Physical Origin of Thermostabilization by a Quadruple Mutation for the Adenosine A_2a Receptor in the Active State

Abstract

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 A receptor (AR) 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 AR. 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.

Theoretical identification of thermostabilizing amino acid mutations for G-protein-coupled receptors

“…For example, for the wild type and an octuple mutant of A 2A in the inactive state shown in Fig. 1 and the wild type and a quadruple mutant (L48A, A54L, T65A, and Q89A) of A 2A in the active state (Lebon et al 2011), we could show that the mutant is more thermostable than the wild type (Kajiwara et al 2016;Kajiwara et al 2018). It follows that our method is capable of correlating the thermostability with the 3D structure.…”

“…The PMF is weaker and shorter ranged than the direct interaction energy. We optimized E NP 5k B T 0 for membrane proteins (Kajiwara et al 2018;Yasuda et al 2017;Yasuda et al 2019). The PMF is a linear, short-ranged function of the distance.…”

How Does a Microbial Rhodopsin RxR Realize Its Exceptionally High Thermostability with the Proton-Pumping Function Being Retained?

“…Furthermore, the remarkable progress of supercomputers has enabled us to perform a molecular dynamics (MD) simulation using all-atom models for the protein and its environment. − Taken together, atomistic details of the structure, properties, and functions of a protein can be obtained both experimentally and computationally. On the other hand, we have been emphasizing that thermodynamics is indispensable to such subjects as the elucidation of protein-folding mechanism − and the assessment of structural stability of a protein. − Let us consider the thermostability as an example. In principle, the thermostability can be assessed from the 3D structure, but the structure-thermostability relation remains quite elusive.…”

Methodology for Further Thermostabilization of an Intrinsically Thermostable Membrane Protein Using Amino Acid Mutations with Its Original Function Being Retained

“…However, its application is limited to a mutation from a nonpolar residue to Ala, primarily because protein intramolecular hydrogen bonds are neglected in the score. In a series of works, − we developed a theoretical method enabling us to identify thermostabilizing mutations for human GPCRs. The original aspects of our method are as follows: (1) the importance of the translational entropy of hydrocarbon groups within the lipid bilayer is taken into account; and (2) all the possible single mutations can be considered with minor computational effort.…”

“…Each of the residues with disordered side chains is mutated to another residue except Ala and Pro, and the resultant mutant structure is constructed by modifying only the local structure around the mutated residue. The stability of the mutant is then evaluated using our FEF first developed for GPCRs − and later extended to rhodopsins . We choose the mutations for which the FEF is much lower than for the WT and test them by experiments.…”