Thermophilic F1-ATPase Research Articles

The rotary catalytic cycle of a thermophilic F1-ATPase

The rotary catalytic cycle of a thermophilic F1-ATPase

ATP-binding affinity of the ε subunit of thermophilic F1-ATPase under label-free conditions

The ε subunits of several bacterial F1-ATPases bind ATP. ATP binding to the ε subunit has been shown to be involved in the regulation of F1-ATPase from thermophilic Bacillus sp. PS3 (TF1). We previously reported that the dissociation constant for ATP of wild-type ε subunit of TF1 at 25 °C is 4.3 μM by measuring changes in the fluorescence of the dye attached to the ε subunit (Kato, S. et al. (2007) J. Biol. Chem.282, 37618). However, we have recently noticed that this varies with the dye used. In this report, to determine the affinity for ATP under label-free conditions, we have measured the competitive displacement of 2′(3′)-O-N′-methylaniloyl-aminoadenosine-5′-triphosphate (Mant-ATP), a fluorescent analog of ATP, by ATP. The dissociation constant for ATP of wild-type ε subunit of TF1 at 25 °C was determined to be 0.29 μM, which is one order of magnitude higher affinity than previously reported values.

Dynamic mechanisms driving conformational conversions of the β and ε subunits involved in rotational catalysis of F<sub>1</sub>-ATPase

F-type ATPase is a ubiquitous molecular motor. Investigations on thermophilic F1-ATPase and its subunits, β and ε, by NMR were reviewed. Using specific isotope labeling, pKa of the putative catalytic carboxylate in β was estimated. Segmental isotope-labeling enabled us to monitor most residues of β, revealing that the conformational conversion from open to closed form of β on nucleotide binding found in ATPase was an intrinsic property of β and could work as a driving force of the rotational catalysis. A stepwise conformational change was driven by switching of the hydrogen bond networks involving Walker A and B motifs. Segmentally labeled ATPase provided a well resolved NMR spectra, revealing while the open form of β was identical for β monomer and ATPase, its closed form could be different. ATP-binding was also a critical factor in the conformational conversion of ε, an ATP hydrolysis inhibitor. Its structural elucidation was described.

High affinity nucleotide-binding mutant of the ε subunit of thermophilic F1-ATPase

Specific ATP binding to the ε subunit of thermophilic F1-ATPase has been utilized for the biosensors of ATP in vivo. I report here that the ε subunit containing R103A/R115A mutations can bind ATP with a dissociation constant at 52 nM, which is two orders of magnitude higher affinity than the wild type. The mutant retained specificity for ATP; ADP and GTP bound to the mutant with dissociation constants 16 and 53 μM, respectively. Thus, the mutant would be a good platform for various types of nucleotide biosensor with appropriate modifications.

Structure of a thermophilic F1-ATPase inhibited by an ε-subunit: deeper insight into the ε-inhibition mechanism.

F1-ATPase (F1) is the catalytic sector in F(o)F1-ATP synthase that is responsible for ATP production in living cells. In catalysis, its three catalytic β-subunits undergo nucleotide occupancy-dependent and concerted open-close conformational changes that are accompanied by rotation of the γ-subunit. Bacterial and chloroplast F1 are inhibited by their own ε-subunit. In the ε-inhibited Escherichia coli F1 structure, the ε-subunit stabilizes the overall conformation (half-closed, closed, open) of the β-subunits by inserting its C-terminal helix into the α3β3 cavity. The structure of ε-inhibited thermophilic F1 is similar to that of E. coli F1, showing a similar conformation of the ε-subunit, but the thermophilic ε-subunit stabilizes another unique overall conformation (open, closed, open) of the β-subunits. The ε-C-terminal helix 2 and hook are conserved between the two structures in interactions with target residues and in their positions. Rest of the ε-C-terminal domains are in quite different conformations and positions, and have different modes of interaction with targets. This region is thought to serve ε-inhibition differently. For inhibition, the ε-subunit contacts the second catches of some of the β- and α-subunits, the N- and C-terminal helices, and some of the Rossmann fold segments. Those contacts, as a whole, lead to positioning of those β- and α- second catches in ε-inhibition-specific positions, and prevent rotation of the γ-subunit. Some of the structural features are observed even in IF1 inhibition in mitochondrial F1.

A novel multigene cloning method for the production of a motile ATPase

With the advent of nanotechnology, new functional modules (e.g., nanomotors, nanoprobes) have become essential in several medical fields. Generally, mechanical modulators systems are the principal components of most cutting-edge technologies in modern biomedical applications. However, the in vivo use of motile probes has raised many concerns due to their low sensitivity and non-biocompatibility. As an alternative, biological enzymatic engines have received increased attention. In particular, ATPases, which belong to a class of motile enzymes that catalyze chemical metabolic reactions, have emerged as a promising motor due to their improved biocompatibility and performance. However, ATPases usually suffer from lower functional activity and are difficult to express recombinantly in bacteria relative to their conventional and synthetic competitors. Here, we report a novel functional modified ATPase with both a simple purification protocol and enhanced motile activity. For this mutant ATPase, a new bacterial subcloning method was established. The ATPase-encoding sequence was redesigned so that the mutant ATPase could be easily produced in an Escherichia coli system. The modified thermophilic F1-ATPase (mTF1-ATPase) demonstrated 17.8unit/mg ATPase activity. We propose that derivatives of our ATPase may enable the development of novel in vitro and in vivo synthetic medical diagnostics, as well as therapeutics.

Analysis of the Open and Closed Conformations of the β Subunits in Thermophilic F1-ATPase by Solution NMR

F(1)-ATPase, composed of alpha, beta, gamma, delta, and epsilon subunits, is a unique enzyme in terms of its rotational catalytic activity. The smallest unit showing this function is the alpha(3)beta(3)gamma complex. We have investigated the alpha(3)beta(3)gamma epsilon(Delta C) (epsilon(Delta C), truncated epsilon) complex from thermophilic Bacillus PS3 (TF(1)', 360 kDa) in the solution state by using the combination of extensive deuteration, segmental-labeling, and CRINEPT (cross-correlated relaxation-enhanced polarization transfer) NMR. Well-resolved CRINEPT-HMQC (heteronuclear multiple-quantum correlation) spectra of partially (15)N-labeled TF(1)' were obtained for this huge and asymmetric protein complex. The spectrum of the C-terminal domain of the beta subunit revealed that the open form of the beta subunit in the TF(1)' complex is similar to that of the free beta monomer. The open beta subunit in the TF(1)' complex does not exhibit high affinity for nucleotides unlike the monomer, but this is in agreement with the results of single-molecule analysis of TF(1)alpha(3)beta(3)gamma. On the other hand, the closed form of the beta subunit in the TF(1)' complex was shown to be distinct from that of the nucleotide-bound beta monomer. This is consistent with a previous report that the closed form of the TF(1)beta monomer could be a catalytically activated state. The loop between the N-terminal beta-barrel and the central domain is highly flexible in the TF(1)' complex, in contrast to that in the alpha(3)beta(3) hexamer, suggesting that it is affected by the presence of the gamma subunit in this area.

Inhibition of thermophilic F1-ATPase by the ε subunit takes different path from the ADP-Mg inhibition.

The F1-ATPase, the soluble part of FoF1-ATP synthase, is a rotary molecular motor consisting of α3β3γδε. The γ and ε subunits rotate relative to the α3β3δ sub-complex on ATP hydrolysis by the β subunit. The ε subunit is known as an endogenous inhibitor of the ATPase activity of the F1-ATPase and is believed to function as a regulator of the ATP synthase. This inhibition by the ε subunit (ε inhibition) of F1-ATPase from thermophilic Bacillus PS3 was analyzed by single molecule measurements. By using a mutant ε subunit deficient in ATP binding, reversible transitions between active and inactive states were observed. Analysis of pause and rotation durations showed that the ε inhibition takes a different path from the ADP-Mg inhibition. Furthermore, the addition of the mutant ε subunit to the α3β3γ sub-complex was found to facilitate recovery of the ATPase activity from the ADP-Mg inhibition. Thus, it was concluded that these two inhibitions are essentially exclusive of each other.

Modulation of nucleotide binding to the catalytic sites of thermophilic F1-ATPase by the ε subunit: Implication for the role of the ε subunit in ATP synthesis

Effect of ε subunit on the nucleotide binding to the catalytic sites of F1-ATPase from the thermophilic Bacillus PS3 (TF1) has been tested by using α3β3γ and α3β3γε complexes of TF1 containing βTyr341 to Trp substitution. The nucleotide binding was assessed with fluorescence quenching of the introduced Trp. The presence of the ε subunit weakened ADP binding to each catalytic site, especially to the highest affinity site. This effect was also observed when GDP or IDP was used. The ratio of the affinity of the lowest to the highest nucleotide binding sites had changed two orders of magnitude by the ε subunit. The differences may relate to the energy required for the binding change in the ATP synthesis reaction and contribute to the efficient ATP synthesis.

Dynamic inter-subunit interactions in thermophilic F1-ATPase subcomplexes studied by cross-correlated relaxation-enhanced polarization transfer NMR

F(1)-ATPase is a unique enzyme in terms of its rotational catalytic activity. The smallest unit showing this property is the alpha(3)beta(3)gamma complex (351 kDa). For investigation of such a huge system by means of solution NMR, we have explored a suitable NMR method using F(1)-ATPase subcomplexes from a thermophilic Bacillus PS3 including an alpha(3)beta(3) hexamer (319 kDa). Pulse sequences for large molecules, effects of deuteration and simplification of the spectra were examined in this work. Since the beta subunit includes the catalytic site, this was the target of the analysis in this work. The combination of [(15)N,(1)H]-CRINEPT-HMQC-[(1)H]-TROSY, deuteration of both alpha and beta subunits, and segmental isotope-labeling was found essential to analyze such a huge and complex molecular system. Utilizing this method, subcomplexes composed of alpha and beta subunits were investigated in terms of inter-subunit interactions. It turned out that there is equilibrium among monomers, heterodimers and the alpha(3)beta(3) hexamers in solution. The rate of exchange between the dimer and hexamer is in the slow regime on the NMR time scale. In chemical shift perturbation experiments, the N-terminal domain was found to be involved in strong inter-subunit interactions. In contrast, the C-terminal domain was found to be mobile even in the hexamer.

Role of the ϵ Subunit of Thermophilic F1-ATPase as a Sensor for ATP

The epsilon subunit of F(1)-ATPase from the thermophilic Bacillus PS3 (TF(1)) has been shown to bind ATP. The precise nature of the regulatory role of ATP binding to the epsilon subunit remains to be determined. To address this question, 11 mutants of the epsilon subunit were prepared, in which one of the basic or acidic residues was substituted with alanine. ATP binding to these mutants was tested by gel-filtration chromatography. Among them, four mutants that showed no ATP binding were selected and reconstituted with the alpha(3)beta(3)gamma complex of TF(1). The ATPase activity of the resulting alpha(3)beta(3)gammaepsilon complexes was measured, and the extent of inhibition by the mutant epsilon subunits was compared in each case. With one exception, weaker binding of ATP correlated with greater inhibition of ATPase activity. These results clearly indicate that ATP binding to the epsilon subunit plays a regulatory role and that ATP binding may stabilize the ATPase-active form of TF(1) by fixing the epsilon subunit into the folded conformation.

Inverse Regulation of Rotation of F1-ATPase by the Mutation at the Regulatory Region on the γ Subunit of Chloroplast ATP Synthase

In F1-ATPase, the rotation of the central axis subunit gamma relative to the surrounding alpha3beta3 subunits is coupled to ATP hydrolysis. We previously reported that the introduced regulatory region of the gamma subunit of chloroplast F1-ATPase can modulate rotation of the gamma subunit of the thermophilic bacterial F1-ATPase (Bald, D., Noji, H., Yoshida, M., Hirono-Hara, Y., and Hisabori, T. (2001) J. Biol. Chem. 276, 39505-39507). The attenuated enzyme activity of this chimeric enzyme under oxidizing conditions was characterized by frequent and long pauses of rotation of gamma. In this study, we report an inverse regulation of the gamma subunit rotation in the newly engineered F1-chimeric complex whose three negatively charged residues Glu210-Asp211-Glu212 adjacent to two cysteine residues of the regulatory region derived from chloroplast F1-ATPase gamma were deleted. ATP hydrolysis activity of the mutant complex was stimulated up to 2-fold by the formation of the disulfide bond at the regulatory region by oxidation. We successfully observed inverse redox switching of rotation of gamma using this mutant complex. The complex exhibited long and frequent pauses in its gamma rotation when reduced, but the rotation rates between pauses remained unaltered. Hence, the suppression or activation of the redox-sensitive F1-ATPase can be explained in terms of the change in the rotation behavior at a single molecule level. These results obtained by the single molecule analysis of the redox regulation provide further insights into the regulation mechanism of the rotary enzyme.

Mg-bound ADPの結合した好熱菌F_1の構造解析

Mg-bound ADPの結合した好熱菌F_1の構造解析

Movement of the Helical Domain of the ε Subunit Is Required for the Activation of Thermophilic F1-ATPase

The inhibitory effect of epsilon subunit in F(1)-ATPase from thermophilic Bacillus PS3 was examined focusing on the structure-function relationship. For this purpose, we designed a mutant for epsilon subunit similar to the one constructed by Schulenberg and Capaldi (Schulenberg, B., and Capaldi, R. A. (1999) J. Biol. Chem. 274, 28351-28355). We introduced two cysteine residues at the interface of N-terminal beta-sandwich domain (S48C) and C-terminal alpha-helical domain (N125C) of epsilon subunit. The alpha(3)beta(3)gammaepsilon complex containing the reduced form of this mutant epsilon subunit showed suppressed ATPase activity and gradual activation during the measurement. This activation pattern was similar to the complex with the wild type epsilon subunit. The conformation of the mutant epsilon subunit must be fixed and similar to the reported three-dimensional structure of the isolated epsilon subunit, when the intramolecular disulfide bridge was formed on this subunit by oxidation. This oxidized mutant epsilon subunit could form the alpha(3)beta(3)gammaepsilon complex but did not show any inhibitory effect. The complex was converted to the activated state, and the cross-link in the mutant epsilon subunit in the complex was efficiently formed in the presence of ATP-Mg, whereas no cross-link was observed without ATP-Mg, suggesting the conformation of the oxidized mutant epsilon subunit must be similar to that in the activated state complex. A non-hydrolyzable analog of ATP, 5'-adenylyl-beta,gamma-imidodiphosphate, could stimulate the formation of the cross-link on the epsilon subunit. Furthermore, the cross-link formation was stimulated by nucleotides even when this mutant epsilon subunit was assembled with a mutant alpha(3)beta(3)gamma complex lacking non-catalytic sites. These results indicate that binding of ATP to the catalytic sites induces a conformational change in the epsilon subunit and triggers transition of the complex from the suppressed state to the activated state.

Cross-linking of Two β Subunits in the Closed Conformation in F1-ATPase

In the crystal structure of mitochondrial F1-ATPase, two beta subunits with a bound Mg-nucleotide are in "closed" conformations, whereas the third beta subunit without bound nucleotide is in an "open" conformation. In this "CCO" (beta-closed beta-closed beta-open) conformational state, Ile-390s of the two closed beta subunits, even though they are separated by an intervening alpha subunit, have a direct contact. We replaced the equivalent Ile of the alpha3beta3gamma subcomplex of thermophilic F1-ATPase with Cys and observed the formation of the beta-beta cross-link through a disulfide bond. The analysis of conditions required for the cross-link formation indicates that: (i) F1-ATPase takes the CCO conformation when two catalytic sites are filled with Mg-nucleotide, (ii) intermediate(s) with the CCO conformation are generated during catalytic cycle, (iii) the Mg-ADP inhibited form is in the CCO conformation, and (iv) F1-ATPase dwells in conformational state(s) other than CCO when only one (or none) of catalytic sites is filled by Mg-nucleotide or when catalytic sites are filled by Mg2+-free nucleotide. The alpha3beta3gamma subcomplex containing the beta-beta cross-link retained the activity of uni-site catalysis but lost that of multiple catalytic turnover, suggesting that open-closed transition of beta subunits is required for the rotation of gamma subunit but not for hydrolysis of a single ATP.

Redox Properties of Iron in the Binding Site(s) of F1ATPase from Mammalian Mitochondria and Thermophilic Bacterium PS3: A Comparative Study

Iron ions in the two iron centers of beef heart mito-chondrial F, ATPase, which we have been recently characterized (FEBS Letters 1996,379, 231–235), exhibit different redox properties. In fact, the ATP-dependent site is able to maintain iron in the redox state of Fe(II) even in the absence of reducing agents, whereas in the nucleotide-independent site iron is oxidized to Fe(III) upon removal of the reductant. Fe(III) ions in the two sites display different reactivity towards H2O2, because only Fe(III) bound in the nucleotide-independent site rapidly reacts with H2O2 thus mediating a 30% enzyme inactivation. Thermophilic bacterium PS3 bears one Fe(III) binding site, which takes up Fe(III) either in the absence or presence of nucleotides and is unable to maintain iron in the redox state of Fe(II) in the absence of ascorbate. Fe(III) bound in thermophilic F1ATPase in a molar ratio 1:1 rapidly reacts with H2O2 mediating a 30% enzyme inactivation. These results support the presence in mitochon-drial and thermophilic F1ATPase of a conserved site involved in iron binding and in oxidative inactivation, in which iron exhibits similar redox properties. On the other hand, at variance with thermophilic F1ATPase, the mitochondrial enzyme has the possibility of maintaining one equivalent of Fe(II) in its peculiar ATP-dependent site, besides one equivalent of Fe(III) in the conserved nucleotide-independent site. In this case mitochondrial F, ATPase undergoes a higher inactivation (75%) upon exposure to H2O2. Under all conditions the inactivation is significantly prevented by PBN and DMSO but not by Cu, Zn superoxide dis-mutase, thus suggesting the formation of OH radicals as mediators of the oxidative damage. No dityrosines, carbonyls or oxidized thiols are formed. In addition, in any cases no protein fragmentation or aggregation is observed upon the treatment with H2O2.

Thermophilic F1-ATPase Is Activated without Dissociation of an Endogenous Inhibitor, ε Subunit

Subunit complexes (alpha3beta3gamma, alpha3beta3gammadelta, alpha3beta3gammaepsilon, and alpha3beta3gammadeltaepsilon) of thermophilic F1-ATPase were prepared, and their catalytic properties were compared to know the role of delta and epsilon subunits in catalysis. The presence of delta subunit in the complexes had slight inhibitory effect on the ATPase activity. The effect of epsilon subunit was more profound. The (-epsilon) complexes, alpha3beta3gamma and alpha3beta3gammadelta, initiated ATP hydrolysis without a lag. In contrast, the (+epsilon) complexes, alpha3beta3gammaepsilon and alpha3beta3gammadeltaepsilon, started hydrolysis of ATP (<700 microM) with a lag phase that was gradually activated during catalytic turnover. As ATP concentration increased, the lag phase of the (+epsilon) complexes became shorter, and it was not observed above 1 mM ATP. Analysis of binding and hydrolysis of the ATP analog, 2',3'-O-(2,4,6-trinitrophenyl)-ATP, suggested that the (+epsilon) complexes bound substrate only slowly. Differing from Escherichia coli F1-ATPase, the activation of the (+epsilon) complexes from the lag phase was not due to dissociation of epsilon subunit since the re-isolated activated complex retained epsilon subunit. This indicates that there are two alternative forms of the (+epsilon) complex, inhibited form and activated form, and the inhibited one is converted to the activated one during catalytic turnover.