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 ∼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 apparentKm of 878 μm and aV max 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 μmKdo2-lipid A. Addition of Kdo2-lipid A increased the V max 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. 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 apparentKm of 878 μm and aV max 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 μmKdo2-lipid A. Addition of Kdo2-lipid A increased the V max 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. ATPase activity of the MsbA lipid flippase ofEscherichia coli.Journal of Biological ChemistryVol. 277Issue 46PreviewPage 36698, Fig. 1: The upper panel of this figure was reproduced incorrectly. The correct figure is shown below. Full-Text PDF Open Access lipopolysaccharide ATP-binding cassette dodecyl maltoside hexahistidine 3-deoxy-d-mannooctulosonic acid N,N-dimethyldodecylamine N-oxide multidrug resistance The envelope of Escherichia coli and other Gram-negative bacteria consists of an inner membrane, a periplasmic space, and an outer membrane (1Nikaido H. Neidhardt F.C. 2nd Ed. Escherichia coli and Salmonella: Cellular and Molecular Biology. 1. American Society for Microbiology, Washington, D. C.1996: 29-47Google Scholar). The outer leaflet of the outer membrane is composed of lipopolysaccharide (LPS),1 a complex glycolipid that is essential for an effective permeability barrier and is a potent stimulator of the innate immune system in mammals (2Raetz C.R.H. Annu. Rev. Biochem. 1990; 59: 129-170Crossref PubMed Scopus (1040) Google Scholar, 3Raetz C.R.H. Neidhardt F.C. 2nd Ed. Escherichia coli and Salmonella: Cellular and Molecular Biology. 1. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google Scholar, 5Raetz C.R.H. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3382) Google Scholar). Lipid A is the hydrophobic anchor of LPS (2Raetz C.R.H. Annu. Rev. Biochem. 1990; 59: 129-170Crossref PubMed Scopus (1040) Google Scholar, 3Raetz C.R.H. Neidhardt F.C. 2nd Ed. Escherichia coli and Salmonella: Cellular and Molecular Biology. 1. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google Scholar, 5Raetz C.R.H. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3382) Google Scholar). Lipid A biosynthesis is essential for survival of most Gram-negative bacteria and is a target for the design of new antibiotics (5Raetz C.R.H. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3382) Google Scholar, 6Galloway S.M. Raetz C.R.H. J. Biol. Chem. 1990; 265: 6394-6402Abstract Full Text PDF PubMed Google Scholar, 7Onishi H.R. Pelak B.A. Gerckens L.S. Silver L.L. Kahan F.M. Chen M.H. Patchett A.A. Galloway S.M. Hyland S.A. Anderson M.S. Raetz C.R.H. Science. 1996; 274: 980-982Crossref PubMed Scopus (357) Google Scholar, 8Jackman J.E. Fierke C.A. Tumey L.N. Pirrung M. Uchiyama T. Tahir S.H. Hindsgaul O. Raetz C.R.H. J. Biol. 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The mechanism by which E. colitransports newly synthesized lipid A from its site of biosynthesis at the inner face of the inner membrane to the outer membrane is not fully understood. Recent studies from our laboratory have demonstrated that export of both lipid A and glycerophospholipids requires the inner membrane protein MsbA (10Zhou Z. White K.A. Polissi A. Georgopoulos C. Raetz C.R.H. J. Biol. Chem. 1998; 273: 12466-12475Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 11Doerrler W.T. Reedy M.C. Raetz C.R.H. J. Biol. Chem. 2001; 276: 11461-11464Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar), originally identified as a multicopy suppressor of the temperature-sensitive growth phenotype oflpxL(htrB) knockouts (12Karow M. Georgopoulos C. Mol. Microbiol. 1993; 7: 69-79Crossref PubMed Scopus (125) Google Scholar), a a in the lipid A T. J. Raetz C.R.H. J. PubMed Scopus Google Scholar, T. Raetz C.R.H. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google MsbA is a of the ATP-binding cassette is to mammalian and is required for growth ofE. coli (12Karow M. Georgopoulos C. Mol. Microbiol. 1993; 7: 69-79Crossref PubMed Scopus (125) Google Scholar, A. Georgopoulos C. Mol. Microbiol. 1996; PubMed Scopus Google Scholar). (10Zhou Z. White K.A. Polissi A. Georgopoulos C. Raetz C.R.H. J. Biol. Chem. 1998; 273: 12466-12475Abstract Full Text Full Text PDF PubMed Scopus (283) Google demonstrated that of MsbA in the accumulation of lipid A and glycerophospholipids in the inner have a temperature-sensitive E. of MsbA and have shown that loss of MsbA function results in of by accumulation of lipid A and phospholipids in the inner membrane and of inner membrane by W.T. Reedy M.C. Raetz C.R.H. J. Biol. Chem. 2001; 276: 11461-11464Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). MsbA in a acid in its evidence that MsbA as a lipid or flippase, a in the of from the inner membrane to the outer membrane of E. coli W.T. Reedy M.C. Raetz C.R.H. J. Biol. Chem. 2001; 276: 11461-11464Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). is evidence that MsbA multidrug resistance in E. coli, the protein in resistance to of the that are by A. 1998; PubMed Scopus Google M. Microbiol. Mol. Biol. Rev. 2000; PubMed Scopus Google Scholar). E. coli a acid inner membrane protein W.T. Reedy M.C. Raetz C.R.H. J. Biol. Chem. 2001; 276: 11461-11464Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, M. Georgopoulos C. Mol. Microbiol. 1993; 7: 69-79Crossref PubMed Scopus (125) Google Scholar). was to a at its and a hydrophobic at its of W.T. Reedy M.C. Raetz C.R.H. J. Biol. Chem. 2001; 276: 11461-11464Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, M. Georgopoulos C. Mol. Microbiol. 1993; 7: 69-79Crossref PubMed Scopus (125) Google Scholar). The proposed and of MsbA by the Science. 2001; PubMed Scopus Google at MsbA an the its Science. 2001; PubMed Scopus Google Scholar). The is with and is in to lipid A or Science. 2001; PubMed Scopus Google Scholar). ATP and hydrolysis a that results in lipid to the periplasmic of the inner membrane Science. 2001; PubMed Scopus Google Scholar). The A270T is at a site the periplasmic in the MsbA with the lipid seen in at 42 °C. studies have shown that the ATPase activity of and other purified and reconstituted into stimulated by of J. I. S. A. PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, H. J. J. Biol. Chem. 274: Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, S. S. A. 2001; PubMed Scopus Google Scholar). for MsbA and to its in lipid export, have the protein as an N-terminal hexahistidine purified to and reconstituted into The ATPase activity of purified MsbA is the presence of phospholipids and is stimulated by Kdo2-lipid A. was from was from E. coli phospholipids from from and was from and from other and from or The E. coli was from by with to the The was site and the was site and in the are These at a of in a containing of and of The as a °C for by of °C for °C for and °C for was by a at °C. The was and and into an and Molecular to containing the temperature-sensitive MsbA was in an from from W.T. Reedy M.C. Raetz C.R.H. J. Biol. Chem. 2001; 276: 11461-11464Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). was by the of the hexahistidine tag and into an and at the and to of was prepared the from was into cells A was into of in Molecular to and containing 30 and at 37 °C The was into of containing and the cells at 37 °C A was to a of was at 30 °C for and cells by at for at °C. in of mm containing and by a at The was by at for prepared by at for with a of the first membrane in 10 of mm mm mm and mm The membrane was in A at a protein of was to the in to a of and with for at °C. was by at for The was to 10 mm and a with of 10 mm and with of mm in A containing protein was with of mm in A containing of the purified E. lipid a of and to for in a at a of in mm containing mm and mm The liposomes at and by a The liposomes to a of in the and was to the system to of of was at M. PubMed Scopus Google Scholar). was to the at a of The was with at for 30 with and at The system was for at °C and for and for at °C the of The by at for at °C. at of in A containing mm of and at °C. ATPase that a and a MsbA protein in or reconstituted into was for ATPase activity at a protein of in a containing mm to with 10 mm mm and mm containing was or in the a of was the at 37 °C. ATPase for the at 37 °C and by the of of The of by a as by S. M. Biochem. PubMed Scopus Google Scholar), as the the of of a containing of acid in and in was and the at for was by the of of an of and a at was at The was in the of cells S. O. H. J. Biochem. PubMed Scopus Google at 37 °C to A in of to and with by at for 10 with an of M. J. PubMed Scopus Google Scholar), and in of and to a of J. Biochem. PubMed Scopus Google Scholar). The cells for at with was by at for 10 and the containing the glycerophospholipids and Kdo2-lipid A, was was to a system by the of and to the J. Biochem. PubMed Scopus Google Scholar). the by as and the upper was with a The with a upper and in a The in 10 of and to a as the in the C.R.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar, C.R.H. S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The was with of and with of or C.R.H. S. J. Biol. Chem. Full Text PDF PubMed Google the was not to the and other phospholipids in the mm The Kdo2-lipid A species in the but the Kdo2-lipid A Z. S. Raetz C.R.H. J. Biol. Chem. 274: Full Text Full Text PDF PubMed Scopus Google Z. A.A. S. Raetz C.R.H. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus (182) Google in the mm The containing the purified Kdo2-lipid A and to a system by the of of and The was with a of and was a of was with for in a in mm and at °C. The of the from the was by by in a containing and and by acid The of purified lipid was by acid hydrolysis in of at °C for by of the the S. M. Biochem. PubMed Scopus Google Scholar). in to a The of Kdo2-lipid A was from of The Kdo2-lipid A from was as by by acid and by not Lipid X was from E. coli M. Raetz C.R.H. J. PubMed Google as by and Raetz Raetz C.R.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Lipid was prepared to the of Raetz C.R.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and was a from of this C.R.H. Neidhardt F.C. 2nd Ed. Escherichia coli and Salmonella: Cellular and Molecular Biology. 1. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google was from and at a of mm in mm hexaacylated lipid A was prepared by hydrolysis at °C from or Kdo2-lipid A (10Zhou Z. White K.A. Polissi A. Georgopoulos C. Raetz C.R.H. J. Biol. 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PubMed Scopus Google Scholar), that the ATPase activity of the MsbA was in the of nmol/min/mg The of E. to the system at stimulated and the ATPase activity at or to in the presence of the of Kdo2-lipid A at in stimulation of ATPase activity this the lipid with the nonionic J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of the results shown in Fig. reconstituted the solubilized MsbA protein into liposomes at a of prepared coli phospholipids and purified MsbA, both solubilized with by with to the as The MsbA-containing to have an ATPase activity that was with at 37 °C for and with a protein to at of A mm a of 10 mm and from to not max of the reconstituted protein was 37 and the Km for ATP was 878 μm These are in the as for the multidrug resistance protein and transporter H. J. J. Biol. Chem. 274: Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, C. M. E. M. D. J.M. J. Biol. 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The ATPase activity of MsbA increased as as the seen in the of Kdo2-lipid A Stimulation was half-maximal at μm Kdo2-lipid A The that MsbA with hexaacylated lipid A with E. in stimulation of ATPase the of the MsbA with other LPS or with lipid A precursors, at as as with and which are not coli. in Fig. the lipid X and lipid not the ATPase activity of MsbA the with glycerophospholipids purified hexaacylated lipid A the and other by hydrolysis of the ATPase activity of MsbA the seen with reconstituted liposomes LPS from E. coli hexaacylated lipid A and a but the ATPase activity of MsbA the seen with These are with that lipid A are not to the outer membrane as as hexaacylated lipid A is J. Biol. Chem. Full Text PDF PubMed Google Scholar, M. Raetz C.R.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The V max for ATP in the presence of μm Kdo2-lipid A increased from 37 to 154 the Km was decreased by of similar for the V max and Km of reconstituted with H. J. J. Biol. 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