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 nons pecific 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 endonuclea ses, 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 ne ither Zn2+ nor Ni2+ is required for DNase activity, which instead requires Mg2+ ions, but binding transition metals to the E9 DNase causes subtle chan ges to both secondary and tertiary structure. Spectroscopic, proteolytic, a nd calorimetric data show that, accompanying the binding of 1 eq of Zn2+, N i2+, or Co2+, the thermodynamic st;ability of the domain increased substant ially, and that the equilibrium dissociation constant for Zn2+ was less tha n or equal to nanomolar, while that for Co2+ and Ni2+ was micromolar, Our d ata demonstrate that the transition metal is not essential for colicin DNas e activity but rather serves a structural role. We speculate that the HNH m otif has been adapted for use by endonuclease colicins because of its invol vement in DNA recognition and because removal of the bound metal ion destab ilizes the DNase domain, a likely prerequisite for its translocation across bacterial membranes.