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 2010, we have continued work on antigens from Plasmodiumand Shigella flexneri, as well as on site-specific recombinases and neurotoxic and anticoagulant phospholipases A2.

Plasmodiumantigens

(a) Apical Membrane antigen 1 (AMA1) (B. Vulliez-Le Normand): 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 AMA1 in complex with an invasion-inhibitory antibody and as well as an invasion-inhibitory peptide.

(b) P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) (G. Bentley): 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 2010, the structure of the functionally important N-terminal double domain of a PfEMP1 variant implicated in rosetting has been determined (see Figure).

Site-specific recombinases and Protein-DNA complexes (D. N. Gopaul)

Site-specific recombinases perform a wide variety of DNA rearrangement tasks in the cell from Prokaryotes to Eukaryotes, and extending to Archaea. In the bacterial world, they are often found associated with mobile DNA elements carrying resistance markers and on the chromosome Integron locus. 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 protein 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.). Continuing work on the biochemistry of the reaction intermediates is ongoing, with the characterization of the Holliday junction arms affinity constants. We are also pursuing the biochemical and structural characterization of other pathogen-related recombinases enzymes.

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.

The interaction between the domain NBD1 of the protein CFTR and cytosolic PLA2, which is implicated in the regulation of eicosanoid-dependent inflammation, has been demonstrated.

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

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

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) Structural mimicry of O-antigen by a peptide revealed in a complex with an antibody raised against Shigella flexneri serotype 2a. J Mol Biol. 388:839-50. PMID: 19328810

Saul FA, Prijatelj-Znidarsic P, Vulliez-le Normand B, Villette B, Raynal B, Pungercar J, Krizaj I, Faure G. (2010). Comparative structural studies of two natural isoforms of ammodytoxin, phospholipases A2 from Vipera ammodytes ammodytes which differ in neurotoxicity and anticoagulant activity. J Struct Biol. 169:360-9. PMID: 19857576.

Srivastava A, Gangnard S, Round A, Dechavanne S, Juillerat A, Raynal B, Faure G, Baron B, Ramboarina S, Singh SK, Belrhali H, England P, Lewit-Bentley A, Scherf A, Bentley GA, Gamain B. (2010) Full-length extracellular region of the var2CSA variant of PfEMP1 is required for specific, high-affinity binding to CSA. Proc. Natl. Acad. Sci. U. S. A. 107:4884-9. PMID: 20194779.