Conformational changes in DNA gyrase revealed by limited proteolysis

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• DNA gyrase is a crucial enzyme for DNA replication and transcription.
• Understanding its conformational changes is key to developing new antibacterial agents.
• Limited proteolysis is a valuable tool for probing enzyme structure and dynamics.

Purpose of the Study:

• To identify conformational changes in DNA gyrase using limited proteolysis.
• To investigate the impact of quinolone binding and ATP-induced dimerization on gyrase conformation.
• To differentiate quinolone-induced conformational changes from other functional states.

Main Methods:

• Limited proteolysis was employed to analyze DNA gyrase structure.
• Proteolytic fingerprints were generated for various gyrase-DNA-ligand complexes.
• Mutant enzymes and non-hydrolyzable ATP analogs were used to probe specific states.

Main Results:

• Quinolone binding to the enzyme-DNA complex protects the C-terminal domain of the B protein.
• A quinolone-resistant mutant lacks the characteristic quinolone-induced proteolytic signature.
• ATP-induced dimerization of B subunits protects the N-terminal domain of the B subunit.
• Quinolones do not prevent ATP-induced dimerization but alter the resulting complex's proteolytic signature.

Conclusions:

• Limited proteolysis effectively distinguishes different conformational states of DNA gyrase.
• Quinolone binding induces a unique conformational change distinct from DNA cleavage.
• The study provides insights into the mechanism of quinolone action and resistance.
• Four distinct conformational states of DNA gyrase were identified based on proteolytic signatures.