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

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

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

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.
Original languageEnglish
JournalJournal of Biological Inorganic Chemistry
Volume19
Issue number8
Pages (from-to)1295-1303
ISSN0949-8257
DOIs
Publication statusPublished - 2014

Keywords

  • HASH(0x482a968)
  • Binding affinity
  • Calorimetry
  • Zinc nuclease
  • Substrate induced folding
  • Protein engineering

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