Article | Published:

Observing cellulose biosynthesis and membrane translocation in crystallo

Nature volume 531, pages 329334 (17 March 2016) | Download Citation

Abstract

Many biopolymers, including polysaccharides, must be translocated across at least one membrane to reach their site of biological function. Cellulose is a linear glucose polymer synthesized and secreted by a membrane-integrated cellulose synthase. Here, in crystallo enzymology with the catalytically active bacterial cellulose synthase BcsA–BcsB complex reveals structural snapshots of a complete cellulose biosynthesis cycle, from substrate binding to polymer translocation. Substrate- and product-bound structures of BcsA provide the basis for substrate recognition and demonstrate the stepwise elongation of cellulose. Furthermore, the structural snapshots show that BcsA translocates cellulose via a ratcheting mechanism involving a ‘finger helix’ that contacts the polymer’s terminal glucose. Cooperating with BcsA’s gating loop, the finger helix moves ‘up’ and ‘down’ in response to substrate binding and polymer elongation, respectively, thereby pushing the elongated polymer into BcsA’s transmembrane channel. This mechanism is validated experimentally by tethering BcsA’s finger helix, which inhibits polymer translocation but not elongation.

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Acknowledgements

We thank T. Rapoport for critical comments on the manuscript and J. Acheson for advice on reducing BcsA–BcsB complexes in crystallo. Diffraction data were collected at the Argonne National Laboratory’s Advanced Photon Source (APS) beam lines 23-ID-D (GM/CA-), 22-ID (SER-) and 24-ID-C (NE-CAT). GM/CA@APS has been funded in whole or in part with Federal funds from the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006). The NE-CAT beam lines are funded by the National Institute of General Medical Sciences from the National Institutes of Health (P41 GM103403). The Pilatus 6M detector on 24-ID-C beam line is funded by a NIH-ORIP HEI grant (S10 RR029205). Data for this research was also in part collected at the APS SER-CAT beam line, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by ANL under Contract No. DE-AC02-06CH11357. J.L.W.M. is supported by a National Science Foundation Graduate Research Fellowship, Grant No. DGE-1315231. M.F. thanks the Austrian Science Fund (FWF) (J3293-B21) for an Erwin Schrödinger postdoctoral fellowship. This research was primarily supported by the National Institutes of Health, Grant 1R01GM101001, awarded to J.Z.; S.G.W. thanks the Natural Sciences and Engineering Research Council of Canada for financial support.

Author information

Author notes

    • Michael Fischer
    •  & Jamie Rich

    Present Addresses: Sandoz GmbH, Biochemiestrasse 10, A-6250 Kundl, Austria (M.F.); Kairos Therapeutics, 2405 Wesbrook Mall, Fourth Floor, Vancouver, British Columbia V6T 1Z3, Canada (J.R.).

    • Jacob L. W. Morgan
    •  & Joshua T. McNamara

    These authors contributed equally to this work.

Affiliations

  1. University of Virginia School of Medicine, Center for Membrane Biology, Molecular Physiology and Biological Physics, 480 Ray C. Hunt Drive, Charlottesville, Virginia 22908, USA

    • Jacob L. W. Morgan
    • , Joshua T. McNamara
    •  & Jochen Zimmer
  2. Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, British Columbia V6T 1Z1, Canada

    • Michael Fischer
    • , Jamie Rich
    • , Hong-Ming Chen
    •  & Stephen G. Withers

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Contributions

J.T.M. and J.L.W.M. purified and crystallized BcsA–BcsB and performed all crystal soaking experiments. J.T.M. cloned and analysed all BcsA cysteine mutants. J.T.M. and J.L.W.M. collected and processed diffraction data and built and refined the BcsA–BcsB models. M.F. synthesized the fluorinated and phosphonate UDP-Glc analogues and J.R. and H.-M.C. synthesized the UDP-thio-galactose analogues. J.T.M., J.L.W.M. and J.Z. analysed the data. J.Z. and J.L.W.M. wrote the paper and all authors edited the text.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Jochen Zimmer.

Structure factors and coordinates have been deposited at the Protein Data Bank under entry codes 5EJ1, 5EIY and 5EJZ.

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    Supplementary Information

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DOI

https://doi.org/10.1038/nature16966

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