2fld

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spinqualitylabelsall models 2fld, resolution 2.00Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

I-MsoI Re-Designed for Altered DNA Cleavage Specificity

Overview

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.

About this Structure

2FLD is a Single protein structure of sequence from Monomastix sp. with NA and CA as ligands. Full crystallographic information is available from OCA.

Reference

Computational redesign of endonuclease DNA binding and cleavage specificity., Ashworth J, Havranek JJ, Duarte CM, Sussman D, Monnat RJ Jr, Stoddard BL, Baker D, Nature. 2006 Jun 1;441(7093):656-9. PMID:16738662

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