Structures of the first and second double-stranded RNA-binding domains of human TAR RNA-binding protein
Structures of the first and second double-stranded RNA-binding domains of human TAR RNA-binding protein
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DOI：
10.1002/pro.543
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发表时间：
2011-01-01
期刊：
影响因子：
8
通讯作者：
Yokoyama, Shigeyuki
中科院分区：
文献类型：
--
作者：
Yamashita, Seisuke;Nagata, Takashi;Yokoyama, Shigeyuki
TAR RNA结合蛋白（TRBP）是一种双链RNA（dsRNA）结合蛋白，它与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。第一和第二dsRBD（分别为dsRBD 1和dsRBD 2）对dsRNA具有亲和力，而第三dsRBD（dsRBD 3）结合Dicer。在本研究中，我们制备了对应于dsRBD 1和dsRBD 2的人TRBP单结构域片段，并解析了dsRBD 1的晶体结构和dsRBD 2的溶液结构。这两种结构含有α-β-α折叠，这是dsRBD共有的。dsRBD 1和dsRBD 2的整体结构彼此相似，除了第一个α螺旋的轻微移位。参与dsRNA结合的残基是保守的。我们研究了小干扰RNA（siRNA）的结合特性，这些dsRBD的等温滴定比色法测量。dsRBD 1和dsRBD 2片段均与siRNA结合，解离常数分别为220和113 nM。相比之下，全长TRBP及其与dsRBD 1和dsRBD 2的片段表现出小得多的解离常数（分别为0.24和0.25 nM），表明串联的dsRBD同时结合一个siRNA分子。另一方面，dsRBD 2的第一个α螺旋和第一个β链之间的环，而不是dsRBD 1，具有Trp残基，其与周围残基形成疏水性和阳离子π相互作用。圆二色性分析表明，dsRBD 2的热稳定性高于dsRBD 1，并依赖于Trp残基。
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 alpha-beta-beta-beta-alpha 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 alpha 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 alpha helix and the first beta strand of dsRBD2, but not dsRBD1, has a Trp residue, which forms hydrophobic and cation-pi 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.