Studying dynamics without explicit dynamics: a structure-based study of the export mechanism by AcrB

RND family proteins are transmembrane proteins identified as large spectrum drug transporters. A prototypical case in this superfamily, responsible for antibiotic resistance in selected gram negative bacteria, is AcrB. AcrB forms a trimer, which uses the proton motive force to efflux drugs, implementing a functional rotation mechanism. Unfortunately, the size of the system (1049 amino-acid per monomer and membrane) has prevented a systematic dynamical exploration, so that the mild understanding of this coupled transport jeopardizes our ability to counter it. To further our understanding, we present a novel strategy based on two key ingredients which are to study dynamics by exploiting information embodied in the numerous crystal structures of AcrB obtained to date, and to systematically consider subdomains, their dynamics, and their interactions. Along the way, we identify the subdomains responsible for dynamic events, refine the states (A,B,E) of the functional rotation mechanism, and analyze the evolution of intra-monomer and inter-monomer interfaces along the functional cycle. Our analysis paves the way to targeted simulations exploiting the most relevant degrees of freedom at certain steps, and also to a targeting of specific interfaces to block the drug efflux. More generally, our work shows that complex dynamics can be unveiled from static snapshots, and our strategy may be used on a variety of molecular machines of large size

MexAB-OprM Efflux Pump Interaction with the Peptidoglycan of Escherichia coli and Pseudomonas aeruginosa.

One of the major families of membrane proteins found in prokaryote genome corresponds to the transporters. Among them, the resistance-nodulation-cell division (RND) transporters are highly studied, as being responsible for one of the most problematic mechanisms used by bacteria to resist to antibiotics, i.e., the active efflux of drugs. In Gram-negative bacteria, these proteins are inserted in the inner membrane and form a tripartite assembly with an outer membrane factor and a periplasmic linker in order to cross the two membranes to expulse molecules outside of the cell. A lot of information has been collected to understand the functional mechanism of these pumps, especially with AcrAB-TolC from Escherichia coli, but one missing piece from all the suggested models is the role of peptidoglycan in the assembly. Here, by pull-down experiments with purified peptidoglycans, we precise the MexAB-OprM interaction with the peptidoglycan from Escherichia coli and Pseudomonas aeruginosa, highlighting a role of the peptidoglycan in stabilizing the MexA-OprM complex and also differences between the two Gram-negative bacteria peptidoglycans

Functional Mechanism of the Efflux Pumps Transcription Regulators From Pseudomonas aeruginosa Based on 3D Structures

Bacterial antibiotic resistance is a worldwide health problem that deserves important research attention in order to develop new therapeutic strategies. Recently, the World Health Organization (WHO) classified Pseudomonas aeruginosa as one of the priority bacteria for which new antibiotics are urgently needed. In this opportunistic pathogen, antibiotics efflux is one of the most prevalent mechanisms where the drug is efficiently expulsed through the cell-wall. This resistance mechanism is highly correlated to the expression level of efflux pumps of the resistance-nodulation-cell division (RND) family, which is finely tuned by gene regulators. Thus, it is worthwhile considering the efflux pump regulators of P. aeruginosa as promising therapeutical targets alternative. Several families of regulators have been identified, including activators and repressors that control the genetic expression of the pumps in response to an extracellular signal, such as the presence of the antibiotic or other environmental modifications. In this review, based on different crystallographic structures solved from archetypal bacteria, we will first focus on the molecular mechanism of the regulator families involved in the RND efflux pump expression in P. aeruginosa, which are TetR, LysR, MarR, AraC, and the two-components system (TCS). Finally, the regulators of known structure from P. aeruginosa will be presented

Activity Monitoring of Functional OprM Using a Biomimetic Microfluidic Device

International audienceThis paper describes the fabrication and use of a biomimetic microfluidic device for the monitoring of a functional porin reconstituted within miniaturized suspended artificial bilayer lipid membrane (BLM). Such a microfluidic device allows for 1) fluidic and electrical access to both sides of the BLM, 2) reproducible membrane protein insertion and long-term electrical monitoring of its conductance (Gi), thanks to the miniaturization of the BLM. We demonstrate here for the first time the feasibility to insert a large trans-membrane protein through its β-barrel, and monitor its functional activity during more than 1 hour (limited by buffer evaporation). In this paper, we specifically used our device for the monitoring of OprM, a bacterial efflux channel involved in the multidrug resistance of the bacteria Pseudomonas aeruginosa. Sub-steps of the OprM channel conductance were detected during the electrical recordings within our device, which might be due to oscillations between several structural conformations (sub-states) adopted by the protein, as part of its opening mechanism. This work is a first step towards the establishing of a genuine platform dedicated to the investigation of bacterial proteins under reconstituted conditions, a very promising tool for the screening of new inhibitors against bacterial channels involved in drug resistance

Solution NMR structure of the SH3 domain of human nephrocystin and analysis of a mutation-causing juvenile nephronophthisis.

Human nephrocystin is a protein associated with juvenile NPH, an autosomal recessive, inherited kidney disease responsible for chronic renal failure in children. It contains an SH3 domain involved in signaling pathways controlling cell adhesion and cytoskeleton organization. The solution structure of this domain was solved by triple resonance NMR spectroscopy. Within the core, the structure is similar to those previously reported for other SH3 domains but exhibits a number of specific noncanonical features within the polyproline ligand binding site. Some of the key conserved residues are missing, and the N-Src loop exhibits an unusual twisted geometry, which results in a narrowing of the binding groove. This is induced by the replacement of a conserved Asp, Asn, or Glu residue by a Pro at one side of the N-Src loop. A systematic survey of other SH3 domains also containing a Pro at this position reveals that most of them belong to proteins involved in cell adhesion or motility. A variant of this domain, which carries a point mutation causing NPH, was also analyzed. This change, L180P, although it corresponds to a nonconserved and solvent-exposed position, causes a complete loss of the tertiary structure. Similar effects are also observed with the L180A variant. This could be a context-dependent effect resulting from an interaction between neighboring charged side-chains

From Vascular Smooth Muscle Cells to Folliculogenesis: What About Vasorin?

First described in 1988, vasorin (VASN) is a transmembrane glycoprotein expressed during early mouse development, and with a less extent, in various organs and tissues (e.g., kidney, aorta, and brain) postnatally. Vasn KO mice die after 3 weeks of life from unknown cause(s). No human disease has been associated with variants of this gene so far, but VASN seems to be a potential biomarker for nephropathies and tumorigenesis. Its interactions with the TGF-β and Notch1 pathways offer the most serious assumptions regarding VASN functions. In this review, we will describe current knowledge about this glycoprotein and discuss its implication in various organ pathophysiology

Transverse-momentum and pseudorapidity distributions of charged hadrons in pp collisions at √s=0.9 and 2.36 TeV

Measurements of inclusive charged-hadron transverse-momentum and pseudorapidity distributions are presented for proton-proton collisions at root s = 0.9 and 2.36 TeV. The data were collected with the CMS detector during the LHC commissioning in December 2009. For non-single-diffractive interactions, the average charged-hadron transverse momentum is measured to be 0.46 +/- 0.01 (stat.) +/- 0.01 (syst.) GeV/c at 0.9 TeV and 0.50 +/- 0.01 (stat.) +/- 0.01 (syst.) GeV/c at 2.36 TeV, for pseudorapidities between -2.4 and +2.4. At these energies, the measured pseudorapidity densities in the central region, dN(ch)/d eta vertical bar(vertical bar eta vertical bar and pp collisions. The results at 2.36 TeV represent the highest-energy measurements at a particle collider to date

Etude structurale du système Prolactine/Récepteur de la prolactine

La prolactine (PRL) est une hormone pituitaire sécrétée par l'hypophyse. Elle est impliquée dans un très grand nombre de fonctions biologiques dont la croissance et la lactation. Elle induit ses fonctions en interagissant au niveau cellulaire avec un dimère de son récepteur (PRLR). Des études sur modèles animaux montrent que la PRL pourrait être impliquée dans les cancers du sein ou de la prostate. Plusieurs antagonistes du PRLR ont été développés, mais leurs effets in vivo est faible. Dans le but d'optimiser ces antagonistes, nous voulons mieux comprendre le mécanisme d'activation du PRLR et abordons cette étude par une approche structurale. Nous avons résolu la structure de la PRL humaine en complexe avec les domaines extracellulaires de deux PRLR de rat. Celle-ci permet de décrire le site d'interaction entre la PRL et rECD 2 et le site d'interaction entre les deux rECD. Mais le mécanisme d'activation reste incompris. Nous avons donc entrepris de produire une forme recombinante du PRLR transmembranaire entier. Des tentatives de production ont été réalisées chez \ecoli et \pichia. Des échantillons purs de PRLR renaturé ont été obtenus dont leur fonctionnalité reste à démontrerProlactin (PRL) is a pituitary hormone secreted by the hypothalamus. It's involved in a wide range of biological functions. The best known are growth and lactation. PRL induces its function upon interaction with a dimmer of its specific receptor (PRLR). Increasing experimental evidence on animal models shows that PRL might be involved in breast or prostate cancer. Several antagonists have been proposed, but their in vivo effects are weak. As we want to improve these antagonists, we are seeking to understand the activation mechanism of the PRLR by a structural approach. We solved the structure of PRL in complex with two extracellular domains of the rat PRLR (rECD). It enables to describe the site 2 interface between PRL and rECD 2, and the stem-stem interface between the two receptors. But the PRLR activation mechanism remains unclear. We therefore tried to produce a recombinant form of the full transmembrane PRLR in two expression system: \ecoli and \pichia. Pure samples of refolded PRLR have been obtained in a non-aggregated form. But its functionality has to be testedPARIS-BIUP (751062107) / SudocSudocFranceF

Structural Motifs in the Extracellular Domain of the Prolactin Receptor Govern Fold and Functionality

The prolactin receptor (PRLR) is an archetype cytokine receptor. It is a single-pass transmembrane receptor with limited complexity that is devoid of enzyme activity. Intracellular signaling involves various receptor-associated kinases including Jak2, Erk1/2, Src and Akt. As the PRLR is emerging as a relevant target in Oncology the understanding of the molecular basis of its activation is crucial. In the frame of an inter-disciplinary consortium involving biophysicists, structural biologists and cell biologists, we have successfully combined complementary approaches such as optical and nuclear magnetic resonance spectroscopic analyses, X-ray crystallography, surface plasmon resonance and cell-based assays to start elucidate the structural features of ligand-receptor interaction. However, the features of the PRLR extracellular domain (ECD) that participate in the transmission of the hormonal message across the cell membrane and/or in selective activation of intracellular signaling cascades remained uncharacterized. In two recently published studies, we identified residues 146 and 170 as two key residues of the PRLR-ECD that control critical receptor properties including basal signaling activity, ligand sensitivity, species specificity, folding, stability and receptor turnover. These two residues are in close proximity of each other in the membrane proximal domain of the PRLR-ECD and participate in a network of interactions with other residues, in particular within a specific residue quartet. Strikingly, these residues are involved in, or close to, the receptor dimerization interface, suggesting that their mechanism of action may involve structural reorientation of the receptor chains that are necessary to (selectively) disseminate the signal from the ECD to the intracellular domain. The identification of such residues in this and other cytokine receptors should affect future structure-directed drug development strategies aimed at providing pathway-selective treatment strategies

Focus on the Outer Membrane Factor OprM, the Forgotten Player from Efflux Pumps Assemblies

International audienceAntibiotics have been used extensively during several decades and we are now facing the emergence of multidrug resistant strains. It has become a major public concern, urging the need to discover new strategies to combat them. Among the different ways used by bacteria to resist antibiotics, the active efflux is one of the main mechanisms. In Gram-negative bacteria the efflux pumps are comprised of three components forming a long edifice crossing the complete cell wall from the inside to the outside of the cell. Blocking these pumps would permit the restoration of the effectiveness of the current antibiotherapy which is why it is important to increase our knowledge on the different proteins involved in these complexes. A tremendous number of experiments have been performed on the inner membrane protein AcrB from Escherichia coli and, to a lesser extent, the protein partners forming the AcrAB-TolC pump, but less information is available concerning the efflux pumps from other virulent Gram-negative bacteria. The present review will focus on the OprM outer membrane protein from the MexAB-OprM pump of Pseudomonas aeruginosa, highlighting similarities and differences compare to the archetypal AcrAB-TolC in terms of structure, function, and assembly properties