Molecular Determinants for OMF Selectivity in Tripartite RND Multidrug Efflux Systems.

Tripartite multidrug RND efflux systems made of an inner membrane transporter, an outer membrane factor (OMF) and a periplasmic adaptor protein (PAP) form a canal to expel drugs across Gram-negative cell wall. Structures of MexA-MexB-OprM and AcrA-AcrB-TolC, from and , respectively, depict a reduced interfacial contact between OMF and PAP, making unclear the comprehension of how OMF is recruited. Here, we show that a Q93R mutation of MexA located in the α-hairpin domain increases antibiotic resistance in the MexA-MexB-OprM-expressed strain.

Structural characterization of the EmrAB-TolC efflux complex from E. coli.

Gram-negative bacteria export a large variety of antimicrobial compounds by forming two-membrane spanning tripartite multidrug efflux systems composed of an inner membrane transporter, an outer membrane channel and a periplasmic adaptor protein. Here we present the co-expression, purification and first electron microscopy insights of the Escherichia coli EmrAB-TolC tripartite Major Facilitator Superfamily (MSF) efflux system as a whole complex stabilized by Amphipol polymer. The structure reveals a 33 nm long complex delineated by the Amphipol belt at both extremities.

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.

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.

Single lipoaminoglycoside promotes efficient intracellular antibody delivery: A comprehensive insight into the mechanism of action.

Delivery of biologically active proteins into cells is emerging as important strategy for many applications. Previous experiments have shown that lipoaminoglycosides were capable of delivery of the anti-cytokeratin8 antibody (anti-K8) but only when formulated with lipid helpers potentially leading to toxicity from excess lipids. Here, we optimized anti-K8 delivery with various lipoaminoglycosides in the absence of a lipid helper.

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.

Tripartite assembly of RND multidrug efflux pumps.

Tripartite multidrug efflux systems of Gram-negative bacteria are composed of an inner membrane transporter, an outer membrane channel and a periplasmic adaptor protein. They are assumed to form ducts inside the periplasm facilitating drug exit across the outer membrane. Here we present the reconstitution of native Pseudomonas aeruginosa MexAB-OprM and Escherichia coli AcrAB-TolC tripartite Resistance Nodulation and cell Division (RND) efflux systems in a lipid nanodisc system.

Multipod-like silica/polystyrene clusters.

Multipod-like clusters composed of a silica core and PS satellites are prepared according to a seeded-growth emulsion polymerization of styrene in the presence of size-monodisperse silica particles previously surface-modified with methacryloxymethyltriethoxysilane. Tuning the diameter and concentration of the silica seeds affords homogeneous batches of tetrapods, hexapods, octopods, nonapods and dodecapods with morphology yields as high as 80%. Three-dimensional reconstructions by cryo-electron tomography are presented on large fields for the first time to show the high symmetry and regularity of the clusters demonstrating the good control of the synthesis process.

Electrokinetic assembly of one-dimensional nanoparticle chains with cucurbit[7]uril controlled subnanometer junctions.

One-dimensional (1D) nanoparticle chains with defined nanojunctions are of strong interest due to their plasmonic and electronic properties. A strategy is presented for the assembly of 1D gold-nanoparticle chains with fixed and rigid cucurbit[n]uril-nanojunctions of 9 Å. The process is electrokinetically accomplished using a nanoporous polycarbonate membrane and controlled by the applied voltage, the nanoparticle/CB[n] concentration ratio, time and temperature.

Spheres growing on a sphere: a model to predict the morphology yields of colloidal molecules obtained through a heterogeneous nucleation route.

Through the heterogeneous nucleation of polymer nodules on a surface-modified silica particle, the high-yield achievement of hybrid colloidal molecules with a well-controlled multipod-like morphology was recently demonstrated. However, as the formation mechanism of these colloidal molecules has not been completely understood yet, some opportunities remain to reduce the tedious empirical process needed to optimize the chemical recipes. In this work, we propose a model to help understand the formation mechanism of almost pure suspensions of well-defined colloidal molecules.

Mouse TSPO in a lipid environment interacting with a functionalized monolayer.

Translocator protein TSPO is a membrane protein highly conserved in evolution which does not belong to any structural known family. TSPO is involved in physiological functions among which transport of molecules such as cholesterol to form steroids and bile salts in mammalian cells. Membrane protein structure determination remains a difficult task and needs concomitant approaches (for instance X-ray- or Electron-crystallography and NMR).

Structure determination of membrane protein by both cryo-electron tomography and single particle analysis.

The structure determination of membrane protein in lipid environment can be carried out using cryo-electron microscopy combined with the recent development of data collection and image processing. We describe a protocol to study assemblies or stacks of membrane protein reconstituted into a lipid membrane using both cryo-electron tomography and single particle analysis, which is an alternative approach to electron crystallography for solving 3D structure. We show the organization of the successive layers of OprM molecules revealing the protein-protein interactions between OprM molecules of two successive lipid bilayers.

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.

Cryo-electron tomography of nanoparticle transmigration into liposome.

Nanoparticle transport across cell membrane plays a crucial role in the development of drug delivery systems as well as in the toxicity response induced by nanoparticles. As hydrophilic nanoparticles interact with lipid membranes and are able to induce membrane perturbations, hypothetic mechanisms based on membrane curvature or hole formation have been proposed for activating their transmigration. We report on the transport of hydrophilic silica nanoparticles into large unilamellar neutral DOPC liposomes via an internalization process.

Structure of artificial and natural VE-cadherin-based adherens junctions.

In vascular endothelium, adherens junctions between endothelial cells are composed of VE-cadherin (vascular endothelial cadherin), an adhesive receptor that is crucial for the proper assembly of vascular structures and the maintenance of vascular integrity. As a classical cadherin, VE-cadherin links endothelial cells together by homophilic interactions mediated by its extracellular part and associates intracellularly with the actin cytoskeleton via catenins. Although, from structural crystallographic data, a dimeric structure arranged in a trans orientation has emerged as a potential mechanism of cell-cell adhesion, the cadherin organization within adherens junctions remains controversial.

New insights into the nucleation and growth of PS nodules on nanoparticles by 3D cryo-electron tomography.

The nucleation and growth of polystyrene (PS) nodules on 170 nm silica seeds under emulsion-polymerization conditions have been investigated for the first time by cryo-electron tomography. 3D arrangements were reconstructed from samples collected at several polymerization times (from 5 to 120 min). Early samples display the presence of small PS nodules bound to silica particles in a random distribution.

Organization of reconstituted lipoprotein MexA onto supported lipid membrane.

MexA, a periplasmic component of OprM-MexA-MexB tripartite multidrug efflux pump from Pseudomonas aeruginosa, is natively anchored via its fatty acid in the bacteria inner membrane protruding into the periplasm. We used supported lipid bilayer (SLB) to attach the protein to a single leaflet mimicking its perisplamic orientation. For that purpose, we studied the solubilization of DOPC lipid bilayer supported on silica surface with beta-octyl glucoside (betaOG).

The basic framework of VE-cadherin junctions revealed by cryo-EM.

Artificial adherens junctions were reconstituted in vitro by assembly of cadherin fragments at the surfaces of liposomes. The architecture of the adherens junctions was revealed by cryo-electron microscopy (cryo-EM). The formation of these artificial adherens junctions was shown to result from the two-dimensional (2D) self-assembly of cadherin fragments at membrane surfaces.

Revisiting the structure of Alvinella pompejana hemoglobin at 20A resolution by cryoelectron microscopy.

The hemoglobin of the polychaete worm Alvinella pompejana was reconstructed at 20A resolution from frozen-hydrated samples observed by electron microscopy according to the random conical tilt series method. This three-dimensional reconstruction was mirror-inverted with respect to a previous volume published by de Haas et al. in 1996.