Structural analysis of the hepatitis C virus RNA polymerase in complex with Ribonucleotides
Structural analysis of the hepatitis C virus RNA polymerase in complex with Ribonucleotides
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DOI：
10.1128/jvi.76.7.3482-3492.2002
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发表时间：
2002-04-01
影响因子：
5.4
通讯作者：
De Francesco, R
中科院分区：
文献类型：
--
作者：
Bressanelli, S;Tomei, L;De Francesco, R
我们在这里报告的结果，一个系统的高分辨率X-射线晶体学分析的复合物的丙型肝炎病毒（HCV）RNA聚合酶与核糖核苷三磷酸（rNTPs）和二价金属离子。这项研究揭示的一个意想不到的观察结果是在酶分子表面的浅口袋中存在特异性rGTP结合位点，距离催化位点30埃。这个以前未识别的rGTP口袋，它位于手指和拇指之间的界面，可能是一个变构调节位点，并可能发挥作用，允许两个域之间的替代相互作用，在可能的构象变化的酶所需的有效启动。1.7埃分辨率的电子密度图清楚地显示了鸟苷部分与酶的结合模式。在催化位点中，与结合到催化金属的核苷酸的三磷酸盐对应的密度在具有核苷酸的每个复合物中是明显的。此外，检测到三磷酸密度的网络;这些密度叠加到在噬菌体phi 6的聚合酶的起始复合物中观察到的核苷酸的相应部分，加强了这两种酶通过类似机制从头开始复制的建议。然而，在HCV聚合酶中不存在对于phi 6酶中的引发核苷酸观察到的蛋白质堆积平台的等同物，这再次表明在模板结合后拇指结构域的构象发生变化以允许RNA合成的有效从头起始。
We report here the results of a systematic high-resolution X-ray crystallographic analysis of complexes of the hepatitis C virus (HCV) RNA polymerase with ribonucleoside triphosphates (rNTPs) and divalent metal ions. An unexpected observation revealed by this study is the existence of a specific rGTP binding site in a shallow pocket at the molecular surface of the enzyme, 30 Angstrom away from the catalytic site. This previously unidentified rGTP pocket, which lies at the interface between fingers and thumb, may be an allosteric regulatory site and could play a role in allowing alternative interactions between the two domains during a possible conformational change of the enzyme required for efficient initiation. The, electron density map at 1.7-Angstrom resolution clearly shows the mode of binding of the guanosine moiety to the enzyme. In the catalytic site, density corresponding to the triphosphates of nucleotides bound to the catalytic metals was apparent in each complex with nucleotides. Moreover, a network of triphosphate densities was detected; these densities superpose to the corresponding moieties of the nucleotides observed in the initiation complex reported for the polymerase of bacteriophage phi6, strengthening the proposal that the two enzymes initiate replication de novo by similar mechanisms. No equivalent of the protein stacking platform observed for the priming nucleotide in the phi6 enzyme is present in HCV polymerase, however, again suggesting that a change in conformation of the thumb domain takes place upon template binding to allow for efficient de novo initiation of RNA synthesis.