Abstract
Escherichia coli ATP synthase (F0F1) couples catalysis and proton transport through subunit rotation. The ϵ subunit, an endogenous inhibitor, lowers F1-ATPase activity by decreasing the rotation speed and extending the duration of the inhibited state (Sekiya, M., Hosokawa, H., Nakanishi-Matsui, M., Al-Shawi, M. K., Nakamoto, R. K., and Futai, M. (2010) Single molecule behavior of inhibited and active states of Escherichia coli ATP synthase F1 rotation. J. Biol. Chem. 285, 42058-42067). In this study, we constructed a series of ϵ subunits truncated successively from the carboxyl-terminal domain (helix 1/loop 2/helix 2) and examined their effects on rotational catalysis (ATPase activity, average rotation rate, and duration of inhibited state). As expected, the ϵ subunit lacking helix 2 caused about ½-fold reduced inhibition, and that without loop 2/helix 2 or helix 1/loop 2/helix 2 showed a further reduced effect. Substitution of ϵSer(108) in loop 2 and ϵTyr(114) in helix 2, which possibly interact with the β and γ subunits, respectively, decreased the inhibitory effect. These results suggest that the carboxyl-terminal domain of the ϵ subunit plays a pivotal role in the inhibition of F1 rotation through interaction with other subunits.
Highlights
These results suggest that the carboxyl-terminal domain of the ⑀ subunit plays a pivotal role in the inhibition of F1 rotation through interaction with other subunits
This structure should lead to inhibition of rotational catalysis
We have shown that the carboxyl-terminal domain of the ⑀ subunit, ⑀CTD, especially loop 2/helix 2, plays pivotal roles in inhibition of F1-ATPase activity
Summary
The recently determined high-resolution crystal structure of the E. coli F1 sector revealed that the ⑀CTD adopts a highly extended conformation, with helix 2 inserted deeply between the  and ␥ subunits (Fig. 1a) [15] This structure should correspond to a transiently inhibited conformation because the ␥ rotor may be connected non-covalently to the stator (␣33) by the ⑀ subunit, thereby preventing ␥ rotation. We prepared a series of truncated ⑀ subunits lacking part of the ⑀CTD and ones with amino acid replacements and examined their effects on ATPase activity, rotation speed, and duration of the inhibitory state These studies indicated the importance of the ⑀CTD interaction with the  and ␥ subunits. The concerted effect of loop 2 and helix 2 was suggested
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