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Structural basis of MsbA-mediated lipopolysaccharide transport

Nature volume 549, pages 233237 (14 September 2017) | Download Citation

Abstract

Lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria is critical for the assembly of their cell envelopes. LPS synthesized in the cytoplasmic leaflet of the inner membrane is flipped to the periplasmic leaflet by MsbA, an ATP-binding cassette transporter. Despite substantial efforts, the structural mechanisms underlying MsbA-driven LPS flipping remain elusive. Here we use single-particle cryo-electron microscopy to elucidate the structures of lipid-nanodisc-embedded MsbA in three functional states. The 4.2 Å-resolution structure of the transmembrane domains of nucleotide-free MsbA reveals that LPS binds deep inside MsbA at the height of the periplasmic leaflet, establishing extensive hydrophilic and hydrophobic interactions with MsbA. Two sub-nanometre-resolution structures of MsbA with ADP-vanadate and ADP reveal an unprecedented closed and an inward-facing conformation, respectively. Our study uncovers the structural basis for LPS recognition, delineates the conformational transitions of MsbA to flip LPS, and paves the way for structural characterization of other lipid flippases.

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Acknowledgements

We thank G. Chang for providing the MsbA expression plasmid. We are grateful to Z. Li and M. Chambers for EM technical support. We thank D. Kahne for making us aware of ClearColi. We thank W. Harper, T. Rapoport and T. Walther for critical reading of the manuscript. We thank members of the Liao group for helpful discussions and comments on the manuscript, and members of the Walz group for help in the initial phases of the project.

Author information

Affiliations

  1. Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Wei Mi
    •  & Maofu Liao
  2. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China

    • Yanyan Li
  3. Department of Microbial Pathogenesis, School of Dentistry, University of Maryland, Baltimore, Maryland 21201, USA

    • Sung Hwan Yoon
    •  & Robert K. Ernst
  4. Laboratory of Molecular Electron Microscopy, The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA

    • Thomas Walz

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Contributions

M.L. conceived the project. T.W. advised on experimental design. W.M. purified MsbA, performed nanodisc and proteoliposome reconstitution, generated MsbA mutants, measured ATPase activities, carried out EM data collection and image processing, and built the atomic models. Y.L., S.H.Y. and R.K.E. performed the mass spectrometry measurement and analysis presented in Extended Data Fig. 2g–j. W.M. and M.L. analysed the data and wrote the manuscript with contributions from T.W.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Maofu Liao.

Reviewer Information Nature thanks D. Levy, D. Slotboom and H. W. van Veen for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Figure 1

    This file contains the uncropped scans with size marker indications

  2. 2.

    Reporting Summary

Videos

  1. 1.

    Proposed mechanism of MsbA-mediated LPS transport

    The video shows a morph between the conformations depicted in Figure 5. The MsbA structures in states 2, 5 and 6 are based on the cryo-EM structures of MsbA in the nucleotide-free, vanadate-trapped and ADP-bound states, respectively. The MsbA structures in states 1, 3 and 4 are based on the crystal structures of inward-facing conformation (3B5X), occluded conformation (4PL0), and outward-facing conformation (3B60), respectively. The boundaries of the lipid membrane are indicated with dashed lines. While the movie shows LPS flipping, ATP hydrolysis and the conformational transition of MsbA as separate steps, these changes likely occur as a concerted process.

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DOI

https://doi.org/10.1038/nature23649

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