ATP Synthase with Its γ Subunit Reduced to the N-terminal Helix Can Still Catalyze ATP Synthesis
2009; Elsevier BV; Volume: 284; Issue: 39 Linguagem: Inglês
10.1074/jbc.m109.030528
ISSN1083-351X
AutoresNelli Mnatsakanyan, Jonathon A. Hook, Leah Quisenberry, Joachim Weber,
Tópico(s)Biochemical and Molecular Research
ResumoATP synthase uses a unique rotary mechanism to couple ATP synthesis and hydrolysis to transmembrane proton translocation. As part of the synthesis mechanism, the torque of the rotor has to be converted into conformational rearrangements of the catalytic binding sites on the stator to allow synthesis and release of ATP. The γ subunit of the rotor, which plays a central role in the energy conversion, consists of two long helices inside the central cavity of the stator cylinder plus a globular portion outside the cylinder. Here, we show that the N-terminal helix alone is able to fulfill the function of full-length γ in ATP synthesis as long as it connects to the rest of the rotor. This connection can occur via the ϵ subunit. No direct contact between γ and the c ring seems to be required. In addition, the results indicate that the ϵ subunit of the rotor exists in two different conformations during ATP synthesis and ATP hydrolysis. ATP synthase uses a unique rotary mechanism to couple ATP synthesis and hydrolysis to transmembrane proton translocation. As part of the synthesis mechanism, the torque of the rotor has to be converted into conformational rearrangements of the catalytic binding sites on the stator to allow synthesis and release of ATP. The γ subunit of the rotor, which plays a central role in the energy conversion, consists of two long helices inside the central cavity of the stator cylinder plus a globular portion outside the cylinder. Here, we show that the N-terminal helix alone is able to fulfill the function of full-length γ in ATP synthesis as long as it connects to the rest of the rotor. This connection can occur via the ϵ subunit. No direct contact between γ and the c ring seems to be required. In addition, the results indicate that the ϵ subunit of the rotor exists in two different conformations during ATP synthesis and ATP hydrolysis. F1Fo-ATP synthase is responsible for the bulk of ATP synthesis from ADP and Pi in most organisms. F1Fo-ATP synthase consists of the membrane-embedded Fo subcomplex with, in most bacteria, a subunit composition of ab2cn (with n = 10–15) and the peripheral F1 subcomplex, with a subunit composition of α3β3γδϵ. The energy necessary for ATP synthesis is derived from an electrochemical transmembrane proton (or, in some organisms, sodium ion) gradient. Proton flow, down the gradient, through Fo is coupled to ATP synthesis on F1 by a unique rotary mechanism. The protons flow through channels at the interface of a and c subunits, which drives rotation of the ring of c subunits. The cn ring, together with F1 subunits γ and ϵ, forms the rotor. Rotation of γ leads to conformational changes in the catalytic nucleotide-binding sites on the β subunits, where ADP and Pi are bound. The conformational changes result in formation and release of ATP. Thus, ATP synthase converts electrochemical energy, the proton gradient, into mechanical energy in the form of subunit rotation and back into chemical energy as ATP. In bacteria, under certain physiological conditions, the process can run in reverse. ATP is hydrolyzed to generate a transmembrane proton gradient that the bacterium requires for such functions as nutrient import and locomotion (for reviews, see Refs. 1Noji H. Yoshida M. J. Biol. Chem. 2001; 276: 1665-1668Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 2Weber J. Senior A.E. FEBS Lett. 2003; 545: 61-70Crossref PubMed Scopus (240) Google Scholar, 3Wilkens S. Adv. Protein Chem. 2005; 71: 345-382Crossref PubMed Scopus (44) Google Scholar, 4Weber J. Biochim. Biophys. Acta. 2006; 1757: 1162-1170Crossref PubMed Scopus (52) Google Scholar, 5Nakamoto R.K. Baylis Scanlon J.A. Al-Shawi M.K. Arch. Biochem. Biophys. 2008; 476: 43-50Crossref PubMed Scopus (131) Google Scholar, 6Junge W. Sielaff H. Engelbrecht S. Nature. 2009; 459: 364-370Crossref PubMed Scopus (302) Google Scholar). F1 (or “F1-ATPase”) has three catalytic nucleotide-binding sites, located on the β subunits, at the interface with the adjacent α subunit. The catalytic sites have pronounced differences in their nucleotide-binding affinity. During rotational catalysis, the sites switch their affinities in a synchronized manner; the position of γ determines which catalytic site is the high affinity site (Kd1 in the nanomolar range), which site is the medium affinity site (Kd2 ≈ 1 μm), and which site is the low affinity site (Kd3 ≈ 30–100 μm; see Refs. 7Weber J. Wilke-Mounts S. Lee R.S. Grell E. Senior A.E. J. Biol. Chem. 1993; 268: 20126-20133Abstract Full Text PDF PubMed Google Scholar, 8Weber J. Senior A.E. Methods Enzymol. 2004; 380: 132-152Crossref PubMed Scopus (17) Google Scholar). Only the high affinity site is catalytically active (9Baylis Scanlon J.A. Al-Shawi M.K. Le N.P. Nakamoto R.K. Biochemistry. 2007; 46: 8785-8797Crossref PubMed Scopus (30) Google Scholar). In the original crystal structure of bovine mitochondrial F1 (10Abrahams J.P. Leslie A.G. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2764) Google Scholar), one of the three catalytic sites was filled with the ATP analog AMPPNP, 3The abbreviations used are: AMPPNP5′-adenylyl-β,γ-imidodiphosphateACMA9-amino-6-chloro-2-methoxyacridineAp5AP1,P5-di(adenosine-5′)pentaphosphate; TF1 or TF1Fo (instead of F1 and F1Fo, respectively) refer specifically to the enzyme from the thermophilic bacterium Bacillus PS3γ′truncated γ subunit. a second one with ADP (plus azide; see Ref. 11Bowler M.W. Montgomery M.G. Leslie A.G. Walker J.E. J. Biol. Chem. 2007; 282: 14238-14242Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar), and the third site was empty. Hence, the β subunits are referred to as βTP, βDP, and βE, respectively. The high affinity site is located on the βTP subunit (12Mao H.Z. Weber J. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 18478-18483Crossref PubMed Scopus (32) Google Scholar). 5′-adenylyl-β,γ-imidodiphosphate 9-amino-6-chloro-2-methoxyacridine P1,P5-di(adenosine-5′)pentaphosphate; TF1 or TF1Fo (instead of F1 and F1Fo, respectively) refer specifically to the enzyme from the thermophilic bacterium Bacillus PS3 truncated γ subunit. The coupling process between ATP synthesis or hydrolysis on β and rotation of γ is not yet fully understood on a residue level. A number of point mutations at the interfaces between α or β and γ and between γ, ϵ, and c have been described that result in varying degrees of uncoupling (13Al-Shawi M.K. Nakamoto R.K. Biochemistry. 1997; 36: 12954-12960Crossref PubMed Scopus (38) Google Scholar, 14Ketchum C.J. Nakamoto R.K. J. Biol. Chem. 1998; 273: 22292-22297Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 15Peskova Y.B. Nakamoto R.K. Biochemistry. 2000; 39: 11830-11836Crossref PubMed Scopus (22) Google Scholar, 16Andrews S.H. Peskova Y.B. Polar M.K. Herlihy V.B. Nakamoto R.K. Biochemistry. 2001; 40: 10664-10670Crossref PubMed Scopus (18) Google Scholar, 17Lowry D.S. Frasch W.D. Biochemistry. 2005; 44: 7275-7281Crossref PubMed Scopus (16) Google Scholar). Some mutations at these interfaces were found that abolish ATP synthesis or hydrolysis activity or both (18Greene M.D. Frasch W.D. J. Biol. Chem. 2003; 278: 51594-51598Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 19Boltz K.W. Frasch W.D. Biochemistry. 2005; 44: 9497-9506Crossref PubMed Scopus (9) Google Scholar, 20Boltz K.W. Frasch W.D. Biochemistry. 2006; 45: 11190-11199Crossref PubMed Scopus (7) Google Scholar). Considering the pronounced effect of these point mutations, some of which were even conservative substitutions, it came as a surprise when it was recently shown that an “axle-less” γ, consisting just of the globular portion, with the portions of the N- and C-terminal helices that reach into the α3β3 cylinder removed, displayed ATP-driven rotation in the correct direction (21Furuike S. Hossain M.D. Maki Y. Adachi K. Suzuki T. Kohori A. Itoh H. Yoshida M. Kinosita Jr., K. Science. 2008; 319: 955-958Crossref PubMed Scopus (93) Google Scholar). Some reports have implicated the ϵ subunit (corresponding to the δ subunit in the mitochondrial enzyme) as being involved in coupling (15Peskova Y.B. Nakamoto R.K. Biochemistry. 2000; 39: 11830-11836Crossref PubMed Scopus (22) Google Scholar, 22Xiao Y. Metzl M. Mueller D.M. J. Biol. Chem. 2000; 275: 6963-6968Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 23Duvezin-Caubet S. Caron M. Giraud M.F. Velours J. di Rago J.P. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 13235-13240Crossref PubMed Scopus (45) Google Scholar, 24Rondelez Y. Tresset G. Nakashima T. Kato-Yamada Y. Fujita H. Takeuchi S. Noji H. Nature. 2005; 433: 773-777Crossref PubMed Scopus (305) Google Scholar, 25Cipriano D.J. Dunn S.D. J. Biol. Chem. 2006; 281: 501-507Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). It was shown that ϵ exists in different conformations that vary in the folding and positioning of the C-terminal domain. The x-ray structure of the mitochondrial enzyme (26Gibbons C. Montgomery M.G. Leslie A.G. Walker J.E. Nat. Struct. Biol. 2000; 7: 1055-1061Crossref PubMed Scopus (437) Google Scholar) shows the two helices of the C-terminal domain folded back on each other like a hairpin and positioned close to the interface between γ and the c ring (“down” conformation). In the crystal structure of a γϵ complex from Escherichia coli the hairpin is unfolded (27Rodgers A.J. Wilce M.C. Nat. Struct. Biol. 2000; 7: 1051-1054Crossref PubMed Scopus (153) Google Scholar); when integrated into F1 or F1Fo, the C terminus would reach “up,” coming close to the DELSEED-loop of the α and/or β subunits. While in this up conformation the angle between both helices of the C-terminal domain of ϵ is ∼90°, it has been postulated that this domain might also exist in a fully extended up conformation, stretching close to the N terminus of γ, with helical regions replacing the turn between the two helices (28Yagi H. Kajiwara N. Tanaka H. Tsukihara T. Kato-Yamada Y. Yoshida M. Akutsu H. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 11233-11238Crossref PubMed Scopus (96) Google Scholar). Fixing ϵ in either up conformation by cross-linking to γ has been shown to impair ATP hydrolysis but not synthesis. Freezing ϵ in the down position inhibited neither reaction (29Tsunoda S.P. Rodgers A.J. Aggeler R. Wilce M.C. Yoshida M. Capaldi R.A. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 6560-6564Crossref PubMed Scopus (164) Google Scholar, 30Suzuki T. Murakami T. Iino R. Suzuki J. Ono S. Shirakihara Y. Yoshida M. J. Biol. Chem. 2003; 278: 46840-46846Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Here, we report a finding that is arguably just as surprising as the rotation of an axle-less γ. In ATP synthase from the thermophilic bacterium Bacillus PS3, enzymes with γ subunit constructs where the globular domain and the C-terminal helix were eliminated, consisting of just the N-terminal 35 or 42 residues, TF1Fo(γQ36stop) 4Bacillus PS3 numbering is used throughout. and TF1Fo(γP43stop), were able to catalyze significant rates of ATP synthesis. According to the crystal structure (26Gibbons C. Montgomery M.G. Leslie A.G. Walker J.E. Nat. Struct. Biol. 2000; 7: 1055-1061Crossref PubMed Scopus (437) Google Scholar), the shorter of the two γ constructs should not make any contact either with c or with ϵ in the down conformation. Thus, the fact that ATP synthesis was observed suggests that ϵ in an up conformation forms a complex with the truncated γ, which is able to catalyze ATP synthesis. Indeed, when the γQ36stop truncation was combined with an ϵ truncation where the C-terminal domain was removed, ATP synthesis was abolished. The functions of the γ and ϵ subunits will be discussed in light of the new findings. Plasmid pTR19-ASDS, which carries the genes for the F1Fo-ATP synthase from thermophilic Bacillus PS3 (31Suzuki T. Ueno H. Mitome N. Suzuki J. Yoshida M. J. Biol. Chem. 2002; 277: 13281-13285Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), was used to generate the mutations γK7stop, γQ36stop, γP43stop, and ϵI88stop. In initial experiments to generate γ-less TF1Fo (TF1FoΔγ) by introducing the γK7stop mutation, we observed that the E. coli host produced small amounts of full-length PS3 γ, probably caused by a termination suppressor mutation in a tRNA. To eliminate this problem, an NheI site was introduced downstream of the stop codon. This allowed removal of the remainder of the gene for γ on an NheI-NheI fragment (the second NheI site is between the genes for γ and β). For construction of the γQ36stop and γP43stop truncation mutants, the same strategy was used. The mutagenic oligonucleotides were designed in such a way that, in addition to the desired mutation, a restriction site would be eliminated or generated to facilitate screening. Deletions were introduced by PCR using the QuikChange II XL mutagenesis kit (Stratagene). Wild-type and mutated plasmids were transformed into E. coli strain DK8, which does not express E. coli ATP synthase (32Klionsky D.J. Brusilow W.S. Simoni R.D. J. Bacteriol. 1984; 160: 1055-1060Crossref PubMed Google Scholar). E. coli strain DK8, harboring wild-type or mutated pTR19-ASDS plasmids, was aerobically cultivated at 37 °C for 18 h in 2× YT medium containing 100 μg/ml ampicillin. Inverted membrane vesicles from E. coli cells expressing thermophilic F1Fo were prepared as described (31Suzuki T. Ueno H. Mitome N. Suzuki J. Yoshida M. J. Biol. Chem. 2002; 277: 13281-13285Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 33Senior A.E. Latchney L.R. Ferguson A.M. Wise J.G. Arch. Biochem. Biophys. 1984; 228: 49-53Crossref PubMed Scopus (54) Google Scholar). The amount of wild-type F1Fo in E. coli membrane preparations was determined by SDS-PAGE, as visualized by staining with Coomassie Brilliant Blue. The relative amount of mutant F1Fo the membranes was estimated via Western blotting, using an anti-β antibody (Agrisera, Vännäs, Sweden) or an anti-α/anti-β antibody (a kind gift from Bill Brusilow, Wayne State University, Detroit, MI). The staining intensity was quantified using a Photodyne imaging system and ImageJ acquisition software (National Institutes of Health). The antibody against the globular portion of γ, used to confirm the absence of this portion of the protein in the truncation mutants, was a kind gift from Toshiharu Suzuki and Masasuke Yoshida (Japan Science and Technology Agency, Tokyo). Growth of strains expressing wild-type or mutant PS3 ATP synthase in limiting glucose was determined as described previously (34Senior A.E. Langman L. Cox G.B. Gibson F. Biochem. J. 1983; 210: 395-403Crossref PubMed Scopus (42) Google Scholar). ATPase activities were measured as described previously (35Mnatsakanyan N. Krishnakumar A.M. Suzuki T. Weber J. J. Biol. Chem. 2009; 284: 11336-11345Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). ATP synthesis activity in vitro was measured as follows. The batch assay used to obtain the data in Table 1 allowed running of the assay at a different temperature (42 °C) than the luciferin/luciferase reaction (room temperature) because the luciferase turned out to be very unstable at 42 °C. For this assay, inverted membrane vesicles were suspended in a solution containing 20 mm Hepes/KOH, 100 mm KCl, 5 mm MgCl2, 2 mm ADP, 5 mm KPi, 2 μm Ap5A, and 10% glycerol (pH 7.5) and incubated at 42 °C; Ap5A was included to suppress adenylate kinase activity of the membranes (36Goelz S.E. Cronan Jr., J.E. Biochemistry. 1982; 21: 189-195Crossref PubMed Scopus (27) Google Scholar). The reaction was initiated by the addition of 2 mm NADH. After 10, 40, and 70 s, aliquots of the reaction mixture (each containing 20 μg of membrane protein) were transferred into boiling buffer of 100 mm Tris/H2SO4, 4 mm EDTA (pH 7.75) for heat denaturation. The samples were incubated at 100 °C for 2 min, cooled on ice, and centrifuged for 1 min at 1000 × g. The amount of ATP produced was determined by the luciferin/luciferase method (CLS II ATP bioluminescence kit, Roche Applied Science). Light emission was measured at 562 nm in a spectrofluorometer type Fluorolog 3 (HORIBA Jovin Yvon, Edison, NY). In control experiments in the presence of the inhibitor oligomycin (20 μg/ml), no ATP synthesis was observed (Table 1).TABLE 1Oxidative phosphorylation in vivo and ATP synthesis activities in vitroStrain/mutationGrowth yields in limiting glucoseAmount of F1Fo on membranesNADH-driven ATP synthesis ctivityTurnover rate kcat% of wild-type% of total proteinmilliunits/mgs−1Wild-type100 ± 420 ± 560 ± 222.7+ oligomycin1.35 ± 0.50<0.1ϵI88stop101 ± 421 ± 6151 ± 336.4Δγ65 ± 29 ± 40.62 ± 0.97<0.1pUC118/DK8 (unc−)65 ± 300.17 ± 0.85γQ36stop75 ± 29 ± 27.5 ± 2.60.7+ oligomycin0.13 ± 0.93<0.1γQ36stop/ϵI88stop62 ± 210 ± 21.03 ± 0.86<0.1γP43stop76 ± 313 ± 414.1 ± 3.41.0+ oligomycin−1.05 ± 1.26<0.1γP43stop/ϵI88stop71 ± 212 ± 49.5 ± 4.50.7+ oligomycin0.04 ± 0.90<0.1 Open table in a new tab The real time ATP synthesis assay was performed in a buffer containing 20 mm Hepes/KOH, 100 mm potassium acetate, 5 mm magnesium acetate, 2 mm ADP, 5 mm KPi, and 10% glycerol (pH 7.5) at 37 °C. Membrane vesicles (containing 100 μg of membrane protein/ml) and 1/20 volume of the luciferin/luciferase mix were added. The reaction was initiated by the addition of 5 mm succinate. The slow but steady decrease in the signal of the negative control is caused by instability of the luciferase at 37 °C. NADH- and ATP-driven H+-pumping in membrane vesicles was measured via fluorescence quenching of ACMA at 42 °C. To a buffer of 10 mm Hepes/KOH, 100 mm KCl, 5 mm MgCl2 (pH 7.5), 0.5 mg/ml membrane vesicles and 0.3 μg/ml ACMA were added. Proton pumping was initiated by adding NADH or ATP to a final concentration of 1 mm and terminated by adding carbonyl cyanide m-chlorophenylhydrazone (final concentration 1 μm). The excitation wavelength was 410 nm, and the emission wavelength was 480 nm. Protein concentrations of membrane vesicles were determined by the Lowry method (37Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) using bovine serum albumin as standard. The γQ36stop and γP43stop truncation mutants remove the globular portion of γ and the C-terminal helix, leaving just a large fragment of the N-terminal helix (Fig. 1). With γP43stop, the contact site between the truncated γ (abbreviated γ′ in the following) and the N-terminal domain of ϵ would be reduced to a small patch; with γQ36stop, there would be no interaction between γ′ and the N-terminal domain of ϵ (Fig. 1, B–E). Neither γ truncation mutant should be able to reach the ring of c subunits (which would be located directly below γ and ϵ in Fig. 1A). To obtain an ϵ subunit consisting of just the N-terminal domain, the C-terminal domain was removed using an ϵI88stop mutation. E. coli membrane vesicles containing TF1Fo, TF1FoΔγ, TF1Fo(γQ36stop), TF1Fo(γP43stop), TF1Fo(ϵI88stop), TF1Fo(γQ36stop/ϵI88stop), and TF1Fo(γP43stop/ϵI88stop) were prepared as described under “Materials and Methods.” Western blotting using an anti-γ antibody raised against a peptide from the globular domain of γ confirmed that this domain was absent from the γ deletion and truncation mutants (for TF1FoΔγ, see Fig. 2). Western blotting using an anti-β or an anti-α/anti-β antibody was used to quantify the amount of TF1Fo in the membranes; the results are included in Table 1. The data (Fig. 2) show that it is possible to generate a γ-less ATP synthase. In contrast, it has been reported, based on in vitro experiments, that δ and ϵ subunits are required for binding of TF1 to TFo (38Yoshida M. Okamoto H. Sone N. Hirata H. Kagawa Y. Proc. Natl. Acad. Sci. U.S.A. 1977; 74: 936-940Crossref PubMed Scopus (104) Google Scholar). To analyze the ability of the truncation mutants to catalyze ATP synthesis in vivo, we measured growth yields on limiting glucose of E. coli cultures containing strain pTR19-ASDS/DK8 with the desired mutation. As expected, growth of the strain expressing a γ-less enzyme was the same as that of the negative control strain, pUC118/DK8, which does not express ATP synthase. In contrast, very surprisingly, TF1Fo containing either truncation mutant, γQ36stop or γP43stop, supported significantly higher growth than the negative control (Table 1), demonstrating that γ requires only its N-terminal helix to effect the conformational changes in β (and perhaps α) required to catalyze ATP synthesis. To confirm these findings, we measured ATP synthesis by membrane vesicles in vitro. Again, although the γ-less enzyme did not show any synthesis activity, both γ truncation mutants displayed significant ATP synthesis rates, corresponding to 25–35% of the activity of the wild-type enzyme (Table 1; for γQ36stop, see also Fig. 3). Given the small size of the contact area between ϵ in the down conformation and γ′ in the γP43stop mutant, and the complete absence of any interaction with γ′ in the γQ36stop mutant, it seemed possible that a complex between γ′ and the C-terminal domain of ϵ in the up conformation might be responsible for the observed ATP synthesis in the truncation mutants. To test this possibility, we combined the γ truncation mutants with a truncation of the C-terminal domain of ϵ, using an ϵI88stop mutation. As shown before (25Cipriano D.J. Dunn S.D. J. Biol. Chem. 2006; 281: 501-507Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), in wild-type enzyme with full-length γ the C-terminal domain of ϵ is not necessary for ATP synthesis; actually, removal of the domain in the ϵI88stop mutant resulted in increased ATP synthesis rates (Table 1). TF1Fo(γP43stop/ϵI88stop) was able to catalyze ATP synthesis, demonstrating that in this case the interaction between γ′ and the N-terminal domain of ϵ is sufficiently strong to couple proton flux-driven rotation of the c ring to ATP synthesis. In contrast, TF1Fo(γQ36stop/ϵI88stop) had lost the ability to catalyze ATP synthesis (Table 1 and Fig. 3). The fact that the TF1Fo(γQ36stop) single mutant catalyzed ATP synthesis showed that, in the absence of any direct interaction between γ′ and the N-terminal domain of ϵ, the C-terminal domain of ϵ can connect the N-terminal domain and γ′ so that γ′ can perform its functional role in ATP synthesis. Compared with their ATP synthase activity, the ATP hydrolysis activity of the truncation mutants was low. In membrane vesicles the ATPase activity was less than 10% of that of the wild-type control. Still, at least in the case of the γP43stop mutant, this was somewhat higher than found for the γ-less enzyme (Table 2). Membrane vesicles containing the truncation mutants did not exhibit any ATP-driven proton pumping (data not shown). The ATPase activity is too low to build up a proton gradient against the natural leak rate of the membranes. NADH-driven proton pumping in vesicles containing mutant ATP synthase resulted in the same degree of quenching as observed for the wild-type enzyme, indicating that truncation or elimination of the γ subunit and/or truncation of the ϵ subunit does not cause assembly problems associated with increased proton leak rates of the membranes.TABLE 2ATPase activities of membrane vesicles of deletion mutantsStrain/mutationMembrane ATPase activityTurnover rate kcatunits/mgs−1Wild type2.4 ± 0.6102ϵI88stop2.5 ± 0.3106Δγ0.051 ± 0.0205pUC118/DK8 (unc−)0.012 ± 0.011γQ36stop0.075 ± 0.0527γQ36stop/ϵI88stop0.076 ± 0.0437γP43stop0.102 ± 0.0207γP43stop/ϵI88stop0.106 ± 0.0128 Open table in a new tab The results of the present study demonstrate that the globular portion and the C-terminal helix of γ, together accounting for about 80% of the molecular mass of γ, are not required for ATP synthesis activity. The N-terminal domain of γ alone is sufficient to generate the conformational changes in the catalytic sites necessary for ATP synthesis and release. If γ is completely absent, in TF1FoΔγ, ATP synthesis is abolished. Recently it was shown that another radically reduced γ construct, this one consisting of just the globular portion, thus missing both the N-terminal and the C-terminal helix inside the α3β3 cylinder (γ-ΔN22C43), did indeed rotate in the correct direction upon ATP hydrolysis (21Furuike S. Hossain M.D. Maki Y. Adachi K. Suzuki T. Kohori A. Itoh H. Yoshida M. Kinosita Jr., K. Science. 2008; 319: 955-958Crossref PubMed Scopus (93) Google Scholar). ATPase activity of α3β3γ′ subcomplex containing γ-ΔN22C43 was not higher than that of α3β3 (21Furuike S. Hossain M.D. Maki Y. Adachi K. Suzuki T. Kohori A. Itoh H. Yoshida M. Kinosita Jr., K. Science. 2008; 319: 955-958Crossref PubMed Scopus (93) Google Scholar), and the torque generated by ATP hydrolysis seemed rather low. When a small portion of both helices inside the α3β3 cylinder was preserved, in the γ-ΔN11C32 construct, the torque reached 50% of that of wild-type (39Hossain M.D. Furuike S. Maki Y. Adachi K. Suzuki T. Kohori A. Itoh H. Yoshida M. Kinosita Jr., K. Biophys. J. 2008; 95: 4837-4844Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). This prompted the authors to conclude that “neither helix in the coiled-coil region of the axle of F1-ATPase plays a significant role in torque production” (39Hossain M.D. Furuike S. Maki Y. Adachi K. Suzuki T. Kohori A. Itoh H. Yoshida M. Kinosita Jr., K. Biophys. J. 2008; 95: 4837-4844Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The results presented here contradict this conclusion because a significant amount of torque is required to effect the conformational changes in the β (and perhaps α) subunits necessary to synthesize and release ATP, and this torque is transmitted just using the N-terminal helix of γ. Combining the outcome of the mentioned studies (21Furuike S. Hossain M.D. Maki Y. Adachi K. Suzuki T. Kohori A. Itoh H. Yoshida M. Kinosita Jr., K. Science. 2008; 319: 955-958Crossref PubMed Scopus (93) Google Scholar, 39Hossain M.D. Furuike S. Maki Y. Adachi K. Suzuki T. Kohori A. Itoh H. Yoshida M. Kinosita Jr., K. Biophys. J. 2008; 95: 4837-4844Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) and the results presented here, it appears that the portion of γ that is most involved in coupling of catalysis and rotation is the stretch of the N-terminal helix between residues ∼20 and ∼40. According to the x-ray structures (10Abrahams J.P. Leslie A.G. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2764) Google Scholar, 26Gibbons C. Montgomery M.G. Leslie A.G. Walker J.E. Nat. Struct. Biol. 2000; 7: 1055-1061Crossref PubMed Scopus (437) Google Scholar, 40Menz R.I. Walker J.E. Leslie A.G. Cell. 2001; 106: 331-341Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar), the contacts of the N-terminal helix of γ with the α3β3 ring are with the DELSEED-loop, a helix-turn-helix structure named after the conserved DELSEED motif, in the C-terminal domains of βDP and βE, as well as with the αE subunit. The finding that the γ truncation mutants catalyze ATP synthesis underlines the importance of the DELSEED-loop for mechanochemical coupling of rotation and catalysis. There is very little, if any, contact between the N-terminal helix of γ and βTP, which carries the catalytically active high affinity nucleotide-binding site (9Baylis Scanlon J.A. Al-Shawi M.K. Le N.P. Nakamoto R.K. Biochemistry. 2007; 46: 8785-8797Crossref PubMed Scopus (30) Google Scholar, 12Mao H.Z. Weber J. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 18478-18483Crossref PubMed Scopus (32) Google Scholar, 41Yang W. Gao Y.Q. Cui Q. Ma J. Karplus M. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 874-879Crossref PubMed Scopus (94) Google Scholar). In some models of the enzyme mechanism (5Nakamoto R.K. Baylis Scanlon J.A. Al-Shawi M.K. Arch. Biochem. Biophys. 2008; 476: 43-50Crossref PubMed Scopus (131) Google Scholar, 42Gao Y.Q. Yang W. Karplus M. Cell. 2005; 123: 195-205Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 43Mao H.Z. Abraham C.G. Krishnakumar A.M. Weber J. J. Biol. Chem. 2008; 283: 24781-24788Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar), a major function of the γ subunit in ATP synthesis is to convert the torque produced by proton gradient-driven rotation to force the βTP subunit open, to release the newly formed ATP. Our results offer strong support for this hypothesis. Rotation of the N-terminal helix of γ by 120° in ATP synthesis direction will bring the convex portion of the helix in contact with the βTP subunit and press against it (Fig. 1F). This should rotate the DELSEED-loop and push it downward, to open the subunit and bring it into βE conformation, thereby releasing product ATP. No other parts of γ except for the N-terminal helix seem to be required for this step, just as observed here. Another important outcome of the present study concerns the role of the ϵ subunit. It had been shown before that the C-terminal domain of ϵ can assume different conformations, with different properties (see the Introduction). Transition between the different conformations was found to be regulated by nucleotides (44Iino R. Murakami T. Iizuka S. Kato-Yamada Y. Suzuki T. Yoshida M. J. Biol. Chem. 2005; 280: 40130-40134Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Here, we present evidence that ϵ is indeed in an up position during ATP synthesis. γ′ in the γQ36stop truncation mutant makes no contact with the N-terminal domain of ϵ (Fig. 1, C–E). Still, the TF1Fo(γQ36stop) enzyme can synthesize ATP, suggesting that the C-terminal domain of ϵ in the up conformation might connect γ′ to the rest of the rotor. Indeed, removal of the C-terminal domain of ϵ abolished ATP synthesis. Of the two up conformations of the C-terminal domain of ϵ observed or postulated, the half-extended conformation seen by x-ray structure analysis (27Rodgers A.J. Wilce M.C. Nat. Struct. Biol. 2000; 7: 1051-1054Crossref PubMed Scopus (153) Google Scholar) is less likely to form a complex with γ′. Unless the N-terminal domain of γ by itself assumes a different position than in the presence of the rest of γ, it seems to have very little contact with the C-terminal domain of ϵ in this conformation. In contrast, cross-linking experiments (30Suzuki T. Murakami T. Iino R. Suzuki J. Ono S. Shirakihara Y. Yoshida M. J. Biol. Chem. 2003; 278: 46840-46846Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar) support the notion that the C terminus of ϵ in a fully extended conformation comes close to the extreme N terminus of γ. Very recently, an antiparallel coiled-coil between the C-terminal helix of ϵ and the N-terminal helix of γ has been detected in the E. coli enzyme (45Duncan, T. M., Cingolani, G., (2009) ASBMB Annual Meeting, New Orleans, LA, April 18–22, 2009, Abstract 504.6Google Scholar). As mentioned above, the experiments with TF1FoΔγ indicate that ϵ alone, in the absence of any portion of γ, cannot catalyze ATP synthesis. It is more difficult to assess whether the C-terminal domain of ϵ also stabilizes the interaction between γ′ and the N-terminal domain of ϵ in the γP43stop mutant. TF1Fo(γP43stop/ϵI88stop) synthesizes ATP at rates slightly lower than those for TF1Fo(γP43stop). However, it should be taken into account that for the wild-type enzyme removal of the C-terminal domain of ϵ actually accelerates ATP synthesis, at least in the in vitro assay. While TF1Fo(γP43stop) has about 35% of the synthesis activity of the wild-type enzyme, TF1Fo(γP43stop/ϵI88stop) has ∼10% of the activity of TF1Fo(ϵI88stop). Thus, one could argue that in this case as well the C-terminal domain of ϵ contributes to the stability of the rotor construct. Based on the crystal structure of the E. coli γϵ complex (28Yagi H. Kajiwara N. Tanaka H. Tsukihara T. Kato-Yamada Y. Yoshida M. Akutsu H. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 11233-11238Crossref PubMed Scopus (96) Google Scholar), direct contacts between γ′ and the N-terminal domain of ϵ might involve residues γN37, γS40, γF41, ϵP11, ϵD12, and ϵP14. Formation of hydrogen bonds appears possible between the hydroxyl group of γS40 and the main chain oxygen of ϵD12, and between the amide nitrogen of γN37 and the carboxyl group of ϵD12. It should be noted that the γ truncations remove two residues, γF205 and γE206, which were identified as important for high affinity binding of γ to the c ring (46Pogoryelov D. Nikolaev Y. Schlattner U. Pervushin K. Dimroth P. Meier T. FEBS J. 2008; 275: 4850-4862Crossref PubMed Scopus (26) Google Scholar). As noted before for other protein-protein interactions in ATP synthase (47Weber J. Wilke-Mounts S. Senior A.E. J. Biol. Chem. 2003; 278: 13409-13416Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), the high binding affinity between full-length γ and the c ring appears to be not absolutely necessary, but an example of “overengineering.” ATP stabilizes the down conformation of ϵ, probably by direct binding to ϵ (28Yagi H. Kajiwara N. Tanaka H. Tsukihara T. Kato-Yamada Y. Yoshida M. Akutsu H. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 11233-11238Crossref PubMed Scopus (96) Google Scholar). Fixating ϵ in this position by cross-linking eliminates inhibition of the ATPase activity by ϵ (29Tsunoda S.P. Rodgers A.J. Aggeler R. Wilce M.C. Yoshida M. Capaldi R.A. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 6560-6564Crossref PubMed Scopus (164) Google Scholar). Thus, the down conformation appears to be the one that ϵ assumes during ATP hydrolysis. Having two different structures of ϵ for ATP synthesis and hydrolysis has far reaching consequences. Above all, it means that the pathways for ATP synthesis and hydrolysis do not necessary have to be the exact reversal of each other (48Vinogradov A.D. J. Exp. Biol. 2000; 203: 41-49Crossref PubMed Google Scholar, 49Feniouk B.A. Suzuki T. Yoshida M. Biochim. Biophys. Acta. 2006; 1757: 326-338Crossref PubMed Scopus (84) Google Scholar). Among others, this could solve one of the main problems encountered during ATP synthesis, the necessity to bind ADP in the presence of an excess of ATP (2Weber J. Senior A.E. FEBS Lett. 2003; 545: 61-70Crossref PubMed Scopus (240) Google Scholar, 50Weber J. Senior A.E. Biochim. Biophys. Acta. 2000; 1458: 300-309Crossref PubMed Scopus (144) Google Scholar). Under ATP hydrolysis conditions, there is no catalytic site that binds ADP with a higher affinity than ATP, at least in the absence of Pi (51Mao H.Z. Gray W.D. Weber J. FEBS Lett. 2006; 580: 4131-4135Crossref PubMed Scopus (7) Google Scholar). ϵ in the up position might change the affinities for nucleotides, generating a site that preferentially binds ADP. It is sometimes stated that ϵ does not inhibit ATP synthesis by F1Fo (28Yagi H. Kajiwara N. Tanaka H. Tsukihara T. Kato-Yamada Y. Yoshida M. Akutsu H. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 11233-11238Crossref PubMed Scopus (96) Google Scholar, 52Feniouk B.A. Suzuki T. Yoshida M. J. Biol. Chem. 2007; 282: 764-772Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), although it is difficult to find experimental data in support of this claim. As discussed above, we show here that TF1Fo(ϵI88stop) has significantly higher ATP synthesis activity than wild-type TF1Fo, suggesting that the C-terminal domain of ϵ, which is removed in the mutant, is indeed inhibiting ATP synthesis. In general, one difficulty in assessing the role of the ϵ subunit is the fact that ϵ from different organisms behaves differently. Even among bacterial ATP synthases, the function of ϵ in the enzymes from E. coli and Bacillus PS3 is not the same. For instance, in E. coli ATP synthase or F1-ATPase the degree of inhibition of ATPase activity is constant over a large range of ATP concentrations, up to millimolar (25Cipriano D.J. Dunn S.D. J. Biol. Chem. 2006; 281: 501-507Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 53Weber J. Dunn S.D. Senior A.E. J. Biol. Chem. 1999; 274: 19124-19128Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). In contrast, in the PS3 enzyme the inhibition is much more effective at low ATP concentrations (54Kato-Yamada Y. Bald D. Koike M. Motohashi K. Hisabori T. Yoshida M. J. Biol. Chem. 1999; 274: 33991-33994Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Finally, it should be noted that the results presented recently (21Furuike S. Hossain M.D. Maki Y. Adachi K. Suzuki T. Kohori A. Itoh H. Yoshida M. Kinosita Jr., K. Science. 2008; 319: 955-958Crossref PubMed Scopus (93) Google Scholar) and here support the notion of ATP synthase being evolved from RNA helicases and RNA or protein translocases (55Walker J.E. Cozens A.L. Chem. Scr. 1986; 268: 263-272Google Scholar, 56Mulkidjanian A.Y. Makarova K.S. Galperin M.Y. Koonin E.V. Nat. Rev. Microbiol. 2007; 5: 892-899Crossref PubMed Scopus (144) Google Scholar). Both studies demonstrate that ATP synthase maintains some functionality with fragments of γ, showing that proteins of much simpler structure than full-length γ can serve as a rotor in ATP synthase.
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