Fonctional and structural studies of hemoproteins (Anne Lecroisey, Clarisse Deniau, Nicolas Wolff, Catherine Simenel, Joël Couprie)
HasA, a 19 kDa protein, is secreted by the Gram-negative bacteria Serratia marcescens, under iron deficiency conditions. HasA has a strong affinity for haem and is able to bind free haem as well as to take it up from hemoglobin allowing bacteria to grow in the absence of any other iron sources. This protein is described as a new kind of proteinic siderophore by capturing the haem from the extracellular medium and delivering it to a specific outer membrane receptor HasR. Only haem is internalized. HasA has no homology to other known proteins excepting three other proteins recently identified in Pseudomonas aeriuginosa Pseudomonas fluorescens and Yersinia Pestis. All these proteins are secreted by a signal peptide-independent pathway which involves a C-terminal secretion signal and an ABC transporter. The main physicochemical properties of HasA, whose knowledge is essential for understanding its function, have been characterized. HasA interacts with heme in a 1:1 molar ratio and the binding occurs with high affinity Kd = 3.10-11 M. Up to now, no structural information for the heme uptake system is known. Thus, the three-dimensional structure of HasA was determined in order to clarify the mechanisms involved in heme uptake and release at the molecular level. Two approaches were used: first the apo-protein structure was determined in solution by NMR while, in first instance, to overcome the problem linked to the presence of a paramagnetic center, the holoprotein structure was determined by X-Ray cristallography. Then to obtain a more complete picture of the haem transfer mechanism each of the three important residues His32, Tyr75 and His83 involved in haem binding was mutated into Ala and theirs physicochemical properties analysed. Finally, considering the central role played by two of the histidines in haem ligation and the probable role of histidine protonation in the mechanism of haem uptake and release, the histidine protonation states in the wild type apo-protein but also in the mutants were studied.
Structure and dynamics of the Bacillus Stearothermophilus tyrosyl-tRNA synthetase C-terminal domain (Inaki Guijarro, Ada Prochnicka-Chalufour, Catherine Simenel, Muriel Delepierre)
Tyrosyl-tRNA synthetase (TyrRS) is a homodimeric protein that catalyzes 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 characterized in solution by biophysical techniques and compared with those of the full-length TyrRS and of its N-terminal fragment. These characterizations 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 suggesting that the disorder observed in the crystal structure is due to a flexible linker between the N- and C-terminal domains. 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. 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
DNA structure and protein :DNA interactions (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 modeling 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 modeling show evidences for a dynamic behavior 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 analyzed 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 emphasizing how a DNA site can play an active role in DNA-protein recognition.
Structural studies of antigenic determinants recognized by a protective monoclonal antibody in view of developping a vaccin 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 developping countries and characterized 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 recognized 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. NMR studies of conformation and dynamic of synthetic oligosaccharides representative of the O-SP of Shigella flexneri serotype 5a and peptide mimics in interaction with protective monoclonal immunoglobulin A specific for Shigella flexneri serotype 5a, were initiated by means of traansferred Nuclear Overhauser Effect as well as saturation transfert experiments.