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

An Extremely Stable Interprotein Tetrahedral Hg(Cys) ₄ Core Forms in the Zinc Hook Domain of Rad50 Protein at Physiological pH

In nature, thiolate-based systems are the primary targets of divalent mercury (HgII ) toxicity. The formation of Hg(Cys)x cores in catalytic and structural protein centers mediates mercury's toxic effects and ultimately leads to cellular damage. Multiple studies have revealed distinct HgII -thiolate coordination preferences, among which linear HgII complexes are the most commonly observed in solution at physiological pH. Trigonal or tetrahedral geometries are formed at basic pH or in tight intraprotein Cys-rich metal sites. So far, no interprotein tetrahedral HgII complex formed at neutral pH has been reported. Rad50 protein is a part of the multiprotein MRN complex, a major player in DNA damage-repair processes. Its central region consists of a conserved CXXC motif that enables dimerization of two Rad50 molecules by coordinating ZnII . Dimerized motifs form a unique interprotein zinc hook domain (Hk) that is critical for the biological activity of the MRN. Using a series of length-differentiated peptide models of the Pyrococcus furiosus zinc hook domain, we investigated its interaction with HgII . Using UV-Vis, CD, PAC, and 199 Hg NMR spectroscopies as well as anisotropy decay, we discovered that all Rad50 fragments preferentially form homodimeric Hg(Hk)2 species with a distorted tetrahedral HgS4 coordination environment at physiological pH; this is the first example of an interprotein mercury site displaying tetrahedral geometry in solution. At higher HgII content, monomeric HgHk complexes with linear geometry are formed. The Hg(Cys)4 core of Rad50 is extremely stable and does not compete with cyanides, NAC, or DTT. Applying ITC, we found that the stability constant of the Rad50 Hg(Hk)2 complex is approximately three orders of magnitude higher than those of the strongest HgII complexes known to date

Oligopeptide Sequences of the Metal Binding Domain of CueR Metalloregulatory Proteins as Candidates for Toxic Metal Ion Capture

Various toxic metal ion resistance systems operate from bacterial level up to higher plants and animals. In bacteria, metalloregulatory proteins are key factors in the control of metal ion level. Inspired by the metal binding domain of these highly sensitive metal ion sensor proteins we have designed artificial oligopeptides, containing two cysteine residues, and investigated their interaction with cadmium(II) and mercury(II) ions. The studied ligands bound both metal ions with a rather high stability. The composition and solution structure of the various metal ion complexes have been determined. The genetic code of one of the oligopeptide sequences has been introduced into E. coli BL21 cells and (over)produced in the form of a fusion protein. Preliminary investigation of the viability and potential metal ion accumulation of the modified bacteria, compared to control cells, in the presence of cadmium(II) and mercury(II) has also been performed

Perspectives for the VITO beam line at ISOLDE, CERN

By using polarized ion beams in combination with the β-NMR technique, the Versatile Ion-polarized Techniques On-line (VITO) experiment at ISOLDE, CERN links together expertise from different fields in an unique experimental setup. An overview of the experimental techniques and a general description of the newly designed beam line are presented. Potential uses in multidisciplinary research and perspectives for future experiments are discussed

Amyloid-β and α-Synuclein Decrease the Level of Metal-Catalyzed Reactive Oxygen Species by Radical Scavenging and Redox Silencing.

The formation of reactive oxygen species (ROS) is linked to the pathogenesis of neurodegenerative diseases. Here we have investigated the effect of soluble and aggregated amyloid-β (Aβ) and α-synuclein (αS), associated with Alzheimer's and Parkinson's diseases, respectively, on the Cu(2+)-catalyzed formation of ROS in vitro in the presence of a biological reductant. We find that the levels of ROS, and the rate by which ROS is generated, are significantly reduced when Cu(2+) is bound to Aβ or αS, particularly when they are in their oligomeric or fibrillar forms. This effect is attributed to a combination of radical scavenging and redox silencing mechanisms. Our findings suggest that the increase in ROS associated with the accumulation of aggregated Aβ or αS does not result from a particularly ROS-active form of these peptides, but rather from either a local increase of Cu(2+) and other ROS-active metal ions in the aggregates or as a downstream consequence of the formation of the pathological amyloid structures.This work was supported by the Villum Foundation (J.T.P., L.H.), the Lundbeck Foundation (J.T.P., K.T.), the Agency for Science, Technology and Research, Singapore (S.W.C.), The Wellcome Trust (C.M.D.) and the Spanish Ministry of Economy and Competitiveness through the Ramon y Cajal ́ program (N.C.).This is the final version of the article. It first appeared from the American Chemical Society via http://dx.doi.org/10.1021/jacs.5b1357