GaMF1.39's antibiotic efficacy and its enhanced antitubercular activity in combination with clofazimine, Telacebec, ND-011992, or TBAJ-876.

IMPORTANCE: New drugs are needed to combat multidrug-resistant tuberculosis. The electron transport chain (ETC) maintains the electrochemical potential across the cytoplasmic membrane and allows the production of ATP, the energy currency of any living cell. The mycobacterial engine F-ATP synthase catalyzes the formation of ATP and has come into focus as an attractive and rich drug target. Recent deep insights into these mycobacterial F1FO-ATP synthase elements opened the door for a renaissance of structure-based target identification and inhibitor design. In this study, we present the GaMF1.39 antimycobacterial compound, targeting the rotary subunit γ of the biological engine. The compound is bactericidal, inhibits infection ex vivo, and displays enhanced anti-tuberculosis activity in combination with ETC inhibitors, which promises new strategies to shorten tuberculosis chemotherapy.

Structural Elements Involved in ATP Hydrolysis Inhibition and ATP Synthesis of Tuberculosis and Nontuberculous Mycobacterial F-ATP Synthase Decipher New Targets for Inhibitors.

The F1FO-ATP synthase is required for the viability of tuberculosis (TB) and nontuberculous mycobacteria (NTM) and has been validated as a drug target. Here, we present the cryo-EM structures of the Mycobacterium smegmatis F1-ATPase and the F1FO-ATP synthase with different nucleotide occupation within the catalytic sites and visualize critical elements for latent ATP hydrolysis and efficient ATP synthesis. Mutational studies reveal that the extended C-terminal domain (αCTD) of subunit α is the main element for the self-inhibition mechanism of ATP hydrolysis for TB and NTM bacteria. Rotational studies indicate that the transition between the inhibition state by the αCTD and the active state is a rapid process. We demonstrate that the unique mycobacterial γ-loop and subunit δ are critical elements required for ATP formation. The data underline that these mycobacterium-specific elements of α, γ, and δ are attractive targets, providing a platform for the discovery of species-specific inhibitors.

ATP synthesis in an ancient ATP synthase at low driving forces.

Hyperthermophilic archaea are close to the origin of life. Some hyperthermophilic anaerobic archaea live under strong energy limitation and have to make a living near thermodynamic equilibrium. Obviously, this requires adaptations of the energy-conserving machinery to harness small energy increments. Their ATP synthases often have an unusual motor subunit c that is predicted to prevent ATP synthesis. We have purified and reconstituted into liposomes such an archaeal ATP synthase found in a mesophilic bacterium. The enzyme indeed synthesized ATP at physiological membrane potentials, despite its unusual c subunit, but the minimal driving force for ATP synthesis was found to be even lower than in ATP synthases with usual c subunits. These data not only reveal an intermediate in the transition from ATP hydrolases to ATP synthases but also give a rationale for a bioenergetic adaptation of microbial growth near the thermodynamic equilibrium.

Mutational Analysis of Mycobacterial F-ATP Synthase Subunit δ Leads to a Potent δ Enzyme Inhibitor.

While many bacteria are able to bypass the requirement for oxidative phosphorylation when grown on carbohydrates, Mycobacterium tuberculosis is unable to do so. Differences of amino acid composition and structural features of the mycobacterial F-ATP synthase (α3:ß3:γ:δ:ε:a:b:b':c9) compared to its prokaryotic or human counterparts were recently elucidated and paved avenues for the discovery of molecules interfering with various regulative mechanisms of this essential energy converter. In this context, the mycobacterial peripheral stalk subunit δ came into focus, which displays a unique N-terminal 111-amino acid extension. Here, mutants of recombinant mycobacterial subunit δ were characterized, revealing significant reduction in ATP synthesis and demonstrating essentiality of this subunit for effective catalysis. These results provided the basis for the generation of a four-feature model forming a δ receptor-based pharmacophore and to identify a potent subunit δ inhibitor DeMF1 via in silico screening. The successful targeting of the δ subunit demonstrates the potential to advance δ's flexible coupling as a new area for the development of F-ATP synthase inhibitors.

Functional production of an archaeal ATP synthase with a V-type c subunit in Escherichia coli.

ATP synthases are the key elements of cellular bioenergetics and present in any life form and the overall structure and function of this rotary energy converter is conserved in all domains of life. However, ancestral microbes, the archaea, have a unique and huge diversity in the size and number of ion-binding sites in their membrane-embedded rotor subunit c. Due to the harsh conditions for ATP synthesis in these life forms it has never been possible to address the consequences of these unusual c subunits for ATP synthesis. Recently, we have found a Na+-dependent archaeal ATP synthase with a V-type c subunit in a mesophilic bacterium and here, we have cloned and expressed the genes in the ATP synthase-negative strain Escherichia coli DK8. The enzyme was present in membranes of E. coli DK8 and catalyzed ATP hydrolysis with a rate of 35 nmol·min-1·mg protein-1. Inverted membrane vesicles of this strain were then checked for their ability to synthesize ATP. Indeed, ATP was synthesized driven by NADH oxidation despite the V-type c subunit. ATP synthesis was dependent on Na+ and inhibited by ionophores. Most importantly, ATPase activity was inhibited by DCCD and this inhibition was relieved by addition of Na+, indicating a functional coupling of the F1 and FO domains, a prerequisite for studies on structure-function relationship. A first step in this direction was the exchange of a conserved arginine (Arg530) in the FO motor subunit a which led to loss of ATP synthesis whereas ATP hydrolysis was retained.

A Na+ A1 AO ATP synthase with a V-type c subunit in a mesophilic bacterium.

A1 AO ATP synthases with a V-type c subunit have only been found in hyperthermophilic archaea which makes bioenergetic analyses impossible due to the instability of liposomes at high temperatures. A search for a potential archaeal A1 AO ATP synthase with a V-type c subunit in a mesophilic organism revealed an A1 AO ATP synthase cluster in the anaerobic, acetogenic bacterium Eubacterium limosum KIST612. The enzyme was purified to apparent homogeneity from cells grown on methanol to a specific activity of 1.2 U·mg-1 with a yield of 12%. The enzyme contained subunits A, B, C, D, E, F, H, a, and c. Subunit c is predicted to be a typical V-type c subunit with only one ion (Na+ )-binding site. Indeed, ATP hydrolysis was strictly Na+ -dependent. N,N'-dicyclohexylcarbodiimide (DCCD) inhibited ATP hydrolysis, but inhibition was relieved by addition of Na+ . Na+ was shown directly to abolish binding of the fluorescence DCCD derivative, NCD-4, to subunit c, demonstrating a competition of Na+ and DCCD/NCD-4 for a common binding site. After incorporation of the A1 AO ATP synthase into liposomes, ATP-dependent primary transport of 22 Na+ as well as ΔµNa+ -driven ATP synthesis could be demonstrated. The Na+ A1 AO ATP synthase from E. limosum is the first ATP synthase with a V-type c subunit from a mesophilic organism. This will enable future bioenergetic analysis of these unique ATP synthases.