Characterization of the interface between gamma and epsilon subunits of Escherichia coli F-1-ATPase

The interaction faces of the gamma and epsilon subunits in the Escherichia coli F-1-ATPase have been explored by a combination of cross-linking and chemical modification experiments using several mutant epsilon subunits as follows: epsilon S10C, epsilon H38C, epsilon T43C, epsilon S65C, epsilon S108C, and epsilon M138C, along with a mutant of the gamma subunit, gamma T106C. The replacement of Ser-10 by a Cys or Met-138 by a Cys reduced the inhibition of ECF(1) by the epsilon subunit, while the mutation S65C increased this inhibitory effect. Modification of the Cys at position 10 with N-ethylmaleimide or fluoroscein maleimide further reduced the binding affinity of, and the maximal inhibition by, the epsilon subunit. Similar chemical modification of the Cys at position 43 of the epsilon subunit (in the mutant epsilon T43C) and a Cys at position 106 of the gamma subunit (gamma T106C) also affected the inhibition of ECF(1) by the epsilon subunit. The various epsilon subunit mutants were reacted with TFPAM3, and the site(s) of cross-linking within the ECF(1) complex was determined. Previous studies have shown cross-linking from the Cys at positions 10 and 38 with the gamma subunit and from a Cys at position 108 to an alpha subunit (Aggeler, R., Chicas-Cruz, K., Cai, S. X., Keana, J. F. W., and Capaldi, R. A. (1992) Biochemistry 31, 2956-2961; 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 gamma subunit and from the Cys at position 138 to a beta subunit. The site of cross-linking from Cys-10 of epsilon to the gamma subunit was localized by peptide mapping to a region of the gamma 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 gamma subunit, between residues 202 and 286. ECF(1) treated with trypsin at pH 7.0 still binds purified epsilon 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 gamma subunit as involved in epsilon subunit binding.