Homing in on the role of transition metals in the HNH motif of colicin endonucleases

The cytotoxic domain of the bacteriocin colicin E9 (the E9 DNase) is a nonspecific endonuclease that must traverse two membranes to reach its cellular target, bacterial DNA. Recent structural studies revealed that the active site of colicin DNases encompasses the HNH motif found in homing endonucleases, and bound within this motif a single transition metal ion (either Zn2+ or Ni2+) the role of which is unknown. In the present work we find that neither Zn2+ nor Ni2+ is required for DNase activity, which instead requires Mg2+ ions, but binding transition metals to the E9 DNase causes subtle changes to both secondary and tertiary structure. Spectroscopic, proteolytic, and calorimetric data show that, accompanying the binding of 1 eq of Zn2+, Ni2+, or Co2+, the thermodynamic st;ability of the domain increased substantially, and that the equilibrium dissociation constant for Zn2+ was less than or equal to nanomolar, while that for Co2+ and Ni2+ was micromolar, Our data demonstrate that the transition metal is not essential for colicin DNase activity but rather serves a structural role. We speculate that the HNH motif has been adapted for use by endonuclease colicins because of its involvement in DNA recognition and because removal of the bound metal ion destabilizes the DNase domain, a likely prerequisite for its translocation across bacterial membranes.