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Probing the role of proline −135 on the structure, stability, and cell proliferation activity of human acidic fibroblast growth factor

Author:
Davis, Julie Eberle, Alghanmi, Arwa, Gundampati, Ravi Kumar, Jayanthi, Srinivas, Fields, Ellen, Armstrong, Monica, Weidling, Vanessa, Shah, Varun, Agrawal, Shilpi, Koppolu, Bhanu prasanth, Zaharoff, David A., Kumar, Thallapuranam Krishnaswamy Suresh
Source:
Archives of biochemistry and biophysics 2018 v.654 pp. 115-125
ISSN:
0003-9861
Subject:
binding capacity, cell growth, cell proliferation, fibroblast growth factor 1, fibroblast growth factor receptors, heparin, humans, lysine, mutants, mutation, proline, thermal stability, tissue repair
Abstract:
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 ∼3-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.
Agid:
6093059