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

Shalini Pathak, Jana Alonso , Marianne Schimpl, Karim Rafie, David E. Blair, Vladimir S. Borodkin, Alexander W. Schüttelkopf, Osama Albarbarawi, Daan M. F. Van Aalten (Lead / Corresponding author)

Research output: Contribution to journalArticlepeer-review

61 Citations (Scopus)

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 â '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.

Original languageEnglish
Pages (from-to)744-749
Number of pages6
JournalNature Structural & Molecular Biology
Volume22
Issue number9
DOIs
Publication statusPublished - Sep 2015

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