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Structure and mechanism of an active lipid-linked oligosaccharide flippase

Nature volume 524, pages 433438 (27 August 2015) | Download Citation

Abstract

The flipping of membrane-embedded lipids containing large, polar head groups is slow and energetically unfavourable, and is therefore catalysed by flippases, the mechanisms of which are unknown. A prominent example of a flipping reaction is the translocation of lipid-linked oligosaccharides that serve as donors in N-linked protein glycosylation. In Campylobacter jejuni, this process is catalysed by the ABC transporter PglK. Here we present a mechanism of PglK-catalysed lipid-linked oligosaccharide flipping based on crystal structures in distinct states, a newly devised in vitro flipping assay, and in vivo studies. PglK can adopt inward- and outward-facing conformations in vitro, but only outward-facing states are required for flipping. While the pyrophosphate-oligosaccharide head group of lipid-linked oligosaccharides enters the translocation cavity and interacts with positively charged side chains, the lipidic polyprenyl tail binds and activates the transporter but remains exposed to the lipid bilayer during the reaction. The proposed mechanism is distinct from the classical alternating-access model applied to other transporters.

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Primary accessions

Data deposits

Atomic coordinates and structure factors were deposited with the RCSB Protein Data Bank (PDB) under accessions 5C78 (apo-inward-1), 5C76 (apo-inward-2) and 5C73 (outward-occluded).

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Acknowledgements

We thank the staff scientists at the PX beamline of the Swiss Light Source for help with data collection, and M. Napiorkowska and A.Ramirez for assistance with PglB assays. This work was supported by the Swiss National Science Foundation (SNF 31003A–146191 to K.P.L. and Transglyco Sinergia program to M.A., J.-L.R. and K.P.L.). C.P. acknowledges support from the ETH postdoctoral fellowship program.

Author information

Author notes

    • Sabina Gerber

    Present address: GlycoVaxyn AG, Grabenstrasse 3, 8952 Schlieren, Switzerland.

Affiliations

  1. Institute of Molecular Biology and Biophysics, ETH Zürich, CH-8093 Zürich, Switzerland

    • Camilo Perez
    • , Sabina Gerber
    • , Monika Bucher
    •  & Kaspar P. Locher
  2. Department of Chemistry and Biochemistry, University of Berne, CH-3012 Berne, Switzerland

    • Jérémy Boilevin
    • , Tamis Darbre
    •  & Jean-Louis Reymond
  3. Institute of Microbiology, ETH Zürich, CH-8093 Zürich, Switzerland

    • Markus Aebi

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Contributions

C.P. determined the structures of PglK, established the in vitro flipping assay, and performed in vivo flipping studies. S.G. crystallized PglK in the apo-inward-2 state, M.B. assisted in expression and purification of PglK. J.B., T.D. and J.-L.R. synthesized LLO analogues. K.P.L., S.G. and C.P. conceived the project. K.P.L., M.A., and C.P. analysed the data. K.P.L. and C.P. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Kaspar P. Locher.

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https://doi.org/10.1038/nature14953

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