6UWG

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

Summary for 6UWG

• Entry DOI: 10.2210/pdb6uwg/pdb
• Descriptor: I-OnuI-e-Therm-E178D, DNA (26-MER), CALCIUM ION, ... (6 entities in total)
• Functional Keywords: meganuclease, homing endonuclease, dna binding protein, dna binding protein-dna complex, dna binding protein/dna
• Biological source: synthetic construct; More
• Total number of polymer chains: 3
• Total formula weight: 51209.41

Authors

Werther, R.,Stoddard, B.L. (deposition date: 2019-11-05, release date: 2019-12-18, Last modification date: 2023-10-11)

Primary citation

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

Experimental method

X-RAY DIFFRACTION (2.22 Å)