CRISPR RNA and anti-CRISPR protein binding to the Xanthomonas albilineans Csy1-Csy2 heterodimer in the type I-F CRISPR-Cas system
CRISPR RNA and anti-CRISPR protein binding to the Xanthomonas albilineans Csy1-Csy2 heterodimer in the type I-F CRISPR-Cas system
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DOI：
10.1074/jbc.ra117.001611
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发表时间：
2018-02-23
影响因子：
4.8
通讯作者：
Bae, Euiyoung
中科院分区：
文献类型：
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作者：
Hong, Suji;Ka, Donghyun;Bae, Euiyoung
成簇的规则间隔的短回文重复序列 (CRISPR) 和 CRISPR 相关 (Cas) 蛋白提供微生物针对噬菌体的适应性免疫。在 I-F 型 CRISPR-Cas 系统中，多个 Cas 蛋白 (Csy1-4) 与 CRISPR RNA (crRNA) 组成监视复合物 (Csy 复合物)，用于目标识别。在这里，我们报告了白色黄单胞菌 Csy1-Csy2 亚复合体的生化特征，包括分析其与 crRNA 和 AcrF2 的相互作用，AcrF2 是一种来自感染铜绿假单胞菌的噬菌体的抗 CRISPR (Acr) 蛋白。 X. albilineans Csy1 和 Csy2 蛋白（分别为 XaCsy1 和 XaCsy2）形成稳定的异二聚体复合物，特异性结合 crRNA 的 8 核苷酸 (nt) 5' 柄。相比之下，XaCsy1-XaCsy2 异二聚体对含有 5' 手柄序列的 28-nt X. albilineans CRISPR 重复 RNA 的亲和力降低。色谱和量热分析揭示了铜绿假单胞菌噬菌体的 Acr 蛋白与白斑杆菌 Csy 复合物的异二聚体亚基之间的紧密结合，表明 AcrF2 识别 Csy1-Csy2 异二聚体的保守特征。我们发现 XaCsy1 和 XaCsy2 单独都不与 AcrF2 和 5'-handle RNA 形成稳定的复合物，表明 XaCsy1-XaCsy2 异二聚化是结合它们所必需的。我们还将 AcrF2 的晶体结构解析至 1.34 埃的分辨率，从而能够对参与与 Csy1-Csy2 异二聚体相互作用的残基进行更详细的结构分析。我们的结果提供了有关多亚基 crRNA 引导的监视复合物形成过程中事件顺序的信息，并表明 Acr 蛋白失活 I-F 型 CRISPR-Cas 系统具有广泛的特异性。
Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins provide microbial adaptive immunity against bacteriophages. In type I-F CRISPR-Cas systems, multiple Cas proteins (Csy1-4) compose a surveillance complex (Csy complex) with CRISPR RNA (crRNA) for target recognition. Here, we report the biochemical characterization of the Csy1-Csy2 subcomplex from Xanthomonas albilineans, including the analysis of its interaction with crRNA and AcrF2, an anti-CRISPR (Acr) protein from a phage that infects Pseudomonas aeruginosa. The X. albilineans Csy1 and Csy2 proteins (XaCsy1 and XaCsy2, respectively) formed a stable heterodimeric complex that specifically bound the 8-nucleotide (nt) 5'-handle of the crRNA. In contrast, the XaCsy1-XaCsy2 heterodimer exhibited reduced affinity for the 28-nt X. albilineans CRISPR repeat RNA containing the 5'-handle sequence. Chromatographic and calorimetric analyses revealed tight binding between the Acr protein from the P. aeruginosa phage and the heterodimeric subunit of the X. albilineans Csy complex, suggesting that AcrF2 recognizes conserved features of Csy1-Csy2 heterodimers. We found that neither XaCsy1 nor XaCsy2 alone forms a stable complex with AcrF2 and the 5'-handle RNA, indicating that XaCsy1-XaCsy2 heterodimerization is required for binding them. We also solved the crystal structure of AcrF2 to a resolution of 1.34 angstrom, enabling a more detailed structural analysis of the residues involved in the interactions with the Csy1-Csy2 heterodimer. Our results provide information about the order of events during the formation of the multisubunit crRNA-guided surveillance complex and suggest that the Acr protein inactivating type I-F CRISPR-Cas systems has broad specificity.