Structural Connection between Activation Microswitch and Allosteric Sodium Site in GPCR Signaling.

Sodium ions are endogenous allosteric modulators of many G-protein-coupled receptors (GPCRs). Mutation of key residues in the sodium binding motif causes a striking effect on G-protein signaling. We report the crystal structures of agonist complexes for two variants in the first sodium coordination shell of the human A2A adenosine receptor, D522.50 N and S913.39 A. Both structures present an overall active-like conformation; however, the variants show key changes in the activation motif NPxxY. Changes in the hydrogen bonding network in this microswitch suggest a possible mechanism for modified G-protein signaling and enhanced thermal stability. These structures, signaling data, and thermal stability analysis with a panel of pharmacological ligands provide a basis for understanding the role of the sodium-coordinating residues on stability and G-protein signaling. Utilizing the D2.50 N variant is a promising method for stabilizing class A GPCRs to accelerate structural efforts and drug discovery.