Abstract
O-linked N-acetylglucosamine (O-GlcNAc) is an essential and dynamic post-translational modification found on hundreds of nucleocytoplasmic proteins in metazoa. Although a single enzyme, O-GlcNAc transferase (OGT), generates the entire cytosolic O-GlcNAc proteome, it is not understood how it recognizes its protein substrates, targeting only a fraction of serines/threonines in the metazoan proteome for glycosylation. We describe a trapped complex of human OGT with the C-terminal domain of TAB1, a key innate immunity-signalling O-GlcNAc protein, revealing extensive interactions with the tetratricopeptide repeats of OGT. Confirmed by mutagenesis, this interaction suggests that glycosylation substrate specificity is achieved by recognition of a degenerate sequon in the active site combined with an extended conformation C-terminal of the O-GlcNAc target site.
Highlights
The attachment of a single b-N-acetylglucosamine (O-GlcNAc) sugar onto serine and threonine residues of nucleocytoplasmic proteins is a dynamic and abundant post-translational modification found in higher eukaryotes [1 –3]
Short peptides derived from these sites can be co-crystallized with O-GlcNAc transferase (OGT) [31,32,33], we have been unsuccessful in using this approach with longer sequences/intact proteins to explore the role of the OGT TPR domain in substrate recognition
We first demonstrated that this fusion approach recapitulates the published HCF1PRO peptide binding mode and used that to reveal how the C-terminus of the OGT glycosylation substrate TAB1 is recognized by the enzyme
Summary
The attachment of a single b-N-acetylglucosamine (O-GlcNAc) sugar onto serine and threonine residues of nucleocytoplasmic proteins is a dynamic and abundant post-translational modification found in higher eukaryotes [1 –3]. These proteins cover a wide range of cellular processes such as transcription and translation [11 –13], trafficking and localization [14,15], as well as cell cycle progression [16 –19] It remains unclear how a single OGT enzyme is able to recognize a limited number of serines/threonines on such a large number of substrates. The first structural insights into the OGT catalytic domain came from an OGT orthologue in the bacterium Xanthomonas campestris [28,29] This structure revealed that the sugar donor binding site is made up of the two lobes of the glycosyl transferase B (GT-B) fold, tightly fused to the superhelical TPR domain [28]. Initial structural studies exploring Michaelis/substrate complexes with short acceptor peptides have
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