|
Structure B-evidence |
|
of O |
|
the O |
|
Dual O |
|
- O |
|
Mode O |
|
Wnt B-protein_type |
|
Regulator O |
|
Kremen1 B-protein |
|
and O |
|
Insight O |
|
into O |
|
Ternary O |
|
Complex O |
|
Formation O |
|
with O |
|
LRP6 B-protein |
|
and O |
|
Dickkopf B-protein_type |
|
|
|
Kremen B-protein_type |
|
1 I-protein_type |
|
and I-protein_type |
|
2 I-protein_type |
|
have O |
|
been O |
|
identified O |
|
as O |
|
co B-protein_type |
|
- I-protein_type |
|
receptors I-protein_type |
|
for O |
|
Dickkopf B-protein_type |
|
( O |
|
Dkk B-protein_type |
|
) O |
|
proteins O |
|
, O |
|
hallmark O |
|
secreted O |
|
antagonists O |
|
of O |
|
canonical O |
|
Wnt B-protein_type |
|
signaling O |
|
. O |
|
|
|
We O |
|
present O |
|
here O |
|
three O |
|
crystal B-evidence |
|
structures I-evidence |
|
of O |
|
the O |
|
ectodomain B-structure_element |
|
of O |
|
human B-species |
|
Kremen1 B-protein |
|
( O |
|
KRM1ECD B-protein |
|
) O |
|
at O |
|
resolutions O |
|
between O |
|
1 O |
|
. O |
|
9 O |
|
and O |
|
3 O |
|
. O |
|
2 O |
|
Å O |
|
. O |
|
KRM1ECD B-protein |
|
emerges O |
|
as O |
|
a O |
|
rigid O |
|
molecule O |
|
with O |
|
tight O |
|
interactions O |
|
stabilizing O |
|
a O |
|
triangular B-protein_state |
|
arrangement I-protein_state |
|
of O |
|
its O |
|
Kringle B-structure_element |
|
, O |
|
WSC B-structure_element |
|
, O |
|
and O |
|
CUB B-structure_element |
|
structural O |
|
domains O |
|
. O |
|
|
|
The O |
|
structures B-evidence |
|
reveal O |
|
an O |
|
unpredicted O |
|
homology O |
|
of O |
|
the O |
|
WSC B-structure_element |
|
domain O |
|
to O |
|
hepatocyte B-protein_type |
|
growth I-protein_type |
|
factor I-protein_type |
|
. O |
|
|
|
We O |
|
further O |
|
report O |
|
the O |
|
general O |
|
architecture O |
|
of O |
|
the O |
|
ternary O |
|
complex O |
|
formed O |
|
by O |
|
the O |
|
Wnt B-protein_type |
|
co B-protein_type |
|
- I-protein_type |
|
receptor I-protein_type |
|
Lrp5 B-protein_type |
|
/ I-protein_type |
|
6 I-protein_type |
|
, O |
|
Dkk B-protein_type |
|
, O |
|
and O |
|
Krm B-protein_type |
|
, O |
|
determined O |
|
from O |
|
a O |
|
low O |
|
- O |
|
resolution O |
|
complex O |
|
crystal B-evidence |
|
structure I-evidence |
|
between O |
|
β B-structure_element |
|
- I-structure_element |
|
propeller I-structure_element |
|
/ I-structure_element |
|
EGF I-structure_element |
|
repeats I-structure_element |
|
( I-structure_element |
|
PE I-structure_element |
|
) I-structure_element |
|
3 I-structure_element |
|
and I-structure_element |
|
4 I-structure_element |
|
of O |
|
the O |
|
Wnt B-protein_type |
|
co B-protein_type |
|
- I-protein_type |
|
receptor I-protein_type |
|
LRP6 B-protein |
|
( O |
|
LRP6PE3PE4 B-protein |
|
), O |
|
the O |
|
cysteine B-structure_element |
|
- I-structure_element |
|
rich I-structure_element |
|
domain I-structure_element |
|
2 I-structure_element |
|
( O |
|
CRD2 B-structure_element |
|
) O |
|
of O |
|
DKK1 B-protein |
|
, O |
|
and O |
|
KRM1ECD B-protein |
|
. O |
|
|
|
DKK1CRD2 B-protein |
|
is O |
|
sandwiched O |
|
between O |
|
LRP6PE3 B-protein |
|
and O |
|
KRM1Kringle B-protein |
|
- B-structure_element |
|
WSC I-structure_element |
|
. O |
|
|
|
Modeling B-experimental_method |
|
studies O |
|
supported O |
|
by O |
|
surface B-experimental_method |
|
plasmon I-experimental_method |
|
resonance I-experimental_method |
|
suggest O |
|
a O |
|
direct O |
|
interaction B-site |
|
site I-site |
|
between O |
|
Krm1CUB B-protein |
|
and O |
|
Lrp6PE2 B-protein |
|
. O |
|
|
|
The O |
|
structure B-evidence |
|
of O |
|
the O |
|
KREMEN B-protein |
|
1 I-protein |
|
ectodomain B-structure_element |
|
is O |
|
solved B-experimental_method |
|
from O |
|
three O |
|
crystal B-evidence |
|
forms I-evidence |
|
|
|
Kringle B-structure_element |
|
, O |
|
WSC B-structure_element |
|
, O |
|
and O |
|
CUB B-structure_element |
|
subdomains O |
|
interact O |
|
tightly O |
|
to O |
|
form O |
|
a O |
|
single O |
|
structural O |
|
unit O |
|
|
|
The O |
|
interface B-site |
|
to O |
|
DKKs B-protein_type |
|
is O |
|
formed O |
|
from O |
|
the O |
|
Kringle B-structure_element |
|
and O |
|
WSC B-structure_element |
|
domains O |
|
|
|
The O |
|
CUB B-structure_element |
|
domain O |
|
is O |
|
found O |
|
to O |
|
interact O |
|
directly O |
|
with O |
|
LRP6PE1PE2 B-protein |
|
|
|
Zebisch O |
|
et O |
|
al O |
|
. O |
|
describe O |
|
the O |
|
ectodomain B-structure_element |
|
structure B-evidence |
|
of O |
|
KREMEN B-protein |
|
1 I-protein |
|
, O |
|
a O |
|
receptor B-protein_type |
|
for O |
|
Wnt B-protein_type |
|
antagonists O |
|
of O |
|
the O |
|
DKK B-protein_type |
|
family O |
|
. O |
|
|
|
Apo B-protein_state |
|
structures B-evidence |
|
and O |
|
a O |
|
complex B-protein_state |
|
with I-protein_state |
|
functional B-protein_state |
|
fragments I-protein_state |
|
of O |
|
DKK1 B-protein |
|
and O |
|
LRP6 B-protein |
|
shed O |
|
light O |
|
on O |
|
the O |
|
function O |
|
of O |
|
this O |
|
dual O |
|
- O |
|
mode O |
|
regulator O |
|
of O |
|
Wnt B-protein_type |
|
signaling O |
|
. O |
|
|
|
Signaling O |
|
by O |
|
Wnt B-protein_type |
|
morphogens O |
|
is O |
|
renowned O |
|
for O |
|
its O |
|
fundamental O |
|
roles O |
|
in O |
|
embryonic O |
|
development O |
|
, O |
|
tissue O |
|
homeostasis O |
|
, O |
|
and O |
|
stem O |
|
cell O |
|
maintenance O |
|
. O |
|
|
|
Due O |
|
to O |
|
these O |
|
functions O |
|
, O |
|
generation O |
|
, O |
|
delivery O |
|
, O |
|
and O |
|
interpretation O |
|
of O |
|
Wnt B-protein_type |
|
signals O |
|
are O |
|
all O |
|
heavily O |
|
regulated O |
|
in O |
|
the O |
|
animal O |
|
body O |
|
. O |
|
|
|
Vertebrate B-taxonomy_domain |
|
Dickkopf B-protein_type |
|
proteins O |
|
( O |
|
Dkk1 B-protein_type |
|
, O |
|
2 B-protein_type |
|
, O |
|
and O |
|
4 B-protein_type |
|
) O |
|
are O |
|
one O |
|
of O |
|
many O |
|
secreted O |
|
antagonists O |
|
of O |
|
Wnt B-protein_type |
|
and O |
|
function O |
|
by O |
|
blocking O |
|
access O |
|
to O |
|
the O |
|
Wnt B-protein_type |
|
co B-protein_type |
|
- I-protein_type |
|
receptor I-protein_type |
|
LRP5 B-protein |
|
/ I-protein |
|
6 I-protein |
|
. O |
|
|
|
Kremen B-protein_type |
|
proteins O |
|
( O |
|
Krm1 B-protein_type |
|
and O |
|
Krm2 B-protein_type |
|
) O |
|
have O |
|
been O |
|
identified O |
|
as O |
|
additional O |
|
high O |
|
- O |
|
affinity O |
|
transmembrane B-protein_type |
|
receptors I-protein_type |
|
for O |
|
Dkk B-protein_type |
|
. O |
|
|
|
Krm B-protein_type |
|
and O |
|
Dkk B-protein_type |
|
synergize O |
|
in O |
|
Wnt B-protein_type |
|
inhibition O |
|
during O |
|
Xenopus B-taxonomy_domain |
|
embryogenesis O |
|
to O |
|
regulate O |
|
anterior O |
|
- O |
|
posterior O |
|
patterning O |
|
. O |
|
|
|
Mechanistically O |
|
it O |
|
is O |
|
thought O |
|
that O |
|
, O |
|
in O |
|
the O |
|
presence B-protein_state |
|
of I-protein_state |
|
Dkk B-protein_type |
|
, O |
|
Krm B-protein_type |
|
forms O |
|
a O |
|
ternary O |
|
complex B-protein_state |
|
with I-protein_state |
|
Lrp6 B-protein_type |
|
, O |
|
which O |
|
is O |
|
then O |
|
rapidly O |
|
endocytosed O |
|
. O |
|
|
|
This O |
|
amplifies O |
|
the O |
|
intrinsic O |
|
Wnt B-protein_type |
|
antagonistic O |
|
activity O |
|
of O |
|
Dkk B-protein_type |
|
by O |
|
efficiently O |
|
depleting O |
|
the O |
|
cell O |
|
surface O |
|
of O |
|
the O |
|
Wnt B-protein_type |
|
co B-protein_type |
|
- I-protein_type |
|
receptor I-protein_type |
|
. O |
|
|
|
In O |
|
accordance O |
|
with O |
|
this O |
|
, O |
|
Krm1 B-protein_type |
|
−/− O |
|
and O |
|
Krm2 B-protein_type |
|
−/− O |
|
double B-experimental_method |
|
knockout I-experimental_method |
|
mice B-taxonomy_domain |
|
show O |
|
a O |
|
high O |
|
bone O |
|
mass O |
|
phenotype O |
|
typical O |
|
of O |
|
increased O |
|
Wnt B-protein_type |
|
signaling O |
|
, O |
|
as O |
|
well O |
|
as O |
|
growth O |
|
of O |
|
ectopic O |
|
forelimb O |
|
digits O |
|
. O |
|
|
|
Growth O |
|
of O |
|
ectopic O |
|
digits O |
|
is O |
|
further O |
|
enhanced O |
|
upon O |
|
additional O |
|
loss O |
|
of O |
|
dkk B-protein_type |
|
expression O |
|
. O |
|
|
|
The O |
|
Wnt B-protein_type |
|
antagonistic O |
|
activity O |
|
of O |
|
Krm1 B-protein_type |
|
is O |
|
also O |
|
linked O |
|
to O |
|
its O |
|
importance O |
|
for O |
|
correct O |
|
thymus O |
|
epithelium O |
|
formation O |
|
in O |
|
mice B-taxonomy_domain |
|
. O |
|
|
|
The O |
|
importance O |
|
of O |
|
intact B-protein_state |
|
KRM1 B-protein |
|
for O |
|
normal O |
|
human B-species |
|
development O |
|
and O |
|
health O |
|
is O |
|
highlighted O |
|
by O |
|
the O |
|
recent O |
|
finding O |
|
that O |
|
a O |
|
homozygous O |
|
mutation O |
|
in O |
|
the O |
|
ectodomain B-structure_element |
|
of O |
|
KRM1 B-protein |
|
leads O |
|
to O |
|
severe O |
|
ectodermal O |
|
dysplasia O |
|
including O |
|
oligodontia O |
|
. O |
|
|
|
Interestingly O |
|
, O |
|
the O |
|
Wnt B-protein_type |
|
antagonistic O |
|
activity O |
|
of O |
|
Krm B-protein_type |
|
is O |
|
context O |
|
dependent O |
|
, O |
|
and O |
|
Krm B-protein_type |
|
proteins O |
|
are O |
|
actually O |
|
dual O |
|
- O |
|
mode O |
|
Wnt B-protein_type |
|
regulators O |
|
. O |
|
|
|
In O |
|
the O |
|
absence B-protein_state |
|
of I-protein_state |
|
Dkk B-protein_type |
|
, O |
|
Krm1 B-protein_type |
|
and O |
|
2 B-protein_type |
|
change O |
|
their O |
|
function O |
|
from O |
|
inhibition O |
|
to O |
|
enhancement O |
|
of O |
|
Lrp6 B-protein_type |
|
- O |
|
mediated O |
|
signaling O |
|
. O |
|
|
|
By O |
|
direct O |
|
binding O |
|
to O |
|
Lrp6 B-protein_type |
|
via O |
|
the O |
|
ectodomains B-structure_element |
|
, O |
|
Krm B-protein_type |
|
proteins O |
|
promote O |
|
Lrp6 B-protein_type |
|
cell O |
|
- O |
|
surface O |
|
localization O |
|
and O |
|
hence O |
|
increase O |
|
receptor O |
|
availability O |
|
. O |
|
|
|
Further O |
|
increasing O |
|
the O |
|
complexity O |
|
of O |
|
Krm B-protein_type |
|
functionality O |
|
, O |
|
it O |
|
was O |
|
recently O |
|
found O |
|
that O |
|
Krm1 B-protein_type |
|
( O |
|
but O |
|
not O |
|
Krm2 B-protein_type |
|
) O |
|
can O |
|
also O |
|
act O |
|
independently O |
|
of O |
|
LRP5 B-protein |
|
/ I-protein |
|
6 I-protein |
|
and O |
|
Wnt B-protein_type |
|
as O |
|
a O |
|
dependence O |
|
receptor O |
|
, O |
|
triggering O |
|
apoptosis O |
|
unless O |
|
bound B-protein_state |
|
to I-protein_state |
|
Dkk B-protein_type |
|
. O |
|
|
|
Structurally O |
|
, O |
|
Krm1 B-protein_type |
|
and O |
|
2 B-protein_type |
|
are O |
|
type B-protein_type |
|
I I-protein_type |
|
transmembrane I-protein_type |
|
proteins I-protein_type |
|
with O |
|
a O |
|
40 O |
|
kDa O |
|
ectodomain B-structure_element |
|
and O |
|
a O |
|
flexible B-protein_state |
|
cytoplasmic B-structure_element |
|
tail I-structure_element |
|
consisting O |
|
of O |
|
60 B-residue_range |
|
– O |
|
75 B-residue_range |
|
residues O |
|
. O |
|
|
|
The O |
|
ectodomain B-structure_element |
|
consists O |
|
of O |
|
three O |
|
similarly O |
|
sized O |
|
structural O |
|
domains O |
|
of O |
|
around O |
|
10 O |
|
kDa O |
|
each O |
|
: O |
|
the O |
|
N O |
|
- O |
|
terminal O |
|
Kringle B-structure_element |
|
domain O |
|
( O |
|
KR B-structure_element |
|
) O |
|
is O |
|
followed O |
|
by O |
|
a O |
|
WSC B-structure_element |
|
domain O |
|
of O |
|
unknown O |
|
fold O |
|
. O |
|
|
|
The O |
|
third O |
|
structural O |
|
domain O |
|
is O |
|
a O |
|
CUB B-structure_element |
|
domain O |
|
. O |
|
|
|
An O |
|
approximately B-residue_range |
|
70 I-residue_range |
|
- I-residue_range |
|
residue I-residue_range |
|
linker B-structure_element |
|
connects O |
|
the O |
|
CUB B-structure_element |
|
domain O |
|
to O |
|
the O |
|
transmembrane B-structure_element |
|
span I-structure_element |
|
. O |
|
|
|
An O |
|
intact B-protein_state |
|
KR B-structure_element |
|
- I-structure_element |
|
WSC I-structure_element |
|
- I-structure_element |
|
CUB I-structure_element |
|
domain O |
|
triplet O |
|
and O |
|
membrane O |
|
attachment O |
|
is O |
|
required O |
|
for O |
|
Wnt B-protein_type |
|
antagonism O |
|
. O |
|
|
|
The O |
|
transmembrane B-structure_element |
|
span I-structure_element |
|
and O |
|
cytoplasmic B-structure_element |
|
tail I-structure_element |
|
can O |
|
be O |
|
replaced O |
|
with O |
|
a O |
|
GPI B-structure_element |
|
linker B-structure_element |
|
without O |
|
impact O |
|
on O |
|
Wnt B-protein_type |
|
antagonism O |
|
. O |
|
|
|
The O |
|
structures B-evidence |
|
presented O |
|
here O |
|
reveal O |
|
the O |
|
unknown O |
|
fold O |
|
of O |
|
the O |
|
WSC B-structure_element |
|
domain O |
|
and O |
|
the O |
|
tight O |
|
interactions O |
|
of O |
|
all O |
|
three O |
|
domains O |
|
. O |
|
|
|
We O |
|
further O |
|
succeeded O |
|
in O |
|
determination O |
|
of O |
|
a O |
|
low O |
|
- O |
|
resolution O |
|
LRP6PE3PE4 B-complex_assembly |
|
- I-complex_assembly |
|
DKK1CRD2 I-complex_assembly |
|
- I-complex_assembly |
|
KRM1ECD I-complex_assembly |
|
complex O |
|
, O |
|
defining O |
|
the O |
|
architecture O |
|
of O |
|
the O |
|
Wnt B-protein_type |
|
inhibitory B-complex_assembly |
|
complex I-complex_assembly |
|
that O |
|
leads O |
|
to O |
|
Lrp6 B-protein |
|
cell O |
|
- O |
|
surface O |
|
depletion O |
|
. O |
|
|
|
The O |
|
recombinant O |
|
production O |
|
of O |
|
the O |
|
extracellular B-structure_element |
|
domain I-structure_element |
|
of O |
|
Krm B-protein_type |
|
for O |
|
structural B-experimental_method |
|
studies I-experimental_method |
|
proved O |
|
challenging O |
|
( O |
|
see O |
|
Experimental O |
|
Procedures O |
|
). O |
|
|
|
We O |
|
succeeded O |
|
in O |
|
purifying O |
|
KRM1ECD B-protein |
|
complexes B-protein_state |
|
with I-protein_state |
|
DKK1fl B-protein |
|
, O |
|
DKK1Linker B-protein |
|
- B-structure_element |
|
CRD2 I-structure_element |
|
, O |
|
and O |
|
DKK1CRD2 B-protein |
|
that O |
|
were O |
|
monodisperse O |
|
and O |
|
stable O |
|
in O |
|
gel B-experimental_method |
|
filtration I-experimental_method |
|
, O |
|
hence O |
|
indicating O |
|
at O |
|
least O |
|
micromolar O |
|
affinity O |
|
( O |
|
data O |
|
not O |
|
shown O |
|
). O |
|
|
|
Several O |
|
crystal B-evidence |
|
forms I-evidence |
|
were O |
|
obtained O |
|
from O |
|
these O |
|
complexes O |
|
, O |
|
however O |
|
, O |
|
crystals B-evidence |
|
always O |
|
contained O |
|
only O |
|
KRM1 B-protein |
|
protein O |
|
. O |
|
|
|
We O |
|
solved B-experimental_method |
|
the O |
|
structure B-evidence |
|
of O |
|
KRM1ECD B-protein |
|
in O |
|
three O |
|
crystal O |
|
forms O |
|
at O |
|
1 O |
|
. O |
|
9 O |
|
, O |
|
2 O |
|
. O |
|
8 O |
|
, O |
|
and O |
|
3 O |
|
. O |
|
2 O |
|
Å O |
|
resolution O |
|
( O |
|
Table O |
|
1 O |
|
). O |
|
|
|
The O |
|
high O |
|
- O |
|
resolution O |
|
structure B-evidence |
|
is O |
|
a O |
|
near O |
|
full B-protein_state |
|
- I-protein_state |
|
length I-protein_state |
|
model O |
|
( O |
|
Figure O |
|
1 O |
|
). O |
|
|
|
The O |
|
small B-protein_state |
|
, O |
|
flexible B-protein_state |
|
, O |
|
and O |
|
charged B-protein_state |
|
98AEHED102 B-structure_element |
|
loop I-structure_element |
|
could O |
|
only O |
|
be O |
|
modeled O |
|
in O |
|
a O |
|
slightly O |
|
lower O |
|
resolution O |
|
structure B-evidence |
|
and O |
|
in O |
|
crystal O |
|
form O |
|
III O |
|
. O |
|
|
|
The O |
|
KR B-structure_element |
|
, O |
|
WSC B-structure_element |
|
, O |
|
and O |
|
CUB B-structure_element |
|
are O |
|
arranged O |
|
in O |
|
a O |
|
roughly O |
|
triangular O |
|
fashion O |
|
with O |
|
tight O |
|
interactions O |
|
between O |
|
all O |
|
three O |
|
domains O |
|
. O |
|
|
|
The O |
|
KR B-structure_element |
|
domain O |
|
, O |
|
which O |
|
bears O |
|
two O |
|
of O |
|
the O |
|
four O |
|
glycosylation B-site |
|
sites I-site |
|
, O |
|
contains O |
|
the O |
|
canonical O |
|
three O |
|
disulfide B-ptm |
|
bridges I-ptm |
|
( O |
|
C32 B-residue_name_number |
|
- O |
|
C114 B-residue_name_number |
|
, O |
|
C55 B-residue_name_number |
|
- O |
|
C95 B-residue_name_number |
|
, O |
|
C84 B-residue_name_number |
|
- O |
|
C109 B-residue_name_number |
|
) O |
|
and O |
|
, O |
|
like O |
|
other O |
|
Kringle B-structure_element |
|
domains O |
|
, O |
|
is O |
|
low O |
|
in O |
|
secondary O |
|
structure O |
|
elements O |
|
. O |
|
|
|
The O |
|
structurally O |
|
most O |
|
similar O |
|
Kringle B-structure_element |
|
domain O |
|
is O |
|
that O |
|
of O |
|
human B-species |
|
plasminogen B-protein |
|
( O |
|
PDB O |
|
: O |
|
1PKR O |
|
) O |
|
with O |
|
an O |
|
root B-evidence |
|
- I-evidence |
|
mean I-evidence |
|
- I-evidence |
|
square I-evidence |
|
deviation I-evidence |
|
( O |
|
RMSD B-evidence |
|
) O |
|
of O |
|
1 O |
|
. O |
|
7 O |
|
Å O |
|
for O |
|
73 O |
|
aligned O |
|
Cα O |
|
( O |
|
Figure O |
|
1B O |
|
). O |
|
|
|
The O |
|
KRM1 B-protein |
|
structure B-evidence |
|
reveals O |
|
the O |
|
fold O |
|
of O |
|
the O |
|
WSC B-structure_element |
|
domain O |
|
for O |
|
the O |
|
first O |
|
time O |
|
. O |
|
|
|
The O |
|
structure B-evidence |
|
is O |
|
best O |
|
described O |
|
as O |
|
a O |
|
sandwich B-structure_element |
|
of O |
|
a O |
|
β1 B-structure_element |
|
- I-structure_element |
|
β5 I-structure_element |
|
- I-structure_element |
|
β3 I-structure_element |
|
- I-structure_element |
|
β4 I-structure_element |
|
- I-structure_element |
|
β2 I-structure_element |
|
antiparallel I-structure_element |
|
β I-structure_element |
|
sheet I-structure_element |
|
and O |
|
a O |
|
single O |
|
α B-structure_element |
|
helix I-structure_element |
|
. O |
|
|
|
The O |
|
structure B-evidence |
|
is O |
|
also O |
|
rich O |
|
in O |
|
loops B-structure_element |
|
and O |
|
is O |
|
stabilized O |
|
by O |
|
four O |
|
disulfide B-ptm |
|
bridges I-ptm |
|
( O |
|
C122 B-residue_name_number |
|
- O |
|
C186 B-residue_name_number |
|
, O |
|
C147 B-residue_name_number |
|
- O |
|
C167 B-residue_name_number |
|
, O |
|
C151 B-residue_name_number |
|
- O |
|
C169 B-residue_name_number |
|
, O |
|
C190 B-residue_name_number |
|
- O |
|
C198 B-residue_name_number |
|
). O |
|
|
|
Using O |
|
the O |
|
PDBeFold B-experimental_method |
|
server I-experimental_method |
|
, O |
|
we O |
|
detected O |
|
a O |
|
surprising O |
|
yet O |
|
significant O |
|
homology O |
|
to O |
|
PAN B-structure_element |
|
module I-structure_element |
|
domains I-structure_element |
|
. O |
|
|
|
The O |
|
closest O |
|
structural O |
|
relative O |
|
is O |
|
hepatocyte B-protein_type |
|
growth I-protein_type |
|
factor I-protein_type |
|
( O |
|
HGF B-protein_type |
|
, O |
|
PDB O |
|
: O |
|
1GP9 O |
|
), O |
|
which O |
|
superposes B-experimental_method |
|
with O |
|
an O |
|
RMSD B-evidence |
|
of O |
|
2 O |
|
. O |
|
3 O |
|
Å O |
|
for O |
|
58 O |
|
aligned O |
|
Cα O |
|
( O |
|
Figure O |
|
1B O |
|
). O |
|
|
|
The O |
|
CUB B-structure_element |
|
domain O |
|
bears O |
|
two O |
|
glycosylation B-site |
|
sites I-site |
|
. O |
|
|
|
Although O |
|
present O |
|
, O |
|
the O |
|
quality O |
|
of O |
|
the O |
|
electron B-evidence |
|
density I-evidence |
|
around O |
|
N217 B-residue_name_number |
|
did O |
|
not O |
|
allow O |
|
modeling O |
|
of O |
|
the O |
|
sugar O |
|
moiety O |
|
. O |
|
|
|
In O |
|
crystal B-evidence |
|
form I-evidence |
|
I I-evidence |
|
, O |
|
a O |
|
calcium B-chemical |
|
ion O |
|
is O |
|
present O |
|
at O |
|
the O |
|
canonical O |
|
position O |
|
coordinated B-bond_interaction |
|
by I-bond_interaction |
|
the O |
|
carboxylates O |
|
of O |
|
D263 B-residue_name_number |
|
, O |
|
D266 B-residue_name_number |
|
( O |
|
bidentate O |
|
), O |
|
and O |
|
D306 B-residue_name_number |
|
, O |
|
as O |
|
well O |
|
as O |
|
the O |
|
carbonyl O |
|
of O |
|
N309 B-residue_name_number |
|
and O |
|
a O |
|
water B-chemical |
|
molecule O |
|
. O |
|
|
|
The O |
|
coordination B-site |
|
sphere I-site |
|
deviates O |
|
significantly O |
|
from O |
|
perfectly O |
|
octahedral O |
|
( O |
|
not O |
|
shown O |
|
). O |
|
|
|
This O |
|
might O |
|
result O |
|
in O |
|
the O |
|
site O |
|
having O |
|
a O |
|
low O |
|
affinity O |
|
and O |
|
may O |
|
explain O |
|
why O |
|
calcium B-chemical |
|
is O |
|
not O |
|
present O |
|
in O |
|
the O |
|
two O |
|
low O |
|
- O |
|
resolution O |
|
crystal B-evidence |
|
forms I-evidence |
|
. O |
|
|
|
Loss B-protein_state |
|
of I-protein_state |
|
calcium B-chemical |
|
has O |
|
led O |
|
to O |
|
loop B-structure_element |
|
rearrangements O |
|
and O |
|
partial O |
|
disorder O |
|
in O |
|
these O |
|
crystal B-evidence |
|
forms I-evidence |
|
. O |
|
|
|
The O |
|
closest O |
|
structural O |
|
relative O |
|
is O |
|
the O |
|
CUB_C B-structure_element |
|
domain O |
|
of O |
|
Tsg B-protein |
|
- I-protein |
|
6 I-protein |
|
( O |
|
PDB O |
|
: O |
|
2WNO O |
|
), O |
|
which O |
|
superposes B-experimental_method |
|
with O |
|
KRMCUB B-protein |
|
with O |
|
an O |
|
RMSD B-evidence |
|
of O |
|
1 O |
|
. O |
|
6 O |
|
Å O |
|
for O |
|
104 O |
|
Cα O |
|
( O |
|
Figure O |
|
1B O |
|
). O |
|
|
|
A O |
|
superposition B-experimental_method |
|
of O |
|
the O |
|
three O |
|
KRM1 B-protein |
|
structures B-evidence |
|
reveals O |
|
no O |
|
major O |
|
structural O |
|
differences O |
|
( O |
|
Figure O |
|
1C O |
|
) O |
|
as O |
|
anticipated O |
|
from O |
|
the O |
|
plethora O |
|
of O |
|
interactions O |
|
between O |
|
the O |
|
three O |
|
domains O |
|
. O |
|
|
|
Minor O |
|
differences O |
|
are O |
|
caused O |
|
by O |
|
the O |
|
collapse O |
|
of O |
|
the O |
|
Ca2 B-site |
|
+ I-site |
|
binding I-site |
|
site I-site |
|
in O |
|
crystal B-evidence |
|
forms I-evidence |
|
II I-evidence |
|
and I-evidence |
|
III I-evidence |
|
and O |
|
loop B-structure_element |
|
flexibility O |
|
in O |
|
the O |
|
KR B-structure_element |
|
domain O |
|
. O |
|
|
|
The O |
|
F207S B-mutant |
|
mutation O |
|
recently O |
|
found O |
|
to O |
|
cause O |
|
ectodermal O |
|
dysplasia O |
|
in O |
|
Palestinian O |
|
families O |
|
maps O |
|
to O |
|
the O |
|
hydrophobic B-site |
|
core I-site |
|
of O |
|
the O |
|
protein O |
|
at O |
|
the O |
|
interface B-site |
|
of O |
|
the O |
|
three O |
|
subdomains O |
|
( O |
|
Figure O |
|
1A O |
|
). O |
|
|
|
Such O |
|
a O |
|
mutation O |
|
is O |
|
bound B-protein_state |
|
to I-protein_state |
|
severely O |
|
destabilize O |
|
the O |
|
protein O |
|
structure O |
|
of O |
|
KRM1 B-protein |
|
, O |
|
leading O |
|
to O |
|
disturbance O |
|
of O |
|
its O |
|
Wnt B-protein_type |
|
antagonistic O |
|
, O |
|
Wnt B-protein_type |
|
stimulatory O |
|
, O |
|
and O |
|
Wnt B-protein_type |
|
independent O |
|
activity O |
|
. O |
|
|
|
Co B-experimental_method |
|
- I-experimental_method |
|
crystallization I-experimental_method |
|
of O |
|
LRP6PE3PE4 B-protein |
|
with O |
|
DKK1CRD2 B-protein |
|
, O |
|
and O |
|
LRP6PE1 B-protein |
|
with O |
|
an O |
|
N O |
|
- O |
|
terminal O |
|
peptide O |
|
of O |
|
DKK1 B-protein |
|
has O |
|
provided O |
|
valuable O |
|
structural O |
|
insight O |
|
into O |
|
direct O |
|
Wnt B-protein_type |
|
inhibition O |
|
by O |
|
Dkk B-protein_type |
|
ligands O |
|
. O |
|
|
|
One O |
|
face O |
|
of O |
|
the O |
|
rather O |
|
flat B-protein_state |
|
DKK1CRD2 B-protein |
|
fragment O |
|
binds B-protein_state |
|
to I-protein_state |
|
the O |
|
third B-structure_element |
|
β I-structure_element |
|
propeller I-structure_element |
|
of O |
|
LRP6 B-protein |
|
. O |
|
|
|
Mutational B-experimental_method |
|
analyses I-experimental_method |
|
further O |
|
implied O |
|
that O |
|
the O |
|
LRP6PE3 B-protein |
|
- O |
|
averted O |
|
face O |
|
of O |
|
DKK1CRD2 B-protein |
|
bears O |
|
the O |
|
Krm B-site |
|
binding I-site |
|
site I-site |
|
, O |
|
hence O |
|
suggesting O |
|
how O |
|
Dkk B-protein_type |
|
can O |
|
recruit O |
|
both O |
|
receptors B-protein_type |
|
into O |
|
a O |
|
ternary O |
|
complex O |
|
. O |
|
|
|
To O |
|
obtain O |
|
direct O |
|
insight O |
|
into O |
|
ternary O |
|
complex O |
|
formation O |
|
by O |
|
Lrp5 B-protein_type |
|
/ I-protein_type |
|
6 I-protein_type |
|
, O |
|
Dkk B-protein_type |
|
, O |
|
and O |
|
Krm B-protein_type |
|
, O |
|
we O |
|
subjected O |
|
an O |
|
LRP6PE3PE4 B-complex_assembly |
|
- I-complex_assembly |
|
DKK1fl I-complex_assembly |
|
- I-complex_assembly |
|
KRM1ECD I-complex_assembly |
|
complex O |
|
to O |
|
crystallization B-experimental_method |
|
trials I-experimental_method |
|
. O |
|
|
|
Diffraction B-evidence |
|
data I-evidence |
|
collected O |
|
from O |
|
the O |
|
resulting O |
|
crystals B-evidence |
|
were O |
|
highly O |
|
anisotropic O |
|
with O |
|
diffraction O |
|
extending O |
|
in O |
|
the O |
|
best O |
|
directions O |
|
to O |
|
3 O |
|
. O |
|
5 O |
|
Å O |
|
and O |
|
3 O |
|
. O |
|
7 O |
|
Å O |
|
but O |
|
only O |
|
to O |
|
6 O |
|
. O |
|
4 O |
|
Å O |
|
in O |
|
the O |
|
third O |
|
direction O |
|
. O |
|
|
|
Despite O |
|
the O |
|
lack O |
|
of O |
|
high O |
|
- O |
|
resolution O |
|
diffraction B-evidence |
|
, O |
|
the O |
|
general O |
|
architecture O |
|
of O |
|
the O |
|
ternary O |
|
complex O |
|
is O |
|
revealed O |
|
( O |
|
Figure O |
|
2A O |
|
). O |
|
|
|
DKK1CRD2 B-protein |
|
binds B-protein_state |
|
to I-protein_state |
|
the O |
|
top O |
|
face O |
|
of O |
|
the O |
|
LRP6 B-protein |
|
PE3 B-structure_element |
|
β B-structure_element |
|
propeller I-structure_element |
|
as O |
|
described O |
|
earlier O |
|
for O |
|
the O |
|
binary O |
|
complex O |
|
. O |
|
|
|
KRM1ECD B-protein |
|
does O |
|
indeed O |
|
bind B-protein_state |
|
on I-protein_state |
|
the O |
|
opposite O |
|
side O |
|
of O |
|
DKK1CRD2 B-protein |
|
with O |
|
only O |
|
its O |
|
KR B-structure_element |
|
and O |
|
WSC B-structure_element |
|
domains O |
|
engaged O |
|
in O |
|
binding O |
|
( O |
|
Figure O |
|
2A O |
|
). O |
|
|
|
Although O |
|
present O |
|
in O |
|
the O |
|
complex O |
|
subjected O |
|
to O |
|
crystallization B-experimental_method |
|
, O |
|
we O |
|
observe O |
|
no O |
|
density B-evidence |
|
that O |
|
could O |
|
correspond O |
|
to O |
|
CRD1 B-structure_element |
|
or O |
|
the O |
|
domain B-structure_element |
|
linker I-structure_element |
|
( O |
|
L B-structure_element |
|
). O |
|
|
|
We O |
|
confirm O |
|
that O |
|
the O |
|
CRD2 B-structure_element |
|
of O |
|
DKK1 B-protein |
|
is O |
|
required O |
|
and O |
|
sufficient O |
|
for O |
|
binding O |
|
to O |
|
KRM1 B-protein |
|
: O |
|
In O |
|
surface B-experimental_method |
|
plasmon I-experimental_method |
|
resonance I-experimental_method |
|
( O |
|
SPR B-experimental_method |
|
), O |
|
we O |
|
measured O |
|
low O |
|
micromolar O |
|
affinity B-evidence |
|
between O |
|
full B-protein_state |
|
- I-protein_state |
|
length I-protein_state |
|
DKK1 B-protein |
|
and O |
|
immobilized O |
|
KRM1ECD B-protein |
|
( O |
|
Figure O |
|
2B O |
|
). O |
|
|
|
A O |
|
SUMO B-experimental_method |
|
fusion I-experimental_method |
|
of O |
|
DKK1L B-structure_element |
|
- I-structure_element |
|
CRD2 I-structure_element |
|
displayed O |
|
a O |
|
similar O |
|
( O |
|
slightly O |
|
higher O |
|
) O |
|
affinity B-evidence |
|
. O |
|
|
|
In O |
|
contrast O |
|
, O |
|
a O |
|
SUMO B-experimental_method |
|
fusion I-experimental_method |
|
of O |
|
DKK1CRD1 B-structure_element |
|
- I-structure_element |
|
L I-structure_element |
|
did O |
|
not O |
|
display O |
|
binding O |
|
for O |
|
concentrations O |
|
tested O |
|
up O |
|
to O |
|
325 O |
|
μM O |
|
( O |
|
Figure O |
|
2B O |
|
). O |
|
|
|
Overall O |
|
, O |
|
the O |
|
DKK1 B-site |
|
- I-site |
|
KRM1 I-site |
|
interface I-site |
|
is O |
|
characterized O |
|
by O |
|
a O |
|
large O |
|
number O |
|
of O |
|
polar B-bond_interaction |
|
interactions I-bond_interaction |
|
but O |
|
only O |
|
few O |
|
hydrophobic B-bond_interaction |
|
contacts I-bond_interaction |
|
( O |
|
Figure O |
|
2C O |
|
). O |
|
|
|
The O |
|
crystal B-evidence |
|
structure I-evidence |
|
gives O |
|
an O |
|
explanation O |
|
for O |
|
DKK1 B-protein |
|
loss O |
|
- O |
|
of O |
|
- O |
|
binding O |
|
mutations O |
|
identified O |
|
previously O |
|
: O |
|
R191 B-residue_name_number |
|
of O |
|
DKK1 B-protein |
|
forms O |
|
a O |
|
double O |
|
salt B-bond_interaction |
|
bridge I-bond_interaction |
|
to O |
|
D125 B-residue_name_number |
|
and O |
|
E162 B-residue_name_number |
|
of O |
|
KRM1 B-protein |
|
( O |
|
Figure O |
|
2C O |
|
). O |
|
|
|
A O |
|
charge B-experimental_method |
|
reversal I-experimental_method |
|
as O |
|
in O |
|
the O |
|
mouse B-taxonomy_domain |
|
Dkk1 B-protein |
|
( O |
|
mDkk1 B-protein |
|
) O |
|
R197E B-mutant |
|
variant O |
|
would O |
|
severely O |
|
disrupt O |
|
the O |
|
binding O |
|
. O |
|
|
|
Similarly O |
|
, O |
|
the O |
|
K226 B-residue_name_number |
|
side O |
|
chain O |
|
of O |
|
DKK1 B-protein |
|
, O |
|
which O |
|
points O |
|
to O |
|
a O |
|
small O |
|
hydrophobic B-site |
|
pocket I-site |
|
on O |
|
the O |
|
surface O |
|
of O |
|
KRM1 B-protein |
|
formed O |
|
by O |
|
Y108 B-residue_name_number |
|
, O |
|
W94 B-residue_name_number |
|
, O |
|
and O |
|
W106 B-residue_name_number |
|
, O |
|
forms O |
|
salt B-bond_interaction |
|
bridges I-bond_interaction |
|
with O |
|
the O |
|
side O |
|
chains O |
|
of O |
|
KRM1 B-protein |
|
D88 B-residue_name_number |
|
and O |
|
D90 B-residue_name_number |
|
. O |
|
|
|
Again O |
|
, O |
|
a O |
|
charge B-experimental_method |
|
reversal I-experimental_method |
|
as O |
|
shown O |
|
before O |
|
for O |
|
mDkk1 B-protein |
|
K232E B-mutant |
|
would O |
|
be O |
|
incompatible O |
|
with O |
|
binding O |
|
. O |
|
|
|
The O |
|
side O |
|
chain O |
|
of O |
|
DKK1 B-protein |
|
S192 B-residue_name_number |
|
was O |
|
also O |
|
predicted O |
|
to O |
|
be O |
|
involved O |
|
in O |
|
Krm B-protein_type |
|
binding O |
|
. O |
|
|
|
Indeed O |
|
, O |
|
we O |
|
found O |
|
( O |
|
Figure O |
|
2C O |
|
) O |
|
that O |
|
the O |
|
side O |
|
chain O |
|
of O |
|
D201 B-residue_name_number |
|
of O |
|
KRM1 B-protein |
|
forms O |
|
two O |
|
hydrogen B-bond_interaction |
|
bonds I-bond_interaction |
|
to O |
|
the O |
|
side O |
|
- O |
|
chain O |
|
hydroxyl O |
|
and O |
|
the O |
|
backbone O |
|
amide O |
|
of O |
|
S192 B-residue_name_number |
|
( O |
|
mouse B-taxonomy_domain |
|
, O |
|
S198 B-residue_name_number |
|
). O |
|
|
|
Additional O |
|
polar B-bond_interaction |
|
interactions I-bond_interaction |
|
are O |
|
formed O |
|
between O |
|
the O |
|
N140 B-residue_name_number |
|
, O |
|
S163 B-residue_name_number |
|
, O |
|
and O |
|
Y165 B-residue_name_number |
|
side O |
|
chains O |
|
of O |
|
KRM1 B-protein |
|
and O |
|
DKK1 B-protein |
|
backbone O |
|
carbonyls O |
|
of O |
|
W206 B-residue_name_number |
|
, O |
|
L190 B-residue_name_number |
|
, O |
|
and O |
|
C189 B-residue_name_number |
|
, O |
|
respectively O |
|
. O |
|
|
|
The O |
|
carbonyl O |
|
of O |
|
DKK1 B-protein |
|
R224 B-residue_name_number |
|
is O |
|
hydrogen B-bond_interaction |
|
bonded I-bond_interaction |
|
to O |
|
Y105 B-residue_name_number |
|
and O |
|
W106 B-residue_name_number |
|
of O |
|
KRM1 B-protein |
|
. O |
|
|
|
We O |
|
suspect O |
|
that O |
|
the O |
|
Dkk B-protein_type |
|
charge B-experimental_method |
|
reversal I-experimental_method |
|
mutations I-experimental_method |
|
performed O |
|
in O |
|
the O |
|
murine B-taxonomy_domain |
|
background O |
|
and O |
|
shown O |
|
to O |
|
diminish O |
|
Krm B-protein_type |
|
binding O |
|
K211E B-mutant |
|
and O |
|
R203E B-mutant |
|
( O |
|
mouse B-taxonomy_domain |
|
K217E B-mutant |
|
and O |
|
R209E B-mutant |
|
) O |
|
do O |
|
so O |
|
likely O |
|
indirectly O |
|
by O |
|
disruption O |
|
of O |
|
the O |
|
Dkk B-protein_type |
|
fold O |
|
. O |
|
|
|
We O |
|
further O |
|
validated O |
|
the O |
|
DKK1 B-site |
|
binding I-site |
|
site I-site |
|
on O |
|
KRM1 B-protein |
|
by O |
|
introducing B-experimental_method |
|
glycosylation B-site |
|
sites I-site |
|
at O |
|
the O |
|
KR B-structure_element |
|
( O |
|
90DVS92 B-mutant |
|
→ I-mutant |
|
NVS I-mutant |
|
) O |
|
and O |
|
WSC B-structure_element |
|
( O |
|
189VCF191 B-mutant |
|
→ I-mutant |
|
NCS I-mutant |
|
) O |
|
domains O |
|
pointing O |
|
toward O |
|
DKK B-protein |
|
( O |
|
Figures O |
|
2A O |
|
and O |
|
2D O |
|
). O |
|
|
|
Introduction O |
|
of O |
|
N B-ptm |
|
- I-ptm |
|
linked I-ptm |
|
glycans I-ptm |
|
in O |
|
protein B-site |
|
- I-site |
|
protein I-site |
|
- I-site |
|
binding I-site |
|
sites I-site |
|
is O |
|
an O |
|
established O |
|
way O |
|
of O |
|
disrupting O |
|
protein B-site |
|
- I-site |
|
binding I-site |
|
interfaces I-site |
|
. O |
|
|
|
Both O |
|
ectodomain B-structure_element |
|
mutants B-protein_state |
|
were O |
|
secreted O |
|
comparably O |
|
with O |
|
the O |
|
wild B-protein_state |
|
- I-protein_state |
|
type I-protein_state |
|
, O |
|
indicating O |
|
correct O |
|
folding O |
|
, O |
|
but O |
|
failed O |
|
to O |
|
achieve O |
|
any O |
|
detectable O |
|
binding O |
|
in O |
|
SPR B-experimental_method |
|
using O |
|
full B-protein_state |
|
- I-protein_state |
|
length I-protein_state |
|
DKK1 B-protein |
|
as O |
|
analyte O |
|
. O |
|
|
|
In O |
|
contrast O |
|
, O |
|
a O |
|
mutant B-protein_state |
|
carrying O |
|
an O |
|
additional O |
|
N B-ptm |
|
- I-ptm |
|
glycan I-ptm |
|
outside O |
|
the O |
|
interface B-site |
|
at O |
|
the O |
|
CUB B-structure_element |
|
domain O |
|
( O |
|
309NQA311 B-mutant |
|
→ I-mutant |
|
NQS I-mutant |
|
), O |
|
was O |
|
wild B-protein_state |
|
- I-protein_state |
|
type I-protein_state |
|
- O |
|
like O |
|
in O |
|
DKK1 B-protein |
|
binding O |
|
( O |
|
Figure O |
|
2D O |
|
). O |
|
|
|
Identification O |
|
of O |
|
a O |
|
Direct O |
|
LRP6 B-site |
|
- I-site |
|
KRM1 I-site |
|
Binding I-site |
|
Site I-site |
|
|
|
The O |
|
LRP6PE3PE4 B-complex_assembly |
|
- I-complex_assembly |
|
DKK1CRD2 I-complex_assembly |
|
- I-complex_assembly |
|
KRM1ECD I-complex_assembly |
|
complex O |
|
structure B-evidence |
|
reveals O |
|
no O |
|
direct O |
|
interactions O |
|
between O |
|
KRM1 B-protein |
|
and O |
|
LRP6 B-protein |
|
. O |
|
|
|
We O |
|
constructed O |
|
in O |
|
silico O |
|
a O |
|
ternary O |
|
complex B-protein_state |
|
with I-protein_state |
|
a O |
|
close O |
|
to O |
|
full B-protein_state |
|
- I-protein_state |
|
length I-protein_state |
|
LRP6 B-protein |
|
ectodomain B-structure_element |
|
( O |
|
PE1PE2PE3PE4 B-structure_element |
|
horse B-structure_element |
|
shoe I-structure_element |
|
) O |
|
similar O |
|
to O |
|
but O |
|
without O |
|
refinement O |
|
against O |
|
electron B-experimental_method |
|
microscopy I-experimental_method |
|
( O |
|
EM B-experimental_method |
|
) O |
|
or O |
|
small B-experimental_method |
|
- I-experimental_method |
|
angle I-experimental_method |
|
X I-experimental_method |
|
- I-experimental_method |
|
ray I-experimental_method |
|
scattering I-experimental_method |
|
data O |
|
. O |
|
|
|
An O |
|
auxiliary O |
|
PE3PE4 B-structure_element |
|
fragment O |
|
was O |
|
superimposed B-experimental_method |
|
via O |
|
PE4 B-structure_element |
|
onto O |
|
PE3 B-structure_element |
|
of O |
|
the O |
|
crystal B-evidence |
|
structure I-evidence |
|
, O |
|
and O |
|
the O |
|
LRP6PE1PE2 B-protein |
|
structure B-evidence |
|
was O |
|
superimposed B-experimental_method |
|
via O |
|
PE2 B-structure_element |
|
onto O |
|
PE3 B-structure_element |
|
of O |
|
this O |
|
auxiliary O |
|
fragment O |
|
( O |
|
Figure O |
|
3A O |
|
). O |
|
|
|
For O |
|
this O |
|
crude O |
|
approximation O |
|
of O |
|
a O |
|
true O |
|
ternary O |
|
complex O |
|
, O |
|
we O |
|
noted O |
|
very O |
|
close O |
|
proximity O |
|
between O |
|
the O |
|
Ca2 B-site |
|
+- I-site |
|
binding I-site |
|
region I-site |
|
of O |
|
KRM1 B-protein |
|
and O |
|
the O |
|
top O |
|
face O |
|
of O |
|
the O |
|
PE2 B-structure_element |
|
β B-structure_element |
|
propeller I-structure_element |
|
of O |
|
LRP6 B-protein |
|
. O |
|
|
|
The O |
|
solvent B-protein_state |
|
- I-protein_state |
|
exposed I-protein_state |
|
residues O |
|
R307 B-residue_name_number |
|
, O |
|
I308 B-residue_name_number |
|
, O |
|
and O |
|
N309 B-residue_name_number |
|
of O |
|
the O |
|
central O |
|
Ca2 B-structure_element |
|
+- I-structure_element |
|
binding I-structure_element |
|
β I-structure_element |
|
connection I-structure_element |
|
loop I-structure_element |
|
of O |
|
KRM1 B-protein |
|
would O |
|
be O |
|
almost O |
|
ideally O |
|
positioned O |
|
for O |
|
binding O |
|
to O |
|
this O |
|
face O |
|
, O |
|
which O |
|
is O |
|
commonly O |
|
used O |
|
as O |
|
a O |
|
binding B-site |
|
site I-site |
|
on O |
|
β B-structure_element |
|
propellers I-structure_element |
|
. O |
|
|
|
Peptides O |
|
containing O |
|
arginine B-residue_name |
|
/ O |
|
lysine B-residue_name |
|
, O |
|
isoleucine B-residue_name |
|
, O |
|
and O |
|
asparagine B-residue_name |
|
( O |
|
consensus O |
|
sequence O |
|
N B-structure_element |
|
- I-structure_element |
|
X I-structure_element |
|
- I-structure_element |
|
I I-structure_element |
|
-( I-structure_element |
|
G I-structure_element |
|
)- I-structure_element |
|
R I-structure_element |
|
/ I-structure_element |
|
K I-structure_element |
|
) O |
|
are O |
|
also O |
|
employed O |
|
by O |
|
DKK1 B-protein |
|
and O |
|
SOST B-protein |
|
to O |
|
bind O |
|
to O |
|
LRP6 B-protein |
|
( O |
|
albeit O |
|
to O |
|
propeller B-structure_element |
|
1 I-structure_element |
|
; O |
|
Figure O |
|
3B O |
|
). O |
|
|
|
To O |
|
support O |
|
the O |
|
hypothesis O |
|
that O |
|
KRM1CUB B-protein |
|
binds B-protein_state |
|
to I-protein_state |
|
LRP6PE2 B-protein |
|
, O |
|
we O |
|
used O |
|
SPR B-experimental_method |
|
and O |
|
compared O |
|
binding O |
|
of O |
|
the O |
|
wild B-protein_state |
|
- I-protein_state |
|
type I-protein_state |
|
and O |
|
the O |
|
GlycoCUB B-protein_state |
|
mutant I-protein_state |
|
of O |
|
KRM1ECD B-protein |
|
( O |
|
bearing O |
|
an O |
|
N B-site |
|
- I-site |
|
glycosylation I-site |
|
site I-site |
|
at O |
|
N309 B-residue_name_number |
|
) O |
|
with O |
|
a O |
|
purified O |
|
LRP6PE1PE2 B-protein |
|
fragment O |
|
. O |
|
|
|
Indeed O |
|
, O |
|
we O |
|
found O |
|
that O |
|
in O |
|
the O |
|
absence B-protein_state |
|
of I-protein_state |
|
Dkk B-protein_type |
|
, O |
|
KRM1ECD B-protein |
|
bound B-protein_state |
|
with O |
|
considerable O |
|
affinity O |
|
to B-protein_state |
|
LRP6PE1PE2 B-protein |
|
( O |
|
Figure O |
|
3C O |
|
). O |
|
|
|
In O |
|
contrast O |
|
, O |
|
no O |
|
saturable O |
|
binding O |
|
was O |
|
observed O |
|
between O |
|
KRM1 B-protein |
|
and O |
|
LRP6PE3PE4 B-protein |
|
. O |
|
|
|
Introduction B-experimental_method |
|
of I-experimental_method |
|
an O |
|
N B-site |
|
- I-site |
|
glycosylation I-site |
|
site I-site |
|
at O |
|
N309 B-residue_name_number |
|
in O |
|
KRM1ECD B-protein |
|
abolished O |
|
LRP6PE1PE2 B-protein |
|
binding O |
|
( O |
|
Figure O |
|
3C O |
|
), O |
|
while O |
|
binding O |
|
to O |
|
DKK1 B-protein |
|
was O |
|
unaffected O |
|
( O |
|
Figure O |
|
2D O |
|
). O |
|
|
|
We O |
|
conclude O |
|
that O |
|
the O |
|
predicted O |
|
binding B-site |
|
site I-site |
|
between O |
|
KRM1CUB B-protein |
|
and O |
|
LRP6PE2 B-protein |
|
is O |
|
a O |
|
strong O |
|
candidate O |
|
for O |
|
mediating O |
|
the O |
|
direct O |
|
Lrp6 B-complex_assembly |
|
- I-complex_assembly |
|
Krm I-complex_assembly |
|
interaction O |
|
, O |
|
which O |
|
is O |
|
thought O |
|
to O |
|
increase O |
|
Wnt B-protein_type |
|
responsiveness O |
|
by O |
|
stabilizing O |
|
Lrp6 B-protein |
|
at O |
|
the O |
|
cell O |
|
surface O |
|
. O |
|
|
|
Further O |
|
experiments O |
|
are O |
|
required O |
|
to O |
|
pinpoint O |
|
the O |
|
exact O |
|
binding B-site |
|
site I-site |
|
. O |
|
|
|
Although O |
|
LRP6PE1 B-protein |
|
appears O |
|
somewhat O |
|
out O |
|
of O |
|
reach O |
|
in O |
|
the O |
|
modeled O |
|
ternary O |
|
complex O |
|
, O |
|
it O |
|
cannot O |
|
be O |
|
excluded O |
|
as O |
|
the O |
|
Krm B-site |
|
binding I-site |
|
site I-site |
|
in O |
|
the O |
|
ternary O |
|
complex O |
|
and O |
|
LRP6 B-complex_assembly |
|
- I-complex_assembly |
|
Krm I-complex_assembly |
|
binary O |
|
complex O |
|
. O |
|
|
|
The O |
|
presence B-protein_state |
|
of I-protein_state |
|
DKK B-protein |
|
may O |
|
govern O |
|
which O |
|
propeller B-structure_element |
|
( O |
|
PE1 B-structure_element |
|
versus O |
|
PE2 B-structure_element |
|
) O |
|
of O |
|
LRP6 B-protein |
|
is O |
|
available O |
|
for O |
|
Krm B-protein_type |
|
binding O |
|
. O |
|
|
|
Apparent O |
|
binding O |
|
across O |
|
the O |
|
proposed O |
|
KRM1CUB B-site |
|
- I-site |
|
LRP6PE2 I-site |
|
interface I-site |
|
is O |
|
expected O |
|
to O |
|
be O |
|
higher O |
|
once O |
|
Krm B-protein_type |
|
is O |
|
also O |
|
cross O |
|
- O |
|
linked O |
|
to O |
|
LRP6PE3 B-protein |
|
via O |
|
DKK1CRD2 B-protein |
|
( O |
|
Figure O |
|
3D O |
|
). O |
|
|
|
Low O |
|
- O |
|
resolution O |
|
negative B-experimental_method |
|
- I-experimental_method |
|
stain I-experimental_method |
|
EM I-experimental_method |
|
and O |
|
small B-experimental_method |
|
- I-experimental_method |
|
angle I-experimental_method |
|
X I-experimental_method |
|
- I-experimental_method |
|
ray I-experimental_method |
|
scattering I-experimental_method |
|
studies O |
|
of O |
|
LRP6PE1PE2PE3PE4 B-protein |
|
, O |
|
in B-protein_state |
|
isolation I-protein_state |
|
and O |
|
in B-protein_state |
|
complex I-protein_state |
|
with I-protein_state |
|
Dkk1 B-protein_type |
|
, O |
|
plus O |
|
negative B-experimental_method |
|
- I-experimental_method |
|
stain I-experimental_method |
|
EM I-experimental_method |
|
of O |
|
full B-protein_state |
|
- I-protein_state |
|
length I-protein_state |
|
LRP6 B-protein |
|
ectodomain B-structure_element |
|
, O |
|
have O |
|
indicated O |
|
curved B-protein_state |
|
, O |
|
platform B-protein_state |
|
- I-protein_state |
|
like I-protein_state |
|
conformations O |
|
but O |
|
also O |
|
potential O |
|
flexibility O |
|
between O |
|
PE2 B-structure_element |
|
and O |
|
PE3 B-structure_element |
|
. O |
|
|
|
It O |
|
is O |
|
therefore O |
|
possible O |
|
that O |
|
the O |
|
interplay O |
|
of O |
|
Krm B-protein_type |
|
and O |
|
Dkk B-protein_type |
|
binding O |
|
can O |
|
promote O |
|
changes O |
|
in O |
|
LRP6 B-protein |
|
ectodomain B-structure_element |
|
conformation O |
|
with O |
|
functional O |
|
consequences O |
|
; O |
|
however O |
|
, O |
|
such O |
|
ideas O |
|
await O |
|
investigation O |
|
. O |
|
|
|
Taken O |
|
together O |
|
, O |
|
the O |
|
structural B-experimental_method |
|
and I-experimental_method |
|
biophysical I-experimental_method |
|
studies I-experimental_method |
|
we O |
|
report O |
|
here O |
|
extend O |
|
our O |
|
mechanistic O |
|
understanding O |
|
of O |
|
Wnt B-protein_type |
|
signal O |
|
regulation O |
|
. O |
|
|
|
We O |
|
describe O |
|
the O |
|
ectodomain B-structure_element |
|
structure B-evidence |
|
of O |
|
the O |
|
dual O |
|
Wnt B-protein_type |
|
regulator O |
|
Krm1 B-protein_type |
|
, O |
|
providing O |
|
an O |
|
explanation O |
|
for O |
|
the O |
|
detrimental O |
|
effect O |
|
on O |
|
health O |
|
and O |
|
development O |
|
of O |
|
a O |
|
homozygous O |
|
KRM1 B-protein |
|
mutation O |
|
. O |
|
|
|
We O |
|
also O |
|
reveal O |
|
the O |
|
interaction O |
|
mode O |
|
of O |
|
Krm B-complex_assembly |
|
- I-complex_assembly |
|
Dkk I-complex_assembly |
|
and O |
|
the O |
|
architecture O |
|
of O |
|
the O |
|
ternary O |
|
complex O |
|
formed O |
|
by O |
|
Lrp5 B-protein_type |
|
/ I-protein_type |
|
6 I-protein_type |
|
, O |
|
Dkk B-protein_type |
|
, O |
|
and O |
|
Krm B-protein_type |
|
. O |
|
|
|
Furthermore O |
|
, O |
|
the O |
|
ternary O |
|
crystal B-evidence |
|
structure I-evidence |
|
has O |
|
guided O |
|
in B-experimental_method |
|
silico I-experimental_method |
|
and I-experimental_method |
|
biophysical I-experimental_method |
|
analyses I-experimental_method |
|
to O |
|
suggest O |
|
a O |
|
direct O |
|
LRP6 B-site |
|
- I-site |
|
KRM1 I-site |
|
interaction I-site |
|
site I-site |
|
. O |
|
|
|
Our O |
|
findings O |
|
provide O |
|
a O |
|
solid O |
|
foundation O |
|
for O |
|
additional O |
|
studies O |
|
to O |
|
probe O |
|
how O |
|
ternary O |
|
complex O |
|
formation O |
|
triggers O |
|
internalization O |
|
, O |
|
whereas O |
|
Krm B-protein_type |
|
binding O |
|
in O |
|
the O |
|
absence B-protein_state |
|
of I-protein_state |
|
Dkk B-protein_type |
|
stabilizes O |
|
the O |
|
Wnt B-protein_type |
|
co B-protein_type |
|
- I-protein_type |
|
receptor I-protein_type |
|
at O |
|
the O |
|
cell O |
|
surface O |
|
. O |
|
|
|
Structure B-evidence |
|
of O |
|
Unliganded B-protein_state |
|
KRM1ECD B-protein |
|
|
|
( O |
|
A O |
|
) O |
|
The O |
|
KRM1ECD B-protein |
|
fold O |
|
( O |
|
crystal B-evidence |
|
form I-evidence |
|
I I-evidence |
|
) O |
|
colored O |
|
blue O |
|
to O |
|
red O |
|
from O |
|
the O |
|
N O |
|
to O |
|
C O |
|
terminus O |
|
. O |
|
|
|
Cysteines B-residue_name |
|
as O |
|
ball O |
|
and O |
|
sticks O |
|
, O |
|
glycosylation B-site |
|
sites I-site |
|
as O |
|
sticks O |
|
. O |
|
|
|
The O |
|
bound O |
|
calcium B-chemical |
|
is O |
|
shown O |
|
as O |
|
a O |
|
gray O |
|
sphere O |
|
. O |
|
|
|
The O |
|
site O |
|
of O |
|
the O |
|
F207S B-mutant |
|
mutation O |
|
associated O |
|
with O |
|
ectodermal O |
|
dysplasia O |
|
in O |
|
humans B-species |
|
is O |
|
shown O |
|
as O |
|
mesh O |
|
. O |
|
|
|
( O |
|
B O |
|
) O |
|
Superposition B-experimental_method |
|
of O |
|
the O |
|
three O |
|
KRM1ECD B-protein |
|
subdomains O |
|
( O |
|
solid O |
|
) O |
|
with O |
|
their O |
|
next O |
|
structurally O |
|
characterized O |
|
homologs O |
|
( O |
|
half O |
|
transparent O |
|
). O |
|
|
|
( O |
|
C O |
|
) O |
|
Superposition B-experimental_method |
|
of O |
|
KRM1ECD B-protein |
|
from O |
|
the O |
|
three O |
|
crystal B-evidence |
|
forms I-evidence |
|
. O |
|
|
|
Alignment B-evidence |
|
scores I-evidence |
|
for O |
|
each O |
|
pairing O |
|
are O |
|
indicated O |
|
on O |
|
the O |
|
dashed O |
|
triangle O |
|
. O |
|
|
|
( O |
|
A O |
|
) O |
|
The O |
|
structure B-evidence |
|
of O |
|
the O |
|
ternary O |
|
LRP6PE3PE4 B-complex_assembly |
|
- I-complex_assembly |
|
DKK1CRD2 I-complex_assembly |
|
- I-complex_assembly |
|
KRM1ECD I-complex_assembly |
|
complex O |
|
. O |
|
|
|
DKK1 B-protein |
|
( O |
|
orange O |
|
) O |
|
is O |
|
sandwiched O |
|
between O |
|
the O |
|
PE3 B-structure_element |
|
module O |
|
of O |
|
LRP6 B-protein |
|
( O |
|
blue O |
|
) O |
|
and O |
|
the O |
|
KR B-structure_element |
|
- I-structure_element |
|
WSC I-structure_element |
|
domain O |
|
pair O |
|
of O |
|
KRM1 B-protein |
|
( O |
|
green O |
|
). O |
|
|
|
Colored O |
|
symbols O |
|
indicate O |
|
introduced O |
|
N B-site |
|
- I-site |
|
glycan I-site |
|
attachment I-site |
|
sites I-site |
|
( O |
|
see O |
|
D O |
|
). O |
|
|
|
( O |
|
B O |
|
) O |
|
SPR B-experimental_method |
|
data O |
|
comparing O |
|
binding O |
|
of O |
|
full B-protein_state |
|
- I-protein_state |
|
length I-protein_state |
|
DKK1 B-protein |
|
and O |
|
SUMO B-experimental_method |
|
fusions I-experimental_method |
|
of O |
|
DKK1 B-protein |
|
truncations O |
|
for O |
|
binding O |
|
to O |
|
immobilized O |
|
wild B-protein_state |
|
- I-protein_state |
|
type I-protein_state |
|
KRM1ECD B-protein |
|
. O |
|
|
|
( O |
|
C O |
|
) O |
|
Close O |
|
- O |
|
up O |
|
view O |
|
of O |
|
the O |
|
DKK1CRD2 B-site |
|
- I-site |
|
KRM1ECD I-site |
|
interface I-site |
|
. O |
|
|
|
Residues O |
|
involved O |
|
in O |
|
interface B-site |
|
formation O |
|
are O |
|
shown O |
|
as O |
|
sticks O |
|
; O |
|
those O |
|
mentioned O |
|
in O |
|
the O |
|
text O |
|
are O |
|
labeled O |
|
. O |
|
|
|
Salt B-bond_interaction |
|
bridges I-bond_interaction |
|
are O |
|
in O |
|
pink O |
|
and O |
|
hydrogen B-bond_interaction |
|
bonds I-bond_interaction |
|
in O |
|
black O |
|
. O |
|
|
|
( O |
|
D O |
|
) O |
|
SPR B-experimental_method |
|
binding B-evidence |
|
data I-evidence |
|
comparing O |
|
DKK1 B-protein |
|
analyte O |
|
binding O |
|
with O |
|
wild B-protein_state |
|
- I-protein_state |
|
type I-protein_state |
|
KRM1ECD B-protein |
|
and O |
|
three O |
|
variants O |
|
bearing O |
|
engineered B-protein_state |
|
glycosylation B-site |
|
sites I-site |
|
on O |
|
the O |
|
KR B-structure_element |
|
and O |
|
WSC B-structure_element |
|
domains O |
|
( O |
|
green O |
|
and O |
|
blue O |
|
pointing O |
|
to O |
|
DKK1 B-protein |
|
) O |
|
and O |
|
on O |
|
the O |
|
CUB B-structure_element |
|
domain O |
|
( O |
|
orange O |
|
). O |
|
|
|
LRP6 B-complex_assembly |
|
- I-complex_assembly |
|
KRM1 I-complex_assembly |
|
Direct O |
|
Interaction O |
|
and O |
|
Summary O |
|
|
|
( O |
|
A O |
|
) O |
|
In O |
|
a O |
|
construction O |
|
of O |
|
a O |
|
ternary O |
|
complex B-protein_state |
|
with I-protein_state |
|
all O |
|
four O |
|
β B-structure_element |
|
propellers I-structure_element |
|
of O |
|
LRP6 B-protein |
|
intact B-protein_state |
|
, O |
|
the O |
|
CUB B-structure_element |
|
domain O |
|
points O |
|
via O |
|
its O |
|
Ca2 B-site |
|
+- I-site |
|
binding I-site |
|
region I-site |
|
toward O |
|
the O |
|
top O |
|
face O |
|
of O |
|
the O |
|
second B-structure_element |
|
β I-structure_element |
|
propeller I-structure_element |
|
. O |
|
|
|
( O |
|
B O |
|
) O |
|
Close O |
|
- O |
|
up O |
|
view O |
|
of O |
|
the O |
|
potential O |
|
interaction B-site |
|
site I-site |
|
. O |
|
|
|
In O |
|
addition O |
|
, O |
|
LRP6PE2 B-protein |
|
has O |
|
been O |
|
superimposed B-experimental_method |
|
with O |
|
DKK1 B-protein |
|
( O |
|
yellow O |
|
) O |
|
and O |
|
SOST B-protein |
|
( O |
|
pink O |
|
) O |
|
peptide O |
|
complexes O |
|
of O |
|
LRP6PE1 B-protein |
|
. O |
|
|
|
( O |
|
C O |
|
) O |
|
SPR B-experimental_method |
|
measurements I-experimental_method |
|
comparing O |
|
LRP6PE1PE2 B-protein |
|
binding O |
|
with O |
|
wild B-protein_state |
|
- I-protein_state |
|
type I-protein_state |
|
KRM1ECD B-protein |
|
and O |
|
the O |
|
GlycoCUB B-protein_state |
|
mutant I-protein_state |
|
bearing O |
|
an O |
|
N B-ptm |
|
- I-ptm |
|
glycan I-ptm |
|
at O |
|
N309 B-residue_name_number |
|
. O |
|
|
|
( O |
|
D O |
|
) O |
|
Schematic O |
|
representation O |
|
of O |
|
structural O |
|
and O |
|
biophysical O |
|
findings O |
|
and O |
|
their O |
|
implications O |
|
for O |
|
Wnt B-protein_type |
|
- O |
|
dependent O |
|
( O |
|
left O |
|
, O |
|
middle O |
|
) O |
|
and O |
|
independent O |
|
( O |
|
right O |
|
) O |
|
signaling O |
|
. O |
|
|
|
Conformational O |
|
differences O |
|
in O |
|
the O |
|
depictions O |
|
of O |
|
LRP6 B-protein |
|
are O |
|
included O |
|
purely O |
|
for O |
|
ease O |
|
of O |
|
representation O |
|
. O |
|
|
|
Diffraction B-evidence |
|
and I-evidence |
|
Refinement I-evidence |
|
Statistics I-evidence |
|
|
|
KRM1ECD B-protein |
|
KRM1ECD B-protein |
|
KRM1ECD B-protein |
|
KRM1ECD B-protein |
|
LRP6PE3PE4 B-complex_assembly |
|
- I-complex_assembly |
|
DKKCRD2 I-complex_assembly |
|
- I-complex_assembly |
|
KRM1ECD I-complex_assembly |
|
Crystal O |
|
form O |
|
I O |
|
I O |
|
II O |
|
III O |
|
I O |
|
X O |
|
- O |
|
ray O |
|
source O |
|
Diamond O |
|
i04 O |
|
Diamond O |
|
i03 O |
|
Diamond O |
|
i03 O |
|
Diamond O |
|
i04 O |
|
Diamond O |
|
i04 O |
|
Wavelength O |
|
( O |
|
Å O |
|
) O |
|
0 O |
|
. O |
|
9793 O |
|
0 O |
|
. O |
|
9700 O |
|
0 O |
|
. O |
|
9700 O |
|
0 O |
|
. O |
|
9795 O |
|
0 O |
|
. O |
|
9795 O |
|
Space O |
|
group O |
|
P3121 O |
|
P3121 O |
|
P43 O |
|
P41212 O |
|
C2221 O |
|
Unit O |
|
cell O |
|
a O |
|
/ O |
|
α O |
|
( O |
|
Å O |
|
/°) O |
|
50 O |
|
. O |
|
9 O |
|
/ O |
|
90 O |
|
50 O |
|
. O |
|
5 O |
|
/ O |
|
90 O |
|
65 O |
|
. O |
|
8 O |
|
/ O |
|
90 O |
|
67 O |
|
. O |
|
8 O |
|
/ O |
|
90 O |
|
86 O |
|
. O |
|
9 O |
|
/ O |
|
90 O |
|
b O |
|
/ O |
|
β O |
|
( O |
|
Å O |
|
/°) O |
|
50 O |
|
. O |
|
9 O |
|
/ O |
|
90 O |
|
50 O |
|
. O |
|
5 O |
|
/ O |
|
90 O |
|
65 O |
|
. O |
|
8 O |
|
/ O |
|
90 O |
|
67 O |
|
. O |
|
8 O |
|
/ O |
|
90 O |
|
100 O |
|
. O |
|
1 O |
|
/ O |
|
90 O |
|
c O |
|
/ O |
|
γ O |
|
( O |
|
Å O |
|
/°) O |
|
188 O |
|
. O |
|
4 O |
|
/ O |
|
120 O |
|
187 O |
|
. O |
|
4 O |
|
/ O |
|
120 O |
|
75 O |
|
. O |
|
0 O |
|
/ O |
|
90 O |
|
198 O |
|
. O |
|
2 O |
|
/ O |
|
90 O |
|
270 O |
|
. O |
|
7 O |
|
/ O |
|
90 O |
|
Wilson O |
|
B O |
|
factor O |
|
( O |
|
Å2 O |
|
) O |
|
31 O |
|
41 O |
|
76 O |
|
77 O |
|
NA O |
|
Resolution O |
|
range O |
|
( O |
|
Å O |
|
) O |
|
47 O |
|
. O |
|
10 O |
|
– O |
|
1 O |
|
. O |
|
90 O |
|
( O |
|
1 O |
|
. O |
|
95 O |
|
– O |
|
1 O |
|
. O |
|
90 O |
|
) O |
|
62 O |
|
. O |
|
47 O |
|
– O |
|
2 O |
|
. O |
|
10 O |
|
( O |
|
2 O |
|
. O |
|
16 O |
|
– O |
|
2 O |
|
. O |
|
10 O |
|
) O |
|
75 O |
|
. O |
|
00 O |
|
– O |
|
2 O |
|
. O |
|
80 O |
|
( O |
|
2 O |
|
. O |
|
99 O |
|
– O |
|
2 O |
|
. O |
|
80 O |
|
) O |
|
67 O |
|
. O |
|
80 O |
|
– O |
|
3 O |
|
. O |
|
20 O |
|
( O |
|
3 O |
|
. O |
|
42 O |
|
– O |
|
3 O |
|
. O |
|
20 O |
|
) O |
|
67 O |
|
. O |
|
68 O |
|
– O |
|
3 O |
|
. O |
|
50 O |
|
( O |
|
7 O |
|
. O |
|
16 O |
|
– O |
|
6 O |
|
. O |
|
40 O |
|
, O |
|
3 O |
|
. O |
|
92 O |
|
– O |
|
3 O |
|
. O |
|
50 O |
|
) O |
|
Unique O |
|
reflections O |
|
23 O |
|
, O |
|
300 O |
|
( O |
|
1 O |
|
, O |
|
524 O |
|
) O |
|
17 O |
|
, O |
|
089 O |
|
( O |
|
1 O |
|
, O |
|
428 O |
|
) O |
|
7 O |
|
, O |
|
964 O |
|
( O |
|
1 O |
|
, O |
|
448 O |
|
) O |
|
8 O |
|
, O |
|
171 O |
|
( O |
|
1 O |
|
, O |
|
343 O |
|
) O |
|
8 O |
|
, O |
|
070 O |
|
( O |
|
723 O |
|
, O |
|
645 O |
|
) O |
|
Average O |
|
multiplicity O |
|
9 O |
|
. O |
|
1 O |
|
( O |
|
9 O |
|
. O |
|
2 O |
|
) O |
|
5 O |
|
. O |
|
2 O |
|
( O |
|
5 O |
|
. O |
|
3 O |
|
) O |
|
3 O |
|
. O |
|
7 O |
|
( O |
|
3 O |
|
. O |
|
7 O |
|
) O |
|
22 O |
|
. O |
|
7 O |
|
( O |
|
12 O |
|
. O |
|
6 O |
|
) O |
|
3 O |
|
. O |
|
8 O |
|
( O |
|
3 O |
|
. O |
|
5 O |
|
, O |
|
4 O |
|
. O |
|
4 O |
|
) O |
|
Completeness O |
|
(%) O |
|
99 O |
|
. O |
|
8 O |
|
( O |
|
98 O |
|
. O |
|
5 O |
|
) O |
|
100 O |
|
( O |
|
100 O |
|
) O |
|
99 O |
|
. O |
|
8 O |
|
( O |
|
100 O |
|
) O |
|
98 O |
|
. O |
|
8 O |
|
( O |
|
93 O |
|
. O |
|
4 O |
|
) O |
|
51 O |
|
. O |
|
6 O |
|
( O |
|
98 O |
|
. O |
|
5 O |
|
, O |
|
14 O |
|
. O |
|
1 O |
|
) O |
|
< O |
|
I O |
|
/ O |
|
σI O |
|
> O |
|
11 O |
|
. O |
|
4 O |
|
( O |
|
1 O |
|
. O |
|
7 O |
|
) O |
|
12 O |
|
. O |
|
0 O |
|
( O |
|
1 O |
|
. O |
|
7 O |
|
) O |
|
14 O |
|
. O |
|
9 O |
|
( O |
|
1 O |
|
. O |
|
5 O |
|
) O |
|
13 O |
|
. O |
|
1 O |
|
( O |
|
1 O |
|
. O |
|
9 O |
|
) O |
|
4 O |
|
. O |
|
6 O |
|
( O |
|
4 O |
|
. O |
|
1 O |
|
, O |
|
2 O |
|
. O |
|
2 O |
|
) O |
|
Rmerge O |
|
(%) O |
|
14 O |
|
. O |
|
8 O |
|
( O |
|
158 O |
|
. O |
|
3 O |
|
) O |
|
9 O |
|
. O |
|
3 O |
|
( O |
|
98 O |
|
. O |
|
0 O |
|
) O |
|
6 O |
|
. O |
|
2 O |
|
( O |
|
98 O |
|
. O |
|
9 O |
|
) O |
|
29 O |
|
. O |
|
8 O |
|
( O |
|
142 O |
|
. O |
|
2 O |
|
) O |
|
44 O |
|
. O |
|
9 O |
|
( O |
|
40 O |
|
. O |
|
5 O |
|
, O |
|
114 O |
|
. O |
|
2 O |
|
) O |
|
Rpim O |
|
(%) O |
|
15 O |
|
. O |
|
7 O |
|
( O |
|
55 O |
|
. O |
|
3 O |
|
) O |
|
10 O |
|
. O |
|
3 O |
|
( O |
|
109 O |
|
. O |
|
0 O |
|
) O |
|
3 O |
|
. O |
|
7 O |
|
( O |
|
53 O |
|
. O |
|
8 O |
|
) O |
|
6 O |
|
. O |
|
3 O |
|
( O |
|
40 O |
|
. O |
|
0 O |
|
) O |
|
24 O |
|
. O |
|
7 O |
|
( O |
|
23 O |
|
. O |
|
9 O |
|
, O |
|
59 O |
|
. O |
|
9 O |
|
) O |
|
Refinement O |
|
Rwork O |
|
(%) O |
|
17 O |
|
. O |
|
9 O |
|
18 O |
|
. O |
|
4 O |
|
21 O |
|
. O |
|
6 O |
|
20 O |
|
. O |
|
2 O |
|
32 O |
|
. O |
|
1 O |
|
Rfree O |
|
(%) O |
|
22 O |
|
. O |
|
7 O |
|
23 O |
|
. O |
|
2 O |
|
30 O |
|
. O |
|
7 O |
|
27 O |
|
. O |
|
1 O |
|
35 O |
|
. O |
|
5 O |
|
No O |
|
. O |
|
of O |
|
Non O |
|
- O |
|
Hydrogen O |
|
Atoms O |
|
Protein O |
|
2 O |
|
, O |
|
260 O |
|
2 O |
|
, O |
|
301 O |
|
2 O |
|
, O |
|
102 O |
|
2 O |
|
, O |
|
305 O |
|
7 O |
|
, O |
|
730 O |
|
N O |
|
- O |
|
glycans O |
|
42 O |
|
42 O |
|
28 O |
|
28 O |
|
0 O |
|
Water B-chemical |
|
79 O |
|
54 O |
|
0 O |
|
2 O |
|
0 O |
|
Ligands O |
|
6 O |
|
6 O |
|
2 O |
|
5 O |
|
0 O |
|
Average O |
|
B O |
|
factor O |
|
( O |
|
Å2 O |
|
) O |
|
Protein O |
|
63 O |
|
65 O |
|
108 O |
|
84 O |
|
– O |
|
N O |
|
- O |
|
glycans O |
|
35 O |
|
46 O |
|
102 O |
|
18 O |
|
– O |
|
Water B-chemical |
|
68 O |
|
85 O |
|
– O |
|
75 O |
|
– O |
|
Ligands O |
|
36 O |
|
47 O |
|
91 O |
|
75 O |
|
66 O |
|
RMSD B-evidence |
|
from O |
|
Ideality O |
|
Bond O |
|
lengths O |
|
( O |
|
Å O |
|
) O |
|
0 O |
|
. O |
|
020 O |
|
0 O |
|
. O |
|
016 O |
|
0 O |
|
. O |
|
019 O |
|
0 O |
|
. O |
|
016 O |
|
0 O |
|
. O |
|
004 O |
|
Bond O |
|
angles O |
|
(°) O |
|
2 O |
|
. O |
|
050 O |
|
1 O |
|
. O |
|
748 O |
|
1 O |
|
. O |
|
952 O |
|
1 O |
|
. O |
|
796 O |
|
0 O |
|
. O |
|
770 O |
|
Ramachandran O |
|
Plot O |
|
Favored O |
|
(%) O |
|
96 O |
|
. O |
|
8 O |
|
95 O |
|
. O |
|
5 O |
|
96 O |
|
. O |
|
9 O |
|
94 O |
|
. O |
|
9 O |
|
92 O |
|
. O |
|
3 O |
|
Allowed O |
|
(%) O |
|
99 O |
|
. O |
|
7 O |
|
100 O |
|
. O |
|
0 O |
|
100 O |
|
. O |
|
0 O |
|
99 O |
|
. O |
|
7 O |
|
99 O |
|
. O |
|
8 O |
|
Number O |
|
of O |
|
outliers O |
|
1 O |
|
0 O |
|
0 O |
|
1 O |
|
2 O |
|
PDB O |
|
code O |
|
5FWS O |
|
5FWT O |
|
5FWU O |
|
5FWV O |
|
5FWW O |
|
|
|
An O |
|
additional O |
|
shell O |
|
given O |
|
for O |
|
the O |
|
ternary O |
|
complex O |
|
corresponds O |
|
to O |
|
the O |
|
last O |
|
shell O |
|
with O |
|
near O |
|
- O |
|
complete O |
|
diffraction B-evidence |
|
data I-evidence |
|
. O |
|
|
|
|