Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Crystal structure of the polysialic acid–degrading endosialidase of bacteriophage K1F

Nature Structural & Molecular Biology volume 12, pages 90–96 (2005)Cite this article

Abstract

Phages infecting the polysialic acid (polySia)-encapsulated human pathogen Escherichia coli K1 are equipped with capsule-degrading tailspikes known as endosialidases, which are the only identified enzymes that specifically degrade polySia. As polySia also promotes cellular plasticity and tumor metastasis in vertebrates, endosialidases are widely applied in polySia-related neurosciences and cancer research. Here we report the crystal structures of endosialidase NF and its complex with oligomeric sialic acid. The structure NF, which reveals three distinct domains, indicates that the unique polySia specificity evolved from a combination of structural elements characteristic of exosialidases and bacteriophage tailspike proteins. The endosialidase assembles into a catalytic trimer stabilized by a triple β-helix. Its active site differs markedly from that of exosialidases, indicating an endosialidase-specific substrate-binding mode and catalytic mechanism. Residues essential for endosialidase activity were identified by structure-based mutational analysis.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Structure of endoNF (residues 245–911).
Figure 2: The active site of endoNF in comparison to conserved active site features of exosialidases.
Figure 3: Expression, complex formation and catalytic activity of active site mutants of endoNF.
Figure 4: Details of the two identified sialic acid–binding sites of endoNF.
Figure 5: Surface representation of the endoNF homotrimer in complex with sialic acid.

Similar content being viewed by others

Accession codes

Accessions

GenBank/EMBL/DDBJ

Protein Data Bank

References

  1. Kleene, R. & Schachner, M. Glycans and neural cell interactions. Nat. Rev. Neurosci. 5, 195–208 (2004).

    Article  CAS  Google Scholar 

  2. Jodar, L., Feavers, I.M., Salisbury, D. & Granoff, D.M. Development of vaccines against meningococcal disease. Lancet 359, 1499–1508 (2002).

    Article  CAS  Google Scholar 

  3. Merril, C.R., Scholl, D. & Adhya, S.L. The prospect for bacteriophage therapy in Western medicine. Nat. Rev. Drug Discov. 2, 489–497 (2003).

    Article  CAS  Google Scholar 

  4. Gross, R.J., Cheasty, T. & Rowe, B. Isolation of bacteriophages specific for the K1 polysaccharide antigen of Escherichia coli. J. Clin. Microbiol. 6, 548–550 (1977).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Smith, H.W. & Huggins, M.B. Successful treatment of experimental Escherichia coli infections in mice using phage: its general superiority over antibiotics. J. Gen. Microbiol. 128, 307–318 (1982).

    CAS  Google Scholar 

  6. Vimr, E.R., McCoy, R.D., Vollger, H.F., Wilkison, N.C. & Troy, F.A. Use of prokaryotic-derived probes to identify poly(sialic acid) in neonatal neuronal membranes. Proc. Natl. Acad. Sci. USA 81, 1971–1975 (1984).

    Article  CAS  Google Scholar 

  7. Miyake, K. et al. Screening of bacteriophages producing endo-N-acetylneuraminidase. J. Ferm. Bioeng. 84, 90–93 (1997).

    Article  CAS  Google Scholar 

  8. Pelkonen, S., Aalto, J. & Finne, J. Differential activities of bacteriophage depoly-merase on bacterial polysaccharide: binding is essential but degradation is inhibitory in phage infection of K1-defective Escherichia coli. J. Bacteriol. 174, 7757–7761 (1992).

    Article  CAS  Google Scholar 

  9. Petter, J.G. & Vimr, E.R. Complete nucleotide sequence of the bacteriophage K1F tail gene encoding endo-N-acylneuraminidase (endo-N) and comparison to an endo-N homolog in bacteriophage PK1E. J. Bacteriol. 175, 4354–4363 (1993).

    Article  CAS  Google Scholar 

  10. Mühlenhoff, M., Stummeyer, K., Grove, M., Sauerborn, M. & Gerardy-Schahn, R. Proteolytic processing and oligomerization of bacteriophage-derived endosialidases. J. Biol. Chem. 278, 12634–12644 (2003).

    Article  Google Scholar 

  11. Gerardy-Schahn, R. et al. Molecular cloning and functional expression of bacteriophage PK1E- encoded endoneuraminidase endo NE. Mol. Microbiol. 16, 441–450 (1995).

    Article  CAS  Google Scholar 

  12. Long, G.S., Bryant, J.M., Taylor, P.W. & Luzio, J.P. Complete nucleotide sequence of the gene encoding bacteriophage E endosialidase: implications for K1E endosialidase structure and function. Biochem. J. 309, 543–550 (1995).

    Article  CAS  Google Scholar 

  13. Scholl, D., Rogers, S., Adhya, S. & Merril, C.R. Bacteriophage K1-5 encodes two different tail fiber proteins, allowing it to infect and replicate on both K1 and K5 strains of Escherichia coli. J. Virol. 75, 2509–2515 (2001).

    Article  CAS  Google Scholar 

  14. Henrissat, B. & Bairoch, A. Updating the sequence-based classification of glycosyl hydrolases. Biochem. J. 316, 695–696 (1996).

    Article  Google Scholar 

  15. Hallenbeck, P.C., Vimr, E.R., Yu, F., Bassler, B. & Troy, F.A. Purification and properties of a bacteriophage-induced endo-N- acetylneuraminidase specific for poly-α-2,8-sialosyl carbohydrate units. J. Biol. Chem. 262, 3553–3561 (1987).

    CAS  PubMed  Google Scholar 

  16. Pelkonen, S., Pelkonen, J. & Finne, J. Common cleavage pattern of polysialic acid by bacteriophage endosialidases of different properties and origins. J. Virol. 63, 4409–4416 (1989).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Tulip, W.R. et al. Refined atomic structures of N9 subtype influenza virus neuraminidase and escape mutants. J. Mol. Biol. 221, 487–497 (1991).

    Article  CAS  Google Scholar 

  18. Varghese, J.N. & Colman, P.M. Three-dimensional structure of the neuraminidase of influenza virus A/Tokyo/3/67 at 2.2 Å resolution. J. Mol. Biol. 221, 473–486 (1991).

    Article  CAS  Google Scholar 

  19. Burmeister, W.P., Ruigrok, R.W. & Cusack, S. The 2.2 Å resolution crystal structure of influenza B neuraminidase and its complex with sialic acid. EMBO J. 11, 49–56 (1992).

    Article  CAS  Google Scholar 

  20. Crennell, S.J., Garman, E.F., Laver, W.G., Vimr, E.R. & Taylor, G.L. Crystal structure of a bacterial sialidase (from Salmonella typhimurium LT2) shows the same fold as an influenza virus neuraminidase. Proc. Natl. Acad. Sci. USA 90, 9852–9856 (1993).

    Article  CAS  Google Scholar 

  21. Crennell, S., Garman, E., Laver, G., Vimr, E. & Taylor, G. Crystal structure of Vibrio cholerae neuraminidase reveals dual lectin-like domains in addition to the catalytic domain. Structure 2, 535–544 (1994).

    Article  CAS  Google Scholar 

  22. Crennell, S., Takimoto, T., Portner, A. & Taylor, G. Crystal structure of the multifunctional paramyxovirus hemagglutinin-neuraminidase. Nat. Struct. Biol. 7, 1068–1074 (2000).

    Article  CAS  Google Scholar 

  23. Gaskell, A., Crennell, S. & Taylor, G. The three domains of a bacterial sialidase: a β-propeller, an immunoglobulin module and a galactose-binding jelly-roll. Structure 3, 1197–1205 (1995).

    Article  CAS  Google Scholar 

  24. Luo, Y., Li, S.C., Chou, M.Y., Li, Y.T. & Luo, M. The crystal structure of an intramolecular trans-sialidase with a NeuAc α2→3Gal specificity. Structure 6, 521–530 (1998).

    Article  CAS  Google Scholar 

  25. Buschiazzo, A. et al. Structural basis of sialyltransferase activity in trypanosomal sialidases. EMBO J. 19, 16–24 (2000).

    Article  CAS  Google Scholar 

  26. Chong, A.K.J., Pegg, M.S., Taylor, N.R. & von Itzstein, M. Evidence for a sialosyl cation transition-state complex in the reaction of sialidase from influenza-virus. Eur. J. Biochem. 207, 335–343 (1992).

    Article  CAS  Google Scholar 

  27. Watts, A.G. et al. Trypanosoma cruzi trans-sialidase operates through a covalent sialyl-enzyme intermediate: tyrosine is the catalytic nucleophile. J. Am. Chem. Soc. 125, 7532–7533 (2003).

    Article  CAS  Google Scholar 

  28. Watson, J.N., Dookhun, V., Borgford, T.J. & Bennet, A.J. Mutagenesis of the conserved active-site tyrosine changes a retaining sialidase into an inverting sialidase. Biochemistry 42, 12682–12690 (2003).

    Article  CAS  Google Scholar 

  29. Amaya, M.F. et al. Structural insights into the catalytic mechanism of Trypanosoma cruzi trans-sialidase. Structure 12, 775–784 (2004).

    Article  CAS  Google Scholar 

  30. Daniel, L. et al. A nude mice model of human rhabdomyosarcoma lung metastases for evaluating the role of polysialic acids in the metastatic process. Oncogene 20, 997–1004 (2001).

    Article  CAS  Google Scholar 

  31. Mushtaq, N., Redpath, M.B., Luzio, J.P. & Taylor, P.W. Prevention and cure of systemic Escherichia coli K1 infection by modification of the bacterial phenotype. Antimicrob. Agents Chemother. 48, 1503–1508 (2004).

    Article  CAS  Google Scholar 

  32. Taylor, G. Sialidases: Structures, biological significance and therapeutic potential. Curr. Opin. Struct. Biol. 6, 830–837 (1996).

    Article  CAS  Google Scholar 

  33. Nissen, P. et al. Crystal structure of the ternary complex of Phe-tRNAPhe, EF-Tu, and a GTP analog. Science 270, 1464–1472 (1995).

    Article  CAS  Google Scholar 

  34. Steinbacher, S. et al. Crystal structure of P22 tailspike protein: interdigitated subunits in a thermostable trimer. Science 265, 383–386 (1994).

    Article  CAS  Google Scholar 

  35. van Raaij, M.J., Schoehn, G., Burda, M.R. & Miller, S. Crystal structure of a heat and protease-stable part of the bacteriophage T4 short tail fiber. J. Mol. Biol. 314, 1137–1146 (2001).

    Article  CAS  Google Scholar 

  36. Kanamaru, S. et al. Structure of the cell-puncturing device of bacteriophage T4. Nature 415, 553–557 (2002).

    Article  CAS  Google Scholar 

  37. Chappell, J.D., Gunn, V.L., Wetzel, J.D., Baer, G.S. & Dermody, T.S. Mutations in type 3 reovirus that determine binding to sialic acid are contained in the fibrous tail domain of viral attachment protein sigma 1. J. Virol. 71, 1834–1841 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Buschiazzo, A., Amaya, M.F., Cremona, M.L., Frasch, A.C. & Alzari, P.M. The crystal structure and mode of action of trans-sialidase, a key enzyme in Trypanosoma cruzi pathogenesis. Mol. Cell 10, 757–768 (2002).

    Article  CAS  Google Scholar 

  39. Kitazume, S., Kitajima, K., Inoue, S. & Inoue, Y. Detection, isolation, and characterization of oligo/poly(sialic acid) and oligo/poly(deaminoneuraminic acid) units in glycoconjugates. Anal. Biochem. 202, 25–34 (1992).

    Article  CAS  Google Scholar 

  40. Manzi, A.E., Higa, H.H., Diaz, S. & Varki, A. Intramolecular self-cleavage of polysialic acid. J. Biol. Chem. 269, 23617–23624 (1994).

    CAS  PubMed  Google Scholar 

  41. Whitfield, C., Vimr, E.R., Costerton, J.W. & Troy, F.A. Protein synthesis is required for in vivo activation of polysialic acid capsule synthesis in Escherichia coli K1. J. Bacteriol. 159, 321–328 (1984).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994).

  43. Sheldrick, G.M., Hauptman, H.A., Weeks, C.M., Miller, R. & Usón, I. In International Tables for Crystallography Vol. F (eds. Rossmann, M.G. & Arnold, E.) 333–351 (Kluwer Academic, Dordrecht, The Netherlands, 2001).

    Google Scholar 

  44. Cowtan, K.D. & Zhang, K.Y. Density modification for macromolecular phase improvement. Prog. Biophys. Mol. Biol. 72, 245–270 (1999).

    Article  CAS  Google Scholar 

  45. Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjeldgaard, M. Improved methods for building protein models in electron-density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991).

    Article  Google Scholar 

  46. McRee, D.E. XtalView/Xfit - a versatile program for manipulating atomic coordinates and electron density. J. Struct. Biol. 125, 156–165 (1999).

    Article  CAS  Google Scholar 

  47. Perrakis, A., Morris, R. & Lamzin, V.S. Automated protein model building combined with iterative structure refinement. Nat. Struct. Biol. 6, 458–463 (1999).

    Article  CAS  Google Scholar 

  48. Murshudov, G.N., Vagin, A.A., Lebedev, A., Wilson, K.S. & Dodson, E.J. Efficient anisotropic refinement of macromolecular structures using FFT. Acta Crystallogr. D 55, 247–255 (1999).

    Article  CAS  Google Scholar 

  49. Schüttelkopf, A.W. & Van Aalten, D.M. PRODRG: a tool for high-throughput crystallography of protein-ligand complexes. Acta Crystallogr. D 60, 1355–1363 (2004).

    Article  Google Scholar 

  50. Holm, L. & Sander, C. Touring protein fold space with Dali/FSSP. Nucleic Acids Res. 26, 316–319 (1998).

    Article  CAS  Google Scholar 

  51. Weisgerber, C. et al. Embryonic neural cell adhesion molecule in cerebrospinal fluid of younger children: age-dependent decrease during the first year. J. Neurochem. 55, 2063–2071 (1990).

    Article  CAS  Google Scholar 

  52. Frosch, M., Gorgen, I., Boulnois, G.J., Timmis, K.N. & Bitter-Suermann, D. NZB mouse system for production of monoclonal antibodies to weak bacterial antigens: isolation of an IgG antibody to the polysaccharide capsules of Escherichia coli K1 and group B meningococci. Proc. Natl. Acad. Sci. USA 82, 1194–1198 (1985).

    Article  CAS  Google Scholar 

  53. Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685 (1970).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank the staff of European Molecular Biology Laboratory beamline at DESY, Hamburg, and the staff of the PSF beamline at BESSY, Berlin, for guidance during data collection. We are grateful to our colleague O. Einsle for help during the crystal structure analysis. This work was supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.

Author information

Authors and Affiliations

  1. Abteilung Zelluläre Chemie, Zentrum Biochemie, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, Hannover, 30625, Germany

    Katharina Stummeyer, Martina Mühlenhoff & Rita Gerardy-Schahn

  2. Abteilung für Molekulare Strukturbiologie, Institut für Mikrobiologie und Genetik, Georg-August-Universität Göttingen, Justus-von-Liebig-Weg 11, Göttingen, 37077, Germany

    Achim Dickmanns & Ralf Ficner

Authors
  1. Katharina Stummeyer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Achim Dickmanns
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Martina Mühlenhoff
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Rita Gerardy-Schahn
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ralf Ficner
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ralf Ficner.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Binding of inactive endoNF mutants to polySia. (PDF 159 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Stummeyer, K., Dickmanns, A., Mühlenhoff, M. et al. Crystal structure of the polysialic acid–degrading endosialidase of bacteriophage K1F. Nat Struct Mol Biol 12, 90–96 (2005). https://doi.org/10.1038/nsmb874

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb874

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing