A new insight into the zinc-dependent DNA-cleavage by the colicin E7 nuclease: a crystallographic and computational study

Abstract

The nuclease domain of colicin E7 metallonuclease (NColE7) contains its active centre at the C-terminus. The mutant ΔN4-NColE7-C* - where the four N-terminal residues including the positively charged K446, R447 and K449 are replaced with eight residues from the GST tag - is catalytically inactive. The crystal structure of this mutant demonstrates that its overall fold is very similar to that of the native NColE7 structure. This implicates the stabilizing effect of the remaining N-terminal sequence on the structure of the C-terminal catalytic site and the essential role of the deleted residues in the mechanism of the catalyzed reaction. Complementary QM/MM calculations on the protein-DNA complexes support the less favourable cleavage by the mutant protein than by NColE7. Furthermore, a water molecule as a possible ligand for the Zn(2+)-ion is proposed to play a role in the catalytic process. These results suggest that the mechanism of the Zn(2+)-containing HNH nucleases needs to be further studied and discussed.

Intrinsic protein disorder could be overlooked in cocrystallization conditions: An SRCD case study

“…The four N‐terminal amino acids (446‐KRNK‐449) are required for catalytic activity of NColE7 . However, they do not affect the overall fold of the protein in the crystal structure . The truncation including only these amino acids does not have significant effect on the DNA and Zn 2+ ‐binding properties, as well .…”

“…This as well as the interaction with the substrate DNA may affect the structure of NColE7 mutants. In the crystal structures published so far, NColE7 and its mutants are present together with either Im7 13, or DNA, with the exception of only two crystals: the Zn 2+ ‐complexes of NColE7 itself and its ΔN4 truncated mutant . Independently of the interacting molecules or ions, the overall nuclease structure is retained in all crystals.…”

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

“…Our results revealed a complex case study on how to influence, destroy and rescue a preorganized catalytic Zn [34,[36][37][38][39], or with DNA [8,16,39,40] while only two were obtained in the absence of Im7 and/or DNA [13,17] The same distribution is valid for the NColE9 crystal structures (see Table S1). As we observed, Im7 or DNA-binding may stabilize the structure.…”

“…It is known that the nuclease activity of NColE7 is completely lost upon deletion of the Nterminal KRNK sequence (residues 446-449) [12], while the structure [13], metal-ion and DNAbinding affinity [12] of the truncated mutant is unchanged. Studies on N-terminal point mutants yielded similar results [14].…”

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

“…Indeed, the presence of the metal ions is essential for the catalytic activity [97]. For NColE7 Zn 2+ [96,105] was suggested to be the physiological metal ion, but there is still a debate about the quality of metal ions in colicin nucleases [89,96,[109][110][111][112][113][114][115][116][117] [97,105]. The metal ion having a free coordination site may have essential multiple roles in DNA-cleavage: it binds to the scissile phosphodiester, polarizes the P-O bond for nucleophylic attack and stabilizes the phosphoanion transition state and/or the leaving group.…”