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

• 成果类型: Article
• 署名作者: Papasergi-Scott, Makaia M.; Perez-Hernandez, Guillermo; Batebi, Hossein; Gao, Yang; Eskici, Goezde; Seven, Alpay B.; Panova, Ouliana; Hilger, Daniel; Casiraghi, Marina; He, Feng; Maul, Luis; Gmeiner, Peter; Kobilka, Brian K.; Hildebrand, Peter W.; Skiniotis, Georgios
• 署名单位: Stanford University; Free University of Berlin; Humboldt University of Berlin; Charite Universitatsmedizin Berlin; Free University of Berlin; Humboldt University of Berlin; Leipzig University; Philipps University Marburg; University of Milan; University of Erlangen Nuremberg; Humboldt University of Berlin; Free University of Berlin; Charite Universitatsmedizin Berlin; Berlin Institute of Health; Stanford University
• 刊物名称: Nature
• ISSN/ISSBN: 0028-4534
• DOI: 10.1038/s41586-024-07153-1
• 发表日期: 2024-05-30
• 关键词: beta(2) adrenergic-receptor; crystal-structure; adenylate-cyclase; dynamic process; force-field; alpha; binding; complex; conformations; interface

摘要

G-protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by stimulating guanine nucleotide exchange in the G alpha 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 Gs protein in complex with the beta 2-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 G alpha switch regions and the alpha 5 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 alpha-helical domain against the nucleotide-bound Ras-homology domain correlates with alpha 5 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. Time-resolved cryo-EM is used to capture structural transitions during G-protein activation stimulated by a G-protein-coupled receptor.