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首页> 美国卫生研究院文献>The Journal of Biological Chemistry >Crystal Structure of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated Csn2 Protein Revealed Ca2+-dependent Double-stranded DNA Binding Activity
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Crystal Structure of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated Csn2 Protein Revealed Ca2+-dependent Double-stranded DNA Binding Activity

机译：簇状规则间隔的短回文重复（CRISPR）相关的Csn2蛋白的晶体结构揭示了Ca2 +依赖的双链DNA结合活性。

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摘要

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated protein genes (cas genes) are widespread in bacteria and archaea. They form a line of RNA-based immunity to eradicate invading bacteriophages and malicious plasmids. A key molecular event during this process is the acquisition of new spacers into the CRISPR loci to guide the selective degradation of the matching foreign genetic elements. Csn2 is a Nmeni subtype-specific cas gene required for new spacer acquisition. Here we characterize the Enterococcus faecalis Csn2 protein as a double-stranded (ds-) DNA-binding protein and report its 2.7 Å tetrameric ring structure. The inner circle of the Csn2 tetrameric ring is ∼26 Å wide and populated with conserved lysine residues poised for nonspecific interactions with ds-DNA. Each Csn2 protomer contains an α/β domain and an α-helical domain; significant hinge motion was observed between these two domains. Ca2+ was located at strategic positions in the oligomerization interface. We further showed that removal of Ca2+ ions altered the oligomerization state of Csn2, which in turn severely decreased its affinity for ds-DNA. In summary, our results provided the first insight into the function of the Csn2 protein in CRISPR adaptation by revealing that it is a ds-DNA-binding protein functioning at the quaternary structure level and regulated by Ca2+ ions.
机译：成簇的规则间隔的短回文重复序列（CRISPR）及其相关的蛋白质基因（cas基因）在细菌和古细菌中广泛分布。它们形成了一系列基于RNA的免疫力，以根除入侵的噬菌体和恶意质粒。在此过程中的关键分子事件是在CRISPR基因座中获得新的间隔子，以指导匹配的外源遗传元件的选择性降解。 Csn2是新间隔区获得所需的Nmeni亚型特异性cas基因。在这里，我们将粪肠球菌Csn2蛋白表征为双链（ds-）DNA结合蛋白，并报告其2.7Å四聚环结构。 Csn2四聚体环的内圈宽约26Å，并充满了保守的赖氨酸残基，准备与ds-DNA进行非特异性相互作用。每个Csn2启动子包含一个α/β结构域和一个α螺旋结构域；在这两个域之间观察到明显的铰链运动。 Ca 2 + 位于低聚界面的关键位置。我们进一步表明，去除Ca 2 + 离子会改变Csn2的低聚状态，进而严重降低其对ds-DNA的亲和力。总之，我们的研究结果揭示了Csn2蛋白是一种ds-DNA结合蛋白，其在四级结构水平上起作用并受Ca 2 + 调控，从而首次了解了CRISPR适应中Csn2蛋白的功能。离子。

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