A Comprehensive Investigation of the Stabilization of Monomeric Hfgf1 by Heparin Hexasaccharide using Microsecond-Level MD Simulations and Enhanced Sampling Techniques

Abstract

Acidic human fibroblast growth factor 1 (hFGF1) is a major signaling molecule that is heavily involved in cell proliferation, angiogenesis, tumor invasion and metastatic progression. Previous experimental studies have demonstrated that hFGF1 is naturally unstable and that it has a near-physiological denaturation temperature. Heparin (a linear sulfated polysaccharide) is known to stabilize hFGF1 and protect it from thermal and proteolytic degradation. Our study used experimental data to set up a rigorous computational investigation of the hFGF1-heparin hexasaccharide complex. Three models were simulated for 4.8 microseconds each. Our equilibrium-MD simulations confirmed that the heparin-free monomer is less stable than the heparin-bound monomer. The decreased stability of the heparin-free monomer is due to a conformational change in the heparin-binding region. This conformational change was not observed in the heparin-bound systems. Important interactions that contribute to the stability of the complex were also characterized. K113 and S117 were identified as important residues of the heparin-binding region that contribute to intramolecular hydrogen bonding. Strong intermolecular hydrogen bonding was also observed between R123 and IdoA(4) of the heparin hexasaccharide. We then used a combination of non-equilibrium pulling and bias exchange umbrella sampling simulations to determine the binding free energy of heparin. Thus far, all published computational studies of the hFGF-1 heparin complex have been based on nanosecond-level simulations. For the very first time, we have used a combination of microsecond-level MD simulations and large-scale enhanced sampling techniques to carry out a more realistic and biochemically relevant assessment of the hFGF1-heparin complex.