Fig. 7: NELL2 trimers signal through multimerization of Robo3 monomers.

Sedimentation velocity (SV) AUC (a) and SEC (b) show that mRobo1 ectodomain is mostly dimeric (red curves), while mRobo3 ectodomain is a monomer (blue curves). Single-concentration SV runs (a) were performed with 2.5 µM Robo1 and 1.8 µM Robo3. See Supplementary Fig. 7 for a series of concentrations, which shows that mRobo1 ectodomain is in an equilibrium of monomer to dimer to oligomer, unlike the stably monomeric mRobo3. c Bead models calculated from SAXS data indicate a fully extended shape for hRobo3 ECD. Guinier plot for SAXS data is in Supplementary Fig. 8a. Pair distance distribution (P(r)) analysis is in agreement with the extended shape of Robo3. d Bead models calculated from SAXS data for hNELL2 indicate a trimer more compact than Robo3 ECD (c). Guinier plot analysis and P(r) plot are in Supplementary Fig. 9a. e DIC images of commissural axons exposed to wild-type NELL2 or NELL2^ΔCC (0 and 2 h). f Quantification of axon turning angles in response to NELL2 and NELL2^ΔCC (n = 3 for all conditions). NELL2^ΔCC repels axons to a lesser degree than wild-type NELL2. g A model for conformational changes in mammalian Robos. Open Robo3 prefers to bind trimeric NELLs, while multiple conformational models exist for Robo1 and Robo2^16,17, which are in “closed” states for NELL binding. The conformational flexibility is created by the IG4-IG5, IG5-FN1, and FN1-FN2 linkers. The lightly shaded area in the NELL–Robo complex model was structurally characterized in this study (shown on the left side of the model). “x3” labels indicate trimerization via the CC domain of NELLs. Scale bar, 10 μm (e).