THE HIGH-RESOLUTION CRYSTAL-STRUCTURE OF A 24-KDA GYRASE B-FRAGMENT FROM ESCHERICHIA-COLI COMPLEXED WITH ONE OF THE MOST POTENT COUMARIN INHIBITORS, CLOROBIOCIN

Coumarin antibiotics, such as clorobiocin, novobiocin, and coumermycin A1, inhibit the supercoiling activity of gyrase by binding to the gyr ase B (GyrB) subunit. Previous crystallographic studies of a 24-kDa N- terminal domain of GyrB from E. coli complexed with novobiocin and a c yclothialidine analogue have shown that both ligands act by binding at the ATP-binding site. Clorobiocin is a natural antibiotic isolated fr om several Streptomyces strains and differs from novobiocin in that th e methyl group at the 8 position in the coumarin ring of novobiocin is replaced by a chlorine atom, and the carbamoyl at the 3' position of the noviose sugar is substituted by a 5-methyl-2-pyrrolylcarbonyl grou p. To understand the difference in affinity, in order that this inform ation might be exploited in rational drug design, the crystal structur e of the 24-kDa GyrB fragment in complex with clorobiocin was determin ed to high resolution. This structure was determined independently in two laboratories, which allowed the validation of equivalent interpret ations. The clorobiocin complex structure is compared with the crystal structures of gyrase complexes with novobiocin and 5'-adenylyl-beta,g amma-imidodiphosphate, and with information on the bound conformation of novobiocin in the pal-novobiocin complex obtained by heteronuclear isotope-filtered NMR experiments in solution. Moreover, to understand the differences in energetics of binding of clorobiocin and novobiocin to the protein, the results from isothermal titration calorimetry are also presented. (C) 1997 Wiley-Liss, Inc.