Structural insights into the formation of repulsive Netrin guidance complexes

ABSTRACT Netrins can dictate attractive and repulsive responses during axon growth and cell migration, where presence of the receptor UNC-5 on target cells results in Netrin-mediated repulsion. Molecular details of Netrin–UNC-5 interactions and how they signal remain elusive. Here, we show that nematode UNC-5 is a heparin-binding protein, and the UNC-5–heparin affinity can be modulated using directed evolution or via rational design using our novel structure of UNC-5 with a heparin fragment. Furthermore, UNC-5 and nematode UNC-6/Netrin form a large, stable and rigid oligomeric complex in the presence of heparin, which can incorporate the attractive UNC-40/DCC receptor, demonstrating binary and ternary ectodomain complexes at preparative scale. C. elegans with a heparin-binding deficient UNC-5 fail to establish proper gonad morphology due to abrogated distal tip cell migration, which relies on repulsive UNC-5 signaling in response to UNC-6. Our findings establish Netrin responses to be mediated through glycosaminoglycan-regulated large macromolecular complexes.