Galvanization of Protein-Protein Interactions in a Dynamic Zinc Interactome.

The presence of Zn2+ at protein-protein interfaces modulates complex function, stability, and introduces structural flexibility/complexity, chemical selectivity, and reversibility driven in a Zn2+-dependent manner. Recent studies have demonstrated that dynamically changing Zn2+ affects numerous cellular processes, including protein-protein communication and protein complex assembly. How Zn2+-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 Zn2+ 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.

Combining Native Mass Spectrometry and Proteomics to Differentiate and Map the Metalloform Landscape in Metallothioneins.

Within the intricate landscape of the proteome, approximately 30% of all proteins bind metal ions. This repertoire is even larger when considering all the different forms of a protein, known as proteoforms. Here, we propose the term "metalloforms" to refer to different structural or functional variations of a protein resulting from the binding of various hetero- or homogeneous metal ions. Using human Cu(I)/Zn(II)-metallothionein-3 as a representative model, we developed a chemical proteomics strategy to simultaneously differentiate and map Zn(II) and Cu(I) metal binding sites. In the first labeling step, N-ethylmaleimide reacts with Cysteine (Cys), resulting in the dissociation of all Zn(II) ions while Cu(I) remains bound to the protein. In the second labeling step, iodoacetamide is utilized to label Cu(I)-bound Cys residues. Native mass spectrometry (MS) was used to determine the metal/labeling protein stoichiometries, while bottom-up/top-down MS was used to map the Cys-labeled residues. Next, we used a developed methodology to interrogate an isolated rabbit liver metallothionein fraction containing three metallothionein-2 isoforms and multiple Cd(II)/Zn(II) metalloforms. The approach detailed in this study thus holds the potential to decode the metalloproteoform diversity within other proteins.

Phytochelatins Bind Zn(II) with Micro- to Picomolar Affinities without the Formation of Binuclear Complexes, Exhibiting Zinc Buffering and Muffling Rather than Storing Functions.

Phytochelatins (PCs) are poly-Cys peptides containing a repeating γ-Glu-Cys motif synthesized in plants, algae, certain fungi, and worms by PC synthase from reduced glutathione. It has been shown that an excess of toxic metal ions induces their biosynthesis and that they are responsible for the detoxification process. Little is known about their participation in essential metal binding under nontoxic, basal conditions under which PC synthase is active. This study presents spectroscopic and thermodynamic interactions with the PC2-PC5 series, mainly focusing on the relations between Zn(II) complex stability and cellular Zn(II) availability. The investigations employed mass spectrometry, UV-vis spectroscopy, potentiometry, competition assays with zinc probes, and isothermal titration calorimetry (ITC). All peptides form ZnL complexes, while ZnL2 was found only for PC2, containing two to four sulfur donors in the coordination sphere. Binuclear species typical of Cd(II)-PC complexes are not formed in the case of Zn(II). Results demonstrate that the affinity for Zn(II) increases linearly from PC2 to PC4, ranging from micro- to low-picomolar. Further elongation does not significantly increase the stability. Stability elevation is driven mainly by entropic factors related to the chelate effect and conformational restriction rather than enthalpic factors related to the increasing number of sulfur donors. The affinity of the investigated PCs falls within the range of exchangeable Zn(II) concentrations (hundreds of pM) observed in plants, supporting for the first time a role of PCs both in buffering and in muffling cytosolic Zn(II) concentrations under normal conditions, not exposed to zinc excess, where short PCs have been identified in numerous studies. Furthermore, we found that Cd(II)-PC complexes demonstrate significantly higher metal capacities due to the formation of polynuclear species, which are lacking for Zn(II), supporting the role of PCs in Cd(II) storage (detoxification) and Zn(II) buffering and muffling. Our results on phytochelatins' coordination chemistry and thermodynamics are important for zinc biology and understanding the molecular basis of cadmium toxicity, leaving room for future studies.

Structural Characterization of Cu(I)/Zn(II)-metallothionein-3 by Ion Mobility Mass Spectrometry and Top-Down Mass Spectrometry.

Mammalian zinc metallothionein-3 (Zn7MT3) plays an important role in protecting against copper toxicity by scavenging free Cu(II) ions or removing Cu(II) bound to ß-amyloid and α-synuclein. While previous studies reported that Zn7MT3 reacts with Cu(II) ions to form Cu(I)4Zn(II)4MT3ox containing two disulfides (ox), the precise localization of the metal ions and disulfides remained unclear. Here, we undertook comprehensive structural characterization of the metal-protein complexes formed by the reaction between Zn7MT3 and Cu(II) ions using native ion mobility mass spectrometry (IM-MS). The complex formation mechanism was found to involve the disassembly of Zn3S9 and Zn4S11 clusters from Zn7MT3 and reassembly into Cu(I)xZn(II)yMT3ox complexes rather than simply Zn(II)-to-Cu(I) exchange. At neutral pH, the ß-domain was shown to be capable of binding up to six Cu(I) ions to form Cu(I)6Zn(II)4MT3ox, although the most predominant species was the Cu(I)4Zn(II)4MT3ox complex. Under acidic conditions, four Zn(II) ions dissociate, but the Cu(I)4-thiolate cluster remains stable, highlighting the MT3 role as a Cu(II) scavenger even at lower than the cytosolic pH. IM-derived collision cross sections (CCS) reveal that Cu(I)-to-Zn(II) swap in Zn7MT3 with concomitant disulfide formation induces structural compaction and a decrease in conformational heterogeneity. Collision-induced unfolding (CIU) experiments estimated that the native-like folded Cu(I)4Zn(II)4MT3ox conformation is more stable than Zn7MT3. Native top-down MS demonstrated that the Cu(I) ions are exclusively bound to the ß-domain in the Cu(I)4Zn(II)4MT3ox complex as well as the two disulfides, serving as a steric constraint for the Cu(I)4-thiolate cluster. In conclusion, this study enhances our comprehension of the structure, stability, and dynamics of Cu(I)xZn(II)yMT3ox complexes.

OaAEP1 Ligase-Assisted Chemoenzymatic Synthesis of Full Cysteine-Rich Metal-Binding Cyanobacterial Metallothionein SmtA.

Among all approaches used for the semisynthesis of natural or chemically modified products, enzyme-assisted ligation is among the most promising and dynamically developing approaches. Applying an efficient C247A mutant of Oldenlandia affinis plant ligase OaAEP1 and solid-phase peptide synthesis chemistry, we present the chemoenzymatic synthesis of a complete sequence of the cysteine-rich and metal-binding cyanobacterial metallothionein Synechococcus metallothionein A (SmtA). Zn(II) and Cd(II) binding to the newly synthesized SmtA showed identical properties to the protein expressed in Escherichia coli. The presented approach is the first example of the use of OaAEP1 mutant for total protein synthesis of metallothionein, which occurs in mild conditions preventing cysteine thiol oxidation. The recognition motif of the applied enzyme could naturally occur in the protein structure or be synthetically or genetically incorporated in some loops or secondary structure elements. Therefore, we envision that this strategy can be used for efficiently obtaining SmtA and for a wide range of proteins and their derivatives.

Nesfatin-3 possesses divalent metal ion binding properties, which remain hidden in the nucleobindin-2 precursor protein.

BACKGROUND: Nucleobindin-2 (Nucb2) is a multidomain protein that, due to its structure, participates in many physiological processes. It was originally identified in several regions of the hypothalamus. However, more recent studies have redefined and extended the function of Nucb2 far beyond its initially observed role as a negative modulator of food intake. RESULTS: Previously, we described Nucb2 as structurally divided into two parts: the Zn2+-sensitive N-terminal half and the Ca2+-sensitive C-terminal half. Here, we investigated the structural and biochemical properties of its C-terminal half, which, after posttranslational processing, yields the formation of a fully uncharacterized peptide product known as nesfatin-3. Nesfatin-3 likely contains all the key respective structural regions of Nucb2. Hence, we expected that its molecular properties and affinity toward divalent metal ions might resemble those of Nucb2. Surprisingly, the obtained results showed that the molecular properties of nesftain-3 were completely different from those of its precursor protein. Moreover, we designed our work as a comparative analysis of two nesfatin-3 homologs. We noticed that in their apo forms, both proteins had similar shapes and existed in solution as extended molecules. They both interacted with divalent metal ions, and this interaction manifested itself in a compaction of the protein molecules. Despite their similarities, the differences between the homologous nesfatin-3s were even more informative. Each of them favored interaction with a different metal cation and displayed unique binding affinities compared either to each other or to Nucb2. CONCLUSIONS: The observed alterations suggested different from Nucb2 physiological roles of nesfatin-3 and different impacts on the functioning of the tissues and on metabolism and its control. Our results clearly demonstrated that nesfatin-3 possessed divalent metal ion binding properties, which remained hidden in the nucleobindin-2 precursor protein.

Calcium-assisted sortase A cleavage of SUMOylated metallothionein constructs leads to high-yield production of human MT3.

BACKGROUND: Mammalian metallothioneins (MTs) are small (6-7 kDa), intracellular, cysteine-rich, metal-binding proteins involved, inter alia, in the homeostasis of zinc and copper, detoxification of heavy metals, antioxidation against reactive oxygen species, and protection against DNA damage. The high cysteine content (~ 30%) in MTs makes them toxic to bacterial cells during protein production, resulting in low yield. To address this issue, we present for the first time a combinatorial approach using the small ubiquitin-like modifier (SUMO) and/or sortase as fusion tags for high-level expression of human MT3 in E. coli and its purification by three different strategies. RESULTS: Three different plasmids were generated using SUMO, sortase A pentamutant (eSrtA), and sortase recognition motif (LPETG) as removable fusion tags for high-level expression and purification of human MT3 from the bacterial system. In the first strategy, SUMOylated MT3 was expressed and purified using Ulp1-mediated cleavage. In the second strategy, SUMOylated MT3 with a sortase recognition motif at the N-terminus of MT3 was expressed and purified using sortase-mediated cleavage. In the final strategy, the fusion protein His6-SUMO-eSrtA-LPETG-MT3 was expressed and purified by one-step sortase-mediated inducible on-bead autocleavage. Using these three strategies the apo-MT3 was purified in a yield of 11.5, 11, and 10.8 mg/L, respectively, which is the highest yield achieved for MT expression and purification to date. No effect of MT3 on Ni2+-containing resin was observed. CONCLUSION: The SUMO/sortase-based strategy used as the production system for MT3 resulted in a very high expression level and protein production yield. The apo-MT3 purified by this strategy contained an additional glycine residue and had similar metal binding properties as WT-MT3. This SUMO-sortase fusion system is a simple, robust, and inexpensive one-step purification approach for various MTs as well as other toxic proteins with very high yield via immobilized metal affinity chromatography (IMAC).

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.

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 Agx(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.

The Bioinorganic Chemistry of Mammalian Metallothioneins.

The functions, purposes, and roles of metallothioneins have been the subject of speculations since the discovery of the protein over 60 years ago. This article guides through the history of investigations and resolves multiple contentions by providing new interpretations of the structure-stability-function relationship. It challenges the dogma that the biologically relevant structure of the mammalian proteins is only the one determined by X-ray diffraction and NMR spectroscopy. The terms metallothionein and thionein are ambiguous and insufficient to understand biological function. The proteins need to be seen in their biological context, which limits and defines the chemistry possible. They exist in multiple forms with different degrees of metalation and types of metal ions. The homoleptic thiolate coordination of mammalian metallothioneins is important for their molecular mechanism. It endows the proteins with redox activity and a specific pH dependence of their metal affinities. The proteins, therefore, also exist in different redox states of the sulfur donor ligands. Their coordination dynamics allows a vast conformational landscape for interactions with other proteins and ligands. Many fundamental signal transduction pathways regulate the expression of the dozen of human metallothionein genes. Recent advances in understanding the control of cellular zinc and copper homeostasis are the foundation for suggesting that mammalian metallothioneins provide a highly dynamic, regulated, and uniquely biological metal buffer to control the availability, fluctuations, and signaling transients of the most competitive Zn(II) and Cu(I) ions in cellular space and time.

Interdependence of the rad50 hook and globular domain functions.

Rad50 contains a conserved Zn(2+) coordination domain (the Rad50 hook) that functions as a homodimerization interface. Hook ablation phenocopies Rad50 deficiency in all respects. Here, we focused on rad50 mutations flanking the Zn(2+)-coordinating hook cysteines. These mutants impaired hook-mediated dimerization, but recombination between sister chromatids was largely unaffected. This may reflect that cohesin-mediated sister chromatid interactions are sufficient for double-strand break repair. However, Mre11 complex functions specified by the globular domain, including Tel1 (ATM) activation, nonhomologous end joining, and DNA double-strand break end resection were affected, suggesting that dimerization exerts a broad influence on Mre11 complex function. These phenotypes were suppressed by mutations within the coiled-coil and globular ATPase domains, suggesting a model in which conformational changes in the hook and globular domains are transmitted via the extended coils of Rad50. We propose that transmission of spatial information in this manner underlies the regulation of Mre11 complex functions.

HP1021 is a redox switch protein identified in Helicobacter pylori.

Helicobacter pylori is a gram-negative, microaerophilic, pathogenic bacterium and a widespread colonizer of humans. H. pylori has developed mechanisms that enable it to overcome the harsh environment of the human stomach, including reactive oxygen species (ROS). Interestingly, up to now no typical regulator dedicated to the oxidative-stress response has been discovered. In this work, we reveal that the inhibitor of replication initiation HP1021 functions as a redox switch protein in H. pylori and plays an important role in response to oxidative stress of the gastric pathogen. Each of the two predicted HP1021 domains contains three cysteine residues. We show that the cysteine residues of HP1021 are sensitive to oxidation both in vitro and in vivo, and we demonstrate that HP1021 DNA-binding activity to oriC depends on the redox state of the protein. Moreover, Zn2+ modulates HP1021 affinity towards oriC template DNA. Transcription analysis of selected H. pylori genes by RT-qPCR indicated that HP1021 is directly involved in the oxygen-dependent control of H. pylori fecA3 and gluP genes, which are implicated in response to oxidative stress. In conclusion, HP1021 is a redox switch protein and could be a target for H. pylori control strategies.

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

In nature, thiolate-based systems are the primary targets of divalent mercury (HgII ) 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 HgII -thiolate coordination preferences, among which linear HgII 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 HgII 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 ZnII . Dimerized motifs form a unique interprotein zinc hook domain (Hk) that is critical for the biological activity of the MRN. Using a series of length-differentiated peptide models of the Pyrococcus furiosus zinc hook domain, we investigated its interaction with HgII . 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 HgS4 coordination environment at physiological pH; this is the first example of an interprotein mercury site displaying tetrahedral geometry in solution. At higher HgII 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 HgII complexes known to date.

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

Invited for the cover of this issue is the group of Artur Krezel at the University of Wroclaw 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.

Does one plus one always equal two? Structural differences between nesfatin-1, -2, and nesfatin-1/2.

Nesfatin-1 and -2 are produced from a reaction in which the N-terminus of human Nucleobindin-2 undergoes proteolytical processing. To date, Nucleobindin-2 and/or nesfatin-1 have only been shown to act as peptide hormones. On the other hand, the purpose of nesfatin-2 remains unknown. Since Nucleobindin-2/nesfatin-1 is thought impact the control of a wide range of physiological processes, including energy homeostasis, neurodegenerative processes and carcinogenesis, its ligands/interactions deserve special studies and attention. However, there are no reports about the molecular properties of the proteolytical products of human Nucleobindin-2 in the literature. Hence, this study aimed to analyze the effect of Zn(II) and Ca(II) on human nesfatin-1, -2, and -1/2 structures. Herein, we report that human nesfatin-1 is a member of the intrinsically disordered protein family, as indicated by circular dichroism and analytical ultracentrifugation experiments. In contrast, we found that the human nesfatin-2 and nesfatin-1/2 structures were globular with intrinsically disordered regions. Under Zn(II) treatment, we observed concentration-dependent structurization and compaction of intrinsically disordered nesfatin-1 and its propensity for oligomerization, as well as destabilization of both nesfatin-2 and nesfatin-1/2. Furthermore, dissociation constants for Zn(II) binding by nesfatin-1, nesfatin-2, and nesfatin-1/2 were also reported. Moreover, structurally distinct nesfatin-1 and -2 seem to be interdependent when linked together, as indicated by the observed molecular properties of nesfatin-1/2, which in turn are not a simple sum of the properties exhibited by the former peptides. Thus, herein, we shed new light on the molecular behavior of human nesfatins, which might help to elucidate the complex function of those peptides. Video abstract.

Relations between Structure and Zn(II) Binding Affinity Shed Light on the Mechanisms of Rad50 Hook Domain Functioning and Its Phosphorylation.

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.

Non-Conserved Amino Acid Residues Modulate the Thermodynamics of Zn(II) Binding to Classical ßßα Zinc Finger Domains.

Classical zinc fingers domains (ZFs) bind Zn(II) ion by a pair of cysteine and histidine residues to adopt a characteristic and stable ßßα fold containing a small hydrophobic core. As a component of transcription factors, they recognize specific DNA sequences to transcript particular genes. The loss of Zn(II) disrupts the unique structure and function of the whole protein. It has been shown that the saturation of ZFs under cellular conditions is strictly related to their affinity for Zn(II). High affinity warrants their constant saturation, while medium affinity results in their transient structurization depending on cellular zinc availability. Therefore, there must be factors hidden in the sequence and structure of ZFs that impact Zn(II)-to-protein affinities to control their function. Using molecular dynamics simulations and experimental spectroscopic and calorimetric approaches, we showed that particular non-conserved residues derived from ZF sequences impact hydrogen bond formation. Our in silico and in vitro studies show that non-conserved residues can alter metal-coupled folding mechanisms and overall ZF stability. Furthermore, we show that Zn(II) binding to ZFs can also be entropically driven. This preference does not correlate either with Zn(II) binding site or with the extent of the secondary structure but is strictly related to a reservoir of interactions within the second coordination shell, which may loosen or tighten up the structure. Our findings shed new light on how the functionality of ZFs is modulated by non-coordinating residues diversity under cellular conditions. Moreover, they can be helpful for systematic backbone alteration of native ZF ßßα scaffold to create artificial foldamers and proteins with improved stability.

An Overlooked Hepcidin-Cadmium Connection.

Hepcidin (DTHFPICIFCCGCCHRSKCGMCCKT), an iron-regulatory hormone, is a 25-amino-acid peptide with four intramolecular disulfide bonds circulating in blood. Its hormonal activity is indirect and consists of marking ferroportin-1 (an iron exporter) for degradation. Hepcidin biosynthesis involves the N-terminally extended precursors prepro-hepcidin and pro-hepcidin, processed by peptidases to the final 25-peptide form. A sequence-specific formation of disulfide bonds and export of the oxidized peptide to the bloodstream follows. In this study we considered the fact that prior to export, reduced hepcidin may function as an octathiol ligand bearing some resemblance to the N-terminal part of the α-domain of metallothioneins. Consequently, we studied its ability to bind Zn(II) and Cd(II) ions using the original peptide and a model for prohepcidin extended N-terminally with a stretch of five arginine residues (5R-hepcidin). We found that both form equivalent mononuclear complexes with two Zn(II) or Cd(II) ions saturating all eight Cys residues. The average affinity at pH 7.4, determined from pH-metric spectroscopic titrations, is 1010.1 M-1 for Zn(II) ions; Cd(II) ions bind with affinities of 1015.2 M-1 and 1014.1 M-1. Using mass spectrometry and 5R-hepcidin we demonstrated that hepcidin can compete for Cd(II) ions with metallothionein-2, a cellular cadmium target. This study enabled us to conclude that hepcidin binds Zn(II) and Cd(II) sufficiently strongly to participate in zinc physiology and cadmium toxicity under intracellular conditions.

Structures of Silver Fingers and a Pathway to Their Genotoxicity.

Recently, we demonstrated that AgI can directly replace ZnII in zinc fingers (ZFs). The cooperative binding of AgI to ZFs leads to a thermodynamically irreversible formation of silver clusters destroying the native ZF structure. Thus, a reported loss of biological function of ZF proteins is a likely consequence of such replacement. Here, we report an X-ray absorption spectroscopy (XAS) study of Agn Sn clusters formed in ZFs to probe their structural features. Selective probing of the local environment around AgI by XAS showed the predominance of digonal AgI coordination to two sulfur donors, coordinated with an average Ag-S distance at 2.41 Å. No Ag-N bonds were present. A mixed AgS2 /AgS3 geometry was found solely in the CCCH AgI -ZF. We also show that cooperative replacement of ZnII ions with the studied Ag2 S2 clusters occurred in a three-ZF transcription factor protein 1MEY#, leading to a dissociation of 1MEY# from the complex with its cognate DNA.

InterMetalDB: A Database and Browser of Intermolecular Metal Binding Sites in Macromolecules with Structural Information.

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 https://intermetaldb.biotech.uni.wroc.pl/. 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.

Mass Spectrometry-Based Structural Analysis of Cysteine-Rich Metal-Binding Sites in Proteins with MetaOdysseus R Software.

Identification of metal-binding sites in proteins and understanding metal-coupled protein folding mechanisms are aspects of high importance for the structure-to-function relationship. Mass spectrometry (MS) has brought a powerful adjunct perspective to structural biology, obtaining from metal-to-protein stoichiometry to quaternary structure information. Currently, the different experimental and/or instrumental setups usually require the use of multiple data analysis software, and in some cases, they lack some of the main data analysis steps (MS processing, scoring, identification). Here, we present a comprehensive data analysis pipeline that addresses charge-state deconvolution, statistical scoring, and mass assignment for native MS, bottom-up, and native top-down with emphasis on metal-protein complexes. We have evaluated all of the approaches using assemblies of increasing complexity, including free and chemically labeled proteins, from low- to high-resolution MS. In all cases, the results have been compared with common software and proved how MetaOdysseus outperformed them.