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Recognition of methamphetamine and other amines by trace amine receptor TAAR1

Nature volume 624, pages 663–671 (2023)Cite this article

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Abstract

Trace amine-associated receptor 1 (TAAR1), the founding member of a nine-member family of trace amine receptors, is responsible for recognizing a range of biogenic amines in the brain, including the endogenous β-phenylethylamine (β-PEA)1 as well as methamphetamine2, an abused substance that has posed a severe threat to human health and society3. Given its unique physiological role in the brain, TAAR1 is also an emerging target for a range of neurological disorders including schizophrenia, depression and drug addiction2,4,5. Here we report structures of human TAAR1–G-protein complexes bound to methamphetamine and β-PEA as well as complexes bound to RO5256390, a TAAR1-selective agonist, and SEP-363856, a clinical-stage dual agonist for TAAR1 and serotonin receptor 5-HT1AR (refs. 6,7). Together with systematic mutagenesis and functional studies, the structures reveal the molecular basis of methamphetamine recognition and underlying mechanisms of ligand selectivity and polypharmacology between TAAR1 and other monoamine receptors. We identify a lid-like extracellular loop 2 helix/loop structure and a hydrogen-bonding network in the ligand-binding pockets, which may contribute to the ligand recognition in TAAR1. These findings shed light on the ligand recognition mode and activation mechanism for TAAR1 and should guide the development of next-generation therapeutics for drug addiction and various neurological disorders.

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Fig. 1: Functional assessment of four TAAR1 agonists in activating TAAR1 and overall structure of METH–TAAR1–Gs complex.
Fig. 2: METH-binding pocket of TAAR1.
Fig. 3: The recognition of β-PEA by TAAR1.
Fig. 4: Structural basis of SEP-363856 polypharmacology and selectivity.
Fig. 5: Conserved and divergent features of agonist recognition at TAAR1.

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Data availability

Density maps and structure coordinates have been deposited in the Electron Microscopy Data Bank (EMDB) and the Protein Data Bank (PDB) with accession codes EMD-37347 and 8W87 for METH–TAAR1–Gs complex; EMD-37348 and 8W88 for SEP-363856-bound TAAR1–Gs complex; and EMD-37349 and 8W89 for β-PEA-bound TAAR1–Gs complex; EMD-37350 and 8W8A for RO5256390–TAAR1–Gs complex; and EMD-37351 and 8W8B for SEP-363856 bounded 5-HT1AR–Gi protein complex. Source data are provided with this paper.

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Acknowledgements

This work was partially supported by the Ministry of Science and Technology of China (2018YFA0507002 to H.E.X., 2020YFA0509102 to S.W.); the National Natural Science Foundation of China (32071194 to F.X., 32130022 and 82121005 to H.E.X., 52272087 to W.L., 82225025 and 32071197 to S.W.); Shanghai Municipal Science and Technology Major Project (2019SHZDZX02 to H.E.X.); CAS Strategic Priority Research Programme (XDB37030103 to H.E.X.); the Lingang Laboratory, grant no. LG-GG-202204-01 (H.E.X.); and the National Key R&D Program of China (2022YFC2703105 to H.E.X.). The cryo-EM data were collected at the Shanghai Advanced Electron Microscope Center, Shanghai Institute of Material Medica, as well as at the Bio-EM platform of ShanghaiTech University. We thank Q. Yuan, K. Wu, W. Hu and S. Zhang for providing technical support and assistance during data collection at the Shanghai Advanced Electron Microscope Center, Shanghai Institute of Material Medica; we thank Q. Sun, Z. Zhang, L. Wang, D. Liu and Y. Liu at the Bio-EM facility at ShanghaiTech University; and Q. Tan, Q. Shi, J. Liu, N. Chen, S. Hu and W. Xiao from ShanghaiTech University for protein cloning, expression, assay and data collection support. We thank S. Zhao from ShanghaiTech University, J. Wang from Institute of Biophysics, CAS, and Q. Li from Shanghai Jiaotong University to provide initial sets of TAAR1 tool compounds and suggestions for this work. We also thank the support from Shanghai Frontiers Science Center for Biomacromolecules and Precision Medicine at ShanghaiTech University.

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Author notes

  1. These authors contributed equally: Heng Liu, You Zheng, Yue Wang, Yumeng Wang

Authors and Affiliations

  1. The State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

    Heng Liu, Yue Wang, Xinheng He, Peiyu Xu, Sijie Huang, Qingning Yuan, Xinyue Zhang, Zhen Li & H. Eric Xu

  2. iHuman Institute, School of Life Science and Technology, ShanghaiTech University, Shanghai, China

    You Zheng, Ling Wang, Kexin Jiang & Fei Xu

  3. University of Chinese Academy of Sciences, Beijing, China

    Yue Wang, Yumeng Wang, Xinheng He, Peiyu Xu, Sijie Huang & H. Eric Xu

  4. State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecule Cell Science, Chinese Academy of Sciences, Shanghai, China

    Yumeng Wang & Sheng Wang

  5. The Shanghai Advanced Electron Microscope Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

    Qingning Yuan

  6. School of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing, China

    Xinyue Zhang & Zhen Li

  7. Shanghai Key Laboratory of Crime Scene Evidence, Shanghai Research Institute of Criminal Science and Technology, Shanghai, China

    Hong Chen & Wenbin Liu

  8. Shanghai Yuansi Standard Science and Technology Co., Ltd, Shanghai, China

    Hong Chen & Wenbin Liu

  9. Key Laboratory of Systems Health Science of Zhejiang Province, School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, China

    Sheng Wang

  10. School of Life Science and Technology, ShanghaiTech University, Shanghai, China

    H. Eric Xu

  11. Shanghai Clinical Research and Trial Center, Shanghai, China

    Fei Xu

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  1. Heng Liu
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  2. You Zheng
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Contributions

F.X. and H.E.X. initiated the project. H.L. designed and screened the expression constructs of TAAR1 and prepared protein samples of TAAR1–METH–Gs, TAAR1–SEP-363856–Gs, 5-HT1AR–SEP-363856–Gi complexes for cryo-EM data collection, performed cryo-EM grids preparation, data acquisition, and structure determination, participated in GloSensor cAMP assay, and prepared the draft of the manuscript and figures. Y.Z. designed and screened the expression constructs of TAAR1 and prepared protein samples of TAAR1–β-PEA-Gs, TAAR1–RO5256390-Gs, complexes for cryo-EM data collection, performed cryo-EM grids preparation, data acquisition and structure determination, and participated in manuscript editing and figure preparation. Y.W. performed GloSensor cAMP assay, G-protein recruitment assay and participated in figure preparation. Y.W. performed the radioligand-binding assay and Gαiβγ dissociation assay. X.H. performed MD simulation analysis. P.X. and S.H. participated in protein sample preparation and structure determination. Q.Y. participated in data acquisition. X.Z. participated in G-protein recruitment assay. L.W. participated in GloSensor cAMP assay. K.J. assisted in some protein preparation work. H.C. assisted in compound preparation and synthesis. Z.L. helped construct the mutations for function assays. W.L. supervised compound preparation and synthesis. S.W. supervised ligand binding and functional assay. F.X. and H.E.X. conceived and supervised the project and participated in manuscript editing. F.X. and H.E.X. wrote the manuscript with input from H.L., Y.Z. and Y.W.

Corresponding authors

Correspondence to Wenbin Liu, Sheng Wang, H. Eric Xu or Fei Xu.

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Nature thanks Thomas Keck, Tao Che and Harald Sitte for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

Extended Data Fig. 1 Functional assessment of TAAR1 by four agonists and structural feature of aminergic receptors.

(a-b) Concentration–response curves of the four agonists activating TAAR1 evaluated by miniGs recruitment assay (a) and radio-ligand-binding assay (b). Data are mean ± S.E.M. from 3 independent experiments (n = 3). (c) Structural comparison of ECL2s from TAAR1 and other aminergic receptors, ECL2 that forms a short helix in the structures is highlighted with red dotted circle.

Extended Data Fig. 2 Purification and structure determination of METH and β-PEA-bound TAAR1-Gs complexes.

(a) Cartoon models of the two TAAR1 constructs used in this study. (b) Representative size exclusion chromatography (SEC) profiles and SDS-PAGE analysis of TAAR1-Gs complex activated by METH. Experiment was repeated at least three times with similar results. (c-d) Representative cryo-EM image from 6,686 movies (c) and 2D classification averages (d) of METH–TAAR1–Gs. (e) Cryo-EM data processing flowcharts of METH-TAAR1-Gs by cyroSPARC 3.2. (f) The Fourier shell correlation (FSC) curves of METH-TAAR1-Gs. The global resolution of the final processed density map estimated at the FSC = 0.143 is 2.8 Å. (g) The global density map of METH-TAAR1-Gs colored by local resolutions. (h) The density maps of helices TM1–TM7 of transmembrane domain, extracellular loop ECL2 of TAAR1, α5 helix of Gαs, and METH in METH-TAAR1-Gs complex. (i) Representative size exclusion chromatography (SEC) profiles and SDS-PAGE analysis of TAAR1–Gs complex activated by β-PEA. Experiment was repeated at least three times with similar results. (j-k) Representative cryo-EM image from 4,371 movies (j) and 2D classification averages (k) of β-PEA-TAAR1-Gs. (l) Cryo-EM data processing flowcharts of β-PEA-TAAR1-Gs by cyroSPARC 3.2. (m) The Fourier shell correlation (FSC) curves of β-PEA-TAAR1-Gs. The global resolution of the final processed density map estimated at the FSC = 0.143 is 3.0 Å. (n) The global density map of β-PEA-TAAR1-Gs colored by local resolutions. (o) The density maps of helices TM1–TM7 of transmembrane domain, extracellular loop ECL2 of TAAR1, α5 helix of Gαs, and β-PEA in β-PEA-TAAR1-Gs complex.

Extended Data Fig. 3 Mutational effects of agonists and MD simulation analysis of TAAR1.

(a) Concentration–response curves of wild-type/mutants of TAAR1 induced by the four agonists. Every response of mutant was normalized to corresponding wild-type as 100%. Data are mean ± S.E.M. from 3 independent experiments (n = 3). (b-c) RMSD plot of METH (b) or β-PEA (c) in TAAR1 pocket during simulations. (d) The free energy contribution of key residues in TAAR1 pocket for METH or β-PEA binding. Data are mean ± S.E.M. from 6 independent experiments (n = 6).

Source Data

Extended Data Fig. 4 Purification and structure determination of SEP-363856 bound TAAR1-Gs and 5HT1AR-Gi complexes.

(a) Representative size exclusion chromatography (SEC) profiles and SDS-PAGE analysis of TAAR1-Gs complex activated by SEP-363856. Experiment was repeated at least three times with similar results. (b-c) Representative cryo-EM image from 7,262 movies (b) and 2D classification averages (c) of SEP-363856-TAAR1-Gs. (d) Cryo-EM data processing flowcharts of SEP-363856-TAAR1-Gs by cyroSPARC 3.2. (e) The Fourier shell correlation (FSC) curves of SEP-363856-TAAR1-Gs. The global resolution of the final processed density map estimated at the FSC = 0.143 is 2.6 Å. (f) The global density map of SEP-363856-TAAR1-Gs colored by local resolutions. (g) The density maps of helices TM1–TM7 of transmembrane domain, extracellular loop ECL2 of TAAR1, α5 helix of Gαs, and SEP-363856 in SEP-363856-TAAR1-Gs complex. (h) Representative size exclusion chromatography (SEC) profiles and SDS-PAGE analysis of 5HT1AR -Gi complex activated by SEP-363856. Experiment was repeated at least three times with similar results. (i-j) Representative cryo-EM image from 6,222 movies (i) and 2D classification averages (j) of SEP-363856-5HT1AR -Gi. (k) Cryo-EM data processing flowcharts of SEP-363856-5HT1AR-Gi complex by cyroSPARC 3.2. (l) The Fourier shell correlation (FSC) curves of SEP-363856-5HT1AR -Gi complex. The global resolution of the final processed density map estimated at the FSC = 0.143 is 3.0 Å. (m) The global density map of SEP-363856-5HT1AR-Gi colored by local resolutions. (n) The density maps of helices TM1–TM7 of transmembrane domain, α5 helix of Gαi, and SEP-363856 in 5HT1AR -Gi complex.

Extended Data Fig. 5 Comparison of the SEP-363856 bound pockets of TAAR1 and 5HT1AR, and purification, structure determination of RO5256390 bound TAAR1-Gs complex.

(a) Binding pockets of SEP-363856, aripiprazole (PDB ID: 7e2z) and serotonin (PDB ID: 7e2y) in 5HT1AR. (b) Binding pocket of SEP-363856 in TAAR1. (c) Alignment of key residues constituting the ligand-binding pockets in SEP-383656 bound 5HT1AR or TAAR1 structures, and serotonin and aripiprazole bound 5HT1AR structures. The green background indicated 100% conserved residues and orange background indicated residues that are conserved in 5HT1AR but not in TAAR1. (d) Representative size exclusion chromatography (SEC) profiles and SDS-PAGE analysis of TAAR1-Gs complex activated by RO5256390. Experiment was repeated at least three times with similar results. (e-f) Representative cryo-EM image from 5,135 movies (e) and 2D classification averages (f) of RO5256390-TAAR1-Gs. (g) Cryo-EM data processing flowcharts of RO5256390-TAAR1-Gs complex by cyroSPARC 3.2. (h) The Fourier shell correlation (FSC) curves of RO5256390-TAAR1-Gs. The global resolution of the final processed density map estimated at the FSC = 0.143 is 2.8 Å. (i) The global density map of RO5256390-TAAR1-Gs colored by local resolutions. (j) The density maps of helices TM1–TM7 of transmembrane domain, extracellular loop ECL2 of TAAR1, α5 helix of Gαs, and RO5256390 in RO5256390-TAAR1-Gs complex.

Extended Data Fig. 6 Molecular mechanism of the high potency and selectivity of RO5256390.

(a) Interactions of RO5256390 and key residues in TAAR1 pocket. (b) RO5256390 inserted deep into the cavity formed by D1033x32, S1073x36, W2646x48, Y2947x43. (c-d) Concentration–response curves for mutations of key residues in TAAR1 induced by RO5256390 using Glo-sensor assay. Data are mean ± S.E.M. from 3 independent experiments (n = 3). (e) and (g) Evaluation of RO5256390 and SEP-363856 activating 5HT1AR using Gi protein dissociation assay (e) or Glo-sensor assay (g), 5HT was used as reference. Data are mean ± S.E.M. from 3 independent experiments (n = 3). (f) and (h) Evaluation of RO5256390 activating 5HT1AR with C120 substituted with alanine or serine, using Gi protein dissociation assay (f) or Glo-sensor assay (h), 5HT was used as reference. Data are mean ± S.E.M. from 3 independent experiments (n = 3).

Source Data

Extended Data Fig. 7 Conservation of key residues for TAAR1 activating and role of ECL2 in aminergic receptors.

(a) Sequence alignment of key residues in ECL2 from representative aminergic receptors. (b) Display of residues in ECL2s from TAAR1 and other aminergic receptors that may take part in ligand recognition and binding pocket formation.

Extended Data Fig. 8 Functional assessment of four agonists activating human TAAR family subtypes.

Human TAAR family subtypes (hTAAR1/2/5/6/8/9) were cloned into pcDNA3.1 vector and the activity was measured by Glo-Sensor assay. The negative control was pcDNA3.1 vector and DMSO solvent. The results showed that only TAAR1 can be activated by the four agonist. Data are mean ± S.E.M. from 3 independent experiments (n = 3).

Source Data

Extended Data Fig. 9 Sequence alignment of human TAAR family, TAAR1 activation mechanism, and analysis of METH binding in human and rodent TAAR1.

(a-b) Sequence alignment of residues important for TAAR1-ligand binding (a) or activating (b) from human TAAR family and mTAAR9. (c-d) Superposition of activated TAAR1 (dark cyan) with active mTAAR9 (light green; PDB ID: 8iw7), β2AR (orange; PDB ID: 6kr8) and inactive β2AR (gray; PDB ID: 5jqh). Notable conformational changes occur at extracellular end of TM1 and intracellular ends of TM6, TM7 and H8 upon receptor activation, side view (c) and bottom view (d). (e) The “toggle switch”, W2646.48, of TAAR1 display relative rotameric change when sensing agonist. (f–h) The key P-I-F6.44 (f), D-R3.50-Y (g), and N-P7.50-xx-Y7.53 (h) motifs displayed conformational rearrangement in activated TAAR1 structure. (i–k) RMSD plot of METH binding in rat, mouse, or human TAAR1 pocket during simulations. (l) Bar graph showing RMSD differences for METH binding in rat, mouse, or human TAAR1 pocket during simulations. Data are mean ± S.E.M. of RMSD from 3 independent experiments (n = 3) for rat and mouse TAAR1 and 6 independent experiments (n = 6) for human TAAR1. (m) Sequence alignment for residues that are important for ligand binding to TAAR1 from rat, mouse or human.

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Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics
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Liu, H., Zheng, Y., Wang, Y. et al. Recognition of methamphetamine and other amines by trace amine receptor TAAR1. Nature 624, 663–671 (2023). https://doi.org/10.1038/s41586-023-06775-1

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