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

The F domain of FF-ATP synthases/ATPases (FF) possesses three catalytic sites on the three αβ interfaces, termed αβ, αβ, and αβ, 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, FF 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) FF 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 FF, 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 αβ, responsible for ATP hydrolysis, was occupied by ADP-Mg, with the absence of Pi. Furthermore, the catalytic site at αβ, where ATP enters the F 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 F domain is more likely due to differences in the nucleotide-binding states at the catalytic sites rather than structural differences.