4AL5

Crystal structure of the Csy4-crRNA product complex

4AL5 の概要

• エントリーDOI: 10.2210/pdb4al5/pdb
• 関連するPDBエントリー: 4AL6 4AL7
• 分子名称: CSY4 ENDORIBONUCLEASE, 5'-R(*UP*UP*CP*AP*CP*UP*GP*CP*CP*GP*UP*AP*UP*AP *GP*GP*CP*AP*GP*C)-3', GLYCEROL, ... (4 entities in total)
• 機能のキーワード: hydrolase-rna complex, crispr, hydrolase/rna
• 由来する生物種: PSEUDOMONAS AERUGINOSA; 詳細
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 28287.89

構造登録者

Haurwitz, R.E.,Sternberg, S.H.,Doudna, J.A. (登録日: 2012-03-02, 公開日: 2012-06-13, 最終更新日: 2023-12-20)

主引用文献

Haurwitz, R.E.,Sternberg, S.H.,Doudna, J.A.
Csy4 Relies on an Unusual Catalytic Dyad to Position and Cleave Crispr RNA.
Embo J., 31:2824-, 2012

PubMed Abstract: CRISPR-Cas adaptive immune systems protect prokaryotes against foreign genetic elements. crRNAs derived from CRISPR loci base pair with complementary nucleic acids, leading to their destruction. In Pseudomonas aeruginosa, crRNA biogenesis requires the endoribonuclease Csy4, which binds and cleaves the repetitive sequence of the CRISPR transcript. Biochemical assays and three co-crystal structures of wild-type and mutant Csy4/RNA complexes reveal a substrate positioning and cleavage mechanism in which a histidine deprotonates the ribosyl 2'-hydroxyl pinned in place by a serine, leading to nucleophilic attack on the scissile phosphate. The active site catalytic dyad lacks a general acid to protonate the leaving group and positively charged residues to stabilize the transition state, explaining why the observed catalytic rate constant is ∼10(4)-fold slower than that of RNase A. We show that this RNA cleavage step is essential for assembly of the Csy protein-crRNA complex that facilitates target recognition. Considering that Csy4 recognizes a single cellular substrate and sequesters the cleavage product, evolutionary pressure has likely selected for substrate specificity and high-affinity crRNA interactions at the expense of rapid cleavage kinetics.

PubMed: 22522703
DOI: 10.1038/EMBOJ.2012.107

実験手法

X-RAY DIFFRACTION (2 Å)