A thioredoxin from the hyperthermophilic archaeon Methanococcus jannaschii has a glutaredoxin-like fold but thioredoxin-like activities

A thioredoxin homologue (Mj0307) from the hyperthermophilic archaeon Methanococcus jannaschii (MjTRX) was cloned, produced in E. coli, and compared to the thioredoxin from E. coli (ETRX). The secondary structure profile of MjTRX obtained by NMR spectroscopy shows that it has four beta-sheets and three alpha-helices arranged in betaalphabetaalphabetabetaalpha, similar to that of glutaredoxin. However, MjTRX supports the growth of T7 bacteriophage in E. coli and is weakly reduced by the thioredoxin reductase from E. coli, indicating that MjTRX is functionally closer to a thioredoxin than a glutaredoxin. MjTRX has higher specific insulin reductase activity than ETRX and retained its full activity over 4 days at 95 degrees C, whereas ETRX lost its activity in 150 min. The standard state redox potential of MjTRX is about -277 mV, which is the lowest value thus far known among redox potentials of the thioredoxin superfamily. This indicates that the lower redox potential is necessary in keeping catalytic disulfide bonds reduced in the cytoplasm and in coping with oxidative stress in an anaerobic hyperthermophile.     

A dominant trifluoperazine resistance gene from Saccharomyces cerevisiae has homology with F0F1 ATP synthase and confers calcium-sensitive growth.

The antipsychotic drug trifluoperazine has been long considered a calmodulin inhibitor from in vitro studies but may function in vivo as a more general inhibitor by disturbing ion fluxes and altering the membrane potential. Resistance to trifluoperazine can arise in Saccharomyces cerevisiae cells by alterations in at least three distinct genetic loci. One locus, defined by a spontaneous dominant trifluoperazine resistance mutation (TFP1-408), was isolated and sequenced. The sequence of the TFP1-408 gene revealed a large open reading frame coding for a large protein of 1,031 amino acids with predicted hydrophobic transmembrane domains. A search of existing amino acid sequences revealed a significant homology with F0F1 ATP synthase. Mutant TFP1-408 cells did not grow efficiently in the presence of 50 mM CaCl2, whereas wild-type cells did. Wild-type cells became resistant to trifluoperazine in the presence of 50 mM CaCl2 or 50 mM MgCl2. Mutant cells showed a higher rate of calcium transport relative to wild-type cells. These data suggest that the TFP1 gene product codes for a transmembrane ATPase-like enzyme possibly involved in Ca2+ transport or in generating a transmembrane ion gradient between two cellular compartments.     

Prediction of transmembrane topology of F0 proteins from Escherichia coli F1F0 ATP synthase using variational and hydrophobic moment analyses.

The a subunit, a membrane protein from the E. coli F1F0 ATP synthase has been examined by Fourier analysis of hydrophobicity and of amino-acid residue variation. The amino-acid sequences of homologous subunits from Vibrio alginolyticus, Saccharomyces cerevisiae, Neurospora crassa, Aspergillus nidulans, Schizosaccharomyces pombe and Candida parapsilosis were used in the variability analysis. By Fourier analysis of sequence variation, two transmembrane helices are predicted to have one face in contact with membrane lipids, while the other spans are predicted to be more shielded from the lipids by protein. By Fourier analysis of hydrophobicity, six amphipathic alpha-helical segments are predicted in extra-membrane regions, including the region from Glu-196 to Asn-214. Fourier analysis of sequence variation in the b- and the c-subunits of the Escherichia coli F1F0 ATP synthase indicates that the single transmembrane span of the b-subunit and the C-terminal span of the c subunit each have a face in contact with membrane lipids. On the basis of this analysis topographical models for the a- and c-subunits and for the F0 complex are proposed.     

New insights into structure-function relationships between archeal ATP synthase (A1A0) and vacuolar type ATPase (V1V0)

Adenosine triphosphate, ATP, is the energy currency of living cells. While ATP synthases of archae and ATP synthases of pro- and eukaryotic organisms operate as energy producers by synthesizing ATP, the eukaryotic V-ATPase hydrolyzes ATP and thus functions as energy transducer. These enzymes share features like the hydrophilic catalytic- and the membrane-embedded ion-translocating sector, allowing them to operate as nano-motors and to transform the transmembrane electrochemical ion gradient into ATP or vice versa. Since archaea are rooted close to the origin of life, the A-ATP synthase is probably more similar in its composition and function to the "original" enzyme, invented by Nature billion years ago. On the contrary, the V-ATPases have acquired specific structural, functional and regulatory features during evolution. This review will summarize the current knowledge on the structure, mechanism and regulation of A-ATP synthases and V-ATPases. The importance of V-ATPase in pathophysiology of diseases will be discussed.     

Molecular mechanisms of rotational catalysis in the F(0)F(1) ATP synthase

Rotation of the F(0)F(1) ATP synthase gamma subunit drives each of the three catalytic sites through their reaction pathways. The enzyme completes three cycles and synthesizes or hydrolyzes three ATP for each 360 degrees rotation of the gamma subunit. Mutagenesis studies have yielded considerable information on the roles of interactions between the rotor gamma subunit and the catalytic beta subunits. Amino acid substitutions, such as replacement of the conserved gammaMet-23 by Lys, cause altered interactions between gamma and beta subunits that have dramatic effects on the transition state of the steady state ATP synthesis and hydrolysis reactions. The mutations also perturb transmission of specific conformational information between subunits which is important for efficient conversion of energy between rotation and catalysis, and render the coupling between catalysis and transport inefficient. Amino acid replacements in the transport domain also affect the steady state catalytic transition state indicating that rotation is involved in coupling to transport.     

Structure of the ATP synthase complex (ECF1F0) of Escherichia coli from cryoelectron microscopy

The structural relationship of the catalytic portion (ECF1) of the Escherichia coli F1F0 ATP synthase (ECF1F0) to the intact, membrane-bound complex has been determined by cryoelectron microscopy and image analysis of single, unordered particles. ECF1F0, reconstituted into membrane structures, has been preserved and examined in its native state in a layer of amorphous ice. Side views of the ECF1F0 show the same elongated bilobed and trilobed projection of the ECF1 views shown previously to be normal to the hexagonal projection. The elongated aqueous cavity of the ECF1 is perpendicular to the membrane bilayer profile in the bilobed view. ECF1 is separated from the membrane-embedded F0 by a narrow stalk approximately 40 A long and approximately 25-30 A thick. The F0 part extends from the lipid bilayer by approximately 10 A on the side facing the ECF1. There is no clear extension of the protein on the opposite side of the membrane.     

Structural interpretations of F(0) rotary function in the Escherichia coli F(1)F(0) ATP synthase

F(1)F(0) ATP synthases are known to synthesize ATP by rotary catalysis in the F(1) sector of the enzyme. Proton translocation through the F(0) membrane sector is now proposed to drive rotation of an oligomer of c subunits, which in turn drives rotation of subunit gamma in F(1). The primary emphasis of this review will be on recent work from our laboratory on the structural organization of F(0), which proves to be consistent with the concept of a c(12) oligomeric rotor. From the NMR structure of subunit c and cross-linking studies, we can now suggest a detailed model for the organization of the c(12) oligomer in F(0) and some of the transmembrane interactions with subunits a and b. The structural model indicates that the H(+)-carrying carboxyl of subunit c is located between subunits of the c(12) oligomer and that two c subunits pack in a front-to-back manner to form the proton (cation) binding site. The proton carrying Asp61 side chain is occluded between subunits and access to it, for protonation and deprotonation via alternate entrance and exit half-channels, requires a swiveled opening of the packed c subunits and stepwise association with different transmembrane helices of subunit a. We suggest how some of the structural information can be incorporated into models of rotary movement of the c(12) oligomer during coupled synthesis of ATP in the F(1) portion of the molecule.     

The Na(+)-translocating F(1)F(0) ATP synthase of Propionigenium modestum: mechanochemical insights into the F(0) motor that drives ATP synthesis

The ATP synthase of Propionigenium modestum encloses a rotary motor involved in the production of ATP from ADP and inorganic phosphate utilizing the free energy of an electrochemical Na(+) ion gradient. This enzyme clearly belongs to the family of F(1)F(0) ATP synthases and uses exclusively Na(+) ions as the physiological coupling ion. The motor domain, F(0), comprises subunit a and the b subunit dimer which are part of the stator and the subunit c oligomer acting as part of the rotor. During ATP synthesis, Na(+) translocation through F(0) proceeds from the periplasm via the stator channel (subunit a) onto a Na(+) binding site of the rotor (subunit c). Upon rotation of the subunit c oligomer versus subunit a, the occupied rotor site leaves the interface with the stator and the Na(+) ion can freely dissociate into the cytoplasm. Recent experiments demonstrate that the membrane potential is crucial for ATP synthesis under physiological conditions. These findings support the view that voltage generates torque in F(0), which drives the rotation of the gamma subunit thus liberating tightly bound ATP from the catalytic sites in F(1). We suggest a mechanochemical model for the transduction of transmembrane Na(+)-motive force into rotary torque by the F(0) motor that can account quantitatively for the experimental data.     

Lengthening the second stalk of F(1)F(0) ATP synthase in Escherichia coli

In Escherichia coli F(1)F(0) ATP synthase, the two b subunits dimerize forming the peripheral second stalk linking the membrane F(0) sector to F(1). Previously, we have demonstrated that the enzyme could accommodate relatively large deletions in the b subunits while retaining function (Sorgen, P. L., Caviston, T. L., Perry, R. C., and Cain, B. D. (1998) J. Biol. Chem. 273, 27873-27878). The manipulations of b subunit length have been extended by construction of insertion mutations into the uncF(b) gene adding amino acids to the second stalk. Mutants with insertions of seven amino acids were essentially identical to wild type strains, and mutants with insertions of up to 14 amino acids retained biologically significant levels of activity. Membranes prepared from these strains had readily detectable levels of F(1)F(0)-ATPase activity and proton pumping activity. However, the larger insertions resulted in decreasing levels of activity, and immunoblot analysis indicated that these reductions in activity correlated with reduced levels of b subunit in the membranes. Addition of 18 amino acids was sufficient to result in the loss of F(1)F(0) ATP synthase function. Assuming the predicted alpha-helical structure for this area of the b subunit, the 14-amino acid insertion would result in the addition of enough material to lengthen the b subunit by as much as 20 A. The results of both insertion and deletion experiments support a model in which the second stalk is a flexible feature of the enzyme rather than a rigid rod-like structure.     

Folding and stability of the b subunit of the F(1)F(0) ATP synthase

The F(1)F(0) ATP synthase is a reversible molecular motor that employs a rotary catalytic cycle to couple a chemiosmotic membrane potential to the formation/hydrolysis of ATP. The multisubunit enzyme contains two copies of the b subunit that form a homodimer as part of a narrow, peripheral stalk structure that connects the membrane (F(0)) and soluble (F(1)) sectors. The three-dimensional structure of the b subunit is unknown making the nature of any interactions or conformational changes within the F(1)F(0) complex difficult to interpret. We have used circular dichroism and analytical ultracentrifugation analyses of a series of N- and C-terminal truncated b proteins to investigate its stability and structure. Thermal denaturation of the b constructs exhibited distinct two-state, cooperative unfolding with T(m) values between 30 and 40 degrees C. CD spectra for the region comprising residues 53-122 (b(53-122)) showed theta;(222)/theta;(208) = 0.99, which reduced to 0.92 in the presence of the hydrophobic solvent trifluoroethanol. Thermodynamic parameters for b(53-122) (DeltaG, DeltaH and DeltaC(p)) were similar to those reported for several nonideal, coiled-coil proteins. Together these results are most consistent with a noncanonical and unstable parallel coiled-coil at the interface of the b dimer.     

The N-terminal region of the Escherichia coli WecA (Rfe) protein, containing three predicted transmembrane helices, is required for function but not for membrane insertion

The correct site for translation initiation for Escherichia coli WecA (Rfe), presumably involved in catalyzing the transfer of N-acetylglucosamine 1-phosphate to undecaprenylphosphate, was determined by using its FLAG-tagged derivatives. The N-terminal region containing three predicted transmembrane helices was found to be necessary for function but not for membrane localization of this protein.     

Conformation-specific antiserum raised against subunit c of ATP synthase (F1F0) from Escherichia coli

Subunit c of the membrane-integrated, proton-translocating F0 portion of the ATP synthase (F1F0) from Escherichia coli has been isolated under nondenaturing conditions (Schneider, E., and Altendorf, K. (1985) EMBO J. 4, 515-518) and antibodies have been raised in rabbits. The primary antisera did not recognize the antigen when present in the same buffer as used for the immunization. Surprisingly, in one of the three antisera a strong antibody binding was observed when intact F0, a.c complex or reconstituted subunit c was provided as the antigen. Incorporation of subunit c into liposomes together with subunits a and b forming an active, H+-translocating complex was not required for the recognition by the antiserum. Subunit c prepared by chloroform/methanol extraction or by chromatography in the presence of sodium dodecyl sulfate was not recognized by the anti-c antiserum when incorporated into liposomes.     

Mechanism of the F(1)F(0)-type ATP synthase, a biological rotary motor

The F(1)F(0)-type ATP synthase is a key enzyme in cellular energy interconversion. During ATP synthesis, this large protein complex uses a proton gradient and the associated membrane potential to synthesize ATP. It can also reverse and hydrolyze ATP to generate a proton gradient. The structure of this enzyme in different functional forms is now being rapidly elucidated. The emerging consensus is that the enzyme is constructed as two rotary motors, one in the F(1) part that links catalytic site events with movements of an internal rotor, and the other in the F(0) part, linking proton translocation to movements of this F(0) rotor. Although both motors can work separately, they must be connected together to interconvert energy. Evidence for the function of the rotary motor, from structural, genetic and biophysical studies, is reviewed here, and some uncertainties and remaining mysteries of the enzyme mechanism are also discussed.     

Chemical modification of the F0 part of the ATP synthase (F1F0) from Escherichia coli. Effects on proton conduction and F1 binding

The purified F0 part of the ATP synthase complex from Escherichia coli was incorporated into liposomes and chemically modified by various reagents. The modified F0-liposomes were assayed for H+ uptake and, after reconstitution with F1, for total and dicyclohexylcarbodiimide-sensitive ATPase activity. The water-soluble carbodiimide, 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide methiodide, (1.2 mM), inhibited H+ uptake to a great extent. Binding of F1 was almost unaffected, but the hydrolysis of ATP was uncoupled from H+ transport. This is reflected by the inhibition of dicyclohexylcarbodiimide-sensitive ATPase activity. Woodward's reagent K, N-ethyl-5-phenylisoxazolium-3'-sulfonate, inhibited both H+ uptake and total ATPase activity. Modification of arginine residues by phenylglyoxal (20 mM) was followed by inhibition of the F1 binding activity by 80% of the control. H+ translocation was reduced to 70%. Diethylpyrocarbonate (3 mM) exhibited a strong inhibiting effect on H+ uptake but not on F1 binding. Modification of tyrosine (by tetranitromethane) as well as lysine residues (by succinic anhydride) did not affect F0 functions. From the data presented we conclude that carboxyl-groups, different from the dicyclohexylcarbodiimide-binding site, are involved in H+ translocation through F0 and, in part, in the functional binding of F1. Furthermore, for the latter function, also arginine residues seem to be important. The role of histidine residues remains unclear at present.     

Proton efflux through the chloroplast ATP synthase (CF0 . CF1) in the presence of sulfhydryl-modifying agents

The rate of photosynthetic electron transport measured in the absence of ADP and Pi is stimulated by low levels of Hg2+ or Ag+ (50% stimulation approximately or equal to 3 Hg2+ or 6 Ag+/100 chlorophyll) to a plateau equal to the transport rate under normal phosphorylating conditions (i.e. +ADP, +Pi). Chloroplasts pretreated in the light under energizing conditions with N-ethylmaleimide show a similar stimulation of non-phosphorylating electron transport. The stimulations of non-phosphorylating electron transport by Hg2+, Ag+ and N-ethylmaleimide are reversed by the CF1 inhibitor phlorizin, the CF0 inhibitor triphenyltin chloride, and can be further stimulated by uncouplers such as methylamine. The Hg2+ and N-ethylmalemide stimulations, but not the Ag+ stimulation, are completely reversed by low levels of ADP (2 microM), ATP (2 microM), AND Pi (400 microM). Ag+, which is a potent inhibitor of ATP synthesis, has little or no effect upon phosphorylating electron transport (+ADP, +Pi). Concomitant with the stimulations of non-phosphorylating electron transport by Hg2+, Ag+ and ADP + Pi, there is a decrease in the level of membrane energization (as measured by atebrin fluorescence quenching) which is reversed when the CF0 channel is blocked by triphenyltin. These results suggest that modification of critical CF1 sulfhydryl residues by Hg2+, Ag+ or N-ethylmalemide leads to the loss of intra-enzyme coupling between the transmembrane proton-transferring and the ATP synthesis activities of the CF0-CF1 ATP synthase complex.     

Defective gamma subunit of ATP synthase (F1F0) from Escherichia coli leads to resistance to aminoglycoside antibiotics.

A strain of Escherichia coli which was derived from a gentamicin-resistant clinical isolate was found to be cross-resistant to neomycin and streptomycin. The molecular nature of the genetic defect was found to be an insertion of two GC base pairs in the uncG gene of the mutant. The insertion led to the production of a truncated gamma subunit of 247 amino acids in length instead of the 286 amino acids that are present in the normal gamma subunit. A plasmid which carried the ATP synthase genes from the mutant produced resistance to aminoglycoside antibiotics when it was introduced into a strain with a chromosomal deletion of the ATP synthase genes. Removal of the genes coding for the beta and epsilon subunits abolished antibiotic resistance coded by the mutant plasmid. The relationship between antibiotic resistance and the gamma subunit was investigated by testing the antibiotic resistance of plasmids carrying various combinations of unc genes. The presence of genes for the F0 portion of the ATP synthase in the presence or absence of genes for the gamma subunit was not sufficient to cause antibiotic resistance. alpha, beta, and truncated gamma subunits were detected on washed membranes of the mutant by immunoblotting. The first 247 amino acid residues of the gamma subunit may be sufficient to allow its association with other F1 subunits in such a way that the proton gate of F0 is held open by the mutant F1.     

Reconstitution of energy-linked activities of the solubilized F1F0 ATP synthase from Bacillus subtilis.

The F1F0 ATP synthases from wild-type Bacillus subtilis and an uncoupler-resistant mutant have comparable subunit structures. In accord with an earlier hypothesis, ATP hydrolysis and ATP-Pi exchange by the two synthases were equally stimulated and inhibited by protonophores, respectively, when reconstituted alone in either wild-type or mutant lipids.     

Energy-dependent conformational changes in the epsilon subunit of the chloroplast ATP synthase (CF0CF1)

Incubation of spinach chloroplast thylakoids with pyridoxal 5'-phosphate modified the epsilon subunit of ATP synthase (CF0CF1). Illumination of thylakoids stimulated the modification of one specific amino acid residue of the epsilon subunit by a factor of 3. Endoproteinase Glu-C treatment of the isolated epsilon subunit and fractionation of the peptides by high performance liquid chromatography revealed a major fluorescent peptide with the sequence GKRQKIE. Further treatment of this peptide with endoproteinase Arg-C gave a strongly fluorescent tripeptide (GXR). From the primary structure of the epsilon subunit, the specifically modified residue was deduced to be Lys-109. This suggests the energy-dependent conformational changes in the epsilon subunit which change the surroundings of Lys-109 and alter the reactivity of this residue.     

The imported preprotein of the proteolipid subunit of the mitochondrial ATP synthase from Neurospora crassa. Molecular cloning and sequencing of the mRNA.

The proteolipid subunit of the mitochondrial ATP synthase from Neurospora crassa is an extremely hydrophobic protein of 81 amino acid residues, which is imported into mitochondria as a precursor of mol. wt. 15 000. The primary structure of the imported form has now been determined by isolating and analyzing cDNA clones of the preproteolipid mRNA. An initial cDNA clone was identified by hybridizing total polyadenylated RNA to pooled cDNA recombinant plasmids from an ordered clone bank and subsequent cell-free translation of hybridization-selected mRNA. Further preproteolipid clones were identified at a frequency of 0.2% by colony filter hybridization. One isolated cDNA represented the major part of the preproteolipid mRNA. The nucleotide sequence showed 243 bases corresponding to the mature proteolipid and, in addition, 178 bases coding for an amino-terminal presequence . Non-coding sequences of 48 bases at the 5' end and of 358 bases at the 3' end plus a poly(A) tail were determined. The long presequence of 66 amino acids is very polar, in contrast to the lipophilic mature proteolipid, and includes 12 basic and no acidic side chains. It is suggested that the presequence is specifically designed to solubilize the proteolipid for post-translational import into the mitochondria.