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

• Published in: Archives of biochemistry and biophysics Vol. 654; no. C; pp. 115 - 125
• Main Authors: 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
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.09.2018; Elsevier
• Subjects: Bioactivity; Biochemistry & Molecular Biology; Biophysics; Cell Proliferation - physiology; Charge-reversal; Fibroblast growth factor; Fibroblast Growth Factor 1 - chemistry; Fibroblast Growth Factor 1 - genetics; Fibroblast Growth Factor 1 - physiology; Heparin - metabolism; Heparin binding; Humans; Mutagenesis, Site-Directed; Proline - physiology; Protein Binding; Protein Stability; Protein Structure, Tertiary; Proton Magnetic Resonance Spectroscopy; Receptors, Fibroblast Growth Factor - metabolism; Stability; Fibroblast growth factor; human acidic fibroblast growth factor-1; Stability; Heparin binding; site directed mutagenesis; circular dichroism; Charge-reversal; isothermal titration calorimetry; wildtype hFGF1; Visual Molecular Dynamics; heteronuclear single quantum coherence; American Type Culture Collection; Bioactivity
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2018.07.017

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. •Mutation of residues P135 and R136 alters heparin-binding affinity of hFGF1.•The cell proliferation activity of hFGF1 is not correlated to its heparin-binding affinity.•Mutation R136E increases hFGF1 thermal stability and activity independent of heparin.