Structural Immunology - CNRS URA 2185  


  HEADDr. BENTLEY Graham / bentley@pasteur.fr
  MEMBERSDr FAURE Grazyna / Dr. GANGNARD Stéphane / Dr GOPAUL Deshmukh / Mme FRAYSSE Jocelyne / Dr. JUILLERAT Alexandre / Mr SAUL Frederick / Mme SOUCHON Hélène / Mr THOMAS Christophe / Mr VILLETTE Benoît / Dr VULLIEZ-LE NORMAND Brigitte / Dr YAO Deqiang


  Annual Report

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 2007, 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. falciparumErythrocyte 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)

Using surface plasmon resonance, protein-protein interaction measurements and a biological test of inhibition of protrombinase activity we showed that Ammodytoxin (Atx), a neurotoxic phospholipase A2from Vipera ammodytes ammodytes, inhibits blood coagulation through 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. Finally, an antivenom protein, Atx-inhibitor, which neutralizes toxicity and inhibits PLA2activity of Atx, has been identified in the blood of the snake Vipera a.ammodytes

Keywords: X-ray crystallography, antibodies, malaria, shigellosis, integrases, site-specific recombinases, Holliday junction, phospholipases A2



  Publications

Saul FA, Arié JP, Vulliez-le Normand B, Kahn R, Betton JM, Bentley GA.(2004). Structural and functional studies of FkpA from Escherichia coli, a cis/trans peptidyl-prolyl isomerase with chaperone activity. J Mol Biol. 335:595-608. PMID: 14672666

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



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Activity Reports 2007 - Institut Pasteur
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