G-protein-coupled receptor (GPCR) signalling occurs through heterotrimeric G proteins, whose selective activation leads to distinct cellular outcomes. Although more than 200 GPCR-G protein complex ...G-protein-coupled receptor (GPCR) signalling occurs through heterotrimeric G proteins, whose selective activation leads to distinct cellular outcomes. Although more than 200 GPCR-G protein complex structures have been determined, 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 and G, 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 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 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 dissociates from the receptor through a pathway that is distinct from that of G, and the canonical and non-canonical NTSR1-G complexes further diverge in their dissociation trajectories. These findings provide a comprehensive framework for understanding GPCR signalling dynamics and guiding signal-targeted therapeutic development.
EMDB-64904, PDB-9vat: Cryo-EM structure of human Neurotensin Receptor 1 (hNTSR1)-Go complex in nucleotide-free C state Method: EM (single particle) / Resolution: 3.37 Å
EMDB-64905, PDB-9vau: Cryo-EM structure of human Neurotensin Receptor 1 (hNTSR1)-Go complex in nucleotide-free NC state 1 Method: EM (single particle) / Resolution: 2.95 Å
EMDB-64906, PDB-9vav: Cryo-EM structure of human Neurotensin Receptor 1 (hNTSR1)-Go complex in nucleotide-free NC state 2 Method: EM (single particle) / Resolution: 2.99 Å
EMDB-64907, PDB-9vaw: Cryo-EM structure of human Neurotensin Receptor 1 (hNTSR1)-Gq (delipidated) complex in nucleotide-free C state Method: EM (single particle) / Resolution: 2.24 Å
EMDB-64908, PDB-9vax: Cryo-EM structure of human Neurotensin Receptor 1 (hNTSR1)-Gi1 complex in nucleotide-free C state 1 Method: EM (single particle) / Resolution: 3.46 Å
EMDB-64909, PDB-9vay: Cryo-EM structure of human Neurotensin Receptor 1 (hNTSR1)-Gi1 complex in nucleotide-free C state 2 Method: EM (single particle) / Resolution: 3.65 Å
EMDB-64910, PDB-9vaz: Cryo-EM structure of human Neurotensin Receptor 1 (hNTSR1)-Gi1 complex in nucleotide-free NC state 1 Method: EM (single particle) / Resolution: 3.38 Å
EMDB-64911, PDB-9vb0: Cryo-EM structure of human Neurotensin Receptor 1 (hNTSR1)-Gi1 complex in nucleotide-free NC state 2 Method: EM (single particle) / Resolution: 3.19 Å
EMDB-64912, PDB-9vb1: Cryo-EM structure of human Neurotensin Receptor 1 (hNTSR1)-Gi1 complex in nucleotide-free NC state 3 Method: EM (single particle) / Resolution: 3.31 Å
EMDB-64913, PDB-9vb2: Cryo-EM structure of the human neurotensin receptor 1 (hNTSR1)-Gi1 complex in the GDP-bound, AHD-closed C state 1, plunge-frozen 8 seconds after GDP addition Method: EM (single particle) / Resolution: 3.08 Å
EMDB-64914, PDB-9vb3: Cryo-EM structure of the human neurotensin receptor 1 (hNTSR1)-Gi1 complex in the GDP-bound, AHD-closed C state 2, plunge-frozen 8 seconds after GDP addition Method: EM (single particle) / Resolution: 3.02 Å
EMDB-64915, PDB-9vb4: Cryo-EM structure of the human neurotensin receptor 1 (hNTSR1)-Gi1 complex in the GDP-bound, AHD-closed C state 1, plunge-frozen 15 seconds after GDP addition Method: EM (single particle) / Resolution: 2.85 Å
EMDB-64916, PDB-9vb5: Cryo-EM structure of the human neurotensin receptor 1 (hNTSR1)-Gi1 complex in the GDP-bound, AHD-closed C state 2, plunge-frozen 15 seconds after GDP addition Method: EM (single particle) / Resolution: 3.14 Å
EMDB-64917, PDB-9vb6: Cryo-EM structure of the human neurotensin receptor 1 (hNTSR1)-Gi1 complex in the GTP-bound, AHD-open C state, plunge-frozen 8 seconds after GTP addition Method: EM (single particle) / Resolution: 3.38 Å
EMDB-64918, PDB-9vb7: Cryo-EM structure of the human neurotensin receptor 1 (hNTSR1)-Gi1 complex in the GTP-bound, AHD-closed C state 1, plunge-frozen 8 seconds after GTP addition Method: EM (single particle) / Resolution: 3.01 Å
EMDB-64919, PDB-9vba: Cryo-EM structure of the human neurotensin receptor 1 (hNTSR1)-Gi1 complex in the GTP-bound, AHD-closed C state 2, plunge-frozen 8 seconds after GTP addition Method: EM (single particle) / Resolution: 3.38 Å
EMDB-67426, PDB-20zc: Cryo-EM structure of human Neurotensin Receptor 1 (hNTSR1)-Gi1 (delipidated) complex in nucleotide-free C state Method: EM (single particle) / Resolution: 2.6 Å
EMDB-67427, PDB-20zd: Cryo-EM structure of human Neurotensin Receptor 1 (hNTSR1)-Gi1 (delipidated) complex in nucleotide-free NC state Method: EM (single particle) / Resolution: 2.8 Å
EMDB-67429, PDB-20zg: Cryo-EM structure of the human neurotensin receptor 1 (hNTSR1)-Gi1 complex in the GTP-bound, AHD-open C state 1, plunge-frozen 0-5 seconds after GTP addition Method: EM (single particle) / Resolution: 2.4 Å
EMDB-67430, PDB-20zh: Cryo-EM structure of the human neurotensin receptor 1 (hNTSR1)-Gi1 complex in the GTP-bound, AHD-open C state 2, plunge-frozen 0-5 seconds after GTP addition Method: EM (single particle) / Resolution: 2.4 Å
EMDB-67431, PDB-20zi: Cryo-EM structure of the human neurotensin receptor 1 (hNTSR1)-Gi1 complex in the GTP-bound, AHD-open NC state 1, plunge-frozen 0-5 seconds after GTP addition Method: EM (single particle) / Resolution: 2.7 Å
EMDB-67432, PDB-20zj: Cryo-EM structure of the human neurotensin receptor 1 (hNTSR1)-Gi1 complex in the GTP-bound, AHD-open NC state 2, plunge-frozen 0-5 seconds after GTP addition Method: EM (single particle) / Resolution: 2.9 Å
EMDB-67433, PDB-20zk: Cryo-EM structure of the human neurotensin receptor 1 (hNTSR1)-Gi1 complex in the GTP-bound, AHD-open NC state 3, plunge-frozen 0-5 seconds after GTP addition Method: EM (single particle) / Resolution: 2.7 Å
EMDB-67434, PDB-20zl: Cryo-EM structure of the human neurotensin receptor 1 (hNTSR1)-Gi1 complex in the GTP-bound, AHD-open NC state 4, plunge-frozen 0-5 seconds after GTP addition Method: EM (single particle) / Resolution: 3.1 Å
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homo sapiens (human)
mus musculus (house mouse)