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抗病毒 Dicer-2 的瞬态动力学研究揭示了 ATP 在自我-非我识别中的作用。

Transient kinetic studies of the antiviral Dicer-2 reveal roles of ATP in self-nonself discrimination.

机构信息

Department of Biochemistry, University of Utah, Salt Lake City, United States.

Department of Chemistry, University of Utah, Salt Lake City, United States.

出版信息

Elife. 2021 Mar 31;10:e65810. doi: 10.7554/eLife.65810.

DOI:10.7554/eLife.65810
PMID:33787495
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC8079148/
Abstract

Some RIG-I-like receptors (RLRs) discriminate viral and cellular dsRNA by their termini, and Dicer-2 (dmDcr-2) differentially processes dsRNA with blunt or 2 nucleotide 3'-overhanging termini. We investigated the transient kinetic mechanism of the dmDcr-2 reaction using a rapid reaction stopped-flow technique and time-resolved fluorescence spectroscopy. Indeed, we found that ATP binding to dmDcr-2's helicase domain impacts association and dissociation kinetics of dsRNA in a termini-dependent manner, revealing termini-dependent discrimination of dsRNA on a biologically relevant time scale (seconds). ATP hydrolysis promotes transient unwinding of dsRNA termini followed by slow rewinding, and directional translocation of the enzyme to the cleavage site. Time-resolved fluorescence anisotropy reveals a nucleotide-dependent modulation in conformational fluctuations (nanoseconds) of the helicase and Platform-PAZ domains that is correlated with termini-dependent dsRNA cleavage. Our study offers a kinetic framework for comparison to other Dicers, as well as all members of the RLRs involved in innate immunity.

摘要

一些 RIG-I 样受体 (RLRs) 通过其末端区分病毒和细胞的 dsRNA，而 Dicer-2 (dmDcr-2) 则以不同的方式处理具有平头或 2 个核苷酸 3'-突出末端的 dsRNA。我们使用快速反应停流技术和时间分辨荧光光谱法研究了 dmDcr-2 反应的瞬态动力学机制。事实上，我们发现 ATP 结合到 dmDcr-2 的解旋酶结构域以依赖末端的方式影响 dsRNA 的结合和解离动力学，从而揭示了 dsRNA 在生物学相关时间尺度（秒）上的依赖末端的区分。ATP 水解促进 dsRNA 末端的短暂解旋，随后缓慢重绕，并且酶向切割位点的定向易位。时间分辨荧光各向异性揭示了解旋酶和 Platform-PAZ 结构域构象波动（纳秒）的核苷酸依赖性调制，该调制与 dsRNA 切割的末端依赖性相关。我们的研究为与其他 Dicer 以及参与先天免疫的所有 RLRs 成员进行比较提供了一个动力学框架。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/370f/8079148/c60661e6c113/elife-65810-fig7.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/370f/8079148/cbc8975750c4/elife-65810-fig6-figsupp2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/370f/8079148/c60661e6c113/elife-65810-fig7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/370f/8079148/e95f7b58a6b6/elife-65810-fig1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/370f/8079148/9b8d558a24aa/elife-65810-fig1-figsupp1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/370f/8079148/e269befa04e0/elife-65810-fig1-figsupp2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/370f/8079148/2ac129c944bd/elife-65810-fig2.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/370f/8079148/0c12ebe4c876/elife-65810-fig5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/370f/8079148/6203543de621/elife-65810-fig5-figsupp1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/370f/8079148/b685a83af367/elife-65810-fig5-figsupp2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/370f/8079148/0a13b65e961f/elife-65810-fig6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/370f/8079148/eec1e7533f56/elife-65810-fig6-figsupp1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/370f/8079148/cbc8975750c4/elife-65810-fig6-figsupp2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/370f/8079148/c60661e6c113/elife-65810-fig7.jpg

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