Time-resolved cryo-EM of G-protein activation by a GPCR

Makaía M Papasergi-Scott; Guillermo Pérez-Hernández; Hossein Batebi; Yang Gao; Gözde Eskici; Alpay B Seven; Ouliana Panova; Daniel Hilger; Marina Casiraghi; Feng He; Luis Maul; Peter Gmeiner; Brian K Kobilka; Peter W Hildebrand; Georgios Skiniotis

doi:10.1038/s41586-024-07153-1

Time-resolved cryo-EM of G-protein activation by a GPCR

Nature. 2024 Mar 13. doi: 10.1038/s41586-024-07153-1. Online ahead of print.

Authors

Makaía M Papasergi-Scott¹, Guillermo Pérez-Hernández², Hossein Batebi³, Yang Gao¹, Gözde Eskici¹, Alpay B Seven¹, Ouliana Panova¹, Daniel Hilger^{1

4}, Marina Casiraghi^{1

5}, Feng He¹, Luis Maul⁶, Peter Gmeiner⁶, Brian K Kobilka¹, Peter W Hildebrand^{2

3

7}, Georgios Skiniotis^{8

9}

Affiliations

¹ Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA.
² Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Institute of Medical Physics and Biophysics, Berlin, Germany.
³ Institute of Medical Physics and Biophysics, Faculty of Medicine, Leipzig University, Leipzig, Germany.
⁴ Institute of Pharmaceutical Chemistry, Philipps-University of Marburg, Marburg, Germany.
⁵ Dipartimento di Bioscienze, Università degli Studi di Milano, Milan, Italy.
⁶ Department of Chemistry and Pharmacy, Medicinal Chemistry, Friedrich-Alexander University Erlangen-Nürnberg, Erlangen, Germany.
⁷ Berlin Institute of Health at Charité-Universitätsmedizin Berlin, Berlin, Germany.
⁸ Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. yiorgo@stanford.edu.
⁹ Department of Structural Biology, Stanford University School of Medicine, Stanford, CA, USA. yiorgo@stanford.edu.

PMID: 38480881
DOI: 10.1038/s41586-024-07153-1

Abstract

G-protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by stimulating guanine nucleotide exchange in the Gα subunit¹. To visualize this mechanism, we developed a time-resolved cryo-EM approach that examines the progression of ensembles of pre-steady-state intermediates of a GPCR-G-protein complex. By monitoring the transitions of the stimulatory G_s protein in complex with the β₂-adrenergic receptor at short sequential time points after GTP addition, we identified the conformational trajectory underlying G-protein activation and functional dissociation from the receptor. Twenty structures generated from sequential overlapping particle subsets along this trajectory, compared to control structures, provide a high-resolution description of the order of main events driving G-protein activation in response to GTP binding. Structural changes propagate from the nucleotide-binding pocket and extend through the GTPase domain, enacting alterations to Gα switch regions and the α5 helix that weaken the G-protein-receptor interface. Molecular dynamics simulations with late structures in the cryo-EM trajectory support that enhanced ordering of GTP on closure of the α-helical domain against the nucleotide-bound Ras-homology domain correlates with α5 helix destabilization and eventual dissociation of the G protein from the GPCR. These findings also highlight the potential of time-resolved cryo-EM as a tool for mechanistic dissection of GPCR signalling events.