Structure of a low-population binding intermediate in protein-RNA recognition
Structure of a low-population binding intermediate in protein-RNA recognition
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DOI：
10.1073/pnas.1521349113
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发表时间：
2016-06-28
影响因子：
11.1
通讯作者：
Vendruscolo, Michele
中科院分区：
文献类型：
--
作者：
Borkar, Aditi N.;Bardaro, Michael F., Jr.;Vendruscolo, Michele
HIV-1蛋白质转录反式激活因子（达特）及其同源的反式激活应答元件（TAR）RNA的相互作用反式激活病毒转录，并且代表蛋白质-RNA识别中构象重排广泛发生的范例。虽然自由和结合形式的TAR的结构得到了很好的表征，在结合过程中的中间体的构象仍然是未知的。通过确定的自由能景观的复杂使用NMR残余偶极耦合在复制平均metadaptics模拟，我们观察到两个低人口中间体。然后，我们理性地设计了两个突变体，一个在蛋白质中，另一个在RNA中，它们削弱了稳定中间体之一的特定非天然相互作用。通过使用表面等离子体共振，我们表明，这些突变降低释放速率的达特，如预测的。这些结果确定了RNA-蛋白质结合的中间体的结构，并说明了以高分辨率实现这一目标的一般策略。
The interaction of the HIV-1 protein transactivator of transcription (Tat) and its cognate transactivation response element (TAR) RNA transactivates viral transcription and represents a paradigm for the widespread occurrence of conformational rearrangements in protein-RNA recognition. Although the structures of free and bound forms of TAR are well characterized, the conformations of the intermediates in the binding process are still unknown. By determining the free energy landscape of the complex using NMR residual dipolar couplings in replica-averaged metadynamics simulations, we observe two low-population intermediates. We then rationally design two mutants, one in the protein and another in the RNA, that weaken specific nonnative interactions that stabilize one of the intermediates. By using surface plasmon resonance, we show that these mutations lower the release rate of Tat, as predicted. These results identify the structure of an intermediate for RNA-protein binding and illustrate a general strategy to achieve this goal with high resolution.