Limits...
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.

Show MeSH

Related in: MedlinePlus

Representation of the longest rotational correlation time Ф3 in the absence and in the presence of NC(11–55). Ф3 values for the 2-Ap residues inserted in the lower half of cTAR stem (hatched white bars) are significantly shorter than the values observed in the upper part of the sequence (hatched grey bars), indicating significant segmental motions in the lower part of the cTAR stem. In the presence of saturating concentrations of NC(11–55) (non hatched bars), NC(11–55) largely decreases the segmental mobility of the DNA segments containing 2-Ap residues at position 9, 45 and 49, whereas segmental motions persist in the last ds-segments of the cTAR stem (dark grey).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3643577&req=5

gkt164-F4: Representation of the longest rotational correlation time Ф3 in the absence and in the presence of NC(11–55). Ф3 values for the 2-Ap residues inserted in the lower half of cTAR stem (hatched white bars) are significantly shorter than the values observed in the upper part of the sequence (hatched grey bars), indicating significant segmental motions in the lower part of the cTAR stem. In the presence of saturating concentrations of NC(11–55) (non hatched bars), NC(11–55) largely decreases the segmental mobility of the DNA segments containing 2-Ap residues at position 9, 45 and 49, whereas segmental motions persist in the last ds-segments of the cTAR stem (dark grey).

Mentions: Beside the snapshot picture of the 2-Ap conformations distribution given by the time-resolved fluorescence decays, rotational dynamics of 2-Ap monitored through time-resolved fluorescence anisotropy provide information about the local and global dynamics of the cTAR sequences. Anisotropy decays were satisfactorily fitted using a three exponential model, with the exception of 2-Ap at position 55 for which only two components were sufficient. The short correlation time (Ф1), 0.1–0.5 ns, was assigned to the local motion of 2-Ap with respect to the strand backbone, whereas the longest rotational correlation time (Ф3) described the whole tumbling of the molecule. The presence of an intermediate rotational correlation time Ф2 (∼1 ns) with an amplitude almost constant of about ∼ 30% evidenced additional conformational fluctuations in cTAR. The amplitude of the short rotational correlation time reflected the conformational space explored by the 2-Ap residue during the excited time. The extent of the explored space was quantified by the cone semi-angle value θ0 (Table 1). The β1 and θ0 values were low for 2-Ap residues located in ds-segments, but large for 2-Ap9 and 2-Ap49, indicating a high rotational freedom for the latter, in line with the higher propensity of mismatched bases to explore geometries outside from the helix. The values of the long rotational correlation time (Ф3) ranged from 4 to 9 ns, depending on the labelled position. For 2-Ap residues in the upper half-part of the hairpin loop (at positions 17, 21, 28 or 35) (Figure 4), the Ф3 value was ∼8–9 ns, in full line with the theoretical correlation time calculated for the tumbling motion of a cylinder rod of length 95 Å and diameter 20 Å (66). In contrast, the Ф3 values were substantially lower (∼4–5 ns) for 2-Ap residues in the bottom of the cTAR sequence (positions 45, 49, 53 or 55) and were thus likely a combination of the overall tumbling of the cTAR sequence and of the segmental ‘fraying’ motion of the cTAR ends (30,34). Similarly to the Ф3 value, the Ф2 value was probably shortened in 2-Ap55 because of the larger segmental motions in the cTAR terminal ds-segment, making it impossible to resolve from Ф1. This hypothesis was further supported by the unexpectedly high β1 value observed for 2-Ap at position 55.Figure 4.


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)

Representation of the longest rotational correlation time Ф3 in the absence and in the presence of NC(11–55). Ф3 values for the 2-Ap residues inserted in the lower half of cTAR stem (hatched white bars) are significantly shorter than the values observed in the upper part of the sequence (hatched grey bars), indicating significant segmental motions in the lower part of the cTAR stem. In the presence of saturating concentrations of NC(11–55) (non hatched bars), NC(11–55) largely decreases the segmental mobility of the DNA segments containing 2-Ap residues at position 9, 45 and 49, whereas segmental motions persist in the last ds-segments of the cTAR stem (dark grey).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt164-F4: Representation of the longest rotational correlation time Ф3 in the absence and in the presence of NC(11–55). Ф3 values for the 2-Ap residues inserted in the lower half of cTAR stem (hatched white bars) are significantly shorter than the values observed in the upper part of the sequence (hatched grey bars), indicating significant segmental motions in the lower part of the cTAR stem. In the presence of saturating concentrations of NC(11–55) (non hatched bars), NC(11–55) largely decreases the segmental mobility of the DNA segments containing 2-Ap residues at position 9, 45 and 49, whereas segmental motions persist in the last ds-segments of the cTAR stem (dark grey).
Mentions: Beside the snapshot picture of the 2-Ap conformations distribution given by the time-resolved fluorescence decays, rotational dynamics of 2-Ap monitored through time-resolved fluorescence anisotropy provide information about the local and global dynamics of the cTAR sequences. Anisotropy decays were satisfactorily fitted using a three exponential model, with the exception of 2-Ap at position 55 for which only two components were sufficient. The short correlation time (Ф1), 0.1–0.5 ns, was assigned to the local motion of 2-Ap with respect to the strand backbone, whereas the longest rotational correlation time (Ф3) described the whole tumbling of the molecule. The presence of an intermediate rotational correlation time Ф2 (∼1 ns) with an amplitude almost constant of about ∼ 30% evidenced additional conformational fluctuations in cTAR. The amplitude of the short rotational correlation time reflected the conformational space explored by the 2-Ap residue during the excited time. The extent of the explored space was quantified by the cone semi-angle value θ0 (Table 1). The β1 and θ0 values were low for 2-Ap residues located in ds-segments, but large for 2-Ap9 and 2-Ap49, indicating a high rotational freedom for the latter, in line with the higher propensity of mismatched bases to explore geometries outside from the helix. The values of the long rotational correlation time (Ф3) ranged from 4 to 9 ns, depending on the labelled position. For 2-Ap residues in the upper half-part of the hairpin loop (at positions 17, 21, 28 or 35) (Figure 4), the Ф3 value was ∼8–9 ns, in full line with the theoretical correlation time calculated for the tumbling motion of a cylinder rod of length 95 Å and diameter 20 Å (66). In contrast, the Ф3 values were substantially lower (∼4–5 ns) for 2-Ap residues in the bottom of the cTAR sequence (positions 45, 49, 53 or 55) and were thus likely a combination of the overall tumbling of the cTAR sequence and of the segmental ‘fraying’ motion of the cTAR ends (30,34). Similarly to the Ф3 value, the Ф2 value was probably shortened in 2-Ap55 because of the larger segmental motions in the cTAR terminal ds-segment, making it impossible to resolve from Ф1. This hypothesis was further supported by the unexpectedly high β1 value observed for 2-Ap at position 55.Figure 4.

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