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Abstract
Mutants of ECF1-ATPase were generated, containing cysteine residues in one or more of the following positions: alphaSer-411, betaGlu-381, and epsilonSer-108, after which disulfide bridges could be created by CuCl2 induced oxidation in high yield between alpha and epsilon, beta and epsilon, alpha and gamma, beta and gamma (endogenous Cys-87), and alpha and beta. All of these cross-links lead to inhibition of ATP hydrolysis activity. In the two double mutants, containing a cysteine in epsilonSer-108 along with either the DELSEED region of beta (Glu-381) or the homologous region in alpha (Ser-411), there was a clear nucleotide dependence of the cross-link formation with the epsilon subunit. In betaE381C/epsilonS108C the beta-epsilon cross-link was obtained preferentially when Mg2+ and ADP + Pi (addition of MgCl2 + ATP) was present, while the alpha-epsilon cross-link product was strongly favored in the alphaS411C/epsilonS108C mutant in the Mg2+ ATP state (addition of MgCl2 + 5'-adenylyl-beta,gamma-imidodiphosphate). In the triple mutant alphaS411C/betaE381C/epsilonS108C, the epsilon subunit bound to the beta subunit in Mg2+-ADP and to the alpha subunit in Mg2+-ATP, indicating a significant movement of this subunit. The gamma subunit cross-linked to the beta subunit in higher yield in Mg2+-ATP than in Mg2+-ADP, and when possible, i.e. in the triple mutant, always preferred the interaction with the beta over the alpha subunit.
Highlights
Nucleotide-binding domain of 9  strands and 9 ␣ helices, and a COOH-terminal, predominantly ␣-helical domain, that provides the binding site for the ⑀ subunit, probably the ␦ subunit, as well as the b subunit of the F0
The part of the ␥ subunit within the ring of ␣ and  subunits is arranged as two ␣ helices: a long COOH-terminal helix extending from the top NH2-terminal domain of the ␣ and  subunits into the stalk region, and a short NH2-terminal helix running from the catalytic site domain into the stalk
Four different mutants were used in this study: (i) E381C/ ⑀S108C, (ii) ␣S411C/⑀S108C, (iii) ␣S411C/E381C, and (iv) ␣S411C/E381C/⑀S108C
Summary
Nucleotide-binding domain of 9  strands and 9 ␣ helices, and a COOH-terminal, predominantly ␣-helical domain, that provides the binding site for the ⑀ subunit, probably the ␦ subunit, as well as the b subunit of the F0. The yield of disulfide bond formation between  and ␥ is higher in Mg2ϩ AMP-PNP than in Mg2ϩ ADP ϩ Pi. Cross-linking Studies with the Mutant ␣S411C/⑀S108C— The ␣ and  subunits of F1-ATPases have a very similar fold (Abrahams et al, 1994) and residue 411 of the ␣ subunit is the equivalent of Glu-381 in the DELSEED region of the  subunit.
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