©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Disulfide Bond Formation between the COOH-terminal Domain of the Subunits and the and Subunits of the Escherichia coli F-ATPase
STRUCTURAL IMPLICATIONS AND FUNCTIONAL CONSEQUENCES (*)

Robert Aggeler , Margaret A. Haughton , Roderick A. Capaldi (§)

From the (1) Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

A set of mutants of the Escherichia coli FF-type ATPase has been generated by site-directed mutagenesis as follows: E381C, S383C, E381C/S108C, and S383C/S108C. Treatment of ECFisolated from any of these mutants with CuClinduces disulfide bond formation. For the single mutants, E381C and S383C, a disulfide bond is formed in essentially 100% yield between a subunit and the subunit, probably at Cysbased on the recent structure determination of F(Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628). In the double mutants, two [Medline] disulfide bonds are formed, again in essentially full yield, one between and , the other between a and the subunit via Cys. The same two cross-links are produced with CuCltreatment of ECFFisolated from either of the double mutants. These results show that the parts of around residue 87 (a short -helix) and the subunit interact with different subunits.

The yield of covalent linkage of to is nucleotide dependent and highest in ATP and much lower with ADP in catalytic sites. The yield of covalent linkage of to is also nucleotide dependent but in this case is highest in ADP and much lower in ATP. Disulfide bond formation between either and , or and inhibits the ATPase activity of the enzyme in proportion to the yield of the cross-linked product. Chemical modification of the Cys at either position 381 or 383 of the subunit inhibits ATPase activity in a manner that appears to be dependent on the size of the modifying reagent. These results are as expected if movements of the catalytic site-containing subunits relative to the and subunits are an essential part of the cooperativity of the enzyme.


INTRODUCTION

An H-ATPase is found in the plasma membrane of bacteria, the inner membrane of mitochondria, and the thylakoid membrane of chloroplasts. This enzyme catalyzes both ATP synthesis coupled to an electrochemical gradient and ATP hydrolysis-driven proton translocation. The enzyme complex is made up of two parts: an Fpart composed of five different subunits, , , , , and in the molar ratio 3:3:1:1:1 and an Fsector which, in bacterial enzymes such as that from Escherichia coli (ECFF),() contains subunits a, b, and c in the ratio 1:2:10-12 (reviewed in Cross, 1988; Senior, 1990; Boyer, 1993).

Low resolution cryoelectron microscopy studies show the Fpart separated from the Fby a narrow stalk of around 45 Å in length (Gogol et al., 1987; Lücken et al., 1990). This stalk region includes parts of the , , and subunits of Fand b subunits of the Fpart (Capaldi et al., 1994; Wilkens et al., 1994). Catalytic site events (ATP synthesis and ATP hydrolysis) appear to be linked to proton translocation via conformational changes in these stalk-forming subunits. Electron microscopy studies have also shown that the and subunits alternate in a hexagonal arrangement around a central cavity containing the subunit (Gogol et al., 1989a, 1989b). The recent high resolution structure of MF(mitochondrial F) (Abrahams et al., 1994) adds detail to this picture. The and subunits have a very similar fold, each made up of three domains. At the top (farthest from the Fpart) is an amino-terminal six-stranded barrel. The more central domain contains the nucleotide-binding domain, which in the subunits includes the (three) catalytic sites, while the subunits contain the (three) non-catalytic sites. The third and bottom domain (closest to the Fmoiety) consists of a carboxyl-terminal bundle of seven helices in the subunit and six helices in the subunit. In the COOH-terminal domain of the subunits is the so-called ``DELSEED'' region which has been implicated in binding of the subunit (Dallmann et al., 1992) and which is seen from the x-ray data to interact tightly with a segment of the subunit (Abrahams et al., 1994).

We have been examining structure-function relationships in ECFFby introducing Cys residues at various sites in the enzyme complex, which can be used to incorporate reporter groups of the environment of the site, as well as (conformational) changes that occur around the site during functioning of the enzyme. In recent work, we have introduced Cys residues into the and subunits, and by using fluorescent probes (Turina and Capaldi, 1994a, 1994b) as well as cross-linking reagents ( e.g. Aggeler et al., 1992, 1993), have provided evidence for large conformational changes in these subunits during coupling of ATP hydrolysis to proton translocation. Here we describe experiments in which we have introduced Cys residues into the DELSEED region of the subunit in conjunction with mutating a Cys residue into the COOH-terminal region of the subunit (S108C). With these mutants, we were able to reversibly generate disulfide bonds between subunits and both the and subunits. The reactivity of the Cys residues in the DELSEED region with maleimides has been studied. In addition, the functional consequences of such modifications, and of disulfide bond formation between and and between and subunits both together and independently, have been examined.


EXPERIMENTAL PROCEDURES

Construction of Plasmids Containing Mutations in the unc D and unc C genes

A 1.01-kb NcoI fragment encoding the COOH-terminal part of the subunit was isolated from pRA100 (Aggeler et al., 1992) and inserted into M13mp18 (New England Biolabs) in an NcoI site that was created by introduction of an NcoI linker (Boehringer Mannheim, GmbH.) into the SmaI site.

In the site-directed mutagenesis step (Kunkel et al., 1987) the oligonucleotides CTTCAGACAGACAATCCATACCC and TTGTCTTCTTCACACAGTTCATCCAT were used for replacing a glutamate at position 381 and a serine at position 383 each with cysteine. The successful incorporation of the mutations was determined by sequencing (Sequenase from United States Biochemical Corp., Cleveland, OH ).

The mutations were inserted in an unc operon containing plasmid in two steps: (i) the 5.8-kb XhoI/ NsiI fragment from pRA100 and pRA102, respectively, was inserted in pBluescript SK+ (Stratagene), which had been previously modified by placing an NsiI linker (New England Biolabs) in the EagI site. These plasmids, pRA13 and pRA14, contained the unc C coding for wild-type subunit and mutant S108C, respectively; (ii) the NcoI fragments containing mutations E381C and S383C were then inserted in pRA13 and pRA14. Finally, the 5.8-kb XhoI/ NsiI fragments from these four constructs were ligated to the 6.8-kb XhoI/ NsiI portion of pRA100. The resulting plasmids were pRA133, pRA134, pRA135, and pRA136, containing the mutations E381C, E381C/S108C, S383C, and S383C/S108C, respectively.

CuClInduced Cross-linking of ECFand ECFF

ECF-ATPase was precipitated for 1 h in 70% ammonium sulfate at 4 °C and collected by centrifugation at 10,000 g for 15 min. The protein was dissolved in 50 m M MOPS, pH 7.0, 0.5 m M EDTA, and 10% glycerol and passed through two consecutive centrifuge columns in Sephadex G-50 equilibrated in the same buffer at a concentration between 2-5 mg/ml, as described in Aggeler et al. (1992). Cross-linking was carried out in either of two ways: (i) the enzyme was passed through two centrifuge columns, equilibrated in 50 m M MOPS, pH 7.0, 10% glycerol, and 20 µ M CuCl(Bragg and Hou, 1986; Tozer and Dunn, 1986); (ii) the ATPase solution was diluted to 0.1 m M EDTA, supplemented with 2.5 m M MgCland 2 m M nucleotide, followed by incubation with 20 or 50 µ M CuClfor 3 h at room temperature.

ATP synthase was reconstituted into egg-lecithin vesicles by first adding 10% sodium deoxycholate to ECFFfrom the sucrose gradient at 1 mg/ml to a final concentration of 0.5%. After 10 min on ice, 25 µl of 20 mg/ml egg-lecithin (in 1.5% sodium deoxycholate) was added per ml, followed by another 10-min incubation on ice. The mixture was then passed through a Sephadex G-50 column (medium, 1 45 cm) (Brunner et al., 1978), equilibrated in 50 m M Tris-HCl, pH 7.5, 2 m M MgCl, 2 m M DTT, and 10% glycerol. Turbid fractions containing ATPase activity were pooled and vesicles pelleted by centrifugation for 1 h at 45,000 revolutions/min at 4 °C in a Beckman Ti60 rotor. The pellets were resuspended in the same buffer and kept frozen in liquid nitrogen. For cross-linking with CuCl, 1 mg of ECFFwas brought to a volume of 2 ml with 50 m M MOPS, pH 7.0, 2 m M MgCl, and 10% glycerol and centrifuged twice at 20 °C for 25 min at 70,000 revolutions/min in a Beckman TLA100.2 rotor to remove reducing agent. The pellet was resuspended in the same buffer at 0.7-1 mg/ml, samples were supplemented with 2 m M nucleotide, and cross-linked with 20 µ M CuClfor 3 h at room temperature. ATPase activity was measured with or without prior treatment with 10-20 m M DTT for 2 to 3 h at room temperature.

Maleimide Modifications of ECF

The rate of incorporation of [C]NEM into Cysof the subunit of E381C/S108C was determined by reacting ATPase at a concentration of 0.4 mg/ml in 50 m M MOPS, pH 7.0, 0.5 m M EDTA, 5 m M ATP, and 10% glycerol with 25 µ M [C]NEM (specific activity 40 mCi/mmol) at room temperature. Aliquots were withdrawn at time intervals of 1, 10, 30, 60, and 150 min and quenched by the addition of 10 m M L-cysteine. For determination of stoichiometry of incorporation (mol [C]NEM/mol F), an additional aliquot denatured by 2% SDS was reacted with 200 µ M [C]NEM for 150 min. The aliquots were then electrophoresed on a 10-18% SDS-polyacrylamide gel and the radioactivity in individual subunits measured as described in Aggeler et al. (1987). The change of ATPase activity, due to the incorporation of the maleimides NEM or CM into Cys, was determined in a parallel experiment under identical conditions. Aliquots of a reaction mixture containing 25 µ M maleimide were quenched by the addition of 20 m M DTT at the time intervals 1, 10, 30, 60, and 150 min and ATPase activity determined.

Other Methods

E. coli strains used for routine cloning procedures (Davis et al., 1986; Maniatis et al., 1982) and site-directed mutagenesis according to Kunkel et al. (1987) were XL1-Blue from Stratagene and CJ236 from New England Biolabs. For expression of mutant ATPase and synthase, the plasmids pRA133-pRA136 were used to transform the uncstrain AN888 (Aggeler et al. 1992). ECFand ECFFwere isolated as described by Wise et al. (1981), modified by Gogol et al. (1989a) and Aggeler et al. (1987). -Depleted ECFwas prepared by the use of the monoclonal antibody -4 as described by Dunn (1986). Protein concentrations were determined using the BCA protein assay from Pierce. ATPase activity was measured with a regenerating system described by Lötscher et al. (1984). For the analysis of cross-link products, SDS containing 10-18% polyacrylamide gels were run (Laemmli, 1970), and polypeptides were blotted on nitrocellulose membranes and identified with monoclonal antibodies (Aggeler et al., 1990; Mendel-Hartvig and Capaldi, 1991a). Protein bands on gels were visualized by staining with Coomassie Brilliant Blue R according to Downer et al. (1976).


RESULTS

Mutants in the DELSEED region were prepared in which a cysteine was introduced in place of a glutamate at residue 381 (E381C) or a cysteine replacing a serine at residue 383 (S383C). In addition, double mutants were generated with the same mutations in the DELSEED region along with the mutation of a serine to a cysteine at position 108 of the subunit (E381C/S108C; S383C/S108C). The four different mutants grew well, and the activities of the isolated Fof each and FFfrom the two double mutants were similar to wild-type enzyme, i.e. in the range 9-15 µmol ATP/min/mg for the Fand 25-35 µmol/min/mg for the FFpreparations. All four mutants showed normal LDAO activation, i.e. 8-fold for the Fand 3-4-fold for the FF.

The DELSEED Region Interacts with Both the and Subunits

CuClwas used to induce disulfide bond formation using the isolated Fand FFfrom the various mutants with or without gel filtration centrifugation. Fig. 1 shows data for the two double mutants. Passage of ECFthrough a centrifuge column in the presence of 20 µ M of CuClgenerated four major cross-link products, as revealed by SDS-polyacrylamide gel electrophoresis of samples in the absence of reducing agents (Fig. 1), which Western blotting with monoclonal antibodies to the individual subunits of the enzyme showed to involve +, +, +, and + (result not shown). The effect of cross-linking on ATPase activity is listed above each lane. The residual activity of both mutants was less than 10% of that of the untreated enzyme. The only cross-linked products seen in wild-type ECFtreated similarly were between the and subunits (results not shown, but see Mendel-Hartvig and Capaldi, 1991b). This cross-linking of to had no effect on ATPase activity.


Figure 1: Cross-linking of ECF-ATPase from double mutants on CuCl containing centrifuge columns. 1 mg of ATPase, precipitated with ammonium sulfate, was dissolved in 0.2 ml of 50 m M MOPS, pH 7.0, 0.5 m M EDTA, and 10% glycerol and passed twice through a centrifuge column in the same buffer. Half of the sample was then applied on two consecutive columns containing 20 µ M CuCland no EDTA. The other half was left untreated. After addition of 5 m M EDTA, ATPase activities were measured and expressed as relative values with the untreated sample as 100%. 50-µg samples were electrophoresed on an 10-18% polyacrylamide gel after addition of 20 m M NEM and dissociation buffer without reducing agent. E381C/S108C without ( lane 1) and with CuCltreatment ( lane 2); S383C/S108C without ( lane 3) and with CuCltreatment ( lane 4).



Cross-linking of to via disulfide bond formation must involve the introduced Cys in the DELSEED region and the introduced Cysof the subunit. The yield of this product is close to 100% in both mutants, based on the disappearance of the subunit. The cross-linking between and must involve the Cys introduced into the DELSEED region and either Cysor Cys, the two endogenous cysteines of the subunit. Based on the recently published x-ray structure of MF(Abrahams et al., 1994), it is more likely that Cysis involved. The yield of the - cross-link is 90-95% in the mutant E381C/S108C and somewhat less in the mutant S383C/S108C. In wild-type ECF, the yield of the + cross-linked product is essentially 100%. In the DELSEED mutants, the yield of + is significantly less because of the presence of a + cross-link product (discussed later). In the mutant S383C/S108C, the + cross-linked product migrates differently from +; this is not the case for the mutant E381C/S108C (Fig. 1).

Disulfide Bond Formation between and Inhibits ATPase Activity and Is Nucleotide-dependent

When ECFfrom either double mutant was reacted with CuClin the absence of the column centrifugation step, the same cross-link products were obtained. However, the yield in which the disulfide bonds were formed was now determined by the concentration of CuClused, and the length of time for which samples were incubated before the reaction was stopped by addition of EDTA to chelate the metal ion. Preliminary experiments showed that disulfide bond formation between and was much greater than that between +, +, or + under the conditions of cross-linking chosen. This made it possible to examine both the nucleotide dependence of this specific cross-link and its effect on ATPase activity (Fig. 2). As shown for the mutant E381C/S108C, the yield of the + cross-link product was high in the absence of nucleotides, i.e. in Mgalone ( lane 1) and high in Mg+ADP, either added directly ( lane 3) or as generated by addition of ATP+Mgfollowed by turnover of the enzyme ( lane 2). However, the yield of this cross-link was very low (less than 10%) in the presence of AMPPNP+Mg( lane 4) or with ATP+Mgwhen azide was added to prevent enzyme turnover (result not shown). The cross-linking yield obtained with the mutant S383C/S108C was much lower (lessthan 10%). The inhibition of ATPase activity was in proportion to the yield of cross-linked products, i.e. in the range 70-75% in ADP for the mutant E381C/S108C, but below 10% in the presence of AMPPNP. Fig. 2shows also the important finding that the cross-linking could be reversed by adding DTT. This is true, whether the cross-linking was conducted by adding CuClwith or without the gel filtration centrifugation step. The activity determination reported above each lane in Fig. 2, and in subsequent figures, is the rate of ATPase hydrolysis of the cross-linked enzyme as a percentage of the activity of the same sample after adding DTT to break the linkages.


Figure 2: Cross-linking of ECF from E381C/S108C without the use of centrifuge columns. 2.5 m M MgClwas added to ATPase in 50 m M MOPS, pH 7.0, 0.1 m M EDTA, and 10% glycerol at 0.3 mg/ml. 0.2 ml samples were supplemented with no nucleotides ( lanes 1 and 5), 2 m M ATP ( lanes 2 and 6), 2 m M ADP ( lanes 3 and 7) and 2 m M AMPPNP ( lanes 4 and 8), respectively, incubated for 10 min at room temperature, and treated with 20 µ M CuClfor 3 h. 5 m M EDTA was added, and activities were measured after 2 h in the absence ( lanes 1-4) or presence of 10 m M DTT ( lanes 5-8). 30 µg of enzyme was applied per lane after addition of 40 m M NEM and DTT-free dissociation buffer. ATPase activities are expressed as percentage of DTT-treated samples. The ATPase activities of the control samples in lanes 5-7 were 11 µmol of ATP hydrolyzed/min/mg. The activity of the sample reacted with AMPPNP and then diluted in the assay buffer was 5 µmol of ATP hydrolyzed/min/mg.



Disulfide Bond Formation between and Inhibits ATPase Activity and Has the Opposite Nucleotide Dependence of the + Linkage

The nucleotide dependence and activity effects of the + cross-link could be examined independently of the + cross-link by using the single mutants in which an introduced cysteine was present in the DELSEED region, but the subunit was wild-type. Fig. 3 A shows cross-linking of the single mutants E381C and S383C using the CuClgel filtration centrifugation procedure. The 90% yield of cross-linking of to gave a 90% inhibition of ATPase activity. Fig. 3 B shows cross-linking of to in the mutant E381C generated by addition of 50 µ M CuClwithout a column step. The yield of + was low in Mg+ADP ( lane 1), or in Mg+ADP+P( lane 2). In contrast, the yield of this cross-linked product was high with Mg+ATP added in the presence of azide to prevent enzyme turnover ( lane 3) or with Mg+AMPPNP added ( lane 4). Under all nucleotide conditions, the inhibition of activity mirrored the extent of + cross-linking, being around 10% with ADP in catalytic sites (the experiment in Fig. 3), and 70% with ATP bound. The overall yield of cross-linked products obtained with the mutant S383C was much lower than that for E381C, when experiments such as those in Fig. 3 B were conducted under the identical conditions of CuClreaction.


Figure 3: Cross-linking of ECF from single mutants E381C and S383C. A, 0.7 mg of ATPase from mutants E381C ( lanes 1 and 2) and S383C ( lanes 3 and 4) in 0.2 ml was passed, as described in Fig. 1, twice through EDTA containing columns, followed by two CuCl-containing columns. The cross-linked material (1.6 mg/ml) was either incubated in the absence ( lanes 1 and 3) or presence of 20 m M DTT ( lanes 2 and 4). 60 µg of ATPase was applied per lane. B, 2.5 m M MgClwas added to 0.2-ml aliquots of 0.3 mg/ml ATPase from mutant E381C in 50 m M MOPS, pH 7.0, 0.1 m M EDTA and 10% glycerol, followed by 2 m M ADP ( lane 1), 2 m M ADP + 2 m M P( lane 2), 2 m M NaN+ 2 m M ATP ( lane 3), and 2 m M AMPPNP ( lane 4). Samples were treated for 4 h with 50 µ M CuClat room temperature and 30 µg of protein applied on a gel as described in Fig. 2. The ATPase activities of samples used in the experiments in lanes 2 and 4 of part A and the controls for lanes 1 and 2 of part B were in the range 7-9 µmol of ATP hydrolyzed/min/mg. The activity of the control for lane 3B, in which the sample with azide had been diluted in the assay buffer, was 4.3 and that for lane 4B where AMPPNP had been added was 4.9 µmol of ATP hydrolyzed/min/mg.



The Nucleotide Dependence of the + Cross-linked Product Depends on the Subunit

Our previous work has shown that nucleotide-dependent conformational changes involving the subunit depend on the presence of the subunit (Aggeler and Capaldi, 1993; Turina and Capaldi, 1994a). To examine the effect of the subunit on + cross-linking, ECFfrom the mutant E381C was depleted of the subunit by affinity chromatography using a monoclonal antibody against the subunit (Dunn, 1986). A comparison of the gel profiles of enzyme cross-linked under different nucleotide conditions by CuCl(Fig. 4) shows that there was essentially no nucleotide dependence of cross-linking of to in -free ECF. This was confirmed by activity measurements which gave around 90% residual activity for all samples.

Chemical Modification of the DELSEED Region Inhibits ATPase Activity

To examine the relative exposure of the Cys residues introduced into the DELSEED region, ECFfrom the mutants E381C and S383C was reacted with [C]NEM and with the larger maleimide, CM. [C]NEM was found to label the introduced Cys at either 381 or 383 in all three subunits. Fig. 5 correlates ATPase activity and the incorporation of NEM into E381C. Incorporation of close to 3 mol of the maleimide caused no inhibition of ATPase activity. Modification of the Cys at 381 with CM at the same concentration and for the same time intervals had a much more dramatic effect on activity. With this bulky maleimide, inhibition occurred rapidly in a time course that suggests that only 1 mol is necessary for maximal inhibition (95%).

Experiments similar to those shown in Fig. 5 were conducted using -free ECFfrom the mutant E381C, and identical results were obtained. Therefore, any observed effects of maleimide modification must be due to changes at the interface between and , rather than the and subunits. In one set of experiments, the effect of modifying the free subunit, i.e. the one not associated with either the or subunit, was examined. ECFfrom the mutant E381C/S108C was reacted with CuClto induce cross-linking of one subunit to and a second to in essentially 100% yield. The cross-linked sample was then reacted with CM to modify the fraction of Cys of the free subunit that had not been cross-linked with the subunit (estimated at around 60%). Cross-links were then broken by DTT treatment, and the effect of incorporating the maleimide into the free subunit was measured. The reaction with CM gave 60% inhibition of the ATPase activity.


Figure 5: Correlation of ATPase activity of ECF from E381C/S108C with incorporation of [C]NEM. ATPase from the / double mutant E381C/S108C was reacted at 0.4 mg/ml in 50 m M MOPS, pH 7.0, 0.5 m M EDTA, 5 m M ATP, and 10% glycerol with 25 m M [C] N-ethylmaleimide. The incorporation of [C]NEM into Cyswas determined at 1, 10, 30, 60, and 150 min (mol [C]NEM/mol F( open circles)). In a parallel experiment the change of ATPase activity due to the incorporation of 25 µ M NEM ( circles) or 25 µ M CM ( squares) into Cysof E381C/S108C was determined at 1, 10, 30, 60, and 150 min.



Disulfide Bond Formation in ECFF

FFwas isolated from both double mutants and examined for disulfide bond formation upon CuCladdition. Fig. 6 A shows data for the mutant S383C/S108C on treatment with 20 µ M CuClin the presence of ATP+Mgand without the column centrifugation step (which is difficult to perform with the vesicular enzyme preparations). Both the - and - subunit cross-links were formed. However, no cross-linking between or plus was obtained in the intact ECFF. As with ECF, cross-linking between and , and and , caused inhibition of ATPase activity in proportion to the yield of the two cross-linked products. The cross-links were destroyed and activity restored to greater than 90% by addition of 20 m M DTT. Fig. 6B shows disulfide bond formation induced by 20 µ M CuClin the mutant E381C/S108C under different nucleotide conditions. In the presence of ADP+Mg( lanes 1 and 2), the yield of the - cross-linked product was much greater than that of -, while in the presence of ATP+Mg(as ATP+Mg+azide in lane 5 and AMPPNP+Mgin lane 6), the yield of - was much higher than that of - subunits. Therefore, the same nucleotide dependence of cross-linked products is seen in ECFFas in ECFalone.


Figure 6: CuCl induced cross-linking of ECFF from the two / double mutants. A, 0.12 mg of reconstituted ECFFfrom S383C/S108C in 0.2 ml of 50 m M MOPS, pH 7.0, 2 m M MgCl2 m M ATP, and 10% glycerol was incubated with 20 µ M CuClfor 3 h at room temperature. The sample was split in half and, after incubation for another 3 h in the absence ( lane 2) or presence of 20 m M DTT ( lane 3), 10 µl of 1 M NEM in MeSO was added to each 100-µl aliquot. Dissociation buffer without reducing agent was added, and 60 µg of protein was loaded on each lane of an 10-18% polyacrylamide gel. As a control, CuCl-treated ECF(see Fig. 1) was applied ( lane 1). B, 1.5 mg of reconstituted ECFFfrom E381C/S108C was suspended in 1.5 ml of 50 m M MOPS, pH 7.0, 2 m M MgCl, and 10% glycerol. To 0.3-ml aliquots, 2 m M ATP ( lanes 1 and 3), 2 m M ADP + 2 m M P( lanes 2 and 4), 2 m M NaN+ 2 m M ATP ( lanes 5 and 7), and 2 m M AMPPNP ( lanes 6 and 8) were added, respectively. After incubation for 10 min and CuCltreatment for 3 h, 100-µl samples were taken and not reduced ( lanes 1, 2, 5, and 6) or reduced with 20 m M DTT for 2 h ( lanes 3, 4, 7, and 8) and then quenched with 100 m M NEM. 80 µg of protein was loaded per lane. Only the top portion of the 10-18% gel is shown. The ATPase activity of the control lanes was 19 µmol of ATP hydrolyzed/min/mg for lanes 3 and 4, and 10 and 14 for lanes 7 and 8 which had seen azide and AMPPNP, respectively.




DISCUSSION

The studies presented here, using mutants in which Cys residues have been incorporated into the DELSEED region of the subunit, provide new data on the structure of ECFand help describe nucleotide-dependent conformational changes occurring in the complex. Using single mutants, in which Cys residues have been introduced into the DELSEED region, and double mutants with alterations of the DELSEED region along with introduction of a Cys into the subunit at position 108, it was possible to obtain disulfide bond formation between and and/or and . The cross-linking of to is not surprising, based on the recent x-ray structure which shows the proximity of Cysof to the DELSEED region (Abrahams et al., 1994). The cross-linking of to from a Cys at 108 of to the DELSEED region was predicted from earlier work (Lötscher and Capaldi, 1984; Dallmann et al., 1992). Dallmann et al. (1992) have shown that the cross-link of to by the water-soluble carbodiimide 1-ethyl-3-[3-(dimethylamino)-propyl]carbodiimide involves the DELSEED region and that part of the subunit COOH-terminal to residue 96. The cross-link, which was shown to be an ester linkage, was not seen in the mutant S108C, implying that 1 of the 3 serines, either at 106, 107, or most likely at 108, was involved. The data presented here confirm that it is the region of near residue 108 that binds to the subunit.

Importantly, the fact that both + and + cross-links can be obtained in near 100% yield in the same ECFmolecule when the double mutants E381C/S108C or S383C/S108C were used, establishes that the small -helix of the subunit (around residue 87) and the COOH-terminal part of the subunit, interact with different subunits. Cross-linking of these mutants in the high yields obtained here means that it is now possible to experimentally distinguish the three subunits in any Fmolecule, based on the interaction of these catalytic subunits with the small subunits. This will be very useful in future studies to examine the affinity of the three different subunits for ADP, ATP, etc.

When cross-linking on ECFwas conducted by addition of CuClwithout the column procedure, the rate and/or yield of the cross-linking of both - and - was much less in S383C/S108C than in E381C/S108C. It appears, therefore, that the Glu at 381 is better positioned for interaction with both the endogenous Cys of (Cys) and Cysof the subunit than is the Ser at 383 of the subunit.

The recently published 2.8-Å resolution structure of MF(Abrahams et al., 1994) has provided details of the arrangement of the , , and subunits and new insights into catalysis by the enzyme. Briefly, this structure confirmed that the three subunits are in different conformations and are structurally different, with the asymmetry related both to nucleotide occupancy of catalytic sites and interactions of the subunit. Thus, in one subunit the catalytic site is closed around an MgADP () and the subunit is closest to the NH-terminal -helical region of the subunit. A second subunit has a partly open catalytic site containing AMPPNP+Mg(), and it is this subunit which interacts with the short -helix of the subunit containing Cys. The third subunit is empty of nucleotide (), and this has interactions with the long COOH-terminal -helix of the subunit. It is interesting then that there is nucleotide dependence of (the rate of) cross-linking between the DELSEED region and the and subunits. The disulfide link between the DELSEED region of that subunit which interacts with the short central -helix of the subunit is generated in higher yield with uncleaved ATP (either ATP+azide, or AMPPNP) in catalytic sites. This suggests that the interaction between the region of and is more stable with ATP than ADP in the catalytic site. In contrast, the interaction between the subunit and another subunit is more stable in ADP than ATP, based on the data presented here, and on our previous studies of the interaction of the with the core ECFcomplex (Mendel-Hartvig and Capaldi, 1991a; Aggeler et al., 1992).

The important role of the subunit in determining the properties of the Fis now well established. It has been shown that -free ECFhas both altered rates of unisite ATP hydrolysis, due to an increased off rate of product, P(Dunn et al., 1987), and a greatly enhanced (10-fold increased) rate of multisite ATP hydrolysis (Dunn et al., 1987; Aggeler et al., 1990; Mendel-Hartvig and Capaldi, 1991a). The functional effect of the subunit could result from the direct interaction of the with a subunit, or with the subunit, or both. We have previously found that the nucleotide-dependent conformational changes in the subunit, as measured by fluorescence probes bound to a cysteine introduced at position 8 of the subunit, are lost on removal of the subunit (Turina and Capaldi, 1994a), as is the nucleotide dependence of cross-linking from this site to the subunit by the tetrafluorophenylazide maleimide series of bifunctional reagents (Aggeler and Capaldi, 1993). Here we show that the nucleotide dependence of disulfide bond formation between the short central -helix of and is lost on removal of the subunit. Taken together, these data suggest that the subunit may regulate the functioning of the F, at least in part, by controlling the conformation of the subunit.

The key observation related to function is that covalent linkage of a subunit to the subunit through a single disulfide bond, from either the Cys at position 381 or position 383 in to a Cys at position 108 of , blocks ATPase activity. Similarly, the linkage of the Cys at 381 or 383 in to a Cys at position 87 in the subunit completely inhibits enzyme activity. When the disulfide bonds are broken, full activity is restored.

The DELSEED region of the subunit is arranged as a loop linking two -helices and is in a domain distinct from that containing the catalytic site. As with much of the subunit, the DELSEED region appears unaltered (and the structures can be superimposed) whether ATP or ADP are bound in catalytic sites (Abrahams et al., 1994). It is unlikely, therefore, that disulfide bond formation between and and the DELSEED region causes conformational changes within the subunit which alter nucleotide binding in the catalytic sites. Rather, the most straightforward interpretation of the inhibitory effect of covalent bond formation between - or - is that these linkages block movements of the (+) subunits relative to and that are necessary for catalytic cooperativity. Based on our previous studies ( e.g. Gogol et al., 1990; Wilkens and Capaldi, 1994) and on the features of the structure of the Fmoiety (Abrahams et al., 1994), we believe that there are translocations of the and subunits between different subunits driven by ATP hydrolysis (and ATP synthesis in the reverse direction of enzyme functioning) which, as first suggested by Boyer and Kohlbrenner (1981), cause alternation of catalytic sites between the three nucleotide-binding states ( i.e. between , , and ). The chemical modification studies described here are similarly best interpreted in terms of an effect on movement of subunits relative to one another. Incorporation of a small molecule (NEM) into the DELSEED region has no significant effect on the functioning of the enzyme even when close to 3 mol of reagent are bound and cannot therefore be inducing major conformational changes in the subunit. In contrast, when the more bulky maleimide CM was bound into the subunit at position 381 at 1 mol equivalent ( i.e. 1 mol of CM/mol of ECF), activity was dramatically reduced. Inhibition of activity was observed when free subunit ( i.e. that not in contact with or ) was modified. Such a result would be expected if the incorporation of CM prevents the free subunit from ``switching'' to become associated with the or subunits during catalytic site alternation.

In addition to the cross-linked products between and , and and , discussed above, CuCltreatment of the double mutant led to disulfide bond formation between and , and and . The cross-linking of to by disulfide bond formation has been observed before and obtained in essentially 100% yield (Bragg and Hou, 1986; Tozer and Dunn, 1986; Mendel-Hartvig and Capaldi, 1991b). It involves Cysof the subunit (Mendel-Hartvig and Capaldi, 1991b). In the DELSEED mutants, there is competition between the and subunits for the subunit. In contrast to the linkage of to , or to , there is no disulfide bond formation between and , or and in ECFF, implying that the association of the subunit in ECFis not physiological.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant HL 24526. 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.

§
To whom correspondence should be addressed.

The abbreviations used are: ECF, soluble portion of the E. coli FFATP synthase; CM, N-[4-[7-(diethylamino)-4 methyl]coumarin-3-yl]maleimide; NEM, N-ethylmaleimide; DTT, dithiothreitol; AMPPNP, 5`-adenylyl-,-imidodiphosphate; LDAO, N, N-dimethyldodecylamine- N-oxide; kb, kilobase(s).


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