The ATP synthase of many anaerobic archaea have the unusual motor subunit c, otherwise only found in eukaryotic V1VO ATPases. The evolutionary transition from synthase to hydrolase is thought to be caused by the doubling of the rotor subunit c, followed by the loss of an ion-binding site. By purifying and reconstituting an ATP synthase with a V-type c subunit, we have unambiguously demonstrated, unexpectedly, the ability of this enzyme to synthesize ATP under physiologically relevant driving forces ranging from 90 to 150 mV. This is a long-awaited answer to an important question in microbial energetics and physiology, especially for life near the thermodynamic limit of ATP synthesis.
Abstract
The structure has been determined by electron cryomicroscopy of adenosine triphosphate (ATP) synthase from Mycobacterium smegmatis. This analysis confirms features in previous descriptions of the enzyme structure, but it also describes other, previously unrecognized, highly important properties, These properties are critical for understanding the mechanism and regulation of mycobacterial enzymes. First, we address not only the three main states in the catalytic cycle previously described, but also eight sub-states that describe the structural and mechanical changes that occur during the 360° catalytic cycle. Second, the auto-inhibitory mechanism of ATP hydrolysis involves not only the engagement of the C-terminal region of the α-subunit in the loop of the γ-subunit, as previously described, but also "fail-safe" mechanisms, including in peripheral stems that enhance engagement the b'-subunit. A third unreported feature is that the fused bδ-subunit contains a repeating domain in its N-terminal region, where two copies of this domain are involved in two of the three N-terminal regions of the α-subunit. Similar attachment mode. Autoinhibition coupled with an associated "fail-safe" mechanism and manner of attachment of the α subunit provide targets for the development of innovative anti-TB drugs. The structure also supports the observations made in bovine ATP synthase that the transmembrane proton motive force is transmitted directly and tangentially to the rotor through the Grotthuss water chain in the polar L-shaped tunnel.
Adenosine triphosphate (ATP) synthase in Mycobacterium tuberculosis is an antibacterial target for the treatment of tuberculosis (TB) with the drug bedaquiline (BD), but despite the drug's effectiveness in the treatment of many extremely and fully resistant tuberculosis mycobacteria Bacillus strains are effective. Resistance to BD has been observed in clinical isolates (1). Additionally, patients treated with BD had a 5-fold higher risk of death than placebo controls, likely due to its effects on human ATP synthase (2). Therefore, one possible strategy to combat the growing threat of tuberculosis is to identify additional inhibitors that act at novel sites of mycobacterial ATP synthase that could be developed into antibiotics without these side effects. A detailed understanding of the structure and properties of this enzyme will aid this quest, and the closely related ATP synthase in the non-pathogenic species M. smegmatis provides an excellent model for the enzyme from pathogens. Like other mycobacteria, M. smegmatis is covered by a complex cellular envelope with three distinct layers, including the inner plasma membrane (IPM), the peptidoglycan-arabinogalactan complex, and the outer membrane, which is associated with Arabinogalactan is covalently linked (3). The membrane domain of mycobacterial ATP synthase is embedded in the IPM, and the catalytic F1 domain of the enzyme extends into the bacterial cytoplasm and attaches to the membrane domain via a central stem and a peripheral stem (PS). Like other ATP synthases in eubacteria, chloroplasts, and mitochondria, this enzyme couples proton motive force (pmf) through respiration-generated IPM to the F1 domain of ATPase synthesis from adenosine diphosphate (ADP) and phosphate via Mechanical rotation mechanism (4). The mycobacterial rotor consists of 9 c-subunits (5), attached to an elongated central stem, consisting of a single copy of the γ- and ε-subunits. This central stalk is common to all F-type ATP synthases and penetrates into the spherical portion of the F1 domain, an aggregate of three α and three β subunits arranged alternately around the central axis (6). Rotation of the asymmetric rotor results in a series of conformational changes in the three catalytic sites located in three of the six interfaces between the α- and β-subunits, leading in turn to the substrates ADP and phosphate Binding, the formation of ATP, and finally the release of ATP.