A model of the quaternary structure of the Escherichia coli F₁ ATPase from X-ray solution scattering and evidence for structural changes in the Delta subunit during ATP hydrolysis

The shape and subunit arrangement: of the Escherichia coli F₁ ATPase (ECF₁ ATPase) was investigated by synchrotron radiation x-ray solution scattering. The radius of gyration and the maximum dimension of the enzyme complex are 4.61 ± 0.03 nm and 15.5 ± 0.05 nm, respectively. The shape of the complex was determined ab initio from the scattering data at a resolution of 3 nm, which allowed unequivocal identification of the volume occupied by the α₃β₃ subassembly and further positioning of the atomic models of the smaller subunits. The δ subunit was positioned near the bottom of the α₃β₃ hexamer in a location consistent with a β-δ disulfide formation in the mutant ECF₁ ATPase, βY331W:βY381C:εS108C, when MgADP is bound to the enzyme. The position and orientation of the ε subunit were found by interactively fitting the solution scattering data to maintain connection of the two-helix hairpin with the α₃β₃ complex and binding of the β- sandwich domain to the γ subunit. Nucleotide-dependent changes of the δ subunit were investigated by stopped-flow fluorescence technique at 12°C using N-[4-[7-(dimethylamino)-4-methyl]coumarin-3-yl]maleimide (CM) as a label. Fluorescence quenching monitored after addition of MgATP was rapid [k = 6.6 s^-1] and then remained constant. Binding of MgADP and the noncleavable nucleotide analog AMP · PNP caused an initial fluorescent quenching followed by a slower decay back to the original level. This suggests that the δ subunit undergoes conformational changes and/or rearrangements in the ECF₁ ATPase during ATP hydrolysis.