Original Paper

European Biophysics Journal

, Volume 36, Issue 8, pp 1029-1037

Organization of reconstituted lipoprotein MexA onto supported lipid membrane

  • Sylvain TrépoutAffiliated withLaboratoire d’Imagerie Moléculaire et Nano-Bio-Technologie, UMR 5248 CBMN, CNRS, Université Bordeaux 1, ENITAB, IECB
  • , Jean-Christophe TaveauAffiliated withLaboratoire d’Imagerie Moléculaire et Nano-Bio-Technologie, UMR 5248 CBMN, CNRS, Université Bordeaux 1, ENITAB, IECB
  • , Stéphane MornetAffiliated withLaboratoire d’Imagerie Moléculaire et Nano-Bio-Technologie, UMR 5248 CBMN, CNRS, Université Bordeaux 1, ENITAB, IECBEuropean Commission Joint Research Centre Institute for Health and Consumer Protection
  • , Houssain BenabdelhakAffiliated withLaboratoire de Cristallographie et RMN Biologiques, UMR 8015 CNRS, Faculté de Pharmacie, Université Paris Descartes
  • , Arnaud DucruixAffiliated withLaboratoire de Cristallographie et RMN Biologiques, UMR 8015 CNRS, Faculté de Pharmacie, Université Paris Descartes
  • , Olivier LambertAffiliated withLaboratoire d’Imagerie Moléculaire et Nano-Bio-Technologie, UMR 5248 CBMN, CNRS, Université Bordeaux 1, ENITAB, IECB Email author 

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Abstract

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.

Keywords

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