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

DOI: 10.1038/nsmb.3063 DOI Help
PMID: 26237509 PMID Help

Authors: Shalini Pathak (University of Dundee) , Jana Alonso (University of Dundee) , Marianne Schimpl (University of Dundee) , Karim Rafie (University of Dundee) , David E. Blair (University of Dundee) , Vladimir S. Borodkin (University of Dundee) , Alexander W. Schüttelkopf (University of Dundee) , Osama Albarbarawi (University of Dundee) , Daan M. F. Van Aalten (University of Dundee)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Nature Structural & Molecular Biology , VOL 22 (9) , PAGES 744 - 750

State: Published (Approved)
Published: October 2015

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.

Journal Keywords: Enzyme mechanisms; Glycobiology; Glycosylation

Diamond Keywords: Enzymes

Subject Areas: Biology and Bio-materials

Instruments: I04-1-Macromolecular Crystallography (fixed wavelength)

Other Facilities: ID23-2, ID30A-3 at ESRF

Added On: 20/11/2015 14:50

Discipline Tags:

Structural biology Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)