title: Tethering an N-Glycosylation Sequon-Containing Peptide Creates a Catalytically Competent Oligosaccharyltransferase Complex.
abstract: Oligosaccharyltransferase (OST) transfers an oligosaccharide chain to the Asn residue in the Asn-X-Ser/Thr sequon in proteins, where X is not proline. A sequon was tethered to an archaeal OST enzyme via a disulfide bond. The positions of the cysteine residues in the OST protein and the sequon-containing acceptor peptide were selected by reference to the eubacterial OST structure in a noncovalent complex with an acceptor peptide. We determined the crystal structure of the cross-linked OST-sequon complex. The Ser/Thr-binding pocket recognizes the Thr residue in the sequon, and the catalytic structure termed the "carboxylate dyad" interacted with the Asn residue. Thus, the recognition and the catalytic mechanism of the sequon are conserved between the archaeal and eubacterial OSTs. We found that the tethered peptides in the complex were efficiently glycosylated in the presence of the oligosaccharide donor. The stringent requirements are greatly relaxed in the cross-linked state. The two conserved acidic residues in the catalytic structure were each dispensable, although the double mutation abolished the activity. A Gln residue at the Asn position in the sequon functioned as an acceptor, and the hydroxy group at position +2 was not required. In the standard assay using short free peptides, strong amino acid preferences were observed at the X position, but the preferences, except for Pro, completely disappeared in the cross-linked state. By skipping the initial binding process and stabilizing the complex state, the catalytically competent cross-linked complex offers a unique system for studying the oligosaccharyl transfer reaction.
authors.affiliation: Division of Structural Biology, Medical Institute of Bioregulation, ‡Research Center for Advanced Immunology, and §Research Center for Live-Protein Dynamics, Kyushu University , Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan.
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Matsumoto
authors.affiliation: Division of Structural Biology, Medical Institute of Bioregulation, ‡Research Center for Advanced Immunology, and §Research Center for Live-Protein Dynamics, Kyushu University , Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan.
authors.forename: Yuya
authors.initials: Y
authors.lastname: Taguchi
authors.affiliation: Division of Structural Biology, Medical Institute of Bioregulation, ‡Research Center for Advanced Immunology, and §Research Center for Live-Protein Dynamics, Kyushu University , Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Shimada
authors.affiliation: Division of Structural Biology, Medical Institute of Bioregulation, ‡Research Center for Advanced Immunology, and §Research Center for Live-Protein Dynamics, Kyushu University , Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan.
authors.forename: Mayumi
authors.initials: M
authors.lastname: Igura
authors.affiliation: Division of Structural Biology, Medical Institute of Bioregulation, ‡Research Center for Advanced Immunology, and §Research Center for Live-Protein Dynamics, Kyushu University , Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan.
authors.forename: Daisuke
authors.identifier: 0000-0001-8234-3776
authors.initials: D
authors.lastname: Kohda
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019611:Archaeoglobus fulgidus / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D006031:Glycosylation; D006602:Hexosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D019843:Archaeal Proteins; D001426:Bacterial Proteins; D008565:Membrane Proteins; D010455:Peptides; D011994:Recombinant Proteins; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase; D003545:Cysteine