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87 a.a.
131 a.a.
_ZN
Key reference
DOI no: 10.1110/ps.0220602 Protein Sci 11:2947-2957 (2002) PubMed id: 12441392
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. M.J.Sui, L.C.Tsai, K.C.Hsia, L.G.Doudeva, W.Y.Ku, G.W.Han, H.S.Yuan. 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 A to 2.0 A. 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 beta-strands linked to a C-terminal alpha-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.
Selected figure(s)
Figure 4.
Fig. 4. Ribbon model of nuclease-ColE7/Im7 bound with a Zn2+ ion and a phosphate. The four-helices in Im7 are displayed in red and the nuclease-ColE7 is shown in blue, with only the H-N-H motif in green.
Figure 7.
Fig. 7. Structural model of nuclease-ColE7 bound to DNA. The model was constructed by overlapping the two ß-strands and oneThe above figures are reprinted by permission from the Protein Society: Protein Sci (2002, 11, 2947-2957) copyright 2002. Figures were selected by an automated process.
Literature references that cite this PDB file's key reference
PubMed id Reference
18439318 Q.Song, and X.Zhang (2008).
Characterization of a novel non-specific nuclease from thermophilic bacteriophage GBSV1.BMC Biotechnol, 8, 43.
17175542 Y.T.Wang, W.J.Yang, C.L.Li, L.G.Doudeva, and H.S.Yuan (2007).
Structural basis for sequence-dependent DNA cleavage by nonspecific endonucleases.Nucleic Acids Res, 35, 584-594.
PDB codes: 2ivh 2ivk
16470805 C.H.Lu, Y.S.Lin, Y.C.Chen, C.S.Yu, S.Y.Chang, and J.K.Hwang (2006).
The fragment transformation method to detect the protein structural motifs.Proteins, 63, 636-643.
16434744 L.G.Doudeva, H.Huang, K.C.Hsia, Z.Shi, C.L.Li, Y.Shen, Y.S.Cheng, and H.S.Yuan (2006).
Crystal structural analysis and metal-dependent stability and activity studies of the ColE7 endonuclease domain in complex with DNA/Zn2+ or inhibitor/Ni2+.Protein Sci, 15, 269-280.
PDB codes: 1zns 1znv
16395523 M.M.Zhao, D.R.An, J.Zhao, G.H.Huang, Z.H.He, and J.Y.Chen (2006).
Transiently expressed short hairpin RNA targeting 126 kDa protein of tobacco mosaic virus interferes with virus infection.Acta Biochim Biophys Sin (Shanghai), 38, 22-28.
15726170 E.T.van den Bremer, A.H.Keeble, C.Kleanthous, and A.J.Heck (2005).
Metal induced selectivity in phosphate ion binding in E9 DNase.Chem Commun (Camb), 0, 1137-1139.
12881435 C.L.Li, L.I.Hor, Z.F.Chang, L.C.Tsai, W.Z.Yang, and H.S.Yuan (2003).
DNA binding and cleavage by the periplasmic nuclease Vvn: a novel structure with a known active site.EMBO J, 22, 4014-4025.
PDB codes: 1ouo 1oup The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB codes are shown on the right.
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