Structural and biochemical insights into the mechanism of the anti-CRISPR protein AcrIE3

IF 4.4 2区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Structure Pub Date : 2024-11-13 DOI:10.1016/j.str.2024.10.024

Jasung Koo, Gyujin Lee, Changkon Park, Hyejin Oh, Sung-Hyun Hong, Jeong-Yong Suh, Euiyoung Bae

引用次数: 0

Abstract

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.

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从结构和生化角度揭示抗 CRISPR 蛋白 AcrIE3 的作用机制

抗 CRISPR（Acr）蛋白是噬菌体和其他遗传物质中 CRISPR-Cas 系统的天然抑制剂。在假单胞菌噬菌体中发现的 AcrIE3 通过与 Cascade 复合物结合，使铜绿假单胞菌中的 I-E 型 CRISPR-Cas 系统失活。然而，其精确的抑制机制一直难以捉摸。在本研究中，我们对 AcrIE3 进行了全面的结构和生化分析，从机理上揭示了它的抗 CRISPR 功能。我们的研究结果表明，AcrIE3 能选择性地与级联复合物的 Cas8e 亚基结合。AcrIE3 的晶体结构呈现全螺旋折叠，表面带负电荷。通过广泛的突变分析，我们发现 AcrIE3 通过其带负电荷的表面残基与 Cas8e 中的原位相邻基序（PAM）识别位点相互作用。这些发现加深了我们对 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 抑制剂的主要靶点。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Structure 生物-生化与分子生物学

CiteScore

8.90

自引率

1.80%

发文量

155

审稿时长

3-8 weeks

期刊介绍： Structure aims to publish papers of exceptional interest in the field of structural biology. The journal strives to be essential reading for structural biologists, as well as biologists and biochemists that are interested in macromolecular structure and function. Structure strongly encourages the submission of manuscripts that present structural and molecular insights into biological function and mechanism. Other reports that address fundamental questions in structural biology, such as structure-based examinations of protein evolution, folding, and/or design, will also be considered. We will consider the application of any method, experimental or computational, at high or low resolution, to conduct structural investigations, as long as the method is appropriate for the biological, functional, and mechanistic question(s) being addressed. Likewise, reports describing single-molecule analysis of biological mechanisms are welcome. In general, the editors encourage submission of experimental structural studies that are enriched by an analysis of structure-activity relationships and will not consider studies that solely report structural information unless the structure or analysis is of exceptional and broad interest. Studies reporting only homology models, de novo models, or molecular dynamics simulations are also discouraged unless the models are informed by or validated by novel experimental data; rationalization of a large body of existing experimental evidence and making testable predictions based on a model or simulation is often not considered sufficient.