Identification of a\nPolypeptide Inhibitor of <i>O</i>‑GlcNAc Transferase\nwith Picomolar Affinity

<i>O</i>-GlcNAc transferase (OGT) is an essential\nmammalian\nenzyme that binds thousands of different proteins, including substrates\nthat it glycosylates and nonsubstrate interactors that regulate its\nbiology. OGT also has one proteolytic substrate, the transcriptional\ncoregulator host cell factor 1 (HCF-1), which it cleaves in a process\ninitiated by glutamate side chain glycosylation at a series of central\nrepeats. Although HCF-1 is OGT’s most prominent binding partner,\nits affinity for the enzyme has not been quantified. Here, we report\na time-resolved Förster resonance energy transfer assay to\nmeasure ligand binding to OGT and show that an HCF-1-derived polypeptide\n(HCF3R) binds with picomolar affinity to the enzyme (<i>K</i><sub>D</sub> ≤ 85 pM). This high affinity is driven in large\npart by conserved asparagines in OGT’s tetratricopeptide repeat\ndomain, which form bidentate contacts to the HCF-1 peptide backbone;\nreplacing any one of these asparagines with alanine reduces binding\nby more than 5 orders of magnitude. Because the HCF-1 polypeptide\nbinds so tightly to OGT, we tested its ability to inhibit enzymatic\nfunction. We found that HCF3R potently inhibits OGT both in vitro\nand in cells and used this finding to develop a genetically encoded,\ninducible OGT inhibitor that can be degraded with a small molecule,\nallowing for reversible and tunable inhibition of OGT.