title: Enzymatic basis for N-glycan sialylation: structure of rat α2,6-sialyltransferase (ST6GAL1) reveals conserved and unique features for glycan sialylation.
abstract: Glycan structures on glycoproteins and glycolipids play critical roles in biological recognition, targeting, and modulation of functions in animal systems. Many classes of glycan structures are capped with terminal sialic acid residues, which contribute to biological functions by either forming or masking glycan recognition sites on the cell surface or secreted glycoconjugates. Sialylated glycans are synthesized in mammals by a single conserved family of sialyltransferases that have diverse linkage and acceptor specificities. We examined the enzymatic basis for glycan sialylation in animal systems by determining the crystal structures of rat ST6GAL1, an enzyme that creates terminal α2,6-sialic acid linkages on complex-type N-glycans, at 2.4 Å resolution. Crystals were obtained from enzyme preparations generated in mammalian cells. The resulting structure revealed an overall protein fold broadly resembling the previously determined structure of pig ST3GAL1, including a CMP-sialic acid-binding site assembled from conserved sialylmotif sequence elements. Significant differences in structure and disulfide bonding patterns were found outside the sialylmotif sequences, including differences in residues predicted to interact with the glycan acceptor. Computational substrate docking and molecular dynamics simulations were performed to predict and evaluate the CMP-sialic acid donor and glycan acceptor interactions, and the results were compared with kinetic analysis of active site mutants. Comparisons of the structure with pig ST3GAL1 and a bacterial sialyltransferase revealed a similar positioning of donor, acceptor, and catalytic residues that provide a common structural framework for catalysis by the mammalian and bacterial sialyltransferases.
authors.affiliation: From the Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602.
authors.forename: Lu
authors.initials: L
authors.lastname: Meng
authors.forename: Farhad
authors.initials: F
authors.lastname: Forouhar
authors.forename: David
authors.initials: D
authors.lastname: Thieker
authors.forename: Zhongwei
authors.initials: Z
authors.lastname: Gao
authors.forename: Annapoorani
authors.initials: A
authors.lastname: Ramiah
authors.forename: Heather
authors.initials: H
authors.lastname: Moniz
authors.forename: Yong
authors.initials: Y
authors.lastname: Xiang
authors.forename: Jayaraman
authors.initials: J
authors.lastname: Seetharaman
authors.forename: Sahand
authors.initials: S
authors.lastname: Milaninia
authors.forename: Min
authors.initials: M
authors.lastname: Su
authors.forename: Robert
authors.initials: R
authors.lastname: Bridger
authors.forename: Lucas
authors.initials: L
authors.lastname: Veillon
authors.forename: Parastoo
authors.initials: P
authors.lastname: Azadi
authors.forename: Gregory
authors.initials: G
authors.lastname: Kornhaber
authors.forename: Lance
authors.initials: L
authors.lastname: Wells
authors.forename: Gaetano T
authors.initials: GT
authors.lastname: Montelione
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Woods
authors.forename: Liang
authors.initials: L
authors.lastname: Tong
authors.forename: Kelley W
authors.initials: KW
authors.lastname: Moremen
mesh_terms: D000818:Animals; D001419:Bacteria / Q000201:enzymology / Q000235:genetics; D001665:Binding Sites; D018360:Crystallography, X-Ray; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D011134:Polysaccharides / Q000096:biosynthesis / Q000737:chemistry*; D011487:Protein Conformation; D051381:Rats; D012794:Sialic Acids / Q000737:chemistry / Q000378:metabolism*; D012799:Sialyltransferases / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013552:Swine / Q000235:genetics; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
chemical_list: D011134:Polysaccharides; D012794:Sialic Acids; D012799:Sialyltransferases; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
keywords: AMBER; Carbohydrate Biosynthesis; Carbohydrate Glycoprotein; Enzyme Structure; GLYCAM; Glycobiology; Glycoprotein Biosynthesis; Molecular Dynamics