The defective proton-ATPase of uncA mutants of Escherichia coli: ATP-binding and ATP-induced conformational change in mutant α-subunits

Mutations in the uncA gene of Escherichia coli cause loss of both oxidative phosphorylation and ATP-driven generation of the transmembrane proton gradient. The uncA gene encodes the α-subunit of the F₁-sector of the E. coli membrane proton-ATPase. F₁-α-subunit from normal (unc⁺) E. coli binds ATP tightly (K_D = 0.1 μm) and undergoes a large ATP-induced conformational change, but the functional role of the ATP-binding site is currently unknown. There is disagreement in the literature as to whether the ATP-binding site is present or lacking in F₁-α-subunit from uncA mutant strains. One obstacle in studying this question is the difficulty of purifying mutant α-subunits in native form. In order to circumvent this difficulty we have studied ATP binding and ATP-induced conformational changes in mixtures of F₁ subunits obtained by dissociating uncA mutant F₁. Anti-α antibody was used in conjunction with immunoblotting to identify the α-subunits in the mixtures. Retention of native conformation by the α-subunits was demonstrated by the fact that the dissociated α-subunits were fully competent to repolymerize with other F₁ subunits to yield intact F₁ aggregate. The results show that, contrary to previous reports, α-subunits from three catalytically defective uncA mutants do indeed bind ATP and do undergo an ATP-induced conformational change. The binding affinity of α-subunit for ATP was lower than normal in each of the three mutants, but this is not likely to be a significant factor under physiological conditions.