Mechanistic Picture for Monomeric Human Fibroblast Growth Factor 1 Stabilization by Heparin Binding

Abstract

Human fibroblast growth factor (FGF) 1 or hFGF1 is a member of the FGF family that isare involved in various vital processes such as cell proliferation, cell differentiation, angiogenesis and wound healing. hFGF1, which is associated with low stability in vivo, is known to be stabilized by binding heparin sulfate, a glycosaminoglycan that aids the protein in the activation of its cell surface receptor. The poor thermal and proteolytic stability of hFGF1 and the stabilizing role of heparin have long been observed experimentally; however, the mechanistic details of these phenomena either phenomenon are still not well understood. Here, we have used a combination of microsecond-level equilibrium molecular dynamics (MD) simulations, and state-of-the-art enhanced sampling MD simulations to quantitatively characterize the structural dynamics of monomeric hFGF1 in the presence and absence of heparin hexasaccharide. We have observed a local conformational changeconformational change in the heparin-binding pocketregion of hFGF1 that only occurs only in the absence of heparin. Several intramolecular hydrogen bonds were also identified within the heparin-binding pocket region, that form only when hFGF1 interacts with heparin. The loss of both intermolecular and intramolecular electrostatic interactions in the absence of heparin plausibly leads to the observed conformational change. This conformational transition results in increased flexibility of the heparin-binding pocket region and provides an explanation for the susceptibility of apo hFGF1 to proteolytic degradation and thermal instability . The hFGF1-heparin interactions) has also been quantified using absolute binding free energy calculations. Binding affinity (Kd) estimates determined computationally using our novel MD approach are in a good quantitative agreement with experimental Kd values from isothermal titration calorimetry experiments. The successful application of a combination of rigorous physics-based simulation techniques microsecond-level MD and accurate free energy calculations and its ability to accurately explain the biomolecular phenomena such as the heparin-mediated stabilization of hFGF1 at a quantitative level, provides open new opportunitiesrepresents a promising approach for studying complex towards understanding biological processesbiomolecular interactions between proteins and their binding partners at a detailed molecular level. using rigorous physics-based simulation techniques.

Binding Affinity Estimation From Restrained Umbrella Sampling Simulations

“…The initial runs lasted 15 nanoseconds, followed by the productions run on the supercomputer Anton 2 (Pittsburgh Supercomputing Center) for 4.8 μs, with a timestep of 2.5 fs. These equilibrium simulations have previously been described in a related study 95 . We used these equilibrium simulations to construct the PMF in terms of the RMSD of the protein ( r P ) both for the apo 95 and holo proteins (for bulk and binding pocket, respectively).…”

“…We used these equilibrium simulations to construct the PMF in terms of the RMSD of the protein ( r P ) both for the apo 95 and holo proteins (for bulk and binding pocket, respectively). We also used the holo protein simulations 95 to construct the PMF in terms of the RMSD of the ligand ( r L ) in the binding pocket.…”

“…The final conformation of the hFGF1-heparin equilibrium simulation 95 was used to generate starting conformations for the non-equilibrium pulling simulations. Two collective variables 96 were used for SMD simulations 97 : (1) distance between the heavy-atom center of mass of heparin and that of the protein ( d ) and (2) the orientation angle of heparin with respect to the protein (Ω).…”