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The ABC transporter MsbA interacts with lipid A and amphipathic drugs at different sites

By Alena Siarheyeva and Frances J. Sharom

Abstract

MsbA is an essential ABC (ATP-binding cassette) transporter involved in lipid A transport across the cytoplasmic membrane of Gram-negative bacteria. The protein has also been linked to efflux of amphipathic drugs. Purified wild-type MsbA was labelled stoichiometrically with the fluorescent probe MIANS [2-(4′-maleimidylanilino)naphthalene-6-sulfonic acid] on C315, which is located within the intracellular domain connecting transmembrane helix 6 and the nucleotide-binding domain. MsbA–MIANS displayed high ATPase activity, and its folding and stability were unchanged. The initial rate of MsbA labelling by MIANS was reduced in the presence of amphipathic drugs, suggesting that binding of these compounds alters the protein conformation. The fluorescence of MsbA–MIANS was saturably quenched by nucleotides, lipid A and various drugs, and estimates of the Kd values for binding fell in the range of 0.35–10 μM. Lipid A and daunorubicin were able to bind to MsbA–MIANS simultaneously, implying that they occupy different binding sites. The effects of nucleotide and lipid A/daunorubicin binding were additive, and binding was not ordered. The Kd of MsbA for binding lipid A was substantially decreased when the daunorubicin binding site was occupied first, and prior binding of nucleotide also modulated lipid A binding affinity. These results indicate that MsbA contains two substrate-binding sites that communicate with both the nucleotide-binding domain and with each other. One is a high affinity binding site for the physiological substrate, lipid A, and the other site interacts with drugs with comparable affinity. Thus MsbA may function as both a lipid flippase and a multidrug transporter

Topics: Research Article
Publisher: Portland Press Ltd.
OAI identifier: oai:pubmedcentral.nih.gov:2662489
Provided by: PubMed Central
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    Citations

    1. (1998). A bacterial antibiotic-resistance gene that complements the human multidrug-resistance P-glycoprotein gene.
    2. (1998). A combined Hansch/Free-Wilson approach as predictive tool in QSAR studies on propafenone-type modulators of multidrug resistance.
    3. (2005). A critical role of a carboxylate in proton conduction by the ATP-binding cassette multidrug transporter LmrA.
    4. (1977). A simplification of the protein assay method of Lowry et al. which is more generally applicable.
    5. (2006). A structural analysis of asymmetry required for catalytic activity of an ABC-ATPase domain dimer.
    6. (2001). ABC transporters: physiology, structure and mechanism – an overview.
    7. (2004). Amino acid type selective isotope labelling of the multidrug ABC transporter LmrA for solid-state NMR studies.
    8. (2001). An Escherichia coli mutant defective in lipid export.
    9. (2003). Analyses of circular dichroism spectra of membrane proteins.
    10. (2002). ATP binding to the motor domain from an ABC transporter drives formation of a nucleotide sandwich dimer.
    11. (2004). ATP-binding cassette transporters in bacteria.
    12. (2002). ATPase activity of the MsbA lipid flippase of Escherichia coli.
    13. (2007). Catalytic cycle of ATP hydrolysis by P-glycoprotein: evidence for formation of the E.S reaction intermediate with ATP-γ-S, a nonhydrolyzable analogue of ATP.
    14. (1995). Characterization of the ATPase activity of P-glycoprotein from multidrug-resistant Chinese hamster ovary cells.
    15. (2006). Characterization of the LSGGQ and H motifs from the Escherichia coli lipid A transporter MsbA.
    16. (2005). Characterization of the Walker A motif of MsbA using site-directed spin labeling electron paramagnetic resonance spectroscopy.
    17. (2007). Cholesterol fill-in model: mechanism for substrate recognition by ABC proteins.
    18. (2007). Conformational motion of the ABC transporter MsbA induced by ATP hydrolysis.
    19. (2004). Conformational stability and domain coupling in D-glucose/D-galactose-binding protein from Escherichia coli.
    20. (2007). Crystal structure of a catalytic intermediate of the maltose transporter. Nature 450, 515–521 c The Authors Journal compilation c 2009 Biochemical Society ©
    21. (2002). DICHROWEB: an interactive website for the analysis of protein secondary structure from circular dichroism spectra.
    22. (2005). Drug-lipid A interactions on the Escherichia coli ABC transporter MsbA.
    23. (1998). Evidence for a role of the multidrug resistance protein (MRP) in the outward translocation of NBD-phospholipids in the erythrocyte membrane.
    24. (2007). Flexibility in the ABC transporter MsbA: alternating access with a twist.
    25. (1997). Fluorescence studies on the nucleotide binding domains of the P-glycoprotein multidrug transporter.
    26. (1998). Function of Escherichia coli MsbA, an essential ABC family transporter, in lipid A and phospholipid biosynthesis.
    27. (2008). Functional characterization of Escherichia coli MsbA: interaction with nucleotides and substrates.
    28. (2005). Interaction of LDS-751 and rhodamine 123 with P-glycoprotein: evidence for simultaneous binding of both drugs.
    29. (2005). Interaction of LDS-751 with P-glycoprotein and mapping of the location of the R drug binding site.
    30. (2008). Interaction of the P-glycoprotein multidrug efflux pump with cholesterol: effects on ATPase activity, drug binding and transport.
    31. (2000). Intrinsic fluorescence of the P-glycoprotein multidrug transporter: Sensitivity of tryptophan residues to binding of drugs and nucleotides.
    32. (2005). Involvement of the “occluded nucleotide conformation” of P-glycoprotein in the catalytic pathway.
    33. (1992). Is the multidrug transporter a flippase? Trends Biochem.
    34. (1992). Movement of Cys-697 in myosin ATPase associated with ATP hydrolysis.
    35. (2005). MsbA is not required for phospholipid transport
    36. (2004). MsbA-dependent translocation of lipids across the inner membrane of Escherichia coli.J .B i o l .C h e m .279,
    37. (1996). Multidrug resistance mediated by a bacterial homolog of the human multidrug transporter MDR1.
    38. (2001). Multidrug transport by ATP binding cassette transporters: a proposed two-cylinder engine mechanism.
    39. (1994). Phosphatidylcholine translocase: a physiological role for the mdr2 gene.
    40. (2001). Phospholipid flippase activity of the reconstituted P-glycoprotein multidrug transporter.
    41. (2002). Proximity of bound Hoechst 33342 to the ATPase catalytic sites places the drug binding site of P-glycoprotein within the cytoplasmic membrane leaflet.
    42. (1993). Reaction of the ArsA adenosinetriphosphatase with 2-(4 -maleimidoanilino)naphthalene-6-sulfonic acid.
    43. (2004). Resting state conformation of the MsbA homodimer as studied by site-directed spin labeling.
    44. (2003). Simultaneous binding of two different drugs in the binding pocket of the human multidrug resistance P-glycoprotein.
    45. (1996). Site-directed fluorescence labeling of P-glycoprotein on cysteine residues in the nucleotide binding domains.
    46. (2001). Site-directed sulfhydryl labeling of the lactose permease of Escherichia coli: helices IV and V that contain the major determinants for substrate binding.
    47. (2005). Structural basis of energy transduction in the transport cycle of MsbA.
    48. (2003). Substrate-induced conformational, changes in the transmembrane segments of human P-glycoprotein – direct evidence for the substrate-induced fit mechanism for drug binding.
    49. (2001). The ABC of ABCs: a phylogenetic and functional classification of ABC systems in living organisms.
    50. (2003). The ATP binding cassette multidrug transporter LmrA and lipid transporter MsbA have overlapping substrate specificities.
    51. (2004). The ATP switch model for ABC transporters.
    52. (1995). The catalytic cycle of P-glycoprotein.
    53. (1993). The essential Escherichia coli msbA gene, a multicopy suppressor of null mutations in the htrB gene, is related to the universally conserved family of ATP-dependent translocators.
    54. (1999). The membrane lipid environment modulates drug interactions with the P-glycoprotein multidrug transporter.
    55. (1997). The multi-drug resistance reversal agent SR33557 and modulation of vinca alkaloid binding to P-glycoprotein by an allosteric interaction.
    56. (1999). The purified and functionally reconstituted multidrug transporter LmrA of Lactococcus lactis mediates the transbilayer movement of specific fluorescent phospholipids.
    57. (2005). The reconstituted P-glycoprotein multidrug transporter is a flippase for glucosylceramide and other simple glycosphingolipids.
    58. (2003). Translocation of phospholipids is facilitated by a subset of membrane-spanning proteins of the bacterial cytoplasmic membrane.
    59. (2007). Uptake or extrusion: crystal structures of full ABC transporters suggest a common mechanism.

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