Abstract
Four double mutants in the epsilon subunit were generated, each containing two cysteines, which, based on the NMR structure of this subunit, should form internal disulfide bonds. Two of these were designed to generate interdomain cross-links that lock the C-terminal alpha-helical domain against the beta-sandwich (epsilonM49C/A126C and epsilonF61C/V130C). The second set should give cross-linking between the two C-terminal alpha-helices (epsilonA94C/L128C and epsilonA101C/L121C). All four mutants cross-linked with 90-100% efficiency upon CuCl(2) treatment in isolated Escherichia coli ATP synthase. This shows that the structure obtained for isolated epsilon is essentially the same as in the assembled complex. Functional studies revealed increased ATP hydrolysis after cross-linking between the two domains of the subunit but not after cross-linking between the C-terminal alpha-helices. None of the cross-links had any effect on proton pumping-coupled ATP hydrolysis, on DCCD sensitivity of this activity, or on ATP synthesis rates. Therefore, big conformational changes within epsilon can be ruled out as a part of the enzyme function. Protease digestion studies, however, showed that subtle changes do occur, since the epsilon subunit could be locked in an ADP or 5'-adenylyl-beta,gamma-imidodiphosphate conformation by the cross-linking with resulting differences in cleavage rates.
Highlights
Functional studies revealed increased ATP hydrolysis after cross-linking between the two domains of the subunit but not after cross-linking between the C-terminal ␣-helices
With ECF1F0 purified from each mutant, disulfide bond formation was readily obtained in essentially 100% yield when high enough levels of Cu2ϩ were used
The present study offers important insights into the arrangement and functioning of the ⑀ subunit in ECF1F0
Summary
Functional studies revealed increased ATP hydrolysis after cross-linking between the two domains of the subunit but not after cross-linking between the C-terminal ␣-helices. The structure of the ⑀ subunit when isolated from the E. coli enzyme has recently been solved by both NMR and x-ray crystallography (19 –21). This subunit is a two-domain protein with an N-terminal part arranged in a -sandwich structure and the C-terminal portion in a helix-loop-helix motif. To understand how the ⑀ subunit functions in the intact ECF1F0, it is necessary to know whether the structure of ⑀ is the same in the assembled complex as when isolated To examine this question, we have created four different mutants, each containing two cysteines, that should form disulfide bridges if the structure of the ⑀ subunit is the same as in solution.
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