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

•The W464A mutation causes structural changes and decreased Zn2 + binding of NColE7.•The structure and Zn2 +-binding of W464A NColE7 mutants are rescued by Im7 protein.•The Zn2 +-binding site was found to be preorganized in the active centre of NColE7.•The allosteric effect of the N-terminal loop can be applied in design of regulated nucleases.

The structure of the active site in a metalloenzyme can be a key determinant of its metal ion binding affinity and catalytic activity. In this study, the conformational features of the Zn2 +-binding HNH motif were investigated by CD-spectroscopy in combination with isothermal microcalorimetric titrations. Various point mutations, including T454A, K458A and W464A, were introduced into the N-terminal loop of the nuclease domain of colicin E7 (NColE7). We show that the folding of the proteins was severely disturbed by the mutation of the tryptophan residue. This points to the importance of W464, being a part of the hydrophobic core located close to the HNH-motif. ITC demonstrated that the Zn2 +-binding of the mutants including the W464 site became weak, and according to CD-spectroscopic measurements the addition of the metal ion itself cannot fully recover the functional structure. Titrations with Zn2 +-ion in the presence and absence of the Im7 protein proved that the structural changes in the unfolded mutant included the HNH-motif itself. The metal-binding of the NColE7 mutants could be, however, fully rescued by the complexation of Im7. This suggests that the formation of a preorganized metal-binding site – existing in the wild-type enzyme but not in the W464 mutants – was induced by Im7. The low nuclease activity of all W464A mutants, however, implies that the interactions of this tryptophan residue are required for precise location of the catalytic residues, i.e. for stabilization of the fine-structure and of the tertiary structure. Our results contribute to the understanding of the metal binding site preorganization.

Graphical abstractThe stabilization of the preorganized Zn2 +-binding site within the HNH catalytic centre was shown to be promoted by the hydrophobic protein environment in the NColE7 nuclease.Figure optionsDownload full-size imageDownload as PowerPoint slide