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1 Graduate Institute of Life Science, National Defense Medical Center, Taipei, Taiwan 11472, ROC
2 Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan 11529, ROC
3 Molecular Biology Institute, University of California at Los Angeles, California 90095-1570, USA
Reprint requests to: Hanna S. Yuan, Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan 11529; e-mail: hanna{at}sinica.edu.tw; fax: 886-2-2782-6085.
H-N-H is a motif found in the nuclease domain of a subfamily of bacteria toxins, including colicin E7, that are capable of cleaving DNA nonspecifically. This H-N-H motif has also been identified in a subfamily of homing endonucleases, which cleave DNA site specifically. To better understand the role of metal ions in the H-N-H motif during DNA hydrolysis, we crystallized the nuclease domain of colicin E7 (nuclease-ColE7) in complex with its inhibitor Im7 in two different crystal forms, and we resolved the structures of EDTA-treated, Zn2+-bound and Mn2+-bound complexes in the presence of phosphate ions at resolutions of 2.6 Å to 2.0 Å. This study offers the first determination of the structure of a metal-free and substrate-free enzyme in the H-N-H family. The H-N-H motif contains two antiparallel ß-strands linked to a C-terminal 
-helix, with a divalent metal ion located in the center. Here we show that the metal-binding sites in the center of the H-N-H motif, for the EDTA-treated and Mg2+-soaked complex crystals, were occupied by water molecules, indicating that an alkaline earth metal ion does not reside in the same position as a transition metal ion in the H-N-H motif. However, a Zn2+ or Mn2+ ions were observed in the center of the H-N-H motif in cases of Zn2+ or Mn2+-soaked crystals, as confirmed in anomalous difference maps. A phosphate ion was found to bridge between the divalent transition metal ion and His545. Based on these structures and structural comparisons with other nucleases, we suggest a functional role for the divalent transition metal ion in the H-N-H motif in stabilizing the phosphoanion in the transition state during hydrolysis.
Keywords: Metal binding in proteins; divalent metal ions; magnesium ion; zinc ion; endonuclease; DNase; DNA hydrolysis mechanism
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