The crystal structure of netrin-1 in complex with DCC reveals the bifunctionality of netrin-1 as a guidance cue

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.

Figure 1. Domain architecture and crystal structure of the netrin-1/DCC complex

(A) Domain diagram of DCC and netrin-1, with the domains present in the crystal structure colored red. (B) Ribbon diagram of the Netrin_VIV/DCC_FN56 complex is depicted showing two DCC fragments (in green) bound to the V domain (in salmon red) of one netrin-1 molecule. Glycosylation sites on the VI domain of netrin-1 (in orange) are shown as sticks.

Figure 2. The DCC specific binding site on netrin-1

(A) Close-up view of binding site 1 with DCC_FN56 represented in green as a ribbon and the EGF-3 domain of Netrin_VIV in salmon represented as a surface. (B) Close-up view of the hotspot interface on site 1 with DCC in green and Netrin_VIV colored salmon, with several tentative hydrogen bonds drawn as dotted lines. (C) Cell binding assays for full-length DCC Val848, Met933 and Netrin_VIV Gln443 mutants, showing the percentage of DCC presenting COS-7 cells that bind netrin-1 or Netrin_VIV. (D) DCC multimerization assays for DCC Val 848, Met933 and Netrin_VIV Gln443 mutants showing enrichment of oppositely tagged receptors. (E) Axon guidance assays for DCC Val848, Met933 and Netrin_VIV Gln443 mutants showing percentage of beads attracting axons.

Figure 3. Detailed molecular view of generic receptor binding site 2 on netrin-1

(A) An open book view of the regions on netrin-1 and DCC involved in binding site 2, showing the electrostatic surface potential to emphasize the positively charged regions within the binding site interface. (B) View of the sulfate cluster at binding site 2, with sulfate ions shows as sticks, and the surface of the netrin-1 molecule shown with its electrostatic surface potential. The FN5 and FN6 domains of DCC are shown as a ribbon diagram in green. (C) Identification of a chloride ion (green ball) at the interface of binding site 2, between the EGF-1 domain of netrin-1 colored salmon and the FN5 domain of DCC colored light green. An F_o-F_c omit map was calculated using the CCP4 suite (Winn et al., 2011) for a model lacking the chloride ion, and the electron density is displayed at a contour level of 5δ

Figure 4. Mapping residues on the generic receptor binding site 2 by mutagenesis that contribute to DCC binding, multimerization and axon guidance

(A) Cell binding assays for DCC His857 and Asp858 and Netrin_VIV Arg349/Arg351 mutants, showing the percentage of DCC presenting COS-7 cells that bind netrin-1 or Netrin_VIV. (B) Receptor multimerization assays for DCC His857 and Asp858 and Netrin_VIV Arg349/Arg351 mutants showing enrichment of oppositely tagged receptors. (C) Axon guidance assays for DCC His857 and Asp858 and Netrin_VIV Arg349/Arg351 mutants showing percentage of beads attracting axons.

Figure 5. Molecular envelopes of Netrin_VIV and DCC_FN56 and SAXS analysis of Netrin_VIV/DCC_FN56 complex formation in solution

Ab initio molecular envelopes (grey spheres) calculated from the SAXS data overlaid onto the crystal structures (ribbons) of the individual (A) DCC_FN56 and (B) Netrin_VIV molecules with the glycosylation sites on netrin-1 depicted (red and black balls). (C) Bar diagrams of the volume fractions of components contributing to the overall scattering of a Netrin_VIV/DCC_FN56 and (D) a mutant Netrin_VIV/DCC_FN56-M933R mixture in solution. At a molar Netrin_VIV concentration of 40 PM, DCC_FN56 or DCC_FN56-M933R was mixed to achieve final molar stoichiometries of 1:1, 1:5 and 1:10.

Figure 6. Repulsion/attraction assays involving UNC5A and DCC

Radar plots showing the distribution of axon growth around the nucleus, with the netrin-1 coated beads at the origin. Repulsion/attraction assays were performed with DCC-/- neurons injected with DNA of UNC5A, DCC and DCC mutants, showing that DCC is required for UNC5A dependent repulsion. DCC mutants affecting site 1 (V858R and M933R) abolish repulsion, whereas DCC mutants affecting site 2 (H857A and D858R) still manifest repulsion, indicating UNC5A has outcompeted DCC on site 2.

Figure 7. Model of heparan sulfate dependent formation of the DCC/DCC and DCC/UNC5 complex with netrin-1

(A) Composite model of an extended netrin-1/DCC cluster based on the superimposition of the crystal structure presented here with the crystal structure of Netrin_VIV in complex with the FN4 and FN5 domains of DCC (Xu et al., 2014). The Netrin_VIV molecule are colored in cyan, the DCC molecules (FN4-FN5-FN6) occupying binding site 1 are colored green, and the DCC molecules (shown only FN5-FN6) occupying site 2 are colored purple. (B) DCC binds specifically to the EGF-3 domain of netrin-1 (binding site 1). Netrin-1 associates with a heparan sulfate molecule that is selective for DCC (HP1) or UNC5 (HP2). When heparan sulfate HP1 is bound to netrin-1, a second DCC molecule is recruited to binding site 2, and the cytosolic P3 domains of the two DCC molecules associate to form a signaling complex, leading to attraction of the growth cone. When heparan sulfate HP2 binds to netrin-1, UNC5A is recruited to the EGF-1/EGF-2 domains, and the cytosolic P1 domain of DCC and the region between the ZU5 and DB domain of UNC5A form a signaling complex, leading to repulsion of the growth cone.