Physical Origin of Thermostabilization by a Quadruple Mutation for the Adenosine A 2a Receptor in the Active State.

The G protein-coupled receptors (GPCRs) form a large, physiologically important family of membrane proteins and are currently the most attractive targets for drug discovery. We investigate the physical origin of thermostabilization of the adenosine A2a receptor (A2a R) in the active state, which was experimentally achieved by another research group using the four point mutations: L48A, A54L, T65A, and Q89A. The investigation is performed on the basis of our recently developed physics-based free-energy function (FEF), which has been quite successful for the thermodynamics of GPCRs in the inactive state. The experimental condition for solving the wild-type and mutant crystal structures was substantially different from that for comparing their thermostabilities. Therefore, all-atom molecular dynamics simulations are necessitated, which also allows us to account for the structural fluctuations of the membrane protein. We show that the quadruple mutation leads to the enlargement of the solvent-entropy gain upon protein folding. The solvent is formed by hydrocarbon groups constituting nonpolar chains within the lipid bilayer, and the entropy is relevant to the thermal motion of the hydrocarbon groups. From an energetic point of view (e.g., in terms of protein intramolecular hydrogen bonds), the mutation confers no improvement upon the structural stability of A2a R. The reliability of our FEF and the crucial importance of the solvent-entropy effect have thus been demonstrated for a GPCR in the active state. We are now ready to identify thermostabilizing mutations of GPCRs not only in the inactive state but also in the active one.