Activation of Bacterial F-ATPase by LDAO: Deciphering the Molecular Mechanism

Abstract

Proton F_OF₁ ATP synthase catalyzes the formation of ATP from ADP and inorganic phosphate coupled with transmembrane proton transfer using the energy of the protonmotive force (pmf). As pmf decreases, the direction of the reaction is reversed and the enzyme generates pmf, transferring protons across the membrane using the energy of ATP hydrolysis. ATPase activity of the enzyme can be suppressed by ADP in a non-competitive manner (ADP-inhibition), and in a number of bacteria, it can be inhibited by conformational changes in the regulatory C-terminal domain of the ε subunit. Lauryldimethylamine oxide (LDAO), a zwitterionic detergent, is known to attenuate both of these inhibitory mechanisms, significantly increasing the ATPase activity of the enzyme. For this reason, LDAO is sometimes used for semi-quantitative estimation of the enzyme’s susceptibility to these regulatory mechanisms. However, the binding site of LDAO in ATP synthase remains unknown. The mechanism by which the detergent counteracts ADP-inhibition and the inhibition involving the ε subunit is also unclear. We performed molecular docking and predicted that LDAO binding might occur at the catalytic site of ATP synthase, whether empty or containing nucleotides. Molecular dynamics simulations showed that LDAO could affect the mobility of the loop in the β subunit (residues β404-415 in Escherichia coli ATP synthase) near the catalytic site. Mutagenesis of residue β409 in the E. coli enzyme and the corresponding β419 residue in the Bacillus subtilis ATP synthase revealed that the type of side chain of this residue indeed affects LDAO-dependent stimulation of ATPase activity. We also found that LDAO activates the enzyme more strongly in the presence of 100 mM sulfate compared to sulfate-free medium. This phenomenon is likely due to the enhancement of ADP-inhibition of the enzyme by sulfate.