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PDBsum entry 2fld
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Hydrolase/DNA
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PDB id
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2fld
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References listed in PDB file
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Key reference
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Title
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Computational redesign of endonuclease DNA binding and cleavage specificity.
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Authors
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J.Ashworth,
J.J.Havranek,
C.M.Duarte,
D.Sussman,
R.J.Monnat,
B.L.Stoddard,
D.Baker.
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Ref.
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Nature, 2006,
441,
656-659.
[DOI no: ]
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PubMed id
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Abstract
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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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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.
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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.
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The above figures are
reprinted
by permission from Macmillan Publishers Ltd:
Nature
(2006,
441,
656-659)
copyright 2006.
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