1. Structural Biology and Molecular Biophysics

The crystal structure of bromide-bound GtACR1 reveals a pre-activated state in the transmembrane anion tunnel

  1. Hai Li
  2. Chia-Ying Huang
  3. Elena G Govorunova
  4. Oleg A Sineshchekov
  5. Adrian Yi
  6. Kenneth J Rothschild
  7. Meitian Wang
  8. Lei Zheng  Is a corresponding author
  9. John L Spudich  Is a corresponding author
  1. University of Texas Health Science Center at Houston, McGovern Medical School, United States
  2. Swiss Light Source, Paul Scherrer Institute, Switzerland
  3. Boston University, United States
https://doi.org/10.7554/eLife.65903
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Cite this article
  1. Hai Li
  2. Chia-Ying Huang
  3. Elena G Govorunova
  4. Oleg A Sineshchekov
  5. Adrian Yi
  6. Kenneth J Rothschild
  7. Meitian Wang
  8. Lei Zheng
  9. John L Spudich
(2021)
The crystal structure of bromide-bound GtACR1 reveals a pre-activated state in the transmembrane anion tunnel
eLife 10:e65903.
https://doi.org/10.7554/eLife.65903
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Abstract

The crystal structure of the light-gated anion channel GtACR1 reported in our previous Research Article (Li et al., 2019) revealed a continuous tunnel traversing the protein from extracellular to intracellular pores. We proposed the tunnel as the conductance channel closed by three constrictions: C1 in the extracellular half, mid-membrane C2 containing the photoactive site, and C3 on the cytoplasmic side. Reported here, the crystal structure of bromide-bound GtACR1 reveals structural changes that relax the C1 and C3 constrictions, including a novel salt-bridge switch mechanism involving C1 and the photoactive site. These findings indicate that substrate binding induces a transition from an inactivated state to a pre-activated state in the dark that facilitates channel opening by reducing free energy in the tunnel constrictions. The results provide direct evidence that the tunnel is the closed form of the channel of GtACR1 and shed light on the light-gated channel activation mechanism.

Data availability

Diffraction data have been deposited in PDB under the accession code 7L1E.

The following data sets were generated

Article and author information

Author details

  1. Hai Li

    Department of Biochemistry and Molecular Biology, McGovern Medical School, University of Texas Health Science Center at Houston, McGovern Medical School, Houston, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3969-6709

  2. Chia-Ying Huang

    Macromolecular Crystallography, Swiss Light Source, Paul Scherrer Institute, Villigen, Switzerland
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7676-0239

  3. Elena G Govorunova

    Center for Membrane Biology, Department of Biochemistry and Molecular Biology, University of Texas Health Science Center at Houston, McGovern Medical School, Houston, United States
    Competing interests
    Elena G Govorunova, as an inventor and The University of Texas Health Science Center at Houston has been granted a patent titled: Compositions and Methods for Use of Anion Channel Rhodopsins Patent # 10,519,205 granted Dec 31, 2019 by the US Patent and Trademark Office..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0522-9683

  4. Oleg A Sineshchekov

    Center for Membrane Biology, Department of Biochemistry and Molecular Biology, University of Texas Health Science Center at Houston, McGovern Medical School, Houston, United States
    Competing interests
    Oleg A Sineshchekov, as an inventor and The University of Texas Health Science Center at Houston has been granted a patent titled: Compositions and Methods for Use of Anion Channel Rhodopsins Patent # 10,519,205 granted Dec 31, 2019 by the US Patent and Trademark Office..

  5. Adrian Yi

    Boston University, Boston, United States
    Competing interests
    No competing interests declared.

  6. Kenneth J Rothschild

    Boston University, Boston, United States
    Competing interests
    No competing interests declared.

  7. Meitian Wang

    Macromolecular Crystallography, Swiss Light Source, Paul Scherrer Institute, Villigen, Switzerland
    Competing interests
    No competing interests declared.

  8. Lei Zheng

    Center for Membrane Biology, Department of Biochemistry and Molecular Biology, University of Texas Health Science Center at Houston, McGovern Medical School, Houston, United States
    For correspondence
    Lei.Zheng@uth.tmc.edu
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7789-5234

  9. John L Spudich

    Center for Membrane Biology, Department of Biochemistry and Molecular Biology, University of Texas Health Science Center at Houston, McGovern Medical School, Houston, United States
    For correspondence
    John.L.Spudich@uth.tmc.edu
    Competing interests
    John L Spudich, as an inventor and The University of Texas Health Science Center at Houston has been granted a patent titled: Compositions and Methods for Use of Anion Channel Rhodopsins Patent # 10,519,205 granted Dec 31, 2019 by the US Patent and Trademark Office..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4167-8590

Funding

National Institute of General Medical Sciences (R01GM027750)

  • John L Spudich

National Institute of General Medical Sciences (R35GM140838)

  • John L Spudich

Robert A. Welch Foundation (Endowed Chair AU-0009)

  • John L Spudich

American Heart Association (18TPA34230046)

  • Lei Zheng

National Science Foundation (CBET-1264434)

  • Kenneth J Rothschild

European Union's Horizon 2020 (Marie-Skłodowska-Curie grant agreement No. 701647)

  • Chia-Ying Huang

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

Copyright

© 2021, Li 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.

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  1. Hai Li
  2. Chia-Ying Huang
  3. Elena G Govorunova
  4. Oleg A Sineshchekov
  5. Adrian Yi
  6. Kenneth J Rothschild
  7. Meitian Wang
  8. Lei Zheng
  9. John L Spudich
(2021)
The crystal structure of bromide-bound GtACR1 reveals a pre-activated state in the transmembrane anion tunnel
eLife 10:e65903.
https://doi.org/10.7554/eLife.65903

Share this article

https://doi.org/10.7554/eLife.65903

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Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics

    Crystal structure of a natural light-gated anion channelrhodopsin

    Hai Li, Chia-Ying Huang ... John L Spudich
    Research Article Updated

    The anion channelrhodopsin GtACR1 from the alga Guillardia theta is a potent neuron-inhibiting optogenetics tool. Presented here, its X-ray structure at 2.9 Å reveals a tunnel traversing the protein from its extracellular surface to a large cytoplasmic cavity. The tunnel is lined primarily by small polar and aliphatic residues essential for anion conductance. A disulfide-immobilized extracellular cap facilitates channel closing and the ion path is blocked mid-membrane by its photoactive retinylidene chromophore and further by a cytoplasmic side constriction. The structure also reveals a novel photoactive site configuration that maintains the retinylidene Schiff base protonated when the channel is open. These findings suggest a new channelrhodopsin mechanism, in which the Schiff base not only controls gating, but also serves as a direct mediator for anion flux.