Crystal structural analysis and metal-dependent stability and activity studies of the ColE7 endonuclease domain in complex with DNA/Zn2+ or inhibitor/Ni2+
Journal
Protein Science
Journal Volume
15
Journal Issue
2
Pages
269-280
Date Issued
2006
Author(s)
Abstract
The nuclease domain of ColE7 (N-ColE7) contains an H-N-H motif that folds in a £]£]£\-metal topology. Here we report the crystal structures of a Zn2+-bound N-ColE7 (H545E mutant) in complex with a 12-bp duplex DNA and a Ni2+ -bound N-ColE7 in complex with the inhibitor Im7 at a resolution of 2.5? and 2.0 ?, respectively. Metal-dependent cleavage assays showed that N-ColE7 cleaves double-stranded DNA with a singlemetal ion cofactor, Ni2+, Mg2+, Mn2+, and Zn2+. ColE7 purified from Escherichia coli contains an endogenous zinc ion that was not replaced by Mg2+ at concentrations of <25 mM, indicating that zinc is the 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, Ni2+ 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 Mg2+ in I-PpoI, Serratia nuclease and Vvn, five ligands or four ligands, such as Ni2+ or Zn2+ 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. Published by Cold Spring Harbor Laboratory Press. Copyright ? 2006 The Protein Society.
Subjects
£]£]£\-metal motif
Colicin E7
DNA hydrolysis mechanism
DNase
H-N-H motif
Nonspecific nuclease
Protein nucleic acid interactions
Type
journal article
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