title: Crystal structural analysis and metal-dependent stability and activity studies of the ColE7 endonuclease domain in complex with DNA/Zn2+ or inhibitor/Ni2+.
abstract: The nuclease domain of ColE7 (N-ColE7) contains an H-N-H motif that folds in a beta beta alpha-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 A and 2.0 A, respectively. Metal-dependent cleavage assays showed that N-ColE7 cleaves double-stranded DNA with a single metal 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.
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan 11529, Republic of China.
authors.forename: Lyudmila G
authors.initials: LG
authors.lastname: Doudeva
authors.forename: Hsinchin
authors.initials: H
authors.lastname: Huang
authors.forename: Kuo-Chiang
authors.initials: KC
authors.lastname: Hsia
authors.forename: Zhonghao
authors.initials: Z
authors.lastname: Shi
authors.forename: Chia-Lung
authors.initials: CL
authors.lastname: Li
authors.forename: Yongliang
authors.initials: Y
authors.lastname: Shen
authors.forename: Yi-Sheng
authors.initials: YS
authors.lastname: Cheng
authors.forename: Hanna S
authors.initials: HS
authors.lastname: Yuan
mesh_terms: D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002413:Cations, Divalent / Q000378:metabolism; D003087:Colicins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D009532:Nickel / Q000737:chemistry* / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D015032:Zinc / Q000737:chemistry* / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; D002413:Cations, Divalent; C508089:ColE7 protein, E coli; D003087:Colicins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C007104:colicin immunity proteins; D006639:Histidine; D009532:Nickel; D015032:Zinc