The ins and outs of viral RNA polymerase translocation.
The ins and outs of viral RNA polymerase translocation.
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病毒 RNA 聚合酶易位的来龙去脉。
DOI：
10.1016/j.jmb.2013.12.030
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发表时间：
2014
影响因子：
5.6
通讯作者：
Boehr,DavidD
中科院分区：
文献类型：
--
作者：
Boehr,DavidD
RNA病毒复制的中心是RNA依赖的RNA聚合酶(RdRp)[1]，它是重要的抗病毒药物靶标[2-4]。RdRps属于模板导向的核酸聚合酶超家族，包括DNA聚合酶和逆转录酶[1]。这些聚合酶具有相似的结构折叠，被描述为具有手指、拇指和手掌亚域的“杯状右手”[5](图1)，以及类似的化学机制，涉及至少两个金属离子[6]。聚合酶的功能可以分为三个阶段，包括核苷酸选择、磷酸二酯键的形成和移位到下一个模板碱基，为下一轮核苷酸加成做准备。结构生物学为多类聚合酶的每个催化阶段的结构变化提供了巨大的洞察力[7-10]。最不了解的是聚合酶的构象变化如何与易位相结合。在A家族DNA聚合酶中，涉及“O-螺旋”和指亚区其他区域临时移位的活性部位的部分开放对于沿DNA的移动可能是重要的[11-15]。这样的机制不太可能帮助RdRp中的易位；这些聚合酶包含手指通过与拇指结构域相互作用的“指尖”延伸，很可能排除了活性部位的类似“打开”[16](图1)。在这一期中，Sholes和Peersen对RdRps中的另一种易位机制提供了诱人的见解，涉及一种称为“Motif-B”的手掌结构基序[17]。Motif-B环和模板RNA之间的空间碰撞可能会促进RNA沿着RNA移动到下一个催化寄存器。考虑到Motif-B环参与RdRp催化的多个阶段，并且可能是聚合酶功能的主要调节因子，这些发现特别有趣[18]。近年来，对RdRps[9，10，19]有了巨大的结构洞察力。特别是，Peersen实验室已经能够在延长复合体[20-22]中解析脊髓灰质炎病毒(PV)和其他病毒RdRp的多种X射线晶体结构，使他们能够捕获核苷酸添加前后RdRp的快照。与DNA聚合酶中的一些观察结果相比，X射线结晶学观察到的RdRps的构象变化相当微妙。手指和拇指亚区没有大的构象变化，相反，酶的激活需要手掌亚区的局部变化来重新定位对核苷酸结合和/或磷酸二酯键形成至关重要的关键残基[22]。正确的核苷酸结合诱导棕榈亚区β-链的重新对齐，包括结构基序A和基序C，导致绝对保守的Asp233(PV编号)的重新定位，以允许与RdRp功能所需的两种金属离子相互作用[22]。这种构象变化可能是当基序B残基Ser288和Asn297与进入的核苷酸的核糖羟基发生氢键作用时触发的。通过这种方式，RdRp的构象变化有效地将核苷酸选择与磷酸二酯键的形成结合在一起。
Central to the replication of RNA viruses is the RNA-dependent RNA polymerase (RdRp)[1], an important antiviral drug target [2–4]. RdRps belong to a large superfamily of template-directed nucleic acid polymerases, including DNA polymerases and reverse transcriptases [1]. These polymerases have a similar structural fold, which has been described as a “cupped right hand” with fingers, thumb and palm subdomains [5](Fig. 1), and a similar chemical mechanism, involving at least two metal ions [6]. Polymerase function can be divided into three phases, including nucleotide selection, phosphodiester bond formation and translocation to the next templating nucleobase to prepare for the next round of nucleotide addition. Structural biology has provided tremendous insight into the structural changes that accompany each stage of catalysis for multiple classes of polymerases [7–10]. Least understood is how conformational changes in the polymerases may couple to translocation. In A-family DNA polymerases, a partial opening of the active site involving the temporary displacement of the “O-helix” and other regions in the fingers subdomain is likely important for movement along the DNA [11–15]. Such a mechanism is unlikely to aid translocation in RdRps; these polymerases contain an extension of the fingers through the “fingertips” that interacts with the thumb domain, likely precluding a similar “opening” of the active site [16](Fig. 1). In this issue, Sholders and Peersen have provided tantalizing insight into an alternative mechanism for translocation in RdRps, involving a palm structural motif known as “motif-B”[17]. Steric clashes between the motif-B loop and the template RNA may promote movement along the RNA to the next catalytic register. These findings are especially intriguing considering that the motif-B loop is involved in multiple stages of RdRp catalysis and may be a master regulator of polymerase function [18].Recent years have brought tremendous structural insight into RdRps [9, 10, 19]. The Peersen laboratory, in particular, has been able to solve multiple X-ray crystal structures of poliovirus (PV) and other viral RdRps in elongation complexes [20–22], enabling them to capture snapshots of the RdRp before and after nucleotide addition. In contrast to some observations in DNA polymerases, the conformational changes in RdRps observed by X-ray crystallography are rather subtle. There are no grand conformational changes in the fingers and thumb subdomains, but instead, activation of the enzyme requires localized changes to the palm subdomain to reposition key residues important for nucleotide binding and/or phosphodiester bond formation [22]. Correct nucleotide binding induces a re-alignment of β-strands in the palm subdomain, including structural motif-A and motif-C, resulting in the repositioning of the absolutely conserved Asp233 (PV numbering) to allow interactions with both metal ions required for RdRp function [22]. This conformational change is likely triggered when motif-B residues Ser288 and Asn297 make hydrogen-bonding interactions with the ribose hydroxyls of the incoming nucleotide. In this way, conformational changes in the RdRp efficiently couple nucleotide selection to phosphodiester bond formation.