环状寡腺苷酸与转录因子 Csa3 结合的结构基础概述了 III 型和 I 型 CRISPR 系统之间的串扰。,Journal of Biological Chemistry

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环状寡腺苷酸与转录因子 Csa3 结合的结构基础概述了 III 型和 I 型 CRISPR 系统之间的串扰。
Journal of Biological Chemistry ( IF 4.0 ) Pub Date : 2022-01-14 , DOI: 10.1016/j.jbc.2022.101591
Pengjun Xia ₁ , Anirudha Dutta ₂ , Kushol Gupta ₃ , Mona Batish ₄ , Vijay Parashar ₄

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III 型 CRISPR 系统的 RNA 干扰导致环状寡腺苷酸 (coA) 第二信使的合成，已知该第二信使结合并调节各种含有 CARF 结构域的核酸酶受体。含有 CARF 结构域的转录因子 Csa3 家族与 DNA 靶向 I 型 CRISPR 系统相关，调节许多原核生物中各种 CRISPR 和 DNA 修复基因的表达。在这项研究中，我们通过证明环四腺苷酸 (cA4) 与硫磺糖酵母 Csa3 (Csa3Sso) 的特异性结合，将已知的 cOA 信使受体库扩展到包括转录因子。我们对 cA4 结合的全长 Csa3Sso 的 2.0-Å 分辨率 X 射线晶体结构揭示了其 CARF 结构域与 cA4 的细长构象的结合。使用针对观察到的Csa3Sso·cA4结构界面的Csa3Sso突变体的cA4结合亲和力分析，我们鉴定了与更广泛保守的cOA结合CARF基序不同的Csa3特异性cA4结合基序。使用合理的表面工程方法，我们将 Csa3Sso 的 cA4 结合亲和力提高到野生型的约 145 倍，这对于未来第二信使驱动的 CRISPR 基因表达和编辑系统具有潜在的应用。我们的溶液内 Csa3Sso 结构分析确定了 cA4 诱导的 C 端翼状螺旋-转角-螺旋效应结构域的变构和不对称构象重排，这可能与 DNA 结合不相容。然而，纯化的 Csa3Sso 与其推定启动子 (PCas4a) 的体外特异性结合被发现不依赖于 cA4，这表明 Csa3Sso 调节的复杂模式。总体而言，我们的结果支持 cA4 和 Csa3 介导的 III 型和 I 型 CRISPR 系统之间的串扰。

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Structural basis of cyclic oligoadenylate binding to the transcription factor Csa3 outlines cross talk between type III and type I CRISPR systems.

RNA interference by type III CRISPR systems results in the synthesis of cyclic oligoadenylate (cOA) second messengers, which are known to bind and regulate various CARF domain-containing nuclease receptors. The CARF domain-containing Csa3 family of transcriptional factors associated with the DNA-targeting type I CRISPR systems regulate expression of various CRISPR and DNA repair genes in many prokaryotes. In this study, we extend the known receptor repertoire of cOA messengers to include transcriptional factors by demonstrating specific binding of cyclic tetra-adenylate (cA4) to Saccharolobus solfataricus Csa3 (Csa3Sso). Our 2.0-Å resolution X-ray crystal structure of cA4-bound full-length Csa3Sso reveals the binding of its CARF domain to an elongated conformation of cA4. Using cA4 binding affinity analyses of Csa3Sso mutants targeting the observed Csa3Sso•cA4 structural interface, we identified a Csa3-specific cA4 binding motif distinct from a more widely conserved cOA-binding CARF motif. Using a rational surface engineering approach, we increased the cA4 binding affinity of Csa3Sso up to ∼145-fold over the wildtype, which has potential applications for future second messenger-driven CRISPR gene expression and editing systems. Our in-solution Csa3Sso structural analysis identified cA4-induced allosteric and asymmetric conformational rearrangement of its C-terminal winged helix-turn-helix effector domains, which could potentially be incompatible to DNA binding. However, specific in vitro binding of the purified Csa3Sso to its putative promoter (PCas4a) was found to be cA4 independent, suggesting a complex mode of Csa3Sso regulation. Overall, our results support cA4-and Csa3-mediated cross talk between type III and type I CRISPR systems.

更新日期：2022-01-14

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