Probing the rotor subunit interface of the ATP synthase from Ilyobacter tartaricus

The interaction between the c(11) ring and the gamma epsilon complex, forming the rotor of the Ilyobacter tartaricus ATP synthase, was probed by surface plasmon resonance spectroscopy and in vitro reconstitution analysis. The results provide, for the first time, a direct and quantitative assessment of the stability of the rotor. The data indicated very tight binding between the c(11) ring and the gamma epsilon complex, with an apparent K(d) value of approximately 7.4 nM. The rotor assembly was primarily dependent on the interaction of the c ring with the gamma subunit, and binding of the c ring to the free epsilon subunit was not observed. Mutagenesis of selected conserved amino acid residues of all three rotor components (cR45, cQ46, gamma E204, gamma F203 and epsilon H38) severely affected rotor assembly. The interaction kinetics between the gamma epsilon complex and c(11) ring mutants suggested that the assembly of the c(11)gamma epsilon complex was governed by interactions of low and high affinity. Low-affinity binding was observed between the polar loops of the c ring subunits and the bottom part of the gamma subunit. High-affinity interactions, involving the two residues gamma E204 and epsilon H38, stabilized the holo-c(11)gamma epsilon complex. NMR experiments indicated the acquisition of conformational order in otherwise flexible C- and N-terminal regions of the gamma subunit on rotor assembly. The results of this study suggest that docking of the central stalk of the F(1) complex to the rotor ring of F(o) to form tight, but reversible, contacts provides an explanation for the relative ease of dissociation and reconstitution of F(1)F(o) complexes.