Substrate binding activates the designed triple mutant of the colicin E7 metallonuclease

Eszter Németh; Tamás Körtvélyesi; Milan Kožíšek; Peter Waaben Thulstrup; Hans Erik Mølager Christensen; Masamitsu N. Asaka; Kyosuke Nagata; Béla Gyurcsik

doi:10.1007/s00775-014-1186-6

Substrate binding activates the designed triple mutant of the colicin E7 metallonuclease

Eszter Németh, Tamás Körtvélyesi, Milan Kožíšek, Peter Waaben Thulstrup, Hans Erik Mølager Christensen, Masamitsu N. Asaka, Kyosuke Nagata, Béla Gyurcsik^*

^*Corresponding author af dette arbejde

Kemisk Institut

6 Citationer (Scopus)

Abstract

The nuclease domain of colicin E7 (NColE7) cleaves DNA nonspecifically. The active center is a Zn²⁺-containing HNH motif at the C-terminus. The N-terminal loop is essential for the catalytic activity providing opportunity for allosteric modulation of the enzyme. To identify the key residues responsible for the structural integrity of NColE7, a virtual alanine scan was performed on a semiempirical quantum chemical level within the 25 residue long N-terminal sequence (446-470). Based on the calculations the T454A/K458A/W464A-NColE7 triple mutant (TKW) was expressed and purified. According to the agarose gel electrophoresis experiments and linear dichroism spectra the catalytic activity of the TKW mutant decreased in comparison with wild-type NColE7. The distorted structure and weakened Zn²⁺ binding may account for this as revealed by circular dichroism spectra, mass spectrometry, fluorescence-based thermal analysis and isothermal microcalorimetric titrations. Remarkably, the substrate induced the folding of the mutant protein.

Originalsprog	Engelsk
Tidsskrift	Journal of Biological Inorganic Chemistry
Vol/bind	19
Udgave nummer	8
Sider (fra-til)	1295-1303
Antal sider	9
ISSN	0949-8257
DOI	https://doi.org/10.1007/s00775-014-1186-6
Status	Udgivet - 2014

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10.1007/s00775-014-1186-6

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title = "Substrate binding activates the designed triple mutant of the colicin E7 metallonuclease",

abstract = "The nuclease domain of colicin E7 (NColE7) cleaves DNA nonspecifically. The active center is a Zn2+-containing HNH motif at the C-terminus. The N-terminal loop is essential for the catalytic activity providing opportunity for allosteric modulation of the enzyme. To identify the key residues responsible for the structural integrity of NColE7, a virtual alanine scan was performed on a semiempirical quantum chemical level within the 25 residue long N-terminal sequence (446-470). Based on the calculations the T454A/K458A/W464A-NColE7 triple mutant (TKW) was expressed and purified. According to the agarose gel electrophoresis experiments and linear dichroism spectra the catalytic activity of the TKW mutant decreased in comparison with wild-type NColE7. The distorted structure and weakened Zn2+ binding may account for this as revealed by circular dichroism spectra, mass spectrometry, fluorescence-based thermal analysis and isothermal microcalorimetric titrations. Remarkably, the substrate induced the folding of the mutant protein.",

keywords = "Binding affinity, Calorimetry, Protein engineering, Substrate induced folding, Zinc nuclease",

author = "Eszter N{\'e}meth and Tam{\'a}s K{\"o}rtv{\'e}lyesi and Milan Ko{\v z}{\'i}{\v s}ek and Thulstrup, {Peter Waaben} and Christensen, {Hans Erik M{\o}lager} and Asaka, {Masamitsu N.} and Kyosuke Nagata and B{\'e}la Gyurcsik",

year = "2014",

doi = "10.1007/s00775-014-1186-6",

language = "English",

volume = "19",

pages = "1295--1303",

journal = "Journal of Biological Inorganic Chemistry",

issn = "0949-8257",

publisher = "Springer",

number = "8",

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TY - JOUR

T1 - Substrate binding activates the designed triple mutant of the colicin E7 metallonuclease

AU - Németh, Eszter

AU - Körtvélyesi, Tamás

AU - Kožíšek, Milan

AU - Thulstrup, Peter Waaben

AU - Christensen, Hans Erik Mølager

AU - Asaka, Masamitsu N.

AU - Nagata, Kyosuke

AU - Gyurcsik, Béla

PY - 2014

Y1 - 2014

N2 - The nuclease domain of colicin E7 (NColE7) cleaves DNA nonspecifically. The active center is a Zn2+-containing HNH motif at the C-terminus. The N-terminal loop is essential for the catalytic activity providing opportunity for allosteric modulation of the enzyme. To identify the key residues responsible for the structural integrity of NColE7, a virtual alanine scan was performed on a semiempirical quantum chemical level within the 25 residue long N-terminal sequence (446-470). Based on the calculations the T454A/K458A/W464A-NColE7 triple mutant (TKW) was expressed and purified. According to the agarose gel electrophoresis experiments and linear dichroism spectra the catalytic activity of the TKW mutant decreased in comparison with wild-type NColE7. The distorted structure and weakened Zn2+ binding may account for this as revealed by circular dichroism spectra, mass spectrometry, fluorescence-based thermal analysis and isothermal microcalorimetric titrations. Remarkably, the substrate induced the folding of the mutant protein.

AB - The nuclease domain of colicin E7 (NColE7) cleaves DNA nonspecifically. The active center is a Zn2+-containing HNH motif at the C-terminus. The N-terminal loop is essential for the catalytic activity providing opportunity for allosteric modulation of the enzyme. To identify the key residues responsible for the structural integrity of NColE7, a virtual alanine scan was performed on a semiempirical quantum chemical level within the 25 residue long N-terminal sequence (446-470). Based on the calculations the T454A/K458A/W464A-NColE7 triple mutant (TKW) was expressed and purified. According to the agarose gel electrophoresis experiments and linear dichroism spectra the catalytic activity of the TKW mutant decreased in comparison with wild-type NColE7. The distorted structure and weakened Zn2+ binding may account for this as revealed by circular dichroism spectra, mass spectrometry, fluorescence-based thermal analysis and isothermal microcalorimetric titrations. Remarkably, the substrate induced the folding of the mutant protein.

KW - Binding affinity

KW - Calorimetry

KW - Protein engineering

KW - Substrate induced folding

KW - Zinc nuclease

U2 - 10.1007/s00775-014-1186-6

DO - 10.1007/s00775-014-1186-6

M3 - Journal article

C2 - 25156149

AN - SCOPUS:84904753004

SN - 0949-8257

VL - 19

SP - 1295

EP - 1303

JO - Journal of Biological Inorganic Chemistry

JF - Journal of Biological Inorganic Chemistry

IS - 8

ER -

Substrate binding activates the designed triple mutant of the colicin E7 metallonuclease

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