Abstract
Mycobacterium tuberculosis DNA gyrase, an indispensable nanomachine involved in the regulation of DNA topology, is the only type II topoisomerase present in this organism and is hence the sole target for quinolone action, a crucial drug active against multidrug-resistant tuberculosis. To understand at an atomic level the quinolone resistance mechanism, which emerges in extensively drug resistant tuberculosis, we performed combined functional, biophysical and structural studies of the two individual domains constituting the catalytic DNA gyrase reaction core, namely the Toprim and the breakage-reunion domains. This allowed us to produce a model of the catalytic reaction core in complex with DNA and a quinolone molecule, identifying original mechanistic properties of quinolone binding and clarifying the relationships between amino acid mutations and resistance phenotype of M. tuberculosis DNA gyrase. These results are compatible with our previous studies on quinolone resistance. Interestingly, the structure of the entire breakage-reunion domain revealed a new interaction, in which the Quinolone-Binding Pocket (QBP) is blocked by the N-terminal helix of a symmetry-related molecule. This interaction provides useful starting points for designing peptide based inhibitors that target DNA gyrase to prevent its binding to DNA.
Highlights
Type II topoisomerases are essential and ubiquitous nucleic aciddependent nanomachines involved in the regulation of DNA topology and especially in the regulation of DNA supercoiling [1]
The GA57BK-TopBK complex has DNA cleavage activity, showing that these domains possess all determinants for DNA cleavage and confirming that these two domains form the catalytic reaction core of the M. tuberculosis DNA gyrase (Figure 2A and B)
The structure of the breakage-reunion domain reveals a new interaction promising for drug design, whilst the high resolution structures of the Toprim domain highlights two disordered regions that play a crucial role during the catalytic reaction of DNA gyrase
Summary
Type II topoisomerases are essential and ubiquitous nucleic aciddependent nanomachines involved in the regulation of DNA topology and especially in the regulation of DNA supercoiling [1]. Except archaeal topoisomerase VI [2,3], they all belong to a single protein superfamily, the type IIA topoisomerases, sharing homologous sequences and overall structures [4]. They have acquired distinct functions during evolution [1]. Bacterial genomes usually encode two type IIA enzymes, DNA gyrase and topoisomerase IV. DNA gyrase facilitates DNA unwinding at replication forks and topoisomerase IV has a specialized function in mediating the decatenation of interlocked daughter chromosomes [5]. The M. tuberculosis DNA gyrase exhibits a different activity spectrum as compared to other DNA gyrases, namely it supercoils DNA with an efficiency comparable to that of other DNA gyrases but shows enhanced relaxation, DNA cleavage, and decatenation activities [7]
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