The Subunit δ-Subunit b Domain of the Escherichia coli F1F0 ATPase
1997; Elsevier BV; Volume: 272; Issue: 49 Linguagem: Inglês
10.1074/jbc.272.49.31058
ISSN1083-351X
AutoresAndrew Rodgers, Stephan Wilkens, Robert Aggeler, Michael B. Morris, Susan M. Howitt, Roderick Capaldi,
Tópico(s)RNA modifications and cancer
ResumoThe δ and b subunits are both involved in binding the F1 to the F0 part in theEscherichia coli ATP synthase (ECF1F0). The interaction of the purified δ subunit and the isolated hydrophilic domain of the b subunit (bsol) has been studied here. Purified δ binds to bsol weakly in solution, as indicated by NMR studies and protease protection experiments. On F1,i.e. in the presence of ECF1-δ, δ, and bsol interact strongly, and a complex of ECF1·bsol can be isolated by native gel electrophoresis. Both δ subunit and bsol are protected from trypsin cleavage in this complex. In contrast, the δ subunit is rapidly degraded by the protease when bound to ECF1 when bsol is absent. The interaction of bsol with ECF1 involves the C-terminal domain of δ as δ(1–134) cannot replace intact δ in the binding experiments.As purified, bsol is a stable dimer with 80% α helix. A monomeric form of bsol can be obtained by introducing the mutation A128D (Howitt, S. M., Rodgers, A. J.,W., Jeffrey, P. D., and Cox, G. B. (1996) J. Biol. Chem.271, 7038–7042). Monomeric bsol has less α helix,i.e. only 58%, is much more sensitive to trypsin cleavage than dimer, and unfolds at much lower temperatures than the dimer in circular dichroism melting studies, indicating a less stable structure. The bsol dimer, but not monomer, binds to δ in ECF1.To examine whether subunit b is a monomor or dimer in intact ECF1F0, CuCl2 was used to induce cross-link formation in the mutants bS60C, bQ104C, bA128C, bG131C, and bS146C. With the exception of bS60C, CuCl2 treatment resulted in formation of b subunit dimers in all mutants. Cross-linking yield was independent of nucleotide conditions and did not affect ATPase activity. These results show the b subunit to be dimeric for a large portion of the C terminus, with residues 124–131 likely forming a pair of parallel α helices. The δ and b subunits are both involved in binding the F1 to the F0 part in theEscherichia coli ATP synthase (ECF1F0). The interaction of the purified δ subunit and the isolated hydrophilic domain of the b subunit (bsol) has been studied here. Purified δ binds to bsol weakly in solution, as indicated by NMR studies and protease protection experiments. On F1,i.e. in the presence of ECF1-δ, δ, and bsol interact strongly, and a complex of ECF1·bsol can be isolated by native gel electrophoresis. Both δ subunit and bsol are protected from trypsin cleavage in this complex. In contrast, the δ subunit is rapidly degraded by the protease when bound to ECF1 when bsol is absent. The interaction of bsol with ECF1 involves the C-terminal domain of δ as δ(1–134) cannot replace intact δ in the binding experiments. As purified, bsol is a stable dimer with 80% α helix. A monomeric form of bsol can be obtained by introducing the mutation A128D (Howitt, S. M., Rodgers, A. J.,W., Jeffrey, P. D., and Cox, G. B. (1996) J. Biol. Chem.271, 7038–7042). Monomeric bsol has less α helix,i.e. only 58%, is much more sensitive to trypsin cleavage than dimer, and unfolds at much lower temperatures than the dimer in circular dichroism melting studies, indicating a less stable structure. The bsol dimer, but not monomer, binds to δ in ECF1. To examine whether subunit b is a monomor or dimer in intact ECF1F0, CuCl2 was used to induce cross-link formation in the mutants bS60C, bQ104C, bA128C, bG131C, and bS146C. With the exception of bS60C, CuCl2 treatment resulted in formation of b subunit dimers in all mutants. Cross-linking yield was independent of nucleotide conditions and did not affect ATPase activity. These results show the b subunit to be dimeric for a large portion of the C terminus, with residues 124–131 likely forming a pair of parallel α helices. An F1F0 type ATPase is located in mitochondrial, chloroplast, and bacterial membranes, where it catalyzes the terminal step in oxidative- and photo-phosphorylation. InEscherichia coli, the enzyme contains five different subunits in the F1 part, α, β, γ, δ, and ε, in the stoichiometry 3:3:1:1:1, and three different subunits in the F0 part, a, b and c, in the ratio 1:2:9–12. The F1 part contains three catalytic sites on β subunits and is an ATPase when released from the F0, while the F0 part forms a proton pore (1Senior A.E. Physiol. Rev. 1988; 68: 177-231Crossref PubMed Scopus (461) Google Scholar, 2Boyer P.D. Biochim. Biophys. Acta. 1993; 1140: 215-250Crossref PubMed Scopus (924) Google Scholar, 3Capaldi R.A. Aggeler R. Turina P. Wilkens S. Trends Biochem. Sci. 1994; 19: 284-289Abstract Full Text PDF PubMed Scopus (132) Google Scholar). It is now generally accepted that the F1 and F0parts are joined by a relatively narrow stalk of 40–45 Å in length (4Gogol E.P. Lücken U. Capaldi R.A. FEBS Lett. 1980; 219: 274-278Crossref Scopus (96) Google Scholar, 5Lücken U. Gogol E.P. Capaldi R.A. Biochemistry. 1990; 29: 5339-5343Crossref PubMed Scopus (63) Google Scholar) that is constituted by the γ and ε subunits (3Capaldi R.A. Aggeler R. Turina P. Wilkens S. Trends Biochem. Sci. 1994; 19: 284-289Abstract Full Text PDF PubMed Scopus (132) Google Scholar, 6Capaldi R.A. Aggeler R. Wilkens S. Grüber G. J. Bioenerg. Biomemb. 1996; 28: 397-401Crossref PubMed Scopus (61) Google Scholar). Two other subunits, δ of the F1 part and the two copies of the b subunit of the F0 part, are also involved in binding the F1 to the F0 (7Perlin D.S. Senior A.E. Arch. Biochem. Biophys. 1985; 236: 603-611Crossref PubMed Scopus (31) Google Scholar, 8Sternweis P.C. Smith J.B. Biochemistry. 1977; 16: 4020-4025Crossref PubMed Scopus (71) Google Scholar). They do not appear to be a part of the main stalk, and it has been suggested that they form a second connection, a stator that fixes the α3β3 subdomain to the a subunit to allow rotation of a γ-ε-c subunit subdomain during functioning (9Wilkens S. Dunn S.D. Chandler J. Dahlquist F.W. Capaldi R.A. Nat. Struct. Biol. 1997; 4: 198-201Crossref PubMed Scopus (110) Google Scholar). We have recently obtained a structure for a major part of the δ subunit by NMR (9Wilkens S. Dunn S.D. Chandler J. Dahlquist F.W. Capaldi R.A. Nat. Struct. Biol. 1997; 4: 198-201Crossref PubMed Scopus (110) Google Scholar). The polypeptide forms two domains. The N-terminal domain, composed of residues 1–105, is a six-helix bundle. The C-terminal domain of residues 106–176 contains at least one α helix (residues 118–129), which can pack into a cleft in the N-terminal part, but this domain is partly unfolded in the isolated δ subunit. Our recent cross-linking studies in ECF1F0 1The abbreviations used are: ECF1, soluble portion of the Escherichia coliF1F0 ATP synthase; ECF1F0 Escherichia coliF1F0 ATP synthase; MOPS, 3-[N-morpholino]propanesulfonic acid; AMP·PNP, 5′-adenylyl-β,γ-imidodiphosphate; MRW, mean residue weight. (10Ogilvie I. Aggeler R. Capaldi R.A. J. Biol. Chem. 1997; 272: 16652-16656Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) and the work of Lill et al. (11Lill H. Hensel F. Junge W. Engelbrecht S. J. Biol. Chem. 1996; 271: 32737-32742Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) in chloroplasts place the δ subunit near the top of the F1 on the outside of the complex, interacting with one of the three α subunits. How the b subunits are arranged in ECF1F0 is less well understood. The b subunits of bacterial F1F0-ATPase, and the equivalent polypeptides in the mitochondrial enzyme, are characterized by an N-terminal membrane intercalated region and a large hydrophilic C-terminal domain (12Walker J.E. Saraste M. Gay N.J. Biochim. Biophys. Acta. 1984; 768: 164-200Crossref PubMed Scopus (371) Google Scholar). Based on trypsin digestion studies (7Perlin D.S. Senior A.E. Arch. Biochem. Biophys. 1985; 236: 603-611Crossref PubMed Scopus (31) Google Scholar, 13Hermolin J. Gallant J. Fillingame R.H. J. Biol. Chem. 1983; 258: 14550-14555Abstract Full Text PDF PubMed Google Scholar, 14Hoppe J. Friedl P. Schairer H.U. Sebald W. von Meyenburg K. Jørgensen B.B. EMBO J. 1983; 2: 105-110Crossref PubMed Scopus (46) Google Scholar), it is this cytoplasmic domain of the b subunit that is involved in the binding of F1 to F0. A truncated form of the b subunit containing the C-terminal domain has been obtained genetically and purified (15Dunn S.D. J. Biol. Chem. 1992; 267: 7630-7636Abstract Full Text PDF PubMed Google Scholar, 16Howitt S.M. Rodgers A.J.W. Jeffrey P.D. Cox G.B. J. Biol. Chem. 1996; 271: 7038-7042Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). The construct generated by Dunn (15Dunn S.D. J. Biol. Chem. 1992; 267: 7630-7636Abstract Full Text PDF PubMed Google Scholar) included residues 25–156 and an N-terminal octapeptide extension derived from the vector pUC8. The polypeptide produced by Howitt et al.(16Howitt S.M. Rodgers A.J.W. Jeffrey P.D. Cox G.B. J. Biol. Chem. 1996; 271: 7038-7042Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), purified initially as a C-terminal fusion with glutathioneS-transferase, includes residues 29–156, with Gly-Ser introduced at the N terminus to allow cleavage of the b subunit cytoplasmic domain from the fusion by thrombin. Both forms of the C-terminal domain, here called bsol as proposed by Dunn (15Dunn S.D. J. Biol. Chem. 1992; 267: 7630-7636Abstract Full Text PDF PubMed Google Scholar), are highly soluble in aqueous buffer. Sedimentation velocity centrifugation and circular dichroism spectroscopy studies show that bsol is an elongated dimer with a high α-helical content (15Dunn S.D. J. Biol. Chem. 1992; 267: 7630-7636Abstract Full Text PDF PubMed Google Scholar, 16Howitt S.M. Rodgers A.J.W. Jeffrey P.D. Cox G.B. J. Biol. Chem. 1996; 271: 7038-7042Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Recent mutational analysis has established that residues Val124, Ala128, and Gly131 lie on one face of an α helix formed by a conserved hydrophobic region (Val124 to Ala132) near the C terminus of the b subunit (16Howitt S.M. Rodgers A.J.W. Jeffrey P.D. Cox G.B. J. Biol. Chem. 1996; 271: 7038-7042Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). The mutation bG131D had previously been shown to allow assembly of the b subunit into the membrane but to prevent assembly of the whole F1F0·ATPase complex (17Jans D.A. Fimmel A.L. Langman L. James L.B. Downie J.A. Senior A.E. Ash G.R. Gibson F. Cox G.B. Biochem. J. 1983; 211: 717-726Crossref PubMed Scopus (35) Google Scholar). Replacement of Ala128 by Asp is now known to disrupt dimer formation: bsol with the mutation A128D is a stable monomer (16Howitt S.M. Rodgers A.J.W. Jeffrey P.D. Cox G.B. J. Biol. Chem. 1996; 271: 7038-7042Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Whether the dimeric structure seen in bsol is representative of the structural organization of the b subunit in the F1F0 complex is not known. Here, we have explored the arrangement of δ and the b subunits in ECF1F0 in some detail. Clear evidence that the b subunit binds to F1 via the δ subunit is reported, and it is shown that the dimer is both required for, and represents, the arrangement of subunit b in the complex. Site-directed mutagenesis was carried out on an M13 mp18 clone containing the unc genes B, E,F and H on a 2356-base pairHindIII/EcoRI fragment, according to the method of Kunkel et al. (18Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4558) Google Scholar). Residues Ser60, Gln104, Ala128, Gly131, and Ser146 were replaced by Cys using the following oligonucleotides (sequence changes underlined): GCAAAGGCCTGCGCGACCGAC (bS60C), ATCGTGGCCTGTGCGCAGGCG (bQ104C), GCTATCCTGTGTGTTGCTGGCG (bA128C), GGCTGTTGCTTGCGCCGAGAAG (bG131C) and GCTGCTAACTGTGACATCGTG (bS146C). 909-base pairBsrGI/BssHII fragments carrying the mutateduncF genes were excised from the M13 mp18 replicative form and ligated into the vector pRA100 (described in Aggeler et al. (19Aggeler R. Chicas-Cruz K. Cai S.X. Keana J.F.W. Capaldi R.A. Biochemistry. 1992; 31: 2956-2961Crossref PubMed Scopus (95) Google Scholar)), creating plasmids pRA165 (bS60C), pRA166 (bQ104C), pRA167 (bA128C), pRA168 (bG131C), and pRA169 (bS146C). These plasmids were used to transform the unc − E. coli strain AN888 (20Gibson F. Downie J.A. Cox G.B. Radik J. J. Bacteriol. 1978; 134: 728-736Crossref PubMed Google Scholar). Strains XL1-Blue (Stratagene) and CJ236 (New England Biolabs) were used in cloning and mutagenesis procedures. Bacterial strains used in the overexpression of glutathione S-transferase fusion proteins (16Howitt S.M. Rodgers A.J.W. Jeffrey P.D. Cox G.B. J. Biol. Chem. 1996; 271: 7038-7042Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar) were grown in Luria broth supplemented with 33 mmglycerol. Wild-type and mutant (A128D) forms of bsol were purified as described previously (16Howitt S.M. Rodgers A.J.W. Jeffrey P.D. Cox G.B. J. Biol. Chem. 1996; 271: 7038-7042Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), using glutathione-linked agarose resin and thrombin obtained from Sigma. Strains used for the preparation of F1F0-ATPase were grown in minimal medium with supplements (21Gibson F. Cox G.B. Downie J.A. Radik J. Biochem. J. 1977; 164: 193-198Crossref PubMed Scopus (51) Google Scholar). Circular dichroism spectra were obtained using a Jasco J-720 spectropolarimeter. For the determination of the relative percentages of secondary structure, wild-type b subunit cytoplasmic domain (0.242 g/liter) and the corresponding mutant A128D (0.255 g/liter) were dissolved in 5 mm MgSO4, 10 mm sodium phosphate, pH 7.0. The concentration of protein was determined using amino acid analysis. The samples were placed in 0.1-mm path length cells at 20 °C, and spectra were acquired using a scan speed of 20 nm/min, response time of 1 s, bandwidth of 1 nm, and resolution of 0.5 nm. For each sample, 10 acquisitions were collected between 260 and 200 nm and co-added, and 50 acquisitions were collected between 200 and 182 nm and co-added. The two sets were concatenated and the spectrum of the buffer, collected in the same way, was subtracted. The raw data were converted to mean residue ellipticity ([θ]MRW) using a mean residue weight of 109.5 calculated from the amino acid sequence. Data were analyzed using the computer program VARSLC1 (22Johnson W.C. Proteins Struct. Funct. Genet. 1990; 7: 205-214Crossref PubMed Scopus (894) Google Scholar) to obtain estimates of the percentages of α helix, parallel and antiparallel β sheet, turns, and "other" structure. For the temperature dependence of unfolding, both wild-type cytoplasmic domain (0.21 g/liter) and the mutant A128D (0.114 g/liter) were placed in 1-mm path length cells. The cells were placed in the jacketed cell holder of the spectropolarimeter. The temperature in the cell was controlled through the Jasco software and a Neslab RTE-111 water bath. The water bath temperature was raised at a rate of 1 °C/min over the range 7 to 95 °C, and data were collected at 222 nm every 0.2 °C as a measure of the loss of α helix. Corrections for the difference in the temperature between the water bath and the cell were made based upon previous calibrations using a thermocouple probe placed in the cell. Plots of [θ]MRW at 222 nm were analyzed assuming a two-state transition between the native state and the unfolded state. The melting temperature, t m , was estimated using nonlinear regression of the plots with the following equation θt=θ0+at+[(θ∞+bt)−(θ0+at)] ×exp[d(t−tm]/{1+exp[d(t−tm)]}Equation 1 where the parameters θ0 and θ∞ are the starting and ending ellipticity values, respectively, aand b are the slopes of the initial and final linear parts of the plots, respectively, d is the dispersion of the data, and t is the temperature at a given ellipticity, θ t . The fitting program returned estimates of the values of the parameters (including t m ) ± the standard errors. ECF1F0 was isolated and reconstituted into egg-lecithin vesicles as described in Aggeler et al. (23Aggeler R. Haughton M.A. Capaldi R.A. J. Biol. Chem. 1995; 270: 9185-9191Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). ATP synthase-containing vesicles collected from the Sephadex G-50 column were pelleted by centrifugation for 1 h at 45,000 rpm at 4 °C in a Beckman Ti60 rotor. The pellets were resuspended in 50 mm Tris-HCl, pH 7.5, 2 mm MgCl2, 2 mm dithiothreitol, and 10% glycerol and stored in liquid nitrogen. Prior to cross-linking experiments with CuCl2, the reducing agent was removed by pelleting the enzyme at 175,000 × g for 30 min at 4 °C in a Beckman TLA100.2 rotor. The sample was then washed twice by successive resuspension and centrifugation steps in 50 mm MOPS, pH 7.0, 2 mm MgCl2, and 10% glycerol. Final resuspension was in the same buffer at a protein concentration of 1 mg/ml. Cross-linking was carried out at 22 °C for 2 h using concentrations of between 5 μm and 200 μm CuCl2. All cross-linking reactions were stopped by the addition of 5 mm EDTA, and ATPase activities (24Lötscher H.-R. deJong C. Capaldi R.A. Biochemistry. 1984; 23: 4140-4143Crossref PubMed Scopus (99) Google Scholar) were measured with and without prior incubation of the samples with 20 mm dithiothreitol for 2 h at 22 °C. In experiments comparing cleavage patterns of wild-type and mutant bsol, these proteins (1 mg/ml) were prepared in 50 mm Tris-HCl, pH 7.0, 1 mm EDTA, and 2 m glucose. The ratio of trypsin to bsol (w/w) was 1:3000. The reaction, conducted at 13 °C, was stopped by adding 4 mmphenylmethylsulfonyl fluoride from a freshly prepared stock solution. Trypsin cleavage products were analyzed using 16% SDS-polyacrylamide gel electrophoresis gels prepared and run according to the method of Schägger and von Jagow (25Schägger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10480) Google Scholar). N-terminal sequences of tryptic fragments excised from Coomassie stained SDS-polyacrylamide gel electrophoresis gels were determined by Dr. Denis Shaw, Australian National University, Canberra, after passive transfer from the gel pieces onto polyvinylidene difluoride membrane. Experiments comparing cleavage of bsol, the δ subunit and ECF1, alone or in combination, were carried out in 50 mmTris-HCl, pH 7.4, 1 mm EDTA, 10% glycerol. In experiments comparing cleavage of bsol and ECF1 in the presence and absence of bsol, the ratio of trypsin to bsol (w/w) was 1:5000. In experiments comparing cleavage of bsol and δ in the presence and absence of bsol, the ratio of trypsin to bsol (w/w) was 1:300. These reactions were conducted at 22 °C and were stopped by adding 4 mm phenylmethylsulfonyl fluoride. The δ subunit and δ(1–134)were produced from pJCI kindly provided by Dr. Stanley Dunn (University of Western Ontario). Polypeptides were purified, and NMR spectra were obtained as described previously (9Wilkens S. Dunn S.D. Chandler J. Dahlquist F.W. Capaldi R.A. Nat. Struct. Biol. 1997; 4: 198-201Crossref PubMed Scopus (110) Google Scholar). Atebrin fluorescence quenching activities were assayed as described by Hatch et al. (26Hatch L. Fimmel A.L. Gibson F. Biochim. Biophys. Acta. 1993; 1141: 183-189Crossref PubMed Scopus (6) Google Scholar). Protein concentrations were determined using the BCA protein assay from Pierce, with bovine serum albumin as a standard. F1-ATPase was prepared from membranes of strain AN1460 (27Downie J.A. Langman L. Cox G.B. Yanofsky C. Gibson F. J. Bacteriol. 1980; 143: 8-17Crossref PubMed Google Scholar) as described by Coxet al. (28Cox G.B. Jans D.A. Gibson F. Langman L. Senior A.E. Fimmel A.L. Biochem. J. 1983; 216: 143-150Crossref PubMed Scopus (27) Google Scholar). Stripped membranes were prepared from strain AN2840 (29Howitt S.M. Cox G.B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9799-9803Crossref PubMed Scopus (31) Google Scholar) using the method described by Wise et al.(30Wise J.G. Duncan T.M. Latchney L.R. Cox D.N. Senior A.E. Biochem. J. 1983; 215: 343-350Crossref PubMed Scopus (81) Google Scholar). The bsol used here was obtained by thrombin release from a fusion protein that includes glutathioneS-transferase at the N terminus (16Howitt S.M. Rodgers A.J.W. Jeffrey P.D. Cox G.B. J. Biol. Chem. 1996; 271: 7038-7042Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). It contains residues 25–156 with a Gly-Ser N-terminal extension. Both intact δ and δ(1–134) were obtained as described previously (9Wilkens S. Dunn S.D. Chandler J. Dahlquist F.W. Capaldi R.A. Nat. Struct. Biol. 1997; 4: 198-201Crossref PubMed Scopus (110) Google Scholar). All of the isolated subunits and subunit fragments were pure, based on SDS-polyacrylamide gel electrophoresis (Fig.1). Interactions between F1-δ, δ, δ(1–134), and bsolwere examined first by native gel electrophoresis. In a typical experiment, such as shown in Fig. 1 A, fractions were mixed and then electrophoresed through agarose. Protein was detected on the native gel by Coomassie Brilliant Blue staining. Bands containing protein were excised, dissolved in SDS, and then subjected to SDS-polyacrylamide gel electrophoresis (Fig. 1 B). As shown in Fig. 1 A, F1-δ, δ, δ(1–134), and bsol each migrated very differently based on the combination of size and charge, while complexes of F1-δ, F1 + δ, F1 + δ(1–134), and even F1 + δ + bsol migrated similarly. No complex formation was observed between bsol and either pure δ or δ(1–134) in native gel electrophoresis (Fig.1 A, lane IV). However, both δ and δ(1–134) bound to F1-δ to form a complex that was retained through the electrophoresis step (Fig. 1 A,lanes VII and IX). Moreover, a complex could be formed between F1-δ, δ, and bsol (Fig.1 A, lane VIII), but not between F1-δ and bsol (Fig. 1 A, lane VI), or between F1 + δ(1–134) + bsol (Fig. 1 A, lane X). These results indicate that bsol binds tightly only to F1when δ is present, and that the C-terminal 43 residues are important for this interaction. The native gel electrophoresis experiments rule out a strong interaction between δ and bsol in the absence of F1, but do not preclude that there was weak interaction between the two subunits that was destabilized by the electrophoresis step. We have recently reported a structure determination of the δ subunit by NMR (9Wilkens S. Dunn S.D. Chandler J. Dahlquist F.W. Capaldi R.A. Nat. Struct. Biol. 1997; 4: 198-201Crossref PubMed Scopus (110) Google Scholar). All of the backbone resonances of residues 1–134 were assigned. As a second approach to examining interactions of δ and bsol in solution, pure 15N-labeled δ subunit was titrated with purified bsol, and the resonance broadening of the δ subunit spectrum was monitored. Many of the individual resonances of the δ subunit progressively disappeared as the ratio of bsol to δ was increased (Fig.2) as clearly evident for Ser23, Gly72, Gly83, and Asn86, four residues that are widely distributed in the N-terminal domain. These results indicate that an interaction occurs between the two polypeptides with a slow to intermediate rate of exchange between a free δ subunit and δ that is bound to bsol. The binding constant between δ and bsolfrom these NMR studies must be larger than 2 μm. This is consistent with the interaction between the two polypeptides being weaker in solution than on F1, where a binding affinity of 2 μm was estimated by Dunn (15Dunn S.D. J. Biol. Chem. 1992; 267: 7630-7636Abstract Full Text PDF PubMed Google Scholar). The disappearance of resonances, then, represents the lowing of the tumbling rate of the δ subunit by binding of bsol. The approach does not pinpoint the site of interaction, except that when the experiment was repeated with δ(1–134), there was significantly less broadening of resonances of the N-terminal part, as would be expected if the interaction between δ and bsol is mainly through the C-terminal 42 residues of the δ subunit. Other individual subunits of ECF1 failed to alter the NMR spectrum of the δ. Both the δ subunit and bsol are highly protease sensitive in pure form (shown for bsol in Fig.3 A). Moreover, the δ subunit is still highly protease-sensitive when bound to the core ECF1 (Fig. 3 C). However, as shown in Fig.3 B, when bsol is reacted with ECF1, both the δ subunit and bsol are protected from trypsin cleavage. These results confirm the role of the δ subunit in binding bsol. Fig. 3 E presents data for the trypsin cleavage of the δ·bsol complex formed in solution. It is evident that there is some protection from cleavage of both δ and bsol by complex formation, but this interaction is clearly weak as proteolysis still proceeds quite rapidly. To examine if bsol was binding to F1 at its functionally important site, the ability of the polypeptide to block the reconstitution of coupled F1F0 ATPase activity was measured by atebrin fluorescence quenching assays. In the absence of added bsol, rebinding of F1 to the membrane preparation gave ATP-dependent quenching that was 53% of the original fluorescence level (Fig.4 C) compared with 51% after preincubation of ECF1 with monomeric bsol at the same concentration (Fig. 4 B). When bsol was incubated with F1 and then the complex reconstituted, ATP-dependent quenching was only 12% of the original fluorescence level (Fig. 4 A). Thus, the binding of bsol blocks the functional rebinding of F1 to F0. A CD spectrum of the wild-type (dimeric) form of the bsolover the wavelength range 182–260 nm is presented in Fig.5 A. The data indicate 80% ± 3% (SD) α helix. The CD spectrum of the mutant (A128D) form of bsol is also shown in Fig. 5 A, from which 58 ± 2% α-helix was estimated. The loss of α-helical structure observed for the mutant protein is compensated largely by an increase in "other" (random coil) structure and parallel β sheet. The trypsin digestion patterns of the dimeric and monomeric forms of bsol are additional evidence of loss of secondary structure in the monomer. Thus, with trypsin to bsol at a ratio of 1:5000 (cf. 1:3000 in Fig. 3) as well as with 2m glucose present to stabilize the protein, there was only a single cut of bsol in 30 min, the product of which had the N-terminal sequence QKEIAD (cutting after residue Arg36). Under equivalent conditions, the monomeric protein was cleaved at Arg36, Arg49, Lys58, Arg83, and Lys100, indicating increased accessibility of the protease, not only in the vicinity of the mutation but at a number of points throughout the protein (results not shown). The increased susceptibility of monomeric bsol to proteolytic cleavage correlates with decreased thermal stability as shown by CD melting studies. CD scans at a fixed wavelength of 222 nm over the temperature range 6–82 °C (Fig. 5 B) showed a clear secondary structure transition with midpoint at 40 °C for the wild-type dimeric bsol. In this transition the polypeptide goes from a folded conformation to an unstructured state. The unfolding of the mutant monomeric bsol is centered at a significantly lower temperature (32 °C). The stability of the dimer, and importance of the dimer in binding to F1, supports the idea that b is a dimer in the ECF1F0 complex. To test this more directly, mutants were constructed in which a cysteine was introduced in place of the following b subunit residues, Ser60, Gln104, Ala128, Gly131, and Ser146. With the exception of the mutant bS60C, which grew poorly, all of the strains grew well on solid succinate minimal medium. All of the strains exhibited growth yields in limiting (5 mm) glucose similar to the wild-type control (data not shown). The specific activities of ECF1F0purified from each of the mutants were similar to the wild-type enzyme,i.e. in the range 22–27 μmol of ATP hydrolyzed/min/mg. Mutant enzyme preparations were treated with CuCl2 to induce formation of disulfide bonds. Experiments were carried out under both ATP conditions (following preincubation with AMP·PNP) and ADP conditions (addition of ATP + Mg2+ followed by enzyme turnover), over a range of CuCl2 concentrations (5–200 μm). A cross-linked product in the range 32–35 kDa was observed with ECF1F0 from mutants Q104C, A128C, G131C, and S146C. No cross linking was obtained with mutant S60C. Data for the mutant bQ104C under ATP conditions are shown in Fig.6 A in the form of a concentration dependence of CuCl2 on cross-linking yield. The maximal yields obtained with this mutant were in the range of 70%. Fig. 6 B shows data for all mutants using the minimum CuCl2 concentration required for maximum cross-link formation under ATP conditions. The appearance of cross-linked product seen in Fig. 6, A andB, was in each case accompanied by a commensurate loss of the b subunit band. That this new band was a b subunit dimer was confirmed by antibody blotting. It reacted with anti-b subunit mAbs but not with antibodies to any of the other subunits of the complex (data not shown). Note the altered migration of both the monomeric and cross-linked dimeric forms of the mutant S146C b subunit. There was no nucleotide dependence of the yield of cross-linking for any of the mutants examined. Subunit b dimer formation had only a small effect on ATPase activity with any of the mutants, e.g. for bQ104C, ATPase activity was 25.1 μmol/min/mg before and 19.2 μmol/min/mg after cross-linking in 70% yield. This is the same loss of ATPase activity obtained by treating wild-type enzyme with CuCl2. There is emerging evidence that the F1 and F0 parts of the ATP synthase are joined not only by the narrow and 40–45-Å long stalk seen in electron micrographs (5Lücken U. Gogol E.P. Capaldi R.A. Biochemistry. 1990; 29: 5339-5343Crossref PubMed Scopus (63) Google Scholar), and now known to be made up of the γ and ε subunits (3Capaldi R.A. Aggeler R. Turina P. Wilkens S. Trends Biochem. Sci. 1994; 19: 284-289Abstract Full Text PDF PubMed Scopus (132) Google Scholar), but also by a second stalk provided by the δ and b subunits (9Wilkens S. Dunn S.D. Chandler J. Dahlquist F.W. Capaldi R.A. Nat. Struct. Biol. 1997; 4: 198-201Crossref PubMed Scopus (110) Google Scholar). There is clear evidence from cross-linking experiments that the δ subunit is bound at the outside of the α3β3 subunit barrel near the top of the F1 and away from the F0(10Ogilvie I. Aggeler R. Capaldi R.A. J. Biol. Chem. 1997; 272: 16652-16656Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 11Lill H. Hensel F. Junge W. Engelbrecht S. J. Biol. Chem. 1996; 271: 32737-32742Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The δ subunit is a two-domain protein, with an N-terminal domain of around 105 residues, which is roughly globular and contains a six α-helical bundle (9Wilkens S. Dunn S.D. Chandler J. Dahlquist F.W. Capaldi R.A. Nat. Struct. Biol. 1997; 4: 198-201Crossref PubMed Scopus (110) Google Scholar) and a less ordered C-terminal domain. The C-terminal domain must be in close contact with the N-terminal domain in the complex as a cross link is readily formed between intrinsic Cys64 and Cys140 (31Ziegler M. Xiao R. Penefsky H.S. J. Biol. Chem. 1994; 269: 4233-4239Abstract Full Text PDF PubMed Google Scholar). A number of studies have shown that the δ subunit and its equivalent in the mitochondrial enzyme, OSCP, are involved in the interaction of F1 with the F0 part. For example, ECF1 does not bind to F0 in the absence of δ. The key sites for this interaction appear to be in the C terminus of δ. C-terminal truncations of as few as 4–6 residues from either δ or OSCP prevent the rebinding of ECF1 or MF1, respectively, to F0 (32Joshi S. Cao G.-J. Nath C. Shah J. Biochemistry. 1996; 35: 12094-12103Crossref PubMed Scopus (26) Google Scholar, 33Jounouchi M. Takeyama M. Chaiprasert P. Noumi T. Moriyama Y. Maeda M. Futai M. Arch. Biochem. Biophys. 1992; 292: 376-381Crossref PubMed Scopus (27) Google Scholar). The studies presented here show conclusively that the δ subunit binds to b subunits, and that this interaction involves mainly the C-terminal domain of δ. This arrangement had been speculated upon based on cross-linking of δ to subunit CF0I in the chloroplast enzyme (34Beckers G. Berzborn R.J. Strotmann H. Biochim. Biophys. Acta. 1992; 1101: 97-104Crossref PubMed Scopus (42) Google Scholar). However, the data reported here are the first direct binding experiments. It is shown that δ subunit is required for the interaction of the cytoplasmic domain of subunit b with F1. This binding requires the C-terminal domain of δ, as the interaction was lost when δ(1–134) was used in the reconstitution experiments. A complex was obtained between F1 + δ + bsol which was stable to native gel electrophoresis. No such stable complex was formed between δ and bsol in the absence of the F1. It is likely that the α3β3γ domain helps stabilize the interactions between the N- and C-terminal domains of δ, which are required for the tight binding of b subunits. Weak interaction between δ and bsol in solution was observed by NMR and in protease digestion studies. Both the δ and bsol are protected from trypsin digestion in the ECF1 + δ + bsol complex, while the protection of δ and bsol by mixing the two in solution is much less. The binding of bsol blocks rebinding of F1 + δ to F0, consistent with the functional interaction between the cytoplasmic domain of b and δ in the reconstitution experiments. Purified wild-type bsol is a stable dimer, and only a dimer, not monomer, is able to block F1 + δ binding to F0. A priori, the two copies of subunit b could provide separate connections between the F1 and F0parts. However, the results presented here argue against this. First, dimer formation appears to stabilize the secondary structure of bsol. In the monomer form created by introducing an Asp for Gly at position 128, bsol is much less α-helical, because it is highly protease sensitive and denatures at lower temperatures. More direct evidence that b subunits are close in F1F0 was sought by cross-linking studies. Mutants were created with Cys residues at several sites along the C-terminal domain. On the addition of the oxidizing agent Cu2+, disulfide bonds were generated in high yield between Cys at positions 104, 128, 131, and 146, but not at 60. This could happen only if the two b subunits are paired for a significant length. As the δ subunit is near the top of the F1 (10Ogilvie I. Aggeler R. Capaldi R.A. J. Biol. Chem. 1997; 272: 16652-16656Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 11Lill H. Hensel F. Junge W. Engelbrecht S. J. Biol. Chem. 1996; 271: 32737-32742Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), the obvious arrangement of the b subunits is an extended one running up one side of the F1F0 structure. In several of the mutants described here, it is possible to react the introduced Cys with bulky maleimides. This should allow us to label the b subunits,e.g. with gold particles, and then visualize them in side views of F1F0 by cryoelectron microscopy. The expert technical assistance of Kathy Chicas-Cruz is gratefully acknowledged. We thank our colleagues Lyndall Hatch, Prof. Graeme Cox, and Prof. Frank Gibson for helpful discussions.
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