4ES3

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

4ES3 の概要

• エントリーDOI: 10.2210/pdb4es3/pdb
• 関連するPDBエントリー: 4ES1 4ES2
• 分子名称: BH0342 protein, 1,2-ETHANEDIOL (3 entities in total)
• 機能のキーワード: ferredoxin, nuclease, hydrolase
• 由来する生物種: Bacillus halodurans
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 11370.08

構造登録者

Ke, A.,Nam, K.H. (登録日: 2012-04-21, 公開日: 2012-08-22, 最終更新日: 2024-02-28)

主引用文献

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

実験手法

X-RAY DIFFRACTION (1.704 Å)