Conformational Dynamics of DNA Binding and Cas3 Recruitment by the CRISPR RNA-Guided Cascade Complex

Bacteria and archaea rely on CRISPR (clustered regularly interspaced short palindromic repeats) RNA-guided adaptive immune systems for sequence specific elimination of foreign nucleic acids. In Escherichia coli, short CRISPR-derived RNAs (crRNAs) assemble with Cas (CRISPR-associated) proteins into a 405-kilodalton multisubunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Cascade binds foreign DNA complementary to the crRNA guide and recruits Cas3, a trans-acting nuclease-helicase required for target degradation. Structural models of Cascade have captured static snapshots of the complex in distinct conformational states, but conformational dynamics of the 11-subunit surveillance complex have not been measured. Here, we use hydrogen–deuterium exchange coupled to mass spectrometry (HDX-MS) to map conformational dynamics of Cascade onto the three-dimensional structure. New insights from structural dynamics are used to make functional predictions about the mechanisms of the R-loop coordination and Cas3 recruitment. We test these predictions in vivo and in vitro. Collectively, we show how mapping conformational dynamics onto static 3D-structures adds an additional dimension to the functional understanding of this biological machine.

细菌与古菌依赖CRISPR（成簇规律间隔短回文重复序列，clustered regularly interspaced short palindromic repeats）RNA引导的适应性免疫系统，实现对外源核酸的序列特异性清除。在大肠杆菌（Escherichia coli）中，短CRISPR衍生RNA（CRISPR-derived RNAs，缩写crRNAs）与Cas（CRISPR相关蛋白，CRISPR-associated）组装形成分子量为405千道尔顿的多亚基监测复合物，命名为Cascade（CRISPR相关抗病毒防御复合物，CRISPR-associated complex for antiviral defense）。Cascade可结合与crRNA引导序列互补的外源DNA，并招募Cas3——一种介导靶标降解的反式作用核酸酶-解旋酶。此前的Cascade结构模型已捕捉到该复合物处于不同构象状态的静态快照，但这一11亚基监测复合物的构象动态尚未被测定。本研究利用氢氘交换耦合质谱（hydrogen–deuterium exchange coupled to mass spectrometry，缩写HDX-MS）技术，将Cascade的构象动态映射至其三维结构中。基于结构动态分析获得的新认知，我们对R环协调与Cas3招募的机制做出了功能预测。我们分别在体内与体外验证了这些预测。综上，本研究阐明了将构象动态映射至静态三维结构的方法，如何为这一生物机器的功能认知增添全新维度。