Structural basis of EGF-repeat O-glucosylation by the protein O-glucosyltransferase POGLUT2

Human protein O-glucosyltransferase 2 (POGLUT2) catalyzes the O-glucosylation of Notch receptors and extracellular matrix proteins, with its dysfunction linked to human disorders. Despite its physiological importance, the structural and mechanistic basis of POGLUT2 has remained elusive. Here, we report the first 1.79 Å structure of POGLUT2 in complex with UDP, revealing a three-domain architecture stabilized by an N-terminal filamin domain, which is unique in Notch-modifying enzymes. Integrated structural, computational, and functional analyses demonstrate that POGLUT2 recognizes structural features within epidermal growth factor-like repeats, including a conserved hydrophobic patch, which explains its stringent substrate selectivity. Our findings further identify Asp238 as the catalytic base, supporting an S_N2-type inverting mechanism. Furthermore, we show that cancer-associated mutations impair enzymatic activity through distinct structural and mechanistic disruptions. By delineating conserved and divergent features between POGLUT2 and POGLUT1, our study advances the mechanistic understanding of epidermal growth factor-like-repeat O-glucosylation and establishes a framework for investigating its dysregulation in human diseases.