Bacterial DNA gyrase and topoisomerase IV are selective targets of fluoroquinolones. Topoisomerase IV versus gyrase and Gram-positive versus Gram-negative behavior was studied based on the different recognition of DNA sequences by topoisomerase-quinolone complexes. A careful statistical analysis of preferred bases was performed on a large number (>400) of cleavage sites. We found discrete preferred sequences that were similar when using different enzymes (i.e. gyrase and topoisomerase IV) from the same bacterial source, but in part diverse when employing enzymes from different origins (i.e. Escherichia coli and Streptococcus pneumoniae). Subsequent analysis on the wild-type and mutated consensus sequences showed that: (i) Gn/Cn-rich sequences at and around the cleavage site are hot spots for quinolone-mediated strand breaks, especially for E. coli topoisomerases: we elucidated positions required for quinolone and enzyme recognition; (ii) for S. pneumoniae enzymes only, A and T at positions -2 and +6 are discriminating cleavage determinants; (iii) symmetry of the target sequence is a key trait to promote cleavage and (iv) the consensus sequence adopts a heteronomous A/B conformation, which may trigger DNA processing by the enzyme-drug complex.

The Multiple Copy Simultaneous Search method (MCSS) was used to construct consensus functionality maps for functional group binding in the ATP binding site of DNA gyrase B. To account for the conformational flexibility of the protein active site, which involves small side chain fluctuations as well as large-scale loop motions, the calculations were done for three different conformations of the 24 kDa subdomain of DNA gyrase B. A postprocessing procedure that employs a continuum dielectric model to include solvent effects was used to calculate the binding free energy for every functional group. These results were ranked according to their affinity for DNA gyrase B and clustered using a new procedure based on van der Waals contacts that is better adapted for cases where multiple conformations are being considered. A total of 23 different functional groups were tested. The results gave consensus maps that indicate those functional group binding sites that are insensitive to the specific protein conformation. The maps also demonstrate that functional groups other than those found in the known ligands may bind competitively in the binding sites of known ligands. This suggests numerous scaffolds that can be used in the development of new ligands for the ATP and coumarinic binding sites in DNA gyrase B. Finally, the calculations show the existence of alternative binding sites near the known binding sites that could be targeted in the rational design for new inhibitors.

A secondary structure has been predicted for the heat shock protein HSP90 family from an aligned set of homologous protein sequences by using a transparent method in both manual and automated implementation that extracts conformational information from patterns of variation and conservation within the family. No statistically significant sequence similarity relates this family to any protein with known crystal structure. However, the secondary structure prediction, together with the assignment of active site positions and possible biochemical properties, suggest that the fold is similar to that seen in N-terminal domain of DNA gyrase B (the ATPase fragment).