Structure-based principles underlying ligand recognition of xanthine-II riboswitch

Area of Science:

• Molecular Biology
• RNA Biology
• Structural Biology

Background:

• Riboswitches are RNA molecules that regulate gene expression in response to specific metabolites.
• Two classes of xanthine-responsive riboswitches are known, crucial for maintaining xanthine homeostasis.
• The xanthine-II riboswitch, derived from the guanine riboswitch, possesses unique structural modifications.

Purpose of the Study:

• To determine the complex structure of the xanthine-II riboswitch bound to xanthine.
• To understand the molecular basis for xanthine recognition and specificity.
• To explore the application of the xanthine-II riboswitch in developing a xanthine biosensor.

Main Methods:

• X-ray crystallography to determine the three-dimensional structure of the xanthine-II riboswitch-xanthine complex.
• Isothermal titration calorimetry (ITC) to validate ligand-binding specificity through structure-based mutations.
• Fusion of the xanthine-II riboswitch with the Pepper fluorogenic aptamer to create a biosensor.

Main Results:

• The xanthine-II riboswitch adopts a three-way junction structure, similar to the guanine riboswitch.
• A unique binding pocket, formed by a specific mutation and nucleotide insertions, confers high xanthine specificity.
• Xanthine forms a base triple with C64 and G37, and is further stabilized by base pairing and a base triple.
• Ligand-binding assays confirmed the specificity of the xanthine-II riboswitch.
• A sensitive and specific xanthine biosensor was successfully developed.

Conclusions:

• The structural elucidation of the xanthine-II riboswitch reveals a distinct mechanism for xanthine binding.
• The unique structural features enable high specificity, differentiating it from guanine riboswitches.
• The xanthine-II riboswitch holds significant potential for developing diagnostic tools for xanthine metabolism disorders.