Chemical crosslinking of alpha subunits in the F1 adenosine triphosphatase of Escherichia coli

The arrangement of the subunits in the F1 adenosine triphosphatase of Escherichia coli has been investigated using bifunctional chemical crosslinking agents to covalently link adjacent subunits in the enzyme molecule. The synthesis of the new cleavable crosslinking agent 2,2'-dithiobis(succinimidyl propionate) is described. The crosslinked products resulting from the reaction of the enzyme with 2,2'- and 3,3'-dithiobis(succinimidyl propionate), 3,3'-dithiobis(sulfosuccinimidyl propionate), disuccinimidyl tartrate, dimethyl adipimidate, 1-ethyl-3[3-(dimethylamino)propyl]carbodiimide, and 1,2:3,4-diepoxybutane were analyzed by "three-dimensional" polyacrylamide gel electrophoresis in which they were resolved first in a two-dimensional system. Following cleavage of the crosslinking bridge in the separated products, the constituent subunits were identified by a further one-dimensional gel electrophoresis step. This procedure greatly improved the precision with which crosslinked subunits could be identified. It largely overcame problems due to abnormal migration of crosslinked species on gel electrophoresis and to the formation of multiple species of the same crosslinked subunit dimers. The following crosslinked subunit dimers were identified: alpha alpha, alpha beta, beta gamma, alpha delta, beta epsilon, and gamma epsilon. The trimer alpha alpha delta was recognized. The formation of alpha alpha over alpha beta dimers was favored when more polar crosslinking agents were used. The constraints placed by the finding of adjacent alpha subunits upon current models for the arrangement of the subunits in the F1 ATPase are discussed.     

Sulfhydryl groups of the F1 adenosine triphosphatase of Escherichia coli and the stoichiometry of the subunits

The distribution and total number of sulfhydryl groups present in the F1 adenosine triphosphatase of Escherichia coli were used to calculate the stoichiometry of the alpha-delta subunits. Titration with 5,5'-dithiobis (2-nitrobenzoate) gave 19.1 +/- 2.2 sulfhydryl groups/mol ATPase. Labeling with [14C]iodoacetamide and [14C]N-ethylmaleimide showed that 11.9, 3.1, 1.9, and 1.8 sulfhydryl groups per molecule of ATPase were associated with the alpha, beta, gamma, and delta subunits, respectively. The epsilon subunit was not labeled. Application of the method of Creighton [Nature (London) (1980) 284, 487-489] showed that 4, 1, and 2 sulfhydryl groups were present in the alpha, beta, and gamma subunits, respectively. This, together with published data for the delta subunit, allowed a subunit stoichiometry of alpha 3 beta 3 gamma delta to be calculated. The presence of four cysteinyl residues in the alpha subunit, as shown by several different methods, does not agree with the results of DNA sequencing of the ATPase genes [H. Kanazawa, T. Kayano, K. Mabuchi, and M. Futai (1981) Biochem. Biophys. Res. Commun. 103, 604-612; N. J. Gay and J. E. Walker (1981) Nucl. Acids Res. 9, 2187-2194] where three cysteinyl residues/alpha subunit have been found. It is suggested that post-translational modification of the alpha subunit to add a fourth cysteinyl residue might occur.     

Assembly of the adenosine triphosphatase complex in Escherichia coli: assembly of F0 is dependent on the formation of specific F1 subunits.

A strain of Escherichia coli (AN1007) carrying the polar uncD436 allele which affects the operon coding for the F1-F0 adenosine triphosphatase (ATPase) complex was isolated and characterized. The uncD436 allele affected the two genes most distal to the operon promoter, i.e., uncD and uncC. Although the genes coding for the F0 portion of the ATPase complex were not affected in strains carrying this mutant allele, the lack of reconstitution of washed membranes by normal F1 ATPase suggested that a functional F0 might not be formed. This conclusion was supported by the observation that the 18,000-molecular-weight F0 subunit, coded for by the uncF gene, was absent from the membranes. Plasmid pAN36 (uncD+C+), when inserted into a strain carrying the uncD436 allele, resulted in the incorporation of the 18,000-molecular-weight F0 subunit into the membrane. A further series of experiments with Mu-induced polarity mutants, with and without plasmid pAN36, showed that the formation of both the alpha- and beta-subunits of F1 ATPase was an essential prerequisite to the incorporation into the membrane of the 18,000-molecular-weight F0 subunit and to the formation of a functional F0. Examination of the polypeptide composition of membranes from various unc mutants allowed a sequence for the normal assembly of the F1-F0 ATPase complex to be proposed.     

Structural characterization of the interaction of the delta and alpha subunits of the Escherichia coli F1F0-ATP synthase by NMR spectroscopy

A critical point of interaction between F(1) and F(0) in the bacterial F(1)F(0)-ATP synthase is formed by the alpha and delta subunits. Previous work has shown that the N-terminal domain (residues 3-105) of the delta subunit forms a 6 alpha-helix bundle [Wilkens, S., Dunn, S. D., Chandler, J., Dahlquist, F. W., and Capaldi, R. A. (1997) Nat. Struct. Biol. 4, 198-201] and that the majority of the binding energy between delta and F(1) is provided by the interaction between the N-terminal 22 residues of the alpha- and N-terminal domain of the delta subunit [Weber, J., Muharemagic, A., Wilke-Mounts, S., and Senior, A. E. (2003) J. Biol. Chem. 278, 13623-13626]. We have now analyzed a 1:1 complex of the delta-subunit N-terminal domain and a peptide comprising the N-terminal 22 residues of the alpha subunit by heteronuclear protein NMR spectroscopy. A comparison of the chemical-shift values of delta-subunit residues with and without alpha N-terminal peptide bound indicates that the binding interface on the N-terminal domain of the delta subunit is formed by alpha helices I and V. NOE cross-peak patterns in 2D (12)C/(12)C-filtered NOESY spectra of the (13)C-labeled delta-subunit N-terminal domain in complex with unlabeled peptide verify that residues 8-18 in the alpha-subunit N-terminal peptide are folded as an alpha helix when bound to delta N-terminal domain. On the basis of intermolecular contacts observed in (12)C/(13)C-filtered NOESY experiments, we describe structural details of the interaction of the delta-subunit N-terminal domain with the alpha-subunit N-terminal alpha helix.     

Structure-function relationships of domains of the delta subunit in Escherichia coli adenosine triphosphatase.

The topology of the and subunit of the Escherichia coli adenosinetriphosphatase (ECF1) has been explored by proteinase digestion and chemical labeling methods. The delta subunit of ECF1 could be cleaved selectively by reaction of the enzyme complex with very low amounts of trypsin (1:5000, w/w). Cleavage of the delta subunit occurred serially from the C-terminus. The N-terminal fragments of the delta subunit remained bound to the core ECF1 complex through sucrose gradient centrifugation, indicating that part of the binding of this subunit involves the N-terminal segment. ECF1, in which around 20 amino acids had been removed from the C-terminus of delta, still bound to ECF0 but DCCD sensitivity of the ATPase activity was lost. When ECF1 was reacted with N-ethyl[14C]maleimide ([14C]NEM) in the native state, only one of the two Cys residues on the delta subunit was modified. This residue, Cys-140, was also labeled in ECF1F0. Cys-140 was shown to be involved in the disulfide bridge between alpha and delta subunits that is generated when ECF1 is treated with CuCl2. Thus, the C-terminal part of the delta subunit around Cys-140 can interact with the core ECF1 complex. These results suggest a model for the delta subunit in which the central part of polypeptide is a part of the stalk, with both N- and C-termini associated with ECF1.     

Reaction of 2-azido-ATP with beta subunits in the F1-adenosine triphosphatase of Escherichia coli

The three beta subunits of the Escherichia coli F1-ATPase react independently with chemical reagents (Stan Lotter, H. and Bragg, P.D. (1986) Arch. Biochem. Biophys. 248, 116-120). Thus, one beta subunit is readily cross-linked to the epsilon subunit, another reacts with N,N'-dicyclohexylcarbodiimide (DCCD), and the third one is modified by 4-chloro-7-nitrobenzofurazan (NbfCl). The relationship of the binding site for 2-azido-ATP to the three types of beta subunit recognized by chemical labeling was examined. The binding site for 2-azido-ATP was not associated with a specific type of beta-subunit. There was no relationship between the site of nucleotide and the association of the epsilon subunit with a particular beta subunit. It is concluded that the presence of the epsilon subunit (possibly in association with the other minor subunits) does not determine the position of the catalytic site. The possibility that the lack of a specific relationship between the 2-azido-ATP binding site and a specific beta subunit was due to turnover of the enzyme, making each beta a catalytic site in turn, could not be entirely rejected. However, the rate of hydrolysis of 2-azido-ATP by the DCCD-modified ATPase was very low in the presence of EDTA, and was likely due to catalysis at single sites.     

Ligand-induced conformational changes in the Escherichia coli F1 adenosine triphosphatase probed by trypsin digestion

Digestion of the F1-ATPase of Escherichia coli with trypsin stimulated ATP hydrolytic activity and removed the delta and epsilon subunits of the enzyme. A species represented by the formula alpha 1(3) beta 1(3) gamma 1, where alpha 1, beta 1 and gamma 1 are forms of the native alpha, beta and gamma subunits which have been attacked by trypsin, was formed by trypsin digestion in the presence of ATP. In the presence of ATP and MgCl2, conversion of gamma to gamma 1 was retarded and the enzyme retained the epsilon subunit. These results imply that binding of ATP to the beta subunits alters the conformation of ECF1 to increase the accessibility of the gamma subunit to trypsin. The likely trypsin cleavage sites in the alpha, beta and gamma subunits are discussed. ECF1 from the alpha subunit-defective mutant uncA401, or after treatment with N,N'-dicyclohexylcarbodiimide or 4-chloro-7-nitrobenzofurazan, was present in a conformation in which the gamma subunit was readily accessible to trypsin and could not be protected by the presence of ATP and MgCl2. In a similar manner to native E. coli F1-ATPase, the hydrolytic activity of the trypsin-digested enzyme was stimulated by the detergent lauryldimethylamine N-oxide. Since the digested enzyme lacked the epsilon subunit, a putative inhibitor of hydrolytic activity, a mechanism for the stimulation which involves loss or movement of this subunit is untenable.     

Purification of membrane attachment and inhibitory subunits of the proton translocating adenosine triphosphatase from Escherichia coli.

The portion of Escherichia coli adenosine triphosphatase (ATPase) which is peripheral to the membrane (ECFl) is composed of five separate polypeptides referred to as alpha, beta, gamma, delta, and epsilon. Treating purified ECFl with pyridine precipitated the three larger polypeptides (alpha, beta, and gamma), but the two smaller ones (delta and epsilon), which represent only about 10% of ECFl, remained in solution. After removing the pyridine, both delta and epsilon were active and both were obtained in essentially pure form after chromatography on a single molecular-seive column. epsilon strongly inhibited the ATPase activity of ECFl, indicating that epsilon has a regulatory role in the enzyme. epsilon inhibited ECFl missing delta, indicating that delta is not required for inhibition by epsilon. However, enzyme containing just the alpha and beta subunits, which was prepared by treating ECFl with a protease, was fully active hydrolytically but not at all sensitive to inhibition by epsilon. This result suggests that the gamma polypeptide is required for the inhibition of the ATPase by epsilon. delta restored the capacity of ECFl missing delta to recombine with ECFl-depleted membrane vesicles. The ECFl, which became attached to the vesicles by the added delta, was functional in energy transduction, as evidenced by the coupling of ATP hydrolysis to the transhydrogenase reaction in the vesicles. The rebinding of ECFl missing delta was directly proportional to the amount of delta added until all the ECFl receptors in the membranes were occupied. delta may be a stalk which connects the Fl headpiece to the membrane, since the attachment of ECFl to the membrane exhibited an absolute dependence on delta. Although delta is known to have an apparent molecular weight of about 20,000 by gel electrophoresis in the presence of sodium dodecyl sulfate, the active delta eluted from a molecular-seive column with an apparent molecular weight of about 35,000, suggesting that in the active form delta is a dimer or rather elongated in shape. The active epsilon subunit eluted from the same column with an apparent molecular weight of about 16,000.     

Reaction of membrane-bound F1-adenosine triphosphatase of Escherichia coli with chemical ligands and the asymmetry of beta subunits

The three beta subunits of the isolated Escherichia coli F1-ATPase react independently with chemical reagents (Stan-Lotter, H. and Bragg, P.D. (1986) Arch. Biochem. Biophys. 284, 116-120). Thus, one beta subunit is readily cross-linked to the epsilon subunit, Another reacts with N,N'-dicyclohexylcarbodiimide (DCCD), and the third one is modified on a lysine residue by 4-chloro-7-nitrobenzofurazan (NbfCl). The binding site for the ATP analog, 2-azido-ATP, was not associated with a specific type of beta subunit (Bragg, P.D. and Hou, C. (1989) Biochim. Biophys. Acta 974, 24-29). We now show that this binding site is a catalytic site as opposed to a noncatalytic nucleotide-binding site. NbfCl reacted with a tyrosine residue on the DCCD-reacting beta subunit in contrast to the different subunit location of the lysine residue labeled by the reagent. Thus, O to N transfer of the Nbf group in the free F1-ATPase involves transfer between subunits. The chemical labelling pattern of membrane-bound F1-ATPase differed from that of free F1. The strict asymmetry of labeling of the free F1-ATPase was not observed. Thus, double labeling of beta subunits by several reagents was found. This suggests that the asymmetry was not induced by chemical modification, but is inherent in the structure of the ATPase.     

Site-directed cross-linking of b to the alpha, beta, and a subunits of the Escherichia coli ATP synthase

The b subunit dimer of the Escherichia coli ATP synthase, along with the delta subunit, is thought to act as a stator to hold the alpha(3)beta(3) hexamer stationary relative to the a subunit as the gammaepsilonc(9-12) complex rotates. Despite their essential nature, the contacts between b and the alpha, beta, and a subunits remain largely undefined. We have introduced cysteine residues individually at various positions within the wild type membrane-bound b subunit, or within b(24-156), a truncated, soluble version consisting only of the hydrophilic C-terminal domain. The introduced cysteine residues were modified with a photoactivatable cross-linking agent, and cross-linking to subunits of the F(1) sector or to complete F(1)F(0) was attempted. Cross-linking in both the full-length and truncated forms of b was obtained at positions 92 (to alpha and beta), and 109 and 110 (to alpha only). Mass spectrometric analysis of peptide fragments derived from the b(24-156)A92C cross-link revealed that cross-linking took place within the region of alpha between Ile-464 and Met-483. This result indicates that the b dimer interacts with the alpha subunit near a non-catalytic alpha/beta interface. A cysteine residue introduced in place of the highly conserved arginine at position 36 of the b subunit could be cross-linked to the a subunit of F(0) in membrane-bound ATP synthase, implying that at least 10 residues of the polar domain of b are adjacent to residues of a. Sites of cross-linking between b(24-156)A92C and beta as well as b(24-156)I109C and alpha are proposed based on the mass spectrometric data, and these sites are discussed in terms of the structure of b and its interactions with the rest of the complex.     

Insights into the rotary catalytic mechanism of F0F1 ATP synthase from the cross-linking of subunits b and c in the Escherichia coli enzyme

The transmembrane sector of the F(0)F(1) rotary ATP synthase is proposed to organize with an oligomeric ring of c subunits, which function as a rotor, interacting with two b subunits at the periphery of the ring, the b subunits functioning as a stator. In this study, cysteines were introduced into the C-terminal region of subunit c and the N-terminal region of subunit b. Cys of N2C subunit b was cross-linked with Cys at positions 74, 75, and 78 of subunit c. In each case, a maximum of 50% of the b subunit could be cross-linked to subunit c, which suggests that either only one of the two b subunits lie adjacent to the c-ring or that both b subunits interact with a single subunit c. The results support a topological arrangement of these subunits, in which the respective N- and C-terminal ends of subunits b and c extend to the periplasmic surface of the membrane and cAsp-61 lies at the center of the membrane. The cross-linking of Cys between bN2C and cV78C was shown to inhibit ATP-driven proton pumping, as would be predicted from a rotary model for ATP synthase function, but unexpectedly, cross-linking did not lead to inhibition of ATPase activity. ATP hydrolysis and proton pumping are therefore uncoupled in the cross-linked enzyme. The c subunit lying adjacent to subunit b was shown to be mobile and to exchange with c subunits that initially occupied non-neighboring positions. The movement or exchange of subunits at the position adjacent to subunit b was blocked by dicyclohexylcarbodiimide. These experiments provide a biochemical verification that the oligomeric c-ring can move with respect to the b-stator and provide further support for a rotary catalytic mechanism in the ATP synthase.     

Size and molecular parameters of adenosine triphosphatase from Escherichia coli.

The Mg2+- and Ca2+-stimulated ATPase (bacterial coupling factor) has been investigated in solution with different independent techniques. The molecular weight of the five-subunit enzyme was found to be 345,000 +/- 5,000 by means of light scattering, 350,000 by sedimentation equilibrium experiments, and 358,000 by means of small-angle x-ray scattering. The radius of gyration was found to be 41.9 A, the volume 7.39 x 10(5) A3, and the surface to volume ratio 5.5 x 10(-2) A-1 from small-angle x-ray scattering measurements of the enzyme in solution. The degree of hydration was found to be 0.62 ml of H2O/g of ATPase. The translational diffusion coefficient was determined to be 3.47 x 10(-7) cm2 s-1 by means of inelastic light scattering. The distribution of the scattered intensity near the origin appears to be bimodal, suggesting that the ATPase molecule is composed of spherical parts bound together by a flexible polypeptide chain. The largest dimension of the ATPase in solution is 120.0 A, determined from the pair distribution function.     

Kinetic properties of soluble adenosine triphosphatase of escherichia coli

Summary Bound and solubilized ATPase from Escherichia coli show similar kinetic properties. The saturation curves for MgATP are hyperbolic with both preparations. The straight lines in the Line-weaver–Burk plot indicate that MgATP is the true substrate, that one molecule MgATP is bound per enzyme molecule, and that there is no cooperativity. Presence of EDTA leads to sigmoidal saturation curves. This effect could be reversed by adding MgCl₂ stoichiometrically to EDTA.Different results in other publications, especially in that ofCarreira andMuñoz can be explained as being primarily the consequence of complexing agent contaminations in the assay.