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Escherichia coli MsbA, the proposed inner membrane lipid flippase, is an essential ATP-binding cassette transporter protein with homology to mammalian multidrug resistance proteins. Depletion or loss of function of MsbA results in the accumulation of lipopolysaccharide and phospholipids in the inner membrane of E. coli. MsbA modified with an N-terminal hexahistidine tag was overexpressed, solubilized with a nonionic detergent, and purified by nickel affinity chromatography to ∼95% purity. The ATPase activity of the purified protein was stimulated by phospholipids. When reconstituted into liposomes prepared from E. coli phospholipids, MsbA displayed an apparent Km of 878 μM and a Vmax of 37 nmol/min/mg for ATP hydrolysis in the presence of 10 mM Mg2+. Preincubation of MsbA-containing liposomes with 3-deoxy-D-mannooctulosonic acid (Kdo)2-lipid A increased the ATPase activity 4-5-fold, with half-maximal stimulation seen at 21 μM Kdo2-lipid A. Addition of Kdo2-lipid A increased the Vmax to 154 nmol/min/mg and decreased the Km to 379 μM. Stimulation was only seen with hexaacylated lipid A species and not with precursors, such as diacylated lipid X or tetraacylated lipid IVA. MsbA containing the A270T substitution, which renders cells temperature-sensitive for growth and lipid export, displayed ATPase activity similar to that of the wild type protein at 30°C but was significantly reduced at 42°C. These results provide the first in vitro evidence that MsbA is a lipid-activated ATPase and that hexaacylated lipid A is an especially potent activator.

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Journal of Biological Chemistry

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