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

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Abstract

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.

Introduction

FoF1-ATPase/synthase (FoF1) consists of two rotary molecular motors: a water-soluble, ATP-driven F1 motor and a membrane embedded, H+- (or Na+-, in some species) driven Fo motor. These molecular motors are connected together to couple ATP synthesis/hydrolysis and ion flow [reviewed in [1], [2]]. The F1-ATPase (α3β3γδε) hydrolyzes ATP into ADP and inorganic phosphate, and the hydrolysis of one ATP drives discrete 120° rotation of the γε subunits relative to the other subunits. The asymmetric γ subunit makes three β subunits in different conformations [3]. In the crystal structure, the differences between conformations of three β subunits can be described as the relative motion of the C-terminal domain to the N-terminal domain. When the C-terminal domain of the β subunit takes closed conformation, the β subunit has high affinity for the nucleotide. On the other hand, when the β subunit takes open conformation, the affinity for the nucleotide is reduced. The binding change makes most of the torque required for the rotation of the γ subunit in the ATP hydrolysis reaction. It has been revealed that the major 80° sub-step of F1-ATPase is driven by the ATP binding and minor 40° sub-step by Pi release [4], [5]. In the ATP synthesis reaction, the energy from the electrochemical gradient of H+ drives Fo motor, makes γ subunit turn, results in changes in the conformation of the β subunits and their affinity for the nucleotide, to facilitate the substrate ADP and Pi binding and the product ATP release.

The smallest subunit of F1-ATPase, the ε subunit acts as an endogenous inhibitor of the ATPase activity in both bacterial and chloroplast F1-ATPase, where it is believed to play a regulatory role in FoF1[6], [7]. A recent single molecule study revealed its importance not only in the regulation but also in the efficient coupling of rotation of γ subunit and ATP synthesis [8]. The ε subunit consists of two distinct domains, an N-terminal β sandwich domain and a C-terminal α helical domain. Structural and biochemical studies have shown that the ε subunit adopts at least two different conformational states in F1 and FoF1[9], [10], [11], [12], [13], [14], [15], [16], [17]. The conformation that causes inhibition of ATP hydrolysis activity is an extended one, in which the C-terminal α helices of the ε subunit extended and run parallel to the coiled-coil of the γ subunit. The other non-inhibitory conformation is characterized by a hairpin-fold of C-terminal α helices. These two states of the ε subunit, termed as an extended ε and folded ε hereafter, are controlled by the concentration of ATP, as well as the membrane potential [16], [17]. It has been suggested that when FoF1 catalyzes ATP synthesis the ε subunit takes extended-conformation and that ATP synthesis is not inhibited by the extended ε[18]. The folded ε itself can bind ATP, but not ADP or other nucleotides, and is stabilized by this ATP binding in the FoF1’s from some bacteria including thermophilic Bacillus PS3 [19].

There are several reports on the reaction step that is suppressed by the ε subunit. Dunn et al. reported that, in EF1, ε subunit mainly affects the product release of ATPase reaction [20]. Effect of the ε subunit on the nucleotide binding to the catalytic sites of Escherichia coli F1-ATPase (EF1) was examined by using a tryptophan mutant, which allows measurement of nucleotide binding to catalytic sites through fluorescent quenching of the introduced tryptophans [21]. In the case of EF1, the binding was not significantly affected by the ε subunit, also suggesting that ε subunit mainly suppress the catalytic events after the binding in ATP hydrolysis reaction. On the other hand, in the case of F1-ATPase from thermophilic Bacillus PS3 (TF1), ε subunit has been reported to suppress the sub-stoichiometric 2′,3′,-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) binding to the high affinity site [16], [22] and this inhibitory effect is released when ε subunit changes its conformation into folded-state by the exposure to high concentration of ATP [14], [17].

Here, to determine the effect of the ε subunit on the nucleotide binding to the catalytic sites of TF1, we have carried out fluorescence measurement with a mutant (αW463F/βY341W) α3β3γ complex with and without ε subunit. Strong inhibitory effect of the ε subunit on nucleotide binding was observed and this may relate to the efficient ATP synthesis in the presence of the ε subunit.

Section snippets

Materials and methods

Proteins. A mutant (αW463F, βY341W) α3β3γ complex of TF1 was prepared as described previously [23], [24]. Wild type and truncated (εΔC, Val90 to stop) mutant ε subunit of TF1 were prepared as described previously [25], [26].

Fluorescence measurement. Fluorescent change by the nucleotide binding was assayed as follows: the assay mixture consisted of 50 mM Tris–HCl (pH 8.0), 100 mM KCl, 2 mM MgCl2 and 0.17% or 0.5% lauryl dimethyl amine oxide (LDAO) was transferred to a quartz cuvette (2 ml). LDAO was

ATP and ADP binding to the catalytic sites

As reported previously, nucleotide binding to the catalytic sites can be monitored as the decrease in the tryptophan fluorescence of the mutant (αW463F/βY341W) of TF1[24], [28], [31], [32]. Time-courses of the decrease in the fluorescence induced by the addition of 10 μM ATP and ADP are shown in Fig. 1. Upon addition of ATP to the α3β3γ complex, the fluorescence dropped immediately down to about 25% and no further decrease was observed (Fig. 1). In the case of the α3β3γε complex, ATP binding was

Acknowledgments

We thank Yamasa Corporation for providing us with IDP. This work was supported in parts by Grants-in-Aid for Scientific Research on Priority Areas (No. 18074002) and for Young Scientists (B) (No. 18770118) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to Y. K.-Y.). We thank members of Kato-Yamada’s laboratory in Rikkyo University for their help and fruitful discussion.

References (36)

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    Consistent with this assumption, an increase of the binding affinity of Mg-ATP and Mg-ADP to the high affinity catalytic site(s) of α3β3γ of EF1 caused by the addition of the ϵ subunit has already been reported (47). In contrast, the ϵ subunit weakened the binding affinity of Mg-ADP to each catalytic site, especially to the high affinity site(s) in the case of the α3β3γ complex of TF1 (48). Hence, there must be a variety of ϵ effects on the nucleotide binding affinity based on the difference of the origin of the ϵ subunits (47, 48).

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