Wzy 3D structural models correlate with inter-repeat unit glycosidic bond configuration in pneumococcal capsule polysaccharides

ABSTRACT Bacterial surface glycan polymerases, such as Wzy, are integral membrane-bound glycosyltransferases that synthesize various surface-bound glycopolymers by linking repeat units via α-glycosidic or β-glycosidic bonds. Despite its central role in the widely employed “Wzy/Wzx-dependent pathway” for glycan synthesis, Wzy remains poorly understood, largely due to its high sequence variability. Using Streptococcus pneumoniae (pneumococcus) capsules as a model, we leveraged AlphaFold and Orientation of Proteins in Membranes computational tools to predict 3D molecular architectures of pneumococcal Wzys and elucidate their correlation with glycosidic linkage stereochemistry. Predicted structures revealed two distinct Wzy types with high confidence (average predicted local distance difference test score ≥85; 95% confidence interval: 85.86–86.01): type-A and type-B, distinguished by the cavity orientation and partly by differing C-terminus topology. Predictions showed that type-A Wzy models featured a cavity extending toward the cytoplasm and were predominantly associated with α-glycosidic bonds. In contrast, type-B Wzy models displayed a cavity oriented toward the extracellular interface and primarily catalyze β-glycosidic bonds. To facilitate sequence-based classification of type-A and type-B Wzys, we identified two location-specific conserved motifs, GN1 and GN2, predominantly found in type-B Wzys and strongly associated with β-configuration. Our findings offer critical insights into the Wzy structure-function relationship, highlighting its role in the stereochemical control of glycan polymerization. Although focused on pneumococci, these findings may extend to other bacteria. Further studies across species are needed to confirm and expand this structural-glycosidic bond relationship with experimental evidence. IMPORTANCE Bacterial surface glycan polymerases, like Wzy, are key players in the synthesis of complex sugar-based polymers that form protective layers on the bacterial surfaces. However, the structure-function relationship of Wzy remains poorly defined due to its high sequence variability and membrane-bound nature. This study provides the first structural framework for classifying pneumococcal Wzy enzymes using AlphaFold-predicted 3D models in combination with Orientation of Proteins in Membranes computational tool, revealing two distinct structural types (type-A and type-B) that strongly correlate with glycosidic linkage stereochemistry. The identification of conserved sequence motifs further enables sequence-based classification, offering a valuable tool for studying Wzy across bacterial species. These findings not only advance our fundamental understanding of glycan polymerization but also have broader implications for bacterial pathogenesis and vaccine design. By establishing a direct link between Wzy structure and glycosidic bond formation, these findings provide a blueprint for exploring Wzy-dependent glycopolymer synthesis in other bacterial species, paving the way for targeted interventions in bacterial surface glycan biosynthesis.