Liu Su, Yuan Zuanning, Yuan Y Adam
Department of Biological Sciences and Centre for Bioimaging Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore.
Department of Biological Sciences and Centre for Bioimaging Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore; National University of Singapore (Suzhou) Research Institute, 377 Lin Quan Street, Suzhou Industrial Park, Jiangsu 215123, China.
J Struct Biol. 2015 May;190(2):122-34. doi: 10.1016/j.jsb.2015.03.001. Epub 2015 Mar 16.
CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats)-mediated defense against invading nucleic acids is a process recently discovered in prokaryotes, which includes recognition and incorporation of invading genetic elements, transcription and processing of CRISPR-RNA (crRNA) and targeting the invaders through base pair recognition. In the type I-E CRISPR-Cas system, Cse2 is proposed to provide a platform to facilitate the targeting of the invading dsDNA by crRNA. Here we report the crystal structure of Meiothermus ruber Cse2 at 2.8Å. M. ruber Cse2 adopts an α-helical bundle scaffold, harbors a positive surface for nucleic acid binding and a conserved dimer interface with strikingly low buried surface area. M. ruber Cse2 selectively binds to G-rich crRNA sequence, which is stripped off from the Cse2-crRNA and Cascade-crRNA complexes by ssDNA or dsDNA with complementary sequence. Stable M. ruber Cascade is readily formed by co-expression of M. ruber Cascade proteins together with G-rich crRNA in vitro. Docking of M. ruber Cse2 structures into the Escherichia coli Cascade Cryo-EM envelope reveals a curved elongated shallow groove for ssRNA binding, which adopts a similar dimer interface discovered by high-resolution crystal structure of Cse2 within E. Coli Cascade. Taken together, our data provides the structural insights into crRNA G-rich sequence recognition by M. ruber Cse2 and reveals the potential structural mechanism for M. ruber Cascade assembly and function.
CRISPR（成簇规律间隔短回文重复序列）介导的抵御入侵核酸的防御机制是最近在原核生物中发现的一个过程，该过程包括识别和整合入侵的遗传元件、CRISPR-RNA（crRNA）的转录和加工以及通过碱基对识别靶向入侵者。在I-E型CRISPR-Cas系统中，有人提出Cse2提供了一个平台，以促进crRNA对入侵双链DNA的靶向作用。在此，我们报道了嗜热栖热菌Cse2在2.8埃分辨率下的晶体结构。嗜热栖热菌Cse2采用α-螺旋束支架结构，具有用于核酸结合的正电荷表面以及埋藏表面积极低的保守二聚体界面。嗜热栖热菌Cse2选择性地结合富含G的crRNA序列，该序列可被具有互补序列的单链DNA或双链DNA从Cse2-crRNA和Cascade-crRNA复合物中剥离。通过在体外共表达嗜热栖热菌Cascade蛋白和富含G的crRNA，可轻松形成稳定的嗜热栖热菌Cascade。将嗜热栖热菌Cse2结构对接至大肠杆菌Cascade冷冻电镜包膜中，揭示了一个用于单链RNA结合的弯曲细长浅沟，其采用了在大肠杆菌Cascade中通过Cse2的高分辨率晶体结构发现的类似二聚体界面。综上所述，我们的数据提供了嗜热栖热菌Cse2对crRNA富含G序列识别的结构见解，并揭示了嗜热栖热菌Cascade组装和功能的潜在结构机制。
