Etudes structurales du transporteur mitochondrial d'ADP et d'ATP

Le transporteur mitochondrial d'ADP et d'A TP (AAC) permet l'échange des nucléotides adényliques au niveau de la membrane interne des mitochondries. Cette protéine essentielle, qui comporte six hélices transmembranaires, est le représentant le plus abondant et le mieux connu d'une large famille de transporteurs mitochondriaux. Afin de comprendre le mécanisme de ce transport, nous avons entrepris une étude structurale avec un double objectif: d'une part déterminer l'organisation oligomérique de l'AAC, et d'autre part en obtenir une nouvelle structure à l'échelle atomique. Nos expériences de diffraction de neutrons, d'ultracentrifugation analytique, couplées aux résultats de cristallographie permettent de remettre en cause sur des bases solides le dogme affirmant que l'AAC est dimérique en solution. En présence de Lapao, comme probablement la plupart du temps en présence de détergent, la protéine est un monomère. Nos résultats sont néanmoins compatibles avec une organisation dense dans les membranes. Nous avons mis en évidence l'importance structurale de trois cardiolipides qui entourent la protéine et participent à la stabilité de son repliement. Nous avons tenté de purifier des complexes entre l'AAC et deux de ses partenaires protéiques connus: la cyclophiline D, une protéine de la matrice, et Vpr, un peptide viral du Vlli. Nous montrons par co-purification et résonance plasmonique de surface que, dans nos conditions expérimentales, l'interaction entre ces protéines est inexistante.ADP-ATP carriers (AACs) are major and essential constituents of the inner mitochondrial membrane. They drive the import of ADP and the export of neo-synthesized ATP. AAC belongs to the mitochondrial carrier family (MCF). There are more than 45 MCF members in human that handle a tremendous diversity of substrates implicated in the mitochondrial metabolic cycles over the inner membrane. ln order to get a precise insight into the molecular mechanism of transport, we aimed to (i) characterize the controversial oligomeric state of the protein (ii) solve its atomic structure in a new conformation. Our neutron scattering and analytical ultracentrifugation experiments -coupled to crystallography- give a rational basis to de fend a monomeric protein in the presence of Lapao. This is in contradiction with the dogma that the protein is dimeric in solution. Rowever our results are compatible with the existence of dense patches. We evidenced the structural importance of three cardiolipin molecules bound to the protein that enhance the folding's stability. We tried to purify complexes of AAC with known partner protein: cyclophilin D which is a soluble matrix protein and Vpr which a RN peptide. We showed, using co purification and surface plasmon resonance, that no interaction exists in our experimental conditions.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Structure atomique de deux anesthésiques généraux liés à leur cible principale

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Distinct classes of potassium channels fused to GPCRs as electrical signaling biosensors

International audienceLigand-gated ion channels (LGICs) are natural biosensors generating electrical signals in response to the binding of specific ligands. Creating de novo LGICs for biosensing applications is technically challenging. We have previously designed modified LGICs by linking G protein-coupled receptors (GPCRs) to the Kir6.2 channel. In this article, we extrapolate these design concepts to other channels with different structures and oligomeric states, namely a tetrameric viral Kcv channel and the dimeric mouse TREK-1 channel. After precise engineering of the linker regions, the two ion channels were successfully regulated by a GPCR fused to their N-terminal domain. Two-electrode voltage-clamp recordings showed that Kcv and mTREK-1 fusions were inhibited and activated by GPCR agonists, respectively, and antagonists abolished both effects. Thus, dissimilar ion channels can be allosterically regulated through their N-terminal domains, suggesting that this is a generalizable approach for ion channel engineering

A release of local subunit conformational heterogeneity underlies gating in a muscle nicotinic acetylcholine receptor

Abstract Synaptic receptors respond to neurotransmitters by opening an ion channel across the post-synaptic membrane to elicit a cellular response. Here we use recent Torpedo acetylcholine receptor structures and functional measurements to delineate a key feature underlying allosteric communication between the agonist-binding extracellular and channel-gating transmembrane domains. Extensive mutagenesis at this inter-domain interface re-affirms a critical energetically coupled role for the principal α subunit β1-β2 and M2-M3 loops, with agonist binding re-positioning a key β1-β2 glutamate/valine to facilitate the outward motions of a conserved M2-M3 proline to open the channel gate. Notably, the analogous structures in non-α subunits adopt a locally active-like conformation in the apo state even though each L9’ hydrophobic gate residue in each pore-lining M2 α-helix is closed. Agonist binding releases local conformational heterogeneity transitioning all five subunits into a conformationally symmetric open state. A release of conformational heterogeneity provides a framework for understanding allosteric communication in pentameric ligand-gated ion channels

Structural basis for lipid-mediated interactions between mitochondrial ADP/ATP carrier monomers.

International audienceThe oligomerization state of the ADP/ATP carrier is an important issue in understanding the mechanism underlying nucleotide exchange across the inner mitochondrial membrane. The first high resolution structure obtained in the presence of carboxyatractyloside revealed a large cavity formed within a monomer in which the inhibitor is strongly bound. Whereas the protein-protein interactions implicated in the first crystal form are not biologically relevant, the new crystal form described herein, highlights favorable protein-protein interactions. The interactions are mediated by endogenous cardiolipins, which are tightly bound to the protein, two cardiolipins being sandwiched between the monomers on the matrix side. The putative dimerization interface evidenced here is consistent with other structural, biochemical or functional data published so far

Sedimentation velocity analytical ultracentrifugation in hydrogenated and deuterated solvents for the characterization of membrane proteins.

International audienceThis chapter is a step-by-step protocol for setting up, realizing, and analyzing sedimentation velocity experiments in hydrogenated and deuterated solvents, in the context of the characterization of membrane protein, in terms of homogeneity, association state, and amount of bound detergent, based on a real case study of the membrane protein BmrA solubilized in n-Dodecyl-β-D-Maltopyranoside) detergent

Large scale expression and purification of the mouse 5-HT3 receptor

Receptors of the Cys-loop family are central to neurotransmission and primary therapeutic targets. In order to decipher their gating and modulation mechanisms, structural data is essential. However, structural studies require large amounts of pure, functional receptors. Here, we present the expression and purification of the mouse serotonin 5-HT3 receptor to high purity and homogeneity levels. Inducible expression in human embryonic kidney 293 cells in suspension cultures with orbital shaking resulted in yields of 6-8 mg receptor per liter of culture. Affinity purification using a strep tag provided pure protein in active form. Further deglycosylation and removal of the purification tag led to a pentameric receptor after size-exclusion chromatography, at the milligram scale. This material is suitable for crystallography, as demonstrated by X-ray diffraction of receptor crystals at low resolution. (C) 2013 Elsevier B.V. All rights reserved