Tuberculosis is caused by the pathogen Mycobacterium tuberculosis (M. tb). This microbe can invade various organs of the human body, such as the lungs, intestines and spine. The most common form is lung tuberculosis.Tuberculosis claims about 2 million lives each year. It is estimated that about one-third of the world’s population is infected with M. tb, and there are around 10 million new cases of TB every year. In China, about 44.5% of the population is infected with M. tb. The devastating impact of this pathogen on world health has been exacerbated since the early 1990s due to the emergence of multidrug-resistant tuberculosis, and in 1993 the World Health Organization (WHO) declared tuberculosis a global emergency.

Fluoroquinolones are the most promising second-line antibiotics for TB treatment and have the potential to become part of a new first-line treatment against TB. They have been widely used to treat TB patients infected with M. tb strains that are resistant to first-line drugs. The drug target of fluoroquinolones is gyrase and mutations in gyrase coding genes gyrA and gyrB are correlated with its resistance. DNA gyrase, which can introduce negative supercoils and remove positive supercoils ahead of replication form, belongs to the type IIA enzymes. The type IIA topoisomerases include prokaryotic DNA topoisomerase II (DNA gyrase and topo IV) and eukaryotic DNA topoisomerase II, Eukaryotic topoisomerase II is a homodimer whereas bacterial DNA gyrase exists as an A2B2 heterotetramer. DNA gyrase is the primary target of many important antibacterial agents. Quinlones can trap the complex of the enzyme and DNA and stabilize the cleavage complex, thereby blocking DNA replication and leading to cell death.

Structural and functional studies, particularly with the enzymes from Escherichia coli and Saccharomyces cerevisiae, have established a two-gate mechanism for type IIA topoisomerase catalysis, but the role of GyrB C-terminal domain during the catalysis is not well understood. It is known that DNA gyrase can processively introduces negative supercoils. A simple mechanochemical model was proposed that processivity depends on a kinetic competition between dissociation and rapid, tension-sensitive DNA wrapping. However, the molecular basis for the mechanism of processivity is not known.

Professor Lijun Bi and their colleagues at the Institute of Biophysics and Wuhan Institute of Virology, Chinese Academy of Sciences carried out studies on the structure and function of DNA gyrase from M. tuberculosis. They first investigated GyrA DNA-binding activity (Nucleic Acides Research, 2006，34(19):5650-9) and then Professor Lijun Bi and Professor Dacheng Wang co-presented the dimer structure of GyrB C-terminal domain from M. tuberculosis (Nucleic Acids Res. 2009, 37, 5908-5916). Further functional analyses were done in this study (Nucleic Acids Res. (2011) 39 (19): 8488-8502. doi: 10.1093/nar/gkr553). GyrB was observed to exist as a slow monomer-dimer equilibrium in solution using cross-linking and analytical ultracentrifugation assays. The cross-linked dimer of GyrB bound GyrA very weakly, but bound dsDNA with a much higher affinity than that of the monomer state. Moreover, cross-linking and far-Western experiments imply that the dimer state of GyrB is involved in the ternary GyrA/GyrB/DNA complex. These results suggest that the dimer state of GyrB is an active form. Mutational studies reveal that the dimer structure of GyrB represents a state before DNA cleavage, involving in the initial complex, but also possibly forms between present between the cleavage and reunion steps during processive transport, which may provide a structural basis for the mechanism of processivity in DNA gyrase (see Figure). These results highlight the role of the dimer structure in regulating the enzyme activities and suggest that the dimer interface might be a new potential target for drug design.

Fig. A working model for strand passage in DNA gyrase. The ATPase, B′ and A′ domains are in pink, red and blue, respectively; the G and T segment are both shown in yellow. For clarity GyrA–CTD is not included. In (a) the dimer structures of GyrB (ATPase and B′ domains) and the A′ domain bind the G segment to form the initial complex. The ATPase domains dimerize upon the binding of ATP, thereby capturing the T segment; the B′ dimer is opened, accompanying the distortion of the G segment, and finally converts to the cleavage–competent complex (b), where the G segment is cleaved by the active centre formed by the B′ and A′ domains. A series of conformational changes occur and the cleaved G segment is separated (c), including a conformational change shown in (d). Then an electrostatic potential gradient within the enzyme might be generated, thereby drawing the T segment through the break and towards the central hole (d). In (e) the enzyme recovers the conformation of the initial complex as shown in (a) and thus b–c–d–e–b forms a cycle of consecutive strand passage. The G segment is resealed and the T segment released from the exit gate (f).