Nucleotide-dependent movement of the epsilon subunit between alpha and beta subunits in the Escherichia coli F1F0-type ATPase.
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.
Aggeler
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Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403-1229, USA.
Capaldi
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RA
D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D017354:Point Mutation; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D003432:Cross-Linking Reagents; D009838:Oligodeoxyribonucleotides; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D003545:Cysteine