Inhibition mechanisms of AcrF9, AcrF8, and AcrF6 against type I-F CRISPR-Cas complex revealed by cryo-EM
Inhibition mechanisms of AcrF9, AcrF8, and AcrF6 against type I-F CRISPR-Cas complex revealed by cryo-EM
复制标题
通过冷冻电镜揭示 AcrF9、AcrF8 和 AcrF6 对 I-F 型 CRISPR-Cas 复合物的抑制机制。
DOI：
10.1073/pnas.1922638117
复制
发表时间：
2020-03-31
影响因子：
11.1
通讯作者：
Chiu, Wah
中科院分区：
文献类型：
--
作者：
Zhang, Kaiming;Wang, Shuo;Chiu, Wah
原核生物和病毒之间进行了长期的斗争。原核生物使用CRISPR-Cas介导的适应性免疫，而相反，病毒进化出多种抗crispr （Acr）蛋白来击败这些CRISPR-Cas系统。铜绿假单胞菌中的I-F型CRISPR-Cas系统需要crrna引导的监视复合体（Csy复合体）来识别入侵的DNA。虽然已经报道了一些抗Csy复合物的Acr蛋白，但其他相关的Acr蛋白仍需要研究以了解其机制。在这里，我们使用电子冷冻显微镜（cro - em）获得了三个先前未解析的Acr蛋白（AcrF9， AcrF8和AcrF6）与Csy配合物结合的结构，分辨率分别为2.57埃，3.42埃和3.15埃。2.57埃的结构揭示了Csy复合体中每个分子成分的精细细节，以及蛋白质与CRISPR RNA （crRNA）之间直接和水介导的相互作用。我们的结构也明确地显示了这些Acr蛋白如何以不同的方式与Csy复合物结合。AcrF9与Csy螺旋主链上的关键dna结合位点结合。AcrF6结合在Cas7.6f和Cas8f之间的连接处，这对DNA双工分裂至关重要。AcrF8结合到Csy螺旋主链上的一个独特位置，并与crRNA形成相互作用，这在其他抗Csy复合物的Acr蛋白中尚未发现。我们的结构导向诱变和生物化学实验进一步支持了这些Acr蛋白的抗crispr机制。我们的研究结果支持这些Acr蛋白抑制入侵DNA降解的趋同结果，尽管与I-F型CRISPR-Cas系统的相互作用模式不同。
Prokaryotes and viruses have fought a long battle against each other. Prokaryotes use CRISPR-Cas-mediated adaptive immunity, while conversely, viruses evolve multiple anti-CRISPR (Acr) proteins to defeat these CRISPR-Cas systems. The type I-F CRISPR-Cas system in Pseudomonas aeruginosa requires the crRNA-guided surveillance complex (Csy complex) to recognize the invading DNA. Although some Acr proteins against the Csy complex have been reported, other relevant Acr proteins still need studies to understand their mechanisms. Here, we obtain three structures of previously unresolved Acr proteins (AcrF9, AcrF8, and AcrF6) bound to the Csy complex using electron cryo-microscopy (cryo-EM), with resolution at 2.57 angstrom, 3.42 angstrom, and 3.15 angstrom, respectively. The 2.57-angstrom structure reveals fine details for each molecular component within the Csy complex as well as the direct and water-mediated interactions between proteins and CRISPR RNA (crRNA). Our structures also show unambiguously how these Acr proteins bind differently to the Csy complex. AcrF9 binds to key DNA-binding sites on the Csy spiral backbone. AcrF6 binds at the junction between Cas7.6f and Cas8f, which is critical for DNA duplex splitting. AcrF8 binds to a distinct position on the Csy spiral backbone and forms interactions with crRNA, which has not been seen in other Acr proteins against the Csy complex. Our structure-guided mutagenesis and biochemistry experiments further support the anti-CRISPR mechanisms of these Acr proteins. Our findings support the convergent consequence of inhibiting degradation of invading DNA by these Acr proteins, albeit with different modes of interactions with the type I-F CRISPR-Cas system.