Crystal Structure of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated Csn2 Protein Revealed Ca2+-dependent Double-stranded DNA Binding Activity
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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DOI：
10.1074/jbc.m111.256263
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发表时间：
2011-09-02
影响因子：
4.8
通讯作者：
Ke, Ailong
中科院分区：
文献类型：
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作者：
Ki Hyun Nam;Kurinov, Igor;Ke, Ailong
规则间隔短回文重复序列（CRISPR）及其相关蛋白基因（cas基因）广泛存在于细菌和古细菌中。它们形成一种基于RNA的免疫系统，以根除入侵的噬菌体和恶意质粒。在这个过程中的一个关键分子事件是在CRISPR基因座中获得新的间隔区，以指导匹配的外源遗传元件的选择性降解。Csn 2是获得新间隔区所需的Nmeni亚型特异性cas基因。在这里，我们的特点是粪肠球菌Csn 2蛋白作为一个双链（ds-）DNA结合蛋白，并报告其2.7埃四聚体环结构。Csn 2四聚体环的内环类似于26埃宽，并且填充有保守的赖氨酸残基，其准备与ds-DNA非特异性相互作用。每个Csn 2原聚体含有α/β结构域和α-螺旋结构域;在这两个结构域之间观察到显著的铰链运动。Ca ~（2+）位于齐聚反应界面的重要位置。我们进一步表明，去除Ca 2+离子改变了Csn 2的寡聚化状态，这反过来又严重降低了其对ds-DNA的亲和力。总之，我们的研究结果首次揭示了Csn 2蛋白在CRISPR适应中的功能，揭示了它是一种在四级结构水平上发挥作用并受Ca 2+离子调控的ds-DNA结合蛋白。
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 angstrom tetrameric ring structure. The inner circle of the Csn2 tetrameric ring is similar to 26 angstrom wide and populated with conserved lysine residues poised for nonspecific interactions with ds-DNA. Each Csn2 protomer contains an alpha/beta domain and an alpha-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.