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.)).
The authors declare no competing financial interests.
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Chung, K., Rasmussen, S., Liu, T. et al. Conformational changes in the G protein Gs induced by the β2 adrenergic receptor. Nature 477, 611–615 (2011). https://doi.org/10.1038/nature10488
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