Photoactivatable O-GlcNAc Transferase Library Enables Covalent Chemical Capture of Solvent-Exposed TPR Domain Interactions

O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) is an essential, stress-sensing enzyme responsible for adding the O-GlcNAc monosaccharide to thousands of nuclear and cytoplasmic proteins to regulate cellular homeostasis. OGT substrates are found in almost all intracellular processes, and perturbations in protein O-GlcNAc levels have been implicated in proteostatic diseases, such as cancers, metabolic disorders, and neurodegeneration. This broad disease activity makes OGT an attractive therapeutic target; however, the substrate diversity makes pan-inhibition as a therapeutic strategy unfeasible. Rather, a substrate-specific approach to targeting is more advantageous, but how OGT chooses its substrates remains poorly understood. Substrate specificity is controlled by the interactions between OGT's non-catalytic tetratricopeptide repeat (TPR) domain, rather than its glycosyltransferase domain. OGT's TPR domain forms a 100 Å superhelical structure, containing a lumenal surface, known as the substrate-binding surface, and a solvent-exposed surface. To date, there are no tools to site-selectively target regions of the domain and differentiate between the two binding surfaces. Here, we developed a library of recombinant OGT constructs containing site-specifically incorporated photoactivatable unnatural amino acids (UAAs) along the solvent-exposed surface of the TPR domain to covalently capture and map OGT's interactome.