Mutual regulation mechanism of the O-GlcNAcylation enzyme pair revealed by Cryo-EM structure of human OGT–OGA complex

Abstract O-GlcNAcylation is a conserved post-translational modification that attaches N-acetyl glucosamine (GlcNAc) to myriad cellular proteins1–4. In response to nutritional and hormonal signals, O-GlcNAcylation regulates diverse cellular processes by modulating the stability, structure, and function of target proteins. Misregulation of O-GlcNAcylation is implicated in cancer, diabetes, and neurodegeneration5–7. A single pair of enzymes, the O-GlcNAc transferase (OGT) and the O-GlcNAcase (OGA), catalyzes the addition and removal of O-GlcNAc on over 3,000 proteins in the human proteome8,9. How OGT selects its native substrate(s) and maintains the homeostatic control of O-GlcNAcylation of so many substrates against OGA are not understood. Here we show that chemically induced degradation of OGT co-depletes OGA in human cells, suggesting the existence of a stable OGT–OGA complex in vivo. The cryo-electron microscopy (cryo-EM) structures of human OGT and the OGT–OGA complex reveal that OGT forms a functionally important scissor-shaped dimer. A long flexible OGA segment occupies the extended substrate-binding groove of OGT and positions a serine for O-GlcNAcylation, thus preventing OGT from modifying other substrates. Conversely, OGT disrupts the functional dimerization of OGA and occludes its active site, resulting in the blocking of access by other substrates. This unexpected but direct mutual inhibition between OGT and OGA limits futile O-GlcNAcylation cycles and maintains O-GlcNAc homeostasis.