Beyond copper: examining the significance of His-mutations in mycobacterial GroEL1 HRCT for Ni(II) complex stability and formation.

Recently, we have studied the coordination chemistry of the Cu(II)-histidine-rich C-terminal tail (HRCT) complex of the mycobacterial GroEL1 protein. The structure of this domain differs significantly compared to the well-known methionine-glycine-rich GroEL chaperonin - it was predicted that mycobacterial GroEL1 could play a significant role in the metal homeostasis of Mycobacteria, especially copper. However, we found that this particular domain's pattern also repeats in a number of Ni(II)-binding proteins. Here, we present the studies concerning the properties of GroEL1 HRCT as a ligand for Ni(II) ions. For this purpose, we chose eight model peptides: L1 - Ac-DHDHHHGHAH, L2 - Ac-DKPAKAEDHDHHHGHAH, and 6 mutants of the latter in the pH range of 2-11. We examined the stoichiometry, stability, and spectroscopic features of copper complexes. We noticed that similar to the Cu(II)-complex, the presence of a Lys5 residue significantly increases the stability of the system. The impact of His mutations was also examined and carefully studied using NMR spectroscopy. His9 and His13 are the crucial residues for Ni(II) binding, whereas His12 has minimal relevance in complex formation.

Determination of the role of specific amino acids in the binding of Zn(II), Ni(II), and Cu(II) to the active site of the M10 family metallopeptidase.

Metallopeptidases are a group of metal-dependent enzymes that hydrolyze peptide bonds. These enzymes found in Streptococcus pneumoniae assist the pathogen in infecting the host by breaking down host tissues and extracellular matrix proteins. Considering metallopeptidases' significant role in bacterial virulence, inhibiting this enzyme represents a promising avenue for research. These enzymes are characterized by the presence of Zn(II) in the active site, proper coordination of which is essential for their catalytic function. This work aims to determine the significance of the specific amino acids in the metal binding domain of metallopeptidase from S. pneumoniae. For this purpose, we investigated the coordination chemistry of Zn(II), Ni(II), and Cu(II) ions with point-mutated peptide models of the metal-binding domain. Mutations were introduced at His-2 (L1) and Glu-1. Studies have shown that at pH 7.14 (pH of infected lungs by S. pneumoniae), point mutation on glutamic acid caused only minor effects on the binding of Zn(II) and Ni(II), while significantly improving Cu(II) binding. The stability of copper complexes is greater with the mutant Glu-1 â†’ Gln-1 than with the original domain due to a hydrogen bonding network created by the Gln backbone with its side chain. Substituting histidine resulted in a significant reduction in metal binding for all metal ions, highlighting the crucial role of His-2 in metal coordination. Introduced mutations at neutral pH did not significantly affect the secondary structure of metal complexes. However, at alkaline pH, the peptides showed a higher percentage of antiparallel ß-sheet structures upon the addition of Cu(II), Ni(II) and Zn(II).

Exploring binding preferences: Cu(II), Ni(II), and Zn(II) complexes of mycobacterial GroEL1 His-rich and Glu/His-rich domains.

Mycobacterial histidine-rich GroEL1 protein significantly differs from the well-known methionine-glycine-rich GroEL chaperonin and most preferably participates in Cu(II) homeostasis. Some GroEL1 proteins, however, do not possess six but only three histidine residues and more acidic residues that can function as binding sites for metal ions. To evaluate the importance of this difference, we examined and compared the properties of GroEL1 His-rich or Glu/His-rich C-terminal domains as ligands for Cu(II), Ni(II), and Zn(II) ions. We studied the stoichiometry, stability, and binding sites of Cu(II)/Ni(II)/Zn(II) complexes of two model peptides: XEN = Ac-DKPEEEEDGHGHAH (M. xenopi) and ABS = Ac-DKPAEEADHGHGHHGHAH (M. abscessus) in the pH range 2-11. In the case of Cu(II), Ni(II), and Zn(II) complexes of XEN and ABS, ABS always formed more stable complexes. For XEN, there seemed to be no preference for Ni(II) or Zn(II) ions. In contrast, for ABS, Zn(II) formed a complex that was slightly more stable than the one formed by Ni(II). This may be due to the 6 His residues, which preferentially interact with Zn(II) rather than Ni(II). The study identified that an equilibrium of complexes-known as polymorphism-may occur in ABS complexes. Therefore, distinct sets of histidine residues may be involved in metal binding.

Copper Forms a PPII Helix-Like Structure with the Catalytic Domains of Bacterial Zinc Metalloproteases.

The rapid spread of antibiotic-resistant bacteria continuously raises concerns about the future ineffectiveness of current antimicrobial treatments against infectious diseases. To address this problem, new therapeutic strategies and antimicrobial drugs with unique modes of action are urgently needed. Inhibition of metalloproteases, bacterial virulence factors, is a promising target for the development of antibacterial treatments. In this study, the interaction among Zn(II), Cu(II), and the metal-binding domains of two metalloproteases, AprA (Pseudomonas aureginosa) and CpaA (Acinetobacter baumanii), was investigated. The objective was to determine the coordination sphere of Zn(II) with a peptide model of two zinc-dependent metalloproteases. Additionally, the study explored the formation of Cu(II) complexes with the domains, as Cu(II) has been shown to inhibit metalloproteases. The third aim was to understand the role of nonbinding amino acids in stabilizing the metal complexes formed by these proteases. This work identified specific coordination patterns (HExxHxxxxxH) for both Zn(II) and Cu(II) complexes, with AprA and CpaA exhibiting a higher affinity for Cu(II) compared to Zn(II). The study also found that the CpaA domain has greater stability for both Zn(II) and Cu(II) complexes compared to AprA. The nonbinding amino acids of CpaA surrounding the metal ion contribute to the increased thermodynamic stability of the metal-peptide complex through various intramolecular interactions. These interactions can also influence the secondary structures of the peptides. The presence of certain amino acids, such as tyrosine, arginine, and glutamic acid, and their interactions contribute to the stability and, only in the case of Cu(II) complexes, the formation of a rare protein structure called a left-handed polyproline II helix (PPII), which is known to play a role in the stability and function of various proteins. These findings provide valuable insights into the coordination chemistry of bacterial metalloproteases and expand our understanding of potential mechanisms for inhibiting these enzymes.

Fe(II), Mn(II), and Zn(II) Binding to the C-Terminal Region of FeoB Protein: An Insight into the Coordination Chemistry and Specificity of the Escherichia coli Fe(II) Transporter.

The interactions between two peptide ligands [Ac763CCAASTTGDCH773 (P1) and Ac743RRARSRVDIELLATRKSVSSCCAASTTGDCH773 (P2)] derived from the cytoplasmic C-terminal region of Eschericha coli FeoB protein and Fe(II), Mn(II), and Zn(II) ions were investigated. The Feo system is regarded as the most important bacterial Fe(II) acquisition system, being one of the key virulence factors, especially in anaerobic conditions. Located in the inner membrane of Gram-negative bacteria, FeoB protein transports Fe(II) from the periplasm to the cytoplasm. Despite its crucial role in bacterial pathogenicity, the mechanism in which the metal ion is trafficked through the membrane is not yet elucidated. In the gammaproteobacteria class, the cytoplasmic C-terminal part of FeoB contains conserved cysteine, histidine, and glutamic and aspartic acid residues, which could play a vital role in Fe(II) binding in the cytoplasm, receiving the metal ion from the transmembrane helices. In this work, we characterized the complexes formed between the whole cytosolic C-terminal sequence of E. coli FeoB (P2) and its key polycysteine region (P1) with Fe(II), Mn(II), and Zn(II) ions, exploring the specificity of the C-terminal region of FeoB. With the help of a variety of potentiometric, spectroscopic (electron paramagnetic resonance and NMR), and spectrometric (electrospray ionization mass spectrometry) techniques and molecular dynamics, we propose the metal-binding modes of the ligands, compare their affinities toward the metal ions, and discuss the possible physiological role of the C-terminal region of E. coli FeoB.

Histidine-Rich C-Terminal Tail of Mycobacterial GroEL1 and Its Copper Complex─The Impact of Point Mutations.

The mycobacterial histidine-rich GroEL1 protein differs significantly compared to the well-known methionine/glycine-rich GroEL chaperonin. It was predicted that mycobacterial GroEL1 can play a significant role in the metal homeostasis of Mycobacteria but not, as its analogue, in protein folding. In this paper, we present the properties of the GroEL1 His-rich C-terminus as a ligand for Cu(II) ions. We studied the stoichiometry, stability, and spectroscopic features of copper complexes of the eight model peptides: L1─Ac-DHDHHHGHAH, L2─Ac-DKPAKAEDHDHHHGHAH, and six mutants of L2 in the pH range of 2-11. We revealed the impact of adjacent residues to the His-rich fragment on the complex stability: the presence of Lys and Asp residues significantly increases the stability of the system. The impact of His mutations was also examined: surprisingly, the exchange of each single His to the Gln residue did not disrupt the ability of the ligand to provide three binding sites for Cu(II) ions. Despite the most possible preference of the Cu(II) ion for the His9-His13 residues (Ac-DKPAKAEDHDHHH-) of the model peptide, especially the His11 residue, the study shows that there is not only one possible binding mode for Cu(II). The significance of this phenomenon is very important for the GroEL1 function─if the single mutation occurs naturally, the protein would be still able to interact with the metal ion.

Bacterial M10 metallopeptidase as a medicinal target - coordination chemistry of possible metal-based inhibition.

Streptococcus pneumoniae is the most frequent cause of fatal bacterial pneumonia infection worldwide. Due to the spreading of antibiotic-resistant pathogens, it is important to search for new therapeutic and prevention strategies against bacterial infections. It is believed that the search for effective inhibitors of bacterial and pathogenic metallopeptidases could be one of the innovative strategies for the design of new antibiotics. Most of them contain zinc in the metal-binding site of the protein, which is a critical component for the biological activity of the enzyme. The main goal of this work is to determine the specificity of the interactions between the binding domain of the metallopeptidase from S. pneumoniae, and Zn(II). Considering the observed inhibitory role of copper towards the metallopeptidases, the next step is to analyze the formation of complexes with Cu(II) and Ni(II). The thermodynamic properties of Zn(II), Cu(II), and Ni(II) complexes were examined by potentiometry, NMR, MS, UV-Vis, CD, and EPR. The results show a similar coordination pattern, HExxHxxxxxH, for all three studied metals below pH 7. Moreover, the primary binding sites were established as the N-terminus in all cases. However, at a pH value of 7.4, the coordination and geometry of the formed complexes differ. The comparison of the stability of the formed complexes reveals that both Cu(II) and Ni(II) are able to displace Zn(II) from its binding site in the whole studied pH range. It opens a discussion on the catalytic zinc ion displacement possibilities by other divalent metal ions and the importance of this process in enzymatic inhibition.

Zn(II) and Cd(II) Complexes of AMT1/MAC1 Homologous Cys/His-Rich Domains: So Similar yet So Different.

Infections caused by Candida species are becoming seriously dangerous and difficult to cure due to their sophisticated mechanisms of resistance. The host organism defends itself from the invader, e.g., by increasing the concentration of metal ions. Therefore, there is a need to understand the overall mechanisms of metal homeostasis in Candida species. One of them is associated with AMT1, an important virulence factor derived from Candida glabrata, and another with MAC1, present in Candida albicans. Both of the proteins possess a homologous Cys/His-rich domain. In our studies, we have chosen two model peptides, L680 (Ac-10ACMECVRGHRSSSCKHHE27-NH2, MAC1, Candida albicans) and L681 (Ac-10ACDSCIKSHKAAQCEHNDR28-NH2, AMT1, Candida glabrata), to analyze and compare the properties of their complexes with Zn(II) and Cd(II). We studied the stoichiometry, thermodynamic stability, and spectroscopic parameters of the complexes in a wide pH range. When competing for the metal ion in the equimolar mixture of two ligands and Cd(II)/Zn(II), L680 forms more stable complexes with Cd(II) while L681 forms more stable complexes with Zn(II) in a wide pH range. Interestingly, a Glu residue was responsible for the additional stability of Cd(II)-L680. Despite a number of scientific reports suggesting Cd(II) as an efficient surrogate of Zn(II), we showed significant differences between the Zn(II) and Cd(II) complexes of the studied peptides.

Coordination Properties of the Zinc Domains of BigR4 and SmtB Proteins in Nickel Systems─Designation of Key Donors.

The increasing number of antibiotic-resistant pathogens has become one of the foremost health problems of modern times. One of the most lethal and multidrug-resistant bacteria is Mycobacterium tuberculosis (Mtb), which causes tuberculosis (TB). TB continues to engulf health systems due to the significant development of bacterial multidrug-resistant strains. Mammalian immune system response to mycobacterial infection includes, but is not limited to, increasing the concentration of zinc(II) and other divalent metal ions in phagosome vesicles up to toxic levels. Metal ions are necessary for the survival and virulence of bacteria but can be highly toxic to organisms if their concentrations are not strictly controlled. Therefore, understanding the mechanisms of how bacteria use metal ions to maintain their optimum concentrations and survive under lethal environmental conditions is essential. The mycobacterial SmtB protein, one of the metal-dependent transcription regulators of the ArsR/SmtB family, dissociates from DNA in the presence of high concentrations of metals, activating the expression of metal efflux proteins. In this work, we explore the properties of α5 metal-binding domains of SmtB/BigR4 proteins (the latter being the SmtB homolog from nonpathogenic Mycobacterium smegmatis), and two mutants of BigR4 as ligands for nickel(II) ions. The study focuses on the specificity of metal-ligand interactions and describes the effect of mutations on the coordination properties of the studied systems. The results of this research reveal that the Ni(II)-BigR4 α5 species are more stable than the Ni(II)-SmtB α5 complexes. His mutations, exchanging one of the histidines for alanine, cause a decrease in the stability of Ni(II) complexes. Surprisingly, the lack of His102 resulted also in increased involvement of acidic amino acids in the coordination. The results of this study may help to understand the role of critical mycobacterial virulence factor─SmtB in metal homeostasis. Although SmtB prefers Zn(II) binding, it may also bind metal ions that prefer other coordination modes, for example, Ni(II). We characterized the properties of such complexes in order to understand the nature of mycobacterial SmtB when acting as a ligand for metal ions, given that nickel and zinc ArsR family proteins possess analogous metal-binding motifs. This may provide an introduction to the design of a new antimicrobial strategy against the pathogenic bacterium M. tuberculosis.

Sometimes less is more-the impact of the number of His residues on the stability of Zn(II)-SmtB and BigR4 α-5 domain complexes.

The increasing number of antibiotic-resistant pathogens has become one of the major health problems of modern times, including infections caused by Mycobacterium tuberculosis. One of the possible mammalian immune system responses to mycobacterial infection is the increase of the zinc(II) concentration in phagosomes to a toxic level. The mycobacterial SmtB protein belongs to the family of ArsR/SmtB transcription regulators. In the presence of high concentrations of metals, SmtB dissociates from DNA and activates the expression of metal efflux proteins. In this work, we focus on the α5 zinc(II) binding domains of SmtB/BigR4 proteins (the latter being the SmtB homolog from non-pathogenic M. smegmatis) and two mutants of BigR4. We will be taking a closer look at the coordination modes and thermodynamic properties of their zinc(II) complexes. The study points out the specificity of metal-ligand interactions and describes the effect of mutations on the coordination properties of the studied systems. The stabilities of the zinc(II) complexes were determined by potentiometry. The coordination sites were determined by NMR experiments and DFT calculations. The comparison of complex stabilities reveals that the Zn(II)-BigR4 species are more stable than the Zn(II)-SmtB complexes. His mutations strongly affect the stability of the complexes and the coordination modes of the metal ion. Exchanging one of the histidines for alanine causes, surprisingly, an increase in the stability of zinc(II) complexes with the studied domain. This was confirmed by potentiometric and DFT methods. This work can be considered as a bioinorganic introduction to the discovery of new strategies in M. tuberculosis infection treatment based on zinc(II)-sensitive mechanisms.

Sensing of Proteins by ICD Response of Iron(II) Clathrochelates Functionalized by Carboxyalkylsulfide Groups.

Recognition of elements of protein tertiary structure is crucial for biotechnological and biomedical tasks; this makes the development of optical sensors for certain protein surface elements important. Herein, we demonstrated the ability of iron(II) clathrochelates (1-3) functionalized with mono-, di- and hexa-carboxyalkylsulfide to induce selective circular dichroism (CD) response upon binding to globular proteins. Thus, inherently CD-silent clathrochelates revealed selective inducing of CD spectra when binding to human serum albumin (HSA) (1, 2), beta-lactoglobuline (2) and bovine serum albumin (BSA) (3). Hence, functionalization of iron(II) clathrochelates with the carboxyalkylsulfide group appears to be a promising tool for the design of CD-probes sensitive to certain surface elements of proteins tertiary structure. Additionally, interaction of 1-3 with proteins was also studied by isothermal titration calorimetry, protein fluorescence quenching, electrospray ionization mass spectrometry (ESI-MS) and computer simulations. Formation of both 1:1 and 1:2 assemblies of HSA with 1-3 was evidenced by ESI-MS. A protein fluorescence quenching study suggests that 3 binds with both BSA and HSA via the sites close to Trp residues. Molecular docking calculations indicate that for both BSA and HSA, binding of 3 to Site I and to an "additional site" is more favorable energetically than binding to Site II.

Zinc(II)-The Overlooked Éminence Grise of Chloroquine's Fight against COVID-19?

Zn(II) is an inhibitor of SARS-CoV-2's RNA-dependent RNA polymerase, and chloroquine and hydroxychloroquine are Zn(II) ionophores-this statement gives a curious mind a lot to think about. We show results of the first clinical trials on chloroquine (CQ) and hydroxychloroquine (HCQ) in the treatment of COVID-19, as well as earlier reports on the anticoronaviral properties of these two compounds and of Zn(II) itself. Other FDA-approved Zn(II) ionophores are given a decent amount of attention and are thought of as possible COVID-19 therapeutics.

Triplet of cysteines - Coordinational riddle?

Polythiol binding of metal ions plays crucial role in the proper functioning of cysteine-rich proteins that are responsible for metal homeostasis and defending processes against metal toxicity (including heavy metals detoxification). The coordination properties of cysteine residues involved in specific sequencional patterns in proteins (like those present in e.g. metallothioneins) are interesting not only from a chemical point of view but may also lead to a better understanding of the purpose and allocation of metal ions in various biomolecules. In this study, the interaction of Zn2+, Cd2+ and Ni2+ ions with four peptides containing cysteine triplet motif were studied by potentiometric and spectroscopic methods. The main goal of this research was to answer the question how effectively three thiols, each being next to other, are able to bind single metal ion. Two of peptides contain additional, fourth cysteine residue, separated from triplet by two and three other amino acid residues. As results show, all three cysteine residues in the CCC motif are able to participate in the coordination of the metal ion (Cd2+, Zn2+). Except cysteine thiol groups, amide nitrogen atoms are also involved in the coordination of Ni2+.

Induced CD of iron(ii) clathrochelates: sensing of the structural and conformational alterations of serum albumins.

An ability of inherently achiral macrobicyclic metal complexes iron(ii) clathrochelates to acquire an induced CD (ICD) output in the visible spectral range upon interaction with bovine serum albumin (BSA) was recently discovered. In the present work, the CD-reporting properties of iron(ii) clathrochelates to proteins and the thermodynamic parameters of their binding to albumins are evaluated. It is shown that iron(ii) clathrochelates functionalized by six ribbed carboxyphenylsulfide groups are able to discriminate between serum albumins of relative structure (here human and bovine albumins) by giving distinct ICD spectra. Besides, by the variation of the shape and intensity of CD bands, these cage metal complexes reflect the pH-triggered alterations of the tertiary structure of albumins. The constitutional isomerism (ortho-, meta- or para-isomers) of terminal carboxyphenylsulfide groups of iron(ii) clathrochelates strongly affects both the character of their ICD output upon binding with proteins and the parameters of the formed guest-host associates. Using isothermal titration calorimetry, it was determined that cage metal complexes bearing meta- and ortho-isomers of carboxyphenylsulfide groups possess higher association constants (Ka ∼ 2 × 104 M-1) and clathrochelate-to-BSA binding ratios (n = 2) than the para-isomer (Ka ∼ 5 × 103 M-1, n = 1). The iron(ii) clathrochelates are suggested to be potential molecular three-dimensional scaffolds for the design of CD-sensitive reporters able to recognize specific elements of protein surfaces.

Pneumococcal histidine triads - involved not only in Zn2+, but also Ni2+ binding?

Polyhistidine triad proteins, which participate in Zn2+ uptake in Streptococcus pneumoniae, contain multiple copies of the HxxHxH (histidine triad motif) sequence. We focus on three such motifs from one of the most common and well-conserved polyhistidine triad proteins, PhtA, in order to understand their bioinorganic chemistry; particular focus is given to (i) understanding which of the PhtA triads binds Zn2+ with the highest affinity (and why) and (ii) explaining whether Ni2+ (also crucial for bacterial survival and virulence) could potentially outcompete Zn2+ at its native binding site. There is no significant difference in the stability of zinc(ii) complexes with the three studied protein fragments, but one of the nickel(ii)-polyhistidine triads is remarkably stable; we explain why and hypothesize about the biological importance of this finding.

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.

HENRYK - An endless source of metal coordination surprises.

The basic knowledge about biological inorganic chemistry, thermodynamics and metal binding sites of metalloproteins is crucial for the understanding of their metal binding-structure-function relationship. Metal-peptide complexes are useful and commonly used models of metal-enzyme active sites, among which copper and zinc models are one of the most extensively studied. HENRYK is a peptide sequence present in numerous proteins, and serves as a potentially tempting binding site for Cu2+ and Zn2+. Maybe more importantly, HENRYK also happens to be the first name of our group leader. The results of this work, which, at the first glance, might seem to be a 'chemical scrabble', went far beyond our expectations and surprised us with a novel, uncommon behavior of a Cu2+ complex with a peptide with a histidine in position one. At low pH, the binding is a typical histamine-like coordination, but with the increase of pH, the imidazole nitrogen is moved to the axial position and replaced with an amide; at basic pH, the binding mode is a {NH2, 3N-} one in the equatorial plane. It is important to note, that no dimeric species are formed in between. Such binding is thermodynamically much more stable than a simple complex with histamine, and quite comparable to complexes with several possible imidazole anchoring sites.

Specificity of the Zn(2+), Cd(2+) and Ni(2+) ion binding sites in the loop domain of the HypA protein.

The zinc binding loop domain of the HypA protein of Helicobacter pylori consists of two CXXC motifs with flanking His residues. These motifs bind metal ions, and thus they are crucial for the functioning of the whole protein. The N-terminal site, where His is separated from CXXC by Ser residue is more effective in binding Zn(2+) and Ni(2+) ions than the C-terminal site, in which His is adjacent to the CXXC motif. Studies on various modifications of the peptide sequence within the Ac-ELECKDCSHVFKPNALDYGVCEKCHS-NH2 loop show the role of the residues in the linker between the CXXC motifs and the effect of the length of the linker on the stability of the complexes it forms with Zn(2+), Cd(2+) and Ni(2+) ions. The proline residue in the linker between the two CXXC binding sites plays a distinct role in the metal ion binding ability of the loop, lowering the efficacy of the metal ion coordination. The deletion of the aliphatic residues from the linker between the CXXC motifs remarkably improves the binding efficacy of the loop.

Metal ion mediated transition from random coil to ß-sheet and aggregation of Bri2-23, a natural inhibitor of Aß aggregation.

Furin-dependent maturation of the BRI2 protein generates the Bri2-23 fragment that is able to arrest the aggregation of amyloidß, the peptide implicated in Alzheimer's disease (AD). Bri2-23 contains cysteines at positions 5 and 22, which are likely to bind to metal ions such as Cu(i). Metal ions may play a role in the etiology of neurodegenerative disorders such as AD, and in this work we explore the metal ion induced folding and aggregation of Bri2-23 using Hg(ii) and Ag(i) as spectroscopic probes with structural and ligand preferences similar to those of Cu(i), while not displaying redox activity under the experimental conditions. In general, interaction of Bri2-23 with soft metal ions changes the structural properties and solution behavior of the peptide that tune to increasing metal to peptide stoichiometry. Potentiometric, (199m)Hg PAC and ESI-MS data indicate that addition of up to 0.5 equivalents of Hg(ii) to Bri2-23 yields a two-coordinated HgS2 structure at the metal site. While the free peptide is inherently unstructured, the presence of Ag(i) and Hg(ii) gives rise to ß-sheet formation. NMR spectroscopy supports the formation of ß-sheet structure in the presence of 0.5 equivalents of Hg(ii), and displays an interesting and marked change in the TOCSY spectra when increasing the Hg(ii) to peptide stoichiometry from 0.5 to 0.7 equivalents, indicating the equilibrium between two structural analogues of the complex. Addition of more than 0.7 equivalents of Hg(ii) gives rise to line broadening, presumably reflecting aggregation. This is further supported by ThT fluorescence studies showing that the Bri2-23 peptide does not aggregate over 24 hours, while addition of over 0.7 equivalents of Ag(i) or Hg(ii) leads to increase of fluorescence, indicating that these metal ions induce aggregation. Thus, a model integrating all data into a coherent picture is that the metal ion binding to the two thiolates gives rise to folding of the peptide into a structure that is prone to aggregation, forming aggregates with a considerable amount of ß-sheets. Molecular dynamics simulations initiated with structures that agree with NMR data additionally support this model.

The unusual binding mechanism of Cu(II) ions to the poly-histidyl domain of a peptide found in the venom of an African viper.

Copper complexes of a poly-His/poly-Gly peptide (EDDHHHHHHHHHGVGGGGGGGGGG-NH2), a natural component of a snake venom, were studied by means of both experimental (thermodynamic, spectroscopic and MS) techniques and molecular dynamics (MD) simulations and density functional theory (DFT) calculations. This peptide proved to be an exceptionally effective copper chelator, forming complexes which are thermodynamically more stable than those formed by both the albumin-like ATCUN motif and several other poly-histidine protein fragments. We show that, in a poly-histidine stretch, copper seems to prefer binding to residues separated by one amino acid and that a correlation between an α-helical structure of the predicted complexes and their thermodynamic stability is observed.