Structures of coxsackievirus, rhinovirus, and poliovirus polymerase elongation complexes solved by engineering RNA mediated crystal contacts.
Structures of coxsackievirus, rhinovirus, and poliovirus polymerase elongation complexes solved by engineering RNA mediated crystal contacts.
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DOI：
10.1371/journal.pone.0060272
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Peersen OB
中科院分区：
文献类型：
--
作者：
Gong P;Kortus MG;Nix JC;Davis RE;Peersen OB
依赖RNA的RNA聚合酶在RNA病毒的生长中起着至关重要的作用，它们负责基因组的复制，但这样做的保真度相当低，允许快速适应不同的宿主细胞环境。这些聚合酶也是抗病毒药物开发的靶标。然而，由于缺乏关于功能性聚合酶- rna复合物的详细结构信息以及在延伸周期中发生的结构变化，药物发现工作和我们对保真度决定因素的理解都受到了阻碍。在我们最近的脊髓灰质炎病毒聚合酶- rna复合物的结构中揭示了与核苷酸选择和催化有关的许多分子细节，该结构首先纯化，然后结晶停滞伸长复合物。在这里提出的工作中，我们扩展了基本方法，以2.2-2.9 Å分辨率确定脊髓灰质炎病毒、柯萨奇病毒和鼻病毒延伸复合物的九种新结构。这些结构突出了小核糖核酸病毒聚合酶的保守特征，以及它们与模板和产物RNA链的相互作用，包括紧紧抓住新生双链的八个碱基对，完全预先定位的模板核苷酸，以及+2位置模板链碱基的保守结合袋。在活性位点，我们看到了一个预结合的镁离子，并且在相互作用中模板链的非标准主干构象保存，这可能有助于通过与保守聚合酶基序b的接触触发RNA易位。此外，通过将可塑性工程转化为RNA-RNA接触，我们获得了能够进行多轮晶内催化和RNA易位的晶体形式。总之，这些数据表明，工程柔性RNA接触促进晶格形成是一个通用的平台，可用于解决病毒RdRP延伸配合物及其催化循环中间体的结构。
RNA-dependent RNA polymerases play a vital role in the growth of RNA viruses where they are responsible for genome replication, but do so with rather low fidelity that allows for the rapid adaptation to different host cell environments. These polymerases are also a target for antiviral drug development. However, both drug discovery efforts and our understanding of fidelity determinants have been hampered by a lack of detailed structural information about functional polymerase-RNA complexes and the structural changes that take place during the elongation cycle. Many of the molecular details associated with nucleotide selection and catalysis were revealed in our recent structure of the poliovirus polymerase-RNA complex solved by first purifying and then crystallizing stalled elongation complexes. In the work presented here we extend that basic methodology to determine nine new structures of poliovirus, coxsackievirus, and rhinovirus elongation complexes at 2.2–2.9 Å resolution. The structures highlight conserved features of picornaviral polymerases and the interactions they make with the template and product RNA strands, including a tight grip on eight basepairs of the nascent duplex, a fully pre-positioned templating nucleotide, and a conserved binding pocket for the +2 position template strand base. At the active site we see a pre-bound magnesium ion and there is conservation of a non-standard backbone conformation of the template strand in an interaction that may aid in triggering RNA translocation via contact with the conserved polymerase motif B. Moreover, by engineering plasticity into RNA-RNA contacts, we obtain crystal forms that are capable of multiple rounds of in-crystal catalysis and RNA translocation. Together, the data demonstrate that engineering flexible RNA contacts to promote crystal lattice formation is a versatile platform that can be used to solve the structures of viral RdRP elongation complexes and their catalytic cycle intermediates.
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影响因子：
14.9
作者：
Lavery R;Moakher M;Maddocks JH;Petkeviciute D;Zakrzewska K
通讯作者：
Zakrzewska K
影响因子：
4.8
作者：
Ferrer-Orta, C;Arias, A;Verdaguer, N
通讯作者：
Verdaguer, N
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
4.8
作者：
Arnold, JJ;Cameron, CE
通讯作者：
Cameron, CE
影响因子：
3.7
作者：
Hong, Z;Cameron, CE;Zhong, WD
通讯作者：
Zhong, WD