|Structural Immunology - CNRS URA 2185|
|HEAD||Dr. BENTLEY Graham / firstname.lastname@example.org|
|MEMBERS||Dr FAURE--KUZMINSKA Grazyna / Mme FRAYSSE Jocelyne / Dr. GANGNARD Stéphane / Mr. GHORBAL Medhi / Dr GOPAUL Deshmukh / Dr. JUILLERAT Alexandre / Dr. LEWIT-BENTLEY Anita / Dr. RAMBOARINA Stéphanie / Mr. RANDAMI Tarik / Mr SAUL Frederick / Mme SOUCHON Hélène / Mr THOMAS Christophe / Dr VULLIEZ-LE NORMAND Brigitte / Dr YAO Deqiang
Research themes of the Unit of Structural Immunology centre on the structural and functional characterization of proteins with a biomedical interest. Particular emphasis is given to antigens from infectious agents that are vaccine candidates or targets for drug design. During 2008, we have continued work on antigens from Plasmodiumand Shigella flexneri, as well as on site-specific recombinases and neurotoxic and anticoagulant phospholipases A2.
Plasmodium antigens(G. Bentley)
(a) Apical Membrane antigen 1 (AMA1): AMA1, a Plasmodiummembrane protein involved in host cell invasion, is a malaria vaccine candidate currently in clinical trials. Following the structure determination of the AMA1 ectoplasmic region (Pizarro et al., Science, 2005), we have set out to provide a more comprehensive structural analysis of dominant B-cell epitopes that contribute to immune protection. Detailed knowledge of their location and the presence of polymorphic residues can provide useful information for optimizing AMA1 vaccine constructs. We have determined structure of an invasion-inhibitory AMA1-antibody complex, and crystallographic analysis of other antibody complexes is currently in progress.
(b) P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1): PfEMP1 forms a family of variant parasite adhesion proteins expressed on the surface of infected erythrocytes (IE). These are complex multi-domain proteins that cause cyto-adhesion of IE in diverse tissues and organs. We are studying two PfEMP1 variants implicated in severe malaria: one causing rosetting (adhesion of IE to healthy erythrocytes), the other causing sequestration of IE in the placenta. We have expressed individual domains of these two PfEMP1 variants and have identified those that are directly involved in adhesion.
Shigellosis vaccine development(B. Vulliez-Le Normand)
The O-antigen polysaccharide of LPS is a major determinant of immune protection against Shigella flexneri. We have determined the structure of a protecting anti-O-antigen mAb in complex with a synthetic O-antigen oligosaccharide and as a cross-reaction complex with an affinity-selected peptide. The peptide was shown to be a structural mimic of the saccharide epitope.
Site-specific recombinases and Protein-DNA complexes (D. N. Gopaul)
Site specific recombinases are often found associated with mobile DNA elements carrying resistance markers. We have characterized the Integron VchIntIa recombinase in complex with a folded stem loop bottom strand substrate of the attC site from V. cholerae (MacDonald et al, Nature 2006). We are pursuing mutagenesis experiments, associated partner search and biochemical characterization of the enzyme reaction, as well as the structural characterization of the Int1 recombinase and an attI double stranded substrate (Demarre et al, NAR 2007).
Neurotoxic and anticoagulant phospholipase A2(G. Faure)
To elaborate a new anticoagulant or antineurotoxic agents we are studying grIIA PLA2s from snake venom (Viperidaefamily). Using surface plasmon resonance to study protein-protein interactions and a biological inhibition test (prothrombinase activity) we showed that Ammodytoxin (Atx), a neurotoxic phospholipase A2from Vipera a.a. and the CB subunit of crotoxin from Crotalus d.t, inhibit blood coagulation by direct binding to human blood coagulation factor FXa via a non-catalytic, PL-independent mechanism. By site-directed mutagenesis, affinity binding studies and theoretical bioinformatics methods we mapped the interaction sites on both PLA2and FXa. The crystal structures of two ammodytoxin isoforms, AtxA and AtxC, which differ in neurotoxicity and anticoagulant potency, have been determined to understand their functional differences. Finally, we proposed that natural PLA2inhibitors present in the blood of the snakes, neutralize toxicity and inhibits PLA2activity of Atx and crotoxin possessingstructural homology with some proteins of the innate immune system.
Keywords: X-ray crystallography, antibodies, malaria, shigellosis, integrases, site-specific recombinases, Holliday junction, phospholipases A2
Pizarro JC, Vulliez-Le Normand B, Chesne-Seck ML, Collins CR, Withers-Martinez C, Hackett F, Blackman MJ, Faber BW, Remarque EJ, Kocken CH, Thomas AW, Bentley GA. (2005) Crystal structure of the malaria vaccine candidate apical membrane antigen 1. Science. 308:408-11. PMID: 15731407
MacDonald D, Demarre G, Bouvier M, Mazel D, Gopaul DN. (2006) Structural basis for broad DNA-specificity in integron recombination. Nature. 440:1157-62. PMID: 16641988
Igonet S, Vulliez-Le Normand B, Faure G, Riottot MM, Kocken CH, Thomas AW, Bentley GA. (2007) Cross-reactivity studies of an anti-Plasmodium vivax apical membrane antigen 1 monoclonal antibody: binding and structural characterisation. J Mol Biol. 366:1523-37. PMID: 17229439
Faure G, Gowda VT, Maroun RC. (2007) Characterization of human coagulation factor Xa-binding site on Viperidae snake venom phospholipases A2 by affinity binding studies and molecular bioinformatics. BMC Struct Biol. 7:82 PMID: 18062812
Vulliez-Le Normand B, Saul FA, Phalipon A, Bélot F, Guerreiro C, Mulard LA, Bentley GA. (2008) Structures of synthetic O-antigen fragments from serotype 2a Shigella flexneri in complex with a protective monoclonal antibody. Proc Natl Acad Sci U S A. 105:9976-81. PMID: 18621718
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