Baca Christian F, Majumder Puja, Hickling James H, Ye Linzhi, Teplova Marianna, Brady Sean F, Patel Dinshaw J, Marraffini Luciano A
Laboratory of Bacteriology, The Rockefeller University, New York, NY 10065, USA; Tri-Institutional PhD Program in Chemical Biology, Weill Cornell Medical College, Rockefeller University and Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.
Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.
Cell. 2024 Dec 12;187(25):7183-7195.e24. doi: 10.1016/j.cell.2024.10.002. Epub 2024 Oct 28.
Type III CRISPR systems provide immunity against genetic invaders through the production of cyclic oligo-adenylate (cA) molecules that activate effector proteins that contain CRISPR-associated Rossman fold (CARF) domains. Here, we characterized the function and structure of an effector in which the CARF domain is fused to an adenosine deaminase domain, CRISPR-associated adenosine deaminase 1 (Cad1). We show that upon binding of cA or cA to its CARF domain, Cad1 converts ATP to ITP, both in vivo and in vitro. Cryoelectron microscopy (cryo-EM) structural studies on full-length Cad1 reveal an hexameric assembly composed of a trimer of dimers, with bound ATP at inter-domain sites required for activity and ATP/ITP within deaminase active sites. Upon synthesis of cA during phage infection, Cad1 activation leads to a growth arrest of the host that prevents viral propagation. Our findings reveal that CRISPR-Cas systems employ a wide range of molecular mechanisms beyond nucleic acid degradation to provide adaptive immunity in prokaryotes.
III型CRISPR系统通过产生环状寡腺苷酸（cA）分子来抵御基因入侵者，这些cA分子可激活含有CRISPR相关罗斯曼折叠（CARF）结构域的效应蛋白。在此，我们对一种效应蛋白的功能和结构进行了表征，该效应蛋白中CARF结构域与腺苷脱氨酶结构域——CRISPR相关腺苷脱氨酶1（Cad1）融合。我们发现，在体内和体外，当cA或cA与其CARF结构域结合时，Cad1会将ATP转化为ITP。对全长Cad1的冷冻电子显微镜（cryo-EM）结构研究揭示了一种由三聚体二聚体组成的六聚体组装结构，在结构域间位点结合有活性所需的ATP，在脱氨酶活性位点有ATP/ITP。在噬菌体感染期间cA合成时，Cad1的激活会导致宿主生长停滞，从而阻止病毒繁殖。我们的研究结果表明，CRISPR-Cas系统采用了广泛的分子机制，而非仅限于核酸降解，以在原核生物中提供适应性免疫。
