The dynamic basis of G-protein recognition and activation by a GPCR | Nature

Subjects Cryoelectron microscopy G protein-coupled receptors Abstract G-protein-coupled receptor (GPCR) signalling occurs through heterotrimeric G proteins, whose selective activation leads to distinct cellular outcomes 1 . Although more than 200 GPCR–G protein complex structures have been determined 2 , these static snapshots provide limited insight into the dynamics of G-protein association and dissociation. Here we present cryo-electron microscopy structures of human neurotensin receptor type 1 (NTSR1) with minimally modified G o and G q , showing how the receptor’s intracellular surface dynamically rearranges to accommodate each G-protein subtype. Furthermore, time-resolved cryo-electron microscopy analyses of NTSR1–G i visualized G-protein dissociation processes on GDP/GTP binding. Characterization of more than 20 intermediates, complemented by mutational and computational analyses, identifies four key mechanistic features. First, GDP/GTP induces G i release from both canonical and non-canonical active conformations with distinct kinetics. Second, NTSR1 uses common intracellular rearrangements to recognize different G-protein subtypes and to promote activation of a single subtype. Third, separation from Gβγ involves stepwise remodelling of the Gα switches I–III. Finally, G i dissociates from the receptor through a pathway that is distinct from that of G s , and the canonical and non-canonical NTSR1–G i complexes further diverge in their dissociation trajectories. These findings provide a comprehensive framework for understanding GPCR signalling dynamics and guiding signal-targeted therapeutic development. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution Access options Access through your institution Access Nature and 54 other Nature Portfolio journals Get Nature+, our best-value online-access subscription $32.99 / 30 days cancel any time Learn more Subscribe to this journal Receive 51 print issues and online access $199.00 per year only $3.90 per issue Learn more Buy this article Purchase on SpringerLink Instant access to the full article PDF. USD 39.95 Prices may be subject to local taxes which are calculated during checkout Fig. 1: Cryo-EM structures of NTSR1 complexes with G o or G q in the nucleotide-free state. Fig. 2: Time-resolved cryo-EM analysis of the NTSR1–G i complex with GDP. Fig. 3: 3DVA captures stepwise GDP-induced G-protein dissociation from the receptor. Fig. 4: Time-resolved cryo-EM analysis of the NTSR1–G i complex with GTP. Fig. 5: GTP-induced G-protein dissociation in the NC state. Data availability The cryo-EM density maps and atomic coordinates have been deposited in the Electron Microscopy Data Bank (EMDB) and the PDB with accession codes EMD-64904 and 9VAT for NTSR1–G o , C (nucleotide-free, AHD-open); EMD-64905 and 9VAU for NTSR1–G o , NC1 (nucleotide-free, AHD-open); EMD-64906 and 9VAV for NTSR1–G o , NC2 (nucleotide-free, AHD-open); EMD-64907 and 9VAW for NTSR1–G q , C (nucleotide-free, AHD-open); EMD-64908 and 9VAX for NTSR1–G i GDP 8s -C1 (GDP-unbound, AHD-open); EMD-64909 and 9VAY for NTSR1–G i GDP 8s -C2 (GDP-unbound, AHD-open); EMD-64910 and 9VAZ for NTSR1–G i GDP 8s -NC1 (GDP-unbound, AHD-open); EMD-64911 and 9VB0 for NTSR1–G i GDP 8s -NC2 (GDP-unbound, AHD-open); EMD-64912 and 9VB1 for NTSR1–G i GDP 8s -NC3 (GDP-unbound, AHD-open); EMD-64913 and 9VB2 for NTSR1–G i GDP 8s -C3 (GDP-bound, AHD-closed); EMD-64914 and 9VB3 for NTSR1–G i GDP 8s -C4 (GDP-bound, AHD-closed); EMD-64915 and 9VB4 for NTSR1–G i GDP 15s -C1 (GDP-bound, AHD-closed); EMD-64916 and 9VB5 for NTSR1–G i GDP 15s -C2 (GDP-bound, AHD-closed); EMD-64917 and 9VB6 for NTSR1–G i GTP 8s -C1 (GTP-bound, AHD-open); EMD-64918 and 9VB7 for NTSR1–G i GTP 8s -C2 (GTP-bound, AHD-closed); EMD-64919 and 9VBA for NTSR1–G i GTP 8s -C3 (GTP-bound, AHD-closed); EMD-67429 and 20ZG for i GTP 0–5s -C1 (GTP-unbound, AHD-open); EMD-67430 and 20ZH for i GTP 0–5s -C2 (GTP-unbound, AHD-open); EMD-67431 and 20ZI for i GTP 0–5s -NC1 (GTP-unbound, AHD-open); EMD-67432 and 20ZJ for i GTP 0–5s -NC2 (GTP-unbound, AHD-open); EMD-67433 and 20ZK for NTSR1–G i GTP 0–5s -NC3 (GTP-unbound, AHD-open); EMD-67434 and 20ZL for NTSR1–G i GTP 0–5s -NC4 (GTP-bound, AHD-open); EMD-67426 and 20ZC for NTSR1-delipidated G i GTP 0–5s -C (GTP-unbound, AHD-open); EMD-67427 and 20ZD for NTSR1-delipidated G i GTP 0–5s -NC (GTP-unbound, AHD-open). The raw cryo-EM images of all datasets have been deposited in the Electron Microscopy Public Image Archive (EMPIAR). Maps and atomic coordinates for GTP 0–5s -NC5 and the 3DVA results of GDP 8s are available at Zenodo ( https://doi.org/10.5281/zenodo.15755069 and https://doi.org/10.5281/zenodo.17731629 ) 70 , 71 . All other data are available on request from the corresponding author. Source data are provided with this paper. References Hilger, D., Masureel, M. & Kobilka, B. K. Structure and dynamics of GPCR signaling complexes. Nat. Struct. M