Quantitative real-time analysis of the efflux by the MacAB-TolC tripartite efflux pump clarifies the role of ATP hydrolysis within mechanotransmission mechanism

摘要

a Schematic representation of the efflux pump assembly in E. coli membranes. The MacAB-TolC pump encompasses the two-membrane barrier the Gram negative bacterium. A trimer of TolC, embedded in the outer membrane, protrudes into the periplasm and docks onto a hexamer of MacA that mediates the interaction with the periplasmic domain of MacB, which is a dimer. b Schematic representation of the in vitro assay. MacA and MacB, in lipid nanodiscs and TolC in proteoliposomes form the tripartite complex upon mixing and incubation. Transport through the whole efflux pump is studied by monitoring ATP hydrolysis and antibiotic transport from the solution, representing the periplasmic space to the interior of the liposome representing the exterior of the bacteria. c Stern–Volmer plot. Quenching of the quantum dots fluorescence in the presence of increasing concentrations of roxithromycin. Results are best described by the Stern–Volmer type equation: F0/F = 1+ KSV[S], where F and F0 are the fluorescent intensities of the QDs at a given roxithromycin concentration and in a roxithromycin-free solution, respectively. d NADH-coupled ATPase assay. Hydrolysis of ATP is coupled to the oxidation of NADH as a consequence of two consecutive enzymatic reactions. First, ADP generated upon ATP hydrolysis is regenerated to ATP by pyruvate kinase (PK) that catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate. Pyruvate is then reduced to lactate by lactate dehydrogenase (LDH), which concomitantly catalyzes the oxidation of NADH. Hence, decrease in ATP concentration is directly and stoichiometrically correlated to the decrease in NADH concentration.