|PDF Version||Structural Immunology|
|Director : BENTLEY Graham (firstname.lastname@example.org)|
Research projects of the Unit of Structural Immunology are focussed on the structural study of antigenic recognition using the techniques of X-ray crystallography. Certain of our projects include molecules that are of interest for the development of vaccines and drugs. Particular emphasis is placed on surface antigens from Plasmodium that are promising malaria vaccine candidates. In many cases, the function of Plasmodium antigens is poorly characterised, and we aim to contribute to understanding their biological role in the life cycle of the parasite by determining their 3-dimensional structure. Moreover, these structural results can be usefully exploited for vaccine development by providing a framework for analysing polymorhism and the distribution of important epitopes induced by these proteins when used as immunogens.
Structural Studies of Merozoite Surface Protein 1 (MSP1) from Plasmodium(G.A.Bentley, G. Boulot, L. Larret, J.C. Pizarro, M.M. Riottot, and B. Vulliez-le Normand in collaboration with S. Longacre (Immunologie Moléculaire des Parasites, I.P.) and F. Nato, Plateaux technique 6: Production de Protéines Recombinantes et d'Anticorps, I.P.))
Surface proteins of the merozoite, the erythrocyte-invading form of Plasmodium, are promising malaria vaccine candidates. In this regard, Merozoite Surface Protein 1 (MSP1) is one of the most widely studied of the Plasmodium surface antigens. This protein, which is implicated in the erythrocyte invasion process of the parasite, is subjected to several proteolytic cleavages during maturation of the merozoite. During the first phase, it is cleaved to yield four peptide fragments, of which the 42 kDa C-terminal fragment (MSP1-42) remains fixed to the merozoite membrane. During the second phase, occurring at the moment of erythrocyte invasion, MSP1-42 itself is cleaved to yield a polypeptide of molecular weight 11 kDa (MSP1-19). The latter cleavage is essential for erythrocyte invasion, although the mechanism of this process has yet to be elucidated. In order to understand the biological role of MSP1 in the infection of erythrocytes by Plasmodium, we are studying the structure of the recombinant fragments of this protein. Firstly, the structural comparison of MSP1-19 with MSP1-42 could help to explain the importance of the second cleavage step for the penetration of the red blood cell by the merozoite. Secondly, their 3-dimensional structure would give the spatial distribution of polymorphic (dimorphic) residues and the position of protecting epitopes, which is essential information for the optimal conception of candidate vaccine products. We have determined the structure of MSP1-19 from P. cynomolgi and MSP1-19 from P. falciparum as a complex with the Fab fragment of a specific monoclonal antibody, and studies of other Fab complexes with the falciparum homologue are in progress.
Structural studies of a monoclonal antibody specific for a ribosomal protein from T. cruzi(G.A. Bentley, G. Boulot et J.C. Pizarro, en collaboration M. Hontebeyrie, (Repliement et Modéllisation des Protéines, I.P.).
Chagas' disease, for which the etiological agent is Trypanosoma cruzi, is a tropical disease that affects many Latin American countries. Antibodies induced by the ribosomal protein, P2b, from T. cruzi appears to be associated with the phase of chronic cardiomyopathology of this disease that can lead to death. This pathology seems to be linked to the presence of antibodies specific for the C-terminal region of the ribosomal protein P2b of the parasite, of which certain can interact with peptides from the second loop of the human b1-adrenalic receptor present on cardiomyocytes.
Our objective is to study the structure of these cross-reacting antibodies and their interactions with the protein P2b and epitpoic peptides derived from the b1-adrenergic receptor. One such monoclonal antibody, 17.2, shows a positive chronotropic effect on cardiocytes in vitro via the recognition of an epitope located in the second external loop of the b1-adrenergic receptor. This system therefore seems to be a good model to study the hypothesis of the role of auto-immunity in Chagas' disease. To date, we have determined the crystal structure of Fab-17.2 as a complex both with recombinant P2b as well as with a peptide carrying the epitope from this ribosomal protein. We are attempting to crystallise Fab-17.2 as a complex with the cross-reacting epitope from the b1-adrenergic receptor.
Structural studies of FkpA, a peptidyl-prolyl isomerase of E. coli(G.A. Bentley, F. Saul and B. Vulliez-Le Normand, in collaboration with J.P. Arié and J.M. Betton, (Repliement et Modéllisation des Protéines, I.P.)).
FkpA is a peptidyl-prolyl isomerase present in the periplasmic region of E. coli. The protein is divided into two domains: an N-terminal domain of unknown function and a C-terminal domain homologous to FKBP (or FK506-binding protein) which is able to bind the immuno-suppressor ligand FK506. FkpA is a heat-shock protein that catalyses certain steps in the folding of proteins. We have determined the crystal structure of FkpA in three different forms: the complete molecule comprising 245 residues, FkpA truncated by 25 residues in the C-terminus, and truncated FkpA as a complex with FK506 (FK506 inhibits the isomerase function but not the chaperon activity). FkpA forms a dimer through the interlacing of helices in the N-terminal domain of the protein. In the structure of the complete molecule, the last 25 C-terminal residues are not visible, implying that they are disordered in the crystal. This observation lead us to produce a modified recombinant protein lacking these residues in order to improve crystal quality. The complex of truncated FkpA with FK506 shows the ligand to be bound in the same manner as in FKBP, an observation compatible with its isomerase function.
We have also determined the structure of MalE31, a mutant of MalE (or Maltose-binding protein) from E. coli that forms inclusion bodies during expression owing to a defective folding. In the presence of FkpA, however, MalE31 is correctly folded, and the crystal structure of this mutant shows that its conformation is very close to that of the wild-type protein. This result is concordant with other observations that suggest that the misfolding of MalE31 is governed by kinetic factors.
Figure 1: The dimmer of FkpA complexed with FK506 (in red).
Keywords: structural biology, X-ray crystallography, antigenic recognition, antibody structure, Plasmodium antigens
|Publications of the unit on Pasteur's references database|
|Office staff||Researchers||Scientific trainees||Other personnel|
|FEJT Françoise (email@example.com)||BENTLEY Graham, researcher I.P. (firstname.lastname@example.org)
LEMA Fernando, researcher I.P. (email@example.com)
SAUL Frederick, researcher I.P. (firstname.lastname@example.org)
|BADAUT Cyril, postdoc
PIZARRO Juan-Carlos, postdoc
IGONET Sébastien, Ph. D. student
|BOULOT Ginette, engineer, CNRS (email@example.com)
LARRET Laurent, technician (firstname.lastname@example.org)
RIOTTOT Marie-Madeleine, engineer, CNRS (email@example.com)
VULLIEZ-LE NORMAND Brigitte, engineer I.P. (firstname.lastname@example.org)