RESUMEN
Human acidic fibroblast growth factor 1 (hFGF1) is a protein intricately involved in cell growth and tissue repair. In this study, we investigate the effect(s) of understanding the role of a conserved proline (P135), located in the heparin binding pocket, on the structure, stability, heparin binding affinity, and cell proliferation activity of hFGF1. Substitution of proline-135 with a positively charged lysine (P135K) resulted in partial destabilization of the protein; however, the overall structural integrity of the protein was maintained upon substitution of proline-135 with either a negative charge (P135E) or a polar amino acid (P135Q). Interestingly, upon heparin binding, an increase in thermal stability equivalent to that of wt-hFGF1 was observed when P135 was replaced with a positive (P135K) or a negative charge (P135E), or with a polar amino acid (P135Q). Surprisingly, introduction of negative charge in the heparin-binding pocket at position 135 (P135E) increased hFGF1's affinity for heparin by 3-fold, while the P135K mutation, did not alter the heparin-binding affinity. However, the enhanced heparin-binding affinity of mutant P135E did not translate to an increase in cell proliferation activity. Interestingly, the P135K and P135E double mutations, P135K/R136E and P135/R136E, reduced the heparin binding affinity by â¼3-fold. Furthermore, the cell proliferation activity was increased when the charge reversal mutation R136E was paired with both P135E (P135E/R136E) and P135K (P135K/R136E). Overall, the results of this study suggest that while heparin is useful for stabilizing hFGF1 on the cell surface, this interaction is not mandatory for activation of the FGF receptor.