Nucleotide channel of RNA-dependent RNA polymerase used for intermolecular uridylylation of protein primer.
Nucleotide channel of RNA-dependent RNA polymerase used for intermolecular uridylylation of protein primer.
复制标题
用于蛋白质引物分子间尿苷酰化的 RNA 依赖性 RNA 聚合酶的核苷酸通道。
DOI：
10.1016/j.jmb.2005.12.044
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发表时间：
2006
期刊：
影响因子：
--
通讯作者：
Kirkegaard,Karla
中科院分区：
文献类型：
--
作者：
Tellez,AndresB;Crowder,Scott;Spagnolo,JeannieF;Thompson,AaronA;Peersen,OlveB;Brutlag,DouglasL;Kirkegaard,Karla
脊髓灰质炎病毒VPg是一种22个氨基酸残基的肽，作为病毒RNA基因组复制的蛋白质引物。已知VPg直接与病毒RNA依赖性RNA聚合酶3D结合，用于共价尿苷酰化，产生单和二尿苷酰化产物VPg-pU和VPg-pUpU，其随后被延长。为了模拟VPg底物与3D聚合酶分子上假定的VPg结合位点的对接，我们进行了各种基于结构的计算，然后进行了实验验证。首先，鉴定了潜在的VPg折叠结构，产生了一套预测的β-发夹结构。这些推定的VPg结构，然后对接到该区域的聚合酶的基因实验所涉及的结合VPg，使用网格为基础的和片段为基础的方法。通过分子动力学模拟确定了预测影响结合的VPg中的残基，并通过计算和生物化学测试了它们对3D-VPg相互作用的影响。使用突变体VPg和突变体聚合酶分子的实验证实了在尿苷酰化反应期间聚合酶分子背面上的VPg的预测结合位点，与预测结合延伸RNA引物的结合位点相反。
Poliovirus VPg is a 22 amino acid residue peptide that serves as the protein primer for replication of the viral RNA genome. VPg is known to bind directly to the viral RNA-dependent RNA polymerase, 3D, for covalent uridylylation, yielding mono and di-uridylylated products, VPg-pU and VPg-pUpU, which are subsequently elongated. To model the docking of the VPg substrate to a putative VPg-binding site on the 3D polymerase molecule, we performed a variety of structure-based computations followed by experimental verification. First, potential VPg folded structures were identified, yielding a suite of predicted β-hairpin structures. These putative VPg structures were then docked to the region of the polymerase implicated by genetic experiments to bind VPg, using grid-based and fragment-based methods. Residues in VPg predicted to affect binding were identified through molecular dynamics simulations, and their effects on the 3D-VPg interaction were tested computationally and biochemically. Experiments with mutant VPg and mutant polymerase molecules confirmed the predicted binding site for VPg on the back side of the polymerase molecule during the uridylylation reaction, opposite to that predicted to bind elongating RNA primers.
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