Probing the binding of coumarins and cyclothialidines to DNA gyrase

DNA gyrase is the target of a number of antibacterial agents, including the coumarins and the cyclothialidines. To extend our understanding of the mec hanism of action of these compounds, we have examined the previously publis hed crystal structures of the complexes between the 24 kDa fragment of GyrB and coumarin and cyclothialidine drugs and made mutations by site-directed mutagenesis. We used proteolysis as a probe of drug binding to wild-type a nd mutant proteins. Limited proteolysis of gyrase revealed that binding of these antibiotics is associated with a characteristic proteolytic fingerpri nt, suggesting a drug-induced conformational change. The ability of the mut ants to bind the drugs was studied by testing their ability to induce the c oumarin-associated proteolytic signature and to bind to a novobiocin-affini ty column. To analyze further the interaction of the drugs with gyrase, we studied the binding using surface plasmon resonance. Mutation of Asn(46) to Asp has only a modest effect on the binding of coumarins, while an Asn(46) to Leu mutation results in a 10-fold decrease in the affinity. Mutation of Asp(73) to Asn completely abolishes binding to both coumarins and cyclothi alidines. Mutations at these residues also abolish ATP hydrolysis, explaini ng the inability of such mutations to occur spontaneously.