Abstract
Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (cas) proteins provide RNA-mediated adaptive immunity against foreign invading nucleic acids such as phages and plasmids in archaea and bacteria. Cas2 protein is one of the two core Cas proteins are present in all types of CRISPR-Cas systems and is required for new spacer integration into CRISPR loci. Cas2 homologues from several CRISPR-Cas subtypes were characterized as metal-dependent nucleases with different substrate preferences, and it was proposed that a pH-dependent conformational change mediates metal binding and catalysis. Here, we report the X-ray crystal structures of Streptococcus pyogenes Cas2 protein at three different pHs (5.6, 6.5, and 7.5), and the results of its nuclease activity assay at varying pHs (6.0-9.0). While S. pyogenes Cas2 exhibited strongly pH-dependent catalytic activity, there was no significant conformational difference among the three crystal structures. However, structural comparisons with other Cas2 homologues suggest structural variability and the flexible nature of its putative hinge regions, supporting the supposition that conformational change is important for catalysis.
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