Netrins dictate attractive and repulsive responses during axon growth and cell migration, where the presence of the receptor Uncoordinated-5 (UNC-5) on target cells results in repulsion. Here, we showed that UNC-5 is a heparin-binding protein, determined its structure bound to a heparin fragment, and could modulate UNC-5–heparin affinity using a directed evolution platform or structure-based rational design. We demonstrated that UNC-5 and UNC-6/netrin form a large, stable, and rigid complex in the presence of heparin, and heparin and UNC-5 exclude the attractive UNC-40/DCC receptor from binding to UNC-6/netrin to a large extent. Caenorhabditis elegans with a heparin-binding–deficient UNC-5 fail to establish proper gonad morphology due to abrogated cell migration, which relies on repulsive UNC-5 signaling in response to UNC-6. Combining UNC-5 mutations targeting heparin and UNC-6/netrin contacts results in complete cell migration and axon guidance defects. Our findings establish repulsive netrin responses to be mediated through a glycosaminoglycan-regulated macromolecular complex.
Details
Title
Structural insights into the formation of repulsive netrin guidance complexes
Data availability statement
All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials, except the following: The coordinates and crystallographic structure factors have been deposited in the PDB under the accession codes 8EDC (UNC-5 IG1 + 2) and 8EDI (UNC-5 IG1 + 2 with heparin-dp4), and 8EDK (UNC-6ΔC). SAXS datasets are deposited at the SASBDB under the accession codes SASDQJ9 (UNC-6ΔC–heparin–UNC-5 ECD) and SASDQK9 (UNC-6ΔC–heparin–UNC-5 ECD–UNC-40 ECD).
Funding Information
National Science Foundation, DGE-1656518 National Institutes of Health, S10OD021527 U.S. Department of Energy, DE-AC02-05CH11231 U.S. Department of Energy, DE-AC02-06CH11357 National Cancer Institute, ACB-12002 National Institute of General Medical Sciences, P30 GM138395 National Institute of General Medical Sciences, AGM-12006 National Institute of General Medical Sciences, P30GM138396 National Institute of General Medical Sciences, P30 GM124165 National Institute of Neurological Disorders and Stroke, R01 NS097161 National Institute of General Medical Sciences, R35 GM147179 National Institute of General Medical Sciences, T32 GM138826 H2020 European Research Council, MCSA-IF 702346
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