PDBsum entry: 3fbd

Hydrolase/DNA

PDB id: 3fbd

Contents

Protein chains

132 a.a. *

DNA/RNA

Waters ×71

* Residue conservation analysis

PDB id:

3fbd

Name:

Hydrolase/DNA

Title:

Crystal structure of the nuclease domain of cole7(d493q mutant) in complex with an 18-bp duplex DNA

Structure:

Colicin-e7. Chain: a, d. Fragment: nuclease domain. Engineered: yes. Mutation: yes. 5'- d( Dgp Dgp Dap Dap Dtp Dtp Dcp Dgp Dap Dtp Dcp Dgp Dap Dap Dtp Dtp Dcp Dc)-3'. Chain: b, c, e, f.

Source:

Escherichia coli. Organism_taxid: 562. Strain: w3110. Gene: cea, cole7. Expressed in: escherichia coli. Expression_system_taxid: 562. Synthetic: yes

Resolution:

2.90Å R-factor: 0.201 R-free: 0.264

Authors:

Y.T.Wang,L.G.Doudeva,H.S.Yuan

Key ref:

Y.T.Wang et al. (2009). Redesign of high-affinity nonspecific nucleases with altered sequence preference. J Am Chem Soc, 131, 17345-17353. PubMed id: 19929021 DOI: 10.1021/ja907160r

Date:

19-Nov-08 Release date: 03-Nov-09

Protein chains

Q47112  (CEA7_ECOLX) -  Colicin-E7

Seq: 576 a.a.

Struc: 132 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

 Gene Ontology (GO) functional annotation 

• Biological process: cytolysis (3 terms)
• Biochemical function: nucleic acid binding (3 terms)

DOI no: 10.1021/ja907160r

J Am Chem Soc 131:17345-17353 (2009)

PubMed id: 19929021

Redesign of high-affinity nonspecific nucleases with altered sequence preference.

Y.T.Wang, J.D.Wright, L.G.Doudeva, H.C.Jhang, C.Lim, H.S.Yuan.

ABSTRACT

It is of crucial importance to elucidate the underlying principles that govern the binding affinity and selectivity between proteins and DNA. Here we use the nuclease domain of Colicin E7 (nColE7) as a model system to generate redesigned nucleases with improved DNA-binding affinities. ColE7 is a bacterial toxin, bearing a nonspecific endonuclease domain with a preference for hydrolyzing DNA phosphodiester bonds at the 3'O-side after thymine and adenine; i.e., it prefers Thy and Ade at the -1 site. Using systematic computational screening, six nColE7 mutants were predicted to bind DNA with high affinity. Five of the redesigned single-point mutants were constructed and purified, and four mutants had a 3- to 5-fold higher DNA binding affinity than wild-type nColE7 as measured by fluorescence kinetic assays. Moreover, three of the designed mutants, D493N, D493Q, and D493R, digested DNA with an increased preference for guanine at +3 sites compared to the wild-type enzyme, as shown by DNA footprint assays. X-ray structure determination of the ColE7 mutant D493Q-DNA complex in conjunction with structural and free energy decomposition analyses provides a physical basis for the improved protein-DNA interactions: Replacing D493 at the protein-DNA interface with an amino acid residue that can maintain the native hydrogen bonds removes the unfavorable electrostatic repulsion between the negatively charged carboxylate and DNA phosphate groups. These results show that computational screening combined with biochemical, structural, and free energy analyses provide a useful means for generating redesigned nucleases with a higher DNA-binding affinity and altered sequence preferences in DNA cleavage.