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ATP synthase is a multi-subunit protein complex that synthesizes ATP, the universal biological energy carrier. Proton movement along the electrochemical gradient rotates the membrane embedded F0 motor that causes conformation changes in the soluble F1 motor, synthesizing ATP from ADP and inorganic phosphate. Given its critical role in cellular energy and cell death, ATP synthase has become a promising drug target against bacteria, especially with the rise of drug-resistant strains. Thus, understanding ATP synthase's subunit interactions is pivotal for identifying novel targets for future drug innovation. Using cryo-electron microscopy models of E. coli ATP synthase, we identified hydrogen bond formations involving D124 of subunit a, Q10 of subunit b, and H15 of subunit a. To understand the importance of this hydrogen bonding network, we generated four mutations for D124 and studied their impact on the in-vitro ATP synthesis, ATP hydrolysis, proton pumping, and their ability to grow on a succinate medium. The results indicated that the non-polar residue substitutions showed moderate to no inhibition of the in-vitro activities. However, D124A moderately inhibited growth on the succinate medium. Surprisingly, D124E significantly inhibited the ATP synthase in-vitro functions and growth on a succinate medium. These results suggest that the D124 may not be necessary for a hydrogen bonding network but may be involved in supporting subunit b in the membrane. The authors acknowledge support from ORAU Ralph E. Powe Junior Faculty Enhancement Award, Berea College start-up fund, and Undergraduate Research and Creative Projects Program. The authors would also acknowledge Prof. Ryan Steed (UNCA) for supplying the unc operon plasmid and Berea College Chemistry and Biology departments for instrumentations.
Shrestha et al. (Fri,) studied this question.