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

Nature Structural & Molecular Biology volume 22, pages 744–750 (2015)Cite this article

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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.

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Figure 1: OGT shows substrate selectivity at the peptide level.
Figure 2: OGT modifies specific sites on peptide substrates.
Figure 3: Substrate peptides bind the active site of OGT with similar conformations in the −3 to +2 subsites.
Figure 4: OGT hexapeptide sequon derived from the peptide-library hits.

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Acknowledgements

We thank the European Synchrotron Radiation Facility (ESRF) and Diamond Light Source for beam time and assistance. This work was funded by a Wellcome Trust Senior Research Fellowship (WT087590MA) to D.M.F.v.A.

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Author notes

  1. Shalini Pathak, Jana Alonso and Marianne Schimpl: These authors contributed equally to this work.

Authors and Affiliations

  1. Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee, UK

    Shalini Pathak, Jana Alonso, Marianne Schimpl, Karim Rafie, David E Blair, Vladimir S Borodkin, Alexander W Schüttelkopf, Osama Albarbarawi & Daan M F van Aalten

  2. Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee, UK

    Daan M F van Aalten

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Contributions

S.P. and D.M.F.v.A. conceived the study; S.P., D.E.B. and K.R. performed the peptide assays; M.S., K.R. and D.M.F.v.A. performed structural biology; V.S.B. performed peptide synthesis; J.A. and O.A. performed MS studies; S.P., M.S., A.W.S. and D.M.F.v.A. interpreted the data; S.P., M.S. and D.M.F.v.A. wrote the manuscript.

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Correspondence to Daan M F van Aalten.

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Integrated supplementary information

Supplementary Figure 1 OGT modifies specific sites on peptide substrates.

Peptides emerging as hits from the screen, ranked by OGT activity. The O-GlcNAc serine or threonine residue in each peptide is highlighted in red. Green shading marks peptides in which the site identified from the screen matches previous reports. Peptides derived from proteins that have been published in the literature as O-GlcNAcylated proteins (with unknown modification sites) are highlighted in blue.

Supplementary Figure 2 Individual structures for all OGT–substrate complexes including electron density maps.

Unbiased Fo-Fc difference electron density for ligands (UDP-5S-GlcNAc and peptide) contoured at 2.25 σ (a, b) or contoured at 3.5 σ after NCS averaging (c, d). E, f Previously reported OGT substrate complexes (PDB ID 4AY6, Schimpl, M. et al., Nat.Chem.Biol. 8: 969, 2012; and PDB ID 4GYY, Lazarus, M.B. et al., Nat.Chem.Biol. 8: 966-968, 2012). The entire sequence of the peptides used in the study is given; underlined residues are represented in the final model, and the amino acid in bold is the O-GlcNAc-modified serine or threonine.

Supplementary Figure 3 Tolerance of different OGT isoforms for single amino acid substitutions.

OGT activity on the reference peptide KKVPVSRA was measured with two different constructs of the enzyme possessing a different number of TPR repeats. Nucleocytoplasmic OGT (ncOGT) is the longest (full length) natural OGT isoform, whereas the truncated construct, OGT (312—1031), was used for crystallographic studies and library screening due to its increased stability. The reference peptide KKVPVSRA represents the optimal OGT hexapeptide sequon except for position −3, where Val was used in order to avoid a potential second O-GlcNAc acceptor. Two N-terminal Lys residues were added, in order to aid peptide solubility. Assay details are given in the Online Methods section. The average of three measurements is shown, with error bars depicting the s.e.m. Activity for each enzyme isoform was normalized to the reference peptide. Full-length human OGT was expressed and purified as described for the truncated construct in Schimpl, M. et al., Nat.Chem.Biol. 8: 969, 2012.

Supplementary Figure 4 List of 32 hexapeptides derived from the peptide-library hits used to generate the sequon for Figure 4.

Experimentally determined O-GlcNAc sites were analyzed for sequence similarities. Peptides were aligned by the modified serine or threonine, and the sequence truncated to include only 3 residues to the N-terminus of the GlcNAc site (“−3”) and 2 residues to the C-terminus of the site (“+2”) as dictated by the ordered binding of peptides in only these subsites from the structural data. The hexapeptides highlighted in yellow were used for the generation of the sequence logo shown in Fig. 4b.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 (PDF 478 kb)

Supplementary Table 1

Peptide library sequences (XLS 134 kb)

Supplementary Data Set 1

Mass spectra for O-GlcNAc site determination of 26 peptides (PDF 3745 kb)

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Pathak, S., Alonso, J., Schimpl, M. et al. The active site of O-GlcNAc transferase imposes constraints on substrate sequence. Nat Struct Mol Biol 22, 744–750 (2015). https://doi.org/10.1038/nsmb.3063

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