ADP-inhibited structure of non-catalytic site-depleted FoF1-ATPase from thermophilic Bacillus sp. PS-3

The F1 domain of FoF1-ATP synthases/ATPases (FoF1) possesses three catalytic sites on the three αβ interfaces, termed αEβE, αDβD, and αTβT, located mainly on the β subunits. The enzyme also has three non-catalytic ATP-binding sites on the three αβ interfaces, located mainly on the α subunits. When ATP does not bind to the non-catalytic site, FoF1 becomes significantly prone to ADP inhibition, ultimately resulting in the loss of ATPase activity. However, the underlying mechanism of ADP inhibition remains unclear. Here, we report the cryo-EM structure of the non-catalytic site-depleted (ΔNC) FoF1 from thermophilic Bacillus sp. PS-3, which completely lacks the ability to bind ATP (and ADP) upon transitioning to the ADP-inhibited form. The structure closely resembled the 81° rotated structure of the wild-type FoF1, except for minor movements in the C-terminal region of the α subunit. In this structure, unlike the wild-type enzyme, the catalytic site at αDβD, responsible for ATP hydrolysis, was occupied by ADP-Mg, with the absence of Pi. Furthermore, the catalytic site at αEβE, where ATP enters the F1 domain during steady-state catalysis, is occupied by ADP, seemingly impeding further ATP binding to the enzyme. The structure suggests that the ADP-inhibited form of the F1 domain is more likely due to differences in the nucleotide-binding states at the catalytic sites rather than structural differences. •Cryo-EM structure of non-catalytic site-depleted thermophilic bacterium (ΔNC) FoF1 reveals ADP inhibited structure of the FoF1.•In this structure, unlike the wild-type enzyme, the αDβD was occupied by ADP-Mg without the presence of Pi, and the αEβE is occupied by ADP-Mg•In the ADP-inhibited form of the F1 domain, is more likely due to differences in the nucleotide-binding states at the catalytic sites rather than structural differences.