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Glutathione binding to the plant At Atm3 transporter and implications for the conformational coupling of ABC transporters

Fan, Chengcheng and Rees, Douglas C. (2021) Glutathione binding to the plant At Atm3 transporter and implications for the conformational coupling of ABC transporters. . (Unpublished)

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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 structures of AtAtm3 in multiple conformational states. These structures not only provide a structural framework for defining the alternating access transport cycle, but also highlight an unappreciated feature of the glutathione binding site, namely the paucity of cysteine residues that could potentially form inhibitory mixed disulfides with glutathione. Despite extensive efforts, we were unable to prepare the ternary complex of AtAtm3 with bound 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 and outward-facing conformations. In contrast, transport substrates have only been observed associated with inward-facing conformations. The absence of structures containing both nucleotide and transport substrate suggests that this ternary complex exists only transiently during the transport cycle.

Item Type:Report or Paper (Discussion Paper)
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URLURL TypeDescription Paper
Fan, Chengcheng0000-0003-4213-5758
Rees, Douglas C.0000-0003-4073-1185
Additional Information:The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. This version posted December 14, 2021. We thank Andrey Malyutin, Songye Chen and Corey Hecksel for their support during single particle cryo-EM data collections. Cryo-electron microscopy 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 cryo-EM facility at Caltech. D.C.R. is a Howard Hughes Medical Institute Investigator. Author Contributions: C.F. and D.C.R. designed the research; C.F. performed the research; C.F. and D.C.R. analyzed the data; and C.F and D.C.R. prepared the manuscript. The authors declare no competing interests. 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.
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Howard Hughes Medical Institute (HHMI)UNSPECIFIED
Record Number:CaltechAUTHORS:20211217-513701700
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Official Citation:Glutathione binding to the plant AtAtm3 transporter and implications for the conformational coupling of ABC transporters. Chengcheng Fan, Douglas C. Rees. bioRxiv 2021.12.13.472443; doi:
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:112498
Deposited By: Tony Diaz
Deposited On:17 Dec 2021 17:14
Last Modified:17 Dec 2021 17:14

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