G-protein-coupled receptor (GPCR)-mediated signal transduction is central to human physiology and disease intervention, yet the molecular mechanisms responsible for ligand-dependent signalling responses remain poorly understood. In class A GPCRs, receptor activation and G-protein coupling entail outward movements of transmembrane helix 6 (TM6). Here, using single-molecule fluorescence resonance energy transfer imaging, we examine TM6 movements in the β2 adrenergic receptor (β2AR) upon exposure to orthosteric ligands with different efficacies, in the absence and presence of the Gs heterotrimer. We show that partial and full agonists differentially affect TM6 motions to regulate the rate at which GDP-bound β2AR–Gs complexes are formed and the efficiency of nucleotide exchange leading to Gs activation. These data also reveal transient nucleotide-bound β2AR–Gs species that are distinct from known structures, and provide single-molecule perspectives on the allosteric link between ligand- and nucleotide-binding pockets that shed new light on the G-protein activation mechanism.
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We thank M. Howarth for the gift of trans-divalent streptavidin, and C. Stern in the laboratory of J. Chodera for constructing the CHARMM-consistent parameters for the dyes used in the molecular dynamics simulations. Computational resources are gratefully acknowledged: an XSEDE allocation at the Texas Advanced Computing Center at the University of Texas at Austin (Stampede supercomputer, project TG MCB120008), support from resources at the Oak Ridge Leadership Computing Facility (ALCC allocation BIP109) at the Oak Ridge National Laboratory that is supported by the Office of Science of the US Department of Energy under contract no. DE-AC05-00OR22725; and the resources of the David A. Cofrin Center for Biomedical Information in the HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine at Weill Cornell Medicine. This work was supported in part by National Institutes of Health (NIH) grants GM098859 (S.C.B.), R21DA0354585 (J.A.J., S.C.B. and G.G.G.), K05DA022413 and R01 MH54137 (J.A.J.), R01GM083118 and R01NS028471 (B.K.K.), and U54GM087519 (H.W. and J.M.P.-A.), the German Academic Exchange Service (DAAD) (D.H.), the American Heart Association Postdoctoral fellowship (15POST22700020) (M.M.), and the Novo Nordisk Foundation Center for Basic Metabolic Research (M.H.).
Extended data figures
This file contains Supplementary Methods, additional references and the uncropped gels.
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Biophysical Reviews (2019)