Tracking Membrane Protein Association in Model Membranes

Abstract

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.

Assembly and Channel Opening of Outer Membrane Protein in Tripartite Drug Efflux Pumps of Gram-negative Bacteria

“…The crystal structure of the MexA in the lipid moiety-free form exhibited the six-or seven-membered twisted horseshoe assembly (21,22), which does not directly explain the MexA assembly in the membrane bound state. However, cryo-microscopic results in the reconstructed membrane layer showed that the MexA forms a hexameric complex with a dimension of 13-nm height (15,23,24). This finding is consistent with the hexameric model of MexA proposed in the previous report (14).…”

“…The hexameric MexA could be observed only when it anchors to the membrane and/or binds to OprM (15,23,24). To obtain the hexameric form of MexA, we initially attempted to determine the crystal structures of the complex of MexA and OprM.…”

“…It was reported previously that the stoichiometry of the MexA-OprM complex in a reconstituted membrane bilayer shows strong pH dependence (15). To examine the pH dependence of the MexA-OprM complex formation under detergentfree condition, we carried out the in vitro binding assay.…”

Trinity revealed: Stoichiometric complex assembly of a bacterial multidrug efflux pump

“…Recent crystallographic studies with the adapter MacA of the ATPdriven MacAB-TolC system suggests a MacA:TolC stoichiometry of 2, and the interactions appear to be much more stable compared with the AcrAB-TolC RND system in vitro and possibly also in vivo (16,17). Whether this stoichiometry is exclusive to this particular type of pump remains to be tested as well, because another very recent study (18) provides evidence for yet another 2:1 ratio between the MexA/OrpM subunits of the RND-type MexAB-OprM tripartite efflux pump from P. aeruginosa. Previous studies on AcrA, however, seem to be in accordance with its trimeric appearance (19).…”

Structure and Mechanism of RND‐Type Multidrug Efflux Pumps

“…Another significant advance in the study of component interaction was the in vitro examination of association between MexA and OprM (62). Such studies are difficult because both are membrane proteins (although some adaptor proteins appear to work in intact cells without the lipid modification at the N-terminus (52)), and the adaptor inserted into one membrane must interact with the OM channel inserted into another membrane (see Fig.…”