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A structural framework for unidirectional transport by a bacterial ABC exporter

Fan, Chengcheng and Kaiser, Jens T. and Rees, Douglas C. (2020) A structural framework for unidirectional transport by a bacterial ABC exporter. Proceedings of the National Academy of Sciences of the United States of America, 117 (32). pp. 19228-19236. ISSN 0027-8424. PMCID PMC7430982. https://resolver.caltech.edu/CaltechAUTHORS:20200724-090008939

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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.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1073/pnas.2006526117DOIArticle
https://www.pnas.org/content/suppl/2020/07/23/2006526117.DCSupplementalPublisherSupporting Information
http://www.ncbi.nlm.nih.gov/pmc/articles/pmc7430982/PubMed CentralArticle
ORCID:
AuthorORCID
Fan, Chengcheng0000-0003-4213-5758
Kaiser, Jens T.0000-0002-5948-5212
Rees, Douglas C.0000-0003-4073-1185
Additional Information:© 2020 the Author(s). Published by PNAS. This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND). Contributed by Douglas C. Rees, May 28, 2020 (sent for review April 7, 2020; reviewed by Susan K. Buchanan and Dirk-Jan Slotboom). PNAS first published July 23, 2020. We thank the staffs of the Stanford Synchrotron Radiation Lightsource (SSRL) beamline 12-2 and of the Advanced Photon Source GM/CA beamline for support during X-ray diffraction data collection; Paul Adams, Kaspar Locher, Oded Lewinson, Gabriele Meloni, William Clemons, the organizers and speakers at the Cold Spring Harbor X-ray Method in Structural Biology Course (2018), the CCP4/APS School for Macromolecular Crystallography (2017), and the SBGrid/NE-CAT Phenix Workshop (2016) for discussions; Haoqing Wang, Andrey Malyutin, Songye Chen, and Megan Meyer for their support during single-particle cryo-EM data collection; and the Gordon and Betty Moore Foundation and the Beckman Institute for their generous support of the Molecular Observatory at Caltech. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the US Department of Energy (DOE), Office of Science, Office of Basic Energy Sciences, under contract no. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE of Biological and Environmental Research and by the NIH National Institute of General Medical Sciences (NIGMS) (Grant P41GM103393). GM/CA@APS was funded in whole or in part by federal funds from the National Cancer Institute (Grant ACB-12002) and the NIGMS (Grant AGM-12006). This research used resources of the Advanced Photon Source, a DOE Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. The Eiger 16M detector was funded by NIH Office of Research Infrastructure Programs High-End Instrumentation Grant 1S10OD012289-01A1. The cryo-EM was performed in the Beckman Institute Resource Center for Transmission Electron Microscopy at Caltech and at the Stanford SLAC Cryo-EM Center (S2C2). The S2C2 is supported by the NIH Health Common Fund Transformative High Resolution Cryo-Electron Microscopy program. Author contributions: C.F. and D.C.R. designed research; C.F. performed research; C.F., J.T.K., and D.C.R. analyzed data; and C.F. and D.C.R. wrote the paper. Reviewers: S.K.B., National Institutes of Health; and D.-J.S., University of Groningen. The authors declare no competing interest. Data deposition: Data for this article have been deposited in the RCSB Protein Data Bank under IDs 6PAM, 6PAN, 6PAO, 6PAQ, 6PAR, 6VQT, and 6VQU. The two cryo-EM single-particle structures in nanodiscs have also been deposited in the Electron Microscopy Data Bank under accession codes EMD-21356 (NaAtm1-MgADPVO4) and EMD-21357 (NaAtm1). This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2006526117/-/DCSupplemental.
Funders:
Funding AgencyGrant Number
Department of Energy (DOE)DE-AC02-76SF00515
NIHP41GM103393
NIHACB-12002
National Cancer InstituteUNSPECIFIED
NIHAGM-12006
Department of Energy (DOE)DE-AC02-06CH11357
NIH1S10OD012289-01A1
Subject Keywords:ATP-binding cassette transporters; ABC transporters; alternating access mechanism; glutathione transport; transport cycle
Issue or Number:32
PubMed Central ID:PMC7430982
Record Number:CaltechAUTHORS:20200724-090008939
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20200724-090008939
Official Citation:A structural framework for unidirectional transport by a bacterial ABC exporter. Chengcheng Fan, Jens T. Kaiser, Douglas C. Rees. Proceedings of the National Academy of Sciences Aug 2020, 117 (32) 19228-19236; DOI: 10.1073/pnas.2006526117
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:104556
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:24 Jul 2020 16:46
Last Modified:10 Sep 2020 21:30

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