Abstract
Recognition of the secreted protein Slit by transmembrane receptors of the Robo family provides important signals in the development of the nervous system and other organs, as well as in tumor metastasis and angiogenesis. Heparan sulfate (HS) proteoglycans serve as essential co-receptors in Slit-Robo signaling. Previous studies have shown that the second leucinerich repeat domain of Slit, D2, binds to the N-terminal immunoglobulin-like domains of Robo, IG1-2. Here we present two crystal structures of Drosophila Robo IG1-2, one of which contains a bound heparin-derived oligosaccharide. Using structure-based mutagenesis of a Robo IG1-5 construct we identified key Slit binding residues (Thr-74, Phe-114, Arg-117) forming a conserved patch on the surface of IG1; mutation of similarly conserved residues in IG2 had no effect on Slit binding. Mutation of conserved basic residues in IG1 (Lys-69, Arg-117, Lys-122, Lys-123), but not in IG2, reduced binding of Robo IG1-5 to heparin, in full agreement with the Robo-heparin co-crystal structure. Our collective results, together with a recent crystal structure of a minimal human Slit-Robo complex ( Morlot, C., Thielens, N. M., Ravelli, R. B., Hemrika, W., Romijn, R. A., Gros, P., Cusack, S., and McCarthy, A. A. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 14923-14928 ), reveal a contiguous HS/heparin binding surface extending across the Slit-Robo interface. Based on the size of this composite binding site, we predict that at least five HS disaccharide units are required to support Slit-Robo signaling.
Highlights
We previously identified Slit residues involved in heparin binding and demonstrated that one important function of heparan sulfate (HS)/heparin is to promote the formation of a ternary Slit-Robo-HS signaling
Using structure-based mutagenesis, we have identified Robo residues involved in Slit and heparin binding
Robo IG1-2 Structure—We determined the structure of Drosophila Robo IG1-2 in two crystal forms: a tetragonal form containing the protein with a C-terminal His tag, and a monoclinic form containing the untagged protein in the presence of a heparin octasaccharide
Summary
Expression Vectors—Initial constructs were made by PCR amplification from complete cDNA clones of Drosophila Slit and Robo, kindly provided by Guy Tear (King’s College, London, UK). Untagged Robo IG1-2 and His-tagged Slit D1– 4 were purified using 5-ml HiTrap Heparin HP affinity columns (GE Healthcare). The Robo IG1-5 Fc proteins (wild-type and mutants) were purified using 1-ml HiTrap rProtein-A FF columns (GE Healthcare). The Robo IG1-2 proteins used for crystallization were further purified on a 24-ml Superdex column (GE Healthcare) in TBS (Histagged protein) or 0.02 M Na-HEPES pH 7.5, 0.15 M NaCl (untagged protein). The monoclinic Robo IG1-2 structure was solved by molecular replacement using the partially refined structure of the tetragonal form as a search model; the IG domains had to be located individually to obtain a solution. Heparin Affinity Chromatography—300 g of each Robo IG1-5 Fc protein (wild-type and mutants) were injected onto a 5-ml HiTrap Heparin HP column (GE Healthcare) equilibrated in 0.05 M Tris-HCl, pH 7.5. Data collection and reduction Space group Unit cell dimensions Beamline Wavelength (Å) Resolution range (Å) Copies/asymmetric unit Solvent content (%) Unique reflections Multiplicity Average I/(I) Completeness (%) Rmerge
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