Nagata T, Kanno R, Kurihara Y, Uesugi S, Imai T, Sakakibara S, Okano H, Katahira M
Department of Chemistry and Biotechnology, Faculty of Engineering, Yokohama National University, 79-5 Tokiwadai, Hodogaya-ku, Yokohama, 240-8501, Japan.
J Mol Biol. 1999 Mar 26;287(2):315-30. doi: 10.1006/jmbi.1999.2596.
Musashi1 is an RNA-binding protein abundantly expressed in the developing mouse central nervous system. Its restricted expression in neural precursor cells suggests that it is involved in the regulation of asymmetric cell division. Musashi1 contains two ribonucleoprotein (RNP)-type RNA-binding domains (RBDs), RBD1 and RBD2. Our previous studies showed that RBD1 alone binds to RNA, while the binding of RBD2 is not detected under the same conditions. Joining of RBD2 to RBD1, however, increases the affinity to greater than that of RBD1 alone, indicating that RBD2 contributes to RNA-binding. We have determined the three-dimensional solution structure of the C-terminal RBD (RBD2) of Musashi1 by NMR. It folds into a compact alpha beta structure comprising a four-stranded antiparallel beta-sheet packed against two alpha-helices, which is characteristic of RNP-type RBDs. Special structural features of RBD2 include a beta-bulge in beta2 and a shallow twist of the beta-sheet. The smaller 1H-15N nuclear Overhauser enhancement values for the residues of loop 3 between beta2 and beta3 suggest that this loop is flexible in the time-scale of nano- to picosecond order. The smaller 15N T2 values for the residues around the border between alpha2 and the following loop (loop 5) suggest this region undergoes conformational exchange in the milli- to microsecond time-scale. Chemical shift perturbation analysis indicated that RBD2 binds to an RNA oligomer obtained by in vitro selection under the conditions for NMR measurements, and thus the nature of the weak RNA-binding of RBD2 was successfully characterized by NMR, which is otherwise difficult to assess. Mainly the residues of the surface composed of the four-stranded beta-sheet, loops and C-terminal region are involved in the interaction. The appearance of side-chain NH proton resonances of arginine residues of loop 3 and imino proton resonances of RNA bases upon complex formation suggests the formation of intermolecular hydrogen bonds. The structural arrangement of the rings of the conserved aromatic residues of beta2 and beta3 is suitable for stacking interaction with RNA bases, known to be one of the major protein-RNA interactions, but a survey of the perturbation data suggested that the stacking interaction is not ideally achieved in the complex, which may be related to the weaker RNA-binding of RBD2.
Musashi1是一种在发育中的小鼠中枢神经系统中大量表达的RNA结合蛋白。它在神经前体细胞中的特异性表达表明其参与不对称细胞分裂的调控。Musashi1包含两个核糖核蛋白（RNP）型RNA结合结构域（RBD），即RBD1和RBD2。我们之前的研究表明，单独的RBD1能与RNA结合，而在相同条件下未检测到RBD2的结合。然而，将RBD2与RBD1连接后，其对RNA的亲和力比单独的RBD1更高，这表明RBD2有助于RNA结合。我们通过核磁共振（NMR）确定了Musashi1的C端RBD（RBD2）的三维溶液结构。它折叠成一个紧凑的αβ结构，由一个四链反平行β折叠片与两个α螺旋堆积而成，这是RNP型RBD的特征。RBD2的特殊结构特征包括β2中的β凸起和β折叠片的浅扭曲。β2和β3之间环3残基的较小的1H-15N核Overhauser增强值表明该环在纳秒到皮秒时间尺度上是灵活的。α2与后续环（环5）边界周围残基的较小的15N T2值表明该区域在毫秒到微秒时间尺度上经历构象交换。化学位移扰动分析表明，在核磁共振测量条件下，RBD2与通过体外筛选获得的RNA寡聚物结合，因此通过核磁共振成功表征了RBD2弱RNA结合的性质，否则很难评估。主要是由四链β折叠片、环和C端区域组成的表面残基参与了相互作用。复合物形成时环3精氨酸残基的侧链NH质子共振和RNA碱基的亚氨基质子共振的出现表明形成了分子间氢键。β2和β3保守芳香族残基环的结构排列适合与RNA碱基进行堆积相互作用，已知这是主要的蛋白质-RNA相互作用之一，但对扰动数据的研究表明，复合物中堆积相互作用并未理想实现，这可能与RBD2较弱的RNA结合有关。
