Structural insights into mechanism and specificity of O-GlcNAc transferase

• Andrew J Clarke ^[1] ; Ramon Hurtado-Guerrero ^[1] ; Shalini Pathak ^[1] ; Alexander W Schüttelkopf ^[1] ; Vladimir Borodkin ^[1] ; Sharon M Shepherd ^[1] ; Adel F M Ibrahim ^[1] ; Daan M F van Aalten ^[1]
  1. [1] University of Dundee

     University of Dundee

     Reino Unido

     
• Localización: EMBO journal: European Molecular Biology Organization, ISSN 0261-4189, Vol. 27, Nº. 20, 2008, págs. 2780-2788
• Idioma: inglés
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• Resumen

  • 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 Å 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.