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Structural probing of the HIV-1 polypurine tract RNA:DNA hybrid using classic nucleic acid ligands.

Turner KB, Brinson RG, Yi-Brunozzi HY, Rausch JW, Miller JT, Le Grice SF, Marino JP, Fabris D - Nucleic Acids Res. (2008)

Bottom Line: In contrast, neomycin bound preferentially and selectively to the PPT near the 5'(rA)(4):(dT)(4) tract and the 3' PPT-U3 junction.Nuclear magnetic resonance data from a complex between HIV-1 RT and the PPT indicate RT contacts within the same regions highlighted on the PPT by neomycin.These observations, together with the fact that the sites are correctly spaced to allow interaction with residues in the ribonuclease H (RNase H) active site and thumb subdomain of the p66 RT subunit, suggest that despite the long cleft employed by RT to make contact with nucleic acids substrates, these sites provide discrete binding units working in concert to determine not only specific PPT recognition, but also its orientation on the hybrid structure.

View Article: PubMed Central - PubMed

Affiliation: University of Maryland Baltimore County, Baltimore, MD, USA.

ABSTRACT
The interactions of archetypical nucleic acid ligands with the HIV-1 polypurine tract (PPT) RNA:DNA hybrid, as well as analogous DNA:DNA, RNA:RNA and swapped hybrid substrates, were used to probe structural features of the PPT that contribute to its specific recognition and processing by reverse transcriptase (RT). Results from intercalative and groove-binding ligands indicate that the wild-type PPT hybrid does not contain any strikingly unique groove geometries and/or stacking arrangements that might contribute to the specificity of its interaction with RT. In contrast, neomycin bound preferentially and selectively to the PPT near the 5'(rA)(4):(dT)(4) tract and the 3' PPT-U3 junction. Nuclear magnetic resonance data from a complex between HIV-1 RT and the PPT indicate RT contacts within the same regions highlighted on the PPT by neomycin. These observations, together with the fact that the sites are correctly spaced to allow interaction with residues in the ribonuclease H (RNase H) active site and thumb subdomain of the p66 RT subunit, suggest that despite the long cleft employed by RT to make contact with nucleic acids substrates, these sites provide discrete binding units working in concert to determine not only specific PPT recognition, but also its orientation on the hybrid structure.

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One-dimensional water flip-back watergate 1H NMR spectra of the imino region of the PPTwt duplex after titration with neomycin B at 10°C, or E478Q RT at 30°C, in 80 mM NaCl and 10 mM NaH2PO4/Na2HPO4 at pH 7.0. (a) For the neomycin titration, the concentration of the PPTwt duplex was ∼200 μM. 1D 1H NMR spectra are shown in the absence (upper trace) and presence of 1.0 equivalents (middle trace) and 2.0 equivalents (lower trace) of neomycin B. The imino resonances for G(+2), T(+1), and g(−1) where chemical shift changes can be tracked are highlighted in bold. Dotted lines and arrows indicate the shift in position of the resonances. (b) For the E478Q RT titration, the concentration of PPTwt was ∼40 μM. 1D 1H NMR spectra are shown for PPTwt in the absence (upper trace) and presence of 1.0 equivalent (lower trace) of E478Q RT. Imino protons were assigned using 2D NOESY experiments; assignments are listed above each peak.
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Figure 6: One-dimensional water flip-back watergate 1H NMR spectra of the imino region of the PPTwt duplex after titration with neomycin B at 10°C, or E478Q RT at 30°C, in 80 mM NaCl and 10 mM NaH2PO4/Na2HPO4 at pH 7.0. (a) For the neomycin titration, the concentration of the PPTwt duplex was ∼200 μM. 1D 1H NMR spectra are shown in the absence (upper trace) and presence of 1.0 equivalents (middle trace) and 2.0 equivalents (lower trace) of neomycin B. The imino resonances for G(+2), T(+1), and g(−1) where chemical shift changes can be tracked are highlighted in bold. Dotted lines and arrows indicate the shift in position of the resonances. (b) For the E478Q RT titration, the concentration of PPTwt was ∼40 μM. 1D 1H NMR spectra are shown for PPTwt in the absence (upper trace) and presence of 1.0 equivalent (lower trace) of E478Q RT. Imino protons were assigned using 2D NOESY experiments; assignments are listed above each peak.

Mentions: Further insights into the position of the specific binding sites were revealed by high-resolution 1H NMR. Ligand binding to the wild-type PPT was followed by monitoring changes in the imino proton spectrum of PPTwt upon titration with neomycin B in increments of 0.25 equivalents to a final 2:1 ligand:substrate ratio (Figure 6a). The imino proton chemical shift assignments are indicated for the free PPTwt and were determined using 2D NOESY spectra (50). Upon addition of neomycin B to PPTwt, perturbations are observed for a subset of imino proton resonances. After addition of one equivalent of ligand, a shift in the imino proton signals was detected for g(−1), T(+1) and G(+2) (indicated by the arrows), consistent with selective binding at the PPT-U3 junction. In addition, uniformly subtle downfield shifts were observed for imino protons assigned to the G-tract 5′ of the PPT-U3 junction, which further localizes binding of neomycin B to this end of the PPT. After the primary site was saturated, addition of a second equivalent did not introduce any detectable shift of the imino protons, but caused only a broadening of their signals. This behavior is typical of intermediate chemical exchange processes on the NMR timescale and is suggestive of a weaker, more non-specific binding interaction(s) between ligand and PPT substrate. While the NMR data does not allow mapping of this secondary site(s), the observation of consecutive binding events in the NMR titrations is consistent with the observation of high and low affinity sites in the ESI-FTICR experiments.Figure 6.


Structural probing of the HIV-1 polypurine tract RNA:DNA hybrid using classic nucleic acid ligands.

Turner KB, Brinson RG, Yi-Brunozzi HY, Rausch JW, Miller JT, Le Grice SF, Marino JP, Fabris D - Nucleic Acids Res. (2008)

One-dimensional water flip-back watergate 1H NMR spectra of the imino region of the PPTwt duplex after titration with neomycin B at 10°C, or E478Q RT at 30°C, in 80 mM NaCl and 10 mM NaH2PO4/Na2HPO4 at pH 7.0. (a) For the neomycin titration, the concentration of the PPTwt duplex was ∼200 μM. 1D 1H NMR spectra are shown in the absence (upper trace) and presence of 1.0 equivalents (middle trace) and 2.0 equivalents (lower trace) of neomycin B. The imino resonances for G(+2), T(+1), and g(−1) where chemical shift changes can be tracked are highlighted in bold. Dotted lines and arrows indicate the shift in position of the resonances. (b) For the E478Q RT titration, the concentration of PPTwt was ∼40 μM. 1D 1H NMR spectra are shown for PPTwt in the absence (upper trace) and presence of 1.0 equivalent (lower trace) of E478Q RT. Imino protons were assigned using 2D NOESY experiments; assignments are listed above each peak.
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Figure 6: One-dimensional water flip-back watergate 1H NMR spectra of the imino region of the PPTwt duplex after titration with neomycin B at 10°C, or E478Q RT at 30°C, in 80 mM NaCl and 10 mM NaH2PO4/Na2HPO4 at pH 7.0. (a) For the neomycin titration, the concentration of the PPTwt duplex was ∼200 μM. 1D 1H NMR spectra are shown in the absence (upper trace) and presence of 1.0 equivalents (middle trace) and 2.0 equivalents (lower trace) of neomycin B. The imino resonances for G(+2), T(+1), and g(−1) where chemical shift changes can be tracked are highlighted in bold. Dotted lines and arrows indicate the shift in position of the resonances. (b) For the E478Q RT titration, the concentration of PPTwt was ∼40 μM. 1D 1H NMR spectra are shown for PPTwt in the absence (upper trace) and presence of 1.0 equivalent (lower trace) of E478Q RT. Imino protons were assigned using 2D NOESY experiments; assignments are listed above each peak.
Mentions: Further insights into the position of the specific binding sites were revealed by high-resolution 1H NMR. Ligand binding to the wild-type PPT was followed by monitoring changes in the imino proton spectrum of PPTwt upon titration with neomycin B in increments of 0.25 equivalents to a final 2:1 ligand:substrate ratio (Figure 6a). The imino proton chemical shift assignments are indicated for the free PPTwt and were determined using 2D NOESY spectra (50). Upon addition of neomycin B to PPTwt, perturbations are observed for a subset of imino proton resonances. After addition of one equivalent of ligand, a shift in the imino proton signals was detected for g(−1), T(+1) and G(+2) (indicated by the arrows), consistent with selective binding at the PPT-U3 junction. In addition, uniformly subtle downfield shifts were observed for imino protons assigned to the G-tract 5′ of the PPT-U3 junction, which further localizes binding of neomycin B to this end of the PPT. After the primary site was saturated, addition of a second equivalent did not introduce any detectable shift of the imino protons, but caused only a broadening of their signals. This behavior is typical of intermediate chemical exchange processes on the NMR timescale and is suggestive of a weaker, more non-specific binding interaction(s) between ligand and PPT substrate. While the NMR data does not allow mapping of this secondary site(s), the observation of consecutive binding events in the NMR titrations is consistent with the observation of high and low affinity sites in the ESI-FTICR experiments.Figure 6.

Bottom Line: In contrast, neomycin bound preferentially and selectively to the PPT near the 5'(rA)(4):(dT)(4) tract and the 3' PPT-U3 junction.Nuclear magnetic resonance data from a complex between HIV-1 RT and the PPT indicate RT contacts within the same regions highlighted on the PPT by neomycin.These observations, together with the fact that the sites are correctly spaced to allow interaction with residues in the ribonuclease H (RNase H) active site and thumb subdomain of the p66 RT subunit, suggest that despite the long cleft employed by RT to make contact with nucleic acids substrates, these sites provide discrete binding units working in concert to determine not only specific PPT recognition, but also its orientation on the hybrid structure.

View Article: PubMed Central - PubMed

Affiliation: University of Maryland Baltimore County, Baltimore, MD, USA.

ABSTRACT
The interactions of archetypical nucleic acid ligands with the HIV-1 polypurine tract (PPT) RNA:DNA hybrid, as well as analogous DNA:DNA, RNA:RNA and swapped hybrid substrates, were used to probe structural features of the PPT that contribute to its specific recognition and processing by reverse transcriptase (RT). Results from intercalative and groove-binding ligands indicate that the wild-type PPT hybrid does not contain any strikingly unique groove geometries and/or stacking arrangements that might contribute to the specificity of its interaction with RT. In contrast, neomycin bound preferentially and selectively to the PPT near the 5'(rA)(4):(dT)(4) tract and the 3' PPT-U3 junction. Nuclear magnetic resonance data from a complex between HIV-1 RT and the PPT indicate RT contacts within the same regions highlighted on the PPT by neomycin. These observations, together with the fact that the sites are correctly spaced to allow interaction with residues in the ribonuclease H (RNase H) active site and thumb subdomain of the p66 RT subunit, suggest that despite the long cleft employed by RT to make contact with nucleic acids substrates, these sites provide discrete binding units working in concert to determine not only specific PPT recognition, but also its orientation on the hybrid structure.

Show MeSH
Related in: MedlinePlus