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Dynamical assembly of efflux pump membrane proteins in a biomimetic bilayer – Efflux

Submission summary

Originally described in bacteria, drug transporters (or efflux pumps) are now recognized as major determinants in the modulation of the accumulation and efflux of antibacterials in virtually all cell types, from prokaryotes to superior eukaryotes. Based on sequence similarities with known transporters and with proteins possessing at least two transmembrane segments, it has been calculated that 15–20% of the genome of E. coli may code for this type of protein. Transport is protein-mediated and performed either by a uniport mechanism coupled to ATP hydrolysis or, more commonly, by an antiport mechanism in which drug efflux is linked to proton influx i.e. energized by the proton motive force (pmf). The efflux systems of Gram-negative bacteria tend to have a complex arrangement. P. aeruginosa possesses many ways to resist antibiotics. Active efflux is a recently described mechanism causing very concerning multidrug-resistant phenotypes in P. aeruginosa. Twelve genetically distinct systems have been described in P. aeruginosa genome among which four have been biochemically characterised. Each system is composed of three proteins. A cytoplasmic membrane protein is thought to act as an energy (H+) dependent pump (pmf) with broad substrate specificity. A second protein is located in the outer membrane whereas a third one, anchored in the inner membrane, is located in the periplasmic space and thought to make a link between the two other proteins. Recently, a fourth efflux pump system OprM/MexX-MexY, specific for aminoglycoside has been identified in P. aeruginosa . The four pumps display different resistance phenotypes. Only the MexAB-OprM system is expressed constitutively whereas the others are expressed under special circumstances. The present project, at the interface of physics and biology, gathers molecular biologists, biochemists, chemists, structural biologists, and specialists of bilayer physics. The aim of the project is to fully reconstitute in vitro, using micromanipulation, an efflux pump. The project will be carried out with three workpackages: WP1. Co-expression and purification of proteins from efflux pumps (team 1) WP2. Understanding the assembly of one efflux pump (team 2) In order to characterize the interaction between two interacting proteins we will determine: <sum> 2.1. The distance and mode of interaction between two interacting proteins. <sum> 2.2. The stoechiometry in order to deduce the association state of two interacting proteins. <sum> 2.3. The energies, the forces of connections, and of the constants of association between three proteins of the pump taken two by two in order to understand the stability of the unit. WP3. Assembly and activation of an efflux pump in giant unilamelar vesicles G.U.V. (teams 1, 2) The short-term objective is to reconstitute the protein machinery of an efflux pump responsible for the resistance to antibiotics of Gram negative bacteria, in order to investigate their structure-function activities. The activity of the pump will be tested in giant vesicles (GUV). This would be the first time that an in vitro double layer system is used to reconstitute an efflux pump. On the long range, one may expect that the design of a novel pharmacological in vitro assay may lead to new antibiotic candidates. Despite an intense international competition, reconstitution of protein machineries made of membrane proteins from the inner and outer membranes (and possibly) of periplasmic proteins has not yet been achieved. The complementarity of competences acquired by each group participating to this interdisciplinary project is: Team 1. A. Ducruix, Faculty of Pharmacy in Paris The molecular biology difficulties and preparative biochemistry (mg of pure proteins) of individual components have been overcome. Team 1 has a strong experience in overexpressing, purifying, crystallizing membrane proteins and more generally in structural biology. The functional aspects of the active efflux pump will be addresses by team 1. Team 2. W. Urbach, LPS at ENS in Paris: Team 2 has a strong experience in micromanipulation, handling unique molecules and measuring forces and mobility in giant vesicles, lamellar or sponge phases (e.g. L3). It has the competence to characterize lyotropic phases by SAXS or DLS/DQLS. In order to characterize the interactions between the proteins that constitute an efflux pump, we will use a sponge phase (i.e. L3 type) constituted from non-ionic bilayers in which it is possible to insert protein. The thickness of the bilayer was proved to be adjustable as well as the distance d between two bilayers (60 to 300 Å). The sequence of assembly being currently unknown, it is proposed to study the formation of the "pre-complex (i.e. a complex formed of two interacting proteins) using FRAPP.

Project coordination


The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.



Help of the ANR 350,000 euros
Beginning and duration of the scientific project: - 36 Months

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