(334k) Molecular Dynamics (Md) Study of T to R Transition in 2ZN-Insulin Hexamer

Insulin is a small (51 amino acids, 5800Da) polypeptide hormone that regulates carbohydrate metabolism, and is pharmacologically important in the treatment of type-1 diabetes mellitus. At high concentrations insulin aggregates to form hexamers which exist in one of the three allosteric states in solution, and are related by dynamic equilibrium: T6T3R3R6. Two kinds of important binding pockets exist in hexamers for various allosteric ligands: (1) Two ?Zn binding pockets' which can bind single anions such as chloride, phenolate, carboxylates or thiocyanate (2) Six ?hydrophobic binding pockets' located on dimer-dimer interfaces in hexamers which can bind small organic ligands such as phenol. Both kinds of allosteric ligands play a central role in TR transition. Before binding to its transmembrane receptor, insulin must dissociate from hexameric storage form to the bioactive monomer. Hence, it is not difficult to infer that TR equilibrium lies at the heart of insulin dissolution and anything affecting this rightly deserves scrutiny for pharmaceutical preparations of insulin. In this contribution, we use molecular simulations to investigate and understand this allosteric R to T transition in the presence of aforesaid ligands. We have carried out explicit-solvent, long (~20ns simulation time) molecular dynamics (MD) simulations of T-state insulin hexamers at two different concentrations of chloride as ligands to explore Zn binding domains. Our results show that Cl- ions bind more strongly as the concentration field is switched from 1.0 M to 1.5M, and are able to displace more water from Zn (II) pockets. This means, at increased concentration of Cl-, high-probability regions of binding are formed. Conformation of this stable binding by anions is an important step in understanding this transition. Further, in order to explore hydrophobic binding pockets, and establish importance of phenolic ligands, we carried out MD simulations again in explicit-solvent, both with and without phenol. Our hypothesis was that, as phenols stabilize R-state, a phenol free hexamer would make a transition to the T state. Indeed, we observed significant reduction in helical content of B1-B8 residues of each monomer in a ~20ns long MD simulation in NVT ensemble without all phenolic content. The melting of these helices is hallmark of this TR transition. In order to strengthen this hypothesis and to eliminate the concern that the observed transition is due to thermostat or any other artifacts, we are presently carrying out long NVE simulations with explicit-solvent for the same cases as mentioned above. Interestingly, one of these ~10ns long run shows again the onset of melting of helices in three monomers out of six in the absence of phenols. This observation can be related to the important concept of co-operativity between subunits in all proteins. The work is underway to investigate this negative co-operativity and understand communication among subunits, and also to quantify residues involved in interaction of helix with phenols. The results of the present work will further serve to provide significant insights into structure-function relationship in insulin hormone and explain the mechanism of dissociation of insulin hexamer assembly to bioactive monomers.