Conformational changes in the epsilon subunit of the Escherichia coli F1F0 ATP synthase.

摘要

E. coli F1F0 ATP synthase, the central energy transduction enzyme, catalyzes the proton-driven ATP synthesis and ATP hydrolysis-driven proton translocation. The epsilon subunit, which is a part of F1, plays a crucial role in binding F1 to F0, together with the gamma subunit.; It is a two-domain protein in which the N-terminal domain is a 10-stranded beta-sandwich and the C-terminal domain is a pair of alpha-helices in an anti-parallel coiled coil. epsilon is known to undergo conformational changes in response to the binding of nucleotides at the catalytic sites. These changes involve movement and unpacking of the C-terminal alpha-helices. In the "up"-state the helices are extended towards the alpha and beta subunits of F 1and in the "down"-state they are packed against the N-terminal domain of epsilon.; In this study, site-specific mutagenesis was performed to introduce unique cysteines at various positions of epsilon subunit. The accessibility of the cysteine residues was probed by labeling with N-maleimidylpropionyl biocytin (MPB). The results indicated that lot of small positional changes occur in the beta-sheet region along the gamma-epsilon-c interface. Labeling also showed that the cysteine residues in the C-terminal alpha-helices labeled well under all conditions, which means that the alpha-helices must open up, at least transiently. The accessibility pattern of the corresponding residues in the isolated epsilon and gamma'-epsilon co-crystal structures does not match the labeling results obtained here. Effect of inhibition by ATP + MgSO 4 and ADP-AlF4 on the labeling were also studied. Most of the effect was found to be in the N-terminal region, especially near the gamma-epsilon interface. This means that the conformational changes observed here are mostly at the gamma-epsilon and gamma-epsilon-c interfaces (regions interacting with other subunits), supporting the idea that the binding of nucleotides at the catalytic sites causes conformational changes in epsilon through the gamma subunit. ATP did not have much effect on the residues in the alpha-helical region. This means that in the resting state the epsilon subunit spends some of its time in an intermediate conformation, in which most of the C-terminal residues are exposed.