Insights into the mechanisms and structure of the key coupling subunits ? and ? of the Mycobacterium tuberculosis F1FO ATP synthase, and their potential as novel TB drug target

Tuberculosis is in focus of interest due to the emergence of multi- and extensive drug-resistant TB strains. The Mycobacterium tuberculosis F-ATP synthase is one of the essential enzymes in energy requirement of both proliferating aerobic and hypoxic dormant stage of mycobacterium life cycle. The MtF-ATP synthase, which does not show detectable ATP hydrolysis activity, consists of an FO domain with the subunits a:b:b’:c9-12 and an F1 part (?3:?3:?:?:?:). Subunit ? plays an important role in coupling, whereas subunit ? is also proposed to be responsible for the suppression of hydrolytic activity, preventing excess ATP consumption under low oxygen tensions. Stimulation of latent ATP hydrolysis activity would lead to depleted cellular ATP levels during dormancy and decrease the bacteria’s viability. In our effort to understand the biological role of this, we will (i) determine the NMR structure of M. tuberculosis subunit ? (MT?) using our recombinant MT?, (ii) to see the effect(s) of our generated and ATPase-active Mt?3:?3:?-complex in the presence of the proposed regulator MT? and its truncated forms, and (iii) to identify critical C-terminal residues of MT? in vivo. In addition, we described a key role of MT? and subunit c in the interaction of the TB drug candidate TMC207, which we aim to further understand in vivo. Furthermore, we revealed a low resolution structure of subunit ? of the MtF-ATP synthase and the unique loop of ?165-178, representing a novel TB drug target. We aim to determine the 3D structure of this subunit by NMR spectroscopy, which will be essential to describe the arrangement of the loop inside Mt?1-204 and its interaction with the H+-pumping c-ring. To assess the functional role of Mt?165-178 during catalysis, we mutagenize Mt? in the entire F-ATP synthase by deleting the loop or substitution specific residues, present in the loop of pathogenic forms.