Functional Characterization of Escherichia coli MsbA

The Escherichia coli MsbA protein is a 65-kDa member of the ATP-binding cassette superfamily. It is thought to function as an ATP-dependent lipid translocase that transports lipid A from the inner to the outer leaflet of the cytoplasmic membrane. MsbA with high ATPase activity was isolated and found to be homodimeric in detergent solution. The protein ATPase activity was inhibited by vanadate and showed variable patterns of stimulation and inhibition by lipid A and other compounds. The intrinsic tryptophan fluorescence of the protein was characterized, and dynamic quenching using acrylamide showed that a conformational change took place on binding of lipid A. Fluorescence quenching was used to characterize the interactions of MsbA with nucleotides and various putative substrates, including lipids, lipid-like compounds, and drugs. MsbA had an apparent binding affinity for ATP of ∼2 mm and also bound nonhydrolyzable ATP analogs and fluorescent ATP derivatives. The putative substrate lipid A interacted with the protein with an affinity of 6.4 μm. Drugs that are known to be substrates for ABC multidrug transporters also interacted with MsbA with affinities in the range 0.25–50 μm. This study represents the first use of fluorescence approaches to estimate MsbA binding affinities for nucleotides and putative transport substrates. The Escherichia coli MsbA protein is a 65-kDa member of the ATP-binding cassette superfamily. It is thought to function as an ATP-dependent lipid translocase that transports lipid A from the inner to the outer leaflet of the cytoplasmic membrane. MsbA with high ATPase activity was isolated and found to be homodimeric in detergent solution. The protein ATPase activity was inhibited by vanadate and showed variable patterns of stimulation and inhibition by lipid A and other compounds. The intrinsic tryptophan fluorescence of the protein was characterized, and dynamic quenching using acrylamide showed that a conformational change took place on binding of lipid A. Fluorescence quenching was used to characterize the interactions of MsbA with nucleotides and various putative substrates, including lipids, lipid-like compounds, and drugs. MsbA had an apparent binding affinity for ATP of ∼2 mm and also bound nonhydrolyzable ATP analogs and fluorescent ATP derivatives. The putative substrate lipid A interacted with the protein with an affinity of 6.4 μm. Drugs that are known to be substrates for ABC multidrug transporters also interacted with MsbA with affinities in the range 0.25–50 μm. This study represents the first use of fluorescence approaches to estimate MsbA binding affinities for nucleotides and putative transport substrates. The outer membrane of Gram-negative bacteria possesses a unique asymmetric structure. The inner leaflet is composed primarily of glycerophospholipids, although with a higher proportion of phosphatidylethanolamine (PE) 3The abbreviations used are: PE, phosphatidylethanolamine; ABC, ATP-binding cassette; AMP-PCP, adenylylmethylenediphosphonate; AMP-PNP, adenosine 5′-[β,γ-imido]triphosphate; DM, dodecyl maltoside; DTE, dithioerythritol; FRET, Forster resonance energy transfer; GdnHCl, guanidine hydrochloride; H33342, Hoechst 33342; LPS, lipopolysaccharide; MDR, multidrug resistance; NATA, N-acetyl-l-tryptophanamide; NB, nucleotide binding; Pgp, P-glycoprotein; TM, transmembrane; TNP, 2′(3′)-O-(2,4,6-trinitrophenyl); Vi, vanadate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. 3The abbreviations used are: PE, phosphatidylethanolamine; ABC, ATP-binding cassette; AMP-PCP, adenylylmethylenediphosphonate; AMP-PNP, adenosine 5′-[β,γ-imido]triphosphate; DM, dodecyl maltoside; DTE, dithioerythritol; FRET, Forster resonance energy transfer; GdnHCl, guanidine hydrochloride; H33342, Hoechst 33342; LPS, lipopolysaccharide; MDR, multidrug resistance; NATA, N-acetyl-l-tryptophanamide; NB, nucleotide binding; Pgp, P-glycoprotein; TM, transmembrane; TNP, 2′(3′)-O-(2,4,6-trinitrophenyl); Vi, vanadate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. and saturated acyl chains than the cytoplasmic membrane (reviewed in Ref. 1Huijbregts R.P. de Kroon A.I. de Kruijff B. Biochim. Biophys. Acta. 2000; 1469: 43-61Crossref PubMed Scopus (96) Google Scholar). The outer leaflet of the outer membrane is composed mainly of lipid A (Fig. 1), the hexa-acylated hydrophobic core lipid of lipopolysaccharide (LPS) (reviewed in Ref. 2Raetz C.R. Reynolds C.M. Trent M.S. Bishop R.E. Annu. Rev. Biochem. 2007; 76: 295-329Crossref PubMed Scopus (876) Google Scholar). Our understanding of how these lipids traffic to the outer membrane, and how lipid asymmetry is maintained, is limited. One important component of this lipid trafficking system appears to be MsbA (Fig. 1), an essential 64.5-kDa protein found in the cytoplasmic membrane. MsbA is a member of the ATP-binding cassette (ABC) superfamily of proteins, and is proposed to translocate the LPS precursor lipid A (and possibly also phospholipids) from the inner to the outer leaflet of the cytoplasmic membrane. MsbA-mediated translocation is thought to be the first step in the movement of lipid A from its site of synthesis on the inner surface of the cytoplasmic membrane to its final location in the outer leaflet of the outer membrane. MsbA is a 582-residue integral protein with six putative transmembrane (TM) helices and one ABC-type nucleotide-binding (NB) domain at its C-terminal end (Fig. 1). It is presumed to function as a dimer. The sequence of MsbA is 30% identical and 46% similar to that of the N-terminal half of P-glycoprotein (Pgp; ABCB1; MDR1) (3Karow M. Georgopoulos C. Mol. Microbiol. 1993; 7: 69-79Crossref PubMed Scopus (124) Google Scholar), a mammalian efflux pump implicated in multidrug resistance to anti-cancer agents (reviewed in Ref. 4Sharom F.J. Biochem. Cell Biol. 2006; 84: 979-992Crossref PubMed Google Scholar). MsbA is a further 51% identical and 66% similar to Pgp in the NB regions (3Karow M. Georgopoulos C. Mol. Microbiol. 1993; 7: 69-79Crossref PubMed Scopus (124) Google Scholar), and it is one of the prokaryotic proteins most homologous to a mammalian ABC protein. The x-ray crystal structures of MsbA from three bacterial species in different conformations were recently reported (5Ward A. Reyes C.L. Yu J. Roth C.B. Chang G. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 19005-19010Crossref PubMed Scopus (627) Google Scholar), after the original MsbA structures were withdrawn due to the discovery of a flaw in the software used to solve them (6Chang G. Roth C.B. Reyes C.L. Pornillos O. Chen Y.J. Chen A.P. Science. 2006; 314: 1875Crossref PubMed Scopus (89) Google Scholar). A new AMP-PNP-bound structure of MsbA was solved to 3.7 Å, and showed a series of interacting helices that span the bilayer and extend from the membrane into the cytosol, where they are coupled to two interdigitated NB domains (5Ward A. Reyes C.L. Yu J. Roth C.B. Chang G. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 19005-19010Crossref PubMed Scopus (627) Google Scholar). The overall shape and domain organization of MsbA resemble that of the 3.0 Å structure of the putative bacterial multidrug transporter Sav1866 (7Dawson R.J. Locher K.P. Nature. 2006; 443: 180-185Crossref PubMed Scopus (1052) Google Scholar) and the 8 Å cryo-EM structure of Pgp (8Rosenberg M.F. Callaghan R. Modok S. Higgins C.F. Ford R.C. J. Biol. Chem. 2005; 280: 2857-2862Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). The ATPase activity of MsbA into Escherichia coli lipids be in the of A C.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), that MsbA is a G. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) reported that MsbA and bacterial of had similar substrate MsbA in coli resistance to by apparent transport of the from The protein be by the substrate and and Pgp, its ATPase activity in membrane was by and Hoechst ATP-dependent transport of be in A study reported that transport of and by MsbA in was inhibited by lipid A B. G. S. S. J. 2005; PubMed Scopus Google Scholar). of a for MsbA in the translocation of lipid and possibly from the inner to the outer leaflet of the cytoplasmic membrane. in MsbA in of lipid A and in the inner membrane of coli C.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). coli MsbA at the and lipid A were to and from the cytoplasmic than the C.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), with a of MsbA-mediated translocation membrane than from the ABC proteins translocate from the inner to the outer leaflet of the membrane, that MsbA function in the Pgp, and of lipids in a system A. M. PubMed Scopus Google F.J. PubMed Scopus Google F.J. Biochem. J. 2005; PubMed Scopus Google Chang Biochim. Biophys. Acta. PubMed Scopus Google Scholar). the of MsbA with high of ATPase activity of ATP of in detergent it appears to as a and MsbA ATPase activity was by lipid lipid-like compounds, and other known to be substrates for ABC multidrug This the intrinsic fluorescence of MsbA for the first and conformational from nucleotide and substrate Fluorescence quenching are used to the of lipids, and with the protein and binding are a first step to understanding the transport and of this protein. was was from was from AMP-PCP, AMP-PNP, lipid and were from coli lipids were from H33342, and were from and were from was from the and the were as in the were as in coli lipid were in and at MsbA and coli MsbA was by of the of PubMed Scopus Google Scholar). The protein was in as PubMed Scopus Google Scholar). the was into for and the were in in the of A at were with mm for membrane were by using a at a of by and high of the The were with DM, and MsbA was using a with was by the of Biochem. PubMed Scopus Google Scholar) using a was to Nature. PubMed Scopus Google Scholar), using a acrylamide with of membrane protein of MsbA in DM, by with was to a membrane and with by were using the system of the of MsbA by was on a mm using an protein with mm mm mm and DM, a of MsbA protein was to the were at and was by at The of MsbA was by of its to of the protein of MsbA into was into of coli lipids by of lipid with of and of MsbA at the in mm mm mm mm DTE, three were of of with for on The was from the using a and for at and the was in were using a ATPase activity with membrane MsbA in DM, and MsbA into was by the of S. A. R. S. Biochem. PubMed Scopus Google Scholar) as F.J. Biochem. Cell Biol. 2006; 84: PubMed Google Scholar), using mm mm mm DTE, mm as the ATPase and a final of mm of by Fluorescence affinity was for a of lipids, lipid-like compounds, and other using quenching of the intrinsic fluorescence of The protein was in at a of in ATPase Fluorescence were as F.J. Biochem. Cell Biol. 2006; 84: PubMed Google R. A. F.J. 2000; PubMed Scopus Google Scholar) using a with a for and lipid and were as in ATPase and other were as were after at for and lipids for nucleotides by the Fluorescence were for inner and where R. F.J. PubMed Scopus Google Scholar), and to binding to a where is the fluorescence quenching change in fluorescence to the of substrate at a and is the for binding to the protein. was using and of and the fluorescence were were with to the quenching of fluorescence by lipids, and drugs. Fluorescence of MsbA by of acrylamide in ATPase was in to of MsbA in ATPase with DM, in the of mm ATP and lipid A Fluorescence was at at of were after at to of where and to Fluorescence were for inner and were using to acrylamide quenching of the fluorescence of in solution. were using the where and are the fluorescence in the and of is the of and is the quenching a quenching a of a with a of and of coli MsbA was in coli and by bacterial membrane MsbA as by and protein in detergent was (Fig. A from of bacterial of membrane protein with activity of of and of MsbA in detergent with activity of of protein. ATPase activity was at ATP than mm and mm A was ATP was at this The of to the at a of mm the ATPase activity of the protein with the in its The of MsbA in was using protein showed that with protein of known the of the MsbA to an of (Fig. bound to the protein a of it that MsbA as a these of a with to the MsbA also be on of the protein (Fig. of the NB domains is for in ABC proteins, it is that MsbA ATPase MsbA was into of coli lipids using to the lipid and and for detergent using dynamic that coli using this were with a of The of the was and of be a be in this the activity of the protein by of the by the detergent the ATPase activity by that of the MsbA are with NB domains on the outer surface of of and on the ATPase of ATPase activity of ABC proteins be inhibited by substrates. The activity of MsbA in was by and to by a of coli lipids PE, and and of other species as lipid A. of lipid A to MsbA ATPase activity than and the activity of the protein to the that be and the activity by a at and inhibited the activity at higher (Fig. 1). The ATPase activity of MsbA was also by that are substrates for efflux as and Pgp (Fig. 1). different patterns of were as and inhibited the activity of MsbA in the range for Pgp (Fig. as (Fig. activity at and inhibited it at higher The (Fig. and with and 1), inhibited ATPase activity it at stimulation inhibition of MsbA ATPase activity to a of mm (Fig. 1). inhibition of of the activity at a high of of bacterial lipids, lipid-like and on MsbA ATPase at ATPase activity is of the stimulation is stimulation of ATPase activity by the is at ATPase activity is inhibited by is inhibition of ATPase activity by the is lipids is in stimulation is in is in is in is in at ATPase activity is of the stimulation is stimulation of ATPase activity by the is at ATPase activity is inhibited by is inhibition of ATPase activity by the is is in stimulation in a new of the ATPase activity of MsbA in coli lipids by drugs. of and were to MsbA into coli lipids and on for ATPase activity was at in the of mm the and where are the of on the ATPase of is a to ABC proteins (and other in a that is thought to resemble the one of ATP from the to a that ATPase isolated from coli MsbA inhibition of ATPase activity with of (Fig. The activity of at high of the activity of other in the coli membrane that are to MsbA into of coli lipids also inhibition by at similar that most of the ATPase activity in be to MsbA (Fig. The activity at high is for MsbA than coli and a of the activity in be to the be an intrinsic of the protein. The inhibition of MsbA ATPase activity was μm. The of the in the the of ATP by MsbA in the of with an of with activity and Pgp in similar thought to resemble the and of the F.J. Biochem. J. 2006; PubMed Scopus Google Scholar). of these also inhibited MsbA ATPase activity Fluorescence of the MsbA MsbA the N-terminal end of the protein (Fig. 1). the x-ray crystal structure of AMP-PNP-bound these are to the end of the (5Ward A. Reyes C.L. Yu J. Roth C.B. Chang G. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 19005-19010Crossref PubMed Scopus (627) Google Scholar). It is to that the are in in this in the membrane the intrinsic fluorescence of MsbA in detergent with at to from the in (Fig. The of was with the that the to fluorescence in a of MsbA with guanidine the the fluorescence and a in the that of of the the for MsbA and fluorescence are for MsbA in MsbA with MsbA with and for at and Fluorescence was at at of MsbA by is as a dynamic of is and into the protein acrylamide quenching an of the of was an of fluorescence the fluorescence of MsbA The for MsbA was that a of is and are to The of for was higher than the for MsbA that the to MsbA intrinsic fluorescence are to the of lipid lipid A and acrylamide quenching also The for acrylamide quenching in the of of lipid A the by that the to in the of of ATP and lipid A the by the as of lipid A appears to be a conformational binding of ATP binding in the of lipid A a on of the substrate the to a than lipid A (and the fluorescence to a than lipid it also showed a similar in the of ATP of acrylamide quenching on the fluorescence of MsbA and MsbA in ATPase were at with of acrylamide in the of mm ATP and lipid A quenching from the of a of of as lipid ATP lipid ATP MsbA in ATPase were at with of acrylamide in the of mm ATP and lipid A quenching from the of a of of as in a new of MsbA Fluorescence by and MsbA is an that binding of nucleotides to its NB domains the intrinsic in ATP MsbA fluorescence in a the quenching to an the of the nucleotide with a of binding site on the protein in the for and the quenching ATP bound to MsbA with a of This ATP binding than quenching was at where is of the activity at in the of also bound to with similar affinity to ATP (Fig. The nonhydrolyzable a quenching with similar affinity and The nonhydrolyzable ATP appears to to MsbA with higher affinity than ATP The fluorescent nucleotides and also bound to MsbA with higher with fluorescence quenching of It is that quenching by these analogs place by Forster resonance energy the and the fluorescent binding to the for quenching of MsbA intrinsic fluorescence by lipids, and of the and quenching were by the fluorescence quenching to an for a binding site as are as Fluorescence were at for lipids and and for nucleotides to by the of the and quenching were by the fluorescence quenching to an for a binding site as are as Fluorescence were at for lipids and and for nucleotides to by the and lipid-like are in are in was with MsbA into coli lipid than in detergent of the and quenching were by the fluorescence quenching to an for a binding site as are as Fluorescence were at for lipids and and for nucleotides to by the are in was with MsbA into coli lipid than in detergent solution. in a new Pgp, MsbA is to at a than its ATPase activity was of the for mm ATP and at a of higher than the of the the activity at a higher than the for The of mm inhibited MsbA ATPase activity by The for inhibition of ATPase activity by was than the for binding to the protein that the binding and AMP-PCP, with the nucleotide-binding site in the as than by an of that MsbA fluorescence also quenching binding of substrates. of of lipid A to MsbA in in a quenching of the intrinsic fluorescence (Fig. in lipid A appears to to the protein with an affinity of 6.4 μm. of and also to quenching of MsbA fluorescence (Fig. and with binding affinities of and MsbA ATPase activity and be substrates for the protein. a of and bound to as by intrinsic fluorescence quenching (Fig. a range of for the various of of from to than μm. higher quenching of fluorescence for fluorescent as and than with substrates This that at a of the quenching for fluorescent is a of from to the bound lipid substrates had the quenching The quenching in this is due to conformational in the protein binding that the of the of various quenching of intrinsic MsbA of and were at to MsbA in MsbA in coli lipid Fluorescence at was at The quenching was to an binding to a the and where are the the ATPase activity of MsbA the protein with the affinity of the and as and bound to MsbA with high as be for a lipid The Pgp bound to MsbA with the affinity of the the ATPase activity of the protein at high The fluorescence of MsbA into of coli lipid was by (Fig. with a of is similar to higher than that for binding to MsbA in detergent MsbA was as a of to in LPS (3Karow M. Georgopoulos C. Mol. Microbiol. 1993; 7: 69-79Crossref PubMed Scopus (124) Google Scholar). The protein of coli is a in the of lipid A C.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). in an of of of lipid and the to M. O. Georgopoulos C. J. PubMed Google M. O. A. Georgopoulos C. J. PubMed Scopus Google M. Georgopoulos C. Mol. Microbiol. PubMed Scopus Google Scholar). The was as one of two of and (3Karow M. Georgopoulos C. Mol. Microbiol. 1993; 7: 69-79Crossref PubMed Scopus (124) Google M. Georgopoulos C. J. PubMed Scopus Google Scholar). of MsbA for the of lipid A in the cytoplasmic membrane and in in the outer membrane, that it be in the transport of lipid A and species to the outer membrane. ATPase activity is a of ABC MsbA was in the membrane high ATPase activity that was inhibited by It is that a of this activity from the of MsbA in the cytoplasmic membrane proteins of the isolated MsbA from this membrane to and the protein into of coli lipids, are for a of of MsbA is to be a for ATPase the nucleotide-binding site of ABC proteins is at the the two NB domains J. Mol. Full Text Full Text PDF PubMed Scopus Google Scholar). that MsbA using a with that of a and a with the for the was also on The activity of MsbA was is higher than reported C.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google PubMed Scopus Google Scholar) in with (5Ward A. Reyes C.L. Yu J. Roth C.B. Chang G. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 19005-19010Crossref PubMed Scopus (627) Google Scholar), and that the protein is activity at mm with activity at ∼2 is to the reported for ATP of and mm C.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google PubMed Scopus Google Scholar). on other ABC proteins that different and detergent to in and for ATP The ATPase activity of MsbA was inhibited by by Vi, with This is in with the of and C.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), reported that of MsbA ATPase activity was in with inhibition at μm. from for other ABC the and also inhibited MsbA ATPase The ATPase activity of ABC proteins be inhibited by the of substrates. The activity of MsbA in was than by the of the putative lipid that this lipid with the protein. stimulation of MsbA ATPase activity by lipid A C.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google PubMed Scopus Google Scholar). that are known substrates for other ABC efflux were found to the ATPase activity of that they also with the protein. and C.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) reported stimulation by in G. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) reported that the activity of MsbA be by and in bacterial membrane stimulation of MsbA in for inhibition at high of stimulation and inhibition by H33342, stimulation inhibition by and are to with the protein as by The intrinsic fluorescence of MsbA was the protein was using and high the for a The MsbA that the to fluorescence are to be in a the protein. This is with proposed location the of the membrane the as acrylamide on in the of and conformational that place on binding of substrates. of coli MsbA a in the NB domains of the two are by Å in the of substrate and nucleotide (5Ward A. Reyes C.L. Yu J. Roth C.B. Chang G. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 19005-19010Crossref PubMed Scopus (627) Google Scholar). A movement of protein domains nucleotide binding be to ATP in ABC proteins at the of the two NB PubMed Scopus Google Scholar) used and to for this that on of ATP to MsbA in the for quenching by acrylamide showed that ATP binding a protein the fluorescent of lipid A to MsbA the that the to in the of the putative the of ATP and lipid the was by the as for of lipid A Our the of a conformational change binding of substrates. ATP binding in the of substrate to a of the to the NB domains Å the that is a change in the protein as a domain substrate ATP binding that the of the of intrinsic fluorescence be a for binding to this for the first of the binding affinity of MsbA for lipids, lipid-like compounds, and a of drugs. AMP-PNP, and showed quenching of MsbA and of the to an in the range nucleotides bound to MsbA with higher with of similar to affinity for binding to Pgp R. A. F.J. 2000; PubMed Scopus Google Scholar). The binding by interactions in the nucleotide-binding the nucleotides to with MsbA in the as they were with ATP for The putative lipid also MsbA fluorescence with a for binding of 6.4 and and and in the range μm. The high binding affinities found for lipid A and lipid-like the that MsbA be a lipid transporter G. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) were the first to binding to MsbA of the Pgp substrate This study is the first to estimate the binding affinity of MsbA for a of with in the range 0.25–50 μm. and with affinity than lipids and other as reported transport and that MsbA fluorescence and MsbA ATPase that they with the protein in a different from that ATPase as MsbA a substrate binding of the proposed for Pgp, be to the NB The fluorescence were high for the that quenching of by place for these compounds, as for Pgp R. A. F.J. 2000; PubMed Scopus Google Scholar). The integral of and is and for Pgp is a and R. A. F.J. 2000; PubMed Scopus Google Scholar), is to be for MsbA as were for lipid A and other compounds, they were higher for including and It is that also to quenching by these drugs. the for binding of to MsbA the for binding to Pgp F.J. Biochem. Cell Biol. 2006; 84: PubMed Google R. A. F.J. 2000; PubMed Scopus Google Scholar). the for binding to Pgp is for and and for binding to Pgp and are other of for binding of to transport G. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) ATP-dependent transport of in and B. G. S. S. J. 2005; PubMed Scopus Google Scholar) further the transport of a for of in membrane B. G. S. S. J. 2005; PubMed Scopus Google Scholar). found an of for ATPase stimulation of MsbA in and a for binding to MsbA in detergent of μm. this that substrates with MsbA at an binding It is that lipid species with MsbA and its binding The binding to an site with lipid for binding to an to be the structure and function of MsbA and The ATPase activity of proteins is by lipid and as and in F.J. Biochem. Cell Biol. 2006; 84: PubMed Google Scholar). ATPase activity be by and binding of nucleotides and the protein to be to as lipid and The of these ABC proteins of the and membrane of substrates. It that MsbA its substrates in a similar to Pgp, be to the of its lipid A of of for the

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