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, T. Körtvélyesi, Kyosuke Nagata, Hans E M Christensen, B. Gyurcsik

Research output: Contribution to journalArticle

12 Citations (Scopus)

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 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.

Original languageEnglish
Pages (from-to)309-321
Number of pages13
JournalJournal of Biological Inorganic Chemistry
Volume18
Issue number3
DOIs
Publication statusPublished - Mar 2013

Fingerprint

Colicins
Amino Acids
Ions
Circular dichroism spectroscopy
Synchrotrons
Proteins
DNA
Fluorescence Spectrometry
Fluorescence spectroscopy
Electrophoretic Mobility Shift Assay
Mutant Proteins
Molecular Dynamics Simulation
Circular Dichroism
Synchrotron radiation
Sepharose
Escherichia coli
Mass spectrometry
Metal ions
Arginine
Molecular dynamics

Keywords

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

ASJC Scopus subject areas

  • Biochemistry
  • Inorganic Chemistry

Cite this

The role of the N-terminal loop in the function of the colicin E7 nuclease domain. / Czene, Anikó; Németh, Eszter; Zóka, István G.; Jakab-Simon, Noémi I.; Körtvélyesi, T.; Nagata, Kyosuke; Christensen, Hans E M; Gyurcsik, B.

In: Journal of Biological Inorganic Chemistry, Vol. 18, No. 3, 03.2013, p. 309-321.

Research output: Contribution to journalArticle

Czene, Anikó ; Németh, Eszter ; Zóka, István G. ; Jakab-Simon, Noémi I. ; Körtvélyesi, T. ; Nagata, Kyosuke ; Christensen, Hans E M ; Gyurcsik, B. / The role of the N-terminal loop in the function of the colicin E7 nuclease domain. In: Journal of Biological Inorganic Chemistry. 2013 ; Vol. 18, No. 3. pp. 309-321.
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T1 - The role of the N-terminal loop in the function of the colicin E7 nuclease domain

AU - Czene, Anikó

AU - Németh, Eszter

AU - Zóka, István G.

AU - Jakab-Simon, Noémi I.

AU - Körtvélyesi, T.

AU - Nagata, Kyosuke

AU - Christensen, Hans E M

AU - Gyurcsik, B.

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N2 - 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.

AB - 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.

KW - Colicin E7

KW - Metallonuclease

KW - N-terminally truncated mutants

KW - Zinc(II) binding

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