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

Author

Emeline Vernhes, Linda Larbi Chérif, Nicolas Ducrot, Clément Vanbergue, Malika Ouldali, Lena Zig, N’diaye Sidibe, Sylviane Hoos, Luis Ramirez-Chamorro, Madalena Renouard, Ombeline Rossier, Patrick England, Guy Schoehn, Pascale Boulanger, and Karim Benihoud

Subjects
Immunologic diseases. Allergy, RC581-607, Neoplasms. Tumors. Oncology. Including cancer and carcinogens, RC254-282
Abstract

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

Published
2024
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2. Dynamics and structural changes of calmodulin upon interaction with the antagonist calmidazolium

Author

Corentin Léger, Irène Pitard, Mirko Sadi, Nicolas Carvalho, Sébastien Brier, Ariel Mechaly, Dorothée Raoux-Barbot, Maryline Davi, Sylviane Hoos, Patrick Weber, Patrice Vachette, Dominique Durand, Ahmed Haouz, J. Iñaki Guijarro, Daniel Ladant, and Alexandre Chenal

Subjects
Calmodulin, CaM, Calmidazolium, CDZ, Calmodulin antagonist, Structure, Biology (General), QH301-705.5
Abstract

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

Published
2022
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3. 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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4. Structural basis for haem piracy from host haemopexin by Haemophilus influenzae

Author

Silvia Zambolin, Bernard Clantin, Mohamed Chami, Sylviane Hoos, Ahmed Haouz, Vincent Villeret, and Philippe Delepelaire

Subjects
Science
Abstract

Haemophilus influenzae requires haem, and acquires it from host haemoproteins including haemopexin. Here, the authors examine the haem transport system consisting of HxuA, HxuB and HxuC via the structures of HxuA in complex with haemopexin.

Published
2016
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5. 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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6. Cross-reactivity between apical membrane antgen 1 and rhoptry neck protein 2 in P. vivax and P. falciparum: A structural and binding study.

Author

Brigitte Vulliez-Le Normand, Frederick A Saul, Sylviane Hoos, Bart W Faber, and Graham A Bentley

Subjects
Medicine, Science
Abstract

Malaria, a disease endemic in many tropical and subtropical regions, is caused by infection of the erythrocyte by the apicomplexan parasite Plasmodium. Host-cell invasion is a complex process but two Plasmodium proteins, Apical Membrane Antigen 1 (AMA1) and the Rhoptry Neck protein complex (RON), play a key role. AMA1, present on the surface of the parasite, binds tightly to the RON2 component of the RON protein complex, which is inserted into the erythrocyte membrane during invasion. Blocking the AMA1-RON2 interaction with antibodies or peptides inhibits invasion, underlining its importance in the Plasmodium life cycle and as a target for therapeutic strategies. We describe the crystal structure of the complex formed between AMA1 from P. vivax (PvAMA1) and a peptide derived from the externally exposed region of P. vivax RON2 (PvRON2sp1), and of the heterocomplex formed between P. falciparum AMA1 (PfAMA1) and PvRON2sp1. Binding studies show that the affinity of PvRON2sp1 for PvAMA1 is weaker than that previously reported for the PfRON2sp1-PfAMA1 association. Moreover, while PvRON2sp1 shows strong cross-reactivity with PfAMA1, PfRON2sp1 displays no detectable interaction with PvAMA1. The structures show that the equivalent residues PvRON2-Thr2055 and PfRON2-Arg2041 largely account for this pattern of reactivity.

Published
2017
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7. 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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8. Structural and functional insights into the malaria parasite moving junction complex.

Author

Brigitte Vulliez-Le Normand, Michelle L Tonkin, Mauld H Lamarque, Susann Langer, Sylviane Hoos, Magali Roques, Frederick A Saul, Bart W Faber, Graham A Bentley, Martin J Boulanger, and Maryse Lebrun

Subjects
Immunologic diseases. Allergy, RC581-607, Biology (General), QH301-705.5
Abstract

Members of the phylum Apicomplexa, which include the malaria parasite Plasmodium, share many features in their invasion mechanism in spite of their diverse host cell specificities and life cycle characteristics. The formation of a moving junction (MJ) between the membranes of the invading apicomplexan parasite and the host cell is common to these intracellular pathogens. The MJ contains two key parasite components: the surface protein Apical Membrane Antigen 1 (AMA1) and its receptor, the Rhoptry Neck Protein (RON) complex, which is targeted to the host cell membrane during invasion. In particular, RON2, a transmembrane component of the RON complex, interacts directly with AMA1. Here, we report the crystal structure of AMA1 from Plasmodium falciparum in complex with a peptide derived from the extracellular region of PfRON2, highlighting clear specificities of the P. falciparum RON2-AMA1 interaction. The receptor-binding site of PfAMA1 comprises the hydrophobic groove and a region that becomes exposed by displacement of the flexible Domain II loop. Mutations of key contact residues of PfRON2 and PfAMA1 abrogate binding between the recombinant proteins. Although PfRON2 contacts some polymorphic residues, binding studies with PfAMA1 from different strains show that these have little effect on affinity. Moreover, we demonstrate that the PfRON2 peptide inhibits erythrocyte invasion by P. falciparum merozoites and that this strong inhibitory potency is not affected by AMA1 polymorphisms. In parallel, we have determined the crystal structure of PfAMA1 in complex with the invasion-inhibitory peptide R1 derived by phage display, revealing an unexpected structural mimicry of the PfRON2 peptide. These results identify the key residues governing the interactions between AMA1 and RON2 in P. falciparum and suggest novel approaches to antimalarial therapeutics.

Published
2012
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9. 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

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

Author

Emeline Vernhes, Linda Larbi Chérif, Nicolas Ducrot, Malika Ouldali, Lena Zig, N’diaye Sidibe, Sylviane Hoos, Luis Ramirez-Chamorro, Madalena Renouard, Ombeline Rossier, Patrick England, Guy Schoehn, Pascale Boulanger, Karim Benihoud, Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Aspects métaboliques et systémiques de l'oncogénèse pour de nouvelles approches thérapeutiques (METSY), Institut Gustave Roussy (IGR)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Biophysique Moléculaire (Plate-forme), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), This work was supported by the Centre National de la Recherche Scientifique and the University Paris-Saclay. L. L.C., E.V. and N.D. received a fellowship from the French Ministère de l’Enseignement Supérieur et de la Recherche. L. L.C.received an additional fellowship from the Ligue Nationale contre le cancer. L.R.-C received a fellowship 'Becas Don Carlos Antonio Lopez' from the Paraguayan Ministry of Education. This work benefited from the Cryo-EM platform of I2BC, supported by the French Infrastructure for Integrated Structural Biology (FRISBI) [ANR-10-INSB-05-05] and member of IBISA. This work used the platforms of the Grenoble Instruct-ERIC center (ISBG, UAR3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB), supported by FRISBI (ANR-10-INSB-05-02 & project ID 160 to P. B.) and GRAL, financed within the University Grenoble Alpes graduate school (Écoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003). The electron microscope facility is supported by the Auvergne-Rhône-Alpes Region, the Fondation pour la Recherche Médicale (FRM), the fonds FEDER and the GIS-Infrastructures en Biologie Santé et Agronomie (IBiSA)., ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), and ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017)

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

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

Published
2022

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

Author

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

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

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

Published
2022

16. Dynamics and structural changes of calmodulin upon interaction with its potent antagonist calmidazolium

Author

Corentin Léger, Irène Pitard, Mirko Sadi, Nicolas Carvalho, Sébastien Brier, Ariel Mechaly, Dorothée Raoux-Barbot, Maryline Davi, Sylviane Hoos, Patrick Weber, Patrice Vachette, Dominique Durand, Ahmed Haouz, J. Iñaki Guijarro, Daniel Ladant, and Alexandre Chenal

Abstract

Calmodulin (CaM) is a eukaryotic multifunctional, calcium-modulated protein that regulates the activity of numerous effector proteins involved in a variety of physiological processes. Calmidazolium (CDZ) is a potent small molecule antagonist of CaM and one the most widely used inhibitors of CaM in cell biology. Here, we report the structural characterization of CaM:CDZ complexes using combined SAXS, X-ray crystallography, HDX-MS and NMR approaches. Our results provide molecular insights into the CDZ-induced dynamics and structural changes of CaM leading to its inhibition. CDZ-binding induces an open-to-closed conformational change of CaM and results in a strong stabilization of its structural elements associated with a reduction of protein dynamics over a large time range. These CDZ-triggered CaM changes mimic those induced by CaM-binding peptides derived from protein targets, despite their distant chemical nature. CaM residues in close contact with CDZ and involved in the stabilization of the CaM:CDZ complex have been identified. These results open the way to rationally design new CaM-selective drugs.Figure and text for the Table of Contents (ToC)Calmidazolium is a potent and widely used inhibitor of calmodulin, a major mediator of calcium-signaling in eukaryotic cells. Structural characterization of calmidazolium-binding to calmodulin reveals that it triggers open-to-closed conformational changes similar to those induced by calmodulin-binding peptides derived from enzyme targets. These results open the way to rationally design new and more selective inhibitors of calmodulin.

Published
2022

17. 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

19. 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

20. 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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21. 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

22. A Druggable Pocket at the Nucleocapsid/Phosphoprotein Interaction Site of Human Respiratory Syncytial Virus

Author

Mohamed Ouizougun-Oubari, Patrick England, Patrick Couvreur, Sylviane Hoos, Safa Lassoued, Jenna Fix, Félix A. Rey, Christina Sizun, M. Alejandra Tortorici, Bogdan Tarus, Jean-François Eléouët, François Bontems, Bruno Baron, Anny Slama-Schwok, Marie Galloux, N. Pereira, Stéphane Duquerroy, Didier Desmaële, Virologie Structurale - Structural Virology, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Institut de Chimie des Substances Naturelles (ICSN), Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Unité de recherche Virologie et Immunologie Moléculaires (VIM (UR 0892)), Institut National de la Recherche Agronomique (INRA), Protéopole, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut Galien Paris-Sud (IGPS), Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut de Chimie du CNRS (INC), Université Paris-Sud - Paris 11 (UP11), This work was supported by the Agence Nationale de la Recherche (grant ANR 11 BSV8 024 02 to S.D., F.A.R., J.-F.E., and C.S.) and the Grand Equipement National de Calcul Intensif (grant 2012-076378 to A.S.-S. and B.T. for access to IDRIS HPC resources). Doctoral fellowships from Université Pierre et Marie Curie (Ecole Doctorale Iviv) to M.O.-O., Institut de Chimie des Substances Naturelles to N.P., and Région Ile-de-France DIM Malinf to S.L. are acknowledged., We thank the staffs at beam lines PROXIMA-1 at the Soleil synchrotron (St Aubin, France), ID14-4 at the European Synchrotron Radiation Facility (Grenoble, France), and PX06SA at the SLS synchrotron (Villigen, Switzerland), Ahmed Haouz and Patrick Weber (Protein Crystallization Platform, Institut Pasteur Paris), Origène Nyanguile and Vanessa Gaillard (HES-SO Valais-Wallis, Sion, Switzerland) for providing the P12 peptide, and Jacques Perrault (San Diego, CA) and Christophe Chevalier (Jouy-en-Josas, France) for providing and amplifying VSV-GFP, respectively., ANR-11-BSV8-0024,BRONCHIOLITEASER,Etude structurale et fonctionnelle du complexe polymérase du virus respiratoire syncytial et recherche d'inhibiteurs(2011), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), and Université Paris-Sud - Paris 11 (UP11)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Models, Molecular, MESH: Antiviral Agents, Magnetic Resonance Spectroscopy, Protein Conformation, Viral protein, MESH: Nucleocapsid, [SDV]Life Sciences [q-bio], In silico, Immunology, Respiratory Syncytial Virus Infections, MESH: Respiratory Syncytial Virus, Human, Calorimetry, MESH: Drug Design, Biology, Crystallography, X-Ray, medicine.disease_cause, Antiviral Agents, MESH: Phosphoproteins, Microbiology, Virus, MESH: Respiratory Syncytial Virus Infections, MESH: Protein Conformation, X-Ray Diffraction, Virology, medicine, Humans, Binding site, Nucleocapsid, MESH: Calorimetry, Polymerase, MESH: Humans, MESH: Magnetic Resonance Spectroscopy, Structure and Assembly, MESH: X-Ray Diffraction, Phosphoproteins, MESH: Crystallography, X-Ray, 3. Good health, Nucleoprotein, Luminescent Proteins, Viral replication, Drug Design, Respiratory Syncytial Virus, Human, Insect Science, Phosphoprotein, biology.protein, MESH: Luminescent Proteins, MESH: Models, Molecular
Abstract

Presently, respiratory syncytial virus (RSV), the main cause of severe respiratory infections in infants, cannot be treated efficiently with antivirals. However, its RNA-dependent polymerase complex offers potential targets for RSV-specific drugs. This includes the recognition of its template, the ribonucleoprotein complex (RNP), consisting of genomic RNA encapsidated by the RSV nucleoprotein, N. This recognition proceeds via interaction between the phosphoprotein P, which is the main polymerase cofactor, and N. The determinant role of the C terminus of P, and more particularly of the last residue, F241, in RNP binding and viral RNA synthesis has been assessed previously. Here, we provide detailed structural insight into this crucial interaction for RSV polymerase activity. We solved the crystallographic structures of complexes between the N-terminal domain of N (N-NTD) and C-terminal peptides of P and characterized binding by biophysical approaches. Our results provide a rationale for the pivotal role of F241, which inserts into a well-defined N-NTD pocket. This primary binding site is completed by transient contacts with upstream P residues outside the pocket. Based on the structural information of the N-NTD:P complex, we identified inhibitors of this interaction, selected by in silico screening of small compounds, that efficiently bind to N and compete with P in vitro . One of the compounds displayed inhibitory activity on RSV replication, thereby strengthening the relevance of N-NTD for structure-based design of RSV-specific antivirals. IMPORTANCE Respiratory syncytial virus (RSV) is a widespread pathogen that is a leading cause of acute lower respiratory infections in infants worldwide. RSV cannot be treated efficiently with antivirals, and no vaccine is presently available, with the development of pediatric vaccines being particularly challenging. Therefore, there is a need for new therapeutic strategies that specifically target RSV. The interaction between the RSV phosphoprotein P and the ribonucleoprotein complex is critical for viral replication. In this study, we identified the main structural determinants of this interaction, and we used them to screen potential inhibitors in silico . We found a family of molecules that were efficient competitors of P in vitro and showed inhibitory activity on RSV replication in cellular assays. These compounds provide a basis for a pharmacophore model that must be improved but that holds promises for the design of new RSV-specific antivirals.

Published
2015

23. 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

24. The catalytic domains ofClostridium sordelliilethal toxin and related large clostridial glucosylating toxins specifically recognize the negatively charged phospholipids phosphatidylserine and phosphatidic acid

Author

Georges Michel Haustant, Serge Pauillac, D. Borden Lacy, Sylviane Hoos, Arnaud Blondel, Alexandre Chenal, Michel R. Popoff, Carolina Varela Chavez, and Patrick England

Subjects
Immunology, Phosphatidylserine, GTPase, Phosphatidic acid, Biology, Endocytosis, Microbiology, Sphingolipid, chemistry.chemical_compound, Cytosol, chemistry, Biochemistry, Virology, Phospholipid Binding, Lipid bilayer
Abstract

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

Published
2015

25. A Residue Quartet in the Extracellular Domain of the Prolactin Receptor Selectively Controls Mitogen-activated Protein Kinase Signaling

Author

Sylviane Hoos, Chi Zhang, Gitte W. Haxholm, Patrick England, Birthe B. Kragelund, Isabelle Broutin, Florence Boutillon, Mads Nygaard, Marie Bernadet, Vincent Goffin, Institut Necker Enfants-Malades (INEM - UM 111 (UMR 8253 / U1151)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Université Paris Descartes - Paris 5 (UPD5), University of Copenhagen = Københavns Universitet (KU), Biophysique des macromolécules et leurs interactions, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Laboratoire de cristallographie et RMN biologiques (LCRB - UMR 8015), Université Paris Descartes - Paris 5 (UPD5)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), This work was supported by the Danish Cancer Society grants (to B. B. K.), Danish Research Councils for Health and Disease Grants 09-072179 and 12-125862 (to B. B. K.), the Novo Nordic Foundation (to B. B. K.), Ligue Contre le Cancer Grant RS11/75-26 (to I. B.), University Paris Descartes Grant 990 UMRS 845/2011/02 (to V. G. and I. B.), and the Agence Nationale de la Recherche Grant ANR-07-PCVI-0029 (to V. G.)., ANR-07-PCVI-0029,PROLACTUMOR,Processus physiopathologiques impliquant le récepteur de la prolactine : approches physico-chimiques dynamiques et structurales(2007), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), University of Copenhagen = Københavns Universitet (UCPH), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), broutin, isabelle, and Physique et chimie du vivant - Processus physiopathologiques impliquant le récepteur de la prolactine : approches physico-chimiques dynamiques et structurales - - PROLACTUMOR2007 - ANR-07-PCVI-0029 - PCVI - VALID

Subjects
MESH: Signal Transduction, MAPK/ERK pathway, Magnetic Resonance Spectroscopy, Nuclear Magnetic Resonance (NMR), [SDV]Life Sciences [q-bio], Ligands, STAT Transcription Factor, Biochemistry, MESH: Circular Dichroism, MESH: Protein Structure, Tertiary, 0302 clinical medicine, STAT5 Transcription Factor, MESH: Ligands, Extracellular Signal-Regulated MAP Kinases, Receptor, MESH: Extracellular Signal-Regulated MAP Kinases, MESH: Cytokines, 0303 health sciences, MESH: Protein Multimerization, Circular Dichroism, Cell Surface Receptor, Cell biology, [SDV] Life Sciences [q-bio], Extracellular Signal-regulated Kinase (ERK), 030220 oncology & carcinogenesis, Mitogen-activated protein kinase, Cytokines, Signal transduction, Cytokine receptor, MESH: Spectrometry, Fluorescence, Protein Binding, Signal Transduction, MAP Kinase Signaling System, Receptors, Prolactin, Biology, Cell Line, 03 medical and health sciences, MESH: Receptors, Prolactin, Cell surface receptor, MESH: Cell Proliferation, Extracellular, Humans, MESH: Protein Binding, Cytokine, Molecular Biology, Cell Proliferation, 030304 developmental biology, MESH: Humans, MESH: MAP Kinase Signaling System, MESH: Magnetic Resonance Spectroscopy, MESH: STAT5 Transcription Factor, Prolactin receptor, Cell Membrane, Cell Biology, Protein Structure, Tertiary, MESH: Cell Line, Spectrometry, Fluorescence, biology.protein, Protein Multimerization, MESH: Cell Membrane
Abstract

Cytokine receptors elicit several signaling pathways, but it is poorly understood how they select and discriminate between them. We have scrutinized the prolactin receptor as an archetype model of homodimeric cytokine receptors to address the role of the extracellular membrane proximal domain in signal transfer and pathway selection. Structure-guided manipulation of residues involved in the receptor dimerization interface identified one residue (position 170) that in cell-based assays profoundly altered pathway selectivity and species-specific bio-characteristics. Subsequent in vitro spectroscopic and nuclear magnetic resonance analyses revealed that this residue was part of a residue quartet responsible for specific local structural changes underlying these effects. This included alteration of a novel aromatic T-stack within the membrane proximal domain, which promoted selective signaling affecting primarily the MAPK (ERK1/2) pathway. Importantly, activation of the MAPK pathway correlated with in vitro stabilities of ternary ligand·receptor complexes, suggesting a threshold mean lifetime of the complex necessary to achieve maximal activation. No such dependence was observed for STAT5 signaling. Thus, this study establishes a residue quartet in the extracellular membrane proximal domain of homodimeric cytokine receptors as a key regulator of intracellular signaling discrimination. Background: Discrimination of cytokine receptor signaling pathways is poorly understood. Results: Manipulation of extracellular prolactin receptor interface residues selectively affected activation of the MAPK pathway but not the STAT5 pathway. Conclusion: MAPK pathway activation correlated with apparent ligand·receptor complex lifetimes suggesting pathway-specific lifetime thresholds for activation. Significance: Discrimination of prolactin receptor signaling is controlled by the extracellular homodimer receptor interface.

Published
2015

26. Erratum: High affinity anchoring of the decoration protein pb10 onto the bacteriophage T5 capsid

Author

Emeline Vernhes, Madalena Renouard, Bernard Gilquin, Philippe Cuniasse, Dominique Durand, Patrick England, Sylviane Hoos, Alexis Huet, James F. Conway, Anatoly Glukhov, Vladimir Ksenzenko, Eric Jacquet, Naïma Nhiri, Sophie Zinn-Justin, Pascale Boulanger, 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), Bactériophage T5 (T5PHAG), Département Virologie (Dpt Viro), 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), Enveloppe Nucléaire, Télomères et Réparation de l’ADN (INTGEN), Département Biochimie, Biophysique et Biologie Structurale (B3S), and Fonction et Architecture des Assemblages Macromoléculaires (FAAM)

Subjects
0301 basic medicine, 03 medical and health sciences, 030104 developmental biology, Multidisciplinary, [SDV]Life Sciences [q-bio]
Abstract

Scientific Reports 7: Article number: 41662; published online: 06 February 2017; updated: 28 April 2017 This Article contains typographical errors. In the results section under subheading ‘The C-terminal domain adopts an immunoglobulin fold in solution’, “A search for structurally similar proteins possessing using the Dali server identified many proteins containing an Ig-like fold, including immunoglobulin receptors, fibroblast growth factor receptors and the T-cell surface glycoprotein CD4.

Published
2017

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

Author

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

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

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

Published
2016

28. Characterization of the elongasome core PBP2 : MreC complex of Helicobacter pylori

Author

Sylviane Hoos, Christine Ebel, Alexandre Martins, Christine Schmitt, Frank Gabel, Chantal Ecobichon, Patrick England, Andréa Dessen, Pierre-Jean Matteï, Ivo G. Boneca, Meriem El Ghachi, Marie-Christine Prévost, and Frédéric Colland

Subjects
chemistry.chemical_classification, 0303 health sciences, 030306 microbiology, Cell, Periplasmic space, biochemical phenomena, metabolism, and nutrition, Biology, Cell morphology, Microbiology, Yeast, Bacterial cell structure, Amino acid, law.invention, 03 medical and health sciences, medicine.anatomical_structure, chemistry, Biochemistry, law, polycyclic compounds, Recombinant DNA, Biophysics, medicine, Surface plasmon resonance, Molecular Biology, 030304 developmental biology
Abstract

Summary The definition of bacterial cell shape is a complex process requiring the participation of multiple components of an intricate macromolecular machinery. We aimed at characterizing the determinants involved in cell shape of the helical bacterium Helicobacter pylori. Using a yeast two-hybrid screen with the key cell elongation protein PBP2 as bait, we identified an interaction between PBP2 and MreC. The minimal region of MreC required for this interaction ranges from amino acids 116 to 226. Using recombinant proteins, we showed by affinity and size exclusion chromatographies and surface plasmon resonance that PBP2 and MreC form a stable complex. In vivo, the two proteins display a similar spatial localization and their complex has an apparent 1:1 stoichiometry; these results were confirmed in vitro by analytical ultracentrifugation and chemical cross-linking. Small angle X-ray scattering analyses of the PBP2 : MreC complex suggest that MreC interacts directly with the C-terminal region of PBP2. Depletion of either PBP2 or MreC leads to transition into spherical cells that lose viability. Finally, the specific expression in trans of the minimal interacting domain of MreC with PBP2 in the periplasmic space leads to cell rounding, suggesting that the PBP2/MreC complex formation in vivo is essential for cell morphology.

Published
2011

29. Cadherin-23, myosin VIIa and harmonin, encoded by Usher syndrome type I genes, form a ternary complex and interact with membrane phospholipids

Author

Sylviane Hoos, Jean-Pierre Hardelin, Christine Petit, Vincent Michel, Patrick England, Anne Houdusse, Amel Bahloul, Sylvie Nouaille, Génétique et Physiologie de l'Audition, Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Biophysique des Macromolécules et de leurs Interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut Curie [Paris], Institut Pasteur [Paris] (IP), Collège de France (CdF (institution)), This work was supported by LHW-Stiftung, Fondation Orange, Conny Maeva Foundation, ANR-05-MRAR-015-01, Raymonde and Guy Strittmatter Foundation (under the aegis of Fondation de France), FAUN Stiftung (Suchert Foundation). Funding to pay the Open Access Charge was provided by Unite de Genetique et Physiologie de l'Audition, Institut Pasteur, France., The authors thank Muriel Delepierre for NMR spectra, Raphaël Etournay and Alexandre Chenal for their advice in the preparation of LUV, Beatrice Amigues for myosin VIIa tail preparation, Bruno Baron for circular dichroism experiments, Jacqueline Levilliers for her help in the manuscript preparation and the staff of Dynamic Imaging platform of the Pasteur Institute., ANR-05-MRAR-0015,Usher type I,Physiopathologie du syndrome de Usher de type I : de la structure de la myosine VIIa et de l'harmonine à leur fonction dans les cellules ciliées auditives(2005), ENGLAND, Patrick, and Programme pluriannuel de recherche sur les maladies rares - Physiopathologie du syndrome de Usher de type I : de la structure de la myosine VIIa et de l'harmonine à leur fonction dans les cellules ciliées auditives - - Usher type I2005 - ANR-05-MRAR-0015 - MRAR - VALID

Subjects
Male, MESH: Cytoskeletal Proteins, [SDV]Life Sciences [q-bio], Cell Cycle Proteins, Plasma protein binding, MESH: Mice, Knockout, MESH: Cadherins, Mice, MESH: Protein Structure, Tertiary, Myosin, MESH: Hair Cells, Auditory, MESH: Animals, Ternary complex, Phospholipids, Genetics (clinical), Mice, Knockout, MESH: Protein Multimerization, Articles, General Medicine, Cadherins, [SDV] Life Sciences [q-bio], Biochemistry, Myosin VIIa, Female, Usher Syndromes, Protein Binding, Gene isoform, PDZ domain, MESH: Carrier Proteins, macromolecular substances, Myosins, Biology, Cell Line, MESH: Cell Cycle Proteins, Hair Cells, Auditory, otorhinolaryngologic diseases, Genetics, Molecular motor, Animals, Humans, MESH: Myosin VIIa, MESH: Protein Binding, MESH: Usher Syndromes, MESH: Mice, Molecular Biology, MESH: Phospholipids, MESH: Humans, Cadherin, MESH: Myosins, MESH: Male, Protein Structure, Tertiary, MESH: Cell Line, Cytoskeletal Proteins, Disease Models, Animal, Cytoplasm, Biophysics, sense organs, Protein Multimerization, MESH: Disease Models, Animal, Carrier Proteins, MESH: Female
Abstract

International audience; Cadherin-23 is a component of early transient lateral links of the auditory sensory cells' hair bundle, the mechanoreceptive structure to sound. This protein also makes up the upper part of the tip links that control gating of the mechanoelectrical transduction channels. We addressed the issue of the molecular complex that anchors these links to the hair bundle F-actin core. By using surface plasmon resonance assays, we show that the cytoplasmic regions of the two cadherin-23 isoforms that do or do not contain the exon68encoded peptide directly interact with harmonin, a submembrane PDZ (post-synaptic density, disc large, zonula occludens) domain-containing protein, with unusually high affinity. This interaction involves the harmonin Nter-PDZ1 supramodule, but not the C-terminal PDZ-binding motif of cadherin-23. We establish that cadherin-23 directly binds to the tail of myosin VIIa. Moreover, cadherin-23, harmonin and myosin VIIa can form a ternary complex, which suggests that myosin VIIa applies tension forces on hair bundle links. We also show that the cadherin-23 cytoplasmic region, harmonin and myosin VIIa interact with phospholipids on synthetic liposomes. Harmonin and the cytoplasmic region of cadherin-23, both independently and as a binary complex, can bind specifically to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2), which may account for the role of this phospholipid in the adaptation of mechanoelectrical transduction in the hair bundle. The distributions of cadherin-23, harmonin, myosin VIIa and PI(4,5)P 2 in the growing and mature auditory hair bundles as well as the abnormal locations of harmonin and myosin VIIa in cadherin-23 null mutant mice strongly support the functional relevance of these interactions.

Published
2010

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

Author

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

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

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

Published
2015

31. Quality assessment and optimization of purified protein samples: why and how?

Author

Bruno Baron, Patrick England, Bertrand Raynal, Sylviane Hoos, Pascal Lenormand, Biophysique des macromolécules et leurs interactions, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Quality Control, Electrophoresis, Recombinant protein, Bioengineering, Review, Biology, Applied Microbiology and Biotechnology, Consistency (database systems), Size-exclusion chromatography, Surface plasmon resonance, Diagnosis, Comparability, UV/visible spectroscopy, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Mass spectrometry, Quality assessment, Homogeneity (statistics), Light scattering, Recombinant Proteins, Biochemistry, Formulation, Biochemical engineering, Therapeutic, Structural biology, Protein quality, Biotechnology
Abstract

International audience; Purified protein quality control is the final and critical check-point of any protein production process. Unfortunately, it is too often overlooked and performed hastily, resulting in irreproducible and misleading observations in downstream applications. In this review, we aim at proposing a simple-to-follow workflow based on an ensemble of widely available physico-chemical technologies, to assess sequentially the essential properties of any protein sample: purity and integrity, homogeneity and activity. Approaches are then suggested to optimize the homogeneity, time-stability and storage conditions of purified protein preparations, as well as methods to rapidly evaluate their reproducibility and lot-to-lot consistency.

Published
2014

32. Structural and functional insights into the malaria parasite moving junction complex

Author

Maryse Lebrun, Magali Roques, Graham A. Bentley, Sylviane Hoos, Martin J. Boulanger, Susann Langer, Bart W. Faber, Mauld H. Lamarque, Brigitte Vulliez-Le Normand, Frederick Saul, Michelle L. Tonkin, Immunologie structurale, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Department of Biochemistry and Microbiology, University of Victoria [Canada] (UVIC), Dynamique des interactions membranaires normales et pathologiques (DIMNP), Université Montpellier 1 (UM1)-Université Montpellier 2 - Sciences et Techniques (UM2)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Biophysique des Macromolécules et de leurs Interactions, Department of Parasitology, Biomedical Primate Research Centre [Rijswijk] (BPRC), This work was supported by Canadian Institutes of Health Research (CIHR) grant MOP82915 to MJB and ANR-2009 MIEN-0222 grant to ML and GAB. MJB is a CIHR New Investigator and a Michael Smith Foundation for Health Research Scholar. MLT is supported by the Alexander Graham Bell Natural Sciences and Engineering Research Council Scholarship. MHL is supported by an ANR-2009 grant. FVO 25-545 protein and the different strains of PfAMA1 domains I and II were produced with funding of the European (Malaria) Vaccine Initiative., and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Models, Molecular, MESH: Protein Structure, Quaternary, [SDV]Life Sciences [q-bio], Protozoan Proteins, MESH: Amino Acid Sequence, Biochemistry, Protein structure, Molecular Cell Biology, MESH: Animals, Peptide sequence, lcsh:QH301-705.5, MESH: Protozoan Proteins, MESH: Plasmodium falciparum, MESH: Crystallization, Host cell membrane, 0303 health sciences, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Transmembrane protein, 3. Good health, Cell biology, MESH: Surface Plasmon Resonance, Rhoptry neck, MESH: Membrane Proteins, Crystallization, MESH: Models, Molecular, Protein Binding, Research Article, lcsh:Immunologic diseases. Allergy, Protein Structure, Immunology, Molecular Sequence Data, Plasmodium falciparum, Antigens, Protozoan, MESH: Host-Parasite Interactions, Microbiology, Host-Parasite Interactions, 03 medical and health sciences, Virology, MESH: Polymorphism, Genetic, parasitic diseases, Genetics, MESH: Protein Binding, Animals, [SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, Amino Acid Sequence, Apical membrane antigen 1, Protein Structure, Quaternary, Protein Interactions, Molecular Biology, Biology, 030304 developmental biology, MESH: Molecular Sequence Data, Polymorphism, Genetic, 030306 microbiology, Intracellular parasite, Cell Membrane, Membrane Proteins, Proteins, Surface Plasmon Resonance, biology.organism_classification, lcsh:Biology (General), Parasitology, lcsh:RC581-607, MESH: Antigens, Protozoan, MESH: Cell Membrane
Abstract

Members of the phylum Apicomplexa, which include the malaria parasite Plasmodium, share many features in their invasion mechanism in spite of their diverse host cell specificities and life cycle characteristics. The formation of a moving junction (MJ) between the membranes of the invading apicomplexan parasite and the host cell is common to these intracellular pathogens. The MJ contains two key parasite components: the surface protein Apical Membrane Antigen 1 (AMA1) and its receptor, the Rhoptry Neck Protein (RON) complex, which is targeted to the host cell membrane during invasion. In particular, RON2, a transmembrane component of the RON complex, interacts directly with AMA1. Here, we report the crystal structure of AMA1 from Plasmodium falciparum in complex with a peptide derived from the extracellular region of PfRON2, highlighting clear specificities of the P. falciparum RON2-AMA1 interaction. The receptor-binding site of PfAMA1 comprises the hydrophobic groove and a region that becomes exposed by displacement of the flexible Domain II loop. Mutations of key contact residues of PfRON2 and PfAMA1 abrogate binding between the recombinant proteins. Although PfRON2 contacts some polymorphic residues, binding studies with PfAMA1 from different strains show that these have little effect on affinity. Moreover, we demonstrate that the PfRON2 peptide inhibits erythrocyte invasion by P. falciparum merozoites and that this strong inhibitory potency is not affected by AMA1 polymorphisms. In parallel, we have determined the crystal structure of PfAMA1 in complex with the invasion-inhibitory peptide R1 derived by phage display, revealing an unexpected structural mimicry of the PfRON2 peptide. These results identify the key residues governing the interactions between AMA1 and RON2 in P. falciparum and suggest novel approaches to antimalarial therapeutics., Author Summary Malaria arises from infection of erythrocytes by single-cell parasites belonging to the genus Plasmodium, the species P. falciparum causing the most severe forms of the disease. The formation of a moving junction (MJ) between the membranes of the parasite and its host cell is essential for invasion. Two important components of the MJ are Apical Membrane Antigen 1 (AMA1) on the parasite surface and the Plasmodium rhoptry neck (RON) protein complex that is translocated to the erythrocyte membrane during invasion. The extra-cellular region of RON2, a component of this complex, interacts with AMA1, providing a bridge between the parasite and its host cell that is crucial for successful invasion. The parasite thus provides its own receptor for AMA1 and accordingly this critical interaction is not subject to evasive adaptations by the host. We present atomic details of the interaction of PfAMA1 with the carboxy-terminal region of RON2 and shed light on structural adaptations by each apicomplexan parasite to maintain an interaction so crucial for invasion. The structure of the RON2 ligand bound to AMA1 thus provides an ideal basis for drug design as such molecules may be refractory to the development of drug resistance in P. falciparum.

Published
2011

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

Author

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

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

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

Published
2011

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

Author

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

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

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

Published
2011

35. Structural characterization of the stem-stem dimerization interface between prolactin receptor chains complexed with the natural hormone

Author

Vincent Goffin, Johannes F. Van Agthoven, Chi Zhang, Isabelle Broutin, Bruno Baron, Bertrand Raynal, Patrick England, Estelle Tallet, Sylviane Hoos, Laboratoire de cristallographie et RMN biologiques (LCRB - UMR 8015), Université Paris Descartes - Paris 5 (UPD5)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Centre de recherche Croissance et signalisation (UMR_S 845), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Descartes - Paris 5 (UPD5), Biophysique des Macromolécules et de leurs Interactions, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), This work was supported, in part, by University Paris Descartes and the Agence Nationale de la Recherche (grant ANR-07-PCVI-0029). J.v.A., C.Z., and E.T. acknowledge the Ministère de l'Education Nationale de la Recherche et de la Technologie for fellowship support., ANR-07-PCVI-0029,PROLACTUMOR,Processus physiopathologiques impliquant le récepteur de la prolactine : approches physico-chimiques dynamiques et structurales(2007), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Models, Molecular, MESH: Protein Structure, Quaternary, Receptors, Prolactin, [SDV]Life Sciences [q-bio], Protein Data Bank (RCSB PDB), MESH: Prolactin, Growth hormone receptor, Biology, Crystallography, X-Ray, 03 medical and health sciences, Paracrine signalling, MESH: Receptors, Prolactin, 0302 clinical medicine, Structural Biology, MESH: Protein Binding, Receptor, Autocrine signalling, Protein Structure, Quaternary, Molecular Biology, 030304 developmental biology, 0303 health sciences, MESH: Protein Multimerization, Prolactin receptor, Mutagenesis, Surface Plasmon Resonance, MESH: Crystallography, X-Ray, MESH: Amino Acid Substitution, MESH: Surface Plasmon Resonance, Cell biology, Prolactin, MESH: Mutagenesis, Site-Directed, Biochemistry, Amino Acid Substitution, 030220 oncology & carcinogenesis, Growth hormone receptor complex, Mutagenesis, Site-Directed, Protein Multimerization, MESH: Models, Molecular, Protein Binding
Abstract

International audience; The most promising approach to targeting the tumor-growth-promoting actions of prolactin (PRL) mediated by its autocrine/paracrine pathway has been the development of specific PRL receptor (PRLR) antagonists. However, the optimization of such antagonists requires a thorough understanding of the activation mechanism of PRLR. We have thus conducted a systematic X-ray crystallographic study in order to visualize the successive steps of PRLR activation by PRL. We report here the structure at 3.35 Å resolution of the 1:2 complex between natural PRL and two PRLR chains (PRLR1 and PRLR2), corresponding to the final activated state of PRLR. Further than our previously published structure involving an affinity-matured PRL variant, this structure allowed to visualize for the first time the loop L5 spanning PRLR2 residues Thr133-Phe140, revealing its central implication for the three intermolecular interfaces of the complex. We equally succeeded in obtaining a comprehensive picture of the PRLR-PRLR dimerization interface, also called stem-stem interface. Site-directed mutagenesis was conducted to probe the energetic importance of stem-stem contacts highlighted by the structure. Surprisingly, in spite of significant structural differences between the PRL/PRLR(2) complex and the 1:2 growth hormone/growth hormone receptor complex, our mutational data suggest that hot-spot residues that stabilize the receptor dimerization interface are equivalent in the two complexes. This study provides a new overall picture of the structural features of PRLR involved in stabilizing its complex with PRL.

Published
2010

36. AIF promotes chromatinolysis and caspase-independent programmed necrosis by interacting with histone H2AX

Author

Hanan Boujrad, Pascal Lenormand, Sylviane Hoos, Cédric Artus, Marie Noëlle Brunelle, Hans K. Lorenzo, Santos A. Susin, Patrick England, Ada Bouharrour, Abdelkader Namane, Jean Claude Rousselle, Victor J. Yuste, Centre de Recherche des Cordeliers (CRC (UMR_S 872)), Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Biophysique des Macromolécules et de leurs Interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Universitat Autònoma de Barcelona (UAB), Protéomique (Plate-Forme), Institut Pasteur [Paris] (IP), Institut André Lwoff [Villejuif] ( IAL), Université Paris-Sud - Paris 11 - Faculté de médecine (UP11 UFR Médecine), Université Paris-Sud - Paris 11 (UP11), AP-HP Hôpital Bicêtre (Le Kremlin-Bicêtre), SAS is supported by INSERM, UPMC-Paris 6, CNRS, ANR (contract ANR-09-BLAN-0247-01), ARC (contracts 4043, 5104, and 7987), and Fondation de France., We thank A Nussenzweig (NIH, NCI, Bethesda) for immortalized H2AX-wt and H2AX−/− MEFs, NV Tomilin (Institute of Cytology, St Petersburg) for the H2AX and H2AXS139A cDNA, and M Segade and S Krantic for critical comments, ANR-09-BLAN-0247,AIFCICD(2009), ENGLAND, Patrick, and Blanc - - AIFCICD2009 - ANR-09-BLAN-0247 - Blanc - VALID

Subjects
Models, Molecular, MESH: Calpain, [SDV]Life Sciences [q-bio], Poly (ADP-Ribose) Polymerase-1, Cypa, MESH: Poly (ADP-Ribose) Polymerase-1, MESH: Down-Regulation, Histones, Mice, H2AX, MESH: Animals, Caspase, bcl-2-Associated X Protein, MESH: Histones, biology, Calpain, General Neuroscience, MESH: Cyclophilin A, Apoptosis Inducing Factor, Chromatin, Cell biology, [SDV] Life Sciences [q-bio], Histone, Caspases, Apoptosis-inducing factor, biological phenomena, cell phenomena, and immunity, Poly(ADP-ribose) Polymerases, programmed necrosis, Cyclophilin A, MESH: Models, Molecular, Programmed cell death, Methylnitronitrosoguanidine, Poly ADP ribose polymerase, Down-Regulation, PARP-1, General Biochemistry, Genetics and Molecular Biology, chromatin & transcription, Article, MESH: Chromatin, Cell Line, Necrosis, Bcl-2-associated X protein, Animals, MESH: bcl-2-Associated X Protein, cardiovascular diseases, Molecular Biology, MESH: Mice, MESH: Necrosis, MESH: DNA Damage, MESH: Caspases, General Immunology and Microbiology, MESH: Poly(ADP-ribose) Polymerases, MESH: Methylnitronitrosoguanidine, Fibroblasts, biology.organism_classification, MESH: Cell Line, MESH: Apoptosis Inducing Factor, MESH: Gene Deletion, MESH: Fibroblasts, differentiation & death AIF, biology.protein, Gene Deletion, DNA Damage
Abstract

International audience; Programmed necrosis induced by DNA alkylating agents, such as MNNG, is a caspase-independent mode of cell death mediated by apoptosis-inducing factor (AIF). After poly(ADP-ribose) polymerase 1, calpain, and Bax activation, AIF moves from the mitochondria to the nucleus where it induces chromatinolysis and cell death. The mechanisms underlying the nuclear action of AIF are, however, largely unknown. We show here that, through its C-terminal proline-rich binding domain (PBD, residues 543-559), AIF associates in the nucleus with histone H2AX. This interaction regulates chromatinolysis and programmed necrosis by generating an active DNA-degrading complex with cyclophilin A (CypA). Deletion or directed mutagenesis in the AIF C-terminal PBD abolishes AIF/H2AX interaction and AIF-mediated chromatinolysis. H2AX genetic ablation or CypA downregulation confers resistance to programmed necrosis. AIF fails to induce chromatinolysis in H2AX or CypA-deficient nuclei. We also establish that H2AX is phosphorylated at Ser139 after MNNG treatment and that this phosphorylation is critical for caspase-independent programmed necrosis. Overall, our data shed new light in the mechanisms regulating programmed necrosis, elucidate a key nuclear partner of AIF, and uncover an AIF apoptogenic motif.

Published
2010

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

Author

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

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

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

Published
2010

38. Attenuation of rabies virulence: takeover by the cytoplasmic domain of its envelope protein

Author

Muriel Delepierre, Gene S. Tan, Françoise Mégret, Christophe Prehaud, Pauline Ménager, Matthias J. Schnell, Nicolas Wolff, Damien Chopy, Mireille Lafage, Elouan Terrien, Florence Cordier, Elodie Maitrepierre, Nicolas Babault, Monique Lafon, Patrick England, Sylviane Hoos, Henri Buc, Neuro-Immunologie Virale, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Résonance Magnétique Nucléaire des Biomolécules, Department of Microbiology and Immunology, Thomas Jefferson University-Jefferson Vaccine Centre, Biophysique des Macromolécules et de leurs Interactions, Institut Pasteur [Paris] (IP), This work was supported by grants from Institut Pasteur (IP) and Agence Nationale pour la Recherche (ANR) MIME. E.T., D.C., and N.B. are recipients of Ministère de l’Education et de la Recherche fellowships, and P.M. is a recipient of an ANR fellowship., We thank P. Roux, J. D’Alayer, V. Bondet, E. Frachon, J. Bellalou, and J.-F. Charles from IP core facilities for their help. We thank S. Loh and P. Nicotera for the gift of the SH-SY5Y cells, B. Dietzschold for fruitful discussions, E. Vinolo and B. Ruggiero for help in two-hybrid result interpretation, and F. Rey for his support., Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Jefferson (Philadelphia University + Thomas Jefferson University), and Institut Pasteur [Paris]

Subjects
Cytoplasm, [SDV]Life Sciences [q-bio], MESH: Neurons, PDZ Domains, Apoptosis, Protein tyrosine phosphatase, MESH: Virulence, medicine.disease_cause, Biochemistry, Mice, 0302 clinical medicine, Viral Envelope Proteins, MESH: PDZ Domains, MESH: Animals, chemistry.chemical_classification, Neurons, 0303 health sciences, Gene knockdown, Virulence, Kinase, Protein Tyrosine Phosphatase, Non-Receptor Type 4, MESH: Amino Acid Substitution, MESH: Rabies virus, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Rabies, PDZ domain, Biology, Protein Serine-Threonine Kinases, MESH: Protein-Serine-Threonine Kinases, 03 medical and health sciences, MESH: Rabies, medicine, Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, MESH: Mice, 030304 developmental biology, MESH: Protein Tyrosine Phosphatase, Non-Receptor Type 4, MESH: Apoptosis, MESH: Cytoplasm, Rabies virus, Cell Biology, Molecular biology, chemistry, Amino Acid Substitution, MESH: Viral Envelope Proteins, sense organs, Glycoprotein, 030217 neurology & neurosurgery
Abstract

International audience; The capacity of a rabies virus to promote neuronal survival (a signature of virulence) or death (a marker of attenuation) depends on the cellular partners recruited by the PDZ-binding site (PDZ-BS) of its envelope glycoprotein (G). Neuronal survival requires the selective association of the PDZ-BS of G with the PDZ domains of two closely related serine-threonine kinases, MAST1 and MAST2. Here, we found that a single amino acid change in the PDZ-BS triggered the apoptotic death of infected neurons and enabled G to interact with additional PDZ partners, in particular the tyrosine phosphatase PTPN4. Knockdown of PTPN4 abrogated virus-mediated apoptosis. Thus, we propose that attenuation of rabies virus requires expansion of the set of host PDZ proteins with which G interacts, which interferes with the finely tuned homeostasis required for survival of the infected neuron.

Published
2010

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

Author

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

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

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

Published
2009

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

Author

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

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

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

Published
2009

41. A global benchmark study using affinity-based biosensors

Author

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

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

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

Published
2009

42. The FHA-containing protein GarA acts as a phosphorylation-dependent molecular switch in mycobacterial signaling

Author

Andrea Villarino, Gwenaëlle André-Leroux, Sonia Martins, Annemarie Wehenkel, Sylviane Hoos, Patrick England, Pedro M. Alzari, Alzari, Pedro M, Institut Pasteur [Paris], Institut Pasteur [Paris] (IP), Biochimie Structurale, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Biophysique des macromolécules et de leurs interactions (Plate-forme), This work was supported by grants from the Institut Pasteur, the CNRS (France) and the European Union (NM4TB, Contract No. LSHP-CT-2005-018923)., The authors gratefully acknowledge the professional technical assistance of J. d'Alayer, F. Groh, I. Miras and V. Bondet. Finally we thank Dr. M. Bellinzoni for helpful advice and constructive criticism, and European Project: LSHP-CT

Subjects
Circular dichroism, Protein Conformation, [SDV]Life Sciences [q-bio], Biophysics, Regulator, Glutamic Acid, Signal transduction, Calorimetry, Biochemistry, Mycobacterium tuberculosis, 03 medical and health sciences, Bacterial Proteins, Structural Biology, Genetics, Phosphorylation, Molecular Biology, Polyacrylamide gel electrophoresis, 030304 developmental biology, 0303 health sciences, biology, Kinase, Chemistry, 030302 biochemistry & molecular biology, Forkhead Transcription Factors, Cell Biology, biology.organism_classification, Protein Structure, Tertiary, Physical chemistry, FHA domain, Glycogen, Bacteria
Abstract

Fork-head associated (FHA) domains are widely found in bacteria, but their cellular functions remain unclear. Here, we focus on Mycobacterium tuberculosis GarA, an FHA-containing protein conserved in actinomycetes that is phosphorylated by different Ser/Thr protein kinases. Using various physicochemical approaches, we show that phosphorylation significantly stabilizes GarA, and that its FHA domain interacts strongly with the phosphorylated N-terminal extension. Altogether, our results indicate that phosphorylation triggers an intra-molecular protein closure, blocking the phosphothreonine-binding site and switching off the regulatory properties of GarA. The model can explain the reported functions of this mycobacterial protein as regulator of glycogen degradation and glutamate metabolism.Structured summary:MINT-6804218: GarA (uniprotkb:P64897) and GarA (uniprotkb:P64897) bind (MI:0407) by isothermal titration calorimetry (MI:0065)

Published
2009

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

Author

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

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

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

Published
2007

44. Structural and thermodynamic bases for the design of pure prolactin receptor antagonists: X-ray structure of Del1-9-G129R-hPRL

Author

Jean-Baptiste, Jomain, Estelle, Tallet, Isabelle, Broutin, Sylviane, Hoos, Jan, van Agthoven, Arnaud, Ducruix, Paul A, Kelly, Birthe B, Kragelund, Patrick, England, and Vincent, Goffin

Subjects
Models, Molecular, Protein Denaturation, Hot Temperature, Receptors, Prolactin, Circular Dichroism, Molecular Sequence Data, Glycine, Crystallography, X-Ray, Cell Line, Prolactin, Protein Structure, Tertiary, Rats, Kinetics, Structural Homology, Protein, Drug Design, Mutation, Animals, Humans, Thermodynamics, Amino Acid Sequence, Protein Binding
Abstract

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

Published
2007

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

Author

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

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

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

Published
2019

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

Author

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

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

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

Published
2016

47. Binding of the unorthodox transcription activator, Crl, to the components of the transcription machinery

Author

Véronique Robbe-Saule, Gouzel Karimova, Annie Kolb, Françoise Norel, Patrick England, Lars F. Westblade, Institut Pasteur [Paris] (IP), Biophysique des Macromolécules et de leurs Interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Rockefeller University [New York], Biochimie des Interactions Macromoléculaires, Génétique Moléculaire, and We thank Samita Sally Goyal for performing the β-galactosidase assays and Christelle Talhouarne and Sylviane Hoos for monitoring the first SPR experiments. We thank Henri Buc for helpful suggestions.

Subjects
BACTH, Transcription, Genetic, [SDV]Life Sciences [q-bio], bacterial adenylate cyclase two-hybrid, response units, SPR, RU, Sigma Factor, Plasma protein binding, Biology, Biochemistry, chemistry.chemical_compound, Bacterial Proteins, Transcription (biology), Sigma factor, MESH: Catalase, RNA polymerase, MESH: Promoter Regions, Genetic, MESH: Protein Binding, Transcription, Chromatin, and Epigenetics, Mode of action, Promoter Regions, Genetic, MESH: Bacterial Proteins, Molecular Biology, Gene, Transcription factor, Regulation of gene expression, MESH: Transcription, Genetic, fungi, MESH: Sigma Factor, food and beverages, Salmonella enterica, MESH: Transcription Factors, Cell Biology, DNA-Directed RNA Polymerases, Surface Plasmon Resonance, Catalase, MESH: DNA-Directed RNA Polymerases, MESH: Surface Plasmon Resonance, Cell biology, ciated holoenzyme, chemistry, MESH: Salmonella enterica, Protein Binding, Transcription Factors
Abstract

International audience; The small regulatory protein Crl binds to sigmaS, the RNA polymerase stationary phase sigma factor. Crl facilitates the formation of the sigmaS-associated holoenzyme (EsigmaS) and thereby activates sigmaS-dependent genes. Using a real time surface plasmon resonance biosensor, we characterized in greater detail the specificity and mode of action of Crl. Crl specifically forms a 1:1 complex with sigmaS, which results in an increase of the association rate of sigmaS to core RNA polymerase without any effect on the dissociation rate of EsigmaS. Crl is also able to associate with preformed EsigmaS with a higher affinity than with sigmaS alone. Furthermore, even at saturating sigmaS concentrations, Crl significantly increases EsigmaS association with the katN promoter and the productive isomerization of the EsigmaS-katN complex, supporting a direct role of Crl in transcription initiation. Finally, we show that Crl does not bind to sigma70 itself but is able at high concentrations to form a weak and transient 1:1 complex with both core RNA polymerase and the sigma70-associated holoenzyme, leaving open the possibility that Crl might also exert a side regulatory role in the transcriptional activity of additional non-sigmaS holoenzymes.

Published
2008

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