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

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.

[1]  L. Tabak,et al.  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. , 1999, The Journal of biological chemistry.

[2]  M. Palcic,et al.  A continuous spectrophotometric assay for glycosyltransferases. , 1994, Analytical biochemistry.

[3]  S. Crennell,et al.  Crystal structure of a bacterial sialidase (from Salmonella typhimurium LT2) shows the same fold as an influenza virus neuraminidase. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[4]  P. Alzari,et al.  The high resolution structures of free and inhibitor-bound Trypanosoma rangeli sialidase and its comparison with T. cruzi trans-sialidase. , 2003, Journal of molecular biology.

[5]  G J Kleywegt,et al.  Model building and refinement practice. , 1997, Methods in enzymology.

[6]  S. Crennell,et al.  The three domains of a bacterial sialidase: a beta-propeller, an immunoglobulin module and a galactose-binding jelly-roll. , 1995, Structure.

[7]  S Cusack,et al.  The 2.2 A resolution crystal structure of influenza B neuraminidase and its complex with sialic acid. , 1992, The EMBO journal.

[8]  B. Duim,et al.  Molecular Characterization of Campylobacter jejunifrom Patients with Guillain-Barré and Miller Fisher Syndromes , 2000, Journal of Clinical Microbiology.

[9]  B. Horenstein,et al.  The N-Acetyl Neuraminyl Oxecarbenium Ion Is an Intermediate in the Presence of Anionic Nucleophiles , 1998 .

[10]  P. Argos,et al.  The taxonomy of binding sites in proteins , 1978, Molecular and Cellular Biochemistry.

[11]  Andrew G. Watts,et al.  The synthesis of some mechanistic probes for sialic acid processing enzymes and the labeling of a sialidase from Trypanosoma rangeli , 2004 .

[12]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[13]  C. Szymanski,et al.  Phase Variation of Campylobacter jejuni 81-176 Lipooligosaccharide Affects Ganglioside Mimicry and Invasiveness In Vitro , 2002, Infection and Immunity.

[14]  D E McRee,et al.  XtalView/Xfit--A versatile program for manipulating atomic coordinates and electron density. , 1999, Journal of structural biology.

[15]  M. Chou,et al.  The crystal structure of an intramolecular trans-sialidase with a NeuAc alpha2-->3Gal specificity. , 1998, Structure.

[16]  K. Furukawa,et al.  Biosynthesis and functions of gangliosides: recent advances , 1998, Glycoconjugate Journal.

[17]  S. Ley,et al.  Chemo-enzymatic synthesis of fluorinated sugar nucleotide: useful mechanistic probes for glycosyltransferases. , 2000, Bioorganic & medicinal chemistry.

[18]  D. Stuart,et al.  Comparison of AMP and NADH binding to glycogen phosphorylase b. , 1983, Journal of molecular biology.

[19]  S. Crennell,et al.  The structures of Salmonella typhimurium LT2 neuraminidase and its complexes with three inhibitors at high resolution. , 1996, Journal of molecular biology.

[20]  A. Frasch,et al.  Structural basis of sialyltransferase activity in trypanosomal sialidases , 2000 .

[21]  Ruth Lloyd,et al.  Insights into trehalose synthesis provided by the structure of the retaining glucosyltransferase OtsA. , 2002, Chemistry & biology.

[22]  Stephen G. Withers,et al.  Crystal structure of the retaining galactosyltransferase LgtC from Neisseria meningitidis in complex with donor and acceptor sugar analogs , 2001, Nature Structural Biology.

[23]  B. Horenstein.  QUANTUM MECHANICAL ANALYSIS OF AN ALPHA -CARBOXYLATE-SUBSTITUTED OXOCARBENIUM ION. ISOTOPE EFFECTS FOR FORMATION OF THE SIALYL CATION AND THE ORIGIN O F AN UNUSUALLY LARGE SECONDARY 14C ISOTOPE EFFECT , 1997 .

[24]  P. Freemont,et al.  Crystal structure of the DNA modifying enzyme beta‐glucosyltransferase in the presence and absence of the substrate uridine diphosphoglucose. , 1994, The EMBO journal.

[25]  K. Sharp,et al.  Protein folding and association: Insights from the interfacial and thermodynamic properties of hydrocarbons , 1991, Proteins.

[26]  Eduardo N. Taboada,et al.  The Genetic Bases for the Variation in the Lipo-oligosaccharide of the Mucosal Pathogen, Campylobacter jejuni , 2002, The Journal of Biological Chemistry.

[27]  柴山 恵吾.  Conserved structural regions involved in the catalytic mechanism of Escherichia coli K-12 WaaO (RfaI) , 2000 .

[28]  V. N. Molchanov,et al.  Superconducting Single Crystals of Tl2Ba2CaCu2O8 and YBa2Cu4O8: Crystal Structures in the Vicinity of Tc , 1998 .

[29]  J. Navaza,et al.  AMoRe: an automated package for molecular replacement , 1994 .

[30]  T. Darden,et al.  Heparan/Chondroitin Sulfate Biosynthesis , 2000, The Journal of Biological Chemistry.

[31]  S. Withers,et al.  Substrate-induced inactivation of a crippled beta-glucosidase mutant: identification of the labeled amino acid and mutagenic analysis of its role. , 1995, Biochemistry.

[32]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[33]  Z. Otwinowski,et al.  Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[34]  Robert A. Harris,et al.  Crystal structure of thiamin pyrophosphokinase. , 2001, Journal of molecular biology.

[35]  Bernard Henrissat,et al.  An evolving hierarchical family classification for glycosyltransferases. , 2003, Journal of molecular biology.

[36]  Y. Li,et al.  The 1.8 A structures of leech intramolecular trans-sialidase complexes: evidence of its enzymatic mechanism. , 1999, Journal of molecular biology.

[37]  E A Merritt,et al.  Raster3D: photorealistic molecular graphics. , 1997, Methods in enzymology.

[38]  S. Crennell,et al.  Crystal structure of the multifunctional paramyxovirus hemagglutinin-neuraminidase , 2001, Nature Structural Biology.

[39]  Yigong Shi,et al.  The 1.9 Å crystal structure of Escherichia coli MurG, a membrane‐associated glycosyltransferase involved in peptidoglycan biosynthesis , 2000, Protein science : a publication of the Protein Society.

[40]  S. Munro,et al.  A Common Motif of Eukaryotic Glycosyltransferases Is Essential for the Enzyme Activity of Large Clostridial Cytotoxins* , 1998, The Journal of Biological Chemistry.

[41]  Thomas C. Terwilliger,et al.  Electronic Reprint Biological Crystallography Maximum-likelihood Density Modification Using Pattern Recognition of Structural Motifs Pattern Recognition of Structural Motifs , 2022 .

[42]  S. Munro,et al.  Activity of the yeast MNN1 alpha-1,3-mannosyltransferase requires a motif conserved in many other families of glycosyltransferases. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[43]  S. Doublié.  Preparation of selenomethionyl proteins for phase determination. , 1997, Methods in enzymology.

[44]  G. Davies,et al.  Structure of the nucleotide-diphospho-sugar transferase, SpsA from Bacillus subtilis, in native and nucleotide-complexed forms. , 1999, Biochemistry.

[45]  P. Roach,et al.  Crystal structure of the autocatalytic initiator of glycogen biosynthesis, glycogenin. , 2002, Journal of molecular biology.

[46]  R. Schnaar,et al.  Brain gangliosides: functional ligands for myelin stability and the control of nerve regeneration. , 2001, Biochimie.

[47]  A. Frasch,et al.  Structural basis of sialyltransferase activity in trypanosomal sialidases. , 2000, The EMBO journal.

[48]  E Garman,et al.  Crystal structure of Vibrio cholerae neuraminidase reveals dual lectin-like domains in addition to the catalytic domain. , 1994, Structure.

[49]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

[50]  J. Penner,et al.  Diversity of lipopolysaccharide structures in Campylobacter jejuni. , 1997, The Journal of infectious diseases.

[51]  J. Rini,et al.  X‐ray crystal structure of rabbit N‐acetylglucosaminyltransferase I: catalytic mechanism and a new protein superfamily , 2000, The EMBO journal.

[52]  J. Brisson,et al.  Biosynthesis of Ganglioside Mimics in Campylobacter jejuni OH4384 , 2000, The Journal of Biological Chemistry.

[53]  S. Withers,et al.  X-ray structures along the reaction pathway of cyclodextrin glycosyltransferase elucidate catalysis in the α-amylase family , 1999, Nature Structural Biology.

[54]  J. Paulson,et al.  The Sialyltransferase Sialylmotif Participates in Binding the Donor Substrate CMP-NeuAc (*) , 1995, The Journal of Biological Chemistry.