NMR solution structure of subunit F of the methanogenic A1AO adenosine triphosphate synthase and its interaction with the nucleotide-binding subunit B

The A1AO adenosine triphosphate (ATP) synthase from archaea uses the ion gradients generated across the membrane sector (AO) to synthesize ATP in the A3B3 domain of the A1 sector. The energy coupling between the two active domains occurs via the so-called stalk part(s), to which the 12 kDa subunit F does belong. Here, we present the solution structure of the F subunit of the A1AO ATP synthase from Methanosarcina mazei G?1. Subunit F exhibits a distinct two-domain structure, with the N-terminal having 78 residues and residues 79-101 forming the flexible C-terminal part. The well-ordered N-terminal domain is composed of a four-stranded parallel beta-sheet structure and three alpha-helices placed alternately. The two domains are loosely associated with more flexibility relative to each other. The flexibility of the C-terminal domain is further confirmed by dynamics studies. In addition, the affinity of binding of mutant subunit F, with a substitution of Trp100 against Tyr and Ile at the very C-terminal end, to the nucleotide-binding subunit B was determined quantitatively using the fluorescence signals of natural subunit B (Trp430). Finally, the arrangement of subunit F within the complex is presented.     

Small-angle X-ray scattering reveals the solution structure of the peripheral stalk subunit H of the A1AO ATP synthase from Methanocaldococcus jannaschii and its binding to the catalytic A subunit

The H subunit of the A1AO ATP synthase is a component of one of the peripheral stalks connecting the A1 and AO domain. Subunit H of the Methanocaldococcus jannaschii A1AO ATP synthase was analyzed by small-angle X-ray scattering (SAXS) in order to determine the first low-resolution structure of this molecule in solution. Independent to the concentration used, the protein is dimeric and has a boomerang-like shape, divided into two arms of 12.0 and 6.8 nm in length. Circular dichroism (CD) spectroscopy revealed that subunit H is comprised of 78% alpha-helix and a coiled-coil arrangement. To understand the orientation of the helices and the localization of the N- and C-termini inside the dimer, three truncated forms of subunit H (H8-104, H1-98, and H8-98) were expressed, purified, and analyzed by CD. SAXS experiments of H1-98 show that the maximum dimension of the truncated protein dropped to 15.1 nm. Comparison of the low-resolution shapes of H and H1-98 indicates that this goes along with structural changes in the C-terminal arm of the boomerang-like structure. Together with the result of a disulfide formation of a fourth truncated form, H1-47, with a cysteine at position 47, the data suggest a parallel alpha-helical interaction. In addition, all four truncated proteins are dimeric in solution. Tryptophan emission spectra showed specific binding of H and H8-104 to the neighboring, catalytic A subunit, which could not be detected in the presence of H1-98. Finally, the arrangement of H within the A1AO ATP synthase is presented.     

Defining the domain of binding of F1 subunit epsilon with the polar loop of F0 subunit c in the Escherichia coli ATP synthase.

We have previously shown that the E31C-substituted epsilon subunit of F1 can be cross-linked by disulfide bond formation to the Q42C-substituted c subunit of F0 in the Escherichia coli F1F0-ATP synthase complex (Zhang, Y., and Fillingame, R. H. (1995) J. Biol. Chem. 270, 24609-24614). The interactions of subunits epsilon and c are thought to be central to the coupling of H+ transport through F0 to ATP synthesis in F1. To further define the domains of interaction, we have introduced additional Cys into subunit epsilon and subunit c and tested for cross-link formation following sulfhydryl oxidation. The results show that Cys, in a continuous stretch of residues 26-33 in subunit epsilon, can be cross-linked to Cys at positions 40, 42, and 44 in the polar loop region of subunit c. The results are interpreted, and the subunit interaction is modeled using the NMR and x-ray diffraction structures of the monomeric subunits together with information on the packing arrangement of subunit c in a ring of 12 subunits. In the model, residues 26-33 form a turn of antiparallel beta-sheet which packs between the polar loop regions of adjacent subunit c at the cytoplasmic surface of the c12 oligomer.     

Quantitative determination of direct binding of b subunit to F1 in Escherichia coli F1F0-ATP synthase

The stator in F(1)F(0)-ATP synthase resists strain generated by rotor torque. In Escherichia coli, the b(2)delta subunit complex comprises the stator, bound to subunit a in F(0) and to the alpha(3)beta(3) hexagon of F(1). To quantitatively characterize binding of b subunit to the F(1) alpha(3)beta(3) hexagon, we developed fluorimetric assays in which wild-type F(1), or F(1) enzymes containing introduced Trp residues, were titrated with a soluble portion of the b subunit (b(ST34-156)). With five different F(1) enzymes, K(d)(b(ST34-156)) ranged from 91 to 157 nm. Binding was strongly Mg(2+)-dependent; in EDTA buffer, K(d)(b(ST34-156)) was increased to 1.25 microm. The addition of the cytoplasmic portion of the b subunit increases the affinity of binding of delta subunit to delta-depleted F(1). The apparent K(d)(b(ST34-156)) for this effect was increased from 150 nm in Mg(2+) buffer to 1.36 microm in EDTA buffer. This work demonstrates quantitatively how binding of the cytoplasmic portion of the b subunit directly to F(1) contributes to stator resistance and emphasizes the importance of Mg(2+) in stator interactions.     

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.     

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.     

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.     

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.     

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.     

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.     

Modification of subunit b of the F0 complex from Escherichia coli ATP synthase by a hydrophobic maleimide and its effects on F0 functions

Purified F0 from Escherichia coli ATP synthase was labelled with N-(7-dimethylamino-4-methyl-coumarinyl)-maleimide (DACM), a hydrophobic reagent which forms a stable, strongly fluorescent adduct with SH groups. Sodium dodecyl sulfate gel electrophoresis clearly demonstrated that subunit b was exclusively labelled, most likely at Cys-21, the only cysteine residue in E. coli F0. The amount of two molecules of DACM bound per F0, which was calculated from the absorption spectrum at 380 nm, is in full agreement with the postulated stoichiometry of two copies of subunit b/F0 complex. Thus the label provides a useful tool for simply detecting subunit b in protein chemical studies. DACM-labelled F0 was incorporated into liposomes and assayed for H+ translocating activity and its capacity to bind purified F1. Whereas the initial rate of H+ uptake was inhibited about 40% the reconstitution of a dicyclohexylcarbodiimide-sensitive F1F0 ATPase activity was completely unaffected. In a second set of experiments we reconstituted an F0 complex from DACM-labelled purified subunit b and an ac complex. In contrast to the results obtained with intact, DACM-labelled F0, both H+ translocating activity and F1 binding capacity were greatly reduced. Our data indicate that cysteine-21, probably together with other amino acids, is involved in maintaining a proper interaction of the hydrophobic N-terminal region of subunit b with the ac complex. This interplay seems to be a prerequisite for at least the in vitro assembly of a functional F0 complex.     

Subunit a facilitates aqueous access to a membrane-embedded region of subunit c in Escherichia coli F1F0 ATP synthase

Rotary catalysis in F(1)F(0) ATP synthase is powered by proton translocation through the membrane-embedded F(0) sector. Proton binding and release occurs in the middle of the membrane at Asp-61 on transmembrane helix 2 of subunit c. Previously, the reactivity of cysteines substituted into F(0) subunit a revealed two regions of aqueous access, one extending from the periplasm to the middle of the membrane and a second extending from the middle of the membrane to the cytoplasm. To further characterize aqueous accessibility at the subunit a-c interface, we have substituted Cys for residues on the cytoplasmic side of transmembrane helix 2 of subunit c and probed the accessibility to these substituted positions using thiolate-reactive reagents. The Cys substitutions tested were uniformly inhibited by Ag(+) treatment, which suggested widespread aqueous access to this generally hydrophobic region. Sensitivity to N-ethylmaleimide (NEM) and methanethiosulfonate reagents was localized to a membrane-embedded pocket surrounding Asp-61. The cG58C substitution was profoundly inhibited by all the reagents tested, including membrane impermeant methanethiosulfonate reagents. Further studies of the highly reactive cG58C substitution revealed that NEM modification of a single c subunit in the oligomeric c-ring was sufficient to cause complete inhibition. In addition, NEM modification of subunit c was dependent upon the presence of subunit a. The results described here provide further evidence for an aqueous-accessible region at the interface of subunits a and c extending from the middle of the membrane to the cytoplasm.     

Temperature-sensitive mutations at the carboxy terminus of the alpha subunit of the Escherichia coli F1F0 ATP synthase.

Mutations were constructed in the a subunit of the F1F0 ATP synthase from Escherichia coli. Truncated forms of this subunit showed a temperature sensitivity phenotype. We conclude that the carboxy terminus of the a subunit is not involved directly with proton translocation but that it has an important structural role.     

F1F0 ATP synthase subunit c is a substrate of the novel YidC pathway for membrane protein biogenesis

The Escherichia coli YidC protein belongs to the Oxa1 family of membrane proteins that have been suggested to facilitate the insertion and assembly of membrane proteins either in cooperation with the Sec translocase or as a separate entity. Recently, we have shown that depletion of YidC causes a specific defect in the functional assembly of F1F0 ATP synthase and cytochrome o oxidase. We now demonstrate that the insertion of in vitro-synthesized F1F0 ATP synthase subunit c (F0c) into inner membrane vesicles requires YidC. Insertion is independent of the proton motive force, and proteoliposomes containing only YidC catalyze the membrane insertion of F0c in its native transmembrane topology whereupon it assembles into large oligomers. Co-reconstituted SecYEG has no significant effect on the insertion efficiency. Remarkably, signal recognition particle and its membrane-bound receptor FtsY are not required for the membrane insertion of F0c. In conclusion, a novel membrane protein insertion pathway in E. coli is described in which YidC plays an exclusive role.     

1H nuclear magnetic resonance studies of an integral membrane protein: subunit c of the F1F0 ATP synthase

The membrane-traversing subunit c parallel from the F0 part of the ATP synthase molecule has been studied in chloroform/methanol by high-resolution 1H n.m.r. Various one-dimensional and two-dimensional techniques have been used for assignment purposes, some NOE connectivities were established and some 3JHN alpha coupling constants were measured from spin--echo experiments. The effects of varying pH, solvent composition, lanthanide concentration and temperature have been investigated. Evidence is presented that the molecule has extensive alpha-helical segments, and the hairpin structure suggested by other groups is supported by our n.m.r. data. Only one ionizable group, assigned to the C-terminal carboxyl, is observed to titrate in the pH range 2 to 10; so the conserved residue, Asp61, which binds dicyclohexylcarbodiimide, presumably has (at least in this solvent system) an abnormally high pK value.     

Construction and plasmid-borne complementation of strains lacking the epsilon subunit of the Escherichia coli F1F0 ATP synthase.

Two strains of Escherichia coli that lack the epsilon subunit of the F1F0 ATP synthase have been constructed. They are shown to be viable but with very low growth yields (28%). These strains can be complemented by plasmids carrying wild-type uncC, but not when epsilon is overproduced. These results indicate that epsilon is not essential for growth on minimal glucose medium and that the level of its expression affects the assembly of the ATP synthase.     

Energy-linked binding of Pi is required for continuous steady-state proton-translocating ATP hydrolysis catalyzed by F0.F1 ATP synthase

The presence of medium Pi (half-maximal concentration of 20 microM at pH 8.0) was found to be required for the prevention of the rapid decline in the rate of proton-motive force (pmf)-induced ATP hydrolysis by Fo.F1 ATP synthase in coupled vesicles derived from Paracoccus denitrificans. The initial rate of the reaction was independent of Pi. The apparent affinity of Pi for its "ATPase-protecting" site was strongly decreased with partial uncoupling of the vesicles. Pi did not reactivate ATPase when added after complete time-dependent deactivation during the enzyme turnover. Arsenate and sulfate, which was shown to compete with Pi when Fo.F1 catalyzed oxidative phosphorylation, substituted for Pi as the protectors of ATPase against the turnover-dependent deactivation. Under conditions where the enzyme turnover was not permitted (no ATP was present), Pi was not required for the pmf-induced activation of ATPase, whereas the presence of medium Pi (or sulfate) delayed the spontaneous deactivation of the enzyme which was induced by the membrane de-energization. The data are interpreted to suggest that coupled and uncoupled ATP hydrolysis catalyzed by Fo.F1 ATP synthases proceeds via different intermediates. Pi dissociates after ADP if the coupling membrane is energized (no E.ADP intermediate exists). Pi dissociates before ADP during uncoupled ATP hydrolysis, leaving the E.ADP intermediate which is transformed into the inactive ADP(Mg2+)-inhibited form of the enzyme (latent ATPase).     

Membrane topography of the coupling ion binding site in Na+-translocating F1F0 ATP synthase.

A carbodiimide with a photoactivatable diazirine substituent was synthesized and incubated with the Na(+)-translocating F(1)F(0) ATP synthase from both Propionigenium modestum and Ilyobacter tartaricus. This caused severe inhibition of ATP hydrolysis activity in the absence of Na(+) ions but not in its presence, indicating the specific reaction with the Na(+) binding c-Glu(65) residue. Photocross-linking was investigated with the substituted ATP synthase from both bacteria in reconstituted 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC)-containing proteoliposomes. A subunit c/POPC conjugate was found in the illuminated samples but no a-c cross-links were observed, not even after ATP-induced rotation of the c-ring. Our substituted diazirine moiety on c-Glu(65) was therefore in close contact with phospholipid but does not contact subunit a. Na(+)in/(22)Na(+)out exchange activity of the ATP synthase was not affected by modifying the c-Glu(65) sites with the carbodiimide, but upon photoinduced cross-linking, this activity was abolished. Cross-linking the rotor to lipids apparently arrested rotational mobility required for moving Na(+) ions back and forth across the membrane. The site of cross-linking was analyzed by digestions of the substituted POPC using phospholipases C and A(2) and by mass spectroscopy. The substitutions were found exclusively at the fatty acid side chains, which indicates that c-Glu(65) is located within the core of the membrane.     

Second stalk of ATP synthase. Cross-linking of gamma subunit in F1 to truncated Fob subunit prevents ATP hydrolysis

ATP synthase consists of two portions, F(1) and F(o), connected by two stalks: a central rotor stalk containing gamma and epsilon subunits and a peripheral, second stalk formed by delta and two copies of F(o)b subunits. The second stalk is expected to keep the stator subunits from spinning along with the rotor. We isolated a TF(1)-b'(2) complex (alpha(3)beta(3)gammadeltaepsilonb'(2)) of a thermophilic Bacillus PS3, in which b' was a truncated cytoplasmic fragment of F(o)b subunit, and introduced a cysteine at its N terminus (bc'). Association of b'(2) or bc'(2) with TF(1) did not have significant effect on ATPase activity. A disulfide bond between the introduced cysteine of bc' and cysteine 109 of gamma subunit was readily formed, and this cross-link caused inactivation of ATPase. This implies that F(o)b subunit bound to stator subunits of F(1) with enough strength to resist rotation of gamma subunit and to prevent catalysis. Contrary to this apparent tight binding, some detergents such as lauryldodecylamine oxide tend to cause release of b'(2) from TF(1).