Organization of reconstituted lipoprotein MexA onto supported lipid membrane

MexA, a periplasmic component of OprMMexA- 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 b-octyl glucoside (bOG). First we showed that SLBs resist to bOG concentrations that usually solubilize liposomes. Native form of MexA was directly inserted in the outer leaflet at (bOG concentrations in a range of 20–25 mM). Second, observations by cryo-electron microscopy (cryo- EM) 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.