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The structures of F-ATPases have predominantly been determined from mitochondrial enzymes, and those of the enzymes in eubacteria have been less studied. Paracoccus denitrificans is a member of the α-proteobacteria and is related to the extinct protomitochondrion that became engulfed by the ancestor of eukaryotic cells. The P. denitrificans F-ATPase is an example of a eubacterial F-ATPase that can carry out ATP synthesis only, whereas many others can catalyse both the synthesis and the hydrolysis of ATP. Inhibition of the ATP hydrolytic activity of the P. denitrificans F-ATPase involves the ζ inhibitor protein, an α-helical protein that binds to the catalytic F1 domain of the enzyme. This domain is a complex of three α-subunits and three β-subunits, and one copy of each of the γ-, δ- and ɛ-subunits. Attempts to crystallize the F1-ζ inhibitor complex yielded crystals of a subcomplex of the catalytic domain containing the α- and β-subunits only. Its structure was determined to 2.3 Å resolution and consists of a heterodimer of one α-subunit and one β-subunit. It has no bound nucleotides, and it corresponds to the `open' or `empty' catalytic interface found in other F-ATPases. The main significance of this structure is that it aids in the determination of the structure of the intact membrane-bound F-ATPase, which has been crystallized. [ABSTRACT FROM AUTHOR]

The ATP synthases have been isolated by affinity chromatography from the mitochondria of the fungal species Yarrowia lipolytica, Pichia pastoris, Pichia angusta and Saccharomyces cerevisiae. The subunit compositions of the purified enzyme complexes depended on the detergent used to solubilize and purify the complex, and the presence or absence of exogenous phospholipids. All four enzymes purified in the presence of n-dodecyl-β-D-maltoside had a complete complement of core subunits involved directly in the synthesis of ATP, but they were deficient to different extents in their supernumerary membrane subunits. In contrast, the enzymes from P. angusta and S. cerevisiae purified in the presence of n-decyl-β-maltose neopentyl glycol and the phospholipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, cardiolipin (diphosphatidylglycerol) and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] had a complete complement of core subunits and also contained all of the known supernumerary membrane subunits, e, f, g, j, k and ATP8 (or Aap1), plus an additional new membrane component named subunit l, related in sequence to subunit k. The catalytic domain of the enzyme from P. angusta was more resistant to thermal denaturation than the enzyme from S. cerevisiae, but less stable than the catalytic domain of the bovine enzyme, but the stator and the integrity of the transmembrane proton pathway were most stable in the enzyme from P. angusta. The P. angusta enzyme provides a suitable source of enzyme for studying the structure of the membrane domain and properties associated with that sector of the enzyme complex. [ABSTRACT FROM AUTHOR]

The rotation of the central stalk of F1-ATPase is driven by energy derived from the sequential binding of an ATP molecule to its three catalytic sites and the release of the products of hydrolysis. In human F1-ATPase, each 360° rotation consists of three 120° steps composed of substeps of about 65°, 25°, and 30°, with intervening ATP binding, phosphate release, and catalytic dwells, respectively. The F1-ATPase inhibitor protein, IF1, halts the rotary cycle at the catalytic dwell. The human and bovine enzymes are essentially identical, and the structure of bovine F1-ATPase inhibited by IF1 represents the catalytic dwell state. Another structure, described here, of bovine F1-ATPase inhibited by an ATP analog and the phosphate analog, thiophosphate, represents the phosphate binding dwell. Thiophosphate is bound to a site in the αEβE-catalytic interface, whereas in F1-ATPase inhibited with IF1, the equivalent site is changed subtly and the enzyme is incapable of binding thiophosphate. These two structures provide a molecular mechanism of how phosphate release generates a rotary substep as follows. In the active enzyme, phosphate release from the βE-subunit is accompanied by a rearrangement of the structure of its binding site that prevents released phosphate from rebinding. The associated extrusion of a loop in the βE-subunit disrupts interactions in the αEβE-catalytic interface and opens it to its fullest extent. Other rearrangements disrupt interactions between the γ-subunit and the C-terminal domain of the αE-subunit. To restore most of these interactions, and to make compensatory new ones, the γ-subunit rotates through 25°-30°. [ABSTRACT FROM AUTHOR]

The hydrolysis of ATP by the ATP synthase in mitochondria is inhibited by a protein called IF1. Bovine IF1 has 84 amino acids, and its N-terminal inhibitory region is intrinsically disordered. In a known structure of bovine F1-ATPase inhibited with residues 1-60 of IF1, the inhibitory region from residues 1-50 is mainly a-helical and buried deeply at the αDPβDP-catalytic interface, where it forms extensive interactions with five of the nine subunits of F1-ATPase but mainly with the βDP-subunit. As described here, on the basis of two structures of inhibited complexes formed in the presence of large molar excesses of residues 1-60 of IF1 and of a version of IF, with the mutation K39A, it appears that the intrinsically disordered inhibitory region interacts first with the αEβE-catalytic interface, the most open of the three catalytic interfaces, where the available interactions with the enzyme allow it to form an α-helix from residues 31-49. Then, in response to the hydrolysis of an ATP molecule and the associated partial closure of the interface to the αTPβTP state, the extent of the folded α-helical region of IF1 increases to residues 23-50 as more interactions with the enzyme become possible. Finally, in response to the hydrolysis of a second ATP molecule and a concomitant 120° rotation of the γ-subunit, the interface closes further to the αDPβDP-state, allowing more interactions to form between the enzyme and IF1. The structure of IF1 now extends to its maximally folded state found in the previously observed inhibited complex. [ABSTRACT FROM AUTHOR]

The molecular description of the mechanism of F,-ATPase is based mainly on high-resolution structures of the enzyme from mitochondria, coupled with direct observations of rotation in bacterial enzymes. During hydrolysis of ATP, the rotor turns counterclockwise (as viewed from the membrane domain of the intact enzyme) in 120° steps. Because the rotor is asymmetric, at any moment the three catalytic sites are at different points in the catalytic cycle. In a "ground-state" structure of the bovine enzyme, one site (ßE) is devoid of nucleotide and represents a state that has released the products of ATP hydrolysis. A second site (pTp) has bound the substrate, magnesium. ATP, in a precatalytic state, and in the third site (PDP). the substrate is about to undergo hydrolysis. Three successive 120° turns of the rotor interconvert the sites through these three states, hydrolyzing three ATP molecules, releasing the products and leaving the enzyme with two bound nucleotides. A transition-state analog structure, F,-TS, displays intermediate states between those observed in the ground state. For example, in the ß0P-site of FTS, the terminal phosphate of an ATP molecule is undergoing in-line nucleophilic attack by a water molecule. As described here, we have captured another intermediate in the catalytic cycle, which helps to define the order of substrate release. In this structure, the ßE-site is occupied by the product ADP, but without a magnesium ion or phosphate, providing evidence that the nucleotide is the last of the products of ATP hydrolysis to be released. [ABSTRACT FROM AUTHOR]

The structure of bovine F1-ATPase inhibited by a monomeric form of the inhibitor protein, IF1, known as I1-60His, lacking most of the dimerization region, has been determined at 2.1-Å resolution. The resolved region of the inhibitor from residues 8–50 consists of an extended structure from residues 8–13, followed by two α-helices from residues 14–18 and residues 21–50 linked by a turn. The binding site in the βDP-αDP catalytic interface is complex with contributions from five different subunits of F1-ATPase. The longer helix extends from the external surface of F1 via a deep groove made from helices and loops in the C-terminal domains of subunits βDP, αDP, βTP, and αTP to the internal cavity surrounding the central stalk. The linker and shorter helix interact with the γ-subunit in the central stalk, and the N-terminal region extends across the central cavity to interact with the nucleotide binding domain of the αE subunit. To form these complex interactions and penetrate into the core of the enzyme, it is likely that the initial interaction of the inhibitor with F1 forms via the open conformation of the βE subunit. Then, as two ATP molecules are hydrolyzed, the βE-αE interface converts to the βDP-αDP interface via the βTP-αTP interface, trapping the inhibitor progressively in its binding site and a nucleotide in the catalytic site of subunit βDP. The inhibition probably arises by IF1 imposing the structure and properties of the βTP-αTP interface on the βDP-αDP interface, thereby preventing it from hydrolyzing the bound ATP. [ABSTRACT FROM AUTHOR]

The structures of F1-ATPase from bovine heart mitochondria inhibited with the dietary phytopolyphenol, resveratrol, and with the related polyphenols quercetin and piceatannol have been determined at 2.3-, 2.4- and 2.7-Å resolution, respectively. The inhibitors bind to a common site in the inside surface of an annulus made from loops in the three α- and three β-subunits beneath the "crown" of β-strands in their N-terminal domains. This region of F1-ATPase forms a bearing to allow the rotation of the tip of the γ-subunit inside the annulus during catalysis. The binding site is a hydrophobic pocket between the C-terminal tip of the γ-subunit and the βTP subunit, and the inhibitors are bound via H-bonds mostly to their hydroxyl moieties mediated by bound water molecules and by hydrophobic interactions. There are no equivalent sites between the γ-subunit and either the βDP or the βE subunit. The inhibitors probably prevent both the synthetic and hydrolytic activities of the enzyme by blocking both senses of rotation of the γ-subunit. The beneficial effects of dietary resveratrol may derive in part by preventing mitochondrial ATP synthesis in tumor cells, thereby inducing apoptosis. [ABSTRACT FROM AUTHOR]

Orthorhombic crystals of bovine F1-ATPase have been subjected to controlled dehydration. A decrease in the relative humidity surrounding the crystals to 90% reproducibly reduced their unit-cell volume by 22% (950 000 Å3) and improved the diffraction limit and mosaic spread of the crystals significantly. These dehydrated crystals diffracted X-­rays to 1.8 Å resolution at a synchrotron source, the best diffraction limit yet attained with these crystals, although radiation damage limited the resolution of a complete data set to 1.95 Å. [ABSTRACT FROM AUTHOR]

The structure of bovine F1-ATPase inhibited with ADP and beryllium fluoride at 2.0 Å resolution contains two ADP.BeF3- complexes mimicking ATP, bound in the catalytic sites of the ßTP and ßDP subunits. Except for a 1 Å shift in the guanidinium of aArg373, the conformations of catalytic side chains are very similar in both sites. However, the ordered water molecule that carries out nucleophilic attack on the ?-phosphate of ATP during hydrolysis is 2.6 Å from the beryllium in the ßDP subunit and 3.8 Å away in the ßTP subunit, strongly indicating that the ßDP subunit is the catalytically active conformation. In the structure of F1-ATPase with five bound ADP molecules (three in a-subunits, one each in the ßTP and ßDP subunits), which has also been determined, the conformation of aArg373 suggests that it senses the presence (or absence) of the ?-phosphate of ATP. Two catalytic schemes are discussed concerning the various structures of bovine F1-ATPase. [ABSTRACT FROM AUTHOR]

In mitochondria, the hydrolytic activity of ATP synthase is prevented by an inhibitor protein, IF1. The active bovine protein (84 amino acids) is an a-helical dimer with monomers associated via an antiparallel a-helical coiled coil composed of residues 49-81. The N-terminal inhibitory sequences in the active dimer bind to two F1-ATPases in the presence of ATP. In the crystal structure of the F1-IF1 complex at 2.8 Å resolution, residues 1-37 of IF1 bind in the aDP-ßDP interface of F1-ATPase, and also contact the central ? subunit. The inhibitor opens the catalytic interface between the aDP and ßDP subunits relative to previous structures. The presence of ATP in the catalytic site of the ßDP subunit implies that the inhibited state represents a pre-hydrolysis step on the catalytic pathway of the enzyme. [ABSTRACT FROM AUTHOR]

In mitochondria, the hydrolytic activity of ATP synthase is prevented by an inhibitor protein, IF1. The active bovine protein (84 amino acids) is an a-helical dimer with monomers associated via an antiparallel a-helical coiled coil composed of residues 49-81. The N-terminal inhibitory sequences in the active dimer bind to two F1-ATPases in the presence of ATP. In the crystal structure of the F1-IF1 complex at 2.8 Å resolution, residues 1-37 of IF1 bind in the aDP-ßDP interface of F1-ATPase, and also contact the central ? subunit. The inhibitor opens the catalytic interface between the aDP and ßDP subunits relative to previous structures. The presence of ATP in the catalytic site of the ßDP subunit implies that the inhibited state represents a pre-hydrolysis step on the catalytic pathway of the enzyme. [ABSTRACT FROM AUTHOR]

The central stalk in ATP synthase, made of γ, δ and ε subunits in the mitochondrial enzyme, is the key rotary element in the enzyme's catalytic mechanism. The γ subunit penetrates the catalytic (αβ)3 domain and protrudes beneath it, interacting with a ring of c subunits in the membrane that drives rotation of the stalk during ATP synthesis. In other crystals of F1-ATPase, the protrusion was disordered, but with crystals of F1-ATPase inhibited with dicyclohexylcarbodiimide, the complete structure was revealed. The δ and ε subunits interact with a Rossmann fold in the γ subunit, forming a foot. In ATP synthase, this foot interacts with the c-ring and couples the transmembrane proton motive force to catalysis in the (αβ)3 domain. [ABSTRACT FROM AUTHOR]

The structure of F₁-ATPase from Saccharomyces cerevisiae inhibited by the yeast IF₁ has been determined at 2.5 Å resolution. The inhibitory region of IF₁ from residues 1 to 36 is entrapped between the C-terminal domains of the α(DP)- and β(DP)-subunits in one of the three catalytic interfaces of the enzyme. Although the structure of the inhibited complex is similar to that of the bovine-inhibited complex, there are significant differences between the structures of the inhibitors and their detailed interactions with F₁-ATPase. However, the most significant difference is in the nucleotide occupancy of the catalytic β(E)-subunits. The nucleotide binding site in β(E)-subunit in the yeast complex contains an ADP molecule without an accompanying magnesium ion, whereas it is unoccupied in the bovine complex. Thus, the structure provides further evidence of sequential product release, with the phosphate and the magnesium ion released before the ADP molecule.