Understanding the roles of intermediate states in signaling is pivotal to unraveling the activation processes of G protein-coupled receptors (GPCRs). However, the field is still struggling to define these conformational states with sufficient resolution to study their individual functions. Here, we demonstrate the feasibility of enriching the populations of discrete states via conformation-biased mutants, guided by a conformational landscape profiled by 19F-NMR. These mutants adopt distinct distributions among five states, from the fully inactive state S1 to the fully activated state S5, which lie along the activation pathway of adenosine A 2A receptor (A 2A R), a class A GPCR. Our study reveals a structurally conserved cation-π lock between transmembrane helix VI (TM6) and Helix8 that regulates cytoplasmic cavity opening as a “gatekeeper” for G protein penetration. A GPCR activation process based on the well-discerned conformational states is thus proposed, allosterically micro-modulated by the cation-π lock and a previously well-defined ionic interaction between TM3 and TM6. The function of these trapped intermediate states and their complexes were also studied, indicating that nucleotide exchange occurs at the stage of forming the intermediate GPCR-G protein complexes instead of the fully activated end-state complex. These advances fill up the structural and functional gaps of our understanding of GPCR activation and signaling complexity.
Ye et al. (Sun,) studied this question.