Crystallization and preliminary crystallographic analysis of an Escherichia coli-selected mutant of the nuclease domain of the metallonuclease colicin E7

Aniko Czene, Eszter Toth, Bela Gyurcsik, Harm Otten, Jens-Christian N. Poulsen, Leila Lo Leggio, Sine Larsen, Hans Erik Mølager Christensen, Kyosuke Nagata

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

The metallonuclease colicin E7 is a member of the HNH family of endonucleases. It serves as a bacterial toxin in Escherichia coli, protecting the host cell from other related bacteria and bacteriophages by degradation of their chromosomal DNA under environmental stress. Its cell-killing activity is attributed to the nonspecific nuclease domain (NColE7), which possesses the catalytic ββα-type metal ion-binding HNH motif at its C-terminus. Mutations affecting the positively charged amino acids at the N-terminus of NColE7 (444-576) surprisingly showed no or significantly reduced endonuclease activity [Czene et al. (2013), J. Biol. Inorg. Chem. 18, 309-321]. The necessity of the N-terminal amino acids for the function of the C-terminal catalytic centre poses the possibility of allosteric activation within the enzyme. Precise knowledge of the intramolecular interactions of these residues that affect the catalytic activity could turn NColE7 into a novel platform for artificial nuclease design. In this study, the N-terminal deletion mutant ΔN4-NColE7-C* of the nuclease domain of colicin E7 selected by E. coli was overexpressed and crystallized at room temperature by the sitting-drop vapour-diffusion method. X-ray diffraction data were collected to 1.6 Å resolution and could be indexed and averaged in the trigonal space group P3121 or P3221, with unit-cell parameters a = b = 55.4, c = 73.1 Å. Structure determination by molecular replacement is in progress.

Original languageEnglish
JournalActa Crystallographica. Section F: Structural Biology and Crystallization Communications Online
VolumeF69
Issue number5
Pages (from-to)551-554
ISSN2053-230X
DOIs
Publication statusPublished - 2013

Keywords

  • Colicin E7
  • Nuclease domain
  • Metallonucleases

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