CARF and WYL domains: ligand-binding regulators of prokaryotic defense systems

Abstract

CRISPR-Cas adaptive immunity systems of bacteria and archaea insert fragments of virus or plasmid DNA as spacer sequences into CRISPR repeat loci. Processed transcripts encompassing these spacers guide the cleavage of the cognate foreign DNA or RNA. Most CRISPR-Cas loci, in addition to recognized cas genes, also include genes that are not directly implicated in spacer acquisition, CRISPR transcript processing or interference. Here we comprehensively analyze sequences, structures and genomic neighborhoods of one of the most widespread groups of such genes that encode proteins containing a predicted nucleotide-binding domain with a Rossmann-like fold, which we denote CARF (CRISPR-associated Rossmann fold). Several CARF protein structures have been determined but functional characterization of these proteins is lacking. The CARF domain is most frequently combined with a C-terminal winged helix-turn-helix DNA-binding domain and “effector” domains most of which are predicted to possess DNase or RNase activity. Divergent CARF domains are also found in RtcR proteins, sigma-54 dependent regulators of the rtc RNA repair operon. CARF genes frequently co-occur with those coding for proteins containing the WYL domain with the Sm-like SH3 β-barrel fold, which is also predicted to bind ligands. CRISPR-Cas and possibly other defense systems are predicted to be transcriptionally regulated by multiple ligand-binding proteins containing WYL and CARF domains which sense modified nucleotides and nucleotide derivatives generated during virus infection. We hypothesize that CARF domains also transmit the signal from the bound ligand to the fused effector domains which attack either alien or self nucleic acids, resulting, respectively, in immunity complementing the CRISPR-Cas action or in dormancy/programmed cell death.

Recognition of a pseudo-symmetric RNA tetranucleotide by Csx3, a new member of the CRISPR associated Rossmann fold superfamily

“…Specifically, they looked for protein families associated with genes encoding CARF domains, and found that genes for Csx3 are enriched in the gene neighborhoods of the CARF superfamily. 11 This suggests Csx3 and the previously identified CARF domain proteins may be functionally linked. One potential connection is the shared CARF domain common to each of these proteins.…”

“…Bioinformatics efforts suggest that similar domains are also present in the Csm6 family, and other nonCas proteins associated with antiviral defense or a more general stress response. 11 These "CRISPR Associated Rossmann Folds," or CARF domains are frequently fused to potential effector domains that can include winged helix-turn-helix (wHTH) DNA binding domains, DNases of the restriction endonuclease fold and RNases of the RelE and HEPN families. 9,11-13 Collectively, these observations have led to the suggestion that the CRISPR associated Rossmann fold serves to provide ligand binding sites for nucleotides or modified nucleotides that may signal invading nucleic acid, and in cases where they are bound by a homodimer, the ligand may be 2-fold or pseudo-2-fold symmetric.…”

“…9,11-13 Collectively, these observations have led to the suggestion that the CRISPR associated Rossmann fold serves to provide ligand binding sites for nucleotides or modified nucleotides that may signal invading nucleic acid, and in cases where they are bound by a homodimer, the ligand may be 2-fold or pseudo-2-fold symmetric. 9,11 While Csx3 was not identified in the bioinformatics study as a member of the CARF domain superfamily, the tertiary structure certainly bears some resemblance. Further, at the quaternary level, the presence of the nucleotide binding domains of Csx3 arranged within the context a symmetric homodimer is very reminiscent of the arrangement of the proposed ligand binding domains Csa3 and Csx1.…”

“…It remains to be determined whether the role of these CARF domain proteins is to regulate CRISPR/Cas, or as Makarova et al suggest, form an additional arm of CRISPR/ Cas, or both. 9,11 However, it is increasingly clear that these domains are of significant importance in CRISPR/Cas, and the observation of Yan et al that Csx3 is a manganese-dependent deadenylation exoribonuclease capable of binding a pseudosymmetric RNA tetranucleotide is a significant step toward understanding the roles of these CARF domain proteins in CRISPR/Cas.…”

Structural basis for the endoribonuclease activity of the type III-A CRISPR-associated protein Csm6

“…The remainder of the polypeptide, connected to the CARF domain via a short linker, is almost completely α-helical and is composed of two domains. The middle region of the TtCsm6 polypeptide chain (residues 191-292) consists of five α-helices and forms a right-handed solenoid domain previously denoted as the 6H domain (Makarova et al 2014). The C-terminal region of TtCsm6 (residues 293-450) comprises eight α-helices that form the HEPN domain.…”