title: Structural insights into mechanism and specificity of O-GlcNAc transferase.
abstract: Post-translational modification of protein serines/threonines with N-acetylglucosamine (O-GlcNAc) is dynamic, inducible and abundant, regulating many cellular processes by interfering with protein phosphorylation. O-GlcNAcylation is regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase, both encoded by single, essential, genes in metazoan genomes. It is not understood how OGT recognises its sugar nucleotide donor and performs O-GlcNAc transfer onto proteins/peptides, and how the enzyme recognises specific cellular protein substrates. Here, we show, by X-ray crystallography and mutagenesis, that OGT adopts the (metal-independent) GT-B fold and binds a UDP-GlcNAc analogue at the bottom of a highly conserved putative peptide-binding groove, covered by a mobile loop. Strikingly, the tetratricopeptide repeats (TPRs) tightly interact with the active site to form a continuous 120 A putative interaction surface, whereas the previously predicted phosphatidylinositide-binding site locates to the opposite end of the catalytic domain. On the basis of the structure, we identify truncation/point mutants of the TPRs that have differential effects on activity towards proteins/peptides, giving first insights into how OGT may recognise its substrates.
authors.affiliation: Division of Biological Chemistry & Drug Discovery, College of Life Sciences, University of Dundee, Dundee, UK.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Clarke
authors.forename: Ramon
authors.initials: R
authors.lastname: Hurtado-Guerrero
authors.forename: Shalini
authors.initials: S
authors.lastname: Pathak
authors.forename: Alexander W
authors.initials: AW
authors.lastname: Schüttelkopf
authors.forename: Vladimir
authors.initials: V
authors.lastname: Borodkin
authors.forename: Sharon M
authors.initials: SM
authors.lastname: Shepherd
authors.forename: Adel F M
authors.initials: AF
authors.lastname: Ibrahim
authors.forename: Daan M F
authors.initials: DM
authors.lastname: van Aalten
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D018360:Crystallography, X-Ray; D006801:Humans; D008954:Models, Biological; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013379:Substrate Specificity; D014981:Xenopus
chemical_list: D017351:N-Acetylglucosaminyltransferases; C449880:O-GlcNAc transferase