|
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 |
|
|
|
|