Engineering rotor ring stoichiometries in the ATP synthase

Abstract

ATP synthase membrane rotors consist of a ring of c-subunits whose stoichiometry is constant for a given species but variable across different ones. We investigated the importance of c/c-subunit contacts by site-directed mutagenesis of a conserved stretch of glycines (GxGxGxGxG) in a bacterial c11 ring. Structural and biochemical studies show a direct, specific influence on the c-subunit stoichiometry, revealing c <11, c 12, c 13, c 14, and c >14 rings. Molecular dynamics simulations rationalize this effect in terms of the energetics and geometry of the c-subunit interfaces. Quantitative data from a spectroscopic interaction study demonstrate that the complex assembly is independent of the c-ring size. Real-time ATP synthesis experiments in proteoliposomes show the mutant enzyme, harboring the larger c 12 instead of c 11, is functional at lower ion motive force. The high degree of compliance in the architecture of the ATP synthase rotor offers a rationale for the natural diversity of c-ring stoichiometries, which likely reflect adaptations to specific bioenergetic demands. These results provide the basis for bioengineering ATP synthases with customized ion-to-ATP ratios, by sequence modifications.