4ES2

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

Summary for 4ES2

• Entry DOI: 10.2210/pdb4es2/pdb
• Related: 4ES1 4ES3
• Descriptor: BH0342 protein (2 entities in total)
• Functional Keywords: ferredoxin, nuclease, hydrolase
• Biological source: Bacillus halodurans
• Total number of polymer chains: 1
• Total formula weight: 11308.01

Authors

Ke, A.,Nam, K.H. (deposition date: 2012-04-21, release date: 2012-08-22, Last modification date: 2024-02-28)

Primary citation

Nam, K.H.,Ding, F.,Haitjema, C.,Huang, Q.,DeLisa, M.P.,Ke, A.
Double-stranded endonuclease activity in Bacillus halodurans clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas2 protein.
J. Biol. Chem., 287:35943-35952, 2012

PubMed Abstract: The CRISPR (clustered regularly interspaced short palindromic repeats) system is a prokaryotic RNA-based adaptive immune system against extrachromosomal genetic elements. Cas2 is a universally conserved core CRISPR-associated protein required for the acquisition of new spacers for CRISPR adaptation. It was previously characterized as an endoribonuclease with preference for single-stranded (ss)RNA. Here, we show using crystallography, mutagenesis, and isothermal titration calorimetry that the Bacillus halodurans Cas2 (Bha_Cas2) from the subtype I-C/Dvulg CRISPR instead possesses metal-dependent endonuclease activity against double-stranded (ds)DNA. This activity is consistent with its putative function in producing new spacers for insertion into the 5'-end of the CRISPR locus. Mutagenesis and isothermal titration calorimetry studies revealed that a single divalent metal ion (Mg(2+) or Mn(2+)), coordinated by a symmetric Asp pair in the Bha_Cas2 dimer, is involved in the catalysis. We envision that a pH-dependent conformational change switches Cas2 into a metal-binding competent conformation for catalysis. We further propose that the distinct substrate preferences among Cas2 proteins may be determined by the sequence and structure in the β1-α1 loop.

PubMed: 22942283
DOI: 10.1074/jbc.M112.382598

Experimental method

X-RAY DIFFRACTION (1.299 Å)