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R A Capaldi

Publications and source records attributed to R A Capaldi.

At least 37 records · Page 2Linked to original sources
PubMed1999

The gammaepsilon-c subunit interface in the ATP synthase of Escherichia coli. cross-linking of the epsilon subunit to the c subunit ring does not impair enzyme function, that of gamma to c subunits leads to uncoupling.

Mutants with a cysteine residue in the gamma subunit at position 207 and the epsilon subunit at position 31 were expressed in combination with a c-dimer construct, which contains a single cysteine at position 42 of the second c subunit. These mutants are called gammaY207C/cc'Q42C and epsilonE31C/cc'Q42C, respectively. Cross-linking of epsilon to the c subunit ring was obtained almost to completion without significant effect on any enzyme function, i.e. ATP hydrolysis, ATP synthesis, and ATP hydrolysis-driven proton translocation were all close to that of wild type. The gamma subunit could also be linked to the c subunit ring in more than 90% yield, but this affected coupling. Thus, ATP hydrolysis was increased 2. 5-fold, ATP synthesis was dramatically decreased, and ATP hydrolysis-driven proton translocation was abolished, as measured by the 9-amino-6-chloro-2-methoxyacridinequenching method. These results for epsilonE31C/cc'Q42C indicate that the c subunit ring rotates with the central stalk element. That the gamma-epsilon cross-linked enzyme retains ATPase activity also argues for a gammaepsilon-c subunit rotor. However, the uncoupling induced by cross-linking of gamma to the c subunit ring points to important conformational changes taking place in the gammaepsilon-c subunit interface during this. Blocking these structural changes by cross-linking leads to a proton leak within the F(0).

Amino Acid Substitution↗
PubMed1999

Structural features of the gamma subunit of the Escherichia coli F(1) ATPase revealed by a 4.4-A resolution map obtained by x-ray crystallography.

The F(1) part of the F(1)F(O) ATP synthase from Escherichia coli has been crystallized and its structure determined to 4.4-A resolution by using molecular replacement based on the structure of the beef-heart mitochondrial enzyme. The bacterial F(1) consists of five subunits with stoichiometry alpha(3), beta(3), gamma, delta, and epsilon. delta was removed before crystallization. In agreement with the structure of the beef-heart mitochondrial enzyme, although not that from rat liver, the present study suggests that the alpha and beta subunits are arranged in a hexagonal barrel but depart from exact 3-fold symmetry. In the structures of both beef heart and rat-liver mitochondrial F(1), less than half of the structure of the gamma subunit was seen because of presumed disorder in the crystals. The present electron-density map includes a number of rod-shaped features which appear to correspond to additional alpha-helical regions within the gamma subunit. These suggest that the gamma subunit traverses the full length of the stalk that links the F(1) and F(O) parts and makes significant contacts with the c subunit ring of F(O).

Amino Acid Sequence↗
PubMed1999

The epsilon subunit of the F(1)F(0) complex of Escherichia coli. cross-linking studies show the same structure in situ as when isolated.

Four double mutants in the epsilon subunit were generated, each containing two cysteines, which, based on the NMR structure of this subunit, should form internal disulfide bonds. Two of these were designed to generate interdomain cross-links that lock the C-terminal alpha-helical domain against the beta-sandwich (epsilonM49C/A126C and epsilonF61C/V130C). The second set should give cross-linking between the two C-terminal alpha-helices (epsilonA94C/L128C and epsilonA101C/L121C). All four mutants cross-linked with 90-100% efficiency upon CuCl(2) treatment in isolated Escherichia coli ATP synthase. This shows that the structure obtained for isolated epsilon is essentially the same as in the assembled complex. Functional studies revealed increased ATP hydrolysis after cross-linking between the two domains of the subunit but not after cross-linking between the C-terminal alpha-helices. None of the cross-links had any effect on proton pumping-coupled ATP hydrolysis, on DCCD sensitivity of this activity, or on ATP synthesis rates. Therefore, big conformational changes within epsilon can be ruled out as a part of the enzyme function. Protease digestion studies, however, showed that subtle changes do occur, since the epsilon subunit could be locked in an ADP or 5'-adenylyl-beta,gamma-imidodiphosphate conformation by the cross-linking with resulting differences in cleavage rates.

Adenine Nucleotides↗
PubMed1999

Mutation of the mitochrondrially encoded ATPase 6 gene modeled in the ATP synthase of Escherichia coli.

Defects of respiratory chain protein complexes and the ATP synthase are becoming increasingly implicated in human disease. Recently, mutations in the ATPase 6 gene have been shown to cause several different neurological disorders. The product of this gene is homologous to the a subunit of the ATP synthase of Escherichia coli. Here, mutations equivalent to those described in humans have been introduced into the a subunit of E. coli by site-directed mutagenesis, and the effects of these mutations on the ATPase activity, ATP synthesis and ability of the enzyme to pump protons studied in detail. The effects of the mutations varied considerably. The mutation L262P (9185 T-C equivalent) caused a 70% loss of ATP synthesis activity, reduced DCCD sensitivity, and lowered proton pumping activity. The L207P (8993 T-C equivalent) reduced ATP synthesis by 50%, affected DCCD sensitivity, while proton pumping was only marginally affected when measured by the standard AMCA quenching assay. The other mutations studied affected the functioning of the ATP synthase much less. The results confirm that modeling of these point mutations in the E. coli enzyme is a useful approach to determining how alterations in the ATPase 6 gene affect enzyme function and, therefore, how a pathogenic effect can be exerted.

Bacterial Proteins↗
PubMed1999

A missense mutation of cytochrome oxidase subunit II causes defective assembly and myopathy.

We report the first missense mutation in the mtDNA gene for subunit II of cytochrome c oxidase (COX). The mutation was identified in a 14-year-old boy with a proximal myopathy and lactic acidosis. Muscle histochemistry and mitochondrial respiratory-chain enzymology demonstrated a marked reduction in COX activity. Immunohistochemistry and immunoblot analyses with COX subunit-specific monoclonal antibodies showed a pattern suggestive of a primary mtDNA defect, most likely involving CO II, for COX subunit II (COX II). mtDNA-sequence analysis demonstrated a novel heteroplasmic T-->A transversion at nucleotide position 7,671 in CO II. This mutation changes a methionine to a lysine residue in the middle of the first N-terminal membrane-spanning region of COX II. The immunoblot studies demonstrated a severe reduction in cross-reactivity, not only for COX II but also for the mtDNA-encoded subunit COX III and for nuclear-encoded subunits Vb, VIa, VIb, and VIc. Steady-state levels of the mtDNA-encoded subunit COX I showed a mild reduction, but spectrophotometric analysis revealed a dramatic decrease in COX I-associated heme a3 levels. These observations suggest that, in the COX protein, a structural association of COX II with COX I is necessary to stabilize the binding of heme a3 to COX I.

Acidosis, Lactic↗
PubMed1998

The second stalk composed of the b- and delta-subunits connects F0 to F1 via an alpha-subunit in the Escherichia coli ATP synthase.

The b- and delta-subunits of the Escherichia coli ATP synthase are critical for binding ECF1 to the F0 part, and appear to constitute the stator necessary for holding the alpha3beta3 hexamer as the c-epsilon-gamma domain rotates during catalysis. Previous studies have determined that the b-subunits are dimeric for a large part of their length, and interact with the F1 part through the delta-subunit (Rodgers, A. J. W., Wilkens, S., Aggeler, R., Morris, M. B., Howitt, S. M., and Capaldi, R. A. (1997) J. Biol. Chem. 272, 31058-31064). To further study b-subunit interactions, three mutants were constructed in which Ser-84, Ala-144, and Leu-156, respectively, were replaced by Cys. Treatment of purified ECF1F0 from all three mutants with CuCl2 induced disulfide formation resulting in b-subunit dimer cross-link products. In addition, the mutant bL156C formed a cross-link from a b-subunit to an alpha-subunit via alphaCys90. Neither b-b nor b-alpha cross-linking had significant effect on ATPase activities in any of the mutants. Proton pumping activities were measured in inner membranes from the three mutants. Dimerization of the b-subunit did not effect proton pumping in mutants bS84C or bA144C. In the mutant bL156C, CuCl2 treatment reduced proton pumping markedly, probably because of uncoupling caused by the b-alpha cross-link formation. The results show that the alpha-subunit forms part of the binding site on ECF1 for the b2delta domain and that the b-subunit extends all the way from the membrane to the top of the F1 structure. Some conformational flexibility in the connection between the second stalk and F1 appears to be required for coupled catalysis.

Base Sequence↗
PubMed1998

Solution structure of the epsilon subunit of the F1-ATPase from Escherichia coli and interactions of this subunit with beta subunits in the complex.

The solution structure of the epsilon subunit of the Escherichia coli F1-ATPase has been determined by NMR spectroscopy. This subunit has a two-domain structure with an N-terminal 10-stranded beta sandwich and a C-terminal antiparallel two alpha-helix hairpin, as described previously (Wilkens, S., Dahlquist, F. W., McIntosh, L. P., Donaldson, L. W., and Capaldi, R. A. (1995) Nat. Struct. Biol. 2, 961-967). New data show that the two domains interact in solution in an interface formed by beta strand 7 and the very C-terminal alpha-helix. This interface involves only hydrophobic interactions. The dynamics of the epsilon subunit in solution were examined. The two domains are relatively tightly associated with little or no flexibility relative to one another. The epsilon subunit can exist in two states in the ECF1F0 complex depending on whether ATP or ADP occupies catalytic sites. Proteolysis of the epsilon subunit in solution and when bound to the core F1 complex indicates that the conformation of the polypeptide in solution closely resembles the conformation of epsilon when bound to the F1 in the ADP state. Chemical and photo-cross-linking show that the epsilon subunit spans and interacts with two beta subunits in the ADP state. These interactions are disrupted on binding of ATP + Mg2+, as is the interaction between the N- and C-terminal domains of the epsilon subunit.

Escherichia coli↗
PubMed1998

Unisite catalysis without rotation of the gamma-epsilon domain in Escherichia coli F1-ATPase.

Unisite [gamma-32P]ATP hydrolysis was studied in ECF1 from the mutant betaE381C after generating a single disulfide bond between beta and gamma subunits to prevent the rotation of the gamma/epsilon domain. The single beta-gamma cross-link was obtained by removal of the delta subunit from F1 and then treating with CuCl2 as described previously (Aggeler, R., Haughton, M. A., and Capaldi, R. A. (1996) J. Biol. Chem. 270, 9185-9191). The mutant enzyme, betaE381C, had an increased overall rate of unisite hydrolysis of [gamma-32P]ATP compared with the wild type ECF1 due to increases in the rate of ATP binding (k+1), Pi release (k+3), and ADP release (k+4). Release of bound substrate ([gamma-32P]ATP) was also increased in the betaE381C mutant. Cross-linking between Cys-381 and the intrinsic Cys-87 of gamma caused a further increase in the rate of unisite catalysis, mainly by additional effects on nucleotide binding in the high affinity catalytic site (k+1 and k+4). In delta-subunit-free ECF1 from wild type or betaE381C F1, addition of an excess of ATP accelerated unisite catalysis. After cross-linking, unisite catalysis of betaE381C was not enhanced by the cold chase. The covalent linkage of gamma to beta increased the rate of unisite catalysis to that obtained by cold chase of ATP of the noncross-linked enzyme. It is concluded that the conversion of Glu-381 of beta to Cys induces an activated conformation of the high affinity catalytic site with low affinity for substrate and products. This state is stabilized by cross-linking the Cys at beta381 to Cys-87 of gamma. We infer from the data that rotation of the gamma/epsilon rotor in ECF1 is not linked to unisite hydrolysis of ATP at the high affinity catalytic site but to ATP binding to a second or third catalytic site on the enzyme.

Adenosine Triphosphate↗
PubMed1998

Electron microscopic evidence of two stalks linking the F1 and F0 parts of the Escherichia coli ATP synthase.

The structure of monodisperse ATP synthase from Escherichia coli (ECF1F0) has been examined by electron microscopy after negative staining of specimens. The F1 part is seen to be connected by two stalks. One is more centrally located and includes the gamma and epsilon subunits. The second stalk, observed here in ECF1F0, is arranged peripherally. It probably contains the delta and b subunits which, in addition to gamma and epsilon, are required for binding of the F1 and F0 parts of the complex. Other novel features of the F1F0 complex can be discerned. There is a cap at the top of the F1 part at which the second stalk may bind. This likely includes N-terminal stretches of the three copies of the alpha subunit and a part of the delta subunit. The F0 part is clearly asymmetric. The presence of two stalks in the complex has important functional implications. There is good evidence that the more central stalk of gamma and epsilon subunits is a mobile domain that rotates to link the three catalytic sites on beta subunits in turn, with the proton channel of the F0 part. The second stalk of delta and b subunits is then the stator which makes this rotation possible.

Animals↗
PubMed1998

Trapping of conformations of the Escherichia coli F1 ATPase by disulfide bond formation. A state of the enzyme with all three catalytic sites of equal and low affinity for nucleotides.

A mutant of Escherichia coli F1F0-ATPase, alphaS411C/betaY331W/betaE381C/gammaC87S, has been generated. CuCl2 treatment of this mutant led to cross-linking between alpha and beta subunits in yields of up to 90%. This cross-linking across non-catalytic site interfaces inhibited ATP hydrolysis activity. In the absence of cross-linking, MgATP bound in catalytic sites of the mutant with three different affinities of 0.1 microM, 6 microM and 60 microM, respectively, values that are comparable to wild-type. For MgADP, there was one tight site (0.34 microM) and two sites of lower affinity (each 27 microM), again comparable to wild-type enzyme. After cross-linking all three catalytic sites bound MgATP or MgADP with the same relatively low affinity (approximately 60 microM). Thus cross-linking fixed all three catalytic sites in the same conformation. Trypsin cleavage experiments showed that cross-linking fixed the epsilon subunit in the ATP+EDTA conformation.

Bacterial Proteins↗
PubMed1998

The second stalk: the delta-b subunit connection in ECF1F0.

The ATP synthase F1F0 is the smallest molecular motor yet studied. ATP hydrolysis drives the rotary motion of the primary stalk subunits gamma and epsilon relative to the alpha 3 beta 3 part of F1. Evidence is reviewed to show that the delta and b subunits provide a second stalk that can act as a stator to facilitate these rotational movements.

Isoenzymes↗
PubMed1997

Expression of mtDNA and nDNA encoded respiratory chain proteins in chemically and genetically-derived Rho0 human fibroblasts: a comparison of subunit proteins in normal fibroblasts treated with ethidium bromide and fibroblasts from a patient with mtDNA depletion syndrome.

Although much progress has been made in identifying genetic defects associated with mitochondrial diseases, the protein expression patterns of most disorders are poorly understood. Here we use immunochemical techniques to describe subunit expression patterns of respiratory chain enzyme complexes II (succinate dehydrogenase: SD) and IV (cytochrome c oxidase: COX) in cultured cells lacking mtDNA (Rho0 cells) derived either chemically by exposure of normal cells to ethidium bromide, or genetically in cells derived from a patient with mtDNA depletion syndrome. Both control cells and early passage patient-derived cells express a normal complement of SD and COX subunit proteins. Ethidium bromide treatment of normal cells and in vitro cell proliferation of patient-derived cells caused both populations to acquire identical Rho0 phenotypes. As expected, they lack mtDNA-encoded subunits COX-I and COX-II. In contrast, nDNA-encoded subunits are affected differentially, with some (COX-VIc) lacking and others (COX-IV, COX-Va, SD 30 and SD 70) maintained at somewhat reduced levels. We suggest that the differential stability of nDNA-encoded subunits in the absence of intact enzyme complexes is due to the ability of some, but not all, subunits to associate as partial complexes in the absence of mtDNA-encoded subunits.

Cells, Cultured↗
PubMed1997

The subunit delta-subunit b domain of the Escherichia coli F1F0 ATPase. The B subunits interact with F1 as a dimer and through the delta subunit.

The delta and b subunits are both involved in binding the F1 to the F0 part in the Escherichia coli ATP synthase (ECF1F0). The interaction of the purified delta subunit and the isolated hydrophilic domain of the b subunit (bsol) has been studied here. Purified delta binds to bsol weakly in solution, as indicated by NMR studies and protease protection experiments. On F1, i.e. in the presence of ECF1-delta, delta, and bsol interact strongly, and a complex of ECF1.bsol can be isolated by native gel electrophoresis. Both delta subunit and bsol are protected from trypsin cleavage in this complex. In contrast, the delta subunit is rapidly degraded by the protease when bound to ECF1 when bsol is absent. The interaction of bsol with ECF1 involves the C-terminal domain of delta as delta(1-134) cannot replace intact delta in the binding experiments. As purified, bsol is a stable dimer with 80% alpha helix. A monomeric form of bsol can be obtained by introducing the mutation A128D (Howitt, S. M., Rodgers, A. J.,W., Jeffrey, P. D., and Cox, G. B. (1996) J. Biol. Chem. 271, 7038-7042). Monomeric bsol has less alpha helix, i.e. only 58%, is much more sensitive to trypsin cleavage than dimer, and unfolds at much lower temperatures than the dimer in circular dichroism melting studies, indicating a less stable structure. The bsol dimer, but not monomer, binds to delta in ECF1. To examine whether subunit b is a monomor or dimer in intact ECF1F0, CuCl2 was used to induce cross-link formation in the mutants bS60C, bQ104C, bA128C, bG131C, and bS146C. With the exception of bS60C, CuCl2 treatment resulted in formation of b subunit dimers in all mutants. Cross-linking yield was independent of nucleotide conditions and did not affect ATPase activity. These results show the b subunit to be dimeric for a large portion of the C terminus, with residues 124-131 likely forming a pair of parallel alpha helices.

Circular Dichroism↗
PubMed1997

Rotation of a gamma-epsilon subunit domain in the Escherichia coli F1F0-ATP synthase complex. The gamma-epsilon subunits are essentially randomly distributed relative to the alpha3beta3delta domain in the intact complex.

A triple mutant of Escherichia coli F1F0-ATP synthase, alphaQ2C/alphaS411C/epsilonS108C, has been generated for studying movements of the gamma and epsilon subunits during functioning of the enzyme. It includes mutations that allow disulfide bond formation between the Cys at alpha411 and both Cys-87 of gamma and Cys-108 of epsilon, two covalent cross-links that block enzyme function (Aggeler, R., and Capaldi, R. A. (1996) J. Biol. Chem. 271, 13888-13891). A cross-link is also generated between the Cys at alpha2 and Cys-140 of the delta subunit, which has no effect on functioning (Ogilvie, I., Aggeler, R., and Capaldi, R. A. (1997) J. Biol. Chem. 272, 16652-16656). CuCl2 treatment of the mutant alphaQ2C/alphaS411C/epsilonS108C generated five major cross-linked products. These are alpha-gamma-delta, alpha-gamma, alpha-delta-epsilon, alpha-delta, and alpha-epsilon. The ratio of alpha-gamma-delta to the alpha-gamma product was close to 1:2, i.e. in one-third of the ECF1F0 molecules the gamma subunit was attached to the alpha subunit at which the delta subunit is bound. Also, 20% of the epsilon subunit was present as a alpha-delta-epsilon product. With regard to the delta subunit, 30% was in the alpha-gamma-delta, 20% in the alpha-delta-epsilon, and 50% in the alpha-delta products when the cross-linking was done after incubation in ATP + MgCl2. The amounts of these three products were 40, 22, and 38%, respectively, in experiments where Cu2+ was added after preincubation in ATP + Mg2+ + azide. The delta subunit is fixed to, and therefore identifies, one specific alpha subunit (alphadelta). A distribution of the gamma and epsilon subunits, which is essentially random with respect to the alpha subunits, can only be explained by rotation of gamma-epsilon relative to the alpha3beta3 domain in ECF1F0.

Adenosine Triphosphate↗
PubMed1997

An improved purification of ECF1 and ECF1F0 by using a cytochrome bo-deficient strain of Escherichia coli facilitates crystallization of these complexes.

A novel strategy, which employs a cytochrome bo-lacking strain (GO104) and a modified isolation procedure provides an effective approach for obtaining much purer preparations of ECF1F0 than described previously, as well as for isolating homogeneous and protein-chemically pure ECF1. ECF1 obtained in this way could be crystallized by vapor-diffusion using polyethylene glycol (PEG) as a precipitant in a form suitable for X-ray diffraction analysis. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with lattice parameters a = 110, h = 134, and c = 269 A, and diffract to a resolution of at least 6.4 A.

Crystallization↗
PubMed1997

Cross-linking of the delta subunit to one of the three alpha subunits has no effect on functioning, as expected if delta is a part of the stator that links the F1 and F0 parts of the Escherichia coli ATP synthase.

A mutant of the Escherichia coli F1F0-ATPase has been generated (alphaQ2C) in which the glutamine at position 2 of the alpha subunit has been replaced with a cysteine residue. Cu2+ treatment of ECF1 from this mutant cross-linked an alpha subunit to the delta subunit in high yield. Two different sites of disulfide bond formation were involved, i.e. between Cys90 (or the closely spaced Cys47) of alpha with Cys140 of delta, and between Cys2 of alpha and Cys140 of delta. Small amounts of other cross-linked products, including alpha-alpha, delta internal, and alpha-alpha-delta were obtained. In ECF1F0, there was no cross-linking between the intrinsic Cys of alpha and Cys140. Instead, the product generated between Cys2 of alpha and Cys140 of delta was obtained at near 90% yield. Small amounts of alpha-alpha and delta internal were present, and under high Cu2+ concentrations, alpha-alpha-delta was also formed. The ATPase activity of ECF1 and ECF1F0 was not significantly affected by the presence of these cross-links. When Cys140 of delta was first modified with N-ethylmaleimide in ECF1F0, an alpha-delta cross-link was still produced, although in lower yield, between Cys64 of delta and Cys2 of alpha. ATP hydrolysis-linked proton pumping of inner membranes from the mutant alpha2QC was only marginally affected by cross-linking of the alpha to the delta subunit. These results indicate that Cys140 and Cys64 of the delta subunit and Cys2 of the alpha subunit are in close proximity. This places the delta subunit near the top of the alpha-beta hexagon and not in the stalk region. As fixing the delta to the alpha by cross-linking does not greatly impair either the ATPase function of the enzyme, or coupled proton translocation, we argue that the delta subunit forms a portion of the stator linking F1 to F0.

Adenosine Triphosphate↗
PubMed1997

Interactions between the F1 and F0 parts in the Escherichia coli ATP synthase. Associations involving the loop region of C subunits.

The N-ethylmaleimide reactivity of c subunits in Escherichia coli F1F0 ATP synthase (ECF1F0) isolated from five mutants, each with a cysteine at a different position in the polar loop region (positions 39, 40, 42, 43, and 44), has been investigated. The maleimide was found to react with Cys placed at positions 42, 43, and 44 but not at 39 or 40. All copies of the c subunit reacted similarly when the Cys was at position 43 or 44. In contrast, the Cys in the mutant cQ42C reacted as two classes, with 60% reacting relatively rapidly and 40% reacting at a rate 40-fold slower. After removing F1, all copies of the c subunit in this mutant reacted equally fast. Therefore, the slow class in the cQ42C mutant represents c subunits shielded by, and probably involved directly in, the interaction of the F0 with gamma and epsilon subunits of the F1 part. Based on the estimated stoichiometry of c subunits in the ECF1F0 complex, 4 or 5 c subunits are involved in this F1 interaction. N-Ethylmaleimide modification of all of the c subunits reduced ATPase activity by only 30% in ECF1F0 from mutant cQ42C. Modification of the more rapidly reacting class had little effect on ATP hydrolysis-driven proton translocation, and did not alter the DCCD inhibition of ATPase activity. However, as those c subunits involved in the F1 interaction became modified, DCCD inhibition was progressively lost, as was coupling between ATP hydrolysis and proton translocation.

Cysteine↗