Tetracycline-tet repressor binding specificity: insights from experiments and simulations.

Area of Science:

• Biochemistry
• Molecular Biology
• Computational Chemistry

Background:

• Tetracycline (Tc) antibiotics are used in artificial gene regulation systems by binding to the Tet repressor protein (TetR).
• Tc variants are developed to combat bacterial resistance and tune TetR binding affinity.
• Understanding TetR-Tc interactions is crucial for designing precise gene control systems.

Purpose of the Study:

• To investigate the binding interactions between a library of 16 tetracycline variants and the Tet repressor protein (TetR).
• To computationally predict and validate the binding free energies of various Tc-TetR complexes.
• To elucidate the contributions of specific Tc chemical substituents to binding affinity and specificity.

Main Methods:

• Utilized fluorescence experiments to measure binding affinities.
• Employed molecular dynamics free energy (MDFE) simulations to compute relative TetR binding free energies.
• Performed reversible transformations between Tc variants within simulations to quantify binding energy differences.

Main Results:

• MDFE simulations accurately reproduced experimental binding free energies within experimental uncertainty (+/-0.5 kcal/mol).
• Binding constants for the studied Tc variants spanned five orders of magnitude.
• Detailed insights into the contributions of individual Tc substituents and their additivity were revealed.

Conclusions:

• Molecular dynamics free energy simulations are a powerful tool for understanding and engineering protein-ligand recognition, complementing experimental methods.
• The study provides a detailed molecular understanding of TetR-Tc interactions, valuable for synthetic biology applications.
• This approach can guide the rational design of novel TetR-Tc systems with tailored binding properties.