Yan Xinfu, Guo Wei, Yuan Y Adam
a Department of Biological Sciences and Center for Bioimaging Sciences; National University of Singapore ; Singapore , Singapore.
RNA Biol. 2015;12(7):749-60. doi: 10.1080/15476286.2015.1051300.
In prokaryotes, the CRISPR/Cas system is known to target and degrade invading phages and foreign genetic elements upon subsequent infection. However, the structure and function of many Cas proteins remain largely unknown, due to the high diversity of Cas proteins. Here we report 3 crystal structures of Archaeoglobus fulgidus Csx3 (AfCsx3) in free form, in complex with manganese ions and in complex with a single-stranded RNA (ssRNA) fragment, respectively. AfCsx3 harbors a ferredoxin-like fold and forms dimer both in the crystal and in solution. Our structure-based biochemical analysis demonstrates that the RNA binding sites and cleavage sites are located at 2 separate surfaces within the AfCsx3 dimer, suggesting a model to bind, tether and cleave the incoming RNA substrate. In addition, AfCsx3 displays robust 3'-deadenylase activity in the presence of manganese ions, which strongly suggests that AfCsx3 functions as a deadenylation exonuclease. Taken together, our results indicate that AfCsx3 is a Cas protein involved in RNA deadenylation and provide a framework for understanding the role of AfCsx3 in the Type III-B CRISPR/Cas system.
在原核生物中，已知CRISPR/Cas系统会在后续感染时靶向并降解入侵的噬菌体和外来遗传元件。然而，由于Cas蛋白的高度多样性，许多Cas蛋白的结构和功能仍 largely unknown。在此，我们分别报道了嗜热栖热放线菌Csx3（AfCsx3）的3种晶体结构，分别为游离形式、与锰离子结合以及与单链RNA（ssRNA）片段结合的形式。AfCsx3具有类铁氧化还原蛋白折叠结构，在晶体和溶液中均形成二聚体。我们基于结构的生化分析表明，RNA结合位点和切割位点位于AfCsx3二聚体内的2个不同表面，这提示了一种结合、系留并切割进入的RNA底物的模型。此外，AfCsx3在存在锰离子的情况下表现出强大的3'-去腺苷酸化酶活性，这强烈表明AfCsx3作为一种去腺苷酸化外切核酸酶发挥作用。综上所述，我们的结果表明AfCsx3是一种参与RNA去腺苷酸化的Cas蛋白，并为理解AfCsx3在III-B型CRISPR/Cas系统中的作用提供了一个框架。
