Two potential sites of protein‐protein interaction within the Mycobacterium tuberculosis DNA gyrase complex

Abstract

Recent World Health Organization studies have shown that one‐third of the global population are carriers of latent or active Mycobacterium tuberculosis (TB) infection. With the emergence of antibiotic resistant TB, it is crucial to understand the current drug targets in the treatment of this disease. One of these targets is DNA gyrase, the target of fluoroquinolones and a protein vital to cell function and replication. The goal of this project is to characterize binding interactions between the GyrA and GyrB subunits of M. tuberculosis DNA gyrase. Molecular modeling of the DNA gyrase complex was performed by superimposition of solved GyrA and GyrB M. tuberculosis structures (PDB ID's 3IFZ, 3M4I) over a solved structure of the highly homologous S. pneumoniae Topoisomerase IV protein/DNA complex (PDB ID 3K9F). Structural overlay was performed by the Magic Fit tool in SwissPdb Viewer. The resulting model was used to identify potential sites of charge‐charge interaction between the GyrA and GyrB subunits. Molecular cloning, site‐directed mutagenesis, functional, and structural assays have been utilized to investigate the two identified potential sites of interaction. Initial results from supercoiling assays suggest that the GyrB K658 residue is important for function, as well as a possible interaction occurring between the GyrA R26 residue and the GyrB residues D684 and E685. Gel shift and pull down assays investigating the strength of interaction between wild type and mutant subunits are ongoing.