Glu-96 of basic fibroblast growth factor is essential for high affinity receptor binding. Identification by structure-based site-directed mutagenesis.

The importance of basic fibroblast growth factor (bFGF) in several pathophysiological processes has stimulated interest in the design of receptor antagonists to mitigate such effects. Of key importance in this connection is the characterization of the functional binding epitopes of the growth factor for its receptor. Based on peptide mapping and molecular dynamics calculations of the three-dimensional structure of basic fibroblast growth factor, we employed site-directed mutagenesis to investigate the effect of altering residues at positions 107, 109-114, and 96 on bFGF on receptor binding affinity. All muteins were cloned and expressed in Escherichia coli, purified to homogeneity employing heparin-Sepharose columns, and evaluated for receptor binding affinity. We found that replacement of residues at positions 107 and 109-114 by alanine or phenylalanine had little effect on receptor binding affinities compared with wild type bFGF, in agreement with previous evidence that bFGF residues 109-114 comprise a low affinity binding site. By contrast, substitution of Glu-96 with alanine yielded a molecule having about 0.1% of the affinity of the wild type bFGF. The affinity of the corresponding lysine and glutamine muteins was 0.3 and 10%, respectively, emphasizing the importance of a negative charge at this position. Our findings are consistent with the view that residues 106-115 on bFGF represent a low affinity binding site on bFGF. In addition, we identify Glu-96 as a crucial residue for binding to fibroblast growth factor receptor-1.