Letter | Published:

Wza the translocon for E. coli capsular polysaccharides defines a new class of membrane protein

Abstract

Many types of bacteria produce extracellular polysaccharides (EPSs). Some are secreted polymers and show only limited association with the cell surface, whereas others are firmly attached to the cell surface and form a discrete structural layer, the capsule, which envelopes the cell and allows the bacteria to evade or counteract the host immune system1. EPSs have critical roles in bacterial colonization of surfaces2, such as epithelia and medical implants; in addition some EPSs have important industrial and biomedical applications in their own right3. Here we describe the 2.26 Å resolution structure of the 340 kDa octamer of Wza, an integral outer membrane lipoprotein, which is essential for group 1 capsule export in Escherichia coli. The transmembrane region is a novel α-helical barrel. The bulk of the Wza structure is located in the periplasm and comprises three novel domains forming a large central cavity. Wza is open to the extracellular environment but closed to the periplasm. We propose a route and mechanism for translocation of the capsular polysaccharide. This work may provide insight into the export of other large polar molecules such as DNA and proteins.

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References

  1. 1

    Roberts, I. S. The biochemistry and genetics of capsular polysaccharide production in bacteria. Annu. Rev. Microbiol. 50, 285–315 (1996)

  2. 2

    Hall-Stoodley, L., Costerton, J. W. & Stoodley, P. Bacterial biofilms: from the natural environment to infectious diseases. Nature Rev. Microbiol. 2, 95–108 (2004)

  3. 3

    Sutherland, I. W. Novel and established applications of microbial polysaccharides. Trends Biotechnol. 16, 41–46 (1998)

  4. 4

    Whitfield, C. Biosynthesis and assembly of capsular polysaccharides in Escherichia coli.. Annu. Rev. Biochem. 75, 39–68 (2006)

  5. 5

    Drummelsmith, J. & Whitfield, C. Gene products required for surface expression of the capsular form of the group 1 K antigen in Escherichia coli (O9a:K30). Mol. Microbiol. 31, 1321–1332 (1999)

  6. 6

    Drummelsmith, J. & Whitfield, C. Translocation of group 1 capsular polysaccharide to the surface of Escherichia coli requires a multimeric complex in the outer membrane. EMBO J. 19, 57–66 (2000)

  7. 7

    Nesper, J. et al. Translocation of group 1 capsular polysaccharide in Escherichia coli serotype K30. Structural and functional analysis of the outer membrane lipoprotein Wza. J. Biol. Chem. 278, 49763–49772 (2003)

  8. 8

    Beis, K. et al. Three-dimensional structure of Wza, the protein required for translocation of group 1 capsular polysaccharide across the outer membrane of Escherichia coli.. J. Biol. Chem. 279, 28227–28232 (2004)

  9. 9

    Reid, A. N. & Whitfield, C. Functional analysis of conserved gene products involved in assembly of Escherichia coli capsules and exopolysaccharides: evidence for molecular recognition between Wza and Wzc for colanic acid biosynthesis. J. Bacteriol. 187, 5470–5481 (2005)

  10. 10

    Collins, R. F. et al. Periplasmic protein–protein contacts in the inner membrane protein Wzc form a tetrameric complex required for the assembly of Escherichia coli group 1 capsules. J. Biol. Chem. 281, 2144–2150 (2006)

  11. 11

    Wugeditsch, T. et al. Phosphorylation of Wzc, a tyrosine autokinase, is essential for assembly of group 1 capsular polysaccharides in Escherichia coli.. J. Biol. Chem. 276, 2361–2371 (2001)

  12. 12

    Paiment, A., Hocking, J. & Whitfield, C. Impact of phosphorylation of specific residues in the tyrosine autokinase, Wzc, on its activity in assembly of group 1 capsules in Escherichia coli.. J. Bacteriol. 184, 6437–6447 (2002)

  13. 13

    Xu, Z. H., Horwich, A. L. & Sigler, P. B. The crystal structure of the asymmetric GroEL–GroES–(ADP)(7) chaperonin complex. Nature 388, 741–750 (1997)

  14. 14

    Koronakis, V., Sharff, A., Koronakis, E., Luisi, B. & Hughes, C. Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export. Nature 405, 914–919 (2000)

  15. 15

    Tokuda, H. & Matsuyama, S. Sorting of lipoproteins to the outer membrane in E. coli. Biochim. Biophys. Acta. 1693, 5–13 (2004)

  16. 16

    Ruiz, N., Kahne, D. & Silhavy, T. J. Advances in understanding bacterial outer-membrane biogenesis. Nature Rev. Microbiol. 4, 57–66 (2006)

  17. 17

    Bulet, P., Stocklin, R. & Menin, L. Anti-microbial peptides: from invertebrates to vertebrates. Immunol. Rev. 198, 169–184 (2004)

  18. 18

    Cowan, S. W. et al. Crystal structures explain functional properties of two E. coli porins. Nature 358, 727–733 (1992)

  19. 19

    Ferguson, A. D., Hofmann, E., Coulton, J. W., Diederichs, K. & Welte, W. Siderophore-mediated iron transport: crystal structure of FhuA with bound lipopolysaccharide. Science 282, 2215–2220 (1998)

  20. 20

    Christie, P. J., Atmakuri, K., Krishnamoorthy, V., Jakubowski, S. & Cascales, E. Biogenesis, architecture, and function of bacterial type IV secretion systems. Annu. Rev. Microbiol. 59, 451–485 (2005)

  21. 21

    Linderoth, N. A., Simon, M. N. & Russel, M. The filamentous phage pIV multimer visualized by scanning transmission electron microscopy. Science 278, 1635–1638 (1997)

  22. 22

    Nouwen, N. et al. Secretin PulD: association with pilot PulS, structure, and ion-conducting channel formation. Proc. Natl Acad. Sci. USA 96, 8173–8177 (1999)

  23. 23

    Collins, R. F. et al. Structure of the Neisseria meningitidis outer membrane PilQ secretin complex at 12 Å resolution. J. Biol. Chem. 279, 39750–39756 (2004)

  24. 24

    Zhou, Y. F., Morais-Cabral, J. H., Kaufman, A. & MacKinnon, R. Chemistry of ion coordination and hydration revealed by a K+ channel-Fab complex at 2.0 Å resolution. Nature 414, 43–48 (2001)

  25. 25

    Khademi, S. et al. Mechanism of ammonia transport by Amt/MEP/Rh: structure of AmtB at 1.35 Å. Science 305, 1587–1594 (2004)

  26. 26

    Abramson, J. et al. Structure and mechanism of the lactose permease of Escherichia coli.. Science 301, 610–615 (2003)

  27. 27

    Reyes, C. L. & Chang, G. Structure of the ABC transporter MsbA in complex with ADP-vanadate and lipopolysaccharide. Science 308, 1028–1031 (2005)

  28. 28

    Moothoo, D. N. & Naismith, J. H. Concanavalin A distorts the β-GlcNAc-(1→2)-Man linkage of β-GlcNAc-(1→2)-α-Man-(1→3)-[β-GLcNAc-(1→2)-α-Man-(1→6)]-Man upon binding. Glycobiology 8, 173–181 (1998)

  29. 29

    Rahn, A., Drummelsmith, J. & Whitfield, C. Conserved organization in the cps gene clusters for expression of Escherichia coli group 1 K antigens: Relationship to the colanic acid biosynthesis locus and the cps genes from Klebsiella pneumoniae.. J. Bacteriol. 181, 2307–2313 (1999)

  30. 30

    Beis, K., Nesper, J., Whitfield, C. & Naismith, J. H. Crystallization and preliminary X-ray diffraction analysis of Wza outer-membrane lipoprotein from Escherichia coli serotype O9a:K30. Acta Crystallogr. D. 60, 558–560 (2004)

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Acknowledgements

J.H.N. is a Biotechnology and Biology Sciences Research Council (BBSRC) Career Development Fellow, and acknowledges funding from a Wellcome Trust programme grant for biological aspects of the research. C.W. holds a Canada Research Chair and acknowledges funding from the Canadian Institutes of Health Research. The experimental structural biology was performed by the Scottish Structural Proteomics Facility, which is funded by the Scottish Higher Education Funding Council and the BBSRC. We acknowledge the use of ESRF beamlines and are grateful for assistance from D. Bourgeois and G. Leonard with data collection. We thank L. Cuthbertson for assistance with bioinformatic analyses, R. Clarke for fluorescence-activated cell sorting, and G. Taylor, M. White and B. Hunter for a critical review of the manuscript.

Author Contributions C.D. optimized crystals, collected data, and solved, refined and analysed the Wza structure. K.B. and J.N. grew the first crystals. K.B. devised and refined the SeMet protocol. A.L.B.-L. and B.R.C. made and analysed the site-directed mutants and Wza–Flag fusion, respectively. C.W. oversaw the project, analysed the data and wrote the paper. J.H.N. oversaw the project, collected X-ray data, solved and refined the structure, analysed the data and wrote the paper.

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Competing interests

Coordinates and data are available from the Worldwide Protein Data Bank, accession code 2j58. Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.

Correspondence to James H. Naismith.

Supplementary information

Supplementary Notes

This file contains Supplementary Methods, Supplementary Figures 1–7 and additional references. The Supplementary Methods detail the molecular biology and crystallography. The Supplementary Figures give different views of the structure including experimental density in wall eye stereo. Extra experimental data is also shown in the Supplementary Figures. (PDF 4670 kb)

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Further reading

Figure 1: Group 1 capsular polysaccharide export in Gram-negative bacteria.
Figure 2: The structure of Wza.
Figure 3: The surfaces of Wza.

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