Characterization of the stromal cell-derived factor-l alpha-heparin complex

The binding of chemokines to glycosaminoglycans is thought to play a crucia l role in chemokine functions. It has recently been shown that stromal cell -derived factor-1 alpha (SDF-1 alpha), a CXC chemokine with potent anti-hum an immunodeficiency virus activity, binds to heparan sulfate through a typi cal consensus sequence for heparin recognition (BBXB, where B is a basic re sidue KHLK, amino acids 24-27). Calculation of the accessible surface, toge ther with the electrostatic potential of the SDF-1 alpha dimer, revealed th at other amino acids (Arg-41 and Lys-43) are found in the same surface area and contribute to the creation of a positively charged crevice, located at the dimer interface. GRID calculations confirmed that this binding site wi ll be the most energetically favored area for the interaction with sulfate groups, Site-directed mutagenesis and surface plasmon resonance-based bindi ng assays were used to investigate the structural basis for SDF-1 alpha bin ding to heparin. Among the residues clustered in this basic surface area, L ys-24 and Lys-27 have dominant roles and are essential for interaction with heparin. Amino acids Arg-41 and Lys-43 participate in the binding but are not strictly required for the interaction to take place. Direct binding ass ays and competition analysis with monoclonal antibodies also permitted us t o show that the N-terminal residue (Lys-1), an amino acid critical for rece ptor activation, is involved in complex formation. Binding studies with sel ectively desulfated heparin, heparin oligo saccharides, and heparitinase-re sistant heparan sulfate fragments showed that a minimum size of 12-14 monos accharide units is required for efficient binding and that 2-O- and N-sulfa te groups have a dominant role in the interaction. Finally, the heparin-bin ding site was identified on the crystal structure of SDF-1 alpha, and a doc king study was undertaken. During the energy minimization process, heparin lost its perfect ribbon shape and fitted the protein surface perfectly. In the model, Lys-1, Lys-24, Lys-27, and Arg-41 were found to have the major r ole in binding a polysaccharide fragment consisting of 13 monosaccharide un its.