Author: "Bertrand Raynal" / Publication Year Range: Last 50 years - Searchworks@Jio Institute Digital Library Search Results

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107 results on '"Bertrand Raynal"'

1. Acetylation regulates the oligomerization state and activity of RNase J, the Helicobacter pylori major ribonuclease

Author: Alejandro Tejada-Arranz, Aleksei Lulla, Maxime Bouilloux-Lafont, Evelyne Turlin, Xue-Yuan Pei, Thibaut Douché, Mariette Matondo, Allison H. Williams, Bertrand Raynal, Ben F. Luisi, and Hilde De Reuse
Subjects: Science
Abstract: Abstract In the gastric pathogen Helicobacter pylori, post-transcriptional regulation relies strongly on the activity of the essential ribonuclease RNase J. Here, we elucidated the crystal and cryo-EM structures of RNase J and determined that it assembles into dimers and tetramers in vitro. We found that RNase J extracted from H. pylori is acetylated on multiple lysine residues. Alanine substitution of several of these residues impacts on H. pylori morphology, and thus on RNase J function in vivo. Mutations of Lysine 649 modulates RNase J oligomerization in vitro, which in turn influences ribonuclease activity in vitro. Our structural analyses of RNase J reveal loops that gate access to the active site and rationalizes how acetylation state of K649 can influence activity. We propose acetylation as a regulatory level controlling the activity of RNase J and its potential cooperation with other enzymes of RNA metabolism in H. pylori.
Published: 2023
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2. Prodomain-driven enzyme dimerization: a pH-dependent autoinhibition mechanism that controls Plasmodium Sub1 activity before merozoite egress

Author: Mariano Martinez, Anthony Bouillon, Sébastien Brûlé, Bertrand Raynal, Ahmed Haouz, Pedro M. Alzari, and Jean-Christophe Barale
Subjects: malaria, Plasmodium, subtilin-like protease, pro-domain, enzyme regulation, 3D structure, Microbiology, QR1-502
Abstract: ABSTRACT Malaria symptoms are associated with the asexual multiplication of Plasmodium falciparum within human red blood cells (RBCs) and fever peaks coincide with the egress of daughter merozoites following the rupture of the parasitophorous vacuole (PV) and the RBC membranes. Over the last two decades, it has emerged that the release of competent merozoites is tightly regulated by a complex cascade of events, including the unusual multi-step activation mechanism of the pivotal subtilisin-like protease 1 (Sub1) that takes place in three different cellular compartments and remains poorly understood. Following an initial auto-maturation in the endoplasmic reticulum (ER) between its pro- and catalytic domains, the Sub1 prodomain (PD) undergoes further cleavages by the parasite aspartic protease plasmepsin X (PmX) within acidic secretory organelles that ultimately lead to full Sub1 activation upon discharge into the PV. Here, we report the crystal structure of full-length P. falciparum Sub1 (PfS1FL) and demonstrate, through structural, biochemical, and biophysical studies, that the atypical Plasmodium-specific Sub1 PD directly promotes the assembly of inactive enzyme homodimers at acidic pH, whereas Sub1 is primarily monomeric at neutral pH. Our results shed new light into the finely tuned Sub1 spatiotemporal activation during secretion, explaining how PmX processing and full activation of Sub1 can occur in different cellular compartments, and uncover a robust mechanism of pH-dependent subtilisin autoinhibition that plays a key role in P. falciparum merozoites egress from infected host cells.IMPORTANCEMalaria fever spikes are due to the rupture of infected erythrocytes, allowing the egress of Plasmodium sp. merozoites and further parasite propagation. This fleeting tightly regulated event involves a cascade of enzymes, culminating with the complex activation of the subtilisin-like protease 1, Sub1. Differently than other subtilisins, Sub1 activation strictly depends upon the processing by a parasite aspartic protease within acidic merozoite secretory organelles. However, Sub1 biological activity is required in the pH neutral parasitophorous vacuole, to prime effectors involved in the rupture of the vacuole and erythrocytic membranes. Here, we show that the unusual, parasite-specific Sub1 prodomain is directly responsible for its acidic-dependent dimerization and autoinhibition, required for protein secretion, before its full activation at neutral pH in a monomeric form. pH-dependent Sub1 dimerization defines a novel, essential regulatory element involved in the finely tuned spatiotemporal activation of the egress of competent Plasmodium merozoites.
Published: 2024
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3. DNA-binding mechanism and evolution of replication protein A

Author: Clément Madru, Markel Martínez-Carranza, Sébastien Laurent, Alessandra C. Alberti, Maelenn Chevreuil, Bertrand Raynal, Ahmed Haouz, Rémy A. Le Meur, Marc Delarue, Ghislaine Henneke, Didier Flament, Mart Krupovic, Pierre Legrand, and Ludovic Sauguet
Subjects: Science
Abstract: Abstract Replication Protein A (RPA) is a heterotrimeric single stranded DNA-binding protein with essential roles in DNA replication, recombination and repair. Little is known about the structure of RPA in Archaea, the third domain of life. By using an integrative structural, biochemical and biophysical approach, we extensively characterize RPA from Pyrococcus abyssi in the presence and absence of DNA. The obtained X-ray and cryo-EM structures reveal that the trimerization core and interactions promoting RPA clustering on ssDNA are shared between archaea and eukaryotes. However, we also identified a helical domain named AROD (Acidic Rpa1 OB-binding Domain), and showed that, in Archaea, RPA forms an unanticipated tetrameric supercomplex in the absence of DNA. The four RPA molecules clustered within the tetramer could efficiently coat and protect stretches of ssDNA created by the advancing replisome. Finally, our results provide insights into the evolution of this primordial replication factor in eukaryotes.
Published: 2023
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4. Interactions of the protein tyrosine phosphatase PTPN3 with viral and cellular partners through its PDZ domain: insights into structural determinants and phosphatase activity

Author: Mariano Genera, Baptiste Colcombet-Cazenave, Anastasia Croitoru, Bertrand Raynal, Ariel Mechaly, Joël Caillet, Ahmed Haouz, Nicolas Wolff, and Célia Caillet-Saguy
Subjects: protein tyrosine phosphatase PTPN3, X-ray crystallography, PDZ domains, PDZ-binding motif, enzyme kinetics, bioinformatics, Biology (General), QH301-705.5
Abstract: The human protein tyrosine phosphatase non-receptor type 3 (PTPN3) is a phosphatase containing a PDZ (PSD-95/Dlg/ZO-1) domain that has been found to play both tumor-suppressive and tumor-promoting roles in various cancers, despite limited knowledge of its cellular partners and signaling functions. Notably, the high-risk genital human papillomavirus (HPV) types 16 and 18 and the hepatitis B virus (HBV) target the PDZ domain of PTPN3 through PDZ-binding motifs (PBMs) in their E6 and HBc proteins respectively. This study focuses on the interactions between the PTPN3 PDZ domain (PTPN3-PDZ) and PBMs of viral and cellular protein partners. We solved the X-ray structures of complexes between PTPN3-PDZ and PBMs of E6 of HPV18 and the tumor necrosis factor-alpha converting enzyme (TACE). We provide new insights into key structural determinants of PBM recognition by PTPN3 by screening the selectivity of PTPN3-PDZ recognition of PBMs, and by comparing the PDZome binding profiles of PTPN3-recognized PBMs and the interactome of PTPN3-PDZ. The PDZ domain of PTPN3 was known to auto-inhibit the protein’s phosphatase activity. We discovered that the linker connecting the PDZ and phosphatase domains is involved in this inhibition, and that the binding of PBMs does not impact this catalytic regulation. Overall, the study sheds light on the interactions and structural determinants of PTPN3 with its cellular and viral partners, as well as on the inhibitory role of its PDZ domain on its phosphatase activity.
Published: 2023
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5. Deciphering the Molecular Interaction Between the Adhesion G Protein-Coupled Receptor ADGRV1 and its PDZ-Containing Regulator PDZD7

Author: Baptiste Colcombet-Cazenave, Florence Cordier, Yanlei Zhu, Guillaume Bouvier, Eleni Litsardaki, Louise Laserre, Marie S. Prevost, Bertrand Raynal, Célia Caillet-Saguy, and Nicolas Wolff
Subjects: deafness, G protein-coupled receptor, PDZ-containing protein, ADGRV1, PDZD7, PDZ tandem, Biology (General), QH301-705.5
Abstract: Hearing relies on the transduction of sound-evoked vibrations into electrical signals, occurring in the stereocilia bundle of inner ear hair cells. The G protein-coupled receptor (GPCR) ADGRV1 and the multi-PDZ protein PDZD7 play a critical role in the formation and function of stereocilia through their scaffolding and signaling properties. During hair cell development, the GPCR activity of ADGRV1 is specifically inhibited by PDZD7 through an unknown mechanism. Here, we describe the key interactions mediated by the two N-terminal PDZ domains of PDZD7 and the cytoplasmic domain of ADGRV1. Both PDZ domains can bind to the C-terminal PDZ binding motif (PBM) of ADGRV1 with the critical contribution of atypical C-terminal β extensions. The two PDZ domains form a supramodule in solution, stabilized upon PBM binding. Interestingly, we showed that the stability and binding properties of the PDZ tandem are affected by two deafness-causing mutations located in the binding grooves of PDZD7 PDZ domains.
Published: 2022
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6. Quality control of protein reagents for the improvement of research data reproducibility

Author: Ario de Marco, Nick Berrow, Mario Lebendiker, Maria Garcia-Alai, Stefan H. Knauer, Blanca Lopez-Mendez, André Matagne, Annabel Parret, Kim Remans, Stephan Uebel, and Bertrand Raynal
Subjects: Science
Abstract: Proteins and peptides are amongst the most widely used research reagents but often their quality is inadequate and can result in poor data reproducibility. Here we propose a simple set of guidelines that, when correctly applied to protein reagents should provide more reliable experimental data.
Published: 2021
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7. Molecular Mechanisms Underpinning the Circulation and Cellular Uptake of Mycobacterium ulcerans Toxin Mycolactone

Author: Bruno Tello Rubio, Florence Bugault, Blandine Baudon, Bertrand Raynal, Sébastien Brûlé, Jean-David Morel, Sarah Saint-Auret, Nicolas Blanchard, Caroline Demangel, and Laure Guenin-Macé
Subjects: mycolactone, Mycobacterium ulcerans, lipid carriers, SR-B1, uptake, Therapeutics. Pharmacology, RM1-950
Abstract: Mycolactone is a diffusible lipid toxin produced by Mycobacterium ulcerans, the causative agent of Buruli ulcer disease. Altough bacterially derived mycolactone has been shown to traffic from cutaneous foci of infection to the bloodstream, the mechanisms underpinning its access to systemic circulation and import by host cells remain largely unknown. Using biophysical and cell-based approaches, we demonstrate that mycolactone specific association to serum albumin and lipoproteins is necessary for its solubilization and is a major mechanism to regulate its bioavailability. We also demonstrate that Scavenger Receptor (SR)-B1 contributes to the cellular uptake of mycolactone. Overall, we suggest a new mechanism of transport and cell entry, challenging the dogma that the toxin enters host cells via passive diffusion.
Published: 2021
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8. A High‐Affinity Calmodulin‐Binding Site in the CyaA Toxin Translocation Domain is Essential for Invasion of Eukaryotic Cells

Author: Alexis Voegele, Mirko Sadi, Darragh Patrick O'Brien, Pauline Gehan, Dorothée Raoux‐Barbot, Maryline Davi, Sylviane Hoos, Sébastien Brûlé, Bertrand Raynal, Patrick Weber, Ariel Mechaly, Ahmed Haouz, Nicolas Rodriguez, Patrice Vachette, Dominique Durand, Sébastien Brier, Daniel Ladant, and Alexandre Chenal
Subjects: adenylate cyclase, Bordetella pertussis, calmodulin, CyaA toxin, membrane translocation, protein membrane interaction, Science
Abstract: Abstract The molecular mechanisms and forces involved in the translocation of bacterial toxins into host cells are still a matter of intense research. The adenylate cyclase (CyaA) toxin from Bordetella pertussis displays a unique intoxication pathway in which its catalytic domain is directly translocated across target cell membranes. The CyaA translocation region contains a segment, P454 (residues 454–484), which exhibits membrane‐active properties related to antimicrobial peptides. Herein, the results show that this peptide is able to translocate across membranes and to interact with calmodulin (CaM). Structural and biophysical analyses reveal the key residues of P454 involved in membrane destabilization and calmodulin binding. Mutational analysis demonstrates that these residues play a crucial role in CyaA translocation into target cells. In addition, calmidazolium, a calmodulin inhibitor, efficiently blocks CyaA internalization. It is proposed that after CyaA binding to target cells, the P454 segment destabilizes the plasma membrane, translocates across the lipid bilayer and binds calmodulin. Trapping of CyaA by the CaM:P454 interaction in the cytosol may assist the entry of the N‐terminal catalytic domain by converting the stochastic motion of the polypeptide chain through the membrane into an efficient vectorial chain translocation into host cells.
Published: 2021
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9. Characterization of neutralizing antibodies reacting with the 213-224 amino-acid segment of human galectin-9.

Author: Claire Lhuillier, Clément Barjon, Valentin Baloche, Toshiro Niki, Aurore Gelin, Rami Mustapha, Laetitia Claër, Sylviane Hoos, Yoichi Chiba, Masaki Ueno, Mitsuomi Hirashima, Ming Wei, Olivier Morales, Bertrand Raynal, Nadira Delhem, Olivier Dellis, and Pierre Busson
Subjects: Medicine, Science
Abstract: Extra-cellular galectin-9 (gal-9) is an immuno-modulatory protein with predominant immunosuppressive effects. Inappropriate production of gal-9 has been reported in several human malignancies and viral diseases like nasopharyngeal, pancreatic and renal carcinomas, metastatic melanomas and chronic active viral hepatitis. Therefore therapeutic antibodies neutralizing extra-cellular gal-9 are expected to contribute to immune restoration in these pathological conditions. Two novel monoclonal antibodies targeting gal-9 -Gal-Nab 1 and 2-have been produced and characterized in this study. We report a protective effect of Gal-Nab1 and Gal-Nab2 on the apoptotic cell death induced by gal-9 in primary T cells. In addition, they inhibit late phenotypic changes observed in peripheral T cells that survive gal-9-induced apoptosis. Gal-Nab1 and Gal-Nab2 bind nearly identical, overlapping linear epitopes contained in the 213-224 amino-acid segments of gal-9. Nevertheless, they have some distinct functional characteristics suggesting that their three-dimensional epitopes are distinct. These differences are best demonstrated when gal-9 is applied on Jurkat cells where Gal-Nab1 is less efficient than Gal-Nab2 in the prevention of apoptotic cell death. In addition, Gal-Nab1 stimulates non-lethal phosphatidylserine translocation at the plasma membrane and calcium mobilization triggered by gal-9 in these cells. Both Gal-Nab1 and 2 cross-react with murine gal-9. They bind its natural as well as its recombinant form. This cross-species recognition will be an advantage for their assessment in pre-clinical tumor models.
Published: 2018
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10. Two classes of bacterial IMPDHs according to their quaternary structures and catalytic properties.

Author: Thomas Alexandre, Bertrand Raynal, and Hélène Munier-Lehmann
Subjects: Medicine, Science
Abstract: Inosine-5'-monophosphate dehydrogenase (IMPDH) occupies a key position in purine nucleotide metabolism. In this study, we have performed the biochemical and physico-chemical characterization of eight bacterial IMPDHs, among which six were totally unexplored. This study led to a classification of bacterial IMPDHs according to the regulation of their catalytic properties and their quaternary structures. Class I IMPDHs are cooperative enzymes for IMP, which are activated by MgATP and are octameric in all tested conditions. On the other hand, class II IMPDHs behave as Michaelis-Menten enzymes for both substrates and are tetramers in their apo state or in the presence of IMP, which are shifted to octamers in the presence of NAD or MgATP. Our work provides new insights into the IMPDH functional regulation and a model for the quaternary structure modulation is proposed.
Published: 2015
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11. Correction: Two Classes of Bacterial IMPDHs according to Their Quaternary Structures and Catalytic Properties.

Author: Thomas Alexandre, Bertrand Raynal, and Hélène Munier-Lehmann
Subjects: Medicine, Science
Published: 2015
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12. Interaction of a partially disordered antisigma factor with its partner, the signaling domain of the TonB-dependent transporter HasR.

Author: Idir Malki, Catherine Simenel, Halina Wojtowicz, Gisele Cardoso de Amorim, Ada Prochnicka-Chalufour, Sylviane Hoos, Bertrand Raynal, Patrick England, Alain Chaffotte, Muriel Delepierre, Philippe Delepelaire, and Nadia Izadi-Pruneyre
Subjects: Medicine, Science
Abstract: Bacteria use diverse signaling pathways to control gene expression in response to external stimuli. In Gram-negative bacteria, the binding of a nutrient is sensed by an outer membrane transporter. This signal is then transmitted to an antisigma factor and subsequently to the cytoplasm where an ECF sigma factor induces expression of genes related to the acquisition of this nutrient. The molecular interactions involved in this transmembrane signaling are poorly understood and structural data on this family of antisigma factor are rare. Here, we present the first structural study of the periplasmic domain of an antisigma factor and its interaction with the transporter. The study concerns the signaling in the heme acquisition system (Has) of Serratia marcescens. Our data support unprecedented partially disordered periplasmic domain of an anti-sigma factor HasS in contact with a membrane-mimicking environment. We solved the 3D structure of the signaling domain of HasR transporter and identified the residues at the HasS-HasR interface. Their conservation in several bacteria suggests wider significance of the proposed model for the understanding of bacterial transmembrane signaling.
Published: 2014
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13. Correction: The Carboxy-Terminal αN Helix of the Archaeal XerA Tyrosine Recombinase Is a Molecular Switch to Control Site-Specific Recombination.

Author: Marie-Claude Serre, Toufic El Arnaout, Mark A. Brooks, Dominique Durand, Johnny Lisboa, Noureddine Lazar, Bertrand Raynal, Herman van Tilbeurgh, and Sophie Quevillon-Cheruel
Subjects: Medicine, Science
Published: 2014
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14. The carboxy-terminal αN helix of the archaeal XerA tyrosine recombinase is a molecular switch to control site-specific recombination.

Author: Marie-Claude Serre, Toufic El Arnaout, Mark A Brooks, Dominique Durand, Johnny Lisboa, Noureddine Lazar, Bertrand Raynal, Herman van Tilbeurgh, and Sophie Quevillon-Cheruel
Subjects: Medicine, Science
Abstract: Tyrosine recombinases are conserved in the three kingdoms of life. Here we present the first crystal structure of a full-length archaeal tyrosine recombinase, XerA from Pyrococcus abyssi, at 3.0 Å resolution. In the absence of DNA substrate XerA crystallizes as a dimer where each monomer displays a tertiary structure similar to that of DNA-bound Tyr-recombinases. Active sites are assembled in the absence of dif except for the catalytic Tyr, which is extruded and located equidistant from each active site within the dimer. Using XerA active site mutants we demonstrate that XerA follows the classical cis-cleavage reaction, suggesting rearrangements of the C-terminal domain upon DNA binding. Surprisingly, XerA C-terminal αN helices dock in cis in a groove that, in bacterial tyrosine recombinases, accommodates in trans αN helices of neighbour monomers in the Holliday junction intermediates. Deletion of the XerA C-terminal αN helix does not impair cleavage of suicide substrates but prevents recombination catalysis. We propose that the enzymatic cycle of XerA involves the switch of the αN helix from cis to trans packing, leading to (i) repositioning of the catalytic Tyr in the active site in cis and (ii) dimer stabilisation via αN contacts in trans between monomers.
Published: 2013
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15. Structural basis for the ABO blood-group dependence of Plasmodium falciparum rosetting.

Author: Inès Vigan-Womas, Micheline Guillotte, Alexandre Juillerat, Audrey Hessel, Bertrand Raynal, Patrick England, Jacques H Cohen, Olivier Bertrand, Thierry Peyrard, Graham A Bentley, Anita Lewit-Bentley, and Odile Mercereau-Puijalon
Subjects: Immunologic diseases. Allergy, RC581-607, Biology (General), QH301-705.5
Abstract: The ABO blood group influences susceptibility to severe Plasmodium falciparum malaria. Recent evidence indicates that the protective effect of group O operates by virtue of reduced rosetting of infected red blood cells (iRBCs) with uninfected RBCs. Rosetting is mediated by a subgroup of PfEMP1 adhesins, with RBC binding being assigned to the N-terminal DBL1α₁ domain. Here, we identify the ABO blood group as the main receptor for VarO rosetting, with a marked preference for group A over group B, which in turn is preferred to group O RBCs. We show that recombinant NTS-DBL1α₁ and NTS-DBL1α₁-CIDR1γ reproduce the VarO-iRBC blood group preference and document direct binding to blood group trisaccharides by surface plasmon resonance. More detailed RBC subgroup analysis showed preferred binding to group A₁, weaker binding to groups A₂ and B, and least binding to groups A(x) and O. The 2.8 Å resolution crystal structure of the PfEMP1-VarO Head region, NTS-DBL1α₁-CIDR1γ, reveals extensive contacts between the DBL1α₁ and CIDR1γ and shows that the NTS-DBL1α₁ hinge region is essential for RBC binding. Computer docking of the blood group trisaccharides and subsequent site-directed mutagenesis localized the RBC-binding site to the face opposite to the heparin-binding site of NTS-DBLα₁. RBC binding involves residues that are conserved between rosette-forming PfEMP1 adhesins, opening novel opportunities for intervention against severe malaria. By deciphering the structural basis of blood group preferences in rosetting, we provide a link between ABO blood grouppolymorphisms and rosette-forming adhesins, consistent with the selective role of falciparum malaria on human genetic makeup.
Published: 2012
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16. Raynals, an online tool for the analysis of dynamic light scattering

Author: Osvaldo Burastero, George Draper-Barr, Bertrand Raynal, Maelenn Chevreuil, Patrick England, and Maria M. Garcia-Alai
Abstract: Dynamic light scattering (DLS) is routinely employed to assess the homogeneity and size distribution profile of samples containing microscopic particles in suspension or solubilised polymers. In this work, we introduce Raynals, an user-friendly software for the analysis of single-angle DLS data that uses the Tikhonov-Phillips regularisation. Performance is evaluated on simulated and experimental data, generated by different DLS instruments, for several proteins and gold nanoparticles. DLS data can be easily misinterpreted and the simulation tools available in Raynals allow understanding of the limitations of the measurement and its resolution. It has been designed as a tool to address quality control of biological samples, during sample preparation and optimisation, and it helps in the detection of aggregates showing the influence of large particles. Last, Raynals provides flexibility in the way the data is presented, allows exporting publication-quality figures, it is free for academic use, and can be accessed online on the eSPC data analysis platform at spc.embl-hamburg.de.
Published: 2023
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17. The c‐di‐ <scp>AMP</scp> ‐binding protein <scp>CbpB</scp> modulates the level of <scp>ppGpp</scp> alarmone in Streptococcus agalactiae

Author: Giovanni Covaleda‐Cortés, Ariel Mechaly, Terry Brissac, Heike Baehre, Laura Devaux, Patrick England, Bertrand Raynal, Sylviane Hoos, Myriam Gominet, Arnaud Firon, Patrick Trieu‐Cuot, Pierre Alexandre Kaminski, Biologie des Bactéries pathogènes à Gram-positif - Biology of Gram-Positive Pathogens, Université Paris Cité (UPCité)-Microbiologie Intégrative et Moléculaire (UMR6047), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Cristallographie (Plateforme) - Crystallography (Platform), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Hannover Medical School [Hannover] (MHH), Biophysique Moléculaire (plateforme) - Molecular Biophysics (platform), This work was supported by grants from the French Government ‘Laboratory of Excellence - Integrative Biology of Emerging Infectious Diseases’ (LabEx IBEID, grant number ANR-10-LABX-62-IBEID to PTC), the Fondation pour la Recherche Médicale (FRM grant number DEQ20181039599 to PTC)., We acknowledge SOLEIL for provision of synchrotron radiation facilities, and we thank the staff of beamlines PROXIMA-1 and PROXIMA-2A for assistance. The authors are grateful to the Staff of the Crystallography platform at the Institut Pasteur for robot-driven crystallization screenings. We acknowledge Eduard Baquero Salazar for his help during the early stage of this work. We also acknowledge the help of Mariette Matondo and Thibault Chaze from the Proteomics facility of Institut Pasteur., and ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010)
Subjects: CbpB protein, Cyclic diadenosine monophosphate (c‐di‐AMP), [SDV]Life Sciences [q-bio], X‐ray crystallography & binding, Streptococcus, Cell Biology, Potassium homeostasis, CBS domain, Molecular Biology, Biochemistry
Abstract: International audience; Cyclic di-AMP is an essential signaling molecule in Gram-positive bacteria. This second messenger regulates the osmotic pressure of the cell by interacting directly with the regulatory domains, either RCK_C or CBS domains, of several potassium and osmolyte uptake membrane protein systems. Cyclic di-AMP also targets stand-alone CBS domain proteins such as DarB in Bacillus subtilis and CbpB in Listeria monocytogenes. We show here that the CbpB protein of Group B Streptococcus binds c-di-AMP with a very high affinity. Crystal structures of CbpB reveal the determinants of binding specificity and significant conformational changes occurring upon c-di-AMP binding. Deletion of the cbpB gene alters bacterial growth in low potassium conditions most likely due to a decrease in the amount of ppGpp caused by a loss of interaction between CbpB and Rel, the GTP/GDP pyrophosphokinase.
Published: 2023
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18. Author response for 'The <scp>c‐di‐AMP</scp> ‐binding protein <scp>CbpB</scp> modulates the level of <scp>ppGpp</scp> alarmone in Streptococcus agalactiae'

Author: null Giovanni Covaleda‐Cortés, null Ariel Mechaly, null Terry Brissac, null Heike Baehre, null Laura Devaux, null Patrick England, null Bertrand Raynal, null Sylviane Hoos, null Myriam Gominet, null Arnaud Firon, null Patrick Trieu‐Cuot, and null Pierre Alexandre Kaminski
Published: 2022
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19. DNA-binding mechanism and evolution of Replication Protein A

Author: Clément Madru, Markel Martinez-Carranza, Sébastien Laurent, Alessandra C. Alberti, Maelenn Chevreuil, Bertrand Raynal, Ahmed Haouz, Rémy A. Le Meur, Marc Delarue, Didier Flament, Mart Krupovic, Pierre Legrand, and Ludovic Sauguet
Abstract: Replication Protein A (RPA) is a heterotrimeric single stranded DNA-binding protein with essential roles in DNA replication, recombination and repair, in both eukaryotic and archaeal cells. By using an integrative approach that combines three crystal structures, four cryo-EM structures in complex with single-stranded DNA (ssDNA) of different lengths, we extensively characterized RPA from Pyrococcus abyssi in different states. These structures show two essential features conserved in eukaryotes: a trimeric core and a module that promotes cooperative binding to ssDNA, as well as a newly identified archaeal-specific domain. These structures reveal for the first time how ssDNA is handed over from one RPA complex to the other, and uncover an unanticipated mechanism of self-association on ssDNA tracts. This work constitutes a significant step forward in the molecular understanding of the structure and DNA-binding mechanism of RPA, with far-reaching implications for the evolution of this primordial replication factor in Archaea and Eukarya.
Published: 2022
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20. Quality control of protein reagents for the improvement of research data reproducibility

Author: Bertrand Raynal, Maria Garcia-Alai, Kim Remans, Stefan H. Knauer, Stephan Uebel, Ario de Marco, Mario Lebendiker, Annabel H. A. Parret, André Matagne, Blanca López-Méndez, Nick Berrow, University of Nova Gorica, Barcelona Institute of Science and Technology (BIST), The Hebrew University of Jerusalem (HUJ), European Molecular Biology Laboratory [Hamburg] (EMBL), Universität Bayreuth, Novo Nordisk Foundation Center for Protein Research (CPR), Faculty of Health and Medical Sciences, University of Copenhagen = Københavns Universitet (UCPH)-University of Copenhagen = Københavns Universitet (UCPH), Université de Liège, European Molecular Biology Laboratory [Heidelberg] (EMBL), Charles River, Biophysique Moléculaire (Plate-forme), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and ARBRE-MOBIEU is supported by European CO-operation in Science and Technology (COST) Action number CA15126.
Subjects: Quality Control, 0301 basic medicine, Computer science, media_common.quotation_subject, [SDV]Life Sciences [q-bio], Science, General Physics and Astronomy, 02 engineering and technology, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, Animals, Humans, Quality (business), Analytical biochemistry, Research data, media_common, Reproducibility, Multidisciplinary, Comment, Proteins, Reproducibility of Results, Experimental data, General Chemistry, Molecular biophysics, 021001 nanoscience & nanotechnology, 030104 developmental biology, Indicators and Reagents, Biochemical engineering, 0210 nano-technology
Abstract: Proteins and peptides are amongst the most widely used research reagents but often their quality is inadequate and can result in poor data reproducibility. Here we propose a simple set of guidelines that, when correctly applied to protein reagents should provide more reliable experimental data.
Published: 2021
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21. Dengue virus NS1 protein conveys pro‐inflammatory signals by docking onto high‐density lipoproteins

Author: Souheyla Benfrid, Kyu‐Ho Park, Mariano Dellarole, James E Voss, Carole Tamietti, Gérard Pehau‐Arnaudet, Bertrand Raynal, Sébastien Brûlé, Patrick England, Xiaokang Zhang, Anastassia Mikhailova, Milena Hasan, Marie‐Noëlle Ungeheuer, Stéphane Petres, Scott B Biering, Eva Harris, Anavaj Sakuntabhai, Philippe Buchy, Veasna Duong, Philippe Dussart, Fasséli Coulibaly, François Bontems, Félix A Rey, Marie Flamand, Virologie Structurale - Structural Virology, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Université Paris Descartes, Sorbonne Paris Cité, Plateforme BioImagerie Ultrastructurale – Ultrastructural BioImaging Platform (UTechS UBI), Institut Pasteur [Paris] (IP), Biophysique Moléculaire (plateforme) - Molecular Biophysics (platform), HIV, Inflammation et persistance - HIV, Inflammation and Persistence, Cytometrie et Biomarqueurs – Cytometry and Biomarkers (UTechS CB), Investigation Clinique et d’Accès aux Ressources Biologiques (Plate-forme) - Clinical Investigation and Access to BioResources (ICAReB), Plateforme technologique Production et purification de protéines recombinantes – Production and Purification of Recombinant Proteins Technological Platform (PPR), University of California [Berkeley] (UC Berkeley), University of California (UC), Génétique du Développement humain - Human developmental genetics, Institut Pasteur du Cambodge, Réseau International des Instituts Pasteur (RIIP), Monash University [Clayton], Institut de Chimie des Substances Naturelles (ICSN), Institut de Chimie du CNRS (INC)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), This study benefited from the financial support of the Institut Pasteur ACIP-27-16 (P.D., V.D., M.F.), the Institut Pasteur Dengue Task Force (to MF), the Institut Pasteur INNOV-44-19 (M.F), the National Natural Science Foundation of China 31600606 (X.Z.), the National Key R&D Program of China 2016YFA0501100 (X.Z.), Guangdong Provincial Key Laboratory of Brain Connectome and Behavior 2017B030301017 (X.Z.), CAS Key Laboratory of Brain Connectome and Manipulation 2019DP173024 (X.Z.), the NIAID/NIH R01 AI24493 (E.H.) and R21 AI146464 (E.H.), ANR Equipex CACSICE ANR-11-EQPX-0008 (G.P.-A.)., The authors gratefully acknowledge the staff of the Kampong Cham Referral Hospital, the patients and parents who participated in the study, and the Arbovirus Team in the Virology Unit at the Institut Pasteur du Cambodge who contributed to this study. We acknowledge the participation of the ICAReB facility in setting up the recruitment of donors and the acquisition of blood samples, in particular Gloria Morizot, Bianca Liliana Perlaza, Sophie Chaouche, Linda Sangari, Céline Chapel, Philippe Esterre and Hélène Laude. We are most grateful to Christine Girard-Blanc and Evelyne Dufour for their contribution in producing and purifying the recombinant DENV NS1 protein, to Béatrice Poirier-Beaudouin and Cartini Mardi for their help in setting up the cytokine quantification assay, to Arvind Sharma for providing purified anti-E MAbs, to Mathilde Ban for preparing Fab-bound NS1-HDL complexes and to Xavier Montagutelli and Etienne Simon-Lorière for testing an in vivo protection assay. We thank M. Nilges and the Equipex CACSICE for providing the Falcon II direct detector and David Veesler for his help in acquiring the first electron microscopy images of the bovine NS1-HDL complex. Finally, we thank Sébastien Quesney, Alexandre Pachot and Karine Kaiser for their support. The synopsis figure was created with BioRender.com., and ANR-11-EQPX-0008,CACSICE,Centre d'analyse de systèmes complexes dans les environnements complexes(2011)
Subjects: Arbovirus, lipoprotein particle, viruses, accessory protein, virus diseases, Dengue Virus, Viral Nonstructural Proteins, biochemical phenomena, metabolism, and nutrition, Biochemistry, virulence factor, Dengue, molecular pathogenesis, Phagocytosis, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Genetics, Humans, [SDV.IMM]Life Sciences [q-bio]/Immunology, lipids (amino acids, peptides, and proteins), Lipoproteins, HDL, Molecular Biology, hemorrhagic fever
Abstract: International audience; The dengue virus nonstructural protein 1 (NS1) is a secreted virulence factor that modulates complement, activates immune cells and alters endothelial barriers. The molecular basis of these events remains incompletely understood. Here we describe a functional high affinity complex formed between NS1 and human high-density lipoproteins (HDL). Collapse of the soluble NS1 hexamer upon binding to the lipoprotein particle leads to the anchoring of amphipathic NS1 dimeric subunits into the HDL outer layer. The stable complex can be visualized by electron microscopy as a spherical HDL with rod-shaped NS1 dimers protruding from the surface. We further show that the assembly of NS1-HDL complexes triggers the production of pro-inflammatory cytokines in human primary macrophages while NS1 or HDL alone do not. Finally, we detect NS1 in complex with HDL and low-density lipoprotein (LDL) particles in the plasma of hospitalized dengue patients and observe NS1-apolipoprotein E-positive complexes accumulating overtime. The functional reprogramming of endogenous lipoprotein particles by NS1 as a means to exacerbate systemic inflammation during viral infection provides a new paradigm in dengue pathogenesis.
Published: 2022
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22. Review for 'In‐solution structure and oligomerization of human histone deacetylase 6 ‐ an integrative approach'

Author: null Bertrand Raynal
Published: 2022
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23. CyaA toxin translocation across host cell plasma membrane requires hijacking of calmodulin

Author: Alexis Voegele, Mirko Sadi, Darragh P. O'Brien, Pauline Gehan, Sylviane Hoos, Sébastien Brûlé, Bertrand Raynal, Ariel Mechaly, Ahmed Haouz, Nicolas Rodriguez, Patrice Vachette, Dominique Durand, Sébastien Brier, Daniel Ladant, and Alexandre Chenal
Subjects: Biophysics
Published: 2023
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24. Actinobacteria challenge the paradigm: A unique protein architecture for a well-known, central metabolic complex

Author: Marco Bellinzoni, Pedro M. Alzari, Lu Yang, Eduardo M. Bruch, Bertrand Raynal, Alexandra Boyko, Norik Lexa-Sapart, Pierre Vilela, Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Wuhan Institute of Biological Products, Biophysique Moléculaire (plateforme) - Molecular Biophysics (platform), This work was funded by the Agence Nationale de la Recherche (ANR) through the projects SUPERCPLX (ANR-13-JSV8-0003) and METACTINO (ANR-18-CE92-0003), both granted to M.B., and by institutional grants from the Institut Pasteur and the CNRS., We are grateful to the core facilities at Institut Pasteur C2RT (Centre for Technological Resources and Research), in particular to A. Haouz, P. Weber, and C. Pissis (Crystallography), S. Brûlé and B. Baron (Molecular Biophysics), M. Matondo, T. Chaze, and T. Douché (Proteomics), and J.-M. Winter, S. Tachon, and M. Vos (NanoImaging). We also gratefully acknowledge F. Gubellini (Structural Microbiology Unit) for her help in EM sample preparation and J.J. Pierella Karlusich (Ecole Normale Superieure, Paris), A. Thureau (Synchrotron Soleil), and V. Bunik (Lomonosov University, Moscow) for many helpful discussions. The NanoImaging Core at Institut Pasteur was created with the help of a grant from the French Government’s 'Investissements d’Avenir' program (EQUIPEX CACSICE—'Centre d’analyse de systèmes complexes dans les environnements complexes,' ANR-11-EQPX-0008) and is acknowledged for support with cryo-EM sample preparation, image acquisition, and analysis. We also acknowledge the synchrotron sources Soleil (Saint-Aubin, France) and ESRF (Grenoble, France) for granting access to their facilities and their staff for helpful assistance on the respective beamlines., ANR-13-JSV8-0003,SUPERCPLX,Structure, fonction et régulation d'un supercomplexe métabolique clé chez les Actinobacteries(2013), ANR-18-CE92-0003,METACTINO,Déchiffrer la plasticité métabolique des Actinobactéries(2018), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)
Subjects: Biochemical Phenomena, Molecular Conformation, Dehydrogenase, Computational biology, Crystallography, X-Ray, Actinobacteria, Metabolic engineering, 03 medical and health sciences, Pyruvate dehydrogenase complex, pyruvate dehydrogenase, Integrative structural biology, Pyruvic Acid, Protein oligomerization, Ketoglutarate Dehydrogenase Complex, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Gene, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, Multidisciplinary, Bacteria, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 030302 biochemistry & molecular biology, Computational Biology, Mycobacterium tuberculosis, Biological Sciences, biology.organism_classification, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Kinetics, Enzyme, chemistry, Acyltransferase, Macromolecular complexes, central metabolism, Plasmids
Abstract: International audience; α-oxoacid dehydrogenase complexes are large, tripartite enzymatic machineries carrying out key reactions in central metabolism. Extremely conserved across the tree of life, they have been, so far, all considered to be structured around a high–molecular weight hollow core, consisting of up to 60 subunits of the acyltransferase component. We provide here evidence that Actinobacteria break the rule by possessing an acetyltranferase component reduced to its minimally active, trimeric unit, characterized by a unique C-terminal helix bearing an actinobacterial specific insertion that precludes larger protein oligomerization. This particular feature, together with the presence of an odhA gene coding for both the decarboxylase and the acyltransferase domains on the same polypetide, is spread over Actinobacteria and reflects the association of PDH and ODH into a single physical complex. Considering the central role of the pyruvate and 2-oxoglutarate nodes in central metabolism, our findings pave the way to both therapeutic and metabolic engineering applications.
Published: 2021
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25. Acetylation regulates the oligomerization state and activity of RNase J, the major ribonuclease of Helicobacter pylori

Author: Mariette Matondo, Ben F. Luisi, Xue Yuan Pei, Bertrand Raynal, M. Bouilloux-Lafont, Alejandro Tejada-Arranz, H. De Reuse, Allison H. Williams, and Thibaut Douché
Subjects: biology, Biochemistry, Chemistry, Acetylation, RNase P, biology.protein, Active site, Ribonuclease, Degradosome, RNA Helicase A, In vitro, Function (biology)
Abstract: In Helicobacter pylori, post-transcriptional regulation strongly relies on the activity of an RNA degradosome, composed of the essential ribonuclease RNase J and the DEAD-box RNA helicase RhpA. Here, we describe post-translational modifications of this protein complex that affect its activity. Cell-extracted RNase J is acetylated on multiple residues, one of which, K649, strongly impacts RNase J oligomerization, which in turn influences ribonuclease activity. Corroborating the link between oligomerization and activity, mutations targeting K649 and other residues affect the dimerization and in vitro activity of RNase J. Our crystal structure of RNase J reveals three loops that gate access to the active site and rationalizes how oligomerization state influences activity. The acetylated residues of RNase J are important for H. pylori morphology, highlighting that the modifications affect the RNase J cellular function. We propose acetylation as a regulatory level controlling the activity of RNase J and the H. pylori RNA degradosome.
Published: 2021
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26. PDZ Sample Quality Assessment by Biochemical and Biophysical Characterizations

Author: Célia Caillet-Saguy, Bertrand Raynal, Sébastien Brûlé, Nicolas Wolff, Récepteurs Canaux - Channel Receptors, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Biophysique Moléculaire (plateforme) - Molecular Biophysics (platform), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Jean-Paul Borg, and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0303 health sciences, Computer science, Quality assessment, [SDV]Life Sciences [q-bio], 030303 biophysics, PDZ domain, Quality control, Structure, Folding, Computational biology, Purity, Protein sample, Sample quality, Folding (chemistry), 03 medical and health sciences, Biophysical techniques, Identity, Quantification, Human proteome project, Stability, 030304 developmental biology
Abstract: International audience; PDZ domains are small globular domains involved in protein–protein interactions. They participate in a wide range of critical cellular processes. These domains, very abundant in the human proteome, are widely studied by high-throughput interactomics approaches and by biophysical and structural methods. However, the quality of the results is strongly related to the optimal folding and solubility of the domains. We provide here a detailed description of protocols for a strict quality assessment of the PDZ constructs. We describe appropriate experimental approaches that have been selected to overcome the small size of such domains to check the purity, identity, homogeneity, stability, and folding of samples.
Published: 2021
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27. PDZ Sample Quality Assessment by Biochemical and Biophysical Characterizations

Author: Célia, Caillet-Saguy, Sébastien, Brûlé, Nicolas, Wolff, and Bertrand, Raynal
Subjects: Models, Molecular, Protein Folding, Binding Sites, Protein Conformation, Biophysics, Electrophoresis, Capillary, Humans, PDZ Domains, Protein Serine-Threonine Kinases, Microtubule-Associated Proteins, Mass Spectrometry, Protein Binding
Abstract: PDZ domains are small globular domains involved in protein-protein interactions. They participate in a wide range of critical cellular processes. These domains, very abundant in the human proteome, are widely studied by high-throughput interactomics approaches and by biophysical and structural methods. However, the quality of the results is strongly related to the optimal folding and solubility of the domains. We provide here a detailed description of protocols for a strict quality assessment of the PDZ constructs. We describe appropriate experimental approaches that have been selected to overcome the small size of such domains to check the purity, identity, homogeneity, stability, and folding of samples.
Published: 2021

28. The dengue virus NS1 protein conveys pro-inflammatory signals by docking onto human high-density lipoproteins

Author: X Zhang, Tamietti C, Stéphane Petres, Anastassia Mikhailova, Fasséli Coulibaly, Benfrid S, Park K, Eva Harris, Duong, Mariano Dellarole, James E. Voss, Marie Flamand, Philippe Buchy, Sakunthabaï A, Gérard Pehau-Arnaudet, Patrick England, Philippe Dussart, Félix A. Rey, Scott B. Biering, Sébastien Brûlé, Marie-Noëlle Ungeheuer, François Bontems, and Bertrand Raynal
Subjects: biology, Chemistry, viruses, virus diseases, biochemical phenomena, metabolism, and nutrition, Dengue virus, medicine.disease_cause, medicine.disease, Virulence factor, Complement system, Dengue fever, Cell biology, Pathogenesis, Immune system, medicine, biology.protein, lipids (amino acids, peptides, and proteins), Antibody, Lipoprotein
Abstract: The nonstructural NS1 protein is a virulence factor secreted by dengue virus (DENV)-infected cells. NS1 is known to alter the complement system, activate immune cells and perturb endothelial barriers. Here we show that pro-inflammatory signals are triggered by a high affinity complex formed between NS1 and human high-density lipoproteins (HDL). Electron microscopy images of the NS1-HDL complexes show spherical HDL particles with rod-shaped NS1 protrusions on their surface. These complexes are readily detectable in the plasma of hospitalized dengue patients using anti-apolipoprotein A-I (ApoA-I) antibodies specific of the HDL moiety. The functional reprogramming of HDL particles by the NS1 protein as a means to exacerbate systemic inflammation during DENV infection provides a new paradigm linking the human lipoprotein network to dengue pathogenesis.
Published: 2021
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29. A High‐Affinity Calmodulin‐Binding Site in the CyaA Toxin Translocation Domain is Essential for Invasion of Eukaryotic Cells

Author: Nicolas Rodriguez, Daniel Ladant, Maryline Davi, Ahmed Haouz, Bertrand Raynal, Darragh P. O'Brien, Alexis Voegele, Ariel E. Mechaly, Patrick Weber, Sébastien Brûlé, Pauline Gehan, Dominique Durand, Mirko Sadi, Sylviane Hoos, Sébastien Brier, Alexandre Chenal, Dorothée Raoux-Barbot, Patrice Vachette, Biochimie des Interactions Macromoléculaires / Biochemistry of Macromolecular Interactions, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Université de Paris (UP), Laboratoire des biomolécules (LBM UMR 7203), Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Département de Chimie - ENS Paris, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Chimie Moléculaire de Paris Centre (FR 2769), Institut de Chimie du CNRS (INC)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM), Biophysique Moléculaire (plateforme) - Molecular Biophysics (platform), Cristallographie (Plateforme) - Crystallography (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), Plateforme technologique de RMN biologique - Biological NMR Technological Platform, A.V. was supported by a DIM MalInf grant from the region Ile‐de‐France. M.S. was supported by the Pasteur – Paris University (PPU) International PhD Program. D.P.O.B. was supported by Institut Pasteur (grants PasteurInnoV15006‐01A and PTR451). P.G. was supported by Sorbonne Université. Funding is acknowledged from Agence Nationale de la Recherche (grant number CACSICE Equipex ANR‐11‐EQPX‐0008). Region Ile de France (grant number DIM MalInf 2016), CNRS (UMR 3528), Institut Pasteur (grant numbers PasteurInnoV15006‐01A, PTR451 and PTR166‐19, PPUIP program). The funders have no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript., The authors acknowledge SOLEIL and ESRF for provision of synchrotron radiation facilities. The authors thank the staffs of the DISCO, PROXIMA‐1, and SWING beamlines for constant support and help during data collection (Synchrotron SOLEIL, St Aubin, France) and MASSIF (Synchrotron ESRF, Grenoble, France) beamlines for assistance during the X‐ray diffraction data collection., ANR-11-EQPX-0008,CACSICE,Centre d'analyse de systèmes complexes dans les environnements complexes(2011), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Université Paris Cité (UPCité), Chimie Moléculaire de Paris Centre (FR 2769), École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Département de Chimie - ENS Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Chenal, Alexandre, and Equipements d'excellence - Centre d'analyse de systèmes complexes dans les environnements complexes - - CACSICE2011 - ANR-11-EQPX-0008 - EQPX - VALID
Subjects: calmodulin, General Chemical Engineering, General Physics and Astronomy, Medicine (miscellaneous), 02 engineering and technology, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, 01 natural sciences, Bordetella pertussis, protein membrane interaction, General Materials Science, Internalization, Lipid bilayer, [SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], media_common, biology, Full Paper, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, General Engineering, Full Papers, 021001 nanoscience & nanotechnology, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Protein Transport, Eukaryotic Cells, Adenylate Cyclase Toxin, 0210 nano-technology, Protein Binding, adenylate cyclase, Calmodulin, [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], CyaA toxin, media_common.quotation_subject, Science, Antimicrobial peptides, [SDV.BBM.BP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, protein–protein interactions, 010402 general chemistry, Biochemistry, Genetics and Molecular Biology (miscellaneous), Protein–protein interaction, Protein Domains, membrane translocation, Binding site, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Binding Sites, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, cyaA, 0104 chemical sciences, Cytosol, Biophysics, biology.protein
Abstract: The molecular mechanisms and forces involved in the translocation of bacterial toxins into host cells are still a matter of intense research. The adenylate cyclase (CyaA) toxin from Bordetella pertussis displays a unique intoxication pathway in which its catalytic domain is directly translocated across target cell membranes. The CyaA translocation region contains a segment, P454 (residues 454–484), which exhibits membrane‐active properties related to antimicrobial peptides. Herein, the results show that this peptide is able to translocate across membranes and to interact with calmodulin (CaM). Structural and biophysical analyses reveal the key residues of P454 involved in membrane destabilization and calmodulin binding. Mutational analysis demonstrates that these residues play a crucial role in CyaA translocation into target cells. In addition, calmidazolium, a calmodulin inhibitor, efficiently blocks CyaA internalization. It is proposed that after CyaA binding to target cells, the P454 segment destabilizes the plasma membrane, translocates across the lipid bilayer and binds calmodulin. Trapping of CyaA by the CaM:P454 interaction in the cytosol may assist the entry of the N‐terminal catalytic domain by converting the stochastic motion of the polypeptide chain through the membrane into an efficient vectorial chain translocation into host cells., The mechanism of cell invasion by bacterial toxins is still poorly understood. The adenylate cyclase (CyaA) toxin from Bordetella pertussis, the causative agent of whooping cough, directly translocates its catalytic domain across plasma membranes. The membrane‐permeabilizing segment (yellow) of CyaA translocates across the plasma membrane and binds calmodulin (red), which assists the entry of the catalytic domain (blue) into host cells while the hydrophobic and acylation domains (green) interact with the membrane and the C‐terminal Repeat‐in‐Toxin domain (grey) remains in the extra‐cellular milieu.
Published: 2021
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30. RNase R is associated in a functional complex with the RhpA DEAD-box RNA helicase in Helicobacter pylori

Author: Etienne Kornobis, Valérie Briolat, Maxime Bouilloux-Lafont, Cecília M. Arraiano, Bertrand Raynal, Eloïse Galtier, Mariette Matondo, Yves Quentin, Thibaut Douché, Alejandro Tejada-Arranz, Hilde De Reuse, Rute G. Matos, Pathogenèse de Helicobacter / Helicobacter Pathogenesis, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Université de Paris (UP), Instituto de Tecnologia Química e Biológica António Xavier (ITQB), Universidade Nova de Lisboa = NOVA University Lisbon (NOVA), Laboratoire de microbiologie et génétique moléculaires (LMGM), Centre de Biologie Intégrative (CBI), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Pôle Biomics (C2RT), Centre de Ressources et de Recherche Technologique - Center for Technological Resources and Research (C2RT), Institut Pasteur [Paris]-Institut Pasteur [Paris], Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Plateforme de Protéomique / Proteomics platform, Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Biophysique Moléculaire (plateforme) - Molecular Biophysics (platform), European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie [665807], Institut Carnot Pasteur Microbes & Santé, Fondation pour la Recherche Médicale [DBF20161136767 to H.D.R.], Pasteur-Weizmann Consortium of ‘The Roles of Noncoding RNAs in Regulation of Microbial Life Styles and Virulence’ to HDR, work at ITQB NOVA was financially supported by: Project UIDB/04612/2020 and UIDP/04612/2020 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER funds through COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI) and by national funds through FCT – Fundação para a Ciência e a Tecnologia, project PTDC/BIA-BQM/28479/2017 to R.G.M. R.G.M. was also financed by an FCT contract [CEECIND/02065/2017]. Funding for open access charge: Institut Pasteur., European Project: 665807,H2020,H2020-MSCA-COFUND-2014,PASTEURDOC(2015), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Université Paris Cité (UPCité), Laboratoire de microbiologie et génétique moléculaires - UMR5100 (LMGM), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Institut Pasteur [Paris] (IP), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), and Laboratoire de Microbiologie et de Génétique Moléculaires (LMGM) , Centre de Biologie Intégrative (CBI), Université de Toulouse, UMR CNRS 5100, 31062 TOULOUSE Cedex 9, France.
Subjects: DEAD box, AcademicSubjects/SCI00010, [SDV]Life Sciences [q-bio], Amino Acid Motifs, RNase R, DEAD-box RNA Helicases, 03 medical and health sciences, 5S ribosomal RNA, Exoribonuclease, Genetics, RNA, Double-Stranded, 030304 developmental biology, Regulation of gene expression, 0303 health sciences, Helicobacter pylori, biology, Nucleic Acid Enzymes, 030302 biochemistry & molecular biology, RNA, Ribosomal, 5S, Helicase, RNA, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], RNA Helicase A, Molecular biology, Exoribonucleases, biology.protein, Epsilonproteobacteria
Abstract: Ribonucleases are central players in post-transcriptional regulation, a major level of gene expression regulation in all cells. Here, we characterized the 3′-5′ exoribonuclease RNase R from the bacterial pathogen Helicobacter pylori. The ‘prototypical’ Escherichia coli RNase R displays both exoribonuclease and helicase activities, but whether this latter RNA unwinding function is a general feature of bacterial RNase R had not been addressed. We observed that H. pylori HpRNase R protein does not carry the domains responsible for helicase activity and accordingly the purified protein is unable to degrade in vitro RNA molecules with secondary structures. The lack of RNase R helicase domains is widespread among the Campylobacterota, which include Helicobacter and Campylobacter genera, and this loss occurred gradually during their evolution. An in vivo interaction between HpRNase R and RhpA, the sole DEAD-box RNA helicase of H. pylori was discovered. Purified RhpA facilitates the degradation of double stranded RNA by HpRNase R, showing that this complex is functional. HpRNase R has a minor role in 5S rRNA maturation and few targets in H. pylori, all included in the RhpA regulon. We concluded that during evolution, HpRNase R has co-opted the RhpA helicase to compensate for its lack of helicase activity.
Published: 2021
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31. Assessing and Improving Protein Sample Quality

Author: Bertrand, Raynal, Sébastien, Brûlé, Stephan, Uebel, and Stefan H, Knauer
Subjects: Protein Stability, Proteins, Reproducibility of Results, Electrophoresis, Polyacrylamide Gel, Dynamic Light Scattering, Mass Spectrometry, Workflow
Abstract: One essential prerequisite of any experiment involving a purified protein, such as interaction studies or structural and biophysical characterization, is to work with a "good-quality" sample in order to ensure reproducibility and reliability of the data. Here, we define a "good-quality" sample as a protein preparation that fulfills three criteria: (1) the preparation contains a protein that is pure and soluble and exhibits structural and functional integrity, (2) the protein must be structurally homogeneous, and (3) the preparation must be reproducible. To ensure effective quality control (QC) of all these parameters, we suggest to follow a simple workflow involving the use of gel electrophoresis, light scattering, and spectroscopic experiments. We describe the techniques used in every step of this workflow and provide easy-to-use standard protocols for each step.
Published: 2021

32. Protein intrinsic viscosity determination with the Viscosizer TD instrument: reaching beyond the initially expected applications

Author: Patrick England, Bertrand Raynal, Raffaele Leroux, Sébastien Brûlé, Biophysique Moléculaire (plateforme) - Molecular Biophysics (platform), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, 030103 biophysics, Materials science, Polymers, Capillary action, Intrinsic viscosity, Taylor dispersion, Biophysics, Thermodynamics, Fluorescence spectroscopy, Absorbance, 03 medical and health sciences, Viscosizer TD, U-tube, Huggins’ constant, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], chemistry.chemical_classification, Viscosity, Viscometer, Kraemers’ constant, Serum Albumin, Bovine, General Medicine, Polymer, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, 030104 developmental biology, chemistry, Bovine serum albumin (BSA), Macromolecule
Abstract: International audience; Intrinsic viscosity is a key hydrodynamic parameter to understand molecular structure and hydration, as well as intramolecular interactions. Commercially available instruments measure intrinsic viscosity by recording the macromolecular mobility in a capillary. These instruments monitor Taylor dispersion using an absorbance or fluorescence detector. By design, these instruments behave like U-tube viscometers. To our knowledge, there are no studies to date showing that the Viscosizer TD instrument (Malvern-Panalytical) is able to measure the intrinsic viscosity of macromolecules. In this study, we then performed our assays on the Poly(ethylene oxide) polymer (PEO), used classically as a standard for viscometry measurements and on three model proteins: the bovine serum albumin (BSA), the bevacizumab monoclonal antibody, and the RTX Repeat Domain (RD) of the adenylate cyclase toxin of Bordetella pertussis (CyaA). The presence of P20 in the samples is critical to get reliable results. The data obtained with our in-house protocol show a strong correlation with intrinsic viscosity values obtained using conventional techniques. However, with respect to them, our measurements could be performed at relatively low concentrations, between 2 and 5 mg/ml, using only 7 µL per injection. Altogether, our results show that the Viscosizer TD instrument is able to measure intrinsic viscosities in a straightforward manner. This simple and innovative approach should give a new boost to intrinsic viscosity measurements and should reignite the interest of biophysicists, immunologists, structural biologists and other researchers for this key physicochemical parameter.
Published: 2021
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33. Assessing and Improving Protein Sample Quality

Author: Bertrand Raynal, Sébastien Brûlé, Stefan H. Knauer, and Stephan Uebel
Subjects: 0303 health sciences, Computer science, Sample (material), 030303 biophysics, A protein, Interaction studies, Sample quality, 03 medical and health sciences, Functional integrity, Workflow, Homogeneous, Biological system, Reliability (statistics), 030304 developmental biology
Abstract: One essential prerequisite of any experiment involving a purified protein, such as interaction studies or structural and biophysical characterization, is to work with a "good-quality" sample in order to ensure reproducibility and reliability of the data. Here, we define a "good-quality" sample as a protein preparation that fulfills three criteria: (1) the preparation contains a protein that is pure and soluble and exhibits structural and functional integrity, (2) the protein must be structurally homogeneous, and (3) the preparation must be reproducible. To ensure effective quality control (QC) of all these parameters, we suggest to follow a simple workflow involving the use of gel electrophoresis, light scattering, and spectroscopic experiments. We describe the techniques used in every step of this workflow and provide easy-to-use standard protocols for each step.
Published: 2021
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34. Quality control of purified proteins to improve data quality and reproducibility: results from a large-scale survey

Author: Maria Garcia-Alai, Stephan Uebel, Stefan H. Knauer, Nick Berrow, Mario Lebendiker, Annabel Parret, Blanca López-Méndez, André Matagne, Bertrand Raynal, Kim Remans, Ario de Marco, Barcelona Institute of Science and Technology (BIST), University of Nova Gorica, The Hebrew University of Jerusalem (HUJ), European Molecular Biology Laboratory [Hamburg] (EMBL), Universität Bayreuth, Novo Nordisk Foundation Center for Protein Research (CPR), Faculty of Health and Medical Sciences, University of Copenhagen = Københavns Universitet (UCPH)-University of Copenhagen = Københavns Universitet (UCPH), Université de Liège, European Molecular Biology Laboratory [Heidelberg] (EMBL), Max-Planck-Institut für Biochemie = Max Planck Institute of Biochemistry (MPIB), Max-Planck-Gesellschaft, Biophysique Moléculaire (plateforme) - Molecular Biophysics (platform), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and ARBRE-MOBIEU is supported by European CO-operation in Science and Technology (COST) Action number CA15126.
Subjects: 0301 basic medicine, Quality Control, 030103 biophysics, Reproducibility, Computer science, Process (engineering), media_common.quotation_subject, Scale (chemistry), [SDV]Life Sciences [q-bio], Control (management), Biophysics, Reproducibility of Results, Context (language use), General Medicine, Data Accuracy, 03 medical and health sciences, 030104 developmental biology, Risk analysis (engineering), Data quality, Quality (business), Reliability (statistics), media_common
Abstract: International audience; As the scientific community strives to make published results more transparent and reliable, it has become obvious that poor data reproducibility can often be attributed to insufficient quality control of experimental reagents. In this context, proteins and peptides reagents require much stricter quality controls than those routinely performed on them in a significant proportion of research laboratories. Members of the ARBRE-MOBIEU and the P4EU networks have combined their expertise to generate guidelines for the evaluation of purified proteins used in life sciences and medical trials. These networks, representing more than 150 laboratories specialized in protein production and/or protein molecular biophysics, have implemented such guidelines in their respective laboratories. Over a one-year period, the network members evaluated the contribution these guidelines made toward obtaining more productive, robust and reproducible research by correlating the applied quality controls to given samples with the reliability and reproducibility of the scientific data obtained using these samples in follow-up experiments. The results indicate that QC guideline implementation facilitates the optimization of the protein purification process and improves the reliability of downstream experiments. It seems, therefore, that investing in protein QC might be advantageous to all the stakeholders in life sciences (researchers, editors, and funding agencies alike), because this practice improves data veracity and minimizes loss of valuable time and resources. In the light of these conclusions, the network members suggest that the implementation of these simple QC guidelines should become minimal reporting practice in the publication of data derived from the use of protein and peptide reagents.
Published: 2020
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35. A high-affinity calmodulin-binding site in the CyaA toxin translocation domain is essential for invasion into eukaryotic cells

Author: Alexandre Chenal, Mirko Sadi, Patrick Weber, Pauline Gehan, Ariel E. Mechaly, Nicolas Rodriguez, Alexis Voegele, Sébastien Brier, Patrice Vachette, Daniel Ladant, Darragh P. O'Brien, Maryline Davi, Dorothée Raoux-Barbot, Sylviane Hoos, Bertrand Raynal, Sébastien Brûlé, Dominique Durand, and Ahmed Haouz
Subjects: Calmodulin, biology, Chemistry, media_common.quotation_subject, Antimicrobial peptides, Chromosomal translocation, cyaA, Cell biology, Cytosol, biology.protein, Binding site, Internalization, Lipid bilayer, media_common
Abstract: The molecular mechanisms and forces involved in the translocation of bacterial toxins into host cells have thus far remained elusive. The adenylate cyclase (CyaA) toxin fromBordetella pertussisdisplays a unique intoxication pathway in which its catalytic domain is directly translocated across target cell membranes. We have previously identified a translocation region in CyaA that contains a segment, P454 (residues 454–484), exhibiting membrane-active properties related to antimicrobial peptides. Herein, we show that this peptide is able to translocate across membranes and interact with calmodulin. Structural and biophysical analyses have revealed the key residues of P454 involved in membrane destabilization and calmodulin binding. Mutational analysis demonstrated that these residues play a crucial role in CyaA translocation into target cells. We have also shown that calmidazolium, a calmodulin inhibitor, efficiently blocks CyaA internalization. We propose that after CyaA binding to target cells, the P454 segment destabilizes the plasma membrane, translocates across the lipid bilayer and binds calmodulin. Trapping of the CyaA polypeptide chain by the CaM:P454 interaction in the cytosol may assist the entry of the N-terminal catalytic domain by converting the stochastic process of protein translocation into an efficient vectorial chain transfer into host cells.
Published: 2020
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36. Actinobacteria challenge the paradigm: a unique protein architecture for a well-known central metabolic complex

Author: Norik Lexa-Sapart, Pedro M. Alzari, Pierre Vilela, Marco Bellinzoni, Bertrand Raynal, Lu Yang, and Eduardo M. Bruch
Subjects: biology, Structural biology, Chemistry, Acyltransferase, Acetyltransferase, Dehydrogenase, Computational biology, Pyruvate dehydrogenase complex, biology.organism_classification, Mega, Archaea, Actinobacteria
Abstract: α-ketoacid dehydrogenase complexes are one the biggest enzymatic complexes found in nature with molecular mass ranging from several to more than 10 mega Daltons. This tripartite enzymatic machinery carries out key reactions in central metabolism. With an architecture extremely conserved, they have so far all considered to be structured around a hollow, highly symmetric core made by up to 60 subunits of the acyltransferase component. By using an integrative structural biology approach, we provide here evidence that Actinobacteria break the rule by possessing an acetyltranferase component reduced to its minimally active, trimeric unit, due to a unique C-terminus structure that has a three-residue insertion. This insertion was found in organisms over the whole Actinobacteria class. We show how this insertion affects the oligomerization properties and the whole three-dimensional architecture of the acetyltransferase AceF, proposed to serve as the central component of a mixed supercomplex that bears together the pyruvate dehydrogenase (PDH) and 2-oxoglutarate dehydrogenase (ODH) activities. As a counterexample, we also show how the acyltransferase component of the branched-chain ketoacid dehydrogenase (BCKDH) complex from Mycobacterium tuberculosis does not possess such insertion, and shows a more canonical cubic architecture. This new architecture challenges the widely accepted paradigm for 2-oxoacid dehydrogenases, based in all available structures from Eubacteria, Archaea and Eukaryotes, and that was assumed to be universally conserved. Our results shed new light on the structure and evolution of α-ketoacid dehydrogenase complexes involving one of the biggest phyla in Eubacteria, a phylum that includes species relevant for biotechnology as well as many human pathogens. Moreover, components of this PDH/ODH supercomplex are key for M. tuberculosis survival in the human host, and its unique core and protein-protein interactions represent potentially “druggable” targets.
Published: 2020
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37. Deciphering the Unexpected Binding Capacity of the Third PDZ Domain of Whirlin to Various Cochlear Hair Cell Partners

Author: Baptiste Colcombet-Cazenave, Ariel E. Mechaly, Sylviane Hoos, Nicolas Wolff, Florence Cordier, Célia Caillet-Saguy, Susanne Lüchow, Ahmed Haouz, Ylva Ivarsson, Bertrand Raynal, Amel Bahloul, Yanlei Zhu, Elise Pepermans, Florent Delhommel, Sébastien Brûlé, Candice Gautier, Virginie Girault, Récepteurs Canaux - Channel Receptors, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Ecole Doctorale Complexité du Vivant (ED515), Sorbonne Université (SU), Bioinformatique structurale - Structural Bioinformatics, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Uppsala Universitet [Uppsala], Cristallographie (Plateforme) - Crystallography (Platform), Génétique et Physiologie de l'Audition, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Institut Pasteur, Fondation Cenci Bolognetti - Istituto Pasteur Italia, Fondazione Cenci Bolognetti, Réseau International des Instituts Pasteur (RIIP), Biophysique Moléculaire (plateforme) - Molecular Biophysics (platform), The project has received funding from the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement PDZnet No 675341. The authors also acknowledge the Conseil Régional d'Ile de France (France) for financial support through the SESAME 2014 NMRCHR program No. 14014526 (800 MHz spectrometer)., European Project: 675341,H2020-MSCA-ITN-2015,PDZNet(2016), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)
Subjects: Scaffold protein, Stereocilia (inner ear), [SDV]Life Sciences [q-bio], PDZ domain, PDZ Domains, Cell Cycle Proteins, Myosins, Stereocilia, 03 medical and health sciences, 0302 clinical medicine, Structural Biology, Myosin, Hair Cells, Auditory, medicine, otorhinolaryngologic diseases, Humans, Inner ear, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, CASK, Molecular Biology, Biology, 030304 developmental biology, 0303 health sciences, Stereocilium, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, Membrane Proteins, Proteins, Cell biology, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Cytoskeletal Proteins, medicine.anatomical_structure, Hair cell, sense organs, Guanylate Kinases, 030217 neurology & neurosurgery, Protein Binding
Abstract: Hearing is a mechanical and neurochemical process, which occurs in the hair cells of inner ear that converts the sound vibrations into electrical signals transmitted to the brain. The multi-PDZ scaffolding protein whirlin plays a critical role in the formation and function of stereocilia exposed at the surface of hair cells. In this article, we reported seven stereociliary proteins that encode PDZ binding motifs (PBM) and interact with whirlin PDZ3, where four of them are first reported. We solved the atomic resolution structures of complexes between whirlin PDZ3 and the PBMs of myosin 15a, CASK, harmonin a1 and taperin. Interestingly, the PBM of CASK and taperin are rare non-canonical PBM, which are not localized at the extreme C terminus. This large capacity to accommodate various partners could be related to the distinct functions of whirlin at different stages of the hair cell development. (C) 2020 Published by Elsevier Ltd.
Published: 2020
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38. Cell invasion involves membrane-active and calmodulin-binding properties of the CyaA toxin translocation domain

Author: Alexis Voegele, Mirko Sadi, Darragh P. O’Brien, Pauline Gehan, Dorothée Raoux-Barbot, Sylviane Hoos, Sébastien Brûlé, Bertrand Raynal, Ahmed Haouz, Nicolas Rodriguez, Patrice Vachette, Dominique Durand, Sébastien Brier, Daniel Ladant, and Alexandre Chenal
Subjects: Biophysics
Published: 2022
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39. Structural and functional characterization of the PDZ domain of the human phosphatase PTPN3 and its interaction with the human papillomavirus E6 oncoprotein

Author: Bruno Baron, Nicolas Wolff, Mariano Genera, Bertrand Raynal, Célia Caillet-Saguy, Damien Samson, Ahmed Haouz, Catherine Simenel, Raphael Guerois, ED 515 - Complexité du vivant, Sorbonne Université (SU), Récepteurs Canaux - Channel Receptors, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Résonance Magnétique Nucléaire des Biomolécules, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Biophysique Moléculaire (Plate-forme), Cristallographie (Plateforme) - Crystallography (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), Assemblage moléculaire et intégrité du génome (AMIG), Département Biochimie, Biophysique et Biologie Structurale (B3S), 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), Mariano Genera was supported by the Pasteur - Paris University (PPU) International PhD Program, and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, Oncogene Proteins, [SDV]Life Sciences [q-bio], Phosphatase, PDZ domain, lcsh:Medicine, PDZ Domains, Protein tyrosine phosphatase, Plasma protein binding, Crystallography, X-Ray, Ligands, Article, Structure-Activity Relationship, 03 medical and health sciences, Mitogen-Activated Protein Kinase 12, NMR spectroscopy, 0302 clinical medicine, Humans, Structure–activity relationship, Amino Acid Sequence, lcsh:Science, Peptide sequence, X-ray crystallography, Human papillomavirus 16, Multidisciplinary, Human papillomavirus 18, biology, Protein Stability, Chemistry, lcsh:R, Papillomavirus Infections, Protein Tyrosine Phosphatase, Non-Receptor Type 3, fungi, Oncogene Proteins, Viral, Molecular conformation, Phosphoric Monoester Hydrolases, 3. Good health, Cell biology, DNA-Binding Proteins, Repressor Proteins, 030104 developmental biology, Mitogen-activated protein kinase, biology.protein, lcsh:Q, Structural biology, 030217 neurology & neurosurgery, Protein Binding
Abstract: The human protein tyrosine phosphatase non-receptor type 3 (PTPN3) is a PDZ (PSD-95/Dlg/ZO-1) domain-containing phosphatase with a tumor-suppressive or a tumor-promoting role in many cancers. Interestingly, the high-risk genital human papillomavirus (HPV) types 16 and 18 target the PDZ domain of PTPN3. The presence of a PDZ binding motif (PBM) on E6 confers interaction with a number of different cellular PDZ domain-containing proteins and is a marker of high oncogenic potential. Here, we report the molecular basis of interaction between the PDZ domain of PTPN3 and the PBM of the HPV E6 protein. We combined biophysical, NMR and X-ray experiments to investigate the structural and functional properties of the PDZ domain of PTPN3. We showed that the C-terminal sequences from viral proteins encompassing a PBM interact with PTPN3-PDZ with similar affinities to the endogenous PTPN3 ligand MAP kinase p38γ. PBM binding stabilizes the PDZ domain of PTPN3. We solved the X-ray structure of the PDZ domain of PTPN3 in complex with the PBM of the HPV E6 protein. The crystal structure and the NMR chemical shift mapping of the PTPN3-PDZ/peptide complex allowed us to pinpoint the main structural determinants of recognition of the C-terminal sequence of the E6 protein and the long-range perturbations induced upon PBM binding.
Published: 2019
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40. Calcium-dependent disorder-to-order transitions are central to the secretion and folding of the CyaA toxin of Bordetella pertussis, the causative agent of whooping cough

Author: Dominique Durand, Christian Malosse, Dorothée Raoux-Barbot, Véronique Hourdel, Mahmoud Ghomi, Bertrand Raynal, Darragh P. O'Brien, Daniel Ladant, Ana Cristina Sotomayor Pérez, Bruno Baron, Alexis Voegele, Patrick England, Véronique Yvette Ntsogo Enguéné, Orso Subrini, Alexandre Chenal, Sara E. Cannella, Patrice Vachette, Marilyne Davi, Johanna C. Karst, Belén Hernández, Sébastien Brier, Audrey Hessel, J. Iñaki Guijarro, Julia Chamot-Rooke, Biochimie des Interactions Macromoléculaires / Biochemistry of Macromolecular Interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Résonance Magnétique Nucléaire des Biomolécules, Biophysique Moléculaire (Plate-forme), Université Paris 13 (UP13), Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-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), Département Biochimie, Biophysique et Biologie Structurale (B3S), 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), Fonction et Architecture des Assemblages Macromoléculaires (FAAM), 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), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], and Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, Protein Folding, Bordetella pertussis, FAAM, CyaA toxin, [SDV]Life Sciences [q-bio], chemistry.chemical_element, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Calcium, Toxicology, Models, Biological, 03 medical and health sciences, Protein Domains, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Secretion, Whooping cough, biology, Disorder-to-order transition, Chemistry, Folding, cyaA, biology.organism_classification, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Protein Transport, Cytosol, Chaotropic agent, Eukaryotic Cells, 030104 developmental biology, Secretory protein, Adenylate Cyclase Toxin, Protein Translocation Systems, Biophysics, Protein secretion, B3S
Abstract: International audience; The adenylate cyclase toxin (CyaA) plays an essential role in the early stages of respiratory tract colonization by Bordetella pertussis, the causative agent of whooping cough. Once secreted, CyaA invades eukaryotic cells, leading to cell death. The cell intoxication process involves a unique mechanism of translocation of the CyaA catalytic domain directly across the plasma membrane of the target cell. Herein, we review our recent results describing how calcium is involved in several steps of this intoxication process. In conditions mimicking the low calcium environment of the crowded bacterial cytosol, we show that the C-terminal, calcium-binding Repeat-in-ToXin (RTX) domain of CyaA, RD, is an extended, intrinsically disordered polypeptide chain with a significant level of local, secondary structure elements, appropriately sized for transport through the narrow channel of the secretion system. Upon secretion, the high calcium concentration in the extracellular milieu induces the refolding of RD, which likely acts as a scaffold to favor the refolding of the upstream domains of the full-length protein. Due to the presence of hydrophobic regions, CyaA is prone to aggregate into multimeric forms in vitro, in the absence of a chaotropic agent. We have recently defined the experimental conditions required for CyaA folding, comprising both calcium binding and molecular confinement. These parameters are critical for CyaA folding into a stable, monomeric and functional form. The monomeric, calcium-loaded (holo) toxin exhibits efficient liposome permeabilization and hemolytic activities in vitro, even in a fully calcium-free environment. By contrast, the toxin requires sub-millimolar calcium concentrations in solution to translocate its catalytic domain across the plasma membrane, indicating that free calcium in solution is actively involved in the CyaA toxin translocation process. Overall, this data demonstrates the remarkable adaptation of bacterial RTX toxins to the diversity of calcium concentrations it is exposed to in the successive environments encountered in the course of the intoxication process.
Published: 2018
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41. Biochemical characterization of the respiratory syncytial virus N0-P complex in solution

Author: Jean-François Eléouët, Sébastien Brûlé, Charles-Adrien Richard, Pierre Roblin, Jenna Fix, Camille Esneau, Marie Galloux, Bertrand Raynal, Centre National de la Recherche Scientifique - CNRS (FRANCE), Institut National Polytechnique de Toulouse - Toulouse INP (FRANCE), Institut Pasteur (FRANCE), Université Toulouse III - Paul Sabatier - UT3 (FRANCE), Unité de recherche Virologie et Immunologie Moléculaires (VIM (UR 0892)), Institut National de la Recherche Agronomique (INRA), Centre de Ressources et de Recherche Technologique - Center for Technological Resources and Research (C2RT), Institut Pasteur [Paris], Synchrotron SOLEIL (SSOLEIL), Centre National de la Recherche Scientifique (CNRS), Laboratoire de Génie Chimique (LGC), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), French Agence Nationale de la Recherche, specific program ANR Blanc ANR-13-IVS3-0007, Institut Pasteur [Paris] (IP), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT), Unité de recherche Virologie et Immunologie Moléculaires (VIM), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées, Institut National Polytechnique de Toulouse - INPT (FRANCE), Eleouet, Jean Francois, and Galloux, Marie
Subjects: 0301 basic medicine, Biochimie, Biologie Moléculaire, Respiratory syncytial virus, Biochemistry, Virus, 03 medical and health sciences, Human metapneumovirus, N0-P complex, Viral replication, Génie chimique, Nucleoprotein N, Mononegavirales, Génie des procédés, Molecular Biology, protein folding, structural model, small-angle X-ray scattering (saxs), mutagenesis, analytical ultracentrifugation, n0-p complex, nucleoprotein n, respiratory syncytial virus, structure-function, viral replication, 030102 biochemistry & molecular biology, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Structure-function, Chemistry, RNA, Cell Biology, biology.organism_classification, Nucleoprotein, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], 030104 developmental biology, Phosphoprotein, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Protein folding
Abstract: International audience; As all the viruses belonging to the Mononegavirales order, the non-segmented negative strand RNA genome of respiratory syncytial virus (RSV) is encapsidated by the viral nucleoprotein N. N protein polymerizes along the genomic and anti-genomic RNAs during replication. This requires the maintenance of the neosynthesized N protein in a monomeric and RNA-free form by the viral phosphoprotein P that plays the role of a chaperone protein, forming a soluble N0-P complex. We have previously demonstrated that residues 1-30 of P specifically bind to N0. Here, to isolate a stable N0-P complex suitable for structural studies, we used the N-terminal peptide of P (P40) to purify truncated forms of the N protein. We show that to purify a stable N0-P-like complex, a deletion of the first 30 N-terminal residues of N (NΔ30) is required to impair N oligomerization, whereas the presence of a full-length C-arm of N is required to inhibit RNA binding. We generated structural models of the RSV N0-P with biophysical approaches, including hydrodynamic measurements and small-angle X-ray scattering (SAXS), coupled with biochemical and functional analyses of human RSV (hRSV) NΔ30 mutants. These models suggest a strong structural homology between the hRSV and the human metapneumovirus (hMPV) N0-P complexes. In both complexes, the P40-binding sites on N0 appear to be similar, and the C-arm of N provides a high flexibility and a propensity to interact with the N RNA groove. These findings reveal two potential sites to target on N0-P for the development of RSV antivirals.
Published: 2019
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42. Regulation of measles virus gene expression by P protein coiled-coil properties

Author: Louis-Marie, Bloyet, Antoine, Schramm, Carine, Lazert, Bertrand, Raynal, Maggy, Hologne, Olivier, Walker, Sonia, Longhi, Denis, Gerlier, Immunobiologie des infections virales – Immunobiology of Viral Infections, Centre International de Recherche en Infectiologie - UMR (CIRI), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-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), Biophysique Moléculaire (Plate-forme), Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Biophysics of complex systems, Institut des Sciences Analytiques (ISA), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), This work was supported, in part, by French ANR NITRODEP (project ANR-13-PDOC-0010-01) (www.agence-nationale-recherche.fr) and LABEX ECOFECT (ANR-11-LABX-0048) of Lyon University, within the program 'Investissements d’Avenir' (ANR-11-IDEX-0007) operated by the French National Research Agency (ANR). The studies described here were carried out with the financial support of the CNRS.A.S. is a recipient of a Ph.D. fellowship from the French Ministry of National Education, Research and Technology allotted to the Ecole Doctorale des Sciences de la Vie et de la Santé (Aix-Marseille Université)., ANR-13-PDOC-0010,NITRODEP,Nouvelle exploration du tropisme et de la dissémination des Paramyxoviridae encephalitogènes(2013), ANR-11-IDEX-0007-02/11-LABX-0048,ECOFECT,Dynamiques eco-évolutives des maladies infectieuses(2011), Immunobiologie des infections virales – Immunobiology of Viral Infections (IbIV), Institut National de la Santé et de la Recherche Médicale (INSERM)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), ANR-11-LABX-0048,ECOFECT,Dynamiques eco-évolutives des maladies infectieuses(2011), Centre International de Recherche en Infectiologie (CIRI), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Bussy, Agnès, Retour Post-Doctorant - Nouvelle exploration du tropisme et de la dissémination des Paramyxoviridae encephalitogènes - - NITRODEP2013 - ANR-13-PDOC-0010 - PDOC - VALID, and Dynamiques eco-évolutives des maladies infectieuses - - ECOFECT2011 - ANR-11-LABX-0048 - LABX - VALID
Subjects: Gene Expression Regulation, Viral, Protein Conformation, alpha-Helical, Protein Folding, Molecular Dynamics Simulation, Viral Proteins, Protein Domains, Virology, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acid Sequence, RNA, Small Interfering, Research Articles, [SDV.MP.VIR] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Binding Sites, SciAdv r-articles, Phosphoproteins, Recombinant Proteins, HEK293 Cells, Measles virus, Mutagenesis, Paramyxoviridae, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, lipids (amino acids, peptides, and proteins), RNA Interference, Protein Multimerization, Research Article
Abstract: Measles virus gene expression requires a precise range of cohesiveness and a kink in the coiled coil of its phosphoprotein., The polymerase of negative-stranded RNA viruses consists of the large protein (L) and the phosphoprotein (P), the latter serving both as a chaperon and a cofactor for L. We mapped within measles virus (MeV) P the regions responsible for binding and stabilizing L and showed that the coiled-coil multimerization domain (MD) of P is required for gene expression. MeV MD is kinked as a result of the presence of a stammer. Both restoration of the heptad regularity and displacement of the stammer strongly decrease or abrogate activity in a minigenome assay. By contrast, P activity is rather tolerant of substitutions within the stammer. Single substitutions at the “a” or “d” hydrophobic anchor positions with residues of variable hydrophobicity revealed that P functionality requires a narrow range of cohesiveness of its MD. Results collectively indicate that, beyond merely ensuring P oligomerization, the MD finely tunes viral gene expression through its cohesiveness.
Published: 2019
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43. 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
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44. Biochemical characterization of the respiratory syncytial virus N

Author: Camille, Esneau, Bertrand, Raynal, Pierre, Roblin, Sébastien, Brûlé, Charles-Adrien, Richard, Jenna, Fix, Jean-François, Eléouët, and Marie, Galloux
Subjects: Models, Molecular, Solutions, Viral Proteins, Binding Sites, Nucleoproteins, Protein Conformation, Surface Properties, Respiratory Syncytial Virus, Human, Mutation, Protein Structure and Folding, DNA-Directed RNA Polymerases
Abstract: As all the viruses belonging to the Mononegavirales order, the nonsegmented negative-strand RNA genome of respiratory syncytial virus (RSV) is encapsidated by the viral nucleoprotein N. N protein polymerizes along the genomic and anti-genomic RNAs during replication. This requires the maintenance of the neosynthesized N protein in a monomeric and RNA-free form by the viral phosphoprotein P that plays the role of a chaperone protein, forming a soluble N(0)-P complex. We have previously demonstrated that residues 1–30 of P specifically bind to N(0). Here, to isolate a stable N(0)-P complex suitable for structural studies, we used the N-terminal peptide of P (P40) to purify truncated forms of the N protein. We show that to purify a stable N(0)-P–like complex, a deletion of the first 30 N-terminal residues of N (N(Δ30)) is required to impair N oligomerization, whereas the presence of a full-length C-arm of N is required to inhibit RNA binding. We generated structural models of the RSV N(0)-P with biophysical approaches, including hydrodynamic measurements and small-angle X-ray scattering (SAXS), coupled with biochemical and functional analyses of human RSV (hRSV) N(Δ30) mutants. These models suggest a strong structural homology between the hRSV and the human metapneumovirus (hMPV) N(0)-P complexes. In both complexes, the P40-binding sites on N(0) appear to be similar, and the C-arm of N provides a high flexibility and a propensity to interact with the N RNA groove. These findings reveal two potential sites to target on N(0)-P for the development of RSV antivirals.
Published: 2018

45. Overall Structures of Mycobacterium tuberculosis DNA Gyrase Reveal the Role of a Corynebacteriales GyrB-Specific Insert in ATPase Activity

Author: Claudine Mayer, Pedro M. Alzari, Clément Giffard, Aurélien Thureau, Stéphanie Petrella, Françoise Bonneté, Alexandra Aubry, Estelle Capton, Bertrand Raynal, CCSD, Accord Elsevier, Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Centre d'Immunologie et des Maladies Infectieuses (CIMI), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Biophysique Moléculaire (Plate-forme), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Synchrotron SOLEIL (SSOLEIL), Centre National de la Recherche Scientifique (CNRS), 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)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), This work was funded by Institut Pasteur, Université Paris Diderot, and Sorbonne-Université (France)., Université Paris Diderot - Paris 7 (UPD7)-Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Centre d'Immunologie et de Maladies Infectieuses (CIMI), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), and Institut Pasteur [Paris]-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS)
Subjects: type IIA topoisomerases, Models, Molecular, Protein Conformation, alpha-Helical, ATPase, Gene Expression, Crystallography, X-Ray, ATPase activity, DNA gyrase, Adenosine Triphosphate, Structural Biology, Cloning, Molecular, Adenosine Triphosphatases, molecular machine, 0303 health sciences, biology, Chemistry, Drug discovery, 030302 biochemistry & molecular biology, Molecular machine, Recombinant Proteins, 3. Good health, Biochemistry, DNA Gyrase, Protein Binding, Adenylyl Imidodiphosphate, Genetic Vectors, DNA-binding protein, Corynebacterium, fluoroquinolone, Cleavage (embryo), [PHYS.PHYS.PHYS-CHEM-PH] Physics [physics]/Physics [physics]/Chemical Physics [physics.chem-ph], Mycobacterium tuberculosis, Corynebacteriales, 03 medical and health sciences, Escherichia coli, Atpase activity, Protein Interaction Domains and Motifs, Amino Acid Sequence, Protein Structure, Quaternary, SAXS experiments, Molecular Biology, 030304 developmental biology, Binding Sites, Sequence Homology, Amino Acid, DNA, biology.organism_classification, Kinetics, biology.protein, Protein Conformation, beta-Strand, X-ray structure, [PHYS.PHYS.PHYS-CHEM-PH]Physics [physics]/Physics [physics]/Chemical Physics [physics.chem-ph], Apoproteins, Sequence Alignment
Abstract: International audience; Despite sharing common features, previous studies have shown that gyrases from different species have been modified throughout evolution to modulate their properties. Here, we report two crystal structures of Mycobacterium tuberculosis DNA gyrase, an apo and AMPPNP-bound form at 2.6-Å and 3.3-Å resolution, respectively. These structures provide high-resolution structural data on the quaternary organization and interdomain connections of a gyrase (full-length GyrB-GyrA57)2 thus providing crucial inputs on this essential drug target. Together with small-angle X-ray scattering studies, they revealed an "extremely open" N-gate state, which persists even in the DNA-free gyrase-AMPPNP complex and an unexpected connection between the ATPase and cleavage core domains mediated by two Corynebacteriales-specific motifs, respectively the C-loop and DEEE-loop. We show that the C-loop participates in the stabilization of this open conformation, explaining why this gyrase has a lower ATPase activity. Our results image a conformational state which might be targeted for drug discovery.
Published: 2018
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46. Caractérisation d'anticorps neutralisants réagissant avec le segment des acides aminés 213-224 de la galectine-9 humaine

Author: Claire Lhuillier, Clément Barjon, Valentin Baloche, Toshiro Niki, Aurore Gelin, Rami Mustapha, Laetitia Claër, Sylviane Hoos, Yoichi Chiba, Masaki Ueno, Mitsuomi Hirashima, Ming Wei, Olivier Morales, Bertrand Raynal, Nadira Delhem, Olivier Dellis, Pierre Busson, Signalisation, noyaux et innovations en cancérologie (UMR8126), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Kagawa University [Japan], Institut de biologie de Lille - IBL (IBLI), Université de Lille, Sciences et Technologies-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Université de Lille, Droit et Santé-Centre National de la Recherche Scientifique (CNRS), H-Immune Therapeutics [Paris], Biophysique Moléculaire (Plate-forme), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Cellvax, École nationale vétérinaire d'Alfort (ENVA), Intéractions cellulaires et physiopathologie hépathique (Orsay, Essonne) UMRS 1174 (ICPH ), Institut National de la Santé et de la Recherche Médicale (INSERM), This work was supported by Idex Saclay - Satt Saclay, Prematuration Grant 2015, https://www.satt-paris-saclay.fr, and BMS Foundation for Immuno-Oncology- Grant 2016-2017, http://fondation-bms.fr/. Several authors were employed by commercial companies: Cellvax (CL, CB, MW)
Subjects: MESH: T-Lymphocytes/metabolism, MESH: Apoptosis/drug effects, MESH: Biological Transport, [SDV]Life Sciences [q-bio], Galectins, T-Lymphocytes, MESH: T-Lymphocytes/drug effects, MESH: Galectins/adverse effects, lcsh:Medicine, Lactose, MESH: Galectins/immunology, [SDV.CAN]Life Sciences [q-bio]/Cancer, Apoptosis, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Phosphatidylserines, MESH: Antibodies, Neutralizing/pharmacology, Antibodies, Epitopes, Jurkat Cells, Mice, MESH: T-Lymphocytes/cytology, MESH: Jurkat Cells, Animals, Humans, MESH: Animals, Flow cytometry, Enzyme-linked immunoassays, lcsh:Science, MESH: Mice, MESH: Antibodies, Monoclonal/pharmacology, Antibody isotypes, MESH: Humans, lcsh:R, MESH: Calcium/metabolism, Antibodies, Monoclonal, Biological Transport, MESH: Epitopes/immunology, Cell staining, Antibodies, Neutralizing, MESH: Galectins/chemistry, Apoptosis T cells, MESH: Immunization, [SDV.IMM]Life Sciences [q-bio]/Immunology, lcsh:Q, Calcium, Immunization, MESH: Phosphatidylserines/metabolism
Abstract: International audience; Extra-cellular galectin-9 (gal-9) is an immuno-modulatory protein with predominant immuno-suppressive effects. Inappropriate production of gal-9 has been reported in several human malignancies and viral diseases like nasopharyngeal, pancreatic and renal carcinomas, metastatic melanomas and chronic active viral hepatitis. Therefore therapeutic antibodies neutralizing extra-cellular gal-9 are expected to contribute to immune restoration in these pathological conditions. Two novel monoclonal antibodies targeting gal-9-Gal-Nab 1 and 2-have been produced and characterized in this study. We report a protective effect of Gal-Nab1 and Gal-Nab2 on the apoptotic cell death induced by gal-9 in primary T cells. In addition, they inhibit late phenotypic changes observed in peripheral T cells that survive gal-9-induced apoptosis. Gal-Nab1 and Gal-Nab2 bind nearly identical, overlapping linear epi-topes contained in the 213-224 amino-acid segments of gal-9. Nevertheless, they have some distinct functional characteristics suggesting that their three-dimensional epitopes are distinct. These differences are best demonstrated when gal-9 is applied on Jurkat cells where Gal-Nab1 is less efficient than Gal-Nab2 in the prevention of apoptotic cell death. In addition, Gal-Nab1 stimulates non-lethal phosphatidylserine translocation at the plasma membrane and calcium mobilization triggered by gal-9 in these cells. Both Gal-Nab1 and 2 cross-react with murine gal-9. They bind its natural as well as its recombinant form. This cross-species recognition will be an advantage for their assessment in pre-clinical tumor models.
Published: 2018
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47. The stress sigma factor of RNA polymerase RpoS/σ S is a solvent-exposed open molecule in solution

Author: Christina Sizun, Julia Chamot-Rooke, Françoise Bonneté, Sébastien Brier, Claudine Mayer, Bertrand Raynal, Françoise Norel, Patrick England, Paola Cavaliere, Fabienne Levi-Acobas, Petr Filipenko, Jacques Bellalou, Systèmes macromoléculaires et Signalisation, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Spectrométrie de Masse structurale et protéomique, Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Department of Computer Science [New York], New York University [New York] (NYU), NYU System (NYU)-NYU System (NYU), Institut de Chimie des Substances Naturelles (ICSN), Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Biophysique Moléculaire (Plate-forme), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut des Biomolécules Max Mousseron [Pôle Chimie Balard] (IBMM), Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université de Montpellier (UM)-Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM), Production de Protéines Recombinantes (Plate-Forme) (PRPF), Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris]-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Biochimie des Interactions Macromoléculaires / Biochemistry of Macromolecular Interactions, This work was supported by the French National Research Agency [grant ANR-11-BSV3-009] and by grants from the Institut Pasteur and the Centre National de la Recherche Scientifique. The MS work was supported by the Investissements d’Avenir through the CACSICE project [grant ANR-11-EQPX-008]. Access to high-field NMR spectrometers financially supported by IR-RMN-THC FR3050 CNRS is gratefully acknowledged., ANR-11-BSV3-0009,SIGMADAPT,Rôle de SigmaS dans la compétitivité et l'adaptation des bactéries à l'environnement(2011), ANR-11-EQPX-0008,CACSICE,Centre d'analyse de systèmes complexes dans les environnements complexes(2011), Centre National de la Recherche Scientifique (CNRS), Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Biochimie des Interactions Macromoléculaires, Centre National de la Recherche Scientifique (CNRS)-Centre de Ressources et de Recherche Technologique - Center for Technological Resources and Research (C2RT), Institut Pasteur [Paris]-Institut Pasteur [Paris], ANR-11-EQPX-0008/11-EQPX-0008,CACSICE,Centre d'analyse de systèmes complexes dans les environnements complexes(2011), Centre de Ressources et de Recherche Technologique - Center for Technological Resources and Research (C2RT), Institut Pasteur [Paris]-Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Université Paris Diderot - Paris 7 (UPD7)-Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique ( CNRS ), New York University [New York], Institut de Chimie des Substances Naturelles ( ICSN ), Centre National de la Recherche Scientifique ( CNRS ), Institut des Biomolécules Max Mousseron [Pôle Chimie Balard] ( IBMM ), Ecole Nationale Supérieure de Chimie de Montpellier ( ENSCM ) -Université de Montpellier ( UM ) -Centre National de la Recherche Scientifique ( CNRS ), Production de Protéines Recombinantes (Plate-Forme) ( PRPF ), Microbiologie structurale, Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology ( UTechS MSBio ), ANR-11-BSV3-0009,SIGMADAPT,Rôle de SigmaS dans la compétitivité et l'adaptation des bactéries à l'environnement ( 2011 ), ANR-11-EQPX-0008/11-EQPX-0008,CACSICE,Centre d'analyse de systèmes complexes dans les environnements complexes ( 2011 ), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)-Institut de Chimie du CNRS (INC)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Avignon Université (AU), and Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, [SDV]Life Sciences [q-bio], 030106 microbiology, Biochemistry, 03 medical and health sciences, Molecular dynamics, chemistry.chemical_compound, Protein structure, RpoS, Sigma factor, protein conformation, Salmonella, RNA polymerase, Molecule, protein structure, Molecular Biology, [ SDV ] Life Sciences [q-bio], biology, Cell Biology, Crystallography, 030104 developmental biology, chemistry, Chaperone (protein), biology.protein, Hydrogen–deuterium exchange, sigma factor, rpoS
Abstract: International audience; In bacteria, one primary and multiple alternative sigma (σ) factors associate with the RNA polymerase core enzyme (E) to form holoenzymes (Eσ) with different promoter recognition specificities. The alternative σ factor RpoS/σ S is produced in stationary phase and under stress conditions and reprograms global gene expression to promote bacterial survival. To date, the three-dimensional structure of a full-length free σ factor remains elusive. The current model suggests that extensive interdomain contacts in a free σ factor result in a compact conformation that masks the DNA-binding determinants of σ, explaining why a free σ factor does not bind double-stranded promoter DNA efficiently. Here, we explored the solution conformation of σ S using amide hydrogen/deuterium exchange coupled with mass spectrometry, NMR, analytical ultracentrifugation and molecular dynamics. Our data strongly argue against a compact conformation of free σ S. Instead, we show that σ S adopts an open conformation in solution in which the folded σ 2 and σ 4 domains are interspersed by domains with a high degree of disorder. These findings suggest that E binding induces major changes in both the folding and domain arrangement of σ S and provide insights into the possible mechanisms of regulation of σ S activity by its chaperone Crl.
Published: 2018
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48. Deciphering the unconventional peptide binding to the PDZ domain of MAST2

Author: Florence Cordier, Nicolas Wolff, Henri Buc, Bertrand Raynal, Elouan Terrien, Muriel Delepierre, Alain Chaffotte, Florent Delhommel, Résonance Magnétique Nucléaire des Biomolécules, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Cellule Pasteur UPMC, Institut Pasteur [Paris]-Sorbonne Université (SU), Biophysique Moléculaire (Plate-forme), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris], This work was supported by the Institut Pasteur and Institut Carnot Pasteur Maladies Infectieuses [grant number 13/10/120], and the Ministère de l’Enseignement Supérieur [grant number 883/2013 (to F.D.)]., We thank Jane Hardy and Terence Strick for careful proofreading of the manuscript and Patrick England for valuable discussions., Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Sorbonne Université (SU), and Institut Pasteur [Paris] (IP)
Subjects: Scaffold protein, MESH: Protein Structure, Quaternary, [SDV]Life Sciences [q-bio], Peptide binding, MESH: Protein Multimerization, Biochemistry, MESH: Protein Structure, Tertiary, MESH: Protein-Serine-Threonine Kinases / metabolism, 0302 clinical medicine, MESH: Nuclear Magnetic Resonance, Biomolecular, Tensin, MESH: Rabies virus / metabolism, MESH: Viral Proteins / chemistry, pre-steady-state kinetics, 0303 health sciences, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], biology, MESH: Molecular Dynamics Simulation, Cell biology, DLG1, Microtubule-Associated Proteins, Protein Binding, PDZ domain, MESH: Rabies virus / chemistry, protein–protein interactions, Molecular Dynamics Simulation, Protein Serine-Threonine Kinases, MESH: Protein-Serine-Threonine Kinases / chemistry, Viral Proteins, 03 medical and health sciences, MESH: Microtubule-Associated Proteins / chemistry, Humans, MESH: Protein Binding, PTEN, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Protein Structure, Quaternary, Nuclear Magnetic Resonance, Biomolecular, Molecular Biology, Protein kinase B, 030304 developmental biology, Serine/threonine-specific protein kinase, MESH: Humans, PDZ domains, Cell Biology, MESH: Viral Proteins / metabolism, Protein Structure, Tertiary, MESH: Microtubule-Associated Proteins / metabolism, Rabies virus, intradomain energetic connectivity, biology.protein, sense organs, Protein Multimerization, 030217 neurology & neurosurgery
Abstract: Phosphatase and tensin homologue (PTEN) and microtubule-associated serine threonine kinase 2 (MAST2) are key negative regulators of survival pathways in neuronal cells. The two proteins interact via the PDZ (PSD-95, Dlg1, Zo-1) domain of MAST2 (MAST2–PDZ). During infection by rabies virus, the viral glycoprotein competes with PTEN for interaction with MAST2–PDZ and promotes neuronal survival. The C-terminal PDZ-binding motifs (PBMs) of the two proteins bind similarly to MAST2–PDZ through an unconventional network of connectivity involving two anchor points. Combining stopped-flow fluorescence, analytical ultracentrifugation (AUC), microcalorimetry and NMR, we document the kinetics of interaction between endogenous and viral ligands to MAST2–PDZ as well as the dynamic and structural effects of these interactions. Viral and PTEN peptide interactions to MAST2–PDZ occur via a unique kinetic step which involves both canonical C-terminal PBM binding and N-terminal anchoring. Indirect effects induced by the PBM binding include modifications to the structure and dynamics of the PDZ dimerization surface which prevent MAST2–PDZ auto-association. Such an energetic communication between binding sites and distal surfaces in PDZ domains provides interesting clues for protein regulation overall. * Akt, : protein kinase B; AUC, : analytical ultracentrifugation; Δδ, : chemical shift perturbation; Dlg1, : disk large homologue 1; GRASP, : GRP-1-associated scaffold protein; GRP-1, : general receptor of phosphoinositides 1; IDP, : intrinsically disordered protein; ITC, : isothermal titration calorimetry; MAST, : microtubule-associated serine threonine kinase; PBM, : PDZ-binding motif; PDZ, : PSD-95, Dlg1, Zo-1; PSD-95, : post synaptic density 95; PTEN, : phosphatase and tensin homologue; Zo-1, : zona occludens 1
Published: 2015
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49. Structural Characterization of Whirlin Reveals an Unexpected and Dynamic Supramodule Conformation of Its PDZ Tandem

Author: Christine Petit, Florent Delhommel, Benjamin Bardiaux, Julia Chamot-Rooke, Michael Nilges, Sébastien Brier, Florence Cordier, Amel Bahloul, Bertrand Raynal, Sylvie Nouaille, Nicolas Wolff, Baptiste Colcombet-Cazenave, Guillaume Bouvier, ED 515 - Complexité du vivant, Université Pierre et Marie Curie - Paris 6 (UPMC), Résonance Magnétique Nucléaire des Biomolécules, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Bioinformatique structurale - Structural Bioinformatics, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Spectrométrie de Masse structurale et protéomique, Biophysique Moléculaire (Plate-forme), Chaire Génétique et physiologie cellulaire, Collège de France (CdF (institution)), Bioinformatique Structurale, Financial support from TGIR-RMN-THC Fr3050 CNRS for conducting the research is gratefully acknowledged. We acknowledge SOLEIL and ESRF for provision of synchrotron radiation facilities, and we would like to thank the staff of the SWING and BM29 BioSAXS beamlines for assistance during the SAXS measurements. This work was supported by the Program Transversal de Recherche from the Institut Pasteur (PTR grant no. 483 to N.W.), the European Union (FP7-IDEAS-ERC 294809 to M.N.), the Ministère de l’Enseignement Supérieur et de la Recherche (grant no. 883/2013 to F.D), and the Comité Berthe Fouassier – Maladies de l’œil de la Fondation de France (no. 00071779 to F.D)., The authors are grateful to Bradley Worley for careful proofreading of the manuscript and to Muriel Delepierre for useful discussions. We thank Olivier Lequin, Patrick England, and Alain Chaffotte for their technical expertise., European Project: 294809,EC:FP7:ERC,ERC-2011-ADG_20110310,BAYCELLS(2012), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Collège de France - Chaire Génétique et physiologie cellulaire, Wolff, Nicolas, and A Bayesian Framework for Cellular Structural Biology - BAYCELLS - - EC:FP7:ERC2012-05-01 - 2017-04-30 - 294809 - VALID
Subjects: Protein Conformation, alpha-Helical, 0301 basic medicine, Scaffold protein, Protein Folding, MESH: Sequence Homology, Amino Acid, MESH: PDZ Domains, MESH: Nerve Tissue Proteins / genetics, Gene Expression, PDZ Domains, MESH: Amino Acid Sequence, MESH: Escherichia coli / genetics, 01 natural sciences, Mice, Transduction (genetics), Structural Biology, MESH: Hair Cells, Auditory / metabolism, MESH: Animals, MESH: Molecular Dynamics Simulation, MESH: Membrane Proteins / metabolism, Cloning, Molecular, MESH: Peptides / chemistry, MESH: Membrane Proteins / genetics, Tandem, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], SAXS, Recombinant Proteins, Thermodynamics, Stereocilia bundle, MESH: Protein Conformation, beta-Strand, MESH: Escherichia coli / metabolism, MESH: Thermodynamics, MESH: Hair Cells, Auditory / cytology, Usher syndrome, Protein Binding, MESH: Recombinant Proteins / chemistry, supramodule, MESH: Gene Expression, [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH: Protein Folding, Genetic Vectors, PDZ domain, MESH: Sequence Alignment, Nerve Tissue Proteins, MESH: Nerve Tissue Proteins / chemistry, Molecular Dynamics Simulation, Biology, 010402 general chemistry, Closed conformation, MESH: Membrane Proteins / chemistry, Electric signal, 03 medical and health sciences, NMR spectroscopy, Hair Cells, Auditory, Escherichia coli, otorhinolaryngologic diseases, Animals, MESH: Protein Binding, MESH: Cloning, Molecular, Amino Acid Sequence, MESH: Genetic Vectors / chemistry, Molecular Biology, MESH: Mice, MESH: Nerve Tissue Proteins / metabolism, MESH: Protein Conformation, alpha-Helical, MESH: Peptides / chemical synthesis, Binding Sites, whirlin, Sequence Homology, Amino Acid, Stereocilia, Membrane Proteins, 0104 chemical sciences, Crystallography, 030104 developmental biology, MESH: Genetic Vectors / metabolism, MESH: Binding Sites, Biophysics, Protein Conformation, beta-Strand, sense organs, MESH: Recombinant Proteins / metabolism, Peptides, Sequence Alignment, MESH: Recombinant Proteins / genetics
Abstract: International audience; Hearing relies on the transduction of sound-evoked vibrations into electric signals, occurring in the stereocilia bundle of hair cells. The bundle is organized in a staircase pattern formed by rows of packed stereocilia. This architecture is pivotal to transduction and involves a network of scaffolding proteins with hitherto uncharacterized features. Key interactions in this network are mediated by PDZ domains. Here, we describe the architecture of the first two PDZ domains of whirlin, a protein involved in these assemblies and associated with congenital deaf-blindness. C-terminal hairpin extensions of the PDZ domains mediate the transient supramodular assembly, which improves the binding capacity of the first domain. We determined a detailed structural model of the closed conformation of the PDZ tandem and characterized its equilibrium with an ensemble of open conformations. The structural and dynamic behavior of this PDZ tandem provides key insights into the regulatory mechanisms involved in the hearing machinery.
Published: 2017
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50. The stress sigma factor of RNA polymerase RpoS/σ

Author: Paola, Cavaliere, Sébastien, Brier, Petr, Filipenko, Christina, Sizun, Bertrand, Raynal, Françoise, Bonneté, Fabienne, Levi-Acobas, Jacques, Bellalou, Patrick, England, Julia, Chamot-Rooke, Claudine, Mayer, and Françoise, Norel
Subjects: Protein Conformation, alpha-Helical, Salmonella typhimurium, Protein Folding, Binding Sites, Recombinant Fusion Proteins, Deuterium Exchange Measurement, Sigma Factor, Gene Expression Regulation, Bacterial, Molecular Dynamics Simulation, Crystallography, X-Ray, Protein Structure, Tertiary, Kinetics, Bacterial Proteins, Escherichia coli, Solvents, Thermodynamics, Protein Conformation, beta-Strand, Protein Interaction Domains and Motifs, Holoenzymes, Promoter Regions, Genetic, Protein Binding
Abstract: In bacteria, one primary and multiple alternative sigma (σ) factors associate with the RNA polymerase core enzyme (E) to form holoenzymes (Eσ) with different promoter recognition specificities. The alternative σ factor RpoS/σ
Published: 2017

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