title: The role of the N-terminal loop in the function of the colicin E7 nuclease domain.
abstract: Colicin E7 (ColE7) is a metallonuclease toxin of Escherichia coli belonging to the HNH superfamily of nucleases. It contains highly conserved amino acids in its HHX(14)NX(8)HX(3)H ββα-type metal ion binding C-terminal active centre. However, the proximity of the arginine at the N-terminus of the nuclease domain of ColE7 (NColE7, 446-576) is necessary for the hydrolytic activity. This poses a possibility of allosteric activation control in this protein. To obtain more information on this phenomenon, two protein mutants were expressed, i.e. four and 25 N-terminal amino acids were removed from NColE7. The effect of the N-terminal truncation on the Zn(2+) ion and DNA binding as well as on the activity was investigated in this study by mass spectrometry, synchrotron-radiation circular dichroism and fluorescence spectroscopy and agarose gel mobility shift assays. The dynamics of protein backbone movement was simulated by molecular dynamics. Semiempirical quantum chemical calculations were performed to obtain better insight into the structure of the active centre. The longer protein interacted with both Zn(2+) ion and DNA more strongly than its shorter counterpart. The results were explained by the structural stabilization effect of the N-terminal amino acids on the catalytic centre. In agreement with this, the absence of the N-terminal sequences resulted in significantly increased movement of the backbone atoms compared with that in the native NColE7: in ΔN25-NColE7 the amino acid strings between residues 485-487, 511-515 and 570-571, and in ΔN4-NColE7 those between residues 467-468, 530-535 and 570-571.
authors: Department of Inorganic and Analytical Chemistry, University of Szeged, Dóm tér 7, Szeged, 6720, Hungary.
authors: Anikó
authors: A
authors: Czene
authors: Eszter
authors: E
authors: Németh
authors: István G
authors: IG
authors: Zóka
authors: Noémi I
authors: NI
authors: Jakab-Simon
authors: Tamás
authors: T
authors: Körtvélyesi
authors: Kyosuke
authors: K
authors: Nagata
authors: Hans E M
authors: HE
authors: Christensen
authors: Béla
authors: B
authors: Gyurcsik
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D003087:Colicins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism; D004720:Endonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D015032:Zinc / Q000378:metabolism
chemical_list: C508089:ColE7 protein, E coli; D003087:Colicins; D004269:DNA, Bacterial; D004720:Endonucleases; D015032:Zinc