title: Crystal structure of clustered regularly interspaced short palindromic repeats (CRISPR)-associated Csn2 protein revealed Ca2+-dependent double-stranded DNA binding activity.
abstract: Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated protein genes (cas genes) are widespread in bacteria and archaea. They form a line of RNA-based immunity to eradicate invading bacteriophages and malicious plasmids. A key molecular event during this process is the acquisition of new spacers into the CRISPR loci to guide the selective degradation of the matching foreign genetic elements. Csn2 is a Nmeni subtype-specific cas gene required for new spacer acquisition. Here we characterize the Enterococcus faecalis Csn2 protein as a double-stranded (ds-) DNA-binding protein and report its 2.7 Å tetrameric ring structure. The inner circle of the Csn2 tetrameric ring is ∼26 Å wide and populated with conserved lysine residues poised for nonspecific interactions with ds-DNA. Each Csn2 protomer contains an α/β domain and an α-helical domain; significant hinge motion was observed between these two domains. Ca(2+) was located at strategic positions in the oligomerization interface. We further showed that removal of Ca(2+) ions altered the oligomerization state of Csn2, which in turn severely decreased its affinity for ds-DNA. In summary, our results provided the first insight into the function of the Csn2 protein in CRISPR adaptation by revealing that it is a ds-DNA-binding protein functioning at the quaternary structure level and regulated by Ca(2+) ions.
authors.affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14850.
authors.forename: Ki Hyun
authors.initials: KH
authors.lastname: Nam
authors.affiliation: Northeastern Collaborative Access Team (NE-CAT), Department of Chemistry and Chemical Biology, Cornell University, Argonne, Illinois 60439.
authors.forename: Igor
authors.initials: I
authors.lastname: Kurinov
authors.affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14850. Electronic address: ak425@cornell.edu.
authors.forename: Ailong
authors.initials: A
authors.lastname: Ke
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray / Q000379:methods*; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D013293:Enterococcus faecalis / Q000378:metabolism; D008957:Models, Genetic; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D005810:Multigene Family; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D017386:Sequence Homology, Amino Acid
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D004247:DNA