A new NMR solution structure of the SL1 HIV-1(Lai) loop–loop dimer

Dimerization of genomic RNA is directly related with the event of encapsidation and maturation of the virion. The initiating sequence of the dimerization is a short autocomplementary region in the hairpin loop SL1. We describe here a new solution structure of the RNA dimerization initiation site (DIS) of HIV-1(Lai). NMR pulsed field-gradient spin-echo techniques and multidimensional heteronuclear NMR spectroscopy indicate that this structure is formed by two hairpins linked by six Watson–Crick GC base pairs. Hinges between the stems and the loops are stabilized by intra and intermolecular interactions involving the A8, A9 and A16 adenines. The coaxial alignment of the three A-type helices present in the structure is supported by previous crystallography analysis but the A8 and A9 adenines are found in a bulged in position. These data suggest the existence of an equilibrium between bulged in and bulged out conformations in solution

NMR studies of telomeric nucleoprotein complexes involving the Myb-like domain of the human telomeric protein TRF2

In order to study the binding of the Myb-like domain of the human telomeric protein TRF2 (Myb-TRF2) with different structural components of the t-loop model, we report NMR studies of the binding of Myb-TRF2 protein with two repeats human telomeric DNA under three conformations. Our results showed that Myb-TRF2 binds to the duplex and even to the quadruplex and the random coil G-rich strand. The solution structure of Myb-TRF2 reported here looks like Myb-TRF1 suggesting similar DNA binding mode. As a matter of fact, we have shown that its binding to the human telomeric duplex presents great similarities with this of Myb-TRF1

Toll-Like Receptor 3 (TLR3) Plays a Major Role in the Formation of Rabies Virus Negri Bodies

Human neurons express the innate immune response receptor, Toll-like receptor 3 (TLR3). TLR3 levels are increased in pathological conditions such as brain virus infection. Here, we further investigated the production, cellular localisation, and function of neuronal TLR3 during neuronotropic rabies virus (RABV) infection in human neuronal cells. Following RABV infection, TLR3 is not only present in endosomes, as observed in the absence of infection, but also in detergent-resistant perinuclear inclusion bodies. As well as TLR3, these inclusion bodies contain the viral genome and viral proteins (N and P, but not G). The size and composition of inclusion bodies and the absence of a surrounding membrane, as shown by electron microscopy, suggest they correspond to the previously described Negri Bodies (NBs). NBs are not formed in the absence of TLR3, and TLR3−/− mice—in which brain tissue was less severely infected—had a better survival rate than WT mice. These observations demonstrate that TLR3 is a major molecule involved in the spatial arrangement of RABV–induced NBs and viral replication. This study shows how viruses can exploit cellular proteins and compartmentalisation for their own benefit

Séminaire invité : Unraveling the interaction between the capsular polysaccharide of Streptococcus pneumoniae type 6B and the anti-6B Monoclonal Antibody using an NMR approach

International audienc

Intramolecular oximercuration-demercuration reaction: a new stereocontrolled approach to sialic acid containing disaccharides

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NMR Studies of lac Operator and lac Repressor

International audienceFor many years, and even now, the lac operon of Escherichia coli has been a model for gene regulation. The lac repressor is a tetrameric protein that binds strongly the lac operator and the affinity of which is modulated by an inducer molecule. The lac repressor–lac operator complex was the first nucleic acid–protein complex studied with success by NMR. The NMR studies began in 1973 and gave publications until now. 1H, 13C, 15N, 19F, 31P NMR and photo-CIDNP studies increased progressively the level of understanding of the system during these decades. At first, it appeared to be possible to duplicate the basic lac repressor–lac operator interaction by using a smaller protein: the lac repressor N-terminal fragment. In 1987, the Kaptein's group reported the first model of interaction where the motif helix-turn-helix plays a central role in the recognition process of the large groove of the operator by the protein. Later, heteronuclear NMR studies refined the NMR solution structure of both free protein and complexed protein. 13C and 15N relaxation rate measurements showed dynamic processes in both nucleic acid and protein moieties during the complexation. By studying a larger headpiece (1-62) Kaptein's group showed the fundamental role played by the hinge helix, which interacts, in the minor groove of the DNA. The protection factor of a dimeric lac repressor headpiece mutant, which has a high affinity for DNA, was measured and used to qualify the folding of this protein during the complexation. The role played by the asymmetry of the lac operator was studied and explained. NMR studies allowed proposing a scheme to explain the loss in affinity of the lac repressor for its operator when binding to an inducer molecule. Comparison of these data with crystal structures showed that, until now, the more detailed view of the lac repressor–lac operator interaction comes from NMR studies. The NMR solution structures and the dynamic processes revealed by relaxation are in agreement with all the genetic and biochemical data

A new stereocontrolled approach to KDO containing disaccharides

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Etude de complexes télomériques par résonance magnétique nucléaire

Les télomères sont des complexes nucléoprotéiques localisés à l extrémité des chromosomes des cellules eucaryotes. L ADN télomérique humain contient entre 500 et 3000 répétitions de la séquence d(T2AG3)n (brin-G) et de son brin complémentaire C3TA2 (brin-C). Le brin G est terminé par une extrémité simple brin contenant de 50 à 200 nucléotides. Les télomères jouent des rôles essentiels au niveau cellulaire en permettant la protection des chromosomes. Un modèle proposé pour expliquer le rôle protecteur des télomères est le modèle de la boucle-T (télomérique) / boucle-D (déplacement). Dans ce modèle, on observe le repliement de l ADN télomérique double brin et l insertion de la partie simple brin dans la partie double brin. La formation de ces boucles fait intervenir, directement ou indirectement, de nombreuses protéines comme les protéines TRF1 et TRF2. Néanmoins les mécanismes moléculaires et notamment le rôle de la protéine TRF2 dans la formation de ces boucles restent spéculatifs. Le travail de thèse présenté dans ce manuscrit expose l étude par Résonance Magnétique Nucléaire du domaine de fixation à l ADN de la protéine TRF2 (domaine Myb) et de ses interactions avec l ADN télomérique ainsi que l étude de modèles de boucles-D. Après la détermination de la structure tridimensionnelle et l étude de la dynamique interne du domaine Myb de TRF2, nous avons réalisé l étude des interactions du domaine Myb avec l ADN télomérique. Ainsi, deux complexes entre le domaine Myb de TRF2 et l ADN télomérique ont été étudiés par RMN et ont permis de mieux comprendre le rôle du domaine Myb en montrant sa grande spécificité pour l ADN télomérique double brin. Ensuite quatre modèles de boucles D différents ont été étudiés par RMN afin d en déterminer les structures tridimensionnelles.ORLEANS-BU Sciences (452342104) / SudocSudocFranceF