Hrle Ajla, Maier Lisa-Katharina, Sharma Kundan, Ebert Judith, Basquin Claire, Urlaub Henning, Marchfelder Anita, Conti Elena
a Structural Cell Biology Department; Max Planck Institute of Biochemistry ; Martinsried , Germany.
RNA Biol. 2014;11(8):1072-82. doi: 10.4161/rna.29893.
Upon pathogen invasion, bacteria and archaea activate an RNA-interference-like mechanism termed CRISPR (clustered regularly interspaced short palindromic repeats). A large family of Cas (CRISPR-associated) proteins mediates the different stages of this sophisticated immune response. Bioinformatic studies have classified the Cas proteins into families, according to their sequences and respective functions. These range from the insertion of the foreign genetic elements into the host genome to the activation of the interference machinery as well as target degradation upon attack. Cas7 family proteins are central to the type I and type III interference machineries as they constitute the backbone of the large interference complexes. Here we report the crystal structure of Thermofilum pendens Csc2, a Cas7 family protein of type I-D. We found that Csc2 forms a core RRM-like domain, flanked by three peripheral insertion domains: a lid domain, a Zinc-binding domain and a helical domain. Comparison with other Cas7 family proteins reveals a set of similar structural features both in the core and in the peripheral domains, despite the absence of significant sequence similarity. T. pendens Csc2 binds single-stranded RNA in vitro in a sequence-independent manner. Using a crosslinking - mass-spectrometry approach, we mapped the RNA-binding surface to a positively charged surface patch on T. pendens Csc2. Thus our analysis of the key structural and functional features of T. pendens Csc2 highlights recurring themes and evolutionary relationships in type I and type III Cas proteins.
在病原体入侵时，细菌和古生菌会激活一种类似RNA干扰的机制，称为CRISPR（成簇规律间隔短回文重复序列）。一大类Cas（CRISPR相关）蛋白介导这种复杂免疫反应的不同阶段。生物信息学研究已根据Cas蛋白的序列和各自功能将其分类为不同家族。这些功能从将外来遗传元件插入宿主基因组到激活干扰机制以及在受到攻击时进行靶标降解。Cas7家族蛋白对于I型和III型干扰机制至关重要，因为它们构成了大型干扰复合物的主干。在此，我们报道了嗜热栖热放线菌Csc2（一种I-D型Cas7家族蛋白）的晶体结构。我们发现Csc2形成一个核心的类RRM结构域，两侧有三个外围插入结构域：一个帽结构域、一个锌结合结构域和一个螺旋结构域。与其他Cas7家族蛋白的比较揭示了在核心结构域和外围结构域中都存在一组相似的结构特征，尽管缺乏显著的序列相似性。嗜热栖热放线菌Csc2在体外以序列非依赖的方式结合单链RNA。使用交联-质谱方法，我们将RNA结合表面定位到嗜热栖热放线菌Csc2上一个带正电荷的表面区域。因此，我们对嗜热栖热放线菌Csc2关键结构和功能特征的分析突出了I型和III型Cas蛋白中反复出现的主题和进化关系。
