The RNA-binding protein KhpB binds dsRNA and retains binding activity at elevated temperatures

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• RNA-binding proteins regulate gene expression post-transcriptionally.
• The roles of less-characterized domains like KH and R3H in dsRNA interaction require further investigation.

Purpose of the Study:

• To characterize the dsRNA binding capabilities of engineered variants of the thermostable bacterial RNA-binding protein KhpB.
• To elucidate the contributions of KH and R3H domains to dsRNA binding and stability.

Main Methods:

• Engineered recombinant variants of KhpB (containing KH, R3H, or both domains).
• In vitro electrophoretic mobility shift assays to assess dsRNA binding capacity.
• In silico modeling, physicochemical analyses, and molecular dynamics simulations.

Main Results:

• Engineered KhpB variants demonstrated stable dsRNA binding up to 95°C.
• The R3H-only variant exhibited the strongest dsRNA interaction, suggesting it as a minimal functional module.
• Molecular dynamics confirmed basic residues are crucial for maintaining the dsRNA:KhpB complex.

Conclusions:

• Thermostable KhpB is a novel dsRNA-binding protein with potential biotechnological applications.
• Insights into KH and R3H domain interactions with dsRNA expand understanding of modular RNA-binding architectures.