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Crystal structural analysis and metal-dependent stability
and activity studies of the ColE7 endonuclease domain in complex with DNA/Zn2+
or inhibitor/Ni2+
LYUDMILA G. DOUDEVA,1 HSINCHIN HUANG,1,2
KUO-CHIANG HSIA,1
ZHONGHAO SHI,1 CHIA-LUNG LI,1
YONGLIANG SHEN,1,3 YI-SHENG CHENG,1
AND HANNA S. YUAN1,2,3
1Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan,
Republic of China
2Institute of Biochemistry, National
Yang-Ming University,
Taipei, Taiwan,
Republic of China
3Institute of Biochemistry and Molecular Biology, College of Medicine,
National Taiwan
University, Taipei,
Taiwan, Republic of
China
(RECEIVED October 10, 2005; FINAL REVISION November
10, 2005; ACCEPTED November 14, 2005)
Abstract
Abstract
The
nuclease domain of ColE7 (N-ColE7) contains an H-N-H motif that folds in a bba-metal
topology.Here we report the crystal structures of a Zn2+-bound N-ColE7 (H545E
mutant) in complex with a 12-bp duplexDNAand aNi2+-bound N-ColE7 in complex with the inhibitor Im7 at a
resolution of 2.5A˚ and 2.0A˚ , respectively.Metal-dependent
cleavage assays showed thatN-ColE7 cleaves double-strandedDNAwith
a
singlemetal ion cofactor, Ni2+,Mg2+,Mn2+, andZn2+. ColE7 purified from Escherichia
coli contains an endogenous zinc ion that was not replaced byMg2+ at concentrations
of<25mM,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.Keywords: Protein nucleic acid
interactions; nonspecific nuclease; DNase; DNA hydrolysis mechanism; H-N-H
motif; bba-metal motif; colicin E7
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