4193 - Biophysical Journal Volume 95 November 2008 41934204...

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Ligand Escape Pathways and (Un)Binding Free Energy Calculations for the Hexameric Insulin-Phenol Complex Harish Vashisth and Cameron F. Abrams Department of Chemical and Biological Engineering, Drexel University, Philadelphia, Pennsylvania ABSTRACT Cooperative binding of phenolic species to insulin hexamers is known to stabilize pharmaceutical preparations of the hormone. Phenol dissociation is rapid on hexamer dissolution timescales, and phenol unbinding upon dilution is likely the ±rst step in the conversion of (pharmaceutical) hexameric insulin to the active monomeric form upon injection. However, a clear understanding of the determinants of the rates of phenol unbinding remains obscure, chiefly because residues implicated in phenol exchange as determined by NMR are not all associated with likely unbinding routes suggested by the best-resolved hexamer structures. We apply random acceleration molecular dynamics simulation to identify potential escape routes of phenol from hydrophobic cavities in the hexameric insulin-phenol complex. We ±nd three major pathways, which provide new insights into (un)binding mechanisms for phenol. We identify several residues directly participating in escape events that serve to resolve ambiguities from recent NMR experiments. Reaction coordinates for dissociation of phenol are developed based on these exit pathways. Potentials of mean force along the reaction coordinate for each pathway are resolved using multiple independent steered molecular dynamics simulations with second-order cumulant expansion of Jarzynski’s equality. Our results for D F agree reasonably well within the range of known experimental and previous simulation magnitudes of this quantity. Based on structural analysis and energetic barriers for each pathway, we suggest a plausible preferred mechanism of phenolic exchange that differs from previous mechanisms. Several weakly-bound metastable states are also observed for the ±rst time in the phenol dissociation reaction. INTRODUCTION The insulin-phenol complex is a pharmacologically impor- tant protein-ligand system. Insulin is a dual chain hormone (A-chain with 21 residues, and B-chain with 30 residues) responsible for carbohydrate metabolism and is used in the treatment of insulin-dependent (type 1) diabetes mellitus. Insulin monomers (51 residues and 5800 Da each) self- associate at physiological concentration (1 ng/ml) to form torus-shaped hexamers in the presence of zinc ions (1). Hexamers exist in three allosteric states termed T 6 , T 3 R 3 , and R 6 (2–11), related by a T 6 4 T 3 R 3 4 R 6 dynamic equilib- rium, which is shifted to R 6 only by phenolic derivatives (12,13). However, the T 3 R 3 state can be achieved either by concentrated anionic medium (Cl ÿ , SCN ÿ , etc.) or phenolic species or both. Phenolic compounds act as antimicrobial agents and increase the shelf-life of industrial formulations by stabilizing the R 6 state (14). Six hydrophobic cavities are present for the phenolic ligands in the R 6 state and none in the T 6 state. The conformation of the N-terminal B-chain resi-
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4193 - Biophysical Journal Volume 95 November 2008 41934204...

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