6UWG

Engineered variant of I-OnuI meganuclease with improved thermostability and E178D mutation at catalytic site

6UWG の概要

• エントリーDOI: 10.2210/pdb6uwg/pdb
• 分子名称: I-OnuI-e-Therm-E178D, DNA (26-MER), CALCIUM ION, ... (6 entities in total)
• 機能のキーワード: meganuclease, homing endonuclease, dna binding protein, dna binding protein-dna complex, dna binding protein/dna
• 由来する生物種: synthetic construct; 詳細
• タンパク質・核酸の鎖数: 3
• 化学式量合計: 51209.41

構造登録者

Werther, R.,Stoddard, B.L. (登録日: 2019-11-05, 公開日: 2019-12-18, 最終更新日: 2023-10-11)

主引用文献

Lambert, A.R.,Hallinan, J.P.,Werther, R.,Glow, D.,Stoddard, B.L.
Optimization of Protein Thermostability and Exploitation of Recognition Behavior to Engineer Altered Protein-DNA Recognition.
Structure, 28:760-775.e8, 2020

PubMed Abstract: The redesign of a macromolecular binding interface and corresponding alteration of recognition specificity is a challenging endeavor that remains recalcitrant to computational approaches. This is particularly true for the redesign of DNA binding specificity, which is highly dependent upon bending, hydrogen bonds, electrostatic contacts, and the presence of solvent and counterions throughout the molecular interface. Thus, redesign of protein-DNA binding specificity generally requires iterative rounds of amino acid randomization coupled to selections. Here, we describe the importance of scaffold thermostability for protein engineering, coupled with a strategy that exploits the protein's specificity profile, to redesign the specificity of a pair of meganucleases toward three separate genomic targets. We determine and describe a series of changes in protein sequence, stability, structure, and activity that accumulate during the engineering process, culminating in fully retargeted endonucleases.

PubMed: 32359399
DOI: 10.1016/j.str.2020.04.009

実験手法

X-RAY DIFFRACTION (2.22 Å)