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Molecular basis of lipid-linked oligosaccharide recognition and processing by bacterial oligosaccharyltransferase

Nature Structural & Molecular Biology volume 24, pages 11001106 (2017) | Download Citation

Abstract

Oligosaccharyltransferase (OST) is a membrane-integral enzyme that catalyzes the transfer of glycans from lipid-linked oligosaccharides (LLOs) onto asparagine side chains, the first step in protein N-glycosylation. Here, we report the X-ray structure of a single-subunit OST, PglB from Campylobacter lari, trapped in an intermediate state bound to an acceptor peptide and a synthetic LLO analog. The structure reveals the role of the external loop EL5, present in all OST enzymes, in substrate recognition. Whereas the N-terminal half of EL5 binds LLO, the C-terminal half interacts with the acceptor peptide. The glycan moiety of LLO must thread under EL5 to access the active site. Reducing EL5 mobility decreases the catalytic rate of OST when full-size heptasaccharide LLO is provided, but not for a monosaccharide-containing LLO analog. Our results define the chemistry of a ternary complex state, assign functional roles to conserved OST motifs, and provide opportunities for glycoengineering by rational design of PglB.

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Acknowledgements

This research was supported by the Swiss National Science Foundation (SNF 310030B_166672 to K.P.L. and Transglyco Sinergia grant to M.A., J.-L.R. and K.P.L.). We thank the beamline staff at the Swiss Light Source for assistance with data collection, C. Lizak for preparing a cysteineless construct of PglB, and A. Ramírez for assistance with the chemo-enzymatic synthesis of farnesyl-PP-GlcNAc-1,3α-GalNAc.

Author information

Author notes

    • Tina Sovdat

    Present address: Rolic Technologies Ltd., Allschwil, Switzerland.

Affiliations

  1. Institute of Molecular Biology and Biophysics, ETH Zurich, Zurich, Switzerland.

    • Maja Napiórkowska
    •  & Kaspar P Locher
  2. Department of Chemistry and Biochemistry, University of Berne, Berne, Switzerland.

    • Jérémy Boilevin
    • , Tina Sovdat
    • , Tamis Darbre
    •  & Jean-Louis Reymond
  3. Institute of Microbiology, ETH Zurich, Zurich, Switzerland.

    • Markus Aebi

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Contributions

M.N. performed the overexpression, purification, disulfide cross-linking, functional characterization, and crystallization of PglB. K.P.L. and M.N. performed X-ray data collection, structure determination, and model building and refinement. J.B., T.S., T.D., and J.-L.R. synthesized LLO analogs. M.N., M.A., and K.P.L. devised experiments and analyzed the data. K.P.L. and M.N. wrote the manuscript with input from all authors.

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/nsmb.3491

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