PDBsum entry 2fld

Hydrolase/DNA

PDB id: 2fld

Contents

Protein chains

161 a.a. *

DNA/RNA

Metals

_NA

_CA ×2

Waters ×97

* Residue conservation analysis

PDB id:

2fld

Name:

Hydrolase/DNA

Title:

I-msoi re-designed for altered DNA cleavage specificity

Structure:

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'. Chain: c. Engineered: yes. Mutation: yes. 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'. Chain: d. Engineered: yes.

Source:

Synthetic: yes. Monomastix sp.. Organism_taxid: 141716. Strain: oke-1. Expressed in: escherichia coli bl21. Expression_system_taxid: 511693.

Biol. unit:

Tetramer (from PQS)

Resolution:

2.00Å R-factor: 0.229 R-free: 0.271

Authors:

J.Ashworth,C.M.Duarte,J.J.Havranek,D.Sussman,R.J.Monnat,B.L.Stoddard, D.Baker

Key ref:

J.Ashworth et al. (2006). Computational redesign of endonuclease DNA binding and cleavage specificity. Nature, 441, 656-659. PubMed id: 16738662 DOI: 10.1038/nature04818

Date:

05-Jan-06 Release date: 06-Jun-06

Protein chains

C0JWR6  (C0JWR6_MONSK) -  Site-specific DNA endonuclease I-MsoI from Monomastix sp. (strain OKE-1)

Seq: 170 a.a.

Struc: 161 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

DNA/RNA chains

G-C-A-G-A-A-G-G-T-C-G-T-G-A-G-A-C-C-G-T-T-C-C-G 24 bases

C-G-G-A-A-C-G-G-T-C-T-C-A-C-G-A-C-C-T-T-C-T-G-C 24 bases

DOI no: 10.1038/nature04818

Nature 441:656-659 (2006)

PubMed id: 16738662

Computational redesign of endonuclease DNA binding and cleavage specificity.

J.Ashworth, J.J.Havranek, C.M.Duarte, D.Sussman, R.J.Monnat, B.L.Stoddard, D.Baker.

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.

Selected figure(s)

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^16 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 3.
Figure 3: Crystal structure of the designed enzyme–DNA complex. Left, F[o]–F[c] 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, +2.2 ). 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.

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nature (2006, 441, 656-659) copyright 2006.

Figures were selected by an automated process.