Multi-domain O-GlcNAcase structures reveal allosteric regulatory mechanisms

Nucleocytoplasmic protein O-GlcNAcylation is an essential modification catalysed by O-GlcNAc transferase (OGT) and reversed by O-GlcNAc hydrolase (OGA), a multi-domain enzyme that also contains a C-terminal pseudo-histone acetyltransferase (pHAT) domain. OGA and OGT are tightly regulated using O-GlcNAc-dependent feedback mechanisms that are largely unknown. Although the structure of the OGA homodimeric catalytic domain has been reported, the structure and function of the pHAT domain remains poorly understood. We describe a crystal structure of the Trichoplax adhaerens pHAT domain and cryo-EM data of the multi-domain T. adhaerens and human OGAs, together with surface plasmon resonance and small-angle X-ray scattering studies. Our findings show that the eukaryotic OGA pHAT domains affect O-GlcNAc homeostasis, and form catalytically incompetent, symmetric homodimers, projecting a partially conserved putative peptide binding site available for interactions with binding partners. While there is evidence for symmetric OGA multi-domain dimers in solution, interactions between the linkers to the pHAT domains allow these to adopt a limited range of positions. In hOGA, the positions of the pHAT domains determine the wider active site environment through a key conformational change involving a tryptophan in a flexible arm region. Taken together, these multi-domain OGA structures reveal allosteric mechanisms of regulation.