Transient kinetic studies of the antiviral Drosophila Dicer-2 reveal roles of ATP in self-nonself discrimination.
Transient kinetic studies of the antiviral Drosophila Dicer-2 reveal roles of ATP in self-nonself discrimination.
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DOI：
10.7554/elife.65810
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发表时间：
2021-03-31
期刊：
影响因子：
7.7
通讯作者：
Bass BL
中科院分区：
文献类型：
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作者：
Singh RK;Jonely M;Leslie E;Rejali NA;Noriega R;Bass BL
一些RIG-I样受体（RIG-I-like receptor，RLR）通过末端区分病毒和细胞dsRNA，并且果蝇Dicer-2（Drosophila melanogaster Dicer-2，dmDcr-2）通过平端或2个核苷酸的3 '-悬垂末端来差异处理dsRNA。我们采用快速反应停流技术和时间分辨荧光光谱研究了dmDcr-2反应的瞬态动力学机制。事实上，我们发现ATP与dmDcr-2的解旋酶结构域的结合以末端依赖性方式影响dsRNA的缔合和解离动力学，揭示了dsRNA在生物学相关时间尺度（秒）上的末端依赖性辨别。ATP水解促进dsRNA末端的瞬时解旋，随后缓慢重绕，并将酶定向易位至切割位点。时间分辨荧光各向异性揭示了与末端依赖性dsRNA切割相关的解旋酶和平台-PAZ结构域的构象波动（纳秒）中的核苷酸依赖性调制。我们的研究提供了一个动力学框架，用于与其他Dicers以及参与先天免疫的所有RLR成员进行比较。
Some RIG-I-like receptors (RLRs) discriminate viral and cellular dsRNA by their termini, and Drosophila melanogaster 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.