Graphical Abstract Highlights d Crystal structure of the intact human Robo2 ectodomain at 3.6 Å d Dimerization through domain 4 (D4) is required for Robo axon guidance d Robo receptors have an auto-inhibited conformation in which D4 is blocked d Slit may relieve Robo auto-inhibition, followed by dimerization and signaling SUMMARYProper brain function requires high-precision neuronal expansion and wiring, processes controlled by the transmembrane Roundabout (Robo) receptor family and their Slit ligands. Despite their great importance, the molecular mechanism by which Robos' switch from ''off'' to ''on'' states remains unclear.Here, we report a 3.6 Å crystal structure of the intact human Robo2 ectodomain (domains D1-8). We demonstrate that Robo cis dimerization via D4 is conserved through hRobo1, 2, and 3 and the C. elegans homolog SAX-3 and is essential for SAX-3 function in vivo. The structure reveals two levels of auto-inhibition that prevent premature activation: (1) cis blocking of the D4 dimerization interface and (2) trans interactions between opposing Robo receptors that fasten the D4-blocked conformation. Complementary experiments in mouse primary neurons and C. elegans support the auto-inhibition model. These results suggest that Slit stimulation primarily drives the release of Robo auto-inhibition required for dimerization and activation.

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In this issue of Structure,Aleksandrova et al. (2018) present low- and high-resolution structures of Robo1, a key player in axonal guidance. The structures shed light on the arrangement of Robo1 at the plasma membrane and provide evidence for back-to-back trans Robo1 contacts.