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Conformational transitions of a neurotensin receptor 1–Gi1 complex


Neurotensin receptor 1 (NTSR1) is a G-protein-coupled receptor (GPCR) that engages multiple subtypes of G protein, and is involved in the regulation of blood pressure, body temperature, weight and the response to pain. Here we present structures of human NTSR1 in complex with the agonist JMV449 and the heterotrimeric Gi1 protein, at a resolution of 3 Å. We identify two conformations: a canonical-state complex that is similar to recently reported GPCR–Gi/o complexes (in which the nucleotide-binding pocket adopts more flexible conformations that may facilitate nucleotide exchange), and a non-canonical state in which the G protein is rotated by about 45 degrees relative to the receptor and exhibits a more rigid nucleotide-binding pocket. In the non-canonical state, NTSR1 exhibits features of both active and inactive conformations, which suggests that the structure may represent an intermediate form along the activation pathway of G proteins. This structural information, complemented by molecular dynamics simulations and functional studies, provides insights into the complex process of G-protein activation.

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Fig. 1: Cryo-EM structures of hNTSR1–Gi1 complex.
Fig. 2: Structural comparison of hNTSR1 in C and NC states.
Fig. 3: Structural comparison of Gαi1 in C and NC states.
Fig. 4: Comparison of receptor–G-protein interfaces in C and NCstates.
Fig. 5: Interactions specifically observed in NC-state hNTSR1–Gi1 complex.
Fig. 6: Proposed model of hNTSR1 activation.

Data availability

All data generated or analysed during this study are included in the published Article and Supplementary Information. The cryo-EM density maps for the hNTSR1–Gi1 complex in C and NC states have been deposited in the Electron Microscopy Data Bank (EMDB) under accession codes EMD-20180 and EMD-20181, respectively. The coordinates for the models of hNTSR1–Gi1 in both states have been deposited in the PDB under accession numbers 6OS9 and 6OSA, respectively. All other data are available upon request to the corresponding authors.


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We thank Y. S. Kim for assistance with HEK cell maintenance and transfection; B. White for assistance with Sf9 insect cell maintenance and mini-preparation of plasmids; S. Maeda for the P1 virus of scFv16; K. Geiselhart and M. Lima for administrative support of the project; and M. Masureel and S. Lavington for useful discussions on the manuscript. C.-M.S. acknowledges the Sigrid Jusélius Foundation and the International Human Frontier Science Program (Long-Term Fellowship LT000916-2018-L). R.F. was funded by grant NNF15OC0015268 from the Novo Nordisk Foundation and the Stanford Bio-X Program. This work was supported by National Institutes of Health (NIH) grants R01GM127359 (R.O.D.), R01GM083118 (B.K.K. and G.S.) and R01NS028471 (B.K.K.), the PRIME JP17gm5910013 (A.I.) and the LEAP JP17gm0010004 (A.I. and J.A.) from the Japan Agency for Medical Research and Development, JSPS KAKENHI 19H03163 (H.E.K.) and 17K08264 (A.I.), and the Mathers Foundation (G.S. and B.K.K.). B.K.K. is a Chan–Zuckerberg Biohub Investigator.

Peer review information

Nature thanks Shangyu Dang, Daniel Wacker and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information




H.E.K. started the project, performed the molecular cloning, expressed and purified proteins, prepared the NTSR1–Gi1 complexes, refined the structure from cryo-EM density maps, performed FSEC-TS and GTPase-Glo assays, and analysed GPCR–G-protein interactions, with R.F. Y.Z. obtained cryo-EM images with the help of H.H. and processed cryo-EM data to generate 3D maps. C.-M.S. and N.R.L. performed and analysed the molecular dynamics simulations under the supervision of R.O.D. R.F. performed in silico analysis of GPCR–G-protein complexes. A.I. and F.M.N.K. performed nano-BiT G-protein dissociation assay under supervision of J.A. K.K.K. helped with G protein and scFv16 purification. D.H. provided critical input on structural analysis. W.H. contributed to the early stages of the project, including HEK cell transfection. H.E.K. prepared the initial manuscript and H.E.K., G.S. and B.K.K. wrote the paper with input from all the authors.

Corresponding authors

Correspondence to Brian K. Kobilka or Georgios Skiniotis.

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Competing interests

B.K.K. is a founder of and consultant for ConfometRx, Inc.

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Extended data figures and tables

Extended Data Fig. 1 Structure-based sequence alignment of class-A GPCRs.

The sequences shown are those are for hNTSR1, rNTSR1, mouse μOR, human cannabinoid receptor 1 (CB1), human rhodopsin, human 5-hydroxytryptamine receptor 1B (5HT1B), human A1 adenosine receptor (A1AR), human β2AR and human A2 adenosine receptor (A2AR). The sequence alignment was created using GPCRdb ( and ESPript 373 servers. Secondary structure elements for hNTSR1 are shown as coils and arrows. PIF, PAF, PLF or LVF, DRY or ERY, and NPXXY motifs are highlighted in green, red and blue, respectively. The truncated sequences of hNTSR1(∆ICL3) are highlighted in grey.

Extended Data Fig. 2 Preparation and cryo-EM of the full-length hNTSR1–Gi1–scFv16 and the hNTSR1(∆ICL3)–Gi1–scFv16 complexes.

a, Representative elution profile (out of more than three independent runs) of full-length hNTSR1 (hNTSR1(FL), comprising residues 20–418 and A85L mutation) in complex with Gi1 and scFv16, on a Superdex 200 Increase 10/300 GL. b, Representative 3D classifications of the hNTSR1(FL)–Gi1–scFv16 complex. The C-state and NC-state complex maps are coloured in cyan and red, respectively. The black arrow indicates the partially disordered α-helical domain. c, Representative cryo-EM micrograph of the hNTSR1(∆ICL3) in complex with Gi1 and scFv16. d, Representative 2D averages showing different views of the hNTSR1(∆ICL3)–Gi1–scFv16 complex. e, Flow chart of cryo-EM data processing. f, Local resolutions of C1 state (left) and NC1 state (right). Full view of the RELION local-resolution-filtered map coloured by local resolution. g, Representative 3D classifications of the hNTSR1(∆ICL3)–Gi1–scFv16 complex. The black arrow indicates the partially disordered α-helical domain. h, Gold-standard Fourier shell correlation plots. i, Fourier shell correlation curves for the final model versus the final map and the half maps.

Extended Data Fig. 3 Functional comparison between hNTSR1(FL) and hNTSR1(∆ICL3).

a, FSEC-TS74 for hNTSR1(FL)–Gi1 (left) and hNTSR1(∆ICL3)–Gi1 (right). Each profile is a representative of two independent experiments. Only about 50% of the hNTSR1(FL)–Gi1 complex survives after a 45 °C incubation for 10 min, whereas over 90% of the hNTSR1(FL)–Gi1 complex survives after the same heat stress. b, GTPase-Glo assay39 of hNTSR1(FL) and hNTSR1(∆ICL3). Sample sizes for both hNTSR1(FL) and hNTSR1(∆ICL3) are 3. The intrinsic GTP hydrolysis activities of Gi1 heterotrimer and Gq heterotrimer are enhanced by hNTSR1. The guanine-nucleotide exchange factor activities of hNTSR1(FL) and hNTSR1(∆ICL3) proteins are equally potent. Symbols and bars represent individual value and mean of a single experiment performed in triplicate. c, Cell-surface expression level. HEK293 cells transiently expressing a Flag-epitope-tagged NTSR1 construct were analysed by flow cytometry. Sample sizes are shown in parentheses. Centre lines and error bars represent mean and s.e.m. of the indicated experiments. One-way analysis of variance (ANOVA) with Dunnett’s post hoc test was used to assess statistical analyses (ANOVA P value = 0.90, not significantly different (NS) among the four samples). dg, NanoBiT G-protein dissociation assay. Concentration–response curves for G-protein dissociation signals (d, top) and their summary (d, bottom), for hNTSR1(FL) and hNTSR1(∆ICL3). Symbols and error bars represent mean and s.e.m. of indicated independent numbers of experiments, each performed in duplicate. e, Heat map of NanoBiT G-protein dissociation signals for hNTSR1(∆ICL3) (10 μM JMV449), β2AR (10 μM isoproterenol) and μOR (10 μM DAMGO). Mean values of test GPCR-specific signal-changes (differences in NanoBiT-G protein dissociation signal between test GPCR-transfected cells and mock-transfected cells) are shown. Sample sizes for Gs, Gi1, Go, Gq and G13 are as follows: 5, 5, 5, 5 and 5 (hNTSR1), 6, 5, 3, 3 and 3 (β2AR) and 7, 7, 6, 5 and 5 (μOR). Unlike β2AR and μOR, the NTSR1 agonist (JMV449) causes a signal decrease for all G-proteins (e), which suggest that all G proteins can be recognized and activated by hNTSR1, and dissociated into Gα and Gβγ subunits. f, The summary of NanoBiT G-protein dissociation assay of wild-type hNTSR1 and the hNTSR1(S93A/L94A/R294A/H373A) mutant for full-length constructs. Concentration–response curves are shown in Fig. 5b. We used an unpaired t-test with correction for multiple comparisons using the Holm–Sidak method. NS, not significantly different from wild type; **P < 0.01. g, NanoBiT G-protein dissociation assay of wild-type hNTSR1 and the hNTSR1(S93A/L94A/R294A/H373A) mutant for ∆ICL3 constructs. Concentration–response curves of Gs, Gi1, Go, Gq and G13 signalling (top), and the summary of the assay result (bottom). Symbols and error bars (top) represent mean and s.e.m. of indicated independent numbers of experiments (bottom), each performed in duplicate. We used an unpaired t-test with correction for multiple comparisons using the Holm–Sidak method. NS, not significantly different from wild type; *P < 0.05, **P < 0.01, ***P < 0.001.

Extended Data Fig. 4 Structural comparisons of micro-conformers observed in NC- and C-state hNTSR1(∆ICL3)–Gi1 complexes.

a, Side (top) and extracellular (bottom) views of the superposed structures of three conformers in the C state. b, Side (top) and extracellular (bottom view (bottom) of the superimposed structures of two conformers in the NC state. In each micro-conformer, the G protein is 4–5° rotated relative to the receptor.

Extended Data Fig. 5 Cryo-EM map quality.

a, b, Density and model for transmembrane helices of hNTSR1, α5 helix of Gαi1 and JMV449 in C-state (a) and NC-state (b) complexes. c, Putative cholesterol observed near TM6 and TM7, and the neighbouring side chains in the putative binding site of C-state (left) and NC-state (right) complexes.

Extended Data Fig. 6 Agonist-peptide binding to NTSR1.

ac, Agonist peptide and the neighbouring side chains in the ligand-binding site of active rNTSR1 (rNTSR1(ELF)) (a), C-state complex (b) and NC-state complex (c). Black dashed lines represent hydrogen bonds.

Extended Data Fig. 7 Structural comparison of NTSR1, β2AR and μOR.

a, b, Comparison of TM6, DRY motif and NPXXY motif between C-state hNTSR1 (blue), NC-state hNTSR1 (red), active β2AR (orange) (PDB 3SN6) and active μOR (purple) (PDB 6DDF). Black double-headed arrow represents the conformational difference of TM6 between the receptor in the Gs complex and the receptor in the Gi complex (a). Y7.53 in the NPXXY motif packs against R3.50 in C-state hNTSR1, active β2AR and active μOR, but there is no direct interaction between Y7.53 and R3.50 in NC-state hNTSR1 (b). c, Comparison of the cytoplasmic half of TM7 between C-state hNTSR1 (blue), NC-state hNTSR1 (red), inactive-state rNTSR1 (rNTSR1-inact, grey), and active-state rNTSR1 (rNTSR1-act, green). NC-state hNTSR1 is superimposes well onto rNTSR1(TM86V/ΔIC3A), which suggests that TM7 adopts an inactive-like conformation in NC-state hNTSR1.

Extended Data Fig. 8 Structural comparison of G proteins and GPCR–G-protein complexes.

a, Overall structures of Gαi from C-state hNTSR1–Gi1 (yellow), NC-state hNTSR1–Gi1 (grey), μOR–Gi1 (green), rhodopsin–Gi1 (purple) and A1AR–Gi2 (pink) complexes. The α-helical domain of the rhodopsin–Gi1 complex is removed for clarity. b, The π–π stacking interaction between the α5 helix and β6 strand, which is specifically observed in Gi complexes. c, d, Side view and extracellular view of the superimposed structures of C-state hNTSR1–Gi1 complex (blue, hNTSR1; yellow, Gi1) and β2AR-Gs complex (grey, β2AR; orange, Gs) (c), and C-state hNTSR1–Gi1 complex (blue, hNTSR1; yellow, Gi1) and μOR–Gi1 complex (green, μOR; grey, Gi1) (d).

Extended Data Fig. 9 Dynamics of the nucleotide-binding pocket in NC and C states.

a, Cryo-EM density, shown in two different contour levels, corresponding to the α5–β6 loop of Gαi1 from C-state (top) and NC-state (bottom) hNTSR1–Gi1 complex. b, Summary of molecular dynamics simulation conditions. c, Dynamics of the P-loop and switch II regions during molecular dynamics simulations of the C-state (left) and NC-state (right) complexes. The figures show superposed frames sampled every 50 ns from 5 independent simulations for each state. In these simulations, the P-loop and switch-II regions show similar flexibility in both the C-state and NC-state complexes. d, Representative molecular dynamics simulations initiated from the C-state hNTSR1–Gi1 complex (left) and the NC-state hNTSR1–Gi1 complex (right). The r.m.s.d. of the NPXXY motif relative to the NC-state structure (top) and the distance between TM3 and TM6 (bottom) are plotted for each simulation. In both C-state and NC-state hNTSR1–Gi1 complexes, the NPXXY region and TM6 consistently retain the conformations that are observed in the cryo-EM structures. e, The r.m.s.d. of α5 to the cryo-EM C-state Gi1 for each simulation of the NC-state and C-state complexes. The trajectories were aligned on TM1–TM4 of the receptor, and the r.m.s.d. was calculated for the backbone atoms of residues 329 to 354 of α5. f, Dynamics of the α5–β6 loop for each independent simulation of the C- and NC-state complexes. Frames are sampled every 20 ns from each individual simulation. In these simulations, the α5–β6 loop shows enhanced conformational variability in the C-state complex compared to the NC-state complex. g, The calculated solvent-accessible surface area (SASA) of the nucleotide-binding pocket in the NC and C states. The solvent accessibility of the pocket is consistently larger for the C state (P = 0.00027976943074583155, using Welch’s two-sided t-test and treating each simulation as an independent data point).

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics

Supplementary information

Supplementary Information

Supplementary Discussion about a putative cholesterol binding site in hNTSR1, and information about the amino acid sequences of the constructs used in the NanoBiT G-protein dissociation assay.

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Kato, H.E., Zhang, Y., Hu, H. et al. Conformational transitions of a neurotensin receptor 1–Gi1 complex. Nature 572, 80–85 (2019).

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