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

Area of Science:

• Microbiology
• Structural Biology
• Biochemistry

Background:

• Bacterial surface glycan polymerases, such as Wzy, are crucial integral membrane glycosyltransferases for synthesizing surface glycopolymers.
• The Wzy/Wzx-dependent pathway is widely used for glycan synthesis, but Wzy's structure-function relationship is poorly understood due to high sequence variability.
• Pneumococcal capsules serve as a model to investigate Wzy's role in linking repeat units via α- or β-glycosidic bonds.

Purpose of the Study:

• To predict the 3D molecular architectures of pneumococcal Wzy enzymes using computational tools.
• To elucidate the correlation between Wzy structure and glycosidic linkage stereochemistry (α vs. β).
• To identify sequence-based motifs for classifying Wzy types and predicting their function.

Main Methods:

• Utilized AlphaFold for predicting 3D molecular structures of pneumococcal Wzy enzymes.
• Employed Orientation of Proteins in Membranes (OPM) computational tool to analyze protein structures in membrane context.
• Correlated predicted Wzy structures with known glycosidic linkage stereochemistry (α- and β-).

Main Results:

• Two distinct Wzy types (type-A and type-B) were predicted with high confidence, differing in cavity orientation and C-terminus topology.
• Type-A Wzy models, with cavities extending toward the cytoplasm, were associated with α-glycosidic bonds.
• Type-B Wzy models, with cavities oriented toward the extracellular interface, were primarily associated with β-glycosidic bonds. Conserved motifs GN1 and GN2 were identified in type-B Wzys, linked to β-configuration.

Conclusions:

• The study provides the first structural framework for pneumococcal Wzy enzymes, linking distinct structural types to specific glycosidic bond stereochemistry.
• Identified conserved sequence motifs (GN1, GN2) enable sequence-based classification of Wzy types, facilitating broader studies in other bacteria.
• Findings advance the understanding of Wzy-dependent glycopolymer synthesis, with implications for bacterial pathogenesis and vaccine development.