Dynamic interplay between catalytic and lectin domains of GalNAc-transferases modulates protein O-glycosylation

Protein O -glycosylation is controlled by polypeptide GalNAc-transferases (GalNAc-Ts) that uniquely feature both a catalytic and lectin domain. The underlying molecular basis of how the lectin domains of GalNAc-Ts contribute to glycopeptide specificity and catalysis remains unclear. Here we present the first crystal structures of complexes of GalNAc-T2 with glycopeptides that together with enhanced sampling molecular dynamics simulations demonstrate a cooperative mechanism by which the lectin domain enables free acceptor sites binding of glycopeptides into the catalytic domain. Atomic force microscopy and small-angle X-ray scattering experiments further reveal a dynamic conformational landscape of GalNAc-T2 and a prominent role of compact structures that are both required for efficient catalysis. Our model indicates that the activity profile of GalNAc-T2 is dictated by conformational heterogeneity and relies on a flexible linker located between the catalytic and the lectin domains. Our results also shed light on how GalNAc-Ts generate dense decoration of proteins with O -glycans. Polypeptide GalNAc-transferases decorate proteins with dense arrays of O -glycans, which in the case of mucins are essential for their barrier functions. Here the authors present comprehensive structural studies that shed light on the molecular attributes that allow GalNAc-T2 to efficiently carry out dense O -glycosylation.