Using macromolecular phasing and refinement techniques for a small molecule gyrase inhibitor

Abstract

Topoisomerases resolve DNA topoisomers, which is essential for faithful replication and transcription. Gyrases are type II topoisomerases unique to bacteria and well-known targets for antibiotics. Unfortunately, currently used gyrase inhibitors suffer from increased resistance, necessitating structure-based drug design on novel chemical classes. During such efforts, a crystal of a small molecule gyrase inhibitor grew fortuitously from an experiment aimed at a protein/ligand complex. The crystal diffracted insufficiently for standard direct phasing methods to work, and the diffraction data to 1.3 Å resolution did not contain anomalous signal for experimental phasing. Using macromolecular replacement techniques with fragments of the ligand as search models that were predicted to be planar, an initial electron density map was obtained that allowed building of a complete structure. While standard small molecule refinement was unstable, macromolecular refinement protocols were applied successfully to an Rfree of ∼20%, likely aided by the appreciably large solvent content of the small molecule crystal (34-51%, depending on how it is calculated). Comparison of the small molecule structure to a chemically related ligand in complex with the ATPase domain of P. aeruginosa gyrase reveals quite different conformations due to variations in torsion angles connecting aromatic and aliphatic moieties. Such information may prove useful in drug design. While the present case may be among the first in using “macromolecular” methods for phasing and refinement of “small-molecule data,” we hope that in the future this approach proves useful in extracting information from sub-standard small-molecule diffraction data.