Binding of subunit E into the A–B interface of the A1AO ATP synthase

Abstract

Two of the distinct diversities of the engines A1AO ATP synthase and F1FO ATP synthase are the existence of two peripheral stalks and the 24kDa stalk subunit E inside the A1AO 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 A1AO 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 A3B3 hexamer with an affinity (Kd) of 6.1±0.2μM. FCS experiments with single A and B, respectively, demonstrated unequivocally that subunit E binds stronger to subunit B (Kd=18.9±3.7μM) than to the catalytic A subunit (Kd=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.