epsilon-binding regions of the gamma subunit of Escherichia coli ATP synthase

The interaction between the gamma and epsilon subunits of the F-1-ATPase sector of Escherichia coli ATP synthase has been investigated using monoclonal antibodies directed against the gamma subunit and ligand blotting using I-125-epsilon. Monoclonal antibody (MAb) gamma-1 was able to bind to epsilon-depleted F-1-ATPase but not to epsilon-replete F-1, implying that epsilon blocked access to the epitope. A ligand blot assay for the binding of I-125-epsilon to gamma was developed. Both MAb gamma-1 and a second antibody, MAb gamma(II), inhibited binding of I-125-epsilon to gamma in this assay while two other anti-gamma monoclonal antibodies did not. The epitope recognized by MAb gamma-1 was mapped between residues R49 and R70, quite distant in sequence from that of MAb gamma(II), which is located C-terminal to residue K199 of the 286-residue polypeptide. The competition of these antibodies with epsilon for binding to gamma implies that their epitopes, quite separate in sequence, are both located in parts of the subunit involved in binding epsilon.