Metal ion mediated transition from random coil to β-sheet and aggregation of Bri2-23, a natural inhibitor of Aβ aggregation

Furin-dependent maturation of the BRI2 protein generates the Bri2-23 fragment that is able to arrest the aggregation of amyloidβ, 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 β-sheet formation. NMR spectroscopy supports the formation of β-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 β-sheets. Molecular dynamics simulations initiated with structures that agree with NMR data additionally support this model

The specificity of interaction of Zn(2+), Ni(2+) and Cu(2+) ions with the histidine-rich domain of the TjZNT1 ZIP family transporter

The Zrt/Irt-like protein (ZIP) family contributes to the metal homeostasis by regulating the transport of divalent metal cations such as Fe(2+), Zn(2+), Mn(2+), Cd(2+) and sometimes even Cu(2+). Most ZIP members have a long variable loop between transmembrane domains (TMDs) III and IV; this region is predicted to be located in the cytoplasm and is postulated to be the metal ion binding site. In this study, we looked at the thermodynamic behavior and coordination chemistry of Zn(2+), Ni(2+) and Cu(2+) complexes with the histidine-rich domain, Ac-(185)RAHAAHHRHSH(195)-NH2 (HRD), from the yeast TjZNT1 protein, located between TMDs III and IV. The sequence is conserved also in higher species like Thlaspi japonicum. The stability of complexes increases in the series Ni(2+) < Zn(2+)≪ Cu(2+). The geometry of complexes is very different for each metal and in the case of Zn(2+) complexes, high specificity in binding is observed. Moreover, the stability of HRD-Cu(2+) complexes was compared with the five His residues containing peptide from Hpn protein (Helicobacter pylori). The results suggest a high ability of HRD in the binding of all three studied metals

The extracellular loop of IRT1 ZIP protein--the chosen one for zinc?

Zinc complexes with the extracellular loop of IRT1 (iron-regulated transporter 1), a ZIP (ZRT/IRT - Related Protein) family protein from Arabidopsis thaliana, have been studied. This unstructured fragment is responsible for metal selectivity and is located between the II and III transmembrane domains of IRT1. Zinc complexes with the Ac-(95)MHVLPDSFEMLSSICLEENPWHK(117)-NH2 peptide (IRT1), revealed surprisingly high thermodynamic stability. Additionally, an N-terminal fragment of human/mouse ZIP 13 zinc transporter (MPGCPCPGCGMACPR-NH2, later called ZIP13+C), has been chosen for the thermodynamic stability comparison studies. The relative ZIP13+C stability has been shown using several Zn(2+) complexes with artificially arranged multi-cysteine sequences. An interesting coordination mode has been proposed for the IRT1-Zn(2+) complex, in which imidazoles from two histidines (His-96 and His-116), a cysteine thiolate (Cys-109) and one of a glutamic acid carboxyl group are involved. All data were collected using potentiometric, NMR and mass spectrometric methods

Specific metal ion binding sites in unstructured regions of proteins

In this review, we summarise the most recent findings on some very effective binding sites (e.g. ATCUN motif, poly-His, poly-Cys, or Met-containing sequences) for biologically relevant metals in proteins and peptides. In addition, the influence of the specific sequence on the binding stability, besides the donor atoms, is described (e.g. Pro residues as in PrP or poly-Gln sequences). It is well-known that some disorders are connected with proteins which need metal ions for biological activity. Often metal ions coordinate to binding sites located in loops or unstructured regions of those proteins. These rather recent discoveries make metal ion binding to proteins slightly more enigmatic than in the case of an insertion of metal into an “organized” site. Although in the latter cases, we still may need chaperons helping to select the proper metal ion, some selectivity is provided by the pre-organized structure of the donor site itself in the protein. The metal ion binding usually exerts a distinct impact on the binding pocket structure due to the secondary or tertiary structure donors from the residues being often very far away in the peptide sequence. Recently, several metallo-proteins were discovered whose structures are rather disordered (e.g. α-synuclein, prions or β-amyloid peptide involved in Alzheimer disease). Also some specific metal chaperons, consisting of long poly-His sequences being very effective binders of metals, do not show any specific secondary structure. Examples of these might be bacterial nickel accessory proteins, involved in the complicated pathway of metal uptake, delivery and regulation in microorganisms. Recently, several findings were reported on the homeostasis of nickel in Helicobacter pylori, a Gram-negative bacterium that colonizes the gastric mucosa in humans, and is the causative agent of acute and chronic gastritis, peptic ulcer disease, gastric carcinoma, and gastric lymphoma. The homeostasis of nickel is crucial for the survival of this bacterium in the extremely acidic environment of the stomach; the metal is delivered to urease and hydrogenase by a set of accessory proteins. Zinc often plays a structural or regulatory role in those nickel chaperones. It can also be one of the metal ion which interferes with the homeostasis of Ni2+ , since the affinity of the two metals towards His- and Cys- rich sequences can sometimes be comparable

The zinc-binding fragment of HypA from Helicobacter pylori: a tempting site also for nickel ions

HypA, a nickel accessory protein from H. pylori, binds a zinc ion in it's structural site, a loop with two conserved CXXC motifs (Ac-ELECKDCSHVFKPNALDYGVCEKCHS-NH(2)). There are at least three hypotheses on the binding mode of this ion. In this paper, we try to understand how Zn(2+) binds to this fragment and why Ni(2+), a metal with quite a high affinity towards thiolic sites, doesn't compete with zinc in the binding to this motif. Potentiometric titrations, mass spectrometry, NMR, UV-Vis and CD spectroscopy help us to compare the coordination modes in both metal complexes and discuss their thermodynamic stabilities

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

The zinc-binding fragment of HypA from Helicobacter pylori: a tempting site also for nickel ions

HypA, a nickel accessory protein from H. pylori, binds a zinc ion in it's structural site, a loop with two conserved CXXC motifs (Ac-ELECKDCSHVFKPNALDYGVCEKCHS-NH(2)). There are at least three hypotheses on the binding mode of this ion. In this paper, we try to understand how Zn(2+) binds to this fragment and why Ni(2+), a metal with quite a high affinity towards thiolic sites, doesn't compete with zinc in the binding to this motif. Potentiometric titrations, mass spectrometry, NMR, UV-Vis and CD spectroscopy help us to compare the coordination modes in both metal complexes and discuss their thermodynamic stabilities