New capsaicin analogs as molecular rulers to define the permissive conformation of the mouse TRPV1 ligand-binding pocket

  1. Simon Vu
  2. Vikrant Singh
  3. Heike Wulff
  4. Vladimir Yarov-Yarovoy
  5. Jie Zheng  Is a corresponding author
  1. University of California, Davis, United States

Abstract

The capsaicin receptor TRPV1 is an outstanding representative of ligand-gated ion channels in ligand selectivity and sensitivity. However, molecular interactions that stabilize the ligand-binding pocket in its permissive conformation, and how many permissive conformations the ligand-binding pocket may adopt, remain unclear. To answer these questions, we designed a pair of novel capsaicin analogs to increase or decrease the ligand size by about 1.5 Å without altering ligand chemistry. Together with capsaicin, these ligands form a set of molecular rulers for investigating ligand-induced conformational changes. Computational modeling and functional tests revealed that structurally these ligands alternate between drastically different binding poses but stabilize the ligand-binding pocket in nearly identical permissive conformations; functionally they all yielded a stable open state despite varying potencies. Our study suggests the existence of an optimal ligand-binding pocket conformation for capsaicin-mediated TRPV1 activation gating, and reveals multiple ligand-channel interactions that stabilize this permissive conformation.

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files.

Article and author information

Author details

  1. Simon Vu

    Physiology and Membrane Biology, University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1529-8220
  2. Vikrant Singh

    Pharmacology, University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Heike Wulff

    Pharmacology, University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Vladimir Yarov-Yarovoy

    Physiology and Membrane Biology, University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2325-4834
  5. Jie Zheng

    Department of Physiology and Membrane Biology, University of California, Davis, Davis, United States
    For correspondence
    jzheng@ucdavis.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4161-627X

Funding

National Institutes of Health (R01NS103954)

  • Jie Zheng

National Institutes of Health (R01NS103954)

  • Vladimir Yarov-Yarovoy

American Heart Association (16PRE29340002)

  • Simon Vu

NIH Office of the Director (U54NS079202)

  • Heike Wulff

National Institute of Neurological Disorders and Stroke (U54NS079202)

  • Heike Wulff

NIH Office of the Director (U54NS079202)

  • Vikrant Singh

National Institute of Neurological Disorders and Stroke (U54NS079202)

  • Vikrant Singh

National Institutes of Health (R01GM132110)

  • Jie Zheng

National Institutes of Health (R01GM132110)

  • Vladimir Yarov-Yarovoy

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Stephan A. Pless, University of Copenhagen, Denmark

Publication history

  1. Received: August 12, 2020
  2. Accepted: November 8, 2020
  3. Accepted Manuscript published: November 9, 2020 (version 1)
  4. Version of Record published: November 17, 2020 (version 2)

Copyright

© 2020, Vu et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 1,808
    Page views
  • 204
    Downloads
  • 3
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Simon Vu
  2. Vikrant Singh
  3. Heike Wulff
  4. Vladimir Yarov-Yarovoy
  5. Jie Zheng
(2020)
New capsaicin analogs as molecular rulers to define the permissive conformation of the mouse TRPV1 ligand-binding pocket
eLife 9:e62039.
https://doi.org/10.7554/eLife.62039

Further reading

    1. Structural Biology and Molecular Biophysics
    Daan B Boltje, Jacob P Hoogenboom ... Sander den Hoedt
    Tools and Resources Updated

    Cryogenic electron tomography (cryo-ET) combined with subtomogram averaging, allows in situ visualization and structure determination of macromolecular complexes at subnanometre resolution. Cryogenic focused ion beam (cryo-FIB) micromachining is used to prepare a thin lamella-shaped sample out of a frozen-hydrated cell for cryo-ET imaging, but standard cryo-FIB fabrication is blind to the precise location of the structure or proteins of interest. Fluorescence-guided focused ion beam (FIB) milling at target locations requires multiple sample transfers prone to contamination, and relocation and registration accuracy is often insufficient for 3D targeting. Here, we present in situ fluorescence microscopy-guided FIB fabrication of a frozen-hydrated lamella to address this problem: we built a coincident three-beam cryogenic correlative microscope by retrofitting a compact cryogenic microcooler, custom positioning stage, and an inverted widefield fluorescence microscope (FM) on an existing FIB scanning electron microscope. We show FM controlled targeting at every milling step in the lamella fabrication process, validated with transmission electron microscope tomogram reconstructions of the target regions. The ability to check the lamella during and after the milling process results in a higher success rate in the fabrication process and will increase the throughput of fabrication for lamellae suitable for high-resolution imaging.

    1. Cell Biology
    2. Structural Biology and Molecular Biophysics
    Johannes Elferich, Giulia Schiroli ... Nikolaus Grigorieff
    Tools and Resources Updated

    A major goal of biological imaging is localization of biomolecules inside a cell. Fluorescence microscopy can localize biomolecules inside whole cells and tissues, but its ability to count biomolecules and accuracy of the spatial coordinates is limited by the wavelength of visible light. Cryo-electron microscopy (cryo-EM) provides highly accurate position and orientation information of biomolecules but is often confined to small fields of view inside a cell, limiting biological context. In this study, we use a new data-acquisition scheme called Defocus-Corrected Large-Area cryo-EM (DeCo-LACE) to collect high-resolution images of entire sections (100- to 250-nm-thick lamellae) of neutrophil-like mouse cells, representing 1–2% of the total cellular volume. We use 2D template matching (2DTM) to determine localization and orientation of the large ribosomal subunit in these sections. These data provide maps of ribosomes across entire sections of mammalian cells. This high-throughput cryo-EM data collection approach together with 2DTM will advance visual proteomics and provide biological insight that cannot be obtained by other methods.