Key Laboratory of RNA Biology, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
Key Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
Nucleic Acids Res. 2023 Feb 28;51(4):1984-1995. doi: 10.1093/nar/gkad052.
Anti-CRISPR proteins are encoded by phages to inhibit the CRISPR-Cas systems of the hosts. AcrIIC5 inhibits several naturally high-fidelity type II-C Cas9 enzymes, including orthologs from Neisseria meningitidis (Nme1Cas9) and Simonsiella muelleri (SmuCas9). Here, we solve the structure of AcrIIC5 in complex with Nme1Cas9 and sgRNA. We show that AcrIIC5 adopts a novel fold to mimic the size and charge distribution of double-stranded DNA, and uses its negatively charged grooves to bind and occlude the protospacer adjacent motif (PAM) binding site in the target DNA cleft of Cas9. AcrIIC5 is positioned into the crevice between the WED and PI domains of Cas9, and one end of the anti-CRISPR interacts with the phosphate lock loop and a linker between the RuvC and BH domains. We employ biochemical and mutational analyses to build a model for AcrIIC5's mechanism of action, and identify residues on both the anti-CRISPR and Cas9 that are important for their interaction and inhibition. Together, the structure and mechanism of AcrIIC5 reveal convergent evolution among disparate anti-CRISPR proteins that use a DNA-mimic strategy to inhibit diverse CRISPR-Cas surveillance complexes, and provide new insights into a tool for potent inhibition of type II-C Cas9 orthologs.
抗 CRISPR 蛋白由噬菌体编码，以抑制宿主的 CRISPR-Cas 系统。AcrIIC5 抑制几种天然高保真的 II-C Cas9 酶，包括脑膜炎奈瑟菌（Nme1Cas9）和西蒙氏菌（SmuCas9）的同源物。在这里，我们解决了 AcrIIC5 与 Nme1Cas9 和 sgRNA 复合物的结构。我们表明，AcrIIC5 采用了一种新的折叠方式来模拟双链 DNA 的大小和电荷分布，并利用其带负电荷的凹槽来结合并封闭 Cas9 靶 DNA 裂隙中的原间隔基序（PAM）结合位点。AcrIIC5 位于 Cas9 的 WED 和 PI 结构域之间的缝隙中，反 CRISPR 的一端与磷酸锁环和 RuvC 与 BH 结构域之间的连接子相互作用。我们采用生化和突变分析来构建 AcrIIC5 作用机制的模型，并确定反 CRISPR 和 Cas9 上对其相互作用和抑制至关重要的残基。AcrIIC5 的结构和机制揭示了不同抗 CRISPR 蛋白之间趋同进化，它们使用 DNA 模拟策略来抑制不同的 CRISPR-Cas 监视复合物，并为强效抑制 II-C Cas9 同源物提供了新的见解。
