Article | Published:

Structural analysis of the sialyltransferase CstII from Campylobacter jejuni in complex with a substrate analog

Nature Structural & Molecular Biology volume 11, pages 163170 (2004) | Download Citation

Subjects

Abstract

Sialic acid terminates oligosaccharide chains on mammalian and microbial cell surfaces, playing critical roles in recognition and adherence. The enzymes that transfer the sialic acid moiety from cytidine-5′-monophospho-N-acetyl-neuraminic acid (CMP-NeuAc) to the terminal positions of these key glycoconjugates are known as sialyltransferases. Despite their important biological roles, little is understood about the mechanism or molecular structure of these membrane-associated enzymes. We report the first structure of a sialyltransferase, that of CstII from Campylobacter jejuni, a highly prevalent foodborne pathogen. Our structural, mutagenesis and kinetic data provide support for a novel mode of substrate binding and glycosyl transfer mechanism, including essential roles of a histidine (general base) and two tyrosine residues (coordination of the phosphate leaving group). This work provides a framework for understanding the activity of several sialyltransferases, from bacterial to human, and for the structure-based design of specific inhibitors.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

References

  1. 1.

    & Brain gangliosides: functional ligands for myelin stability and the control of nerve regeneration. Biochimie 83, 677–682 (2001).

  2. 2.

    & Biosynthesis and functions of gangliosides: recent advances. Glycoconj. J. 15, 627–636 (1998).

  3. 3.

    et al. Phase variation of Campylobacter jejuni 81–176 lipooligosaccharide affects ganglioside mimicry and invasiveness in vitro. Infect. Immun. 70, 787–793 (2002).

  4. 4.

    & Diversity of lipopolysaccharide structures in Campylobacter jejuni. J. Infect. Dis. 176 Suppl 2, S135–S138 (1997).

  5. 5.

    et al. Molecular characterization of Campylobacter jejuni from patients with Guillain-Barre and Miller Fisher syndromes. J. Clin. Microbiol. 38, 2297–2301 (2000).

  6. 6.

    et al. Biosynthesis of ganglioside mimics in Campylobacter jejuni OH4384. Identification of the glycosyltransferase genes, enzymatic synthesis of model compounds, and characterization of nanomole amounts by 600-MHz 1H and 13C NMR analysis. J. Biol. Chem. 275, 3896–3906 (2000).

  7. 7.

    et al. The genetic bases for the variation in the lipo-oligosaccharide of the mucosal pathogen, Campylobacter jejuni. Biosynthesis of sialylated ganglioside mimics in the core oligosaccharide. J. Biol. Chem. 277, 327–337 (2002).

  8. 8.

    , , & An evolving hierarchical family classification for glycosyltransferases. J. Mol. Biol. 328, 307–317 (2003).

  9. 9.

    & The taxonomy of binding sites in proteins. Mol. Cell. Biochem. 21, 161–182 (1978).

  10. 10.

    & Structure of the nucleotide-diphospho-sugar transferase, SpsA from Bacillus subtilis, in native and nucleotide-complexed forms. Biochemistry 38, 6380–6385 (1999).

  11. 11.

    et al. Crystal structure of the retaining galactosyltransferase LgtC from Neisseria meningitidis in complex with donor and acceptor sugar analogs. Nat. Struct. Biol. 8, 166–175 (2001).

  12. 12.

    et al. X-ray crystal structure of rabbit N-acetylglucosaminyltransferase I: catalytic mechanism and a new protein superfamily. EMBO J. 19, 5269–5280 (2000).

  13. 13.

    , , & Crystal structure of the DNA modifying enzyme β-glucosyltransferase in the presence and absence of the substrate uridine diphosphoglucose. EMBO J. 13, 3413–3422 (1994).

  14. 14.

    , , & The 1.9 Å crystal structure of Escherichia coli MurG, a membrane-associated glycosyltransferase involved in peptidoglycan biosynthesis. Protein Sci. 9, 1045–1052 (2000).

  15. 15.

    , , , & Insights into trehalose synthesis provided by the structure of the retaining glucosyltransferase OtsA. Chem. Biol. 9, 1337–1346 (2002).

  16. 16.

    , & Crystal structure of the autocatalytic initiator of glycogen biosynthesis, glycogenin. J. Mol. Biol. 319, 463–477 (2002).

  17. 17.

    , , & Crystal structure of thiamin pyrophosphokinase. J. Mol. Biol. 310, 195–204 (2001).

  18. 18.

    et al. Comparison of AMP and NADH binding to glycogen phosphorylase b. J. Mol. Biol. 170, 529–565 (1983).

  19. 19.

    et al. Chemo-enzymatic synthesis of fluorinated sugar nucleotide: useful mechanistic probes for glycosyltransferases. Bioorg. Med. Chem. 8, 1937–1946 (2000).

  20. 20.

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

  21. 21.

    , & The 2.2 Å resolution crystal structure of influenza B neuraminidase and its complex with sialic acid. EMBO J. 11, 49–56 (1992).

  22. 22.

    , , , & Crystal structure of Vibrio cholerae neuraminidase reveals dual lectin-like domains in addition to the catalytic domain. Structure 2, 535–544 (1994).

  23. 23.

    , , & Crystal structure of the multifunctional paramyxovirus hemagglutinin-neuraminidase. Nat. Struct. Biol. 7, 1068–1074 (2000).

  24. 24.

    , , , & 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).

  25. 25.

    et al. The structures of Salmonella typhimurium LT2 neuraminidase and its complexes with three inhibitors at high resolution. J. Mol. Biol. 259, 264–280 (1996).

  26. 26.

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

  27. 27.

    , , , & The crystal structure of an intramolecular trans-sialidase with a NeuAc[α]2→3Gal specificity. Structure 6, 521–530 (1998).

  28. 28.

    , , & The 1.8 Å structures of leech intramolecular trans-sialidase complexes: evidence of its enzymatic mechanism. J. Mol. Biol. 285, 323–332 (1999).

  29. 29.

    , , & The high resolution structures of free and inhibitor-bound Trypanosoma rangeli sialidase and its comparison with T. cruzi trans-sialidase. J. Mol. Biol. 325, 773–784 (2003).

  30. 30.

    & The sialyltransferase “sialylmotif” participates in binding the donor substrate CMP-NeuAc. J. Biol. Chem. 270, 1497–1500 (1995).

  31. 31.

    & Activity of the yeast MNN1 α-1,3-mannosyltransferase requires a motif conserved in many other families of glycosyltransferases. Proc. Natl. Acad. Sci. USA 95, 7945–7950 (1998).

  32. 32.

    , , , & Structure-function analysis of the UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase. Essential residues lie in a predicted active site cleft resembling a lactose repressor fold. J. Biol. Chem. 274, 6797–6803 (1999).

  33. 33.

    et al. A common motif of eukaryotic glycosyltransferases is essential for the enzyme activity of large clostridial cytotoxins. J. Biol. Chem. 273, 19566–19572 (1998).

  34. 34.

    , , , & Conserved structural regions involved in the catalytic mechanism of Escherichia coli K-12 WaaO (RfaI). J. Bacteriol. 180, 5313–5318 (1998).

  35. 35.

    et al. X-ray structures along the reaction pathway of cyclodextrin glycosyltransferase elucidate catalysis in the α-amylase family. Nat. Struct. Biol. 6, 432–436 (1999).

  36. 36.

    et al. Substrate-induced inactivation of a crippled β-glucosidase mutant: identification of the labeled amino acid and mutagenic analysis of its role. Biochemistry 34, 14547–14553 (1995).

  37. 37.

    et al. Heparan/chondroitin sulfate biosynthesis. Structure and mechanism of human glucuronyltransferase I. J. Biol. Chem. 275, 34580–34585 (2000).

  38. 38.

    Quantum mechanical analysis of an α-carboxylate-substituted oxocarbenium ion. Isotope effects for formation of the sialyl cation and the origin of an unusually large 14C secondary isotope effect. J. Am. Chem. Soc. 119, 1101–1107 (1997).

  39. 39.

    & The N-acetyl neuraminyl oxocarbenium ion is an intermediate in the presence of anionic nucleophiles. J. Am. Chem. Soc. 120, 1357–1362 (1998).

  40. 40.

    Preparation of selenomethionyl proteins for phase determination. Methods Enzymol. 276, 523–530 (1997).

  41. 41.

    & The synthesis of some mechanistic probes for sialic acid processing enzymes and the labeling of a sialidase from Trypanosoma rangeli. Can. J. Chem. (in the press).

  42. 42.

    & Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

  43. 43.

    et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. A 54, 905–921 (1998).

  44. 44.

    Maximun likelihood density modification. Acta Crystallogr. D. 56, 965–972 (2000).

  45. 45.

    DM: An automated procedure for phase improvement by density modification. Joint CCP4 and ESF-EACBM Newslett. Protein Crystallogr. 31, 34–38 (1994).

  46. 46.

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

  47. 47.

    AMoRe: an automated package for molecular replacement. Acta Crystallogr A 50, 157–163 (1994).

  48. 48.

    & Model building and refinement practice. Methods Enzymol. 277, 208–230 (1997).

  49. 49.

    , , & PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291 (1993).

  50. 50.

    MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946–950 (1991).

  51. 51.

    , & Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11, 281–296 (1991).

  52. 52.

    & Raster3D: Photorealistic molecular graphics. Methods Enzymol. 277, 505–524 (1997).

  53. 53.

    , , , & A continuous spectrophotometric assay for glycosyltransferases. Anal. Biochem. 220, 92–97 (1994).

Download references

Acknowledgements

We thank the US Department of Energy for access to data collection facilities at the NSLS, and also M. Karwaski and S. Ryan for technical help. We thank Neose Pharmaceuticals for providing sialyllactose and CMP-NeuAc for the kinetic studies. The work was funded by the Howard Hughes Medical Institute, the Canadian Institute of Health Research and the Burroughs Wellcome Foundation (to N.S.), the Natural Sciences and Engineering Research Council and a Human Frontiers Science Grant (to S.W.) and by the National Research Council–GH (to W.W. and M.G.).

Author information

Author notes

    • Andrew G Watts
    •  & Luke L Lairson

    These authors contributed equally to this work.

Affiliations

  1. Department of Biochemistry and Molecular Biology, University of British Columbia, 2146 Health Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada.

    • Cecilia P C Chiu
    • , Daniel Lim
    •  & Natalie C J Strynadka
  2. Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, British Columbia V6T 1Z1, Canada.

    • Andrew G Watts
    • , Luke L Lairson
    •  & Stephen G Withers
  3. Institute for Biological Sciences, National Research Council, Room 3157, 100 Sussex Drive, Ottawa, Ontario K1A OR6, Canada.

    • Michel Gilbert
    •  & Warren W Wakarchuk

Authors

  1. Search for Cecilia P C Chiu in:

  2. Search for Andrew G Watts in:

  3. Search for Luke L Lairson in:

  4. Search for Michel Gilbert in:

  5. Search for Daniel Lim in:

  6. Search for Warren W Wakarchuk in:

  7. Search for Stephen G Withers in:

  8. Search for Natalie C J Strynadka in:

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Natalie C J Strynadka.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nsmb720

Further reading