Interactions between β and γ subunits of ATP synthase are critical in nucleotide binding and energy coupling

ATP synthase serves as a molecular engine, coupling ATP catalysis and proton translocation. To achieve this function, interactions between β and γ subunits are essential. In this study, the γ subunit was fixed at two signature angles, the ATP waiting dwell and the catalytic dwell. We found that the nucleotide binding patterns in these two time frames were greatly different; three catalytic sites were occupied at the catalytic dwell, meanwhile only two were occupied at the ATP waiting dwell. These observations further confirm that nucleotide binding affinity and conformation alternation of the catalytic sites are dependent on the rotational angle of the γ subunit. In addition, two segments of the γ subunit, γ5-15 and γ256-265, were further investigated with alanine replacement; the results revealed that the helical backbones, but not individual amino acid side chains, play important roles in determination of nucleotide binding affinity. Finally, γC87K mutation was identified to uncouple the ATP catalysis and proton translocation due to its positive charge, and a secondary γR242C/S mutation was found capable to suppress this uncoupling phenotype. More pieces of evidence suggest that γC87 could interact with both the C-terminus of γ and the βDELSEED motif.