On the other hand, given the lower frequency of complex V deficiency compared to the other OXPHOS deficiencies, routine screening of all nuclear structural genes is rarely implemented in a diagnostic setting. Whole genome or whole exome screening could counter this problem and possibly solve some of the hitherto unknown genetic defects causing complex V deficiency.
Finally, the biggest challenge will be to find a tailored curative therapy for this patient group. Large-scale and high-throughput compound screening is needed to find a possible pharmacological approach.
For mtDNA defects, gene-shifting and germline techniques are promising, but much more and thorough experimental research is needed before this can be implemented in the patient setting. In conclusion, mitochondrial ATP synthase has been and still is a popular research topic.
Thanks to sustained effort, many aspects of this intriguing protein have been elucidated. This knowledge will guide further physio patho logical studies, paving the way for future therapeutic interventions. The authors confirm independence from the sponsors; the content of the article has not been influenced by the sponsors. This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author s and source are credited.
Competing interest: None declared. National Center for Biotechnology Information , U. Journal of Inherited Metabolic Disease. J Inherit Metab Dis.
Published online Aug Jonckheere , Jan A. Smeitink , and Richard J. Jan A. Richard J. Author information Article notes Copyright and License information Disclaimer. Corresponding author. This article has been cited by other articles in PMC.
Abstract Human mitochondrial mt ATP synthase, or complex V consists of two functional domains: F 1 , situated in the mitochondrial matrix, and F o , located in the inner mitochondrial membrane. ATP synthase: architecture Fig. Open in a separate window. Table 1 Subunit composition of human, yeast and E.
Stoichiometry Bacteria Mitochondria E. Complex V assembly Current knowledge about the assembly of ATP synthase is mainly based on research performed on assembly-deficient yeast mutants Kucharczyk et al. Complex V di- and oligomerization An important role of subunits a and A6L is the stabilization of holocomplex V Wittig et al. Complex V and mitochondrial morphology The association of ATP synthase dimers as generating the tubular cristae has been hypothesized by Allen Allen Biochemical diagnosis Measurement of the mitochondrial energy-generating system MEGS capacity in fresh muscle tissue is a powerful tool to assess mitochondrial function and to detect deficiencies of complex V and other OXPHOS complexes.
Modifiers Phenotypical variations between patients harboring the same mtDNA mutation have classically been attributed to mtDNA heteroplasmy. Therapy Current available treatment options for patients with mitochondrial diseases are mainly supportive. Antioxidants As mentioned above, complex V mutations can increase ROS production which is deleterious for the cell.
Affecting heteroplasmy of the mtDNA gene-shifting This genetic approach aims to force a shift in heteroplasmy, reducing the ratio of mutant to wild-type genomes also called gene-shifting DiMauro et al. Allotopic expression Here, a normal version of a mutant mtDNA-encoded protein is imported into the nucleus. Xenotopic expression The correction here implies the transfection of mammalian cells with either mitochondrial or nuclear genes from other organisms encoding the protein of interest DiMauro et al.
Oligomycin It has been shown that culturing heteroplasmic m. Germline therapy It has been proposed that nuclear transfer techniques may be an approach for the prevention of transmission of human mtDNA disease Sato et al.
Metaphase II spindle transfer between unfertilized metaphase II oocytes It has been demonstrated in mature non-human primate oocytes Macaca mulatta that the mitochondrial genome can be efficiently replaced by spindle-chromosomal complex transfer from one egg to an enucleated, mitochondrial-replete egg Tachibana et al.
Pronuclear transfer between zygotes This is essentially the same procedure, except that the nuclear material, both the male and female pronucleus, is removed after fertilization Tachibana et al.
Prenatal and preimplantation diagnosis Since current therapeutic options for mitochondrial diseases are insufficient, the possibility of prenatal diagnosis for fetuses at risk is a valuable alternative. Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author s and source are credited.
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Biochemistry 38, — Mitchell, P. Coupling of phosphorylation to electron and hydrogen transfer by a chemiosmotic mechanism. Mohlmann, T. Moore, A. Douce and D. Day Berlin: Springer , — Cellular respiration in the mitochondria is a widely studied process that incorporates chemiosmosis for the production of ATP. Mitochondria, the chief organelles producing ATP, are absent in prokaryotic organisms.
In the absence of mitochondria, archaea and bacteria maneuver chemiosmosis to produce ATP through photophosphorylation. The electrochemical energy built through the difference in proton concentration and separation of charge across inner mitochondrial membrane translates to the proton motive force PMF.
This also satisfies a main criterion stated by Mitchell for the chemiosmotic coupling to occur: the inner mitochondrial membrane must be impermeable to protons.
Thus, protons are compelled to re-enter matrix through F 0 while F 1 catalyzes the synthesis of ATP [ 16 ]. The Electron transport chain composed of four different multi-subunit complexes transfer electrons e- in a sequential manner ultimately reducing O 2 to H 2 O. Electron transfer is coupled to a vectorial proton translocation outdoor into the matrix via three of the four complexes I, III and IV.
Protons gather and create an electrochemical gradient throughout the inner mitochondrial membrane. This osmotic potential is used to power ATP synthesis when protons re-enter the mitochondrial matrix through ATP synthase [ 13 ].
The equation for reaction catalyzed is:. There are only slight variations in its structure in the chloroplast and in the mitochondria. The chloroplast ATPase has two isoforms and in the mitochondria it has additional subunits. Besides these differences, ATPases are structurally and functionally similar. The F 0 part, bound to inner mitochondrial membrane is involved in proton translocation, whereas the F 1 part found in the mitochondrial matrix is the water soluble catalytic domain.
F 1 is the first factor recognized and isolated from bovine heart mitochondria and is involved in oxidative phosphorylation. F 0 was named so as it is a factor that conferred oligomycin sensitivity to soluble F 1 [ 18 ].
Schematic subunit composition of ATP synthase. The structure of enzyme ATP synthase mimics an assembly of two motors with a shared common rotor shaft and stabilized by a peripheral stator stalk. Bacterial F 0 has the simplest subunit structure consisting a 1 , b 2 and c subunits. Other additional subunits such as subunit e, f, g, and A6L extending over the membrane cohort with F 0 [ 5 , 10 , 20 ].
Paul Boyer proposed a simple catalytic scheme, commonly known as the binding change mechanism, which predicted that F-ATPase implements a rotational mechanism in the catalysis of ATP [ 21 ]. The movement of subunits within the ATP synthase complex plays essential roles in both transport and catalytic mechanisms. Another subsequent change in conformation brings about the release of ATP. These conformational changes are accomplished by rotating the inner core of the enzyme. The core itself is powered by the proton motive force conferred by protons crossing the mitochondrial membrane.
The binding-change mechanism as seen from the top of the F 1 complex. There are three catalytic sites in three different conformations: loose, open, and tight.
As a result, ATP is released from the enzyme. In step 2, substrate again binds to the open site, and another ATP is synthesized at the tight site [ 25 ]. Masamitsu et. Conformational transitions that are significant in rotational catalysis are directed by the passage of protons through the F 0 assembly of ATP synthase. On the other hand, when the proton concentration is higher in the mitochondrial matrix, the F 1 motor reverses the F 0 motor bringing about the hydrolysis of ATP to power translocation of protons to the other side of membrane.
A team of Japanese scientists have succeeded in attaching magnetic beads to the stalks of F 1 -ATPase isolated in vitro , which rotated in presence of a rotating magnetic field.
Additionally, ATP was hydrolyzed when the stalks were rotated in the counterclockwise direction or when they were not rotated at all [ 26 ]. Defects or mutations in this enzyme are known to cause many diseases in humans. The first defect in ATP synthase was reported by Houstek et. It was postulated that mutations in some factors explicitly involved in the assembly of ATP synthase could have caused the defect [ 27 ].
Kucharczyk et. A mutation in one or many of the subunits in ATPase synthase can cause these diseases [ 28 ]. These diseases also result decrement in intermediary metabolism and functioning of the kidneys in removing acid from the body due to increased production of free oxygen radicals.
Dysfunction of F 1 specific nuclear encoded assembly factors causes selective ATPase deficiency [ 31 ]. Similar inborn defects in the mitochondrial F-ATP synthase, termed ATP synthase deficiency, have been noted where newborns die within few months or a year. Current research on ATP synthase as a potential molecular target for the treatment for some human diseases have produced positive consequences.
Recently, ATPase has emerged as appealing molecular target for the development of new treatment options for several diseases. ATP synthase is regarded as one of the oldest and most conserved enzymes in the molecular world and it has a complex structure with the possibility of inhibition by a number of inhibitors. In addition, structure elucidation has opened new horizons for development of novel ATP synthase-directed agents with plausible therapeutic effects.
More than natural and synthetic inhibitors have been classified to date, with reports of their known or proposed inhibitory sites and modes of action [ 30 ]. We look to explore a few important inhibitors of ATP synthase in this paper. A drug, diarylquinoline also known as TMC developed against tuberculosis is known to block the synthesis of ATP by targeting subunit c of ATP synthase of tuberculosis bacteria.
Whereas the N-terminal region of H5 a dark green is kinked towards the c-ring in the yeast complex, it extends towards the lumen in the E. Unlike its yeast homolog, the N-terminus of E. Since no homologs were reported for any of these subunits in E.
Based on the matching position and topology of the transmembrane helices as well as conserved positions of interactions with subunit a , we identified all six associated subunits, which are structurally conserved, but display no significant sequence similarity to yeast counterparts Figure 3. These data show that despite sequence divergence, the assembly of the central F o subunits around subunit a is architecturally conserved between Euglenozoa and Metazoa.
A Side view of the conserved E. Transmembrane helices with structural equivalents in yeast are labelled. B Top view of the superimposed conserved F o subcomplexes from E. Although subunit k does not superimpose well, it occupies the same position relative to the H5 a. The striking architectural divergence of the E. Only subunit b is truncated. The extensions of the conserved F o subunits are mostly involved in forming interactions with the euglenozoa-specific subunits, thus providing a platform for the observed increased molecular mass of the F o Figure 4—figure supplement 1A,C.
The additional 13 euglenozoa-specific F o subunits determine the architecture of the ATP synthase dimer, giving the F o a markedly different overall shape, making it almost three times the size of its yeast counterpart Figure 4—figure supplement 1. They contribute to the dimerization interface, the peripheral stalk and F o periphery.
A similar lumenal interaction has previously been reported in the bovine ATP synthase, where subunit e extends from the membrane to contact the c-ring Zhou et al. In the porcine ATP synthase tetramer, subunit e has been proposed to interact with the 6.
Other euglenozoa-specific F o subunits contain structural domains that were shown to be functionally important in mitochondria Figure 1E to G. ATPTB1 is a membrane-associated protein on the matrix side of the F o periphery that adopts an Mdmlike fold, which was shown to associate with yeast mitochondrial ribosomes at the inner mitochondrial membrane Frazier et al. ATPTB3 is an isocitrate dehydrogenase ortholog that adopts a Rossman fold located at the tip of the peripheral stalk. The defining feature of mitochondrial ATP synthases is the formation of dimers in the crista membrane.
Our atomic model of the E. The extensive dimer contacts are stacked across three layers: the matrix side, the transmembrane region and the lumenal side Figure 4A and Figure 4—figure supplement 1A,C. ATPEG2 contributes to the dimer interface with both a transmembrane helix that interacts with ATPEG1 and its termini, which link the two F o -parts, each extending into the rotor-stator interface of one monomer.
Thus, despite the presence of conserved F o subunits and the contribution to their extensions to the dimer interface Figure 4—figure supplement 2 , the E. A and B Views of the dimer interface along A and perpendicular B to the membrane plane. The dimerisation motifs interacting subunits coloured are stacked along the C 2 -symmetry axis and formed by two copies of subunit d red and ATPEG1 blue , which interacts with its symmetry-related copy, as well as ATPEG2 green and subunit f yellow.
Asterisks in B indicate positions of lipid-binding sites. C to G Close-ups of the lipid-binding sites indicated in B. Interacting residues subunits coloured include at least one arginine residue. Density shown as grey mesh. In addition to the described protein-protein interactions, we identified nine bound phospholipids occupying the dimer interface Figure 4C to G, Figure 4—figure supplement 2. Five of them are cardiolipin molecules linking dimerising subunits close to the C 2 -symmetry axis Figure 4C to E.
These protein-lipid interactions indicate a functionale role of cardiolipin in the stabilisation of the dimer contacts, which is consistent with its proposed role in mediating subunit interactions between the transmembrane helices in mitochondrial supercomplexes Mileykovskaya and Dowhan, ; Wu et al. The cryo-EM map of the membrane region is shown as mesh.
Proton translocation occurs at the rotor-stator-interface, which is canonically formed in the membrane by horizontal helices H5 a , H6 a and the c-ring Allegretti et al. In the E. A View from the c-ring towards the membrane-embedded stator subunits. H5 a and H6 a are augmented by the tilted, amphipathic H1 EG4 brown. Cardiolipin molecules flanking subunit a are shown in red tails of acyl chains are mostly disordered and shown only for illustration.
Proton half-channels on the lumen and matrix side are shown in orange and red respectively. Remaining subunits not shown for clarity. The conserved R and the H at the lumen channel exit are shown with interacting residues. Inside the F o , the lumen channel is confined by transmembrane helices of subunits f and b.
Arrows indicate proposed path of proton flow. C Polar and protonatable residues between R and the matrix-side half channel red mesh. Subunit k contributes a horizontal helix H1 k to the rotor-stator interface. The E. Protons enter the membrane region via the lumenal half-channel, which we traced as an internal cavity of the atomic model.
Inside the F o region, the lumenal half-channel is lined by the conserved transmembrane helices of subunits f and b and extends between H5 a , H6 a , as previously suggested Guo et al.
Proton translocation to the rotor-stator interface results in protonation of the conserved glutamate residue E86 in E. This proton transfer has been suggested to be ultimately mediated by a glutamate of H6 a E in S. This residue pair facing the lumenal half-channel is conserved in yeast, mammals and Polytomella Gu et al.
The absence of an acidic residue from the exit of the lumenal proton half-channel indicates that it is not strictly required for proton transfer to the c-ring in mitochondrial ATP synthases.
Instead, this function appears to be compensated in our structure by H of H5 a , which extends towards the c-ring and interacts with Y of H6 a , thus forming an alternative residue pair at the lumenal channel exit Figure 5B. After almost a full rotation of the c-ring, the glutamate residue E86 is deprotonated by R of subunit a.
The translocated proton is then released into the matrix half-channel. In other ATP synthases, H5 a bends around the c -ring, thereby determining the channel path Guo et al. Due to the unusually kinked H5 a , E. Thus, the reduced interface between subunits a and c is compensated by species-specific structural elements forming the matrix half channel Figure 5C. As a consequence, the E.
Adjacent to the proton-half channels, the E. The head groups of both lipids are bound around the middle of the membrane plane, with their acyl chains extending towards the rotor-stator interface. Together, these two bound cardiolipins enclose the two horizontal membrane helices H5 a and H6 a , possibly acting to seal the F o against proton leakage by recruiting a high density of acyl chains, as well as separating lipid and aqueous environments in the vicinity of the two half-channels.
Thus, in addition to previous studies suggesting transient interactions of the metazoan c-ring rotor with cardiolipin Duncan et al. A cluster of phylum-specific subunits is located at the F o periphery, away from the dimer interface. On the lumenal side, it is flanked by the terminal extensions of subunit a and subunit k Figure 6—figure supplement 1A,B.
On the matrix side, ATPTB1 anchors the subcomplex to the conserved core through multiple contact sites. Thus, a protein-enclosed membrane cavity is formed. In the cavity, we identified six bound cardiolipins Figure 6B.
The simulations indicate that the cavity is filled with a bilayer lipid array in which the lipid molecules can freely diffuse in or out of within the membrane Video 3. To probe the dynamics of lipid binding, we calculated the probabilities of entering and remaining in the cavity. The lipid exhibiting the highest probability, especially over longer time intervals, is cardiolipin Figure 6—figure supplement 2C,D , which is consistent with the assignment in the cryo-EM density maps Figure 6B.
A Euglenozoa-specific subunits form a peripheral F o subcomplex. Density of the F o with proteins of the peripheral region coloured, c-ring model shown in grey, outline of the detergent belt yellow dashed lines with 2 nm offset towards the lumen indicated as determined by the density transparent gold B Atomic model of the F o periphery, cavity lipids are shown in magenta.
C to E Attachment of the peripheral stalk to F 1. D Side view of the peripheral stalk tip and F 1 white, crown domain light grey. A sliced view of the membrane bilayer is initially shown for reference, but later removed to allow viewing of the binding and unbinding phospholipids.
Cardiolipin is indicated with purple, phosphatidic acid with yellow, phophatidylethanolamine with red, phosphatidylcholine with cyan acyl chains respectively. Lipids considered to be bound in the beginning or end of the simulation are visualized, demonstrating that cardiolipin replaces other lipids in the cavity during the simulation.
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