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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InterMetalDB is a free-of-charge database and browser of intermolecular metal binding sites that are present on the interfaces of macromolecules forming larger assemblies based on structural information deposited in Protein Data Bank (PDB). It can be found and freely used at . InterMetalDB collects the interfacial binding sites with involvement of metal ions and clusters them on the basis of 50% sequence similarity and the nearest metal environment (5 Å radius). The data are available through the web interface where they can be queried, viewed, and downloaded. Complexity of the query depends on the user, because the questions in the query are connected with each other by a logical AND. InterMetalDB offers several useful options for filtering records including searching for structures by particular parameters such as structure resolution, structure description, and date of deposition. Records can be filtered by coordinated metal ion, number of bound amino acid residues, coordination sphere, and other features. InterMetalDB is regularly updated and will continue to be regularly updated with new content in the future. InterMetalDB is a useful tool for all researchers interested in metalloproteins, protein engineering, and metal-driven oligomerization.

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.

The widespread application of silver nanoparticles in medicinal and daily life products increases the exposure to Ag(I) of thiol-rich biological environments, which help control the cellular metallome. A displacement of native metal cofactors from their cognate protein sites is a known phenomenon for carcinogenic and otherwise toxic metal ions. Here, we examined the interaction of Ag(I) with the peptide model of the interprotein zinc hook (Hk) domain of Rad50 protein from Pyrococcus furiosus, a key player in DNA double-strand break (DSB) repair. The binding of Ag(I) to 14 and 45 amino acid long peptide models of apo- and Zn(Hk)2 was experimentally investigated by UV–vis spectroscopy, circular dichroism, isothermal titration calorimetry, and mass spectrometry. The Ag(I) binding to the Hk domain was found to disrupt its structure via the replacement of the structural Zn(II) ion by multinuclear Ag x (Cys) y complexes. The ITC analysis indicated that the formed Ag(I)–Hk species are at least 5 orders of magnitude stronger than the otherwise extremely stable native Zn(Hk)2 domain. These results show that Ag(I) ions may easily disrupt the interprotein zinc binding sites as an element of silver toxicity at the cellular level.

HgII interacting with a naturally ZnII‐containing Rad50 protein interface yields HgS4 species, the strongest metal–protein interaction known to date. It also introduces the novel manifestation of HgII mechanism of prokaryotic and eukaryotic cells. Furthermore, the substitution of naturally occurring ZnII possibly has an impact on the quaternary structure and the protein assembly process, thus disrupting the DNA‐repair mechanisms that engage the Mre11–Rad50 complex. More information can be found in the Research Article by A. Krężel and co‐workers. (DOI: 10.1002/chem.202202738).

Invited for the cover of this issue is the group of Artur Krężel at the University of Wrocław in collaboration with Lars Hemmingsen at The University of Copenhagen and Eva Freisinger at the University of Zürich. The image depicts the outcomes of HgII interactions with Rad50 protein. Read the full text of the article at 10.1002/chem.202202738.

The metal binding at protein–protein interfaces is still uncharted territory in intermolecular interactions. To date, only a few protein complexes binding Zn(II) in an intermolecular manner have been deeply investigated. The most notable example of such interfaces is located in the highly conserved Rad50 protein, part of the Mre11-Rad50-Nbs1 (MRN) complex, where Zn(II) is required for homodimerization (Zn(Rad50)2). The high stability of Zn(Rad50)2 is conserved not only for the protein derived from the thermophilic archaeon Pyrococcus furiosus (logK12 = 20.95 for 130-amino-acid-long fragment), which was the first one studied, but also for the human paralog studied here (logK12 = 19.52 for a 183-amino-acid-long fragment). As we reported previously, the extremely high stability results from the metal-coupled folding process where particular Rad50 protein fragments play a critical role. The sequence–structure–stability analysis based on human Rad50 presented here separates the individual structural components that increase the stability of the complex, pointing to amino acid residues far away from the Zn(II) binding site as being largely responsible for the complex stabilization. The influence of the individual components is very well reflected by the previously published crystal structure of the human Rad50 zinc hook (PDB: 5GOX). In addition, we hereby report the effect of phosphorylation of the zinc hook domain, which exerts a destabilizing effect on the domain. This study identifies factors governing the stability of metal-mediated protein–protein interactions and illuminates their molecular basis.