ATPase activity of the MsbA lipid flippase of Escherichia coli.
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 approximately 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 K(m) of 878 microm and a V(max) of 37 nmol/min/mg for ATP hydrolysis in the presence of 10 mm Mg(2+). 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 microm Kdo(2)-lipid A. Addition of Kdo(2)-lipid A increased the V(max) to 154 nmol/min/mg and decreased the K(m) to 379 microm. Stimulation was only seen with hexaacylated lipid A species and not with precursors, such as diacylated lipid X or tetraacylated lipid IV(A). 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 degrees C but was significantly reduced at 42 degrees 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.
Doerrler
William T
WT
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Raetz
Christian R H
CR
D018528:ATP-Binding Cassette Transporters; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry*; D002240:Carbohydrate Sequence; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D017353:Gene Deletion; D006868:Hydrolysis; D007700:Kinetics; D008070:Lipopolysaccharides / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D013696:Temperature; D013997:Time Factors; D014638:Vanadates / Q000494:pharmacology
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D003902:Detergents; D008070:Lipopolysaccharides; D008565:Membrane Proteins; C079726:MsbA protein, Bacteria; D014638:Vanadates; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D008274:Magnesium