Fine tuning of the catalytic activity of colicin E7 nuclease domain by systematic N‐terminal mutations

Abstract

The nuclease domain of colicin E7 (NColE7) promotes the nonspecific cleavage of nucleic acids at its C-terminal HNH motif. Interestingly, the deletion of four N-terminal residues (446-449 NColE7 5 KRNK) resulted in complete loss of the enzyme activity. R447A mutation was reported to decrease the nuclease activity, but a detailed analysis of the role of the highly positive and flexible N-terminus is still missing. Here, we present the study of four mutants, with a decreased activity in the following order: NColE7 >> KGNK > KGNG~GGNK > GGNG. At the same time, the folding, the metal-ion, and the DNA-binding affinity were unaffected by the mutations as revealed by linear and circular dichroism spectroscopy, isothermal calorimetric titrations, and gel mobility shift experiments. Semiempirical quantum chemical calculations and molecular dynamics simulations revealed that K446, K449, and/or the N-terminal amino group are able to approach the active centre in the absence of the other positively charged residues. The results suggested a complex role of the N-terminus in the catalytic process that could be exploited in the design of a controlled nuclease.

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

“…Studies on N-terminal point mutants yielded similar results [14]. It is intriguing why and how the seemingly disordered N-terminuslying outside the active site and DNA-binding helices -influences the catalytic reaction.…”

“…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. The same procedure as described earlier [14] was applied for protein purification. All the proteins were stored in 20 mM HEPES, at pH = 7.7.…”

“…Synchrotron radiation circular dichroism (SRCD) spectra were recorded at the CD1 beamline of the storage ring ASTRID at the Institute for Storage Ring Facilities (ISA), University of Aarhus, Denmark [18,19], as described before [14]. In parallel CD-measurements were performed on a Jasco-815 spectropolarimeter in the wavelength range of 180-260 nm, in a 0.2 mm pathlength quartz cell.…”

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

“…R57 has been identified by a mutational analysis in the Serratia nuclease to be essential for catalysis [74,128]. Previous studies also demonstrated that the catalytic activity of NColE7 drops to a low level or is completely cancelled upon modification or deletion of the positively charged N-terminal amino acids [116,129,130]. The arginine residue being distal in the amino acid sequence, but becoming spatially close to the active site is a conserved amino acid, a common feature of the HNH nucleases.…”

“…It has been suggested to bind and stabilize the cleaved phosphate product [131]. As the presence of this residue or any positively charged residue potentially replacing proved to be essential for the catalytic activity of the HNH motif [129,130,132] it can also be considered to participate directly in the catalytic mechanism. Being a flexible residue, it may facilitate the proton transfer from the histidine general base towards the leaving alcoholate [133].…”