Binding of subunit e into the A-B interface of the A₁A _O ATP synthase

Two of the distinct diversities of the engines A₁A_O ATP synthase and F₁F_O ATP synthase are the existence of two peripheral stalks and the 24 kDa stalk subunit E inside the A ₁A_O ATP synthase. Crystallographic structures of subunit E have been determined recently, but the epitope(s) and the strength to which this subunit does bind in the enzyme complex are still a puzzle. Using the recombinant A₃B₃D complex and the major subunits A and B of the methanogenic A₁A_O ATP synthase in combination with fluorescence correlation spectroscopy (FCS) we demonstrate, that the stalk subunit E does bind to the catalytic headpiece formed by the A₃B ₃ hexamer with an affinity (K_d) of 6.1 ± 0.2 μM. FCS experiments with single A and B, respectively, demonstrated unequivocally that subunit E binds stronger to subunit B (K_d = 18.9 ± 3.7 μM) than to the catalytic A subunit (K_d = 53.1 ± 4.4). Based on the crystallographic structures of the three subunits A, B and E available, the arrangement of the peripheral stalk subunit E in the A-B interface has been modeled, shining light into the A-B-E assembly of this enzyme.