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J. Biol. Chem., Vol. 283, Issue 23, 16226-16234, June 6, 2008

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Structural and Functional Analysis of Slit and Heparin Binding to Immunoglobulin-like Domains 1 and 2 of Drosophila Robo^*

From the Department of Life Sciences, Imperial College London, London SW7 2AZ, United Kingdom

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[Abstract/Free Full Text] ), 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.

Received for publication, January 25, 2008 , and in revised form, March 17, 2008.

The atomic coordinates and structure factors (codes 2vr9 and 2vra) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by a Wellcome Senior Research Fellowship (to E. H.) and an EMBO Postdoctoral Fellowship (to N. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Howard Florey Institute, University of Melbourne, Parkville, VIC, 3010, Australia.

² To whom correspondence should be addressed: Biophysics Section, Blackett Laboratory, Imperial College London, London SW7 2AZ, United Kingdom. Tel.: 44-20-7594-7701; Fax: 44-20-7589-0191; E-mail: e.hohenester{at}imperial.ac.uk.

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