Mass Spectrometry Reveals That the Antibiotic Simocyclinone D8 Binds to DNA Gyrase in a “Bent-Over” Conformation: Evidence of Positive Cooperativity in Binding

摘要

DNA topoisomerases are enzymes that control DNA topology and are vital targetsnfor antimicrobial and anticancer drugs. Here we present a mass spectrometry study of complexesnformed between the A subunit of the topoisomerase DNA gyrase and the bifunctional inhibitornsimocyclinone D8 (SD8), an antibiotic isolated from Streptomyces. These studies show that, in annalternative mode of interaction to that found by X-ray crystallography, each subunit binds a singlenbifunctional inhibitor with separate binding pockets for the two ends of SD8. The gyrase subunitsnform constitutive dimers, and fractional occupancies of inhibitor-bound states show that there isnstrong allosteric cooperativity in the binding of two bifunctional ligands to the dimer.We show thatnthe mass spectrometry data can be fitted to a general model of cooperative binding via an extensionnof the “tight-binding” approach, providing a rigorous determination of the dissociation constantsnand degree of cooperativity. This general approach will be applicable to other systems withmultiple binding sites and highlightsmassnspectrometry’s role as a powerful emerging tool for unraveling the complexities of biomolecular interactions.