Structural basis of cyclic oligoadenylate binding to the transcription factor Csa3 outlines cross talk between type III and type I CRISPR systems.
Structural basis of cyclic oligoadenylate binding to the transcription factor Csa3 outlines cross talk between type III and type I CRISPR systems.
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DOI：
10.1016/j.jbc.2022.101591
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发表时间：
2022-03
期刊：
影响因子：
--
通讯作者：
Parashar V
中科院分区：
文献类型：
--
作者：
Xia P;Dutta A;Gupta K;Batish M;Parashar V
III型CRISPR系统的RNA干扰导致环低聚腺苷酸（cOA）第二信使的合成，已知其结合并调节各种CARF结构域核酸酶受体。含有CARF结构域的Csa3转录因子家族与DNA靶向I型CRISPR系统相关，在许多原核生物中调节各种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Saccharolobus solfataricus Csa3 （Csa3Sso）的特异性结合，扩展了已知的cOA信使受体库，以包括转录因子。我们的2.0-Å分辨率的cA4结合全长Csa3Sso的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。通过对Csa3Sso突变体针对Csa3Sso•cA4结构界面的cA4结合亲和力分析，我们发现了一个csa3特异性的cA4结合基序，不同于更广泛保守的coa结合CARF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，这在未来的第二信使驱动CRISPR基因表达和编辑系统中具有潜在的应用前景。我们在溶液中对Csa3Sso进行了结构分析，发现ca4诱导其c端有翼螺旋-螺旋-螺旋效应域的变构重排和不对称构象重排，这可能与DNA结合不相容。然而，纯化的Csa3Sso在体外特异性结合其假定的启动子（PCas4a）被发现是cA4独立的，表明Csa3Sso调控的复杂模式。总体而言，我们的研究结果支持III型和I型CRISPR系统之间ca4和csa3介导的串扰。
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.
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影响因子：
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作者：
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DOI：
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发表时间：
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影响因子：
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