An Extremely Stable Interprotein Tetrahedral Hg(Cys)₄ Core Forms in the Zinc Hook Domain of Rad50 Protein at...

In nature, thiolate-based systems are the primary targets of divalent mercury (Hg^II ) toxicity. The formation of Hg(Cys)_x cores in catalytic and structural protein centers mediates mercury's toxic effects and ultimately leads to cellular damage. Multiple studies have revealed distinct Hg^II -thiolate coordination preferences, among which linear Hg^II complexes are the most commonly observed in solution at physiological pH. Trigonal or tetrahedral geometries are formed at basic pH or in tight intraprotein Cys-rich metal sites. So far, no interprotein tetrahedral Hg^II complex formed at neutral pH has been reported. Rad50 protein is a part of the multiprotein MRN complex, a major player in DNA damage-repair processes. Its central region consists of a conserved CXXC motif that enables dimerization of two Rad50 molecules by coordinating Zn^II . Dimerized motifs form a unique interprotein zinc hook domain (Hk) that is critical for the biological activity of the MRN. Using a series of length-differentiated peptide models of the Pyrococcus furiosus zinc hook domain, we investigated its interaction with Hg^II . Using UV-Vis, CD, PAC, and ¹⁹⁹ Hg NMR spectroscopies as well as anisotropy decay, we discovered that all Rad50 fragments preferentially form homodimeric Hg(Hk)₂ species with a distorted tetrahedral HgS₄ coordination environment at physiological pH; this is the first example of an interprotein mercury site displaying tetrahedral geometry in solution. At higher Hg^II content, monomeric HgHk complexes with linear geometry are formed. The Hg(Cys)₄ core of Rad50 is extremely stable and does not compete with cyanides, NAC, or DTT. Applying ITC, we found that the stability constant of the Rad50 Hg(Hk)₂ complex is approximately three orders of magnitude higher than those of the strongest Hg^II complexes known to date.