Conformational Dynamics and Function of an Intermediate GPCR-Gαβγ Complex

Understanding the roles of intermediate states in signaling is pivotal for G protein-coupled receptor (GPCR) drug development. Despite the cryo-EM revolution leading to the resolution of hundreds of GPCR-Gαβγ complex structures, these snapshots primarily capture the fully activated end-state. Consequently, a comprehensive understanding of the conformational transitions during GPCR activation and the roles of GPCR-G protein intermediate complexes in signal transduction remains elusive. In response, we created a series of conformation-biased constructs of the adenosine A2A receptor (A2AR), a neurodegenerative GPCR, under the guidance of energy landscape profiled by 19F quantitative NMR (19F-qNMR). These mutants adopt distinct distributions among five states (S1 through S5) that lie along the activation pathway of the receptor. In particular, a single mutation R291A was able to trap the receptor into an intermediate state S4 in a ligand-independent manner, allowing us to yield a high-resolution (2.8 Å) cryo-EM structure of an intermediate ligand-free (apo) GPCR-Gαβγ complex. The intermediate-trapped mutant also aided us to unravel the functioning of the intermediate complex by eliminating the cross-effects confounded by the end-state complex. Our data provided direct proof that the GPCR intermediate can form a complex with the Gαsβγ and regulate a rate-limited nucleotide exchange without transitioning to the fully activated end-state complex. This advancement fills critical conformational and functional roles of intermediate complexes in GPCR signaling, thereby deconvoluting our understanding of the complexity of GPCR signaling efficacy, which will facilitate the design of drugs that selectively target a disease-related conformation. I will also discuss two recently developed platforms in my lab for conformation-based in-membrane protein drug screening and design with a broad application to any protein of interest.