Nucleic Acids Res

Site-directed spin labeling is emerging as an essential tool to investigate the structural and dynamical features of RNA. We propose here an enzymatic method, which allows the insertion of a paramagnetic center at a specific position in an RNA molecule. The technique is based on a segmental approach using a ligation protocol with T4 RNA ligase 2. One transcribed acceptor RNA is ligated to a donor RNA in which a thio-modified nucleotide is introduced at its 5'-end by in vitro transcription with T7 RNA polymerase. The paramagnetic thiol-specific reagent is subsequently attached to the RNA ligation product. This novel strategy is demonstrated by introducing a paramagnetic probe into the 55 nucleotides long RNA corresponding to K-turn and Specifier Loop domains from the Bacillus subtilis tyrS T-Box leader RNA. The efficiency of the coupling reaction and the quality of the resulting spin-labeled RNA were assessed by Mass Spectrometry, Electron Paramagnetic Resonance (EPR) and Nuclear Magnetic Resonance (NMR). This method enables various combinations of isotopic segmental labeling and spin labeling schemes, a strategy that will be of particular interest to investigate the structural and dynamical properties of large RNA complexes by NMR and EPR spectroscopies

Self-Association of an Activating Natural Killer Cell Receptor, KIR2DS1

As a major component of the innate immune system, natural killer cells are responsible for activating the cytolytic killing of certain pathogen-infected or tumor cells. The self-recognition of natural killer cells is achieved in part by the killer cell immunoglobulin-like receptors (KIRs) protein family. In the current study, using a suite of biophysical methods, we investigate the self-association of an activating KIR, KIR2DS1. This KIR is of particular interest because when in the presence of the HLA-Cw6 protein, KIR2DS1 becomes a major risk factor for psoriasis, an autoimmune chronic skin disease. Using circular dichroism spectroscopy, dynamic light scattering, and atomic force microscopy, we reveal that KIR2DS1 self-associates in a well-defined fashion. Our novel results on an activating KIR allow us to suggest a working model for the KIR2DS1- HLA class I molecular mechanism

Etude structurale par résonance magnétique nucléaire de deux protéines du VIH-1 interagissant avec les acides nucléiques, Vpr et NCp7

Le cycle de réplication du VIH l'interaction de protéines virales avec l'ARN et l'ADN viraux. Nous avons étudié l'interaction entre Vpr ou la NCp7 et des petits oligonucleotides et le primer binding site (PBS), respectivement. Le PBS forme une tige-boucle et interagit avec la NCp7 lors de la transcription inverse, induisant un transfert de brin. Notre étude montre que la NCp7 glisse entre ses deux sites de liaison sur le PBS, déstabilisant ainsi sa structure pour faciliter le transfert de brin. Vpr interagit avec l'ADN viral et participe à son import dans le noyau des cellules. Après avoir déterminé la structure en "leucine zipper" de Vpr(52-96), nous avons montré par RMN, fluorescence et Biacore une interaction entre Vpr(52-96) et les acides nucléiques. La résolution des éléments importants pour l'interaction entre la NCp7 et Vpr et leurs cibles nucléotidiques, pourrait être utilisée dans le cadre de la recherche d'agents anti-viraux.HIV replication cycle requires viral protein interactions with viral RNA and DNA. We studied the interaction between Vpr, regulation protein and NCp7, nucleocapsid protein and short oligonucleotides and Primer Binding Site (PBS) respectively. The PBS structures as a stem loop and interacts with the NCp7 during reverse transcription. This interaction induces strand transfer. Our study proves the capacity f NCp7 to slide between its two binding sites on the PBS, thus opening its secondary structure, which is necessary to the strand transfer. Vpr interacts with viral DNA and facilitates its import in cell nucleus. Having determined a "leucine zipper" dimer structure of Vpr(52-96), we showed by NMR, fluorescence and surface plasmon resonance an interaction between Vpr(52-96) and nucleic acids. Finding out structural features necessary for the interaction between the NCp7 and Vpr and their nucleotidic targets could be used to design new antiviral agentsPARIS-BIUP (751062107) / SudocSudocFranceF

How the HIV-1 Nucleocapsid Protein Binds and Destabilises the (−)Primer Binding Site During Reverse Transcription

International audienceThe human immunodeficiency virus type 1 nucleocapsid protein (NCp7) plays an important role in the second strand transfer during reverse transcription. It promotes annealing of the 18-nucleotide complementary DNA primer-binding site (PBS) sequences at the 3' ends of (-)DNA and (+)DNA. NMR studies show that NCp7(12-55) and NCp7(1-55) interact at the 5' end of the loop of DeltaP(-)PBS, a (-)PBS derivative without the 3' protruding sequence, in a slow-exchange equilibrium. This interaction is mediated through the binding of the hydrophobic plateau (Val13, Phe16, Thr24, Ala25, Trp37, and Met46) on the zinc finger domain of both peptides to the 5-CTG-7 sequence of DeltaP(-)PBS. The stacking of the Trp37 aromatic ring with the G7 residue likely constitutes the determinant factor of the interaction. Although NCp7(12-55) does not melt the DeltaP(-)PBS stem-loop structure, it opens the loop and weakens the C5.G11 base pair next to the loop. Moreover, NCp7(12-55) was also found to bind but with lower affinity to the 10-CGG-12 sequence in an intermediate-exchange equilibrium on the NMR time scale. The loop modifications may favour a kissing interaction with the complementary (+)PBS loop. Moreover, the weakening of the upper base pair of the stem likely promotes the melting of the stem that is required to convert the kissing complex into the final (+/-)PBS extended duplex

The C-terminal domain of the HIV-1 regulatory protein Vpr adopts an antiparallel dimeric structure in solution via its leucine-zipper-like domain

HIV-1 Vpr is a highly conserved accessory protein that is involved in many functions of the virus life cycle. Vpr facilitates the entry of the HIV pre-integration complex through the nuclear pore, induces G2 cell cycle arrest, regulates cell apoptosis, increases transcription from the long terminal repeat and enhances viral replication. Vpr contains a Leu/Ile-rich domain (amino acids 60–81) in its C-terminal part, which is critical for dimerization. The sequence comprising residues 52–96 is implicated in properties of the protein such as DNA interaction and apoptosis via interaction with the adenine nucleotide translocator. To understand the specific interactions of Vpr-(52–96), the ability of this peptide to dimerize via a leucine-zipper mechanism has been investigated, by NMR and fluorescence spectroscopy. In contrast with results from a study performed in the presence of trifluoroethanol, our results, obtained in 30% (v/v) [(2)H]acetonitrile, show that Vpr-(52–96) in solution still forms an α-helix spanning residues 53–75, but dimerizes in an antiparallel orientation, through hydrophobic interactions between leucine and isoleucine residues and stacking between His(71) and Trp(54). Moreover, to demonstrate the physiological relevance of the dimer structure, fluorescence spectroscopy experiments have been performed in a Mes buffer, which confirmed the formation of the dimer in aqueous solution and highlighted the spatial proximity between Trp(54) and His(71). Surprisingly, the leucine-zipper structure shown in the present work for Vpr-(52–96) mimics the structure of full-length Vpr-(1–96), and this could explain why some of the properties of Vpr-(52–96) and Vpr-(1–96) are identical, while some are even enhanced for Vpr-(52–96), particularly in the case of DNA transfection experiments