Dynamic Allostery Controls Coat Protein Conformer Switching during MS2 Phage Assembly
Dynamic Allostery Controls Coat Protein Conformer Switching during MS2 Phage Assembly
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DOI：
10.1016/j.jmb.2009.11.016
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发表时间：
2010-02-05
影响因子：
5.6
通讯作者：
Twarock, R.
中科院分区：
文献类型：
--
作者：
Dykeman, E. C.;Stockley, P. G.;Twarock, R.
以前，RNA茎环（TR），包括19个核苷酸的噬菌体MS 2的基因组被证明是作为一个变构效应的外壳蛋白在体外衣壳组装过程中的构象转换。TR RNA与对称外壳蛋白二聚体的结合导致构象变化，主要是在连接每个亚基中的F和G β链的FG环处，产生不对称结构。FG环定义了T=3衣壳中外壳蛋白亚基（A、B和C）的准等价构象异构体。该衣壳的体外有效组装需要溶液中存在对称和不对称形式的外壳蛋白二聚体，这意味着它们与最终衣壳中观察到的准等效二聚体（A/B和C/C）非常相似。实验表明，装配可以触发由一些RNA茎环无关TR的序列和详细的二级结构，这表明有很少的序列特异性的变构效应。由于外壳蛋白二聚体上的茎环结合位点远离FG环，因此需要研究这种转换效应的机制。我们已经分析了TR结合和RNA自由外壳蛋白二聚体的振动模式，使用全原子正常模式分析。结果表明，A-双链体RNA磷酸二酯骨架和一个外壳蛋白亚基中的EF-环之间的不对称接触导致该亚基的FG-环变得更具动态性，而另一个单体上的等效环降低了其流动性。当采用准等价B构象时，增加的动态行为发生在需要经历最大构象变化的亚基的FG环中。因此，与未结合的亚基相比，形成这种新结构的途径上的自由能垒将降低。我们的研究结果还意味着，变构效应应该是独立的绑定茎环的碱基序列，如实验所观察到的。作为这个模型的测试，我们还研究了一个已知的组装突变体，W82 R，它不能组装超过二聚体的振动模式。该突变导致TR结合后DE环而不是FG环的迁移率增加，这与该突变蛋白的非组装表型一致。(C)2009爱思唯尔有限公司保留所有权利
Previously, an RNA stem-loop (TR) encompassing 19 nt of the genome of bacteriophage MS2 was shown to act as an allosteric effector of conformational switching in the coat protein during in vitro capsid assembly. TR RNA binding to symmetric coat protein dimers results in conformational changes, principally at the FG-loop connecting the F and G beta-strands in each subunit, yielding an asymmetric structure. The FG-loops define the quasi-equivalent conformers of the coat protein subunit (A, B, and C) in the T=3 capsid. Efficient assembly of this capsid in vitro requires that both symmetrical and asymmetrical forms of the coat protein dimer be present in solution, implying that they closely resemble the quasi-equivalent dimers (A/B and C/C) seen in the final capsid. Experiments show that assembly can be triggered by a number of RNA stem-loops unrelated to TR in sequence and detailed secondary structure, suggesting that there is little sequence specificity to the allosteric effect. Since the stem-loop binding site on the coat protein dimer is distal to the FG-loops the mechanism of this switching effect needs to be investigated. We have analyzed the vibrational modes of both TR-bound and RNA-free coat protein dimers using an all-atom normal-mode analysis. The results suggest that asymmetric contacts between the A-duplex RNA phosphodiester backbone and the EF-loop in one coat protein Subunit result in the FG-loop of that subunit becoming more dynamic, whilst the equivalent loop on the other monomer decreases its mobility. The increased dynamic behaviour occurs in the FG-loop of the subunit required to undergo the largest conformational change when adopting the quasi-equivalent B conformation. The free energy barrier on the pathway to form this new structure would consequently be reduced compared to the unbound subunit. Our results also imply that the allosteric effect should be independent of the base sequence of the bound stem-loop, as observed experimentally. As a test of this model, we also examined the vibrational modes of a known assembly mutant, W82R, which cannot assemble beyond dimer. This mutation leads to an increased mobility of the DE-loop rather than the FG-loop after TR binding, consistent with the non-assembling phenotype of this mutant protein. (C) 2009 Elsevier Ltd All rights reserved