annotation_IOB/test.tsv

In	O
particular	O
,	O
the	O
molecular	O
basis	O
of	O
complex	B-chemical
polysaccharide	I-chemical
recognition	O
,	O
an	O
essential	O
prerequisite	O
to	O
hydrolysis	O
by	O
cell	O
surface	O
glycosidases	B-protein_type
and	O
subsequent	O
metabolism	O
,	O
is	O
generally	O
poorly	O
understood	O
.	O

The	O
unique	O
,	O
tetra	B-structure_element
-	I-structure_element
modular	I-structure_element
structure	B-evidence
of	O
SGBP	B-protein
-	I-protein
B	I-protein
is	O
comprised	O
of	O
tandem	B-structure_element
Ig	I-structure_element
-	I-structure_element
like	I-structure_element
folds	I-structure_element
,	O
with	O
XyG	B-chemical
binding	O
mediated	O
at	O
the	O
distal	O
C	B-structure_element
-	I-structure_element
terminal	I-structure_element
domain	I-structure_element
.	O

A	O
remarkable	O
feature	O
of	O
the	O
Bacteroidetes	B-taxonomy_domain
is	O
the	O
packaging	O
of	O
genes	O
for	O
carbohydrate	O
catabolism	O
into	O
discrete	O
polysaccharide	B-gene
utilization	I-gene
loci	I-gene
(	O
PUL	B-gene
),	O
which	O
are	O
transcriptionally	O
regulated	O
by	O
specific	O
substrate	O
signatures	O
.	O

The	O
importance	O
of	O
PUL	B-gene
as	O
a	O
successful	O
evolutionary	O
strategy	O
is	O
underscored	O
by	O
the	O
observation	O
that	O
Bacteroidetes	B-taxonomy_domain
such	O
as	O
B	B-species
.	I-species
thetaiotaomicron	I-species
and	O
Bacteroides	B-species
ovatus	I-species
devote	O
~	O
18	O
%	O
of	O
their	O
genomes	O
to	O
these	O
systems	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

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

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

SGBP	B-protein
-	I-protein
A	I-protein
and	O
SGBP	B-protein
-	I-protein
B	I-protein
visualized	O
by	O
immunofluorescence	B-experimental_method
.	O

(	O
D	O
)	O
FITC	B-evidence
images	I-evidence
of	O
ΔSGBP	B-mutant
-	I-mutant
B	I-mutant
cells	O
labeled	O
with	O
anti	O
-	O
SGBP	O
-	O
B	O
antibodies	O
.	O

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

ITC	B-experimental_method
demonstrates	O
that	O
SGBP	B-protein
-	I-protein
A	I-protein
binds	O
to	O
XyG	B-chemical
polysaccharide	B-chemical
and	O
XyGO2	B-chemical
(	O
based	O
on	O
a	O
Glc8	B-structure_element
backbone	I-structure_element
)	O
with	O
essentially	O
equal	O
affinities	B-evidence
,	O
while	O
no	O
binding	O
of	O
XyGO1	B-chemical
(	O
Glc4	B-structure_element
backbone	I-structure_element
)	O
was	O
detectable	O
(	O
Table	O
1	O
;	O
see	O
Fig	O
.	O
S2	O
and	O
S3	O
in	O
the	O
supplemental	O
material	O
).	O

Together	O
,	O
these	O
data	O
clearly	O
suggest	O
that	O
polysaccharide	B-chemical
binding	O
of	O
both	O
SGBPs	B-protein_type
is	O
fulfilled	O
by	O
a	O
dimer	B-oligomeric_state
of	O
the	O
minimal	B-structure_element
repeat	I-structure_element
,	O
corresponding	O
to	O
XyGO2	B-chemical
(	O
cf	O
.	O

Summary	O
of	O
thermodynamic	O
parameters	O
for	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
SGBP	B-protein
-	I-protein
A	I-protein
and	O
SGBP	B-protein
-	I-protein
B	I-protein
obtained	O
by	O
isothermal	B-experimental_method
titration	I-experimental_method
calorimetry	I-experimental_method
at	O
25	O
°	O
Ca	O

Specifically	O
,	O
SGBP	B-protein
-	I-protein
A	I-protein
overlays	B-experimental_method
B	B-species
.	I-species
thetaiotaomicron	I-species
SusD	B-protein
(	O
BtSusD	B-protein
)	O
with	O
a	O
root	B-evidence
mean	I-evidence
square	I-evidence
deviation	I-evidence
(	O
RMSD	B-evidence
)	O
value	O
of	O
2	O
.	O
2	O
Å	O
for	O
363	O
Cα	O
pairs	O
,	O
which	O
is	O
notable	O
given	O
the	O
26	O
%	O
amino	O
acid	O
identity	O
(	O
40	O
%	O
similarity	O
)	O
between	O
these	O
homologs	O
(	O
Fig	O
.	O
4C	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

Binding	O
thermodynamics	O
are	O
based	O
on	O
the	O
concentration	O
of	O
the	O
binding	O
unit	O
,	O
XyGO2	B-chemical
.	O

The	O
structure	B-experimental_method
-	I-experimental_method
based	I-experimental_method
alignment	I-experimental_method
of	O
these	O
proteins	O
reveals	O
17	O
%	O
sequence	O
identity	O
,	O
with	O
a	O
core	O
RMSD	B-evidence
of	O
3	O
.	O
6	O
Å	O
for	O
253	O
aligned	O
residues	O
.	O

While	O
there	O
is	O
no	O
substrate	O
-	O
complexed	O
structure	O
of	O
Bacova_04391	B-protein
available	O
,	O
the	O
binding	B-site
site	I-site
is	O
predicted	O
to	O
include	O
W241	B-residue_name_number
and	O
Y404	B-residue_name_number
,	O
which	O
are	O
proximal	O
to	O
the	O
XyGO	B-site
binding	I-site
site	I-site
in	O
SGBP	B-protein
-	I-protein
B	I-protein
.	O
However	O
,	O
the	O
opposing	B-protein_state
,	I-protein_state
clamp	I-protein_state
-	I-protein_state
like	I-protein_state
arrangement	I-protein_state
of	O
these	B-structure_element
residues	I-structure_element
in	O
Bacova_04391	B-protein
is	O
clearly	O
distinct	O
from	O
the	O
planar	B-site
surface	I-site
arrangement	I-site
of	O
the	O
residues	B-structure_element
that	O
interact	O
with	O
XyG	B-chemical
in	O
SGBP	B-protein
-	I-protein
B	I-protein
(	O
described	O
below	O
).	O

Inspection	O
of	O
the	O
tertiary	O
structure	B-evidence
indicates	O
that	O
domains	O
C	B-structure_element
and	O
D	B-structure_element
are	O
effectively	O
inseparable	O
,	O
with	O
a	O
contact	O
interface	O
of	O
396	O
Å2	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

The	O
aromatic	B-site
platform	I-site
created	O
by	O
W330	B-residue_name_number
,	O
W364	B-residue_name_number
,	O
and	O
Y363	B-residue_name_number
spans	O
four	O
glucosyl	B-chemical
residues	O
,	O
compared	O
to	O
the	O
longer	B-protein_state
platform	B-site
of	O
SGBP	B-protein
-	I-protein
A	I-protein
,	O
which	O
supports	O
six	O
glucosyl	B-chemical
residues	O
(	O
Fig	O
.	O
5E	O
).	O

Additional	O
residues	B-structure_element
surrounding	O
the	O
binding	B-site
site	I-site
,	O
including	O
Y369	B-residue_name_number
and	O
E412	B-residue_name_number
,	O
may	O
contribute	O
to	O
the	O
recognition	O
of	O
more	O
highly	O
decorated	O
XyG	B-chemical
,	O
but	O
precisely	O
how	O
this	O
is	O
mediated	O
is	O
presently	O
unclear	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

SGBP	B-protein
-	I-protein
A	I-protein
and	O
SGBP	B-protein
-	I-protein
B	I-protein
have	O
distinct	O
,	O
coordinated	O
functions	O
in	O
vivo	O
.	O

To	O
disentangle	O
the	O
functions	O
of	O
SGBP	B-protein
-	I-protein
A	I-protein
and	O
SGBP	B-protein
-	I-protein
B	I-protein
in	O
XyG	B-chemical
recognition	O
and	O
uptake	O
,	O
we	O
created	O
individual	O
in	B-experimental_method
-	I-experimental_method
frame	I-experimental_method
deletion	I-experimental_method
and	I-experimental_method
complementation	I-experimental_method
mutant	I-experimental_method
strains	O
of	O
B	B-species
.	I-species
ovatus	I-species
.	O

A	O
strain	O
in	O
which	O
the	O
entire	O
XyGUL	B-gene
is	O
deleted	B-experimental_method
displays	O
a	O
lag	B-evidence
of	O
24	O
.	O
5	O
h	O
during	O
growth	O
on	O
glucose	B-chemical
compared	O
to	O
the	O
isogenic	O
parental	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
(	O
WT	B-protein_state
)	O
Δtdk	B-mutant
strain	O
,	O
for	O
which	O
exponential	O
growth	O
lags	B-evidence
for	O
19	O
.	O
8	O
h	O
(	O
see	O
Fig	O
.	O
S8D	O
).	O

Complementation	B-experimental_method
of	O
the	O
ΔSGBP	B-mutant
-	I-mutant
A	I-mutant
strain	O
(	O
ΔSGBP	B-mutant
-	I-mutant
A	I-mutant
::	O
SGBP	B-protein
-	I-protein
A	I-protein
)	O
restores	O
growth	O
to	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
rates	O
on	O
xyloglucan	B-chemical
and	O
XyGO1	B-chemical
,	O
yet	O
the	O
calculated	O
rate	O
of	O
the	O
complemented	O
strain	O
is	O
~	O
72	O
%	O
that	O
of	O
the	O
WT	B-protein_state
Δtdk	B-mutant
strain	O
on	O
XyGO2	B-chemical
;	O
similar	O
results	O
were	O
obtained	O
for	O
the	O
SGBP	B-protein
-	I-protein
B	I-protein
complemented	O
strain	O
despite	O
the	O
fact	O
that	O
the	O
growth	O
curves	O
do	O
not	O
appear	O
much	O
different	O
(	O
see	O
Fig	O
.	O
S8C	O
and	O
F	O
).	O

This	O
result	O
mirrors	O
our	O
previous	O
data	O
for	O
the	O
canonical	O
Sus	B-complex_assembly
of	O
B	B-species
.	I-species
thetaiotaomicron	I-species
,	O
which	O
revealed	O
that	O
a	O
homologous	O
ΔsusD	B-mutant
mutant	B-protein_state
is	O
unable	O
to	O
grow	O
on	O
starch	B-chemical
or	O
malto	B-chemical
-	I-chemical
oligosaccharides	I-chemical
,	O
despite	O
normal	O
cell	O
surface	O
expression	O
of	O
all	O
other	O
PUL	B-gene
-	O
encoded	O
proteins	O
.	O

More	O
recently	O
,	O
we	O
demonstrated	O
that	O
this	O
phenotype	O
is	O
due	O
to	O
the	O
loss	O
of	O
the	O
physical	O
presence	O
of	O
SusD	B-protein
;	O
complementation	B-experimental_method
of	O
ΔsusD	B-mutant
with	O
SusD	B-mutant
*,	I-mutant
a	O
triple	B-protein_state
site	I-protein_state
-	I-protein_state
directed	I-protein_state
mutant	I-protein_state
(	O
W96A	B-mutant
W320A	B-mutant
Y296A	B-mutant
)	O
that	O
ablates	B-protein_state
glycan	I-protein_state
binding	I-protein_state
,	O
restores	O
B	B-species
.	I-species
thetaiotaomicron	I-species
growth	O
on	O
malto	B-chemical
-	I-chemical
oligosaccharides	I-chemical
and	O
starch	B-chemical
when	O
sus	B-gene
transcription	O
is	O
induced	O
by	O
maltose	B-chemical
addition	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

The	O
precise	O
reason	O
for	O
this	O
lag	B-evidence
is	O
unclear	O
,	O
but	O
recapitulating	O
our	O
findings	O
on	O
the	O
role	O
of	O
SusD	B-protein
in	O
malto	B-chemical
-	I-chemical
oligosaccharide	I-chemical
sensing	O
in	O
B	B-species
.	I-species
thetaiotaomicron	I-species
,	O
this	O
extended	O
lag	B-evidence
may	O
be	O
due	O
to	O
inefficient	O
import	O
and	O
thus	O
sensing	O
of	O
xyloglucan	B-chemical
in	O
the	O
environment	O
in	O
the	O
absence	O
of	O
glycan	B-chemical
binding	O
by	O
essential	O
SGBPs	B-protein_type
.	O

Our	O
previous	O
work	O
demonstrates	O
that	O
B	B-species
.	I-species
ovatus	I-species
cells	O
grown	O
in	O
minimal	O
medium	O
plus	O
glucose	B-chemical
express	O
low	O
levels	O
of	O
the	O
XyGUL	B-gene
transcript	O
.	O

Thus	O
,	O
in	O
our	O
experiments	O
,	O
we	O
presume	O
that	O
each	O
strain	O
,	O
initially	O
grown	O
in	O
glucose	B-chemical
,	O
expresses	O
low	O
levels	O
of	O
the	O
XyGUL	B-gene
transcript	O
and	O
thus	O
low	O
levels	O
of	O
the	O
XyGUL	B-gene
-	O
encoded	O
surface	O
proteins	O
,	O
including	O
the	O
vanguard	O
GH5	B-protein
.	O

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

Likewise	O
,	O
such	O
cognate	O
interactions	O
between	O
homologous	O
protein	O
pairs	O
such	O
as	O
SGBP	B-protein
-	I-protein
A	I-protein
and	O
its	O
TBDT	B-protein_type
may	O
underlie	O
our	O
observation	O
that	O
a	O
ΔSGBP	B-mutant
-	I-mutant
A	I-mutant
mutant	B-protein_state
cannot	O
grow	O
on	O
xyloglucan	B-chemical
.	O

The	O
ability	O
of	O
gut	O
-	O
adapted	O
microorganisms	B-taxonomy_domain
to	O
thrive	O
in	O
the	O
gastrointestinal	O
tract	O
is	O
critically	O
dependent	O
upon	O
their	O
ability	O
to	O
efficiently	O
recognize	O
,	O
cleave	O
,	O
and	O
import	O
glycans	B-chemical
.	O

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

On	O
the	O
other	O
hand	O
,	O
there	O
is	O
clear	O
evidence	O
for	O
independent	O
TBDTs	B-protein_type
in	O
Bacteroidetes	B-taxonomy_domain
that	O
do	O
not	O
require	O
SGBP	B-protein_type
association	O
for	O
activity	O
.	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

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

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

The	O
PduL	B-protein_type
fold	B-structure_element
is	O
unrelated	O
to	O
that	B-structure_element
of	O
Pta	B-protein_type
;	O
it	O
contains	O
a	O
dimetal	B-site
active	I-site
site	I-site
involved	O
in	O
a	O
catalytic	O
mechanism	O
distinct	O
from	O
that	O
of	O
the	O
housekeeping	B-protein_state
PTAC	B-protein_type
.	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

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

Remarkably	O
,	O
after	O
removing	B-experimental_method
the	O
N	O
-	O
terminal	O
putative	O
EP	B-structure_element
(	O
27	B-residue_range
amino	I-residue_range
acids	I-residue_range
),	O
most	O
of	O
the	O
sPduLΔEP	B-mutant
protein	O
was	O
in	O
the	O
soluble	O
fraction	O
upon	O
cell	O
lysis	O
.	O

Similar	O
differences	O
in	O
solubility	O
were	O
observed	O
for	O
pPduL	B-protein
and	O
rPduL	B-protein
when	O
comparing	O
EP	B-protein_state
-	I-protein_state
truncated	I-protein_state
forms	O
to	O
the	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
protein	O
,	O
but	O
none	O
were	O
quite	O
as	O
dramatic	O
as	O
for	O
sPduL	B-protein
.	O
We	O
confirmed	O
that	O
all	O
homologs	O
were	O
active	B-protein_state
(	O
S1a	O
and	O
S1b	O
Fig	O
).	O

Structural	O
overview	O
of	O
R	B-species
.	I-species
palustris	I-species
PduL	B-protein_type
from	O
the	O
grm3	B-gene
locus	I-gene
.	O

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

(	O
b	O
)	O
Cartoon	O
representation	O
of	O
the	O
structure	B-evidence
colored	O
by	O
domains	O
and	O
including	O
secondary	O
structure	B-evidence
numbering	O
.	O

Residues	O
100	O
%	O
conserved	O
across	O
all	O
PduL	B-protein_type
homologs	O
in	O
our	O
dataset	O
are	O
noted	O
with	O
an	O
asterisk	O
,	O
and	O
residues	O
conserved	O
in	O
over	O
90	O
%	O
of	O
sequences	O
are	O
noted	O
with	O
a	O
colon	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

Distances	O
between	O
atom	O
centers	O
are	O
indicated	O
in	O
Å	O
.	O
(	O
a	O
)	O
Coenzyme	B-chemical
A	I-chemical
containing	O
,	O
(	O
b	O
)	O
phosphate	B-protein_state
-	I-protein_state
bound	I-protein_state
structure	B-evidence
.	O

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

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

The	O
phosphate	B-protein_state
-	I-protein_state
bound	I-protein_state
structure	B-evidence
aligns	B-experimental_method
well	O
with	O
the	O
CoA	B-protein_state
-	I-protein_state
bound	I-protein_state
structure	B-evidence
(	O
0	O
.	O
43	O
Å	O
rmsd	B-evidence
over	O
2	O
,	O
361	O
atoms	O
for	O
the	O
monomer	B-oligomeric_state
,	O
0	O
.	O
83	O
Å	O
over	O
5	O
,	O
259	O
aligned	O
atoms	O
for	O
the	O
dimer	B-oligomeric_state
).	O

Upon	O
deletion	B-experimental_method
of	O
the	O
putative	O
EP	B-structure_element
(	O
residues	O
1	B-residue_range
–	I-residue_range
47	I-residue_range
for	O
rPduL	B-protein
,	O
and	O
1	B-residue_range
–	I-residue_range
20	I-residue_range
for	O
pPduL	B-protein
),	O
there	O
was	O
a	O
distinct	O
change	O
in	O
the	O
elution	O
profiles	O
(	O
Fig	O
5b	O
and	O
5c	O
respectively	O
,	O
blue	O
curves	O
).	O

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

Curiously	O
,	O
while	O
the	O
housekeeping	B-protein_state
Pta	B-protein_type
could	O
provide	O
this	O
function	O
,	O
and	O
indeed	O
does	O
so	O
in	O
the	O
case	O
of	O
one	O
type	O
of	O
ethanolamine	B-complex_assembly
-	I-complex_assembly
utilizing	I-complex_assembly
(	I-complex_assembly
EUT	I-complex_assembly
)	I-complex_assembly
BMC	I-complex_assembly
,	O
the	O
evolutionarily	O
unrelated	O
PduL	B-protein_type
fulfills	O
this	O
function	O
for	O
the	O
majority	O
of	O
metabolosomes	B-complex_assembly
using	O
a	O
novel	O
structure	B-evidence
and	O
active	B-site
site	I-site
for	O
convergent	O
evolution	O
of	O
function	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

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

All	O
of	O
the	O
metal	B-site
-	I-site
coordinating	I-site
residues	I-site
(	O
Fig	O
2a	O
)	O
are	O
absolutely	B-protein_state
conserved	I-protein_state
,	O
implicating	O
them	O
in	O
catalysis	O
or	O
the	O
correct	O
spatial	O
orientation	O
of	O
the	O
substrates	O
.	O

However	O
,	O
for	O
the	O
clostripain	B-protein_type
family	I-protein_type
(	O
denoted	O
C11	B-protein_type
),	O
little	O
is	O
currently	O
known	O
.	O

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

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

Sequences	O
around	O
the	O
catalytic	B-site
site	I-site
of	O
clostripain	B-protein
and	O
PmC11	B-protein
align	O
well	O
.	O

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

Biochemical	B-experimental_method
and	I-experimental_method
structural	I-experimental_method
characterization	I-experimental_method
of	O
PmC11	B-protein
.	O

A	O
,	O
ribbon	O
representation	O
of	O
the	O
overall	O
structure	O
of	O
PmC11	B-protein
illustrating	O
the	O
catalytic	B-site
site	I-site
,	O
cleavage	O
site	O
displacement	O
,	O
and	O
potential	O
S1	B-site
binding	I-site
site	I-site
.	O

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

Five	O
of	O
the	O
α	B-structure_element
-	I-structure_element
helices	I-structure_element
surrounding	O
the	O
β	B-structure_element
-	I-structure_element
sheet	I-structure_element
of	O
PmC11	B-protein
(	O
α1	B-structure_element
,	O
α2	B-structure_element
,	O
α4	B-structure_element
,	O
α6	B-structure_element
,	O
and	O
α7	B-structure_element
)	O
are	O
found	O
in	O
similar	O
positions	O
to	O
the	O
five	O
structurally	B-protein_state
conserved	I-protein_state
helices	B-structure_element
in	O
caspases	B-protein_type
and	O
other	O
members	O
of	O
clan	B-protein_type
CD	I-protein_type
,	O
apart	O
from	O
family	O
C80	B-protein_type
.	O

Autoprocessing	B-ptm
of	O
PmC11	B-protein

Substrate	O
Specificity	O
of	O
PmC11	B-protein

The	O
autocatalytic	B-ptm
cleavage	I-ptm
of	O
PmC11	B-protein
at	O
Lys147	B-residue_name_number
(	O
sequence	O
KLK	O
∧	O
A	O
)	O
demonstrates	O
that	O
the	O
enzyme	O
accepts	O
substrates	O
with	O
Lys	B-residue_name
in	O
the	O
P1	B-residue_number
position	O
.	O

This	O
pocket	B-site
is	O
lined	O
with	O
the	O
potential	O
functional	O
side	O
chains	O
of	O
Asn50	B-residue_name_number
,	O
Asp177	B-residue_name_number
,	O
and	O
Thr204	B-residue_name_number
with	O
Gly134	B-residue_name_number
,	O
Asp207	B-residue_name_number
,	O
and	O
Met205	B-residue_name_number
also	O
contributing	O
to	O
the	O
pocket	B-site
(	O
Fig	O
.	O
2A	O
).	O

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

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
a	O
fluorometric	B-experimental_method
activity	I-experimental_method
assay	I-experimental_method
with	O
(+,	O
purple	O
)	O
and	O
without	O
(−,	O
red	O
)	O
Z	B-chemical
-	I-chemical
VRPR	I-chemical
-	I-chemical
FMK	I-chemical
.	O

A	O
three	B-experimental_method
-	I-experimental_method
dimensional	I-experimental_method
structural	I-experimental_method
overlay	I-experimental_method
of	O
Z	B-chemical
-	I-chemical
VRPR	I-chemical
-	I-chemical
FMK	I-chemical
from	O
the	O
MALT1	B-protein
-	I-protein
P	I-protein
complex	O
onto	O
PmC11	B-protein
.	O

Comparison	O
with	O
Clostripain	B-protein

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

Several	O
other	O
members	O
of	O
clan	B-protein_type
CD	I-protein_type
require	O
processing	B-ptm
for	O
full	B-protein_state
activation	I-protein_state
including	O
legumain	B-protein
,	O
gingipain	B-protein
-	I-protein
R	I-protein
,	O
MARTX	B-protein
-	I-protein
CPD	I-protein
,	O
and	O
the	O
effector	B-protein_type
caspases	I-protein_type
,	O
e	O
.	O
g	O
.	O
caspase	B-protein
-	I-protein
7	I-protein
.	O

Structural	O
insights	O
into	O
the	O
regulatory	O
mechanism	O
of	O
the	O
Pseudomonas	B-species
aeruginosa	I-species
YfiBNR	B-complex_assembly
system	O

In	O
response	O
to	O
cell	O
stress	O
,	O
YfiB	B-protein
in	O
the	O
outer	O
membrane	O
can	O
sequester	O
the	O
periplasmic	O
protein	O
YfiR	B-protein
,	O
releasing	O
its	O
inhibition	O
of	O
YfiN	B-protein
on	O
the	O
inner	O
membrane	O
and	O
thus	O
provoking	O
the	O
diguanylate	O
cyclase	O
activity	O
of	O
YfiN	B-protein
to	O
induce	O
c	B-chemical
-	I-chemical
di	I-chemical
-	I-chemical
GMP	I-chemical
production	O
.	O

Biofilm	O
formation	O
protects	O
pathogenic	O
bacteria	B-taxonomy_domain
from	O
antibiotic	O
treatment	O
,	O
and	O
c	O
-	O
di	O
-	O
GMP	O
-	O
regulated	O
biofilm	O
formation	O
has	O
been	O
extensively	O
studied	O
in	O
P	B-species
.	I-species
aeruginosa	I-species
(	O
Evans	O
,;	O
Kirisits	O
et	O
al	O
.,;	O
Malone	O
,;	O
Reinhardt	O
et	O
al	O
.,).	O

Recently	O
,	O
Malone	O
and	O
coworkers	O
identified	O
the	O
tripartite	B-protein_state
c	B-chemical
-	I-chemical
di	I-chemical
-	I-chemical
GMP	I-chemical
signaling	O
module	O
system	O
YfiBNR	B-complex_assembly
(	O
also	O
known	O
as	O
AwsXRO	B-complex_assembly
(	O
Beaumont	O
et	O
al	O
.,;	O
Giddens	O
et	O
al	O
.,)	O
or	O
Tbp	B-complex_assembly
(	O
Ueda	O
and	O
Wood	O
,))	O
by	O
genetic	B-experimental_method
screening	I-experimental_method
for	O
mutants	O
that	O
displayed	O
SCV	O
phenotypes	O
in	O
P	B-species
.	I-species
aeruginosa	I-species
PAO1	I-species
(	O
Malone	O
et	O
al	O
.,;	O
Malone	O
et	O
al	O
.,).	O

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

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

Recently	O
,	O
we	O
solved	O
the	O
crystal	B-evidence
structure	I-evidence
of	O
YfiR	B-protein
in	O
both	O
the	O
non	B-protein_state
-	I-protein_state
oxidized	I-protein_state
and	O
the	O
oxidized	B-protein_state
states	O
,	O
revealing	O
breakage	O
/	O
formation	O
of	O
one	O
disulfide	B-ptm
bond	I-ptm
(	O
Cys71	B-residue_name_number
-	O
Cys110	B-residue_name_number
)	O
and	O
local	O
conformational	O
change	O
around	O
the	O
other	O
one	O
(	O
Cys145	B-residue_name_number
-	O
Cys152	B-residue_name_number
),	O
indicating	O
that	O
Cys145	B-residue_name_number
-	O
Cys152	B-residue_name_number
plays	O
an	O
important	O
role	O
in	O
maintaining	O
the	O
correct	O
folding	O
of	O
YfiR	B-protein
(	O
Yang	O
et	O
al	O
.,).	O

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

The	O
“	O
back	B-protein_state
to	I-protein_state
back	I-protein_state
”	O
dimer	B-oligomeric_state
presents	O
a	O
Y	B-protein_state
shape	I-protein_state
.	O

Therefore	O
,	O
we	O
constructed	B-experimental_method
two	I-experimental_method
such	I-experimental_method
single	I-experimental_method
mutants	I-experimental_method
of	O
YfiB	B-protein
(	O
YfiBL43P	B-mutant
and	O
YfiBF48S	B-mutant
).	O

The	O
N	O
-	O
terminal	O
structural	O
conformation	O
of	O
YfiBL43P	B-mutant
,	O
from	O
the	O
foremost	O
N	O
-	O
terminus	O
to	O
residue	O
D70	B-residue_name_number
,	O
is	O
significantly	O
altered	O
compared	O
with	O
that	O
of	O
the	O
apo	B-protein_state
YfiB	B-protein
.	O
The	O
majority	O
of	O
the	O
α1	B-structure_element
helix	I-structure_element
(	O
residues	O
34	B-residue_range
–	I-residue_range
43	I-residue_range
)	O
is	O
invisible	O
on	O
the	O
electron	B-evidence
density	I-evidence
map	I-evidence
,	O
and	O
the	O
α2	B-structure_element
helix	I-structure_element
and	O
β1	B-structure_element
and	O
β2	B-structure_element
strands	I-structure_element
are	O
rearranged	O
to	O
form	O
a	O
long	O
loop	B-structure_element
containing	O
two	O
short	O
α	B-structure_element
-	I-structure_element
helix	I-structure_element
turns	I-structure_element
(	O
Fig	O
.	O
3B	O
and	O
3C	O
),	O
thus	O
embracing	O
the	O
YfiR	B-protein
dimer	B-oligomeric_state
.	O

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

Similarly	O
,	O
in	O
the	O
YfiR	B-protein_state
-	I-protein_state
bound	I-protein_state
YfiBL43P	B-mutant
structure	B-evidence
,	O
the	O
sulfate	B-chemical
ion	O
interacts	O
with	O
the	O
side	O
-	O
chain	O
atoms	O
of	O
D102	B-residue_name_number
(	O
corresponding	O
to	O
D71	B-residue_name_number
in	O
Pal	B-protein_type
)	O
and	O
R117	B-residue_name_number
(	O
corresponding	O
to	O
R86	B-residue_name_number
in	O
Pal	B-protein_type
)	O
and	O
the	O
main	O
-	O
chain	O
amide	O
of	O
N68	B-residue_name_number
(	O
corresponding	O
to	O
D37	B-residue_name_number
in	O
Pal	B-protein_type
).	O

Therefore	O
,	O
we	O
proposed	O
that	O
the	O
PG	B-chemical
-	O
binding	O
ability	O
of	O
inactive	B-protein_state
YfiB	B-protein
might	O
be	O
weaker	O
than	O
that	O
of	O
active	B-protein_state
YfiB	B-protein
.	O
To	O
validate	O
this	O
,	O
we	O
performed	O
a	O
microscale	B-experimental_method
thermophoresis	I-experimental_method
(	O
MST	B-experimental_method
)	O
assay	O
to	O
measure	O
the	O
binding	B-evidence
affinities	I-evidence
of	O
PG	B-chemical
to	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
YfiB	B-protein
and	O
YfiBL43P	B-mutant
,	O
respectively	O
.	O

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

Malone	O
JG	O
et	O
al	O
.	O
have	O
reported	O
that	O
F151	B-residue_name_number
,	O
E163	B-residue_name_number
,	O
I169	B-residue_name_number
and	O
Q187	B-residue_name_number
,	O
located	O
near	O
the	O
C	O
-	O
terminus	O
of	O
YfiR	B-protein
,	O
comprise	O
a	O
putative	O
YfiN	B-site
binding	I-site
site	I-site
(	O
Malone	O
et	O
al	O
.,).	O

Interestingly	O
,	O
these	O
residues	O
are	O
part	O
of	O
the	O
conserved	B-site
surface	I-site
of	O
YfiR	B-protein
(	O
Fig	O
.	O
3G	O
).	O

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

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

(	O
C	O
and	O
D	O
)	O
BIAcore	B-experimental_method
data	O
and	O
analysis	O
for	O
binding	B-evidence
affinities	I-evidence
of	O
(	O
C	O
)	O
VB6	B-chemical
and	O
(	O
D	O
)	O
L	B-chemical
-	I-chemical
Trp	I-chemical
with	O
YfiR	B-protein
.	O
(	O
E	O
–	O
G	O
)	O
ITC	B-experimental_method
data	O
and	O
analysis	O
for	O
titration	B-experimental_method
of	O
(	O
E	O
)	O
YfiB	B-protein
wild	B-protein_state
-	I-protein_state
type	I-protein_state
,	O
(	O
F	O
)	O
YfiBL43P	O
,	O
and	O
(	O
G	O
)	O
YfiBL43P	B-mutant
/	O
F57A	B-mutant
into	O
YfiR	B-protein

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

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

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

A	O
direct	O
link	O
between	O
ACC	B-protein_type
and	O
cancer	O
is	O
provided	O
by	O
cancer	O
-	O
associated	O
mutations	B-mutant
in	O
the	O
breast	B-protein
cancer	I-protein
susceptibility	I-protein
gene	I-protein
1	I-protein
(	O
BRCA1	B-protein
),	O
which	O
relieve	O
inhibitory	O
interactions	O
of	O
BRCA1	B-protein
with	O
ACC	B-protein_type
.	O

Structural	B-experimental_method
studies	I-experimental_method
on	O
the	O
functional	O
architecture	O
of	O
intact	B-protein_state
ACCs	B-protein_type
have	O
been	O
hindered	O
by	O
their	O
huge	O
size	O
and	O
pronounced	O
dynamics	O
,	O
as	O
well	O
as	O
the	O
transient	B-protein_state
assembly	O
mode	O
of	O
bacterial	B-taxonomy_domain
ACCs	B-protein_type
.	O

Phosphorylated	B-protein_state
Ser80	B-residue_name_number
,	O
which	O
is	O
highly	B-protein_state
conserved	I-protein_state
only	O
in	O
higher	B-taxonomy_domain
eukaryotes	I-taxonomy_domain
,	O
presumably	O
binds	O
into	O
the	O
Soraphen	B-site
A	I-site
-	I-site
binding	I-site
pocket	I-site
.	O

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

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

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

To	O
define	O
the	O
functional	O
state	O
of	O
insect	B-experimental_method
-	I-experimental_method
cell	I-experimental_method
-	I-experimental_method
expressed	I-experimental_method
ACC	B-protein_type
variants	O
,	O
we	O
employed	O
mass	B-experimental_method
spectrometry	I-experimental_method
(	O
MS	B-experimental_method
)	O
for	O
phosphorylation	B-experimental_method
site	I-experimental_method
detection	I-experimental_method
.	O

The	O
N	O
-	O
terminal	O
region	O
of	O
the	O
regulatory	B-structure_element
loop	I-structure_element
also	O
directly	O
contacts	O
the	O
C	O
-	O
terminal	O
region	O
of	O
CDC2	B-structure_element
leading	O
into	O
CT	B-structure_element
.	O

Phosphoserine	B-residue_name_number
1157	I-residue_name_number
is	O
tightly	O
bound	O
by	O
two	O
highly	B-protein_state
conserved	I-protein_state
arginines	B-residue_name
(	O
Arg1173	B-residue_name_number
and	O
Arg1260	B-residue_name_number
)	O
of	O
CDC1	B-structure_element
(	O
Fig	O
.	O
1d	O
).	O

The	O
values	O
obtained	O
for	O
dephosphorylated	B-protein_state
SceACC	B-protein
are	O
comparable	O
to	O
earlier	O
measurements	O
of	O
non	B-protein_state
-	I-protein_state
phosphorylated	I-protein_state
yeast	B-taxonomy_domain
ACC	B-protein_type
expressed	B-experimental_method
in	I-experimental_method
E	B-species
.	I-species
coli	I-species
.	O

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

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

Each	O
of	O
the	O
four	O
CD	B-structure_element
domains	O
in	O
HsaBT	B-mutant
-	I-mutant
CD	I-mutant
individually	O
resembles	O
the	O
corresponding	O
SceCD	B-species
domain	O
;	O
however	O
,	O
human	B-species
and	O
yeast	B-taxonomy_domain
CDs	B-structure_element
exhibit	O
distinct	O
overall	O
structures	B-evidence
.	O

The	O
BT	B-structure_element
domain	O
of	O
HsaBT	B-mutant
-	I-mutant
CD	I-mutant
consists	O
of	O
a	O
helix	B-structure_element
that	O
is	O
surrounded	O
at	O
its	O
N	O
terminus	O
by	O
an	O
antiparallel	B-structure_element
eight	I-structure_element
-	I-structure_element
stranded	I-structure_element
β	I-structure_element
-	I-structure_element
barrel	I-structure_element
.	O

It	O
resembles	O
the	O
BT	B-structure_element
of	O
propionyl	B-protein_type
-	I-protein_type
CoA	I-protein_type
carboxylase	I-protein_type
;	O
only	O
the	O
four	O
C	O
-	O
terminal	O
strands	B-structure_element
of	I-structure_element
the	I-structure_element
β	I-structure_element
-	I-structure_element
barrel	I-structure_element
are	O
slightly	O
tilted	O
.	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

However	O
,	O
residual	O
phosphorylation	B-ptm
levels	O
were	O
detected	O
for	O
Ser1204	B-residue_name_number
(	O
7	O
%)	O
and	O
Ser1218	B-residue_name_number
(	O
7	O
%)	O
in	O
the	O
same	B-structure_element
loop	I-structure_element
.	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

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

For	O
CthΔBCCP	B-mutant
,	O
crystals	B-evidence
diffracting	O
to	O
8	O
.	O
4	O
Å	O
resolution	O
were	O
obtained	O
.	O

On	O
the	O
basis	O
of	O
the	O
occurrence	O
of	O
related	O
conformational	O
changes	O
between	O
fungal	B-taxonomy_domain
and	O
human	B-species
ACC	B-mutant
fragments	I-mutant
,	O
the	O
observed	O
set	O
of	O
conformations	O
may	O
well	O
represent	O
general	O
states	O
present	O
in	O
all	O
eukaryotic	B-taxonomy_domain
ACCs	B-protein_type
.	O

To	O
obtain	O
a	O
comprehensive	O
view	O
of	O
fungal	B-taxonomy_domain
ACC	B-protein_type
dynamics	O
in	B-protein_state
solution	I-protein_state
,	O
we	O
employed	O
SAXS	B-experimental_method
and	O
EM	B-experimental_method
.	O

They	O
identify	O
the	O
connections	O
between	O
CDN	B-structure_element
/	O
CDL	B-structure_element
and	O
between	O
CDC2	B-structure_element
/	O
CT	B-structure_element
as	O
major	O
contributors	O
to	O
conformational	O
heterogeneity	O
(	O
Supplementary	O
Fig	O
.	O
4a	O
,	O
b	O
).	O

Furthermore	O
,	O
based	O
on	O
an	O
average	O
length	O
of	O
the	O
BCCP	B-structure_element
–	I-structure_element
CD	I-structure_element
linker	I-structure_element
in	O
fungal	B-taxonomy_domain
ACC	B-protein_type
of	O
26	B-residue_range
amino	I-residue_range
acids	I-residue_range
,	O
mobility	O
of	O
the	O
BCCP	B-structure_element
alone	O
would	O
not	O
be	O
sufficient	O
to	O
bridge	O
the	O
active	B-site
sites	I-site
of	O
BC	B-structure_element
and	O
CT	B-structure_element
.	O

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

In	O
fungal	B-taxonomy_domain
ACC	B-protein_type
,	O
however	O
,	O
Ser1157	B-residue_name_number
in	O
the	O
regulatory	B-structure_element
loop	I-structure_element
of	O
the	O
CD	B-structure_element
is	O
the	O
only	O
phosphorylation	B-site
site	I-site
that	O
has	O
been	O
demonstrated	O
to	O
be	O
both	O
phosphorylated	B-protein_state
in	O
vivo	O
and	O
involved	O
in	O
the	O
regulation	O
of	O
ACC	B-protein_type
activity	O
.	O

A	O
comparison	O
between	O
fungal	B-taxonomy_domain
and	O
human	B-species
ACC	B-protein_type
will	O
help	O
to	O
further	O
discriminate	O
mechanistic	O
differences	O
that	O
contribute	O
to	O
the	O
extended	O
control	O
and	O
polymerization	O
of	O
human	B-species
ACC	B-protein_type
.	O

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

In	O
flACC	B-mutant
,	O
CDC2	B-structure_element
rotates	O
∼	O
120	O
°	O
with	O
respect	O
to	O
the	O
CT	B-structure_element
domain	O
.	O

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

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

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

Architecture	O
of	O
the	O
CD	B-structure_element
–	O
CT	B-structure_element
core	O
of	O
fungal	B-taxonomy_domain
ACC	B-protein_type
.	O

Flexibility	O
of	O
the	O
CDC2	B-structure_element
/	O
CT	B-structure_element
and	O
CDN	B-structure_element
/	O
CDL	B-structure_element
hinges	B-structure_element
is	O
illustrated	O
by	O
arrows	O
.	O

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

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

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

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

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

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

An	O
inhibitor	O
of	O
the	O
LdcI	B-protein
and	O
LdcC	B-protein
activity	O
,	O
the	O
stringent	B-chemical
response	I-chemical
alarmone	I-chemical
ppGpp	B-chemical
,	O
is	O
known	O
to	O
bind	O
at	O
the	O
interface	B-site
between	O
neighboring	O
monomers	B-oligomeric_state
within	O
each	O
ring	B-structure_element
(	O
Fig	O
.	O
S4	O
).	O

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

The	O
third	O
and	O
most	O
remarkable	O
finding	O
was	O
that	O
exactly	O
the	O
same	O
separation	O
into	O
“	O
LdcI	B-protein_type
-	I-protein_type
like	I-protein_type
”	O
and	O
“	O
LdcC	B-protein_type
”-	I-protein_type
like	I-protein_type
groups	O
can	O
be	O
obtained	O
based	O
on	O
a	O
comparison	O
of	O
the	O
C	O
-	O
terminal	O
β	B-structure_element
-	I-structure_element
sheets	I-structure_element
only	O
,	O
without	O
taking	O
the	O
rest	O
of	O
the	O
primary	O
sequence	O
into	O
account	O
.	O

Together	O
with	O
the	O
apo	B-protein_state
-	O
LdcI	B-protein
and	O
ppGpp	B-complex_assembly
-	I-complex_assembly
LdcIi	I-complex_assembly
crystal	B-evidence
structures	I-evidence
,	O
our	O
cryoEM	B-experimental_method
reconstructions	B-evidence
provide	O
a	O
structural	O
framework	O
for	O
future	O
studies	O
of	O
structure	O
-	O
function	O
relationships	O
of	O
lysine	B-protein_type
decarboxylases	I-protein_type
from	O
other	O
enterobacteria	B-taxonomy_domain
and	O
even	O
of	O
their	O
homologues	O
outside	O
Enterobacteriaceae	B-taxonomy_domain
.	O
For	O
example	O
,	O
the	O
lysine	B-protein_type
decarboxylase	I-protein_type
of	O
Eikenella	B-species
corrodens	I-species
is	O
thought	O
to	O
play	O
a	O
major	O
role	O
in	O
the	O
periodontal	O
disease	O
and	O
its	O
inhibitors	O
were	O
shown	O
to	O
retard	O
gingivitis	O
development	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
A	O
)	O
LdcIi	B-protein
crystal	B-evidence
structure	I-evidence
,	O
with	O
one	O
ring	B-structure_element
represented	O
as	O
a	O
grey	O
surface	O
and	O
the	O
second	O
as	O
a	O
cartoon	O
.	O

Analysis	O
of	O
the	O
LdcIC	B-mutant
and	O
LdcCI	B-mutant
chimeras	B-mutant
.	O

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

Ammonium	B-chemical
transport	O
is	O
tightly	O
regulated	O
.	O

By	O
binding	O
tightly	O
to	O
Amt	B-protein_type
proteins	I-protein_type
without	O
inducing	O
a	O
conformational	O
change	O
in	O
the	O
transporter	B-protein_type
,	O
GlnK	B-protein_type
sterically	O
blocks	O
ammonium	B-chemical
conductance	O
when	O
nitrogen	O
levels	O
are	O
sufficient	O
.	O

Under	O
conditions	O
of	O
nitrogen	B-chemical
limitation	O
,	O
GlnK	B-protein_type
becomes	O
uridylated	B-protein_state
,	O
blocking	O
its	O
ability	O
to	O
bind	O
and	O
inhibit	O
Amt	B-protein_type
proteins	I-protein_type
.	O

General	O
architecture	O
of	O
Mep2	B-protein_type
ammonium	B-protein_type
transceptors	I-protein_type

The	O
Mep2	B-protein
protein	O
of	O
S	B-species
.	I-species
cerevisiae	I-species
(	O
ScMep2	B-protein
)	O
was	O
overexpressed	B-experimental_method
in	O
S	B-species
.	I-species
cerevisiae	I-species
in	O
high	O
yields	O
,	O
enabling	O
structure	B-experimental_method
determination	I-experimental_method
by	O
X	B-experimental_method
-	I-experimental_method
ray	I-experimental_method
crystallography	I-experimental_method
using	O
data	O
to	O
3	O
.	O
2	O
Å	O
resolution	O
by	O
molecular	B-experimental_method
replacement	I-experimental_method
(	O
MR	B-experimental_method
)	O
with	O
the	O
archaebacterial	B-taxonomy_domain
Amt	B-protein
-	I-protein
1	I-protein
structure	B-evidence
(	O
see	O
Methods	O
section	O
).	O

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

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

Finally	O
,	O
the	O
important	O
ICL3	B-structure_element
linking	O
the	O
pseudo	B-structure_element
-	I-structure_element
symmetrical	I-structure_element
halves	I-structure_element
(	O
TM1	B-structure_element
-	I-structure_element
5	I-structure_element
and	O
TM6	B-structure_element
-	I-structure_element
10	I-structure_element
)	O
of	O
the	O
transporter	B-protein_type
is	O
also	O
shifted	O
up	O
to	O
∼	O
10	O
Å	O
and	O
forms	O
an	O
additional	O
barrier	O
that	O
closes	O
the	O
channel	B-site
on	O
the	O
cytoplasmic	O
side	O
(	O
Fig	O
.	O
5	O
).	O

In	O
Amt	B-protein
-	I-protein
1	I-protein
and	O
other	O
bacterial	B-taxonomy_domain
ammonium	B-protein_type
transporters	I-protein_type
,	O
these	O
CTR	B-structure_element
residues	O
interact	O
with	O
residues	O
within	O
the	O
N	B-structure_element
-	I-structure_element
terminal	I-structure_element
half	I-structure_element
of	O
the	O
protein	O
.	O

For	O
ScMep2	B-protein
,	O
Ser457	B-residue_name_number
is	O
the	O
most	O
C	O
-	O
terminal	O
residue	O
for	O
which	O
electron	B-evidence
density	I-evidence
is	O
visible	O
,	O
indicating	O
that	O
the	O
region	O
beyond	O
Ser457	B-residue_name_number
is	O
disordered	B-protein_state
.	O

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

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

We	O
therefore	O
predict	O
that	O
phosphorylation	B-ptm
of	O
Ser453	B-residue_name_number
will	O
result	O
in	O
steric	O
clashes	O
as	O
well	O
as	O
electrostatic	O
repulsion	O
,	O
which	O
in	O
turn	O
might	O
cause	O
substantial	O
conformational	O
changes	O
within	O
the	O
CTR	B-structure_element
.	O

To	O
supplement	O
the	O
crystal	B-evidence
structures	I-evidence
,	O
we	O
also	O
performed	O
modelling	B-experimental_method
and	O
MD	B-experimental_method
studies	O
of	O
WT	B-protein_state
CaMep2	B-protein
,	O
the	O
DD	B-mutant
mutant	I-mutant
and	O
phosphorylated	B-protein_state
protein	O
(	O
S453J	B-mutant
).	O

The	O
protein	O
is	O
structurally	B-protein_state
stable	I-protein_state
throughout	O
the	O
simulation	B-experimental_method
with	O
little	O
deviation	O
in	O
the	O
other	O
parts	O
of	O
the	O
protein	O
.	O

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

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

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

In	O
addition	O
,	O
ICL1	B-structure_element
has	O
shifted	O
inwards	O
to	O
contribute	O
to	O
the	O
channel	B-site
closure	O
by	O
engaging	O
His2	B-residue_name_number
from	O
the	O
twin	B-structure_element
-	I-structure_element
His	I-structure_element
motif	I-structure_element
via	O
hydrogen	O
bonding	O
with	O
a	O
highly	B-protein_state
conserved	I-protein_state
tyrosine	B-residue_name
hydroxyl	O
group	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

With	O
regards	O
to	O
plant	B-taxonomy_domain
AMTs	B-protein_type
,	O
it	O
has	O
been	O
proposed	O
that	O
phosphorylation	B-ptm
at	O
T460	B-residue_name_number
generates	O
conformational	O
changes	O
that	O
would	O
close	O
the	O
neighbouring	O
pore	B-site
via	O
the	O
C	B-structure_element
terminus	I-structure_element
.	O

This	O
assumption	O
was	O
based	O
partly	O
on	O
a	O
homology	B-experimental_method
model	I-experimental_method
for	O
Amt	B-protein
-	I-protein
1	I-protein
;	I-protein
1	I-protein
based	O
on	O
the	O
(	O
open	B-protein_state
)	O
archaebacterial	B-taxonomy_domain
AfAmt	B-protein
-	I-protein
1	I-protein
structure	B-evidence
,	O
which	O
suggested	O
that	O
the	O
C	B-structure_element
terminus	I-structure_element
of	O
Amt	B-protein
-	I-protein
1	I-protein
;	I-protein
1	I-protein
would	O
extend	O
further	O
to	O
the	O
neighbouring	O
monomer	B-oligomeric_state
.	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

We	O
propose	O
that	O
intra	B-site
-	I-site
monomeric	I-site
CTR	I-site
-	I-site
ICL1	I-site
/	I-site
ICL3	I-site
interactions	I-site
lie	O
at	O
the	O
basis	O
of	O
regulation	O
of	O
both	O
fungal	B-taxonomy_domain
and	O
plant	B-taxonomy_domain
ammonium	B-protein_type
transporters	I-protein_type
;	O
close	O
interactions	O
generate	O
open	B-protein_state
channels	B-site
,	O
whereas	O
the	O
lack	B-protein_state
of	I-protein_state
‘	O
intra	O
-'	O
interactions	O
leads	O
to	O
inactive	B-protein_state
states	O
.	O

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

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

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

The	O
grey	O
sequences	O
at	O
the	O
C	O
termini	O
of	O
CaMep2	B-protein
and	O
ScMep2	B-protein
are	O
not	O
visible	O
in	O
the	O
structures	B-evidence
and	O
are	O
likely	B-protein_state
disordered	I-protein_state
.	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

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

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

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

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

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

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

(	O
b	O
)	O
Monomer	B-oligomeric_state
side	O
-	O
view	O
superposition	B-experimental_method
of	O
WT	B-protein_state
CaMep2	B-protein
and	O
the	O
DD	B-mutant
mutant	I-mutant
,	O
showing	O
the	O
conformational	O
change	O
and	O
disorder	O
around	O
the	O
ExxGxD	B-structure_element
motif	I-structure_element
.	O

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

Upon	O
phosphorylation	B-ptm
and	O
mimicked	B-protein_state
by	O
the	O
CaMep2	B-protein
S453D	B-mutant
and	O
DD	B-mutant
mutants	I-mutant
(	O
ii	O
),	O
the	O
region	O
around	O
the	O
ExxGxD	B-structure_element
motif	I-structure_element
undergoes	O
a	O
conformational	O
change	O
that	O
results	O
in	O
the	O
CTR	B-structure_element
interacting	O
with	O
the	O
inward	O
-	O
moving	O
ICL3	B-structure_element
,	O
opening	O
the	O
channel	B-site
(	O
full	O
circle	O
)	O
(	O
iii	O
).	O

The	O
open	B-protein_state
-	O
channel	B-site
Mep2	B-protein
structure	B-evidence
is	O
represented	O
by	O
archaebacterial	B-taxonomy_domain
Amt	B-protein
-	I-protein
1	I-protein
and	O
shown	O
in	O
lighter	O
colours	O
consistent	O
with	O
Fig	O
.	O
4	O
.	O

An	O
extended	B-protein_state
U2AF65	B-structure_element
–	I-structure_element
RNA	I-structure_element
-	I-structure_element
binding	I-structure_element
domain	I-structure_element
recognizes	O
the	O
3	B-site
′	I-site
splice	I-site
site	I-site
signal	O

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

The	O
differential	O
skipping	O
or	O
inclusion	O
of	O
alternatively	O
spliced	O
pre	B-structure_element
-	I-structure_element
mRNA	I-structure_element
regions	I-structure_element
is	O
a	O
major	O
source	O
of	O
diversity	O
for	O
nearly	O
all	O
human	B-species
gene	O
transcripts	O
.	O

High	O
-	O
resolution	O
structures	B-evidence
of	O
intact	B-protein_state
splicing	B-complex_assembly
factor	I-complex_assembly
–	I-complex_assembly
RNA	I-complex_assembly
complexes	O
would	O
offer	O
key	O
insights	O
regarding	O
the	O
juxtaposition	O
of	O
the	O
distinct	O
splice	B-site
site	I-site
consensus	O
sequences	O
and	O
their	O
relationship	O
to	O
disease	O
-	O
causing	O
point	O
mutations	O
.	O

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

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

An	O
extended	B-protein_state
conformation	I-protein_state
of	O
the	O
U2AF65	B-protein
inter	B-structure_element
-	I-structure_element
RRM	I-structure_element
linker	I-structure_element
traverses	O
across	O
the	O
α	B-structure_element
-	I-structure_element
helical	I-structure_element
surface	I-structure_element
of	O
RRM1	B-structure_element
and	O
the	O
central	O
β	B-structure_element
-	I-structure_element
strands	I-structure_element
of	O
RRM2	B-structure_element
and	O
is	O
well	O
defined	O
in	O
the	O
electron	B-evidence
density	I-evidence
(	O
Fig	O
.	O
2b	O
).	O

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

The	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
RRM2	B-structure_element
,	O
the	O
inter	B-structure_element
-	I-structure_element
RRM	I-structure_element
linker	I-structure_element
and	O
RRM1	B-structure_element
concomitantly	O
recognize	O
the	O
three	O
central	O
nucleotides	B-chemical
of	O
the	O
Py	B-chemical
tract	I-chemical
,	O
which	O
are	O
likely	O
to	O
coordinate	O
the	O
conformational	O
arrangement	O
of	O
these	O
disparate	O
portions	O
of	O
the	O
protein	O
.	O

Residues	O
in	O
the	O
C	B-structure_element
-	I-structure_element
terminal	I-structure_element
region	I-structure_element
of	O
the	O
U2AF65	B-protein
inter	B-structure_element
-	I-structure_element
RRM	I-structure_element
linker	I-structure_element
comprise	O
a	O
centrally	O
located	O
binding	B-site
site	I-site
for	O
the	O
fifth	B-residue_number
nucleotide	B-chemical
on	O
the	O
RRM2	B-site
surface	I-site
and	O
abutting	O
the	O
RRM1	B-site
/	I-site
RRM2	I-site
interface	I-site
(	O
Fig	O
.	O
3d	O
).	O

Otherwise	O
,	O
the	O
rU4	B-residue_name_number
nucleotide	B-chemical
packs	O
against	O
F304	B-residue_name_number
in	O
the	O
signature	O
ribonucleoprotein	B-structure_element
consensus	I-structure_element
motif	I-structure_element
(	I-structure_element
RNP	I-structure_element
)-	I-structure_element
2	I-structure_element
of	O
RRM2	B-structure_element
.	O

This	O
nucleotide	B-chemical
twists	O
to	O
face	O
away	O
from	O
the	O
U2AF65	B-protein
linker	B-structure_element
and	O
instead	O
inserts	O
the	O
rU6	B-residue_name_number
-	O
uracil	B-residue_name
into	O
a	O
sandwich	O
between	O
the	O
β2	B-structure_element
/	I-structure_element
β3	I-structure_element
loops	I-structure_element
of	O
RRM1	B-structure_element
and	O
RRM2	B-structure_element
.	O

The	O
Q147	B-residue_name_number
residue	O
participates	O
in	O
hydrogen	O
bonds	O
with	O
the	O
-	O
N3H	O
of	O
the	O
eighth	B-residue_number
uracil	B-residue_name
and	O
-	O
O2	O
of	O
the	O
ninth	B-residue_number
pyrimidine	B-chemical
.	O

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

We	O
compare	B-experimental_method
U2AF65	B-protein
interactions	O
with	O
uracil	B-residue_name
relative	O
to	O
cytosine	B-residue_name
pyrimidines	B-chemical
at	O
the	O
ninth	B-site
binding	I-site
site	I-site
in	O
Fig	O
.	O
3g	O
,	O
h	O
and	O
the	O
Supplementary	O
Discussion	O
.	O

Versatile	O
primary	O
sequence	O
of	O
the	O
U2AF65	B-protein
inter	B-structure_element
-	I-structure_element
RRM	I-structure_element
linker	I-structure_element

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

The	O
adjacent	O
V249	B-residue_name_number
and	O
V250	B-residue_name_number
are	O
notable	O
for	O
their	O
respective	O
interactions	O
that	O
connect	O
RRM1	B-structure_element
and	O
RRM2	B-structure_element
at	O
this	O
distal	O
interface	B-site
from	O
the	O
RNA	B-site
-	I-site
binding	I-site
site	I-site
(	O
Fig	O
.	O
4a	O
,	O
top	O
).	O

Few	O
direct	O
contacts	O
are	O
made	O
between	O
the	O
remaining	O
residues	O
of	O
the	O
linker	B-structure_element
and	O
the	O
U2AF65	B-protein
RRM2	B-structure_element
;	O
instead	O
,	O
the	O
C	O
-	O
terminal	O
conformation	O
of	O
the	O
linker	B-structure_element
appears	O
primarily	O
RNA	B-chemical
mediated	O
(	O
Fig	O
.	O
3c	O
,	O
d	O
).	O

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

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

We	O
found	O
that	O
the	O
affinity	B-evidence
of	O
dU2AF651	B-mutant
,	I-mutant
2L	I-mutant
for	O
the	O
AdML	B-gene
RNA	B-chemical
was	O
significantly	O
reduced	O
relative	O
to	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
(	O
four	O
-	O
fold	O
,	O
Figs	O
1b	O
and	O
4b	O
;	O
Supplementary	O
Fig	O
.	O
4i	O
).	O

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

Addition	O
of	O
the	O
AdML	B-gene
RNA	B-chemical
to	O
tethered	B-protein_state
U2AF651	B-mutant
,	I-mutant
2LFRET	I-mutant
(	O
Cy3	B-chemical
/	O
Cy5	B-chemical
)	O
selectively	O
increases	O
a	O
fraction	O
of	O
molecules	O
showing	O
an	O
∼	O
0	O
.	O
45	O
apparent	O
FRET	B-evidence
efficiency	I-evidence
,	O
suggesting	O
that	O
RNA	O
binding	O
stabilizes	O
a	O
single	O
conformation	O
,	O
which	O
corresponds	O
to	O
the	O
0	O
.	O
45	O
FRET	B-evidence
state	I-evidence
(	O
Fig	O
.	O
6e	O
,	O
f	O
).	O

To	O
assess	O
the	O
possible	O
contributions	O
of	O
RNA	B-protein_state
-	I-protein_state
free	I-protein_state
conformations	O
of	O
U2AF65	B-protein
and	O
/	O
or	O
structural	O
heterogeneity	O
introduced	O
by	O
tethering	B-experimental_method
of	O
U2AF651	B-mutant
,	I-mutant
2LFRET	I-mutant
(	O
Cy3	B-chemical
/	O
Cy5	B-chemical
)	O
to	O
the	O
slide	O
to	O
the	O
observed	O
distribution	B-evidence
of	I-evidence
FRET	I-evidence
values	I-evidence
,	O
we	O
reversed	B-experimental_method
the	I-experimental_method
immobilization	I-experimental_method
scheme	I-experimental_method
.	O

We	O
tethered	B-protein_state
the	O
AdML	B-gene
RNA	B-chemical
to	O
the	O
slide	O
via	O
a	O
biotinylated	B-chemical
oligonucleotide	I-chemical
DNA	I-chemical
handle	O
and	O
added	B-experimental_method
U2AF651	B-mutant
,	I-mutant
2LFRET	I-mutant
(	O
Cy3	B-chemical
/	O
Cy5	B-chemical
)	O
in	O
the	O
absence	B-protein_state
of	I-protein_state
biotin	B-chemical
-	I-chemical
NTA	I-chemical
resin	I-chemical
(	O
Fig	O
.	O
6g	O
,	O
h	O
;	O
Supplementary	O
Fig	O
.	O
7c	O
–	O
g	O
).	O

Nevertheless	O
,	O
in	O
the	O
presence	O
of	O
saturating	O
concentrations	O
of	O
rArA	B-chemical
-	O
interrupted	O
RNA	B-chemical
slide	B-protein_state
-	I-protein_state
tethered	I-protein_state
U2AF651	B-mutant
,	I-mutant
2LFRET	I-mutant
(	O
Cy3	B-chemical
/	O
Cy5	B-chemical
)	O
showed	O
a	O
prevalent	O
∼	O
0	O
.	O
45	O
apparent	O
FRET	B-evidence
value	I-evidence
(	O
Fig	O
.	O
6i	O
,	O
j	O
),	O
which	O
was	O
also	O
predominant	O
in	O
the	O
presence	O
of	O
continuous	O
Py	B-chemical
tract	I-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
majority	O
of	O
traces	B-evidence
that	O
show	O
fluctuations	O
began	O
at	O
high	O
(	O
0	O
.	O
65	O
–	O
0	O
.	O
8	O
)	O
FRET	B-evidence
value	I-evidence
and	O
transitioned	O
to	O
a	O
∼	O
0	O
.	O
45	O
FRET	B-evidence
value	I-evidence
(	O
Supplementary	O
Fig	O
.	O
7c	O
–	O
g	O
).	O

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

Likewise	O
,	O
deletion	B-experimental_method
of	O
20	B-residue_range
inter	B-structure_element
-	I-structure_element
RRM	I-structure_element
linker	I-structure_element
residues	I-structure_element
significantly	O
reduces	O
U2AF65	B-protein
–	O
RNA	B-chemical
binding	O
only	O
when	O
introduced	O
in	O
the	O
context	O
of	O
the	O
longer	B-protein_state
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
construct	O
comprising	O
the	O
RRM	B-structure_element
extensions	I-structure_element
,	O
which	O
in	O
turn	O
position	O
the	O
linker	B-structure_element
for	O
RNA	B-chemical
interactions	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

Examples	O
of	O
‘	O
extended	B-protein_state
conformational	O
selection	O
'	O
during	O
ligand	O
binding	O
have	O
been	O
characterized	O
for	O
a	O
growing	O
number	O
of	O
macromolecules	O
(	O
for	O
example	O
,	O
adenylate	B-protein_type
kinase	I-protein_type
,	O
LAO	B-protein_type
-	I-protein_type
binding	I-protein_type
protein	I-protein_type
,	O
poly	B-protein_type
-	I-protein_type
ubiquitin	I-protein_type
,	O
maltose	B-protein_type
-	I-protein_type
binding	I-protein_type
protein	I-protein_type
and	O
the	O
preQ1	B-protein_type
riboswitch	I-protein_type
,	O
among	O
others	O
).	O

Similar	O
interdisciplinary	O
structural	O
approaches	O
are	O
likely	O
to	O
illuminate	O
whether	O
similar	O
mechanistic	O
bases	O
for	O
RNA	O
binding	O
are	O
widespread	O
among	O
other	O
members	O
of	O
the	O
vast	O
multi	O
-	O
RRM	B-structure_element
family	O
.	O

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

The	O
apparent	O
equilibrium	B-evidence
dissociation	I-evidence
constants	I-evidence
(	O
KD	B-evidence
)	O
for	O
binding	O
the	O
AdML	B-gene
13mer	O
are	O
as	O
follows	O
:	O
flU2AF65	B-protein
,	O
30	O
±	O
3	O
nM	O
;	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
,	O
35	O
±	O
6	O
nM	O
;	O
U2AF651	B-mutant
,	I-mutant
2	I-mutant
,	O
3	O
,	O
600	O
±	O
300	O
nM	O
.	O
(	O
c	O
)	O
Comparison	O
of	O
the	O
RNA	B-evidence
sequence	I-evidence
specificities	I-evidence
of	O
flU2AF65	B-protein
and	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
constructs	O
binding	O
C	B-structure_element
-	I-structure_element
rich	I-structure_element
Py	B-chemical
tracts	I-chemical
with	O
4U	O
'	O
s	O
embedded	O
in	O
either	O
the	O
5	O
′-	O
(	O
light	O
grey	O
fill	O
)	O
or	O
3	O
′-	O
(	O
dark	O
grey	O
fill	O
)	O
regions	O
.	O

The	O
KD	B-evidence
'	O
s	O
for	O
binding	O
5	B-chemical
′-	I-chemical
CCUUUUCCCCCCC	I-chemical
-	I-chemical
3	I-chemical
′	I-chemical
are	O
:	O
flU2AF65	B-protein
,	O
41	O
±	O
2	O
nM	O
;	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
,	O
31	O
±	O
3	O
nM	O
.	O
The	O
KD	B-evidence
'	O
s	O
for	O
binding	O
5	B-chemical
′-	I-chemical
CCCCCCCUUUUCC	I-chemical
-	I-chemical
3	I-chemical
′	I-chemical
are	O
:	O
flU2AF65	B-protein
,	O
414	O
±	O
12	O
nM	O
;	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
,	O
417	O
±	O
10	O
nM	O
.	O
Bar	O
graphs	O
are	O
hatched	O
to	O
match	O
the	O
constructs	O
shown	O
in	O
a	O
.	O
The	O
average	B-evidence
apparent	I-evidence
equilibrium	I-evidence
affinity	I-evidence
(	O
KA	B-evidence
)	O
and	O
s	O
.	O
e	O
.	O
m	O
.	O
for	O
three	O
independent	O
titrations	O
are	O
plotted	O
.	O

Representative	O
views	O
of	O
the	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
interactions	O
with	O
each	O
new	O
nucleotide	B-chemical
of	O
the	O
bound	B-protein_state
Py	B-chemical
tract	I-chemical
.	O

The	O
average	O
fitted	O
fluorescence	O
anisotropy	O
RNA	B-evidence
-	I-evidence
binding	I-evidence
curves	I-evidence
are	O
shown	O
in	O
Supplementary	O
Fig	O
.	O
4a	O
–	O
c	O
.	O

RNA	O
binding	O
stabilizes	O
the	O
side	B-protein_state
-	I-protein_state
by	I-protein_state
-	I-protein_state
side	I-protein_state
conformation	O
of	O
U2AF65	B-protein
RRMs	B-structure_element
.	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

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

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

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

Hydrophobic	O
contacts	O
and	O
a	O
hydrogen	B-site
-	I-site
bond	I-site
network	I-site
mediate	O
the	O
interaction	O
between	O
HAESA	B-protein
and	O
the	O
peptide	B-protein_type
hormone	I-protein_type
IDA	B-protein
.	O

HAESA	B-site
interface	I-site
residues	I-site
are	O
shown	O
as	O
sticks	O
,	O
selected	O
hydrogen	O
bond	O
interactions	O
are	O
denoted	O
as	O
dotted	O
lines	O
(	O
in	O
magenta	O
).	O
(	O
B	O
)	O
View	O
of	O
the	O
complete	O
IDA	B-protein
(	O
in	O
bonds	O
representation	O
,	O
in	O
yellow	O
)	O
binding	B-site
pocket	I-site
in	O
HAESA	B-protein
(	O
surface	O
view	O
,	O
in	O
blue	O
).	O

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

Details	O
of	O
the	O
interactions	O
between	O
the	O
central	O
Hyp	B-structure_element
anchor	I-structure_element
in	O
IDA	B-protein
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
with	O
HAESA	B-protein
are	O
highlighted	O
in	O
(	O
E	O
)	O
and	O
(	O
F	O
),	O
respectively	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

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

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

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

A	O
Hyp	B-protein_state
-	I-protein_state
modified	I-protein_state
dodecamer	B-structure_element
comprising	O
the	O
highly	B-protein_state
conserved	I-protein_state
PIP	B-structure_element
motif	I-structure_element
in	O
IDA	B-protein
(	O
Figure	O
1A	O
)	O
interacts	O
with	O
HAESA	B-protein
with	O
1	O
:	O
1	O
stoichiometry	O
(	O
N	O
)	O
and	O
with	O
a	O
dissociation	B-evidence
constant	I-evidence
(	O
Kd	B-evidence
)	O
of	O
~	O
20	O
μM	O
(	O
Figure	O
1B	O
).	O

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

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

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

As	O
all	O
five	O
SERK	B-protein_type
family	I-protein_type
members	I-protein_type
appear	O
to	O
be	O
expressed	O
in	O
the	O
Arabidopsis	B-taxonomy_domain
abscission	O
zone	O
,	O
we	O
quantified	O
their	O
relative	O
contribution	O
to	O
floral	O
abscission	O
in	O
Arabidopsis	B-taxonomy_domain
using	O
a	O
petal	B-experimental_method
break	I-experimental_method
-	I-experimental_method
strength	I-experimental_method
assay	I-experimental_method
.	O

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

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

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

SERK1	B-protein
loop	B-structure_element
residues	O
establish	O
multiple	O
hydrophobic	O
and	O
polar	O
contacts	O
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

(	O
C	O
)	O
Quantitative	O
petal	B-experimental_method
break	I-experimental_method
-	I-experimental_method
strength	I-experimental_method
assay	I-experimental_method
for	O
Col	O
-	O
0	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
flowers	O
and	O
35S	B-gene
::	O
IDA	B-protein
wild	B-protein_state
-	I-protein_state
type	I-protein_state
and	O
35S	B-gene
::	O
IDA	B-mutant
K66A	I-mutant
/	I-mutant
R67A	I-mutant
mutant	B-protein_state
flowers	O
.	O

We	O
thus	O
assessed	O
their	O
contribution	O
to	O
HAESA	B-complex_assembly
–	I-complex_assembly
SERK1	I-complex_assembly
complex	O
formation	O
.	O

In	O
contrast	O
,	O
over	B-experimental_method
-	I-experimental_method
expression	I-experimental_method
of	O
the	O
IDA	B-mutant
Lys66IDA	I-mutant
/	I-mutant
Arg67IDA	I-mutant
→	I-mutant
Ala	I-mutant
double	B-protein_state
mutant	I-protein_state
significantly	O
delays	O
floral	O
abscission	O
when	O
compared	O
to	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
control	O
plants	B-taxonomy_domain
,	O
suggesting	O
that	O
the	O
mutant	B-protein_state
IDA	B-chemical
peptide	I-chemical
has	O
reduced	O
activity	O
in	O
planta	B-taxonomy_domain
(	O
Figure	O
5C	O
–	O
E	O
).	O

Comparison	O
of	O
35S	B-gene
::	O
IDA	B-protein
wild	B-protein_state
-	I-protein_state
type	I-protein_state
and	O
mutant	B-protein_state
plants	B-taxonomy_domain
further	O
indicates	O
that	O
mutation	B-experimental_method
of	O
Lys66IDA	B-mutant
/	I-mutant
Arg67IDA	I-mutant
→	I-mutant
Ala	I-mutant
may	O
cause	O
a	O
weak	O
dominant	O
negative	O
effect	O
(	O
Figure	O
5C	O
–	O
E	O
).	O

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

In	O
both	O
cases	O
however	O
,	O
the	O
co	O
-	O
receptor	O
completes	O
the	O
hormone	B-site
binding	I-site
pocket	I-site
.	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

Biogenesis	O
of	O
the	O
20S	B-complex_assembly
proteasome	I-complex_assembly
is	O
tightly	O
regulated	O
.	O

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

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

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

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

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

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

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

Surprisingly	O
,	O
Gly	B-residue_name_number
(-	I-residue_name_number
1	I-residue_name_number
)	I-residue_name_number
is	O
completely	O
extended	O
and	O
forces	O
the	O
histidine	B-residue_name
side	O
chain	O
at	O
position	O
(-	B-residue_number
2	I-residue_number
)	I-residue_number
to	O
occupy	O
the	O
S2	B-site
instead	O
of	O
the	O
S1	B-site
pocket	I-site
,	O
thereby	O
disrupting	O
the	O
antiparallel	B-structure_element
β	I-structure_element
-	I-structure_element
sheet	I-structure_element
.	O

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

As	O
proven	O
by	O
the	O
β2	B-mutant
-	I-mutant
T1A	I-mutant
crystal	B-evidence
structures	I-evidence
,	O
Thr	B-residue_name_number
(-	I-residue_name_number
2	I-residue_name_number
)	I-residue_name_number
hydrogen	O
bonds	O
to	O
Gly	B-residue_name_number
(-	I-residue_name_number
1	I-residue_name_number
)	I-residue_name_number
O	O
.	O
Although	O
this	O
interaction	O
was	O
not	O
observed	O
for	O
the	O
β5	B-mutant
-	I-mutant
H	I-mutant
(-	I-mutant
2	I-mutant
)	I-mutant
T	I-mutant
-	I-mutant
T1A	I-mutant
mutant	B-protein_state
(	O
Fig	O
.	O
2c	O
and	O
Supplementary	O
Fig	O
.	O
4c	O
,	O
i	O
),	O
exchange	B-experimental_method
of	O
Thr	B-residue_name_number
(-	I-residue_name_number
2	I-residue_name_number
)	I-residue_name_number
by	O
Val	B-residue_name
in	O
β2	B-protein
,	O
a	O
conservative	O
mutation	O
regarding	O
size	O
but	O
drastic	O
with	O
respect	O
to	O
polarity	O
,	O
was	O
found	O
to	O
inhibit	O
maturation	O
of	O
this	O
subunit	O
(	O
Fig	O
.	O
2d	O
and	O
Supplementary	O
Fig	O
.	O
4e	O
,	O
j	O
).	O

Instead	O
,	O
Lys33NH2	B-residue_name_number
,	O
which	O
is	O
in	O
hydrogen	O
-	O
bonding	O
distance	O
to	O
Thr1Oγ	B-residue_name_number
(	O
2	O
.	O
7	O
Å	O
)	O
in	O
all	O
catalytically	B-protein_state
active	I-protein_state
β	B-protein
subunits	I-protein
(	O
Fig	O
.	O
3a	O
,	O
b	O
),	O
was	O
proposed	O
to	O
serve	O
as	O
the	O
proton	O
acceptor	O
.	O

A	O
proposed	O
catalytic	B-site
tetrad	I-site
model	O
involving	O
Thr1OH	B-residue_name_number
,	O
Thr1NH2	B-residue_name_number
,	O
Lys33NH2	B-residue_name_number
and	O
Asp17Oδ	B-residue_name_number
,	O
as	O
well	O
as	O
a	O
nucleophilic	O
water	B-chemical
molecule	O
as	O
the	O
proton	O
shuttle	O
appeared	O
to	O
accommodate	O
all	O
possible	O
views	O
of	O
the	O
proteasomal	O
active	B-site
site	I-site
.	O

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

Since	O
no	O
acetylation	B-ptm
of	O
the	O
Thr1	B-residue_name_number
N	O
terminus	O
was	O
observed	O
for	O
the	O
β5	B-mutant
-	I-mutant
K33A	I-mutant
pp	B-chemical
trans	B-protein_state
apo	B-protein_state
crystal	B-evidence
structure	I-evidence
,	O
the	O
reduced	O
reactivity	O
towards	O
substrates	O
and	O
inhibitors	O
indicates	O
that	O
Lys33NH2	B-residue_name_number
,	O
rather	O
than	O
Thr1NH2	B-residue_name_number
,	O
deprotonates	O
and	O
activates	O
Thr1OH	B-residue_name_number
.	O

Autolysis	B-ptm
and	O
residual	O
catalytic	O
activity	O
of	O
the	O
β5	B-mutant
-	I-mutant
D17N	I-mutant
mutants	O
may	O
originate	O
from	O
the	O
carbonyl	O
group	O
of	O
Asn17	B-residue_name_number
,	O
which	O
albeit	O
to	O
a	O
lower	O
degree	O
still	O
can	O
polarize	O
Lys33	B-residue_name_number
for	O
the	O
activation	O
of	O
Thr1	B-residue_name_number
.	O

Together	O
,	O
these	O
observations	O
suggest	O
that	O
efficient	O
peptide	O
-	O
bond	O
hydrolysis	O
requires	O
that	O
Lys33NH2	B-residue_name_number
hydrogen	O
bonds	O
to	O
the	O
active	O
site	O
nucleophile	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

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

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

In	O
eukaryotes	B-taxonomy_domain
,	O
deletion	O
of	O
or	O
failure	O
to	O
cleave	O
the	O
β1	B-protein
and	O
β2	B-protein
propeptides	B-structure_element
is	O
well	O
tolerated	O
.	O

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

We	O
propose	O
a	O
catalytic	B-site
triad	I-site
for	O
the	O
active	B-site
site	I-site
of	O
the	O
CP	B-complex_assembly
consisting	O
of	O
residues	O
Thr1	B-residue_name_number
,	O
Lys33	B-residue_name_number
and	O
Asp	B-residue_name
/	O
Glu17	B-residue_name_number
,	O
which	O
are	O
conserved	O
among	O
all	O
proteolytically	O
active	O
eukaryotic	B-taxonomy_domain
,	O
bacterial	B-taxonomy_domain
and	O
archaeal	B-taxonomy_domain
proteasome	B-complex_assembly
subunits	O
.	O

Analogously	O
,	O
Thr1NH3	B-residue_name_number
+	O
might	O
promote	O
the	O
bivalent	O
reaction	O
mode	O
of	O
epoxyketone	O
inhibitors	O
by	O
protonating	O
the	O
epoxide	O
moiety	O
to	O
create	O
a	O
positively	O
charged	O
trivalent	O
oxygen	O
atom	O
that	O
is	O
subsequently	O
nucleophilically	O
attacked	O
by	O
Thr1NH2	B-residue_name_number
.	O

The	O
residues	O
Ser129	B-residue_name_number
and	O
Asp166	B-residue_name_number
are	O
expected	O
to	O
increase	O
the	O
pKa	O
value	O
of	O
Thr1N	B-residue_name_number
,	O
thereby	O
favouring	O
its	O
charged	O
state	O
.	O

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

Notably	O
,	O
in	O
the	O
Ntn	B-protein_type
hydrolase	I-protein_type
penicillin	B-protein_type
acylase	I-protein_type
,	O
substitution	B-experimental_method
of	O
the	O
catalytic	B-protein_state
N	O
-	O
terminal	O
Ser	B-residue_name
residue	O
by	O
Cys	B-residue_name
also	O
inactivates	B-protein_state
the	O
enzyme	B-protein_type
but	O
still	O
enables	O
precursor	B-ptm
processing	I-ptm
.	O

Notably	O
,	O
proteolytically	B-protein_state
active	I-protein_state
proteasome	B-complex_assembly
subunits	O
from	O
archaea	B-taxonomy_domain
,	O
yeast	B-taxonomy_domain
and	O
mammals	B-taxonomy_domain
,	O
including	O
constitutive	O
,	O
immuno	O
-	O
and	O
thymoproteasome	O
subunits	O
,	O
either	O
encode	O
Thr	B-residue_name
or	O
Ile	B-residue_name
at	O
position	O
3	B-residue_number
,	O
indicating	O
the	O
importance	O
of	O
the	O
Cγ	O
for	O
fixing	O
the	O
position	O
of	O
the	O
nucleophilic	O
Thr1	B-residue_name_number
.	O

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

The	O
resulting	O
deprotonated	O
Thr1NH2	B-residue_name_number
finally	O
activates	O
a	O
water	B-chemical
molecule	O
for	O
hydrolysis	O
of	O
the	O
acyl	O
-	O
enzyme	O
.	O

The	O
prosegment	B-structure_element
is	O
cleaved	B-protein_state
but	O
still	B-protein_state
bound	I-protein_state
in	O
the	O
substrate	B-site
-	I-site
binding	I-site
channel	I-site
.	O

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

Note	O
that	O
IC50	B-evidence
values	I-evidence
depend	O
on	O
time	O
and	O
enzyme	O
concentration	O
.	O

The	O
WT	B-protein_state
proteasome	B-complex_assembly
:	I-complex_assembly
inhibitor	I-complex_assembly
complex	I-complex_assembly
structures	B-evidence
(	O
inhibitor	O
in	O
grey	O
;	O
Thr1	B-residue_name_number
in	O
black	O
)	O
are	O
superimposed	B-experimental_method
and	O
demonstrate	O
that	O
mutation	B-experimental_method
of	O
Thr1	B-residue_name_number
to	O
Ser	B-residue_name
does	O
not	O
affect	O
the	O
binding	O
mode	O
of	O
bortezomib	B-chemical
or	O
carfilzomib	B-chemical
.	O