The CRISPR-associated Cas4 protein Pcal_0546 from Pyrobaculum calidifontis contains a [2Fe-2S] cluster: crystal structure and nuclease activity

Abstract

Cas4 nucleases constitute a core family of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) associated proteins, but little is known about their structure and activity. Here we report the crystal structure of the Cas4 protein Pcal_0546 from Pyrobaculum calidifontis, which revealed a monomeric protein with a RecB-like fold and one [2Fe-2S] cluster coordinated by four conserved Cys residues. Pcal_0546 exhibits metal-dependent 5′ to 3′ exonuclease activity against ssDNA substrates, whereas the Cas4 protein SSO1391 from Sulfolobus solfataricus can cleave ssDNA in both the 5′ to 3′ and 3′ to 5′ directions. The active site of Pcal_0546 contains a bound metal ion coordinated by the side chains of Asp123, Glu136, His146, and the main chain carbonyl of Ile137. Site-directed mutagenesis of Pcal_0546 and SSO1391 revealed that the residues of RecB motifs II, III and QhXXY are critical for nuclease activity, whereas mutations of the conserved Cys residues resulted in a loss of the iron-sulfur cluster, but had no effect on DNA cleavage. Our results revealed the biochemical diversity of Cas4 nucleases, which can have different oligomeric states, contain [4Fe-4S] or [2Fe-2S] clusters, and cleave single stranded DNA in different directions producing single-stranded DNA overhangs, which are potential intermediates for the synthesis of new CRISPR spacers.

CRISPR-Cas in Streptococcus pyogenes

“…In addition to Cas1 and Cas2, these systems also encode Cas4 [44,45], which was recently shown to be involved in adaptation [46][47][48]. Cas4 from Bacillus halodurans type I-C system forms a complex with the Cas1 integrase and, in the presence of the Cas1-Cas2 complex, specifically cleaves the 3′ overhangs of prespacers in a PAM-dependent manner.…”

Cas4 Nucleases Can Effect Specific Integration of CRISPR Spacers

“…Cas4 family proteins occur widely within CRISPR-Cas adaptation modules of subtype I-A, I-B, I-C, I-D, I-U, and II-B and type V systems (2), and they include Csa1 of Sulfolobus subtype I-A systems and Csn2 of the subtype II-A systems of S. thermophilus (20) and Streptococcus pyogenes (21). High-resolution crystal structures of Cas4 proteins from S. solfataricus (33) and Pyrobaculum calidifontis (38) indicate that they carry two domains, an N-terminal RecB-like nuclease domain and a C-terminal domain containing an Fe-S cluster coordinated by four conserved cysteine residues. Moreover, biochemical studies have shown that Cas4 can exhibit 5=-to-3= and 3=-to-5= DNA exonuclease activity, as well as ATP-independent DNA unwinding activity (33,34,38), which suggests that they produce single-strand DNA overhangs that are potential intermediates for insertion of new CRISPR spacers.…”

“…High-resolution crystal structures of Cas4 proteins from S. solfataricus (33) and Pyrobaculum calidifontis (38) indicate that they carry two domains, an N-terminal RecB-like nuclease domain and a C-terminal domain containing an Fe-S cluster coordinated by four conserved cysteine residues. Moreover, biochemical studies have shown that Cas4 can exhibit 5=-to-3= and 3=-to-5= DNA exonuclease activity, as well as ATP-independent DNA unwinding activity (33,34,38), which suggests that they produce single-strand DNA overhangs that are potential intermediates for insertion of new CRISPR spacers. Previously, we have demonstrated that both Cas4 and Csa1 proteins are essential for de novo spacer acquisition (19).…”

Cas4 nucleases can effect specific integration of CRISPR spacers

“…Cas4 family proteins occur widely within CRISPR-Cas adaptation modules of subtype I-A, I-B, I-C, I-D, I-U, II-B, and type V systems (2) and they include Csa1 of Sulfolobus subtype I-A systems and Csn2 of the subtype II-A systems of S. thermophiles (20) and S. pyogenes (21). High resolution crystal structures of Cas4 proteins from S. solfataricus (33) and P. calidifontis (38) indicate that they carry two domains, an N-terminal RecB-like nuclease domain and a C-terminal domain containing an Fe-S cluster coordinated by four conserved cysteine residues. Moreover, biochemical studies have shown that Cas4 can exhibit 5’ to 3’ and 3’ to 5’ DNA exonuclease activity, as well as ATP independent DNA unwinding activity (33, 34, 38) which suggests that they produce single-strand DNA overhangs that are potential intermediates for insertion of new CRISPR spacers.…”

“…High resolution crystal structures of Cas4 proteins from S. solfataricus (33) and P. calidifontis (38) indicate that they carry two domains, an N-terminal RecB-like nuclease domain and a C-terminal domain containing an Fe-S cluster coordinated by four conserved cysteine residues. Moreover, biochemical studies have shown that Cas4 can exhibit 5’ to 3’ and 3’ to 5’ DNA exonuclease activity, as well as ATP independent DNA unwinding activity (33, 34, 38) which suggests that they produce single-strand DNA overhangs that are potential intermediates for insertion of new CRISPR spacers. Previously, we have demonstrated that both Cas4 and Csa1 proteins are essential for de novo spacer acquisition (19).…”