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Mapping the conformational landscape of the stimulatory heterotrimeric G protein

Nature Structural & Molecular Biology volume 30, pages 502–511 (2023)Cite this article

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Abstract

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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Fig. 1: Labeling sites for probing the conformational landscape of Gsα.
Fig. 2: Chosen labeling sites are allosterically engaged with the nucleotide binding pocket.
Fig. 3: Gsα reveals a complex landscape characterized by both global and local conformational dynamics.
Fig. 4: Distinct conformational signatures are observed for the N-terminal helix in the combined presence of Gβγ and A2AR.
Fig. 5: Hinge region connecting the RHD and the AHD undergoes order-disorder transitions upon nucleotide release.
Fig. 6: Y358C exhibits chemical shift sensitivity toward nucleotide.
Fig. 7: Gsαβγ heterotrimer undergoes structural rearrangement rather than complete physical dissociation in the presence of GDP-[AlF4]− and GTPγS.
Fig. 8: ‘Open’ conformation of Gsα is stabilized by membrane and Gβγ.

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Data availability

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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Acknowledgements

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.

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Authors and Affiliations

  1. Department of Chemistry, University of Toronto, UTM, Mississauga, Ontario, Canada

    Shuya Kate Huang, Louis-Philippe Picard, Rima S. M. Rahmatullah, Aditya Pandey & R. Scott Prosser

  2. Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada

    Ned Van Eps, Oliver P. Ernst & R. Scott Prosser

  3. Department of Pharmacology, University of California San Diego School of Medicine, La Jolla, CA, USA

    Roger K. Sunahara

  4. Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada

    Oliver P. Ernst

  5. RIKEN Center for Advanced Intelligence Project, RIKEN, Tokyo, Japan

    Adnan Sljoka

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Contributions

S.K.H. and R.S.P. designed the research. S.K.H. and R.S.M.R. carried out expression and purification of various Gsα constructs. S.K.H. carried out expression and purification of A2AR. S.K.H. and A.P. carried out expression and purification of Gβγ. N.V.E., R.S.M.R. and S.K.H. carried out cloning for the various Gsα constructs. S.K.H. and R.S.M.R. performed the NMR experiments and the GTP hydrolysis experiments. L.-P.P. produced the double cysteine mutant of Gsα and carried out the FRET experiments. A.S. performed the RTA analysis. R.K.S. provided assistance with data interpretation and the manuscript. S.K.H. and R.S.P. prepared the manuscript. R.S.P. and O.P.E. supervised the project.

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Correspondence to Adnan Sljoka or R. Scott Prosser.

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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

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