Large conformational changes of the epsilon subunit in the bacterial F1F0 ATP synthase provide a ratchet action to regulate this rotary motor enzyme.

The F(1)F(0) ATP synthase is the smallest motor enzyme known. Previous studies had established that the central stalk, made of the gamma and epsilon subunits in the F(1) part and c subunit ring in the F(0) part, rotates relative to a stator composed of alpha(3)beta(3)deltaab(2) during ATP hydrolysis and synthesis. How this rotation is regulated has been less clear. Here, we show that the epsilon subunit plays a key role by acting as a switch of this motor. Two different arrangements of the epsilon subunit have been visualized recently. The first has been observed in beef heart mitochondrial F(1)-ATPase where the C-terminal portion is arranged as a two-alpha-helix hairpin structure that extends away from the alpha(3)beta(3) region, and toward the position of the c subunit ring in the intact F(1)F(0). The second arrangement was observed in a structure determination of a complex of the gamma and epsilon subunits of the Escherichia coli F(1)-ATPase. In this, the two C-terminal helices are apart and extend along the gamma to interact with the alpha and beta subunits in the intact complex. We have been able to trap these two arrangements by cross-linking after introducing appropriate Cys residues in E. coli F(1)F(0), confirming that both conformations of the epsilon subunit exist in the enzyme complex. With the C-terminal domain of epsilon toward the F(0), ATP hydrolysis is activated, but the enzyme is fully coupled in both ATP hydrolysis and synthesis. With the C-terminal domain toward the F(1) part, ATP hydrolysis is inhibited and yet the enzyme is fully functional in ATP synthesis; i.e., it works in one direction only. These results help explain the inhibitory action of the epsilon subunit in the F(1)F(0) complex and argue for a ratchet function of this subunit.

Structure of the gamma-epsilon complex of ATP synthase.

ATP synthases (F(1)F(o)-ATPases) use energy released by the movement of protons down a transmembrane electrochemical gradient to drive the synthesis of ATP, the universal biological energy currency. Proton flow through F(o) drives rotation of a ring of c-subunits and a complex of the gamma and epsilon-subunits, causing cyclical conformational changes in F(1) that are required for catalysis. The crystal structure of a large portion of F(1) has been resolved. However, the structure of the central portion of the enzyme, through which conformational changes in F(o) are communicated to F(1), has until now remained elusive. Here we report the crystal structure of a complex of the epsilon-subunit and the central domain of the gamma-subunit refined at 2.1 A resolution. The structure reveals how rotation of these subunits causes large conformational changes in F(1), and thereby provides new insights into energy coupling between F(o) and F(1).

The second stalk composed of the b- and delta-subunits connects F0 to F1 via an alpha-subunit in the Escherichia coli ATP synthase.

The b- and delta-subunits of the Escherichia coli ATP synthase are critical for binding ECF1 to the F0 part, and appear to constitute the stator necessary for holding the alpha3beta3 hexamer as the c-epsilon-gamma domain rotates during catalysis. Previous studies have determined that the b-subunits are dimeric for a large part of their length, and interact with the F1 part through the delta-subunit (Rodgers, A. J. W., Wilkens, S., Aggeler, R., Morris, M. B., Howitt, S. M., and Capaldi, R. A. (1997) J. Biol. Chem. 272, 31058-31064). To further study b-subunit interactions, three mutants were constructed in which Ser-84, Ala-144, and Leu-156, respectively, were replaced by Cys. Treatment of purified ECF1F0 from all three mutants with CuCl2 induced disulfide formation resulting in b-subunit dimer cross-link products. In addition, the mutant bL156C formed a cross-link from a b-subunit to an alpha-subunit via alphaCys90. Neither b-b nor b-alpha cross-linking had significant effect on ATPase activities in any of the mutants. Proton pumping activities were measured in inner membranes from the three mutants. Dimerization of the b-subunit did not effect proton pumping in mutants bS84C or bA144C. In the mutant bL156C, CuCl2 treatment reduced proton pumping markedly, probably because of uncoupling caused by the b-alpha cross-link formation. The results show that the alpha-subunit forms part of the binding site on ECF1 for the b2delta domain and that the b-subunit extends all the way from the membrane to the top of the F1 structure. Some conformational flexibility in the connection between the second stalk and F1 appears to be required for coupled catalysis.

The second stalk: the delta-b subunit connection in ECF1F0.

The ATP synthase F1F0 is the smallest molecular motor yet studied. ATP hydrolysis drives the rotary motion of the primary stalk subunits gamma and epsilon relative to the alpha 3 beta 3 part of F1. Evidence is reviewed to show that the delta and b subunits provide a second stalk that can act as a stator to facilitate these rotational movements.

The subunit delta-subunit b domain of the Escherichia coli F1F0 ATPase. The B subunits interact with F1 as a dimer and through the delta subunit.

The delta and b subunits are both involved in binding the F1 to the F0 part in the Escherichia coli ATP synthase (ECF1F0). The interaction of the purified delta subunit and the isolated hydrophilic domain of the b subunit (bsol) has been studied here. Purified delta binds to bsol weakly in solution, as indicated by NMR studies and protease protection experiments. On F1, i.e. in the presence of ECF1-delta, delta, and bsol interact strongly, and a complex of ECF1.bsol can be isolated by native gel electrophoresis. Both delta subunit and bsol are protected from trypsin cleavage in this complex. In contrast, the delta subunit is rapidly degraded by the protease when bound to ECF1 when bsol is absent. The interaction of bsol with ECF1 involves the C-terminal domain of delta as delta(1-134) cannot replace intact delta in the binding experiments. As purified, bsol is a stable dimer with 80% alpha helix. A monomeric form of bsol can be obtained by introducing the mutation A128D (Howitt, S. M., Rodgers, A. J.,W., Jeffrey, P. D., and Cox, G. B. (1996) J. Biol. Chem. 271, 7038-7042). Monomeric bsol has less alpha helix, i.e. only 58%, is much more sensitive to trypsin cleavage than dimer, and unfolds at much lower temperatures than the dimer in circular dichroism melting studies, indicating a less stable structure. The bsol dimer, but not monomer, binds to delta in ECF1. To examine whether subunit b is a monomor or dimer in intact ECF1F0, CuCl2 was used to induce cross-link formation in the mutants bS60C, bQ104C, bA128C, bG131C, and bS146C. With the exception of bS60C, CuCl2 treatment resulted in formation of b subunit dimers in all mutants. Cross-linking yield was independent of nucleotide conditions and did not affect ATPase activity. These results show the b subunit to be dimeric for a large portion of the C terminus, with residues 124-131 likely forming a pair of parallel alpha helices.

The coupling of the relative movement of the a and c subunits of the F0 to the conformational changes in the F1-ATPase.

F0F1-ATPase structural information gained from X-ray crystallography and electron microscopy has activated interest in a rotational mechanism for the F0F1-ATPase. Because of the subunit stoichiometry and the involvement of both a- and c-subunits in the mechanism of proton movement, it is argued that relative movement must occur between the subunits. Various options for the arrangement and structure of the subunits involved are discussed and a mechanism proposed.

A mutation in which alanine 128 Is replaced by aspartic acid abolishes dimerization of the b-subunit of the F0F1-ATPase from Escherichia coli.

Site-directed mutagenesis was used to investigate the roles of a short series of hydrophobic amino acids in the b-subunit of the Escherichia coli F0F1-ATPase. A mutation affecting one of these, G131D, had been previously characterized and was found to interrupt assembly of the F0F1-ATPase (Jans, D. A., Hatch, L., Fimmel, A. L., Gibson, D., and Cox, G. B. (1985) J. Bacteriol. 162, 420-426). To extend this work, aspartic acid was substituted for each one of the residues from positions 124 to 132. The properties of mutants in this series are consistent with the region from Val124 to Gly131 forming an alpha-helix. Two of the mutations, V124D and A128D, resulted in a similar phenotype to the G131D mutation. This suggested that Val124, Ala128, and Gly131 form a helical face which may have a role in inter- or intrasubunit interactions. This was tested by overexpressing and purifying the cytoplasmic domains of the wild type and A128D mutant b-subunits. Sedimentation equilibrium centrifugation indicated that the wild type domain formed a dimer whereas the mutant was present as a monomer.

The chloroplast CF0I subunit can replace the b-subunit of the F0F1-ATPase in a mutant strain of Escherichia coli K12.

The amino acid sequence of the CF0I subunit from the chloroplast F0F1-ATPase has only a low similarity to the amino acid sequence of the b-subunit of the E. coli F0F1-ATPase. However, secondary and tertiary structure predictions plus the distribution of hydrophobic and hydrophilic amino acids have indicated that these two subunits serve a similar function. This proposition was investigated directly. A cDNA clone for the chloroplast atpF gene, encoding the CF0I subunit, was altered by site-directed mutagensis such that the translation start site corresponded to the N-terminus of the mature protein. An E. coli mutant strain carrying a chain-terminating mutation in the uncF gene, encoding the b-subunit, was transformed with the plasmid carrying the altered atpF gene. The resultant transformant was able to grow on succinate and gave a growth yield similar to that of a wild-type control. Assays on membrane preparations from the transformant also clearly indicated that the mature CF0I subunit from spinach chloroplasts was able to replace the E. coli b-subunit in the E. coli F0F1-ATPase.