CO-CRYSTAL STRUCTURES OF BASIC FIBROBLAST GROWTH FACTOR COMPLEXED WITH DI- AND TRISACCHARIDE HEPARIN ANALOGS. Andrew B. Herr, David M. Ornitz^*, and Gabriel Waksman, Department of Biochemistry and Molecular Biophysics and ^*Department of Molecular Biology and Pharmacology, Washington University Medical School, St. Louis, MO 63110, USA

Basic fibroblast growth factor (bFGF) has been co-crystallized with two different nonsulfated heparin-like oligosaccharides, providing the first view of the binding sites of biologically active heparin analogs (Ornitz, Herr, et al., Science 268:432, 1995). Both oligosaccharides are nonsulfated, isomerically pure analogs that show significant biological activity. The co-crystal structures revealed three saccharide binding sites per bFGF monomer, designated sites 1, 2, and 2'. The co-crystals of each bFGF/saccharide complex show identical binding sites. Site 1 is identical to the previously proposed high-affinity heparin binding site. Binding sites 2 and 2', however, have not been previously identified. These two sites occur at a crystal packing interface between two neighboring bFGF monomers, and a single saccharide ligand bridges the two binding sites. Analysis of the number of hydrogen bonds between the ligand and each site indicates that each is an independent binding site and not merely an artifact of crystal packing. The observation of a single ligand bridging two binding sites in adjacent bFGF molecules suggests that these ligands may induce bFGF self-association.

Analytical ultracentrifugation was used to test whether small heparin analogs can induce self-association of bFGF in solution. Sedimentation equilibrium studies were conducted using bFGF and a number of heparin analogs. The results have shown that several different heparin analogs do indeed cause association of bFGF in solution. Unliganded bFGF was run as a control in each experiment, and no association was seen. These studies are being continued in order to further characterize the nature of the ligand-induced association caused by an active nonsulfated trisaccharide (Tri-3), an inactive sulfated disaccharide (sucrose octasulfate), and a sulfated heparin octasaccharide fragment. These ultracentrifugation studies provide support for the model of ligand-induced association of bFGF as suggested by the co-crystal structures.