FGF2-HEPARIN CO-CRYSTAL COMPLEX-ASSISTED DESIGN OF MUTANTS FGF1 AND FGF7 WITH PREDICTABLE HEPARIN AFFINITIES

The co-crystal structures of FGF2 and heparin derived tetra- and hexas accharides demonstrated the existence of high and low affinity contact residues that are likely to be involved in heparin binding (Faham, S. , Hileman, R, E., Fromm, J. R., Linhardt, R. J., and Rees, D. C. (1996 ) Science 271, 1116-1120), To study the role of these putative contact residues, me chose three fibroblast growth factor family members with distinct heparin affinities for comparative mutagenesis studies. Only one amino acid significantly differed between FGF1 and FGF2 and was m utated, FGF1-31K. FGF7, also called keratinocyte growth factor, was mu tated to mimic either FGF1 or FGF2 at two of the putative high contact points termed FGF7-1 and FGF7-2, respectively, FGF2 has higher appare nt heparin affinity than FGF1 or FGF7, and FGF1 has higher heparin aff inity than FGF7, All three mutants showed an increase in apparent hepa rin affinity compared with wild types, FGF7-1 has a lower apparent hep arin affinity than FGF7-2, analogous to wild type FGF1 and FGF2, The F GF1-31K mutant showed no change in mitogenic activity, whereas the FGF 7 mutants exhibited a decrease in activity, These results indicate tha t the co-crystal structure of the FGF2-heparin complexes can be used t o design a rational approach to the generation of mutants with defined affinities for heparin or heparan sulfate proteoglycans.