Glutathione binding to the plant AtAtm3 transporter and implications for the conformational coupling of ABC transporters

  1. Chengcheng Fan
  2. Douglas C Rees  Is a corresponding author
  1. Howard Hughes Medical Institute, California Institute of Technology, United States

Abstract

The ATP Binding Cassette (ABC) transporter of mitochondria (Atm) from Arabidopsis thaliana (AtAtm3) has been implicated in the maturation of cytosolic iron-sulfur proteins and heavy metal detoxification, plausibly by exporting glutathione derivatives. Using single-particle cryo-electron microscopy, we have determined four structures of AtAtm3 in three different conformational states: two inward-facing conformations (with and without bound oxidized glutathione (GSSG)), together with closed and outward-facing states stabilized by MgADP-VO4. These structures not only provide a structural framework for defining the alternating access transport cycle, but also reveal the paucity of cysteine residues in the glutathione binding site that could potentially form inhibitory mixed disulfides with GSSG. Despite extensive efforts, we were unable to prepare the ternary complex of AtAtm3 containing both GSSG and MgATP. A survey of structurally characterized type IV ABC transporters that includes AtAtm3 establishes that while nucleotides are found associated with all conformational states, they are effectively required to stabilize occluded, closed, and outward-facing conformations. In contrast, transport substrates have only been observed associated with inward-facing conformations. The absence of structures with dimerized nucleotide binding domains containing both nucleotide and transport substrate suggests that this form of the ternary complex exists only transiently during the transport cycle.

Data availability

The atomic coordinates for inward-facing, inward-facing with GSSG bound, closed and outward-facing conformations were separately deposited in the Protein Data Bank (PDB) and the Electron Microscopy Data Bank (EMDB) with accession codes: PDB 7N58, 7N59, 7N5A and 7N5B; EMDB EMD-24182, EMD-24183, EMD-24184 and EMD-24185. The plasmid encoding full-length AtAtm3 and the AtAtm3 with N-terminal 80 residue deletion were deposited in Addgene with Addgene ID 172321 and 173045, respectively. The raw data for ATPase assays presented in Figure 1 are provided in Supplementary File 1, while the essdyn.f Fortran source code used for the PCA analysis is provided as Source Code 1.

Article and author information

Author details

  1. Chengcheng Fan

    Division of Chemistry and Chemical Engineering, Howard Hughes Medical Institute, California Institute of Technology, Pasadena, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4213-5758
  2. Douglas C Rees

    Division of Chemistry and Chemical Engineering, Howard Hughes Medical Institute, California Institute of Technology, Pasadena, United States
    For correspondence
    dcrees@caltech.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4073-1185

Funding

Howard Hughes Medical Institute

  • Douglas C Rees

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

Reviewing Editor

  1. David Drew, Stockholm University, Sweden

Publication history

  1. Received: December 6, 2021
  2. Preprint posted: December 14, 2021 (view preprint)
  3. Accepted: March 23, 2022
  4. Accepted Manuscript published: March 25, 2022 (version 1)
  5. Version of Record published: April 11, 2022 (version 2)

Copyright

© 2022, Fan & Rees

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. Chengcheng Fan
  2. Douglas C Rees
(2022)
Glutathione binding to the plant AtAtm3 transporter and implications for the conformational coupling of ABC transporters
eLife 11:e76140.
https://doi.org/10.7554/eLife.76140
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