Giovanni La Penna

Metal ions and intrinsically disordered proteins and peptides: from Cu/Zn amyloid-β to general principles.

The interaction of d-block metal ions (Cu, Zn, Fe, etc.) with intrinsically disordered proteins (IDPs) has gained interest, partly due to their proposed roles in several diseases, mainly neurodegenerative. A prominent member of IDPs is the peptide amyloid-β (Aβ) that aggregates into metal-enriched amyloid plaques, a hallmark of Alzheimers disease, in which Cu and Zn are bound to Aβ. IDPs are a class of proteins and peptides that lack a unique 3D structure when the protein is isolated. This disordered structure impacts their interaction with metal ions compared with structured metalloproteins. Metalloproteins either have a preorganized metal binding site or fold upon metal binding, resulting in defined 3D structure with a well-defined metal site. In contrast, for Aβ and likely most of the other IDPs, the affinity for Cu(I/II) and Zn(II) is weaker and the interaction is flexible with different coordination sites present. Coordination of Cu(I/II) with Aβ is very dynamic including fast Cu-exchange reactions (milliseconds or less) that are intrapeptidic between different sites as well as interpeptidic. This highly dynamic metal-IDP interaction has a strong impact on reactivity and potential biological role: (i) Due to the low affinity compared with classical metalloproteins, IDPs likely bind metals only at special places or under special conditions. For Aβ, this is likely in the neurons that expel Zn or Cu into the synapse and upon metal dysregulation occurring in Alzheimers disease. (ii) Amino acid substitutions (mutations) on noncoordinating residues can change drastically the coordination sphere. (iii) Considering the Cu/Zn-Aβ aberrant interaction, therapeutic strategies can be based on removal of Cu/Zn or precluding their binding to the peptide. The latter is very difficult due to the multitude of metal-binding sites, but the fast koff facilitates removal. (iv) The high flexibility of the Cu-Aβ complex results in different conformations with different redox activity. Only some conformations are able to produce reactive oxygen species. (v) Other, more specific catalysis (like enzymes) is very unlikely for Cu/Zn-Aβ. (vi) The Cu/Zn exchange reactions with Aβ are faster than the aggregation process and can hence have a strong impact on this process. In conclusion, the coordination chemistry is fundamentally different for most of IDPs compared with the classical, structured metalloproteins or with (bio)-inorganic complexes. The dynamics is a key parameter to understand this interaction and its potential biological impact.

Modeling the Cu+ Binding in the 1-16 Region of the Amyloid-β Peptide Involved in Alzheimer's Disease

The coordination of copper to the amyloid-β (1-16) (Aβ) peptide has been investigated because of its relevance for understanding Cu redox activity when the ion is embedded in peptides involved in neurodegenerative diseases. In this work, several reasonable models of Cu(+) coordination were built on the basis of experimental information and investigated by first-principles molecular dynamics simulations in the Car-Parrinello scheme. The propensity of a linear Nδ (His)-Cu-Nδ (His) coordination for Cu(+) is shown by all the models investigated here, with distortions due to weak interactions with the carbonyl O of His 6 and His 13 and with the amide N of His 14. Though the His 6-Cu-His 14 linear coordination is favored in truncated models, the His 13-Cu-His 14 linear coordination is favored by interactions present in the complete solvated and in vacuo models of Cu-Aβ (1-16). These interactions include steric hindrance for the expulsion of His 13, hydrogen bonds between Asp and His side chains and a network of electrostatic interactions stabilizing two separated 1-10 and 11-16 peptide regions. The role of linear His 13-Cu-His 14 coordination in stabilizing Cu(I) and in increasing the Cu(II)/Cu(I) reorganization energy can be therefore modulated by boundary conditions acting on the Aβ (1-16) ligand.

Free Superoxide is an Intermediate in the Production of H2O2 by Copper(I)‐Aβ Peptide and O2

Oxidative stress is considered as an important factor and an early event in the etiology of Alzheimers disease (AD). Cu bound to the peptide amyloid-β (Aβ) is found in AD brains, and Cu-Aβ could contribute to this oxidative stress, as it is able to produce in vitro H2O2 and HO˙ in the presence of oxygen and biological reducing agents such as ascorbate. The mechanism of Cu-Aβ-catalyzed H2O2 production is however not known, although it was proposed that H2O2 is directly formed from O2 via a 2-electron process. Here, we implement an electrochemical setup and use the specificity of superoxide dismutase-1 (SOD1) to show, for the first time, that H2O2 production by Cu-Aβ in the presence of ascorbate occurs mainly via a free O2˙(-) intermediate. This finding radically changes the view on the catalytic mechanism of H2O2 production by Cu-Aβ, and opens the possibility that Cu-Aβ-catalyzed O2˙(-) contributes to oxidative stress in AD, and hence may be of interest.

Identifying, By First-Principles Simulations, Cu[Amyloid-β] Species Making Fenton-Type Reactions in Alzheimer’s Disease

According to the amyloid cascade hypothesis, amyloid-β peptides (Aβ) play a causative role in Alzheimers disease (AD), of which oligomeric forms are proposed to be the most neurotoxic by provoking oxidative stress. Copper ions seem to play an important role as they are bound to Aβ in amyloid plaques, a hallmark of AD. Moreover, Cu-Aβ complexes are able to catalyze the production of hydrogen peroxide and hydroxyl radicals, and oligomeric Cu-Aβ was reported to be more reactive. The flexibility of the unstructured Aβ peptide leads to the formation of a multitude of different forms of both Cu(I) and Cu(II) complexes. This raised the question of the structure-function relationship. We address this question for the biologically relevant Fenton-type reaction. Computational models for the Cu-Aβ complex in monomeric and dimeric forms were built, and their redox behavior was analyzed together with their reactivity with peroxide. A set of 16 configurations of Cu-Aβ was studied and the configurations were classified into 3 groups: (A) configurations that evolve into a linearly bound and nonreactive Cu(I) coordination; (B) reactive configurations without large reorganization between the two Cu redox states; and (C) reactive configurations with an open structure in the Cu(I)-Aβ coordination, which have high water accessibility to Cu. All the structures that showed high reactivity with H2O2 (to form HO(•)) fall into class C. This means that within all the possible configurations, only some pools are able to produce efficiently the deleterious HO(•), while the other pools are more inert. The characteristics of highly reactive configurations consist of a N-Cu(I)-N coordination with an angle far from 180° and high water crowding at the open side. This allows the side-on entrance of H2O2 and its cleavage to form a hydroxyl radical. Interestingly, the reactive Cu(I)-Aβ states originated mostly from the dimeric starting models, in agreement with the higher reactivity of oligomers. Our study gives a rationale for the Fenton-type reactivity of Cu-Aβ and how dimeric Cu-Aβ could lead to a higher reactivity. This opens a new therapeutic angle of attack against Cu-Aβ-based reactive oxygen species production.

Ab initio simulations of Cu binding sites on the N-terminal region of prion protein

The human prion protein binds Cu2+ ions in the octarepeat domain of the N-terminal tail up to full occupancy at pH 7.4. Recent experiments have shown that the HGGG octarepeat subdomain is responsible for holding the metal bound in a square-planar configuration. By using first principle ab initio molecular dynamics simulations of the Car–Parrinello type, the coordination of copper to the binding sites of the prion protein octarepeat region is investigated. Simulations are carried out for a number of structured binding sites. Results for the complexes Cu(HGGGW)(wat), Cu(HGGG), and [Cu(HGGG)]2 are presented. While the presence of a Trp residue and a water molecule does not seem to affect the nature of the copper coordination, high stability of the bond between copper and the amide nitrogen of deprotonated Gly residues is confirmed in all cases. For the more interesting [Cu(HGGG)]2 complex, a dynamically entangled arrangement of the two domains with exchange of amide nitrogen bonds between the two copper centers emerges, which is consistent with the short Cu–Cu distance observed in experiments at full copper occupancy.

Modeling of the Zn2+ binding in the 1–16 region of the amyloid β peptide involved in Alzheimer’s disease

Zinc ions are found at mM concentration in amyloid plaques of Alzheimers disease and the role of zinc in protein oligomerization is the object of intense investigations. As an in vitro model for studying interactions between Zn(2+) and the Abeta peptide, that is the main component of plaques, the N- and C-termini protected Abeta(1-16) fragment has been chosen because reliable spectroscopic studies in water solution are possible due to the low propensity for oligomerization at pH approximately 6.5, and because all the Zn binding sites of Abeta have been identified in the 1-16 region. In this work we present the results of first principle simulations of several initial models of Zn-Abeta(1-16) complexes. The NMR results about the same system, where His 6, 13, 14 and Glu 11 side-chains coordinate the Zn ion, are strongly supported by these models. Coordination of Asp 1 to Zn drives the complex towards the expulsion of one of initially bonded His side-chains. Coordination of Tyr 10 to Zn is possible only when Tyr 10 is deprotonated. The interplay between physico-chemical properties of the Abeta ligand and the Zn coordination is discussed.

Exploring the reactions of β-amyloid (Aβ) peptide 1-28 with Al(III) and Fe(III) ions.

The reactions of human β-amyloid peptide 1-28 (Aβ28) with Al(III) and Fe(III) ions were investigated by (1)H NMR and electrospray ionization mass spectrometry (ESI-MS) under pH conditions close to physiological ones. (1)H NMR titrations, performed in the 5.3-8.0 pH range, revealed that no measurable amounts of Aβ28-Al(III) or Aβ28-Fe(III) adducts are formed; such metal adducts could not be obtained even by changing a number of experimental conditions, e.g., temperature, buffer, nature of the salt, etc. These observations were later confirmed by ESI-MS. It is thus demonstrated that Aβ28, at physiological pH, is not able to form binary complexes with Al(III) and Fe(III) ions of sufficient stability to compete with metal hydroxide precipitation. The biological implications of these findings are discussed in the frame of current literature.

Modeling Copper Binding to the Amyloid-β Peptide at Different pH: Toward a Molecular Mechanism for Cu Reduction

Oxidative stress, including the production of reactive oxygen species (ROS), has been reported to be a key event in the etiology of Alzheimers disease (AD). Cu has been found in high concentrations in amyloid plaques, a hallmark of AD, where it is bound to the main constituent amyloid-β (Aβ) peptide. Whereas it has been proposed that Cu-Aβ complexes catalyze the production of ROS via redox-cycling between the Cu(I) and Cu(II) state, the redox chemistry of Cu-Aβ and the precise mechanism of redox reactions are still unclear. Because experiments indicate different coordination environments for Cu(II) and Cu(I), it is expected that the electron is not transferred between Cu-Aβ and reactants in a straightforward manner but involves structural rearrangement. In this work the structures indicated by experimental data are modeled at the level of modern density-functional theory approximations. Possible pathways for Cu(II) reduction in different coordination sites are investigated by means of first-principles molecular dynamics simulations in the water solvent and at room temperature. The models of the ligand reorganization around Cu allow the proposal of a preferential mechanism for Cu-Aβ complex reduction at physiological pH. Models reveal that for efficient reduction the deprotonated amide N in the Ala 2-Glu 3 peptide bond has to be protonated and that interactions in the second coordination sphere make important contributions to the reductive pathway, in particular the interaction between COO(-) and NH(2) groups of Asp 1. The proposed mechanism is an important step forward to a clear understanding of the redox chemistry of Cu-Aβ, a difficult task for spectroscopic approaches as the Cu-peptide interactions are weak and dynamical in nature.

A rigid core‐flexible chain model for mesogenic molecules in molecular dynamics simulations of liquid crystals

A model of a mesogenic molecule, built up as a rigid anisotropic Gay–Berne site mimicking the aromatic core, connected to an array of isotropic sites mimicking a flexible chain, is proposed and tested in molecular dynamics calculations. Simulations have been performed on a system composed of 256 molecules with three different numbers of methylenic units in the chain, in order to explore the effect of chain length on static and dynamic properties. The systems are all at the same mass density and temperature and result in nematic liquid crystalline phases. The order parameters for various molecular fragments and the T1z nuclear magnetic resonance (NMR) relaxation times of deuterons are in agreement with previous molecular dynamics simulations on atomistic systems and, at least qualitatively, with 2H‐NMR experimental results. The intermolecular interactions are always dominated by the anisotropic site simulating the molecular core. The influence of the phase order on the chain static and dynamic properties is put in evidence. Extensions of the model are suggested in order to have a better reproduction of the dynamical features of such systems.A model of a mesogenic molecule, built up as a rigid anisotropic Gay–Berne site mimicking the aromatic core, connected to an array of isotropic sites mimicking a flexible chain, is proposed and tested in molecular dynamics calculations. Simulations have been performed on a system composed of 256 molecules with three different numbers of methylenic units in the chain, in order to explore the effect of chain length on static and dynamic properties. The systems are all at the same mass density and temperature and result in nematic liquid crystalline phases. The order parameters for various molecular fragments and the T1z nuclear magnetic resonance (NMR) relaxation times of deuterons are in agreement with previous molecular dynamics simulations on atomistic systems and, at least qualitatively, with 2H‐NMR experimental results. The intermolecular interactions are always dominated by the anisotropic site simulating the molecular core. The influence of the phase order on the chain static and dynamic properties i...

Designing generalized statistical ensembles for numerical simulations of biopolymers.

Conformational properties of polymers, such as average dihedral angles or molecular alpha-helicity, display a rather weak dependence on the detailed arrangement of the elementary constituents (atoms). We propose a computer simulation method to explore the polymer phase space using a variant of the standard multicanonical method, in which the density of states associated to suitably chosen configurational variables is considered in place of the standard energy density of states. This configurational density of states is used in the Metropolis acceptance/rejection test when configurations are generated with the help of a hybrid Monte Carlo algorithm. The resulting configurational probability distribution is then modulated by exponential factors derived from the general principle of the maximal constrained entropy by requiring that certain average configurational quantities take preassigned (possibly temperature dependent) values. Thermal averages of other configurational quantities can be computed by using the probability distributions obtained in this way. Moments of the energy distribution require an extra canonical sampling of the system phase space at the desired temperature, in order to locally thermalize the configurational degrees of freedom. As an application of these ideas we present the study of the structural properties of two simple models: a bead-and-spring model of polyethylene with independent hindered torsions and an all-atom model of alanine and glycine oligomers with 12 amino acids in vacuum.