Hydrolytic Mechanism of a Metalloenzyme Is Modified by the Nature of the Coordinated Metal Ion.

The nuclease domain of colicin E7 cleaves double-strand DNA non-specifically. Zn2+ ion was shown to be coordinated by the purified NColE7 as its native metal ion. Here, we study the structural and catalytic aspects of the interaction with Ni2+, Cu2+ and Cd2+ non-endogenous metal ions and the consequences of their competition with Zn2+ ions, using circular dichroism spectroscopy and intact protein mass spectrometry. An R447G mutant exerting decreased activity allowed for the detection of nuclease action against pUC119 plasmid DNA via agarose gel electrophoresis in the presence of comparable metal ion concentrations. It was shown that all of the added metal ions could bind to the apoprotein, resulting in a minor secondary structure change, but drastically shifting the charge distribution of the protein. Zn2+ ions could not be replaced by Ni2+, Cu2+ and Cd2+. The nuclease activity of the Ni2+-bound enzyme was extremely high in comparison with the other metal-bound forms, and could not be inhibited by the excess of Ni2+ ions. At the same time, this activity was significantly decreased in the presence of equivalent Zn2+, independent of the order of addition of each component of the mixture. We concluded that the Ni2+ ions promoted the DNA cleavage of the enzyme through a more efficient mechanism than the native Zn2+ ions, as they directly generate the nucleophilic OH- ion.

Zinc binding of a Cys2His2-type zinc finger protein is enhanced by the interaction with DNA.

Zinc finger proteins specifically recognize DNA sequences and, therefore, play a crucial role in living organisms. In this study the Zn(II)-, and DNA-binding of 1MEY#, an artificial zinc finger protein consisting of three finger units was characterized by multiple methods. Fluorimetric, circular dichroism and isothermal calorimetric titrations were applied to determine the accurate stability constant of a zinc finger protein. Assuming that all three zinc finger subunits behave identically, the obtained thermodynamic data for the Zn(II) binding were ΔHbinding site = - (23.5 - 28.0) kcal/mol (depending on the applied protonation state of the cysteines) and logß'pH 7.4 = 12.2 ± 0.1, being similar to those of the CP1 consensus zinc finger peptide. The specific DNA binding of the protein can be characterized by logß'pH 7.4 = 8.20 ± 0.08, which is comparable to the affinity of the natural zinc finger proteins (Sp1, WT1, TFIIIA) toward DNA. This value is ~ 1.9 logß' unit higher than those determined for semi- or nonspecific DNA binding. Competitive circular dichroism and electrophoretic mobility shift measurements revealed that the conditional stability constant characteristic for Zn(II) binding of 1MEY# protein increased by 3.4 orders of magnitude in the presence of its target DNA sequence.

The coordination modes of (thio)semicarbazone copper(II) complexes strongly modulate the solution chemical properties and mechanism of anticancer activity.

Thiosemicarbazones are promising candidates for anticancer therapy and their mechanism of action is often linked to their metal chelating ability. In this study, five (thio)semicarbazones with different donor sets (NNS, NNO, ONS, ONO) were selected and their behaviour in aqueous solution, the stability of their copper(II) complexes in addition to their cytotoxicity, DNA-binding, DNA cleavage ability and inhibition of topoisomerase IIα were investigated and compared. We aimed to reveal relationships between the structural variations, the significantly different physico-chemical properties, solution speciation and biological activity. The cytotoxicity of the ligands did not show correlation with the solubility, lipophilicity and permeability; and the decreased activity of the oxygen donor containing compounds was explained by their stronger preference towards chelation of iron(III) over iron(II). Meanwhile, among the copper complexes the most lipophilic species with the highest stability and membrane permeability exhibited the highest cytotoxicity. The studied copper(II) complexes interact with DNA, and reaction with glutathione led to heavy DNA cleavage in the case of the highly stable complexes which could be reduced in a reversible reaction with moderate rate. All the tested copper complexes inhibited topoisomerase IIα, however, this property of the complexes with low stability is most probably linked to the liberated free copper(II).

Structures of Silver Fingers and a Pathway to Their Genotoxicity.

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

Estradiol-Based Salicylaldehyde (Thio)Semicarbazones and Their Copper Complexes with Anticancer, Antibacterial and Antioxidant Activities.

A series of novel estradiol-based salicylaldehyde (thio)semicarbazones ((T)SCs) bearing (O,N,S) and (O,N,O) donor sets and their Cu(II) complexes were developed and characterized in detail by 1H and ¹³C nuclear magnetic resonance spectroscopy, UV-visible and electron paramagnetic resonance spectroscopy, electrospray ionization mass spectrometry and elemental analysis. The structure of the Cu(II)-estradiol-semicarbazone complex was revealed by X-ray crystallography. Proton dissociation constants of the ligands and stability constants of the metal complexes were determined in 30% (v/v) DMSO/H2O. Estradiol-(T)SCs form mono-ligand complexes with Cu(II) ions and exhibit high stability with the exception of estradiol-SC. The Cu(II) complexes of estradiol-TSC and its N,N-dimethyl derivative displayed the highest cytotoxicity among the tested compounds in MCF-7, MCF-7 KCR, DU-145, and A549 cancer cells. The complexes do not damage DNA according to both in vitro cell-free and cellular assays. All the Cu(II)-TSC complexes revealed significant activity against the Gram-positive Staphylococcus aureus bacteria strain. Estradiol-TSCs showed efficient antioxidant activity, which was decreased by complexation with Cu(II) ions. The exchange of estrone moiety to estradiol did not result in significant changes to physico-chemical and biological properties.

Modulation of the catalytic activity of a metallonuclease by tagging with oligohistidine.

Peptide tags are extensively used for affinity purification of proteins. In an optimal case, these tags can be completely removed from the purified protein by a specific protease mediated hydrolysis. However, the interactions of these tags with the target protein may also be utilized for the modulation of the protein function. Here we show that the C-terminal hexahistidine (6 × His) tag can influence the catalytic activity of the nuclease domain of the Colicin E7 metallonuclease (NColE7) used by E. coli to kill competing bacteria under stress conditions. This enzyme non-specifically cleaves the DNA that results in cytotoxicity. We have successfully cloned the genes of NColE7 protein and its R447G mutant into a modified pET-21a DNA vector fusing the affinity tag to the protein upon expression, which would be otherwise not possible in the absence of the gene of the Im7 inhibitory protein. This reflects the inhibitory effect of the 6 × His fusion tag on the nuclease activity, which proved to be a complex process via both coordinative and non-specific steric interactions. The modulatory effect of Zn2+ ion was observed in the catalytic activity experiments. The DNA cleavage ability of the 6 × His tagged enzyme was first enhanced by an increase of metal ion concentration, while high excess of Zn2+ ions caused a lower rate of the DNA cleavage. Modelling of the coordinative effect of the fusion tag by external chelators suggested ternary complex formation instead of removal of the metal ion from the active center.

Purification of proteins with native terminal sequences using a Ni(II)-cleavable C-terminal hexahistidine affinity tag.

The role of the termini of protein sequences is often perturbed by remnant amino acids after the specific protease cleavage of the affinity tags and/or by the amino acids encoded by the plasmid at/around the restriction enzyme sites used to insert the genes. Here we describe a method for affinity purification of a metallonuclease with its precisely determined native termini. First, the gene encoding the target protein is inserted into a newly designed cloning site, which contains two self-eliminating BsmBI restriction enzyme sites. As a consequence, the engineered DNA code of Ni(II)-sensitive Ser-X-His-X motif is fused to the 3'-end of the inserted gene followed by the gene of an affinity tag for protein purification purpose. The C-terminal segment starting from Ser mentioned above is cleaved off from purified protein by a Ni(II)-induced protease-like action. The success of the purification and cleavage was confirmed by gel electrophoresis and mass spectrometry, while structural integrity of the purified protein was checked by circular dichroism spectroscopy. Our new protein expression DNA construct is an advantageous tool for protein purification, when the complete removal of affinity or other tags, without any remaining amino acid residue is essential. The described procedure can easily be generalized and combined with various affinity tags at the C-terminus for chromatographic applications.

Algal cell response to laboratory-induced cadmium stress: a multimethod approach.

We examined the response of algal cells to laboratory-induced cadmium stress in terms of physiological activity, autonomous features (motility and fluorescence), adhesion dynamics, nanomechanical properties, and protein expression by employing a multimethod approach. We develop a methodology based on the generalized mathematical model to predict free cadmium concentrations in culture. We used algal cells of Dunaliella tertiolecta, which are widespread in marine and freshwater systems, as a model organism. Cell adaptation to cadmium stress is manifested through cell shape deterioration, slower motility, and an increase of physiological activity. No significant change in growth dynamics showed how cells adapt to stress by increasing active surface area against toxic cadmium in the culture. It was accompanied by an increase in green fluorescence (most likely associated with cadmium vesicular transport and/or beta-carotene production), while no change was observed in the red endogenous fluorescence (associated with chlorophyll). To maintain the same rate of chlorophyll emission, the cell adaptation response was manifested through increased expression of the identified chlorophyll-binding protein(s) that are important for photosynthesis. Since production of these proteins represents cell defence mechanisms, they may also signal the presence of toxic metal in seawater. Protein expression affects the cell surface properties and, therefore, the dynamics of the adhesion process. Cells behave stiffer under stress with cadmium, and thus, the initial attachment and deformation are slower. Physicochemical and structural characterizations of algal cell surfaces are of key importance to interpret, rationalize, and predict the behaviour and fate of the cell under stress in vivo.

Nickel(ii)-promoted specific hydrolysis of zinc finger proteins.

In this work we demonstrate that the previously described reaction of sequence specific Ni(ii)-dependent hydrolytic peptide bond cleavage can be performed in complex metalloprotein molecules, such as the Cys2His2 zinc finger proteins. The cleavage within a zinc finger unit possessing a (Ser/Thr)-X-His sequence is not hindered by the presence of the Zn(ii) ions. It results in loss of the Zn(ii) ion, oxidation of the SH groups and thus, in a collapse of the functional structure. We show that such natural Ni(ii)-cleavage sites in zinc finger domains can be edited out without compromising the DNA binding specificity. Inserting a Ni(ii)-susceptible sequence between the edited zinc finger and an affinity tag allows for removal of the latter sequence by Ni(ii) ions after the protein purification. We have shown that this reaction can be executed even when a metal ion binding N-terminal His-tag is present. The cleavage product maintains the native zinc finger structure involving Zn(ii) ions. Mass spectra revealed that a Ni(ii) ion remains coordinated to the hydrolyzed protein product through the N-terminal (Ser/Thr)-X-His tripeptide segment. The fact that the Ni(ii)-dependent protein hydrolysis is influenced by the Ni(ii) concentration, pH and temperature of the reaction provides a platform for novel regulated DNA effector design.

Benchmark ab Initio Characterization of the Complex Potential Energy Surfaces of the X- + NH2Y [X, Y = F, Cl, Br, I] Reactions.

We report a comprehensive high-level explicitly correlated ab initio study on the X- + NH2Y [X,Y = F, Cl, Br, I] reactions characterizing the stationary points of the SN2 (Y- + NH2X) and proton-transfer (HX + NHY-) pathways as well as the reaction enthalpies of various endothermic additional product channels such as H- + NHXY, XY- + NH2, XY + NH2-, and XHY- + NH. Benchmark structures and harmonic vibrational frequencies are obtained at the CCSD(T)-F12b/aug-cc-pVTZ(-PP) level of theory, followed by CCSD(T)-F12b/aug-cc-pVnZ(-PP) [n = Q and 5] and core correlation energy computations. In the entrance and exit channels we find two equivalent hydrogen-bonded C1 minima, X-···HH'NY and X-···H'HNY connected by a Cs first-order saddle point, X-···H2NY, as well as a halogen-bonded front-side complex, X-···YNH2. SN2 reactions can proceed via back-side attack Walden inversion and front-side attack retention pathways characterized by first-order saddle points, submerged [X-NH2-Y]- and high-energy [H2NXY]-, respectively. Product-like stationary points below the HX + NHY- asymptotes are involved in the proton-transfer processes.