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

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The interaction between the c11 ring and the γη 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 c11 ring and the γη complex, with an apparent Kd value of approximately 7.4 nM. The rotor assembly was primarily dependent on the interaction of the c ring with the γ subunit, and binding of the c ring to the free η subunit was not observed. Mutagenesis of selected conserved amino acid residues of all three rotor components (cR45, cQ46, γE204, γF203 and ηH38) severely affected rotor assembly. The interaction kinetics between the γη complex and c11 ring mutants suggested that the assembly of the c11γη 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 γ subunit. High-affinity interactions, involving the two residues γE204 and ηH38, stabilized the holo-c11γη complex. NMR experiments indicated the acquisition of conformational order in otherwise flexible C- and N-terminal regions of the γ subunit on rotor assembly. The results of this study suggest that docking of the central stalk of the F1 complex to the rotor ring of Fo to form tight, but reversible, contacts provides an explanation for the relative ease of dissociation and reconstitution of F1Fo complexes.

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