Alpha 3 beta 3 complex of thermophilic ATP synthase. Catalysis without the gamma-subunit

A complex of the alpha- and beta-subunits of thermophilic ATP synthase showed about 25% of the ATPase activity of the alpha beta gamma complex. The alpha 3 beta 3 hexamer structure was analyzed by sedimentation (11.2 S) and gel filtration (310 kDa). Dilution of the alpha beta complex caused dissociation of the complex and rapid loss of ATPase activity which was restored by addition of the gamma-subunit. A previous method using urea for isolating the subunits resulted in an alpha beta complex with lower activity than that prepared by over-expression of the genes. The alpha beta-ATP complex was formed from the alpha beta complex, ADP and Pi in the presence of dimethyl sulfoxide.     

The alpha 1 beta 1 heterodimer of ATP synthase

The alpha 3 beta 3 hexamer was reconstituted from the alpha and beta subunits of TF1 portion of ATP synthase of thermophilic bacterium (Kagawa et al. (1989) FEBS Lett. 249, 67). The alpha 1 beta 1 heterodimer of ATP synthase was isolated by high performance liquid chromatography (HPLC) of the alpha 3 beta 3 hexamer in the presence of AT(D)P-Mg. On polyacrylamide gel electrophoresis, both bands corresponding to the dimer and hexamer showed ATPase activity. The alpha 1 beta 1 dimer was dissociated into the equal amounts of the alpha and beta monomers by sodium dodecyl sulfate. The alpha and beta monomers were practically inactive. The alpha 2 and beta 2 homodimers were not detected by electrophoresis and HPLC.     

Small-angle X-ray scattering studies of Mg.AT(D)P-induced hexamer to dimer dissociation in the reconstituted alpha 3 beta 3 complex of ATP synthase from thermophilic bacterium PS3.

The alpha 3 beta 3 complex of ATP synthase obtained from a thermophilic bacterium PS3 was isolated and found to show the ATPase activity (Kagawa, Y., Ohta, S., and Otawara-Hamamoto, Y. (1989) FEBS Lett. 249, 67-69). The structure and the nucleotide binding effects of the alpha 3 beta 3 complex were investigated by means of small-angle x-ray scattering and high performance liquid chromatography. The scattering profile from the alpha 3 beta 3 complex was explained with a model in which the complex is made of an ellipsoid of revolution with the axes of 121.8, 121.8, and 72.0 A having an elliptical hollow cavity with the axes of 35.4, 35.4, and 72.0 A. By the addition of Mg.AT(D)P, significant changes in the scattering profile were observed, in which the radius of gyration decreased from 44 to 35 A. This change was found by gel filtration to be caused by the dissociation reaction from the alpha 3 beta 3 hexamer to the alpha beta dimer. The dissociation of the alpha 3 beta 3 complex was not induced by unhydrolyzable ATP analogue, nor by Pi, Mg2+, and Pi + Mg2+. The structure of the dimer was well explained by the triaxial ellipsoidal model with the axes of 105.2, 39.4, and 108.2 A. The dissociation into the dimer is considered to be related to the ATPase activity because the AT(D)P-induced dissociation is observed only in the presence of Mg2+ ions.     

Three-dimensional structure of the tryptophan synthase alpha 2 beta 2 multienzyme complex from Salmonella typhimurium

The three-dimensional structure of the alpha 2 beta 2 complex of tryptophan synthase from Salmonella typhimurium has been determined by x-ray crystallography at 2.5 A resolution. The four polypeptide chains are arranged nearly linearly in an alpha beta beta alpha order forming a complex 150 A long. The overall polypeptide fold of the smaller alpha subunit, which cleaves indole glycerol phosphate, is that of an 8-fold alpha/beta barrel. The alpha subunit active site has been located by difference Fourier analysis of the binding of indole propanol phosphate, a competitive inhibitor of the alpha subunit and a close structural analog of the natural substrate. The larger pyridoxal phosphate-dependent beta subunit contains two domains of nearly equal size, folded into similar helix/sheet/helix structures. The binding site for the coenzyme pyridoxal phosphate lies deep within the interface between the two beta subunit domains. The active sites of neighboring alpha and beta subunits are separated by a distance of about 25 A. A tunnel with a diameter matching that of the intermediate substrate indole connects these active sites. The tunnel is believed to facilitate the diffusion of indole from its point of production in the alpha subunit active site to the site of tryptophan synthesis in the beta active site and thereby prevent its escape to the solvent during catalysis.     

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.     

Microspectrophotometric studies on single crystals of the tryptophan synthase alpha 2 beta 2 complex demonstrate formation of enzyme-substrate intermediates

Microspectrophotometry of single crystals of the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium is used to compare the catalytic and regulatory properties of the enzyme in the soluble and crystalline states. Polarized absorption spectra demonstrate that chromophoric intermediates are formed between pyridoxal phosphate at the active site of the beta subunit and added substrates, substrate analogs, and reaction intermediate analogs. Although the crystalline and soluble forms of the enzyme produce some of the same enzyme-substrate intermediates, including Schiff base and quinonoid intermediates, in some cases the equilibrium distribution of these intermediates differs in the two states of the enzyme. Ligands which bind to the active site of the alpha subunit alter the distribution of intermediates formed at the active site of the beta subunit in both the crystalline and soluble states. The three-dimensional structures of the tryptophan synthase alpha 2 beta 2 complex and of a derivative with indole-3-propanol phosphate bound at the active site of the alpha subunit have recently been reported (Hyde, C. C., Ahmed, S. A., Padlan, E. A., Miles, E. W., and Davies, D. R. (1988) J. Biol. Chem. 264, 17857-17871). Our present findings help to establish experimental conditions for selecting defined intermediates for future x-ray crystallographic analysis of the alpha 2 beta 2 complex with ligands bound at the active sites of both alpha and beta subunits. These crystallographic studies should explain how catalysis occurs at the active site of the beta subunit and how the binding of a ligand to one active site affects the binding of a ligand to the other active site which is 25 A away.     

Conformational Changes in the tryptophan synthase from a hyperthermophile upon alpha2beta2 complex formation: crystal structure of the complex

The three-dimensional structure of the bifunctional tryptophan synthase alpha(2)beta(2) complex from Pyrococcus furiosus was determined by crystallographic analysis. This crystal structure, with the structures of an alpha subunit monomer and a beta(2) subunit dimer that have already been reported, is the first structural set in which changes in structure that occur upon the association of the individual tryptophan synthase subunits were observed. To elucidate the structural basis of the stimulation of the enzymatic activity of each of the alpha and beta(2) subunits upon alpha(2)beta(2) complex formation, the conformational changes due to complex formation were analyzed in detail compared with the structures of the alpha monomer and beta(2) subunit dimer. The major conformational changes due to complex formation occurred in the region correlated with the catalytic function of the enzyme as follows. (1) Structural changes in the beta subunit were greater than those in the alpha subunit. (2) Large movements of A46 and L165 in the alpha subunit due to complex formation caused a more open conformation favoring the entry of the substrate at the alpha active site. (3) The major changes in the beta subunit were the broadening of a long tunnel through which the alpha subunit product (indole) is transferred to the beta active site and the opening of an entrance at the beta active site. (4) The changes in the conformations of both the alpha and beta subunits due to complex formation contributed to the stabilization of the subunit association, which is critical for the stimulation of the enzymatic activities.     

Failure to assemble the alpha 3 beta 3 subcomplex of the ATP synthase leads to accumulation of the alpha and beta subunits within inclusion bodies and the loss of mitochondrial cristae in Saccharomyces cerevisiae

The F(1) component of mitochondrial ATP synthase is an oligomeric assembly of five different subunits, alpha, beta, gamma, delta, and epsilon. In terms of mass, the bulk of the structure ( approximately 90%) is provided by the alpha and beta subunits, which form an (alphabeta)(3) hexamer with adenine nucleotide binding sites at the alpha/beta interfaces. We report here ultrastructural and immunocytochemical analyses of yeast mutants that are unable to form the alpha(3)beta(3) oligomer, either because the alpha or the beta subunit is missing or because the cells are deficient for proteins that mediate F assembly (e.g. Atp11p, Atp12p, or Fmc1p). The F(1) alpha(1) and beta subunits of such mutant strains are detected within large electron-dense particles in the mitochondrial matrix. The composition of the aggregated species is principally full-length F(1) alpha and/or beta subunit protein that has been processed to remove the amino-terminal targeting peptide. To our knowledge this is the first demonstration of mitochondrial inclusion bodies that are formed largely of one particular protein species. We also show that yeast mutants lacking the alpha(3)beta(3) oligomer are devoid of mitochondrial cristae and are severely deficient for respiratory complexes III and IV. These observations are in accord with other studies in the literature that have pointed to a central role for the ATP synthase in biogenesis of the mitochondrial inner membrane.     

Confinement and crowding effects on tryptophan synthase alpha2beta2 complex

Biological molecules experience in vivo a highly crowded environment. The investigation of the functional properties of the tryptophan synthase alpha(2)beta(2) complex either entrapped in wet nanoporous silica gels or in the presence of the crowding agents dextran 70 and ficoll 70 indicates that the rates of the conformational transitions associated to catalysis and regulation are reduced, and an open and less catalytically active conformation is stabilized.     

Isolation and characterisation of a functional alpha beta heterodimer from the ATP synthase of Rhodospirillum rubrum.

An alpha beta heterodimer of the F1-ATPase of Rhodospirillum rubrum was isolated by extraction of chromatophores with LiCl. Each alpha beta heterodimer contains one tightly bound ADP, which is released upon removal of medium Mg2+. The dimer can be reversibly dissociated by removal of Mg(2+)-ions. The alpha beta heterodimer restores both ATP-synthetic and -hydrolytic activities to LiCl-treated chromatophores, saturation being achieved at approximately 2 mmol alpha beta.mol BChl-1. The heterodimer itself hydrolyses Mg-ATP with an activity distinct from RF1, being unaffected by azide or sulphite ions. The Vmax and Km (ATP) for this Mg(2+)-dependent activity were 110 +/- 10 nmol.min-1.mg protein-1 and 100 +/- 30 microM, respectively. The Km did not differ significantly from that of RF1.     

Structure of the gamma-epsilon complex of ATP synthase

ATP synthases (F(1)F(o)-ATPases) use energy released by the movement of protons down a transmembrane electrochemical gradient to drive the synthesis of ATP, the universal biological energy currency. Proton flow through F(o) drives rotation of a ring of c-subunits and a complex of the gamma and epsilon-subunits, causing cyclical conformational changes in F(1) that are required for catalysis. The crystal structure of a large portion of F(1) has been resolved. However, the structure of the central portion of the enzyme, through which conformational changes in F(o) are communicated to F(1), has until now remained elusive. Here we report the crystal structure of a complex of the epsilon-subunit and the central domain of the gamma-subunit refined at 2.1 A resolution. The structure reveals how rotation of these subunits causes large conformational changes in F(1), and thereby provides new insights into energy coupling between F(o) and F(1).     

Structures of the genes encoding the alpha and beta subunits of the Neurospora crassa mitochondrial ATP synthase.

We have isolated and sequenced cDNA and genomic clones encoding the alpha and beta subunits of the Neurospora crassa ATP synthase. The genes are not linked to each other: atp-1(alpha) maps to either linkage group I or V, and atp-2(beta) lies on linkage group II. The two genes resemble each other in having a large number of introns, five in atp-1 and seven in atp-2, mostly positioned near their 5' ends and varying in length from 60-332 bp. The coding regions of both genes have a high G+C content (59%) and use a low number of codons, 46 (atp-1) and 44 (atp-2), a feature associated with highly expressed genes. Northern-blot analysis shows both genes are expressed at high levels during mycelial growth. Comparison of the exon-intron structures of the beta-subunit-encoding gene with those from human and tobacco showed a similar number of introns, several closely positioned, but no exact conservation in position, size or sequence of introns.     

Efficient ATP synthesis by thermophilic Bacillus FoF1-ATP synthase

F(o)F(1)-ATP synthase (F(o)F(1)) synthesizes ATP in the F(1) portion when protons flow through F(o) to rotate the shaft common to F(1) and F(o). Rotary synthesis in isolated F(1) alone has been shown by applying external torque to F(1) of thermophilic origin. Proton-driven ATP synthesis by thermophilic Bacillus PS3 F(o)F(1) (TF(o)F(1)), however, has so far been poor in vitro, of the order of 1 s(-1) or less, hampering reliable characterization. Here, by using a mutant TF(o)F(1) lacking an inhibitory segment of the ε-subunit, we have developed highly reproducible, simple procedures for the preparation of active proteoliposomes and for kinetic analysis of ATP synthesis, which was driven by acid-base transition and K(+)-diffusion potential. The synthesis activity reached ～ 16 s(-1) at 30 °C with a Q(10) temperature coefficient of 3-4 between 10 and 30 °C, suggesting a high level of activity at the physiological temperature of ～ 60 °C. The Michaelis-Menten constants for the substrates ADP and inorganic phosphate were 13 μM and 0.55 mM, respectively, which are an order of magnitude lower than previous estimates and are suited to efficient ATP synthesis.     

5-Azidoindole binding to the Escherichia coli tryptophan synthase alpha 2 beta 2 complex

The tryptophan synthase alpha 2 beta 2 complex catalyzes tryptophan (Trp) biosynthesis from serine plus either indole (IN) or indole-3-glycerol phosphate (InGP). The photoreactive 5-azido analog in IN (AzIN), itself a substrate in the dark, was utilized to examine the substrate binding sites on this enzyme. When irradiated with AzIN at concentrations approaching IN saturation for the IN----Trp activity (0.1 mM), in the absence of serine, the enzyme was increasingly inactivated (up to 70-80%) concomitant with the progressive binding of a net of 2 mol AzIN per alpha beta equivalent. Little or no cooperativity in the binding of the 2 mol AzIN was observed. In contrast, there was minimal effect on the IN----InGP activity. Under these conditions AzIN appeared to be incorporated equally into each subunit. No significant inactivation nor binding occurred in the presence of serine. A quantitatively similar inactivation of InGP----Trp activity was observed over the same AzIN concentration range, suggesting common IN sites for Trp biosynthesis from either indole substrate. At higher concentrations (0.1-0.7 mM), no further inactivation occurred, although there was extensive additional binding (up to 10 mol/alpha beta equivalent). These data are consistent, although more clear-cut quantitatively, with the high- and low-affinity sites proposed from equilibrium dialysis studies. AzIN binding studies utilizing the isolated beta 2 subunit confirmed earlier reports suggesting the existence of many nonspecific IN binding sites on this subunit.     

Nucleotide sequences of the genes for the alpha,beta and epsilon subunits of wheat chloroplast ATP synthase

Summary The nucleotide sequences of the chloroplast genes for the alpha, beta and epsilon subunits of wheat chloroplast ATP synthase have been determined. Open reading frames of 1512 bp, 1494 bp and 411 bp are deduced to code for polypeptides of molecular weights 55201, 53796 and 15200, identified as the alpha, beta and epsilon subunits respectively by homology with the subunits from other sources and by amino acid sequencing of the epsilon subunit. The genes for the beta and epsilon subunits overlap by 4 bp. The gene for methionine tRNA is located 118 bp downstream from the epsilon subunit gene. Comparisons of the deduced amino acid sequences of the alpha and beta subunits with those from other species suggest regions of the proteins involved in adenine nucleotide binding.     

Site-directed cross-linking of b to the alpha, beta, and a subunits of the Escherichia coli ATP synthase

The b subunit dimer of the Escherichia coli ATP synthase, along with the delta subunit, is thought to act as a stator to hold the alpha(3)beta(3) hexamer stationary relative to the a subunit as the gammaepsilonc(9-12) complex rotates. Despite their essential nature, the contacts between b and the alpha, beta, and a subunits remain largely undefined. We have introduced cysteine residues individually at various positions within the wild type membrane-bound b subunit, or within b(24-156), a truncated, soluble version consisting only of the hydrophilic C-terminal domain. The introduced cysteine residues were modified with a photoactivatable cross-linking agent, and cross-linking to subunits of the F(1) sector or to complete F(1)F(0) was attempted. Cross-linking in both the full-length and truncated forms of b was obtained at positions 92 (to alpha and beta), and 109 and 110 (to alpha only). Mass spectrometric analysis of peptide fragments derived from the b(24-156)A92C cross-link revealed that cross-linking took place within the region of alpha between Ile-464 and Met-483. This result indicates that the b dimer interacts with the alpha subunit near a non-catalytic alpha/beta interface. A cysteine residue introduced in place of the highly conserved arginine at position 36 of the b subunit could be cross-linked to the a subunit of F(0) in membrane-bound ATP synthase, implying that at least 10 residues of the polar domain of b are adjacent to residues of a. Sites of cross-linking between b(24-156)A92C and beta as well as b(24-156)I109C and alpha are proposed based on the mass spectrometric data, and these sites are discussed in terms of the structure of b and its interactions with the rest of the complex.