RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
Protein Sci. 2011 Jan;20(1):118-30. doi: 10.1002/pro.543.
The TAR RNA-binding Protein (TRBP) is a double-stranded RNA (dsRNA)-binding protein, which binds to Dicer and is required for the RNA interference pathway. TRBP consists of three dsRNA-binding domains (dsRBDs). The first and second dsRBDs (dsRBD1 and dsRBD2, respectively) have affinities for dsRNA, whereas the third dsRBD (dsRBD3) binds to Dicer. In this study, we prepared the single domain fragments of human TRBP corresponding to dsRBD1 and dsRBD2 and solved the crystal structure of dsRBD1 and the solution structure of dsRBD2. The two structures contain an α-β-β-β-α fold, which is common to the dsRBDs. The overall structures of dsRBD1 and dsRBD2 are similar to each other, except for a slight shift of the first α helix. The residues involved in dsRNA binding are conserved. We examined the small interfering RNA (siRNA)-binding properties of these dsRBDs by isothermal titration colorimetry measurements. The dsRBD1 and dsRBD2 fragments both bound to siRNA, with dissociation constants of 220 and 113 nM, respectively. In contrast, the full-length TRBP and its fragment with dsRBD1 and dsRBD2 exhibited much smaller dissociation constants (0.24 and 0.25 nM, respectively), indicating that the tandem dsRBDs bind simultaneously to one siRNA molecule. On the other hand, the loop between the first α helix and the first β strand of dsRBD2, but not dsRBD1, has a Trp residue, which forms hydrophobic and cation-π interactions with the surrounding residues. A circular dichroism analysis revealed that the thermal stability of dsRBD2 is higher than that of dsRBD1 and depends on the Trp residue.
TAR RNA 结合蛋白（TRBP）是一种双链 RNA（dsRNA）结合蛋白，可与 Dicer 结合，是 RNA 干扰途径所必需的。TRBP 由三个 dsRNA 结合结构域（dsRBD）组成。第一个和第二个 dsRBD（分别称为 dsRBD1 和 dsRBD2）对 dsRNA 具有亲和力，而第三个 dsRBD（dsRBD3）与 Dicer 结合。在这项研究中，我们制备了与人 TRBP 相对应的 dsRBD1 和 dsRBD2 的单结构域片段，并解析了 dsRBD1 的晶体结构和 dsRBD2 的溶液结构。这两个结构都包含一个α-β-β-β-α 折叠，这是 dsRBD 所共有的。dsRBD1 和 dsRBD2 的整体结构彼此相似，除了第一个α螺旋稍有移动。dsRNA 结合的残基保守。我们通过等温滴定量热法测量研究了这些 dsRBD 对小干扰 RNA（siRNA）的结合特性。dsRBD1 和 dsRBD2 片段都与 siRNA 结合，解离常数分别为 220 和 113 nM。相比之下，全长 TRBP 及其包含 dsRBD1 和 dsRBD2 的片段表现出更小的解离常数（分别为 0.24 和 0.25 nM），表明串联 dsRBD 同时结合一个 siRNA 分子。另一方面，dsRBD2 中第一个α螺旋和第一个β链之间的环，但不是 dsRBD1 ，具有一个色氨酸残基，它与周围残基形成疏水和阳离子-π 相互作用。圆二色性分析表明，dsRBD2 的热稳定性高于 dsRBD1，并且依赖于色氨酸残基。
