Structure of a CRISPR-associated protein Cas2 fromDesulfovibrio vulgaris

Abstract

PDB Reference: Cas2, 3oq2.CRISPRs (clustered regularly interspaced short palindromic repeats) provide bacteria and archaea with RNA-guided acquired immunity to invasive DNAs. CRISPR-associated (Cas) proteins carry out the immune effector functions. Cas2 is a universal component of the CRISPR system. Here, a 1.35 Å resolution crystal structure of Cas2 from the bacterium Desulfovibrio vulgaris (DvuCas2) is reported. DvuCas2 is a homodimer, with each protomer consisting of an N-terminal ferredoxin fold (amino acids 1-78) to which is appended a C-terminal segment (amino acids 79-102) that includes a short 3 10 -helix and a fifth -strand. The 5 strands align with the 4 strands of the opposite protomers, resulting in two five-stranded antiparallel -sheets that form a sandwich at the dimer interface. The DvuCas2 dimer is stabilized by a distinctive network of hydrophilic cross-protomer side-chain interactions.

Nuclease Activity of Legionella pneumophila Cas2 Promotes Intracellular Infection of Amoebal Host Cells

“…3D), indicating that L. pneumophila Cas2 has a strong preference for Mg 2ϩ . Members of the Cas2 family contain both a ferredoxin fold that is often found in RNA-binding proteins and an N-terminal ␤ strand followed by a polar amino acid (32,33,44,45). L. pneumophila Cas2 has both an N-terminal ␤ strand followed by an aspartate and a ferredoxin fold that is analogous to what exists in the Cas2 proteins of B. halodurans, D. vulgaris, Pyrococcus furiosus, S. solfataricus, and T. thermophilus (15).…”

“…However, it was more difficult to posit the target of the activity, given the various results that had been reported for other Cas2 proteins. Initially, purified Cas2 proteins from the bacteria Nitrosomonas europaea and Thermotoga maritima and the archaea Archaeoglobus fulgidus, Methanobacterium thermoautotrophicum, and Sulfolobus solfataricus were found to have activity against single-stranded RNA (ssRNA) but not against single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA) (32), yet no RNase activity was detected when the Cas2 proteins from the bacteria Desulfovibrio vulgaris and Bacillus halodurans were examined, and only weak RNase activity was reported for Cas2 from Thermus thermophilus (33,34). Moreover, further testing revealed that the Cas2 proteins of B. halodurans, Streptococcus pyogenes, T. thermophilus, and Xanthomonas oryzae have activity against dsDNA (34,35).…”

Characterization of the CRISPR/Cas Subtype I-A System of the Hyperthermophilic Crenarchaeon Thermoproteus tenax

“…In contrast, Cas1 of S. solfataricus bound DNA and RNA with high affinity in a nonspecific manner, but no nuclease activity was detected (33). Cas2 of S. solfataricus was characterized as an Mg-dependent endoribonuclease with a ferredoxin-like fold, specific for single-stranded RNA, but not for crRNAs with a preferential cleavage site within U-rich regions (6,63). Cas4 and Csa1 are not yet enzymatically characterized, but both proteins belong to a restriction endonuclease-like superfamily, defined by a conserved PD-(D/E)XK-motif and are functional similar to RecB exonucleases (42).…”

Double-stranded Endonuclease Activity in Bacillus halodurans Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated Cas2 Protein

“…A recent study further classified the cas2 genes among different subtypes into three different clades based on phyloge-netic analysis (34). The crystal structures of Cas2 from Sulfolobus solfataricus (Sso_Cas2) and Desulfovibrio vulgaris (Dvu_ Cas2) revealed that Cas2 contains a ferredoxin domain and assemblies into a symmetric dimer (27,37) A conserved N-terminal aspartate residue in the S. solfataricus Cas2 was hypothesized to coordinate the catalytic divalent metal ion(s) in the Cas2 dimer. The S. solfataricus Cas2 was further characterized as a metal-dependent single-strand (ss) endoribonuclease with preference for Uracil-rich ssRNA (27); however, this activity was not observed in the D. vulgaris Cas2 (37).…”