TY - JOUR

T1 - Probing the binding of coumarins and cyclothialidines to DNA gyrase

AU - Kampranis, S C

AU - Gormley, N A

AU - Tranter, R

AU - Orphanides, G

AU - Maxwell, A

PY - 1999/2/16

Y1 - 1999/2/16

N2 - DNA gyrase is the target of a number of antibacterial agents, including the coumarins and the cyclothialidines. To extend our understanding of the mechanism of action of these compounds, we have examined the previously published 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 and mutant proteins. Limited proteolysis of gyrase revealed that binding of these antibiotics is associated with a characteristic proteolytic fingerprint, suggesting a drug-induced conformational change. The ability of the mutants to bind the drugs was studied by testing their ability to induce the coumarin-associated proteolytic signature and to bind to a novobiocin-affinity column. To analyze further the interaction of the drugs with gyrase, we studied the binding using surface plasmon resonance. Mutation of Asn46 to Asp has only a modest effect on the binding of coumarins, while an Asn46 to Leu mutation results in a 10-fold decrease in the affinity. Mutation of Asp73 to Asn completely abolishes binding to both coumarins and cyclothialidines. Mutations at these residues also abolish ATP hydrolysis, explaining the inability of such mutations to occur spontaneously.

AB - DNA gyrase is the target of a number of antibacterial agents, including the coumarins and the cyclothialidines. To extend our understanding of the mechanism of action of these compounds, we have examined the previously published 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 and mutant proteins. Limited proteolysis of gyrase revealed that binding of these antibiotics is associated with a characteristic proteolytic fingerprint, suggesting a drug-induced conformational change. The ability of the mutants to bind the drugs was studied by testing their ability to induce the coumarin-associated proteolytic signature and to bind to a novobiocin-affinity column. To analyze further the interaction of the drugs with gyrase, we studied the binding using surface plasmon resonance. Mutation of Asn46 to Asp has only a modest effect on the binding of coumarins, while an Asn46 to Leu mutation results in a 10-fold decrease in the affinity. Mutation of Asp73 to Asn completely abolishes binding to both coumarins and cyclothialidines. Mutations at these residues also abolish ATP hydrolysis, explaining the inability of such mutations to occur spontaneously.

KW - Adenosine Triphosphate

KW - Arginine

KW - Asparagine

KW - Aspartic Acid

KW - Binding Sites

KW - Coumarins

KW - DNA Topoisomerases, Type II

KW - Enzyme Inhibitors

KW - Hydrolysis

KW - Kinetics

KW - Mutagenesis, Site-Directed

KW - Peptides, Cyclic

KW - Surface Plasmon Resonance

KW - Topoisomerase II Inhibitors

KW - Trypsin

U2 - 10.1021/bi982320p

DO - 10.1021/bi982320p

M3 - Journal article

C2 - 10026280

VL - 38

SP - 1967

EP - 1976

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 7

ER -