Structure Of ATP Synthase Research Articles

Structure of dimeric mitochondrial ATP synthase: novel F0 bridging features and the structural basis of mitochondrial cristae biogenesis.

The F1F0-ATP synthase exists as a dimer in mitochondria, where it is essential for the biogenesis of the inner membrane cristae. How two ATP synthase complexes dimerize to promote cristae formation is unknown. Here we resolved the structure of the dimeric F1F0 ATP synthase complex isolated from bovine heart mitochondria by transmission electron microscopy. The structure of the ATP synthase dimer has an overall conic appearance that is consistent with the proposed role of the dimeric enzyme in mitochondrial cristae biogenesis. The ATP synthase dimer interface is formed by contacts on both the F0 and F1 domains. A cross-bridging protein density was resolved which connects the two F0 domains on the intermembrane space side of the membrane. On the matrix side of the complex, the two F1 moieties are connected by a protein bridge, which is attributable to the IF1 inhibitor protein.

The Modulation in Subunits e and g Amounts of Yeast ATP Synthase Modifies Mitochondrial Cristae Morphology

Subunits e and g of Saccharomyces cerevisiae ATP synthase are required to maintain ATP synthase dimeric forms. Mutants devoid of these subunits display anomalous mitochondrial morphologies. An expression system regulated by doxycycline was used to modulate the expression of the genes encoding the subunits e and g. A decrease in the amount of subunit e induces a decrease in the amount of subunit g, but a decrease in the amount of subunit g does not affect subunit e. The loss of subunit e or g leads to the loss of supramolecular structures of ATP synthase, which is fully reversible upon removal of doxycycline. In the absence of doxycycline, mitochondria present poorly defined cristae. In the presence of doxycycline, onion-like structures are formed after five generations. When doxycycline is removed after five generations, cristae are mainly observed. The data demonstrate that the inner structure of mitochondria depends upon the ability of ATP synthase to make supramolecular structures.

Pathogenesis of primary defects in mitochondrial ATP synthesis

Maternally inherited mutations in the mtDNA-encoded ATPase 6 subunit of complex V (ATP synthase) of the respiratory chain/oxidative phosphorylation system are responsible for a subgroup of severe and often-fatal disorders characterized predominantly by lesions in the brain, particularly in the striatum. These include NARP (neuropathy, ataxia, and retinitis pigmentosa), MILS (maternally inherited Leigh syndrome), and FBSN (familial bilateral striatal necrosis). Of the five known pathogenic mutations causing these disorders, four are located at two codons (156 and 217), each of which can suffer mutations converting a conserved leucine to either an arginine or a proline. Based on the accumulating data on both the structure of ATP synthase and the mechanism by which rotary catalysis couples proton flow to ATP synthesis, we propose a model that may help explain why mutations at codons 156 and 217 are pathogenic.

Mitochondrial F0F1 ATP Synthase: SUBUNIT REGIONS ON THE F1 MOTOR SHIELDED BY F0, FUNCTIONAL SIGNIFICANCE, AND EVIDENCE FOR AN INVOLVEMENT OF THE UNIQUE F0 SUBUNIT F6

Studies reported here were undertaken to gain greater molecular insight into the complex structure of mitochondrial ATP synthase (F(0)F(1)) and its relationship to the enzyme's function and motor-related properties. Significantly, these studies, which employed N-terminal sequence, mass spectral, proteolytic, immunological, and functional analyses, led to the following novel findings. First, at the top of F(1) within F(0)F(1), all six N-terminal regions derived from alpha + beta subunits are shielded, indicating that one or more F(0) subunits forms a "cap." Second, at the bottom of F(1) within F(0)F(1), the N-terminal region of the single delta subunit and the C-terminal regions of all three alpha subunits are shielded also by F(0). Third, and in contrast, part of the gamma subunit located at the bottom of F(1) is already shielded in F(1), indicating that there is a preferential propensity for interaction with other F(1) subunits, most likely delta and epsilon. Fourth, and consistent with the first two conclusions above that specific regions at the top and bottom of F(1) are shielded by F(0), further proteolytic shaving of alpha and beta subunits at these locations eliminates the capacity of F(1) to couple a proton gradient to ATP synthesis. Finally, evidence was obtained that the F(0) subunit called "F(6)," unique to animal ATP synthases, is involved in shielding F(1). The significance of the studies reported here, in relation to current views about ATP synthase structure and function in animal mitochondria, is discussed.

Insights into ATP synthase structure and function using affinity and site-specific spin labeling.

A variety of different approaches has been used during the last couple of decades to investigate structure and function relationships within the catalytic portion of the F0F1-ATP synthase and of its interactions with the proton-translocator F0. In our group, we employ ESR spectroscopy with the use of stable organic radicals, so-called spin labels, as reporter groups. The radicals are either attached to substrates/ligands or specifically inserted into the protein structure by "site-specific spin labeling." Both approaches bear intrinsic advantages for their special uses and result in the specific information that is available through ESR, e.g., structural changes due to binding of effector molecules (e.g., Mg2+ ions), conformational transitions during catalytic turnover, distance information on radicals bound at 20 A or less, and information on the binding characteristics of labeled substrates. This review summarizes the results of a variety of different approaches we have used during the last years to study, with the help of ESR spectroscopy, the structure of the nucleotide binding sites of F1-ATPases of different origins as well as interactions with F0 subunits.

Structure of ATP synthase by SFM and single-particle image analysis

F1FoATP synthases are the proteins responsible for the synthesis of ATP in oxidative phosphorylation, and are present in some form in all aerobic organisms, both prokaryotic and eukaryotic. They use the energy stored in a transmembrane proton gradient (which is generated by other members of the oxidative phosphorylation pathway) to synthesize ATP from ADP and Pior, working in reverse, to pump protons across the membrane using the energy of ATP hydrolysis. The full protein has two sectors, F1and Fo. F1is normally bound to Fo(which is membrane integrated), but is water soluble when dissociated. The F1sector contains the sites which bind ADP and catalyze its conversion to ATP. The Fo sector contains a channel which allows protons to to cross the membrane, dissipating the transmembrane chemical potential. By an unknown mechanism this translocation of protons through Fo is coupled to the hydrolysis or synthesis of ATP in F1, so that the energy released in hydrolysis of ATP can drive the motion of protons against an electrochemical potential, or the energy of translocating protons can be used to form high energy ADP-Pibonds.

Quaternary structure of ATP synthases: Symmetry and asymmetry in the F1 moiety

It has been proposed that during ATP synthesis/hydrolysis F1 ATPases experience a complex pattern of nucleotide binding and release during the catalytic cycle (binding change mechanism). This type of mechanism has implications that can be correlated with the structure of the enzyme. F1-ATPases (stoichiometry alpha 3 beta 3 gamma delta epsilon) are essentially a symmetrical trimer of pairs of the major subunits (alpha and beta); the minor subunits (gamma, delta and epsilon) are in single copies and interact with the trimer in an asymmetrical fashion. The asymmetry introduced by the minor subunits has important structural and functional consequences: (1) it introduces differences between the potentially equivalent binding and catalytic sites in the major subunits, (2) it restricts the ways in which a binding change mechanism can occur, and (3) it governs the way in which the F1 interacts with the (asymmetrical) F0 sector.

The structure of ATP synthase from chloroplasts. Conformational changes of CF 1 studied by electron microscopy

The structure of the hydrophilic part of the ATP synthase from chloroplasts (CF1) has been studied by electron microscopy and image analysis of negatively stained samples. The projections of different types of CF1 were analyzed: the holoenzyme with five different subunits and CF1 depleted of either delta or epsilon and of both delta and epsilon. We studied 11 sets of 1000 projections of oxidized and reduced forms of these particles. Comparisons between the 11 average images of the various types of CF1 were made by measuring the distances between the mass centers of the alpha,beta and gamma-subunits. In the holoenzyme projections the three alpha-alpha-distances are on the average 0.9 nm larger than the beta-beta-distances. This discrepancy is caused by the presence of the small subunits in the lower half of CF1, which is close to the membrane and becomes smaller when delta is removed. The differences between the alpha-alpha and beta-beta distances enables the localization of all copies of the 5 different subunits in the hexagonal projections. It is concluded that subunits-delta and epsilon are close to one alpha-subunit and two beta-subunits. The stronger staining halo of the alpha subuints indicates that they are on a lower level in CF1 than the beta-s. Upon reduction of the holoenzyme with dithiothreitol the distances between the mass centers of gamma and the alpha-s and beta-s do not change significantly. This means that permanent conformational changes upon reduction in the plane of the hexagonal projection parallel to the membrane are relatively small. Although the alpha, beta and gamma-subunits do not change in their position, it is shown that epsilon is more flexible; it can be close to two different beta-subunits. The movements of epsilon are thought to play a role in the catalytic mechanism and regulation of ATP synthesis/hydrolysis.

Structure of the ATP-synthase studied by electron microscopy and image processing

The structure of ATP synthase from beef heart mitochondria has been studied by electron microscopy and image processing using negatively stained specimens of detergent-solubilized and membrane-bound molecules. The F 1-ATPase and the membrane-embedded F 0 sector of ATP synthase are found to be connected by a narrow stalk, 4–4.5 nm long and 3–3.5 nm wide, projecting about 4.2 nm from the membrane surface. The F 0 sector has a globular shape, 6–8 nm in diameter and, in part, extends from the membrane lipid bilayer.