9BGI

Activated wild-type SgrAI endonuclease DNA-bound dimer with Mg2+ and cleaved primary site DNA

9BGI の概要

• エントリーDOI: 10.2210/pdb9bgi/pdb
• EMDBエントリー: 44513
• 分子名称: SgraIR restriction enzyme, 40-1 DNA, MAGNESIUM ION, ... (5 entities in total)
• 機能のキーワード: restriction endonuclease, dnase, allostery, bacterial innate immunity, filament, hyper-activation, substrate specificity, hydrolase-dna complex, dna binding protein, hydrolase/dna
• 由来する生物種: Streptomyces griseus; 詳細
• タンパク質・核酸の鎖数: 6
• 化学式量合計: 104204.87

構造登録者

Shan, Z.,Lyumkis, D.,Horton, N.C. (登録日: 2024-04-18, 公開日: 2025-03-05)

主引用文献

Shan, Z.,Rivero-Gamez, A.,Lyumkis, D.,Horton, N.C.
Two-metal ion mechanism of DNA cleavage by activated, filamentous SgrAI.
J.Biol.Chem., 300:107576-107576, 2024

PubMed Abstract: Enzymes that form filamentous assemblies with modulated enzymatic activities have gained increasing attention in recent years. SgrAI is a sequence specific type II restriction endonuclease that forms polymeric filaments with accelerated DNA cleavage activity and expanded DNA sequence specificity. Prior studies have suggested a mechanistic model linking the structural changes accompanying SgrAI filamentation to its accelerated DNA cleavage activity. In this model, the conformational changes that are specific to filamentous SgrAI maximize contacts between different copies of the enzyme within the filament and create a second divalent cation binding site in each subunit, which in turn facilitates the DNA cleavage reaction. However, our understanding of the atomic mechanism of catalysis is incomplete. Herein, we present two new structures of filamentous SgrAI solved using cryo-EM. The first structure, resolved to 3.3 Å, is of filamentous SgrAI containing an active site mutation that is designed to stall the DNA cleavage reaction, which reveals the enzymatic configuration prior to DNA cleavage. The second structure, resolved to 3.1 Å, is of WT filamentous SgrAI containing cleaved substrate DNA, which reveals the enzymatic configuration at the end of the enzymatic cleavage reaction. Both structures contain the phosphate moiety at the cleavage site and the biologically relevant divalent cation cofactor Mg and define how the Mg cation reconfigures during enzymatic catalysis. The data support a model for the activation mechanism that involves binding of a second Mg in the SgrAI active site as a direct result of filamentation induced conformational changes.

PubMed: 39009341
DOI: 10.1016/j.jbc.2024.107576

実験手法

ELECTRON MICROSCOPY (3.05 Å)