2FLD

I-MsoI Re-Designed for Altered DNA Cleavage Specificity

Summary for 2FLD

• Entry DOI: 10.2210/pdb2fld/pdb
• Related: 1M5X
• Descriptor: 5'-D(*GP*CP*AP*GP*AP*AP*GP*GP*TP*CP*GP*TP*GP*AP*GP*AP*CP*CP*GP*TP*TP*CP*CP*G)-3', 5'-D(*CP*GP*GP*AP*AP*CP*GP*GP*TP*CP*TP*CP*AP*CP*GP*AP*CP*CP*TP*TP*CP*TP*GP*C)-3', DNA ENDONUCLEASE I-MSOI, ... (6 entities in total)
• Functional Keywords: homing endonuclease, dna, hydrolase-dna complex, hydrolase/dna
• Biological source: Monomastix sp.
• Total number of polymer chains: 4
• Total formula weight: 52715.23

Authors

Ashworth, J.,Duarte, C.M.,Havranek, J.J.,Sussman, D.,Monnat, R.J.,Stoddard, B.L.,Baker, D. (deposition date: 2006-01-05, release date: 2006-06-06, Last modification date: 2023-08-30)

Primary citation

Ashworth, J.,Havranek, J.J.,Duarte, C.M.,Sussman, D.,Monnat, R.J.,Stoddard, B.L.,Baker, D.
Computational redesign of endonuclease DNA binding and cleavage specificity.
Nature, 441:656-659, 2006

PubMed Abstract: The reprogramming of DNA-binding specificity is an important challenge for computational protein design that tests current understanding of protein-DNA recognition, and has considerable practical relevance for biotechnology and medicine. Here we describe the computational redesign of the cleavage specificity of the intron-encoded homing endonuclease I-MsoI using a physically realistic atomic-level forcefield. Using an in silico screen, we identified single base-pair substitutions predicted to disrupt binding by the wild-type enzyme, and then optimized the identities and conformations of clusters of amino acids around each of these unfavourable substitutions using Monte Carlo sampling. A redesigned enzyme that was predicted to display altered target site specificity, while maintaining wild-type binding affinity, was experimentally characterized. The redesigned enzyme binds and cleaves the redesigned recognition site approximately 10,000 times more effectively than does the wild-type enzyme, with a level of target discrimination comparable to the original endonuclease. Determination of the structure of the redesigned nuclease-recognition site complex by X-ray crystallography confirms the accuracy of the computationally predicted interface. These results suggest that computational protein design methods can have an important role in the creation of novel highly specific endonucleases for gene therapy and other applications.

PubMed: 16738662
DOI: 10.1038/nature04818

Experimental method

X-RAY DIFFRACTION (2 Å)