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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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Proposed mechanism for NC-induced destabilization of cTAR. NC(11–55) preferentially recognizes G10 and G50 and initiate the destabilization of cTAR. In the presence of higher concentrations, NC(11–55) can adjust to the heterogeneous structures and dynamics to bind all along cTAR and generate the reactive species needed to anneal the complementary TAR sequence.
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gkt164-F9: Proposed mechanism for NC-induced destabilization of cTAR. NC(11–55) preferentially recognizes G10 and G50 and initiate the destabilization of cTAR. In the presence of higher concentrations, NC(11–55) can adjust to the heterogeneous structures and dynamics to bind all along cTAR and generate the reactive species needed to anneal the complementary TAR sequence.

Mentions: Binding of NC(11–55)-induced local fluorescence changes that depended on the location of the 2-Ap residue within the sequence and on the amount of protein molecules bound to cTAR. Although at a 1:1 NC(11–55)/cTAR ratio, the peptide shows a preferential binding to the ds-segments encompassing residues 9 and 49, NC(11–55) molecules were shown to bind all over the cTAR sequence at saturating concentrations, suggesting that they can adjust to the heterogeneous structures and dynamics present all along cTAR (Figure 9). Nevertheless, major NC(11–55)-induced changes in the cTAR structure and dynamics were observed in the lower half of the stem and notably at positions 9 and 49. The dramatic changes in the 2-Ap fluorescence signals at these two positions were similar to those attributed to the stacking interaction of the aromatic Trp37 residue of NC with a Guanine flanking a 2-Ap residue on the interaction of NC(11–55) with small 2-Ap-labelled oligonucleotides or (−)primer-binding site (44,45,77). Thus, 2-Ap9 and 2-Ap49 fluorescence parameters strongly suggest that G10 and G50 residues directly interact with NC(11–55), likely through a stacking interaction with the Trp37 residue of NC. The resulting ‘freezing’ of cTAR at these two positions is thought to constitute a prerequisite for the NC-induced destabilization of cTAR stem. Indeed, this NC-induced restriction of the local and segmental flexibility of the NA likely prevents fast transitions back to the stably stacked conformations, and thus allows the formation of locally destabilized domains with longer lifetime and probably with exposed bases that are competent for annealing with the complementary TAR sequence. The specific restriction of the local DNA mobility by NC ZFs likely constitutes a key component of the NC destabilization mechanism that is required to further facilitate the cTAR/TAR hybridization. This hypothesis is confirmed by the fact that the (SSHS)2NC(11–55) mutant, which is unable to constrain the cTAR structure and dynamics, seems to be much less efficient than NC(11–55) to promote the cTAR/TAR annealing reaction.Figure 9.


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)

Proposed mechanism for NC-induced destabilization of cTAR. NC(11–55) preferentially recognizes G10 and G50 and initiate the destabilization of cTAR. In the presence of higher concentrations, NC(11–55) can adjust to the heterogeneous structures and dynamics to bind all along cTAR and generate the reactive species needed to anneal the complementary TAR sequence.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt164-F9: Proposed mechanism for NC-induced destabilization of cTAR. NC(11–55) preferentially recognizes G10 and G50 and initiate the destabilization of cTAR. In the presence of higher concentrations, NC(11–55) can adjust to the heterogeneous structures and dynamics to bind all along cTAR and generate the reactive species needed to anneal the complementary TAR sequence.
Mentions: Binding of NC(11–55)-induced local fluorescence changes that depended on the location of the 2-Ap residue within the sequence and on the amount of protein molecules bound to cTAR. Although at a 1:1 NC(11–55)/cTAR ratio, the peptide shows a preferential binding to the ds-segments encompassing residues 9 and 49, NC(11–55) molecules were shown to bind all over the cTAR sequence at saturating concentrations, suggesting that they can adjust to the heterogeneous structures and dynamics present all along cTAR (Figure 9). Nevertheless, major NC(11–55)-induced changes in the cTAR structure and dynamics were observed in the lower half of the stem and notably at positions 9 and 49. The dramatic changes in the 2-Ap fluorescence signals at these two positions were similar to those attributed to the stacking interaction of the aromatic Trp37 residue of NC with a Guanine flanking a 2-Ap residue on the interaction of NC(11–55) with small 2-Ap-labelled oligonucleotides or (−)primer-binding site (44,45,77). Thus, 2-Ap9 and 2-Ap49 fluorescence parameters strongly suggest that G10 and G50 residues directly interact with NC(11–55), likely through a stacking interaction with the Trp37 residue of NC. The resulting ‘freezing’ of cTAR at these two positions is thought to constitute a prerequisite for the NC-induced destabilization of cTAR stem. Indeed, this NC-induced restriction of the local and segmental flexibility of the NA likely prevents fast transitions back to the stably stacked conformations, and thus allows the formation of locally destabilized domains with longer lifetime and probably with exposed bases that are competent for annealing with the complementary TAR sequence. The specific restriction of the local DNA mobility by NC ZFs likely constitutes a key component of the NC destabilization mechanism that is required to further facilitate the cTAR/TAR hybridization. This hypothesis is confirmed by the fact that the (SSHS)2NC(11–55) mutant, which is unable to constrain the cTAR structure and dynamics, seems to be much less efficient than NC(11–55) to promote the cTAR/TAR annealing reaction.Figure 9.

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