Structure of bovine mitochondrial F 1-ATPase inhibited by Mg 2+ADP and aluminium fluoride

Abstract

Background: The globular domain of the membrane-associated F 1F o-ATP synthase complex can be detached intact as a water-soluble fragment known as F 1-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 F 1-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, F 1-ATPase was inhibited with Mg 2+ADP and aluminium fluoride and the structure of the inhibited complex was determined by X-ray crystallography. Results: The structure of bovine F 1-ATPase inhibited with Mg 2+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 Mg 2+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.