Structures of netrin-1 bound to two receptors provide insight into its axon guidance mechanism.
ABSTRACT Netrins are secreted proteins that regulate axon guidance and neuronal migration. Deleted in colorectal cancer (DCC) is a well-established netrin-1 receptor mediating attractive responses. We provide evidence that its close relative neogenin is also a functional netrin-1 receptor that acts with DCC to mediate guidance in vivo. We determined the structures of a functional netrin-1 region, alone and in complexes with neogenin or DCC. Netrin-1 has a rigid elongated structure containing two receptor-binding sites at opposite ends through which it brings together receptor molecules. The ligand/receptor complexes reveal two distinct architectures: a 2:2 heterotetramer and a continuous ligand/receptor assembly. The differences result from different lengths of the linker connecting receptor domains fibronectin type III domain 4 (FN4) and FN5, which differs among DCC and neogenin splice variants, providing a basis for diverse signaling outcomes.
- SourceAvailable from: Nina Krüger[Show abstract] [Hide abstract]
ABSTRACT: Netrin-1 is a guidance cue that can trigger either attraction or repulsion effects on migrating axons of neurons, depending on the repertoire of receptors available on the growth cone. How a single chemotropic molecule can act in such contradictory ways has long been a puzzle at the molecular level. Here we present the crystal structure of netrin-1 in complex with the Deleted in Colorectal Cancer (DCC) receptor. We show that one netrin-1 molecule can simultaneously bind to two DCC molecules through a DCC-specific site and through a unique generic receptor binding site, where sulfate ions staple together positively charged patches on both DCC and netrin-1. Furthermore, we demonstrate that UNC5A can replace DCC on the generic receptor binding site to switch the response from attraction to repulsion. We propose that the modularity of binding allows for the association of other netrin receptors at the generic binding site, eliciting alternative turning responses.Neuron 08/2014; · 15.98 Impact Factor
competition through multiple mating and a higher
relative testes mass than humans (0.27% of av-
in male mutational bias are to explain observed
patterns of divergence, then gorillas would have
a male mutational bias lower than that of hu-
mans arising from decreased sperm competition
(12). Our results suggest that variation in mating
patterns between species can affect the sex bias
of mutation and motivate the wider study of
mutation rates and relationship to parental age
REFERENCES AND NOTES
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21. G. H. Perry, J. C. Marioni, P. Melsted, Y. Gilad, Mol. Ecol. 19,
22. C. Hvilsom et al., Proc. Natl. Acad. Sci. U.S.A. 109, 2054–2059
23. N. Patterson, D. J. Richter, S. Gnerre, E. S. Lander, D. Reich,
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25. A. P. Møller, J. Hum. Evol. 17, 479–488 (1988).
Funded by Wellcome Trust grants 086786/Z/08/Z to O.V. and
090532/Z/09/Z to the Wellcome Trust Centre for Human
Genetics and by MRC hub grant G0900747 91070. We thank
M. Przeworski and D. Reich for discussion and comments
on the manuscript and A. Kong for providing data on request
from reference (6). Samples were provided through the
Transnational Access Activity in the European Primate Network
(EUPRIM-NET) under the Convention on International Trade of
Endangered Species (CITES) authorization and a Material Transfer
Agreement between the University of Oxford and the Foundation
Biomedical Primate Research Centre. Read-level data are
accessible under SRA Study accession no. PRJEB5937 from www.
ebi.ac.uk/ena/data/view/PRJEB5937. All other project data are
available from ftp://birch.well.ox.ac.uk.
Materials and Methods
Figs. S1 to S12
Tables S1 to S11
23 January 2014; accepted 20 May 2014
Structures of netrin-1 bound to two
receptors provide insight into its axon
Kai Xu,1* Zhuhao Wu,2* Nicolas Renier,2* Alexander Antipenko,1
Dorothea Tzvetkova-Robev,1Yan Xu,1Maria Minchenko,1
Vincenzo Nardi-Dei,1† Kanagalaghatta R. Rajashankar,3Juha Himanen,1
Marc Tessier-Lavigne,2‡ Dimitar B. Nikolov1‡
Netrins are secreted proteins that regulate axon guidance and neuronal migration. Deleted
in colorectal cancer (DCC) is a well-established netrin-1 receptor mediating attractive
responses. We provide evidence that its close relative neogenin is also a functional
netrin-1 receptor that acts with DCC to mediate guidance in vivo. We determined the
structures of a functional netrin-1 region, alone and in complexes with neogenin or DCC.
Netrin-1 has a rigid elongated structure containing two receptor-binding sites at
opposite ends through which it brings together receptor molecules. The ligand/receptor
complexes reveal two distinct architectures: a 2:2 heterotetramer and a continuous
ligand/receptor assembly. The differences result from different lengths of the linker
connecting receptor domains fibronectin type III domain 4 (FN4) and FN5, which differs
among DCC and neogenin splice variants, providing a basis for diverse signaling outcomes.
and invasion, leukocyte migration, angiogenesis,
and cell survival (5). Netrins contain an N-terminal
laminin domain (LN, also known as domain VI),
followed by three cysteine-rich LN-type epidermal
growth factor (EGF)–like modules (LE1, LE2, and
LE3; also known as domain V), and a small posi-
tively charged C-terminal domain (LC). In mam-
mals, the secreted netrins-1, -3 and -4 are only
distantly related to the glycosylphosphatidylinositol–
anchored G netrins (6, 7).
Netrin actions are mediated by distinct recep-
tors (4). Deleted in colorectal cancer (DCC) me-
diates attractive responses to netrin-1, whereas
Unc5 proteins, alone or with DCC, are required
for its repulsive effects (4, 8). The ectodomain of
DCC is composed of four immunoglobulin-like
domains and six fibronectin type III (FNIII)
region, when added as an Fc-fusion protein, is
sufficient to mimic the axon outgrowth activity
of full-length netrin-1 (12). Neogenin is structur-
but also binds the structurally distinct repulsive
guidance molecule (RGM) (14, 15). Knockdown
etrins, acting as both attractants and re-
pellents, regulate neuronal migration, axon
guidance, and synaptogenesis (1–4). In non-
neural tissues, netrins have a variety of func-
tions, including promoting cell adhesion
in mediating axonal attraction to netrin (16), but
this role has not been established in mammals,
where it has mostly been studied as an adhesive
factor (17) and a putative guidance receptor for
We revisited the role of neogenin in netrin
attraction while studying commissural axon at-
traction to a netrin-1 source at the spinal cord
to neurological syndromes, some of which result
from mutations in DCC (18–20). Prior analysis
suggested that DCC mediates the entire attract-
ive effect of netrin-1 because the phenotype ob-
4D7to TAG-1, appeared stronger in Dcc mutant
than in netrin-1 mutant embryos (21). As new
markers became available (22), we reevaluated
embryos mutant for Dcc or netrin-1. Commis-
sural projections develop between embryonic
days 10.5 (E10.5) and E12.5, when spinal cord
shape changes rapidly. To minimize artifacts
from variation in embryo size and stage across
litters, we compared size-matched embryos that
were littermates from intercrosses of compound
heterozygous animals. By using an antibody to
Robo3 (22) in E11.5 Dcc mutant embryos, we
observed only a 55% reduction in width of the
ventral commissure compared with wild-type
littermates, less than in netrin-1−/−embryos,
which had a 78% reduction (Fig. 1, A and E).
The same was seen with a new antibody to
marker neurofilament-M (Fig. 1A). The differ-
ence with the prior study appears to result from
4D7 giving weaker labeling of commissural axons
that is also influenced by Dcc expression (fig. S1).
Thus, the guidance phenotype is actually less
severe than in Dcc than in netrin-1 mutants, sug-
gesting an additional netrin-1 receptor(s) con-
tributes to residual attraction in Dcc−/−embryos.
1Structural Biology Program, Memorial Sloan-Kettering Cancer
Center, New York, NY 10065, USA.2Laboratory of Brain
Development and Repair, Rockefeller University, New York, NY
10065, USA.3Department of Chemistry and Chemical Biology,
Cornell University and Northeastern Collaborative Access
Team, Advanced Photon Source, Argonne, IL 60439, USA.
*These authors contributed equally to this work. †Present address:
Novartis Vaccines, Siena, Italy. ‡Corresponding author. E-mail:
firstname.lastname@example.org (D.B.N.); email@example.com
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Totestthis,we examined whether Dcc mutant
vitro. We cultured dorsal spinal cord explants
from E11 wild-type and Dcc−/−embryos (Fig. 1, B
and C). In control explants, netrin-1 application
induced robust axonal outgrowth that peaked at
250 ng/ml. The peak response was reduced sig-
nificantly (by ~97%) when explants from Dcc−/−
embryos were used, confirming DCC’s central role
as a netrin-1 receptor, but a dose-dependent re-
sponse of Dcc mutant axons was still consistently
observed (Fig. 1, B and C). To determine which re-
ceptor mediates the residual netrin-1 response, we
screened known and putative netrin-1 receptors by
in situ hybridization and immunohistochemistry
in E11.5 spinal cord. We observed neogenin im-
munoreactivity on commissural axons (23), which
was lost in neogenin (Neo1) mutant spinal cords
rate with DCC in guiding these axons. Consistent
with this, whereas commissural axon trajectories
in transverse sections from Neo1−/−embryos were
apparently normal (Fig. 1D), removing neogenin
as well as DCC in Dcc−/−;Neo1−/−double mutants
resulted in an 84% reduction in ventral commis-
sure size, that is, greater than Dcc−/−but compa-
rable to netrin-1−/−embryos (Fig. 1, D and E).
Moreover, we observed abnormal Robo3+ com-
embryos;fewer are seen inDcc−/−single mutants,
but a comparable number was seen in Dcc−/−;
the Neo1−/−and netrin-1−/−alleles are severely
hypomorphic rather than complete null alleles
so our finding that commissural axon guidance
defects in Dcc−/−;Neo1−/−embryos are greater
than in Dcc−/−mutants but comparable to those in
netrin-1−/−mutants are consistent with the model
that neogenin is a functional netrin-1 receptor
that acts in concert with DCC to direct commis-
sural axons to the midline netrin source.
To study how neogenin and DCC function as
netrin-1 receptors, we investigated the structural
basis of the netrin-1/neogenin and netrin-1/DCC
interactions. There are conflicting reports regard-
ing which DCC FNIII domains mediate interac-
tions with the netrin-1 LN-LE region (9–11), so we
(materials and methods) to clarify this. Our re-
sults (Fig. 2A) show that domains FN4 and FN5
both interact with this ligand and that they ac-
count for the full in vitro binding affinity. Ac-
construct that contains the LN and LE(1-3) do-
mains and neogenin/DCC constructs containing
FN4 and FN5. We did not include LC [also known
as C345C, suggested to bind heparan sulfate (24)],
because it is attached via a flexible linker and not
strictly required for receptor binding (9–11) and
because a netrin-1–Fc fusion construct lacking
this domain induces similar axon outgrowth in
vitro as full-length netrin-1 (12). Splice variants
Fig. 1. Neogenin and Dcc collaborate to mediate the attraction of com-
missural neurons by netrin-1. (A) Cross sections of E11 wild-type (WT),
Dcc−/−and Netrin-1−/−littermate mouse embryos at the level of brachial spinal
ganglia, stained for TAG-1, Robo3, and neurofilament, medium chain (NF-M).
Bottom two rows show details of the ventral commissural axon bundle. Dcc
mutants have a reduced ventral commissure, but a large number of axons still
cross. Netrin-1−/−embryos have a much-reduced ventral commissure. (B)
Axon outgrowth (arrows) in E11 wild-type or Dcc−/−littermate mouse dorsal
spinal cord explants cultured in three-dimensional collagen gels with increasing
concentrations of netrin-1 and stained for Tuj1. (C) Quantification of the response
of axon outgrowth from wild-type and Dcc−/−dorsal spinal cord explants to
increasing netrin-1 concentrations, normalized to wild type at 250 ng/m of netrin-1.
Dcc−/−mutants show a residual response to netrin-1 application (arrow). ns,
not significant. (D) Cross sections of E11 wild-type, Dcc−/−, Neo1−/−and Dcc−/−;
Neo1−/−littermate mouse embryos at the level of brachial spinal ganglia,
stained for TAG-1, Robo3, and NF-M. Bottom two rows show details of the
motor column and the ventral commissural axon bundle.The Dcc−/−; Neo1−/−
double mutant has a much reduced ventral commissure and numerous axons
in the motor column. (E) Ratio of the commissural axon bundle size to the
dorsoventral spinal cord length of wild-type, Dcc−/−, and Netrin1−/−embryos,
normalized to wild types (left). Ratio of commissural axon bundle size to the
dorsoventral spinal cord length of Dcc−/−, Neo1−/−, and Dcc−/−; Neo1−/−
embryos normalized to wild type (right).The quantification shows the mean
and SEM of five sections taken in brachial spinal cord in littermates and is
representative of three litters. Scale bars are 200 mm (Robo3) and 100 mm
(TAG-1 and NF-M).
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(isoforms) of both neogenin and DCC with dif-
ferent length of the FN4-FN5 linker have been
reported in most species. Both shorter and longer
isoforms bind netrin-1 with high affinity (Fig. 2B).
For our structural studies, we used the shorter
The structure of the netrin-1 LN-LE region
was determined at 2.8 Å resolution (table S1
and figs. S3 to S7), revealing an elongated mol-
ecule with the same flowerlike shape as laminin
and netrin-G (Fig. 2C). The LN domain forms
the head, and LE the stalk. The disulfide bond
network throughout the molecule and the short
linkers between the individual netrin domains
result in a rigidmoleculararchitecture with little
interdomain flexibility. The globular LN domain
has the canonical laminin LN fold, including a
conserved Ca2+binding site. The LE region con-
tains three EGF repeats, and its structure is sim-
ilar to those of laminin-a5, laminin-g1 (26, 27) and
netrin-G (6, 7), although the latter lacks the third
EGF repeat (LE3).
The structure of the netrin/neogenin complex
(Fig. 3A) was determined at 3.2 Å resolution and
reveals a 2:2 heterotetramer, consistent with its
gel-filtration elution profile. At the heart of the
complex are two netrin molecules forming a
head-to-head X-shaped dimer and interacting
via an extensive LE2/LE2 interface. This dimer
brings together two neogenin molecules, with
and their C-termini facing the same direction,
presumably toward the neuronal membrane.
The two receptor binding regions are located
about 90 Å apart on the two ends of the rigid
netrin structure, but the distance between netrin-
binding surfaces of neogenin FN4 and FN5 do-
mains cannot exceed 55 Å, so the two receptor
binding sites on netrin must interact with two
different receptor molecules. Netrin does not
undergo any significant conformational changes
upon receptor binding, and the bound and un-
bound netrin structures could be superposed
with root mean square deviation (RMSD) of
0.9 Å over 353 Ca atoms.
arranged linearly, with the linker between them
would be flexible in the absence of bound ligand.
The netrin/neogenin 2:2 complex (Fig. 3) con-
tains five protein-protein interfaces that fall in
three categories: Interface-1, between neogenin-
FN4 and netrin-LN, buries ~680 Å2in each inter-
acting domain and is dominated by van der Waals
interactions between two largely hydrophobic
hydrogen bonds and a salt bridge (Fig. 3B and
figs. S3, S4, and S8). The LN Ca2+binding site is
immediately adjacent to interface-1, and bound
Ca2+would be required to maintain its proper
conformation. Indeed, EDTA reduces the netrin/
receptor binding affinities (Fig. 2B).
Interface-2, between neogenin-FN5 and netrin-
LE3, buries ~610 Å2in each interacting domain
and contains a mix of hydrophobic and polar con-
tacts, including hydrogen bonds involving main-
S4, and S8). This interface is centered around
Met985of neogenin, the hydrophobic side chain
of which is buried in a netrin surface hydropho-
bic pocket. Interface-1 is slightly larger and more
binding affinity measurements (Fig. 2A).
the netrin/neogenin 2:2 complex, burying ~1020 Å2
thanthe other netrin LE domains, containing an
Fig. 2. Structure and receptor-binding affin-
ity of the netrin-1 LN-LE region. (A) Binding of
netrin-1 (LN-LE1-LE2-LE3) to different DCC con-
structs documenting that the receptor FN4-FN5
region is necessary and sufficient for netrin binding.
Kd, dissociation constant (in mM). (B) Binding of
netrin-1 (LN-LE1-LE2-LE3) to the FN4-FN5 region
of the different neogenin and DCC isoforms (fig.
S4).To evaluate the role of the netrin-bound Ca++,
10 mM EDTA was added in one of the measure-
ments. (C) Structure of unbound netrin-1. The in-
dividual netrin domains are labeled and colored in
blue (LN), green (LE1), pink (LE2), and red (LE3).
The glycosylation moieties at the three glycosyl-
ated Asn residues are shown as gray spheres.The
N- and C-termini are labeled. (Inset) A close-up
view of the calcium-binding sire in the LN domain.
The calcium ion is drawn in magenta and two bound
water molecules in red. D, Asp; F, Phe; S, Ser;T,Thr;
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extra strand-helix-strand motif, which provides
most of the dimerization contacts. This netrin
region is conserved between the canonical netrins
(netrin1 to5)butisvery differentinthe Gnetrins,
suggesting that the latter might not support this
netrin-dimer architecture. Interface-3 is twofold
symmetric, although it is not on a crystallographic
symmetry axis because the crystal asymmetric
unit contains the full 2:2 heterotetramer. Un-
like interface-1 and -2, the vast majority of the
interface-3 residues are polar, forming several
hydrogen bonds and four salt bridges (Fig. 3D
and figs. S3 and S8).
The netrin LN-LE region is positively charged
FN4-FN5 region (pI ~9.2). The main positively
charged surfaces on netrin (on its LE2 domain)
and receptors (on FN5) are exposed to solvent in
the complex, making them potentially available
for interactions with negatively charged entities
like proteoglycans (28, 29).
The structure of the netrin-1/DCC complex
(Fig. 4A) was determined at 2.9 Å resolution and
shows a different overall architecture than the
netrin-1/neogeninstructure,namely a continuous
-DCC-netrin-DCC-netrin-DCC- assembly. Each netrin
molecule still interacts, via its two receptor binding
sites on the LN and LE3 domains, with two dif-
ferent DCC molecules. At the same time, each
DCC receptor interacts with two netrin mole-
cules via its two distinct netrin-binding sites on
FN4 and FN5, but these two netrins are shared
with two other DCC neighbors (Fig. 4D). The
reason the netrin/neogenin complex architec-
ture cannot be replicatedinthe netrin/DCC com-
plexis thatthe FN4-FN5 linker is slightly shorter
in DCC than in neogenin (fig. S4) and the DCC
linker also forms a short a helix. Formation of
the 2:2 netrin-1/neogenin signaling complex
aroundthe X-shaped netrin dimer requires full
extension of neogenin FN4-FN5 linkers, but the
DCC FN4-FN5 linker is not long enough to ac-
commodate this architecture. As in the netrin/
neogenin complex,theDCCreceptor molecules
the same direction.
The individual DCC and neogenin FNIII do-
mains share about 70% sequence identity and
their structures are very similar, with RMSDs be-
tween equivalent Ca positions of 0.47 Å for FN4
and 0.38 Å for FN5. The netrin-1 structure is also
very similar in the complexes with its two recep-
tors, with RMSDs between Ca positions of
0.73 Å. The netrin/DCC interfaces are nearly
identical to the netrin/neogenin interfaces (Fig. 4
andfig.S4).Interface-1 is again formed between
the netrin LN domain and the DCC FN4 do-
main, whereas interface-2 is formed between the
netrin LE3 domain and the DCC FN5 domain.
Figure 4, B and C, illustrates the similarly of the
interacting LN-FN4 and LE3-FN5 domains in
the two complexes.
On the basis of the structures reported here,
we propose that netrin induces signaling by
binding to and bringing together receptor mol-
ecules via its two binding sites, thus creating
ligand/receptor signaling assemblies at the neu-
bivalent complex with a single receptor mole-
cule, even with the long isoforms, because the
distance between the two receptor binding sites
on netrin is larger than the distance between
the two ligand-binding sites on receptors. Our
structures illustrate the potential for netrins to
cross-link different receptor types via distinct
receptor binding sites: for example, DCC or
on one end and neogenin the other. The netrin
LC domain might further concentrate or cluster
assemblies, for example, via interactions with
heparan sulfate, because the degree of axon out-
to a dimeric Fc tag is similar to that with full-
length netrin-1 and much greater than with the
LN-LE region alone (12).
The differences in the two signaling architec-
tures result from different lengths of the linker
connecting the receptor FN4 and FN5 domains,
which differs between the DCC and neogenin iso-
forms studied here. In most species with these
molecules, two different isoforms for each recep-
tor, short and long, arise from alternative splicing
of the FN4-FN5 linker sequence (25) (fig. S4). The
Fig. 3. Structure of the netrin-1/neogenin com-
plex. (A) Structure of the 2:2 netrin-1/neogenin
complex. The netrin molecules are in green and
blue and the neogenin in orange and magenta.
The N- and C-termini of the molecules are labeled.
(B) Close-up view of the netrin-LN/neogenin-FN4
interface (interface-1). Interacting residues and
the netrin-bound calcium are labeled. (C) Close-
up view of the netrin-LE3/neogenin-FN5 interface
(interface-2). Interacting residues are labeled. (D)
Close-up view of the netrin-LE2/netrin-LE2 inter-
face (interface-3). Interacting residues are labeled.
C, Cys; G, Gly; H, His; M, Met; P, Pro; Q, Gln; V,Val.
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two neogenin isoforms and the long DCC isoform
all contain linkers long enough to support the 2:2
signaling-complex architecture,whereasthe shorter
DCC isoform does not allow this formation. Our
assembly is energetically favored over the contin-
large interface between the two netrin molecules
that it permits. The short neogenin and DCC iso-
(30) and could have distinct signaling properties
mediated by distinct signaling-complex architec-
in any given cell type to determine the potential
of distinct assemblies to elicit particular cellular
responses to netrin.
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30. C. Manitt, K. M. Thompson, T. E. Kennedy, J. Neurosci. Res. 77,
We thank M. Kolev for technical support, M. Himanen for
illustrations, Y. Goldgur for help with data collection and
processing, and O. Olsen for help and support. X-ray diffraction
studies were conducted at the Advanced Photon Source on the
Northeastern Collaborative Access Team beamlines, which are
supported by a grant from the National Institute of General Medical
Sciences (P41 GM103403) from NIH. Use of the Advanced
Photon Source, an Office of Science User Facility operated for
the U.S. Department of Energy (DOE) Office of Science by
Argonne National Laboratory, was supported by the U.S. DOE
under contract no. DE-AC02-06CH11357. Z.W. was supported in
part by a Bristol-Myers Squibb postdoctoral fellowship at the
Rockefeller University; N.R. was supported by a European
Molecular Biology Organization long-term postdoctoral fellowship.
The netrin-1, netrin-1/neogenin, and netrin-1/DCC structures
have been deposited in the Protein Data Bank under codes 4PLM,
4PLN, and 4PLO, respectively.
Materials and Methods
Figs. S1 to S9
23 April 2014; accepted 20 May 2014
Published online 29 May 2014;
Fig. 4. Structure of the netrin-1/DCC complex. (A) Structure of the netrin-1/
DCC complex. Netrin-1 is colored in blue, and DCC in red. (B) Superposition of
the LN-FN4 interaction site (interface-1) in the netrin-1/DCC (blue/red) and
netrin-1/neogenin (gray/yellow) complexes. (C) Superimposition of the LE3-
FN5 interaction site (interface-2) in the netrin-1/DCC (blue/red) and netrin-1/
neogenin (gray/yellow) complexes. (D) Schematic drawing comparing the
distinct netrin-1/neogenin and netrin-1/DCC signaling assemblies. The in-
dividual netrin and receptor domains are labeled. Ig, immunoglobulin. (Top)
Schematic representation of the 2:2 netrin-1/neogenin complex.The netrin
molecules are colored in blue and green, and the neogenin in yellow and
magenta. (Bottom) Schematic representation of the continuous netrin-1/
DCC assembly.The netrin molecules are colored in blue, and DCC in red. In
both netrin-1/DCC and netrin-1/neogenin assemblies, the positively charged
netrin LC domain would be positioned toward the negatively charged plasma
membrane, thus potentially further stabilizing the signaling complexes at
the neuronal surface.
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