Role of the epsilon subunit of thermophilic F1-ATPase as a sensor for ATP

The epsilon subunit of F(1)-ATPase from the thermophilic Bacillus PS3 (TF(1)) has been shown to bind ATP. The precise nature of the regulatory role of ATP binding to the epsilon subunit remains to be determined. To address this question, 11 mutants of the epsilon subunit were prepared, in which one of the basic or acidic residues was substituted with alanine. ATP binding to these mutants was tested by gel-filtration chromatography. Among them, four mutants that showed no ATP binding were selected and reconstituted with the alpha(3)beta(3)gamma complex of TF(1). The ATPase activity of the resulting alpha(3)beta(3)gammaepsilon complexes was measured, and the extent of inhibition by the mutant epsilon subunits was compared in each case. With one exception, weaker binding of ATP correlated with greater inhibition of ATPase activity. These results clearly indicate that ATP binding to the epsilon subunit plays a regulatory role and that ATP binding may stabilize the ATPase-active form of TF(1) by fixing the epsilon subunit into the folded conformation.     

ATP-dependent rotation of mutant ATP synthases defective in proton transport

During ATP hydrolysis, the gammaepsilon c10 complex (gamma and epsilon subunits and a c subunit ring formed from 10 monomers) of F0F1 ATPase (ATP synthase) rotates relative to the alpha3beta3delta ab2 complex, leading to proton transport through the interface between the a subunit and the c subunit ring. In this study, we replaced the two pertinent residues for proton transport, cAsp-61 and aArg-210 of the c and a subunits, respectively. The mutant enzymes exhibited lower ATPase activities than that of the wild type but exhibited ATP-dependent rotation in planar membranes, in which their original assemblies are maintained. The mutant enzymes were defective in proton transport, as shown previously. These results suggest that proton transport can be separated from rotation in ATP hydrolysis, although rotation ensures continuous proton transport by bringing the cAsp-61 and aArg-210 residues into the correct interacting positions.     

gammaepsilon Sub-complex of thermophilic ATP synthase has the ability to bind ATP

The isolated epsilon subunit of F(1)-ATPase from thermophilic Bacillus PS3 (TF(1)) binds ATP [Y. Kato-Yamada, M. Yoshida, J. Biol. Chem. 278 (2003) 36013]. The obvious question is whether the ATP binding concern with the regulation of ATP synthase activity or not. If so, the epsilon subunit even in the ATP synthase complex should have the ability to bind ATP. To check if the ATP binding to the epsilon subunit within the ATP synthase complex may occur, the gammaepsilon sub-complex of TF(1) was prepared and ATP binding was examined. The results clearly showed that the gammaepsilon sub-complex can bind ATP.     

Thiol modification as a probe of conformational forms of the F1 ATPase of Escherichia coli and of the structural asymmetry of its beta subunits

The sulfhydryl groups of soluble and membrane-bound F1 adenosine triphosphatase of Escherichia coli were modified by reaction with the fluorescent thiol reagents 5-iodoacetamidofluorescein, 2-[(4'-iodoacetamido)anilino]naphthalene-6-sulfonic acid 4-[N-(iodoacetoxy)ethyl-N-methyl]amino-7-nitrobenzo-2-oxa-1,3-d iaz ole and 2-[(4'-maleimidyl)anilino]naphthalene-6-sulfonic acid. Whereas gamma and delta subunits were always labeled by these reagents, the beta subunit reacted preferentially in the soluble enzyme, and the alpha subunit in the membrane-bound enzyme. This suggests that the soluble enzyme undergoes a conformational change on binding to the membrane. The three beta subunits of the soluble ATPase did not react with chemical reagents in a similar manner. One beta subunit was cross-linked to the epsilon subunit on treatment of the ATPase with 1-ethyl-3-[3-(dimethyl-amino)propyl]carbodiimide, as observed previously by L?tscher et al. [Biochemistry (1984) 23, 4134-4140]. A second beta subunit, which did not cross-link to the epsilon subunit, was modified preferentially by the fluorescent thiol reagents and by 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole. The third beta subunit was less chemically reactive than the others. Both alpha and beta subunits of the soluble 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole-modified enzyme were labeled by the fluorescent thiol reagents. Thus, the modified enzyme, which is inactive, probably has a different conformation from the native soluble ATPase.     

Determination of the 1-ethyl-3-[(3-dimethylamino)propyl]-carbodiimide- induced cross-link between the beta and epsilon subunits of Escherichia coli F1-ATPase.

The zero-length cross-link between the inhibitory epsilon subunit and one of three catalytic beta subunits of Escherichia coli F1-ATPase (alpha 3 beta 3 gamma delta epsilon), induced by a water-soluble carbodiimide, 1-ethyl-3-[(3-dimethylamino) propyl]-carbodiimide (EDC), has been determined at the amino acid level. Lability of cross-linked beta-epsilon to base suggested an ester cross-link rather than the expected amide. A 10-kDa cross-linked CNBr fragment derived from beta-epsilon was identified by electrophoresis on high percentage polyacrylamide gels. Sequence analysis of this peptide revealed the constituent peptides to be Asp-380 to Met-431 of beta and Glu-96 to Met-138 of epsilon. Glu-381 of beta was absent from cycle 2 indicating that it was one of the cross-linked residues, but no potential cross-linked residue in epsilon was identified in this analysis. A form of epsilon containing a methionine residue in place of Val-112 (epsilon V112M) was produced by site-directed mutagenesis. epsilon V112M was incorporated into F1-ATPase which was then cross-linked with EDC. An 8-kDa cross-linked CNBr fragment of beta-epsilon V112M was shown to contain the peptide of epsilon between residues Glu-96 and Met-112 and the peptide of beta between residues Asp-380 and Met-431. Again residue Glu-381 of beta was notably reduced and no missing residue from the epsilon peptide could be identified, but the peptide sequence limited the possible choices to Ser-106, Ser-107, or Ser-108. Furthermore, an epsilon mutant in which Ser-108 was replaced by cysteine could no longer be cross-linked to a beta subunit in F1-ATPase by EDC. Both mutant forms of epsilon supported growth of an uncC-deficient E. coli strain and inhibited F1-ATPase. These results indicate that the EDC-induced cross-link between the beta and epsilon subunits of F1-ATPase is an ester linkage between beta-Glu-381 and, likely, epsilon-Ser-108. As these residues must be located immediately adjacent to one another in F1-ATPase, our results define a site of subunit-subunit contact between beta and epsilon.     

Cross-linking of two beta subunits in the closed conformation in F1-ATPase

In the crystal structure of mitochondrial F1-ATPase, two beta subunits with a bound Mg-nucleotide are in "closed" conformations, whereas the third beta subunit without bound nucleotide is in an "open" conformation. In this "CCO" (beta-closed beta-closed beta-open) conformational state, Ile-390s of the two closed beta subunits, even though they are separated by an intervening alpha subunit, have a direct contact. We replaced the equivalent Ile of the alpha3beta3gamma subcomplex of thermophilic F1-ATPase with Cys and observed the formation of the beta-beta cross-link through a disulfide bond. The analysis of conditions required for the cross-link formation indicates that: (i) F1-ATPase takes the CCO conformation when two catalytic sites are filled with Mg-nucleotide, (ii) intermediate(s) with the CCO conformation are generated during catalytic cycle, (iii) the Mg-ADP inhibited form is in the CCO conformation, and (iv) F1-ATPase dwells in conformational state(s) other than CCO when only one (or none) of catalytic sites is filled by Mg-nucleotide or when catalytic sites are filled by Mg2+-free nucleotide. The alpha3beta3gamma subcomplex containing the beta-beta cross-link retained the activity of uni-site catalysis but lost that of multiple catalytic turnover, suggesting that open-closed transition of beta subunits is required for the rotation of gamma subunit but not for hydrolysis of a single ATP.     

Ligand-induced conformational changes in the Escherichia coli F1 adenosine triphosphatase probed by trypsin digestion

Digestion of the F1-ATPase of Escherichia coli with trypsin stimulated ATP hydrolytic activity and removed the delta and epsilon subunits of the enzyme. A species represented by the formula alpha 1(3) beta 1(3) gamma 1, where alpha 1, beta 1 and gamma 1 are forms of the native alpha, beta and gamma subunits which have been attacked by trypsin, was formed by trypsin digestion in the presence of ATP. In the presence of ATP and MgCl2, conversion of gamma to gamma 1 was retarded and the enzyme retained the epsilon subunit. These results imply that binding of ATP to the beta subunits alters the conformation of ECF1 to increase the accessibility of the gamma subunit to trypsin. The likely trypsin cleavage sites in the alpha, beta and gamma subunits are discussed. ECF1 from the alpha subunit-defective mutant uncA401, or after treatment with N,N'-dicyclohexylcarbodiimide or 4-chloro-7-nitrobenzofurazan, was present in a conformation in which the gamma subunit was readily accessible to trypsin and could not be protected by the presence of ATP and MgCl2. In a similar manner to native E. coli F1-ATPase, the hydrolytic activity of the trypsin-digested enzyme was stimulated by the detergent lauryldimethylamine N-oxide. Since the digested enzyme lacked the epsilon subunit, a putative inhibitor of hydrolytic activity, a mechanism for the stimulation which involves loss or movement of this subunit is untenable.     

Probing conformations of the beta subunit of F0F1-ATP synthase in catalysis

A subcomplex of F0F1-ATP synthase (F0F1), alpha3beta3gamma, was shown to undergo the conformation(s) during ATP hydrolysis in which two of the three beta subunits have the "Closed" conformation simultaneously (CC conformation) [S.P. Tsunoda, E. Muneyuki, T. Amano, M. Yoshida, H. Noji, Cross-linking of two beta subunits in the closed conformation in F1-ATPase, J. Biol. Chem. 274 (1999) 5701-5706]. This was examined by the inter-subunit disulfide cross-linking between two mutant beta(I386C)s that was formed readily only when the enzyme was in the CC conformation. Here, we adopted the same method for the holoenzyme F0F1 from Bacillus PS3 and found that the CC conformation was generated during ATP hydrolysis but barely during ATP synthesis. The experiments using F0F1 with the epsilon subunit lacking C-terminal helices further suggest that this difference is related to dynamic nature of the epsilon subunit and that ATP synthesis is accelerated when it takes the pathway involving the CC conformation.     

Catalytic control and coupling efficiency of the Escherichia coli FoF1 ATP synthase: influence of the Fo sector and epsilon subunit on the catalytic transition state

The rate-limiting transition state of steady-state ATP hydrolysis and synthesis reactions in the F(o)F(1) ATP synthase involves the rotation of the gamma, epsilon, and c subunits. To probe the role of the transport and coupling mechanisms in controlling catalysis, kinetic and thermodynamic parameters of ATP hydrolysis were determined for enzymes in the presence of the detergent lauryldimethylamine oxide (LDAO), which uncouples active transport and disables the inhibitory effect of the epsilon subunit. At 5 mM LDAO or greater, the inhibitory effects of epsilon subunit are abrogated in both purified F(1) and membranous F(o)F(1). In these conditions, LDAO solubilized F(o)F(1) has a higher k(cat) for ATP hydrolysis than F(1). These results indicate an influence of F(o) on F(1) even though catalysis is uncoupled from transport. The alpha(3)beta(3)gamma complex free of the epsilon subunit is activated at a lower concentration of 0.5 mM LDAO. Significantly, the gammaY205C mutant enzyme is similarly activated at 0.5 mM LDAO, suggesting that the mutant enzyme lacks epsilon inhibition. The gammaY205C F(o)F(1), which has a k(cat) for ATP hydrolysis 2-fold higher than wild type, has an ATP synthesis rate 3-fold lower than wild type, showing that coupling is inefficient. Arrhenius and isokinetic analyses indicate that enzymes that are free of epsilon subunit inhibition have a different transition-state structure from those under the influence of the epsilon subunit. We propose that the epsilon subunit is one of the factors that determines the proper transition-state structure, which is essential for efficient coupling.     

A homologous sequence between H+-ATPase (F0F1) and cation-transporting ATPases. Thr-285----Asp replacement in the beta subunit of Escherichia coli F1 changes its catalytic properties

A sequence of 10 amino acids (I-C-S-D-K-T-G-T-L-T) of ion motive ATPases such as Na+/K+-ATPase is similar to the sequence of the beta subunit of H+-ATPases, including that of Escherichia coli (I-T-S-T-K-T-G-S-I-T) (residues 282-291). The Asp (D) residue phosphorylated in ion motive ATPase corresponds to Thr (T) of the beta subunit. This substitution may be reasonable because there is no phosphoenzyme intermediate in the catalytic cycle of F1-ATPase. We replaced Thr-285 of the beta subunit by an Asp residue by in vitro mutagenesis and reconstituted the alpha beta gamma complex from the mutant (or wild-type) beta and wild-type alpha and gamma subunits. The uni- and multisite ATPase activities of the alpha beta gamma complex with mutant beta subunits were about 20 and 30% of those with the wild-type subunit. The rate of ATP binding (k1) of the mutant complex under uni-site conditions was about 10-fold less than that of the wild-type complex. These results suggest that Thr-285, or the region in its vicinity, is essential for normal catalysis of the H+-ATPase. The mutant complex could not form a phosphoenzyme under the conditions where the H+/K+-ATPase is phosphorylated, suggesting that another residue(s) may also be involved in formation of the intermediate in ion motive ATPase. The wild-type alpha beta gamma complex had slightly different kinetic properties from the wild-type F1, possibly because it did not contain the epsilon subunit.     

The ATPase activity of the alpha 3 beta 3 complex of the F1-ATPase of the thermophilic bacterium PS3 is inactivated on modification of tyrosine 307 in a single beta subunit by 7-chloro-4-nitrobenzofurazan

The catalytically active alpha 3 beta 3 complex, assembled as described (Miwa, K., and Yoshida, M. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 6484-6487) from the isolated alpha and beta subunits of the F1-ATPase of the thermophilic bacterium PS3 (TF1), is inactivated by 7-chloro-4-nitrobenzofurazan (Nbf-Cl) with characteristics very similar to those observed when TF1, which has the subunit composition, alpha 3 beta 3 gamma delta epsilon, is inactivated by the reagent under the same conditions. Both native TF1 and the alpha 3 beta 3 complex are inactivated by 200 microM Nbf-Cl with a pseudo-first order rate constant of 3.7 x 10(-2) min-1 in the presence of 0.2 M Na2SO4 at pH 7.6 and 23 degrees C. The rate of increase in absorbance at 385 nm of reaction mixtures containing 200 microM [14C]Nbf-Cl and TF1, the wild-type alpha 3 beta 3 complex, or the mutant alpha 3(beta Y307----F)3 complex, each at 18 microM was also examined. Since the alpha 3(beta y307----F)3 complex is resistant to inactivation by Nbf-Cl, difference spectrophotometry revealed that inactivation of native TF1 and the wild-type alpha 3 beta 3 complex could be correlated with formation of about 1 mol of Nbf-O-Tyr/mol of enzyme or complex. Fractionation of peptic digests of the labeled enzyme and complexes by reversed-phase high performance liquid chromatography resolved a major radioactive peptide that was common to labeled TF1 and the labeled alpha 3 beta 3 complex but was absent in the digest of the labeled alpha 3(beta Y307----F)3 complex. This labeled peptide was shown to contain Tyr-beta 307 derivatized with [14C]Nbf-Cl by automatic amino acid sequence analyses. From these results, it is concluded that one-third of the sites' reactivity of Nbf-Cl with Tyr-beta 307 in TF1 or its equivalent in other F1-ATPases is not influenced by the presence of the gamma, delta, or epsilon subunits. It has also been shown that Tyr-307 is not modified to an appreciable extent when the isolated beta subunit is treated with [14C]Nbf-Cl under conditions in which this residue is nearly completely labeled in a single beta subunit when TF1 or the alpha 3 beta 3 complex is inactivated by the reagent.     

ATPase activity of a highly stable alpha(3)beta(3)gamma subcomplex of thermophilic F(1) can be regulated by the introduced regulatory region of gamma subunit of chloroplast F(1)

A mutant F(1)-ATPase alpha(3)beta(3)gamma subcomplex from the thermophilic Bacillus PS3 was constructed, in which 111 amino acid residues (Val(92) to Phe(202)) from the central region of the gamma subunit were replaced by the 148 amino acid residues of the homologous region from spinach chloroplast F(1)-ATPase gamma subunit, including the regulatory stretch, and were designated as alpha(3)beta(3)gamma((TCT)) (Thermophilic-Chloroplast-Thermophilic). By the insertion of this regulatory region into the gamma subunit of thermophilic F(1), we could confer the thiol modulation property to the thermophilic alpha(3)beta(3)gamma subcomplex. The overexpressed alpha(3)beta(3)gamma((TCT)) was easily purified in large scale, and the ATP hydrolyzing activity of the obtained complex was shown to increase up to 3-fold upon treatment with chloroplast thioredoxin-f and dithiothreitol. No loss of thermostability compared with the wild type subcomplex was found, and activation by dithiothreitol was functional at temperatures up to 80 degrees C. alpha(3)beta(3)gamma((TCT)) was inhibited by the epsilon subunit from chloroplast F(1)-ATPase but not by the one from the thermophilic F(1)-ATPase, indicating that the introduced amino acid residues from chloroplast F(1)-gamma subunit are important for functional interaction with the epsilon subunit.     

The G226A mutant of Gs alpha highlights the requirement for dissociation of G protein subunits.

Adenylylcyclase cannot be activated by hormones or guanine nucleotide analogs in membranes from cells that express the G226A mutant form Gs alpha instead of the wild-type protein. The mutant Gs alpha protein appears incapable of undergoing the conformational change necessary for guanine nucleotide-induced dissociation of the G protein alpha subunit from the beta gamma subunit complex (Miller, R.T., Masters, S.B., Sullivan, K.A., Beiderman, B., and Bourne, H.R. (1988) Nature 334, 712-715). G226A Gs alpha was synthesized in Escherichia coli, purified, and characterized. Examination of the kinetics of dissociation of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) suggests that G226A Gs alpha is incapable of assuming the conformation necessary for high affinity binding of Mg2+ to the alpha subunit-GTP gamma S complex. Associated changes include the failure of Mg2+ and GTP gamma S to confer resistance to tryptic proteolysis upon the protein, to enhance intrinsic tryptophan fluorescence, or to cause dissociation of alpha from beta gamma. However, the GTPase activity of the mutant protein is near normal (at high Mg2+ concentrations), and the protein is capable of activating adenylylcyclase. A similar defect is present in G49V Gs alpha. Failure of G protein subunit dissociation appears to be the explanation for the phenotypic properties of cells that express G226A Gs alpha, and this mutation thus highlights the crucial nature of this reaction as a component of G protein action.     

Formation and properties of hybrid photosynthetic F1-ATPases. Demonstration of different structural requirements for stimulation and inhibition by tentoxin.

A hybrid ATPase composed of cloned chloroplast ATP synthase beta and gamma subunits (betaC and gammaC) and the cloned alpha subunit from the Rhodospirillum rubrum ATP synthase (alphaR) was assembled using solubilized inclusion bodies and a simple single-step folding procedure. The catalytic properties of the assembled alpha3Rbeta3CgammaC were compared to those of the core alpha3Cbeta3CgammaC complex of the native chloroplast coupling factor 1 (CF1) and to another recently described hybrid enzyme containing R. rubrum alpha and beta subunits and the CF1 gamma subunit (alpha3Rbeta3RgammaC). All three enzymes were similarly stimulated by dithiothreitol and inhibited by copper chloride in response to reduction and oxidation, respectively, of the disulfide bond in the chloroplast gamma subunit. In addition, all three enzymes exhibited the same concentration dependence for inhibition by the CF1 epsilon subunit. Thus the CF1 gamma subunit conferred full redox regulation and normal epsilon binding to the two hybrid enzymes. Only the native CF1 alpha3Cbeta3CgammaC complex was inhibited by tentoxin, confirming the requirement for both CF1 alpha and beta subunits for tentoxin inhibition. However, the alpha3Rbeta3CgammaC complex, like the alpha3Cbeta3CgammaC complex, was stimulated by tentoxin at concentrations in excess of 10 microm. In addition, replacement of the aspartate at position 83 in betaC with leucine resulted in the loss of stimulation in the alpha3Rbeta3CgammaC hybrid. The results indicate that both inhibition and stimulation by tentoxin require a similar structural contribution from the beta subunit, but differ in their requirements for alpha subunit structure.     

Role of the intracellular domain of the beta subunit in Na,K pump function

The catalytic alpha subunit of the (Na,K)- and (H,K)-ATPases needs to be coexpressed with a beta subunit in order to produce cation transport activity. Although the isoform of the beta subunit is known to influence the functional characteristics of the Na,K pump, the role of the different domains of the beta subunit is not fully understood. We have studied the function of a Na,K pump resulting from the expression of a wild-type alpha subunit with a N-terminally truncated mutant of the beta subunit using the two-electrode voltage clamp and the cut-open oocyte techniques. While the maximal activity, measured as the K+-activated outward current, was not significantly altered, the beta N-terminal truncation induced an ouabain-sensitive conductance in the absence of extracellular K+. The voltage dependence of the ouabain-sensitive charge distribution indicated that in the Na/Na exchange conditions, the E1-E2 conformation equilibrium was shifted towards the E2 conformation, a change resulting from alteration of both the forward and the backward reaction rate. Removal of the intracellular domain of the beta subunit modifies several aspects of the whole enzyme function by a mechanism that must imply the state of the extracellular and/or transmembrane parts of the alpha/beta subunit complex.     

Site-specific mutagenesis of the alpha subunit of tryptophan synthase from Salmonella typhimurium. Changing arginine 179 to leucine alters the reciprocal transmission of substrate-induced conformational changes between the alpha and beta 2 subunits

Arginine 179 of the alpha subunit of tryptophan synthase of Salmonella typhimurium was changed to leucine by site-directed mutagenesis. The mutant alpha subunit was expressed in S. typhimurium, purified and crystallized as the alpha 2 beta 2 complex, and characterized by kinetic studies under steady-state reaction conditions. The rate of cleavage of indole 3-glycerol phosphate (alpha reaction) is reduced by 60% in the mutant alpha 2 beta 2 complex, whereas the rate of L-tryptophan synthesis from indole and L-serine (beta reaction) is unchanged. Thus, arginine 179 is not obligatory for catalysis, for binding of indole 3-glycerol phosphate, or for interaction of the alpha and beta 2 subunits. However, changing arginine 179 to leucine does have striking effects on ligand-dependent properties of this multienzyme complex. Ligands of the alpha subunit (DL-alpha-glycerophosphate and indole 3-propanol phosphate) which strongly inhibit the beta reaction of the native alpha 2 beta 2 complex have a slight stimulatory effect on the beta reaction of the mutant alpha 2 beta 2 complex. Likewise, L-serine, a ligand of the beta subunit which produces a 5-fold reduction in the Km for the alpha ligand indole 3-glycerol phosphate in the native alpha 2 beta 2 complex, has no effect on the mutant alpha 2 beta 2 complex. These results suggest that arginine 179 of the alpha subunit plays a role in the reciprocal transmission of substrate-induced conformational changes which occur between native alpha and beta 2 subunits in the alpha 2 beta 2 complex.     

Locking the beta3 integrin I-like domain into high and low affinity conformations with disulfides

Although integrin alpha subunit I domains exist in multiple conformations, it is controversial whether integrin beta subunit I-like domains undergo structurally analogous movements of the alpha7-helix that are linked to affinity for ligand. Disulfide bonds were introduced into the beta(3) integrin I-like domain to lock its beta6-alpha7 loop and alpha7-helix in two distinct conformations. Soluble ligand binding, ligand mimetic mAb binding and cell adhesion studies showed that disulfide-bonded receptor alpha(IIb)beta(3)(T329C/A347C) was locked in a low affinity state, and dithiothreitol treatment restored the capability of being activated to high affinity binding; by contrast, disulfide-bonded alpha(IIb)beta(3)(V332C/M335C) was locked in a high affinity state. The results suggest that activation of the beta subunit I-like domain is analogous to that of the alpha subunit I domain, i.e. that axial movement in the C-terminal direction of the alpha7-helix is linked to rearrangement of the I-like domain metal ion-dependent adhesion site into a high affinity conformation.     

The structure of the chloroplast F1-ATPase at 3.2 A resolution

The structure of the F(1)-ATPase from spinach chloroplasts was determined to 3.2 A resolution by molecular replacement based on the homologous structure of the bovine mitochondrial enzyme. The crystallized complex contains four different subunits in a stoichiometry of alpha(3)beta(3)gammaepsilon. Subunit delta was removed before crystallization to improve the diffraction of the crystals. The overall structure of the noncatalytic alpha-subunits and the catalytic beta-subunits is highly similar to those of the mitochondrial and thermophilic subunits. However, in the crystal structure of the chloroplast enzyme, all alpha- and beta-subunits adopt a closed conformation and appear to contain no bound adenine nucleotides. The superimposed crystallographic symmetry in the space group R32 impaired an exact tracing of the gamma- and epsilon-subunits in the complex. However, clear electron density was present at the core of the alpha(3)beta(3)-subcomplex, which probably represents the C-terminal domain of the gamma-subunit. The structure of the spinach chloroplast F(1) has a potential binding site for the phytotoxin, tentoxin, at the alphabeta-interface near betaAsp(83) and an insertion from betaGly(56)-Asn(60) in the N-terminal beta-barrel domain probably increases the thermal stability of the complex. The structure probably represents an inactive latent state of the ATPase, which is unique to chloroplast and cyanobacterial enzymes.     

Hexagonal structure of two-dimensional crystals of the alpha 3 beta 3 complex thermophilic ATP synthase

The highly dissociable alpha 3 beta 3 subunit complex (Mr = 319,582) of thermophilic ATP synthase was crystallized on a mercury surface under oxygen. The two-dimensional crystal was compared with that of TF1 (Mr = 385,351, alpha 3 beta 3 gamma delta epsilon subunit complex) by means of computer image processing. The crystals showed the same hexagonal lattice (a = b = 10 nm), despite the difference in their molecular weights. The color images of the two protein molecules were also hexagonal. However, there was an open hole in the image of the alpha 3 beta 3 complex, where small subunits (gamma, delta, and epsilon) of TF1 may have been located. The structure of this heterohexamer is consistent with that deduced from other physical parameters.     

Chemical cross-linking studies of chloroplast coupling factor 1.

Cross-linking reagents have been used to link covalently adjacent subunits of solubilized spinach chloroplast coupling factor 1, which is a latent ATPase. 1,5-Difluoro-2,4-dinitrobenzene, dimethyl-3,3'-dithiobispropionimidate, and dimethylsuberimidate are able to form bridges of 3 to 11 A between amino groups, and hydrogen peroxide and the o-phenanthroline-cupric ion complex catalyze the oxidation of intrinsic sulfhydryl groups. The five individual subunit bands (alpha, beta, gamma, delta, and epsilon) and several new aggregate bands can be separated by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The same four fastest moving aggregate bands, as characterized by their mobilities, migrate more slowly than the heaviest subunit band and appear with all of the cross-linkers employed. The subunit composition of the aggregate bands has been determined through the use of the reversible cross-linkers, dimethyldithiobispropionimidate, (o-phenanthroline)2Cu(II), and H2O2, and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis in which aggregates are separated in the first dimension, the disulfide cross-links are cleaved, and the individual subunits present in the aggregates are separated in the second dimension. The subunits are detected by Coomassie brilliant blue staining and by labeling some of the sulfhydryl groups of the gamma and epsilon subunits with radioactive N-ethylmaleimide. The results obtained indicate that the alpha and beta subunits can cross-link directly with each of the other subunits, that two beta subunits are adjacent, and that gamma epsilon, gamma epsilon 2, alpha delta, and beta delta aggregates are present. A minimal subunit stoichiometry consistent with these results is alpha 2 beta 2 gamma delta epsilon 2. A possible structural model of the coupling factor is derived from the data. Similar, but less extensive, experiments have been carried out with the heat-activated coupling factor (which is an ATPase); no differences in the spatial arrangement of subunits are detected from the two-dimensional gel electrophoresis analysis of the cross-linked aggregates.