Ligand-Dependent Structural Changes in the V1 ATPase from Manduca sexta

The response of V(1) ATPase of the tobacco hornworm Manduca sexta to Mg(2+) and nucleotide binding in the presence of the enhancer methanol has been studied by CuCl(2)-induced disulfide formation, fluorescence spectroscopy, and small-angle X-ray scattering. When the V(1) complex was supplemented with CuCl(2) nucleotide-dependence of A-B-E and A-B-E-D cross-linking products was observed in absence of nucleotides and presence of MgADP+Pi but not when MgAMP.PNP or MgADP were added. A zero-length cross-linking product of subunits D and E was formed, supporting their close proximity in the V(1) complex. The catalytic subunit A was reacted with N-4[4-[7-(dimethylamino)-4-methyl]coumarin-3-yl]maleimide (CM) and spectral shifts and changes in fluorescence intensity were detected upon addition of MgAMP.PNP, -ATP, -ADP+Pi, or -ADP. Differences in the fluorescence emission of these nucleotide-binding states were monitored using the intrinsic tryptophan fluorescence. The structural composition of the V(1) ATPase from M. sexta and conformational alterations in this enzyme due to Mg(2+) and nucleotide binding are discussed on the basis of these and previous observations.     

Cysteine-directed cross-linking to subunit B suggests that subunit E forms part of the peripheral stalk of the vacuolar H+-ATPase.

We have employed a combination of site-directed mutagenesis and covalent cross-linking to identify subunits in close proximity to subunit B in the vacuolar H(+)-ATPase (V-ATPase) complex. Unique cysteine residues were introduced into a Cys-less form of subunit B, and the V-ATPase complex in isolated vacuolar membranes from each mutant strain was reacted with the bifunctional, photoactivable maleimide reagent 4-(N-maleimido)benzophenone. Photoactivation resulted in cross-linking of the unique sulfhydryl groups on subunit B with other subunits in the complex. Four of the eight mutants constructed containing a unique cysteine residue at Ala(15), Lys(45), Glu(494), or Thr(501) resulted in the formation of cross-linked products, which were recognized by Western blot analysis using antibodies against both subunits B and E. These products had a molecular mass of 84 kDa, consistent with a cross-linked product of subunits B and E. Molecular modeling of subunit B places Ala(15) and Lys(45) near the top of the V(1) structure (i.e. farthest from the membrane), whereas Glu(494) and Thr(501) are predicted to reside near the bottom of V(1), with all four residues predicted to be oriented toward the external surface of the complex. A model incorporating these and previous data is presented in which subunit E exists in an extended conformation on the outer surface of the A(3)B(3) hexamer that forms the core of the V(1) domain. This location for subunit E suggests that this subunit forms part of the peripheral stalk of the V-ATPase that links the V(1) and V(0) domains.     

Structure of beef heart mitochondrial F1-ATPase. Arrangement of subunits as disclosed by cross-linking reagents and selective labeling by radioactive ligands.

1. The following bifunctional reagents, dimethylsuberimidiate, dimethyladipimidate, methylmercaptobutyrimidate have been used to produce dimers between the neighboring subunits of beef heart F1-ATPase. 2. Treatment of beef heart F1-ATPase with dimethylsuberimidate or dimethyladipimidate resulted in the formation of four cross-linked products. Their molecular weights determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 11 500, 105 000, 95 000 and 80 000, respectively. The products of molecular weight 115 000 and 105 000 were predominant and could be detected at the early stage of the cross-linking reaction. Treatment of beef heart F1-ATPase with methylmercaptobutyrimidate resulted in the accumulation of the product of molecular weight 115 000 and in traces of products of lower molecular weight. When the cross-linked products obtained with methylmercaptobutyrimidate were cleaved by beta-mercaptoethanol, the original gel electrophoresis pattern was restored. 3. Cross-linking of beef heart F1-ATPase by dimethylsuberimidate, dimethyladipimidate and methylmercaptobutyrimidate was accompanied by a loss of the ATPase activity. Cleavage of the cross-linked products obtained with methylmercaptobutyrimidate did not restore the original ATPase activity. 4. Identification of subunits A and B in the products of molecular weight 115 000 and 105 000 was achieved by specific labeling of subunit A with N-[14C]ethylmaleimide and of subunit B by chloronitro [14C]benzooxodiazole. Both products were able to bind N-[14C]ethylmaleimide; only the 105 000 dalton product was able to bind chloronitro [14C]benzooxodiazole. 5. The product of molecular weight 115 000 obtained by treatment of beef heart ATPase with methylmercaptobutyrimidate could bind N-[14C]ethylmaleimide. Its cleavage, following N-[14C]ethylmaleimide binding, yielded one labeled peptide identified with subunit A by polyacrylamide gel electrophoresis. 6. The above results indicate that the product of molecular weight 115 000 is a dimer containing two subunits A and that the product of molecular weight 105 000 is a dimer containing one subunit A and one subunit B. It can therefore be concluded that, in beef heart F1-ATPase, the A subunits are close to each other and that subunit A is close to subunit B. In contrast the B sublnits are probably too far from each other to be cross-linked by dimethylsuberimidate, dimethyladipimidate or methylmercaptobutyrimidate.     

Subunit interactions in the clathrin-coated vesicle vacuolar (H(+))-ATPase complex.

The vacuolar (H(+))-ATPases (or V-ATPases) are structurally related to the F(1)F(0) ATP synthases of mitochondria, chloroplasts and bacteria, being composed of a peripheral (V(1)) and an integral (V(0)) domain. To further investigate the arrangement of subunits in the V-ATPase complex, covalent cross-linking has been carried out on the V-ATPase from clathrin-coated vesicles using three different cross-linking reagents. Cross-linked products were identified by molecular weight and by Western blot analysis using polyclonal antibodies raised against individual V-ATPase subunits. In the intact V(1)V(0) complex, evidence for cross-linking of subunits C and E, D and F, as well as E and G by disuccinimidyl glutarate was obtained, while in the free V(1) domain, cross-linking of subunits H and E was also observed. Subunits C and E as well as D and E could be cross-linked by 1-ethyl-3-(dimethylaminopropyl)carbodiimide, while subunits a and E could be cross-linked by 4-(N-maleimido)benzophenone. It was further demonstrated that it is possible to treat the V-ATPase with potassium iodide and MgATP in such a way that while subunits A, B, and H are nearly quantitatively removed, significant amounts of subunits C, D, E, and F remain attached to the membrane, suggesting that one or more of these latter subunits are in contact with the V(0) domain. In addition, treatment of the V-ATPase with cystine, which modifies Cys-254 of the catalytic A subunit, results in dissociation of subunit H, suggesting communication between the catalytic nucleotide binding site and subunit H. Finally, the stoichiometry of subunits F, G, and H were determined by quantitative amino acid analysis. Based on these and previous observations, a new structural model of the V-ATPase from clathrin-coated vesicles is proposed.     

Three-dimensional organization of the archaeal A1-ATPase from Methanosarcina mazei Gö1

A modified isolation procedure provides a homogeneous A(1)-ATPase from the archaeon Methanosarcina mazei G?1, containing the five subunits in stoichiometric amounts of A(3):B(3):C:D:F. A(1) obtained in this way was characterized by three-dimensional electron microscopy of single particles, resulting in the first three-dimensional reconstruction of an A(1)-ATPase at a resolution of 3.2 nm. The A(1) consists of a headpiece of 10.2 nm in diameter and 10.8 nm in height, formed by the six elongated subunits A(3) and B(3). At the bottom of the A(3)B(3) complex, a stalk of 3.0 nm in length can be seen. The A(3)B(3) domain surrounds a large cavity that extends throughout the length of the A(3)B(3) barrel. A part of the stalk penetrates inside this cavity and is displaced toward an A-B-A triplet. To investigate further the topology of the stalk subunits C-F in A(1), cross-linking has been carried out by using dithiobis[sulfosuccinimidylpropionate] (DSP) and 1-ethyl-3-(dimethylaminopropyl)-carbodiimide (EDC). In experiments where DSP was added the cross-linked products B-F, A(x)-D, A-B-D, and A(x)-B(x)-D were formed. Subunits B-F, A-D, A-B-D, and A-B-C-D could be cross-linked by EDC. The subunit-subunit interaction in the presence of DSP was also studied as a function of nucleotide binding, demonstrating movements of subunits C, D, and F during ATP cleavage. Finally, the three-dimensional organization of this A(1) complex is discussed in terms of the relationship to the F(1)- and V(1)-ATPases at a resolution of 3.2 nm.     

Identification of a domain in the V0 subunit d that is critical for coupling of the yeast vacuolar proton-translocating ATPase

Vacuolar proton-translocating ATPase pumps consist of two domains, V(1) and V(o). Subunit d is a component of V(o) located in a central stalk that rotates during catalysis. By generating mutations, we showed that subunit d couples ATP hydrolysis and proton transport. The mutation F94A strongly uncoupled the enzyme, preventing proton transport but not ATPase activity. C-terminal mutations changed coupling as well; ATPase activity was decreased by 59-72%, whereas proton transport was not measurable (E328A) or was moderately reduced (E317A and C329A). Except for W325A, which had low levels of V(1)V(o), mutations allowed wild-type assembly regardless of the fact that subunits E and d were reduced at the membrane. N- and C-terminal deletions of various lengths were inhibitory and gradually destabilized subunit d, limiting V(1)V(o) formation. Both N and C terminus were required for V(o) assembly. The N-terminal truncation 2-19Delta prevented V(1)V(o) formation, although subunit d was available. The C terminus was required for retention of subunits E and d at the membrane. In addition, the C terminus of its bacterial homolog (subunit C from T. thermophilus) stabilized the yeast subunit d mutant 310-345Delta and allowed assembly of the rotor structure with subunits A and B. Structural features conserved between bacterial and eukaryotic subunit d and the significance of domain 3 for vacuolar proton-translocating ATPase function are discussed.     

Reconstitution of vacuolar-type rotary H+-ATPase/synthase from Thermus thermophilus

Vacuolar-type rotary H(+)-ATPase/synthase (V(o)V(1)) from Thermus thermophilus, composed of nine subunits, A, B, D, F, C, E, G, I, and L, has been reconstituted from individually isolated V(1) (A(3)B(3)D(1)F(1)) and V(o) (C(1)E(2)G(2)I(1)L(12)) subcomplexes in vitro. A(3)B(3)D and A(3)B(3) also reconstituted with V(o), resulting in a holoenzyme-like complexes. However, A(3)B(3)D-V(o) and A(3)B(3)-V(o) did not show ATP synthesis and dicyclohexylcarbodiimide-sensitive ATPase activity. The reconstitution process was monitored in real time by fluorescence resonance energy transfer (FRET) between an acceptor dye attached to subunit F or D in V(1) or A(3)B(3)D and a donor dye attached to subunit C in V(o). The estimated dissociation constants K(d) for V(o)V(1) and A(3)B(3)D-V(o) were ～0.3 and ～1 nm at 25 °C, respectively. These results suggest that the A(3)B(3) domain tightly associated with the two EG peripheral stalks of V(o), even in the absence of the central shaft subunits. In addition, F subunit is essential for coupling of ATP hydrolysis and proton translocation and has a key role in the stability of whole complex. However, the contribution of the F subunit to the association of A(3)B(3) with V(o) is much lower than that of the EG peripheral stalks.     

Catalytic site nucleotide and inorganic phosphate dependence of the conformation of the epsilon subunit in Escherichia coli adenosinetriphosphatase.

The rate of trypsin cleavage of the epsilon subunit of Escherichia coli F1 (ECF1) has been found to be ligand-dependent, as measured indirectly by the activation of the enzyme that occurs on protease digestion, or when followed directly by monitoring the cleavage of this subunit using monoclonal antibodies. The cleavage of the epsilon subunit was fast in the presence of ADP alone, ADP + MG2+, ATP + EDTA, or AMP-PNP, but slow when Pi was added along with ADP + Mg2+ or when ATP + Mg2+ was added to generate ADP + Pi (+Mg2+) in the catalytic site(s). The half-maximal concentration of Pi required in the presence of ADP + Mg2+ to protect the epsilon subunit from cleavage by trypsin was 50 microM, which is in the range measured for the high-affinity binding of Pi to F1. The ligand-dependent conformational changes in the epsilon subunit were also examined in cross-linking experiments using the water-soluble carbodiimide 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). In the presence of ATP + Mg2+ or ADP + Mg2+ + Pi, the epsilon subunit cross-linked to beta in high yield. With ATP + EDTA or ADP + Mg2+ (no Pi), the yield of the beta-epsilon cross-linked product was much reduced. We conclude that the epsilon subunit undergoes a conformational change dependent on the presence of Pi. It has been found previously that binding of the epsilon subunit to ECF1 inhibits ATPase activity by decreasing the off rate of Pi [Dunn, S. D., Zadorozny, V. D., Tozer, R. G., & Orr, L. E. (1987) Biochemistry 26, 4488-4493]. This reciprocal relationship between Pi binding and epsilon-subunit conformation has important implications for energy transduction by the E. coli ATP synthase.     

Structure of the yeast vacuolar ATPase

The subunit architecture of the yeast vacuolar ATPase (V-ATPase) was analyzed by single particle transmission electron microscopy and electrospray ionization (ESI) tandem mass spectrometry. A three-dimensional model of the intact V-ATPase was calculated from two-dimensional projections of the complex at a resolution of 25 angstroms. Images of yeast V-ATPase decorated with monoclonal antibodies against subunits A, E, and G position subunit A within the pseudo-hexagonal arrangement in the V1, the N terminus of subunit G in the V1-V0 interface, and the C terminus of subunit E at the top of the V1 domain. ESI tandem mass spectrometry of yeast V1-ATPase showed that subunits E and G are most easily lost in collision-induced dissociation, consistent with a peripheral location of the subunits. An atomic model of the yeast V-ATPase was generated by fitting of the available x-ray crystal structures into the electron microscopy-derived electron density map. The resulting atomic model of the yeast vacuolar ATPase serves as a framework to help understand the role the peripheral stalk subunits are playing in the regulation of the ATP hydrolysis driven proton pumping activity of the vacuolar ATPase.     

Reconstitution in vitro of V1 complex of Thermus thermophilus V-ATPase revealed that ATP binding to the A subunit is crucial for V1 formation

Vacuolar-type H(+)-ATPase (V-ATPase or V-type ATPase) is a multisubunit complex comprised of a water-soluble V(1) complex, responsible for ATP hydrolysis, and a membrane-embedded V(o) complex, responsible for proton translocation. The V(1) complex of Thermus thermophilus V-ATPase has the subunit composition of A(3)B(3)DF, in which the A and B subunits form a hexameric ring structure. A central stalk composed of the D and F subunits penetrates the ring. In this study, we investigated the pathway for assembly of the V(1) complex by reconstituting the V(1) complex from the monomeric A and B subunits and DF subcomplex in vitro. Assembly of these components into the V(1) complex required binding of ATP to the A subunit, although hydrolysis of ATP is not necessary. In the absence of the DF subcomplex, the A and B monomers assembled into A(1)B(1) and A(3)B(3) subcomplexes in an ATP binding-dependent manner, suggesting that ATP binding-dependent interaction between the A and B subunits is a crucial step of assembly into V(1) complex. Kinetic analysis of assembly of the A and B monomers into the A(1)B(1) heterodimer using fluorescence resonance energy transfer indicated that the A subunit binds ATP prior to binding the B subunit. Kinetics of binding of a fluorescent ADP analog, N-methylanthraniloyl ADP (mant-ADP), to the monomeric A subunit also supported the rapid nucleotide binding to the A subunit.     

Mutational analysis of the stator subunit E of the yeast V-ATPase

Subunit E is a component of the peripheral stalk(s) that couples membrane and peripheral subunits of the V-ATPase complex. In order to elucidate the function of subunit E, site-directed mutations were performed at the amino terminus and carboxyl terminus. Except for S78A and D233A/T202A, which exhibited V(1)V(o) assembly defects, the function of subunit E was resistant to mutations. Most mutations complemented the growth phenotype of vma4Delta mutants, including T6A and D233A, which only had 25% of the wild-type ATPase activity. Residues Ser-78 and Thr-202 were essential for V(1)V(o) assembly and function. The mutation S78A destabilized subunit E and prevented assembly of V(1) subunits at the membranes. Mutant T202A membranes exhibited 2-fold increased V(max) and about 2-fold less of V(1)V(o) assembly; the mutation increased the specific activity of V(1)V(o) by enhancing the k(cat) of the enzyme 4-fold. Reduced levels of V(1)V(o) and V(o) complexes at T202A membranes suggest that the balance between V(1)V(o) and V(o) was not perturbed; instead, cells adjusted the amount of assembled V-ATPase complexes in order to compensate for the enhanced activity. These results indicated communication between subunit E and the catalytic sites at the A(3)B(3) hexamer and suggest potential regulatory roles for the carboxyl end of subunit E. At the carboxyl end, alanine substitution of Asp-233 significantly reduced ATP hydrolysis, although the truncation 229-233Delta and the point mutation K230A did not affect assembly and activity. The implication of these results for the topology and functions of subunit E within the V-ATPase complex are discussed.     

The B1 subunit of the H+ATPase is a PDZ domain-binding protein. Colocalization with NHE-RF in renal B-intercalated cells

The 56-kDa B1 subunit of the vacuolar H(+)ATPase has a C-terminal DTAL amino acid motif typical of PDZ-binding proteins that associate with the PDZ protein, NHE-RF (Na(+)/H(+) exchanger regulatory factor). This B1 isoform is amplified in renal intercalated cells, which play a role in distal urinary acid-base transport. In contrast, proximal tubules express the B2 isoform that lacks the C-terminal PDZ-binding motif. Both the B1 56-kDa subunit and the 31-kDa (E) subunit of the H(+)ATPase are pulled down by glutathione S-transferase NHE-RF bound to GSH-Sepharose beads. These subunits associate in vivo as part of the cytoplasmic V1 portion of the H(+)ATPase, and the E subunit was co-immunoprecipitated from rat kidney cytosol with NHE-RF antibodies. The interaction of H(+)ATPase subunits with NHE-RF was inhibited by a peptide derived from the C terminus of the B1 but not the B2 isoform. NHE-RF colocalized with H(+)ATPase in either the apical or the basolateral region of B-type intercalated cells, whereas NHE-RF staining was undetectable in A-intercalated cells. In proximal tubules, NHE-RF was located in the apical brush border. In contrast, H(+)ATPase was concentrated in a distinct membrane domain at the base of the brush border, from which NHE-RF was absent, consistent with the expression of the truncated B2 subunit isoform in this tubule segment. The colocalization of NHE-RF and H(+)ATPase in B- but not A-intercalated cells suggests a role in generating, maintaining, or modulating the variable H(+)ATPase polarity that characterizes the B-cell phenotype.     

Expression,purification, and characterization of subunit E,an essential subunit of the vacuolar ATPase

A recombinant form of subunit E (Vma4p) from yeast vacuolar ATPases (V-ATPases) has been overexpressed in Escherichia coli, purified to homogeneity, and explored by mass spectrometry. Analysis of the secondary structure of Vma4p by circular dichroism spectroscopy indicated 32% alpha-helix and 23% beta-sheet content. Vma4p formed a hybrid-complex with the nucleotide-binding subunits alpha and beta of the closely related F(1) ATPase of the thermophilic bacterium PS3 (TF(1)). The alpha(3)beta(3)E-hybrid-complex had 56% of the ATPase activity of the native TF(1). By comparison, an alpha(3)beta(3)-formation without Vma4p showed about 24% of total TF(1) ATPase activity. This is the first demonstration of a hydrolytically active hybrid-complex consisting of F(1) and V(1) subunits. The arrangement of subunit E in V(1) has been probed using the recombinant Vma4p, the alpha(3)beta(3)E-hybrid-complex together with V(1) and an A(3)B(3)HEG-subcomplex of the V(1) ATPase from Manduca sexta, respectively, indicating that subunit E is shielded in V(1).     

Propagation of Acto-S-1 ATPase reaction-coupled conformational change in actin along the filament

F-Actin was partially cross-linked to myosin subfragment-1 (S-1) at various molar ratios (r = S-1/actin) with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The cross-linked acto-S-1 ATPase showed so called "super-activation," Vx. S-1 was added further to the cross-linked acto-S-1 and the ATPase activity, Vy, was measured. Since the added S-1 can interact only with the bare actin protomers within the cross-linked actin filament, the difference, delta V = Vy - Vx - Vs (where Vs is the ATPase activity of the additional S-1 alone), can indicate the state of the bare actin protomers while the cross-linked acto-S-1 is hydrolyzing ATP. With increasing r, delta V decreased much more rapidly than delta Vo(1 - r) (where delta Vo is delta V at r = 0) and reached a minimum around r = 0.15. As r increased further, delta V approached the level of delta Vo(1 - r). When SH1/SH2-blocked S-1 was cross-linked to F-actin, delta V decreased according to delta Vo(1 - r). Therefore, the large reduction of delta V, observed when intact S-1 was cross-linked, was coupled to the high ATPase activity of the cross-linked acto-S-1. Combining these data with other kinetic data, we could deduce that structural distortion in a cross-linked actin induced by the ATPase reaction of the S-1 partner propagated over several bare actin protomers along the filament and reduced their affinity for the S-1-ADP-Pi complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cross-talk in the A1-ATPase from Methanosarcina mazei Go1 due to nucleotide binding

Changes in the A(3)B(3)CDF-complex of the Methanosarcina mazei G?1 A(1)-ATPase in response to ligand binding have been studied by small-angle x-ray scattering, protease digestion, fluorescence spectroscopy, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and CuCl(2)-induced disulfide formation. The value of the radius of gyration, R(g), increases slightly when MgATP, MgADP, or MgADP + P(i) (but not MgAMP-PNP) is present. The nucleotide-binding subunits A and B were reacted with N-4[4-[7-(dimethylamino)-4-methyl]coumarin-3-yl]maleimide, and spectral shifts and changes in fluorescence intensity were detected upon addition of MgAMP-PNP, MgATP, MgADP + P(i), or MgADP. Trypsin treatment of A(1) resulted in cleavage of the stalk subunits C and F, which was rapid in the presence of MgAMP-PNP but slow when MgATP or MgADP were added to the enzyme. When A(1) was supplemented with CuCl(2) a clear nucleotide dependence of an A-A-D cross-linking product was generated in the presence of MgADP and MgATP but not when MgAMP-PNP or MgADP + P(i) was added. The site of cross-link formation was located in the region of the N and C termini of subunit D. The data suggest that the stalk subunits C, D, and F in A(1) undergo conformational changes during ATP hydrolysis.     

Distinct expression patterns of different subunit isoforms of the V-ATPase in the rat epididymis

In the epididymis and vas deferens, the vacuolar H(+)ATPase (V-ATPase), located in the apical pole of narrow and clear cells, is required to establish an acidic luminal pH. Low pH is important for the maturation of sperm and their storage in a quiescent state. The V-ATPase also participates in the acidification of intracellular organelles. The V-ATPase contains many subunits, and several of these subunits have multiple isoforms. So far, only subunits ATP6V1B1, ATP6V1B2, and ATP6V1E2, previously identified as B1, B2, and E subunits, have been described in the rat epididymis. Here, we report the localization of V-ATPase subunit isoforms ATP6V1A, ATP6V1C1, ATP6V1C2, ATP6V1G1, ATP6V1G3, ATP6V0A1, ATP6V0A2, ATP6V0A4, ATP6V0D1, and ATP6V0D2, previously labeled A, C1, C2, G1, G3, a1, a2, a4, d1, and d2, in epithelial cells of the rat epididymis and vas deferens. Narrow and clear cells showed a strong apical staining for all subunits, except the ATP6V0A2 isoform. Subunits ATP6V0A2 and ATP6V1A were detected in intracellular structures closely associated but not identical to the TGN of principal cells and narrow/clear cells, and subunit ATP6V0D1 was strongly expressed in the apical membrane of principal cells in the apparent absence of other V-ATPase subunits. In conclusion, more than one isoform of subunits ATP6V1C, ATP6V1G, ATP6V0A, and ATP6V0D of the V-ATPase are present in the epididymal and vas deferens epithelium. Our results confirm that narrow and clear cells are well fit for active proton secretion. In addition, the diverse functions of the V-ATPase may be established through the utilization of specific subunit isoforms. In principal cells, the ATP6V0D1 isoform may have a physiological function that is distinct from its role in proton transport via the V-ATPase complex.     

V-Type H+-ATPase/synthase from a thermophilic eubacterium, Thermus thermophilus. Subunit structure and operon

V-type ATPase (V(o)V(1)) capable of ATP-driven H(+) pumping and of H(+) gradient driven ATP synthesis was isolated from a thermophilic eubacterium, Thermus thermophilus. When the enzyme was analyzed by gel electrophoresis in the presence of sodium dodecyl sulfate, it showed eight polypeptide bands of which four were subunits of V(1). We also isolated the V(o)V(1) operon, containing nine genes in the order of atpG-I-L-E-X-F-A-B-D, which encoded proteins with molecular sizes of 13, 43, 10, 20, 35, 11, 64, 53, and 25 kDa, respectively. The last four genes were identified as those for V(1) subunits; atpA, B, D, and F encoded the A, B, gamma, and delta subunits, respectively. The first five genes, atpG-atpX, were identified as genes for the V(o) subunits. The product of atpL, the proteolipid subunit, lacked a 19-amino acid presequence and, unlike V-type ATPases, contained two membrane-spanning domains rather than four. The hydrophobic 43-kDa product of atpI is the smallest member so far found of the eukaryotic 100-kDa subunit family. Its electrophoretic band overlapped with the band of the A subunit. Therefore, all the gene products were found in our purified V(o)V(1). We isolated the A(3)B(3) subcomplex reconstituted from the isolated subunits and the A(3)B(3)gamma subcomplex from subunit-expressing Escherichia coli. Electron microscopic observation of these subcomplexes revealed that the gamma subunit of V(1) filled the central cavity of A(3)B(3) and might be central subunit, similar to the gamma subunit of F(1)-ATPase.     

Oxidation of the alpha(3)(betaD311C/R333C)(3)gamma subcomplex of the thermophilic Bacillus PS3 F(1)-ATPase indicates that only two beta subunits can exist in the closed conformation simultaneously.

In the crystal structure of the bovine heart mitochondrial F(1)-ATPase (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628), the two liganded beta subunits, one with MgAMP-PNP bound to the catalytic site (beta(T)) and the other with MgADP bound (beta(D)) have closed conformations. The empty beta subunit (beta(E)) has an open conformation. In beta(T) and beta(D), the distance between the carboxylate of beta-Asp(315) and the guanidinium of beta-Arg(337) is 3.0-4.0 A. These side chains are at least 10 A apart in beta(E). The alpha(3)(betaD311C/R333C)(3)gamma subcomplex of TF(1) with the corresponding residues substituted with cysteine has very low ATPase activity unless it is reduced prior to assay or assayed in the presence of dithiothreitol. The reduced subcomplex hydrolyzes ATP at 50% the rate of wild-type and is rapidly inactivated by oxidation by CuCl(2) with or without magnesium nucleotides bound to catalytic sites. Titration of the subcomplex with iodo[(14)C]acetamide after prolonged treatment with CuCl(2) in the presence or absence of 1 mM MgADP revealed nearly two free sulfhydryl groups/mol of enzyme. Therefore, one pair of introduced cysteines is located on a beta subunit that exists in the open or partially open conformation even when catalytic sites are saturated with MgADP. Since V(max) of ATP hydrolysis is attained when three catalytic sites of F(1) are saturated, the catalytic site that binds ATP must be closing as the catalytic site that releases products is opening.     

Interaction of Escherichia coli adenosine triphosphatase with aurovertin and citreoviridin: inhibition and fluorescence studies.

Aurovertins B and D inhibited the adenosine triphosphatase (ATPase) activity of soluble Escherichia coli coupling factor ATPase (BF1) isolated from wile-type E. coli K-12. Half inhibition was obtained with 2 microns aurovertin B and 0.9 microns aurovertin D. Aurovertins B and D had no inhibitory effect on BF1 isolated from the aurovertin-resistant E. coli mutant MA12. Acetylation or saponification of aurovertin D yielded a derivative which was devoid of inhibitory effect on BF1. Citreoviridin also inhibited wild-type BF1 but with much less efficiency (half inhibition at 60 microns) than aurovertin. Citreoviridin had no effect on the aurovertin-resistant BF1. The fluorescence intensity of aurovertins B and D was markedly enhanced upon addition to purified BF1. There was no enhancement of fluorescence when the aurovertins were added to BF1 isolated from the aurovertin-resistant mutant. The fluorescence of the aurovertin-BF1 complex was enhanced by adenosine 5'-diphosphate and by low concentrations of adenosine 5'-triphosphate. The adenosine 5'-diphosphate-enhanced fluorescence of the aurovertin-BF1 complex was quenched by high concentrations of adenosine 5'-triphosphate or by MG2+. Aurovertin bound selectively to the beta subunit of BF1 isolated from wile-type cells. By complementation assays in vitro, using a reconstituted system made of subunits isolated from wild-type and aurovertin-resistant BF1, it was shown that the altered peptide in aurovertin-resistant BF1 was the beta subunit.     

Nucleotide-dependent and dicyclohexylcarbodiimide-sensitive conformational changes in the epsilon subunit of Escherichia coli ATP synthase.

The rate of trypsin cleavage of the epsilon subunit of Escherichia coli F1F0 (ECF1F0) is shown to be ligand-dependent as measured by Western analysis using monoclonal antibodies. The cleavage of the epsilon subunit was rapid in the presence of ADP alone, ATP + EDTA, or AMP-PNP + Mg2+, but slow when Pi was added along with ADP + Mg2+ or when ATP + Mg2+ was added to generate ADP + Pi (+Mg2+) in the catalytic site. Trypsin treatment of ECF1Fo was also shown to increase enzymic activity on a time scale corresponding to that of the cleavage of the epsilon subunit, indicating that the epsilon subunit inhibits ATPase activity in ECF1Fo. The ligand-dependent conformational changes in the epsilon subunit were also examined in cross-linking experiments using the water-soluble carbodiimide 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide (EDC). In the presence of ATP + Mg2+ or ADP + Pi + Mg2+, the epsilon subunit cross-linked product was much reduced. Prior reaction of ECF1Fo with dicyclohexylcarbodiimide (DCCD), under conditions in which only the Fo part was modified, blocked the conformational changes induced by ligand binding. When the enzyme complex was reacted with DCCD in ATP + EDTA, the cleavage of the epsilon subunit was rapid and yield of cross-linking of beta to epsilon subunit low, whether trypsin cleavage was conducted in ATP + EDTA or ATP + Mg2+. When enzyme was reacted with DCCD in ATP + Mg2+, cleavage of the epsilon subunit was slow and yield of cross-linking of beta to epsilon high, under all nucleotide conditions for proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)