Theoretical Prediction of Mutations Improving Thermal Stability of Adenosine A2a Receptor

Abstract

G protein-coupled receptors (GPCRs) are physiologically important membrane proteins possessing seven transmembrane domains, which are responsible for signal transduction pathways. Therefore, they form the most important target for drug design. However, their mass production and structure determination by the X-ray crystallography are quite difficult to achieve due to the low thermal stability in detergents. Though the stability can be enhanced by introducing mutations into GPCRs, a random search accompanying a heavy experimental burden is currently employed to obtain mutations leading to sufficient enhancement. In the present study, through mutations for the antagonist-binding structure of the adenosine A2a receptor, we investigate how to predict the mutants which lead to enhanced thermal stability using our free-energy function (FEF) recently developed for membrane proteins. The FEF comprises two components: the energetic term, which is focused on the energy decrease arising from formation of intramolecular hydrogen bonds, and the entropic term, which originates from the translational displacement of hydrocarbon groups constituting nonpolar chains of the lipid bilayer. After calculations of the FEF for all mutants, we have chosen some candidate mutants whose thermal stability would be most improved, and then their stabilities are experimentally examined. The findings are as follows. The success rate of the prediction focused on the entropic term alone is about 1/3 that is much higher than that reached by the trial-and-error prediction. This result implies that the entropic effect of hydrocarbon groups is critical for the structural stability of GPCRs. Moreover, when the energetic term is also considered, the success rate is improved to 1/2. Since the calculation of the FEF can be accomplished quite rapidly, we can theoretically examine a large number of mutations.