(653b) Non-Aqueous Environment Disrupts the Allosteric Network in Subtilisin

The stability and activity of a widely used serine protease in industry, subtilisin, depends on calcium ion. However, the X-ray structure of subtilisin shows that the active site and the calcium-binding site are separated by 30 Å. Although large-scale conformational change is not involved in the functioning of subtilisin, calcium binding could induce long-range communication through the protein matrix. By using all-atom molecular dynamics simulations with and without calcium ion and coarse-grain modeling based on the elastic network model, we resolve the allosteric network in subtilisin conformation. Two routes of inter-residue interactions through which the strain induced by calcium binding affects the structure and fluctuations at sites far away from the calcium-binding site are identified. In addition to changes in inter-residue distances, the allosteric network also involves residues whose mechanical coupling with the surrounding amino acids changes significantly as a function of calcium binding. The key inter-residue interactions responsible for long-range coupling are also consistent with the results of sequence alignment. To further analyze the functional roles of the allosteric network, we performed all-atom MD simulation of subtilisin in hexane since enzyme activity has been shown to reduce by 3-6 orders when solvating in a non-aqueous environment. We show that the flexibility of subtilisin conformation is significantly reduced in hexane due to enhanced electrostatic interactions between polar groups. Changing solvent also perturbs the communication through the allosteric network. Our studies demonstrate that long-range communications through protein matrix is a strong function of ligand binding and solvent and is in turn correlated with enzyme activity. The residues identified in the allosteric network can be employed as protein engineering sites to validate their functional roles.