Retroviruses are characterized by their ability to gain resistance from therapeutic or vaccinal intervention through the accumulation of escape mutations in the viral genome and/or the interaction of regulatory viral proteins with cellular pathways. Understanding the structural determinants of the functions of the viral proteins is crucial for the development of efficient antiviral therapies.
Our research are conducted on both animal and human retroviruses to take advantage of the presence in the Biodistrict Lyon-Gerland of the CIRI (International Center for Infectiology Research) and of the world-leading companies Merial and Sanofi, which are dedicated to animal and human health, respectively.
Hence, we have characterized the crystal structure of an epitope of the trans-activator Tat of the human virus HIV-1 in complex with its cognate antibody (Serrière et al., J. Mol. Biol. 2011), observed a new dimeric form of an avian integrase domain (Ballandras et al., PloS One 2011), characterized the biophysical properties of the capsid protein of the Feline Immunodeficiency Virus FIV (Serrière et al., PloS One 2013) and determined the crystal structure of the matrix protein of FIV (Serrière et al., Retrovirology 2013).
We also have a genuine interest for some non-viral therapeutic targets and we have carried out structural studies on taurocyamine kinases in trematodes (Merceron et al., J. Biol. Chem. 2015) and the bacterial type 2 secretion system (Pineau et al., Mol. Microbiol. 2014).
Finally, we have created the joint Web server ENDscript / ESPript for protein sequence and structure analyses (>12,000 users per year; Robert & Gouet, Nucleic Acids Res. 2014).
On-going projects on retroviral integrases aim to rationally develop new classes of protein-protein inhibitors to fight AIDS (P. Gouet, H. Yajjou in coll. with C. Ronfort and L. Guy) and to make safer the use of pig tissues and organs in human xenotransplantation (P. Gouet, M. Chahpazoff in coll. with Y. Blanchard). Molecular and structural studies of chromosomal targeting by integrases are underway (P. Gouet, X. Robert in coll. with V. Parissi) and we have started observing the concerted integration mechanism at the single molecule scale with magnetic tweezers (S. Réty, F. Fiorini in coll. with P. Jalinot).
We also work on the molecular mechanisms underlying the biological function of the transactivating protein Tax from Human T-Lymphotropic Virus (HTLV), whose structure is unknown (C. Guillon, C. Folio, M. Dujardin in coll. with R. Mahieux and G. Schoehn). Tax is a modular protein with disordered regions and we use a structural biological integrative approach combining X-ray crystallography, NMR and cryo-EM.
Furthermore, we aim at the identification of therapeutic molecules targeting assembly of the capsid protein of FIV by a combination of in vitro screening, bio-guided design process and structural studies (C. Guillon in coll. with G. Álvarez Touron).
By clicking on the PDB logo, you will be redirected to the Protein Data Bank website presenting the extensive list of the structures we solved @IBCP.
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Biophysical characterization and crystal structure of the Feline Immunodeficiency Virus p15 matrix protein
Our biochemical study of the p15 matrix protein of the Feline Immunodeficiency Virus (FIV) revealed that it forms a stable dimer in solution under acidic conditions and at high concentration, unlike other retroviral matrix proteins. We determined the crystal structure of full-length FIV p15 to 2 Å resolution and observed a helical organization of the protein, typical for retroviral matrix proteins. A hydrophobic pocket that could accommodate a myristoyl group was identified, and the C-terminal end of FIV p15, which is mainly unstructured, was visible in electron density maps. As FIV p15 crystallizes in acidic conditions but with one monomer in the asymmetric unit, we searched for the presence of a biological dimer in the crystal. No biological assembly was detected by the PISA server, but the three most buried crystallographic interfaces have interesting features: the first one displays a highly conserved tryptophan acting as a binding platform; the second one is located along a 2-fold symmetry axis and the third one resembles the dimeric interface of EIAV p15. Because the C-terminal end of p15 is involved in two of these three interfaces, we investigated the structure and assembly of a C-terminal-truncated form of p15 lacking 14 residues. The truncated FIV p15 dimerizes in solution at a lower concentration and crystallizes with two molecules in the asymmetric unit. The EIAV-like dimeric interface is the only one to be retained in the new crystal form and could therefore correspond to the one of FIV p15 in solution.
Serrière J, Robert X, Perez M, Gouet P & Guillon C (2013). Retrovirology. 10:64
A crystal structure of the catalytic core domain of an avian sarcoma and leukemia virus integrase suggests an alternate dimeric assembly
Integrase (IN) is an important therapeutic target in the search for anti-Human Immunodeficiency Virus (HIV) inhibitors. This modular enzyme is hard to crystallize. A first structural result was obtained with the IN catalytic core domain (CCD) of the avian Rous Sarcoma Virus (RSV). A ribonuclease-H like motif was revealed as well as a dimeric interface stabilized by two pairs of α-helices. These structural features have been validated in other structures of IN CCDs. We have determined the crystal structure of the Rous-associated virus type-1 (RAV-1) IN CCD to 1.8 Å resolution. RAV-1 IN shows a standard activity for integration and its CCD differs in sequence from that of RSV by a single accessible residue in position 182 (substitution A182T). Surprisingly, the CCD of RAV-1 IN associates itself with an unexpected dimeric interface characterized by three pairs of α-helices. A182 is not involved in this novel interface, which results from a rigid body rearrangement of the protein at its dimeric surface. A new basic groove that is suitable for single-stranded nucleic acid binding is observed at the surface of the dimer. We have determined the structure of the mutant A182T of RAV-1 IN CCD and obtained a RSV IN CCD-like structure with two pairs of buried α-helices at the interface. Our results suggest that the CCD of avian INs can dimerize in more than one state. Such flexibility can further explain the multifunctionality of the retroviral IN, which beside integration of dsDNA is implicated in different steps of the retroviral cycle in presence of viral ssRNA.
Ballandras A, Moreau K, Robert X, Confort MP, Merceron R, Haser R, Ronfort C & Gouet P (2011). PLoS One. 6:e23032
The substrate-free and -bound crystal structures of the duplicated taurocyamine kinase from the human parasite Schistosoma mansoni
The taurocyamine kinase from the blood fluke Schistosoma mansoni (SmTK) belongs to the phosphagen kinase (PK) family and catalyzes the reversible Mg2+-dependent transfer of a phosphoryl group between ATP and taurocyamine. SmTK is derived from gene duplication, as are all known trematode TKs. Our crystallographic study of SmTK reveals the first atomic structure of both a TK and a PK with a bilobal structure. The two unliganded lobes present a canonical open conformation and interact via their respective C- and N-terminal domains at a helix-mediated interface. This spatial arrangement differs from that observed in true dimeric PKs, in which both N-terminal domains make contact. Our structures of SmTK complexed with taurocyamine or L-arginine compounds explain the mechanism by which an arginine residue of the phosphagen specificity loop is crucial for substrate specificity. A SmTK crystal was soaked with the dead-end transition state analog (TSA) components taurocyamine-NO3--MgADP. One SmTK monomer was observed with two bound TSAs and an asymmetric conformation, with the first lobe semiclosed and the second closed. However, isothermal titration calorimetry and enzyme kinetics experiments showed that the two lobes function independently. A small angle X-ray scattering model of SmTK-TSA in solution with two closed active sites was generated.
Merceron R, Awama AM, Montserret R, Marcillat O & Gouet P (2015). J. Biol. Chem. 290:12951-63
Deciphering key features in protein structures with the new ENDscript server
ENDscript 2 is a friendly webserver for extracting and rendering a comprehensive analysis of primary to quaternary protein structure information in an automated way. This major upgrade has been fully re-engineered to enhance speed, accuracy and usability with interactive 3D visualization. It takes advantage of the new version 3 of ESPript, our well-known sequence alignment renderer, improved to handle a large number of data with reduced computation time. From a single PDB entry or file, ENDscript produces high quality figures displaying multiple sequence alignment of proteins homologous to the query, colored according to residue conservation. Furthermore, the experimental secondary structure elements and a detailed set of relevant biophysical and structural data are depicted. All this information and more are now mapped on interactive 3D PyMOL representations. Thanks to its adaptive and rigorous algorithm, beginner to expert users can modify settings to fine-tune ENDscript to their needs. ENDscript has also been upgraded as an open platform for the visualization of multiple biochemical and structural data coming from external biotool webservers, with both 2D and 3D representations. ENDscript 2 and ESPript 3 are freely available at http://endscript.ibcp.fr and http://espript.ibcp.fr.
Robert X & Gouet P (2014). Nucleic Acids Res. 42:W320-4
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Guzmán Álvarez TouronUniversidad de la República - Paysandú, Uruguay
Yannick BlanchardViral Genetics and Biosafety - ANSES Ploufragan, France
Laure GuyÉcole Normale Supérieure - Lyon, France
Renaud MahieuxInternational Center for Infectiology Research - Lyon, France
Vincent ParissiFundamental Microbiology and Pathogenicity Lab - CNRS Bordeaux, France
Corinne RonfortPathogenicity and Virus Vaccine Lab - INRA Grenoble, France
Maria-Cristina de RosaInstitute of Chemistry of Molecular Recognition - Rome, Italy
Guy SchoehnInstitute of Structural Biology - Grenoble, France
Vladimir ShevchikMicrobiology, Adaptation and Pathogenesis Lab - INSA Villeurbanne, France
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* Corresponding authors
• Research articles @IBCP
Chen WF, Dai YX, Duan XL, Liu NN, Shi W, Li N, Li M, Dou SX, Dong YH, Réty S*, Xi XG* (2016). Crystal structures of the BsPif1 helicase reveal that a major movement of the 2B SH3 domain is required for DNA unwinding. Nucleic Acids Res.44:2949-61
Liu NN, Duan XL, Ai X, Yang YT, Li M, Dou SX, Réty S, Deprez E & Xi XG* (2015). The Bacteroides sp. 3_1_23 Pif1 protein is a multifunctional helicase. Nucleic Acids Res.43:8942-54
Demange A, Yajjou-Hamalian H, Gallay K, Luengo C, Beven V, Leroux A, Confort MP, Al Andary E, Gouet P, Moreau K, Ronfort C* & Blanchard Y* (2015). Porcine Endogenous Retrovirus (PERV)-A/C: biochemical properties of its Integrase and susceptibility to Raltegravir. J Gen Virol.96:3124-30
Merceron R, Awama AM, Montserret R, Marcillat O* & Gouet P* (2015). The substrate-free and -bound crystal structures of the duplicated taurocyamine kinase from the human parasite Schistosoma mansoni. J Biol Chem.290:12951-63
Guillon C*, Bigouagou UM, Folio C, Jeannin P, Delneste Y & Gouet P (2014). A staggered decameric assembly of human C-reactive protein stabilized by zinc ions revealed by X-ray crystallography. Protein Pept Lett.22:248-55
Pineau C, Guschinskaya N, Robert X, Gouet P, Ballut L* & Shevchik VE* (2014). Substrate recognition by the bacterial type II secretion system: more than a simple interaction. Mol Microbiol.94:126-40
Robert X & Gouet P* (2014). Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res.42:W320-4
Hassan S, Shevchik VE*, Robert X & Hugouvieux-Cotte-Pattat N (2013). PelN is a new pectate lyase of Dickeya dadantii with unusual characteristics. J Bacteriol.195:2197-206
Lallemand M, Login FH, Guschinskaya N, Pineau C, Effantin G, Robert X & Shevchik VE* (2013). Dynamic interplay between the periplasmic and transmembrane domains of GspL and GspM in the type II secretion system. PLoS One 8:e79562-e79562
Serrière J, Robert X, Perez M, Gouet P & Guillon C* (2013). Biophysical characterization and crystal structure of the Feline Immunodeficiency Virus p15 matrix protein. Retrovirology.10:64
Cellier C, Moreau K, Gallay K, Ballandras A, Gouet P & Ronfort C* (2013). In vitro functional analyses of the human immunodeficiency virus type 1 (HIV-1) integrase mutants give new insights into the intasome assembly. Virology.439:97-104
Serrière J, Fenel D, Schoehn G, Gouet P & Guillon C* (2013). Biophysical characterization of the feline immunodeficiency virus p24 capsid protein conformation and in vitro capsid assembly. PLoS One.8:e56424
Merceron R, Foucault M, Haser R, Mattes R, Watzlawick H* & Gouet P* (2012). The molecular mechanism of thermostable α-galactosidases AgaA and AgaB explained by X-ray crystallography and mutational studies. J Biol Chem.287:39642-52
Ballandras A, Moreau K, Robert X, Confort MP, Merceron R, Haser R, Ronfort C* & Gouet P* (2011). A crystal structure of the catalytic core Domain of an avian sarcoma and leukemia virus integrase suggests an alternate dimeric assembly. PloS One.6:e23032
Serrière J, Dugua JM, Bossus M, Vérrier B, Haser R, Gouet P & Guillon C* (2011). Fab'-induced folding of antigenic N-terminal peptides from intrinsically disordered HIV-1 Tat revealed by X-ray crystallography. J Mol Biol.405:33-42
Charmetant J, Moreau K, Gallay K, Ballandras A, Gouet P & Ronfort C* (2011). Functional analyses of mutants of the central core domain of an Avian Sarcoma/Leukemia Virus integrase. Virology.421:42-50
Paracuellos P, Ballandras A, Robert X, Kahn R, Hervé M, Mengin-Lecreulx D, Cozzone AJ, Duclos B & Gouet P* (2010). The extended conformation of the 2.9-Å crystal structure of the three-PASTA domain of a Ser/Thr kinase from the human pathogen Staphylococcus aureus. J Mol Biol.404:847-58
Foucault M, Mayol K, Receveur-Bréchot V, Bussat MC, Klinguer-Hamour C, Vérrier B, Beck A, Haser R, Gouet P & Guillon C* (2010). UV and X-ray structural studies of a 101-residue long Tat protein from a HIV-1 primary isolate and of its mutated, detoxified, vaccine candidate. Proteins.78:1441-56
Paracuellos P, Ballandras A, Robert X, Cozzone AJ, Duclos B & Gouet P* (2009). Crystallization and initial X-ray diffraction study of the three PASTA domains of the Ser/Thr kinase Stk1 from the human pathogen Staphylococcus aureus. Acta Crystallogr Sect F.65:1187-9
Andreoletti P*, Mouesca JM, Gouet P, Jaquinod M, Capeillère-Blandin C & Jouve HM (2009). Verdoheme formation in Proteus mirabilis catalase. Biochim Biophys Acta.1790:741-53
Awama AM, Paracuellos P, Laurent S, Dissous C, Marcillat O & Gouet P* (2008). Crystallization and X-ray analysis of the Schistosoma mansoni guanidino kinase. Acta Crystallogr Sect F.64:854-7
Crézé C, Castang S, Dérivery E, Haser R, Hugouvieux-Cotte-Pattat N, Shevchik VE & Gouet P* (2008). The crystal structure of pectate lyase pelI from soft rot pathogen Erwinia chrysanthemi in complex with its substrate. J Biol Chem.283:18260-8
Rakotobe D, Violot S, Hong SS, Gouet P & Boulanger P* (2008). Mapping of immunogenic and protein-interacting regions at the surface of the seven-bladed β-propeller domain of the HIV-1 cellular interactor EED. Virol J.27;5:32
Soulat D, Jault JM, Geourjon C, Gouet P, Cozzone AJ & Grangeasse C* (2007). Tyrosine-kinase Wzc from Escherichia coli possesses an ATPase activity regulated by autophosphorylation. FEMS Microbiol Lett.274:252-9
Stephanidis B, Adichtchev S, Gouet P, McPherson A & Mermet A* (2007). Elastic properties of viruses. Biophys J.93:1354-9
Crézé C, Rinaldi B, Haser R, Bouvet P & Gouet P* (2007). Structure of a d(TGGGGT) quadruplex crystallized in the presence of Li+ ions. Acta Crystallogr D.63:682-8
Castang S, Reverchon S, Gouet P & Nasser W* (2006). Direct evidence for the modulation of the activity of the Erwinia chrysanthemi quorum-sensing regulator ExpR by acylhomoserine lactone pheromone. J Biol Chem.281:29972-87
Foucault M, Watzlawick H, Mattes R, Haser R & Gouet P* (2006). Crystallization and preliminary X-ray diffraction studies of two thermostable α-galactosidases from glycoside hydrolase family 36. Acta Crystallogr Sect F.62:100-3
Castang S, Chantegrel B, Deshayes C, Dolmazon R, Gouet P, Haser R, Reverchon S, Nasser W, Hugouvieux-Cotte-Pattat N & Doutheau A* (2004). N-Sulfonyl homoserine lactones as antagonists of bacterial quorum sensing. Bioorg Med Chem Lett.14:5145-9
Moreau K, Faure C, Violot S, Gouet P, Verdier G & Ronfort C* (2004). Mutational analyses of the core domain of Avian Leukemia and Sarcoma Viruses integrase: critical residues for concerted integration and multimerization. Virology.318:566-81
Castang S, Shevchik VE, Hugouvieux-Cotte-Pattat N, Legrand P, Haser R & Gouet P* (2004). Crystallization of the pectate lyase PelI from Erwinia chrysanthemi and SAD phasing of a gold derivative. Acta Crystallogr D.60:190-2
Andreoletti P, Pernoud A, Sainz G, Gouet P & Jouve HM* (2003). Structural studies of Proteus mirabilis catalase in its ground state, oxidized state and in complex with formic acid. Acta Crystallogr D.59:2163-8
Gouet P*, Robert X & Courcelle E (2003). ESPript/ENDscript: Extracting and rendering sequence and 3D information from atomic structures of proteins. Nucleic Acids Res.31:3320-3
Gouet P* & Courcelle E (2002). ENDscript: a workflow to display sequence and structure information. Bioinformatics.18:767-8
• Review & Proceedings
Marcillat O, Laurent S, Awama A, Paracuellos P & Gouet P* (2009). "Structure of the Schistosoma mansoni guanidino kinase." FEBS J.276:151-152 (Proceedings)
Gouet P* (2005). « L'exemple de la détermination de la structure cristallographique de la nucléocapside du bluetongue virus. » J Phys IV France. 130:203-207 (Review)
• Commercial licence
CNRS Commercial license DV 62804 for the webserver ESPript/ENDscript
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Biochemistry room 1
Biochemistry room 2
X-ray diffraction setup
Cryo data collection
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Margaux CHAHPAZOFF PhD student ARC1 Santé Région
Marie DUJARDIN Post-doc ANR
Francesca FIORINI Post-doc ANRS
Christelle FOLIO PhD student MERS
Patrice GOUET PR1 Lyon 1
Christophe GUILLON CR1 CNRS
Stéphane RÉTY CR1 CNRS
Xavier ROBERT IE1 CNRS
Halima YAJJOU PhD student ANRS
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