Letter | Published:

Subnanometre-resolution electron cryomicroscopy structure of a heterodimeric ABC exporter

Nature volume 517, pages 396400 (15 January 2015) | Download Citation

Abstract

ATP-binding cassette (ABC) transporters translocate substrates across cell membranes, using energy harnessed from ATP binding and hydrolysis at their nucleotide-binding domains1,2. ABC exporters are present both in prokaryotes and eukaryotes, with examples implicated in multidrug resistance of pathogens and cancer cells, as well as in many human diseases3,4. TmrAB is a heterodimeric ABC exporter from the thermophilic Gram-negative eubacterium Thermus thermophilus; it is homologous to various multidrug transporters and contains one degenerate site with a non-catalytic residue next to the Walker B motif5. Here we report a subnanometre-resolution structure of detergent-solubilized TmrAB in a nucleotide-free, inward-facing conformation by single-particle electron cryomicroscopy. The reconstructions clearly resolve characteristic features of ABC transporters, including helices in the transmembrane domain and nucleotide-binding domains. A cavity in the transmembrane domain is accessible laterally from the cytoplasmic side of the membrane as well as from the cytoplasm, indicating that the transporter lies in an inward-facing open conformation. The two nucleotide-binding domains remain in contact via their carboxy-terminal helices. Furthermore, comparison between our structure and the crystal structures of other ABC transporters suggests a possible trajectory of conformational changes that involves a sliding and rotating motion between the two nucleotide-binding domains during the transition from the inward-facing to outward-facing conformations.

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Accessions

Primary accessions

Electron Microscopy Data Bank

Data deposits

All three-dimensional cryo-EM density maps have been deposited in the Electron Microscopy Data Bank under accession numbers EMD-6085 (TmrAB–AH5), EMD-6086 (TmrAB–BA6) and EMD-6087 (TmrAB).

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Acknowledgements

This work was supported by grants from the National Institutes of Health (R01GM098672, S10RR026814 and P50GM082250 to Y.C., 1P41CA196276-01 to C.S.C., P50GM073210 to R.M.S. and C.S.C., and R37GM024485 to R.M.S.), the University of California San Francisco Program for Breakthrough Biomedical Research (to Y.C.), and the German Research Foundation (SFB 807, SFB 902 and TA157/7 to R.T.) as well as the European Drug Initiative on Channels and Transporters (EDICT to R.T.) funded by the European Commission Seventh Framework Program.

Author information

Author notes

    • JungMin Kim
    • , Shenping Wu
    •  & Thomas M. Tomasiak

    These authors contributed equally to this work.

Affiliations

  1. Department of Pharmaceutical Chemistry, University of California San Francisco, 600 16th Street, San Francisco, California 94158, USA

    • JungMin Kim
    • , Michael B. Winter
    • , Robert M. Stroud
    •  & Charles S. Craik
  2. Department of Biochemistry and Biophysics, University of California San Francisco, 600 16th Street, San Francisco, California 94158, USA

    • Shenping Wu
    • , Thomas M. Tomasiak
    • , Yaneth Robles-Colmanares
    • , Robert M. Stroud
    •  & Yifan Cheng
  3. Institute of Biochemistry, Biocenter, Goethe-University Frankfurt, Max-von-Laue-Strasse 9, D-60438 Frankfurt am Main, Germany

    • Claudia Mergel
    • , Sebastian B. Stiller
    •  & Robert Tampé
  4. Cluster of Excellence – Macromolecular Complexes, Goethe-University Frankfurt, Max-von-Laue-Strasse 9, D-60438 Frankfurt am Main, Germany

    • Robert Tampé

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Contributions

J.K. identified, expressed, purified and characterized all Fabs used in this study, and generated TmrAB–Fab complexes. S.W. performed all cryo-EM experiments, including data acquisition and processing. T.M.T. and C.M. expressed and purified TmrAB, and purified TmrAB–Fab complexes. T.M.T. performed cross-linking experiments. C.M. expressed and purified TmrAB for the generation and initial screening of all Fabs. S.B.S. performed initial characterization of all Fabs. M.B.W. performed high-performance liquid chromatography (HPLC) experiments. Y.R.-C. performed mutagenesis experiments. J.K., S.W., T.M.T. and Y.C. analysed data. J.K., S.W., T.M.T., M.B.W., R.M.S., R.T., C.S.C. and Y.C. participated in discussion and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Robert M. Stroud or Robert Tampé or Charles S. Craik or Yifan Cheng.

Extended data

Supplementary information

Videos

  1. 1.

    3D reconstruction of TmrAB-AH5 complex at a resolution of 8.2Å

    3D reconstruction of TmrAB-AH5 complex at a resolution of 8.2Å.

  2. 2.

    Slicing through TMDs of TmrAB, showing micelle density and the separation of TM helices

    Slicing through TMDs of TmrAB, showing micelle density and the separation of TM helices.

  3. 3.

    Conformational change from apo (TmrAB) through TM287/288 to Sav1866.

    Morph video shows a conformational change from apo (TmrAB) through TM287/288 to Sav1866. In this orientation, closing of the lateral gate is shown.

  4. 4.

    Conformational change from a different orientation

    Morph video shows the same conformational change from a different orientation to Supplementary video 3. Transition from inward-facing to outward-facing conformations is shown.

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DOI

https://doi.org/10.1038/nature13872

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