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

Abstract

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 IIthiolate 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 lengthdifferentiated 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 HgS 4 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.

Synthesis, Characterization, and Antimicrobial Activity of New Metal Complexes Derived from 4-chloro-N'-[(E)-(3,5-dichloro-2-hydroxyphenyl) methylidene] benzohydrazide

“…A square planar geometry was attributed to these changes [37]. However, the d-d band is not present in the Zn(II), Cd(II), and Hg(II) complexes because the d 10 transition is filled in these complexes [38][39][40].…”

Zn(II) to Ag(I) Swap in Rad50 Zinc Hook Domain Leads to Interprotein Complex Disruption through the Formation of Highly Stable Agx(Cys)y Cores

“…45,46 Nevertheless, in this case, the hyperstability is not associated with selectivity, and the Zn(II) ion could be easily displaced by Cd(II) or Hg(II), yielding complexes with altered structure and enhanced stability and making Rad50 a potential target in Cd(II)-or Hg(II)-induced genotoxicity. 47,48 The present work investigates the influence of Ag(I) ions on the Rad50 hook domain dimerization and its structure. For this purpose, we examined the Ag(I) interaction with two lengthdifferentiated peptide models of the Pyrococcus furiosus hook domain, which is the best-described hook domain model so far.…”

“…The interprotein zinc sites are particularly interesting, as they achieve highly distinctive properties upon the Zn­(II) binding. − Their formation, stability, and reactivity are driven by multiple factors, such as cellular or extracellular Zn­(II) and protein subunit concentration, local redox environment, pH, and the presence of competitive ligands . The biological, physicochemical, and structural properties of the Zn­(II)-dependent assembly of the Rad50 zinc hook complex have been investigated very thoroughly. − Rad50 is a member of the Mre11–Rad50–Nbs1 (MRN) complex orchestrating the DNA double-strand break (DSB) repair. , The functional Rad50 homodimer is stabilized by a tetrahedral Zn­(II)–thiolate complex established by the two CXXC motifs. This motif is highly conserved in Rad50 homologs and was found across all forms of life, including bacteria, higher eukaryotes, and even viruses (Figure S1).…”

“…They are hitched together by a short β-hairpin motif and stabilized further by a set of interactions derived from the coiled-coil fragments (Figure ). , To ensure the activity of the whole MRN complex, the Rad50 zinc hook has to be hyperstable. , Nevertheless, in this case, the hyperstability is not associated with selectivity, and the Zn­(II) ion could be easily displaced by Cd­(II) or Hg­(II), yielding complexes with altered structure and enhanced stability and making Rad50 a potential target in Cd­(II)- or Hg­(II)-induced genotoxicity. , …”

“…Crystal structure representation of the P. furiosus Rad50 coiled-coil zinc hook dimer (396–498) (PDB: 1L8D) with marked hook domain model fragments − used in this study: Hk14 (440–453, violet) and Hk45 (426–470, green + violet).…”