title: Synergy of peptide and sugar in O-GlcNAcase substrate recognition.
abstract: Protein O-GlcNAcylation is an essential reversible posttranslational modification in higher eukaryotes. O-GlcNAc addition and removal is catalyzed by O-GlcNAc transferase and O-GlcNAcase, respectively. We report the molecular details of the interaction of a bacterial O-GlcNAcase homolog with three different synthetic glycopeptides derived from characterized O-GlcNAc sites in the human proteome. Strikingly, the peptides bind a conserved O-GlcNAcase substrate binding groove with similar orientation and conformation. In addition to extensive contacts with the sugar, O-GlcNAcase recognizes the peptide backbone through hydrophobic interactions and intramolecular hydrogen bonds, while avoiding interactions with the glycopeptide side chains. These findings elucidate the molecular basis of O-GlcNAcase substrate specificity, explaining how a single enzyme achieves cycling of the complete O-GlcNAc proteome. In addition, this work will aid development of O-GlcNAcase inhibitors that target the peptide binding site.
authors.affiliation: Division of Cell Signalling & Immunology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland.
authors.forename: Marianne
authors.initials: M
authors.lastname: Schimpl
authors.forename: Vladimir S
authors.initials: VS
authors.lastname: Borodkin
authors.forename: Lindsey J
authors.initials: LJ
authors.lastname: Gray
authors.forename: Daan M F
authors.initials: DM
authors.lastname: van Aalten
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002241:Carbohydrates / Q000737:chemistry*; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020543:Proteome / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D002241:Carbohydrates; D004791:Enzyme Inhibitors; D010455:Peptides; D020543:Proteome; D017351:N-Acetylglucosaminyltransferases; C449880:O-GlcNAc transferase