Site-directed mutagenesis and molecular modeling identify a crucial amino acid in specifying the heparin affinity of FGF-1.

Heparin potentiates the mitogenic activity of FGF-1 by increasing the affinity for its receptor and by extending its biological half-life. During the course of labeling human FGF-1 with Na(125)I and chloramine T, it was observed that the protein lost its ability to bind to heparin. In contrast, bovine FGF-1 retained its heparin affinity even after iodination. To localize the region responsible for the lost heparin affinity, chimeric FGF-1 proteins were constructed from human and bovine FGF-1 expression constructs and tested for their heparin affinity after iodination. The results showed that the C-terminal region of human FGF-1 was responsible for the loss of heparin affinity. This region harbors a single tyrosine residue in human FGF-1 in contrast to a phenylalanine at this position in bovine FGF-1. Mutating this tyrosine residue in the human FGF-1 sequence to phenylalanine did not restore the heparin affinity of the iodinated protein. Likewise, changing the phenylalanine to tyrosine in the bovine FGF-1 did not reduce the ability of the iodinated protein to bind to heparin. In contrast, a mutant human FGF-1 that has cysteine-131 replaced with serine (C131S) was able to bind to heparin even after iodination while bovine FGF-1 (S131C) lost its binding affinity to heparin upon iodination. In addition, the human FGF-1 C131S mutant showed a decrease in homodimer formation when exposed to CuCl(2). Molecular modeling showed that the heparin-binding domain of FGF-1 includes cysteine-131 and that cysteine-131, upon oxidation to cysteic acid during the iodination procedures, would interact with lysine-126 and lysine-132. This interaction alters the conformation of the basic residues such that they no longer bind to heparin.