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