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1. Membrane-remodeling protein ESCRT-III homologs incarnate the evolution and morphogenesis of multicellular magnetotactic bacteria

Author

Wenyan Zhang, Jianwei Chen, Jie Dai, Shiwei Zhu, Hugo Le Guenno, Artemis Kosta, Hongmiao Pan, Xin-Xin Qian, Claire-Lise Santini, Nicolas Menguy, Xuegong Li, Yiran Chen, Jia Liu, Kaixuan Cui, Yicong Zhao, Guilin Liu, Eric Durand, Wei-Jia Zhang, Alain Roussel, Tian Xiao, Long-Fei Wu, Helmholtz-Zentrum Geesthacht (GKSS), Ming Hsieh Department of Electrical Engineering [Los Angeles], USC Viterbi School of Engineering, University of Southern California (USC)-University of Southern California (USC), Research Center for Proteome Analysis Key Lab of Proteomics, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Chinese Academy of Sciences [Beijing] (CAS), Yale School of Medicine [New Haven, Connecticut] (YSM), Centre d'Immunologie de Marseille - Luminy (CIML), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), CAS Key Laboratory of Marine Ecology and Environmental Science (KLMEES), CAS Institute of Oceanology (IOCAS), Chinese Academy of Sciences [Beijing] (CAS)-Chinese Academy of Sciences [Beijing] (CAS), Laboratoire de chimie bactérienne (LCB), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Institut de minéralogie, de physique des matériaux et de cosmochimie (IMPMC), Muséum national d'Histoire naturelle (MNHN)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de recherche pour le développement [IRD] : UR206-Centre National de la Recherche Scientifique (CNRS), International Associated Laboratory of Evolution and Development of Magnetotactic Multicellular Organisms ( LIA-MagMC), Chinese Academy of Sciences [Beijing] (CAS)-Centre National de la Recherche Scientifique (CNRS), Xi'an Jiaotong University (Xjtu), Endothélium, valvulopathies et insuffisance cardiaque (EnVI), Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM), Architecture et fonction des macromolécules biologiques (AFMB), and Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)

Subjects
[SDV.BID.EVO]Life Sciences [q-bio]/Biodiversity/Populations and Evolution [q-bio.PE]
Abstract

Endosomal sorting complex required transport (ESCRT) III proteins are essential for membrane remodeling and repair across all domains of life. Eukaryotic ESCRT-III and the cyanobacterial homologs PspA and Vipp1/Imm30 remodel membrane into vesicles, rings, filaments and tubular rods structures. Here our microscopy analysis showed that multicellular bacteria, referred to as magnetoglobules, possess multiple compartments including magnetosome organelles, polyphosphate granules, vesicles, rings, tubular rods, filaments and MVB-like structures. Therefore, membrane remodeling protein PspA might be required for the formation of these compartments, and contribute to the morphogenesis and evolution of multicellularity. To assess these hypotheses, we sequenced nine genomes of magnetoglobules and found a significant genome expansion compared to unicellular magnetotactic bacteria. Moreover, PspA was ubiquitous in magnetoglobules and formed a distinct clade on the tree of eubacterial and archaeal ESCRT-III. The phylogenetic feature suggested the evolution of magnetoglobules from a unicellular ancestor of deltaproteobacterium. Hetero-expression of ellipsoidal magnetoglobulepspA2gene alone inEscherichia coliresulted in intracellular membrane aggregation. GFP fusion labeling revealed polar location of PspA2 in rod-shaped unicells and regular interval location in filamentous cells. Cryo-electron tomography analysis showed filament bundle, membrane sacculus, vesicles and MVB-like structure in the cells expressing PspA2. Moreover, electron-dense area with a similar distribution as GFP-PspA2 foci in filamentous cells changed the inward orientation of the septum, which might interfere with the cell division. Collectively, these results show the membrane remodeling function of magnetoglobule PspA proteins, which may contribute to morphogenesis and the evolution of multicellularity of magnetotactic bacteria.

Published
2022

2. Sensing of COVID-19 spike protein in nasopharyngeal samples using a portable surface plasmon resonance diagnostic system

Author

Hiba Saada, Quentin Pagneux, James Wei, Ludovic Live, Alain Roussel, Alexis Dogliani, Lycia Die Morini, Ilka Engelmann, Enagnon Kazali Alidjinou, Anne Sophie Rolland, Emmanuel Faure, Julien Poissy, Julien Labreuche, Gil Lee, Peng Li, Gerard Curran, Anass Jawhari, Jhonny A. Yunda, Sorin Melinte, Axel Legay, Jean-Luc Gala, David Devos, Rabah Boukherroub, Sabine Szunerits, Institut d’Électronique, de Microélectronique et de Nanotechnologie - UMR 8520 (IEMN), Centrale Lille-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Université Polytechnique Hauts-de-France (UPHF)-JUNIA (JUNIA), Université catholique de Lille (UCL)-Université catholique de Lille (UCL), Affinité Instruments, Laboratoire d'ingénierie des systèmes macromoléculaires (LISM), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Pathogenèse virale du diabète de type 1 - ULR 3610 (Laboratoire de Virologie), Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille), Lille Neurosciences & Cognition - U 1172 (LilNCog), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille), 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), Evaluation des technologies de santé et des pratiques médicales - ULR 2694 (METRICS), Magnostics, Coherent Optical Microscopy and X-rays (COMiX), Institut FRESNEL (FRESNEL), Aix Marseille Université (AMU)-École Centrale de Marseille (ECM)-Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-École Centrale de Marseille (ECM)-Centre National de la Recherche Scientifique (CNRS), Biosensing Diagnostics, Université Catholique de Louvain = Catholic University of Louvain (UCL), NanoBioInterfaces - IEMN (NBI - IEMN), Université catholique de Lille (UCL)-Université catholique de Lille (UCL)-Centrale Lille-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Université Polytechnique Hauts-de-France (UPHF)-JUNIA (JUNIA), The project is funded by the Horizon 2020 framework programme of the European Union under grant agreement No. 101016038 (CorDial-S). This work was supported by theBelgian F.R.S. – FNRS under Grant No. R.8008.21. Financial support by ANR via CorDial-Flu is in addition acknowledged., European Project: 101016038,H2020,H2020-SC1-PHE-CORONAVIRUS-2020-2-CNECT, CorDial-S(2020), IEMN, Collection, Portable and fast surface plasmon resonance point-of-care test for COVID-19 - CorDial-S - - H20202020-12-01 - 2021-11-30 - 101016038 - VALID, UCL - SST/ICTM/ELEN - Pôle en ingénierie électrique, and UCL - SSS/IREC/CTMA - Centre de technologies moléculaires appliquées (plate-forme technologique)

Subjects
Machine Learning, VHH-72-Fc, [SPI]Engineering Sciences [physics], Diagnostic system, Plasmon resonance, COVID-19 spike protein, [SPI] Engineering Sciences [physics], SARS-CoV-2, COVID-19, Surface Plasmon Resonance
Abstract

International audience; Rapid, yet sensitive and accurate testing concepts are critical in the control of spreading diseases. With the COVID-19 pandemic still ongoing, the need for efficient, fast and accurate testing of the infection state of children and the elderly traveling as well as people with symptoms has not declined. Most current methods, which are highly sensitive, are rather slow and cannot be applied at the point of care. While rapid antigenic tests ascertain only high viral burden, here, we demonstrate an alternative, rapid point-of-care diagnostics with the ability to sense low viral loads. The goal of a portable and fast quantitative diagnostic device has been achieved via the use of VHH-72-Fc, a nanobody featuring high binding strength to the spike 1 glycoprotein of the SARS-CoV-2 viral envelope, a surface plasmon resonance sensing approach, and machine learning for predicting the cut-off value between positive and negative nasopharyngeal swab samples. The concept was validated on 119 nasopharyngeal samples, 50 positive and 69 negative, as identified by reverse transcription quantitative polymerase chain reaction (RT-qPCR) tests, showing a 88% positive percentage agreement and a 92% negative percentage agreement, as compared to RT-qPCR. Simple artificial neural network data processing revealed the influence in the sampling time to achieve unique performance in terms of speed, specificity and sensitivity. These sensing features combined with no sample preparation and portability of the diagnostic device suggest that this approach is well-adapted to be operated in hospital or laboratory located diagnostic centres.

Published
2022

3. The role of the surface ligand on the performance of electrochemical SARS-CoV-2 antigen biosensors

Author

David Devos, Christian Cambillau, Henri Happy, Quentin Pagneux, Vladyslav Mishyn, Enagnon Kazali Alidjinou, Ilka Engelmann, Rabah Boukherroub, Abir Swaidan, Sabine Szunerits, Alain Roussel, Institut d’Électronique, de Microélectronique et de Nanotechnologie - UMR 8520 (IEMN), Centrale Lille-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Université Polytechnique Hauts-de-France (UPHF)-JUNIA (JUNIA), NanoBioInterfaces - IEMN (NBI - IEMN), Centrale Lille-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Université Polytechnique Hauts-de-France (UPHF)-JUNIA (JUNIA)-Centrale Lille-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Université Polytechnique Hauts-de-France (UPHF)-JUNIA (JUNIA), Architecture et fonction des macromolécules biologiques (AFMB), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Lille Neurosciences & Cognition - U 1172 (LilNCog (ex-JPARC)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille), Centre d'Immunologie de Marseille - Luminy (CIML), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU), Pathogenèse virale du diabète de type 1 - ULR 3610 (Laboratoire de Virologie), Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille), Carbon - IEMN (CARBON-IEMN), Centre National de la Recherche Scientifique (CNRS)Centre National de la Recherche Scientifique (CNRS), University of Lille, I-SITE, via the COVID task force, Hauts-de-France region via ANR Resilience (CorDial-FLU), French Infrastructure for Integrated Structural Biology (FRISBI) [ANR-10-INBS-0005], ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-21-HDF1-0003,CorDial-FLU,Dispositive portable de diagnostic pour différencier le virus la grippe de celui de Covid-19(2021), Université catholique de Lille (UCL)-Université catholique de Lille (UCL), Université catholique de Lille (UCL)-Université catholique de Lille (UCL)-Centrale Lille-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Université Polytechnique Hauts-de-France (UPHF)-JUNIA (JUNIA), Lille Neurosciences & Cognition - U 1172 (LilNCog), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Carbon - IEMN (CARBON - IEMN), IEMN, Collection, Infrastructure Française pour la Biologie Structurale Intégrée - - FRISBI2010 - ANR-10-INBS-0005 - INBS - VALID, and Dispositive portable de diagnostic pour différencier le virus la grippe de celui de Covid-19 - - CorDial-FLU2021 - ANR-21-HDF1-0003 - PRHDF - VALID

Subjects
Coronavirus disease 2019 (COVID-19), Computer science, [SPI] Engineering Sciences [physics], Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Point-of-care testing, Point-of-Care Systems, Medical laboratory, Economic shortage, 02 engineering and technology, Review, Biosensing Techniques, point-of-care (poc) technologies, Ligands, 01 natural sciences, Biochemistry, Analytical Chemistry, [SPI]Engineering Sciences [physics], COVID-19 Testing, Health care, Humans, field effect transistors, Antigens, Viral, Disease surveillance, business.industry, 010401 analytical chemistry, COVID-19, Electrochemical Techniques, 021001 nanoscience & nanotechnology, biosensors, 0104 chemical sciences, 3. Good health, sars-cov-2, Risk analysis (engineering), electrochemistry, surface ligands, Analytics, Point-of-Care Testing, 0210 nano-technology, business
Abstract

International audience; Point-of-care (POC) technologies and testing programs hold great potential to significantly improve diagnosis and disease surveillance. POC tests have the intrinsic advantage of being able to be performed near the patient or treatment facility, owing to their portable character. With rapid results often in minutes, these diagnostic platforms have a high positive impact on disease management. POC tests are, in addition, advantageous in situations of a shortage of skilled personnel and restricted availability of laboratory-based analytics. While POC testing programs are widely considered in addressing health care challenges in low-income health systems, the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections could largely benefit from fast, efficient, accurate, and cost-effective point-of-care testing (POCT) devices for limiting COVID-19 spreading. The unrestrained availability of SARS-CoV-2 POC tests is indeed one of the adequate means of better managing the COVID-19 outbreak. A large number of novel and innovative solutions to address this medical need have emerged over the last months. Here, we critically elaborate the role of the surface ligands in the design of biosensors to cope with the current viral outbreak situation. Their notable effect on electrical and electrochemical sensors' design will be discussed in some given examples.

Published
2021

4. Cas9 Allosteric Inhibition by the Anti-CRISPR Protein AcrIIA6

Author

Christian Cambillau, Béatrice Amigues, Geneviève M. Rousseau, Sylvain Moineau, Claire Zimberger, Alain Roussel, Olivier Fuchsbauer, Antonio Chaves-Sanjuan, Yannick Doyon, Alexis Duringer, Adeline Goulet, Sébastien Lévesque, Minja Velimirovic, Paolo Swuec, Silvia Spinelli, Daniel Agudelo, Martino Bolognesi, Architecture et fonction des macromolécules biologiques (AFMB), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Università degli Studi di Milano [Milano] (UNIMI), Centre de recherche du CHU de Québec-Université Laval (CRCHUQ), CHU de Québec–Université Laval, Université Laval [Québec] (ULaval)-Université Laval [Québec] (ULaval), Faculté de médecine dentaire [Université Laval, Québec], Université Laval [Québec] (ULaval), Dpt de Microbiologie-Infectiologie et d’Immunologie [Laval], Department of Biosciences [Milano], Félix d'Hérelle Reference Center for Bacterial Viruses, Groupe de Recherche en Écologie Buccale (GREB), Centre National de la Recherche Scientifique (CNRS), Aix Marseille Université (AMU), NSERC of Canada, Canada RGPIN-2014-05132/RGPIN-2014-05698Fonds de la Recherche en Sante du Quebec FRQS 254294United States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA R01-GM129325/P41-GM103311, ANR-18-CE11-0016,PHARE,Vengeance de phages: analyses structurales et fonctionnelles de protéines anti CRISPR-Cas9(2018), ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), Hôpital de l'Enfant-Jésus [CHU Québec] (HEJ), Faculté de médecine de l'Université Laval [Québec] (ULaval), ANR-10-INSB-05-01 INSB ANR-10-INSB-0501, Fonds de la recherche du Québec-Santé (FRQS 254294)NIH R01-GM129325 and P41-GM103311, Discovery program, RGPIN-2014-05132, RGPIN-2014-05698, Università degli Studi di Milano = University of Milan (UNIMI), Laboratoire d'ingénierie des systèmes macromoléculaires (LISM), and Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
bacteriophages, Protein Conformation, [SDV]Life Sciences [q-bio], Allosteric regulation, anti-CRISPR protein, Virulence, cryo-electron microscopy, Biology, Genome, Structure-Activity Relationship, Viral Proteins, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Allosteric Regulation, CRISPR-Associated Protein 9, Hydrolase, Escherichia coli, Humans, Streptococcus thermophilus, CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats, Streptococcus thermophilus Cas9, Molecular Biology, 030304 developmental biology, 0303 health sciences, Binding Sites, Cas9, DNA, Cell Biology, biology.organism_classification, 3. Good health, Cell biology, St1Cas9, Kinetics, chemistry, Mutation, CRISPR-Cas Systems, CRISPR-Cas9, K562 Cells, 030217 neurology & neurosurgery, Bacteria, Protein Binding
Abstract

International audience; Molecular CellArticleCas9 Allosteric Inhibitionby the Anti-CRISPR Protein AcrIIA6Olivier Fuchsbauer,1,2,9Paolo Swuec,3,4,9Claire Zimberger,1,2Be ́atrice Amigues,1,2Se ́bastien Levesque,5Daniel Agudelo,5Alexis Duringer,5Antonio Chaves-Sanjuan,4Silvia Spinelli,1,2Genevie`ve M. Rousseau,6,7Minja Velimirovic,5Martino Bolognesi,3,4Alain Roussel,1,2Christian Cambillau,1,2Sylvain Moineau,6,7,8Yannick Doyon,5and Adeline Goulet1,2,10,*1Architecture et Fonction des Macromole ́cules Biologiques, Centre National de la Recherche Scientifique (CNRS), Campus de Luminy, Case932, 13288 Marseille Cedex 09, France2Architecture et Fonction des Macromole ́cules Biologiques, Aix-Marseille Universite ́, Campus de Luminy, Case 932, 13288 Marseille Cedex09, France3Dipartimento di Bioscienze, Universita`degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy4Centro di Ricerca Pediatrica Romeo ed Enrica Invernizzi, Universita`degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy5Centre Hospitalier Universitaire de Que ́bec–Universite ́Laval Research Center, Que ́bec City, QC G1V 4G2, Canada6De ́partement de biochimie, de microbiologie, et de bio-informatique, Faculte ́des sciences et de ge ́nie, Universite ́Laval, Que ́bec City, QC,G1V 0A6, Canada7Groupe de recherche en e ́cologie buccale, Faculte ́de me ́decine dentaire, Universite ́Laval, Que ́bec City, QC, G1V 0A6, Canada8Fe ́lix d’He ́relle Reference Center for Bacterial Viruses, Faculte ́de me ́decine dentaire, Universite ́Laval, Que ́bec City, QC, G1V 0A6, Canada9These authors contributed equally10Lead Contact*Correspondence:adeline.goulet@afmb.univ-mrs.frhttps://doi.org/10.1016/j.molcel.2019.09.012SUMMARYIn the arms race against bacteria, bacteriophageshave evolved diverse anti-CRISPR proteins (Acrs)that block CRISPR-Cas immunity. Acrs play key rolesin the molecular coevolution of bacteria with theirpredators, use a variety of mechanisms of action,and provide tools to regulate Cas-based genomemanipulation. Here, we present structural and func-tional analyses of AcrIIA6, an Acr from virulentphages, exploring its unique anti-CRISPR action.Our cryo-EM structures and functional data ofAcrIIA6 binding toStreptococcus thermophilusCas9 (St1Cas9) show that AcrIIA6 acts as an allo-steric inhibitor and induces St1Cas9 dimerization.AcrIIA6 reduces St1Cas9 binding affinity for DNAand prevents DNA binding within cells. The PAMand AcrIIA6 recognition sites are structurally closeand allosterically linked. Mechanistically, AcrIIA6 af-fects the St1Cas9 conformational dynamics associ-ated with PAM binding. Finally, we identify a naturalSt1Cas9 variant resistant to AcrIIA6 illustratingAcr-driven mutational escape and molecular diversi-fication of Cas9 proteins

Published
2019

5. Towards a complete structural deciphering of Type VI secretion system

Author

Badreddine Douzi, Van Son Nguyen, Eric Cascales, Eric Durand, Alain Roussel, Christian Cambillau, Laboratoire de l'intégration, du matériau au système (IMS), Université Sciences et Technologies - Bordeaux 1-Institut Polytechnique de Bordeaux-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'ingénierie des systèmes macromoléculaires (LISM), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), 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), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU), and Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA)

Subjects
0301 basic medicine, Models, Molecular, Future studies, Cryo-electron microscopy, Bacterial Proteins/*chemistry/ultrastructure, Protein Conformation, [SDV]Life Sciences [q-bio], 030106 microbiology, Crystallography, X-Ray, 03 medical and health sciences, Protein structure, Bacterial Proteins, Structural Biology, Gram-Negative Bacteria, Prokaryotic cells, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Molecular Biology, ComputingMilieux_MISCELLANEOUS, Type VI secretion system, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Effector, Chemistry, Cryoelectron Microscopy, Gram-Negative Bacteria/*chemistry/ultrastructure, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, [SDV.BBM.MN]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular Networks [q-bio.MN], Type VI Secretion Systems, Type VI Secretion Systems/*chemistry/ultrastructure, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Medium resolution, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Biophysics, Tail structure
Abstract

International audience; The Type VI secretion system (T6SS) is a dynamic nanomachine present in many Gram-negative bacteria. Using a contraction mechanism similar to that of myophages, bacteriocins or anti-feeding prophages, it injects toxic effectors into both eukaryotic and prokaryotic cells. T6SS assembles three large ensembles: the trans-membrane complex (TMC), the baseplate and the tail. Recently, the tail structure has been elucidated by cryo electron microscopy (cryoEM) in extended and contracted forms. The structure of the trans-membrane complex has been deciphered using a combination of X-ray crystallography and EM. However, the structural characterisation of the baseplate lags behind and should be the target of future studies. Finally, cryo-tomography should provide low/medium resolution maps allowing to assemble the different parts ultimately leading to a complete structural description of T6SS.

Published
2018

6. Type IX secretion system PorM and gliding machinery GldM form extended arches spanning the periplasmic space

Author

Alain Roussel, Christian Cambillau, Eric Cascales, Jennifer Roche, Christine Kellenberger, Aline Desmyter, Maxence S. Vincent, Philippe Leone, Quang Hieu Tran, 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), Physiologie de la reproduction et des comportements [Nouzilly] (PRC), 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), Laboratoire d'ingénierie des systèmes macromoléculaires (LISM), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), and Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU)

Subjects
0301 basic medicine, crystal structure, bacterial pathogenesis, Operon, Protein Conformation, Science, [SDV]Life Sciences [q-bio], 030106 microbiology, General Physics and Astronomy, Flavobacterium, General Biochemistry, Genetics and Molecular Biology, Article, gliding machinery, 03 medical and health sciences, Protein structure, Bacterial Proteins, Escherichia coli, Inner membrane, Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], lcsh:Science, Porphyromonas gingivalis, Bacterial Secretion Systems, ComputingMilieux_MISCELLANEOUS, Multidisciplinary, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, [SDV.BBM.MN]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular Networks [q-bio.MN], General Chemistry, Periplasmic space, biology.organism_classification, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Cell biology, Transport protein, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], 030104 developmental biology, Helix, Periplasm, lcsh:Q, Bacterial outer membrane, Camelids, New World, dental diseases, type IX secretion system
Abstract

Type IX secretion system (T9SS), exclusively present in the Bacteroidetes phylum, has been studied mainly in Flavobacterium johnsoniae and Porphyromonas gingivalis. Among the 18 genes, essential for T9SS function, a group of four, porK-N (P. gingivalis) or gldK-N (F. johnsoniae) belongs to a co-transcribed operon that expresses the T9SS core membrane complex. The central component of this complex, PorM (or GldM), is anchored in the inner membrane by a trans-membrane helix and interacts through the outer membrane PorK-N complex. There is a complete lack of available atomic structures for any component of T9SS, including the PorKLMN complex. Here we report the crystal structure of the GldM and PorM periplasmic domains. Dimeric GldM and PorM, each contain four domains of ~180-Å length that span most of the periplasmic space. These and previously reported results allow us to propose a model of the T9SS core membrane complex as well as its functional behavior., No structural data for the bacterial type IX secretion system (T9SS) are available so far. Here, the authors present the crystal structures of the periplasmic domains from two major T9SS components PorM and GldM, which span most of the periplasmic space, and propose a putative model of the T9SS core membrane complex.

Published
2018

7. TssA: The cap protein of the Type VI secretion system tail

Author

Laure Journet, Yoann G. Santin, Eric Durand, Abdelrahim Zoued, Alain Roussel, Christian Cambillau, Eric Cascales, Laboratoire d'ingénierie des systèmes macromoléculaires (LISM), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU), Architecture et fonction des macromolécules biologiques (AFMB), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, [SDV]Life Sciences [q-bio], 030106 microbiology, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, Escherichia coli, Bacteriophages, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], ComputingMilieux_MISCELLANEOUS, Type VI secretion system, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Effector, Chemistry, Escherichia coli Proteins, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, [SDV.BBM.MN]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular Networks [q-bio.MN], Type VI Secretion Systems, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Cell biology, Transport protein, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], 030104 developmental biology, Biochemistry, Cytoplasm, Enteroaggregative Escherichia coli, Chaperone (protein), biology.protein, Biogenesis, Catabolite activator protein
Abstract

The Type VI secretion system (T6SS) is a multiprotein and mosaic apparatus that delivers protein effectors into prokaryotic or eukaryotic cells. Recent data on the enteroaggregative Escherichia coli (EAEC) T6SS have provided evidence that the TssA protein is a key component during T6SS biogenesis. The T6SS comprises a trans-envelope complex that docks the baseplate, a cytoplasmic complex that represents the assembly platform for the tail. The T6SS tail is structurally, evolutionarily and functionally similar to the contractile tails of bacteriophages. We have shown that TssA docks to the membrane complex, recruits the baseplate complex and initiates and coordinates the polymerization of the inner tube with that of the sheath. Here, we review these recent findings, discuss the variations within TssA-like proteins, speculate on the role of EAEC TssA in T6SS biogenesis and propose future research perspectives.

Published
2017

8. Camelid nanobodies used as crystallization chaperones for different constructs of PorM, a component of the type IX secretion system from Porphyromonas gingivalis

Author

Christian Cambillau, Christine Kellenberger, Alain Roussel, Aline Desmyter, Anaïs Gaubert, Jennifer Roche, Philippe Leone, Thi Trang Nhung Trinh, Yoan Duhoo, 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), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), Architecture et fonction des macromolécules biologiques ( AFMB ), and Centre National de la Recherche Scientifique ( CNRS ) -Aix Marseille Université ( AMU ) -Institut National de la Recherche Agronomique ( INRA )

Subjects
MESH: Sequence Homology, Amino Acid, [SDV.NEU.NB]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, Plasma protein binding, MESH: Amino Acid Sequence, Biochemistry, MESH: Recombinant Proteins, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, MESH: Animals, [ SDV.BIBS ] Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], Peptide sequence, MESH: Bacterial Proteins, MESH : Porphyromonas gingivalis, MESH : Protein Conformation, alpha-Helical, [SDV.MHEP.ME]Life Sciences [q-bio]/Human health and pathology/Emerging diseases, [ SDV.MHEP.ME ] Life Sciences [q-bio]/Human health and pathology/Emerging diseases, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH: Escherichia coli, MESH : Amino Acid Sequence, MESH : Protein Binding, MESH: Camelus, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], MESH : Sequence Homology, Amino Acid, MESH : Genetic Vectors, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], [ SDV.MHEP.MI ] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, MESH : Crystallization, [ SDV.NEU.NB ] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, MESH: Camelids, New World, MESH: Porphyromonas gingivalis, MESH: Models, Molecular, Camelus, MESH: Gene Expression, MESH : Cloning, Molecular, Biophysics, MESH: Sequence Alignment, [ SDV.MP.VIR ] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, MESH: Single-Domain Antibodies, Microbiology, 03 medical and health sciences, Bacterial Proteins, Genetics, MESH: Protein Binding, Secretion, Molecular replacement, Protein Interaction Domains and Motifs, 5fwo, MESH: Cloning, Molecular, Amino Acid Sequence, Porphyromonas gingivalis, MESH: Protein Conformation, alpha-Helical, [ SDV.IMM.II ] Life Sciences [q-bio]/Immunology/Innate immunity, MESH: Protein Interaction Domains and Motifs, MESH : Molecular Chaperones, Periplasmic space, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, MESH : Camelids, New World, MESH : Gene Expression, 030104 developmental biology, MESH: Binding Sites, Protein Conformation, beta-Strand, PorM, Molecular Chaperones, type IX secretion system, 0301 basic medicine, Models, Molecular, Protein Conformation, alpha-Helical, MESH : Escherichia coli, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, Gene Expression, [ SDV.MP.BAC ] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Crystallography, X-Ray, [SDV.IMM.II]Life Sciences [q-bio]/Immunology/Innate immunity, MESH : Bacterial Secretion Systems, Research Communications, [ SDV.BBM.BC ] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Structural Biology, MESH: Genetic Vectors, 5lmj, MESH : Bacterial Proteins, Cloning, Molecular, MESH: Bacterial Secretion Systems, Bacterial Secretion Systems, MESH : Protein Conformation, beta-Strand, MESH: Crystallization, 5lmw, MESH: Kinetics, MESH : Sequence Alignment, MESH : Camelus, Condensed Matter Physics, Recombinant Proteins, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], MESH : Single-Domain Antibodies, Thermodynamics, MESH: Protein Conformation, beta-Strand, nb02, MESH : Kinetics, nb01, MESH: Thermodynamics, MESH: Molecular Chaperones, Crystallization, Camelids, New World, Protein Binding, crystallization chaperones, MESH : Recombinant Proteins, MESH : Models, Molecular, Genetic Vectors, Context (language use), Biology, MESH : Peptide Library, Peptide Library, Escherichia coli, Animals, nb130, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Peptide library, MESH : Thermodynamics, nb19, Binding Sites, Sequence Homology, Amino Acid, [ SDV.SP.PHARMA ] Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, [ SDV.BIO ] Life Sciences [q-bio]/Biotechnology, Single-Domain Antibodies, biology.organism_classification, MESH: Crystallography, X-Ray, Kinetics, [SDV.SP.PHARMA]Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, MESH : Animals, MESH: Peptide Library, MESH : Crystallography, X-Ray, camelid nanobodies, Sequence Alignment, 5lz0, MESH : Binding Sites, MESH : Protein Interaction Domains and Motifs, [ SDV.BBM.BS ] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM]
Abstract

PorM is a membrane protein that is involved in the assembly of the type IX secretion system (T9SS) inPorphyromonas gingivalis, a major bacterial pathogen that is responsible for periodontal disease in humans. In the context of structural studies of PorM to better understand T9SS assembly, four camelid nanobodies were selected, produced and purified, and their specific interaction with the N-terminal or C-terminal part of the periplasmic domain of PorM was investigated. Diffracting crystals were also obtained, and the structures of the four nanobodies were solved by molecular replacement. Furthermore, two nanobodies were used as crystallization chaperones and turned out to be valuable tools in the structure-determination process of the periplasmic domain of PorM.

Published
2017

9. Characterization of the Porphyromonas gingivalis Type IX Secretion Trans-envelope PorKLMNP Core Complex

Author

Julien Stathopulos, Abdelrahim Zoued, Christine Kellenberger, Maxence S. Vincent, Alain Roussel, Philippe Leone, Mickaël J. Canestrari, Christian Cambillau, Eric Cascales, Bérengère Ize, Laboratoire d'ingénierie des systèmes macromoléculaires (LISM), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU), Architecture et fonction des macromolécules biologiques (AFMB), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Gliding motility, [SDV]Life Sciences [q-bio], 030106 microbiology, channel, membrane proteins, Biochemistry, Flavobacterium, 03 medical and health sciences, membrane complex, protein secretion, Tannerella forsythia, Secretion, Type IX secretion, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Porphyromonas, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Molecular Biology, Porphyromonas gingivalis, periodontitis, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, toxins, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, [SDV.BBM.MN]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular Networks [q-bio.MN], Cell Biology, biology.organism_classification, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Cell biology, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Bacterial adhesin, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], stomatognathic diseases, Secretory protein, Membrane protein, T9SS, protein transport, gingipains, gliding motility, Cell envelope, gingivitis
Abstract

International audience; The transport of proteins at the cell surface of Bacteriodetes depends on a secretory apparatus known as Type IX secretion system (T9SS). This machine is responsible for the cell surface exposition of various proteins such as adhesins required for gliding motility in Flavobacteria, S-layer components in Tannerella forsythia and tooth tissue-degrading enzymes in the oral pathogen Porphyromonas gingivalis. While a number of subunits of the T9SS have been identified, we lack details on the architecture of this secretion apparatus. Here we provide evidence that five of the genes encoding the core complex of the T9SS are co-transcribed, and that the gene products are distributed in the cell envelope. Protein-protein interaction studies then revealed that these proteins oligomerize and interact through a dense network of contacts.

Published
2017

10. Probing Conformational Changes and Interfacial Recognition Site of Lipases With Surfactants and Inhibitors

Author

Christian Cambillau, Sonia Longhi, Eduardo Mateos-Diaz, Frédéric Carrière, Alain Roussel, Sawsan Amara, Architecture et fonction des macromolécules biologiques (AFMB), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), Enzymologie interfaciale et de physiologie de la lipolyse (EIPL), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU), Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), and TADEO, DANIELE

Subjects
0301 basic medicine, chemistry.chemical_classification, 030102 biochemistry & molecular biology, biology, Variable size, Combined use, Active site, Site-directed spin labeling, Substrate docking, Combinatorial chemistry, 03 medical and health sciences, chemistry.chemical_compound, 030104 developmental biology, Monomer, Enzyme, chemistry, biology.protein, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Organic chemistry, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Lipase, ComputingMilieux_MISCELLANEOUS
Abstract

Structural studies on lipases by X-ray crystallography have revealed conformational changes occurring in the presence of surfactants/inhibitors and the pivotal role played by a molecular "lid" of variable size and structure depending on the enzyme. Besides controlling the access to the enzyme active site, the lid is involved in lipase activation, formation of the interfacial recognition site (IRS), and substrate docking within the active site. The combined use of surfactants and inhibitors has been critical for a better understanding of lipase structure-function relationships. An overview of crystal structures of lipases in complex with surfactants and inhibitors reveals common structural features and shows how surfactants monomers interact with the lid in its open conformation. The location of surfactants, inhibitors, and hydrophobic residues exposed upon lid opening provides insights into the IRS of lipases. The mechanism by which surfactants promote the lid opening can be further investigated in solution by site-directed spin labeling of lipase coupled to electron paramagnetic resonance spectroscopy. These experimental approaches are illustrated here by results obtained with mammalian digestive lipases, fungal lipases, and cutinases.

Published
2017

11. Cytokine Diedel and a viral homologue suppress the IMD pathway in Drosophila

Author

Cordula Kemp, Olivier Lamiable, Alain Roussel, Laurent Daeffler, Christine Kellenberger, Nadège Pelte, Laurent Troxler, João Trindade Marques, Jules A. Hoffmann, Michael Boutros, Jean-Luc Imler, Architecture et fonction des macromolécules biologiques (AFMB), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), and Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Sindbis virus, viruses, Molecular Sequence Data, Mutant, Cell, 03 medical and health sciences, chemistry.chemical_compound, Immune system, RNA interference, medicine, Animals, Drosophila Proteins, Amino Acid Sequence, Gene, ComputingMilieux_MISCELLANEOUS, Multidisciplinary, Sequence Homology, Amino Acid, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Alphavirus Infections, Intracellular parasite, fungi, Immunity, Biological Sciences, biology.organism_classification, Survival Analysis, Virology, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], Up-Regulation, 3. Good health, Cell biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Drosophila melanogaster, 030104 developmental biology, medicine.anatomical_structure, chemistry, Mutation, Cytokines, Sindbis Virus, DNA, Signal Transduction
Abstract

Viruses are obligatory intracellular parasites that suffer strong evolutionary pressure from the host immune system. Rapidly evolving viral genomes can adapt to this pressure by acquiring genes that counteract host defense mechanisms. For example, many vertebrate DNA viruses have hijacked cellular genes encoding cytokines or cytokine receptors to disrupt host cell communication. Insect viruses express suppressors of RNA interference or apoptosis, highlighting the importance of these cell intrinsic antiviral mechanisms in invertebrates. Here, we report the identification and characterization of a family of proteins encoded by insect DNA viruses that are homologous to a 12-kDa circulating protein encoded by the virus-induced Drosophila gene diedel (die). We show that die mutant flies have shortened lifespan and succumb more rapidly than controls when infected with Sindbis virus. This reduced viability is associated with deregulated activation of the immune deficiency (IMD) pathway of host defense and can be rescued by mutations in the genes encoding the homolog of IKKγ or IMD itself. Our results reveal an endogenous pathway that is exploited by insect viruses to modulate NF-κB signaling and promote fly survival during the antiviral response.

Published
2016

12. Biogenesis and structure of a Type VI secretion membrane core complex

Author

Abdelrahim Zoued, Annick Dujeancourt, Laureen Logger, Rémi Fronzes, Benjamin Bardiaux, Eric Cascales, Van Son Nguyen, Silvia Spinelli, Aline Desmyter, Marie-Stéphanie Aschtgen, Eric Durand, Christian Cambillau, Gérard Pehau-Arnaudet, Alain Roussel, Laboratoire d'ingénierie des systèmes macromoléculaires (LISM), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU), Architecture et fonction des macromolécules biologiques (AFMB), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), Biologie Structurale de la Sécrétion Bactérienne, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Bioinformatique structurale - Structural Bioinformatics, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], This work was funded by Agence Nationale de la Recherche (ANR) grants ANR-10-JCJC-1303-03 to E.C., Bip:Bip to R.F., ANR-14-CE14-0006-02 to C.C. and E.C., and supported by the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INSB-05-01. E.D. was supported by a post-doctoral fellowship from the Fondation pour la Recherche Médicale (SPF20101221116) and ANR grants Bip:Bip and ANR-10-JCJC-1303-03. V.S.N. was supported by a PhD grant from the French Embassy in Vietnam (792803C). A.Z., L.L. and M.S.A. were recipients of doctoral fellowships from the French Ministère de la Recherche. A.Z. received a Fondation pour la Recherche Médicale fellowship (FDT20140931060)., ANR-10-JCJC-1303,A Fun T6SS,Assemblage et Fonction des Systèmes de Sécrétion de Type VI bactériens(2010), ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-14-CE14-0006,B-War,Guerre bactérienne: Architecture et Fonction du Système de Sécrétion de Type VI(2014), Institut Pasteur [Paris] - Centre National de la Recherche Scientifique (CNRS), Aix Marseille Université (AMU) - Institut National de la Santé et de la Recherche Médicale (INSERM) - Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS) - Aix Marseille Université (AMU) - Institut National de la Recherche Agronomique (INRA), Microscopie ultrastructurale - Ultrapole (CITECH), Institut Pasteur [Paris], Bioinformatique structurale, This work was funded by Agence Nationale de la Recherche (ANR) grants ANR-10-JCJC-1303-03 to E.C., Bip:Bip to R.F., ANR-14-CE14-0006-02 to C.C. and E.C., and supported by the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INSB-05-01. E.D. was supported by a post-doctoral fellowship from the Fondation pour la Recherche Médicale (SPF20101221116) and ANR grants Bip:Bip and ANR-10-JCJC-1303-03. V.S.N. was supported by a PhD grant from the French Embassy in Vietnam (792803C). A.Z., L.L. and M.S.A. were recipients of doctoral fellowships from the French Ministère de la Recherche. A.Z. received a Fondation pour la Recherche Médicale fellowship (FDT20140931060), We thank O. Francetic for providing anti-DglA and anti-OmpF antibodies. We thank E. Marza, P. Violinova Krasteva and H. Remaut for comments on the manuscript, and T. Mignot, M. Guzzo and L. Espinosa for advice about the fluorescence microscopy experiments and the statistical analyses. We also thank the members of the R.F. and E.C. research groups for discussions and suggestions, and R. Lloubès, J. Sturgis and A. Galinier for encouragement. We thank the ERSF and Soleil Synchrotron radiation facilities for beamline allocation, ANR-10-JCJC-1303, A Fun T6SS, Assemblage et Fonction des Systèmes de Sécrétion de Type VI bactériens(2010), ANR-14-CE14-0006, B-War, Guerre bactérienne: Architecture et Fonction du Système de Sécrétion de Type VI(2014), ANR-10-INBS-05-01/10-INBS-0005, FRISBI, Infrastructure Française pour la Biologie Structurale Intégrée(2010), CASCALES, ERIC, Jeunes Chercheuses et Jeunes Chercheurs - Assemblage et Fonction des Systèmes de Sécrétion de Type VI bactériens - - A Fun T6SS2010 - ANR-10-JCJC-1303 - JCJC - VALID, Infrastructure Française pour la Biologie Structurale Intégrée - - FRISBI2010 - ANR-10-INBS-0005 - INBS - VALID, Appel à projets générique - Guerre bactérienne: Architecture et Fonction du Système de Sécrétion de Type VI - - B-War2014 - ANR-14-CE14-0006 - Appel à projets générique - VALID, Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and ANR-10-INBS-05-01/10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010)

Subjects
Models, Molecular, Cytoplasm, MESH : Bacterial Secretion Systems, MESH : Escherichia coli/chemistry, MESH: Escherichia coli Proteins, MESH : Cytoplasm/chemistry, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Crystallography, X-Ray, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, MESH : Multiprotein Complexes/biosynthesis, MESH: Protein Structure, Tertiary, Bacterial secretion, MESH : Membrane Proteins/biosynthesis, MESH: Lipopeptides, MESH : Escherichia coli Proteins/chemistry, MESH: Bacterial Secretion Systems, Bacterial Secretion Systems, [SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], MESH : Cell Membrane/chemistry, "structure of a type VI", MESH : Membrane Proteins/chemistry, MESH : Escherichia coli Proteins/biosynthesis, 0303 health sciences, [SDV.BIBS] Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], Multidisciplinary, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH: Escherichia coli, Escherichia coli Proteins, MESH: Periplasm, MESH : Escherichia coli/metabolism, MESH: Protein Subunits, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Biochemistry, Periplasm, MESH : Cytoplasm/metabolism, MESH: Membrane Proteins, Cell envelope, Bacterial outer membrane, Porosity, MESH : Lipopeptides/biosynthesis, MESH : Protein Structure, Tertiary, MESH: Models, Molecular, [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH : Models, Molecular, MESH : Lipopeptides/chemistry, MESH: Microscopy, Electron, Biology, MESH : Protein Subunits/biosynthesis, Lipopeptides, 03 medical and health sciences, MESH: Porosity, Escherichia coli, [SDV.BC.BC] Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Electron microscopy, Secretion, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], "Biogenesis", MESH : Cell Membrane/metabolism, "secretion membrane", MESH : Protein Subunits/chemistry, 030304 developmental biology, Type VI secretion system, X-ray crystallography, MESH : Periplasm/metabolism, MESH : Periplasm/chemistry, 030306 microbiology, MESH: Cytoplasm, Cell Membrane, Membrane Proteins, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Periplasmic space, MESH: Multiprotein Complexes, MESH: Crystallography, X-Ray, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, MESH : Microscopy, Electron, MESH : Multiprotein Complexes/chemistry, Protein Structure, Tertiary, Microscopy, Electron, Protein Subunits, Membrane protein, Multiprotein Complexes, Biophysics, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, MESH : Porosity, MESH : Crystallography, X-Ray, Biogenesis, MESH: Cell Membrane
Abstract

International audience; Bacteria share their ecological niches with other microbes. The bacterial type VI secretion system is one of the key players in microbial competition, as well as being an important virulence determinant during bacterial infections. It assembles a nano-crossbow-like structure in the cytoplasm of the attacker cell that propels an arrow made of a haemolysin co-regulated protein (Hcp) tube and a valine–glycine repeat protein G (VgrG) spike and punctures the prey’s cell wall. The nano-crossbow is stably anchored to the cell envelope of the attacker by a membrane core complex. Here we show that this complex is assembled by the sequential addition of three type VI subunits (Tss)— TssJ, TssM and TssL—and present a structure of the fully assembled complex at 11.6 A ̊ resolution, determined by negative-stain electron microscopy. With overall C5 symmetry, this 1.7-megadalton complex comprises a large base in the cytoplasm. It extends in the periplasm via ten arches to form a double-ring structure containing the carboxy-terminal domain of TssM (TssMct) and TssJ that is anchored in the outer membrane. The crystal structure of the TssMct–TssJ complex coupled to whole-cell accessibility studies suggest that large conformational changes induce transient pore formation in the outer membrane, allowing passage of the attacking Hcp tube/VgrG spike.

Published
2015

13. Development and evaluation of a sublingual tablet based on recombinant Bet v 1 in birch pollen-allergic patients

Author

B. Robin, M. Le Mignon, Emmanuel Nony, P. Lemoine, Sabina Rak, Alain Roussel, Julien Bouley, Thierry Batard, Laurent Mascarell, Philippe Leone, Marc Pallardy, O. de Beaumont, Renaud Vincentelli, K. Abiteboul, Philippe Moingeon, K. Jain, S. Horiot, Monica Arvidsson, Architecture et fonction des macromolécules biologiques (AFMB), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), Stallergenes SA (Stallergenes), Stallergenes, Université de Paris - UFR Pharmacie [Santé] (UP UFR Pharmacie), Université de Paris (UP), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), UFR Pharmacie [Santé] - Université Paris Cité (UFR Pharmacie UPCité), and Université Paris Cité (UPCité)

Subjects
Male, Models, Molecular, Allergy, Protein Conformation, Pharmacology, medicine.disease_cause, law.invention, 0302 clinical medicine, Allergen, Risk Factors, law, Immunology and Allergy, birch pollen, ComputingMilieux_MISCELLANEOUS, 0303 health sciences, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Middle Aged, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], Recombinant Proteins, Respiratory Function Tests, 3. Good health, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Recombinant DNA, Pollen, Female, Adult, Adolescent, Immunology, Placebo, Peripheral blood mononuclear cell, sublingual immunotherapy, Sublingual administration, Young Adult, 03 medical and health sciences, Bet v 1, medicine, [CHIM.CRIS]Chemical Sciences/Cristallography, Humans, 030304 developmental biology, Sublingual Tablet, business.industry, Rhinitis, Allergic, Seasonal, Allergens, Antigens, Plant, medicine.disease, Birch pollen, 030228 respiratory system, business, recombinant allergen
Abstract

International audience; BackgroundSublingual immunotherapy (SLIT) applied to type I respiratory allergies is commonly performed with natural allergen extracts. Herein, we developed a sublingual tablet made of pharmaceutical-grade recombinant Bet v 1.0101 (rBet v 1) and investigated its clinical safety and efficacy in birch pollen (BP)-allergic patients.MethodsFollowing expression in Escherichia coli and purification, rBet v 1 was characterized using chromatography, capillary electrophoresis, circular dichroism, mass spectrometry and crystallography. Safety and efficacy of rBet v 1 formulated as a sublingual tablet were assessed in a multicentre, double-blind, placebo-controlled study conducted in 483 patients with BP-induced rhinoconjunctivitis.ResultsIn-depth characterization confirmed the intact product structure and high purity of GMP-grade rBet v 1. The crystal structure resolved at 1.2 angstrom documented the natural conformation of the molecule. Native or oxidized forms of rBet v 1 did not induce the production of any proinflammatory cytokine by blood dendritic cells or mononuclear cells. Bet v 1 tablets were well tolerated by patients, consistent with the known safety profile of SLIT. The average adjusted symptom scores were significantly decreased relative to placebo in patients receiving once daily for 5months rBet v 1 tablets, with a mean difference of 17.0-17.7% relative to the group treated with placebo (P

Published
2015

14. Camelid nanobodies: killing two birds with one stone

Author

Silvia Spinelli, Christian Cambillau, Alain Roussel, Aline Desmyter, Architecture et fonction des macromolécules biologiques (AFMB), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), and Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Models, Molecular, Protein Conformation, Computational biology, Biology, Epitope, 03 medical and health sciences, Protein stability, Protein structure, Structural Biology, Animals, Humans, Molecular Biology, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Protein Stability, 030302 biochemistry & molecular biology, Proteins, Single-Domain Antibodies, Combinatorial chemistry, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], Enzyme inhibition, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Membrane protein, Structural biology, Crystallization, Camelids, New World
Abstract

In recent years, the use of single-domain camelid immunoglobulins, termed vHHs or nanobodies, has seen increasing growth in biotechnology, pharmaceutical applications and structure/function research. The usefulness of nanobodies in structural biology is now firmly established, as they provide access to new epitopes in concave and hinge regions - and stabilize them. These sites are often associated with enzyme inhibition or receptor neutralization, and, at the same time, provide favorable surfaces for crystal packing. Remarkable results have been achieved by using nanobodies with flexible multi-domain proteins, large complexes and, last but not least, membrane proteins. While generating nanobodies is still a rather long and expensive procedure, the advent of naive libraries might be expected to facilitate the whole process.

Published
2015

15. X-ray and Cryo-electron Microscopy Structures of Monalysin Pore-forming Toxin Reveal Multimerization of the Pro-form

Author

Philippe Leone, Cecilia Bebeacua, Bruno Lemaitre, Christine Kellenberger, Alain Roussel, Onya Opota, Igor Orlov, Bruno P. Klaholz, Christian Cambillau, 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), and Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA)

Subjects
Models, Molecular, Pore Forming Cytotoxic Proteins, animal structures, Cryo-electron microscopy, Stereochemistry, Bacterial Toxins, Molecular Sequence Data, Aerolysin, Biology, Crystallography, X-Ray, Cleavage (embryo), Biochemistry, Cell membrane, Protein structure, Pseudomonas, medicine, Amino Acid Sequence, Molecular Biology, ComputingMilieux_MISCELLANEOUS, Pore-forming toxin, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Sequence Homology, Amino Acid, Cell Membrane, Cryoelectron Microscopy, Cell Biology, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], Transmembrane protein, Protein Structure, Tertiary, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], medicine.anatomical_structure, Membrane, Protein Structure and Folding, Mutation, Protein Multimerization, Crystallization, Protein Binding
Abstract

beta-Barrel pore-forming toxins (beta-PFT), a large family of bacterial toxins, are generally secreted as water-soluble monomers and can form oligomeric pores in membranes following proteolytic cleavage and interaction with cell surface receptors. Monalysin has been recently identified as a beta-PFT that contributes to the virulence of Pseudomonas entomophila against Drosophila. It is secreted as a pro-protein that becomes active upon cleavage. Here we report the crystal and cryo-electron microscopy structure of the pro-form of Monalysin as well as the crystal structures of the cleaved form and of an inactive mutant lacking the membrane-spanning region. The overall structure of Monalysin displays an elongated shape, which resembles those of beta-poreforming toxins, such as Aerolysin, but is devoid of a receptorbinding domain. X-ray crystallography, cryo-electron microscopy, and light-scattering studies show that pro-Monalysin forms a stable doughnut-like 18-mer complex composed of two disk-shaped nonamers held together by N-terminal swapping of the pro-peptides. This observation is in contrast with the monomeric pro-form of the other beta-PFTs that are receptor-dependent for membrane interaction. The membrane-spanning region of pro-Monalysin is fully buried in the center of the doughnut, suggesting that upon cleavage of pro-peptides, the two disk-shaped nonamers can, and have to, dissociate to leave the transmembrane segments free to deploy and lead to pore formation. In contrast with other toxins, the delivery of 18 subunits at once, nearby the cell surface, may be used to bypass the requirement of receptor-dependent concentration to reach the threshold for oligomerization into the pore-forming complex.

Published
2015

16. Production, crystallization and X-ray diffraction analysis of a complex between a fragment of the TssM T6SS protein and a camelid nanobody

Author

Christian Cambillau, Aline Desmyter, Thi Thu Hang Le, Alain Roussel, Van Son Nguyen, Silvia Spinelli, Eric Cascales, Christine Kellenberger, 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), Laboratoire d'ingénierie des systèmes macromoléculaires (LISM), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-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
[SDV]Life Sciences [q-bio], Molecular Sequence Data, Biophysics, Myoviridae, Biology, Crystallography, X-Ray, medicine.disease_cause, Biochemistry, Research Communications, Structural Biology, Escherichia coli, Genetics, medicine, Animals, Inner membrane, Molecular replacement, Amino Acid Sequence, Bacterial Secretion Systems, ComputingMilieux_MISCELLANEOUS, Type VI secretion system, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Escherichia coli Proteins, Membrane Proteins, Single-Domain Antibodies, Condensed Matter Physics, biology.organism_classification, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], Peptide Fragments, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Membrane, Membrane protein, biological sciences, health occupations, bacteria, Crystallization, Bacterial outer membrane, Camelids, New World
Abstract

The type VI secretion system (T6SS) is a machine evolved by Gram-negative bacteria to deliver toxin effectors into target bacterial or eukaryotic cells. The T6SS is functionally and structurally similar to the contractile tail of theMyoviridaefamily of bacteriophages and can be viewed as a syringe anchored to the bacterial membrane by a transenvelope complex. The membrane complex is composed of three proteins: the TssM and TssL inner membrane components and the TssJ outer membrane lipoprotein. The TssM protein is central as it interacts with both TssL and TssJ, therefore linking the membranes. Using controlled trypsinolysis, a 32.4 kDa C-terminal fragment of enteroaggregativeEscherichia coliTssM (TssM32Ct) was purified. A nanobody obtained from llama immunization, nb25, exhibited subnanomolar affinity for TssM32Ct. Crystals of the TssM32Ct–nb25 complex were obtained and diffracted to 1.9 Å resolution. The crystals belonged to space groupP64, with unit-cell parametersa = b = 95.23,c= 172.95 Å. Molecular replacement with a model nanobody indicated the presence of a dimer of TssM32Ct–nb25 in the asymmetric unit.

Published
2015

17. Crystallization and preliminary X-ray analysis of the C-terminal fragment of PorM, a subunit of the Porphyromonas gingivalis type IX secretion system

Author

Philippe Leone, Julien Stathopulos, Christian Cambillau, Alain Roussel, Eric Cascales, Laboratoire d'ingénierie des systèmes macromoléculaires (LISM), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU), 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), and Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA)

Subjects
Proteolysis, Protein subunit, [SDV]Life Sciences [q-bio], Biophysics, macromolecular substances, Crystallography, X-Ray, medicine.disease_cause, Biochemistry, Research Communications, 03 medical and health sciences, Bacterial Proteins, Structural Biology, Genetics, medicine, Secretion, Bacterial Secretion Systems, Escherichia coli, Porphyromonas gingivalis, Pathogen, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, medicine.diagnostic_test, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 030306 microbiology, Periplasmic space, Condensed Matter Physics, biology.organism_classification, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], Protein Structure, Tertiary, Protein Subunits, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Membrane protein, Chromatography, Gel, Crystallization, human activities
Abstract

PorM is a membrane protein involved in the assembly of the type IX secretion system (T9SS) fromPorphyromonas gingivalis, a major bacterial pathogen responsible for periodontal disease in humans. The periplasmic domain of PorM was overexpressed inEscherichia coliand purified. A fragment of the purified protein was obtained by limited proteolysis. Crystals of this fragment belonged to the tetragonal space groupP43212. Native and MAD data sets were recorded to 2.85 and 3.1 Å resolution, respectively, using synchrotron radiation.

Published
2015

18. A Cutinase from Trichoderma reesei with a Lid-Covered Active Site and Kinetic Properties of True Lipases

Author

Antti Nyyssölä, Alain Roussel, Ann Westerholm-Parvinen, Stéphanie Blangy, Frédéric Carrière, Sawsan Amara, Eduardo Mateos-Diaz, Hanna Kontkanen, Christian Cambillau, 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), Enzymologie interfaciale et de physiologie de la lipolyse (EIPL), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU), Bioénergie et Microalgues (EBM), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Environnement, Bioénergie, Microalgues et Plantes (EBMP), and Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA))

Subjects
Models, Molecular, Cutinase, Protein Conformation, Lipolysis, Trichoderma reesei, Molecular Sequence Data, Organophosphonates, Cutin, Crystallography, X-Ray, Catalysis, Structural Biology, Catalytic Domain, Hydrolase, Catalytic triad, lipase, Amino Acid Sequence, Enzyme Inhibitors, Lipase, interfacial activation, Molecular Biology, cutinase, ComputingMilieux_MISCELLANEOUS, Trichoderma, chemistry.chemical_classification, Binding Sites, Sequence Homology, Amino Acid, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, Active site, [SDV.BBM.MN]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular Networks [q-bio.MN], [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, biology.organism_classification, Kinetics, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Enzyme, Biochemistry, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, biology.protein, X-ray structure, Carboxylic Ester Hydrolases
Abstract

Cutinases belong to the α/β-hydrolase fold family of enzymes and degrade cutin and various esters, including triglycerides, phospholipids and galactolipids. Cutinases are able to degrade aggregated and soluble substrates because, in contrast with true lipases, they do not have a lid covering their catalytic machinery. We report here the structure of a cutinase from the fungus Trichoderma reesei (Tr) in native and inhibitor-bound conformations, along with its enzymatic characterization. A rare characteristic of Tr cutinase is its optimal activity at acidic pH. Furthermore, Tr cutinase, in contrast with classical cutinases, possesses a lid covering its active site and requires the presence of detergents for activity. In addition to the presence of the lid, the core of the Tr enzyme is very similar to other cutinase cores, with a central five-stranded β-sheet covered by helices on either side. The catalytic residues form a catalytic triad involving Ser164, His229 and Asp216 that is covered by the two N-terminal helices, which form the lid. This lid opens in the presence of surfactants, such as β-octylglucoside, and uncovers the catalytic crevice, allowing a C11Y4 phosphonate inhibitor to bind to the catalytic serine. Taken together, these results reveal Tr cutinase to be a member of a new group of lipolytic enzymes resembling cutinases but with kinetic and structural features of true lipases and a heightened specificity for long-chain triglycerides.

Published
2014

19. Crystal structure and self-interaction of the type VI secretion tail-tube protein from enteroaggregative Escherichia coli

Author

Christian Cambillau, Yannick R. Brunet, Badreddine Douzi, Silvia Spinelli, Eric Cascales, Alain Roussel, Stéphanie Blangy, Eric Durand, Architecture et fonction des macromolécules biologiques (AFMB), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), Laboratoire d'ingénierie des systèmes macromoléculaires (LISM), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), ANR-10-INBS-05-01/10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-10-JCJC-1303,A Fun T6SS,Assemblage et Fonction des Systèmes de Sécrétion de Type VI bactériens(2010), Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU), and Douzi, Badreddine

Subjects
Macromolecular Assemblies, Models, Molecular, Mutant Proteins/chemistry/metabolism, Light, [SDV]Life Sciences [q-bio], lcsh:Medicine, Plasma protein binding, medicine.disease_cause, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Crystallography, X-Ray, Biochemistry, Transmembrane Transport Proteins, Protein structure, Escherichia coli/*metabolism, Escherichia coli Proteins/*chemistry/*metabolism/ultrastructure, Scattering, Radiation, Bacteriophages, Disulfides, lcsh:Science, Bacterial Secretion Systems, Peptide sequence, [SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], ComputingMilieux_MISCELLANEOUS, 0303 health sciences, Multidisciplinary, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, Escherichia coli Proteins, 030302 biochemistry & molecular biology, Recombinant Proteins, Bacterial Biochemistry, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Disulfides/metabolism, Pseudomonas aeruginosa, Chromatography, Gel, Research Article, Protein Binding, Plasmons, Virulence Factors, [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Molecular Sequence Data, Biophysics, [SDV.BBM.BP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Myoviridae, Microbiology, Crystals, 03 medical and health sciences, Secretion systems, Caudovirales, Escherichia coli, medicine, Protein structure comparison, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acid Sequence, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Protein Interactions, Biology, 030304 developmental biology, Type VI secretion system, Crystal structure, lcsh:R, Proteins, Bacteriology, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Surface Plasmon Resonance, biology.organism_classification, Virulence Factors/*chemistry/*metabolism, Structural Homology, Protein, biology.protein, lcsh:Q, Mutant Proteins, Protein G, Protein Multimerization, Sequence Alignment
Abstract

International audience; The type VI secretion system (T6SS) is a widespread machine used by bacteria to control their environment and kill or disable bacterial species or eukaryotes through toxin injection. The T6SS comprises a central tube formed of stacked hexamers of hemolysin co-regulated proteins (Hcp) and terminated by a trimeric valine-glycine repeat protein G (VgrG) component, the cell puncturing device. A contractile tail sheath, formed by the TssB and TssC proteins, surrounds this tube. This syringe-like machine has been compared to an inverted phage, as both Hcp and VgrG share structural homology with tail components of Caudovirales. Here we solved the crystal structure of a tryptophan-substituted double mutant of Hcp1 from enteroaggregative Escherichia coli and compared it to the structures of other Hcps. Interestingly, we observed that the purified Hcp native protein is unable to form tubes in vitro. To better understand the rationale for observation, we measured the affinity of Hcp1 hexamers with themselves by surface plasmon resonance. The intra-hexamer interaction is weak, with a K D value of 7.2 mM. However, by engineering double cysteine mutants at defined positions, tubes of Hcp1 gathering up to 15 stacked hexamers formed in oxidative conditions. These results, together with those available in the literature regarding TssB and TssC, suggest that assembly of the T6SS tube differs significantly from that of Sipho-or Myoviridae.

Published
2014

20. Bile-mediated activation of the acrAB and tolC multidrug efflux genes occurs mainly through transcriptional derepression of ramA in [i]Salmonella enterica[/i] serovar Typhimurium

Author

Sylvie Baucheron, Axel Cloeckaert, Etienne Giraud, Franck Coste, Alain Roussel, Sylvie Canepa, Marie-Christine Maurel, Kunihiko Nishino, Isabelle Monchaux, Infectiologie et Santé Publique (UMR ISP), Institut National de la Recherche Agronomique (INRA)-Université de Tours, Laboratory of Microbiology and Infectious Diseases, Institute of Scientific and Industrial Research, Osaka University [Osaka], 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-Centre National de la Recherche Scientifique (CNRS), Plateforme d’Analyse Intégrative des Biomolécules et de Phénomique des Animaux d’Intérêt Bio-agronomique, Institut National de la Recherche Agronomique (INRA), Centre de biophysique moléculaire (CBM), Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), nstitut National pour la Recherche Agronomique (INRA) as an INRA-JSPS (Japan Society for the Promotion of Science) 2010 – 2 012 joint research project. French Region Centre (grant 2008 00036085). European Union with the European Regional Development Fund (grant 1634 – 32245), Institut National de la Recherche Agronomique (INRA)-Université de Tours (UT), 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), UR Infectiologie animale et Santé publique (UR IASP), 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), and Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Microbiology (medical), DNA, Bacterial, Salmonella typhimurium, [SDV.OT]Life Sciences [q-bio]/Other [q-bio.OT], Transcription, Genetic, Electrophoretic Mobility Shift Assay, Real-Time Polymerase Chain Reaction, digestive system, 03 medical and health sciences, Bacterial Proteins, Transcription (biology), Transcriptional regulation, Bile, Humans, Pharmacology (medical), transcriptional regulation, ramr, Gene, Derepression, 030304 developmental biology, Pharmacology, Genetics, 0303 health sciences, promoter, biology, 030306 microbiology, Activator (genetics), Gene Expression Profiling, Promoter, Gene Expression Regulation, Bacterial, biochemical phenomena, metabolism, and nutrition, biology.organism_classification, Cell biology, Infectious Diseases, Salmonella enterica, Trans-Activators, [SDV.SPEE]Life Sciences [q-bio]/Santé publique et épidémiologie, Efflux, Multidrug Resistance-Associated Proteins, Carrier Proteins, surface plasmon resonance, Protein Binding
Abstract

Objectives In Salmonella Typhimurium, the genes encoding the AcrAB-TolC multidrug efflux system are mainly regulated by the ramRA locus, composed of the divergently transcribed ramA and ramR genes. The acrAB and tolC genes are transcriptionally activated by RamA, the gene for which is itself transcriptionally repressed by RamR. Previous studies have reported that bile induces acrAB in a ramA-dependent manner, but none provided evidence for an induction of ramA expression by bile. Therefore, the objective of this study was to clarify the regulatory mechanism by which bile activates acrAB and tolC. Methods qRT-PCR was used to address the effects of bile (using choleate, an ox-bile extract) on the expression of ramA, ramR, acrB and tolC. Electrophoretic mobility shift assays and surface plasmon resonance experiments were used to measure the effect of bile on RamR binding to the ramA promoter (PramA) region. Results We show that ramA is transcriptionally activated by bile and is strictly required for the bile-mediated activation of acrB and tolC. Additionally, bile is shown to specifically inhibit the binding of RamR to the PramA region, which overlaps the putative divergent ramR promoter, thereby explaining our observation that bile also activates ramR transcription. Conclusions We propose a regulation model whereby the bile-mediated activation of the acrAB and tolC multidrug efflux genes occurs mainly through the transcriptional derepression of the ramA activator gene.

Published
2014

21. Crystallization and preliminary X-ray analysis of monalysin, a novel β-pore-forming toxin from the entomopathogen Pseudomonas entomophila

Author

Christine Kellenberger, Bruno Lemaitre, Alain Roussel, Onya Opota, Philippe Leone, Renaud Vincentelli, Maryline Blemont, Architecture et fonction des macromolécules biologiques (AFMB), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Department of Fundamental Microbiology [Lausanne], Université de Lausanne = University of Lausanne (UNIL), Ecole Polytechnique Fédérale de Lausanne (EPFL), and Université de Lausanne (UNIL)

Subjects
Pore Forming Cytotoxic Proteins, Stereochemistry, 030303 biophysics, Bacterial Toxins, pore-forming toxins, Biophysics, macromolecular substances, Crystallography, X-Ray, Biochemistry, law.invention, 03 medical and health sciences, chemistry.chemical_compound, Structural Biology, law, Pseudomonas, Genetics, [CHIM.CRIS]Chemical Sciences/Cristallography, monalysin, Crystallization, 030304 developmental biology, 0303 health sciences, Pore-forming toxin, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Resolution (electron density), Condensed Matter Physics, biology.organism_classification, Crystallography, Monomer, Pseudomonas entomophila, chemistry, Crystallization Communications, human activities, Derivative (chemistry), Monoclinic crystal system
Abstract

International audience; Monalysin was recently described as a novel pore-forming toxin (PFT) secreted by the Drosophila pathogen Pseudomonas entomophila. Recombinant monalysin is multimeric in solution, whereas PFTs are supposed to be monomeric until target membrane association. Monalysin crystals were obtained by the hanging-drop vapour-diffusion method using PEG 8000 as precipitant. Preliminary X-ray diffraction analysis revealed that monalysin crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 162.4, b = 146.2, c = 144.4 Å, [beta] = 122.8°, and diffracted to 2.85 Å resolution using synchrotron radiation. Patterson self-rotation analysis and Matthews coefficient calculation indicate that the asymmetric unit contains nine copies of monalysin. Heavy-atom derivative data were collected and a Ta6Br14 cluster derivative data set confirmed the presence of ninefold noncrystallographic symmetry.

Published
2013

22. Molecular engineering of fungal GH5 and GH26 beta-(1,4)-mannanases toward improvement of enzyme activity

Author

Jean-Guy Berrin, Sophie Bozonnet, Marie Couturier, Alain Roussel, Julia Feliu, Biodiversité et Biotechnologie Fongiques (BBF), Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA)-École Centrale de Marseille (ECM), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Architecture et fonction des macromolécules biologiques (AFMB), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), Region Midi-Pyrenees, European Regional Development Fund, Institut National de la Recherche Agronomique (INRA), OSEO Innovation, Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-École Centrale de Marseille (ECM), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), and Berrin, Jean-Guy

Subjects
0106 biological sciences, Glycoside Hydrolases, Science, [SDV]Life Sciences [q-bio], Mutant, champignon, Yarrowia, 01 natural sciences, Mannans, 03 medical and health sciences, 010608 biotechnology, Catalytic Domain, [SDV.IDA]Life Sciences [q-bio]/Food engineering, Glycoside hydrolase, [SPI.GPROC]Engineering Sciences [physics]/Chemical and Process Engineering, Enzyme kinetics, 030304 developmental biology, 0303 health sciences, Multidisciplinary, biology, Beta-mannosidase, activité enzymatique, beta-Mannosidase, Active site, Galactose, biology.organism_classification, Directed evolution, Enzyme assay, Biochemistry, biology.protein, Medicine, mannanase, Research Article
Abstract

International audience; Microbial mannanases are biotechnologically important enzymes since they target the hydrolysis of hemicellulosic polysaccharides of softwood biomass into simple molecules like manno-oligosaccharides and mannose. In this study, we have implemented a strategy of molecular engineering in the yeast Yarrowia lipolytica to improve the specific activity of two fungal endo-mannanases, PaMan5A and PaMan26A, which belong to the glycoside hydrolase (GH) families GH5 and GH26, respectively. Following random mutagenesis and two steps of high-throughput enzymatic screening, we identified several PaMan5A and PaMan26A mutants that displayed improved kinetic constants for the hydrolysis of galactomannan. Examination of the three-dimensional structures of PaMan5A and PaMan26A revealed which of the mutated residues are potentially important for enzyme function. Among them, the PaMan5A-G311S single mutant, which displayed an impressive 8.2-fold increase in k(cat)/K-M due to a significant decrease of K-M, is located within the core of the enzyme. The PaMan5A-K139R/Y223H double mutant revealed modification of hydrolysis products probably in relation to an amino-acid substitution located nearby one of the positive subsites. The PaMan26A-P140L/D416G double mutant yielded a 30% increase in k(cat)/K-M compared to the parental enzyme. It displayed a mutation in the linker region (P140L) that may confer more flexibility to the linker and another mutation (D416G) located at the entrance of the catalytic cleft that may promote the entrance of the substrate into the active site. Taken together, these results show that the directed evolution strategy implemented in this study was very pertinent since a straightforward round of random mutagenesis yielded significantly improved variants, in terms of catalytic efiiciency (k(cat)/K-M).

Published
2013

23. Structural and biochemical analyses of glycoside hydrolase families 5 and 26 β-(1,4)-mannanases from Podospora anserina reveal differences upon manno-oligosaccharide catalysis

Author

Marie Couturier, Philippe Leone, Alain Roussel, Henrik Stålbrand, Anna Rosengren, Jean-Guy Berrin, Biodiversité et Biotechnologie Fongiques (BBF), Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA)-École Centrale de Marseille (ECM), Architecture et fonction des macromolécules biologiques (AFMB), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), Department of biochemistry and structural biology, Lund University [Lund], FUTUROL project, OSEO Innovation, FORMAS, VINNOVA, Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-École Centrale de Marseille (ECM), Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Berrin, Jean-Guy, and École Centrale de Marseille (ECM)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA)

Subjects
0106 biological sciences, Glycosylation, Oligosaccharides, biomasse lignocellulosique, 7. Clean energy, 01 natural sciences, Biochemistry, Podospora anserina, Substrate Specificity, dégradation enzymatique, chemistry.chemical_compound, hydrolyse, Cell Wall, Catalytic Domain, site actif, structure cristalline, glycoside hydrolase, Glycoside hydrolase, analyse structurale, Polysaccharide, [SDV.BDD]Life Sciences [q-bio]/Development Biology, 0303 health sciences, Podospora, Vegetal Biology, biology, Hydrolysis, Microbiology and Parasitology, Biologie du développement, analyse comparative, Development Biology, Mannan, Microbiologie et Parasitologie, Enzyme structure, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, structure enzymatique, substrat, Carbohydrate-binding module, mannane, paroi cellulaire végétale, CAZymes, Carbohydrate, Enzyme Structure, Fungi, Glycoside Hydrolases, Plant Cell Wall, Proline, Catalysis, 03 medical and health sciences, podospora anserina, Polysaccharides, 010608 biotechnology, Hydrolase, [SDV.BV]Life Sciences [q-bio]/Vegetal Biology, Molecular Biology, 030304 developmental biology, mannose, ascomycète, analyse biochimique, Active site, Cell Biology, biology.organism_classification, Protein Structure, Tertiary, manno-oligosaccharide, chemistry, biocarburant, Mutagenesis, mannotétraose, biology.protein, Enzymology, mannanase, Biologie végétale, transglycosylation
Abstract

The microbial deconstruction of the plant cell wall is a key biological process that is of increasing importance with the development of a sustainable biofuel industry. The glycoside hydrolase families GH5 (PaMan5A) and GH26 (PaMan26A) endo-β-1,4-mannanases from the coprophilic ascomycete Podospora anserina contribute to the enzymatic degradation of lignocellulosic biomass. In this study, P. anserina mannanases were further subjected to detailed comparative analysis of their substrate specificities, active site organization, and transglycosylation capacity. Although PaMan5A displays a classical mode of action, PaMan26A revealed an atypical hydrolysis pattern with the release of mannotetraose and mannose from mannopentaose resulting from a predominant binding mode involving the -4 subsite. The crystal structures of PaMan5A and PaMan26A were solved at 1.4 and 2.85 Å resolution, respectively. Analysis of the PaMan26A structure supported strong interaction with substrate at the -4 subsite mediated by two aromatic residues Trp-244 and Trp-245. The PaMan26A structure appended to its family 35 carbohydrate binding module revealed a short and proline-rich rigid linker that anchored together the catalytic and the binding modules.

Published
2013

24. Crystal structure of greglin, a novel non-classical Kazal inhibitor, in complex with subtilisin

Author

Guillaume Gabant, Alain Roussel, Christine Kellenberger, Christophe Epinette, Brice Korkmaz, Chrystelle Derache, Martine Cadene, Francis Gauthier, Centre de biophysique moléculaire (CBM), Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Pathologies Respiratoires : Protéolyse et Aérosolthérapie, Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Université de Tours-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
Models, Molecular, MESH: Ovary, Proteases, Stereochemistry, Molecular Sequence Data, Protein Data Bank (RCSB PDB), Grasshoppers, MESH: Amino Acid Sequence, Crystallography, X-Ray, Biochemistry, Mass Spectrometry, Serine, 03 medical and health sciences, MESH: Insect Proteins, Animals, MESH: Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acid Sequence, Phosphorylation, Molecular Biology, Pancreatic elastase, 030304 developmental biology, Serine protease, MESH: Mass Spectrometry, 0303 health sciences, Chymotrypsin, MESH: Molecular Sequence Data, biology, Protease inhibitor complex, MESH: Phosphorylation, Chemistry, 030302 biochemistry & molecular biology, Ovary, Subtilisin, MESH: Grasshoppers, Cell Biology, MESH: Crystallography, X-Ray, biology.protein, Insect Proteins, Female, MESH: Subtilisin, MESH: Female, MESH: Models, Molecular
Abstract

International audience; Greglin is an 83-residue serine protease inhibitor purified from the ovaries of the locust Schistocerca gregaria. Greglin is a strong inhibitor of subtilisin and human neutrophil elastase, acting at sub-nanomolar and nanomolar concentrations, respectively; it also inhibits neutrophil cathepsin G, α-chymotrypsin and porcine pancreatic elastase, but to a lesser extent. In the present study, we show that greglin resists denaturation at high temperature (95 °C) and after exposure to acetonitrile and acidic or basic pH. Greglin is composed of two domains consisting of residues 1-20 and 21-83. Mass spectrometry indicates that the N-terminal domain (1-20) is post-translationally modified by phosphorylations at three sites and probably contains a glycosylation site. The crystal structure of the region of greglin comprising residues 21-78 in complex with subtilisin was determined at 1.75 Å resolution. Greglin represents a novel member of the non-classical Kazal inhibitors, as it has a unique additional C-terminal region (70-83) connected to the core of the molecule via a supplementary disulfide bond. The stability of greglin was compared with that of an ovomucoid inhibitor. The thermostability and inhibitory specificity of greglin are discussed in light of its structure. In particular, we propose that the C-terminal region is responsible for non-favourable interactions with the autolysis loop (140-loop) of serine proteases of the chymotrypsin family, and thus governs specificity. DATABASE: The atomic coordinates and structure factors for the greglin-subtilisin complex have been deposited with the RCSB Protein Data Bank under accession number 4GI3. STRUCTURED DIGITAL ABSTRACT: Greglin and Subtilisin Carlsberg bind by X-ray crystallography (View interaction).

Published
2012

25. Design of a star, planet and exo-zodiacal cloud simulator for the nulling testbench PERSEE

Author

Bruno Lopez, Jean-Michel Reess, K. Houairi, Yves Rabbia, Emilie Lhomé, Frédéric Cassaing, François Hénault, N. Mauclert, Jean-Michel Le Duigou, Jean-Tristan Buey, Sophie Jacquinod, A. Marcotto, Laurie Pham, Marc Ollivier, P. Girard, J. Montri, Florentin Millour, Julien Lozi, Vincent Coudé du Foresto, Marc Barillot, C. Bailet, B. Sorrente, Alain Roussel, Laboratoire d'études spatiales et d'instrumentation en astrophysique (LESIA), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut national des sciences de l'Univers (INSU - CNRS)-Observatoire de Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Haute résolution angulaire en astrophysique, Laboratoire d'études spatiales et d'instrumentation en astrophysique = Laboratory of Space Studies and Instrumentation in Astrophysics (LESIA), Institut national des sciences de l'Univers (INSU - CNRS)-Observatoire de Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)-Institut national des sciences de l'Univers (INSU - CNRS)-Observatoire de Paris, and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)

Subjects
Physics, Test bench, Zodiacal light, Planet, business.industry, law, A* search algorithm, Cloud computing, business, [PHYS.ASTR]Physics [physics]/Astrophysics [astro-ph], Exoplanet, Simulation, law.invention
Abstract

On-going developments on the PERSEE nulling testbench include the realization of a focal plane simulator featuring one central star, an extra-solar planet orbiting around it, and an Exo-Zodiacal Cloud (EZC) surrounding the observed stellar system. PERSEE (Pegase Experiment for Research and Stabilization of Extreme Extinction) is a laboratory testbench jointly developed by a Consortium of six French institutes and companies, incorporating Observatoire de la Cote d'Azur (OCA) who is in charge of the manufacturing and procurement of the future Star and Planet Simulator (SPS). In this communication is presented a complete description of the SPS, including general requirements, techniques employed for simulating the observed planet and EZC, opto-mechanical design and expected performance. The current status of the SPS activities is summarized in the conclusion, pending final integration on the PERSEE test bench in September 2011.

Published
2011

26. Structure-Function Analysis of Grass Clip Serine Protease Involved in Drosophila Toll Pathway Activation

Author

Jean-Marc Reichhart, Philippe Leone, Alain Roussel, Christine Kellenberger, Laurent Coquet, Thierry Jouenne, 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), Polymères, biopolymères, membranes (PBM), Centre National de la Recherche Scientifique (CNRS)-Institut national des sciences appliquées Rouen Normandie (INSA Rouen Normandie), Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Université de Rouen Normandie (UNIROUEN), Normandie Université (NU), Polymères Biopolymères Surfaces (PBS), Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Institut national des sciences appliquées Rouen Normandie (INSA Rouen Normandie), Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA), Réponse immunitaire et developpement chez les insectes (RIDI - UPR 9002), Université de Strasbourg (UNISTRA)-Institut de biologie moléculaire et cellulaire (IBMC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Université de Rouen Normandie (UNIROUEN), Institut national des sciences appliquées Rouen Normandie (INSA Rouen Normandie), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut de Chimie du CNRS (INC)-Institut Normand de Chimie Moléculaire Médicinale et Macromoléculaire (INC3M), Institut de Chimie du CNRS (INC)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Normandie Université (NU)-Institut national des sciences appliquées Rouen Normandie (INSA Rouen Normandie), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Université Le Havre Normandie (ULH), Normandie Université (NU)-Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Université de Caen Normandie (UNICAEN), Normandie Université (NU)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Université Le Havre Normandie (ULH), and Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Proteases, medicine.medical_treatment, [SDV]Life Sciences [q-bio], Biology, Biochemistry, Cell Line, Serine, Structure-Activity Relationship, 03 medical and health sciences, 0302 clinical medicine, Catalytic Domain, Zymogen, medicine, Animals, Drosophila Proteins, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Molecular Biology, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Serine protease, chemistry.chemical_classification, 0303 health sciences, Protease, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Toll-Like Receptors, Proteolytic enzymes, food and beverages, Cell Biology, Enzyme, chemistry, Protein Structure and Folding, biology.protein, Drosophila, Serine Proteases, 030217 neurology & neurosurgery, MASP1, MESH: Animals, Drosophila Proteins / chemistry, Drosophila Proteins / genetics, Drosophila Proteins / metabolism, Serine Proteases / chemistry, Serine Proteases / genetics, Serine Proteases / metabolism, Signal Transduction / genetics, Signal Transduction / physiology, Toll-Like Receptors / genetics, Toll-Like Receptors / metabolism, Signal Transduction
Abstract

International audience; Grass is a clip domain serine protease (SP) involved in a proteolytic cascade triggering the Toll pathway activation of Drosophila during an immune response. Epistasic studies position it downstream of the apical protease ModSP and upstream of the terminal protease Spaetzle-processing enzyme. Here, we report the crystal structure of Grass zymogen. We found that Grass displays a rather deep active site cleft comparable with that of proteases of coagulation and complement cascades. A key distinctive feature is the presence of an additional loop (75-loop) in the proximity of the activation site localized on a protruding loop. All biochemical attempts to hydrolyze the activation site of Grass failed, strongly suggesting restricted access to this region. The 75-loop is thus proposed to constitute an original mechanism to prevent spontaneous activation. A comparison of Grass with clip serine proteases of known function involved in analogous proteolytic cascades allowed us to define two groups, according to the presence of the 75-loop and the conformation of the clip domain. One group (devoid of the 75-loop) contains penultimate proteases whereas the other contains terminal proteases. Using this classification, Grass appears to be a terminal protease. This result is evaluated according to the genetic data documenting Grass function.

Published
2011

27. The Drosophila peptidoglycan-recognition protein LF interacts with peptidoglycan-recognition protein LC to downregulate the Imd pathway

Author

Julien Royet, Franck Coste, Philippe Leone, Nada Basbous, Renaud Vincentelli, Christine Kellenberger, Alain Roussel, Centre de biophysique moléculaire (CBM), Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), 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), Réponse immunitaire et developpement chez les insectes (RIDI - UPR 9002), Institut de biologie moléculaire et cellulaire (IBMC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université de Strasbourg (UNISTRA)-Institut de biologie moléculaire et cellulaire (IBMC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), and Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Molecular Sequence Data, Peptidoglycan, Plasma protein binding, Biology, Crystallography, X-Ray, Biochemistry, ACTIVATION, 03 medical and health sciences, chemistry.chemical_compound, Tracheal cytotoxin, Genetics, Animals, Drosophila Proteins, IMMUNE-RESPONSE, structural biology, Amino Acid Sequence, Molecular Biology, Peptide sequence, innate immunity, 030304 developmental biology, 0303 health sciences, Sequence Homology, Amino Acid, RECEPTOR, Scientific Reports, 030302 biochemistry & molecular biology, Surface Plasmon Resonance, 3. Good health, Structural biology, chemistry, Ectodomain, PGRP-LC, BACTERIA, Drosophila, Signal transduction, Carrier Proteins, Drosophila Protein, Protein Binding, Signal Transduction
Abstract

International audience; The peptidoglycan (PGN)-recognition protein LF (PGRP-LF) is a specific negative regulator of the immune deficiency (Imd) pathway in Drosophila. We determine the crystal structure of the two PGRP domains constituting the ectodomain of PGRP-LF at 1.72 and 1.94 angstrom resolution. The structures show that the LFz and LFw domains do not have a PGN-docking groove that is found in other PGRP domains, and they cannot directly interact with PGN, as confirmed by biochemical-binding assays. By using surface plasmon resonance analysis, we show that the PGRP-LF ectodomain interacts with the PGRP-LCx ectodomain in the absence and presence of tracheal cytotoxin. Our results suggest a mechanism for downregulation of the Imd pathway on the basis of the competition between PRGP-LCa and PGRP-LF to bind to PGRP-LCx.

Published
2011

28. Study of the atmospheric refraction in a single mode instrument - Application to AMBER/VLTI

Author

Stephane Lagarde, Isabelle Tallon-Bosc, Romain Petrov, Sylvie Robbe-Dubois, P. Antonelli, Martin Vannier, F. Rantakyrö, G. Martinot-Lagarde, Daniel Tasso, Alain Roussel, Yves Bresson, Etienne Le-Coarer, Marcel Carbillet, Laboratoire Hippolyte Fizeau (FIZEAU), Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut national des sciences de l'Univers (INSU - CNRS)-Observatoire de la Côte d'Azur, Université Côte d'Azur (UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Côte d'Azur (UCA)-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'Astrophysique de Grenoble (LAOG), Université Joseph Fourier - Grenoble 1 (UJF)-Institut national des sciences de l'Univers (INSU - CNRS)-Centre National de la Recherche Scientifique (CNRS), Gemini Observatory [Southern Operations Center], Association of Universities for Research in Astronomy (AURA), European Southern Laboratory, European Southern Observatory (ESO), Centre de Recherche Astrophysique de Lyon (CRAL), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut national des sciences de l'Univers (INSU - CNRS), Géoazur (GEOAZUR 6526), Observatoire de la Côte d'Azur, Université Côte d'Azur (UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Côte d'Azur (UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut national des sciences de l'Univers (INSU - CNRS)-Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Recherche pour le Développement (IRD)-Centre National de la Recherche Scientifique (CNRS), Division technique INSU/SDU (DTI), Institut national des sciences de l'Univers (INSU - CNRS)-Centre National de la Recherche Scientifique (CNRS), Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Côte d'Azur (UCA)-Université Côte d'Azur (UCA)-Centre National de la Recherche Scientifique (CNRS), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut national des sciences de l'Univers (INSU - CNRS)-Centre National de la Recherche Scientifique (CNRS), Institut de Recherche pour le Développement (IRD)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Nice Sophia Antipolis (1965 - 2019) (UNS), and Joseph Louis LAGRANGE (LAGRANGE)

Subjects
[PHYS.ASTR.IM]Physics [physics]/Astrophysics [astro-ph]/Instrumentation and Methods for Astrophysic [astro-ph.IM], FOS: Physical sciences, Physics::Optics, 01 natural sciences, law.invention, 010309 optics, Telescope, Optics, law, 0103 physical sciences, Atmospheric refraction, Astrophysics::Solar and Stellar Astrophysics, Adaptive optics, 010303 astronomy & astrophysics, Instrumentation and Methods for Astrophysics (astro-ph.IM), Zenith, ComputingMilieux_MISCELLANEOUS, Physics, Very Large Telescope, business.industry, Single-mode optical fiber, Astrophysics::Instrumentation and Methods for Astrophysics, Astronomy and Astrophysics, Refraction, [SDU.ASTR.IM]Sciences of the Universe [physics]/Astrophysics [astro-ph]/Instrumentation and Methods for Astrophysic [astro-ph.IM], Interferometry, Space and Planetary Science, Astrophysics::Earth and Planetary Astrophysics, business, Astrophysics - Instrumentation and Methods for Astrophysics
Abstract

This paper presents a study of the atmospheric refraction and its effect on the light coupling efficiency in an instrument using single-mode optical fibers. We show the analytical approach which allowed us to assess the need to correct the refraction in J- and H-bands while observing with an 8-m Unit Telescope. We then developed numerical simulations to go further in calculations. The hypotheses on the instrumental characteristics are those of AMBER (Astronomical Multi BEam combineR), the near infrared focal beam combiner of the Very Large Telescope Interferometric mode (VLTI), but most of the conclusions can be generalized to other single-mode instruments. We used the software package caos (Code for Adaptive Optics Systems) to take into account the atmospheric turbulence effect after correction by the ESO system MACAO (Multi-Application Curvature Adaptive Optics). The opto-mechanical study and design of the system correcting the atmospheric refraction on AMBER is then detailed. We showed that the atmospheric refraction becomes predominant over the atmospheric turbulence for some zenith angles z and spectral conditions: for z larger than 30{\circ} in J-band for example. The study of the optical system showed that it allows to achieve the required instrumental performance in terms of throughput in J- and H-bands. First observations in J-band of a bright star, alpha Cir star, at more than 30{\circ} from zenith clearly showed the gain to control the atmospheric refraction in a single mode instrument, and validated the operating law., Mon. Not. R. Astron. Soc. (2009) accepted

Published
2009

29. First results from a laboratory hypertelescope using single-mode fibers

Author

F. Patru, François Reynaud, Laurent Delage, Denis Mourard, Alain Roussel, D. Bonneau, Y. Hugues, S. Bosio, Yves Bresson, Jean-Michel Clausse, M. Dubreuil, O. Lardière, Laboratoire d'Astrophysique de Grenoble (LAOG), Université Joseph Fourier - Grenoble 1 (UJF)-Institut national des sciences de l'Univers (INSU - CNRS)-Centre National de la Recherche Scientifique (CNRS), Laboratoire Hippolyte Fizeau (FIZEAU), Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut national des sciences de l'Univers (INSU - CNRS)-Observatoire de la Côte d'Azur, Université Côte d'Azur (UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Côte d'Azur (UCA)-Centre National de la Recherche Scientifique (CNRS), PHOTONIQUE, XLIM (XLIM), Université de Limoges (UNILIM)-Centre National de la Recherche Scientifique (CNRS)-Université de Limoges (UNILIM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire Gemini (LG), Institut national des sciences de l'Univers (INSU - CNRS)-Observatoire de la Côte d'Azur, and Université Côte d'Azur (UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Côte d'Azur (UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Physics, business.industry, Astrophysics::Instrumentation and Methods for Astrophysics, Single-mode optical fiber, Extrapolation, Astronomy and Astrophysics, Context (language use), 01 natural sciences, 010309 optics, Photometry (optics), Interferometry, Optics, Space and Planetary Science, 0103 physical sciences, business, Focus (optics), 010303 astronomy & astrophysics, Optical path length, Beam (structure), ComputingMilieux_MISCELLANEOUS
Abstract

Context. In the future, giant optical interferometric arrays will be developed with kilometric baselines and a large number of telescopes. Such arrays could have direct imaging capabilities if optimized beam combiners are used. Aims. This paper aims at studying the performance of an interferometric beam combiner using single mode fibers and in the frame of a hypertelescope. Methods. A laboratory testbed called SIRIUS was developed. We describe the general concept, the technical specifications and the results obtained. These results are analyzed with the help of a numerical simulator. Results. Direct images were obtained at the densified focus of SIRIUS. We show that the fibers greatly ease the pupil rearrangement. They also greatly improve the quality and the stability of the direct image. The computed images allow us to reproduce the effects of differential photometry and the influence of optical path difference variations. Optical path difference errors less than $\lambda/10$ and differential photometries less than 60% are required to keep the quality of the direct image. Conclusions. These results demonstrate the great potential of direct imaging interferometric beam combiners for future optical large arrays. The excellent comparison between experience and simulation clearly shows the simplicity of the fibered pupil densifier. It also gives us a great confidence in the extrapolation of these results and specifications for future arrays with a very large number of apertures.

Published
2008

30. Crystal structure of Drosophila PGRP-SD suggests binding to DAP-type but not lysine-type peptidoglycan

Author

Alain Roussel, Charles Hetru, Christine Kellenberger, Vincent Bischoff, Philippe Leone, Julien Royet, Institut de Biologie du Développement de Marseille (IBDM), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Architecture et fonction des macromolécules biologiques (AFMB), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), Institut de biologie moléculaire et cellulaire (IBMC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Centre de biophysique moléculaire (CBM), Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and Aix Marseille Université (AMU)-Collège de France (CdF)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Toll signaling pathway, Immunology, Mutant, Antimicrobial peptides, Molecular Sequence Data, PGRP, Peptidoglycan, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, Crystallography, X-Ray, Diaminopimelic Acid, Microbiology, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Catalytic Domain, Pattern recognition, Animals, Drosophila Proteins, Amino Acid Sequence, Molecular Biology, Toll, 030304 developmental biology, Innate immunity, 0303 health sciences, Innate immune system, Binding Sites, Bacteria, Binding protein, Pattern recognition receptor, 3. Good health, Cell biology, Protein Structure, Tertiary, chemistry, Drosophila, Signal transduction, Carrier Proteins, Sequence Alignment, 030217 neurology & neurosurgery, Signal Transduction
Abstract

In Drosophila the synthesis of antimicrobial peptides in response to microbial infections is under the control of the Toll and immune deficiency (Imd) signaling pathways. The Toll signaling pathway responds mainly to Gram-positive bacterial and fungal infection while the Imd pathway mediates the response to Gram-negative bacteria. Microbial recognition upstream of Toll involves, at least in part, peptidoglycan recognition proteins (PGRPs). The sensing of Gram-positive bacteria is mediated by the pattern recognition receptors PGRP-SA and Gram-negative binding protein 1 (GNBP1) that cooperate to detect the presence of lysine-type peptidoglycan in the host. Recently it has been shown that a loss-of-function mutation in peptidoglycan recognition protein SD (PGRP-SD) severely exacerbates the PGRP-SA and GNBP1 mutant phenotypes. Here we have solved the crystal structure of PGRP-SD at 1.5A resolution. Comparison with available structures of PGRPs in complex with their peptidoglycan (PGN) ligand strongly suggests a diaminopimelic acid (DAP) specificity for PGRP-SD. This result is supported by pull-down assays with insoluble PGNs. In addition we show that Toll pathway activation after infection by DAP-type PGN containing bacteria is clearly reduced in PGRP-SD mutant flies. Our hypothesis is that the role of PGRP-SD is the recognition of DAP-type PGNs responsible for the activation of the Toll pathway by Gram-negative bacteria.

Published
2008

31. How much can a T-cell antigen receptor adapt to structurally distinct antigenic peptides?

Author

Nathalie Auphan-Anezin, P. Anton van der Merwe, Bernard Malissen, Alice Kearney, Anne-Marie Schmitt-Verhulst, Annick Guimezanes, Claude Grégoire, Alain Roussel, Catherine Mazza, Christine Kellenberger, Centre d'Immunologie de Marseille - Luminy (CIML), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Architecture et fonction des macromolécules biologiques (AFMB), Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU), and Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA)

Subjects
Models, Molecular, MESH: Protein Structure, Secondary, Complementarity determining region, MESH: Amino Acid Sequence, Crystallography, X-Ray, Ligands, Protein Structure, Secondary, MESH: Tyrosine, Mice, MESH: Mutant Proteins, 0302 clinical medicine, Protein structure, MESH: Ligands, Degeneracy (biology), MESH: Animals, Peptide sequence, 0303 health sciences, biology, MESH: Peptides, General Neuroscience, MESH: Receptors, Antigen, T-Cell, Cell biology, medicine.anatomical_structure, Biochemistry, MESH: Complementarity Determining Regions, Thermodynamics, [SDV.IMM]Life Sciences [q-bio]/Immunology, MESH: Antigens, MESH: Thermodynamics, MESH: Models, Molecular, T cell, Molecular Sequence Data, Receptors, Antigen, T-Cell, chemical and pharmacologic phenomena, Major histocompatibility complex, MESH: H-2 Antigens, General Biochemistry, Genetics and Molecular Biology, Article, 03 medical and health sciences, Antigen, medicine, Animals, Amino Acid Sequence, Antigens, Molecular Biology, MESH: Mice, 030304 developmental biology, MESH: Molecular Sequence Data, General Immunology and Microbiology, T-cell receptor, H-2 Antigens, MESH: Crystallography, X-Ray, Complementarity Determining Regions, biology.protein, Tyrosine, Mutant Proteins, Peptides, 030215 immunology
Abstract

Binding degeneracy is thought to constitute a fundamental property of the T-cell antigen receptor (TCR), yet its structural basis is poorly understood. We determined the crystal structure of a complex involving the BM3.3 TCR and a peptide (pBM8) bound to the H-2K(bm8) major histocompatibility complex (MHC) molecule, and compared it with the structures of the BM3.3 TCR bound to H-2K(b) molecules loaded with two peptides that had a minimal level of primary sequence identity with pBM8. Our findings provide a refined structural view of the basis of BM3.3 TCR cross-reactivity and a structural explanation for the long-standing paradox that a TCR antigen-binding site can be both specific and degenerate. We also measured the thermodynamic features and biological penalties that incurred during cross-recognition. Our data illustrate the difficulty for a given TCR in adapting to distinct peptide-MHC surfaces while still maintaining affinities that result in functional in vivo responses. Therefore, when induction of protective effector T cells is used as the ultimate criteria for adaptive immunity, TCRs are probably much less degenerate than initially assumed.

Published
2007

32. Crystal structure at 1.45-A resolution of the major allergen endo-beta-1,3-glucanase of banana as a molecular basis for the latex-fruit syndrome

Author

Pierre Rougé, Véronique Receveur-Bréchot, Annick Barre, Willy J. Peumans, Mirjam Czjzek, Els J.M. Van Damme, Alain Roussel, Interactions et Modulateurs de Réponses (IMR), Centre National de la Recherche Scientifique (CNRS), Résonance Magnétique Nucléaire des Biomolécules, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Surfaces Cellulaires et Signalisation chez les Végétaux (SCSV), Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Laboratoire Hippolyte Fizeau (FIZEAU), Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut national des sciences de l'Univers (INSU - CNRS)-Observatoire de la Côte d'Azur, COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Côte d'Azur (UCA)-Université Côte d'Azur (UCA)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, Université Nice Sophia Antipolis (... - 2019) (UNS), and Université Côte d'Azur (UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Côte d'Azur (UCA)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Models, Molecular, Proteomics, MESH: Epitopes, B-Lymphocyte, Time Factors, Latex, Protein Conformation, Molecular Conformation, MESH: Protein Structure, Secondary, MESH: Catalytic Domain, MESH: Amino Acid Sequence, Crystallography, X-Ray, Biochemistry, Protein Structure, Secondary, Substrate Specificity, MESH: Tyrosine, MESH: Cross Reactions, Epitopes, Protein structure, MESH: Protein Conformation, Structural Biology, Catalytic Domain, Glycoside hydrolase, MESH: Proteins, MESH: Syndrome, Trisaccharide, chemistry.chemical_classification, 0303 health sciences, Chemistry, Glucan Endo-1,3-beta-D-Glucosidase, MESH: Proteomics, 030302 biochemistry & molecular biology, MESH: Immunoglobulin E, food and beverages, Syndrome, MESH: Glutamic Acid, MESH: Glucan Endo-1,3-beta-D-Glucosidase, MESH: Glucose, Epitopes, B-Lymphocyte, MESH: Fruit, Food Hypersensitivity, MESH: Models, Molecular, Protein Binding, Triose-Phosphate Isomerase, MESH: Allergens, MESH: Epitopes, Stereochemistry, Phenylalanine, Molecular Sequence Data, Glutamic Acid, Cross Reactions, Catalysis, 03 medical and health sciences, MESH: Phenylalanine, Polysaccharides, MESH: Musa, Hydrolase, MESH: Protein Binding, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acid Sequence, Binding site, Molecular Biology, 030304 developmental biology, Glucan, MESH: Molecular Conformation, Binding Sites, MESH: Molecular Sequence Data, MESH: Time Factors, MESH: Latex, Proteins, Musa, Glucanase, Allergens, Immunoglobulin E, MESH: Catalysis, MESH: Crystallography, X-Ray, Glucose, MESH: Polysaccharides, MESH: Binding Sites, Docking (molecular), Fruit, MESH: Food Hypersensitivity, Tyrosine, MESH: Substrate Specificity, MESH: Triose-Phosphate Isomerase
Abstract

International audience; Resolution of the crystal structure of the banana fruit endo-beta-1,3-glucanase by synchrotron X-ray diffraction at 1.45-A resolution revealed that the enzyme possesses the eightfold beta/alpha architecture typical for family 17 glycoside hydrolases. The electronegatively charged catalytic central cleft harbors the two glutamate residues (Glu94 and Glu236) acting as hydrogen donor and nucleophile residue, respectively. Modeling using a beta-1,3 linked glucan trisaccharide as a substrate confirmed that the enzyme readily accommodates a beta-1,3-glycosidic linkage in the slightly curved catalytic groove between the glucose units in positions -2 and -1 because of the particular orientation of residue Tyr33 delimiting subsite -2. The location of Phe177 in the proximity of subsite +1 suggested that the banana glucanase might also cleave beta-1,6-branched glucans. Enzymatic assays using pustulan as a substrate demonstrated that the banana glucanase can also cleave beta-1,6-glucans as was predicted from docking experiments. Similar to many other plant endo-beta-1,3-glucanases, the banana glucanase exhibits allergenic properties because of the occurrence of well-conserved IgE-binding epitopes on the surface of the enzyme. These epitopes might trigger some cross-reactions toward IgE antibodies and thus account for the IgE-binding cross-reactivity frequently reported in patients with the latex-fruit syndrome.

Published
2006

33. The H-2Kk MHC peptide-binding groove anchors the backbone of an octameric antigenic peptide in an unprecedented mode

Author

Bernard Malissen, Christine Kellenberger, Alain Roussel, Centre d'Immunologie de Marseille - Luminy (CIML), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Guglietta, Noëlle, and Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)

Subjects
[SDV.IMM] Life Sciences [q-bio]/Immunology, Protein Conformation, Stereochemistry, Immunology, Hemagglutinin Glycoproteins, Influenza Virus, Peptide binding, Peptide, Simian virus 40, Plasma protein binding, Crystallography, X-Ray, Mice, Protein structure, MHC class I, Animals, Humans, Immunology and Allergy, Binding site, Histocompatibility Antigen H-2D, Antigens, Viral, chemistry.chemical_classification, Antigen Presentation, Binding Sites, biology, Chemistry, T-cell receptor, H-2 Antigens, Peptide Fragments, Biochemistry, biology.protein, [SDV.IMM]Life Sciences [q-bio]/Immunology, Oligopeptides, Groove (joinery), Protein Binding
Abstract

A wealth of data has accumulated on the structure of mouse MHC class I (MHCI) molecules encoded by the H-2b and H-2d haplotypes. In contrast, there is a dearth of structural data regarding H-2k-encoded molecules. Therefore, the structures of H-2Kk complexed to an octameric peptide from influenza A virus (HA259–266) and to a nonameric peptide from SV40 (SV40560–568) have been determined by x-ray crystallography at 2.5 and 3.0 Å resolutions, respectively. The structure of the H-2Kk-HA259–266 complex reveals that residues located on the floor of the peptide-binding groove contact directly the backbone of the octameric peptide and force it to lie deep within the H-2Kk groove. This unprecedented mode of peptide binding occurs despite the presence of bulky residues in the middle of the floor of the H-2Kk peptide-binding groove. As a result, the Cα atoms of peptide residues P5 and P6 are more buried than the corresponding residues of H-2Kb-bound octapeptides, making them even less accessible to TCR contact. When bound to H-2Kk, the backbone of the SV40560–568 nonapeptide bulges out of the peptide-binding groove and adopts a conformation reminiscent of that observed for peptides bound to H-2Ld. This structural convergence occurs despite the totally different architectures of the H-2Ld and H-2Kk peptide-binding grooves. Therefore, these two H-2Kk-peptide complexes provide insights into the mechanisms through which MHC polymorphism outside primary peptide pockets influences the conformation of the bound peptides and have implications for TCR recognition and vaccine design.

Published
2005

34. The Dual Nature of the Wheat Xylanase Protein Inhibitor XIP-I

Author

Alain Roussel, S. Porciero, Paloma Manzanares, Gary Williamson, Françoise Payan, Caroline S.M. Furniss, Nathalie Juge, Philippe Leone, Anne Durand, Harry J. Gilbert, T. Tahir, Architecture et fonction des macromolécules biologiques (AFMB), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Department of Radiology, Norfolk and Norwich University Hospital, Colney Lane, Norwich, Instituto de Agroquímica y Tecnología de Alimentos - Institute of Agrochemistry and Food Technology [Valencia] (IATA-CSIC), University of Newcastle [Australia] (UoN), Sciences en Société (SenS), Institut National de la Recherche Agronomique (INRA), and University of Newcastle [Callaghan, Australia] (UoN)

Subjects
2. Zero hunger, 0303 health sciences, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 030302 biochemistry & molecular biology, Active site, Cell Biology, biology.organism_classification, Biochemistry, 03 medical and health sciences, Protein structure, Aspergillus nidulans, biology.protein, Xylanase, [CHIM.CRIS]Chemical Sciences/Cristallography, Structure–activity relationship, Glycoside hydrolase, Penicillium funiculosum, Molecular Biology, Peptide sequence, 030304 developmental biology
Abstract

International audience; The xylanase inhibitor protein I (XIP-I) from wheat Triticum aestivum is the prototype of a novel class of cereal protein inhibitors that inhibit fungal xylanases belonging to glycoside hydrolase families 10 (GH10) and 11 (GH11). The crystal structures of XIP-I in complex with Aspergillus nidulans (GH10) and Penicillium funiculosum (GH11) xylanases have been solved at 1.7 and 2.5 Angstrom resolution, respectively. The inhibition strategy is novel because XIP-I possesses two independent enzyme-binding sites, allowing binding to two glycoside hydrolases that display a different fold. Inhibition of the GH11 xylanase is mediated by the insertion of an XIP-I Pi-shaped loop (Lalpha(4)beta(5)) into the enzyme active site, whereas residues in the helix alpha7 of XIP-I, pointing into the four central active site subsites, are mainly responsible for the reversible inactivation of GH10 xylanases. The XIP-I strategy for inhibition of xylanases involves substrate-mimetic contacts and interactions occluding the active site. The structural determinants of XIP-I specificity demonstrate that the inhibitor is able to interact with GH10 and GH11 xylanases of both fungal and bacterial origin. The biological role of the xylanase inhibitors is discussed in light of the present structural data.

Published
2004

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