Nucleic-acid-triggered NADase activation of a short prokaryotic Argonaute
Nucleic-acid-triggered NADase activation of a short prokaryotic Argonaute
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DOI：
10.1038/s41586-023-06665-6
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发表时间：
2023-10-02
期刊：
影响因子：
64.8
通讯作者：
Cui, Sheng
中科院分区：
文献类型：
--
作者：
Gao, Xiaopan;Shang, Kun;Cui, Sheng
Argonaute (Ago) 蛋白介导 RNA 或 DNA 引导的核酸抑制1,2。尽管真核 Ago 蛋白和长原核 Ago 蛋白 (pAgos) 使用的机制是已知的，但短 pAgos 使用的机制仍然难以捉摸。在这里，我们确定了来自 Crenotalea thermophila (Crt) 的短 pAgo 和相关 TIR-APAZ 蛋白 (SPARTA) 的冷冻电子显微镜结构：游离态 Crt-SPARTA；负载引导 RNA 的 DNA 的 Crt-SPARTA；两个具有不同 TIR 组织的 Crt-SPARTA 二聚体；和 Crt-SPARTA 四聚体。这些结构表明，Crt-SPARTA 由与 TIR 叶相连的双叶折叠 Ago 叶组成。 Crt-Ago 包含 MID 和 PIWI 结构域，而 Crt-TIR-APAZ 则具有 TIR 结构域、N 样结构域、链接结构域和触发结构域。结合的 RNA-DNA 双链体采用 B 型构象，可被碱基特异性接触识别。核酸结合会引起构象变化，因为触发结构域充当“路障”，阻止向导 RNA 5' 末端和目标 DNA 3' 末端到达其规范口袋；这会扰乱 MID 结构域并促进 Crt-SPARTA 二聚化。两个装载 RNA-DNA 的 Crt-SPARTA 二聚体通过其 TIR 结构域形成四聚体。四个 Crt-TIR 结构域组装成两个平行的头尾相连的 TIR 二聚体，表明 NADase 活性构象，这得到了我们的诱变研究的支持。我们的研究结果揭示了短pAgo介导的针对入侵核酸的防御的结构基础，并为优化基于SPARTA的可编程DNA序列的检测提供了见解。短原核Argonaute (pAgo)和由指导RNA和靶DNA结合的相关TIR-APAZ蛋白的冷冻电镜结构揭示了pAgo系统的组装和激活机制。
Argonaute (Ago) proteins mediate RNA- or DNA-guided inhibition of nucleic acids1,2. Although the mechanisms used by eukaryotic Ago proteins and long prokaryotic Ago proteins (pAgos) are known, that used by short pAgos remains elusive. Here we determined the cryo-electron microscopy structures of a short pAgo and the associated TIR-APAZ proteins (SPARTA) from Crenotalea thermophila (Crt): a free-state Crt-SPARTA; a guide RNA-target DNA-loaded Crt-SPARTA; two Crt-SPARTA dimers with distinct TIR organization; and a Crt-SPARTA tetramer. These structures reveal that Crt-SPARTA is composed of a bilobal-fold Ago lobe that connects with a TIR lobe. Whereas the Crt-Ago contains a MID and a PIWI domain, Crt-TIR-APAZ has a TIR domain, an N-like domain, a linker domain and a trigger domain. The bound RNA-DNA duplex adopts a B-form conformation that is recognized by base-specific contacts. Nucleic acid binding causes conformational changes because the trigger domain acts as a 'roadblock' that prevents the guide RNA 5 ' ends and the target DNA 3 ' ends from reaching their canonical pockets; this disorders the MID domain and promotes Crt-SPARTA dimerization. Two RNA-DNA-loaded Crt-SPARTA dimers form a tetramer through their TIR domains. Four Crt-TIR domains assemble into two parallel head-to-tail-organized TIR dimers, indicating an NADase-active conformation, which is supported by our mutagenesis study. Our results reveal the structural basis of short-pAgo-mediated defence against invading nucleic acids, and provide insights for optimizing the detection of SPARTA-based programmable DNA sequences.Cryo-electron microscopy structures of a short prokaryotic Argonaute (pAgo) and the associated TIR-APAZ proteins bound by guide RNA and target DNA shed light on the mechanisms of assembly and activation of pAgo systems.