©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Asymmetry of Escherichia coli F-ATPase as a Function of the Interaction of - Subunit Pairs with the and Subunits (*)

(Received for publication, May 12, 1995; and in revised form, June 16, 1995)

Margaret A. Haughton Roderick A. Capaldi (§)

From the Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403-1229

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The asymmetry of Escherichia coli F(1) -ATPase (ECF(1)) has been explored in chemical modification experiments involving two mutant enzyme preparations. One mutant contains a cysteine (Cys) at position 149 of the beta subunit, along with conversion of a Val to Ala at residue 198 to suppress the deleterious effect of the Cys for Gly at 149 mutation (mutant betaG149C:V198A). The second mutant has these mutations and also Cys residues at positions 381 of beta and 108 of the subunit (mutant betaG149C:V198A:E381C/S108C). On CuCl(2) treatment of this second mutant, there is cross-linking of one copy of the beta subunit to via the Cys at 381, a second to the subunit (between betaCys and Cys), while the third beta subunit in the ECF(1) complex is mostly free (some cross-linking to ); thereby distinguishing the three beta subunits as beta, beta, and beta, respectively. Both mutants have ATPase activities similar to wild-type enzyme.

Under all nucleotide conditions, including with essentially nucleotide-free enzyme, the three different beta subunits were found to react differently with N-ethylmaleimide (NEM) which reacts with Cys, dicyclohexyl carbodiimide (DCCD) which reacts with Glu, and 7-chloro-4-nitrobenzofurazan (NbfCl) which reacts with Tyr. Thus, beta reacted with DCCD but not NEM or NbfCl; beta was reactive with all three reagents; beta reacted with NEM, but was poorly reactive to DCCD or NbfCl. There was a strong nucleotide dependence of the reaction of Cys in beta (but not in beta) with NEM, indicative of the important role that the subunit plays in functioning of the enzyme.


INTRODUCTION

An F(1)F(0) type ATPase is found in the plasma membrane of bacteria, as well as chloroplast thylakoid and mitochondrial inner membranes, and this enzyme functions to synthesize ATP in response to a light or respiratory chain-driven proton gradient. It is a reversible enzyme, also working as an ATPase, using the hydrolysis of ATP to establish a pH gradient for subsequent use in ion transport processes. The simplest F(1)F(0) type ATPases structurally are those from bacteria. The enzyme from Escherichia coli (ECF(1)F(0)) (^1)is made up of a total of eight different subunits, five in the ECF(1) part (alpha, beta, , , and ) and three in the F(0) part (a, b(2), c) (Futai and Kanazawa 1983; Walker et al. 1984). The F(1) part of the enzyme from bacteria, mitochondria, and chloroplasts is remarkably similar, particularly with respect to alpha, beta, and subunits (Cross, 1988; Senior, 1990; Boyer, 1993). Earlier chemical studies had pointed to an intrinsic asymmetry of F(1), which is expected given a stoichiometry of three copies each of alpha and beta subunits but only one copy of , , and subunits. For example, it was shown that one of the beta subunits, which contain the catalytic sites, could be cross-linked to the subunit by the water-soluble carbodiimide EDC (Lötscher et al., 1984a). DCCD was found to react with only two of the three beta subunits, excluding the one that is linked to the subunit (Lötscher et al., 1984a, 1984b; Tommasino and Capaldi, 1985; Stan-Lotter and Bragg, 1986a). Also, it has been shown that NbfCl and DCCD can be reacted with different beta subunits (Stan-Lotter and Bragg, 1986b). In addition, clear asymmetry of the F(1) was observed on examination of F(1) by cryoelectron microscopy (Gogol et al., 1989a; Wilkens and Capaldi, 1994), as well as in studies of negatively stained specimens of the enzyme (Boekema and Böttcher, 1992).

The recent high resolution x-ray structure of MF(1) (Abrahams et al., 1994) confirms and provides more details of the asymmetry of the enzyme molecule in relation to the interaction of small subunits and nucleotide occupancy of catalytic sites. In the crystal form examined, one of the beta subunits (beta) contains ADP + Mg, and this beta is close to and may make contact with the N-terminal alpha-helical part of the subunit. A second beta subunit, containing bound AMP-PNP + Mg (beta), is linked via the DELSEED region to a short alpha-helix (residues 82-99 in the numbering system of ECF(1)) in the central part of the subunit. The third beta subunit, which is empty, (beta(E)), has several interactions with the C-terminal alpha-helical part of the subunit.

Unanswered, as yet, is whether differences in the structure of alpha-beta subunit pairs such as observed by Abrahams et al.(1994) are due to the association of small subunits, nucleotide binding, or both interactions. This is particularly relevant as a crystal form of rat liver F(1) has been reported which is claimed to be symmetrical with respect to features of the alpha and beta subunits and appears to have the small subunits, including and , scrambled (Bianchet et al., 1991; Pedersen et al., 1995).

We are using a combination of molecular biological and biophysical approaches to examine structure-function relationships in ECF(1)F(0) (e.g. Aggeler and Capaldi, 1992; Turina and Capaldi, 1994a, 1994b) Here, we describe studies in a mutant betaG149C:V198A, a functioning enzyme with a Cys residue in the catalytic site region (Iwamoto et al., 1993), thereby allowing probing of the conformation of this region under different nucleotide conditions. We have used this mutant, and a second mutant which includes the above mutations along with a Cys introduced into the DELSEED region of the beta subunit (through which different beta subunits can be cross-linked to and subunits, respectively) to study the asymmetry of the enzyme in relation to different nucleotide occupancies of catalytic sites.


EXPERIMENTAL PROCEDURES

Materials

[^14C]NEM and [^14C]DCCD were purchased from NEN DuPont, and Amersham Corp. respectively; Sephadex (G-50 DNA grade, fine, and G-25, medium) was from Pharmacia Biotech Inc.; the BCA protein assay was from Pierce; ATP assay kit was from BioOrbit (Finland); restriction enzymes were from Boehringer Mannheim and New England Biolabs; and NEM, Nbf-Cl (NBD-Cl), AMP-PNP, and other chemicals were from Sigma.

Strains and Construction of Plasmids Containing Mutations in the uncD, uncC, and uncG Genes

A 1.44-kb RsrII/EagI fragment containing the uncD mutation betaG149C:V198A was isolated from pBMUD14 (a generous gift from Dr. M. Futai, Osaka University, Japan) (Iwamoto et al., 1993) and inserted into the 11.23-kb RsrII/EagI fragment of the plasmid pRA100 coding for wild-type (derivitized pAN45 vector described in Aggeler et al., 1992), or pRA112 containing the uncG mutation S8C (Aggeler and Capaldi, 1992). The resulting plasmids pRA123 and pRA124 contain the mutations betaG149C:V198A and betaG149C:V198A/S8C, respectively, and all of the genes encoding the ECF(1)F(0) ATPase. Experiments showed that the presence of a Cys at position 8 of the subunit (for subsequent studies in which this site is reacted with a fluorophore) had no effect on modification by any of the reagents used here. Therefore, in some experiments, enzyme including this mutation was used. The plasmid pRA137, containing the mutation betaG149C:V198A:E381C/S108C, was constructed by ligation of the 6.7-kb EagI/EagI fragment of pRA123 (containing betaG149C:V198A) to the 5.9-kb fragment of pRA134 (containing betaE381C/S108C, Aggeler et al., 1995). The uncE. coli strain AN888 (uncB Mu::416 argH pyrE entA nalA recA) (Aggeler et al., 1992) was transformed with the plasmids pRA134 (Aggeler et al., 1995), pRA123, pRA124, and pRA137 (this work) for expression of mutant ECF(1). The wild-type E. coli strain AN1460 (pAN45/unc413::Mu argH pyrE entA nalA recA) is described in Aggeler et al.(1992). The plasmid pAN45, and strains AN1460 and AN888, were the generous gift of Graeme B. Cox, The Australian National University, Canberra). The E. coli strain XL1 Blue (Stratagene) was used for routine subcloning procedures (Davis et al., 1986; Sambrook et al., 1989).

Preparation of ECF(1) and ECF(1)F(0)

ECF(1) and ECF(1)F(0) were isolated by a modification of the methods of Wise et al.,(1981), Foster and Fillingame, (1979), described in Aggeler et al.(1987) and Gogol et al. (1989b). ECF(1), either wild-type or mutant, was precipitated in 70% (NH(4))(2)SO(4) for 1 h at 4 °C, then pelleted by centrifugation at 10,000 g for 20 min. The protein was dissolved in 50 mM MOPS, pH 7.0, 0.5 mM EDTA, and 10% glycerol (v/v) (MOPS pH 7.0 buffer) then passed through two consecutive centrifuge columns (Sephadex G-50, fine, 0.5 5.5 cm) (Penefsky, 1977), equilibrated in the same buffer at a concentration of 8-26 µM (3-10 mg/ml) in order to remove loosely bound nucleotides, as described in Aggeler et al.(1992).

Removal of Endogenous Nucleotides and Analysis of Bound ATP and ADP

ECF(1) (pRA124) was freed of endogenous nucleotides as described by Senior et al.(1992). Briefly, protein (10 mg) was precipitated twice in 70% saturated (NH(4))(2)SO(4) then passed through a column of Sephadex G-25, medium, (1 cm 100 cm, flow rate 0.8 ml/h, 23 °C) equilibrated in 100 mM Tris-H(2)SO(4), pH 8.0, 4 mM EDTA, and 50% (v/v) glycerol (Tris pH 8.0 buffer). Peak fractions were precipitated in 70% saturated (NH(4))(2)SO(4), centrifuged, dissolved in 100 mM MOPS, pH 7.0, 1 mM EDTA, and 50% (v/v) glycerol, and stored in liquid N(2). The concentration of endogenous nucleotide (ATP + ADP) in denatured F(1) plus standard ATP was determined by the luciferin/luciferase technique as described in Fromme and Gräber(1990), using the BioOrbit ATP assay kit and a FlowTech Engineering Luminometer model 1030. Total ATP content was estimated following incubation of denatured protein with phosphoenol pyruvate and pyruvate kinase (Fromme and Gräber, 1990; Senior et al., 1992). Mutant ECF(1) was found to contain 1.1-1.4 mol of nucleotide (ATP + ADP) cf. 1.3-1.5 mol of nucleotide/mol in wild-type enzyme after two gel centrifugation columns (and initial precipitation with (NH(4))(2)SO(4)). Enzyme treated to remove all nucleotides was found to contain 0.2-0.3 mol of (ATP + ADP)/mol.

Maleimide Reaction of ECF(1) and ECF(1)F(0)

For modification by maleimides, mutant ECF(1), either betaG149C:V198A or betaG149C:V198A:S8C (1-2 µM) from which loosely bound nucleotides had been removed (as above), was equilibrated for 30 min-1 h at room temperature in a buffer of 50 mM MOPS, pH 7.0, 0.5 mM EDTA, and 10% glycerol, with or without the addition of nucleotide. As specified in individual experiments, nucleotide was added to give aliquots containing 0.5 mM EDTA (EDTA), 5 mM ATP + 0.5 mM EDTA (ATP + EDTA), 5 mM ADP + 5.5 mM MgCl(2) + 5 mM NaH(2)PO(4) (ADP + Mg + P(i)), or 5 mM ATP + 5.5 mM MgCl(2) (ATP + Mg) (equivalent to ADP + Mg + P(i) after catalysis). [^14C]NEM was incorporated by the addition of 25 µM [^14C]NEM (2.6 mM stock in Me(2)SO, specific activity 40.0 mCi/mmol) or 200 µM [^14C]NEM (10 mM stock in Me(2)SO, specific activity 8.3 mCi/mmol) to 100-200-µl aliquots of F(1), where the final concentration of Me(2)SO did not exceed 2-4% (v/v). At specific time intervals (1-120 min), aliquots were withdrawn, and the reaction quenched by the addition of 10 mML-cysteine or 10 mM NEM. 20-50 µg of samples were dissociated in a DTT-containing buffer, pH 6.2, and subjected to 10-18% linear gradient SDS-PAGE (see below). For the analysis of cross-linked products, DTT was omitted from the dissociation buffer. The radioactivity in beta subunits was determined from gel slices and expressed as moles of [^14C]NEM incorporated per mole of enzyme (as described in Aggeler et al., 1987). The stoichiometry of incorporation of [^14C]NEM into the beta subunits was determined from an aliquot which was denatured in 2% SDS prior to reaction with 200 µM [^14C]NEM.

CuCl(2)-induced Cross-link Formation

ECF(1) (7.5-12.5 µM) from which loosely bound nucleotides had been removed (as above) was passed through two consecutive centrifuge columns equilibrated in 50 mM MOPS, pH 7.0, 30 µM CuCl(2), and 10% glycerol to induce disulfide bond formation between beta-, beta-, beta-, and alpha- (Aggeler et al., 1995). After incubation at room temperature for a further 30 min, the cross-linking reaction was stopped with 1 mM EDTA. For the analysis of cross-linked products, samples were dissolved in DTT-free dissociation buffer prior to 10-18% linear gradient SDS-PAGE.

Modification by DCCD and NbfCl

ECF(1) (2 µM) was reacted with 200 µM DCCD (10 mM ethanolic stock), or 200 µM [^14C]DCCD (10 mM ethanolic stock, 26.6 mCi/mmol) in MOPS pH 7.0 buffer (50 mM MOPS, 0.5 mM EDTA, and 10% (v/v) glycerol) for up to 3 h. For determination of ATPase activity, aliquots were withdrawn at time intervals of 30 s, 2, 5, 10, 15, 30, 60, 120, and 180 min and assayed immediately (5 µg/ml). In a parallel experiment, the rate of incorporation of [^14C]DCCD into betaGlu was determined from incorporation of ^14C into gel slices (as described above), at the same time intervals. Where indicated in figure legends, ECF(1) was equilibrated with nucleotides ATP, or ADP + P(i) + Mg, prior to reaction with DCCD, or [^14C]DCCD. Unreacted [^14C]DCCD was removed by passage through a centrifuge column of Sephadex G-50, fine, equilibrated in MOPS pH 7.0 buffer when subsequent chemical modification was required, or by addition of 10 mM NaOAc, pH 5.2 if loaded directly on SDS-polyacrylamide gels. Formation of cross-links between beta-, beta-, beta-, and alpha- (as described above) was induced both prior to, and subsequent to, incorporation of [^14C]DCCD.

Prior to modification by NbfCl, ECF(1) was reacted with CuCl(2) to induce formation of cross-links and the reaction stopped by addition of 1 mM EDTA. NEM (200 µM) was then added (for 1 h) to modify accessible Cys residues prior to reaction with NbfCl. Excess NEM was removed by transfer to a buffer containing 50 mM Tris-H(2)SO(4), 1 mM EDTA, and 10% (v/v) glycerol (Tris pH 7.5 buffer) by column centrifugation. Cross-linked ECF(1) (2-5 µM) was reacted with 500 µM NbfCl (10 mM ethanolic stock) in Tris pH 7.5 buffer for 1 h at 30 °C then unreacted NbfCl was removed by passage through a centrifuge column equilibrated in MOPS pH 7.0 buffer. The Nbf-modified F(1) was split into two aliquots, one of which was further modified by reaction with [^14C]DCCD (200 µM) for 3 h at room temperature, after which time unreacted [^14C]DCCD was quenched by addition of 10 mM NaOAc, pH 5.2. Aliquots were subjected to modified SDS-PAGE where the pH of the separating gel was lowered from 8.6 to 8.0 in order to increase the stability of the Tyr-Nbf adduct.

Calculation of Kinetic Constants

Kinetic constants describing the incorporation of [^14C]NEM and [^14C]DCCD were determined assuming pseudo-first-order reactions of one, two, or three independently reacting cysteinyl residues on ECF(1) using KaleidaGraph data analysis and graphics program for personal computer. For example, the following equation describes the reaction of two cysteinyl residues with NEM,

where P(1) = Cys-1-[^14C]NEM adduct, P(2) = Cys-2-[^14C]NEM adduct, C = concentration of protein (ECF(1)), B = initial concentration of ligand ([^14C]NEM), k(1), k(2) = second-order reaction constants for Cys-1, Cys-2, respectively, and t = time.

Other Methods

ATPase activity was measured with a regenerating system described by Lötscher et al. (1984b). Protein concentrations were determined using the BCA protein assay (Pierce). Trypsin cleavage (1:50 w/w, protease/protein) of wild-type and mutant (betaG149C:V198A) ECF(1) (3 µM) was carried out in 50 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, and 5 mM ATP, for 1 h at room temperature. SDS-polyacrylamide gel electrophoresis was routinely performed with a 4% stacking gel and 10-18% linear gradient separating gel (Laemmli, 1970) stained with Coomassie Brilliant Blue R (Downer et al., 1976). Samples were dissociated in 0.12 M Tris-HCl buffer, pH 6.2, 2% SDS, 5% glycerol, and 50 mM DTT prior to electrophoresis. For quantitation of yield of cross-linked products, Coomassie Brilliant Blue-stained gels were scanned with a Microtek flat-bed scanner and the intensity of protein bands digitized by the NIH Image 1.53 processing and analysis program for Macintosh. For identification of subunit composition of cross-linked products, gels were electroblotted to Immobilon polyvinylidene difluoride membranes (Millipore), incubated with monoclonal antibodies, and visualized as described previously (Gogol et al., 1989a; Aggeler et al., 1990; Mendel-Hartvig and Capaldi, 1991).


RESULTS

The double mutant betaG149C:V198A was obtained by Futai and colleagues as an enzymatically active revertant of the deleterious mutation betaG149C (Iwamoto et al., 1993). For our purposes, these two mutations were inserted into the plasmid pRA100 as described under ``Experimental Procedures.'' The ATPase activity of ECF(1) isolated from the mutant was 8-14 µmol of ATP hydrolyzed/min/mg as assayed at pH 7.5 in the presence of 2 mM ATP and 5 mM MgCl(2), an activity in the same range as that of the wild-type enzyme when measured under the same conditions. After removal of the subunit by trypsin cleavage, the activity of the mutant betaG149C:V198A increased to 40-55 µmol of ATP hydrolyzed/min/mg, again the same value obtained with wild-type ECF(1).

The Different Reactivities of the Three betaCys149 Residues Indicates Asymmetry of ECF(1) under All Nucleotide Conditions

The presence of Cys allows probing of the catalytic site region of each of the three beta subunits under various nucleotide conditions. For most experiments, enzyme was subjected to two column centrifugation steps to remove loosely bound nucleotide, and then reacted with [^14C]NEM, either 25 or 200 µM, for varying lengths of time. Such preparations were found to retain 1.1-1.4 mol of tightly bound nucleotide which, based on other studies, were assumed to be in non-catalytic sites. The time courses of labeling of the enzyme prepared under these conditions showed incorporation of around 2 mol of [^14C]NEM. This involves modification of only two of three beta subunits (see later) which react with rate constants differing by a factor of around 2 (Fig. 1, trace A). In the experiment in Fig. 1, the rate of incorporation of the first mole of NEM was calculated to be 88 M s and that of the second mole 38 M s. As the endogenous Cys in the beta subunits is buried, based on the lack of reactivity of this site in the wild-type enzyme, this labeling is in the introduced Cys. Some modification of the third copy of Cys could be achieved by prolonged incubation with high concentration of [^14C]NEM (500 µM), when incorporation of as much as 2.6 mol of maleimide/mol ECF(1) was obtained. However, the rate constant for reaction of this third beta subunit (0.2 M s) is negligible under the conditions used in the experiments reported here.


Figure 1: Kinetics of incorporation of [^14C]NEM into betaCys of mutant ECF(1) (betaG149C:V198A) under different nucleotide conditions. ECF(1) (2 µM) in MOPS pH 7.0 buffer containing EDTA (A, crosses), ATP + EDTA (B, circles), AMP-PNP + Mg (C, triangles), or ADP + Mg + P(i) (D, diamonds) was incubated with 25 µM [^14C]NEM for varying times up to 2 h. Aliquots were withdrawn at the times indicated, quenched with 10 mML-Cys, and electrophoresed on a 10-18% linear gradient SDS-polyacrylamide gel. The incorporation of radioactivity into betaCys (mol [^14C]NEM/mol F(1)) was determined in gel slices of beta subunits (described under ``Experimental Procedures''). The curve fits represent modification of 2 Cys residues according to .



One possible explanation of the observed asymmetry described above is that 1 mol of nucleotide is tightly bound in catalytic sites rather than in non-catalytic sites after two passages through centrifuge columns. To avoid this ambiguity, labeling studies were also conducted with ECF(1) freed of nucleotide according to Senior et al.(1992). In this procedure, the enzyme is twice precipitated from solution in (NH(4))(2)SO(4) and then subjected to gel filtration in a buffer containing 50% glycerol, 4 mM EDTA. After such treatment, the betaG149C:V198A mutant was found to retain less than 0.3 mol of nucleotide/mol of enzyme. Reaction of this essentially nucleotide-free enzyme with [^14C]NEM still gave the same labeling profiles as that obtained with enzyme retaining from 1 to 2 mol of tightly bound nucleotide (result not shown).

The profile of [^14C]NEM reaction with Cys of ECF(1) in the presence of 5 mM ATP (trace B) or 5 mM AMP-PNP + 5 mM MgCl(2) (trace C), conditions in which all three catalytic sites would be occupied by nucleotide, are also shown in Fig. 1. The rates of incorporation of NEM under these conditions were 84 M s and 16 M s in ATP, and 81 M s and 17 M s in AMP-PNP, respectively in Fig. 1. The data are similar to those obtained for ECF(1) without added nucleotides (trace A), except for an approximately 2-fold lower rate of incorporation of the second mole of NEM.

[^14C]NEM modification of ECF(1) in the presence of 5 mM ADP + 5 mg Mg + 5 mM P(i), both added directly or generated by catalytic turnover of added ATP (Fig. 1, trace D), also resulted in incorporation of only 2 mol of reagent. However, with all three catalytic sites occupied by ADP, the rates of NEM incorporation were considerably slower, i.e. 10 and 1 M s leading to incorporation of less that 1 mol of reagent under the conditions described in Fig. 1(i.e. 25 µM, 2 h of incubation). The stoichiometry of incorporation and calculation of kinetic constants (notably k(2)), under this nucleotide condition, was therefore determined using a higher concentration of [^14C]NEM (200 µM) when 2 mol of reagent could be reacted with the enzyme.

In different experiments, the absolute rates of NEM incorporation varied by a factor of around 20% with the relative differences observed under different nucleotide conditions always maintained. From Fig. 1, it is clear that the conformation of one or both of the NEM reactive, catalytic site regions is significantly different when ADP is bound compared with when ATP is bound or when the catalytic sites are empty.

[^14C]NEM Reaction with ECF(1)F(0) from the Mutant betaG149C:V198A

Profiles of [^14C]NEM incorporation into Cys in ECF(1)F(0) isolated from the mutant betaG149C:V198A are shown in Fig. 2. Again, only 2 mol of NEM were incorporated when experiments were conducted as described. For the two nucleotide conditions, AMP-PNP + Mg and ADP + Mg + P(i) (generated by turnover of the enzyme), the rates of NEM incorporation were similar to that in ECF(1). Importantly, the difference in rates of modification of Cys in ATP compared with ADP is retained in the intact ATP synthase complex (ECF(1)F(0)).


Figure 2: Kinetics of incorporation of [^14C] NEM into betaCys of mutant ECF(1)F(0) (betaG149C:V198A) under different nucleotide conditions. F(1)F(0) (5 µM) in MOPS pH 7.0 buffer containing AMP-PNP + Mg (triangles), or ATP + Mg (ADP + P(i) + Mg after catalysis) (squares), was incubated with 200 µM [^14C]NEM for up to 2 h. At the times indicated, the incorporation of radioactivity into Cys of beta subunits (mol [^14C]NEM/mol F(1)) was determined as described in Fig. 1.



The Catalytic Site Asymmetry of ECF(1) is Related to Interaction of beta Subunits with the and Subunits

We have recently described a mutant, betaE381C:S108C, in which CuCl(2) treatment induces essentially quantitative cross-linking between one beta subunit and the subunit (via the Cys at 381 of beta and probably the intrinsic Cys of the subunit) (Aggeler et al., 1995). A second beta subunit is cross-linked, again in near 100% yield, to the subunit (via Cys at 381 of beta and Cys of the subunit). A portion of the third beta subunit becomes cross-linked to the subunit, but the majority migrates on gels as free beta subunit (Aggeler et al., 1995). With this mutant, it is possible to differentiate the three beta subunits by their interactions with small subunits. By combining the mutants betaG149C:V198A and betaE381C/S108C, a novel mutant was constructed in which the different reactivities of Cys residues could be related to the association of the different beta subunits with the single copy and subunits.

The mutant betaG149C:V198A:betaE381C/S108C had an ATPase activity of 12-16 µmol of ATP hydrolyzed/min/mg, within the range obtained for wild-type enzyme. ECF(1) for this mutant was reacted first with CuCl(2) to generate disulfide bonds between beta Cys residues and the and subunits, respectively (Fig. 3). The cross-linked enzyme was then reacted with [^14C]NEM which becomes incorporated into betaCys along with any betaCys not involved in disulfide bond formation with or . The rate of reaction of NEM with the different beta subunits was followed by slicing and counting ^14C incorporated into the cross-linked products as well as into the non-cross-linked beta subunit, as a function of time. As shown in Fig. 4A, there was no incorporation of [^14C]NEM into the beta- cross-linked product under any of the nucleotide conditions tested. Therefore, the beta subunit, to which the short central alpha-helix of is bound, is the one most shielded from maleimide reaction. There was incorporation of up to 1 mol of ^14C into the beta- cross-linked product, the rate of which was nucleotide dependent (Fig. 4B). NEM modification of Cys in this beta subunit occurred at the fast rate observed for the reaction of Cys in the mutant betaG149C:V198A when ATP + EDTA was present, but occurred at the slower of the two rates in this mutant when ADP + Mg + P(i) was present. The nucleotide dependent switching in rates of modification of Cys in the beta subunit linked to is, therefore, more than 50-fold. In the time course of the labeling experiment, two NEM molecules were bound into the free beta subunit, 1 mol into Cys and the second into the (free) Cys. There was no significant nucleotide dependence of the rates of modification of these sites in the free beta subunit (Fig. 4C). Experiments with the mutant betaE381C have shown no alteration in the rate of [^14C]NEM modification of the Cys at 381 under different nucleotide conditions (results not shown).


Figure 3: Disulfide bond formation between betaCys and , , and subunits of mutant ECF(1) (betaG149C:V198A:E381C/S108C). Coomassie Brilliant Blue-stained 10-18% linear gradient SDS-PAGE of untreated (lane 1) and cross-linked mutant (lane 2) ECF(1). Cross-links were induced by passage of ECF(1) (7.5-12.5 µM) through two consecutive CuCl(2)-containing centrifuge columns then the reaction stopped by the addition of 1 mM EDTA. 40 µg aliquots were incubated in dissociation buffer in the absence of DTT prior to SDS-PAGE.




Figure 4: Kinetics of incorporation of [^14C]NEM into cross-linked beta subunits of mutant ECF(1) (betaG149C:V198A:E381C/S108C). Cross-linked mutant ECF(1) in MOPS pH 7.0 buffer containing ATP + EDTA (circles), or ADP + Mg + P(i) (diamonds), was incubated with 200 µM [^14C]NEM for varying times up to 2.5 h. At the times indicated, the incorporation of radioactivity into betaCys (mol [^14C]NEM/mol F(1)) was determined as described in Fig. 1for beta- (A), beta- (B), and free beta subunit (C).



Activity Effects of the Modification of Cys by NEM

Fig. 5plots the residual ATPase activity of ECF(1) in the mutant betaG149C:V198A as a function of incorporation of NEM into the Cys at 149. Reaction of 1 mol of NEM occurring predominantly in the beta subunit linked to when the reaction was done in ATP + EDTA (from Fig. 4) caused only a 60% inhibition of ATPase activity. At 2 mol of NEM incorporated, and therefore with modification of both the beta linked to and free beta subunit, ATPase activity was reduced to less than 2% of the untreated enzyme.


Figure 5: Correlation of ATPase activity with incorporation of [^14C]NEM into beta subunits of mutant ECF(1) (betaG149C:V198A). ECF(1), 1 µM or 2 µM, was equilibrated in MOPS pH 7.0 buffer containing ATP + EDTA (circles) or ATP + Mg (ADP + P(i) + Mg after catalysis) (squares), and incubated with 25 µM [^14C]NEM (ATP + EDTA) or 200 µM [^14C]NEM (ATP + Mg for up to 2 h. The incorporation of radioactivity into beta149 (mol [^14C]NEM/mol F(1)) was determined at various times from gel slices of the beta subunit isolated by SDS-PAGE. In a parallel experiment, the residual ATPase activity (% basal activity) was determined at the same time intervals under the same conditions. The dashed and dotted lines plot for full inhibition at 1 and 2 mol of NEM incorporated, respectively.



DCCD Modification of ECF(1) Examined with the Mutant betaE381C:S108C

Reaction of F(1) with DCCD has been shown to lead to incorporation of 1 or 2 mol/mol of enzyme (Satre et al., 1979; Esch et al., 1981; Yoshida et al., 1981; Tommasino and Capaldi, 1985), with evidence presented that the beta subunit linked to (based on cross-linking with EDC) is only poorly reactive with the reagent (Lötscher and Capaldi, 1984). The use of the mutant betaE381C/S108C allows the specificity of DCCD for the different beta mutants to be examined in more detail than previously possible. Fig. 6A shows the time dependence and Fig. 6B the molar incorporation of DCCD in relation to inhibition of ATPase activity of the mutant. The results are essentially the same as for wild-type (not shown, but see Tommasino and Capaldi, 1985) and show a Mg dependence but no nucleotide dependence of the modification of the enzyme by DCCD.


Figure 6: DCCD modification of mutant ECF(1) (betaE381C/S108C) under different nucleotide conditions and correlation of ATPase activity with incorporation of [^14C]DCCD. A, mutant ECF (2 µM) was equilibrated in MOPS pH 7.0 buffer containing EDTA (crosses), ATP + EDTA (circles), AMP-PNP + Mg (triangles), or ATP + Mg (ADP + P + Mg after catalysis) (squares), and incubated with 200 µM DCCD. Aliquots were withdrawn for assay of residual ATPase activity at various time intervals up to 3 h. B, in a parallel experiment, aliquots of ECF (2 µM) were equilibrated in MOPS pH 7.0 buffer containing ATP + EDTA (circles), AMP-PNP + Mg (triangles), or ATP + Mg (ADP + P + Mg after catalysis) (squares), and incubated with 200 µM [C]DCCD. At the times indicated in A, and at 2 and 3 h, aliquots were withdrawn and the reaction was quenched by addition of 10mM NaOAc, pH 5.2. The incorporation of radioactivity into betaGlu (mol [C]DCCD/mol F) was determined as described in Fig. 1and plotted against the corresponding ATPase activity.



Table 1gives data on incorporation of DCCD into the different beta subunits assessed by CuCl(2)-induced disulfide bond formation in the betaE381C/S108C mutant. In these experiments, enzyme was reacted for 1 h in EDTA-containing buffer, by which time between 1.3 and 1.6 mol of [^14C]DCCD had become incorporated into the enzyme with more than 90% inhibition of activity. The modification of ECF(1) by the hydrophobic carbodiimide occurred predominantly in the beta subunit linked to and in the free beta subunit, with no major preference between the two. Incorporation of DCCD into the beta linked to was low, as expected from previous studies, and may in part represent background labeling, given that there was a proportionally small labeling of and subunits in our experiments (result not shown).



Importantly, the same distribution of label, mainly into beta- and into the free beta subunit, was observed whether the DCCD reaction occurred first followed by disulfide bond formation to link beta subunits to their partner small subunits, or if cross-linking was performed first, followed by DCCD labeling.

NbfCl Reaction and DCCD Labeling of NbfCl-reacted ECF(1)

NbfCl has been found to inhibit F(1) (95%) when reacted under conditions that modify a Tyr in the beta subunit, i.e. Tyr on ECF(1) (Andrews et al., 1984). Recently, Weber et al. (1994) have shown that the NbfCl effect requires binding of 1 mol of reagent and that the resulting modification causes selective loss of the lowest affinity binding site for nucleotide, i.e. that with a K for MgATP of around 25 µM.

Fig. 7shows the labeling of ECF(1) from the mutant betaE381C/S108C with NbfCl. The reagent, detected by its fluorescence, binds predominantly to the free beta subunit (seen both in the beta subunit band and in the beta- cross-linked product), with a small amount of reaction in beta-, but no labeling of that beta linked to the subunit.


Figure 7: NbfCl modification of cross-linked mutant ECF(1) (betaE381C/S108C) followed by incorporation of [^14C]DCCD. A, cross-links were induced by passage of mutant ECF (7.5-12.5 µM) through two consecutive CuCl-containing centrifuge columns and the reaction stopped by the addition of 1 mM EDTA. Cross-linked mutant ECF was transferred to a Tris pH 7.5 buffer, and incubated with 500 µM NbfCl for 1 h at 30 °C. Excess NbfCl was removed by column centrifugation and an 80-µg aliquot subjected to SDS-PAGE in the absence of DTT. Lane 1, Coomassie Brilliant Blue-stained gel; lane 2, fluorogram of the same gel. B, relative intensities of NbfCl incorporation into the different beta subunits as observed upon UV illumination (302 nm) (A, lane 2).



DCCD reaction of NbfCl-labeled ECF(1) led to incorporation of [^14C]DCCD into the beta subunit linked to , and into the free beta subunit, to the same extent as with enzyme that had no prior reaction with NbfCl (Table 1).


DISCUSSION

The studies reported here exploit the recently described mutant of ECF(1), betaE381C/S108C, in which CuCl(2) induces high yield cross-linking between one beta subunit and , and a second beta subunit and the subunit. As a consequence, it is possible to distinguish the three beta subunits by their interaction with the small subunits. Additionally, a mutant was constructed that contained a Cys introduced into the catalytic site region (beta Cys) as well as Cys at beta residue 381 and at residue 108. Chemical modification studies were conducted: (i) with NEM to modify Cys, (ii) with DCCD, which reacts with betaGlu, and (iii) with NbfCl, which reacts at betaTyr.

With DCCD, the modification was introduced both prior to, and following, induction of disulfide bond formation between beta subunits and and subunits, respectively. Up to 2 mol of reagent were incorporated under both conditions with the same distribution of reagent between the three different beta subunits. This result establishes that the asymmetrical distribution of DCCD is not induced by the incorporation of the reagent, but reflects an intrinsic asymmetry of the enzyme. Furthermore, the data with DCCD are reassurance that cross-linked enzyme fairly reflects the structure of ECF(1), which is not significantly altered by disulfide bond formation between beta with , and beta with subunits.

The key finding of the work presented here is that the three different beta subunits have different conformations (shown schematically in Fig. 8), in the absence of nucleotides in catalytic sites and even when both catalytic and non-catalytic nucleotide-binding sites are empty. Therefore, there must be an intrinsic asymmetry of F(1) induced by interactions of the alpha-beta pairs with the small subunits and . One beta subunit, that which interacts directly with the short central alpha helix of the subunit (residues 82-99 in E. coli) (beta), is reactive to the hydrophobic carbodiimide DCCD but does not react with NbfCl. A Cys introduced at residue 149 in place of Gly in this copy of the beta subunit is shielded from reaction with NEM. The beta subunit linked to the subunit (beta) does not appear to bind DCCD at Glu and has very poor reactivity to NbfCl. Cys in this beta subunit is the most reactive of the three to NEM. The third beta subunit, the free beta subunit (beta), reacts readily with DCCD, Cys is modified by NEM, and this copy of the beta subunit is the primary site of reaction of NbfCl.


Figure 8: Schematic representation of the different conformations of the three beta subunits of ECF(1), designated beta, beta, and beta, relative to interactions of the alpha-beta subunit pairs with the single-copy subunits and . beta forms a cross-link between betaCys and Cys, reacts with DCCD but not with NbfCl, and residue Gly Cys (P-loop) of this beta subunit is shielded from reaction with NEM; beta forms a cross-link between betaCys and Cys, incorporates NEM into Cys, but reacts only very poorly with DCCD and NbfCl; beta is the primary site of reaction with NbfCl, reacts with DCCD, and Cys of this beta subunit is modified by NEM.



Asymmetry of the enzyme, as reflected by the different reactivities of the three beta subunits, is retained after binding of nucleotides into catalytic (and non-catalytic) sites under conditions where all sites would be occupied (i.e. 5 mM nucleotide). With either ATP + EDTA, AMP-PNP + Mg, or ADP + Mg + P(i) bound, DCCD reacted with beta and beta but not beta, while NEM reacted with beta, beta but not beta. The recent structure determination of Abrahams et al.(1994) establishes an asymmetry of F(1) under conditions different from those used here, in this case for enzyme with ADP in one catalytic site, AMP-PNP in a second catalytic site (that which is in the beta subunit linked to the short alpha helix of and, therefore beta in our terminology), and a third catalytic site empty of nucleotide. It is interesting to note that Weber et al.(1994) have shown that the three catalytic sites have the same affinity for ATP or ADP in the absence of Mg (i.e. in EDTA). Therefore, the catalytic sites can become equivalent without loss of the asymmetry induced by binding of the small subunits.

The chemical modification studies presented here provide interesting data on nucleotide-dependent conformational changes occurring in ECF(1). Binding of ATP to the enzyme under conditions that prevent hydrolysis of the substrate (ATP + EDTA or AMP-PNP + Mg) does not greatly alter the reactivity of Cys compared with when catalytic sites are empty of nucleotide. However, binding of ADP + Mg + P(i) has a profound effect. The reactivity of the enzyme to DCCD, in contrast, is not sensitive to nucleotide conditions. It is modulated by Mg, probably indirectly, by interaction of the cation with carboxyl groups, resulting in some shielding from the carbodiimide. Importantly, the nucleotide-dependent conformational change reflected in the altered reactivity of Cys occurs specifically in that beta subunit linked to the subunit. Conformational changes have been detected in the subunit during ATP hydrolysis (Mendel-Hartvig and Capaldi, 1991; Aggeler et al., 1992; Turina and Capaldi, 1994a) and ATP synthesis (Richter and McCarty, 1987), in concert with translocation of this subunit between an alpha and a beta subunit (Wilkens and Capaldi, 1994). The results presented here, then, add evidence to the proposal (Capaldi et al., 1994) that the subunit plays an important role in the functioning of ECF(1)F(0).


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. This work was supported by National Institutes of Health Grant HL 24526.

§
To whom correspondence should be addressed. Tel.: 503-346-5881.

(^1)
The abbreviations used are: ECF(1), soluble portion of the Escherichia coli F(1)F(0) ATP synthase; ECF(1)F(0), Escherichia coli F(1)F(0) ATP synthase; MOPS, 3-(N-morpholino)propanesulfonic acid; NEM, N-ethylmaleimide; DCCD, dicyclohexyl carbodiimide; NbfCl, 7-chloro-4-nitrobenzofurazan; AMP-PNP, 5`-adenylyl-beta,-imidodiphosphate; DTT, dithiothreitol; Me(2)SO, dimethyl sulfoxide; kb, kilobase(s); PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Kathy Chicas-Cruz for expert technical assistance and Dr. R. Aggeler for helpful discussions.


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