Structural Dynamics as a Contributor to Error-prone Replication by an RNA-dependent RNA Polymerase
Structural Dynamics as a Contributor to Error-prone Replication by an RNA-dependent RNA Polymerase
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DOI：
10.1074/jbc.m114.616193
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发表时间：
2014-12-26
影响因子：
4.8
通讯作者：
Cameron, Craig E.
中科院分区：
文献类型：
--
作者：
Moustafa, Ibrahim M.;Korboukh, Victoria K.;Cameron, Craig E.
编码高保真或低保真RNA依赖性RNA聚合酶（RdRp）的RNA病毒被减毒。预测忠实掺入核苷酸所需的RdRp残基的能力代表了任何旨在利用扰动保真度作为合理设计候选疫苗基础的管道中的重要步骤。我们使用X射线晶体学，分子动力学模拟，核磁共振光谱，和前稳态动力学比较脊髓灰质炎病毒的突变体（H273 R）RdRp野生型（WT）酶。我们发现，核苷酸结合位点之间的核苷酸结合封闭和核苷酸结合能力的状态切换。这些状态之间的构象动力学通过与引发的模板RNA结合而增强。对于WT，封闭构象是有利的;对于H273 R，感受态构象是有利的。在我们的NMR光谱中Met-187的共振报告了酶检查结合核苷酸的正确性的能力。动力学实验与有助于建立的掺入前构象变化和保真度检查点的构象动力学一致。对于H273 R，包含活性位点的残基在催化活性构象中花费更多的时间，并且比WT更正相关。我们建议，通过连接的结合封闭和结合能力的构象的核苷酸结合口袋和其他活性位点的动态的正确性的结合核苷酸之间的平衡，忠实的核苷酸掺入实现。这些研究强调需要应用多种生物物理和生物化学方法来阐明聚合酶保真度的物理基础。
RNA viruses encoding high- or low-fidelity RNA-dependent RNA polymerases (RdRp) are attenuated. The ability to predict residues of the RdRp required for faithful incorporation of nucleotides represents an essential step in any pipeline intended to exploit perturbed fidelity as the basis for rational design of vaccine candidates. We used x-ray crystallography, molecular dynamics simulations, NMRspectroscopy, and pre-steady-state kinetics to compare a mutator (H273R) RdRp from poliovirus to the wild-type (WT) enzyme. We show that the nucleotide-binding site toggles between the nucleotide binding-occluded and nucleotide binding-competent states. The conformational dynamics between these states were enhanced by binding to primed template RNA. For the WT, the occluded conformation was favored; for H273R, the competent conformation was favored. The resonance for Met-187 in our NMR spectra reported on the ability of the enzyme to check the correctness of the bound nucleotide. Kinetic experiments were consistent with the conformational dynamics contributing to the established pre-incorporation conformational change and fidelity checkpoint. For H273R, residues comprising the active site spent more time in the catalytically competent conformation and were more positively correlated than the WT. We propose that by linking the equilibrium between the binding-occluded and binding-competent conformations of the nucleotide-binding pocket and other active-site dynamics to the correctness of the bound nucleotide, faithful nucleotide incorporation is achieved. These studies underscore the need to apply multiple biophysical and biochemical approaches to the elucidation of the physical basis for polymerase fidelity.