Aca 蛋白介导 Anti-CRISPR 抑制的分子机制研究

CRISPR-Cas系统是原核生物进化出来的用于对抗外源可移动遗传元件的获得性免疫系统。同时，外源核酸进化出了Anti-CRISPR（Acr）蛋白来对抗宿主菌的CRISPR-Cas系统。Acr蛋白种类繁多，在序列和结构上没有共同特征，然而生物信息学分析发现大多数acr基因都位于保守的Anti-CRISPR-associated（aca）基因的上游。Aca基因编码的Aca蛋白是很保守的，因此可以用来识别新的acr基因。目前为止已经鉴定出了10个Aca蛋白家族。Aca1和Aca2蛋白可以结合acr-aca操纵子启动子区的反向重复序列，进而抑制acr基因的转录，这揭示了Aca蛋白作为抑制子的功能，并提示它可以作为一种潜在的Anti-anti-CRISPR机理被开发利用。另外，这种抑制因子的自调节机制已经被一些Aca-Acr融合蛋白所利用，比如AcrIIA1及其同源物AcrIIA6，AcrIIA13-15以及AcrIF24。这些蛋白都包含一个N末端的，作为acr启动子抑制因子的HTH结构域，和一个起Anti-CRISPR作用的C末端区域，表明这些Acrs具有Acr和Aca蛋白的双重作用，暗示了更加广泛的Anti-anti-CRISPR机制。有趣的是在一些细菌的自身基因组中也发现了Aca蛋白的同源基因，提示细菌宿主利用了这种Anti-anti-CRISPR机理来拮抗潜在的噬菌体。在使用CRISPR-Cas基因编辑工具对细菌基因组进行编辑时，细菌体内的原噬菌体可能通过自身表达的Acr蛋白对宿主的基因组编辑起抑制作用。最近人们利用外源表达Aca1蛋白，克服了Acr蛋白对CRISPR-Cas3编辑活性的抑制，从而提高了编辑效率，这是Aca的Anti-anti-CRISPR机理被应用于基因编辑的一个例子，暗示了Aca蛋白在基因编辑领域的重要应用价值。Aca蛋白的抑制子功能以及Anti-anti-CRISPR机理均依赖于它与acr-aca启动子区反向重复DNA序列的结合，但是Aca蛋白与这些DNA序列结合的分子机制还不清楚。

本论文计划利用生物化学和结构生物学手段阐明Aca蛋白介导Anti-CRISPR抑制的分子机制。本研究首先使用大肠杆菌异源表达的方法成功表达纯化出了铜绿假单胞菌噬菌体JBD30编码的Aca1蛋白和果胶杆菌原噬菌体ZF40编码的Aca2蛋白，随后根据文献报道设计合成了不同长度的DNA底物，与纯化好的Aca1和Aca2蛋白混合孵育形成蛋白-DNA复合物。接下来将单独的Aca蛋白以及Aca蛋白-DNA复合物进行晶体生长。生长好的晶体进行X-射线衍射数据的收集，首先使用Se-Met标记的方法获得了初始相位，随后使用分子置换的方法分别解析了Apo-Aca1和Apo-Aca2蛋白的结构以及Aca1-19 bp JBD30 IR2 DNA和Aca2-26 bp ZF40 IR1 DNA的复合物结构。

本论文首先对Apo-Aca1和Apo-Aca2蛋白的结构进行分析，结构分析显示Aca1和Aca2蛋白都包含一个保守的HTH DNA结合结构域并通过不同的分子间作用模式形成同源二聚体。虽然Aca1和Aca2蛋白具有不同的二聚化机制，但是结构和序列的保守性表明二聚化是Aca1和Aca2家族蛋白的固有特征。随后重点分析了Aca1和Aca2蛋白与其特异性DNA底物的复合物结构。复合物结构显示Aca1二聚体结合19 bp操纵子DNA后将DNA整体弯曲了42°，这种弯曲依赖于DNA侧翼区和间隔区的相互作用；而Aca2主要与26 bp操纵子DNA的核心基序进行特异性识别，因此DNA没有发生明显的弯曲。详细的结构分析表明，虽然Aca1和Aca2蛋白采用不同的二聚机制，识别不同的底物DNA，但两者都利用其HTH结构域的a3螺旋上保守的底物识别氨基酸特异性识别各自的底物DNA，进而抑制下游基因的转录。本研究用ITC实验对参与特异性识别DNA的氨基酸进行了突变验证。另外本研究还设计了体内荧光报告实验验证了Aca1和Aca2的关键氨基酸对acr-aca启动子的抑制作用。将Aca1和Aca2的结构在Dali数据库中进行了搜索，结果显示Aca1的结构与V. cholerae和E. coli中的抗毒素和抑制因子HigA2和MqsA具有相似性，暗示Aca1家族可能起源于抗毒素因子。而Aca2的结构与YdiL和SO_3848具有很高相似性，表明这两个未知功能的蛋白可能像Aca2蛋白一样起转录抑制因子的作用。本研究对YdiL和SO_3848蛋白的编码基因的基因组背景进行了分析，遗憾的是并没有找到结合的启动子序列。

总的来说，本研究首次解析了Aca家族蛋白与底物DNA的复合物结构，揭示了Aca蛋白介导的Anti-CRISPR抑制的结构基础。揭示了Aca1和Aca2家族保守的二聚化机制，虽然两个家族蛋白采用不同的二聚化方式，但具有保守的底物识别位点，因此可以识别各自的底物DNA进而发挥抑制因子的作用。有助于理解其它Aca家族（Aca3-Aca10家族）蛋白的作用机制。另外，本研究的结构分析可以作为模板设计更强的Aca抑制子（与靶DNA有更高的亲和力），这为包括基因编辑在内的不同应用提供更强大的Anti-anti-CRISPR抑制机制，为在应用中进一步准确调控CRISPR-Cas系统提供研究基础。

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