Biochemical	B-experimental_method
analysis	I-experimental_method
reveals	O
that	O
these	O
outer	B-protein_type
membrane	I-protein_type
-	I-protein_type
anchored	I-protein_type
proteins	I-protein_type
are	O
in	O
fact	O
exquisitely	O
specific	O
for	O
the	O
highly	O
branched	O
xyloglucan	B-chemical
(	O
XyG	B-chemical
)	O
polysaccharide	B-chemical
.	O

However	O
,	O
there	O
is	O
a	O
paucity	O
of	O
data	O
regarding	O
how	O
the	O
vast	O
array	O
of	O
complex	B-chemical
carbohydrate	I-chemical
structures	O
are	O
selectively	O
recognized	O
and	O
imported	O
by	O
members	O
of	O
the	O
microbiota	B-taxonomy_domain
,	O
a	O
critical	O
process	O
that	O
enables	O
these	O
organisms	O
to	O
thrive	O
in	O
the	O
competitive	O
gut	O
environment	O
.	O

The	O
location	O
of	O
SGBP	B-protein
-	I-protein
A	I-protein
/	O
B	B-protein
is	O
presented	O
in	O
this	O
work	O
;	O
the	O
location	O
of	O
GH5	B-protein
has	O
been	O
empirically	O
determined	O
,	O
and	O
the	O
enzymes	O
have	O
been	O
placed	O
based	O
upon	O
their	O
predicted	O
cellular	O
location	O
.	O

These	O
data	O
extend	O
our	O
current	O
understanding	O
of	O
the	O
Sus	O
-	O
like	O
glycan	B-chemical
uptake	O
paradigm	O
within	O
the	O
Bacteroidetes	B-taxonomy_domain
and	O
reveals	O
how	O
the	O
complex	O
dietary	O
polysaccharide	B-chemical
xyloglucan	B-chemical
is	O
recognized	O
at	O
the	O
cell	O
surface	O
.	O

In	O
contrast	O
,	O
SGBP	B-protein
-	I-protein
B	I-protein
,	O
encoded	O
by	O
locus	O
tag	O
Bacova_02650	B-gene
,	O
displays	O
little	O
sequence	O
similarity	O
to	O
the	O
products	O
of	O
similarly	O
positioned	O
genes	O
in	O
syntenic	O
XyGUL	B-gene
nor	O
to	O
any	O
other	O
gene	O
product	O
among	O
the	O
diversity	O
of	O
Bacteroidetes	B-taxonomy_domain
PUL	B-gene
.	O

Formalin	O
-	O
fixed	O
,	O
nonpermeabilized	O
B	B-species
.	I-species
ovatus	I-species
cells	O
were	O
grown	O
in	O
minimal	O
medium	O
plus	O
XyG	B-chemical
,	O
probed	O
with	O
custom	O
rabbit	O
antibodies	O
to	O
SGBP	B-protein
-	I-protein
A	I-protein
or	O
SGBP	B-protein
-	I-protein
B	I-protein
,	O
and	O
then	O
stained	O
with	O
Alexa	O
Fluor	O
488	O
goat	O
anti	O
-	O
rabbit	O
IgG	O
.	O
(	O
A	O
)	O
Overlay	B-experimental_method
of	O
bright	B-evidence
-	I-evidence
field	I-evidence
and	I-evidence
FITC	I-evidence
images	I-evidence
of	O
B	B-species
.	I-species
ovatus	I-species
cells	O
labeled	O
with	O
anti	O
-	O
SGBP	O
-	O
A	O
.	O
(	O
B	O
)	O
Overlay	B-experimental_method
of	O
bright	B-evidence
-	I-evidence
field	I-evidence
and	I-evidence
FITC	I-evidence
images	I-evidence
of	O
B	B-species
.	I-species
ovatus	I-species
cells	O
labeled	O
with	O
anti	O
-	O
SGBP	O
-	O
B	O
.	O
(	O
C	O
)	O
Bright	B-evidence
-	I-evidence
field	I-evidence
image	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

The	O
vanguard	O
endo	B-protein_type
-	I-protein_type
xyloglucanase	I-protein_type
of	O
the	O
XyGUL	B-gene
,	O
BoGH5	B-protein
,	O
preferentially	O
cleaves	O
the	O
polysaccharide	B-chemical
at	O
unbranched	O
glucosyl	B-chemical
residues	O
to	O
generate	O
xylogluco	B-chemical
-	I-chemical
oligosaccharides	I-chemical
(	O
XyGOs	B-chemical
)	O
comprising	O
a	O
Glc4	B-structure_element
backbone	I-structure_element
with	O
variable	B-structure_element
side	I-structure_element
-	I-structure_element
chain	I-structure_element
galactosylation	I-structure_element
(	O
XyGO1	B-chemical
)	O
(	O
Fig	O
.	O
1A	O
;	O
n	O
=	O
1	O
)	O
as	O
the	O
limit	O
of	O
digestion	O
products	O
in	O
vitro	O
;	O
controlled	B-experimental_method
digestion	I-experimental_method
and	I-experimental_method
fractionation	I-experimental_method
by	O
size	B-experimental_method
exclusion	I-experimental_method
chromatography	I-experimental_method
allow	O
the	O
production	O
of	O
higher	O
-	O
order	O
oligosaccharides	B-chemical
(	O
e	O
.	O
g	O
.,	O
XyGO2	B-chemical
)	O
(	O
Fig	O
.	O
1A	O
;	O
n	O
=	O
2	O
).	O

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

Most	O
surprising	O
in	O
light	O
of	O
the	O
saccharide	B-evidence
-	I-evidence
binding	I-evidence
data	I-evidence
,	O
however	O
,	O
was	O
a	O
lack	O
of	O
extensive	O
recognition	O
of	O
the	O
XyG	B-chemical
side	O
chains	O
;	O
only	O
Y84	B-residue_name_number
appeared	O
to	O
provide	O
a	O
hydrophobic	B-site
interface	I-site
for	O
a	O
xylosyl	B-chemical
residue	O
(	O
Xyl1	B-residue_name_number
).	O

Domains	O
A	B-structure_element
,	O
B	B-structure_element
,	O
and	O
C	B-structure_element
display	O
similar	O
β	B-structure_element
-	I-structure_element
sandwich	I-structure_element
folds	I-structure_element
;	O
domains	O
B	B-structure_element
(	O
residues	O
134	B-residue_range
to	I-residue_range
230	I-residue_range
)	O
and	O
C	B-structure_element
(	O
residues	O
231	B-residue_range
to	I-residue_range
313	I-residue_range
)	O
can	O
be	O
superimposed	B-experimental_method
onto	O
domain	O
A	B-structure_element
(	O
residues	O
34	B-residue_range
to	I-residue_range
133	I-residue_range
)	O
with	O
RMSDs	B-evidence
of	O
1	O
.	O
1	O
and	O
1	O
.	O
2	O
Å	O
,	O
respectively	O
,	O
for	O
47	O
atom	O
pairs	O
(	O
23	O
%	O
and	O
16	O
%	O
sequence	O
identity	O
,	O
respectively	O
).	O

While	O
neither	O
the	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
protein	O
nor	O
domain	O
D	B-structure_element
displays	O
structural	O
homology	O
to	O
known	O
XyG	B-protein_type
-	I-protein_type
binding	I-protein_type
proteins	I-protein_type
,	O
the	O
topology	O
of	O
SGBP	B-protein
-	I-protein
B	I-protein
resembles	O
the	O
xylan	B-protein_type
-	I-protein_type
binding	I-protein_type
protein	I-protein_type
Bacova_04391	B-protein
(	O
PDB	O
3ORJ	O
)	O
encoded	O
within	O
a	O
xylan	B-chemical
-	O
targeting	O
PUL	B-gene
of	O
B	B-species
.	I-species
ovatus	I-species
(	O
Fig	O
.	O
5C	O
).	O

Six	O
glucosyl	B-chemical
residues	O
,	O
comprising	O
the	O
main	O
chain	O
,	O
and	O
three	O
branching	O
xylosyl	B-chemical
residues	O
of	O
XyGO2	B-chemical
can	O
be	O
modeled	O
in	O
the	O
density	B-evidence
(	O
Fig	O
.	O
5D	O
;	O
cf	O
.	O

Complementation	B-experimental_method
of	O
ΔSGBP	B-mutant
-	I-mutant
A	I-mutant
with	O
the	O
SGBP	B-mutant
-	I-mutant
A	I-mutant
*	I-mutant
(	O
W82A	B-mutant
W283A	B-mutant
W306A	B-mutant
)	O
variant	O
,	O
which	O
does	O
not	B-protein_state
bind	I-protein_state
XyG	B-chemical
,	O
supports	O
growth	O
on	O
XyG	B-chemical
and	O
XyGOs	B-chemical
(	O
Fig	O
.	O
6	O
;	O
ΔSGBP	B-mutant
-	I-mutant
A	I-mutant
::	O
SGBP	B-mutant
-	I-mutant
A	I-mutant
*),	I-mutant
with	O
growth	O
rates	O
that	O
are	O
~	O
70	O
%	O
that	O
of	O
the	O
WT	B-protein_state
.	O

However	O
,	O
combined	B-experimental_method
deletion	I-experimental_method
of	I-experimental_method
the	I-experimental_method
genes	I-experimental_method
encoding	I-experimental_method
GH9	B-protein
(	O
encoded	O
by	O
Bacova_02649	B-gene
)	O
and	O
SGBP	B-protein
-	I-protein
B	I-protein
does	O
not	O
exacerbate	O
the	O
growth	O
defect	O
on	O
XyGO1	B-chemical
(	O
Fig	O
.	O
6	O
;	O
ΔSGBP	B-mutant
-	I-mutant
B	I-mutant
/	O
ΔGH9	B-mutant
).	O

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

Our	O
observation	O
here	O
that	O
the	O
physical	O
presence	O
of	O
the	O
SusD	B-protein
homolog	O
SGBP	B-protein
-	I-protein
A	I-protein
,	O
independent	O
of	O
XyG	B-chemical
-	O
binding	O
ability	O
,	O
is	O
both	O
necessary	O
and	O
sufficient	O
for	O
XyG	B-chemical
utilization	O
further	O
supports	O
a	O
model	O
of	O
glycan	B-chemical
import	O
whereby	O
the	O
SusC	B-protein_type
-	I-protein_type
like	I-protein_type
TBDTs	I-protein_type
and	O
the	O
SusD	B-protein_type
-	I-protein_type
like	I-protein_type
SGBPs	I-protein_type
must	O
be	O
intimately	O
associated	O
to	O
support	O
glycan	B-chemical
uptake	O
(	O
Fig	O
.	O
1C	O
).	O

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

In	O
conclusion	O
,	O
the	O
present	O
study	O
further	O
illuminates	O
the	O
essential	O
role	O
that	O
surface	B-protein_type
-	I-protein_type
glycan	I-protein_type
binding	I-protein_type
proteins	I-protein_type
play	O
in	O
facilitating	O
the	O
catabolism	O
of	O
complex	O
dietary	O
carbohydrates	B-chemical
by	O
Bacteroidetes	B-taxonomy_domain
.	O

The	O
PTAC	B-protein_type
predominantly	O
associated	O
with	O
metabolosomes	B-complex_assembly
(	O
PduL	B-protein_type
)	O
has	O
no	O
sequence	O
homology	O
to	O
the	O
PTAC	B-protein_type
ubiquitous	O
among	O
fermentative	B-taxonomy_domain
bacteria	I-taxonomy_domain
(	O
Pta	B-protein_type
).	O

Here	O
,	O
we	O
report	O
two	O
high	O
-	O
resolution	O
PduL	B-protein_type
crystal	B-evidence
structures	I-evidence
with	B-protein_state
bound	I-protein_state
substrates	I-protein_state
.	O

Recently	O
,	O
a	O
new	O
type	O
of	O
phosphotransacylase	B-protein_type
was	O
described	O
that	O
shares	O
no	O
evolutionary	O
relation	O
to	O
Pta	B-protein_type
.	O

Not	O
only	O
does	O
PduL	B-protein_type
facilitate	O
substrate	O
level	O
phosphorylation	O
,	O
but	O
it	O
also	O
is	O
critical	O
for	O
cofactor	O
recycling	O
within	O
,	O
and	O
product	O
efflux	O
from	O
,	O
the	O
organelle	O
.	O

The	O
reactions	O
carried	O
out	O
in	O
the	O
majority	O
of	O
catabolic	B-protein_state
BMCs	B-complex_assembly
(	O
also	O
known	O
as	O
metabolosomes	B-complex_assembly
)	O
fit	O
a	O
generalized	O
biochemical	O
paradigm	O
for	O
the	O
oxidation	O
of	O
aldehydes	B-chemical
(	O
Fig	O
1	O
).	O

Reaction	O
1	O
:	O
acetyl	B-chemical
-	I-chemical
S	I-chemical
-	I-chemical
CoA	I-chemical
+	O
Pi	B-chemical
←→	O
acetyl	B-chemical
phosphate	I-chemical
+	O
CoA	B-chemical
-	I-chemical
SH	I-chemical
(	O
PTAC	B-protein_type
)	O

The	O
canonical	O
PTAC	B-protein_type
,	O
Pta	B-protein_type
,	O
is	O
an	O
ancient	O
enzyme	O
found	O
in	O
some	O
eukaryotes	B-taxonomy_domain
and	O
archaea	B-taxonomy_domain
,	O
and	O
widespread	O
among	O
the	O
bacteria	B-taxonomy_domain
;	O
90	O
%	O
of	O
the	O
bacterial	B-taxonomy_domain
genomes	O
in	O
the	O
Integrated	O
Microbial	O
Genomes	O
database	O
contain	O
a	O
gene	O
encoding	O
the	O
PTA_PTB	B-protein_type
phosphotransacylase	I-protein_type
(	O
Pfam	O
domain	O
PF01515	B-structure_element
).	O

This	O
protein	O
,	O
PduL	B-protein_type
(	O
Pfam	O
domain	O
PF06130	B-structure_element
),	O
was	O
shown	O
to	O
catalyze	O
the	O
conversion	O
of	O
propionyl	B-chemical
-	I-chemical
CoA	I-chemical
to	O
propionyl	B-chemical
-	I-chemical
phosphate	I-chemical
and	O
is	O
associated	O
with	O
a	O
BMC	B-complex_assembly
involved	O
in	O
propanediol	O
utilization	O
,	O
the	O
PDU	B-complex_assembly
BMC	I-complex_assembly
.	O

In	O
contrast	O
,	O
the	O
structure	B-evidence
of	O
PduL	B-protein_type
,	O
the	O
PTAC	B-protein_type
found	O
in	O
the	O
vast	O
majority	O
of	O
catabolic	B-protein_state
BMCs	B-complex_assembly
,	O
has	O
not	O
been	O
determined	O
.	O

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

Structure	B-experimental_method
Determination	I-experimental_method
of	O
PduL	B-protein_type

(	O
a	O
)	O
Primary	O
and	O
secondary	O
structure	O
of	O
rPduL	B-protein
(	O
tubes	O
represent	O
α	B-structure_element
-	I-structure_element
helices	I-structure_element
,	O
arrows	O
β	B-structure_element
-	I-structure_element
sheets	I-structure_element
and	O
dashed	O
line	O
residues	O
disordered	O
in	O
the	O
structure	B-evidence
.	O

Coenzyme	B-chemical
A	I-chemical
is	O
shown	O
in	O
magenta	O
sticks	O
and	O
Zinc	B-chemical
(	O
grey	O
)	O
as	O
spheres	O
.	O

There	O
are	O
two	O
PduL	B-protein_type
molecules	O
in	O
the	O
asymmetric	O
unit	O
of	O
the	O
P212121	O
unit	O
cell	O
.	O

Structurally	O
,	O
PduL	B-protein_type
consists	O
of	O
two	O
domains	B-structure_element
(	O
Fig	O
2	O
,	O
blue	O
/	O
red	O
),	O
each	O
a	O
beta	B-structure_element
-	I-structure_element
barrel	I-structure_element
that	O
is	O
capped	O
on	O
both	O
ends	O
by	O
short	O
α	B-structure_element
-	I-structure_element
helices	I-structure_element
.	O

Residues	O
in	O
this	O
region	O
(	O
Gln42	B-residue_name_number
,	O
Pro43	B-residue_name_number
,	O
Gly44	B-residue_name_number
),	O
covering	O
the	O
active	B-site
site	I-site
,	O
are	O
strongly	B-protein_state
conserved	I-protein_state
(	O
Fig	O
3	O
).	O

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

The	O
folds	O
of	O
the	O
two	O
chains	O
in	O
the	O
asymmetric	O
unit	O
are	O
very	O
similar	O
,	O
superimposing	B-experimental_method
with	O
a	O
rmsd	B-evidence
of	O
0	O
.	O
16	O
Å	O
over	O
2	O
,	O
306	O
aligned	O
atom	O
pairs	O
.	O

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

The	O
large	O
differences	O
between	O
the	O
anomalous	O
signals	O
confirm	O
the	O
presence	O
of	O
zinc	B-chemical
at	O
both	O
metal	O
sites	O
(	O
S3	O
Fig	O
).	O

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

It	O
has	O
been	O
proposed	O
that	O
the	O
catabolic	B-protein_state
BMCs	B-complex_assembly
may	O
assemble	O
in	O
a	O
core	O
-	O
first	O
manner	O
,	O
with	O
the	O
luminal	O
enzymes	O
(	O
signature	O
enzyme	O
,	O
aldehyde	B-protein_type
,	I-protein_type
and	I-protein_type
alcohol	I-protein_type
dehydrogenases	I-protein_type
and	O
the	O
BMC	B-complex_assembly
PTAC	B-protein_type
)	O
forming	O
an	O
initial	O
bolus	O
,	O
or	O
prometabolosome	O
,	O
around	O
which	O
a	O
shell	B-structure_element
assembles	O
.	O

However	O
,	O
when	O
the	O
putative	O
EP	B-structure_element
(	O
residues	O
1	B-residue_range
–	I-residue_range
27	I-residue_range
)	O
was	O
removed	B-experimental_method
(	O
sPduL	B-mutant
ΔEP	I-mutant
),	O
the	O
truncated	B-protein_state
protein	O
was	O
stable	O
and	O
eluted	O
as	O
a	O
single	O
peak	O
(	O
Fig	O
5a	O
)	O
consistent	O
with	O
the	O
size	O
of	O
a	O
monomer	B-oligomeric_state
(	O
Fig	O
5d	O
,	O
blue	O
curve	O
).	O

rPduL	B-protein
full	B-protein_state
length	I-protein_state
runs	O
as	O
Mw	B-evidence
=	O
140	O
.	O
3	O
kDa	O
+/−	O
1	O
.	O
2	O
%	O
and	O
Mn	B-evidence
=	O
140	O
.	O
5	O
kDa	O
+/−	O
1	O
.	O
2	O
%.	O

Our	O
structure	B-evidence
reveals	O
that	O
the	O
two	O
assigned	O
PF06130	B-structure_element
domains	O
(	O
Fig	O
3	O
)	O
do	O
not	O
form	O
structurally	O
discrete	O
units	O
;	O
this	O
reduces	O
the	O
apparent	O
sequence	O
conservation	O
at	O
the	O
level	O
of	O
primary	O
structure	O
.	O

The	O
closest	O
structural	O
homolog	O
of	O
the	O
PduL	B-protein_type
barrel	B-structure_element
domain	I-structure_element
is	O
a	O
subdomain	O
of	O
a	O
multienzyme	O
complex	O
,	O
the	O
alpha	B-structure_element
subunit	I-structure_element
of	O
ethylbenzene	B-protein_type
dehydrogenase	I-protein_type
(	O
S5b	O
Fig	O
,	O
rmsd	B-evidence
of	O
2	O
.	O
26	O
Å	O
over	O
226	O
aligned	O
atoms	O
consisting	O
of	O
one	O
beta	B-structure_element
barrel	I-structure_element
and	O
one	O
capping	B-structure_element
helix	I-structure_element
).	O

The	O
PduL	B-protein_type
signature	O
primary	O
structure	O
,	O
two	O
PF06130	B-structure_element
domains	O
,	O
occurs	O
in	O
some	O
multidomain	O
proteins	O
,	O
most	O
of	O
them	O
annotated	O
as	O
Acks	B-protein_type
,	O
suggesting	O
that	O
PduL	B-protein_type
may	O
also	O
replace	O
Pta	B-protein_type
in	O
variants	O
of	O
the	O
phosphotransacetylase	B-protein_type
-	O
Ack	B-protein_type
pathway	O
.	O

These	O
PduL	B-protein_type
homologs	O
lack	B-protein_state
EPs	B-structure_element
,	O
and	O
their	B-protein_type
fusion	O
to	O
Ack	B-protein_type
may	O
have	O
evolved	O
as	O
a	O
way	O
to	O
facilitate	O
substrate	O
channeling	O
between	O
the	O
two	O
enzymes	O
.	O

sPduL	B-protein
has	O
also	O
previously	O
been	O
reported	O
to	O
localize	O
to	O
inclusion	O
bodies	O
when	O
overexpressed	B-experimental_method
;	O
we	O
show	O
here	O
that	O
this	O
is	O
dependent	O
on	O
the	O
presence	O
of	O
the	O
EP	B-structure_element
.	O

Structured	O
aggregation	O
of	O
the	O
core	O
enzymes	O
has	O
been	O
proposed	O
to	O
be	O
the	O
initial	O
step	O
in	O
metabolosome	B-complex_assembly
assembly	O
and	O
is	O
known	O
to	O
be	O
the	O
first	O
step	O
of	O
β	O
-	O
carboxysome	O
biogenesis	O
,	O
where	O
the	O
core	O
enzyme	O
Ribulose	B-protein_type
Bisphosphate	I-protein_type
Carboxylase	I-protein_type
/	I-protein_type
Oxygenase	I-protein_type
(	O
RuBisCO	B-protein_type
)	O
is	O
aggregated	O
by	O
the	O
CcmM	B-protein_type
protein	O
.	O

As	O
expected	O
,	O
the	O
amino	O
acid	O
sequence	O
conservation	O
is	O
highest	O
in	O
the	O
region	O
around	O
the	O
proposed	O
active	B-site
site	I-site
(	O
Fig	O
4d	O
);	O
highly	B-protein_state
conserved	I-protein_state
residues	O
are	O
also	O
involved	O
in	O
CoA	B-chemical
binding	O
(	O
Figs	O
2a	O
and	O
3	O
,	O
residues	O
Ser45	B-residue_name_number
,	O
Lys70	B-residue_name_number
,	O
Arg97	B-residue_name_number
,	O
Leu99	B-residue_name_number
,	O
His204	B-residue_name_number
,	O
Asn211	B-residue_name_number
).	O

In	O
contrast	O
,	O
in	O
the	O
rPduL	B-protein
structure	B-evidence
,	O
there	O
are	O
no	O
conserved	O
aspartate	B-residue_name
residues	O
in	O
or	O
around	O
the	O
active	B-site
site	I-site
,	O
and	O
the	O
only	O
well	B-protein_state
-	I-protein_state
conserved	I-protein_state
glutamate	B-residue_name
residue	O
in	O
the	O
active	B-site
site	I-site
is	O
involved	O
in	O
coordinating	O
one	O
of	O
the	O
metal	O
ions	O
.	O

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

Our	O
structures	B-evidence
provide	O
the	O
foundation	O
for	O
studies	O
to	O
elucidate	O
the	O
details	O
of	O
the	O
catalytic	O
mechanism	O
of	O
PduL	B-protein_type
.	O
Conserved	B-protein_state
residues	O
in	O
the	O
active	B-site
site	I-site
that	O
may	O
contribute	O
to	O
substrate	O
binding	O
and	O
/	O
or	O
transition	O
state	O
stabilization	O
include	O
Ser127	B-residue_name_number
,	O
Arg103	B-residue_name_number
,	O
Arg194	B-residue_name_number
,	O
Gln107	B-residue_name_number
,	O
Gln74	B-residue_name_number
,	O
and	O
Gln	B-residue_name_number
/	O
Glu77	B-residue_name_number
.	O

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

PduL	B-protein_type
and	O
Pta	B-protein_type
are	O
mechanistically	O
and	O
structurally	O
distinct	O
enzymes	O
that	O
catalyze	O
the	O
same	O
reaction	O
,	O
a	O
prime	O
example	O
of	O
evolutionary	O
convergence	O
upon	O
a	O
function	O
.	O

However	O
,	O
apparently	O
less	O
frequent	O
is	O
functional	O
convergence	O
that	O
is	O
supported	O
by	O
distinctly	O
different	O
active	B-site
sites	I-site
and	O
accordingly	O
catalytic	O
mechanism	O
,	O
as	O
revealed	O
by	O
comparison	O
of	O
the	O
structures	O
of	O
Pta	B-protein_type
and	O
PduL	B-protein_type
.	O
One	O
well	O
-	O
studied	O
example	O
of	O
this	O
is	O
the	O
β	B-protein_type
-	I-protein_type
lactamase	I-protein_type
family	O
of	O
enzymes	O
,	O
in	O
which	O
the	O
active	B-site
site	I-site
of	O
Class	O
A	O
and	O
Class	O
C	O
enzymes	O
involve	O
serine	O
-	O
based	O
catalysis	O
,	O
but	O
Class	O
B	O
enzymes	O
are	O
metalloproteins	B-protein_type
.	O

This	O
is	O
not	O
surprising	O
,	O
as	O
β	B-protein_type
-	I-protein_type
lactamases	I-protein_type
are	O
not	O
so	O
widespread	O
among	O
bacteria	B-taxonomy_domain
and	O
therefore	O
would	O
be	O
expected	O
to	O
have	O
evolved	O
independently	O
several	O
times	O
as	O
a	O
defense	O
mechanism	O
against	O
β	O
-	O
lactam	O
antibiotics	O
.	O

Cysteine	B-protein_type
peptidases	I-protein_type
play	O
crucial	O
roles	O
in	O
the	O
virulence	O
of	O
bacterial	B-taxonomy_domain
and	O
other	O
eukaryotic	B-taxonomy_domain
pathogens	O
.	O

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

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

Six	O
of	O
the	O
central	O
β	B-structure_element
-	I-structure_element
strands	I-structure_element
in	O
PmC11	B-protein
(	O
β1	B-structure_element
–	I-structure_element
β2	I-structure_element
and	O
β5	B-structure_element
–	I-structure_element
β8	I-structure_element
)	O
share	O
the	O
same	O
topology	O
as	O
the	O
six	B-structure_element
-	I-structure_element
stranded	I-structure_element
β	I-structure_element
-	I-structure_element
sheet	I-structure_element
found	O
in	O
caspases	B-protein_type
,	O
with	O
strands	B-structure_element
β3	B-structure_element
,	O
β4	B-structure_element
,	O
and	O
β9	B-structure_element
located	O
on	O
the	O
outside	O
of	O
this	O
core	B-structure_element
structure	I-structure_element
(	O
Fig	O
.	O
1B	O
,	O
box	O
).	O

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

B	O
,	O
size	B-experimental_method
exclusion	I-experimental_method
chromatography	I-experimental_method
of	O
PmC11	B-protein
.	O

PmC11C179A	O
(	O
20	O
μg	O
)	O
was	O
incubated	O
overnight	O
at	O
37	O
°	O
C	O
with	O
increasing	O
amounts	O
of	O
processed	O
PmC11	B-protein
and	O
analyzed	O
on	O
a	O
10	O
%	O
SDS	B-experimental_method
-	I-experimental_method
PAGE	I-experimental_method
gel	O
.	O

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

Incubation	B-experimental_method
of	O
PmC11	B-protein
at	O
37	O
°	O
C	O
for	O
16	O
h	O
,	O
resulted	O
in	O
a	O
fully	B-protein_state
processed	I-protein_state
enzyme	O
that	O
remained	O
as	O
an	O
intact	B-protein_state
monomer	B-oligomeric_state
when	O
applied	O
to	O
a	O
size	O
-	O
exclusion	O
column	O
(	O
Fig	O
.	O
2B	O
).	O

The	O
single	O
cleavage	B-site
site	I-site
of	O
PmC11	B-protein
at	O
Lys147	B-residue_name_number
is	O
found	O
immediately	O
after	O
α3	B-structure_element
,	O
in	O
loop	B-structure_element
L5	B-structure_element
within	O
the	O
central	O
β	B-structure_element
-	I-structure_element
sheet	I-structure_element
(	O
Figs	O
.	O
1	O
,	O
A	O
and	O
B	O
,	O
and	O
2A	O
).	O

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

Initial	O
SDS	B-experimental_method
-	I-experimental_method
PAGE	I-experimental_method
and	O
Western	B-experimental_method
blot	I-experimental_method
analysis	O
of	O
both	O
mutants	O
revealed	O
no	O
discernible	O
processing	O
occurred	O
as	O
compared	O
with	O
active	B-protein_state
PmC11	B-protein
(	O
Fig	O
.	O
2C	O
).	O

C	O
,	O
divalent	O
cations	O
do	O
not	O
increase	O
the	O
activity	O
of	O
PmC11	B-protein
.	O

Furthermore	O
,	O
Cu2	B-chemical
+,	I-chemical
Fe2	B-chemical
+,	I-chemical
and	O
Zn2	B-chemical
+	I-chemical
appear	O
to	O
inhibit	B-protein_state
PmC11	B-protein
.	O

In	O
addition	O
,	O
the	O
predicted	O
primary	O
S1	B-site
-	I-site
binding	I-site
residue	I-site
in	O
PmC11	B-protein
Asp177	B-residue_name_number
also	O
overlays	B-experimental_method
with	O
the	O
residue	O
predicted	O
to	O
be	O
the	O
P1	B-site
specificity	I-site
determining	I-site
residue	I-site
in	O
clostripain	B-protein
(	O
Asp229	B-residue_name_number
,	O
Fig	O
.	O
1A	O
).	O

Surprisingly	O
,	O
Ca2	B-chemical
+	I-chemical
did	O
not	O
enhance	O
PmC11	B-protein
activity	O
and	O
,	O
furthermore	O
,	O
other	O
divalent	O
cations	O
,	O
Mg2	B-chemical
+,	I-chemical
Mn2	B-chemical
+,	I-chemical
Co2	B-chemical
+,	I-chemical
Fe2	B-chemical
+,	I-chemical
Zn2	B-chemical
+,	I-chemical
and	O
Cu2	B-chemical
+,	I-chemical
were	O
not	O
necessary	O
for	O
PmC11	B-protein
activity	O
(	O
Fig	O
.	O
3D	O
).	O

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

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

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

Here	O
,	O
we	O
report	O
the	O
crystal	B-evidence
structures	I-evidence
of	O
YfiB	B-protein
alone	B-protein_state
and	O
of	O
an	O
active	B-protein_state
mutant	B-protein_state
YfiBL43P	B-mutant
complexed	B-protein_state
with	I-protein_state
YfiR	B-protein
with	O
2	O
:	O
2	O
stoichiometry	O
.	O

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

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

It	O
has	O
been	O
reported	O
that	O
the	O
activation	O
of	O
YfiN	B-protein
may	O
be	O
induced	O
by	O
redox	O
-	O
driven	O
misfolding	O
of	O
YfiR	B-protein
(	O
Giardina	O
et	O
al	O
.,;	O
Malone	O
et	O
al	O
.,;	O
Malone	O
et	O
al	O
.,).	O

It	O
is	O
also	O
proposed	O
that	O
the	O
sequestration	O
of	O
YfiR	B-protein
by	O
YfiB	B-protein
can	O
be	O
induced	O
by	O
certain	O
YfiB	B-protein
-	O
mediated	O
cell	O
wall	O
stress	O
,	O
and	O
mutagenesis	B-experimental_method
studies	I-experimental_method
revealed	O
a	O
number	O
of	O
activation	B-structure_element
residues	I-structure_element
of	O
YfiB	B-protein
that	O
were	O
located	O
in	O
close	O
proximity	O
to	O
the	O
predicted	B-protein_state
first	B-structure_element
helix	I-structure_element
of	O
the	O
periplasmic	B-structure_element
domain	I-structure_element
(	O
Malone	O
et	O
al	O
.,).	O

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

Compared	O
with	O
the	O
reported	O
complex	O
structure	O
,	O
YfiBL43P	B-mutant
in	O
our	O
YfiB	B-complex_assembly
-	I-complex_assembly
YfiR	I-complex_assembly
complex	O
structure	B-evidence
has	O
additional	O
visible	O
N	O
-	O
terminal	O
residues	O
44	B-residue_range
–	I-residue_range
58	I-residue_range
that	O
are	O
shown	O
to	O
play	O
essential	O
roles	O
in	O
YfiB	B-protein
activation	O
and	O
biofilm	O
formation	O
.	O

In	O
addition	O
,	O
we	O
found	O
that	O
the	O
YfiBL43P	B-mutant
shows	O
a	O
much	O
higher	O
PG	B-evidence
-	I-evidence
binding	I-evidence
affinity	I-evidence
than	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
YfiB	B-protein
,	O
most	O
likely	O
due	O
to	O
its	O
more	O
compact	O
PG	B-site
-	I-site
binding	I-site
pocket	I-site
.	O

Overall	O
structure	B-evidence
of	O
YfiB	B-protein

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

As	O
expected	O
,	O
both	O
mutants	O
form	O
a	O
stable	B-protein_state
complex	B-protein_state
with	I-protein_state
YfiR	B-protein
.	O
Finally	O
,	O
we	O
crystalized	B-experimental_method
YfiR	B-protein
in	B-protein_state
complex	I-protein_state
with	I-protein_state
the	O
YfiBL43P	B-mutant
mutant	B-protein_state
and	O
solved	O
the	O
structure	B-evidence
at	O
1	O
.	O
78	O
Å	O
resolution	O
by	O
molecular	B-experimental_method
replacement	I-experimental_method
using	O
YfiR	B-protein
and	O
YfiB	B-protein
as	O
models	O
.	O

The	O
YfiB	B-site
-	I-site
YfiR	I-site
interface	I-site
can	O
be	O
divided	O
into	O
two	O
regions	O
(	O
Fig	O
.	O
3A	O
and	O
3D	O
).	O

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

Apo	B-protein_state
YfiB	B-protein
is	O
shown	O
in	O
yellow	O
and	O
YfiR	B-protein_state
-	I-protein_state
bound	I-protein_state
YfiBL43P	B-mutant
in	O
cyan	O
.	O
(	O
E	O
and	O
F	O
)	O
MST	B-experimental_method
data	O
and	O
analysis	O
for	O
binding	B-evidence
affinities	I-evidence
of	O
(	O
E	O
)	O
YfiB	B-protein
wild	B-protein_state
-	I-protein_state
type	I-protein_state
and	O
(	O
F	O
)	O
YfiBL43P	B-mutant
with	O
PG	B-chemical
.	O
(	O
G	O
)	O
The	O
sequence	B-experimental_method
alignment	I-experimental_method
of	O
P	B-species
.	I-species
aeruginosa	I-species
and	O
E	B-species
.	I-species
coli	I-species
sources	O
of	O
YfiB	B-protein
,	O
Pal	B-protein_type
and	O
the	O
periplasmic	B-structure_element
domain	I-structure_element
of	O
OmpA	B-protein_type

In	O
parallel	O
,	O
to	O
better	O
understand	O
the	O
putative	O
functional	O
role	O
of	O
VB6	B-chemical
and	O
L	B-chemical
-	I-chemical
Trp	I-chemical
,	O
yfiB	B-gene
was	O
deleted	B-experimental_method
in	O
a	O
PAO1	B-species
wild	B-protein_state
-	I-protein_state
type	I-protein_state
strain	O
,	O
and	O
a	O
construct	B-experimental_method
expressing	I-experimental_method
the	O
YfiBL43P	B-mutant
mutant	B-protein_state
was	O
transformed	B-experimental_method
into	I-experimental_method
the	O
PAO1	B-species
ΔyfiB	B-mutant
strain	O
to	O
trigger	O
YfiBL43P	B-mutant
-	O
induced	O
biofilm	O
formation	O
.	O

To	O
answer	O
the	O
question	O
whether	O
competition	O
of	O
VB6	B-chemical
or	O
L	B-chemical
-	I-chemical
Trp	I-chemical
for	O
the	O
YfiB	B-protein
F57	B-site
-	I-site
binding	I-site
pocket	I-site
of	O
YfiR	B-protein
plays	O
an	O
essential	O
role	O
in	O
inhibiting	O
biofilm	O
formation	O
,	O
we	O
measured	O
the	O
binding	B-evidence
affinities	I-evidence
of	O
VB6	B-chemical
and	O
L	B-chemical
-	I-chemical
Trp	I-chemical
for	O
YfiR	B-protein
via	O
BIAcore	B-experimental_method
experiments	O
.	O

In	O
addition	O
to	O
the	O
preceding	B-residue_range
8	I-residue_range
aa	I-residue_range
loop	B-structure_element
(	O
from	O
the	O
lipid	O
acceptor	O
Cys26	B-residue_range
to	I-residue_range
Gly34	I-residue_range
),	O
the	O
full	B-protein_state
length	I-protein_state
of	O
the	O
periplasmic	O
portion	O
of	O
apo	B-protein_state
YfiB	B-protein
can	O
reach	O
approximately	O
60	O
Å	O
.	O
It	O
was	O
reported	O
that	O
the	O
distance	O
between	O
the	O
outer	O
membrane	O
and	O
the	O
cell	O
wall	O
is	O
approximately	O
50	O
Å	O
and	O
that	O
the	O
thickness	O
of	O
the	O
PG	O
layer	O
is	O
approximately	O
70	O
Å	O
(	O
Matias	O
et	O
al	O
.,).	O

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

Regulatory	O
model	O
of	O
the	O
YfiBNR	B-complex_assembly
tripartite	B-protein_state
system	O
.	O

The	O
lipid	O
acceptor	O
Cys26	B-residue_name_number
is	O
indicated	O
as	O
blue	O
ball	O
.	O

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

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

These	O
results	O
,	O
together	O
with	O
our	O
observation	O
that	O
activated	B-protein_state
YfiB	B-protein
has	O
a	O
much	O
higher	O
cell	B-evidence
wall	I-evidence
binding	I-evidence
affinity	I-evidence
,	O
and	O
previous	O
mutagenesis	O
data	O
showing	O
that	O
(	O
1	O
)	O
both	O
PG	B-chemical
binding	O
and	O
membrane	O
anchoring	O
are	O
required	O
for	O
YfiB	B-protein
activity	O
and	O
(	O
2	O
)	O
activating	O
mutations	O
possessing	O
an	O
altered	O
N	O
-	O
terminal	O
loop	B-structure_element
length	O
are	O
dominant	O
over	O
the	O
loss	O
of	O
PG	B-chemical
binding	O
(	O
Malone	O
et	O
al	O
.,),	O
suggest	O
an	O
updated	O
regulatory	O
model	O
of	O
the	O
YfiBNR	B-complex_assembly
system	O
(	O
Fig	O
.	O
7	O
).	O

In	O
this	O
model	O
,	O
in	O
response	O
to	O
a	O
particular	O
cell	O
stress	O
that	O
is	O
yet	O
to	O
be	O
identified	O
,	O
the	O
dimeric	B-oligomeric_state
YfiB	B-protein
is	O
activated	B-protein_state
from	O
a	O
compact	B-protein_state
,	O
inactive	B-protein_state
conformation	B-protein_state
to	O
a	O
stretched	B-protein_state
conformation	I-protein_state
,	O
which	O
possesses	O
increased	O
PG	B-chemical
binding	O
affinity	O
.	O

A	O
regulatory	B-structure_element
loop	I-structure_element
,	O
which	O
is	O
phosphorylated	B-protein_state
at	O
the	O
key	O
functional	O
phosphorylation	B-site
site	I-site
of	O
fungal	B-taxonomy_domain
ACC	B-protein_type
,	O
wedges	O
into	O
a	O
crevice	O
between	O
two	O
domains	O
of	O
CD	B-structure_element
.	O

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

By	O
catalysing	O
this	O
rate	O
-	O
limiting	O
step	O
in	O
fatty	O
-	O
acid	O
biosynthesis	O
,	O
ACC	B-protein_type
plays	O
a	O
key	O
role	O
in	O
anabolic	O
metabolism	O
.	O

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

Human	B-species
ACC	B-protein_type
occurs	O
in	O
two	O
closely	O
related	O
isoforms	B-protein_state
,	O
ACC1	B-protein
and	O
2	B-protein
,	O
located	O
in	O
the	O
cytosol	O
and	O
at	O
the	O
outer	O
mitochondrial	O
membrane	O
,	O
respectively	O
.	O

However	O
,	O
crystal	B-evidence
structures	I-evidence
of	O
individual	O
components	O
or	O
domains	O
from	O
prokaryotic	B-taxonomy_domain
and	O
eukaryotic	B-taxonomy_domain
ACCs	B-protein_type
,	O
respectively	O
,	O
have	O
been	O
solved	O
.	O

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

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

First	O
,	O
we	O
focused	O
on	O
structure	B-experimental_method
determination	I-experimental_method
of	O
the	O
82	O
-	O
kDa	O
CD	B-structure_element
.	O

The	O
overall	O
extent	O
of	O
the	O
SceCD	B-species
is	O
70	O
by	O
75	O
Å	O
(	O
Fig	O
.	O
1b	O
and	O
Supplementary	O
Fig	O
.	O
1a	O
,	O
b	O
),	O
and	O
the	O
attachment	O
points	O
of	O
the	O
N	O
-	O
terminal	O
26	B-structure_element
-	I-structure_element
residue	I-structure_element
linker	I-structure_element
to	O
the	O
BCCP	B-structure_element
domain	O
and	O
the	O
C	O
-	O
terminal	O
CT	B-structure_element
domain	O
are	O
separated	O
by	O
46	O
Å	O
(	O
the	O
N	O
-	O
and	O
C	O
termini	O
are	O
indicated	O
with	O
spheres	O
in	O
Fig	O
.	O
1b	O
).	O

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

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

MS	B-experimental_method
analysis	O
of	O
dissolved	B-experimental_method
crystals	I-experimental_method
confirmed	O
the	O
phosphorylated	B-protein_state
state	O
of	O
Ser1157	B-residue_name_number
also	O
in	O
SceCD	B-species
crystals	B-evidence
.	O

Owing	O
to	O
the	O
limited	O
resolution	O
the	O
discussion	O
of	O
structures	B-evidence
of	O
CthCD	B-mutant
-	I-mutant
CT	I-mutant
and	O
CthΔBCCP	B-mutant
is	O
restricted	O
to	O
the	O
analysis	O
of	O
domain	O
localization	O
.	O

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

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

The	O
CDN	B-structure_element
domain	O
positioning	O
relative	O
to	O
CDL	B-structure_element
/	O
CDC1	B-structure_element
is	O
highly	O
variable	O
with	O
three	O
main	O
orientations	O
observed	O
in	O
the	O
structures	B-evidence
of	O
SceCD	B-species
and	O
the	O
larger	B-mutant
CthACC	I-mutant
fragments	I-mutant
:	O
CDN	B-structure_element
tilts	O
,	O
resulting	O
in	O
a	O
displacement	O
of	O
its	O
N	O
terminus	O
by	O
23	O
Å	O
(	O
Fig	O
.	O
4a	O
,	O
observed	O
in	O
both	O
protomers	B-oligomeric_state
of	O
CthCD	B-mutant
-	I-mutant
CT	I-mutant
and	O
one	O
protomer	B-oligomeric_state
of	O
CthΔBCCP	B-mutant
,	O
denoted	O
as	O
CthCD	B-mutant
-	I-mutant
CT1	I-mutant
/	I-mutant
2	I-mutant
and	O
CthΔBCCP1	B-mutant
,	O
respectively	O
).	O

Most	O
recently	O
,	O
a	O
crystal	B-evidence
structure	I-evidence
of	O
near	B-protein_state
full	I-protein_state
-	I-protein_state
length	I-protein_state
non	B-protein_state
-	I-protein_state
phosphorylated	I-protein_state
ACC	B-protein_type
from	O
S	B-species
.	I-species
cerevisae	I-species
(	O
lacking	B-protein_state
only	I-protein_state
21	B-residue_range
N	O
-	O
terminal	O
amino	O
acids	O
,	O
here	O
denoted	O
as	O
flACC	B-mutant
)	O
was	O
published	O
by	O
Wei	O
and	O
Tong	O
.	O

In	O
flACC	B-mutant
,	O
the	O
ACC	B-protein_type
dimer	B-oligomeric_state
obeys	O
twofold	O
symmetry	O
and	O
assembles	O
in	O
a	O
triangular	B-protein_state
architecture	I-protein_state
with	O
dimeric	B-oligomeric_state
BC	B-structure_element
domains	O
(	O
Supplementary	O
Fig	O
.	O
5a	O
).	O

Comparison	B-experimental_method
of	O
flACC	B-mutant
with	O
our	O
CthΔBCCP	B-mutant
structure	B-evidence
reveals	O
the	O
CDC2	B-structure_element
/	I-structure_element
CT	I-structure_element
hinge	I-structure_element
as	O
a	O
major	O
contributor	O
to	O
conformational	O
flexibility	O
(	O
Supplementary	O
Fig	O
.	O
5b	O
,	O
c	O
).	O

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

Applying	B-experimental_method
the	O
conformation	O
of	O
the	O
CDC1	B-structure_element
/	I-structure_element
CDC2	I-structure_element
hinge	I-structure_element
observed	O
in	O
SceCD	B-species
on	O
flACC	B-mutant
leads	O
to	O
CDN	B-structure_element
sterically	O
clashing	O
with	O
CDC2	B-structure_element
and	O
BT	B-structure_element
/	O
CDN	B-structure_element
clashing	O
with	O
CT	B-structure_element
(	O
Supplementary	O
Fig	O
.	O
6a	O
,	O
b	O
).	O

In	O
addition	O
,	O
EM	B-experimental_method
micrographs	B-evidence
of	O
phosphorylated	B-protein_state
and	O
dephosphorylated	B-protein_state
SceACC	B-protein
display	O
for	O
both	O
samples	O
mainly	O
elongated	B-protein_state
and	I-protein_state
U	I-protein_state
-	I-protein_state
shaped	I-protein_state
conformations	I-protein_state
and	O
reveal	O
no	O
apparent	O
differences	O
in	O
particle	B-evidence
shape	I-evidence
distributions	I-evidence
(	O
Supplementary	O
Fig	O
.	O
7	O
).	O

It	O
disfavours	O
the	O
adoption	O
of	O
a	O
rare	B-protein_state
,	I-protein_state
compact	I-protein_state
conformation	I-protein_state
,	O
in	O
which	O
intramolecular	O
dimerization	O
of	O
the	O
BC	B-structure_element
domains	O
results	O
in	O
catalytic	O
turnover	O
.	O

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

(	O
a	O
)	O
Hinge	B-structure_element
properties	O
of	O
the	O
CDC2	B-structure_element
–	I-structure_element
CT	I-structure_element
connection	I-structure_element
analysed	O
by	O
a	O
CT	B-experimental_method
-	I-experimental_method
based	I-experimental_method
superposition	I-experimental_method
of	O
eight	O
instances	O
of	O
the	O
CDC2	B-mutant
-	I-mutant
CT	I-mutant
segment	I-mutant
.	O

The	O
range	O
of	O
hinge	O
bending	O
is	O
indicated	O
and	O
the	O
connection	O
points	O
between	O
CDC2	B-structure_element
and	O
CT	B-structure_element
(	O
blue	O
)	O
as	O
well	O
as	O
between	O
CDC1	B-structure_element
and	O
CDC2	B-structure_element
(	O
green	O
and	O
grey	O
)	O
are	O
marked	O
as	O
spheres	O
.	O

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

Comparison	B-experimental_method
with	O
each	O
other	O
and	O
with	O
available	O
structures	B-evidence
uncovers	O
differences	O
between	O
LdcI	B-protein
and	O
LdcC	B-protein
explaining	O
why	O
only	O
the	O
acid	B-protein_type
stress	I-protein_type
response	I-protein_type
enzyme	I-protein_type
is	O
capable	O
of	O
binding	O
RavA	B-protein
.	O
We	O
identify	O
interdomain	O
movements	O
associated	O
with	O
the	O
pH	B-protein_state
-	I-protein_state
dependent	I-protein_state
enzyme	O
activation	O
and	O
with	O
the	O
RavA	B-protein
binding	O
.	O

Enterobacterial	B-taxonomy_domain
inducible	B-protein_state
decarboxylases	B-protein_type
of	O
basic	B-protein_state
amino	B-chemical
acids	I-chemical
lysine	B-residue_name
,	O
arginine	B-residue_name
and	O
ornithine	B-residue_name
have	O
a	O
common	O
evolutionary	O
origin	O
and	O
belong	O
to	O
the	O
α	B-protein_type
-	I-protein_type
family	I-protein_type
of	O
pyridoxal	B-chemical
-	I-chemical
5	I-chemical
′-	I-chemical
phosphate	I-chemical
(	O
PLP	B-chemical
)-	O
dependent	O
enzymes	O
.	O

Each	O
decarboxylase	B-protein_type
is	O
induced	O
by	O
an	O
excess	O
of	O
the	O
target	O
amino	B-chemical
acid	I-chemical
and	O
a	O
specific	O
range	O
of	O
extracellular	O
pH	O
,	O
and	O
works	O
in	O
conjunction	O
with	O
a	O
cognate	O
inner	B-protein_type
membrane	I-protein_type
antiporter	I-protein_type
.	O

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

This	O
effect	O
is	O
attributed	O
to	O
cadaverine	B-chemical
,	O
the	O
diamine	O
produced	O
by	O
decarboxylation	O
of	O
lysine	B-residue_name
by	O
LdcI	B-protein
and	O
LdcC	B-protein
,	O
that	O
was	O
shown	O
to	O
enhance	O
UPEC	B-species
colonisation	O
of	O
the	O
bladder	O
.	O

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

Each	O
monomer	B-oligomeric_state
is	O
composed	O
of	O
three	O
domains	O
–	O
an	O
N	O
-	O
terminal	O
wing	B-structure_element
domain	I-structure_element
(	O
residues	O
1	B-residue_range
–	I-residue_range
129	I-residue_range
),	O
a	O
PLP	B-structure_element
-	I-structure_element
binding	I-structure_element
core	I-structure_element
domain	I-structure_element
(	O
residues	O
130	B-residue_range
–	I-residue_range
563	I-residue_range
),	O
and	O
a	O
C	B-structure_element
-	I-structure_element
terminal	I-structure_element
domain	I-structure_element
(	O
CTD	B-structure_element
,	O
residues	O
564	B-residue_range
–	I-residue_range
715	I-residue_range
).	O

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

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

As	O
a	O
first	O
step	O
of	O
a	O
comparative	O
analysis	O
,	O
we	O
superimposed	B-experimental_method
the	O
three	O
cryoEM	B-experimental_method
reconstructions	B-evidence
(	O
LdcIa	B-protein
,	O
LdcI	B-complex_assembly
-	I-complex_assembly
LARA	I-complex_assembly
and	O
LdcC	B-protein
)	O
and	O
the	O
crystal	B-evidence
structure	I-evidence
of	O
the	O
LdcIi	B-protein
decamer	B-oligomeric_state
(	O
Fig	O
.	O
2	O
and	O
Movie	O
S1	O
).	O

This	O
superposition	B-experimental_method
reveals	O
that	O
the	O
densities	B-evidence
lining	O
the	O
central	B-structure_element
hole	I-structure_element
of	O
the	O
toroid	B-structure_element
are	O
roughly	O
at	O
the	O
same	O
location	O
,	O
while	O
the	O
rest	O
of	O
the	O
structure	B-evidence
exhibits	O
noticeable	O
changes	O
.	O

In	O
addition	O
,	O
our	O
earlier	O
biochemical	B-experimental_method
observation	I-experimental_method
that	O
the	O
enzymatic	O
activity	O
of	O
LdcIa	B-protein
is	O
unaffected	O
by	O
RavA	B-protein
binding	O
is	O
consistent	O
with	O
the	O
relatively	O
small	O
changes	O
undergone	O
by	O
the	O
active	B-site
site	I-site
upon	O
transition	O
from	O
LdcIa	B-protein
to	O
LdcI	B-complex_assembly
-	I-complex_assembly
LARA	I-complex_assembly
.	O

Rearrangements	O
of	O
the	O
ppGpp	B-site
binding	I-site
pocket	I-site
upon	O
pH	B-protein_state
-	I-protein_state
dependent	I-protein_state
enzyme	O
activation	O
and	O
LARA	B-structure_element
binding	O

Indeed	O
,	O
all	O
CTDs	B-structure_element
have	O
very	O
similar	O
structures	O
(	O
RMSDmin	B-evidence
<	O
1	O
Å	O
).	O

In	O
our	O
previous	O
contribution	O
,	O
based	O
on	O
the	O
fit	O
of	O
the	O
LdcIi	B-protein
and	O
the	O
LARA	B-structure_element
crystal	B-evidence
structures	I-evidence
into	O
the	O
LdcI	B-complex_assembly
-	I-complex_assembly
LARA	I-complex_assembly
cryoEM	B-experimental_method
density	B-evidence
,	O
we	O
predicted	O
that	O
the	O
LdcI	B-complex_assembly
-	I-complex_assembly
RavA	I-complex_assembly
interaction	O
should	O
involve	O
the	O
C	O
-	O
terminal	O
two	B-structure_element
-	I-structure_element
stranded	I-structure_element
β	I-structure_element
-	I-structure_element
sheet	I-structure_element
of	O
the	O
LdcI	B-protein
.	O
Our	O
present	O
cryoEM	B-experimental_method
maps	B-evidence
and	O
pseudoatomic	B-evidence
models	I-evidence
provide	O
first	O
structure	O
-	O
based	O
insights	O
into	O
the	O
differences	O
between	O
the	O
inducible	B-protein_state
and	O
the	O
constitutive	B-protein_state
lysine	B-protein_type
decarboxylases	I-protein_type
.	O

Our	O
current	O
analysis	O
shows	O
that	O
Y697	B-residue_name_number
is	O
strictly	B-protein_state
conserved	I-protein_state
in	O
the	O
“	O
LdcI	B-protein_type
-	I-protein_type
like	I-protein_type
”	O
group	O
whereas	O
the	O
“	O
LdcC	B-protein_type
-	I-protein_type
like	I-protein_type
”	O
enzymes	O
always	B-protein_state
have	I-protein_state
a	O
lysine	B-residue_name
in	O
this	O
position	O
;	O
it	O
also	O
uncovers	O
several	O
other	O
residues	O
potentially	O
essential	O
for	O
the	O
interaction	O
with	O
RavA	B-protein
which	O
can	O
now	O
be	O
addressed	O
by	O
site	B-experimental_method
-	I-experimental_method
directed	I-experimental_method
mutagenesis	I-experimental_method
.	O

The	O
conformational	O
rearrangements	O
of	O
LdcI	B-protein
upon	O
enzyme	O
activation	O
and	O
RavA	B-protein
binding	O
revealed	O
in	O
this	O
work	O
,	O
and	O
our	O
amazing	O
finding	O
that	O
the	O
molecular	O
determinant	O
of	O
the	O
LdcI	B-complex_assembly
-	I-complex_assembly
RavA	I-complex_assembly
interaction	O
is	O
the	O
one	O
that	O
straightforwardly	O
determines	O
if	O
a	O
particular	O
enterobacterial	B-taxonomy_domain
lysine	B-protein_type
decarboxylase	I-protein_type
belongs	O
to	O
“	O
LdcI	B-protein_type
-	I-protein_type
like	I-protein_type
”	O
or	O
“	O
LdcC	B-protein_type
-	I-protein_type
like	I-protein_type
”	O
proteins	O
,	O
should	O
give	O
a	O
new	O
impetus	O
to	O
functional	O
studies	O
of	O
the	O
unique	O
LdcI	B-complex_assembly
-	I-complex_assembly
RavA	I-complex_assembly
cage	O
.	O

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

Only	O
one	O
of	O
the	O
two	O
rings	B-structure_element
of	O
the	O
double	B-structure_element
toroid	I-structure_element
is	O
shown	O
for	O
clarity	O
.	O

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

Stretching	O
of	O
the	O
LdcI	B-protein
monomer	B-oligomeric_state
upon	O
pH	B-protein_state
-	I-protein_state
dependent	I-protein_state
enzyme	O
activation	O
and	O
LARA	B-structure_element
binding	O
.	O

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

(	O
A	O
)	O
Maximum	B-evidence
likelihood	I-evidence
tree	I-evidence
with	O
the	O
“	O
LdcC	B-protein_type
-	I-protein_type
like	I-protein_type
”	O
and	O
the	O
“	O
LdcI	B-protein_type
-	I-protein_type
like	I-protein_type
”	O
groups	O
highlighted	O
in	O
green	O
and	O
pink	O
,	O
respectively	O
.	O

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

(	O
C	O
)	O
Signature	O
sequences	O
of	O
LdcI	B-protein
and	O
LdcC	B-protein
in	O
the	O
C	O
-	O
terminal	O
β	B-structure_element
-	I-structure_element
sheet	I-structure_element
.	O

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

The	O
crystal	B-evidence
structure	I-evidence
of	O
phosphorylation	B-protein_state
-	I-protein_state
mimicking	I-protein_state
Mep2	B-mutant
variants	I-mutant
from	O
C	B-species
.	I-species
albicans	I-species
show	O
large	O
conformational	O
changes	O
in	O
a	O
conserved	B-protein_state
and	O
functionally	O
important	O
region	O
of	O
the	O
CTR	B-structure_element
.	O

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

One	O
hypothesis	O
is	O
that	O
downstream	O
signalling	O
is	O
dependent	O
on	O
a	O
specific	O
conformation	O
of	O
the	O
transporter	B-protein_type
.	O

Mep2	B-protein_type
(	B-protein_type
methylammonium	I-protein_type
(	I-protein_type
MA	I-protein_type
)	I-protein_type
permease	I-protein_type
)	I-protein_type
proteins	I-protein_type
are	O
ammonium	B-protein_type
transceptors	I-protein_type
that	O
are	O
ubiquitous	O
in	O
fungi	B-taxonomy_domain
.	O

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

The	O
structures	B-evidence
are	O
similar	O
to	O
each	O
other	O
but	O
show	O
considerable	O
differences	O
to	O
all	O
other	O
ammonium	B-protein_type
transporter	I-protein_type
structures	B-evidence
.	O

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

In	O
the	O
remainder	O
of	O
the	O
manuscript	O
,	O
we	O
will	O
specifically	O
discuss	O
CaMep2	B-protein
due	O
to	O
the	O
superior	O
resolution	O
of	O
the	O
structure	B-evidence
.	O

Moreover	O
,	O
the	O
N	O
terminus	O
of	O
one	O
monomer	B-oligomeric_state
interacts	O
with	O
the	O
extended	O
extracellular	B-structure_element
loop	I-structure_element
ECL5	B-structure_element
of	O
a	O
neighbouring	O
monomer	B-oligomeric_state
.	O

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

However	O
,	O
given	O
that	O
an	O
N	O
-	O
terminal	O
deletion	B-protein_state
mutant	I-protein_state
(	O
2	B-mutant
-	I-mutant
27Δ	I-mutant
)	O
grows	O
as	O
well	O
as	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
(	O
WT	B-protein_state
)	O
Mep2	B-protein
on	O
minimal	O
ammonium	B-chemical
medium	O
(	O
Fig	O
.	O
3	O
and	O
Supplementary	O
Fig	O
.	O
1	O
),	O
the	O
importance	O
of	O
the	O
N	O
terminus	O
for	O
Mep2	B-protein
activity	O
is	O
not	O
clear	O
.	O

In	O
the	O
vicinity	O
of	O
the	O
Mep2	B-protein
channel	B-site
exit	I-site
,	O
the	O
cytoplasmic	O
end	O
of	O
TM2	B-structure_element
has	O
unwound	O
,	O
generating	O
a	O
longer	O
ICL1	B-structure_element
even	O
though	O
there	O
are	O
no	O
insertions	O
in	O
this	O
region	O
compared	O
to	O
the	O
bacterial	B-taxonomy_domain
proteins	O
(	O
Figs	O
2	O
and	O
4	O
).	O

At	O
the	O
C	O
-	O
terminal	O
end	O
of	O
TM1	B-structure_element
,	O
the	O
side	O
-	O
chain	O
hydroxyl	O
group	O
of	O
the	O
relatively	B-protein_state
conserved	I-protein_state
Tyr49	B-residue_name_number
(	O
Tyr53	B-residue_name_number
in	O
ScMep2	B-protein
)	O
makes	O
a	O
strong	O
hydrogen	O
bond	O
with	O
the	O
ɛ2	O
nitrogen	O
atom	O
of	O
the	O
absolutely	B-protein_state
conserved	I-protein_state
His342	B-residue_name_number
of	O
the	O
twin	B-structure_element
-	I-structure_element
His	I-structure_element
motif	I-structure_element
(	O
His348	B-residue_name_number
in	O
ScMep2	B-protein
),	O
closing	O
the	O
channel	B-site
(	O
Figs	O
4	O
and	O
5	O
).	O

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

The	O
closed	B-protein_state
state	O
of	O
the	O
channel	B-site
might	O
also	O
explain	O
why	O
no	B-evidence
density	I-evidence
,	O
which	O
could	O
correspond	O
to	O
ammonium	B-chemical
(	O
or	O
water	B-chemical
),	O
is	O
observed	O
in	O
the	O
hydrophobic	O
part	O
of	O
the	O
Mep2	B-protein
channel	B-site
close	O
to	O
the	O
twin	B-structure_element
-	I-structure_element
His	I-structure_element
motif	I-structure_element
.	O

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

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

The	O
data	O
behind	O
this	O
hypothesis	O
is	O
the	O
observation	O
that	O
a	O
ScMep2	B-protein
449	B-mutant
-	I-mutant
485Δ	I-mutant
deletion	B-protein_state
mutant	I-protein_state
lacking	B-protein_state
the	O
AI	B-structure_element
region	I-structure_element
is	O
highly	B-protein_state
active	I-protein_state
in	O
MA	B-chemical
uptake	O
both	O
in	O
the	O
triple	B-mutant
mepΔ	I-mutant
and	O
triple	B-mutant
mepΔ	I-mutant
npr1Δ	I-mutant
backgrounds	O
,	O
implying	O
that	O
this	O
Mep2	B-mutant
variant	I-mutant
has	O
a	O
constitutively	B-protein_state
open	I-protein_state
channel	B-site
.	O

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

The	O
side	O
-	O
chain	O
hydroxyl	O
of	O
Ser457	B-residue_name_number
/	O
453	B-residue_number
is	O
located	O
in	O
a	O
well	O
-	O
defined	O
electronegative	B-site
pocket	I-site
that	O
is	O
solvent	B-protein_state
accessible	I-protein_state
(	O
Fig	O
.	O
6	O
).	O

The	O
closest	O
atoms	O
to	O
the	O
serine	B-residue_name
hydroxyl	O
group	O
are	O
the	O
backbone	O
carbonyl	O
atoms	O
of	O
Asp419	B-residue_name_number
,	O
Glu420	B-residue_name_number
and	O
Glu421	B-residue_name_number
,	O
which	O
are	O
3	O
–	O
4	O
Å	O
away	O
.	O

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

By	O
contrast	O
,	O
the	O
conserved	B-protein_state
part	O
of	O
the	O
CTR	B-structure_element
has	O
undergone	O
a	O
large	O
conformational	O
change	O
involving	O
formation	O
of	O
a	O
12	B-structure_element
-	I-structure_element
residue	I-structure_element
-	I-structure_element
long	I-structure_element
α	I-structure_element
-	I-structure_element
helix	I-structure_element
from	O
Leu427	B-residue_range
to	I-residue_range
Asp438	I-residue_range
.	O

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

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

The	O
short	B-structure_element
helix	I-structure_element
formed	O
by	O
residues	O
Leu427	B-residue_range
to	I-residue_range
Asp438	I-residue_range
unravels	O
during	O
the	O
simulations	B-experimental_method
to	O
a	O
disordered	B-protein_state
state	O
.	O

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

Interestingly	O
,	O
phosphomimetic	B-mutant
mutations	I-mutant
introduced	O
into	O
one	O
monomer	B-oligomeric_state
inactivate	O
the	O
entire	O
trimer	B-oligomeric_state
,	O
indicating	O
that	O
(	O
i	O
)	O
heterotrimerization	O
occurs	O
and	O
(	O
ii	O
)	O
the	O
CTR	B-structure_element
mediates	O
allosteric	O
regulation	O
of	O
ammonium	B-chemical
transport	O
activity	O
via	O
phosphorylation	B-ptm
.	O

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

However	O
,	O
the	O
absence	B-protein_state
of	I-protein_state
GlnK	B-protein_type
proteins	I-protein_type
in	O
eukaryotes	B-taxonomy_domain
suggests	O
that	O
phosphorylation	B-ptm
-	O
based	O
regulation	O
of	O
ammonium	B-chemical
transport	O
may	O
be	O
widespread	O
.	O

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

Coloured	O
residues	O
are	O
functionally	O
important	O
and	O
correspond	O
to	O
those	O
of	O
the	O
Phe	B-site
gate	I-site
(	O
blue	O
),	O
the	O
binding	B-site
site	I-site
Trp	B-residue_name
residue	O
(	O
magenta	O
)	O
and	O
the	O
twin	O
-	O
His	O
motif	O
(	O
red	O
).	O

(	O
a	O
)	O
The	O
triple	B-mutant
mepΔ	I-mutant
strain	O
(	O
black	O
)	O
and	O
triple	O
mepΔ	O
npr1Δ	O
strain	O
(	O
grey	O
)	O
containing	O
plasmids	O
expressing	O
WT	B-protein_state
and	O
variant	B-mutant
ScMep2	I-mutant
were	O
grown	B-experimental_method
on	I-experimental_method
minimal	I-experimental_method
medium	I-experimental_method
containing	O
1	O
mM	O
ammonium	B-chemical
sulphate	I-chemical
.	O

Channel	O
closures	O
in	O
Mep2	B-protein
.	O

(	O
c	O
)	O
Cytoplasmic	O
view	O
of	O
the	O
Mep2	B-protein
trimer	B-oligomeric_state
indicating	O
the	O
large	O
distance	O
between	O
Ser453	B-residue_name_number
and	O
the	O
channel	B-site
exits	I-site
(	O
circles	O
;	O
Ile52	B-residue_name_number
lining	O
the	O
channel	B-site
exit	I-site
is	O
shown	O
).	O

Side	O
chains	O
for	O
residues	O
452	B-residue_number
and	O
453	B-residue_number
are	O
shown	O
as	O
stick	O
models	O
.	O

(	O
a	O
)	O
In	O
the	O
closed	B-protein_state
,	O
non	B-protein_state
-	I-protein_state
phosphorylated	I-protein_state
state	O
(	O
i	O
),	O
the	O
CTR	B-structure_element
(	O
magenta	O
)	O
and	O
ICL3	B-structure_element
(	O
green	O
)	O
are	O
far	O
apart	O
with	O
the	O
latter	O
blocking	O
the	O
intracellular	O
channel	B-site
exit	I-site
(	O
indicated	O
with	O
a	O
hatched	O
circle	O
).	O

We	O
determined	B-experimental_method
four	I-experimental_method
structures	I-experimental_method
of	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
bound	B-protein_state
to	I-protein_state
Py	B-chemical
-	I-chemical
tract	I-chemical
oligonucleotides	I-chemical
at	O
resolutions	O
between	O
2	O
.	O
0	O
and	O
1	O
.	O
5	O
Å	O
.	O
These	O
structures	B-evidence
together	O
with	O
RNA	B-experimental_method
binding	I-experimental_method
and	I-experimental_method
splicing	I-experimental_method
assays	I-experimental_method
reveal	O
unforeseen	O
roles	O
for	O
U2AF65	B-protein
inter	B-site
-	I-site
domain	I-site
residues	I-site
in	O
recognizing	O
a	O
contiguous	B-structure_element
,	O
nine	O
-	O
nucleotide	B-chemical
Py	B-chemical
tract	I-chemical
.	O

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

Biochemical	B-experimental_method
characterizations	I-experimental_method
of	O
U2AF65	B-protein
demonstrated	O
that	O
tandem	O
RNA	B-structure_element
recognition	I-structure_element
motifs	I-structure_element
(	O
RRM1	B-structure_element
and	O
RRM2	B-structure_element
)	O
recognize	O
the	O
Py	B-chemical
tract	I-chemical
(	O
Fig	O
.	O
1a	O
).	O

Milestone	O
crystal	B-evidence
structures	I-evidence
of	O
the	O
core	B-protein_state
U2AF65	B-protein
RRM1	B-structure_element
and	O
RRM2	B-structure_element
connected	O
by	O
a	O
shortened	B-protein_state
inter	B-structure_element
-	I-structure_element
RRM	I-structure_element
linker	I-structure_element
(	O
dU2AF651	B-mutant
,	I-mutant
2	I-mutant
)	O
detailed	O
a	O
subset	O
of	O
nucleotide	O
interactions	O
with	O
the	O
individual	O
U2AF65	B-protein
RRMs	B-structure_element
.	O

The	O
RNA	B-evidence
affinity	I-evidence
of	O
the	O
minimal	B-protein_state
U2AF651	B-mutant
,	I-mutant
2	I-mutant
domain	O
comprising	O
the	O
core	B-protein_state
RRM1	B-structure_element
–	O
RRM2	B-structure_element
folds	B-structure_element
(	O
U2AF651	B-mutant
,	I-mutant
2	I-mutant
,	O
residues	O
148	B-residue_range
–	I-residue_range
336	I-residue_range
)	O
is	O
relatively	O
weak	O
compared	O
with	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
U2AF65	B-protein
(	O
Fig	O
.	O
1a	O
,	O
b	O
;	O
Supplementary	O
Fig	O
.	O
1	O
).	O

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

Based	O
on	O
dU2AF651	B-mutant
,	I-mutant
2	I-mutant
structures	B-evidence
,	O
we	O
originally	O
hypothesized	O
that	O
the	O
U2AF65	B-protein
RRMs	B-structure_element
would	O
bind	O
the	O
minimal	B-protein_state
seven	O
nucleotides	B-chemical
observed	O
in	O
these	O
structures	B-evidence
.	O

The	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
structures	B-evidence
characterize	O
ribose	B-chemical
(	O
r	B-chemical
)	O
nucleotides	B-chemical
at	O
all	O
of	O
the	O
binding	B-site
sites	I-site
except	O
the	O
seventh	B-residue_number
and	O
eighth	B-residue_number
deoxy	B-chemical
-(	I-chemical
d	I-chemical
)	I-chemical
U	I-chemical
,	O
which	O
are	O
likely	O
to	O
lack	O
2	O
′-	O
hydroxyl	O
contacts	O
based	O
on	O
the	O
RNA	B-protein_state
-	I-protein_state
bound	I-protein_state
dU2AF651	B-mutant
,	I-mutant
2	I-mutant
structure	B-evidence
.	O

The	O
rU6	B-residue_name_number
base	O
edge	O
is	O
relatively	O
solvent	B-protein_state
exposed	I-protein_state
;	O
accordingly	O
,	O
the	O
rU6	B-residue_name_number
hydrogen	O
bonds	O
with	O
U2AF65	B-protein
are	O
water	B-chemical
mediated	O
apart	O
from	O
a	O
single	O
direct	O
interaction	O
by	O
the	O
RRM1	B-structure_element
-	O
N196	B-residue_name_number
side	O
chain	O
.	O

The	O
energetic	O
penalties	O
due	O
to	O
these	O
mutations	O
(	O
ΔΔG	B-evidence
0	O
.	O
8	O
–	O
0	O
.	O
9	O
kcal	O
mol	O
−	O
1	O
)	O
are	O
consistent	O
with	O
the	O
loss	O
of	O
each	O
hydrogen	O
bond	O
with	O
the	O
rU5	B-residue_name_number
base	O
and	O
support	O
the	O
relevance	O
of	O
the	O
central	O
nucleotide	O
interactions	O
observed	O
in	O
the	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
structures	B-evidence
.	O

Despite	O
12	B-experimental_method
concurrent	I-experimental_method
mutations	I-experimental_method
,	O
the	O
AdML	B-gene
RNA	B-evidence
affinity	I-evidence
of	O
the	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
-	I-mutant
12Gly	I-mutant
variant	B-protein_state
was	O
reduced	O
by	O
only	O
three	O
-	O
fold	O
relative	O
to	O
the	O
unmodified	B-protein_state
protein	B-protein
(	O
Fig	O
.	O
4b	O
),	O
which	O
is	O
less	O
than	O
the	O
penalty	O
of	O
the	O
V254P	B-mutant
mutation	O
that	O
disrupts	O
the	O
rU5	B-residue_name_number
hydrogen	O
bond	O
(	O
Fig	O
.	O
3d	O
,	O
i	O
).	O

To	O
test	O
the	O
interplay	O
of	O
the	O
U2AF65	B-protein
inter	B-structure_element
-	I-structure_element
RRM	I-structure_element
linker	I-structure_element
with	O
its	O
N	O
-	O
and	O
C	O
-	O
terminal	O
RRM	B-structure_element
extensions	I-structure_element
,	O
we	O
constructed	B-experimental_method
an	O
internal	O
linker	B-experimental_method
deletion	I-experimental_method
of	O
20	B-residue_range
-	I-residue_range
residues	I-residue_range
within	O
the	O
extended	B-protein_state
RNA	B-structure_element
-	I-structure_element
binding	I-structure_element
domain	I-structure_element
(	O
dU2AF651	B-mutant
,	I-mutant
2L	I-mutant
).	O

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

This	O
difference	O
indicates	O
that	O
the	O
linearly	B-protein_state
distant	I-protein_state
regions	B-structure_element
of	O
the	O
U2AF65	B-protein
primary	O
sequence	O
,	O
including	O
Q147	B-residue_name_number
in	O
the	O
N	O
-	O
terminal	O
RRM1	B-structure_element
extension	I-structure_element
and	O
R227	B-residue_name_number
/	O
V254	B-residue_name_number
in	O
the	O
N	O
-/	O
C	O
-	O
terminal	O
linker	B-structure_element
regions	I-structure_element
at	O
the	O
fifth	B-site
nucleotide	I-site
site	I-site
,	O
cooperatively	O
recognize	O
the	O
Py	B-chemical
tract	I-chemical
.	O

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

The	O
inter	B-structure_element
-	I-structure_element
RRM	I-structure_element
dynamics	O
of	O
U2AF65	B-protein
were	O
followed	O
using	O
FRET	B-experimental_method
between	O
fluorophores	B-chemical
attached	O
to	O
RRM1	B-structure_element
and	O
RRM2	B-structure_element
(	O
Fig	O
.	O
6a	O
,	O
b	O
,	O
Methods	O
).	O

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

Double	O
-	O
cysteine	B-residue_name
variant	B-protein_state
of	O
U2AF651	B-mutant
,	I-mutant
2	I-mutant
was	O
modified	B-experimental_method
with	O
equimolar	O
amount	O
of	O
Cy3	B-chemical
and	O
Cy5	B-chemical
.	O

We	O
first	O
characterized	O
the	O
conformational	O
dynamics	O
spectrum	O
of	O
U2AF65	B-protein
in	O
the	O
absence	B-protein_state
of	I-protein_state
RNA	B-chemical
(	O
Fig	O
.	O
6c	O
,	O
d	O
;	O
Supplementary	O
Fig	O
.	O
7a	O
,	O
b	O
).	O

The	O
FRET	B-evidence
distribution	I-evidence
histogram	I-evidence
built	O
from	O
more	O
than	O
a	O
thousand	O
traces	B-evidence
of	O
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
ligand	B-chemical
showed	O
an	O
extremely	O
broad	O
distribution	O
centred	O
at	O
a	O
FRET	B-evidence
efficiency	I-evidence
of	O
∼	O
0	O
.	O
4	O
(	O
Fig	O
.	O
6d	O
).	O

Despite	O
the	O
large	O
width	O
of	O
the	O
FRET	B-evidence
-	I-evidence
distribution	I-evidence
histogram	I-evidence
,	O
the	O
majority	O
(	O
80	O
%)	O
of	O
traces	B-evidence
that	O
showed	O
fluctuations	O
sampled	O
only	O
two	O
distinct	O
FRET	B-evidence
states	I-evidence
(	O
for	O
example	O
,	O
Supplementary	O
Fig	O
.	O
7a	O
).	O

We	O
introduced	B-experimental_method
an	O
rArA	B-chemical
purine	B-chemical
dinucleotide	I-chemical
within	O
a	O
variant	O
of	O
the	O
AdML	B-gene
Py	B-chemical
tract	I-chemical
(	O
detailed	O
in	O
Methods	O
).	O

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

Notably	O
,	O
a	O
triple	B-protein_state
mutation	I-protein_state
of	O
three	O
residues	O
(	O
V254P	B-mutant
,	O
Q147A	B-mutant
and	O
R227A	B-mutant
)	O
in	O
the	O
respective	O
inter	B-structure_element
-	I-structure_element
RRM	I-structure_element
linker	I-structure_element
,	O
N	B-structure_element
-	I-structure_element
and	I-structure_element
C	I-structure_element
-	I-structure_element
terminal	I-structure_element
extensions	I-structure_element
non	O
-	O
additively	O
reduce	O
RNA	B-evidence
binding	I-evidence
by	O
150	O
-	O
fold	O
.	O

Altogether	O
,	O
these	O
data	O
indicate	O
that	O
interactions	O
among	O
the	O
U2AF65	B-protein
RRM1	B-structure_element
/	O
RRM2	B-structure_element
,	O
inter	B-structure_element
-	I-structure_element
RRM	I-structure_element
linker	I-structure_element
,	O
N	B-structure_element
-	I-structure_element
and	I-structure_element
C	I-structure_element
-	I-structure_element
terminal	I-structure_element
extensions	I-structure_element
are	O
mutually	O
inter	O
-	O
dependent	O
for	O
cognate	O
Py	B-chemical
-	I-chemical
tract	I-chemical
recognition	O
.	O

These	O
transitions	O
could	O
correspond	O
to	O
rearrangement	O
from	O
the	O
‘	O
closed	B-protein_state
'	O
NMR	B-experimental_method
/	O
PRE	B-experimental_method
-	O
based	O
U2AF65	B-protein
conformation	O
in	O
which	O
the	O
RNA	B-site
-	I-site
binding	I-site
surface	I-site
of	O
only	O
a	O
single	B-protein_state
RRM	B-structure_element
is	O
exposed	O
and	O
available	O
for	O
RNA	O
binding	O
,	O
to	O
the	O
structural	O
state	O
seen	O
in	O
the	O
side	B-protein_state
-	I-protein_state
by	I-protein_state
-	I-protein_state
side	I-protein_state
,	O
RNA	B-protein_state
-	I-protein_state
bound	I-protein_state
crystal	B-evidence
structure	I-evidence
.	O

The	O
regions	O
of	O
RRM1	B-structure_element
,	O
RRM2	B-structure_element
and	O
linker	B-structure_element
contacts	O
are	O
indicated	O
above	O
by	O
respective	O
black	O
and	O
blue	O
arrows	O
from	O
N	O
-	O
to	O
C	O
-	O
terminus	O
.	O

Crystallographic	O
statistics	O
are	O
given	O
in	O
Table	O
1	O
and	O
the	O
overall	O
conformations	O
of	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
and	O
prior	O
dU2AF651	B-mutant
,	I-mutant
2	I-mutant
/	O
U2AF651	B-mutant
,	I-mutant
2	I-mutant
structures	B-evidence
are	O
compared	O
in	O
Supplementary	O
Fig	O
.	O
2	O
.	O

Residues	O
V249	B-residue_name_number
,	O
V250	B-residue_name_number
,	O
V254	B-residue_name_number
(	O
yellow	O
)	O
are	O
mutated	B-experimental_method
to	O
V249G	B-mutant
/	O
V250G	B-mutant
/	O
V254G	B-mutant
in	O
the	O
3Gly	B-mutant
mutant	I-mutant
;	O
residues	O
S251	B-residue_name_number
,	O
T252	B-residue_name_number
,	O
V253	B-residue_name_number
,	O
P255	B-residue_name_number
(	O
red	O
)	O
along	O
with	O
V254	B-residue_name_number
are	O
mutated	B-experimental_method
to	O
S251G	B-mutant
/	O
T252G	B-mutant
/	O
V253G	B-mutant
/	O
V254G	B-mutant
/	O
P255G	B-mutant
in	O
the	O
5Gly	B-mutant
mutant	I-mutant
or	O
to	O
S251N	B-mutant
/	O
T252L	B-mutant
/	O
V253A	B-mutant
/	O
V254L	B-mutant
/	O
P255A	B-mutant
in	O
the	O
NLALA	B-mutant
mutant	I-mutant
;	O
residues	O
M144	B-residue_name_number
,	O
L235	B-residue_name_number
,	O
M238	B-residue_name_number
,	O
V244	B-residue_name_number
,	O
V246	B-residue_name_number
(	O
orange	O
)	O
along	O
with	O
V249	B-residue_name_number
,	O
V250	B-residue_name_number
,	O
S251	B-residue_name_number
,	O
T252	B-residue_name_number
,	O
V253	B-residue_name_number
,	O
V254	B-residue_name_number
,	O
P255	B-residue_name_number
are	O
mutated	B-experimental_method
to	O
M144G	B-mutant
/	O
L235G	B-mutant
/	O
M238G	B-mutant
/	O
V244G	B-mutant
/	O
V246G	B-mutant
/	O
V249G	B-mutant
/	O
V250G	B-mutant
/	O
S251G	B-mutant
/	O
T252G	B-mutant
/	O
V253G	B-mutant
/	O
V254G	B-mutant
/	O
P255G	B-mutant
in	O
the	O
12Gly	B-mutant
mutant	I-mutant
.	O

Other	O
linker	B-structure_element
residues	O
are	O
coloured	O
either	O
dark	O
blue	O
for	O
new	O
residues	O
in	O
the	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
structure	O
or	O
light	O
blue	O
for	O
the	O
remaining	O
inter	B-structure_element
-	I-structure_element
RRM	I-structure_element
residues	O
.	O

The	O
central	O
panel	O
shows	O
an	O
overall	O
view	O
with	O
stick	O
diagrams	O
for	O
mutated	O
residues	O
;	O
boxed	O
regions	O
are	O
expanded	O
to	O
show	O
the	O
C	O
-	O
terminal	O
(	O
bottom	O
left	O
)	O
and	O
central	B-structure_element
linker	I-structure_element
regions	I-structure_element
(	O
top	O
)	O
at	O
the	O
inter	B-structure_element
-	I-structure_element
RRM	I-structure_element
interfaces	I-structure_element
,	O
and	O
N	O
-	O
terminal	O
linker	O
region	O
contacts	O
with	O
RRM1	B-structure_element
(	O
bottom	O
right	O
).	O

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

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

(	O
b	O
)	O
Following	O
binding	O
to	O
the	O
Py	B-chemical
-	I-chemical
tract	I-chemical
RNA	I-chemical
,	O
a	O
conformation	O
corresponding	O
to	O
high	B-evidence
FRET	I-evidence
and	O
consistent	O
with	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
apo	B-protein_state
-	O
U2AF65	B-protein
model	O
resulting	O
from	O
PRE	B-experimental_method
/	O
NMR	B-experimental_method
characterization	O
(	O
PDB	O
ID	O
2YH0	O
)	O
often	O
transitions	O
to	O
a	O
conformation	O
corresponding	O
to	O
∼	O
0	O
.	O
45	O
FRET	B-evidence
value	I-evidence
,	O
which	O
is	O
consistent	O
with	O
‘	O
open	B-protein_state
',	O
side	B-protein_state
-	I-protein_state
by	I-protein_state
-	I-protein_state
side	I-protein_state
RRMs	B-structure_element
such	O
as	O
the	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
crystal	B-evidence
structures	I-evidence
.	O

Floral	O
abscission	O
is	O
controlled	O
by	O
the	O
leucine	B-protein_type
-	I-protein_type
rich	I-protein_type
repeat	I-protein_type
receptor	I-protein_type
kinase	I-protein_type
(	O
LRR	B-protein_type
-	I-protein_type
RK	I-protein_type
)	O
HAESA	B-protein
and	O
the	O
peptide	B-protein_type
hormone	I-protein_type
IDA	B-protein
.	O

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

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

However	O
,	O
the	O
molecular	O
details	O
of	O
how	O
IDA	B-protein
triggers	O
organ	O
shedding	O
are	O
not	O
clear	O
.	O

Santiago	O
et	O
al	O
.	O
used	O
protein	B-experimental_method
biochemistry	I-experimental_method
,	O
structural	B-experimental_method
biology	I-experimental_method
and	O
genetics	B-experimental_method
to	O
uncover	O
how	O
the	O
IDA	B-protein
hormone	B-chemical
activates	O
HAESA	B-protein
.	O

HAESA	B-protein
is	O
shown	O
in	O
blue	O
(	O
ribbon	O
diagram	O
),	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
(	O
left	O
panel	O
),	O
the	O
central	O
Hyp	B-structure_element
anchor	I-structure_element
(	O
center	O
)	O
and	O
the	O
N	O
-	O
terminal	O
Pro	B-structure_element
-	I-structure_element
rich	I-structure_element
motif	I-structure_element
in	O
IDA	B-protein
(	O
right	O
panel	O
)	O
are	O
shown	O
in	O
yellow	O
(	O
in	O
bonds	O
representation	O
).	O

Structure	B-experimental_method
-	I-experimental_method
based	I-experimental_method
sequence	I-experimental_method
alignment	I-experimental_method
of	O
the	O
HAESA	B-protein_type
family	I-protein_type
members	I-protein_type
:	O
Arabidopsis	B-species
thaliana	I-species
HAESA	B-protein
(	O
Uniprot	O
(	O
http	O
://	O
www	O
.	O
uniprot	O
.	O
org	O
)	O
ID	O
P47735	O
),	O
Arabidopsis	B-species
thaliana	I-species
HSL2	B-protein
(	O
Uniprot	O
ID	O
C0LGX3	O
),	O
Capsella	B-species
rubella	I-species
HAESA	B-protein
(	O
Uniprot	O
ID	O
R0F2U6	O
),	O
Citrus	B-species
clementina	I-species
HSL2	B-protein
(	O
Uniprot	O
ID	O
V4U227	O
),	O
Vitis	B-species
vinifera	I-species
HAESA	B-protein
(	O
Uniprot	O
ID	O
F6HM39	O
).	O

The	O
alignment	O
includes	O
a	O
secondary	O
structure	O
assignment	O
calculated	O
with	O
the	O
program	O
DSSP	O
and	O
colored	O
according	O
to	O
Figure	O
1	O
,	O
with	O
the	O
N	O
-	O
and	O
C	O
-	O
terminal	O
caps	B-structure_element
and	O
the	O
21	O
LRR	B-structure_element
motifs	I-structure_element
indicated	O
in	O
orange	O
and	O
blue	O
,	O
respectively	O
.	O

HAESA	B-protein
residues	O
interacting	O
with	O
the	O
IDA	B-chemical
peptide	I-chemical
and	O
/	O
or	O
the	O
SERK1	B-protein
co	B-protein_type
-	I-protein_type
receptor	I-protein_type
kinase	I-protein_type
ectodomain	B-structure_element
are	O
highlighted	O
in	O
blue	O
and	O
orange	O
,	O
respectively	O
.	O

The	O
PKGV	B-structure_element
motif	I-structure_element
present	O
in	O
our	O
N	B-protein_state
-	I-protein_state
terminally	I-protein_state
extended	I-protein_state
IDA	B-chemical
peptide	I-chemical
is	O
highlighted	O
in	O
red	O
.	O
(	O
B	O
)	O
Isothermal	B-experimental_method
titration	I-experimental_method
calorimetry	I-experimental_method
of	O
the	O
HAESA	B-protein
ectodomain	B-structure_element
vs	O
.	O
IDA	B-protein
and	O
including	O
the	O
synthetic	B-protein_state
peptide	B-chemical
sequence	O
.	O

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

Active	B-protein_state
IDA	B-protein_type
-	I-protein_type
family	I-protein_type
peptide	I-protein_type
hormones	I-protein_type
are	O
hydroxyprolinated	B-protein_state
dodecamers	B-structure_element
.	O

Void	O
(	O
V0	O
)	O
volume	O
and	O
total	O
volume	O
(	O
Vt	O
)	O
are	O
shown	O
,	O
together	O
with	O
elution	O
volumes	O
for	O
molecular	O
mass	O
standards	O
(	O
A	O
,	O
Thyroglobulin	B-protein
,	O
669	O
,	O
000	O
Da	O
;	O
B	O
,	O
Ferritin	B-protein
,	O
440	O
,	O
00	O
Da	O
,	O
C	O
,	O
Aldolase	B-protein
,	O
158	O
,	O
000	O
Da	O
;	O
D	O
,	O
Conalbumin	B-protein
,	O
75	O
,	O
000	O
Da	O
;	O
E	O
,	O
Ovalbumin	B-protein
,	O
44	O
,	O
000	O
Da	O
;	O
F	O
,	O
Carbonic	B-protein
anhydrase	I-protein
,	O
29	O
,	O
000	O
Da	O
).	O

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

We	O
next	O
determined	O
crystal	B-evidence
structures	I-evidence
of	O
the	O
apo	B-protein_state
HAESA	B-protein
ectodomain	B-structure_element
and	O
of	O
a	O
HAESA	B-complex_assembly
-	I-complex_assembly
IDA	I-complex_assembly
complex	O
,	O
at	O
1	O
.	O
74	O
and	O
1	O
.	O
86	O
Å	O
resolution	O
,	O
respectively	O
(	O
Figure	O
1C	O
;	O
Figure	O
1	O
—	O
figure	O
supplement	O
1B	O
–	O
D	O
;	O
Tables	O
1	O
,	O
2	O
).	O

The	O
COO	O
-	O
group	O
of	O
Asn69IDA	B-residue_name_number
is	O
in	O
direct	O
contact	O
with	O
Arg407HAESA	B-residue_name_number
and	O
Arg409HAESA	B-residue_name_number
and	O
HAESA	B-protein
cannot	O
bind	O
a	O
C	B-protein_state
-	I-protein_state
terminally	I-protein_state
extended	I-protein_state
IDA	B-mutant
-	I-mutant
SFVN	I-mutant
peptide	O
(	O
Figures	O
1D	O
,	O
F	O
,	O
2D	O
).	O

A	O
2	O
.	O
56	O
Å	O
co	B-evidence
-	I-evidence
crystal	I-evidence
structure	I-evidence
with	O
IDL1	B-protein
reveals	O
that	O
different	O
IDA	B-protein_type
family	I-protein_type
members	I-protein_type
use	O
a	O
common	O
binding	O
mode	O
to	O
interact	O
with	O
HAESA	B-protein_type
-	I-protein_type
type	I-protein_type
receptors	I-protein_type
(	O
Figure	O
2A	O
–	O
C	O
,	O
E	O
,	O
Table	O
2	O
).	O

The	O
N	O
-	O
terminal	O
capping	B-structure_element
domain	I-structure_element
of	O
SERK1	B-protein
(	O
in	O
orange	O
)	O
directly	O
contacts	O
the	O
C	O
-	O
terminal	O
part	O
of	O
IDA	B-protein
(	O
in	O
yellow	O
,	O
in	O
bonds	O
representation	O
)	O
and	O
the	O
receptor	B-protein_type
HAESA	B-protein
(	O
in	O
blue	O
).	O

The	O
SERK1	B-protein
ectodomain	B-structure_element
interacts	O
with	O
the	O
IDA	B-site
peptide	I-site
binding	I-site
site	I-site
using	O
a	O
loop	B-structure_element
region	I-structure_element
(	O
residues	O
51	B-residue_range
-	I-residue_range
59SERK1	I-residue_range
)	O
from	O
its	O
N	O
-	O
terminal	O
cap	B-structure_element
(	O
Figure	O
4A	O
,	O
C	O
).	O

SERK1	B-protein
binds	O
HAESA	B-protein
using	O
these	O
two	O
distinct	O
interaction	B-site
surfaces	I-site
(	O
Figure	O
1	O
—	O
figure	O
supplement	O
3	O
),	O
with	O
the	O
N	B-structure_element
-	I-structure_element
cap	I-structure_element
of	O
the	O
SERK1	B-protein
LRR	B-structure_element
domain	I-structure_element
partially	O
covering	O
the	O
IDA	B-site
peptide	I-site
binding	I-site
cleft	I-site
.	O

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

Purified	B-experimental_method
HAESA	B-protein
and	O
SERK1	B-protein
are	O
~	O
75	O
and	O
~	O
28	O
kDa	O
,	O
respectively	O
.	O

Note	O
that	O
the	O
HAESA	B-protein
and	O
SERK1	B-protein
input	O
lanes	O
have	O
already	O
been	O
shown	O
in	O
Figure	O
3D	O
.	O
(	O
B	O
)	O
Isothermal	B-evidence
titration	I-evidence
thermographs	I-evidence
of	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
and	O
mutant	B-protein_state
IDA	B-chemical
peptides	I-chemical
titrated	B-experimental_method
into	O
a	O
HAESA	B-protein
-	O
SERK1	B-protein
mixture	O
in	O
the	O
cell	O
.	O

Table	O
summaries	O
for	O
calorimetric	B-evidence
binding	I-evidence
constants	I-evidence
and	O
stoichoimetries	O
for	O
different	O
IDA	B-chemical
peptides	I-chemical
binding	O
to	O
the	O
HAESA	B-protein
–	O
SERK1	B-protein
ectodomain	B-structure_element
mixture	O
(	O
±	O
fitting	O
errors	O
;	O
n	O
.	O
d	O
.	O

Up	O
to	O
inflorescence	O
position	O
4	O
,	O
petal	O
break	O
in	O
35S	B-gene
::	O
IDA	B-mutant
K66A	I-mutant
/	I-mutant
R67A	I-mutant
mutant	B-protein_state
plants	B-taxonomy_domain
was	O
significantly	O
increased	O
compared	O
to	O
both	O
Col	O
-	O
0	O
control	O
plants	B-taxonomy_domain
(	O
b	O
)	O
and	O
35S	B-gene
::	O
IDA	B-protein
plants	B-taxonomy_domain
(	O
c	O
)	O
(	O
D	O
)	O
Normalized	O
expression	O
levels	O
(	O
relative	O
expression	O
±	O
standard	O
error	O
;	O
ida	O
:	O
-	O
0	O
.	O
02	O
±	O
0	O
.	O
001	O
;	O
Col	O
-	O
0	O
:	O
1	O
±	O
0	O
.	O
11	O
;	O
35S	B-gene
::	O
IDA	B-protein
124	O
±	O
0	O
.	O
75	O
;	O
35S	B-gene
::	O
IDA	B-mutant
K66A	I-mutant
/	I-mutant
R67A	I-mutant
:	O
159	O
±	O
0	O
.	O
58	O
)	O
of	O
IDA	B-protein
wild	B-protein_state
-	I-protein_state
type	I-protein_state
and	O
mutant	B-protein_state
transcripts	O
in	O
the	O
35S	B-experimental_method
promoter	I-experimental_method
over	I-experimental_method
-	I-experimental_method
expression	I-experimental_method
lines	I-experimental_method
analyzed	O
in	O
(	O
C	O
).	O
(	O
E	O
)	O
Magnified	O
view	O
of	O
representative	O
abscission	O
zones	O
from	O
35S	B-gene
::	O
IDA	B-protein
,	O
Col	O
-	O
0	O
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
double	B-protein_state
-	I-protein_state
mutant	I-protein_state
T3	B-experimental_method
transgenic	I-experimental_method
lines	I-experimental_method
.	O

The	O
four	O
C	O
-	O
terminal	O
residues	O
in	O
IDA	B-protein
(	O
Lys66IDA	B-residue_range
-	I-residue_range
Asn69IDA	I-residue_range
)	O
are	O
conserved	B-protein_state
among	O
IDA	B-protein_type
family	I-protein_type
members	I-protein_type
and	O
are	O
in	O
direct	O
contact	O
with	O
SERK1	B-protein
(	O
Figures	O
1A	O
,	O
4C	O
).	O

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

SERK1	O
has	O
been	O
previously	O
reported	O
as	O
a	O
positive	O
regulator	O
in	O
plant	B-taxonomy_domain
embryogenesis	O
,	O
male	O
sporogenesis	O
,	O
brassinosteroid	O
signaling	O
and	O
in	O
phytosulfokine	O
perception	O
.	O

The	O
fact	O
that	O
SERK1	B-protein
specifically	O
interacts	O
with	O
the	O
very	O
C	O
-	O
terminus	O
of	O
IDLs	B-protein_type
may	O
allow	O
for	O
the	O
rational	O
design	O
of	O
peptide	B-chemical
hormone	I-chemical
antagonists	I-chemical
,	O
as	O
previously	O
demonstrated	O
for	O
the	O
brassinosteroid	O
pathway	O
.	O

These	O
residues	O
are	O
not	O
involved	O
in	O
the	O
sensing	O
of	O
the	O
steroid	B-chemical
hormone	I-chemical
brassinolide	B-chemical
.	O

Structure	B-experimental_method
-	I-experimental_method
guided	I-experimental_method
multiple	I-experimental_method
sequence	I-experimental_method
alignment	I-experimental_method
of	O
IDA	B-protein
and	O
IDA	B-chemical
-	I-chemical
like	I-chemical
peptides	I-chemical
with	O
other	O
plant	B-taxonomy_domain
peptide	B-protein_type
hormone	I-protein_type
families	I-protein_type
,	O
including	O
CLAVATA3	B-protein_type
–	I-protein_type
EMBRYO	I-protein_type
SURROUNDING	I-protein_type
REGION	I-protein_type
-	I-protein_type
RELATED	I-protein_type
(	O
CLV3	B-protein_type
/	I-protein_type
CLE	I-protein_type
),	O
ROOT	B-protein_type
GROWTH	I-protein_type
FACTOR	I-protein_type
–	I-protein_type
GOLVEN	I-protein_type
(	O
RGF	B-protein_type
/	I-protein_type
GLV	I-protein_type
),	O
PRECURSOR	B-protein_type
GENE	I-protein_type
PROPEP1	I-protein_type
(	O
PEP1	B-protein_type
)	O
from	O
Arabidopsis	B-species
thaliana	I-species
.	O

Our	O
experiments	O
reveal	O
that	O
SERK1	B-protein
recognizes	O
a	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

A	O
unified	O
mechanism	O
for	O
proteolysis	O
and	O
autocatalytic	B-ptm
activation	I-ptm
in	O
the	O
20S	B-complex_assembly
proteasome	I-complex_assembly

Here	O
we	O
use	O
mutagenesis	B-experimental_method
,	O
X	B-experimental_method
-	I-experimental_method
ray	I-experimental_method
crystallography	I-experimental_method
and	O
biochemical	B-experimental_method
assays	I-experimental_method
to	O
suggest	O
that	O
Lys33	B-residue_name_number
initiates	O
nucleophilic	O
attack	O
of	O
the	O
propeptide	B-structure_element
by	O
deprotonating	O
the	O
Thr1	B-residue_name_number
hydroxyl	O
group	O
and	O
that	O
both	O
residues	O
together	O
with	O
Asp17	B-residue_name_number
are	O
part	O
of	O
a	O
catalytic	B-site
triad	I-site
.	O

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

In	O
the	O
last	O
stage	O
of	O
CP	B-complex_assembly
biogenesis	O
,	O
the	O
prosegments	B-structure_element
are	O
autocatalytically	B-ptm
removed	I-ptm
through	O
nucleophilic	O
attack	O
by	O
the	O
active	B-site
site	I-site
residue	I-site
Thr1	B-residue_name_number
on	O
the	O
preceding	O
peptide	O
bond	O
involving	O
Gly	B-residue_name_number
(-	I-residue_name_number
1	I-residue_name_number
).	I-residue_name_number

Release	O
of	O
the	O
propeptides	B-structure_element
creates	O
a	O
functionally	O
active	B-protein_state
CP	B-complex_assembly
that	O
cleaves	O
proteins	O
into	O
short	O
peptides	O
.	O

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

In	O
the	O
final	O
steps	O
of	O
proteasome	B-complex_assembly
biogenesis	O
,	O
the	O
propeptides	B-structure_element
are	O
autocatalytically	B-ptm
cleaved	I-ptm
from	O
the	O
mature	B-protein_state
β	B-protein
-	I-protein
subunit	I-protein
domains	I-protein
.	O

Instead	O
,	O
the	O
plasticity	O
of	O
the	O
β5	B-protein
S1	B-site
pocket	I-site
caused	O
by	O
the	O
rotational	O
flexibility	O
of	O
Met45	B-residue_name_number
might	O
prevent	O
stable	O
accommodation	O
of	O
His	B-residue_name_number
(-	I-residue_name_number
2	I-residue_name_number
)	I-residue_name_number
in	O
the	O
S1	B-site
site	I-site
and	O
thus	O
also	O
promote	O
its	O
immediate	O
release	O
after	O
autolysis	B-ptm
.	O

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

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

The	O
active	B-site
site	I-site
of	O
the	O
proteasome	B-complex_assembly

Twenty	O
years	O
later	O
,	O
with	O
a	O
plethora	O
of	O
yCP	B-complex_assembly
X	B-evidence
-	I-evidence
ray	I-evidence
structures	I-evidence
in	O
hand	O
,	O
we	O
decided	O
to	O
re	O
-	O
analyse	O
the	O
active	B-site
site	I-site
of	O
the	O
proteasome	B-complex_assembly
and	O
to	O
resolve	O
the	O
uncertainty	O
regarding	O
the	O
nature	O
of	O
the	O
general	O
base	O
.	O

As	O
determined	O
by	O
crystallographic	B-experimental_method
analysis	I-experimental_method
,	O
this	O
mutant	B-protein_state
β5	B-protein
subunit	O
was	O
partially	B-protein_state
processed	I-protein_state
(	O
Table	O
1	O
)	O
but	O
displayed	O
impaired	O
reactivity	O
towards	O
the	O
proteasome	B-complex_assembly
inhibitor	O
carfilzomib	B-chemical
compared	O
with	O
the	O
subunits	O
β1	B-protein
and	O
β2	B-protein
,	O
and	O
with	O
WT	B-protein_state
β5	B-protein
(	O
Supplementary	O
Fig	O
.	O
7a	O
).	O

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

In	O
agreement	O
,	O
an	O
E17A	B-mutant
mutant	B-protein_state
in	O
the	O
proteasomal	O
β	B-protein
-	I-protein
subunit	I-protein
of	O
the	O
archaeon	B-taxonomy_domain
Thermoplasma	B-species
acidophilum	I-species
prevents	O
autolysis	B-ptm
and	O
catalysis	O
.	O

This	O
model	O
is	O
also	O
consistent	O
with	O
the	O
fact	O
that	O
no	O
defined	O
water	B-chemical
molecule	O
is	O
observed	O
in	O
the	O
mature	B-protein_state
WT	B-protein_state
proteasomal	O
active	B-site
site	I-site
that	O
could	O
shuttle	O
the	O
proton	O
from	O
Thr1Oγ	B-residue_name_number
to	O
Thr1NH2	B-residue_name_number
.	O

The	O
β5	B-mutant
-	I-mutant
D166N	I-mutant
pp	B-chemical
cis	B-protein_state
yeast	B-taxonomy_domain
mutant	B-protein_state
is	O
significantly	O
impaired	O
in	O
growth	O
and	O
its	O
ChT	O
-	O
L	O
activity	O
is	O
drastically	O
reduced	O
(	O
Supplementary	O
Fig	O
.	O
6a	O
,	O
b	O
and	O
Table	O
1	O
).	O

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

In	O
one	O
of	O
the	O
two	O
β5	B-protein
subunits	O
,	O
however	O
,	O
we	O
found	O
the	O
cleaved	B-protein_state
propeptide	B-structure_element
still	B-protein_state
bound	I-protein_state
in	O
the	O
substrate	B-site
-	I-site
binding	I-site
channel	I-site
(	O
Fig	O
.	O
4c	O
).	O

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

However	O
,	O
the	O
apo	B-protein_state
crystal	B-evidence
structure	I-evidence
revealed	O
that	O
Ser1Oγ	B-residue_name_number
is	O
turned	O
away	O
from	O
the	O
substrate	B-site
-	I-site
binding	I-site
channel	I-site
(	O
Fig	O
.	O
4g	O
).	O

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

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

The	O
resulting	O
uncharged	O
Thr1NH2	B-residue_name_number
is	O
hydrogen	O
-	O
bridged	O
to	O
the	O
C3	O
-	O
OH	O
group	O
.	O

The	O
greater	O
suitability	O
of	O
threonine	B-residue_name
for	O
the	O
proteasome	B-complex_assembly
active	B-site
site	I-site
,	O
which	O
has	O
been	O
noted	O
in	O
biochemical	O
as	O
well	O
as	O
in	O
kinetic	O
studies	O
,	O
constitutes	O
a	O
likely	O
reason	O
for	O
the	O
conservation	B-protein_state
of	O
the	O
Thr1	B-residue_name_number
residue	O
in	O
all	O
proteasomes	B-complex_assembly
from	O
bacteria	B-taxonomy_domain
to	O
eukaryotes	B-taxonomy_domain
.	O

(	O
c	O
)	O
Structural	B-experimental_method
superposition	I-experimental_method
of	O
the	O
β1	B-mutant
-	I-mutant
T1A	I-mutant
,	O
the	O
β2	B-mutant
-	I-mutant
T1A	I-mutant
and	O
the	O
β5	B-mutant
-	I-mutant
T1A	I-mutant
-	I-mutant
K81R	I-mutant
propeptide	B-structure_element
remnants	O
depict	O
their	O
differences	O
in	O
conformation	O
.	O

While	O
residue	O
(-	B-residue_number
2	I-residue_number
)	I-residue_number
of	O
the	O
β1	B-protein
and	O
β2	B-protein
prosegments	B-structure_element
fit	O
the	O
S1	B-site
pocket	I-site
,	O
His	B-residue_name_number
(-	I-residue_name_number
2	I-residue_name_number
)	I-residue_name_number
of	O
the	O
β5	B-protein
propeptide	B-structure_element
occupies	O
the	O
S2	B-site
pocket	I-site
.	O

(	O
a	O
)	O
Structural	B-experimental_method
superposition	I-experimental_method
of	O
the	O
β1	B-mutant
-	I-mutant
T1A	I-mutant
propeptide	B-structure_element
and	O
the	O
β5	B-mutant
-	I-mutant
H	I-mutant
(-	I-mutant
2	I-mutant
)	I-mutant
L	I-mutant
-	I-mutant
T1A	I-mutant
mutant	B-protein_state
propeptide	B-structure_element
.	O

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

(	O
d	O
)	O
Structural	B-experimental_method
superposition	I-experimental_method
of	O
the	O
matured	B-protein_state
β2	B-protein
active	B-site
site	I-site
,	O
the	O
WT	B-protein_state
β2	B-mutant
-	I-mutant
T1A	I-mutant
propeptide	B-structure_element
and	O
the	O
β2	B-mutant
-	I-mutant
T	I-mutant
(-	I-mutant
2	I-mutant
)	I-mutant
V	I-mutant
mutant	B-protein_state
propeptide	B-structure_element
.	O

(	O
a	O
)	O
Hydrogen	B-site
-	I-site
bonding	I-site
network	I-site
at	O
the	O
mature	B-protein_state
WT	B-protein_state
β5	B-protein
proteasomal	O
active	B-site
site	I-site
(	O
dotted	O
lines	O
).	O

The	O
Thr1	B-residue_name_number
N	O
terminus	O
is	O
engaged	O
in	O
hydrogen	O
bonds	O
with	O
Ser129Oγ	B-residue_name_number
,	O
the	O
carbonyl	O
oxygen	O
of	O
residue	O
168	B-residue_number
,	O
Ser169Oγ	B-residue_name_number
and	O
Asp166Oδ	B-residue_name_number
.	O
(	O
b	O
)	O
The	O
orientations	O
of	O
the	O
active	B-site
-	I-site
site	I-site
residues	I-site
involved	O
in	O
hydrogen	O
bonding	O
are	O
strictly	B-protein_state
conserved	I-protein_state
in	O
each	O
proteolytic	B-site
centre	I-site
,	O
as	O
shown	O
by	O
superposition	B-experimental_method
of	O
the	O
β	B-protein
subunits	I-protein
.	O

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

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

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

Notably	O
,	O
His	B-residue_name_number
(-	I-residue_name_number
2	I-residue_name_number
)	I-residue_name_number
does	O
not	O
occupy	O
the	O
S1	B-site
pocket	I-site
formed	O
by	O
Met45	B-residue_name_number
,	O
similar	O
to	O
what	O
was	O
observed	O
for	O
the	O
β5	B-mutant
-	I-mutant
T1A	I-mutant
-	I-mutant
K81R	I-mutant
mutant	B-protein_state
.	O

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

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