Immunochemical analysis of Micrococcus lysodeikticus (luteus) F1-ATPase and its subunits

The F₁-ATPase from Micrococcus lysodeikticus has been purified to 95% protein homogeneity in this laboratory and as all other bacterial F₁s, possesses five distinct subunits with molecular weights ranging from 60 000 to 10 000 (Huberman, M. and Salton, M.R.J. (1979) Biochim. Biophys. Acta 547, 230–240). In this communication, we demonstrate the immunochemical reactivities of antibodies to native and SDS-dissociated subunits with the native and dissociated F₁-ATPase and show that: (1) the antibodies generated to the native or SDS-dissociated subunits react with the native molecule; (2) all of the subunits comprising the F₁ are antigenically unique as determined by crossed immunoelectrophoresis and the Ouchterlony double-diffusion techniques; (3) antibodies to the SDS-denatured individual δ- and ?-subunits can be used to destabilize the interaction of these specific subunits with the rest of the native F₁; and (4) all subunit antibodies as well as anti-native F₁ were found to inhibit ATPase activity to varying degrees, the strongest inhibition being seen with antibodies to the total F₁ and anti-α- and anti-β-subunit antibodies. The interaction of specific subunit antibodies may provide a new and novel way to study further and characterize the catalytic portions of F₁-ATPases and in general may offer an additional method for the examination of multimeric proteins.     

Immunochemistry of membrane F1-Adenosine triphosphatase fromMicrococcus lysodeikticus. Role of the alpha subunit

The membrane-bound ATPase activity from two substrains ofMicrococcus lysodeikticus, designated as A and B, was inhibited by antibodies raised against the two forms of purified F₁-ATPase. Form B of the enzyme, which behaved as a poorer immunogen than form A, also showed less reactivity as an antigen, independent of the physical state of the F₁-ATPase form. Antibodies were raised against the two major subunits ( and ) isolated fromM. lysodeikticus F₁-ATPase form A, which was the most stable form of the enzyme. Anti-(-subunit) serum strongly inhibited the ATPase activity of membrane-bound ATPase but showed little inhibition of the purified, soluble F₁-ATPase. The anti-(-subunit) serum inhibited the soluble F₁-ATPase, but to a lesser extent than the membrane-bound enzyme. In any event, the effect of anti- antibodies on the membrane-bound ATPase was smaller than that of anti- antibodies. It was postulated that the subunit ofM. lysodeikticus F₁-ATPase plays an essential and regulatory role in the expression of the immunochemical properties of the protein.     

Reconstitution of ATPase from the isolated subunits of coupling factor F1's of Escherichia coli and thermophilic bacterium PS3

ATPase was reconstituted from mixtures of isolated subunits of coupling factor, F₁ ATPase of E. coli (EF₁) and thermophilic bacterium PS3 (TF₁); ability to hydrolyze ATP was attained from the combination of α and β subunits from EF₁ and γ subunit from TF₁, α and β from TF₁ and γ from EF₁, and α and γ from EF₁ and β from TF₁. The β subunit of TF₁ also could complement the EF₁ from an E. coli mutant defective in this subunit. This is the first demonstration of interspecies in vitro recombination of ATPase activity from isolated subunits.     

Purification and properties of the latent F1-ATPase of Micrococcus lysodeikticus

The latent coupling factor (F₁)-ATPase of Micrococcus lysodeikticus has been purified to homogeneity as determined by a number of criteria including, non-denaturing polyacrylamide gel electrophoresis, crossed immunoelectrophoresis and analytical ultracentrifugation. By inclusion of 1 mM phenylmethyl sulfonyl fluoride, a serine protease inhibitor, in the shock-wash step of release of F₁ from the membranes, the spontaneous activation of both crude and purified ATPase by endogenous membrane protease(s) can be prevented, thereby yielding a highly latent ATPase preparation. Equilibrium ultracentrifugation of the latent ATPase gave a molecular weight of 400 000. The ATPase contained five different subunits α, β, γ, δ, and ? and their molecular weights determined by SDS-polyacrylamide gel electrophoresis were 60 000, 54 000, 37 000, 27 000 and 9000, respectively. The subunit composition was determined with ¹⁴C-labelled, F₁-ATPase prepared from cells grown on medium containing [U-¹⁴C]-labelled algal protein hydrolysate. Within the limitations of this method the results tentatively suggest a subunit composition of 3 : 3 : 1 : 1 : 3.     

Direct identification of the rotary angle of ATP cleavage in F1-ATPase from Bacillus PS3

F₁-ATPase is the world’s smallest biological rotary motor driven by ATP hydrolysis at three catalytic β subunits. The 120° rotational step of the central shaft γ consists of 80° substep driven by ATP binding and a subsequent 40° substep. In order to correlate timing of ATP cleavage at a specific catalytic site with a rotary angle, we designed a new F₁-ATPase (F₁) from thermophilic Bacillus PS3 carrying β(E190D/F414E/F420E) mutations, which cause extremely slow rates of both ATP cleavage and ATP binding. We produced an F₁ molecule that consists of one mutant β and two wild-type βs (hybrid F₁). As a result, the new hybrid F₁ showed two pausing angles that are separated by 200°. They are attributable to two slowed reaction steps in the mutated β, thus providing the direct evidence that ATP cleavage occurs at 200° rather than 80° subsequent to ATP binding at 0°. This scenario resolves the long-standing unclarified issue in the chemomechanical coupling scheme and gives insights into the mechanism of driving unidirectional rotation.     

Insertion of a “chloroplast-like” regulatory segment responsible for thiol modulation into γ-subunit of F0F1-ATPase of the cyanobacterium Synechocystis 6803 by mutagenesis of atpC

A regulatory sequence in the γ subunit of the F₀F₁-ATPase complex of higher plant chloroplasts, responsible for so-called thiol modulation, is absent in the corresponding polypeptides of the cyanobacterial complexes analysed so far. We have modified the atpC gene encoding this γ subunit in Synechocystis 6803 by site-directed mutagenesis. A segment was introduced coding for nine additional amino acids, including the two functional cysteines, which constitutes the sequence of the respective element in the chloroplast γ subunit. The growth rate as well as the rate of photosynthesis of the transformant was comparable to that of the wild-type, but the transitory increase in respiration observed immediately after a period of illumination was significantly lower in the mutant than in the wild-type. The F₁ subcomplex solubilized from thylakoid membranes of both the wild-type and the transformant can be activated by trypsin to yield Ca²⁺-dependent ATPase activity, but only the F₁ from the transformant can be activated by the thiol reagent dithiothreitol.     

Inactivation and the site of labeling of F1-ATPase from Mycobacterium phlei by dicyclohexylcarbodiimide, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and quinacrine mustard

The F₁-ATPase from Mycobacterium phlei is inactivated by dicyclohexylcarbodiimide (DCCD), 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) and quinacrine mustard. The inactivation is both time-and concentration-dependent and in the case of DCCD being more pronounced at acidic pH. The minimum inactivation half-time ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) for DCCD, NBD-Cl and quinacrine mustard was observed to be 14, 6 and 7 min, respectively. Inactivation of F₁-ATPase resulted in the incorporation of [¹⁴C]DCCD as well as [¹⁴C]NBD-Cl into α and γ subunits. The incorporation of label into α and γ subunits, utilizing [¹⁴C]NBD-Cl, was reversible by dithiothreitol. Complete inactivation, by linear extrapolation to zero activity, revealed that 4 mol [¹⁴C]DCCD and 4 mol [¹⁴C]NBD-Cl bind per mol F₁-ATPase. Kinetic and binding studies show that these probes bind to site(s) distinct from ATP-binding site in F₁-ATPase from M. phlei.     

Substructure of F1-ATPase (BF1 factor) from Micrococcus lysodeikticus

Summary Dimethyl suberimidate and dithiobis (succinimidyl propionate) have been used to explore the nearest neighbor relationship of the subunits (, , and by decreasing molecular weight) of F₁-ATPase or BF₁ factor of Micrococcus lysodeikticus. Cross-linking with the two diimido esters inhibited the ATPase activity but this inhibition never exceeded 50% of the initial value. The cross-linking pattern of this BF₁ factor, as revealed by sodium dodecyl sulfate gel electrophoresis, shows a relative low proportion of high molecular weight aggregates which move slowly than the heaviest subunit (). They are resolved as three components of molecular weights 200,000, 130,000 and 100,000 in 5% acrylamide gels, plus an additional component (mol. wt 80,000) identified in 10% acrylamide gels. The other aggregate bands represent cross-linking products of the smaller subunits ( and ) that may travel to the conventional position of the heavier subunits.The subunit composition of the aggregate bands has been determined through the reversion of dithiobis (succinimidyl propionate) cross-linking of the BF₁ factor by dithiothreitol and analysis in second dimension by gel electrophoresis. The results indicate that subunit can cross-link with itself and with each of the other subunits except . The subunit is also able to cross-link with itself and with the other subunits although to a minor extent than , and that ₂ aggregates are present. These results represent a specific pattern of cross-linking for this BF₁ factor as compared to other F₁ coupling factors. It suggests a certain asymmetry in the spatial organization of the major subunits of M. lysodeikticus F₁-ATPase where the subunit must play a central role. A subunit stoichiometry _{3 3 2 2} is proposed for whole F₁-ATPase which leads to a molecular weight 440,000 consistent with the 430,000 value estimated by sedimentation equilibrium at low speed. A tentative structural model of M. lysodeikticus BF₁ factor is derived from these data. The significance of the results in relation to the possible generalization of the molecular architecture of F₁ factors is discussed.     

Reconstitution of ATPase activity from the isolated alpha, beta, and gamma subunits of the coupling factor, F1, of Escherichia coli.

The three major subunits (α, β and γ) of the coupling factor, F₁ ATPase, of $\text{Escherichia coli}$ were separated and purified by hydrophobic column chromatography after the enzyme was dissociated by cold inactivation. The ability to hydrolyze ATP was reconstituted by dialyzing the mixture of subunits against 0.05 M Tris-succinate, pH 6.0, containing 2 mM ATP and 2 mM MgCl₂. A mixture containing α, β and γ regained ATP hydrolyzing activity. Individual subunits alone or mixtures of any two subunits did not develop ATPase activity, except for a low but significant activity with α plus β. The reconstituted ATPase had a K_m of 0.23 mM for ATP and a molecular weight by sucrose gradient density centrifugation of about 280,000.     

Does F1-ATPase subunit γ turn in the wrong direction?

Analyzing the direction of F₁-ATPase subunit γ rotation, its shape and non-random distribution of surface residues, a mechanism is proposed for how γ induces the closing/opening of the catalytic sites at β/α interfaces: by keeping contact with the mobile domain of subunits β at the ‘jaw’ (D386, the seven consecutive hydrophobic residues and D394/E395), rotating γ works as a screw conveyer within the barrel of (α,β)₃. Mutations of the conveyer contacts are predicted to inhibit. Rotating wheel cartoons illustrate enzyme turnover and conformational changes. Steric clashes, polar interactions and also substrate limitations lead to specific stops. Because it is constructed as a stepper, γ prevents uncoupling at high energy charge.     

Normal‐mode‐based modeling of allosteric couplings that underlie cyclic conformational transition in F1 ATPase

F₁ ATPase, a rotary motor comprised of a central stalk ( γ subunit) enclosed by three α and β subunits alternately arranged in a hexamer, features highly cooperative binding and hydrolysis of ATP. Despite steady progress in biophysical, biochemical, and computational studies of this fascinating motor, the structural basis for cooperative ATPase involving its three catalytic sites remains not fully understood. To illuminate this key mechanistic puzzle, we have employed a coarse‐grained elastic network model to probe the allosteric couplings underlying the cyclic conformational transition in F₁ ATPase at a residue level of detail. We will elucidate how ATP binding and product (ADP and phosphate) release at two catalytic sites are coupled with the rotation of γ subunit via various domain motions in α ₃ β ₃ hexamer (including intrasubunit hinge‐bending motions in β subunits and intersubunit rigid‐body rotations between adjacent α and β subunits). To this end, we have used a normal‐mode‐based correlation analysis to quantify the allosteric couplings of these domain motions to local motions at catalytic sites and the rotation of γ subunit. We have then identified key amino acid residues involved in the above couplings, some of which have been validated against past studies of mutated and γ ‐truncated F₁ ATPase. Our finding strongly supports a binding change mechanism where ATP binding to the empty catalytic site triggers a series of intra‐ and intersubunit domain motions leading to ATP hydrolysis and product release at the other two closed catalytic sites. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Nucleotide Sequence of the F(1)-ATPase alpha Subunit Gene from Maize Mitochondria

The α subunit of the F₁-ATPase complex of maize is a mitochondrial translational product, presumably encoded by the mitochondrial genome. Based on nucleotide and amino acid homology, we have identified a mitochondrial gene, designated atpα, that appears to code for the F₁-ATPase α subunit of Zea mays. The atpα gene is present as a single copy in the maize. Texas cytoplasm and is actively transcribed. The maize α polypeptide has a predicted length of 508 amino acids and a molecular mass of 55,187 daltons. Amino acid homologies between the maize mitochondrial α subunit and the tobacco chloroplast CF₁ and Escherichia coli α subunits are 54 and 51%, respectively. The origin of the atpα gene is discussed.     

Subunit composition, function, and spatial arrangement in the Ca2+-and Mg2+-activated adenosine triphosphatases of Escherichia coli and Salmonella typhimurium.

The Ca²⁺- and Mg²⁺-activated ATPases of Escherichia coli NRC 482 and Salmonella typhimurium LT2 were purified to homogeneity. Both enzymes consisted of five polypeptides (α-?). The molecular weights of the α, β, and ? polypeptides were 56,800, 51,800 and 13,200 for both enzymes. The molecular weights of the γ and δ polypeptides of the E. coli and S. typhimurium ATPases were 32,000 and 20,700, and 30,900 and 21,500, respectively. In both ATPases the stoichiometry of the subunits was α₃β₃γδ? as determined with the ¹⁴C-labeled enzymes. The ATPases of either organism reacted with equal effectiveness with ATPase-deficient particles of the other organism to reconstitute energy-dependent transhydrogenase activity. Treatment of the homogeneous ATPases of both organisms with TPCK-trypsin stimulated ATPase activity but resulted in destruction of coupling factor activity. Trypsin treatment completely digested the δ and ? polypeptides, and removed up to 70% of the γ polypeptide. In the presence of the bifunctional cross-linking reagent dithiobis(succinimidyl propionate) ATPase activity was lost and cross-linking of α to β polypeptides occurred. Crosslinking of α to α or β to β polypeptides was not detected. The function of the individual polypeptides of the ATPase is discussed and a model for their spatial arrangement in the enzyme is presented.     

α and β subunits of F1-ATPase are required for survival of petite mutants in Saccharomyces cerevisiae

Although Saccharomyces cerevisiae can form petite mutants with deletions in mitochondrial DNA (mtDNA) (ρ^?) and can survive complete loss of the organellar genome (ρ^o), the genetic factor(s) that permit(s) survival of ρ^? and ρ^o mutants remain(s) unknown. In this report we show that a function associated with the F₁-ATPase, which is distinct from its role in energy transduction, is required for the petite-positive phenotype of S. cerevisiae. Inactivation of either the α or β subunit, but not the γ, δ, or ? subunit of F₁, renders cells petite-negative. The F₁ complex, or a subcomplex composed of the α and β subunits only, is essential for survival of ρ^o cells and those impaired in electron transport. The activity of F₁ that suppresses ρ^o lethality is independent of the membrane F_o complex, but is associated with an intrinsic ATPase activity. A further demonstration of the ability of F₁ subunits to suppress ρ^o lethality has been achieved by simultaneous expression of S. cerevisiae F₁α and γ subunit genes in Kluyveromyces lactis– which allows this petite-negative yeast to survive the loss of its mtDNA. Consequently, ATP1 and ATP2, in addition to the previously identified AAC2, YME1 and PEL1/PGS1 genes, are required for establishment of ρ^? or ρ^o mutations in S. cerevisiae.     

Suppression of ρ 0 lethality by mitochondrial ATP synthase F1 mutations in Kluyveromyces lactis occurs in the absence of F0

Specific mgi mutations in the α, β or γ subunits of the mitochondrial F₁-ATPase have previously been found to suppress ρ⁰ lethality in the petite-negative yeast Kluyveromyces lactis. To determine whether the suppressive activity of the altered F₁ is dependent on the F₀ sector of ATP synthase, we isolated and disrupted the genes KlATP4, 5 and 7, the three nuclear genes encoding subunits b, OSCP and d. Strains disrupted for any one, or all three of these genes are respiration deficient and have reduced viability. However a strain devoid of the three nuclear genes is still unable to lose mitochondrial DNA, whereas a mgi mutant with the three genes inactivated remains petite-positive. In the latter case, ρ⁰ mutants can be isolated, upon treatment with ethidium bromide, that lack six major F₀ subunits, namely the nucleus-encoded subunits b, OSCP and d, and the mitochondrially encoded Atp6, 8 and 9p. Production of ρ⁰ mutants indicates that an F₁-complex carrying a mgi mutation can assemble in the absence of F₀ subunits and that suppression of ρ⁰ lethality is an intrinsic property of the altered F₁ particle.     

Amputation of a C-terminal helix of the γ subunit increases ATP-hydrolysis activity of cyanobacterial F1 ATP synthase

F₁ is a soluble part of F_oF₁-ATP synthase and performs a catalytic process of ATP hydrolysis and synthesis. The γ subunit, which is the rotary shaft of F₁ motor, is composed of N-terminal and C-terminal helices domains, and a protruding Rossman-fold domain located between the two major helices parts. The N-terminal and C-terminal helices domains of γ assemble into an antiparallel coiled-coil structure, and are almost embedded into the stator ring composed of α₃β₃ hexamer of the F₁ molecule. Cyanobacterial and chloroplast γ subunits harbor an inserted sequence of 30 or 39 amino acids length within the Rossman-fold domain in comparison with bacterial or mitochondrial γ. To understand the structure–function relationship of the γ subunit, we prepared a mutant F₁-ATP synthase of a thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1, in which the γ subunit is split into N-terminal α-helix along with the inserted sequence and the remaining C-terminal part. The obtained mutant showed higher ATP-hydrolysis activities than those containing the wild-type γ. Contrary to our expectation, the complexes containing the split γ subunits were mostly devoid of the C-terminal helix. We further investigated the effect of post-assembly cleavage of the γ subunit. We demonstrate that insertion of the nick between two helices of the γ subunit imparts resistance to ADP inhibition, and the C-terminal α-helix is dispensable for ATP-hydrolysis activity and plays a crucial role in the assembly of F₁-ATP synthase.     

On chemomechanical coupling of the F1-ATPase molecular motor

F₁-ATPase catalyzes ATP hydrolysis to drive the central γ-shaft rotating inside a hexameric cylinder composed of alternating α and β subunits. Experiments showed that the rotation of γ-shaft proceeds in steps of 120° and each 120°-rotation is composed of an 80° substep and a 40° substep. Here, based on the previously proposed models, an improved physical model for chemomechanical coupling of F₁-ATPase is presented, with which the two-substep rotation is well explained. One substep is driven by the power stroke upon ATP binding, while the other one resulted from the passage of γ-shaft from previous to next adjacent β subunits via free diffusion. Using the model, the dynamics and kinetics of F₁-ATPase, such as the rotating time of each substep, the dwell time at each pause and the rotation rate, are analytically studied. The theoretical results obtained with only three adjustable parameters reproduce the available experimental data well.     

The mechanism of rotating proton pumping ATPases

Two proton pumps, the F-ATPase (ATP synthase, F_oF₁) and the V-ATPase (endomembrane proton pump), have different physiological functions, but are similar in subunit structure and mechanism. They are composed of a membrane extrinsic (F₁ or V₁) and a membrane intrinsic (F_o or V_o) sector, and couple catalysis of ATP synthesis or hydrolysis to proton transport by a rotational mechanism. The mechanism of rotation has been extensively studied by kinetic, thermodynamic and physiological approaches. Techniques for observing subunit rotation have been developed. Observations of micron-length actin filaments, or polystyrene or gold beads attached to rotor subunits have been highly informative of the rotational behavior of ATP hydrolysis-driven rotation. Single molecule FRET experiments between fluorescent probes attached to rotor and stator subunits have been used effectively in monitoring proton motive force-driven rotation in the ATP synthesis reaction. By using small gold beads with diameters of 40-60 nm, the E. coli F₁ sector was found to rotate at surprisingly high speeds (> 400 rps). This experimental system was used to assess the kinetics and thermodynamics of mutant enzymes. The results revealed that the enzymatic reaction steps and the timing of the domain interactions among the β subunits, or between the β and γ subunits, are coordinated in a manner that lowers the activation energy for all steps and avoids deep energy wells through the rotationally-coupled steady-state reaction. In this review, we focus on the mechanism of steady-state F₁-ATPase rotation, which maximizes the coupling efficiency between catalysis and rotation.     

Nucleotide sequence of the genes for β and ε subunits of proton-translocating ATPase from Escherichia coli

The 1855-nucleotide long DNA sequence of part of the gene cluster for the proton-translocating ATPase from $\text{E. coli}$ was determined by the method of Maxam-Gilbert. The sequence covers the genes for the β and ε subunits of F₁ along with the flanking region. The amino acid sequence of these subunits deduced from the nucleotide sequence indicates that the β and ε subunits have 459 and 138 amino acids, respectively. The possible secondary structure of the both subunits was estimated from the deduced primary structures. A possible nucleotide binding site in the β subunit is also discussed on the basis of the primary and secondary structures. The codons used in the genes for all the components of F₁F₀ were different in different genes, suggesting that the amount of each subunit in the F₁F₀ is determined to some extent on a translational level.     

Binding of the inhibitor protein IF(1) to bovine F(1)-ATPase

In the structure of bovine F₁-ATPase inhibited with residues 1-60 of the bovine inhibitor protein IF₁, the α-helical inhibitor interacts with five of the nine subunits of F₁-ATPase. In order to understand the contributions of individual amino acid residues to this complex binding mode, N-terminal deletions and point mutations have been introduced, and the binding properties of each mutant inhibitor protein have been examined. The N-terminal region of IF₁ destabilizes the interaction of the inhibitor with F₁-ATPase and may assist in removing the inhibitor from its binding site when F₁F_o-ATPase is making ATP. Binding energy is provided by hydrophobic interactions between residues in the long α-helix of IF₁ and the C-terminal domains of the β_DP-subunit and β_TP-subunit and a salt bridge between residue E30 in the inhibitor and residue R408 in the C-terminal domain of the β_DP-subunit. Several conserved charged amino acids in the long α-helix of IF₁ are also required for establishing inhibitory activity, but in the final inhibited state, they are not in contact with F₁-ATPase and occupy aqueous cavities in F₁-ATPase. They probably participate in the pathway from the initial interaction of the inhibitor and the enzyme to the final inhibited complex observed in the structure, in which two molecules of ATP are hydrolysed and the rotor of the enzyme turns through two 120° steps. These findings contribute to the fundamental understanding of how the inhibitor functions and to the design of new inhibitors for the systematic analysis of the catalytic cycle of the enzyme.