Structural Immunology - CNRS URA 2185  


  HEADDr. BENTLEY Graham / bentley@pasteur.fr
  MEMBERSMr. DECHAVANNE Sébastien / Dr FAURE-KUZMINSKA Grazyna / Mme FRAYSSE Jocelyne
r. GANGNARD Stéphane / M. 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


  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 2008, we have continued work on antigens from Plasmodiumand Shigella flexneri, as well as on site-specific recombinases and neurotoxic and anticoagulant phospholipases A2.

Plasmodiumantigens (G. Bentley)

(a) Apical Membrane antigen 1 (AMA1): AMA1, a Plasmodiummembrane protein involved in host cell invasion, is a malaria vaccine candidate currently undergoing 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 is 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. Of note for 2009, the structure of the functionally important N-terminal domain of a PfEMP1 variant implicated in rosetting has been determined (see Figure).

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). We have shown that base matching in the central exchange area of the DNA site is crucial for efficient recombination to occur (Frumerie et al,Nucleic Acids Res. 2010 38:559-69.).

Structural and functional characterization of neurotoxic and anticoagulant phospholipase A2 (G. Faure)

To elaborate a new anticoagulant or antineurotoxic agents we study the functional sites of group IIA neurotoxic and anticoagulant PLA2s from snake venom (Viperidae family). We have determined the crystal structure of two isoforms of ammodytoxin, AtxA and AtxC, which differ in sequence by only two amino-acid residues but display significant differences in toxicity and anticoagulant properties. The crystal structures reveal how these two mutations lead to differences in neurotoxicity and anticoagulant activity and help to explain their role in the binding mode of PLA2 with FXa. We also showed a novel dimeric conformation of Atx, with the two N-terminal α-helices oriented in a parallel fashion making important hydrophobic contacts between the two monomers. Formation of the Atx dimer could create more accessible surface for interaction with neurotoxic receptor by the presence of two binding sites in C-terminal regions, fully exposed and accessible for this interaction. Knowledge of the three-dimensional structure of the anticoagulant snake venom PLA2s and their sites of interaction with FXa at the level of specific amino acid residues could lead to an understanding of hemostatic processes at the molecular level.

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

bentley.jpg

Figure:Crystal structure of the rosetting domain, DBL1a1, of the PfEMP1 variant varO. The structure is shown in ribbon form to highlight the high a-helix content and the disulphide bridges are indicated in yellow.



  Publications

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

Theillet FX, Saul FA, Vulliez-Le Normand B, Hoos S, Felici F, Weintraub A, Mulard LA, Phalipon A, Delepierre M, Bentley GA. (2009) J Mol Biol. 388:839-50. PMID: 19328810



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Activity Reports 2009 - Institut Pasteur
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