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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

  • Olga Kerber
    Olga Kerber
    Department of Chemical Biology, Faculty of Biotechnology, University of Wrocław, Joliot-Curie 14a, 50-383 Wrocław, Poland
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  • , 
  • Józef Tran
    Józef Tran
    Department of Chemical Biology, Faculty of Biotechnology, University of Wrocław, Joliot-Curie 14a, 50-383 Wrocław, Poland
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  • , 
  • Alicja Misiaszek
    Alicja Misiaszek
    Department of Chemical Biology, Faculty of Biotechnology, University of Wrocław, Joliot-Curie 14a, 50-383 Wrocław, Poland
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  • , 
  • Aleksandra Chorążewska
    Aleksandra Chorążewska
    Department of Chemical Biology, Faculty of Biotechnology, University of Wrocław, Joliot-Curie 14a, 50-383 Wrocław, Poland
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  • , 
  • Wojciech Bal
    Wojciech Bal
    Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland
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    Orcidhttps://orcid.org/0000-0003-3780-083X
  • , and 
  • Artur Krężel*
    Artur Krężel
    Department of Chemical Biology, Faculty of Biotechnology, University of Wrocław, Joliot-Curie 14a, 50-383 Wrocław, Poland
    *Email: [email protected]
    More by Artur Krężel
    Orcidhttps://orcid.org/0000-0001-9252-5784
Cite this: Inorg. Chem. 2023, 62, 10, 4076–4087
Publication Date (Web):March 2, 2023
https://doi.org/10.1021/acs.inorgchem.2c03767

Copyright © 2022 The Authors. Published by American Chemical Society. This publication is licensed under

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Abstract

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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.

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Synopsis

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.

Introduction

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Metallic silver Ag(0) and Ag(I) compounds have a long history of use as antimicrobial and antifungal agents owing to their unique biocidal properties. (1) In recent years, metallic silver-based biocides gained immense popularity in topical applications due to widespread antibiotic resistance. (2,3) Since these biocides are not regulated, they have also become extensively used beyond medicine, including food preservation and a vast range of consumer goods, such as cosmetics, clothes, and more. (4) Silver metal nanoparticles (AgNPs) are most common in these applications due to their favorable activity stemming from a high surface-to-volume ratio. The biocidal activity of AgNPs arises mainly from their oxidative dissolution in biological environments and subsequent release of the actual bioactive Ag(I) species. (5,6) Such species are then able to induce ROS production and form dysfunctional complexes with peptides, proteins, and DNA. (7−11) The expanding use of AgNPs raised concern about the increased bioavailability of silver, as the high mobility of NPs inside the body may lead to silver accumulation and toxicity in organs distant from the site of contact, including the liver. (12−17) Detailed studies of Ag(I) speciation and its interaction with biomolecules, such as binding to different protein motifs, are therefore critically important to assess and minimize the potential hazards of AgNP use.
In biological systems, cysteine residues and low-molecular-weight (LMW) thiols, such as glutathione, are found to be common targets of Ag(I). (6,8,17,18) This is not surprising. Due to a strong preference toward soft bases, Ag(I) ions display a very high affinity toward thiol ligands. For example, K1 for the Ag(I) complex of cysteine is more than 8 orders of magnitude more stable than that of methionine and nearly 10 orders stronger than that of histidine (log K1 of 11.9 vs 3.29 vs 2.5). (19−21) Solid-state and solution studies of LMW Ag(I)–thiolate structures revealed a strong tendency of thiolate sulfur to bridge Ag(I) ions in a two- and three-coordinate manner, demonstrating either linear AgS2 or trigonal planar AgS3 geometry, respectively. (22,23) These studies also evidenced that the architecture of Ag(I)–thiolate complexes is determined by the steric hindrances between the ligands. (23) Accordingly, the steric effects are likely the important factor driving the organization of Ag(I)–thiolate sites inside the crowded protein scaffolds. In fact, Ag(I) is known for its relatively flexible coordination sphere, not constrained by d-orbital directionality, due to the spherical symmetry of the filled-shell d10 configuration. (24) Such flexibility allows the ion to be coordinated by a variety of ligands and to adopt various geometries. The coordination sphere of Ag(I) in proteins is, therefore, often completed by noncysteine ligands, such as imidazole nitrogen or methionine sulfur, leading to the adoption of various coordination numbers (generally 2–4) and geometries by the Ag(I) ion. (25) Nevertheless, in structures where the Ag(I)–thiolate bonding dominates, Ag(Cys)2 and Ag(Cys)3 coordination spheres are mostly formed, with the characteristic bond lengths of 2.40 and 2.49 Å, respectively. (26) Structural examples of Ag(I)–thiolate sites demonstrate the formation of mono- (PDB ID: 1Q06, (27)5NXL, (28)6XTL, (29)2MZC (30)) and multinuclear (PDB ID: 1AOO, (31)5F0W (32)) centers, localized either within a single polypeptide chain (27,28,31) or at protein interfaces. (30,32) Such diversity of binding architectures reflects the mentioned plasticity of Ag(I)–thiolate sites, which enables them to accommodate different protein conformations and may also relate to the Ag(I) binding strength.
The binding of Ag(I) may abrogate the protein structure dynamics, (33) prevent the formation of the enzyme–substrate complex through the catalytic site blockage, (25) or compete for the physiological metal ions’ cognate sites. (11,26,34−37) This leads to protein malfunction and metal dyshomeostasis. Structural and catalytic effects are interwoven in cases when the native metal ion modulates protein structure stability and dynamics. (36,37) Recently, we demonstrated a spontaneous Zn(II) to Ag(I) swap in the main variants (CCHH, CCCH/CCHC, and CCCC) of the consensus peptide-1 zinc finger (ZF). (36) Substituting for Zn(II) ions, Ag(I) ions destroyed the native ZF fold in the course of cooperative formation of Agn(Cys)n clusters that were saturated at 1:1 Ag(I)/Cys stoichiometry. In the following in vitro study, we also showed that the Zn(II)-to-Ag(I) exchange in a ZF protein derived from the Sp1 transcription factor (PDB ID: 1MEY) resulted in a loss of structure and dissociation of the protein from its cognate DNA. This may constitute a mechanism of silver genotoxicity. (37) Despite the ubiquitous presence of cysteine thiols within Zn(II) binding sites, (38,39) the interaction of Ag(I) with zinc proteome remains largely unexplored. The interprotein zinc sites are particularly interesting, as they achieve highly distinctive properties upon the Zn(II) binding. (39−41) Their formation, stability, and reactivity are driven by multiple factors, such as cellular or extracellular Zn(II) and protein subunit concentration, local redox environment, pH, and the presence of competitive ligands. (40) The biological, physicochemical, and structural properties of the Zn(II)-dependent assembly of the Rad50 zinc hook complex have been investigated very thoroughly. (42−48) Rad50 is a member of the Mre11–Rad50–Nbs1 (MRN) complex orchestrating the DNA double-strand break (DSB) repair. (42,43) The functional Rad50 homodimer is stabilized by a tetrahedral Zn(II)–thiolate complex established by the two CXXC motifs. This motif is highly conserved in Rad50 homologs and was found across all forms of life, including bacteria, higher eukaryotes, and even viruses (Figure S1). In contrast to the classical zinc finger domain (ββα), where Zn(II) is bound by four donors (Cys2His2) from this domain, in the hook domain, each pair of Cys residues is donated by two separate Rad50 protomers. They are hitched together by a short β-hairpin motif and stabilized further by a set of interactions derived from the coiled-coil fragments (Figure 1). (43,44) To ensure the activity of the whole MRN complex, the Rad50 zinc hook has to be hyperstable. (45,46) Nevertheless, in this case, the hyperstability is not associated with selectivity, and the Zn(II) ion could be easily displaced by Cd(II) or Hg(II), yielding complexes with altered structure and enhanced stability and making Rad50 a potential target in Cd(II)- or Hg(II)-induced genotoxicity. (47,48)

Figure 1

Figure 1. Crystal structure representation of the P. furiosus Rad50 coiled-coil zinc hook dimer (396–498) (PDB: 1L8D) (43) with marked hook domain model fragments (45−48) used in this study: Hk14 (440–453, violet) and Hk45 (426–470, green + violet).

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The present work investigates the influence of Ag(I) ions on the Rad50 hook domain dimerization and its structure. For this purpose, we examined the Ag(I) interaction with two length-differentiated peptide models of the Pyrococcus furiosus hook domain, which is the best-described hook domain model so far. The peptide models were previously characterized with Zn(II) and toxic metals Cd(II) and Hg(II) (Hk14 and Hk45, Figure 1). Hk14 is a minimal model (violet in Figure 1) of Zn(II)-induced β-hairpin, while Hk45 is a full domain model (green + violet) covering the β-hairpin region and the adjacent coiled-coil fragments. These two constructs allowed us to determine the Ag(I) coordination properties of the metal binding center and correlate them with the structural stability of the hook domain fold. We used spectrophotometry, circular dichroism (CD), calorimetry, and mass spectrometry to demonstrate that Ag(I) can destabilize the Rad50 hook domain through Zn(II) displacement and disruption of the domain fold. This is the first report of Ag(I)-related destruction of the interprotein metal binding domain composed of two CXXC motifs. This result is likely to be generalized over other Zn(II)-based protein complexes, setting silver as a likely genotoxic agent with a broad range of deleterious effects.

Experimental Methods

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Peptide Synthesis

The investigated Rad50 hook peptides were synthesized via solid-phase synthesis on TentaGel S Ram resin (substitution 0.22 mmol/g) using Fmoc-strategy (49) and Liberty1 Microwave Peptide Synthesizer (CEM). The reagent excess, cleavage, and purification were performed as previously described. (45) Briefly, the synthesized peptides were N-terminally acetylated using acetic anhydride and cleaved from the resin with a mixture of TFA/anisole/thioanisole/EDT/TIPS (88/2/3/5/2 v/v/v/v/v) over a period of 2.5 h followed by precipitation in cold (−80 °C) diethyl ether. The crude peptide was collected by centrifugation, dried, and purified via RP-HPLC (Dionex Ultimate 3000) using Phenomenex C18 columns and a linear gradient of ACN/water with 0.1% TFA from 1 to 90% of ACN over 25 min. The purified peptides were lyophilized, and their identity was confirmed by ESI-Q-TOF MS (Bruker Daltonik GmbH). The RP-HPLC chromatograms of purified peptides and the corresponding MS spectra are shown in Figures S2 and S3, respectively.

Spectrophotometric Titration of Hk Peptides with Ag(I)

The UV–vis spectra of AgNO3-titrated Hk14 and Hk45 peptides were recorded at 25 °C under constant nitrogen flow in the wavelength range of 220–400 nm and a 1 cm pathlength quartz cuvette. Three accumulations were averaged using 200 nm·min–1 scanning speed and 2 nm bandwidth. Briefly, 25 μM of each Hk peptide in a degassed and chelexed 20 mM TES (100 mM NaF, pH 7.4) was titrated with small aliquots of concentrated Ag(I) solution from 0 to 4 molar Ag(I)/Hk ratio. The Hk solution was equilibrated 1 min after the addition of each portion of Ag(I). The AgNO3 titrations of Zn(Hk14)2 and Zn(Hk45)2 were performed analogously. All measurements were performed using a Jasco V-650 spectrophotometer with a Peltier heating/cooling system (Jasco).

CD Titration of Hk Peptides with Ag(I)

The CD spectra of AgNO3-titrated Hk14 and Hk45 peptides were recorded at 25 °C under constant nitrogen flow in the wavelength range of 200–400 nm and a 2 mm pathlength quartz cuvette. Three accumulations were averaged using 200 nm min–1 scanning speed, 2 s digital integration time, and 2 nm bandwidth. Briefly, 25 μM of each Hk peptide in a degassed and chelexed 20 mM TES (100 mM NaF, pH 7.4) was titrated with small aliquots of concentrated AgNO3 solution from 0 to 4 molar Ag(I)/Hk ratio. The Hk solution was equilibrated 1 min after the addition of each portion of AgNO3. The AgNO3 titrations of Zn(Hk14)2 and Zn(Hk45)2 were performed analogously. All measurements were performed using a Jasco-1500 spectropolarimeter with a Peltier heating/cooling system (Jasco).

Spectrophotometric PAR (4-(2-pyridylazo)resorcinol) Assay of Zn(II) to Ag(I) Swap

The Ag(I)-induced transfer of Zn(II) from either Zn(Hk14)2 or Zn(Hk45)2 to PAR was monitored spectrophotometrically at 25 °C under constant nitrogen flow and stirring (300 rpm) in a 1 cm quartz cuvette. The absorbance signal was monitored continuously using a kinetic mode at a fixed wavelength of 492 nm. The AgNO3 titrations of 10 μM Zn(Hk)2 complexes were performed in a degassed and chelexed 20 mM TES buffer (100 mM NaF, pH 7.4) in the presence of 100 μM PAR from 0 to 20 μM of added AgNO3. The Hk solution was equilibrated after each portion of added Ag(I) until the appearing curve, resulting from the Zn(II) to PAR transfer, reached a plateau. Finally, the molar concentration of the released Zn(II) was calculated using the effective molar absorption coefficient of the Zn(PAR)2 complex at pH 7.4, which is 71 150 M–1·cm–1. (50) The measurements were performed using a Jasco V-650 spectrophotometer with a Peltier heating/cooling system (Jasco).

Size-Exclusion Analysis of Ag(I)–Hk Complexes

Size-exclusion chromatography analyses were performed at room temperature using an NGC Quest 10 Plus Chromatography System (Bio-Rad). Samples of Hk14 (or Zn(Hk14)2) and Hk45 (or Zn(Hk45)2) were prepared in 20 mM TES, 100 mM NaF, pH 7.4 and run at 0.8 mL/min flow rate in the same buffer using Superdex 75 Increase 10/300 GL and Superdex 30 Increase 10/300 GL columns, respectively. Each sample was prepared immediately before the run by adding different molar equivalents of AgNO3. The column was equilibrated with at least two column volumes of the running buffer and calibrated with a mix of standard proteins prior to each experiment.

ESI-MS of Ag(I)–Hk Complexes

The (+)ESI-MS-monitored AgNO3 titration of Hk peptides was performed in a degassed 50 mM ammonium acetate solution pH 7.4. The 2 μM samples of Hk14 or Hk45 were mixed with 0–4.0 mol equiv of AgNO3, incubated for 1 min, and injected by a syringe pump (10 μL/min) into an ESI-Q-ToF mass spectrometer (Compact Q-TOF, Bruker Daltonik GmbH). The MS spectra were recorded over 1 min in the positive ion mode within the 300–3000 m/z range at a 1 Hz acquisition rate. The following parameters were optimized to preserve peptide–metal ion complexes: capillary voltage of 3500 V, end plate offset potential of 500 V, nebulizer gas (N2) pressure of 0.4 bar, drying gas (N2) flow rate of 4 L/min, and drying temperature of 180 °C. The AgNO3 titrations of Zn(Hk14)2 and Zn(Hk45)2 complexes were performed analogously using the above conditions and parameters. The obtained mass spectrometry data were processed and analyzed using Compass DataAnalysis software (Version 5.1, Bruker Daltonik GmbH).

Isothermal Titration Calorimetry (ITC)

The binding of Ag(I) to Hk14 and Zn(Hk14)2 was monitored with a Nano-ITC calorimeter (TA Instruments) at 25 °C in a Hastelloy cell of active volume of 1 mL in an overflow mode. All experiments were performed in 50 mM HEPES buffer, containing 100 mM NaF to establish the ionic strength at pH 7.4 under an argon atmosphere. (46) In normal-mode experiments, the titrant (AgNO3) concentration was 6.03 mM and the titrate (Hk14 or Zn(Hk14)2) concentration was 150 μM. In the reverse mode, the titrant (Hk14 or Zn(Hk14)2) and titrate (AgNO3) were 1.3 mM and 36.2 μM, respectively. All titrant solutions were prepared freshly just before the measurement. After the initial temperature equilibration, a titrant solution was injected in a stepwise manner in 3 μL aliquots into the cell at 350 s intervals with stirring at 250 rpm. The control experiment, to determine the heat of titrant dilutions, was performed in the case of reverse-mode titration. Then, the net reaction heat was obtained by subtraction of the dilution heat from the corresponding total heat of injections. In the normal mode, the heat of titrant dilution was modeled from the final part of the isotherm. The data were analyzed in NanoAnalyze (version 3.12, TA Instruments), where the baseline and control heat subtractions were performed. Such preprocessed data were fitted to a one-site or multiple-site model supplied with the software. The error analysis was carried out by a Monte-Carlo approach with 1000 trials and a 0.95 level of confidence.

Results and Discussion

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Previous studies on the central fragments of the Rad50 protein from P. furiosus showed a strong relationship between the amino acid sequence, the tertiary/quaternary zinc hook domain structure, and the Zn(II) complex stability. (39,45,46) The 14 amino acid long central fragment (Hk14, Figure 1) was found to form a highly structured minimal zinc hook domain upon the Zn(II) coordination. The 45 amino acid fragment (Hk45) was shown to form a full zinc hook domain, with all hydrophobic and electrostatic interactions. Therefore, herein, we used these two Rad50 fragments to study the principles governing the Rad50 hook domain interaction with Ag(I) ions, including their structural impact.

UV–Vis and CD Study of Ag(I) Binding to Hk14 and Hk45 Metal-Free Peptides

UV–vis and CD-monitored AgNO3 titrations were performed to detect and characterize the Ag(I) binding to Hk14 and Hk45 peptides. The changes in the near-UV spectral range enabled us to follow the formation of Ag(I)–thiolate complexes, while the accompanying changes in the peptide secondary structure were followed at shorter wavelengths. Figure 2 presents the evolution of electronic absorption and CD spectra of Hk14 and Hk45 at pH 7.4 upon the additions of 0–3.0 mol equiv of Ag(I). Several ligand-to-metal charge transfer (LMCT) bands originating in Ag(I)–thiol bonds appeared in the 230–300 nm region similar to those reported for Ag(I) complexes of metallothionein (MT) and Ag(I)–ZF peptides. (36,51) The alterations of band positions indicated the formation of various Ag(I) coordination species.

Figure 2

Figure 2. Spectrophotometric- (A, B) and CD- (C, D) monitored AgNO3 titrations of 25 μM Hk14 (left) and Hk45 (right) peptides in 20 mM TES, 100 mM NaF, pH 7.4. Black, red, cyan, green, and violet lines indicate 0, 1.0, 1.5, 2.0, and 3.0 Ag(I) mol equiv, respectively. Asterisk refers to signal changes presented in the inset of Figure 5C. The corresponding titration curves are presented in Figure 3.

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Hk14 and Hk45 displayed similar Ag(I)-induced UV–vis spectra, although, for Hk45, the LMCT bands were somewhat obscured by the absorbance of the Tyr residue. The first added portions of Ag(I) induced the characteristic bands at 255–258 and 290–295 nm, which became saturated at ∼1.0 mol equiv of Ag(I), thus indicating the formation of either 1:1 or 2:2 Ag(I)-to-Hk complexes (see Figure 2A,B for the UV–vis spectra, Figure 2C,D for the CD spectra, Figure 3 for the corresponding titration curves, and Figure S5 presents the differential spectra, obtained by subtraction of the apo-Hk spectra). Upon further Ag(I) additions, the entire spectrum gained intensity, which reached a broad maximum within at ca. 1.5 Ag(I) mol equiv. Although the stoichiometries of Agx(Hk)y complexes formed at this point are unclear, the two observed bands become less evident. The one at 295 nm is visibly blue-shifted, pointing to a change in the Ag(I) coordination environment. At the 2:1 Ag(I)-to-Hk molar ratio, the absorbance drops significantly and the plot minimum suggests a major complex rearrangement. It is even more evident when looking at the differential absorption spectra obtained by stepwise subtractions at 1, 1.5, 2, and 3 Ag(I) mol equiv (Figure S6). They show a decrease of the molar absorbance coefficient at 2 Ag(I) mol equiv. A further addition of AgNO3 caused the absorbance to increase again, but changes in the UV–vis spectra at 3.0 Ag(I) mol equiv and above are probably due to Hk aggregation initiated by such a high AgNO3 concentration. It could also be the result of the gradual oxidation of cysteine residues in the absence of the reducing agents that would hinder the interpretation of the data. However, this was ruled out by the DTNB assay, which demonstrated the stability of thiols during the time of the titration experiment (Figure S4). The characteristic bands at ∼250–260 and 280–295 nm observed throughout the spectroscopic titrations were previously noted in Ag(I) complexes with MT, Cys-bearing Cu(I)–chelators mimicking MT and Atox-1 sites, and in ZFs. (26,36,51) Although they have been assigned as CT transitions of thiolate–Ag(I) bonds, they were not interpreted in terms of Ag(I) coordination geometry.

Figure 3

Figure 3. Spectrophotometric- (A, B) and CD- (C–F) monitored AgNO3 titrations of 25 μM Hk14 (left) and Hk45 (right) peptides in 20 mM TES, 100 mM NaF, pH 7.4 within 0–4.5 Ag(I) mol equiv. The corresponding absorbance and CD spectra are presented in Figure 2. The wavelengths in all graphs correspond to the region, where the signal change is most pronounced, thus providing the best signal-to-noise ratio.

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The Ag(I)-induced changes in molar ellipticity of Hk peptides coincide with the ones observed in the UV–vis titrations. They display similar LMCT bands at the same Ag(I)-to-Hk stoichiometries (Figures 2C,D, 3C,D, and S5C,D). The first one appears after adding 1.0 Ag(I) mol equiv and corresponds to the formation of the 1:1 (or 2:2) Ag(I)-to-Hk species. The CD spectrum of Hk14 features a strong positive band at 218 nm, possibly coming from structural rearrangements, and two negative bands at 232 and 257 nm (Figures 2C and 3C,E). A broad positive band is also noticeable in the 280–320 nm range with a maximum at 295 nm. Further Ag(I) additions induced significant ellipticity changes, with turning points at ∼1.6, 2.0, and ∼3.0 Ag(I) mol equiv, above which no more characteristic bands were observed in the CD spectrum, like in the absorption spectra. Again, at 1.6 Ag(I) mol equiv, a weak positive band at 295 nm was shifted toward shorter wavelengths, which can be related to the formation of complexes with the larger nuclearity, favoring the diagonal AgS2 coordination mode. The negative bands at 232 and 257 nm disappeared above 1.6 Ag(I) mol equiv, indicating changes in S → Ag(I) band energies. Moreover, in the Hk45 variant, the addition of 1.0 Ag(I) mol equiv resulted in the partial α-helical peptide structurization, as evidenced by negative bands at 210 and 222 nm (Figures 2D and S5D). The turning point of the 222 nm CD signal (Figure 3F) illustrates the formation of a predominant AgHk45 (or Ag2(Hk45)2) species with high α-helical content, which suggests that at this ratio, Ag(I) is stabilizing some secondary structure in the Hk45 domain. The spectrum is, however, significantly different from that of the native Zn(II) complex (see Figure 4D). (46) Additional Ag(I) ions caused a progressive α-helix loss (possibly by the coiled-coil structure disruption). These changes reach a plateau above 2.0 mol. equiv, whereas the LMCT changes are similar to those observed for Hk14 (Figure 3C–F). Unfortunately, the absence of theoretical analysis of S → Ag(I) in the literature precludes even tentative assignments of these bands to specific complex geometries.

Figure 4

Figure 4. Spectrophotometric- (A, B) and CD- (C, D) monitored AgNO3 titrations of 25 μM Zn(Hk14)2 (left) and Zn(Hk45)2 (right) peptides in 20 mM TES, 100 mM NaF, pH 7.4. Black, blue, red, cyan, green, and violet lines indicate apo-Hk, Zn(Hk)2, 1.0, 1.5, 2.0, and 3.0 Ag(I) mol equiv, respectively. Asterisk refers to signal changes presented in the inset. The corresponding titration curves are presented in Figure 5.

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The similar behavior of Hk14 and Hk45 in AgNO3 titrations suggests that the same types of Agx(Hk)y complexes were formed regardless of the Hk peptide length. The gradual loss of secondary structure demonstrated by far-UV CD, accompanied by abrupt CD and absorption changes in the LMCT region, indicates that Agx(Hk)y complexes of various stoichiometries were formed and that Ag(I) geometry evolved along the number of Ag(I) ions bound in a given complex. Nevertheless, the apparent formation of a mixed Agx(Hk)y population makes it difficult to define the specific species.

Ag(I) Binding to Zn(Hk14)2 and Zn(Hk45)2 Peptides: UV–Vis and CD Study

To investigate whether the Zn(II) to Ag(I) swap observed in ZFs (36) can also take place in the hook domain, its peptide models were first saturated with Zn(II) and then titrated with Ag(I) followed by UV–vis and CD spectroscopies. As with AgNO3 titration of the metal-free peptides, the absorbance spectra of Zn(Hk14)2 and Zn(Hk45)2 displayed Ag(I)-dependent changes between 230 and 300 nm, including the appearance of S → Ag(I) LMCT bands at ∼255 and 290 nm (Figures 4 and S7). Ag(I) can then bind to the cysteinyl ligands previously occupied by Zn(II), likely displacing it from its coordination site. When plotted against the Ag(I) mol equiv, the intensity of the two observed bands continuously increases up to ∼1.5, without a shoulder at 1.0 that was previously observed in titrated apo-peptides (Figure 5A,B). The characteristic broad maximum between 1 and 2 Ag(I) mol equiv was also absent. These initial differences in the UV range absorption trace for apo- and holo-Hk peptides suggest that the initial Zn(II) presence induced the preferable formation of specific Agx(Hk)y complexes. This phenomenon can originate from the already preformed metal coordination center and the dimeric structure of Hk induced by Zn(II). Another possibility is that certain Agx(Hk)y species are significantly less stable from the Zn(Hk14)2 complex (46) and simply will not form in the presence of Zn(II). Upon further Ag(I) increase, both peptides displayed analogous changes to their zinc-free counterparts and after reaching minimum at 2.0 Ag(I) mol equiv, the signal continuously grew up to 3.0 mol equiv, over which no more new specific bands appeared that could be assigned to S → Ag(I).

Figure 5

Figure 5. Spectrophotometric- (A, B) and CD- (C–F) monitored AgNO3 titrations of 25 μM Zn(Hk14)2 (left) and Zn(Hk45)2 (right) peptides in 20 mM TES, 100 mM NaF, pH 7.4 within 0–4.5 Ag(I) mol equiv. The x-axis represents the molar ratio of AgNO3 to the Hk monomer being in the Zn(II) complex. The corresponding absorbance and CD spectra are presented in Figure 4. The wavelengths in all graphs correspond to the region, where the signal change is most pronounced, thus providing the best signal-to-noise ratio.

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CD-monitored AgNO3 titration of Zn(Hk)2 complexes partially clarified this issue (Figures 4C,D, 5C–F, and S7C,D). When looking at ellipticity changes within the CT range, the CD signal at 250–255 nm gradually increased up to ∼1.5 Ag(I) mol equiv. A minimum at 1.0 molar ratio did not appear (Figure 5C,D) and its lack coincided with negligible CT bands (compare the insets of Figures 2 and 4, red line) and the general loss of CD signal when compared to the corresponding changes observed for apo-Hk (Figure 4C,D, red line). This, again, indicates that the 1:1 complex either did not form or was not a predominant one during the Zn(II) substitution by Ag(I). Zn(Hk)2 is nevertheless significantly less stable than certain other Agx(Hk)y complexes, which is indicated not only by the presence of S → Ag(I) CT bands but also by the continuous disappearance of minima characterizing β-fold and α-helical (coiled-coil) structure (Figure 5E,F). Indeed, the CD spectra characteristic for Zn(Hk14)2 and Zn(Hk45)2 disappeared along the Ag(I) additions, indicating the Ag(I)-induced structure disruption. This was somewhat expected due to a strong Ag(I) affinity toward “soft” sulfur donors. As a consequence, at a sufficiently high ratio, the added Ag(I) ions were able to disrupt the Zn(II)-induced native fold of not only the Zn(Hk14)2 β-hairpin complex but also the Zn(Hk45)2 dimer that is further stabilized by additional interactions within two monomers.
To correlate the Ag(I) complexation process with Zn(II) release from Zn(Hk)2 complexes, the AgNO3 titration of holopeptides was repeated in the presence of Zn(II)-sensitive chromophore 4-(2-pyridylazo)resorcinol (PAR). (50) The concomitant Zn(II) dissociation steps were monitored in a continuous mode at 492 nm to observe the formation of the Zn(PAR)2 complex (Figure 6). For both Hk14 and Hk45 peptides, the addition of 2.0 mol equiv of Ag(I) was sufficient to fully displace Zn(II) from its coordination site, as evidenced by the absorption increase. More than 90% of bound Zn(II) was released upon the addition of 1.5 Ag(I) mol equiv, which correlates with the maximum present in UV–vis and CD titration plots at 255 nm. Visibly slower swap kinetics for Hk14 than for the longer Hk45 peptide is noticed, which may arise from a higher fraction of various Agx(Hk)y complexes that are formed upon the Ag(I) addition, which may require more time to form and rearrange due to a larger structuring.

Figure 6

Figure 6. Zn(II) transfer from 10 μM Zn(Hk14)2 (A) and 10 μM Zn(Hk45)2 complex (B) to 100 μM PAR upon an addition of 0.5–2.0 mol equiv of Ag(I). The formation of the Zn(PAR)2 complex was monitored spectroscopically by measuring an increase in absorbance at 492 nm. AgNO3 titration was performed in 20 mM TES, 100 NaF, pH 7.4. Asterisk denotes the initial absorbance of Zn(Hk)2 before the addition of Ag(I).

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Oligomeric Ag(I)–Hk Complexes Demonstrated by Size-Exclusion Chromatography

The observations from spectroscopic experiments strongly suggest the formation of Hk complexes with different Ag(I)-to-Hk (or rather Ag(I)-to-Cys) stoichiometries. However, these data are not sufficient to tell monomeric from oligomeric Ag(I)/Hk complexes that may form in solution. We therefore performed size-exclusion analysis to assess the oligomerization state of the Ag(I)–Hk complexes depending on the Ag(I)-to-Cys molar ratio. Samples of Hk14 and Hk45 peptides either in apo- or Zn(II)-loaded forms were briefly incubated with different mol equiv of Ag(I) and run on SEC at room temperature. Figure 7A shows that the addition of 1.0 Ag(I) equivalent to Hk14 resulted in two signals markedly shifted from the apo-Hk14 monomer peak, a weak one at ∼11.5 min and the intense one at ∼10 min. The former one corresponds to the retention time of a dimeric Zn(Hk14)2, thus indicating the formation of Ag2Hk2. The latter one comes from the higher oligomeric complex. Lastly, a small intensity peak at 12.6 min points to the formation of AgHk species. These outcomes stay in agreement with the UV–vis and CD results (Figure 3A,C,E), where the titration curves revealed the formation of AgHk complexes. Especially, the CD data shows clearly that the AgHk─or rather─Ag2Hk2 dimer is formed, as it gains a certain level of structurization. The addition of another 0.5 Ag(I) equivalent caused the weaker signals to disappear, while the oligomer peak became dominant. This again corresponds to the maxima observed in spectroscopic titration curves at ca. 1.5 ratio. The addition of further Ag(I) equivalents weakened and shifted the peak slightly to ∼10.2 min. At this ratio, at which the minimum of the signal was observed in both UV–vis and CD titrations, the oligomeric complex likely rearranges into different species, in which either the oligomerization state has changed or the number of Ag(I) bound in the cysteine-rich core altered. Further addition of AgNO3 altered the overall signal shape and shifted it to ∼9.5 min, pointing to the formation of larger aggregates, as expected from UV–vis observations. The elution profiles obtained for Ag(I)-titrated Zn(Hk)2 were quite similar to that of the apo-peptide. The major peak at ∼10 min. (Figure 7C, cyan) likely corresponds to the maximum observed in the UV–vis and CD titration curves at ∼1.5 Ag(I)/Hk14 ratio (Figure 5A,C,E). An almost linear increase of the intensity of the UV–vis and CD bands up to this value indicates that this complex is preferably formed from the already preformed and structurally organized Zn(Hk14)2. It further agrees with the PAR assay results, where the addition of slightly more than 1.5 mol equiv of Ag(I) is sufficient to displace Zn(II) from its binding site (Figure 6). Again, at 2.0 mol equiv of Ag(I), the oligomeric complex rearranges into the complex with 2:1 Ag(I)-to-Hk stoichiometry (as indicated by the marked shift in the retention time) that transforms into higher aggregates upon further increase of Ag(I) concentration. The AgNO3 titration profiles of metal-free and Zn(II)-loaded Hk45 were similar to the ones observed for Hk14 and Zn(Hk14)2 (Figure 7B,D), although the formation of Ag2(Hk45)2 is much more pronounced than for Hk14, likely due to structurally determined arrangement of Cys residues of the longer polypeptide chain. For apo-Hk45, an addition of 1.0 Ag(I) mol equiv resulted in the major peak at 13.4 min, with the relative intensity much higher compared to the signal at 11.5 min for Hk14 at the same metal-to-peptide ratio (Figure 7B). The presence of AgHk45 (or partially structured Ag2(Hk45)2) was observed in UV–vis and CD titrations (Figure 3B,D,F). At 1.5 Ag(I) mol equiv, the signal from the larger complex increased, although, in contrast to the shorter Hk14 model, some signals corresponding to the dimeric and monomeric forms were still present. At 2.0 Ag(I) mol equiv, the main peak shifted, as for Hk14, indicating analogous reorganization of the complex. A similar trend was observed for Zn(Hk45)2 (Figure 7D), where the addition of 1.0 Ag(I) mol equiv disrupted the Zn(II)-bridged dimer into a monomer and higher oligomeric complex. Such disruption of Zn(Hk45)2 also demonstrates that the direct displacement of Zn(II) by the two Ag(I) ions in its coordination site would not preserve the proper structure of the hook dimer (see also Figures 4D and 5F).

Figure 7

Figure 7. Size-exclusion chromatography analysis of Ag(I)–Hk14 and Ag(I)–Hk45 complexes. Elution profiles of metal-free Hk14 (A), Hk45 (B), Zn(Hk14)2 (C), and Zn(Hk45)2 (D) at different molar AgNO3 equivalents. Absorbance was measured at 220 nm. Dashed lines indicate the main peak signals.

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It should be underlined that SEC is not fully quantitative because it separates particular species with respect to their hydrodynamic radii (i.e., size and shape), breaking the state of equilibrium. However, it is very convenient for examining various species formation or their transformation. SEC profiles of AgNO3-titrated Hk14 and H45 show that at the very first stage of Ag(I) complexation, a species with possible 2:2 stoichiometry is formed, which elutes with the same time as the Zn(Hk)2 species. Based on SEC profiles, this species turns at the 1.5 Ag(I)-to-Hk molar ratio into a species, which gives the highest signal for Hk14 and Hk45, respectively. This peak likely corresponds to higher Ag(I)-nuclearity. At a Ag(I)/Hk ratio of 2:1, the complex rearranges into another oligomeric form (and likely with a different number of bound Ag(I) ions), which either turns to higher aggregates (for Hk14) or starts to tail (for Hk45) along with further addition of AgNO3. One clear conclusion from SEC analysis is the subsequent Ag(I)-induced oligomerization of both peptides regardless of the original form.

Identification of Ag(I)–Hk Complexes by ESI-MS

Solution studies of Ag(I) binding to metal-free and Zn(II)-saturated Hk peptides were followed by electrospray ionization mass spectrometry (ESI-MS) analysis to identify the resulting Agx(Hk)y products. ESI-MS preserves the noncovalent metal–protein interactions to some extent, especially those with large enthalpic components (52) (e.g., soft metals with soft ligands). Hence, the specific Agx(Hk)y complexes present in solution are likely to remain intact under gas-phase conditions. Apo-Hk peptides were initially dissolved in ammonium acetate pH 7.4 (50 mM salt concentration was used to minimize ESI-induced pH drop) and then either incubated with 0.5 Zn(II) mol equiv or directly saturated with increasing Ag(I) amounts, followed by a short incubation prior to the MS injection. Figure 8 presents the MS spectra obtained for Hk14, while the MS results for Hk45 and all of the assigned m/z signals are included in Figures S8 and S9 and Tables S1–S4, respectively.

Figure 8

Figure 8. (+)ESI-MS-monitored AgNO3 titration of apo-Hk14 and Zn(Hk14)2 complexes. Mass spectra of 2 μM peptide solutions were recorded for 0–4.0 mol equiv of Ag(I) in 50 mM ammonium acetate, pH 7.4. (A) Red, green, and cyan labels denote [AgL]2+, [Ag5L3]5+, and [Ag5L3]4+ species, respectively. Dashed lines indicate substrates (black) and the final (red) AgNO3 titration product. (B) Comparison of the calculated and experimental isotopic patterns of the observed metal complexes.

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Hk14 and Zn(Hk14)2 were measured first to assign the major m/z signals of the AgNO3 titration substrates (Figure 8A, black dashed lines). The initial addition of 0.5 Ag(I) mol equiv to either sample produced the Ag(Hk14) complex, appearing as a 2+ ion, accompanied by several low intensity peaks of other complexes, including Ag2(Hk14) and Ag2(Hk14)2 (Tables S1 and S2). In the presence of 1.0 Ag(I) mol equiv, the abundance of Ag(Hk14) significantly increased (the highest intensity), and new minor complexes were noticed, including the apo-Hk14 substrate. The predominance of Ag(Hk14), as well as the presence of Ag2(Hk14)2 signal, coincided with the turning point observed at 1.0 Ag(I) mol equiv in UV and CD titrations, confirming that 1:1 molar species were specifically formed in solution. At 1.5 Ag(I) mol equiv, a mixed population of Ag(Hk14), Ag4(Hk14)2, and Ag5(Hk14)3 complexes was detected for both Hk14 and Zn(Hk14)2, whereas signals from other species were vanishing. Such coexistence of various Ag(I) complex stoichiometries is consistent with spectroscopic and SEC results and further explains the noted difficulties in determining the Ag(I)-to-Hk binding stoichiometry. Ultimately, upon continuous Ag(I) additions, the two m/z signals corresponding to the Ag4(Hk14)2 complex dominated the mass spectra, especially at and above 2.0 mol equiv for both apo- and Zn(II)-bound peptides (Figure 8A, red dashed lines). This finding is consistent with the Zn(II) displacement experiment and demonstrates that at least at given gas-phase conditions, Ag4(Hk14)2 is the most stable complex that Ag(I) may form with Hk14.
The MS-monitored AgNO3 titration of Hk45 and Zn(Hk45)2 showed the formation of species with the same molar stoichiometries and the Ag4(Hk45)2 predominance, providing further evidence that Ag(I) readily replaces Zn(II) in the hook domain (Figures S8 and S9 and Tables S3 and S4). Table 1 summarizes major Agx(Hk)y complexes detected by MS. The detection of the Ag4(Hk)2 complex is particularly interesting, as it is consistent with the result obtained for the Ag(I)-titrated CCCC ZF, for which the AgNO3 titration experiments revealed a stable Ag4ZF complex. (37) The molecular dynamics and well-tempered metadynamics showed the formation of a Ag4(Cys)4 cluster in the ZF complex core. Analogously to the CCCC ZF, the two Hk monomers, each donating two cysteinyl residues, establish a tetrahedral coordination site for the Zn(II) ion. It is then possible, and consistent with the ESI-MS stoichiometry, that the Ag4(Cys)4 core can also be formed in the dimeric Hk system. It should be of course emphasized that ESI-MS is a qualitative rather than a quantitative method and that the appearing signals and their intensities corresponding to the metal–peptide complex present in the gas phase do not necessarily represent the presence and relative abundance of species existing in solution. (52,53) Nevertheless, under the controlled pH and ionization conditions, the specific Ag(I)-to-Hk complexes that form in solution should be retained when transferred into the gas phase. The other potential risk to consider is the supermetallization (54) of peptides during electrospray ionization. Yet, the observed 1:1 and 2:1 Ag(I)-to-Hk stoichiometries dominating in ESI-MS results converge with the stoichiometries observed in gel permeation and spectroscopic data.
Table 1. Summary of the Major Agx(Hk14)y and Agx(Hk45)y Complexes Observed in (+) ESI-MS Experimenta
AgxLyHk14 (Da)Hk45 (Da)Ag(I) mol equiv
L1516.7b5161.8b0
1516.7c5161.7c
AgL1622.6b5267.6b0.5–1.5
1621.6c5266.6c
Ag5L35079.8b16 017.6b1.0–2.0
5078.8c16 013.6c
Ag4L23457.1b10 747.0b1.5–4.0
3457.1c10 747.0c
a

Monoisotopic masses were derived from experimental isotopic patterns of the corresponding ion signals.

b

Experimental m/z value.

c

Calculated m/z value.

Determination of Ag(I) Binding Affinity to Hk and Zn(Hk)2 by ITC

As shown recently, Zn(II) is easily displaced by Ag(I) from all ZF types, regardless of the number of Cys residues present in the zinc coordination site. (36,37) Since Rad50 binds Zn(II) in a different stoichiometry than the single-molecule ZFs, a direct comparison of the respective affinities is not possible. It can be done, however, using the competitivity index (CI) method. (55) The zinc hook domain from P. furiosus was shown to form complexes with subattomolar affinity, which in turn translates into CI that outperforms most of familiar ZFs, whose affinities do not exceed femtomolar values (10–12–10–15 M). (38,46) Ag(I) titration to Hk14 and Hk45 (monitored by UV–vis, CD, and SEC) showed the formation of Ag(I)–Hk complexes of various stoichiometries, while the UV–vis-, CD-, and PAR-monitored titrations of Ag(I) to-Zn(Hk)2 complexes showed that the Zn(II)-to-Ag(I) substitution occurred spontaneously. The apparently full completion of the metal ion exchange for less than 2.0 molar excess of Ag(I) over the Zn(II) amount indicates that the Ag(I) affinity to the studied peptides must be at least 100-fold higher than that of Zn(II) (assuming that the noticeable amount of unreacted Zn(II) complex is less than 1% of the total signal). We applied ITC to estimate the stability difference of the hook complexes more precisely. The titration of Hk14 with Ag(I) ions showed two transitions at Ag(I)-to-Hk14 molar ratios 0.8 and 2.1. The latter, taken together with the UV–vis, CD, and MS data, strongly suggests the formation of the Ag(I)–Hk complex of 2:1 stoichiometry (Figure 9A and Table S5). The first step of complexation is a proton-linked process; thus, the observed ΔHITC is a sum of several effects: deprotonation of the Hk14 peptide upon metal complexation, protonation of buffer component, and the binding of Ag(I) to deprotonated thiol groups (thiolates). The second transition represents the end of Ag(I) binding to Hk14. The reverse Hk14-to-AgNO3 titration (Figure 9C) confirms this scenario. Interestingly, compared to the previously used methods, ITC does not show the formation of any other Ag(I)–Hk complexes, which may be due to the possibility that their formation is not accompanied by distinguishable changes in enthalpy. What is worth noting is the fact that AgNO3 addition to the preformed Zn(Hk14)2 complex yielded only one simultaneous step of Ag(I) complexation (at the inflection point of ∼2) and Zn(II) dissociation from the already deprotonated sulfur donors (Figure 9B). Direct Ag(I)-to-Hk14 and Hk14-to-Ag(I) titration experiments were fitted to multiple site model while Ag(I)-to-Zn(Hk14)2 titrations to independent site model (Table S5, control titrations Figure S10). Dissociation constants for the first cannot be considered because very high Ag(I)–Hk complex affinity is out of the effective range of the ITC method. Kd obtained from the fitting of Ag(I) titration to the preformed Zn(Hk14)2 complex is 4.1 μM and remains in the effective range of ITC. In fact, the obtained Kd should be considered here as an exchange constant (Kex), (50) which allows estimating affinity of Ag(I)–Hk species by this value multiplying by K12 of Zn(Hk)2. The affinity of the Ag(I) complex is therefore ∼240 000-fold higher (low zeptomolar range) than the Zn(II) one taking into account known −log K12 = 19.9 for Zn(Hk14)2. (46)

Figure 9

Figure 9. ITC analysis of Ag(I) binding to Hk14. ITC profile for titration of Ag(I) into Hk14 (A), Zn(Hk14)2 (B), and Hk14 into Ag(I) (C) in 20 mM HEPES, 100 mM NaF, pH 7.4. Top panels show the baseline-subtracted thermogram. The bottom panels represent the binding isotherm (see Table S5). The x-axis represents the molar ratio of AgNO3 to the Hk monomer being in the Zn(II) complex (A, B), and the molar ratio of Hk monomer to AgNO3 (C). Points not included in the fits were marked with white fill.

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Rad50 Activity Disruption by Ag(I) Relies on Zn(II) Swap

The AgNP uptake into the cell is an active process; apart from this, there is no clear consensus on the AgNP uptake pathway. (17) Inside the cell, silver species coexist as Ag(0) that form AgNPs and as organothiol-Ag(I). The ratio of the species depends on various factors, such as duration of exposure, dose, and cell type. The process of Ag(I) leaching from AgNPs is conducted inside the endolysosomal vesicles; the decrease of pH and the accompanying chloride concentration cause the release of soluble AgClx. (17) Within the endoplasm, Ag(I) is present mostly as organothiol complexes, from which the exchange of Ag(I) to Cu(I) or Zn(II) from various metalloproteins can occur. (17,18) Ag(I) ions can be translocated to the nucleus and interact with physiologically occurring Zn(II)–thiolate binding sites, i.e., zinc binding sites in ZFs in transcription factors, as well as in the intermolecular zinc binding site in Rad50 (Figure 10). (15,18) The possible Rad50 activity disruption by Ag(I) may rely on Zn(II) swap and formation of Ag(I)–Hk species, which significantly affect the structure of the Rad50 hook. As has been established by other studies, even a small mutation in the Rad50 hook may lead to a huge biological impact, thus by analogy, the Rad50 mismetallation by Ag(I) may lead to impairment of at least some of the Rad50 functions or even the whole MRN complex. (44,56)

Figure 10

Figure 10. Proposed pathway of AgNP transfer to the cell, their dissolution and Ag(I) release in nuclear peripheries. Ag(I) ions are translocated to the nucleus and interact with Zn(II)–thiolate binding sites, such as Rad50 being a part of the MRN(X) complex. Zn(II)-to-Ag(I) swap alters the structure of the dimeric hook domain and generates dysfunctional MRN complexes. Graph has been prepared using Servier Medical Art https://smart.servier.com.

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Conclusions

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Our study demonstrates that Ag(I) ions can directly replace Zn(II) not only in zinc finger domains but also in unique binding sites located on the interface between interacting proteins. Binding of Ag(I) to the Rad50 hook domain exhibited complicated speciation of the formed Ag(I)–Hk complexes, which likely coexisted in solution during the direct titration experiments. Complexes of various stoichiometries were found, among which Ag(I)–Hk species of 1:1 and 2:1 molar ratio were formed preferentially when a sufficient amount of Ag(I) ions was available. (36,37) The stability of the latter one was at least 5 orders of magnitude higher than the high attomolar one of Zn(Hk)2. This indicates the Ag(I)-to-hook domain affinity is in the low zeptomolar range. The formation of the underlying Ag4(Cys)4 core results in the native structure disruption of the Zn(Hk)2 complex, which is highly important for the proper functioning of the MRN complex in DNA DSB repair. The observed association tendency of Ag(I)–Hk species also indicates that the disruption of Rad50 may occur due to its irreversible oligomerization. The presence of many Cys-rich binding sites in DNA and RNA processing proteins strongly suggests that the loss of their native function and structure can be the basis of silver genotoxicity.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.inorgchem.2c03767.

  • Materials; RP-HPLC chromatograms of purified Hk14 and Hk45 peptides and the corresponding ESI-MS spectra; figures with spectra from UV–vis, CD, and ESI-MS experiments; tables with all the assigned m/z values in ESI-MS experiments; supplementary table with values calculated in the ITC experiment (PDF)

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Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

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  • Corresponding Author
  • Authors
    • Olga Kerber - Department of Chemical Biology, Faculty of Biotechnology, University of Wrocław, Joliot-Curie 14a, 50-383 Wrocław, Poland
    • Józef Tran - Department of Chemical Biology, Faculty of Biotechnology, University of Wrocław, Joliot-Curie 14a, 50-383 Wrocław, Poland
    • Alicja Misiaszek - Department of Chemical Biology, Faculty of Biotechnology, University of Wrocław, Joliot-Curie 14a, 50-383 Wrocław, Poland
    • Aleksandra Chorążewska - Department of Chemical Biology, Faculty of Biotechnology, University of Wrocław, Joliot-Curie 14a, 50-383 Wrocław, Poland
    • Wojciech Bal - Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland;  Orcidhttps://orcid.org/0000-0003-3780-083X
  • Author Contributions

    Experiments were performed by O.K., J.T., A.M., and A.C. This manuscript was written by O.K., J.T., W.B., and A.K. All authors have given approval to the final version of the manuscript.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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The research was supported by the Polish National Science Centre of Poland under grants: Opus No. 2016/21/B/NZ1/02847 (to A.K.) and Preludium No. 2020/37/N/NZ1/03319 (to O.K.).

References

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    Silver nanoparticles: Partial oxidation and antibacterial activities
    Lok, Chun-Nam; Ho, Chi-Ming; Chen, Rong; He, Qing-Yu; Yu, Wing-Yiu; Sun, Hongzhe; Tam, Paul Kwong-Hang; Chiu, Jen-Fu; Che, Chi-Ming
    JBIC, Journal of Biological Inorganic Chemistry (2007), 12 (4), 527-534CODEN: JJBCFA; ISSN:0949-8257. (Springer GmbH)
    The phys. and chem. properties of silver nanoparticles that are responsible for their antimicrobial activities have been studied with spherical silver nanoparticles (av. diam. approx. 9 nm) synthesized by the borohydride redn. of Ag+ ions, in relation to their sensitivity to oxidn., activities towards silver-resistant bacteria, size-dependent activities, and dispersal in electrolytic solns. Partially (surface) oxidized silver nanoparticles have antibacterial activities, but zero-valent nanoparticles do not. The levels of chemisorbed Ag+ that form on the particle's surface, as revealed by changes in the surface plasmon resonance absorption during oxidn. and redn., correlate well with the obsd. antibacterial activities. Silver nanoparticles, like Ag+ in the form of AgNO3 soln., are tolerated by the bacteria strains resistant to Ag+. The antibacterial activities of silver nanoparticles are related to their size, with the smaller particles having higher activities on the basis of equiv. silver mass content. The silver nanoparticles aggregate in media with a high electrolyte content, resulting in a loss of antibacterial activities. However, complexation with albumin can stabilize the silver nanoparticles against aggregation, leading to a retention of the antibacterial activities. Taken together, the results show that the antibacterial activities of silver nanoparticles are dependent on chemisorbed Ag+, which is readily formed owing to extreme sensitivity to oxygen. The antibacterial activities of silver nanoparticles are dependent on optimally displayed oxidized surfaces, which are present in well-dispersed suspensions.
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  6. 6
    Hsiao, I.-L.; Hsieh, Y.-K.; Wang, C.-F.; Chen, I.-C.; Huang, Y.-J. Trojan-horse mechanism in the cellular uptake of silver nanoparticles verified by direct intra- and extracellular silver speciation analysis. Environ. Sci. Technol. 2015, 49, 3813– 3821,  DOI: 10.1021/es504705p
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    6
    Trojan-Horse Mechanism in the Cellular Uptake of Silver Nanoparticles Verified by Direct Intra- and Extracellular Silver Speciation Analysis
    Hsiao, I-Lun; Hsieh, Yi-Kong; Wang, Chu-Fang; Chen, I-Chieh; Huang, Yuh-Jeen
    Environmental Science & Technology (2015), 49 (6), 3813-3821CODEN: ESTHAG; ISSN:0013-936X. (American Chemical Society)
    The so-called "Trojan-horse" mechanism, in which nanoparticles are internalized within cells and then release high levels of toxic ions, has been proposed as a behavior in the cellular uptake of Ag nanoparticles (AgNPs). While several reports claim to have proved this mechanism by measuring AgNPs and Ag ions (I) in cells, it cannot be fully proven without examg. those two components in both intra- and extracellular media. In our study, we found that even though cells take up AgNPs similarly to (microglia (BV-2)) or more rapidly than (astrocyte (ALT)) Ag (I), the ratio of AgNPs to total Ag (AgNPs+Ag (I)) in both cells was lower than that in outside media. It could be explained that H2O2, a major intracellular reactive oxygen species (ROS), reacts with AgNPs to form more Ag (I). Moreover, the major speciation of Ag (I) in cells was Ag(cysteine) and Ag(cysteine)2, indicating the possible binding of monomer cysteine or vital thiol proteins/peptides to Ag ions. Evidence we found indicates that the Trojan-horse mechanism really exists.
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  7. 7
    Betts, H. D.; Whitehead, C.; Harris, H. H. Silver in biology and medicine: opportunities for metallomics researchers. Metallomics 2021, 13, mfaa001  DOI: 10.1093/mtomcs/mfaa001
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  8. 8
    Betts, H. D.; Neville, S. L.; McDevitt, C. A.; Sumby, C. J.; Harris, H. H. The biochemical fate of Ag+ ions in Staphylococcus aureus, Escherichia coli, and biological media. J. Inorg. Biochem. 2021, 225, 111598  DOI: 10.1016/j.jinorgbio.2021.111598
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    8
    The biochemical fate of Ag+ ions in Staphylococcus aureus, Escherichia coli, and biological media
    Betts, Harley D.; Neville, Stephanie L.; McDevitt, Christopher A.; Sumby, Christopher J.; Harris, Hugh H.
    Journal of Inorganic Biochemistry (2021), 225 (), 111598CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier Inc.)
    Silver is commonly included in a range of household and medical items to provide bactericidal action. Despite this, the chem. fate of the metal in both mammalian and bacterial systems remains poorly understood. Here, we applied a metallomics approach using X-ray absorption spectroscopy (XAS) and size-exclusion chromatog. hyphenated with inductively coupled plasma mass spectrometry (SEC-ICP-MS) to advance our understanding of the biochem. fate of silver ions in bacterial culture and cells, and the chem. assocd. with these interactions. When silver ions were added to lysogeny broth, silver was exclusively assocd. with moderately-sized species (∼30 kDa) and bound by thiolate ligands. In two representative bacterial pathogens cultured in lysogeny broth including sub-lethal concns. of ionic silver, silver was found in cells to be predominantly coordinated by thiolate species. The silver biomacromol.-binding profile in Staphylococcus aureus and Escherichia coli was complex, with silver bound by a range of species spanning from 20 kDa to >1220 kDa. In bacterial cells, silver was nonuniformly colocalised with copper-bound proteins, suggesting that cellular copper processing may, in part, confuse silver for nutrient copper. Notably, in the treated cells, silver was not detected bound to low mol. wt. compds. such as glutathione or bacillithiol.
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  9. 9
    Liao, X.; Yang, F.; Wang, R.; He, X.; Li, H.; Kao, R. Y. T.; Xia, W.; Sun, H. Identification of catabolite control protein A from Staphylococcus aureus as a target of silver ions. Chem. Sci. 2017, 8, 8061– 8066,  DOI: 10.1039/c7sc02251d
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    9
    Identification of catabolite control protein A from Staphylococcus aureus as a target of silver ions
    Liao, Xiangwen; Yang, Fang; Wang, Runming; He, Xiaojun; Li, Hongyan; Kao, Richard Y. T.; Xia, Wei; Sun, Hongzhe
    Chemical Science (2017), 8 (12), 8061-8066CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
    Staphylococcus aureus is one of the most common pathogenic bacteria that causes human infectious diseases. The emergence of antibiotic-resistant strains of S. aureus promotes the development of new anti-bacterial strategies. Silver ions (Ag+) have attracted profound attention due to their broad-spectrum antimicrobial activities. Although the antibacterial properties of silver have been well known for many centuries, its mechanism of action remains unclear and its protein targets are rarely reported. Herein, we identify the catabolite control protein A (CcpA) of S. aureus as a putative target for Ag+. CcpA binds 2 molar equivalents of Ag+via its two cysteine residues (Cys216 and Cys242). Importantly, Ag+ binding induces CcpA oligomerization and abolishes its DNA binding capability, which further attenuates S. aureus growth and suppresses a-hemolysin toxicity. This study extends our understanding of the bactericidal effects of silver.
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  10. 10
    Wang, H.; Wang, M.; Xu, X.; Gao, P.; Xu, Z.; Zhang, Q.; Li, H.; Yan, A.; Kao, R. Y.-T.; Sun, H. Multi-target mode of action of silver against Staphylococcus aureus endows it with capability to combat antibiotic resistance. Nat. Commun. 2021, 12, 3331  DOI: 10.1038/s41467-021-23659-y
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    10
    Multi-target mode of action of silver against Staphylococcus aureus endows it with capability to combat antibiotic resistance
    Wang, Haibo; Wang, Minji; Xu, Xiaohan; Gao, Peng; Xu, Zeling; Zhang, Qi; Li, Hongyan; Yan, Aixin; Kao, Richard Yi-Tsun; Sun, Hongzhe
    Nature Communications (2021), 12 (1), 3331CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
    Abstr.: The rapid emergence of drug resistant Staphylococcus aureus (S. aureus) poses a serious threat to public health globally. Silver (Ag)-based antimicrobials are promising to combat antibiotic resistant S. aureus, yet their mol. targets are largely elusive. Herein, we sep. and identify 38 authentic Ag+-binding proteins in S. aureus at the whole-cell scale. We then capture the mol. snapshot on the dynamic action of Ag+ against S. aureus and further validate that Ag+ could inhibit a key target 6-phosphogluconate dehydrogenase through binding to catalytic His185 by X-ray crystallog. Significantly, the multi-target mode of action of Ag+ (and nanosilver) endows its sustainable antimicrobial efficacy, leading to enhanced efficacy of conventional antibiotics and resensitization of MRSA to antibiotics. Our study resolves the long-standing question of the mol. targets of silver in S. aureus and offers insights into the sustainable bacterial susceptibility of silver, providing a potential approach for combating antimicrobial resistance.
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  11. 11
    Zhang, Q.; Wang, R.; Wang, M.; Liu, C.; Koohi-Moghadam, M.; Wang, H.; Ho, P.-L.; Li, H.; Sun, H. Re-sensitization of mcr carrying multidrug resistant bacteria to colistin by silver. Proc. Natl. Acad. Sci. U.S.A. 2022, 119, e2119417119  DOI: 10.1073/pnas.2119417119
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    11
    Re-sensitization of mcr carrying multidrug resistant bacteria to colistin by silver
    Zhang, Qi; Wang, Runming; Wang, Minji; Liu, Chunjiao; Koohi-Moghadam, Mohamad; Wang, Haibo; Ho, Pak-Leung; Li, Hongyan; Sun, Hongzhe
    Proceedings of the National Academy of Sciences of the United States of America (2022), 119 (11), e2119417119CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Colistin is considered the last-line antimicrobial for the treatment of multidrug-resistant gram-neg. bacterial infections. The emergence and spread of superbugs carrying the mobile colistin resistance gene (mcr) have become the most serious and urgent threat to healthcare. Here, we discover that silver (Ag+), including silver nanoparticles, could restore colistin efficacy against mcr-pos. bacteria. We show that Ag+ inhibits the activity of the MCR-1 enzyme via substitution of Zn2+ in the active site. Unexpectedly, a tetra-silver center was found in the active-site pocket of MCR-1 as revealed by the X-ray structure of the Ag-bound MCR-1, resulting in the prevention of substrate binding. Moreover, Ag+ effectively slows down the development of higher-level resistance and reduces mutation frequency. Importantly, the combined use of Ag+ at a low concn. with colistin could relieve dermonecrotic lesions and reduce the bacterial load of mice infected with mcr-1-carrying pathogens. This study depicts a mechanism of Ag+ inhibition of MCR enzymes and demonstrates the potentials of Ag+ as broad-spectrum inhibitors for the treatment of mcr-pos. bacterial infection in combination with colistin.
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  12. 12
    Jiménez-Lamana, J.; Laborda, F.; Bolea, E.; Abad-Álvaro, I.; Castillo, J. R.; Bianga, J.; He, M.; Bierla, K.; Mounicou, S.; Ouerdane, L.; Gaillet, S.; Rouanet, J. M.; Szpunar, J. An insight into silver nanoparticles bioavailability in rats. Metallomics 2014, 6, 2242– 2249,  DOI: 10.1039/c4mt00200h
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    12
    An insight into silver nanoparticles bioavailability in rats
    Jimenez-Lamana, Javier; Laborda, Francisco; Bolea, Eduardo; Abad-Alvaro, Isabel; Castillo, Juan R.; Bianga, Juliusz; He, Man; Bierla, Katarzyna; Mounicou, Sandra; Ouerdane, Laurent; Gaillet, Sylvie; Rouanet, Jean-Max; Szpunar, Joanna
    Metallomics (2014), 6 (12), 2242-2249CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)
    A comprehensive study of the bioavailability of orally administered silver nanoparticles (AgNPs) was carried out using a rat model. The silver uptake was monitored in liver and kidney tissues, as well as in urine and in feces. Significant accumulation of silver was found in both organs, the liver being the principal target of AgNPs. A significant (∼50%) fraction of silver was found in feces whereas the fraction excreted via urine was negligible (<0.01%). Intact silver nanoparticles were found in feces by asym. flow field-flow fractionation (AsFlFFF) coupled with UV-Vis anal. Laser ablation-ICP MS imaging showed that AgNPs were able to penetrate into the liver, in contrast to kidneys where they were retained in the cortex. Silver speciation anal. in cytosols from kidneys showed the metallothionein complex as the major species whereas in the liver the majority of silver was bound to high-mol. (70-25 kDa) proteins. These findings demonstrate the presence of Ag(I), released by the oxidn. of AgNPs in the biol. environment.
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    Yang, L.; Kuang, H.; Zhang, W.; Aguilar, Z. P.; Wei, H.; Xu, H. Comparisons of the biodistribution and toxicological examinations after repeated intravenous administration of silver and gold nanoparticles in mice. Sci. Rep. 2017, 7, 3303  DOI: 10.1038/s41598-017-03015-1
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    13
    Comparisons of the biodistribution and toxicological examinations after repeated intravenous administration of silver and gold nanoparticles in mice
    Yang Lin; Kuang Huijuan; Wei Hua; Xu Hengyi; Zhang Wanyi; Aguilar Zoraida P
    Scientific reports (2017), 7 (1), 3303 ISSN:.
    Nanoparticles (NPs) size, surface functionalization, and concentration were claimed to contribute to distribution and toxicity outcomes of NPs in vivo. However, intrinsic chemical compositions of NPs caused inconsistent biodistribution and toxic profiles which attracted little attention. In this study, silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs) were used to determine the biodistribution, toxickinetic, and genotoxicity variances in murine animals. The results demonstrated AgNPs and AuNPs were primarily deposited in the mononuclear phagocyte system (MPS) such as the liver and spleen. In particular, AuNPs seemed to be prominently stored in the liver, whereas AgNPs preferentially accumulated in more organs such as the heart, lung, kidney, etc. Also, the circulation in the blood and fecal excretions showed higher AgNPs contents in comparison with the AuNPs. Measurements of the mouse body and organ mass, hematology and biochemistry evaluation, and histopathological examinations indicated slight toxic difference between the AgNPs and AuNPs over a period of two months. RT-qPCR data revealed that AgNPs induced greater changes in gene expression with relevance to oxidative stress, apoptosis, and ion transport. Our observations proved that the NPs chemical composition played a critical role in their in vivo biodistribution and toxicity.
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  14. 14
    Poznański, J.; Sołdacki, D.; Czarkowska-Pączek, B.; Bonna, A.; Kornasiewicz, O.; Krawczyk, M.; Bal, W.; Pączek, L. Cirrhotic liver of liver transplant recipients accumulate silver and co-accumulate copper. Int. J. Mol. Sci. 2021, 22, 1782,  DOI: 10.3390/ijms22041782
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    Suárez, V. T.; Karepina, E.; Chevallet, M.; Gallet, B.; Cottet-Rousselle, C.; Charbonnier, P.; Moriscot, C.; Michaud-Soret, I.; Bal, W.; Fuchs, A.; Tucoulou, R.; Jouneau, P.-H.; Veronesi, G.; Deniaud, A. Nuclear translocation of silver ions and hepatocyte nuclear Receptor impairment upon exposure to silver nanoparticles. Environ. Sci.: Nano 2020, 7, 1373– 1387,  DOI: 10.1039/c9en01348b
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    Wiemann, M.; Vennemann, A.; Blaske, F.; Sperling, M.; Karst, U. Silver nanoparticles in the lung: Toxic effects and focal accumulation of silver in remote organs. Nanomaterials 2017, 7, 441,  DOI: 10.3390/nano7120441
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    16
    Silver nanoparticles in the lung: toxic effects and focal accumulation of silver in remote organs
    Wiemann, Martin; Vennemann, Antje; Blaske, Franziska; Sperling, Michael; Karst, Uwe
    Nanomaterials (2017), 7 (12), 441/1-441/26CODEN: NANOKO; ISSN:2079-4991. (MDPI AG)
    The distribution of silver (Ag) into remote organs secondary to the application of Ag nanoparticles (Ag-NP) to the lung is still incompletely understood and was investigated in the rat with imaging methods. Dose-finding expts. were carried out with 50 nm- or 200 nm-sized polyvinyl pyrrolidine (PVP)-coated Ag-NP using alveolar macrophages in vitro and female rats, which received Ag-NP via intratracheal instillation. In the main study, we administered 37.5-300 μg per rat lung of the more toxic Ag50-PVP and assessed the broncho-alveolar lavage fluid (BALF) for inflammatory cells, total protein and fibronectin after three and 21 days. In parallel, lung tissue was analyzed for DNA double-strand breaks and altered cell proliferation. While 75-150 μg Ag50-PVP per rat lung caused a reversible inflammation, 300 μg led to DNA damage, accelerated cell proliferation and progressively increasing nos. of neutrophilic granulocytes. Ag accumulation was significant in homogenates of liver and other peripheral organs upon lung dose of ≥75 μg. Quant. laser-ablation inductively-coupled plasma mass spectrometry (LA-ICP-MS) combined with enhanced dark field microscopy and autometallog. revealed focal accumulations of Ag and/or Ag-NP in sections of peripheral organs: mediastinal lymph nodes contained Ag-NP esp. in peripheral macrophages and Ag in argyrophilic fibers. In the kidney, Ag had accumulated within proximal tubuli, while renal filter structures contained no Ag. Discrete localizations were also obsd. in immune cells of liver and spleen. Overall, the study shows that concns. of Ag-NP, which elicit a transient inflammation in the rat lung, lead to focal accumulations of Ag in peripheral organs, and this might pose a risk to particular cell populations in remote sites.
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    Marchioni, M.; Jouneau, P.-H.; Chevallet, M.; Michaud-Soret, I.; Deniaud, A. Silver nanoparticle fate in mammals: Bridging in vitro and in vivo studies. Coord. Chem. Rev. 2018, 364, 118– 136,  DOI: 10.1016/j.ccr.2018.03.008
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    17
    Silver nanoparticle fate in mammals: Bridging in vitro and in vivo studies
    Marchioni, Marianne; Jouneau, Pierre-Henri; Chevallet, Mireille; Michaud-Soret, Isabelle; Deniaud, Aurelien
    Coordination Chemistry Reviews (2018), 364 (), 118-136CODEN: CCHRAM; ISSN:0010-8545. (Elsevier B.V.)
    Silver nanoparticles (AgNPs) are exponentially used in various consumer products including medical devices. This prodn. leads to an increasing human exposure to silver in different forms. Indeed, AgNPs are subject to various transformations in aq. aerobic conditions that trigger the prodn. of Ag(I) species. The main environmental transformation produces the non-toxic species silver sulfide. Transformations occurring in mammals are more diverse and mainly depend on the interaction of AgNPs with thiol, chloride and proteins. Any of these species have a different impact on AgNPs and induces AgNP dissoln. into Ag(I) species, aggregation and/or stabilization. The transformations occurring also depend on the exposure route. The main one is dietary but medical exposure is also growing with the massive use of nanosilver as biocide in medical devices. For the former, AgNP modifications and Ag distribution has been extensively studied using in vitro and in vivo models, while data related to medical use of nanosilver are scarce. However, most of the in vitro and in vivo data often remain inconsistent. In this review, we describe both in vitro, in cellulo and in vivo data about AgNP transformations, silver speciation and biodistribution. We try to reconcile all these data and describe the latest methods for the future studies of AgNP fate in mammals.
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  18. 18
    Malysheva, A.; Ivask, A.; Doolette, C. L.; Voelcker, N. H.; Lombi, E. Cellular binding, uptake and biotransformation of silver nanoparticles in human T lymphocytes. Nat. Nanotechnol. 2021, 16, 926– 932,  DOI: 10.1038/s41565-021-00914-3
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    18
    Cellular binding, uptake and biotransformation of silver nanoparticles in human T lymphocytes
    Malysheva, Anzhela; Ivask, Angela; Doolette, Casey L.; Voelcker, Nicolas H.; Lombi, Enzo
    Nature Nanotechnology (2021), 16 (8), 926-932CODEN: NNAABX; ISSN:1748-3387. (Nature Portfolio)
    Our knowledge of uptake, toxicity and detoxification mechanisms as related to nanoparticles' (NPs') characteristics remains incomplete. Here we combine the anal. power of three advanced techniques to study the cellular binding and uptake and the intracellular transformation of silver nanoparticles (AgNPs): single-particle inductively coupled mass spectrometry, mass cytometry and synchrotron X-ray absorption spectrometry. Our results show that although intracellular and extracellularly bound AgNPs undergo major transformation depending on their primary size and surface coating, intracellular Ag in 24 h AgNP-exposed human lymphocytes exists in nanoparticulate form. Biotransformation of AgNPs is dominated by sulfidation, which can be viewed as one of the cellular detoxification pathways for Ag. These results also show that the toxicity of AgNPs is primarily driven by internalized Ag. In fact, when toxicity thresholds are expressed as the intracellular mass of Ag per cell, differences in toxicity between NPs of different coatings and sizes are minimized. The anal. approach developed here has broad applicability in different systems where the aim is to understand and quantify cell-NP interactions and biotransformation.
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  19. 19
    Adams, N. W. H.; Kramer, J. R. Potentiometric determination of silver thiolate formation constants using a Ag2S electrode. Aquat. Geochem. 1999, 5, 1– 11,  DOI: 10.1023/A:1009699617808
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    19
    Potentiometric determination of silver thiolate formation constants using a Ag2S electrode
    Adams, Nicholas W. H.; Kramer, James R.
    Aquatic Geochemistry (1999), 5 (1), 1-11CODEN: AQGEFP; ISSN:1380-6165. (Kluwer Academic Publishers)
    Formation consts. for silver thiolates were obtained by titrn. of the ligand in a const. temp., ionic strength and pH medium and measuring the potential change at a Ag2S electrode. A non-linear equation was derived from which the first and second silver formation consts., β'1 and β'2, and the sulfide group acid dissocn. const., K'a, were detd. An overall est. of the uncertainty in the derived parameters was obtained using a Monte Carlo approach. The procedure was compared to a previous work on AgHS°. Log β'1, log β'2 and -log K'a results were obtained for cysteine (11.9 ± 0.5, 15.2 ± 0.4, 7.8 ± 0.1), glutathione (12.3 ± 0.3, 14.3 ± 0.8, 8.8 ± 0.3) and 3-mercaptopropanoic acid (12.0 ± 0.4, 14.0 ± 0.4, 10.5 ± 0.3) at 20° and 0.01 m ionic strength.
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  20. 20
    Pettit, L. D.; Siddiqui, K. F.; Kozłowski, H.; Kowalik, T. Potentiometric and 1H NMR studies on silver(I) interaction with S-methyl-L-cysteine, L-methionine and L-ethionine. Inorg. Chim. Acta 1981, 55, 87– 91,  DOI: 10.1016/S0020-1693(00)90787-4
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    20
    Potentiometric and proton NMR studies on silver(I) interaction with S-methyl-L-cysteine, L-methionine and L-ethionine
    Pettit, Leslie D.; Siddiqui, Kaniz F.; Kozlowski, Henryk; Kowalik, Teresa
    Inorganica Chimica Acta (1981), 55 (3), 87-91CODEN: ICHAA3; ISSN:0020-1693.
    Potentiometric titrn. (pH, pAg) studies at 25° and ionic strength 0.1(KNO3) gave stability consts. for Ag(HL), Ag(HL)2, AgL (or Ag2L2), and AgL2 complexes (H2L = title ligands). The NMR data indicate S-bonding to Ag with NH2 group bonding only at pH >4.5. Rotamer populations of free and coordinated S-methyl-L-cysteine were also calcd.
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  21. 21
    Legler, E. V.; Kazbanov, V. I.; Kazachenko, A. S. Study of complexing equilibrium in the Ag(I)-histidine system. Zh. Neorg. Khim. 2002, 47, 150– 153
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    Dance, I. G. The Structural Chemistry of Metal Thiolate Complexes. Polyhedron 1986, 5, 1037– 1104,  DOI: 10.1016/S0277-5387(00)84307-7
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    22
    The structural chemistry of metal thiolate complexes
    Dance, Ian G.
    Polyhedron (1986), 5 (5), 1037-104CODEN: PLYHDE; ISSN:0277-5387.
    A review with 346 refs.
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    Leung, B. O.; Jalilehvand, F.; Mah, V.; Parvez, M.; Wu, Q. Silver(I) complex formation with cysteine, penicillamine, and glutathione. Inorg. Chem. 2013, 52, 4593– 4602,  DOI: 10.1021/ic400192c
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    23
    Silver(I) Complex Formation with Cysteine, Penicillamine, and Glutathione
    Leung, Bonnie O.; Jalilehvand, Farideh; Mah, Vicky; Parvez, Masood; Wu, Qiao
    Inorganic Chemistry (2013), 52 (8), 4593-4602CODEN: INOCAJ; ISSN:0020-1669. (American Chemical Society)
    The complex formation between silver(I) and cysteine (H2Cys), penicillamine (H2Pen), and glutathione (H3Glu) in alk. aq. soln. was examd. using extended x-ray absorption fine structure (EXAFS) and 109Ag NMR spectroscopic techniques. The complexes formed in 0.1 mol dm-3 Ag(I) solns. with cysteine and penicillamine were investigated for ligand/Ag(I) (L/Ag) mole ratios increasing from 2.0 to 10.0. For cysteine solns. (pH 10-11) a mean Ag-S bond distance of 2.45 ± 0.02 Å consistently emerged, while for penicillamine (pH 9) the av. Ag-S bond distance gradually increased from 2.40 to 2.44 ± 0.02 Å. EXAFS and 109Ag NMR spectra of a concd. Ag(I)-cysteine soln. (CAg(I) = 0.8 mol dm-3, L/Ag = 2.2) showed a mean Ag-S bond distance of 2.47 ± 0.02 Å and δ(109Ag) 1103 ppm, consistent with prevailing, partially oligomeric AgS3 coordinated species, while for penicillamine (CAg(I) = 0.5 mol dm-3, L/Ag = 2.0) the mean Ag-S bond distance of 2.40 ± 0.02 Å and δ(109Ag) 922 ppm indicate that mononuclear AgS2 coordinated complexes dominate. For Ag(I)-glutathione solns. (CAg(I) = 0.01 mol dm-3, pH ∼11), mononuclear AgS2 coordinated species with a mean Ag-S bond distance of 2.36 ± 0.02 Å dominate for L/Ag mole ratios from 2.0 to 10.0. The crystal structure of the silver(I)-cysteine compd. (NH4)Ag2(HCys)(Cys)·H2O (1) pptg. at pH ∼10 was solved and showed a layer structure with both AgS3 and AgS3N coordination to the cysteinate ligands. A redetn. of the crystal structure of Ag(HPen)·H2O (2) confirmed the proposed digonal AgS2 coordination environment to bridging thiolate sulfur atoms in polymeric intertwining chains forming a double helix. A survey of Ag-S bond distances for cryst. Ag(I) complexes with S-donor ligands in different AgS2, AgS2(O/N), and AgS3 coordination environments was used, together with a survey of 109Ag NMR chem. shifts, to assist assignments of the Ag(I) coordination in soln.
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    Young, A. G.; Hanton, L. R. Square planar silver(I) complexes: A rare but increasingly observed stereochemistry for silver(I). Coord. Chem. Rev. 2008, 252, 1346– 1386,  DOI: 10.1016/j.ccr.2007.07.017
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    Square planar silver(I) complexes: A rare but increasingly observed stereochemistry for silver(I)
    Young, Aidan G.; Hanton, Lyall R.
    Coordination Chemistry Reviews (2008), 252 (12-14), 1346-1386CODEN: CCHRAM; ISSN:0010-8545. (Elsevier B.V.)
    A review. The square planar Ag(I) stereochem. is generally acknowledged as rare, with only ∼2% of all reported silver complexes possessing this stereochem. Many researchers reporting such complexes often mistakenly believe that their example is one of only a handful of previously reported examples. This is despite the fact that there are currently around 65 well characterized complexes contg. square planar Ag(I) ions, about half of which are coordination polymers. In this review, the authors critically examine each example and draw attention to trends that arise in their formation. The scope is limited to traditional coordination complexes. Inorg. complexes contg. extended mineral like structures, and complexes contg. silver-π or silver-arene motifs are not considered in this review.
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    Wang, H.; Yang, X.; Wang, M.; Hu, M.; Xu, X.; Yan, A.; Hao, Q.; Li, H.; Sun, H. Atomic differentiation of silver binding preference in protein targets: Escherichia colimalate dehydrogenase as a paradigm. Chem. Sci. 2020, 11, 11714– 11719,  DOI: 10.1039/d0sc04151c
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    Atomic differentiation of silver binding preference in protein targets: Escherichia coli malate dehydrogenase as a paradigm
    Wang, Haibo; Yang, Xinming; Wang, Minji; Hu, Menglong; Xu, Xiaohan; Yan, Aixin; Hao, Quan; Li, Hongyan; Sun, Hongzhe
    Chemical Science (2020), 11 (43), 11714-11719CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
    Understanding how metallodrugs interact with their protein targets is of vital importance for uncovering their mol. mode of actions as well as overall pharmacol./toxicol. profiles, which in turn facilitates the development of novel metallodrugs. Silver has been used as an antimicrobial agent since antiquity, yet there is limited knowledge about silver-binding proteins. Given the multiple dispersed cysteine residues and histidine-methionine pairs, Escherichia coli malate dehydrogenase (EcMDH) represents an excellent model to investigate silver coordination chem. as well as its targeting sites in enzymes. We show by systematic biochem. characterizations that silver ions (Ag+) bind EcMDH at multiple sites including three cysteine-contg. sites. By X-ray crystallog., we unravel the binding preference of Ag+ to multiple binding sites in EcMDH, i.e., Cys113 > Cys251 > Cys109 > Met227. Silver exhibits preferences to the donor atoms and residues in the order of S > N > O and Cys > Met > His > Lys > Val, resp., in EcMDH. For the first time, we report the coordination of silver to a lysine in proteins. Besides, we also obsd. argentophilic interactions (AgU+00B7U+00B7U+00B7Ag, 2.7 to 3.3 U+00C5) between two silver ions coordinating to one thiolate. Combined with site-directed mutagenesis and an enzymic activity test, we unveil that the binding of Ag+ to the site IV (His177-Ag-Met227 site) plays a vital role in Ag+mediated MDH inactivation. This work stands as the first unusual and explicit study of silver binding preference to multiple binding sites in its authentic protein target at the at. resoln. These findings enrich our knowledge on the biocoordination chem. of silver(I), which in turn facilitates the prediction of the unknown silver-binding proteins and extends the pharmaceutical potentials of metal-based drugs.
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    Veronesi, G.; Gallon, T.; Deniaud, A.; Boff, B.; Gateau, C.; Lebrun, C.; Vidaud, C.; Rollin-Genetet, F.; Carrière, M.; Kieffer, I.; Mintz, E.; Delangle, P.; Michaud-Soret, I. XAS investigation of silver(I) coordination in copper(I) biological binding sites. Inorg. Chem. 2015, 54, 11688– 11696,  DOI: 10.1021/acs.inorgchem.5b01658
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    26
    XAS Investigation of Silver(I) Coordination in Copper(I) Biological Binding Sites
    Veronesi, Giulia; Gallon, Thomas; Deniaud, Aurelien; Boff, Bastien; Gateau, Christelle; Lebrun, Colette; Vidaud, Claude; Rollin-Genetet, Francoise; Carriere, Marie; Kieffer, Isabelle; Mintz, Elisabeth; Delangle, Pascale; Michaud-Soret, Isabelle
    Inorganic Chemistry (2015), 54 (24), 11688-11696CODEN: INOCAJ; ISSN:0020-1669. (American Chemical Society)
    Silver(I) is an unphysiol. ion that, as the physiol. copper(I) ion, shows high binding affinity for thiolate ligands; its toxicity has been proposed to be due to its capability to replace Cu(I) in the thiolate binding sites of proteins involved in copper homeostasis. Nevertheless, the nature of the Ag(I)-thiolate complexes formed within cells is poorly understood, and the details of Ag(I) coordination in such complexes in physiol. relevant conditions are mostly unknown. By making use of X-ray absorption spectroscopy (XAS), we characterized the Ag(I) binding sites in proteins related to copper homeostasis, such as the chaperone Atox1 and metallothioneins (MTs), as well as in bioinspired thiolate Cu(I) chelators mimicking these proteins, in soln. and at physiol. pH. Different Ag(I) coordination environments were revealed: the Ag-S bond length was found to correlate to the Ag(I) coordination no., with characteristic values of 2.40 and 2.49 Å in AgS2 and AgS3 sites, resp., comparable to the values reported for cryst. Ag(I)-thiolate compds. The bioinspired Cu(I) chelator L1 is proven to promote the unusual trigonal AgS3 coordination and, therefore, can serve as a ref. compd. for this environment. In the Cu(I)-chaperone Atox1, Ag(I) binds in digonal coordination to the two Cys residues of the Cu(I) binding loop, with the AgS2 characteristic bond length of 2.40 ± 0.01 Å. In the multinuclear Ag(I) clusters of rabbit and yeast metallothionein, the av. Ag-S bond lengths are 2.48 ± 0.01 Å and 2.47 ± 0.01 Å, resp., both indicative of the predominance of trigonal AgS3 sites. This work lends insight into the coordination chem. of silver in its most probable intracellular targets and might help in elucidating the mechanistic aspects of Ag(I) toxicity.
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    Changela, A.; Chen, K.; Xue, Y.; Holschen, J.; Outten, C. E.; O’Halloran, T. V.; Mondragón, A. Molecular basis of metal-ion selectivity and zeptomolar sensitivity by CueR. Science 2003, 301, 1383– 1387,  DOI: 10.1126/science.1085950
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    Molecular Basis of Metal-Ion Selectivity and Zeptomolar Sensitivity by CueR
    Changela, Anita; Chen, Kui; Xue, Yi; Holschen, Jackie; Outten, Caryn E.; O'Halloran, Thomas V.; Mondragon, Alfonso
    Science (Washington, DC, United States) (2003), 301 (5638), 1383-1387CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    The earliest of a series of copper efflux genes in Escherichia coli are controlled by CueR, a member of the MerR family of transcriptional activators. Thermodn. calibration of CueR reveals a zeptomolar (10-21 molar) sensitivity to free Cu+, which is far less than one atom per cell. At. details of this extraordinary sensitivity and selectivity for +1 transition-metal ions are revealed by comparing the crystal structures of CueR and a Zn2+-sensing homolog, ZntR. An unusual buried metal-receptor site in CueR restricts the metal to a linear, two-coordinate geometry and uses helix-dipole and hydrogen-bonding interactions to enhance metal binding. This binding mode is rare among metalloproteins but well suited for an ultrasensitive genetic switch.
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    Meury, M.; Knop, M.; Seebeck, F. P. Structural basis for copper–oxygen mediated C–H bond activation by the formylglycine-generating enzyme. Angew. Chem., Int. Ed. 2017, 56, 8115– 8119,  DOI: 10.1002/anie.201702901
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    Structural Basis for Copper-Oxygen Mediated C-H Bond Activation by the Formylglycine-Generating Enzyme
    Meury, Marcel; Knop, Matthias; Seebeck, Florian P.
    Angewandte Chemie, International Edition (2017), 56 (28), 8115-8119CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The formylglycine-generating enzyme (FGE) is a unique copper protein that catalyzes oxygen-dependent C-H activation. We describe 1.66 Å- and 1.28 Å-resoln. crystal structures of FGE from Thermomonospora curvata in complex with either AgI or CdII providing definitive evidence for a high-affinity metal-binding site in this enzyme. The structures reveal a bis-cysteine linear coordination of the monovalent metal, and tetrahedral coordination of the bivalent metal. Similar coordination changes may occur in the active enzyme as a result of CuI/II redox cycling. Complexation of copper atoms by two cysteine residues is common among copper-trafficking proteins, but is unprecedented for redox-active copper enzymes or synthetic copper catalysts.
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    Leisinger, F.; Miarzlou, D. A.; Seebeck, F. P. Non-coordinative binding of O2 at the active center of a copper-dependent Enzyme. Angew. Chem., Int. Ed. 2021, 60, 6154– 6159,  DOI: 10.1002/anie.202014981
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    29
    Non-Coordinative Binding of O2 at the Active Center of a Copper-Dependent Enzyme
    Leisinger, Florian; Miarzlou, Dzmitry A.; Seebeck, Florian P.
    Angewandte Chemie, International Edition (2021), 60 (11), 6154-6159CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Mol. oxygen (O2) is a sustainable oxidn. reagent. O2 is strongly oxidizing but kinetically stable and its final reaction product is water. For these reasons learning how to activate O2 and how to steer its reactivity along desired reaction pathways is a longstanding challenge in chem. research. Activation of ground-state diradical O2 can occur either via conversion to singlet oxygen or by one-electron redn. to superoxide. Many enzymes facilitate activation of O2 by direct formation of a metal-oxygen coordination complex concomitant with inner sphere electron transfer. The formylglycine generating enzyme (FGE) is an unusual mononuclear copper enzyme that appears to follow a different strategy. Atomic-resoln. crystal structures of the precatalytic complex of FGE demonstrate that this enzyme binds O2 juxtaposed, but not coordinated to the catalytic CuI. Isostructural complexes that contain AgI instead of CuI or nitric oxide instead of O2 confirm that formation of the initial oxygenated complex of FGE does not depend on redox activity. A stepwise mechanism that decouples binding and activation of O2 is unprecedented for metal-dependent oxidases, but is reminiscent of flavin-dependent enzymes.
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    Bilinovich, S. M.; Morris, D. L.; Prokop, J. W.; Caporoso, J. A.; Taraboletti, A.; Duangjumpa, N.; Panzner, M. J.; Shriver, L. P.; Leeper, T. C. Silver binding to bacterial glutaredoxins observed by NMR. Biophysica 2021, 1, 359– 376,  DOI: 10.3390/biophysica1040027
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    Peterson, C. W.; Narula, S. S.; Armitage, I. M. 3D Solution structure of copper and silver-substituted yeast metallothioneins. FEBS Lett. 1996, 379, 85– 93,  DOI: 10.1016/0014-5793(95)01492-6
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    3D solution structure of copper and silver-substituted yeast metallothioneins
    Peterson, Cynthia W.; Narula, Surinder S.; Armitage, Ian M.
    FEBS Letters (1996), 379 (1), 85-93CODEN: FEBLAL; ISSN:0014-5793. (Elsevier)
    3D soln. structural calcns. for yeast silver(I)-substituted metallothionein (MT) and native copper(I) MT were completed using exptl. detd. NOE and dihedral angle constraints, in conjunction with exptl. derived metal-to-Cys connectivities for AgMT which were assumed identical for CuMT. For the first 40 residues in both structures, the polypeptide backbone wraps around the metal cluster in two large parallel loops sepd. by a deep cleft contg. the metal cluster. Minor differences between the two structures include differences in hydrogen bonds and the orientation of the N-terminus with the overall protein vol. conserved to within 6.5%.
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    Wang, X.; Han, Z.-C.; Wei, W.; Hu, H.; Li, P.; Sun, P.; Liu, X.; Lv, Z.; Wang, F.; Cao, Y.; Guo, Z.; Li, J.; Zhao, J. An unexpected all-metal aromatic tetranuclear silver cluster in human copper chaperone Atox1. Chem. Sci. 2022, 13, 7269– 7275,  DOI: 10.1039/d1sc07122j
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    An unexpected all-metal aromatic tetranuclear silver cluster in human copper chaperone Atox1
    Wang, Xiuxiu; Han, Zong-Chang; Wei, Wei; Hu, Hanshi; Li, Pengfei; Sun, Peiqing; Liu, Xiangzhi; Lv, Zhijia; Wang, Feng; Cao, Yi; Guo, Zijian; Li, Jun; Zhao, Jing
    Chemical Science (2022), 13 (24), 7269-7275CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
    Metal clusters, such as iron-sulfur clusters, play key roles in sustaining life and are intimately involved in the functions of metalloproteins. Herein we report the formation and crystal structure of a planar square tetranuclear silver cluster when silver ions were mixed with human copper chaperone Atox1. Quantum chem. studies reveal that two Ag 5s1 electrons in the tetranuclear silver cluster fully occupy the one bonding MO, with the assumption that this Ag4 cluster is Ag42+, leading to extensive electron delocalization over the planar square and significant stabilization. This bonding pattern of the tetranuclear silver cluster represents an arom. all-metal structure that follows a 4n + 2 electron counting rule (n = 0). This is the first time an all-metal arom. silver cluster was obsd. in a protein.
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    Mazzei, L.; Cianci, M.; Gonzalez Vara, A.; Ciurli, S. The structure of urease inactivated by Ag(I): a new paradigm for enzyme inhibition by heavy metals. Dalton Trans. 2018, 47, 8240– 8247,  DOI: 10.1039/c8dt01190g
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    The structure of urease inactivated by Ag(I): a new paradigm for enzyme inhibition by heavy metals
    Mazzei, Luca; Cianci, Michele; Gonzalez Vara, Antonio; Ciurli, Stefano
    Dalton Transactions (2018), 47 (25), 8240-8247CODEN: DTARAF; ISSN:1477-9226. (Royal Society of Chemistry)
    The nickel-dependent enzyme urease is a virulence factor for a large no. of human pathogens, as well as a neg. element for the efficiency of soil nitrogen fertilization for crop prodn. The use of urease inhibitors to contrast these effects requires the knowledge, at the mol. level, of their mode of action. Among these, silver is an efficient antimicrobial agent and an established inhibitor of this enzyme. The 1.91 Å resoln. structure of Sporosarcina pasteurii urease inhibited by silver reveals the presence of two Ag(I) ions bound to the largely conserved triad αCys322/αHis323/αMet367: the first two residues are located on the mobile flap that is essential in modulating the size of the active site cavity and the position of key residues for enzyme catalysis, while αMet367 is on a loop facing the flap at the entrance of the active site cavity. The two Ag(I) ions are bridged by the thiolate Sγ atom of αCys322, and are coordinated, resp., to the Nδ1 atom of the αHis323 imidazole ring and to the Sδ of αMet367. The binding of the Ag(I) ions at the edge of the active site channel supposedly blocks the movement of the flap, inhibiting the catalytic activity of urease. The structure of the silver-inhibited urease allows us to understand and rationalize all previously acquired kinetic and calorimetric data on this phenomenon, but also provides the details of how silver can exert its antimicrobial action with respect to ureolytic bacteria, a step forward against antibiotic-resistant pathogens.
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    Panzner, M. J.; Bilinovich, S. M.; Parker, J. A.; Bladholm, E. L.; Ziegler, C. J.; Berry, S. M.; Leeper, T. C. Isomorphic deactivation of a Pseudomonas aeruginosa oxidoreductase: The crystal structure of Ag(I) metallated azurin at 1.7 Å. J. Inorg. Biochem. 2013, 128, 11– 16,  DOI: 10.1016/j.jinorgbio.2013.07.011
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    Isomorphic deactivation of a Pseudomonas aeruginosa oxidoreductase: The crystal structure of Ag(I) metallated azurin at 1.7 Å
    Panzner, Matthew J.; Bilinovich, Stephanie M.; Parker, Jillian A.; Bladholm, Erika L.; Ziegler, Christopher J.; Berry, Steven M.; Leeper, Thomas C.
    Journal of Inorganic Biochemistry (2013), 128 (), 11-16CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier)
    Multiple biophys. methods demonstrate that Ag(I) effectively metalates P. aeruginosa apo-azurin in soln. X-ray crystallog. of the Ag(I)-modified protein revealed that Ag(I) bound to azurin at the traditional Cu-mediated active site with nearly identical geometry. Cyclic voltammetry indicated that the Ag(I) adduct was redox inert. The results suggested that a potential mechanism for the microbial toxicity of Ag(I) is the deactivation of Cu-contg. oxidoreductases by the effective binding and structural mimicry by Ag(I) without the corresponding function.
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    Liu, W.; Worms, I. A. M.; Herlin-Boime, N.; Truffier-Boutry, D.; Michaud-Soret, I.; Mintz, E.; Vidaud, C.; Rollin-Genetet, F. Interaction of silver nanoparticles with metallothionein and ceruloplasmin: impact on metal substitution by Ag(I), corona formation and enzymatic activity. Nanoscale 2017, 9, 6581– 6594,  DOI: 10.1039/c7nr01075c
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    Interaction of silver nanoparticles with metallothionein and ceruloplasmin: impact on metal substitution by Ag(I), corona formation and enzymatic activity
    Liu, Wei; Worms, Isabelle A. M.; Herlin-Boime, Nathalie; Truffier-Boutry, Delphine; Michaud-Soret, Isabelle; Mintz, Elisabeth; Vidaud, Claude; Rollin-Genetet, Francoise
    Nanoscale (2017), 9 (19), 6581-6594CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)
    The release of Ag(I) from silver nanoparticles (AgNPs) unintentionally spread in the environment is suspected to impair some key biol. functions. In comparison with AgNO3, in-depth investigations were carried out into the interactions between citrate-coated AgNPs (20 nm) and two metalloproteins, intracellular metallothionein 1 (MT1) and plasmatic ceruloplasmin (Cp), both involved in metal homeostasis. These were chosen for their physiol. relevance and the diversity of their various native metals bound because of thiol groups and/or their structural differences. Transmission electron microscopy (TEM), and dynamic light scattering (DLS), UV-vis and CD (CD) spectroscopies were used to study the effects of such intricate interactions on AgNP dissoln. and proteins in terms of metal exchanges and structural modifications. The isolation of the different populations formed together with online quantifications of their metal content were performed by asym. flow field-flow fractionation (AF4) linked to inductively coupled plasma mass spectrometry (ICP-MS). For the 2 proteins, Ag(I) dissolved from the AgNPs, substituted for the native metal, to different extents and with different types of dynamics for the corona formed: the MT1 rapidly surrounded the AgNPs with the transient reticulate corona thus promoting their dissoln. assocd. with the metal substitution, whereas the Cp established a more stable layer around the AgNPs, with a limited substitution of Cu and a decrease in its ferroxidase activity. The accessibility and lability of the metal binding sites inside these proteins and their relative affinities for Ag(I) are discussed, taking into account the structural characteristics of the proteins.
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    Kluska, K.; Peris-Díaz, M. D.; Płonka, D.; Moysa, A.; Dadlez, M.; Deniaud, A.; Bal, W.; Krężel, A. Formation of highly stable multinuclear AgnSn clusters in zinc fingers disrupts their structure and function. Chem. Commun. 2020, 56, 1329– 1332,  DOI: 10.1039/c9cc09418k
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    Formation of highly stable multinuclear AgnSn clusters in zinc fingers disrupts their structure and function
    Kluska, Katarzyna; Peris-Diaz, Manuel D.; Plonka, Dawid; Moysa, Alexander; Dadlez, Michal; Deniaud, Aurelien; Bal, Wojciech; Krezel, Artur
    Chemical Communications (Cambridge, United Kingdom) (2020), 56 (9), 1329-1332CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    Silver (Ag(I)) binding to consensus zinc fingers (ZFs) causes Zn(II) release inducing a gradual disruption of the hydrophobic core, followed by an overall conformational change and formation of highly stable AgnSn clusters. A compact eight-membered Ag4S4 structure formed by a CCCC ZF is the first cluster example reported for a single biol. mol. Ag(I)-induced conformational changes of ZFs can, as a consequence, affect transcriptional regulation and other cellular processes.
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    Kluska, K.; Veronesi, G.; Deniaud, A.; Hajdu, B.; Gyurcsik, B.; Bal, W.; Krężel, A. Structures of silver fingers and a pathway to their genotoxicity. Angew. Chem., Int. Ed. 2022, 61, e202116621  DOI: 10.1002/anie.202116621
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    Structures of Silver Fingers and a Pathway to Their Genotoxicity
    Kluska, Katarzyna; Veronesi, Giulia; Deniaud, Aurelien; Hajdu, Balint; Gyurcsik, Bela; Bal, Wojciech; Krezel, Artur
    Angewandte Chemie, International Edition (2022), 61 (12), e202116621CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    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 biol. function of ZF proteins is a likely consequence of such replacement. Here, we report an X-ray absorption spectroscopy (XAS) study of AgnSn 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 av. Ag-S distance at 2.41 S. 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 Ag2S2 clusters occurred in a three-ZF transcription factor protein 1MEY#, leading to a dissocn. of 1MEY# from the complex with its cognate DNA.
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    Kluska, K.; Adamczyk, J.; Krężel, A. Metal binding properties, stability and reactivity of zinc fingers. Coord. Chem. Rev. 2018, 367, 18– 64,  DOI: 10.1016/j.ccr.2018.04.009
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    Metal binding properties, stability and reactivity of zinc fingers
    Kluska, Katarzyna; Adamczyk, Justyna; Krezel, Artur
    Coordination Chemistry Reviews (2018), 367 (), 18-64CODEN: CCHRAM; ISSN:0010-8545. (Elsevier B.V.)
    A review. Zinc fingers (ZFs) are among the most structurally diverse protein domains. They interact with nucleic acids, other proteins and lipids to facilitate a multitude of biol. processes. Currently, there are more than 10 known classes of ZFs, with various architectures, metal binding modes, functions and reactivity. The versatility, selectivity and stability of these short amino acid sequences is achieved mainly by (i) residues participating in Zn(II) coordination (mostly Cys and His), (ii) hydrophobic core and ZF structure formation, and (iii) variable residues responsible for inter- and intramol. interactions. Since their discovery, ZFs have been extensively studied in terms of their structure, stability and recognition targets by the application of various methodologies. Studies based on interactions with other metal ions and their complexes have contributed to the understanding of their chem. properties and the discovery of new types of ZF complexes, such as gold fingers or lead fingers. Moreover, due to the presence of nucleophilic thiolates, ZFs are targets for reactive oxygen and nitrogen species as well as alkylating agents. Interactions with many reactive mols. lead to disturb the native Zn(II) coordination site which further result in structural and functional damage of the ZFs. The post-translational modifications including phosphorylation, acetylation, methylation or nitrosylation frequently affect ZFs function via changes in the protein structure and dynamics. Even though the literature is replete with structural and stability data regarding classical (ββα) ZFs, there is still a huge gap in the knowledge on physicochem. properties and reactivity of other ZF types. In this review, metal binding properties of ZFs and stability factors that modulate their functions are reviewed. These include interactions of ZFs with biogenic and toxic metal ions as well as damage occurring upon reaction with reactive oxygen and nitrogen species, the methodol. used for ZFs characterization, and aspects related to coordination chem.
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    Padjasek, M.; Kocyła, A.; Kluska, K.; Kerber, O.; Tran, J. B.; Krężel, A. Structural zinc binding sites shaped for greater works: Structure-function relations in classical zinc finger, hook and clasp domains. J. Inorg. Biochem. 2020, 204, 110955  DOI: 10.1016/j.jinorgbio.2019.110955
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    Structural zinc binding sites shaped for greater works: Structure-function relations in classical zinc finger, hook and clasp domains
    Padjasek, Michal; Kocyla, Anna; Kluska, Katarzyna; Kerber, Olga; Tran, Jozef Ba; Krezel, Artur
    Journal of Inorganic Biochemistry (2020), 204 (), 110955CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier)
    A review. Metal ions are essential elements present in biol. systems able to facilitate many cellular processes including proliferation, signaling, DNA synthesis and repair. Zinc ion (Zn(II)) is an important cofactor for numerous biochem. reactions. Commonly, structural zinc sites demonstrate high Zn(II) affinity and compact architecture required for sequence-specific macromol. binding. However, how Zn(II)-dependent proteins fold, how their dissocn. occurs, and which factors modulate zinc protein affinity as well as stability remains not fully understood. The mol. rules governing precise regulation of zinc proteins function are hidden in the relationship between sequence and structure, and hence require deep understanding of their folding mechanism under metal load, reactivity and metal-to-protein affinity. Even though, this sequence-structure relationship has an impact on zinc proteins function, it has been shown that other biol. factors including cellular localization and Zn(II) availability influence overall protein behavior. Taking into account all of the mentioned factors, in this review, we aim to describe the relationship between structure-function-stability of zinc structural sites, found in a zinc finger, zinc hook and zinc clasps, and reach far beyond a structural point of view in order to appreciate the balance between chem. and biol. that govern the protein world.
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    Kocyła, A.; Tran, J. B.; Krężel, A. Galvanization of protein–protein interactions in a dynamic zinc interactome. Trends Biochem. Sci. 2021, 46, 64– 79,  DOI: 10.1016/j.tibs.2020.08.011
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    Galvanization of Protein-Protein Interactions in a Dynamic Zinc Interactome
    Kocyla, Anna; Tran, Jozef Ba; Krezel, Artur
    Trends in Biochemical Sciences (2021), 46 (1), 64-79CODEN: TBSCDB; ISSN:0968-0004. (Elsevier Ltd.)
    A review The presence of Zn2+ at protein-protein interfaces modulates complex function, stability, and introduces structural flexibility/complexity, chem. 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 dissoc. and how their stability and reactivity are driven in a zinc interactome remain poorly understood, mostly due to exptl. 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 exptl. challenges required for the identification and investigation of ZPPIs.
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    Tran, J. B.; Krężel, A. InterMetalDB: A database and browser of intermolecular metal binding sites in macromolecules with structural information. J. Proteome Res. 2021, 20, 1889– 1901,  DOI: 10.1021/acs.jproteome.0c00906
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    41
    InterMetalDB: A Database and Browser of Intermolecular Metal Binding Sites in Macromolecules with Structural Information
    Tran, Jozef Ba; Krezel, Artur
    Journal of Proteome Research (2021), 20 (4), 1889-1901CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
    InterMetalDB is a free-of-charge database and browser of intermol. metal binding sites that are present on the interfaces of macromols. 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 resoln., structure description, and date of deposition. Records can be filtered by coordinated metal ion, no. 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.
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    Stracker, T. H.; Petrini, J. H. J. The MRE11 complex: starting from the ends. Nat. Rev. Mol. Cell Biol. 2011, 12, 90– 103,  DOI: 10.1038/nrm3047
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    The MRE11 complex: Starting from the ends
    Stracker, Travis H.; Petrini, John H. J.
    Nature Reviews Molecular Cell Biology (2011), 12 (2), 90-103CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)
    A review. The maintenance of genome stability depends on the DNA damage response (DDR), which is a functional network comprising signal transduction, cell cycle regulation and DNA repair. The metab. of DNA double-strand breaks governed by the DDR is important for preventing genomic alterations and sporadic cancers, and hereditary defects in this response cause debilitating human pathologies, including developmental defects and cancer. The MRE11 complex, composed of the meiotic recombination 11 (MRE11), RAD50 and Nijmegen breakage syndrome 1 (NBS1; also known as nibrin) proteins is central to the DDR, and recent insights into its structure and function were gained from in vitro structural anal. and studies of animal models in which the DDR response is deficient.
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    Hopfner, K.-P.; Craig, L.; Moncalian, G.; Zinkel, R. A.; Usui, T.; Owen, B. A. L.; Karcher, A.; Henderson, B.; Bodmer, J.-L.; McMurray, C. T.; Carney, J. P.; Petrini, J. H. J.; Tainer, J. A. The Rad50 zinc-hook is a structure joining Mre11 complexes in DNA recombination and repair. Nature 2002, 418, 562– 566,  DOI: 10.1038/nature00922
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    The Rad50 zinc-hook is a structure joining Mre11 complexes in DNA recombination and repair
    Hopfner, Karl-Peter; Craig, Lisa; Moncalian, Gabriel; Zinkel, Robert A.; Usui, Takehiko; Owen, Barbara A. L.; Karcher, Annette; Henderson, Brendan; Bodmer, Jean-Luc; McMurray, Cynthia T.; Carney, James P.; Petrini, John H. J.; Tainer, John A.
    Nature (London, United Kingdom) (2002), 418 (6897), 562-566CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    The Mre11 complex (Mre11-Rad50-Nbs1) is central to chromosomal maintenance and functions in homologous recombination, telomere maintenance and sister chromatid assocn. These functions all imply that the linked binding of two DNA substrates occurs, although the mol. basis for this process remains unknown. Here we present a 2.2 Å crystal structure of the Rad50 coiled-coil region that reveals an unexpected dimer interface at the apex of the coiled coils in which pairs of conserved Cys-X-X-Cys motifs form interlocking hooks that bind one Zn2+ ion. Biochem., X-ray and electron microscopy data indicate that these hooks can join oppositely protruding Rad50 coiled-coil domains to form a flexible bridge of up to 1,200 Å. This suggests a function for the long insertion in the Rad50 ABC-ATPase domain. The Rad50 hook is functional, because mutations in this motif confer radiation sensitivity in yeast and disrupt binding at the distant Mre11 nuclease interface. These data support an architectural role for the Rad50 coiled coils in forming metal-mediated bridging complexes between two DNA-binding heads. The resulting assemblies have appropriate lengths and conformational properties to link sister chromatids in homologous recombination and DNA ends in non-homologous end-joining.
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    Park, Y. B.; Hohl, M.; Padjasek, M.; Jeong, E.; Jin, K. S.; Krężel, A.; Petrini, J. H. J.; Cho, Y. Eukaryotic Rad50 functions as a rod-shaped dimer. Nat. Struct. Mol. Biol. 2017, 24, 248– 257,  DOI: 10.1038/nsmb.3369
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    Eukaryotic Rad50 functions as a rod-shaped dimer
    Park, Young Bong; Hohl, Marcel; Padjasek, Michal; Jeong, Eunyoung; Jin, Kyeong Sik; Krezel, Artur; Petrini, John H. J.; Cho, Yunje
    Nature Structural & Molecular Biology (2017), 24 (3), 248-257CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)
    The Rad50 hook interface is crucial for assembly and various functions of the Mre11 complex. Previous analyses suggested that Rad50 mols. interact within (intracomplex) or between (intercomplex) dimeric complexes. In this study, we detd. the structure of the human Rad50 hook and coiled-coil domains. The data suggest that the predominant structure is the intracomplex, in which the two parallel coiled coils proximal to the hook form a rod shape, and that a novel interface within the coiled-coil domains of Rad50 stabilizes the interaction of Rad50 protomers in the dimeric assembly. In yeast, removal of the coiled-coil interface compromised Tel1 activation without affecting DNA repair, while simultaneous disruption of that interface and the hook phenocopied a null mutation. The results demonstrate that the hook and coiled-coil interfaces coordinately promote intracomplex assembly and define the intracomplex as the functional form of the Mre11 complex.
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    Kochańczyk, T.; Jakimowicz, P.; Krężel, A. Femtomolar Zn(II) affinity of minimal zinc hook peptides – a promising small tag for protein engineering. Chem. Commun. 2013, 49, 1312– 1314,  DOI: 10.1039/c2cc38174e
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    Femtomolar Zn(II) affinity of minimal zinc hook peptides - a promising small tag for protein engineering
    Kochanczyk, Tomasz; Jakimowicz, Piotr; Krezel, Artur
    Chemical Communications (Cambridge, United Kingdom) (2013), 49 (13), 1312-1314CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    The minimal zinc hook peptide of Rad50 and its alanine mutants form highly stable Zn(II) complexes. These peptides were successfully used as a small, efficient tag for reversible Zn(II)-mediated protein homodimerization. The high stability, its biol. consequences and potential applications in protein engineering are discussed.
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    Kochańczyk, T.; Nowakowski, M.; Wojewska, D.; Kocyła, A.; Ejchart, A.; Koźmiński, W.; Krężel, A. Metal-coupled folding as the driving force for the extreme stability of Rad50 zinc hook dimer assembly. Sci. Rep. 2016, 6, 36346  DOI: 10.1038/srep36346
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    Metal-coupled folding as the driving force for the extreme stability of Rad50 zinc hook dimer assembly
    Kochanczyk, Tomasz; Nowakowski, Michal; Wojewska, Dominika; Kocyla, Anna; Ejchart, Andrzej; Kozminski, Wiktor; Krezel, Artur
    Scientific Reports (2016), 6 (), 36346CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
    The binding of metal ions at the interface of protein complexes presents a unique and poorly understood mechanism of mol. 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 anal. of the structural and thermodn. 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 const. 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 crit. for the tight assocn. of protein subunits.
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    Padjasek, M.; Maciejczyk, M.; Nowakowski, M.; Kerber, O.; Pyrka, M.; Koźmiński, W.; Krężel, A. Metal exchange in the interprotein Zn(II)-binding site of the Rad50 hook domain: Structural insights into Cd(II)-induced DNA-repair inhibition. Chem. – Eur. J. 2020, 26, 3297– 3313,  DOI: 10.1002/chem.201904942
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    Metal exchange in the interprotein ZnII-binding site of the Rad50 hook domain: Structural insights into CdII-induced DNA-repair inhibition
    Padjasek, Michal; Maciejczyk, Maciej; Nowakowski, Michal; Kerber, Olga; Pyrka, Maciej; Kozminski, Wiktor; Krezel, Artur
    Chemistry - A European Journal (2020), 26 (15), 3297-3313CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
    CdII is a major genotoxic agent that readily displaces ZnII in a multitude of zinc proteins, abrogates redox homeostasis, and deregulates cellular metalloproteome. To date, this displacement has been described mostly for cysteine(Cys)-rich intraprotein binding sites in certain zinc finger domains and metallothioneins. To visualize how a ZnII-to-CdII swap can affect the target protein's status and thus understand the mol. basis of CdII-induced genotoxicity an intermol. ZnII-binding site from the crucial DNA repair protein Rad50 and its zinc hook domain were examd. By using a length-varied peptide base, ZnII-to-CdII displacement in Rad50's hook domain is demonstrated to alter it in a bimodal fashion: (1) CdII induces around a two-orders-of-magnitude stabilization effect (log K12ZnII=20.8 vs. log K12CdII=22.7), which defines an extremely high affinity of a peptide towards a metal ion, and (2) the displacement disrupts the overall assembly of the domain, as shown by NMR spectroscopic and anisotropy decay data. Based on the results, a new model explaining the mol. mechanism of CdII genotoxicity that underlines CdII's impact on Rad50's dimer stability and quaternary structure that could potentially result in abrogation of the major DNA damage response pathway is proposed.
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    Łuczkowski, M.; Padjasek, M.; Tran, J.; Hemmingsen, L.; Kerber, O.; Habjanič, J.; Freisinger, E.; Krężel, A. An extremely stable interprotein tetrahedral Hg(Cys)4 core formed in the zinc hook domain of Rad50 protein at physiological pH. Chem. – Eur. J. 2022, 28, e202202738  DOI: 10.1002/chem.202202738
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    An Extremely Stable Interprotein Tetrahedral Hg(Cys)4 Core Forms in the Zinc Hook Domain of Rad50 Protein at Physiological pH
    Luczkowski, Marek; Padjasek, Michal; Ba Tran, Jozef; Hemmingsen, Lars; Kerber, Olga; Habjanic, Jelena; Freisinger, Eva; Krezel, Artur
    Chemistry - A European Journal (2022), 28 (66), e202202738CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
    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 mercurys 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 obsd. in soln. at physiol. 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 mols. by coordinating ZnII. Dimerized motifs form a unique interprotein zinc hook domain (Hk) that is crit. for the biol. 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 199Hg 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 physiol. pH; this is the first example of an interprotein mercury site displaying tetrahedral geometry in soln. 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 const. of the Rad50 Hg(Hk)2 complex is approx. three orders of magnitude higher than those of the strongest HgII complexes known to date.
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    Fields, G. B.; Noble, R. L. Solid phase peptide synthesis utilizing 9 -fluorenylmethoxycarbonyl amino acids. Int. J. Pept. Protein Res. 2009, 35, 161– 214,  DOI: 10.1111/j.1399-3011.1990.tb00939.x
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    Kocyła, A.; Pomorski, A.; Krężel, A. Molar absorption coefficients and stability constants of metal complexes of 4-(2-pyridylazo) resorcinol (PAR): Revisiting common chelating probe for the study of metalloproteins. J. Inorg. Biochem. 2015, 152, 82– 92,  DOI: 10.1016/j.jinorgbio.2015.08.024
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    Molar absorption coefficients and stability constants of metal complexes of 4-(2-pyridylazo)resorcinol (PAR): Revisiting common chelating probe for the study of metalloproteins
    Kocyla, Anna; Pomorski, Adam; Krezel, Artur
    Journal of Inorganic Biochemistry (2015), 152 (), 82-92CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier)
    4-(2-Pyridylazo)resorcinol (PAR) is one of the most popular chromogenic chelators used in the detn. of the concns. of various metal ions from the d, p and f blocks and their affinities for metal ion-binding biomols. The most important characteristics of such a sensor are the molar absorption coeff. and the metal-ligand complex dissocn. const. However, it must be remembered that these values are dependent on the specific exptl. conditions (e.g. pH, solvent components, and reactant ratios). If one uses these values to process data obtained in different conditions, the final result can be under- or overestimated. We aimed to establish the spectral properties and the stability of PAR and its complexes accurately with Zn2+, Cd2+, Hg2+, Co2+, Ni2+, Cu2+, Mn2+ and Pb2+ at a multiple pH values. The obtained results account for the presence of different species of metal-PAR complexes in the physiol. pH range of 5 to 8 and have been frequently neglected in previous studies. The effective molar absorption coeff. at 492 nm for the ZnHx(PAR)2 complex at pH 7.4 in buffered water soln. is 71,500 M-1 cm- 1, and the dissocn. const. of the complex in these conditions is 7.08 × 10-13 M2. To confirm these values and est. the range of the dissocn. consts. of zinc-binding biomols. that can be measured using PAR, we performed several titrns. of zinc finger peptides (ZF133-11 C@e, TC motif, and MTFI-1) and zinc chelators (cyclam and EGTA). Taken together, our results provide the updated parameters that are applicable to any expt. conducted using inexpensive and com. available PAR.
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    Zelazowski, A. J.; Stillman, M. J. Silver binding to rabbit liver zinc metallothionein and zinc α and β fragments. Formation of silver metallothionein with silver(I):protein ratios of 6, 12, and 18 observed using circular dichroism spectroscopy. Inorg. Chem. 1992, 31, 3363– 3370,  DOI: 10.1021/ic00042a008
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    Silver binding to rabbit liver zinc metallothionein and zinc α and β fragments. Formation of silver metallothionein with silver(I):protein ratios of 6, 12, and 18 observed using circular dichroism spectroscopy
    Zelazowski, Andrzej J.; Stillman, Martin J.
    Inorganic Chemistry (1992), 31 (16), 3363-70CODEN: INOCAJ; ISSN:0020-1669.
    Formation of a series of complexes between Ag(I) and the cysteine thiolate groups in rabbit liver zinc metallothionein (MT) and the Zn3-β MT 1 and Zn4-α MT 1 fragments is reported from anal. of the CD spectral data recorded between 5 and 55° during titrns. of the protein with Ag(I). The spectral envelopes reveal formation of Ag12-MT, Ag18-MT, Ag6-α MT, and Ag6-β MT. Silver(I)-thiolate complex formation is assocd. with characteristic CD spectral envelopes and depends on the stoichiometric ratio of Ag:MT, the temp., and the pH. The presence of the tetrahedrally-coordinated Zn(II) in Zn7-MT 2 inhibits formation of the Ag6-MT and Ag12-MT species previously obsd. when Ag+ binds to apo-MT 2 at 20°, and formation of Ag18-MT dominates the spectral traces. At 55°, the Ag12-MT species does form. Addn. of Ag+ at pH 3.8 and 55° to Zn7-MT 2 (nominally apo-MT 2) results in a different sequence of complexes forming in the range Ag+:MT = 1-18. Anal. of the CD spectral data suggests that the low pH enhances formation of Ag6-S9 clusters in the β domain, characterized by a single band at 254 nm, inhibits formation of Ag6-S11 clusters in the α domain, and sats. the binding sites with the formation of Ag18-MT. The CD spectral envelopes obtained as Ag+ was added to solns. of the Zn4-α MT and Zn3-β MT fragments clearly show for the first time spectral signatures assocd. with formation of both Ag6-α MT 1 and Ag6-β MT 1 complexes, resp. The CD spectral characteristics of the Ag6 fragments match the spectral patterns obsd. for Agn-MT (n = 6, 12) formed from Zn7-MT 2. A new species forms at high mole ratios of Ag:MT with Zn4-α MT 1. Tentatively written as Ag12-α MT 1, this complex shows an intense CD spectrum which suggests that its structure may be similar to the supercoil postulated previously for Hg18-MT 2 (Cai, W.; et al., 1988). Metal anal. for titrns. of Zn7-MT 2 at pH 7 shows that each Zn2+ is displaced by 2 Ag+ ions, so that stoichiometric amts. of Zn2+ remain bound to the protein up to the 14 Ag+ point.
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    Journal of the American Society for Mass Spectrometry (2021), 32 (12), 2766-2776CODEN: JAMSEF; ISSN:1879-1123. (American Chemical Society)
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    Journal of the American Chemical Society (2021), 143 (40), 16486-16501CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Mammalian metallothioneins (MTs) are a group of cysteine-rich proteins that bind metal ions in two α- and β-domains and represent a major cellular Zn(II)/Cu(I) buffering system in the cell. At cellular free Zn(II) concns. (10-11-10-9 M), MTs do not exist in fully loaded forms with seven Zn(II)-bound ions (Zn7MTs). Instead, MTs exist as partially metal-depleted species (Zn4-6MT) because their Zn(II) binding affinities are on the nano- to picomolar range comparable to the concns. of cellular Zn(II). The mode of action of MTs remains poorly understood, and thus, the aim of this study is to characterize the mechanism of Zn(II) (un)binding to MTs, the thermodn. properties of the Zn1-6MT2 species, and their mechanostability properties. To this end, native mass spectrometry (MS) and label-free quant. bottom-up and top-down MS in combination with steered mol. dynamics simulations, well-tempered metadynamics (WT-MetaD), and parallel-bias WT-MetaD (amounting to 3.5μs) were integrated to unravel the chem. coordination of Zn(II) in all Zn1-6MT2 species and to explain the differences in binding affinities of Zn(II) ions to MTs. Differences are the result of the degree of water participation in MT (un)folding and the hyper-reactive character of Cys21 and Cys29 residues. The thermodn. properties of Zn(II) (un)binding to MT2 differ from those of Cd(II), justifying their distinctive roles. The potential of this integrated strategy in the study of numerous unexplored metalloproteins is attested by the results highlighted.
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    GSH and L-His are abundant biomols. and likely biol. ligands for Zn(II) under certain conditions. Potentiometric titrns. provide evidence of formation of ternary Zn(II) complexes with GSH and L-His or D-His with slight stereoselectivity in favor of L-His (ca. 1 log unit of stability const.). The soln. structure of the ZnH(GSH)(L-His)(H2O) complex at pH 6.8, detd. by NMR, includes tridentate L-His, monodentate (sulfur) GSH, and weak interligand interactions. Calcns. of competitiveness of this complex for Zn(II) binding at pH 7.4 indicate that it is likely to be formed in vivo under conditions of GSH depletion. Otherwise, GSH alone emerges as a likely Zn(I) carrier.
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  • Abstract

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    Figure 1

    Figure 1. Crystal structure representation of the P. furiosus Rad50 coiled-coil zinc hook dimer (396–498) (PDB: 1L8D) (43) with marked hook domain model fragments (45−48) used in this study: Hk14 (440–453, violet) and Hk45 (426–470, green + violet).

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    Figure 2

    Figure 2. Spectrophotometric- (A, B) and CD- (C, D) monitored AgNO3 titrations of 25 μM Hk14 (left) and Hk45 (right) peptides in 20 mM TES, 100 mM NaF, pH 7.4. Black, red, cyan, green, and violet lines indicate 0, 1.0, 1.5, 2.0, and 3.0 Ag(I) mol equiv, respectively. Asterisk refers to signal changes presented in the inset of Figure 5C. The corresponding titration curves are presented in Figure 3.

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    Figure 3

    Figure 3. Spectrophotometric- (A, B) and CD- (C–F) monitored AgNO3 titrations of 25 μM Hk14 (left) and Hk45 (right) peptides in 20 mM TES, 100 mM NaF, pH 7.4 within 0–4.5 Ag(I) mol equiv. The corresponding absorbance and CD spectra are presented in Figure 2. The wavelengths in all graphs correspond to the region, where the signal change is most pronounced, thus providing the best signal-to-noise ratio.

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    Figure 4

    Figure 4. Spectrophotometric- (A, B) and CD- (C, D) monitored AgNO3 titrations of 25 μM Zn(Hk14)2 (left) and Zn(Hk45)2 (right) peptides in 20 mM TES, 100 mM NaF, pH 7.4. Black, blue, red, cyan, green, and violet lines indicate apo-Hk, Zn(Hk)2, 1.0, 1.5, 2.0, and 3.0 Ag(I) mol equiv, respectively. Asterisk refers to signal changes presented in the inset. The corresponding titration curves are presented in Figure 5.

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    Figure 5

    Figure 5. Spectrophotometric- (A, B) and CD- (C–F) monitored AgNO3 titrations of 25 μM Zn(Hk14)2 (left) and Zn(Hk45)2 (right) peptides in 20 mM TES, 100 mM NaF, pH 7.4 within 0–4.5 Ag(I) mol equiv. The x-axis represents the molar ratio of AgNO3 to the Hk monomer being in the Zn(II) complex. The corresponding absorbance and CD spectra are presented in Figure 4. The wavelengths in all graphs correspond to the region, where the signal change is most pronounced, thus providing the best signal-to-noise ratio.

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    Figure 6

    Figure 6. Zn(II) transfer from 10 μM Zn(Hk14)2 (A) and 10 μM Zn(Hk45)2 complex (B) to 100 μM PAR upon an addition of 0.5–2.0 mol equiv of Ag(I). The formation of the Zn(PAR)2 complex was monitored spectroscopically by measuring an increase in absorbance at 492 nm. AgNO3 titration was performed in 20 mM TES, 100 NaF, pH 7.4. Asterisk denotes the initial absorbance of Zn(Hk)2 before the addition of Ag(I).

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    Figure 7

    Figure 7. Size-exclusion chromatography analysis of Ag(I)–Hk14 and Ag(I)–Hk45 complexes. Elution profiles of metal-free Hk14 (A), Hk45 (B), Zn(Hk14)2 (C), and Zn(Hk45)2 (D) at different molar AgNO3 equivalents. Absorbance was measured at 220 nm. Dashed lines indicate the main peak signals.

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    Figure 8

    Figure 8. (+)ESI-MS-monitored AgNO3 titration of apo-Hk14 and Zn(Hk14)2 complexes. Mass spectra of 2 μM peptide solutions were recorded for 0–4.0 mol equiv of Ag(I) in 50 mM ammonium acetate, pH 7.4. (A) Red, green, and cyan labels denote [AgL]2+, [Ag5L3]5+, and [Ag5L3]4+ species, respectively. Dashed lines indicate substrates (black) and the final (red) AgNO3 titration product. (B) Comparison of the calculated and experimental isotopic patterns of the observed metal complexes.

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    Figure 9

    Figure 9. ITC analysis of Ag(I) binding to Hk14. ITC profile for titration of Ag(I) into Hk14 (A), Zn(Hk14)2 (B), and Hk14 into Ag(I) (C) in 20 mM HEPES, 100 mM NaF, pH 7.4. Top panels show the baseline-subtracted thermogram. The bottom panels represent the binding isotherm (see Table S5). The x-axis represents the molar ratio of AgNO3 to the Hk monomer being in the Zn(II) complex (A, B), and the molar ratio of Hk monomer to AgNO3 (C). Points not included in the fits were marked with white fill.

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    Figure 10

    Figure 10. Proposed pathway of AgNP transfer to the cell, their dissolution and Ag(I) release in nuclear peripheries. Ag(I) ions are translocated to the nucleus and interact with Zn(II)–thiolate binding sites, such as Rad50 being a part of the MRN(X) complex. Zn(II)-to-Ag(I) swap alters the structure of the dimeric hook domain and generates dysfunctional MRN complexes. Graph has been prepared using Servier Medical Art https://smart.servier.com.

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    8. 8
      Betts, H. D.; Neville, S. L.; McDevitt, C. A.; Sumby, C. J.; Harris, H. H. The biochemical fate of Ag+ ions in Staphylococcus aureus, Escherichia coli, and biological media. J. Inorg. Biochem. 2021, 225, 111598  DOI: 10.1016/j.jinorgbio.2021.111598
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      8
      The biochemical fate of Ag+ ions in Staphylococcus aureus, Escherichia coli, and biological media
      Betts, Harley D.; Neville, Stephanie L.; McDevitt, Christopher A.; Sumby, Christopher J.; Harris, Hugh H.
      Journal of Inorganic Biochemistry (2021), 225 (), 111598CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier Inc.)
      Silver is commonly included in a range of household and medical items to provide bactericidal action. Despite this, the chem. fate of the metal in both mammalian and bacterial systems remains poorly understood. Here, we applied a metallomics approach using X-ray absorption spectroscopy (XAS) and size-exclusion chromatog. hyphenated with inductively coupled plasma mass spectrometry (SEC-ICP-MS) to advance our understanding of the biochem. fate of silver ions in bacterial culture and cells, and the chem. assocd. with these interactions. When silver ions were added to lysogeny broth, silver was exclusively assocd. with moderately-sized species (∼30 kDa) and bound by thiolate ligands. In two representative bacterial pathogens cultured in lysogeny broth including sub-lethal concns. of ionic silver, silver was found in cells to be predominantly coordinated by thiolate species. The silver biomacromol.-binding profile in Staphylococcus aureus and Escherichia coli was complex, with silver bound by a range of species spanning from 20 kDa to >1220 kDa. In bacterial cells, silver was nonuniformly colocalised with copper-bound proteins, suggesting that cellular copper processing may, in part, confuse silver for nutrient copper. Notably, in the treated cells, silver was not detected bound to low mol. wt. compds. such as glutathione or bacillithiol.
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    9. 9
      Liao, X.; Yang, F.; Wang, R.; He, X.; Li, H.; Kao, R. Y. T.; Xia, W.; Sun, H. Identification of catabolite control protein A from Staphylococcus aureus as a target of silver ions. Chem. Sci. 2017, 8, 8061– 8066,  DOI: 10.1039/c7sc02251d
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      9
      Identification of catabolite control protein A from Staphylococcus aureus as a target of silver ions
      Liao, Xiangwen; Yang, Fang; Wang, Runming; He, Xiaojun; Li, Hongyan; Kao, Richard Y. T.; Xia, Wei; Sun, Hongzhe
      Chemical Science (2017), 8 (12), 8061-8066CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
      Staphylococcus aureus is one of the most common pathogenic bacteria that causes human infectious diseases. The emergence of antibiotic-resistant strains of S. aureus promotes the development of new anti-bacterial strategies. Silver ions (Ag+) have attracted profound attention due to their broad-spectrum antimicrobial activities. Although the antibacterial properties of silver have been well known for many centuries, its mechanism of action remains unclear and its protein targets are rarely reported. Herein, we identify the catabolite control protein A (CcpA) of S. aureus as a putative target for Ag+. CcpA binds 2 molar equivalents of Ag+via its two cysteine residues (Cys216 and Cys242). Importantly, Ag+ binding induces CcpA oligomerization and abolishes its DNA binding capability, which further attenuates S. aureus growth and suppresses a-hemolysin toxicity. This study extends our understanding of the bactericidal effects of silver.
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    10. 10
      Wang, H.; Wang, M.; Xu, X.; Gao, P.; Xu, Z.; Zhang, Q.; Li, H.; Yan, A.; Kao, R. Y.-T.; Sun, H. Multi-target mode of action of silver against Staphylococcus aureus endows it with capability to combat antibiotic resistance. Nat. Commun. 2021, 12, 3331  DOI: 10.1038/s41467-021-23659-y
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      10
      Multi-target mode of action of silver against Staphylococcus aureus endows it with capability to combat antibiotic resistance
      Wang, Haibo; Wang, Minji; Xu, Xiaohan; Gao, Peng; Xu, Zeling; Zhang, Qi; Li, Hongyan; Yan, Aixin; Kao, Richard Yi-Tsun; Sun, Hongzhe
      Nature Communications (2021), 12 (1), 3331CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      Abstr.: The rapid emergence of drug resistant Staphylococcus aureus (S. aureus) poses a serious threat to public health globally. Silver (Ag)-based antimicrobials are promising to combat antibiotic resistant S. aureus, yet their mol. targets are largely elusive. Herein, we sep. and identify 38 authentic Ag+-binding proteins in S. aureus at the whole-cell scale. We then capture the mol. snapshot on the dynamic action of Ag+ against S. aureus and further validate that Ag+ could inhibit a key target 6-phosphogluconate dehydrogenase through binding to catalytic His185 by X-ray crystallog. Significantly, the multi-target mode of action of Ag+ (and nanosilver) endows its sustainable antimicrobial efficacy, leading to enhanced efficacy of conventional antibiotics and resensitization of MRSA to antibiotics. Our study resolves the long-standing question of the mol. targets of silver in S. aureus and offers insights into the sustainable bacterial susceptibility of silver, providing a potential approach for combating antimicrobial resistance.
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    11. 11
      Zhang, Q.; Wang, R.; Wang, M.; Liu, C.; Koohi-Moghadam, M.; Wang, H.; Ho, P.-L.; Li, H.; Sun, H. Re-sensitization of mcr carrying multidrug resistant bacteria to colistin by silver. Proc. Natl. Acad. Sci. U.S.A. 2022, 119, e2119417119  DOI: 10.1073/pnas.2119417119
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      11
      Re-sensitization of mcr carrying multidrug resistant bacteria to colistin by silver
      Zhang, Qi; Wang, Runming; Wang, Minji; Liu, Chunjiao; Koohi-Moghadam, Mohamad; Wang, Haibo; Ho, Pak-Leung; Li, Hongyan; Sun, Hongzhe
      Proceedings of the National Academy of Sciences of the United States of America (2022), 119 (11), e2119417119CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Colistin is considered the last-line antimicrobial for the treatment of multidrug-resistant gram-neg. bacterial infections. The emergence and spread of superbugs carrying the mobile colistin resistance gene (mcr) have become the most serious and urgent threat to healthcare. Here, we discover that silver (Ag+), including silver nanoparticles, could restore colistin efficacy against mcr-pos. bacteria. We show that Ag+ inhibits the activity of the MCR-1 enzyme via substitution of Zn2+ in the active site. Unexpectedly, a tetra-silver center was found in the active-site pocket of MCR-1 as revealed by the X-ray structure of the Ag-bound MCR-1, resulting in the prevention of substrate binding. Moreover, Ag+ effectively slows down the development of higher-level resistance and reduces mutation frequency. Importantly, the combined use of Ag+ at a low concn. with colistin could relieve dermonecrotic lesions and reduce the bacterial load of mice infected with mcr-1-carrying pathogens. This study depicts a mechanism of Ag+ inhibition of MCR enzymes and demonstrates the potentials of Ag+ as broad-spectrum inhibitors for the treatment of mcr-pos. bacterial infection in combination with colistin.
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    12. 12
      Jiménez-Lamana, J.; Laborda, F.; Bolea, E.; Abad-Álvaro, I.; Castillo, J. R.; Bianga, J.; He, M.; Bierla, K.; Mounicou, S.; Ouerdane, L.; Gaillet, S.; Rouanet, J. M.; Szpunar, J. An insight into silver nanoparticles bioavailability in rats. Metallomics 2014, 6, 2242– 2249,  DOI: 10.1039/c4mt00200h
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      12
      An insight into silver nanoparticles bioavailability in rats
      Jimenez-Lamana, Javier; Laborda, Francisco; Bolea, Eduardo; Abad-Alvaro, Isabel; Castillo, Juan R.; Bianga, Juliusz; He, Man; Bierla, Katarzyna; Mounicou, Sandra; Ouerdane, Laurent; Gaillet, Sylvie; Rouanet, Jean-Max; Szpunar, Joanna
      Metallomics (2014), 6 (12), 2242-2249CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)
      A comprehensive study of the bioavailability of orally administered silver nanoparticles (AgNPs) was carried out using a rat model. The silver uptake was monitored in liver and kidney tissues, as well as in urine and in feces. Significant accumulation of silver was found in both organs, the liver being the principal target of AgNPs. A significant (∼50%) fraction of silver was found in feces whereas the fraction excreted via urine was negligible (<0.01%). Intact silver nanoparticles were found in feces by asym. flow field-flow fractionation (AsFlFFF) coupled with UV-Vis anal. Laser ablation-ICP MS imaging showed that AgNPs were able to penetrate into the liver, in contrast to kidneys where they were retained in the cortex. Silver speciation anal. in cytosols from kidneys showed the metallothionein complex as the major species whereas in the liver the majority of silver was bound to high-mol. (70-25 kDa) proteins. These findings demonstrate the presence of Ag(I), released by the oxidn. of AgNPs in the biol. environment.
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    13. 13
      Yang, L.; Kuang, H.; Zhang, W.; Aguilar, Z. P.; Wei, H.; Xu, H. Comparisons of the biodistribution and toxicological examinations after repeated intravenous administration of silver and gold nanoparticles in mice. Sci. Rep. 2017, 7, 3303  DOI: 10.1038/s41598-017-03015-1
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      13
      Comparisons of the biodistribution and toxicological examinations after repeated intravenous administration of silver and gold nanoparticles in mice
      Yang Lin; Kuang Huijuan; Wei Hua; Xu Hengyi; Zhang Wanyi; Aguilar Zoraida P
      Scientific reports (2017), 7 (1), 3303 ISSN:.
      Nanoparticles (NPs) size, surface functionalization, and concentration were claimed to contribute to distribution and toxicity outcomes of NPs in vivo. However, intrinsic chemical compositions of NPs caused inconsistent biodistribution and toxic profiles which attracted little attention. In this study, silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs) were used to determine the biodistribution, toxickinetic, and genotoxicity variances in murine animals. The results demonstrated AgNPs and AuNPs were primarily deposited in the mononuclear phagocyte system (MPS) such as the liver and spleen. In particular, AuNPs seemed to be prominently stored in the liver, whereas AgNPs preferentially accumulated in more organs such as the heart, lung, kidney, etc. Also, the circulation in the blood and fecal excretions showed higher AgNPs contents in comparison with the AuNPs. Measurements of the mouse body and organ mass, hematology and biochemistry evaluation, and histopathological examinations indicated slight toxic difference between the AgNPs and AuNPs over a period of two months. RT-qPCR data revealed that AgNPs induced greater changes in gene expression with relevance to oxidative stress, apoptosis, and ion transport. Our observations proved that the NPs chemical composition played a critical role in their in vivo biodistribution and toxicity.
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    14. 14
      Poznański, J.; Sołdacki, D.; Czarkowska-Pączek, B.; Bonna, A.; Kornasiewicz, O.; Krawczyk, M.; Bal, W.; Pączek, L. Cirrhotic liver of liver transplant recipients accumulate silver and co-accumulate copper. Int. J. Mol. Sci. 2021, 22, 1782,  DOI: 10.3390/ijms22041782
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    15. 15
      Suárez, V. T.; Karepina, E.; Chevallet, M.; Gallet, B.; Cottet-Rousselle, C.; Charbonnier, P.; Moriscot, C.; Michaud-Soret, I.; Bal, W.; Fuchs, A.; Tucoulou, R.; Jouneau, P.-H.; Veronesi, G.; Deniaud, A. Nuclear translocation of silver ions and hepatocyte nuclear Receptor impairment upon exposure to silver nanoparticles. Environ. Sci.: Nano 2020, 7, 1373– 1387,  DOI: 10.1039/c9en01348b
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      Wiemann, M.; Vennemann, A.; Blaske, F.; Sperling, M.; Karst, U. Silver nanoparticles in the lung: Toxic effects and focal accumulation of silver in remote organs. Nanomaterials 2017, 7, 441,  DOI: 10.3390/nano7120441
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      16
      Silver nanoparticles in the lung: toxic effects and focal accumulation of silver in remote organs
      Wiemann, Martin; Vennemann, Antje; Blaske, Franziska; Sperling, Michael; Karst, Uwe
      Nanomaterials (2017), 7 (12), 441/1-441/26CODEN: NANOKO; ISSN:2079-4991. (MDPI AG)
      The distribution of silver (Ag) into remote organs secondary to the application of Ag nanoparticles (Ag-NP) to the lung is still incompletely understood and was investigated in the rat with imaging methods. Dose-finding expts. were carried out with 50 nm- or 200 nm-sized polyvinyl pyrrolidine (PVP)-coated Ag-NP using alveolar macrophages in vitro and female rats, which received Ag-NP via intratracheal instillation. In the main study, we administered 37.5-300 μg per rat lung of the more toxic Ag50-PVP and assessed the broncho-alveolar lavage fluid (BALF) for inflammatory cells, total protein and fibronectin after three and 21 days. In parallel, lung tissue was analyzed for DNA double-strand breaks and altered cell proliferation. While 75-150 μg Ag50-PVP per rat lung caused a reversible inflammation, 300 μg led to DNA damage, accelerated cell proliferation and progressively increasing nos. of neutrophilic granulocytes. Ag accumulation was significant in homogenates of liver and other peripheral organs upon lung dose of ≥75 μg. Quant. laser-ablation inductively-coupled plasma mass spectrometry (LA-ICP-MS) combined with enhanced dark field microscopy and autometallog. revealed focal accumulations of Ag and/or Ag-NP in sections of peripheral organs: mediastinal lymph nodes contained Ag-NP esp. in peripheral macrophages and Ag in argyrophilic fibers. In the kidney, Ag had accumulated within proximal tubuli, while renal filter structures contained no Ag. Discrete localizations were also obsd. in immune cells of liver and spleen. Overall, the study shows that concns. of Ag-NP, which elicit a transient inflammation in the rat lung, lead to focal accumulations of Ag in peripheral organs, and this might pose a risk to particular cell populations in remote sites.
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      Marchioni, M.; Jouneau, P.-H.; Chevallet, M.; Michaud-Soret, I.; Deniaud, A. Silver nanoparticle fate in mammals: Bridging in vitro and in vivo studies. Coord. Chem. Rev. 2018, 364, 118– 136,  DOI: 10.1016/j.ccr.2018.03.008
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      17
      Silver nanoparticle fate in mammals: Bridging in vitro and in vivo studies
      Marchioni, Marianne; Jouneau, Pierre-Henri; Chevallet, Mireille; Michaud-Soret, Isabelle; Deniaud, Aurelien
      Coordination Chemistry Reviews (2018), 364 (), 118-136CODEN: CCHRAM; ISSN:0010-8545. (Elsevier B.V.)
      Silver nanoparticles (AgNPs) are exponentially used in various consumer products including medical devices. This prodn. leads to an increasing human exposure to silver in different forms. Indeed, AgNPs are subject to various transformations in aq. aerobic conditions that trigger the prodn. of Ag(I) species. The main environmental transformation produces the non-toxic species silver sulfide. Transformations occurring in mammals are more diverse and mainly depend on the interaction of AgNPs with thiol, chloride and proteins. Any of these species have a different impact on AgNPs and induces AgNP dissoln. into Ag(I) species, aggregation and/or stabilization. The transformations occurring also depend on the exposure route. The main one is dietary but medical exposure is also growing with the massive use of nanosilver as biocide in medical devices. For the former, AgNP modifications and Ag distribution has been extensively studied using in vitro and in vivo models, while data related to medical use of nanosilver are scarce. However, most of the in vitro and in vivo data often remain inconsistent. In this review, we describe both in vitro, in cellulo and in vivo data about AgNP transformations, silver speciation and biodistribution. We try to reconcile all these data and describe the latest methods for the future studies of AgNP fate in mammals.
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    18. 18
      Malysheva, A.; Ivask, A.; Doolette, C. L.; Voelcker, N. H.; Lombi, E. Cellular binding, uptake and biotransformation of silver nanoparticles in human T lymphocytes. Nat. Nanotechnol. 2021, 16, 926– 932,  DOI: 10.1038/s41565-021-00914-3
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      18
      Cellular binding, uptake and biotransformation of silver nanoparticles in human T lymphocytes
      Malysheva, Anzhela; Ivask, Angela; Doolette, Casey L.; Voelcker, Nicolas H.; Lombi, Enzo
      Nature Nanotechnology (2021), 16 (8), 926-932CODEN: NNAABX; ISSN:1748-3387. (Nature Portfolio)
      Our knowledge of uptake, toxicity and detoxification mechanisms as related to nanoparticles' (NPs') characteristics remains incomplete. Here we combine the anal. power of three advanced techniques to study the cellular binding and uptake and the intracellular transformation of silver nanoparticles (AgNPs): single-particle inductively coupled mass spectrometry, mass cytometry and synchrotron X-ray absorption spectrometry. Our results show that although intracellular and extracellularly bound AgNPs undergo major transformation depending on their primary size and surface coating, intracellular Ag in 24 h AgNP-exposed human lymphocytes exists in nanoparticulate form. Biotransformation of AgNPs is dominated by sulfidation, which can be viewed as one of the cellular detoxification pathways for Ag. These results also show that the toxicity of AgNPs is primarily driven by internalized Ag. In fact, when toxicity thresholds are expressed as the intracellular mass of Ag per cell, differences in toxicity between NPs of different coatings and sizes are minimized. The anal. approach developed here has broad applicability in different systems where the aim is to understand and quantify cell-NP interactions and biotransformation.
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    19. 19
      Adams, N. W. H.; Kramer, J. R. Potentiometric determination of silver thiolate formation constants using a Ag2S electrode. Aquat. Geochem. 1999, 5, 1– 11,  DOI: 10.1023/A:1009699617808
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      19
      Potentiometric determination of silver thiolate formation constants using a Ag2S electrode
      Adams, Nicholas W. H.; Kramer, James R.
      Aquatic Geochemistry (1999), 5 (1), 1-11CODEN: AQGEFP; ISSN:1380-6165. (Kluwer Academic Publishers)
      Formation consts. for silver thiolates were obtained by titrn. of the ligand in a const. temp., ionic strength and pH medium and measuring the potential change at a Ag2S electrode. A non-linear equation was derived from which the first and second silver formation consts., β'1 and β'2, and the sulfide group acid dissocn. const., K'a, were detd. An overall est. of the uncertainty in the derived parameters was obtained using a Monte Carlo approach. The procedure was compared to a previous work on AgHS°. Log β'1, log β'2 and -log K'a results were obtained for cysteine (11.9 ± 0.5, 15.2 ± 0.4, 7.8 ± 0.1), glutathione (12.3 ± 0.3, 14.3 ± 0.8, 8.8 ± 0.3) and 3-mercaptopropanoic acid (12.0 ± 0.4, 14.0 ± 0.4, 10.5 ± 0.3) at 20° and 0.01 m ionic strength.
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    20. 20
      Pettit, L. D.; Siddiqui, K. F.; Kozłowski, H.; Kowalik, T. Potentiometric and 1H NMR studies on silver(I) interaction with S-methyl-L-cysteine, L-methionine and L-ethionine. Inorg. Chim. Acta 1981, 55, 87– 91,  DOI: 10.1016/S0020-1693(00)90787-4
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      20
      Potentiometric and proton NMR studies on silver(I) interaction with S-methyl-L-cysteine, L-methionine and L-ethionine
      Pettit, Leslie D.; Siddiqui, Kaniz F.; Kozlowski, Henryk; Kowalik, Teresa
      Inorganica Chimica Acta (1981), 55 (3), 87-91CODEN: ICHAA3; ISSN:0020-1693.
      Potentiometric titrn. (pH, pAg) studies at 25° and ionic strength 0.1(KNO3) gave stability consts. for Ag(HL), Ag(HL)2, AgL (or Ag2L2), and AgL2 complexes (H2L = title ligands). The NMR data indicate S-bonding to Ag with NH2 group bonding only at pH >4.5. Rotamer populations of free and coordinated S-methyl-L-cysteine were also calcd.
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      Legler, E. V.; Kazbanov, V. I.; Kazachenko, A. S. Study of complexing equilibrium in the Ag(I)-histidine system. Zh. Neorg. Khim. 2002, 47, 150– 153
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      Dance, I. G. The Structural Chemistry of Metal Thiolate Complexes. Polyhedron 1986, 5, 1037– 1104,  DOI: 10.1016/S0277-5387(00)84307-7
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      22
      The structural chemistry of metal thiolate complexes
      Dance, Ian G.
      Polyhedron (1986), 5 (5), 1037-104CODEN: PLYHDE; ISSN:0277-5387.
      A review with 346 refs.
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      Leung, B. O.; Jalilehvand, F.; Mah, V.; Parvez, M.; Wu, Q. Silver(I) complex formation with cysteine, penicillamine, and glutathione. Inorg. Chem. 2013, 52, 4593– 4602,  DOI: 10.1021/ic400192c
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      23
      Silver(I) Complex Formation with Cysteine, Penicillamine, and Glutathione
      Leung, Bonnie O.; Jalilehvand, Farideh; Mah, Vicky; Parvez, Masood; Wu, Qiao
      Inorganic Chemistry (2013), 52 (8), 4593-4602CODEN: INOCAJ; ISSN:0020-1669. (American Chemical Society)
      The complex formation between silver(I) and cysteine (H2Cys), penicillamine (H2Pen), and glutathione (H3Glu) in alk. aq. soln. was examd. using extended x-ray absorption fine structure (EXAFS) and 109Ag NMR spectroscopic techniques. The complexes formed in 0.1 mol dm-3 Ag(I) solns. with cysteine and penicillamine were investigated for ligand/Ag(I) (L/Ag) mole ratios increasing from 2.0 to 10.0. For cysteine solns. (pH 10-11) a mean Ag-S bond distance of 2.45 ± 0.02 Å consistently emerged, while for penicillamine (pH 9) the av. Ag-S bond distance gradually increased from 2.40 to 2.44 ± 0.02 Å. EXAFS and 109Ag NMR spectra of a concd. Ag(I)-cysteine soln. (CAg(I) = 0.8 mol dm-3, L/Ag = 2.2) showed a mean Ag-S bond distance of 2.47 ± 0.02 Å and δ(109Ag) 1103 ppm, consistent with prevailing, partially oligomeric AgS3 coordinated species, while for penicillamine (CAg(I) = 0.5 mol dm-3, L/Ag = 2.0) the mean Ag-S bond distance of 2.40 ± 0.02 Å and δ(109Ag) 922 ppm indicate that mononuclear AgS2 coordinated complexes dominate. For Ag(I)-glutathione solns. (CAg(I) = 0.01 mol dm-3, pH ∼11), mononuclear AgS2 coordinated species with a mean Ag-S bond distance of 2.36 ± 0.02 Å dominate for L/Ag mole ratios from 2.0 to 10.0. The crystal structure of the silver(I)-cysteine compd. (NH4)Ag2(HCys)(Cys)·H2O (1) pptg. at pH ∼10 was solved and showed a layer structure with both AgS3 and AgS3N coordination to the cysteinate ligands. A redetn. of the crystal structure of Ag(HPen)·H2O (2) confirmed the proposed digonal AgS2 coordination environment to bridging thiolate sulfur atoms in polymeric intertwining chains forming a double helix. A survey of Ag-S bond distances for cryst. Ag(I) complexes with S-donor ligands in different AgS2, AgS2(O/N), and AgS3 coordination environments was used, together with a survey of 109Ag NMR chem. shifts, to assist assignments of the Ag(I) coordination in soln.
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      Young, A. G.; Hanton, L. R. Square planar silver(I) complexes: A rare but increasingly observed stereochemistry for silver(I). Coord. Chem. Rev. 2008, 252, 1346– 1386,  DOI: 10.1016/j.ccr.2007.07.017
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      24
      Square planar silver(I) complexes: A rare but increasingly observed stereochemistry for silver(I)
      Young, Aidan G.; Hanton, Lyall R.
      Coordination Chemistry Reviews (2008), 252 (12-14), 1346-1386CODEN: CCHRAM; ISSN:0010-8545. (Elsevier B.V.)
      A review. The square planar Ag(I) stereochem. is generally acknowledged as rare, with only ∼2% of all reported silver complexes possessing this stereochem. Many researchers reporting such complexes often mistakenly believe that their example is one of only a handful of previously reported examples. This is despite the fact that there are currently around 65 well characterized complexes contg. square planar Ag(I) ions, about half of which are coordination polymers. In this review, the authors critically examine each example and draw attention to trends that arise in their formation. The scope is limited to traditional coordination complexes. Inorg. complexes contg. extended mineral like structures, and complexes contg. silver-π or silver-arene motifs are not considered in this review.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXlvFyntrs%253D&md5=50d9bd99731f2cba74a502a3bc16910d
    25. 25
      Wang, H.; Yang, X.; Wang, M.; Hu, M.; Xu, X.; Yan, A.; Hao, Q.; Li, H.; Sun, H. Atomic differentiation of silver binding preference in protein targets: Escherichia colimalate dehydrogenase as a paradigm. Chem. Sci. 2020, 11, 11714– 11719,  DOI: 10.1039/d0sc04151c
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      Atomic differentiation of silver binding preference in protein targets: Escherichia coli malate dehydrogenase as a paradigm
      Wang, Haibo; Yang, Xinming; Wang, Minji; Hu, Menglong; Xu, Xiaohan; Yan, Aixin; Hao, Quan; Li, Hongyan; Sun, Hongzhe
      Chemical Science (2020), 11 (43), 11714-11719CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
      Understanding how metallodrugs interact with their protein targets is of vital importance for uncovering their mol. mode of actions as well as overall pharmacol./toxicol. profiles, which in turn facilitates the development of novel metallodrugs. Silver has been used as an antimicrobial agent since antiquity, yet there is limited knowledge about silver-binding proteins. Given the multiple dispersed cysteine residues and histidine-methionine pairs, Escherichia coli malate dehydrogenase (EcMDH) represents an excellent model to investigate silver coordination chem. as well as its targeting sites in enzymes. We show by systematic biochem. characterizations that silver ions (Ag+) bind EcMDH at multiple sites including three cysteine-contg. sites. By X-ray crystallog., we unravel the binding preference of Ag+ to multiple binding sites in EcMDH, i.e., Cys113 > Cys251 > Cys109 > Met227. Silver exhibits preferences to the donor atoms and residues in the order of S > N > O and Cys > Met > His > Lys > Val, resp., in EcMDH. For the first time, we report the coordination of silver to a lysine in proteins. Besides, we also obsd. argentophilic interactions (AgU+00B7U+00B7U+00B7Ag, 2.7 to 3.3 U+00C5) between two silver ions coordinating to one thiolate. Combined with site-directed mutagenesis and an enzymic activity test, we unveil that the binding of Ag+ to the site IV (His177-Ag-Met227 site) plays a vital role in Ag+mediated MDH inactivation. This work stands as the first unusual and explicit study of silver binding preference to multiple binding sites in its authentic protein target at the at. resoln. These findings enrich our knowledge on the biocoordination chem. of silver(I), which in turn facilitates the prediction of the unknown silver-binding proteins and extends the pharmaceutical potentials of metal-based drugs.
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    26. 26
      Veronesi, G.; Gallon, T.; Deniaud, A.; Boff, B.; Gateau, C.; Lebrun, C.; Vidaud, C.; Rollin-Genetet, F.; Carrière, M.; Kieffer, I.; Mintz, E.; Delangle, P.; Michaud-Soret, I. XAS investigation of silver(I) coordination in copper(I) biological binding sites. Inorg. Chem. 2015, 54, 11688– 11696,  DOI: 10.1021/acs.inorgchem.5b01658
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      26
      XAS Investigation of Silver(I) Coordination in Copper(I) Biological Binding Sites
      Veronesi, Giulia; Gallon, Thomas; Deniaud, Aurelien; Boff, Bastien; Gateau, Christelle; Lebrun, Colette; Vidaud, Claude; Rollin-Genetet, Francoise; Carriere, Marie; Kieffer, Isabelle; Mintz, Elisabeth; Delangle, Pascale; Michaud-Soret, Isabelle
      Inorganic Chemistry (2015), 54 (24), 11688-11696CODEN: INOCAJ; ISSN:0020-1669. (American Chemical Society)
      Silver(I) is an unphysiol. ion that, as the physiol. copper(I) ion, shows high binding affinity for thiolate ligands; its toxicity has been proposed to be due to its capability to replace Cu(I) in the thiolate binding sites of proteins involved in copper homeostasis. Nevertheless, the nature of the Ag(I)-thiolate complexes formed within cells is poorly understood, and the details of Ag(I) coordination in such complexes in physiol. relevant conditions are mostly unknown. By making use of X-ray absorption spectroscopy (XAS), we characterized the Ag(I) binding sites in proteins related to copper homeostasis, such as the chaperone Atox1 and metallothioneins (MTs), as well as in bioinspired thiolate Cu(I) chelators mimicking these proteins, in soln. and at physiol. pH. Different Ag(I) coordination environments were revealed: the Ag-S bond length was found to correlate to the Ag(I) coordination no., with characteristic values of 2.40 and 2.49 Å in AgS2 and AgS3 sites, resp., comparable to the values reported for cryst. Ag(I)-thiolate compds. The bioinspired Cu(I) chelator L1 is proven to promote the unusual trigonal AgS3 coordination and, therefore, can serve as a ref. compd. for this environment. In the Cu(I)-chaperone Atox1, Ag(I) binds in digonal coordination to the two Cys residues of the Cu(I) binding loop, with the AgS2 characteristic bond length of 2.40 ± 0.01 Å. In the multinuclear Ag(I) clusters of rabbit and yeast metallothionein, the av. Ag-S bond lengths are 2.48 ± 0.01 Å and 2.47 ± 0.01 Å, resp., both indicative of the predominance of trigonal AgS3 sites. This work lends insight into the coordination chem. of silver in its most probable intracellular targets and might help in elucidating the mechanistic aspects of Ag(I) toxicity.
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    27. 27
      Changela, A.; Chen, K.; Xue, Y.; Holschen, J.; Outten, C. E.; O’Halloran, T. V.; Mondragón, A. Molecular basis of metal-ion selectivity and zeptomolar sensitivity by CueR. Science 2003, 301, 1383– 1387,  DOI: 10.1126/science.1085950
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      Molecular Basis of Metal-Ion Selectivity and Zeptomolar Sensitivity by CueR
      Changela, Anita; Chen, Kui; Xue, Yi; Holschen, Jackie; Outten, Caryn E.; O'Halloran, Thomas V.; Mondragon, Alfonso
      Science (Washington, DC, United States) (2003), 301 (5638), 1383-1387CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      The earliest of a series of copper efflux genes in Escherichia coli are controlled by CueR, a member of the MerR family of transcriptional activators. Thermodn. calibration of CueR reveals a zeptomolar (10-21 molar) sensitivity to free Cu+, which is far less than one atom per cell. At. details of this extraordinary sensitivity and selectivity for +1 transition-metal ions are revealed by comparing the crystal structures of CueR and a Zn2+-sensing homolog, ZntR. An unusual buried metal-receptor site in CueR restricts the metal to a linear, two-coordinate geometry and uses helix-dipole and hydrogen-bonding interactions to enhance metal binding. This binding mode is rare among metalloproteins but well suited for an ultrasensitive genetic switch.
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      Meury, M.; Knop, M.; Seebeck, F. P. Structural basis for copper–oxygen mediated C–H bond activation by the formylglycine-generating enzyme. Angew. Chem., Int. Ed. 2017, 56, 8115– 8119,  DOI: 10.1002/anie.201702901
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      Structural Basis for Copper-Oxygen Mediated C-H Bond Activation by the Formylglycine-Generating Enzyme
      Meury, Marcel; Knop, Matthias; Seebeck, Florian P.
      Angewandte Chemie, International Edition (2017), 56 (28), 8115-8119CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The formylglycine-generating enzyme (FGE) is a unique copper protein that catalyzes oxygen-dependent C-H activation. We describe 1.66 Å- and 1.28 Å-resoln. crystal structures of FGE from Thermomonospora curvata in complex with either AgI or CdII providing definitive evidence for a high-affinity metal-binding site in this enzyme. The structures reveal a bis-cysteine linear coordination of the monovalent metal, and tetrahedral coordination of the bivalent metal. Similar coordination changes may occur in the active enzyme as a result of CuI/II redox cycling. Complexation of copper atoms by two cysteine residues is common among copper-trafficking proteins, but is unprecedented for redox-active copper enzymes or synthetic copper catalysts.
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    29. 29
      Leisinger, F.; Miarzlou, D. A.; Seebeck, F. P. Non-coordinative binding of O2 at the active center of a copper-dependent Enzyme. Angew. Chem., Int. Ed. 2021, 60, 6154– 6159,  DOI: 10.1002/anie.202014981
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      29
      Non-Coordinative Binding of O2 at the Active Center of a Copper-Dependent Enzyme
      Leisinger, Florian; Miarzlou, Dzmitry A.; Seebeck, Florian P.
      Angewandte Chemie, International Edition (2021), 60 (11), 6154-6159CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Mol. oxygen (O2) is a sustainable oxidn. reagent. O2 is strongly oxidizing but kinetically stable and its final reaction product is water. For these reasons learning how to activate O2 and how to steer its reactivity along desired reaction pathways is a longstanding challenge in chem. research. Activation of ground-state diradical O2 can occur either via conversion to singlet oxygen or by one-electron redn. to superoxide. Many enzymes facilitate activation of O2 by direct formation of a metal-oxygen coordination complex concomitant with inner sphere electron transfer. The formylglycine generating enzyme (FGE) is an unusual mononuclear copper enzyme that appears to follow a different strategy. Atomic-resoln. crystal structures of the precatalytic complex of FGE demonstrate that this enzyme binds O2 juxtaposed, but not coordinated to the catalytic CuI. Isostructural complexes that contain AgI instead of CuI or nitric oxide instead of O2 confirm that formation of the initial oxygenated complex of FGE does not depend on redox activity. A stepwise mechanism that decouples binding and activation of O2 is unprecedented for metal-dependent oxidases, but is reminiscent of flavin-dependent enzymes.
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    30. 30
      Bilinovich, S. M.; Morris, D. L.; Prokop, J. W.; Caporoso, J. A.; Taraboletti, A.; Duangjumpa, N.; Panzner, M. J.; Shriver, L. P.; Leeper, T. C. Silver binding to bacterial glutaredoxins observed by NMR. Biophysica 2021, 1, 359– 376,  DOI: 10.3390/biophysica1040027
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      Peterson, C. W.; Narula, S. S.; Armitage, I. M. 3D Solution structure of copper and silver-substituted yeast metallothioneins. FEBS Lett. 1996, 379, 85– 93,  DOI: 10.1016/0014-5793(95)01492-6
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      3D solution structure of copper and silver-substituted yeast metallothioneins
      Peterson, Cynthia W.; Narula, Surinder S.; Armitage, Ian M.
      FEBS Letters (1996), 379 (1), 85-93CODEN: FEBLAL; ISSN:0014-5793. (Elsevier)
      3D soln. structural calcns. for yeast silver(I)-substituted metallothionein (MT) and native copper(I) MT were completed using exptl. detd. NOE and dihedral angle constraints, in conjunction with exptl. derived metal-to-Cys connectivities for AgMT which were assumed identical for CuMT. For the first 40 residues in both structures, the polypeptide backbone wraps around the metal cluster in two large parallel loops sepd. by a deep cleft contg. the metal cluster. Minor differences between the two structures include differences in hydrogen bonds and the orientation of the N-terminus with the overall protein vol. conserved to within 6.5%.
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      Wang, X.; Han, Z.-C.; Wei, W.; Hu, H.; Li, P.; Sun, P.; Liu, X.; Lv, Z.; Wang, F.; Cao, Y.; Guo, Z.; Li, J.; Zhao, J. An unexpected all-metal aromatic tetranuclear silver cluster in human copper chaperone Atox1. Chem. Sci. 2022, 13, 7269– 7275,  DOI: 10.1039/d1sc07122j
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      An unexpected all-metal aromatic tetranuclear silver cluster in human copper chaperone Atox1
      Wang, Xiuxiu; Han, Zong-Chang; Wei, Wei; Hu, Hanshi; Li, Pengfei; Sun, Peiqing; Liu, Xiangzhi; Lv, Zhijia; Wang, Feng; Cao, Yi; Guo, Zijian; Li, Jun; Zhao, Jing
      Chemical Science (2022), 13 (24), 7269-7275CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
      Metal clusters, such as iron-sulfur clusters, play key roles in sustaining life and are intimately involved in the functions of metalloproteins. Herein we report the formation and crystal structure of a planar square tetranuclear silver cluster when silver ions were mixed with human copper chaperone Atox1. Quantum chem. studies reveal that two Ag 5s1 electrons in the tetranuclear silver cluster fully occupy the one bonding MO, with the assumption that this Ag4 cluster is Ag42+, leading to extensive electron delocalization over the planar square and significant stabilization. This bonding pattern of the tetranuclear silver cluster represents an arom. all-metal structure that follows a 4n + 2 electron counting rule (n = 0). This is the first time an all-metal arom. silver cluster was obsd. in a protein.
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      Mazzei, L.; Cianci, M.; Gonzalez Vara, A.; Ciurli, S. The structure of urease inactivated by Ag(I): a new paradigm for enzyme inhibition by heavy metals. Dalton Trans. 2018, 47, 8240– 8247,  DOI: 10.1039/c8dt01190g
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      33
      The structure of urease inactivated by Ag(I): a new paradigm for enzyme inhibition by heavy metals
      Mazzei, Luca; Cianci, Michele; Gonzalez Vara, Antonio; Ciurli, Stefano
      Dalton Transactions (2018), 47 (25), 8240-8247CODEN: DTARAF; ISSN:1477-9226. (Royal Society of Chemistry)
      The nickel-dependent enzyme urease is a virulence factor for a large no. of human pathogens, as well as a neg. element for the efficiency of soil nitrogen fertilization for crop prodn. The use of urease inhibitors to contrast these effects requires the knowledge, at the mol. level, of their mode of action. Among these, silver is an efficient antimicrobial agent and an established inhibitor of this enzyme. The 1.91 Å resoln. structure of Sporosarcina pasteurii urease inhibited by silver reveals the presence of two Ag(I) ions bound to the largely conserved triad αCys322/αHis323/αMet367: the first two residues are located on the mobile flap that is essential in modulating the size of the active site cavity and the position of key residues for enzyme catalysis, while αMet367 is on a loop facing the flap at the entrance of the active site cavity. The two Ag(I) ions are bridged by the thiolate Sγ atom of αCys322, and are coordinated, resp., to the Nδ1 atom of the αHis323 imidazole ring and to the Sδ of αMet367. The binding of the Ag(I) ions at the edge of the active site channel supposedly blocks the movement of the flap, inhibiting the catalytic activity of urease. The structure of the silver-inhibited urease allows us to understand and rationalize all previously acquired kinetic and calorimetric data on this phenomenon, but also provides the details of how silver can exert its antimicrobial action with respect to ureolytic bacteria, a step forward against antibiotic-resistant pathogens.
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    34. 34
      Panzner, M. J.; Bilinovich, S. M.; Parker, J. A.; Bladholm, E. L.; Ziegler, C. J.; Berry, S. M.; Leeper, T. C. Isomorphic deactivation of a Pseudomonas aeruginosa oxidoreductase: The crystal structure of Ag(I) metallated azurin at 1.7 Å. J. Inorg. Biochem. 2013, 128, 11– 16,  DOI: 10.1016/j.jinorgbio.2013.07.011
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      34
      Isomorphic deactivation of a Pseudomonas aeruginosa oxidoreductase: The crystal structure of Ag(I) metallated azurin at 1.7 Å
      Panzner, Matthew J.; Bilinovich, Stephanie M.; Parker, Jillian A.; Bladholm, Erika L.; Ziegler, Christopher J.; Berry, Steven M.; Leeper, Thomas C.
      Journal of Inorganic Biochemistry (2013), 128 (), 11-16CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier)
      Multiple biophys. methods demonstrate that Ag(I) effectively metalates P. aeruginosa apo-azurin in soln. X-ray crystallog. of the Ag(I)-modified protein revealed that Ag(I) bound to azurin at the traditional Cu-mediated active site with nearly identical geometry. Cyclic voltammetry indicated that the Ag(I) adduct was redox inert. The results suggested that a potential mechanism for the microbial toxicity of Ag(I) is the deactivation of Cu-contg. oxidoreductases by the effective binding and structural mimicry by Ag(I) without the corresponding function.
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    35. 35
      Liu, W.; Worms, I. A. M.; Herlin-Boime, N.; Truffier-Boutry, D.; Michaud-Soret, I.; Mintz, E.; Vidaud, C.; Rollin-Genetet, F. Interaction of silver nanoparticles with metallothionein and ceruloplasmin: impact on metal substitution by Ag(I), corona formation and enzymatic activity. Nanoscale 2017, 9, 6581– 6594,  DOI: 10.1039/c7nr01075c
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      35
      Interaction of silver nanoparticles with metallothionein and ceruloplasmin: impact on metal substitution by Ag(I), corona formation and enzymatic activity
      Liu, Wei; Worms, Isabelle A. M.; Herlin-Boime, Nathalie; Truffier-Boutry, Delphine; Michaud-Soret, Isabelle; Mintz, Elisabeth; Vidaud, Claude; Rollin-Genetet, Francoise
      Nanoscale (2017), 9 (19), 6581-6594CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)
      The release of Ag(I) from silver nanoparticles (AgNPs) unintentionally spread in the environment is suspected to impair some key biol. functions. In comparison with AgNO3, in-depth investigations were carried out into the interactions between citrate-coated AgNPs (20 nm) and two metalloproteins, intracellular metallothionein 1 (MT1) and plasmatic ceruloplasmin (Cp), both involved in metal homeostasis. These were chosen for their physiol. relevance and the diversity of their various native metals bound because of thiol groups and/or their structural differences. Transmission electron microscopy (TEM), and dynamic light scattering (DLS), UV-vis and CD (CD) spectroscopies were used to study the effects of such intricate interactions on AgNP dissoln. and proteins in terms of metal exchanges and structural modifications. The isolation of the different populations formed together with online quantifications of their metal content were performed by asym. flow field-flow fractionation (AF4) linked to inductively coupled plasma mass spectrometry (ICP-MS). For the 2 proteins, Ag(I) dissolved from the AgNPs, substituted for the native metal, to different extents and with different types of dynamics for the corona formed: the MT1 rapidly surrounded the AgNPs with the transient reticulate corona thus promoting their dissoln. assocd. with the metal substitution, whereas the Cp established a more stable layer around the AgNPs, with a limited substitution of Cu and a decrease in its ferroxidase activity. The accessibility and lability of the metal binding sites inside these proteins and their relative affinities for Ag(I) are discussed, taking into account the structural characteristics of the proteins.
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    36. 36
      Kluska, K.; Peris-Díaz, M. D.; Płonka, D.; Moysa, A.; Dadlez, M.; Deniaud, A.; Bal, W.; Krężel, A. Formation of highly stable multinuclear AgnSn clusters in zinc fingers disrupts their structure and function. Chem. Commun. 2020, 56, 1329– 1332,  DOI: 10.1039/c9cc09418k
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      Formation of highly stable multinuclear AgnSn clusters in zinc fingers disrupts their structure and function
      Kluska, Katarzyna; Peris-Diaz, Manuel D.; Plonka, Dawid; Moysa, Alexander; Dadlez, Michal; Deniaud, Aurelien; Bal, Wojciech; Krezel, Artur
      Chemical Communications (Cambridge, United Kingdom) (2020), 56 (9), 1329-1332CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      Silver (Ag(I)) binding to consensus zinc fingers (ZFs) causes Zn(II) release inducing a gradual disruption of the hydrophobic core, followed by an overall conformational change and formation of highly stable AgnSn clusters. A compact eight-membered Ag4S4 structure formed by a CCCC ZF is the first cluster example reported for a single biol. mol. Ag(I)-induced conformational changes of ZFs can, as a consequence, affect transcriptional regulation and other cellular processes.
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      Kluska, K.; Veronesi, G.; Deniaud, A.; Hajdu, B.; Gyurcsik, B.; Bal, W.; Krężel, A. Structures of silver fingers and a pathway to their genotoxicity. Angew. Chem., Int. Ed. 2022, 61, e202116621  DOI: 10.1002/anie.202116621
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      Structures of Silver Fingers and a Pathway to Their Genotoxicity
      Kluska, Katarzyna; Veronesi, Giulia; Deniaud, Aurelien; Hajdu, Balint; Gyurcsik, Bela; Bal, Wojciech; Krezel, Artur
      Angewandte Chemie, International Edition (2022), 61 (12), e202116621CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      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 biol. function of ZF proteins is a likely consequence of such replacement. Here, we report an X-ray absorption spectroscopy (XAS) study of AgnSn 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 av. Ag-S distance at 2.41 S. 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 Ag2S2 clusters occurred in a three-ZF transcription factor protein 1MEY#, leading to a dissocn. of 1MEY# from the complex with its cognate DNA.
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      Kluska, K.; Adamczyk, J.; Krężel, A. Metal binding properties, stability and reactivity of zinc fingers. Coord. Chem. Rev. 2018, 367, 18– 64,  DOI: 10.1016/j.ccr.2018.04.009
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      Metal binding properties, stability and reactivity of zinc fingers
      Kluska, Katarzyna; Adamczyk, Justyna; Krezel, Artur
      Coordination Chemistry Reviews (2018), 367 (), 18-64CODEN: CCHRAM; ISSN:0010-8545. (Elsevier B.V.)
      A review. Zinc fingers (ZFs) are among the most structurally diverse protein domains. They interact with nucleic acids, other proteins and lipids to facilitate a multitude of biol. processes. Currently, there are more than 10 known classes of ZFs, with various architectures, metal binding modes, functions and reactivity. The versatility, selectivity and stability of these short amino acid sequences is achieved mainly by (i) residues participating in Zn(II) coordination (mostly Cys and His), (ii) hydrophobic core and ZF structure formation, and (iii) variable residues responsible for inter- and intramol. interactions. Since their discovery, ZFs have been extensively studied in terms of their structure, stability and recognition targets by the application of various methodologies. Studies based on interactions with other metal ions and their complexes have contributed to the understanding of their chem. properties and the discovery of new types of ZF complexes, such as gold fingers or lead fingers. Moreover, due to the presence of nucleophilic thiolates, ZFs are targets for reactive oxygen and nitrogen species as well as alkylating agents. Interactions with many reactive mols. lead to disturb the native Zn(II) coordination site which further result in structural and functional damage of the ZFs. The post-translational modifications including phosphorylation, acetylation, methylation or nitrosylation frequently affect ZFs function via changes in the protein structure and dynamics. Even though the literature is replete with structural and stability data regarding classical (ββα) ZFs, there is still a huge gap in the knowledge on physicochem. properties and reactivity of other ZF types. In this review, metal binding properties of ZFs and stability factors that modulate their functions are reviewed. These include interactions of ZFs with biogenic and toxic metal ions as well as damage occurring upon reaction with reactive oxygen and nitrogen species, the methodol. used for ZFs characterization, and aspects related to coordination chem.
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      Padjasek, M.; Kocyła, A.; Kluska, K.; Kerber, O.; Tran, J. B.; Krężel, A. Structural zinc binding sites shaped for greater works: Structure-function relations in classical zinc finger, hook and clasp domains. J. Inorg. Biochem. 2020, 204, 110955  DOI: 10.1016/j.jinorgbio.2019.110955
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      Structural zinc binding sites shaped for greater works: Structure-function relations in classical zinc finger, hook and clasp domains
      Padjasek, Michal; Kocyla, Anna; Kluska, Katarzyna; Kerber, Olga; Tran, Jozef Ba; Krezel, Artur
      Journal of Inorganic Biochemistry (2020), 204 (), 110955CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier)
      A review. Metal ions are essential elements present in biol. systems able to facilitate many cellular processes including proliferation, signaling, DNA synthesis and repair. Zinc ion (Zn(II)) is an important cofactor for numerous biochem. reactions. Commonly, structural zinc sites demonstrate high Zn(II) affinity and compact architecture required for sequence-specific macromol. binding. However, how Zn(II)-dependent proteins fold, how their dissocn. occurs, and which factors modulate zinc protein affinity as well as stability remains not fully understood. The mol. rules governing precise regulation of zinc proteins function are hidden in the relationship between sequence and structure, and hence require deep understanding of their folding mechanism under metal load, reactivity and metal-to-protein affinity. Even though, this sequence-structure relationship has an impact on zinc proteins function, it has been shown that other biol. factors including cellular localization and Zn(II) availability influence overall protein behavior. Taking into account all of the mentioned factors, in this review, we aim to describe the relationship between structure-function-stability of zinc structural sites, found in a zinc finger, zinc hook and zinc clasps, and reach far beyond a structural point of view in order to appreciate the balance between chem. and biol. that govern the protein world.
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    40. 40
      Kocyła, A.; Tran, J. B.; Krężel, A. Galvanization of protein–protein interactions in a dynamic zinc interactome. Trends Biochem. Sci. 2021, 46, 64– 79,  DOI: 10.1016/j.tibs.2020.08.011
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      Galvanization of Protein-Protein Interactions in a Dynamic Zinc Interactome
      Kocyla, Anna; Tran, Jozef Ba; Krezel, Artur
      Trends in Biochemical Sciences (2021), 46 (1), 64-79CODEN: TBSCDB; ISSN:0968-0004. (Elsevier Ltd.)
      A review The presence of Zn2+ at protein-protein interfaces modulates complex function, stability, and introduces structural flexibility/complexity, chem. 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 dissoc. and how their stability and reactivity are driven in a zinc interactome remain poorly understood, mostly due to exptl. 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 exptl. challenges required for the identification and investigation of ZPPIs.
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      Tran, J. B.; Krężel, A. InterMetalDB: A database and browser of intermolecular metal binding sites in macromolecules with structural information. J. Proteome Res. 2021, 20, 1889– 1901,  DOI: 10.1021/acs.jproteome.0c00906
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      InterMetalDB: A Database and Browser of Intermolecular Metal Binding Sites in Macromolecules with Structural Information
      Tran, Jozef Ba; Krezel, Artur
      Journal of Proteome Research (2021), 20 (4), 1889-1901CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
      InterMetalDB is a free-of-charge database and browser of intermol. metal binding sites that are present on the interfaces of macromols. 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 resoln., structure description, and date of deposition. Records can be filtered by coordinated metal ion, no. 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.
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      Stracker, T. H.; Petrini, J. H. J. The MRE11 complex: starting from the ends. Nat. Rev. Mol. Cell Biol. 2011, 12, 90– 103,  DOI: 10.1038/nrm3047
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      The MRE11 complex: Starting from the ends
      Stracker, Travis H.; Petrini, John H. J.
      Nature Reviews Molecular Cell Biology (2011), 12 (2), 90-103CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)
      A review. The maintenance of genome stability depends on the DNA damage response (DDR), which is a functional network comprising signal transduction, cell cycle regulation and DNA repair. The metab. of DNA double-strand breaks governed by the DDR is important for preventing genomic alterations and sporadic cancers, and hereditary defects in this response cause debilitating human pathologies, including developmental defects and cancer. The MRE11 complex, composed of the meiotic recombination 11 (MRE11), RAD50 and Nijmegen breakage syndrome 1 (NBS1; also known as nibrin) proteins is central to the DDR, and recent insights into its structure and function were gained from in vitro structural anal. and studies of animal models in which the DDR response is deficient.
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    43. 43
      Hopfner, K.-P.; Craig, L.; Moncalian, G.; Zinkel, R. A.; Usui, T.; Owen, B. A. L.; Karcher, A.; Henderson, B.; Bodmer, J.-L.; McMurray, C. T.; Carney, J. P.; Petrini, J. H. J.; Tainer, J. A. The Rad50 zinc-hook is a structure joining Mre11 complexes in DNA recombination and repair. Nature 2002, 418, 562– 566,  DOI: 10.1038/nature00922
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      The Rad50 zinc-hook is a structure joining Mre11 complexes in DNA recombination and repair
      Hopfner, Karl-Peter; Craig, Lisa; Moncalian, Gabriel; Zinkel, Robert A.; Usui, Takehiko; Owen, Barbara A. L.; Karcher, Annette; Henderson, Brendan; Bodmer, Jean-Luc; McMurray, Cynthia T.; Carney, James P.; Petrini, John H. J.; Tainer, John A.
      Nature (London, United Kingdom) (2002), 418 (6897), 562-566CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      The Mre11 complex (Mre11-Rad50-Nbs1) is central to chromosomal maintenance and functions in homologous recombination, telomere maintenance and sister chromatid assocn. These functions all imply that the linked binding of two DNA substrates occurs, although the mol. basis for this process remains unknown. Here we present a 2.2 Å crystal structure of the Rad50 coiled-coil region that reveals an unexpected dimer interface at the apex of the coiled coils in which pairs of conserved Cys-X-X-Cys motifs form interlocking hooks that bind one Zn2+ ion. Biochem., X-ray and electron microscopy data indicate that these hooks can join oppositely protruding Rad50 coiled-coil domains to form a flexible bridge of up to 1,200 Å. This suggests a function for the long insertion in the Rad50 ABC-ATPase domain. The Rad50 hook is functional, because mutations in this motif confer radiation sensitivity in yeast and disrupt binding at the distant Mre11 nuclease interface. These data support an architectural role for the Rad50 coiled coils in forming metal-mediated bridging complexes between two DNA-binding heads. The resulting assemblies have appropriate lengths and conformational properties to link sister chromatids in homologous recombination and DNA ends in non-homologous end-joining.
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    44. 44
      Park, Y. B.; Hohl, M.; Padjasek, M.; Jeong, E.; Jin, K. S.; Krężel, A.; Petrini, J. H. J.; Cho, Y. Eukaryotic Rad50 functions as a rod-shaped dimer. Nat. Struct. Mol. Biol. 2017, 24, 248– 257,  DOI: 10.1038/nsmb.3369
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      Eukaryotic Rad50 functions as a rod-shaped dimer
      Park, Young Bong; Hohl, Marcel; Padjasek, Michal; Jeong, Eunyoung; Jin, Kyeong Sik; Krezel, Artur; Petrini, John H. J.; Cho, Yunje
      Nature Structural & Molecular Biology (2017), 24 (3), 248-257CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)
      The Rad50 hook interface is crucial for assembly and various functions of the Mre11 complex. Previous analyses suggested that Rad50 mols. interact within (intracomplex) or between (intercomplex) dimeric complexes. In this study, we detd. the structure of the human Rad50 hook and coiled-coil domains. The data suggest that the predominant structure is the intracomplex, in which the two parallel coiled coils proximal to the hook form a rod shape, and that a novel interface within the coiled-coil domains of Rad50 stabilizes the interaction of Rad50 protomers in the dimeric assembly. In yeast, removal of the coiled-coil interface compromised Tel1 activation without affecting DNA repair, while simultaneous disruption of that interface and the hook phenocopied a null mutation. The results demonstrate that the hook and coiled-coil interfaces coordinately promote intracomplex assembly and define the intracomplex as the functional form of the Mre11 complex.
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      Kochańczyk, T.; Jakimowicz, P.; Krężel, A. Femtomolar Zn(II) affinity of minimal zinc hook peptides – a promising small tag for protein engineering. Chem. Commun. 2013, 49, 1312– 1314,  DOI: 10.1039/c2cc38174e
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      Femtomolar Zn(II) affinity of minimal zinc hook peptides - a promising small tag for protein engineering
      Kochanczyk, Tomasz; Jakimowicz, Piotr; Krezel, Artur
      Chemical Communications (Cambridge, United Kingdom) (2013), 49 (13), 1312-1314CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      The minimal zinc hook peptide of Rad50 and its alanine mutants form highly stable Zn(II) complexes. These peptides were successfully used as a small, efficient tag for reversible Zn(II)-mediated protein homodimerization. The high stability, its biol. consequences and potential applications in protein engineering are discussed.
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      Kochańczyk, T.; Nowakowski, M.; Wojewska, D.; Kocyła, A.; Ejchart, A.; Koźmiński, W.; Krężel, A. Metal-coupled folding as the driving force for the extreme stability of Rad50 zinc hook dimer assembly. Sci. Rep. 2016, 6, 36346  DOI: 10.1038/srep36346
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      Metal-coupled folding as the driving force for the extreme stability of Rad50 zinc hook dimer assembly
      Kochanczyk, Tomasz; Nowakowski, Michal; Wojewska, Dominika; Kocyla, Anna; Ejchart, Andrzej; Kozminski, Wiktor; Krezel, Artur
      Scientific Reports (2016), 6 (), 36346CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
      The binding of metal ions at the interface of protein complexes presents a unique and poorly understood mechanism of mol. 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 anal. of the structural and thermodn. 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 const. 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 crit. for the tight assocn. of protein subunits.
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    47. 47
      Padjasek, M.; Maciejczyk, M.; Nowakowski, M.; Kerber, O.; Pyrka, M.; Koźmiński, W.; Krężel, A. Metal exchange in the interprotein Zn(II)-binding site of the Rad50 hook domain: Structural insights into Cd(II)-induced DNA-repair inhibition. Chem. – Eur. J. 2020, 26, 3297– 3313,  DOI: 10.1002/chem.201904942
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      Metal exchange in the interprotein ZnII-binding site of the Rad50 hook domain: Structural insights into CdII-induced DNA-repair inhibition
      Padjasek, Michal; Maciejczyk, Maciej; Nowakowski, Michal; Kerber, Olga; Pyrka, Maciej; Kozminski, Wiktor; Krezel, Artur
      Chemistry - A European Journal (2020), 26 (15), 3297-3313CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
      CdII is a major genotoxic agent that readily displaces ZnII in a multitude of zinc proteins, abrogates redox homeostasis, and deregulates cellular metalloproteome. To date, this displacement has been described mostly for cysteine(Cys)-rich intraprotein binding sites in certain zinc finger domains and metallothioneins. To visualize how a ZnII-to-CdII swap can affect the target protein's status and thus understand the mol. basis of CdII-induced genotoxicity an intermol. ZnII-binding site from the crucial DNA repair protein Rad50 and its zinc hook domain were examd. By using a length-varied peptide base, ZnII-to-CdII displacement in Rad50's hook domain is demonstrated to alter it in a bimodal fashion: (1) CdII induces around a two-orders-of-magnitude stabilization effect (log K12ZnII=20.8 vs. log K12CdII=22.7), which defines an extremely high affinity of a peptide towards a metal ion, and (2) the displacement disrupts the overall assembly of the domain, as shown by NMR spectroscopic and anisotropy decay data. Based on the results, a new model explaining the mol. mechanism of CdII genotoxicity that underlines CdII's impact on Rad50's dimer stability and quaternary structure that could potentially result in abrogation of the major DNA damage response pathway is proposed.
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    48. 48
      Łuczkowski, M.; Padjasek, M.; Tran, J.; Hemmingsen, L.; Kerber, O.; Habjanič, J.; Freisinger, E.; Krężel, A. An extremely stable interprotein tetrahedral Hg(Cys)4 core formed in the zinc hook domain of Rad50 protein at physiological pH. Chem. – Eur. J. 2022, 28, e202202738  DOI: 10.1002/chem.202202738
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      An Extremely Stable Interprotein Tetrahedral Hg(Cys)4 Core Forms in the Zinc Hook Domain of Rad50 Protein at Physiological pH
      Luczkowski, Marek; Padjasek, Michal; Ba Tran, Jozef; Hemmingsen, Lars; Kerber, Olga; Habjanic, Jelena; Freisinger, Eva; Krezel, Artur
      Chemistry - A European Journal (2022), 28 (66), e202202738CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
      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 mercurys 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 obsd. in soln. at physiol. 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 mols. by coordinating ZnII. Dimerized motifs form a unique interprotein zinc hook domain (Hk) that is crit. for the biol. 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 199Hg 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 physiol. pH; this is the first example of an interprotein mercury site displaying tetrahedral geometry in soln. 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 const. of the Rad50 Hg(Hk)2 complex is approx. three orders of magnitude higher than those of the strongest HgII complexes known to date.
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      Fields, G. B.; Noble, R. L. Solid phase peptide synthesis utilizing 9 -fluorenylmethoxycarbonyl amino acids. Int. J. Pept. Protein Res. 2009, 35, 161– 214,  DOI: 10.1111/j.1399-3011.1990.tb00939.x
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      Kocyła, A.; Pomorski, A.; Krężel, A. Molar absorption coefficients and stability constants of metal complexes of 4-(2-pyridylazo) resorcinol (PAR): Revisiting common chelating probe for the study of metalloproteins. J. Inorg. Biochem. 2015, 152, 82– 92,  DOI: 10.1016/j.jinorgbio.2015.08.024
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      Molar absorption coefficients and stability constants of metal complexes of 4-(2-pyridylazo)resorcinol (PAR): Revisiting common chelating probe for the study of metalloproteins
      Kocyla, Anna; Pomorski, Adam; Krezel, Artur
      Journal of Inorganic Biochemistry (2015), 152 (), 82-92CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier)
      4-(2-Pyridylazo)resorcinol (PAR) is one of the most popular chromogenic chelators used in the detn. of the concns. of various metal ions from the d, p and f blocks and their affinities for metal ion-binding biomols. The most important characteristics of such a sensor are the molar absorption coeff. and the metal-ligand complex dissocn. const. However, it must be remembered that these values are dependent on the specific exptl. conditions (e.g. pH, solvent components, and reactant ratios). If one uses these values to process data obtained in different conditions, the final result can be under- or overestimated. We aimed to establish the spectral properties and the stability of PAR and its complexes accurately with Zn2+, Cd2+, Hg2+, Co2+, Ni2+, Cu2+, Mn2+ and Pb2+ at a multiple pH values. The obtained results account for the presence of different species of metal-PAR complexes in the physiol. pH range of 5 to 8 and have been frequently neglected in previous studies. The effective molar absorption coeff. at 492 nm for the ZnHx(PAR)2 complex at pH 7.4 in buffered water soln. is 71,500 M-1 cm- 1, and the dissocn. const. of the complex in these conditions is 7.08 × 10-13 M2. To confirm these values and est. the range of the dissocn. consts. of zinc-binding biomols. that can be measured using PAR, we performed several titrns. of zinc finger peptides (ZF133-11 C@e, TC motif, and MTFI-1) and zinc chelators (cyclam and EGTA). Taken together, our results provide the updated parameters that are applicable to any expt. conducted using inexpensive and com. available PAR.
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      Zelazowski, A. J.; Stillman, M. J. Silver binding to rabbit liver zinc metallothionein and zinc α and β fragments. Formation of silver metallothionein with silver(I):protein ratios of 6, 12, and 18 observed using circular dichroism spectroscopy. Inorg. Chem. 1992, 31, 3363– 3370,  DOI: 10.1021/ic00042a008
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      Silver binding to rabbit liver zinc metallothionein and zinc α and β fragments. Formation of silver metallothionein with silver(I):protein ratios of 6, 12, and 18 observed using circular dichroism spectroscopy
      Zelazowski, Andrzej J.; Stillman, Martin J.
      Inorganic Chemistry (1992), 31 (16), 3363-70CODEN: INOCAJ; ISSN:0020-1669.
      Formation of a series of complexes between Ag(I) and the cysteine thiolate groups in rabbit liver zinc metallothionein (MT) and the Zn3-β MT 1 and Zn4-α MT 1 fragments is reported from anal. of the CD spectral data recorded between 5 and 55° during titrns. of the protein with Ag(I). The spectral envelopes reveal formation of Ag12-MT, Ag18-MT, Ag6-α MT, and Ag6-β MT. Silver(I)-thiolate complex formation is assocd. with characteristic CD spectral envelopes and depends on the stoichiometric ratio of Ag:MT, the temp., and the pH. The presence of the tetrahedrally-coordinated Zn(II) in Zn7-MT 2 inhibits formation of the Ag6-MT and Ag12-MT species previously obsd. when Ag+ binds to apo-MT 2 at 20°, and formation of Ag18-MT dominates the spectral traces. At 55°, the Ag12-MT species does form. Addn. of Ag+ at pH 3.8 and 55° to Zn7-MT 2 (nominally apo-MT 2) results in a different sequence of complexes forming in the range Ag+:MT = 1-18. Anal. of the CD spectral data suggests that the low pH enhances formation of Ag6-S9 clusters in the β domain, characterized by a single band at 254 nm, inhibits formation of Ag6-S11 clusters in the α domain, and sats. the binding sites with the formation of Ag18-MT. The CD spectral envelopes obtained as Ag+ was added to solns. of the Zn4-α MT and Zn3-β MT fragments clearly show for the first time spectral signatures assocd. with formation of both Ag6-α MT 1 and Ag6-β MT 1 complexes, resp. The CD spectral characteristics of the Ag6 fragments match the spectral patterns obsd. for Agn-MT (n = 6, 12) formed from Zn7-MT 2. A new species forms at high mole ratios of Ag:MT with Zn4-α MT 1. Tentatively written as Ag12-α MT 1, this complex shows an intense CD spectrum which suggests that its structure may be similar to the supercoil postulated previously for Hg18-MT 2 (Cai, W.; et al., 1988). Metal anal. for titrns. of Zn7-MT 2 at pH 7 shows that each Zn2+ is displaced by 2 Ag+ ions, so that stoichiometric amts. of Zn2+ remain bound to the protein up to the 14 Ag+ point.
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      Płonka, D.; Kotuniak, R.; Dąbrowska, K.; Bal, W. Electrospray-induced mass spectrometry is not suitable for determination of peptidic Cu(II) complexes. J. Am. Soc. Mass Spectrom. 2021, 32, 2766– 2776,  DOI: 10.1021/jasms.1c00206
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      Electrospray-Induced Mass Spectrometry Is Not Suitable for Determination of Peptidic Cu(II) Complexes
      Plonka, Dawid; Kotuniak, Radoslaw; Dabrowska, Katarzyna; Bal, Wojciech
      Journal of the American Society for Mass Spectrometry (2021), 32 (12), 2766-2776CODEN: JAMSEF; ISSN:1879-1123. (American Chemical Society)
      The toolset of mass spectrometry (MS) is still expanding, and the no. of metal ion complexes researched this way is growing. The Cu(II) ion forms particularly strong peptide complexes of biol. interest which are frequent objects of MS studies, but quant. aspects of some reported results are at odds with those of expts. performed in soln. Cu(II) complexes are usually characterized by fast ligand exchange rates, despite their high affinity, and we speculated that such kinetic lability could be responsible for the obsd. discrepancies. In order to resolve this issue, we selected peptides belonging to the ATCUN family characterized with high and thoroughly detd. Cu(II) binding consts. and re-estd. them using two ESI-MS techniques: std. conditions in combination with serial diln. expts. and very mild conditions for competition expts. The sample acidification, which accompanies the electrospray formation, was simulated with the pH-jump stopped-flow technique. Our results indicate that ESI-MS should not be used for quant. studies of Cu(II)-peptide complexes because the electrospray formation process compromises the entropic contribution to the complex stability, yielding underestimations of complex stability consts.
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    53. 53
      Peris-Díaz, M. D.; Guran, R.; Domene, C.; de los Rios, V.; Zitka, O.; Adam, V.; Krężel, A. An integrated mass spectrometry and molecular dynamics simulations approach reveals the spatial organization impact of metal-binding sites on the stability of metal-depleted metallothionein-2 Species. J. Am. Chem. Soc. 2021, 143, 16486– 16501,  DOI: 10.1021/jacs.1c05495
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      An Integrated Mass Spectrometry and Molecular Dynamics Simulations Approach Reveals the Spatial Organization Impact of Metal-Binding Sites on the Stability of Metal-Depleted Metallothionein-2 Species
      Peris-Diaz, Manuel David; Guran, Roman; Domene, Carmen; de los Rios, Vivian; Zitka, Ondrej; Adam, Vojtech; Krezel, Artur
      Journal of the American Chemical Society (2021), 143 (40), 16486-16501CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Mammalian metallothioneins (MTs) are a group of cysteine-rich proteins that bind metal ions in two α- and β-domains and represent a major cellular Zn(II)/Cu(I) buffering system in the cell. At cellular free Zn(II) concns. (10-11-10-9 M), MTs do not exist in fully loaded forms with seven Zn(II)-bound ions (Zn7MTs). Instead, MTs exist as partially metal-depleted species (Zn4-6MT) because their Zn(II) binding affinities are on the nano- to picomolar range comparable to the concns. of cellular Zn(II). The mode of action of MTs remains poorly understood, and thus, the aim of this study is to characterize the mechanism of Zn(II) (un)binding to MTs, the thermodn. properties of the Zn1-6MT2 species, and their mechanostability properties. To this end, native mass spectrometry (MS) and label-free quant. bottom-up and top-down MS in combination with steered mol. dynamics simulations, well-tempered metadynamics (WT-MetaD), and parallel-bias WT-MetaD (amounting to 3.5μs) were integrated to unravel the chem. coordination of Zn(II) in all Zn1-6MT2 species and to explain the differences in binding affinities of Zn(II) ions to MTs. Differences are the result of the degree of water participation in MT (un)folding and the hyper-reactive character of Cys21 and Cys29 residues. The thermodn. properties of Zn(II) (un)binding to MT2 differ from those of Cd(II), justifying their distinctive roles. The potential of this integrated strategy in the study of numerous unexplored metalloproteins is attested by the results highlighted.
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      Kostyukevich, Y.; Kononikhin, A.; Popov, I.; Indeykina, M.; Kozin, S. A.; Makarov, A. A.; Nikolaev, E. Supermetallization of peptides and proteins during electrospray ionization. J. Mass Spectrom. 2015, 50, 1079– 1087,  DOI: 10.1002/jms.3622
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      Supermetallization of peptides and proteins during electrospray ionization
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      Journal of Mass Spectrometry (2015), 50 (9), 1079-1087CODEN: JMSPFJ; ISSN:1076-5174. (John Wiley & Sons Ltd.)
      The formation of metal-peptide complexes during electrospray ionization (ESI) is a widely known phenomenon and is often considered to be undesirable. Such effect considerably limits the use of ESI mass spectrometry for the study of biol. relevant metal-peptide compds. that are present in the soln. and play crit. roles in many bioprocesses such as progression of neurodegenerative diseases. Under specific conditions such as high temp. of the desolvating capillary, an interesting effect, which can be called supermetallization, occurs. Using a model peptide Aβ amyloid domain 1-16, an increase in the temp. of the desolvating capillary results in multiple substitutions of hydrogen atoms by Zn atoms in this peptide. At high temps. (T ∼ 400°), up to 11 zinc atoms can be covalently bound to (1-16) Aβ. Supermetallization of (1-16) Aβ depends on the solvent compn. and pH. Supermetallization was also demonstrated for proteins, such as ubiquitin and cytochrome C. That proves that the supermetallization is a general phenomenon for peptides and proteins. For the structural study of supermetallized complexes, electron-capture dissocn. (ECD) fragmentation was applied. The effect of hydrogen rearranging during ECD was obsd. In addn., quantum chem. calcns. were used to est. the possible structures of different supermetallized complexes. These results allow a more deep understanding of the limitations of the use of ESI mass spectrometry for the study of biol. relevant metal-peptide complexes.
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      Krężel, A.; Wójcik, J.; Maciejczyk, M.; Bal, W. May GSH and L-His contribute to intracellular binding of zinc? Thermodynamic and solution structural study of a ternary complex. Chem. Commun. 2003, 704– 705,  DOI: 10.1039/b300632h
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      Chemical Communications (Cambridge, United Kingdom) (2003), (6), 704-705CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      GSH and L-His are abundant biomols. and likely biol. ligands for Zn(II) under certain conditions. Potentiometric titrns. provide evidence of formation of ternary Zn(II) complexes with GSH and L-His or D-His with slight stereoselectivity in favor of L-His (ca. 1 log unit of stability const.). The soln. structure of the ZnH(GSH)(L-His)(H2O) complex at pH 6.8, detd. by NMR, includes tridentate L-His, monodentate (sulfur) GSH, and weak interligand interactions. Calcns. of competitiveness of this complex for Zn(II) binding at pH 7.4 indicate that it is likely to be formed in vivo under conditions of GSH depletion. Otherwise, GSH alone emerges as a likely Zn(I) carrier.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXhslOks70%253D&md5=5de4e1520fa0b79b9d8ca2304aee41d2
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      Lafrance-Vanasse, J.; Williams, G. J.; Tainer, J. A. Envisioning the dynamics and flexibility of Mre11-Rad50-Nbs1 complex to decipher its roles in DNA replication and repair. Prog. Biophys. Mol. Biol. 2015, 117, 182– 193,  DOI: 10.1016/j.pbiomolbio.2014.12.004
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      Envisioning the dynamics and flexibility of Mre11-Rad50-Nbs1 complex to decipher its roles in DNA replication and repair
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      The Mre11-Rad50-Nbs1 (MRN) complex is a dynamic macromol. machine that acts in the first steps of DNA double strand break repair, and each of its components has intrinsic dynamics and flexibility properties that are directly linked with their functions. As a result, deciphering the functional structural biol. of the MRN complex is driving novel and integrated technologies to define the dynamic structural biol. of protein machinery interacting with DNA. Rad50 promotes dramatic long-range allostery through its coiled-coil and zinc-hook domains. Its ATPase activity drives dynamic transitions between monomeric and dimeric forms that can be modulated with mutants modifying the ATPase rate to control end joining vs. resection activities. The biol. functions of Mre11's dual endo- and exonuclease activities in repair pathway choice were enigmatic until recently, when they were unveiled by the development of specific nuclease inhibitors. Mre11 dimer flexibility, which may be regulated in cells to control MRN function, suggests new inhibitor design strategies for cancer intervention. Nbs1 has FHA and BRCT domains to bind multiple interaction partners that further regulate MRN. One of them, CtIP, modulates the Mre11 excision activity for homologous recombination repair. Overall, these combined properties suggest novel therapeutic strategies. Furthermore, they collectively help to explain how MRN regulates DNA repair pathway choice with implications for improving the design and anal. of cancer clin. trials that employ DNA damaging agents or target the DNA damage response.
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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.inorgchem.2c03767.

    • Materials; RP-HPLC chromatograms of purified Hk14 and Hk45 peptides and the corresponding ESI-MS spectra; figures with spectra from UV–vis, CD, and ESI-MS experiments; tables with all the assigned m/z values in ESI-MS experiments; supplementary table with values calculated in the ITC experiment (PDF)


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