title: Crystallization and preliminary crystallographic analysis of an Escherichia coli-selected mutant of the nuclease domain of the metallonuclease colicin E7.
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.
authors.affiliation: MTA-SZTE Bioinorganic Chemistry Research Group, Dóm tér 7, H-6720 Szeged, Hungary.
authors.forename: Anikó
authors.initials: A
authors.lastname: Czene
authors.forename: Eszter
authors.initials: E
authors.lastname: Tóth
authors.forename: Béla
authors.initials: B
authors.lastname: Gyurcsik
authors.forename: Harm
authors.initials: H
authors.lastname: Otten
authors.forename: Jens Christian N
authors.initials: JC
authors.lastname: Poulsen
authors.forename: Leila
authors.initials: L
authors.lastname: Lo Leggio
authors.forename: Sine
authors.initials: S
authors.lastname: Larsen
authors.forename: Hans E M
authors.initials: HE
authors.lastname: Christensen
authors.forename: Kyosuke
authors.initials: K
authors.lastname: Nagata
mesh_terms: D000595:Amino Acid Sequence; D003087:Colicins / Q000737:chemistry* / Q000235:genetics*; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*
chemical_list: C508089:ColE7 protein, E coli; D003087:Colicins
keywords: colicin E7; metallonucleases; nuclease domain