Anti-CRISPR (Acr) proteins are natural inhibitors of CRISPR-Cas systems, found in bacteriophages and other genetic elements. AcrIE3, identified in a Pseudomonas phage, inactivates the type I-E CRISPR-Cas system in Pseudomonas aeruginosa by engaging with the Cascade complex. However, its precise inhibition mechanism has remained elusive. In this study, we present a comprehensive structural and biochemical analysis of AcrIE3, providing mechanistic insight into its anti-CRISPR function. Our results reveal that AcrIE3 selectively binds to the Cas8e subunit of the Cascade complex. The crystal structure of AcrIE3 exhibits an all-helical fold with a negatively charged surface. Through extensive mutational analyses, we show that AcrIE3 interacts with the protospacer adjacent motif (PAM) recognition site in Cas8e through its negatively charged surface residues. These findings enhance our understanding of the structure and function of type I-E Acr proteins, suggesting PAM interaction sites as primary targets for divergent Acr inhibitors.

中文翻译：

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对抗 CRISPR 蛋白 AcrIE3 机制的结构和生化见解


抗 CRISPR （Acr） 蛋白是 CRISPR-Cas 系统的天然抑制剂，存在于噬菌体和其他遗传元件中。在假单胞菌噬菌体中鉴定的 AcrIE3 通过与 Cascade 复合物结合来灭活铜绿假单胞菌中的 I-E CRISPR-CAS 系统。然而，其确切的抑制机制仍然难以捉摸。在这项研究中，我们提出了 AcrIE3 的全面结构和生化分析，提供了对其抗 CRISPR 功能的机制见解。我们的结果表明，AcrIE3 选择性地与 Cascade 复合物的 Cas8e 亚基结合。AcrIE3 的晶体结构表现出全螺旋折叠和带负电荷的表面。通过广泛的突变分析，我们表明 AcrIE3 通过其带负电荷的表面残基与 Cas8e 中的前间隔区相邻基序 （PAM） 识别位点相互作用。这些发现增强了我们对 I-E 型 Acr 蛋白结构和功能的理解，表明 PAM 相互作用位点是不同 Acr 抑制剂的主要靶标。