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.

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The presence of Zn 2+ at protein-protein interfaces modulates complex function, stability, and introduces structural flexibility/complexity, chemical selectivity, and reversibility driven in a Zn 2+ -dependent manner. Recent studies have demonstrated that dynamically changing Zn 2+ affects numerous cellular processes, including protein-protein communication and protein complex assembly. How Zn 2+ -involved protein-protein interactions (ZPPIs) are formed and dissociate and how their stability and reactivity are driven in a zinc interactome remain poorly understood, mostly due to experimental obstacles. Here, we review recent research advances on the role of Zn 2+ in the formation of interprotein sites, their architecture, function, and stability. Moreover, we underline the importance of zinc networks in intersystemic communication and highlight bioinformatic and experimental challenges required for the identification and investigation of ZPPIs. Protein-Protein Interactome and the Role of Metal IonsA complete picture of protein-protein interactions (PPIs), also known as the protein-protein interactome (see Glossary), is crucial to understand cell molecular machinery at the system biology level [1-3]. Identification of protein molecular complexes requires the description of various aspects, such as composition, component affinity, and lifetime with series of spatial and physicochemical properties describing the interface between proteins. Considering both the affinity and lifetime of proteins to interact, obligate and nonobligate complexes can be found, where the latter are divided into permanent (mostly strong, irreversible) and transient (reversible, frequently with medium affinity). Transient complexes are characterized by smaller interfaces, frequently dependent on specific recognition patterns, where the limited surface meets the requirement of constituents to fold and exist independently without aggregation [4,5]. Their interfaces contain more residues of higher polarity stabilized by salt bridges and hydrogen bonds with more chain turns to gain flexibility [6][7][8]. The specificity of such interactions is also elevated by the utilization of the same interface for multiple interactions across evolution, where the local environment has been affecting the fast adaptation of mutations [9,10].

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