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Structure of the MacAB–TolC ABC-type tripartite multidrug efflux pump

Nature Microbiology volume 2, Article number: 17070 (2017) Cite this article

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Abstract

The MacA–MacB–TolC assembly of Escherichia coli is a transmembrane machine that spans the cell envelope and actively extrudes substrates, including macrolide antibiotics and polypeptide virulence factors. These transport processes are energized by the ATPase MacB, a member of the ATP-binding cassette (ABC) superfamily. We present an electron cryo-microscopy structure of the ABC-type tripartite assembly at near-atomic resolution. A hexamer of the periplasmic protein MacA bridges between a TolC trimer in the outer membrane and a MacB dimer in the inner membrane, generating a quaternary structure with a central channel for substrate translocation. A gating ring found in MacA is proposed to act as a one-way valve in substrate transport. The MacB structure features an atypical transmembrane domain with a closely packed dimer interface and a periplasmic opening that is the likely portal for substrate entry from the periplasm, with subsequent displacement through an allosteric transport mechanism.

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Figure 1: A pseudo-atomic model for the MacAB–TolC pump.
Figure 2: Structure of MacB.
Figure 3: Interactions between MacA and MacB.

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Acknowledgements

This work was supported by the Wellcome Trust (to B.F.L.), the Human Frontiers Science Program (to B.F.L., H.W.v.V. and S.M.), a Marie Curie International Outgoing Fellowship (to A.W.P.F.), the UK Medical Research Council (MC_UP_A025_1013, to S.H.W.S.), a Wellcome Trust ISSF award (grant no. WT097818MF), the Scottish Universities’ Physics Alliance (to U.Z. and S.L.) and the MRC Mitochondrial Biology Unit (grant no. U105663141). A.N. is the recipient of a Herchel–Smith Scholarship. The authors thank J. Skepper for help using the EM microscope and the imaging facility at Cambridge University, H. Zhou for providing access to the electron microscope at the University of California at Los Angeles, J. Grimmett and T. Darling for support with high-performance computing at the MRC Laboratory of Molecular Biology, S. Rankin for help with computing on the High-Performance Computing System at the University of Cambridge, and L. Packman for mass spectrometry analyses. The authors thank the staff of the Diamond Light Source for access to the eBIC facility. The EM maps have been deposited in the Electron Microscopy Databank.

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Author notes

  1. Anthony W. P. Fitzpatrick and Dijun Du: These authors contributed equally to this work.

Authors and Affiliations

  1. Medical Research Council Laboratory of Molecular Biology, Cambridge Biomedical Campus, Francis Crick Avenue, Cambridge, CB2 0QH, UK

    Anthony W. P. Fitzpatrick, Xiao-Chen Bai & Sjors H. W. Scheres

  2. Computational Biology, School of Life Sciences, University of Dundee, Dow Street, Dundee, DD1 5EH, UK

    Salomé Llabrés & Ulrich Zachariae

  3. Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1PD, UK

    Arthur Neuberger & Hendrik W. van Veen

  4. MRC Mitochondrial Biology Unit, Cambridge Biomedical Campus, Hills Road, Cambridge, CB2 0XY, UK

    James N. Blaza

  5. Department of Life Science, Tokyo Institute of Technology, 4259-J2-17 Nagatsuta, Midori-ku, 226-8501, Yokohama, Japan

    Ui Okada & Satoshi Murakami

  6. Physics, School of Science and Engineering, University of Dundee, Nethergate, Dundee, DD1 4NH, UK

    Ulrich Zachariae

  7. Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1GA, UK

    Ben F. Luisi & Dijun Du

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Contributions

D.D., B.F.L. and S.H.W.S. designed the project. D.D. purified the fusion and disulfide-linkage-stabilized MacAB–TolC complexes. D.D., A.W.P.F., X.C.B. and J.N.B. obtained and analysed the single-particle cryo-EM data. U.O. and S.M. built the homology model of MacB. D.D. and B.F.L. devised a model of MacAB–TolC based on the cryo-EM map. A.N. and H.W.v.V. conducted MIC assays on the MacAB–TolC pump. S.L. and U.Z. carried out molecular dynamics simulations of MacA. D.D., B.F.L. and S.H.W.S. wrote the paper. All authors contributed to editing the manuscript.

Corresponding authors

Correspondence to Sjors H. W. Scheres, Ben F. Luisi or Dijun Du.

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The authors declare no competing financial interests.

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Fitzpatrick, A., Llabrés, S., Neuberger, A. et al. Structure of the MacAB–TolC ABC-type tripartite multidrug efflux pump. Nat Microbiol 2, 17070 (2017). https://doi.org/10.1038/nmicrobiol.2017.70

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