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

Abstract

The F₁ domain of F_oF₁-ATP synthases/ATPases (F_oF₁) possesses three catalytic sites on the three αβ interfaces, termed α_Eβ_E, α_Dβ_D, and α_Tβ_T, 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, F_oF₁ 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) F_oF₁ 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 F_oF₁, 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 α_Dβ_D, responsible for ATP hydrolysis, was occupied by ADP-Mg, with the absence of Pi. Furthermore, the catalytic site at α_Eβ_E, 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.

Keywords: ATP synthase; ATPase; Cryo-electron microscopy; Cryo-snapshot; Enzyme mechanism; F(o)F(1); H(+)-ATPase; Molecular motor.