Structural insights into mechanism and specificity of O-GlcNAc transferase.
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.
Clarke
Andrew J
AJ
Division of Biological Chemistry & Drug Discovery, College of Life Sciences, University of Dundee, Dundee, UK.
Hurtado-Guerrero
Ramon
R
Pathak
Shalini
S
Schüttelkopf
Alexander W
AW
Borodkin
Vladimir
V
Shepherd
Sharon M
SM
Ibrahim
Adel F M
AF
van Aalten
Daan M F
DM
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
D017351:N-Acetylglucosaminyltransferases; C449880:O-GlcNAc transferase