A Thermodynamic Study Of Ligand Access/escape From Protein Cavities

Abstract

Insulin, a protein hormone, regulates glucose homeostasis and carbohydrate metabolism in higher organisms. Therapeutic formulations of hormone are preserved against degradation and denaturation by antimicrobial preservatives such as phenols, cooperative binding of which stabilizes hexameric complexes of insulin. Dissociation of hexameric species (on minutes to days time scale) into biologically active monomers is facilitated by rapid unbinding of phenols (on milliseconds time scale). However, a clear understanding of dissolution kinetics and determinants of the rates of ligand unbinding remains obscure, chiefly due to unresolved ambiguities in NMR results. We have used random acceleration molecular dynamics (RAMD) to identify and characterize a variety of potential ligand dissociation mechanisms. We observe three distinct exit routes for the ligand and resolve potentials of mean force (PMFs) along them by performing free energy calculations. Free energy profiles for each mechanism are computed with the help of second order cumulant expansion of Jarzynski's equality and non-equilibrium work statistics gathered from multiple independent steered molecular dynamics (SMD) simulations. Based on energetic barriers and structural properties, we suggest a plausible preferred mechanism for the ligand exchange. The most likely pathway with the lowest free energy barrier involves a leap over the “gate” formed by HisF5 and IleA10, with simultaneous passage of the ligand through a narrow channel existing between LeuA13, LeuH17, and the “gate”. Free energy profiles also display several weakly-bound metastable states for the ligand during entry and exit from R6 insulin hexamer.