Metal-coupled folding as the driving force for the extreme stability of Rad50 zinc hook dimer assembly

Abstract

The binding of metal ions at the interface of protein complexes presents a unique and poorly understood mechanism of molecular assembly. A remarkable example is the Rad50 zinc hook domain, which is highly conserved and facilitates the Zn2+-mediated homodimerization of Rad50 proteins. Here, we present a detailed analysis of the structural and thermodynamic effects governing the formation and stability (logK12 = 20.74) of this evolutionarily conserved protein assembly. We have dissected the determinants of the stability contributed by the small β-hairpin of the domain surrounding the zinc binding motif and the coiled-coiled regions using peptides of various lengths from 4 to 45 amino acid residues, alanine substitutions and peptide bond-to-ester perturbations. In the studied series of peptides, an >650 000-fold increase of the formation constant of the dimeric complex arises from favorable enthalpy because of the increased acidity of the cysteine thiols in metal-free form and the structural properties of the dimer. The dependence of the enthalpy on the domain fragment length is partially compensated by the entropic penalty of domain folding, indicating enthalpy-entropy compensation. This study facilitates understanding of the metal-mediated protein-protein interactions in which the metal ion is critical for the tight association of protein subunits.

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

“…However, in the case of Hk27, Hk31, and Hk45, the formation of CdHk (ML) complexes also occurs. The largest difference in stability between ML and ML 2 complexes is observed for Hk27, which is reflected by two visible inflection points, whereas the Hk6, Hk10, and Hk14 peptides demonstrate the strongest tendency to form only dimeric species, similarly to the study of Zn II complexes (Figure and Figure S2, Supporting Information). In the case of Hk130, the CD spectra changes slightly (Figure a), which complicates stoichiometric analysis.…”

“…To assess if the structure and stability of Cd II complexes rely on a similar structural basis to that of Zn II , we used a series of zinc hook (Hk) peptides ranging in length from 4 to 130 amino acid residues (Hk4–Hk130) (Figure ). Peptide Hk45 has been recognized as a fragment which, as a Zn II complex, possesses all residues that form intermolecular contacts and contains both α‐helical and β‐hairpin regions of the domain . Peptides Hk4, Hk6, Hk10, and Hk14 were used to investigate metal‐coupled formation of the β‐hairpin.…”

“…Previous studies on the central fragment of the zinc hook domain of the Rad50 protein from P. furiosus demonstrated that this fold forms an extremely stable complex with Zn II . The molecular basis of this high stability has been analyzed by using a set of hook model peptides (Hk) containing a ‐CPVC‐ binding motif with increased peptide flanks from both ends (Hk4–Hk45) .…”

“…The existence of an equimolar complex being in equilibrium with a dimer could be of high importance and suggests a possibility of readily exchangeable Rad50 molecules during cellular processes, which is believed to be the requirement for MRN native functions . Keeping in mind the toxic effect of Cd II on zinc finger domains of transcription factors and other zinc–sulfur proteins associated with DNA processing, we aimed to examine the impact of Cd II binding on the Rad50 protein using a well‐described model from P. furiosus , which would be the first intermolecular zinc‐binding site analyzed in terms of Cd II attack. The aim of this study was threefold: 1) to demonstrate how strongly Cd II ions bind to the Rad50 protein, 2) to show whether or not Cd II ions are able to replace Zn II in the cellular timescale, and finally, 3) to analyze how Cd II binding affects the Rad50 hook structure in a way that could translate into functional alterations of Rad50 and the entire MRN complex.…”

The Functions of Metamorphic Metallothioneins in Zinc and Copper Metabolism

“…We showed that cysteines of certain CXXC motifs in zinc binding sites possess significantly different acid-base properties due to the presence of S⋯H–N hydrogen bonds [36,89]. Such hydrogen bonds exist in MTs [9,90,91] and could be responsible for the observed structure of T despite the apparent lack of a defined overall structure of the protein.…”