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. 2012 Sep;40(16):8119-28.
doi: 10.1093/nar/gks512. Epub 2012 Jun 4.

High-resolution structures of two complexes between thrombin and thrombin-binding aptamer shed light on the role of cations in the aptamer inhibitory activity

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High-resolution structures of two complexes between thrombin and thrombin-binding aptamer shed light on the role of cations in the aptamer inhibitory activity

Irene Russo Krauss et al. Nucleic Acids Res. 2012 Sep.

Abstract

The G-quadruplex architecture is a peculiar structure adopted by guanine-rich oligonucleotidic sequences, and, in particular, by several aptamers, including the thrombin-binding aptamer (TBA) that has the highest inhibitory activity against human α-thrombin. A crucial role in determining structure, stability and biological properties of G-quadruplexes is played by ions. In the case of TBA, K(+) ions cause an enhancement of the aptamer clotting inhibitory activity. A detailed picture of the interactions of TBA with the protein and with the ions is still lacking, despite the importance of this aptamer in biomedical field for detection and inhibition of α-thrombin. Here, we fill this gap by presenting a high-resolution crystallographic structural characterization of the thrombin-TBA complex formed in the presence of Na(+) or K(+) and a circular dichroism study of the structural stability of the aptamer both free and complexed with α-thrombin, in the presence of the two ionic species. The results indicate that the different effects exerted by Na(+) and K(+) on the inhibitory activity of TBA are related to a subtle perturbation of a few key interactions at the protein-aptamer interface. The present data, in combination with those previously obtained on the complex between α-thrombin and a modified aptamer, may allow the design of new TBA variants with a pharmacological performance enhancement.

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Figures

Figure 1.
Figure 1.
CD spectra of 7 μM TBA in 25 mM M-phosphate buffer pH 7.1 and 0.1 M MCl, where M is either Na+ (A) and K+ (B) at 10°C before (blue line) and after (red line) annealing at 90°C.
Figure 2.
Figure 2.
Thermal denaturation of TBA as followed by CD spectroscopy at 295 nm, in the absence (dashed line) and in the presence (bold line) of an equimolar amount of thrombin at a heating rate of 1°C/min. Measurements were carried out in 25 mM M-phosphate buffer pH 7.1 and 0.1 M MCl, where M is either Na+ (A) and K+ (B), using a 7 μM aptamer concentration.
Figure 3.
Figure 3.
Overall structure of the thrombin–TBA complex in the presence of sodium (A) and potassium (B) ions. Thrombin molecule is represented as cartoon, TBA molecule is represented as sticks.
Figure 4.
Figure 4.
Omit Fo–Fc electron density map (6.0 σ level) of the ion stacked between aptamer quartets in the thrombin–TBA–Na (A) and thrombin–TBA–K (B) structures.
Figure 5.
Figure 5.
(A) Comparison between the two alternative binding modes of TBA: the one observed in the present structures (aptamer is colored in orange) and the one observed in thrombin–mTBA and 1HAO structures (aptamer is colored blue). Omit Fo–Fc electron density map (3σ level) of the TGT loop is also shown. (B) Zoomed vision of the TGT loop with its omit Fo–Fc electron density map (3σ level).
Figure 6.
Figure 6.
Different position of His71 with respect to Thy3 in thrombin–TBA–Na (purple) and thrombin–TBA–K (orange) upon superposition of (A) Thy3 and (B) protein backbone atoms.

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