F1F0-ATP synthase -: Binding of δ subunit to a 22-residue peptide mimicking the N-terminal region of α subunit

The stator in F1F0-ATP synthase resists strain generated by rotor torque. In Escherichia coli the b(2)delta subunit complex comprises the stator, bound to subunit a in F-0 and to alpha(3)beta(3) hexagon of F-1. Proteolysis and cross-linking had suggested that N-terminal residues of a subunit are involved in binding delta. Here we demonstrate that a synthetic peptide consisting of the first 22 residues of a ("alphaN1-22") binds specifically to isolated wild-type delta subunit with high affinity (K-d = 130 nM), accounting for a major portion of the binding energy when delta-depleted F-1 and isolated delta bind together (K-d = 1.4 nM). Stoichiometry of binding of alphaN1-22 to delta at saturation was 1/1, showing that in intact F1F0-ATP synthase only one of the three a subunits is involved in delta binding. When alphaN1-22 was incubated with delta subunits containing mutations in helices I or 5 on the F-1-binding face of delta, peptide binding was impaired as was binding of delta-depleted F-2. Residues alpha6-18 are predicted to be helical, and a potential helix capping box occurs at residues alpha3-8. Circular dichroism measurements showed that alphaN1-22 had significant helical content. Hypothetically a helical region of residues alphaN1-22 packs with helices 1 and 5 on the F-1-binding face of delta, forming the alpha/delta interface.