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Structural basis of amine odorant perception by a mammal olfactory receptor

Nature volume 618, pages 193–200 (2023)Cite this article

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Abstract

Odorants are detected as smell in the nasal epithelium of mammals by two G-protein-coupled receptor families, the odorant receptors and the trace amine-associated receptors1,2 (TAARs). TAARs emerged following the divergence of jawed and jawless fish, and comprise a large monophyletic family of receptors that recognize volatile amine odorants to elicit both intraspecific and interspecific innate behaviours such as attraction and aversion3,4,5. Here we report cryo-electron microscopy structures of mouse TAAR9 (mTAAR9) and mTAAR9–Gs or mTAAR9–Golf trimers in complex with β-phenylethylamine, N,N-dimethylcyclohexylamine or spermidine. The mTAAR9 structures contain a deep and tight ligand-binding pocket decorated with a conserved D3.32W6.48Y7.43 motif, which is essential for amine odorant recognition. In the mTAAR9 structure, a unique disulfide bond connecting the N terminus to ECL2 is required for agonist-induced receptor activation. We identify key structural motifs of TAAR family members for detecting monoamines and polyamines and the shared sequence of different TAAR members that are responsible for recognition of the same odour chemical. We elucidate the molecular basis of mTAAR9 coupling to Gs and Golf by structural characterization and mutational analysis. Collectively, our results provide a structural basis for odorant detection, receptor activation and Golf coupling of an amine olfactory receptor.

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Fig. 1: The overall structure of the mTAAR9–Gs and mTAAR9–Golf complexes.
Fig. 2: A conserved TAAR motif for recognition of monoamines and polyamines.
Fig. 3: Structural features determining polyamine recognition by mTAAR9.
Fig. 4: Potential structural mechanism of SPE and PEA recognition in mTAAR9.
Fig. 5: Coupling of mTAAR9 with Gs and Golf.

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Data availability

All data produced or analysed in this study are included in the main text or the supplementary materials. The cryo-EM density maps and atomic coordinates have been deposited at the Electron Microscopy Data Bank under accession codes EMD-35762 (SPE–mTAAR9–Gs complex), EMD-35763 (PEA–mTAAR9–Gs complex), EMD-35705 (DMCHA–mTAAR9–Gs complex), EMD-35764 (CAD–mTAAR9–Gs complex), EMD-35761 (PEA–mTAAR9–Golf complex), EMD-35765 (local refinement for the SPE–mTAAR9 complex) and EMD-35771 (local refinement for PEA–mTAAR9 in the PEA–mTAAR9–Gs complex), and Protein Data Bank under accession codes 8IW4 (SPE–mTAAR9–Gs complex), 8IW7 (PEA–mTAAR9–Gs complex), 8ITF (DMCHA–mTAAR9–Gs complex), 8IW9 (CAD–mTAAR9–Gs complex), 8IW1 (PEA–mTAAR9–Golf complex), 8IWE (local refinement for the SPE–mTAAR9 complex) and 8IWM (local refinement for PEA–mTAAR9 in the PEA–mTAAR9–Gs complex). All other data are available upon request from the corresponding authors.

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Acknowledgements

This work was supported by National Key R&D Program of China (2022YFC2702600 to F.Y., 2018YFC1003600 to X.Y. and J.-P.S., 2019YFA0904200 to J.-P.S., 2021ZD0203100 to Q. Li and 2022YFA1104003 to Y.X.), National Science Fund for Distinguished Young Scholars Grant (81825022 to J.-P.S.), National Science Fund for Excellent Young Scholars (82122070 to F.Y. and 32122038 to Q.L.), National Natural Science Foundation of China (81773704 to J.-P.S. and 92057121 to X.Y.), the Key Research Project of the Beijing Natural Science Foundation, China (Z200019 to J.-P.S.), Major Fundamental Research Program of Natural Science Foundation of Shandong Province, China (ZR2021ZD18 to X.Y. and ZR2020MH132 to S. Liu), the Basic Research Project from the Science and Technology Commission of Shanghai Municipality (21JC1404500 to Q.L.), Shandong Province Key Research and Development Project (2021CXGC011105 to Y.X.) and Shuguang Program supported by Shanghai Education Development Foundation and Shanghai Municipal Education Commission (21SG16 to Q.L.). The molecular simulations were performed on the HPC Cloud Platform of Shandong University. We thank Y. Yin and W. Shui for data collection. S.D.L. is an investigator of the Howard Hughes Medical Institute.

Author information

Author notes

  1. These authors contributed equally: Lulu Guo, Jie Cheng, Shuo Lian, Qun Liu, Yan Lu, Yuan Zheng, Kongkai Zhu

Authors and Affiliations

  1. Advanced Medical Research Institute, Meili Lake Translational Research Park, Cheeloo College of Medicine, Shandong University, Jinan, China

    Lulu Guo, Jie Cheng, Kongkai Zhu, Xiang Han, Shangming Liu, Fan Yang & Jin-Peng Sun

  2. Department of Biochemistry and Molecular Biology, Shandong University School of Medicine, Jinan, China

    Lulu Guo, Qun Liu, Yan Lu, Chao Zhang, Naikang Rong, Yuming Zhuang, Guoxing Fang, Jingjing Jiang, Tianyao Zhang, Xiang Han, Zili Liu, Fan Yang & Jin-Peng Sun

  3. Key Laboratory Experimental Teratology of the Ministry of Education and Department of Physiology, School of Basic Medical Sciences, Shandong University, Jinan, China

    Jie Cheng & Xiao Yu

  4. Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Peking University, Key Laboratory of Molecular Cardiovascular Science, Ministry of Education, Beijing, China

    Jie Cheng, Yuan Zheng & Jin-Peng Sun

  5. Department of General Surgery, Qilu Hospital of Shandong University, Jinan, China

    Shuo Lian, Minghui Zhang & Yunfei Xu

  6. Songjiang Institute and Songjiang Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Yalei Kong & Qian Li

  7. Center for Brain Science, Shanghai Children’s Medical Center, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

    Yalei Kong & Qian Li

  8. Department of Anatomy and Physiology, Ministry of Education-Shanghai Key Laboratory of Children’s Environmental Health in Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Yalei Kong & Qian Li

  9. Department of Otolaryngology, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Shandong, China

    Ming Xia

  10. MOE Key Laboratory for Nonequilibrium Synthesis and Modulation of Condensed Matter, School of Physics, Xi’an Jiaotong University, Xi’an, China

    Lei Zhang

  11. Howard Hughes Medical Institute, Department of Cell Biology, Harvard Medical School, Boston, MA, USA

    Stephen D. Liberles

  12. Shanghai Research Center for Brain Science and Brain-Inspired Intelligence, Shanghai, China

    Qian Li

Authors
  1. Lulu Guo
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  2. Jie Cheng
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  3. Shuo Lian
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Contributions

J.-P.S. and Q. Li initiated the study of TAAR olfactory receptors. J.-P.S., Y.X. and F.Y. organized the project. J.-P.S., X.Y. and F.Y. designed and supervised the overall experimental design and execution. J.-P.S., Y.X., F.Y., L.G. and J.C. participated in data analysis and interpretation. J.-P.S. and F.Y. guided all structural analysis. Q. Li and S.D.L. provided original and modified TAAR plasmids, and organized functional experiments together with J.-P.S., X.Y. and F.Y. L.G. and Q. Liu designed and executed receptor screening for structural studies. Y.K., L.G., Q. Liu and Q. Li generated the mTAAR9 insect cell expression construct. L.G., Y. Zheng and Y.X. established the PEA–mTAAR–Gs–scFv16, SPE–mTAAR–Gs–scFv16 and PEA–mTAAR9–Golf–scFv16 complex purification protocols and prepared samples for cryo-EM. J.C., S. Lian and Y.L. generated mTAAR9 constructs and mutants for the cell-based G-protein activity assays. J.C., S. Lian, X.H., S. Liu and Y.L. designed ligand-binding pocket and G-protein interaction mutants. J.C., S. Lian, X.Y. and Y.L. established the Golf–Gβγ dissociation assay. Y.L. performed the mass spectrometry assay. K.Z. built the binding modes of octanal and propionate within the ligand pocket of the corresponding reported odorant receptors Olfr2 and Olfr78. K.K.Z. and M.Z. performed molecular dynamics simulations for ligands within the mTAAR9 complex structures. C.Z. performed molecular dynamics simulations for the apo-mTAAR9 model and PEA–mTAAR9–Gαsβ1γ13 model. L.G., M.Z., N.R., J.J., Q. Liu, T.Z., G.F., Y. Zhuang, L.Z. and Y. Zheng prepared the cryo-EM grids, collected the cryo-EM data for the SPE–mTAAR9–Gs–scFv16, PEA–mTAAR9–Gs–scFv16, PEA–mTAAR9–Golf–scFv16, DMCHA–mTAAR9–Gs–scFv16 and CAD–mTAAR9–Gs–scFv16 complexes. J.-P.S. wrote the initial manuscript. F.Y., Q. Li, L.G., M.X. and X.Y. provided important discussions and essential revisions. Y. Zheng and J.C. provided Fig. 1a–c. F.Y., L.G., Y.L., M.X and Y.X. provided Figs. 1–5. F.Y., J.C., L.G., S. Lian and M.Z. provided extended data figures.

Corresponding authors

Correspondence to Yunfei Xu, Fan Yang, Qian Li or Jin-Peng Sun.

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Extended data figures and tables

Extended Data Fig. 1 SDS-PAGE analysis of protein expression levels of TAAR receptors and size-exclusion chromatography.

a, The protein levels of TAAR receptors in the plasma membrane fraction, including 6 hTAARs, 13 mTAARs, and 16 rTAARs that were overexpressed in Sf9 cell lines using a Bac-to-Bac® (Invitrogen) baculovirus expression system. Data are represented as mean ± SEM from 3 independent experiments (n = 3). b-e, Most olfactory receptors, including TAARs, are well known for very low expression levels using heterologous expression systems, either in baculovirus- insect cells or in mammalian HEK293 cells.We therefore screened the protein levels of 35 TAAR members in the plasma membrane fraction, including 6 in humans (hTAARs), 13 in mice (mTAARs, except mTAAR8b and mTAAR8c) and 16 in rats (rTAARs, expect rTAAR8a), using Spodoptera frugiperda (Sf9) insect cells and Bac-to-Bac® (Invitrogen) baculovirus system. Notably, only mTAAR1, mTAAR9, rTAAR7d, hTAAR5, and hTAAR6 were detected on the plasma membrane, with yields ranging from approximately 0.11-0.21 mg/L. f, Representative size-exclusion chromatography elution profiles of the purified mTAAR9-Golf, mTAAR1-Golf, rTAAR7d-Golf, hTAAR6-Golf, and hTAAR5-Golf complexes using Superose 6 Increase 10/300 GL. Size exclusion chromatography showed that only mTAAR9 generated homogenous and stable complexes with the Golf trimer (Gβ1-Gγ2), whereas all 4 other TAAR-Gαolf-Gβ1-Gγ2 complexes and TAAR in complex with Gαolf-Gβ1-Gγ13 were unstable and often underwent constant dissociation. The size-exclusion chromatography peaks are marked by triangles. g, Representative size-exclusion chromatography elution profiles of the purified mTAAR9-Gs, mTAAR1-Gs, rTAAR7d-Gs, hTAAR6-Gs, and hTAAR5-Gs complexes using Superose 6 Increase 10/300 GL. Size exclusion chromatography showed that only mTAAR9 and mTAAR1 generated homogenous and stable complexes with the Gs trimer (Gβ1-Gγ2), whereas all 3 other TAAR-Gs complexes were unstable and often underwent constant dissociation. The size-exclusion chromatography peaks are marked by triangles.

Extended Data Fig. 2 Activation of mTAAR9 by different odorant agonists.

a, The potency and efficacy of Gα (including Golf, Gs, Gi1-3, Go, and Gq)-Gβγ dissociation in response to stimulation with different agonists in mTAAR9-overexpressing HEK293 cells. The heatmap is colored according to the value of pEC50 and Emax. ND, signal not detectable. Values were averaged from 3 independent experiments for mTAAR9 (n = 3). Abbreviations: SPE, spermidine; T1AM, 3-iodothyronamine; DMCHA, N, N-dimethylcyclohexylamine; IAA, isoamylamine; IBA, isobutylamine; TMA, trimethylamine; CHA, cyclohexylamine; 1-MPD, 1-methylpiperidine; PEA, β-phenylethylamine; CAD, cadaverine; PTC, putrescine. b, Dose response curves of Gα (including Golf, Gs, Gi1-3, Go, and Gq)-Gβγ dissociation in mTAAR9-overexpressing HEK293 cells in response to stimulation with different odorant agonists (SPE, SEP-363856, T1AM, DMCHA, IAA, IBA, TMA, CHA, 1-MPD, PEA, CAD, and PTC). Values are represented as mean ± SEM of 3 independent experiments (n = 3).

Extended Data Fig. 3 Purification of the mTAAR9–Gs–scFv16 complex and the mTAAR9–Golf–scFv16 complex.

a-d, Dose response curves of SPE (a), DMCHA (b), PEA (c), and CAD (d) induced Gαs-Gβγ dissociation in mTAAR9 or BRIL-mTAAR9 overexpressing cells. Values are represented as mean ± SEM of 3 independent experiments (n = 3). To increase receptor expression, thermostabilized cytochrome b562RIL (BRIL) was incorporated at the N-terminus of full-length mTAAR9, and the chimeric protein was found to exhibit G protein coupling activity similar to that of wild-type mTAAR9. e-h, Dose response curves of SPE (e), DMCHA (f), PEA (g), and CAD (h) induced Gαs-Gβγ or modified Gαs (modGαs)-Gβγ dissociation in mTAAR9 overexpressing cells. Values are represented as mean ± SEM of 3 independent experiments (n = 3). A modified Gαs chimera (modGαs) was generated on the basis of the mini-Gs scaffold with replacement of the N terminus of Gs (residue 1 to residue 25) with Gαi1 (residue 1 to residue 18) to facilitate the binding of scFv16 and replacing the GGSGGSGG linker at the position of original Gαs α-helical domain (AHD, V65-L203) with that of human Gi1 (G60-K180). In HEK293 cells, the modGαs chimera has similar pharmacological profile in G protein dissociation assay downstream of mTAAR9 activation in response to stimulations of SPE, DMCHA, PEA, or CAD. i-l, Dose response curves of SPE (i), DMCHA (j), PEA (k), and CAD (l) induced Gαolf-Gβγ or modified Gαolf (modGαolf)-Gβγ dissociation in mTAAR9 overexpressing cells. Values are represented as mean ± SEM of 3 independent experiments (n = 3). A modified Gαolf chimera was generated on the basis of the mini-Golf scaffold with replacement of the N terminus of Golf (residue 1 to residue 27) with Gαi1 (residue 1 to residue 18) to facilitate the binding of scFv16. The modGαolf was expressed and purified in insect cells to stabilize the mTAAR9 complexes for cryo-EM study. In HEK293 cells, the modified Gαolf chimera has similar pharmacological profile in G protein dissociation assay downstream of mTAAR9 activation in response to SPE, DMCHA, PEA, or CAD. m-q, Representative elution profiles of in vitro reconstituted SPE–mTAAR9–Gs–scFv16 complex (m), PEA–mTAAR9–Gs–scFv16 complex (n), PEA–mTAAR9–Golf–scFv16 complex (o), DMCHA–mTAAR9–Gs–scFv16 complex (p), and CAD–mTAAR9–Gs–scFv16 complex (q). on Superose 6 Increase 10/300 column and representative SDS-PAGE results of the size-exclusion chromatography peaks.

Extended Data Fig. 4 Cryo-EM images and single particle reconstruction of the EM map of the SPE-mTAAR9-Gs and PEA-mTAAR9-Gs complexes.

a, Representative Cryo-EM micrograph and two-dimensional (2D) class averages of SPE-mTAAR9-Gs particles (Scale bar: 100 nm). Representative Cryo-EM micrograph from 7132 movies and representative two-dimensional class averages determined using approximately 193217 particles after 3D classification were shown. b, Flow chart for three-dimensional (3D) classification of SPE-mTAAR9-Gs particles. Representative Cryo-EM micrographs from 7,132 movies and representative 2D class averages determined using approximately 193,217 particles after 3D classification are shown. We have performed mask for receptor-alone for SPE-mTAAR9-Gs complex structures. The result indicated that mask of receptor region in SPE-mTAAR9-Gs complexes significantly improved the map quality of specific region of mTAAR9. c, Fourier shell correlation curves for the final 3D density map of SPE-mTAAR9-Gs particles. At the FSC 0.143 cut-off, the overall resolution for the map is 3.5 Å. d, 3D density map colored according to local resolution (Å) of the SPE-mTAAR9-Gs trimer complex. e, Representative Cryo-EM micrograph and 2D class averages of PEA-mTAAR9-Gs particles (Scale bar: 100 nm). Representative Cryo-EM micrograph from 8025 movies and representative two-dimensional class averages determined using approximately 463012 particles after 3D classification were shown. f, Flow chart for 3D classification of PEA-mTAAR9-Gs particles. Representative Cryo-EM micrographs from 8,025 movies and representative 2D class averages determined using approximately 463,012 particles after 3D classification are shown. We have performed mask for receptor-alone for PEA-mTAAR9-Gs complex structures. The result indicated that mask of receptor region in PEA-mTAAR9-Gs complexes significantly improved the map quality of specific region of mTAAR9. g, Fourier shell correlation curves for the final 3D density map of PEA-mTAAR9-Gs particles. At the FSC 0.143 cut-off, the overall resolution for the map is 3.0 Å. h, 3D density map colored according to local resolution (Å) of PEA-mTAAR9-Gs trimer complex. i, Three-dimensional (3D) representation of N-terminus, ECL1 and ECL2 of mTAAR9 in CAD-mTAAR9-Gs complex, Olfr78 (predicted by AlphaFold 2), β2AR (PDB: 3SN6) and D1R (PDB: 7X2F). The electron density or EM density of N-terminus are not observed in the crystal structure of β2AR-Gs complex and in the EM structure of D1R complex. The N-terminal of β2AR and D1R are supposed disordered. In the Alphafold2 predicted Olfr78 structure, the N-terminus is inserted between the ECL1 and ECL2. In contrast, the N-terminus of mTAAR9 traverses across the ECL1 and reaches the tip of ECL2.

Extended Data Fig. 5 Cryo-EM density maps of the SPE-mTAAR9-Gs, PEA-mTAAR9-Gs and PEA-mTAAR9-Golf complexes.

a-c, Cryo-EM density maps and models are shown for TM1, TM2, TM3, TM4, TM5, TM6, TM7 of mTAAR9 and α5 helix of Gαs/Gαolf in the SPE-mTAAR9-Gs complex using the global map of SPE-mTAAR9-Gs complex with map threshold at 0.0255 (a), in the PEA-mTAAR9-Gs complex using the global map of PEA-mTAAR9-Gs complex with map threshold at 0.0160 (b), in the PEA-mTAAR9-Golf complex using the global map of PEA-mTAAR9-Golf complex with map threshold at 0.0600 (c). When using “Color Zone” tool in Chimera, we set the color radius at 3 Å.

Extended Data Fig. 6 The function of disulfide bonds in TAAR members.

a, Summary of analyses of the C22Nter:C186ECL2 disulfide bond pair that was observed or potentially observed in the Cryo-EM density and identified by Mass Spectrometry (MS). This conserved disulfide bond was also predicted by Alphafold2 for most of TAAR members except for TAAR1. Despite the EM density for extracellular regions of TAARs were not good enough to identify the potential disulfide bonds in PEA-mTAAR9-Gs and PEA-mTAAR9-Golf structures, our mass spectrometry data suggested that the apo-mTAAR9, PEA-mTAAR9-Golf complex, PEA-, SPE-, DMCHA-mTAAR9-Gs and SPE-mTAAR5 complexes all have this conserved disulfide bonds. The potential Cryo-EM densities for the disulfide bond of C22Nter:C186ECL2 pair in SPE-mTAAR9-Gs and DMCHA-mTAAR9-Gs structures are shown in Supplementary Fig. 9. b-g, Representative higher-energy collisional dissociation (HCD) spectra showing the identification of the disulfide bond C22Nter:C186ECL2 pair in PEA-mTAAR9-Gs complex (b) SPE-mTAAR9-Gs complex (c), DMCHA-mTAAR9-Gs complex (d), PEA-mTAAR9-Golf complex(e), apo-mTAAR9 (g) and the disulfide bond C24Nter:C188ECL2 pair in TMA-mTAAR5 (f) by MS. NEM, nethylmaleimide. h, Schematic overview of 3 disulfide patterns (including the C22Nter:C186ECL2 pair) of apo-mTAAR9 predicted by AlphaFold2. i, Effects of key residue mutations of the C14Nter:C97ECL1 disulfide pair and C22Nter:C186ECL2 disulfide pair of mTAAR9, mTAAR4, mTAAR5, and mTAAR8c on receptor activities induced by their respective agonists (SPE, PEA, TMA, and 1-MPD). The heatmap is colored according to the value of ΔpEC50 (ΔpEC50 = pEC50 of mutant-pEC50 of wild type). ND, signal not detectable. Values were averaged from 3 independent experiments (n = 3). j-m, Dose response curves of key residue mutations of the disulfide pairs, C14Nter:C97ECL1 pair and C22Nter:C186ECL2 pair of mTAAR9 on their activities induced by SPE, CAD, PEA and DMCHA, respectively. Values are represented as mean ± SEM of 3 independent experiments (n = 3). n-p, Dose response curves of key residue mutations of the disulfide pairs, C14Nter:C97ECL1 pair and C22Nter:C186ECL2 pair of mTAAR8c (n), mTAAR5 (o), and mTAAR4 (p) on their activities induced by respective agonists (1-MPD, TMA and PEA). Values are represented as mean ± SEM of 3 independent experiments (n = 3).

Extended Data Fig. 7 The ligand binding mode of SPE-mTAAR9-Gs structure.

The EM densities of SPE and DMCHA could be positioned in 3 different modes, and PEA in 2 potential modes in the SPE-, DMCHA- and PEA-mTAAR9-Gs complex structures. After molecular dynamics (MD) simulations, only 1 mode of PEA, SPE, and DMCHA was used for further structural analysis and biochemical characterizations (also see supplementary Figs. 11–12). However, the CAD binding mode within the mTAAR9 ligand pocket could not be precisely defined, in which multiple conformations of CAD could be fit with current EM density. a, EM density corresponding to the three different modes of SPE in SPE-mTAAR9-Gs complex. SPE-mode 1: green, SPE-mode 2: orange, SPE-mode 3: blue. b, 3D representation of the detailed interactions between SPE and mTAAR9 in mode 1, mode 2 and mode 3. Hydrogen bonds are shown as red dashed lines. c, Barcode representation of interaction patterns in three binding modes of mTAAR9 bound by SPE. Residues that interact with SPE are indicated in green circles, those that interact only in mode 1 or mode 2 are shown in light blue circles, and those that interact only in mode 3 are shown in orange circles. d, The binding energy contribution comparison of binding site residues of mTAAR9 in the two modes of SPE-mTAAR9-Gs complex as calculated by the molecular mechanics MM-PBSA method57. SPE-mode 1: green, SPE-mode 2: orange. e, Effects of mutations of interacting residues in three SPE-binding modes of mTAAR9 in response to the stimulation with SPE. The heatmap is colored according to the value of ΔpEC50. Values were from 3 independent experiments (n = 3). Mutations of residues that interacted in mode 1 but not in mode 3 are shown in light blue background, and those that interact only in mode 3 are shown in orange background. f, The average RMSD value of SPE and binding site residues in the three modes of SPE-mTAAR9-Gs complex during triplicate 200 ns MD simulations. Notably, the binding mode 1 and mode 2 of SPE were very similar, except for the N5 of SPE in SPE-mTAAR9-Gs could be positioned differently that all fit well with EM density, potentially due to the linear configuration of SPE. Compared to the other mode (mode 2), the binding mode with SPE (mode 1) exhibited lower energy when the ligand was in proximity to F1173.37 and the middle N5 was closer to Y2746.51. Moreover, mutating F1173.37A significantly decreased mTAAR9 activation in response to SPE stimulation. Further analysis of the binding energy contributions of each residue located in the binding pocket by MD analysis confirmed that binding mode 1 exhibited a greater stability than that of mode 2. In mode 3, the SPE lost specific interactions with the T1093.29, F1684.61, W2716.48 and V3007.42, and formed new contact with Y2937.35. Notably, despite Y293A mutation showed no significant effects on SPE induced mTAAR9 activation, mutations of T1093.29A, F1684.61A, W2716.48A or V3007.42A either significantly reduced or totally abolished SPE induced mTAAR9 activation. Moreover, the MD simulation suggested that mode 3 is less stable than both mode 1 and mode 2. We therefore mainly used mode 1 of SPE for further biochemical characterizations.

Extended Data Fig. 8 Ligand pocket prediction of two classic OR receptors.

We have used AlphaFold2 to predict the structure of mouse Olfr2 and Olfr78 and used the SiteMap algorithm to predict their corresponding ligand pockets. We then performed molecular docking and MD simulation to further probe the interaction between the octanal and propionate within the ligand pockets of their reported odorant receptors, the Olfr2 and Olfr78, respectively. a, Cut-away view of the ligand-binding pocket in the dopamine-D1R-Gs complex, (PDB:7CKZ, left), Octanal-Olfr2 complex generated by AlphaFold2 and MD simulation (middle) and MS47134-MRGPRX4-Gq (PDB:7S8P, right). b, Illustration of the Olfr2 and Olfr78 structures predicted by AlphaFold2. c, Predicted ligand-binding pockets in Olfr2 (left panel) and Olfr78 (right panel) determined using the SiteMap algorithm. The Olfr2 predicted binding pockets are filled with green, yellow, orange, cyan or blue balls. The Olfr78 predicted binding pockets are filled with red, green, pink, blue or cyan balls. d, The average RMSD value of octanal (upper panel) and key residues in Olfr2 that directly interact with octanal (lower panel) during triplicate 200 ns of MD simulation. e, The average RMSD value of propionate (upper panel) and key residues in Olfr78 that directly interact with propionate (lower panel) during triplicate 200 ns of MD simulation. f, Structural comparison of binding pocket of mTAAR9 with simulated structure of mouse octanal-Olfr2 and propionate Olfr78 which were generated by molecular docking based on the AlphaFold2 predicted structures of Olfr2 and Olfr78. The ligand pocket of both odorant receptors were defined by 4 helices, TM3-TM6. In contrast, the ligand pocket of mTAAR9 are defined by additional TM7, which potentially provide more interactions. Another notable difference is that the ligands of mTAAR9 binds deeper than those of predicted ligands within Olfr2 or Olfr78, forming direct contact with large hydrophobic residue W6.48. In Olfr2 or Olfr78, the large hydrophobic residue W6.48 was replaced by smaller residue Y6.48, and don’t form direct contact with corresponding ligands.

Extended Data Fig. 9 The function of D/E3.32-W6.48-Y7.43 motif in TAAR members.

a, Sequence alignment of the D/E3.32-W6.48-Y7.43 motif in different human and mouse TAAR members. These 3 conserved residues are highlighted in light blue background. b, Effects of key residue mutations for the D/E3.32-W6.48-Y7.43 motif and C/S3.36 of mTAAR9, mTAAR4, 5, and 8c in response to stimulation with respective agonist (SPE, PEA, TMA, and 1-MPD). The heatmap is colored according to the value of ΔpEC50 (ΔpEC50 = pEC50 of mutant - pEC50 of wild type). ND, signal not detectable. Values were averaged from 3 independent experiments (n = 3). c, Dose response curves of key residue mutations for the D3.32-W6.48-Y7.43 motif and C/S3.36 of mTAAR9 (d), mTAAR8c (e), mTAAR5 (f), and mTAAR4 (g) in response to stimulation with respective agonists (SPE, 1-MPD, TMA, and PEA). Values are represented as mean ± SEM of 3 independent experiments (n = 3). Although the conserved C/S3.36 could form H-bond interactions with the main chain of D/E3.32, mutating C3.36 to A in mTAAR9, mTAAR4, mTAAR5, and mTAAR8c caused no significant effects on the odor-induced activation of these receptors, suggesting that the role of the C/S3.36-mediated H-bond with the main chain of D/E3.32 is dispensable in ordinary TAAR receptor activation.

Extended Data Fig. 10 Potential structural mechanism of SPE recognition among TAAR members.

a, Dose response curves of Gαs-Gβγ dissociation in receptor (mTAAR1-6, 7d, 8c, and 9)-overexpressing HEK293 cells in response to stimulation with SPE. Values are represented as mean ± SEM of 3 independent experiments (n = 3). b, The average RMSD value of SPE (upper panel) and binding site residues in SPE-mTAAR5 complex during triplicate 200 ns MD simulations. The overall MD simulated structure was shown in Supplementary Fig. 14b. We built the coordinates of mTAAR5 using our solved mTAAR9 structure as a template via SWISS-MODEL. c, 3D representation of the detailed interactions between SPE and D1143.32, Y2957.34 residues of mTAAR5. d-e, Dose response curves of key residue mutations in the polyamine recognition pocket of mTAAR9 (d) and mTAAR5 (e) in response to stimulation with SPE. Values are represented as mean ± SEM of 3 independent experiments (n = 3). f, Structural representation of the potential effects of structurally equivalent mutations of mTAAR6, mTAAR7d, and mTAAR8c. Conserved interactions of in the polyamine recognition pocket (T3.29 and T1133.33) were mimicked by generating mutations of V1133.33 to T in mTAAR6, S1243.39 and G1283.33 to T in mTAAR7d, and S1083.29 and V1123.33 to T in mTAAR8c. g, Dose response curves of HEK293 cells overexpressing mTAAR6, mTAAR7d, mTAAR8c, and the indicated mutants in the polyamine recognition pocket according to Fig. 4f in response to SPE stimulation. Values are represented as mean ± SEM of 3 independent experiments (n = 3).

Extended Data Fig. 11 Potential structural mechanism of SPE and PEA recognition among TAAR members.

a, The potency and efficacy of Gαs-Gβγ dissociation in receptor (mTAAR2-6, 7d, 8c, and 9)-overexpressing HEK293 cells in response to stimulation with PEA. The heatmap is colored according to the value of pEC50 and Emax from 3 independent experiments (n = 3). ND, signal not detectable. b, Dose response curves of Gαs-Gβγ dissociation in receptor (mTAAR2-6, 7d, 8c, and 9)-overexpressing HEK293 cells in response to stimulation with PEA. Values are represented as mean ± SEM of 3 independent experiments (n = 3). c, Effects of mutations of key residues in SPE-binding hydrophobic pocket of mTAAR9 in response to the stimulation with SPE. The heatmap is colored according to the value of ΔpEC50 (ΔpEC50 = pEC50 of mutant - pEC50 of wild type). Values were from 3 independent experiments (n = 3). d, Dose response curves of mutations of key residues in SPE-binding hydrophobic pocket of mTAAR9 in response to stimulation with SPE. Values are represented as mean ± SEM of 3 independent experiments (n = 3). Consistently, structurally equivalent mutations of these 4 hydrophobic residues of F1173.37T, F1684.61V, Y2746.51C, V2977.39T/C, and V3007.42A/G decreased SPE-induced mTAAR9 activation. e-f, Dose response curves of HEK293 cells overexpressing wild type and mutants (F1684.61L and V3007.42A) of mTAAR9 (c) or wild type and mutants (L170F4.61 and A2947.42V) of mTAAR5 (d) in response to SPE stimulation. Values are represented as mean ± SEM of 3 independent experiments (n = 3). Mutation of F1684.61L and V3007.42A in mTAAR9 indeed decreased SPE-induced receptor activation by 5.13 ± 0.66- and 27.2 ± 5.04-fold, respectively (e), whereas mutation of L1704.61F and A2947.42V in mTAAR5 increased SPE-stimulated receptor activation by 5.43 ± 0.55- and 9.26 ± 0.68-fold, respectively(f). These results suggest that the binding of these 2 residues contributes to the potency difference of SPE that acts between the 2 mTAAR members. g, Effects of mutations of key residues in PEA-binding pocket of mTAAR9 in response to the stimulation with PEA. The heatmap is colored according to the value of ΔpEC50 (ΔpEC50 = pEC50 of mutant - pEC50 of wild type). Values were from 3 independent experiments (n = 3). h, Dose response curves of mutations of key residues in PEA-binding hydrophobic pocket of mTAAR9 in response to stimulation with PEA. Values are represented as mean ± SEM of 3 independent experiments (n = 3). Notably, mutating the four residues in mTAAR4/6/7d/9 to structurally equivalent residues present in other mTAARs, including F/Y3.37L/T, Y6.51C, Y7.35L, or V7.39T/C, significantly decreased the PEA-induced mTAAR9 activities. i, Effects of mutations of key residues in PEA-binding pocket of mTAAR7d in response to the stimulation with PEA. The heatmap is colored according to the value of ΔpEC50 (ΔpEC50 = pEC50 of mutant - pEC50 of wild type). Values were from 3 independent experiments (n = 3). j, Dose response curves of mutations of key residues of mTAAR7d in response to stimulation with PEA by Gαs-Gβγ dissociation assay. Values are represented as mean ± SEM of 3 independent experiments (n = 3). Notably, mutating the four residues in mTAAR4/6/7d/9 to structurally equivalent residues present in other mTAARs, including F/Y3.37L/T, Y6.51C, Y7.35L, or V7.39T/C, significantly decreased the PEA-induced mTAAR7d activities.

Extended Data Fig. 12 The activation mechanism of mTAAR9.

a, Comparison of the simulated structure of apo-mTAAR9 (cyan) with the model of apo-mTAAR9 generated by AlphaFold2 (grey). b, The average RMSD value of apo-mTAAR9 during triplicate 200 ns of MD simulation. c, Comparison of the cryo-EM structure of SPE-mTAAR9 (blue) with simulated model of apo-mTAAR9 (grey). Whereas the cytoplasmic end of TM6 in agonist-bound structures of mTAAR9 shows a significant outward shift, the extracellular end of TM1 shows an inward movement. d, Structural comparison of TM3 and TM6 in inactive β2AR (cyan), β2AR-Gs complex (light green, PDB: 3SN6), inactive mTAAR9 (grey), mTAAR9-Gs complex (light blue). Comparison of the corresponding structures in the mTAAR9 and β2AR shows that the direct interaction of these agonists with toggle switch W2716.48 pushed its downward movement, accompanied with downward shift of the F2676.44 and A2636.40 located at two adjacent helices using TM3 as a reference. And the separation angle between TM3 and TM6 in mTAAR9 was larger than that in active β2AR due to the substitution of I2786.40 with β2AR by A2636.40 in mTAAR9 as this substitution disrupts its interaction with conserved R1303.50. e, Sequence alignment of TAAR member and selected class A GPCR family members on key residues of TM6 important for receptor activation. The conserved residues are highlighted with red fonts. Importantly, W2716.48 and F2676.44 are conserved across the TAAR family, except that TAAR5 members have Y6.44, suggesting that the conformational changes in the conserved toggle switches W6.48 and F/Y6.44, as well as the packing changes between TM3 and TM6, are potential common mechanisms that mediate Gs/Golf-coupled TAAR activation. f, Mutational effects of the A6.40, including A2636.40I and A2636.40L on PEA-induced mTAAR9-Golf or mTAAR9-Gs activity, and A2576.40I and A2576.40L on TMA-induced mTAAR5-Golf or mTAAR5-Gs activity were assessed via Gαs-Gβγ dissociation assay. The heatmap is colored according to the value of ΔpEC50 (ΔpEC50 = pEC50 of mutant - pEC50 of wild type). Values were from 3 independent experiments (n = 3). g-j, Dose response curves of residue mutations for A6.40, including A6.40I and A6.40L in mTAAR9 (g-h) or mTAAR5 (i-j) in response to stimulation with PEA or TMA by Gαs/olf-Gβγ dissociation assay. Values are represented as mean ± SEM of 3 independent experiments (n = 3).

Extended Data Fig. 13 The mechanism of Gs/Golf coupling with mTAAR9.

a, Detailed representation of the mTAAR9-binding interface with Gs. The interface is composed by ICL2, cytoplasmic ends of TM3, TM5, TM6, helix 8 of mTAAR9 (medium aquamarine) and α4-β6 loop, α5 helix, β2-β3 loop of Gs (pink). b, 3D representation of the detailed interactions between the C-terminal end of the α5 helix in the Gαs and mTAAR9.The Y391G.H5.23, and L388G.H5.20 form extensive contacts with the hydrophobic surface created by V1343.54, I2205.61, A2245.65 and Q2275.68 of mTAAR9. c, 3D representation of the detailed interactions between Y391G.H5.23, E392G.H5.24, C-terminal carboxyoxylate ends of the α5 helix of Gαs and R1303.50, K2556.32 of mTAAR9. Hydrogen bond distances are shown as red dashed lines. Y391G.H5.23 engages in π: cation interaction with R1303.50. The E392G.H5.24 and C-terminal carboxylate ends of the α5 helix of Gαs formed H-bond or charge-charge interactions with K2556.32 of mTAAR9. d, Overall structure of binding interface of the ICL2 of mTAAR9 with αN, β2-β3 loop, and α5 helix of Gs. e, 3D representation of the detailed interactions between Y140ICL2, P137ICL2, L138ICL2 of mTAAR9 and H41G.S1.02, V217G.S3.01, F376G.H5.08, C379G.H5.11, R380G.H5.11, I383G.H5.15, Q384G.H5.16, H387G.H5.19, Y391G.H5.23 of Gαs. The P137ICL2 and L138ICL2 are located in a hydrophobic crater created by H41G.S1.02, V217G.S3.01, C379G.H5.11, F376G.H5.08, R380G.H5.12, I383G.H5.15 and Q384G.H5.16 of Gαs. Y140ICL2 is involved in π:π interaction with H387G.H5.19 and Y391G.H5.23. The P137ICL2 and L138ICL2 are located in a hydrophobic crater created by β1-β3 strands and α5 helix of Gαs. f, 3D representation of the detailed interactions between the C-terminal α5 helix of Gαs and the initial segment in the H8 of mTAAR9. Hydrogen bond distances are shown as red dashed lines. g, Compared to V287G.S5.02 in Gαs, I274G.S5.02 in Gαolf has closer association with I263G.H3.12, W264G.H3.13, and F260G.H3.0. At the initial segment in the H8 of mTAAR9, Y3158.47 and W3178.49 constitute one sidewall that accommodates the hook end of the C-terminal α5 helix of Gαs and forms a hydrogen bond with R1303.50 to stabilize the active structure of mTAAR9. h, 3D structural representation of differences between mTAAR9-Golf interface (Golf in medium purple) and mTAAR9-Gs interface (Gs in pink). The comparison of K343G.h4s6.12/D341G.h4s6.10 in Gαolf and R356G.h4s6.12/D354G.h4s6.10 in Gαs. Hydrogen bond distances are shown as red dashed lines. The K343G.h4s6.12 and D341G.h4s6.10 in Gαolf formed weaker charge-charge interactions and are more separated compared with the structural equivalent R356G.h4s6.12 and D354G.h4s6.10 pair in Gαs. i, 3D representation of the detailed interactions between V134ICL2 and V2235.64 of mTAAR9 and L375G.H5.20 and Q371G.H5.16 of Gαolf and superimposition of these interactions with those between V134ICL2 and V2235.64 of mTAAR9 and L388G.H5.20 and Q371G.H5.16 of Gαs. The conformational changes in cytoplasmic ends of TMD enabled tighter interaction of V2235.64 and V1343.54 in mTAAR9 with Q371G.H5.16 and L375G.H5.20 in Gαolf. mTAAR9 bound to Gs is shown in medium aquamarine, mTAAR9 bound to Golf is shown in hot pink, Gs is shown in pink, Golf is shown in medium purple. j, 3D representation of the detailed interactions between R3198.51 of mTAAR9 and E379G.H5.24of Gαolf or E392G.H5.24 of Gαs. At the C-terminus of the α5 helix of Gαolf, E379G.H5.24 forms stronger charge-charge interactions with R3198.51 of mTAAR9 compared with those of Gαs. k, 3D representation of the detailed interactions between P141ICL2, F144ICL2 of mTAAR9 and K31G.hns1.02 of Gαolf compared to those between P141ICL2, F144ICL2 of mTAAR9 and R38G.hns1.02 of Gαs (f). Hydrogen bond distances are shown as red dashed lines. Although ICL2 of mTAAR9 forms similar interaction patterns with both Gαs and Gαolf, substituting R38G.hns1.02 in Gαs by K31G.hns1.02 in Gαolf abolished the interaction with P141ICL2 and main chain F144ICL2 of mTAAR9.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics
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Guo, L., Cheng, J., Lian, S. et al. Structural basis of amine odorant perception by a mammal olfactory receptor. Nature 618, 193–200 (2023). https://doi.org/10.1038/s41586-023-06106-4

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