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Bacterial polysaccharide co-polymerases share a common framework for control of polymer length

Nature Structural & Molecular Biology volume 15, pages 130–138 (2008)Cite this article

Abstract

The chain length distribution of complex polysaccharides present on the bacterial surface is determined by polysaccharide co-polymerases (PCPs) anchored in the inner membrane. We report crystal structures of the periplasmic domains of three PCPs that impart substantially different chain length distributions to surface polysaccharides. Despite very low sequence similarities, they have a common protomer structure with a long central α-helix extending 100 Å into the periplasm. The protomers self-assemble into bell-shaped oligomers of variable sizes, with a large internal cavity. Electron microscopy shows that one of the full-length PCPs has a similar organization as that observed in the crystal for its periplasmic domain alone. Functional studies suggest that the top of the PCP oligomers is an important region for determining polysaccharide modal length. These structures provide a detailed view of components of the bacterial polysaccharide assembly machinery.

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Figure 1: Structures of the periplasmic regions of FepE, WzzE and WzzB(ST).
Figure 2: Stereoview of the clusters of conserved residues.
Figure 3: The PCP oligomers.
Figure 4: Electron microscopy model of full-length WzzE in negative stain.
Figure 5: Mutations in FepE and Wzz and their effects on Oag modal length.
Figure 6: Stereo view of molecular surface of FepE with locations of mutations marked in different colors.

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Acknowledgements

We thank J.D. Schrag, M. Whiteway, J. Paton and T. Vernet for comments on the manuscript, S. Gruenheid (McGill University, Montréal) for E. coli O157:H7 EDL933 genomic DNA and L. Flaks, A. Soares and H. Robinson for assistance in synchrotron X-ray data collection. Data for this study were measured at beamlines X8C, X12B and X29 of the National Synchrotron Light Source. Financial support comes principally from the Offices of Biological and Environmental Research and Basic Energy Sciences of the US Department of Energy and from the National Center for Research Resources of the US National Institutes of Health. Use of the SGX Collaborative Access Team (SGX-CAT) beamline facilities at Sector 31 of the Advanced Photon Source was provided by SGX Pharmaceuticals, Inc., who constructed and operate the facility. Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357. This work was supported in part by the National Research Council of Canada and the Canadian Institutes of Health Research (CIHR) grant MOP-48370 (M.C.) and by an Australian National Health and Medical Research Council Program Grant (R.M.).

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Authors and Affiliations

  1. Department of Biochemistry, McGill University, 3655 Promenade Sir William Osler, Montréal, H3G 1Y6, Québec, Canada

    Ante Tocilj, Christine Munger, Ariane Proteau, Eunice Ajamian & Miroslaw Cygler

  2. Australian Bacterial Pathogenesis Program, School of Molecular and Biomedical Science, Discipline of Microbiology and Immunology, University of Adelaide, North Terrace, Adelaide, 5005, South Australia, Australia

    Renato Morona, Leanne Purins, Magdalene Papadopoulos & Luisa Van Den Bosch

  3. Biotechnology Research Institute, National Research Council, 6100 Royalmount Avenue, Montréal, H4P 2R2, Québec, Canada

    John Wagner, Allan Matte & Miroslaw Cygler

  4. Molecular Structure and Function Program, the Hospital for Sick Children, 555 University Avenue, Toronto, M5G 1X8, Ontario, Canada

    John L Rubinstein

  5. Institut de Recherche en Immunologie et Cancerologie and Department of Pharmacology, Pavillon Marcelle Coutu, University de Montréal, 2900 Edouard-Montpetit, Montréal, H3T 1J4, Québec, Canada

    James Féthière

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Contributions

A.T. determined and refined crystal structures of FepE, WzzE and WzzB, purified full-length WzzE; J.W. cloned the constructs; C.M., A.P. and E.A. purified and crystallized the PCPs, WzzE; J.F. analyzed oligomerization states and purified full-length FepE; A.M. collected data for all the crystals and contributed to writing of the manuscript; L.P. constructed and characterized FepE(O157) and WzzpHS2 mutants; M.P. constructed and characterized WzzB(SF) mutants; L.V.D.B. prepared antiserum to FepE, isolated and characterized WzzB(ST) mutants; J.L.R. performed EM experiments and data analysis; M.C. and R.M. planned experiments, analyzed data and contributed to writing of the manuscript.

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Correspondence to Miroslaw Cygler.

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Tocilj, A., Munger, C., Proteau, A. et al. Bacterial polysaccharide co-polymerases share a common framework for control of polymer length. Nat Struct Mol Biol 15, 130–138 (2008). https://doi.org/10.1038/nsmb.1374

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