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J. Biol. Chem., Vol. 282, Issue 13, 10018-10027, March 30, 2007

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Structural and Functional Basis of CXCL12 (Stromal Cell-derived Factor-1) Binding to Heparin^*

James W. Murphy¹, Yoonsang Cho, Aristidis Sachpatzidis, Chengpeng Fan, Michael E. Hodsdon, and Elias Lolis²

From the Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520-8066 and the Department of Laboratory Medicine, Yale University School of Medicine, New Haven, Connecticut 06520-8035

CXCL12 (SDF-1) and CXCR4 are critical for embryonic development and cellular migration in adults. These proteins are involved in HIV-1 infection, cancer metastasis, and WHIM disease. Sequestration and presentation of CXCL12 to CXCR4 by glycosaminoglycans (GAGs) is proposed to be important for receptor activation. Mutagenesis has identified CXCL12 residues that bind to heparin. However, the molecular details of this interaction have not yet been determined. Here we demonstrate that soluble heparin and heparan sulfate negatively affect CXCL12-mediated in vitro chemotaxis. We also show that a cluster of basic residues in the dimer interface is required for chemotaxis and is a target for inhibition by heparin. We present structural evidence for binding of an unsaturated heparin disaccharide to CXCL12 attained through solution NMR spectroscopy and x-ray crystallography. Increasing concentrations of the disaccharide altered the two-dimensional ¹H-¹⁵N-HSQC spectra of CXCL12, which identified two clusters of residues. One cluster corresponds to -strands in the dimer interface. The second includes the amino-terminal loop and the -helix. In the x-ray structure two unsaturated disaccharides are present. One is in the dimer interface with direct contacts between residues His²⁵, Lys²⁷, and Arg⁴¹ of CXCL12 and the heparin disaccharide. The second disaccharide contacts Ala²⁰, Arg²¹, Asn³⁰, and Lys⁶⁴. This is the first x-ray structure of a CXC class chemokine in complex with glycosaminoglycans. Based on the observation of two heparin binding sites, we propose a mechanism in which GAGs bind around CXCL12 dimers as they sequester and present CXCL12 to CXCR4.

Received for publication, September 12, 2006 , and in revised form, January 19, 2007.

The atomic coordinates and structure factors (code 2NWG) 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 in part by National Institutes of Health Grant RO1 AI065029 (to E. L.), the Pilot Project Funding from the Yale Cancer Center (to M. E. H.), the Campbell Foundation (to E. L.), and the Patterson Foundation (E. L.). 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 Supported by a Neuropharmacology Training Program by National Institutes of Health Grant T32 NS007136.

² To whom correspondence should be addressed: 333 Cedar St., New Haven, CT 06510-8066. Tel.: 203-785-6233; Fax: 203-737-2027; E-mail: elias.lolis{at}yale.edu.

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