Figure 2: Binding comparison of Net4 and Net1 to netrin receptors and laminin.

(a) Biolayer interferometry binding kinetic analysis of DCC, neogenin, and UNC5B to Net4 (red) as well as Net1 (green). AHC sensors were coated with the Fc chimera proteins. Association (from 0 to 300 s) and dissociation (from 300 to 600 s) phases are shown for 50 nM of both netrins. (b) The SAXS model for the Net4-FL-γ1LN-LEa1-4 complex was generated by firstly calculating models for Net4-ΔC-γ1LN-LEa1-4 complex (Supplementary Fig. 5d) followed by the addition of the LE and globular domain C. This model highlights the role of N-terminal globular domains in the interaction. The agreement between experimentally collected SAXS data and model-derived SAXS data is presented in Supplementary Table 1. (c) Rotary-shadowing electron microscopy image of Net4-ΔC-γ1LN-LEa1-4 clearly showing a 1:1 complex mediated via the globular domains. (d) Microscale thermophoresis binding analysis of different Net4 mutants to labelled γ1LN-LEa1-4. Binding of Net4 lacking the netrin-specific C domain (Net4-ΔC). Binding of full-length Net4 (Net4-FL). Analysis of the binding of a Net4 mutant (Net4-ΔC^ΔKAPGA) in which the specific loop b (KAPGA) within LE1 was deleted. Binding of a combined Net4 mutant (Net4-ΔC^{E195A,R199A,ΔKAPGA}). Error bars, s.d. (n=3 independent technical replicates). K_D values are shown in the graph (n.b., no binding). (e,f) Laminin-binding epitopes within Net4 (green) compared with the equivalent positions in Net1 (marine). Note loop b of LE1 of Net4 has an extensive contact area with the LN domain, and the KAPGA motif (shown in stick notation) is highly flexible. (g) Sequence alignment of Net4, Net1 and netrin-3 showing the laminin-binding region. (h) Solid-phase binding study of Net4 and Net1 to immobilized laminin γ1 (γ1LN-LEa1-4). Error bars, s.d. (n=3 independent technical replicates). Scale bar, (c) 100 nm.