Quinolone-Binding Pocket of DNA Gyrase: Role of GyrB

Autor: Anthony Maxwell, Jonathan G. Heddle
Rok vydání: 2002
Předmět:
Zdroj: Antimicrobial Agents and Chemotherapy. 46:1805-1815
ISSN: 1098-6596
0066-4804
Popis: Quinolone drugs are a large and widely used class of synthetic antibacterial compounds (10, 11, 19). First-generation (acidic) quinolones include nalidixic acid and oxolinic acid (OXO).Subsequent generations have been modified to increase spectrum and potency. The most significant modification has been the addition of a fluorine atom at position C-6 in drugs such as ciprofloxacin (CFX), which results in a considerable increase in activity (30). The newer drugs also commonly contain a secondary amine in addition to the carboxylic acid group common to most quinolones, making them amphoteric rather than acidic (Fig. ​(Fig.1A).1A). More recent modifications that increase drug potency include the presence of a methoxy group at C-8 (48). FIG. 1. (A) Structures of oxolinic acid and ciprofloxacin. (B) Crystal structure of the 92-kDa fragment of yeast topoisomerase II (5). A′, region corresponding to GyrA; B′, region corresponding to GyrB. Residues corresponding to quinolone resistance ... In Escherichia coli the major target for the quinolones is DNA gyrase (14, 35). DNA gyrase is a type II topoisomerase that is able to alter the topological state of DNA by cleaving both strands, passing a double strand of DNA through the gap and resealing the ends (6). In the presence of ATP, this results in negative supercoiling, a reaction unique to gyrase. The crystal structures of the 43-kDa N-terminal domain of GyrB (responsible for ATP hydrolysis and capturing a DNA strand) and a 59-kDa fragment of the 64-kDa N-terminal domain of GyrA (containing the residues for DNA binding and cleavage and the quinolone resistance-determining region [QRDR; residues 67 to 106]) have been determined (24, 39). The structure of the 47-kDa C-terminal domain of GyrB is not known but can be inferred from the analogous enzyme from Saccharomyces cerevisiae, topoisomerase II, for which the crystal structures of a 92-kDa region have been solved (5, 12). Resistance to quinolones commonly arises in DNA gyrase via mutations in the QRDR (44). Such mutations include GyrA(Ser83→Trp), which gives ≈20-fold resistance to a wide range of quinolones (9, 46). This region of GyrA is close to the active site, where the DNA is bound, and close to the tyrosine at position 122, where the phosphotyrosine link between enzyme and DNA is formed. Quinolone drugs bind to gyrase-DNA complexes, but only weakly to either gyrase or DNA (34, 37, 38, 41, 47). It is thought that quinolones bind to a pocket consisting of the QRDR of GyrA and the region of distorted DNA bound to it (17). In such a pocket, the quinolone would interact with elements of both the DNA and the enzyme. Two clinical isolates resistant to quinolones have mutations in the 47-kDa C-terminal region of GyrB (42, 43). These mutations are GyrB(Asp426→Asn), which has been shown to confer resistance to both acidic and amphoteric quinolones, and GyrB(Lys447→Glu), which has been shown to confer resistance to acidic drugs but slight (≈4-fold) hypersensitivity to amphoteric drugs. Modeling these residues on the analogous parts of the 92-kDa yeast topoisomerase II crystal structure (5, 12) reveals that they lie over 40 A from the QRDR and the active site and so appear too far from this region to have a direct effect on drug binding (Fig. ​(Fig.1B1B). There are several possible explanations for the changes in quinolone sensitivity in these mutants: the mutations may change the conformation of the QRDR indirectly, via conformational changes transmitted through the peptide chain or through effects on the reaction cycle of the enzyme that could alter the frequency of a quinolone-sensitive conformation. A further possibility is that these residues of GyrB are directly involved in the quinolone-binding pocket, something which, based on the topoisomerase II structures, would require a large conformational change. Evidence for such a change can be seen in differences between the positions of the equivalent region in the two topoisomerase II structures (5, 12). The fact that residues in this region, in particular Asp424, are required for DNA cleavage (20), which takes place at the active site of GyrA, is further evidence for major changes in the position of the 47-kDa region of GyrB relative to the active site. It is possible that the two residues (Asp426 and Lys447) are themselves part of a quinolone-binding pocket (45) and cause the observed resistance or hypersensitivity through electrostatic interactions with the drug molecules. In this scheme, residues 426 and 447 are hypothesized to lie close to each other. In the wild-type enzyme, the negatively charged Asp residue at position 426 is proposed to interact with the positively charged C-7 substituent of amphoteric quinolones while having no effect on the equivalent region of acidic quinolones. The positively charged Lys at 447 is proposed to interact with the negatively charged carboxyl group of Asp426, providing a neutral environment for binding hydrophobic groups. Thus, the wild-type pocket binds both types of quinolone. In the quinolone resistance mutation GyrB(Asp426→Asn), a negative charge is neutralized, and so binding of both types of drug is reduced. In GyrB(Lys447→Glu), the charge on the residue is reversed, repelling the acidic drugs but actually increasing the affinity for the positively charged C-7 substituent of amphoteric drugs. While providing an explanation for the observed patterns of resistance and hypersensitivity of these two mutants, this model leaves several questions unanswered. The role of DNA, for example, known to be required for quinolone binding to gyrase, is not apparent. It has been suggested that the complete quinolone-binding pocket may involve regions of both GyrB and the QRDR of GyrA (45, 47). In addition, experiments to quantitate binding of quinolones to both GyrB mutants have shown almost identical amounts of enoxacin binding to A2B2447 as to the wild-type protein, while in vitro supercoiling experiments showed that this protein was twofold more sensitive to enoxacin (47) and in vivo experiments suggested it to be fourfold more sensitive (45, 47). The details of the interaction between quinolone drugs, DNA, and gyrase remain unclear. Such information will be useful for designing new and improved drugs to combat the ongoing threat posed by bacterial pathogens. The aim of this work was to further investigate the in vitro resistance and hypersensitivity of the two GyrB mutants and to establish a more complete model of how these residues are involved in interaction with quinolone drugs.
Databáze: OpenAIRE
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