Department of Biochemistry, University of Toronto, 661 University Avenue, Suite 1600, Toronto, Ontario M5G 1M1, Canada. Electronic address: https://twitter.com/s1hwang_21.
Department of Biochemistry, University of Toronto, 661 University Avenue, Suite 1600, Toronto, Ontario M5G 1M1, Canada.
J Mol Biol. 2023 Apr 1;435(7):167991. doi: 10.1016/j.jmb.2023.167991. Epub 2023 Feb 2.
Anti-CRISPR proteins inhibit CRISPR-Cas immune systems through diverse mechanisms. Previously, the anti-CRISPR protein AcrIIC5 was shown to potently inhibit a type II-C Cas9 from Neisseria meningitidis (Nme1Cas9). In this work, we explore the mechanism of activity of the AcrIIC5 homologue from Neisseria chenwenguii (AcrIIC5) and show that it prevents Cas9 binding to target DNA. We show that AcrIIC5 targets the PAM-interacting domain (PID) of Nme1Cas9 for inhibition, agreeing with previous findings for AcrIIC5, and newly establish that strong binding of the anti-CRISPR requires guide RNA be pre-loaded on Cas9. We determined the crystal structure of AcrIIC5 using X-ray crystallography and identified amino acid residues that are critical for its function. Using a protein docking algorithm we show that AcrIIC5 likely occupies the Cas9 DNA binding pocket, thereby inhibiting target DNA binding through a mechanism similar to that previously described for AcrIIA2 and AcrIIA4.
抗 CRISPR 蛋白通过多种机制抑制 CRISPR-Cas 免疫系统。以前，抗 CRISPR 蛋白 AcrIIC5 被证明能够强烈抑制脑膜炎奈瑟菌（Nme1Cas9）的 II-C 型 Cas9。在这项工作中，我们探索了 Neisseria chenwenguii（AcrIIC5）的 AcrIIC5 同源物的活性机制，并表明它阻止 Cas9 与靶 DNA 结合。我们表明，AcrIIC5 针对 Nme1Cas9 的 PAM 相互作用结构域（PID）进行抑制，这与之前针对 AcrIIC5 的发现一致，并新建立了抗 CRISPR 的强结合需要预先加载 Cas9 的向导 RNA。我们使用 X 射线晶体学确定了 AcrIIC5 的晶体结构，并鉴定了对其功能至关重要的氨基酸残基。使用蛋白质对接算法，我们表明 AcrIIC5 可能占据 Cas9 的 DNA 结合口袋，从而通过类似于先前描述的 AcrIIA2 和 AcrIIA4 的机制抑制靶 DNA 结合。
