Ligand-Specified Signaling Efficacy Defined by Unique Transitions in G Protein Conformations

Despite decades of research into the signaling efficacy of G protein-coupled receptors (GPCRs), including the resolution of numerous receptor structures in complex with nucleotide-free G proteins1,2, mini-G proteins3,4, and G protein carboxyl-terminal peptides5-7, our understanding of the underlying mechanisms remains elusive, because a fluidic, dynamic view of the conformational transitions during GPCR activation is still missing. Benefiting from its supersensitization to microenvironmental changes, recent advances in 19F-quantitative NMR (19F-qNMR)8 enable the dissection and quantification of the conformational energy landscape and dynamics of receptors and their signaling partners, offering new perspectives for deciphering the signaling machinery through a novel lens. Herein, we first established a 3-state model of Gs conformational landscape and then quantitatively investigated the conformational transitions and dynamics of Gs proteins in response to different ligand-bound receptors. Our research demonstrated that Gs substates are not only differentially populated in a ligand-dependent manner but also undergo distinct conformational-cycling kinetics across sub-states in response to different ligand bindings. These discoveries enable us to propose a novel GPCR model that integrates quantitative G protein conformational dynamics at the atomic level to explain GPCR pharmacological efficacy, emphasizing that unique equilibrium and transitions of G protein conformational states in response to ligands are critical determinants of signaling specificity.