European Biophysics Journal

, Volume 36, Issue 8, pp 1029–1037 | Cite as

Organization of reconstituted lipoprotein MexA onto supported lipid membrane

  • Sylvain Trépout
  • Jean-Christophe Taveau
  • Stéphane Mornet
  • Houssain Benabdelhak
  • Arnaud Ducruix
  • Olivier Lambert
Original Paper


MexA, a periplasmic component of OprM-MexA-MexB tripartite multidrug efflux pump from Pseudomonas aeruginosa, is natively anchored via its fatty acid in the bacteria inner membrane protruding into the periplasm. We used supported lipid bilayer (SLB) to attach the protein to a single leaflet mimicking its perisplamic orientation. For that purpose, we studied the solubilization of DOPC lipid bilayer supported on silica surface with β-octyl glucoside (βOG). First we showed that SLBs resist to βOG concentrations that usually solubilize liposomes. Native form of MexA was directly inserted in the outer leaflet at (βOG concentrations in a range of 20–25 mM). Second, observations by cryo-electron microscopy (cryoEM) revealed a dense protein layer attached to the surface corresponding to a 13-nm layer of MexA proteins. Analysis of protein densities allows proposing a schematic organization of native MexA inserted in lipid membrane. This structural organization provides further insights with respect to the partially solved structure of the soluble form.


Membrane protein Multidrug resistance Cryo-electron microscopy Membrane protein on solid support SLB QCM-D 



This work has been supported in part by EC grants “NMP4-CT2003-505868- Nanocues”, EC grants “QLR-2000-01339” and program ACI “Dynamique et réactivité des assemblages biologiques” DRAB04/136. The authors wish to thank Xavier Moreel, Dimitri Lerouge and Joséphine Lai Kee Him for technical assistance with MexA and QCM-D. Sylvain Trépout is a recipient of a PhD fellowship of French Ministry of Education and Research and Technology (MENRT).


  1. Akama H, Kanemaki M, Yoshimura M, Tsukihara T, Kashiwagi T, Yoneyama H, Narita S, Nakagawa A, Nakae T (2004a) Crystal structure of the drug discharge outer membrane protein, OprM, of Pseudomonas aeruginosa: dual modes of membrane anchoring and occluded cavity end. J Biol Chem 279:52816–52819CrossRefGoogle Scholar
  2. Akama H, Matsuura T, Kashiwagi S, Yoneyama H, Narita S, Tsukihara T, Nakagawa A, Nakae T (2004b) Crystal structure of the membrane fusion protein, MexA, of the multidrug transporter in Pseudomonas aeruginosa. J Biol Chem 279:25939–25942CrossRefGoogle Scholar
  3. Andersen C, (2003) Channel-tunnels: outer membrane components of type I secretion systems and multidrug efflux pumps of Gram-negative bacteria. Rev Physiol Biochem Pharmacol 147:122–165CrossRefGoogle Scholar
  4. Angrand M, Briolay A, Ronzon F, Roux B (1997) Detergent-mediated reconstitution of a glycosyl-phosphatidylinositolprotein into liposomes. Eur J Biochem 250:168–176CrossRefGoogle Scholar
  5. Ataka K, Giess F, Knoll W, Haber-Pohlmeier S, Richter B, Heberle J (2004) Oriented attachment and membrane reconstitution of His-tagged cytochrome c oxidase to a gold electrode: in situ monitoring by surface-enhanced infrared absorption spectroscopy. J Am Chem Soc 126:16199–16206CrossRefGoogle Scholar
  6. Bader B, Kuhn K, Owen DJ, Waldmann H, Wittinghofer A, Kuhlmann J (2000) Bioorganic synthesis of lipid-modified proteins for the study of signal transduction. Nature 403:223–226CrossRefADSGoogle Scholar
  7. Fralick JA (1996) Evidence that TolC is required for functioning of the Mar/AcrAB efflux pump of Escherichia coli. J Bacteriol 178:5803–5805Google Scholar
  8. Grogan MJ, Kaizuka Y, Conrad RM, Groves JT, Bertozzi CR (2005) Synthesis of lipidated green fluorescent protein and its incorporation in supported lipid bilayers. J Am Chem Soc 127:14383–14387CrossRefGoogle Scholar
  9. Guzman LM, Belin D, Carson MJ, Beckwith J (1995) Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 177:4121–4130Google Scholar
  10. Higgins MK, Bokma E, Koronakis E, Hughes C, Koronakis V (2004) Structure of the periplasmic component of a bacterial drug efflux pump. Proc Natl Acad Sci USA 101:9994–9999CrossRefADSGoogle Scholar
  11. Hetzer M, Heinz S, Grage S, Bayerl TM (1998) Asymmetric molecular friction in supported phospholipid bilayers revealed by NMR measurements of lipid diffusion. Langmuir 14:982–984CrossRefGoogle Scholar
  12. Keller CA, Kasemo K (1998) Surface specific kinetics of lipid vesicle adsorption measured with a quartz crystal microbalance. Biophys J 75:1397–1402CrossRefGoogle Scholar
  13. Keller M, Kerth A, Blume A (1997) Thermodynamics of interaction of octyl glucoside with phosphatidylcholine vesicles: partitioning and solubilization as studied by high sensitivity titration calorimetry. Biochim Biophys Acta 1326:178–192CrossRefGoogle Scholar
  14. Koronakis V, Sharff A, Koronakis E, Luisi B, Hughes C (2000) Crystal structure of the bacterial embrane protein TolC central to multidrug efflux and protein export. Nature 405:914–919CrossRefADSGoogle Scholar
  15. Lambert O, Benabdelhak H, Chami M, Jouan L, Nouaille E, Ducruix A, Brisson A (2005) Trimeric structure of OprN and OprM efflux proteins from Pseudomonas aeruginosa, by 2D electron crystallography. J Struct Biol 150:50–57CrossRefGoogle Scholar
  16. Lehto MT, Sharom FJ (1998) Release of the glycosylphosphatidylinositol-anchored enzyme ecto-5’-nucleotidase by phospholipase C: catalytic activation and modulation by the lipid bilayer. Biochem J 332:101–109Google Scholar
  17. leMaire M, Moller JV, Champeil P (1987) Binding of a non-ionic detergent to membranes: flip–flop rate and location on the bilayer. Biochemistry 26:4803–4810CrossRefGoogle Scholar
  18. Mikolosko J, Bobyk K, Zgurskaya HI, Ghosh P (2006) Conformational flexibility in the multidrug efflux system protein AcrA. Structure 14:577–587CrossRefGoogle Scholar
  19. Mornet S, Lambert O, Duguet E, Brisson A (2005) The formation of supported lipid bilayers on silica nanoparticles revealed by Cryoelectron microscopy. Nano Lett 5:281–285CrossRefADSGoogle Scholar
  20. Murakami S, Nakashima R, Yamashita E, Yamaguchi A (2002) Crystal structure of bacterial multidrug efflux transporter AcrB. Nature 419:587–593CrossRefADSGoogle Scholar
  21. Murakami S, Nakashima R, Yamashita E, Matsumoto T, Yamaguchi A (2006) Crystal structures of a multidrug transporter reveal a functionally rotating mechanism. Nature 443:173–179CrossRefADSGoogle Scholar
  22. Nosjean S, Roux B (2003) Anchoring of glycosylphosphatidylinosiyol-proteins to liposomes. Methods Enzymol 12:216–232CrossRefGoogle Scholar
  23. Paternostre MT, Roux M, Rigaud JL (1988) Mechanisms of membrane protein insertion into liposomes during reconstitution procedures involving the use of detergents. 1. Solubilization of large unilamellar liposomes (prepared by reverse-phase evaporation) by triton X-100, octyl glucoside, and sodium cholate. Biochemistry 27:2668–2677CrossRefGoogle Scholar
  24. Rasband WS 1997–2005 ImageJ U. S. National Institutes of Health, Bethesda,
  25. Reid-Taylor KL, Chu JWK, Sharom FJ (1999) Reconstitution of the glycosylphosphatidylinositol-anchored protein Thy-1: interaction with membrane phospholipids and galactosylceramide. Biochem Cell Biol 77:189–200CrossRefGoogle Scholar
  26. Richter R, Brisson A (2004) QCM-D on mica for parallel QCM-D-AFM studies. Langmuir 20:4609–4613CrossRefGoogle Scholar
  27. Richter R, Mukhopadhay A, Brisson A (2003) Pathways of lipid vesicle deposition on solid surfaces: a combined QCM-D and AFM study. Biophys J 85:3035–3047Google Scholar
  28. Rigaud JL, Pitard B, Levy D (1995) Reconstitution of membrane proteins into liposomes: application to energy-transducing membrane proteins. Biochim Biophys Acta 1231:223–246CrossRefGoogle Scholar
  29. Rigaud JL, Mosser G, Lacapere JJ, Olofsson A, Levy D, Ranck JL (1997) Bio-Beads: an efficient strategy for two-dimensional crystallization of membrane proteins. J Struct Biol 118:226–235CrossRefGoogle Scholar
  30. Ronzon F, Morandat S, Roux B, Bortolato M (2004) Insertion of a glycosylphosphatidylinosiyol –anchored enzyme into liposomes. J Membrane Biol 197:167–177CrossRefGoogle Scholar
  31. Salafsky J, Groves JT, Boxer SG (1996) Architecture and function of membrane proteins in planar supported bilayers: a study with photosynthetic reaction centers. Biochemistry 35:14773–14781CrossRefGoogle Scholar
  32. Sennhauser G, Amstutz P, Briand C, Storchenegger O, Gruetter MG (2007) Drug export pathway of multidrug exporter AcrB revealed by DARPin inhibitors. PLoS Biol 5:e7CrossRefGoogle Scholar
  33. Sharom FJ, Lehto MT (2002) Glycosylphosphatidylinositol-anchored proteins: structure, function, and cleavage by phosphatidylinositol-specific phospholipase C. Biochem Cell Biol 80:535–549CrossRefGoogle Scholar
  34. Silvius JR (1992) Solubilization and Functional Reconstitution of Biomembrane Components. Annu Rev Biophys Biomol Struct 21:323–348CrossRefGoogle Scholar
  35. Tamura N, Murakami S, Oyama Y, Ishiguro M, Yamaguchi A (2005) Direct interaction of multidrug efflux transporter AcrB and outer membrane channel TolC detected via site-directed disulfide crosslinking. Biochemistry 44:11115–11121CrossRefGoogle Scholar
  36. Trépout S, Mornet S, Benabdelhak H, Ducruix A, Brisson AR, Lambert O (2007) Membrane protein selectively oriented on solid support and reconstituted into a lipid membrane. Langmuir 23:2647–2654CrossRefGoogle Scholar
  37. Wenk MR, Alt T, Seelig A, Seelig J (1997) Octyl-β-Dglucopyranoside partitioning into lipid bilayers: thermodynamics of binding and structural changes of the bilayer. Biophys J 72:1719–1731Google Scholar
  38. Yoneyama H, Maseda H, Kamiguchi H, Nakae T (2000) Function of the membrane fusion protein, MexA, of the MexAB-OprM efflux pump in Pseudomonas aeruginosa without an anchoring membrane. J Biol Chem 275:4628–4634CrossRefGoogle Scholar

Copyright information

© EBSA 2007

Authors and Affiliations

  • Sylvain Trépout
    • 1
  • Jean-Christophe Taveau
    • 1
  • Stéphane Mornet
    • 1
    • 3
  • Houssain Benabdelhak
    • 2
  • Arnaud Ducruix
    • 2
  • Olivier Lambert
    • 1
  1. 1.Laboratoire d’Imagerie Moléculaire et Nano-Bio-TechnologieUMR 5248 CBMN, CNRS, Université Bordeaux 1, ENITAB, IECBPessacFrance
  2. 2.Laboratoire de Cristallographie et RMN Biologiques, UMR 8015 CNRS, Faculté de PharmacieUniversité Paris DescartesParis Cedex 06France
  3. 3.European Commission Joint Research Centre Institute for Health and Consumer ProtectionIspraItaly

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