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

CHAPTER 6. Prion DiseaseMechanisms and Metal Involvement in Neurodegenerative Diseases

Prion diseases (derived either from infection, germline mutations or most often occurring sporadically), both in humans and animals, are fatal neurodegenerative disorders characterized by progressive brain degeneration. It is widely accepted that they are caused by protein‐only infectious agents propagating disease by inducing protein conformational changes. The molecular mechanism of prion pathologies is not yet entirely understood but some aspects seem to be generally accepted, such as spongiform degeneration, non‐classical inflammation of the brain, progressive neuron loss, accumulation of protein aggregates and synaptic alterations.</jats:p

Impact of SDS surfactant on the interactions of Cu(2+) ions with the amyloidogenic region of human prion protein

Prion diseases, known as Transmissible Spongiform Encephalopathies (TSEs), are a group of fatal neuronal, and to some extent infectious disorders, associated with a pathogenic protein agent called prion protein (PrP). The human prion protein (hPrP) fragment encompassing the 91-127 region, also known as the amyloidogenic domain, comprises two copper-binding sites corresponding to His-96 and His-111 residues that act as anchors for Cu(2+) binding. In this work, we investigated Cu(2+) interaction with hPrP91-127 in the presence of the anionic surfactant sodium dodecyl sulfate (SDS), which induces a partial α-helix folding of the peptide. Our data indicate that the Cu(2+) coordination ability of the amyloidogenic fragment in the presence of SDS micelles is significantly different to that observed in aqueous solution. This is mainly due to the fact that SDS micelles strongly stabilize the formation of the α-helical structure of the peptide backbone, which is well conserved also upon Cu(2+) binding, contrary to the random coil conformation mainly assumed by hPrP91-127 in aqueous solutions. Potentiometric and spectroscopic studies clearly indicate that in the case of SDS containing solutions, Cu(2+) ions coordinate simultaneously to both imidazoles, while in the case of water solutions, metal ion coordination involves only a single His side chain, which individually acts as an independent Cu(2+) anchoring site

Specific binding modes of Cu(I) and Ag(I) with neurotoxic domain of the human prion protein

Prion diseases are neurodegenerative disorders associated with a conformational change of the normal cellular isoform of the prion protein (PrPC) to an abnormal scrapie isoform (PrPSc). human prion protein (hPrPC) is able to bind up to six Cu(II) ions. Four of them are distributed in the octarepeat domain, containing four tandem-repetitions of the sequence PHGGGWGQ. Immediately outside the octarepeat domain, in so called PrP amyloidogenic region, two additional and independent Cu(II) binding sites, encompassing His96 and His111 residues, respectively, are present. Considering the potential involvement of PrP in cellular redox homeostasis, investigations on Cu(I)-PrP interaction might be also biologically relevant. Interestingly, the amyloidogenic fragment of PrP contains a -M(X)nM- motif, known to act as Cu(I) binding site in different proteins. In order to shed more light on this issue, copper(I) and silver(I) interactions with model peptides derived from that region were analyzed. The results of our studies reveal that both metal ions are anchored to two thioether sulfurs of Met109 and Met112, respectively. Subsequent metal interaction and coordination to His96 and His111 imidazoles are primarily found for Cu(I) at physiological pH. Metal binding was also investigated in the presence of negatively charged micelles formed by the anionic surfactant, sodium dodecyl sulfate (SDS). Our results strongly support that metal binding mode strongly depends on the protein backbone structure. In particular we show that α-helix structuring of the amyloid PrP domain influences both the metal coordination sphere and the binding affinity

The role of His-50 of alfa-synuclein in binding Cu(II): pH dependence, speciation, thermodinamics and structure

7siCopper interaction with alpha synuclein (aS) has been shown to accelerate aggregation and oligomerization of the protein. Three different aS copper binding domains have been proposed: (i) the N-terminal residues (1–9) that represent the minimal copper binding domain; (ii) the His-50 imidazole and (iii) the Asp and Glu residues within the acidic C-terminal domain. The copper coordination at the N-terminus has been extensively characterized and it is generally accepted that it provides the highest affinity site. The same does not hold for the role played by His-50 in copper binding. In this work Cu(II) coordination to peptide fragments encompassing residues 45–55 of aS has been exhaustively characterized, including systems containing the inherited mutations E46K and A53T, as model peptides of the His-50 site. Through potentiometric titrations all the speciation profiles have been determined and the stability constants have been used to estimate the dissociation constants of complexes corresponding to the binding modes at pH 6.5 and 7.5. Spectroscopic analyses allowed determination of (i) the copper coordination sphere, (ii) its geometry and (iii) the constraints wherefrom the 3D structural models of the copper complexes could be obtained.reservedmixedValensin, Daniela; Camponeschi, Francesca; Luczkowski, M.; Baratto, MARIA CAMILLA; Remelli, M.; Valensin, Gianni; Kozlowski, H.Valensin, Daniela; Camponeschi, Francesca; M., Luczkowski; Baratto, MARIA CAMILLA; M., Remelli; Valensin, Gianni; H., Kozlowsk

Design of thiolate rich metal binding sites within a peptidic framework

A de novo protein design strategy provides a powerful tool to elucidate how heavy metals interact with proteins. Cysteine derivatives of the TRI peptide family (Ac-G(LKALEEK)(4)G-NH(2)) have been shown to bind heavy metals in an unusual trigonal geometry. Our present objective was to design binding sites in α-helical scaffolds that are able to form higher coordination number complexes with Cd(II) and Hg(II). Herein, we evaluate the binding of Cd(II) and Hg(II) to double cysteine substituted TRI peptides lacking intervening leucines between sulfurs in the heptads. We compare a -Cys(d)-X-X-X-Cys(a)- binding motif found in TRIL12CL16C to the more common -Cys(a)-X-X-Cys(d)- sequence of native proteins found in TRIL9CL12C. Compared to TRI, these substitutions destabilize the helical aggregates, leading to mixtures of two and three stranded bundles. The three stranded coiled coils are stabilized by the addition of metals. TRIL9CL12C forms distorted tetrahedral complexes with both Cd(II) and Hg(II), as supported by UV-vis, CD, (113)Cd NMR, (199)Hg NMR and (111m)Cd PAC spectroscopy. Additionally, these signatures are very similar to those found for heavy metal substituted rubredoxin. These results suggest that in terms of Hg(II) binding, TRIL9CL12C can be considered as a good mimic of the metallochaperone HAH1, that has previously been shown to form protein dimers. TRIL12CL16C has limited ability to generate homoleptic tetrahedral complexes (Cd(SR)(4)(2−)). These type of complexes were identified only for Hg(II). However, the spectroscopic signatures suggest a different geometry around the metal ion, demonstrating that effective metal sequestration into the hydrophobic interior of the bundle requires more than simply adding two sulfur residues in adjacent layers of the peptide core. Thus, proper design of metal binding sites must also consider the orientation of cysteine sidechains in a vs d positions of the heptads

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