Cryo-EM structures of a LRRC8 chimera with native functional properties reveal heptameric assembly

  1. Hirohide Takahashi
  2. Toshiki Yamada
  3. Jerod S Denton
  4. Kevin Strange
  5. Erkan Karakas  Is a corresponding author
  1. Vanderbilt University, United States
  2. Vanderbilt University Medical Center, United States

Abstract

Volume-regulated anion channels (VRACs) mediate volume regulatory Cl- and organic solute efflux from vertebrate cells. VRACs are heteromeric assemblies of LRRC8A-E proteins with unknown stoichiometries. Homomeric LRRC8A and LRRC8D channels have a small pore, hexameric structure. However, these channels are either non-functional nor exhibit abnormal regulation and pharmacology, limiting their utility for structure-function analyses. We circumvented these limitations by developing novel homomeric LRRC8 chimeric channels with functional properties consistent with those of native VRAC/LRRC8 channels. We demonstrate here that the LRRC8C-LRRC8A(IL125) chimera comprising LRRC8C and 25 amino acids unique to the first intracellular loop (IL1) of LRRC8A has a heptameric structure like that of homologous pannexin channels. Unlike homomeric LRRC8A and LRRC8D channels, heptameric LRRC8C-LRRC8A(IL125) channels have a large-diameter pore similar to that estimated for native VRACs, exhibit normal DCPIB pharmacology, and have higher permeability to large organic anions. Lipid-like densities are located between LRRC8C-LRRC8A(IL125) subunits and occlude the channel pore. Our findings provide new insights into VRAC/LRRC8 channel structure and suggest that lipids may play important roles in channel gating and regulation.

Data availability

Cryo-EM maps and atomic coordinates are deposited to the Electron Microscopy Data Bank (EMDB) and Protein Data Bank (PDB) databases. The accession codes are EMD-27770 and 8DXN for class 1; EMD-27771 and 8DXO for class 2; EMD-27772 and 8DXP for class 3; EMD-27773 and 8DXQ for class 4; EMD-27774 and 8DXR for class 5.

Article and author information

Author details

  1. Hirohide Takahashi

    Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2553-8806
  2. Toshiki Yamada

    Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, United States
    Competing interests
    No competing interests declared.
  3. Jerod S Denton

    Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, United States
    Competing interests
    No competing interests declared.
  4. Kevin Strange

    Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, United States
    Competing interests
    Kevin Strange, is cofounder and principal scientist of Revidia Therapeutics, Inc..
  5. Erkan Karakas

    Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, United States
    For correspondence
    erkan.karakas@vanderbilt.edu
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6552-3185

Funding

National Institute of Diabetes and Digestive and Kidney Diseases (DK51610)

  • Jerod S Denton

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

Reviewing Editor

  1. Merritt Maduke, Stanford University School of Medicine, United States

Version history

  1. Preprint posted: July 28, 2022 (view preprint)
  2. Received: August 3, 2022
  3. Accepted: March 9, 2023
  4. Accepted Manuscript published: March 10, 2023 (version 1)
  5. Version of Record published: March 28, 2023 (version 2)

Copyright

© 2023, Takahashi 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

  • 1,510
    views
  • 271
    downloads
  • 7
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

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. Hirohide Takahashi
  2. Toshiki Yamada
  3. Jerod S Denton
  4. Kevin Strange
  5. Erkan Karakas
(2023)
Cryo-EM structures of a LRRC8 chimera with native functional properties reveal heptameric assembly
eLife 12:e82431.
https://doi.org/10.7554/eLife.82431

Share this article

https://doi.org/10.7554/eLife.82431

Further reading

    1. Structural Biology and Molecular Biophysics
    Hitendra Negi, Aravind Ravichandran ... Ranabir Das
    Research Article

    The proteasome controls levels of most cellular proteins, and its activity is regulated under stress, quiescence, and inflammation. However, factors determining the proteasomal degradation rate remain poorly understood. Proteasome substrates are conjugated with small proteins (tags) like ubiquitin and Fat10 to target them to the proteasome. It is unclear if the structural plasticity of proteasome-targeting tags can influence substrate degradation. Fat10 is upregulated during inflammation, and its substrates undergo rapid proteasomal degradation. We report that the degradation rate of Fat10 substrates critically depends on the structural plasticity of Fat10. While the ubiquitin tag is recycled at the proteasome, Fat10 is degraded with the substrate. Our results suggest significantly lower thermodynamic stability and faster mechanical unfolding in Fat10 compared to ubiquitin. Long-range salt bridges are absent in the Fat10 structure, creating a plastic protein with partially unstructured regions suitable for proteasome engagement. Fat10 plasticity destabilizes substrates significantly and creates partially unstructured regions in the substrate to enhance degradation. NMR-relaxation-derived order parameters and temperature dependence of chemical shifts identify the Fat10-induced partially unstructured regions in the substrate, which correlated excellently to Fat10-substrate contacts, suggesting that the tag-substrate collision destabilizes the substrate. These results highlight a strong dependence of proteasomal degradation on the structural plasticity and thermodynamic properties of the proteasome-targeting tags.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Amy H Andreotti, Volker Dötsch
    Editorial

    The articles in this special issue highlight how modern cellular, biochemical, biophysical and computational techniques are allowing deeper and more detailed studies of allosteric kinase regulation.