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Site-selective probing of cTAR destabilization highlights the necessary plasticity of the HIV-1 nucleocapsid protein to chaperone the first strand transfer.

Godet J, Kenfack C, Przybilla F, Richert L, Duportail G, Mély Y - Nucleic Acids Res. (2013)

Bottom Line: NC(11-55), a truncated NCp7 version corresponding to its zinc-finger domain, was found to bind all over the sequence and to preferentially destabilize the penultimate double-stranded segment in the lower part of the cTAR stem.Sequence comparison further revealed that C•A mismatches close to the two G residues were critical for fine tuning the stability of the lower part of the cTAR stem and conferring to G(10) and G(50) the appropriate mobility and accessibility for specific recognition by NC.Our data also highlight the necessary plasticity of NCp7 to adapt to the sequence and structure variability of cTAR to chaperone its annealing with TAR through a specific pathway.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Biophotonique et Pharmacologie, Faculté de Pharmacie, UMR 7213 CNRS, Université de Strasbourg, 67401 Illkirch, France.

ABSTRACT
The HIV-1 nucleocapsid protein (NCp7) is a nucleic acid chaperone required during reverse transcription. During the first strand transfer, NCp7 is thought to destabilize cTAR, the (-)DNA copy of the TAR RNA hairpin, and subsequently direct the TAR/cTAR annealing through the zipping of their destabilized stem ends. To further characterize the destabilizing activity of NCp7, we locally probe the structure and dynamics of cTAR by steady-state and time resolved fluorescence spectroscopy. NC(11-55), a truncated NCp7 version corresponding to its zinc-finger domain, was found to bind all over the sequence and to preferentially destabilize the penultimate double-stranded segment in the lower part of the cTAR stem. This destabilization is achieved through zinc-finger-dependent binding of NC to the G(10) and G(50) residues. Sequence comparison further revealed that C•A mismatches close to the two G residues were critical for fine tuning the stability of the lower part of the cTAR stem and conferring to G(10) and G(50) the appropriate mobility and accessibility for specific recognition by NC. Our data also highlight the necessary plasticity of NCp7 to adapt to the sequence and structure variability of cTAR to chaperone its annealing with TAR through a specific pathway.

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Dependence of the populations of the stacked-conformations (A) and the local mobility expressed as a cone semi-angle (B) of the 2-Ap residues as a function of NC(11–55) concentrations. Points from dark to light grey correspond to 0, 1, 4, 7, 10 and 15 equivalents of NC(11–55), respectively. Error bars correspond to SD of at least three independent experiments. (A) NC(11–55) generally decreased the stacked conformations, albeit to different extents, being the most efficient for residues at positions 9 and 49. (B) The cone semi-angle of the conformational space explored by 2-Ap is largely decreased by NC(11–55), when the 2-Ap residue was located in the lower part of the cTAR stem (at positions 9, 49, 53 and 55).
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gkt164-F5: Dependence of the populations of the stacked-conformations (A) and the local mobility expressed as a cone semi-angle (B) of the 2-Ap residues as a function of NC(11–55) concentrations. Points from dark to light grey correspond to 0, 1, 4, 7, 10 and 15 equivalents of NC(11–55), respectively. Error bars correspond to SD of at least three independent experiments. (A) NC(11–55) generally decreased the stacked conformations, albeit to different extents, being the most efficient for residues at positions 9 and 49. (B) The cone semi-angle of the conformational space explored by 2-Ap is largely decreased by NC(11–55), when the 2-Ap residue was located in the lower part of the cTAR stem (at positions 9, 49, 53 and 55).

Mentions: Addition of NC(11–55) also increased the 2-Ap QY. In the presence of saturating concentrations of NC(11–55), the 2-Ap quantum yields varied from almost unchanged (2-Ap28) to an increase by a factor up to nearly 14 (2-Ap49) (Figure 2). The fluorescence changes were dependent on the amount of NC bound and on the position of the 2-Ap within the sequence. Time-resolved parameters further show that NC(11–55) did marginally change the lifetime values but induced a redistribution of the 2-Ap conformational states towards the longer-lived conformations (Table 1). The extent of this redistribution was dependent on the labelling position and on NC(11–55) concentration, being the most important at positions 9 and 49, showing that NC(11–55) strongly reduced base stacking at these two positions (Figure 5A). Stacked conformations were also decreased in positions 17, 21, 45, 53 and 55, albeit to a lesser extent, showing that NC(11–55) was able to bind and to decrease the base stacking all along the cTAR sequence (67). NCp7 seemed to be thus efficient to prevent the stacking of 2-Ap with its flanking bases, especially when 2-Ap is located close to a G. This likely resulted from the insertion of the neighbouring guanine into the hydrophobic platform at the top of the folded zinc-fingers of NC, which in turn prevents its collision with 2-Ap and thus, reduces its quenching efficiency (38,39,42,44). Moreover, the reduction of the amplitude associated with the shortest rotational correlation time Φ1, further indicates that NC(11–55) additionally restricts the base mobility on binding. Again, the Φ1 amplitude was the most decreased for 2-Ap at position 9 and 49, suggesting stable interactions of NC(11–55) with bases in the surrounding of 2-Ap in these two positions. The fluorescence parameters associated to 2-Ap9 and 2-Ap49 suggested that the collisions of the 2-Ap with their neighbouring guanines (G10 and G50, respectively) were strongly prevented by NC(11–55).Figure 5.


Site-selective probing of cTAR destabilization highlights the necessary plasticity of the HIV-1 nucleocapsid protein to chaperone the first strand transfer.

Godet J, Kenfack C, Przybilla F, Richert L, Duportail G, Mély Y - Nucleic Acids Res. (2013)

Dependence of the populations of the stacked-conformations (A) and the local mobility expressed as a cone semi-angle (B) of the 2-Ap residues as a function of NC(11–55) concentrations. Points from dark to light grey correspond to 0, 1, 4, 7, 10 and 15 equivalents of NC(11–55), respectively. Error bars correspond to SD of at least three independent experiments. (A) NC(11–55) generally decreased the stacked conformations, albeit to different extents, being the most efficient for residues at positions 9 and 49. (B) The cone semi-angle of the conformational space explored by 2-Ap is largely decreased by NC(11–55), when the 2-Ap residue was located in the lower part of the cTAR stem (at positions 9, 49, 53 and 55).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3643577&req=5

gkt164-F5: Dependence of the populations of the stacked-conformations (A) and the local mobility expressed as a cone semi-angle (B) of the 2-Ap residues as a function of NC(11–55) concentrations. Points from dark to light grey correspond to 0, 1, 4, 7, 10 and 15 equivalents of NC(11–55), respectively. Error bars correspond to SD of at least three independent experiments. (A) NC(11–55) generally decreased the stacked conformations, albeit to different extents, being the most efficient for residues at positions 9 and 49. (B) The cone semi-angle of the conformational space explored by 2-Ap is largely decreased by NC(11–55), when the 2-Ap residue was located in the lower part of the cTAR stem (at positions 9, 49, 53 and 55).
Mentions: Addition of NC(11–55) also increased the 2-Ap QY. In the presence of saturating concentrations of NC(11–55), the 2-Ap quantum yields varied from almost unchanged (2-Ap28) to an increase by a factor up to nearly 14 (2-Ap49) (Figure 2). The fluorescence changes were dependent on the amount of NC bound and on the position of the 2-Ap within the sequence. Time-resolved parameters further show that NC(11–55) did marginally change the lifetime values but induced a redistribution of the 2-Ap conformational states towards the longer-lived conformations (Table 1). The extent of this redistribution was dependent on the labelling position and on NC(11–55) concentration, being the most important at positions 9 and 49, showing that NC(11–55) strongly reduced base stacking at these two positions (Figure 5A). Stacked conformations were also decreased in positions 17, 21, 45, 53 and 55, albeit to a lesser extent, showing that NC(11–55) was able to bind and to decrease the base stacking all along the cTAR sequence (67). NCp7 seemed to be thus efficient to prevent the stacking of 2-Ap with its flanking bases, especially when 2-Ap is located close to a G. This likely resulted from the insertion of the neighbouring guanine into the hydrophobic platform at the top of the folded zinc-fingers of NC, which in turn prevents its collision with 2-Ap and thus, reduces its quenching efficiency (38,39,42,44). Moreover, the reduction of the amplitude associated with the shortest rotational correlation time Φ1, further indicates that NC(11–55) additionally restricts the base mobility on binding. Again, the Φ1 amplitude was the most decreased for 2-Ap at position 9 and 49, suggesting stable interactions of NC(11–55) with bases in the surrounding of 2-Ap in these two positions. The fluorescence parameters associated to 2-Ap9 and 2-Ap49 suggested that the collisions of the 2-Ap with their neighbouring guanines (G10 and G50, respectively) were strongly prevented by NC(11–55).Figure 5.

Bottom Line: NC(11-55), a truncated NCp7 version corresponding to its zinc-finger domain, was found to bind all over the sequence and to preferentially destabilize the penultimate double-stranded segment in the lower part of the cTAR stem.Sequence comparison further revealed that C•A mismatches close to the two G residues were critical for fine tuning the stability of the lower part of the cTAR stem and conferring to G(10) and G(50) the appropriate mobility and accessibility for specific recognition by NC.Our data also highlight the necessary plasticity of NCp7 to adapt to the sequence and structure variability of cTAR to chaperone its annealing with TAR through a specific pathway.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Biophotonique et Pharmacologie, Faculté de Pharmacie, UMR 7213 CNRS, Université de Strasbourg, 67401 Illkirch, France.

ABSTRACT
The HIV-1 nucleocapsid protein (NCp7) is a nucleic acid chaperone required during reverse transcription. During the first strand transfer, NCp7 is thought to destabilize cTAR, the (-)DNA copy of the TAR RNA hairpin, and subsequently direct the TAR/cTAR annealing through the zipping of their destabilized stem ends. To further characterize the destabilizing activity of NCp7, we locally probe the structure and dynamics of cTAR by steady-state and time resolved fluorescence spectroscopy. NC(11-55), a truncated NCp7 version corresponding to its zinc-finger domain, was found to bind all over the sequence and to preferentially destabilize the penultimate double-stranded segment in the lower part of the cTAR stem. This destabilization is achieved through zinc-finger-dependent binding of NC to the G(10) and G(50) residues. Sequence comparison further revealed that C•A mismatches close to the two G residues were critical for fine tuning the stability of the lower part of the cTAR stem and conferring to G(10) and G(50) the appropriate mobility and accessibility for specific recognition by NC. Our data also highlight the necessary plasticity of NCp7 to adapt to the sequence and structure variability of cTAR to chaperone its annealing with TAR through a specific pathway.

Show MeSH
Related in: MedlinePlus