Mutations in the delta subunit influence the assembly of F1F0 ATP synthase in Escherichia coli.

Missense mutations affecting Asp-161 and Ser-163 in the delta subunit of F1F0 ATP synthase have been generated. Although most substitutions allowed substantial enzyme function, the delta Asp-161-->Pro substitution resulted in a loss of enzyme activity. The loss of activity was attributable to a structural failure altering assembly of the enzyme complex.     

Structure of the membrane domain of subunit b of the Escherichia coli F0F1 ATP synthase.

The structure of the N-terminal transmembrane domain (residues 1-34) of subunit b of the Escherichia coli F0F1-ATP synthase has been solved by two-dimensional 1H NMR in a membrane mimetic solvent mixture of chloroform/methanol/H2O (4:4:1). Residues 4-22 form an alpha-helix, which is likely to span the hydrophobic domain of the lipid bilayer to anchor the largely hydrophilic subunit b in the membrane. The helical structure is interrupted by a rigid bend in the region of residues 23-26 with alpha-helical structure resuming at Pro-27 at an angle offset by 20 degrees from the transmembrane helix. In native subunit b, the hinge region and C-terminal alpha-helical segment would connect the transmembrane helix to the cytoplasmic domain. The transmembrane domains of the two subunit b in F0 were shown to be close to each other by cross-linking experiments in which single Cys were substituted for residues 2-21 of the native subunit and b-b dimer formation tested after oxidation with Cu(II)(phenanthroline)2. Cys residues that formed disulfide cross-links were found with a periodicity indicative of one face of an alpha-helix, over the span of residues 2-18, where Cys at positions 2, 6, and 10 formed dimers in highest yield. A model for the dimer is presented based upon the NMR structure and distance constraints from the cross-linking data. The transmembrane alpha-helices are positioned at a 23 degrees angle to each other with the side chains of Thr-6, Gln-10, Phe-14, and Phe-17 at the interface between subunits. The change in direction of helical packing at the hinge region may be important in the functional interaction of the cytoplasmic domains.     

Secondary structure composition of reconstituted subunit b of the Escherichia coli ATP synthase.

Subunit b of the Escherichia coli ATP synthase was isolated by preparative gel electrophoresis, acetone precipitated and after ion-pair extraction redissolved in a buffer either containing n-dodecyl-beta-D-maltoside or sodium cholate. The secondary structure of isolated subunit b was shown to be the same as within the FO complex, but was strongly dependent on the detergent used for replacement of the phospholipid environment. This was shown by an identical tryptic digestion pattern, which was strongly influenced by the detergent used for solubilization. An influence of the detergent n-dodecyl-beta-D-maltoside on the secondary structure of the hydrophilic part of subunit b was also shown for the soluble part of the polypeptide comprising residues Val25 to Leu156 (bsol) using CD spectroscopy. In order to determine the secondary structure of subunit b in its native conformation, isolated subunit b was reconstituted into E. coli lipid vesicles and analyzed with CD spectroscopy. The resulting spectrum revealed a secondary structure composition of 80% alpha helix together with 14% beta turn conformation. These results suggest that subunit b is not a rigid rod-like alpha helix simply linking F1 to FO, but rather provides an inherent flexibility for the storage of elastic energy within the second stalk generated by rotational movements within the F1FO complex.     

ATP synthase b subunit dimerization domain: a right-handed coiled coil with offset helices

The dimerization domain of Escherichia coli ATP synthase b subunit forms an atypical parallel two-stranded coiled coil. Sequence analysis reveals an 11-residue abcdefghijk repeat characteristic of right-handed coiled coils, but no other naturally occurring parallel dimeric structure of this class has been identified. The arrangement of the helices was studied by their propensity to form interhelix disulfide linkages and analysis of the stability and shape of disulfide-linked dimers. Disulfides formed preferentially between cysteine residues in an a position of one helix and either of the adjacent h positions of the partner. Such heterodimers were far more stable to thermal denaturation than homodimers and, on the basis of gel-filtration chromatography studies, were similar in shape to both non-covalent dimers and dimers linked through flexible Gly(1-3)Cys C-terminal extensions. The results indicate a right-handed coiled-coil structure with intrinsic asymmetry, the two helices being offset rather than in register. A function for the right-handed coiled coil in rotational catalysis is proposed.     

Fo portion of Escherichia coli ATP synthase. Further resolution of trypsin-generated fragments from subunit b

F1-stripped everted membrane vesicles of the ATP synthase-overproducing Escherichia coli strain KY 7485 were treated with trypsin for different lengths of time. Subsequently, the Fo complex was isolated and analyzed by sodium dodecyl sulfate-gel electrophoresis, as well as immunoblotting using antibodies raised against subunit b. By these techniques 3 degradation products with apparent molecular masses of about 16 kDa could be detected in accordance with previous findings (Perlin, D.S., and Senior, A.E. (1985) Arch. Biochem. Biophys. 236, 603-611). Labeling of isolated trypsin-treated Fo fractions with the thiol-specific reagent N-(7-dimethylamino-4-methylcoumarinyl)-maleimide, which has been demonstrated recently to specifically modify subunit b (Schneider, E., and Altendorf, K. (1985) Eur. J. Biochem. 153, 105-109) revealed that the 16-kDa digestion products were degraded into two stable fragments of 12 and 8.3 kDa. These polypeptides do not react with the anti-b antibodies. Treatment of purified liposome-integrated Fo with trypsin resulted in a similar cleavage pattern. In both cases protease digestion inhibited F1 binding while proton-translocating activity remained unaffected. However, liposomes reconstituted with Fo isolated from trypsin-treated membranes were impaired in both binding of F1 and proton translocation. These activities could be restored when reconstitution was carried out in the presence of native subunit b. From this we conclude that the C-terminal region of subunit b is necessary for proper reconstitution of Fo into liposomes.     

Labeling of subunit b of the ATP synthase from Escherichia coli with a photoreactive phospholipid analogue

Purified ATP synthase (F1F0) from Escherichia coli K12 was labeled with the hydrophobic photoreactive label 1-palmitoyl 2-(2-azido-4-nitro)benzoyl sn-glycero-3-[3H]phosphocholine in reconstituted proteoliposomes. The F0-subunit b was predominantly labeled. A very low amount of label was detected on the other F0-subunits a and c. The label in subunit b could be traced back by proteolytic digestion to the NH2-terminal fragment 1 to 53 which contains the stretch of hydrophobic amino acid residues 1 to 32. By sequencing the intact protein, the distribution of label among the amino acids in this segment was determined. Cysteine 21 was predominantly labeled. Other labeled amino acids occurred at the NH2-terminal (Asn-2) and at position 26 (tryptophan). Due to the restricted mobility of the label in the lipid bilayer, these residues are suggested to be located in or close to the polar head of the lipid bilayer. These results will be compared with predictions for the arrangement of the polypeptide b derived from the hydrophobicity profile.     

Substitution of the cysteinyl residue (Cys21) of subunit b of the ATP synthase from Escherichia coli.

The Fo complex of the ATP synthase (F1Fo) of Escherichia coli contains only two cysteinyl residues, Cys21, of the two copies of subunit b. Modification of Cys21 with the hydrophobic maleimide N-(7-dimethylamino-4-methyl-coumarinyl)maleimide resulted in impairment of Fo functions [Schneider, E. & Altendorf, K. (1985) Eur. J. Biochim. 153, 105-109]. We replaced this residue (via cassette mutagenesis) by Ser, Gly, Ala, Thr, Asp and Pro. None of the replacements resulted in detectable alterations of the function of the ATP synthase, making a functional role for these sulfhydryl residues unlikely. Due to its high tolerance towards amino acid substitutions, the region around Cys21 seems not to be a protein-protein contact area.     

The "second stalk" of Escherichia coli ATP synthase: structure of the isolated dimerization domain

The b subunit of E. coli F(0)F(1)-ATPase links the peripheral F(1) subunits to the membrane-integral F(0) portion and functions as a "stator", preventing rotation of F(1). The b subunit is present as a dimer in ATP synthase, and residues 62-122 are required to mediate dimerization. To understand how the b subunit dimer is formed, we have studied the structure of the isolated dimerization domain, b(62-122). Analytical ultracentrifugation and solution small-angle X-ray scattering (SAXS) indicate that the b(62-122) dimer is extremely elongated, with a frictional ratio of 1.60, a maximal dimension of 95 A, and a radius of gyration of 27 A, values that are consistent with an alpha-helical coiled-coil structure. The crystal structure of b(62-122) has been solved and refined to 1.55 A. The protein crystallized as an isolated, monomeric alpha helix with a length of 90 A. Combining the crystal structure of monomeric b(62-122) with SAXS data from the dimer in solution, we have constructed a model for the b(62-122) dimer in which the two helices form a coiled coil with a right-handed superhelical twist. Analysis of b sequences from E. coli and other prokaryotes indicates conservation of an undecad repeat, which is characteristic of a right-handed coiled coil and consistent with our structural model. Mutation of residue Arg-83, which interrupts the undecad pattern, to alanine markedly stabilized the dimer, as expected for the proposed two-stranded, right-handed coiled-coil structure.     

Expression of subunit III of the ATP synthase from spinach chloroplasts in Escherichia coli

Expression of subunit III of the ATP synthase from spinach chloroplasts in Escherichia coli has been achieved. Although the protein is inserted into the bacterial cytoplasmic membrane, formation of a functional Fo complex was not observed.     

ATP synthesis without R210 of subunit a in the Escherichia coli ATP synthase

Interactions between subunit a and oligomeric subunit c are essential for the coupling of proton translocation to rotary motion in the ATP synthase. A pair of previously described mutants, R210Q/Q252R and P204T/R210Q/Q252R [L.P. Hatch, G.B. Cox and S.M. Howitt, The essential arginine residue at position 210 in the a subunit of the Escherichia coli ATP synthase can be transferred to position 252 with partial retention of activity, J. Biol. Chem. 270 (1995) 29407-29412] has been constructed and further analyzed. These mutants, in which the essential arginine of subunit a, R210, was switched with a conserved glutamine residue, Q252, are shown here to be capable of both ATP synthesis by oxidative phosphorylation, and ATP-driven proton translocation. In addition, lysine can replace the arginine at position 252 with partial retention of both activities. The pH dependence of ATP-driven proton translocation was determined after purification of mutant enzymes, and reconstitution into liposomes. Proton translocation by the lysine mutant, and to a lesser extent the arginine mutant, dropped off sharply above pH 7.5, consistent with the requirement for a positive charge during function. Finally, the rates of ATP synthesis and of ATP-driven proton translocation were completely inhibited by treatment with DCCD (N,N'-dicyclohexylcarbodiimide), while rates of ATP hydrolysis by the mutants were not significantly affected, indicating that DCCD modification disrupts the F(1)-F(o) interface. The results suggest that minimal requirements for proton translocation by the ATP synthase include a positive charge in subunit a and a weak interface between subunit a and oligomeric subunit c.     

Disulfide linkage of the b and delta subunits does not affect the function of the Escherichia coli ATP synthase

The ATP synthase of Escherichia coli is believed to act through a rotational mechanism in which the b(2)delta subcomplex holds the alphabeta hexamer stationary relative to the rotating gamma and epsilon subunits. We have engineered a disulfide bond between cysteines introduced at position 158 of the delta subunit and at a position just beyond the normal C-terminus of the b subunit. The formation of this disulfide bond verifies that the C-terminal region of b is proximal to residue 158 of delta. The disulfide bond does not affect the ability of the F(1)F(0) complex to hydrolyze ATP, couple ATP hydrolysis to the establishment of a proton gradient, or maintain a proton gradient generated by the electron transport chain. These results are consistent with a permanent association of b(2) with delta as suggested by the rotational model of enzyme function.     

Deletions in hydrophilic domains of subunit a from the Escherichia coli F1F0-ATP synthase interfere with membrane insertion or F0 assembly.

The a subunit is a membrane component of the F1F0-ATP synthase from Escherichia coli. Regions of a which appear important for membrane insertion or F0 assembly have been identified by analysis of both deletion mutants and fusion proteins which link the mutant a subunits to alkaline phosphatase. This analysis suggests the hydrophilic, amino-terminal domain of a is required for proper membrane targeting and/or insertion of the nascent polypeptide. In addition, the subcellular fractionation of four different a subunit-beta-galactosidase fusion proteins suggests this domain is localized to the periplasm, in agreement with a proposed topological model of the protein (Lewis, M.J., Chang, J.A., and Simoni, R.D. (1990) J. Biol. Chem. 265, 10541-10550). Deletions within the next three putative loops of a appear to have no significant effect on membrane targeting or insertion. Rather, they seem to interfere with the subsequent assembly of a functional enzyme.     

Rotor/Stator interactions of the epsilon subunit in Escherichia coli ATP synthase and implications for enzyme regulation

The H(+)-translocating F(0)F(1)-ATP synthase of Escherichia coli functions as a rotary motor, coupling the transmembrane movement of protons through F(0) to the synthesis of ATP by F(1). Although the epsilon subunit appears to be tightly associated with the gamma subunit in the central stalk region of the rotor assembly, several studies suggest that the C-terminal domain of epsilon can undergo significant conformational change as part of a regulatory process. Here we use disulfide cross-linking of substituted cysteines on functionally coupled ATP synthase to characterize interactions of epsilon with an F(0) component of the rotor (subunit c) and with an F(1) component of the stator (subunit beta). Oxidation of the engineered F(0)F(1) causes formation of two disulfide bonds, betaD380C-S108C epsilon and epsilonE31C-cQ42C, to give a beta-epsilon-c cross-linked product in high yield. The results demonstrate the ability of epsilon to span the central stalk region from the surface of the membrane (epsilon-c) to the bottom of F(1) (beta-epsilon) and suggest that the conformation detected here is distinct from both the "closed" state seen with isolated epsilon (Uhlin, U., Cox, G. B., and Guss, J. M. (1997) Structure 5, 1219-1230) and the "open" state seen in a complex with a truncated form of the gamma subunit (Rodgers, A. J., and Wilce, M. C. (2000) Nat. Struct. Biol. 7, 1051-1054). The kinetics of beta-epsilon and epsilon-c cross-linking were studied separately using F(0)F(1) containing one or the other matched cysteine pair. The rate of cross-linking at the epsilon/c (rotor/rotor) interface is not influenced by the type of nucleotide added. In contrast, the rate of beta-epsilon cross-linking is fastest under ATP hydrolysis conditions, intermediate with MgADP, and slowest with MgAMP-PNP. This is consistent with a regulatory role for a reversible beta/epsilon (stator/rotor) interaction that blocks rotation and inhibits catalysis. Furthermore, the rate of beta-epsilon cross-linking is much faster than that indicated by previous studies, allowing for the possibility of a rapid response to regulatory signals.     

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)     

The dimerization domain of the b subunit of the Escherichia coli F(1)F(0)-ATPase.

In this study a series of N- and/or C-terminal truncations of the cytoplasmic domain of the b subunit of the Escherichia coli F(1)F(0) ATP synthase were tested for their ability to form dimers using sedimentation equilibrium ultracentrifugation. The deletion of residues between positions 53 and 122 resulted in a strongly decreased tendency to form dimers, whereas all the polypeptides that included that sequence exhibited high levels of dimer formation. b dimers existed in a reversible monomer-dimer equilibrium and when mixed with other b truncations formed heterodimers efficiently, provided both constructs included the 53-122 sequence. Sedimentation velocity and (15)N NMR relaxation measurements indicated that the dimerization region is highly extended in solution, consistent with an elongated second stalk structure. A cysteine introduced at position 105 was found to readily form intersubunit disulfides, whereas other single cysteines at positions 103-110 failed to form disulfides either with the identical mutant or when mixed with the other 103-110 cysteine mutants. These studies establish that the b subunit dimer depends on interactions that occur between residues in the 53-122 sequence and that the two subunits are oriented in a highly specific manner at the dimer interface.     

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).     

Identification of a subunit assembly domain in the alpha subunit of Escherichia coli RNA polymerase

The alpha subunit of Escherichia coli DNA-dependent RNA polymerase is encoded by the rpoA gene and plays a major role in enzyme assembly. A set of C-terminal deletion mutations of the rpoA gene was constructed. The results of mixed reconstitution experiments in vitro, using the truncated alpha polypeptides encoded by the rpoA deletion mutants, suggest that the amino-terminal two-thirds of alpha subunit is sufficient for the formation of pseudo-core complexes containing both beta and beta' subunits.