Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, South Korea.
Protein Structure Research Team, Korea Basic Science Institute, 162 Yeongudanji-Ro, Ochang, Chungbuk 28119, South Korea.
Nucleic Acids Res. 2020 Jul 27;48(13):7584-7594. doi: 10.1093/nar/gkaa512.
Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins provide adaptive immunity to prokaryotes against invading phages and plasmids. As a countermeasure, phages have evolved anti-CRISPR (Acr) proteins that neutralize the CRISPR immunity. AcrIIA5, isolated from a virulent phage of Streptococcus thermophilus, strongly inhibits diverse Cas9 homologs, but the molecular mechanism underlying the Cas9 inhibition remains unknown. Here, we report the solution structure of AcrIIA5, which features a novel α/β fold connected to an N-terminal intrinsically disordered region (IDR). Remarkably, truncation of the N-terminal IDR abrogates the inhibitory activity against Cas9, revealing that the IDR is essential for Cas9 inhibition by AcrIIA5. Progressive truncations and mutations of the IDR illustrate that the disordered region not only modulates the association between AcrIIA5 and Cas9-sgRNA, but also alters the catalytic efficiency of the inhibitory complex. The length of IDR is critical for the Cas9-sgRNA recognition by AcrIIA5, whereas the charge content of IDR dictates the inhibitory activity. Conformational plasticity of IDR may be linked to the broad-spectrum inhibition of Cas9 homologs by AcrIIA5. Identification of the IDR as the main determinant for Cas9 inhibition expands the inventory of phage anti-CRISPR mechanisms.
成簇规律间隔短回文重复序列 (CRISPRs) 和 CRISPR 相关 (Cas) 蛋白为原核生物提供了针对入侵噬菌体和质粒的适应性免疫。作为一种对策，噬菌体进化出了抗 CRISPR (Acr) 蛋白，以中和 CRISPR 免疫。AcrIIA5 是从嗜热链球菌的一种毒性噬菌体中分离出来的，它强烈抑制多种 Cas9 同源物，但 Cas9 抑制的分子机制尚不清楚。在这里，我们报告了 AcrIIA5 的溶液结构，其特征是一种新颖的α/β折叠与 N 端无规卷曲结构域 (IDR) 相连。值得注意的是，N 端 IDR 的截断消除了对 Cas9 的抑制活性，表明 IDR 对于 AcrIIA5 对 Cas9 的抑制是必不可少的。IDR 的逐步截断和突变表明，无规卷曲结构域不仅调节了 AcrIIA5 与 Cas9-sgRNA 之间的结合，而且改变了抑制复合物的催化效率。IDR 的长度对于 AcrIIA5 识别 Cas9-sgRNA 至关重要，而 IDR 的电荷含量决定了抑制活性。IDR 的构象灵活性可能与 AcrIIA5 对 Cas9 同源物的广谱抑制有关。确定 IDR 是 Cas9 抑制的主要决定因素，扩展了噬菌体抗 CRISPR 机制的清单。
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