the unusual metal ion binding ability of histidyl tags and their mutated derivatives

Peptides that consist of repeated sequences of alternating histidines and alanines strongly bind Cu(ii) and form α-helical structures

AGHLDDLPGALSAL: A hemoglobin fragment potentially competing with albumin to bind transition metal ions

Protein degradation leads to the formation of endogenous peptides, the biological activity of which is most often unknown. The peptide AGHLDDLPGALSAL, recently isolated from mouse brain homogenates, has been recognized as a fragment of the α-chain of hemoglobin. AGHLDDLPGALSAL has the ability of inhibiting the peripheral hyperalgesic inflammatory responses through the indirect activation of the μ-opioid receptors. A peculiarity of AGHLDDLPGALSAL is the presence, at its N-terminus of a strong binding site for divalent transition metal ions, similar to that characterizing the human albumin and called “ATCUN motif”. The consequential metal binding ability of AGHLDDLPGALSAL can be connected to its biological activity. For this reason, we decided to investigate the coordination properties of AGHLDDLPGALSAL towards Cu(II), Ni(II) and Zn(II) ions, reported here for the first time. The results confirm that AGHLDDLPGALSAL is a strong ligand for those metals: it can even compete with albumin under suitable conditions. In vitro assays on the inhibition of Cu(II) toxicity towards different cell lines confirmed that the binding ability of AGHLDDLPGALSAL can imply relevant biological consequences

Zn(II) and Ni(II) complexes with poly-histidyl peptides derived from a snake venom

The snake venoms are complex mixtures containing many bioactive peptides and proteins; some of them are aimed to protect the snake glands, where the venom is stored, until the latter is inoculated in the victim. In the venom of some vipers of the genus Atheris, a set of peptides containing poly-His and poly-Gly segments was recently found. Poly-His peptides are not rare in Nature. Although their exact biological function is most often unknown, one thing is certain: they have good binding properties towards the transition metal ions. As a matter of fact, the imidazole side chain of histidine is one of the groups most frequently involved in metal complexation in the active sites of metallo-enzymes. This is also true for snake-venom metallo-proteases, which contain Zn(II) and Ca(II) ions.In the present paper, the complex-formation ability of the poly-His-poly-Gly peptide found in the venom of Atheris squamigera (EDDH9GVG10-NH2) towards the Zn(II) and Ni(II) ions was investigated by means of thermodynamic and spectroscopic techniques. Two model peptides, derived from the poly-His portion of this peptide but where His residues were alternated with alanines (Ac-EDDAHAHAHAHAG-NH2, and Ac-EDDHAHAHAHAHG-NH2) were also studied, for the sake of comparison. The high affinity of these peptides for the metal ions under investigation was confirmed. In addition, it was demonstrated that the number of His residues in the peptide and their relative position play a main role in the complex-formation ability of the ligand. The very high affinity of EDDH9GVG10-NH2 for Zn(II) can be the key for its role in the inactivation of the venom in the snake glands

DOES hemopressin bind metal ions in vivo?

Hemopressin is a neuropeptide, derived from the degradation of the α(1)-chain of hemoglobin, and possesses several pharmacologic properties, such as the ability to block cannabinoid CB1 receptor activity, to cause dose-dependent hypotension and to inhibit food intake. Actually, human hemopressin (PVNFKLLSH) is only the precursor of a class of longer peptides, called "Pepcans", which bear additional residues at their amino-terminus and possess slightly different chemical and biological properties with respect to hemopressin. The presence of a histidyl residue and the free terminal amine imparts to hemopressin and its derivatives good binding properties towards transition metal ions. In this paper, we present a wide investigation on the complex-formation equilibria of human hemopressin and three analogues towards the Cu(ii) and Ni(ii) ions. The study showed that the main coordination site is always the amino terminus (if not protected), while the C-terminal histidine acts only as an anchoring site for the metal ions at acidic pH, with the formation of a macrochelate complex. The presence of additional residues in N-terminal position produces significant differences in the protonation and complex-formation behaviors of these peptides, which can be explained in terms of charge of the ligand and coordination environment. Although the participation of metal ions in the biological activity of hemopressin and Pepcans has not yet been demonstrated, the data reported here can help to shed light on the mechanisms governing the action of these neuropeptides in vivo

Insight into the Coordination and the Binding Sites of Cu²⁺ by the Histidyl-6-Tag using Experimental and Computational Tools

His-tags are specific sequences containing six to nine subsequent histydyl residues, and they are used for purification of recombinant proteins by use of IMAC chromatography. Such polyhistydyl tags, often used in molecular biology, can be also found in nature. Proteins containing histidine-rich domains play a critical role in many life functions in both prokaryote and eukaryote organisms. Binding mode and the thermodynamic properties of the system depend on the specific metal ion and the histidine sequence. Despite the wide application of the His-tag for purification of proteins, little is known about the properties of metal-binding to such tag domains. This inspired us to undertake detailed studies on the coordination of Cu²⁺ ion to hexa-His-tag. Experiments were performed using the potentiometric, UV–visible, CD, and EPR techniques. In addition, molecular dynamics (MD) simulations and density functional theory (DFT) calculations were applied. The experimental studies have shown that the Cu²⁺ ion binds most likely to two imidazoles and one, two, or three amide nitrogens, depending on the pH. The structures and stabilities of the complexes for the Cu²⁺-Ac-(His)₆-NH₂ system using experimental and computational tools were established. Polymorphic binding states are suggested, with a possibility of the formation of α-helix structure induced by metal ion coordination. Metal ion is bound to various pairs of imidazole moieties derived from the tag with different efficiencies. The coordination sphere around the metal ion is completed by molecules of water. Finally, the Cu²⁺ binding by Ac-(His)₆-NH₂ is much more efficient compared to other multihistidine protein domains

African Viper Poly-His Tag Peptide Fragment Efficiently Binds Metal Ions and Is Folded into an α‑Helical Structure

Snake venoms are complex mixtures of toxic and often spectacularly biologically active components. Some African vipers contain polyhistidine and polyglycine peptides, which play a crucial role in the interaction with metal ions during the inhibition of snake metalloproteases. Polyhistidine peptide fragments, known as poly-His tags, play many important functions, e.g., in metal ion transport in bacterial chaperon proteins. In this paper, we report a detailed characterization of Cu²⁺, Ni²⁺, and Zn²⁺ complexes with the EDDHHHHHHHHHG peptide fragment (pHG) derived from the venom of the rough scale bush viper (Atheris squamigera). In order to determine the thermodynamic properties, stoichiometry, binding sites, and structures of the metal–pHG complexes, we used a combination of experimental techniques (potentiometric titrations, electrospray ionization mass spectrometry, UV–vis spectroscopy, circular dichroism spectroscopy, and electron paramagnetic resonance spectroscopy) and extensive computational tools (molecular dynamics simulations and density functional theory calculations). The results showed that pHG has a high affinity toward metal ions. The numerous histidine residues located along this sequence are efficient metal ion chelators with high affinities toward Cu²⁺, Ni²⁺, and Zn²⁺ ions. The formation of an α-helical structure induced by metal ion coordination and the occurrence of polymorphic binding states were observed. It is proposed that metal ions can “move along” the poly-His tag, which serves as a metal ion transport pathway. The coordination of Cu²⁺, Ni²⁺, and Zn²⁺ ions to the histidine tag is very effective in comparison with other histidine-rich peptides. The stabilities of the metal–pHG complexes increase in the order Zn²⁺ < Ni²⁺≪ Cu²⁺