In F1-ATPase, a rotary motor enzyme, the region of the conserved DELSEED motif in the β subunit moves and contacts the rotor γ subunit when the nucleotide fills the catalytic site, and the acidic nature of the motif was previously assumed to play a critical role in rotation. Our previous work, however, disproved the assumption (Hara, K. Y., Noji, H., Bald, D., Yasuda, R., Kinosita, K., Jr., and Yoshida, M. (2000) J. Biol. Chem. 275, 14260–14263), and the role of this motif remained unknown. Here, we found that the ε subunit, an intrinsic inhibitor, was unable to inhibit the ATPase activity of a mutant thermophilic F1-ATPase in which all of the five acidic residues in the DELSEED motif were replaced with alanines, although the ε subunit in the mutant F1-ATPase assumed the inhibitory form. In addition, the replacement of basic residues in the C-terminal region of the ε subunit by alanines caused a decrease of the inhibitory effect. Partial replacement of the acidic residues in the DELSEED motif of the β subunit or of the basic residues in the C-terminal α-helix of the ε subunit induced a partial effect. We here conclude that the ε subunit exerts its inhibitory effect through the electrostatic interaction with the DELSEED motif of the β subunit.

# EXPERIMENTAL PROCEDURES

### Construction of Expression Plasmid for α3β3γ Complex and ε Subunit of TF1

The α3β3γ complex of TF1 containing the mutations (αC193S, 10 His at N termini of β, γS107C; wt′) was considered to be a wild-type complex in this work. The expression plasmid for the wt′ complex was pKABG1 (31), and the mutations in the βDELSDED sequence were introduced into the plasmid as described previously (15). The expression plasmid for the wild-type ε subunit (εWT) was pTE2 (27), and that for the mutant ε subunit (S48C/N125C; εNCX) of TF1 was prepared as described previously (23). Alanine mutations (K133A, εP1A; K121A/R122A/R126A, εP3A; K121A/R122A/R126A/K133A, εP4A) were introduced into pTE2 by the method of Kunkel et al. (32). Using a polymerase chain reaction method with rTaqpolymerase (Takara Inc.), NdeI and HindIII sites were introduced into the 5′ and 3′ termini of the mutant ε subunit gene, respectively. The polymerase chain reaction products were digested by NdeI and HindIII, and the fragments obtained were cloned into pET21c vector (Novagen Inc.) for efficient expression.

### Proteins

The mutant α3β3γ complexes of TF1 were purified as described (31). The wild-type and mutant ε subunits of TF1 were prepared as described previously (23, 33). The α3β3γε complexes were reconstituted from α3β3γ and the ε subunit by incubation for 30 min at room temperature. To remove the unbound ε subunit, the mixture was concentrated several times with a centrifuge concentrator (Microcon 100; 100,000 M
                              r cut-off; Millipore Amicon), and their purity was analyzed by 6% (w/v) PAGE without SDS (native-PAGE) in the presence of 20 μm ATP and 2 mm MgCl2.

### Measurement of ATPase Activity

ATPase activity was measured spectrophotometrically with an ATP regenerating system at 25 °C (34). The assay mixture contained 50 mm Tris-HCl (pH 8.0), 100 mm KCl, 2.5 mmphosphoenolpyruvate, 2 mm MgCl2, 0.2 mm NADH, 50 μg/ml pyruvate kinase, 50 μg/ml lactate dehydrogenase, and 20 μm or 2 mm of ATP-Mg. The reaction was initiated by the addition of the enzyme solution into 1 ml of assay mixture. The change in absorbance at 340 nm was monitored in a spectrophotometer V-550 (Jasco, Japan) and stored in an on-line computer. In the experiments shown in Table I
                              , the rates of ATP hydrolysis at 20 μm ATP in the period 5–6 min after the addition of the α3β3γ and α3β3γε complexes were compared.

### Analysis of Stability of Mutant α3β3γε Complex in the Presence of ATP-Mg

The mutant α3β3γε complex (1 mg/ml) was incubated with 20 μm ATP, 2 mmMgCl2, and 100 mm KCl in 50 mmTris-HCl (pH 8.0) for 30 min at 25 °C. 100 μl of the complex was subjected to gel filtration HPLC (G-3000SWXL (Tosoh, Japan)) equilibrated with 20 μm ATP, 2 mmMgCl2, 100 mm KCl, and 50 mmTris-HCl (pH 7.0). The flow rate was 0.5 ml/min. A peak fraction was concentrated with Microcon 100, and their subunit composition was analyzed by 6% (w/v) native-PAGE and 14% (w/v) SDS-PAGE.

### Conformational Transition of the ε Subunit in α3β3γε Complex

Formation of the intramolecular cross-link in the mutant ε subunit in α3β3γε complex was examined in the presence or the absence of 20 μm or 2 mmATP-Mg. The isolated α3β3γεNCX-RED complex (1 mg/ml) was incubated with 40 μm or 4 mm ATP and 4 mm MgCl2 for 10 min at 25 °C in 50 mm TES-NaOH (pH 7.0) and 100 mm NaCl. As a control, 4 mm MgCl2 was added in the absence of ATP. Cross-linking was induced by the addition of equal volumes of 100 μm of CuCl2 solution (in 50 mmTES-NaOH (pH 7.0) and 100 mm NaCl), which gives the final concentration of ATP-Mg at 0 and 20 μm and 2 mm, respectively. After incubation for 1 h at 25 °C, the reaction was quenched by the addition of 10 mm EDTA. After 10 min, 0.1% (w/v) SDS and 25 μm TMR maleimide were added to label cysteines that did not form disulfide bonds, and the solution was incubated at 25 °C for 10 min. Then 10 mm 
                              N-ethylmaleimide was added to terminate the labeling. Incorporation of TMR maleimide into the ε subunit was analyzed on 15% (w/v) SDS-PAGE without reducing reagent. Other conditions were the same as those described by Kato-Yamada et al. (23).

### Other Procedures

Protein concentrations were determined by the method of Bradford (35) using bovine serum albumin as a standard.

# RESULTS

### Effect of the ε Subunit on ATPase Activity of N5A Mutant Complex

The ATPase activity of the N5A mutant complex, in which all of the five acidic residues in the βDELSDED sequence of the wt′ complex were replaced with alanines, was measured and compared with the activity of the wt′ complex. The reaction was initiated by the addition of the α3β3γ or the α3β3γε complex, designated as the −ε complex and the +ε complex, respectively, to the reaction mixture. As reported previously (27), ATP hydrolysis at low ATP concentration by the wt′(+ε) complex was very slow in comparison with the wt′(−ε) complex, and the inhibitory role of the ε subunit is thus evident (Fig. 1
                               
                              A). It should be noted that the inhibition by the ε subunit was relieved slowly and that the ATPase activity was recovered gradually by the reaction period. At 2 mm ATP, this recovery was much faster, and the inhibitory effect of the ε subunit was only observed at the very initial phase of the reaction (Fig. 1 
                              B). As stated previously, this recovery is due to the nucleotide-dependent conformational transition of the ε subunit (23). On the contrary, the N5A(+ε) complex hydrolyzed ATP at the same rate as the N5A(−ε) complex at both 20 μm and 2 mm ATP (Fig. 1,C and D). Apparently, the N5A complex lost sensitivity to the inhibition by ε subunit.

### The ε Subunit Does Not Dissociate from the N5A(+ε) Complex during ATP Hydrolysis

The results shown in Fig. 1 could be explained even if it were by the dissociation of the ε subunit from the N5A complex rapidly during ATP hydrolysis. To confirm that this is not the case, we incubated the N5A(+ε) complex with 20 μm ATP-Mg under the same conditions used for the measurement of the ATPase activities. After a 30-min incubation, the complex was isolated by gel filtration HPLC in the presence of 20 μm ATP-Mg (Fig.2
                              ). The homogeneity of the N5A(+ε) complex was further demonstrated by native-PAGE, because the complex electrophoresed as a single band (Fig. 2,inset b). The electrophoretic mobility of the N5A(+ε) complex was slightly slower than that of the N5A(-ε) complex. SDS-PAGE analysis of the isolated complex showed that the N5A(+ε) complex contained all of the α, β, γ, and ε subunits with the correct stoichiometry (Fig. 2, inset a). These results strongly suggest that the N5A complex lost its sensitivity to inhibition by the ε subunit without dissociation of this subunit.

### Conformational Transition of εNCX in the N5A(+ε) Complex

To confirm the nucleotide-dependent conformational change of the ε subunit in the N5A(+ε) complex, we used a mutant ε subunit, εNCX, in which two cysteine residues were introduced at the interface of the two domains (S48C/N125C) (23). εNCX inhibited the wt′ complex but not the N5A complex as wild-type ε (Fig. 1, A andC). When the N-terminal β strand domain and C-terminal α-helical domain packed closely in εNCX (“closed” form) as determined by NMR and x-ray crystallography (24-26), an interdomain disulfide is readily formed by the assistance of the low concentrations of CuCl2. However, if these two domains assume the “open” form, a disulfide bond is no longer formed, and the free sulfide residues are susceptible to labeling by fluorescent maleimide. This efficiency of labeling by TMR maleimide is therefore a good indicator of the existence of the open form of εNCXin which a disulfide bond is very rarely formed. Electrophoretic mobility of εNCX containing a disulfide is slightly faster than the εNCX with TMR-labeled sulfides, and this distinction was also adopted as a means to assess the open and closed forms. In the presence of 2 mm ATP and 50 μmCuCl2, εNCX both in the wt′ and the N5A complex was largely unlabeled by TMR maleimide, and the protein bands corresponding to εNCX with a disulfide were dominant (Fig. 3
                              ). On the other hand, disulfide formation in εNCX in the N5A complex did not occur the presence or the absence of 20 μm ATP, similar to wt′ complex. These results imply that the ε subunit in the complex can undergo a nucleotide-dependent conformational transition to the noninhibitory state, although 20 μm ATP-Mg was insufficient to this transition .

### Effect of Partial Elimination of Negative Charges in the βDELSEED Motif

To clarify the important residues in the βDELSEED motif for inhibition by the ε subunit, we generated seven mutant α3β3γ complexes in which the acidic residues in the βDELSDED sequence were substituted by alanines. Substitution of the last three acidic residues (DELSAAA) efficiently suppressed the propagation of the inhibitory effect of the ε subunit (Table I). The replacement of the first two acidic residues (AALSDED) also resulted in suppression of the ε-induced inhibition, although the mutation showed a partial effect (Table I). A single replacement at Asp390, Glu391, or Asp396 also has some suppression on the ε-induced inhibition. Taken together, nearly all of the acidic residues in the βDELSDED sequence cumulatively contribute to the inhibitory effect of the ε subunit, and the last three are the most critical.

### Effect of Partial Elimination of Positive Charges in the C-terminal α-Helix of the ε Subunit

Next we investigated whether the positively charged residues in the C-terminal α-helix of the ε subunit contribute to the inhibitory role of the ε subunit. For this purpose, we designed three new mutant ε subunits of TF1in which some positively charged residues in the C-terminal α-helix were replaced with Ala, and they were designated εP1A, εP3A, and εP4A according to the number of the substituted Ala from the positively charged residue(s). The ATPase activities of the wt′ complex with these mutant ε subunits were measured in the presence of 20 μm ATP (Fig.4
                              ). The wt′ (+εP4A) complex hydrolyzed ATP at the almost same rate as the wt′ (−ε) complex. The wt′ (+εP4A) complex was isolated by gel filtration HPLC in the presence of 20 μm ATP-Mg (Fig.5
                              ), suggesting that the complex obtained with this mutant ε subunit was stable. The wt′ (+εP1A) and wt′ (+εP3A) complexes showed partial effects on the inhibitory role of the ε subunit. These results suggested that the ε subunit exerts the inhibitory role through the positive charges in the C-terminal α-helix of the ε subunit.

# DISCUSSION

### Inhibitory Signal from ε Subunit Is Transmitted through the βDELSEED Motif

The ATPase activity of the N5A(+ε) mutant complex is resistant to inhibition by the ε subunit. There are two possible explanations for this observation. Either the mutated βDELSEED motif interferes with the conformational transition of the ε subunit to an inhibitory state, or the β subunit with the mutated βDELSEED motif is no longer affected by the ε subunit in the inhibitory state. We conclude that the latter is the case for the following reasons. First, nucleotide-dependent formation of the interdomain disulfide bond in εNCX, indicative of the conformational conversion of ε from the inhibitory state to the noninhibitory state, occurred in the N5A(+ε) complex in the same manner as in the wt′ complex. Second, in the N5A(+ε) complex, the ε subunit did not inhibit ATP hydrolysis at 20 μm ATP (Fig. 1), although εNCX existed in the inhibitory conformation (Fig. 3). Therefore, we here propose that the inhibitory effect of the ε subunit propagates through the interaction with the βDELSEED motif.

### Large Conformational Transition of the ε Subunit Is Further Supported

Because the resolution of the yeast mitochondrial F1+Foc structure was limited to 3.9 Å (36), the details of the ε subunit (called δ in mitochondrial F1) could not be clearly defined. Nevertheless, the electron density of the δ subunit (bacterial ε subunit) can readily accommodate the structure of the isolated ε subunit from EF1 (24, 26). The δ subunit (bacterial ε subunit), however, lies on the c subunit ring, far from the α3β3 structure, where a part of the ε subunit cannot interact with the DELSEED motif of the β subunit. When the ε subunit acts as an inhibitor, a large conformational transition must occur in the ε subunit to achieve interaction with the catalytic portion. Cross-linking experiments showed that the C-terminal domain of the ε subunit is close to the βDELSEED motif under certain conditions (28-30). A mutant ε subunit whose C-terminal domain was truncated could not inhibit the ATPase activity of the FoF1-ATP synthase (22). A nucleotide-dependent conformational change in the ε subunit of EF1 was reported by Wilkens and Capaldi (26). We also have reported the nucleotide-dependent formation of an interdomain cross-link in the ε subunit of TF1 (23). Hence there are several results that strongly suggest the direct interaction of the βDELSEED motif with the C-terminal domain of the ε subunit and a large conformational transition of the ε subunit. Finally, these possibilities were supported recently by the newly reported three-dimensional structures of the MF1 (37) and the γ'-ε complex from EF1 (38) as described below.

### Interaction between the βDELSEED Motif and the C-terminal α-Helix of the ε Subunit Involves Electrostatic Interactions

Elimination of negative charges in the βDELSEED motif cumulatively desensitized F1 from ε-induced inhibition (Table I), indicating that the βDELSEED motif interacts with the ε subunit not by specific interactions between certain residues but through a cluster of negative charges. In addition, elimination of positive charges in the C-terminal α-helix of the ε subunit also gave similar results (Fig. 4). The cumulative contribution of the negative charges in the βDELSEED motif and the positive charges in the C-terminal region of the ε subunit strongly suggest that the electrostatic interaction between these two regions dominates the inhibition of the F1-ATPase activity.

### A Model for the Conformational Transition of the ε Subunit and Activation of α3β3γε Complex

Just when we concluded the present study, the crystal structure of the γ′ (residues γ11-γ258)-ε complex in EF1 (38) and that of the α3β3γδε complex in MF1 (37) were reported. These two structures revealed that the ε subunit in EF1 and the corresponding subunit in MF1, δ, adopted very different conformations. The two helices of EF1-ε extended and entwined the coiled-coil structure of the γ subunit, and the C-terminal end reached out toward the α3β3 ring. In contrast, the two α-helices of MF1-δ abutted the β-sandwich structure and the C termini located on the surface of c subunits.

The interaction between the βDELSEED motif and the C-terminal α-helix of the ε subunit must be possible if the conformation of the ε subunit in the complex is similar to that reported in the γ′-ε complex of EF1 (38). In contrast, the cross-linking in εNCX must occur when the structure of the ε subunit in the complex is in the same conformation as that of the δ subunit in MF1 (37). If this is the case, our results should be well explained by the large conformational transition of the ε subunit. The conformation of the ε subunit in the γ′-ε complex of EF1 (38) and the δ subunit in MF1(37) correspond to the inhibitory state and noninhibitory state, respectively.

Interestingly, the ε subunit of MF1 lay between the γ subunit and the δ subunit like a wedge (37). This subunit seems to mimic the structure of the C-terminal α-helical domain of the bacterial ε subunit in the complex. The C-terminal part of the MF1-ε subunit contains four positively charged residues at the counterpart of the C-terminal α-helix of the MF1-δ subunit, which is shorter and contains only two positively charged residues. It is therefore an intriguing assumption that the C-terminal region of the ε subunit of MF1interacts with βDELSEED motif and inhibits the activity in MF1-ATPase under certain conditions, although the role of this subunit is not known very well.

Here we propose a model for the conformational transition of the bacterial ε subunit to explain the activation of α3β3γε complex (Fig.6
                              ). When the ε subunit is in the inhibitory form, the C-terminal α-helix of the ε subunit interacts with βDELSEED motif through their respective charges. The apparent bridge formed between the ε subunit and a β subunit make the α3β3γε complex inactive. When the conformational change is induced in the β subunit by ATP binding, the electrostatic interaction is broken, and the ε subunit resumes the noninhibitory form. Then the α3β3γε complex becomes active. From the present results and our previous reports, it is evident that ATP-Mg stimulates the conformational transition of the ε subunit from the inhibitory state to the noninhibitory state. The information on the trigger for this conformational transition of the ε subunit is requisite for further understanding of the internal regulation of the F1-ATPase.

We thank R. Suno, Y. Hirono, J. Suzuki, S. P. Tsunoda, H. Noji, H. Taguchi, E. Muneyuki, T. Masaike, H. Konno, K. Sugiyama, T. Suzuki, and D. Bald for technical assistance and helpful discussion. We also thank J. Hardy for critical reading of the manuscript.