Antibiotic export by MexB multidrug efflux transporter is allosterically controlled by a MexA-OprM chaperone-like complex.

The tripartite multidrug efflux system MexAB-OprM is a major actor in Pseudomonas aeruginosa antibiotic resistance by exporting a large variety of antimicrobial compounds. Crystal structures of MexB and of its Escherichia coli homolog AcrB had revealed asymmetric trimers depicting a directional drug pathway by a conformational interconversion (from Loose and Tight binding pockets to Open gate (LTO) for drug exit). It remains unclear how MexB acquires its LTO form. Here by performing functional and cryo-EM structural investigations of MexB at various stages of the assembly process, we unveil that MexB inserted in lipid membrane is not set for active transport because it displays an inactive LTC form with a Closed exit gate. In the tripartite complex, OprM and MexA form a corset-like platform that converts MexB into the active form. Our findings shed new light on the resistance nodulation cell division (RND) cognate partners which act as allosteric factors eliciting the functional drug extrusion.

Rationale for the Quantitative Reconstitution of Membrane Proteins into Proteoliposomes.

Proteoliposome reconstitution is a method of choice for the investigation of membrane proteins as it allows their manipulation in the desired hydrophobic environment and allows one to tackle their study from both functional and structural points of view. Methods for their rapid and efficient reconstitution have been known for a long time but the quality and dispersity of the resulting suspensions is often overlooked. Here we describe our routine for the obtention of monodisperse populations of proteoliposomes as well as for the quantitation of protein per liposome.

Minimal nanodisc without exogenous lipids for stabilizing membrane proteins in detergent-free buffer.

Membrane protein stabilization after detergent solubilization presents drawbacks for structural and biophysical studies, in particular that of a reduced stability in detergent micelles. Therefore, alternative methods are required for efficient stabilization. Lipid nanodisc made with the membrane scaffold protein MSP is a valuable system but requires a fine optimization of the lipid to protein ratio. We present here the use of the scaffold protein MSP without added lipids as a minimal system to stabilize membrane proteins. We show that this method is applicable to α-helical and ß-strands transmembrane proteins. This method allowed cryo-electron microscopy structural study of the bacterial transporter MexB. A protein quantification indicates that MexB is stabilized by two MSP proteins. This simplified and efficient method proposes a new advance in harnessing the MSP potential to stabilize membrane proteins.

Reconstitution of Membrane Proteins into Nanodiscs for Single-Particle Electron Microscopy.

The structure determination of integral membrane protein (IMP) in lipid environment is particularly challenging. Among emerging methods for exchanging detergent required for IMP purification by original compounds, the use of lipid nanodisc preserves a lipid environment. Compared with the classical method of proteoliposome formation, the nanodisc technology provides a better control of IMP molecules inserted in lipid membrane, therefore giving access to structural methodologies developed for soluble proteins. Here, we present the reconstitution of OprM membrane protein into nanodisc associated with a step of size-exclusion chromatography, an approach applicable to prepare IMPs for subsequent visualization by single-particle electron microscopy.