|
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 |
|
|
|
Here O |
|
, O |
|
we O |
|
provide O |
|
the O |
|
first O |
|
biochemical B-experimental_method |
|
, I-experimental_method |
|
crystallographic I-experimental_method |
|
, I-experimental_method |
|
and I-experimental_method |
|
genetic I-experimental_method |
|
insight I-experimental_method |
|
into O |
|
how O |
|
two O |
|
surface B-protein_type |
|
glycan I-protein_type |
|
- I-protein_type |
|
binding I-protein_type |
|
proteins I-protein_type |
|
from O |
|
the O |
|
complex O |
|
Bacteroides B-species |
|
ovatus I-species |
|
xyloglucan B-gene |
|
utilization I-gene |
|
locus I-gene |
|
( O |
|
XyGUL B-gene |
|
) O |
|
enable O |
|
recognition O |
|
and O |
|
uptake O |
|
of O |
|
this O |
|
ubiquitous O |
|
vegetable B-taxonomy_domain |
|
polysaccharide B-chemical |
|
. O |
|
|
|
This O |
|
microbial B-taxonomy_domain |
|
community O |
|
is O |
|
largely O |
|
bacterial B-taxonomy_domain |
|
, O |
|
with O |
|
the O |
|
Bacteroidetes B-taxonomy_domain |
|
, O |
|
Firmicutes B-taxonomy_domain |
|
, O |
|
and O |
|
Actinobacteria B-taxonomy_domain |
|
comprising O |
|
the O |
|
dominant O |
|
phyla O |
|
. O |
|
|
|
In O |
|
the O |
|
archetypal O |
|
starch B-complex_assembly |
|
utilization I-complex_assembly |
|
system I-complex_assembly |
|
of O |
|
B B-species |
|
. I-species |
|
thetaiotaomicron I-species |
|
, O |
|
starch O |
|
binding O |
|
to O |
|
the O |
|
cell O |
|
surface O |
|
is O |
|
mediated O |
|
at O |
|
eight O |
|
distinct O |
|
starch B-site |
|
- I-site |
|
binding I-site |
|
sites I-site |
|
distributed O |
|
among O |
|
four O |
|
surface B-protein_type |
|
glycan I-protein_type |
|
- I-protein_type |
|
binding I-protein_type |
|
proteins I-protein_type |
|
( O |
|
SGBPs B-protein_type |
|
): O |
|
two O |
|
within O |
|
the O |
|
amylase B-protein_type |
|
SusG B-protein |
|
, O |
|
one O |
|
within O |
|
SusD B-protein |
|
, O |
|
two O |
|
within O |
|
SusE B-protein |
|
, O |
|
and O |
|
three O |
|
within O |
|
SusF B-protein |
|
. O |
|
The O |
|
functional O |
|
redundancy O |
|
of O |
|
many O |
|
of O |
|
these O |
|
sites O |
|
is O |
|
high O |
|
: O |
|
whereas O |
|
SusD B-protein |
|
is O |
|
essential O |
|
for O |
|
growth O |
|
on O |
|
starch B-chemical |
|
, O |
|
combined O |
|
mutations O |
|
of O |
|
the O |
|
SusE B-protein |
|
, O |
|
SusF B-protein |
|
, O |
|
and O |
|
SusG B-protein |
|
binding B-site |
|
sites I-site |
|
are O |
|
required O |
|
to O |
|
impair O |
|
growth O |
|
on O |
|
the O |
|
polysaccharide B-chemical |
|
. O |
|
|
|
Combined O |
|
biochemical B-experimental_method |
|
, I-experimental_method |
|
structural I-experimental_method |
|
, I-experimental_method |
|
and I-experimental_method |
|
reverse I-experimental_method |
|
- I-experimental_method |
|
genetic I-experimental_method |
|
approaches I-experimental_method |
|
clearly O |
|
illuminate O |
|
the O |
|
distinct O |
|
, O |
|
yet O |
|
complementary O |
|
, O |
|
functions O |
|
that O |
|
these O |
|
two O |
|
proteins O |
|
play O |
|
in O |
|
XyG B-chemical |
|
recognition O |
|
as O |
|
it O |
|
impacts O |
|
the O |
|
physiology O |
|
of O |
|
B B-species |
|
. I-species |
|
ovatus I-species |
|
. O |
|
|
|
Hence O |
|
, O |
|
there O |
|
is O |
|
a O |
|
critical O |
|
need O |
|
for O |
|
the O |
|
elucidation O |
|
of O |
|
detailed O |
|
structure O |
|
- O |
|
function O |
|
relationships O |
|
among O |
|
PUL B-gene |
|
SGBPs B-protein_type |
|
, O |
|
in O |
|
light O |
|
of O |
|
the O |
|
manifold O |
|
glycan B-chemical |
|
structures O |
|
in O |
|
nature O |
|
. 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 |
|
|
|
In O |
|
our O |
|
initial O |
|
study O |
|
focused O |
|
on O |
|
the O |
|
functional O |
|
characterization O |
|
of O |
|
the O |
|
glycoside B-protein_type |
|
hydrolases I-protein_type |
|
of O |
|
the O |
|
XyGUL B-gene |
|
, O |
|
we O |
|
reported O |
|
preliminary O |
|
affinity B-experimental_method |
|
PAGE I-experimental_method |
|
and O |
|
isothermal B-experimental_method |
|
titration I-experimental_method |
|
calorimetry I-experimental_method |
|
( O |
|
ITC B-experimental_method |
|
) O |
|
data O |
|
indicating O |
|
that O |
|
both O |
|
SGBP B-protein |
|
- I-protein |
|
A I-protein |
|
and O |
|
SGBP B-protein |
|
- I-protein |
|
B I-protein |
|
are O |
|
competent O |
|
xyloglucan B-protein_type |
|
- I-protein_type |
|
binding I-protein_type |
|
proteins I-protein_type |
|
( O |
|
affinity B-evidence |
|
constant I-evidence |
|
[ O |
|
Ka B-evidence |
|
] O |
|
values O |
|
of O |
|
3 O |
|
. O |
|
74 O |
|
× O |
|
105 O |
|
M O |
|
− O |
|
1 O |
|
and O |
|
4 O |
|
. O |
|
98 O |
|
× O |
|
104 O |
|
M O |
|
− O |
|
1 O |
|
, O |
|
respectively O |
|
[ O |
|
23 O |
|
]). O |
|
|
|
Cocrystallization B-experimental_method |
|
of O |
|
SGBP B-protein |
|
- I-protein |
|
A I-protein |
|
with O |
|
XyGO2 B-chemical |
|
generated O |
|
a O |
|
substrate B-complex_assembly |
|
complex I-complex_assembly |
|
structure B-evidence |
|
( O |
|
2 O |
|
. O |
|
3 O |
|
Å O |
|
, O |
|
Rwork B-evidence |
|
= O |
|
21 O |
|
. O |
|
8 O |
|
%, O |
|
Rfree B-evidence |
|
= O |
|
24 O |
|
. O |
|
8 O |
|
%, O |
|
residues O |
|
36 B-residue_range |
|
to I-residue_range |
|
546 I-residue_range |
|
) O |
|
( O |
|
Fig O |
|
. O |
|
4A O |
|
and O |
|
B O |
|
; O |
|
Table O |
|
2 O |
|
) O |
|
that O |
|
revealed O |
|
the O |
|
distinct O |
|
binding B-site |
|
- I-site |
|
site I-site |
|
architecture O |
|
of O |
|
the O |
|
XyG B-protein_type |
|
binding I-protein_type |
|
protein I-protein_type |
|
. O |
|
|
|
Molecular O |
|
structure B-evidence |
|
of O |
|
SGBP B-protein |
|
- I-protein |
|
A I-protein |
|
( O |
|
Bacova_02651 B-gene |
|
). O |
|
( O |
|
A O |
|
) O |
|
Overlay B-experimental_method |
|
of O |
|
SGBP B-protein |
|
- I-protein |
|
A I-protein |
|
from O |
|
the O |
|
apo B-protein_state |
|
( O |
|
rainbow O |
|
) O |
|
and O |
|
XyGO2 B-chemical |
|
( O |
|
gray O |
|
) O |
|
structures B-evidence |
|
. O |
|
|
|
The O |
|
backbone O |
|
glucose B-chemical |
|
residues O |
|
are O |
|
numbered O |
|
from O |
|
the O |
|
nonreducing O |
|
end O |
|
; O |
|
xylose B-chemical |
|
residues O |
|
are O |
|
labeled O |
|
X1 B-residue_name_number |
|
and O |
|
X2 B-residue_name_number |
|
. 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 |
|
|
|
Protein O |
|
name O |
|
Ka B-evidence |
|
ΔG O |
|
( O |
|
kcal O |
|
⋅ O |
|
mol O |
|
− O |
|
1 O |
|
) O |
|
ΔH B-evidence |
|
( O |
|
kcal O |
|
⋅ O |
|
mol O |
|
− O |
|
1 O |
|
) O |
|
TΔS B-evidence |
|
( O |
|
kcal O |
|
⋅ O |
|
mol O |
|
− O |
|
1 O |
|
) O |
|
Fold O |
|
changeb O |
|
M O |
|
− O |
|
1 O |
|
SGBP B-protein |
|
- I-protein |
|
A I-protein |
|
( O |
|
W82A B-mutant |
|
W283A B-mutant |
|
W306A B-mutant |
|
) O |
|
ND O |
|
NB O |
|
NB O |
|
NB O |
|
NB O |
|
SGBP B-protein |
|
- I-protein |
|
A I-protein |
|
( O |
|
W82A B-mutant |
|
) O |
|
c O |
|
4 O |
|
. O |
|
9 O |
|
9 O |
|
. O |
|
1 O |
|
× O |
|
104 O |
|
− O |
|
6 O |
|
. O |
|
8 O |
|
− O |
|
6 O |
|
. O |
|
3 O |
|
0 O |
|
. O |
|
5 O |
|
SGBP B-protein |
|
- I-protein |
|
A I-protein |
|
( O |
|
W306 B-residue_name_number |
|
) O |
|
ND O |
|
NB O |
|
NB O |
|
NB O |
|
NB O |
|
SGBP B-protein |
|
- I-protein |
|
B I-protein |
|
( O |
|
230 B-residue_range |
|
– I-residue_range |
|
489 I-residue_range |
|
) O |
|
0 O |
|
. O |
|
7 O |
|
( O |
|
8 O |
|
. O |
|
6 O |
|
± O |
|
0 O |
|
. O |
|
20 O |
|
) O |
|
× O |
|
104 O |
|
− O |
|
6 O |
|
. O |
|
7 O |
|
− O |
|
14 O |
|
. O |
|
9 O |
|
± O |
|
0 O |
|
. O |
|
1 O |
|
− O |
|
8 O |
|
. O |
|
2 O |
|
SGBP B-protein |
|
- I-protein |
|
B I-protein |
|
( O |
|
Y363A B-mutant |
|
) O |
|
19 O |
|
. O |
|
7 O |
|
( O |
|
2 O |
|
. O |
|
9 O |
|
± O |
|
0 O |
|
. O |
|
10 O |
|
) O |
|
× O |
|
103 O |
|
− O |
|
4 O |
|
. O |
|
7 O |
|
− O |
|
18 O |
|
. O |
|
1 O |
|
± O |
|
0 O |
|
. O |
|
1 O |
|
− O |
|
13 O |
|
. O |
|
3 O |
|
SGBP B-protein |
|
- I-protein |
|
B I-protein |
|
( O |
|
W364A B-mutant |
|
) O |
|
ND O |
|
Weak O |
|
Weak O |
|
Weak O |
|
Weak O |
|
SGBP B-protein |
|
- I-protein |
|
B I-protein |
|
( O |
|
F414A B-mutant |
|
) O |
|
3 O |
|
. O |
|
2 O |
|
( O |
|
1 O |
|
. O |
|
80 O |
|
± O |
|
0 O |
|
. O |
|
03 O |
|
) O |
|
× O |
|
104 O |
|
− O |
|
5 O |
|
. O |
|
8 O |
|
− O |
|
11 O |
|
. O |
|
4 O |
|
± O |
|
0 O |
|
. O |
|
1 O |
|
− O |
|
5 O |
|
. O |
|
6 O |
|
|
|
Weak O |
|
binding O |
|
represents O |
|
a O |
|
Ka B-evidence |
|
of O |
|
< O |
|
500 O |
|
M O |
|
− O |
|
1 O |
|
. O |
|
|
|
SGBP B-protein |
|
- I-protein |
|
B I-protein |
|
has O |
|
a O |
|
multimodular O |
|
structure O |
|
with O |
|
a O |
|
single O |
|
, O |
|
C O |
|
- O |
|
terminal O |
|
glycan B-structure_element |
|
- I-structure_element |
|
binding I-structure_element |
|
domain I-structure_element |
|
. O |
|
|
|
The O |
|
crystal B-evidence |
|
structure I-evidence |
|
of O |
|
full B-protein_state |
|
- I-protein_state |
|
length I-protein_state |
|
SGBP B-protein |
|
- I-protein |
|
B I-protein |
|
in B-protein_state |
|
complex I-protein_state |
|
with I-protein_state |
|
XyGO2 B-chemical |
|
( O |
|
2 O |
|
. O |
|
37 O |
|
Å O |
|
, O |
|
Rwork B-evidence |
|
= O |
|
19 O |
|
. O |
|
9 O |
|
%, O |
|
Rfree B-evidence |
|
= O |
|
23 O |
|
. O |
|
9 O |
|
%, O |
|
residues O |
|
34 B-residue_range |
|
to I-residue_range |
|
489 I-residue_range |
|
) O |
|
( O |
|
Table O |
|
2 O |
|
) O |
|
revealed O |
|
an O |
|
extended O |
|
structure B-evidence |
|
composed O |
|
of O |
|
three O |
|
tandem B-structure_element |
|
immunoglobulin I-structure_element |
|
( I-structure_element |
|
Ig I-structure_element |
|
)- I-structure_element |
|
like I-structure_element |
|
domains I-structure_element |
|
( O |
|
domains O |
|
A B-structure_element |
|
, O |
|
B B-structure_element |
|
, O |
|
and O |
|
C B-structure_element |
|
) O |
|
followed O |
|
at O |
|
the O |
|
C O |
|
terminus O |
|
by O |
|
a O |
|
novel O |
|
xyloglucan B-structure_element |
|
- I-structure_element |
|
binding I-structure_element |
|
domain I-structure_element |
|
( O |
|
domain O |
|
D B-structure_element |
|
) O |
|
( O |
|
Fig O |
|
. O |
|
5A O |
|
). O |
|
|
|
Analogously O |
|
, O |
|
the O |
|
outer B-protein_state |
|
membrane I-protein_state |
|
- I-protein_state |
|
anchored I-protein_state |
|
endo B-protein_type |
|
- I-protein_type |
|
xyloglucanase I-protein_type |
|
BoGH5 B-protein |
|
of O |
|
the O |
|
XyGUL B-gene |
|
contains O |
|
a O |
|
100 B-structure_element |
|
- I-structure_element |
|
amino I-structure_element |
|
- I-structure_element |
|
acid I-structure_element |
|
, I-structure_element |
|
all I-structure_element |
|
- I-structure_element |
|
β I-structure_element |
|
- I-structure_element |
|
strand I-structure_element |
|
, O |
|
N B-structure_element |
|
- I-structure_element |
|
terminal I-structure_element |
|
module I-structure_element |
|
and O |
|
flexible B-structure_element |
|
linker I-structure_element |
|
that O |
|
imparts O |
|
conformational O |
|
flexibility O |
|
and O |
|
distances O |
|
the O |
|
catalytic B-structure_element |
|
module I-structure_element |
|
from O |
|
the O |
|
cell O |
|
surface O |
|
. O |
|
|
|
The O |
|
Y363A B-mutant |
|
site B-experimental_method |
|
- I-experimental_method |
|
directed I-experimental_method |
|
mutant I-experimental_method |
|
of O |
|
SGBP B-protein |
|
- I-protein |
|
B I-protein |
|
displays O |
|
a O |
|
20 O |
|
- O |
|
fold O |
|
decrease O |
|
in O |
|
the O |
|
Ka B-evidence |
|
for O |
|
XyG B-chemical |
|
, O |
|
while O |
|
the O |
|
W364A B-mutant |
|
mutant B-protein_state |
|
lacks B-protein_state |
|
XyG I-protein_state |
|
binding I-protein_state |
|
( O |
|
Table O |
|
3 O |
|
; O |
|
see O |
|
Fig O |
|
. O |
|
S6 O |
|
in O |
|
the O |
|
supplemental O |
|
material O |
|
). O |
|
|
|
Hoping O |
|
to O |
|
achieve O |
|
a O |
|
higher O |
|
- O |
|
resolution O |
|
view O |
|
of O |
|
the O |
|
SGBP B-protein |
|
- I-protein |
|
B I-protein |
|
– O |
|
xyloglucan B-chemical |
|
interaction O |
|
, O |
|
we O |
|
solved B-experimental_method |
|
the O |
|
crystal B-evidence |
|
structure I-evidence |
|
of O |
|
the O |
|
fused B-mutant |
|
CD I-mutant |
|
domains I-mutant |
|
in B-protein_state |
|
complex I-protein_state |
|
with I-protein_state |
|
XyGO2 B-chemical |
|
( O |
|
1 O |
|
. O |
|
57 O |
|
Å O |
|
, O |
|
Rwork B-evidence |
|
= O |
|
15 O |
|
. O |
|
6 O |
|
%, O |
|
Rfree B-evidence |
|
= O |
|
17 O |
|
. O |
|
1 O |
|
%, O |
|
residues O |
|
230 B-residue_range |
|
to I-residue_range |
|
489 I-residue_range |
|
) O |
|
( O |
|
Table O |
|
2 O |
|
). O |
|
|
|
Growth O |
|
on O |
|
glucose B-chemical |
|
displayed O |
|
the O |
|
shortest O |
|
lag B-evidence |
|
time I-evidence |
|
for O |
|
each O |
|
strain O |
|
, O |
|
and O |
|
so O |
|
lag B-evidence |
|
times I-evidence |
|
were O |
|
normalized O |
|
for O |
|
each O |
|
carbohydrate B-chemical |
|
by O |
|
subtracting O |
|
the O |
|
lag B-evidence |
|
time I-evidence |
|
of O |
|
that O |
|
strain O |
|
in O |
|
glucose B-chemical |
|
( O |
|
Fig O |
|
. O |
|
6 O |
|
; O |
|
see O |
|
Fig O |
|
. O |
|
S8 O |
|
in O |
|
the O |
|
supplemental O |
|
material O |
|
). O |
|
|
|
Complementation B-experimental_method |
|
of O |
|
the O |
|
ΔSGBP B-mutant |
|
- I-mutant |
|
A I-mutant |
|
strain O |
|
( O |
|
ΔSGBP B-mutant |
|
- I-mutant |
|
A I-mutant |
|
:: O |
|
SGBP B-protein |
|
- I-protein |
|
A I-protein |
|
) O |
|
restores O |
|
growth O |
|
to O |
|
wild B-protein_state |
|
- I-protein_state |
|
type I-protein_state |
|
rates O |
|
on O |
|
xyloglucan B-chemical |
|
and O |
|
XyGO1 B-chemical |
|
, O |
|
yet O |
|
the O |
|
calculated O |
|
rate O |
|
of O |
|
the O |
|
complemented O |
|
strain O |
|
is O |
|
~ O |
|
72 O |
|
% O |
|
that O |
|
of O |
|
the O |
|
WT B-protein_state |
|
Δtdk B-mutant |
|
strain O |
|
on O |
|
XyGO2 B-chemical |
|
; O |
|
similar O |
|
results O |
|
were O |
|
obtained O |
|
for O |
|
the O |
|
SGBP B-protein |
|
- I-protein |
|
B I-protein |
|
complemented O |
|
strain O |
|
despite O |
|
the O |
|
fact O |
|
that O |
|
the O |
|
growth O |
|
curves O |
|
do O |
|
not O |
|
appear O |
|
much O |
|
different O |
|
( O |
|
see O |
|
Fig O |
|
. O |
|
S8C O |
|
and O |
|
F O |
|
). O |
|
|
|
Fig O |
|
. O |
|
1B O |
|
) O |
|
was O |
|
completely O |
|
incapable O |
|
of O |
|
growth O |
|
on O |
|
XyG B-chemical |
|
, O |
|
XyGO1 B-chemical |
|
, O |
|
and O |
|
XyGO2 B-chemical |
|
, O |
|
indicating O |
|
that O |
|
SGBP B-protein |
|
- I-protein |
|
A I-protein |
|
is O |
|
essential O |
|
for O |
|
XyG B-chemical |
|
utilization O |
|
( O |
|
Fig O |
|
. O |
|
6 O |
|
). O |
|
|
|
Intriguingly O |
|
, O |
|
the O |
|
ΔSGBP B-mutant |
|
- I-mutant |
|
B I-mutant |
|
strain O |
|
( O |
|
ΔBacova_02650 B-mutant |
|
) O |
|
( O |
|
cf O |
|
. O |
|
|
|
In O |
|
the O |
|
BtSus B-gene |
|
, O |
|
SusD B-protein |
|
and O |
|
the O |
|
TBDT B-protein_type |
|
SusC B-protein |
|
interact O |
|
, O |
|
and O |
|
we O |
|
speculate O |
|
that O |
|
this O |
|
interaction O |
|
is O |
|
necessary O |
|
for O |
|
glycan B-chemical |
|
uptake O |
|
, O |
|
as O |
|
suggested O |
|
by O |
|
the O |
|
fact O |
|
that O |
|
a O |
|
ΔsusD B-mutant |
|
mutant B-protein_state |
|
cannot O |
|
grow O |
|
on O |
|
starch B-chemical |
|
, O |
|
but O |
|
a O |
|
ΔsusD B-mutant |
|
:: O |
|
SusD B-mutant |
|
* I-mutant |
|
strain O |
|
regains O |
|
this O |
|
ability O |
|
if O |
|
a O |
|
transcriptional B-protein_type |
|
activator I-protein_type |
|
of O |
|
the O |
|
sus B-gene |
|
operon I-gene |
|
is O |
|
supplied O |
|
. O |
|
|
|
Recent O |
|
work O |
|
has O |
|
elucidated O |
|
that O |
|
Bacteroidetes B-taxonomy_domain |
|
cross O |
|
- O |
|
feed O |
|
during O |
|
growth O |
|
on O |
|
many O |
|
glycans B-chemical |
|
; O |
|
the O |
|
glycoside B-protein_type |
|
hydrolases I-protein_type |
|
expressed O |
|
by O |
|
one O |
|
species O |
|
liberate O |
|
oligosaccharides B-chemical |
|
for O |
|
consumption O |
|
by O |
|
other O |
|
members O |
|
of O |
|
the O |
|
community O |
|
. O |
|
|
|
In O |
|
this O |
|
instance O |
|
, O |
|
coexpression O |
|
of O |
|
the O |
|
susD B-gene |
|
- O |
|
like O |
|
gene O |
|
nanU B-gene |
|
was O |
|
not O |
|
required O |
|
, O |
|
nor O |
|
did O |
|
the O |
|
expression O |
|
of O |
|
the O |
|
nanU B-gene |
|
gene O |
|
enhance O |
|
growth O |
|
kinetics O |
|
. O |
|
|
|
However O |
|
, O |
|
the O |
|
natural O |
|
diversity O |
|
of O |
|
these O |
|
proteins O |
|
represents O |
|
a O |
|
rich O |
|
source O |
|
for O |
|
the O |
|
discovery O |
|
of O |
|
unique O |
|
carbohydrate B-structure_element |
|
- I-structure_element |
|
binding I-structure_element |
|
motifs I-structure_element |
|
to O |
|
both O |
|
inform O |
|
gut O |
|
microbiology O |
|
and O |
|
generate O |
|
new O |
|
, O |
|
specific O |
|
carbohydrate B-chemical |
|
analytical O |
|
reagents O |
|
. O |
|
|
|
The O |
|
ability O |
|
of O |
|
our O |
|
resident O |
|
gut O |
|
bacteria B-taxonomy_domain |
|
to O |
|
recognize O |
|
polysaccharides B-chemical |
|
is O |
|
the O |
|
first O |
|
committed O |
|
step O |
|
of O |
|
glycan B-chemical |
|
consumption O |
|
by O |
|
these O |
|
organisms O |
|
, O |
|
a O |
|
critical O |
|
process O |
|
that O |
|
influences O |
|
the O |
|
community O |
|
structure O |
|
and O |
|
thus O |
|
the O |
|
metabolic O |
|
output O |
|
( O |
|
i O |
|
. O |
|
e O |
|
., O |
|
short O |
|
- O |
|
chain O |
|
fatty O |
|
acid O |
|
and O |
|
metabolite O |
|
profile O |
|
) O |
|
of O |
|
these O |
|
organisms O |
|
. O |
|
|
|
Mucosal O |
|
glycan B-chemical |
|
foraging O |
|
enhances O |
|
fitness O |
|
and O |
|
transmission O |
|
of O |
|
a O |
|
saccharolytic O |
|
human O |
|
gut O |
|
bacterial O |
|
symbiont O |
|
|
|
Neisseria B-protein |
|
adhesin I-protein |
|
A I-protein |
|
( O |
|
NadA B-protein |
|
) O |
|
present O |
|
on O |
|
the O |
|
meningococcal B-taxonomy_domain |
|
surface O |
|
can O |
|
mediate O |
|
binding O |
|
to O |
|
human B-species |
|
cells O |
|
and O |
|
is O |
|
one O |
|
of O |
|
the O |
|
three O |
|
MenB B-species |
|
vaccine O |
|
protein O |
|
antigens O |
|
. O |
|
|
|
MarR B-protein_type |
|
family O |
|
proteins O |
|
can O |
|
promote O |
|
bacterial B-taxonomy_domain |
|
survival O |
|
in O |
|
the O |
|
presence O |
|
of O |
|
antibiotics O |
|
, O |
|
toxic O |
|
chemicals O |
|
, O |
|
organic O |
|
solvents O |
|
or O |
|
reactive O |
|
oxygen O |
|
species O |
|
and O |
|
can O |
|
regulate O |
|
virulence O |
|
factor O |
|
expression O |
|
. O |
|
|
|
MarR B-protein_type |
|
homologues O |
|
can O |
|
act O |
|
either O |
|
as O |
|
transcriptional O |
|
repressors O |
|
or O |
|
as O |
|
activators O |
|
. O |
|
|
|
A O |
|
potentially O |
|
interesting O |
|
exception O |
|
comes O |
|
from O |
|
the O |
|
ligand B-protein_state |
|
- I-protein_state |
|
free I-protein_state |
|
and O |
|
salicylate B-protein_state |
|
- I-protein_state |
|
bound I-protein_state |
|
forms O |
|
of O |
|
the O |
|
Methanobacterium B-species |
|
thermoautotrophicum I-species |
|
protein O |
|
MTH313 B-protein |
|
which O |
|
revealed O |
|
that O |
|
two O |
|
salicylate B-chemical |
|
molecules O |
|
bind O |
|
to O |
|
one O |
|
MTH313 B-protein |
|
dimer B-oligomeric_state |
|
and O |
|
induce O |
|
large O |
|
conformational O |
|
changes O |
|
, O |
|
apparently O |
|
sufficient O |
|
to O |
|
prevent O |
|
DNA O |
|
binding O |
|
. O |
|
|
|
We O |
|
obtained O |
|
detailed O |
|
new O |
|
insights O |
|
into O |
|
ligand O |
|
specificity O |
|
, O |
|
how O |
|
the O |
|
ligand O |
|
allosterically O |
|
influences O |
|
the O |
|
DNA O |
|
- O |
|
binding O |
|
ability O |
|
of O |
|
NadR B-protein |
|
, O |
|
and O |
|
the O |
|
regulation O |
|
of O |
|
nadA B-gene |
|
expression O |
|
, O |
|
thus O |
|
also O |
|
providing O |
|
a O |
|
deeper O |
|
structural O |
|
understanding O |
|
of O |
|
the O |
|
ligand O |
|
- O |
|
responsive O |
|
MarR B-protein_type |
|
super O |
|
- O |
|
family O |
|
. O |
|
|
|
Since O |
|
ligand O |
|
- O |
|
binding O |
|
often O |
|
increases O |
|
protein O |
|
stability O |
|
, O |
|
we O |
|
also O |
|
investigated O |
|
the O |
|
effect O |
|
of O |
|
various O |
|
HPAs B-chemical |
|
( O |
|
Fig O |
|
1A O |
|
) O |
|
on O |
|
the O |
|
melting B-evidence |
|
temperature I-evidence |
|
( O |
|
Tm B-evidence |
|
) O |
|
of O |
|
NadR B-protein |
|
. O |
|
As O |
|
a O |
|
control O |
|
of O |
|
specificity O |
|
, O |
|
we O |
|
also O |
|
tested O |
|
salicylate B-chemical |
|
, O |
|
a O |
|
known O |
|
ligand O |
|
of O |
|
some O |
|
MarR B-protein_type |
|
proteins O |
|
previously O |
|
reported O |
|
to O |
|
increase O |
|
the O |
|
Tm B-evidence |
|
of O |
|
ST1710 B-protein |
|
and O |
|
MTH313 B-protein |
|
. O |
|
|
|
However O |
|
, O |
|
an O |
|
increased O |
|
thermal O |
|
stability O |
|
was O |
|
induced O |
|
by O |
|
4 B-chemical |
|
- I-chemical |
|
HPA I-chemical |
|
and O |
|
, O |
|
to O |
|
a O |
|
lesser O |
|
extent O |
|
, O |
|
by O |
|
3 B-chemical |
|
- I-chemical |
|
HPA I-chemical |
|
. O |
|
|
|
( O |
|
A O |
|
) O |
|
Molecular O |
|
structures O |
|
of O |
|
3 B-chemical |
|
- I-chemical |
|
HPA I-chemical |
|
( O |
|
MW O |
|
152 O |
|
. O |
|
2 O |
|
), O |
|
4 B-chemical |
|
- I-chemical |
|
HPA I-chemical |
|
( O |
|
MW O |
|
152 O |
|
. O |
|
2 O |
|
), O |
|
3Cl B-chemical |
|
, I-chemical |
|
4 I-chemical |
|
- I-chemical |
|
HPA I-chemical |
|
( O |
|
MW O |
|
186 O |
|
. O |
|
6 O |
|
) O |
|
and O |
|
salicylic B-chemical |
|
acid I-chemical |
|
( O |
|
MW O |
|
160 O |
|
. O |
|
1 O |
|
). O |
|
( O |
|
B O |
|
) O |
|
DSC B-experimental_method |
|
profiles B-evidence |
|
, O |
|
colored O |
|
as O |
|
follows O |
|
: O |
|
apo B-protein_state |
|
- O |
|
NadR B-protein |
|
( O |
|
violet O |
|
), O |
|
NadR B-complex_assembly |
|
+ I-complex_assembly |
|
salicylate I-complex_assembly |
|
( O |
|
red O |
|
), O |
|
NadR B-complex_assembly |
|
+ I-complex_assembly |
|
3 I-complex_assembly |
|
- I-complex_assembly |
|
HPA I-complex_assembly |
|
( O |
|
green O |
|
), O |
|
NadR B-complex_assembly |
|
+ I-complex_assembly |
|
4 I-complex_assembly |
|
- I-complex_assembly |
|
HPA I-complex_assembly |
|
( O |
|
blue O |
|
), O |
|
NadR B-complex_assembly |
|
+ I-complex_assembly |
|
3Cl I-complex_assembly |
|
, I-complex_assembly |
|
4 I-complex_assembly |
|
- I-complex_assembly |
|
HPA I-complex_assembly |
|
( O |
|
pink O |
|
). O |
|
|
|
NadR B-protein |
|
displays O |
|
distinct O |
|
binding B-evidence |
|
affinities I-evidence |
|
for O |
|
hydroxyphenylacetate B-chemical |
|
ligands O |
|
|
|
To O |
|
fully O |
|
characterize O |
|
the O |
|
NadR B-protein |
|
/ O |
|
HPA B-chemical |
|
interactions O |
|
, O |
|
we O |
|
sought O |
|
to O |
|
determine O |
|
crystal B-evidence |
|
structures I-evidence |
|
of O |
|
NadR B-protein |
|
in O |
|
ligand B-protein_state |
|
- I-protein_state |
|
bound I-protein_state |
|
( O |
|
holo B-protein_state |
|
) O |
|
and O |
|
ligand B-protein_state |
|
- I-protein_state |
|
free I-protein_state |
|
( O |
|
apo B-protein_state |
|
) O |
|
forms O |
|
. O |
|
|
|
First O |
|
, O |
|
we O |
|
crystallized B-experimental_method |
|
NadR B-protein |
|
( O |
|
a O |
|
selenomethionine B-experimental_method |
|
- I-experimental_method |
|
labelled I-experimental_method |
|
derivative I-experimental_method |
|
) O |
|
in O |
|
the O |
|
presence O |
|
of O |
|
a O |
|
200 O |
|
- O |
|
fold O |
|
molar O |
|
excess O |
|
of O |
|
4 B-chemical |
|
- I-chemical |
|
HPA I-chemical |
|
. O |
|
|
|
A O |
|
single O |
|
conserved B-protein_state |
|
leucine B-residue_name |
|
residue O |
|
( O |
|
L130 B-residue_name_number |
|
) O |
|
is O |
|
crucial O |
|
for O |
|
dimerization O |
|
|
|
The O |
|
NadR B-protein |
|
dimer B-site |
|
interface I-site |
|
is O |
|
formed O |
|
by O |
|
at O |
|
least O |
|
32 O |
|
residues O |
|
, O |
|
which O |
|
establish O |
|
numerous O |
|
inter O |
|
- O |
|
chain O |
|
salt O |
|
bridges O |
|
or O |
|
hydrogen O |
|
bonds O |
|
, O |
|
and O |
|
many O |
|
hydrophobic O |
|
packing O |
|
interactions O |
|
( O |
|
Fig O |
|
3A O |
|
and O |
|
3B O |
|
). O |
|
|
|
Only O |
|
the O |
|
L130K B-mutant |
|
mutation O |
|
induced O |
|
a O |
|
notable O |
|
change O |
|
in O |
|
the O |
|
oligomeric O |
|
state O |
|
of O |
|
NadR B-protein |
|
( O |
|
Fig O |
|
3C O |
|
). O |
|
|
|
Chain B-structure_element |
|
B I-structure_element |
|
, O |
|
grey O |
|
surface O |
|
, O |
|
is O |
|
marked O |
|
blue O |
|
to O |
|
highlight O |
|
residues O |
|
probed O |
|
by O |
|
site B-experimental_method |
|
- I-experimental_method |
|
directed I-experimental_method |
|
mutagenesis I-experimental_method |
|
( O |
|
E136 B-residue_name_number |
|
only O |
|
makes O |
|
a O |
|
salt O |
|
bridge O |
|
with O |
|
K126 B-residue_name_number |
|
, O |
|
therefore O |
|
it O |
|
was O |
|
sufficient O |
|
to O |
|
make O |
|
the O |
|
K126A B-mutant |
|
mutation O |
|
to O |
|
assess O |
|
the O |
|
importance O |
|
of O |
|
this O |
|
ionic O |
|
interaction O |
|
; O |
|
the O |
|
H7 B-residue_name_number |
|
position O |
|
is O |
|
labelled O |
|
for O |
|
monomer B-oligomeric_state |
|
A B-structure_element |
|
, O |
|
since O |
|
electron B-evidence |
|
density I-evidence |
|
was O |
|
lacking O |
|
for O |
|
monomer B-oligomeric_state |
|
B B-structure_element |
|
). O |
|
( O |
|
B O |
|
) O |
|
A O |
|
zoom O |
|
into O |
|
the O |
|
environment O |
|
of O |
|
helix B-structure_element |
|
α6 B-structure_element |
|
to O |
|
show O |
|
how O |
|
residue O |
|
L130 B-residue_name_number |
|
chain B-structure_element |
|
B I-structure_element |
|
( O |
|
blue O |
|
side O |
|
chain O |
|
) O |
|
is O |
|
a O |
|
focus O |
|
of O |
|
hydrophobic O |
|
packing O |
|
interactions O |
|
with O |
|
L130 B-residue_name_number |
|
, O |
|
L133 B-residue_name_number |
|
, O |
|
L134 B-residue_name_number |
|
and O |
|
L137 B-residue_name_number |
|
of O |
|
chain B-structure_element |
|
A I-structure_element |
|
( O |
|
red O |
|
side O |
|
chains O |
|
). O |
|
|
|
* O |
|
Bond O |
|
distance O |
|
between O |
|
the O |
|
ligand O |
|
carboxylate O |
|
group O |
|
and O |
|
the O |
|
water B-chemical |
|
molecule O |
|
, O |
|
which O |
|
in O |
|
turn O |
|
makes O |
|
H O |
|
- O |
|
bond O |
|
to O |
|
the O |
|
SerA9 B-residue_name_number |
|
and O |
|
AsnA11 B-residue_name_number |
|
side O |
|
chains O |
|
. O |
|
|
|
Consequently O |
|
, O |
|
residues O |
|
in O |
|
the O |
|
4 B-site |
|
- I-site |
|
HPA I-site |
|
binding I-site |
|
pocket I-site |
|
are O |
|
mostly O |
|
contributed O |
|
by O |
|
NadR B-protein |
|
chain B-structure_element |
|
B I-structure_element |
|
, O |
|
and O |
|
effectively O |
|
created O |
|
a O |
|
polar O |
|
‘ O |
|
floor O |
|
’ O |
|
and O |
|
a O |
|
hydrophobic O |
|
‘ O |
|
ceiling O |
|
’, O |
|
which O |
|
house O |
|
the O |
|
ligand O |
|
. O |
|
|
|
Collectively O |
|
, O |
|
this O |
|
mixed O |
|
network O |
|
of O |
|
polar O |
|
and O |
|
hydrophobic O |
|
interactions O |
|
endows O |
|
NadR B-protein |
|
with O |
|
a O |
|
strong O |
|
recognition O |
|
pattern O |
|
for O |
|
HPAs B-chemical |
|
, O |
|
with O |
|
additional O |
|
medium O |
|
- O |
|
range O |
|
interactions O |
|
potentially O |
|
established O |
|
with O |
|
the O |
|
hydroxyl O |
|
group O |
|
at O |
|
the O |
|
4 O |
|
- O |
|
position O |
|
. O |
|
|
|
Structure O |
|
- O |
|
activity O |
|
relationships O |
|
: O |
|
molecular O |
|
basis O |
|
of O |
|
enhanced O |
|
stabilization O |
|
by O |
|
3Cl B-chemical |
|
, I-chemical |
|
4 I-chemical |
|
- I-chemical |
|
HPA I-chemical |
|
|
|
We O |
|
modelled B-experimental_method |
|
the O |
|
binding O |
|
of O |
|
other O |
|
HPAs B-chemical |
|
by O |
|
in B-experimental_method |
|
silico I-experimental_method |
|
superposition I-experimental_method |
|
onto O |
|
4 B-chemical |
|
- I-chemical |
|
HPA I-chemical |
|
in O |
|
the O |
|
holo B-protein_state |
|
- O |
|
NadR B-protein |
|
structure B-evidence |
|
, O |
|
and O |
|
thereby O |
|
obtained O |
|
molecular O |
|
explanations O |
|
for O |
|
the O |
|
binding O |
|
specificities O |
|
of O |
|
diverse O |
|
ligands O |
|
. O |
|
|
|
For O |
|
example O |
|
, O |
|
similar O |
|
to O |
|
4 B-chemical |
|
- I-chemical |
|
HPA I-chemical |
|
, O |
|
the O |
|
binding O |
|
of O |
|
3Cl B-chemical |
|
, I-chemical |
|
4 I-chemical |
|
- I-chemical |
|
HPA I-chemical |
|
could O |
|
involve O |
|
multiple O |
|
bonds O |
|
towards O |
|
the O |
|
carboxylate O |
|
group O |
|
of O |
|
the O |
|
ligand O |
|
and O |
|
some O |
|
to O |
|
the O |
|
4 O |
|
- O |
|
hydroxyl O |
|
group O |
|
. O |
|
|
|
Finally O |
|
, O |
|
salicylate B-chemical |
|
is O |
|
presumably O |
|
unable O |
|
to O |
|
specifically O |
|
bind O |
|
NadR B-protein |
|
due O |
|
to O |
|
the O |
|
2 O |
|
- O |
|
hydroxyl O |
|
substitution O |
|
and O |
|
the O |
|
shorter O |
|
aliphatic O |
|
chain O |
|
connecting O |
|
its O |
|
carboxylate O |
|
group O |
|
( O |
|
Fig O |
|
1A O |
|
): O |
|
the O |
|
compound O |
|
simply O |
|
seems O |
|
too O |
|
small O |
|
to O |
|
simultaneously O |
|
establish O |
|
the O |
|
network O |
|
of O |
|
beneficial O |
|
bonds O |
|
observed O |
|
in O |
|
the O |
|
NadR B-protein |
|
/ O |
|
HPA B-chemical |
|
interactions O |
|
. O |
|
|
|
The O |
|
stoichiometry O |
|
of O |
|
the O |
|
NadR B-complex_assembly |
|
- I-complex_assembly |
|
HPA I-complex_assembly |
|
interactions O |
|
was O |
|
determined O |
|
using O |
|
Eq O |
|
1 O |
|
( O |
|
see O |
|
Materials O |
|
and O |
|
Methods O |
|
), O |
|
and O |
|
revealed O |
|
stoichiometries B-evidence |
|
of O |
|
1 O |
|
. O |
|
13 O |
|
for O |
|
4 B-chemical |
|
- I-chemical |
|
HPA I-chemical |
|
, O |
|
1 O |
|
. O |
|
02 O |
|
for O |
|
3 B-chemical |
|
- I-chemical |
|
HPA I-chemical |
|
, O |
|
and O |
|
1 O |
|
. O |
|
21 O |
|
for O |
|
3Cl B-chemical |
|
, I-chemical |
|
4 I-chemical |
|
- I-chemical |
|
HPA I-chemical |
|
, O |
|
strongly O |
|
suggesting O |
|
that O |
|
one O |
|
NadR B-protein |
|
dimer B-oligomeric_state |
|
bound B-protein_state |
|
to I-protein_state |
|
1 O |
|
HPA B-chemical |
|
analyte O |
|
molecule O |
|
. O |
|
|
|
The O |
|
crystallographic B-evidence |
|
data I-evidence |
|
, O |
|
supported O |
|
by O |
|
the O |
|
SPR B-experimental_method |
|
studies O |
|
of O |
|
binding B-evidence |
|
stoichiometry I-evidence |
|
, O |
|
revealed O |
|
the O |
|
lack O |
|
of O |
|
a O |
|
second O |
|
4 B-chemical |
|
- I-chemical |
|
HPA I-chemical |
|
molecule O |
|
in O |
|
the O |
|
homodimer B-oligomeric_state |
|
, O |
|
suggesting O |
|
negative O |
|
co O |
|
- O |
|
operativity O |
|
, O |
|
a O |
|
phenomenon O |
|
previously O |
|
described O |
|
for O |
|
the O |
|
MTH313 B-protein |
|
/ O |
|
salicylate B-chemical |
|
interaction O |
|
and O |
|
for O |
|
other O |
|
MarR B-protein_type |
|
family O |
|
proteins O |
|
. O |
|
|
|
However O |
|
, O |
|
since O |
|
residues O |
|
of O |
|
helix B-structure_element |
|
α6 B-structure_element |
|
were O |
|
not O |
|
directly O |
|
involved O |
|
in O |
|
ligand O |
|
binding O |
|
, O |
|
an O |
|
explanation O |
|
for O |
|
the O |
|
lack O |
|
of O |
|
4 B-chemical |
|
- I-chemical |
|
HPA I-chemical |
|
in O |
|
monomer B-oligomeric_state |
|
A B-structure_element |
|
did O |
|
not O |
|
emerge O |
|
by O |
|
analyzing O |
|
only O |
|
these O |
|
backbone O |
|
atom O |
|
positions O |
|
, O |
|
suggesting O |
|
that O |
|
a O |
|
more O |
|
complex O |
|
series O |
|
of O |
|
allosteric O |
|
events O |
|
may O |
|
occur O |
|
. O |
|
|
|
The O |
|
broad O |
|
spectral O |
|
dispersion O |
|
and O |
|
the O |
|
number O |
|
of O |
|
peaks O |
|
observed O |
|
, O |
|
which O |
|
is O |
|
close O |
|
to O |
|
the O |
|
number O |
|
of O |
|
expected O |
|
backbone O |
|
amide O |
|
N O |
|
- O |
|
H O |
|
groups O |
|
for O |
|
this O |
|
polypeptide O |
|
, O |
|
confirmed O |
|
that O |
|
apo B-protein_state |
|
- O |
|
NadR B-protein |
|
is O |
|
well B-protein_state |
|
- I-protein_state |
|
folded I-protein_state |
|
under O |
|
these O |
|
conditions O |
|
and O |
|
exhibits O |
|
one O |
|
conformation O |
|
appreciable O |
|
on O |
|
the O |
|
NMR B-experimental_method |
|
timescale O |
|
, O |
|
i O |
|
. O |
|
e O |
|
. O |
|
in O |
|
the O |
|
NMR B-experimental_method |
|
experiments O |
|
at O |
|
25 O |
|
° O |
|
C O |
|
, O |
|
two O |
|
or O |
|
more O |
|
distinct O |
|
conformations O |
|
of O |
|
apo B-protein_state |
|
- O |
|
NadR B-protein |
|
monomers B-oligomeric_state |
|
were O |
|
not O |
|
readily O |
|
apparent O |
|
. O |
|
|
|
( O |
|
A O |
|
) O |
|
Superposition B-experimental_method |
|
of O |
|
two O |
|
1H B-experimental_method |
|
- I-experimental_method |
|
15N I-experimental_method |
|
TROSY I-experimental_method |
|
- I-experimental_method |
|
HSQC I-experimental_method |
|
spectra B-evidence |
|
recorded O |
|
at O |
|
25 O |
|
° O |
|
C O |
|
on O |
|
apo B-protein_state |
|
- O |
|
NadR B-protein |
|
( O |
|
cyan O |
|
) O |
|
and O |
|
on O |
|
NadR B-protein |
|
in O |
|
the O |
|
presence B-protein_state |
|
of I-protein_state |
|
4 B-chemical |
|
- I-chemical |
|
HPA I-chemical |
|
( O |
|
red O |
|
). O |
|
|
|
Considering O |
|
the O |
|
small O |
|
size O |
|
, O |
|
fast O |
|
diffusion O |
|
and O |
|
relatively O |
|
low O |
|
binding B-evidence |
|
affinity I-evidence |
|
of O |
|
4 B-chemical |
|
- I-chemical |
|
HPA I-chemical |
|
, O |
|
it O |
|
would O |
|
not O |
|
be O |
|
surprising O |
|
if O |
|
the O |
|
ligand O |
|
associates O |
|
and O |
|
dissociates O |
|
rapidly O |
|
on O |
|
the O |
|
NMR B-experimental_method |
|
time O |
|
scale O |
|
, O |
|
resulting O |
|
in O |
|
only O |
|
one O |
|
set O |
|
of O |
|
peaks O |
|
whose O |
|
chemical O |
|
shifts O |
|
represent O |
|
the O |
|
average O |
|
environment O |
|
of O |
|
the O |
|
bound B-protein_state |
|
and O |
|
unbound B-protein_state |
|
states O |
|
. O |
|
|
|
Overall O |
|
apo B-protein_state |
|
- O |
|
and O |
|
holo B-protein_state |
|
- O |
|
NadR B-protein |
|
structures B-evidence |
|
are O |
|
similar O |
|
. O |
|
|
|
To O |
|
further O |
|
investigate O |
|
the O |
|
conformational O |
|
rearrangements O |
|
of O |
|
NadR B-protein |
|
, O |
|
we O |
|
performed O |
|
local B-experimental_method |
|
structural I-experimental_method |
|
alignments I-experimental_method |
|
using O |
|
only O |
|
a O |
|
subset O |
|
of O |
|
residues O |
|
in O |
|
the O |
|
DNA B-structure_element |
|
- I-structure_element |
|
binding I-structure_element |
|
helix I-structure_element |
|
( O |
|
α4 B-structure_element |
|
). O |
|
|
|
By O |
|
selecting B-experimental_method |
|
and O |
|
aligning B-experimental_method |
|
residues O |
|
Arg64 B-residue_range |
|
- I-residue_range |
|
Ala77 I-residue_range |
|
of O |
|
one O |
|
α4 B-structure_element |
|
helix I-structure_element |
|
per O |
|
dimer B-oligomeric_state |
|
, O |
|
superposition B-experimental_method |
|
of O |
|
the O |
|
holo B-protein_state |
|
- O |
|
homodimer B-oligomeric_state |
|
onto O |
|
the O |
|
two O |
|
apo B-protein_state |
|
- O |
|
homodimers B-oligomeric_state |
|
revealed O |
|
differences O |
|
in O |
|
the O |
|
monomer B-oligomeric_state |
|
conformations O |
|
of O |
|
each O |
|
structure B-evidence |
|
. O |
|
|
|
The O |
|
three O |
|
homodimers B-oligomeric_state |
|
( O |
|
chains O |
|
AB B-structure_element |
|
holo B-protein_state |
|
, O |
|
AB B-structure_element |
|
apo B-protein_state |
|
, O |
|
and O |
|
CD B-structure_element |
|
apo B-protein_state |
|
) O |
|
were O |
|
overlaid B-experimental_method |
|
by O |
|
structural B-experimental_method |
|
alignment I-experimental_method |
|
exclusively O |
|
of O |
|
all O |
|
heavy O |
|
atoms O |
|
in O |
|
residues O |
|
R64 B-residue_range |
|
- I-residue_range |
|
A77 I-residue_range |
|
( O |
|
shown O |
|
in O |
|
red O |
|
, O |
|
with O |
|
side O |
|
chain O |
|
sticks O |
|
) O |
|
of O |
|
chains O |
|
A B-structure_element |
|
holo B-protein_state |
|
, O |
|
A B-structure_element |
|
apo B-protein_state |
|
, O |
|
and O |
|
C B-structure_element |
|
apo B-protein_state |
|
, O |
|
belonging O |
|
to O |
|
helix B-structure_element |
|
α4 B-structure_element |
|
( O |
|
left O |
|
). O |
|
|
|
However O |
|
, O |
|
structural B-experimental_method |
|
comparisons I-experimental_method |
|
revealed O |
|
that O |
|
the O |
|
shift O |
|
of O |
|
holo B-protein_state |
|
- O |
|
NadR B-protein |
|
helix B-structure_element |
|
α4 B-structure_element |
|
induced O |
|
by O |
|
the O |
|
presence B-protein_state |
|
of I-protein_state |
|
4 B-chemical |
|
- I-chemical |
|
HPA I-chemical |
|
was O |
|
also O |
|
accompanied O |
|
by O |
|
several O |
|
changes O |
|
at O |
|
the O |
|
holo B-protein_state |
|
dimer B-site |
|
interface I-site |
|
, O |
|
while O |
|
such O |
|
extensive O |
|
structural O |
|
differences O |
|
were O |
|
not O |
|
observed O |
|
in O |
|
the O |
|
apo B-protein_state |
|
dimer B-site |
|
interfaces I-site |
|
, O |
|
particularly O |
|
notable O |
|
when O |
|
comparing O |
|
the O |
|
α6 B-structure_element |
|
helices I-structure_element |
|
( O |
|
S3 O |
|
Fig O |
|
). O |
|
|
|
Pairwise B-experimental_method |
|
superpositions I-experimental_method |
|
showed O |
|
that O |
|
the O |
|
NadR B-protein |
|
apo B-protein_state |
|
- O |
|
homodimer B-oligomeric_state |
|
AB B-structure_element |
|
was O |
|
the O |
|
most O |
|
similar O |
|
to O |
|
OhrR B-protein |
|
( O |
|
rmsd B-evidence |
|
2 O |
|
. O |
|
6 O |
|
Å O |
|
), O |
|
while O |
|
the O |
|
holo B-protein_state |
|
- O |
|
homodimer B-oligomeric_state |
|
was O |
|
the O |
|
most O |
|
divergent O |
|
( O |
|
rmsd B-evidence |
|
3 O |
|
. O |
|
3 O |
|
Å O |
|
) O |
|
( O |
|
Fig O |
|
8C O |
|
). O |
|
|
|
Interestingly O |
|
, O |
|
and O |
|
on O |
|
the O |
|
contrary O |
|
, O |
|
the O |
|
nadR B-gene |
|
N11A B-mutant |
|
complemented O |
|
strain O |
|
showed O |
|
hypo O |
|
- O |
|
repression O |
|
( O |
|
i O |
|
. O |
|
e O |
|
. O |
|
exhibited O |
|
high O |
|
expression O |
|
of O |
|
nadA B-gene |
|
both O |
|
in O |
|
absence O |
|
and O |
|
presence O |
|
of O |
|
4 B-chemical |
|
- I-chemical |
|
HPA I-chemical |
|
). O |
|
|
|
Western B-experimental_method |
|
blot I-experimental_method |
|
analyses O |
|
of O |
|
wild B-protein_state |
|
- I-protein_state |
|
type I-protein_state |
|
( O |
|
WT B-protein_state |
|
) O |
|
strain O |
|
( O |
|
lanes O |
|
1 O |
|
– O |
|
2 O |
|
) O |
|
or O |
|
isogenic O |
|
nadR B-gene |
|
knockout O |
|
strains O |
|
( O |
|
ΔNadR B-mutant |
|
) O |
|
complemented O |
|
to O |
|
express O |
|
the O |
|
indicated O |
|
NadR B-protein |
|
WT B-protein_state |
|
or O |
|
mutant B-protein_state |
|
proteins O |
|
( O |
|
lanes O |
|
3 O |
|
– O |
|
12 O |
|
) O |
|
or O |
|
not O |
|
complemented O |
|
( O |
|
lanes O |
|
13 O |
|
– O |
|
14 O |
|
), O |
|
grown O |
|
in O |
|
the O |
|
presence O |
|
( O |
|
even O |
|
lanes O |
|
) O |
|
or O |
|
absence O |
|
( O |
|
odd O |
|
lanes O |
|
) O |
|
of O |
|
5mM O |
|
4 B-chemical |
|
- I-chemical |
|
HPA I-chemical |
|
, O |
|
showing O |
|
NadA B-protein |
|
and O |
|
NadR B-protein |
|
expression O |
|
. O |
|
|
|
Complementation O |
|
of O |
|
ΔNadR B-mutant |
|
with O |
|
WT B-protein_state |
|
NadR B-protein |
|
enables O |
|
induction O |
|
of O |
|
nadA B-gene |
|
expression O |
|
by O |
|
4 B-chemical |
|
- I-chemical |
|
HPA I-chemical |
|
. O |
|
|
|
Here O |
|
, O |
|
we O |
|
determined O |
|
the O |
|
first O |
|
crystal B-evidence |
|
structures I-evidence |
|
of O |
|
apo B-protein_state |
|
- O |
|
NadR B-protein |
|
and O |
|
holo B-protein_state |
|
- O |
|
NadR B-protein |
|
. O |
|
These O |
|
experimentally O |
|
- O |
|
determined O |
|
structures B-evidence |
|
enabled O |
|
a O |
|
new O |
|
detailed O |
|
characterization O |
|
of O |
|
the O |
|
ligand B-site |
|
- I-site |
|
binding I-site |
|
pocket I-site |
|
. O |
|
|
|
Subsequently O |
|
, O |
|
we O |
|
established O |
|
the O |
|
functional O |
|
importance O |
|
of O |
|
His7 B-residue_name_number |
|
, O |
|
Ser9 B-residue_name_number |
|
, O |
|
Asn11 B-residue_name_number |
|
and O |
|
Phe25 B-residue_name_number |
|
in O |
|
the O |
|
in O |
|
vitro O |
|
response O |
|
of O |
|
meningococcus B-taxonomy_domain |
|
to O |
|
4 B-chemical |
|
- I-chemical |
|
HPA I-chemical |
|
, O |
|
via O |
|
site B-experimental_method |
|
- I-experimental_method |
|
directed I-experimental_method |
|
mutagenesis I-experimental_method |
|
. O |
|
|
|
( O |
|
B O |
|
) O |
|
A O |
|
structural B-experimental_method |
|
alignment I-experimental_method |
|
of O |
|
MTH313 B-protein |
|
chain B-structure_element |
|
A I-structure_element |
|
and O |
|
ST1710 B-protein |
|
( O |
|
pink O |
|
) O |
|
( O |
|
Cα O |
|
rmsd B-evidence |
|
2 O |
|
. O |
|
3Å O |
|
), O |
|
shows O |
|
that O |
|
they O |
|
bind O |
|
salicylate B-chemical |
|
in O |
|
equivalent O |
|
sites O |
|
( O |
|
differing O |
|
by O |
|
only O |
|
~ O |
|
3Å O |
|
) O |
|
and O |
|
with O |
|
the O |
|
same O |
|
orientation O |
|
. O |
|
|
|
In O |
|
an O |
|
alternative O |
|
and O |
|
less O |
|
extensive O |
|
manner O |
|
, O |
|
the O |
|
binding O |
|
of O |
|
two O |
|
salicylate B-chemical |
|
molecules O |
|
to O |
|
the O |
|
M B-species |
|
. I-species |
|
thermoautotrophicum I-species |
|
protein O |
|
MTH313 B-protein |
|
appeared O |
|
to O |
|
induce O |
|
large O |
|
changes O |
|
in O |
|
the O |
|
wHTH B-structure_element |
|
domain I-structure_element |
|
, O |
|
which O |
|
was O |
|
associated O |
|
with O |
|
reduced O |
|
DNA O |
|
- O |
|
binding O |
|
activity O |
|
. 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 |
|
|
|
This O |
|
reaction O |
|
directly O |
|
links O |
|
an O |
|
acyl B-chemical |
|
- I-chemical |
|
CoA I-chemical |
|
with O |
|
ATP B-chemical |
|
generation O |
|
via O |
|
substrate O |
|
- O |
|
level O |
|
phosphorylation O |
|
, O |
|
producing O |
|
short B-chemical |
|
- I-chemical |
|
chain I-chemical |
|
fatty I-chemical |
|
acids I-chemical |
|
( O |
|
e O |
|
. O |
|
g O |
|
., O |
|
acetate B-chemical |
|
), O |
|
and O |
|
also O |
|
provides O |
|
a O |
|
path O |
|
for O |
|
short B-chemical |
|
- I-chemical |
|
chain I-chemical |
|
fatty I-chemical |
|
acids I-chemical |
|
to O |
|
enter O |
|
central O |
|
metabolism O |
|
. 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 |
|
|
|
More O |
|
recently O |
|
, O |
|
bioinformatic B-experimental_method |
|
studies I-experimental_method |
|
have O |
|
demonstrated O |
|
the O |
|
widespread O |
|
distribution O |
|
of O |
|
BMCs B-complex_assembly |
|
among O |
|
diverse O |
|
bacterial B-taxonomy_domain |
|
phyla I-taxonomy_domain |
|
and O |
|
grouped O |
|
them O |
|
into O |
|
23 O |
|
different O |
|
functional O |
|
types O |
|
. O |
|
|
|
They O |
|
can O |
|
also O |
|
work O |
|
in O |
|
the O |
|
reverse O |
|
direction O |
|
to O |
|
activate O |
|
acetate B-chemical |
|
to O |
|
the O |
|
CoA B-chemical |
|
- I-chemical |
|
thioester I-chemical |
|
. O |
|
|
|
Another O |
|
distinctive O |
|
feature O |
|
of O |
|
BMC B-protein_state |
|
- I-protein_state |
|
associated I-protein_state |
|
PduL B-protein_type |
|
homologs O |
|
is O |
|
an O |
|
N O |
|
- O |
|
terminal O |
|
encapsulation B-structure_element |
|
peptide I-structure_element |
|
( O |
|
EP B-structure_element |
|
) O |
|
that O |
|
is O |
|
thought O |
|
to O |
|
“ O |
|
target O |
|
” O |
|
proteins O |
|
for O |
|
encapsulation O |
|
by O |
|
the O |
|
BMC B-complex_assembly |
|
shell B-structure_element |
|
. O |
|
|
|
The O |
|
primary O |
|
structure O |
|
of O |
|
PduL B-protein_type |
|
homologs O |
|
is O |
|
subdivided O |
|
into O |
|
two O |
|
PF06130 B-structure_element |
|
domains O |
|
, O |
|
each O |
|
roughly O |
|
80 B-residue_range |
|
residues I-residue_range |
|
in I-residue_range |
|
length I-residue_range |
|
. O |
|
|
|
Structure B-experimental_method |
|
Determination I-experimental_method |
|
of O |
|
PduL B-protein_type |
|
|
|
While O |
|
purifying O |
|
full B-protein_state |
|
- I-protein_state |
|
length I-protein_state |
|
sPduL B-protein |
|
, O |
|
we O |
|
observed O |
|
a O |
|
tendency O |
|
to O |
|
aggregation O |
|
as O |
|
described O |
|
previously O |
|
, O |
|
with O |
|
a O |
|
large O |
|
fraction O |
|
of O |
|
the O |
|
expressed O |
|
protein O |
|
found O |
|
in O |
|
the O |
|
insoluble O |
|
fraction O |
|
in O |
|
a O |
|
white O |
|
, O |
|
cake O |
|
- O |
|
like O |
|
pellet O |
|
. O |
|
|
|
( 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 |
|
|
|
The O |
|
first B-residue_range |
|
33 I-residue_range |
|
amino I-residue_range |
|
acids I-residue_range |
|
are O |
|
present O |
|
only O |
|
in O |
|
the O |
|
wildtype O |
|
construct O |
|
and O |
|
contains O |
|
the O |
|
predicted O |
|
EP B-structure_element |
|
alpha B-structure_element |
|
helix I-structure_element |
|
, O |
|
α0 B-structure_element |
|
); O |
|
the O |
|
truncated B-protein_state |
|
rPduLΔEP B-mutant |
|
that O |
|
was O |
|
crystallized B-experimental_method |
|
begins O |
|
with O |
|
M B-residue_name |
|
- O |
|
G B-residue_name |
|
- O |
|
V B-residue_name |
|
. O |
|
Coloring O |
|
is O |
|
according O |
|
to O |
|
structural O |
|
domains O |
|
( O |
|
domain B-structure_element |
|
1 I-structure_element |
|
D36 B-residue_range |
|
- I-residue_range |
|
N46 I-residue_range |
|
/ O |
|
Q155 B-residue_range |
|
- I-residue_range |
|
C224 I-residue_range |
|
, O |
|
blue O |
|
; O |
|
loop B-structure_element |
|
insertion I-structure_element |
|
G61 B-residue_range |
|
- I-residue_range |
|
E81 I-residue_range |
|
, O |
|
grey O |
|
; O |
|
domain B-structure_element |
|
2 I-structure_element |
|
R47 B-residue_range |
|
- I-residue_range |
|
F60 I-residue_range |
|
/ O |
|
E82 B-residue_range |
|
- I-residue_range |
|
A154 I-residue_range |
|
, O |
|
red O |
|
). O |
|
|
|
Using O |
|
a O |
|
mercury B-experimental_method |
|
- I-experimental_method |
|
derivative I-experimental_method |
|
crystal I-experimental_method |
|
form O |
|
diffracting O |
|
to O |
|
1 O |
|
. O |
|
99 O |
|
Å O |
|
( O |
|
Table O |
|
2 O |
|
), O |
|
we O |
|
obtained O |
|
high O |
|
quality O |
|
electron B-evidence |
|
density I-evidence |
|
for O |
|
model O |
|
building O |
|
and O |
|
used O |
|
the O |
|
initial O |
|
model O |
|
to O |
|
refine O |
|
against O |
|
the O |
|
native O |
|
data O |
|
to O |
|
Rwork B-evidence |
|
/ O |
|
Rfree B-evidence |
|
values O |
|
of O |
|
18 O |
|
. O |
|
9 O |
|
/ O |
|
22 O |
|
. O |
|
1 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 |
|
|
|
Consistent O |
|
with O |
|
this O |
|
, O |
|
results O |
|
from O |
|
size B-experimental_method |
|
exclusion I-experimental_method |
|
chromatography I-experimental_method |
|
of O |
|
rPduLΔEP B-mutant |
|
suggest O |
|
that O |
|
it O |
|
is O |
|
a O |
|
dimer B-oligomeric_state |
|
in O |
|
solution O |
|
( O |
|
Fig O |
|
5e O |
|
). O |
|
|
|
The O |
|
asterisk O |
|
and O |
|
double O |
|
arrow O |
|
marks O |
|
the O |
|
location O |
|
of O |
|
the O |
|
π O |
|
– O |
|
π O |
|
interaction O |
|
between O |
|
F116 B-residue_name_number |
|
and O |
|
the O |
|
CoA B-chemical |
|
base O |
|
of O |
|
the O |
|
other O |
|
dimer B-oligomeric_state |
|
chain O |
|
. O |
|
|
|
CoA B-chemical |
|
and O |
|
the O |
|
metal O |
|
ions O |
|
bind O |
|
between O |
|
the O |
|
two O |
|
domains O |
|
, O |
|
presumably O |
|
in O |
|
the O |
|
active B-site |
|
site I-site |
|
( O |
|
Figs O |
|
2b O |
|
and O |
|
4a 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 |
|
|
|
The O |
|
first O |
|
zinc B-chemical |
|
ion O |
|
( O |
|
Zn1 B-chemical |
|
) O |
|
is O |
|
in O |
|
a O |
|
tetrahedral O |
|
coordination O |
|
state O |
|
with O |
|
His48 B-residue_name_number |
|
, O |
|
His50 B-residue_name_number |
|
, O |
|
Glu109 B-residue_name_number |
|
, O |
|
and O |
|
the O |
|
CoA B-chemical |
|
sulfur B-chemical |
|
( O |
|
Fig O |
|
4a O |
|
). O |
|
|
|
Oligomeric O |
|
States O |
|
of O |
|
PduL B-protein_type |
|
Orthologs O |
|
Are O |
|
Influenced O |
|
by O |
|
the O |
|
EP B-structure_element |
|
|
|
In O |
|
contrast O |
|
, O |
|
both O |
|
full B-protein_state |
|
- I-protein_state |
|
length I-protein_state |
|
rPduL B-protein |
|
and O |
|
pPduL B-protein |
|
appeared O |
|
to O |
|
exist O |
|
in O |
|
two O |
|
distinct O |
|
oligomeric O |
|
states O |
|
( O |
|
Fig O |
|
5b O |
|
and O |
|
5c O |
|
respectively O |
|
, O |
|
orange O |
|
curves O |
|
), O |
|
one O |
|
form O |
|
of O |
|
the O |
|
approximate O |
|
size O |
|
of O |
|
a O |
|
dimer B-oligomeric_state |
|
and O |
|
the O |
|
second O |
|
, O |
|
a O |
|
higher O |
|
molecular O |
|
weight O |
|
oligomer B-oligomeric_state |
|
(~ O |
|
150 O |
|
kDa O |
|
). O |
|
|
|
In O |
|
contrast O |
|
, O |
|
rPduLΔEP B-mutant |
|
eluted O |
|
as O |
|
one O |
|
smaller O |
|
oligomer O |
|
, O |
|
possibly O |
|
a O |
|
dimer B-oligomeric_state |
|
. O |
|
|
|
Homologs O |
|
of O |
|
the O |
|
predominant O |
|
cofactor O |
|
utilizer O |
|
( O |
|
aldehyde B-protein_type |
|
dehydrogenase I-protein_type |
|
) O |
|
and O |
|
NAD B-chemical |
|
+ I-chemical |
|
regenerator O |
|
( O |
|
alcohol B-protein_type |
|
dehydrogenase I-protein_type |
|
) O |
|
have O |
|
been O |
|
structurally O |
|
characterized O |
|
, O |
|
but O |
|
until O |
|
now O |
|
structural O |
|
information O |
|
was O |
|
lacking O |
|
for O |
|
PduL B-protein_type |
|
, O |
|
which O |
|
recycles O |
|
CoA B-chemical |
|
in O |
|
the O |
|
organelle O |
|
lumen O |
|
. O |
|
|
|
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 |
|
|
|
For O |
|
BMC B-complex_assembly |
|
- O |
|
encapsulated O |
|
proteins O |
|
to O |
|
properly O |
|
function O |
|
together O |
|
, O |
|
they O |
|
must O |
|
be O |
|
targeted O |
|
to O |
|
the O |
|
lumen O |
|
and O |
|
assemble O |
|
into O |
|
an O |
|
organization O |
|
that O |
|
facilitates O |
|
substrate O |
|
/ O |
|
product O |
|
channeling O |
|
among O |
|
the O |
|
different O |
|
catalytic B-site |
|
sites I-site |
|
of O |
|
the O |
|
signature O |
|
and O |
|
core O |
|
enzymes O |
|
. O |
|
|
|
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 |
|
|
|
The O |
|
close O |
|
resemblance O |
|
between O |
|
the O |
|
structures O |
|
binding O |
|
CoA B-chemical |
|
and O |
|
phosphate B-chemical |
|
likely O |
|
indicates O |
|
that O |
|
no O |
|
large O |
|
changes O |
|
in O |
|
protein O |
|
conformation O |
|
are O |
|
involved O |
|
in O |
|
catalysis O |
|
, O |
|
and O |
|
that O |
|
our O |
|
crystal B-evidence |
|
structures I-evidence |
|
are O |
|
representative O |
|
of O |
|
the O |
|
active B-protein_state |
|
form O |
|
. O |
|
|
|
This O |
|
hypothesis O |
|
is O |
|
strengthened O |
|
by O |
|
the O |
|
fact O |
|
that O |
|
the O |
|
CoA B-protein_state |
|
- I-protein_state |
|
bound I-protein_state |
|
crystals B-evidence |
|
were O |
|
obtained O |
|
without O |
|
added O |
|
CoA B-chemical |
|
, O |
|
indicating O |
|
that O |
|
the O |
|
protein O |
|
bound B-protein_state |
|
CoA B-chemical |
|
from O |
|
the O |
|
E B-species |
|
. I-species |
|
coli I-species |
|
expression O |
|
strain O |
|
and O |
|
retained O |
|
it O |
|
throughout O |
|
purification O |
|
and O |
|
crystallization O |
|
. 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 |
|
|
|
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 |
|
|
|
These O |
|
results O |
|
suggest O |
|
that O |
|
Regnase B-protein |
|
- I-protein |
|
1 I-protein |
|
RNase B-protein_type |
|
activity O |
|
is O |
|
tightly O |
|
controlled O |
|
by O |
|
both O |
|
intramolecular O |
|
( O |
|
NTD B-structure_element |
|
- O |
|
PIN B-structure_element |
|
) O |
|
and O |
|
intermolecular O |
|
( O |
|
PIN B-structure_element |
|
- O |
|
PIN B-structure_element |
|
) O |
|
interactions O |
|
. O |
|
|
|
The O |
|
initial O |
|
sensing O |
|
of O |
|
infection O |
|
is O |
|
mediated O |
|
by O |
|
a O |
|
set O |
|
of O |
|
pattern B-protein_type |
|
- I-protein_type |
|
recognition I-protein_type |
|
receptors I-protein_type |
|
( O |
|
PRRs B-protein_type |
|
) O |
|
such O |
|
Toll B-protein_type |
|
- I-protein_type |
|
like I-protein_type |
|
receptors I-protein_type |
|
( O |
|
TLRs B-protein_type |
|
) O |
|
and O |
|
the O |
|
intracellular O |
|
signaling O |
|
cascades O |
|
triggered O |
|
by O |
|
TLRs B-protein_type |
|
evoke O |
|
transcriptional O |
|
expression O |
|
of O |
|
inflammatory O |
|
mediators O |
|
that O |
|
coordinate O |
|
the O |
|
elimination O |
|
of O |
|
pathogens O |
|
and O |
|
infected O |
|
cells O |
|
. O |
|
|
|
Our O |
|
data O |
|
revealed O |
|
that O |
|
the O |
|
catalytic O |
|
activity O |
|
of O |
|
Regnase B-protein |
|
- I-protein |
|
1 I-protein |
|
is O |
|
regulated O |
|
through O |
|
both O |
|
intra O |
|
and O |
|
intermolecular O |
|
domain O |
|
interactions O |
|
in O |
|
vitro O |
|
. O |
|
|
|
X B-experimental_method |
|
- I-experimental_method |
|
ray I-experimental_method |
|
crystallography I-experimental_method |
|
was O |
|
attempted O |
|
for O |
|
the O |
|
fragment O |
|
containing O |
|
both O |
|
the O |
|
PIN B-structure_element |
|
and O |
|
ZF B-structure_element |
|
domains O |
|
, O |
|
however O |
|
, O |
|
electron B-evidence |
|
density I-evidence |
|
was O |
|
observed O |
|
only O |
|
for O |
|
the O |
|
PIN B-structure_element |
|
domain O |
|
( O |
|
Fig O |
|
. O |
|
1c O |
|
), O |
|
consistent O |
|
with O |
|
a O |
|
previous O |
|
report O |
|
on O |
|
Regnase B-protein |
|
- I-protein |
|
1 I-protein |
|
derived O |
|
from O |
|
Homo B-species |
|
sapiens I-species |
|
. O |
|
|
|
The O |
|
domain O |
|
structures B-evidence |
|
of O |
|
NTD B-structure_element |
|
, O |
|
ZF B-structure_element |
|
, O |
|
and O |
|
CTD B-structure_element |
|
were O |
|
determined O |
|
by O |
|
NMR B-experimental_method |
|
( O |
|
Fig O |
|
. O |
|
1b O |
|
, O |
|
d O |
|
, O |
|
e O |
|
). O |
|
|
|
Although O |
|
the O |
|
PIN B-structure_element |
|
domain O |
|
is O |
|
responsible O |
|
for O |
|
the O |
|
catalytic O |
|
activity O |
|
of O |
|
Regnase B-protein |
|
- I-protein |
|
1 I-protein |
|
, O |
|
the O |
|
roles O |
|
of O |
|
the O |
|
other O |
|
domains O |
|
are O |
|
largely O |
|
unknown O |
|
. O |
|
|
|
Upon O |
|
addition O |
|
of O |
|
a O |
|
larger O |
|
amount O |
|
of O |
|
Regnase B-protein |
|
- I-protein |
|
1 I-protein |
|
, O |
|
the O |
|
fluorescence B-evidence |
|
of O |
|
free B-protein_state |
|
RNA B-chemical |
|
decreased O |
|
, O |
|
indicating O |
|
that O |
|
Regnase B-protein |
|
- I-protein |
|
1 I-protein |
|
bound B-protein_state |
|
to I-protein_state |
|
the O |
|
RNA B-chemical |
|
. O |
|
|
|
Direct O |
|
binding O |
|
of O |
|
the O |
|
ZF B-structure_element |
|
domain O |
|
and O |
|
RNA B-chemical |
|
were O |
|
confirmed O |
|
by O |
|
NMR B-experimental_method |
|
spectral B-evidence |
|
changes I-evidence |
|
. O |
|
|
|
Dimer B-oligomeric_state |
|
formation O |
|
of O |
|
the O |
|
PIN B-structure_element |
|
domains O |
|
|
|
Mutation B-experimental_method |
|
of O |
|
Arg215 B-residue_name_number |
|
, O |
|
whose O |
|
side O |
|
chain O |
|
faces O |
|
to O |
|
the O |
|
opposite O |
|
side O |
|
of O |
|
the O |
|
oligomeric B-site |
|
surface I-site |
|
, O |
|
to O |
|
Glu B-residue_name |
|
preserved O |
|
the O |
|
monomer B-oligomeric_state |
|
/ O |
|
dimer B-oligomeric_state |
|
equilibrium O |
|
, O |
|
similar O |
|
to O |
|
the O |
|
wild B-protein_state |
|
type I-protein_state |
|
. O |
|
|
|
Based O |
|
on O |
|
the O |
|
titration B-evidence |
|
curve I-evidence |
|
for O |
|
the O |
|
chemical B-evidence |
|
shift I-evidence |
|
changes I-evidence |
|
of O |
|
L58 B-residue_name_number |
|
, O |
|
the O |
|
apparent O |
|
Kd B-evidence |
|
between O |
|
the O |
|
isolated O |
|
NTD B-structure_element |
|
and O |
|
PIN B-structure_element |
|
was O |
|
estimated O |
|
to O |
|
be O |
|
110 O |
|
± O |
|
5 O |
|
. O |
|
8 O |
|
μM O |
|
. O |
|
Considering O |
|
the O |
|
fact O |
|
that O |
|
the O |
|
NTD B-structure_element |
|
and O |
|
PIN B-structure_element |
|
domains O |
|
are O |
|
attached O |
|
by O |
|
a O |
|
linker B-structure_element |
|
, O |
|
the O |
|
actual O |
|
binding B-evidence |
|
affinity I-evidence |
|
is O |
|
expected O |
|
much O |
|
higher O |
|
in O |
|
the O |
|
native B-protein_state |
|
protein O |
|
. O |
|
|
|
An O |
|
in B-experimental_method |
|
silico I-experimental_method |
|
docking I-experimental_method |
|
of O |
|
the O |
|
NTD B-structure_element |
|
and O |
|
PIN B-structure_element |
|
domains O |
|
using O |
|
chemical B-evidence |
|
shift I-evidence |
|
restraints I-evidence |
|
provided O |
|
a O |
|
model O |
|
consistent O |
|
with O |
|
the O |
|
NMR B-experimental_method |
|
experiments O |
|
( O |
|
Fig O |
|
. O |
|
3c O |
|
). O |
|
|
|
When O |
|
any O |
|
members O |
|
of O |
|
the O |
|
two O |
|
groups O |
|
are O |
|
mixed O |
|
, O |
|
two O |
|
kinds O |
|
of O |
|
heterodimers B-oligomeric_state |
|
can O |
|
be O |
|
formed O |
|
: O |
|
one O |
|
is O |
|
composed O |
|
of O |
|
a O |
|
DDNN B-mutant |
|
primary B-protein_state |
|
PIN B-structure_element |
|
and O |
|
a O |
|
basic O |
|
residue O |
|
mutant B-protein_state |
|
secondary B-protein_state |
|
PIN B-structure_element |
|
and O |
|
is O |
|
expected O |
|
to O |
|
exhibit O |
|
no O |
|
RNase B-protein_type |
|
activity O |
|
; O |
|
the O |
|
other O |
|
is O |
|
composed O |
|
of O |
|
a O |
|
basic O |
|
residue O |
|
mutant B-protein_state |
|
primary B-protein_state |
|
PIN B-structure_element |
|
and O |
|
a O |
|
DDNN B-mutant |
|
secondary B-protein_state |
|
PIN B-structure_element |
|
and O |
|
is O |
|
predicted O |
|
to O |
|
rescue O |
|
RNase B-protein_type |
|
activity O |
|
( O |
|
Fig O |
|
. O |
|
5a O |
|
). O |
|
|
|
When O |
|
we O |
|
compared O |
|
the O |
|
fluorescence B-evidence |
|
intensity I-evidence |
|
of O |
|
uncleaved B-protein_state |
|
IL B-protein_type |
|
- I-protein_type |
|
6 I-protein_type |
|
mRNA B-chemical |
|
, O |
|
basic O |
|
residue O |
|
mutants B-protein_state |
|
W182A B-mutant |
|
, O |
|
K184A B-mutant |
|
, O |
|
R214A B-mutant |
|
, O |
|
and O |
|
R220A B-mutant |
|
were O |
|
rescued O |
|
upon O |
|
addition O |
|
of O |
|
the O |
|
DDNN B-mutant |
|
mutant B-protein_state |
|
( O |
|
Fig O |
|
. O |
|
5b O |
|
). O |
|
|
|
Rescue O |
|
of O |
|
K184A B-mutant |
|
and O |
|
R214A B-mutant |
|
by O |
|
the O |
|
DDNN B-mutant |
|
mutant B-protein_state |
|
was O |
|
also O |
|
confirmed O |
|
by O |
|
a O |
|
significant O |
|
increase O |
|
in O |
|
the O |
|
cleaved O |
|
products O |
|
. O |
|
|
|
R214 B-residue_name_number |
|
is O |
|
an O |
|
important O |
|
residue O |
|
for O |
|
dimer B-oligomeric_state |
|
formation O |
|
as O |
|
shown O |
|
in O |
|
Fig O |
|
. O |
|
2 O |
|
, O |
|
therefore O |
|
, O |
|
R214A B-mutant |
|
in O |
|
the O |
|
secondary B-protein_state |
|
PIN B-structure_element |
|
cannot O |
|
dimerize O |
|
. O |
|
|
|
Due O |
|
to O |
|
this O |
|
limitation O |
|
, O |
|
it O |
|
is O |
|
difficult O |
|
to O |
|
perform O |
|
further O |
|
structural B-experimental_method |
|
analyses I-experimental_method |
|
of O |
|
mRNA B-complex_assembly |
|
- I-complex_assembly |
|
Regnase I-complex_assembly |
|
- I-complex_assembly |
|
1 I-complex_assembly |
|
complexes O |
|
by O |
|
X B-experimental_method |
|
- I-experimental_method |
|
ray I-experimental_method |
|
crystallography I-experimental_method |
|
or O |
|
NMR B-experimental_method |
|
. O |
|
|
|
Moreover O |
|
, O |
|
our O |
|
structure B-experimental_method |
|
- I-experimental_method |
|
based I-experimental_method |
|
mutational I-experimental_method |
|
analyses I-experimental_method |
|
showed O |
|
these O |
|
two O |
|
Regnase B-protein |
|
- I-protein |
|
1 I-protein |
|
specific O |
|
basic O |
|
regions O |
|
were O |
|
essential O |
|
for O |
|
target O |
|
mRNA B-chemical |
|
cleavage O |
|
in O |
|
vitro O |
|
. O |
|
|
|
The O |
|
affinity B-evidence |
|
of O |
|
the O |
|
domain O |
|
- O |
|
domain O |
|
interaction O |
|
between O |
|
two O |
|
PIN B-structure_element |
|
domains O |
|
( O |
|
Kd B-evidence |
|
= O |
|
~ O |
|
10 O |
|
− O |
|
4 O |
|
M O |
|
) O |
|
is O |
|
similar O |
|
to O |
|
that O |
|
of O |
|
the O |
|
NTD B-structure_element |
|
- O |
|
PIN B-structure_element |
|
( O |
|
Kd B-evidence |
|
= O |
|
110 O |
|
± O |
|
5 O |
|
. O |
|
8 O |
|
μM O |
|
) O |
|
interactions O |
|
; O |
|
however O |
|
, O |
|
the O |
|
covalent O |
|
connection O |
|
corresponding O |
|
to O |
|
residues O |
|
90 B-residue_range |
|
– I-residue_range |
|
133 I-residue_range |
|
between O |
|
the O |
|
NTD B-structure_element |
|
and O |
|
the O |
|
primary B-protein_state |
|
PIN B-structure_element |
|
will O |
|
greatly O |
|
enhance O |
|
the O |
|
intramolecular O |
|
domain O |
|
interaction O |
|
in O |
|
the O |
|
case O |
|
of O |
|
full B-protein_state |
|
- I-protein_state |
|
length I-protein_state |
|
Regnase B-protein |
|
- I-protein |
|
1 I-protein |
|
. O |
|
|
|
In O |
|
this O |
|
context O |
|
, O |
|
it O |
|
is O |
|
interesting O |
|
that O |
|
, O |
|
in O |
|
response O |
|
to O |
|
TCR O |
|
stimulation O |
|
, O |
|
Malt1 B-protein |
|
cleaves O |
|
Regnase B-protein |
|
- I-protein |
|
1 I-protein |
|
at O |
|
R111 B-residue_name_number |
|
to O |
|
control O |
|
immune O |
|
responses O |
|
in O |
|
vivo O |
|
. O |
|
|
|
Two O |
|
PIN B-structure_element |
|
molecules O |
|
in O |
|
the O |
|
crystal B-evidence |
|
were O |
|
colored O |
|
white O |
|
and O |
|
green O |
|
, O |
|
respectively O |
|
. O |
|
|
|
Eukaryotic B-taxonomy_domain |
|
ribosome O |
|
biogenesis O |
|
is O |
|
highly O |
|
complex O |
|
and O |
|
requires O |
|
a O |
|
large O |
|
number O |
|
of O |
|
non O |
|
- O |
|
ribosomal O |
|
proteins O |
|
and O |
|
small B-chemical |
|
non I-chemical |
|
- I-chemical |
|
coding I-chemical |
|
RNAs I-chemical |
|
in O |
|
addition O |
|
to O |
|
ribosomal B-chemical |
|
RNAs I-chemical |
|
( O |
|
rRNAs B-chemical |
|
) O |
|
and O |
|
proteins O |
|
. O |
|
|
|
In O |
|
addition O |
|
, O |
|
18S B-chemical |
|
and O |
|
25S B-chemical |
|
( O |
|
yeast B-taxonomy_domain |
|
)/ O |
|
28S B-chemical |
|
( O |
|
humans B-species |
|
) O |
|
rRNAs B-chemical |
|
contain O |
|
several O |
|
base O |
|
modifications O |
|
catalyzed O |
|
by O |
|
site O |
|
- O |
|
specific O |
|
and O |
|
snoRNA B-chemical |
|
- O |
|
independent O |
|
enzymes O |
|
. O |
|
|
|
In O |
|
Saccharomyces B-species |
|
cerevisiae I-species |
|
18S B-chemical |
|
rRNA I-chemical |
|
contains O |
|
four O |
|
base O |
|
methylations B-ptm |
|
, O |
|
two O |
|
acetylations B-ptm |
|
and O |
|
a O |
|
single O |
|
3 B-chemical |
|
- I-chemical |
|
amino I-chemical |
|
- I-chemical |
|
3 I-chemical |
|
- I-chemical |
|
carboxypropyl I-chemical |
|
( O |
|
acp B-chemical |
|
) O |
|
modification O |
|
, O |
|
whereas O |
|
six O |
|
base O |
|
methylations B-ptm |
|
are O |
|
present O |
|
in O |
|
the O |
|
25S B-chemical |
|
rRNA I-chemical |
|
. O |
|
|
|
Defects O |
|
of O |
|
rRNA B-chemical |
|
modification O |
|
enzymes O |
|
often O |
|
lead O |
|
to O |
|
disturbed O |
|
ribosome O |
|
biogenesis O |
|
or O |
|
functionally O |
|
impaired O |
|
ribosomes O |
|
, O |
|
although O |
|
the O |
|
lack O |
|
of O |
|
individual O |
|
rRNA B-chemical |
|
modifications O |
|
often O |
|
has O |
|
no O |
|
or O |
|
only O |
|
a O |
|
slight O |
|
influence O |
|
on O |
|
the O |
|
cell O |
|
. O |
|
|
|
Wild B-protein_state |
|
type I-protein_state |
|
( O |
|
WT B-protein_state |
|
) O |
|
and O |
|
plasmid O |
|
encoded O |
|
18S B-chemical |
|
rRNA I-chemical |
|
( O |
|
U1191U B-mutant |
|
) O |
|
show O |
|
the O |
|
14C B-chemical |
|
- I-chemical |
|
acp I-chemical |
|
signal O |
|
, O |
|
whereas O |
|
the O |
|
14C B-chemical |
|
- I-chemical |
|
acp I-chemical |
|
signal O |
|
is O |
|
missing O |
|
in O |
|
the O |
|
U1191A B-mutant |
|
mutant B-protein_state |
|
plasmid O |
|
encoded O |
|
18S B-chemical |
|
rRNA I-chemical |
|
( O |
|
U1191A B-mutant |
|
) O |
|
and O |
|
Δtsr3 B-mutant |
|
mutants O |
|
( O |
|
Δtsr3 B-mutant |
|
). O |
|
|
|
The O |
|
primer O |
|
extension O |
|
arrest O |
|
is O |
|
reduced O |
|
in O |
|
HTC116 O |
|
cells O |
|
transfected O |
|
with O |
|
siRNAs B-chemical |
|
544 O |
|
and O |
|
545 O |
|
. O |
|
|
|
For O |
|
the O |
|
Δtsr3 B-mutant |
|
deletion O |
|
strain O |
|
the O |
|
HPLC B-evidence |
|
elution I-evidence |
|
profile I-evidence |
|
of O |
|
18S B-chemical |
|
rRNA I-chemical |
|
nucleosides B-chemical |
|
( O |
|
Figure O |
|
1B O |
|
) O |
|
was O |
|
very O |
|
similar O |
|
to O |
|
that O |
|
of O |
|
the O |
|
pseudouridine B-protein_type |
|
- I-protein_type |
|
N1 I-protein_type |
|
methyltransferase I-protein_type |
|
mutant B-protein_state |
|
Δnep1 B-mutant |
|
, O |
|
where O |
|
a O |
|
shoulder O |
|
at O |
|
∼ O |
|
7 O |
|
. O |
|
4 O |
|
min O |
|
elution O |
|
time O |
|
was O |
|
missing O |
|
in O |
|
the O |
|
elution O |
|
profile O |
|
. O |
|
|
|
As O |
|
previously O |
|
reported O |
|
this O |
|
shoulder O |
|
was O |
|
identified O |
|
by O |
|
ESI B-experimental_method |
|
- I-experimental_method |
|
MS I-experimental_method |
|
as O |
|
corresponding O |
|
to O |
|
m1acp3Ψ B-chemical |
|
. O |
|
|
|
Similar O |
|
to O |
|
yeast B-taxonomy_domain |
|
, O |
|
siRNA B-experimental_method |
|
- I-experimental_method |
|
mediated I-experimental_method |
|
depletion I-experimental_method |
|
of O |
|
the O |
|
Ψ1248 B-protein_type |
|
N1 I-protein_type |
|
- I-protein_type |
|
methyltransferase I-protein_type |
|
Nep1 B-protein |
|
/ O |
|
Emg1 B-protein |
|
had O |
|
no O |
|
influence O |
|
on O |
|
the O |
|
primer B-evidence |
|
extension I-evidence |
|
arrest I-evidence |
|
( O |
|
Figure O |
|
1E O |
|
). O |
|
|
|
However O |
|
, O |
|
the O |
|
Δtsr3 B-mutant |
|
deletion O |
|
was O |
|
synthetic O |
|
sick O |
|
with O |
|
a O |
|
Δsnr35 B-mutant |
|
deletion O |
|
preventing O |
|
pseudouridylation B-ptm |
|
and O |
|
Nep1 B-protein |
|
- O |
|
catalyzed O |
|
methylation O |
|
of O |
|
nucleotide O |
|
1191 B-residue_number |
|
( O |
|
Figure O |
|
2A O |
|
). O |
|
|
|
Phenotypic O |
|
characterization O |
|
of O |
|
yeast B-taxonomy_domain |
|
TSR3 B-protein |
|
deletion O |
|
( O |
|
Δtrs3 B-mutant |
|
) O |
|
and O |
|
human B-species |
|
TSR3 B-protein |
|
depletion O |
|
( O |
|
siRNAs B-chemical |
|
544 O |
|
and O |
|
545 O |
|
) O |
|
and O |
|
cellular O |
|
localization O |
|
of O |
|
yeast B-taxonomy_domain |
|
Tsr3 B-protein |
|
. O |
|
( O |
|
A O |
|
) O |
|
Growth O |
|
of O |
|
yeast B-taxonomy_domain |
|
wild B-protein_state |
|
type I-protein_state |
|
, O |
|
Δtsr3 B-mutant |
|
, O |
|
Δsnr35 B-mutant |
|
and O |
|
Δtsr3 B-mutant |
|
Δsnr35 I-mutant |
|
segregants O |
|
after O |
|
meiosis O |
|
and O |
|
tetrad O |
|
dissection O |
|
of O |
|
Δtsr3 B-mutant |
|
/ O |
|
TSR3 B-protein |
|
Δsnr35 B-mutant |
|
/ O |
|
SNR35 B-protein |
|
heterozygous O |
|
diploids O |
|
. O |
|
|
|
Consistent O |
|
with O |
|
its O |
|
role O |
|
in O |
|
late O |
|
18S B-chemical |
|
rRNA I-chemical |
|
processing O |
|
, O |
|
TSR3 B-protein |
|
deletion O |
|
leads O |
|
to O |
|
a O |
|
ribosomal O |
|
subunit O |
|
imbalance O |
|
with O |
|
a O |
|
reduced O |
|
40S B-complex_assembly |
|
to O |
|
60S B-complex_assembly |
|
ratio O |
|
of O |
|
0 O |
|
. O |
|
81 O |
|
( O |
|
σ O |
|
= O |
|
0 O |
|
. O |
|
024 O |
|
) O |
|
which O |
|
was O |
|
further O |
|
increased O |
|
in O |
|
a O |
|
Δtsr3 B-mutant |
|
Δsnr35 I-mutant |
|
recombinant O |
|
to O |
|
0 O |
|
. O |
|
73 O |
|
( O |
|
σ O |
|
= O |
|
0 O |
|
. O |
|
023 O |
|
) O |
|
( O |
|
Supplementary O |
|
Figure O |
|
S2F O |
|
). O |
|
|
|
N O |
|
- O |
|
terminal O |
|
deletions B-experimental_method |
|
of O |
|
36 B-residue_range |
|
or O |
|
45 B-residue_range |
|
amino O |
|
acids O |
|
and O |
|
C O |
|
- O |
|
terminal O |
|
deletions B-experimental_method |
|
of O |
|
43 B-residue_range |
|
or O |
|
76 B-residue_range |
|
residues O |
|
show O |
|
a O |
|
primer B-evidence |
|
extension I-evidence |
|
stop I-evidence |
|
comparable O |
|
to O |
|
the O |
|
wild B-protein_state |
|
type I-protein_state |
|
. O |
|
|
|
Strong O |
|
20S O |
|
rRNA O |
|
accumulation O |
|
similar O |
|
to O |
|
that O |
|
of O |
|
the O |
|
Δtsr3 B-mutant |
|
deletion B-experimental_method |
|
is O |
|
observed O |
|
for O |
|
Tsr3 B-protein |
|
fragments O |
|
37 B-residue_range |
|
– I-residue_range |
|
223 I-residue_range |
|
or O |
|
46 B-residue_range |
|
– I-residue_range |
|
223 I-residue_range |
|
. O |
|
|
|
Well O |
|
diffracting O |
|
crystals B-evidence |
|
were O |
|
obtained O |
|
for O |
|
Tsr3 B-protein |
|
homologs O |
|
from O |
|
the O |
|
two O |
|
crenarchaeal B-taxonomy_domain |
|
species O |
|
Vulcanisaeta B-species |
|
distributa I-species |
|
( O |
|
VdTsr3 B-protein |
|
) O |
|
and O |
|
Sulfolobus B-species |
|
solfataricus I-species |
|
( O |
|
SsTsr3 B-protein |
|
) O |
|
which O |
|
share O |
|
36 O |
|
% O |
|
( O |
|
VdTsr3 B-protein |
|
) O |
|
and O |
|
38 O |
|
% O |
|
( O |
|
SsTsr3 B-protein |
|
) O |
|
identity O |
|
with O |
|
the O |
|
ScTsr3 B-protein |
|
core B-structure_element |
|
region I-structure_element |
|
( O |
|
ScTsr3 B-protein |
|
aa O |
|
46 B-residue_range |
|
– I-residue_range |
|
223 I-residue_range |
|
). O |
|
|
|
Crystals B-evidence |
|
of O |
|
VdTsr3 B-protein |
|
diffracted O |
|
to O |
|
a O |
|
resolution O |
|
of O |
|
1 O |
|
. O |
|
6 O |
|
Å O |
|
whereas O |
|
crystals B-evidence |
|
of O |
|
SsTsr3 B-protein |
|
diffracted O |
|
to O |
|
2 O |
|
. O |
|
25 O |
|
Å O |
|
. O |
|
Serendipitously O |
|
, O |
|
VdTsr3 B-protein |
|
was O |
|
purified O |
|
and O |
|
crystallized B-experimental_method |
|
in B-protein_state |
|
complex I-protein_state |
|
with I-protein_state |
|
endogenous B-protein_state |
|
( O |
|
E B-species |
|
. I-species |
|
coli I-species |
|
) O |
|
SAM B-chemical |
|
( O |
|
Supplementary O |
|
Figure O |
|
S4 O |
|
) O |
|
while O |
|
SsTsr3 B-protein |
|
crystals B-evidence |
|
contained O |
|
the O |
|
protein O |
|
in O |
|
the O |
|
apo B-protein_state |
|
state O |
|
. O |
|
|
|
The O |
|
structure B-evidence |
|
of O |
|
VdTsr3 B-protein |
|
can O |
|
be O |
|
divided O |
|
into O |
|
two O |
|
domains O |
|
( O |
|
Figure O |
|
4A O |
|
). O |
|
|
|
A O |
|
red O |
|
arrow O |
|
marks O |
|
the O |
|
location O |
|
of O |
|
the O |
|
topological B-structure_element |
|
knot I-structure_element |
|
in O |
|
the O |
|
structure B-evidence |
|
. O |
|
( O |
|
B O |
|
) O |
|
Secondary O |
|
structure O |
|
representation O |
|
of O |
|
the O |
|
VdTsr3 B-protein |
|
structure B-evidence |
|
. O |
|
|
|
Gel B-experimental_method |
|
filtration I-experimental_method |
|
experiments O |
|
with O |
|
both O |
|
VdTsr3 B-protein |
|
and O |
|
SsTsr3 B-protein |
|
( O |
|
Figure O |
|
4E O |
|
) O |
|
showed O |
|
that O |
|
both O |
|
proteins O |
|
are O |
|
monomeric B-oligomeric_state |
|
in O |
|
solution O |
|
thereby O |
|
extending O |
|
the O |
|
structural O |
|
similarities O |
|
to O |
|
Trm10 B-protein |
|
. O |
|
|
|
This O |
|
enzyme O |
|
, O |
|
Tyw2 B-protein |
|
, O |
|
is O |
|
part O |
|
of O |
|
the O |
|
biosynthesis O |
|
pathway O |
|
of O |
|
wybutosine B-chemical |
|
nucleotides I-chemical |
|
in O |
|
tRNAs B-chemical |
|
. O |
|
|
|
SAM B-chemical |
|
- O |
|
binding O |
|
by O |
|
Tsr3 B-protein |
|
. O |
|
|
|
3xHA B-protein_state |
|
tagged I-protein_state |
|
Tsr3 B-protein |
|
mutants B-protein_state |
|
are O |
|
expressed O |
|
comparable O |
|
to O |
|
the O |
|
wild B-protein_state |
|
type I-protein_state |
|
as O |
|
shown O |
|
by O |
|
western B-experimental_method |
|
blot I-experimental_method |
|
( O |
|
lower O |
|
left O |
|
). O |
|
|
|
Accordingly O |
|
, O |
|
a O |
|
W66A B-mutant |
|
- O |
|
mutation B-experimental_method |
|
( O |
|
W73 B-residue_name_number |
|
in O |
|
VdTsr3 B-protein |
|
) O |
|
of O |
|
SsTsr3 B-protein |
|
significantly O |
|
diminished O |
|
SAM B-evidence |
|
- I-evidence |
|
binding I-evidence |
|
in O |
|
a O |
|
filter B-experimental_method |
|
binding I-experimental_method |
|
assay I-experimental_method |
|
compared O |
|
to O |
|
the O |
|
wild B-protein_state |
|
type I-protein_state |
|
( O |
|
Figure O |
|
5E O |
|
). O |
|
|
|
Furthermore O |
|
, O |
|
a O |
|
W B-experimental_method |
|
to I-experimental_method |
|
A I-experimental_method |
|
mutation I-experimental_method |
|
at O |
|
the O |
|
equivalent O |
|
position O |
|
W114 B-residue_name_number |
|
in O |
|
ScTsr3 B-protein |
|
strongly O |
|
reduced O |
|
the O |
|
in O |
|
vivo O |
|
acp B-protein_type |
|
transferase I-protein_type |
|
activity O |
|
( O |
|
Figure O |
|
5F O |
|
). O |
|
|
|
Furthermore O |
|
, O |
|
a O |
|
negatively O |
|
charged O |
|
MES B-chemical |
|
- O |
|
ion O |
|
is O |
|
found O |
|
in O |
|
the O |
|
crystal B-evidence |
|
structure I-evidence |
|
of O |
|
VdTsr3 B-protein |
|
complexed B-protein_state |
|
to I-protein_state |
|
the O |
|
side O |
|
chain O |
|
of O |
|
K22 B-residue_name_number |
|
in O |
|
helix B-structure_element |
|
α1 B-structure_element |
|
. O |
|
|
|
In O |
|
the O |
|
C B-structure_element |
|
- I-structure_element |
|
terminal I-structure_element |
|
domain I-structure_element |
|
, O |
|
the O |
|
surface O |
|
exposed O |
|
α B-structure_element |
|
- I-structure_element |
|
helices I-structure_element |
|
α5 B-structure_element |
|
and O |
|
α7 B-structure_element |
|
carry O |
|
a O |
|
significant O |
|
amount O |
|
of O |
|
positively O |
|
charged O |
|
amino O |
|
acids O |
|
. O |
|
|
|
For O |
|
S B-species |
|
. I-species |
|
solfataricus I-species |
|
the O |
|
chemical O |
|
identity O |
|
of O |
|
the O |
|
hypermodified B-protein_state |
|
nucleotide B-chemical |
|
is O |
|
not O |
|
known O |
|
but O |
|
the O |
|
existence O |
|
of O |
|
NEP1 B-protein |
|
and O |
|
TSR3 B-protein |
|
homologs O |
|
suggest O |
|
that O |
|
it O |
|
is O |
|
indeed O |
|
N1 B-chemical |
|
- I-chemical |
|
methyl I-chemical |
|
- I-chemical |
|
N3 I-chemical |
|
- I-chemical |
|
acp I-chemical |
|
- I-chemical |
|
pseudouridine I-chemical |
|
. O |
|
|
|
5 O |
|
′- O |
|
fluoresceine B-chemical |
|
labeled O |
|
RNA B-chemical |
|
oligonucleotides O |
|
corresponding O |
|
either O |
|
to O |
|
the O |
|
native B-protein_state |
|
( O |
|
20mer B-oligomeric_state |
|
– O |
|
see O |
|
inset O |
|
) O |
|
or O |
|
a O |
|
stabilized B-protein_state |
|
( O |
|
20mer_GC B-oligomeric_state |
|
- O |
|
inset O |
|
) O |
|
helix B-structure_element |
|
31 I-structure_element |
|
of O |
|
the O |
|
small O |
|
ribosomal O |
|
subunit O |
|
rRNA B-chemical |
|
from O |
|
S B-species |
|
. I-species |
|
solfataricus I-species |
|
were O |
|
titrated B-experimental_method |
|
with I-experimental_method |
|
increasing I-experimental_method |
|
amounts I-experimental_method |
|
of O |
|
SsTsr3 B-protein |
|
and O |
|
the O |
|
changes O |
|
in O |
|
the O |
|
fluoresceine B-chemical |
|
fluorescence B-evidence |
|
anisotropy I-evidence |
|
were O |
|
measured O |
|
and O |
|
fitted O |
|
to O |
|
a O |
|
binding B-evidence |
|
curve I-evidence |
|
( O |
|
20mer B-oligomeric_state |
|
– O |
|
red O |
|
, O |
|
20mer_GC B-oligomeric_state |
|
– O |
|
blue O |
|
). O |
|
|
|
This O |
|
suggests O |
|
that O |
|
Tsr3 B-protein |
|
is O |
|
not O |
|
stably O |
|
incorporated O |
|
into O |
|
pre B-complex_assembly |
|
- I-complex_assembly |
|
ribosomal I-complex_assembly |
|
particles I-complex_assembly |
|
and O |
|
that O |
|
its O |
|
binding O |
|
to O |
|
the O |
|
nascent O |
|
ribosomal B-complex_assembly |
|
subunit I-complex_assembly |
|
possibly O |
|
requires O |
|
additional O |
|
interactions O |
|
with O |
|
other O |
|
pre O |
|
- O |
|
ribosomal O |
|
components O |
|
. O |
|
|
|
The O |
|
cleavage O |
|
step O |
|
most O |
|
likely O |
|
acts O |
|
as O |
|
a O |
|
quality O |
|
control O |
|
check O |
|
that O |
|
ensures O |
|
the O |
|
proper O |
|
40S B-complex_assembly |
|
subunit I-complex_assembly |
|
assembly O |
|
with O |
|
only O |
|
completely O |
|
processed O |
|
precursors O |
|
. O |
|
|
|
Finally O |
|
, O |
|
termination B-protein_type |
|
factor I-protein_type |
|
Rli1 B-protein |
|
, O |
|
an O |
|
ATPase B-protein_type |
|
, O |
|
promotes O |
|
the O |
|
dissociation O |
|
of O |
|
assembly O |
|
factors O |
|
and O |
|
the O |
|
80S B-complex_assembly |
|
- I-complex_assembly |
|
like I-complex_assembly |
|
complex I-complex_assembly |
|
dissociates O |
|
and O |
|
releases O |
|
the O |
|
mature B-protein_state |
|
40S B-complex_assembly |
|
subunit I-complex_assembly |
|
. O |
|
|
|
Thus O |
|
, O |
|
the O |
|
acp B-chemical |
|
transfer O |
|
to O |
|
m1Ψ1191 B-residue_name_number |
|
occurs O |
|
during O |
|
the O |
|
step O |
|
at O |
|
which O |
|
Rio2 B-protein |
|
leaves O |
|
the O |
|
pre B-complex_assembly |
|
- I-complex_assembly |
|
40S I-complex_assembly |
|
particle I-complex_assembly |
|
. O |
|
|
|
The O |
|
current O |
|
data O |
|
together O |
|
with O |
|
the O |
|
finding O |
|
that O |
|
acp B-chemical |
|
modification O |
|
takes O |
|
place O |
|
at O |
|
the O |
|
very O |
|
last O |
|
step O |
|
in O |
|
pre B-complex_assembly |
|
- I-complex_assembly |
|
40S I-complex_assembly |
|
subunit I-complex_assembly |
|
maturation O |
|
indicate O |
|
that O |
|
the O |
|
acp B-chemical |
|
modification O |
|
probably O |
|
supports O |
|
the O |
|
formation O |
|
of O |
|
the O |
|
decoding B-site |
|
site I-site |
|
and O |
|
efficient O |
|
20S B-chemical |
|
pre I-chemical |
|
- I-chemical |
|
rRNA I-chemical |
|
D B-site |
|
- I-site |
|
site I-site |
|
cleavage O |
|
. O |
|
|
|
These O |
|
studies O |
|
also O |
|
revealed O |
|
a O |
|
well O |
|
ordered O |
|
break O |
|
in O |
|
the O |
|
polypeptide O |
|
chain O |
|
at O |
|
Lys147 B-residue_name_number |
|
, O |
|
resulting O |
|
in O |
|
a O |
|
large O |
|
conformational O |
|
rearrangement O |
|
close O |
|
to O |
|
the O |
|
active B-site |
|
site I-site |
|
. O |
|
|
|
PmC11 B-protein |
|
has O |
|
an O |
|
acidic B-site |
|
binding I-site |
|
pocket I-site |
|
and O |
|
a O |
|
preference O |
|
for O |
|
basic O |
|
substrates O |
|
, O |
|
and O |
|
accepts O |
|
substrates O |
|
with O |
|
Arg B-residue_name |
|
and O |
|
Lys B-residue_name |
|
in O |
|
P1 B-residue_number |
|
and O |
|
does O |
|
not O |
|
require O |
|
Ca2 B-chemical |
|
+ I-chemical |
|
for O |
|
activity O |
|
. O |
|
|
|
Clan B-protein_type |
|
CD I-protein_type |
|
families I-protein_type |
|
are O |
|
typically O |
|
described O |
|
using O |
|
the O |
|
name O |
|
of O |
|
their O |
|
archetypal O |
|
, O |
|
or O |
|
founding O |
|
, O |
|
member O |
|
and O |
|
also O |
|
given O |
|
an O |
|
identification O |
|
number O |
|
preceded O |
|
by O |
|
a O |
|
“ O |
|
C O |
|
,” O |
|
to O |
|
denote O |
|
cysteine B-protein_type |
|
peptidase I-protein_type |
|
. O |
|
|
|
Although O |
|
seven O |
|
families O |
|
( O |
|
C14 O |
|
is O |
|
additionally O |
|
split O |
|
into O |
|
three O |
|
subfamilies O |
|
) O |
|
have O |
|
been O |
|
described O |
|
for O |
|
this O |
|
clan O |
|
, O |
|
crystal B-evidence |
|
structures I-evidence |
|
have O |
|
only O |
|
been O |
|
determined O |
|
from O |
|
four O |
|
: O |
|
legumain B-protein |
|
( O |
|
C13 B-protein_type |
|
), O |
|
caspase B-protein |
|
( O |
|
C14a B-protein_type |
|
), O |
|
paracaspase B-protein |
|
( O |
|
C14b B-protein_type |
|
( I-protein_type |
|
P I-protein_type |
|
), O |
|
metacaspase B-protein |
|
( O |
|
C14b B-protein_type |
|
( I-protein_type |
|
M I-protein_type |
|
), O |
|
gingipain B-protein |
|
( O |
|
C25 B-protein_type |
|
), O |
|
and O |
|
the O |
|
cysteine B-structure_element |
|
peptidase I-structure_element |
|
domain I-structure_element |
|
( O |
|
CPD B-structure_element |
|
) O |
|
of O |
|
various O |
|
toxins O |
|
( O |
|
C80 B-protein_type |
|
). O |
|
|
|
Clan B-protein_type |
|
CD I-protein_type |
|
enzymes I-protein_type |
|
have O |
|
a O |
|
highly B-protein_state |
|
conserved I-protein_state |
|
His B-site |
|
/ I-site |
|
Cys I-site |
|
catalytic I-site |
|
dyad I-site |
|
and O |
|
exhibit O |
|
strict O |
|
specificity O |
|
for O |
|
the O |
|
P1 B-residue_number |
|
residue O |
|
of O |
|
their O |
|
substrates O |
|
. O |
|
|
|
Structure B-evidence |
|
of O |
|
PmC11 B-protein |
|
|
|
The O |
|
position O |
|
of O |
|
the O |
|
catalytic B-site |
|
dyad I-site |
|
( O |
|
H B-residue_name |
|
, O |
|
C B-residue_name |
|
) O |
|
and O |
|
the O |
|
processing B-site |
|
site I-site |
|
( O |
|
Lys147 B-residue_name_number |
|
) O |
|
are O |
|
highlighted O |
|
. O |
|
|
|
Helices O |
|
( O |
|
1 O |
|
– O |
|
14 O |
|
) O |
|
and O |
|
β B-structure_element |
|
- I-structure_element |
|
strands I-structure_element |
|
( O |
|
1 O |
|
– O |
|
9 O |
|
and O |
|
A O |
|
- O |
|
F O |
|
) O |
|
are O |
|
numbered O |
|
from O |
|
the O |
|
N O |
|
terminus O |
|
. O |
|
|
|
The O |
|
N O |
|
and O |
|
C O |
|
termini O |
|
( O |
|
N O |
|
and O |
|
C O |
|
) O |
|
of O |
|
PmC11 B-protein |
|
along O |
|
with O |
|
the O |
|
central O |
|
β B-structure_element |
|
- I-structure_element |
|
sheet I-structure_element |
|
( O |
|
1 O |
|
– O |
|
9 O |
|
), O |
|
helix B-structure_element |
|
α5 B-structure_element |
|
, O |
|
and O |
|
helices B-structure_element |
|
α8 B-structure_element |
|
, O |
|
α11 B-structure_element |
|
, O |
|
and O |
|
α13 B-structure_element |
|
from O |
|
the O |
|
C B-structure_element |
|
- I-structure_element |
|
terminal I-structure_element |
|
domain I-structure_element |
|
, O |
|
are O |
|
all O |
|
labeled O |
|
. O |
|
|
|
Of O |
|
the O |
|
interacting O |
|
secondary O |
|
structure O |
|
elements O |
|
, O |
|
α5 B-structure_element |
|
is O |
|
perhaps O |
|
the O |
|
most O |
|
interesting O |
|
. O |
|
|
|
This B-structure_element |
|
helix I-structure_element |
|
makes O |
|
a O |
|
total O |
|
of O |
|
eight O |
|
hydrogen O |
|
bonds O |
|
with O |
|
the O |
|
CTD B-structure_element |
|
, O |
|
including O |
|
one O |
|
salt O |
|
bridge O |
|
( O |
|
Arg191 B-residue_name_number |
|
- O |
|
Asp255 B-residue_name_number |
|
) O |
|
and O |
|
is O |
|
surrounded O |
|
by O |
|
the O |
|
CTD B-structure_element |
|
on O |
|
one O |
|
side O |
|
and O |
|
the O |
|
main B-structure_element |
|
core I-structure_element |
|
of O |
|
the O |
|
enzyme O |
|
on O |
|
the O |
|
other O |
|
, O |
|
acting O |
|
like O |
|
a O |
|
linchpin O |
|
holding O |
|
both O |
|
components O |
|
together O |
|
( O |
|
Fig O |
|
. O |
|
1C O |
|
). O |
|
|
|
The O |
|
two O |
|
ends O |
|
of O |
|
the O |
|
autolytic B-site |
|
cleavage I-site |
|
site I-site |
|
( O |
|
Lys147 B-residue_name_number |
|
and O |
|
Ala148 B-residue_name_number |
|
, O |
|
green O |
|
) O |
|
are O |
|
displaced O |
|
by O |
|
19 O |
|
. O |
|
5 O |
|
Å O |
|
( O |
|
thin O |
|
black O |
|
line O |
|
) O |
|
from O |
|
one O |
|
another O |
|
and O |
|
residues O |
|
in O |
|
the O |
|
potential O |
|
substrate B-site |
|
binding I-site |
|
pocket I-site |
|
are O |
|
highlighted O |
|
in O |
|
blue O |
|
. O |
|
|
|
B O |
|
, O |
|
size B-experimental_method |
|
exclusion I-experimental_method |
|
chromatography I-experimental_method |
|
of O |
|
PmC11 B-protein |
|
. O |
|
|
|
Elution O |
|
fractions O |
|
across O |
|
the O |
|
major O |
|
peak O |
|
( O |
|
1 O |
|
– O |
|
6 O |
|
) O |
|
were O |
|
analyzed O |
|
by O |
|
SDS B-experimental_method |
|
- I-experimental_method |
|
PAGE I-experimental_method |
|
on O |
|
a O |
|
4 O |
|
– O |
|
12 O |
|
% O |
|
gel O |
|
in O |
|
MES O |
|
buffer O |
|
. O |
|
|
|
E O |
|
, O |
|
intermolecular B-ptm |
|
processing I-ptm |
|
of O |
|
PmC11C179A B-mutant |
|
by 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 |
|
|
|
A O |
|
single O |
|
lane O |
|
of O |
|
20 O |
|
μg O |
|
of O |
|
active B-protein_state |
|
PmC11 B-protein |
|
( O |
|
labeled O |
|
20 O |
|
) O |
|
is O |
|
shown O |
|
for O |
|
comparison O |
|
. O |
|
|
|
The O |
|
position O |
|
of O |
|
the O |
|
catalytic B-site |
|
dyad I-site |
|
, O |
|
one O |
|
potential O |
|
key B-site |
|
substrate I-site |
|
binding I-site |
|
residue I-site |
|
Asp177 B-residue_name_number |
|
, O |
|
and O |
|
the O |
|
ends O |
|
of O |
|
the O |
|
cleavage B-site |
|
site I-site |
|
Lys147 B-residue_name_number |
|
and O |
|
Ala148 B-residue_name_number |
|
are O |
|
indicated O |
|
. O |
|
|
|
To O |
|
investigate O |
|
this O |
|
possibility O |
|
, O |
|
two O |
|
mutant O |
|
forms O |
|
of O |
|
the O |
|
enzyme O |
|
were O |
|
created O |
|
: O |
|
PmC11C179A B-mutant |
|
( O |
|
a O |
|
catalytically B-protein_state |
|
inactive I-protein_state |
|
mutant I-protein_state |
|
) O |
|
and O |
|
PmC11K147A B-mutant |
|
( O |
|
a O |
|
cleavage B-protein_state |
|
- I-protein_state |
|
site I-protein_state |
|
mutant I-protein_state |
|
). O |
|
|
|
The O |
|
PmC11K147A B-mutant |
|
mutant B-protein_state |
|
enzyme O |
|
had O |
|
a O |
|
markedly O |
|
different O |
|
reaction B-evidence |
|
rate I-evidence |
|
( O |
|
Vmax B-evidence |
|
) O |
|
compared O |
|
with O |
|
WT B-protein_state |
|
, O |
|
where O |
|
the O |
|
reaction B-evidence |
|
velocity I-evidence |
|
of O |
|
PmC11 B-protein |
|
was O |
|
10 O |
|
times O |
|
greater O |
|
than O |
|
that O |
|
of O |
|
PmC11K147A B-mutant |
|
( O |
|
Fig O |
|
. O |
|
2D O |
|
). O |
|
|
|
Thus O |
|
, O |
|
Asn50 B-residue_name_number |
|
, O |
|
Asp177 B-residue_name_number |
|
, O |
|
and O |
|
Asp207 B-residue_name_number |
|
are O |
|
most O |
|
likely O |
|
responsible O |
|
for O |
|
the O |
|
substrate O |
|
specificity O |
|
of O |
|
PmC11 B-protein |
|
. O |
|
|
|
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 |
|
|
|
The O |
|
structural O |
|
similarity O |
|
of O |
|
PmC11 B-protein |
|
with O |
|
its O |
|
nearest O |
|
structural O |
|
neighbors O |
|
in O |
|
the O |
|
PDB O |
|
is O |
|
decidedly O |
|
low O |
|
, O |
|
overlaying O |
|
better O |
|
with O |
|
six O |
|
- O |
|
stranded O |
|
caspase B-protein |
|
- I-protein |
|
7 I-protein |
|
than O |
|
any O |
|
of O |
|
the O |
|
other O |
|
larger O |
|
members O |
|
of O |
|
the O |
|
clan O |
|
( O |
|
Table O |
|
2 O |
|
). O |
|
|
|
PmC11 B-protein |
|
differs O |
|
from O |
|
clostripain B-protein |
|
in O |
|
that O |
|
is O |
|
does O |
|
not O |
|
appear O |
|
to O |
|
require O |
|
divalent O |
|
cations O |
|
for O |
|
activation O |
|
. O |
|
|
|
In O |
|
addition O |
|
, O |
|
several O |
|
members O |
|
of O |
|
clan B-protein_type |
|
CD I-protein_type |
|
exhibit O |
|
self O |
|
- O |
|
inhibition O |
|
, O |
|
whereby O |
|
regions B-structure_element |
|
of O |
|
the O |
|
enzyme O |
|
block O |
|
access O |
|
to O |
|
the O |
|
active B-site |
|
site I-site |
|
. O |
|
|
|
Recently O |
|
, O |
|
we O |
|
solved O |
|
the O |
|
crystal B-evidence |
|
structure I-evidence |
|
of O |
|
YfiR B-protein |
|
in O |
|
both O |
|
the O |
|
non B-protein_state |
|
- I-protein_state |
|
oxidized I-protein_state |
|
and O |
|
the O |
|
oxidized B-protein_state |
|
states O |
|
, O |
|
revealing O |
|
breakage O |
|
/ O |
|
formation O |
|
of O |
|
one O |
|
disulfide B-ptm |
|
bond I-ptm |
|
( O |
|
Cys71 B-residue_name_number |
|
- O |
|
Cys110 B-residue_name_number |
|
) O |
|
and O |
|
local O |
|
conformational O |
|
change O |
|
around O |
|
the O |
|
other O |
|
one O |
|
( O |
|
Cys145 B-residue_name_number |
|
- O |
|
Cys152 B-residue_name_number |
|
), O |
|
indicating O |
|
that O |
|
Cys145 B-residue_name_number |
|
- O |
|
Cys152 B-residue_name_number |
|
plays O |
|
an O |
|
important O |
|
role O |
|
in O |
|
maintaining O |
|
the O |
|
correct O |
|
folding O |
|
of O |
|
YfiR B-protein |
|
( O |
|
Yang O |
|
et O |
|
al O |
|
.,). O |
|
|
|
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 |
|
there O |
|
is O |
|
a O |
|
short O |
|
helix B-structure_element |
|
turn I-structure_element |
|
connecting O |
|
the O |
|
β4 B-structure_element |
|
strand I-structure_element |
|
and O |
|
α4 B-structure_element |
|
helix I-structure_element |
|
( O |
|
Fig O |
|
. O |
|
1A O |
|
and O |
|
1B O |
|
). O |
|
|
|
The O |
|
YfiR B-protein |
|
molecules O |
|
are O |
|
shown O |
|
in O |
|
green O |
|
and O |
|
magenta O |
|
. O |
|
|
|
YfiBL43P B-mutant |
|
and O |
|
YfiR B-protein |
|
are O |
|
shown O |
|
in O |
|
cyan O |
|
and O |
|
green O |
|
, O |
|
respectively O |
|
. O |
|
( O |
|
E O |
|
and O |
|
F O |
|
) O |
|
The O |
|
conserved B-site |
|
surface I-site |
|
in O |
|
YfiR B-protein |
|
contributes O |
|
to O |
|
the O |
|
interaction O |
|
with O |
|
YfiB B-protein |
|
. O |
|
( O |
|
G O |
|
) O |
|
The O |
|
residues B-structure_element |
|
of O |
|
YfiR B-protein |
|
responsible O |
|
for O |
|
interacting O |
|
with O |
|
YfiB B-protein |
|
are O |
|
shown O |
|
in O |
|
green O |
|
sticks O |
|
, O |
|
and O |
|
the O |
|
proposed O |
|
YfiN B-site |
|
- I-site |
|
interacting I-site |
|
residues I-site |
|
are O |
|
shown O |
|
in O |
|
yellow O |
|
sticks O |
|
. O |
|
|
|
The O |
|
YfiB B-complex_assembly |
|
- I-complex_assembly |
|
YfiR I-complex_assembly |
|
complex O |
|
is O |
|
a O |
|
2 O |
|
: O |
|
2 O |
|
heterotetramer B-oligomeric_state |
|
( O |
|
Fig O |
|
. O |
|
3A O |
|
) O |
|
in O |
|
which O |
|
the O |
|
YfiR B-protein |
|
dimer B-oligomeric_state |
|
is O |
|
clamped O |
|
by O |
|
two O |
|
separated O |
|
YfiBL43P B-mutant |
|
molecules O |
|
with O |
|
a O |
|
total O |
|
buried O |
|
surface O |
|
area O |
|
of O |
|
3161 O |
|
. O |
|
2 O |
|
Å2 O |
|
. O |
|
|
|
The O |
|
observed O |
|
changes O |
|
in O |
|
conformation O |
|
of O |
|
YfiB B-protein |
|
and O |
|
the O |
|
results O |
|
of O |
|
mutagenesis B-experimental_method |
|
suggest O |
|
a O |
|
mechanism O |
|
by O |
|
which O |
|
YfiB B-protein |
|
sequesters O |
|
YfiR B-protein |
|
. O |
|
|
|
Region B-structure_element |
|
I I-structure_element |
|
is O |
|
formed O |
|
by O |
|
numerous O |
|
main O |
|
- O |
|
chain O |
|
and O |
|
side O |
|
- O |
|
chain O |
|
hydrophilic O |
|
interactions O |
|
between O |
|
residues O |
|
E45 B-residue_name_number |
|
, O |
|
G47 B-residue_name_number |
|
and O |
|
E53 B-residue_name_number |
|
from O |
|
the O |
|
N O |
|
- O |
|
terminal O |
|
extended O |
|
loop B-structure_element |
|
of O |
|
YfiB B-protein |
|
and O |
|
residues O |
|
S57 B-residue_name_number |
|
, O |
|
R60 B-residue_name_number |
|
, O |
|
A89 B-residue_name_number |
|
and O |
|
H177 B-residue_name_number |
|
from O |
|
YfiR B-protein |
|
( O |
|
Fig O |
|
. O |
|
3D O |
|
- O |
|
I O |
|
( O |
|
i O |
|
)). O |
|
|
|
Additionally O |
|
, O |
|
three O |
|
hydrophobic B-site |
|
anchoring I-site |
|
sites I-site |
|
exist O |
|
in O |
|
region B-structure_element |
|
I I-structure_element |
|
. O |
|
The O |
|
residues O |
|
F48 B-residue_name_number |
|
and O |
|
W55 B-residue_name_number |
|
of O |
|
YfiB B-protein |
|
are O |
|
inserted O |
|
into O |
|
the O |
|
hydrophobic B-site |
|
cores I-site |
|
mainly O |
|
formed O |
|
by O |
|
the O |
|
main O |
|
chain O |
|
and O |
|
side O |
|
chain O |
|
carbon O |
|
atoms O |
|
of O |
|
residues O |
|
S57 B-residue_name_number |
|
/ O |
|
Q88 B-residue_name_number |
|
/ O |
|
A89 B-residue_name_number |
|
/ O |
|
N90 B-residue_name_number |
|
and O |
|
R60 B-residue_name_number |
|
/ O |
|
R175 B-residue_name_number |
|
/ O |
|
H177 B-residue_name_number |
|
of O |
|
YfiR B-protein |
|
, O |
|
respectively O |
|
; O |
|
and O |
|
F57 B-residue_name_number |
|
of O |
|
YfiB B-protein |
|
is O |
|
inserted O |
|
into O |
|
the O |
|
hydrophobic B-site |
|
pocket I-site |
|
formed O |
|
by O |
|
L166 B-residue_name_number |
|
/ O |
|
I169 B-residue_name_number |
|
/ O |
|
V176 B-residue_name_number |
|
/ O |
|
P178 B-residue_name_number |
|
/ O |
|
L181 B-residue_name_number |
|
of O |
|
YfiR B-protein |
|
( O |
|
Fig O |
|
. O |
|
3D O |
|
- O |
|
I O |
|
( O |
|
ii O |
|
)). O |
|
|
|
The O |
|
results O |
|
indicated O |
|
that O |
|
the O |
|
PG B-evidence |
|
- I-evidence |
|
binding I-evidence |
|
affinity I-evidence |
|
of O |
|
YfiBL43P B-mutant |
|
is O |
|
65 O |
|
. O |
|
5 O |
|
μmol O |
|
/ O |
|
L O |
|
, O |
|
which O |
|
is O |
|
about O |
|
16 O |
|
- O |
|
fold O |
|
stronger O |
|
than O |
|
that O |
|
of O |
|
wild B-protein_state |
|
- I-protein_state |
|
type I-protein_state |
|
YfiB B-protein |
|
( O |
|
Kd O |
|
= O |
|
1 O |
|
. O |
|
1 O |
|
mmol O |
|
/ O |
|
L O |
|
) O |
|
( O |
|
Fig O |
|
. O |
|
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|
– O |
|
F O |
|
). O |
|
|
|
As O |
|
the O |
|
experiment O |
|
is O |
|
performed O |
|
in B-protein_state |
|
the I-protein_state |
|
absence I-protein_state |
|
of I-protein_state |
|
YfiR B-protein |
|
, O |
|
it O |
|
suggests O |
|
that O |
|
an O |
|
increase O |
|
in O |
|
the O |
|
PG B-evidence |
|
- I-evidence |
|
binding I-evidence |
|
affinity I-evidence |
|
of O |
|
YfiB B-protein |
|
is O |
|
not O |
|
a O |
|
result O |
|
of O |
|
YfiB B-complex_assembly |
|
- I-complex_assembly |
|
YfiR I-complex_assembly |
|
interaction O |
|
and O |
|
is O |
|
highly O |
|
coupled O |
|
to O |
|
the O |
|
activation O |
|
of O |
|
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|
by O |
|
a O |
|
stretched B-protein_state |
|
N I-protein_state |
|
- I-protein_state |
|
terminal I-protein_state |
|
conformation I-protein_state |
|
. O |
|
|
|
Calculation O |
|
using O |
|
the O |
|
ConSurf B-experimental_method |
|
Server I-experimental_method |
|
( O |
|
http O |
|
:// O |
|
consurf O |
|
. O |
|
tau O |
|
. O |
|
ac O |
|
. O |
|
il O |
|
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|
which O |
|
estimates O |
|
the O |
|
evolutionary B-evidence |
|
conservation I-evidence |
|
of O |
|
amino O |
|
acid O |
|
positions O |
|
and O |
|
visualizes O |
|
information O |
|
on O |
|
the O |
|
structure B-site |
|
surface I-site |
|
, O |
|
revealed O |
|
a O |
|
conserved B-site |
|
surface I-site |
|
on O |
|
YfiR B-protein |
|
that O |
|
contributes O |
|
to O |
|
the O |
|
interaction O |
|
with O |
|
YfiB B-protein |
|
( O |
|
Fig O |
|
. O |
|
3E O |
|
and O |
|
3F O |
|
). O |
|
|
|
F151 B-residue_name_number |
|
, O |
|
E163 B-residue_name_number |
|
and O |
|
I169 B-residue_name_number |
|
form O |
|
a O |
|
hydrophobic B-site |
|
core I-site |
|
while O |
|
, O |
|
Q187 B-residue_name_number |
|
is O |
|
located O |
|
at O |
|
the O |
|
end O |
|
of O |
|
the O |
|
α6 B-structure_element |
|
helix I-structure_element |
|
. O |
|
|
|
YfiR B-protein |
|
binds O |
|
small O |
|
molecules O |
|
|
|
The O |
|
results O |
|
showed O |
|
Kd B-evidence |
|
values O |
|
of O |
|
1 O |
|
. O |
|
4 O |
|
× O |
|
10 O |
|
− O |
|
7 O |
|
mol O |
|
/ O |
|
L O |
|
and O |
|
5 O |
|
. O |
|
3 O |
|
× O |
|
10 O |
|
− O |
|
7 O |
|
mol O |
|
/ O |
|
L O |
|
for O |
|
YfiBL43P B-mutant |
|
and O |
|
YfiBL43P B-mutant |
|
/ O |
|
F57A B-mutant |
|
, O |
|
respectively O |
|
, O |
|
revealing O |
|
that O |
|
the O |
|
YfiBL43P B-mutant |
|
/ O |
|
F57A B-mutant |
|
mutant B-protein_state |
|
caused O |
|
a O |
|
3 O |
|
. O |
|
8 O |
|
- O |
|
fold O |
|
reduction O |
|
in O |
|
the O |
|
binding B-evidence |
|
affinity I-evidence |
|
compared O |
|
with O |
|
the O |
|
YfiBL43P B-mutant |
|
mutant B-protein_state |
|
( O |
|
Fig O |
|
. O |
|
6F O |
|
and O |
|
6G O |
|
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|
|
|
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|
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|
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|
report O |
|
the O |
|
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|
structures I-evidence |
|
of O |
|
YfiB B-protein |
|
alone B-protein_state |
|
and O |
|
an O |
|
active B-protein_state |
|
mutant B-protein_state |
|
YfiBL43P B-mutant |
|
in B-protein_state |
|
complex I-protein_state |
|
with I-protein_state |
|
YfiR B-protein |
|
, O |
|
indicating O |
|
that O |
|
YfiR B-protein |
|
forms O |
|
a O |
|
2 O |
|
: O |
|
2 O |
|
complex B-protein_state |
|
with I-protein_state |
|
YfiB B-protein |
|
via O |
|
a O |
|
region O |
|
composed O |
|
of O |
|
conserved O |
|
residues O |
|
. O |
|
|
|
Thus O |
|
, O |
|
YfiB B-protein |
|
alone B-protein_state |
|
represents O |
|
an O |
|
inactive B-protein_state |
|
form O |
|
that O |
|
may O |
|
only O |
|
partially O |
|
insert O |
|
into O |
|
the O |
|
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|
matrix O |
|
. O |
|
|
|
The O |
|
periplasmic B-structure_element |
|
domain I-structure_element |
|
of O |
|
YfiB B-protein |
|
and O |
|
the O |
|
YfiB B-complex_assembly |
|
- I-complex_assembly |
|
YfiR I-complex_assembly |
|
complex O |
|
are O |
|
depicted O |
|
according O |
|
to O |
|
the O |
|
crystal B-evidence |
|
structures I-evidence |
|
. O |
|
|
|
The O |
|
lipid O |
|
acceptor O |
|
Cys26 B-residue_name_number |
|
is O |
|
indicated O |
|
as O |
|
blue O |
|
ball O |
|
. O |
|
|
|
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 |
|
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|
binding O |
|
( O |
|
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|
et O |
|
al O |
|
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|
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 |
|
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|
|
|
In O |
|
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|
model O |
|
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|
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|
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|
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|
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|
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|
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 |
|
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|
binding O |
|
affinity O |
|
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|
|
|
Ligands O |
|
that O |
|
regulate O |
|
the O |
|
dynamics O |
|
and O |
|
stability O |
|
of O |
|
the O |
|
coactivator B-site |
|
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|
binding I-site |
|
site I-site |
|
in O |
|
the O |
|
C O |
|
‐ O |
|
terminal O |
|
ligand B-structure_element |
|
‐ I-structure_element |
|
binding I-structure_element |
|
domain I-structure_element |
|
, O |
|
called O |
|
activation B-structure_element |
|
function I-structure_element |
|
‐ I-structure_element |
|
2 I-structure_element |
|
( O |
|
AF B-structure_element |
|
‐ I-structure_element |
|
2 I-structure_element |
|
), O |
|
showed O |
|
similar O |
|
activity O |
|
profiles O |
|
in O |
|
different O |
|
cell O |
|
types O |
|
. O |
|
|
|
For O |
|
some O |
|
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|
series O |
|
, O |
|
a O |
|
single O |
|
inter B-evidence |
|
‐ I-evidence |
|
atomic I-evidence |
|
distance I-evidence |
|
in O |
|
the O |
|
ligand B-structure_element |
|
‐ I-structure_element |
|
binding I-structure_element |
|
domain I-structure_element |
|
predicted O |
|
their O |
|
proliferative O |
|
effects O |
|
. O |
|
|
|
AF B-structure_element |
|
‐ I-structure_element |
|
1 I-structure_element |
|
binds O |
|
a O |
|
separate O |
|
surface O |
|
on O |
|
these O |
|
coactivators O |
|
( O |
|
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|
et O |
|
al O |
|
, O |
|
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|
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|
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|
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|
al O |
|
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|
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|
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|
|
|
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|
, O |
|
ERα B-protein |
|
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|
mediated O |
|
proliferative O |
|
responses O |
|
vary O |
|
in O |
|
a O |
|
ligand O |
|
‐ O |
|
dependent O |
|
manner O |
|
( O |
|
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|
et O |
|
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|
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|
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|
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|
thus O |
|
, O |
|
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|
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|
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|
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|
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|
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|
canonical O |
|
model O |
|
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|
widely O |
|
applicable O |
|
across O |
|
diverse O |
|
ERα B-protein |
|
ligands O |
|
. O |
|
|
|
Summary O |
|
of O |
|
ligand B-experimental_method |
|
screening I-experimental_method |
|
assays I-experimental_method |
|
used O |
|
to O |
|
measure O |
|
ER O |
|
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|
mediated O |
|
activities O |
|
. O |
|
|
|
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|
wide O |
|
variance O |
|
enabled O |
|
us O |
|
to O |
|
probe O |
|
specific O |
|
features O |
|
of O |
|
ERα B-protein |
|
signaling O |
|
using O |
|
ligand B-experimental_method |
|
class I-experimental_method |
|
analyses I-experimental_method |
|
, O |
|
and O |
|
identify O |
|
signaling O |
|
patterns O |
|
shared O |
|
by O |
|
specific O |
|
ligand O |
|
series O |
|
or O |
|
scaffolds O |
|
. O |
|
|
|
The O |
|
ERα B-protein |
|
ligand O |
|
library O |
|
contains O |
|
241 O |
|
ligands O |
|
representing O |
|
15 O |
|
indirect O |
|
modulator O |
|
scaffolds O |
|
, O |
|
plus O |
|
4 O |
|
direct O |
|
modulator O |
|
scaffolds O |
|
. O |
|
|
|
Ligand O |
|
‐ O |
|
specific O |
|
signaling O |
|
underlies O |
|
ERα B-protein |
|
‐ O |
|
mediated O |
|
cell O |
|
proliferation O |
|
|
|
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|
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|
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|
idea O |
|
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|
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|
compared O |
|
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|
average B-evidence |
|
L I-evidence |
|
‐ I-evidence |
|
Luc I-evidence |
|
activities I-evidence |
|
of O |
|
each O |
|
scaffold O |
|
in O |
|
HepG2 O |
|
cells O |
|
co B-experimental_method |
|
‐ I-experimental_method |
|
transfected I-experimental_method |
|
with O |
|
wild B-protein_state |
|
‐ I-protein_state |
|
type I-protein_state |
|
ERα B-protein |
|
or O |
|
with O |
|
ERα B-protein |
|
lacking B-protein_state |
|
the I-protein_state |
|
AB B-structure_element |
|
domain O |
|
( O |
|
Figs O |
|
1B O |
|
and O |
|
EV1 O |
|
). O |
|
|
|
Deletion B-experimental_method |
|
of I-experimental_method |
|
the O |
|
AB B-structure_element |
|
domain O |
|
significantly O |
|
reduced O |
|
the O |
|
average B-evidence |
|
L I-evidence |
|
‐ I-evidence |
|
Luc I-evidence |
|
activities I-evidence |
|
of O |
|
14 O |
|
scaffolds O |
|
( O |
|
Student B-experimental_method |
|
' I-experimental_method |
|
s I-experimental_method |
|
t I-experimental_method |
|
‐ I-experimental_method |
|
test I-experimental_method |
|
, O |
|
P B-evidence |
|
≤ O |
|
0 O |
|
. O |
|
05 O |
|
) O |
|
( O |
|
Fig O |
|
3B O |
|
). O |
|
|
|
For O |
|
example O |
|
, O |
|
3 B-chemical |
|
, I-chemical |
|
4 I-chemical |
|
‐ I-chemical |
|
DTP I-chemical |
|
, O |
|
furan B-chemical |
|
, O |
|
and O |
|
S B-chemical |
|
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|
OBHS I-chemical |
|
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|
2 I-chemical |
|
drove O |
|
positively O |
|
correlated O |
|
GREB1 B-protein |
|
levels O |
|
and O |
|
E B-experimental_method |
|
‐ I-experimental_method |
|
Luc I-experimental_method |
|
but O |
|
not O |
|
L B-experimental_method |
|
‐ I-experimental_method |
|
Luc I-experimental_method |
|
ERα B-protein |
|
‐ O |
|
WT B-protein_state |
|
activity O |
|
( O |
|
Fig O |
|
3C O |
|
lanes O |
|
5 O |
|
– O |
|
7 O |
|
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|
|
|
This O |
|
is O |
|
demonstrated O |
|
by O |
|
directly O |
|
comparing O |
|
the O |
|
signaling O |
|
specificities O |
|
of O |
|
matched O |
|
OBHS B-chemical |
|
( O |
|
indirect O |
|
modulator O |
|
, O |
|
cluster O |
|
1 O |
|
) O |
|
and O |
|
OBHS B-chemical |
|
‐ I-chemical |
|
BSC I-chemical |
|
analogs O |
|
( O |
|
direct O |
|
modulator O |
|
, O |
|
cluster O |
|
3 O |
|
), O |
|
which O |
|
differ O |
|
only O |
|
in O |
|
the O |
|
basic O |
|
side O |
|
chain O |
|
( O |
|
Fig O |
|
2E O |
|
). O |
|
|
|
Thus O |
|
, O |
|
examining O |
|
the O |
|
correlated O |
|
patterns O |
|
of O |
|
ERα B-protein |
|
activity O |
|
within O |
|
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|
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|
demonstrates O |
|
that O |
|
an O |
|
extended O |
|
side O |
|
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|
is O |
|
not O |
|
required O |
|
for O |
|
cell O |
|
‐ O |
|
specific O |
|
signaling O |
|
. O |
|
|
|
Similarly O |
|
, O |
|
deletion B-experimental_method |
|
of I-experimental_method |
|
the O |
|
F B-structure_element |
|
domain O |
|
did O |
|
not O |
|
abolish O |
|
correlations O |
|
between O |
|
the O |
|
L B-experimental_method |
|
‐ I-experimental_method |
|
Luc I-experimental_method |
|
and O |
|
E B-experimental_method |
|
‐ I-experimental_method |
|
Luc I-experimental_method |
|
or O |
|
GREB1 B-protein |
|
levels O |
|
induced O |
|
by O |
|
OBHS B-chemical |
|
analogs O |
|
( O |
|
Fig O |
|
EV3F O |
|
). O |
|
|
|
Thus O |
|
, O |
|
ligands O |
|
in O |
|
cluster O |
|
2 O |
|
rely O |
|
on O |
|
AF B-structure_element |
|
‐ I-structure_element |
|
1 I-structure_element |
|
for O |
|
both O |
|
activity O |
|
( O |
|
Fig O |
|
3B O |
|
) O |
|
and O |
|
signaling O |
|
specificity O |
|
( O |
|
Fig O |
|
3D O |
|
). O |
|
|
|
Direct O |
|
modulators O |
|
showed O |
|
low O |
|
NCOA1 B-protein |
|
/ I-protein |
|
2 I-protein |
|
/ I-protein |
|
3 I-protein |
|
recruitment O |
|
( O |
|
Fig O |
|
EV2F O |
|
– O |
|
H O |
|
), O |
|
but O |
|
only O |
|
OBHS B-chemical |
|
‐ I-chemical |
|
ASC I-chemical |
|
analogs O |
|
had O |
|
NCOA2 B-protein |
|
recruitment O |
|
profiles O |
|
that O |
|
predicted O |
|
a O |
|
full O |
|
range O |
|
of O |
|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
|
|
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|
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|
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|
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|
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|
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|
|
|
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|
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|
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|
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GREB1 B-protein |
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|
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|
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|
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|
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|
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|
|
|
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ChIP I-experimental_method |
|
assay I-experimental_method |
|
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recruitment O |
|
of O |
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NCOA3 B-protein |
|
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GREB1 B-protein |
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|
|
|
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|
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|
assay I-experimental_method |
|
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|
NCOA3 B-protein |
|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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GREB1 B-protein |
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|
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|
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|
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|
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|
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|
|
|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
slope O |
|
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|
|
|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
their O |
|
L B-experimental_method |
|
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|
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|
ERα B-protein |
|
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|
WT B-protein_state |
|
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|
L B-experimental_method |
|
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|
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|
ERβ B-protein |
|
activities O |
|
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|
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|
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|
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|
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|
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|
|
|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
|
|
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|
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|
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|
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|
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|
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|
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|
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|
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|
N I-chemical |
|
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|
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|
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|
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|
ethylene I-chemical |
|
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|
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|
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|
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|
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|
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|
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|
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|
groups O |
|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
|
|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
|
|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
|
|
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|
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|
ethylene I-chemical |
|
analogs O |
|
bound B-protein_state |
|
to I-protein_state |
|
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|
superposed B-experimental_method |
|
crystal B-evidence |
|
structures I-evidence |
|
of O |
|
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|
ERα B-protein |
|
LBD B-structure_element |
|
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|
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|
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|
|
|
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|
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|
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|
analogs O |
|
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|
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|
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|
ERα B-protein |
|
conformations O |
|
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|
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|
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|
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|
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|
region O |
|
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|
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|
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|
|
|
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|
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|
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|
side O |
|
groups O |
|
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|
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|
h12 B-structure_element |
|
Leu540 B-residue_name_number |
|
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|
|
|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
|
|
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|
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|
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|
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|
ERα B-protein |
|
LBD B-structure_element |
|
bound B-protein_state |
|
to I-protein_state |
|
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|
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|
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|
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|
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|
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|
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|
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|
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|
|
|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
|
|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
|
|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
analog O |
|
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|
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|
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|
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|
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|
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|
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|
NCOA2 B-protein |
|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
|
|
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|
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|
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|
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|
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|
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|
analogs O |
|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
surface I-site |
|
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|
|
|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
t I-experimental_method |
|
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|
test I-experimental_method |
|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
|
|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
|
|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
analogs O |
|
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|
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|
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|
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|
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|
patterns O |
|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
|
|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
/ I-protein |
|
3 I-protein |
|
contain O |
|
distinct O |
|
binding B-site |
|
sites I-site |
|
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|
interaction O |
|
with O |
|
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|
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|
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|
and O |
|
AF B-structure_element |
|
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|
2 I-structure_element |
|
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|
McInerney O |
|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
nucleate O |
|
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|
– I-complex_assembly |
|
NCOA1 I-complex_assembly |
|
/ I-complex_assembly |
|
2 I-complex_assembly |
|
/ I-complex_assembly |
|
3 I-complex_assembly |
|
interaction O |
|
through O |
|
AF B-structure_element |
|
‐ I-structure_element |
|
2 I-structure_element |
|
, O |
|
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|
reinforce O |
|
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|
interaction O |
|
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|
additional O |
|
binding O |
|
to O |
|
AF B-structure_element |
|
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|
1 I-structure_element |
|
. O |
|
|
|
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|
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|
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|
have O |
|
used O |
|
siRNA B-experimental_method |
|
screening I-experimental_method |
|
to O |
|
identify O |
|
a O |
|
number O |
|
of O |
|
coregulators O |
|
required O |
|
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|
ERα B-protein |
|
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|
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|
repression O |
|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
|
|
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|
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|
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|
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|
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|
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|
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|
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|
compounds O |
|
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|
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|
rely O |
|
of O |
|
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|
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|
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|
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|
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|
efficacy O |
|
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|
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|
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|
the O |
|
slight O |
|
contacts O |
|
with O |
|
h12 B-structure_element |
|
observed O |
|
in O |
|
crystal B-evidence |
|
structures I-evidence |
|
( O |
|
Figs O |
|
3B O |
|
and O |
|
5H O |
|
), O |
|
unlike O |
|
other O |
|
compounds O |
|
in O |
|
cluster O |
|
1 O |
|
that O |
|
dislocate O |
|
h11 B-structure_element |
|
and O |
|
rely O |
|
on O |
|
AF B-structure_element |
|
‐ I-structure_element |
|
1 I-structure_element |
|
for O |
|
signaling O |
|
efficacy O |
|
( O |
|
Figs O |
|
3B O |
|
and O |
|
5C O |
|
, O |
|
and O |
|
EV5B O |
|
). O |
|
|
|
We O |
|
have O |
|
also O |
|
investigated O |
|
the O |
|
binding O |
|
of O |
|
the O |
|
TOCA B-protein |
|
HR1 B-structure_element |
|
domain O |
|
to O |
|
Cdc42 B-protein |
|
and O |
|
the O |
|
potential O |
|
ternary O |
|
complex O |
|
between O |
|
Cdc42 B-protein |
|
and O |
|
the O |
|
G B-site |
|
protein I-site |
|
- I-site |
|
binding I-site |
|
regions I-site |
|
of O |
|
TOCA1 B-protein |
|
and O |
|
a O |
|
member O |
|
of O |
|
the O |
|
Wiskott B-protein_type |
|
- I-protein_type |
|
Aldrich I-protein_type |
|
syndrome I-protein_type |
|
protein I-protein_type |
|
family I-protein_type |
|
, O |
|
N B-protein |
|
- I-protein |
|
WASP I-protein |
|
. O |
|
|
|
The O |
|
guanine B-protein_type |
|
nucleotide I-protein_type |
|
exchange I-protein_type |
|
factors I-protein_type |
|
mediate O |
|
formation O |
|
of O |
|
the O |
|
active B-protein_state |
|
state O |
|
by O |
|
promoting O |
|
the O |
|
dissociation O |
|
of O |
|
GDP B-chemical |
|
, O |
|
allowing O |
|
GTP B-chemical |
|
to O |
|
bind O |
|
. O |
|
|
|
The O |
|
Rho B-protein_type |
|
family I-protein_type |
|
comprises O |
|
20 O |
|
members O |
|
, O |
|
of O |
|
which O |
|
three O |
|
, O |
|
RhoA B-protein |
|
, O |
|
Rac1 B-protein |
|
, O |
|
and O |
|
Cdc42 B-protein |
|
, O |
|
have O |
|
been O |
|
relatively O |
|
well O |
|
studied O |
|
. O |
|
|
|
Following O |
|
their O |
|
release O |
|
, O |
|
the O |
|
C B-structure_element |
|
- I-structure_element |
|
terminal I-structure_element |
|
regions I-structure_element |
|
of O |
|
N B-protein |
|
- I-protein |
|
WASP I-protein |
|
are O |
|
free O |
|
to O |
|
interact O |
|
with O |
|
G B-protein_type |
|
- I-protein_type |
|
actin I-protein_type |
|
and O |
|
a O |
|
known O |
|
nucleator O |
|
of O |
|
actin O |
|
assembly O |
|
, O |
|
the O |
|
Arp2 B-complex_assembly |
|
/ I-complex_assembly |
|
3 I-complex_assembly |
|
complex O |
|
. O |
|
|
|
The O |
|
structures B-evidence |
|
of O |
|
the O |
|
PRK1 B-protein |
|
HR1a B-structure_element |
|
domain O |
|
in O |
|
complex B-protein_state |
|
with I-protein_state |
|
RhoA B-protein |
|
and O |
|
the O |
|
HR1b B-structure_element |
|
domain O |
|
in O |
|
complex B-protein_state |
|
with I-protein_state |
|
Rac1 B-protein |
|
show O |
|
that O |
|
the O |
|
HR1 B-structure_element |
|
domain O |
|
comprises O |
|
an O |
|
anti B-structure_element |
|
- I-structure_element |
|
parallel I-structure_element |
|
coiled I-structure_element |
|
- I-structure_element |
|
coil I-structure_element |
|
that O |
|
interacts O |
|
with O |
|
its O |
|
G B-protein_type |
|
protein I-protein_type |
|
binding O |
|
partner O |
|
via O |
|
both O |
|
helices B-structure_element |
|
. O |
|
|
|
Both O |
|
of O |
|
the O |
|
G B-site |
|
protein I-site |
|
switch I-site |
|
regions I-site |
|
are O |
|
involved O |
|
in O |
|
the O |
|
interaction O |
|
. O |
|
|
|
The O |
|
coiled B-structure_element |
|
- I-structure_element |
|
coil I-structure_element |
|
fold I-structure_element |
|
is O |
|
shared O |
|
by O |
|
the O |
|
HR1 B-structure_element |
|
domain O |
|
of O |
|
the O |
|
TOCA B-protein_type |
|
family I-protein_type |
|
protein I-protein_type |
|
, O |
|
CIP4 B-protein |
|
, O |
|
and O |
|
, O |
|
based O |
|
on O |
|
sequence O |
|
homology O |
|
, O |
|
by O |
|
TOCA1 B-protein |
|
itself O |
|
. O |
|
|
|
How O |
|
different O |
|
HR1 B-structure_element |
|
domain O |
|
proteins O |
|
distinguish O |
|
their O |
|
specific O |
|
G B-protein_type |
|
protein I-protein_type |
|
partners O |
|
remains O |
|
only O |
|
partially O |
|
understood O |
|
, O |
|
and O |
|
structural O |
|
characterization O |
|
of O |
|
a O |
|
novel O |
|
G B-protein_type |
|
protein I-protein_type |
|
- O |
|
HR1 B-structure_element |
|
domain O |
|
interaction O |
|
would O |
|
add O |
|
to O |
|
the O |
|
growing O |
|
body O |
|
of O |
|
information O |
|
pertaining O |
|
to O |
|
these O |
|
protein O |
|
complexes O |
|
. O |
|
|
|
The O |
|
interactions O |
|
of O |
|
TOCA1 B-protein |
|
and O |
|
N B-protein |
|
- I-protein |
|
WASP I-protein |
|
with O |
|
Cdc42 B-protein |
|
as O |
|
well O |
|
as O |
|
with O |
|
each O |
|
other O |
|
have O |
|
raised O |
|
questions O |
|
as O |
|
to O |
|
whether O |
|
the O |
|
two O |
|
Cdc42 B-protein |
|
effectors O |
|
can O |
|
interact O |
|
with O |
|
a O |
|
single O |
|
molecule O |
|
of O |
|
Cdc42 B-protein |
|
simultaneously O |
|
. O |
|
|
|
We O |
|
also O |
|
present O |
|
data O |
|
pertaining O |
|
to O |
|
binding O |
|
of O |
|
the O |
|
TOCA B-protein_type |
|
HR1 B-structure_element |
|
domain O |
|
to O |
|
Cdc42 B-protein |
|
, O |
|
which O |
|
is O |
|
the O |
|
first O |
|
biophysical O |
|
description O |
|
of O |
|
an O |
|
HR1 B-structure_element |
|
domain O |
|
binding O |
|
this O |
|
particular O |
|
Rho B-protein_type |
|
family I-protein_type |
|
small I-protein_type |
|
G I-protein_type |
|
protein I-protein_type |
|
. O |
|
|
|
Finally O |
|
, O |
|
we O |
|
investigate O |
|
the O |
|
potential O |
|
ternary O |
|
complex O |
|
between O |
|
Cdc42 B-protein |
|
and O |
|
the O |
|
G B-site |
|
protein I-site |
|
- I-site |
|
binding I-site |
|
regions I-site |
|
of O |
|
TOCA1 B-protein |
|
and O |
|
N B-protein |
|
- I-protein |
|
WASP I-protein |
|
, O |
|
contributing O |
|
to O |
|
our O |
|
understanding O |
|
of O |
|
G B-protein_type |
|
protein I-protein_type |
|
- O |
|
effector O |
|
interactions O |
|
as O |
|
well O |
|
as O |
|
the O |
|
roles O |
|
of O |
|
Cdc42 B-protein |
|
, O |
|
N B-protein |
|
- I-protein |
|
WASP I-protein |
|
, O |
|
and O |
|
TOCA1 B-protein |
|
in O |
|
the O |
|
pathways O |
|
that O |
|
govern O |
|
actin O |
|
dynamics O |
|
. O |
|
|
|
TOCA1 B-protein |
|
was O |
|
identified O |
|
in O |
|
Xenopus B-taxonomy_domain |
|
extracts O |
|
as O |
|
a O |
|
protein O |
|
necessary O |
|
for O |
|
Cdc42 B-protein |
|
- O |
|
dependent O |
|
actin O |
|
assembly O |
|
and O |
|
was O |
|
shown O |
|
to O |
|
bind O |
|
to O |
|
Cdc42 B-complex_assembly |
|
· I-complex_assembly |
|
GTPγS I-complex_assembly |
|
but O |
|
not O |
|
to O |
|
Cdc42 B-complex_assembly |
|
· I-complex_assembly |
|
GDP I-complex_assembly |
|
or O |
|
to O |
|
Rac1 B-protein |
|
and O |
|
RhoA B-protein |
|
. O |
|
Given O |
|
its O |
|
homology O |
|
to O |
|
other O |
|
Rho B-site |
|
family I-site |
|
binding I-site |
|
modules I-site |
|
, O |
|
it O |
|
is O |
|
likely O |
|
that O |
|
the O |
|
HR1 B-structure_element |
|
domain O |
|
of O |
|
TOCA1 B-protein |
|
is O |
|
sufficient O |
|
to O |
|
bind O |
|
Cdc42 B-protein |
|
. O |
|
|
|
The O |
|
binding O |
|
of O |
|
TOCA1 B-protein |
|
HR1 B-structure_element |
|
to O |
|
Cdc42 B-protein |
|
was O |
|
unexpectedly O |
|
weak O |
|
, O |
|
with O |
|
a O |
|
Kd B-evidence |
|
of O |
|
> O |
|
1 O |
|
μm O |
|
. O |
|
|
|
A O |
|
, O |
|
curves O |
|
derived O |
|
from O |
|
direct B-experimental_method |
|
binding I-experimental_method |
|
assays I-experimental_method |
|
in O |
|
which O |
|
the O |
|
indicated O |
|
concentrations O |
|
of O |
|
Cdc42Δ7Q61L B-complex_assembly |
|
·[ I-complex_assembly |
|
3H I-complex_assembly |
|
] I-complex_assembly |
|
GTP I-complex_assembly |
|
were O |
|
incubated B-experimental_method |
|
with O |
|
30 O |
|
nm O |
|
GST B-mutant |
|
- I-mutant |
|
PAK I-mutant |
|
or O |
|
HR1 B-mutant |
|
- I-mutant |
|
His6 I-mutant |
|
in O |
|
SPAs B-experimental_method |
|
. O |
|
|
|
The O |
|
data O |
|
were O |
|
fitted O |
|
to O |
|
a O |
|
binding B-evidence |
|
isotherm I-evidence |
|
to O |
|
give O |
|
an O |
|
apparent O |
|
Kd B-evidence |
|
and O |
|
are O |
|
expressed O |
|
as O |
|
a O |
|
percentage O |
|
of O |
|
the O |
|
maximum O |
|
signal O |
|
; O |
|
B O |
|
and O |
|
C O |
|
, O |
|
competition B-experimental_method |
|
SPA I-experimental_method |
|
experiments O |
|
were O |
|
carried O |
|
out O |
|
with O |
|
the O |
|
indicated O |
|
concentrations O |
|
of O |
|
ACK B-protein |
|
GBD B-structure_element |
|
( O |
|
B O |
|
) O |
|
or O |
|
HR1 B-structure_element |
|
domain O |
|
( O |
|
C O |
|
) O |
|
titrated B-experimental_method |
|
into O |
|
30 O |
|
nm O |
|
GST B-mutant |
|
- I-mutant |
|
ACK I-mutant |
|
and O |
|
either O |
|
30 O |
|
nm O |
|
Cdc42Δ7Q61L B-complex_assembly |
|
·[ I-complex_assembly |
|
3H I-complex_assembly |
|
] I-complex_assembly |
|
GTP I-complex_assembly |
|
or O |
|
full B-protein_state |
|
- I-protein_state |
|
length I-protein_state |
|
Cdc42Q61L B-complex_assembly |
|
·[ I-complex_assembly |
|
3H I-complex_assembly |
|
] I-complex_assembly |
|
GTP I-complex_assembly |
|
. O |
|
|
|
Isothermal B-experimental_method |
|
titration I-experimental_method |
|
calorimetry I-experimental_method |
|
was O |
|
carried O |
|
out O |
|
, O |
|
but O |
|
no O |
|
heat O |
|
changes O |
|
were O |
|
observed O |
|
at O |
|
a O |
|
range O |
|
of O |
|
concentrations O |
|
and O |
|
temperatures O |
|
( O |
|
data O |
|
not O |
|
shown O |
|
), O |
|
suggesting O |
|
that O |
|
the O |
|
interaction O |
|
is O |
|
predominantly O |
|
entropically O |
|
driven O |
|
. O |
|
|
|
The O |
|
binding B-experimental_method |
|
experiments I-experimental_method |
|
were O |
|
repeated O |
|
with O |
|
full B-protein_state |
|
- I-protein_state |
|
length I-protein_state |
|
[ B-complex_assembly |
|
3H I-complex_assembly |
|
] I-complex_assembly |
|
GTP I-complex_assembly |
|
· I-complex_assembly |
|
Cdc42 I-complex_assembly |
|
, O |
|
but O |
|
the O |
|
affinity B-evidence |
|
of O |
|
the O |
|
HR1 B-structure_element |
|
domain O |
|
for O |
|
full B-protein_state |
|
- I-protein_state |
|
length I-protein_state |
|
Cdc42 B-protein |
|
was O |
|
similar O |
|
to O |
|
its O |
|
affinity B-evidence |
|
for O |
|
truncated B-protein_state |
|
Cdc42 B-protein |
|
( O |
|
Kd B-evidence |
|
≈ O |
|
5 O |
|
μm O |
|
; O |
|
Fig O |
|
. O |
|
1C O |
|
). O |
|
|
|
Full B-protein_state |
|
- I-protein_state |
|
length I-protein_state |
|
TOCA1 B-protein |
|
and O |
|
ΔSH3 B-mutant |
|
TOCA1 B-protein |
|
bound B-protein_state |
|
with O |
|
micromolar O |
|
affinity O |
|
( O |
|
Fig O |
|
. O |
|
2B O |
|
), O |
|
in O |
|
a O |
|
similar O |
|
manner O |
|
to O |
|
the O |
|
isolated O |
|
HR1 B-structure_element |
|
domain O |
|
( O |
|
Fig O |
|
. O |
|
1A O |
|
). O |
|
|
|
There O |
|
were O |
|
1 O |
|
, O |
|
845 O |
|
unambiguous O |
|
NOEs B-evidence |
|
and O |
|
757 O |
|
ambiguous O |
|
NOEs B-evidence |
|
after O |
|
eight O |
|
iterations O |
|
. O |
|
|
|
a O |
|
< O |
|
SA O |
|
>, O |
|
the O |
|
average B-evidence |
|
root I-evidence |
|
mean I-evidence |
|
square I-evidence |
|
deviations I-evidence |
|
for O |
|
the O |
|
ensemble O |
|
± O |
|
S O |
|
. O |
|
D O |
|
. O |
|
|
|
B O |
|
, O |
|
a O |
|
sequence B-experimental_method |
|
alignment I-experimental_method |
|
of O |
|
the O |
|
HR1 B-structure_element |
|
domains O |
|
from O |
|
TOCA1 B-protein |
|
, O |
|
CIP4 B-protein |
|
, O |
|
and O |
|
PRK1 B-protein |
|
. O |
|
|
|
A O |
|
series O |
|
of O |
|
15N B-experimental_method |
|
HSQC I-experimental_method |
|
experiments O |
|
was O |
|
recorded O |
|
on O |
|
15N B-chemical |
|
- O |
|
labeled B-protein_state |
|
TOCA1 B-protein |
|
HR1 B-structure_element |
|
domain O |
|
in O |
|
the O |
|
presence B-protein_state |
|
of I-protein_state |
|
increasing B-experimental_method |
|
concentrations I-experimental_method |
|
of O |
|
unlabeled B-protein_state |
|
Cdc42Δ7Q61L B-complex_assembly |
|
· I-complex_assembly |
|
GMPPNP I-complex_assembly |
|
to O |
|
map O |
|
the O |
|
Cdc42 B-site |
|
- I-site |
|
binding I-site |
|
surface I-site |
|
. O |
|
|
|
Residues O |
|
with O |
|
significantly O |
|
affected O |
|
backbone O |
|
or O |
|
side O |
|
chain O |
|
chemical O |
|
shifts O |
|
when O |
|
Cdc42 B-protein_state |
|
bound I-protein_state |
|
and O |
|
that O |
|
are O |
|
buried O |
|
are O |
|
colored O |
|
dark O |
|
blue O |
|
, O |
|
whereas O |
|
those O |
|
that O |
|
are O |
|
solvent B-protein_state |
|
- I-protein_state |
|
accessible I-protein_state |
|
are O |
|
colored O |
|
yellow O |
|
. O |
|
|
|
Therefore O |
|
, O |
|
13C B-experimental_method |
|
HSQC I-experimental_method |
|
and O |
|
methyl B-experimental_method |
|
- I-experimental_method |
|
selective I-experimental_method |
|
SOFAST I-experimental_method |
|
- I-experimental_method |
|
HMQC I-experimental_method |
|
experiments O |
|
were O |
|
also O |
|
recorded O |
|
on O |
|
15N B-chemical |
|
, O |
|
13C B-chemical |
|
- O |
|
labeled B-protein_state |
|
TOCA1 B-protein |
|
HR1 B-structure_element |
|
to O |
|
yield O |
|
more O |
|
information O |
|
on O |
|
side O |
|
chain O |
|
involvement O |
|
. O |
|
|
|
As O |
|
was O |
|
the O |
|
case O |
|
when O |
|
labeled B-protein_state |
|
HR1 B-structure_element |
|
was O |
|
observed O |
|
, O |
|
several O |
|
peaks O |
|
were O |
|
shifted O |
|
in O |
|
the O |
|
complex O |
|
, O |
|
but O |
|
many O |
|
disappeared O |
|
, O |
|
indicating O |
|
exchange O |
|
on O |
|
an O |
|
unfavorable O |
|
, O |
|
millisecond O |
|
time O |
|
scale O |
|
( O |
|
Fig O |
|
. O |
|
5A O |
|
). O |
|
|
|
B O |
|
, O |
|
CSPs B-experimental_method |
|
are O |
|
shown O |
|
for O |
|
backbone O |
|
NH O |
|
groups O |
|
. O |
|
|
|
C O |
|
, O |
|
the O |
|
residues O |
|
with O |
|
significantly O |
|
affected O |
|
backbone O |
|
and O |
|
side O |
|
chain O |
|
groups O |
|
are O |
|
highlighted O |
|
on O |
|
an O |
|
NMR B-experimental_method |
|
structure B-evidence |
|
of O |
|
free B-protein_state |
|
Cdc42Δ7Q61L B-complex_assembly |
|
· I-complex_assembly |
|
GMPPNP I-complex_assembly |
|
; O |
|
those O |
|
that O |
|
are O |
|
buried O |
|
are O |
|
colored O |
|
dark O |
|
blue O |
|
, O |
|
whereas O |
|
those O |
|
that O |
|
are O |
|
solvent B-protein_state |
|
- I-protein_state |
|
accessible I-protein_state |
|
are O |
|
colored O |
|
red O |
|
. O |
|
|
|
Although O |
|
the O |
|
binding B-site |
|
interface I-site |
|
may O |
|
be O |
|
overestimated O |
|
, O |
|
this O |
|
suggests O |
|
that O |
|
the O |
|
switch B-site |
|
regions I-site |
|
are O |
|
involved O |
|
in O |
|
binding O |
|
to O |
|
TOCA1 B-protein |
|
. O |
|
|
|
HADDOCK B-experimental_method |
|
was O |
|
therefore O |
|
used O |
|
to O |
|
perform O |
|
rigid O |
|
body B-experimental_method |
|
docking I-experimental_method |
|
based O |
|
on O |
|
the O |
|
structures B-evidence |
|
of O |
|
free B-protein_state |
|
HR1 B-structure_element |
|
domain O |
|
and O |
|
Cdc42 B-protein |
|
and O |
|
ambiguous O |
|
interaction O |
|
restraints O |
|
derived O |
|
from O |
|
the O |
|
titration B-experimental_method |
|
experiments I-experimental_method |
|
described O |
|
above O |
|
. O |
|
|
|
The O |
|
cluster O |
|
with O |
|
the O |
|
lowest O |
|
root B-evidence |
|
mean I-evidence |
|
square I-evidence |
|
deviation I-evidence |
|
from O |
|
the O |
|
lowest O |
|
energy O |
|
structure B-evidence |
|
is O |
|
assumed O |
|
to O |
|
be O |
|
the O |
|
best O |
|
model O |
|
. O |
|
|
|
Residues O |
|
of O |
|
Cdc42 B-protein |
|
that O |
|
are O |
|
affected O |
|
in O |
|
the O |
|
presence B-protein_state |
|
of I-protein_state |
|
the O |
|
HR1 B-structure_element |
|
domain O |
|
but O |
|
are O |
|
not O |
|
in O |
|
close O |
|
proximity O |
|
to O |
|
it O |
|
are O |
|
colored O |
|
in O |
|
red O |
|
and O |
|
labeled O |
|
. O |
|
|
|
B O |
|
, O |
|
structure B-evidence |
|
of O |
|
Rac1 B-protein |
|
in B-protein_state |
|
complex I-protein_state |
|
with I-protein_state |
|
the O |
|
HR1b B-structure_element |
|
domain O |
|
of O |
|
PRK1 B-protein |
|
( O |
|
PDB O |
|
code O |
|
2RMK O |
|
). O |
|
|
|
Cdc42 O |
|
is O |
|
shown O |
|
in O |
|
cyan O |
|
, O |
|
and O |
|
TOCA1 B-protein |
|
is O |
|
shown O |
|
in O |
|
purple O |
|
. O |
|
|
|
D O |
|
, O |
|
selected O |
|
regions O |
|
of O |
|
the O |
|
15N B-experimental_method |
|
HSQC I-experimental_method |
|
of O |
|
600 O |
|
μm O |
|
TOCA1 B-protein |
|
HR1 B-structure_element |
|
domain O |
|
in B-protein_state |
|
complex I-protein_state |
|
with I-protein_state |
|
Cdc42 B-protein |
|
in O |
|
the O |
|
absence B-protein_state |
|
and O |
|
presence B-protein_state |
|
of I-protein_state |
|
the O |
|
N B-protein |
|
- I-protein |
|
WASP I-protein |
|
GBD B-structure_element |
|
, O |
|
showing O |
|
displacement O |
|
of O |
|
Cdc42 B-protein |
|
from O |
|
the O |
|
HR1 B-structure_element |
|
domain O |
|
by O |
|
N B-protein |
|
- I-protein |
|
WASP I-protein |
|
. O |
|
|
|
The O |
|
Kd B-evidence |
|
that O |
|
was O |
|
determined O |
|
( O |
|
37 O |
|
nm O |
|
) O |
|
is O |
|
consistent O |
|
with O |
|
the O |
|
previously O |
|
reported O |
|
affinity B-evidence |
|
. O |
|
|
|
A O |
|
comparison O |
|
of O |
|
the O |
|
HSQC B-experimental_method |
|
experiments O |
|
recorded O |
|
on O |
|
15N B-chemical |
|
- O |
|
Cdc42 B-protein |
|
alone B-protein_state |
|
, O |
|
in O |
|
the O |
|
presence B-protein_state |
|
of I-protein_state |
|
TOCA1 B-protein |
|
HR1 B-structure_element |
|
, O |
|
N B-protein |
|
- I-protein |
|
WASP I-protein |
|
GBD B-structure_element |
|
, O |
|
or O |
|
both O |
|
, O |
|
shows O |
|
that O |
|
the O |
|
spectra B-evidence |
|
in O |
|
the O |
|
presence B-protein_state |
|
of I-protein_state |
|
N B-protein |
|
- I-protein |
|
WASP I-protein |
|
and O |
|
in O |
|
the O |
|
presence B-protein_state |
|
of I-protein_state |
|
both O |
|
N B-protein |
|
- I-protein |
|
WASP I-protein |
|
and O |
|
TOCA1 B-protein |
|
HR1 B-structure_element |
|
are O |
|
identical O |
|
( O |
|
Fig O |
|
. O |
|
7C O |
|
). O |
|
|
|
These O |
|
assays O |
|
, O |
|
described O |
|
in O |
|
detail O |
|
elsewhere O |
|
, O |
|
were O |
|
carried O |
|
out O |
|
using O |
|
pyrene B-chemical |
|
actin I-chemical |
|
- O |
|
supplemented O |
|
Xenopus B-taxonomy_domain |
|
extracts O |
|
into O |
|
which O |
|
exogenous O |
|
TOCA1 B-protein |
|
HR1 B-structure_element |
|
domain O |
|
or O |
|
N B-protein |
|
- I-protein |
|
WASP I-protein |
|
GBD B-structure_element |
|
was O |
|
added O |
|
, O |
|
to O |
|
assess O |
|
their O |
|
effects O |
|
on O |
|
actin B-protein_type |
|
polymerization O |
|
. O |
|
|
|
The O |
|
corresponding O |
|
sequence O |
|
in O |
|
CIP4 B-protein |
|
also O |
|
includes O |
|
a O |
|
series O |
|
of O |
|
turns O |
|
but O |
|
is O |
|
flexible O |
|
, O |
|
whereas O |
|
in O |
|
the O |
|
HR1a B-structure_element |
|
domain O |
|
of O |
|
PRK1 B-protein |
|
, O |
|
the O |
|
equivalent O |
|
region O |
|
adopts O |
|
an O |
|
α B-structure_element |
|
- I-structure_element |
|
helical I-structure_element |
|
structure I-structure_element |
|
that O |
|
packs O |
|
against O |
|
the O |
|
coiled B-structure_element |
|
- I-structure_element |
|
coil I-structure_element |
|
. O |
|
|
|
The O |
|
lowest O |
|
energy O |
|
model B-evidence |
|
produced O |
|
by O |
|
HADDOCK B-experimental_method |
|
using O |
|
ambiguous O |
|
interaction O |
|
restraints O |
|
from O |
|
the O |
|
titration B-evidence |
|
data O |
|
resembled O |
|
the O |
|
NMR B-experimental_method |
|
structures B-evidence |
|
of O |
|
RhoA B-protein |
|
and O |
|
Rac1 B-protein |
|
in B-protein_state |
|
complex I-protein_state |
|
with I-protein_state |
|
their O |
|
HR1 B-structure_element |
|
domain O |
|
partners O |
|
. O |
|
|
|
For O |
|
example O |
|
, O |
|
Phe B-residue_name_number |
|
- I-residue_name_number |
|
56Cdc42 I-residue_name_number |
|
, O |
|
which O |
|
is O |
|
not O |
|
visible O |
|
in O |
|
free B-protein_state |
|
Cdc42 B-protein |
|
or O |
|
Cdc42 B-complex_assembly |
|
· I-complex_assembly |
|
HR1TOCA1 I-complex_assembly |
|
, O |
|
is O |
|
close O |
|
to O |
|
the O |
|
TOCA1 B-protein |
|
HR1 B-structure_element |
|
( O |
|
Fig O |
|
. O |
|
6A O |
|
). O |
|
|
|
Phe B-residue_name_number |
|
- I-residue_name_number |
|
56Cdc42 I-residue_name_number |
|
is O |
|
therefore O |
|
likely O |
|
to O |
|
be O |
|
involved O |
|
in O |
|
the O |
|
Cdc42 B-protein |
|
- O |
|
TOCA1 B-protein |
|
interaction O |
|
, O |
|
probably O |
|
by O |
|
stabilizing O |
|
the O |
|
position O |
|
of O |
|
switch B-site |
|
I I-site |
|
. O |
|
|
|
Thr B-residue_name_number |
|
- I-residue_name_number |
|
52Cdc42 I-residue_name_number |
|
, O |
|
which O |
|
has O |
|
also O |
|
been O |
|
identified O |
|
as O |
|
making O |
|
minor O |
|
contacts O |
|
with O |
|
ACK B-protein |
|
, O |
|
falls O |
|
near O |
|
the O |
|
side O |
|
chains O |
|
of O |
|
HR1TOCA1 B-structure_element |
|
helix B-structure_element |
|
1 I-structure_element |
|
, O |
|
particularly O |
|
Lys B-residue_name_number |
|
- I-residue_name_number |
|
372TOCA1 I-residue_name_number |
|
, O |
|
whereas O |
|
the O |
|
equivalent O |
|
position O |
|
in O |
|
Rac1 B-protein |
|
is O |
|
Asn B-residue_name_number |
|
- I-residue_name_number |
|
52Rac1 I-residue_name_number |
|
. O |
|
|
|
In O |
|
contrast O |
|
, O |
|
the O |
|
best O |
|
estimate O |
|
of O |
|
the O |
|
affinity B-evidence |
|
of O |
|
full B-protein_state |
|
- I-protein_state |
|
length I-protein_state |
|
WASP B-protein_type |
|
for O |
|
Cdc42 B-protein |
|
is O |
|
low O |
|
micromolar O |
|
. O |
|
|
|
WIP B-protein |
|
inhibits O |
|
the O |
|
activation O |
|
of O |
|
N B-protein |
|
- I-protein |
|
WASP I-protein |
|
by O |
|
Cdc42 B-protein |
|
, O |
|
an O |
|
effect O |
|
that O |
|
is O |
|
reversed O |
|
by O |
|
TOCA1 B-protein |
|
. O |
|
|
|
It O |
|
has O |
|
been O |
|
postulated O |
|
that O |
|
the O |
|
initial O |
|
interactions O |
|
between O |
|
this O |
|
basic O |
|
region O |
|
and O |
|
Cdc42 B-protein |
|
could O |
|
stabilize O |
|
the O |
|
active B-protein_state |
|
conformation O |
|
of O |
|
WASP B-protein |
|
, O |
|
leading O |
|
to O |
|
high O |
|
affinity O |
|
binding O |
|
between O |
|
the O |
|
core O |
|
CRIB B-structure_element |
|
and O |
|
Cdc42 B-protein |
|
. O |
|
|
|
We O |
|
envisage O |
|
a O |
|
complex O |
|
interplay O |
|
of O |
|
equilibria O |
|
between O |
|
free B-protein_state |
|
and O |
|
bound B-protein_state |
|
, O |
|
active B-protein_state |
|
and O |
|
inactive B-protein_state |
|
Cdc42 B-protein |
|
, O |
|
TOCA B-protein_type |
|
family I-protein_type |
|
, O |
|
and O |
|
WASP B-protein_type |
|
family O |
|
proteins O |
|
, O |
|
facilitating O |
|
a O |
|
tightly O |
|
spatially O |
|
and O |
|
temporally O |
|
regulated O |
|
pathway O |
|
requiring O |
|
numerous O |
|
simultaneous O |
|
events O |
|
in O |
|
order O |
|
to O |
|
achieve O |
|
appropriate O |
|
and O |
|
robust O |
|
activation O |
|
of O |
|
the O |
|
downstream O |
|
pathway O |
|
. O |
|
|
|
It O |
|
is O |
|
clear O |
|
from O |
|
the O |
|
data O |
|
presented O |
|
here O |
|
that O |
|
TOCA1 B-protein |
|
and O |
|
N B-protein |
|
- I-protein |
|
WASP I-protein |
|
do O |
|
not O |
|
bind O |
|
Cdc42 B-protein |
|
simultaneously O |
|
and O |
|
that O |
|
N B-protein |
|
- I-protein |
|
WASP I-protein |
|
is O |
|
likely O |
|
to O |
|
outcompete O |
|
TOCA1 B-protein |
|
for O |
|
Cdc42 B-protein |
|
binding O |
|
. O |
|
|
|
Furthermore O |
|
, O |
|
elevated O |
|
ACC B-protein_type |
|
activity O |
|
is O |
|
observed O |
|
in O |
|
malignant O |
|
tumours 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 |
|
ACC1 B-protein |
|
is O |
|
further O |
|
regulated O |
|
by O |
|
specific O |
|
phosphorylation B-ptm |
|
- O |
|
dependent O |
|
binding O |
|
of O |
|
BRCA1 B-protein |
|
to O |
|
Ser1263 B-residue_name_number |
|
in O |
|
the O |
|
CD B-structure_element |
|
. O |
|
|
|
Furthermore O |
|
, O |
|
phosphorylation B-ptm |
|
by O |
|
AMP B-protein |
|
- I-protein |
|
activated I-protein |
|
protein I-protein |
|
kinase I-protein |
|
( O |
|
AMPK B-protein |
|
) O |
|
and O |
|
cAMP B-protein |
|
- I-protein |
|
dependent I-protein |
|
protein I-protein |
|
kinase I-protein |
|
( O |
|
PKA B-protein |
|
) O |
|
leads O |
|
to O |
|
a O |
|
decrease O |
|
in O |
|
ACC1 B-protein |
|
activity O |
|
. O |
|
|
|
The O |
|
regulatory O |
|
Ser1201 B-residue_name_number |
|
shows O |
|
only O |
|
moderate B-protein_state |
|
conservation I-protein_state |
|
across O |
|
higher B-taxonomy_domain |
|
eukaryotes I-taxonomy_domain |
|
, O |
|
while O |
|
the O |
|
phosphorylated B-protein_state |
|
Ser1216 B-residue_name_number |
|
is O |
|
highly B-protein_state |
|
conserved I-protein_state |
|
across O |
|
all O |
|
eukaryotes B-taxonomy_domain |
|
. O |
|
|
|
In O |
|
yeast B-taxonomy_domain |
|
ACC B-protein_type |
|
, O |
|
phosphorylation B-site |
|
sites I-site |
|
have O |
|
been O |
|
identified O |
|
at O |
|
Ser2 B-residue_name_number |
|
, O |
|
Ser735 B-residue_name_number |
|
, O |
|
Ser1148 B-residue_name_number |
|
, O |
|
Ser1157 B-residue_name_number |
|
and O |
|
Ser1162 B-residue_name_number |
|
( O |
|
ref O |
|
.). O |
|
|
|
SceCD B-species |
|
comprises O |
|
four O |
|
distinct O |
|
domains O |
|
, O |
|
an O |
|
N O |
|
- O |
|
terminal O |
|
α B-structure_element |
|
- I-structure_element |
|
helical I-structure_element |
|
domain I-structure_element |
|
( O |
|
CDN B-structure_element |
|
), O |
|
and O |
|
a O |
|
central O |
|
four B-structure_element |
|
- I-structure_element |
|
helix I-structure_element |
|
bundle I-structure_element |
|
linker I-structure_element |
|
domain I-structure_element |
|
( O |
|
CDL B-structure_element |
|
), O |
|
followed O |
|
by O |
|
two O |
|
α B-structure_element |
|
– I-structure_element |
|
β I-structure_element |
|
- I-structure_element |
|
fold I-structure_element |
|
C I-structure_element |
|
- I-structure_element |
|
terminal I-structure_element |
|
domains I-structure_element |
|
( O |
|
CDC1 B-structure_element |
|
/ O |
|
CDC2 B-structure_element |
|
). O |
|
|
|
In O |
|
insect B-experimental_method |
|
- I-experimental_method |
|
cell I-experimental_method |
|
- I-experimental_method |
|
expressed I-experimental_method |
|
full B-protein_state |
|
- I-protein_state |
|
length I-protein_state |
|
SceACC B-protein |
|
, O |
|
the O |
|
highly B-protein_state |
|
conserved I-protein_state |
|
Ser1157 B-residue_name_number |
|
is O |
|
the O |
|
only O |
|
fully B-protein_state |
|
occupied I-protein_state |
|
phosphorylation B-site |
|
site I-site |
|
with O |
|
functional O |
|
relevance O |
|
in O |
|
S B-species |
|
. I-species |
|
cerevisiae I-species |
|
. O |
|
|
|
Additional O |
|
phosphorylation B-ptm |
|
was O |
|
detected O |
|
for O |
|
Ser2101 B-residue_name_number |
|
and O |
|
Tyr2179 B-residue_name_number |
|
; O |
|
however O |
|
, O |
|
these O |
|
sites O |
|
are O |
|
neither B-protein_state |
|
conserved I-protein_state |
|
across O |
|
fungal B-taxonomy_domain |
|
ACC B-protein_type |
|
nor B-protein_state |
|
natively I-protein_state |
|
phosphorylated I-protein_state |
|
in O |
|
yeast B-taxonomy_domain |
|
. O |
|
|
|
Already O |
|
the O |
|
binding O |
|
of O |
|
phosphorylated B-protein_state |
|
Ser1157 B-residue_name_number |
|
apparently O |
|
stabilizes O |
|
the O |
|
regulatory B-structure_element |
|
loop I-structure_element |
|
conformation O |
|
; O |
|
the O |
|
accessory O |
|
phosphorylation B-site |
|
sites I-site |
|
Ser1148 B-residue_name_number |
|
and O |
|
Ser1162 B-residue_name_number |
|
in O |
|
the O |
|
same B-structure_element |
|
loop I-structure_element |
|
may O |
|
further O |
|
modulate O |
|
the O |
|
strength O |
|
of O |
|
interaction O |
|
between O |
|
the O |
|
regulatory B-structure_element |
|
loop I-structure_element |
|
and O |
|
the O |
|
CDC1 B-structure_element |
|
and O |
|
CDC2 B-structure_element |
|
domains O |
|
. O |
|
|
|
The O |
|
variable O |
|
CD B-structure_element |
|
is O |
|
conserved B-protein_state |
|
between O |
|
yeast B-taxonomy_domain |
|
and O |
|
human B-species |
|
|
|
To O |
|
compare O |
|
the O |
|
organization O |
|
of O |
|
fungal B-taxonomy_domain |
|
and O |
|
human B-species |
|
ACC B-protein_type |
|
CD B-structure_element |
|
, O |
|
we O |
|
determined B-experimental_method |
|
the I-experimental_method |
|
structure I-experimental_method |
|
of O |
|
a O |
|
human B-species |
|
ACC1 B-mutant |
|
fragment I-mutant |
|
that O |
|
comprises O |
|
the O |
|
BT B-structure_element |
|
and O |
|
CD B-structure_element |
|
domains O |
|
( O |
|
HsaBT B-mutant |
|
- I-mutant |
|
CD I-mutant |
|
), O |
|
but O |
|
lacks B-protein_state |
|
the O |
|
mobile O |
|
BCCP B-structure_element |
|
in O |
|
between O |
|
( O |
|
Fig O |
|
. O |
|
1a O |
|
). O |
|
|
|
Besides O |
|
the O |
|
regulatory B-structure_element |
|
loop I-structure_element |
|
, O |
|
also O |
|
the O |
|
phosphopeptide B-site |
|
target I-site |
|
region I-site |
|
for O |
|
BRCA1 B-protein |
|
interaction O |
|
is O |
|
not O |
|
resolved O |
|
presumably O |
|
because O |
|
of O |
|
pronounced O |
|
flexibility O |
|
. O |
|
|
|
The O |
|
neighbouring O |
|
loop B-structure_element |
|
on O |
|
the O |
|
CT B-structure_element |
|
side O |
|
( O |
|
between O |
|
CT B-structure_element |
|
β1 B-structure_element |
|
/ O |
|
β2 B-structure_element |
|
) O |
|
is O |
|
displaced O |
|
by O |
|
2 O |
|
. O |
|
5 O |
|
Å O |
|
compared O |
|
to O |
|
isolated B-protein_state |
|
CT B-structure_element |
|
structures B-evidence |
|
( O |
|
Supplementary O |
|
Fig O |
|
. O |
|
3c O |
|
). O |
|
|
|
The O |
|
interface B-site |
|
between O |
|
CDC2 B-structure_element |
|
and O |
|
CDL B-structure_element |
|
/ O |
|
CDC1 B-structure_element |
|
, O |
|
which O |
|
is O |
|
mediated O |
|
by O |
|
the O |
|
phosphorylated B-protein_state |
|
regulatory B-structure_element |
|
loop I-structure_element |
|
in O |
|
the O |
|
SceCD B-species |
|
structure B-evidence |
|
, O |
|
is O |
|
less O |
|
variable O |
|
than O |
|
the O |
|
CD B-structure_element |
|
– I-structure_element |
|
CT I-structure_element |
|
junction I-structure_element |
|
, O |
|
and O |
|
permits O |
|
only O |
|
limited O |
|
rotation O |
|
and O |
|
tilting O |
|
( O |
|
Fig O |
|
. O |
|
3b O |
|
). O |
|
|
|
The O |
|
BC B-structure_element |
|
domain O |
|
is O |
|
not O |
|
completely O |
|
disordered O |
|
, O |
|
but O |
|
laterally O |
|
attached O |
|
to O |
|
BT B-structure_element |
|
/ O |
|
CDN B-structure_element |
|
in O |
|
a O |
|
generally B-protein_state |
|
conserved I-protein_state |
|
position I-protein_state |
|
, O |
|
albeit O |
|
with O |
|
increased O |
|
flexibility O |
|
. O |
|
|
|
The O |
|
most O |
|
relevant O |
|
candidate O |
|
site O |
|
for O |
|
mediating O |
|
such O |
|
additional O |
|
flexibility O |
|
and O |
|
permitting O |
|
an O |
|
extended O |
|
set O |
|
of O |
|
conformations O |
|
is O |
|
the O |
|
CDC1 B-site |
|
/ I-site |
|
CDC2 I-site |
|
interface I-site |
|
, O |
|
which O |
|
is O |
|
rigidified O |
|
by O |
|
the O |
|
Ser1157 B-residue_name_number |
|
- O |
|
phosphorylated B-protein_state |
|
regulatory B-structure_element |
|
loop I-structure_element |
|
, O |
|
as O |
|
depicted O |
|
in O |
|
the O |
|
SceCD B-species |
|
crystal B-evidence |
|
structure I-evidence |
|
. 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 |
|
|
|
On O |
|
the O |
|
basis O |
|
of O |
|
a O |
|
superposition B-experimental_method |
|
of O |
|
CDC2 B-structure_element |
|
, O |
|
CDC1 B-structure_element |
|
of O |
|
the O |
|
phosphorylated B-protein_state |
|
SceCD B-species |
|
is O |
|
rotated O |
|
by O |
|
30 O |
|
° O |
|
relative O |
|
to O |
|
CDC1 B-structure_element |
|
of O |
|
the O |
|
non B-protein_state |
|
- I-protein_state |
|
phosphorylated I-protein_state |
|
flACC B-mutant |
|
( O |
|
Supplementary O |
|
Fig O |
|
. O |
|
5d O |
|
), O |
|
similar O |
|
to O |
|
what O |
|
we O |
|
have O |
|
observed O |
|
for O |
|
the O |
|
non B-protein_state |
|
- I-protein_state |
|
phosphorylated I-protein_state |
|
HsaBT B-mutant |
|
- I-mutant |
|
CD I-mutant |
|
( O |
|
Supplementary O |
|
Fig O |
|
. O |
|
1d O |
|
). O |
|
|
|
When O |
|
inspecting B-experimental_method |
|
all O |
|
individual O |
|
protomer B-oligomeric_state |
|
and O |
|
fragment B-mutant |
|
structures B-evidence |
|
in O |
|
their O |
|
study O |
|
, O |
|
Wei O |
|
and O |
|
Tong O |
|
also O |
|
identify O |
|
the O |
|
CDN B-structure_element |
|
/ I-structure_element |
|
CDC1 I-structure_element |
|
connection I-structure_element |
|
as O |
|
a O |
|
highly B-protein_state |
|
flexible I-protein_state |
|
hinge B-structure_element |
|
, O |
|
in O |
|
agreement O |
|
with O |
|
our O |
|
observations O |
|
. O |
|
|
|
The O |
|
only O |
|
bona O |
|
fide O |
|
regulatory B-protein_state |
|
phophorylation B-site |
|
site I-site |
|
of O |
|
fungal B-taxonomy_domain |
|
ACC B-protein_type |
|
in O |
|
the O |
|
regulatory B-structure_element |
|
loop I-structure_element |
|
is O |
|
directly O |
|
participating O |
|
in O |
|
CDC1 B-structure_element |
|
/ O |
|
CDC2 B-structure_element |
|
domain O |
|
interactions O |
|
and O |
|
thus O |
|
stabilizes O |
|
the O |
|
hinge B-structure_element |
|
conformation I-structure_element |
|
. O |
|
|
|
The O |
|
phosphorylated B-protein_state |
|
regulatory B-structure_element |
|
loop I-structure_element |
|
binds O |
|
to O |
|
an O |
|
allosteric B-site |
|
site I-site |
|
at O |
|
the O |
|
interface B-site |
|
of O |
|
two O |
|
non B-protein_state |
|
- I-protein_state |
|
catalytic I-protein_state |
|
domains O |
|
and O |
|
restricts O |
|
conformational O |
|
freedom O |
|
at O |
|
several O |
|
hinges B-structure_element |
|
in O |
|
the O |
|
dynamic B-protein_state |
|
ACC B-protein_type |
|
. O |
|
|
|
However O |
|
, O |
|
the O |
|
example O |
|
of O |
|
ACC B-protein_type |
|
now O |
|
demonstrates O |
|
the O |
|
possibility O |
|
of O |
|
regulating O |
|
activity O |
|
by O |
|
controlled O |
|
dynamics O |
|
of O |
|
non B-structure_element |
|
- I-structure_element |
|
enzymatic I-structure_element |
|
linker I-structure_element |
|
regions I-structure_element |
|
also O |
|
in O |
|
other O |
|
families O |
|
of O |
|
carrier B-protein_type |
|
- I-protein_type |
|
dependent I-protein_type |
|
multienzymes I-protein_type |
|
. O |
|
|
|
CDN B-structure_element |
|
is O |
|
linked O |
|
by O |
|
a O |
|
four B-structure_element |
|
- I-structure_element |
|
helix I-structure_element |
|
bundle I-structure_element |
|
( O |
|
CDL B-structure_element |
|
) O |
|
to O |
|
two B-structure_element |
|
α I-structure_element |
|
– I-structure_element |
|
β I-structure_element |
|
- I-structure_element |
|
fold I-structure_element |
|
domains I-structure_element |
|
( O |
|
CDC1 B-structure_element |
|
and O |
|
CDC2 B-structure_element |
|
). O |
|
|
|
( O |
|
e O |
|
) O |
|
Structural O |
|
overview O |
|
of O |
|
HsaBT B-mutant |
|
- I-mutant |
|
CD I-mutant |
|
. O |
|
|
|
( O |
|
a O |
|
– O |
|
c O |
|
) O |
|
Large O |
|
- O |
|
scale O |
|
conformational O |
|
variability O |
|
of O |
|
the O |
|
CDN B-structure_element |
|
domain O |
|
relative O |
|
to O |
|
the O |
|
CDL B-structure_element |
|
/ O |
|
CDC1 B-structure_element |
|
domain O |
|
. O |
|
|
|
Domains O |
|
other O |
|
than O |
|
CDN B-structure_element |
|
and O |
|
CDL B-structure_element |
|
/ O |
|
CDC1 B-structure_element |
|
are O |
|
omitted O |
|
for O |
|
clarity O |
|
. O |
|
|
|
Structural O |
|
insights O |
|
into O |
|
the O |
|
Escherichia B-species |
|
coli I-species |
|
lysine B-protein_type |
|
decarboxylases I-protein_type |
|
and O |
|
molecular O |
|
determinants O |
|
of O |
|
interaction O |
|
with O |
|
the O |
|
AAA B-protein_type |
|
+ I-protein_type |
|
ATPase I-protein_type |
|
RavA B-protein |
|
|
|
Previously O |
|
, O |
|
we O |
|
proposed O |
|
a O |
|
pseudoatomic B-evidence |
|
model I-evidence |
|
of O |
|
the O |
|
LdcI B-complex_assembly |
|
- I-complex_assembly |
|
RavA I-complex_assembly |
|
cage O |
|
based O |
|
on O |
|
its O |
|
cryo B-experimental_method |
|
- I-experimental_method |
|
electron I-experimental_method |
|
microscopy I-experimental_method |
|
map B-evidence |
|
and O |
|
crystal B-evidence |
|
structures I-evidence |
|
of O |
|
an O |
|
inactive B-protein_state |
|
LdcI B-protein |
|
decamer B-oligomeric_state |
|
and O |
|
a O |
|
RavA B-protein |
|
monomer B-oligomeric_state |
|
. O |
|
|
|
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 |
|
|
|
Inducible B-protein_state |
|
enterobacterial B-taxonomy_domain |
|
amino B-protein_type |
|
acid I-protein_type |
|
decarboxylases I-protein_type |
|
have O |
|
been O |
|
intensively O |
|
studied O |
|
since O |
|
the O |
|
early O |
|
1940 O |
|
because O |
|
the O |
|
ability O |
|
of O |
|
bacteria B-taxonomy_domain |
|
to O |
|
withstand O |
|
acid O |
|
stress O |
|
can O |
|
be O |
|
linked O |
|
to O |
|
their O |
|
pathogenicity O |
|
in O |
|
humans B-species |
|
. 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 |
|
|
|
Monomers B-oligomeric_state |
|
tightly O |
|
associate O |
|
via O |
|
their O |
|
core B-structure_element |
|
domains I-structure_element |
|
into O |
|
2 B-protein_state |
|
- I-protein_state |
|
fold I-protein_state |
|
symmetrical I-protein_state |
|
dimers B-oligomeric_state |
|
with O |
|
two O |
|
complete O |
|
active B-site |
|
sites I-site |
|
, O |
|
and O |
|
further O |
|
build O |
|
a O |
|
toroidal B-structure_element |
|
D5 I-structure_element |
|
- I-structure_element |
|
symmetrical I-structure_element |
|
structure I-structure_element |
|
held O |
|
by O |
|
the O |
|
wing B-structure_element |
|
and O |
|
core B-structure_element |
|
domain I-structure_element |
|
interactions O |
|
around O |
|
the O |
|
central B-structure_element |
|
pore I-structure_element |
|
, O |
|
with O |
|
the O |
|
CTDs B-structure_element |
|
at O |
|
the O |
|
periphery O |
|
. O |
|
|
|
Furthermore O |
|
, O |
|
we O |
|
recently O |
|
solved B-experimental_method |
|
the I-experimental_method |
|
structure I-experimental_method |
|
of O |
|
the O |
|
E B-species |
|
. I-species |
|
coli I-species |
|
LdcI B-complex_assembly |
|
- I-complex_assembly |
|
RavA I-complex_assembly |
|
complex O |
|
by O |
|
cryo B-experimental_method |
|
- I-experimental_method |
|
electron I-experimental_method |
|
microscopy I-experimental_method |
|
( O |
|
cryoEM B-experimental_method |
|
) O |
|
and O |
|
combined O |
|
it O |
|
with O |
|
the O |
|
crystal B-evidence |
|
structures I-evidence |
|
of O |
|
the O |
|
individual O |
|
proteins O |
|
. O |
|
|
|
To O |
|
solve O |
|
this O |
|
discrepancy O |
|
, O |
|
in O |
|
the O |
|
present O |
|
work O |
|
we O |
|
provided O |
|
a O |
|
three O |
|
- O |
|
dimensional O |
|
( O |
|
3D O |
|
) O |
|
cryoEM B-experimental_method |
|
reconstruction B-evidence |
|
of O |
|
LdcC B-protein |
|
and O |
|
compared O |
|
it O |
|
with O |
|
the O |
|
available O |
|
LdcI B-protein |
|
and O |
|
LdcI B-complex_assembly |
|
- I-complex_assembly |
|
RavA I-complex_assembly |
|
structures B-evidence |
|
. O |
|
|
|
Given O |
|
that O |
|
the O |
|
LdcI B-protein |
|
crystal B-evidence |
|
structures I-evidence |
|
were O |
|
obtained O |
|
at O |
|
high B-protein_state |
|
pH I-protein_state |
|
where O |
|
the O |
|
enzyme O |
|
is O |
|
inactive B-protein_state |
|
( O |
|
LdcIi B-protein |
|
, O |
|
pH B-protein_state |
|
8 I-protein_state |
|
. I-protein_state |
|
5 I-protein_state |
|
), O |
|
whereas O |
|
the O |
|
cryoEM B-experimental_method |
|
reconstructions B-evidence |
|
of O |
|
LdcI B-complex_assembly |
|
- I-complex_assembly |
|
RavA I-complex_assembly |
|
and O |
|
LdcI B-complex_assembly |
|
- I-complex_assembly |
|
LARA I-complex_assembly |
|
were O |
|
done O |
|
at O |
|
acidic B-protein_state |
|
pH I-protein_state |
|
optimal I-protein_state |
|
for O |
|
the O |
|
enzymatic O |
|
activity O |
|
, O |
|
for O |
|
a O |
|
meaningful O |
|
comparison O |
|
, O |
|
we O |
|
also O |
|
produced O |
|
a O |
|
3D B-evidence |
|
reconstruction I-evidence |
|
of O |
|
the O |
|
LdcI B-protein |
|
at O |
|
active B-protein_state |
|
pH I-protein_state |
|
( O |
|
LdcIa B-protein |
|
, O |
|
pH B-protein_state |
|
6 I-protein_state |
|
. I-protein_state |
|
2 I-protein_state |
|
). O |
|
|
|
As O |
|
common O |
|
for O |
|
the O |
|
α B-protein_type |
|
family I-protein_type |
|
of O |
|
the O |
|
PLP B-protein_type |
|
- I-protein_type |
|
dependent I-protein_type |
|
decarboxylases I-protein_type |
|
, O |
|
dimerization O |
|
is O |
|
required O |
|
for O |
|
the O |
|
enzymatic O |
|
activity O |
|
because O |
|
the O |
|
active B-site |
|
site I-site |
|
is O |
|
buried O |
|
in O |
|
the O |
|
dimer B-site |
|
interface I-site |
|
( O |
|
Fig O |
|
. O |
|
3A O |
|
, O |
|
B O |
|
). O |
|
|
|
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 |
|
|
|
Second O |
|
, O |
|
the O |
|
phylogenetic B-experimental_method |
|
analysis I-experimental_method |
|
clearly O |
|
split O |
|
the O |
|
lysine B-protein_type |
|
decarboxylases I-protein_type |
|
into O |
|
two O |
|
groups O |
|
( O |
|
Fig O |
|
. O |
|
6A O |
|
). O |
|
|
|
Inspection O |
|
of O |
|
these O |
|
consensus B-evidence |
|
sequences I-evidence |
|
revealed O |
|
important O |
|
differences O |
|
between O |
|
the O |
|
groups O |
|
regarding O |
|
charge O |
|
, O |
|
size O |
|
and O |
|
hydrophobicity O |
|
of O |
|
several O |
|
residues O |
|
precisely O |
|
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 |
|
that O |
|
is O |
|
responsible O |
|
for O |
|
the O |
|
interaction O |
|
with O |
|
RavA B-protein |
|
( O |
|
Fig O |
|
. O |
|
6B O |
|
– O |
|
D O |
|
). O |
|
|
|
For O |
|
example O |
|
, O |
|
in O |
|
our O |
|
previous O |
|
study O |
|
, O |
|
site B-experimental_method |
|
- I-experimental_method |
|
directed I-experimental_method |
|
mutations I-experimental_method |
|
identified O |
|
Y697 B-residue_name_number |
|
as O |
|
critically O |
|
required O |
|
for O |
|
the O |
|
RavA B-protein |
|
binding O |
|
. 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 |
|
|
|
Superposition B-experimental_method |
|
of O |
|
the O |
|
pseudoatomic B-evidence |
|
models I-evidence |
|
of O |
|
LdcC B-protein |
|
, O |
|
LdcI B-protein |
|
from O |
|
LdcI B-complex_assembly |
|
- I-complex_assembly |
|
LARA I-complex_assembly |
|
and O |
|
LdcIa B-protein |
|
colored O |
|
as O |
|
in O |
|
Fig O |
|
. O |
|
1 O |
|
, O |
|
and O |
|
the O |
|
crystal B-evidence |
|
structure I-evidence |
|
of O |
|
LdcIi B-protein |
|
in O |
|
shades O |
|
of O |
|
yellow O |
|
. O |
|
|
|
( O |
|
B O |
|
) O |
|
The O |
|
LdcIi B-protein |
|
dimer B-oligomeric_state |
|
extracted O |
|
from O |
|
the O |
|
crystal B-evidence |
|
structure I-evidence |
|
of O |
|
the O |
|
decamer B-oligomeric_state |
|
. O |
|
|
|
( O |
|
A O |
|
) O |
|
A O |
|
slice O |
|
through O |
|
the O |
|
pseudoatomic B-evidence |
|
models I-evidence |
|
of O |
|
the O |
|
LdcIa B-protein |
|
( O |
|
purple O |
|
) O |
|
and O |
|
LdcC B-protein |
|
( O |
|
green O |
|
) O |
|
monomers B-oligomeric_state |
|
extracted O |
|
from O |
|
the O |
|
superimposed B-experimental_method |
|
decamers B-oligomeric_state |
|
( O |
|
Fig O |
|
. O |
|
2 O |
|
). O |
|
( O |
|
B O |
|
) O |
|
The O |
|
C O |
|
- O |
|
terminal O |
|
β B-structure_element |
|
- I-structure_element |
|
sheet I-structure_element |
|
in O |
|
LdcIa B-protein |
|
and O |
|
LdcC B-protein |
|
enlarged O |
|
from O |
|
( O |
|
A O |
|
, O |
|
C O |
|
) O |
|
Exchanged O |
|
primary O |
|
sequences O |
|
( O |
|
capital O |
|
letters O |
|
) O |
|
and O |
|
their O |
|
immediate O |
|
vicinity O |
|
( O |
|
lower O |
|
case O |
|
letters O |
|
) O |
|
colored O |
|
as O |
|
in O |
|
( O |
|
A O |
|
, O |
|
B O |
|
), O |
|
with O |
|
the O |
|
corresponding O |
|
secondary O |
|
structure O |
|
elements O |
|
and O |
|
the O |
|
amino O |
|
acid O |
|
numbering O |
|
shown O |
|
. O |
|
|
|
Sequence B-experimental_method |
|
alignment I-experimental_method |
|
suggests O |
|
that O |
|
both O |
|
kinases B-protein_type |
|
belong O |
|
to O |
|
the O |
|
ribulokinase B-protein_type |
|
- I-protein_type |
|
like I-protein_type |
|
carbohydrate I-protein_type |
|
kinases I-protein_type |
|
, O |
|
a O |
|
sub O |
|
- O |
|
family O |
|
of O |
|
FGGY B-protein_type |
|
family I-protein_type |
|
carbohydrate I-protein_type |
|
kinases I-protein_type |
|
. O |
|
|
|
In O |
|
addition O |
|
, O |
|
our O |
|
enzymatic B-experimental_method |
|
assays I-experimental_method |
|
suggested O |
|
that O |
|
SePSK B-protein |
|
has O |
|
the O |
|
capability O |
|
to O |
|
phosphorylate O |
|
D B-chemical |
|
- I-chemical |
|
ribulose I-chemical |
|
. O |
|
|
|
These O |
|
kinases B-protein_type |
|
exhibit O |
|
considerable O |
|
differences O |
|
in O |
|
their O |
|
folding O |
|
pattern O |
|
and O |
|
substrate O |
|
specificity O |
|
. O |
|
|
|
Domain B-structure_element |
|
I I-structure_element |
|
exhibits O |
|
a O |
|
ribonuclease B-structure_element |
|
H I-structure_element |
|
- I-structure_element |
|
like I-structure_element |
|
folding I-structure_element |
|
pattern I-structure_element |
|
, O |
|
and O |
|
is O |
|
responsible O |
|
for O |
|
the O |
|
substrate O |
|
binding O |
|
, O |
|
while O |
|
domain B-structure_element |
|
II I-structure_element |
|
possesses O |
|
an O |
|
actin B-structure_element |
|
- I-structure_element |
|
like I-structure_element |
|
ATPase I-structure_element |
|
domain I-structure_element |
|
that O |
|
binds O |
|
cofactor O |
|
ATP B-chemical |
|
. O |
|
|
|
2 B-residue_range |
|
– I-residue_range |
|
228 I-residue_range |
|
and O |
|
aa O |
|
. O |
|
|
|
Domain B-structure_element |
|
II I-structure_element |
|
is O |
|
comprised O |
|
of O |
|
aa O |
|
. O |
|
|
|
229 B-residue_range |
|
– I-residue_range |
|
401 I-residue_range |
|
and O |
|
classified O |
|
into O |
|
B2 B-structure_element |
|
( O |
|
β31 B-structure_element |
|
/ O |
|
β29 B-structure_element |
|
/ O |
|
β22 B-structure_element |
|
/ O |
|
β23 B-structure_element |
|
/ O |
|
β25 B-structure_element |
|
/ O |
|
β24 B-structure_element |
|
) O |
|
and O |
|
A3 B-structure_element |
|
( O |
|
α26 B-structure_element |
|
/ O |
|
α27 B-structure_element |
|
/ O |
|
α28 B-structure_element |
|
/ O |
|
α30 B-structure_element |
|
) O |
|
( O |
|
Fig O |
|
1A O |
|
and O |
|
S1 O |
|
Fig O |
|
). O |
|
|
|
( O |
|
A O |
|
) O |
|
Three O |
|
- O |
|
dimensional O |
|
structure B-evidence |
|
of O |
|
apo B-protein_state |
|
- O |
|
SePSK B-protein |
|
. O |
|
|
|
( O |
|
B O |
|
) O |
|
Three O |
|
- O |
|
dimensional O |
|
structure B-evidence |
|
of O |
|
apo B-protein_state |
|
- O |
|
AtXK B-protein |
|
- I-protein |
|
1 I-protein |
|
. O |
|
|
|
In O |
|
order O |
|
to O |
|
understand O |
|
the O |
|
function O |
|
of O |
|
these O |
|
two O |
|
kinases O |
|
, O |
|
we O |
|
performed O |
|
structural B-experimental_method |
|
comparison I-experimental_method |
|
using O |
|
Dali B-experimental_method |
|
server I-experimental_method |
|
. O |
|
|
|
Both O |
|
SePSK B-protein |
|
and O |
|
AtXK B-protein |
|
- I-protein |
|
1 I-protein |
|
showed O |
|
ATP B-chemical |
|
hydrolysis O |
|
activity O |
|
in O |
|
the O |
|
absence B-protein_state |
|
of I-protein_state |
|
substrate O |
|
. O |
|
|
|
This O |
|
result O |
|
was O |
|
consistent O |
|
with O |
|
our O |
|
enzymatic B-experimental_method |
|
activity I-experimental_method |
|
assays I-experimental_method |
|
where O |
|
SePSK B-protein |
|
and O |
|
AtXK B-protein |
|
- I-protein |
|
1 I-protein |
|
showed O |
|
ATP B-chemical |
|
hydrolysis O |
|
activity O |
|
without O |
|
adding O |
|
any O |
|
substrates O |
|
( O |
|
Fig O |
|
2A O |
|
and O |
|
2C O |
|
). O |
|
|
|
( O |
|
A O |
|
) O |
|
The O |
|
electron B-evidence |
|
density I-evidence |
|
of O |
|
AMP B-chemical |
|
- I-chemical |
|
PNP I-chemical |
|
. O |
|
|
|
To O |
|
better O |
|
understand O |
|
the O |
|
interaction O |
|
pattern O |
|
between O |
|
SePSK B-protein |
|
and O |
|
D B-chemical |
|
- I-chemical |
|
ribulose I-chemical |
|
, O |
|
the O |
|
apo B-protein_state |
|
- O |
|
SePSK B-protein |
|
crystals B-experimental_method |
|
were I-experimental_method |
|
soaked I-experimental_method |
|
into I-experimental_method |
|
the O |
|
reservoir B-experimental_method |
|
with O |
|
10 O |
|
mM O |
|
D B-chemical |
|
- I-chemical |
|
ribulose I-chemical |
|
( O |
|
RBL B-chemical |
|
) O |
|
and O |
|
the O |
|
RBL B-complex_assembly |
|
- I-complex_assembly |
|
SePSK I-complex_assembly |
|
structure B-evidence |
|
was O |
|
solved B-experimental_method |
|
. O |
|
|
|
As O |
|
shown O |
|
in O |
|
Fig O |
|
4A O |
|
, O |
|
the O |
|
nearest O |
|
distance O |
|
between O |
|
the O |
|
carbon O |
|
skeleton O |
|
of O |
|
two O |
|
D B-chemical |
|
- I-chemical |
|
ribulose I-chemical |
|
molecules O |
|
are O |
|
approx O |
|
. O |
|
|
|
Furthermore O |
|
, O |
|
the O |
|
O2 O |
|
of O |
|
RBL1 B-residue_name_number |
|
interacts O |
|
with O |
|
the O |
|
main O |
|
chain O |
|
amide O |
|
nitrogen O |
|
of O |
|
Ser72 B-residue_name_number |
|
( O |
|
Fig O |
|
4B O |
|
). O |
|
|
|
( O |
|
A O |
|
) O |
|
The O |
|
electrostatic B-evidence |
|
potential I-evidence |
|
surface I-evidence |
|
map I-evidence |
|
of O |
|
RBL B-complex_assembly |
|
- I-complex_assembly |
|
SePSK I-complex_assembly |
|
and O |
|
a O |
|
zoom O |
|
- O |
|
in O |
|
view O |
|
of O |
|
RBL B-site |
|
binding I-site |
|
site I-site |
|
. O |
|
|
|
The O |
|
RBL B-chemical |
|
molecules O |
|
( O |
|
carbon O |
|
atoms O |
|
colored O |
|
yellow O |
|
) O |
|
and O |
|
amino O |
|
acid O |
|
residues O |
|
of O |
|
SePSK B-protein |
|
( O |
|
carbon O |
|
atoms O |
|
colored O |
|
green O |
|
) O |
|
involved O |
|
in O |
|
RBL B-chemical |
|
interaction O |
|
are O |
|
shown O |
|
as O |
|
sticks O |
|
. O |
|
|
|
Structural B-experimental_method |
|
comparison I-experimental_method |
|
of O |
|
SePSK B-protein |
|
and O |
|
AtXK B-protein |
|
- I-protein |
|
1 I-protein |
|
showed O |
|
that O |
|
while O |
|
the O |
|
RBL1 B-site |
|
binding I-site |
|
pocket I-site |
|
is O |
|
conserved B-protein_state |
|
, O |
|
the O |
|
RBL2 B-site |
|
pocket I-site |
|
is O |
|
disrupted O |
|
in O |
|
AtXK B-protein |
|
- I-protein |
|
1 I-protein |
|
structure B-evidence |
|
, O |
|
despite O |
|
the O |
|
fact O |
|
that O |
|
the O |
|
residues O |
|
interacting O |
|
with O |
|
RBL2 B-residue_name_number |
|
are O |
|
highly B-protein_state |
|
conserved I-protein_state |
|
between O |
|
the O |
|
two O |
|
proteins O |
|
. O |
|
|
|
To O |
|
further O |
|
verified O |
|
this O |
|
result O |
|
, O |
|
we O |
|
measured O |
|
the O |
|
binding B-evidence |
|
affinity I-evidence |
|
for O |
|
D B-chemical |
|
- I-chemical |
|
ribulose I-chemical |
|
of O |
|
both O |
|
wild B-protein_state |
|
type I-protein_state |
|
( O |
|
WT B-protein_state |
|
) O |
|
and O |
|
D8A B-mutant |
|
mutant B-protein_state |
|
of O |
|
SePSK B-protein |
|
using O |
|
a O |
|
surface B-experimental_method |
|
plasmon I-experimental_method |
|
resonance I-experimental_method |
|
method I-experimental_method |
|
. O |
|
|
|
Dissociation B-evidence |
|
rate I-evidence |
|
constant I-evidence |
|
( O |
|
Kd B-evidence |
|
) O |
|
of O |
|
wild B-protein_state |
|
type I-protein_state |
|
and O |
|
D8A B-mutant |
|
- O |
|
SePSK B-protein |
|
are O |
|
3 O |
|
ms O |
|
- O |
|
1 O |
|
and O |
|
9 O |
|
ms O |
|
- O |
|
1 O |
|
, O |
|
respectively O |
|
. O |
|
|
|
Simulated O |
|
conformational O |
|
change O |
|
of O |
|
SePSK B-protein |
|
during O |
|
the O |
|
catalytic O |
|
process 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 |
|
|
|
Mep2 B-protein_type |
|
proteins I-protein_type |
|
are O |
|
tightly O |
|
regulated O |
|
fungal B-taxonomy_domain |
|
ammonium B-protein_type |
|
transporters I-protein_type |
|
. O |
|
|
|
While O |
|
most O |
|
studies O |
|
have O |
|
focused O |
|
on O |
|
the O |
|
Saccharomyces B-species |
|
cerevisiae I-species |
|
transceptors B-protein_type |
|
for O |
|
phosphate B-chemical |
|
( O |
|
Pho84 B-protein |
|
), O |
|
amino B-chemical |
|
acids I-chemical |
|
( O |
|
Gap1 B-protein |
|
) O |
|
and O |
|
ammonium B-chemical |
|
( O |
|
Mep2 B-protein |
|
), O |
|
transceptors B-protein_type |
|
are O |
|
found O |
|
in O |
|
higher B-taxonomy_domain |
|
eukaryotes I-taxonomy_domain |
|
as O |
|
well O |
|
( O |
|
for O |
|
example O |
|
, O |
|
the O |
|
mammalian B-taxonomy_domain |
|
SNAT2 B-protein |
|
amino B-protein_type |
|
- I-protein_type |
|
acid I-protein_type |
|
transporter I-protein_type |
|
and O |
|
the O |
|
GLUT2 B-protein |
|
glucose B-protein_type |
|
transporter I-protein_type |
|
). O |
|
|
|
With O |
|
the O |
|
exception O |
|
of O |
|
the O |
|
human B-species |
|
RhCG B-protein |
|
structure B-evidence |
|
, O |
|
no O |
|
structural O |
|
information O |
|
is O |
|
available O |
|
for O |
|
eukaryotic B-taxonomy_domain |
|
ammonium B-protein_type |
|
transporters I-protein_type |
|
. O |
|
|
|
Ammonium B-chemical |
|
transport O |
|
is O |
|
tightly O |
|
regulated O |
|
. O |
|
|
|
In O |
|
bacteria B-taxonomy_domain |
|
, O |
|
amt B-gene |
|
genes O |
|
are O |
|
present O |
|
in O |
|
an O |
|
operon O |
|
with O |
|
glnK B-gene |
|
, O |
|
encoding O |
|
a O |
|
PII B-protein_type |
|
- I-protein_type |
|
like I-protein_type |
|
signal I-protein_type |
|
transduction I-protein_type |
|
class I-protein_type |
|
protein I-protein_type |
|
. O |
|
|
|
In O |
|
plants B-taxonomy_domain |
|
, O |
|
transporter B-protein_type |
|
phosphorylation B-ptm |
|
and O |
|
dephosphorylation B-ptm |
|
are O |
|
known O |
|
to O |
|
regulate O |
|
activity O |
|
. O |
|
|
|
In O |
|
S B-species |
|
. I-species |
|
cerevisiae I-species |
|
, O |
|
phosphorylation B-ptm |
|
of O |
|
Ser457 B-residue_name_number |
|
within O |
|
the O |
|
C B-structure_element |
|
- I-structure_element |
|
terminal I-structure_element |
|
region I-structure_element |
|
( O |
|
CTR B-structure_element |
|
) O |
|
in O |
|
the O |
|
cytoplasm O |
|
was O |
|
recently O |
|
proposed O |
|
to O |
|
cause O |
|
Mep2 B-protein_type |
|
opening O |
|
, O |
|
possibly O |
|
via O |
|
inducing O |
|
a O |
|
conformational O |
|
change O |
|
. O |
|
|
|
The O |
|
most O |
|
striking O |
|
difference O |
|
is O |
|
the O |
|
fact O |
|
that O |
|
the O |
|
Mep2 B-protein_type |
|
proteins I-protein_type |
|
have O |
|
closed B-protein_state |
|
conformations O |
|
. O |
|
|
|
Electron B-evidence |
|
density I-evidence |
|
is O |
|
visible O |
|
for O |
|
the O |
|
entire O |
|
polypeptide O |
|
chains O |
|
, O |
|
with O |
|
the O |
|
exception O |
|
of O |
|
the O |
|
C O |
|
- O |
|
terminal O |
|
43 B-residue_range |
|
( O |
|
ScMep2 B-protein |
|
) O |
|
and O |
|
25 B-residue_range |
|
residues O |
|
( O |
|
CaMep2 B-protein |
|
), O |
|
which O |
|
are O |
|
poorly B-protein_state |
|
conserved I-protein_state |
|
and O |
|
presumably O |
|
disordered B-protein_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 |
|
|
|
The O |
|
largest O |
|
backbone O |
|
movements O |
|
of O |
|
equivalent O |
|
residues O |
|
within O |
|
ICL1 B-structure_element |
|
are O |
|
∼ O |
|
10 O |
|
Å O |
|
, O |
|
markedly O |
|
affecting O |
|
the O |
|
conserved B-protein_state |
|
basic B-protein_state |
|
RxK B-structure_element |
|
motif I-structure_element |
|
( O |
|
Fig O |
|
. O |
|
4 O |
|
). O |
|
|
|
The O |
|
head O |
|
group O |
|
of O |
|
Arg54 B-residue_name_number |
|
has O |
|
moved O |
|
∼ O |
|
11 O |
|
Å O |
|
relative O |
|
to O |
|
that O |
|
in O |
|
Amt B-protein |
|
- I-protein |
|
1 I-protein |
|
, O |
|
whereas O |
|
the O |
|
shift O |
|
of O |
|
the O |
|
head O |
|
group O |
|
of O |
|
the O |
|
variable O |
|
Lys55 B-residue_name_number |
|
residue O |
|
is O |
|
almost O |
|
20 O |
|
Å O |
|
. O |
|
The O |
|
side O |
|
chain O |
|
of O |
|
Lys56 B-residue_name_number |
|
in O |
|
the O |
|
basic B-protein_state |
|
motif B-structure_element |
|
points O |
|
in O |
|
an O |
|
opposite O |
|
direction O |
|
in O |
|
the O |
|
Mep2 B-protein |
|
structures B-evidence |
|
compared O |
|
with O |
|
that O |
|
of O |
|
, O |
|
for O |
|
example O |
|
, O |
|
Amt B-protein |
|
- I-protein |
|
1 I-protein |
|
( O |
|
Fig O |
|
. O |
|
4 O |
|
). O |
|
|
|
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 |
|
final O |
|
region O |
|
in O |
|
Mep2 B-protein |
|
that O |
|
shows O |
|
large O |
|
differences O |
|
compared O |
|
with O |
|
the O |
|
bacterial B-taxonomy_domain |
|
transporters B-protein_type |
|
is O |
|
the O |
|
CTR B-structure_element |
|
. O |
|
|
|
In O |
|
Mep2 B-protein |
|
, O |
|
the O |
|
CTR B-structure_element |
|
has O |
|
moved O |
|
away O |
|
and O |
|
makes O |
|
relatively O |
|
few O |
|
contacts O |
|
with O |
|
the O |
|
main B-structure_element |
|
body I-structure_element |
|
of O |
|
the O |
|
transporter B-protein_type |
|
, O |
|
generating O |
|
a O |
|
more O |
|
elongated B-protein_state |
|
protein O |
|
( O |
|
Figs O |
|
1 O |
|
and O |
|
4 O |
|
). O |
|
|
|
This O |
|
is O |
|
illustrated O |
|
by O |
|
the O |
|
positions O |
|
of O |
|
the O |
|
five O |
|
universally B-protein_state |
|
conserved I-protein_state |
|
residues O |
|
within O |
|
the O |
|
CTR B-structure_element |
|
, O |
|
that O |
|
is O |
|
, O |
|
Arg415 B-residue_name_number |
|
( O |
|
370 B-residue_number |
|
), O |
|
Glu421 B-residue_name_number |
|
( O |
|
376 B-residue_number |
|
), O |
|
Gly424 B-residue_name_number |
|
( O |
|
379 B-residue_number |
|
), O |
|
Asp426 B-residue_name_number |
|
( O |
|
381 B-residue_number |
|
) O |
|
and O |
|
Tyr B-residue_name_number |
|
435 I-residue_name_number |
|
( O |
|
390 B-residue_number |
|
) O |
|
in O |
|
CaMep2 B-protein |
|
( O |
|
Amt B-protein |
|
- I-protein |
|
1 I-protein |
|
) O |
|
( O |
|
Fig O |
|
. O |
|
2 O |
|
). O |
|
|
|
On O |
|
one O |
|
side O |
|
, O |
|
the O |
|
Tyr390 B-residue_name_number |
|
hydroxyl O |
|
in O |
|
Amt B-protein |
|
- I-protein |
|
1 I-protein |
|
is O |
|
hydrogen O |
|
bonded O |
|
with O |
|
the O |
|
side O |
|
chain O |
|
of O |
|
the O |
|
conserved B-protein_state |
|
His185 B-residue_name_number |
|
at O |
|
the O |
|
C O |
|
- O |
|
terminal O |
|
end O |
|
of O |
|
loop B-structure_element |
|
ICL3 B-structure_element |
|
. O |
|
|
|
In O |
|
the O |
|
Mep2 B-protein |
|
structures B-evidence |
|
, O |
|
none O |
|
of O |
|
the O |
|
interactions O |
|
mentioned O |
|
above O |
|
are O |
|
present O |
|
. O |
|
|
|
In O |
|
the O |
|
absence B-protein_state |
|
of I-protein_state |
|
Npr1 B-protein |
|
, O |
|
plasmid B-experimental_method |
|
- I-experimental_method |
|
encoded I-experimental_method |
|
WT B-protein_state |
|
Mep2 B-protein |
|
in O |
|
a O |
|
S B-species |
|
. I-species |
|
cerevisiae I-species |
|
mep1 B-mutant |
|
- I-mutant |
|
3Δ I-mutant |
|
strain O |
|
( O |
|
triple B-mutant |
|
mepΔ I-mutant |
|
) O |
|
does O |
|
not O |
|
allow O |
|
growth O |
|
on O |
|
low O |
|
concentrations O |
|
of O |
|
ammonium B-chemical |
|
, O |
|
suggesting O |
|
that O |
|
the O |
|
transporter B-protein_type |
|
is O |
|
inactive B-protein_state |
|
( O |
|
Fig O |
|
. O |
|
3 O |
|
and O |
|
Supplementary O |
|
Fig O |
|
. O |
|
1 O |
|
). O |
|
|
|
Conversely O |
|
, O |
|
the O |
|
phosphorylation B-protein_state |
|
- I-protein_state |
|
mimicking I-protein_state |
|
S457D B-mutant |
|
variant O |
|
is O |
|
active B-protein_state |
|
both O |
|
in O |
|
the O |
|
triple B-mutant |
|
mepΔ I-mutant |
|
background O |
|
and O |
|
in O |
|
a O |
|
triple B-mutant |
|
mepΔ I-mutant |
|
npr1Δ I-mutant |
|
strain O |
|
( O |
|
Fig O |
|
. O |
|
3 O |
|
). O |
|
|
|
Mutation B-experimental_method |
|
of O |
|
other O |
|
potential O |
|
phosphorylation B-site |
|
sites I-site |
|
in O |
|
the O |
|
CTR B-structure_element |
|
did O |
|
not O |
|
support O |
|
growth O |
|
in O |
|
the O |
|
npr1Δ B-mutant |
|
background O |
|
. O |
|
|
|
In O |
|
CaMep2 B-protein |
|
, O |
|
the O |
|
visible O |
|
part O |
|
of O |
|
the O |
|
sequence O |
|
extends O |
|
for O |
|
two O |
|
residues O |
|
beyond O |
|
Ser453 B-residue_name_number |
|
( O |
|
Fig O |
|
. O |
|
6 O |
|
). O |
|
|
|
Boeckstaens O |
|
et O |
|
al O |
|
. O |
|
proposed O |
|
that O |
|
phosphorylation B-ptm |
|
does O |
|
not O |
|
affect O |
|
channel O |
|
activity O |
|
directly O |
|
, O |
|
but O |
|
instead O |
|
relieves O |
|
inhibition O |
|
by O |
|
the O |
|
AI B-structure_element |
|
region I-structure_element |
|
. O |
|
|
|
The O |
|
first O |
|
one O |
|
is O |
|
that O |
|
the O |
|
open B-protein_state |
|
state O |
|
is O |
|
disfavoured O |
|
by O |
|
crystallization B-experimental_method |
|
because O |
|
of O |
|
lower O |
|
stability O |
|
or O |
|
due O |
|
to O |
|
crystal O |
|
packing O |
|
constraints O |
|
. 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 |
|
|
|
In O |
|
the O |
|
WT B-protein_state |
|
structure B-evidence |
|
, O |
|
the O |
|
acidic O |
|
residues O |
|
Asp419 B-residue_name_number |
|
, O |
|
Glu420 B-residue_name_number |
|
and O |
|
Glu421 B-residue_name_number |
|
are O |
|
within O |
|
hydrogen O |
|
bonding O |
|
distance O |
|
of O |
|
Ser453 B-residue_name_number |
|
. O |
|
|
|
Finally O |
|
, O |
|
the O |
|
S453J B-mutant |
|
mutant B-protein_state |
|
is O |
|
also O |
|
stable B-protein_state |
|
throughout O |
|
the O |
|
200 O |
|
- O |
|
ns O |
|
simulation B-experimental_method |
|
and O |
|
has O |
|
an O |
|
average O |
|
backbone O |
|
deviation O |
|
of O |
|
∼ O |
|
3 O |
|
. O |
|
8 O |
|
Å O |
|
, O |
|
which O |
|
is O |
|
similar O |
|
to O |
|
the O |
|
DD B-mutant |
|
mutant I-mutant |
|
. O |
|
|
|
The O |
|
distance B-evidence |
|
between O |
|
the O |
|
phosphate B-chemical |
|
of O |
|
Sep453 B-residue_name_number |
|
and O |
|
the O |
|
acidic O |
|
oxygen O |
|
atoms O |
|
of O |
|
Glu420 B-residue_name_number |
|
is O |
|
initially O |
|
∼ O |
|
11 O |
|
Å O |
|
, O |
|
but O |
|
increases O |
|
to O |
|
> O |
|
30 O |
|
Å O |
|
after O |
|
200 O |
|
ns O |
|
. O |
|
|
|
These O |
|
efforts O |
|
have O |
|
advanced O |
|
our O |
|
knowledge O |
|
considerably O |
|
but O |
|
have O |
|
not O |
|
yet O |
|
yielded O |
|
atomic O |
|
- O |
|
level O |
|
answers O |
|
to O |
|
several O |
|
important O |
|
mechanistic O |
|
questions O |
|
, O |
|
including O |
|
how O |
|
ammonium B-chemical |
|
transport O |
|
is O |
|
regulated O |
|
in O |
|
eukaryotes B-taxonomy_domain |
|
and O |
|
the O |
|
mechanism O |
|
of O |
|
ammonium B-chemical |
|
signalling O |
|
. O |
|
|
|
In O |
|
Arabidopsis B-species |
|
thaliana I-species |
|
Amt B-protein |
|
- I-protein |
|
1 I-protein |
|
; I-protein |
|
1 I-protein |
|
, O |
|
phosphorylation B-ptm |
|
of O |
|
the O |
|
CTR B-structure_element |
|
residue O |
|
T460 B-residue_name_number |
|
under O |
|
conditions O |
|
of O |
|
high O |
|
ammonium B-chemical |
|
inhibits O |
|
transport O |
|
activity O |
|
, O |
|
that O |
|
is O |
|
, O |
|
the O |
|
default O |
|
( O |
|
non B-protein_state |
|
- I-protein_state |
|
phosphorylated I-protein_state |
|
) O |
|
state O |
|
of O |
|
the O |
|
plant B-taxonomy_domain |
|
transporter B-protein_type |
|
is O |
|
open B-protein_state |
|
. O |
|
|
|
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 |
|
|
|
More O |
|
specifically O |
|
, O |
|
the O |
|
close O |
|
interactions O |
|
between O |
|
the O |
|
CTR B-structure_element |
|
and O |
|
ICL1 B-structure_element |
|
/ O |
|
ICL3 B-structure_element |
|
present O |
|
in O |
|
open B-protein_state |
|
transporters B-protein_type |
|
are O |
|
disrupted O |
|
, O |
|
causing O |
|
ICL3 B-structure_element |
|
to O |
|
move O |
|
outwards O |
|
and O |
|
block O |
|
the O |
|
channel B-site |
|
( O |
|
Figs O |
|
4 O |
|
and O |
|
9a O |
|
). O |
|
|
|
How O |
|
exactly O |
|
the O |
|
channel B-site |
|
opens O |
|
and O |
|
whether O |
|
opening O |
|
is O |
|
intra O |
|
- O |
|
monomeric O |
|
are O |
|
still O |
|
open B-protein_state |
|
questions O |
|
; O |
|
it O |
|
is O |
|
possible O |
|
that O |
|
the O |
|
change O |
|
in O |
|
the O |
|
CTR B-structure_element |
|
may O |
|
disrupt O |
|
its O |
|
interactions O |
|
with O |
|
ICL3 B-structure_element |
|
of O |
|
the O |
|
neighbouring O |
|
monomer B-oligomeric_state |
|
( O |
|
Fig O |
|
. O |
|
9b O |
|
), O |
|
which O |
|
could O |
|
result O |
|
in O |
|
opening O |
|
of O |
|
the O |
|
neighbouring O |
|
channel B-site |
|
via O |
|
inward O |
|
movement O |
|
of O |
|
its O |
|
ICL3 B-structure_element |
|
. O |
|
|
|
Is O |
|
our O |
|
model O |
|
for O |
|
opening O |
|
and O |
|
closing O |
|
of O |
|
Mep2 B-protein |
|
channels B-site |
|
valid O |
|
for O |
|
other O |
|
eukaryotic B-taxonomy_domain |
|
ammonium B-protein_type |
|
transporters I-protein_type |
|
? O |
|
Our O |
|
structural B-evidence |
|
data I-evidence |
|
support O |
|
previous O |
|
studies O |
|
and O |
|
clarify O |
|
the O |
|
central O |
|
role O |
|
of O |
|
the O |
|
CTR B-structure_element |
|
and O |
|
cytoplasmic B-structure_element |
|
loops I-structure_element |
|
in O |
|
the O |
|
transition O |
|
between O |
|
closed B-protein_state |
|
and O |
|
open B-protein_state |
|
states O |
|
. O |
|
|
|
There O |
|
is O |
|
generally O |
|
no O |
|
equivalent O |
|
for O |
|
CaMep2 B-protein |
|
Tyr49 B-residue_name_number |
|
in O |
|
plant B-taxonomy_domain |
|
AMTs B-protein_type |
|
, O |
|
indicating O |
|
that O |
|
a O |
|
Tyr B-site |
|
– I-site |
|
His2 I-site |
|
hydrogen I-site |
|
bond I-site |
|
as O |
|
observed O |
|
in O |
|
Mep2 B-protein |
|
may O |
|
not O |
|
contribute O |
|
to O |
|
the O |
|
closed B-protein_state |
|
state O |
|
in O |
|
plant B-taxonomy_domain |
|
transporters B-protein_type |
|
. O |
|
|
|
The O |
|
need O |
|
to O |
|
regulate O |
|
in O |
|
opposite O |
|
ways O |
|
may O |
|
be O |
|
the O |
|
reason O |
|
why O |
|
the O |
|
phosphorylation B-site |
|
sites I-site |
|
are O |
|
in O |
|
different O |
|
parts O |
|
of O |
|
the O |
|
CTR B-structure_element |
|
, O |
|
that O |
|
is O |
|
, O |
|
centrally O |
|
located O |
|
close O |
|
to O |
|
the O |
|
ExxGxD B-structure_element |
|
motif I-structure_element |
|
in O |
|
AMTs B-protein_type |
|
and O |
|
peripherally O |
|
in O |
|
Mep2 B-protein |
|
. O |
|
|
|
With O |
|
respect O |
|
to O |
|
ammonium B-chemical |
|
transport O |
|
, O |
|
phosphorylation B-ptm |
|
has O |
|
thus O |
|
far O |
|
only O |
|
been O |
|
shown O |
|
for O |
|
A B-species |
|
. I-species |
|
thaliana I-species |
|
AMTs B-protein_type |
|
and O |
|
for O |
|
S B-species |
|
. I-species |
|
cerevisiae I-species |
|
Mep2 B-protein |
|
( O |
|
refs O |
|
). O |
|
|
|
In O |
|
one O |
|
model O |
|
, O |
|
signalling O |
|
is O |
|
proposed O |
|
to O |
|
depend O |
|
on O |
|
the O |
|
nature O |
|
of O |
|
the O |
|
transported O |
|
substrate O |
|
, O |
|
which O |
|
might O |
|
be O |
|
different O |
|
in O |
|
certain O |
|
subfamilies O |
|
of O |
|
ammonium B-protein_type |
|
transporters I-protein_type |
|
( O |
|
for O |
|
example O |
|
, O |
|
Mep1 B-protein |
|
/ O |
|
Mep3 B-protein |
|
versus O |
|
Mep2 B-protein |
|
). O |
|
|
|
In O |
|
the O |
|
other O |
|
model O |
|
, O |
|
signalling O |
|
is O |
|
thought O |
|
to O |
|
require O |
|
a O |
|
distinct O |
|
conformation O |
|
of O |
|
the O |
|
Mep2 B-protein |
|
transporter B-protein_type |
|
occurring O |
|
during O |
|
the O |
|
transport O |
|
cycle O |
|
. O |
|
|
|
The O |
|
region O |
|
showing O |
|
ICL1 B-structure_element |
|
( O |
|
blue O |
|
), O |
|
ICL3 B-structure_element |
|
( O |
|
green O |
|
) O |
|
and O |
|
the O |
|
CTR B-structure_element |
|
( O |
|
red O |
|
) O |
|
is O |
|
boxed O |
|
for O |
|
comparison O |
|
. O |
|
|
|
( O |
|
a O |
|
) O |
|
ICL1 B-structure_element |
|
in O |
|
AfAmt B-protein |
|
- I-protein |
|
1 I-protein |
|
( O |
|
light O |
|
blue O |
|
) O |
|
and O |
|
CaMep2 B-protein |
|
( O |
|
dark O |
|
blue O |
|
), O |
|
showing O |
|
unwinding O |
|
and O |
|
inward O |
|
movement O |
|
in O |
|
the O |
|
fungal B-taxonomy_domain |
|
protein O |
|
. O |
|
( O |
|
b O |
|
) O |
|
Stereo O |
|
diagram O |
|
viewed O |
|
from O |
|
the O |
|
cytosol O |
|
of O |
|
ICL1 B-structure_element |
|
, O |
|
ICL3 B-structure_element |
|
( O |
|
green O |
|
) O |
|
and O |
|
the O |
|
CTR B-structure_element |
|
( O |
|
red O |
|
) O |
|
in O |
|
AfAmt B-protein |
|
- I-protein |
|
1 I-protein |
|
( O |
|
light O |
|
colours O |
|
) O |
|
and O |
|
CaMep2 B-protein |
|
( O |
|
dark O |
|
colours O |
|
). O |
|
|
|
The O |
|
labelled O |
|
residues O |
|
are O |
|
analogous O |
|
within O |
|
both O |
|
structures B-evidence |
|
. O |
|
|
|
Channel O |
|
closures O |
|
in O |
|
Mep2 B-protein |
|
. O |
|
|
|
The O |
|
Npr1 B-protein |
|
kinase B-protein_type |
|
target O |
|
Ser453 B-residue_name_number |
|
is O |
|
dephosphorylated B-protein_state |
|
and O |
|
located O |
|
in O |
|
an O |
|
electronegative B-site |
|
pocket I-site |
|
. O |
|
|
|
( O |
|
a O |
|
) O |
|
Stereoviews O |
|
of O |
|
CaMep2 B-protein |
|
showing O |
|
2Fo O |
|
– O |
|
Fc O |
|
electron O |
|
density O |
|
( O |
|
contoured O |
|
at O |
|
1 O |
|
. O |
|
0 O |
|
σ O |
|
) O |
|
for O |
|
CTR B-structure_element |
|
residues O |
|
Asp419 B-residue_range |
|
- I-residue_range |
|
Met422 I-residue_range |
|
and O |
|
for O |
|
Tyr446 B-residue_range |
|
- I-residue_range |
|
Thr455 I-residue_range |
|
of O |
|
the O |
|
AI B-structure_element |
|
region I-structure_element |
|
. O |
|
|
|
Phosphorylation B-ptm |
|
causes O |
|
conformational O |
|
changes O |
|
in O |
|
the O |
|
CTR B-structure_element |
|
. O |
|
|
|
( 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 |
|
|
|
Visualizing O |
|
chaperone B-protein_type |
|
- O |
|
assisted O |
|
protein O |
|
folding O |
|
|
|
READ B-experimental_method |
|
enabled O |
|
us O |
|
to O |
|
visualize O |
|
even O |
|
sparsely O |
|
populated O |
|
conformations O |
|
of O |
|
the O |
|
substrate O |
|
protein O |
|
immunity B-protein |
|
protein I-protein |
|
7 I-protein |
|
( O |
|
Im7 B-protein |
|
) O |
|
in B-protein_state |
|
complex I-protein_state |
|
with I-protein_state |
|
the O |
|
E B-species |
|
. I-species |
|
coli I-species |
|
chaperone B-protein_type |
|
Spy B-protein |
|
. O |
|
|
|
It O |
|
is O |
|
clear O |
|
that O |
|
molecular O |
|
chaperones B-protein_type |
|
aid O |
|
in O |
|
protein O |
|
folding O |
|
. O |
|
|
|
Structural B-evidence |
|
models I-evidence |
|
of O |
|
chaperone B-protein_type |
|
- O |
|
substrate O |
|
complexes O |
|
have O |
|
recently O |
|
begun O |
|
to O |
|
provide O |
|
information O |
|
as O |
|
to O |
|
how O |
|
a O |
|
chaperone B-protein_type |
|
can O |
|
recognize O |
|
its O |
|
substrate O |
|
. O |
|
|
|
For O |
|
most O |
|
chaperones B-protein_type |
|
, O |
|
it O |
|
is O |
|
still O |
|
unclear O |
|
whether O |
|
the O |
|
chaperone B-protein_type |
|
actively O |
|
participates O |
|
in O |
|
and O |
|
affects O |
|
the O |
|
folding O |
|
of O |
|
the O |
|
substrate O |
|
proteins O |
|
, O |
|
or O |
|
merely O |
|
provides O |
|
a O |
|
suitable O |
|
microenvironment O |
|
enabling O |
|
the O |
|
substrate O |
|
to O |
|
fold O |
|
on O |
|
its O |
|
own O |
|
. O |
|
|
|
We O |
|
therefore O |
|
screened B-experimental_method |
|
crystallization B-experimental_method |
|
conditions I-experimental_method |
|
for O |
|
Spy B-protein |
|
with O |
|
four O |
|
different O |
|
substrate O |
|
proteins O |
|
: O |
|
a O |
|
fragment O |
|
of O |
|
the O |
|
largely O |
|
unfolded B-protein_state |
|
bovine B-taxonomy_domain |
|
α B-chemical |
|
- I-chemical |
|
casein I-chemical |
|
protein O |
|
, O |
|
wild B-protein_state |
|
- I-protein_state |
|
type I-protein_state |
|
( O |
|
WT B-protein_state |
|
) O |
|
E B-species |
|
. I-species |
|
coli I-species |
|
Im7 B-protein |
|
, O |
|
an O |
|
unfolded B-protein_state |
|
variant O |
|
of O |
|
Im7 B-protein |
|
( O |
|
L18A B-mutant |
|
L19A B-mutant |
|
L37A B-mutant |
|
), O |
|
and O |
|
the O |
|
N B-structure_element |
|
- I-structure_element |
|
terminal I-structure_element |
|
half I-structure_element |
|
of O |
|
Im7 B-protein |
|
( O |
|
Im76 B-mutant |
|
- I-mutant |
|
45 I-mutant |
|
), O |
|
which O |
|
encompasses O |
|
the O |
|
entire O |
|
Spy B-structure_element |
|
- I-structure_element |
|
binding I-structure_element |
|
portion I-structure_element |
|
of O |
|
Im7 B-protein |
|
. O |
|
|
|
Subsequent O |
|
crystal B-experimental_method |
|
washing I-experimental_method |
|
and I-experimental_method |
|
dissolution I-experimental_method |
|
experiments O |
|
confirmed O |
|
the O |
|
presence O |
|
of O |
|
the O |
|
substrates O |
|
in O |
|
the O |
|
co B-experimental_method |
|
- I-experimental_method |
|
crystals I-experimental_method |
|
( O |
|
Supplementary O |
|
Fig O |
|
. O |
|
2 O |
|
). O |
|
|
|
We O |
|
split O |
|
this O |
|
approach O |
|
into O |
|
five O |
|
steps O |
|
: O |
|
( O |
|
1 O |
|
) O |
|
By O |
|
using O |
|
a O |
|
well O |
|
- O |
|
diffracting O |
|
Spy B-protein |
|
: O |
|
substrate O |
|
co B-evidence |
|
- I-evidence |
|
crystal I-evidence |
|
, O |
|
we O |
|
first O |
|
determined O |
|
the O |
|
structure B-evidence |
|
of O |
|
the O |
|
folded B-protein_state |
|
domain B-structure_element |
|
of O |
|
Spy B-protein |
|
and O |
|
obtained O |
|
high O |
|
quality O |
|
residual B-evidence |
|
electron I-evidence |
|
density I-evidence |
|
within O |
|
the O |
|
dynamic B-protein_state |
|
regions O |
|
of O |
|
the O |
|
substrate O |
|
. O |
|
|
|
The O |
|
READ B-experimental_method |
|
sample B-experimental_method |
|
- I-experimental_method |
|
and I-experimental_method |
|
- I-experimental_method |
|
select I-experimental_method |
|
algorithm I-experimental_method |
|
is O |
|
diagrammed O |
|
in O |
|
Fig O |
|
. O |
|
2 O |
|
. O |
|
|
|
To O |
|
make O |
|
the O |
|
electron B-experimental_method |
|
density I-experimental_method |
|
selection I-experimental_method |
|
practical O |
|
, O |
|
we O |
|
needed O |
|
to O |
|
develop O |
|
a O |
|
method O |
|
to O |
|
rapidly O |
|
evaluate O |
|
the O |
|
agreement O |
|
between O |
|
the O |
|
selected O |
|
sub O |
|
- O |
|
ensembles O |
|
and O |
|
the O |
|
experimental O |
|
electron B-evidence |
|
density I-evidence |
|
on O |
|
- O |
|
the O |
|
- O |
|
fly O |
|
during O |
|
the O |
|
selection O |
|
procedure O |
|
. O |
|
|
|
To O |
|
reduce O |
|
the O |
|
extent O |
|
of O |
|
3D O |
|
space O |
|
to O |
|
be O |
|
explored O |
|
, O |
|
this O |
|
compressed O |
|
map B-evidence |
|
was O |
|
created O |
|
by O |
|
only O |
|
using O |
|
density B-evidence |
|
from O |
|
regions O |
|
of O |
|
space O |
|
significantly O |
|
sampled O |
|
by O |
|
Im76 B-mutant |
|
- I-mutant |
|
45 I-mutant |
|
in O |
|
the O |
|
Spy B-complex_assembly |
|
: I-complex_assembly |
|
Im76 I-complex_assembly |
|
- I-complex_assembly |
|
45 I-complex_assembly |
|
MD B-experimental_method |
|
simulations B-experimental_method |
|
. O |
|
|
|
We O |
|
were O |
|
particularly O |
|
interested O |
|
in O |
|
finding O |
|
answers O |
|
to O |
|
one O |
|
of O |
|
the O |
|
most O |
|
fundamental O |
|
questions O |
|
in O |
|
chaperone B-protein_type |
|
biology O |
|
— O |
|
how O |
|
does O |
|
chaperone B-protein_type |
|
binding O |
|
affect O |
|
substrate O |
|
structure O |
|
and O |
|
vice O |
|
versa O |
|
. O |
|
|
|
We O |
|
constructed O |
|
a O |
|
contact B-evidence |
|
map I-evidence |
|
of O |
|
the O |
|
complex O |
|
, O |
|
which O |
|
shows O |
|
the O |
|
frequency O |
|
of O |
|
interactions O |
|
for O |
|
chaperone B-protein_type |
|
- O |
|
substrate O |
|
residue O |
|
pairs O |
|
( O |
|
Fig O |
|
. O |
|
4 O |
|
). O |
|
|
|
This O |
|
twist O |
|
yields O |
|
asymmetry O |
|
and O |
|
results O |
|
in O |
|
substantially O |
|
different O |
|
interaction O |
|
patterns O |
|
in O |
|
the O |
|
two O |
|
Spy B-protein |
|
monomers B-oligomeric_state |
|
( O |
|
Fig O |
|
. O |
|
4b O |
|
). O |
|
|
|
Additionally O |
|
, O |
|
we O |
|
observed O |
|
that O |
|
the O |
|
linker B-structure_element |
|
region I-structure_element |
|
( O |
|
residues O |
|
47 B-residue_range |
|
– I-residue_range |
|
57 I-residue_range |
|
) O |
|
of O |
|
Spy B-protein |
|
, O |
|
which O |
|
participates O |
|
in O |
|
substrate O |
|
interaction O |
|
, O |
|
becomes O |
|
mostly O |
|
disordered B-protein_state |
|
upon O |
|
binding O |
|
the O |
|
substrate O |
|
. O |
|
|
|
Importantly O |
|
, O |
|
we O |
|
observed O |
|
the O |
|
same O |
|
structural O |
|
changes O |
|
in O |
|
Spy B-protein |
|
regardless O |
|
of O |
|
which O |
|
of O |
|
the O |
|
four O |
|
substrates O |
|
was O |
|
bound O |
|
( O |
|
Fig O |
|
. O |
|
5b O |
|
, O |
|
Table O |
|
1 O |
|
). O |
|
|
|
We O |
|
recently O |
|
showed O |
|
that O |
|
Im7 B-protein |
|
can O |
|
fold O |
|
while O |
|
remaining O |
|
continuously B-protein_state |
|
bound I-protein_state |
|
to I-protein_state |
|
Spy B-protein |
|
. O |
|
|
|
This O |
|
model O |
|
is O |
|
consistent O |
|
with O |
|
previous O |
|
studies O |
|
postulating O |
|
that O |
|
the O |
|
flexible O |
|
binding O |
|
of O |
|
chaperones B-protein_type |
|
allows O |
|
for O |
|
substrate O |
|
protein O |
|
folding O |
|
. O |
|
|
|
The O |
|
amphipathic O |
|
concave B-site |
|
surface I-site |
|
of O |
|
Spy B-protein |
|
likely O |
|
facilitates O |
|
this O |
|
flexible O |
|
binding O |
|
and O |
|
may O |
|
be O |
|
a O |
|
crucial O |
|
feature O |
|
for O |
|
Spy B-protein |
|
and O |
|
potentially O |
|
other O |
|
chaperones B-protein_type |
|
, O |
|
allowing O |
|
them O |
|
to O |
|
bind O |
|
multiple O |
|
conformations O |
|
of O |
|
many O |
|
different O |
|
substrates O |
|
. O |
|
|
|
The O |
|
negatively O |
|
charged O |
|
Im7 B-protein |
|
residues O |
|
Glu21 B-residue_name_number |
|
, O |
|
Asp32 B-residue_name_number |
|
, O |
|
and O |
|
Asp35 B-residue_name_number |
|
reside O |
|
on O |
|
the O |
|
surface O |
|
of O |
|
Im7 B-protein |
|
and O |
|
form O |
|
interactions O |
|
with O |
|
Spy B-protein |
|
’ O |
|
s O |
|
positively O |
|
charged O |
|
cradle B-site |
|
in O |
|
both O |
|
the O |
|
unfolded B-protein_state |
|
and O |
|
native B-protein_state |
|
- I-protein_state |
|
like I-protein_state |
|
states O |
|
. O |
|
|
|
This O |
|
selection O |
|
resulted O |
|
in O |
|
“ O |
|
Super O |
|
Spy B-protein |
|
” O |
|
variants B-protein_state |
|
that O |
|
were O |
|
more O |
|
effective O |
|
at O |
|
both O |
|
preventing O |
|
aggregation O |
|
and O |
|
promoting O |
|
protein O |
|
folding O |
|
. O |
|
|
|
By O |
|
sampling O |
|
multiple O |
|
conformations O |
|
, O |
|
this O |
|
linker B-structure_element |
|
region I-structure_element |
|
may O |
|
allow O |
|
diverse O |
|
substrate O |
|
conformations O |
|
to O |
|
be O |
|
accommodated O |
|
. O |
|
|
|
Overall O |
|
, O |
|
comparison O |
|
of O |
|
our O |
|
ensemble B-evidence |
|
to O |
|
the O |
|
Super O |
|
Spy B-protein |
|
variants B-protein_state |
|
provides O |
|
specific O |
|
examples O |
|
to O |
|
corroborate O |
|
the O |
|
importance O |
|
of O |
|
conformational O |
|
flexibility O |
|
in O |
|
chaperone B-protein_type |
|
- O |
|
substrate O |
|
interactions O |
|
. O |
|
|
|
ATP B-chemical |
|
and O |
|
co O |
|
- O |
|
chaperone B-protein_type |
|
dependencies O |
|
may O |
|
have O |
|
emerged O |
|
later O |
|
through O |
|
evolution O |
|
to O |
|
better O |
|
modulate O |
|
and O |
|
control O |
|
chaperone B-protein_type |
|
action O |
|
. O |
|
|
|
As O |
|
the O |
|
residues O |
|
involved O |
|
in O |
|
contacts O |
|
are O |
|
more O |
|
evenly O |
|
distributed O |
|
in O |
|
Im76 B-mutant |
|
- I-mutant |
|
45 I-mutant |
|
compared O |
|
to O |
|
Spy B-protein |
|
, O |
|
its O |
|
contact B-evidence |
|
map I-evidence |
|
was O |
|
amplified O |
|
. O |
|
( O |
|
b O |
|
) O |
|
Detailed O |
|
contact B-evidence |
|
maps I-evidence |
|
of O |
|
Spy B-complex_assembly |
|
: I-complex_assembly |
|
Im76 I-complex_assembly |
|
- I-complex_assembly |
|
45 I-complex_assembly |
|
. O |
|
|
|
The O |
|
Super O |
|
Spy B-protein |
|
mutants O |
|
F115L B-mutant |
|
, O |
|
F115I B-mutant |
|
, O |
|
and O |
|
L32P B-mutant |
|
are O |
|
proposed O |
|
to O |
|
gain O |
|
activity O |
|
by O |
|
increasing O |
|
the O |
|
flexibility O |
|
or O |
|
size O |
|
of O |
|
this O |
|
linker B-structure_element |
|
region I-structure_element |
|
. O |
|
|
|
To O |
|
support O |
|
antibody B-protein_type |
|
therapeutic O |
|
development O |
|
, O |
|
the O |
|
crystal B-evidence |
|
structures I-evidence |
|
of O |
|
a O |
|
set O |
|
of O |
|
16 O |
|
germline O |
|
variants O |
|
composed O |
|
of O |
|
4 O |
|
different O |
|
kappa B-structure_element |
|
light I-structure_element |
|
chains I-structure_element |
|
paired O |
|
with O |
|
4 O |
|
different O |
|
heavy B-structure_element |
|
chains I-structure_element |
|
have O |
|
been O |
|
determined O |
|
. O |
|
|
|
The O |
|
longer B-protein_state |
|
CDRs B-structure_element |
|
with O |
|
tandem O |
|
glycines B-residue_name |
|
or O |
|
serines B-residue_name |
|
have O |
|
more O |
|
conformational O |
|
diversity O |
|
than O |
|
the O |
|
others O |
|
. O |
|
|
|
Two O |
|
of O |
|
16 O |
|
structures B-evidence |
|
showed O |
|
particularly O |
|
large O |
|
variations O |
|
in O |
|
the O |
|
tilt B-evidence |
|
angles I-evidence |
|
when O |
|
compared O |
|
with O |
|
the O |
|
other O |
|
pairings O |
|
. O |
|
|
|
These O |
|
domains O |
|
have O |
|
a O |
|
common O |
|
folding O |
|
pattern O |
|
often O |
|
referred O |
|
to O |
|
as O |
|
the O |
|
“ O |
|
immunoglobulin B-structure_element |
|
fold I-structure_element |
|
,” O |
|
formed O |
|
by O |
|
the O |
|
packing O |
|
together O |
|
of O |
|
2 O |
|
anti B-structure_element |
|
- I-structure_element |
|
parallel I-structure_element |
|
β I-structure_element |
|
- I-structure_element |
|
sheets I-structure_element |
|
. O |
|
|
|
All O |
|
immunoglobulin B-protein_type |
|
chains I-protein_type |
|
have O |
|
an O |
|
N O |
|
- O |
|
terminal O |
|
V B-structure_element |
|
domain I-structure_element |
|
followed O |
|
by O |
|
1 O |
|
to O |
|
4 O |
|
C B-structure_element |
|
domains I-structure_element |
|
, O |
|
depending O |
|
upon O |
|
the O |
|
chain O |
|
type O |
|
. O |
|
|
|
The O |
|
cataloging O |
|
and O |
|
development O |
|
of O |
|
the O |
|
rules O |
|
for O |
|
predicting O |
|
the O |
|
conformation O |
|
of O |
|
the O |
|
anchor B-structure_element |
|
region I-structure_element |
|
of O |
|
CDR B-structure_element |
|
H3 B-structure_element |
|
continue O |
|
to O |
|
be O |
|
refined O |
|
, O |
|
producing O |
|
new O |
|
insight O |
|
into O |
|
the O |
|
CDR B-structure_element |
|
H3 B-structure_element |
|
conformations O |
|
and O |
|
new O |
|
tools O |
|
for O |
|
antibody B-protein_type |
|
engineering O |
|
. O |
|
|
|
One O |
|
important O |
|
finding O |
|
of O |
|
the O |
|
antibody B-experimental_method |
|
modeling I-experimental_method |
|
assessments I-experimental_method |
|
was O |
|
that O |
|
errors O |
|
in O |
|
the O |
|
structural O |
|
templates O |
|
that O |
|
are O |
|
used O |
|
as O |
|
the O |
|
basis O |
|
for O |
|
homology B-experimental_method |
|
models I-experimental_method |
|
can O |
|
propagate O |
|
into O |
|
the O |
|
final O |
|
models O |
|
, O |
|
producing O |
|
inaccuracies O |
|
that O |
|
may O |
|
negatively O |
|
influence O |
|
the O |
|
predictive O |
|
nature O |
|
of O |
|
the O |
|
V B-structure_element |
|
region I-structure_element |
|
model O |
|
. O |
|
|
|
The O |
|
structures B-evidence |
|
and O |
|
their O |
|
analyses O |
|
provide O |
|
a O |
|
foundation O |
|
for O |
|
future O |
|
antibody B-protein_type |
|
engineering O |
|
and O |
|
structure O |
|
determination O |
|
efforts O |
|
. O |
|
|
|
The O |
|
similarity O |
|
in O |
|
the O |
|
crystal B-evidence |
|
forms I-evidence |
|
is O |
|
attributed O |
|
in O |
|
part O |
|
to O |
|
cross O |
|
- O |
|
seeding O |
|
using O |
|
the O |
|
microseed B-experimental_method |
|
matrix I-experimental_method |
|
screening I-experimental_method |
|
for O |
|
groups O |
|
2 O |
|
and O |
|
3 O |
|
. O |
|
|
|
The O |
|
number O |
|
of O |
|
Fab B-structure_element |
|
molecules O |
|
in O |
|
the O |
|
crystallographic O |
|
asymmetric O |
|
unit O |
|
varies O |
|
from O |
|
1 O |
|
( O |
|
for O |
|
12 O |
|
Fabs B-structure_element |
|
) O |
|
to O |
|
2 O |
|
( O |
|
for O |
|
4 O |
|
Fabs B-structure_element |
|
). O |
|
|
|
For O |
|
the O |
|
LC B-structure_element |
|
, O |
|
the O |
|
disorder B-protein_state |
|
is O |
|
observed O |
|
at O |
|
2 O |
|
of O |
|
the O |
|
C O |
|
- O |
|
terminal O |
|
residues O |
|
with O |
|
few O |
|
exceptions O |
|
. O |
|
|
|
CDR B-structure_element |
|
H1 B-structure_element |
|
|
|
The O |
|
canonical O |
|
structures O |
|
of O |
|
CDR B-structure_element |
|
H2 B-structure_element |
|
have O |
|
fairly O |
|
consistent O |
|
conformations O |
|
( O |
|
Table O |
|
2 O |
|
, O |
|
Fig O |
|
. O |
|
2 O |
|
). O |
|
|
|
In O |
|
one O |
|
case O |
|
, O |
|
in O |
|
the O |
|
second O |
|
Fab B-structure_element |
|
of O |
|
H1 B-complex_assembly |
|
- I-complex_assembly |
|
69 I-complex_assembly |
|
: I-complex_assembly |
|
L3 I-complex_assembly |
|
- I-complex_assembly |
|
20 I-complex_assembly |
|
, O |
|
CDR B-structure_element |
|
H2 B-structure_element |
|
is O |
|
partially B-protein_state |
|
disordered I-protein_state |
|
( O |
|
Δ55 B-mutant |
|
- I-mutant |
|
60 I-mutant |
|
). O |
|
|
|
Despite O |
|
this O |
|
, O |
|
the O |
|
conformations O |
|
are O |
|
tightly O |
|
clustered O |
|
( O |
|
rmsd B-evidence |
|
is O |
|
0 O |
|
. O |
|
20 O |
|
Å O |
|
). O |
|
|
|
L3 B-mutant |
|
- I-mutant |
|
20 I-mutant |
|
is O |
|
the O |
|
most O |
|
variable O |
|
in O |
|
CDR B-structure_element |
|
L1 B-structure_element |
|
among O |
|
the O |
|
4 O |
|
germlines O |
|
as O |
|
indicated O |
|
by O |
|
an O |
|
rmsd B-evidence |
|
of O |
|
0 O |
|
. O |
|
54 O |
|
Å O |
|
( O |
|
Fig O |
|
. O |
|
3C O |
|
). O |
|
|
|
The O |
|
CDR B-structure_element |
|
L2 B-structure_element |
|
conformations O |
|
for O |
|
each O |
|
of O |
|
the O |
|
LCs B-structure_element |
|
paired O |
|
with O |
|
the O |
|
4 O |
|
HCs B-structure_element |
|
are O |
|
clustered O |
|
more O |
|
tightly O |
|
than O |
|
any O |
|
of O |
|
the O |
|
other O |
|
CDRs B-structure_element |
|
( O |
|
rmsd B-evidence |
|
values O |
|
are O |
|
in O |
|
the O |
|
range O |
|
0 O |
|
. O |
|
09 O |
|
- O |
|
0 O |
|
. O |
|
16 O |
|
Å O |
|
), O |
|
and O |
|
all O |
|
4 O |
|
sets O |
|
have O |
|
virtually O |
|
the O |
|
same O |
|
conformation O |
|
despite O |
|
the O |
|
sequence O |
|
diversity O |
|
of O |
|
the O |
|
loop B-structure_element |
|
. O |
|
|
|
The O |
|
superposition B-experimental_method |
|
of O |
|
CDR B-structure_element |
|
L3 B-structure_element |
|
backbones O |
|
for O |
|
all O |
|
HC B-complex_assembly |
|
: I-complex_assembly |
|
LC I-complex_assembly |
|
pairs O |
|
with O |
|
light B-structure_element |
|
chains I-structure_element |
|
: O |
|
( O |
|
A O |
|
) O |
|
L1 B-mutant |
|
- I-mutant |
|
39 I-mutant |
|
, O |
|
( O |
|
B O |
|
) O |
|
L3 B-mutant |
|
- I-mutant |
|
11 I-mutant |
|
, O |
|
( O |
|
C O |
|
) O |
|
L3 B-mutant |
|
- I-mutant |
|
20 I-mutant |
|
and O |
|
( O |
|
D O |
|
) O |
|
L4 B-mutant |
|
- I-mutant |
|
1 I-mutant |
|
. O |
|
|
|
An O |
|
interesting O |
|
feature O |
|
of O |
|
these O |
|
CDR B-structure_element |
|
H3 B-structure_element |
|
structures B-evidence |
|
is O |
|
the O |
|
presence O |
|
of O |
|
a O |
|
water B-chemical |
|
molecule O |
|
that O |
|
interacts O |
|
with O |
|
the O |
|
peptide O |
|
nitrogens O |
|
and O |
|
carbonyl O |
|
oxygens O |
|
near O |
|
the O |
|
bridging O |
|
loop B-structure_element |
|
connecting O |
|
the O |
|
2 O |
|
β B-structure_element |
|
- I-structure_element |
|
strands I-structure_element |
|
. O |
|
|
|
A O |
|
representative O |
|
CDR B-structure_element |
|
H3 B-structure_element |
|
structure B-evidence |
|
for O |
|
H1 B-complex_assembly |
|
- I-complex_assembly |
|
69 I-complex_assembly |
|
: I-complex_assembly |
|
L1 I-complex_assembly |
|
- I-complex_assembly |
|
39 I-complex_assembly |
|
illustrating O |
|
this O |
|
is O |
|
shown O |
|
in O |
|
Fig O |
|
. O |
|
7A O |
|
. O |
|
|
|
The O |
|
stem B-structure_element |
|
regions I-structure_element |
|
in O |
|
these O |
|
3 O |
|
cases O |
|
are O |
|
in O |
|
the O |
|
‘ O |
|
kinked B-protein_state |
|
’ O |
|
conformation O |
|
consistent O |
|
with O |
|
that O |
|
observed O |
|
for O |
|
4DN3 O |
|
. O |
|
|
|
The O |
|
stem B-structure_element |
|
regions I-structure_element |
|
of O |
|
CDR B-structure_element |
|
H3 B-structure_element |
|
for O |
|
the O |
|
H5 B-complex_assembly |
|
- I-complex_assembly |
|
51 I-complex_assembly |
|
: I-complex_assembly |
|
L4 I-complex_assembly |
|
- I-complex_assembly |
|
1 I-complex_assembly |
|
Fabs B-structure_element |
|
are O |
|
in O |
|
the O |
|
‘ O |
|
kinked B-protein_state |
|
’ O |
|
conformation O |
|
while O |
|
, O |
|
surprisingly O |
|
, O |
|
those O |
|
of O |
|
the O |
|
H1 B-complex_assembly |
|
- I-complex_assembly |
|
69 I-complex_assembly |
|
: I-complex_assembly |
|
L3 I-complex_assembly |
|
- I-complex_assembly |
|
20 I-complex_assembly |
|
pair O |
|
and O |
|
H3 B-complex_assembly |
|
- I-complex_assembly |
|
53 I-complex_assembly |
|
: I-complex_assembly |
|
L4 I-complex_assembly |
|
- I-complex_assembly |
|
1 I-complex_assembly |
|
are O |
|
in O |
|
the O |
|
‘ O |
|
extended B-protein_state |
|
’ O |
|
conformation O |
|
( O |
|
Fig O |
|
. O |
|
7B O |
|
). O |
|
|
|
The O |
|
two O |
|
domains O |
|
pack O |
|
together O |
|
such O |
|
that O |
|
the O |
|
5 B-structure_element |
|
- I-structure_element |
|
stranded I-structure_element |
|
β I-structure_element |
|
- I-structure_element |
|
sheets I-structure_element |
|
, O |
|
which O |
|
have O |
|
hydrophobic O |
|
surfaces O |
|
, O |
|
interact O |
|
with O |
|
each O |
|
other O |
|
bringing O |
|
the O |
|
CDRs B-structure_element |
|
from O |
|
both O |
|
the O |
|
VH B-structure_element |
|
and O |
|
VL B-structure_element |
|
domains O |
|
into O |
|
close O |
|
proximity O |
|
. O |
|
|
|
VH B-site |
|
: I-site |
|
VL I-site |
|
interface I-site |
|
amino O |
|
acid O |
|
residue O |
|
interactions O |
|
|
|
Position O |
|
43 B-residue_number |
|
may O |
|
be O |
|
alternatively O |
|
occupied O |
|
by O |
|
Ser B-residue_name |
|
, O |
|
Val B-residue_name |
|
or O |
|
Pro B-residue_name |
|
( O |
|
as O |
|
in O |
|
L4 B-mutant |
|
- I-mutant |
|
1 I-mutant |
|
), O |
|
but O |
|
the O |
|
hydrophobic O |
|
interaction O |
|
with O |
|
H B-structure_element |
|
- O |
|
Tyr91 B-residue_name_number |
|
is O |
|
preserved O |
|
. O |
|
|
|
These O |
|
core O |
|
interactions O |
|
provide O |
|
enough O |
|
stability O |
|
to O |
|
the O |
|
VH B-complex_assembly |
|
: I-complex_assembly |
|
VL I-complex_assembly |
|
dimer B-oligomeric_state |
|
so O |
|
that O |
|
additional O |
|
VH B-site |
|
- I-site |
|
VL I-site |
|
contacts I-site |
|
can O |
|
tolerate O |
|
amino O |
|
acid O |
|
sequence O |
|
variations O |
|
in O |
|
CDRs B-structure_element |
|
H3 B-structure_element |
|
and O |
|
L3 B-structure_element |
|
that O |
|
form O |
|
part O |
|
of O |
|
the O |
|
VH B-site |
|
: I-site |
|
VL I-site |
|
interface I-site |
|
. O |
|
|
|
One O |
|
notable O |
|
exception O |
|
is O |
|
H B-structure_element |
|
- O |
|
Trp47 B-residue_name_number |
|
, O |
|
which O |
|
exhibits O |
|
2 O |
|
conformations O |
|
of O |
|
the O |
|
indole O |
|
ring O |
|
. O |
|
|
|
Apparently O |
|
, O |
|
residues O |
|
flanking O |
|
CDR B-structure_element |
|
H3 B-structure_element |
|
in O |
|
the O |
|
2 O |
|
VH B-complex_assembly |
|
: I-complex_assembly |
|
VL I-complex_assembly |
|
pairings O |
|
are O |
|
inconsistent O |
|
with O |
|
any O |
|
stable B-protein_state |
|
conformation O |
|
of O |
|
CDR B-structure_element |
|
H3 B-structure_element |
|
, O |
|
which O |
|
translates O |
|
into O |
|
a O |
|
less O |
|
restricted O |
|
conformational O |
|
space O |
|
for O |
|
some O |
|
of O |
|
them O |
|
, O |
|
including O |
|
H B-structure_element |
|
- O |
|
Trp47 B-residue_name_number |
|
. O |
|
|
|
Differences O |
|
in O |
|
VH B-complex_assembly |
|
: I-complex_assembly |
|
VL I-complex_assembly |
|
tilt B-evidence |
|
angles I-evidence |
|
. O |
|
|
|
The O |
|
differences B-evidence |
|
in O |
|
the O |
|
tilt B-evidence |
|
angle I-evidence |
|
are O |
|
shown O |
|
for O |
|
all O |
|
pairs O |
|
of O |
|
V B-structure_element |
|
regions I-structure_element |
|
in O |
|
Table O |
|
3 O |
|
. O |
|
|
|
Residues O |
|
in O |
|
CDR B-structure_element |
|
H3 B-structure_element |
|
are O |
|
missing O |
|
: O |
|
YGE B-structure_element |
|
in O |
|
H5 B-complex_assembly |
|
- I-complex_assembly |
|
51 I-complex_assembly |
|
: I-complex_assembly |
|
L3 I-complex_assembly |
|
- I-complex_assembly |
|
11 I-complex_assembly |
|
, O |
|
GIY B-structure_element |
|
in O |
|
H5 B-complex_assembly |
|
- I-complex_assembly |
|
51 I-complex_assembly |
|
: I-complex_assembly |
|
L3 I-complex_assembly |
|
- I-complex_assembly |
|
20 I-complex_assembly |
|
. O |
|
|
|
Interestingly O |
|
, O |
|
the O |
|
2 O |
|
structures B-evidence |
|
that O |
|
have O |
|
the O |
|
largest O |
|
tilt B-evidence |
|
angle I-evidence |
|
differences I-evidence |
|
with O |
|
the O |
|
other O |
|
variants O |
|
, O |
|
H3 B-complex_assembly |
|
- I-complex_assembly |
|
23 I-complex_assembly |
|
: I-complex_assembly |
|
L3 I-complex_assembly |
|
- I-complex_assembly |
|
20 I-complex_assembly |
|
and O |
|
H1 B-complex_assembly |
|
- I-complex_assembly |
|
69 I-complex_assembly |
|
: I-complex_assembly |
|
L3 I-complex_assembly |
|
- I-complex_assembly |
|
20 I-complex_assembly |
|
, O |
|
have O |
|
the O |
|
smallest O |
|
VH B-site |
|
: I-site |
|
VL I-site |
|
interfaces I-site |
|
, O |
|
684 O |
|
and O |
|
725 O |
|
Å2 O |
|
, O |
|
respectively O |
|
. O |
|
|
|
It O |
|
appears O |
|
that O |
|
for O |
|
each O |
|
given O |
|
LC B-structure_element |
|
, O |
|
the O |
|
Fabs B-structure_element |
|
with O |
|
germlines O |
|
H1 B-mutant |
|
- I-mutant |
|
69 I-mutant |
|
and O |
|
H3 B-mutant |
|
- I-mutant |
|
23 I-mutant |
|
are O |
|
substantially O |
|
more O |
|
stable B-protein_state |
|
than O |
|
those O |
|
with O |
|
germlines O |
|
H3 B-mutant |
|
- I-mutant |
|
53 I-mutant |
|
and O |
|
H5 B-mutant |
|
- I-mutant |
|
51 I-mutant |
|
. O |
|
|
|
Parts O |
|
of O |
|
CDR B-structure_element |
|
H3 B-structure_element |
|
main O |
|
chain O |
|
are O |
|
completely O |
|
disordered B-protein_state |
|
, O |
|
and O |
|
were O |
|
not O |
|
modeled O |
|
in O |
|
Fabs B-structure_element |
|
H5 B-complex_assembly |
|
- I-complex_assembly |
|
51 I-complex_assembly |
|
: I-complex_assembly |
|
L3 I-complex_assembly |
|
- I-complex_assembly |
|
20 I-complex_assembly |
|
and O |
|
H5 B-complex_assembly |
|
- I-complex_assembly |
|
51 I-complex_assembly |
|
: I-complex_assembly |
|
L3 I-complex_assembly |
|
- I-complex_assembly |
|
11 I-complex_assembly |
|
that O |
|
have O |
|
the O |
|
lowest O |
|
Tms B-evidence |
|
in O |
|
the O |
|
set O |
|
. O |
|
|
|
All O |
|
those O |
|
molecules O |
|
are O |
|
relatively O |
|
unstable O |
|
, O |
|
as O |
|
is O |
|
reflected O |
|
in O |
|
their O |
|
low O |
|
Tms B-evidence |
|
. O |
|
|
|
Of O |
|
the O |
|
4 O |
|
HCs B-structure_element |
|
, O |
|
H1 B-mutant |
|
- I-mutant |
|
69 I-mutant |
|
has O |
|
the O |
|
greatest O |
|
number O |
|
of O |
|
canonical O |
|
structure O |
|
assignments O |
|
( O |
|
Table O |
|
2 O |
|
). O |
|
|
|
The O |
|
remaining O |
|
8 O |
|
structures B-evidence |
|
exhibit O |
|
“ O |
|
non O |
|
- O |
|
parental O |
|
” O |
|
conformations O |
|
, O |
|
indicating O |
|
that O |
|
the O |
|
VH B-structure_element |
|
and O |
|
VL B-structure_element |
|
context O |
|
can O |
|
also O |
|
be O |
|
a O |
|
dominating O |
|
factor O |
|
influencing O |
|
CDR B-structure_element |
|
H3 B-structure_element |
|
. O |
|
|
|
Thus O |
|
, O |
|
no O |
|
patterns O |
|
of O |
|
conformational O |
|
preference O |
|
for O |
|
a O |
|
particular O |
|
HC B-structure_element |
|
or O |
|
LC B-structure_element |
|
emerge O |
|
to O |
|
shed O |
|
any O |
|
direct O |
|
light O |
|
on O |
|
what O |
|
drives O |
|
the O |
|
conformational O |
|
differences O |
|
. O |
|
|
|
One O |
|
of O |
|
the O |
|
variants O |
|
, O |
|
H3 B-complex_assembly |
|
- I-complex_assembly |
|
23 I-complex_assembly |
|
: I-complex_assembly |
|
L3 I-complex_assembly |
|
- I-complex_assembly |
|
20 I-complex_assembly |
|
, O |
|
has O |
|
the O |
|
CDR B-structure_element |
|
H3 B-structure_element |
|
conformation O |
|
similar O |
|
to O |
|
the O |
|
parent O |
|
, O |
|
but O |
|
the O |
|
other O |
|
, O |
|
H1 B-complex_assembly |
|
- I-complex_assembly |
|
69 I-complex_assembly |
|
: I-complex_assembly |
|
L3 I-complex_assembly |
|
- I-complex_assembly |
|
20 I-complex_assembly |
|
, O |
|
is O |
|
different O |
|
. O |
|
|
|
These O |
|
differences O |
|
undoubtedly O |
|
influence O |
|
the O |
|
conformation O |
|
of O |
|
the O |
|
CDRs B-structure_element |
|
, O |
|
in O |
|
particular O |
|
CDR B-structure_element |
|
H1 B-structure_element |
|
( O |
|
Fig O |
|
. O |
|
1A O |
|
) O |
|
and O |
|
CDR B-structure_element |
|
L1 B-structure_element |
|
( O |
|
Fig O |
|
. O |
|
3C O |
|
), O |
|
especially O |
|
with O |
|
the O |
|
tandem O |
|
glycines B-residue_name |
|
and O |
|
multiple O |
|
serines B-residue_name |
|
present O |
|
, O |
|
respectively O |
|
. O |
|
|
|
Pairing O |
|
of O |
|
different O |
|
germlines O |
|
yields O |
|
antibodies B-protein_type |
|
with O |
|
various O |
|
degrees O |
|
of O |
|
stability O |
|
. O |
|
|
|
Other O |
|
germlines O |
|
have O |
|
bulky O |
|
residues O |
|
, O |
|
Tyr B-residue_name |
|
, O |
|
Arg B-residue_name |
|
and O |
|
Trp B-residue_name |
|
, O |
|
at O |
|
these O |
|
positions O |
|
, O |
|
whereas O |
|
L1 B-mutant |
|
- I-mutant |
|
39 I-mutant |
|
has O |
|
Ser B-residue_name |
|
and O |
|
Thr B-residue_name |
|
. O |
|
|
|
The O |
|
set O |
|
of O |
|
16 O |
|
germline O |
|
Fab B-structure_element |
|
structures B-evidence |
|
offers O |
|
a O |
|
unique O |
|
dataset O |
|
to O |
|
facilitate O |
|
software O |
|
development O |
|
for O |
|
antibody B-protein_type |
|
modeling O |
|
. O |
|
|
|
An O |
|
extended B-protein_state |
|
U2AF65 B-structure_element |
|
– I-structure_element |
|
RNA I-structure_element |
|
- I-structure_element |
|
binding I-structure_element |
|
domain I-structure_element |
|
recognizes O |
|
the O |
|
3 B-site |
|
′ I-site |
|
splice I-site |
|
site I-site |
|
signal O |
|
|
|
The O |
|
U2AF65 B-protein |
|
linker B-structure_element |
|
residues O |
|
between O |
|
the O |
|
dual O |
|
RNA B-structure_element |
|
recognition I-structure_element |
|
motifs I-structure_element |
|
( O |
|
RRMs B-structure_element |
|
) O |
|
recognize O |
|
the O |
|
central O |
|
nucleotide B-chemical |
|
, O |
|
whereas O |
|
the O |
|
N O |
|
- O |
|
and O |
|
C O |
|
- O |
|
terminal O |
|
RRM B-structure_element |
|
extensions I-structure_element |
|
recognize O |
|
the O |
|
3 B-site |
|
′ I-site |
|
terminus I-site |
|
and O |
|
third B-residue_number |
|
nucleotide B-chemical |
|
. O |
|
|
|
The O |
|
splice B-site |
|
sites I-site |
|
are O |
|
marked O |
|
by O |
|
relatively O |
|
short B-structure_element |
|
consensus I-structure_element |
|
sequences I-structure_element |
|
and O |
|
are O |
|
regulated O |
|
by O |
|
additional O |
|
pre B-structure_element |
|
- I-structure_element |
|
mRNA I-structure_element |
|
motifs I-structure_element |
|
( O |
|
reviewed O |
|
in O |
|
ref O |
|
.). O |
|
|
|
The O |
|
early O |
|
- O |
|
stage O |
|
pre B-protein_type |
|
- I-protein_type |
|
mRNA I-protein_type |
|
splicing I-protein_type |
|
factor I-protein_type |
|
U2AF65 B-protein |
|
is O |
|
essential O |
|
for O |
|
viability O |
|
in O |
|
vertebrates B-taxonomy_domain |
|
and O |
|
other O |
|
model O |
|
organisms O |
|
( O |
|
for O |
|
example O |
|
, O |
|
ref O |
|
.). O |
|
|
|
A O |
|
tightly O |
|
controlled O |
|
assembly B-complex_assembly |
|
among O |
|
U2AF65 B-protein |
|
, O |
|
the O |
|
pre B-chemical |
|
- I-chemical |
|
mRNA I-chemical |
|
, O |
|
and O |
|
partner O |
|
proteins O |
|
sequentially O |
|
identifies O |
|
the O |
|
3 B-site |
|
′ I-site |
|
splice I-site |
|
site I-site |
|
and O |
|
promotes O |
|
association O |
|
of O |
|
the O |
|
spliceosome B-complex_assembly |
|
, O |
|
which O |
|
ultimately O |
|
accomplishes O |
|
the O |
|
task O |
|
of O |
|
splicing O |
|
. O |
|
|
|
We O |
|
use O |
|
single B-experimental_method |
|
- I-experimental_method |
|
molecule I-experimental_method |
|
Förster I-experimental_method |
|
resonance I-experimental_method |
|
energy I-experimental_method |
|
transfer I-experimental_method |
|
( O |
|
smFRET B-experimental_method |
|
) O |
|
to O |
|
characterize O |
|
the O |
|
conformational B-evidence |
|
dynamics I-evidence |
|
of O |
|
this O |
|
extended B-protein_state |
|
U2AF65 B-structure_element |
|
– I-structure_element |
|
RNA I-structure_element |
|
- I-structure_element |
|
binding I-structure_element |
|
domain I-structure_element |
|
during O |
|
Py B-chemical |
|
- I-chemical |
|
tract I-chemical |
|
recognition O |
|
. O |
|
|
|
Likewise O |
|
, O |
|
both O |
|
U2AF651 B-mutant |
|
, I-mutant |
|
2L I-mutant |
|
and O |
|
full B-protein_state |
|
- I-protein_state |
|
length I-protein_state |
|
U2AF65 B-protein |
|
showed O |
|
similar O |
|
sequence B-evidence |
|
specificity I-evidence |
|
for O |
|
U B-structure_element |
|
- I-structure_element |
|
rich I-structure_element |
|
stretches I-structure_element |
|
in O |
|
the O |
|
5 B-site |
|
′- I-site |
|
region I-site |
|
of O |
|
the O |
|
Py B-chemical |
|
tract I-chemical |
|
and O |
|
promiscuity O |
|
for O |
|
C B-structure_element |
|
- I-structure_element |
|
rich I-structure_element |
|
regions I-structure_element |
|
in O |
|
the O |
|
3 B-site |
|
′- I-site |
|
region I-site |
|
( O |
|
Fig O |
|
. O |
|
1c O |
|
, O |
|
Supplementary O |
|
Fig O |
|
. O |
|
1e O |
|
– O |
|
h O |
|
). O |
|
|
|
We O |
|
compare O |
|
the O |
|
global O |
|
conformation O |
|
of O |
|
the O |
|
U2AF651 B-mutant |
|
, I-mutant |
|
2L I-mutant |
|
structures B-evidence |
|
with O |
|
the O |
|
prior O |
|
dU2AF651 B-mutant |
|
, I-mutant |
|
2 I-mutant |
|
crystal B-evidence |
|
structure I-evidence |
|
and O |
|
U2AF651 B-mutant |
|
, I-mutant |
|
2 I-mutant |
|
NMR B-experimental_method |
|
structure B-evidence |
|
in O |
|
the O |
|
Supplementary O |
|
Discussion O |
|
and O |
|
Supplementary O |
|
Fig O |
|
. O |
|
2 O |
|
. O |
|
|
|
Otherwise O |
|
, O |
|
the O |
|
rU4 B-residue_name_number |
|
nucleotide B-chemical |
|
packs O |
|
against O |
|
F304 B-residue_name_number |
|
in O |
|
the O |
|
signature O |
|
ribonucleoprotein B-structure_element |
|
consensus I-structure_element |
|
motif I-structure_element |
|
( I-structure_element |
|
RNP I-structure_element |
|
)- I-structure_element |
|
2 I-structure_element |
|
of O |
|
RRM2 B-structure_element |
|
. O |
|
|
|
This O |
|
nucleotide B-chemical |
|
twists O |
|
to O |
|
face O |
|
away O |
|
from O |
|
the O |
|
U2AF65 B-protein |
|
linker B-structure_element |
|
and O |
|
instead O |
|
inserts O |
|
the O |
|
rU6 B-residue_name_number |
|
- O |
|
uracil B-residue_name |
|
into O |
|
a O |
|
sandwich O |
|
between O |
|
the O |
|
β2 B-structure_element |
|
/ I-structure_element |
|
β3 I-structure_element |
|
loops I-structure_element |
|
of O |
|
RRM1 B-structure_element |
|
and O |
|
RRM2 B-structure_element |
|
. O |
|
|
|
The O |
|
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 |
|
|
|
Consistent O |
|
with O |
|
loss O |
|
of O |
|
a O |
|
hydrogen O |
|
bond O |
|
with O |
|
the O |
|
ninth B-residue_number |
|
pyrimidine B-chemical |
|
- O |
|
O2 O |
|
( O |
|
ΔΔG B-evidence |
|
1 O |
|
. O |
|
0 O |
|
kcal O |
|
mol O |
|
− O |
|
1 O |
|
), O |
|
mutation B-experimental_method |
|
of O |
|
the O |
|
Q147 B-residue_name_number |
|
to O |
|
an O |
|
alanine B-residue_name |
|
reduced O |
|
U2AF651 B-evidence |
|
, I-evidence |
|
2L I-evidence |
|
affinity I-evidence |
|
for O |
|
the O |
|
AdML B-gene |
|
Py B-chemical |
|
tract I-chemical |
|
by O |
|
five O |
|
- O |
|
fold O |
|
( O |
|
Fig O |
|
. O |
|
3i O |
|
; O |
|
Supplementary O |
|
Fig O |
|
. O |
|
4c O |
|
). O |
|
|
|
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 |
|
|
|
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 |
|
|
|
When O |
|
transfected B-experimental_method |
|
into O |
|
HEK293T O |
|
cells O |
|
containing O |
|
only O |
|
endogenous B-protein_state |
|
U2AF65 B-protein |
|
, O |
|
the O |
|
PY B-site |
|
splice I-site |
|
site I-site |
|
is O |
|
used O |
|
and O |
|
the O |
|
remaining O |
|
transcript O |
|
remains O |
|
unspliced O |
|
. O |
|
|
|
Co B-experimental_method |
|
- I-experimental_method |
|
transfection I-experimental_method |
|
of O |
|
the O |
|
U2AF65 B-mutant |
|
- I-mutant |
|
3Mut I-mutant |
|
with O |
|
the O |
|
pyPY B-chemical |
|
splicing O |
|
substrate O |
|
significantly O |
|
reduced O |
|
splicing O |
|
of O |
|
the O |
|
weak O |
|
‘ B-site |
|
py I-site |
|
' I-site |
|
splice I-site |
|
site I-site |
|
relative O |
|
to O |
|
wild B-protein_state |
|
- I-protein_state |
|
type I-protein_state |
|
U2AF65 B-protein |
|
( O |
|
Fig O |
|
. O |
|
5b O |
|
, O |
|
c O |
|
). O |
|
|
|
Paramagnetic B-experimental_method |
|
resonance I-experimental_method |
|
enhancement I-experimental_method |
|
( O |
|
PRE B-experimental_method |
|
) O |
|
measurements O |
|
previously O |
|
had O |
|
suggested O |
|
a O |
|
predominant O |
|
back B-protein_state |
|
- I-protein_state |
|
to I-protein_state |
|
- I-protein_state |
|
back I-protein_state |
|
, O |
|
or O |
|
‘ O |
|
closed B-protein_state |
|
' O |
|
conformation O |
|
of O |
|
the O |
|
apo B-protein_state |
|
- O |
|
U2AF651 B-mutant |
|
, I-mutant |
|
2 I-mutant |
|
RRM1 B-structure_element |
|
and O |
|
RRM2 B-structure_element |
|
in O |
|
equilibrium O |
|
with O |
|
a O |
|
minor O |
|
‘ O |
|
open B-protein_state |
|
' O |
|
conformation O |
|
resembling O |
|
the O |
|
RNA B-protein_state |
|
- I-protein_state |
|
bound I-protein_state |
|
inter B-structure_element |
|
- I-structure_element |
|
RRM I-structure_element |
|
arrangement O |
|
. O |
|
|
|
To O |
|
complement O |
|
the O |
|
static O |
|
portraits O |
|
of O |
|
U2AF651 B-mutant |
|
, I-mutant |
|
2L I-mutant |
|
structure B-evidence |
|
that O |
|
we O |
|
had O |
|
determined O |
|
by O |
|
X B-experimental_method |
|
- I-experimental_method |
|
ray I-experimental_method |
|
crystallography I-experimental_method |
|
, O |
|
we O |
|
used O |
|
smFRET B-experimental_method |
|
to O |
|
characterize O |
|
the O |
|
probability B-evidence |
|
distribution I-evidence |
|
functions I-evidence |
|
and O |
|
time O |
|
dependence O |
|
of O |
|
U2AF65 B-protein |
|
inter B-structure_element |
|
- I-structure_element |
|
RRM I-structure_element |
|
conformational O |
|
dynamics O |
|
in O |
|
solution O |
|
. O |
|
|
|
The O |
|
positions O |
|
of O |
|
single O |
|
cysteine B-residue_name |
|
mutations B-experimental_method |
|
for O |
|
fluorophore B-chemical |
|
attachment O |
|
( O |
|
A181C B-mutant |
|
in O |
|
RRM1 B-structure_element |
|
and O |
|
Q324C B-mutant |
|
in O |
|
RRM2 B-structure_element |
|
) O |
|
were O |
|
chosen O |
|
based O |
|
on O |
|
inspection O |
|
of O |
|
the O |
|
U2AF651 B-mutant |
|
, I-mutant |
|
2L I-mutant |
|
structures B-evidence |
|
and O |
|
the O |
|
‘ O |
|
closed B-protein_state |
|
' O |
|
model O |
|
of O |
|
apo B-protein_state |
|
- O |
|
U2AF651 B-mutant |
|
, I-mutant |
|
2 I-mutant |
|
. O |
|
|
|
Approximately O |
|
70 O |
|
% O |
|
of O |
|
observed O |
|
fluctuations O |
|
were O |
|
interchanges O |
|
between O |
|
the O |
|
∼ O |
|
0 O |
|
. O |
|
65 O |
|
and O |
|
∼ O |
|
0 O |
|
. O |
|
45 O |
|
FRET B-evidence |
|
values I-evidence |
|
( O |
|
Supplementary O |
|
Fig O |
|
. O |
|
7b O |
|
). O |
|
|
|
However O |
|
, O |
|
the O |
|
presence O |
|
of O |
|
repetitive O |
|
fluctuations O |
|
between O |
|
particular O |
|
FRET B-evidence |
|
values I-evidence |
|
supports O |
|
the O |
|
hypothesis O |
|
that O |
|
RNA B-protein_state |
|
- I-protein_state |
|
free I-protein_state |
|
U2AF65 B-protein |
|
samples O |
|
several O |
|
distinct O |
|
conformations O |
|
. O |
|
|
|
U2AF65 B-protein |
|
conformational O |
|
selection O |
|
and O |
|
induced O |
|
fit O |
|
by O |
|
bound B-protein_state |
|
RNA B-chemical |
|
|
|
Insertion B-experimental_method |
|
of O |
|
adenine B-chemical |
|
nucleotides I-chemical |
|
decreased O |
|
binding B-evidence |
|
affinity I-evidence |
|
of O |
|
U2AF65 B-protein |
|
to O |
|
RNA B-chemical |
|
by O |
|
approximately O |
|
five O |
|
- O |
|
fold O |
|
. O |
|
|
|
Further O |
|
research O |
|
will O |
|
be O |
|
needed O |
|
to O |
|
understand O |
|
the O |
|
roles O |
|
of O |
|
SF1 B-protein |
|
and O |
|
U2AF35 B-protein |
|
subunits O |
|
in O |
|
the O |
|
conformational O |
|
equilibria O |
|
underlying O |
|
U2AF65 B-protein |
|
association O |
|
with O |
|
Py B-chemical |
|
tracts I-chemical |
|
. O |
|
|
|
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 |
|
fitted O |
|
fluorescence O |
|
anisotropy O |
|
RNA B-evidence |
|
- I-evidence |
|
binding I-evidence |
|
curves I-evidence |
|
are O |
|
shown O |
|
in O |
|
Supplementary O |
|
Fig O |
|
. O |
|
4d O |
|
– O |
|
j O |
|
. O |
|
( O |
|
c O |
|
) O |
|
Close O |
|
view O |
|
of O |
|
the O |
|
U2AF65 B-protein |
|
RRM1 B-site |
|
/ I-site |
|
RRM2 I-site |
|
interface I-site |
|
following O |
|
a O |
|
two O |
|
- O |
|
fold O |
|
rotation O |
|
about O |
|
the O |
|
x O |
|
- O |
|
axis O |
|
relative O |
|
to O |
|
a O |
|
. O |
|
|
|
( O |
|
a O |
|
, O |
|
b O |
|
) O |
|
Views O |
|
of O |
|
FRET B-experimental_method |
|
pairs O |
|
chosen O |
|
to O |
|
follow O |
|
the O |
|
relative O |
|
movement O |
|
of O |
|
RRM1 B-structure_element |
|
and O |
|
RRM2 B-structure_element |
|
on O |
|
the O |
|
crystal B-evidence |
|
structure I-evidence |
|
of O |
|
‘ O |
|
side B-protein_state |
|
- I-protein_state |
|
by I-protein_state |
|
- I-protein_state |
|
side I-protein_state |
|
' O |
|
U2AF651 B-mutant |
|
, I-mutant |
|
2L I-mutant |
|
RRMs B-structure_element |
|
bound B-protein_state |
|
to I-protein_state |
|
a O |
|
Py B-chemical |
|
- I-chemical |
|
tract I-chemical |
|
oligonucleotide I-chemical |
|
( O |
|
a O |
|
, O |
|
representative O |
|
structure O |
|
iv O |
|
) O |
|
or O |
|
‘ O |
|
closed B-protein_state |
|
' O |
|
NMR B-experimental_method |
|
/ O |
|
PRE B-experimental_method |
|
- O |
|
based O |
|
model O |
|
of O |
|
U2AF651 B-mutant |
|
, I-mutant |
|
2 I-mutant |
|
( O |
|
b O |
|
, O |
|
PDB O |
|
ID O |
|
2YH0 O |
|
) O |
|
in O |
|
identical O |
|
orientations O |
|
of O |
|
RRM2 B-structure_element |
|
. O |
|
|
|
( O |
|
c O |
|
– O |
|
f O |
|
, O |
|
i O |
|
, O |
|
j O |
|
) O |
|
The O |
|
U2AF651 B-mutant |
|
, I-mutant |
|
2LFRET I-mutant |
|
( O |
|
Cy3 B-chemical |
|
/ O |
|
Cy5 B-chemical |
|
) O |
|
protein O |
|
was O |
|
immobilized O |
|
on O |
|
the O |
|
microscope O |
|
slide O |
|
via O |
|
biotin B-chemical |
|
- I-chemical |
|
NTA I-chemical |
|
/ I-chemical |
|
Ni I-chemical |
|
+ I-chemical |
|
2 I-chemical |
|
( O |
|
orange O |
|
line O |
|
) O |
|
on O |
|
a O |
|
neutravidin O |
|
( O |
|
black O |
|
X O |
|
)- O |
|
biotin O |
|
- O |
|
PEG O |
|
( O |
|
orange O |
|
triangle O |
|
)- O |
|
treated O |
|
surface O |
|
and O |
|
imaged O |
|
either O |
|
in O |
|
the O |
|
absence B-protein_state |
|
of I-protein_state |
|
ligands B-chemical |
|
( O |
|
c O |
|
, O |
|
d O |
|
), O |
|
in O |
|
the O |
|
presence O |
|
of O |
|
5 O |
|
μM O |
|
AdML B-gene |
|
Py B-chemical |
|
- I-chemical |
|
tract I-chemical |
|
RNA I-chemical |
|
( O |
|
5 B-chemical |
|
′- I-chemical |
|
CCUUUUUUUUCC I-chemical |
|
- I-chemical |
|
3 I-chemical |
|
′) I-chemical |
|
( O |
|
e O |
|
, O |
|
f O |
|
), O |
|
or O |
|
in O |
|
the O |
|
presence O |
|
of O |
|
10 O |
|
μM O |
|
adenosine B-residue_name |
|
- O |
|
interrupted O |
|
variant O |
|
RNA B-chemical |
|
( O |
|
5 B-chemical |
|
′- I-chemical |
|
CUUUUUAAUUUCCA I-chemical |
|
- I-chemical |
|
3 I-chemical |
|
′) I-chemical |
|
( O |
|
i O |
|
, O |
|
j O |
|
). O |
|
|
|
The O |
|
untethered B-protein_state |
|
U2AF651 B-mutant |
|
, I-mutant |
|
2LFRET I-mutant |
|
( O |
|
Cy3 B-chemical |
|
/ O |
|
Cy5 B-chemical |
|
) O |
|
protein O |
|
( O |
|
1 O |
|
nM O |
|
) O |
|
was O |
|
added O |
|
to O |
|
AdML B-gene |
|
RNA B-chemical |
|
– I-chemical |
|
polyethylene I-chemical |
|
- I-chemical |
|
glycol I-chemical |
|
- I-chemical |
|
linker I-chemical |
|
– I-chemical |
|
DNA I-chemical |
|
oligonucleotide I-chemical |
|
( O |
|
10 O |
|
nM O |
|
), O |
|
which O |
|
was O |
|
immobilized O |
|
on O |
|
the O |
|
microscope O |
|
slide O |
|
by O |
|
annealing O |
|
with O |
|
a O |
|
complementary O |
|
biotinyl B-chemical |
|
- I-chemical |
|
DNA I-chemical |
|
oligonucleotide I-chemical |
|
( O |
|
black O |
|
vertical O |
|
line O |
|
). 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 |
|
|
|
RRM1 B-structure_element |
|
, O |
|
green O |
|
; O |
|
RRM2 B-structure_element |
|
, O |
|
pale O |
|
blue O |
|
; O |
|
RRM B-structure_element |
|
extensions I-structure_element |
|
/ O |
|
linker B-structure_element |
|
, O |
|
blue O |
|
. O |
|
|
|
Systematic O |
|
analysis O |
|
of O |
|
radiation O |
|
damage O |
|
within O |
|
a O |
|
protein B-complex_assembly |
|
– I-complex_assembly |
|
RNA I-complex_assembly |
|
complex O |
|
over O |
|
a O |
|
large O |
|
dose O |
|
range O |
|
( O |
|
1 O |
|
. O |
|
3 O |
|
– O |
|
25 O |
|
MGy O |
|
) O |
|
reveals O |
|
significant O |
|
differential O |
|
susceptibility O |
|
of O |
|
RNA B-chemical |
|
and O |
|
protein O |
|
. O |
|
|
|
With O |
|
the O |
|
wide O |
|
use O |
|
of O |
|
high O |
|
- O |
|
flux O |
|
third O |
|
- O |
|
generation O |
|
synchrotron O |
|
sources O |
|
, O |
|
radiation O |
|
damage O |
|
( O |
|
RD O |
|
) O |
|
has O |
|
once O |
|
again O |
|
become O |
|
a O |
|
dominant O |
|
reason O |
|
for O |
|
the O |
|
failure O |
|
of O |
|
structure B-experimental_method |
|
determination I-experimental_method |
|
using O |
|
macromolecular B-experimental_method |
|
crystallography I-experimental_method |
|
( O |
|
MX B-experimental_method |
|
) O |
|
in O |
|
experiments O |
|
conducted O |
|
both O |
|
at O |
|
room O |
|
temperature O |
|
and O |
|
under O |
|
cryocooled O |
|
conditions O |
|
( O |
|
100 O |
|
K O |
|
). O |
|
|
|
Significant O |
|
progress O |
|
has O |
|
been O |
|
made O |
|
in O |
|
recent O |
|
years O |
|
in O |
|
understanding O |
|
the O |
|
inevitable O |
|
manifestations O |
|
of O |
|
X O |
|
- O |
|
ray O |
|
- O |
|
induced O |
|
RD O |
|
within O |
|
protein O |
|
crystals B-evidence |
|
, O |
|
and O |
|
there O |
|
is O |
|
now O |
|
a O |
|
body O |
|
of O |
|
literature O |
|
on O |
|
possible O |
|
strategies O |
|
to O |
|
mitigate O |
|
the O |
|
effects O |
|
of O |
|
RD O |
|
( O |
|
e O |
|
. O |
|
g O |
|
. O |
|
Zeldin O |
|
, O |
|
Brockhauser O |
|
et O |
|
al O |
|
., O |
|
2013 O |
|
; O |
|
Bourenkov O |
|
& O |
|
Popov O |
|
, O |
|
2010 O |
|
). O |
|
|
|
There O |
|
are O |
|
a O |
|
number O |
|
of O |
|
cases O |
|
where O |
|
SRD O |
|
manifestations O |
|
have O |
|
compromised O |
|
the O |
|
biological O |
|
information O |
|
extracted O |
|
from O |
|
MX B-experimental_method |
|
- I-experimental_method |
|
determined I-experimental_method |
|
structures B-evidence |
|
at O |
|
much O |
|
lower O |
|
doses O |
|
than O |
|
the O |
|
recommended O |
|
30 O |
|
MGy O |
|
limit O |
|
, O |
|
leading O |
|
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|
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|
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|
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|
of O |
|
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|
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|
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|
|
|
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|
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|
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|
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|
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|
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|
|
|
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|
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|
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|
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|
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|
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|
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|
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|
|
|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
|
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|
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|
|
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|
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|
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assembly O |
|
over O |
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|
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|
dose O |
|
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|
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|
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|
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|
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|
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|
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|
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complex O |
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|
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|
|
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8 O |
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|
base O |
|
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|
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|
binding I-site |
|
interfaces I-site |
|
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and O |
|
has O |
|
indistinguishable O |
|
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|
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|
D O |
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|
dose I-evidence |
|
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dynamics I-evidence |
|
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|
RNA B-protein_state |
|
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|
bound I-protein_state |
|
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|
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|
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|
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|
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at O |
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|
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|
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|
and O |
|
holes O |
|
produced O |
|
directly O |
|
within O |
|
the O |
|
protein B-complex_assembly |
|
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|
RNA I-complex_assembly |
|
components O |
|
or O |
|
in O |
|
closely O |
|
associated O |
|
solvent O |
|
. O |
|
|
|
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|
results O |
|
presented O |
|
here O |
|
suggest O |
|
that O |
|
biologically O |
|
relevant O |
|
nucleoprotein B-complex_assembly |
|
complexes O |
|
also O |
|
exhibit O |
|
prolonged O |
|
life O |
|
- O |
|
doses O |
|
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|
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|
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|
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|
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|
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|
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|
structural O |
|
changes O |
|
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|
involving O |
|
direct O |
|
physical O |
|
protection O |
|
of O |
|
key O |
|
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|
- I-site |
|
binding I-site |
|
residues I-site |
|
. O |
|
|
|
Such O |
|
reduced O |
|
radiation O |
|
- O |
|
sensitivity O |
|
in O |
|
this O |
|
case O |
|
ensures O |
|
that O |
|
the O |
|
interacting O |
|
protein O |
|
remains O |
|
bound B-protein_state |
|
long O |
|
enough O |
|
to O |
|
the O |
|
RNA B-chemical |
|
to O |
|
complete O |
|
its O |
|
function O |
|
, O |
|
even O |
|
whilst O |
|
exposed O |
|
to O |
|
ionizing O |
|
radiation O |
|
. O |
|
|
|
RNA B-chemical |
|
is O |
|
shown O |
|
is O |
|
yellow O |
|
. O |
|
|
|
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|
a O |
|
subset O |
|
of O |
|
key O |
|
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|
residue O |
|
types O |
|
are O |
|
included O |
|
. O |
|
|
|
D O |
|
loss O |
|
calculated O |
|
for O |
|
all O |
|
side O |
|
- O |
|
chain O |
|
carboxyl O |
|
group O |
|
Glu B-residue_name |
|
Cδ O |
|
and O |
|
Asp B-residue_name |
|
Cγ O |
|
atoms O |
|
within O |
|
the O |
|
TRAP B-complex_assembly |
|
– I-complex_assembly |
|
RNA I-complex_assembly |
|
complex O |
|
for O |
|
a O |
|
dose O |
|
of O |
|
19 O |
|
. O |
|
3 O |
|
MGy O |
|
( O |
|
d O |
|
8 O |
|
). O |
|
|
|
Another O |
|
challenge O |
|
will O |
|
be O |
|
to O |
|
find O |
|
out O |
|
where O |
|
IDA B-protein |
|
is O |
|
produced O |
|
in O |
|
the O |
|
plant B-taxonomy_domain |
|
and O |
|
what O |
|
causes O |
|
it O |
|
to O |
|
accumulate O |
|
in O |
|
specific O |
|
places O |
|
in O |
|
preparation O |
|
for O |
|
organ O |
|
shedding O |
|
. O |
|
|
|
The O |
|
HAESA B-protein |
|
ectodomain B-structure_element |
|
folds O |
|
into O |
|
a O |
|
superhelical B-structure_element |
|
assembly I-structure_element |
|
of O |
|
21 O |
|
leucine B-structure_element |
|
- I-structure_element |
|
rich I-structure_element |
|
repeats I-structure_element |
|
. O |
|
|
|
( O |
|
A O |
|
) O |
|
Details O |
|
of O |
|
the O |
|
IDA B-site |
|
binding I-site |
|
pocket I-site |
|
. O |
|
|
|
The O |
|
IDA B-complex_assembly |
|
- I-complex_assembly |
|
HAESA I-complex_assembly |
|
and O |
|
SERK1 B-complex_assembly |
|
- I-complex_assembly |
|
HAESA I-complex_assembly |
|
complex O |
|
interfaces B-site |
|
are O |
|
conserved B-protein_state |
|
among O |
|
HAESA B-protein |
|
and O |
|
HAESA B-protein_type |
|
- I-protein_type |
|
like I-protein_type |
|
proteins I-protein_type |
|
from O |
|
different O |
|
plant B-taxonomy_domain |
|
species O |
|
. O |
|
|
|
The O |
|
peptide B-site |
|
binding I-site |
|
pocket I-site |
|
covers O |
|
HAESA B-protein |
|
LRRs B-structure_element |
|
2 I-structure_element |
|
– I-structure_element |
|
14 I-structure_element |
|
. O |
|
( O |
|
D O |
|
) O |
|
Close O |
|
- O |
|
up O |
|
view O |
|
of O |
|
the O |
|
entire O |
|
IDA B-protein |
|
( O |
|
in O |
|
yellow O |
|
) O |
|
peptide B-site |
|
binding I-site |
|
site I-site |
|
in O |
|
HAESA B-protein |
|
( O |
|
in O |
|
blue O |
|
). O |
|
|
|
Hydrogren O |
|
bonds O |
|
are O |
|
depicted O |
|
as O |
|
dotted O |
|
lines O |
|
( O |
|
in O |
|
magenta O |
|
), O |
|
a O |
|
water B-chemical |
|
molecule O |
|
is O |
|
shown O |
|
as O |
|
a O |
|
red O |
|
sphere O |
|
. O |
|
|
|
This B-structure_element |
|
sequence I-structure_element |
|
motif I-structure_element |
|
is O |
|
highly B-protein_state |
|
conserved I-protein_state |
|
among O |
|
IDA B-protein_type |
|
family I-protein_type |
|
members I-protein_type |
|
( O |
|
IDA B-protein_type |
|
- I-protein_type |
|
LIKE I-protein_type |
|
PROTEINS I-protein_type |
|
, O |
|
IDLs B-protein_type |
|
) O |
|
and O |
|
contains O |
|
a O |
|
central O |
|
Pro B-residue_name |
|
residue O |
|
, O |
|
presumed O |
|
to O |
|
be O |
|
post B-protein_state |
|
- I-protein_state |
|
translationally I-protein_state |
|
modified I-protein_state |
|
to O |
|
hydroxyproline B-residue_name |
|
( O |
|
Hyp B-residue_name |
|
; O |
|
Figure O |
|
1A 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 |
|
|
|
no O |
|
detectable O |
|
binding O |
|
). O |
|
( O |
|
E O |
|
) O |
|
Structural B-experimental_method |
|
superposition I-experimental_method |
|
of O |
|
the O |
|
active B-protein_state |
|
IDA B-protein |
|
( O |
|
in O |
|
bonds O |
|
representation O |
|
, O |
|
in O |
|
gray O |
|
) O |
|
and O |
|
IDL1 B-chemical |
|
peptide I-chemical |
|
( O |
|
in O |
|
yellow O |
|
) O |
|
hormones O |
|
bound B-protein_state |
|
to I-protein_state |
|
the O |
|
HAESA B-protein |
|
ectodomain B-structure_element |
|
. O |
|
|
|
Petal O |
|
break O |
|
- O |
|
strength O |
|
was O |
|
found O |
|
significantly O |
|
increased O |
|
in O |
|
almost O |
|
all O |
|
positions O |
|
( O |
|
indicated O |
|
with O |
|
a O |
|
*) O |
|
for O |
|
haesa B-gene |
|
/ O |
|
hsl2 B-gene |
|
and O |
|
serk1 B-gene |
|
- I-gene |
|
1 I-gene |
|
mutant B-protein_state |
|
plants B-taxonomy_domain |
|
with O |
|
respect O |
|
to O |
|
the O |
|
Col O |
|
- O |
|
0 O |
|
control O |
|
. O |
|
|
|
( O |
|
B O |
|
) O |
|
Analytical B-experimental_method |
|
size I-experimental_method |
|
- I-experimental_method |
|
exclusion I-experimental_method |
|
chromatography I-experimental_method |
|
. O |
|
|
|
The O |
|
central O |
|
Hyp64IDA B-residue_name_number |
|
is O |
|
buried O |
|
in O |
|
a O |
|
specific O |
|
pocket B-site |
|
formed O |
|
by O |
|
HAESA B-protein |
|
LRRs B-structure_element |
|
8 I-structure_element |
|
– I-structure_element |
|
10 I-structure_element |
|
, O |
|
with O |
|
its O |
|
hydroxyl O |
|
group O |
|
establishing O |
|
hydrogen O |
|
bonds O |
|
with O |
|
the O |
|
strictly B-protein_state |
|
conserved I-protein_state |
|
Glu266HAESA B-residue_name_number |
|
and O |
|
with O |
|
a O |
|
water B-chemical |
|
molecule O |
|
, O |
|
which O |
|
in O |
|
turn O |
|
is O |
|
coordinated O |
|
by O |
|
the O |
|
main O |
|
chain O |
|
oxygens O |
|
of O |
|
Phe289HAESA B-residue_name_number |
|
and O |
|
Ser311HAESA B-residue_name_number |
|
( O |
|
Figure O |
|
1E O |
|
; O |
|
Figure O |
|
1 O |
|
— O |
|
figure O |
|
supplement O |
|
3 O |
|
). O |
|
|
|
In O |
|
this O |
|
structure B-evidence |
|
, O |
|
no O |
|
additional O |
|
electron B-evidence |
|
density I-evidence |
|
accounts O |
|
for O |
|
the O |
|
PKGV B-structure_element |
|
motif I-structure_element |
|
at O |
|
the O |
|
IDA B-protein |
|
N O |
|
- O |
|
terminus O |
|
( O |
|
Figure O |
|
2A O |
|
, O |
|
B O |
|
). O |
|
|
|
We O |
|
do O |
|
not O |
|
detect O |
|
interaction O |
|
between O |
|
HAESA B-protein |
|
and O |
|
a O |
|
synthetic B-protein_state |
|
peptide B-chemical |
|
missing B-protein_state |
|
the I-protein_state |
|
C I-protein_state |
|
- I-protein_state |
|
terminal I-protein_state |
|
Asn69IDA B-residue_name_number |
|
( O |
|
ΔN69 B-mutant |
|
), O |
|
highlighting O |
|
the O |
|
importance O |
|
of O |
|
the O |
|
polar O |
|
interactions O |
|
between O |
|
the O |
|
IDA B-protein |
|
carboxy O |
|
- O |
|
terminus O |
|
and O |
|
Arg407HAESA B-residue_name_number |
|
/ O |
|
Arg409HAESA B-residue_name_number |
|
( O |
|
Figures O |
|
1F O |
|
, O |
|
2D O |
|
). O |
|
|
|
The O |
|
co B-protein_type |
|
- I-protein_type |
|
receptor I-protein_type |
|
kinase I-protein_type |
|
SERK1 B-protein |
|
allows O |
|
for O |
|
high O |
|
- O |
|
affinity O |
|
IDA O |
|
sensing O |
|
|
|
We O |
|
found O |
|
that O |
|
the O |
|
force O |
|
required O |
|
to O |
|
remove O |
|
the O |
|
petals O |
|
of O |
|
serk1 B-gene |
|
- I-gene |
|
1 I-gene |
|
mutants B-protein_state |
|
is O |
|
significantly O |
|
higher O |
|
than O |
|
that O |
|
needed O |
|
for O |
|
wild B-protein_state |
|
- I-protein_state |
|
type I-protein_state |
|
plants B-taxonomy_domain |
|
, O |
|
as O |
|
previously O |
|
observed O |
|
for O |
|
haesa B-gene |
|
/ O |
|
hsl2 B-gene |
|
mutants B-protein_state |
|
, O |
|
and O |
|
that O |
|
floral O |
|
abscission O |
|
is O |
|
delayed O |
|
in O |
|
serk1 B-gene |
|
- I-gene |
|
1 I-gene |
|
( O |
|
Figure O |
|
3A O |
|
). O |
|
|
|
In O |
|
vitro O |
|
, O |
|
the O |
|
LRR B-structure_element |
|
ectodomain I-structure_element |
|
of O |
|
SERK1 B-protein |
|
( O |
|
residues O |
|
24 B-residue_range |
|
– I-residue_range |
|
213 I-residue_range |
|
) O |
|
forms O |
|
stable B-protein_state |
|
, O |
|
IDA B-protein_state |
|
- I-protein_state |
|
dependent I-protein_state |
|
heterodimeric B-oligomeric_state |
|
complexes B-protein_state |
|
with I-protein_state |
|
HAESA B-protein |
|
in O |
|
size B-experimental_method |
|
exclusion I-experimental_method |
|
chromatography I-experimental_method |
|
experiments O |
|
( O |
|
Figure O |
|
3B O |
|
). O |
|
|
|
We O |
|
next O |
|
titrated B-experimental_method |
|
SERK1 B-protein |
|
into O |
|
a O |
|
solution O |
|
containing O |
|
only O |
|
the O |
|
HAESA B-protein |
|
ectodomain B-structure_element |
|
. O |
|
|
|
In O |
|
this O |
|
case O |
|
, O |
|
there O |
|
was O |
|
no O |
|
detectable O |
|
interaction O |
|
between O |
|
receptor O |
|
and O |
|
co O |
|
- O |
|
receptor O |
|
, O |
|
while O |
|
in O |
|
the O |
|
presence B-protein_state |
|
of I-protein_state |
|
IDA B-protein |
|
, O |
|
SERK1 B-protein |
|
strongly O |
|
binds O |
|
HAESA B-protein |
|
with O |
|
a O |
|
dissociation B-evidence |
|
constant I-evidence |
|
in O |
|
the O |
|
mid O |
|
- O |
|
nanomolar O |
|
range O |
|
( O |
|
Figure O |
|
3C O |
|
). O |
|
|
|
Upon O |
|
IDA B-protein |
|
binding O |
|
at O |
|
the O |
|
cell O |
|
surface O |
|
, O |
|
the O |
|
kinase B-structure_element |
|
domains I-structure_element |
|
of O |
|
HAESA B-protein |
|
and O |
|
SERK1 B-protein |
|
, O |
|
which O |
|
have O |
|
been O |
|
shown O |
|
to O |
|
be O |
|
active B-protein_state |
|
protein B-protein_type |
|
kinases I-protein_type |
|
, O |
|
may O |
|
interact O |
|
in O |
|
the O |
|
cytoplasm O |
|
to O |
|
activate O |
|
each O |
|
other O |
|
. O |
|
|
|
Consistently O |
|
, O |
|
the O |
|
HAESA B-protein |
|
kinase B-structure_element |
|
domain I-structure_element |
|
can O |
|
transphosphorylate O |
|
SERK1 B-protein |
|
and O |
|
vice O |
|
versa O |
|
in O |
|
in O |
|
vitro O |
|
transphosphorylation B-experimental_method |
|
assays I-experimental_method |
|
( O |
|
Figure O |
|
3E O |
|
). O |
|
|
|
Crystal B-evidence |
|
structure I-evidence |
|
of O |
|
a O |
|
HAESA B-complex_assembly |
|
– I-complex_assembly |
|
IDA I-complex_assembly |
|
– I-complex_assembly |
|
SERK1 I-complex_assembly |
|
signaling O |
|
complex O |
|
. O |
|
|
|
( O |
|
A O |
|
) O |
|
Overview O |
|
of O |
|
the O |
|
ternary O |
|
complex O |
|
with O |
|
HAESA B-protein |
|
in O |
|
blue O |
|
( O |
|
surface O |
|
representation O |
|
), O |
|
IDA B-protein |
|
in O |
|
yellow O |
|
( O |
|
bonds O |
|
representation O |
|
) O |
|
and O |
|
SERK1 B-protein |
|
in O |
|
orange O |
|
( O |
|
surface O |
|
view O |
|
). O |
|
( O |
|
B O |
|
) O |
|
The O |
|
HAESA B-protein |
|
ectodomain B-structure_element |
|
undergoes O |
|
a O |
|
conformational O |
|
change O |
|
upon O |
|
SERK1 B-protein |
|
co O |
|
- O |
|
receptor O |
|
binding O |
|
. O |
|
|
|
Polar O |
|
contacts O |
|
of O |
|
SERK1 B-protein |
|
with O |
|
IDA B-protein |
|
are O |
|
shown O |
|
in O |
|
magenta O |
|
, O |
|
with O |
|
the O |
|
HAESA B-protein |
|
LRR B-structure_element |
|
domain I-structure_element |
|
in O |
|
gray O |
|
. O |
|
( O |
|
D O |
|
) O |
|
Details O |
|
of O |
|
the O |
|
zipper B-structure_element |
|
- I-structure_element |
|
like I-structure_element |
|
SERK1 B-site |
|
- I-site |
|
HAESA I-site |
|
interface I-site |
|
. 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 |
|
|
|
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 |
|
|
|
In O |
|
contrast O |
|
to O |
|
animal B-taxonomy_domain |
|
LRR B-protein_type |
|
receptors I-protein_type |
|
, O |
|
plant B-taxonomy_domain |
|
LRR B-structure_element |
|
- I-structure_element |
|
RKs I-structure_element |
|
harbor O |
|
spiral B-protein_state |
|
- I-protein_state |
|
shaped I-protein_state |
|
ectodomains B-structure_element |
|
and O |
|
thus O |
|
they O |
|
require O |
|
shape B-protein_state |
|
- I-protein_state |
|
complementary I-protein_state |
|
co B-protein_type |
|
- I-protein_type |
|
receptor I-protein_type |
|
proteins I-protein_type |
|
for O |
|
receptor O |
|
activation O |
|
. O |
|
|
|
However O |
|
our O |
|
results O |
|
show O |
|
that O |
|
SERK1 B-protein |
|
also O |
|
can O |
|
activate O |
|
this O |
|
process O |
|
upon O |
|
IDA B-protein |
|
sensing O |
|
, O |
|
indicating O |
|
that O |
|
SERKs B-protein_type |
|
may O |
|
fulfill O |
|
several O |
|
different O |
|
functions O |
|
in O |
|
the O |
|
course O |
|
of O |
|
the O |
|
abscission O |
|
process O |
|
. O |
|
|
|
The O |
|
central O |
|
Hyp B-residue_name |
|
residue O |
|
in O |
|
IDA B-protein |
|
is O |
|
found O |
|
buried O |
|
in O |
|
the O |
|
HAESA B-protein |
|
peptide B-site |
|
binding I-site |
|
surface I-site |
|
and O |
|
thus O |
|
this O |
|
post O |
|
- O |
|
translational O |
|
modification O |
|
may O |
|
regulate O |
|
IDA B-protein |
|
bioactivity O |
|
. O |
|
|
|
In O |
|
our O |
|
quantitative B-experimental_method |
|
biochemical I-experimental_method |
|
assays I-experimental_method |
|
, O |
|
the O |
|
presence B-protein_state |
|
of I-protein_state |
|
SERK1 B-protein |
|
dramatically O |
|
increases O |
|
the O |
|
HAESA B-protein |
|
binding O |
|
specificity O |
|
and O |
|
affinity O |
|
for O |
|
IDA B-protein |
|
. O |
|
|
|
A O |
|
ribbon O |
|
diagram O |
|
of O |
|
SERK1 B-protein |
|
in O |
|
the O |
|
same O |
|
orientation O |
|
is O |
|
shown O |
|
alongside 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 |
|
|
|
Different O |
|
plant B-taxonomy_domain |
|
peptide B-protein_type |
|
hormone I-protein_type |
|
families I-protein_type |
|
contain O |
|
a O |
|
C O |
|
- O |
|
terminal O |
|
( B-structure_element |
|
Arg I-structure_element |
|
)- I-structure_element |
|
His I-structure_element |
|
- I-structure_element |
|
Asn I-structure_element |
|
motif I-structure_element |
|
, O |
|
which O |
|
in O |
|
IDA B-protein |
|
represents O |
|
the O |
|
co B-site |
|
- I-site |
|
receptor I-site |
|
recognition I-site |
|
site I-site |
|
. 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 |
|
|
|
Among O |
|
these O |
|
are O |
|
the O |
|
CLE B-chemical |
|
peptides I-chemical |
|
regulating O |
|
stem O |
|
cell O |
|
maintenance O |
|
in O |
|
the O |
|
shoot O |
|
and O |
|
the O |
|
root O |
|
. O |
|
|
|
The O |
|
crotonyllysine B-residue_name |
|
mark O |
|
on O |
|
histone B-protein_type |
|
H3K18 B-protein_type |
|
is O |
|
produced O |
|
by O |
|
p300 B-protein |
|
, O |
|
a O |
|
histone B-protein_type |
|
acetyltransferase I-protein_type |
|
also O |
|
responsible O |
|
for O |
|
acetylation B-ptm |
|
of O |
|
histones O |
|
. O |
|
|
|
The O |
|
family O |
|
of O |
|
acetyllysine B-residue_name |
|
readers O |
|
has O |
|
been O |
|
expanded O |
|
with O |
|
the O |
|
discovery O |
|
that O |
|
the O |
|
YEATS B-structure_element |
|
( O |
|
Yaf9 B-protein |
|
, O |
|
ENL B-protein |
|
, O |
|
AF9 B-protein |
|
, O |
|
Taf14 B-protein |
|
, O |
|
Sas5 B-protein |
|
) O |
|
domains O |
|
of O |
|
human B-species |
|
AF9 B-protein |
|
and O |
|
yeast B-taxonomy_domain |
|
Taf14 B-protein |
|
are O |
|
capable O |
|
of O |
|
recognizing O |
|
the O |
|
histone B-protein_type |
|
mark O |
|
H3K9ac B-protein_type |
|
. O |
|
|
|
Similarly O |
|
, O |
|
activation O |
|
of O |
|
a O |
|
subset O |
|
of O |
|
genes O |
|
and O |
|
DNA O |
|
damage O |
|
repair O |
|
in O |
|
yeast B-taxonomy_domain |
|
require O |
|
the O |
|
acetyllysine B-residue_name |
|
binding O |
|
activity O |
|
of O |
|
the O |
|
Taf14 B-protein |
|
YEATS B-structure_element |
|
domain I-structure_element |
|
. O |
|
|
|
However O |
|
, O |
|
Taf14 B-protein |
|
is O |
|
also O |
|
found O |
|
in O |
|
a O |
|
number O |
|
of O |
|
chromatin O |
|
- O |
|
remodeling O |
|
complexes O |
|
( O |
|
i O |
|
. O |
|
e O |
|
., O |
|
INO80 B-complex_assembly |
|
, O |
|
SWI B-complex_assembly |
|
/ I-complex_assembly |
|
SNF I-complex_assembly |
|
and O |
|
RSC B-complex_assembly |
|
) O |
|
and O |
|
the O |
|
histone B-protein_type |
|
acetyltransferase I-protein_type |
|
complex O |
|
NuA3 B-complex_assembly |
|
, O |
|
indicating O |
|
a O |
|
multifaceted O |
|
role O |
|
of O |
|
Taf14 B-protein |
|
in O |
|
transcriptional O |
|
regulation O |
|
and O |
|
chromatin O |
|
biology O |
|
. O |
|
|
|
We O |
|
found O |
|
that O |
|
H3K9cr B-protein_type |
|
is O |
|
present O |
|
in O |
|
yeast B-taxonomy_domain |
|
and O |
|
is O |
|
dynamically O |
|
regulated O |
|
. O |
|
|
|
This O |
|
distinctive O |
|
mechanism O |
|
was O |
|
corroborated O |
|
through O |
|
mapping O |
|
the O |
|
Taf14 B-protein |
|
YEATS B-site |
|
- I-site |
|
H3K9cr I-site |
|
binding I-site |
|
interface I-site |
|
in O |
|
solution O |
|
using O |
|
NMR B-experimental_method |
|
chemical I-experimental_method |
|
shift I-experimental_method |
|
perturbation I-experimental_method |
|
analysis I-experimental_method |
|
( O |
|
Supplementary O |
|
Fig O |
|
. O |
|
2a O |
|
, O |
|
b O |
|
). O |
|
|
|
Binding O |
|
of O |
|
the O |
|
Taf14 B-protein |
|
YEATS B-structure_element |
|
domain I-structure_element |
|
to O |
|
H3K9cr B-protein_type |
|
is O |
|
robust O |
|
. O |
|
|
|
We O |
|
concluded O |
|
that O |
|
H3K9cr B-protein_type |
|
is O |
|
the O |
|
preferred O |
|
target O |
|
of O |
|
this O |
|
domain O |
|
. O |
|
|
|
However O |
|
, O |
|
bromodomains B-structure_element |
|
did O |
|
not O |
|
interact O |
|
( O |
|
or O |
|
associated O |
|
very O |
|
weakly O |
|
) O |
|
with O |
|
longer O |
|
acyl O |
|
modifications O |
|
, O |
|
including O |
|
crotonyllysine B-residue_name |
|
, O |
|
as O |
|
in O |
|
the O |
|
case O |
|
of O |
|
BDs B-structure_element |
|
of O |
|
TAF1 B-protein |
|
and O |
|
BRD2 B-protein |
|
, O |
|
supporting O |
|
recent O |
|
reports O |
|
. O |
|
|
|
As O |
|
we O |
|
previously O |
|
showed O |
|
the O |
|
importance O |
|
of O |
|
acyllysine B-residue_name |
|
binding O |
|
by O |
|
the O |
|
Taf14 B-protein |
|
YEATS B-structure_element |
|
domain I-structure_element |
|
for O |
|
the O |
|
DNA O |
|
damage O |
|
response O |
|
and O |
|
gene O |
|
transcription O |
|
, O |
|
it O |
|
will O |
|
be O |
|
essential O |
|
in O |
|
the O |
|
future O |
|
to O |
|
define O |
|
the O |
|
physiological O |
|
role O |
|
of O |
|
crotonyllysine B-residue_name |
|
recognition O |
|
and O |
|
to O |
|
differentiate O |
|
the O |
|
activities O |
|
of O |
|
Taf14 B-protein |
|
that O |
|
are O |
|
due O |
|
to O |
|
binding O |
|
to O |
|
crotonyllysine B-residue_name |
|
and O |
|
acetyllysine B-residue_name |
|
modifications O |
|
. O |
|
|
|
Furthermore O |
|
, O |
|
the O |
|
functional O |
|
significance O |
|
of O |
|
crotonyllysine B-residue_name |
|
recognition O |
|
by O |
|
other O |
|
YEATS B-protein_type |
|
proteins O |
|
will O |
|
be O |
|
of O |
|
great O |
|
importance O |
|
to O |
|
elucidate O |
|
and O |
|
compare O |
|
. O |
|
|
|
The O |
|
structural O |
|
mechanism O |
|
for O |
|
the O |
|
recognition O |
|
of O |
|
H3K9cr B-protein_type |
|
|
|
Total O |
|
H3 B-protein_type |
|
was O |
|
used O |
|
as O |
|
a O |
|
loading O |
|
control O |
|
. O |
|
|
|
( O |
|
c O |
|
) O |
|
Superimposed O |
|
1H B-experimental_method |
|
, I-experimental_method |
|
15N I-experimental_method |
|
HSQC I-experimental_method |
|
spectra B-evidence |
|
of O |
|
Taf14 B-protein |
|
YEATS B-structure_element |
|
recorded O |
|
as O |
|
H3K9cr5 B-chemical |
|
- I-chemical |
|
13 I-chemical |
|
and O |
|
H3K9ac5 B-chemical |
|
- I-chemical |
|
13 I-chemical |
|
peptides O |
|
were O |
|
titrated B-experimental_method |
|
in O |
|
. O |
|
|
|
Although O |
|
a O |
|
Thr1Ser B-mutant |
|
mutant B-protein_state |
|
is O |
|
active B-protein_state |
|
, O |
|
it O |
|
is O |
|
less O |
|
efficient O |
|
compared O |
|
with O |
|
wild B-protein_state |
|
type I-protein_state |
|
because O |
|
of O |
|
the O |
|
unfavourable O |
|
orientation O |
|
of O |
|
Ser1 B-residue_name_number |
|
towards O |
|
incoming O |
|
substrates O |
|
. O |
|
|
|
The O |
|
proteasome B-complex_assembly |
|
, O |
|
an O |
|
essential O |
|
molecular O |
|
machine O |
|
, O |
|
is O |
|
a O |
|
threonine B-protein_type |
|
protease I-protein_type |
|
, O |
|
but O |
|
the O |
|
evolution O |
|
and O |
|
the O |
|
components O |
|
of O |
|
its O |
|
proteolytic O |
|
centre O |
|
are O |
|
unclear 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 |
|
|
|
These O |
|
results O |
|
indicate O |
|
that O |
|
the O |
|
β1 B-protein |
|
and O |
|
β2 B-protein |
|
proteolytic O |
|
activities O |
|
are O |
|
not O |
|
essential O |
|
for O |
|
cell O |
|
survival O |
|
. O |
|
|
|
Our O |
|
present O |
|
crystallographic B-experimental_method |
|
analysis I-experimental_method |
|
of O |
|
the O |
|
β5 B-mutant |
|
- I-mutant |
|
T1A I-mutant |
|
pp B-chemical |
|
trans B-protein_state |
|
mutant B-protein_state |
|
demonstrates O |
|
that O |
|
the O |
|
mutation B-experimental_method |
|
per O |
|
se O |
|
does O |
|
not O |
|
structurally O |
|
alter O |
|
the O |
|
catalytic B-site |
|
active I-site |
|
site I-site |
|
and O |
|
that O |
|
the O |
|
trans B-experimental_method |
|
- I-experimental_method |
|
expressed I-experimental_method |
|
β5 B-protein |
|
propeptide B-structure_element |
|
is O |
|
not B-protein_state |
|
bound I-protein_state |
|
in O |
|
the O |
|
β5 B-protein |
|
substrate B-site |
|
- I-site |
|
binding I-site |
|
channel I-site |
|
( O |
|
Supplementary O |
|
Fig O |
|
. O |
|
1a O |
|
). O |
|
|
|
Sequencing B-experimental_method |
|
of I-experimental_method |
|
the I-experimental_method |
|
plasmids I-experimental_method |
|
, O |
|
testing O |
|
them O |
|
in O |
|
both O |
|
published O |
|
yeast B-taxonomy_domain |
|
strain O |
|
backgrounds O |
|
and O |
|
site B-experimental_method |
|
- I-experimental_method |
|
directed I-experimental_method |
|
mutagenesis I-experimental_method |
|
revealed O |
|
that O |
|
the O |
|
β5 B-mutant |
|
- I-mutant |
|
T1A I-mutant |
|
mutant B-protein_state |
|
pp B-chemical |
|
cis B-protein_state |
|
is O |
|
viable O |
|
, O |
|
but O |
|
suffers O |
|
from O |
|
a O |
|
marked O |
|
growth O |
|
defect O |
|
that O |
|
requires O |
|
extended O |
|
incubation O |
|
of O |
|
4 O |
|
– O |
|
5 O |
|
days O |
|
for O |
|
initial O |
|
colony O |
|
formation O |
|
( O |
|
Table O |
|
1 O |
|
and O |
|
Supplementary O |
|
Methods O |
|
). O |
|
|
|
In O |
|
subunit O |
|
β1 B-protein |
|
, O |
|
we O |
|
found O |
|
that O |
|
Gly B-residue_name_number |
|
(- I-residue_name_number |
|
1 I-residue_name_number |
|
) I-residue_name_number |
|
indeed O |
|
forms O |
|
a O |
|
sharp B-structure_element |
|
turn I-structure_element |
|
, O |
|
which O |
|
relaxes O |
|
on O |
|
prosegment B-ptm |
|
cleavage I-ptm |
|
( O |
|
Fig O |
|
. O |
|
1a O |
|
and O |
|
Supplementary O |
|
Fig O |
|
. O |
|
2a O |
|
). O |
|
|
|
Regarding O |
|
the O |
|
β2 B-protein |
|
propeptide B-structure_element |
|
, O |
|
Thr B-residue_name_number |
|
(- I-residue_name_number |
|
2 I-residue_name_number |
|
) I-residue_name_number |
|
occupies O |
|
the O |
|
S1 B-site |
|
pocket I-site |
|
but O |
|
is O |
|
less O |
|
deeply O |
|
anchored O |
|
compared O |
|
with O |
|
Leu B-residue_name_number |
|
(- I-residue_name_number |
|
2 I-residue_name_number |
|
) I-residue_name_number |
|
in O |
|
β1 B-protein |
|
, O |
|
which O |
|
might O |
|
be O |
|
due O |
|
to O |
|
the O |
|
rather O |
|
large O |
|
β2 B-protein |
|
- O |
|
S1 B-site |
|
pocket I-site |
|
created O |
|
by O |
|
Gly45 B-residue_name_number |
|
. O |
|
|
|
Nevertheless O |
|
, O |
|
both O |
|
Leu B-residue_name_number |
|
(- I-residue_name_number |
|
2 I-residue_name_number |
|
) I-residue_name_number |
|
and O |
|
Thr B-residue_name_number |
|
(- I-residue_name_number |
|
2 I-residue_name_number |
|
) I-residue_name_number |
|
were O |
|
found O |
|
to O |
|
occupy O |
|
the O |
|
S1 B-site |
|
specificity I-site |
|
pocket I-site |
|
formed O |
|
by O |
|
Met45 B-residue_name_number |
|
( O |
|
Fig O |
|
. O |
|
2a O |
|
, O |
|
b O |
|
and O |
|
Supplementary O |
|
Fig O |
|
. O |
|
4f O |
|
– O |
|
h O |
|
). O |
|
|
|
Bearing O |
|
in O |
|
mind O |
|
that O |
|
in O |
|
contrast O |
|
to O |
|
Thr B-residue_name_number |
|
(- I-residue_name_number |
|
2 I-residue_name_number |
|
) I-residue_name_number |
|
in O |
|
β2 B-protein |
|
, O |
|
Leu B-residue_name_number |
|
(- I-residue_name_number |
|
2 I-residue_name_number |
|
) I-residue_name_number |
|
in O |
|
subunit O |
|
β1 B-protein |
|
is O |
|
not B-protein_state |
|
conserved I-protein_state |
|
among O |
|
species O |
|
( O |
|
Supplementary O |
|
Fig O |
|
. O |
|
3a O |
|
), O |
|
we O |
|
created B-experimental_method |
|
a O |
|
β2 B-mutant |
|
- I-mutant |
|
T I-mutant |
|
(- I-mutant |
|
2 I-mutant |
|
) I-mutant |
|
V I-mutant |
|
proteasome B-complex_assembly |
|
mutant B-protein_state |
|
. O |
|
|
|
Notably O |
|
, O |
|
the O |
|
2FO B-evidence |
|
– I-evidence |
|
FC I-evidence |
|
electron I-evidence |
|
- I-evidence |
|
density I-evidence |
|
map I-evidence |
|
displays O |
|
a O |
|
different O |
|
orientation O |
|
for O |
|
the O |
|
β2 B-protein |
|
propeptide B-structure_element |
|
than O |
|
has O |
|
been O |
|
observed O |
|
for O |
|
the O |
|
β2 B-mutant |
|
- I-mutant |
|
T1A I-mutant |
|
proteasome B-complex_assembly |
|
. O |
|
|
|
The O |
|
active B-site |
|
site I-site |
|
of O |
|
the O |
|
proteasome B-complex_assembly |
|
|
|
Instead O |
|
, O |
|
Lys33NH2 B-residue_name_number |
|
, O |
|
which O |
|
is O |
|
in O |
|
hydrogen O |
|
- O |
|
bonding O |
|
distance O |
|
to O |
|
Thr1Oγ B-residue_name_number |
|
( O |
|
2 O |
|
. O |
|
7 O |
|
Å O |
|
) O |
|
in O |
|
all O |
|
catalytically B-protein_state |
|
active I-protein_state |
|
β B-protein |
|
subunits I-protein |
|
( O |
|
Fig O |
|
. O |
|
3a O |
|
, O |
|
b O |
|
), O |
|
was O |
|
proposed O |
|
to O |
|
serve O |
|
as O |
|
the O |
|
proton O |
|
acceptor O |
|
. O |
|
|
|
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 |
|
|
|
Strikingly O |
|
, O |
|
although O |
|
the O |
|
X B-evidence |
|
- I-evidence |
|
ray I-evidence |
|
data I-evidence |
|
on O |
|
the O |
|
β5 B-mutant |
|
- I-mutant |
|
D17N I-mutant |
|
mutant B-protein_state |
|
with O |
|
the O |
|
propeptide B-structure_element |
|
expressed B-experimental_method |
|
in O |
|
cis B-protein_state |
|
and O |
|
in O |
|
trans B-protein_state |
|
looked O |
|
similar O |
|
, O |
|
there O |
|
was O |
|
a O |
|
pronounced O |
|
difference O |
|
in O |
|
their O |
|
growth O |
|
phenotypes O |
|
observed O |
|
( O |
|
Supplementary O |
|
Fig O |
|
. O |
|
6a O |
|
and O |
|
Supplementary O |
|
Fig O |
|
. O |
|
7b O |
|
). O |
|
|
|
Whereas O |
|
Asn B-residue_name |
|
can O |
|
to O |
|
some O |
|
degree O |
|
replace O |
|
Asp166 B-residue_name_number |
|
due O |
|
to O |
|
its O |
|
carbonyl O |
|
group O |
|
in O |
|
the O |
|
side O |
|
chain O |
|
, O |
|
Ala B-residue_name |
|
at O |
|
this O |
|
position O |
|
was O |
|
found O |
|
to O |
|
prevent O |
|
both O |
|
autolysis B-ptm |
|
and O |
|
catalysis O |
|
. O |
|
|
|
His B-residue_name_number |
|
(- I-residue_name_number |
|
2 I-residue_name_number |
|
) I-residue_name_number |
|
occupies O |
|
the O |
|
S2 B-site |
|
pocket I-site |
|
like 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 |
|
but O |
|
in O |
|
contrast O |
|
to O |
|
the O |
|
latter O |
|
, O |
|
the O |
|
propeptide B-structure_element |
|
in O |
|
the O |
|
T1C B-mutant |
|
mutant B-protein_state |
|
adopts O |
|
an O |
|
antiparallel B-structure_element |
|
β I-structure_element |
|
- I-structure_element |
|
sheet I-structure_element |
|
conformation O |
|
as O |
|
known O |
|
from O |
|
inhibitors O |
|
like O |
|
MG132 B-chemical |
|
( O |
|
Fig O |
|
. O |
|
4c O |
|
– O |
|
e O |
|
and O |
|
Supplementary O |
|
Fig O |
|
. O |
|
9b O |
|
). O |
|
|
|
Activity B-experimental_method |
|
assays I-experimental_method |
|
with O |
|
the O |
|
β5 B-protein |
|
- O |
|
specific O |
|
substrate O |
|
Suc B-chemical |
|
- I-chemical |
|
LLVY I-chemical |
|
- I-chemical |
|
AMC I-chemical |
|
demonstrated O |
|
that O |
|
the O |
|
ChT O |
|
- O |
|
L O |
|
activity O |
|
of O |
|
the O |
|
T1S B-mutant |
|
mutant B-protein_state |
|
is O |
|
reduced O |
|
by O |
|
40 O |
|
– O |
|
45 O |
|
% O |
|
compared O |
|
with O |
|
WT B-protein_state |
|
proteasomes B-complex_assembly |
|
depending O |
|
on O |
|
the O |
|
incubation O |
|
temperature O |
|
( O |
|
Fig O |
|
. O |
|
4b O |
|
and O |
|
Supplementary O |
|
Fig O |
|
. O |
|
9c O |
|
). O |
|
|
|
In O |
|
vitro O |
|
, O |
|
the O |
|
mutant B-protein_state |
|
proteasome B-complex_assembly |
|
is O |
|
less O |
|
susceptible O |
|
to O |
|
proteasome B-complex_assembly |
|
inhibition O |
|
by O |
|
bortezomib B-chemical |
|
( O |
|
3 O |
|
. O |
|
7 O |
|
- O |
|
fold O |
|
) O |
|
and O |
|
carfilzomib B-chemical |
|
( O |
|
1 O |
|
. O |
|
8 O |
|
- O |
|
fold O |
|
; O |
|
Fig O |
|
. O |
|
5 O |
|
). O |
|
|
|
The O |
|
20S B-complex_assembly |
|
proteasome I-complex_assembly |
|
CP B-complex_assembly |
|
is O |
|
the O |
|
major O |
|
non B-protein_type |
|
- I-protein_type |
|
lysosomal I-protein_type |
|
protease I-protein_type |
|
in O |
|
eukaryotic B-taxonomy_domain |
|
cells O |
|
, O |
|
and O |
|
its O |
|
assembly O |
|
is O |
|
highly O |
|
organized O |
|
. O |
|
|
|
The O |
|
β B-protein |
|
- I-protein |
|
subunit I-protein |
|
propeptides B-structure_element |
|
, O |
|
particularly O |
|
that O |
|
of O |
|
β5 B-protein |
|
, O |
|
are O |
|
key O |
|
factors O |
|
that O |
|
help O |
|
drive O |
|
proper O |
|
assembly O |
|
of O |
|
the O |
|
CP B-complex_assembly |
|
complex O |
|
. O |
|
|
|
We O |
|
propose O |
|
a O |
|
catalytic B-site |
|
triad I-site |
|
for O |
|
the O |
|
active B-site |
|
site I-site |
|
of O |
|
the O |
|
CP B-complex_assembly |
|
consisting O |
|
of O |
|
residues O |
|
Thr1 B-residue_name_number |
|
, O |
|
Lys33 B-residue_name_number |
|
and O |
|
Asp B-residue_name |
|
/ O |
|
Glu17 B-residue_name_number |
|
, O |
|
which O |
|
are O |
|
conserved O |
|
among O |
|
all O |
|
proteolytically O |
|
active O |
|
eukaryotic B-taxonomy_domain |
|
, O |
|
bacterial B-taxonomy_domain |
|
and O |
|
archaeal B-taxonomy_domain |
|
proteasome B-complex_assembly |
|
subunits O |
|
. O |
|
|
|
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 |
|
|
|
In O |
|
agreement O |
|
, O |
|
acetylation B-ptm |
|
of O |
|
the O |
|
Thr1 B-residue_name_number |
|
N O |
|
terminus O |
|
irreversibly O |
|
blocks O |
|
hydrolytic O |
|
activity O |
|
, O |
|
and O |
|
binding O |
|
of O |
|
substrates O |
|
is O |
|
prevented O |
|
for O |
|
steric O |
|
reasons O |
|
. O |
|
|
|
This O |
|
interpretation O |
|
agrees O |
|
with O |
|
the O |
|
strongly O |
|
reduced O |
|
catalytic O |
|
activity O |
|
of O |
|
the O |
|
β5 B-mutant |
|
- I-mutant |
|
D166N I-mutant |
|
mutant B-protein_state |
|
on O |
|
the O |
|
one O |
|
hand O |
|
, O |
|
and O |
|
the O |
|
ability O |
|
to O |
|
react O |
|
readily O |
|
with O |
|
carfilzomib B-chemical |
|
on O |
|
the O |
|
other O |
|
. O |
|
|
|
We O |
|
also O |
|
observed O |
|
slightly O |
|
lower O |
|
affinity O |
|
of O |
|
the O |
|
β5 B-mutant |
|
- I-mutant |
|
T1S I-mutant |
|
mutant B-protein_state |
|
yCP B-complex_assembly |
|
for O |
|
the O |
|
Food O |
|
and O |
|
Drug O |
|
Administration O |
|
- O |
|
approved O |
|
proteasome B-complex_assembly |
|
inhibitors O |
|
bortezomib B-chemical |
|
and O |
|
carfilzomib B-chemical |
|
. O |
|
|
|
In O |
|
contrast O |
|
to O |
|
Thr1 B-residue_name_number |
|
, O |
|
the O |
|
hydroxyl O |
|
group O |
|
of O |
|
Ser1 B-residue_name_number |
|
occupies O |
|
the O |
|
position O |
|
of O |
|
the O |
|
Thr1 B-residue_name_number |
|
methyl O |
|
side O |
|
chain O |
|
in O |
|
the O |
|
WT B-protein_state |
|
enzyme B-complex_assembly |
|
, O |
|
which O |
|
requires O |
|
its O |
|
reorientation O |
|
relative O |
|
to O |
|
the O |
|
substrate O |
|
to O |
|
allow O |
|
cleavage O |
|
( O |
|
Fig O |
|
. O |
|
4g O |
|
, O |
|
h O |
|
). O |
|
|
|
Architecture O |
|
and O |
|
proposed O |
|
reaction O |
|
mechanism O |
|
of O |
|
the O |
|
proteasomal O |
|
active B-site |
|
site I-site |
|
. O |
|
|
|
Thr1OH B-residue_name_number |
|
is O |
|
hydrogen O |
|
- O |
|
bonded O |
|
to O |
|
Lys33NH2 B-residue_name_number |
|
( O |
|
2 O |
|
. O |
|
7 O |
|
Å O |
|
), O |
|
which O |
|
in O |
|
turn O |
|
interacts O |
|
with O |
|
Asp17Oδ B-residue_name_number |
|
. O |
|
|
|
Autolysis B-ptm |
|
( O |
|
left O |
|
set O |
|
of O |
|
structures O |
|
) O |
|
is O |
|
initiated O |
|
by O |
|
deprotonation O |
|
of O |
|
Thr1OH B-residue_name_number |
|
via O |
|
Lys33NH2 B-residue_name_number |
|
and O |
|
the O |
|
formation O |
|
of O |
|
a O |
|
tetrahedral O |
|
transition O |
|
state O |
|
. O |
|
|
|
Next O |
|
, O |
|
Thr1NH2 B-residue_name_number |
|
polarizes O |
|
a O |
|
water B-chemical |
|
molecule O |
|
for O |
|
the O |
|
nucleophilic O |
|
attack O |
|
of O |
|
the O |
|
acyl O |
|
- O |
|
enzyme O |
|
intermediate O |
|
. O |
|
|
|
( O |
|
c O |
|
) O |
|
Illustration O |
|
of O |
|
the O |
|
2FO B-evidence |
|
– I-evidence |
|
FC I-evidence |
|
electron I-evidence |
|
- I-evidence |
|
density I-evidence |
|
map I-evidence |
|
( O |
|
blue O |
|
mesh O |
|
contoured O |
|
at O |
|
1σ O |
|
) O |
|
for O |
|
the O |
|
β5 B-mutant |
|
- I-mutant |
|
T1C I-mutant |
|
propeptide B-structure_element |
|
fragment O |
|
. O |
|
|
|
( O |
|
h O |
|
) O |
|
The O |
|
methyl O |
|
group O |
|
of O |
|
Thr1 B-residue_name_number |
|
is O |
|
anchored O |
|
by O |
|
hydrophobic O |
|
interactions O |
|
with O |
|
Ala46Cβ B-residue_name_number |
|
and O |
|
Thr3Cγ B-residue_name_number |
|
. O |
|
|
|
|