The role of the betaDELSEED motif of F1-ATPase: propagation of the inhibitory effect of the epsilon subunit.

In F(1)-ATPase, a rotary motor enzyme, the region of the conserved DELSEED motif in the beta subunit moves and contacts the rotor gamma 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 epsilon subunit, an intrinsic inhibitor, was unable to inhibit the ATPase activity of a mutant thermophilic F(1)-ATPase in which all of the five acidic residues in the DELSEED motif were replaced with alanines, although the epsilon subunit in the mutant F(1)-ATPase assumed the inhibitory form. In addition, the replacement of basic residues in the C-terminal region of the epsilon subunit by alanines caused a decrease of the inhibitory effect. Partial replacement of the acidic residues in the DELSEED motif of the beta subunit or of the basic residues in the C-terminal alpha-helix of the epsilon subunit induced a partial effect. We here conclude that the epsilon subunit exerts its inhibitory effect through the electrostatic interaction with the DELSEED motif of the beta subunit.

Movement of the helical domain of the epsilon subunit is required for the activation of thermophilic F1-ATPase.

The inhibitory effect of epsilon subunit in F(1)-ATPase from thermophilic Bacillus PS3 was examined focusing on the structure-function relationship. For this purpose, we designed a mutant for epsilon subunit similar to the one constructed by Schulenberg and Capaldi (Schulenberg, B., and Capaldi, R. A. (1999) J. Biol. Chem. 274, 28351-28355). We introduced two cysteine residues at the interface of N-terminal beta-sandwich domain (S48C) and C-terminal alpha-helical domain (N125C) of epsilon subunit. The alpha(3)beta(3)gammaepsilon complex containing the reduced form of this mutant epsilon subunit showed suppressed ATPase activity and gradual activation during the measurement. This activation pattern was similar to the complex with the wild type epsilon subunit. The conformation of the mutant epsilon subunit must be fixed and similar to the reported three-dimensional structure of the isolated epsilon subunit, when the intramolecular disulfide bridge was formed on this subunit by oxidation. This oxidized mutant epsilon subunit could form the alpha(3)beta(3)gammaepsilon complex but did not show any inhibitory effect. The complex was converted to the activated state, and the cross-link in the mutant epsilon subunit in the complex was efficiently formed in the presence of ATP-Mg, whereas no cross-link was observed without ATP-Mg, suggesting the conformation of the oxidized mutant epsilon subunit must be similar to that in the activated state complex. A non-hydrolyzable analog of ATP, 5'-adenylyl-beta,gamma-imidodiphosphate, could stimulate the formation of the cross-link on the epsilon subunit. Furthermore, the cross-link formation was stimulated by nucleotides even when this mutant epsilon subunit was assembled with a mutant alpha(3)beta(3)gamma complex lacking non-catalytic sites. These results indicate that binding of ATP to the catalytic sites induces a conformational change in the epsilon subunit and triggers transition of the complex from the suppressed state to the activated state.

Epsilon subunit, an endogenous inhibitor of bacterial F(1)-ATPase, also inhibits F(0)F(1)-ATPase.

Since the report by Sternweis and Smith (Sternweis, P. C., and Smith, J. B. (1980) Biochemistry 19, 526-531), the epsilon subunit, an endogenous inhibitor of bacterial F(1)-ATPase, has long been thought not to inhibit activity of the holo-enzyme, F(0)F(1)-ATPase. However, we report here that the epsilon subunit is exerting inhibition in F(0)F(1)-ATPase. We prepared a C-terminal half-truncated epsilon subunit (epsilon(DeltaC)) of the thermophilic Bacillus PS3 F(0)F(1)-ATPase and reconstituted F(1)- and F(0)F(1)-ATPase containing epsilon(DeltaC). Compared with F(1)- and F(0)F(1)-ATPase containing intact epsilon, those containing epsilon(DeltaC) showed uninhibited activity; severalfold higher rate of ATP hydrolysis at low ATP concentration and the start of ATP hydrolysis without an initial lag at high ATP concentration. The F(0)F(1)-ATPase containing epsilon(DeltaC) was capable of ATP-driven H(+) pumping. The time-course of pumping at low ATP concentration was faster than that by the F(0)F(1)-ATPase containing intact epsilon. Thus, the comparison with noninhibitory epsilon(DeltaC) mutant shed light on the inhibitory role of the intact epsilon subunit in F(0)F(1)-ATPase.

Direct observation of the rotation of epsilon subunit in F1-ATPase.

Rotation of the epsilon subunit in F1-ATPase from thermophilic Bacillus strain PS3 (TF1) was observed under a fluorescence microscope by the method used for observation of the gamma subunit rotation (Noji, H., Yasuda, R., Yoshida, M., and Kinosita, K., Jr. (1997) Nature 386, 299-302). The alpha3 beta3 gamma epsilon complex of TF1 was fixed to a solid surface, and fluorescently labeled actin filament was attached to the epsilon subunit through biotin-streptavidin. In the presence of ATP, the filament attached to epsilon subunit rotated in a unidirection. The direction of the rotation was the same as that observed for the gamma subunit. The rotational velocity was slightly slower than the filament attached to the gamma subunit, probably due to the experimental setup used. Thus, as suggested from biochemical studies (Aggeler, R., Ogilvie, I. , and Capaldi, R. A. (1997) J. Biol. Chem. 272, 19621-19624), the epsilon subunit rotates with the gamma subunit in F1-ATPase during catalysis.