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Nat Struct Mol Biol. 2015 Sep;22(9):744-750. doi: 10.1038/nsmb.3063. Epub 2015 Aug 3.

The active site of O-GlcNAc transferase imposes constraints on substrate sequence.

Author information

1
MRC Protein Phosphorylation and Ubiquitylation Unit and College of Life Sciences, University of Dundee, Dundee, UK.
2
Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee, UK.
#
Contributed equally

Abstract

O-GlcNAc transferase (OGT) glycosylates a diverse range of intracellular proteins with O-linked N-acetylglucosamine (O-GlcNAc), an essential and dynamic post-translational modification in metazoans. Although this enzyme modifies hundreds of proteins with O-GlcNAc, it is not understood how OGT achieves substrate specificity. In this study, we describe the application of a high-throughput OGT assay to a library of peptides. We mapped sites of O-GlcNAc modification by electron transfer dissociation MS and found that they correlate with previously detected O-GlcNAc sites. Crystal structures of four acceptor peptides in complex with Homo sapiens OGT suggest that a combination of size and conformational restriction defines sequence specificity in the -3 to +2 subsites. This work reveals that although the N-terminal TPR repeats of OGT may have roles in substrate recognition, the sequence restriction imposed by the peptide-binding site makes a substantial contribution to O-GlcNAc site specificity.

PMID:
26237509
PMCID:
PMC4979681
DOI:
10.1038/nsmb.3063
[Indexed for MEDLINE]
Free PMC Article

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