Ni(II) binding to the Human Tool Like Receptor (HTLR4)

Nickel allergy is the most frequent cause of contact hypersensitivity (burning, redness, itching, swelling and even blisters) in industrialized countries, with 30% of population being affected. Contact allergy is commonly induced by nickel ions present in nickel-containing jewelry such as rings and earrings, as well as in nickel-containing cellular telephones. Ni(II) seems to trigger an inflammatory response by activating human Toll-like-Receptor 4 (hTLR4) [1-4]. Species-specific activation, as in this case, requires distinct sequence motifs that are present in humans but not in mouse, a species not sensitive to nickel-induced allergies. A sequence containing three histidine residues, H431, and the non-conserved H456 and H458, localized in the C-terminus, could be identified as the specific region of human TLR4 responsible for nickel responses. It has been proposed that the imidazole side chain of the histidine residues H456 and H458 may provide a potential binding site for this metal because they are located at an optimal distance to interact with Ni(II) ions, whereas H431 is located further apart. The aim of our research was to verify the possibility of metal binding to the sequence containing the three histidines supposedly involved in nickel response. The chosen segment was the 32aa peptide FQH431SNLKQMSEFSVFLSLRNLIYLDISH456TH458TR, which was studied in order to understand both its binding properties and the thermodynamic stability of its metal complexes. Formation equilibria of Ni(II) complexes have been investigated in aqueous solution and in a wide pH range. Protonation and complex-formation constants have been potentiometrically determined; complex-formation models and species stoichiometry have been checked by means of UV-Vis absorption and CD spectroscopy and investigation through multidimensional and eteronuclear NMR spectroscopy. The predominant species for a 1:1 peptide/Ni(II) molar ratio was obtained at physiological pH and showed an effective binding of the metal to the target sequence

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

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

Ni(II) binding to 429-460 peptide fragment from human toll-like receptor (hTLR4)

Contact allergy, commonly induced by nickel, is the most frequent cause of contact hypersensitivity in industrialized countries, with 30% of population being affected. Ni(II) seems to trigger an inflammatory response by activating human Tool-like-Receptor 4 (hTLR4). Species-specific activation, as in this case, required distinct sequence motifs that are present in human but not in mouse, a species not sensitive to nickel-induced allergies. The specific region of human TLR4 responsible for nickel responses could be a sequence containing three histidine residues, H431, and the non- conserved H456 and H458, localized in the C-terminus. It has been proposed that the imidazole side chains of the histidine residues H456 and H458 provide a potential binding site for nickel because they were located at an optimal distance to interact with Ni(II) ions, whereas H431 was further apart. We decided to verify the possibility of metal binding to FQH431SNLKQMSEFSVFLSLRNLIYLDISH456TH458TR sequence, containing the three histidines supposedly involved in nickel response, in order to study the binding properties of the peptide fragment and on the thermodynamic stability of its metal complexes. Formation equilibria of Ni(II) complexes have been investigated in aqueous solution and in a wide pH range. Protonation and complex-formation constants have been potentiometrically determined; complex-formation models and species stoichiometry have been checked by means of UV-Vis absorption and CD spectroscopy and investigation through NMR is currently being carried out. The predominant species for a 1:1 peptide/Ni(II) molar ratio was obtained at physiological pH and showed an effective binding of the metal to the target sequence

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-DKPAKAED H D HHH-) 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. The histidine-rich C-terminus (HRCT) of mycobacterial GroEL1 seems to be involved in Cu(II) homeostasis. We studied the coordination properties of model peptides: Ac-DKPAKAEDHDHHHGHAH and its six mutants in which one His residue is replaced with a Gln residue. The His9−His13 fragment has the highest affinity for Cu(II), and most probably, His11 is the main binding site for the metal ion. Interestingly, His residue rearrangement also impacts the formation of binuclear species

Ni(II) binding to the 429–460 peptide fragment from human Toll like receptor (hTLR4): a crucial role for nickel-induced contact allergy?

The FQH431SNLKQMSEFSVFLSLRNLIYLDISH456TH458TR fragment, containing three histidine residues, the conserved H431 and the non-conserved H456 and H458, located from 429 to 460 amino acid residues in the C-terminal portion of human Toll-like-receptor 4 (hTLR4), which is directly activated by nickel, a well known contact allergen, has been tested for Ni(II) binding. The complex formation capability of the 32-amino acid sequence with Ni(II) ions has been followed by potentiometric, UV-Vis, CD, MS and NMR measurements. Ni(II) is able to bind to all three histidines by forming macrocycle complexes at low and physiological pH. From pH 9 on, a 4N diamagnetic species (Nim, 3Nam−) with the participation of an imidazole nitrogen and three deprotonated nitrogens from His28, Ser27 and Ile26 amides from the backbone of the model peptide has been determined. From the NMR results it was possible to determine that His28, which mimics the H456 residue in the protein, together with the environment around it, was mainly involved in the binding

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

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

Bacterial strategies for the use of metal ions – backstage of coordiantion chemistry

In the Biological Inorganic Chemistry Group we are inspired to better understand metal ions acquisition and homeostasis in pathogenic bacteria, and in this review we present three different approaches to the role of these processes. The growing importance of a full understanding of the iron transport system in pathogens prompted us to study synthetic analogs of siderophores, used as structural probes in the process of iron uptake by microorganisms. The ferrichrome biomimetic analogs allowed efficient Fe(III) chelation under biological conditions and were recognized better by P. putida. than E. coli, suggesting differences in uptake mechanisms. Addition of a fluorescent probe to the compound allowed to track biological fate of studied complexes [1, 2]. Biomimetics of ferrioxamine E revealed their potential as radioactive 68Ga(III)-based probes [3], and studies of Zr(IV) complexes permitted to explain the in vivo behavior of desferrioxamine B as 89Zr(IV) radionuclide carrier [4], as well as design better chelators for this metal ion [5]. One of the possible mammalian immune system responsesto mycobacterial infection is the increase of Zn(II) concentration in phagosomes to a toxic level [6-8]. The mycobacterial SmtB protein is a transcription regulator that in the presence of high concentrations of metals, dissociates from DNA and activates the expression of metal efflux proteins. We focused on α5 Zn(II) binding domains of SmtB/BigR4 proteins [9], looking at the coordination modes and thermodynamics of their Zn(II) and Ni(II) complexes. The study points out the specificity of metal-ligand interactions and the effect of mutations on the coordination properties of studied systems. The project can be considered as an introduction to the new strategies in tuberculosis treatment based on Zn(II)/Ni(II)-sensitive mechanisms. F. nucleatum is an anaerobic bacteria present in the plaque. It leads not only to periodontal diseases but also, angina, purulent inflammation of the lung tissue or reproductive organs [10]. Moreover, F. nucleatum promotes colon cancer growth [11]. This bacteria strain promotes inflammation and tumorigenesis by modulating the tumor immune microenvironment [12, 13]. Microbial pathogens drive tumorigenesis in 15–20% of cancer cases [14]. However, not only microorganisms are considered a major risk factor, but also metal ions play an important role in tumor promotion [15, 16]. Therefore, our primary research goal is to investigate the effect of metal ions coordination on the activity of outer-membrane proteins from F. nucleatum and to answer whether these proteins increase the prooxidative activity of Cu(II) and Fe(II) ions [16-18]

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