Beijing Advanced Innovation Center for Soft Matter Science and Engineering, Beijing Key Laboratory of Bioprocess, State Key Laboratory of Chemical Resource Engineering, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, China.
Beijing Advanced Innovation Center for Soft Matter Science and Engineering, Beijing Key Laboratory of Bioprocess, State Key Laboratory of Chemical Resource Engineering, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, China; Jiangxi Provincial Key Laboratory of Natural Active Pharmaceutical Constituents, Department of Chemistry and Bioengineering, Yichun University, Yichun, China.
J Biol Chem. 2022 Jul;298(7):102124. doi: 10.1016/j.jbc.2022.102124. Epub 2022 Jun 10.
Prokaryotes evolved clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins as a kind of adaptive immune defense against mobile genetic elements including harmful phages. To counteract this defense, many mobile genetic elements in turn encode anti-CRISPR proteins (Acrs) to inactivate the CRISPR-Cas system. While multiple mechanisms of Acrs have been uncovered, it remains unknown whether other mechanisms are utilized by uncharacterized Acrs. Here, we report a novel mechanism adopted by recently identified AcrIF23. We show that AcrIF23 interacts with the Cas2/3 helicase-nuclease in the type I-F CRISPR-Cas system, similar to AcrIF3. The structure of AcrIF23 demonstrated a novel fold and structure-based mutagenesis identified a surface region of AcrIF23 involved in both Cas2/3-binding and its inhibition capacity. Unlike AcrIF3, however, we found AcrIF23 only potently inhibits the DNA cleavage activity of Cas2/3 but does not hinder the recruitment of Cas2/3 to the CRISPR RNA-guided surveillance complex (the Csy complex). Also, in contrast to AcrIF3 which hinders substrate DNA recognition by Cas2/3, we show AcrIF23 promotes DNA binding to Cas2/3. Taken together, our study identifies a novel anti-CRISPR mechanism used by AcrIF23 and highlights the diverse mechanisms adopted by Acrs.
原核生物进化出了成簇规律间隔短回文重复序列 (CRISPR) 和 CRISPR 相关 (Cas) 蛋白，作为一种针对包括有害噬菌体在内的移动遗传元件的适应性免疫防御机制。为了对抗这种防御，许多移动遗传元件转而编码抗 CRISPR 蛋白 (Acr) 来使 CRISPR-Cas 系统失活。虽然已经揭示了多种 Acr 的作用机制，但仍不清楚未被表征的 Acr 是否利用了其他机制。在这里，我们报告了最近鉴定的 AcrIF23 采用的一种新机制。我们表明，AcrIF23 与 I-F 型 CRISPR-Cas 系统中的 Cas2/3 解旋酶-核酸酶相互作用，类似于 AcrIF3。AcrIF23 的结构展示了一种新颖的折叠，基于结构的突变分析确定了 AcrIF23 中参与 Cas2/3 结合及其抑制能力的表面区域。然而，与 AcrIF3 不同，我们发现 AcrIF23 仅强烈抑制 Cas2/3 的 DNA 切割活性，但不阻碍 Cas2/3 招募到 CRISPR RNA 引导的监视复合物 (Csy 复合物)。此外，与 AcrIF3 阻碍 Cas2/3 对底物 DNA 的识别不同，我们表明 AcrIF23 促进 Cas2/3 与 DNA 的结合。总之，我们的研究确定了 AcrIF23 采用的一种新的抗 CRISPR 机制，并强调了 Acrs 采用的多种机制。
