Metal Exchange in the Interprotein ZnII -Binding Site of the Rad50 Hook Domain: Structural Insights into CdII -Induced DNA-Repair Inhibition

Abstract

Cd^II is a major genotoxic agent that readily displaces Zn^II in a multitude of zinc proteins, abrogates redox homeostasis, and deregulates cellular metalloproteome. To date, this displacement has been described mostly for cysteine(Cys)-rich intraprotein binding sites in certain zinc finger domains and metallothioneins. To visualize how a Zn^II -to-Cd^II swap can affect the target protein's status and thus understand the molecular basis of Cd^II -induced genotoxicity an intermolecular Zn^II -binding site from the crucial DNA repair protein Rad50 and its zinc hook domain were examined. By using a length-varied peptide base, Zn^II -to-Cd^II displacement in Rad50's hook domain is demonstrated to alter it in a bimodal fashion: 1) Cd^II induces around a two-orders-of-magnitude stabilization effect (log $K\frac{12}{\mathrm{Zn}{}^{\mathrm{II}}}$ =20.8 vs. log $K\frac{12}{\mathrm{Cd}{}^{\mathrm{II}}}$ =22.7), which defines an extremely high affinity of a peptide towards a metal ion, and 2) the displacement disrupts the overall assembly of the domain, as shown by NMR spectroscopic and anisotropy decay data. Based on the results, a new model explaining the molecular mechanism of Cd^II genotoxicity that underlines Cd^II 's impact on Rad50's dimer stability and quaternary structure that could potentially result in abrogation of the major DNA damage response pathway is proposed.

Keywords: DNA damage; Rad50; cadmium; zinc; zinc hook.