Unit: Nuclear Magnetic Resonance of Biomolecules - URA CNRS 2185
Director: DELEPIERRE Muriel
Our research is mainly dedicated to the determination of structures of proteins, peptides, nucleic acids and oligosaccharides in solution in relation with their function as well as to the study of molecular interactions between partners such as DNA-protein, protein-protein and ligand-macromolecule. We work 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, Julien Lefrèvre, 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, conformational as well as paramagnetic 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).
A dimeric form of HasASm, HasAD, also present has been characterised and its structure determined (collaboration Mirjam Czjzek station biologique de Roscoff). The tertiary structure of each monomer in the dimer is similar to that in the monomer, however the 2-49 polypeptide fragment is exchanged in the two polypeptidique chains " domain swapping " (figure 1). One of the heme ligand (His32) belongs to the exchanged fragment and thus could contribute to the decrease of affinity for heme as compared to that of the monomer (9. 108 M-2 vs 5,3 1010M-1). Nevertheless, the affinity remains higher than that observed for most of the heme transporters present in the host. Therefore, HasAD could be a heme reservoir in the hemophore system.
If S. marcescens Has system is now well characterized at a molecular level, the different steps for heme acquisition and internalisation remain unknown. HasASm binds free heme or hemoprotein bound heme with a very high affinity and then delivers it to the specific outer membrane receptor HasR that binds heme with a much lower affinity. Recently, we have shown that a complex is formed between HasA and HasR and that heme transfer was energy free. However, heme internalisation, apo-hemophore release and signal transduction leading to system regulation, all require the energy given by the internal membrane protein HasB. HasB is homologous to TonB a protein providing the energy derived from the proton motrice force and necessary to the functioning of a wide range of external membrane receptors homologous to HasR. While HasB and HasR share 25% sequence identity and are functionally partially redundant, HasB is specific of heme acquisition via the hemophore. Molecular basis of this specificity are not known. The aim here is to understand HasB specificity in the Has system via the structure determination in solution of its periplasmic region and to compare it to that of TonB. The HasB periplasmic domain corresponding to the 130 C-terminal residues has been cloned, expressed and isotopicaly labelled and its structure determination started (Collaboration : Unité des Membranes bactériennes Institut Pasteur)
Structural studies of antigenic determinants recognised by a protective monoclonal antibody in view of developing a vaccine against shigellosis (François Theillet, 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. These studies are now extended to the epitope caractérisation of serotype 2a (collaboration with Unités de Pathologie Microbienne and Chimie Organique).
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)
Structure and function of transcription regulators from hyperthermophile-Archaea viruses (Florence Guillière, Iñaki Guijarro)
Hyperthermophile-archaea viruses show an exceptional diversity and are very different from viruses that infect bacteria and eukaryotes. The particular position of their hosts in evolution, their diversity and their differences relative to all other organisms (90% of their predicted proteins do not show any homologue in databases), render the study of these viruses very attractive. We started a collaboration with the group of D. Prangishvili (Molecular Biology of Extremophiles) to study the structure and function of proteins that could be involved in transcription regulation. The current knowledge on transcription in archaea is still rudimentary, when compared to what is known for bacteria and eukaryotic organisms. Interestingly, the basal transcription machinery of archaea is a simplified version of that of eukaryotes (RNA polymerase and associated TBP, TFB, and TFE factors) but transcription regulators resemble those found in bacteria. This project is developed within the frame of a larger project aimed at understanding Hyperthermophile-archaea viruses and their relations with their hosts.
We started this project in 2005 working with two proteins. The first one, called D56, belongs to virus SIRV1 (" Sulfolobus islandicus rudivirus 1 "). This protein is expressed throughout the infection cycle of the virus and interacts specifically with the regions of its own promoter and of the promoter of a neighboring gene. The second protein, Sta1, is from the host of SIRV1 (S. islandicus). Sta1, which is expressed under stress conditions, stimulates the transcription of SIRV1 genes upon infection. We established a model of the structure of Sta1 and we are currently refining the structure of D56 to study its interaction with its cognate DNA.
Tridimensionnal structure of the dimeric HasASm determined by X-ray diffraction the polypeptide fragment interghanged are indicated with color
Keywords: Biophysics, NMR, structure, interaction, biomolecules, molecular modelling