Heterotrimeric G proteins serve as membrane-associated signaling hubs, in concert with their cognate G-protein-coupled receptors. Fluorine nuclear magnetic resonance spectroscopy was employed to monitor the conformational equilibria of the human stimulatory G-protein α subunit (Gsα) alone, in the intact Gsαβ1γ2 heterotrimer or in complex with membrane-embedded human adenosine A2A receptor (A2AR). The results reveal a concerted equilibrium that is strongly affected by nucleotide and interactions with the βγ subunit, the lipid bilayer and A2AR. The α1 helix of Gsα exhibits significant intermediate timescale dynamics. The α4β6 loop and α5 helix undergo membrane/receptor interactions and order–disorder transitions respectively, associated with G-protein activation. The αN helix adopts a key functional state that serves as an allosteric conduit between the βγ subunit and receptor, while a significant fraction of the ensemble remains tethered to the membrane and receptor upon activation.
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Source data are available on Figshare: https://doi.org/10.6084/m9.figshare.21733997.v2. Source data are provided with this paper.
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We thank D. Pichugin for NMR maintenance and L. Chen for technical support. This work was supported by the Canadian Institutes of Health Research (CIHR) Operating Grants MOP-43998 and PJT-183778 to R.S.P., CIHR Operating Grant PJT-159464 to O.P.E. and N.V.E., the National Institute of General Medical Sciences grants GM106990 and GM083118 to R.K.S. S.K.H. was supported by Alexander Graham Bell Canada Graduate Scholarship-Doctoral from NSERC. A.S. was supported by CREST, Japan Science and Technology Agency (JST), Japan, JPMJCR1402.
The authors declare no competing interests.
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Huang, S.K., Picard, LP., Rahmatullah, R.S.M. et al. Mapping the conformational landscape of the stimulatory heterotrimeric G protein. Nat Struct Mol Biol 30, 502–511 (2023). https://doi.org/10.1038/s41594-023-00957-1