Structural and dynamic characterization of the upper part of the HIV-1 cTAR DNA hairpin

First strand transfer is essential for HIV-1 reverse transcription. During this step, the TAR RNA hairpin anneals to the cTAR DNA hairpin; this annealing reaction is promoted by the nucleocapsid protein and involves an initial loop–loop interaction between the apical loops of TAR and cTAR. Using NMR and probing methods, we investigated the structural and dynamic properties of the top half of the cTAR DNA (mini-cTAR). We show that the upper stem located between the apical and the internal loops is stable, but that the lower stem of mini-cTAR is unstable. The residues of the internal loop undergo slow motions at the NMR time-scale that are consistent with conformational exchange phenomena. In contrast, residues of the apical loop undergo fast motions. The lower stem is destabilized by the slow interconversion processes in the internal loop, and thus the internal loop is responsible for asymmetric destabilization of mini-cTAR. These findings are consistent with the functions of cTAR in first strand transfer: its apical loop is suitably exposed to interact with the apical loop of TAR RNA and its lower stem is significantly destabilized to facilitate the subsequent action of the nucleocapsid protein which promotes the annealing reaction

An Unusual Helix Turn Helix Motif in the Catalytic Core of HIV-1 Integrase Binds Viral DNA and LEDGF

Background: Integrase (IN) of the type 1 human immunodeficiency virus (HIV-1) catalyzes the integration of viral DNA into host cellular DNA. We identified a bi-helix motif (residues 149–186) in the crystal structure of the catalytic core (CC) of the IN-Phe185Lys variant that consists of the a 4 and a 5 helices connected by a 3 to 5-residue turn. The motif is embedded in a large array of interactions that stabilize the monomer and the dimer. Principal Findings: We describe the conformational and binding properties of the corresponding synthetic peptide. This displays features of the protein motif structure thanks to the mutual intramolecular interactions of the a4 and a5 helices that maintain the fold. The main properties are the binding to: 1- the processing-attachment site at the LTR (long terminal repeat) ends of virus DNA with a Kd (dissociation constant) in the sub-micromolar range; 2- the whole IN enzyme; and 3- the IN binding domain (IBD) but not the IBD-Asp366Asn variant of LEDGF (lens epidermal derived growth factor) lacking the essential Asp366 residue. In our motif, in contrast to the conventional HTH (helix-turn-helix), it is the N terminal helix (a 4) which has the role of DNA recognition helix, while the C terminal helix (a 5) would rather contribute to the motif stabilization by interactions with the a4 helix. Conclusion: The motif, termed HTHi (i, for inverted) emerges as a central piece of the IN structure and function. It coul

Structural insights into the cTAR DNA recognition by the HIV-1 nucleocapsid protein: role of sugar deoxyriboses in the binding polarity of NC

An essential step of the reverse transcription of the HIV-1 genome is the first strand transfer that requires the annealing of the TAR RNA hairpin to the cTAR DNA hairpin. HIV-1 nucleocapsid protein (NC) plays a crucial role by facilitating annealing of the complementary hairpins. Using nuclear magnetic resonance and gel retardation assays, we investigated the interaction between NC and the top half of the cTAR DNA (mini-cTAR). We show that NC(11-55) binds the TGG sequence in the lower stem that is destabilized by the adjacent internal loop. The 5′ thymine interacts with residues of the N-terminal zinc knuckle and the 3′ guanine is inserted in the hydrophobic plateau of the C-terminal zinc knuckle. The TGG sequence is preferred relative to the apical and internal loops containing unpaired guanines. Investigation of the DNA–protein contacts shows the major role of hydrophobic interactions involving nucleobases and deoxyribose sugars. A similar network of hydrophobic contacts is observed in the published NC:DNA complexes, whereas NC contacts ribose differently in NC:RNA complexes. We propose that the binding polarity of NC is related to these contacts that could be responsible for the preferential binding to single-stranded nucleic acids

Structure and Function of Adsorbed Hemoglobin on Silica Nanoparticles: Relationship between the Adsorption Process and the Oxygen Binding Properties

International audienceThe connection between the mechanisms of protein adsorption on nanoparticles and the structural and functional properties of the adsorbed protein often remains unclear. We investigate porcine hemoglobin adsorption on silica nanoparticles and we analyze the structural and functional modifications of adsorbed hemoglobin by UV-vis spectrophotometry, circular dichroism and oxygen binding measurement. The structural analysis of adsorbed hemoglobin on silica nanoparticles reveals a significant loss of secondary structure and a preservation of the heme electronic structure. However, adsorbed hemoglobin retains its quaternary structure and exhibits an enhanced oxygen affinity with cooperative binding. Moreover, the structural and functional modifications are fully reversible after complete desorption from silica nanoparticles at pH 8.7. The tunable adsorption and desorption of hemoglobin on SNPs with pH change, and the full control of hemoglobin activity by pH, temperature and the addition of inorganic phosphate effectors opens the way to an interesting system whereby protein adsorption on nanoparticles can allow for full control over hemoglobin oxygen binding activity. Our results suggest that adsorption of hemoglobin on silica nanoparticles leads to a new structural, functional and dynamic state with full reversibility in a way that significantly differs from protein denaturation. TEX

1H, 13C and 15N backbone and partial side-chain resonance assignments of the C-terminal domain of HIV-1 Pr55Gag encompassed in NCp15

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Structural and dynamic characterization of the upper part of the HIV-1 cTAR DNA hairpin. Nucleic Acids Res

First strand transfer is essential for HIV-1 reverse transcription. During this step, the TAR RNA hairpin anneals to the cTAR DNA hairpin; this annealing reaction is promoted by the nucleocapsid protein and involves an initial loop–loop interaction between the apical loops of TAR and cTAR. Using NMR and probing methods, we investigated the structural and dynamic properties of the top half of the cTAR DNA (mini-cTAR). We show that the upper stem located between the apical and the internal loops is stable, but that the lower stem of mini-cTAR is unstable. The residues of the internal loop undergo slow motions at the NMR time-scale that are consistent with conformational exchange phenomena. In contrast, residues of the apical loop undergo fast motions. The lower stem is destabilized by the slow interconversion processes in the internal loop, and thus the internal loop is responsible for asymmetric destabilization of mini-cTAR. These findings are consistent with the functions of cTAR in first strand transfer: its apical loop is suitably exposed to interact with the apical loop of TAR RNA and its lower stem is significantly destabilized to facilitate the subsequent action of the nucleocapsid protein which promotes the annealing reaction

The intrinsic mechanics of B-DNA in solution characterized by NMR

International audienceExperimental characterization of the structural couplings in free B-DNA in solution has been elusive, because of subtle effects that are challenging to tackle. Here, the exploitation of the NMR measurements collected on four dodecamers containing a substantial set of dinucleotide sequences provides new, consistent correlations revealing the DNA intrinsic mechanics. The difference between two successive residual dipolar couplings (RDCs) involving C6/8-H6/8, C3-H3 and C4-H4 vectors are correlated to the 31 P chemical shifts (␦P), which reflect the populations of the BI and BII backbone states. The ␦Ps are also correlated to the internucleotide distances (D inter) involving H6/8, H2 and H2 protons. Calculations of NMR quantities on high resolution X-ray structures and controlled models of DNA enable to interpret these couplings: the studied RDCs depend mostly on roll, while D inter are mainly sensitive to twist or slide. Overall, these relations demonstrate how ␦P measurements inform on key inter base parameters, in addition to probe the BI↔BII backbone equilibrium , and shed new light into coordinated motions of phosphate groups and bases in free B-DNA in solution. Inspection of the 5 and 3 ends of the dode-camers also supplies new information on the fraying events, otherwise neglected