ANALYSIS OF HIGH-AFFINITY BINDING DETERMINANTS IN THE RECEPTOR-BINDING EPITOPE OF BASIC FIBROBLAST GROWTH-FACTOR

Basic fibroblast growth factor (bFGF) is implicated in the pathogenesi s of several vascular and connective diseases, A key step in the disco very of bFGF receptor antagonists to mitigate these actions is to defi ne the functional epitope required for receptor binding of the growth factor. In previous studies, we identified Glu96 as an essential resid ue in this epitope using site-directed mutagenesis. Here we examined t he role of solvent accessible neighboring residues of Glu96 of bFGF on receptor binding affinity. Wild-type bFGF and its muteins were cloned and expressed in Escherichia coli and evaluated for FGF receptor bind ing affinity, Replacement of Asn104 of bFGF by alanine reduced recepto r binding affinity over 400-fold compared with wild-type bFGF. We next explored the effect of neighboring residues of Asn104 on receptor bin ding affinity. Muteins in which Arg97, Leu98, Glu99, Asn101, Asn102, T hr105 and Pro141 were individually replaced by alanine exhibited recep tor binding similar to wild-type bFGF, By contrast, substitution of Ty r103 or Leu140 by alanine reduced receptor binding affinity about 400- and 150-fold, respectively, in accord with a previous report. We conc lude that at least six solvent-accessible residues in bFGF are crucial for high-affinity receptor binding, as evidenced by at least a 10-fol d diminution in the affinity of the corresponding alanine muteins. The polar residues Glu96 and Asn104 appear to form an area important for facilitating the initial contact between ligand and receptor, whereas Tyr24, Tyr103, Leu140 and Met142 form a hydrophobic patch that may sta bilize the complex. The detailed structure of this functional epitope can be employed in the discovery and design of bFGF antagonists using computational methods.