Crystal structure of the RNA-guided immune surveillance Cascade complex in Escherichia coli
Crystal structure of the RNA-guided immune surveillance Cascade complex in Escherichia coli
复制标题
大肠杆菌中 RNA 引导的免疫监视级联复合物的晶体结构
DOI：
10.1038/nature13733
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发表时间：
2014-11-06
期刊：
影响因子：
64.8
通讯作者：
Wang, Yanli
中科院分区：
文献类型：
--
作者：
Zhao, Hongtu;Sheng, Gang;Wang, Yanli
成簇的规则间隔的短回文重复序列 (CRISPR) 与 CRISPR 相关 (Cas) 蛋白一起形成 CRISPR/Cas 系统，以防御细菌和古细菌来源的外来核酸 (1-9)。在 I-E 亚型 CRISPR/Cas 系统中，来自 5 个 Cas 蛋白 (CasA(1)B(2)C(6)D(1)E(1)) 的 11 个亚基沿着 CRISPR RNA (crRNA) 组装形成级联复合物 (10-13)。在这里，我们报告了 405 千道尔顿大肠杆菌级联复合物的 3.05 埃晶体结构，该复合物提供了超出早期低分辨率冷冻电子显微镜结构所能提供的分子细节。结合的 61 个核苷酸的 crRNA 跨越整个包含 11 个蛋白质亚基的复合物，它与所有六个 CasC 亚基（称为 CasC1-6）相互作用，其 5' 和 3' 末端重复序列分别由 CasD 和 CasE 锚定。 crRNA 间隔区沿着对齐的 CasC1-6 亚基生成的凹面上的连续凹槽定位。从各个 CasC2-6 亚基伸出的五个长 β-发夹延伸穿过 crRNA，每个 β-发夹插入最后堆叠的碱基与其相邻的展开对应部分之间的间隙，并位于前一个 CasC 亚基的凹槽内。因此，crRNA 间隔区不是连续堆叠，而是分成五个相等的片段，每个片段包含五个堆叠的碱基，两侧有一个翻转的碱基。每个 crRNA 间隔片段都以类似的方式与 CasC 相互作用。此外，我们的结构解释了为什么种子序列及其向外定向的碱基在目标 DNA 识别中具有关键作用。总之，我们的级联复合物结构提供了蛋白质-蛋白质和蛋白质-RNA 排列和相互作用的新颖分子细节，这些细节是生成介导 RNA 引导的免疫监视的复合物所需的。
Clustered regularly interspaced short palindromic repeats (CRISPR) together with CRISPR-associated (Cas) proteins form the CRISPR/Cas system to defend against foreign nucleic acids of bacterial and archaeal origin(1-9). In the I-E subtype CRISPR/Cas system, eleven subunits from five Cas proteins (CasA(1)B(2)C(6)D(1)E(1)) assemble along a CRISPR RNA (crRNA) to form the Cascade complex(10-13). Here we report on the 3.05 angstrom crystal structure of the 405-kilodalton Escherichia coli Cascade complex that provides molecular details beyond those available from earlier lower-resolution cryo-electron microscopy structures. The bound 61-nucleotide crRNA spans the entire 11-protein subunit-containing complex, where it interacts with all six CasC subunits (named CasC1-6), with its 5 ' and 3 ' terminal repeats anchored by CasD and CasE, respectively. The crRNA spacer region is positioned along a continuous groove on the concave surface generated by the aligned CasC1-6 subunits. The five long beta-hairpins that project from individual CasC2-6 subunits extend across the crRNA, with each beta-hairpin inserting into the gap between the last stacked base and its adjacent splayed counterpart, and positioned within the groove of the preceding CasC subunit. Therefore, instead of continuously stacking, the crRNA spacer region is divided into five equal fragments, with each fragment containing five stacked bases flanked by one flipped-out base. Each of those crRNA spacer fragments interacts with CasC in a similar fashion. Furthermore, our structure explains why the seed sequence, with its outward directed bases, has a critical role in target DNA recognition. Inconclusion, our structure of the Cascade complex provides novel molecular details of protein-protein and protein-RNA alignments and interactions required for generation of a complex mediating RNA-guided immune surveillance.