Dynamic ensemble of HIV-1 RRE stem IIB reveals non-native conformations that disrupt the Rev-binding site
Dynamic ensemble of HIV-1 RRE stem IIB reveals non-native conformations that disrupt the Rev-binding site
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DOI：
10.1093/nar/gkz498
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发表时间：
2019-07-26
影响因子：
14.9
通讯作者：
Al-Hashimi, Hashim M.
中科院分区：
文献类型：
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作者：
Chu, Chia-Chieh;Plangger, Raphael;Al-Hashimi, Hashim M.
HIV-1 Rev response element (RRE) RNA元件通过与病毒蛋白Rev形成寡聚复合物介导含内含子的病毒RNA的核输出。通过两个Rev分子的协同结合，Stem IIB和附近的Stem II三方结核寡聚化。RRE区域的构象柔韧性已被证明对Rev结合很重要。然而，这种灵活性的本质仍然难以捉摸。在这里，使用核磁共振弛散，包括一种直接观察大rna瞬态构象状态的新策略，我们发现stem IIB单独存在，或者当较大的RREII三向结的一部分与非天然激发态（ES）构象稳定地存在于动态平衡中，这些构象的总数约为20%。ESs通过改变局部二级结构破坏Rev-结合位点，并且通过点取代突变使其稳定性降低了与Rev- arginine-rich motif （ARM）的结合亲和力，降低了15- 80倍。该集合阐明了在茎IIB中观察到的构象灵活性，揭示了茎IIB与可能在协同Rev结合中发挥作用的三向结之间的远程构象耦合，并确定了非天然RRE构象状态作为开发抗hiv治疗的新靶点。
The HIV-1 Rev response element (RRE) RNA element mediates the nuclear export of intron containing viral RNAs by forming an oligomeric complex with the viral protein Rev. Stem IIB and nearby stem II three-way junction nucleate oligomerization through cooperative binding of two Rev molecules. Conformational flexibility at this RRE region has been shown to be important for Rev binding. However, the nature of the flexibility has remained elusive. Here, using NMR relaxation dispersion, including a new strategy for directly observing transient conformational states in large RNAs, we find that stem IIB alone or when part of the larger RREII three-way junction robustly exists in dynamic equilibrium with non-native excited state (ES) conformations that have a combined population of similar to 20%. The ESs disrupt the Rev-binding site by changing local secondary structure, and their stabilization via point substitution mutations decreases the binding affinity to the Rev arginine-rich motif (ARM) by 15- to 80-fold. The ensemble clarifies the conformational flexibility observed in stem IIB, reveals long-range conformational coupling between stem IIB and the three-way junction that may play roles in cooperative Rev binding, and also identifies non-native RRE conformational states as new targets for the development of anti-HIV therapeutics.