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. 2006 Jun 1;441(7093):656-9.
doi: 10.1038/nature04818.

Computational redesign of endonuclease DNA binding and cleavage specificity

Justin Ashworth  1 , James J Havranek, Carlos M Duarte, Django Sussman, Raymond J Monnat Jr, Barry L Stoddard, David Baker
Affiliations

Computational redesign of endonuclease DNA binding and cleavage specificity

Justin Ashworth et al. Nature. .

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.

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Figures

Figure 1
Figure 1. Comparison of the predicted interactions in cognate and non-cognate binding complexes, illustrating the designed specificity switch
a, Wild-type I-MsoI, −6C · G (wild type). A water molecule present in the original structure is shown. b, Wild-type I-MsoI, −6G · C. c, I-MsoI-K28L/T83R, −6C · G. d, I-MsoI-K28L/T83R, −6G · C. In parts c and d, the van der Waals surfaces of Leu 28 and +6C are shown in grey. Figures were generated using the molecular graphics program PyMOL (Delano Scientific). WT, wild type; DES, designed; blue strands, protein backbone; beige spheres and sticks, DNA backbone; other spheres, constant nucleotides; dashed lines, hydrogen bonds.
Figure 2
Figure 2. Switch in nuclease cleavage specificity
Equimolar amounts of linearized plasmid DNAs containing wild-type (WT) or designed (DES) I-MsoI cleavage sites were digested by serial dilutions of wild-type or designed I-MsoI endonuclease, and analysed by gel electrophoresis. The switch in sequence specificity is defined as (wild type vs. DES/wild type vs. WT) × (designed vs. WT/designed vs. DES), where quantities in parentheses indicate the lowest enzyme concentration at which significant cleavage of the site is observed. Here, the wild-type enzyme favours the WT site by >27-fold, the designed enzyme favours the DES site by ~25-fold, and hence the specificity switch is greater than 27 × 25 (>4,000-fold).
Figure 3
Figure 3. Crystal structure of the designed enzyme–DNA complex
Left, Fo−Fc electron-density map of the redesigned region calculated from a refinement model lacking the redesigned side chains and bases (cyan). The computational design model (grey) fits well into the unassigned density (blue mesh, +2.2σ). Right, superposition of the design model (salmon) and the refined crystal structure (cyan) confirms the accuracy of the design. A new coordinated water molecule (red sphere) is also apparent.
See this image and copyright information in PMC

Comment in

  • Specificity by design.
    Pabo CO. Pabo CO. Nat Biotechnol. 2006 Aug;24(8):954-5. doi: 10.1038/nbt0806-954. Nat Biotechnol. 2006. PMID: 16900141 No abstract available.

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