title: Structure of reconstituted bacterial membrane efflux pump by cryo-electron tomography.
abstract: Complexes of OprM and MexA, two proteins of the MexA-MexB-OprM multidrug efflux pump from Pseudomonas aeruginosa, an opportunistic Gram-negative bacterium, were reconstituted into proteoliposomes by detergent removal. Stacks of protein layers with a constant height of 21nm, separated by lipid bilayers, were obtained at stoichiometry of 1:1 (w/w). Using cryo-electron microscopy and tomography, we showed that these protein layers were composed of MexA-OprM complexes self-assembled into regular arrays. Image processing of extracted sub-tomograms depicted the architecture of the bipartite complex sandwiched between two lipid bilayers, representing an environment close to that of the native whole pump (i.e. anchored between outer and inner membranes of P. aeruginosa). The MexA-OprM complex appeared as a cylindrical structure in which we were able to identify the OprM molecule and the MexA moiety. MexA molecules have a cylindrical shape prolonging the periplasmic helices of OprM, and widening near the lipid bilayer. The flared part is likely composed of two MexA domains adjacent to the lipid bilayer, although their precise organization was not reachable mainly due to their flexibility. Moreover, the intermembrane distance of 21nm indicated that the height of the bipartite complex is larger than that of the tripartite AcrA-AcrB-TolC built-up model in which TolC and AcrB are docked into contact. We proposed a model of MexA-OprM taking into account features of previous models based on AcrA-AcrB-TolC and our structural results providing clues to a possible mechanism of tripartite system assembly.
authors.affiliation: CBMN UMR 5248 CNRS-Université Bordeaux-ENITAB, IECB, Avenue des Facultés, F-33405 Talence, France.
authors.forename: Sylvain
authors.initials: S
authors.lastname: Trépout
authors.forename: Jean-Christophe
authors.initials: JC
authors.lastname: Taveau
authors.forename: Houssain
authors.initials: H
authors.lastname: Benabdelhak
authors.forename: Thierry
authors.initials: T
authors.lastname: Granier
authors.forename: Arnaud
authors.initials: A
authors.lastname: Ducruix
authors.forename: Achilleas S
authors.initials: AS
authors.lastname: Frangakis
authors.forename: Olivier
authors.initials: O
authors.lastname: Lambert
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D020285:Cryoelectron Microscopy / Q000379:methods*; D018432:Drug Resistance, Multiple; D055032:Electron Microscope Tomography / Q000379:methods; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D011485:Protein Binding; D011550:Pseudomonas aeruginosa / Q000378:metabolism
chemical_list: D001425:Bacterial Outer Membrane Proteins; D008051:Lipid Bilayers; D008081:Liposomes; D026901:Membrane Transport Proteins; C118598:MexA protein, Pseudomonas aeruginosa; D046912:Multiprotein Complexes; C402263:OprM protein, Pseudomonas aeruginosa