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We report the high-resolution (1.9 Å) crystal structure of oligomycin bound to the subunit c ₁₀ ring of the yeast mitochondrial ATP synthase. Oligomycin binds to the surface of the c ₁₀ ring making contact with two neighboring molecules at a position that explains the inhibitory effect on ATP synthesis. The carboxyl side chain of Glu59, which is essential for proton translocation, forms an H-bond with oligomycin via a bridging water molecule but is otherwise shielded from the aqueous environment. The remaining contacts between oligomycin and subunit c are primarily hydrophobic. The amino acid residues that form the oligomycin-binding site are 100% conserved between human and yeast but are widely different from those in bacterial homologs, thus explaining the differential sensitivity to oligomycin. Prior genetics studies suggest that the oligomycin-binding site overlaps with the binding site of other antibiotics, including those effective against Mycobacterium tuberculosis , and thereby frames a common “drug-binding site.” We anticipate that this drug-binding site will serve as an effective target for new antibiotics developed by rational design.

The incidence of tuberculosis has been increasing substantially on a worldwide basis over the past decade, but no tuberculosis-specific drugs have been discovered in 40 years. We identified a diarylquinoline, R207910, that potently inhibits both drug-sensitive and drug-resistant Mycobacterium tuberculosis in vitro (minimum inhibitory concentration 0.06 [micro]g/ml). In mice, R207910 exceeded the bactericidal activities of isoniazid and rifampin by at least 1 log unit. Substitution of drugs included in the World Health Organization's first-line tuberculosis treatment regimen (rifampin, isoniazid, and pyrazinamide) with R207910 accelerated bactericidal activity, leading to complete culture conversion after 2 months of treatment in some combinations. A single dose of R207910 inhibited mycobacterial growth for 1 week. Plasma levels associated with efficacy in mice were well tolerated in healthy human volunteers. Mutants selected in vitro suggest that the drug targets the proton pump of adenosine triphosphate (ATP) synthase.

The F 0 F 1 -adenosin triphosphate (ATP) synthase energy conversion under a proton gradient is fundamental in living cells. However, the detailed mechanism has been poorly understood due to the lack of structural information near subunit a. Here, we explored the free energy landscape of the proton transfer pathway at F 0 in the recent cryoelectron microscopy cF 1 F 0 structure of spinach chloroplast ATP synthase by using our coarse-grained model. Our calculated landscape reproduced the correct unidirectionality of the rotation of F 0 . It is found that the directionality is mainly due to the coupling between the change in the electrostatic energy of the c-ring conformational change and the proton transfer pathway. This work provides guidance for investigating other proton-driven mechanochemical processes. The elucidation of the detailed mechanism used by F 0 to convert proton gradient to torque and rotational motion presents a major puzzle despite significant biophysical and structural progress. Although the conceptual model has advanced our understanding of the working principles of such systems, it is crucial to explore the actual mechanism using structure-based models that actually reproduce a unidirectional proton-driven rotation. Our previous work used a coarse-grained (CG) model to simulate the action of F 0 . However, the simulations were based on a very tentative structural model of the interaction between subunit a and subunit c. Here, we again use a CG model but with a recent cryo-EM structure of cF 1 F 0 and also explore the proton path using our water flooding and protein dipole Langevin dipole semimacroscopic formalism with its linear response approximation version (PDLD/S-LRA) approaches. The simulations are done in the combined space defined by the rotational coordinate and the proton transport coordinate. The study reproduced the effect of the protomotive force on the rotation of the F 0 while establishing the electrostatic origin of this effect. Our landscape reproduces the correct unidirectionality of the synthetic direction of the F 0 rotation and shows that it reflects the combined electrostatic coupling between the proton transport path and the c-ring conformational change. This work provides guidance for further studies in other proton-driven mechanochemical systems and should lead (when combined with studies of F 1 ) to a complete energy transduction picture of the F 0 F 1 -ATPase system.

Abstract The passage of protons across membranes through F1Fo-ATP synthases spins their rotors and drives the synthesis of ATP. While the principle of torque generation by proton transfer is known, the mechanisms and routes of proton access and release and their evolution are not fully understood. Here, we show that the entry site and path of protons in the lumenal half channel of mitochondrial ATP synthases are largely defined by a short N-terminal α-helix of subunit-a. In Trypanosoma brucei and other Euglenozoa, the α-helix is part of another polypeptide chain that is a product of subunit-a gene fragmentation. This α-helix and other elements forming the proton pathway are widely conserved across eukaryotes and in Alphaproteobacteria, the closest extant relatives of mitochondria, but not in other bacteria. The α-helix blocks one of two proton routes found in Escherichia coli, resulting in a single proton entry site in mitochondrial and alphaproteobacterial ATP synthases. Thus, the shape of the access half channel predates eukaryotes and originated in the lineage from which mitochondria evolved by endosymbiosis.

Recognition of lipids by proteins is important for their targeting and activation in many signaling pathways, but the mechanisms that regulate such interactions are largely unknown. Here, we found that binding of proteins to the ubiquitous signaling lipid phosphatidic acid (PA) depended on intracellular pH and the protonation state of its phosphate headgroup. In yeast, a rapid decrease in intracellular pH in response to glucose starvation regulated binding of PA to a transcription factor, Opi1, that coordinately repressed phospholipid metabolic genes. This enabled coupling of membrane biogenesis to nutrient availability.

V-ATPases are rotary motor proteins that convert the chemical energy of ATP into the electrochemical potential of ions across cell membranes. V-ATPases consist of two rotary motors, V o and V 1 , and Enterococcus hirae V-ATPase (EhV o V 1 ) actively transports Na + in V o (EhV o ) by using torque generated by ATP hydrolysis in V 1 (EhV 1 ). Here, we observed ATP-driven stepping rotation of detergent-solubilized EhV o V 1 wild-type, aE634A, and BR350K mutants under various Na + and ATP concentrations ([Na + ] and [ATP], respectively) by using a 40-nm gold nanoparticle as a low-load probe. When [Na + ] was low and [ATP] was high, under the condition that only Na + binding to EhV o is rate limiting, wild-type and aE634A exhibited 10 pausing positions reflecting 10-fold symmetry of the EhV o rotor and almost no backward steps. Duration time before the forward steps was inversely proportional to [Na + ], confirming that Na + binding triggers the steps. When both [ATP] and [Na + ] were low, under the condition that both Na + and ATP bindings are rate limiting, aE634A exhibited 13 pausing positions reflecting 10- and 3-fold symmetries of EhV o and EhV 1 , respectively. The distribution of duration time before the forward step was fitted well by the sum of two exponential decay functions with distinct time constants. Furthermore, occasional backward steps smaller than 36° were observed. Small backward steps were also observed during three long ATP cleavage pauses of BR350K. These results indicate that EhV o and EhV 1 do not share pausing positions, Na + and ATP bindings occur at different angles, and the coupling between EhV o and EhV 1 has a rigid component.

The adenosine triphosphate (ATP) synthase in human mitochondria is a membrane bound assembly of 29 proteins of 18 kinds organized into F1-catalytic, peripheral stalk (PS), and c8-rotor ring modules. All but two membrane components are encoded in nuclear genes, synthesized on cytoplasmic ribosomes, imported into the mitochondrial matrix, and assembled into the complex with the mitochondrial gene products ATP6 and ATP8. Intermediate vestigial ATPase complexes formed by disruption of nuclear genes for individual subunits provide a description of how the various domains are introduced into the enzyme. From this approach, it is evident that three alternative pathways operate to introduce the PS module (including associated membrane subunits e, f, and g). In one pathway, the PS is built up by addition to the core subunit b of membrane subunits e and g together, followed by membrane subunit f. Then this b-e-g-f complex is bound to the preformed F1-c8 module by subunits OSCP and F6. The final component of the PS, subunit d, is added subsequently to form a key intermediate that accepts the two mitochondrially encoded subunits. In another route to this key intermediate, first e and g together and then f are added to a preformed F1-c8-OSCP-F6-b-d complex. A third route involves the addition of the c8-ring module to the complete F1-PS complex. The key intermediate then accepts the two mitochondrially encoded subunits, stabilized by the addition of subunit j, leading to an ATP synthase complex that is coupled to the proton motive force and capable of making ATP.

Mitochondrial ATP synthase is a multiprotein complex, which consists of a matrix-localized F1 domain (F1-ATPase) and an inner membrane-embedded Fo domain (Fo-ATPase). The assembly process of mitochondrial ATP synthase is complex and requires the function of many assembly factors. Although extensive studies on mitochondrial ATP synthase assembly have been conducted on yeast, much less study has been performed on plants. Here, we revealed the function of Arabidopsis prohibitin 3 (PHB3) in mitochondrial ATP synthase assembly by characterizing the phb3 mutant. The blue native PAGE (BN-PAGE) and in-gel activity staining assays showed that the activities of ATP synthase and F1-ATPase were significantly decreased in the phb3 mutant. The absence of PHB3 resulted in the accumulation of the Fo-ATPase and F1-ATPase intermediates, whereas the abundance of the Fo-ATPase subunit a was decreased in the ATP synthase monomer. Furthermore, we showed that PHB3 could interact with the F1-ATPase subunits β and δ in the yeast two-hybrid system (Y2H) and luciferase complementation imaging (LCI) assay and with Fo-ATPase subunit c in the LCI assay. These results indicate that PHB3 acts as an assembly factor required for the assembly and activity of mitochondrial ATP synthase.

The ATPase 6 accessory protein 2 (ATP6AP2)/(pro)renin receptor (PRR) is essential for the biogenesis of active vacuolar H(+)-ATPase (V-ATPase). Genetic deletion of ATP6AP2/PRR causes V-ATPase dysfunction and compromises vesicular acidification. Here, we characterized the domains of ATP6AP2/PRR involved in active V-ATPase biogenesis. Three forms of ATP6AP2/PRR were found intracellularly: full-length protein and the N- and C-terminal fragments of furin cleavage products, with the N-terminal fragment secreted extracellularly. Genetic deletion of ATP6AP2/PRR did not affect the protein stability of V-ATPase subunits. The extracellular domain (ECD) and transmembrane domain (TM) of ATP6AP2/PRR were indispensable for the biogenesis of active V-ATPase. A deletion mutant of ATP6AP2/PRR, which lacks exon 4-encoded amino acids inside the ECD (Δ4M) and causes X-linked mental retardation Hedera type (MRXSH) and X-linked parkinsonism with spasticity (XPDS) in humans, was defective as a V-ATPase-associated protein. Prorenin had no effect on the biogenesis of active V-ATPase. The cleavage of ATP6AP2/PRR by furin seemed also dispensable for the biogenesis of active V-ATPase. We conclude that the N-terminal ECD of ATP6AP2/PRR, which is also involved in binding to prorenin or renin, is required for the biogenesis of active V-ATPase. The V-ATPase assembly occurs prior to its delivery to the trans-Golgi network and hence shedding of ATP6AP2/PRR would not affect the biogenesis of active V-ATPase.

Key Points The V-ATPases are composed of a peripheral domain (V 1 ), which is responsible for ATP hydrolysis, and an integral domain (V 0 ), which is responsible for proton translocation. Electron microscopy has shown the existence of multiple stalks that connect V 1 and V 0 . V-ATPases have an important role in various membrane-transport processes, including both endocytosis and intracellular transport. Moreover, the integral V 0 domain has recently been proposed to have a direct role in membrane fusion. V-ATPases in the plasma membrane of specialized cells function in processes such as renal acidification and bone resorption. Several genetic diseases have now been traced to defects in genes that encode V-ATPase subunits, including renal tubular acidosis and osteopetrosis. The V-ATPases resemble the F-ATPases, which normally function in ATP synthesis, and are believed to operate through a rotary mechanism. Information on subunit interactions and topology and the function of individual residues in activity has begun to emerge from studies using site-directed mutagenesis and covalent modification. The yeast V-ATPase requires a unique set of polypeptides for its assembly in the endoplasmic reticulum. Targeting of the V-ATPase seems to be controlled by signals that are located in the 100-kDa a subunit, although interaction with other cellular proteins, such as PDZ proteins, might be important. Several mechanisms have been proposed to regulate V-ATPase activity, including reversible dissociation, disulphide-bond formation and changes in coupling efficiency. A new ubiquitin-ligase component has recently been shown to have a role in regulated assembly of the V-ATPase. The pH of intracellular compartments in eukaryotic cells is a carefully controlled parameter that affects many cellular processes, including intracellular membrane transport, prohormone processing and transport of neurotransmitters, as well as the entry of many viruses into cells. The transporters responsible for controlling this crucial parameter in many intracellular compartments are the vacuolar (H + )-ATPases (V-ATPases). Recent advances in our understanding of the structure and regulation of the V-ATPases, together with the mapping of human genetic defects to genes that encode V-ATPase subunits, have led to tremendous excitement in this field.