Charge reversal in the heparin-binding pocket enhances the stability and activity of the human FGF1

Human fibroblast growth factor 1 (hFGF1) is a potent signaling molecule whose role in cell growth, differentiation, and migration has made it an excellent candidate for wide-ranging biomedical applications. hFGF1 binds heparan sulfate proteoglycans, which stabilize the protein and play a crucial role in growth factor-induced signaling. Within the heparin-binding region of hFGF1 lies a thrombin cleavage site at arginine-136, which, although not part of the canonical thrombin recognition sequence (LVPRGS), is nevertheless cleaved by thrombin, leading to reduced biological activity. In our previous study, R136 was identified as the primary thrombin cleavage site and was mutated to several hFGF1 variants-R136E, R136K, R136G, R136L, and R136Q-to assess the structural and functional impact of charge and residue substitution. We confirmed that altering the positively charged R136 residue enhances resistance to proteases, thermal stability, and cell proliferation activity. However, the structural basis for the increased stability observed in the R136E mutant remained unclear. Here, NMR experiments reveal that structural perturbations in all variants are largely localized near the mutation site, indicating that the global fold of hFGF1 remains intact. 2D NMR experiments have also identified 14 residues whose amide protons do not undergo exchange with D₂O in the variant, suggesting their contribution to enhancing structural rigidity. Microsecond-level molecular dynamics simulations reveal that the increased structural stability of the variant arises from new electrostatic interactions (E136-R133, E136-K132, and E136-K126) and hydrogen bonds (S130-N128 and K127-N32) within the heparin binding region. A stability comparison study of the R136E and R136D variants further suggests that the side-chain length of the acidic residue at position 136 is critical for conferring inherent stability to the hFGF1 protein. Overall, these findings attribute the enhanced stability of the R136E variant to unique electrostatic and hydrogen-bonding interactions absent in wild-type hFGF1.