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  Director : DELEPIERRE Muriel (murield@pasteur.fr)



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 proteines involved in heme acquisition (Clarisse Deniau, Nadia Izadi-Pruneyre, Nicolas Wolff, Anne Lecroisey)

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 bio-availability 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 (Figure 1) 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. To obtain a more complete picture of the heme transfer mechanism each of 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).

Structure and dynamics of the Bacillus Stearothermophilus tyrosyl-tRNA synthetase C-terminal domain(Inaki Guijarro, Ada Prochnicka-Chalufour, Muriel Delepierre)

Tyrosyl-tRNA synthetase (TyrRS) is a homodimeric protein that catalyses both the activation of the amino acid through its reaction with ATP and the transfer of the aminoacyl-adenylate to the tRNA(tyr). In Bacillus stearothermophilus, each subunit of TyrRS comprises two structural domains, an N-terminal domain (residues 1-319), whose crystal structure is known, and a C-terminal domain (residues 320-419), which appears disordered in the crystals. The binding site of one tRNA-Tyr molecule encompasses both subunits of the TyrRS dimer. The folding state of the C-terminal fragment was characterised in solution by biophysical techniques and compared with those of the full-length TyrRS and of its N-terminal fragment. These characterisations showed that the C-terminal domain was in a condensed, stable, defined state of folding, and that it had similar structures in its isolated form and in the full-length TyrRS.

The 3-dimensional structure and the dynamics of a recombinant protein TyrRS(D4) corresponding to the C-terminal domain of tyrRS was solved by heteronuclear NMR spectroscopy (Figure 2). The TyrRS(D4) structure exhibits a novel fold among the anticodon binding domains of aminoacyl-tRNA synthetases, around two thirds of which appear to be shared with the ribosomal protein S4 and the heat shock protein Hsp15. The common topology involves two a helices packed against an antiparallel b sheet (figure 2). Of six basic residues identified by site-directed mutagenesis as essential for tRNA binding, four are clustered in this domain and are likely to interact with the anticodon arm of tRNA. It is the first structure described for an anticodon-arm-binding domain of a tyrosyl-tRNA synthetase and it completes the structure of the B. stearothermophilus enzyme. Finally 15N-derived order parameters and previous data suggest that the disorder observed in the crystal structure is due to a flexible linker between the N- and C-terminal domains.

The role of BI/BII transition in DNA protein recognition (Karine Wecker, Muriel Delepierre)

NF-kB is involved in the transcriptional regulation of a large number of genes including those of HIV. 1H and 31P NMR spectroscopies have been used together with molecular modelling to determine the fine structure of non-palindromic 16bp DNA duplex containing the HIV-1 kB binding site (16N) together with related mutated sequences (16M1 and 16M2). The 16M duplexes correspond either to mutation of the highly conserved GGG tract (16M1) that is within the kB site into CTC or to mutation of the kB site flanking sequences (16M2) leaving intact the kB site but abolishing the phosphorous BI/BII transition observed in the native duplex (16N). For the native duplex (16N) NMR data and modelling show evidences for a dynamic behaviour of steps flanking the ten base pairs of the NF-kB binding site. A BI-BII equilibrium at these steps is demonstrated and two models for each extreme conformation are proposed in agreement with NMR data. In the refined BII structures, the NF-kB binding site exhibits an intrinsic curvature towards the major groove with the base pairs translated into the major groove. This dynamic intrinsic curvature is in agreement with the DNA curvature observed in the x-ray structure of the P50-DNA complex. While complete loss of binding with NF-kB is observed with the16M1 duplex, the 16M2 duplex, for which the kB site remains intact, still binds to NF-kB but with a decreased affinity as measured by electrophoretic mobility shift assays (collaboration E. Meurs). The decrease or/and the lack of binding are interpreted in relation to the fine solution structure of these duplexes. Overall intrinsic properties of these duplexes were analysed for each family of structures in terms of electrostatic potentials, accessible surfaces energetic and structural effects. It is found that the native sequence (16N) exhibits characteristics that are found neither in the mutated sequences nor in the other DNA fragments already studied. A new recognition mode of kB sites by NF-kB is proposed emphasising how a DNA site can play an active role in DNA-protein recognition.

Structural studies of antigenic determinants recognised by a protective monoclonal antibody in view of developing a vaccine against shigellosis(Marie-Jeanne Clément, 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. While 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.

Photos :

Photo 1: Structure X-ray of holo-HasA from Serratia marcescens.

Photo 2: NMR Structure of the tyrosyl-tRNA synthetase C-terminal domaine


puce Publications of the unit on Pasteur's references database


  Office staff Researchers Scientific trainees Other personnel

LOZANO Liliane llozano@pasteur.fr
Tel : 01 40 61 37 02
Fax : 01 45 68 89 29

DELEPIERRE Muriel, CNRS murield@pasteur.fr

GUIJARRO Inaki, IP guijarro@pasteur.fr

IZADI-PRUNEYRE Nadia, CNRS nizadi@pasteur.fr

LECROISEY Anne, IP alecrois@pasteur.fr

WOLFF Nicolas, IP wolff@pasteur.fr

CLEMENT Marie-Jeanne, thèse mjeanne@pasteur.fr

DENIAU Clarisse, thèse cdeniau@pasteur.fr

STOVEN Véronique, CR1 CNRS, vstoven@pasteur.fr

WECKER Karine, post-doc wecker@pasteur.fr


SIMENEL Catherine, IP simenel@pasteur.fr


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