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A structural framework for unidirectional transport by a bacterial ABC exporter.

ABSTRACT: The ATP-binding cassette (ABC) transporter of mitochondria (Atm1) mediates iron homeostasis in eukaryotes, while the prokaryotic homolog from Novosphingobium aromaticivorans (NaAtm1) can export glutathione derivatives and confer protection against heavy-metal toxicity. To establish the structural framework underlying the NaAtm1 transport mechanism, we determined eight structures by X-ray crystallography and single-particle cryo-electron microscopy in distinct conformational states, stabilized by individual disulfide crosslinks and nucleotides. As NaAtm1 progresses through the transport cycle, conformational changes in transmembrane helix 6 (TM6) alter the glutathione-binding site and the associated substrate-binding cavity. Significantly, kinking of TM6 in the post-ATP hydrolysis state stabilized by MgADPVO4 eliminates this cavity, precluding uptake of glutathione derivatives. The presence of this cavity during the transition from the inward-facing to outward-facing conformational states, and its absence in the reverse direction, thereby provide an elegant and conceptually simple mechanism for enforcing the export directionality of transport by NaAtm1. One of the disulfide crosslinked NaAtm1 variants characterized in this work retains significant glutathione transport activity, suggesting that ATP hydrolysis and substrate transport by Atm1 may involve a limited set of conformational states with minimal separation of the nucleotide-binding domains in the inward-facing conformation.

SUBMITTER: Fan C

PROVIDER: S-EPMC7430982 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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A structural framework for unidirectional transport by a bacterial ABC exporter.

Fan Chengcheng C Kaiser Jens T JT Rees Douglas C DC

Proceedings of the National Academy of Sciences of the United States of America 20200723 32

The ATP-binding cassette (ABC) transporter of mitochondria (Atm1) mediates iron homeostasis in eukaryotes, while the prokaryotic homolog from Novosphingobium aromaticivorans (NaAtm1) can export glutathione derivatives and confer protection against heavy-metal toxicity. To establish the structural framework underlying the NaAtm1 transport mechanism, we determined eight structures by X-ray crystallography and single-particle cryo-electron microscopy in distinct conformational ...[more]

PMID: 32703810

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Project description:Cryo-electron microscopy (cryo-EM) has the capacity to capture molecular machines in action1-3. ATP-binding cassette (ABC) exporters are highly dynamic membrane proteins that extrude a wide range of substances from the cytosol4-6 and thereby contribute to essential cellular processes, adaptive immunity and multidrug resistance7,8. Despite their importance, the coupling of nucleotide binding, hydrolysis and release to the conformational dynamics of these proteins remains poorly resolved, especially for heterodimeric and/or asymmetric ABC exporters that are abundant in humans. Here we present eight high-resolution cryo-EM structures that delineate the full functional cycle of an asymmetric ABC exporter in a lipid environment. Cryo-EM analysis under active turnover conditions reveals distinct inward-facing (IF) conformations-one of them with a bound peptide substrate-and previously undescribed asymmetric post-hydrolysis states with dimerized nucleotide-binding domains and a closed extracellular gate. By decreasing the rate of ATP hydrolysis, we could capture an outward-facing (OF) open conformation-an otherwise transient state vulnerable to substrate re-entry. The ATP-bound pre-hydrolysis and vanadate-trapped states are conformationally equivalent; both comprise co-existing OF conformations with open and closed extracellular gates. By contrast, the post-hydrolysis states from the turnover experiment exhibit asymmetric ATP and ADP occlusion after phosphate release from the canonical site and display a progressive separation of the nucleotide-binding domains and unlocking of the intracellular gate. Our findings reveal that phosphate release, not ATP hydrolysis, triggers the return of the exporter to the IF conformation. By mapping the conformational landscape during active turnover, aided by mutational and chemical modulation of kinetic rates to trap the key intermediates, we resolved fundamental steps of the substrate translocation cycle of asymmetric ABC transporters.

Dataset Information

A structural framework for unidirectional transport by a bacterial ABC exporter.

Publications

A structural framework for unidirectional transport by a bacterial ABC exporter.

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