Abstract
The co-crystal structures of FGF2 and heparin-derived tetra- and hexasaccharides 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, we 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 mutated, FGF1-31K. FGF7, also called keratinocyte growth factor, was mutated to mimic either FGF1 or FGF2 at two of the putative high contact points termed FGF7-1 and FGF7-2, respectively. FGF2 has higher apparent heparin affinity than FGF1 or FGF7, and FGF1 has higher heparin affinity than FGF7. All three mutants showed an increase in apparent heparin affinity compared with wild types. FGF7-1 has a lower apparent heparin affinity than FGF7-2, analogous to wild type FGF1 and FGF2. The FGF1-31K mutant showed no change in mitogenic activity, whereas the FGF7 mutants exhibited a decrease in activity. These results indicate that the co-crystal structure of the FGF2-heparin complexes can be used to design a rational approach to the generation of mutants with defined affinities for heparin or heparan sulfate proteoglycans.
Highlights
The co-crystal structures of FGF2 and heparin-derived tetra- and hexasaccharides demonstrated the existence of high and low affinity contact residues that are likely to be involved in heparin binding
Generation of Wild Type and Mutant FGF1 and FGF7— Specific residues in FGF1 have been identified previously to have a substantial impact on its apparent heparin affinity [20, 29]
FGF2 has higher apparent heparin affinity than FGF1, and from Table I, only one residue was significantly different between the two proteins
Summary
Were entirely conserved throughout the FGF family, yet all members are capable of binding heparin. We chose three members from the FGF family, each with a distinct heparin affinity, to study the role of these contact residues in heparin binding. FGF2 has higher apparent heparin affinity than FGF1 and FGF7, which is known as keratinocyte growth factor. FGF7 has lower apparent heparin affinity than FGF1. We examined the effect on heparin affinity when these putative contact points are mutated to resemble other FGF family members. The results are consistent with the co-crystal structure of heparin-derived tetra- and hexasaccharides and FGF2 and suggest that a rational approach to generating mutants with designed heparin and heparan sulfate proteoglycan affinities is possible
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