The role of the N-terminal loop in the function of the colicin E7 nuclease domain

  • Anikó Czene
  • Eszter Németh
  • István G. Zóka
  • Noémi I. Jakab-Simon
  • Tamás Körtvélyesi
  • Kyosuke Nagata
  • Hans E. M. Christensen
  • Béla Gyurcsik
Original Paper


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 HHX14NX8HX3H 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 Zn2+ 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 Zn2+ 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.


Metallonuclease Colicin E7 N-terminally truncated mutants Zinc(II) binding 



This work has received support through the Hungarian Science Foundation (OTKA-NKTH CK80850), TÁMOP-4.2.1/B-09/1/KONV-2010-0005 and TÁMOP-4.2.2/B-10/1-2010-0012. The computational resources at High Performance Computing of the University of Szeged and financial support from the European Union Research Infrastructure Action FP7 (Integrated Activity on Synchrotron and Free Electron Laser Science, contract no. FP7/2007-2013; no. 226716) are also gratefully acknowledged. B.G. thanks the Japan Society for the Promotion of Science, and I.N.J-S. and A.C. thank the Hungarian Scholarship Board for the fellowships provided.

Supplementary material

775_2013_975_MOESM1_ESM.pdf (992 kb)
Supplementary material 1 (PDF 993 kb)


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Copyright information

© SBIC 2013

Authors and Affiliations

  • Anikó Czene
    • 1
    • 2
  • Eszter Németh
    • 1
    • 3
  • István G. Zóka
    • 1
  • Noémi I. Jakab-Simon
    • 1
    • 4
  • Tamás Körtvélyesi
    • 3
  • Kyosuke Nagata
    • 5
  • Hans E. M. Christensen
    • 4
  • Béla Gyurcsik
    • 1
    • 2
  1. 1.Department of Inorganic and Analytical ChemistryUniversity of SzegedSzegedHungary
  2. 2.Bioinorganic Chemistry Research Group of Hungarian Academy of SciencesSzegedHungary
  3. 3.Department of Physical Chemistry and Material SciencesUniversity of SzegedSzegedHungary
  4. 4.Department of ChemistryTechnical University of DenmarkKongens LyngbyDenmark
  5. 5.Department of Infection Biology, Graduate School of Comprehensive Human Sciences and Faculty of MedicineUniversity of TsukubaTsukubaJapan

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