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.
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.
Sui
Meng-Jiun
MJ
Graduate Institute of Life Science, National Defense Medical Center, Taipei, Taiwan 11472, ROC.
Tsai
Li-Chu
LC
Hsia
Kuo-Chiang
KC
Doudeva
Lyudmila G
LG
Ku
Wen-Yen
WY
Han
Gye Won
GW
Yuan
Hanna S
HS
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D002413:Cations, Divalent / Q000378:metabolism*; D003087:Colicins; D004247:DNA / Q000378:metabolism; D003851:Deoxyribonucleases / Q000737:chemistry* / Q000378:metabolism*; D008670:Metals / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010710:Phosphates / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014961:X-Ray Diffraction
D001426:Bacterial Proteins; D002413:Cations, Divalent; D003087:Colicins; D008670:Metals; D010710:Phosphates; C007104:colicin immunity proteins; D004247:DNA; D003851:Deoxyribonucleases