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

作者：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 · 发表于：bioRxiv (Cold Spring Harbor Laboratory) · 年份：2023 · DOI：10.1101/2023.03.20.533387 · 被引用次数：14 · 研究领域：Receptor Mechanisms and Signaling、Mass Spectrometry Techniques and Applications、Protein Structure and Dynamics

Summary 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 β 2 -adrenergic receptor (β 2 AR) 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 ...