Structural model of the transmembrane Fo rotary sector of H+-transporting ATP synthase derived by solution NMR and intersubunit cross-linking in situ

H(+)-transporting, F(1)F(o)-type ATP synthases utilize a transmembrane H(+) potential to drive ATP formation by a rotary catalytic mechanism. ATP is formed in alternating beta subunits of the extramembranous F(1) sector of the enzyme, synthesis being driven by rotation of the gamma subunit in the center of the F(1) molecule between the alternating catalytic sites. The H(+) electrochemical potential is thought to drive gamma subunit rotation by first coupling H(+) transport to rotation of an oligomeric rotor of c subunits within the transmembrane F(o) sector. The gamma subunit is forced to turn with the c-oligomeric rotor due to connections between subunit c and the gamma and epsilon subunits of F(1). In this essay we will review recent studies on the Escherichia coli F(o) sector. The monomeric structure of subunit c, determined by NMR, shows that subunit c folds in a helical hairpin with the proton carrying Asp(61) centered in the second transmembrane helix (TMH). A model for the structural organization of the c(10) oligomer in F(o) was deduced from extensive cross-linking studies and by molecular modeling. The model indicates that the H(+)-carrying carboxyl of subunit c is occluded between neighboring subunits of the c(10) oligomer and that two c subunits pack in a "front-to-back" manner to form the H(+) (cation) binding site. In order for protons to gain access to Asp(61) during the protonation/deprotonation cycle, we propose that the outer, Asp(61)-bearing TMH-2s of the c-ring and TMHs from subunits composing the inlet and outlet channels must turn relative to each other, and that the swiveling motion associated with Asp(61) protonation/deprotonation drives the rotation of the c-ring. The NMR structures of wild-type subunit c differs according to the protonation state of Asp(61). The idea that the conformational state of subunit c changes during the catalytic cycle is supported by the cross-linking evidence in situ, and two recent NMR structures of functional mutant proteins in which critical residues have been switched between TMH-1 and TMH-2. The structural information is considered in the context of the possible mechanism of rotary movement of the c(10) oligomer during coupled synthesis of ATP.     

Introduction of a carboxyl group in the first transmembrane helix of Escherichia coli F1Fo ATPase subunit c and cytoplasmic pH regulation

The multicopy subunit c of the H(+)-transporting F1Fo ATP synthase of Escherichia coli folds across the membrane as a hairpin of two hydrophobic alpha helices. The subunits interact in a front-to-back fashion, forming an oligomeric ring with helix 1 packing in the interior and helix 2 at the periphery. A conserved carboxyl, Asp(61) in E. coli, centered in the second transmembrane helix is essential for H+ transport. A second carboxylic acid in the first transmembrane helix is found at a position equivalent to Ile28 in several bacteria, some the cause of serious infectious disease. This side chain has been predicted to pack proximal to the essential carboxyl in helix 2. It appears that in some of these bacteria the primary function of the enzyme is H+ pumping for cytoplasmic pH regulation. In this study, Ile28 was changed to Asp and Glu. Both mutants were functional. However, unlike the wild type, the mutants showed pH-dependent ATPase-coupled H+ pumping and passive H+ transport through Fo. The results indicate that the presence of a second carboxylate enables regulation of enzyme function in response to cytoplasmic pH and that the ion binding pocket is aqueous accessible. The presence of a single carboxyl at position 28, in mutants I28D/D61G and I28E/D61G, did not support growth on a succinate carbon source. However, I28E/D61G was functional in ATPase-coupled H+ transport. This result indicates that the side chain at position 28 is part of the ion binding pocket.     

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 highly conserved extracellular peptide, DSYG(893-896), is a critical structure for sodium pump function.

The peptide sequence DSYG(893-896) of the sheep sodium pump alpha 1 subunit is highly conserved among all K(+)-transporting P-type ATPases. To obtain information about its function, single mutations were introduced and the mutants were expressed in yeast and analysed for enzymatic activity, ion recognition, and alpha/beta subunit interactions. Mutants of Ser894 or Tyr895 were all active. Conservative phenylalanine and tryptophan mutants of Tyr895 displayed properties that were similar to the properties of the wild-type enzyme. Replacement of the same amino acid by cysteine, however, produced heat-sensitive enzymes, indicating that the aromatic group contributes to the stability of the enzyme. Mutants of the neighbouring Ser894 recognized K(+) with altered apparent affinities. Thus, the Ser894-->Asp mutant displayed a threefold higher apparent affinity for K(+) (EC(50) = 1.4 +/- 0.06 mm) than the wild-type enzyme (EC(50) = 3.8 +/- 0.33 mm). In contrast, the mutant Ser894-->Ile had an almost sixfold lower apparent affinity for K(+) (EC(50) = 21.95 +/- 1.41 mm). Mutation of Asp893 or Gly896 produced inactive proteins. When an anti-beta 1 subunit immunoglobulin was used to co-immunoprecipitate the alpha 1 subunit, neither the Gly896-->Arg nor the Gly896-->Ile mutant could be visualized by subsequent probing with an anti-alpha 1 subunit immunoglobulin. On the other hand, co-immunoprecipitation was obtained with the inactive Asp893-->Arg and Asp893-->Glu mutants. Thus, it might be that Asp893 is involved in enzyme conformational transitions required for ATP hydrolysis and/or ion translocation. The results obtained here demonstrate the importance of the highly conserved peptide DSYG(893-896) for the function of alpha/beta heterodimeric P-type ATPases.     

Cross-linking and labeling of the Escherichia coli F1F0-ATP synthase reveal a compact hydrophilic portion of F0 close to an F1 catalytic subunit

The subunit arrangement of the F0 sector of the Escherichia coli ATP synthase is examined using hydrophilic and hydrophobic (cleavable) cross-linking reagents and the water-soluble labeling reagent [35S] diazoniumbenzenesulfonate ( [35S]DABS). Cross-linking is performed on purified ATP synthase and inverted minicell membranes. ATP synthase incorporated into liposomes is labeled with [35S]DABS. Three cross-linked products involving the F0 subunits (a, b, and c) are observed with the purified ATP synthase in solution: a-b, b2, and c2 dimers. A cross-link between the F0 and F1 is detected and occurs between the a and beta subunits. A cross-linker independent association between the b and beta subunits is also evident, suggesting that the two subunits are close enough to form a disulfide bridge. A cross-linking reagent stable to reducing agents produces a b-beta dimer, as detected by immunoblotting with anti-beta serum. The c subunit does not cross-link with any F1 polypeptide. Minicell membranes containing ATP synthase polypeptides radioactively labeled in vivo similarly show b2 and c2 dimers after cross-linking. [35S]DABS labels the a and b, but not c, subunits, showing that the a and b, but not c, subunits possess hydrophilic domains. Thus, certain domains of subunits a and b extend from the membrane and are in close proximity to one another and the F1 catalytic subunit beta.     

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

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

A homologous sequence between H+-ATPase (F0F1) and cation-transporting ATPases. Thr-285----Asp replacement in the beta subunit of Escherichia coli F1 changes its catalytic properties

A sequence of 10 amino acids (I-C-S-D-K-T-G-T-L-T) of ion motive ATPases such as Na+/K+-ATPase is similar to the sequence of the beta subunit of H+-ATPases, including that of Escherichia coli (I-T-S-T-K-T-G-S-I-T) (residues 282-291). The Asp (D) residue phosphorylated in ion motive ATPase corresponds to Thr (T) of the beta subunit. This substitution may be reasonable because there is no phosphoenzyme intermediate in the catalytic cycle of F1-ATPase. We replaced Thr-285 of the beta subunit by an Asp residue by in vitro mutagenesis and reconstituted the alpha beta gamma complex from the mutant (or wild-type) beta and wild-type alpha and gamma subunits. The uni- and multisite ATPase activities of the alpha beta gamma complex with mutant beta subunits were about 20 and 30% of those with the wild-type subunit. The rate of ATP binding (k1) of the mutant complex under uni-site conditions was about 10-fold less than that of the wild-type complex. These results suggest that Thr-285, or the region in its vicinity, is essential for normal catalysis of the H+-ATPase. The mutant complex could not form a phosphoenzyme under the conditions where the H+/K+-ATPase is phosphorylated, suggesting that another residue(s) may also be involved in formation of the intermediate in ion motive ATPase. The wild-type alpha beta gamma complex had slightly different kinetic properties from the wild-type F1, possibly because it did not contain the epsilon subunit.     

Nucleotide-binding sites in V-type Na+-ATPase from Enterococcus hirae

Enterococcus hirae V-ATPase, in contrast to most V-type ATPases, is resistant to N-ethylmaleimide (NEM). Alignment of the amino acid sequences of NtpA suggests that the NEM-sensitive Cys of V-type ATPases is replaced by Ala in E. hirae V-ATPase. Consistent with this prediction, the V-ATPase became sensitive upon substitution of the Ala with Cys. The three-dimensional structure of the NtpB subunit of V-ATPase was modeled based on the structure of the corresponding subunit (alpha subunit) of bovine F(1)-ATPase by homology modeling. Overall, the 3D structure of the subunit resembled that of alpha subunit of bovine F(1)-ATPase. The NtpB subunit, which lacks the P-loop consensus sequence for nucleotide binding, was predicted to bind a nucleotide at the modeled nucleotide-binding site. Experimental data supported the prediction that the E. hirae V-ATPase had about six nucleotide-binding sites.     

One intact ATP-binding subunit is sufficient to support ATP hydrolysis and translocation in an ABC transporter, the histidine permease.

The membrane-bound complex of the Salmonella typhimurium histidine permease, a member of the ABC transporters (or traffic ATPases) superfamily, is composed of two integral membrane proteins, HisQ and HisM, and two copies of an ATP-binding subunit, HisP, which hydrolyze ATP, thus supplying the energy for translocation. The three-dimensional structure of HisP has been resolved. Extensive evidence indicates that the HisP subunits form a dimer. We investigated the mechanism of action of such a dimer, both within the complex and in soluble form, by creating heterodimers between the wild type and mutant HisP proteins. The data strongly suggest that within the complex both subunits hydrolyze ATP and that one subunit is activated by the other. In a heterodimer containing one wild type and one hydrolysis defective subunit both hydrolysis and ligand translocation occur at half the rate of the wild type. Soluble HisP also hydrolyzes ATP if one subunit is inactive; its specific activity is identical to that of the wild type, indicating that only one of the subunits in a soluble dimer is involved in hydrolysis. We show that the activating ability varies depending on the nature of the substitution of a well conserved residue, His-211.     

An Exceptional Variability in the Motor of Archaeal A1A0 ATPases: From Multimeric to Monomeric Rotors Comprising 6–13 Ion Binding Sites

The motor domain of A1A0 ATPases is composed of only two subunits, the stator subunit I and the rotor subunit c. Recent studies on the molecular biology of the A0 domains revealed the surprising finding that the gene encoding subunit c underwent several multiplication events leading to rotor subunits comprising 2, 3, or even 13 hairpin domains suggesting multimeric in different stoichiometry as well as monomeric rotors. The number of ion translocating groups per rotor ranges from 13 to 6. Furthermore, as deduced from the gene sequences H(+)-as well as Na(+)-driven rotors are found in archaea. Features previously thought to be distinctive for A0, F0 or V0 are all found in A0 suggesting that the differences encountered in the three classes of ATPases today emerged already very early in evolution. The extraordinary features and exceptional structural and functional variability in the rotor of A1A0 ATPases may have arisen as an adaptation to different cellular needs and the extreme physicochemical conditions in the early history of life.     

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

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

All three subunits are required for the reconstitution of an active proton channel (F0) of Escherichia coli ATP synthase (F1F0).

The membrane-integrated, proton-translocating F0 portion of the ATP synthase (F1F0) from Escherichia coli is built up from three kinds of subunits a, b and c with the proposed stoichiometry of 1:2:10 +/- 1. We have dissociated the F0 complex by treatment with trichloroacetate (3 M) at pH 8.0, in the presence of deoxycholate (1%) and N-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate (Zwittergent 3-14, 5%). The subunits were separated by gel filtration with trichloroacetate (1 M) included in the elution buffer. The homogeneity of the fractions was checked by rechromatography and SDS-gel electrophoresis. After integration into phospholipid vesicles each subunit alone as well as all possible combinations were tested for H+ translocating activity and binding of F1. A functional H+ channel could only be reconstituted by the combination a1b2c10 which corresponds to that of native F0.     

Thermus thermophilus membrane-associated ATPase. Indication of a eubacterial V-type ATPase

An ATPase with Mr of 360,000 was purified from plasma membranes of a thermophilic eubacterium Thermus thermophilus, and was characterized. ATP hydrolytic activity of the purified enzyme was extremely low, 0.07 mumol of Pi released mg-1 min-1, and it was stimulated up to 30-fold by bisulfite. The following properties of the enzyme indicate that it is not a usual F1-ATPase but that it belongs to the V-type ATPase family, another class of ATPases found in membranes of archaebacteria and eukaryotic endomembranes. Among its four kinds of subunits with approximate Mr values of 66,000 (alpha), 55,000 (beta), 30,000 (gamma), and 12,000 (delta), the alpha subunit had a similar molecular size to the catalytic subunits of the V-type ATPases but was significantly larger than the alpha subunit of F1-ATPases. ATP hydrolytic activity was not affected by azide, an inhibitor of F1-ATPases, but was inhibited by nitrate, an inhibitor of the V-type ATPase. N-terminal amino acid sequences determined for the purified alpha and beta subunits showed much higher similarity to those of the V-type ATPases than those of F1-ATPases. Thus the distribution of the V-type ATPase in the prokaryotic kingdom may not be restricted to archaebacteria.     

The Na(+) cycle in Acetobacterium woodii: identification and characterization of a Na(+) translocating F(1)F(0)-ATPase with a mixed oligomer of 8 and 16 kDa proteolipids

The homoacetogenic bacterium Acetobacterium woodii relies on a sodium ion current across its cytoplasmic membrane for energy-dependent reactions. The sodium ion potential is established by a yet to be identified primary, electrogenic pump connected to the Wood-Ljungdahl pathway. Reactions possibly involved in Na(+) export are discussed. The electrochemical sodium ion potential generated is used to drive endergonic reactions such as flagellar rotation and ATP synthesis. Biochemical and molecular data identified the Na(+)-ATPase of A. woodii as a typical member of the F(1)F(0) class of ATPases. Its catalytic properties and the hypothetical sodium ion binding site in subunit c are discussed. The encoding genes were cloned and, surprisingly, the atp operon was shown to contain multiple copies of genes encoding subunit c. Two copies encode identical 8 kDa proteolipids, and a third copy arose by duplication and subsequent fusion of two genes. Furthermore, the duplicated subunit c does not contain the ion binding site in hair pin two. Biochemical and molecular data revealed that all three copies of subunit c constitute a mixed oligomer. The evolution of the structure and function of subunit c in ATPases from eucarya, bacteria, and archaea is discussed.     

The epsilon subunit of bacterial and chloroplast F(1)F(0) ATPases. Structure, arrangement, and role of the epsilon subunit in energy coupling within the complex

Recent studies show that the epsilon subunit of bacterial and chloroplast F(1)F(0) ATPases is a component of the central stalk that links the F(1) and F(0) parts. This subunit interacts with alpha, beta and gamma subunits of F(1) and the c subunit ring of F(0). Along with the gamma subunit, epsilon is a part of the rotor that couples events at the three catalytic sites sequentially with proton translocation through the F(0) part. Structural data on the epsilon subunit when separated from the complex and in situ are reviewed, and the functioning of this polypeptide in coupling within the ATP synthase is considered.     

Structure of Ala24/Asp61 --> Asp24/Asn61 substituted subunit c of Escherichia coli ATP synthase: implications for the mechanism of proton transport and rotary movement in the F0 complex

The structure of the A24D/D61N substituted subunit c of Escherichia coli ATP synthase, in which the essential carboxylate has been switched from residue 61 of the second transmembrane helix (TMH) to residue 24 of the first TMH, has been determined by heteronuclear multidimensional NMR in a monophasic chloroform/methanol/water (4:4:1) solvent mixture. As in the case of the wild-type protein, A24D/D61N substituted subunit c forms a hairpin of two extended alpha-helices (residues 5-39 and 46-78), with residues 40-45 forming a connecting loop at the center of the protein. The structure was determined at pH 5, where Asp24 is fully protonated. The relative orientation of the two extended helices in the A24D/D61N structure is different from that in the protonated form of the wild-type protein, also determined at pH 5. The C-terminal helix is rotated by 150 degrees relative to the wild-type structure, and the N-terminal helix is rotated such that the essential Asp24 carboxyl group packs on the same side of the molecule as Asp61 in the wild-type protein. The changes in helix-helix orientation lead to a structure that is quite similar to that of the deprotonated form of wild-type subunit c, determined at pH 8. When a decameric ring of c subunits was modeled from the new structure, the Asp24 carboxyl group was found to pack in a cavity at the interface between two subunits that is similar to the cavity in which Asp61 of the wild-type protein is predicted to pack. The interacting faces of the packed subunits in this model are also similar to those in the wild-type model. The results provide further evidence that subunit c is likely to fold in at least two conformational states differing most notably in the orientation of the C-terminal helix. Based upon the structure, a mechanistic model is discussed that indicates how the wild-type and A24D/D61N subunits could utilize similar helical movements during H(+) transport-coupled rotation of the decameric c ring.     

Rotation of the c subunit oligomer in EF(0)EF(1) mutant cD61N

ATP synthases (F(0)F(1)-ATPases) mechanically couple ion flow through the membrane-intrinsic portion, F(0), to ATP synthesis within the peripheral portion, F(1). The coupling most probably occurs through the rotation of a central rotor (subunits c(10)epsilon gamma) relative to the stator (subunits ab(2)delta(alpha beta)(3)). The translocation of protons is conceived to involve the rotation of the ring of c subunits (the c oligomer) containing the essential acidic residue cD61 against subunits ab(2). In line with this notion, the mutants cD61N and cD61G have been previously reported to lack proton translocation. However, it has been surprising that the membrane-bound mutated holoenzyme hydrolyzed ATP but without translocating protons. Using detergent-solubilized and immobilized EF(0)F(1) and by application of the microvideographic assay for rotation, we found that the c oligomer, which carried a fluorescent actin filament, rotates in the presence of ATP in the mutant cD61N just as in the wild type enzyme. This observation excluded slippage among subunit gamma, the central rotary shaft, and the c oligomer and suggested free rotation without proton pumping between the oligomer and subunit a in the membrane-bound enzyme.     

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.     

Structural and functional characterization of subunits of the F0 sector of the mitochondrial F0F1-ATP synthase.

Proteolytic digestion of F1-depleted submitochondrial particles (USMP), reconstitution with isolated subunits and titration with inhibitors show that the nuclear-encoded PVP protein, previously identified as an intrinsic component of bovine heart F0 (F01) (Zanotti, F. et al. (1988) FEBS Lett. 237, 9-14), is critically involved in maintaining the proper H+ translocating configuration of this sector and its correct binding to the F1 catalytic moiety. Trypsin digestion of USMP, under conditions leading to cleavage of the carboxyl region of the PVP protein and partial inhibition of transmembrane H+ translocation, results in general loss of sensitivity of this process to F0 inhibitors. This is restored by addition of the isolated PVP protein. Trypsin digestion of USMP causes also loss of oligomycin sensitivity of the catalytic activity of membrane reconstituted soluble F1, which can be restored by the combined addition of PVP and OSCP, or PVP and F6. Amino acid sequence analysis shows that, in USMP, modification by [14C] N,N'-dicyclohexylcarbodiimide of subunit c of F0 induces the formation of a dimer of this protein, which retains the 14C-labelled group. Chemical modification of cysteine-64 of subunit c results in inhibition of H+ conduction by F0. The results indicate that proton conduction in mitochondrial F0 depends on interaction of subunit c with the PVP protein.