Crystal Structure Reveals Dual Antibody Binding Sites

A team of molecular biologists has reported the crystal structure of the complex formed by the antibody simocyclinone D8 with its target the critical Escherichia coli enzyme, DNA gyrase.

While the structure of the simocyclinones includes an aminocoumarin moiety, a key feature of novobiocin, coumermycin A1, and clorobiocin, which also target DNA gyrase, simocyclinones behave strikingly differently from these compounds. Simocyclinone D8 is a potent inhibitor of gyrase supercoiling, with a 50% inhibitory concentration lower than that of novobiocin. However, it does not competitively inhibit the DNA-independent ATPase reaction of GyrB, which is characteristic of other aminocoumarins.

Simocyclinone D8 also inhibits DNA relaxation by gyrase but does not stimulate cleavage complex formation, unlike quinolones, the other major class of gyrase inhibitors; instead, it abrogates both calcium and quinolone-induced cleavage complex formation. Binding studies suggest that simocyclinone D8 interacts with the N-terminal domain of GyrA. Simocyclinones inhibit an early step of the gyrase catalytic cycle by preventing binding of the enzyme to DNA.

Investigators at the John Innes Center (Norwich, United Kingdom) have been studying the simocyclinone family of antibiotics for several years. In the current study, published in the December 4, 2009, issue of the journal Science, they reported that X-ray crystallography revealed two binding pockets that separately accommodated the aminocoumarin and polyketide moieties of the antibiotic. These pockets were close to, but distinct from, the quinolone-binding site, consistent with observations that several mutations in this region confer resistance to both agents. Biochemical studies showed that the individual moieties of simocyclinone D8 were comparatively weak inhibitors of gyrase relative to the parent compound, but their combination generated a more potent inhibitor.

"A completely new way to beat bacteria is an exciting find at a time when resistance to existing antibiotics is growing," said senior author Dr. Anthony Maxwell, professor of biological chemistry at the John Innes Center. "If you can knock out this enzyme, you have a potential new drug. That there are two pockets means that it might require simultaneous mutations in both pockets for the bacteria to acquire full resistance to the drug, which is much less likely."

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