN	B-protein
in	O
response	O
to	O
the	O
redox	O
status	O
of	O
the	O
periplasm	O
,	O
and	O
we	O
have	O
reported	O
YfiR	B-protein
structures	B-evidence
in	O
both	O
the	O
non	B-protein_state
-	I-protein_state
oxidized	I-protein_state
and	O
the	O
oxidized	B-protein_state
states	O
earlier	O
,	O
revealing	O
that	O
the	O
Cys145	B-residue_name_number
-	O
Cys152	B-residue_name_number
disulfide	B-ptm
bond	I-ptm
plays	O
an	O
essential	O
role	O
in	O
maintaining	O
the	O
correct	O
folding	O
of	O
YfiR	B-protein
(	O
Yang	O
et	O
al	O
.,).	O

Interestingly	O
,	O
at	O
a	O
concentration	O
higher	O
than	O
8	O
mmol	O
/	O
L	O
,	O
VB6	B-chemical
lost	O
its	O
ability	O
to	O
inhibit	O
biofilm	O
formation	O
,	O
implying	O
that	O
the	O
VB6	B-chemical
-	O
involving	O
regulatory	O
mechanism	O
is	O
highly	O
complicated	O
and	O
remains	O
to	O
be	O
further	O
investigated	O
.	O

Of	O
note	O
,	O
both	O
VB6	B-chemical
and	O
L	B-chemical
-	I-chemical
Trp	I-chemical
have	O
been	O
reported	O
to	O
correlate	O
with	O
biofilm	O
formation	O
in	O
certain	O
Gram	B-taxonomy_domain
-	I-taxonomy_domain
negative	I-taxonomy_domain
bacteria	I-taxonomy_domain
(	O
Grubman	O
et	O
al	O
.,;	O
Shimazaki	O
et	O
al	O
.,).	O

Based	O
on	O
our	O
results	O
,	O
we	O
concluded	O
that	O
VB6	B-chemical
or	O
L	B-chemical
-	I-chemical
Trp	I-chemical
can	O
bind	O
to	O
YfiR	B-protein
,	O
however	O
,	O
VB6	B-chemical
or	O
L	B-chemical
-	I-chemical
Trp	I-chemical
alone	B-protein_state
may	O
have	O
little	O
effects	O
in	O
interrupting	O
the	O
YfiB	B-complex_assembly
-	I-complex_assembly
YfiR	I-complex_assembly
interaction	O
,	O
the	O
mechanism	O
by	O
which	O
VB6	B-chemical
or	O
L	B-chemical
-	I-chemical
Trp	I-chemical
inhibits	O
biofilm	O
formation	O
remains	O
unclear	O
and	O
requires	O
further	O
investigation	O
.	O

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

The	O
loop	B-structure_element
connecting	O
Cys26	B-residue_name_number
and	O
Gly34	B-residue_name_number
of	O
YfiB	B-protein
is	O
modeled	O
.	O

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

Our	O
study	O
reveals	O
the	O
mechanism	O
for	O
regulation	O
of	O
H3K9me3	B-protein_type
and	O
hm	B-chemical
-	I-chemical
DNA	I-chemical
recognition	O
by	O
URHF1	B-protein
.	O

However	O
,	O
how	O
UHRF1	B-protein
regulates	O
the	O
recognition	O
of	O
these	O
two	O
repressive	O
epigenetic	O
marks	O
and	O
recruits	O
DNMT1	B-protein
for	O
chromatin	O
localization	O
remain	O
largely	O
unknown	O
.	O

Therefore	O
,	O
UHRF1	B-protein
may	O
engage	O
in	O
a	O
sophisticated	O
regulation	O
for	O
its	O
chromatin	O
localization	O
and	O
recruitment	O
of	O
DNMT1	B-protein
through	O
a	O
mechanism	O
yet	O
to	O
be	O
fully	O
elucidated	O
.	O

To	O
test	O
above	O
hypothesis	O
,	O
we	O
performed	O
glutathione	B-experimental_method
S	I-experimental_method
-	I-experimental_method
transferase	I-experimental_method
(	I-experimental_method
GST	I-experimental_method
)	I-experimental_method
pull	I-experimental_method
-	I-experimental_method
down	I-experimental_method
assay	I-experimental_method
using	O
various	O
truncations	B-experimental_method
of	O
UHRF1	B-protein
.	O

The	O
presence	B-protein_state
of	I-protein_state
the	O
Spacer	B-structure_element
markedly	O
impaired	O
the	O
interaction	O
between	O
TTD	B-structure_element
–	I-structure_element
PHD	I-structure_element
and	O
H3K9me3	B-protein_type
(	O
Fig	O
.	O
2c	O
).	O

The	O
results	O
indicate	O
that	O
the	O
Spacer	B-structure_element
directly	O
binds	B-protein_state
to	I-protein_state
the	O
TTD	B-structure_element
and	O
inhibits	O
its	O
interaction	O
with	O
H3K9me3	B-protein_type
.	O

Pre	B-experimental_method
-	I-experimental_method
incubation	I-experimental_method
of	O
the	O
SRA	B-structure_element
also	O
modestly	O
impaired	O
PHD	B-structure_element
–	O
H3K9me0	B-protein_type
interaction	O
.	O

SpacerΔ642	B-mutant
–	I-mutant
651	I-mutant
,	O
SpacerΔ650	B-mutant
–	I-mutant
654	I-mutant
and	O
SpacerΔ655	B-mutant
–	I-mutant
659	I-mutant
also	O
decreased	O
binding	B-evidence
affinities	I-evidence
,	O
indicating	O
that	O
residues	O
642	B-residue_range
–	I-residue_range
674	I-residue_range
are	O
important	O
for	O
TTD	B-structure_element
–	I-structure_element
Spacer	I-structure_element
interaction	O
.	O

Mutations	B-experimental_method
K648D	B-mutant
and	O
S651D	B-mutant
of	O
the	O
Spacer	B-structure_element
decreased	O
their	O
binding	B-evidence
affinities	I-evidence
to	O
the	O
TTD	B-structure_element
,	O
and	O
mutation	B-experimental_method
R649A	B-mutant
of	O
the	O
Spacer	B-structure_element
showed	O
more	O
significant	O
decrease	O
(∼	O
13	O
-	O
fold	O
)	O
in	O
the	O
binding	B-evidence
affinity	I-evidence
(	O
Fig	O
.	O
3f	O
).	O

Thus	O
,	O
the	O
Spacer	B-structure_element
may	O
disrupt	O
the	O
TTD	B-structure_element
–	I-structure_element
Linker	I-structure_element
interaction	O
and	O
inhibits	O
the	O
recognition	O
of	O
H3K9me3	B-protein_type
by	O
TTD	B-structure_element
–	I-structure_element
PHD	I-structure_element
.	O

Notably	O
,	O
although	O
the	O
Linker	B-structure_element
(	O
in	O
the	O
context	O
of	O
TTD	B-structure_element
-	I-structure_element
PHD	I-structure_element
)	O
impairs	O
the	O
TTD	B-structure_element
–	I-structure_element
Spacer	I-structure_element
interaction	O
to	O
some	O
extent	O
,	O
the	O
isolated	O
Spacer	B-structure_element
could	O
still	O
bind	O
to	O
TTD	B-structure_element
–	I-structure_element
PHD	I-structure_element
with	O
moderate	O
binding	B-evidence
affinity	I-evidence
(	O
KD	B-evidence
=	O
10	O
.	O
68	O
μM	O
),	O
supporting	O
the	O
existence	O
of	O
the	O
intramolecular	O
interaction	O
within	O
UHRF1	B-protein
.	O

To	O
test	O
whether	O
TTD	B-structure_element
–	I-structure_element
Spacer	I-structure_element
association	O
exists	O
in	O
the	O
context	O
of	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
UHRF1	B-protein
,	O
we	O
used	O
various	O
truncations	B-experimental_method
of	O
UHRF1	B-protein
in	O
the	O
GST	B-experimental_method
pull	I-experimental_method
-	I-experimental_method
down	I-experimental_method
assay	I-experimental_method
.	O

Taken	O
together	O
,	O
UHRF1	B-protein
adopts	O
a	O
closed	B-protein_state
conformation	O
,	O
in	O
which	O
the	O
Spacer	B-structure_element
binds	B-protein_state
to	I-protein_state
the	O
TTD	B-structure_element
through	O
competing	O
with	O
the	O
Linker	B-structure_element
,	O
and	O
therefore	O
inhibits	O
H3K9me3	B-protein_type
recognition	O
by	O
UHRF1	B-protein
.	O

Because	O
the	O
TTD	B-structure_element
is	O
always	O
associated	O
with	O
the	O
PHD	B-structure_element
,	O
whether	O
the	O
pattern	O
of	O
TTD	B-complex_assembly
–	I-complex_assembly
H3K9me3	I-complex_assembly
interaction	O
exists	O
in	O
vivo	O
remains	O
unknown	O
.	O

Nevertheless	O
,	O
comparison	B-experimental_method
of	O
TTD	B-complex_assembly
–	I-complex_assembly
H3K9me3	I-complex_assembly
and	O
TTD	B-structure_element
–	I-structure_element
Spacer	I-structure_element
structures	B-evidence
indicates	O
that	O
H3K9me3	B-protein_type
and	O
the	O
Spacer	B-structure_element
overlap	O
on	O
the	O
surface	O
of	O
the	O
TTD	B-structure_element
(	O
Supplementary	O
Fig	O
.	O
4d	O
),	O
suggesting	O
that	O
the	O
Spacer	B-structure_element
might	O
block	O
the	O
H3K9me3	B-protein_type
recognition	O
by	O
the	O
isolated	O
TTD	B-structure_element
.	O

We	O
next	O
tested	O
whether	O
such	O
inhibition	O
also	O
occurs	O
in	O
the	O
context	O
of	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
UHRF1	B-protein
.	O

Taken	O
together	O
,	O
the	O
Spacer	B-structure_element
binds	B-protein_state
to	I-protein_state
the	O
TTD	B-structure_element
and	O
inhibits	O
H3K9me3	B-protein_type
recognition	O
by	O
UHRF1	B-protein
through	O
(	O
i	O
)	O
disrupting	O
TTD	B-structure_element
–	I-structure_element
Linker	I-structure_element
interaction	O
,	O
which	O
is	O
essential	O
for	O
H3K9me3	B-protein_type
recognition	O
by	O
TTD	B-structure_element
–	I-structure_element
PHD	I-structure_element
,	O
(	O
ii	O
)	O
prohibiting	O
H3K9me3	B-protein_type
binding	O
to	O
the	O
isolated	O
TTD	B-structure_element
.	O

Residues	O
N228	B-residue_name_number
/	O
R235	B-residue_name_number
from	O
the	O
TTD	B-structure_element
and	O
G653	B-residue_name_number
/	O
G654	B-residue_name_number
from	O
the	O
Spacer	B-structure_element
were	O
chosen	O
according	O
to	O
the	O
TTD	B-structure_element
–	I-structure_element
Spacer	I-structure_element
complex	O
structure	B-evidence
(	O
Supplementary	O
Fig	O
.	O
5c	O
)	O
so	O
that	O
the	O
replaced	O
Cysteine	B-residue_name
residues	O
(	O
one	O
from	O
the	O
TTD	B-structure_element
and	O
one	O
from	O
the	O
Spacer	B-structure_element
)	O
are	O
physically	O
close	O
enough	O
to	O
each	O
other	O
to	O
form	O
a	O
disulphide	B-ptm
bond	I-ptm
in	O
the	O
absence	B-protein_state
of	I-protein_state
reducing	O
reagent	O
(	O
dithiothreitol	B-chemical
,	O
DTT	B-chemical
).	O

These	O
results	O
indicate	O
that	O
the	O
Spacer	B-structure_element
not	O
only	O
binds	B-protein_state
to	I-protein_state
the	O
TTD	B-structure_element
and	O
inhibits	O
H3K9me3	B-protein_type
recognition	O
when	O
UHRF1	B-protein
adopts	O
closed	B-protein_state
conformation	O
,	O
but	O
also	O
facilitates	O
hm	B-chemical
-	I-chemical
DNA	I-chemical
recognition	O
by	O
the	O
SRA	B-structure_element
when	O
UHRF1	B-protein
binds	B-protein_state
to	I-protein_state
hm	B-chemical
-	I-chemical
DNA	I-chemical
.	O

The	O
structure	B-evidence
shows	O
that	O
the	O
SRA	B-structure_element
binds	B-protein_state
to	I-protein_state
hm	B-chemical
-	I-chemical
DNA	I-chemical
in	O
a	O
manner	O
similar	O
to	O
that	O
observed	O
in	O
the	O
previously	O
reported	O
SRA	B-complex_assembly
-	I-complex_assembly
hm	I-complex_assembly
-	I-complex_assembly
DNA	I-complex_assembly
structures	B-evidence
.	O

To	O
investigate	O
the	O
role	O
of	O
the	O
Spacer	B-structure_element
in	O
the	O
regulation	O
of	O
UHRF1	B-protein
function	O
,	O
we	O
transiently	B-experimental_method
overexpressed	I-experimental_method
GFP	B-protein_state
-	I-protein_state
tagged	I-protein_state
wild	B-protein_state
type	I-protein_state
or	O
mutants	B-protein_state
of	O
UHRF1	B-protein
in	O
NIH3T3	O
cells	O
to	O
determine	O
their	O
subcellular	O
localization	O
.	O

For	O
example	O
,	O
UHRF1	B-protein
mutant	B-protein_state
(	O
within	O
TTD	B-structure_element
domain	O
)	O
lacking	B-protein_state
H3K9me3	B-evidence
-	I-evidence
binding	I-evidence
affinity	I-evidence
largely	O
reduces	O
its	O
co	O
-	O
localization	O
with	O
heterochromatin	O
.	O

In	O
the	O
absence	B-protein_state
of	I-protein_state
hm	B-chemical
-	I-chemical
DNA	I-chemical
,	O
only	O
UHRF1ΔTTD	B-mutant
bound	B-protein_state
to	I-protein_state
RFTSDNMT1	B-protein
,	O
whereas	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
UHRF1	B-protein
,	O
UHRF1ΔSRA	B-mutant
and	O
UHRF1Δ627	B-mutant
–	I-mutant
674	I-mutant
showed	O
undetectable	O
interaction	O
(	O
Fig	O
.	O
5e	O
).	O

In	O
addition	O
,	O
this	O
regulatory	O
process	O
should	O
be	O
further	O
characterized	O
using	O
advanced	O
techniques	O
,	O
such	O
as	O
single	B-experimental_method
molecular	I-experimental_method
measurement	I-experimental_method
.	O

Intriguingly	O
,	O
UHRF2	B-protein
(	O
the	O
only	O
mammalian	B-taxonomy_domain
homologue	O
of	O
UHRF1	B-protein
)	O
and	O
UHRF1	B-protein
show	O
very	O
high	O
sequence	O
similarities	O
for	O
all	O
the	O
domains	O
but	O
very	O
low	O
similarity	O
for	O
the	O
Spacer	B-structure_element
(	O
Supplementary	O
Fig	O
.	O
7c	O
).	O

One	O
of	O
the	O
key	O
questions	O
in	O
the	O
field	O
of	O
DNA	B-chemical
methylation	B-ptm
is	O
why	O
UHRF1	B-protein
contains	O
modules	O
recognizing	O
two	O
repressive	O
epigenetic	O
marks	O
:	O
H3K9me3	B-protein_type
(	O
by	O
TTD	B-structure_element
–	I-structure_element
PHD	I-structure_element
)	O
and	O
hm	B-chemical
-	I-chemical
DNA	I-chemical
(	O
by	O
the	O
SRA	B-structure_element
).	O

Previous	O
studies	O
show	O
that	O
chromatin	O
localization	O
of	O
UHRF1	B-protein
is	O
dependent	O
on	O
hm	B-chemical
-	I-chemical
DNA	I-chemical
,	O
whereas	O
other	O
studies	O
indicate	O
that	O
histone	B-protein_type
H3K9me3	B-protein_type
recognition	O
and	O
hm	B-chemical
-	I-chemical
DNA	I-chemical
association	O
are	O
both	O
required	O
for	O
UHRF1	B-protein
-	O
mediated	O
maintenance	O
DNA	B-chemical
methylation	B-ptm
.	O

Therefore	O
,	O
genomic	O
localization	O
of	O
UHRF1	B-protein
is	O
primarily	O
determined	O
by	O
its	O
recognition	O
of	O
hm	B-chemical
-	I-chemical
DNA	I-chemical
,	O
which	O
allows	O
UHRF1	B-protein
to	O
adopt	O
an	O
open	B-protein_state
form	O
and	O
promotes	O
its	O
histone	B-protein_type
tail	O
recognition	O
for	O
proper	O
genomic	O
localization	O
and	O
function	O
.	O

Recent	O
study	O
indicates	O
that	O
histone	B-protein_type
tail	O
association	O
of	O
UHRF1	B-protein
(	O
by	O
the	O
PHD	B-structure_element
domain	O
)	O
is	O
required	O
for	O
histone	B-protein_type
H3	B-protein_type
ubiquitylation	B-ptm
,	O
which	O
is	O
dependent	O
on	O
ubiquitin	B-protein_type
ligase	I-protein_type
activity	O
of	O
the	O
RING	B-structure_element
domain	O
of	O
UHRF1	B-protein
(	O
ref	O
.).	O

Moreover	O
,	O
structural	B-experimental_method
analyses	I-experimental_method
of	O
DNMT1	B-complex_assembly
–	I-complex_assembly
DNA	I-complex_assembly
and	O
SRA	B-complex_assembly
–	I-complex_assembly
DNA	I-complex_assembly
complexes	O
also	O
indicate	O
that	O
it	O
is	O
impossible	O
for	O
DNMT1	B-protein
to	O
methylate	O
the	O
hm	B-chemical
-	I-chemical
DNA	I-chemical
that	O
UHRF1	B-protein
binds	B-protein_state
to	I-protein_state
because	O
of	O
steric	O
hindrance	O
.	O

The	O
S	O
phase	O
-	O
dependent	O
interaction	O
between	O
UHRF1	B-protein
and	O
DNMT1	B-protein
(	O
refs	O
)	O
suggest	O
that	O
DNMT1	B-protein
may	O
also	O
undergo	O
conformation	O
changes	O
so	O
that	O
RFTSDNMT1	B-protein
binds	B-protein_state
to	I-protein_state
UHRF1	B-protein
and	O
the	O
catalytic	B-structure_element
domain	I-structure_element
of	O
DNMT1	B-protein
binds	B-protein_state
to	I-protein_state
hm	B-chemical
-	I-chemical
DNA	I-chemical
for	O
reaction	O
.	O

The	O
bound	O
proteins	O
were	O
analysed	O
in	O
SDS	B-experimental_method
–	I-experimental_method
PAGE	I-experimental_method
followed	O
by	O
Coomassie	O
blue	O
staining	O
.	O

Sequences	O
of	O
the	O
peptides	O
are	O
indicated	O
in	O
Supplementary	O
Table	O
1	O
.	O
(	O
c	O
)	O
Histone	B-protein_type
peptides	O
do	O
not	O
affect	O
hm	B-evidence
-	I-evidence
DNA	I-evidence
-	I-evidence
binding	I-evidence
affinity	I-evidence
of	O
UHRF1	B-protein
.	O

Intramolecular	O
interactions	O
inhibit	O
histone	B-protein_type
recognition	O
by	O
UHRF1	B-protein
.	O

The	O
estimated	O
binding	B-evidence
affinities	I-evidence
(	O
KD	B-evidence
)	O
were	O
listed	O
.	O

TTD	B-complex_assembly
–	I-complex_assembly
PHD	I-complex_assembly
–	I-complex_assembly
H3K9me3	I-complex_assembly
complex	O
is	O
coloured	O
in	O
grey	O
,	O
and	O
the	O
PHD	B-structure_element
and	O
H3K9me3	B-protein_type
are	O
omitted	O
for	O
simplicity	O
.	O

The	O
bound	O
proteins	O
were	O
analysed	O
in	O
SDS	B-experimental_method
–	I-experimental_method
PAGE	I-experimental_method
and	O
Coomassie	B-experimental_method
blue	I-experimental_method
staining	I-experimental_method
(	O
left	O
)	O
and	O
quantified	O
by	O
band	B-experimental_method
densitometry	I-experimental_method
(	O
right	O
).	O

(	O
d	O
)	O
Histone	B-experimental_method
peptide	I-experimental_method
pull	I-experimental_method
-	I-experimental_method
down	I-experimental_method
assay	I-experimental_method
using	O
UHRF1	B-protein
mutants	B-protein_state
as	O
indicated	O
.	O

The	O
estimated	O
binding	B-evidence
affinities	I-evidence
(	O
KD	B-evidence
)	O
are	O
listed	O
above	O
.	O
(	O
d	O
)	O
Subcellular	O
localization	O
of	O
GFP	B-protein_state
-	I-protein_state
tagged	I-protein_state
wild	B-protein_state
-	I-protein_state
type	I-protein_state
or	O
indicated	O
mutants	B-protein_state
of	O
UHRF1	B-protein
in	O
NIH3T3	O
cells	O
.	O

The	O
percentages	O
of	O
cells	O
showing	O
co	O
-	O
localization	O
with	O
DAPI	B-chemical
foci	O
were	O
counted	O
from	O
at	O
least	O
100	O
cells	O
and	O
shown	O
on	O
the	O
left	O
of	O
the	O
corresponding	O
representative	O
confocal	B-experimental_method
microscopy	I-experimental_method
.	O

This	O
paper	O
presents	O
the	O
X	B-evidence
-	I-evidence
ray	I-evidence
crystallographic	I-evidence
structures	I-evidence
of	O
oligomers	B-oligomeric_state
formed	O
by	O
a	O
20	B-residue_range
-	I-residue_range
residue	I-residue_range
peptide	I-residue_range
segment	I-residue_range
derived	O
from	O
Aβ	B-protein
.	O

The	O
development	O
of	O
a	O
peptide	O
in	O
which	O
Aβ17	B-protein
–	B-residue_range
36	I-residue_range
is	O
stabilized	O
as	O
a	O
β	B-structure_element
-	I-structure_element
hairpin	I-structure_element
is	O
described	O
,	O
and	O
the	O
X	B-evidence
-	I-evidence
ray	I-evidence
crystallographic	I-evidence
structures	I-evidence
of	O
oligomers	B-oligomeric_state
it	O
forms	O
are	O
reported	O
.	O

An	O
N	O
-	O
methyl	O
group	O
at	O
position	O
33	B-residue_number
blocks	O
uncontrolled	O
aggregation	O
.	O

The	O
peptide	O
readily	B-evidence
crystallizes	I-evidence
as	O
a	O
folded	B-protein_state
β	B-structure_element
-	I-structure_element
hairpin	I-structure_element
,	O
which	O
assembles	O
hierarchically	O
in	O
the	O
crystal	B-evidence
lattice	I-evidence
.	O

Treatment	O
of	O
the	O
mixture	O
of	O
low	O
molecular	O
weight	O
oligomers	B-oligomeric_state
with	O
hexafluoroisopropanol	B-chemical
resulted	O
in	O
the	O
dissociation	O
of	O
the	O
putative	O
dodecamers	B-oligomeric_state
,	O
nonamers	B-oligomeric_state
,	O
and	O
hexamers	B-oligomeric_state
into	O
trimers	B-oligomeric_state
and	O
monomers	B-oligomeric_state
,	O
suggesting	O
that	O
trimers	B-oligomeric_state
may	O
be	O
the	O
building	O
block	O
of	O
the	O
dodecamers	B-oligomeric_state
,	O
nonamers	B-oligomeric_state
,	O
and	O
hexamers	B-oligomeric_state
.	O

The	O
sizes	O
of	O
APFs	B-complex_assembly
prepared	O
in	O
vitro	O
vary	O
among	O
different	O
studies	O
.	O

Quist	O
et	O
al	O
.	O
observed	O
APFs	B-complex_assembly
with	O
an	O
outer	O
diameter	O
of	O
16	O
nm	O
embedded	O
in	O
a	O
lipid	O
bilayer	O
.	O

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

Locking	O
Aβ	B-protein
into	O
a	O
β	B-structure_element
-	I-structure_element
hairpin	I-structure_element
structure	O
resulted	O
in	O
the	O
formation	O
Aβ	B-protein
oligomers	B-oligomeric_state
,	O
which	O
were	O
observed	O
by	O
size	B-experimental_method
exclusion	I-experimental_method
chromatography	I-experimental_method
(	O
SEC	B-experimental_method
)	O
and	O
SDS	B-experimental_method
-	I-experimental_method
PAGE	I-experimental_method
.	O

We	O
mutated	B-experimental_method
these	O
residues	O
because	O
they	O
occupy	O
the	O
same	O
position	O
as	O
the	O
δOrn	B-structure_element
that	O
connects	O
D23	B-residue_name_number
and	O
A30	B-residue_name_number
in	O
peptide	B-mutant
1	I-mutant
.	O

After	O
determining	O
the	O
X	B-evidence
-	I-evidence
ray	I-evidence
crystallographic	I-evidence
structure	I-evidence
of	O
peptide	B-mutant
4	I-mutant
we	O
reintroduced	B-experimental_method
the	O
native	O
phenylalanine	B-residue_name
at	O
position	O
19	B-residue_number
and	O
the	O
methionine	B-residue_name
at	O
position	O
35	B-residue_number
to	O
afford	O
peptide	B-mutant
2	I-mutant
.	O

Peptides	B-mutant
2	I-mutant
–	I-mutant
4	I-mutant
were	O
purified	O
by	O
RP	B-experimental_method
-	I-experimental_method
HPLC	I-experimental_method
.	O

The	O
optimized	O
conditions	O
consist	O
of	O
0	O
.	O
1	O
M	O
HEPES	B-chemical
at	O
pH	O
6	O
.	O
4	O
with	O
31	O
%	O
Jeffamine	B-chemical
M	I-chemical
-	I-chemical
600	I-chemical
for	O
peptide	B-mutant
4	I-mutant
and	O
0	O
.	O
1	O
M	O
HEPES	B-chemical
pH	O
7	O
.	O
1	O
with	O
29	O
%	O
Jeffamine	B-chemical
M	I-chemical
-	I-chemical
600	I-chemical
for	O
peptide	B-mutant
2	I-mutant
.	O

The	O
X	B-evidence
-	I-evidence
ray	I-evidence
crystallographic	I-evidence
structure	I-evidence
of	O
peptide	B-mutant
2	I-mutant
reveals	O
that	O
it	O
folds	O
to	O
form	O
a	O
twisted	B-structure_element
β	I-structure_element
-	I-structure_element
hairpin	I-structure_element
comprising	O
two	O
β	B-structure_element
-	I-structure_element
strands	I-structure_element
connected	O
by	O
a	O
loop	B-structure_element
(	O
Figure	O
2A	O
).	O

The	O
disulfide	B-ptm
linkages	I-ptm
suffered	O
radiation	O
damage	O
under	O
synchrotron	O
radiation	O
.	O

Two	O
crystallographically	O
distinct	O
trimers	B-oligomeric_state
comprise	O
the	O
peptide	B-chemical
portion	O
of	O
the	O
asymmetric	O
unit	O
.	O

A	O
network	O
of	O
18	O
intermolecular	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
helps	O
stabilize	O
the	O
trimer	B-oligomeric_state
.	O

Hydrophobic	B-bond_interaction
contacts	I-bond_interaction
between	O
residues	O
at	O
the	O
three	O
corners	O
of	O
the	O
trimer	B-oligomeric_state
,	O
where	O
the	O
β	B-structure_element
-	I-structure_element
hairpins	I-structure_element
meet	O
,	O
further	O
stabilize	O
the	O
trimer	B-oligomeric_state
.	O

Each	O
of	O
the	O
12	O
β	B-structure_element
-	I-structure_element
hairpins	I-structure_element
constitutes	O
an	O
edge	O
of	O
the	O
octahedron	B-protein_state
,	O
and	O
the	O
triangular	B-protein_state
trimers	B-oligomeric_state
occupy	O
four	O
of	O
the	O
eight	O
faces	O
of	O
the	O
octahedron	B-protein_state
.	O

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

The	O
exterior	O
of	O
the	O
dodecamer	B-oligomeric_state
displays	O
four	O
F20	B-residue_name_number
faces	O
(	O
Figure	O
S3	O
).	O

Two	O
morphologically	O
distinct	O
interactions	O
between	O
trimers	B-oligomeric_state
occur	O
at	O
the	O
interfaces	B-site
of	O
the	O
five	O
dodecamers	B-oligomeric_state
:	O
one	O
in	O
which	O
the	O
trimers	B-oligomeric_state
are	O
eclipsed	B-protein_state
(	O
Figure	O
5B	O
),	O
and	O
one	O
in	O
which	O
the	O
trimers	B-oligomeric_state
are	O
staggered	B-protein_state
(	O
Figure	O
5C	O
).	O

The	O
annular	B-site
pore	I-site
contains	O
three	O
eclipsed	B-protein_state
interfaces	B-site
and	O
two	O
staggered	B-protein_state
interfaces	B-site
.	O

Ten	O
Aβ25	B-protein
–	B-residue_range
28	I-residue_range
loops	B-structure_element
from	O
the	O
vertices	O
of	O
the	O
five	O
dodecamers	B-oligomeric_state
line	O
the	O
hole	O
in	O
the	O
center	O
of	O
the	O
pore	B-site
.	O

X	B-evidence
-	I-evidence
ray	I-evidence
crystallographic	I-evidence
structure	I-evidence
of	O
the	O
annular	B-site
pore	I-site
formed	O
by	O
peptide	B-mutant
2	I-mutant
.	O
(	O
A	O
)	O
Annular	B-structure_element
porelike	I-structure_element
structure	B-evidence
illustrating	O
the	O
relationship	O
of	O
the	O
five	O
dodecamers	B-oligomeric_state
that	O
form	O
the	O
pore	B-site
(	O
top	O
view	O
).	O

The	O
same	O
staggered	B-site
interface	I-site
also	O
occurs	O
between	O
dodecamers	O
4	O
and	O
5	O
.	O
(	O
D	O
)	O
Eclipsed	B-site
interface	I-site
between	O
dodecamers	B-structure_element
1	I-structure_element
and	I-structure_element
5	I-structure_element
(	O
top	O
view	O
).	O

Rather	O
,	O
the	O
crystal	B-evidence
lattice	I-evidence
is	O
composed	O
of	O
conjoined	O
annular	B-site
pores	I-site
in	O
which	O
all	O
four	O
F20	B-residue_name_number
faces	O
on	O
the	O
surface	O
of	O
each	O
dodecamer	B-oligomeric_state
contact	O
F20	B-residue_name_number
faces	O
on	O
other	O
dodecamers	B-oligomeric_state
(	O
Figure	O
S4	O
).	O

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

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

Three	O
β	B-structure_element
-	I-structure_element
hairpin	I-structure_element
monomers	B-oligomeric_state
assemble	O
to	O
form	O
a	O
triangular	B-protein_state
trimer	B-oligomeric_state
.	O

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

The	O
molecular	O
weights	O
shown	O
correspond	O
to	O
an	O
Aβ42	B-protein
monomer	B-oligomeric_state
(∼	O
4	O
.	O
5	O
kDa	O
),	O
an	O
Aβ42	B-protein
trimer	B-oligomeric_state
(∼	O
13	O
.	O
5	O
kDa	O
),	O
an	O
Aβ42	B-protein
dodecamer	B-oligomeric_state
(∼	O
54	O
kDa	O
),	O
and	O
an	O
Aβ42	B-protein
annular	B-site
pore	I-site
composed	O
of	O
five	O
dodecamers	B-oligomeric_state
(∼	O
270	O
kDa	O
).	O

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

The	O
varying	O
sizes	O
of	O
APFs	B-complex_assembly
formed	O
by	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
Aβ	B-protein
might	O
result	O
from	O
differences	O
in	O
the	O
number	O
of	O
oligomer	B-oligomeric_state
subunits	B-structure_element
comprising	O
each	O
APF	B-complex_assembly
.	O

Although	O
the	O
annular	B-site
pore	I-site
formed	O
by	O
peptide	B-mutant
2	I-mutant
contains	O
five	O
dodecamer	B-oligomeric_state
subunits	B-structure_element
,	O
pores	B-site
containing	O
fewer	O
or	O
more	O
subunits	B-structure_element
can	O
easily	O
be	O
envisioned	O
.	O

Surface	O
views	O
of	O
the	O
annular	B-site
pore	I-site
formed	O
by	O
peptide	B-mutant
2	I-mutant
.	O
(	O
A	O
)	O
Top	O
view	O
.	O

This	O
reactivity	O
suggests	O
that	O
peptide	B-mutant
2	I-mutant
forms	O
oligomers	B-oligomeric_state
in	O
solution	O
that	O
share	O
structural	O
similarities	O
to	O
the	O
nonfibrillar	B-protein_state
oligomers	B-oligomeric_state
formed	O
by	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
Aβ	B-protein
.	O

To	O
identify	O
biophysical	O
determinants	O
of	O
cell	O
‐	O
specific	O
signaling	O
and	O
breast	O
cancer	O
cell	O
proliferation	O
,	O
we	O
synthesized	B-experimental_method
241	O
ERα	B-protein
ligands	O
based	O
on	O
19	O
chemical	O
scaffolds	O
,	O
and	O
compared	O
ligand	O
response	O
using	O
quantitative	B-experimental_method
bioassays	I-experimental_method
for	O
canonical	O
ERα	B-protein
activities	O
and	O
X	B-experimental_method
‐	I-experimental_method
ray	I-experimental_method
crystallography	I-experimental_method
.	O

Many	O
drugs	O
are	O
small	O
‐	O
molecule	O
ligands	O
of	O
allosteric	O
signaling	O
proteins	O
,	O
including	O
G	B-protein_type
protein	I-protein_type
‐	I-protein_type
coupled	I-protein_type
receptors	I-protein_type
(	O
GPCRs	B-protein_type
)	O
and	O
nuclear	B-protein_type
receptors	I-protein_type
such	O
as	O
ERα	B-protein
.	O

Chemical	O
structures	O
of	O
some	O
common	O
ERα	B-protein
ligands	O
.	O

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

We	O
also	O
generated	O
four	O
direct	O
modulator	O
series	O
with	O
side	O
chains	O
designed	O
to	O
directly	O
dislocate	O
h12	B-structure_element
and	O
thereby	O
completely	O
occlude	O
the	O
AF	B-site
‐	I-site
2	I-site
surface	I-site
(	O
Fig	O
2C	O
and	O
E	O
;	O
Dataset	O
EV1	O
)	O
(	O
Kieser	O
et	O
al	O
,	O
2010	O
).	O

ERα	B-protein
ligands	O
induced	O
a	O
range	O
of	O
agonist	O
activity	O
profiles	O

Correlation	O
analysis	O
of	O
OBHS	B-chemical
versus	O
OBHS	B-chemical
‐	I-chemical
BSC	I-chemical
activity	O
across	O
cell	O
types	O
.	O

These	O
results	O
suggest	O
that	O
addition	O
of	O
an	O
extended	O
side	O
chain	O
to	O
an	O
ERα	B-protein
ligand	O
scaffold	O
is	O
sufficient	O
to	O
induce	O
cell	O
‐	O
specific	O
signaling	O
,	O
where	O
the	O
relative	O
activity	O
profiles	O
of	O
the	O
individual	O
ligands	O
change	O
between	O
cell	O
types	O
.	O

Modulation	O
of	O
signaling	O
specificity	O
by	O
AF	B-structure_element
‐	I-structure_element
1	I-structure_element

The	O
positive	O
correlation	O
between	O
the	O
L	B-experimental_method
‐	I-experimental_method
Luc	I-experimental_method
and	O
E	B-experimental_method
‐	I-experimental_method
Luc	I-experimental_method
activities	O
or	O
GREB1	B-protein
levels	O
induced	O
by	O
scaffolds	O
in	O
cluster	O
1	O
was	O
generally	O
retained	O
without	O
the	O
AB	B-structure_element
domain	O
,	O
or	O
the	O
F	B-structure_element
domain	O
(	O
Fig	O
3D	O
lanes	O
1	O
–	O
4	O
).	O

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

These	O
similar	O
patterns	O
of	O
ligand	O
activity	O
in	O
the	O
wild	B-protein_state
‐	I-protein_state
type	I-protein_state
and	O
deletion	O
mutants	B-protein_state
suggest	O
that	O
AF	B-structure_element
‐	I-structure_element
1	I-structure_element
and	O
the	O
F	B-structure_element
domain	O
purely	O
amplify	O
the	O
AF	B-structure_element
‐	I-structure_element
2	I-structure_element
activities	O
of	O
ligands	O
in	O
cluster	O
1	O
.	O

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

Comparing	O
Fig	O
3C	O
and	O
D	O
,	O
the	O
+	O
and	O
−	O
signs	O
indicate	O
where	O
the	O
deletion	B-experimental_method
mutant	I-experimental_method
assays	I-experimental_method
led	O
to	O
a	O
gain	O
or	O
loss	O
of	O
statically	O
significant	O
correlation	O
,	O
respectively	O
.	O

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

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

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

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

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

ERα	B-protein
LBDs	B-structure_element
in	B-protein_state
complex	I-protein_state
with	I-protein_state
diethylstilbestrol	B-chemical
(	O
DES	B-chemical
)	O
or	O
a	O
triaryl	B-chemical
‐	I-chemical
ethylene	I-chemical
analog	O
were	O
superposed	B-experimental_method
to	O
show	O
that	O
the	O
ligand	O
‐	O
induced	O
difference	O
in	O
h11	B-structure_element
conformation	O
is	O
transmitted	O
to	O
the	O
C	O
‐	O
terminus	O
of	O
h12	B-structure_element
(	O
PDB	O
4ZN7	O
,	O
5DMC	O
).	O

The	O
bound	O
ligands	O
are	O
shown	O
,	O
and	O
arrows	O
indicate	O
considerable	O
variation	O
in	O
the	O
orientation	O
of	O
the	O
different	O
h3	B-structure_element
‐,	O
h8	B-structure_element
‐,	O
h11	B-structure_element
‐,	O
or	O
h12	B-structure_element
‐	O
directed	O
ligand	O
side	O
groups	O
.	O

Ligands	O
with	O
cell	O
‐	O
specific	O
activity	O
alter	O
the	O
shape	O
of	O
the	O
AF	B-site
‐	I-site
2	I-site
surface	I-site

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

For	O
instance	O
,	O
S	B-chemical
‐	I-chemical
OBHS	I-chemical
‐	I-chemical
2	I-chemical
and	O
S	B-chemical
‐	I-chemical
OBHS	I-chemical
‐	I-chemical
3	I-chemical
analogs	O
(	O
Fig	O
2	O
)	O
had	O
similar	O
ERα	B-protein
activity	O
profiles	O
in	O
the	O
different	O
cell	O
types	O
(	O
Fig	O
EV2A	O
–	O
C	O
),	O
but	O
the	O
2	O
‐	O
versus	O
3	O
‐	O
methyl	O
substituted	O
phenol	O
rings	O
altered	O
the	O
correlated	O
signaling	O
patterns	O
in	O
different	O
cell	O
types	O
(	O
Fig	O
3B	O
lanes	O
7	O
and	O
12	O
).	O

The	O
2	B-chemical
,	I-chemical
5	I-chemical
‐	I-chemical
DTP	I-chemical
and	O
3	B-chemical
,	I-chemical
4	I-chemical
‐	I-chemical
DTP	I-chemical
scaffolds	O
are	O
isomeric	O
,	O
but	O
with	O
aryl	O
groups	O
at	O
obtuse	O
and	O
acute	O
angles	O
,	O
respectively	O
(	O
Fig	O
2	O
).	O

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

Together	O
,	O
these	O
findings	O
suggest	O
that	O
without	O
an	O
extended	O
side	O
chain	O
,	O
cell	O
‐	O
specific	O
activity	O
stems	O
from	O
different	O
coregulator	O
recruitment	O
profiles	O
,	O
due	O
to	O
unique	O
ligand	O
‐	O
induced	O
conformations	O
of	O
the	O
AF	B-site
‐	I-site
2	I-site
surface	I-site
,	O
in	O
addition	O
to	O
differential	O
usage	O
of	O
AF	B-structure_element
‐	I-structure_element
1	I-structure_element
.	O

Our	O
goal	O
was	O
to	O
identify	O
a	O
minimal	O
set	O
of	O
predictors	O
that	O
would	O
link	O
specific	O
structural	O
perturbations	O
to	O
ERα	B-protein
signaling	O
pathways	O
that	O
control	O
cell	O
‐	O
specific	O
signaling	O
and	O
proliferation	O
.	O

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

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

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

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

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

Significant	O
morbidity	O
and	O
mortality	O
has	O
been	O
associated	O
with	O
an	O
emerging	O
,	O
highly	O
drug	O
-	O
resistant	O
strain	O
of	O
K	B-species
.	I-species
pneumoniae	I-species
characterized	O
as	O
producing	O
the	O
carbapenemase	B-protein_type
enzyme	O
(	O
KPC	O
-	O
producing	O
bacteria	B-taxonomy_domain
;	O
Nordmann	O
et	O
al	O
.,	O
2009	O
).	O

In	O
bacteria	B-taxonomy_domain
,	O
topoisomerase	B-complex_assembly
IV	I-complex_assembly
,	O
a	O
tetramer	B-oligomeric_state
of	O
two	O
ParC	B-protein
and	O
two	O
ParE	B-protein
subunits	O
,	O
unlinks	O
daughter	O
chromosomes	O
prior	O
to	O
cell	O
division	O
,	O
whereas	O
the	O
related	O
enzyme	O
gyrase	B-protein_type
,	O
a	O
GyrA2GyrB2	B-complex_assembly
tetramer	B-oligomeric_state
,	O
supercoils	O
DNA	B-chemical
and	O
helps	O
unwind	O
DNA	B-chemical
at	O
replication	O
forks	O
.	O

Levofloxacin	B-chemical
is	O
a	O
broad	O
-	O
spectrum	O
third	O
-	O
generation	O
fluoro	O
­	O
quinolone	O
antibiotic	O
.	O

The	O
upper	O
region	O
of	O
the	O
topoisomerase	B-protein_type
complex	O
consists	O
of	O
the	O
E	B-protein
-	I-protein
subunit	I-protein
TOPRIM	B-structure_element
metal	I-structure_element
-	I-structure_element
binding	I-structure_element
domain	I-structure_element
formed	O
of	O
four	O
parallel	B-structure_element
β	I-structure_element
-	I-structure_element
sheets	I-structure_element
and	O
the	O
surrounding	O
α	B-structure_element
-	I-structure_element
helices	I-structure_element
.	O

There	O
is	O
41	O
.	O
6	O
%	O
sequence	O
identity	O
and	O
54	O
.	O
4	O
%	O
sequence	O
homology	O
between	O
the	O
ParE	B-protein
subunit	O
of	O
K	B-species
.	I-species
pneumoniae	I-species
and	O
that	O
of	O
S	B-species
.	I-species
pneumoniae	I-species
.	O

Interestingly	O
,	O
for	O
S	B-species
.	I-species
pneumoniae	I-species
we	O
observe	O
only	O
one	O
possible	O
orientation	O
of	O
the	O
C7	O
groups	O
in	O
both	O
sub	O
­	O
units	O
,	O
while	O
for	O
K	B-species
.	I-species
pneumoniae	I-species
we	O
can	O
see	O
two	O
:	O
one	O
with	O
the	O
same	O
orientation	O
as	O
in	O
S	B-species
.	I-species
pneumoniae	I-species
and	O
other	O
rotated	O
180	O
°	O
away	O
.	O

They	O
both	O
exist	O
within	O
the	O
same	O
crystal	B-evidence
in	O
the	O
two	O
different	O
dimers	B-oligomeric_state
in	O
the	O
asymmetric	O
unit	O
.	O

Similar	O
behaviour	O
was	O
observed	O
for	O
the	O
S	B-species
.	I-species
pneumoniae	I-species
topo	B-complex_assembly
­	I-complex_assembly
isomerase	I-complex_assembly
IV	I-complex_assembly
ParE30	B-complex_assembly
-	I-complex_assembly
ParC55	I-complex_assembly
fusion	O
protein	O
.	O

Given	O
the	O
current	O
concerns	O
about	O
drug	O
-	O
resistant	O
strains	O
of	O
Klebsiella	B-taxonomy_domain
,	O
the	O
structure	B-evidence
reported	O
here	O
provides	O
key	O
information	O
in	O
understanding	O
the	O
action	O
of	O
currently	O
used	O
quinolones	B-chemical
and	O
should	O
aid	O
in	O
the	O
development	O
of	O
other	O
topoisomerase	B-protein_type
-	O
targeting	O
therapeutics	O
active	O
against	O
this	O
major	O
human	B-species
pathogen	O
.	O

Bound	B-protein_state
ATP	B-chemical
is	O
indicated	O
by	O
pink	O
circles	O
in	O
the	O
ATPase	B-structure_element
domains	I-structure_element
(	O
reproduced	O
with	O
permission	O
from	O
Fig	O
.	O
1	O
of	O
Lapanogov	O
et	O
al	O
.,	O
2013	O
).	O

After	O
60	O
min	O
incubation	O
,	O
samples	O
were	O
treated	O
with	O
SDS	O
and	O
proteinase	O
K	O
to	O
remove	O
proteins	O
covalent	O
bound	O
to	O
DNA	B-chemical
,	O
and	O
the	O
DNA	B-chemical
products	O
were	O
examined	O
by	O
gel	O
electrophoresis	O
in	O
1	O
%	O
agarose	O
.	O

We	O
illustrate	O
this	O
here	O
using	O
glutamate	B-protein_type
dehydrogenase	I-protein_type
(	O
GDH	B-protein_type
),	O
a	O
336	O
-	O
kDa	O
metabolic	O
enzyme	O
that	O
catalyzes	O
the	O
oxidative	O
deamination	O
of	O
glutamate	B-chemical
.	O

Here	O
,	O
we	O
report	O
near	O
-	O
atomic	O
resolution	O
cryo	B-experimental_method
-	I-experimental_method
EM	I-experimental_method
structures	B-evidence
,	O
at	O
resolutions	O
ranging	O
from	O
3	O
.	O
2	O
Å	O
to	O
3	O
.	O
6	O
Å	O
for	O
GDH	B-protein_type
complexes	O
,	O
including	O
complexes	O
for	O
which	O
crystal	B-evidence
structures	I-evidence
are	O
not	O
available	O
.	O

The	O
first	O
is	O
located	O
near	O
the	O
dimer	B-site
interface	I-site
and	O
forms	O
the	O
core	O
of	O
the	O
hexamer	B-oligomeric_state
.	O

In	O
contrast	O
,	O
in	O
the	O
open	B-protein_state
conformation	O
,	O
the	O
cavity	B-site
present	O
in	O
the	O
closed	B-protein_state
state	O
becomes	O
too	O
narrow	O
for	O
the	O
nicotinamide	O
group	O
;	O
instead	O
,	O
the	O
group	O
is	O
oriented	O
in	O
the	O
opposite	O
direction	O
,	O
parallel	O
to	O
the	O
pivot	B-structure_element
helix	I-structure_element
with	O
the	O
amido	O
group	O
extending	O
toward	O
the	O
C	O
-	O
terminal	O
end	O
of	O
the	O
helix	B-structure_element
.	O

In	O
the	O
open	B-protein_state
conformation	O
,	O
the	O
distance	O
between	O
His209	B-residue_name_number
and	O
the	O
α	O
-	O
phosphate	O
of	O
NADH	B-chemical
is	O
∼	O
4	O
.	O
4	O
Å	O
,	O
which	O
is	O
comparable	O
with	O
the	O
corresponding	O
distance	O
in	O
the	O
ADP	B-protein_state
-	I-protein_state
bound	I-protein_state
conformation	O
.	O

Importantly	O
,	O
the	O
binding	O
of	O
GTP	B-chemical
alone	O
does	O
not	O
appear	O
to	O
drive	O
the	O
transition	O
from	O
the	O
open	B-protein_state
to	O
the	O
closed	B-protein_state
state	O
of	O
GDH	B-protein
.	O

When	O
NADH	B-chemical
and	O
GTP	B-chemical
are	O
both	O
present	O
,	O
classification	B-experimental_method
reveals	O
the	O
presence	B-protein_state
of	I-protein_state
both	O
closed	B-protein_state
and	O
open	B-protein_state
GDH	B-protein
conformations	O
,	O
similar	O
to	O
the	O
condition	O
when	O
only	O
NADH	B-chemical
is	O
present	O
(	O
Fig	O
.	O
4	O
,	O
A	O
and	O
B	O
).	O

When	O
GTP	B-chemical
is	O
present	O
in	O
the	O
GTP	B-site
binding	I-site
site	I-site
,	O
His209	B-residue_name_number
instead	O
interacts	O
with	O
GTP	B-chemical
,	O
probably	O
stabilizing	O
the	O
closed	B-protein_state
conformation	O
(	O
Fig	O
.	O
4	O
,	O
C	O
and	O
D	O
).	O

These	O
structural	O
features	O
provide	O
a	O
potential	O
explanation	O
of	O
the	O
weaker	O
density	B-evidence
for	O
the	O
nicotinamide	O
moiety	O
of	O
NADH	B-chemical
in	O
the	O
open	B-protein_state
state	O
,	O
and	O
may	O
account	O
for	O
the	O
higher	O
reported	O
affinity	O
of	O
NADH	B-chemical
for	O
the	O
closed	B-protein_state
state	O
.	O

It	O
contains	O
a	O
membrane	O
-	O
deforming	O
F	B-structure_element
-	I-structure_element
BAR	I-structure_element
domain	O
as	O
well	O
as	O
a	O
Src	B-structure_element
homology	I-structure_element
3	I-structure_element
(	O
SH3	B-structure_element
)	O
domain	O
and	O
a	O
G	B-structure_element
protein	I-structure_element
-	I-structure_element
binding	I-structure_element
homology	I-structure_element
region	I-structure_element
1	I-structure_element
(	O
HR1	B-structure_element
)	O
domain	O
.	O

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

The	O
structures	B-evidence
of	O
more	O
than	O
60	O
small	O
G	B-protein_type
protein	I-protein_type
·	O
effector	O
complexes	O
have	O
been	O
solved	B-experimental_method
,	O
and	O
,	O
not	O
surprisingly	O
,	O
the	O
switch	B-site
regions	I-site
have	O
been	O
implicated	O
in	O
a	O
large	O
proportion	O
of	O
the	O
G	B-protein_type
protein	I-protein_type
-	O
effector	O
interactions	O
(	O
reviewed	O
in	O
Ref	O
.).	O

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

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

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

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

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

The	O
Kd	B-evidence
values	O
derived	O
for	O
the	O
TOCA1	B-protein
HR1	B-structure_element
with	O
Cdc42Δ7	B-mutant
and	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
Cdc42	B-protein
were	O
6	O
.	O
05	O
±	O
1	O
.	O
96	O
and	O
5	O
.	O
39	O
±	O
1	O
.	O
69	O
μm	O
,	O
respectively	O
.	O

It	O
was	O
possible	O
that	O
the	O
low	O
affinity	O
observed	O
was	O
due	O
to	O
negative	O
effects	O
of	O
immobilization	O
of	O
the	O
HR1	B-structure_element
domain	O
,	O
so	O
other	O
methods	O
were	O
employed	O
,	O
which	O
utilized	O
untagged	B-protein_state
proteins	O
.	O

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

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

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

The	O
affinities	B-evidence
of	O
both	O
the	O
FBP17	B-protein
and	O
CIP4	B-protein
HR1	B-structure_element
domains	O
were	O
also	O
in	O
the	O
low	O
micromolar	O
range	O
(	O
10	O
and	O
5	O
μm	O
,	O
respectively	O
)	O
(	O
Fig	O
.	O
2	O
,	O
D	O
and	O
E	O
),	O
suggesting	O
that	O
low	O
affinity	O
interactions	O
with	O
Cdc42	B-protein
are	O
a	O
common	O
feature	O
within	O
the	O
TOCA	B-protein_type
family	I-protein_type
.	O

100	O
structures	B-evidence
were	O
calculated	B-experimental_method
in	O
the	O
final	O
iteration	O
;	O
the	O
50	O
lowest	O
energy	O
structures	B-evidence
were	O
water	O
-	O
refined	O
;	O
and	O
of	O
these	O
,	O
the	O
35	O
lowest	O
energy	O
structures	B-evidence
were	O
analyzed	O
.	O

The	O
two	O
α	B-structure_element
-	I-structure_element
helices	I-structure_element
of	O
the	O
HR1	B-structure_element
domain	O
interact	O
to	O
form	O
an	O
anti	B-structure_element
-	I-structure_element
parallel	I-structure_element
coiled	I-structure_element
-	I-structure_element
coil	I-structure_element
with	O
a	O
slight	O
left	O
-	O
handed	O
twist	O
,	O
reminiscent	O
of	O
the	O
HR1	B-structure_element
domains	O
of	O
CIP4	B-protein
(	O
PDB	O
code	O
2KE4	O
)	O
and	O
PRK1	B-protein
(	O
PDB	O
codes	O
1CXZ	O
and	O
1URF	O
).	O

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

Long	O
range	O
NOEs	B-evidence
were	O
observed	O
linking	O
Leu	B-residue_name_number
-	I-residue_name_number
334	I-residue_name_number
,	O
Glu	B-residue_name_number
-	I-residue_name_number
335	I-residue_name_number
,	O
and	O
Asp	B-residue_name_number
-	I-residue_name_number
336	I-residue_name_number
with	O
Trp	B-residue_name_number
-	I-residue_name_number
413	I-residue_name_number
of	O
helix	B-structure_element
2	I-structure_element
,	O
Leu	B-residue_name_number
-	I-residue_name_number
334	I-residue_name_number
with	O
Lys	B-residue_name_number
-	I-residue_name_number
409	I-residue_name_number
of	O
helix	B-structure_element
2	I-structure_element
,	O
and	O
Phe	B-residue_name_number
-	I-residue_name_number
337	I-residue_name_number
and	O
Ser	B-residue_name_number
-	I-residue_name_number
338	I-residue_name_number
with	O
Arg	B-residue_name_number
-	I-residue_name_number
345	I-residue_name_number
,	O
Arg	B-residue_name_number
-	I-residue_name_number
348	I-residue_name_number
,	O
and	O
Leu	B-residue_name_number
-	I-residue_name_number
349	I-residue_name_number
of	O
helix	B-structure_element
1	I-structure_element
.	O

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

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

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

Mapping	O
the	O
binding	B-site
surface	I-site
of	O
the	O
HR1	B-structure_element
domain	O
onto	O
Cdc42	B-protein
.	O

Residues	O
with	O
disappeared	O
peaks	O
in	O
13C	B-experimental_method
HSQC	I-experimental_method
experiments	O
are	O
marked	O
on	O
the	O
chart	O
with	O
green	O
asterisks	O
.	O

Residues	O
with	O
either	O
side	O
chain	O
or	O
backbone	O
groups	O
affected	O
are	O
colored	O
blue	O
if	O
buried	O
and	O
yellow	O
if	O
solvent	B-protein_state
-	I-protein_state
accessible	I-protein_state
.	O

The	O
switch	B-site
regions	I-site
of	O
Cdc42	B-protein
did	O
not	O
,	O
however	O
,	O
become	O
visible	O
in	O
the	O
presence	B-protein_state
of	I-protein_state
the	O
TOCA1	B-protein
HR1	B-structure_element
domain	O
.	O

Residues	O
of	O
Cdc42	B-protein
that	O
disappear	O
or	O
show	O
chemical	O
shift	O
changes	O
in	O
the	O
presence	B-protein_state
of	I-protein_state
TOCA1	B-protein
are	O
colored	O
cyan	O
if	O
also	O
identified	O
as	O
contacts	O
in	O
RhoA	B-protein
and	O
Rac1	B-protein
and	O
yellow	O
if	O
they	O
are	O
not	O
.	O

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

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

An	O
N	B-protein
-	I-protein
WASP	I-protein
GBD	B-structure_element
construct	O
was	O
produced	O
,	O
and	O
its	O
affinity	B-evidence
for	O
Cdc42	B-protein
was	O
measured	O
by	O
competition	B-experimental_method
SPA	I-experimental_method
(	O
Fig	O
.	O
7B	O
).	O

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

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

The	O
contacts	B-bond_interaction
between	O
the	O
N	O
-	O
terminal	O
region	O
and	O
the	O
coiled	B-structure_element
-	I-structure_element
coil	I-structure_element
are	O
predominantly	O
hydrophobic	B-bond_interaction
in	O
both	O
cases	O
,	O
but	O
sequence	O
-	O
specific	O
contacts	O
do	O
not	O
appear	O
to	O
be	O
conserved	O
.	O

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

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

The	O
importance	O
of	O
this	O
residue	O
in	O
the	O
Cdc42	B-protein
-	O
TOCA1	B-protein
interaction	O
remains	O
unclear	O
,	O
although	O
its	O
mutation	B-experimental_method
reduces	O
binding	O
to	O
RhoGAP	B-protein
,	O
suggesting	O
that	O
it	O
can	O
be	O
involved	O
in	O
Cdc42	B-protein
interactions	O
.	O

An	O
investigation	O
into	O
the	O
local	O
motions	O
,	O
particularly	O
in	O
the	O
G	B-site
protein	I-site
-	I-site
binding	I-site
regions	I-site
,	O
may	O
offer	O
further	O
insight	O
into	O
the	O
differential	O
specificities	O
and	O
affinities	O
of	O
G	B-protein_type
protein	I-protein_type
-	O
HR1	B-structure_element
domain	O
interactions	O
.	O

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

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

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

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

The	O
region	O
C	O
-	O
terminal	O
to	O
the	O
core	O
CRIB	B-structure_element
,	O
required	O
for	O
maximal	O
affinity	O
binding	O
,	O
would	O
then	O
fully	O
displace	O
the	O
TOCA1	B-protein
HR1	B-structure_element
.	O

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

Our	O
data	O
are	O
therefore	O
easily	O
reconciled	O
with	O
the	O
dynamic	O
instability	O
models	O
described	O
in	O
relation	O
to	O
the	O
formation	O
of	O
endocytic	O
vesicles	O
and	O
with	O
the	O
current	O
data	O
pertaining	O
to	O
the	O
complex	O
activation	O
of	O
WASP	B-protein_type
/	O
N	B-protein
-	I-protein
WASP	I-protein
pathways	O
by	O
allosteric	O
and	O
oligomeric	O
effects	O
.	O

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

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

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

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

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

In	O
these	O
structures	B-evidence
,	O
the	O
BC	B-protein_type
and	O
CT	B-protein_type
active	B-site
sites	I-site
are	O
at	O
distances	O
between	O
40	O
and	O
80	O
Å	O
,	O
such	O
that	O
substrate	O
transfer	O
could	O
be	O
mediated	O
solely	O
by	O
the	O
mobility	O
of	O
the	O
flexibly	B-protein_state
tethered	I-protein_state
BCCP	B-protein_type
.	O

For	O
fungal	B-taxonomy_domain
ACC	B-protein_type
,	O
neither	O
spontaneous	O
nor	O
inducible	O
polymerization	O
has	O
been	O
detected	O
despite	O
considerable	O
sequence	O
conservation	O
to	O
human	B-species
ACC1	B-protein
.	O

Despite	O
the	O
outstanding	O
relevance	O
of	O
ACC	B-protein_type
in	O
primary	O
metabolism	O
and	O
disease	O
,	O
the	O
dynamic	O
organization	O
and	O
regulation	O
of	O
the	O
giant	O
eukaryotic	B-taxonomy_domain
,	O
and	O
in	O
particular	O
fungal	B-taxonomy_domain
ACC	B-protein_type
,	O
remain	O
poorly	O
characterized	O
.	O

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

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

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

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

On	O
the	O
basis	O
of	O
MS	B-experimental_method
analysis	O
of	O
insect	B-experimental_method
-	I-experimental_method
cell	I-experimental_method
-	I-experimental_method
expressed	I-experimental_method
human	B-species
full	B-protein_state
-	I-protein_state
length	I-protein_state
ACC	B-protein_type
,	O
Ser80	B-residue_name_number
shows	O
the	O
highest	O
degree	O
of	O
phosphorylation	B-ptm
(	O
90	O
%).	O

Ser29	B-residue_name_number
and	O
Ser1263	B-residue_name_number
,	O
implicated	O
in	O
insulin	B-ptm
-	I-ptm
dependent	I-ptm
phosphorylation	I-ptm
and	O
BRCA1	B-protein
binding	O
,	O
respectively	O
,	O
are	O
phosphorylated	B-protein_state
at	O
intermediate	O
levels	O
(	O
40	O
%).	O

Accordingly	O
,	O
most	O
of	O
this	B-structure_element
loop	I-structure_element
is	O
not	O
represented	O
in	O
the	O
HsaBT	B-mutant
-	I-mutant
CD	I-mutant
crystal	B-evidence
structure	I-evidence
.	O

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

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

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

The	O
CD	B-structure_element
has	O
no	O
direct	O
role	O
in	O
substrate	O
recognition	O
or	O
catalysis	O
but	O
contributes	O
to	O
the	O
regulation	O
of	O
all	O
eukaryotic	B-taxonomy_domain
ACCs	B-protein_type
.	O

However	O
,	O
flexibility	O
at	O
this	O
hinge	B-structure_element
may	O
be	O
required	O
for	O
full	B-protein_state
ACC	I-protein_state
activity	I-protein_state
,	O
as	O
the	O
distances	O
between	O
the	O
BCCP	B-structure_element
anchor	I-structure_element
points	I-structure_element
and	O
the	O
active	B-site
sites	I-site
of	O
BC	B-structure_element
and	O
CT	B-structure_element
observed	O
here	O
are	O
such	O
large	O
that	O
mobility	O
of	O
the	O
BCCP	B-structure_element
alone	O
is	O
not	O
sufficient	O
for	O
substrate	O
transfer	O
.	O

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

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

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

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

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

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

For	O
other	O
instances	O
,	O
CDC2	B-structure_element
domains	O
are	O
shown	O
in	O
transparent	O
tube	O
representation	O
with	O
only	O
one	O
helix	O
each	O
highlighted	O
.	O

Representation	O
as	O
in	O
a	O
,	O
but	O
the	O
CDC1	B-structure_element
and	O
CDC2	B-structure_element
are	O
superposed	B-experimental_method
based	O
on	O
CDC2	B-structure_element
.	O

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

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

Strikingly	O
,	O
SEL1Lcent	B-structure_element
forms	O
a	O
homodimer	B-oligomeric_state
with	O
two	O
-	O
fold	O
symmetry	O
in	O
a	O
head	B-protein_state
-	I-protein_state
to	I-protein_state
-	I-protein_state
tail	I-protein_state
manner	O
.	O

Particularly	O
,	O
the	O
SLR	B-structure_element
motif	I-structure_element
9	I-structure_element
plays	O
an	O
important	O
role	O
in	O
dimer	B-oligomeric_state
formation	O
by	O
adopting	O
a	O
domain	B-protein_state
-	I-protein_state
swapped	I-protein_state
structure	O
and	O
providing	O
an	O
extensive	O
dimeric	B-site
interface	I-site
.	O

In	O
particular	O
,	O
SEL1L	B-protein
is	O
crucial	O
for	O
translocation	O
of	O
Class	B-complex_assembly
I	I-complex_assembly
major	I-complex_assembly
histocompatibility	I-complex_assembly
complex	I-complex_assembly
(	O
MHC	B-complex_assembly
)	O
heavy	B-protein_type
chains	I-protein_type
(	O
HCs	B-protein_type
).	O

Although	O
there	O
is	O
evidence	O
that	O
the	O
luminal	B-structure_element
domain	I-structure_element
of	O
SEL1L	B-protein
is	O
involved	O
in	O
substrate	O
recognition	O
or	O
in	O
forming	O
complexes	O
with	O
chaperones	B-protein_type
,	O
it	O
is	O
not	O
known	O
how	O
the	O
unique	O
structure	O
of	O
the	O
repeated	O
SLR	B-structure_element
motifs	O
contributes	O
to	O
the	O
molecular	O
function	O
of	O
the	O
HRD1	B-complex_assembly
-	I-complex_assembly
SEL1L	I-complex_assembly
E3	B-protein_type
ligase	I-protein_type
complex	O
and	O
affects	O
ERAD	O
at	O
the	O
molecular	O
level	O
.	O

The	O
11	O
SLR	B-structure_element
motifs	O
are	O
located	O
in	O
the	O
ER	O
lumen	O
and	O
account	O
for	O
more	O
than	O
two	O
thirds	O
of	O
the	O
mass	O
of	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
SEL1L	B-protein
.	O

Sequence	B-experimental_method
alignment	I-experimental_method
of	O
the	O
SLR	B-structure_element
motifs	O
revealed	O
that	O
there	O
is	O
a	O
short	O
linker	B-structure_element
sequence	I-structure_element
(	O
residues	O
336	B-residue_range
–	I-residue_range
345	I-residue_range
)	O
between	O
SLR	B-structure_element
-	I-structure_element
N	I-structure_element
and	O
SLR	B-structure_element
-	I-structure_element
M	I-structure_element
and	O
a	O
long	O
linker	B-structure_element
sequence	I-structure_element
(	O
residues	O
528	B-residue_range
–	I-residue_range
635	I-residue_range
)	O
between	O
SLR	B-structure_element
-	I-structure_element
M	I-structure_element
and	O
SLR	B-structure_element
-	I-structure_element
C	I-structure_element
(	O
Fig	O
.	O
1A	O
).	O

To	O
identify	O
a	O
soluble	O
form	O
of	O
SEL1L	B-protein
,	O
we	O
generated	O
serial	B-experimental_method
truncation	I-experimental_method
constructs	I-experimental_method
of	O
SEL1L	B-protein
based	O
on	O
the	O
predicted	O
SLR	B-structure_element
motifs	O
and	O
highly	B-protein_state
conserved	I-protein_state
regions	O
across	O
several	O
different	O
species	O
.	O

Helices	B-structure_element
A	I-structure_element
and	I-structure_element
B	I-structure_element
are	O
14	O
and	O
13	O
residues	O
long	O
,	O
respectively	O
,	O
and	O
the	O
two	O
helices	B-structure_element
are	O
connected	O
by	O
a	O
short	O
turn	B-structure_element
and	O
loop	B-structure_element
(	O
Fig	O
.	O
1D	O
).	O

The	O
concave	B-site
surface	I-site
of	O
each	O
SEL1L	B-protein
domain	O
comprising	O
helix	B-structure_element
5A	I-structure_element
to	I-structure_element
9A	I-structure_element
encircles	O
its	O
dimer	B-oligomeric_state
counterpart	O
in	O
an	O
interlocking	O
clasp	O
-	O
like	O
arrangement	O
.	O

Leu	B-residue_name_number
521	I-residue_name_number
is	O
located	O
in	O
the	O
dimerization	B-site
center	I-site
of	O
the	O
antiparallel	O
9B	B-structure_element
helices	I-structure_element
in	O
the	O
SEL1Lcent	B-structure_element
dimer	B-oligomeric_state
.	O

For	O
this	O
structural	O
geometry	O
,	O
two	O
adjacent	O
residues	O
,	O
Gly	B-residue_name_number
512	I-residue_name_number
and	O
Gly	B-residue_name_number
513	I-residue_name_number
,	O
in	O
SEL1L	B-protein
confer	O
flexibility	O
at	O
this	O
position	O
by	O
adopting	O
main	O
-	O
chain	O
dihedral	O
angles	O
that	O
are	O
disallowed	O
for	O
non	O
-	O
glycine	O
residues	O
.	O

Gly	B-residue_name_number
513	I-residue_name_number
is	O
conserved	B-protein_state
among	O
other	O
SLR	B-structure_element
motifs	O
in	O
the	O
SEL1Lcent	B-structure_element
,	O
but	O
Gly	B-residue_name_number
512	I-residue_name_number
is	O
present	O
only	O
in	O
the	O
SLR	B-structure_element
motif	I-structure_element
9	I-structure_element
of	O
SEL1Lcent	B-structure_element
(	O
Fig	O
.	O
3A	O
).	O

This	O
means	O
that	O
the	O
effect	O
of	O
the	O
mutation	B-experimental_method
is	O
mainly	O
to	O
generate	O
a	O
more	O
restricted	O
geometry	O
at	O
the	O
hinge	B-structure_element
region	O
.	O

The	O
G512K	B-mutant
/	O
G513K	B-mutant
double	B-protein_state
mutant	I-protein_state
eluted	O
at	O
the	O
monomer	B-oligomeric_state
position	O
in	O
size	B-experimental_method
-	I-experimental_method
exclusion	I-experimental_method
chromatography	I-experimental_method
(	O
Fig	O
.	O
3D	O
).	O

To	O
further	O
examine	O
whether	O
the	O
SEL1Lcent	B-structure_element
domain	O
is	O
sufficient	O
to	O
physically	O
interact	O
with	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
SEL1L	B-protein
,	O
we	O
generated	O
SEL1Lcent	B-structure_element
and	O
SLR	B-structure_element
motif	I-structure_element
9	I-structure_element
deletion	B-experimental_method
(	O
SEL1L348	B-mutant
–	I-mutant
497	I-mutant
)	O
construct	O
,	O
which	O
were	O
fused	B-experimental_method
to	I-experimental_method
the	O
C	O
-	O
terminus	O
of	O
SEL1L	B-protein
signal	B-structure_element
peptides	I-structure_element
.	O

In	O
contrast	O
,	O
SEL1L348	B-mutant
–	I-mutant
497	I-mutant
-	I-mutant
KDEL	I-mutant
and	O
the	O
single	O
-	O
residue	O
mutation	O
L521A	B-mutant
in	O
SEL1Lcent	B-structure_element
did	O
not	O
competitively	O
inhibit	O
the	O
self	O
-	O
association	O
of	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
SEL1L	B-protein
(	O
Fig	O
.	O
4E	O
,	O
F	O
).	O

The	O
fusion	O
proteins	O
were	O
immobilized	O
on	O
glutathione	O
-	O
Sepharose	O
beads	O
and	O
probed	O
for	O
binding	O
to	O
SLR	B-structure_element
-	I-structure_element
N	I-structure_element
,	O
SLR	B-structure_element
-	I-structure_element
M	I-structure_element
,	O
SLR	B-structure_element
-	I-structure_element
C	I-structure_element
,	O
and	O
monomer	B-oligomeric_state
form	O
of	O
SLR	B-structure_element
-	I-structure_element
M	I-structure_element
(	O
SLR	B-mutant
-	I-mutant
ML521A	I-mutant
).	O

One	O
possibility	O
is	O
that	O
SLR	B-structure_element
-	I-structure_element
N	I-structure_element
contributes	O
to	O
substrate	O
recognition	O
of	O
proteins	O
to	O
be	O
degraded	O
because	O
there	O
are	O
a	O
couple	O
of	O
putative	O
glycosylation	B-site
sites	I-site
within	O
the	O
SLR	B-structure_element
-	I-structure_element
N	I-structure_element
domain	O
(	O
Fig	O
.	O
1A	O
).	O

Consistent	O
with	O
the	O
previous	O
data	O
,	O
our	O
crystal	B-evidence
structure	I-evidence
and	O
biochemical	B-evidence
data	I-evidence
demonstrate	O
that	O
mouse	B-taxonomy_domain
SEL1Lcent	B-structure_element
exists	O
as	O
a	O
homodimer	B-oligomeric_state
in	O
the	O
ER	O
lumen	O
via	O
domain	O
swapping	O
of	O
SLR	B-structure_element
motif	I-structure_element
9	I-structure_element
.	O

Rather	O
,	O
recent	O
research	O
shows	O
that	O
a	O
transiently	B-protein_state
expressed	I-protein_state
HRD1	B-complex_assembly
-	I-complex_assembly
SEL1L	I-complex_assembly
complex	O
alone	O
associates	O
with	O
the	O
ERAD	O
lectins	B-protein_type
OS9	B-protein
or	O
XTP	B-protein
-	I-protein
B	I-protein
and	O
is	O
sufficient	O
to	O
facilitate	O
the	O
retrotranslocation	O
and	O
degradation	O
of	O
the	O
model	O
ERAD	O
substrate	O
α	B-protein
-	I-protein
antitrypsin	I-protein
null	I-protein
Hong	I-protein
-	I-protein
Kong	I-protein
(	O
NHK	B-protein
)	O
and	O
its	O
variant	O
,	O
NHK	B-mutant
-	I-mutant
QQQ	I-mutant
,	O
which	O
lacks	B-protein_state
the	O
N	B-site
-	I-site
glycosylation	I-site
sites	I-site
.	O

In	O
yeast	B-taxonomy_domain
,	O
it	O
is	O
unclear	O
whether	O
self	O
-	O
association	O
of	O
Hrd3p	B-protein
is	O
due	O
to	O
SLR	B-structure_element
motifs	O
because	O
the	O
sequence	O
of	O
Hrd3p	B-protein
does	O
not	O
align	O
precisely	O
with	O
the	O
SLR	B-structure_element
motifs	O
in	O
SEL1L	B-protein
.	O

Furthermore	O
,	O
we	O
are	O
uncertain	O
whether	O
self	O
-	O
association	O
of	O
Hrd3p	B-protein
contributes	O
to	O
formation	O
of	O
the	O
active	B-protein_state
form	O
of	O
the	O
Hrd1p	B-protein
complex	O
.	O

This	O
interaction	O
seems	O
to	O
be	O
weak	O
because	O
direct	O
Yos9	B-protein
-	O
Yos9	B-protein
interactions	O
were	O
not	O
detected	O
in	O
immunoprecipitation	B-experimental_method
experiments	I-experimental_method
from	O
yeast	B-taxonomy_domain
cell	O
extracts	O
containing	O
different	O
epitope	B-protein_state
-	I-protein_state
tagged	I-protein_state
variants	O
of	O
Yos9	B-protein
.	O

However	O
,	O
the	O
dimerization	B-oligomeric_state
of	O
Yos9	B-protein
could	O
provide	O
a	O
higher	O
stability	O
for	O
the	O
Hrd1p	B-protein
complex	O
oligomer	B-oligomeric_state
.	O

Crystal	B-evidence
Structure	I-evidence
of	O
SEL1Lcent	B-structure_element
.	O

The	O
11	O
SLR	B-structure_element
motifs	O
were	O
divided	O
into	O
three	O
groups	O
(	O
SLR	B-structure_element
-	I-structure_element
N	I-structure_element
,	O
SLR	B-structure_element
-	I-structure_element
M	I-structure_element
,	O
and	O
SLR	B-structure_element
-	I-structure_element
C	I-structure_element
)	O
due	O
to	O
the	O
presence	O
of	O
linker	B-structure_element
sequences	I-structure_element
that	O
are	O
not	O
predicted	O
SLR	B-structure_element
motifs	O
.	O

(	O
A	O
)	O
The	O
diagram	O
on	O
the	O
left	O
shows	O
the	O
SEL1Lcent	B-structure_element
dimer	B-oligomeric_state
viewed	O
along	O
the	O
two	O
-	O
fold	O
symmetry	O
axis	O
.	O

The	O
elution	O
fractions	O
,	O
indicated	O
by	O
the	O
gray	O
shading	O
,	O
were	O
run	O
on	O
SDS	B-experimental_method
-	I-experimental_method
PAGE	I-experimental_method
and	O
are	O
shown	O
below	O
the	O
gel	B-evidence
-	I-evidence
filtration	I-evidence
elution	I-evidence
profile	I-evidence
.	O

The	O
sequences	O
of	O
SEL1Lcent	B-structure_element
included	O
in	O
the	O
crystal	B-evidence
structure	I-evidence
are	O
highlighted	O
by	O
the	O
blue	O
box	O
.	O

In	O
SLR	B-structure_element
motif	I-structure_element
9	I-structure_element
,	O
the	O
axes	O
for	O
the	O
two	O
helices	B-structure_element
are	O
almost	O
parallel	O
,	O
while	O
the	O
other	O
SLR	B-structure_element
motifs	O
adopt	O
an	O
α	B-structure_element
-	I-structure_element
hairpin	I-structure_element
structure	O
.	O
(	O
C	O
)	O
Stereo	O
view	O
shows	O
that	O
the	O
Gly	B-residue_name_number
512	I-residue_name_number
and	O
Gly	B-residue_name_number
513	I-residue_name_number
residues	O
are	O
surrounded	O
by	O
neighboring	O
residues	O
from	O
helix	B-structure_element
9B	I-structure_element
from	O
the	O
counterpart	O
dimer	B-oligomeric_state
.	O

The	O
Gly	B-residue_name_number
512	I-residue_name_number
and	O
Gly	B-residue_name_number
513	I-residue_name_number
residues	O
are	O
colored	O
green	O
and	O
red	O
,	O
respectively	O
.	O
(	O
D	O
)	O
The	O
following	O
point	B-experimental_method
mutations	I-experimental_method
were	O
generated	O
to	O
check	O
the	O
effect	O
of	O
the	O
Gly	B-residue_name_number
512	I-residue_name_number
and	O
Gly	B-residue_name_number
513	I-residue_name_number
residues	O
in	O
terms	O
of	O
generating	O
the	O
hinge	B-structure_element
of	O
SLR	B-structure_element
motif	I-structure_element
9	I-structure_element
:	O
G512A	B-mutant
,	O
G513A	B-mutant
,	O
G512A	B-mutant
/	O
G513A	B-mutant
,	O
and	O
G512K	B-mutant
/	O
G513K	B-mutant
.	O

The	O
SEL1L348	B-mutant
–	I-mutant
497	I-mutant
fragment	O
was	O
secreted	O
to	O
the	O
culture	O
media	O
but	O
the	O
SEL1Lcent	B-structure_element
was	O
retained	O
in	O
the	O
ER	O
.	O
(	O
C	O
)	O
SEL1Lcent	B-mutant
-	I-mutant
FLAG	I-mutant
-	I-mutant
KDEL	I-mutant
and	O
SEL1L348	B-mutant
–	I-mutant
497	I-mutant
-	I-mutant
FLAG	I-mutant
-	I-mutant
KDEL	I-mutant
localized	O
to	O
the	O
ER	O
.	O

The	O
SEL1L	B-protein
fragments	O
were	O
stained	O
in	O
red	O
.	O
(	O
D	O
)	O
HEK293T	O
cells	O
were	O
transfected	O
with	O
the	O
indicated	O
plasmid	O
constructs	O
and	O
the	O
lysates	O
were	O
immunoprecipitated	B-experimental_method
with	O
an	O
anti	O
-	O
HA	B-experimental_method
antibody	O
followed	O
by	O
Western	B-experimental_method
blot	I-experimental_method
analysis	O
using	O
an	O
anti	O
-	O
FLAG	B-experimental_method
antibody	O
.	O

The	O
red	O
asterisk	O
indicates	O
the	O
expected	O
signal	O
for	O
SEL1L348	B-mutant
–	I-mutant
497	I-mutant
-	I-mutant
FLAG	I-mutant
-	I-mutant
KDEL	I-mutant
.	O

(	O
A	O
)	O
Ribbon	O
diagram	O
showing	O
superimposition	B-experimental_method
of	O
an	O
isolated	O
TPR	B-structure_element
motif	O
from	O
Cdc23	B-protein
and	O
an	O
SLR	B-structure_element
motif	O
from	O
SEL1Lcent	B-structure_element
(	O
left	O
),	O
and	O
SLR	B-structure_element
motifs	O
in	O
HcpC	B-protein
and	O
SEL1Lcent	B-structure_element
(	O
right	O
).	O

The	O
red	O
arrow	O
indicates	O
disulfide	B-ptm
bonds	I-ptm
in	O
the	O
HcpC	B-protein
,	O
and	O
Cys	B-residue_name
residues	O
involved	O
in	O
disulfide	B-ptm
bonding	I-ptm
are	O
shown	O
by	O
a	O
yellow	O
line	O
.	O
(	O
B	O
)	O
Ribbon	O
representation	O
showing	O
superimposition	B-experimental_method
of	O
Cdc23	B-protein
and	O
SEL1Lcent	B-structure_element
(	O
left	O
)	O
or	O
HcpC	B-protein
and	O
SEL1Lcent	B-structure_element
(	O
right	O
)	O
to	O
compare	O
the	O
overall	O
organization	O
of	O
the	O
α	B-structure_element
-	I-structure_element
solenoid	I-structure_element
domain	I-structure_element
.	O

Fragments	O
of	O
the	O
luminal	B-structure_element
loop	I-structure_element
of	O
HRD1	B-protein
fused	O
to	O
GST	B-chemical
were	O
immobilized	O
on	O
glutathione	O
sepharose	O
beads	O
and	O
incubated	O
with	O
purified	O
three	O
clusters	O
of	O
SLR	B-structure_element
motifs	O
and	O
monomer	B-oligomeric_state
form	O
of	O
SLR	B-structure_element
-	I-structure_element
M	I-structure_element
(	O
SLR	B-mutant
-	I-mutant
ML521A	I-mutant
,	O
right	O
panel	O
)	O
in	O
SEL1L	B-protein
.	O

Proteins	O
were	O
analyzed	O
by	O
12	O
%	O
SDS	B-experimental_method
-	I-experimental_method
PAGE	I-experimental_method
and	O
Coomassie	O
blue	O
staining	O
.	O

In	O
order	O
to	O
understand	O
the	O
catalytic	O
mechanism	O
of	O
SePSK	B-protein
,	O
we	O
solved	B-experimental_method
the	O
structure	B-evidence
of	O
SePSK	B-protein
in	B-protein_state
complex	I-protein_state
with	I-protein_state
D	B-chemical
-	I-chemical
ribulose	I-chemical
and	O
found	O
two	O
potential	O
substrate	B-site
binding	I-site
pockets	I-site
in	O
SePSK	B-protein
.	O

Using	O
mutation	B-experimental_method
and	I-experimental_method
activity	I-experimental_method
analysis	I-experimental_method
,	O
we	O
further	O
verified	O
the	O
key	O
residues	O
important	O
for	O
its	O
catalytic	O
activity	O
.	O

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

Carbohydrates	B-chemical
are	O
essential	O
cellular	O
compounds	O
involved	O
in	O
the	O
metabolic	O
processes	O
present	O
in	O
all	O
organisms	O
.	O

The	O
FGGY	B-protein_type
family	I-protein_type
carbohydrate	I-protein_type
kinases	I-protein_type
contain	O
different	O
types	O
of	O
sugar	B-protein_type
kinases	I-protein_type
,	O
all	O
of	O
which	O
possess	O
different	O
catalytic	O
substrates	O
with	O
preferences	O
for	O
short	O
-	O
chained	O
sugar	B-chemical
substrates	O
,	O
ranging	O
from	O
triose	B-chemical
to	O
heptose	B-chemical
.	O

Synpcc7942_2462	B-gene
from	O
the	O
cyanobacteria	B-taxonomy_domain
Synechococcus	B-species
elongatus	I-species
PCC	I-species
7942	I-species
encodes	O
a	O
putative	O
sugar	B-protein_type
kinase	I-protein_type
(	O
SePSK	B-protein
),	O
and	O
this	O
kinase	B-protein_type
contains	O
426	B-residue_range
amino	O
acids	O
.	O

Our	O
structural	B-experimental_method
analysis	I-experimental_method
showed	O
that	O
apo	B-protein_state
-	O
SePSK	B-protein
consists	O
of	O
one	O
SePSK	B-protein
protein	O
molecule	O
in	O
an	O
asymmetric	O
unit	O
.	O

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

This	O
finding	O
is	O
in	O
agreement	O
with	O
a	O
previous	O
result	O
showing	O
that	O
xylulose	B-protein_type
kinase	I-protein_type
(	O
PDB	O
code	O
:	O
2ITM	O
)	O
possessed	O
ATP	B-chemical
hydrolysis	O
activity	O
without	O
adding	O
substrate	O
.	O

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

To	O
understand	O
the	O
catalytic	O
mechanism	O
of	O
SePSK	B-protein
,	O
we	O
performed	O
structural	B-experimental_method
comparisons	I-experimental_method
among	O
xylulose	B-protein_type
kinase	I-protein_type
,	O
glycerol	B-protein_type
kinase	I-protein_type
,	O
ribulose	B-protein_type
kinase	I-protein_type
and	O
SePSK	B-protein
.	O

The	O
extremely	O
weak	O
electron	B-evidence
densities	I-evidence
of	O
ATP	O
γ	O
-	O
phosphate	B-chemical
in	O
both	O
structures	B-evidence
suggest	O
that	O
the	O
γ	O
-	O
phosphate	B-chemical
group	O
of	O
ATP	B-chemical
is	O
either	O
flexible	O
or	O
hydrolyzed	O
by	O
SePSK	B-protein
and	O
AtXK	B-protein
-	I-protein
1	I-protein
.	O

The	O
SePSK	B-protein
structure	B-evidence
is	O
shown	O
in	O
the	O
electrostatic	O
potential	O
surface	O
mode	O
.	O

However	O
,	O
structural	B-experimental_method
comparison	I-experimental_method
shows	O
that	O
the	O
substrate	O
ligating	O
residues	O
between	O
the	O
two	O
structures	B-evidence
are	O
not	B-protein_state
strictly	I-protein_state
conserved	I-protein_state
.	O

Based	O
on	O
the	O
structures	B-evidence
,	O
the	O
ligating	O
residues	O
of	O
RBL1	B-residue_name_number
in	O
RBL	B-complex_assembly
-	I-complex_assembly
SePSK	I-complex_assembly
structure	B-evidence
are	O
Ser72	B-residue_name_number
,	O
Asp221	B-residue_name_number
and	O
Ser222	B-residue_name_number
,	O
and	O
the	O
interacting	O
residues	O
of	O
L	B-chemical
-	I-chemical
ribulose	I-chemical
with	O
L	B-protein
-	I-protein
ribulokinase	I-protein
are	O
Ala96	B-residue_name_number
,	O
Lys208	B-residue_name_number
,	O
Asp274	B-residue_name_number
and	O
Glu329	B-residue_name_number
(	O
S7	O
Fig	O
).	O

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

This	O
break	O
is	O
probably	O
induced	O
by	O
the	O
conformational	O
change	O
of	O
the	O
two	O
β	B-structure_element
-	I-structure_element
sheets	I-structure_element
(	O
β1	B-structure_element
and	O
β2	B-structure_element
),	O
with	O
the	O
result	O
that	O
the	O
linking	B-structure_element
loop	I-structure_element
(	O
loop	B-structure_element
1	I-structure_element
)	O
is	O
located	O
further	O
away	O
from	O
the	O
RBL2	B-site
binding	I-site
site	I-site
.	O

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

However	O
,	O
considering	O
the	O
high	O
concentration	O
of	O
D	B-chemical
-	I-chemical
ribulose	I-chemical
used	O
for	O
crystal	B-experimental_method
soaking	I-experimental_method
,	O
as	O
well	O
as	O
the	O
relatively	O
weak	O
electron	B-evidence
density	I-evidence
of	O
RBL2	B-residue_name_number
,	O
it	O
is	O
also	O
possible	O
that	O
the	O
second	B-site
binding	I-site
site	I-site
of	O
D	B-chemical
-	I-chemical
ribulose	I-chemical
in	O
SePSK	B-protein
is	O
an	O
artifact	O
.	O

Superposing	B-experimental_method
the	O
structures	B-evidence
of	O
RBL	B-complex_assembly
-	I-complex_assembly
SePSK	I-complex_assembly
and	O
AMP	B-complex_assembly
-	I-complex_assembly
PNP	I-complex_assembly
-	I-complex_assembly
SePSK	I-complex_assembly
,	O
the	O
results	O
show	O
that	O
the	O
nearest	O
distance	O
between	O
AMP	B-chemical
-	I-chemical
PNP	I-chemical
γ	O
-	O
phosphate	B-chemical
and	O
RBL1	B-residue_name_number
/	O
RBL2	B-residue_name_number
is	O
7	O
.	O
5	O
Å	O
(	O
RBL1	B-residue_name_number
-	O
O5	O
)/	O
6	O
.	O
7	O
Å	O
(	O
RBL2	B-residue_name_number
-	O
O1	O
)	O
(	O
S8	O
Fig	O
).	O

This	O
distance	O
is	O
too	O
long	O
to	O
transfer	O
the	O
γ	O
-	O
phosphate	B-chemical
group	O
from	O
ATP	B-chemical
to	O
the	O
substrate	O
.	O

The	O
results	O
showed	O
that	O
domain	B-structure_element
I	I-structure_element
and	O
domain	B-structure_element
II	I-structure_element
are	O
closer	O
to	O
each	O
other	O
with	O
Ala228	B-residue_name_number
and	O
Thr401	B-residue_name_number
in	O
A2	B-structure_element
as	O
Hinge	B-structure_element
-	I-structure_element
residues	I-structure_element
.	O

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

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

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

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

In	O
addition	O
,	O
the	O
biosynthetic	B-protein_state
E	B-species
.	I-species
coli	I-species
lysine	B-protein_type
decarboxylase	I-protein_type
LdcC	B-protein
,	O
long	O
thought	O
to	O
be	O
constitutively	O
expressed	O
in	O
low	O
amounts	O
,	O
was	O
demonstrated	O
to	O
be	O
strongly	O
upregulated	O
by	O
fluoroquinolones	B-chemical
via	O
their	O
induction	O
of	O
RpoS	B-protein
.	O
A	O
direct	O
correlation	O
between	O
the	O
level	O
of	O
cadaverine	B-chemical
and	O
the	O
resistance	O
of	O
E	B-species
.	I-species
coli	I-species
to	O
these	O
antibiotics	O
commonly	O
used	O
as	O
a	O
first	O
-	O
line	O
treatment	O
of	O
UTI	O
could	O
be	O
established	O
.	O

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

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

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

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

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

Remarkably	O
,	O
this	O
analysis	O
revealed	O
that	O
several	O
specific	B-structure_element
residues	I-structure_element
in	O
the	O
above	O
-	O
mentioned	O
β	B-structure_element
-	I-structure_element
sheet	I-structure_element
,	O
independently	O
of	O
the	O
rest	O
of	O
the	O
protein	O
sequence	O
,	O
are	O
sufficient	O
to	O
define	O
if	O
a	O
particular	O
lysine	B-protein_type
decarboxylase	I-protein_type
should	O
be	O
classified	O
as	O
an	O
“	O
LdcC	B-protein_type
-	I-protein_type
like	I-protein_type
”	O
or	O
an	O
“	O
LdcI	B-protein_type
-	I-protein_type
like	I-protein_type
”.	O

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

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

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

Between	O
these	O
two	O
extremes	O
,	O
the	O
PLP	B-structure_element
-	I-structure_element
SDs	I-structure_element
of	O
LdcIa	B-protein
and	O
LdcC	B-protein
are	O
similar	O
both	O
in	O
the	O
context	O
of	O
the	O
decamer	B-oligomeric_state
(	O
Fig	O
.	O
3F	O
)	O
and	O
in	O
terms	O
of	O
RMSDmin	B-evidence
=	O
0	O
.	O
9	O
Å	O
,	O
which	O
probably	O
reflects	O
the	O
fact	O
that	O
,	O
at	O
the	O
optimal	B-protein_state
pH	I-protein_state
,	O
these	O
lysine	B-protein_type
decarboxylases	I-protein_type
have	O
a	O
similar	O
enzymatic	O
activity	O
.	O

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

All	O
our	O
current	O
cryoEM	B-experimental_method
reconstructions	B-evidence
of	O
the	O
lysine	B-protein_type
decarboxylases	I-protein_type
were	O
obtained	O
in	O
the	O
absence	B-protein_state
of	I-protein_state
ppGpp	B-chemical
in	O
order	O
to	O
be	O
closer	O
to	O
the	O
active	B-protein_state
state	O
of	O
the	O
enzymes	O
under	O
study	O
.	O

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

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

These	O
small	O
but	O
noticeable	O
swinging	O
and	O
stretching	O
(	O
up	O
to	O
~	O
4	O
Å	O
)	O
may	O
be	O
related	O
to	O
the	O
incorporation	O
of	O
the	O
LdcI	B-protein
decamer	B-oligomeric_state
into	O
the	O
LdcI	B-complex_assembly
-	I-complex_assembly
RavA	I-complex_assembly
cage	O
.	O

The	O
C	O
-	O
terminal	O
β	B-structure_element
-	I-structure_element
sheet	I-structure_element
of	O
a	O
lysine	B-protein_type
decarboxylase	I-protein_type
as	O
a	O
major	O
determinant	O
of	O
the	O
interaction	O
with	O
RavA	B-protein

Thus	O
,	O
to	O
advance	O
beyond	O
our	O
experimental	O
confirmation	O
of	O
the	O
C	O
-	O
terminal	O
β	B-structure_element
-	I-structure_element
sheet	I-structure_element
as	O
a	O
major	O
determinant	O
of	O
the	O
capacity	O
of	O
a	O
particular	O
lysine	B-protein_type
decarboxylase	I-protein_type
to	O
form	O
a	O
cage	O
with	O
RavA	B-protein
,	O
we	O
set	O
out	O
to	O
investigate	O
whether	O
certain	B-structure_element
residues	I-structure_element
in	O
this	O
β	B-structure_element
-	I-structure_element
sheet	I-structure_element
are	O
conserved	B-protein_state
in	O
lysine	B-protein_type
decarboxylases	I-protein_type
of	O
different	O
enterobacteria	B-taxonomy_domain
that	O
have	O
the	O
ravA	B-gene
-	I-gene
viaA	I-gene
operon	I-gene
in	O
their	O
genome	O
.	O

We	O
inspected	B-experimental_method
the	I-experimental_method
genetic	I-experimental_method
environment	I-experimental_method
of	O
lysine	B-protein_type
decarboxylases	I-protein_type
from	O
22	O
enterobacterial	B-taxonomy_domain
species	O
referenced	O
in	O
the	O
NCBI	O
database	O
,	O
corrected	O
the	O
gene	O
annotation	O
where	O
necessary	O
(	O
Tables	O
S3	O
and	O
S4	O
),	O
and	O
performed	O
multiple	B-experimental_method
sequence	I-experimental_method
alignment	I-experimental_method
coupled	O
to	O
a	O
phylogenetic	B-experimental_method
analysis	I-experimental_method
(	O
see	O
Methods	O
).	O

Finally	O
,	O
cadaverine	B-chemical
being	O
an	O
important	O
platform	O
chemical	O
for	O
the	O
production	O
of	O
industrial	O
polymers	O
such	O
as	O
nylon	O
,	O
structural	O
information	O
is	O
valuable	O
for	O
optimisation	O
of	O
bacterial	B-taxonomy_domain
lysine	B-protein_type
decarboxylases	I-protein_type
used	O
for	O
its	O
production	O
in	O
biotechnology	O
.	O

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

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

(	O
D	O
–	O
F	O
)	O
Inserts	O
zooming	O
at	O
the	O
CTD	B-structure_element
part	O
in	O
proximity	O
of	O
the	O
LARA	B-site
binding	I-site
site	I-site
.	O

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

Polarity	O
differences	O
are	O
highlighted	O
.	O
(	O
D	O
)	O
Position	O
and	O
nature	O
of	O
these	O
differences	O
at	O
the	O
surface	O
of	O
the	O
respective	O
cryoEM	B-experimental_method
maps	B-evidence
with	O
the	O
color	O
code	O
as	O
in	O
B	O
.	O
See	O
also	O
Fig	O
.	O
S7	O
and	O
Tables	O
S3	O
and	O
S4	O
.	O

The	O
phosphorylation	B-site
site	I-site
in	O
the	O
CTR	B-structure_element
is	O
solvent	B-protein_state
accessible	I-protein_state
and	O
located	O
in	O
a	O
negatively	B-site
charged	I-site
pocket	I-site
∼	O
30	O
Å	O
away	O
from	O
the	O
channel	B-site
exit	I-site
.	O

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

Fungi	B-taxonomy_domain
typically	O
have	O
more	O
than	O
one	O
Mep	B-protein_type
paralogue	O
,	O
for	O
example	O
,	O
Mep1	B-protein
-	I-protein
3	I-protein
in	O
S	B-species
.	I-species
cerevisiae	I-species
.	O

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

By	O
contrast	O
,	O
several	O
bacterial	B-taxonomy_domain
Amt	B-protein_type
orthologues	O
have	O
been	O
characterized	O
in	O
detail	O
via	O
high	O
-	O
resolution	O
crystal	B-evidence
structures	I-evidence
and	O
a	O
number	O
of	O
molecular	B-experimental_method
dynamics	I-experimental_method
(	O
MD	B-experimental_method
)	O
studies	O
.	O

A	O
highly	B-protein_state
conserved	I-protein_state
pair	O
of	O
channel	B-site
-	O
lining	O
histidine	B-residue_name
residues	O
dubbed	O
the	O
twin	B-structure_element
-	I-structure_element
His	I-structure_element
motif	I-structure_element
may	O
serve	O
as	O
a	O
proton	O
relay	O
system	O
while	O
NH3	B-chemical
moves	O
through	O
the	O
channel	B-site
during	O
NH3	B-chemical
/	O
H	B-chemical
+	I-chemical
symport	O
.	O

Together	O
with	O
a	O
structure	B-evidence
of	O
a	O
C	O
-	O
terminal	O
Mep2	B-mutant
variant	I-mutant
lacking	B-protein_state
the	O
segment	B-structure_element
containing	O
the	O
phosphorylation	B-site
site	I-site
,	O
the	O
results	O
allow	O
us	O
to	O
propose	O
a	O
structural	O
model	O
for	O
phosphorylation	O
-	O
based	O
regulation	O
of	O
eukaryotic	B-taxonomy_domain
ammonium	B-chemical
transport	O
.	O

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

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

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

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

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

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

Despite	O
its	O
location	O
at	O
the	O
periphery	O
of	O
the	O
trimer	B-oligomeric_state
,	O
the	O
electron	B-evidence
density	I-evidence
for	O
the	O
serine	B-residue_name
is	O
well	O
defined	O
in	O
both	O
Mep2	B-protein
structures	B-evidence
and	O
corresponds	O
to	O
the	O
non	B-protein_state
-	I-protein_state
phosphorylated	I-protein_state
state	O
(	O
Fig	O
.	O
6	O
).	O

The	O
same	O
mutant	B-mutant
lacking	B-protein_state
the	I-protein_state
His	I-protein_state
-	I-protein_state
tag	I-protein_state
has	O
WT	B-protein_state
properties	O
(	O
Supplementary	O
Fig	O
.	O
1b	O
),	O
confirming	O
that	O
the	O
region	O
following	O
the	O
phosphorylation	B-site
site	I-site
is	O
dispensable	O
for	O
function	O
.	O

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

This	O
is	O
not	O
unexpected	O
given	O
the	O
fact	O
that	O
the	O
AI	B-structure_element
region	I-structure_element
bridges	O
the	O
CTR	B-structure_element
and	O
the	O
main	B-structure_element
body	I-structure_element
of	O
Mep2	B-protein
(	O
Fig	O
.	O
6	O
).	O

As	O
shown	O
in	O
Supplementary	O
Fig	O
.	O
4	O
,	O
the	O
consequence	O
of	O
the	O
single	B-mutant
D	I-mutant
mutation	B-experimental_method
is	O
very	O
similar	O
to	O
that	O
of	O
the	O
DD	B-mutant
substitution	I-mutant
,	O
with	O
conformational	O
changes	O
and	O
increased	O
dynamics	O
confined	O
to	O
the	O
conserved	B-protein_state
part	O
of	O
the	O
CTR	B-structure_element
(	O
Supplementary	O
Fig	O
.	O
4	O
).	O

As	O
the	O
simulation	B-experimental_method
proceeds	O
,	O
the	O
side	O
chains	O
of	O
the	O
acidic	O
residues	O
move	O
away	O
from	O
Asp452	B-residue_name_number
and	O
Asp453	B-residue_name_number
,	O
presumably	O
to	O
avoid	O
electrostatic	O
repulsion	O
.	O

One	O
possible	O
explanation	O
is	O
that	O
the	O
mutants	B-mutant
do	O
not	O
accurately	O
mimic	O
a	O
phosphoserine	B-residue_name
,	O
but	O
the	O
observation	O
that	O
the	O
S453D	B-mutant
and	O
DD	B-mutant
mutants	I-mutant
are	O
fully	B-protein_state
active	I-protein_state
in	O
the	O
absence	B-protein_state
of	I-protein_state
Npr1	B-protein
suggests	O
that	O
the	O
mutations	B-experimental_method
do	O
mimic	O
the	O
effect	O
of	O
phosphorylation	B-ptm
(	O
Fig	O
.	O
3	O
).	O

In	O
addition	O
,	O
a	O
number	O
of	O
biochemical	B-experimental_method
and	I-experimental_method
genetic	I-experimental_method
studies	I-experimental_method
are	O
available	O
for	O
bacterial	B-taxonomy_domain
,	O
fungal	B-taxonomy_domain
and	O
plant	B-taxonomy_domain
proteins	O
.	O

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

In	O
addition	O
,	O
the	O
AI	B-structure_element
region	I-structure_element
of	O
the	O
CTR	B-structure_element
containing	O
the	O
Npr1	B-site
kinase	I-site
site	I-site
is	O
conserved	B-protein_state
in	O
only	O
a	O
subset	O
of	O
fungal	B-taxonomy_domain
transporters	B-protein_type
,	O
suggesting	O
that	O
the	O
details	O
of	O
the	O
structural	O
changes	O
underpinning	O
regulation	O
vary	O
.	O

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

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

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

The	O
conserved	B-protein_state
RxK	B-structure_element
motif	I-structure_element
in	O
ICL1	B-structure_element
is	O
boxed	O
in	O
blue	O
,	O
the	O
ER	B-structure_element
motif	I-structure_element
in	O
ICL2	B-structure_element
in	O
cyan	O
,	O
the	O
conserved	B-protein_state
ExxGxD	B-structure_element
motif	I-structure_element
of	O
the	O
CTR	B-structure_element
in	O
red	O
and	O
the	O
AI	B-structure_element
region	I-structure_element
in	O
yellow	O
.	O

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

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

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

(	O
a	O
)	O
Stereoviews	O
of	O
CaMep2	B-protein
showing	O
2Fo	O
–	O
Fc	O
electron	O
density	O
(	O
contoured	O
at	O
1	O
.	O
0	O
σ	O
)	O
for	O
CTR	B-structure_element
residues	O
Asp419	B-residue_range
-	I-residue_range
Met422	I-residue_range
and	O
for	O
Tyr446	B-residue_range
-	I-residue_range
Thr455	I-residue_range
of	O
the	O
AI	B-structure_element
region	I-structure_element
.	O

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

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

In	O
this	O
case	O
however	O
,	O
the	O
open	B-protein_state
channel	B-site
corresponds	O
to	O
the	O
non	B-protein_state
-	I-protein_state
phosphorylated	I-protein_state
state	O
;	O
phosphorylation	B-ptm
breaks	O
the	O
CTR	O
–	O
ICL3	O
interactions	O
leading	O
to	O
channel	B-site
closure	O
.	O
(	O
b	O
)	O
Model	O
based	O
on	O
AMT	O
transporter	O
analogy	O
showing	O
how	O
phosphorylation	B-ptm
of	O
a	O
Mep2	B-protein
monomer	B-oligomeric_state
might	O
allosterically	O
open	B-protein_state
channels	B-site
in	O
the	O
entire	O
trimer	B-oligomeric_state
via	O
disruption	O
of	O
the	O
interactions	O
between	O
the	O
CTR	B-structure_element
and	O
ICL3	B-structure_element
of	O
a	O
neighbouring	O
monomer	B-oligomeric_state
(	O
arrow	O
).	O

Furthermore	O
,	O
mutational	B-experimental_method
analyses	I-experimental_method
show	O
that	O
tRNAHis	B-chemical
is	O
bound	B-protein_state
to	I-protein_state
TLP	B-protein_type
in	O
a	O
similar	O
manner	O
as	O
Thg1	B-protein
,	O
thus	O
indicating	O
that	O
TLP	B-protein_type
has	O
a	O
dual	O
binding	O
mode	O
.	O

In	O
3	O
′-	O
5	O
′	O
elongation	O
by	O
Thg1	B-protein
/	O
TLP	B-protein_type
family	O
proteins	O
,	O
the	O
5	B-chemical
′-	I-chemical
monophosphate	I-chemical
of	O
the	O
tRNA	B-chemical
is	O
first	O
activated	O
by	O
ATP	B-chemical
/	O
GTP	B-chemical
,	O
followed	O
by	O
the	O
actual	O
elongation	O
reaction	O
.	O

Furthermore	O
,	O
the	O
structure	B-evidence
of	O
Candida	B-species
albicans	I-species
Thg1	B-protein
(	O
CaThg1	B-protein
)	O
complexed	B-protein_state
with	I-protein_state
tRNAHis	B-chemical
reveals	O
that	O
the	O
tRNA	B-chemical
substrate	O
accesses	O
the	O
reaction	B-site
center	I-site
from	O
a	O
direction	O
opposite	O
to	O
that	O
of	O
canonical	O
DNA	B-protein_type
/	I-protein_type
RNA	I-protein_type
polymerase	I-protein_type
.	O

Therefore	O
,	O
we	O
prepared	O
a	O
crystal	B-evidence
of	O
MaTLP	B-protein
complexed	B-protein_state
with	I-protein_state
ppptRNAPheΔ1	B-chemical
and	O
solved	B-experimental_method
its	O
structure	B-evidence
to	O
study	O
the	O
template	O
-	O
directed	O
3	O
′-	O
5	O
′	O
elongation	O
reaction	O
by	O
TLP	B-protein_type
(	O
fig	O
.	O
S1	O
).	O

(	O
B	O
)	O
Structure	B-evidence
after	O
GDPNP	B-chemical
binding	O
.	O
(	O
C	O
)	O
Superposition	B-experimental_method
of	O
the	O
two	O
structures	B-evidence
showing	O
movement	O
of	O
the	O
5	O
′-	O
end	O
of	O
the	O
tRNA	B-chemical
before	O
(	O
blue	O
)	O
and	O
after	O
(	O
red	O
)	O
insertion	O
of	O
GDPNP	B-chemical
.	O
(	O
D	O
)	O
Superposition	B-experimental_method
of	O
the	O
5	O
′-	O
end	O
of	O
the	O
tRNA	B-chemical
after	O
GDPNP	B-chemical
insertion	O
(	O
red	O
)	O
with	O
GTP	B-chemical
at	O
the	O
activation	O
step	O
(	O
green	O
),	O
showing	O
that	O
both	O
triphosphate	B-chemical
moieties	O
superpose	O
well	O
.	O

The	O
triphosphate	B-chemical
moiety	O
of	O
GDPNP	B-chemical
was	O
at	O
the	O
interface	B-site
between	O
molecules	O
A	B-structure_element
and	O
B	B-structure_element
and	O
was	O
recognized	O
by	O
the	O
side	O
chains	O
of	O
both	O
molecules	O
,	O
including	O
R19	B-residue_name_number
(	O
molecule	O
A	B-structure_element
),	O
R83	B-residue_name_number
(	O
molecule	O
B	B-structure_element
),	O
K86	B-residue_name_number
(	O
molecule	O
B	B-structure_element
),	O
and	O
R114	B-residue_name_number
(	O
molecule	O
A	B-structure_element
)	O
(	O
Fig	O
.	O
3B	O
).	O

On	O
the	O
basis	O
of	O
these	O
structures	B-evidence
,	O
we	O
will	O
discuss	O
the	O
3	O
′-	O
5	O
′	O
addition	O
reaction	O
compared	O
with	O
canonical	O
5	O
′-	O
3	O
′	O
elongation	O
by	O
DNA	B-protein_type
/	I-protein_type
RNA	I-protein_type
polymerases	I-protein_type
.	O

In	O
the	O
first	O
activation	O
step	O
,	O
when	O
GTP	B-chemical
/	O
ATP	B-chemical
is	O
bound	B-protein_state
to	I-protein_state
site	B-site
1	I-site
(	O
Fig	O
.	O
5B	O
),	O
the	O
5	B-chemical
′-	I-chemical
phosphate	I-chemical
of	O
the	O
tRNA	B-chemical
is	O
deprotonated	O
by	O
Mg2	B-chemical
+	I-chemical
A	O
and	O
attacks	O
the	O
α	O
-	O
phosphate	B-chemical
of	O
the	O
GTP	B-chemical
/	O
ATP	B-chemical
,	O
resulting	O
in	O
an	O
activated	O
intermediate	O
(	O
Fig	O
.	O
5C	O
).	O

(	O
D	O
)	O
Structure	B-evidence
of	O
initiation	O
of	O
the	O
elongation	O
reaction	O
(	O
corresponding	O
to	O
Fig	O
.	O
3B	O
).	O

In	O
these	O
reactions	O
,	O
the	O
roles	O
of	O
the	O
two	O
Mg	B-chemical
ions	O
are	O
identical	O
.	O

The	O
role	O
of	O
Mg2	B-chemical
+	I-chemical
B	O
is	O
to	O
position	O
the	O
5	B-chemical
′-	I-chemical
triphosphate	I-chemical
of	O
the	O
tRNA	B-chemical
in	O
TLP	B-protein_type
and	O
the	O
incoming	O
nucleotide	O
in	O
T7	B-protein
RNA	I-protein
polymerase	I-protein
.	O

Structures	B-evidence
of	O
template	O
-	O
dependent	O
nucleotide	O
elongation	O
in	O
the	O
3	O
′-	O
5	O
′	O
and	O
5	O
′-	O
3	O
′	O
directions	O
.	O

Furthermore	O
,	O
the	O
dual	O
binding	O
mode	O
of	O
this	O
protein	O
suggests	O
that	O
it	O
has	O
further	O
evolved	O
to	O
cover	O
G	B-residue_name_number
−	I-residue_name_number
1	I-residue_name_number
addition	O
of	O
tRNAHis	B-chemical
by	O
additional	O
dimerization	O
(	O
dimer	B-oligomeric_state
of	O
dimers	B-oligomeric_state
).	O

Thus	O
,	O
the	O
present	O
structural	B-experimental_method
analysis	I-experimental_method
is	O
consistent	O
with	O
the	O
scenario	O
in	O
which	O
TLP	B-protein_type
began	O
as	O
a	O
5	O
′-	O
end	O
repair	O
enzyme	O
and	O
evolved	O
into	O
a	O
tRNAHis	B-protein_type
-	I-protein_type
specific	I-protein_type
G	I-protein_type
−	I-protein_type
1	I-protein_type
addition	I-protein_type
enzyme	I-protein_type
.	O

Pooled	O
tRNAs	B-chemical
were	O
precipitated	O
with	O
isopropanol	O
and	O
dissolved	O
in	O
buffer	O
E	O
[	O
20	O
mM	O
Hepes	O
-	O
NaOH	O
(	O
pH	O
7	O
.	O
5	O
),	O
100	O
mM	O
NaCl	O
,	O
and	O
10	O
mM	O
MgCl2	O
].	O

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

One	O
conclusion	O
is	O
that	O
the	O
CDR	B-structure_element
H3	B-structure_element
conformations	O
are	O
influenced	O
by	O
both	O
their	O
amino	O
acid	O
sequence	O
and	O
their	O
structural	O
environment	O
determined	O
by	O
the	O
heavy	B-structure_element
and	O
light	B-structure_element
chain	I-structure_element
pairing	O
.	O

In	O
such	O
efforts	O
,	O
the	O
crystal	B-evidence
structure	I-evidence
of	O
the	O
specific	O
antibody	B-protein_type
may	O
not	O
be	O
available	O
,	O
but	O
modeling	O
can	O
be	O
used	O
to	O
guide	O
the	O
engineering	O
efforts	O
.	O

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

The	O
LCs	B-structure_element
that	O
associate	O
with	O
the	O
HCs	B-structure_element
are	O
divided	O
into	O
2	O
functionally	O
indistinguishable	O
classes	O
,	O
κ	B-structure_element
and	O
λ	B-structure_element
.	O

The	O
heavy	B-structure_element
and	O
light	B-structure_element
chains	I-structure_element
are	O
composed	O
of	O
structural	B-structure_element
domains	I-structure_element
that	O
have	O
∼	B-residue_range
110	I-residue_range
amino	I-residue_range
acid	I-residue_range
residues	I-residue_range
.	O

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

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

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

The	O
need	O
for	O
improvement	O
in	O
this	O
area	O
was	O
also	O
highlighted	O
in	O
a	O
recent	O
study	O
reporting	O
an	O
approach	O
and	O
results	O
that	O
may	O
influence	O
future	O
antibody	B-protein_type
modeling	O
efforts	O
.	O

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

This	O
Fab	B-structure_element
library	O
is	O
composed	O
of	O
3	O
HC	B-structure_element
germlines	O
,	O
IGHV1	B-mutant
-	I-mutant
69	I-mutant
(	O
H1	B-mutant
-	I-mutant
69	I-mutant
),	O
IGHV3	B-mutant
-	I-mutant
23	I-mutant
(	O
H3	B-mutant
-	I-mutant
23	I-mutant
)	O
and	O
IGHV5	B-mutant
-	I-mutant
51	I-mutant
(	O
H5	B-mutant
-	I-mutant
51	I-mutant
),	O
and	O
4	O
LC	B-structure_element
germlines	O
(	O
all	O
κ	B-structure_element
),	O
IGKV1	B-mutant
-	I-mutant
39	I-mutant
(	O
L1	B-mutant
-	I-mutant
39	I-mutant
),	O
IGKV3	B-mutant
-	I-mutant
11	I-mutant
(	O
L3	B-mutant
-	I-mutant
11	I-mutant
),	O
IGKV3	B-mutant
-	I-mutant
20	I-mutant
(	O
L3	B-mutant
-	I-mutant
20	I-mutant
)	O
and	O
IGKV4	B-mutant
-	I-mutant
1	I-mutant
(	O
L4	B-mutant
-	I-mutant
1	I-mutant
).	O

The	O
structure	O
analyses	O
include	O
comparisons	O
of	O
the	O
overall	O
structures	B-evidence
,	O
canonical	O
structures	B-evidence
of	O
the	O
L1	B-structure_element
,	O
L2	B-structure_element
,	O
L3	B-structure_element
,	O
H1	B-structure_element
and	O
H2	B-structure_element
CDRs	B-structure_element
,	O
the	O
structures	B-evidence
of	O
all	O
CDR	B-structure_element
H3s	B-structure_element
,	O
and	O
the	O
VH	B-complex_assembly
:	I-complex_assembly
VL	I-complex_assembly
packing	B-bond_interaction
interactions	I-bond_interaction
.	O

In	O
addition	O
to	O
these	O
,	O
2	O
primary	O
disordered	O
stretches	O
of	O
residues	O
are	O
observed	O
in	O
a	O
number	O
of	O
structures	B-evidence
(	O
Table	O
S1	O
).	O

The	O
other	O
is	O
located	O
in	O
CDR	B-structure_element
H3	B-structure_element
(	O
in	O
H5	B-complex_assembly
-	I-complex_assembly
51	I-complex_assembly
:	I-complex_assembly
L3	I-complex_assembly
-	I-complex_assembly
11	I-complex_assembly
,	O
H5	B-complex_assembly
-	I-complex_assembly
51	I-complex_assembly
:	I-complex_assembly
L3	I-complex_assembly
-	I-complex_assembly
20	I-complex_assembly
and	O
in	O
one	O
of	O
2	O
copies	O
of	O
H3	B-complex_assembly
-	I-complex_assembly
23	I-complex_assembly
:	I-complex_assembly
L4	I-complex_assembly
-	I-complex_assembly
1	I-complex_assembly
).	O

The	O
CDR	B-structure_element
H1	B-structure_element
backbone	O
conformations	O
for	O
all	O
variants	O
for	O
each	O
of	O
the	O
HCs	B-structure_element
are	O
shown	O
in	O
Fig	O
.	O
1	O
.	O

The	O
superposition	B-experimental_method
of	O
CDR	B-structure_element
H2	B-structure_element
backbones	O
for	O
all	O
HC	B-complex_assembly
:	I-complex_assembly
LC	I-complex_assembly
pairs	O
with	O
heavy	B-structure_element
chains	I-structure_element
:	O
(	O
A	O
)	O
H1	B-mutant
-	I-mutant
69	I-mutant
,	O
(	O
B	O
)	O
H3	B-mutant
-	I-mutant
23	I-mutant
,	O
(	O
C	O
)	O
H3	B-mutant
-	I-mutant
53	I-mutant
and	O
(	O
D	O
)	O
H5	B-mutant
-	I-mutant
51	I-mutant
.	O

Arg71	B-residue_name_number
in	O
H3	B-mutant
-	I-mutant
23	I-mutant
fills	O
the	O
space	O
between	O
CDRs	B-structure_element
H2	B-structure_element
and	O
H4	B-structure_element
,	O
and	O
defines	O
the	O
conformation	O
of	O
the	O
tip	O
of	O
CDR	B-structure_element
H2	B-structure_element
so	O
that	O
residue	O
54	B-residue_number
points	O
away	O
from	O
the	O
antigen	B-site
binding	I-site
site	I-site
.	O

Some	O
changes	O
in	O
conformation	O
occur	O
between	O
residues	O
30a	B-residue_number
and	O
30f	B-residue_number
(	O
residues	O
8	B-residue_number
and	O
13	B-residue_number
of	O
17	B-residue_number
in	O
CDR	B-structure_element
L1	B-structure_element
).	O

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

The	O
conformation	O
of	O
CDR	B-structure_element
L1	B-structure_element
in	O
these	O
2	O
Fabs	B-structure_element
is	O
slightly	O
different	O
,	O
and	O
both	O
conformations	O
fall	O
somewhere	O
between	O
L1	B-mutant
-	I-mutant
12	I-mutant
-	I-mutant
1	I-mutant
and	O
L1	B-mutant
-	I-mutant
12	I-mutant
-	I-mutant
2	I-mutant
.	O

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

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

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

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

(	O
A	O
)	O
The	O
“	O
kinked	B-protein_state
”	O
CDR	B-structure_element
H3	B-structure_element
of	O
H1	B-complex_assembly
-	I-complex_assembly
69	I-complex_assembly
:	I-complex_assembly
L3	I-complex_assembly
-	I-complex_assembly
11	I-complex_assembly
with	O
purple	O
carbon	O
atoms	O
and	O
yellow	O
dashed	O
lines	O
connecting	O
the	O
H	O
-	O
bond	O
pairs	O
for	O
Leu100b	B-residue_name_number
O	O
and	O
Trp103	B-residue_name_number
NE1	O
,	O
Arg94	B-residue_name_number
NE	O
and	O
Asp101	B-residue_name_number
OD1	O
,	O
and	O
Arg94	B-residue_name_number
NH2	O
and	O
Asp101	B-residue_name_number
OD2	O
.	O

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

VH	B-site
:	I-site
VL	I-site
interface	I-site
amino	O
acid	O
residue	O
interactions	O

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

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

In	O
most	O
of	O
the	O
structures	B-evidence
,	O
it	O
has	O
the	O
χ2	B-evidence
angle	O
of	O
∼	O
80	O
°,	O
while	O
the	O
ring	O
is	O
flipped	O
over	O
(	O
χ2	B-evidence
=	O
−	O
100	O
°)	O
in	O
H5	B-complex_assembly
-	I-complex_assembly
51	I-complex_assembly
:	I-complex_assembly
L3	I-complex_assembly
:	I-complex_assembly
11	I-complex_assembly
and	O
H5	B-complex_assembly
-	I-complex_assembly
51	I-complex_assembly
:	I-complex_assembly
L3	I-complex_assembly
-	I-complex_assembly
20	I-complex_assembly
.	O

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

Residues	O
in	O
CDR	B-structure_element
H3	B-structure_element
are	O
missing	O
:	O
YGE	B-structure_element
in	O
H5	B-complex_assembly
-	I-complex_assembly
51	I-complex_assembly
:	I-complex_assembly
L3	I-complex_assembly
-	I-complex_assembly
11	I-complex_assembly
,	O
GIY	B-structure_element
in	O
H5	B-complex_assembly
-	I-complex_assembly
51	I-complex_assembly
:	I-complex_assembly
L3	I-complex_assembly
-	I-complex_assembly
20	I-complex_assembly
.	O

Melting	B-evidence
temperatures	I-evidence
for	O
the	O
16	O
Fabs	B-structure_element
.	O

Colors	O
:	O
blue	O
(	O
Tm	B-evidence
<	O
70	O
°	O
C	O
),	O
green	O
(	O
70	O
°	O
C	O
<	O
Tm	B-evidence
<	O
73	O
°	O
C	O
),	O
yellow	O
(	O
73	O
°	O
C	O
<	O
Tm	B-evidence
<	O
78	O
°	O
C	O
),	O
orange	O
(	O
Tm	B-evidence
>	O
78	O
°	O
C	O
).	O

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

No	O
electron	B-evidence
density	I-evidence
is	O
observed	O
for	O
a	O
number	O
of	O
side	O
chains	O
in	O
CDRs	B-structure_element
H3	B-structure_element
and	O
L3	B-structure_element
in	O
all	O
Fabs	B-structure_element
with	O
germline	O
H3	B-mutant
-	I-mutant
53	I-mutant
,	O
which	O
indicates	O
loose	O
packing	O
of	O
the	O
variable	B-structure_element
domains	I-structure_element
.	O

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

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

For	O
the	O
18	O
Fabs	B-structure_element
with	O
complete	O
backbone	O
atoms	O
for	O
CDR	B-structure_element
H3	B-structure_element
,	O
10	O
have	O
conformations	O
similar	O
to	O
that	O
of	O
the	O
parent	O
,	O
while	O
the	O
others	O
have	O
significantly	O
different	O
conformations	O
(	O
Fig	O
.	O
6	O
).	O

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

Interestingly	O
,	O
as	O
described	O
earlier	O
,	O
these	O
2	O
pairs	O
differ	O
in	O
the	O
stem	B-structure_element
regions	I-structure_element
with	O
the	O
H1	B-complex_assembly
-	I-complex_assembly
69	I-complex_assembly
:	I-complex_assembly
L3	I-complex_assembly
-	I-complex_assembly
20	I-complex_assembly
pair	O
in	O
the	O
‘	O
extended	B-protein_state
’	O
conformation	O
and	O
H5	B-complex_assembly
-	I-complex_assembly
51	I-complex_assembly
:	I-complex_assembly
L4	I-complex_assembly
-	I-complex_assembly
1	I-complex_assembly
pair	O
in	O
the	O
‘	O
kinked	B-protein_state
’	O
conformation	O
.	O

The	O
CDR	B-structure_element
H3	B-structure_element
conformational	B-experimental_method
analysis	I-experimental_method
shows	O
that	O
,	O
for	O
each	O
set	O
of	O
variants	O
of	O
one	O
HC	B-structure_element
paired	O
with	O
the	O
4	O
different	O
LCs	B-structure_element
,	O
both	O
“	O
parental	O
”	O
and	O
“	O
non	O
-	O
parental	O
”	O
conformations	O
are	O
observed	O
.	O

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

This	O
finding	O
supports	O
the	O
hypothesis	O
of	O
Weitzner	O
et	O
al	O
.	O
that	O
the	O
H3	B-structure_element
conformation	O
is	O
controlled	O
both	O
by	O
its	O
sequence	O
and	O
its	O
environment	O
.	O

Two	O
variants	O
,	O
H1	B-complex_assembly
-	I-complex_assembly
69	I-complex_assembly
:	I-complex_assembly
L3	I-complex_assembly
-	I-complex_assembly
20	I-complex_assembly
and	O
H3	B-complex_assembly
-	I-complex_assembly
23	I-complex_assembly
:	I-complex_assembly
L3	I-complex_assembly
-	I-complex_assembly
20	I-complex_assembly
,	O
have	O
the	O
largest	O
differences	O
in	O
the	O
tilt	O
angles	O
compared	O
to	O
other	O
variants	O
as	O
seen	O
in	O
Table	O
3	O
.	O

One	O
of	O
the	O
variants	O
,	O
H3	B-complex_assembly
-	I-complex_assembly
23	I-complex_assembly
:	I-complex_assembly
L3	I-complex_assembly
-	I-complex_assembly
20	I-complex_assembly
,	O
has	O
the	O
CDR	B-structure_element
H3	B-structure_element
conformation	O
similar	O
to	O
the	O
parent	O
,	O
but	O
the	O
other	O
,	O
H1	B-complex_assembly
-	I-complex_assembly
69	I-complex_assembly
:	I-complex_assembly
L3	I-complex_assembly
-	I-complex_assembly
20	I-complex_assembly
,	O
is	O
different	O
.	O

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

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

The	O
final	O
conformation	O
represents	O
an	O
energetic	O
minimum	O
;	O
however	O
,	O
in	O
most	O
cases	O
it	O
is	O
very	O
shallow	O
,	O
so	O
that	O
a	O
single	O
mutation	O
can	O
cause	O
a	O
dramatic	O
rearrangement	O
of	O
the	O
structure	B-evidence
.	O

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

With	O
the	O
recent	O
advances	O
in	O
expression	B-experimental_method
and	I-experimental_method
crystallization	I-experimental_method
methods	I-experimental_method
,	O
Fab	B-structure_element
structures	B-evidence
can	O
be	O
obtained	O
rapidly	O
.	O

Finally	O
,	O
a	O
model	O
of	O
the	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
NsrR	B-protein
in	O
the	O
active	B-protein_state
and	O
inactive	B-protein_state
state	O
provides	O
insights	O
into	O
protein	O
dimerization	O
and	O
DNA	B-chemical
-	O
binding	O
.	O

One	O
of	O
the	O
potential	O
antibiotic	O
alternatives	O
are	O
lantibiotics	B-chemical
.	O

Thus	O
,	O
the	O
lantibiotic	B-chemical
producer	O
strains	O
have	O
an	O
inbuilt	O
self	O
-	O
protection	O
mechanism	O
(	O
immunity	O
)	O
to	O
prevent	O
cell	O
death	O
caused	O
due	O
to	O
the	O
action	O
of	O
its	O
cognate	O
lantibiotic	B-chemical
.	O

Recently	O
,	O
the	O
structure	B-evidence
of	O
SaNSR	B-protein
from	O
S	B-species
.	I-species
agalactiae	I-species
was	O
solved	O
which	O
provides	O
resistance	O
against	O
nisin	B-chemical
by	O
a	O
protease	O
activity	O
.	O

The	O
expression	O
of	O
the	O
lantibiotic	B-chemical
-	O
resistance	O
genes	O
via	O
TCS	B-complex_assembly
is	O
generally	O
regulated	O
by	O
microorganism	O
-	O
specific	O
lantibiotics	B-chemical
,	O
which	O
act	O
via	O
external	O
stimuli	O
.	O

Interestingly	O
,	O
the	O
histidine	B-protein_type
kinase	I-protein_type
contains	O
two	B-structure_element
-	I-structure_element
transmembrane	I-structure_element
helices	I-structure_element
but	O
lacks	O
an	O
extracellular	B-structure_element
sensory	I-structure_element
domain	I-structure_element
,	O
and	O
are	O
therefore	O
known	O
as	O
‘	B-protein_type
intramembrane	I-protein_type
-	I-protein_type
sensing	I-protein_type
’	I-protein_type
histidine	I-protein_type
kinases	I-protein_type
.	O

All	O
their	O
members	O
are	O
characterized	O
by	O
a	O
winged	B-structure_element
helix	I-structure_element
-	I-structure_element
turn	I-structure_element
-	I-structure_element
helix	I-structure_element
(	O
wHTH	B-structure_element
)	O
motif	O
.	O

The	O
structures	B-evidence
of	O
DrrD	B-protein
and	O
DrrB	B-protein
exist	O
in	O
an	O
open	B-protein_state
conformation	O
,	O
here	O
the	O
recognition	B-structure_element
helix	I-structure_element
is	O
fully	B-protein_state
exposed	I-protein_state
,	O
suggesting	O
that	O
RRs	B-protein_type
are	O
flexible	B-protein_state
in	O
solution	O
and	O
can	O
adopt	O
multiple	O
conformations	O
.	O

NsrR	B-protein
was	O
expressed	B-experimental_method
and	I-experimental_method
purified	I-experimental_method
as	O
described	O
,	O
resulting	O
in	O
a	O
homogenous	O
protein	O
as	O
observed	O
by	O
size	B-experimental_method
exclusion	I-experimental_method
chromatography	I-experimental_method
(	O
Fig	O
1A	O
),	O
with	O
a	O
yield	O
of	O
2	O
mg	O
per	O
liter	O
of	O
cell	O
culture	O
.	O

The	O
purified	O
NsrR	B-protein
protein	O
has	O
a	O
theoretical	O
molecular	B-evidence
mass	I-evidence
of	O
27	O
.	O
7	O
kDa	O
and	O
was	O
>	O
98	O
%	O
pure	O
as	O
assessed	O
by	O
SDS	B-experimental_method
-	I-experimental_method
PAGE	I-experimental_method
(	O
Fig	O
1B	O
,	O
indicated	O
by	O
*).	O

This	O
was	O
also	O
observed	O
by	O
size	B-experimental_method
exclusion	I-experimental_method
chromatography	I-experimental_method
where	O
a	O
peak	O
at	O
an	O
elution	O
time	O
of	O
18	O
min	O
appeared	O
(	O
Fig	O
1A	O
).	O

NsrR	B-protein
was	O
crystallized	B-experimental_method
yielding	O
two	O
crystal	O
forms	O
,	O
which	O
were	O
distinguishable	O
by	O
visual	O
inspection	O
.	O

After	O
the	O
structure	B-evidence
was	O
solved	O
,	O
it	O
became	O
evident	O
that	O
these	O
crystals	B-evidence
contained	O
two	O
monomers	B-oligomeric_state
of	O
the	O
ED	B-structure_element
of	O
NsrR	B-protein
in	O
the	O
asymmetric	O
unit	O
.	O

The	O
Rwork	B-evidence
and	O
Rfree	B-evidence
values	O
after	O
refinement	O
were	O
0	O
.	O
17	O
and	O
0	O
.	O
22	O
,	O
respectively	O
.	O

Although	O
the	O
entire	O
N	O
-	O
terminal	O
receiver	B-structure_element
domain	I-structure_element
is	O
composed	O
of	O
residues	O
Met1	B-residue_range
-	I-residue_range
Leu119	I-residue_range
,	O
only	O
residues	O
Asn4	B-residue_range
to	I-residue_range
Arg121	I-residue_range
of	O
chain	B-structure_element
A	I-structure_element
(	O
including	O
residues	O
Arg120	B-residue_name_number
and	O
Arg121	B-residue_name_number
of	O
the	O
linker	B-structure_element
)	O
and	O
Gln5	B-residue_range
to	I-residue_range
Ser122	I-residue_range
of	O
chain	B-structure_element
B	I-structure_element
(	O
including	O
residues	O
Arg120	B-residue_range
until	I-residue_range
Ser122	I-residue_range
of	O
the	O
linker	B-structure_element
)	O
could	O
be	O
traced	O
in	O
the	O
electron	B-evidence
density	I-evidence
of	O
NsrR	B-protein
-	O
RD	B-structure_element
.	O

The	O
NsrR	B-protein
-	O
RD	B-structure_element
structure	B-evidence
shows	O
a	O
β1	B-structure_element
-	I-structure_element
α1	I-structure_element
-	I-structure_element
β2	I-structure_element
-	I-structure_element
α2	I-structure_element
-	I-structure_element
β3	I-structure_element
-	I-structure_element
α3	I-structure_element
-	I-structure_element
β4	I-structure_element
-	I-structure_element
α4	I-structure_element
-	I-structure_element
β5	I-structure_element
-	I-structure_element
α5	I-structure_element
topology	O
as	O
also	O
observed	O
for	O
other	O
RRs	B-protein_type
.	O

The	O
receiver	B-structure_element
domain	I-structure_element
of	O
NsrR	B-protein
was	O
superimposed	B-experimental_method
with	O
other	O
structurally	O
characterized	O
receiver	B-structure_element
domains	I-structure_element
from	O
the	O
OmpR	B-protein_type
/	I-protein_type
PhoB	I-protein_type
family	I-protein_type
,	O
such	O
as	O
DrrB	B-protein
,	O
KdpE	B-protein
,	O
MtrA	B-protein
,	O
and	O
the	O
crystal	B-evidence
structure	I-evidence
of	O
only	O
the	O
receiver	B-structure_element
domain	I-structure_element
of	O
PhoB	B-protein
.	O
The	O
rmsd	B-evidence
of	O
the	O
overlays	B-experimental_method
and	O
the	O
corresponding	O
PDB	O
codes	O
used	O
are	O
highlighted	O
in	O
Table	O
2	O
.	O

This	O
could	O
explain	O
the	O
flexibility	O
and	O
thereby	O
the	O
different	O
orientation	O
of	O
helix	B-structure_element
α4	B-structure_element
in	O
NsrR	B-protein
.	O

Based	O
on	O
the	O
Dali	B-experimental_method
server	I-experimental_method
,	O
the	O
NsrR	B-protein
-	O
RD	B-structure_element
domain	O
is	O
structurally	O
closely	O
related	O
to	O
KdpE	B-protein
(	O
PDB	O
code	O
:	O
4KNY	O
)	O
from	O
E	B-species
.	I-species
coli	I-species
,	O
displaying	O
a	O
sequence	O
identity	O
of	O
28	O
%.	O

The	O
putative	O
phosphorylation	B-site
site	I-site
of	O
NsrR	B-protein
is	O
Asp55	B-residue_name_number
,	O
which	O
is	O
localized	O
at	O
the	O
end	O
of	O
strand	B-structure_element
β3	B-structure_element
(	O
Fig	O
3	O
,	O
shown	O
in	O
red	O
;	O
Fig	O
4	O
)	O
and	O
lies	O
within	O
an	O
acidic	O
environment	O
composed	O
of	O
the	O
side	O
chains	O
of	O
Glu12	B-residue_name_number
and	O
Asp13	B-residue_name_number
(	O
Fig	O
3	O
,	O
highlighted	O
in	O
pink	O
).	O

A	O
divalent	O
metal	O
ion	O
is	O
usually	O
bound	O
in	O
this	O
acidic	O
environment	O
and	O
is	O
essential	O
for	O
phosphorylation	B-ptm
and	O
de	B-ptm
-	I-ptm
phosphorylation	I-ptm
of	O
RRs	B-protein_type
.	O

In	O
contrast	O
,	O
the	O
switch	B-site
residues	I-site
face	O
towards	O
the	O
active	B-site
site	I-site
in	O
the	O
active	B-protein_state
state	O
conformation	O
(	O
Fig	O
4B	O
).	O

A	O
comparison	O
of	O
the	O
NsrR	B-protein
-	O
RD	B-structure_element
structure	B-evidence
with	O
the	O
available	O
structures	B-evidence
of	O
PhoB	B-protein
(	O
Fig	O
4	O
)	O
in	O
the	O
active	B-protein_state
(	O
PDB	O
code	O
:	O
1ZES	O
)	O
and	O
inactive	B-protein_state
(	O
PDB	O
code	O
:	O
1B00	O
)	O
states	O
demonstrates	O
that	O
Ser82	B-residue_name_number
(	O
NsrR	B-protein
-	O
RD	B-structure_element
)	O
is	O
oriented	O
away	O
from	O
the	O
active	B-site
site	I-site
Asp55	B-residue_name_number
,	O
and	O
that	O
Phe101	B-residue_name_number
is	O
also	O
in	O
an	O
outward	B-protein_state
conformation	O
suggesting	O
an	O
inactive	B-protein_state
state	O
of	O
the	O
NsrR	B-protein
-	O
RD	B-structure_element
(	O
Fig	O
4A	O
).	O

Phosphorylation	B-ptm
shifts	O
the	O
equilibrium	O
towards	O
the	O
active	B-protein_state
conformation	O
and	O
induces	O
the	O
formation	O
of	O
rotationally	O
symmetric	O
dimers	B-oligomeric_state
on	O
the	O
α4	B-site
-	I-site
β5	I-site
-	I-site
α5	I-site
interface	I-site
of	O
RDs	B-structure_element
.	O

It	O
has	O
been	O
suggested	O
that	O
dimerization	O
is	O
crucial	O
for	O
DNA	B-chemical
-	O
binding	O
of	O
RRs	B-protein_type
of	O
the	O
OmpR	B-protein_type
/	I-protein_type
PhoB	I-protein_type
subfamily	I-protein_type
.	O

The	O
putative	O
functional	O
dimer	B-oligomeric_state
of	O
NsrR	B-protein
-	O
RD	B-structure_element
is	O
depicted	O
in	O
Fig	O
5	O
.	O

Majority	O
of	O
these	O
interactions	O
involve	O
residues	O
that	O
are	O
highly	B-protein_state
conserved	I-protein_state
within	O
the	O
OmpR	B-protein_type
/	I-protein_type
PhoB	I-protein_type
subfamily	I-protein_type
of	O
RRs	B-protein_type
.	O

Structurally	O
,	O
a	O
similar	O
set	O
of	O
residues	O
is	O
also	O
found	O
in	O
NsrR	B-protein
:	O
Leu94	B-residue_name_number
(	O
α4	B-structure_element
),	O
Val110	B-residue_name_number
(	O
α5	B-structure_element
)	O
and	O
Ala113	B-residue_name_number
(	O
α5	B-structure_element
),	O
respectively	O
(	O
depicted	O
as	O
spheres	O
in	O
Fig	O
5B	O
),	O
which	O
are	O
conserved	B-protein_state
to	O
some	O
extent	O
on	O
sequence	O
level	O
(	O
highlighted	O
in	O
yellow	O
;	O
Fig	O
3	O
).	O

Here	O
,	O
Asp100	B-residue_name_number
(	O
β5	B-structure_element
)	O
and	O
Lys114	B-residue_name_number
(	O
α5	B-structure_element
)	O
form	O
an	O
interaction	O
within	O
one	O
monomer	B-oligomeric_state
,	O
and	O
an	O
intermolecular	O
interaction	O
can	O
be	O
observed	O
between	O
Asn95	B-residue_name_number
(	O
α4	B-structure_element
)	O
of	O
one	O
monomer	B-oligomeric_state
with	O
Thr116	B-residue_name_number
(	O
α5	B-structure_element
)	O
of	O
the	O
other	O
monomer	B-oligomeric_state
(	O
Fig	O
3	O
,	O
shown	O
in	O
cyan	O
).	O

Ramachandran	B-evidence
validation	I-evidence
was	O
done	O
using	O
MolProbity	O
.	O

Monomer	B-oligomeric_state
A	B-structure_element
contains	O
residue	O
129	B-residue_range
–	I-residue_range
224	I-residue_range
and	O
monomer	B-oligomeric_state
B	B-structure_element
contain	O
residues	O
128	B-residue_range
–	I-residue_range
225	I-residue_range
.	O

The	O
two	O
β	B-structure_element
-	I-structure_element
sheets	I-structure_element
sandwich	O
the	O
three	O
α	B-structure_element
-	I-structure_element
helices	I-structure_element
.	O

Structure	B-evidence
of	O
the	O
C	O
-	O
terminal	O
effector	B-structure_element
domain	I-structure_element
of	O
NsrR	B-protein
.	O

Overall	O
,	O
the	O
structures	B-evidence
are	O
very	O
similar	O
with	O
rmsd	B-evidence
’	O
s	O
ranging	O
from	O
1	O
.	O
7	O
to	O
2	O
.	O
6	O
Å	O
(	O
Table	O
2	O
).	O

This	O
resulted	O
in	O
a	O
list	O
of	O
possible	O
templates	O
for	O
modeling	O
the	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
structure	B-evidence
of	O
NsrR	B-protein
(	O
Table	O
2	O
).	O

This	O
mimics	O
the	O
closed	B-protein_state
inactive	B-protein_state
conformation	O
of	O
NsrR	B-protein
(	O
Fig	O
7A	O
;	O
the	O
missing	B-protein_state
linker	B-structure_element
is	O
represented	O
as	O
dotted	O
line	O
).	O

The	O
RD	B-structure_element
domain	O
of	O
NsrR	B-protein
is	O
highlighted	O
in	O
yellow	O
and	O
the	O
ED	B-structure_element
domain	O
in	O
green	O
with	O
the	O
“	O
recognition	B-structure_element
helix	I-structure_element
”	O
colored	O
in	O
cyan	O
.	O
(	O
a	O
)	O
Inactive	B-protein_state
state	O
conformation	O
:	O
Both	O
domains	O
of	O
NsrR	B-protein
were	O
aligned	B-experimental_method
to	O
the	O
structure	B-evidence
of	O
MtrA	B-protein
(	O
not	O
shown	O
),	O
which	O
adopts	O
a	O
closed	B-protein_state
inactive	B-protein_state
conformation	O
,	O
to	O
obtain	O
a	O
model	O
of	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
NsrR	B-protein
.	O
Phe101	B-residue_name_number
and	O
Asp187	B-residue_name_number
stabilize	O
this	O
closed	B-protein_state
conformation	O
.	O

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

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

Particularly	O
challenging	O
are	O
interactions	O
of	O
intrinsically	B-protein_state
or	I-protein_state
conditionally	I-protein_state
disordered	I-protein_state
sections	O
of	O
proteins	O
with	O
their	O
partner	O
proteins	O
.	O

Structural	O
characterization	O
of	O
chaperone	B-protein_type
-	O
assisted	O
protein	O
folding	O
likely	O
would	O
help	O
bring	O
clarity	O
to	O
this	O
question	O
.	O

To	O
address	O
this	O
question	O
,	O
we	O
investigated	O
the	O
ATP	B-protein_state
-	I-protein_state
independent	I-protein_state
Escherichia	B-species
coli	I-species
periplasmic	O
chaperone	B-protein_type
Spy	B-protein
.	O

We	O
used	O
Spy	B-complex_assembly
:	I-complex_assembly
Im76	I-complex_assembly
-	I-complex_assembly
45	I-complex_assembly
selenomethionine	B-chemical
crystals	B-evidence
for	O
phasing	O
with	O
single	B-experimental_method
-	I-experimental_method
wavelength	I-experimental_method
anomalous	I-experimental_method
diffraction	I-experimental_method
(	O
SAD	B-experimental_method
)	O
experiments	O
,	O
and	O
used	O
this	O
solution	O
to	O
build	O
the	O
well	O
-	O
ordered	O
Spy	B-protein
portions	O
of	O
all	O
four	O
complexes	O
.	O

Moreover	O
,	O
binding	B-experimental_method
experiments	I-experimental_method
indicated	O
that	O
Im76	B-mutant
-	I-mutant
45	I-mutant
comprises	O
the	O
entire	O
Spy	B-site
-	I-site
binding	I-site
region	I-site
.	O

Consistent	O
with	O
the	O
fragmented	O
density	B-evidence
,	O
however	O
,	O
we	O
observed	O
multiple	O
iodine	B-chemical
positions	O
for	O
seven	O
of	O
the	O
eight	O
substituted	O
residues	O
.	O

Simultaneously	O
,	O
it	O
uses	O
the	O
residual	B-evidence
electron	I-evidence
density	I-evidence
to	O
help	O
choose	O
ensembles	O
.	O

By	O
analyzing	O
the	O
individual	O
structures	B-evidence
of	O
the	O
six	O
-	O
member	O
ensemble	O
of	O
Im76	B-mutant
-	I-mutant
45	I-mutant
bound	B-protein_state
to	I-protein_state
Spy	B-protein
,	O
we	O
observed	O
that	O
Im76	B-mutant
-	I-mutant
45	I-mutant
takes	O
on	O
several	O
different	O
conformations	O
while	O
bound	B-protein_state
.	O

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

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

Whereas	O
the	O
least	B-protein_state
-	I-protein_state
folded	I-protein_state
Im76	B-mutant
-	I-mutant
45	I-mutant
pose	O
in	O
the	O
ensemble	O
forms	O
the	O
most	O
hydrophobic	O
contacts	O
with	O
Spy	B-protein
(	O
Fig	O
.	O
3	O
),	O
the	O
two	O
most	B-protein_state
-	I-protein_state
folded	I-protein_state
conformations	O
form	O
the	O
fewest	O
hydrophobic	O
contacts	O
(	O
Fig	O
.	O
3	O
).	O

Spy	B-protein
changes	O
conformation	O
upon	O
substrate	O
binding	O

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

This	O
interaction	O
presumably	O
reduces	O
the	O
mobility	O
of	O
the	O
C	B-structure_element
-	I-structure_element
terminal	I-structure_element
helix	I-structure_element
.	O

The	O
F115I	B-mutant
/	O
L	B-mutant
substitutions	O
would	O
replace	O
these	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
with	O
hydrophobic	B-bond_interaction
interactions	I-bond_interaction
that	O
have	O
little	O
angular	O
dependence	O
.	O

As	O
a	O
result	O
,	O
the	O
C	O
-	O
terminus	O
,	O
and	O
possibly	O
also	O
the	O
flexible	B-protein_state
linker	B-structure_element
,	O
is	O
likely	O
to	O
become	O
more	O
flexible	O
and	O
thus	O
more	O
accommodating	O
of	O
different	O
conformations	O
of	O
substrates	O
.	O

(	O
b	O
)	O
Composites	O
of	O
iodine	B-chemical
positions	O
detected	O
from	O
anomalous	B-evidence
signals	I-evidence
using	O
pI	B-chemical
-	I-chemical
Phe	I-chemical
substitutions	B-experimental_method
,	O
colored	O
and	O
numbered	O
by	O
sequence	O
.	O

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

Flexibility	O
of	O
Spy	B-protein
linker	B-structure_element
region	I-structure_element
and	O
effect	O
of	O
Super	O
Spy	B-protein
mutants	O
.	O
(	O
a	O
)	O
The	O
Spy	B-protein
linker	B-structure_element
region	I-structure_element
adopts	O
one	O
dominant	O
conformation	O
in	O
its	O
apo	B-protein_state
state	O
(	O
PDB	O
ID	O
3039	O
,	O
gray	O
),	O
but	O
expands	O
and	O
adopts	O
multiple	O
conformations	O
in	O
bound	B-protein_state
states	O
(	O
green	O
).	O

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

L32	B-residue_name_number
,	O
F115	B-residue_name_number
,	O
and	O
Y104	B-residue_name_number
are	O
rendered	O
in	O
purple	O
to	O
illustrate	O
residues	O
that	O
are	O
most	O
affected	O
by	O
Super	O
Spy	B-protein
mutations	B-protein_state
;	O
CH	O
⋯	O
π	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
are	O
depicted	O
by	O
orange	O
dashes	O
.	O

Herein	O
,	O
we	O
report	O
X	B-evidence
-	I-evidence
ray	I-evidence
crystal	I-evidence
structures	I-evidence
of	O
ligand	B-protein_state
-	I-protein_state
free	I-protein_state
Tdp2	B-protein
and	O
Tdp2	B-complex_assembly
-	I-complex_assembly
DNA	I-complex_assembly
complexes	O
with	O
alkylated	O
and	O
abasic	O
DNA	B-chemical
that	O
unveil	O
a	O
dynamic	B-protein_state
Tdp2	B-protein
active	B-structure_element
site	I-structure_element
lid	I-structure_element
and	O
deep	O
substrate	B-site
binding	I-site
trench	I-site
well	O
-	O
suited	O
for	O
engaging	O
the	O
diverse	O
DNA	B-chemical
damage	O
triggers	O
of	O
abortive	O
Top2	B-protein_type
reactions	O
.	O

Nuclear	O
DNA	B-chemical
compaction	O
and	O
the	O
action	O
of	O
DNA	B-chemical
and	O
RNA	B-protein_type
polymerases	I-protein_type
create	O
positive	O
and	O
negative	O
DNA	B-chemical
supercoiling	O
—	O
over	O
-	O
and	O
under	O
-	O
winding	O
of	O
DNA	B-chemical
strands	O
,	O
respectively	O
—	O
and	O
the	O
linking	O
together	O
(	O
catenation	O
)	O
of	O
DNA	B-chemical
strands	O
.	O

To	O
promote	O
cancer	O
cell	O
death	O
,	O
Top2	B-protein_type
reactions	O
are	O
‘	O
poisoned	O
’	O
by	O
keystone	O
pharmacological	O
anticancer	O
agents	O
like	O
etoposide	B-chemical
,	O
teniposide	B-chemical
and	O
doxorubicin	B-chemical
.	O

The	O
chemical	O
complexity	O
of	O
DNA	B-chemical
damage	O
-	O
derived	O
Top2cc	B-complex_assembly
necessitates	O
that	O
DNA	B-chemical
repair	O
machinery	O
dedicated	O
to	O
resolving	O
these	O
lesions	O
recognizes	O
both	O
DNA	B-chemical
and	O
protein	O
,	O
whilst	O
accommodating	O
diverse	O
chemical	O
structures	O
that	O
trap	O
Top2cc	B-complex_assembly
.	O

Tdp2	B-protein
processes	O
phosphotyrosyl	B-ptm
linkages	I-ptm
in	O
diverse	O
DNA	B-chemical
damage	O
contexts	O
.	O

Tdp2	B-protein
hydrolyzes	O
the	O
5	O
′–	O
phosphotyrosine	B-residue_name
adduct	O
derived	O
from	O
poisoned	O
Top2	B-protein_type
leaving	O
DNA	B-chemical
ends	O
with	O
a	O
5	B-chemical
′-	I-chemical
phosphate	I-chemical
,	O
which	O
facilitates	O
DNA	B-chemical
end	O
joining	O
through	O
the	O
NHEJ	O
pathway	O
.	O

Phosphotyrosyl	B-ptm
bond	O
hydrolysis	O
catalyzed	O
by	O
mTdp2cat	B-structure_element
releases	O
p	B-chemical
-	I-chemical
nitrophenol	I-chemical
,	O
which	O
is	O
detected	O
by	O
measuring	O
absorbance	O
at	O
415	O
nm	O
.	O
(	O
C	O
)	O
mTdp2cat	B-structure_element
reaction	B-evidence
rates	I-evidence
on	O
p	B-chemical
–	I-chemical
nitrophenol	I-chemical
modified	O
DNA	B-chemical
substrates	O
shown	O
in	O
panel	O
B	O
.	O
Rates	O
are	O
reported	O
as	O
molecules	O
of	O
PNP	B-chemical
s	O
−	O
1	O
produced	O
by	O
mTdp2cat	B-structure_element
.	O

However	O
,	O
important	O
questions	O
regarding	O
the	O
mechanism	O
of	O
Tdp2	B-protein
engagement	O
and	O
processing	O
of	O
DNA	B-chemical
damage	O
remain	O
.	O

Finally	O
,	O
we	O
characterize	O
a	O
Tdp2	B-protein
SNP	O
that	O
ablates	B-protein_state
the	O
Tdp2	B-protein
single	B-site
metal	I-site
binding	I-site
site	I-site
and	O
Tdp2	B-protein
substrate	O
induced	O
conformational	O
changes	O
,	O
and	O
confers	O
Top2	B-protein_type
drug	O
sensitivity	O
in	O
mammalian	B-taxonomy_domain
cells	O
.	O

To	O
test	O
this	O
,	O
we	O
adapted	O
an	O
EDC	B-experimental_method
coupling	I-experimental_method
method	I-experimental_method
to	O
generate	O
5	O
′-	O
terminal	O
p	B-chemical
-	I-chemical
nitrophenol	I-chemical
(	O
PNP	B-chemical
)	O
modified	O
oligonucleotides	O
that	O
also	O
harbored	O
DNA	B-chemical
damage	O
at	O
the	O
5	O
′-	O
nucleotide	O
position	O
(	O
see	O
Materials	O
and	O
Methods	O
).	O

mTdp2cat	B-structure_element
is	O
colored	O
with	O
red	O
(	O
electronegative	O
),	O
blue	O
(	O
electropositive	O
)	O
and	O
gray	O
(	O
hydrophobic	O
)	O
electrostatic	O
surface	O
potential	O
displayed	O
.	O

A	O
structural	B-experimental_method
overlay	I-experimental_method
of	O
damaged	O
and	O
undamaged	O
nucleotides	O
shows	O
no	O
major	O
distortions	O
to	O
nucleotide	O
planarity	O
between	O
different	O
bound	B-protein_state
sequences	O
and	O
DNA	B-chemical
damage	O
(	O
compare	O
ϵA	B-chemical
,	O
dA	B-chemical
and	O
dC	B-chemical
,	O
Supplementary	O
Figure	O
S1A	O
–	O
D	O
).	O

Likewise	O
,	O
the	O
abasic	B-chemical
deoxyribose	I-chemical
analog	O
THF	B-chemical
substrate	O
binds	O
similar	O
to	O
the	O
alkylated	O
and	O
non	O
-	O
alkylated	O
substrates	O
,	O
but	O
with	O
a	O
slight	O
alteration	O
in	O
the	O
approach	O
of	O
the	O
5	O
′-	O
terminus	O
(	O
Figure	O
2C	O
).	O

An	O
intriguing	O
feature	O
of	O
the	O
DNA	B-protein_state
-	I-protein_state
damage	I-protein_state
bound	I-protein_state
conformation	O
of	O
the	O
Tdp2	B-protein
active	B-site
site	I-site
is	O
an	O
underlying	O
network	O
of	O
protein	O
–	O
water	B-chemical
–	O
protein	O
contacts	O
that	O
span	O
a	O
gap	O
between	O
the	O
catalytic	B-site
core	I-site
and	O
the	O
DNA	B-site
binding	I-site
β2Hβ	I-site
-	I-site
grasp	I-site
(	O
Supplementary	O
Figure	O
S2	O
).	O

The	O
β2Hβ	B-site
docking	I-site
pocket	I-site
(	O
circled	O
)	O
is	O
unoccupied	O
and	O
residues	O
N312	B-residue_name_number
,	O
N314	B-residue_name_number
and	O
L315	B-residue_name_number
(	O
orange	O
)	O
are	O
solvent	B-protein_state
-	I-protein_state
exposed	I-protein_state
.	O

Wall	O
-	O
eyed	O
stereo	O
view	O
is	O
displayed	O
.	O
(	O
C	O
)	O
Alignment	O
of	O
active	B-structure_element
site	I-structure_element
loop	I-structure_element
conformers	O
observed	O
in	O
the	O
5	O
promoters	B-oligomeric_state
of	O
the	O
DNA	B-protein_state
-	I-protein_state
free	I-protein_state
mTdp2cat	B-structure_element
(	O
PDB	O
entry	O
5INM	O
,	O
see	O
Table	O
1	O
)	O
crystallographic	O
asymmetric	O
unit	O
(	O
left	O
)	O
and	O
sequence	B-experimental_method
alignment	I-experimental_method
showing	O
residues	O
not	O
observed	O
in	O
the	O
electron	B-evidence
density	I-evidence
as	O
‘∼’	O
(	O
right	O
).	O
(	O
D	O
)	O
Limited	B-experimental_method
trypsin	I-experimental_method
proteolysis	I-experimental_method
probes	O
the	O
solvent	O
accessibility	O
of	O
the	O
flexible	B-protein_state
active	B-structure_element
-	I-structure_element
site	I-structure_element
loop	I-structure_element
.	O

Reactions	O
were	O
separated	O
by	O
SDS	B-experimental_method
-	I-experimental_method
PAGE	I-experimental_method
and	O
proteins	O
visualized	O
by	O
staining	O
with	O
coomassie	O
blue	O
.	O
(	O
E	O
)	O
Limited	B-experimental_method
chymotrypsin	I-experimental_method
proteolysis	I-experimental_method
probes	O
the	O
solvent	O
accessibility	O
of	O
the	O
flexible	B-protein_state
active	B-structure_element
-	I-structure_element
site	I-structure_element
loop	I-structure_element
.	O

Burial	O
of	O
Thr309	B-residue_name_number
is	O
enabled	O
by	O
an	O
unusual	O
main	O
chain	O
cis	B-bond_interaction
–	I-bond_interaction
peptide	I-bond_interaction
bond	I-bond_interaction
between	O
Asp308	B-residue_name_number
-	O
Thr309	B-residue_name_number
and	O
disassembly	O
of	O
the	O
short	B-structure_element
antiparallel	I-structure_element
beta	I-structure_element
-	I-structure_element
strand	I-structure_element
of	O
the	O
β2Hβ	B-structure_element
fold	O
.	O

To	O
transition	O
into	O
the	O
closed	B-protein_state
β2Hβ	B-structure_element
conformation	O
,	O
Thr309	B-residue_name_number
disengages	O
from	O
the	O
EEP	B-structure_element
domain	O
pocket	B-site
,	O
flips	O
peptide	O
backbone	O
conformation	O
cis	O
to	O
trans	O
,	O
and	O
is	O
integral	O
to	O
the	O
β2Hβ	B-structure_element
antiparallel	B-structure_element
β	I-structure_element
-	I-structure_element
sheet	I-structure_element
.	O

Accordingly	O
,	O
in	O
the	O
DNA	B-protein_state
free	I-protein_state
structure	B-evidence
,	O
we	O
observe	O
a	O
trend	O
where	O
the	O
2	O
closed	B-protein_state
monomers	B-oligomeric_state
have	O
an	O
ordered	O
Mg2	B-chemical
+	I-chemical
ion	O
in	O
their	O
active	B-site
sites	I-site
,	O
while	O
the	O
monomers	B-oligomeric_state
with	O
open	B-protein_state
conformations	O
have	O
a	O
poorly	O
ordered	O
or	O
vacant	O
metal	B-site
binding	I-site
site	I-site
.	O

Overall	O
,	O
these	O
observations	O
suggest	O
that	O
engagement	O
of	O
diverse	O
damaged	O
DNA	B-chemical
ends	O
is	O
enabled	O
by	O
an	O
elaborate	O
substrate	O
selected	O
stabilization	O
of	O
the	O
β2Hβ	B-site
DNA	I-site
binding	I-site
grasp	I-site
,	O
and	O
these	O
rearrangements	O
are	O
coordinated	O
with	O
Mg2	B-chemical
+	I-chemical
binding	O
in	O
the	O
Tdp2	B-protein
active	B-site
site	I-site
.	O

While	O
this	O
work	O
was	O
suggestive	O
of	O
a	O
two	O
metal	O
ion	O
mechanism	O
for	O
phosphotyrosyl	B-ptm
bond	O
cleavage	O
by	O
Tdp2	B-protein
,	O
we	O
note	O
that	O
second	O
metal	O
ion	O
titrations	O
can	O
be	O
influenced	O
by	O
metal	B-site
ion	I-site
binding	I-site
sites	I-site
outside	O
of	O
the	O
active	B-site
site	I-site
.	O

This	O
analysis	O
revealed	O
Mg2	B-chemical
+	I-chemical
Kd	B-evidence
values	O
in	O
the	O
sub	O
-	O
millimolar	O
range	O
and	O
Hill	B-evidence
coefficients	I-evidence
which	O
were	O
consistent	O
with	O
a	O
single	O
metal	B-site
binding	I-site
site	I-site
both	O
in	O
the	O
presence	B-protein_state
and	O
absence	B-protein_state
of	I-protein_state
DNA	B-chemical
(	O
Supplementary	O
Table	O
S2	O
).	O

In	O
contrast	O
,	O
while	O
co	B-evidence
-	I-evidence
complex	I-evidence
structures	I-evidence
with	O
Ca2	B-chemical
+	I-chemical
also	O
show	O
a	O
single	O
metal	O
ion	O
,	O
Ca2	B-chemical
+	I-chemical
binds	O
in	O
a	O
slightly	O
different	O
position	O
,	O
shifted	O
∼	O
1	O
Å	O
from	O
the	O
Mg2	B-site
+	I-site
site	I-site
.	O

Residue	O
numbers	O
shown	O
are	O
for	O
the	O
mTdp2	B-protein
homolog	O
.	O
(	O
D	O
)	O
Electrostatic	B-evidence
surface	I-evidence
potential	I-evidence
calculated	O
for	O
5	B-residue_name
′-	I-residue_name
phosphotyrosine	I-residue_name
in	O
isolation	O
(	O
upper	O
panel	O
)	O
and	O
in	O
the	O
presence	B-protein_state
of	I-protein_state
a	O
cation	B-bond_interaction
–	I-bond_interaction
π	I-bond_interaction
interaction	I-bond_interaction
with	O
the	O
guanidinium	O
group	O
of	O
Arg216	B-residue_name_number
(	O
lower	O
panel	O
)	O
shows	O
electron	O
-	O
withdrawing	O
effect	O
of	O
this	O
interaction	O
.	O

Here	O
,	O
the	O
water	B-chemical
proton	O
and	O
the	O
neighboring	O
O	O
of	O
Asp272	B-residue_name_number
participates	O
in	O
a	O
strong	O
hydrogen	B-bond_interaction
bond	I-bond_interaction
(	O
distance	O
of	O
1	O
.	O
58	O
Å	O
)	O
and	O
the	O
phosphotyrosyl	B-ptm
O	O
–	O
P	O
distance	O
is	O
stretched	O
to	O
1	O
.	O
77	O
Å	O
,	O
which	O
is	O
0	O
.	O
1	O
Å	O
beyond	O
an	O
equilibrium	O
bond	O
length	O
.	O

Product	O
formation	O
is	O
coupled	O
to	O
a	O
transfer	O
of	O
a	O
proton	O
from	O
the	O
nucleophillic	O
water	B-chemical
to	O
Asp272	B-residue_name_number
,	O
consistent	O
with	O
the	O
proposed	O
function	O
for	O
this	O
residue	O
as	O
the	O
catalytic	O
base	O
.	O

By	O
analyzing	O
activities	O
on	O
this	O
nested	O
set	O
of	O
chemically	O
related	O
substrates	O
we	O
aimed	O
to	O
dissect	O
structure	O
-	O
activity	O
relationships	O
of	O
Tdp2	B-protein
catalysis	O
.	O

Structural	B-evidence
results	I-evidence
and	O
QM	B-experimental_method
/	I-experimental_method
MM	I-experimental_method
modeling	I-experimental_method
indicate	O
mAsp272	B-residue_name_number
activates	O
a	O
water	B-chemical
molecule	O
for	O
in	O
-	O
line	O
nucleophilic	O
attack	O
of	O
the	O
scissile	O
phosphotyrosyl	B-ptm
linkage	I-ptm
.	O

Mutations	B-experimental_method
hI307A	B-mutant
,	O
hL305A	B-mutant
,	O
hL305F	B-mutant
and	O
hL305W	B-mutant
all	O
impaired	O
catalysis	O
on	O
both	O
nucleotide	O
-	O
containing	O
substrates	O
(<	O
50	O
%	O
activity	O
).	O

Quantification	O
of	O
percent	O
MBP	B-experimental_method
-	O
hTdp2cat	B-structure_element
activity	O
relative	O
to	O
WT	B-protein_state
protein	O
for	O
the	O
5	B-chemical
′-	I-chemical
Y	I-chemical
DNA	I-chemical
oligonucleotide	I-chemical
substrate	O
(	O
blue	O
bars	O
),	O
T5PNP	B-chemical
(	O
red	O
bars	O
)	O
and	O
PNPP	B-chemical
(	O
green	O
bars	O
)	O
is	O
displayed	O
.	O

Accordingly	O
,	O
we	O
demonstrate	O
here	O
that	O
5	B-protein_state
′-	I-protein_state
tyrosylated	I-protein_state
ends	O
are	O
sufficient	O
to	O
severely	O
impair	O
an	O
in	O
vitro	O
reconstituted	O
mammalian	B-taxonomy_domain
NHEJ	O
reaction	O
(	O
Figure	O
7A	O
,	O
lanes	O
3	O
and	O
6	O
),	O
unless	O
supplemented	O
with	O
catalytic	O
quantities	O
of	O
hTdp2FL	B-protein
(	O
Figure	O
7A	O
,	O
lane	O
8	O
).	O

DNA	B-chemical
substrates	O
with	O
5	B-residue_name
′-	I-residue_name
phosphotyrosine	I-residue_name
adducts	O
and	O
4	O
nucleotide	O
5	O
′	O
overhangs	O
were	O
electroporated	O
into	O
cultured	O
mammalian	B-taxonomy_domain
cells	O
.	O

Error	O
bars	O
,	O
s	O
.	O
d	O
,	O
n	O
=	O
3	O
.	O
(	O
E	O
)	O
Clonogenic	B-experimental_method
survival	I-experimental_method
assay	I-experimental_method
of	O
WT	B-protein_state
,	O
Tdp2	B-protein
knockout	O
and	O
complemented	O
MEF	O
cells	O
after	O
treatment	O
with	O
indicated	O
concentrations	O
of	O
etoposide	B-chemical
for	O
3	O
h	O
;	O
error	O
bars	O
,	O
s	O
.	O
d	O
,	O
n	O
=	O
3	O
.	O

Top2	B-protein_type
chemotherapeutic	O
agents	O
remain	O
frontline	O
treatments	O
,	O
and	O
exposure	O
to	O
the	O
chemical	O
and	O
damaged	O
DNA	B-chemical
triggers	O
of	O
Top2	B-protein_type
-	O
DNA	B-chemical
protein	O
crosslink	O
formation	O
are	O
unavoidable	O
.	O

Together	O
with	O
mutagenesis	B-experimental_method
and	O
functional	B-experimental_method
assays	I-experimental_method
,	O
our	O
new	O
Tdp2	B-protein
structures	B-evidence
in	B-protein_state
the	I-protein_state
absence	I-protein_state
of	I-protein_state
ligands	B-chemical
and	O
in	B-protein_state
complex	I-protein_state
with	I-protein_state
DNA	B-chemical
damage	O
reveal	O
four	O
novel	O
facets	O
of	O
Tdp2	B-protein
DNA	B-chemical
-	O
protein	O
conjugate	O
processing	O
:	O
(	O
i	O
)	O
The	O
Tdp2	B-protein
active	B-site
site	I-site
is	O
well	O
-	O
suited	O
for	O
accommodating	O
a	O
variety	O
of	O
DNA	B-chemical
structures	O
including	O
abasic	O
and	O
bulky	O
alkylated	O
DNA	B-chemical
lesions	O
that	O
trigger	O
Top2	B-protein_type
poisoning	O
,	O
(	O
ii	O
)	O
High	O
-	O
resolution	O
structural	B-experimental_method
analysis	I-experimental_method
coupled	O
with	O
mutational	B-experimental_method
studies	I-experimental_method
and	O
QM	B-experimental_method
/	I-experimental_method
MM	I-experimental_method
molecular	I-experimental_method
modeling	I-experimental_method
of	O
the	O
Tdp2	B-protein
reaction	O
coordinate	O
support	O
a	O
single	O
metal	O
-	O
ion	O
mechanism	O
for	O
the	O
diverse	O
clade	O
of	O
EEP	B-structure_element
domain	O
catalyzed	O
phosphoryl	B-protein_type
hydrolase	I-protein_type
reactions	O
,	O
(	O
iii	O
)	O
The	O
Tdp2	B-protein
active	B-site
site	I-site
is	O
conformationally	B-protein_state
plastic	I-protein_state
,	O
and	O
undergoes	O
intricate	O
rearrangements	O
upon	O
DNA	B-chemical
and	O
Mg2	B-chemical
+	I-chemical
cofactor	O
binding	O
and	O
(	O
iv	O
)	O
Naturally	O
occurring	O
Tdp2	B-protein
variants	O
undermine	O
Tdp2	B-protein
active	B-site
site	I-site
chemistry	O
,	O
cellular	O
and	O
biochemical	O
activities	O
.	O

We	O
hypothesize	O
that	O
binding	O
of	O
the	O
Top2	B-protein_type
protein	O
component	O
of	O
a	O
DNA	B-chemical
–	O
protein	O
crosslink	O
and	O
/	O
or	O
other	O
protein	O
-	O
regulated	O
assembly	O
of	O
the	O
Tdp2	B-protein
active	B-site
site	I-site
might	O
also	O
serve	O
to	O
regulate	O
Tdp2	B-protein
activity	O
to	O
restrict	O
it	O
from	O
misplaced	O
Top2	B-protein_type
processing	O
events	O
,	O
such	O
that	O
it	O
cleaves	O
only	O
topologically	O
trapped	O
or	O
poisoned	O
Top2	B-protein_type
molecules	O
when	O
needed	O
.	O

Etoposide	B-chemical
and	O
other	O
Top2	B-protein_type
poisons	O
remain	O
front	O
line	O
anti	O
-	O
cancer	O
drugs	O
,	O
and	O
Tdp2	B-protein
frameshift	O
mutations	O
in	O
the	O
human	B-species
population	O
confer	O
hypersensitivity	O
to	O
Top2	B-protein_type
poisons	O
including	O
etoposide	B-chemical
and	O
doxyrubicin	B-chemical
.	O

Here	O
,	O
we	O
elucidate	O
their	O
mechanisms	O
of	O
extracellular	O
ion	O
recognition	O
and	O
exchange	O
through	O
a	O
structural	B-experimental_method
analysis	I-experimental_method
of	O
the	O
exchanger	B-protein_type
from	O
Methanococcus	B-species
jannaschii	I-species
(	O
NCX_Mj	B-protein
)	O
bound	B-protein_state
to	I-protein_state
Na	B-chemical
+,	I-chemical
Ca2	B-chemical
+	I-chemical
or	O
Sr2	B-chemical
+	I-chemical
in	O
various	O
occupancies	O
and	O
in	O
an	O
apo	B-protein_state
state	O
.	O

The	O
basic	O
functional	O
unit	O
for	O
ion	O
transport	O
in	O
NCX	B-protein_type
consists	O
of	O
ten	O
membrane	B-structure_element
-	I-structure_element
spanning	I-structure_element
segments	I-structure_element
,	O
comprising	O
two	O
homologous	O
halves	B-structure_element
.	O

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

An	O
independent	O
analysis	O
based	O
on	O
molecular	B-experimental_method
-	I-experimental_method
dynamics	I-experimental_method
simulations	I-experimental_method
demonstrates	O
that	O
the	O
structures	B-evidence
capture	O
mechanistically	O
relevant	O
states	O
.	O

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

The	O
Na	B-chemical
+	I-chemical
occupation	O
at	O
SCa	B-site
,	O
compounded	O
with	O
the	O
expected	O
3Na	O
+:	B-chemical
1Ca2	O
+	B-chemical
stoichiometry	O
,	O
implies	O
our	O
previous	O
assignment	O
of	O
the	O
Smid	B-site
site	O
must	O
be	O
re	O
-	O
evaluated	O
.	O

However	O
,	O
when	O
Sext	B-site
becomes	O
empty	B-protein_state
at	O
low	B-protein_state
Na	B-chemical
+	I-chemical
concentrations	O
,	O
TM7a	B-structure_element
and	O
TM7b	B-structure_element
become	O
a	O
continuous	O
straight	O
helix	B-structure_element
(	O
Fig	O
.	O
2a	O
),	O
and	O
the	O
carbonyl	O
group	O
of	O
Ala206	B-residue_name_number
retracts	O
away	O
(	O
Fig	O
.	O
2b	O
-	O
d	O
).	O

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

Sr2	B-chemical
+	I-chemical
is	O
transported	O
by	O
NCX	B-protein_type
similarly	O
to	O
Ca2	B-chemical
+	I-chemical
,	O
and	O
is	O
distinguishable	O
from	O
Na	B-chemical
+	I-chemical
by	O
its	O
greater	O
electron	B-evidence
-	I-evidence
density	I-evidence
intensity	I-evidence
.	O

The	O
Sr2	B-protein_state
+-	I-protein_state
loaded	I-protein_state
NCX_Mj	B-protein
structure	B-evidence
adopts	O
the	O
partially	B-protein_state
open	I-protein_state
conformation	O
observed	O
at	O
low	O
Na	B-chemical
+	I-chemical
concentrations	O
.	O

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

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

Alternatively	O
,	O
this	O
structure	B-evidence
might	O
capture	O
a	O
fully	B-protein_state
protonated	I-protein_state
state	O
of	O
the	O
transporter	B-protein_type
,	O
to	O
which	O
Na	B-chemical
+	I-chemical
and	O
Ca2	B-chemical
+	I-chemical
cannot	O
bind	O
.	O

Ion	O
occupancy	O
determines	O
the	O
free	O
-	O
energy	O
landscape	O
of	O
NCX_Mj	B-protein

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

Specifically	O
,	O
we	O
first	O
simulated	B-experimental_method
the	O
outward	B-protein_state
-	I-protein_state
occluded	I-protein_state
form	O
,	O
in	O
the	O
ion	O
configuration	O
we	O
previously	O
predicted	O
,	O
now	O
confirmed	O
by	O
the	O
high	B-protein_state
-	I-protein_state
Na	I-protein_state
+	I-protein_state
crystal	B-evidence
structure	I-evidence
described	O
above	O
(	O
Fig	O
.	O
1b	O
).	O

That	O
is	O
,	O
Na	B-chemical
+	I-chemical
ions	O
occupy	O
Sext	B-site
,	O
SCa	B-site
,	O
and	O
Sint	B-site
,	O
while	O
D240	B-residue_name_number
is	O
protonated	B-protein_state
and	O
a	O
water	B-chemical
molecule	O
occupies	O
Smid	B-site
.	O

The	O
Na	B-chemical
+	I-chemical
ion	O
at	O
Sext	B-site
was	O
then	O
relocated	O
from	O
the	O
site	O
to	O
the	O
bulk	O
solution	O
(	O
Methods	O
),	O
and	O
this	O
system	O
was	O
then	O
allowed	O
to	O
evolve	O
freely	O
in	O
time	O
.	O

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

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

This	O
semi	B-protein_state
-	I-protein_state
open	I-protein_state
conformation	O
is	O
nearly	O
identical	O
to	O
that	O
found	O
to	O
be	O
the	O
most	O
probable	O
when	O
Na	B-chemical
+	I-chemical
occupies	O
only	O
SCa	B-site
and	O
Sint	B-site
(	O
2	O
×	O
Na	B-chemical
+,	I-chemical
Fig	O
.	O
5a	O
),	O
demonstrating	O
that	O
binding	O
(	O
or	O
release	O
)	O
of	O
Na	B-chemical
+	I-chemical
to	O
Sext	B-site
occurs	O
in	O
this	O
metastable	B-protein_state
conformation	O
.	O

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

For	O
example	O
,	O
in	O
the	O
crystal	B-evidence
of	O
NCX_Mj	B-protein
in	O
LCP	B-experimental_method
,	O
the	O
extracellular	B-structure_element
half	I-structure_element
of	O
the	O
gating	B-structure_element
helices	I-structure_element
(	O
TM6	B-structure_element
and	O
TM1	B-structure_element
)	O
form	O
a	O
lattice	O
contact	O
,	O
which	O
might	O
ultimately	O
restrict	O
the	O
degree	O
of	O
opening	O
of	O
the	O
ion	B-site
-	I-site
binding	I-site
sites	I-site
in	O
some	O
cases	O
(	O
e	O
.	O
g	O
.	O
in	O
the	O
apo	B-protein_state
,	O
low	B-protein_state
pH	I-protein_state
structure	B-evidence
).	O

The	O
simulations	B-experimental_method
also	O
demonstrate	O
how	O
this	O
landscape	O
is	O
drastically	O
re	O
-	O
shaped	O
upon	O
each	O
ion	O
-	O
binding	O
event	O
.	O

We	O
posit	O
that	O
a	O
similar	O
principle	O
might	O
govern	O
the	O
alternating	O
-	O
access	O
mechanism	O
in	O
other	O
transporters	B-protein_type
;	O
that	O
is	O
,	O
we	O
anticipate	O
that	O
for	O
both	O
symporters	B-protein_type
and	O
antiporters	B-protein_type
,	O
it	O
is	O
the	O
feasibility	O
of	O
the	O
occluded	B-protein_state
state	O
,	O
encoded	O
in	O
the	O
protein	B-evidence
conformational	I-evidence
free	I-evidence
-	I-evidence
energy	I-evidence
landscape	I-evidence
and	O
its	O
dependence	O
on	O
substrate	O
binding	O
,	O
that	O
ultimately	O
explains	O
their	O
specific	O
coupling	O
mechanisms	O
.	O

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

Na	B-chemical
+	I-chemical
binding	O
to	O
outward	B-protein_state
-	I-protein_state
facing	I-protein_state
NCX_Mj	B-protein
.	O

(	O
a	O
)	O
Overall	O
structure	B-evidence
of	O
native	B-protein_state
outward	B-protein_state
-	I-protein_state
facing	I-protein_state
NCX_Mj	B-protein
from	O
crystals	B-experimental_method
grown	I-experimental_method
in	O
150	O
mM	O
Na	B-chemical
+.	I-chemical

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

For	O
Ca2	B-chemical
+,	I-chemical
a	O
map	B-evidence
is	O
shown	O
in	O
which	O
a	O
correction	O
for	O
the	O
charge	O
-	O
transfer	O
between	O
the	O
ion	O
and	O
the	O
protein	O
is	O
introduced	O
,	O
alongside	O
the	O
uncorrected	O
map	B-evidence
(	O
see	O
Supplementary	O
Notes	O
3	O
-	O
4	O
and	O
Supplementary	O
Fig	O
.	O
5	O
-	O
6	O
).	O

Asterisks	O
mark	O
the	O
states	O
whose	O
crystal	B-evidence
structures	I-evidence
have	O
been	O
determined	O
in	O
this	O
study	O
.	O

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

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

The	O
discovery	O
of	O
nine	O
U2AF65	B-site
-	I-site
binding	I-site
sites	I-site
for	O
contiguous	B-structure_element
Py	B-chemical
-	I-chemical
tract	I-chemical
nucleotides	I-chemical
was	O
unexpected	O
.	O

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

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

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

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

A	O
second	B-structure_element
kink	I-structure_element
at	O
P236	B-residue_name_number
,	O
coupled	O
with	O
respective	O
packing	O
of	O
the	O
L235	B-residue_name_number
and	O
M238	B-residue_name_number
side	O
chains	O
on	O
the	O
N	O
-	O
terminal	O
α	B-structure_element
-	I-structure_element
helical	I-structure_element
RRM1	I-structure_element
extension	I-structure_element
and	O
the	O
core	B-protein_state
RRM1	B-structure_element
α2	B-structure_element
-	I-structure_element
helix	I-structure_element
,	O
reverses	O
the	O
direction	O
of	O
the	O
inter	B-structure_element
-	I-structure_element
RRM	I-structure_element
linker	I-structure_element
towards	O
the	O
RRM1	B-site
/	I-site
RRM2	I-site
interface	I-site
and	O
away	O
from	O
the	O
RNA	B-site
-	I-site
binding	I-site
site	I-site
.	O

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

Despite	O
12	B-experimental_method
concurrent	I-experimental_method
mutations	I-experimental_method
,	O
the	O
AdML	B-gene
RNA	B-evidence
affinity	I-evidence
of	O
the	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
-	I-mutant
12Gly	I-mutant
variant	B-protein_state
was	O
reduced	O
by	O
only	O
three	O
-	O
fold	O
relative	O
to	O
the	O
unmodified	B-protein_state
protein	B-protein
(	O
Fig	O
.	O
4b	O
),	O
which	O
is	O
less	O
than	O
the	O
penalty	O
of	O
the	O
V254P	B-mutant
mutation	O
that	O
disrupts	O
the	O
rU5	B-residue_name_number
hydrogen	B-bond_interaction
bond	I-bond_interaction
(	O
Fig	O
.	O
3d	O
,	O
i	O
).	O

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

The	O
non	O
-	O
additive	O
effects	O
of	O
the	O
Q147A	B-mutant
/	O
V254P	B-mutant
/	O
R227A	B-mutant
triple	B-experimental_method
mutation	I-experimental_method
,	O
coupled	O
with	O
the	O
context	O
-	O
dependent	O
penalties	O
of	O
an	O
internal	O
U2AF65	B-protein
linker	B-experimental_method
deletion	I-experimental_method
,	O
highlights	O
the	O
importance	O
of	O
the	O
structural	O
interplay	O
among	O
the	O
U2AF65	B-protein
linker	B-structure_element
and	O
the	O
N	B-structure_element
-	I-structure_element
and	I-structure_element
C	I-structure_element
-	I-structure_element
terminal	I-structure_element
extensions	I-structure_element
flanking	O
the	O
core	B-protein_state
RRMs	B-structure_element
.	O

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

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

A	O
handful	O
of	O
inter	B-structure_element
-	I-structure_element
RRM	I-structure_element
hydrogen	B-bond_interaction
bonds	I-bond_interaction
are	O
apparent	O
between	O
the	O
side	O
chains	O
of	O
RRM1	B-structure_element
-	O
N155	B-residue_name_number
and	O
RRM2	B-structure_element
-	O
K292	B-residue_name_number
,	O
RRM1	B-structure_element
-	O
N155	B-residue_name_number
and	O
RRM2	B-structure_element
-	O
D272	B-residue_name_number
as	O
well	O
as	O
the	O
backbone	O
atoms	O
of	O
RRM1	B-structure_element
-	O
G221	B-residue_name_number
and	O
RRM2	B-structure_element
-	O
D273	B-residue_name_number
(	O
Fig	O
.	O
4c	O
).	O

We	O
first	O
characterized	O
the	O
conformational	O
dynamics	O
spectrum	O
of	O
U2AF65	B-protein
in	O
the	O
absence	B-protein_state
of	I-protein_state
RNA	B-chemical
(	O
Fig	O
.	O
6c	O
,	O
d	O
;	O
Supplementary	O
Fig	O
.	O
7a	O
,	O
b	O
).	O

A	O
0	O
.	O
45	O
FRET	B-evidence
value	I-evidence
was	O
again	O
predominant	O
,	O
indicating	O
a	O
similar	O
RNA	B-protein_state
-	I-protein_state
bound	I-protein_state
conformation	O
and	O
structural	O
dynamics	O
for	O
the	O
untethered	B-protein_state
and	O
tethered	B-protein_state
U2AF651	B-mutant
,	I-mutant
2LFRET	I-mutant
(	O
Cy3	B-chemical
/	O
Cy5	B-chemical
).	O

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

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

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

Several	O
observations	O
indicate	O
that	O
the	O
numerous	O
intramolecular	O
contacts	O
,	O
here	O
revealed	O
among	O
the	O
inter	B-structure_element
-	I-structure_element
RRM	I-structure_element
linker	I-structure_element
and	O
RRM1	B-structure_element
,	O
RRM2	B-structure_element
,	O
and	O
the	O
N	O
-	O
terminal	O
RRM1	B-structure_element
extension	I-structure_element
,	O
synergistically	O
coordinate	O
U2AF65	B-protein
–	O
Py	B-chemical
-	I-chemical
tract	I-chemical
recognition	O
.	O

The	O
lesser	O
0	O
.	O
65	O
–	O
0	O
.	O
8	O
and	O
0	O
.	O
2	O
–	O
0	O
.	O
3	O
FRET	B-evidence
values	I-evidence
in	O
the	O
untethered	B-protein_state
U2AF651	B-mutant
,	I-mutant
2LFRET	I-mutant
(	O
Cy3	B-chemical
/	O
Cy5	B-chemical
)	O
experiment	O
could	O
correspond	O
to	O
respective	O
variants	O
of	O
the	O
‘	O
closed	B-protein_state
',	O
back	B-protein_state
-	I-protein_state
to	I-protein_state
-	I-protein_state
back	I-protein_state
U2AF65	B-protein
conformations	O
characterized	O
by	O
NMR	B-experimental_method
/	O
PRE	B-experimental_method
data	O
,	O
or	O
to	O
extended	B-protein_state
U2AF65	B-protein
conformations	O
,	O
in	O
which	O
the	O
intramolecular	O
RRM1	B-structure_element
/	O
RRM2	B-structure_element
interactions	O
have	O
dissociated	O
the	O
protein	B-protein
is	O
bound	B-protein_state
to	I-protein_state
RNA	B-chemical
via	O
single	B-protein_state
RRMs	B-structure_element
.	O

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

Based	O
on	O
(	O
i	O
)	O
similar	O
RNA	B-evidence
affinities	I-evidence
of	O
U2AF65	B-protein
and	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
,	O
(	O
ii	O
)	O
indistinguishable	O
conformations	O
among	O
four	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
structures	B-evidence
in	O
two	O
different	O
crystal	O
packing	O
arrangements	O
and	O
(	O
iii	O
)	O
penalties	B-evidence
of	O
structure	B-experimental_method
-	I-experimental_method
guided	I-experimental_method
mutations	I-experimental_method
in	O
RNA	B-experimental_method
binding	I-experimental_method
and	I-experimental_method
splicing	I-experimental_method
assays	I-experimental_method
,	O
we	O
suggest	O
that	O
the	O
extended	B-protein_state
inter	B-structure_element
-	I-structure_element
RRM	I-structure_element
regions	I-structure_element
of	O
the	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
structures	B-evidence
underlie	O
cognate	O
Py	B-chemical
-	I-chemical
tract	I-chemical
recognition	O
by	O
the	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
U2AF65	B-protein
protein	O
.	O

RRM	B-structure_element
,	O
RNA	B-structure_element
recognition	I-structure_element
motif	I-structure_element
;	O
RS	B-structure_element
,	O
arginine	B-structure_element
-	I-structure_element
serine	I-structure_element
rich	I-structure_element
;	O
UHM	B-structure_element
,	O
U2AF	B-structure_element
homology	I-structure_element
motif	I-structure_element
;	O
ULM	B-structure_element
,	O
U2AF	B-structure_element
ligand	I-structure_element
motif	I-structure_element
.	O

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

New	O
residues	O
of	O
the	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
structures	B-evidence
are	O
coloured	O
a	O
darker	O
shade	O
of	O
blue	O
,	O
apart	O
from	O
residues	O
that	O
were	O
tested	O
by	O
site	B-experimental_method
-	I-experimental_method
directed	I-experimental_method
mutagenesis	I-experimental_method
,	O
which	O
are	O
coloured	O
yellow	O
.	O

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

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

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

The	O
U2AF651	B-mutant
,	I-mutant
2LFRET	I-mutant
proteins	O
were	O
doubly	O
labelled	O
at	O
A181C	B-mutant
/	O
Q324C	B-mutant
such	O
that	O
a	O
mixture	O
of	O
Cy3	B-chemical
/	O
Cy5	B-chemical
fluorophores	B-chemical
are	O
expected	O
to	O
be	O
present	O
at	O
each	O
site	O
.	O

Typical	O
single	B-experimental_method
-	I-experimental_method
molecule	I-experimental_method
FRET	I-experimental_method
traces	B-evidence
(	O
c	O
,	O
e	O
,	O
g	O
,	O
i	O
)	O
show	O
fluorescence	O
intensities	O
from	O
Cy3	B-chemical
(	O
green	O
)	O
and	O
Cy5	B-chemical
(	O
red	O
)	O
and	O
the	O
calculated	B-evidence
apparent	I-evidence
FRET	I-evidence
efficiency	I-evidence
(	O
blue	O
).	O

(	O
a	O
)	O
Diagram	O
of	O
the	O
U2AF65	B-protein
,	O
SF1	B-protein
and	O
U2AF35	B-protein
splicing	O
factors	O
bound	B-protein_state
to	I-protein_state
the	O
consensus	O
elements	O
of	O
the	O
3	B-site
′	I-site
splice	I-site
site	I-site
.	O

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

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

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

At	O
100	O
K	O
,	O
an	O
experimental	O
dose	O
limit	O
of	O
30	O
MGy	O
has	O
been	O
recommended	O
as	O
an	O
upper	O
limit	O
beyond	O
which	O
the	O
biological	O
information	O
derived	O
from	O
any	O
macromolecular	O
crystal	B-evidence
may	O
be	O
compromised	O
(	O
Owen	O
et	O
al	O
.,	O
2006	O
).	O

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

Indeed	O
,	O
the	O
C	O
—	O
Se	B-chemical
bond	O
in	O
selenomethionine	B-chemical
,	O
the	O
stability	O
of	O
which	O
is	O
key	O
for	O
the	O
success	O
of	O
experimental	O
phasing	O
methods	O
,	O
can	O
be	O
cleaved	O
at	O
a	O
dose	O
as	O
low	O
as	O
2	O
MGy	O
for	O
a	O
crystal	B-evidence
maintained	O
at	O
100	O
K	O
(	O
Holton	O
,	O
2007	O
).	O

The	O
significant	O
chemical	O
strain	O
required	O
for	O
catalysis	O
within	O
the	O
active	B-site
site	I-site
of	O
phosphoserine	B-protein_type
aminotransferase	I-protein_type
has	O
been	O
observed	O
to	O
diminish	O
during	O
X	O
-	O
ray	O
exposure	O
(	O
Dubnovitsky	O
et	O
al	O
.,	O
2005	O
).	O

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

Instead	O
,	O
we	O
use	O
here	O
a	O
maximum	B-evidence
density	I-evidence
-	I-evidence
loss	I-evidence
metric	I-evidence
(	O
D	B-evidence
loss	I-evidence
),	O
which	O
is	O
the	O
per	O
-	O
atom	O
equivalent	O
of	O
the	O
magnitude	O
of	O
these	O
negative	B-evidence
Fourier	I-evidence
difference	I-evidence
map	I-evidence
peaks	I-evidence
in	O
units	O
of	O
e	O
Å	O
−	O
3	O
.	O

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

A	O
significant	O
difference	O
was	O
observed	O
between	O
the	O
D	B-evidence
loss	I-evidence
dynamics	I-evidence
for	O
the	O
nonbound	B-protein_state
/	O
bound	B-protein_state
Glu42	B-residue_name_number
O	O
∊	O
1	O
atoms	O
(	O
Fig	O
.	O
5	O
▸	O
c	O
;	O
p	O
=	O
0	O
.	O
007	O
)	O
but	O
not	O
for	O
the	O
Glu42	B-residue_name_number
O	O
∊	O
2	O
atoms	O
(	O
Fig	O
.	O
5	O
▸	O
d	O
;	O
p	O
=	O
0	O
.	O
239	O
),	O
indicating	O
that	O
the	O
stabilizing	O
strength	O
of	O
this	O
salt	B-bond_interaction
-	I-bond_interaction
bridge	I-bond_interaction
interaction	O
was	O
conserved	O
upon	O
RNA	B-chemical
binding	O
and	O
that	O
the	O
water	B-chemical
-	O
mediated	O
hydrogen	B-bond_interaction
bond	I-bond_interaction
had	O
a	O
greater	O
relative	O
susceptibility	O
to	O
atomic	O
disordering	O
in	O
the	O
absence	B-protein_state
of	I-protein_state
RNA	B-chemical
.	O

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

The	O
side	O
chain	O
of	O
Phe32	B-residue_name_number
stacks	O
against	O
the	O
G3	B-residue_name_number
base	O
within	O
the	O
11	O
TRAP	B-site
RNA	I-site
-	I-site
binding	I-site
interfaces	I-site
(	O
Antson	O
et	O
al	O
.,	O
1999	O
).	O

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

At	O
25	O
.	O
0	O
MGy	O
,	O
the	O
magnitude	O
of	O
the	O
RNA	B-chemical
backbone	O
D	B-evidence
loss	I-evidence
was	O
of	O
the	O
same	O
order	O
as	O
for	O
the	O
radiation	O
-	O
insensitive	O
Gly	B-residue_name
Cα	O
atoms	O
and	O
on	O
average	O
less	O
than	O
half	O
that	O
of	O
the	O
acidic	O
residues	O
of	O
the	O
protein	O
(	O
Supplementary	O
Fig	O
.	O
S3	O
).	O

Upon	O
RNA	B-chemical
binding	O
,	O
the	O
Asp39	B-residue_name_number
side	O
-	O
chain	O
carboxyl	O
group	O
solvent	O
-	O
accessible	O
area	O
changes	O
from	O
∼	O
75	O
to	O
35	O
Å2	O
,	O
still	O
allowing	O
a	O
high	O
CO2	B-chemical
-	O
formation	O
rate	B-evidence
K	I-evidence
2	I-evidence
.	O

However	O
,	O
for	O
each	O
of	O
these	O
residues	O
the	O
exact	O
crystal	O
contacts	O
are	O
not	O
preserved	O
between	O
bound	B-protein_state
and	O
nonbound	B-protein_state
TRAP	B-complex_assembly
or	O
even	O
between	O
monomers	O
within	O
one	O
TRAP	B-complex_assembly
ring	B-structure_element
.	O

For	O
example	O
,	O
in	O
bound	B-protein_state
TRAP	B-complex_assembly
,	O
Glu73	B-residue_name_number
hydrogen	O
-	O
bonds	O
to	O
a	O
nearby	O
lysine	B-residue_name
on	O
each	O
of	O
the	O
11	O
subunits	B-structure_element
,	O
whereas	O
in	O
nonbound	B-protein_state
TRAP	B-complex_assembly
no	O
such	O
interaction	O
exists	O
and	O
Glu73	B-residue_name_number
interacts	O
with	O
a	O
variable	O
number	O
of	O
refined	O
waters	B-chemical
in	O
each	O
subunit	B-structure_element
.	O

It	O
has	O
been	O
suggested	O
(	O
Burmeister	O
,	O
2000	O
)	O
that	O
Tyr	B-residue_name
residues	O
can	O
lose	O
their	O
aromatic	O
–	O
OH	O
group	O
owing	O
to	O
radiation	O
-	O
induced	O
effects	O
;	O
however	O
,	O
no	O
energetically	O
favourable	O
pathway	O
for	O
–	O
OH	O
cleavage	O
exists	O
and	O
this	O
has	O
not	O
been	O
detected	O
in	O
aqueous	O
radiation	O
-	O
chemistry	O
studies	O
.	O

Indeed	O
,	O
no	O
convincing	O
reproducible	O
Fourier	B-evidence
difference	I-evidence
peaks	I-evidence
above	O
the	O
background	O
map	B-evidence
noise	O
were	O
observed	O
around	O
any	O
Tyr	B-residue_name
terminal	O
–	O
OH	O
groups	O
.	O

Within	O
the	O
nonbound	B-protein_state
TRAP	B-complex_assembly
macromolecule	O
,	O
the	O
acidic	O
residues	O
within	O
the	O
unoccupied	O
RNA	B-site
-	I-site
binding	I-site
interfaces	I-site
(	O
Asp39	B-residue_name_number
,	O
Glu36	B-residue_name_number
,	O
Glu42	B-residue_name_number
)	O
are	O
notably	O
amongst	O
the	O
most	O
susceptible	O
residues	O
within	O
the	O
asymmetric	O
unit	O
(	O
Fig	O
.	O
4	O
▸).	O

In	O
(	O
a	O
)	O
clear	O
difference	B-evidence
density	I-evidence
is	O
observed	O
around	O
the	O
Glu42	B-residue_name_number
carboxyl	O
side	O
chain	O
in	O
chain	O
H	O
,	O
within	O
the	O
lowest	B-evidence
dose	I-evidence
difference	I-evidence
map	I-evidence
at	O
d	O
2	O
=	O
3	O
.	O
9	O
MGy	O
.	O

The	O
important	O
MAPK	B-protein_type
family	I-protein_type
of	O
signalling	O
proteins	O
is	O
controlled	O
by	O
MAPK	B-protein_type
phosphatases	I-protein_type
(	O
MKPs	B-protein_type
).	O

The	O
best	O
-	O
studied	O
docking	O
interactions	O
are	O
those	O
between	O
MAP	B-protein_type
kinases	I-protein_type
and	O
‘	O
D	B-structure_element
-	I-structure_element
motifs	I-structure_element
',	O
which	O
consists	O
of	O
two	O
or	O
more	O
basic	O
residues	O
followed	O
by	O
a	O
short	B-structure_element
linker	I-structure_element
and	O
a	O
cluster	O
of	O
hydrophobic	O
residues	O
.	O

Here	O
,	O
we	O
present	O
the	O
crystal	B-evidence
structure	I-evidence
of	O
JNK1	B-protein
in	B-protein_state
complex	I-protein_state
with	I-protein_state
the	O
catalytic	B-structure_element
domain	I-structure_element
of	O
MKP7	B-protein
.	O

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

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

To	O
understand	O
the	O
molecular	O
basis	O
of	O
JNK1	B-protein
recognition	O
by	O
MKP7	B-protein
,	O
we	O
determined	O
the	O
crystal	B-evidence
structure	I-evidence
of	O
unphosphorylated	B-protein_state
JNK1	B-protein
in	B-protein_state
complex	I-protein_state
with	I-protein_state
the	O
MKP7	B-protein
-	O
CD	B-structure_element
(	O
Fig	O
.	O
3a	O
,	O
Supplementary	O
Fig	O
.	O
1a	O
and	O
Table	O
1	O
).	O

This	O
loop	B-structure_element
is	O
shortened	O
by	O
nine	O
residues	O
in	O
MKP7	B-protein
-	O
CD	B-structure_element
compared	O
with	O
that	O
in	O
VHR	B-protein
.	O

Although	O
the	O
catalytically	O
important	O
residues	O
in	O
MKP7	B-protein
-	O
CD	B-structure_element
are	O
well	O
aligned	O
with	O
those	O
in	O
VHR	B-protein
,	O
the	O
residues	O
in	O
the	O
P	B-structure_element
-	I-structure_element
loop	I-structure_element
of	O
MKP7	B-protein
are	O
smaller	O
and	O
have	O
a	O
more	O
hydrophobic	O
character	O
than	O
those	O
of	O
VHR	B-protein
(	O
Cys124	B-residue_name_number
-	O
Arg125	B-residue_name_number
-	O
Glu126	B-residue_name_number
-	O
Gly127	B-residue_name_number
-	O
Tyr128	B-residue_name_number
-	O
Gly129	B-residue_name_number
-	O
Arg130	B-residue_name_number
;	O
Fig	O
.	O
3b	O
,	O
c	O
).	O

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

As	O
shown	O
in	O
Fig	O
.	O
4f	O
,	O
all	O
the	O
mutants	B-protein_state
,	O
except	O
F287D	B-mutant
/	I-mutant
A	I-mutant
,	O
showed	O
little	O
or	O
no	O
activity	O
change	O
compared	O
with	O
the	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
MKP7	B-protein
-	O
CD	B-structure_element
.	O

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

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

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

We	O
next	O
tested	O
in	O
vivo	O
interactions	O
between	O
JNK1	B-protein
mutants	B-protein_state
and	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
MKP7	B-protein
by	O
coimmunoprecipitation	B-experimental_method
experiments	I-experimental_method
under	O
unstimulated	O
conditions	O
.	O

When	O
co	B-experimental_method
-	I-experimental_method
expressed	I-experimental_method
in	O
HEK293T	O
cells	O
,	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
(	O
HA	O
)-	O
JNK1	B-protein
was	O
readily	O
precipitated	O
with	O
(	O
Myc	O
)-	O
MKP7	B-protein
(	O
Fig	O
.	O
6d	O
),	O
indicating	O
that	O
MKP7	B-protein
binds	O
dephosphorylated	B-protein_state
JNK1	B-protein
protein	O
in	O
vivo	O
.	O

In	O
contrast	O
,	O
cells	O
transfected	O
with	O
the	O
MKP7	B-protein
FXF	B-structure_element
-	I-structure_element
motif	I-structure_element
mutants	B-protein_state
(	O
F285D	B-mutant
,	O
F287D	B-mutant
and	O
L288D	B-mutant
)	O
showed	O
little	O
protective	O
effect	O
after	O
ultraviolet	O
treatment	O
and	O
similar	O
levels	O
of	O
apoptosis	O
rates	O
were	O
detected	O
to	O
cells	O
transfected	O
with	O
control	O
vectors	O
(	O
Fig	O
.	O
6e	O
,	O
f	O
).	O

MKP5	B-protein
is	O
unique	O
among	O
the	O
MKPs	B-protein_type
in	O
possessing	O
an	O
additional	O
domain	O
of	O
unknown	O
function	O
at	O
the	O
N	O
-	O
terminus	O
(	O
Fig	O
.	O
7a	O
).	O

Deletion	B-experimental_method
of	I-experimental_method
the	O
KBD	B-structure_element
in	O
MKP5	B-protein
leads	O
to	O
a	O
280	O
-	O
fold	O
increase	O
in	O
Km	B-evidence
for	O
p38α	B-protein
substrate	O
.	O

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

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

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

Taken	O
together	O
,	O
our	O
results	O
suggest	O
that	O
MKP5	B-protein
binds	O
JNK1	B-protein
in	O
a	O
docking	O
mode	O
similar	O
to	O
that	O
in	O
the	O
JNK1	B-complex_assembly
–	I-complex_assembly
MKP7	I-complex_assembly
complex	O
,	O
and	O
the	O
detailed	O
interaction	O
model	O
can	O
be	O
generated	O
using	O
molecular	B-experimental_method
dynamics	I-experimental_method
simulation	I-experimental_method
based	O
on	O
the	O
structure	B-evidence
of	O
JNK1	B-complex_assembly
–	I-complex_assembly
MKP7	I-complex_assembly
-	I-complex_assembly
CD	I-complex_assembly
complex	O
(	O
Supplementary	O
Fig	O
.	O
4b	O
,	O
c	O
).	O

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

In	O
addition	O
to	O
the	O
canonical	O
D	B-site
-	I-site
site	I-site
,	O
the	O
MAPK	B-protein_type
ERK2	B-protein
contains	O
a	O
second	B-site
binding	I-site
site	I-site
utilized	O
by	O
transcription	O
factor	O
substrates	O
and	O
phosphatases	B-protein_type
,	O
the	O
FXF	B-site
-	I-site
motif	I-site
-	I-site
binding	I-site
site	I-site
(	O
also	O
called	O
F	B-site
-	I-site
site	I-site
),	O
that	O
is	O
exposed	O
in	O
active	B-protein_state
ERK2	B-protein
and	O
the	O
D	B-structure_element
-	I-structure_element
motif	I-structure_element
peptide	O
-	O
induced	O
conformation	O
of	O
MAPKs	B-protein_type
.	O

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

Mutant	B-protein_state
F285D	B-mutant
and	O
JNK1	B-protein
were	O
eluted	O
as	O
monomers	B-oligomeric_state
,	O
with	O
the	O
molecular	O
masses	O
of	O
∼	O
17	O
and	O
44	O
kDa	O
,	O
respectively	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	B-structure_element
α6	B-structure_element
of	O
KAP	B-protein
(	O
corresponding	O
to	O
helix	B-structure_element
α4	B-structure_element
of	O
MKP7	B-protein
-	O
CD	B-structure_element
)	O
plays	O
little	O
,	O
if	O
any	O
,	O
role	O
in	O
the	O
formation	O
of	O
a	O
stable	B-protein_state
heterodimer	B-oligomeric_state
of	O
CDK2	B-protein
and	O
KAP	B-protein
.	O
(	O
c	O
)	O
Sequence	B-experimental_method
alignment	I-experimental_method
of	O
the	O
JNK	B-site
-	I-site
interacting	I-site
regions	I-site
on	O
MKPs	B-protein_type
.	O

(	O
d	O
)	O
Sequence	B-experimental_method
alignment	I-experimental_method
of	O
the	O
F	B-structure_element
-	I-structure_element
site	I-structure_element
regions	I-structure_element
on	O
MAPKs	B-protein_type
.	O

HEK293T	O
cells	O
were	O
infected	O
with	O
lentiviruses	B-taxonomy_domain
expressing	O
MKP7	B-protein
and	O
its	O
mutants	B-protein_state
(	O
1	O
.	O
0	O
μg	O
).	O

HEK293T	O
cells	O
were	O
co	B-experimental_method
-	I-experimental_method
transfected	I-experimental_method
with	O
MKP7	B-protein
full	B-protein_state
-	I-protein_state
length	I-protein_state
(	O
1	O
.	O
0	O
μg	O
)	O
and	O
JNK1	B-protein
(	O
wild	B-protein_state
type	I-protein_state
or	O
mutants	B-protein_state
as	O
indicated	O
,	O
1	O
.	O
0	O
μg	O
).	O

MKP5	B-protein
-	O
CD	B-structure_element
is	O
crucial	O
for	O
JNK1	B-protein
binding	O
and	O
enzyme	O
catalysis	O
.	O

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

(	O
h	O
)	O
Pull	B-experimental_method
-	I-experimental_method
down	I-experimental_method
assays	I-experimental_method
of	O
MKP5	B-protein
-	O
CD	B-structure_element
by	O
GST	B-protein_state
-	I-protein_state
tagged	I-protein_state
JNK1	B-protein
mutants	B-protein_state
.	O

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

The	O
IDA	B-site
binding	I-site
pocket	I-site
covers	O
LRRs	B-structure_element
2	I-structure_element
–	I-structure_element
14	I-structure_element
and	O
all	O
residues	O
originate	O
from	O
the	O
inner	O
surface	O
of	O
the	O
HAESA	B-protein
superhelix	B-structure_element
.	O

The	O
conserved	B-protein_state
PIP	B-structure_element
motif	I-structure_element
is	O
highlighted	O
in	O
yellow	O
,	O
the	O
central	O
Hyp	B-residue_name
in	O
blue	O
.	O

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

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

Full	B-protein_state
-	I-protein_state
length	I-protein_state
IDA	B-protein
is	O
proteolytically	B-ptm
processed	I-ptm
and	O
a	O
conserved	B-protein_state
stretch	B-residue_range
of	I-residue_range
20	I-residue_range
amino	I-residue_range
-	I-residue_range
acids	I-residue_range
(	O
termed	O
EPIP	B-structure_element
)	O
can	O
rescue	O
the	O
IDA	B-protein
loss	O
-	O
of	O
-	O
function	O
phenotype	O
(	O
Figure	O
1A	O
).	O

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

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

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

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

The	O
COO	O
-	O
group	O
of	O
Asn69IDA	B-residue_name_number
is	O
in	O
direct	O
contact	O
with	O
Arg407HAESA	B-residue_name_number
and	O
Arg409HAESA	B-residue_name_number
and	O
HAESA	B-protein
cannot	O
bind	O
a	O
C	B-protein_state
-	I-protein_state
terminally	I-protein_state
extended	I-protein_state
IDA	B-mutant
-	I-mutant
SFVN	I-mutant
peptide	O
(	O
Figures	O
1D	O
,	O
F	O
,	O
2D	O
).	O

This	O
suggests	O
that	O
the	O
conserved	B-protein_state
Asn69IDA	B-residue_name_number
may	O
constitute	O
the	O
very	O
C	O
-	O
terminus	O
of	O
the	O
mature	B-protein_state
IDA	B-chemical
peptide	I-chemical
in	O
planta	B-taxonomy_domain
and	O
that	O
active	B-protein_state
IDA	B-protein
is	O
generated	O
by	O
proteolytic	O
processing	O
from	O
a	O
longer	O
pre	O
-	O
protein	O
.	O

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

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

It	O
has	O
been	O
recently	O
reported	O
that	O
SOMATIC	B-protein_type
EMBRYOGENESIS	I-protein_type
RECEPTOR	I-protein_type
KINASES	I-protein_type
(	O
SERKs	B-protein_type
)	O
are	O
positive	O
regulators	O
of	O
floral	O
abscission	O
and	O
can	O
interact	O
with	O
HAESA	B-protein
and	O
HSL2	B-protein
in	O
an	O
IDA	O
-	O
dependent	O
manner	O
.	O

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

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

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

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

SERK1	B-protein
loop	B-structure_element
residues	O
establish	O
multiple	O
hydrophobic	B-bond_interaction
and	I-bond_interaction
polar	I-bond_interaction
contacts	I-bond_interaction
with	O
Lys66IDA	B-residue_name_number
and	O
the	O
C	O
-	O
terminal	O
Arg	B-structure_element
-	I-structure_element
His	I-structure_element
-	I-structure_element
Asn	I-structure_element
motif	I-structure_element
in	O
IDA	B-protein
(	O
Figure	O
4C	O
).	O

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

35S	B-gene
::	O
IDA	B-protein
plants	B-taxonomy_domain
showed	O
significantly	O
increased	O
abscission	O
compared	O
to	O
Col	O
-	O
0	O
controls	O
in	O
inflorescence	O
positions	O
2	O
and	O
3	O
(	O
a	O
).	O

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

(	O
A	O
)	O
Structural	B-experimental_method
comparison	I-experimental_method
of	O
plant	B-taxonomy_domain
steroid	B-chemical
and	O
peptide	B-protein_type
hormone	I-protein_type
membrane	B-protein_type
signaling	I-protein_type
complexes	I-protein_type
.	O

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

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

The	O
conserved	B-protein_state
(	B-structure_element
Arg	I-structure_element
)-	I-structure_element
His	I-structure_element
-	I-structure_element
Asn	I-structure_element
motif	I-structure_element
is	O
highlighted	O
in	O
red	O
,	O
the	O
central	O
Hyp	B-residue_name
residue	O
in	O
IDLs	B-protein_type
and	O
CLEs	B-protein_type
is	O
marked	O
in	O
blue	O
.	O

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

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

Diverse	O
plant	B-taxonomy_domain
peptide	B-protein_type
hormones	I-protein_type
may	O
thus	O
also	O
bind	O
their	O
LRR	B-protein_type
-	I-protein_type
RK	I-protein_type
receptors	I-protein_type
in	O
an	O
extended	B-protein_state
conformation	I-protein_state
along	O
the	O
inner	O
surface	O
of	O
the	O
LRR	B-structure_element
domain	I-structure_element
and	O
may	O
also	O
use	O
small	B-protein_state
,	O
shape	B-protein_state
-	I-protein_state
complementary	I-protein_state
co	B-protein_type
-	I-protein_type
receptors	I-protein_type
for	O
high	O
-	O
affinity	O
ligand	O
binding	O
and	O
receptor	O
activation	O
.	O

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

The	O
canonical	O
scenario	O
of	O
cap	O
-	O
dependent	O
and	O
IRES	B-site
-	O
dependent	O
initiation	O
involves	O
positioning	O
of	O
the	O
AUG	O
start	O
codon	O
and	O
the	O
initiator	O
tRNAMet	B-chemical
in	O
the	O
ribosomal	O
peptidyl	B-site
-	I-site
tRNA	I-site
(	I-site
P	I-site
)	I-site
site	I-site
,	O
facilitated	O
by	O
interaction	O
with	O
initiation	B-protein_type
factors	I-protein_type
.	O

Subsequent	O
binding	O
of	O
an	O
elongator	O
aminoacyl	B-chemical
-	I-chemical
tRNA	I-chemical
to	O
the	O
ribosomal	O
A	B-site
site	I-site
transitions	O
the	O
initiation	B-complex_assembly
complex	I-complex_assembly
into	O
the	O
elongation	O
cycle	O
of	O
translation	O
.	O

The	O
IGR	B-structure_element
-	O
IRES	B-site
-	O
driven	O
initiation	B-protein_state
does	O
not	O
involve	O
initiator	O
tRNAMet	B-chemical
and	O
initiation	B-protein_state
factors	O
.	O

The	O
codon	B-structure_element
-	I-structure_element
anticodon	I-structure_element
-	I-structure_element
like	I-structure_element
helix	I-structure_element
of	O
PKI	B-structure_element
is	O
stabilized	O
by	O
interactions	O
with	O
the	O
universally	B-protein_state
conserved	I-protein_state
decoding	B-site
-	I-site
center	I-site
nucleotides	O
G577	B-residue_name_number
,	O
A1755	B-residue_name_number
and	O
A1756	B-residue_name_number
(	O
G530	B-residue_name_number
,	O
A1492	B-residue_name_number
and	O
A1493	B-residue_name_number
in	O
E	B-species
.	I-species
coli	I-species
16S	O
ribosomal	O
RNA	B-chemical
,	O
or	O
rRNA	B-chemical
).	O

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

Intersubunit	O
rotation	O
occurs	O
spontaneously	O
upon	O
peptidyl	O
transfer	O
,	O
and	O
is	O
coupled	O
with	O
formation	O
of	O
hybrid	B-protein_state
tRNA	B-chemical
states	O
.	O

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

The	O
large	O
ribosomal	O
subunit	B-structure_element
is	O
shown	O
in	O
cyan	O
;	O
the	O
small	B-structure_element
subunit	I-structure_element
in	O
light	O
yellow	O
(	O
head	B-structure_element
)	O
and	O
wheat	O
-	O
yellow	O
(	O
body	B-structure_element
);	O
the	O
TSV	B-species
IRES	B-site
in	O
red	O
,	O
eEF2	B-protein
in	O
green	O
.	O

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

Although	O
the	O
mechanism	O
of	O
sordarin	B-chemical
action	O
is	O
not	O
fully	O
understood	O
,	O
the	O
inhibitor	O
does	O
not	O
affect	O
the	O
conformation	O
of	O
eEF2	B-complex_assembly
•	I-complex_assembly
GDPNP	I-complex_assembly
on	O
the	O
ribosome	B-complex_assembly
,	O
rendering	O
it	O
an	O
excellent	O
tool	O
in	O
translocation	O
studies	O
.	O

(	O
a	O
)	O
Rotational	O
states	O
of	O
the	O
40S	B-complex_assembly
subunit	B-structure_element
in	O
the	O
80S	B-complex_assembly
•	I-complex_assembly
IRES	I-complex_assembly
structure	B-evidence
(	O
INIT	B-complex_assembly
;	O
PDB	O
3J6Y	O
)	O
and	O
in	O
80S	B-complex_assembly
•	I-complex_assembly
IRES	I-complex_assembly
•	I-complex_assembly
eEF2	I-complex_assembly
Structures	B-evidence
I	I-evidence
,	I-evidence
II	I-evidence
,	I-evidence
III	I-evidence
,	I-evidence
IV	I-evidence
and	I-evidence
V	I-evidence
(	O
this	O
work	O
).	O

The	O
sizes	O
of	O
the	O
arrows	O
correspond	O
to	O
the	O
extent	O
of	O
the	O
head	B-structure_element
swivel	O
(	O
yellow	O
)	O
and	O
subunit	B-structure_element
rotation	O
(	O
black	O
).	O

Our	O
structures	B-evidence
represent	O
hitherto	O
uncharacterized	O
translocation	O
complexes	O
of	O
the	O
TSV	B-species
IRES	B-site
captured	O
within	O
globally	O
distinct	O
80S	B-complex_assembly
conformations	O
(	O
Figures	O
1b	O
and	O
2	O
).	O

Changes	O
in	O
ribosome	B-complex_assembly
conformation	O
and	O
eEF2	B-protein
positions	O
are	O
coupled	O
with	O
IRES	B-site
movement	O
through	O
the	O
ribosome	B-complex_assembly

Using	O
the	O
post	B-protein_state
-	I-protein_state
translocation	I-protein_state
S	B-species
.	I-species
cerevisiae	I-species
80S	B-complex_assembly
ribosome	I-complex_assembly
bound	B-protein_state
with	I-protein_state
the	O
P	B-site
and	I-site
E	I-site
site	I-site
tRNAs	B-chemical
as	O
a	O
reference	O
(	O
80S	B-complex_assembly
•	I-complex_assembly
2tRNA	I-complex_assembly
•	I-complex_assembly
mRNA	I-complex_assembly
),	O
in	O
which	O
both	O
the	O
subunit	B-structure_element
rotation	O
and	O
the	O
head	B-structure_element
-	O
body	B-structure_element
swivel	O
are	O
0	O
°,	O
we	O
found	O
that	O
the	O
ribosome	B-complex_assembly
adopts	O
four	O
globally	O
distinct	O
conformations	O
in	O
Structures	B-evidence
I	I-evidence
through	I-evidence
V	I-evidence
(	O
Figure	O
1b	O
;	O
see	O
also	O
Figure	O
1	O
—	O
figure	O
supplement	O
1	O
and	O
Figure	O
2	O
—	O
source	O
data	O
1	O
).	O

Structure	B-evidence
V	I-evidence
is	O
in	O
a	O
nearly	O
non	B-protein_state
-	I-protein_state
rotated	I-protein_state
conformation	O
(	O
0	O
.	O
5	O
°),	O
very	O
similar	O
to	O
that	O
of	O
post	B-protein_state
-	I-protein_state
translocation	I-protein_state
ribosome	B-complex_assembly
-	I-complex_assembly
tRNA	I-complex_assembly
complexes	O
.	O

However	O
in	O
the	O
pre	B-protein_state
-	I-protein_state
translocation	I-protein_state
intermediates	O
(	O
from	O
Structure	B-evidence
I	I-evidence
to	I-evidence
IV	I-evidence
),	O
the	O
beak	O
of	O
the	O
head	B-structure_element
domain	O
first	O
turns	O
toward	O
the	O
large	B-structure_element
subunit	I-structure_element
and	O
then	O
backs	O
off	O
(	O
Figure	O
2	O
—	O
figure	O
supplement	O
1	O
).	O

By	O
comparison	O
,	O
the	O
similarly	O
mid	B-protein_state
-	I-protein_state
rotated	I-protein_state
(	O
4	O
°)	O
80S	B-complex_assembly
•	I-complex_assembly
TSV	I-complex_assembly
IRES	I-complex_assembly
initiation	B-protein_state
complex	O
,	O
in	O
the	O
absence	B-protein_state
of	I-protein_state
eEF2	B-protein
,	O
adopts	O
a	O
mid	B-protein_state
-	I-protein_state
swiveled	I-protein_state
position	O
(~	O
10	O
°)	O
(	O
Figure	O
2c	O
).	O

The	O
view	O
shows	O
the	O
vicinity	O
of	O
the	O
ribosomal	O
E	B-site
site	I-site
.	O

Structures	B-evidence
of	O
80S	B-complex_assembly
•	I-complex_assembly
IRES	I-complex_assembly
complexes	O
in	O
the	O
absence	B-protein_state
of	I-protein_state
eEF2	B-protein
(	O
INIT	B-complex_assembly
;	O
PDB	O
3J6Y	O
,)	O
and	O
in	O
the	O
presence	B-protein_state
of	I-protein_state
eEF2	B-protein
(	O
this	O
work	O
)	O
are	O
shown	O
in	O
the	O
lower	O
row	O
and	O
labeled	O
.	O

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

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

We	O
collectively	O
term	O
domains	O
I	B-structure_element
and	O
II	B-structure_element
the	O
5	B-structure_element
’	I-structure_element
domain	I-structure_element
.	O

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

Distances	O
between	O
nucleotides	O
6848	B-residue_number
and	O
6913	B-residue_number
in	O
SL4	B-structure_element
and	O
PKI	B-structure_element
,	O
respectively	O
,	O
are	O
shown	O
(	O
see	O
also	O
Figure	O
2	O
—	O
source	O
data	O
1	O
).	O

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

However	O
,	O
in	O
the	O
extended	B-protein_state
conformations	O
,	O
these	O
parts	O
of	O
the	O
IRES	B-site
and	O
the	O
60S	B-complex_assembly
subunit	B-structure_element
are	O
separated	O
by	O
more	O
than	O
10	O
Å	O
,	O
suggesting	O
that	O
an	O
interaction	O
between	O
them	O
stabilizes	O
the	O
bent	B-protein_state
conformations	O
but	O
not	O
the	O
extended	B-protein_state
ones	O
.	O

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

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

Sordarin	B-chemical
is	O
shown	O
in	O
pink	O
with	O
oxygen	O
atoms	O
in	O
red	O
.	O

The	O
GTPase	B-site
-	I-site
associated	I-site
center	I-site
comprises	O
the	O
P	B-structure_element
stalk	I-structure_element
(	O
L11	B-structure_element
and	O
L7	B-structure_element
/	O
L12	B-structure_element
stalk	B-structure_element
in	O
bacteria	B-taxonomy_domain
)	O
and	O
the	O
sarcin	B-structure_element
-	I-structure_element
ricin	I-structure_element
loop	I-structure_element
(	O
SRL	B-structure_element
,	O
nt	O
3012	B-residue_range
–	I-residue_range
3042	I-residue_range
).	O

The	O
view	O
was	O
obtained	O
by	O
structural	B-experimental_method
alignment	I-experimental_method
of	O
the	O
18S	B-chemical
rRNAs	I-chemical
.	O

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

There	O
are	O
two	O
modest	O
but	O
noticeable	O
domain	O
rearrangements	O
between	O
Structures	B-evidence
I	I-evidence
and	I-evidence
V	I-evidence
.	O
Unlike	O
in	O
free	B-protein_state
eEF2	B-protein
,	O
which	O
can	O
sample	O
large	O
movements	O
of	O
at	O
least	O
50	O
Å	O
of	O
the	O
C	O
-	O
terminal	O
superdomain	B-structure_element
relative	O
to	O
the	O
N	O
-	O
terminal	O
superdomain	B-structure_element
(	O
Figure	O
5c	O
),	O
eEF2	B-protein
undergoes	O
moderate	O
repositioning	O
of	O
domain	O
IV	B-structure_element
(~	O
3	O
Å	O
;	O
Figure	O
5a	O
)	O
and	O
domain	O
III	B-structure_element
(~	O
5	O
Å	O
;	O
Figure	O
6d	O
).	O

Domain	O
IV	B-structure_element
of	O
eEF2	B-protein
binds	O
at	O
the	O
40S	B-complex_assembly
A	B-site
site	I-site
in	O
Structures	B-evidence
I	I-evidence
to	I-evidence
V	I-evidence
but	O
the	O
mode	O
of	O
interaction	O
differs	O
in	O
each	O
complex	O
(	O
Figure	O
6	O
).	O

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

Modest	O
intra	O
-	O
eEF2	B-protein
shifts	O
of	O
domain	O
IV	B-structure_element
between	O
Structures	B-evidence
I	I-evidence
to	I-evidence
V	I-evidence
outline	O
a	O
stochastic	O
trajectory	O
(	O
Figure	O
5a	O
),	O
consistent	O
with	O
local	O
adjustments	O
of	O
the	O
domain	O
in	O
the	O
A	B-site
site	I-site
.	O

We	O
therefore	O
define	O
C1274	B-residue_name_number
as	O
the	O
foundation	O
of	O
the	O
'	O
head	B-structure_element
A	B-site
site	I-site
'.	O

Accordingly	O
,	O
we	O
use	O
U1191	B-residue_name_number
(	O
G966	B-residue_name_number
in	O
E	B-species
.	I-species
coli	I-species
)	O
and	O
C1637	B-residue_name_number
(	O
C1400	B-residue_name_number
in	O
E	B-species
.	I-species
coli	I-species
)	O
as	O
the	O
reference	O
points	O
of	O
the	O
'	O
head	B-structure_element
P	B-site
site	I-site
'	O
and	O
'	O
body	B-structure_element
P	B-site
site	I-site
'	O
(	O
Figure	O
2g	O
),	O
respectively	O
,	O
because	O
these	O
nucleotides	O
form	O
a	O
stacking	O
foundation	O
for	O
the	O
fully	B-protein_state
translocated	I-protein_state
mRNA	B-structure_element
-	I-structure_element
tRNA	I-structure_element
helix	I-structure_element
in	O
tRNA	B-protein_state
-	I-protein_state
bound	I-protein_state
structures	B-evidence
and	O
in	O
our	O
post	B-protein_state
-	I-protein_state
translocation	I-protein_state
Structure	B-evidence
V	I-evidence
discussed	O
below	O
.	O

The	O
interaction	O
of	O
PKI	B-structure_element
with	O
the	O
40S	B-complex_assembly
body	B-structure_element
is	O
substantially	O
rearranged	O
relative	O
to	O
that	O
in	O
the	O
initiation	B-protein_state
state	O
.	O

Diphthamide	B-ptm
is	O
a	O
unique	O
posttranslational	O
modification	O
conserved	B-protein_state
in	O
archaeal	B-taxonomy_domain
and	O
eukaryotic	B-taxonomy_domain
EF2	B-protein
(	O
at	O
residue	O
699	B-residue_number
in	O
S	B-species
.	I-species
cerevisiae	I-species
)	O
and	O
involves	O
addition	O
of	O
a	O
~	O
7	O
-	O
Å	O
long	O
3	O
-	O
carboxyamido	O
-	O
3	O
-(	O
trimethylamino	O
)-	O
propyl	O
moiety	O
to	O
the	O
histidine	B-residue_name
imidazole	O
ring	O
at	O
CE1	O
.	O

Switch	B-structure_element
loop	I-structure_element
II	I-structure_element
(	O
aa	O
105	B-residue_range
–	I-residue_range
110	I-residue_range
),	O
which	O
carries	O
the	O
catalytic	B-protein_state
H108	B-residue_name_number
(	O
H92	B-residue_name_number
in	O
E	B-species
.	I-species
coli	I-species
EF	B-protein
-	I-protein
G	I-protein
;	O
is	O
well	O
resolved	O
in	O
all	O
five	O
structures	B-evidence
.	O

Bulged	B-protein_state
A416	B-residue_name_number
interacts	O
with	O
the	O
switch	B-structure_element
loop	I-structure_element
in	O
the	O
vicinity	O
of	O
D53	B-residue_name_number
.	O

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

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

The	O
head	B-site
interface	I-site
of	O
domain	O
IV	B-structure_element
interacts	O
with	O
the	O
40S	B-complex_assembly
head	B-structure_element
(	O
Figure	O
6a	O
).	O

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

The	O
codon	B-structure_element
-	I-structure_element
anticodon	I-structure_element
–	I-structure_element
like	I-structure_element
helix	I-structure_element
is	O
stacked	O
on	O
P	B-site
-	I-site
site	I-site
residues	O
U1191	B-residue_name_number
and	O
C1637	B-residue_name_number
(	O
Figure	O
3d	O
),	O
analogous	O
to	O
stacking	B-bond_interaction
of	O
the	O
tRNA	B-complex_assembly
-	I-complex_assembly
mRNA	I-complex_assembly
helix	B-structure_element
(	O
Figure	O
3e	O
).	O

As	O
in	O
the	O
preceding	O
Structures	B-evidence
,	O
the	O
histidine	B-site
-	I-site
diphthamide	I-site
tip	I-site
is	O
bound	B-protein_state
in	I-protein_state
the	O
minor	B-site
groove	I-site
of	O
the	O
P	B-site
-	I-site
site	I-site
codon	B-structure_element
-	I-structure_element
anticodon	I-structure_element
helix	I-structure_element
.	O

Animation	O
showing	O
the	O
transition	O
from	O
the	O
initiation	B-protein_state
80S	B-complex_assembly
•	I-complex_assembly
TSV	I-complex_assembly
IRES	I-complex_assembly
structures	B-evidence
(	O
Koh	O
et	O
al	O
.,	O
2014	O
)	O
to	O
eEF2	B-protein_state
-	I-protein_state
bound	I-protein_state
Structures	B-evidence
I	I-evidence
through	I-evidence
V	I-evidence
(	O
this	O
work	O
).	O

In	O
scene	O
4	O
,	O
C1274	B-residue_name_number
and	O
U1191	B-residue_name_number
are	O
labeled	O
and	O
shown	O
in	O
yellow	O
;	O
G577	B-residue_name_number
,	O
A1755	B-residue_name_number
and	O
A1756	B-residue_name_number
of	O
the	O
40S	B-complex_assembly
body	B-structure_element
A	B-site
site	I-site
and	O
C1637	B-residue_name_number
of	O
the	O
body	B-structure_element
P	B-site
site	I-site
are	O
labeled	O
and	O
shown	O
in	O
orange	O
.	O

In	O
this	O
work	O
we	O
have	O
captured	O
the	O
structures	B-evidence
of	O
the	O
TSV	B-species
IRES	B-site
,	O
whose	O
PKI	B-structure_element
samples	O
positions	O
between	O
the	O
A	B-site
and	I-site
P	I-site
sites	I-site
(	O
Structures	B-evidence
I	I-evidence
–	I-evidence
IV	I-evidence
),	O
as	O
well	O
as	O
in	O
the	O
P	B-site
site	I-site
(	O
Structure	B-evidence
V	I-evidence
).	O

In	O
summary	O
,	O
the	O
reported	O
ensemble	O
of	O
structures	B-evidence
substantially	O
enhances	O
our	O
understanding	O
of	O
the	O
translocation	O
mechanism	O
,	O
including	O
that	O
of	O
tRNAs	B-chemical
as	O
discussed	O
below	O
.	O

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

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

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

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

The	O
presence	B-protein_state
of	I-protein_state
several	O
translocation	O
complexes	O
in	O
a	O
single	O
sample	O
suggests	O
that	O
the	O
structures	B-evidence
represent	O
equilibrium	O
states	O
of	O
forward	O
and	O
reverse	O
translocation	O
of	O
the	O
IRES	B-site
,	O
which	O
interconvert	O
among	O
each	O
other	O
.	O

These	O
findings	O
indicate	O
that	O
IRES	B-site
translocation	O
by	O
eEF2	B-protein
is	O
futile	O
:	O
the	O
IRES	B-site
returns	O
to	O
the	O
A	B-site
site	I-site
upon	O
releasing	O
eEF2	B-complex_assembly
•	I-complex_assembly
GDP	I-complex_assembly
unless	O
an	O
amino	B-chemical
-	I-chemical
acyl	I-chemical
tRNA	I-chemical
enters	O
the	O
A	B-site
site	I-site
and	O
blocks	O
IRES	B-site
back	O
-	O
translocation	O
.	O

Various	O
degrees	O
of	O
intersubunit	O
rotation	O
have	O
been	O
observed	O
in	O
cryo	B-experimental_method
-	I-experimental_method
EM	I-experimental_method
studies	I-experimental_method
of	O
the	O
80S	B-complex_assembly
•	I-complex_assembly
IRES	I-complex_assembly
initiation	B-protein_state
complexes	O
.	O

The	O
pre	B-protein_state
-	I-protein_state
translocation	I-protein_state
Structure	B-evidence
I	I-evidence
with	O
eEF2	B-protein
least	O
advanced	O
into	O
the	O
A	B-site
site	I-site
adopts	O
a	O
fully	B-protein_state
rotated	I-protein_state
conformation	I-protein_state
.	O

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

The	O
Structures	B-evidence
reveal	O
hopping	O
of	O
the	O
positive	O
clusters	O
over	O
rRNA	B-chemical
helices	B-structure_element
.	O

Whereas	O
intersubunit	O
rotation	O
of	O
the	O
pre	B-protein_state
-	I-protein_state
translocation	I-protein_state
complex	O
occurs	O
spontaneously	O
,	O
the	O
head	B-structure_element
swivel	O
is	O
induced	O
by	O
the	O
eEF2	B-protein
/	O
EF	B-protein
-	I-protein
G	I-protein
translocase	B-protein_type
,	O
consistent	O
with	O
requirement	O
of	O
eEF2	B-protein
for	O
unlocking	O
.	O

Structural	B-experimental_method
studies	I-experimental_method
revealed	O
large	O
head	B-structure_element
swivels	O
in	O
various	O
70S	B-complex_assembly
•	I-complex_assembly
tRNA	I-complex_assembly
•	I-complex_assembly
EF	I-complex_assembly
-	I-complex_assembly
G	I-complex_assembly
and	O
80S	B-complex_assembly
•	I-complex_assembly
tRNA	I-complex_assembly
•	I-complex_assembly
eEF2	I-complex_assembly
complexes	O
,	O
but	O
not	O
in	O
'	O
locked	B-protein_state
'	O
complexes	B-protein_state
with	I-protein_state
the	O
A	B-site
site	I-site
occupied	O
by	O
the	O
tRNA	B-chemical
in	O
the	O
absence	B-protein_state
of	I-protein_state
the	O
translocase	B-protein_type
.	O

This	O
'	O
locked	B-protein_state
'	O
state	O
is	O
identical	O
to	O
that	O
observed	O
for	O
PKI	B-structure_element
in	O
the	O
80S	B-complex_assembly
•	I-complex_assembly
IRES	I-complex_assembly
initiation	B-protein_state
structures	B-evidence
in	O
the	O
absence	B-protein_state
of	I-protein_state
eEF2	B-protein
.	O

Observation	O
of	O
different	O
PKI	B-structure_element
conformations	O
sampling	O
a	O
range	O
of	O
positions	O
between	O
the	O
A	B-site
and	I-site
P	I-site
sites	I-site
in	O
the	O
presence	B-protein_state
of	I-protein_state
eEF2	B-complex_assembly
•	I-complex_assembly
GDP	I-complex_assembly
implies	O
that	O
thermal	O
fluctuations	O
of	O
the	O
40S	B-complex_assembly
head	B-structure_element
domain	O
are	O
sufficient	O
for	O
translocation	O
along	O
the	O
energetically	O
flat	O
trajectory	O
.	O

In	O
all	O
five	O
structures	B-evidence
,	O
the	O
GTPase	B-structure_element
domain	I-structure_element
is	O
attached	O
to	O
the	O
P	B-structure_element
stalk	I-structure_element
and	O
the	O
sarcin	B-structure_element
-	I-structure_element
ricin	I-structure_element
loop	I-structure_element
.	O

The	O
least	B-protein_state
rotated	I-protein_state
conformation	O
of	O
the	O
post	B-protein_state
-	I-protein_state
translocation	I-protein_state
Structure	B-evidence
V	I-evidence
suggests	O
conformational	O
changes	O
that	O
may	O
trigger	O
eEF2	B-protein
release	O
from	O
the	O
ribosome	B-complex_assembly
at	O
the	O
end	O
of	O
translocation	O
.	O

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

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

Because	O
translocation	O
of	O
tRNA	B-chemical
must	O
involve	O
large	O
-	O
scale	O
dynamics	O
,	O
this	O
step	O
has	O
long	O
been	O
regarded	O
as	O
the	O
most	O
puzzling	O
step	O
of	O
translation	O
.	O

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

Second	O
,	O
the	O
structures	B-evidence
clarify	O
the	O
structural	O
basis	O
of	O
the	O
often	O
-	O
used	O
but	O
structurally	O
undefined	O
terms	O
'	O
locking	O
'	O
and	O
'	O
unlocking	O
'	O
with	O
respect	O
to	O
the	O
pre	B-protein_state
-	I-protein_state
translocation	I-protein_state
complex	O
(	O
Figure	O
6f	O
).	O

Previous	O
studies	O
showed	O
that	O
this	O
movement	O
widens	O
the	O
constriction	B-site
('	O
gate	B-site
')	O
between	O
the	O
P	B-site
and	I-site
E	I-site
sites	I-site
,	O
thus	O
allowing	O
the	O
P	B-site
-	O
tRNA	B-chemical
passage	O
to	O
the	O
E	B-site
site	I-site
.	O

In	O
addition	O
to	O
the	O
'	O
gate	B-site
-	O
opening	O
'	O
role	O
,	O
we	O
now	O
show	O
that	O
the	O
head	B-structure_element
swivel	O
brings	O
the	O
head	B-structure_element
A	B-site
site	I-site
to	O
the	O
body	B-structure_element
P	B-site
site	I-site
,	O
allowing	O
a	O
step	O
-	O
wise	O
conveying	O
of	O
the	O
codon	B-structure_element
-	I-structure_element
anticodon	I-structure_element
helix	I-structure_element
between	O
the	O
A	B-site
and	I-site
P	I-site
sites	I-site
.	O

Movement	O
of	O
PKI	B-structure_element
relative	O
to	O
the	O
head	B-structure_element
occurs	O
during	O
the	O
subsequent	O
reverse	O
swivel	O
in	O
three	O
3	O
–	O
7	O
Å	O
sub	O
-	O
steps	O
(	O
II	B-evidence
to	I-evidence
III	I-evidence
to	I-evidence
IV	I-evidence
to	I-evidence
V	I-evidence
).	O

Our	O
work	O
sheds	O
light	O
on	O
the	O
dynamic	O
mechanism	O
of	O
cap	O
-	O
independent	O
translation	O
by	O
IGR	B-structure_element
IRESs	B-site
,	O
tightly	O
coupled	O
with	O
the	O
universally	B-protein_state
conserved	I-protein_state
dynamic	O
properties	O
of	O
the	O
ribosome	B-complex_assembly
.	O

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

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

Ensemble	O
cryo	B-experimental_method
-	I-experimental_method
EM	I-experimental_method

While	O
the	O
inactive	B-protein_state
α	B-protein
subunits	I-protein
build	O
the	O
two	O
outer	O
rings	B-structure_element
,	O
the	O
β	B-protein
subunits	I-protein
form	O
the	O
inner	O
rings	B-structure_element
.	O

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

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

Although	O
the	O
chemical	O
nature	O
of	O
the	O
substrate	B-site
-	I-site
binding	I-site
channel	I-site
and	O
hence	O
substrate	O
preferences	O
are	O
unique	O
to	O
each	O
of	O
the	O
distinct	O
active	B-protein_state
β	B-protein
subunits	I-protein
,	O
all	O
active	B-site
sites	I-site
employ	O
an	O
identical	O
reaction	O
mechanism	O
to	O
hydrolyse	O
peptide	O
bonds	O
.	O

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

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

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

By	O
contrast	O
,	O
the	O
T1A	B-mutant
mutation	O
in	O
subunit	O
β5	B-protein
has	O
been	O
reported	O
to	O
be	O
lethal	O
or	O
nearly	O
so	O
.	O

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

The	O
extremely	O
weak	O
growth	O
of	O
the	O
β5	B-mutant
-	I-mutant
T1A	I-mutant
mutant	B-protein_state
pp	B-chemical
cis	B-protein_state
described	O
by	O
Chen	O
and	O
Hochstrasser	O
compared	O
with	O
the	O
inviability	O
reported	O
by	O
Heinemeyer	O
et	O
al	O
.	O
prompted	O
us	O
to	O
analyse	O
this	O
discrepancy	O
.	O

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

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

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

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

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

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

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

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

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

On	O
the	O
basis	O
of	O
these	O
results	O
,	O
we	O
propose	O
that	O
CPs	B-complex_assembly
from	O
all	O
domains	O
of	O
life	O
use	O
a	O
catalytic	B-site
triad	I-site
consisting	O
of	O
Thr1	B-residue_name_number
,	O
Lys33	B-residue_name_number
and	O
Asp	B-residue_name
/	O
Glu17	B-residue_name_number
for	O
both	O
autocatalytic	B-ptm
precursor	I-ptm
processing	I-ptm
and	O
proteolysis	O
(	O
Fig	O
.	O
3d	O
).	O

Soaking	B-experimental_method
the	O
β5	B-mutant
-	I-mutant
D166N	I-mutant
crystals	B-experimental_method
with	O
carfilzomib	B-chemical
and	O
MG132	B-chemical
resulted	O
in	O
covalent	O
modification	O
of	O
Thr1	B-residue_name_number
at	O
high	O
occupancy	O
(	O
Supplementary	O
Fig	O
.	O
8c	O
).	O

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

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

Moreover	O
,	O
the	O
structural	B-evidence
data	I-evidence
reveal	O
that	O
the	O
thiol	O
group	O
of	O
Cys1	B-residue_name_number
is	O
rotated	O
by	O
74	O
°	O
with	O
respect	O
to	O
the	O
hydroxyl	O
side	O
chain	O
of	O
Thr1	B-residue_name_number
(	O
Fig	O
.	O
4f	O
and	O
Supplementary	O
Fig	O
.	O
9b	O
).	O

Crystal	B-evidence
structure	I-evidence
analysis	O
of	O
the	O
β5	B-mutant
-	I-mutant
T1S	I-mutant
mutant	B-protein_state
confirmed	O
precursor	B-ptm
processing	I-ptm
(	O
Fig	O
.	O
4g	O
),	O
and	O
ligand	B-complex_assembly
-	I-complex_assembly
complex	I-complex_assembly
structures	B-evidence
with	O
bortezomib	B-chemical
and	O
carfilzomib	B-chemical
unambiguously	O
corroborated	O
the	O
reactivity	O
of	O
Ser1	B-residue_name_number
(	O
Fig	O
.	O
5	O
).	O

Because	O
both	O
conformations	O
of	O
Ser1Oγ	B-residue_name_number
are	O
hydrogen	B-bond_interaction
-	I-bond_interaction
bonded	I-bond_interaction
to	O
Lys33NH2	B-residue_name_number
(	O
Fig	O
.	O
4h	O
),	O
the	O
relay	O
system	O
is	O
capable	O
of	O
hydrolysing	O
peptide	O
substrates	O
,	O
albeit	O
at	O
lower	O
rates	O
compared	O
with	O
Thr1	B-residue_name_number
.	O

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

These	O
observations	O
highlight	O
the	O
unique	O
function	O
and	O
importance	O
of	O
the	O
β5	B-protein
propeptide	B-structure_element
as	O
well	O
as	O
the	O
β5	B-protein
active	B-site
site	I-site
for	O
maturation	O
and	O
function	O
of	O
the	O
eukaryotic	B-taxonomy_domain
CP	B-complex_assembly
.	O

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

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

Consistent	O
with	O
this	O
model	O
,	O
the	O
positively	O
charged	O
Thr1	B-residue_name_number
N	O
terminus	O
is	O
engaged	O
in	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
with	O
inhibitory	O
compounds	O
like	O
fellutamide	B-chemical
B	I-chemical
(	O
ref	O
.),	O
α	B-chemical
-	I-chemical
ketoamides	I-chemical
,	O
homobelactosin	B-chemical
C	I-chemical
(	O
ref	O
.)	O
and	O
salinosporamide	B-chemical
A	I-chemical
(	O
ref	O
.).	O

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

Activity	B-experimental_method
assays	I-experimental_method
with	O
the	O
β5	B-mutant
-	I-mutant
T1S	I-mutant
mutant	B-protein_state
revealed	O
reduced	O
turnover	O
of	O
Suc	B-chemical
-	I-chemical
LLVY	I-chemical
-	I-chemical
AMC	I-chemical
.	O

The	O
greater	O
suitability	O
of	O
threonine	B-residue_name
for	O
the	O
proteasome	B-complex_assembly
active	B-site
site	I-site
,	O
which	O
has	O
been	O
noted	O
in	O
biochemical	O
as	O
well	O
as	O
in	O
kinetic	O
studies	O
,	O
constitutes	O
a	O
likely	O
reason	O
for	O
the	O
conservation	B-protein_state
of	O
the	O
Thr1	B-residue_name_number
residue	O
in	O
all	O
proteasomes	B-complex_assembly
from	O
bacteria	B-taxonomy_domain
to	O
eukaryotes	B-taxonomy_domain
.	O

Conformation	O
of	O
proteasomal	O
propeptides	B-structure_element
.	O

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

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

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

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

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

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

Crotonylation	B-ptm
of	O
lysine	B-residue_name
residues	O
(	O
crotonyllysine	B-residue_name
,	O
Kcr	B-residue_name
)	O
has	O
emerged	O
as	O
one	O
of	O
the	O
fundamental	O
histone	B-protein_type
post	O
-	O
translational	O
modifications	O
(	O
PTMs	O
)	O
found	O
in	O
mammalian	B-taxonomy_domain
chromatin	O
.	O

While	O
a	O
number	O
of	O
acetyllysine	B-residue_name
readers	O
have	O
been	O
identified	O
and	O
characterized	O
,	O
a	O
specific	O
reader	O
of	O
the	O
crotonyllysine	B-residue_name
mark	O
remains	O
unknown	O
(	O
reviewed	O
in	O
).	O

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

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

The	O
planar	O
crotonyl	B-chemical
group	O
is	O
inserted	O
between	O
Trp81	B-residue_name_number
and	O
Phe62	B-residue_name_number
of	O
the	O
protein	O
,	O
the	O
aromatic	O
rings	O
of	O
which	O
are	O
positioned	O
strictly	O
parallel	O
to	O
each	O
other	O
and	O
at	O
equal	O
distance	O
from	O
the	O
crotonyl	B-chemical
group	O
,	O
yielding	O
a	O
novel	O
aromatic	O
-	O
amide	O
/	O
aliphatic	O
-	O
aromatic	O
π	B-bond_interaction
-	I-bond_interaction
π	I-bond_interaction
-	I-bond_interaction
π	I-bond_interaction
-	I-bond_interaction
stacking	I-bond_interaction
system	O
that	O
,	O
to	O
our	O
knowledge	O
,	O
has	O
not	O
been	O
reported	O
previously	O
for	O
any	O
protein	O
-	O
protein	O
interaction	O
(	O
Fig	O
.	O
1d	O
and	O
Supplementary	O
Fig	O
.	O
1c	O
).	O

In	O
addition	O
to	O
π	B-bond_interaction
-	I-bond_interaction
π	I-bond_interaction
-	I-bond_interaction
π	I-bond_interaction
stacking	I-bond_interaction
,	O
the	O
crotonyl	B-chemical
group	O
is	O
stabilized	O
by	O
a	O
set	O
of	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
and	O
electrostatic	B-bond_interaction
interactions	I-bond_interaction
.	O

The	O
fixed	O
position	O
of	O
the	O
Thr61	B-residue_name_number
hydroxyl	O
group	O
,	O
which	O
facilitates	O
interactions	O
with	O
both	O
the	O
amide	O
and	O
Cα	O
of	O
K9cr	B-ptm
,	O
is	O
achieved	O
through	O
a	O
hydrogen	B-bond_interaction
bond	I-bond_interaction
with	O
imidazole	O
ring	O
of	O
His59	B-residue_name_number
.	O

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

Binding	O
of	O
H3K9cr	B-protein_type
induced	O
resonance	B-evidence
changes	I-evidence
in	O
slow	O
exchange	O
regime	O
on	O
the	O
NMR	B-experimental_method
time	O
scale	O
,	O
indicative	O
of	O
strong	O
interaction	O
.	O

To	O
establish	O
whether	O
the	O
Taf14	B-protein
YEATS	B-structure_element
domain	I-structure_element
is	O
able	O
to	O
recognize	O
other	O
recently	O
identified	O
acyllysine	B-residue_name
marks	O
,	O
we	O
performed	O
solution	B-experimental_method
pull	I-experimental_method
-	I-experimental_method
down	I-experimental_method
assays	I-experimental_method
using	O
H3	B-protein_type
peptides	O
acetylated	B-protein_state
,	O
propionylated	B-protein_state
,	O
butyrylated	B-protein_state
,	O
and	O
crotonylated	B-protein_state
at	O
lysine	B-residue_name_number
9	I-residue_name_number
(	O
residues	O
1	B-residue_range
–	I-residue_range
20	I-residue_range
of	O
H3	B-protein_type
).	O

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

In	O
contrast	O
,	O
mutation	B-experimental_method
of	O
Val24	B-residue_name_number
,	O
a	O
residue	O
located	O
on	O
another	O
side	O
of	O
Trp81	B-residue_name_number
,	O
had	O
no	O
effect	O
on	O
binding	O
(	O
Fig	O
.	O
2d	O
and	O
Supplementary	O
Fig	O
.	O
5a	O
,	O
c	O
).	O

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

NMR	B-experimental_method
data	O
was	O
recorded	O
using	O
a	O
Bruker	O
800	O
MHz	O
Data	O
format	O
PDB	O
format	O
text	O
file	O
.	O

Tom1	O
GAT	B-structure_element
structural	O
data	O
is	O
publicly	O
available	O
in	O
the	O
RCSB	O
Protein	O
Data	O
Bank	O
(	O
http	O
://	O
www	O
.	O
rscb	O
.	O
org	O
/)	O
under	O
the	O
accession	O
number	O
PDB	O
:	O
2n9d	O

NMR	B-experimental_method
and	O
refinement	B-evidence
statistics	I-evidence
for	O
the	O
Tom1	B-protein
GAT	B-structure_element
domain	O
.	O

PGRMC1	B-protein
binds	O
to	O
EGFR	B-protein_type
and	O
cytochromes	B-protein_type
P450	I-protein_type
,	O
and	O
is	O
known	O
to	O
be	O
involved	O
in	O
cancer	O
proliferation	O
and	O
in	O
drug	O
resistance	O
.	O

Previous	O
studies	O
showed	O
that	O
deprivation	B-protein_state
of	I-protein_state
iron	B-chemical
or	O
haem	B-chemical
suppresses	O
tumourigenesis	O
.	O

The	O
dimer	B-oligomeric_state
binds	O
to	O
EGFR	B-protein_type
and	O
cytochromes	B-protein_type
P450	I-protein_type
to	O
enhance	O
tumour	O
cell	O
proliferation	O
and	O
chemoresistance	O
.	O

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

We	O
analysed	O
the	O
haem	B-chemical
-	O
dependent	O
dimerization	B-oligomeric_state
of	O
the	O
PGRMC1	B-protein
cytosolic	B-structure_element
domain	I-structure_element
(	O
a	O
.	O
a	O
.	O
44	B-residue_range
–	I-residue_range
195	I-residue_range
)	O
in	O
solution	O
(	O
Fig	O
.	O
2	O
and	O
Table	O
2	O
).	O

The	O
C129S	B-mutant
mutant	B-protein_state
of	O
PGRMC1	B-protein
also	O
interacted	O
with	O
endogenous	B-protein_state
PGRMC1	B-protein
and	O
EGFR	B-protein_type
(	O
Supplementary	O
Fig	O
.	O
16	O
).	O

Similarly	O
,	O
EGFR	B-protein_type
signaling	O
was	O
suppressed	O
by	O
treatment	O
of	O
HCT116	O
cells	O
with	O
SA	B-chemical
(	O
Fig	O
.	O
4g	O
)	O
or	O
CORM3	B-chemical
(	O
Fig	O
.	O
4h	O
).	O

We	O
examined	O
the	O
role	O
of	O
PGRMC1	B-protein
in	O
metastatic	O
progression	O
by	O
xenograft	B-experimental_method
transplantation	I-experimental_method
assays	I-experimental_method
using	O
super	O
-	O
immunodeficient	O
NOD	O
/	O
scid	O
/	O
γnull	O
(	O
NOG	O
)	O
mice	O
.	O

This	O
effect	O
was	O
reversed	O
by	O
co	B-experimental_method
-	I-experimental_method
expression	I-experimental_method
of	O
the	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
PGRMC1	B-protein
but	O
not	O
of	O
the	O
Y113F	B-mutant
mutant	B-protein_state
,	O
suggesting	O
that	O
PGRMC1	B-protein
enhances	O
doxorubicin	B-chemical
resistance	O
of	O
cancer	O
cells	O
by	O
facilitating	O
its	O
degradation	O
via	O
cytochromes	B-protein_type
P450	I-protein_type
.	O

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

Recently	O
,	O
Peluso	O
et	O
al	O
.	O
reported	O
that	O
PGRMC1	B-protein
binds	O
to	O
PGRMC2	B-protein
,	O
suggesting	O
that	O
MAPR	B-protein_type
family	O
members	O
may	O
also	O
undergo	O
haem	B-chemical
-	O
mediated	O
heterodimerization	O
.	O

While	O
the	O
effects	O
of	O
structural	O
diversity	O
of	O
CYP	B-protein_type
family	O
proteins	O
and	O
interactions	O
with	O
different	O
xenobiotic	O
substrates	O
should	O
further	O
be	O
examined	O
,	O
the	O
current	O
results	O
suggest	O
that	O
the	O
interaction	O
of	O
drug	O
-	O
metabolizing	O
CYPs	B-protein_type
with	O
the	O
haem	B-chemical
-	O
mediated	O
dimer	B-oligomeric_state
of	O
PGRMC1	B-protein
plays	O
a	O
crucial	O
role	O
in	O
regulating	O
their	O
activities	O
.	O

Considering	O
microenvironments	O
in	O
and	O
around	O
malignant	O
tumours	O
,	O
the	O
haem	B-chemical
concentration	O
in	O
cancer	O
cells	O
is	O
likely	O
to	O
be	O
elevated	O
through	O
multiple	O
mechanisms	O
,	O
such	O
as	O
(	O
i	O
)	O
an	O
increased	O
intake	O
of	O
haem	B-chemical
,	O
(	O
ii	O
)	O
mutation	O
of	O
enzymes	O
in	O
TCA	O
cycle	O
(	O
for	O
example	O
,	O
fumarate	B-protein_type
hydratase	I-protein_type
)	O
that	O
increases	O
the	O
level	O
of	O
succinyl	B-chemical
CoA	I-chemical
,	O
a	O
substrate	O
for	O
haem	B-chemical
biosynthesis	O
and	O
(	O
iii	O
)	O
metastasis	O
to	O
haem	B-chemical
-	O
rich	O
organs	O
such	O
as	O
liver	O
,	O
brain	O
and	O
bone	O
marrow	O
.	O

Furthermore	O
,	O
Sigma	B-protein
-	I-protein
2	I-protein
ligand	O
-	O
binding	O
is	O
decreased	O
in	O
transgenic	O
amyloid	O
beta	O
deposition	O
model	O
APP	O
/	O
PS1	O
female	O
mice	O
.	O

These	O
results	O
suggest	O
a	O
possible	O
involvement	O
of	O
PGRMC1	B-protein
in	O
Alzheimer	O
'	O
s	O
disease	O
.	O

Comparison	O
of	O
PGRMC1	B-protein
(	O
blue	O
)	O
and	O
cytochrome	B-protein_type
b5	I-protein_type
(	O
yellow	O
,	O
ID	O
:	O
3NER	O
).	O
(	O
c	O
)	O
PGRMC1	B-protein
has	O
a	O
longer	O
helix	B-structure_element
(	O
a	O
.	O
a	O
.	O
147	B-residue_range
–	I-residue_range
163	I-residue_range
),	O
which	O
is	O
shifted	O
away	O
from	O
the	O
haem	B-chemical
(	O
arrow	O
).	O

SV	B-experimental_method
-	I-experimental_method
AUC	I-experimental_method
experiments	O
were	O
performed	O
with	O
1	O
.	O
5	O
mg	O
ml	O
−	O
1	O
of	O
PGRMC1	B-protein
proteins	O
.	O

The	O
major	O
peak	O
with	O
sedimentation	B-evidence
coefficient	I-evidence
S20	B-evidence
,	I-evidence
w	I-evidence
of	O
1	O
.	O
9	O
∼	O
2	O
.	O
0	O
S	O
(	O
monomer	B-oligomeric_state
)	O
or	O
3	O
.	O
1	O
S	O
(	O
dimer	B-oligomeric_state
)	O
was	O
detected	O
.	O

(	O
b	O
)	O
Close	O
-	O
up	O
view	O
of	O
haem	B-bond_interaction
stacking	I-bond_interaction
.	O

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

of	O
10	O
separate	O
experiments	O
.	O
*	B-evidence
P	I-evidence
<	O
0	O
.	O
05	O
using	O
unpaired	O
Student	B-experimental_method
'	I-experimental_method
s	I-experimental_method
t	I-experimental_method
-	I-experimental_method
test	I-experimental_method
.	O

CO	B-chemical
interferes	O
with	O
the	O
stacking	B-bond_interaction
interactions	I-bond_interaction
of	O
the	O
haems	B-chemical
and	O
thereby	O
inhibits	O
PGRMC1	B-protein
functions	O
.	O

Recent	O
data	O
supports	O
the	O
notion	O
that	O
,	O
to	O
perform	O
this	O
role	O
,	O
the	O
highly	B-protein_state
variable	I-protein_state
αβ	B-complex_assembly
T	I-complex_assembly
cell	I-complex_assembly
antigen	I-complex_assembly
receptor	I-complex_assembly
(	O
TCR	B-complex_assembly
)	O
must	O
be	O
able	O
to	O
recognize	O
thousands	O
,	O
if	O
not	O
millions	O
,	O
of	O
different	O
peptide	O
ligands	O
.	O

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

From	O
this	O
large	O
functional	O
scan	O
,	O
we	O
selected	O
7	O
different	O
APLs	B-chemical
that	O
activated	O
the	O
1E6	O
T	O
cell	O
clone	O
across	O
a	O
wide	O
(	O
4	O
-	O
log	O
)	O
functional	O
range	O
(	O
Table	O
1	O
).	O

As	O
with	O
the	O
3D	B-evidence
affinity	I-evidence
measurements	O
,	O
the	O
2D	B-evidence
affinity	I-evidence
measurements	O
correlated	O
well	O
with	O
the	O
EC50	B-evidence
values	O
for	O
each	O
ligand	O
(	O
Figure	O
2K	O
)	O
demonstrating	O
a	O
strong	O
correlation	O
(	O
Pearson	B-evidence
’	I-evidence
s	I-evidence
correlation	I-evidence
=	O
0	O
.	O
8	O
,	O
P	B-evidence
=	O
0	O
.	O
01	O
)	O
between	O
T	O
cell	O
antigen	O
sensitivity	O
and	O
TCR	B-evidence
binding	I-evidence
affinity	I-evidence
.	O

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

In	O
order	O
to	O
examine	O
the	O
mechanism	O
by	O
which	O
the	O
1E6	B-complex_assembly
TCR	I-complex_assembly
engaged	O
a	O
wide	O
range	O
of	O
peptides	O
with	O
divergent	O
binding	B-evidence
affinities	I-evidence
,	O
we	O
solved	B-experimental_method
the	O
structure	B-evidence
of	O
the	O
1E6	B-complex_assembly
TCR	I-complex_assembly
in	B-protein_state
complex	I-protein_state
with	I-protein_state
all	O
7	O
APLs	B-chemical
used	O
in	O
Figure	O
2	O
.	O

The	O
relatively	O
broad	O
range	O
of	O
buried	O
surface	O
areas	O
(	O
1	O
,	O
670	O
–	O
1	O
,	O
920	O
Å2	O
)	O
did	O
not	O
correlate	O
well	O
with	O
TCR	B-evidence
binding	I-evidence
affinity	I-evidence
(	O
Pearson	B-evidence
’	I-evidence
s	I-evidence
correlation	I-evidence
=	O
0	O
.	O
45	O
,	O
P	B-evidence
=	O
0	O
.	O
2	O
).	O

Although	O
the	O
number	O
of	O
peptide	O
contacts	O
was	O
a	O
good	O
predictor	O
of	O
TCR	B-evidence
binding	I-evidence
affinity	I-evidence
for	O
some	O
of	O
the	O
APLs	B-chemical
,	O
for	O
others	O
,	O
the	O
correlation	O
was	O
poor	O
(	O
Pearson	B-evidence
’	I-evidence
s	I-evidence
correlation	I-evidence
=	O
0	O
.	O
045	O
,	O
P	O
=	O
0	O
.	O
92	O
),	O
possibly	O
because	O
of	O
different	O
resolutions	O
for	O
each	O
complex	O
structure	B-evidence
.	O

The	O
stronger	O
ligands	O
all	O
encoded	O
larger	O
side	O
chains	O
(	O
Arg	B-residue_name
or	O
Tyr	B-residue_name
)	O
at	O
peptide	O
position	O
1	B-residue_number
(	O
Figure	O
5	O
,	O
E	O
–	O
H	O
),	O
enabling	O
interactions	O
with	O
1E6	O
that	O
were	O
not	O
present	O
in	O
the	O
weaker	O
APLs	B-chemical
that	O
lacked	O
large	O
side	O
chains	O
in	O
this	O
position	O
(	O
Figure	O
5	O
,	O
A	O
,	O
C	O
,	O
and	O
D	O
).	O

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

The	O
unligated	B-protein_state
structures	B-evidence
of	O
A2	B-chemical
-	I-chemical
AQWGPDAAA	I-chemical
,	O
A2	B-chemical
-	I-chemical
RQWGPDPAAV	I-chemical
,	O
A2	B-chemical
-	I-chemical
YQFGPDFPIA	I-chemical
,	O
and	O
A2	B-chemical
-	I-chemical
RQFGPDFPTI	I-chemical
were	O
virtually	O
identical	O
when	O
in	B-protein_state
complex	I-protein_state
with	I-protein_state
1E6	O
(	O
Figure	O
6	O
,	O
D	O
and	O
F	O
–	O
H	O
).	O

Peptide	O
modifications	O
alter	O
the	O
interaction	O
between	O
the	O
1E6	B-complex_assembly
TCR	I-complex_assembly
and	O
the	O
MHC	B-site
surface	I-site
.	O

An	O
energetic	O
switch	O
from	O
unfavorable	O
to	O
favorable	O
entropy	B-evidence
(	O
order	O
-	O
to	O
-	O
disorder	O
)	O
correlates	O
with	O
antigen	O
potency	O
.	O

The	O
weak	O
binding	B-evidence
affinity	I-evidence
between	O
1E6	O
and	O
A2	B-chemical
-	I-chemical
MVWGPDPLYV	I-chemical
and	O
A2	B-chemical
-	I-chemical
YLGGPDFPTI	I-chemical
generated	O
thermodynamic	O
data	O
that	O
were	O
not	O
robust	O
enough	O
to	O
gain	O
insight	O
into	O
the	O
enthalpic	B-evidence
(	O
ΔH	B-evidence
°)	I-evidence
and	O
entropic	B-evidence
(	O
TΔS	B-evidence
°)	I-evidence
changes	O
that	O
contributed	O
to	O
the	O
different	O
binding	B-evidence
affinities	I-evidence
/	O
potencies	O
for	O
each	O
APL	B-chemical
.	O

Flexibility	O
at	O
the	O
interface	B-site
between	O
the	O
TCR	B-complex_assembly
and	O
pMHC	B-complex_assembly
,	O
demonstrated	O
in	O
various	O
studies	O
,	O
has	O
been	O
suggested	O
as	O
a	O
mechanism	O
mediating	O
T	O
cell	O
cross	O
-	O
reactivity	O
with	O
multiple	O
distinct	O
epitopes	O
.	O

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

Second	O
,	O
molecular	O
studies	O
have	O
not	O
yet	O
revealed	O
a	O
broad	O
set	O
of	O
rules	O
that	O
determine	O
TCR	B-complex_assembly
cross	O
-	O
reactivity	O
because	O
,	O
with	O
the	O
exception	O
of	O
the	O
allo	B-protein_state
–	O
TCR	B-complex_assembly
-	I-complex_assembly
MHC	I-complex_assembly
pair	O
of	O
the	O
42F3	B-protein
TCR	B-complex_assembly
and	O
H2	B-protein
-	I-protein
Ld	I-protein
that	O
did	O
not	O
encounter	O
each	O
other	O
during	O
T	O
cell	O
development	O
,	O
studies	O
have	O
been	O
limited	O
to	O
structures	B-evidence
of	O
a	O
TCR	B-complex_assembly
with	O
only	O
2	O
or	O
3	O
different	O
ligands	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	B-protein_state
this	O
motif	O
,	O
we	O
were	O
unable	O
to	O
robustly	O
measure	O
binding	B-evidence
affinities	I-evidence
or	O
generate	O
complex	O
structures	B-evidence
with	O
these	O
ligands	O
,	O
highlighting	O
the	O
central	O
role	O
of	O
this	O
interaction	O
during	O
1E6	O
T	O
cell	O
antigen	O
recognition	O
.	O

The	O
binding	O
mechanism	O
utilized	O
by	O
the	O
1E6	B-complex_assembly
TCR	I-complex_assembly
during	O
pMHC	B-complex_assembly
recognition	O
is	O
consistent	O
with	O
both	O
of	O
these	O
models	O
.	O

Combined	O
with	O
evidence	O
demonstrating	O
that	O
aromatic	O
side	O
chains	O
are	O
conserved	O
in	O
the	O
CDR2	B-structure_element
loops	I-structure_element
of	O
TCRs	B-complex_assembly
from	O
many	O
species	O
,	O
we	O
speculate	O
that	O
these	O
aromatic	O
residues	O
could	O
impart	O
a	O
level	O
of	O
“	O
stickiness	O
”	O
to	O
TCRs	B-complex_assembly
,	O
which	O
might	O
be	O
enriched	O
in	O
an	O
autoimmune	O
setting	O
when	O
the	O
TCR	B-complex_assembly
often	O
binds	O
in	O
a	O
nonoptimal	O
fashion	O
.	O

Early	O
thermodynamic	B-experimental_method
analysis	I-experimental_method
of	O
TCR	B-complex_assembly
-	I-complex_assembly
pMHC	I-complex_assembly
interactions	O
suggested	O
a	O
common	O
energetic	O
signature	O
,	O
driven	O
by	O
favorable	O
enthalpy	B-evidence
(	O
generally	O
mediated	O
through	O
an	O
increase	O
in	O
electrostatic	O
interactions	O
)	O
and	O
unfavorable	O
entropy	B-evidence
(	O
changes	O
from	O
disorder	O
to	O
order	O
).	O

This	O
analysis	O
demonstrated	O
a	O
strong	O
relationship	O
(	O
according	O
to	O
the	O
Pearson	B-experimental_method
’	I-experimental_method
s	I-experimental_method
correlation	I-experimental_method
analysis	I-experimental_method
)	O
between	O
the	O
energetic	O
signature	O
used	O
by	O
the	O
1E6	B-complex_assembly
TCR	I-complex_assembly
and	O
the	O
sensitivity	O
of	O
the	O
1E6	O
T	O
cell	O
clone	O
to	O
different	O
APLs	B-chemical
.	O

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

Importantly	O
,	O
the	O
preproinsulin	B-protein
-	O
derived	O
epitope	O
was	O
one	O
of	O
the	O
least	O
potent	O
peptides	O
,	O
demonstrating	O
that	O
the	O
1E6	O
T	O
cell	O
clone	O
had	O
the	O
ability	O
to	O
respond	O
to	O
different	O
peptide	O
sequences	O
with	O
far	O
greater	O
potency	O
.	O

Finally	O
,	O
TCR	B-complex_assembly
ligand	O
discrimination	O
was	O
characterized	O
by	O
an	O
energetic	O
shift	O
from	O
an	O
enthalpically	O
to	O
entropically	O
driven	O
interaction	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	B-oligomeric_state
composed	O
of	O
each	O
APL	B-chemical
(	O
colored	O
as	O
above	O
)	O
presented	O
by	O
HLA	B-protein
-	I-protein
A	I-protein
*	I-protein
0201	I-protein
.	O
(	O
D	O
)	O
The	O
stability	O
of	O
each	O
APL	B-chemical
(	O
colored	O
as	O
above	O
)	O
was	O
tested	O
,	O
in	O
duplicate	O
,	O
using	O
CD	B-experimental_method
by	O
recording	O
the	O
peak	O
at	O
218	O
nm	O
absorbance	O
from	O
5	O
°	O
C	O
–	O
90	O
°	O
C	O
.	O

(	O
A	O
)	O
1E6	B-complex_assembly
-	I-complex_assembly
A2	I-complex_assembly
-	I-complex_assembly
MVWGPDPLYV	I-complex_assembly
(	O
approximate	O
value	O
);	O
(	O
B	O
)	O
1E6	B-complex_assembly
-	I-complex_assembly
A2	I-complex_assembly
-	I-complex_assembly
YLGGPDFPTI	I-complex_assembly
(	O
approximate	O
value	O
);	O
(	O
C	O
)	O
1E6	B-complex_assembly
-	I-complex_assembly
A2	I-complex_assembly
-	I-complex_assembly
ALWGPDPAAA	I-complex_assembly
;	O
(	O
D	O
)	O
1E6	B-complex_assembly
-	I-complex_assembly
A2	I-complex_assembly
-	I-complex_assembly
AQWGPDPAAA	I-complex_assembly
;	O
(	O
E	O
)	O
1E6	B-complex_assembly
-	I-complex_assembly
A2	I-complex_assembly
-	I-complex_assembly
RQFGPDWIVA	I-complex_assembly
;	O
(	O
F	O
)	O
1E6	B-complex_assembly
-	I-complex_assembly
A2	I-complex_assembly
-	I-complex_assembly
RQWGPDPAAV	I-complex_assembly
;	O
(	O
G	O
)	O
1E6	B-complex_assembly
-	I-complex_assembly
A2	I-complex_assembly
-	I-complex_assembly
YQFGPDFPTA	I-complex_assembly
;	O
and	O
(	O
H	O
)	O
1E6	B-complex_assembly
-	I-complex_assembly
A2	I-complex_assembly
-	I-complex_assembly
RQFGPDFPTI	I-complex_assembly
.	O
(	O
I	O
)	O
ΔG	B-evidence
values	I-evidence
,	O
calculated	O
from	O
SPR	B-experimental_method
experiments	O
,	O
plotted	O
against	O
1	O
/	O
EC50	B-evidence
(	O
the	O
reciprocal	O
peptide	O
concentration	O
required	O
to	O
reach	O
half	O
-	O
maximal	O
1E6	O
T	O
cell	O
killing	O
)	O
showing	O
Pearson	B-experimental_method
’	I-experimental_method
s	I-experimental_method
coefficient	I-experimental_method
analysis	I-experimental_method
(	O
r	B-evidence
)	O
and	O
P	B-evidence
value	O
(	O
including	O
approximate	O
values	O
from	O
A	O
and	O
B	O
).	O

(	O
A	O
)	O
Interaction	O
between	O
the	O
1E6	B-complex_assembly
TCR	I-complex_assembly
(	O
black	O
illustration	O
and	O
sticks	O
)	O
and	O
A2	B-chemical
-	I-chemical
MVWGPDPLYV	I-chemical
(	O
black	O
illustration	O
and	O
sticks	O
).	O
(	O
B	O
)	O
Interaction	O
between	O
the	O
1E6	B-complex_assembly
TCR	I-complex_assembly
(	O
red	O
illustration	O
and	O
sticks	O
)	O
and	O
A2	B-chemical
-	I-chemical
YLGGPDFPTI	I-chemical
(	O
red	O
illustration	O
and	O
sticks	O
).	O
(	O
C	O
)	O
Interaction	O
between	O
the	O
1E6	B-complex_assembly
TCR	I-complex_assembly
(	O
blue	O
illustration	O
and	O
sticks	O
)	O
and	O
A2	B-chemical
-	I-chemical
ALWGPDPAAA	I-chemical
(	O
blue	O
illustration	O
and	O
sticks	O
)	O
reproduced	O
from	O
previous	O
published	O
data	O
.	O

Superposition	B-experimental_method
of	O
each	O
APL	B-chemical
in	O
unligated	B-protein_state
form	O
and	O
ligated	B-protein_state
to	O
the	O
1E6	B-complex_assembly
TCR	I-complex_assembly
.	O

Interactions	O
between	O
the	O
1E6	B-complex_assembly
TCR	I-complex_assembly
and	O
the	O
MHC	B-complex_assembly
α1	B-structure_element
helix	I-structure_element
residues	O
Arg65	B-residue_name_number
,	O
Lys66	B-residue_name_number
,	O
and	O
Gln72	B-residue_name_number
.	O

Hydrogen	B-bond_interaction
bonds	I-bond_interaction
are	O
shown	O
as	O
red	O
dotted	O
lines	O
;	O
vdW	B-bond_interaction
contacts	O
are	O
shown	O
as	O
black	O
dotted	O
lines	O
.	O

Further	O
dimerization	O
contacts	O
involve	O
switch	B-site
II	I-site
,	O
the	O
G4	B-structure_element
helix	I-structure_element
and	O
the	O
trans	B-structure_element
stabilizing	I-structure_element
loop	I-structure_element
.	O

Extensive	O
biochemical	B-experimental_method
studies	I-experimental_method
suggested	O
that	O
GTP	B-chemical
-	O
induced	O
oligomerization	O
of	O
Irga6	B-protein
requires	O
an	O
interface	B-site
in	O
the	O
GTPase	B-structure_element
domain	I-structure_element
across	O
the	O
nucleotide	B-site
-	I-site
binding	I-site
site	I-site
.	O

Since	O
the	O
signal	B-protein_type
recognition	I-protein_type
particle	I-protein_type
GTPase	I-protein_type
and	O
its	O
homologous	O
receptor	B-protein_type
(	O
called	O
FfH	B-protein
and	O
FtsY	B-protein
in	O
bacteria	B-taxonomy_domain
)	O
also	O
employ	O
the	O
3	O
'-	O
OH	O
ribose	O
group	O
to	O
dimerize	B-oligomeric_state
in	O
an	O
anti	B-protein_state
-	I-protein_state
parallel	I-protein_state
orientation	O
therefore	O
activating	O
its	O
GTPase	B-protein_type
,	O
an	O
analogous	O
dimerization	O
model	O
was	O
proposed	O
for	O
Irga6	B-protein
.	O

The	O
structure	B-evidence
revealed	O
that	O
Irga6	B-protein
can	O
dimerize	B-oligomeric_state
via	O
the	O
G	B-site
interface	I-site
in	O
a	O
parallel	B-protein_state
head	I-protein_state
-	I-protein_state
to	I-protein_state
-	I-protein_state
head	I-protein_state
fashion	O
.	O

Our	O
data	O
suggest	O
that	O
a	O
parallel	B-protein_state
dimerization	O
mode	O
may	O
be	O
a	O
unifying	O
feature	O
in	O
all	O
dynamin	B-protein_type
and	O
septin	B-protein_type
superfamily	O
proteins	O
.	O

Crystals	B-evidence
diffracted	O
to	O
3	O
.	O
2	O
Å	O
resolution	O
and	O
displayed	O
one	O
exceptionally	O
long	O
unit	O
cell	O
axis	O
of	O
1289	O
Å	O
(	O
Additional	O
file	O
1	O
:	O
Table	O
S1	O
).	O

The	O
structure	B-evidence
was	O
solved	O
by	O
molecular	B-experimental_method
replacement	I-experimental_method
and	O
refined	O
to	O
Rwork	B-evidence
/	O
Rfree	B-evidence
of	O
29	O
.	O
7	O
%/	O
31	O
.	O
7	O
%	O
(	O
Additional	O
file	O
1	O
:	O
Table	O
S2	O
).	O

Secondary	O
structure	O
was	O
numbered	O
according	O
to	O
ref	O
..	O
c	O
Top	O
view	O
on	O
the	O
GTPase	B-structure_element
domain	I-structure_element
dimer	B-oligomeric_state
.	O

The	O
structures	B-evidence
of	O
the	O
seven	O
molecules	O
also	O
agree	O
well	O
with	O
the	O
previously	O
determined	O
structure	B-evidence
of	O
native	B-protein_state
GMPPNP	B-protein_state
-	I-protein_state
bound	I-protein_state
Irga6	B-protein
(	O
PDB	O
:	O
1TQ6	O
;	O
rmsd	B-evidence
of	O
1	O
.	O
00	O
-	O
1	O
.	O
13	O
Å	O
over	O
all	O
Cα	O
atoms	O
).	O

This	O
indicates	O
that	O
the	O
introduced	O
mutations	B-experimental_method
in	O
the	O
secondary	B-site
patch	I-site
,	O
from	O
which	O
only	O
Lys176	B-residue_name_number
is	O
part	O
of	O
the	O
backside	B-site
interface	I-site
,	O
do	O
,	O
in	O
fact	O
,	O
not	O
prevent	O
this	O
interaction	O
.	O

Strikingly	O
,	O
molecule	O
A	B-structure_element
of	O
one	O
asymmetric	O
unit	O
assembled	O
with	O
an	O
equivalent	O
molecule	O
of	O
the	O
adjacent	O
asymmetric	O
unit	O
via	O
the	O
G	B-site
-	I-site
interface	I-site
in	O
a	O
symmetric	O
parallel	B-protein_state
fashion	O
via	O
a	O
470	O
Å2	O
interface	O
.	O

Contact	B-site
site	I-site
II	I-site
features	O
polar	B-bond_interaction
and	I-bond_interaction
hydrophobic	I-bond_interaction
interactions	I-bond_interaction
formed	O
by	O
switch	B-site
I	I-site
(	O
V104	B-residue_name_number
,	O
V107	B-residue_name_number
)	O
with	O
a	O
helix	B-structure_element
following	O
the	O
guanine	B-structure_element
specificity	I-structure_element
motif	I-structure_element
(	O
G4	B-structure_element
helix	I-structure_element
,	O
K184	B-residue_name_number
and	O
S187	B-residue_name_number
)	O
and	O
the	O
trans	B-structure_element
stabilizing	I-structure_element
loop	I-structure_element
(	O
T158	B-residue_name_number
)	O
of	O
the	O
opposing	O
GTPase	B-structure_element
domain	I-structure_element
.	O

The	O
GTPase	B-structure_element
domains	I-structure_element
of	O
the	O
left	O
molecules	O
are	O
shown	O
in	O
orange	O
,	O
helical	B-structure_element
domains	I-structure_element
or	O
extensions	O
in	O
blue	O
.	O

Our	O
structural	B-experimental_method
analysis	I-experimental_method
of	O
an	O
oligomerization	B-protein_state
-	I-protein_state
and	I-protein_state
GTPase	I-protein_state
-	I-protein_state
defective	I-protein_state
Irga6	B-protein
mutant	B-protein_state
indicates	O
that	O
Irga6	B-protein
dimerizes	B-oligomeric_state
via	O
the	O
G	B-site
interface	I-site
in	O
a	O
parallel	B-protein_state
orientation	O
.	O

In	O
the	O
crystals	B-evidence
,	O
dimerization	O
via	O
the	O
G	B-site
interface	I-site
is	O
promoted	O
by	O
the	O
high	O
protein	O
concentrations	O
which	O
may	O
mimic	O
a	O
situation	O
when	O
Irga6	B-protein
oligomerizes	O
on	O
a	O
membrane	O
surface	O
.	O

Irga6	B-protein
bears	O
Gly79	B-residue_name_number
at	O
this	O
position	O
,	O
which	O
in	O
the	O
dimerizing	B-oligomeric_state
molecule	O
A	B-structure_element
appears	O
to	O
approach	O
the	O
bridging	O
imido	O
group	O
of	O
GMPPNP	B-chemical
via	O
a	O
main	O
chain	O
hydrogen	B-bond_interaction
bond	I-bond_interaction
.	O

For	O
Irga6	B-protein
,	O
additional	O
interfaces	B-site
in	O
the	O
helical	B-structure_element
domain	I-structure_element
are	O
presumably	O
involved	O
in	O
oligomerization	O
,	O
such	O
as	O
the	O
secondary	B-site
patch	I-site
residues	O
whose	O
mutation	B-experimental_method
prevented	O
oligomerization	O
in	O
the	O
crystallized	B-evidence
mutant	B-protein_state
.	O