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

Fan, Chengcheng and Rees, Douglas C. (2022) Glutathione binding to the plant AtAtm3 transporter and implications for the conformational coupling of ABC transporters. eLife, 11 . Art. No. e76140. ISSN 2050-084X. PMCID PMC9000953. doi:10.7554/eLife.76140. https://resolver.caltech.edu/CaltechAUTHORS:20211217-512156900

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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.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.7554/eLife.76140DOIArticle
http://www.ncbi.nlm.nih.gov/pmc/articles/pmc9000953/PubMed CentralArticle
https://doi.org/10.1101/2021.12.13.472443DOIDiscussion Paper
ORCID:
AuthorORCID
Fan, Chengcheng0000-0003-4213-5758
Rees, Douglas C.0000-0003-4073-1185
Additional Information:© 2022 Fan and Rees. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. Received: December 6, 2021; Preprint posted: December 14, 2021 (view preprint); Accepted: March 23, 2022; Accepted Manuscript published: March 25, 2022 (version 1); Version of Record published: April 11, 2022 (version 2). We thank Andrey Malyutin, Songye Chen, and Corey Hecksel for their support during single-particle cryoEM data collections and the reviewers for their helpful comments, particularly concerning comparisons of AtAtm3 with other ABC transporters. CryoEM was performed in the Beckman Institute Resource Center for cryo-Electron Microscopy at Caltech and at the Stanford-SLAC Cryo-EM Center (S2C2). The S2C2 is supported by the National Institutes of Health Common Fund Transformative High Resolution Cryo-Electron Microscopy program. We thank the Beckman Institute for their support of the cryoEM facility at Caltech. Funding: DCR is a Howard Hughes Medical Institute Investigator. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Author contributions: Chengcheng Fan, Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review and editing; Douglas C Rees, Conceptualization, Funding acquisition, Methodology, Project administration, Software, Supervision, Writing – review and editing. The authors declare that no competing interests exist. 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 plasmids 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 Source Data 1, while the essdyn.f Fortran source code used for the PCA analysis is provided as Source Code 1.
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Funding AgencyGrant Number
NIHUNSPECIFIED
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
PubMed Central ID:PMC9000953
DOI:10.7554/eLife.76140
Record Number:CaltechAUTHORS:20211217-512156900
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20211217-512156900
Official Citation:Glutathione binding to the plant AtAtm3 transporter and implications for the conformational coupling of ABC transporters. Fan and Rees. eLife 2022;11:e76140. DOI: https://doi.org/10.7554/eLife.76140
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:112498
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:17 Dec 2021 17:14
Last Modified:30 Jun 2022 17:22

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