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Bitter taste TAS2R14 activation by intracellular tastants and cholesterol

Nature volume 631, pages 459–466 (2024)Cite this article

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Abstract

Bitter taste receptors, particularly TAS2R14, play central roles in discerning a wide array of bitter substances, ranging from dietary components to pharmaceutical agents1,2. TAS2R14 is also widely expressed in extragustatory tissues, suggesting its extra roles in diverse physiological processes and potential therapeutic applications3. Here we present cryogenic electron microscopy structures of TAS2R14 in complex with aristolochic acid, flufenamic acid and compound 28.1, coupling with different G-protein subtypes. Uniquely, a cholesterol molecule is observed occupying what is typically an orthosteric site in class A G-protein-coupled receptors. The three potent agonists bind, individually, to the intracellular pockets, suggesting a distinct activation mechanism for this receptor. Comprehensive structural analysis, combined with mutagenesis and molecular dynamic simulation studies, elucidate the broad-spectrum ligand recognition and activation of the receptor by means of intricate multiple ligand-binding sites. Our study also uncovers the specific coupling modes of TAS2R14 with gustducin and Gi1 proteins. These findings should be instrumental in advancing knowledge of bitter taste perception and its broader implications in sensory biology and drug discovery.

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Fig. 1: Cryo-EM structures of TAS2R14–G protein complexes.
Fig. 2: Recognition mechanism of AA and cholesterol by TAS2R14.
Fig. 3: Recognition mechanism of FFA and compound 28.1 by TAS2R14.
Fig. 4: The activation features of TAS2R14.
Fig. 5: Gustducin and Gi coupling modes in TAS2R14.

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Data availability

The atomic coordinates for AA150µM–TAS2R14–miniGs/gust, AA50µM–TAS2R14–Gi and AA150µM–TAS2R14–Gi have been deposited in the Protein Data Bank with the accession codes 8XQL, 8XQN and 8XQO, respectively. The EM maps for AA150µM–TAS2R14–miniGs/gust, AA50µM–TAS2R14–Gi and AA150µM–TAS2R14–Gi have been deposited in EMDB with the codes EMD-38580, EMD-38582 and EMD-38583, respectively. The atomic coordinates for FFA1mM–TAS2R14–miniGs/gust and FFA150µM–TAS2R14–Gi, have been deposited in the Protein Data Bank with the accession codes 8XQR and 8XQS, respectively. The EM maps for FFA1mM–TAS2R14–miniGs/gust and FFA150µM–TAS2R14–Gi, have been deposited in EMDB with the codes EMD-38586 and EMD-38587, respectively. The atomic coordinate for AA150µM–TAS2R14–Gg, 28.1150µM–TAS2R14–Gg and TAS2R14–Gi have been deposited in the Protein Data Bank with the accession codes 8XQP, 8YKY and 8XQT. The EM maps for AA150µM–TAS2R14–Gg, 28.1150µM–TAS2R14–Gg and TAS2R14–Gi, have been deposited in EMDB with the code EMD-38584, EMD-39376 and EMD-38588, respectively.  Source data are provided with this paper.

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Acknowledgements

This work was supported by the National Key Research and Development Program of China grant nos. 2022YFA1302902 (T.H.) and 2019YFA0904200 (J.S.), the National Natural Science Foundation of China grant nos. 32230026 (Z.-J.L.), 32271262 (T.H.), 81773704 (J.S.) and 22107071 (Y.-R.W.), the Key Research Project of the Beijing Natural Science Foundation Z200019 (J.S.), the CAS Strategic Priority Research Program XDB37030104 (Z.-J.L.), Shanghai Frontiers Science Center for Biomacromolecules and Precision Medicine at ShanghaiTech University. We thank the Shanghai Municipal Government and ShanghaiTech University for financial support. The cryo-EM data were collected at the Bio-Electron Microscopy Facility of ShanghaiTech University, with the assistance of L. Wang. We thank Q.-Y. Shi for assistance with protein purification core and N. Chen and S.-W. Hu for assistance with cell expression core at iHuman Institute.

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Author notes

  1. These authors contributed equally: Xiaolong Hu, Weizhen Ao, Mingxin Gao, Lijie Wu

Authors and Affiliations

  1. iHuman Institute, ShanghaiTech University, Shanghai, China

    Xiaolong Hu, Weizhen Ao, Lijie Wu, Yuan Pei, Shenhui Liu, Yiran Wu, Fei Zhao, Qianqian Sun, Junlin Liu, Longquan Jiang, Xin Wang, Qiwen Tan, Tian Hua & Zhi-Jie Liu

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

    Xiaolong Hu, Weizhen Ao, Shenhui Liu, Longquan Jiang, Xin Wang, Tian Hua & Zhi-Jie Liu

  3. NHC Key Laboratory of Otorhinolaryngology, Qilu hospital and School of Basic Medical Sciences, Shandong University, Jinan, China

    Mingxin Gao, Jie Cheng, Fan Yang & Jinpeng Sun

  4. Department of Oral Surgery, Shanghai Ninth People’s Hospital and College of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Yan Li & Chi Yang

  5. National Center for Stomatology and National Clinical Research Center for Oral Diseases, Shanghai Key Laboratory of Stomatology and Research Unit of Oral and Maxillofacial Regenerative Medicine, Chinese Academy of Medical Sciences, Shanghai, China

    Yan Li & Chi Yang

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  1. Xiaolong Hu
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Contributions

Z.-J.L. and T.H. initiated the project. X.H. designed the expression constructs, purified the receptor complexes, prepared the final samples for cryo-EM sample and cryo-EM data collection and participated in the figure and manuscript preparation. W.A. performed the calcium mobilization functional assays, assisted by Q.T. and Y.L. M.G. performed the G-protein-dissociation assays and cholesterol activation assays, assisted by J.C. and F.Y. L.W. and X.H. conducted EM data processing and structure determination. Y.P. participated in the EM data processing. S.L. performed the MD simulations. Y.W. performed the molecular docking and electrostatic potential analysis. F.Z. synthesized the compound 28.1. X.W. assisted with the figure preparation for G-protein analysis. Q.S. assisted in EM data collection. J.L. and L.J. performed the insect cell expression. C.Y. participated in project discussion and edited the manuscript. J.S. supervised the functional assays, discussed structure–function relationships and participating in the manuscript editing. Z.-J.L. and T.H. conceived and supervised the research, analysed the structures and wrote the manuscript with the input from all authors.

Corresponding authors

Correspondence to Chi Yang, Jinpeng Sun, Tian Hua or Zhi-Jie Liu.

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Extended data figures and tables

Extended Data Fig. 1 AA150µM-TAS2R14-miniGs/gust-Nb35, AA50µM-TAS2R14-Gi1-scFv16 and AA150µM-TAS2R14-Gi1-scFv16 sample preparation, cryo-EM data processing and structure determination.

(a, f, k) Representative size exclusion chromatography (SEC) profiles, SDS-PAGE analysis, representative cryo-EM micrography (scale bar: 50 nm) and selected 2D classification averages of AA150µM-TAS2R14-miniGs/gust-Nb35 (a), AA50µM-TAS2R14-Gi1-scFv16 (f) and AA150µM-TAS2R14-Gi1-scFv16 (k) complexes. (b, g, l) Cryo-EM data processing flowcharts of AA150µM-TAS2R14-miniGs/gust-Nb35 (b), AA50µM-TAS2R14-Gi1-scFv16 (g) and AA150µM-TAS2R14-Gi1-scFv16 (l) complexes by cryoSPARC 4.3. (c, h, m) Cryo-EM maps are colored by local resolution (Å) for AA150µM-TAS2R14-miniGs/gust-Nb35 (c), AA50µM-TAS2R14-Gi1-scFv16 (h) and AA150µM-TAS2R14-Gi1-scFv16 (m) complexes. (d, i, n) Angular distribution of the particles used for final reconstruction (up) and ‘Gold-standard’ FSC curve at the FSC = 0.143 (down) for AA150µM-TAS2R14-miniGs/gust-Nb35 (d), AA50µM-TAS2R14-Gi1-scFv16 (i) and AA150µM-TAS2R14-Gi1-scFv16 (n) complexes. (e, j, o) The density maps for helices TM1-TM7 of transmembrane domain, helix 8 and α5 helices of Gα for AA150µM-TAS2R14-miniGs/gust-Nb35 (e), AA50µM-TAS2R14-Gi1-scFv16 (j) and AA150µM-TAS2R14-Gi1-scFv16 (o) complexes. The SEC and SDS-PAGE experiments were repeated at least three times with similar results.

Extended Data Fig. 2 AA150µM-TAS2R14-Gg-scFv16, FFA1mM-TAS2R14-miniGs/gust-Nb35 and FFA350µM -TAS2R14-Gi1-scFv16 sample preparation, cryo-EM data processing and structure determination.

(a, f, k) Representative size exclusion chromatography (SEC) profiles, SDS-PAGE analysis, representative cryo-EM micrography (scale bar: 50 nm) and selected 2D classification averages of AA150µM-TAS2R14-Gg-scFv16 (a), FFA1mM-TAS2R14-miniGs/gust-Nb35 (f) and FFA350µM -TAS2R14-Gi1-scFv16 (k) complexes. (b, g, l) Cryo-EM data processing flowcharts of AA150µM-TAS2R14-Gg-scFv16 (b), FFA1mM-TAS2R14-miniGs/gust-Nb35 (g) and FFA350µM -TAS2R14-Gi1-scFv16 (l) complexes by cryoSPARC 4.3. (c, h, m) Cryo-EM maps are colored by local resolution (Å) for AA150µM-TAS2R14-Gg-scFv16 (c), FFA1mM-TAS2R14-miniGs/gust-Nb35 (h) and FFA350µM -TAS2R14-Gi1-scFv16 (m) complexes. (d, i, n) Angular distribution of the particles used for final reconstruction (up) and ‘Gold-standard’ FSC curve at the FSC = 0.143 (down) for AA150µM-TAS2R14-Gg-scFv16 (d), FFA1mM-TAS2R14-miniGs/gust-Nb35 (i) and FFA350µM -TAS2R14-Gi1-scFv16 (n) complexes. (e, j, o) The density maps for helices TM1-TM7 of transmembrane domain, helix 8 and α5 helices of Gα for AA150µM-TAS2R14-Gg-scFv16 (e), FFA1mM-TAS2R14-miniGs/gust-Nb35 (j) and FFA350µM -TAS2R14-Gi1-scFv16 (o) complexes. The SEC and SDS-PAGE experiments were repeated at least three times with similar results.

Extended Data Fig. 3 TAS2R14-Gi-scFv16 and 28.1150µM-TAS2R14-Gg-scFv16 sample preparation, cryo-EM data processing and structure determination.

(a, f) Representative size exclusion chromatography (SEC) profiles, SDS-PAGE analysis, representative cryo-EM micrography (scale bar: 50 nm) and selected 2D classification averages of TAS2R14-Gi-scFv16 (a) and 28.1150µM-TAS2R14-Gg-scFv16 (f) complexes. (b, g) Cryo-EM data processing flowcharts of TAS2R14-Gi-scFv16 (b) and 28.1150µM-TAS2R14-Gg-scFv16 (g) complexes by cryoSPARC 4.3. (c, h) Cryo-EM maps are colored by local resolution (Å) for TAS2R14-Gi-scFv16 (c) and 28.1150µM-TAS2R14-Gg-scFv16 (h) complexes. (d, i) Angular distribution of the particles used for final reconstruction (up) and ‘Gold-standard’ FSC curve at the FSC = 0.143 (down) for TAS2R14-Gi-scFv16 (d) and 28.1150µM-TAS2R14-Gg-scFv16 (i) complexes. (e, j) The density maps for helices TM1-TM7 of transmembrane domain, helix 8 and α5 helices of Gα for TAS2R14-Gi-scFv16 (e) and 28.1150µM-TAS2R14-Gg-scFv16 (j) complexes. The SEC and SDS-PAGE experiments were repeated at least three times with similar results.

Extended Data Fig. 4 Structural comparison of TAS2R14 with TAS2R46 and class A GPCRs.

(a) Schematic representation of constructs design for the G proteins used in the complex structure determination. Red * label indicates the different residues in α5 helix between Gαg and Gαi1. (b) Dose–response curves of Gαi1-Gβγ, Gαi2-Gβγ or Gαi3-Gβγ dissociation in TAS2R14-overexpressing HEK293 cells in response to FFA. Values are represented ad mean ± s.e.m. of 3 independent experiments (n = 3). (c) Structural comparison of AA-TAS2R14 and strychnine-TAS2R46 in side view (c), extracellular view (d) and intracellular view (e). Strychnine is an agonist for TAS2R46. The conformational changes are indicated by red arrows. (f) The sequence alignment of conserved residues in TAS2Rs (TAS2R14 and TAS2R46) and class A GPCRs (β2AR and CB1). (g) The 3D structural comparison of TAS2R14 with TAS2R46 and β2AR in active states. The conserved residues are labelled for TMs1-7 excluding TM4.

Source Data

Extended Data Fig. 5 Structural comparison of different binding pockets and TM6 conformational changes in different TAS2R14 complexes.

(a) Schematic presentation of ligand binding pockets in different TAS2R14 complex structures. The cartoon presentation of receptor is from the AA150µM-TAS2R14-miniGs/gust complex. Cholesterol and ligands are shown as sticks. The oval indicates the location of each pocket. (b) Comparison of cholesterol binding poses in different TAS2R14 complex structures. Cholesterol shows subtle shifts in pocket 1. (c) Dose–response curves of Gαg-Gβγ (up) or Gαi-Gβγ (down) dissociation in WT TAS2R14 or W893.32A mutant in response to cholesterol. Values are represented as mean ± s.e.m. of 3 independent experiments (n = 3). *P < 0.05; ***P < 0.001 (W89A compared with wild-type); All data were analyzed by two-sided one-way ANOVA with Turkey test. (From left to right, P = 0.001, P = 0.022). (d) Comparison of the binding pose of cholesterol in TAS2R14-Gi1, AA in 150µM-TAS2R14-miniGs/gust and AA in AA150µM-TAS2R14-Gg complexes. (e) Conformational changes of cytoplasmic TM6 in TAS2R14-Gi1, 150µM-TAS2R14-miniGs/gust and AA150µM-TAS2R14-Gg structures. (f-h) Depletion of cholesterol in the cell membrane affects TAS2R14 signaling. Representative concentration dependent curves of different signaling including Gg (f) or Gi1 (g) of TAS2R14 in response to simulations with FFA by BRET assay. Values are represented as mean ± s.e.m. of 3 independent experiments (n = 3), as shown in (h). (i) Comparison of binding poses of cholesterol in TAS2R14-Gi1 and AA in 150µM-TAS2R14-miniGs/gust structures, as well as the conformational changes of TM6. (j) Comparison of TM6’s conformational changes in 150µM-TAS2R14-miniGs/gust, AA150µM-TAS2R14-Gg and AA50µM-TAS2R14-Gi1 structures. The zoom-in views of pocket 3 for AA and its steric clash with TM6 in AA150µM-TAS2R14-Gg and AA50µM-TAS2R14-Gi1 structures and sidechain rotation of M2055.65 in different structures are shown. (k) List of key residues in pockets 1–3 in TAS2R14 and the conserved residues in TAS2Rs. The highly conserved residues are labelled in blue. Residues in purple background represent pocket 1, red for pocket 2 and green for pocket 3.

Source Data

Extended Data Fig. 6 Dose responses curves of key residues mutations in ligand binding pockets and receptor activation for aristolochic acid (AA), flufennamic acid (FFA) and 28.1 in TAS2R14 in calcium mobilization assay.

(a-e) Dose–response curves of key residues mutations in TAS2R14 in response to stimulation with AA in calcium mobilization assay. Data are mean ± s.e.m. from 3 independent experiments (n = 3). (f-g) Dose–response curves of key residues mutations in TAS2R14 in response to stimulation with FFA in calcium mobilization assay. Data are mean ± s.e.m. from 3 independent experiments (n = 3). (k) Dose–response curves of key residues mutations of pocket 2 in TAS2R14 in response to stimulation with compound 28.1 in calcium mobilization assay. Data are mean ± s.e.m. from 3 independent experiments (n = 3). Also see Supplementary tables 1–3.

Source Data

Extended Data Fig. 7 The stability of cholesterol or aristolochic acid (AA) binding in different complex structures in molecular dynamic simulations.

Three independent molecular dynamic (MD) simulations runs (500 ns each) were performed for each condition. RMSD values are calculated for each run. (a) RMSD values of AA molecule in AA150µM-TAS2R14-Gg structure in three runs. (b) RMSD values of cholesterol (CLR) in AA150µM-TAS2R14-miniGs/gust structure which AA molecules in pocket 2 and 3 are removed. (c-d) RMSD values of AA in pocket 2 (c) and AA in pocket 3 (d) in AA150µM-TAS2R14-miniGs/gust structure which CLR molecule in pocket 1 is removed. (e-g) RMSD values of CLR in pocket 1 (e), AA in pocket 2 (f) and AA in pocket 3 (g) in AA150µM-TAS2R14-miniGs/gust structure. (h-i) RMSD values of CLR in pocket 1 (h) and AA in pocket 2 (j) in AA50µM-TAS2R14-Gi structure.

Extended Data Fig. 8 Electrostatic surface potential analysis for different binding pockets and molecular docking of representative TAS2R14 agonists.

(a) Electrostatic surface potential of pocket 1, pocket 2 and pocket 3 and representative ligands of TAS2R14. (b) Docking poses of neutral small compounds in pocket 1. (c) Docking poses of organic acids in pocket 2.

Extended Data Fig. 9 The BRET assay of FFA activated TAS2R14 in Gg and Gi pathways and compound 28.1 in Gg pathway.

(a-c) Dose–response curves of single mutations in pocket 1 (a), pocket 2 (b) and double or triple mutations in pocket 1 (c) in TAS2R14 in response to stimulation with FFA by Gαg-Gβγ dissociation assay. Values are represented as mean �� s.e.m. of 3 independent experiments (n = 3). (d-e) Dose–response curves of single mutations in pocket 1 (d) and pocket 2 (e) in TAS2R14 in response to stimulation with FFA by Gαi-Gβγ dissociation assay. Values are represented as mean ± s.e.m. of 3 independent experiments (n = 3). (f) Dose–response curves of single mutations in pocket 2 in TAS2R14 in response to stimulation with compound 28.1 by Gαg-Gβγ dissociation assay. Values are represented as mean ± s.e.m. of 3 independent experiments (n = 3).

Source Data

Extended Data Fig. 10 Structural comparison of active features and G protein coupling in TAS2R14.

(a-c) Structural comparison of key motifs related to activation in TAS2R14 and TAS2R46. Trp3.32 and toggle switch residues (a), HS/P7.50FIL and FY3.50L (b) and Trp3.41 residue (c), respectively. (d) Binding mode between TAS2R14 and Gαg in AA150µM-TAS2R14-Gg structure. (e) Binding mode between TAS2R14 and Gαg in 28.1150µM-TAS2R14-Gg structure. (f) Structural comparison of TM6 and ligand binding poses in AA150µM-TAS2R14-Gg and 28.1150µM-TAS2R14-Gg structures. (g) Structure comparison of the miniGαs/gust and Gαg binding modes in TAS2R14. AA150µM-TAS2R14-miniGs/gust and AA150µM-TAS2R14-Gg complex structures are used for analysis. (h) Structure comparison of the Gαi binding modes in TAS2R14 and CB1. (i) Diagram of the contacts between Gαg and TAS2R14 and Gαi1 and TAS2R14, respectively.

Extended Data Table 1 Cryo-EM data collection, model refinement and validation statistics
Full size table

Supplementary information

Supplementary Information

Supplementary Tables 1–4 and Fig. 1.

Reporting Summary

Supplementary Video 1

The 3D variability videos prepared from cryo-EM data of FFA–TAS2R14–Gi1 to illustrate the multiple conformation features of TM6.

Supplementary Video 2

The MD simulated video shows the unwinding process of TM6 cytosolic end when AA was virtually depleted from the receptor.

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Hu, X., Ao, W., Gao, M. et al. Bitter taste TAS2R14 activation by intracellular tastants and cholesterol. Nature 631, 459–466 (2024). https://doi.org/10.1038/s41586-024-07569-9

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