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Aminoglycoside binding to the HIV-1 RNA dimerization initiation site: thermodynamics and effect on the kissing-loop to duplex conversion.

Bernacchi S, Freisz S, Maechling C, Spiess B, Marquet R, Dumas P, Ennifar E - Nucleic Acids Res. (2007)

Bottom Line: Surprisingly, we found that the affinity of lividomycin and neomycin for the DIS (K(d) approximately 30 nM) is significantly higher than that obtained in the same experimental conditions for their natural target, the bacterial A site (K(d) approximately 1.6 microM).In good agreement with their respective affinity, aminoglycoside increase the melting temperature of the loop-loop interaction and also block the conversion from kissing-loop complex to extended duplex.Taken together, our data might be useful for selecting new molecules with improved specificity and affinity toward the HIV-1 DIS RNA.

View Article: PubMed Central - PubMed

Affiliation: Architecture et Réactivité des ARN, UPR 9002 CNRS, Université Louis Pasteur, Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, 67084 Strasbourg, France.

ABSTRACT
Owing to a striking, and most likely fortuitous, structural and sequence similarity with the bacterial 16 S ribosomal A site, the RNA kissing-loop complex formed by the HIV-1 genomic RNA dimerization initiation site (DIS) specifically binds 4,5-disubstituted 2-deoxystreptamine (2-DOS) aminoglycoside antibiotics. We used chemical probing, molecular modeling, isothermal titration calorimetry (ITC) and UV melting to investigate aminoglycoside binding to the DIS loop-loop complex. We showed that apramycin, an aminoglycoside containing a bicyclic moiety, also binds the DIS, but in a different way than 4,5-disubstituted 2-DOS aminoglycosides. The determination of thermodynamic parameters for various aminoglycosides revealed the role of the different rings in the drug-RNA interaction. Surprisingly, we found that the affinity of lividomycin and neomycin for the DIS (K(d) approximately 30 nM) is significantly higher than that obtained in the same experimental conditions for their natural target, the bacterial A site (K(d) approximately 1.6 microM). In good agreement with their respective affinity, aminoglycoside increase the melting temperature of the loop-loop interaction and also block the conversion from kissing-loop complex to extended duplex. Taken together, our data might be useful for selecting new molecules with improved specificity and affinity toward the HIV-1 DIS RNA.

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(a) Representation of the solvent accessibility surface of aminoglycosides (probe radius 1.4 Å) in the DIS/lividomycin crystal structure (left) and the DIS/apramycin model (right) with the same orientation of the RNA kissing-loop complex. These views clearly depict the different binding geometry of these two aminoglycosides. (b) Two different stereoviews of the apramycin–DIS complex showing direct RNA–drug interactions (black lines). Dotted lines on the top view indicate probable water-mediated drug–RNA interaction. (c) Expected apramycin–RNA direct interactions deduced from the model.
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Figure 3: (a) Representation of the solvent accessibility surface of aminoglycosides (probe radius 1.4 Å) in the DIS/lividomycin crystal structure (left) and the DIS/apramycin model (right) with the same orientation of the RNA kissing-loop complex. These views clearly depict the different binding geometry of these two aminoglycosides. (b) Two different stereoviews of the apramycin–DIS complex showing direct RNA–drug interactions (black lines). Dotted lines on the top view indicate probable water-mediated drug–RNA interaction. (c) Expected apramycin–RNA direct interactions deduced from the model.

Mentions: Structural insight into the apramycin/DIS complex was obtained by 3D molecular modeling starting from the X-ray structure of apramycin bound to the bacterial A site (39). Apramycin was docked into the HIV-1 subtype F DIS kissing-loop complex as observed in the crystal structures with aminoglycosides (26), since docking to unliganded structures of the kissing-loop complex led to a steric clash with the bicyclic moiety of apramycin. The bound structures mainly differ from the unliganded kissing-loop by a shift from a C2′-endo to a C3′-endo conformation of G271 upon drug binding. The model revealed a different binding pattern of apramycin as compared to 4,5-disubstituted 2-DOS aminoglycosides (Figure 3a). First, the latter interact with the loop–loop helix and the major groove of both stems, whereas apramycin interacts also with the loop–loop helix but with the minor groove of both stems (Figure 3a). Second, according to the model, apramycin probably enters the kissing-loop through the minor groove of the RNA whereas, according to the X-ray structures, 4,5-disubstituted 2-DOS aminoglycosides most likely enter through the major groove (Figure 3a).Figure 3.


Aminoglycoside binding to the HIV-1 RNA dimerization initiation site: thermodynamics and effect on the kissing-loop to duplex conversion.

Bernacchi S, Freisz S, Maechling C, Spiess B, Marquet R, Dumas P, Ennifar E - Nucleic Acids Res. (2007)

(a) Representation of the solvent accessibility surface of aminoglycosides (probe radius 1.4 Å) in the DIS/lividomycin crystal structure (left) and the DIS/apramycin model (right) with the same orientation of the RNA kissing-loop complex. These views clearly depict the different binding geometry of these two aminoglycosides. (b) Two different stereoviews of the apramycin–DIS complex showing direct RNA–drug interactions (black lines). Dotted lines on the top view indicate probable water-mediated drug–RNA interaction. (c) Expected apramycin–RNA direct interactions deduced from the model.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175338&req=5

Figure 3: (a) Representation of the solvent accessibility surface of aminoglycosides (probe radius 1.4 Å) in the DIS/lividomycin crystal structure (left) and the DIS/apramycin model (right) with the same orientation of the RNA kissing-loop complex. These views clearly depict the different binding geometry of these two aminoglycosides. (b) Two different stereoviews of the apramycin–DIS complex showing direct RNA–drug interactions (black lines). Dotted lines on the top view indicate probable water-mediated drug–RNA interaction. (c) Expected apramycin–RNA direct interactions deduced from the model.
Mentions: Structural insight into the apramycin/DIS complex was obtained by 3D molecular modeling starting from the X-ray structure of apramycin bound to the bacterial A site (39). Apramycin was docked into the HIV-1 subtype F DIS kissing-loop complex as observed in the crystal structures with aminoglycosides (26), since docking to unliganded structures of the kissing-loop complex led to a steric clash with the bicyclic moiety of apramycin. The bound structures mainly differ from the unliganded kissing-loop by a shift from a C2′-endo to a C3′-endo conformation of G271 upon drug binding. The model revealed a different binding pattern of apramycin as compared to 4,5-disubstituted 2-DOS aminoglycosides (Figure 3a). First, the latter interact with the loop–loop helix and the major groove of both stems, whereas apramycin interacts also with the loop–loop helix but with the minor groove of both stems (Figure 3a). Second, according to the model, apramycin probably enters the kissing-loop through the minor groove of the RNA whereas, according to the X-ray structures, 4,5-disubstituted 2-DOS aminoglycosides most likely enter through the major groove (Figure 3a).Figure 3.

Bottom Line: Surprisingly, we found that the affinity of lividomycin and neomycin for the DIS (K(d) approximately 30 nM) is significantly higher than that obtained in the same experimental conditions for their natural target, the bacterial A site (K(d) approximately 1.6 microM).In good agreement with their respective affinity, aminoglycoside increase the melting temperature of the loop-loop interaction and also block the conversion from kissing-loop complex to extended duplex.Taken together, our data might be useful for selecting new molecules with improved specificity and affinity toward the HIV-1 DIS RNA.

View Article: PubMed Central - PubMed

Affiliation: Architecture et Réactivité des ARN, UPR 9002 CNRS, Université Louis Pasteur, Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, 67084 Strasbourg, France.

ABSTRACT
Owing to a striking, and most likely fortuitous, structural and sequence similarity with the bacterial 16 S ribosomal A site, the RNA kissing-loop complex formed by the HIV-1 genomic RNA dimerization initiation site (DIS) specifically binds 4,5-disubstituted 2-deoxystreptamine (2-DOS) aminoglycoside antibiotics. We used chemical probing, molecular modeling, isothermal titration calorimetry (ITC) and UV melting to investigate aminoglycoside binding to the DIS loop-loop complex. We showed that apramycin, an aminoglycoside containing a bicyclic moiety, also binds the DIS, but in a different way than 4,5-disubstituted 2-DOS aminoglycosides. The determination of thermodynamic parameters for various aminoglycosides revealed the role of the different rings in the drug-RNA interaction. Surprisingly, we found that the affinity of lividomycin and neomycin for the DIS (K(d) approximately 30 nM) is significantly higher than that obtained in the same experimental conditions for their natural target, the bacterial A site (K(d) approximately 1.6 microM). In good agreement with their respective affinity, aminoglycoside increase the melting temperature of the loop-loop interaction and also block the conversion from kissing-loop complex to extended duplex. Taken together, our data might be useful for selecting new molecules with improved specificity and affinity toward the HIV-1 DIS RNA.

Show MeSH