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 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.

Insights into O-Linked N-Acetylglucosamine ([0-9]O-GlcNAc) Processing and Dynamics through Kinetic Analysis of O-GlcNAc Transferase and O-GlcNAcase Activity on Protein Substrates

“…Indeed, knowledge regarding the molecular basis by which OGT and OGA recognize their protein substrates is limited. Structures of a bacterial homologue of hOGT (28,43), in conjunction with a structure of the tetratricopeptide repeats of hOGT (44), and more recently of hOGT itself (45), have suggested that substrates bind within a superhelical groove formed from a series of tetratricopeptide repeats that extends right into the glycosyltransferase active site. There is little understanding, however, of the elements within proteins that are recognized.…”

“…A, top, Coomassie Blue-stained gel of O-GlcNAc-unmodified (Ϫ) and O-GlcNAc-modified (ϩ) TAB1, CaMKIV, CARM1, Tau, and Nup62. TAB1 expression in E. coli results in a major truncated product (residues 1-402) that runs at 43 kDa (43). The truncated product is inseparable from the full-length protein by gel filtration chromatography and is O-GlcNAcylated when expressed using the hOGT coexpression system.…”

Cytoplasmic N-Glycosyltransferase of Actinobacillus pleuropneumoniae Is an Inverting Enzyme and Recognizes the NX(S/T) Consensus Sequence

“…Purification of proteins was done as recommended by the provider. Purification of Xanthomonas campestris O-GlcNAc-transferase (OGT) was performed according to a published procedure (10). Buffer exchange to 25 mM Tris (pH 7.2) and 150 mM NaCl was performed by gel filtration chromatography using HiTrap desalting columns (GE Healthcare).…”

Polycomb repressive complex 2 is necessary for the normal site-specific O -GlcNAc distribution in mouse embryonic stem cells

“…Some of the activities of O-GlcNAcylation may occur as a result of cross-talk between O-GlcNAcylation and other PTMs, a phenomenon documented for protein phosphorylation (4,5) and ubiquitination (6). The cross-talk with phosphorylation is particularly interesting, as there is only one conserved mammalian O-GlcNAc transferase (OGT), with its extensive tetratricopeptide repeat protein-protein interaction domain, that must cooperate and/or compete with hundreds of protein kinases that recognize their substrates individually (7)(8)(9).…”