The binding of metal ions at the interface of protein complexes presents a unique and poorlyudunderstood mechanism of molecular assembly. A remarkable example is the Rad50 zinc hookuddomain, which is highly conserved and facilitates the Zn2+-mediated homodimerization ofudRad50 proteins. Here, we present a detailed analysis of the structural and thermodynamicudeffects governing the formation and stability (logK12 = 20.74) of this evolutionarily conservedudprotein assembly. We have dissected the determinants of the stability contributed by the smalludβ-hairpin of the domain surrounding the zinc binding motif and the coiled-coiled regionsudusing peptides of various lengths from 4 to 45 amino acid residues, alanine substitutions andudpeptide bond-to-ester perturbations. In the studied series of peptides, an >650 000-foldudincrease of the formation constant of the dimeric complex arises from favorable enthalpyudbecause of the increased acidity of the cysteine thiols in metal-free form and the structuraludproperties of the dimer. The dependence of the enthalpy on the domain fragment length isudpartially compensated by the entropic penalty of domain folding, indicating enthalpy-entropyudcompensation. This study facilitates understanding of the metal-mediated protein-proteinudinteractions in which the metal ion is critical for the tight association of protein subunits.
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