PT  - JOURNAL ARTICLE
AU  - Makaía M. Papasergi-Scott
AU  - Guillermo Pérez-Hernández
AU  - Hossein Batebi
AU  - Yang Gao
AU  - Gözde Eskici
AU  - Alpay B. Seven
AU  - Ouliana Panova
AU  - Daniel Hilger
AU  - Marina Casiraghi
AU  - Feng He
AU  - Luis Maul
AU  - Peter Gmeiner
AU  - Brian K. Kobilka
AU  - Peter W. Hildebrand
AU  - Georgios Skiniotis
TI  - Time-resolved cryo-EM of G protein activation by a GPCR
AID  - 10.1101/2023.03.20.533387
DP  - 2023 Jan 01
TA  - bioRxiv
PG  - 2023.03.20.533387
4099  - http://biorxiv.org/content/early/2023/03/21/2023.03.20.533387.short
4100  - http://biorxiv.org/content/early/2023/03/21/2023.03.20.533387.full
AB  - G protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by stimulating the exchange of guanine nucleotide in the Gα subunit. 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. Using variability analysis to monitor the transitions of the stimulatory Gs protein in complex with the β2-adrenergic receptor (β2AR) at short sequential time points after GTP addition, we identified the conformational trajectory underlying G protein activation and functional dissociation from the receptor. Twenty transition structures generated from sequential overlapping particle subsets along this trajectory, compared to control structures, provide a high-resolution description of the order of events driving G protein activation upon 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 (MD) simulations with late structures in the cryo-EM trajectory support that enhanced ordering of GTP upon closure of the alpha-helical domain (AHD) against the nucleotide-bound Ras-homology domain (RHD) correlates with irreversible 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 signaling events.Competing Interest StatementG.S. is a co-founder of and consultant for Deep Apple Therapeutics. B.K.K. is a co-founder of and consultant for ConfometRx.