ATP-Bound Conformation of Topoisomerase IV: a Possible Target for Quinolones in Streptococcus pneumoniae

Abstract

Topoisomerase IV, a C 2 E 2 tetramer, is involved in the topological changes of DNA during replication. This enzyme is the target of antibacterial compounds, such as the coumarins, which target the ATP binding site in the ParE subunit, and the quinolones, which bind, outside the active site, to the quinolone resistance-determining region (QRDR). After site-directed and random mutagenesis, we found some mutations in the ATP binding site of ParE near the dimeric interface and outside the QRDR that conferred quinolone resistance to Streptococcus pneumoniae, a bacterial pathogen. Modeling of the N-terminal, 43-kDa ParE domain of S. pneumoniae revealed that the most frequent mutations affected conserved residues, among them His43 and His103, which are involved in the hydrogen bond network supporting ATP hydrolysis, and Met31, at the dimeric interface. All mutants showed a particular phenotype of resistance to fluoroquinolones and an increase in susceptibility to novobiocin. All mutations in ParE resulted in resistance only when associated with a mutation in the QRDR of the GyrA subunit. Our models of the closed and open conformations of the active site indicate that quinolones preferentially target topoisomerase IV of S. pneumoniae in its ATP-bound closed conformation.Topoisomerase IV and DNA gyrase are bacterial type II topoisomerases, which modify DNA topology during the replication process by unlinking DNA and facilitating chromosome segregation (59). Topoisomerase IV forms a C 2 E 2 tetramer involved in segregation of the chromosome at cell division (1, 30, 59). The ParC subunit contains the site of topoisomerization catalyzing the double-stranded DNA break (30, 53), while the ParE subunit catalyzes the hydrolysis of ATP, providing the free energy necessary for these reactions (2, 6, 11). The ParE and ParC subunits share extensive sequence homology with, respectively, the GyrB and GyrA subunits of the DNA gyrase A 2 B 2 tetramer, which catalyzes negative DNA supercoiling during the initiation and elongation processes of DNA replication (16,53).Both topoisomerases are the targets of antibacterial molecules, such as the quinolones and the coumarins. The coumarins inhibit supercoiling and enzyme turnover by preventing the binding and hydrolysis of ATP (45). The quinolones form a ternary complex with the topoisomerases in the presence of DNA, resulting in lethal double-stranded DNA breaks (11). Enzymatic studies and binding assays have also shown that the quinolones can form a complex with the topoisomerases before binding DNA (15, 29); however, some binding data indicate the occurrence of a specific and higher level of binding to the enzyme-DNA complex rather than to the enzyme alone (43,56). These families of molecules, in particular, the fluoroquinolones (FQs), are under continuous development as resistance to antibiotics in pathogenic bacteria has dramatically increased during the last decade (20,22,49).

Contribution of the ATP Binding Site of ParE to Susceptibility to Novobiocin and Quinolones in Streptococcus pneumoniae

“…A fourfold decrease in topoisomerase activity was also previously observed in E. coli GyrB mutants (6). Our results agree with the suggestion, based on the modeling of the 43-kDa N-terminal ParE domain of S. pneumoniae (26), that the H103Y substitution disturbs the ATP-bound conformation of the ATPase active site. In contrast, there was no difference in the ATP requirement when the WT and the altered topoisomerases were tested for relaxation activity using a wide range of concentrations (40 M to 1.5 mM) of ATP.…”

“…ATP binding is required to close the ATP-dependent clamp of the type II topoisomerases (12). Since the presence of the H103Y substitution in ParE could displace the equilibrium in favor of the open conformation (26), it could result in a defect in dimerization of the ParE subunits. To examine this hypothesis, we performed a protein-protein cross-linking assay exactly as described by Nurse et al (18) to assess the ability of the ParE subunits to dimerize in the presence of ADPNP, a nonhydrolyzable ATP analog.…”

“…In previous studies (11,26), we showed in S. pneumoniae that the H103Y substitution in the N terminus of the ParE subunit of the topoisomerase IV resulted in an original phenotype of resistance only if it was associated with an S84F substitution in GyrA. This phenotype was associated with no change in ciprofloxacin susceptibility, decreased susceptibility to sparfloxacin and grepafloxacin, and increased susceptibility to novobiocin (11,26).…”

“…This phenotype was associated with no change in ciprofloxacin susceptibility, decreased susceptibility to sparfloxacin and grepafloxacin, and increased susceptibility to novobiocin (11,26). This position is located in the 43-kDa ATP binding domain (26), which is homologous to the ATPase catalytic site of the E. coli GyrB subunit of DNA gyrase (2).…”

“…Based on these structures, the modeling of the 43-kDa domain of ParE of S. pneumoniae suggested that the substitutions H103Y and H103F would affect preferentially the ATPbound closed conformation of the ATP active site (26).…”