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

  1. Chengcheng Fan
  2. Douglas C Rees

  • Curated by eLife

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    Evaluation Summary:

    Oxygen consumption in mitochondria by the respiratory chain leads to a major source of reactive oxygen species, and mitochondrial glutathione is an important line of defence against free radical production. The ABC transporter Atm3 exports oxidized glutathione from the mitochondria to help maintain a suitable reducing environment. Here, the authors have determined structure of Atm3 in multiple conformations by single-particle cryo EM and have revealed new insights into local changes coupled to substrate export of oxidised glutathione and ABC exporters in general. The work will be of interest to the mitochondrial biology and transporter communities.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)

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Abstract

The ATP binding cassette (ABC) transporter of mitochondria (Atm) from Arabidopsis thaliana ( At Atm3) 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 At Atm3 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-VO 4 . 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 At Atm3 containing both GSSG and MgATP. A survey of structurally characterized type IV ABC transporters that includes At Atm3 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.

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  1. Version published to 10.7554/elife.76140 on eLife
  2. eLife

    Author Response:

    Reviewer #3 (Public Review):

    The authors aim to understand the functional roles of Atm transporters, a family of ABC transporters with cellular localization of mitochondria and play important roles in transition metal homeostasis. To understand their functions, the author captured the structures of Atm3 from plant Arabidopsis in several functional states by cryo-EM--inward-facing, inward-facing with substrate GSSH bound, nucleotide bound closed state and nucleotide bound outward-facing states. Although many of those functional states have been reported for Atm orthologues in other species, the authors did elegant analyses on how the rareness of cysteine residues in the transport pathways could be very important for efficient glutathione transport. Moreover, the authors systematically show the unlikelihood of capturing an ABC transporter with both substrate and nucleotides bound. Although conceptually, a functional state with substrate and nucleotides bound should be very transient to avoid backflow of the substrate, it is wonderful that the authors put in the effort to capture such state and present a systematic principal component analysis (PCA) to show it may not be possible to attain such state structurally. The usage of PCA on analyzing a plethora of ABC transporters with different functional states could be applicable to other systems. The conclusions of this paper are mostly well supported by data, with some aspects of analysis and discrepancy that need to be addressed.

    1. In Figure 1, the authors report the ATPase activities of AtAtm3 are quite different in detergent and membrane environments, with a more than 10-fold decrease in basal activity from detergent to POPC containing nanodiscs. Is this composition a good representation of Arabidopsis mitochondria membrane? The nanodisc belts in Figure S4 looks quite tight. Could that contribute to the decrease in ATPase activity? In one of the papers you cited, Schaedler et. al, 2014, ATM3's activity is not stimulated by GSH versus in your data, 10mM GSH strongly stimulated AtAtm3. How would you explain the discrepancy? In terms of data analysis, the fit for 10mM GSH and 2.5mM GSSG ATPase activity in nanodiscs is poor visually, the addition of goodness of the fit in the figure could help the reader to assess the real quality of the data.

    In our experience, the ATPase rates of ABC transporters are highly dependent on the precise solubilization conditions, particularly for detergents as some detergents/detergent combinations are stimulatory, while others are inhibitory. In addition, the POPC used in the nanodiscs might not be the best substitute of the natural lipids that are found in the mitochondrial membrane. Given the variability that has been observed in ATPase activities for different solubilization conditions, we do not know which condition more likely reflects the true physiological activity. (As an aside, we would have thought that basal ATPase rate should be zero in the absence of transported substrate to avoid uncoupled (and presumably wasteful) ATPase activity, but this has been rarely reported).

    The Schaedler paper used GSH at two concentrations, 1.7 and 3.3 mM, to test the stimulation of ATPase activity. In our study, we used 10 mM GSSG for the ATPase activity assay to be consistent with our previous studies on NaAtm1. This discrepancy presumably reflects concentration differences utilized in these studies, and other experimental conditions including the nature of the protein construct. Different expression systems were used in the two studies, Schaedler et al employed L. lactis, while we used E. coli; it is possible that different lipids co-purified with the proteins that could influence the ATPase activity. In addition, we used a truncation with an 80-residue deletion, while Schaedler et al utilized a 60-residue truncation; our initial characterization of various constructs showed that the 80-residue deletion had a higher ATPase activity, although we did not characterize these differences in detail – certainly not for publication. While both studies used AtAtm3 solubilized in DDM, it is possible that there are still differences in the solubilization conditions reflecting the precise way in which detergents are introduced. We have added a sentence noting this discrepancy in the GSH stimulation results, without ascribing a particular mechanism.

    We have added the goodness of fit to the figure.

    1. There are a couple of structural studies on Atm transporters available to date, and the authors did a couple of overall structural comparisons in Figure S3 and S5. However, it is still not clear what exactly those systems are similar or dissimilar, and what are new structural insights gained with these structures. It could be helpful to compare the substrate-binding sites side by side or have a cartoon representation of different functional states in different systems. The authors brought this reader's attention to "a ~20 amino acid loop between TM1 and Tm2 of AtAtm3 that would be positioned in the intermembrane space and is absent from the structures of ScAtm1 and NaAtm1" without further explanation. Does this loop have any proposed functional roles? Is it present in other Atm or ABC transporters?

    We included a new figure, Figure S4, comparing different homologs, AtAtm3, human ABCB7 (an Alphafold model), human ABCB6, yeast Atm1 and prokaryotic Atm1. Among these five structures, only AtAtm3 presents a loop between TM1 and TM2. A quick protein sequence blast in PubMed showed the presence of this loop in many other plant atm transporters. This long loop has not been observed in other structure we know so far, but an external helix was observed for PglK, a lipid-linked oligosaccharide flippase, and this loop has been implicated in substrate interaction (Perez et al. Nature 524, 433 (2015); doi: 10.1038/nature14953).

  3. eLife

    Evaluation Summary:

    Oxygen consumption in mitochondria by the respiratory chain leads to a major source of reactive oxygen species, and mitochondrial glutathione is an important line of defence against free radical production. The ABC transporter Atm3 exports oxidized glutathione from the mitochondria to help maintain a suitable reducing environment. Here, the authors have determined structure of Atm3 in multiple conformations by single-particle cryo EM and have revealed new insights into local changes coupled to substrate export of oxidised glutathione and ABC exporters in general. The work will be of interest to the mitochondrial biology and transporter communities.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    Oxygen consumption in mitochondria by the respiratory chain leads to a major source of reactive oxygen species. Mitochondrial glutathione is an important line of defence against free radical production. The ABC transporter Atm3 exports oxidized glutathione from the mitochondria to help maintain a suitable reducing environment. Here, the authors have determined structure of Atm3 in multiple conformations by single-particle cryo EM and have revealed new insights into local changes coupled to substrate export. They conclude that a lack of cysteine residues in Atm3 is a feature to avoid mixed-disulfides that could impair export, and that substrate-bound complexes are only transiently occupied in the presence of ATP.

    Strengths: The structures of Atm3 are well resolved and the paper is very well written. The use of principle component analysis (PC) to assembly the different type IV exporters together with Atm3 is insightful and highlights that i) nucleotides are required for the stabilization of the closed, occluded, and outward-facing conformations and ii) transport substrates and related inhibitors have only been observed associated with inward-facing conformational states.

    Weaknesses: The cysteine-exclusion hypothesis of Atm3 is interesting, but lacks experimental validation. The described local changes by TM6 in Atm3 are appreciated, but the coupling with substrate binding needs to be better clarified.

  5. eLife

    Reviewer #2 (Public Review):

    The structural features are highly reminiscent of previous studies on the Atm subfamily of type I ABC exporters and type I ABC exporters in general, and therefore offer confirmation and reinforcement of current knowledge and not completely novel discoveries. Nevertheless, the in-depth analysis of the obtained data in light of ABC exporters (in general in particular those of the Atm subfamily) make this paper a very interesting read. The manuscript text as well as the figures are logical and easy to grasp. The paper contains two interesting learnings, namely that cysteines are not found close to the GSSG binding site within the Atm1-subfamily and that the closed conformation is not a unique discovery for NaAtm1. At the technical level, the work had been carefully executed (apart some open questions regarding the cryo-EM maps of the closed and outward-facing conformation).

  6. eLife

    Reviewer #3 (Public Review):

    The authors aim to understand the functional roles of Atm transporters, a family of ABC transporters with cellular localization of mitochondria and play important roles in transition metal homeostasis. To understand their functions, the author captured the structures of Atm3 from plant Arabidopsis in several functional states by cryo-EM--inward-facing, inward-facing with substrate GSSH bound, nucleotide bound closed state and nucleotide bound outward-facing states. Although many of those functional states have been reported for Atm orthologues in other species, the authors did elegant analyses on how the rareness of cysteine residues in the transport pathways could be very important for efficient glutathione transport. Moreover, the authors systematically show the unlikelihood of capturing an ABC transporter with both substrate and nucleotides bound. Although conceptually, a functional state with substrate and nucleotides bound should be very transient to avoid backflow of the substrate, it is wonderful that the authors put in the effort to capture such state and present a systematic principal component analysis (PCA) to show it may not be possible to attain such state structurally. The usage of PCA on analyzing a plethora of ABC transporters with different functional states could be applicable to other systems.
    The conclusions of this paper are mostly well supported by data, with some aspects of analysis and discrepancy that need to be addressed.

    1. In Figure 1, the authors report the ATPase activities of AtAtm3 are quite different in detergent and membrane environments, with a more than 10-fold decrease in basal activity from detergent to POPC containing nanodiscs. Is this composition a good representation of Arabidopsis mitochondria membrane? The nanodisc belts in Figure S4 looks quite tight. Could that contribute to the decrease in ATPase activity? In one of the papers you cited, Schaedler et. al, 2014, ATM3's activity is not stimulated by GSH versus in your data, 10mM GSH strongly stimulated AtAtm3. How would you explain the discrepancy? In terms of data analysis, the fit for 10mM GSH and 2.5mM GSSG ATPase activity in nanodiscs is poor visually, the addition of goodness of the fit in the figure could help the reader to assess the real quality of the data.

    2. There are a couple of structural studies on Atm transporters available to date, and the authors did a couple of overall structural comparisons in Figure S3 and S5. However, it is still not clear what exactly those systems are similar or dissimilar, and what are new structural insights gained with these structures. It could be helpful to compare the substrate-binding sites side by side or have a cartoon representation of different functional states in different systems. The authors brought this reader's attention to "a ~20 amino acid loop between TM1 and Tm2 of AtAtm3 that would be positioned in the intermembrane space and is absent from the structures of ScAtm1 and NaAtm1" without further explanation. Does this loop have any proposed functional roles? Is it present in other Atm or ABC transporters?

  7. Version published to 10.1101/2021.12.13.472443v1 on bioRxiv