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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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ITC profiles for the titration of (a) lividomycin on the monomeric DIS U275C mutant, which is typical of unspecific binding, (b) of neamine and (c) of lividomycin on the subtype A DIS kissing-loop, which are typical of weak and strong specific binding, respectively. Each heat burst curve is the result of a 5 μl injection of 200 μM drug in a 2 μM RNA solution (1.42 sample cell). (d) Comparison of the measured free energy (▵Gexp) and the calculated free energy (▵Gcalc) based on charge number and drug/RNA contact surface area observed in crystal structures. Since no crystal structure of the paromomycin/DIS complex was obtained, surface area for paromomycin was considered to be identical to the neomycin one (similar contacts are expected).
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Figure 4: ITC profiles for the titration of (a) lividomycin on the monomeric DIS U275C mutant, which is typical of unspecific binding, (b) of neamine and (c) of lividomycin on the subtype A DIS kissing-loop, which are typical of weak and strong specific binding, respectively. Each heat burst curve is the result of a 5 μl injection of 200 μM drug in a 2 μM RNA solution (1.42 sample cell). (d) Comparison of the measured free energy (▵Gexp) and the calculated free energy (▵Gcalc) based on charge number and drug/RNA contact surface area observed in crystal structures. Since no crystal structure of the paromomycin/DIS complex was obtained, surface area for paromomycin was considered to be identical to the neomycin one (similar contacts are expected).

Mentions: Isothermal titration calorimetry (ITC) was used to characterize aminoglycoside binding to the DIS kissing-loop complex. Unlike other techniques such as chemical footprinting or fluorescence spectroscopy that are indirect methods, ITC directly provides a full thermodynamic profile of the drug/RNA interaction (34) and does not require any modification of the RNA for labeling. In addition, this method was already successfully employed to characterize aminoglycoside/A site interactions (35,37,41–43). We investigated the binding to the HIV-1 subtype-A DIS of neamine, ribostamycin, paromomycin, neomycin, lividomycin, apramycin and tobramycin. Experimental conditions were chosen to be compatible with the DIS kissing-loop complex and to avoid any interference with the spontaneous conversion into the extended duplex form of the DIS in the timescale of the experiment (30): RNA concentration did not exceed 2 µM in a low-salt buffer, and the temperature was set to 25°C. Great care was taken to estimate the unspecific binding of the positively charged aminoglycoside antibiotics on the RNA. For that goal, we monitored their binding to a standard A-form RNA helix of 22 bp with a sequence very similar to that of the DIS, but deprived of unpaired residues and base-pair mismatches and thus of specific binding sites (Figure S3). Unspecific interactions were also investigated with a DIS hairpin monomer using a DIS U275C mutant that cannot form a kissing-loop dimer, as well as with a HIV-1 subtype-B DIS sequence that is not recognized by aminoglycosides (24). The 4,6-disubstituted 2-DOS tobramycin that does not bind the DIS kissing-loop (24) was also used to determine aminoglycoside unspecific binding to the wild-type subtype A DIS kissing-loop complex. As expected, all these situations were characterized by a weak exothermic signal (Figure 4a) and led essentially to superimposable titration curves (Figure S3). They can therefore be considered as typical titration curves for unspecific aminoglycoside/RNA interactions.Figure 4.


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)

ITC profiles for the titration of (a) lividomycin on the monomeric DIS U275C mutant, which is typical of unspecific binding, (b) of neamine and (c) of lividomycin on the subtype A DIS kissing-loop, which are typical of weak and strong specific binding, respectively. Each heat burst curve is the result of a 5 μl injection of 200 μM drug in a 2 μM RNA solution (1.42 sample cell). (d) Comparison of the measured free energy (▵Gexp) and the calculated free energy (▵Gcalc) based on charge number and drug/RNA contact surface area observed in crystal structures. Since no crystal structure of the paromomycin/DIS complex was obtained, surface area for paromomycin was considered to be identical to the neomycin one (similar contacts are expected).
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Related In: Results  -  Collection

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Figure 4: ITC profiles for the titration of (a) lividomycin on the monomeric DIS U275C mutant, which is typical of unspecific binding, (b) of neamine and (c) of lividomycin on the subtype A DIS kissing-loop, which are typical of weak and strong specific binding, respectively. Each heat burst curve is the result of a 5 μl injection of 200 μM drug in a 2 μM RNA solution (1.42 sample cell). (d) Comparison of the measured free energy (▵Gexp) and the calculated free energy (▵Gcalc) based on charge number and drug/RNA contact surface area observed in crystal structures. Since no crystal structure of the paromomycin/DIS complex was obtained, surface area for paromomycin was considered to be identical to the neomycin one (similar contacts are expected).
Mentions: Isothermal titration calorimetry (ITC) was used to characterize aminoglycoside binding to the DIS kissing-loop complex. Unlike other techniques such as chemical footprinting or fluorescence spectroscopy that are indirect methods, ITC directly provides a full thermodynamic profile of the drug/RNA interaction (34) and does not require any modification of the RNA for labeling. In addition, this method was already successfully employed to characterize aminoglycoside/A site interactions (35,37,41–43). We investigated the binding to the HIV-1 subtype-A DIS of neamine, ribostamycin, paromomycin, neomycin, lividomycin, apramycin and tobramycin. Experimental conditions were chosen to be compatible with the DIS kissing-loop complex and to avoid any interference with the spontaneous conversion into the extended duplex form of the DIS in the timescale of the experiment (30): RNA concentration did not exceed 2 µM in a low-salt buffer, and the temperature was set to 25°C. Great care was taken to estimate the unspecific binding of the positively charged aminoglycoside antibiotics on the RNA. For that goal, we monitored their binding to a standard A-form RNA helix of 22 bp with a sequence very similar to that of the DIS, but deprived of unpaired residues and base-pair mismatches and thus of specific binding sites (Figure S3). Unspecific interactions were also investigated with a DIS hairpin monomer using a DIS U275C mutant that cannot form a kissing-loop dimer, as well as with a HIV-1 subtype-B DIS sequence that is not recognized by aminoglycosides (24). The 4,6-disubstituted 2-DOS tobramycin that does not bind the DIS kissing-loop (24) was also used to determine aminoglycoside unspecific binding to the wild-type subtype A DIS kissing-loop complex. As expected, all these situations were characterized by a weak exothermic signal (Figure 4a) and led essentially to superimposable titration curves (Figure S3). They can therefore be considered as typical titration curves for unspecific aminoglycoside/RNA interactions.Figure 4.

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
Related in: MedlinePlus