title: Crystallographic and NMR evidence for flexibility in oligosaccharyltransferases and its catalytic significance.
abstract: Oligosaccharyltransferase (OST) is a membrane-bound enzyme that catalyzes the transfer of an oligosaccharide to an asparagine residue in glycoproteins. It possesses a binding pocket that recognizes Ser and Thr residues at the +2 position in the N-glycosylation consensus, Asn-X-Ser/Thr. We determined the crystal structures of the C-terminal globular domains of the catalytic subunits of two archaeal OSTs. A comparison with previously determined structures identified a segment with remarkable conformational plasticity, induced by crystal contact effects. We characterized its dynamic properties in solution by (15)N NMR relaxation analyses. Intriguingly, the mobile region contains the +2 Ser/Thr-binding pocket. In agreement, the flexibility restriction forced by an engineered disulfide crosslink abolished the enzymatic activity, and its cleavage fully restored activity. These results suggest the necessity of multiple conformational states in the reaction. The dynamic nature of the Ser/Thr pocket could facilitate the efficient scanning of N-glycosylation sequons along nascent polypeptide chains.
authors.affiliation: Division of Structural Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan.
authors.forename: James
authors.initials: J
authors.lastname: Nyirenda
authors.affiliation: Division of Structural Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan.
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Matsumoto
authors.affiliation: Division of Structural Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Saitoh
authors.affiliation: Institute of Enzyme Research, Tokushima University, Tokushima 770-8503, Japan.
authors.forename: Nobuo
authors.initials: N
authors.lastname: Maita
authors.affiliation: Department of Structural Biology, Faculty of Advanced Life Science, Hokkaido University, Sapporo 001-0021, Japan.
authors.forename: Nobuo N
authors.initials: NN
authors.lastname: Noda
authors.affiliation: Department of Structural Biology, Faculty of Advanced Life Science, Hokkaido University, Sapporo 001-0021, Japan.
authors.forename: Fuyuhiko
authors.initials: F
authors.lastname: Inagaki
authors.affiliation: Division of Structural Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan; Research Center for Advanced Immunology, Kyushu University, Fukuoka 812-8582, Japan. Electronic address: kohda@bioreg.kyushu-u.ac.jp.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Kohda
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics; D055162:Biocatalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003553:Cystine / Q000737:chemistry; D006602:Hexosyltransferases / Q000737:chemistry* / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D017433:Protein Structure, Secondary; D043902:Pyrococcus horikoshii / Q000201:enzymology*; D012996:Solutions
chemical_list: D019843:Archaeal Proteins; D008565:Membrane Proteins; D012996:Solutions; D003553:Cystine; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase