title: Structural and functional characterization of Streptococcus pyogenes Cas2 protein under different pH conditions.
abstract: Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins constitute an RNA-guided microbial defense system against invading foreign genetic materials. Cas2 is one of the core Cas proteins found universally in all the subtypes of CRISPR-Cas systems and is required for incorporating new spacers into CRISPR loci. Cas2 homologues from different CRISPR-Cas subtypes were characterized previously 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 crystal structures of Streptococcus pyogenes Cas2 at three different pHs (5.6, 6.5, and 7.5), as well as the results of its nuclease activity assay against double-stranded DNAs at varying pHs (6.0-9.0). Although 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 revealed structural variability and the flexible nature of its putative hinge regions, supporting the hypothesis that conformational switching is important for catalysis. Taken together, our results confirm that Cas2 proteins have pH-dependent nuclease activity against double-stranded DNAs, and provide indirect structural evidence for their conformational changes.
authors: Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Republic of Korea.
authors: Donghyun
authors: D
authors: Ka
authors: Department of Applied Biology and Chemistry, Seoul National University, Seoul 151-921, Republic of Korea.
authors: Dayoun
authors: D
authors: Kim
authors: Department of Applied Biology and Chemistry, Seoul National University, Seoul 151-921, Republic of Korea.
authors: Gyeongyun
authors: G
authors: Baek
authors: Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Republic of Korea; Center for Food and Bioconvergence, Seoul National University, Seoul 151-921, Republic of Korea; Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Republic of Korea. Electronic address: bae@snu.ac.kr.
authors: Euiyoung
authors: E
authors: Bae
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D018360:Crystallography, X-Ray; D004247:DNA / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013297:Streptococcus pyogenes / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D004247:DNA
keywords: CRISPR; Cas; Deoxyribonuclease; Streptococcus pyogenes; Structural biology