Premium
Crystal structural analysis and metal‐dependent stability and activity studies of the ColE7 endonuclease domain in complex with DNA/Zn 2+ or inhibitor/Ni 2+
Author(s) -
Doudeva Lyudmila G.,
Huang Hsinchin,
Hsia KuoChiang,
Shi Zhonghao,
Li ChiaLung,
Shen Yongliang,
Cheng YiSheng,
Yuan Hanna S.
Publication year - 2006
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.051903406
Subject(s) - nuclease , crystallography , chemistry , metal , stereochemistry , dna , metal ions in aqueous solution , zinc , hydrolase , enzyme , biochemistry , organic chemistry
The nuclease domain of ColE7 (N‐ColE7) contains an H‐N‐H motif that folds in a ββα‐metal topology. Here we report the crystal structures of a Zn 2+ ‐bound N‐ColE7 (H545E mutant) in complex with a 12‐bp duplex DNA and a Ni 2+ ‐bound N‐ColE7 in complex with the inhibitor Im7 at a resolution of 2.5 Å and 2.0 Å, respectively. Metal‐dependent cleavage assays showed that N‐ColE7 cleaves double‐stranded DNA with a single metal ion cofactor, Ni 2+ , Mg 2+ , Mn 2+ , and Zn 2+ . ColE7 purified from Escherichia coli contains an endogenous zinc ion that was not replaced by Mg 2+ atconcentrations of <25mM, indicating thatzincisthe physiologically relevant metal ion in N‐ColE7 in host E. coli . In the crystal structure of N‐ColE7/DNA complex, the zinc ion is directly coordinated to three histidines and the DNA scissile phosphate in a tetrahedral geometry. In contrast, Ni 2+ is bound in N‐ColE7 in two different modes, to four ligands (three histidines and one phosphate ion), or to five ligands with an additional water molecule. These data suggest that the divalent metal ion in the His‐metal finger motif can be coordinated to six ligands, such as Mg 2+ in I‐PpoI, Serratia nuclease and Vvn, five ligands or four ligands, such as Ni 2+ or Zn 2+ in ColE7. Universally, the metal ion in the His‐metal finger motif is bound to the DNA scissile phosphate and serves three roles during hydrolysis: polarization of the P–O bond for nucleophilic attack, stabilization of the phosphoanion transition state and stabilization of the cleaved product.