CNRS Institute of Pharmacology and Structural Biology
CNRS Institute of Pharmacology and Structural Biology
Volbeda A.,CNRS Institute of Pharmacology and Structural Biology
Nature Communications | Year: 2017
NsrR from Streptomyces coelicolor (Sc) regulates the expression of three genes through the progressive degradation of its [4Fe–4S] cluster on nitric oxide (NO) exposure. We report the 1.95 Å resolution crystal structure of dimeric holo-ScNsrR and show that the cluster is coordinated by the three invariant Cys residues from one monomer and, unexpectedly, Asp8 from the other. A cavity map suggests that NO displaces Asp8 as a cluster ligand and, while D8A and D8C variants remain NO sensitive, DNA binding is affected. A structural comparison of holo-ScNsrR with an apo-IscR-DNA complex shows that the [4Fe–4S] cluster stabilizes a turn between ScNsrR Cys93 and Cys99 properly oriented to interact with the DNA backbone. In addition, an apo ScNsrR structure suggests that Asn97 from this turn, along with Arg12, which forms a salt-bridge with Asp8, are instrumental in modulating the position of the DNA recognition helix region relative to its major groove. © The Author(s)
Thielens N.M.,CNRS Institute of Pharmacology and Structural Biology
Journal of Immunology | Year: 2013
Complement receptor type 1 (CR1) is a membrane receptor expressed on a wide range of cells. It is involved in immune complex clearance, phagocytosis, and complement regulation. Its ectodomain is composed of 30 complement control protein (CCP) modules, organized into four long homologous repeats (A-D). In addition to its main ligands C3b and C4b, CR1 was reported to interact with C1q and mannan-binding lectin (MBL) likely through its C-terminal region (CCP22-30). To decipher the interaction of human CR1 with the recognition proteins of the lectin complement pathway, a recombinant fragment encompassing CCP22-30 was expressed in eukaryotic cells, and its interaction with human MBL and ficolins was investigated using surface plasmon resonance spectroscopy. MBL and L-ficolin were shown to interact with immobilized soluble CR1 and CR1 CCP22-30 with apparent dissociation constants in the nanomolar range, indicative of high affinity. The binding site for CR1 was located at or near the MBL-associated serine protease (MASP) binding site in the collagen stalks of MBL and L-ficolin, as shown by competition experiments with MASP-3. Accordingly, the mutation of an MBL conserved lysine residue essential for MASP binding (K55) abolished binding to soluble CR1 and CCP22-30. The CR1 binding site for MBL/ficolins was mapped to CCP24-25 of long homologous repeat D using deletion mutants. In conclusion, we show that ficolins are new CR1 ligands and propose that MBL/Lficolin binding involves major ionic interactions between conserved lysine residues of their collagen stalks and surface exposed acidic residues located in CR1 CCP24 and/or CCP25. Copyright © 2013 by The American Association of Immunologists, Inc.
Jensen M.R.,CNRS Institute of Pharmacology and Structural Biology |
Ruigrok R.W.H.,French National Center for Scientific Research |
Blackledge M.,CNRS Institute of Pharmacology and Structural Biology
Current Opinion in Structural Biology | Year: 2013
There is growing interest in the development of physical methods to study the conformational behaviour and biological activity of intrinsically disordered proteins (IDPs). In this review recent advances in the elucidation of quantitative descriptions of disordered proteins from nuclear magnetic resonance spectroscopy are presented. Ensemble approaches are particularly well adapted to map the conformational energy landscape sampled by the protein at atomic resolution. Significant advances in development of calibrated approaches to the statistical representation of the conformational behaviour of IDPs are presented, as well as applications to some biologically important systems where disorder plays a crucial role. © 2013 Elsevier Ltd.
Kollman J.M.,Howard Hughes Medical Institute |
Merdes A.,Pierre Fabre |
Mourey L.,CNRS Institute of Pharmacology and Structural Biology |
Agard D.A.,Howard Hughes Medical Institute
Nature Reviews Molecular Cell Biology | Year: 2011
Microtubule nucleation is regulated by the γ-tubulin ring complex (γTuRC) and related γ-tubulin complexes, providing spatial and temporal control over the initiation of microtubule growth. Recent structural work has shed light on the mechanism of γTuRC-based microtubule nucleation, confirming the long-standing hypothesis that the γTuRC functions as a microtubule template. The first crystallographic analysis of a non-γ-tubulin γTuRC component (γ-tubulin complex protein 4 (GCP4)) has resulted in a new appreciation of the relationships among all γTuRC proteins, leading to a refined model of their organization and function. The structures have also suggested an unexpected mechanism for regulating γTuRC activity via conformational modulation of the complex component GCP3. New experiments on γTuRC localization extend these insights, suggesting a direct link between its attachment at specific cellular sites and its activation. © 2011 Macmillan Publishers Limited. All rights reserved.
Breyton C.,CNRS Institute of Pharmacology and Structural Biology
The European physical journal. E, Soft matter | Year: 2013
Small angle neutron scattering (SANS) is a powerful technique for investigating association states and conformational changes of biological macromolecules in solution. SANS is of particular interest for the study of the multi-component systems, as membrane protein complexes, for which in vitro characterisation and structure determination are often difficult. This article details the important physical properties of surfactants in view of small angle neutron scattering studies and the interest to deuterate membrane proteins for contrast variation studies. We present strategies for the production of deuterated membrane proteins and methods for quality control. We then review some studies on membrane proteins, and focus on the strategies to overcome the intrinsic difficulty to eliminate homogeneously the detergent or surfactant signal for solubilised membrane proteins, or that of lipids for membrane proteins inserted in liposomes.
Girard E.,CNRS Institute of Pharmacology and Structural Biology
Biophysical journal | Year: 2010
Structure-function relationships in the tetrameric enzyme urate oxidase were investigated using pressure perturbation. As the active sites are located at the interfaces between monomers, enzyme activity is directly related to the integrity of the tetramer. The effect of hydrostatic pressure on the enzyme was investigated by x-ray crystallography, small-angle x-ray scattering, and fluorescence spectroscopy. Enzymatic activity was also measured under pressure and after decompression. A global model, consistent with all measurements, discloses structural and functional details of the pressure-induced dissociation of the tetramer. Before dissociating, the pressurized protein adopts a conformational substate characterized by an expansion of its substrate binding pocket at the expense of a large neighboring hydrophobic cavity. This substate should be adopted by the enzyme during its catalytic mechanism, where the active site has to accommodate larger intermediates and product. The approach, combining several high-pressure techniques, offers a new (to our knowledge) means of exploring structural and functional properties of transient states relevant to protein mechanisms. Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.
Ferber M.,CNRS Institute of Pharmacology and Structural Biology
Nature Methods | Year: 2016
Crosslinking mass spectrometry is increasingly used for structural characterization of multisubunit protein complexes. Chemical crosslinking captures conformational heterogeneity, which typically results in conflicting crosslinks that cannot be satisfied in a single model, making detailed modeling a challenging task. Here we introduce an automated modeling method dedicated to large protein assemblies ('XL-MOD' software is available at http://aria.pasteur.fr/supplementary-data/x-links) that (i) uses a form of spatial restraints that realistically reflects the distribution of experimentally observed crosslinked distances; (ii) automatically deals with ambiguous and/or conflicting crosslinks and identifies alternative conformations within a Bayesian framework; and (iii) allows subunit structures to be flexible during conformational sampling. We demonstrate our method by testing it on known structures and available crosslinking data. We also crosslinked and modeled the 17-subunit yeast RNA polymerase III at atomic resolution; the resulting model agrees remarkably well with recently published cryoelectron microscopy structures and provides additional insights into the polymerase structure. © 2016 Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.
Maridonneau-Parini I.,CNRS Institute of Pharmacology and Structural Biology |
Maridonneau-Parini I.,Toulouse 1 University Capitole
Immunological Reviews | Year: 2014
Macrophages are professional migrating cells found in all body tissues from the early embryonic stages till the end of the adult life. Tissue macrophages do not only play beneficial roles. In several diseases, macrophages recruited from blood monocytes have a deleterious action such as favoring cancer progression and destroying tissues in chronic inflammation. To migrate in 3D environments, all leukocytes use the amoeboid movement while macrophages use the amoeboid and the mesenchymal migration modes. Mesenchymal migration takes place in dense matrices and involves podosomes and proteolysis of the extracellular matrix to create paths. Podosome disruption has been correlated with reduced mesenchymal migration of macrophages and unaffected amoeboid migration. Therefore, podosomes are proposed as a therapeutic target. Inhibiting podosome regulators that are only expressed in macrophages and few cell types would avoid collateral effects often encountered when ubiquitous proteins are used as drug targets. With the current status of our knowledge on human macrophage podosomes and 3D migration, the tyrosine kinase Hck appears to be a good candidate. © 2014 John Wiley & Sons A/S.
Un S.,CNRS Institute of Pharmacology and Structural Biology
Inorganic Chemistry | Year: 2013
A common feature of a large majority of the manganese metalloenzymes, as well as many synthetic biomimetic complexes, is the bonding between the manganese ion and imidazoles. This interaction was studied by examining the nature and structure of manganese(II) imidazole complexes in frozen aqueous solutions using 285 GHz high magnet-field continuous-wave electron paramagnetic resonance (cw-HFEPR) and 95 GHz pulsed electron-nuclear double resonance (ENDOR) and pulsed electron-double resonance detected nuclear magnetic resonance (PELDOR-NMR). The 55Mn hyperfine coupling and isotropic g values of MnII in frozen imidazole solutions continuously decreased with increasing imidazole concentration. ENDOR and PELDOR-NMR measurements demonstrated that the structural basis for this behavior arose from the imidazole concentration-dependent distribution of three six-coordinate and two four-coordinate species: [Mn(H2O)6]2+, [Mn(imidazole)(H2O)5]2+, [Mn(imidazole) 2(H2O)4]2+, [Mn(imidazole) 3(H2O)]2+, and [Mn(imidazole)4] 2+. The hyperfine and g values of manganese proteins were also fully consistent with this imidazole effect. Density functional theory methods were used to calculate the structures, spin and charge densities, and hyperfine couplings of a number of different manganese imidazole complexes. The use of density functional theory with large exact-exchange admixture calculations gave isotropic 55Mn hyperfine couplings that were semiquantitative and of predictive value. The results show that the covalency of the Mn-N bonds play an important role in determining not only magnetic spin parameters but also the structure of the metal binding site. The relationship between the isotropic 55Mn hyperfine value and the number of imidazole ligands provides a quick and easy test for determining whether a protein binds an MnII ion using histidine residues and, if so, how many are involved. Application of this method shows that as much as 40% of the MnII ions in Deinococcus radiodurans are ligated to two histidines (Tabares, L. C.; Un, S. J. Biol. Chem2013, in press). © 2013 American Chemical Society.
Erba E.B.,CNRS Institute of Pharmacology and Structural Biology
Proteomics | Year: 2014
MS has emerged as an important tool to investigate noncovalent interactions between proteins and various ligands (e.g. other proteins, small molecules, or drugs). In particular, ESI under so-called "native conditions" (a.k.a. "native MS") has considerably expanded the scope of such investigations. For instance, ESI quadrupole time of flight (Q-TOF) instruments have been used to probe the precise stoichiometry of protein assemblies, the interactions between subunits and the position of subunits within the complex (i.e. defining core and peripheral subunits). This review highlights several illustrative native Q-TOF-based investigations and recent seminal contributions of top-down MS (i.e. Fourier transform (FT) MS) to the characterization of noncovalent complexes. Combined top-down and native MS, recently demonstrated in "high-mass modified" orbitrap mass spectrometers, and further improvements needed for the enhanced investigation of biologically significant noncovalent interactions by MS will be discussed. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.