Structure of bovine mitochondrial F1-ATPase inhibited by Mg2+ADP and aluminium fluoride

Background: The globular domain of the membrane-associated F₁F_o-ATP synthase complex can be detached intact as a water-soluble fragment known as F₁-ATPase. It consists of five different subunits, α, β, γ, δ and ϵ, assembled with the stoichiometry 3:3:1:1:1. In the crystal structure of bovine F₁-ATPase determined previously at 2.8 Å resolution, the three catalytic β subunits and the three noncatalytic α subunits are arranged alternately around a central α-helical coiled coil in the γ subunit. In the crystals, the catalytic sites have different nucleotide occupancies. One contains the triphosphate form of the nucleotide, the second contains the diphosphate, and the third is unoccupied. Fluoroaluminate complexes have been shown to mimic the transition state in several ATP and GTP hydrolases. In order to understand more about its catalytic mechanism, F₁-ATPase was inhibited with Mg²⁺ADP and aluminium fluoride and the structure of the inhibited complex was determined by X-ray crystallography.

Results: The structure of bovine F₁-ATPase inhibited with Mg²⁺ADP and aluminium fluoride determined at 2.5 Å resolution differs little from the original structure with bound AMP-PNP and ADP. The nucleotide occupancies of the α and β subunits are unchanged except that both aluminium trifluoride and Mg²⁺ADP are bound in the nucleotide-binding site of the β_DP subunit. The presence of aluminium fluoride is accompanied by only minor adjustments in the surrounding protein.

Conclusions: The structure appears to mimic a possible transition state. The coordination of the aluminofluoride group has many features in common with other aluminofluoride–NTP hydrolase complexes. Apparently, once nucleotide is bound to the catalytic β subunit, no additional major structural changes are required for catalysis to occur.