Role of the Asymmetry of the Homodimeric b2 Stator Stalk in the Interaction with the F1 Sector of Escherichia coli ATP Synthase
2007; Elsevier BV; Volume: 282; Issue: 44 Linguagem: Inglês
10.1074/jbc.m706259200
ISSN1083-351X
AutoresKristi S. Wood, Stanley D. Dunn,
Tópico(s)Advanced Electron Microscopy Techniques and Applications
ResumoThe b subunit dimer in the peripheral stator stalk of Escherichia coli ATP synthase is essential for enzyme assembly and the rotational catalytic mechanism. Recent protein chemical evidence revealed the dimerization domain of b to contain a novel two-stranded right-handed coiled coil with offset helices. Here, the existence of this structure in more complete constructs of b containing the C-terminal domain, and therefore capable of binding to the peripheral F1-ATPase, was supported by the more efficient formation of intersubunit disulfide bonds between cysteine residues that are proximal only in the offset arrangement and by the greater thermal stabilities of cross-linked heterodimers trapped in the offset configuration as opposed to homodimers with the helices trapped in-register. F1-ATPase binding analyses revealed the offset heterodimers to bind F1 more tightly than in-register homodimers. Mutations near the C terminus of b were incorporated specifically into either the N-terminally or the C-terminally shifted polypeptide, bN or bC, respectively, to determine the contribution of each position to F1 binding. Deletion of the last four residues of bN substantially weakened F1 binding, whereas the effect of the deletion in bC was modest. Similarly, benzophenone maleimide introduced at the C terminus of bN, but not bC, mediated cross-linking to the δ subunit of F1. These results imply that the polypeptide in the bN position is more important for F1 binding than the one in the bC position and illustrate the significance of the asymmetry of the b dimer in the enzyme. The b subunit dimer in the peripheral stator stalk of Escherichia coli ATP synthase is essential for enzyme assembly and the rotational catalytic mechanism. Recent protein chemical evidence revealed the dimerization domain of b to contain a novel two-stranded right-handed coiled coil with offset helices. Here, the existence of this structure in more complete constructs of b containing the C-terminal domain, and therefore capable of binding to the peripheral F1-ATPase, was supported by the more efficient formation of intersubunit disulfide bonds between cysteine residues that are proximal only in the offset arrangement and by the greater thermal stabilities of cross-linked heterodimers trapped in the offset configuration as opposed to homodimers with the helices trapped in-register. F1-ATPase binding analyses revealed the offset heterodimers to bind F1 more tightly than in-register homodimers. Mutations near the C terminus of b were incorporated specifically into either the N-terminally or the C-terminally shifted polypeptide, bN or bC, respectively, to determine the contribution of each position to F1 binding. Deletion of the last four residues of bN substantially weakened F1 binding, whereas the effect of the deletion in bC was modest. Similarly, benzophenone maleimide introduced at the C terminus of bN, but not bC, mediated cross-linking to the δ subunit of F1. These results imply that the polypeptide in the bN position is more important for F1 binding than the one in the bC position and illustrate the significance of the asymmetry of the b dimer in the enzyme. The process of oxidative phosphorylation in mitochondria and bacteria, or photophosphorylation in chloroplasts, requires the enzyme F1F0-ATP synthase to utilize the energy of the transmembrane proton gradient for the production of ATP from ADP and Pi. The enzyme functions as a molecular motor, with rotor and stator complexes consisting of subunits from both the membrane-peripheral F1 and membrane-integral F0 sectors of the protein. In the Escherichia coli enzyme, F0 contains three subunits in an ab2c10 stoichiometry, whereas F1 has five subunits in the stoichiometry of α3β3γδε. The γεc10 subunits compose the rotor, and b2δ forms the stator. As the rotor is driven by the passage of protons through a pore formed by the c and a subunits of F0, rotation of γ within the α3β3 hexamer of F1 causes conformational changes in the catalytic nucleotide-binding sites located on the β subunits, promoting ATP synthesis and release. One function of the b2δ stator is to hold the α3β3 hexamer against the rotational torque, as otherwise α3β3 would simply turn with the rotor rather than undergoing the conformational changes associated with the formation and release of ATP. In anaerobic or facultative bacteria, the enzyme can function as a proton pump, hydrolyzing ATP to drive protons out of the cytoplasm, against the electrochemical gradient. For recent reviews see Refs. 1Senior A.E. Kim S. Weber J. Biochim. Biophys. Acta. 2002; 1553: 188-211Crossref PubMed Scopus (345) Google Scholar, 2Wilkens S. Adv. Protein Chem. 2005; 71: 345-382Crossref PubMed Scopus (44) Google Scholar, 3Duncan T.M. Hackney D.D. Tamanoi F. The Enzymes: Energy Coupling and Molecular Motors. 3rd Ed. Elsevier Academic Press, New York2004: 203-275Google Scholar, 4Nakanishi-Matsui M. Kim M. IUBMB Life. 2006; 58: 318-322Crossref PubMed Scopus (23) Google Scholar, 5Feniouk B.A. Kim T. Yoshida M. Biochim. Biophys. Acta. 2006; 1757: 326-338Crossref PubMed Scopus (84) Google Scholar, 6Dunn S.D. Kim D.J. Del Rizzo P.A. Futai M. Wada Y. Kaplan J.H. Handbook of ATPases: Biochemistry, Cell Biology, Pathophysiology. Wiley-VCH, Weinheim, Germany2004: 311-318Google Scholar, 7Weber J. Trends Biochem. Sci. 2007; 32: 53-56Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 8Weber J. Biochim. Biophys. Acta. 2006; 1757: 1162-1170Crossref PubMed Scopus (52) Google Scholar, 9Walker J.E. Kim V.K. Biochim. Biophys. Acta. 2006; 1757: 286-296Crossref PubMed Scopus (155) Google Scholar, 10Richter M.L. Kim H.S. He F. Giessel A.J. Kuczera K.K. J. Bioenerg. Biomembr. 2005; 37: 467-473Crossref PubMed Scopus (26) Google Scholar. The major component of the stator stalk is the 156-residue b subunit, which forms an elongated dimer extending from the periplasmic side of the cytoplasmic membrane to the top of F1, where it interacts with δ (6Dunn S.D. Kim D.J. Del Rizzo P.A. Futai M. Wada Y. Kaplan J.H. Handbook of ATPases: Biochemistry, Cell Biology, Pathophysiology. Wiley-VCH, Weinheim, Germany2004: 311-318Google Scholar, 7Weber J. Trends Biochem. Sci. 2007; 32: 53-56Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 8Weber J. Biochim. Biophys. Acta. 2006; 1757: 1162-1170Crossref PubMed Scopus (52) Google Scholar, 11Dunn S.D. Kim M. Cipriano D.J. Shilton B.H. J. Bioenerg. Biomembr. 2000; 32: 347-355Crossref PubMed Scopus (41) Google Scholar). Clear roles for some sections of the b subunit have been determined, in particular the transmembrane domain formed by residues 1–24 (12Dmitriev O. Kim P.C. Jiang W. Fillingame R.H. J. Biol. Chem. 1999; 274: 15598-15604Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar), the dimerization domain encompassing residues 53–122 (13Revington M. Kim D.T. Shaw G.S. Dunn S.D. J. Biol. Chem. 1999; 274: 31094-31101Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 14Sorgen P.L. Kim M.R. McCormick K.A. Edison A.S. Cain B.D. Biochemistry. 1998; 37: 923-932Crossref PubMed Scopus (47) Google Scholar), and the C-terminal δ-binding domain composed of residues 123–156 (15Takeyama M. Kim T. Maeda M. Futai M. J. Biol. Chem. 1988; 263: 16106-16112Abstract Full Text PDF PubMed Google Scholar, 16McLachlin D.T. Kim J.A. Dunn S.D. J. Biol. Chem. 1998; 273: 15162-15168Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 17Bhatt D. Kim S.P. Grabar T.B. Claggett S.B. Cain B.D. J. Bioenerg. Biomembr. 2005; 37: 67-74Crossref PubMed Scopus (13) Google Scholar, 18McLachlin D.T. Kim S.D. Biochemistry. 2000; 39: 3486-3490Crossref PubMed Scopus (28) Google Scholar). The tether region (residues 25–52) links the transmembrane and dimerization domains; its function is not well understood, but it is known to interact with the a subunit and to play a role in coupling (19McLachlin D.T. Kim A.M. Clark S.M. Dunn S.D. J. Biol. Chem. 2000; 275: 17571-17577Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 20Caviston T.L. Kim C.J. Sorgen P.L. Nakamoto R.K. Cain B.D. FEBS Lett. 1998; 429: 201-206Crossref PubMed Scopus (37) Google Scholar). The interactions of b2 with F1 occur predominantly through the C-terminal δ-binding domain. Although interactions exist between b and α3β3 (19McLachlin D.T. Kim A.M. Clark S.M. Dunn S.D. J. Biol. Chem. 2000; 275: 17571-17577Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 21Weber J. Kim S. Nadanaciva S. Senior A.E. J. Biol. Chem. 2004; 279: 11253-11258Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), δ subunit is required for the binding of F1 to F0 in membranes (22Futai M. Kim P.C. Heppel L.A. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 2725-2729Crossref PubMed Scopus (210) Google Scholar). The interaction of b and δ, mediated through the C-terminal regions of each subunit, appears to be key to this binding (15Takeyama M. Kim T. Maeda M. Futai M. J. Biol. Chem. 1988; 263: 16106-16112Abstract Full Text PDF PubMed Google Scholar, 16McLachlin D.T. Kim J.A. Dunn S.D. J. Biol. Chem. 1998; 273: 15162-15168Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Dimerization of b is essential for F1 binding and ATP synthase function, although it can be significantly weakened through mutation in the dimerization domain before activity is lost (14Sorgen P.L. Kim M.R. McCormick K.A. Edison A.S. Cain B.D. Biochemistry. 1998; 37: 923-932Crossref PubMed Scopus (47) Google Scholar, 23Cipriano D.J. Kim K.S. Bi Y. Dunn S.D. J. Biol. Chem. 2006; 281: 12408-12413Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). The isolated dimerization domain has been characterized as an atypical, parallel, two-stranded coiled coil (13Revington M. Kim D.T. Shaw G.S. Dunn S.D. J. Biol. Chem. 1999; 274: 31094-31101Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 24McLachlin D.T. Kim S.D. J. Biol. Chem. 1997; 272: 21233-21239Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 25Revington M. Kim S.D. Shaw G.S. Protein Sci. 2002; 11: 1227-1238Crossref PubMed Scopus (27) Google Scholar). Sequence analyses of this region have identified an 11-residue hendecad pattern, with positions denoted abcdefghijk (6Dunn S.D. Kim D.J. Del Rizzo P.A. Futai M. Wada Y. Kaplan J.H. Handbook of ATPases: Biochemistry, Cell Biology, Pathophysiology. Wiley-VCH, Weinheim, Germany2004: 311-318Google Scholar, 26Del Rizzo P.A. Kim Y. Dunn S.D. Shilton B.H. Biochemistry. 2002; 41: 6875-6884Crossref PubMed Scopus (87) Google Scholar). Hendecad patterns are indicative of right-handed coiled coils in which 11 residues make three turns of the helix relative to the interhelical axis (27Lupas A. Trends Biochem. Sci. 1996; 21: 375-382Abstract Full Text PDF PubMed Scopus (1008) Google Scholar, 28Lupas A.N. Kim M. Adv. Protein Chem. 2005; 70: 37-78Crossref PubMed Scopus (580) Google Scholar); the expected distribution of positions for a two-stranded structure of this type is shown on the helical wheel in Fig. 1A. The hendecad pattern seen in b subunit sequences is unusual in that the a and h positions at the center of the interface are usually occupied by alanine or other small amino acids, whereas larger hydrophobic residues are often seen in the d and e positions that are more peripherally situated (6Dunn S.D. Kim D.J. Del Rizzo P.A. Futai M. Wada Y. Kaplan J.H. Handbook of ATPases: Biochemistry, Cell Biology, Pathophysiology. Wiley-VCH, Weinheim, Germany2004: 311-318Google Scholar). In the absence of a high resolution dimeric structure, assignment of the hydrophobic strip defined by the a, d, e, and h positions as the dimerization interface in the isolated dimerization domain has been supported by recent studies of disulfide formation between cysteine residues introduced into the a and h positions between residues 61 and 90 and by assessment of the stabilities of disulfide-linked dimers (29Del Rizzo P.A. Kim Y. Dunn S.D. J. Mol. Biol. 2006; 364: 735-746Crossref PubMed Scopus (47) Google Scholar). Results of these studies further implied that the two helices of the dimer are offset, rather than in-register as in left-handed coiled coils. In this staggered configuration, one of the helices, denoted bN, 2The abbreviations used are: bN and bC, the positions of the b subunits in the offset right-handed coiled coil of the b2 dimer, with bN shifted toward the N terminus and bC shifted toward the C terminus; BPM, benzophenone-4-maleimide; TCEP, Tris(2-carboxyethyl) phosphine hydrochloride. is N-terminally shifted relative to the other helix, denoted bC, by about 5.5 residues (one-half of a hendacad), making the dimer intrinsically asymmetric, as seen in Fig. 1B. A functional significance of the right-handed coiled coil in withstanding the torque imparted by γ rotation within α3β3 was suggested. The goal of this study was to explore the existence of the offset helices in b constructs containing the δ-binding domain and to assess its functional significance in the interaction with F1. The results we present confirm the asymmetric nature of the dimer and reveal the different roles of bN and bC in the binding of F1. Plasmid Construction—Recombinant DNA techniques were performed by standard methods. All generated sequences were verified by sequencing. Plasmids encoding b polypeptides were constructed such that all of the resultant proteins included the R83A mutation, previously shown to stabilize the dimer (26Del Rizzo P.A. Kim Y. Dunn S.D. Shilton B.H. Biochemistry. 2002; 41: 6875-6884Crossref PubMed Scopus (87) Google Scholar), except when cysteine was inserted at this position. Plasmids encoding b residues 53–156 were based on pSD111, which includes a leader sequence of MSYW (24McLachlin D.T. Kim S.D. J. Biol. Chem. 1997; 272: 21233-21239Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), whereas plasmids encoding b residues 34–156 were based on plasmid pJB3, in which the leader sequence is MST (30Dunn S.D. Kim J. J. Biol. Chem. 1998; 273: 8646-8651Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Relevant mutations were introduced into these plasmids by subcloning appropriate sections of DNA from previously described plasmids encoding desired cysteine substitutions (29Del Rizzo P.A. Kim Y. Dunn S.D. J. Mol. Biol. 2006; 364: 735-746Crossref PubMed Scopus (47) Google Scholar) or C-terminal mutations resulting in either premature chain termination, V153Stop, or else addition of GC to the normal C-terminal Leu-156 (16McLachlin D.T. Kim J.A. Dunn S.D. J. Biol. Chem. 1998; 273: 15162-15168Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Protein Expression, Purification, and Production of Disulfide Cross-linked Forms—The various mutant forms of soluble b subunit were expressed and purified by ammonium sulfate fractionation and ion-exchange chromatography, essentially as described (24McLachlin D.T. Kim S.D. J. Biol. Chem. 1997; 272: 21233-21239Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 30Dunn S.D. Kim J. J. Biol. Chem. 1998; 273: 8646-8651Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Purification steps were monitored by SDS-PAGE. To prepare disulfide-linked dimers, purified cysteine-containing b polypeptides, either individually or as equimolar mixtures, were first reduced with 2 mm dithiothreitol for 1 h and then dialyzed overnight against a buffer containing 50 mm Tris-HCl, pH 8.0. Dialysis bags were then transferred to buffer containing 50 mm Tris-HCl, pH 8.0, and 10 μm CuCl2, and dialysis was continued for 3 days. In cases where one of the b subunits contained a C-terminal Gly-Cys addition, as well as a cysteine at either position 83 or 90 in the dimerization domain, a copper/cysteine disulfide exchange buffer that contained 10 μm CuCl2 as well as 10 mm cysteine was used to foster internal disulfide formation, while leaving the C-terminal cysteine residue disulfide-linked to the free amino acid cysteine. The resultant cross-linked forms were purified by anion-exchange chromatography using a Mono Q 5/5 column. Determination of Propensity to Form Disulfides—Purified cysteine-containing b constructs were analyzed for their propensity to form disulfide-linked dimers by a previously described method in which the polypeptides are dialyzed in the presence of air against buffer containing 10 μm CuCl2 and 10 mm cysteine (24McLachlin D.T. Kim S.D. J. Biol. Chem. 1997; 272: 21233-21239Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 29Del Rizzo P.A. Kim Y. Dunn S.D. J. Mol. Biol. 2006; 364: 735-746Crossref PubMed Scopus (47) Google Scholar). Equal volumes of 60 μm protein samples were mixed, giving each a final concentration of 30 μm. Mixtures were dialyzed overnight against 50 mm Tris-HCl, pH 7.5, 0.1 mm EDTA, and 5 mm dithiothreitol at 4 °C to reduce any disulfides. Part of each sample was removed for analysis by nonreducing SDS-PAGE, and the dialysis bags were then transferred to a buffer containing 100 mm Tris-HCl, pH 8.0, 10 mm cysteine, and 10 μm CuCl2 and allowed to dialyze for 24 h at 4 °C with vigorous stirring. Samples were again collected and analyzed by nonreducing SDS-PAGE. To prevent any disulfide exchange, sample buffer for nonreducing SDS-PAGE contained 15 mm N-ethylmaleimide. Chemical Cross-linking with Benzophenone-4-maleimide (BPM)—All steps were performed at room temperature. Samples of purified F1 and disulfide-linked b dimers, in which one of the subunits had a C-terminal glycylcysteine addition, were separately passed through 1-ml centrifuge columns (31Penefsky H.S. Methods Enzymol. 1979; 56: 527-530Crossref PubMed Scopus (343) Google Scholar) containing Bio-Gel P-10 resin (Bio-Rad) equilibrated with 50 mm sodium phosphate, pH 7.5, and 1 mm EDTA. The b dimers at a concentration of 25 μm were incubated for 75 min with 1 mm TCEP to reduce the C-terminal cysteine of those constructs. Chemical cross-linking of the b dimers to F1-ATPase using BPM (Molecular Probes, Eugene, OR) was carried out using the procedure described by McLachlin et al. (16McLachlin D.T. Kim J.A. Dunn S.D. J. Biol. Chem. 1998; 273: 15162-15168Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). BPM dissolved in dimethylformamide was added in a 5-fold molar excess over the b dimers and allowed to incubate for 15 min. Unreacted BPM was quenched by addition of an equimolar amount of β-mercaptoethanol. The b dimer was then mixed with the column-centrifuged F1 at a molar ratio of 2 F1 per b dimer, in the presence of 5 mm MgCl2. Controls were performed in which no BPM was added to the b solution. Samples were exposed to long wave ultraviolet light from an Ultra-Violet Products model TM-36 transilluminator for 5 min. As an additional control, some BPM-modified samples were placed on the transilluminator but removed before it was turned on. After illumination, SDS-PAGE sample buffer was added to the samples, which were then heated at 100 °C for 2 min and analyzed by SDS-PAGE and Western blotting. Other Materials and Methods—Thermal denaturation of protein constructs in 10 mm sodium phosphate, pH 7.0, was followed by circular dichroism spectroscopy at 222 nm using a Jasco J-810 spectropolarimeter equipped with a Peltier temperature control unit. The temperature was increased at the rate of 1 °C per min. Data were converted to mean residue ellipticity and fitted to a two-state model as described (32Briere L.K. Kim S.D. Biochemistry. 2006; 45: 8607-8616Crossref PubMed Scopus (10) Google Scholar, 33Santoro M.M. Kim D.W. Biochemistry. 1988; 27: 8063-8068Crossref PubMed Scopus (1609) Google Scholar, 34Swint L. Kim A.D. Protein Sci. 1993; 2: 2037-2049Crossref PubMed Scopus (131) Google Scholar). F1 binding activity of b dimers was determined using a competitive assay described previously (16McLachlin D.T. Kim J.A. Dunn S.D. J. Biol. Chem. 1998; 273: 15162-15168Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Purified soluble b dimers at the indicated concentrations were mixed with 20 μl of an F1 affinity resin bearing soluble b linked to the resin through a cysteine residue near its N terminus and 31.3 μg of F1 (0.3 μm) for 1 h. The resin was sedimented by centrifugation; the supernatant solution was removed; the pellet was resuspended in SDS-PAGE sample buffer and then analyzed by SDS-PAGE. Bovine serum albumin was added to the competition buffer to serve as a control for trapping of liquid within the resin pellet. As a control, resin with only cysteine linked was used. Protein concentrations were determined using the Advanced Reagent (Cytoskeleton, Inc.), and values for b subunits were corrected using a factor determined by quantitative amino acid analysis as described previously (26Del Rizzo P.A. Kim Y. Dunn S.D. Shilton B.H. Biochemistry. 2002; 41: 6875-6884Crossref PubMed Scopus (87) Google Scholar, 29Del Rizzo P.A. Kim Y. Dunn S.D. J. Mol. Biol. 2006; 364: 735-746Crossref PubMed Scopus (47) Google Scholar). SDS-PAGE was carried out using the Tris-glycine system described by Laemmli (35Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). Sample buffer for nonreducing SDS-PAGE contained 15 mm N-ethylmaleimide. Sample buffer for reducing SDS-PAGE contained 50 mm dithiothreitol. The presence of free thiol groups in polypeptides was determined by treatment with 1.2 mm fluorescein maleimide in SDS sample buffer lacking both dithiothreitol and N-ethylmaleimide for 15 min followed by SDS-PAGE (36McLachlin D.T. Kim S.D. Protein Expression Purif. 1996; 7: 275-280Crossref PubMed Scopus (8) Google Scholar). Before Coomassie Blue staining, the gel was exposed to UV light to visualize the fluorescein-labeled polypeptides. Polyclonal antibodies to the peripheral domain of b and to δ subunit were raised in rabbits. The anti-δ serum was treated with bMERC resin (16McLachlin D.T. Kim J.A. Dunn S.D. J. Biol. Chem. 1998; 273: 15162-15168Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) to remove antibodies that recognized b subunit. The anti-b serum was sufficiently specific for our studies without any special treatment. Blotting was carried out using carbonate blot buffer as described previously (37Dunn S.D. Anal. Biochem. 1986; 157: 144-153Crossref PubMed Scopus (421) Google Scholar). Primary antibodies were subsequently detected using a second antibody conjugated to alkaline phosphatase as described (38Aggeler R. Kim R.A. Dunn S. Gogol E.P. Arch. Biochem. Biophys. 1992; 296: 685-690Crossref PubMed Scopus (10) Google Scholar). Preferential Formation of Intersubunit Disulfides between Positions a and h—The propensity for disulfide formation between cysteine residues introduced into b subunit polypeptides was assessed using a previously developed method involving initial dialysis against buffer containing 5 mm dithiothreitol and 0.1 mm EDTA to reduce any existing disulfides, followed by a second dialysis against a disulfide exchange buffer containing 10 μm Cu2+ and 10 mm free cysteine to foster selective oxygen-dependent disulfide formation. Previous studies of disulfide bond formation using dimerization domain constructs showed that mixed pairs of polypeptides with cysteines in all adjacent a and h positions between Ala-61 and Ala-90 had much stronger propensities to form disulfide-linked heterodimers than any of the individual cysteine-containing constructs to form disulfides (29Del Rizzo P.A. Kim Y. Dunn S.D. J. Mol. Biol. 2006; 364: 735-746Crossref PubMed Scopus (47) Google Scholar). We asked if this result could be extended to forms of b containing the entire C-terminal domain that is involved in binding F1, using b34–156 with cysteines at individual h positions (68, 79, 90) and b53–156 constructs with cysteines at individual a positions (72, 83), as described under "Experimental Procedures" (Fig. 1, C and D). The difference in size between these constructs allowed differentiation between homodimers and heterodimers by their migration on SDS-PAGE. No more than a trace of dimer formed from any pure construct during the dialysis against 5 mm dithiothreitol (Fig. 2A, upper), but surprisingly significant dimer formation occurred between constructs with cysteines in adjacent a and h positions (i.e. 68 × 72, 72 × 79, 79 × 83, and 83 × 90) even during this step (Fig. 2A, lower panel). After oxidizing dialysis in a disulfide-exchange buffer (Fig. 2B), essentially complete dimer formation was observed in those particular mixed samples, whereas disulfide formation was substantially incomplete for the individual polypeptides or the other mixtures. Disulfide-linked Offset Heterodimers Are More Stable than In-register Homodimers—Whereas the results of the disulfide formation studies indicated that interchain disulfide formation is more favorable for each of the four adjacent a/h pairs tested than for any individual construct or for any of the other mixed samples, the distinction is not absolute. We therefore sought additional evidence by determining the thermal stability of purified, disulfide-linked b dimers, following the change in the circular dichroism signal as the temperature was raised. Three in-register homodimers and two offset heterodimers were analyzed (Fig. 3). In comparison with the cysteineless constructs, shown in Fig. 3 as open symbols, homodimers linked through cysteines at positions 79, 83, or 90 all showed broader transitions indicative of lower cooperativity. In contrast, the two offset heterodimers melted with sharp transitions, similar to those of the cysteineless forms, implying that interhelical interactions in the native cysteineless forms are more closely approximated by those in the offset heterodimers. Because the introduction of either type of disulfide converts folding from a bimolecular to a unimolecular reaction, both result in increases in the midpoint of melting, but it is also notable that the melting midpoints of the heterodimers were about 20 °C higher, in the range of 73–75 °C, than those of the in-register homodimers that were between 49 and 54 °C. Overall, the results of these studies imply that the unusual offset relationship of the helices previously seen in the isolated dimerization domain applies also to constructs extending to the C terminus. Offset Heterodimers Preferentially Bind F1—To investigate the functional relevance of the offset relationship of the b subunit helices, we studied the effects of either offset or in-register intersubunit disulfides on the interaction with F1 using a semi-quantitative competitive assay, in which the ability of a soluble b to compete with wild-type b conjugated to agarose beads for binding a limited amount of F1 is tested (16McLachlin D.T. Kim J.A. Dunn S.D. J. Biol. Chem. 1998; 273: 15162-15168Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Results of this experiment are shown in Fig. 4. As seen in the left-hand lanes in Fig. 4 next to the molecular weight standards, control resin lacking conjugated b had only small amounts of F1 trapped within the resin, whereas the affinity resin bound substantial F1 in the absence of soluble competitor, but had only the background level when competing wild-type soluble b was added (upper panel). Both of the tested offset heterodimers, 79 × 83 and 83 × 90, resembled the wild-type b in their ability to compete for F1, whereas the in-register homodimers formed with cysteines at any of the three positions competed less effectively. The stronger binding of the offset heterodimers to F1 implies the relevance of the staggered conformation of b within the enzyme. bN Is More Important for F1 Binding—The final four residues of the b polypeptide have been found previously to be required for the binding of b to F1 through the δ subunit; deletion of these residues completely abolished F1 binding (16McLachlin D.T. Kim J.A. Dunn S.D. J. Biol. Chem. 1998; 273: 15162-15168Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Subsequent studies from the Cain laboratory showed that coexpression of a bR36I mutant, which is also nonfunctional by itself, complemented the four-residue truncation, implying that a heterodimeric form of b with just one of the subunits extending to the C terminus would foster assembly and function of ATP synthase (39Grabar T.B. Kim B.D. J. Biol. Chem. 2004; 279: 31205-31211Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Because formation of a disulfide bond between position 83 of one b subunit and position 90 of the other b subunit locks the polypeptide with 90C in the bN position, and the polypeptide with 83C in the bC position (see Fig. 1C), we were able to ask which of the two b subunits must extend to the normal C terminus, Leu-156, by incorporating the four-residue C-terminal deletion into the constructs bearing either of the cysteine mutations. Through disulfide formation and subsequent purification, we prepared heterodimers in which the final four residues were deleted from bN only, bC only, or from both of the polypeptides. These constructs were tested for their ability to compete for F1 binding using the competitive assay (Fig. 5). As before, both the wild-type control and offset disulfide-linked dimer with both C termini intact (bC × bN) competed effectively, as indicated by the strong reduction in F1 bound to the resin. Notably, the C-terminal truncation of bN strongly reduced competition for F1, whereas the C-terminal truncation of bC had a much less significant effect (the truncation of polypeptides is indicated in the figure by the subscript Δ4). As expected, controls with deletion of the C-terminal residues on both subunits, either offset or in-register, showed no competition for F1. These results indicate that the C-terminal residues of bN play the mor
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