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1. Antigen self-anchoring onto bacteriophage T5 capsid-like particles for vaccine design

Author

Emeline Vernhes, Linda Larbi Chérif, Nicolas Ducrot, Malika Ouldali, Lena Zig, N’diaye Sidibe, Sylviane Hoos, Luis Ramirez-Chamorro, Madalena Renouard, Ombeline Rossier, Patrick England, Guy Schoehn, Pascale Boulanger, Karim Benihoud, 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), Aspects métaboliques et systémiques de l'oncogénèse pour de nouvelles approches thérapeutiques (METSY), Institut Gustave Roussy (IGR)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Biophysique Moléculaire (Plate-forme), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), 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), This work was supported by the Centre National de la Recherche Scientifique and the University Paris-Saclay. L. L.C., E.V. and N.D. received a fellowship from the French Ministère de l’Enseignement Supérieur et de la Recherche. L. L.C.received an additional fellowship from the Ligue Nationale contre le cancer. L.R.-C received a fellowship 'Becas Don Carlos Antonio Lopez' from the Paraguayan Ministry of Education. This work benefited from the Cryo-EM platform of I2BC, supported by the French Infrastructure for Integrated Structural Biology (FRISBI) [ANR-10-INSB-05-05] and member of IBISA. This work used the platforms of the Grenoble Instruct-ERIC center (ISBG, UAR3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB), supported by FRISBI (ANR-10-INSB-05-02 & project ID 160 to P. B.) and GRAL, financed within the University Grenoble Alpes graduate school (Écoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003). The electron microscope facility is supported by the Auvergne-Rhône-Alpes Region, the Fondation pour la Recherche Médicale (FRM), the fonds FEDER and the GIS-Infrastructures en Biologie Santé et Agronomie (IBiSA)., ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), and ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017)

Subjects
[SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, [SDV.IMM]Life Sciences [q-bio]/Immunology, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology
Abstract

The promises of vaccines based on virus-like particles stimulate demand for universal non-infectious virus-like platforms that can be efficiently grafted with large antigens. Here we harnessed the modularity and extreme affinity of the decoration protein pb10 for the capsid of bacteriophage T5. SPR experiments demonstrated that pb10 fused to mCherry or to the model antigen ovalbumin (Ova) retained picomolar affinity for DNA-free T5 capsid-like particles (T5-CLPs), while cryo-EM studies attested to the full occupancy of the 120 capsid binding sites. Mice immunisation with CLP-bound pb10-Ova chimeras elicited strong long-lasting anti-Ova humoral responses involving a large panel of isotypes, as well as CD8+T cell responses, without any extrinsic adjuvant. Therefore, T5-CLP constitutes the first DNA-free bacteriophage capsid able to irreversibly display a regular array of large antigens through highly efficient chemical-free anchoring. Its ability to elicit robust immune responses paves the way for further development of this novel vaccination platform.

Published
2022

2. Dynamics and structural changes of calmodulin upon interaction with the antagonist calmidazolium

Author

Corentin Léger, Irène Pitard, Mirko Sadi, Nicolas Carvalho, Sébastien Brier, Ariel Mechaly, Dorothée Raoux-Barbot, Maryline Davi, Sylviane Hoos, Patrick Weber, Patrice Vachette, Dominique Durand, Ahmed Haouz, J. Iñaki Guijarro, Daniel Ladant, Alexandre Chenal, Biochimie des Interactions Macromoléculaires / Biochemistry of Macromolecular Interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Plateforme Technologique de RMN Biologique et HDX-MS - Biological NMR and HDX-MS Technological Platform, Cristallographie (Plateforme) - Crystallography (Platform), Biophysique Moléculaire (plateforme) - Molecular Biophysics (platform), 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), Agence Nationale de la Recherche (ANR 21-CE11-0014-01-TransCyaA, CACSICE Equipex ANR-11-EQPX-0008). CNRS (UMR 3528). Institut Pasteur (PTR 166-19, DARRI-Emergence S-PI15006-12B, PPUIP program), ANR-21-CE11-0014,3DTransCyaA,Structures et mécanisme de translocation de la toxine CyaA(2021), and ANR-11-EQPX-0008,CACSICE,Centre d'analyse de systèmes complexes dans les environnements complexes(2011)

Subjects
CaM, Physiology, [SDV]Life Sciences [q-bio], Imidazoles, Structure, Cell Biology, Plant Science, Protein dynamics, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, General Biochemistry, Genetics and Molecular Biology, Calmodulin antagonist, X-Ray Diffraction, Calmodulin, Calmidazolium, Structural Biology, CDZ, Calmodulin CaM Calmidazolium CDZ Calmodulin antagonist Structure Protein dynamics, Scattering, Small Angle, Calcium, General Agricultural and Biological Sciences, Ecology, Evolution, Behavior and Systematics, Protein Binding, Developmental Biology, Biotechnology
Abstract

Background Calmodulin (CaM) is an evolutionarily conserved eukaryotic multifunctional protein that functions as the major sensor of intracellular calcium signaling. Its calcium-modulated function regulates the activity of numerous effector proteins involved in a variety of physiological processes in diverse organs, from proliferation and apoptosis, to memory and immune responses. Due to the pleiotropic roles of CaM in normal and pathological cell functions, CaM antagonists are needed for fundamental studies as well as for potential therapeutic applications. Calmidazolium (CDZ) is a potent small molecule antagonist of CaM and one the most widely used inhibitors of CaM in cell biology. Yet, CDZ, as all other CaM antagonists described thus far, also affects additional cellular targets and its lack of selectivity hinders its application for dissecting calcium/CaM signaling. A better understanding of CaM:CDZ interaction is key to design analogs with improved selectivity. Here, we report a molecular characterization of CaM:CDZ complexes using an integrative structural biology approach combining SEC-SAXS, X-ray crystallography, HDX-MS, and NMR. Results We provide evidence that binding of a single molecule of CDZ induces an open-to-closed conformational reorientation of the two domains of CaM and results in a strong stabilization of its structural elements associated with a reduction of protein dynamics over a large time range. These CDZ-triggered CaM changes mimic those induced by CaM-binding peptides derived from physiological protein targets, despite their distinct chemical natures. CaM residues in close contact with CDZ and involved in the stabilization of the CaM:CDZ complex have been identified. Conclusion Our results provide molecular insights into CDZ-induced dynamics and structural changes of CaM leading to its inhibition and open the way to the rational design of more selective CaM antagonists. Graphical abstract Calmidazolium is a potent and widely used inhibitor of calmodulin, a major mediator of calcium-signaling in eukaryotic cells. Structural characterization of calmidazolium-binding to calmodulin reveals that it triggers open-to-closed conformational changes similar to those induced by calmodulin-binding peptides derived from enzyme targets. These results provide molecular insights into CDZ-induced dynamics and structural changes of CaM leading to its inhibition and open the way to the rational design of more selective CaM antagonists.

Published
2022

3. Structural basis for the allosteric inhibition of UMP kinase from Gram‐positive bacteria, a promising antibacterial target

Author

Patrick Walter, Ariel Mechaly, Julien Bous, Ahmed Haouz, Patrick England, Joséphine Lai‐Kee‐Him, Aurélie Ancelin, Sylviane Hoos, Bruno Baron, Stefano Trapani, Patrick Bron, Gilles Labesse, Hélène Munier‐Lehmann, Chimie et Biocatalyse, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Cristallographie (Plateforme) - Crystallography (Platform), Centre de Biologie Structurale [Montpellier] (CBS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Biophysique Moléculaire (Plate-forme), Biophysique Moléculaire (plateforme) - Molecular Biophysics (platform), This work was supported by the European Union’s Framework Programme for Research and Innovation Horizon 2020 (2014-2020) under the Marie Skłodowska-Curie Grant Agreement No. 675555, Accelerated Early staGe drug discovery (AEGIS) project (to HML). The project was also supported in part by the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Santé Et de la Recherche Médicale (INSERM), the Institut Pasteur and the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INBS-05. PW was the recipient of a PhD fellowship from the EU H2020 (2014-2020) under the Marie Skłodowska-Curie Grant Agreement No. 675555, Accelerated Early staGe drug discovery (AEGIS)., ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), and European Project: 675555,H2020,H2020-MSCA-ITN-2015,AEGIS(2016)

Subjects
crystal structure, allostery, [SDV]Life Sciences [q-bio], Cryoelectron Microscopy, Uridine Triphosphate, Cell Biology, nucleotide metabolism, Gram-Positive Bacteria, Biochemistry, Anti-Bacterial Agents, Adenosine Triphosphate, effector, Allosteric Regulation, cryo-EM single particle analysis, Humans, Amino Acid Sequence, Guanosine Triphosphate, enzyme regulation, Nucleoside-Phosphate Kinase, Uridine Monophosphate, Molecular Biology, UMP kinase
Abstract

International audience; Tuberculosis claims significantly more than one million lives each year. A feasible way to face the issue of drug resistance is the development of new antibiotics. Bacterial uridine 5’-monophosphate (UMP) kinase is a promis- ing target for novel antibiotic discovery as it is essential for bacterial sur- vival and has no counterpart in human cells. The UMP kinase from M. tuberculosis is also a model of particular interest for allosteric regula- tion with two effectors, GTP (positive) and UTP (negative). In this study, using X-ray crystallography and cryo-electron microscopy, we report for the first time a detailed description of the negative effector UTP-binding site of a typical Gram-positive behaving UMP kinase. Comparison between this snapshot of low affinity for Mg-ATP with our previous 3D-structure of the GTP-bound complex of high affinity for Mg-ATP led to a better understanding of the cooperative mechanism and the allosteric regulation of UMP kinase. Thermal shift assay and circular dichroism experiments corroborate our model of an inhibition by UTP linked to higher flexibility of the Mg-ATP-binding domain. These new structural insights provide valuable knowledge for future drug discovery strategies targeting bacterial UMP kinases.

Published
2022

4. Structural insights into the functional versatility of an FHA domain protein in mycobacterial signaling

Author

Bertrand Raynal, Brigitte Vulliez-Le Normand, Tristan Wagner, Nathalie Barilone, Sylviane Hoos, María-Natalia Lisa, V. Hindie, Pedro M. Alzari, Helen M. O'Hare, Gwenaëlle André-Leroux, Marco Bellinzoni, Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Université Paris Diderot - Paris 7 (UPD7)-Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Mathématiques et Informatique Appliquées du Génome à l'Environnement [Jouy-En-Josas] (MaIAGE), Institut National de la Recherche Agronomique (INRA), Biophysique Moléculaire (Plate-forme), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Leicester Tuberculosis Research Group (LTBRG) and Leicester Institute of Structural and Chemical Biology (LISCB), Department of Respiratory Science & Department of Molecular and Cell Biology, University of Leicester, This work was partially supported by grants from the Institut Pasteur and the CNRS. MNL received postdoctoral fellowships from EMBO (European Molecular Biology Organization) and FRM (Fondation pour la Recherche Medicale, France), Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris]-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], and Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris]

Subjects
Models, Molecular, Carboxy-Lyases, [SDV]Life Sciences [q-bio], Protein domain, Allosteric regulation, Protein Serine-Threonine Kinases, Crystallography, X-Ray, Biochemistry, 03 medical and health sciences, chemistry.chemical_compound, Bacterial Proteins, Amino Acid Sequence, Phosphorylation, Protein kinase A, Molecular Biology, Central element, 030304 developmental biology, Antigens, Bacterial, 0303 health sciences, Binding Sites, Phosphomimetics, 030306 microbiology, Forkhead Transcription Factors, Mycobacterium tuberculosis, Cell Biology, Protein Structure, Tertiary, Cell biology, chemistry, Docking (molecular), Mutation, Signal transduction, Protein Binding, Signal Transduction
Abstract

Forkhead-associated (FHA) domains are modules that bind to phosphothreonine (pThr) residues in signaling cascades. The FHA-containing mycobacterial protein GarA is a central element of a phosphorylation-dependent signaling pathway that redirects metabolic flux in response to amino acid starvation or cell growth requirements. GarA acts as a phosphorylation-dependent ON/OFF molecular switch. In its nonphosphorylated ON state, the GarA FHA domain engages in phosphorylation-independent interactions with various metabolic enzymes that orchestrate nitrogen flow, such as 2-oxoglutarate decarboxylase (KGD). However, phosphorylation at the GarA N-terminal region by the protein kinase PknB or PknG triggers autoinhibition through the intramolecular association of the N-terminal domain with the FHA domain, thus blocking all downstream interactions. To investigate these different FHA binding modes, we solved the crystal structures of the mycobacterial upstream (phosphorylation-dependent) complex PknB-GarA and the downstream (phosphorylation-independent) complex GarA-KGD. Our results show that the phosphorylated activation loop of PknB serves as a docking site to recruit GarA through canonical FHA-pThr interactions. However, the same GarA FHA–binding pocket targets an allosteric site on nonphosphorylated KGD, where a key element of recognition is a phosphomimetic aspartate. Further enzymatic and mutagenesis studies revealed that GarA acted as a dynamic allosteric inhibitor of KGD by preventing crucial motions in KGD that are necessary for catalysis. Our results provide evidence for physiological phosphomimetics, supporting numerous mutagenesis studies using such approaches, and illustrate how evolution can shape a single FHA-binding pocket to specifically interact with multiple phosphorylated and nonphosphorylated protein partners.

Published
2019

5. Structural basis for haem piracy from host haemopexin by Haemophilus influenzae

Author

Mohamed Chami, Silvia Zambolin, Sylviane Hoos, Ahmed Haouz, Philippe Delepelaire, Vincent Villeret, Bernard Clantin, Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Unité de Glycobiologie Structurale et Fonctionnelle UMR 8576 (UGSF), Université de Lille-Centre National de la Recherche Scientifique (CNRS), Center for Cellular Imaging and Nanoanalytics, University of Basel (Unibas), Biophysique Moléculaire (Plate-forme), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Cristallographie (Plateforme) - Crystallography (Platform), This work was supported by grants from ANR (HEMESTOCKEXCHANGE ANR-12-BSV3-0022-01 and LABEX DYNAMO ANR-11-LABEX-0011-01)., ANR-12-BSV3-0022,HEMESTOCKEXCHANGE,Hème et porphyrine chez des bactéries modèle: des fonctions aux mécanismes(2012), ANR-11-LABX-0011,DYNAMO,Dynamique des membranes transductrices d'énergie : biogénèse et organisation supramoléculaire.(2011), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Recherche Agronomique (INRA)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), CNRS, Université de Lille, Laboratoire de biologie physico-chimique des protéines membranaires [LBPC-PM (UMR_7099)], and Unité de Glycobiologie Structurale et Fonctionnelle UMR 8576 [UGSF]

Subjects
0301 basic medicine, Receptors, Peptide, Science, General Physics and Astronomy, Plasma protein binding, Heme, Biology, blood proteins, medicine.disease_cause, General Biochemistry, Genetics and Molecular Biology, Article, Haemophilus influenzae, 03 medical and health sciences, Bacterial Proteins, Microscopy, Electron, Transmission, X-Ray Diffraction, Haem transport, Heme metabolism, medicine, polycyclic compounds, Escherichia coli, Animals, Multidisciplinary, Molecular Structure, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], digestive, oral, and skin physiology, General Chemistry, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, 3. Good health, 030104 developmental biology, Biochemistry, Rabbits
Abstract

Haemophilus influenzae is an obligate human commensal/pathogen that requires haem for survival and can acquire it from several host haemoproteins, including haemopexin. The haem transport system from haem-haemopexin consists of HxuC, a haem receptor, and the two-partner-secretion system HxuB/HxuA. HxuA, which is exposed at the cell surface, is strictly required for haem acquisition from haemopexin. HxuA forms complexes with haem-haemopexin, leading to haem release and its capture by HxuC. The key question is how HxuA liberates haem from haemopexin. Here, we solve crystal structures of HxuA alone, and HxuA in complex with the N-terminal domain of haemopexin. A rational basis for the release of haem from haem-haemopexin is derived from both in vivo and in vitro studies. HxuA acts as a wedge that destabilizes the two-domains structure of haemopexin with a mobile loop on HxuA that favours haem ejection by redirecting key residues in the haem-binding pocket of haemopexin., Haemophilus influenzae requires haem, and acquires it from host haemoproteins including haemopexin. Here, the authors examine the haem transport system consisting of HxuA, HxuB and HxuC via the structures of HxuA in complex with haemopexin.

Published
2016

6. The catalytic domains of C lostridium sordellii lethal toxin and related large clostridial glucosylating toxins specifically recognize the negatively charged phospholipids phosphatidylserine and phosphatidic acid

Author

Carolina, Varela Chavez, Sylviane, Hoos, Georges Michel, Haustant, Alexandre, Chenal, Patrick, England, Arnaud, Blondel, Serge, Pauillac, D Borden, Lacy, Michel Robert, Popoff, Bactéries anaérobies et Toxines, Institut Pasteur [Paris] (IP), Biophysique Moléculaire (Plate-forme), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Biochimie des Interactions Macromoléculaires, Bioinformatique structurale - Structural Bioinformatics, Vanderbilt University School of Medicine [Nashville], This work was supported by Nucléaires, radiologiques, biologiques, chimiques risques (NRBC) 17.1.2 grant and PTR421 from Institut Pasteur. NRBC and programme transversal de recherche (PTR) fellowship was awarded to CVC., Institut Pasteur [Paris], and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)

Subjects
Anions, Lethal toxin, Binding Sites, Bacterial Toxins, Cell Membrane, Clostridial glucosylating toxins, MESH: Cell Membrane/metabolism, Phosphatidic Acids, MESH: Catalytic Domain, MESH: Enzyme-Linked Immunosorbent Assay, Enzyme-Linked Immunosorbent Assay, Phosphatidylserines, Surface Plasmon Resonance, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], MESH: Bacterial Toxins/metabolism, MESH: Surface Plasmon Resonance, MESH: Phosphatidylserines/metabolism, MESH: Binding Sites, MESH: Phosphatidic Acids/metabolism, MESH: Anions/metabolism, Catalytic Domain, MESH: Protein Binding, Protein Binding, Clostridium sordellii, Catalytic
Abstract

International audience; Clostridium sordellii lethal toxin (TcsL) is a potent virulence factor belonging to the large clostridial glucosylating toxin family. TcsL enters target cells via receptor-mediated endocytosis and delivers the N-terminal catalytic domain (TcsL-cat) into the cytosol upon an autoproteolytic process. TcsL-cat inactivates small GTPases including Rac and Ras by glucosylation with uridine-diphosphate (UDP)-glucose as cofactor leading to drastic changes in cytoskeleton and cell viability. TcsL-cat was found to preferentially bind to phosphatidylserine (PS)-containing membranes and to increase the glucosylation of Rac anchored to lipid membrane. We here report binding affinity measurements of TcsL-cat for brain PS-containing membranes by surface plasmon resonance and enzyme-linked immunosorbent assay (ELISA). In addition, TcsL-cat bound to phosphatidic acid (PA) and, to a lesser extent, to other anionic lipids, but not to neutral lipids, sphingolipids or sterol. We further show that the lipid unsaturation status influenced TcsL-cat binding to phospholipids, PS with unsaturated acyl chains and PA with saturated acyl chains being the preferred bindingsubstrates. Phospholipid binding site is localized at the N-terminal four helical bundle structure (1-93 domain). However, TcsL-1-93 bound to a broad range of substrates, whereas TcsL-cat, which is the active domain physiologically delivered into the cytosol, selectively bound to PS and PA. Similar findings were observed with the other large clostridial glucosylating toxins from C. difficile, C. novyi and C. perfringens.

Published
2015

7. Structural activation of the transcriptional repressor EthR from Mycobacterium tuberculosis by single amino acid change mimicking natural and synthetic ligands

Author

Camille Locht, René Wintjens, Patrick England, Guy Lippens, Xavier Carette, Sameh H. Soror, Nathalie Lecat-Guillet, Frédéric Frénois, Bertrand Dirié, Alain R. Baulard, Vincent Villeret, Alexandre Wohlkonig, Benoit Deprez, Eve Willery, Zoé Lens, Nicolas Blondiaux, Nicolas Willand, Sylviane Hoos, Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), Biophysique des Macromolécules et de leurs Interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut de Recherche Interdisciplinaire [Villeneuve d'Ascq] (IRI), Université de Lille, Sciences et Technologies-Université de Lille, Droit et Santé-Centre National de la Recherche Scientifique (CNRS), Vlaams Instituut voor Biotechnologie [Ghent, Belgique] (VIB), Université libre de Bruxelles (ULB), Université Lille Nord (France), Biostructures et Decouverte de Medicament, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille, Droit et Santé, Institut Pasteur [Paris] (IP), Unité de Glycobiologie Structurale et Fonctionnelle UMR 8576 (UGSF), Université de Lille-Centre National de la Recherche Scientifique (CNRS), We are indebted to the ESRF (Group BAG MX-485). Diffraction data for BDM31343 and EthR-BDM33066 were collected at the Swiss Light Source (Paul Scherrer Institute, Villigen, Switzerland) and at the ESRF (Group BAG MX-485), respectively.We are grateful to the machine and beamline scientists whose outstanding efforts have made these experiments possible., and ENGLAND, Patrick

Subjects
Models, Molecular, Protein Folding, In silico, MESH: Protein Folding, [SDV]Life Sciences [q-bio], Biologie structurale, Repressor, Biology, Crystallography, X-Ray, Ligands, chemistry.chemical_compound, Transcription (biology), MESH: Nuclear Magnetic Resonance, Biomolecular, Genetics, MESH: Ligands, TetR, Binding site, Nuclear Magnetic Resonance, Biomolecular, Molecular Biology, MESH: Mutagenesis, Molecular switch, Binding Sites, MESH: DNA, DNA, MESH: Crystallography, X-Ray, MESH: Amino Acid Substitution, Repressor Proteins, [SDV] Life Sciences [q-bio], Amino Acid Substitution, Biochemistry, chemistry, MESH: Binding Sites, Mutagenesis, MESH: Repressor Proteins, Biophysics, Protein folding, Biologie, MESH: Models, Molecular
Abstract

Ethionamide is an antituberculous drug for the treatment of multidrug-resistant Mycobacterium tuberculosis. This antibiotic requires activation by the monooxygenase EthA to exert its activity. Production of EthA is controlled by the transcriptional repressor EthR, a member of the TetR family. The sensitivity of M. tuberculosis to ethionamide can be artificially enhanced using synthetic ligands of EthR that allosterically inactivate its DNA-binding activity. Comparison of several structures of EthR co-crystallized with various ligands suggested that the structural reorganization of EthR resulting in its inactivation is controlled by a limited portion of the ligand-binding-pocket. In silico simulation predicted that mutation G106W may mimic ligands. X-ray crystallography of variant G106W indeed revealed a protein structurally similar to ligand-bound EthR. Surface plasmon resonance experiments established that this variant is unable to bind DNA, while thermal shift studies demonstrated that mutation G106W stabilizes EthR as strongly as ligands. Proton NMR of the methyl regions showed a lesser contribution of exchange broadening upon ligand binding, and the same quenched dynamics was observed in apo-variant G106W. Altogether, we here show that the area surrounding Gly106 constitutes the molecular switch involved in the conformational reorganization of EthR. These results also shed light on the mechanistic of ligand-induced allosterism controlling the DNA binding properties of TetR family repressors., JOURNAL ARTICLE, SCOPUS: ar.j, info:eu-repo/semantics/published

Published
2011

8. Dynamic aspects of antibody:oligosaccharide complexes characterized by molecular dynamics simulations and saturation transfer difference nuclear magnetic resonance

Author

Laurence A. Mulard, Brigitte Vulliez-Le Normand, Catherine Guerreiro, Muriel Delepierre, François-Xavier Theillet, Farida Nato, Martin Frank, Alain Chaffotte, Catherine Simenel, Armelle Phalipon, Frédéric Bélot, Sylviane Hoos, Résonance Magnétique Nucléaire des Biomolécules, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), German Cancer Research Center - Deutsches Krebsforschungszentrum [Heidelberg] (DKFZ), Immunologie structurale, Biophysique des Macromolécules et de leurs Interactions, Chimie Organique, Chimie des biomolécules, Production de Protéines Recombinantes et d'Anticorps (Plate-Forme), Institut Pasteur [Paris], Pathogénie Microbienne Moléculaire, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), This work was supported by funds from the Institut Pasteur, the Centre National de la Recherche Scientifique and the Ministère National de la Recherche. CPU time for the MD simulations was provided by DFKZ Heidelberg., We thank Dr C. Costachel, Dr F. Segat-Dioury and Dr K. Wright for their contribution to the synthesis of the SF2a oligosaccharides, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP), and Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
medicine.drug_class, Oligosaccharides, Molecular Dynamics Simulation, 010402 general chemistry, Monoclonal antibody, 01 natural sciences, Biochemistry, Epitope, Shigella flexneri, 03 medical and health sciences, Molecular dynamics, Nuclear magnetic resonance, Antigen, medicine, Nuclear Magnetic Resonance, Biomolecular, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, MD simulations, STD-NMR, biology, MESH: Nuclear Magnetic Resonance, Biomolecular, MESH: Antibodies, Monoclonal / chemistry, MESH: Molecular Dynamics Simulation, Antibodies, Monoclonal, Oligosaccharide, O-antigen, biology.organism_classification, Ligand (biochemistry), Affinities, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, 0104 chemical sciences, chemistry, monoclonal antibody, MESH: Oligosaccharides / chemistry
Abstract

International audience; Carbohydrates are likely to maintain significant conformational flexibility in antibody (Ab):carbohydrate complexes. As demonstrated herein for the protective monoclonal Ab (mAb) F22-4 recognizing the Shigella flexneri 2a O-antigen (O-Ag) and numerous synthetic oligosaccharide fragments thereof, the combination of molecular dynamics simulations and nuclear magnetic resonance saturation transfer difference experiments, supported by physicochemical analysis, allows us to determine the binding epitope and its various contributions to affinity without using any modified oligosaccharides. Moreover, the methods used provide insights into ligand flexibility in the complex, thus enabling a better understanding of the Ab affinities observed for a representative set of synthetic O-Ag fragments. Additionally, these complementary pieces of information give evidence to the ability of the studied mAb to recognize internal as well as terminal epitopes of its cognate polysaccharide antigen. Hence, we show that an appropriate combination of computational and experimental methods provides a basis to explore carbohydrate functional mimicry and receptor binding. The strategy may facilitate the design of either ligands or carbohydrate recognition domains, according to needed improvements of the natural carbohydrate:receptor properties.

Published
2011

9. Crystal Structure of an Affinity-matured Prolactin Complexed to Its Dimerized Receptor Reveals the Topology of Hormone Binding Site 2

Author

Vincent Goffin, Isabelle Broutin, Estelle Tallet, Paul A. Kelly, Patrick England, Johannes F. Van Agthoven, Sylviane Hoos, Jean-Baptiste Jomain, Birthe B. Kragelund, Bertrand Raynal, Arnaud Ducruix, Laboratoire de cristallographie et RMN biologiques (LCRB - UMR 8015), Université Paris Descartes - Paris 5 (UPD5)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Centre de recherche Croissance et signalisation (UMR_S 845), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Biophysique des Macromolécules et de leurs Interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), University of Copenhagen = Københavns Universitet (UCPH), This work was supported in part by INSERM, University Paris Descartes, and the Agence Nationale de la Recherche (Grant ANR-PCV07_183953)., Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Descartes - Paris 5 (UPD5), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), and University of Copenhagen = Københavns Universitet (KU)

Subjects
[SDV]Life Sciences [q-bio], MESH: Protein Structure, Secondary, Ligand Binding Protein, MESH: Amino Acid Sequence, MESH: Drug Design, Biochemistry, Protein Structure, Secondary, MESH: Recombinant Proteins, MESH: Protein Structure, Tertiary, 0302 clinical medicine, Protein structure, MESH: Structure-Activity Relationship, X-Ray Diffraction, MESH: Animals, Placental lactogen, Receptor, Peptide sequence, 0303 health sciences, Crystallography, MESH: Crystallography, MESH: X-Ray Diffraction, Ligand (biochemistry), Recombinant Proteins, MESH: Glycine, 3. Good health, MESH: Surface Plasmon Resonance, MESH: Mutagenesis, Site-Directed, Protein Structure and Folding, Dimerization, hormones, hormone substitutes, and hormone antagonists, MESH: Placental Lactogen, endocrine system, MESH: Rats, Stereochemistry, Receptors, Prolactin, Molecular Sequence Data, Glycine, MESH: Prolactin, MESH: Sheep, Biology, 03 medical and health sciences, Structure-Activity Relationship, MESH: Receptors, Prolactin, Structure–activity relationship, Animals, Humans, Amino Acid Sequence, Binding site, Molecular Biology, 030304 developmental biology, Binding Sites, Sheep, MESH: Humans, MESH: Molecular Sequence Data, Cell Biology, Surface Plasmon Resonance, Placental Lactogen, Prolactin, Protein Structure, Tertiary, Rats, MESH: Binding Sites, MESH: Dimerization, Drug Design, Mutagenesis, Site-Directed, 030217 neurology & neurosurgery
Abstract

International audience; We report the first crystal structure of a 1:2 hormone.receptor complex that involves prolactin (PRL) as the ligand, at 3.8-A resolution. Stable ternary complexes were obtained by generating affinity-matured PRL variants harboring an N-terminal tail from ovine placental lactogen, a closely related PRL receptor (PRLR) ligand. This structure allows one to draw up an exhaustive inventory of the residues involved at the PRL.PRLR site 2 interface, consistent with all previously reported site-directed mutagenesis data. We propose, with this description, an interaction model involving three structural components of PRL site 2 ("three-pin plug"): the conserved glycine 129 of helix alpha3, the hydrogen bond network involving surrounding residues (glycine cavity), and the N terminus. The model provides a molecular basis for the properties of the different PRL analogs designed to date, including PRLR antagonists. Finally, comparison of our 1:2 PRL.PRLR(2) structure with those of free PRL and its 1:1 complex indicates that the structure of PRL undergoes significant changes when binding the first, but not the second receptor. This suggests that the second PRLR moiety adapts to the 1:1 complex rather than the opposite. In conclusion, this structure will be a useful guiding tool for further investigations of the molecular mechanisms involved in PRLR dimerization and activation, as well as for the optimization of PRLR antagonists, an emerging class of compounds with high therapeutic potential against breast and prostate cancer.

Published
2010

10. Novel svVEGF isoforms from Macrovipera lebetina venom interact with neuropilins

Author

Zohra Aloui, Hafedh Mejdoub, Habib Kharmachi, Pierre Yves Haumont, Ammar Gasmi, Patrick England, Michael Klagsbrun, Elena Geretti, Sylviane Hoos, Institut Pasteur de Tunis, Réseau International des Instituts Pasteur (RIIP), Biophysique des Macromolécules et de leurs Interactions, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Vascular Biology Program, Department of Surgery and Pathology, Harvard Medical School [Boston] (HMS), Applied Biosystems, Unité de Service Commun pour la Recherche, Faculté des Sciences de Sfax, Université de Sfax - University of Sfax-Université de Sfax - University of Sfax, and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Vascular Endothelial Growth Factor A, Gene isoform, Neuropilins, MESH: Rats, Biophysics, MESH: Viperidae, Vascular permeability, Venom, MESH: Neuropilin-2, Viper Venoms, MESH: Neuropilin-1, Biochemistry, Capillary Permeability, Mice, 03 medical and health sciences, MESH: Mice, Inbred C57BL, Neuropilin 1, Viperidae, Animals, MESH: Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Rats, Wistar, Receptor, Molecular Biology, MESH: Mice, 030304 developmental biology, 0303 health sciences, biology, MESH: Vascular Endothelial Growth Factor Receptor-2, MESH: Vascular Endothelial Growth Factor A, 030302 biochemistry & molecular biology, MESH: Capillary Permeability, Cell Biology, MESH: Rats, Wistar, biology.organism_classification, Vascular Endothelial Growth Factor Receptor-2, Neuropilin-1, Neuropilin-2, Rats, Mice, Inbred C57BL, Snake venom, MESH: Viper Venoms, Macrovipera lebetina
Abstract

International audience; Increased vascular permeability and vasodilation are responses usually elicited by snake envenomation. In this report, we isolated from Macroviperalebetina venom two protein groups designated IC1 (Increasing Capillary1) and IC2 based on their activities on capillary permeability. Mass spectrometry analysis showed that IC1 contained four major proteins of 23,650, 24,306, 24,589 and 24,718Da, whereas IC2 contained three major proteins of 25,101, 25,194 and 25,298Da. N-terminal amino-acid sequencing revealed that IC1 and IC2 belong to the snake venom VEGF (svVEGF) family. IC1 and IC2 had a marked specificity for VEGFR-2, with affinities in the nanomolar range. Interestingly, they also bind to NRP1 and NRP2, with affinities in the micromolar range. This is the first report demonstrating that M. lebetina encodes several distinct svVEGFs, endowed with a capacity to interact with neuropilins. IC1 and IC2 could be valuable tools to understand the molecular properties of angiogenic factors and their receptors.

Published
2009

11. Structural mimicry of O-antigen by a peptide revealed in a complex with an antibody raised against Shigella flexneri serotype 2a

Author

Muriel Delepierre, Armelle Phalipon, Brigitte Vulliez-Le Normand, Frederick Saul, François-Xavier Theillet, Andrej Weintraub, Sylviane Hoos, Graham A. Bentley, Franco Felici, Laurence A. Mulard, Résonance Magnétique Nucléaire des Biomolécules, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Immunologie structurale, Biophysique des Macromolécules et de leurs Interactions, Università degli Studi del Molise (Unimol), Division of Clinical Bacteriology [Stockholm], Karolinska University Hospital [Stockholm]-Department of Laboratory Medicine [Karolinska Institutet], Karolinska Institutet [Stockholm]-Karolinska Institutet [Stockholm], Chimie des biomolécules, Pathogénie Microbienne Moléculaire, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), This work was supported by funds from the Institut Pasteur, Centre National de la Recherche Scientifique, and the Ministère National de la Recherche et de la Technologie., We thank the staff of the ESRF (Grenoble, France) for providing facilities for diffraction measurements and for assistance., We thank Dr. Patrick England for valuable help in the analysis of SPR and ITC data. We thank Dr. Frédéric Bélot and Catherine Guerreiro for their contribution to the synthesis of oligosaccharides., Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Università degli Studi del Molise = University of Molise (UNIMOL), and Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
Phage display, MESH: Shigella flexneri, Protein Conformation, Protein Data Bank (RCSB PDB), Molecular Conformation, Peptide, medicine.disease_cause, Crystallography, X-Ray, 01 natural sciences, Shigella flexneri, Mice, Protein structure, MESH: Protein Conformation, Structural Biology, MESH: Nuclear Magnetic Resonance, Biomolecular, MESH: Animals, chemistry.chemical_classification, 0303 health sciences, MESH: Peptides, [CHIM.ORGA]Chemical Sciences/Organic chemistry, O Antigens, Molecular mimicry, Biochemistry, Carbohydrate Sequence, Thermodynamics, MESH: Molecular Mimicry, [SDV.IMM]Life Sciences [q-bio]/Immunology, carbohydrate–peptide mimicry, MESH: Thermodynamics, crystal structure, Molecular Sequence Data, Biology, 010402 general chemistry, Antibodies, shigellosis, 03 medical and health sciences, Antigen, medicine, Animals, Molecular Biology, Nuclear Magnetic Resonance, Biomolecular, MESH: Mice, 030304 developmental biology, MESH: O Antigens, MESH: Carbohydrate Sequence, MESH: Molecular Conformation, MESH: Molecular Sequence Data, MESH: Antibodies, Molecular Mimicry, solution NMR structure, biology.organism_classification, MESH: Crystallography, X-Ray, 0104 chemical sciences, chemistry, Docking (molecular), Peptides, antibody complex
Abstract

International audience; The use of carbohydrate-mimicking peptides to induce immune responses against surface polysaccharides of pathogenic bacteria offers a novel approach to vaccine development. Factors governing antigenic and immunogenic mimicry, however, are complex and poorly understood. We have addressed this question using the anti-lipopolysaccharide monoclonal antibody F22-4, which was raised against Shigella flexneri serotype 2a and shown to protect against homologous infection in a mouse model. In a previous crystallographic study, we described F22-4 in complex with two synthetic fragments of the O-antigen, the serotype-specific saccharide moiety of lipopolysaccharide. Here, we present a crystallographic and NMR study of the interaction of F22-4 with a dodecapeptide selected by phage display using the monoclonal antibody. Like the synthetic decasaccharide, the peptide binds to F22-4 with micromolar affinity. Although the peptide and decasaccharide use very similar regions of the antigen-binding site, indicating good antigenic mimicry, immunogenic mimicry by the peptide was not observed. The F22-4-antigen interaction is significantly more hydrophobic with the peptide than with oligosaccharides; nonetheless, all hydrogen bonds formed between the peptide and F22-4 have equivalents in the oligosaccharide complex. Two bridging water molecules are also in common, adding to partial structural mimicry. Whereas the bound peptide is entirely helical, its structure in solution, as shown by NMR, is helical in the central region only. Moreover, docking the NMR structure into the antigen-binding site shows that steric hindrance would occur, revealing poor complementarity between the major solution conformation and the antibody that could contribute to the absence of immunogenic mimicry.

Published
2009

12. A global benchmark study using affinity-based biosensors

Author

Aykut Üren, Stephen G. Brohawn, Eva Muñoz, Kenneth Miller, Mike Scott, Heather Hughes, Yasmina Noubia Abdiche, Rohn Lee Millican, Anne W Emerick, Jonathan Brooks, Michael B. Murphy, Charlene S. Lee, Carmelo Di Primo, Joshua Ballard, Quyhn Trinh, Giuseppe A. Papalia, Cynthia S. Hinck, Kevin Thompson, Peter Flynn, Joshua S. Klein, Martin A. Wear, Mark Toews, Richard N. Bohnsack, Mohammed Yousef, Daniel Malashock, Gaetano Barbato, Jill Raymond, Alison Joyce, Min Hsiang Yang, Julie Rothacker, Patrick England, Ashique Rafique, Jacinto López-Sagaseta, Scott L. Klakamp, Daulet Satpaev, Monica Dines, Danny Terwey, Islay Campbell, Joshua Thompson, Ben Busby, Tanmoy Ganguly, Gerardo Gutierrez-Sanchez, Sergei Shikov, Kevin Lindquist, Momchilo Vuyisich, Asya Grinberg, Krista Witte, Yun Hee Cho, Yue Ji Li, Hubert Mantz, Ewa Pol, Gonzalo Obal, Brian J. Pak, María J. Hernáiz, Peter Kainz, Erk Gedig, Mary M. Murphy, Tsafir Bravman, Janus Krarup, Gabrielle Zeder-Lutz, John Quinn, Thomas E. Ryan, Pietro Brandani, Dawn Kernaghan, Anca Clabbers, M. Brent Waddell, Agnes Puskas, Petr Skládal, Sanjay Nilapwar, Kara Herlihy, Gregor Anderluh, Federico Torta, Eric Hommema, Andrej Bavdek, Rejane Guimaraes, Anthony M. Giannetti, Eric Fang, Henrik Anderson, Anne Birgitte Bagge Hagel, Schuyler B Corry, Mark R. Witmer, Phillippe Neuner, Sébastien Wieckowski, Nico Dankbar, Alanna Pinkerton, Suparna Mundodo, Trevor D. Chapman, Sylvie Canepa, Satya P. Yadav, Rostislav Skrabana, Oleksandr Kalyuzhniy, Phini S Katsamba, Vidya Chandrasekaran, Eugene G. Chomey, Dana Reichmann, Katy McGirr, Dotzlaf Joe Edward, Mireille Baltzinger, Ite A. Laird-Offringa, Liann Wang, Erica Boni, Tiffany Tsang, Zaneta Nikolovska-Coleska, Andreas Schoenemann, Yuki Abe, Jamie Furneisen, Kenneth T. Lewis, Eileen M. Lafer, John Corbin, Satyen Gautam, Yuguo Feng, Olan Dolezal, Sylviane Hoos, James R. Horn, Bianca Beusink, Jinlin Peng, Otto Pritsch, Kristian H. Schlick, Ryan James Darling, Malgorzata Mikolajczyk, Quincy L. Carter, Jason T. Schuman, Yang Liu, Abdelkrim Khadir, Loïc Martin, Mark Alan Lewis, Ganeshram Krishnamoorthy, Andrew W. Drake, Jason Baardsnes, George Korza, Jesper Pass, Judie Berlier, Melicia Gainey, Nguyen Ly, James R. Partridge, Rosy Calvert, Roberta D'Agata, Rebecca L. Rich, Monica E. Ferreira, Phillip Page, Frank John Podlaski, Jay Duffner, Ruchira Das Gupta, Melanie Wong, Sergei Bibikov, Jiejin Li, Paola Torreri, Bruce A. Andrien, Peter Spies, Christina Boozer, David G. Myszka, University of Utah School of Medicine [Salt Lake City], KaloBios Pharmaceuticals [San Francisco], Schering–Plough Biopharma [Palo Alto], Nomadics [Oklahoma City], Biochemistry and Molecular Biophysics [Pasadena], California Institute of Technology (CALTECH), Department of Biochemistry and Molecular Biophysics, Columbia University [New York], Hartwell Center for Bioinformatics and Biotechnology [Memphis], St. Jude Children’s Research Hospital [Memphis], Universität Zürich [Zürich] = University of Zurich (UZH), Molecular Probes/Invitrogen [Eugene], Institut de génétique et biologie moléculaire et cellulaire (IGBMC), Université Louis Pasteur - Strasbourg I-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), MerckKGaA, Merck & Co. Inc, Abbott Bioresearch Center [Worcester], Institut de biologie cellulaire et moléculaire [Strasbourg], Université de Strasbourg (UNISTRA), Reichert [Depew}, Momenta Pharmaceuticals [Cambridge], XOMA (U.S.), Department of Chemical and Biomolecular Engineering, National University of Singapore, National University of Singapore (NUS), Department of Biology [Ljubljiana], Biotechnical Faculty, University of Ljubljana, Department of Biological Chemistry (Weizmann Institute of Science), Weizmann Institute of Science [Rehovot, Israël], Molecular Biotechnology Core Laboratory [Cleveland], Cleveland Clinic Foundation, ThermoFisher Scientific [Rockford], Biacore/GE Healthcare [Uppsala], AstraZeneca [Hayward], Neurodegeneration Research Department [Harlow], GlaxoSmithKline [Harlow], MedImmune, Biacore/GE Healthcare [Piscataway], Department of Biochemistry [Milwaukee], Medical College of Wisconsin [Milwaukee] (MCW), Alexion Pharmaceuticals [Cheshire], Applied Biosystems [Foster City], diaDexus [South San Francisco], Eli Lilly and Company [Indianapolis], Center for Applied Medical Research [Plamplona] (CIMA), Universidad de Navarra [Pamplona] (UNAV), EMD Lexigen Research Center [Billerica], Department of Cell Biology and Neuroscience [Rome], Istituto Superiore di Sanita [Rome], Département de Biologie structurale et Chimie - Department of Structural Biology and Chemistry, Institut Pasteur [Paris], Department of Chemistry [Atlanta], Georgia State University, University System of Georgia (USG)-University System of Georgia (USG), Pfizer/Rinat Laboratories [South San Francisco], PDLBioPharma [Fremont], Department of Biochemistry [San Antonio], University of Texas Health Science Center at San Antonio [San Antonio], Akubio [Cambridge], ARN : régulations naturelle et artificielle, Université Bordeaux Segalen - Bordeaux 2-Institut Européen de Chimie et de Biologie-Institut National de la Santé et de la Recherche Médicale (INSERM), Wyeth Research [Cambridge], Department of Biochemistry and Molecular Biology [Odense], University of Southern Denmark (SDU), NovoNordisk [Gentofte], Södertörns University College [Huddinge], Department of Biochemistry [Philadelphia], Temple University [Philadelphia], Pennsylvania Commonwealth System of Higher Education (PCSHE)-Pennsylvania Commonwealth System of Higher Education (PCSHE), U.S. Food and Drug Administration (FDA), Department of Biochemical Engineering [London], University College of London [London] (UCL), Merck [Rome], Roche [Palo Alto], University of Twente [Netherlands], Agensys [Santa Monica], Novartis [Emeryville], Massachusetts Institute of Technology (MIT), Northern Illinois University, Institut Pasteur de Montevideo, Réseau International des Instituts Pasteur (RIIP), University of Manchester [Manchester], Department of Physiology [Baltimore], University of Maryland [Baltimore], Complex Carbohydrate Research Center [Athens, GA, USA], University of Georgia [USA], Adnexus Therapeutics [Waltham}, Physiologie de la reproduction et des comportements [Nouzilly] (PRC), Institut National de la Recherche Agronomique (INRA)-Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS), ForteBio [Menlo Park], University of Michigan [Ann Arbor], University of Michigan System, Human Genome Sciences [Rockville], Department of Chemical Sciences [Catania], University of Catania [Italy], Montana State University (MSU), GKT School of Biomedical Sciences [London], Organic and Pharmaceutical Chemistry Department [Madrid], Universidad Complutense de Madrid = Complutense University of Madrid [Madrid] (UCM), Bio-Rad Haifa, Institute of Chemistry [Taipei], Academia Sinica, Department of Microbiology and Molecular Genetics, The University of Texas Health Science Center at Houston (UTHealth), Fred Hutchinson Cancer Research Center [Seattle] (FHCRC), Division of Molecular Structure [London], National Institute of Medical Research (UK), Centre for Translational and Chemical Biology, Institute of Structural and Molecular Biology [Edinburgh], University of Edinburgh, Acceleron Pharma [Cambridge], Biotechnology Research Institute [Montreal], National Research Council of Canada (NRC), CSIRO Health Sciences and Nutrition [Parkville], Battelle Biomedical Research Center [Columbus], Attana AB [Stockholm], Corning [USA], School of Life Sciences, Institute for Chemistry and Bioanalytics [Muttenz], University of Applied Sciences Northwestern Switzerland (FHNW), Bio-Rad [Hercules], Monsanto [Chesterfield], Genzyme [Cambridge], Saarland University [Saarbrücken], Institute of Neuroimmunology of SAS [Bratislava], Bristol-Myers Squibb [Princeton], Array Biopharma [Boulder], Service d'Ingénierie Moléculaire pour la Santé (ex SIMOPRO) (SIMoS), 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), Department of biochemistry, Masaryk University, Masaryk University [Brno] (MUNI), University of Connecticut Health Center [Farmington], University of Southern California, Los Angeles, GeminX Biotechnologies [Montreal], Hoffman–La Roche [Nutley], IRBM, Pomezia [Rome], Ludwig Institute for Cancer Research, Regeneron Pharmaceuticals [Tarrytown], University of Muenster, Department of Molecular Biology [Salzburg], University of Salzburg, XanTec Bioanalytics [Muenster], Biosciences Division [Los Alamos], Los Alamos National Laboratory (LANL), Lumera [Bothell], Arizona State University [Tempe] (ASU), Dyax [Cambridge], Lombardi Comprehensive Cancer Center, Georgetown University, Department of Biochemistry [Seattle], University of Washington [Seattle], ZymoGenetics, Bio-Rad Canada [Edmonton], Bio-Rad Canada [Toronto], St Jude Children's Research Hospital, University of Ljubljana, Istituto Superiore di Sanità (ISS), Institut Pasteur [Paris] (IP), University of Texas Health Science Center at San Antonio [San Antonio, Tx, USA], University of Twente, Institut National de la Recherche Agronomique (INRA)-Institut Français du Cheval et de l'Equitation [Saumur] (IFCE)-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS), University of Southern California (USC), Georgetown University [Washington] (GU), Pasteur Tunis, Institut, Institut National de la Recherche Agronomique (INRA)-Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours-Centre National de la Recherche Scientifique (CNRS), University of Zürich [Zürich] (UZH), Weizmann Institute of Science, Medical College of Wisconsin [Milwaukee], Centre National de la Recherche Scientifique (CNRS)-Université de Tours-Institut Français du Cheval et de l'Equitation [Saumur]-Institut National de la Recherche Agronomique (INRA), Complutense University of Madrid (UCM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-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), and Masaryk University

Subjects
Analyte, Biophysics, Optical biosensor, Antibodies, Catalytic, Biosensing Techniques, Ligands, 01 natural sciences, Biochemistry, Article, Biacore, Glutathione transferase, 03 medical and health sciences, Surface plasmon resonance, Molecular Biology, Kinetic rate constant, 030304 developmental biology, Glutathione Transferase, 0303 health sciences, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, Chromatography, Binding Sites, Chemistry, 010401 analytical chemistry, A protein, Proteins, Cell Biology, 0104 chemical sciences, Benchmarking, Kinetics, Yield (chemistry), Benchmark (computing), IR-68798, Biosensor, EWI-16945, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract

International audience; To explore the variability in biosensor studies, 150 participants from 20 countries were given the same protein samples and asked to determine kinetic rate constants for the interaction. We chose a protein system that was amenable to analysis using different biosensor platforms as well as by users of different expertise levels. The two proteins (a 50-kDa Fab and a 60-kDa glutathione S-transferase [GST] antigen) form a relatively high-affinity complex, so participants needed to optimize several experimental parameters, including ligand immobilization and regeneration conditions as well as analyte concentrations and injection/dissociation times. Although most participants collected binding responses that could be fit to yield kinetic parameters, the quality of a few data sets could have been improved by optimizing the assay design. Once these outliers were removed, the average reported affinity across the remaining panel of participants was 620 pM with a standard deviation of 980 pM. These results demonstrate that when this biosensor assay was designed and executed appropriately, the reported rate constants were consistent, and independent of which protein was immobilized and which biosensor was used. (C) 2008 Elsevier Inc. All rights reserved.

Published
2009

13. Structural and Thermodynamic Bases for the Design of Pure Prolactin Receptor Antagonists

Author

Estelle Tallet, Patrick England, Arnaud Ducruix, Johannes F. Van Agthoven, Sylviane Hoos, Birthe B. Kragelund, Vincent Goffin, Isabelle Broutin, Paul A. Kelly, Jean-Baptiste Jomain, Centre de recherche Croissance et signalisation (UMR_S 845), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de cristallographie et RMN biologiques (LCRB - UMR 8015), Université Paris Descartes - Paris 5 (UPD5)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Biophysique des Macromolécules et de leurs Interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), University of Copenhagen = Københavns Universitet (UCPH), This work was supported in part by INSERM, University Paris Descartes, the Comité de Paris de la Ligue Nationale Contre le Cancer Grant R05/75-15, and the Danish Natural Research Council Grant 21040604. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked 'advertisement' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact., Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Descartes - Paris 5 (UPD5), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), and University of Copenhagen = Københavns Universitet (KU)

Subjects
MESH: Mutation, MESH: Rats, [SDV]Life Sciences [q-bio], MESH: Prolactin, Context (language use), MESH: Amino Acid Sequence, MESH: Drug Design, MESH: Hot Temperature, Biochemistry, MESH: Circular Dichroism, 03 medical and health sciences, MESH: Protein Structure, Tertiary, MESH: Receptors, Prolactin, MESH: Protein Binding, MESH: Animals, Receptor, Molecular Biology, 030304 developmental biology, MESH: Structural Homology, Protein, 0303 health sciences, MESH: Molecular Sequence Data, MESH: Humans, MESH: Kinetics, Chemistry, Prolactin receptor, 030302 biochemistry & molecular biology, Wild type, Antagonist, Cell Biology, MESH: Crystallography, X-Ray, Affinities, Prolactin, MESH: Glycine, MESH: Cell Line, MESH: Protein Denaturation, MESH: Thermodynamics, Antagonism, MESH: Models, Molecular
Abstract

International audience; Competitive antagonists of the human prolactin (hPRL) receptor are a novel class of molecules of potential therapeutic interest in the context of cancer. We recently developed the pure antagonist Del1-9-G129R-hPRL by deleting the nine N-terminal residues of G129R-hPRL, a first generation partial antagonist. We determined the crystallographic structure of Del1-9-G129R-hPRL, which revealed no major change compared with wild type hPRL, indicating that its pure antagonistic properties are intrinsically due to the mutations. To decipher the molecular bases of pure antagonism, we compared the biological, physicochemical, and structural properties of numerous hPRL variants harboring N-terminal or Gly(129) mutations, alone or combined. The pure versus partial antagonistic properties of the multiple hPRL variants could not be correlated to differences in their affinities toward the hPRL receptor, especially at site 2 as determined by surface plasmon resonance. On the contrary, residual agonism of the hPRL variants was found to be inversely correlated to their thermodynamic stability, which was altered by all the Gly(129) mutations but not by those involving the N terminus. We therefore propose that residual agonism can be abolished either by further disrupting hormone site 2-receptor contacts by N-terminal deletion, as in Del1-9-G129R-hPRL, or by stabilizing hPRL and constraining its intrinsic flexibility, as in G129V-hPRL.

Published
2007

14. Structure-based optimization of a PDZ binding motif within a viral peptide stimulates neurite outgrowth

Author

Cynthia Lefebvre-Omar, Alain Chaffotte, Florent Delhommel, Christophe Prehaud, Elouan Terrien, Sandrine Vitry, Renaud Vincentelli, Clara Bernard, Zakir Khan, Kevin Ricquier, Delphine Bohl, Monique Lafon, Nicolas Wolff, Henri Buc, Juan Ramirez, Neuro-Immunologie Virale - Viral Neuro-immunology, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Résonance Magnétique Nucléaire des Biomolécules, Institut du Cerveau = Paris Brain Institute (ICM), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Sorbonne Université (SU)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Architecture et fonction des macromolécules biologiques (AFMB), Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP), This work was supported by the following grants: ANR (Agence Nationale de la Recherche), BSV8- 0008-01 MAST-2-PTEN. AFM (Association Française contre les Myopathies) Characterization of pathological defects in motor neurons derived from patients Amyotrophic Lateral Sclerosis (ALS). Grand Prix de l’Académie des Sciences/Fondation Générale de Santé, and Incentive grant from Institut Carnot -Pasteur Maladies Infectieuses Unraveling the neuroprotective functions and optimization of new neuroprotective compound . The research leading to the results on IP SC has received funding from the programs Investissements d’Avenir ANR -10- IAIHU -06 and ANR -11- INBS -0011- NeurATRIS : Translational Research Infrastructure for Biotherapies in Neurosciences. ET and FD are recipients of PhD fellowships from the Ministère de l’ Enseignement Supérieur et de la Recherche. CL-O was supported by a PhD fellowship from Revive (ANR -10 -LABX -73). We thank Sylviane Hoos (Biophysics Core, Institut Pasteur) for ITC expertise and the team of the Technology Core of the Center for Translational Science (CRT), Institut Pasteur, for their help our with the Incucyte Technology., ANR-13-BSV8-0008,MAST-2-PTEN,Etude structurale et fonctionnelle du complexe MAST2/PTEN, impliqué dans le contrôle de la survie neuronale.(2013), ANR-11-INBS-0011,NeurATRIS,Infrastructure de Recherche Translationnelle pour les Biothérapies en Neurosciences(2011), ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), Binet, Caroline, Blanc 2013 - Etude structurale et fonctionnelle du complexe MAST2/PTEN, impliqué dans le contrôle de la survie neuronale. - - MAST-2-PTEN2013 - ANR-13-BSV8-0008 - Blanc 2013 - VALID, Infrastructures - Infrastructure de Recherche Translationnelle pour les Biothérapies en Neurosciences - - NeurATRIS2011 - ANR-11-INBS-0011 - INBS - VALID, Laboratoires d'excellence - Stem Cells in Regenerative Biology and Medicine - - REVIVE2010 - ANR-10-LABX-0073 - LABX - VALID, Neuro-Immunologie Virale, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut du Cerveau et de la Moëlle Epinière = Brain and Spine Institute (ICM), Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Pitié-Salpêtrière [APHP]-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris], ANR: 11-INBS-0011,NeurATRIS,Infrastructure de Recherche Translationnelle pour les Biothérapies en Neurosciences(2011), ANR-10-LABX-0073/10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Pitié-Salpêtrière [AP-HP], Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA)

Subjects
0301 basic medicine, viral protein, neurite outgrowth, Neurite, Viral protein, structure function analysis, Nuclear Magnetic Resonance, Induced Pluripotent Stem Cells, Neuronal Outgrowth, PDZ domain, PDZ Domains, Peptide, Serine threonine protein kinase, Protein Serine-Threonine Kinases, medicine.disease_cause, Microtubules, Biochemistry, Protein–protein interaction, protein-protein interaction, Structure-Activity Relationship, Viral Proteins, 03 medical and health sciences, [SCCO]Cognitive science, Neurites, medicine, Humans, Protein Interaction Domains and Motifs, neurodegenerative diseases, Molecular Biology, Neurons, chemistry.chemical_classification, 030102 biochemistry & molecular biology, Kinase, [SCCO.NEUR]Cognitive science/Neuroscience, [SCCO.NEUR] Cognitive science/Neuroscience, Cell Biology, [SCCO] Cognitive science, serine/threonine protein kinase, Cell biology, 030104 developmental biology, chemistry, Rabies virus, Cytoplasm, Central Nervous System Stimulants, Interfering peptide, Peptides, therapeutical drug candidate
Abstract

International audience; Protection of neuronal homeostasis is a major goal in the management of neurodegenerative diseases. Microtubule-associated Ser/Thre kinase 2 (MAST2), inhibits neurite outgrowth, and its inhibition therefore represents a potential therapeutic strategy. We previously reported that a viral protein (G-protein from rabies virus) capable of interfering with protein-protein interactions between the PDZ domain of MAST2 and the C-terminal moieties of its cellular partners counteracts MAST2-mediated suppression of neurite outgrowth. Here, we designed peptides derived from the native viral protein to increase the affinity of these peptides for the MAST2 PDZ domain. Our strategy involved modifying the length and flexibility of the non-interacting sequence linking the two subsites anchoring the peptide to the PDZ domain. Three peptides, Neurovita1 (NV1), NV2, and NV3, were selected, and we found that they all had increased affinities for the MAST2 PDZ domain, with Kd values decreasing from 1300 to 60 nM, while target selectivity was maintained. A parallel biological assay evaluating neurite extension and branching in cell cultures revealed that the NV peptides gradually improved neural activity, with the efficacies of these peptides for stimulating neurite outgrowth mirroring their affinities for MAST2-PDZ. We also show that NVs can be delivered into the cytoplasm of neurons as a gene or peptide. In summary, our findings indicate that virus-derived peptides targeted to MAST2-PDZ stimulate neurite outgrowth in several neuron types, opening up promising avenues for potentially using NVs in the management of neurodegenerative diseases.

Published
2019

15. Real-Time Surface Plasmon Resonance (SPR) for the Analysis of Interactions Between SUMO Traps and Mono- or PolySUMO Moieties

Author

Manuel S. Rodriguez, Patrick England, Wendy Xolalpa, Centro de Investigacion Sobre Enfermedades Infecciosas, National Institute of Public Health = Instituto Nacional de Salud Pública [Cuernavaca, Mexique] (INSP), Inbiomed [Spain], Biophysique des macromolécules et leurs interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), UCMB at Inbiomed was supported by the 'Obra Social KUTXA' and the Diputación Foral de Gipuzkoa. The authors would like to acknowledge networking support by the PROTEOSTASIS action BM1307, supported by COST (European Cooperation in Science and Technology)., and We are grateful with Sylviane Hoos for technical Assistance in SPR experiments.

Subjects
0301 basic medicine, MESH: Gene Expression, [SDV]Life Sciences [q-bio], genetic processes, SPR, Affi nity constants, macromolecular substances, Avidity, environment and public health, Protein–protein interaction, 03 medical and health sciences, 0302 clinical medicine, MESH: Genetic Vectors, MESH: Recombinant Fusion Proteins, Molecule, MESH: Microchip Analytical Procedures, MESH: Protein Binding, MESH: Cloning, Molecular, Surface plasmon resonance, Multivalence, MESH: Glutathione Transferase, MESH: Protein Interaction Domains and Motifs, MESH: Humans, Chemistry, MESH: Escherichia coli, MESH: Antibodies, MESH: SUMO-1 Protein, MESH: Sumoylation, SUMO-binding entities, Protein interactions, Affinities, MESH: Surface Plasmon Resonance, enzymes and coenzymes (carbohydrates), 030104 developmental biology, MESH: Small Ubiquitin-Related Modifier Proteins, Affinity constants, MESH: Protein Processing, Post-Translational, Posttranslational modification, Biophysics, health occupations, 030217 neurology & neurosurgery
Abstract

International audience; Isolating endogenous SUMOylated proteins is a challenging task due to the high reversibility of this posttranslational modifi cation. We have shown that SUMO traps are useful tools for the enrichment and isolation of proteins modifi ed by SUMO in vitro and in vivo. To characterize the affi nity and specifi city of different SUMO chains for these traps, that are based on SUMO-interacting motifs, we have used realtime surface plasmon resonance (SPR), which allows a label-free analysis of protein/protein interactions. Here, a protocol to determine the affi nities of multivalent SUMO traps for polySUMO chains or mono-SUMO molecules by SPR is presented.

Published
2016

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