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22 results on '"Laboratoire d’Enzymologie et Repliement des Protéines"'

1. The Role of Active Site Flexible Loops in Catalysis and of Zinc in Conformational Stability of Bacillus cereus 569/H/9 β-Lactamase*

Author

Andreas Ioannis Karsisiotis, Andr eacute Matagne, Micha eumll Nigen, Christina Redfield, Gordon C. K. Roberts, Nicolas Willet, Caroline Montagner, Christian Damblon, Mireille Dumoulin, Olivier Jacquin, Laboratoire d'Enzymologie et Repliement des Protéines, Centre d'Ingénierie des Protéines, Université de Liège, Ingénierie des Agro-polymères et Technologies Émergentes (UMR IATE), Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), 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)-Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Université Montpellier 2 - Sciences et Techniques (UM2)-Université de Montpellier (UM)-Institut National de la Recherche Agronomique (INRA), School of Biological Sciences, University of Essex, Henry Wellcome Laboratories of Structural Biology, Department of Molecular and Cell Biology [Leicester], University of Leicester-University of Leicester, Département de Chimie, Department of Biochemistry, University of Oxford [Oxford], Laboratoire d'Enzymologie et Repliement des Protéines, Centre d'Ingénierie des Protéines, Liege, Belgique, Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de la Recherche Agronomique (INRA)-Université Montpellier 2 - Sciences et Techniques (UM2)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Université de Montpellier (UM)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), University of Oxford, and Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro)-Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Université Montpellier 2 - Sciences et Techniques (UM2)-Université de Montpellier (UM)-Institut National de la Recherche Agronomique (INRA)

Subjects
0301 basic medicine, Stereochemistry, [SDV]Life Sciences [q-bio], chemistry.chemical_element, Zinc, 010402 general chemistry, 01 natural sciences, Biochemistry, Protein Structure, Secondary, beta-Lactamases, Catalysis, Enzyme catalysis, 03 medical and health sciences, symbols.namesake, Bacillus cereus, Catalytic Domain, Molecular Biology, Protein Unfolding, chemistry.chemical_classification, biology, Chemistry, Active site, Cell Biology, Nuclear magnetic resonance spectroscopy, 0104 chemical sciences, Gibbs free energy, Crystallography, 030104 developmental biology, Enzyme, Protein Structure and Folding, biology.protein, symbols, Protein folding, Hydrophobic and Hydrophilic Interactions
Abstract

Metallo-β-lactamases catalyse the hydrolysis of most β-lactam antibiotics and hence represent a major clinical concern. The development of inhibitors for these enzymes is complicated by the diversity and flexibility of their substrate binding sites, motivating research into their structure and function. In this study, we examined the conformational properties of the Bacillus cereus β-lactamase II in the presence of chemical denaturants using a variety of biochemical and biophysical techniques. The apoenzyme was found to unfold cooperatively, with a Gibbs free energy of stabilization (∆G°) of 32 ± 2 kJ·mol^−1 . For holoBcII, a first noncooperative transition leads to multiple interconverting native-like states, in which both zinc atoms remain bound in an apparently unaltered active site and the protein displays a well-organized compact hydrophobic core with structural changes confined to the enzyme surface, but with no catalytic activity. 2D NMR data revealed that the loss of activity occurs concomitantly with perturbations in two loops that border the enzyme active site. A second cooperative transition, corresponding to global unfolding, is observed at higher denaturant concentrations, with ∆G° value of 65 ± 1.4 kJ·mol^−1 . These combined data highlight the importance of the two zinc ions in maintaining structure as well as a relatively well-defined conformation for both active site loops in order to maintain enzymatic activity.

Published
2016

2. SPOR Proteins Are Required for Functionality of Class A Penicillin-Binding Proteins in Escherichia coli

Author

Pazos, Manuel, Peters, Katharina, Boes, Adrien, Safaei, Yalda, Kenward, Calem, Caveney, Nathanael A., Laguri, Cedric, Breukink, Eefjan, Strynadka, Natalie C.J., Simorre, Jean Pierre, Terrak, Mohammed, Vollmer, Waldemar, Sub Membrane Biochemistry & Biophysics, Membrane Biochemistry and Biophysics, The Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Laboratoire d'Enzymologie et Repliement des Protéines, Centre d'Ingénierie des Protéines, Université de Liège, Department of Biochemistry and Molecular Biology and Centre for Blood Research, University of Northern British Columbia [Prince George] (UNBC), Department of Molecular and Cellular Physiology, Stanford University, Institut de biologie structurale (IBS - UMR 5075), 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), Membrane Biochemistry and Biophysics, Utrecht University [Utrecht], Sub Membrane Biochemistry & Biophysics, and ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017)

Subjects
cell division, Models, Molecular, Molecular Biology and Physiology, Penicillin binding proteins, Cell division, Protein Conformation, DEDD, Peptidoglycan, medicine.disease_cause, Cefsulodin, Microbiology, Peptidoglycan synthases, Bacterial cell structure, 03 medical and health sciences, chemistry.chemical_compound, Virology, medicine, Escherichia coli, Penicillin-Binding Proteins, peptidoglycan synthases, Escherichia coli Infections, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 030306 microbiology, Chemistry, Escherichia coli Proteins, Periplasmic space, QR1-502, 3. Good health, Cell biology, Anti-Bacterial Agents, Enzyme, SPOR domain, Peptidoglycan Glycosyltransferase, Research Article, Protein Binding
Abstract

Escherichia coli has four SPOR proteins that bind peptidoglycan, of which FtsN is essential for cell division. DamX and DedD are suggested to have semiredundant functions in cell division based on genetic evidence. Here, we solved the structure of the SPOR domain of DedD, and we show that both DamX and DedD interact with and stimulate the synthetic activity of the peptidoglycan synthases PBP1A and PBP1B, suggesting that these class A PBP enzymes act in concert with peptidoglycan-binding proteins during cell division., Sporulation-related repeat (SPOR) domains are present in many bacterial cell envelope proteins and are known to bind peptidoglycan. Escherichia coli contains four SPOR proteins, DamX, DedD, FtsN, and RlpA, of which FtsN is essential for septal peptidoglycan synthesis. DamX and DedD may also play a role in cell division, based on mild cell division defects observed in strains lacking these SPOR domain proteins. Here, we show by nuclear magnetic resonance (NMR) spectroscopy that the periplasmic part of DedD consists of a disordered region followed by a canonical SPOR domain with a structure similar to that of the SPOR domains of FtsN, DamX, and RlpA. The absence of DamX or DedD decreases the functionality of the bifunctional transglycosylase-transpeptidase penicillin-binding protein 1B (PBP1B). DamX and DedD interact with PBP1B and stimulate its glycosyltransferase activity, and DamX also stimulates the transpeptidase activity. DedD also binds to PBP1A and stimulates its glycosyltransferase activity. Our data support a direct role of DamX and DedD in enhancing the activity of PBP1B and PBP1A, presumably during the synthesis of the cell division septum.

Published
2020

3. Squalamine and Aminosterol Mimics Inhibit the Peptidoglycan Glycosyltransferase Activity of PBP1b

Author

Boes, Adrien, Brunel, Jean Michel, Derouaux, Adeline, Kerff, Frédéric, Bouhss, Ahmed, Touze, Thierry, Breukink, Eefjan, Terrak, Mohammed, Sub Membrane Biochemistry & Biophysics, Membrane Biochemistry and Biophysics, Brunel, Jean Michel, Laboratoire d'Enzymologie et Repliement des Protéines, Centre d'Ingénierie des Protéines, Université de Liège, Centre de Recherche en Cancérologie de Marseille (CRCM), Aix Marseille Université (AMU)-Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Membranes et cibles thérapeutiques (MCT), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Biomédicale des Armées (IRBA), Centre d’Ingénierie des Protéines [Université de Liège] = Centre for Protein Engineering [University of Liège] (CIP), 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), Structure et activité des biomolécules normales et pathologiques (SABNP), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay, Department of Chemical Biology and Organic Chemistry, Utrecht University [Utrecht], Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Biomédicale des Armées [Brétigny-sur-Orge] (IRBA), 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), Centre d'Ingénierie des Protéines, Université de Liège-Institut de Chimie B6, Sub Membrane Biochemistry & Biophysics, Membrane Biochemistry and Biophysics, Enveloppes Bactériennes et Antibiotiques (ENVBAC), Département Microbiologie (Dpt Microbio), 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), and Department Biochemistry of Membranes

Subjects
0301 basic medicine, Microbiology (medical), Glycan, [SDV]Life Sciences [q-bio], Peptidoglycan, cationic aminosterols, 010402 general chemistry, 01 natural sciences, Microbiology, Biochemistry, Bacterial cell structure, Article, 03 medical and health sciences, chemistry.chemical_compound, Pharmacology, Toxicology and Pharmaceutics(all), squalamine, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Glycosyltransferase, Pharmacology (medical), General Pharmacology, Toxicology and Pharmaceutics, chemistry.chemical_classification, Pharmacology, Squalamine, biology, Lipid II, lcsh:RM1-950, Peptidoglycan glycosyltransferase activity, 0104 chemical sciences, 030104 developmental biology, Enzyme, Toxicology and Pharmaceutics(all), lcsh:Therapeutics. Pharmacology, Infectious Diseases, PBP1b, chemistry, Cationic aminosterols, biology.protein, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases
Abstract

International audience; Peptidoglycan (PG) is an essential polymer of the bacterial cell wall and a major antibacterial target. Its synthesis requires glycosyltransferase (GTase) and transpeptidase enzymes that, respectively, catalyze glycan chain elongation and their cross-linking to form the protective sacculus of the bacterial cell. The GTase domain of bifunctional penicillin-binding proteins (PBPs) of class A, such as Escherichia coli PBP1b, belong to the GTase 51 family. These enzymes play an essential role in PG synthesis, and their specific inhibition by moenomycin was shown to lead to bacterial cell death. In this work, we report that the aminosterol squalamine and mimic compounds present an unexpected mode of action consisting in the inhibition of the GTase activity of the model enzyme PBP1b. In addition, selected compounds were able to specifically displace the lipid II from the active site in a fluorescence anisotropy assay, suggesting that they act as competitive inhibitors.

Published
2020

4. Insight into the dual function of lipid phosphate phosphatase PgpB involved in two essential cell-envelope metabolic pathways in Escherichia coli

Author

Dominique Mengin-Lecreulx, Rodolphe Auger, Xudong Tian, Guillaume Manat, Frédéric Kerff, Thierry Touzé, 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), Enveloppes Bactériennes et Antibiotiques (ENVBAC), Département Microbiologie (Dpt Microbio), 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), Laboratoire d'Enzymologie et Repliement des Protéines, Centre d'Ingénierie des Protéines, Université de Liège, Belgium Program InterUniversity Attraction Poles (IAP n° 7/44), and ANR-11-BSV3-0002,BACTOPRENYL,Elucidation du métabolisme de l'undécaprényl phosphate, un lipide essentiel pour la biosynthèse des polymères de la paroi bactérienne(2011)

Subjects
Models, Molecular, Thermotolerance, 0301 basic medicine, [SDV]Life Sciences [q-bio], Amino Acid Motifs, Phosphatase, Phosphatidate Phosphatase, lcsh:Medicine, Biochemistry, Microbiology, Article, Substrate Specificity, Gene Knockout Techniques, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Polyisoprenyl Phosphates, Biosynthesis, Catalytic triad, Escherichia coli, Penicillin-Binding Proteins, lcsh:Science, Multidisciplinary, Chemistry, Escherichia coli Proteins, Hydrolysis, Cell Membrane, Genetic Complementation Test, lcsh:R, Membrane Proteins, Phosphatidylglycerols, Lipid-phosphate phosphatase, Metabolic pathway, 030104 developmental biology, Membrane protein, lcsh:Q, Peptidoglycan Glycosyltransferase, Peptidoglycan, Cell envelope, Metabolic Networks and Pathways, 030217 neurology & neurosurgery
Abstract

Ubiquitous PAP2 lipid phosphatases are involved in a wide array of central physiological functions. PgpB from Escherichia coli constitutes the archetype of this subfamily of membrane proteins. It displays a dual function by catalyzing the biosynthesis of two essential lipids, the phosphatidylglycerol (PG) and the undecaprenyl phosphate (C55-P). C55-P constitutes a lipid carrier allowing the translocation of peptidoglycan subunits across the plasma membrane. PG and C55-P are synthesized in a redundant manner by PgpB and other PAP2 and/or unrelated membrane phosphatases. Here, we show that PgpB is the sole, among these multiple phosphatases, displaying this dual activity. The inactivation of PgpB does not confer any apparent growth defect, but its inactivation together with another PAP2 alters the cell envelope integrity increasing the susceptibility to small hydrophobic compounds. Evidence is also provided of an interplay between PAP2s and the peptidoglycan polymerase PBP1A. In contrast to PGP hydrolysis, which relies on a His/Asp/His catalytic triad of PgpB, the mechanism of C55-PP hydrolysis appeared as only requiring the His/Asp diad, which led us to hypothesize distinct processes. Moreover, thermal stability analyses highlighted a substantial structural change upon phosphate binding by PgpB, supporting an induced-fit model of action.

Published
2020

5. Mass and charge distributions of amyloid fibers involved in neurodegenerative diseases: mapping heterogeneity and polymorphism

Author

François Legrand, M. Sallanon, C. Marquette, Jonathan Pansieri, Mohammad A. Halim, Vincent Forge, Sabine Chierici, Simona Denti, Philippe Dugourd, Rodolphe Antoine, Mireille Dumoulin, Charlotte Vendrely, Xavier Dagany, Laboratoire de Chimie et Biologie des Métaux (LCBM - UMR 5249), 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 [2016-2019] (UGA [2016-2019]), Institut Lumière Matière [Villeurbanne] (ILM), Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon, Equipe de recherche sur les relations matrice extracellulaire-cellules (ERRMECe), Fédération INSTITUT DES MATÉRIAUX DE CERGY-PONTOISE (I-MAT), Université de Cergy Pontoise (UCP), Université Paris-Seine-Université Paris-Seine-Université de Cergy Pontoise (UCP), Université Paris-Seine-Université Paris-Seine, Laboratoire d'Enzymologie et Repliement des Protéines, Centre d'Ingénierie des Protéines, Université de Liège, Centre de Recherches des Instituts Groupés, Haute Ecole Libre Mosane (CRIG), Radiopharmaceutiques biocliniques (LRB), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Département de Chimie Moléculaire (DCM), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Amyloid Fibres : From Foldopathies to NanoDesign [?-2019] (AFFOND [?-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 Grenoble Alpes [2016-2019] (UGA [2016-2019])-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])-Centre National de la Recherche Scientifique (CNRS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), InBios-Centre for Protein Engineering, Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), 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), and GON, Nathalie

Subjects
0301 basic medicine, chemistry.chemical_classification, education.field_of_study, biology, Tau protein, Population, Peptide, General Chemistry, Mass spectrometry, Charge detection, 03 medical and health sciences, 030104 developmental biology, chemistry, Polymorphism (materials science), mental disorders, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, biology.protein, Biophysics, [CHIM]Chemical Sciences, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], education, Amyloid fibers
Abstract

International audience; Heterogeneity and polymorphism are generic features of amyloid fibers with some important effects on the related disease development. We report here the characterization, by charge detection mass spectrometry, of amyloid fibers made of three polypeptides involved in neurodegenerative diseases: Aβ1–42 peptide, tau and α-synuclein. Beside the mass of individual fibers, this technique enables to characterize the heterogeneity and the polymorphism of the population. In the case of Aβ1–42 peptide and tau protein, several coexisting species could be distinguished and characterized. In the case of α-synuclein, we show how the polymorphism affects the mass and charge distributions.

Published
2018

6. Multimodal imaging Gd-nanoparticles functionalized with Pittsburgh compound B or a nanobody for amyloid plaques targeting

Author

C. Marquette, François Lux, Eric Allémann, Éva Tóth, Mireille Dumoulin, Laurence Heinrich-Balard, Olivier Tillement, Pascaline Rivory, Maria João Saraiva, Jean-François Morfin, Jonathan Pansieri, Nathalie Martine Stransky-Heilkron, Marie Plissonneau, Vincent Forge, 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), Nano-H S.A.S, Nano-H S.A.S, 38070 Saint Quentin Fallavier, France, Institut Lumière Matière [Villeurbanne] (ILM), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), School of Pharmaceutical Sciences, Université de Genève = University of Geneva (UNIGE), Laboratoire d'Enzymologie et Repliement des Protéines, Centre d'Ingénierie des Protéines, Université de Liège, Matériaux, ingénierie et science [Villeurbanne] (MATEIS), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Centre de biophysique moléculaire (CBM), Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Instituto de Inovação e Investigação em Saúde (I3S), University of Porto, Portugal, Molecular Neurobiology Group, IBMC - Institute for Molecular & Cell Biology, University of Porto, 4150-180 Porto, Portugal., GON, Nathalie, 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]), University of Geneva [Switzerland], Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA), and Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)

Subjects
0301 basic medicine, [SDV.IB.IMA]Life Sciences [q-bio]/Bioengineering/Imaging, Medicine (miscellaneous), Nanoparticle, Amylin, Peptide, Gadolinium, Plaque, Amyloid, Multimodal Imaging, chemistry.chemical_compound, Amyloid disease, Mice, General Materials Science, chemistry.chemical_classification, ddc:615, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, Aniline Compounds, biology, Brain, Immunohistochemistry, Islet Amyloid Polypeptide, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], amyloid imaging, Materials science, Amyloid, Biomedical Engineering, [SDV.BBM.BP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Bioengineering, Development, 03 medical and health sciences, Alzheimer Disease, Animals, Humans, [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Amyloid beta-Peptides, PIB, amyloidoses, gadolinium-based nanoparticles, Single-Domain Antibodies, Combinatorial chemistry, In vitro, nanobody, Transthyretin, Thiazoles, [SDV.IB.IMA] Life Sciences [q-bio]/Bioengineering/Imaging, 030104 developmental biology, chemistry, Diabetes Mellitus, Type 2, biology.protein, Biophysics, Nanoparticles, Pittsburgh compound B, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract

Aim: Gadolinium-based nanoparticles were functionalized with either the Pittsburgh compound B or a nanobody (B10AP) in order to create multimodal tools for an early diagnosis of amyloidoses. Materials & methods: The ability of the functionalized nanoparticles to target amyloid fibrils made of β-amyloid peptide, amylin or Val30Met-mutated transthyretin formed in vitro or from pathological tissues was investigated by a range of spectroscopic and biophysics techniques including fluorescence microscopy. Results: Nanoparticles functionalized by both probes efficiently interacted with the three types of amyloid fibrils, with KD values in 10 micromolar and 10 nanomolar range for, respectively, Pittsburgh compound B and B10AP nanoparticles. Moreover, they allowed the detection of amyloid deposits on pathological tissues. Conclusion: Such functionalized nanoparticles could represent promising flexible and multimodal imaging tools for the early diagnostic of amyloid diseases, in other words, Alzheimer's disease, Type 2 diabetes mellitus and the familial amyloidotic polyneuropathy.

Published
2017

7. Biophysical characterization data of the artificial protein Octarellin V.1 and binding test with its X-ray helpers

Author

Erik Goormaghtigh, Julie Vandenameele, Marie Valerio-Lepiniec, Cécile Van de Weerdt, Philippe Minard, André Matagne, Maximiliano Figueroa, Groupe Interdisciplinaire de Génoprotéomique Appliquée ( GIGA-Research ), Université de Liège-Faculté de médecine vétérinaire, Laboratoire d’Enzymologie et Repliement des Protéines, University of Liege, Laboratory for the Structure and Function of Biological Membranes, Université Libre de Bruxelles [Bruxelles] ( ULB ), Modélisation et Ingénierie des Protéines ( MIP ), Département Biochimie, Biophysique et Biologie Structurale ( B3S ), Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ) -Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ), Groupe Interdisciplinaire de Génoprotéomique Appliquée (GIGA-Research), Université de Liège, Université libre de Bruxelles (ULB), Modélisation et Ingénierie des Protéines (MIP), Département Biochimie, Biophysique et Biologie Structurale (B3S), 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 de Biologie Intégrative de la Cellule (I2BC), and 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)

Subjects
0301 basic medicine, Circular dichroism, Isothermal Titration Calorimetry, [SDV]Life Sciences [q-bio], Protein design, lcsh:Computer applications to medicine. Medical informatics, 01 natural sciences, law.invention, 03 medical and health sciences, law, Infra red spectroscopy, Crystallization, lcsh:Science (General), Protein secondary structure, Data Article, Crystallization helpers, Multidisciplinary, [ SDV ] Life Sciences [q-bio], Chemistry, Artificial proteins, 010401 analytical chemistry, X-ray, Isothermal titration calorimetry, Généralités, Fluorescence, 3. Good health, 0104 chemical sciences, Crystallography, 030104 developmental biology, lcsh:R858-859.7, Chemical stability, lcsh:Q1-390
Abstract

The artificial protein Octarellin V.1 (http://dx.doi.org/10.1016/j.jsb.2016.05.004 [1]) was obtained through a direct evolution process over the de novo designed Octarellin V (http://dx.doi.org/10.1016/S0022-2836(02)01206-8 [2]). The protein has been characterized by circular dichroism and fluorescence techniques, in order to obtain data related to its thermo and chemical stability. Moreover, the data for the secondary structure content studied by circular dichroism and infra red techniques is reported for the Octarellin V and V.1. Two crystallization helpers, nanobodies (http://dx.doi.org/10.1038/nprot.2014.039 [3]) and αRep (http://dx.doi.org/10.1016/j.jmb.2010.09.048 [4]), have been used to create stable complexes. Here we present the data obtained of the binding characterization of the Octarellin V.1 with the crystallization helpers by isothermal titration calorimetry., SCOPUS: ar.j, info:eu-repo/semantics/published

Published
2016

8. Gd-nanoparticles functionalization with specific peptides for ß-amyloid plaques targeting

Author

Éva Tόth, François Lux, Cédric Louis, Pierre Mowat, Eric Allémann, Laurence Heinrich-Balard, Mireille Dumoulin, Jonathan Pansieri, Richard Cohen, Nathalie Martine Stransky-Heilkron, Marie Plissonneau, C. Marquette, Olivier Tillement, Pascaline Rivory, Maria João Saraiva, Vincent Forge, Jean-François Morfin, Nano-H SAS, 38070, Saint Quentin Fallavier, France., Nano-H SAS, 38070, Saint Quentin Fallavier, France, Institut Lumière Matière [Villeurbanne] (ILM), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), 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), Matériaux, ingénierie et science [Villeurbanne] (MATEIS), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Centre de biophysique moléculaire (CBM), Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), School of Pharmaceutical Sciences, University of Geneva [Switzerland], Centre for Protein Engineering (CIP), Institut de Chimie (B6) - Sart Tilman, Laboratoire d’Enzymologie et Repliement des Protéines, Université de Liège, Laboratoire de biochimie et biologie moléculaire, Hospices Civils de Lyon (HCL)-Centre Hospitalier Lyon Sud [CHU - HCL] (CHLS), Hospices Civils de Lyon (HCL), Department of Molecular Neurobiology, Institute for Molecular and Cell Biology, Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Universidade do Porto, Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Université de Genève = University of Geneva (UNIGE), Centre d’Ingénierie des Protéines [Université de Liège] = Centre for Protein Engineering [University of Liège] (CIP), and Universidade do Porto = University of Porto

Subjects
0301 basic medicine, Pathology, [SDV.IB.IMA]Life Sciences [q-bio]/Bioengineering/Imaging, [SDV.NEU.NB]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, Gene Expression, Metal Nanoparticles, Pharmaceutical Science, Medicine (miscellaneous), Gadolinium, Plaque, Amyloid, Peptide, Beta-amyloid, Hippocampus, Applied Microbiology and Biotechnology, Mice, chemistry.chemical_compound, 0302 clinical medicine, Peptide-targeting, Prealbumin, MRI contrast agent, Beta-amyloid fibrils, chemistry.chemical_classification, ddc:615, biology, medicine.diagnostic_test, Alzheimer's disease, Magnetic Resonance Imaging, Contrast agent, Positron emission tomography, Molecular Medicine, Female, Alzheimer’s disease, FibrilsMRI, Protein Binding, medicine.medical_specialty, Gadolinium based nanoparticles, Amyloid, Biomedical Engineering, Mice, Transgenic, Bioengineering, macromolecular substances, Fibril, Amyloid imaging, 03 medical and health sciences, Alzheimer Disease, medicine, Animals, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Peptidetargeting, Amyloid beta-Peptides, Research, Surface Plasmon Resonance, medicine.disease, Peptide Fragments, Disease Models, Animal, Kinetics, Transthyretin, 030104 developmental biology, chemistry, Mutation, Biophysics, biology.protein, Peptides, Pittsburgh compound B, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology, 030217 neurology & neurosurgery
Abstract

Background Amyloidoses are characterized by the extracellular deposition of insoluble fibrillar proteinaceous aggregates highly organized into cross-β structure and referred to as amyloid fibrils. Nowadays, the diagnosis of these diseases remains tedious and involves multiple examinations while an early and accurate protein typing is crucial for the patients’ treatment. Routinely used neuroimaging techniques such as magnetic resonance imaging (MRI) and positron emission tomography (PET) using Pittsburgh compound B, [11C]PIB, provide structural information and allow to assess the amyloid burden, respectively, but cannot discriminate between different amyloid deposits. Therefore, the availability of efficient multimodal imaging nanoparticles targeting specific amyloid fibrils would provide a minimally-invasive imaging tool useful for amyloidoses typing and early diagnosis. In the present study, we have functionalized gadolinium-based MRI nanoparticles (AGuIX) with peptides highly specific for Aβ amyloid fibrils, LPFFD and KLVFF. The capacity of such nanoparticles grafted with peptide to discriminate among different amyloid proteins, was tested with Aβ(1–42) fibrils and with mutated-(V30M) transthyretin (TTR) fibrils. Results The results of surface plasmon resonance studies showed that both functionalized nanoparticles interact with Aβ(1–42) fibrils with equilibrium dissociation constant (Kd) values of 403 and 350 µM respectively, whilst they did not interact with V30M-TTR fibrils. Similar experiments, performed with PIB, displayed an interaction both with Aβ(1–42) fibrils and V30M-TTR fibrils, with Kd values of 6 and 10 µM respectively, confirming this agent as a general amyloid fibril marker. Thereafter, the ability of functionalized nanoparticle to target and bind selectively Aβ aggregates was further investigated by immunohistochemistry on AD like-neuropathology brain tissue. Pictures clearly indicated that KLVFF-grafted or LPFFD-grafted to AGuIX nanoparticle recognized and bound the Aβ amyloid plaque localized in the mouse hippocampus. Conclusion These results constitute a first step for considering these functionalized nanoparticles as a valuable multimodal imaging tool to selectively discriminate and diagnose amyloidoses. Electronic supplementary material The online version of this article (doi:10.1186/s12951-016-0212-y) contains supplementary material, which is available to authorized users.

Published
2016

9. The unexpected structure of the designed protein Octarellin V.1 forms a challenge for protein structure prediction tools

Author

Dominique Maes, Philippe Minard, Jens Meiler, Julie Vandenameele, Mike Sleutel, Els Pardon, Christian Damblon, Marie Valerio-Lepiniec, André Matagne, Marylène Vandevenne, Agathe Urvoas, Maximiliano Figueroa, Dominique Durand, Gregory Parvizi, Sophie Attout, Erik Goormaghtigh, Jan Steyaert, Joseph Martial, Axel Fischer, Olivier Jacquin, Cécile Van de Weerdt, Groupe Interdisciplinaire de Génoprotéomique Appliquée (GIGA-Research), Université de Liège, Structural Biology Brussels (SBB), Vrije Universiteit Brussel (VUB), Laboratoire d’Enzymologie et Repliement des Protéines, Department of Chemistry, Center for Structural Biology, Vanderbilt University [Nashville], Department of Chemistry, University of Lie'ge, Lie'ge, Belgium, Department of Chemistry, 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), Groupe Interdisciplinaire de Génoprotéomique Appliquée ( GIGA-Research ), Université de Liège-Faculté de médecine vétérinaire, Structural Biology Brussels ( SBB ), Vrije Universiteit [Brussel] ( VUB ), University of Liege, Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ), Department of Bio-engineering Sciences, Faculty of Sciences and Bioengineering Sciences, and Structural Biology Brussels

Subjects
0301 basic medicine, Protein Folding, In silico, [SDV]Life Sciences [q-bio], Protein design, Molecular modeling, Computational biology, Biology, Crystallography, X-Ray, 03 medical and health sciences, Structural Biology, Humans, Computer Simulation, 030102 biochemistry & molecular biology, [ SDV ] Life Sciences [q-bio], Artificial proteins, Proteins, Evolutionary pressure, Protein structure prediction, Directed evolution, Protein tertiary structure, Recombinant Proteins, Protein Structure, Tertiary, Crystallography, 030104 developmental biology, De novo design, Protein folding, Threading (protein sequence), Directed Molecular Evolution
Abstract

International audience; Despite impressive successes in protein design, designing a well-folded protein of more 100 amino acids de novo remains a formidable challenge. Exploiting the promising biophysical features of the artificial protein Octarellin V, we improved this protein by directed evolution, thus creating a more stable and soluble protein: Octarellin V.1. Next, we obtained crystals of Octarellin V.1 in complex with crystallization chaperons and determined the tertiary structure. The experimental structure of Octarellin V.1 differs from its in silico design: the (αβα) sandwich architecture bears some resemblance to a Rossman-like fold instead of the intended TIM-barrel fold. This surprising result gave us a unique and attractive opportunity to test the state of the art in protein structure prediction, using this artificial protein free of any natural selection. We tested 13 automated webservers for protein structure prediction and found none of them to predict the actual structure. More than 50% of them predicted a TIM-barrel fold, i.e. the structure we set out to design more than 10years ago. In addition, local software runs that are human operated can sample a structure similar to the experimental one but fail in selecting it, suggesting that the scoring and ranking functions should be improved. We propose that artificial proteins could be used as tools to test the accuracy of protein structure prediction algorithms, because their lack of evolutionary pressure and unique sequences features.

Published
2016

10. Structural and functional characterization of Solanum tuberosum VDAC36.

Author

Lopes-Rodrigues M, Matagne A, Zanuy D, Alemán C, Perpète EA, and Michaux C

Subjects
Adenosine Triphosphate metabolism, Binding Sites, Cloning, Molecular, Cross-Linking Reagents chemistry, Escherichia coli genetics, Escherichia coli metabolism, Gene Expression, Genetic Vectors chemistry, Genetic Vectors metabolism, Kinetics, Liposomes metabolism, Models, Molecular, Osmolar Concentration, Phosphatidylcholines chemistry, Phosphatidylcholines metabolism, Plant Proteins genetics, Plant Proteins metabolism, Protein Binding, Protein Conformation, alpha-Helical, Protein Conformation, beta-Strand, Protein Interaction Domains and Motifs, Protein Isoforms chemistry, Protein Isoforms genetics, Protein Isoforms metabolism, Protein Multimerization, Protein Refolding, Protein Structure, Tertiary, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Solanum tuberosum genetics, Voltage-Dependent Anion Channels genetics, Voltage-Dependent Anion Channels metabolism, Adenosine Triphosphate chemistry, Liposomes chemistry, Plant Proteins chemistry, Solanum tuberosum metabolism, Voltage-Dependent Anion Channels chemistry
Abstract

As it forms water-filled channel in the mitochondria outer membrane and diffuses essential metabolites such as NADH and ATP, the voltage-dependent anion channel (VDAC) protein family plays a central role in all eukaryotic cells. In comparison with their mammalian homologues, little is known about the structural and functional properties of plant VDACs. In the present contribution, one of the two VDACs isoforms of Solanum tuberosum, stVDAC36, has been successfully overexpressed and refolded by an in-house method, as demonstrated by the information on its secondary and tertiary structure gathered from circular dichroism and intrinsic fluorescence. Cross-linking and molecular modeling studies have evidenced the presence of dimers and tetramers, and they suggest the formation of an intermolecular disulfide bond between two stVDAC36 monomers. The pore-forming activity was also assessed by liposome swelling assays, indicating a typical pore diameter between 2.0 and 2.7 nm. Finally, insights about the ATP binding inside the pore are given by docking studies and electrostatic calculations., (© 2019 Wiley Periodicals, Inc.)

Published
2020
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11. An NMR and MD study of complexes of bacteriophage lambda lysozyme with tetra- and hexa-N-acetylchitohexaose.

Author

Turupcu A, Bowen AM, Di Paolo A, Matagne A, Oostenbrink C, Redfield C, and Smith LJ

Subjects
Bacteriophage lambda chemistry, Bacteriophage lambda metabolism, Catalytic Domain drug effects, Enzyme Inhibitors chemistry, Enzyme Inhibitors metabolism, Enzyme Inhibitors pharmacology, Molecular Docking Simulation, Molecular Dynamics Simulation, Muramidase chemistry, Nuclear Magnetic Resonance, Biomolecular, Oligosaccharides chemistry, Oligosaccharides pharmacology, Protein Binding, Protein Conformation drug effects, Bacteriophage lambda enzymology, Muramidase antagonists & inhibitors, Muramidase metabolism, Oligosaccharides metabolism
Abstract

The X-ray structure of lysozyme from bacteriophage lambda (λ lysozyme) in complex with the inhibitor hexa-N-acetylchitohexaose (NAG6) (PDB: 3D3D) has been reported previously showing sugar units from two molecules of NAG6 bound in the active site. One NAG6 is bound with four sugar units in the ABCD sites and the other with two sugar units in the E'F' sites potentially representing the cleavage reaction products; each NAG6 cross links two neighboring λ lysozyme molecules. Here we use NMR and MD simulations to study the interaction of λ lysozyme with the inhibitors NAG4 and NAG6 in solution. This allows us to study the interactions within the complex prior to cleavage of the polysaccharide. 1 H N and 15 N chemical shifts of λ lysozyme resonances were followed during NAG4/NAG6 titrations. The chemical shift changes were similar in the two titrations, consistent with sugars binding to the cleft between the upper and lower domains; the NMR data show no evidence for simultaneous binding of a NAG6 to two λ lysozyme molecules. Six 150 ns MD simulations of λ lysozyme in complex with NAG4 or NAG6 were performed starting from different conformations. The simulations with both NAG4 and NAG6 show stable binding of sugars across the D/E active site providing low energy models for the enzyme-inhibitor complexes. The MD simulations identify different binding subsites for the 5th and 6th sugars consistent with the NMR data. The structural information gained from the NMR experiments and MD simulations have been used to model the enzyme-peptidoglycan complex., (© 2019 The Authors. Proteins: Structure, Function, and Bioinformatics published by Wiley Periodicals, Inc.)

Published
2020
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12. P174E Substitution in GES-1 and GES-5 β-Lactamases Improves Catalytic Efficiency toward Carbapenems.

Author

Piccirilli A, Mercuri PS, Galleni M, Aschi M, Matagne A, Amicosante G, and Perilli M

Subjects
Bacterial Proteins antagonists & inhibitors, Cephalosporins pharmacology, Circular Dichroism, Clavulanic Acid pharmacology, Drug Resistance, Multiple, Bacterial genetics, Humans, Klebsiella pneumoniae drug effects, Klebsiella pneumoniae genetics, Klebsiella pneumoniae isolation & purification, Microbial Sensitivity Tests, Molecular Dynamics Simulation, Penicillins pharmacology, Protein Structure, Secondary genetics, Tazobactam pharmacology, beta-Lactamases metabolism, Anti-Bacterial Agents pharmacology, Bacterial Proteins genetics, Imipenem pharmacology, Meropenem pharmacology, beta-Lactamases genetics
Abstract

GES-type β-lactamases are a group of enzymes that have evolved their hydrolytic activity against carbapenems. In this study, the role of residue 174 inside the Ω-loop of GES-1 and GES-5 was investigated. GES-1 P174E and GES-5 P174E mutants, selected by site saturation mutagenesis, were purified and kinetically characterized. In comparison with GES-1 and GES-5 wild-type enzymes, GES-1 P174E and GES-5 P174E mutants exhibited lower k cat and k cat / K m values for cephalosporins and penicillins. Concerning carbapenems, GES-1 P174E shared higher k cat values but lower K m values than those calculated for GES-1. The GES-1 P174E and GES-5 P174E mutants showed high hydrolytic efficiency for imipenem, with k cat / K m values 100- and 660-fold higher, respectively, than those of GES-1. Clavulanic acid and tazobactam are good inhibitors for both GES-1 P174E and GES-5 P174E Molecular dynamic (MD) simulations carried out for GES-1, GES-5, GES-1 P174E , and GES-5 P174E complexed with imipenem and meropenem have shown that mutation at position 174 induces a drastic increase of enzyme flexibility, in particular in the Ω-loop. The circular dichroism (CD) spectroscopy spectra of the four enzymes indicate that the P174E substitution in GES-1 and GES-5 does not affect the secondary structural content of the enzymes., (Copyright © 2018 American Society for Microbiology.)

Published
2018
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13. The proteolytic system of pineapple stems revisited: Purification and characterization of multiple catalytically active forms.

Author

Matagne A, Bolle L, El Mahyaoui R, Baeyens-Volant D, and Azarkan M

Subjects
Bromelains analysis, Chemical Fractionation methods, Cysteine Endopeptidases analysis, Cysteine Proteases analysis, Plant Proteins analysis, Plant Stems chemistry, Ananas chemistry, Cysteine Proteases isolation & purification, Plant Extracts analysis, Plant Proteins isolation & purification, Proteolysis
Abstract

Crude pineapple proteases extract (aka stem bromelain; EC 3.4.22.4) is an important proteolytic mixture that contains enzymes belonging to the cysteine proteases of the papain family. Numerous studies have been reported aiming at the fractionation and characterization of the many molecular species present in the extract, but more efforts are still required to obtain sufficient quantities of the various purified protease forms for detailed physicochemical, enzymatic and structural characterization. In this work, we describe an efficient strategy towards the purification of at least eight enzymatic forms. Thus, following rapid fractionation on a SP-Sepharose FF column, two sub-populations with proteolytic activity were obtained: the unbound (termed acidic) and bound (termed basic) bromelain fractions. Following reversible modification with monomethoxypolyethylene glycol (mPEG), both fractions were further separated on Q-Sepharose FF and SP-Sepharose FF, respectively. This procedure yielded highly purified molecular species, all titrating ca. 1 mol of thiol group per mole of enzyme, with distinct biochemical properties. N-terminal sequencing allowed identifying at least eight forms with proteolytic activity. The basic fraction contained previously identified species, i.e. basic bromelain forms 1 and 2, ananain forms 1 and 2, and comosain (MEROPS identifier: C01.027). Furthermore, a new proteolytic species, showing similarities with basic bomelain forms 1 and 2, was discovered and termed bromelain form 3. The two remaining species were found in the acidic bromelain fraction and were arbitrarily named acidic bromelain forms 1 and 2. Both, acidic bromelain forms 1, 2 and basic bromelain forms 1, 2 and 3 are glycosylated, while ananain forms 1 and 2, and comosain are not. The eight protease forms display different amidase activities against the various substrates tested, namely small synthetic chromogenic compounds (DL-BAPNA and Boc-Ala-Ala-Gly-pNA), fluorogenic compounds (like Boc-Gln-Ala-Arg-AMC, Z-Arg-Arg-AMC and Z-Phe-Arg-AMC), and proteins (azocasein and azoalbumin), suggesting a specific organization of their catalytic residues. All forms are completely inhibited by specific cysteine and cysteine/serine protease inhibitors, but not by specific serine and aspartic protease inhibitors, with the sole exception of pepstatin A that significantly affects acidic bromelain forms 1 and 2. For all eight protease forms, inhibition is also observed with 1,10-phenanthrolin, a metalloprotease inhibitor. Metal ions (i.e. Mn 2+ , Mg 2+ and Ca 2+ ) showed various effects depending on the protease under consideration, but all of them are totally inhibited in the presence of Zn 2+ . Mass spectrometry analyses revealed that all forms have a molecular mass of ca. 24 kDa, which is characteristic of enzymes belonging to the papain-like proteases family. Far-UV CD spectra analysis further supported this analysis. Interestingly, secondary structure calculation proves to be highly reproducible for all cysteine proteases of the papain family tested so far (this work; see also Azarkan et al., 2011; Baeyens-Volant et al., 2015) and thus can be used as a test for rapid identification of the classical papain fold., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Published
2017
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14. The Role of Active Site Flexible Loops in Catalysis and of Zinc in Conformational Stability of Bacillus cereus 569/H/9 β-Lactamase.

Author

Montagner C, Nigen M, Jacquin O, Willet N, Dumoulin M, Karsisiotis AI, Roberts GC, Damblon C, Redfield C, and Matagne A

Subjects
Catalytic Domain, Hydrophobic and Hydrophilic Interactions, Protein Structure, Secondary, Bacillus cereus enzymology, Protein Unfolding, Zinc chemistry, beta-Lactamases chemistry
Abstract

Metallo-β-lactamases catalyze the hydrolysis of most β-lactam antibiotics and hence represent a major clinical concern. The development of inhibitors for these enzymes is complicated by the diversity and flexibility of their substrate-binding sites, motivating research into their structure and function. In this study, we examined the conformational properties of the Bacillus cereus β-lactamase II in the presence of chemical denaturants using a variety of biochemical and biophysical techniques. The apoenzyme was found to unfold cooperatively, with a Gibbs free energy of stabilization (ΔG(0)) of 32 ± 2 kJ·mol(-1) For holoBcII, a first non-cooperative transition leads to multiple interconverting native-like states, in which both zinc atoms remain bound in an apparently unaltered active site, and the protein displays a well organized compact hydrophobic core with structural changes confined to the enzyme surface, but with no catalytic activity. Two-dimensional NMR data revealed that the loss of activity occurs concomitantly with perturbations in two loops that border the enzyme active site. A second cooperative transition, corresponding to global unfolding, is observed at higher denaturant concentrations, with ΔG(0) value of 65 ± 1.4 kJ·mol(-1) These combined data highlight the importance of the two zinc ions in maintaining structure as well as a relatively well defined conformation for both active site loops to maintain enzymatic activity., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Published
2016
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15. Biophysical characterization data of the artificial protein Octarellin V.1 and binding test with its X-ray helpers.

Author

Figueroa M, Vandenameele J, Goormaghtigh E, Valerio-Lepiniec M, Minard P, Matagne A, and Van de Weerdt C

Abstract

The artificial protein Octarellin V.1 (http://dx.doi.org/10.1016/j.jsb.2016.05.004[1]) was obtained through a direct evolution process over the de novo designed Octarellin V (http://dx.doi.org/10.1016/S0022-2836(02)01206-8[2]). The protein has been characterized by circular dichroism and fluorescence techniques, in order to obtain data related to its thermo and chemical stability. Moreover, the data for the secondary structure content studied by circular dichroism and infra red techniques is reported for the Octarellin V and V.1. Two crystallization helpers, nanobodies (http://dx.doi.org/10.1038/nprot.2014.039[3]) and αRep (http://dx.doi.org/10.1016/j.jmb.2010.09.048[4]), have been used to create stable complexes. Here we present the data obtained of the binding characterization of the Octarellin V.1 with the crystallization helpers by isothermal titration calorimetry.

Published
2016
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16. The unexpected structure of the designed protein Octarellin V.1 forms a challenge for protein structure prediction tools.

Author

Figueroa M, Sleutel M, Vandevenne M, Parvizi G, Attout S, Jacquin O, Vandenameele J, Fischer AW, Damblon C, Goormaghtigh E, Valerio-Lepiniec M, Urvoas A, Durand D, Pardon E, Steyaert J, Minard P, Maes D, Meiler J, Matagne A, Martial JA, and Van de Weerdt C

Subjects
Crystallography, X-Ray, Humans, Protein Folding, Protein Structure, Tertiary, Computer Simulation standards, Directed Molecular Evolution methods, Proteins chemistry, Recombinant Proteins chemistry
Abstract

Despite impressive successes in protein design, designing a well-folded protein of more 100 amino acids de novo remains a formidable challenge. Exploiting the promising biophysical features of the artificial protein Octarellin V, we improved this protein by directed evolution, thus creating a more stable and soluble protein: Octarellin V.1. Next, we obtained crystals of Octarellin V.1 in complex with crystallization chaperons and determined the tertiary structure. The experimental structure of Octarellin V.1 differs from its in silico design: the (αβα) sandwich architecture bears some resemblance to a Rossman-like fold instead of the intended TIM-barrel fold. This surprising result gave us a unique and attractive opportunity to test the state of the art in protein structure prediction, using this artificial protein free of any natural selection. We tested 13 automated webservers for protein structure prediction and found none of them to predict the actual structure. More than 50% of them predicted a TIM-barrel fold, i.e. the structure we set out to design more than 10years ago. In addition, local software runs that are human operated can sample a structure similar to the experimental one but fail in selecting it, suggesting that the scoring and ranking functions should be improved. We propose that artificial proteins could be used as tools to test the accuracy of protein structure prediction algorithms, because their lack of evolutionary pressure and unique sequences features., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Published
2016
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17. Kinetic Study of Laboratory Mutants of NDM-1 Metallo-β-Lactamase and the Importance of an Isoleucine at Position 35.

Author

Marcoccia F, Bottoni C, Sabatini A, Colapietro M, Mercuri PS, Galleni M, Kerff F, Matagne A, Celenza G, Amicosante G, and Perilli M

Subjects
Amino Acid Substitution, Anti-Bacterial Agents pharmacology, Biocatalysis, Catalytic Domain, Cephalosporins pharmacology, Cloning, Molecular, Escherichia coli enzymology, Escherichia coli genetics, Gene Expression, Isoleucine metabolism, Kinetics, Models, Molecular, Mutation, Protein Structure, Secondary, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Serine chemistry, Serine metabolism, Threonine chemistry, Threonine metabolism, beta-Lactamases genetics, beta-Lactamases metabolism, Anti-Bacterial Agents chemistry, Cephalosporins chemistry, Escherichia coli drug effects, Isoleucine chemistry, beta-Lactam Resistance genetics, beta-Lactamases chemistry
Abstract

Two laboratory mutants of NDM-1 were generated by replacing the isoleucine at position 35 with threonine and serine residues: the NDM-1(I35T)and NDM-1(I35S)enzymes. These mutants were well characterized, and their kinetic parameters were compared with those of the NDM-1 wild type. Thekcat,Km, andkcat/Kmvalues calculated for the two mutants were slightly different from those of the wild-type enzyme. Interestingly, thekcat/Kmof NDM-1(I35S)for loracarbef was about 14-fold higher than that of NDM-1. Far-UV circular dichroism (CD) spectra of NDM-1 and NDM-1(I35T)and NDM-1(I35S)enzymes suggest local structural rearrangements in the secondary structure with a marked reduction of α-helix content in the mutants., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Published
2016
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18. A novel form of ficin from Ficus carica latex: Purification and characterization.

Author

Baeyens-Volant D, Matagne A, El Mahyaoui R, Wattiez R, and Azarkan M

Subjects
Chromatography, Gel, Cysteine Proteases drug effects, Cysteine Proteinase Inhibitors pharmacology, Electrophoresis, Polyacrylamide Gel, Ficain chemistry, Ficain metabolism, Hydrogen-Ion Concentration, Latex isolation & purification, Leucine analogs & derivatives, Leucine pharmacology, Molecular Weight, Nuclear Magnetic Resonance, Biomolecular, Phenanthrolines pharmacology, Polyethylene Glycols, Protein Structure, Secondary, Sulfhydryl Compounds chemistry, Cysteine Proteases metabolism, Ficain isolation & purification, Ficus chemistry, Latex chemistry
Abstract

A novel ficin form, named ficin E, was purified from fig tree latex by a combination of cation-exchange chromatography on SP-Sepharose Fast Flow, Thiopropyl Sepharose 4B and fplc-gel filtration chromatography. The new ficin appeared not to be sensitive to thiol derivatization by a polyethylene glycol derivative, allowing its purification. The protease is homogeneous according to PAGE, SDS-PAGE, mass spectrometry, N-terminal micro-sequencing analyses and E-64 active site titration. N-terminal sequencing of the first ten residues has shown high identity with the other known ficin (iso)forms. The molecular weight was found to be (24,294±10)Da by mass spectrometry, a lower value than the apparent molecular weight observed on SDS-PAGE, around 27 kDa. Far-UV CD data revealed a secondary structure content of 22% α-helix and 26% β-sheet. The protein is not glycosylated as shown by carbohydrate analysis. pH and temperature measurements indicated maxima activity at pH 6.0 and 50 °C, respectively. Preliminary pH stability analyses have shown that the protease conserved its compact structure in slightly acidic, neutral and alkaline media but at acidic pH (<3), the formation of some relaxed or molten state was evidenced by 8-anilino-1-naphtalenesulfonic acid binding characteristics. Comparison with the known ficins A, B, C, D1 and D2 (iso)forms revealed that ficin E showed activity profile that looked like ficin A against two chromogenic substrates while it resembled ficins D1 and D2 against three fluorogenic substrates. Enzymatic activity of ficin E was not affected by Mg(2+), Ca(2+) and Mn(2+) at a concentration up to 10mM. However, the activity was completely suppressed by Zn(2+) at a concentration of 1mM. Inhibitory activity measurements clearly identified the enzyme as a cysteine protease, being unaffected by synthetic (Pefabloc SC, benzamidine) and by natural proteinaceous (aprotinin) serine proteases inhibitors, by aspartic proteases inhibitors (pepstatin A) and by metallo-proteases inhibitors (EDTA, EGTA). Surprisingly, it was well affected by the metallo-protease inhibitor o-phenanthroline. The enzymatic activity was however completely blocked by cysteine proteases inhibitors (E-64, iodoacetamide), by thiol-blocking compounds (HgCl2) and by cysteine/serine proteases inhibitors (TLCK and TPCK). This is a novel ficin form according to peptide mass fingerprint analysis, specific amidase activity, SDS-PAGE and PAGE electrophoretic mobility, N-terminal sequencing and unproneness to thiol pegylation., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Published
2015
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19. Rapid collapse into a molten globule is followed by simple two-state kinetics in the folding of lysozyme from bacteriophage λ.

Author

Di Paolo A, Balbeur D, De Pauw E, Redfield C, and Matagne A

Subjects
Kinetics, Models, Molecular, Nuclear Magnetic Resonance, Biomolecular, Protein Folding, Protein Structure, Secondary, Spectrometry, Mass, Electrospray Ionization, Bacteriophage lambda enzymology, Muramidase chemistry
Abstract

Stopped-flow fluorescence and circular dichroism spectroscopy have been used in combination with quenched-flow hydrogen exchange labeling, monitored by two-dimensional NMR and electrospray ionization mass spectrometry, to investigate the folding kinetics of lysozyme from bacteriophage λ (λ lysozyme) at pH 5.6, 20 °C. The first step in the folding of λ lysozyme occurs very rapidly (τ < 1 ms) after refolding is initiated and involves both hydrophobic collapse and formation of a high content of secondary structure but only weak protection from (1)H/(2)H exchange and no fixed tertiary structure organization. This early folding step is reflected in the dead-time events observed in the far-UV CD and ANS fluorescence experiments. Following accumulation of this kinetic molten globule species, the secondary structural elements are stabilized and the majority (ca. 88%) of refolding molecules acquire native-like properties in a highly cooperative two-state process, with τ = 0.15 ± 0.03 s. This is accompanied by the acquisition of substantial native-like protection from hydrogen exchange. A double-mixing experiment and the absence of a denaturant effect reveal that slow (τ = 5 ± 1 s) folding of the remaining (ca. 12%) molecules is rate limited by the cis/trans isomerization of prolines that are trans in the folded enzyme. In addition, native state hydrogen exchange and classical denaturant unfolding experiments have been used to characterize the thermodynamic properties of the enzyme. In good agreement with previous crystallographic evidence, our results show that λ lysozyme is a highly dynamic protein, with relatively low conformational stability (ΔG°(N-U) = 25 ± 2 kJ·mol(-1)).

Published
2010
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20. 1H, 13C and 15N backbone resonance assignments for the BS3 class A β-lactamase from Bacillus licheniformis.

Author

Vandenameele J, Matagne A, and Damblon C

Subjects
Carbon Isotopes, Hydrogen, Nitrogen Isotopes, Bacillus enzymology, Nuclear Magnetic Resonance, Biomolecular, beta-Lactamases chemistry
Abstract

Class A β-lactamases (260-280 amino acids; M ( r ) ~ 29,000) are among the largest proteins studied in term of their folding properties. They are composed of two structural domains: an all-α domain formed by five to eight helices and an α/β domain consisting of a five-stranded antiparallel β-sheet covered by three to four α-helices. The α domain (~150 residues) is made up of the central part of the polypeptide chain whereas the α/β domain (111-135 residues) is constituted by the N- and C-termini of the protein. Our goal is to determine in which order the different secondary structure elements are formed during the folding of BS3. With this aim, we will use pulse-labelling hydrogen/deuterium exchange experiments, in combination with 2D-NMR measurements, to monitor the time-course of formation and stabilization of secondary structure elements. Here we report the backbone resonance assignments as the requirement for further hydrogen/deuterium exchange studies.

Published
2010
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21. Folding of class A beta-lactamases is rate-limited by peptide bond isomerization and occurs via parallel pathways.

Author

Vandenameele J, Lejeune A, Di Paolo A, Brans A, Frère JM, Schmid FX, and Matagne A

Subjects
Circular Dichroism, Kinetics, Leucine chemistry, Models, Molecular, Proline chemistry, Protein Conformation, Protein Folding, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Stereoisomerism, Thermodynamics, beta-Lactamases metabolism, beta-Lactamases chemistry
Abstract

Class A beta-lactamases (M(r) approximately 29000) provide good models for studying the folding mechanism of large monomeric proteins. In particular, the highly conserved cis peptide bond between residues 166 and 167 at the active site of these enzymes controls important steps in their refolding reaction. In this work, we analyzed how conformational folding, reactivation, and cis/trans peptide bond isomerizations are interrelated in the folding kinetics of beta-lactamases that differ in the nature of the cis peptide bond, which involves a Pro167 in the BS3 and TEM-1 enzyme, a Leu167 in the NMCA enzyme, and which is missing in the PER-1 enzyme. The analysis of folding by spectroscopic probes and by the regain of enzymatic activity in combination with double-mixing procedures indicates that conformational folding can proceed when the 166-167 bond is still in the incorrect trans form. The very slow trans --> cis isomerization of the Glu166-Xaa167 peptide bond, however, controls the final step of folding and is required for the regain of the enzymatic activity. This very slow phase is absent in the refolding of PER-1, in which the Glu166-Ala167 peptide bond is trans. The double-mixing experiments revealed that a second slow kinetic phase is caused by the cis/trans isomerization of prolines that are trans in the folded proteins. The folding of beta-lactamases is best described by a model that involves parallel pathways. It highlights the role of peptide bond cis/trans isomerization as a kinetic determinant of folding.

Published
2010
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22. Backbone 1H, 13C, and 15N resonance assignments for lysozyme from bacteriophage lambda.

Author

Di Paolo A, Duval V, Matagne A, and Redfield C

Subjects
Carbon Isotopes chemistry, Escherichia coli, Hydrogen chemistry, Muramidase genetics, Nitrogen Isotopes chemistry, Nuclear Magnetic Resonance, Biomolecular, Protein Structure, Secondary, Protein Structure, Tertiary, Recombinant Proteins chemistry, Recombinant Proteins genetics, Viral Proteins genetics, Bacteriophage lambda enzymology, Muramidase chemistry, Viral Proteins chemistry
Abstract

Lysozyme from lambda bacteriophage (lambda lysozyme) is an 18 kDa globular protein displaying some of the structural features common to all lysozymes; in particular, lambda lysozyme consists of two structural domains connected by a helix, and has its catalytic residues located at the interface between these two domains. An interesting feature of lambda lysozyme, when compared to the well-characterised hen egg-white lysozyme, is its lack of disulfide bridges; this makes lambda lysozyme an interesting system for studies of protein folding. A comparison of the folding properties of lambda lysozyme and hen lysozyme will provide important insights into the role that disulfide bonds play in the refolding pathway of the latter protein. Here we report the (1)H, (13)C and (15)N backbone resonance assignments for lambda lysozyme by heteronuclear multidimensional NMR spectroscopy. These assignments provide the starting point for detailed investigation of the refolding pathway using pulse-labelling hydrogen/deuterium exchange experiments monitored by NMR.

Published
2010
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