Distinct conformations of a putative translocation element in poliovirus polymerase.
Distinct conformations of a putative translocation element in poliovirus polymerase.
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DOI：
10.1016/j.jmb.2013.12.031
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发表时间：
2014-04-03
影响因子：
5.6
通讯作者：
Peersen OB
中科院分区：
文献类型：
--
作者：
Sholders AJ;Peersen OB
RNA被单亚基病毒RNA依赖性RNA聚合酶转运的机制尚不清楚。这些酶缺乏“O-螺旋”结构和相关指结构域运动的同源物，这些结构域运动被认为是许多DNA模板聚合酶中易位的原因。多个小核糖核酸病毒聚合酶延伸复合物的结构表明，这些酶使用不同的分子机制，其中易位不强烈耦合到催化后的活性位点的开放。在这里，我们提出了2.0-2.6 μ m分辨率的晶体结构和生物化学数据的12个脊髓灰质炎病毒聚合酶突变体，共同显示如何适当的酶功能和易位活性需要在棕榈结构域B基序的环序列的构象灵活性。在环内，Ser 288-Gly 289-Cys 290序列被证明在基于RNA结合、进行性延伸活性和单核苷酸掺入测定的催化循环中起主要作用。结构表明，Ser 288与Asp 238形成关键的氢键，Gly 289的骨架柔性是易位能力所需的，Cys 290调节酶的整体延伸活性。环的一些构象代表形成催化活性封闭活性位点的途径上的可能中间体，而其他构象与促进新生碱基对从活性位点易位出来的作用一致。环结构和围绕它的关键残基是高度保守的，这表明我们在脊髓灰质炎病毒3Dpol中观察到的结构动力学是病毒RNA依赖性RNA聚合酶的共同特征。
The mechanism whereby RNA is translocated by the single subunit viral RNA-dependent RNA polymerases is not yet understood. These enzymes lack homologs of the “O-helix” structures and associated fingers domain movements thought to be responsible for translocation in many DNA-templated polymerases. The structures of multiple picornavirus polymerase elongation complexes suggest that these enzymes use a different molecular mechanism where translocation is not strongly coupled to the opening of the active site following catalysis. Here we present the 2.0–2.6 Å resolution crystal structures and biochemical data for twelve poliovirus polymerase mutants that together show how proper enzyme function and translocation activity requires conformational flexibility of a loop sequence in the palm domain B-motif. Within the loop, the Ser288–Gly289–Cys290 sequence is shown to play a major role in the catalytic cycle based on RNA binding, processive elongation activity, and single nucleotide incorporation assays. The structures show that Ser288 forms a key hydrogen bond with Asp238, the backbone flexibility of Gly289 is require for translocation competency, and Cys290 modulates the overall elongation activity of the enzyme. Some conformations of the loop represent likely intermediates on the way to forming the catalytically competent closed active site, while others are consistent with a role in promoting translocation of the nascent base pair out of the active site. The loop structure and key residues surrounding it are highly conserved, suggesting the structural dynamics we observe in poliovirus 3Dpol are a common feature of viral RNA-dependent RNA polymerases.