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Metal ions and phosphate binding in the H‐N‐H motif: Crystal structures of the nuclease domain of ColE7/Im7 in complex with a phosphate ion and different divalent metal ions
Author(s) -
Sui MengJiun,
Tsai LiChu,
Hsia KuoChiang,
Doudeva Lyudmila G.,
Ku WenYen,
Han Gye Won,
Yuan Hanna S.
Publication year - 2002
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.0220602
Subject(s) - nuclease , crystallography , colicin , chemistry , metal ions in aqueous solution , metal , stereochemistry , divalent , dna , biochemistry , organic chemistry , plasmid
Abstract 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, Zn 2+ ‐bound and Mn 2+ ‐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 β‐strands linked to a C‐terminal α‐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 Mg 2+ ‐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 Zn 2+ or Mn 2+ ions were observed in the center of the H‐N‐H motif in cases of Zn 2+ or Mn 2+ ‐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.