Probing the rotor subunit interface of the ATP synthase from Ilyobacter tartaricus.
The interaction between the c(11)ring and the gammaepsilon 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 gammaepsilon complex, with an apparent K(d) value of approximately 7.4nm. The rotor assembly was primarily dependent on the interaction of the cring with the gammasubunit, and binding of the cring to the free epsilon subunit was not observed. Mutagenesis of selected conserved amino acid residues of all three rotor components (cR45, cQ46, gammaE204, gammaF203 and epsilonH38) severely affected rotor assembly. The interaction kinetics between the gammaepsilon complex and c(11)ring mutants suggested that the assembly of the c(11)gammaepsiloncomplex was governed by interactions of low and high affinity. Low-affinity binding was observed between the polar loops of the cring subunits and the bottom part of the gamma subunit. High-affinity interactions, involving the two residues gammaE204 and epsilonH38, stabilized the holo-c(11)gammaepsilon 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.
Pogoryelov
Denys
D
Institute of Microbiology, Eidgenössische Technische Hochschule, Zurich, Switzerland.
Nikolaev
Yaroslav
Y
Schlattner
Uwe
U
Pervushin
Konstantin
K
Dimroth
Peter
P
Meier
Thomas
T
D000595:Amino Acid Sequence; D045182:Fusobacteria / Q000201:enzymology*; D007700:Kinetics; D025261:Mitochondrial Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D021122:Protein Subunits / Q000737:chemistry; D016415:Sequence Alignment; D020349:Surface Plasmon Resonance
D021122:Protein Subunits; D025261:Mitochondrial Proton-Translocating ATPases