Unit: Nuclear Magnetic Resonance of Biomolecules
Director: DELEPIERRE Muriel
Our research area is mainly dedicated to the structure determination of proteins, peptides, nucleic acids and oligosaccharides in solution in relation with their function but also to molecular interaction studies such as DNA-protein, protein-protein and ligand-macromolecules, this being developed in close collaboration with the various groups at the Pasteur Institute.
Structural and functional studies of bacterian proteins involved in heme acquisition (Célia Caillet, Nadia Izadi-Pruneyre, Anne Lecroisey, Karine Wecker, Nicolas Wolff)
Free soluble iron, an essential nutrient for microorganisms, is not readily available under biological conditions. Gram-negative bacteria bacteria have developed several ways that can coexist in the same species, to acquire iron. The presence and the use of these different acquisition systems are linked to the iron bioavailability in the host organism. Thus, under iron deficiency conditions opportunist pathogens (Serratia marcescens, Pseudomonas aeruginosa, Pseudomonas fluorescens and Yersinia pestis) secrete proteins or hemophores that allow them to acquire heme from haemoglobin. The hemophore HasA (HasA for Heme acquisition system), once in the extracellular medium, can bind free hem as well as heme bound to haemoglobin and can deliver it to a specific outer membrane receptor HasR. HasA hemophores have no homology to other known proteins and thus form a new family of proteins. In order to understand the mechanism of action of these proteins the first member of this new family HasASm (19 kDa) secreted by Serratia marcescens was studied. The three-dimensional structure of HasASm was determined by X-ray crystallography for the holo-protein and by multidimensional heteronuclear NMR for the apo-protein. Three residues were found to be involved in heme binding: two histidines and a tyrosine. One of the histidine is not directly bound to the iron. Then, to elucidate the role of each of these residues in the mechanism of heme uptake and release, the histidine protonation state was evaluated through pKa measurements in the absence and in the presence of heme, and the three important residues for heme binding was mutated into alanine either one by one or two simultaneously. A triple mutant was also constructed. The physico-chemical properties of all of these mutants were analysed in terms of stability, heme binding and conformational properties. This allowed us to put forward few hypotheses on the mechanism of heme uptake and release that need to be tested now (collaborations: Unité des membranes bactériennes, Institut Pasteur ; AFMB & BIP, CNRS Marseille ; Faculté de Pharmacie, Marseille. Department of Microbiology & Immunology, Emory, University, Atlanta, USA, University of Florence, Italy).
Three-dimensional structure of toxins (Karine Frénal, Manel Ben Aissa, Iñaki Guijarro, Muriel Delepierre, Karine Wecker)
Structural studies of ionic channels were rendered possible through the use of specific ligand of each of the channel. Therefore, the finding of molecules that selectively block a given class of channel is very helpful. Channel ligands are mainly isolated from the venom of vertebrate and/or invertebrate and represent powerful and nice tools for ion channels characterisation. Although the molecular basis of toxin specificity towards species and/or ionic channels has been widely studied, much knowledge is still needed to fully understand their structural and functional characteristics. It is therefore very important to continue to characterize toxins soon these are discovered not only due to the huge public health problem caused by venomous animals stings but also because a large number of new ionic channels are without pharmacology due to the lack of specific ligand. We have determined the structure of three toxins: (i) Pi4 containing 4 disulfide bridges (collaboration Lourival Possani Mexique et Jean-Marc Sabatier Marseille) (ii) the erg toxin that forms a new sub family and possesses a new mode of action (collaboration Lourival Possani) (iii) finally Pa9 a non peptidic toxin and the first molecule active on 2P potassium channels (collaboration Michel Ladzunski Nice). We are investigating now the structure of sodium like toxin acting on potassium channels (collaboration Tunis Pasteur Institute Mohammed El Ayeb).
Structural studies of antigenic determinants recognised by a protective monoclonal antibody in view of developing a vaccine against shigellosis (Marie-Jeanne Clement, Ada Prochnicka-Chalufour, Catherine Simenel, Muriel Delepierre)
Shigella is a Gram-negative bacterium responsible for shigellosis, a dysenteric syndrome causing a high rate of mortality among infant in developing countries and characterised by bacterial invasion of the human colonic mucosa. Shigellosis is thus priority target as defined by the World Health Organisation in its program for the development of vaccines against enteric diseases. Lipopolysaccharide (LPS) and some secreted protein antigens are the major targets of the systemic as well as local humoral immune responses. It has been shown that protection against Shigella infection lies essentially in the local humoral response directed against the O-specific polysaccharide (O-SP). Furthermore, the antibodies conferring this protection are specific for the serotype of Shigella strain defined by the structure of the O-SP. Therefore, a possible strategy for human vaccination is to develop synthetic chemically defined vaccines with simple molecules able to mimic the O-SP and induce then the synthesis of protective antibodies. Two possibilities can be considered. The first one consists in using synthetic oligosaccharides representative of carbohydrate epitopes recognised by protective antibodies. The second one consists in characterising peptide sequences mimicking the protective epitopes, by screening phage-displayed libraries with protective antibodies. Development of either of these options requires a structural study of interaction of peptides with protective antibodies in order to help the design of optimal vaccine. The conformations of oligosaccharides and peptides in interaction with protective monoclonal have been obtained by means of transferred Nuclear Overhauser Effect experiments while the epitopes have been characterised by saturation transfer experiments.
Structure of the dengue virus envelope protein domain III (Iñaki Guijarro Collaboration Hugues Bedouelle)
Infection by the dengue virus is a major public health problem requiring development of antiviral drugs. The murin antibody 4E11 is able to neutralise target cell infection by the four known virus serotypes. The ED protein recognition mechanism of the four virus serotypes by the 4E11 antibody is under investigation in order to increase is protecting power. The antibodies recognizes domain III of the major envelope glycoprotein (ED). We have started the structural study of domain ED-III first to better understand the interaction between this domain and the 4E11 antibody and then to study the interaction of domain III with heparan located at the target cell surface. We have shown by NMR that a hybrid containing domain III of the recombinant protein ED-III (serotype 1) and the maltose binding protein (MalE) leads to a globular and well-structured domain III.
Structure-function studies a DNA repair enzyme expressed by the protozoan parasite Toxoplasma gondii the protein TgDRE, (Karine Frénal, Nicolas Wolff)
The protozoan Toxoplasma gondii is an opportunistic pathogen from the same phylum as Plasmodium falciparum and responsible of toxoplasmose in human. Infection is asymptomatic in most cases although the parasite persists during the lifetime as an encysted latent form in the brain and other organs. When, the protective immunity fails (cancer, sida, graft), the quiescent form can transform into actively replicative and virulent form that can lead to death. Interconversion between these two forms is important in the infection reactivation. It would appear that certain stimuli such as nitrogen oxide or interferon γwould play a role in this process probably inducing a stress for the parasite and causing damage to DNA. A DNA repair enzyme has been recently identified in Toxoplasma gondii and named TgDRE (Toxoplasma gondii DNA Repair Enzyme). It is suggested that this protein would be involved in the interconversion of the parasite two forms. The protein belongs to a large family of proteins containing RNA recognition motifs (RRM) glycine rich motifs, G-patch and a specific motif named SF45 because of its similarity to the human splicing factor 45 protein, which was described as a component of the spliceosome. We have expressed proteins of mono and multidomains (7 kDa to 25 kDa) to determine their structure by NMR and understand their function (Collaborations Laboratoire de Minéralogie-Cristallographie Paris, DIEP, CEA-Saclay and Unité Parasitologie Moléculaire, Université de Lille)
Structural Genomic of Mycobacterium tuberculosis and Mycobacterium leprae (Ada Prochnicka-Chalufour, Iñaki Guijarro Muriel Delepierre)
The Pasteur Institute is involved in a large project of functional and structural genomic of Mycobacterium tuberculosis, and Leprae genomes a project coordinated by Stewart Cole with the aim of developing new drugs to fight tuberculosis and leprae. Indeed, leprae human neuronal disease chronicle is caused by an infection with an intracellular pathogen agent, Mycobacterium leprae (M. leprae), very close to the Mycobacterium tuberculosis (M. tuberculosis). M. leprae and M. tuberculosis genomes are completely sequenced today. They share more then 80 % identity and contain gene specific to each of them, 136 and 470 respectively for M. leprae and M. tuberculosis. Among those 156 have unknown functions. The study of M. leprae specific genes should allow a better understanding of pathogenicity, in particular neurologic, and could provide diagnostic tools for skin test of the disease, quite valuable for an early detection. Then, new target for antibiotics and development of new drugs to avoid neuropathies could be envisaged as well as development of a vaccine through identification of functional protein involved in immunity. Among the 156 genes of unknown function we have chosen those coding proteins of less than 150 amino acids to determine the structure of these proteins by heteronuclear multidimensional NMR (Collaborations Stewart Cole, Jean-Michel Betton et Pedro Alzari).
Keywords: Biophysics, NMR, structure, interaction, biomolecules, molecular modelling