Deciphering How Clustered O‑Glycosylation Shapes Substrate-Binding Preferences in an Intrinsically Disordered Protein...

Area of Science:

• Biochemistry
• Molecular Biology
• Glycobiology

Background:

• Post-translational modifications like O-glycosylation are common in intrinsically disordered regions (IDRs).
• The functional roles of O-glycosylation in IDRs, particularly in ligand binding, are not well understood.

Purpose of the Study:

• To investigate how O-glycosylation patterns in an intrinsically disordered region (IDR) influence its binding affinity to lignin and cellulose.
• To develop a predictive framework for understanding glycosylation-mediated regulation of IDR-ligand interactions.

Main Methods:

• Integrative approach combining chemical synthesis, experimental characterization, and molecular dynamics (MD) simulations.
• Utilized the O-mannosylated linker of *Trichoderma reesei* Family 7 cellobiohydrolase as a model IDR.
• Synthesized and analyzed various glycoforms, from unglycosylated to dimannose-containing structures.

Main Results:

• Unglycosylated and partially glycosylated linkers showed preferential binding to lignin over cellulose, driven by electrostatic interactions.
• Increased glycan density or size weakened lignin binding and shifted preference towards cellulose.
• MD simulations predicted that negatively charged glycans, like phosphorylated mannose, abolish lignin affinity while maintaining cellulose binding.

Conclusions:

• Charge-mediated modulation by O-glycosylation is a key mechanism controlling IDR-ligand interactions.
• This study provides a generalizable framework for predicting how glycosylation patterns regulate IDR function.
• The findings highlight the role of clustered O-glycosylation in tuning protein interactions with different surfaces.