Engineering rotor ring stoichiometries in the ATP synthase

• Published in: Proceedings of the National Academy of Sciences - PNAS 2012-06, Vol.109 (25), p.E1599-E1608
• Main Authors: Pogoryelov, Denys, Klyszejko, Adriana L, Krasnoselska, Ganna O, Heller, Eva-Maria, Leone, Vanessa, Langer, Julian D, Vonck, Janet, Müller, Daniel J, Faraldo-Gómez, José D, Meier, Thomas
• Format: Article
• Language: English
• Subjects: Adenosine triphosphatase; adenosine triphosphate; Adenosine Triphosphate - biosynthesis; ATP Synthetase Complexes - chemistry; ATP Synthetase Complexes - genetics; ATP Synthetase Complexes - metabolism; Bioenergetics; Bioengineering; Biological Sciences; Chemical reactions; compliance; Computer simulation; Electrophoresis, Polyacrylamide Gel; Enzymes; H+/K+-exchanging ATPase; H-transporting ATP synthase; Microscopy, Atomic Force; Microscopy, Electron; Models, Molecular; molecular dynamics; Mutagenesis; mutants; Mutation; PNAS Plus; Protein Conformation; Proteolipids - metabolism; rotors; site-directed mutagenesis; spectroscopy; stoichiometry; Surface Plasmon Resonance
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1120027109

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 c ₁₁ ring. Structural and biochemical studies show a direct, specific influence on the c-subunit stoichiometry, revealing 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.