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.

Phage display uncovers a sequence motif that drives polypeptide binding to a conserved regulatory exosite of O-GlcNAc transferase

“…(42) The roles of the TPR region in recruiting substrates to OGT for glycosylation are gradually emerging. (22, 23, 48) Other regulatory mechanisms are also being defined,(20, 26) including the general concept that binding partners of OGT may influence substrate recruitment. (21, 24, 49) The molecular details of these interactions, however, remain unknown.…”

“…(21, 24, 25) Among these regulatory mechanisms, OGT dimer status has been found to influence its activity(20) as has an interaction with an intrinsically disordered region of OGA. (26) Interactions between OGT and other proteins, mediated through direct binding to the TPR region, have also been proposed to regulate OGT activity. (26) This last idea has gained support through quantitative proteomics studies showing that different cellular conditions alter the set of its interacting partners.…”

“…(26) Interactions between OGT and other proteins, mediated through direct binding to the TPR region, have also been proposed to regulate OGT activity. (26) This last idea has gained support through quantitative proteomics studies showing that different cellular conditions alter the set of its interacting partners. (27, 28) Interestingly, direct binding of candidate proteins identified in these proteomic studies has not yet been observed and how such proteins might modulate OGT activity remains to be elucidated.…”

Phage display uncovers a sequence motif that drives polypeptide binding to a conserved regulatory exosite of O-GlcNAc transferase

“…Because OGT has hundreds of known protein targets, yet lacks a clear consensus sequence governing glycosylation, the activity of OGT is thought to be regulated in various ways by cellular factors ( 21 , 24 , 25 ). Among these regulatory mechanisms, OGT dimer status has been found to influence its activity ( 20 ) as has an interaction with an intrinsically disordered region of OGA ( 26 ). Interactions between OGT and other proteins, mediated through direct binding to the TPR region, have also been proposed to regulate OGT activity ( 26 ).…”

“…Among these regulatory mechanisms, OGT dimer status has been found to influence its activity ( 20 ) as has an interaction with an intrinsically disordered region of OGA ( 26 ). Interactions between OGT and other proteins, mediated through direct binding to the TPR region, have also been proposed to regulate OGT activity ( 26 ). This last idea has gained support through quantitative proteomics studies showing that different cellular conditions alter the set of its interacting partners ( 27 , 28 ).…”