Organization of reconstituted lipoprotein MexA onto supported lipid membrane.
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 beta-octyl glucoside (betaOG). First we showed that SLBs resist to betaOG concentrations that usually solubilize liposomes. Native form of MexA was directly inserted in the outer leaflet at (betaOG 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.
Laboratoire d'Imagerie Moléculaire et Nano-Bio-Technologie, UMR 5248 CBMN, CNRS, Université Bordeaux 1, ENITAB, IECB, 2 rue Robert Escarpit, 33607, Pessac, France.
Sylvain
S
Trépout
Jean-Christophe
JC
Taveau
Stéphane
S
Mornet
Houssain
H
Benabdelhak
Arnaud
A
Ducruix
Olivier
O
Lambert
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D020285:Cryoelectron Microscopy; D003902:Detergents / Q000737:chemistry; D005960:Glucosides / Q000737:chemistry; D007202:Indicators and Reagents; D008051:Lipid Bilayers; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D010713:Phosphatidylcholines; D011550:Pseudomonas aeruginosa / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D003902:Detergents; D005960:Glucosides; D007202:Indicators and Reagents; D008051:Lipid Bilayers; D026901:Membrane Transport Proteins; C118598:MexA protein, Pseudomonas aeruginosa; D010713:Phosphatidylcholines; C018619:octyl-beta-D-glucoside; C017251:1,2-oleoylphosphatidylcholine