Arnaud Ducruix

Structure of reconstituted bacterial membrane efflux pump by cryo-electron tomography.

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.

Tracking membrane protein association in model membranes.

Membrane proteins are essential in the exchange processes of cells. In spite of great breakthrough in soluble proteins studies, membrane proteins structures, functions and interactions are still a challenge because of the difficulties related to their hydrophobic properties. Most of the experiments are performed with detergent-solubilized membrane proteins. However widely used micellar systems are far from the biological two-dimensions membrane. The development of new biomimetic membrane systems is fundamental to tackle this issue. We present an original approach that combines the Fluorescence Recovery After fringe Pattern Photobleaching technique and the use of a versatile sponge phase that makes it possible to extract crucial informations about interactions between membrane proteins embedded in the bilayers of a sponge phase. The clear advantage lies in the ability to adjust at will the spacing between two adjacent bilayers. When the membranes are far apart, the only possible interactions occur laterally between proteins embedded within the same bilayer, whereas when membranes get closer to each other, interactions between proteins embedded in facing membranes may occur as well. After validating our approach on the streptavidin-biotinylated peptide complex, we study the interactions between two membrane proteins, MexA and OprM, from a Pseudomonas aeruginosa efflux pump. The mode of interaction, the size of the protein complex and its potential stoichiometry are determined. In particular, we demonstrate that: MexA is effectively embedded in the bilayer; MexA and OprM do not interact laterally but can form a complex if they are embedded in opposite bilayers; the population of bound proteins is at its maximum for bilayers separated by a distance of about 200 Å, which is the periplasmic thickness of Pseudomonas aeruginosa. We also show that the MexA-OprM association is enhanced when the position and orientation of the protein is restricted by the bilayers. We extract a stoichiometry for the complex that exhibits a strong pH dependance: from 2 to 6 MexA per OprM trimer when the pH decreases from 7.5 to 5.5. Our technique allows to study membrane protein associations in a membrane environment. It provides some challenging information about complexes such as geometry and stoichiometry.

Stoichiometry of the MexA‐OprM binding, as investigated by blue native gel electrophoresis

Multidrug resistance has become a serious concern in the treatment of bacterial infections. A prominent role is ascribed to the active efflux of xenobiotics out of the bacteria by a tripartite protein machinery. The mechanism of drug extrusion is rather well understood, thanks to the X‐ray structures obtained for the Escherichia coli TolC/AcrA/AcrB model system and the related Pseudomonas aeruginosa OprM/MexA/MexB. However, many questions remain unresolved, in particular the stoichiometry of the efflux pump assembly. On the basis of blue native polyacrylamide gel electrophoresis (BN‐PAGE) (Wittig et al., Nat. Protoc. 2006, 1, 418–428), we analyzed the binding stoichiometry of both palmitylated and non‐palmitylated MexA with the cognate partner OprM trimer at different ratios and detergent conditions. We found that β‐octyl glucopyranoside (β‐OG) detergent was not suitable for this technique. Then we proved that MexA has to be palmitylated in order to stabilized the complex formation with OprM. Finally, we provided evidence for a two by two (2, 4, 6, or upper) binding of palmitylated MexA per trimer of OprM.