Impact of metal coordination and pH on the antimicrobial activity of histatin 5 and the products of its hydrolysis.

This work focuses on the relationship between the coordination chemistry and antimicrobial activity of Zn(II) and Cu(II) complexes of histatin 5 and the products of its hydrolysis: its N-terminal fragment (histatin 5-8) and C-terminal fragment (histatin 8). Cu(II) coordinates in an albumin-like binding mode and Zn(II) binds to up to 3 His imidazoles. The antimicrobial activity of histatins and their metal complexes (i) strongly depends on pH - they are more active at pH 5.4 than at 7.4; (ii) the complexes and ligands alone are more effective in eradicating Gram-positive bacteria than the Gram-negative ones, and (iii) Zn(II) coordination is able to change the structure of the N-terminal region of histatin 5 (histatin 5-8) and moderately increase all of the studied histatins' antimicrobial potency.

Characterization of four peptides from milk fermented with kombucha cultures and their metal complexes-in search of new biotherapeutics.

The most common skin diseases include eczema, psoriasis, acne, and fungal infections. There is often no effective cure for them. Increasing antimicrobial drug resistance prompts us to search for new, safe, and effective therapeutics. Among such interesting candidates are peptides derived from milk fermented with specific lactic acid bacteria or with kombucha cultures, which are a potential treasure trove of bioactive peptides. Four of them are discussed in this article. Their interactions with zinc and copper ions, which are known to improve the well-being of the skin, were characterized by potentiometry, MS, ITC, and spectroscopic methods, and their cytostatic potential was analyzed. The results suggest that they are safe for human cells and can be used alone or in complexes with copper for further testing as potential therapeutics for skin diseases.

Aspergillus fumigatus ZrfC Zn(II) transporter scavengers zincophore-bound Zn(II).

Aspergillus fumigatus is an opportunistic pathogen that is able to invade and grow in the lungs of immunosuppressed patients and cause invasive pulmonary aspergillosis. The concentration of free Zn(II) in living tissues is much lower than that required for optimal fungal growth; thus, to obtain Zn(II) from the host, Aspergillus fumigatus uses highly specified Zn(II) transporters: ZrfA, ZrfB and ZrfC. The ZrfC transporter plays the main role in Zn(II) acquisition from the host in neutral and mildly alkaline environment via interacting with the secreted Aspf2 zincophore. Understanding the Aspf2-ZrfC interactions is therefore necessary for explaining the process of Zn(II) acquisition by Aspergillus fumigatus, and identifying Zn(II) binding sites in its transporter and describing the thermodynamics of such binding are the fundamental steps to achieve this goal. We focus on two probable ZrfC Zn(II) binding sites and show that the Ac-MNCHFHAGVEHCIGAGESESGSSQ-NH2 region binds Zn(II) with higher affinity than the Ac-TGCHSHGS-NH2 one and that this binding is much stronger than the binding of Zn(II) to the Aspf2 zincophore, allowing efficient Zn(II) transport from the Aspf2 zincophore to the ZrfC transporter. The same ZrfC fragments also able to bind Ni(II), another metal ion essential for fungi that could also compete with Zn(II) binding, with comparable affinity.

Bioinorganic chemistry of shepherin II complexes helps to fight Candida albicans?

The fungal cell wall and cell membrane are an important target for antifungal therapies, and a needle-like cell wall or membrane disruption may be an entirely novel antifungal mode of action. In this work, we show how the coordination of Zn(II) triggers the antifungal properties of shepherin II, a glycine- and histidine-rich antimicrobial peptide from the root of Capsella bursa-pastoris. We analyze Cu(II) and Zn(II) complexes of this peptide using experimental and theoretical methods, such as: mass spectrometry, potentiometry, UV-Vis and CD spectroscopies, AFM imaging, biological activity tests and DFT calculations in order to understand the correlation between their metal binding mode, structure, morphology and biological activity. We observe that Zn(II) coordinates to Shep II and causes a structural change, resulting in fibril formation, what has a pronounced biological consequence - a strong anticandidal activity. This phenomenon was observed neither for the peptide itself, nor for its copper(II) complex. The Zn(II) - shepherin II complex can be considered as a starting point for further anticandidal drug discovery, which is extremely important in the era of increasing antifungal drug resistance.

-HH and -HAAAH motifs act as fishing nets for biologically relevant metal ions in metallopeptides.

Histidine are one of the most common residues involved in transition metal ion binding in the active sites of metalloenzymes. In order to mimic enzymatic metal binding sites, it is crucial to understand the basic coordination modes of histidine residues, distributed at different positions in the peptide sequence. We show that: (i) the separation of two histidines has a large effect on complex stability - a sequence with adjusting histidine residues forms more stable complexes with Zn(II) than the one in which the residues are separated, while the contrary is observed for Cu(II) complexes, in which amide nitrogens participate in metal binding. No pronounced effect is observed for Ni(II) complexes, where the amides participate in binding at higher pH; (ii) non-coordinating amino acid residues (basic, acidic and aromatic ones) have a significant impact on complex stability; charged and aromatic residues may enhance Zn(II) binding, while the contrary is observed for the amide-binding Cu(II); (iii) cysteine containing sequences are much more effective Zn(II) and Ni(II) binding motifs at pH above 8, while histidine containing ligands are more suitable for effective Zn(II) and Ni(II) binding at lower pH.

Zn(II) Induces Fibril Formation and Antifungal Activity in Shepherin I, An Antimicrobial Peptide from Capsella bursa-pastoris.

Shepherin I is a glycine- and histidine-rich antimicrobial peptide from the root of a shepherd's purse, whose antimicrobial activity was suggested to be enhanced by the presence of Zn(II) ions. We describe Zn(II) and Cu(II) complexes of this peptide, aiming to understand the correlation between their metal binding mode, structure, morphology, and biological activity. We observe a logical sequence of phenomena, each of which is the result of the previous one: (i) Zn(II) coordinates to shepherin I, (ii) causes a structural change, which, in turn, (iii) results in fibril formation. Eventually, this chain of structural changes has a (iv) biological consequence: The shepherin I-Zn(II) fibrils are highly antifungal. What is of particular interest, both fibril formation and strong anticandidal activity are only observed for the shepherin I-Zn(II) complex, linking its structural rearrangement that occurs after metal binding with its morphology and biological activity.

Metal coordination to solute binding proteins - exciting chemistry with potential biological meaning.

Zn(II) is essential for bacterial survival and virulence. In host cells, its abundance is extremely limited, thus, bacteria have evolved transport mechanisms that enable them to take up this essential metal nutrient. Paracoccus denitrificans encodes two solute binding proteins (SBPs) - ZnuA and AztC, which are responsible for zinc acquisition from the host cells. We focus on understanding the interactions of Zn(II) and Ni(II) (zinc's potential competitor, which is a biologically relevant metal ion essential for various bacterial enzymes) with the extracellular ZnuA and AztC's loops from P. denitrificans that are expected to be possible Zn(II) binding sites. In the case of Zn(II) complexes with ZnuA outercellular loop regions, the numerous histidines act as anchoring donors, forming complexes with up to four coordinated His residues, while in the AztC region, three imidazole nitrogens and one water molecule are involved in Zn(II) binding. In Zn(II) complexes with ZnuA His-rich loop regions, so-called polymorphic binding sites are observed. The large number of available imidazoles and carboxylic side chains also strongly affects the structure of Ni(II) complexes; the more histidines in the studied peptide, the higher the affinity to bind Ni(II) and the higher the pH value at which amide nitrogens start to participate in Ni(II) binding. Additionally, for Ni(II)-ZnuA complexes, a more rare octahedral geometry is observed and such complexes are more stable than the corresponding Zn(II) ones, in contrast to what was observed in the AztC region, suggesting that the numerous histidyl and glutamic acid side chains are more tempting for Ni(II) than for Zn(II).The general strong affinity of Zn(II)-zincophore complexes is also discussed.

Impact of C- and N-terminal protection on the stability, metal chelation and antimicrobial properties of calcitermin.

The main limitation to the use of antimicrobial peptides (AMPs) as regular drugs, against antibiotic and antifungal resistance, mainly relates to their rapid degradation by proteolytic enzymes. The introduction of suitable structural changes in the peptide chain can make the peptide less susceptible to the action of proteases, thus overcoming this problem. To improve the plasma stability of calcitermin, a metal-chelating AMP present in the human respiratory tract and investigated in the present study, C- and/or N- terminal modifications have been introduced in the native sequence. Evaluation of peptide stability has been performed to determine the half-life times in human plasma of both native calcitermin and its derivatives. However, the protection of the peptide termini can also affect its metal coordination behaviour. Thus, the characterization of Zn2+ and Cu2+ complexes has been performed by means of several techniques, including potentiometry, high-resolution mass spectrometry, UV-Vis, circular dichroism and EPR. On the basis of the obtained results, it was possible to compare the biological activity of the studied systems, taking into account both the metal-binding ability and the peptide stability to search for a link among them. A significant result of this study is that the N-terminal protection increases the calcitermin half-life over seven times and the formation of metal complexes confers resistance towards degradation almost doubling its half-life.

Semenogelins Armed in Zn(II) and Cu(II): May Bioinorganic Chemistry Help Nature to Cope with Enterococcus faecalis?

Proteolytic degradation of semenogelins, the most abundant proteins from human semen, results in the formation of 26- and 29-amino acid peptides (SgIIA and SgI-29, respectively), which share a common 15 amino acid fragment (Sg-15). All three ligands are effective Zn(II) and Cu(II) binders; in solution, a variety of differently metalated species exist in equilibrium, with the [NH2, 3Nim] donor set prevailing at physiological pH in the case of both metals. For the first time, the Cu(II)-induced antimicrobial activity of Sg-15 against Enterococcus faecalis is shown. In the case of the two native semenogelin fragment metal complexes, the strong local positive charge in the metal-bound HH motif correlates well with their antimicrobial activity. A careful analysis of semenogelins' metal coordination behavior reveals two facts: (i) The histamine-like Cu(II) binding mode of SgI-29 strongly increases the stability of such a complex below pH 6 (with respect to the non-histamine-like binding of SgIIA), while in the case of the SgI-29 Zn(II)-histamine-like species, the stability enhancement is less pronounced. (ii) The HH sequence is a more tempting site for Cu(II) ions than the HXH one.

CH vs. HC-Promiscuous Metal Sponges in Antimicrobial Peptides and Metallophores.

Histidine and cysteine residues, with their imidazole and thiol moieties that deprotonate at approximately physiological pH values, are primary binding sites for Zn(II), Ni(II) and Fe(II) ions and are thus ubiquitous both in peptidic metallophores and in antimicrobial peptides that may use nutritional immunity as a way to limit pathogenicity during infection. We focus on metal complex solution equilibria of model sequences encompassing Cys-His and His-Cys motifs, showing that the position of histidine and cysteine residues in the sequence has a crucial impact on its coordination properties. CH and HC motifs occur as many as 411 times in the antimicrobial peptide database, while similar CC and HH regions are found 348 and 94 times, respectively. Complex stabilities increase in the series Fe(II) < Ni(II) < Zn(II), with Zn(II) complexes dominating at physiological pH, and Ni(II) ones-above pH 9. The stabilities of Zn(II) complexes with Ac-ACHA-NH2 and Ac-AHCA-NH2 are comparable, and a similar tendency is observed for Fe(II), while in the case of Ni(II), the order of Cys and His does matter-complexes in which the metal is anchored on the third Cys (Ac-AHCA-NH2) are thermodynamically stronger than those where Cys is in position two (Ac-ACHA-NH2) at basic pH, at which point amides start to take part in the binding. Cysteine residues are much better Zn(II)-anchoring sites than histidines; Zn(II) clearly prefers the Cys-Cys type of ligands to Cys-His and His-Cys ones. In the case of His- and Cys-containing peptides, non-binding residues may have an impact on the stability of Ni(II) complexes, most likely protecting the central Ni(II) atom from interacting with solvent molecules.

Spectroscopic Characterization and Biological Activity of Hesperetin Schiff Bases and Their Cu(II) Complexes.

The three Schiff base ligands, derivatives of hesperetin, HHSB (N-[2,3-dihydro-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chromen-4-ylidene]isonicotinohydrazide), HIN (N-[2,3-dihydro-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chromen-4-ylidene]benzhydrazide) and HTSC (N-[2,3-dihydro-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chromen-4-ylidene]thiosemicarbazide) and their copper complexes, CuHHSB, CuHIN, and CuHTSC were designed, synthesized and analyzed in terms of their spectral characterization and the genotoxic activity. Their structures were established using several methods: elemental analysis, FT-IR, UV-Vis, EPR, and ESI-MS. Spectral data showed that in the acetate complexes the tested Schiff bases act as neutral tridentate ligand coordinating to the copper ion through two oxygen (or oxygen and sulphur) donor atoms and a nitrogen donor atom. EPR measurements indicate that in solution the complexes keep their structures with the ligands remaining bound to copper(II) in a tridentate fashion with (O-, N, Oket) or (O-, N, S) donor set. The genotoxic activity of the compounds was tested against model tumour (HeLa and Caco-2) and normal (LLC-PK1) cell lines. In HeLa cells the genotoxicity for all tested compounds was noticed, for HHSB and CuHHSB was the highest, for HTSC and CuHTSC-the lowest. Generally, Cu complexes displayed lower genotoxicity to HeLa cells than ligands. In the case of Caco-2 cell line HHSB and HTSC induced the strongest breaks to DNA. On the other side, CuHHSB and CuHTSC induced the highest DNA damage against LLC-PK1.

Zn(II) binding to pramlintide results in a structural kink, fibril formation and antifungal activity.

The antimicrobial properties of amylin, a 37-amino acid peptide hormone, co-secreted with insulin from the pancreas, are far less known than its antidiabetic function. We provide insight into the bioinorganic chemistry of amylin analogues, showing that the coordination of zinc(II) enhances the antifungal properties of pramlintide, a non-fibrillating therapeutic analogue of amylin. Zinc binds to the N-terminal amino group and His18 imidazole, inducing a kink in the peptide structure, which, in turn, triggers a fibrillization process of the complex, resulting in an amyloid structure most likely responsible for the disruption of the fungal cell.

Poly-Gly Region Regulates the Accessibility of Metal Binding Sites in Snake Venom Peptides.

It is supposed that the presence of poly-His regions in close proximity to poly-Gly domains in snake venoms is related to their biological activity; poly-His/poly-Gly (pHpG) peptides inhibit the activity of metalloproteinases during venom storage via the chelation metal ions, necessary for their proper functioning. This work shows that only the histidyl residues from the N-terminal VDHDHDH motif (but not from the poly-His tag) were the primary Zn(II) binding sites and that the poly-Gly domain situated in the proximity of a central proline residue may play a regulatory role in venom gland protection. The proline induces a kink of the peptide, resulting in steric hindrance, which may modulate the accessibility of potential metal binding sites in the poly-His domain and may, in turn, be one of the regulators of Zn(II) accessibility in the venom gland and therefore a modulator of metalloproteinase activity during venom storage.

Specific Zn(II)-binding site in the C-terminus of Aspf2, a zincophore from Aspergillus fumigatus.

Aspergillus fumigatus, one of the most widespread opportunistic human fungal pathogens, adapts to zinc limitation by secreting a 310 amino acid Aspf2 zincophore, able to specifically bind Zn(II) and deliver it to a transmembrane zinc transporter, ZrfC. In this work, we focus on the thermodynamics of Zn(II) complexes with unstructured regions of Aspf2; basing on a variety of spectrometric and potentiometric data, we show that the C-terminal part has the highest Zn(II)-binding affinity among the potential binding sites, and Ni(II) does not compete with Zn(II) binding to this region. The 14 amino acid Aspf2 C-terminus coordinates Zn(II) via two Cys thiolates and two His imidazoles and it could be considered as a promising A. fumigatus targeting molecule.

Correction: Novel insights into the metal binding ability of ZinT periplasmic protein from Escherichia coli and Salmonella enterica.

Correction for 'Novel insights into the metal binding ability of ZinT periplasmic protein from Escherichia coli and Salmonella enterica' by Denise Bellotti et al., Dalton Trans., 2020, 49, 9393-9403, DOI: 10.1039/D0DT01626H.

Zn2+ and Cu2+ Binding to the Extramembrane Loop of Zrt2, a Zinc Transporter of Candida albicans.

Zrt2 is a zinc transporter of the ZIP family. It is predicted to be located in the plasma membrane and it is essential for Candida albicans zinc uptake and growth at acidic pH. Zrt2 from C. albicans is composed of 370 amino acids and contains eight putative transmembrane domains and an extra-membrane disordered loop, corresponding to the amino acid sequence 126-215. This protein region contains at least three possible metal binding motifs: HxHxHxxD (144-153), HxxHxxEHxD (181-193) and the Glu- and Asp- rich sequence DDEEEDxE (161-168). The corresponding model peptides, protected at their termini (Ac-GPHTHSHFGD-NH2, Ac-DDEEEDLE-NH2 and Ac-PSHFAHAQEHQDP-NH2), have been investigated in order to elucidate the thermodynamic and coordination properties of their Zn2+ and Cu2+ complexes, with the further aim to identify the most effective metal binding site among the three fragments. Furthermore, we extended the investigation to the peptides Ac-GPHTHAHFGD-NH2 and Ac-PAHFAHAQEHQDP-NH2, where serine residues have been substituted by alanines in order to check if the presence of a serine residue may favor the displacement of amidic protons by Cu2+. In the native Zrt2 protein, the Ac-GPHTHSHFGD-NH2 region of the Zrt2 loop has the highest metal binding affinity, showing that three alternated histidines separated by only one residue (-HxHxH-) bind Zn2+ and Cu2+ more strongly than the region in which three histidines are separated by two and three His residues (-HxxHxxxH- in Ac-PSHFAHAQEHQDP-NH2). All studied Zrt2 loop fragments have lower affinity towards Zn2+ than the zinc(II) binding site on the Zrt1 transporter; also, all three Zrt2 regions bind Zn2+ and Cu2+ with comparable affinity below pH 5 and, therefore, may equally contribute to the metal acquisition under the most acidic conditions in which the Zrt2 transporter is expressed.

Thermodynamic surprises of Cu(II)-amylin analogue complexes in membrane mimicking solutions.

Membrane environment often has an important effect on the structure, and therefore also on the coordination mode of biologically relevant metal ions. This is also true in the case of Cu(II) coordination to amylin analogues-rat amylin, amylin1-19, pramlintide and Ac-pramlintide, which offer N-terminal amine groups and/or histidine imidazoles as copper(II) anchoring sites. Complex stabilities are comparable, with the exception of the very stable Cu(II)-amylin1-19, which proves that the presence of the amylin C-terminus lowers its affinity for copper(II); although not directly involved, its appropriate arrangement sterically prevents early metal binding. Most interestingly, in membrane-mimicking solution, the Cu(II) affinities of amylin analogues are lower than the ones in water, probably due to the crowding effect of the membrane solution and the fact that amide coordination occurs at higher pH, which happens most likely because the α-helical structure, imposed by the membrane-mimicking solvent, prevents the amides from binding at lower pH, requiring a local unwinding of the α-helix.

A Comparative Study on Nickel Binding to Hpn-like Polypeptides from Two Helicobacter pylori Strains.

Combined potentiometric titration and isothermal titration calorimetry (ITC) methods were used to study the interactions of nickel(II) ions with the N-terminal fragments and histidine-rich fragments of Hpn-like protein from two Helicobacter pylori strains (11637 and 26695). The ITC measurements were performed at various temperatures and buffers in order to extract proton-independent reaction enthalpies of nickel binding to each of the studied protein fragments. We bring up the problem of ITC results of nickel binding to the Hpn-like protein being not always compatible with those from potentiometry and MS regarding the stoichiometry and affinity. The roles of the ATCUN motif and multiple His and Gln residues in Ni(II) binding are discussed. The results provided the possibility to compare the Ni(II) binding properties between N-terminal and histidine-rich part of Hpn-like protein and between N-terminal parts of two Hpn-like strains, which differ mainly in the number of glutamine residues.

Metal specificity of the Ni(II) and Zn(II) binding sites of the N-terminal and G-domain of E. coli HypB.

HypB is one of the chaperones required for proper nickel insertion into [NiFe]-hydrogenase. Escherichia coli HypB has two potential Ni(II) and Zn(II) binding sites-the N-terminal one and the so-called GTPase one. The metal-loaded HypB-SlyD metallochaperone complex activates nickel release from the N-terminal HypB site. In this work, we focus on the metal selectivity of the two HypB metal binding sites and show that (i) the N-terminal region binds Zn(II) and Ni(II) ions with higher affinity than the G-domain and (ii) the lower affinity G domain binds Zn(II) more effectively than Ni(II). In addition, the high affinity N-terminal domain, both in water and membrane mimicking SDS solution, has a larger affinity towards Zn(II) than Ni(II), while an opposite situation is observed at basic pH; at pH 7.4, the affinity of this region towards both metals is almost the same. The N-terminal HypB region is also more effective in Ni(II) binding than the previously studied SlyD metal binding regions. Considering that the nickel chaperone SlyD activates the release of nickel and blocks the release of zinc from the N-terminal high-affinity metal site of HypB, we may speculate that such pH-dependent metal affinity might modulate HypB interactions with SlyD, being dependent on both pH and the protein's metal status.

Chemical "Butterfly Effect" Explaining the Coordination Chemistry and Antimicrobial Properties of Clavanin Complexes.

Can a minor difference in the nonmetal binding sequence of antimicrobial clavanins explain the drastic change in the coordination environment and antimicrobial efficiency? This study answers the question with a definite "yes", showing the details of the bioinorganic chemistry of Zn(II) and Cu(II) complexes with clavanins, histidine-rich, antimicrobial peptides from hemocytes of the tunicate Styela clava.