G protein-coupled receptors represent the largest family of membrane receptors1 that instigate signalling through nucleotide exchange on heterotrimeric G proteins. Nucleotide exchange, or more precisely, GDP dissociation from the G protein α-subunit, is the key step towards G protein activation and initiation of downstream signalling cascades. Despite a wealth of biochemical and biophysical studies on inactive and active conformations of several heterotrimeric G proteins, the molecular underpinnings of G protein activation remain elusive. To characterize this mechanism, we applied peptide amide hydrogen–deuterium exchange mass spectrometry to probe changes in the structure of the heterotrimeric bovine G protein, Gs (the stimulatory G protein for adenylyl cyclase) on formation of a complex with agonist-bound human β2 adrenergic receptor (β2AR). Here we report structural links between the receptor-binding surface and the nucleotide-binding pocket of Gs that undergo higher levels of hydrogen–deuterium exchange than would be predicted from the crystal structure of the β2AR–Gs complex. Together with X-ray crystallographic and electron microscopic data of the β2AR–Gs complex (from refs 2, 3), we provide a rationale for a mechanism of nucleotide exchange, whereby the receptor perturbs the structure of the amino-terminal region of the α-subunit of Gs and consequently alters the ‘P-loop’ that binds the β-phosphate in GDP. As with the Ras family of small-molecular-weight G proteins, P-loop stabilization and β-phosphate coordination are key determinants of GDP (and GTP) binding affinity.
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We thank J.J.G. Tesmer and G. Skiniotis for discussions. This work was supported by an American Lung Association senior research training fellowship (RT-166882-N, to K.Y.C.), the Lundbeck Foundation (a junior group leader fellowship, to S.G.F.R.), a National Institute of General Medical Sciences (NIGMS) molecular biophysics training grant (GM008270, to B.T.D), a National Institute of Neural Disorders and Stroke grant (NS28471, to B.K.K.), a NHLBI grant (HL071078, to B.K.K.), the Mather Charitable Foundation (B.K.K.), NIGMS grants GM083118 (to B.K.K. and R.K.S.) and GM068603 (to R.K.S.), a Michigan Diabetes Research and Training Center grant, the National Institute of Diabetes and Digestive and Kidney Diseases (P60DK-20572, to R.K.S.), the University of Michigan Biological Sciences Scholars Program (R.K.S.), and by the NIH (grants AI076961, AI081982, AI2008031, CA118595, GM20501, GM066170, GM093325 and RR029388 (to V.L.W.)).
This file contains Supplementary Figures 1-12 with legends and additional references.
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Nature Structural & Molecular Biology (2018)