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 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.

Rescue of the activity of HNH nuclease mutants - towards controlled enzymes for gene therapy

“…This raises the question of the quality of the bound metal ion. (ii) The immunity protein, as well as, the DNA substrate has been demonstrated to induce the proper folding of the mutated nuclease domain [125,126]. Thus, even if the mutation would affect the structure of the nuclease domain itself, this can not be detected in the presence of the immunity protein or the substrate.…”

“…It has been demonstrated, that the absence of this residue abolishes the metal ion binding within the active centre [126]. On the other hand, the protein folding and so the metal ion affinity is rescued by the interactions with the immunity protein or the DNA molecule [125,126].…”

“…It has been demonstrated, that the absence of this residue abolishes the metal ion binding within the active centre [126]. On the other hand, the protein folding and so the metal ion affinity is rescued by the interactions with the immunity protein or the DNA molecule [125,126]. It also has to be mentioned that the hydrolytic reaction is furthermore facilitated by the bending of the DNA [96] due to the protein-binding, to which the positively charged residues may contribute, as well.…”

Preorganization of the catalytic Zn2+-binding site in the HNH nuclease motif—A solution study

“…Previously we examined the 25 residues long N-terminal loop [15]. By computational modelling three important residues (T454, K458 and W464) were selected to mutate them to alanines (Fig.…”

“…The PCR fragment was cloned into a pGEX-6P-1 vector that provides an N-terminal GST fusion. The mutations have been introduced 6 by applying the primers described previously for the triple mutant [15]. The constructed plasmids containing the target genes were transformed into E. coli DH10B cells and E. coli BL21 (DE3) cells for cloning and protein expression, respectively.…”