E. Coli F1Fo ATP Synthase Subunit C: Solution NMR Structure by CS-Rosetta

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The ATP synthase is the main source of ATP in cells, whose function is to inter-convert chemical energy and transmembrane electrochemical potential difference. The enzyme contains two domains: a soluble F1 domain where ATP is synthesized/hydrolyzed and a transmembrane FO domain, which functions as an ion pump. Multiple (9-15) copies of subunit c form an oligomeric ring in the FO domain. The ion translocation activity takes place at the interface of c-ring and subunit a where an essential acidic residue in middle of the outer helix in subunit c undergoes a protonation/deprotonation process, which couples with the relative rotation of c-ring to subunit a. Although high-resolution X-ray structures of the c-rings from several organisms have been solved, the mechanism of ion translocation still needs to be clarified at atomic resolution.We aim to solve the structures of E. coli subunit c at both protonation states of the essential Asp61 and elucidate any conformational changes at the active site during the ion translocation process. Due to the difficulty of obtaining unambiguous long-range NOE constraints in membrane proteins, traditional solution NMR methods were unsuccessful in this case. We employed CS-Rosetta, which utilizes minimal NMR constraints, in our study.Our study of the subunit c in the protonated state shows that the choice of proper scoring weights is essential in the application of CS-Rosetta to membrane proteins. Using limited NMR restraints this method converges on a structure for E. coli subunit c that is similar to the X-ray structures of subunit c from other organisms. Further improving the quality of the structure may be achieved by optimizing the weights in the calculation. We are also using this approach on deprotonated subunit c and hoping to model the c-ring from our results.