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γε sub-complex of thermophilic ATP synthase has the ability to bind ATP
BBRC Biochemical and Biophysical Research Communications 349 (2006) 1368–1371 www.elsevier.com/locate/ybbrc
ce sub-complex of thermophilic ATP synthase has the ability to bind ATP q Satoshi Iizuka a, Shigeyuki Kato a, Masasuke Yoshida c, Yasuyuki Kato-Yamada a
a,b,*
Department of Life Science, College of Science, Rikkyo University, 3-34-1, Nishi-Ikebukuro, Toshima-ku, Tokyo 171-8501, Japan b Frontier Project ‘‘Adaptation and Evolution of Extremophile’’, College of Science, Rikkyo University, Tokyo 171-8501, Japan c Chemical Resources Laboratory, Tokyo Institute of Technology, Yokohama 226-8503, Japan Received 30 August 2006 Available online 11 September 2006
Abstract The isolated e subunit of F1-ATPase from thermophilic Bacillus PS3 (TF1) binds ATP [Y. Kato-Yamada, M. Yoshida, J. Biol. Chem. 278 (2003) 36013]. The obvious question is whether the ATP binding concern with the regulation of ATP synthase activity or not. If so, the e subunit even in the ATP synthase complex should have the ability to bind ATP. To check if the ATP binding to the e subunit within the ATP synthase complex may occur, the ce sub-complex of TF1 was prepared and ATP binding was examined. The results clearly showed that the ce sub-complex can bind ATP. 2006 Elsevier Inc. All rights reserved. Keywords: ATP binding; e subunit; F1; ATP synthase; FoF1
ATP synthase (FoF1) catalyzes ATP synthesis coupled with the proton flow across membrane through mechanical rotation of the central rotor shaft subunits relative to the surrounding stator subunits [1,2]. F1 is the water-soluble portion of FoF1 and has ATP hydrolysis activity by itself. It consists of five kinds of subunits with a stoichiometry of a3b3c1d1e1 in which the catalytic nucleotide binding sites are located on the b subunits and the non-catalytic nucleotide binding sites are on the a subunits. Long coiled-coil helices of the c subunit penetrates into the central cavity of the a3b3 cylinder [3] and ATP hydrolysis occurring in the a3b3 drives rotation of the c subunit [reviewed in 2]. The e subunit, associated to the c subunit constituting a central rotor shaft, also rotates [4].
q
Abbreviations: F1-ATPase, soluble portion of FoF1 ATP synthase; TF1, F1-ATPase from thermophilic Bacillus PS3; EF1, F1-ATPase from Escherichia coli; Hepes, 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid; c 0 , a mutant c subunit of TF1 that contains N-terminal Met and residues Ile11–Ile255. * Corresponding author. Fax: +81 3 3985 2386. E-mail address: [email protected] (Y. Kato-Yamada). 0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.09.001
The e subunit in bacterial and chloroplast ATP synthases has been known to be an intrinsic inhibitor of ATP hydrolysis activity since the early stage of studies of ATP synthase [5–7]. It is a small subunit of 130–140 residues consisting of two distinct domains, an N-terminal b sandwich domain and a C-terminal a helical hairpin domain [8,9]. Accumulating biochemical and structural studies have revealed that the e subunit can adopt at least two different conformational states in F1 and FoF1, folded (down)-state and extended (up)-state [10–17]. The structures of the isolated e subunit from Escherichia coli represent the folded-state conformation that does not exhibit the inhibitory effect [8,9]. The exact structure of the extended-state e subunit in F1 and FoF1 is not known but it is most likely that the C-terminal helical hairpin in the folded-state is extended in the extended-state [16] and become in the vicinity of the a and b subunits [18]. The e subunit in the extended-state exerts the inhibitory effect on ATP hydrolysis activity but, apparently, not on ATP synthesis activity of FoF1 [19,20]. We recently found that the isolated e subunit of F1 from thermophilic Bacillus strain PS3 (TF1) can bind ATP
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[21,22]. The binding was specific since GTP and ADP failed to form a complex with e. ATP binding to the e subunit of F1 from Bacillus subtilis was also observed [23]. The recent crystal structure of TF1-e with bound ATP takes the folded conformation (N. Kajiwara, H. Akutsu, Y.K.-Y., M.Y. et al., unpublished data) and C-terminal a helices provide major interactions with ATP [21]. The dissociation constant between the e subunit and ATP is in the range of sub-millimolar to millimolar under physiological temperatures [22,23]. These findings raise an attractive possibility that the e subunit in FoF1 acts not only as a regulator of FoF1 but also as a sensor for cellular ATP level. Under the conditions where DlH+ is poor while ATP is ample, ATP binds to e in FoF1 to stabilize its folded (ATPase-favoring) conformation so that FoF1 can work as an ATPdriven H+ pump to generate DlH+. To confirm the above scenario, direct demonstration of ATP binding to the e subunit in the FoF1 is critical. However, as the F1-ATPase contains six nucleotide binding sites other than the e subunit, it is difficult to demonstrate ATP binding to the e subunit in the FoF1 or even F1, directly by the method such as the gel-filtration assay. The fluorescent titration used in our previous studies [22,23] did not work because fluorescence of the e-bound dye was affected by conformational change in the e subunit triggered by the ATP binding to b subunits [15]. To avoid these problems, we used the central rotor shaft, ce sub-complex of FoF1 because the c subunit is the only subunit among F1 subunits that has interactions with e subunit in the folded conformation. Although Fo c-ring also has interactions with b-sandwich region of e subunit but less with a helices that constitute major part of ATP binding site [24]. Therefore, we expect that the ce sub-complex can be a much better experimental model to test the ATP binding to the e subunit in FoF1 than the isolated e subunit. The results indicate that the ATP binding to the e subunit also occurs in the ce sub-complex, supporting the hypothesis that the ATP binding to the e subunit concerns with the regulation of ATP synthesis/hydrolysis activities of ATP synthase.
of 2·YT medium with vigorous shaking at 37 C. When the optical density at 600 nm of the medium reached to 0.8, an inducer, isopropyl-b-D-thiogalactopyranoside was added to 1 mM. After the further 3 h of cultivation, the cells were harvested by centrifugation. About 2 g per liter culture of the cells were obtained. The cells were suspended in 50 mM Tris–HCl (pH 8.0) and 1 mM EDTA (buffer A) and disrupted by French pressure cell (Aminco). The cell lysate was centrifuged at 1,30,000g for 1 h at 4 C. Following procedures were carried out at room temperature (around 25 C). The supernatant was applied to 30 ml of DEAE-toyopearl (Tosoh, Japan) column equilibrated with buffer A. The c 0 was adsorbed on the column. The column was washed with buffer A and c 0 was eluted by linear gradient of NaCl (0–350 mM) in buffer A. The fractions containing c 0 were collected and solid ammonium sulfate was added to 20 % saturation. Then the samples were subjected to 30 ml of Phenyl-toyopearl (Tosoh, Japan) column equilibrated with buffer A with 20 % saturated ammonium sulfate. After the washing with the same buffer, adsorbed proteins were eluted by a linear gradient of ammonium sulfate (20–0% saturation). The fractions containing c 0 were pooled and solid ammonium sulfate was added to 65 % saturation. The sample was stored at 4 C as ammonium sulfate suspension. About 35 mg of c 0 was obtained from one liter culture. Test of the c 0 e sub-complex formation. c 0 (100 lM) was mixed with 100 or 200 lM of e subunit in 20 mM Hepes–KOH (pH 7.5) and 100 mM KCl (buffer B). After 10 min, 100 ll of the mixture was subjected to a gel-filtration HPLC column (Superdex 75 100/300 GL, GE healthcare) equilibrated with buffer B at room temperature. Flow rate was 0.5 ml/min. The elution was monitored by the absorbance at 280 nm. The peak was collected and analyzed by SDS–PAGE [25]. Test of ATP binding by gel-filtration assay. Various concentrations of ATP (0, 40, 100, and 200 lM) were added to the 100 lM of c 0 or c 0 + e mixture (130 lM c 0 plus 100 lM e) in buffer B at room temperature. After more than 10 min, 100 ll of the mixture was subjected to gel-filtration column equilibrated with buffer B. Flow rate was 0.5 ml/min. The elution was monitored by absorbance at 260 nm. Other procedures. e subunit was prepared as described previously [26]. We have noticed that there is a tendency that when we use large sized butyl-toyopearl column in the purification procedures, there are less (<0.05 mol/mol) amount of the bound ATP in the e subunit preparations. Thus obtained bound nucleotide free e subunit was used. Protein concentrations were determined by the method of Bradford [27] using bovine serum albumin as a standard. The protein concentration of e subunit was corrected by multiplying by a factor 0.54 according to the results of amino acid quantification [21].
Materials and methods
ATP binding to the mutant of c subunit that contained central domain only (c 0 ), that is similar to the one reported for EF1 [16], was tested. As the c subunit contained Rossmann-fold [16,17], one of the typical fold found in nucleotide binding proteins, there is a possibility that the c 0 subunit also binds ATP. However, as tested by gel-filtration analysis, no ATP binding was detected (Fig. 1), although the method can only detect strong binding.
Construction of an expression plasmid for the mutant TF1 c subunit. A mutant form of the c subunit of TF1 that was contained only the central domain of the c subunit (N-terminal Met and residues Ile11–Ile255, denote as c 0 hereafter) was prepared by polymerase chain reaction by ExTaq DNA polymerase (Takara bio, Japan) with the following primers; 5 0 -GGCATATGATCAATGCGACGAAGAAGACAA-3 0 and 5 0 -CCG GATCCTTAAATGAGCTCGTTCGCATTG-3 0 using an expression plasmid for wild type a3b3c complex of TF1 as a template. The resulting fragment contained NdeI site and initiation codon before Ile11 and termination codon after Ile255 and BamHI site at 5 0 and 3 0 termini, respectively. The amplified DNA fragment containing mutated TF1 c 0 subunit gene was cloned into pGEM-T (Promega). Then, the fragment was excised by NdeI and BamHI, and cloned into respective sites of pET21c expression vector (Novagen) to obtain pET21-c 0 . The DNA sequence was confirmed by DNA sequencing. Purification of the c 0 subunit of TF1. The expression of TF1 c 0 subunit was performed by following standard protocol. The pET21-c 0 plasmid was transformed into BL21(DE3) and the transformant was cultivated in 1 L
Results and discussion ATP did not bind to the c 0 subunit
Formation of the c 0 e sub-complex Formation of c 0 e sub-complex was tested by gel-filtration analysis. c 0 alone was eluted at 22 min 20 s while e alone at 23 min 30 s (Fig. 2a). When c 0 was mixed with e, a new peak appeared at 20 min 20 s indicating the formation of c 0 e sub-complex (Fig. 2a). The peak fraction was collected and analyzed by SDS–PAGE (Fig. 2b). The newly
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Fig. 1. Test of ATP binding to TF1-c 0 subunit. The c 0 subunits (100 lM) with and without 100 lM ATP were subjected to Superdex 75HR gelfiltration HPLC analysis. The elution was monitored by the absorbance at 260 nm. The c 0 with and without ATP are marked as +ATP and ATP, respectively. The peak position of the c 0 is marked with an arrow. Other experimental conditions are described in Materials and methods.
Fig. 2. Tests of the formation of TF1-c 0 e sub-complex. (a) Gel-filtration analysis. The c 0 subunit, e subunit and the mixture of c 0 and e subunits were subjected to Superdex 75HR gel-filtration HPLC analysis. The elution was monitored by the absorbance at 280 nm. Each trace represents the result of the sample described in the figure. (b) SDS–PAGE analysis. The peak fractions in (a) (marked with arrows) were subjected to 15% polyacrylamide SDS–PAGE. The sizes of the molecular weight markers were shown on the left in kDa unit. Samples applied were marked on the top. Other experimental conditions are described in Materials and methods.
appeared peak indeed contained both the c 0 and e subunits. The c 0 e sub-complex was stable enough to be isolate by gel-filtration chromatography. Titration of c 0 with e resulted in the saturation at about 1:1 ratio (Fig. 3). Small peak of e appeared in the 1:1 mixture is likely due to the deviations in the protein concentration measurements. The molecular mass of the c 0 e sub-complex was 42,000 as determined by the retention time on the gel-filtration HPLC calibrated with several globular proteins (data not shown) while the calculated molecular mass of the c 0 e sub-complex from their primary structure is 42,100. From these results we concluded that the c 0 e sub-complex is a hetero-dimer as expected from their stoichiometry in holo FoF1 complex and previous observations with EF1 ce sub-complex [16,28]. ATP binding to the c 0 e sub-complex ATP binding to the c 0 e sub-complex was assayed by gelfiltration HPLC (Fig. 4). Up to 1:1 ratio, the peak height of
Fig. 3. Determination of subunit stoichiometry in TF1-c 0 e sub-complex. The 1:1 and 1:2 mixtures of c 0 and e subunits were subjected to Superdex 75HR gel-filtration HPLC analysis. The elution was monitored by the absorbance at 280 nm. The peaks correspond to c 0 e sub-complex were marked with an arrow. Other experimental conditions are described in Materials and methods.
Fig. 4. Test of ATP binding to TF1-c 0 e sub-complex. Various concentrations of ATP (0, 40, 100 and 200 lM) were added to the 100 lM of c 0 + e mixture (130 lM c 0 plus 100 lM e). The mixtures were subjected to Superdex 75HR gel-filtration HPLC analysis. The elution was monitored by the absorbance at 260 nm. The peaks correspond to c 0 e sub-complex were marked with an arrow. Traces were marked with the ratios of the c 0 e sub-complex to added ATP. Other experimental conditions are described in Materials and methods.
the c 0 e sub-complex proportionally grew as the ATP concentration increased. As there was no peak corresponding to free ATP, most of the added ATP bound to the c 0 e sub-complex. When the ATP concentration was increased to 1:2, the peak height of c 0 e sub-complex no longer changed and a peak corresponding to free ATP appeared. These results were similar to those obtained for the isolated e subunit [21]. The present study clearly showed that the e subunit in the c 0 e sub-complex binds ATP at 1:1 ratio. The association of e with c subunits does not disturb the ATP binding. As mentioned, most of the interactions between the e subunit and other subunits in FoF1 are with the c subunit when the e subunit is in the folded state although Fo c-ring interacts with N-terminal domain of the e subunit [17,24]. Therefore, it is highly likely that the ATP binding to the e subunit occurs in FoF1 when the e subunit takes folded conformation. On the other hand, when the e
S. Iizuka et al. / Biochemical and Biophysical Research Communications 349 (2006) 1368–1371
subunit is in the extended state, the C-terminal helices, which constitute most of the ATP binding site [21], are inserted into the cavity of a3b3. There seems to be no room for ATP to bind to the e subunit in the extended state. A likely scenario is that the ATP binding to the b subunits triggers the conformational change in the e subunit from the extended-state to the folded (ATPase active)-state, then another ATP binds to the e subunit and the folded-state is stabilized and the enzyme is kept in the ATPase-active state [15,20,22]. The experiments on the relationships between ATP binding to the e subunit and ATP synthase activity are underway in our group. Acknowledgments We thank Prof. Tsuneyoshi Kuroiwa at Rikkyo University for allowing us to use a DNA sequencing apparatus. This work was supported in part by grants-in-Aid for Young Scientists (B) (No. 18770118) and for Scientific Research on Priority Areas (No. 18074002) from the Ministry of Education, Culture, Sports, Science and Technology and Rikkyo University Special Fund for Research (to Y.K-Y.). References [1] P.D. Boyer, The ATP synthase—a splendid molecular machine, Annu. Rev. Biochem. 66 (1997) 717–749. [2] M. Yoshida, E. Muneyuki, T. Hisabori, ATP synthase—a marvellous rotary engine of the cell, Nat. Rev. Mol. Cell Biol. 2 (2001) 669–677. ˚ [3] J.P. Abrahams, A. Leslie, R. Lutter, J.E. Walker, Structure at 2.8 A
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resolution of F1-ATPase from bovine heart mitochondria, Nature 370 (1994) 621–628. Y. Kato-Yamada, H. Noji, R. Yasuda, K. Kinosita Jr., M. Yoshida, Direct observation of the rotation of epsilon subunit in F1-ATPase, J. Biol. Chem. 273 (1998) 19375–19377. J.B. Smith, P.C. Sternweis, L.A. Heppel, Partial purification of active delta and epsilon subunits of the membrane ATPase from Escherichia coli, J. Supramol. Struct. 3 (1975) 248–255. P.P. Laget, J.B. Smith, Inhibitory properties of endogenous subunit epsilon in the Escherichia coli F1 ATPase, Arch. Biochem. Biophys. 197 (1979) 83–89. P.C. Sternweis, J.B. Smith, Characterization of the inhibitory (epsilon) subunit of the proton-translocating adenosine triphosphatase from Escherichia coli, Biochemistry 19 (1980) 526–531. S. Wilkens, F.W. Dahlquist, L.P. McIntosh, L.W. Donaldson, R.A. Capaldi, Structural features of the epsilon subunit of the Escherichia coli ATP synthase determined by NMR spectroscopy, Nat. Struct. Biol. 2 (1995) 961–967. U. Uhlin, G.B. Cox, J.M. Guss, Crystal structure of the epsilon subunit of the proton-translocating ATP synthase from Escherichia coli, Structure 5 (1997) 1219–1230. R. Aggeler, R.A. Capaldi, Nucleotide-dependent movement of the epsilon subunit between alpha and beta subunits in the Escherichia coli F1Fo-type ATPase, J. Biol. Chem. 271 (1996) 13888–13891.
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[11] G. Gruber, R.A. Capaldi, The trapping of different conformations of the Escherichia coli F1 ATPase by disulfide bond formation. Effect on nucleotide binding affinities of the catalytic sites, J. Biol. Chem. 271 (1996) 32623–32628. [12] Y. Kato, T. Matsui, N. Tanaka, E. Muneyuki, T. Hisabori, M. Yoshida, Thermophilic F1-ATPase is activated without dissociation of an endogenous inhibitor, epsilon subunit, J. Biol. Chem. 272 (1997) 24906–24912. [13] B. Schulenberg, R.A. Capaldi, The epsilon subunit of the F1Fo complex of Escherichia coli cross-linking studies show the same structure in situ as when isolated, J. Biol. Chem. 274 (1999) 28351– 28355. [14] Y. Kato-Yamada, D. Bald, M. Koike, K. Motohashi, T. Hisabori, M. Yoshida, Epsilon subunit, an endogenous inhibitor of bacterial F1-ATPase, also inhibits FoF1-ATPase, J. Biol. Chem. 274 (1999) 33991–33994. [15] Y. Kato-Yamada, M. Yoshida, T. Hisabori, Movement of the helical domain of the epsilon subunit is required for the activation of thermophilic F1-ATPase, J. Biol. Chem. 275 (2000) 35746–35750. [16] A.J.W. Rodgers, M.C.J. Wilce, Structure of the gamma-epsilon complex of ATP synthase, Nat. Struct. Biol. 7 (2000) 1051–1054. [17] C. Gibbons, M.G. Montgomery, A.G.W. Leslie, J.E. Walker, The ˚ resolution, structure of the central stalk in bovine F1-ATPase at 2.4 A Nat. Struct. Biol. 7 (2000) 1055–1061. [18] K.Y. Hara, Y. Kato-Yamada, Y. Kikuchi, T. Hisabori, M. Yoshida, The role of the betaDELSEED motif of F1-ATPase: propagation of the inhibitory effect of the epsilon subunit, J. Biol. Chem. 276 (2001) 23969–23973. [19] S.P. Tsunoda, A.J.W. Rodgers, R. Aggeler, M.C.J. Wilce, M. Yoshida, R.A. Capaldi, Large conformational changes of the epsilon subunit in the bacterial F1Fo ATP synthase provide a ratchet action to regulate this rotary motor enzyme, Proc. Natl. Acad. Sci. USA 98 (2001) 6560–6564. [20] T. Suzuki, T. Murakami, R. Iino, J. Suzuki, S. Ono, Y. Shirakihara, M. Yoshida, FoF1-ATPase/synthase is geared to the synthesis mode by conformational rearrangement of epsilon subunit in response to proton motive force and ADP/ATP balance, J. Biol. Chem. 278 (2003) 46840–46846. [21] Y. Kato-Yamada, M. Yoshida, Isolated epsilon subunit of thermophilic F1-ATPase binds ATP, J. Biol. Chem. 278 (2003) 36013–36016. [22] R. Iino, T. Murakami, S. Iizuka, Y. Kato-Yamada, T. Suzuki, M. Yoshida, Real time monitoring of conformational dynamics of the epsilon subunit in F1-ATPase, J. Biol. Chem. 280 (2005) 40130–40134. [23] Y. Kato-Yamada, Isolated epsilon subunit of Bacillus subtilis F1-ATPase binds ATP, FEBS Lett. 579 (2005) 6875–6878. [24] D. Stock, A.G. Leslie, J.E. Walker, Molecular architecture of the rotary motor in ATP synthase, Science 286 (1999) 1700–1705. [25] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685. [26] T. Hisabori, Y. Kato, K. Motohashi, P. Kroth-Pancic, H. Strotmann, T. Amano, The regulatory functions of the gamma and epsilon subunits from chloroplast CF1are transferred to the core complex, alpha3beta3, from thermophilic bacterial F1, Eur. J. Biochem. 247 (1997) 1158–1165. [27] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. [28] S.D. Dunn, The isolated gamma subunit of Escherichia coli F1 ATPase binds the epsilon subunit, J. Biol. Chem. 257 (1982) 7354– 7359.
ce sub-complex of thermophilic ATP synthase has the ability to bind ATP q Satoshi Iizuka a, Shigeyuki Kato a, Masasuke Yoshida c, Yasuyuki Kato-Yamada a
a,b,*
Department of Life Science, College of Science, Rikkyo University, 3-34-1, Nishi-Ikebukuro, Toshima-ku, Tokyo 171-8501, Japan b Frontier Project ‘‘Adaptation and Evolution of Extremophile’’, College of Science, Rikkyo University, Tokyo 171-8501, Japan c Chemical Resources Laboratory, Tokyo Institute of Technology, Yokohama 226-8503, Japan Received 30 August 2006 Available online 11 September 2006
Abstract The isolated e subunit of F1-ATPase from thermophilic Bacillus PS3 (TF1) binds ATP [Y. Kato-Yamada, M. Yoshida, J. Biol. Chem. 278 (2003) 36013]. The obvious question is whether the ATP binding concern with the regulation of ATP synthase activity or not. If so, the e subunit even in the ATP synthase complex should have the ability to bind ATP. To check if the ATP binding to the e subunit within the ATP synthase complex may occur, the ce sub-complex of TF1 was prepared and ATP binding was examined. The results clearly showed that the ce sub-complex can bind ATP. 2006 Elsevier Inc. All rights reserved. Keywords: ATP binding; e subunit; F1; ATP synthase; FoF1
ATP synthase (FoF1) catalyzes ATP synthesis coupled with the proton flow across membrane through mechanical rotation of the central rotor shaft subunits relative to the surrounding stator subunits [1,2]. F1 is the water-soluble portion of FoF1 and has ATP hydrolysis activity by itself. It consists of five kinds of subunits with a stoichiometry of a3b3c1d1e1 in which the catalytic nucleotide binding sites are located on the b subunits and the non-catalytic nucleotide binding sites are on the a subunits. Long coiled-coil helices of the c subunit penetrates into the central cavity of the a3b3 cylinder [3] and ATP hydrolysis occurring in the a3b3 drives rotation of the c subunit [reviewed in 2]. The e subunit, associated to the c subunit constituting a central rotor shaft, also rotates [4].
q
Abbreviations: F1-ATPase, soluble portion of FoF1 ATP synthase; TF1, F1-ATPase from thermophilic Bacillus PS3; EF1, F1-ATPase from Escherichia coli; Hepes, 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid; c 0 , a mutant c subunit of TF1 that contains N-terminal Met and residues Ile11–Ile255. * Corresponding author. Fax: +81 3 3985 2386. E-mail address: [email protected] (Y. Kato-Yamada). 0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.09.001
The e subunit in bacterial and chloroplast ATP synthases has been known to be an intrinsic inhibitor of ATP hydrolysis activity since the early stage of studies of ATP synthase [5–7]. It is a small subunit of 130–140 residues consisting of two distinct domains, an N-terminal b sandwich domain and a C-terminal a helical hairpin domain [8,9]. Accumulating biochemical and structural studies have revealed that the e subunit can adopt at least two different conformational states in F1 and FoF1, folded (down)-state and extended (up)-state [10–17]. The structures of the isolated e subunit from Escherichia coli represent the folded-state conformation that does not exhibit the inhibitory effect [8,9]. The exact structure of the extended-state e subunit in F1 and FoF1 is not known but it is most likely that the C-terminal helical hairpin in the folded-state is extended in the extended-state [16] and become in the vicinity of the a and b subunits [18]. The e subunit in the extended-state exerts the inhibitory effect on ATP hydrolysis activity but, apparently, not on ATP synthesis activity of FoF1 [19,20]. We recently found that the isolated e subunit of F1 from thermophilic Bacillus strain PS3 (TF1) can bind ATP
S. Iizuka et al. / Biochemical and Biophysical Research Communications 349 (2006) 1368–1371
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[21,22]. The binding was specific since GTP and ADP failed to form a complex with e. ATP binding to the e subunit of F1 from Bacillus subtilis was also observed [23]. The recent crystal structure of TF1-e with bound ATP takes the folded conformation (N. Kajiwara, H. Akutsu, Y.K.-Y., M.Y. et al., unpublished data) and C-terminal a helices provide major interactions with ATP [21]. The dissociation constant between the e subunit and ATP is in the range of sub-millimolar to millimolar under physiological temperatures [22,23]. These findings raise an attractive possibility that the e subunit in FoF1 acts not only as a regulator of FoF1 but also as a sensor for cellular ATP level. Under the conditions where DlH+ is poor while ATP is ample, ATP binds to e in FoF1 to stabilize its folded (ATPase-favoring) conformation so that FoF1 can work as an ATPdriven H+ pump to generate DlH+. To confirm the above scenario, direct demonstration of ATP binding to the e subunit in the FoF1 is critical. However, as the F1-ATPase contains six nucleotide binding sites other than the e subunit, it is difficult to demonstrate ATP binding to the e subunit in the FoF1 or even F1, directly by the method such as the gel-filtration assay. The fluorescent titration used in our previous studies [22,23] did not work because fluorescence of the e-bound dye was affected by conformational change in the e subunit triggered by the ATP binding to b subunits [15]. To avoid these problems, we used the central rotor shaft, ce sub-complex of FoF1 because the c subunit is the only subunit among F1 subunits that has interactions with e subunit in the folded conformation. Although Fo c-ring also has interactions with b-sandwich region of e subunit but less with a helices that constitute major part of ATP binding site [24]. Therefore, we expect that the ce sub-complex can be a much better experimental model to test the ATP binding to the e subunit in FoF1 than the isolated e subunit. The results indicate that the ATP binding to the e subunit also occurs in the ce sub-complex, supporting the hypothesis that the ATP binding to the e subunit concerns with the regulation of ATP synthesis/hydrolysis activities of ATP synthase.
of 2·YT medium with vigorous shaking at 37 C. When the optical density at 600 nm of the medium reached to 0.8, an inducer, isopropyl-b-D-thiogalactopyranoside was added to 1 mM. After the further 3 h of cultivation, the cells were harvested by centrifugation. About 2 g per liter culture of the cells were obtained. The cells were suspended in 50 mM Tris–HCl (pH 8.0) and 1 mM EDTA (buffer A) and disrupted by French pressure cell (Aminco). The cell lysate was centrifuged at 1,30,000g for 1 h at 4 C. Following procedures were carried out at room temperature (around 25 C). The supernatant was applied to 30 ml of DEAE-toyopearl (Tosoh, Japan) column equilibrated with buffer A. The c 0 was adsorbed on the column. The column was washed with buffer A and c 0 was eluted by linear gradient of NaCl (0–350 mM) in buffer A. The fractions containing c 0 were collected and solid ammonium sulfate was added to 20 % saturation. Then the samples were subjected to 30 ml of Phenyl-toyopearl (Tosoh, Japan) column equilibrated with buffer A with 20 % saturated ammonium sulfate. After the washing with the same buffer, adsorbed proteins were eluted by a linear gradient of ammonium sulfate (20–0% saturation). The fractions containing c 0 were pooled and solid ammonium sulfate was added to 65 % saturation. The sample was stored at 4 C as ammonium sulfate suspension. About 35 mg of c 0 was obtained from one liter culture. Test of the c 0 e sub-complex formation. c 0 (100 lM) was mixed with 100 or 200 lM of e subunit in 20 mM Hepes–KOH (pH 7.5) and 100 mM KCl (buffer B). After 10 min, 100 ll of the mixture was subjected to a gel-filtration HPLC column (Superdex 75 100/300 GL, GE healthcare) equilibrated with buffer B at room temperature. Flow rate was 0.5 ml/min. The elution was monitored by the absorbance at 280 nm. The peak was collected and analyzed by SDS–PAGE [25]. Test of ATP binding by gel-filtration assay. Various concentrations of ATP (0, 40, 100, and 200 lM) were added to the 100 lM of c 0 or c 0 + e mixture (130 lM c 0 plus 100 lM e) in buffer B at room temperature. After more than 10 min, 100 ll of the mixture was subjected to gel-filtration column equilibrated with buffer B. Flow rate was 0.5 ml/min. The elution was monitored by absorbance at 260 nm. Other procedures. e subunit was prepared as described previously [26]. We have noticed that there is a tendency that when we use large sized butyl-toyopearl column in the purification procedures, there are less (<0.05 mol/mol) amount of the bound ATP in the e subunit preparations. Thus obtained bound nucleotide free e subunit was used. Protein concentrations were determined by the method of Bradford [27] using bovine serum albumin as a standard. The protein concentration of e subunit was corrected by multiplying by a factor 0.54 according to the results of amino acid quantification [21].
Materials and methods
ATP binding to the mutant of c subunit that contained central domain only (c 0 ), that is similar to the one reported for EF1 [16], was tested. As the c subunit contained Rossmann-fold [16,17], one of the typical fold found in nucleotide binding proteins, there is a possibility that the c 0 subunit also binds ATP. However, as tested by gel-filtration analysis, no ATP binding was detected (Fig. 1), although the method can only detect strong binding.
Construction of an expression plasmid for the mutant TF1 c subunit. A mutant form of the c subunit of TF1 that was contained only the central domain of the c subunit (N-terminal Met and residues Ile11–Ile255, denote as c 0 hereafter) was prepared by polymerase chain reaction by ExTaq DNA polymerase (Takara bio, Japan) with the following primers; 5 0 -GGCATATGATCAATGCGACGAAGAAGACAA-3 0 and 5 0 -CCG GATCCTTAAATGAGCTCGTTCGCATTG-3 0 using an expression plasmid for wild type a3b3c complex of TF1 as a template. The resulting fragment contained NdeI site and initiation codon before Ile11 and termination codon after Ile255 and BamHI site at 5 0 and 3 0 termini, respectively. The amplified DNA fragment containing mutated TF1 c 0 subunit gene was cloned into pGEM-T (Promega). Then, the fragment was excised by NdeI and BamHI, and cloned into respective sites of pET21c expression vector (Novagen) to obtain pET21-c 0 . The DNA sequence was confirmed by DNA sequencing. Purification of the c 0 subunit of TF1. The expression of TF1 c 0 subunit was performed by following standard protocol. The pET21-c 0 plasmid was transformed into BL21(DE3) and the transformant was cultivated in 1 L
Results and discussion ATP did not bind to the c 0 subunit
Formation of the c 0 e sub-complex Formation of c 0 e sub-complex was tested by gel-filtration analysis. c 0 alone was eluted at 22 min 20 s while e alone at 23 min 30 s (Fig. 2a). When c 0 was mixed with e, a new peak appeared at 20 min 20 s indicating the formation of c 0 e sub-complex (Fig. 2a). The peak fraction was collected and analyzed by SDS–PAGE (Fig. 2b). The newly
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Fig. 1. Test of ATP binding to TF1-c 0 subunit. The c 0 subunits (100 lM) with and without 100 lM ATP were subjected to Superdex 75HR gelfiltration HPLC analysis. The elution was monitored by the absorbance at 260 nm. The c 0 with and without ATP are marked as +ATP and ATP, respectively. The peak position of the c 0 is marked with an arrow. Other experimental conditions are described in Materials and methods.
Fig. 2. Tests of the formation of TF1-c 0 e sub-complex. (a) Gel-filtration analysis. The c 0 subunit, e subunit and the mixture of c 0 and e subunits were subjected to Superdex 75HR gel-filtration HPLC analysis. The elution was monitored by the absorbance at 280 nm. Each trace represents the result of the sample described in the figure. (b) SDS–PAGE analysis. The peak fractions in (a) (marked with arrows) were subjected to 15% polyacrylamide SDS–PAGE. The sizes of the molecular weight markers were shown on the left in kDa unit. Samples applied were marked on the top. Other experimental conditions are described in Materials and methods.
appeared peak indeed contained both the c 0 and e subunits. The c 0 e sub-complex was stable enough to be isolate by gel-filtration chromatography. Titration of c 0 with e resulted in the saturation at about 1:1 ratio (Fig. 3). Small peak of e appeared in the 1:1 mixture is likely due to the deviations in the protein concentration measurements. The molecular mass of the c 0 e sub-complex was 42,000 as determined by the retention time on the gel-filtration HPLC calibrated with several globular proteins (data not shown) while the calculated molecular mass of the c 0 e sub-complex from their primary structure is 42,100. From these results we concluded that the c 0 e sub-complex is a hetero-dimer as expected from their stoichiometry in holo FoF1 complex and previous observations with EF1 ce sub-complex [16,28]. ATP binding to the c 0 e sub-complex ATP binding to the c 0 e sub-complex was assayed by gelfiltration HPLC (Fig. 4). Up to 1:1 ratio, the peak height of
Fig. 3. Determination of subunit stoichiometry in TF1-c 0 e sub-complex. The 1:1 and 1:2 mixtures of c 0 and e subunits were subjected to Superdex 75HR gel-filtration HPLC analysis. The elution was monitored by the absorbance at 280 nm. The peaks correspond to c 0 e sub-complex were marked with an arrow. Other experimental conditions are described in Materials and methods.
Fig. 4. Test of ATP binding to TF1-c 0 e sub-complex. Various concentrations of ATP (0, 40, 100 and 200 lM) were added to the 100 lM of c 0 + e mixture (130 lM c 0 plus 100 lM e). The mixtures were subjected to Superdex 75HR gel-filtration HPLC analysis. The elution was monitored by the absorbance at 260 nm. The peaks correspond to c 0 e sub-complex were marked with an arrow. Traces were marked with the ratios of the c 0 e sub-complex to added ATP. Other experimental conditions are described in Materials and methods.
the c 0 e sub-complex proportionally grew as the ATP concentration increased. As there was no peak corresponding to free ATP, most of the added ATP bound to the c 0 e sub-complex. When the ATP concentration was increased to 1:2, the peak height of c 0 e sub-complex no longer changed and a peak corresponding to free ATP appeared. These results were similar to those obtained for the isolated e subunit [21]. The present study clearly showed that the e subunit in the c 0 e sub-complex binds ATP at 1:1 ratio. The association of e with c subunits does not disturb the ATP binding. As mentioned, most of the interactions between the e subunit and other subunits in FoF1 are with the c subunit when the e subunit is in the folded state although Fo c-ring interacts with N-terminal domain of the e subunit [17,24]. Therefore, it is highly likely that the ATP binding to the e subunit occurs in FoF1 when the e subunit takes folded conformation. On the other hand, when the e
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subunit is in the extended state, the C-terminal helices, which constitute most of the ATP binding site [21], are inserted into the cavity of a3b3. There seems to be no room for ATP to bind to the e subunit in the extended state. A likely scenario is that the ATP binding to the b subunits triggers the conformational change in the e subunit from the extended-state to the folded (ATPase active)-state, then another ATP binds to the e subunit and the folded-state is stabilized and the enzyme is kept in the ATPase-active state [15,20,22]. The experiments on the relationships between ATP binding to the e subunit and ATP synthase activity are underway in our group. Acknowledgments We thank Prof. Tsuneyoshi Kuroiwa at Rikkyo University for allowing us to use a DNA sequencing apparatus. This work was supported in part by grants-in-Aid for Young Scientists (B) (No. 18770118) and for Scientific Research on Priority Areas (No. 18074002) from the Ministry of Education, Culture, Sports, Science and Technology and Rikkyo University Special Fund for Research (to Y.K-Y.). References [1] P.D. Boyer, The ATP synthase—a splendid molecular machine, Annu. Rev. Biochem. 66 (1997) 717–749. [2] M. Yoshida, E. Muneyuki, T. Hisabori, ATP synthase—a marvellous rotary engine of the cell, Nat. Rev. Mol. Cell Biol. 2 (2001) 669–677. ˚ [3] J.P. Abrahams, A. Leslie, R. Lutter, J.E. Walker, Structure at 2.8 A
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