The Structure of the CRISPR-Associated Protein Csa3 Provides Insight Into the Regulation of the CRISPR/Cas System

Abstract Adaptive immune systems have recently been recognized in prokaryotic organisms where, in response to viral infection, they incorporate short fragments of invader-derived DNA into loci called clustered regularly interspaced short palindromic repeats ( CRISPR s). In subsequent infections, the CRISPR loci are transcribed and processed into guide sequences for the neutralization of the invading RNA or DNA. The CRISPR-associated protein machinery (Cas) lies at the heart of this process, yet many of the molecular details of the CRISPR/Cas system remain to be elucidated. Here, we report the first structure of Csa3, a CRISPR-associated protein from Sulfolobus solfataricus (Sso1445), which reveals a dimeric two-domain protein. The N-terminal domain is a unique variation on the dinucleotide binding domain that orchestrates dimer formation. In addition, it utilizes two conserved sequence motifs [Thr-h-Gly-Phe-(Asn/Asp)-Glu-X 4 -Arg and Leu-X 2 -Gly-h-Arg] to construct a 2-fold symmetric pocket on the dimer axis. This pocket is likely to represent a regulatory ligand-binding site. The N-terminal domain is fused to a C-terminal MarR-like winged helix–turn–helix domain that is expected to be involved in DNA recognition. Overall, the unique domain architecture of Csa3 suggests a transcriptional regulator under allosteric control of the N-terminal domain. Alternatively, Csa3 may function in a larger complex, with the conserved cleft participating in protein–protein or protein–nucleic acid interactions. A similar N-terminal domain is also identified in Csx1, a second CRISPR-associated protein family of unknown function.