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

VL - 19

SP - 1295

EP - 1303

JO - Journal of Biological Inorganic Chemistry

JF - Journal of Biological Inorganic Chemistry

SN - 0949-8257

IS - 8

ER -