Németh, Eszter8; Körtvélyesi, Tamás8; Kožíšek, Milan4; Thulstrup, Peter W.5; Christensen, Hans Erik Mølager1; Asaka, Masamitsu N.9; Nagata, Kyosuke9; Gyurcsik, Béla7
1 Department of Chemistry, Technical University of Denmark2 Metalloprotein Chemistry and Engineering, Department of Chemistry, Technical University of Denmark3 University of Szeged4 Academy of Sciences of the Czech Republic5 University of Copenhagen6 University of Tsukuba7 MTA-SZTE Bioinorganic Chemistry Research Group8 University of Szeged9 University of Tsukuba
The nuclease domain of colicin E7 (NColE7) cleaves DNA nonspecifically. The active center is a Zn(2+)-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 Zn(2+) 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.
Journal of Biological Inorganic Chemistry, 2014, Vol 19, Issue 8, p. 1295-1303