Orientation of heparin-binding sites in native vitronectin - Analyses of ligand binding to the primary glycosaminoglycan-binding site indicate that putative secondary sites are not functional

A primary heparin-binding site in vitronectin has been localized to a clust er of cationic residues near the C terminus of the protein. More recently, secondary binding sites have been proposed. In order to investigate whether the binding site originally identified on vitronectin functions as an excl usive and independent heparin-binding domain, solution binding methods have been used in combination with NMR and recombinant approaches to evaluate L igand binding to the primary site. Evaluation of the ionic strength depende nce of heparin binding to vitronectin according to classical linkage theory indicates that a single ionic bond is prominent. It had been previously sh own that chemical modification of vitronectin using an arginine reactive pr obe results in a significant reduction in heparin binding (Gibson, A, Babur aj, K, Day, D. E., Verhamme, I., Shore, J, D,, and Peterson, C, B, (1997) J . Biol Chem, 272, 5112-5121), The label has now been localized to arginine residues within the cyanogen bromide fragment-(341-380) that contains the p rimary heparin-binding site on vitronectin. One- and two-dimensional NMR on model peptides based on this primary heparin-binding site indicate that an arginine residue participates in the ionic interaction and that other noni onic interactions may be involved in forming a complex with heparin, A reco mbinant polypeptide corresponding to the C-terminal 129 amino acids of vitr onectin exhibits heparin-binding affinity that is comparable to that of ful l-length vitronectin and is equally effective at neutralizing heparin antic oagulant activity. Results from this broad experimental approach argue that the behavior of the primary site is sufficient to account for the heparin binding activity of vitronectin and support an exposed orientation for the site in the structure of the native protein.