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The structure of lipid nanodisc-reconstituted TRPV3 reveals the gating mechanism

Nature Structural & Molecular Biology volume 27, pages 645–652 (2020)Cite this article

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Abstract

Transient receptor potential vanilloid subfamily member 3 (TRPV3) is a temperature-sensitive cation channel. Previous cryo-EM analyses of TRPV3 in detergent micelles or amphipol proposed that the lower gate opens by α-to-π helical transitions of the nearby S6 helix. However, it remains unclear how physiological lipids are involved in the TRPV3 activation. Here we determined the apo state structure of mouse (Mus musculus) TRPV3 in a lipid nanodisc at 3.3 Å resolution. The structure revealed that lipids bound to the pore domain stabilize the selectivity filter in the narrow state, suggesting that the selectivity filter of TRPV3 affects cation permeation. When the lower gate is closed in nanodisc-reconstituted TRPV3, the S6 helix adopts the π-helical conformation without agonist- or heat-sensitization, potentially stabilized by putative intra-subunit hydrogen bonds and lipid binding. Our findings provide insights into the lipid-associated gating mechanism of TRPV3.

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Fig. 1: Architecture and ion permeation pore of mouse TRPV3 (mTRPV3-ΔN) in a lipid nanodisc.
Fig. 2: Structural changes of the selectivity filter in TRPV3.
Fig. 3: Lower gate architecture of TRPV3 in a nanodisc.
Fig. 4: The hydrogen bond between Asp586 and Thr680.
Fig. 5: Proposed mechanistic models for TRPV3 activation.

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Data availability

Cryo-EM density maps have been deposited in the Electron Microscopy Data Bank (EMDB), under accession number EMD-0882. Model coordinates have been deposited in the Protein Data Bank (wwPDB), under accession code PDB 6LGP. The associated electron microscopy data have been deposited in the Electron Microscopy Public Image Archive, under accession code EMPIAR-10400. Source data for Fig. 4e and Extended Data Fig. 1a are available with the paper online.

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Acknowledgements

We thank T. Nakane for computational support and assistance with the single-particle analysis and model building, K. Yamashita for computational support and model building and Y. Lee for fruitful discussions and encouragement. We also thank the staff scientists at the University of Tokyo’s cryo-EM facility, especially K. Kobayashi, H. Yanagisawa, A. Tsutsumi, M. Kikkawa and R. Danev. This work was supported by a MEXT Grant-in-Aid for Specially Promoted Research (grant no. 16H06294) to O.N. and by MEXT KAKENHI Grant-in-Aid for Scientific Research(C) (18K60156). This research was supported by the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research) from AMED under grant no. JP19am01011115 (support number 1110).

Author information

Author notes

  1. These authors contributed equally: Hiroto Shimada, Tsukasa Kusakizako.

Authors and Affiliations

  1. Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan

    Hiroto Shimada, Tsukasa Kusakizako, Tomohiro Nishizawa & Osamu Nureki

  2. Division of Cell Signaling, National Institute for Physiological Sciences, National Institute of Natural Sciences, Okazaki, Japan

    T. H. Dung Nguyen & Makoto Tominaga

  3. Department of Chemistry and Biotechnology, Graduate School of Engineering, Tottori University, Tottori, Japan

    Tomoya Hino

  4. Center for Research on Green Sustainable Chemistry, Tottori University, Tottori, Japan

    Tomoya Hino

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Contributions

H.S., T.K., T.H. and O.N. designed the project. T.K. investigated the conditions for TRPV3 expression. H.S. performed protein expression, purification and cryo-EM sample preparation, with assistance from T.K. H.S., T.K. and T.N. performed data collection. H.S. processed and analyzed the cryo-EM data with assistance from T.K. T.N., H.S. and T.K. built the models. T.H.D.N. and M.T. performed the electrophysiology, protein biotinylation, western blotting and statistical analysis. H.S., T.K., T.H.D.N., T.N., T.H., M.T. and O.N. wrote the manuscript. O.N. supervised the research.

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Correspondence to Osamu Nureki.

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Peer review information Katarzyna Marcinkiewicz was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Functional characterization and nanodisc reconstitution of truncated mutant TRPV3.

a, Temperature thresholds of mTRPV3WT (light blue) and mTRPV-ΔN (light orange), stimulated by heat, 10 mM camphor, or 300 mM 2-APB. b, c, Electrophysiological comparison between mouse TRPV3 WT (mTRPV3-WT) (b) and N-terminal truncated mutant (mTRPV3ΔN) (c), by whole-cell patch-clamp recordings in HEK293 cells. mTRPV3-ΔN has the same electrophysiological profile as mTRPV3-WT. Graphs show mean and s.e.m. for indicated number of recordings. The source data are available online. d, e, Size exclusion chromatogram (d) and SDS–PAGE (e) of mTRPV3-ΔN in a nanodisc.

Source data

Extended Data Fig. 2 Overview of single-particle cryo-EM for mTRPV3-ΔN in a nanodisc.

a, Cryo-EM micrograph of mTRPV3-ΔN in a nanodisc, with example particles circled in red. b, Reference-free 2D class images of mTRPV3-ΔN in a nanodisc. c, 3D class averages of mTRPV3-ΔN in a nanodisc. d, Refined density, at 3.31 Å resolution (FSC = 0.143). e, FSC curves of the map of TRPV3 in a nanodisc. The horizontal dashed line indicated FSC = 0.143. f, Euler angular distribution for particles used in the final map of TRPV3 in a nanodisc.

Extended Data Fig. 3 The local resolution mapped on the density of mTRPV3-ΔN in a nanodisc.

The map was obtained using CueMol2 at a 0.010 threshold level (http://www.cuemol.org/). The local resolution is higher in the Transmembrane core than the Intracellular region.

Extended Data Fig. 4 Cryo-EM density of mTRPV3-ΔN in a nanodisc.

a, Cryo-EM density (blue mesh) at 2.4σ for a single TRPV3 subunit; the structure, shown as a ribbon, is colored according to the domains. b–i, Fragments of the TRPV3 transmembrane region with the corresponding cryo-EM densities shown as a blue mesh at 2.4σ. j, Cross-validation FSC curves for the refined model versus unfiltered half maps (only half map 1 was used for refinement with PHENIX42) and the summed map.

Extended Data Fig. 5 Lipid-like densities observed in mTRPV3-ΔN in a nanodisc.

a, The seven lipid-like densities (1-7) observed in mTRPV3-ΔN in a nanodisc map. Five densities (1-5) are sufficient to be modeled as phosphatidylcholine. The density is shown at 3.5σ. b, The site occupied by Lipid 1, known as the Vanilloid Binding Pocket, in TRPV1 structures in nanodiscs (PDB: 5IRZ). c, d, The lipid-like densities ascribed to Lipid 2 (c) and Lipid 6 (d) are observed in the TRPV3 structure in digitonin micelles (PDB: 6DVW).

Extended Data Fig. 6 Model validation map of selectivity filter.

a, Ribbon model around the selectivity filter, in stereo. The oxygens of the Gly638 and Gly640 mainchains, and the side chains of Leu639 and Asp641 are shown in stick models. EM density map around Gly638 – Asp641 shown in a gray mesh. b, Superimposition of the pore domain of TRPV3 in a nanodisc (pink) and the TRPV3 open state (light blue) (PDB: 6DVZ), according to the Cα atoms of the S6 helices (Pro651–Ser685), viewed from the extracellular side (same perspective as Fig. 3a). c, Comparison of the phospholipid-binding site in Fig. 3c between TRPV3 in a nanodisc (pink) and the TRPV3 open state (light blue) (same perspective as Fig. 3d). Both models are superimposed on the Cα atoms of the S6 helix (Pro651–Ser685) as a guide. The phospholipid observed in a nanodisc and the residues shown in Fig. 3c in the open state are indicated by stick and CPK models, respectively, revealing that the residues clashed with Lipid 3. d-g, Known TRPV3 structural models fitted to each EM density map (shown in blue mesh). TRPV3 in the closed state in digitonin micelles (purple) (d), closed state in amphipol (yellow) (e), sensitized state (green) (f), and open state (light blue) (g).

Extended Data Fig. 7 Close-up views of Lipid 4, Lipid 5, and Lipid 7.

a, Locations of the densities observed in Lipid 4, Lipid 5, and Lipid 7, indicated as a blue mesh at 3.0σ. The modeled phosphatidylcholine (Lipid 4, Lipid 5), the partially modeled acyl chain (Lipid 7), and the residues near Lipid 4 and Lipid 7 are shown as sticks. b, Interaction between Lipid 4 and the extracellular part of the S5 helix, viewed from the extracellular side and the residues nearby Lipid 4 are shown as sticks. c, Interactions among the intracellular region of the S5 helix, Lipid 5, and Lipid 7, viewed parallel to the membrane, and the residues nearby Lipid 7 are shown as sticks. d, e, Location of the density observed for Lipid 7 in previously reported structures: TRPV3-sensitized state (PDB: 6MHS) (d), TRPV3 open state (PDB: 6DVZ) (e), and the residues nearby the densities are shown as sticks.

Supplementary information

Source data

Source Data Fig. 4

Statistical source data of Fig. 4e

Source Data Extended Data Fig. 1

Statistical source data of Extended data Fig. 1a

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Shimada, H., Kusakizako, T., Dung Nguyen, T.H. et al. The structure of lipid nanodisc-reconstituted TRPV3 reveals the gating mechanism. Nat Struct Mol Biol 27, 645–652 (2020). https://doi.org/10.1038/s41594-020-0439-z

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