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1. Development of an Efficient FRET-Based Ratiometric Uranium Biosensor

Author: Sandrine Sauge-Merle, Morgane Recuerda, Maria Rosa Beccia, David Lemaire, Rym Cherif, Nicolas Bremond, Fabienne Merola, Yasmina Bousmah, and Catherine Berthomieu
Subjects: genetically encoded biosensor, uranium, FRET, sensing, sensitivity, selectivity, Biotechnology, TP248.13-248.65
Abstract: The dispersion of uranium in the environment can pose a problem for the health of humans and other living organisms. It is therefore important to monitor the bioavailable and hence toxic fraction of uranium in the environment, but no efficient measurement methods exist for this. Our study aims to fill this gap by developing a genetically encoded FRET-based ratiometric uranium biosensor. This biosensor was constructed by grafting two fluorescent proteins to both ends of calmodulin, a protein that binds four calcium ions. By modifying the metal-binding sites and the fluorescent proteins, several versions of the biosensor were generated and characterized in vitro. The best combination results in a biosensor that is affine and selective for uranium compared to metals such as calcium or other environmental compounds (sodium, magnesium, chlorine). It has a good dynamic range and should be robust to environmental conditions. In addition, its detection limit is below the uranium limit concentration in drinking water defined by the World Health Organization. This genetically encoded biosensor is a promising tool to develop a uranium whole-cell biosensor. This would make it possible to monitor the bioavailable fraction of uranium in the environment, even in calcium-rich waters.
Published: 2023
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2. Inter-Site Cooperativity of Calmodulin N-Terminal Domain and Phosphorylation Synergistically Improve the Affinity and Selectivity for Uranyl

Author: Maria Rosa Beccia, Sandrine Sauge-Merle, Nicolas Brémond, David Lemaire, Pierre Henri, Christine Battesti, Philippe Guilbaud, Serge Crouzy, and Catherine Berthomieu
Subjects: uranium, protein, cooperativity, metal binding competition, thermodynamics, spectroscopy, Microbiology, QR1-502
Abstract: Uranyl–protein interactions participate in uranyl trafficking or toxicity to cells. In addition to their qualitative identification, thermodynamic data are needed to predict predominant mechanisms that they mediate in vivo. We previously showed that uranyl can substitute calcium at the canonical EF-hand binding motif of calmodulin (CaM) site I. Here, we investigate thermodynamic properties of uranyl interaction with site II and with the whole CaM N-terminal domain by spectrofluorimetry and ITC. Site II has an affinity for uranyl about 10 times lower than site I. Uranyl binding at site I is exothermic with a large enthalpic contribution, while for site II, the enthalpic contribution to the Gibbs free energy of binding is about 10 times lower than the entropic term. For the N–terminal domain, macroscopic binding constants for uranyl are two to three orders of magnitude higher than for calcium. A positive cooperative process driven by entropy increases the second uranyl-binding event as compared with the first one, with ΔΔG = −2.0 ± 0.4 kJ mol−1, vs. ΔΔG = −6.1 ± 0.1 kJ mol−1 for calcium. Site I phosphorylation largely increases both site I and site II affinity for uranyl and uranyl-binding cooperativity. Combining site I phosphorylation and site II Thr7Trp mutation leads to picomolar dissociation constants Kd1 = 1.7 ± 0.3 pM and Kd2 = 196 ± 21 pM at pH 7. A structural model obtained by MD simulations suggests a structural role of site I phosphorylation in the affinity modulation.
Published: 2022
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3. Alain Huck. Drawing History.

Author: David Lemaire
Subjects: Visual arts, N1-9211
Published: 2016
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4. Reduction of Protein Bound Methionine Sulfoxide by a Periplasmic Dimethyl Sulfoxide Reductase

Author: Lionel Tarrago, Sandrine Grosse, David Lemaire, Laetitia Faure, Mathilde Tribout, Marina I. Siponen, Mila Kojadinovic-Sirinelli, David Pignol, Pascal Arnoux, and Monique Sabaty
Subjects: dimethyl sulfoxide reductase, enzyme kinetics, methionine sulfoxide, oxidative stress, protein oxidation, protein quality control, Therapeutics. Pharmacology, RM1-950
Abstract: In proteins, methionine (Met) can be oxidized into Met sulfoxide (MetO). The ubiquitous methionine sulfoxide reductases (Msr) A and B are thiol-oxidoreductases reducing MetO. Reversible Met oxidation has a wide range of consequences, from protection against oxidative stress to fine-tuned regulation of protein functions. Bacteria distinguish themselves by the production of molybdenum-containing enzymes reducing MetO, such as the periplasmic MsrP which protects proteins during acute oxidative stress. The versatile dimethyl sulfoxide (DMSO) reductases were shown to reduce the free amino acid MetO, but their ability to reduce MetO within proteins was never evaluated. Here, using model oxidized proteins and peptides, enzymatic and mass spectrometry approaches, we showed that the Rhodobacter sphaeroides periplasmic DorA-type DMSO reductase reduces protein bound MetO as efficiently as the free amino acid L-MetO and with catalytic values in the range of those described for the canonical Msrs. The identification of this fourth type of enzyme able to reduce MetO in proteins, conserved across proteobacteria and actinobacteria, suggests that organisms employ enzymatic systems yet undiscovered to regulate protein oxidation states.
Published: 2020
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5. Structural basis of cytotoxicity mediated by the type III secretion toxin ExoU from Pseudomonas aeruginosa.

Author: Claire Gendrin, Carlos Contreras-Martel, Stéphanie Bouillot, Sylvie Elsen, David Lemaire, Dimitrios A Skoufias, Philippe Huber, Ina Attree, and Andréa Dessen
Subjects: Immunologic diseases. Allergy, RC581-607, Biology (General), QH301-705.5
Abstract: The type III secretion system (T3SS) is a complex macromolecular machinery employed by a number of Gram-negative pathogens to inject effectors directly into the cytoplasm of eukaryotic cells. ExoU from the opportunistic pathogen Pseudomonas aeruginosa is one of the most aggressive toxins injected by a T3SS, leading to rapid cell necrosis. Here we report the crystal structure of ExoU in complex with its chaperone, SpcU. ExoU folds into membrane-binding, bridging, and phospholipase domains. SpcU maintains the N-terminus of ExoU in an unfolded state, required for secretion. The phospholipase domain carries an embedded catalytic site whose position within ExoU does not permit direct interaction with the bilayer, which suggests that ExoU must undergo a conformational rearrangement in order to access lipids within the target membrane. The bridging domain connects catalytic domain and membrane-binding domains, the latter of which displays specificity to PI(4,5)P₂. Both transfection experiments and infection of eukaryotic cells with ExoU-secreting bacteria show that ExoU ubiquitination results in its co-localization with endosomal markers. This could reflect an attempt of the infected cell to target ExoU for degradation in order to protect itself from its aggressive cytotoxic action.
Published: 2012
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6. Modulating uranium binding affinity in engineered calmodulin EF-hand peptides: effect of phosphorylation.

Author: Romain Pardoux, Sandrine Sauge-Merle, David Lemaire, Pascale Delangle, Luc Guilloreau, Jean-Marc Adriano, and Catherine Berthomieu
Subjects: Medicine, Science
Abstract: To improve our understanding of uranium toxicity, the determinants of uranyl affinity in proteins must be better characterized. In this work, we analyzed the contribution of a phosphoryl group on uranium binding affinity in a protein binding site, using the site 1 EF-hand motif of calmodulin. The recombinant domain 1 of calmodulin from A. thaliana was engineered to impair metal binding at site 2 and was used as a structured template. Threonine at position 9 of the loop was phosphorylated in vitro, using the recombinant catalytic subunit of protein kinase CK2. Hence, the T(9)TKE(12) sequence was substituted by the CK2 recognition sequence TAAE. A tyrosine was introduced at position 7, so that uranyl and calcium binding affinities could be determined by following tyrosine fluorescence. Phosphorylation was characterized by ESI-MS spectrometry, and the phosphorylated peptide was purified to homogeneity using ion-exchange chromatography. The binding constants for uranyl were determined by competition experiments with iminodiacetate. At pH 6, phosphorylation increased the affinity for uranyl by a factor of ∼5, from K(d) = 25±6 nM to K(d) = 5±1 nM. The phosphorylated peptide exhibited a much larger affinity at pH 7, with a dissociation constant in the subnanomolar range (K(d) = 0.25±0.06 nM). FTIR analyses showed that the phosphothreonine side chain is partly protonated at pH 6, while it is fully deprotonated at pH 7. Moreover, formation of the uranyl-peptide complex at pH 7 resulted in significant frequency shifts of the ν(as)(P-O) and ν(s)(P-O) IR modes of phosphothreonine, supporting its direct interaction with uranyl. Accordingly, a bathochromic shift in ν(as)(UO(2))(2+) vibration (from 923 cm(-1) to 908 cm(-1)) was observed upon uranyl coordination to the phosphorylated peptide. Together, our data demonstrate that the phosphoryl group plays a determining role in uranyl binding affinity to proteins at physiological pH.
Published: 2012
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7. Optimized Coordination of Uranyl in Engineered Calmodulin Site 1 Provides a Subnanomolar Affinity for Uranyl and a Strong Uranyl versus Calcium Selectivity

Author: Romain Pardoux, Sandrine Sauge-Merle, Nicolas Bremond, Maria Rosa Beccia, David Lemaire, Christine Battesti, Pascale Delangle, Pier Lorenzo Solari, Philippe Guilbaud, Catherine Berthomieu, Interactions Protéine Métal (IPM), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de Chimie de Nice (ICN), Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), SYstèmes Moléculaires et nanoMatériaux pour l’Energie et la Santé (SYMMES), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Département Interfaces pour l'énergie, la Santé et l'Environnement (DIESE), Synchrotron SOLEIL (SOLEIL), Département de recherche sur les procédés pour la mine et le recyclage du combustible (DMRC), and CEA-Direction des Energies (ex-Direction de l'Energie Nucléaire) (CEA-DES (ex-DEN))
Subjects: Inorganic Chemistry, Calmodulin, Carboxylic Acids, Uranium, Calcium, [CHIM.COOR]Chemical Sciences/Coordination chemistry, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.TOX.TCA]Life Sciences [q-bio]/Toxicology/Toxicology and food chain, Physical and Theoretical Chemistry, Ligands, Peptides, Cyclic
Abstract: As an alpha emitter and chemical toxicant, uranium toxicity in living organisms is driven by its molecular interactions. It is therefore essential to identify main determinants of uranium affinity for proteins. Others and we showed that introducing a phosphoryl group in the coordination sphere of uranyl confers a strong affinity of proteins for uranyl. In this work, using calmodulin site 1 as a template, we modulate the structural organization of a metal-binding loop comprising carboxylate and/or carbonyl ligands and reach affinities for uranyl comparable to that provided by introducing a strong phosphoryl ligand. Shortening the metal binding loop of calmodulin site 1 from 12 to 10 amino acids in CaMΔ increases the uranyl-binding affinity by about 2 orders of magnitude to log
Published: 2022

8. Investigation of the Plutonium(IV) Interactions with Two Variants of the EF-Hand Ca-Binding Site I of Calmodulin

Author: Loïc Daronnat, Vanessa Holfeltz, Nathalie Boubals, Thomas Dumas, Philippe Guilbaud, Diego Moreno Martinez, Philippe Moisy, Sandrine Sauge-Merle, David Lemaire, Pier Lorenzo Solari, Laurence Berthon, Catherine Berthomieu, Interactions Protéine Métal (IPM), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)
Subjects: Inorganic Chemistry, [SDV]Life Sciences [q-bio], Physical and Theoretical Chemistry
Abstract: International audience; Due to its presence in the nuclear industry and its strong radiotoxicity, plutonium is an actinide of major interest in the event of internal contamination. To improve the understanding of its mechanisms of transport and accumulation in the body, the complexation of Pu(IV) to the most common protein calcium-binding motif found in cells, the EF-hand motif of calmodulin, was investigated. Visible and X-ray absorption spectroscopies (XAS) in solution made it possible to investigate the speciation of plutonium at physiological pH (pH 7.4) and pH 6 in two variants of the calmodulin Ca-binding site I and using Pu(IV) in different media: carbonate, chloride, or nitrate solutions. Three different species of Pu were identified in the samples, with formation of 1:1 Pu(IV):calmodulin peptide complexes, Pu(IV) reduction, and formation of peptide-mediated Pu(IV) hexanuclear cluster.
Published: 2023

9. Development of an Affine and Selective Fluorescent Biosensor for Uranium

Author: Sandrine Sauge-Merle, Morgane RECUERDA, Maria-Rosa BECCIA, David LEMAIRE, Rym CHERIF, Nicolas BREMOND, Fabienne MEROLA, Yasmina BOUSMAH, and Catherine BERTHOMIEU
Subjects: History, Polymers and Plastics, Business and International Management, Industrial and Manufacturing Engineering
Published: 2023

10. Correction: Discovery and characterization of UipA, a uranium- and iron-binding PepSY protein involved in uranium tolerance by soil bacteria

Author: Nicolas Gallois, Béatrice Alpha-Bazin, Nicolas Bremond, Philippe Ortet, Mohamed Barakat, Laurie Piette, Abbas Mohamad Ali, David Lemaire, Pierre Legrand, Nicolas Theodorakopoulos, Magali Floriani, Laureline Février, Christophe Den Auwer, Pascal Arnoux, Catherine Berthomieu, Jean Armengaud, Virginie Chapon, Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Service de Pharmacologie et Immunoanalyse (SPI), Médicaments et Technologies pour la Santé (MTS), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Synchrotron SOLEIL (SSOLEIL), Centre National de la Recherche Scientifique (CNRS), Laboratoire de recherche sur les transferts des radionucléides dans les écosystèmes terrestres (IRSN/PSE-ENV/SRTE/LR2T), Service de recherche sur les transferts et les effets des radionucléides sur les écosystèmes (IRSN/PSE-ENV/SRTE), Institut de Radioprotection et de Sûreté Nucléaire (IRSN)-Institut de Radioprotection et de Sûreté Nucléaire (IRSN), Laboratoire d'écotoxicologie des radionucléides (IRSN/PSE-ENV/SRTE/LECO), Institut de Chimie de Nice (ICN), Université Nice Sophia Antipolis (1965 - 2019) (UNS), and COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA)
Subjects: [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Microbiology, Ecology, Evolution, Behavior and Systematics, ComputingMilieux_MISCELLANEOUS
Abstract: International audience
Published: 2022

11. Characterization of the copper chaperone PcuC as substrate of the periplasmic methionine sulfoxide reductase MsrP in Cereibacter sphaeroides

Author: Lionel Tarrago, Lise Molinelli, David Lemaire, Mathilde Tribout, Sandrine Grosse, May Belghazi, Thierry Tron, David Pignol, Monique Sabaty, and Pascal Arnoux
Subjects: Physiology (medical), Biochemistry
Published: 2022

12. Discovery and characterization of UipA, a uranium- and iron-binding PepSY protein involved in uranium tolerance by soil bacteria

Author: David Lemaire, Virginie Chapon, Laurie Piette, Jean Armengaud, Pierre Legrand, Abbas Mohamad Ali, Philippe Ortet, Mohamed Barakat, Béatrice Alpha-Bazin, Laureline Février, Catherine Berthomieu, Nicolas Bremond, Christophe Den Auwer, Nicolas Gallois, Magali Floriani, Nicolas Theodorakopoulos, Pascal Arnoux, Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Interactions Protéine Métal (IPM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Service de Pharmacologie et Immunoanalyse (SPI), Médicaments et Technologies pour la Santé (MTS), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Laboratoire d'Ecologie Microbienne de la Rhizosphère et d'Environnements Extrêmes (LEMIRE), Synchrotron SOLEIL (SSOLEIL), Centre National de la Recherche Scientifique (CNRS), Laboratoire de recherche sur les transferts de radionucléides dans les écosystèmes terrestres (LR2T), Institut de Radioprotection et de Sûreté Nucléaire (IRSN), Laboratoire d'écotoxicologie des radionucléides (PRP-ENV/SERIS/LECO), Institut de Chimie de Nice (ICN), Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), Microbiologie Environnementale et Moléculaire (MEM), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire de recherche sur les transferts des radionucléides dans les écosystèmes terrestres (IRSN/PSE-ENV/SRTE/LR2T), Service de recherche sur les transferts et les effets des radionucléides sur les écosystèmes (IRSN/PSE-ENV/SRTE), Institut de Radioprotection et de Sûreté Nucléaire (IRSN)-Institut de Radioprotection et de Sûreté Nucléaire (IRSN), Laboratoire d'écotoxicologie des radionucléides (IRSN/PRP-ENV/SERIS/LECO), Service de Recherche et d'Expertise sur les Risques environnementaux (IRSN/PRP-ENV/SERIS), Université Nice Sophia Antipolis (1965 - 2019) (UNS), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA)-Université Nice Sophia Antipolis (... - 2019) (UNS), and COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)
Subjects: inorganic chemicals, Iron, chemistry.chemical_element, Biology, Proteomics, Microbiology, complex mixtures, Article, Metal, 03 medical and health sciences, chemistry.chemical_compound, Soil, proteomics, environmental bacteria, Iron-Binding Proteins, metal-binding protein, crystallography, Ecology, Evolution, Behavior and Systematics, 030304 developmental biology, 0303 health sciences, Bacteria, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 030306 microbiology, technology, industry, and agriculture, Correction, Uranium, Phosphate, biology.organism_classification, Uranyl, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Transmembrane protein, chemistry, Membrane protein, Biochemistry, visual_art, visual_art.visual_art_medium, uranium tolerance
Abstract: International audience; Uranium is a naturally occurring radionuclide. Its redistribution, primarily due to human activities, can have adverse effects on human and non-human biota, which poses environmental concerns. The molecular mechanisms of uranium tolerance and the cellular response induced by uranium exposure in bacteria are not yet fully understood. Here, we carried out a comparative analysis of four actinobacterial strains isolated from metal and radionuclide-rich soils that display contrasted uranium tolerance phenotypes. Comparative proteogenomics showed that uranyl exposure affects 39–47% of the total proteins, with an impact on phosphate and iron metabolisms and membrane proteins. This approach highlighted a protein of unknown function, named UipA, that is specific to the uranium-tolerant strains and that had the highest positive fold-change upon uranium exposure. UipA is a single-pass transmembrane protein and its large C-terminal soluble domain displayed a specific, nanomolar binding affinity for UO$_2^{2+}$ and Fe$^{3+}$. ATR-FTIR and XAS-spectroscopy showed that mono and bidentate carboxylate groups of the protein coordinated both metals. The crystal structure of UipA, solved in its apo state and bound to uranium, revealed a tandem of PepSY domains in a swapped dimer, with a negatively charged face where uranium is bound through a set of conserved residues. This work reveals the importance of UipA and its PepSY domains in metal binding and radionuclide tolerance.
Published: 2021

13. In vitro assessment of cobalt oxide particle dissolution in simulated lung fluids for identification of new decorporating agents

Author: Cien Yoong Hee, Véronique Malard, Christelle Gateau, Magali Floriani, Pauline Brunquet, Guillaume Drouet, Sylvie Coudert, Catherine Berthomieu, Célia Gouzerh, Anne Van der Meeren, Bastien Trochaud, Myriam Benameur, David Lemaire, Pascale Delangle, Colette Lebrun, Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Interactions Protéine Métal (IPM), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), ElectRotechnique et MEcanique des Structures (EDF R&D ERMES), EDF R&D (EDF R&D), EDF (EDF)-EDF (EDF), Laboratoire de Radioécologie et d'Ecotoxicologie (DEI/SECRE/LRE), Institut de Radioprotection et de Sûreté Nucléaire (IRSN), Chimie Interface Biologie pour l’Environnement, la Santé et la Toxicologie (CIBEST ), SYstèmes Moléculaires et nanoMatériaux pour l’Energie et la Santé (SYMMES), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Département Interfaces pour l'énergie, la Santé et l'Environnement (DIESE), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire de Radioécologie et d'Ecotoxicologie (IRSN/DEI/SECRE/LRE), Service d'Etude du Comportement des Radionucléides dans les Ecosystèmes (IRSN/DEI/SECRE), and Institut de Radioprotection et de Sûreté Nucléaire (IRSN)-Institut de Radioprotection et de Sûreté Nucléaire (IRSN)
Subjects: chemistry.chemical_element, 02 engineering and technology, 010501 environmental sciences, Toxicology, 01 natural sciences, Redox, Chelation, Solubility, Cobalt Radioisotopes, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Cobalt oxide, Dissolution, Lung, Decontamination, Edetic Acid, ComputingMilieux_MISCELLANEOUS, 0105 earth and related environmental sciences, Chelating Agents, Inhalation, Chemistry, Oxides, General Medicine, Cobalt, Contamination, Pentetic Acid, 021001 nanoscience & nanotechnology, Body Fluids, 0210 nano-technology, Nuclear chemistry
Abstract: Inhalation of 60Co3O4 particles may occur at the work place in nuclear industry. Their low solubility may result in chronic lung exposure to γ rays. Our strategy for an improved therapeutic approach is to enhance particle dissolution to facilitate cobalt excretion, as the dissolved fraction is rapidly eliminated, mainly in urine. In vitro dissolution of Co3O4 particles was assessed with two complementary assays in lung fluid surrogates to mimic a pulmonary contamination scenario. Twenty-one molecules and eleven combinations were selected through an extensive search in the literature, based on dissolution studies of other metal oxides (Fe, Mn, Cu) and tested for dissolution enhancement of cobalt particles after 1–28 days of incubation. DTPA, the recommended treatment following cobalt contamination did not enhance 60Co3O4 particles dissolution when used alone. However, by combining molecules with different properties, such as redox potential and chelating ability, we greatly improved the efficacy of each drug used alone, leading for the highest efficacy, to a 2.7 fold increased dissolution as compared to controls. These results suggest that destabilization of the particle surface is an important initiating event for a good efficacy of chelating drugs, and open new perspectives for the identification of new therapeutic strategies.
Published: 2020

14. Revisiting binding of plutonium to transferrin by CE-ICP-MS

Author: Sandrine Sauge-Merle, Jean Aupiais, Catherine Berthomieu, David Lemaire, Robert W. Evans, Biologie végétale et microbiologie environnementale - UMR7265 (BVME), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Interactions Protéine Métal (IPM), College of Engineering, Design and Physical Sciences, Brunel University London [Uxbridge], DAM Île-de-France (DAM/DIF), Direction des Applications Militaires (DAM), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, [SDV]Life Sciences [q-bio], Bicarbonate, 010402 general chemistry, Mass spectrometry, 01 natural sciences, Mass Spectrometry, Inorganic Chemistry, 03 medical and health sciences, chemistry.chemical_compound, Capillary electrophoresis, Humans, transferrin, Inductively coupled plasma mass spectrometry, chemistry.chemical_classification, Electrophoresis, Capillary, Binding constant, Plutonium, 0104 chemical sciences, EC-ICP-MS, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, [CHIM.THEO]Chemical Sciences/Theoretical and/or physical chemistry, 030104 developmental biology, chemistry, Transferrin, Ethanesulfonic acid, Inductively coupled plasma, Protein Binding, Nuclear chemistry
Abstract: International audience; Capillary electrophoresis coupled with an inductively coupled plasma mass spectrometer was applied for the first time to determine the binding constant of human transferrin (Tf ) for tetravalent plutonium. The experiments were carried out in a buffer 2-(N-morpholino)ethanesulfonic acid (MES) at pH 6, 0.1 M NaCl and at a temperature of 25 °C. The nitrilotriacetate anion (NTA) used in this study prevents the hydrolysis of plutonium and is an ideal competitor with Tf for Pu, both ligands sharing comparable binding strength. The separation revealed unambiguous two peaks associated with the complex Pu(NTA)2 used as the initial species and with Pu–transferrin. Two series of independent experiments were conducted and gave thefirst stepwise conditional bicarbonate-free Pu–transferrin binding constant of log K* = 22:50±0:19. In the absence of bicarbonate the affinity of transferrin for plutonium at pH 6 is about 104 times stronger than that of iron at pH 6.7 (log K1* = 18.06)
Published: 2017

15. Crystal structure of the transcriptional repressor DdrO: insight into the metalloprotease/repressor-controlled radiation response in Deinococcus

Author: Philippe Roche, Fabrice Confalonieri, Pascale Servant, Romaric Magerand, David Lemaire, David Pignol, François Parcy, Pascal Arnoux, Géraldine Brandelet, Raquel Martin-Arevalillo, Laurence Blanchard, Jade Doloy, Nicolas Eugénie, Arjan de Groot, Marina I. Siponen, Renaud Dumas, Microbiologie Environnementale et Moléculaire (MEM), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de Biologie Intégrative de la Cellule (I2BC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Radiorésistance des bactéries et des archées (RBA), Département Biologie des Génomes (DBG), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Régulateurs du développement de la fleur (Flo_RE), Physiologie cellulaire et végétale (LPCV), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Interactions Protéine Métal (IPM), Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC), ANR-19-CE12-0010,NOVOREP,Réparation des dommages ou mort cellulaire chez les bactéries exposées aux stress, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), ANR-19-CE12-0010,NOVOREP,Réparation des dommages ou mort cellulaire chez les bactéries exposées aux stress(2019), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay, Université Paris-Saclay, Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Centre National de la Recherche Scientifique (CNRS), Centre de Recherche en Cancérologie de Marseille (CRCM), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Aix Marseille Université (AMU), MARAVAL, Alexandra, and Réparation des dommages ou mort cellulaire chez les bactéries exposées aux stress - - NOVOREP2019 - ANR-19-CE12-0010 - AAPG2019 - VALID
Subjects: Models, Molecular, DNA damage, [SDV]Life Sciences [q-bio], Repressor, [SDV.CAN]Life Sciences [q-bio]/Cancer, Cleavage (embryo), Crystallography, X-Ray, DNA-binding protein, Protein Structure, Secondary, 03 medical and health sciences, Protein structure, Structural Biology, Stress, Physiological, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Genetics, Deinococcus, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acid Sequence, Peptide sequence, 030304 developmental biology, 0303 health sciences, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, biology, 030306 microbiology, Gene Expression Regulation, Bacterial, biology.organism_classification, Protein Structure, Tertiary, [SDV] Life Sciences [q-bio], Repressor Proteins, Biophysics, Metalloproteases, Alpha helix, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology, DNA Damage, Transcription Factors
Abstract: Exposure to harmful conditions such as radiation and desiccation induce oxidative stress and DNA damage. In radiation-resistant Deinococcus bacteria, the radiation/desiccation response is controlled by two proteins: the XRE family transcriptional repressor DdrO and the COG2856 metalloprotease IrrE. The latter cleaves and inactivates DdrO. Here, we report the biochemical characterization and crystal structure of DdrO, which is the first structure of a XRE protein targeted by a COG2856 protein. DdrO is composed of two domains that fold independently and are separated by a flexible linker. The N-terminal domain corresponds to the DNA-binding domain. The C-terminal domain, containing three alpha helices arranged in a novel fold, is required for DdrO dimerization. Cleavage by IrrE occurs in the loop between the last two helices of DdrO and abolishes dimerization and DNA binding. The cleavage site is hidden in the DdrO dimer structure, indicating that IrrE cleaves DdrO monomers or that the interaction with IrrE induces a structural change rendering accessible the cleavage site. Predicted COG2856/XRE regulatory protein pairs are found in many bacteria, and available data suggest two different molecular mechanisms for stress-induced gene expression: COG2856 protein-mediated cleavage or inhibition of oligomerization without cleavage of the XRE repressor.
Published: 2019

16. Toxicity mechanisms of cobalt oxide particles (Co 3 O 4 P) on human lung cells: impact of solubilization

Author: Thierry Orsière, David Lemaire, Catherine Berthomieu, Véronique Malard, Richard Ortega, Carole Bresson, Anne Van der Meeren, Chiara Uboldi, Pascale Delangle, Carine Darolles, Service de Biochimie et Toxicologie Nucléaire (SBTN), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), CEA-Direction des Energies (ex-Direction de l'Energie Nucléaire) (CEA-DES (ex-DEN)), Institut méditerranéen de biodiversité et d'écologie marine et continentale (IMBE), Avignon Université (AU)-Aix Marseille Université (AMU)-Institut de recherche pour le développement [IRD] : UMR237-Centre National de la Recherche Scientifique (CNRS), Chimie Interface Biologie pour l’Environnement, la Santé et la Toxicologie (CIBEST ), SYstèmes Moléculaires et nanoMatériaux pour l’Energie et la Santé (SYMMES), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Centre d'Etudes Nucléaires de Bordeaux Gradignan (CENBG), Université Sciences et Technologies - Bordeaux 1 (UB)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS), Interactions Protéine Métal (IPM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Institut de Chimie du CNRS (INC)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), and Université Sciences et Technologies - Bordeaux 1-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Environmental Engineering, lcsh:QP1-981, [SDV]Life Sciences [q-bio], lcsh:QR1-502, 02 engineering and technology, 010501 environmental sciences, 021001 nanoscience & nanotechnology, 01 natural sciences, lcsh:Microbiology, lcsh:Physiology, Industrial and Manufacturing Engineering, 3. Good health, Human lung, medicine.anatomical_structure, Biochemistry, Solubilization, [SDV.TOX]Life Sciences [q-bio]/Toxicology, lcsh:Zoology, Toxicity, medicine, lcsh:QL1-991, 0210 nano-technology, Cobalt oxide, ComputingMilieux_MISCELLANEOUS, 0105 earth and related environmental sciences
Abstract: International audience
Published: 2019

17. $Rhodobacter\ sphaeroides$ methionine sulfoxide reductase P reduces $R$ ‑ and $S$ ‑diastereomers of methionine sulfoxide from a broad‑spectrum of protein substrates

Author: Sandrine Grosse, Guylaine Miotello, Pascal Arnoux, David Lemaire, Lionel Tarrago, Marina I. Siponen, Jean Armengaud, David Pignol, Béatrice Alonso, Monique Sabaty, Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Microbiologie Environnementale et Moléculaire (MEM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Interactions Protéine Métal (IPM), Laboratoire Innovations technologiques pour la Détection et le Diagnostic (LI2D), Service de Pharmacologie et Immunoanalyse (SPI), Médicaments et Technologies pour la Santé (MTS), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Médicaments et Technologies pour la Santé (MTS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Commissariat a l'Energie Atomique et aux Energies Alternatives (CEA) [ANR 16-CE11-0012], ANR-16-CE11-0012,METOXIC,Enzyme de réparation des protéines oxydées: un nouveau système de contrôle qualité dans le périplasme(2016), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, proteomics 15, [SDV]Life Sciences [q-bio], Protein oxidation, Biochemistry, 03 medical and health sciences, chemistry.chemical_compound, Rhodobacter sphaeroides, protein oxidation, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, periplasm, Methionine, biology, Methionine sulfoxide, microbiology, Enzyme kinetics, molybdoenzyme, Cell Biology, Periplasmic space, biology.organism_classification, oxidative 14 stress, 030104 developmental biology, chemistry, methionine sulfoxide reductase, Methionine sulfoxide reductase, Photosynthetic bacteria, MSRA
Abstract: International audience; Methionine (Met) is prone to oxidation and can be converted to Met sulfoxide (MetO), which exists as $R$- and $S$-diastereomers. MetO can be reduced back to Met by the ubiquitous methionine sulfoxide reductase (Msr) enzymes. Canonical MsrA and MsrB were shown to be absolutely stereospecific for the reduction of S ‑ and R‑ diastereomer, respectively. Recently, a new enzymatic system, MsrQ/MsrP which is conserved in all gram‑negative bacteria, was identified as a key actor in the reduction of oxidized periplasmic proteins. The haem‑binding membrane protein MsrQ transmits reducing power from the electron transport chains to the molybdoenzyme MsrP, which acts as a protein‑MetO reductase. The MsrQ/MsrP function was well established genetically, but the identity and biochemical properties of MsrP substrates remain unknown. In this work, using the purified MsrP enzyme from the photosynthetic bacteria $Rhodobacter\ sphaeroides$ as a model, we show that it can reduce a broad spectrum of protein substrates. The most efficiently reduced MetO are found in clusters of amino acid sequences devoid of threonine and proline on the C‑terminal side. Moreover, $R.\ sphaeroides$ MsrP lacks stereospecificity as it can reduce both R‑ and S‑ diastereomers of MetO, similarly to its $Escherichia\ coli$ homolog, and preferentially acts on unfolded oxidized proteins. Overall, these results provide important insights into the function of a bacterial envelop protecting system, which should help understand how bacteria cope in harmful environments.
Published: 2018

18. The Rhodobacter sphaeroides methionine sulfoxide reductase MsrP can reduce R- and S-diastereomers of methionine sulfoxide from a broad-spectrum of protein substrates

Author: David Lemaire, Monique Sabaty, Jean Armengaud, David Pignol, Lionel Tarrago, Pascal Arnoux, Sandrine Grosse, Guylaine Miotello, Marina I. Siponen, and Béatrice Alonso
Subjects: Methionine, biology, Chemistry, Methionine sulfoxide, Sulfoxide, Periplasmic space, medicine.disease_cause, biology.organism_classification, chemistry.chemical_compound, Rhodobacter sphaeroides, Biochemistry, medicine, Methionine sulfoxide reductase, Escherichia coli, MSRA
Abstract: SummaryMethionine (Met) is prone to oxidation and can be converted to Met sulfoxide (MetO), which exists as R- and S-diastereomers. MetO can be reduced back to Met by the ubiquitous methionine sulfoxide reductase (Msr) enzymes. Canonical MsrA and MsrB were shown as absolutely stereospecific for the reduction of S- and R-diastereomer, respectively. Recently, the molybdenum-containing protein MsrP, conserved in all gram-negative bacteria, was shown to be able to reduce MetO of periplasmic proteins without apparent stereospecificity inEscherichia coli.Here, we describe the substrate specificity of theRhodobacter sphaeroidesMsrP. Proteomics analysis coupled to enzymology approaches indicate that it reduces a broad spectrum of periplasmic oxidized proteins. Moreover, using model proteins, we demonstrated that RsMsrP preferentially reduces unfolded oxidized proteins and we confirmed that this enzyme, like itsE. colihomolog, can reduce bothR-andS-diastereomers of MetO with similar efficiency.
Published: 2018
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19. Structural Analysis of Uranyl Complexation by the EF-Hand Motif of Calmodulin: Effect of Phosphorylation

Author: Romain Pardoux, Sandrine Sauge-Merle, Mohamed L. Merroun, Philippe Guilbaud, Samir Safi, E. Simoni, Pier Lorenzo Solari, Catherine Berthomieu, David Lemaire, Florian Brulfert, Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Interactions Protéine Métal (IPM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Institut de Physique Nucléaire d'Orsay (IPNO), Université Paris-Sud - Paris 11 (UP11)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS), Synchrotron SOLEIL (SSOLEIL), Centre National de la Recherche Scientifique (CNRS), Laboratory of Interactions Ligand-Actinide (LILA), Département de recherche sur les procédés pour la mine et le recyclage du combustible (DMRC), CEA-Direction des Energies (ex-Direction de l'Energie Nucléaire) (CEA-DES (ex-DEN)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-CEA-Direction des Energies (ex-Direction de l'Energie Nucléaire) (CEA-DES (ex-DEN)), Universidad de Granada = University of Granada (UGR), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Université Paris-Sud - Paris 11 (UP11), and University of Granada [Granada]
Subjects: Denticity, Calmodulin, Absorption spectroscopy, Inorganic chemistry, Amino Acid Motifs, Molecular Dynamics Simulation, 010402 general chemistry, 01 natural sciences, Catalysis, Fluorescence spectroscopy, chemistry.chemical_compound, Coordination Complexes, Spectroscopy, Fourier Transform Infrared, [CHIM]Chemical Sciences, Carboxylate, Phosphorylation, Binding Sites, biology, 010405 organic chemistry, Chemistry, Ligand, Organic Chemistry, General Chemistry, Uranyl, 0104 chemical sciences, Crystallography, Spectrometry, Fluorescence, X-Ray Absorption Spectroscopy, biology.protein, Hydroxide, Uranium, Paramecium tetraurelia
Abstract: International audience; Better understanding of uranyl–protein interactions is a prerequisite to predict uranium chemical toxicity in cells. The EF‐hand motif of the calmodulin site I is about thousand times more affine for uranyl than for calcium, and threonine phosphorylation increases the uranyl affinity by two orders of magnitude at pH 7. In this study, we confront X‐ray absorption spectroscopy with Fourier transform infrared (FTIR) spectroscopy, time‐resolved laser‐induced fluorescence spectroscopy (TRLFS), and structural models obtained by molecular dynamics simulations to analyze the uranyl coordination in the native and phosphorylated calmodulin site I. For the native site I, extended X‐ray absorption fine structure (EXAFS) data evidence a short U−Oeq distance, in addition to distances compatible with mono‐ and bidentate coordination by carboxylate groups. Further analysis of uranyl speciation by TRLFS and thorough investigation of the fluorescence decay kinetics strongly support the presence of a hydroxide uranyl ligand. For a phosphorylated site I, the EXAFS and FTIR data support a monodentate uranyl coordination by the phosphoryl group and strong interaction with mono‐ and bidentate carboxylate ligands. This study confirms the important role of a phosphoryl ligand in the stability of uranyl–protein interactions. By evidencing a hydroxide uranyl ligand in calmodulin site I, this study also highlights the possible role of less studied ligands as water or hydroxide ions in the stability of protein–uranyl complexes.
Published: 2017

20. Radiation response inDeinococcus deserti: IrrE is a metalloprotease that cleaves repressor protein DdrO

Author: David Lemaire, David Pignol, Laurent Bellanger, Arjan de Groot, Géraldine Brandelet, Laurence Blanchard, Monika Ludanyi, and Rémi Dulermo
Subjects: 0303 health sciences, Metalloproteinase, biology, 030306 microbiology, DNA repair, Repressor, Deinococcus deserti, medicine.disease_cause, biology.organism_classification, Microbiology, 03 medical and health sciences, Biochemistry, parasitic diseases, medicine, bacteria, population characteristics, Deinococcus, Molecular Biology, Psychological repression, Gene, Escherichia coli, 030304 developmental biology
Abstract: Deinococcus bacteria are famous for their extreme radiation tolerance. The IrrE protein was shown to be essential for radiation tolerance and, in an unelucidated manner, for induction of a number of genes in response to radiation, including recA and other DNA repair genes. Earlier studies indicated that IrrE could be a zinc peptidase, but proteolytic activity was not demonstrated. Here, using several in vivo and in vitro experiments, IrrE from Deinococcus deserti was found to interact with DdrO, a predicted regulator encoded by a radiation-induced gene that is, like irrE, highly conserved in Deinococcus. Moreover, IrrE was found to cleave DdrO in vitro and when the proteins were coexpressed in Escherichia coli. This cleavage was not observed in the presence of metal chelator EDTA or when IrrE contains a mutation in the conserved active-site motif of metallopeptidases. In D. deserti, IrrE-dependent cleavage of DdrO was observed after exposure to radiation. Furthermore, DdrO-dependent repression of the promoter of a radiation-induced gene was shown. These results demonstrate that IrrE is a metalloprotease and we propose that IrrE-mediated cleavage inactivates repressor protein DdrO, leading to transcriptional induction of various genes required for repair and survival after exposure of Deinococcus to radiation.
Published: 2014

21. An alternative plant-like cyanobacterial ferredoxin with unprecedented structural and functional properties

Author: David Lemaire, Jian Ren Shen, Miwa Sugiura, Alain Boussac, Yasufumi Umena, Taiki Motomura, Lidia Zuccarello, Pierre Sétif, Rainer Hienerwadel, Jatindra N. Tripathy, and Catherine Berthomieu
Subjects: Models, Molecular, Stereochemistry, Thermosynechococcus, Biophysics, Cyanobacteria, Photosynthesis, Photosystem I, Biochemistry, Redox, 03 medical and health sciences, Electron transfer, Bacterial Proteins, Oxidoreductase, Amino Acid Sequence, Phylogeny, Ferredoxin, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, Chemistry, 030302 biochemistry & molecular biology, Hydrogen Bonding, Cell Biology, Nitrite reductase, Electron transport chain, Kinetics, Ferredoxins, Sequence Alignment
Abstract: Photosynthetic [2Fe-2S] plant-type ferredoxins have a central role in electron transfer between the photosynthetic chain and various metabolic pathways. Several genes are coding for [2Fe 2S] ferredoxins in cyanobacteria, with four in the thermophilic cyanobacterium Thermosynechococcus elongatus. The structure and functional properties of the major ferredoxin Fd1 are well known but data on the other ferredoxins are scarce. We report the structural and functional properties of a novel minor type ferredoxin, Fd2 of T. elongatus, homologous to Fed4 from Synechocystis sp. PCC 6803. Remarkably, the midpoint potential of Fd2, Em = −440 mV, is lower than that of Fd1, Em = −372 mV. However, while Fd2 can efficiently react with photosystem I or nitrite reductase, time-resolved spectroscopy shows that Fd2 has a very low capacity to reduce ferredoxin-NADP+ oxidoreductase (FNR). These unique Fd2 properties are discussed in relation with its structure, solved at 1.38 A resolution. The Fd2 structure significantly differs from other known ferredoxins structures in loop 2, N-terminal region, hydrogen bonding networks and surface charge distributions. UV–Vis, EPR, and Mid- and Far-IR data also show that the electronic properties of the [2Fe 2S] cluster of Fd2 and its interaction with the protein differ from those of Fd1 both in the oxidized and reduced states. The structural analysis allows to propose that valine in the motif Cys53ValAsnCys56 of Fd2 and the specific orientation of Phe72, explain the electron transfer properties of Fd2. Strikingly, the nature of these residues correlates with different phylogenetic groups of cyanobacterial Fds. With its low redox potential and its discrimination against FNR, Fd2 exhibits a unique capacity to direct efficiently photosynthetic electrons to metabolic pathways not dependent on FNR.
Published: 2019

22. Biosynthesis of a broad-spectrum nicotianamine-like metallophore in Staphylococcus aureus

Author: Ryszard Lobinski, Elise Borezée-Durant, Felipe Cava, David Lemaire, Ghassan Ghssein, Christine Hajjar, Catherine Brutesco, Amélie Izaute, David Pignol, Romé Voulhoux, Pierre Richaud, Pascal Arnoux, Akbar Espaillat, Laurent Ouerdane, Shuanglong Wang, Clémentine Fojcik, Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Microbiologie Environnementale et Moléculaire (MEM), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut des sciences analytiques et de physico-chimie pour l'environnement et les materiaux (IPREM), Université de Pau et des Pays de l'Adour (UPPA)-Centre National de la Recherche Scientifique (CNRS), MICrobiologie de l'ALImentation au Service de la Santé (MICALIS), Institut National de la Recherche Agronomique (INRA)-AgroParisTech, Biologie végétale et microbiologie environnementale - UMR7265 (BVME), Interactions Protéine Métal (IPM), Bioénergie et Microalgues (EBM), Laboratoire d'ingénierie des systèmes macromoléculaires (LISM), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Umeå University, CEA program ToxNuc-E, HélioBiotec platform, ANR-14-CE09-0007,ANIBAL,Un nouveau metallophore dérivé de la nicotianamine chez des bactéries pathogènes(2014), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Chimie Analytique Bio-Inorganique et Environnement (LCABIE), Laboratory for Molecular Infection Medicine Sweden, Department of Molecular Biology, Umeå Centre of Microbial Research, Umeå, Sweden, French Atomic Energy CommissionCEA program ToxNuc-E HelioBiotec platform - European Regional Development Fund Région Provence-Alpes-Côte d'AzurHelioBiotec platform - French Ministry of Research HelioBiotec platform - 'Commissariat a l'Energie Atomique et aux Energies Alternatives' Région Provence-Alpes-Côte d'AzurLaboratory for Molecular Infection Medicine Sweden Knut & Alice Wallenberg FoundationSwedish Research CouncilEuropean CommissionKempe foundation China Scholarship Council, Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Lobinski, Ryszard, and Appel à projets générique - Un nouveau metallophore dérivé de la nicotianamine chez des bactéries pathogènes - - ANIBAL2014 - ANR-14-CE09-0007 - Appel à projets générique - VALID
Subjects: 0301 basic medicine, Operon, [SDV]Life Sciences [q-bio], transferase, Dehydrogenase, Nicotianamine synthase, chemistry.chemical_compound, iron, Transferase, pathogenicity, Nicotianamine, oxidoreductase, azetidine 2 carboxylic acid, Amino Acid Isomerases, chemistry.chemical_classification, Multidisciplinary, metallophore, nicotianamine synthase, zinc, Imidazoles, Cobalt, [SDV] Life Sciences [q-bio], Biochemistry, Metabolome, Oxidoreductases, Azetidinecarboxylic Acid, Staphylococcus aureus, enzymology, 030106 microbiology, Biology, Biosynthesis, microbial activity, nicotine derivative, 03 medical and health sciences, nickel, Oxidoreductase, [CHIM]Chemical Sciences, Histidine, Alkyl and Aryl Transferases, nicotianamine, mutagenicity, Gene Expression Regulation, Bacterial, histidine, heavy metal, operon, Biosynthetic Pathways, staphylopine, 030104 developmental biology, chemistry, Gene Expression Regulation, copper, biology.protein, phytochemistry, imidazole derivative
Abstract: A new metal scavenger for bacteria All cells must find a way to acquire trace metals. Bacteria and plants scavenge iron, for instance, by synthesizing and releasing iron-chelating compounds called siderophores. Ghssein et al. describe three enzymes in Staphylococcus aureus that are responsible for the biosynthesis of another type of metallophore (see the Perspective by Nolan). Metabolomics and a range of biochemical assays show that this compound, named staphylopine, is involved in the uptake of a range of metals, depending on the growth environment. The genes required for staphylopine biosynthesis are conserved across a number of pathogenic bacteria and are similar to those for a broad-spectrum metallophore produced by plants. Science , this issue p. 1105 ; see also p. 1055
Published: 2016

23. The Zn center of the anaerobic ribonucleotide reductase from E. coli

Author: Etienne Mulliez, Bernard Dublet, David Lemaire, Florence Luttringer, Marc Fontecave, Laboratoire de Chimie et Biologie des Métaux (LCBM - UMR 5249), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Interactions Protéine Métal (IPM), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Collège de France - Chaire Chimie des processus biologiques, Laboratoire de Chimie des Processus Biologiques (LCPB), Collège de France (CdF (institution))-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Collège de France (CdF (institution))-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), and Chaire Chimie des processus biologiques
Subjects: Anaerobic ribonucleotide reductase, Ribonucleotide, Free Radicals, Stereochemistry, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Reductase, 010402 general chemistry, medicine.disease_cause, 01 natural sciences, Biochemistry, Substrate Specificity, Inorganic Chemistry, 03 medical and health sciences, chemistry.chemical_compound, Biosynthesis, Enzyme Stability, Ribonucleotide Reductases, Escherichia coli, medicine, Glycyl radical, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Anaerobiosis, Disulfides, Sulfhydryl Compounds, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, Binding Sites, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 0104 chemical sciences, Zinc, Enzyme, Ribonucleotide reductase, chemistry, Radical S-adenosylmethionine, Thioredoxin, Holoenzymes, Cysteine
Abstract: International audience; Strict and facultative anaerobes depend on a class III ribonucleotide reductase for their growth. These enzymes are the sole cellular catalysts for de novo biosynthesis of the deoxyribonucleotides needed for DNA chain elongation and repair. In its active form, the class III ribonucleotide reductase from Escherichia coli contains a free radical located on the G681 residue which is essential for the activation of the ribonucleotide substrate toward its reduction. The 3D structure of the homologous enzyme from bacteriophage T4 has revealed the presence of a metal center bound to four conserved cysteine residues. In this report we identify the metal of the E. coli enzyme as Zn. We show that the presence of Zn in this site protects the protein from proteolysis and prevents the formation of disulfide bridges within it. Finally, we show with the fully Zn-loaded reductase that thioredoxin or small thiols are dispensable for the formation of the glycyl radical. However, they are necessary for obtaining high turnover numbers, suggesting that they intervene in radical transfer steps subsequent to the formation of the glycyl radical.
Published: 2009

24. The H-bond network surrounding the pyranopterins modulates redox cooperativity in the molybdenum-bisPGD cofactor in arsenite oxidase

Author: Simon Duval, Florence Chaspoul, Michael J. Russell, Wolfgang Nitschke, Barbara Schoepp-Cothenet, Stéphane Grimaldi, David Lemaire, Joanne M. Santini, Bioénergétique et Ingénierie des Protéines (BIP ), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU), Institute of Structural and Molecular Biology, University College of London [London] (UCL), Biologie végétale et microbiologie environnementale - UMR7265 (BVME), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Interactions Protéine Métal (IPM), Aix-Marseille Université - Faculté de médecine (AMU MED), Aix Marseille Université (AMU), Jet Propulsion Laboratory (JPL), NASA-California Institute of Technology (CALTECH), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)
Subjects: 0301 basic medicine, Stereochemistry, [SDV]Life Sciences [q-bio], Inorganic chemistry, Biophysics, Optical spectroscopy, chemistry.chemical_element, Cooperativity, 010402 general chemistry, 01 natural sciences, Biochemistry, Redox, Cofactor, Catalysis, Redox titrations, 03 medical and health sciences, chemistry.chemical_compound, [SPI]Engineering Sciences [physics], Redox titration, Molybdenum, Arsenite oxidase, biology, Electron Spin Resonance Spectroscopy, Hydrogen Bonding, Cell Biology, 0104 chemical sciences, Pterins, 030104 developmental biology, chemistry, Molybdenum enzyme, biology.protein, Titration, Molybdenum cofactor, Oxidoreductases, Oxidation-Reduction, EPR spectroscopy
Abstract: International audience; While the molybdenum cofactor in the majority of bisPGD enzymes goes through two consecutive 1-electron redox transitions, previous protein-film voltammetric results indicated the possibility of cooperative (n = 2) redox behavior in the bioenergetic enzyme arsenite oxidase (Aio). Combining equilibrium redox titrations, optical and EPR spectroscopies on concentrated samples obtained via heterologous expression, we unambiguously confirm this claim and quantify Aio's redox cooperativity. The stability constant, Ks, of the MoV semi-reduced intermediate is found to be lower than 10− 3. Site-directed mutagenesis of residues in the vicinity of the Mo-cofactor demonstrates that the degree of redox cooperativity is sensitive to H-bonding interactions between the pyranopterin moieties and amino acid residues. Remarkably, in particular replacing the Gln-726 residue by Gly results in stabilization of (low-temperature) EPR-observable MoV with KS = 4. As evidenced by comparison of room temperature optical and low temperature EPR titrations, the degree of stabilization is temperature-dependent. This highlights the importance of room-temperature redox characterizations for correctly interpreting catalytic properties in this group of enzymes.Geochemical and phylogenetic data strongly indicate that molybdenum played an essential biocatalytic roles in early life. Molybdenum's redox versatility and in particular the ability to show cooperative (n = 2) redox behavior provide a rationale for its paramount catalytic importance throughout the evolutionary history of life. Implications of the H-bonding network modulating Molybdenum's redox properties on details of a putative inorganic metabolism at life's origin are discussed.
Published: 2015

25. Thermodynamics of Calcium binding to the Calmodulin N-terminal domain to evaluate site-specific affinity constants and cooperativity

Author: Maria Rosa Beccia, Stéphanie Blangy, Romain Pardoux, Sandrine Sauge-Merle, Nicolas Bremond, Catherine Berthomieu, Philippe Guilbaud, David Lemaire, Instituto de Química, Universidade Federal do Rio de Janeiro (UFRJ), Biologie végétale et microbiologie environnementale - UMR7265 (BVME), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Aix Marseille Université (AMU), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Centre National de la Recherche Scientifique (CNRS), CEA Cadarache, Environnement, Bioénergie, Microalgues et Plantes (EBMP), Département RadioChimie et Procédés (DRCP), CEA-Direction des Energies (ex-Direction de l'Energie Nucléaire) (CEA-DES (ex-DEN)), Interactions Protéine Métal (IPM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), and Bioénergie et Microalgues (EBM)
Subjects: Models, Molecular, Calmodulin, [SDV]Life Sciences [q-bio], 030303 biophysics, Molecular Sequence Data, chemistry.chemical_element, Cooperativity, Calcium, Protein Engineering, Biochemistry, Inorganic Chemistry, 03 medical and health sciences, Protein structure, Amino Acid Sequence, 030304 developmental biology, 0303 health sciences, Binding Sites, biology, Mutagenesis, Tryptophan, Cooperative binding, Isothermal titration calorimetry, Protein Structure, Tertiary, Crystallography, chemistry, Biophysics, biology.protein, Mutagenesis, Site-Directed, Thermodynamics
Abstract: International audience; Calmodulin (CaM) is an essential Ca(II)-dependent regulator of cell physiology. To understand its interaction with Ca(II) at a molecular level, it is essential to examine Ca(II) binding at each site of the protein, even if it is challenging to estimate the site-specific binding properties of the interdependent CaM-binding sites. In this study, we evaluated the site-specific Ca(II)-binding affinity of sites I and II of the N-terminal domain by combining site-directed mutagenesis and spectrofluorimetry. The mutations had very low impact on the protein structure and stability. We used these binding constants to evaluate the inter-site cooperativity energy and compared it with its lower limit value usually reported in the literature. We found that site I affinity for Ca(II) was 1.5 times that of site II and that cooperativity induced an approximately tenfold higher affinity for the second Ca(II)-binding event, as compared to the first one. We further showed that insertion of a tryptophan at position 7 of site II binding loop significantly increased site II affinity for Ca(II) and the intra-domain cooperativity. ΔH and ΔS parameters were studied by isothermal titration calorimetry for Ca(II) binding to site I, site II and to the entire N-terminal domain. They showed that calcium binding is mainly entropy driven for the first and second binding events. These findings provide molecular information on the structure-affinity relationship of the individual sites of the CaM N-terminal domain and new perspectives for the optimization of metal ion binding by mutating the EF-hand loops sequences.
Published: 2015

26. Use of hydrogen/deuterium exchange mass spectrometry and mutagenesis as a tool to identify the binding region of inhibitors targeting the human mitotic kinesin Eg5

Author: David Lemaire, Frank Kozielski, Salvatore DeBonis, Sébastien Brier, and Eric Forest
Subjects: Spectrometry, Mass, Electrospray Ionization, Molecular Sequence Data, Drug Evaluation, Preclinical, Kinesins, Mitosis, Analytical Chemistry, chemistry.chemical_compound, Humans, Amino Acid Sequence, Enzyme Inhibitors, Binding site, Protein secondary structure, Spectroscopy, Binding Sites, Molecular Structure, Chemistry, Organic Chemistry, Mutagenesis, Deuterium Exchange Measurement, Kinetics, Monastrol, Deuterium, Biochemistry, Helix, Kinesin, Hydrogen–deuterium exchange
Abstract: An experimental procedure associating both hydrogen/deuterium exchange mass spectrometry (H/D-MS) and mutagenesis was developed to identify the protein-binding region of small inhibitors targeting the motor domain of the human mitotic kinesin Eg5. All the tested inhibitors decrease the deuterium incorporation rate of the same peptides corresponding to the following secondary structure elements: loop L5/helix alpha 2 (region Tyr125-Glu145) and strand beta 5/helix alpha 3 (region Ile202-Leu227). Replacement of these two regions by the equivalent ones from N. crassa conventional kinesin heavy chain completely abolishes the modification of the deuterium incorporation rate by the inhibitors as well as their effects on the basal ATPase activity. The six tested inhibitors thus share a common binding site on Eg5. The strategy reported here allows the regions of a protein involved in ligand binding to be rapidly pinpointed and can be applied to other proteins and used as a general in vitro screening procedure to identify compounds targeting specific binding regions
Published: 2006

27. Crystal Structure of Phosphorylcholine Esterase Domain of the Virulence Factor Choline-binding Protein E from Streptococcus pneumoniae

Author: Otto Dideberg, David Lemaire, Gianpiero Garau, Anne Marie Di Guilmi, and Thierry Vernet
Subjects: chemistry.chemical_classification, Teichoic acid, biology, Phosphorylcholine, Active site, Cell Biology, medicine.disease_cause, Biochemistry, Esterase, Microbiology, chemistry.chemical_compound, Enzyme, chemistry, Streptococcus pneumoniae, Hydrolase, medicine, biology.protein, Molecular Biology, Choline binding
Abstract: Streptococcus pneumoniae is the worldwide leading cause of deaths from invasive infections such as pneumoniae, sepsis, and meningitidis in children and the elderly. Nasopharyngeal colonization, which plays a key role in the development of pneumococcal disease, is highly dependent on a family of surface-exposed proteins, the choline-binding proteins (CBPs). Here we report the crystal structure of phosphorylcholine esterase (Pce), the catalytic domain of choline-binding protein E (CBPE), which has been shown to be crucial for host/pathogen interaction processes. The unexpected features of the Pce active site reveal that this enzyme is unique among the large family of hydrolases harboring the metallo-β-lactamase fold. The orientation and calcium stabilization features of an elongated loop, which lies on top of the active site, suggest that the cleft may be rearranged. Furthermore, the structure of Pce complexed with phosphorylcholine, together with the characterization of the enzymatic role played by two iron ions located in the active site allow us to propose a reaction mechanism reminiscent of that of purple acid phosphatase. This mechanism is supported by site-directed mutagenesis experiments. Finally, the interactions of the choline binding domain and the Pce region of CBPE with chains of teichoic acids have been modeled. The ensemble of our biochemical and structural results provide an initial understanding of the function of CBPE.
Published: 2005

28. Increased pressure at the electrospray interface dramatically raises sensitivity in analysis of denaturated proteins

Author: Laetitia Cravello, Eric Forest, David Lascoux, and David Lemaire
Subjects: Protein Denaturation, Spectrometry, Mass, Electrospray Ionization, Electrospray, Chromatography, Myoglobin, Chemistry, Interface (computing), Organic Chemistry, Cytochromes c, Proteins, Serum Albumin, Bovine, Nanotechnology, Analytical Chemistry, Molecular Weight, Phosphopyruvate Hydratase, Pressure, Animals, Cattle, Sensitivity (control systems), Spectroscopy
Published: 2005

29. Crystallographic and Spectroscopic Evidence for High Affinity Binding of FeEDTA(H2O)- to the Periplasmic Nickel Transporter NikA

Author: David Lemaire, Jacques Gaillard, Christine Cavazza, Mickaël V. Cherrier, Lilian Jacquamet, Juan C. Fontecilla-Camps, and Lydie Martin
Subjects: Operon, Stereochemistry, Iron, Fluorescence spectrometry, chemistry.chemical_element, Crystallography, X-Ray, medicine.disease_cause, Biochemistry, Mass Spectrometry, Catalysis, Colloid and Surface Chemistry, Nickel, Chemical affinity, medicine, Ferrous Compounds, Binding site, Escherichia coli, Edetic Acid, Binding Sites, Escherichia coli Proteins, General Chemistry, Periplasmic space, Protein Structure, Tertiary, Transport protein, Crystallography, chemistry, Periplasm, ATP-Binding Cassette Transporters
Abstract: Because nickel is both essential and toxic to a great variety of organisms, its detection and transport is highly regulated. In Escherichia coli and other related Gram-negative bacteria, high affinity nickel transport depends on proteins expressed by the nik operon. A central actor of this process is the periplasmic NikA transport protein. A previous structural report has proposed that nickel binds to NikA as a pentahydrate species. However, both stereochemical considerations and X-ray absorption spectroscopic results are incompatible with that interpretation. Here, we report the 1.8 A resolution structure of NikA and show that it binds FeEDTA(H2O)- with very high affinity. In addition, we provide crystallographic evidence that a metal-EDTA complex was also bound to the previously reported NikA structure. Our observations strongly suggest that nickel transport in E. coli requires the binding of this metal ion to a metallophore that bears significant resemblance to EDTA. They also provide a basis for the potential use of NikA in the bioremediation of toxic transition metals and the design of artificial metalloenzymes.
Published: 2005

30. The Structural Modifications Induced by the M339F Substitution in PBP2x from Streptococcus pneumoniae Further Decreases the Susceptibility to β-Lactams of Resistant Strains

Author: Otto Dideberg, André Zapun, Lucile Pernot, Jacques Croize, David Lemaire, Thierry Vernet, Laurent Chesnel, and Dominique Champelovier
Subjects: Models, Molecular, medicine.drug_class, Acylation, Molecular Sequence Data, Antibiotics, Cefotaxime, beta-Lactams, medicine.disease_cause, Biochemistry, beta-Lactam Resistance, In vivo, Streptococcus pneumoniae, β lactams, Serine, medicine, Penicillin-Binding Proteins, Point Mutation, Single amino acid, Molecular Biology, Genetics, Binding Sites, Molecular Structure, biology, Double mutant, Active site, Penicillin G, Cell Biology, Hydrogen-Ion Concentration, In vitro, Kinetics, Phenotype, Mutagenesis, Site-Directed, biology.protein, Carrier Proteins, Crystallization
Abstract: PBP2x is a primary determinant of beta-lactams resistance in Streptococcus pneumoniae. Altered PBP2x with multiple mutations have a reduced "affinity" for the antibiotics. An important polymorphism is found in PBP2x sequences from clinical resistant strains. To understand the mechanism of resistance, it is necessary to identify and characterize the relevant substitutions. Many similar PBP2x sequences from resistant isolates have the previously studied T338A mutation, adjacent to the active site Ser337. We report here the structural and functional analysis of the M339F substitution that is found in a subset of these sequences, originating from highly resistant strains. The M339F mutation causes a 4-10-fold reduction of the reaction rate with beta-lactams, depending on the molecular context. In addition, release of the inactivated antibiotic from the active site is up to 3-fold faster as a result from the M339F mutation. These effects measured in vitro are correlated with the level of beta-lactam resistance in vivo conferred by several PBP2x variants. Thus, a single amino acid difference between similar PBP2x from clinical isolates can strongly modulate the degree of beta-lactam resistance. The crystal structure of the double mutant T338A/M339F solved to a resolution of 2.4 A shows a distortion of the active site and a reorientation of the hydroxyl group of the active site Ser337, which can explain the kinetic effects of the mutations.
Published: 2003

31. Collision efficiency in an electrospray source interfaced with a magnetic mass spectrometer

Author: Olivier Laprévote, Laurent Serani, and David Lemaire
Subjects: Electrospray, Ion beam, Chemistry, Condensed Matter Physics, Mass spectrometry, Tandem mass spectrometry, Dissociation (chemistry), Ion source, Ion, Ion beam deposition, Physical and Theoretical Chemistry, Atomic physics, Instrumentation, Spectroscopy
Abstract: The collision efficiency in two distinct areas of an electrospray ion source has been compared on the basis of diagnostic ion intensities related to the voltages applied on three different lenses. The dissociation of covalent or non-covalent bonds and the shift of the charge state distribution of multiple-charged protein ions were used as indicators of the internal energy amount acquired by collisional activation. The results show that, for a given voltage difference between two successive lenses on the ion beam axis, the more efficient collision-induced dissociation processes are observed after the skimmer and not before. Although the enhancement of the fragmentation rate by increasing the skimmer voltage could be accompanied by a loss of sensitivity, this procedure should be of interest for analytical purposes.
Published: 2002

32. Radiation response in Deinococcus deserti: IrrE is a metalloprotease that cleaves repressor protein DdrO

Author: Monika, Ludanyi, Laurence, Blanchard, Rémi, Dulermo, Géraldine, Brandelet, Laurent, Bellanger, David, Pignol, David, Lemaire, and Arjan, de Groot
Subjects: Repressor Proteins, Stress, Physiological, Molecular Sequence Data, Proteolysis, Escherichia coli, Metalloproteases, Gene Expression, Amino Acid Sequence, Deinococcus, Gene Expression Regulation, Bacterial, Sequence Alignment
Abstract: Deinococcus bacteria are famous for their extreme radiation tolerance. The IrrE protein was shown to be essential for radiation tolerance and, in an unelucidated manner, for induction of a number of genes in response to radiation, including recA and other DNA repair genes. Earlier studies indicated that IrrE could be a zinc peptidase, but proteolytic activity was not demonstrated. Here, using several in vivo and in vitro experiments, IrrE from Deinococcus deserti was found to interact with DdrO, a predicted regulator encoded by a radiation-induced gene that is, like irrE, highly conserved in Deinococcus. Moreover, IrrE was found to cleave DdrO in vitro and when the proteins were coexpressed in Escherichia coli. This cleavage was not observed in the presence of metal chelator EDTA or when IrrE contains a mutation in the conserved active-site motif of metallopeptidases. In D. deserti, IrrE-dependent cleavage of DdrO was observed after exposure to radiation. Furthermore, DdrO-dependent repression of the promoter of a radiation-induced gene was shown. These results demonstrate that IrrE is a metalloprotease and we propose that IrrE-mediated cleavage inactivates repressor protein DdrO, leading to transcriptional induction of various genes required for repair and survival after exposure of Deinococcus to radiation.
Published: 2014

33. Kinetics of solvent addition on electrosprayed ions in an electrospray source and in a quadrupole ion trap

Author: Olivier Laprévote, Valérie Gabelica, E De Pauwa, and David Lemaire
Subjects: Addition reaction, Electrospray, Chemistry, Kinetics, Analytical chemistry, Condensed Matter Physics, Mass spectrometry, Ion, chemistry.chemical_compound, Ion trap, Physical and Theoretical Chemistry, Quadrupole ion trap, Acetonitrile, Instrumentation, Spectroscopy
Abstract: Benzylpyridinium cations readily fragment in the electrospray source by loss of pyridine to give benzyl cations (M−79). The full-scan spectra obtained with some instruments also show, in addition, an m/z (M−38) peak corresponding to the addition of acetonitrile, being present in the solvent mixture, on the benzyl cations. Here we report that the addition reaction can occur in the source region of electrospray mass spectrometry instruments, and in a quadrupole ion trap. The kinetics of acetonitrile addition was monitored in an ion trap, acetonitrile being provided by leakage from the source, through the heated capillary. For benzyl ions with different substituents, the addition kinetics has been found positively correlated with the Brown parameter σ + of the benzyl radical, and therefore with the effective charge density on the α-carbon atom of the benzyl ion. This is consistent with the Langevin or average-dipole-orientation (ADO) theory of ion–molecule reaction kinetics.
Published: 2001

34. Modulating uranium binding affinity in engineered calmodulin EF-hand peptides: effect of phosphorylation

Author: Catherine Berthomieu, Sandrine Sauge-Merle, Jean-Marc Adriano, Pascale Delangle, Romain Pardoux, David Lemaire, Luc Guilloreau, Centre National de la Recherche Scientifique (CNRS), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Aix Marseille Université (AMU), Biologie végétale et microbiologie environnementale - UMR7265 (BVME), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Interactions Protéine Métal (IPM), This work was funded in part by the 'Toxicologie' program of the Commissariat à l’énergie atomique et aux énergies alternatives (CEA) (CalUMo). RP acknowledges a PhD grant from the CEA international program IRTELIS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript., and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Arabidopsis, lcsh:Medicine, Protein Engineering, Toxicology, Heavy Metals, Biochemistry, 01 natural sciences, Analytical Chemistry, chemistry.chemical_compound, Biological Systems Engineering, Phosphorylation, Biomacromolecule-Ligand Interactions, Threonine, Casein Kinase II, lcsh:Science, Multidisciplinary, biology, Chemistry, EF hand, [SDE.IE]Environmental Sciences/Environmental Engineering, Hydrogen-Ion Concentration, Uranyl, Recombinant Proteins, peptides uranyl binding site phosphoryl group, Dissociation constant, Uranium, Phosphothreonine, Bioremediation, Protein Binding, Research Article, Biotechnology, Pollutants, Calmodulin, Stereochemistry, Toxic Agents, Biophysics, Bioengineering, 010402 general chemistry, Protein Chemistry, Inorganic Chemistry, Environmental Biotechnology, Chemical Biology, Environmental Chemistry, Binding site, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Biology, Bioinorganic Chemistry, Arabidopsis Proteins, 010405 organic chemistry, Cofactors, lcsh:R, Proteins, Phosphorylated Peptide, Protein Structure, Tertiary, 0104 chemical sciences, biology.protein, lcsh:Q
Abstract: To improve our understanding of uranium toxicity, the determinants of uranyl affinity in proteins must be better characterized. In this work, we analyzed the contribution of a phosphoryl group on uranium binding affinity in a protein binding site, using the site 1 EF-hand motif of calmodulin. The recombinant domain 1 of calmodulin from A. thaliana was engineered to impair metal binding at site 2 and was used as a structured template. Threonine at position 9 of the loop was phosphorylated in vitro, using the recombinant catalytic subunit of protein kinase CK2. Hence, the T(9)TKE(12) sequence was substituted by the CK2 recognition sequence TAAE. A tyrosine was introduced at position 7, so that uranyl and calcium binding affinities could be determined by following tyrosine fluorescence. Phosphorylation was characterized by ESI-MS spectrometry, and the phosphorylated peptide was purified to homogeneity using ion-exchange chromatography. The binding constants for uranyl were determined by competition experiments with iminodiacetate. At pH 6, phosphorylation increased the affinity for uranyl by a factor of ∼5, from K(d) = 25±6 nM to K(d) = 5±1 nM. The phosphorylated peptide exhibited a much larger affinity at pH 7, with a dissociation constant in the subnanomolar range (K(d) = 0.25±0.06 nM). FTIR analyses showed that the phosphothreonine side chain is partly protonated at pH 6, while it is fully deprotonated at pH 7. Moreover, formation of the uranyl-peptide complex at pH 7 resulted in significant frequency shifts of the ν(as)(P-O) and ν(s)(P-O) IR modes of phosphothreonine, supporting its direct interaction with uranyl. Accordingly, a bathochromic shift in ν(as)(UO(2))(2+) vibration (from 923 cm(-1) to 908 cm(-1)) was observed upon uranyl coordination to the phosphorylated peptide. Together, our data demonstrate that the phosphoryl group plays a determining role in uranyl binding affinity to proteins at physiological pH.
Published: 2012
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35. Structural Basis of Cytotoxicity Mediated by the Type III Secretion Toxin ExoU from $Pseudomonas\ aeruginosa$

Author: David Lemaire, Ina Attree, Claire Gendrin, Philippe Huber, Andréa Dessen, Sylvie Elsen, Dimitrios A. Skoufias, Carlos Contreras-Martel, Stéphanie Bouillot, Laboratoire Adaptation et pathogénie des micro-organismes [Grenoble] (LAPM), Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Physiopathologie vasculaire : interactions cellulaires, signalisation et vieillissement, Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Biologie du Cancer et de l'Infection (BCI ), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Interactions Protéine Métal (IPM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Biochimie et biophysique des systèmes intégrés (BBSI), Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF), and MARAVAL, Alexandra
Subjects: Phosphatidylinositol 4,5-Diphosphate, Protein Folding, [SDV]Life Sciences [q-bio], Biochemistry, Type three secretion system, Eukaryotic cells, Toxins, Biology (General), Bacterial Secretion Systems, 0303 health sciences, Effector, Transfection, 3. Good health, Transport protein, Cell biology, Enzymes, Bacterial Pathogens, [SDV] Life Sciences [q-bio], Phospholipases, Pseudomonas aeruginosa, Research Article, Endosome, QH301-705.5, Immunology, Bacterial Toxins, Biophysics, Biology, Microbiology, 03 medical and health sciences, Structure-Activity Relationship, Secretion systems, Bacterial Proteins, Virology, Genetics, Humans, Secretion, Pseudomonas Infections, Molecular Biology, 030304 developmental biology, Membrane potential, 030306 microbiology, Ubiquitination, Proteins, RC581-607, Protein Structure, Tertiary, Cell membranes, Cytoplasm, Chaperone (protein), biology.protein, Parasitology, Immunologic diseases. Allergy, HeLa Cells, Molecular Chaperones
Abstract: The type III secretion system (T3SS) is a complex macromolecular machinery employed by a number of Gram-negative pathogens to inject effectors directly into the cytoplasm of eukaryotic cells. ExoU from the opportunistic pathogen Pseudomonas aeruginosa is one of the most aggressive toxins injected by a T3SS, leading to rapid cell necrosis. Here we report the crystal structure of ExoU in complex with its chaperone, SpcU. ExoU folds into membrane-binding, bridging, and phospholipase domains. SpcU maintains the N-terminus of ExoU in an unfolded state, required for secretion. The phospholipase domain carries an embedded catalytic site whose position within ExoU does not permit direct interaction with the bilayer, which suggests that ExoU must undergo a conformational rearrangement in order to access lipids within the target membrane. The bridging domain connects catalytic domain and membrane-binding domains, the latter of which displays specificity to PI(4,5)P2. Both transfection experiments and infection of eukaryotic cells with ExoU-secreting bacteria show that ExoU ubiquitination results in its co-localization with endosomal markers. This could reflect an attempt of the infected cell to target ExoU for degradation in order to protect itself from its aggressive cytotoxic action., Author Summary Pseudomonas aeruginosa is a leading cause of nosocomial infections and is a particular threat for cystic fibrosis and immunodepressed patients. One of the most aggressive toxins in its arsenal is ExoU, injected directly into target cells by a needle-like complex located on the surface of the bacterium, the type III secretion system. P. aeruginosa strains that express ExoU cause rapid cell death as a consequence of the membrane-destruction (phospholipase) potential of the toxin. In this work, we report the three-dimensional structure of ExoU in complex with a partner molecule, SpcU. ExoU contains three distinct regions, and the fold suggests how ExoU binds to the membrane or other molecules within the target cell and becomes activated. In addition, we also show that once it is translocated into the cell, ExoU co-localizes with intracellular organelles of the endosomal pathway, potentially in an attempt of the target cell to destroy the toxin. This work provides new insight into the cellular destruction mechanism of this aggressive toxin and could be a basis for the development of new inhibitors of P. aeruginosa pathogenesis.
Published: 2012

36. Crystallographic snapshots of iterative substrate translocations during nicotianamine synthesis in Archaea

Author: Pascal Arnoux, Stéphane Mari, David Pignol, Cyril Dreyfus, David Lemaire, Microbiologie Environnementale et Moléculaire (MEM), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Biochimie et Physiologie Moléculaire des Plantes (BPMP), Université de Montpellier (UM)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro)-Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Institut National de la Recherche Agronomique (INRA)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Institut National de la Recherche Agronomique (INRA)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), and Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)
Subjects: Models, Molecular, 0106 biological sciences, Protein Folding, S-Adenosylmethionine, MESH: Sequence Homology, Amino Acid, Azetidine, MESH: Protein Structure, Secondary, Crystal structure, MESH: Amino Acid Sequence, NICOTIANAMINE, METAL HOMEOSTASIS, REACTION MECHANISM, BIOLOGIE DU DEVELOPPEMENT, BIOLOGIE MOLECULAIRE, Crystallography, X-Ray, 01 natural sciences, Mass Spectrometry, Protein Structure, Secondary, Substrate Specificity, Nicotianamine synthase, structure moléculaire, chemistry.chemical_compound, MESH: Protein Structure, Tertiary, Transferase, Nicotianamine, MESH: Cyclization, 0303 health sciences, Multidisciplinary, Molecular Structure, biology, plants, MESH: Models, Chemical, MESH: Archaeal Proteins, Biological Sciences, Small molecule, MESH: Azetidinecarboxylic Acid, synthase, Azetidinecarboxylic Acid, MESH: Models, Molecular, Methanobacteriaceae, MESH: Mutation, metal, Stereochemistry, Archaeal Proteins, MESH: Protein Folding, Molecular Sequence Data, MESH: Molecular Structure, cloning, arabidopsis-thaliana, MESH: Alkyl and Aryl Transferases, Catalysis, physiologie végétale, 03 medical and health sciences, nickel, [SDV.BV]Life Sciences [q-bio]/Vegetal Biology, Amino Acid Sequence, transport membranaire, MESH: Hydrogen Bonding, Binding site, 030304 developmental biology, MESH: Mass Spectrometry, Alkyl and Aryl Transferases, Binding Sites, MESH: Molecular Sequence Data, Sequence Homology, Amino Acid, MESH: Methanobacteriaceae, iron uptake, ACL, Active site, crystal-structure, MESH: S-Adenosylmethionine, Hydrogen Bonding, reaction mechanism converting-enzyme-inhibitor, MESH: Catalysis, MESH: Crystallography, X-Ray, Protein Structure, Tertiary, arabidopsis, Crystallography, Models, Chemical, chemistry, MESH: Binding Sites, Cyclization, Mutation, transport, biology.protein, metal homeostasis, MESH: Substrate Specificity, 010606 plant biology & botany
Abstract: Nicotianamine (NA), a small molecule ubiquitous in plants, is an important divalent metal chelator and the main precursor of phytosiderophores. Nicotianamine synthase (NAS) is the enzyme catalyzing NA synthesis by the condensation of three aminopropyl moieties of S-adenosylmethionine (SAM) and the cyclization of one of them to form an azetidine ring. Here we report five crystal structures of an archaeal NAS from Methanothermobacter thermautotrophicus , either free or in complex with its product(s) and substrate(s). These structures reveal a two-domains fold arrangement of MtNAS, a small molecule related to NA (named here thermoNicotianamine or tNA), and an original mechanism of synthesis in a buried reaction chamber. This reaction chamber is open to the solvent through a small inlet, and a single active site allows the selective entrance of only one substrate at a time that is then processed and translocated stepwise.
Published: 2009

37. Structural characterization of a putative endogenous metal chelator in the periplasmic nickel transporter NikA

Author: Christine Cavazza, David Lemaire, Mickaël V. Cherrier, Juan C. Fontecilla-Camps, and Constance Bochot
Subjects: chemistry.chemical_classification, Hydrogenase, Ligand, Stereochemistry, Escherichia coli Proteins, chemistry.chemical_element, Periplasmic space, medicine.disease_cause, Crystallography, X-Ray, Biochemistry, Metal Chelator, Protein Structure, Secondary, Metal, Nickel, Enzyme, chemistry, visual_art, Periplasm, visual_art.visual_art_medium, medicine, ATP-Binding Cassette Transporters, Escherichia coli, Chelating Agents
Abstract: Escherichia coli and related bacteria require nickel for the synthesis of hydrogenases, enzymes involved in hydrogen oxidation and proton reduction. Nickel transport to the cytoplasm depends on five proteins, NikA-E. We have previously reported the three-dimensional structure of the soluble periplasmic nickel transporter NikA in a complex with FeEDTA(H 2O) (-). We have now determined the structure of EDTA-free NikA and have found that it binds a small organic molecule that contributes three ligands to the coordination of a transition metal ion. Unexpectedly, His416, which was far from the metal-binding site in the FeEDTA(H 2O) (-)-NikA complex, becomes the fourth observed ligand to the metal. The best match to the omit map electron density is obtained for butane-1,2,4-tricarboxylate (BTC). Our attempts to obtain a BTC-Ni-NikA complex using apo protein and commercial reagents resulted in nickel-free BTC-NikA. Overall, our results suggest that nickel transport in vivo requires a specific metallophore that may be BTC.
Published: 2008

38. Trapping of an acyl-enzyme intermediate in a penicillin-binding protein (PBP)-catalyzed reaction

Author: Pauline, Macheboeuf, David, Lemaire, Nathalie, Teller, Alexandre Dos Santos, Martins, André, Luxen, Otto, Dideberg, Marc, Jamin, and Andréa, Dessen
Subjects: Models, Molecular, Penicillin binding proteins, Glycosylation, Stereochemistry, Protein Conformation, Acylation, Static Electricity, Peptide, Thioester, Crystallography, X-Ray, Ligands, Spectrum Analysis, Raman, Catalysis, Mass Spectrometry, Protein Structure, Secondary, Substrate Specificity, chemistry.chemical_compound, Bacterial Proteins, Structural Biology, polycyclic compounds, Penicillin-Binding Proteins, Molecular Biology, chemistry.chemical_classification, Alanine, Binding Sites, biology, Lipid II, Molecular Structure, Hydrolysis, Active site, Hydrogen Bonding, biochemical phenomena, metabolism, and nutrition, Protein Structure, Tertiary, Molecular Weight, Kinetics, Streptococcus pneumoniae, chemistry, Models, Chemical, Covalent bond, biology.protein, Peptidoglycan, Oxyanion hole, Hydrophobic and Hydrophilic Interactions, Protein Binding
Abstract: Class A penicillin-binding proteins (PBPs) catalyze the last two steps in the biosynthesis of peptidoglycan, a key component of the bacterial cell wall. Both reactions, glycosyl transfer (polymerization of glycan chains) and transpeptidation (cross-linking of stem peptides), are essential for peptidoglycan stability and for the cell division process, but remain poorly understood. The PBP-catalyzed transpeptidation reaction is the target of β-lactam antibiotics, but their vast employment worldwide has prompted the appearance of highly resistant strains, thus requiring concerted efforts towards an understanding of the transpeptidation reaction with the goal of developing better antibacterials. This goal, however, has been elusive, since PBP substrates are rapidly deacylated. In this work, we provide a structural snapshot of a “trapped” covalent intermediate of the reaction between a class A PBP with a pseudo-substrate, N-benzoyl- d -alanylmercaptoacetic acid thioester, which partly mimics the stem peptides contained within the natural, membrane-associated substrate, lipid II. The structure reveals that the d -alanyl moiety of the covalent intermediate (N-benzoyl- d -alanine) is stabilized in the cleft by a network of hydrogen bonds that place the carbonyl group in close proximity to the oxyanion hole, thus mimicking the spatial arrangement of β-lactam antibiotics within the PBP active site. This arrangement allows the target bond to be in optimal position for attack by the acceptor peptide and is similar to the structural disposition of β-lactam antibiotics with PBP clefts. This information yields a better understanding of PBP catalysis and could provide key insights into the design of novel PBP inhibitors.
Published: 2007

39. The marine natural product adociasulfate-2 as a tool to identify the MT-binding region of kinesins

Author: David Lemaire, Elisabeth Hewat, Frank Kozielski, Sébastien Brier, Salvatore DeBonis, and Eugénie Carletti
Subjects: Chromosomal Proteins, Non-Histone, Dynein, Molecular Sequence Data, Kinesin 13, Kinesins, macromolecular substances, Biology, Sulfuric Acid Esters, Xenopus Proteins, Biochemistry, Microtubules, Motor protein, Xenopus laevis, Microtubule, ATP hydrolysis, Molecular motor, Animals, Drosophila Proteins, Humans, Amino Acid Sequence, Protein secondary structure, Adenosine Triphosphatases, Binding Sites, Haliclona, Drosophila melanogaster, Biophysics, Kinesin, Cattle
Abstract: Kinesins are molecular motors that transport cargo along microtubules (MTs). To move forward the motor must attach to the MT in a defined orientation and detach from it in a process that is driven by ATP hydrolysis. The knowledge of the motor-MT interface is essential for a detailed understanding of how kinesins move along MTs and how they are related to other molecular motors such as myosins or dyneins. We have used the marine natural product adociasulfate-2 (AS-2), previously identified as a MT-competitive inhibitor of conventional kinesin, to infer the secondary structure elements forming the MT interface of two human mitotic kinesins, namely, CENP-E and Eg5. AS-2 inhibits both basal and MT-stimulated ATPase activities of CENP-E (IC50 of 8.6 and 1.3 microM, respectively) and Eg5 (IC50 of 3.5 and 5.3 microM, respectively) and is a MT-competitive inhibitor of CENP-E with a Ki of 0.35 microM. Binding of AS-2 to CENP-E also stimulates the ADP release from the nucleotide-binding pocket. AS-2 is a nonspecific kinesin inhibitor targeting several superfamily members including KHC, MPP1, MKLP1, RabK6, KIFC1, KIFC3, CENP-E, and Eg5. By measuring hydrogen/deuterium exchange with mass spectrometry we have shown that the formation of the CENP-E/AS-2 complex decreases the solvent accessibility of three neighboring peptides on the same face of CENP-E. We deduce that this is the site of MT attachment and conclude that loop L11, helix alpha4, loop L12, helix alpha5, loop L8, and strand beta5 constitute the main MT interface of the CENP-E motor domain. Similarly for Eg5/AS-2, a region of increased solvent accessibility locates the MT interface of Eg5.
Published: 2006

40. Molecular dissection of the inhibitor binding pocket of mitotic kinesin Eg5 reveals mutants that confer resistance to antimitotic agents

Author: Salvatore DeBonis, David Lemaire, Eric Forest, Sébastien Brier, and Frank Kozielski
Subjects: Models, Molecular, Mutant, Molecular Sequence Data, Drug Resistance, Kinesins, Mitosis, Plasma protein binding, Biology, Antimitotic Agents, Mass Spectrometry, chemistry.chemical_compound, Structural Biology, Microtubule, Point Mutation, Amino Acid Sequence, Cysteine, Binding site, Molecular Biology, Binding Sites, Thiones, Kinetics, Monastrol, Pyrimidines, Biochemistry, chemistry, Mutagenesis, Kinesin, Antimitotic Agent, Protein Binding
Abstract: The mitotic kinesin Eg5 plays an essential role in establishing the bipolar spindle. Recently, several antimitotic inhibitors have been shown to share a common binding region on Eg5. Considering the importance of Eg5 as a potential drug target for cancer chemotherapy it is essential to understand the molecular mechanism, by which these agents block Eg5 activity, and to determine the "key residues" crucial for inhibition. Eleven residues in the inhibitor binding pocket were mutated and the effects were monitored by kinetic analysis and mass spectrometry. Mutants R119A, D130A, P131A, I136A, V210A, Y211A and L214A abolish the inhibitory effect of monastrol. Results for W127A and R221A are less striking, but inhibitor constants are still considerably modified compared to wild-type Eg5. Only one residue, Leu214, was found to be essential for inhibition by STLC. W127A, D130A, V210A lead to increased K(i)(app) values, but binding of STLC is still tight. R119A, P131A, Y211A and R221A convert STLC into a classical rather than a tight-binding inhibitor with increased inhibitor constants. These results demonstrate that monastrol and STLC interact with different amino acids within the same binding region, suggesting that this site is highly flexible to accommodate different types of inhibitors. The drug specificity is due to multiple interactions not only with loop L5, but also with residues located in helices alpha2 and alpha3. These results suggest that tumour cells might develop resistance to Eg5 inhibitors, by expressing Eg5 point mutants that retain the enzyme activity, but prevent inhibition, a feature that is observed for certain tubulin inhibitors.
Published: 2005

41. The PscE-PscF-PscG complex controls type III secretion needle biogenesis in Pseudomonas aeruginosa

Author: Sylvie Elsen, Emmanuelle Neumann, Andréa Dessen, Jacqueline Chabert, Eric Faudry, David Lemaire, Manuelle Quinaud, Alexandrine Pastor, Ina Attree, Laboratoire des Protéines Membranaires ( LPM ), Institut de biologie structurale ( IBS - UMR 5075 ), Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Centre National de la Recherche Scientifique ( CNRS ) -Université Grenoble Alpes ( UGA ) -Université Joseph Fourier - Grenoble 1 ( UJF ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Centre National de la Recherche Scientifique ( CNRS ) -Université Grenoble Alpes ( UGA ) -Université Joseph Fourier - Grenoble 1 ( UJF ), Biochimie et biophysique des systèmes intégrés ( BBSI ), Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Joseph Fourier - Grenoble 1 ( UJF ), Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Centre National de la Recherche Scientifique ( CNRS ) -Université Grenoble Alpes ( UGA ) -Université Joseph Fourier - Grenoble 1 ( UJF ), Laboratoire de Chimie et de Biochimie Pharmacologiques et Toxicologiques ( LCBPT - UMR 8601 ), Université Paris Descartes - Paris 5 ( UPD5 ) -Centre National de la Recherche Scientifique ( CNRS ), Laboratoire des Protéines Membranaires (LPM), Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Biochimie et biophysique des systèmes intégrés (BBSI), Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF), Laboratoire de Chimie et de Biochimie Pharmacologiques et Toxicologiques (LCBPT - UMR 8601), Université Paris Descartes - Paris 5 (UPD5)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie structurale (IBS - UMR 5075), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS), and Université Paris Descartes - Paris 5 (UPD5)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Cytoplasm, Circular dichroism, Time Factors, MESH : Molecular Sequence Data, Polymers, Protein Conformation, Mutant, MESH: Amino Acid Sequence, MESH : Blotting, Western, [ SDV.MP.BAC ] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, medicine.disease_cause, Biochemistry, Mass Spectrometry, MESH: Membrane Transport Proteins, MESH: Circular Dichroism, MESH: Protein Conformation, MESH : Membrane Transport Proteins, MESH: Genetic Vectors, MESH : Bacterial Proteins, Transgenes, MESH: Bacterial Proteins, MESH : Temperature, MESH : Immunoblotting, MESH : Protein Conformation, 0303 health sciences, MESH: Immunoblotting, Circular Dichroism, MESH : Amino Acid Sequence, MESH : Antigens, Bacterial, Temperature, MESH : Pseudomonas aeruginosa, MESH: Temperature, MESH : Bacterial Physiological Phenomena, MESH : Genetic Vectors, Cell biology, MESH: Polymers, Pseudomonas aeruginosa, MESH: Pseudomonas aeruginosa, Intercellular Signaling Peptides and Proteins, MESH: Molecular Chaperones, MESH : Mutation, MESH : Transgenes, MESH : Carrier Proteins, Bacterial Outer Membrane Proteins, MESH : Time Factors, MESH : Cytoplasm, MESH: Mutation, Protein subunit, Blotting, Western, Genetic Vectors, Immunoblotting, Molecular Sequence Data, MESH: Transgenes, MESH: Carrier Proteins, MESH: Microscopy, Electron, Biology, Bacterial Physiological Phenomena, 03 medical and health sciences, MESH : Mass Spectrometry, Bacterial Proteins, medicine, MESH: Blotting, Western, Secretion, Amino Acid Sequence, MESH : Polymers, MESH : Bacterial Outer Membrane Proteins, Molecular Biology, 030304 developmental biology, MESH: Mass Spectrometry, Antigens, Bacterial, MESH: Molecular Sequence Data, 030306 microbiology, MESH: Cytoplasm, MESH: Bacterial Outer Membrane Proteins, MESH: Time Factors, MESH : Molecular Chaperones, Membrane Transport Proteins, MESH : Circular Dichroism, Cell Biology, biology.organism_classification, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, MESH : Microscopy, Electron, Microscopy, Electron, MESH: Bacterial Physiological Phenomena, Mutation, Carrier Proteins, Bacteria, Biogenesis, MESH: Antigens, Bacterial, Molecular Chaperones
Abstract: International audience; Type III secretion (T3S) systems play key roles in pathogenicity of many Gram-negative bacteria and are employed to inject toxins directly into the cytoplasm of target cells. They are composed of over 20 different proteins that associate into a basal structure that traverses both inner and outer bacterial membranes and a hollow, needle-like structure through which toxins travel. The PscF protein is the main component of the Pseudomonas aeruginosa T3S needle. Here we demonstrate that PscF, when purified on its own, is able to form needle-like fibers of 8 nm in width and >1 microm in length. In addition, we demonstrate for the first time that the T3S needle subunit requires two cytoplasmic partners, PscE and PscG, in P. aeruginosa, which trap PscF in a ternary, 1:1:1 complex, thus blocking it in a monomeric state. Knock-out mutants deficient in PscE and PscG are non-cytotoxic, lack PscF, and are unable to export PscF encoded extrachromosomally. Temperature-scanning circular dichroism measurements show that the PscE-PscF-PscG complex is thermally stable and displays a cooperative unfolding/refolding pattern. Thus, PscE and PscG prevent PscF from polymerizing prematurely in the P. aeruginosa cytoplasm and keep it in a secretion prone conformation, strategies which may be shared by other pathogens that employ the T3S system for infection.
Published: 2005

42. Crystal structure of phosphorylcholine esterase domain of the virulence factor choline-binding protein e from streptococcus pneumoniae: new structural features among the metallo-beta-lactamase superfamily

Author: Gianpiero, Garau, David, Lemaire, Thierry, Vernet, Otto, Dideberg, and Anne Marie, Di Guilmi
Subjects: Models, Molecular, Binding Sites, Streptococcus pneumoniae, Bacterial Proteins, Virulence, Protein Conformation, Catalytic Domain, Molecular Sequence Data, Amino Acid Sequence, Crystallography, X-Ray, Carboxylic Ester Hydrolases
Abstract: Streptococcus pneumoniae is the worldwide leading cause of deaths from invasive infections such as pneumoniae, sepsis, and meningitidis in children and the elderly. Nasopharyngeal colonization, which plays a key role in the development of pneumococcal disease, is highly dependent on a family of surface-exposed proteins, the choline-binding proteins (CBPs). Here we report the crystal structure of phosphorylcholine esterase (Pce), the catalytic domain of choline-binding protein E (CBPE), which has been shown to be crucial for host/pathogen interaction processes. The unexpected features of the Pce active site reveal that this enzyme is unique among the large family of hydrolases harboring the metallo-beta-lactamase fold. The orientation and calcium stabilization features of an elongated loop, which lies on top of the active site, suggest that the cleft may be rearranged. Furthermore, the structure of Pce complexed with phosphorylcholine, together with the characterization of the enzymatic role played by two iron ions located in the active site allow us to propose a reaction mechanism reminiscent of that of purple acid phosphatase. This mechanism is supported by site-directed mutagenesis experiments. Finally, the interactions of the choline binding domain and the Pce region of CBPE with chains of teichoic acids have been modeled. The ensemble of our biochemical and structural results provide an initial understanding of the function of CBPE.
Published: 2005

43. S-trityl-L-cysteine Inhibition of the mitotic kinesin Eg5

Author: Salvatore DeBonis, David Lemaire, Eric Forest, Sébastien Brier, and Frank Kozielski
Subjects: Biochemistry, Chemistry, Kinesin, S-Trityl-L-Cysteine, Kinesin 8, Mitosis
Published: 2005

44. Structural characterization of the feline-immunodeficiency-virus envelope glycoprotein 36 ectodomain for the development of new antivirals

Author: Benoît Chatrenet, Florestan Desmaris, David Lemaire, Eric Forest, Sylvie Ricard-Blum, and Deleage, Gilbert
Subjects: Feline immunodeficiency virus, Enfuvirtide, viruses, Biology, Immunodeficiency Virus, Feline, Gp41, Biochemistry, Antiviral Agents, Virus, Viral envelope, Viral Envelope Proteins, medicine, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acid Sequence, Molecular Biology, Peptide sequence, chemistry.chemical_classification, Binding Sites, Sequence Homology, Amino Acid, virus diseases, Cell Biology, biology.organism_classification, Virology, HIV Envelope Protein gp41, Protein Structure, Tertiary, Kinetics, Ectodomain, chemistry, Glycoprotein, Peptides, medicine.drug, Research Article
Abstract: In the fight against the human HIV, new targets are being explored, such as the proteins involved in the process of fusion of the virus with the host cell. Recently, the first generation of fusion inhibitors (enfuvirtide), targeting gp41 (virus envelope glycoprotein 41), has become commercially available. However, this promising class of drugs has to be improved in respect of their efficacy and bioavailability. Considering the strong homologies between HIV and FIV (feline immunodeficiency virus), as well as the highly conserved structure of the transmembrane envelope protein among species, FIV represents a relevant model of pre-screening studies for HIV. Taking into account (i) sequence homologies between the ectodomain of HIV gp41 and FIV gp36 (envelope glycoprotein 36), (ii) structural data available for gp41 and (iii) the fact that synthetic peptides derived from gp36 are effective inhibitors of FIV infection, we designed several peptides derived from gp36 sequence. We checked that these peptides had the same structural features as the corresponding peptides from gp41 HIV by CD, analytical ultracentrifugation and 1H-2H (hydrogen-deuterium) exchange combined with MS. By combining this latter technique with surface-plasmon-resonance assays, we identified the amino acid residues of the C-terminal region of the ectodomain of gp36 that are critical for interaction with the N-terminal region. This gave clues for therapy and vaccines against FIV, thus providing helpful data for treatments against HIV.In the fight against the human HIV, new targets are being explored, such as the proteins involved in the process of fusion of the virus with the host cell. Recently, the first generation of fusion inhibitors (enfuvirtide), targeting gp41 (virus envelope glycoprotein 41), has become commercially available. However, this promising class of drugs has to be improved in respect of their efficacy and bioavailability. Considering the strong homologies between HIV and FIV (feline immunodeficiency virus), as well as the highly conserved structure of the transmembrane envelope protein among species, FIV represents a relevant model of pre-screening studies for HIV. Taking into account (i) sequence homologies between the ectodomain of HIV gp41 and FIV gp36 (envelope glycoprotein 36), (ii) structural data available for gp41 and (iii) the fact that synthetic peptides derived from gp36 are effective inhibitors of FIV infection, we designed several peptides derived from gp36 sequence. We checked that these peptides had the same structural features as the corresponding peptides from gp41 HIV by CD, analytical ultracentrifugation and 1H-2H (hydrogen-deuterium) exchange combined with MS. By combining this latter technique with surface-plasmon-resonance assays, we identified the amino acid residues of the C-terminal region of the ectodomain of gp36 that are critical for interaction with the N-terminal region. This gave clues for therapy and vaccines against FIV, thus providing helpful data for treatments against HIV.
Published: 2005

45. Identification of the protein binding region of S-trityl-L-cysteine, a new potent inhibitor of the mitotic kinesin Eg5

Author: Eric Forest, Sébastien Brier, David Lemaire, Frank Kozielski, and Salvatore DeBonis
Subjects: Spectrometry, Mass, Electrospray Ionization, Genetic Vectors, Molecular Sequence Data, Kinesins, Mitosis, Antineoplastic Agents, Plasma protein binding, Biology, Biochemistry, Neurospora crassa, chemistry.chemical_compound, Protein structure, Humans, Amino Acid Sequence, Cysteine, Binding site, Adenosine Triphosphatases, Molecular Motor Proteins, Deuterium Exchange Measurement, Thiones, biology.organism_classification, Growth Inhibitors, Peptide Fragments, Spindle apparatus, Cell biology, Protein Structure, Tertiary, Monastrol, Pyrimidines, chemistry, Kinesin, Protein Binding
Abstract: Human Eg5, a mitotic motor of the kinesin superfamily, is involved in the formation and maintenance of the mitotic spindle. The recent discovery of small molecules that inhibit HsEg5 by binding to its catalytic motor domain leading to mitotic arrest has attracted more interest in Eg5 as a potential anticancer drug target. We have used hydrogen-deuterium exchange mass spectrometry and directed mutagenesis to identify the secondary structure elements that form the binding sites of new Eg5 inhibitors, in particular for S-trityl-l-cysteine, a potent inhibitor of Eg5 activity in vitro and in cell-based assays. The binding of this inhibitor modifies the deuterium incorporation rate of eight peptides that define two areas within the motor domain: Tyr125-Glu145 and Ile202-Leu227. Replacement of the Tyr125-Glu145 region with the equivalent region in the Neurospora crassa conventional kinesin heavy chain prevents the inhibition of the Eg5 ATPase activity by S-trityl-l-cysteine. We show here that S-trityl-l-cysteine and monastrol both bind to the same region on Eg5 by induced fit in a pocket formed by helix alpha3-strand beta5 and loop L5-helix alpha2, and both inhibitors trigger similar local conformational changes within the interaction site. It is likely that S-trityl-l-cysteine and monastrol inhibit HsEg5 by a similar mechanism. The common inhibitor binding region appears to represent a "hot spot" for HsEg5 that could be exploited for further inhibitor screening.
Published: 2004

46. Oligomerization of type III secretion proteins PopB and PopD precedes pore formation in Pseudomonas

Author: Winfried Weissenhorn, Anne Marie Di Guilmi, Andréa Dessen, Ina Attree, David Lemaire, and Guy Schoehn
Subjects: Pore Forming Cytotoxic Proteins, Molecular Sequence Data, medicine.disease_cause, General Biochemistry, Genetics and Molecular Biology, Bacterial Proteins, Pseudomonas, medicine, Secretion, Amino Acid Sequence, Molecular Biology, General Immunology and Microbiology, biology, Pseudomonas aeruginosa, General Neuroscience, Cell Membrane, Temperature, Articles, Translocon, biology.organism_classification, Cell biology, Microscopy, Electron, Membrane, Membrane protein, Chaperone (protein), Host cell cytoplasm, Liposomes, biology.protein, Sequence Alignment
Abstract: Pseudomonas aeruginosa is the agent of opportunistic infections in immunocompromised individuals and chronic respiratory illnesses in cystic fibrosis patients. Pseudomonas aeruginosa utilizes a type III secretion system for injection of toxins into the host cell cytoplasm through a channel on the target membrane (the 'translocon'). Here, we have functionally and structurally characterized PopB and PopD, membrane proteins implicated in the formation of the P.aeruginosa translocon. PopB and PopD form soluble complexes with their common chaperone, PcrH, either as stable heterodimers or as metastable heterooligomers. Only oligomeric forms are able to bind to and disrupt cholesterol-rich membranes, which occurs within a pH range of 5-7 in the case of PopB/PcrH, and only at acidic pH for PcrH-free PopD. Electron microscopy reveals that upon membrane association PopB and PopD form 80 A wide rings which encircle 40 A wide cavities. Thus, formation of metastable oligomers precedes membrane association and ring generation in the formation of the Pseudomonas translocon, a mechanism which may be similar for other pathogens that employ type III secretion systems.
Published: 2003

47. Active glottal closure during anoxic gasping in lambs

Author: David Lemaire, François Thuot, Dominique Dorion, Patrick Létourneau, and Jean-Paul Praud
Subjects: Pulmonary and Respiratory Medicine, Glottis, Physiology, Hemodynamics, Blood Pressure, Biology, Heart Rate, Respiration, medicine, Animals, Lung volumes, Hypoxia, Lung, Eupnea, Sheep, Electromyography, Apnea, Electroencephalography, respiratory system, Carbon Dioxide, Respiratory Muscles, Oxygen, medicine.anatomical_structure, Animals, Newborn, Anesthesia, Laryngeal Muscle, Lactates, Respiratory Mechanics, Arterial blood, Ketamine, medicine.symptom, Blood Gas Analysis, Lung Volume Measurements, Excitatory Amino Acid Antagonists
Abstract: The present study was aimed at assessing laryngeal dynamics and their consequences during anoxic gasping in ketamine-sedated lambs. We first verified that the glottis was closed between gasps during anoxic gasping in seven chronically instrumented lambs, aged 11-15 days. Recording of glottal constrictor muscle electrical activity, subglottal pressure and lung volume, together with endoscopic observation, confirmed the presence of active glottal closure with maintenance of a high lung volume between gasps. Secondly, we tested whether maintenance of a high lung volume between gasps improved autoresuscitation efficiency. Six sedated lambs aged 8-11 days underwent two anoxic runs, including one with an open tracheostomy to prevent maintenance of a high lung volume. Access back to air was allowed for gasping. No significant difference was found in time to eupnea resumption, hemodynamic parameters or arterial blood gases. We conclude that a high lung volume is actively maintained by glottal closure between anoxic gasps in sedated lambs. Further studies are however needed to define the importance of laryngeal dynamics during gasping.
Published: 2002

48. Stabilization of gas-phase noncovalent macromolecular complexes in electrospray mass spectrometry using aqueous triethylammonium bicarbonate buffer

Author: Laurent Serani, Gerald Marie, David Lemaire, and Olivier Laprévote
Subjects: Electrospray, Spectrometry, Mass, Electrospray Ionization, Chromatography, Aqueous solution, Chemistry, Electrospray ionization, Inorganic chemistry, Proteins, Buffer solution, Buffers, Mass spectrometry, Analytical Chemistry, chemistry.chemical_compound, Bicarbonates, Ammonium bicarbonate, Mass spectrum, Indicators and Reagents, Macromolecule
Abstract: The use of triethylammonium bicarbonate (TEAB) solution in electrospray mass spectrometry proved to be a very efficient way for studying proteins or noncovalent protein complexes under “nondenaturing” conditions. The low charge states observed in the mass spectra improve the separation of ions arising from macromolecular species of close masses. Moreover, the multiply charged ions generated in a TEAB solution are significantly more stable than those formed under more conventional conditions (for example, with ammonium bicarbonate or acetate solution). The analytical interest of TEAB for the analysis of macromolecular species that can easily dissociate in the gas phase, such as hemoglobin or other macromolecular noncovalent complexes, is demonstrated.
Published: 2001

49. Crystallographic snapshots of iterative substrate translocations during nicotianamine synthesis in archaea

Author: David Pignol, David Lemaire, Pascal Arnoux, and Cyril Dreyfus
Subjects: Crystallography, biology, Structural Biology, Chemistry, Substrate (chemistry), Nicotianamine synthesis, biology.organism_classification, Archaea
Published: 2009

50. Corrigendum to 'Trapping of an Acyl-Enzyme Intermediate in a Penicillin-Binding Protein (PBP)-Catalyzed Reaction' [J. Mol. Biol. 376 (2008) 405–413]

Author: Pauline Macheboeuf, Andréa Dessen, David Lemaire, Otto Dideberg, Marc Jamin, and Alexandre Martins
Subjects: chemistry.chemical_classification, Crystallography, Penicillin binding proteins, Enzyme, Structural Biology, Chemistry, Stereochemistry, Molecular Biology, Catalysis
Abstract: Institut de Biologie Structurale Jean-Pierre Ebel, UMR 5075 (CEA, CNRS, UJF, PSB), 41 rue Jules Horowitz, F-38027 Grenoble, France Laboratoire des Interactions Proteine Metal, CEA-Cadarache, IBEB/SBVME, Bât 185, 13108 Saint Paul-lezDurance, France Unit of Virus Host Cell Interactions, UMR 5233 UJF-EMBL-CNRS, 6 rue Jules Horowitz, 38042 Grenoble Cedex 9, France Universite de Liege, Centre de recherches du Cyclotron B30, 4000 Liege, Belgium
Published: 2008

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