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.

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.

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.

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.

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.

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.

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.

Zn-Enhanced Asp-Rich Antimicrobial Peptides: N-Terminal Coordination by Zn(II) and Cu(II), Which Distinguishes Cu(II) Binding to Different Peptides.

The antimicrobial activity of surfactant-associated anionic peptides (SAAPs), which are isolated from the ovine pulmonary surfactant and are selective against the ovine pathogen Mannheimia haemolytica, is strongly enhanced in the presence of Zn(II) ions. Both calorimetry and ITC measurements show that the unique Asp-only peptide SAAP3 (DDDDDDD) and its analogs SAAP2 (GDDDDDD) and SAAP6 (GADDDDD) have a similar micromolar affinity for Zn(II), which binds to the N-terminal amine and Asp carboxylates in a net entropically-driven process. All three peptides also bind Cu(II) with a net entropically-driven process but with higher affinity than they bind Zn(II) and coordination that involves the N-terminal amine and deprotonated amides as the pH increases. The parent SAAP3 binds Cu(II) with the highest affinity; however, as shown with potentiometry and absorption, CD and EPR spectroscopy, Asp residues in the first and/or second positions distinguish Cu(II) binding to SAAP3 and SAAP2 from their binding to SAAP6, decreasing the Cu(II) Lewis acidity and suppressing its square planar amide coordination by two pH units. We also show that these metal ions do not stabilize a membrane disrupting ability nor do they induce the antimicrobial activity of these peptides against a panel of human pathogens.

Metal Complexes of Two Specific Regions of ZnuA, a Periplasmic Zinc(II) Transporter from Escherichia coli.

The crystal structure of ZnZnuA from Escherichia coli reveals two metal binding sites. (i) The primary binding site, His143, is located close the His-rich loop (residues 116-138) and plays a significant role in Zn(II) acquisition. (ii) The secondary binding site involves His224. In this work, we focus on understanding the interactions of two metal ions, Zn(II) and Cu(II), with two regions of ZnuA, which are possible anchoring sites for Zn(II): Ac-115MKSIHGDDDDHDHAEKSDEDHHHGDFNMHLW145-NH2 (primary metal binding site) and Ac-223GHFTVNPEIQPGAQRLHE240-NH2 (secondary metal binding site). The histidine-rich loop (residues 116-138) has a role in the capture of zinc(II), which is then further delivered into other regions of the protein. For both Zn(II) complexes, histidine residues constitute the main anchoring donors. In the longer, His-rich fragment, a tetrahedral complex with four His residues is formed, while in the second ligand, two imidazole nitrogens are involved in zinc(II) binding. In both cases, so-called loop structures are formed. One consists of a 125HxHxExxxExHxH137 motif with seven amino acid residues in the loop between the two central histidines, while the other is formed by a 224HFTVNPEIQPGAQRLH239 motif with 14 amino acid residues in the loop between the two nearest coordinating histidines. The number of available imidazoles also strongly affects the structure of copper(II) complexes; the more histidines in the studied region, the higher the pH in which amide nitrogens will participate in Cu(II) binding.

Zinc Binding Sites Conserved in Short Neuropeptides Containing a Diphenylalanine Motif.

A diphenylalanine motif in peptides plays a crucial role in supramolecular systems. The current work represents a novel strategy in which a diphenylalanine motif in the central domain of neuropeptides conserves the specific Zn2+ binding site and prevents "hopping" of the Zn2+ ion between alternative metal binding sites. Alternative metal binding sites may also include carboxylic atoms in the terminal domains of a peptide. Therefore, one needs to design a peptide in which the metal will not bind the carboxylic groups in the terminal domains. Herein, we propose that engineering and designing peptides with a diphenylalanine motif in the central domain may yield excellent metal chelators.

Copper(II) and Amylin Analogues: A Complicated Relationship.

Protein aggregation has attracted substantial interest because of its role in causing many serious illnesses, such as neurodegenerative diseases and type II diabetes. Recent studies have shown that protein aggregation can be prevented by forming metal ion complexes with a target protein, which affects their conformation in solution and their physical properties, such as aggregation. Thus, understanding the interactions between aggregating molecules and bioactive metal ions such as Cu2+ is beneficial for new drug discovery. Pramlintide, a synthetic peptide drug, and its natural counterpart rat amylin are known to be resistant to aggregation because of the presence of proline residues, which are usually ß-sheet "breakers" within their amino acid sequence. Here, we investigate the Cu2+ coordination properties of pramlintide and rat amylin using nuclear magnetic resonance, circular dichroism, electron paramagnetic resonance, ultraviolet-visible spectroscopy, potentiometry, and mass spectrometry. We test the influence of Cu2+ on the aggregation properties of these amylin analogues with thioflavin T assays. We find that both peptides form stable complexes with Cu2+ with similar affinities at a 1:1 ratio. The N-termini of both peptides are involved in Cu2+ binding; His18 imidazole is an equally attractive binding site in the case of pramlintide. Our results show that Cu2+ ions influence the aggregation of pramlintide, but not that of rat amylin.

Copper(II)-Induced Restructuring of ZnuD, a Zinc(II) Transporter from Neisseria meningitidis.

Cluster 2 (288HDDDNAHAHTH298) from Neisseria meningitidis ZnuD is a flexible loop that captures zinc(II) ions, acting as a "fishing net". We describe its Zn(II) and Cu(II) binding capabilities, focusing on the thermodynamics of such interactions and comparing them with the complexes of the 1MAHHHHHHL9-NH2 region. Copper(II) complexes with the studied ZnuD regions are thermodynamically more stable than the zinc(II) ones-Cu(II) complexes dominate in solution even in close to physiological ratios of the studied metal ions (a 10-fold excess of Zn(II) over Cu(II)). While the binding of native Zn(II) has no significant impact on the structure of its transporter, Cu(II) binding induces a conformational change of cluster 2 to a polyproline II-like helix. To the best of our knowledge, this is the first evidence of a copper(II)-induced formation of a polyproline II-like structure in a sequence that does not contain proline residues. Cu(II) coordination also changes the structure of an intracellular, N-terminal, His-rich region, folding it to an α helix.

Copper(II)-Binding Induces a Unique Polyproline Type II Helical Structure within the Ion-Binding Segment in the Intrinsically Disordered F-Domain of Ecdysteroid Receptor from Aedes aegypti.

Reproduction of the dominant vector of Zika and dengue diseases, Aedes aegypti mosquito, is controlled by an active heterodimer complex composed of the 20-hydroxyecdysone receptor (EcR) and ultraspiracle protein. Although A. aegypti EcR shares the structural and functional organization with other nuclear receptors, its C-terminus has an additional long F domain (AaFEcR). Recently, we showed that the full length AaFEcR is intrinsically disordered with the ability to specifically bind divalent metal ions. Here, we describe the details of the exhaustive structural and thermodynamic properties of Zn2+- and Cu2+-complexes with the AaFEcR domain, based on peptide models of its two putative metal binding sites (Ac-HGPHPHPHG-NH2 and Ac-QQLTPNQQQHQQQHSQLQQVHANGS-NH2). Unexpectedly, only in the presence of increasing concentrations of Cu2+ ions, the Ac-HGPHPHPHG-NH2 peptide gained a metal ion-induced poly-l-proline type II helical structure, which is unique for members of the family of nuclear receptors.

Secondary structure confirmation and localization of Mg2+ ions in the mammalian CPEB3 ribozyme.

Most of today's knowledge of the CPEB3 ribozyme, one of the few small self-cleaving ribozymes known to occur in humans, is based on comparative studies with the hepatitis delta virus (HDV) ribozyme, which is highly similar in cleavage mechanism and probably also in structure. Here we present detailed NMR studies of the CPEB3 ribozyme in order to verify the formation of the predicted nested double pseudoknot in solution. In particular, the influence of Mg(2+), the ribozyme's crucial cofactor, on the CPEB3 structure is investigated. NMR titrations, Tb(3+)-induced cleavage, as well as stoichiometry determination by hydroxyquinoline sulfonic acid fluorescence and equilibrium dialysis, are used to evaluate the number, location, and binding mode of Mg(2+)ions. Up to eight Mg(2+)ions interact site-specifically with the ribozyme, four of which are bound with high affinity. The global fold of the CPEB3 ribozyme, encompassing 80%-90% of the predicted base pairs, is formed in the presence of monovalent ions alone. Low millimolar concentrations of Mg(2+)promote a more compact fold and lead to the formation of additional structures in the core of the ribozyme, which contains the inner small pseudoknot and the active site. Several Mg(2+)binding sites, which are important for the functional fold, appear to be located in corresponding locations in the HDV and CPEB3 ribozyme, demonstrating the particular relevance of Mg(2+)for the nested double pseudoknot structure.

Histidine tracts in human transcription factors: insight into metal ion coordination ability.

Consecutive histidine repeats are chosen both by nature and by molecular biologists due to their high affinity towards metal ions. Screening of the human genome showed that transcription factors are extremely rich in His tracts. In this work, we examine two of such His-rich regions from forkhead box and MAFA proteins-MB3 (contains 18 His) and MB6 (with 21 His residues), focusing on the affinity and binding modes of Cu2+ and Zn2+ towards the two His-rich regions. In the case of Zn2+ species, the availability of imidazole nitrogen donors enhances metal complex stability. Interestingly, an opposite tendency is observed for Cu2+ complexes at above physiological pH, in which amide nitrogens participate in binding.

Zinc Homeostasis at the Bacteria/Host Interface-From Coordination Chemistry to Nutritional Immunity.

Zinc is one of the most important metal nutrients for species from all kingdoms, being a key structural or catalytic component of hundreds of enzymes, crucial for the survival of both pathogenic microorganisms and their hosts. This work is an overview of the homeostasis of zinc in bacteria and humans. It explains the importance of this metal nutrient for pathogens, describes the roles of zinc sensors, regulators, and transporters, and summarizes various uptake systems and different proteins involved in zinc homeostasis-both those used for storage, buffering, and signaling inside the cell and those excreted in order to obtain ZnII from the host. The human zinc-dependent immune system response is explained, with a special focus given to 'zinc nutritional immunity', a process that describes the competition between the bacteria or fungus and the host for this metal, during which both the pathogen and host make huge efforts to control zinc availability. This sophisticated tug of war over ZnII might be considered as a possible target for novel antibacterial therapies.

Solution structure and metal ion binding sites of the human CPEB3 ribozyme's P4 domain.

Three ribozymes are known to occur in humans, the CPEB3 ribozyme, the CoTC ribozyme, and the hammerhead ribozyme. Here, we present the NMR solution structure of a well-conserved motif within the CPEB3 ribozyme, the P4 domain. In addition, we discuss the binding sites and impact of Mg(2+) and [Co(NH3)6](3+), a spectroscopic probe for [Mg(H2O)6](2+), on the structure. The well-defined P4 region is a hairpin closed with a UGGU tetraloop that shows a distinct electrostatic surface potential and a characteristic, strongly curved backbone trajectory. The P4 hairpin contains two specific Mg(2+) binding sites: one outer-sphere binding site close to the proposed CPEB3 ribozyme active site with potential relevance for maintaining a compact fold of the ribozyme core, and one inner-sphere binding site, probably stabilizing the tetraloop structure. The structure of the tetraloop resembles an RNase III recognition structure, as previously described for an AGUU tetraloop. The detailed knowledge of the P4 domain and its metal ion binding preferences thus brings us closer to understanding the importance of Mg(2+) binding for the CPEB3 ribozyme's fold and function in the cell.

-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.

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.

Metal coordination governs the antimicrobial efficacy of calcitermin derivatives.

Antimicrobial peptides are promising alternatives to classical antibiotics. Their microbicidal activity can arise from different mechanisms, one of which is known as nutritional immunity and has metal micronutrients and metal-binding biomolecules as its main players. Calcitermin is an antimicrobial peptide and an effective metal chelator. Its properties as an antibacterial and anti-Candida agent have been recently studied both as a free peptide and in the presence of zinc and copper ions, with which it forms stable complexes. Calcitermin derivatives have also gained attention thanks to the possibility of improving their properties, like metal-binding affinity and/or stability in biological fluids, through ad hoc modifications of the native peptide sequence. In this work, the Ala-to-Ser substitutions close to the coordination site of calcitermin have been introduced to study the impact on the biological activity and metal-binding properties. Our results show that metal coordination has a clear impact on the bioactivity of the studied compounds, to the point that the truncated fragment of calcitermin, solely containing the main metal-binding residues, also shows antimicrobial activity.