title: Structural insights into specific crRNA G-rich sequence binding by Meiothermus ruber Cse2.
abstract: 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.
authors: Department of Biological Sciences and Centre for Bioimaging Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore.
authors: Su
authors: S
authors: Liu
authors: Department of Biological Sciences and Centre for Bioimaging Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore.
authors: Zuanning
authors: Z
authors: Yuan
authors: 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. Electronic address: dbsyya@nus.edu.sg.
authors: Y Adam
authors: YA
authors: Yuan
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015152:Blotting, Northern; D064113:CRISPR-Cas Systems / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D006094:Gram-Positive Bacteria / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D011487:Protein Conformation; D012313:RNA / Q000378:metabolism*; D014461:Ultracentrifugation
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D012313:RNA
keywords: CRISPR G-rich crRNA sequence; Cascade; Crystal structure; Cse2