Characterization of the interface between γ and ε subunits of Escherichia coli F1-ATPase

Abstract

The interaction faces of the γ and ε subunits in the Escherichia coli F1-ATPase have been explored by a combination of cross-linking and chemical modification experiments using several mutant ε subunits as follows: εS10C, εH38C, εT43C, εS65C, εS108C, and εM138C, along with a mutant of the γ subunit, γT106C. The replacement of Ser-10 by a Cys or Met-138 by a Cys reduced the inhibition of ECF1 by the ε subunit, while the mutation S65C increased this inhibitory effect. Modification of the Cys at position 10 with N-ethylmaleimide or fluorescein maleimide further reduced the binding affinity of, and the maximal inhibition by, the ε subunit. Similar chemical modification of the Cys at position 43 of the ε subunit (in the mutant εT43C) and a Cys at position 106 of the γ subunit (γT106C) also affected the inhibition of ECF1 by the ε subunit. The various ε subunit mutants were reacted with TF-PAM3, and the site(s) of cross-linking within the ECF1 complex was determined. Previous studies have shown cross-linking from the Cys at positions 10 and 38 with the γ subunit and from a Cys at position 108 to an subunit (Aggeler, R., Chicas-Cruz, K., Cai, S. X., Keana, J. F. W., and Capaldi, R. A. (1992) Biochemistry 31, 29562961; Aggeler, R., Weinreich, F., and Capaldi, R. A. (1995) Biochim. Biophys. Acta 1230, 62-68). Here, cross- linking was found from a Cys at position 43 to the γ subunit and from the Cys at position 138 to a β subunit. The site of cross-linking from Cys-10 of ε to the γ subunit was localized by peptide mapping to a region of the γ subunit between residues 222 and 242. Cross-linking from a Cys at position 38 and at position 43 was with the C-terminal part of the γ subunit, between residues 202 and 286. ECF1 treated with trypsin at pH 7.0 still binds purified ε subunit, while enzyme treated with the protease at pH 8.0 does not. This identifies sites around residue 70 and/or between 202 and 212 of the γ subunit as involved in ε subunit binding.