The O-linked β-N-acetylglucosamine modification is a core signalling mechanism, with erroneous patterns leading to cancer and neurodegeneration. Although thousands of proteins are subject to this modification, only a single essential glycosyltransferase catalyses its installation, the O-GlcNAc transferase, OGT. Previous studies have provided truncated structures of OGT through X-ray crystallography, but the full-length protein has never been observed. Here, we report a 5.3 Å cryo-EM model of OGT. We show OGT is a dimer, providing a structural basis for how some X-linked intellectual disability mutations at the interface may contribute to disease. We observe that the catalytic section of OGT abuts a 13.5 tetratricopeptide repeat unit region and find the relative positioning of these sections deviate from the previously proposed, X-ray crystallography-based model. We also note that OGT exhibits considerable heterogeneity in tetratricopeptide repeat units N-terminal to the dimer interface with repercussions for how OGT binds protein ligands and partners.
Glycosyltransferases carry out important cellular functions in species ranging from bacteria to humans.Despite their essential roles in biology,simple and robust activity assays that can be easily applied to high-throughput screening for inhibitors of these enzymes have been challenging to develop. Herein, we report abead-based strategy to measure the grouptransfer activity of glycosyltransferases sensitively using simple fluorescence measurements,w ithout the need for coupled enzymes or secondary reactions.W ev alidate the performance and accuracy of the assayusing O-GlcNAc transferase (OGT) as am odel system through detailed Michaelis-Menten kinetic analysis of various substrates and inhibitors.O ptimization of this assay and application to high-throughput screening enabled screening for inhibitors of OGT,l eading to an ovel inhibitory scaffold. We believe this assay will prove valuable not only for the study of OGT,b ut also more widely as ag eneral approach for the screening of glycosyltransferases and other group-transfer enzymes.Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.
Glycosyltransferases are a superfamily of enzymes that are notoriously difficult to inhibit. Here we apply an mRNA display technology integrated with genetic code reprogramming, referred to as the RaPID (random non-standard peptides integrated discovery) system, to identify macrocyclic peptides with high binding affinities for O-GlcNAc transferase (OGT). These macrocycles inhibit OGT activity through an allosteric mechanism that is driven by their binding to the tetratricopeptide repeats of OGT. Saturation mutagenesis in a maturation screen using 39 amino acids, including 22 non-canonical residues, led to an improved unnatural macrocycle that is � 40 times more potent than the parent compound (K i app = 1.5 nM). Subsequent derivatization delivered a biotinylated derivative that enabled one-step affinity purification of OGT from complex samples. The high potency and novel mechanism of action of these OGT ligands should enable new approaches to elucidate the specificity and regulation of OGT.
Owing to its roles in human health and disease, the modification of nuclear, cytoplasmic, and mitochondrial proteins with Olinked N-acetylglucosamine residues (O-GlcNAc) has emerged as a topic of great interest. Despite the presence of O-GlcNAc on hundreds of proteins within cells, only two enzymes regulate this modification. One of these enzymes is O-GlcNAcase (OGA), a dimeric glycoside hydrolase that has a deep active site cleft in which diverse substrates are accommodated. Chemical tools to control OGA are emerging as essential resources for helping to decode the biochemical and cellular functions of the O-GlcNAc pathway. Here we describe rationally designed bicyclic thiazolidine inhibitors that exhibit superb selectivity and picomolar inhibition of human OGA. Structures of these inhibitors in complex with human OGA reveal the basis for their exceptional potency and show that they extend out of the enzyme active site cleft. Leveraging this structure, we create a high affinity chemoproteomic probe that enables simple one-step purification of endogenous OGA from brain and targeted proteomic mapping of its post-translational modifications. These data uncover a range of new modifications, including some that are less-known, such as O-ubiquitination and N-formylation. We expect that these inhibitors and chemoproteomics probes will prove useful as fundamental tools to decipher the mechanisms by which OGA is regulated and directed to its diverse cellular substrates. Moreover, the inhibitors and structures described here lay out a blueprint that will enable the creation of chemical probes and tools to interrogate OGA and other carbohydrate active enzymes.
Glycosyltransferases carry out important cellular functions in species ranging from bacteria to humans. Despite their essential roles in biology, simple and robust activity assays that can be easily applied to high‐throughput screening for inhibitors of these enzymes have been challenging to develop. Herein, we report a bead‐based strategy to measure the group‐transfer activity of glycosyltransferases sensitively using simple fluorescence measurements, without the need for coupled enzymes or secondary reactions. We validate the performance and accuracy of the assay using O‐GlcNAc transferase (OGT) as a model system through detailed Michaelis–Menten kinetic analysis of various substrates and inhibitors. Optimization of this assay and application to high‐throughput screening enabled screening for inhibitors of OGT, leading to a novel inhibitory scaffold. We believe this assay will prove valuable not only for the study of OGT, but also more widely as a general approach for the screening of glycosyltransferases and other group‐transfer enzymes.
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