Drug design based on the complex structure of HIV-Tat and its RNA aptamer

含有两个结合位点的RNA适体对HIV Tat蛋白具有极高的亲和力。我们现在已经通过核磁共振确定了与精氨酸酰胺络合的适体的结构，精氨酸酰胺是Tat蛋白最简单的类似物。我们发现每个适体结合两个精氨酸酰胺分子。两个相邻的U: A: U碱基三元组形成，这使得主槽变宽，为两个精氨酸酰胺分子腾出空间。精氨酸酰胺分子通过氢键与G基结合。通过堆叠相互作用使结合稳定。该适配体与一种由tat衍生的富含精氨酸肽络合而成的结构也被表征。tat衍生的肽以1:1的摩尔比与适体结合。尽管在化学计量学上存在差异，但在复合物形成和分子间接触时适体的化学位移扰动中，精氨酸酰胺和肽结合形式的适体是相似的。这些结果表明，肽的两个不同的精氨酸残基与适配体的两个结合位点相互作用的方式与两个精氨酸酰胺分子相同。该适体与Tat的两个精氨酸残基同时相互作用，可以解释其与真实的TAR RNA相比具有高亲和力。两个相邻碱基三元组的形成通过为两个精氨酸残基和Tat的连接残基的容纳创造空间，使得同时相互作用成为可能，并且也有助于通过堆叠相互作用稳定结合。该适体结构的阐明为合理设计对Tat具有更高亲和力的新适体提供了线索。该结构也为设计一种副作用较小的新型适配体提供了途径。

An RNA aptamer containing two binding sites exhibits extremely high affinity to the HIV Tat protein. We have now determined the structure of the aptamer complexed with argininamide, the simplest analogue of the Tat protein, by NMR. We found that two argininamide molecules bind per aptamer. Two adjacent U : A : U base triples were formed, which widens the major groove to make space for the two argininamide molecules. The argininamide molecules bound to the G bases through hydrogen bonds. The binding is stabilized through stacking interactions.The structure of the aptamer complexed with a Tat-derived argmine-rich peptide was also characterized. The Tat-derived peptide bound to the aptamer in a 1: 1 molar ratio. In spite of the difference in stoichiometry, similarity was noted between the argininamide- and peptide-bound forms of the aptamer in chemical shift perturbations of the aptamer upon complex formation and intermolecular contacts. These results suggest that two different arginine residues of the peptide interact with the two binding sites of the aptamer in the same way as two argininamide molecules do. Simultaneous interactions of the aptamer with two arginine residues of Tat could explain its high affinity compared to the authentic TAR RNA. The formation of the two adjacent base triples makes the simultaneous interactions possible by creating space for the accommodation of two arginine residues and linking residues of Tat, and also contributes to the stabilization of the binding through stacking interactions.The elucidated structure of the aptamer gives clues to rationally design a new aptamer that exhibits even higher affinity to Tat. The way to design a new aptamer with a less side effect is also provided by the elucidated structure.