Crystallographic and modeling studies of RNase III suggest a mechanism for double-stranded RNA cleavage
Crystallographic and modeling studies of RNase III suggest a mechanism for double-stranded RNA cleavage
复制标题
DOI：
10.1016/s0969-2126(01)00685-2
复制
发表时间：
2001-12-01
期刊：
影响因子：
5.7
通讯作者：
Ji, XH
中科院分区：
文献类型：
--
作者：
Blaszczyk, J;Tropea, JE;Ji, XH
背景：aquifex aeolicus riboniclease III（AA-RNase III）属于Mg2（+） - 依赖性核酸内切酶的家族，这些家族显示出对双链RNA（DSRNA）的特异性。 RNase III在所有已知细菌和真核生物中均保守，并具有9个残留共识序列的1-2副本，称为RNase III签名基序。细菌RNase III蛋白是最简单的，由两个结构域组成：N末端核酸酶结构域，其次是双链RNA结合结构域（DSRBD）。大肠杆菌III中DSRBD的三维结构已阐明。没有任何RNase III的核酸内切酶域可用的结构信息：我们介绍了无配体形式的AA-RNase III核酸内切酶域的晶体结构，并与MN2+复杂。这些结构揭示了一种新型的蛋白质折叠，并提出了DSRNA裂解的机制。在结构，遗传和生物学数据的基础上，我们构建了与DsRNA和MG2+离子复合物中AA-RNase III的假设模型，该模型在作用中提供了RNase III的首次瞥见。结论：功能性AA-RNase III III二聚体是通过主要是疏水相互作用形成的，包括“球插座”结，可确保两个单体的准确对齐。多肽链的褶皱及其二聚化在山谷的两端创建了一个带有两个复合活性中心的山谷。山谷可以容纳DSRNA底物。 MN2+结合对晶体堆积，分子间相互作用，热稳定性以及每个化合物活跃中心内两个RNA接扣位点的形成具有显着影响。
Background: Aquifex aeolicus Ribonuclease III (Aa-RNase III) belongs to the family of Mg2(+)-dependent endonucleases that show specificity for double-stranded RNA (dsRNA). RNase III is conserved in all known bacteria and eukaryotes and has 1-2 copies of a 9-residue consensus sequence, known as the RNase III signature motif. The bacterial RNase III proteins are the simplest, consisting of two domains: an N-terminal endonuclease domain, followed by a double-stranded RNA binding domain (dsRBD). The three-dimensional structure of the dsRBD in Escherichia coli RNase III has been elucidated; no structural information is available for the endonuclease domain of any RNase III.Results: We present the crystal structures of the Aa-RNase III endonuclease domain in its ligand-free form and in complex with Mn2+. The structures reveal a novel protein fold and suggest a mechanism for dsRNA cleavage. On the basis of structural, genetic, and biological data, we have constructed a hypothetical model of Aa-RNase III in complex with dsRNA and Mg2+ ion, which provides the first glimpse of RNase III in action.Conclusions: The functional Aa-RNase III dimer is formed via mainly hydrophobic interactions, including a "ball-and-socket" junction that ensures accurate alignment of the two monomers. The fold of the polypeptide chain and its dimerization create a valley with two compound active centers at each end of the valley. The valley can accommodate a dsRNA substrate. Mn2+ binding has significant impact on crystal packing, intermolecular interactions, thermal stability, and the formation of two RNA-cuffing sites within each compound active center.