Copper coordination to the prion protein: Insights from theoretical studies

Coordination Chemistry Reviews 257 (2013) 429–444

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Review

Copper coordination to the prion protein: Insights from theoretical studies Liliana Quintanar ∗ , Lina Rivillas-Acevedo, Rafael Grande-Aztatzi, Carlos Z. Gómez-Castro, Trinidad Arcos-López, Alberto Vela Departamento de Química, Centro de Investigación y de Estudios Avanzados (Cinvestav), Mexico City, Mexico

Contents 1. 2.

3.

4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Copper binding to PrPC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Copper binding to the octarepeat region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1. Structural properties of the OR–Cu(II) complex (Component 1): the role of water networks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2. Electronic structure of the OR–Cu(II) complex (Component 1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.3. Structural properties of multiple histidine coordination modes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Cu binding outside the octarepeat region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Cu binding to His96 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. Cu binding to His111 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.3. Cu binding to the C-terminal domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects of copper binding to PrPC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Redox properties of Cu–PrPC complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Effects of Cu–PrPC interactions in protein folding and aggregation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion and closing remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

a r t i c l e

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Article history: Received 22 May 2012 Received in revised form 11 June 2012 Accepted 12 June 2012 Available online 1 July 2012 Dedicated to Prof. Edward I. Solomon on the occasion of his 65th birthday. Keywords: Prion protein Copper Coordination chemistry Electronic structure DFT Molecular dynamics

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a b s t r a c t The cellular prion protein (PrPC ) has emerged as an important copper binding protein. The interaction of copper with PrPC may play important roles in both the physiological function of the protein, and in the pathogenesis of prion diseases. The copper coordination chemistry of PrPC is complex, as different Cu(II) coordination modes can be formed depending on pH and copper to protein ratios, and involving six different His residues in the N-terminal region of the protein. The nature of these Cu(II) binding sites has been studied using theoretical tools, which expand on the information obtained from experimental results and provide important insights into Cu–PrPC interactions. This article provides a general overview of the different Cu(II) binding sites in PrPC and their redox properties, highlighting the contributions from electronic structure calculations and molecular dynamics simulations. Particular emphasis is placed in discussing the electronic structure of each Cu binding mode, as it is intimately related to redox properties. For most Cu binding modes, the dominating Cu(II) bonding interactions involve deprotonated amide nitrogens, which yield Cu Namide bonds that are significantly more covalent than the Cu NHis bond. The key factors that determine the direction of Cu(II) binding to backbone amides in the vicinity of the anchoring His are discussed. Additionally, the impact of Cu–PrPC interactions in protein folding and in the potential initiation of PrPC aggregation is discussed. © 2012 Elsevier B.V. All rights reserved.

Abbreviations: AA, ascorbic acid; BSE, Bovine spongiform encephalopathies; B3LYP, Becke 3 parameter Lee Yang Parr; BP86, Becke Perdew86; CD, Circular dichroism; CJD, Creutzfeldt–Jakob disease; CPMD, Car–Parrinello molecular dynamics; DFT, Density functional theory; DZVP, Double-␨ valence polarization; EPR, Electron paramagnetic resonance; ESEEM, Electron spin echo envelope modulation; ESI, Electrospray ionization; ENDOR, Electron nuclear double resonance; ffMD, Force field molecular dynamics; GGA, Generalized gradient approximation; GPI, Glycosyl-phosphatidylinositol; GTO, Gaussian type orbitals; LDA, Local density approximation; LMCT, Ligand to metal charge transfer; MAD, Mean absolute deviation; MD, Molecular dynamics; NMDA, N-methyl-d-aspartate; NHE, Normal hydrogen electrode; NMR, Nuclear magnetic resonance; OR, Octarepeat; PBE, Perdew Burke Ernzerhof; PrPC , Cellular prion protein; PrPSc , Scrapie prion protein; PW91, Perdew Wang91; QM/MM, Quantum mechanics/molecular mechanics; ROS, Reactive oxygen species; SOD, Superoxide dismutase; STO, Slater type orbitals; SVWN, Dirac-Slater Vosko Wlik Nusair; TSE, Transmissible spongiform encephalopathies; TZVP, Triple-␨ valence polarization; UV–visible, Ultraviolet–visible. ∗ Corresponding author at: Departamento de Química, Cinvestav, Av. Instituto Politécnico Nacional No. 2508, Col. San Pedro Zacatenco, México, 07360, D.F., Mexico City, Mexico. Tel.: +52 55 57473723; fax: +52 55 57473389. E-mail address: [email protected] (L. Quintanar). 0010-8545/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ccr.2012.06.026

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1. Introduction The cellular prion protein (PrPC ) is associated to a group of fatal neurodegenerative diseases known as transmissible spongiform encephalopathies (TSEs) or prion diseases. These include bovine spongiform encephalopathy (BSE) or mad cow disease, scrapie in sheep and goats, chronic wasting disease in deer and elk, and kuru and Creutzfeldt–Jakob disease (CJD) in humans. While CJD generally involves progressive dementia, scrapie and BSE are ataxic illnesses, and both produce death of the host without any sign of an immune response to a foreign infectious agent [1]. Prion diseases can be genetic (10%), infectious (5%), or sporadic (85%) disorders [2]; but in all cases, they involve the conversion of the soluble functional PrPC into a proteinase K resistant and pathogenic isoform known as PrPSc (scrapie). Prions are infectious pathogens, devoid of nucleic acids, and they seem to be composed exclusively of the infectious isoform PrPSc [1]. This structural transition is accompanied by profound changes in the physicochemical properties of PrPC , but the amino acid sequence of PrPSc corresponds to that encoded by the PrPC gene of the mammalian host in which it last replicated [3]. Transgenic studies argue that PrPSc acts as a template upon which PrPC is refolded into a nascent PrPSc molecule [4]. Although PrPC is expressed in a wide variety of species, including avians, turtles, amphibians, mammals, yeast and fish [5], prion diseases have only been observed in mammals [6]. PrPC is a membrane bound glycoprotein found mainly in the central nervous system, although it is expressed throughout the body [1]. Human PrPC consists of 253 amino acids, with as many as two attached carbohydrates and a glycosyl-phosphatidylinositol (GPI) anchor in the C-terminal domain. After proteolytic processing of the N and C terminals, human PrPC and PrPSc consist of 209 residues that include from residues 23 to 231 [7,8]. Nuclear magnetic resonance (NMR) [9,10] and X-ray crystallography [11] studies have indicated that PrPC contains a predominantly ␣-helical structure at the C-terminal domain spanning residues 120–231, while the N-terminal domain is mostly unstructured [1,4] (Fig. 1). Although there are no high-resolution structures of PrPSc , electroncrystallography experiments suggest that PrPSc only differs from PrPC in conformation, as it is rich in ␤-sheet content [12]. The deposits of aggregated protein are enriched by a 27–30 kDa fraction that lacks 67 amino acids from the N-terminus [8,13]. This fraction is called PrP(27–30), and it is the protease-resistant core of PrPSc [14,15]. The origins of the conversion of PrPC to PrPSc are not fully understood, however, it has been proposed that the conversion occurs in the partially denaturing environment of the endocytic compartment, where high proton concentrations may induce conformational changes that facilitate amyloid and prion self-assembly [16,17]. In general, several factors such as pH, temperature, ionic strength, presence of chaotropic agents, oxidative stress and metal ions, can strongly influence the conformation adopted by PrPC [6,18]. Metal ions such as Cu(II), Zn(II), Mn(II), Fe(II), Ca(II) and Cd(II) can bind to different regions of PrPC in solution [19]. Moreover, disturbances in the levels of copper and manganese have been described in prion-infected brain tissue [20–23], and changes in Cu levels have been suggested to influence incubation time in experimental prion disease [23,24]. Several in vivo and in vitro studies have shown a correlation between PrPSc formation and altered levels of several divalent metal cations including Cu, Zn, Mn, and Fe, suggesting a possible role for these ions in prion pathogenesis [25]. Among these metals, PrPC shows higher affinity for Cu(II). At this point, the role of copper–PrP interactions in prion disease is not fully understood. In particular, there is no consensus as to whether copper impedes or promotes prion disease. Some studies have shown that copper inhibits the in vitro formation of fibrils

in full-length recombinant protein [26]. Conversely, reduction of total brain copper in animals can delay disease [24]. Thus, binding of copper or other metal ions to PrPC may play a relevant role in the pathogenesis of prion diseases. Specifically, variations in the occupancy of metal binding sites may directly influence the structural conversion and aggregation of PrPC that takes place during prion propagation [25]. The wide distribution of PrPC among mammalian species and the high conservation of this protein indicate a role of general importance. However, the physiological function of PrPC in healthy tissues is not fully known. PrP-knockout mice exhibit normal development and behavior as compared to wild type mice, although they seem to be more susceptible to oxidative stress [27]. Indeed, studies show that PrP may protect cells against apoptosis [28] and oxidative stress [29]. Other proposed functions involve PrPC as a superoxide dismutase (SOD) [30], in membrane excitability and synaptic transmission [31], and copper transport and metabolism [32]. In particular, the connection between PrPC and copper has received a lot of attention in the last decade [33–37], since the discovery that Cu(II) and Zn(II) can stimulate endocytosis of PrPC [38], thus linking metal binding to a physiological response. Moreover, a functional connection between PrPC and copper has been recently revealed as an important interaction for the modulation of N-methyl-d-aspartate (NMDA) receptor’s activity [39]. Thus, there is considerable evidence supporting the notion that the interaction of metal ions with PrPC may play important roles in both, the physiological function of the protein, and in the pathogenesis of prion diseases. In particular, the discovery that PrPC can bind copper in vivo [32] has underscored the importance of understanding how this metal ion coordinates to PrPC . The following sections provide a brief overview of what is known about copper binding to PrPC from experimental work, with a detailed discussion of the insights that have been gained on Cu–PrPC interactions from theoretical studies. 2. Copper binding to PrPC Copper has emerged as a strongly interacting divalent metal ion to PrPC . It is generally accepted that PrPC can bind several Cu(II) ions in different regions of the protein; six of these binding sites are located in its flexible N-terminal domain [40,41]. The most studied binding sites are located in the octarepeat region (OR), spanning residues 60–91 in the human sequence (Fig. 1). Four Cu(II) ions bind to this region containing four repeats of the highly conserved octapeptide PHGGGWGQ. Two additional Cu(II) binding sites are located between the OR region and the C-terminal domain [42], involving His96 and His111 as binding anchors in this region spanning residues 92–115 [40–44]. Finally, His residues at positions 177 and 187 in the structured C-terminal region of PrPC have been identified as potential Cu binding sites [45–48]. The nature of the Cu(II) binding sites in PrPC has been explored in the recent decade using electronic structure calculations and/or molecular dynamics, most extensively for the OR region. A brief summary of experimental findings along with the most relevant theoretical studies that have unraveled key features of Cu binding to each of these sites will be discussed. 2.1. Copper binding to the octarepeat region The octarepeat (OR) region contains four repeats of the octapeptide PHGGGWGQ, and it can bind a total of four Cu(II) ions. However, different binding modes have been identified, depending on pH and the relative ratio of copper to protein concentrations [35,36]. At physiological pH, three distinct Cu(II) coordination modes have been identified by electron paramagnetic resonance

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Fig. 1. Schematic representation of PrPC and its Cu(II) binding sites. A sequence alignment for the Cu binding regions is shown including sequences from human [155], mouse [156], hamster [157], bovine [9] and sheep [158] (A). A model of the protein was constructed using the NMR-derived structure adapted from PDB 1QLX [10], and by adding the unstructured N-terminal residues using the TraDES suite program [159] (B).

(EPR) and electron spin echo envelope modulation (ESEEM) [49], as shown in Fig. 2. At low Cu:protein ratios, a mode with multiple histidine coordination, where the metal ion binds three or four His residues from the OR region, can be distinguished (Component 3, Fig. 2). At higher Cu:protein ratios, an intermediate component with a 2N2O coordination mode is observed, where Cu(II) is likely bound by two His residues (Component 2, Fig. 2) [36,49–52]. Finally, at high Cu(II) occupancy, when there is enough Cu(II) to bind each PHGGGWGQ fragment, a 3NO equatorial coordination mode dominates (Component 1, Fig. 2) [40,41]. X-ray crystallography, continuous wave and pulsed EPR studies have shown that Component 1 involves residues HGGGW, where the coordinating atoms are provided by the N␦ nitrogen of the His imidazole ring, two deprotonated amide nitrogens and a carbonyl moiety from the Gly residues that follow the His. Finally, a water molecule coordinates in the axial position to lead to a penta-coordinated Cu(II) that is stabilized by a hydrogen bonding network involving the Trp residue with several water molecules in the vicinity [40,53].

2.1.1. Structural properties of the OR–Cu(II) complex (Component 1): the role of water networks The Cu coordination mode in the OR region that has been most extensively studied by theoretical tools is by far Component 1 (Fig. 2), as the construction of models for geometry optimizations has been greatly facilitated by the crystal structure of the Cu(II)–HGGGW complex [53]. Different structural models for this binding site have been built using truncated peptides containing residues HG [54], HGG [55,56], HGGG [54,57–60], HGGGW [55,58,60–64], or PHGGGWGQ [62]. In most models, the N-terminal part of the peptide is acetylated and the C-terminal is amidated to prevent their participation in Cu binding. Alternatively, the peptide terminals can be capped by stable methyl groups or by forming C H bonds. Reported electronic structure calculations performed within the framework of density functional theory (DFT) have used a wide range of exchange-correlation functionals including the local density approximation (LDA), such as SVWN [65,66], the generalized

Fig. 2. Copper binding modes identified in the octarepeat region of PrP at physiological pH. Component 1 represents the main coordination mode at high occupancy (Cu:protein ratio 4:1); Component 3 is a coordination mode favored at low occupancy (Cu:protein ratio 1:4); Component 2 represents the intermediate coordination mode. The X in Component 2 could be a second deprotonated amide [50,51], or two water molecules coordinating in the equatorial direction along with the His imidazole and deprotonated amide; in the latter, the second His imidazole would coordinate axially [49,52].

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gradient approximation (GGA), such as BP86 [67,68] and PBE [69], and global hybrids (B3LYP [70–73], B3PW91 [67,72,74], BHandHLYP [67,73], PBE0 [75] and mPW91PW91 [76,77]). Several different basis sets have also been used, ranging from very large (IGLO-III) to minimal (STO-3G) ones. A recent quantum chemical investigation reports a careful evaluation of eight different geometry optimization methods, using the coordinates from the crystal structure as the initial configuration of the HGGGW model [62]. The authors find that the method that best reproduces the experimental geometry of the site (from crystal structure) is the mPW91PW91 functional using the basis set 6-31G(d) with diffuse functions and an extra set of d functions in Cu and first coordination shell atoms (mPW91PW91/6-31+G(2d)|6-31G(d)). This method yields the lowest mean absolute deviation (MAD) for ˚ and a reasonable MAD for angles Cu ligand distances (0.030 A) (1.4◦ ) with respect to experimental parameters [62]. It should be noted though, that reasonable MAD values are also obtained with the mPW91PW91 functional using a TZVP basis set [78] (0.060 A˚ for distances and 1.7◦ for angles). Most notably, LDA with a 631+G(d) basis set yields a reasonably good MAD values for distances ˚ and a remarkably low MAD value for angles (0.3◦ ) that (0.060 A), is significantly better than that obtained with the mPW91PW91/631+G(2d)|6-31G(d) methodology. These results indicate that a good geometric description of this Cu binding site can be achieved using either hybrid or LDA functionals with appropriate basis sets. In particular, the best description of Cu ligand distances is achieved with the mPW91PW91/6-31+G(2d)|6-31G(d) method, while the best description of angles is achieved with the SVWN/6-31+G(d) method [62]. Thus, the choice of method for geometry optimization is important, as some of the properties described for these systems can be highly dependent on the methodology. One of the most explored aspects of Component 1 is the relative stability of the penta-coordinated form of Cu(II) as described in the crystal structure, compared to tetra-coordinated Cu(II) models that lack the axial water molecule [58,59,62,63]. It was found that when the peptide model does not include the Trp residue, such as HGGG, the penta-coordinated model lies very close in energy to tetracoordinated models where the water molecule has moved into an equatorial position, displacing the backbone carbonyl ligand. In fact, such a tetra-coordinated model is the most stable structure when the B3LYP and B3PW91 functionals are used [58,59], while the penta-coordinated structure is the most stable one when BP86 is used [58]. Thus, it becomes difficult to discriminate between the two coordination models since their relative energies are very sensitive to the selection of functional and/or basis set. However, beyond these methodological subtleties, an important conclusion of such studies is that the absence of the Trp residue yields a small stability difference between the penta-coordinated Cu(II) model and a tetra-coordinated model where the water molecule moves into the equatorial plane. Indeed, when the larger model HGGGW is used, the most stable structure corresponds to a penta-coordinated Cu(II) model with an axial water molecule that is H-bonded to the Trp residue [58,63]. These results highlight the crucial role that the Trp residue plays in stabilizing the formation of an H-bond network that favors the binding of a water molecule in an axial position. As shown in the crystal structure of Cu(II)–HGGGW [53], there are six water molecules in the vicinity of the metal center: one water molecule is axially coordinated to Cu(II); two molecules are H-bonded on the same side as the coordinated water and the Trp residue, and three water molecules are H-bonded on the opposite side, as shown in Fig. 3A. It has been proposed that the participation of a water molecule as an axial ligand is favored by the H-bonding network that includes the Trp residue, backbone carbonyl groups and several water molecules. Thus, the role of these explicit water molecules in the geometric and electronic structure of this binding

site has also been studied [55,57]. A comparison of HGGG models with and without the axial water ligand, having identical equatorial coordination modes, revealed that the axial water molecule induces a square-pyramidal geometry where the metal ion is pulled out of the equatorial ligand plane [57]. Another study found that the equatorial coordination geometry does not deviate significantly with respect to the crystal structure by the absence of the axial water ligand (compare structures in Fig. 3A and C) [62]. Furthermore, in the models that include explicit water molecules, it becomes evident that a very stable H-bond network is formed, and it prevents the axial water ligand from occupying the equatorial position to yield a four-coordinate Cu. The role of different water networks has been systematically tested in a series of Cu(II)–HGGGW models [62]. In geometry optimizations that use as an initial structure the complete unit cell of the X-ray structure containing six water molecules, small geometric deviations are observed (Fig. 3A) and the axial water molecule remains coordinated to the Cu ion with a Cu O distance of 2.366 A˚ (this distance is 2.380 A˚ in the crystal structure [53]). A model that keeps the axial water and two water molecules on the same side of the complex, and lacks the three waters that appear on the other side of the complex in the crystal structure, reproduces the Cu coordination geometry and preserves the axial water lig˚ In contrast, when all explicit and with a Cu O distance of 2.358 A. water molecules are removed from the model except for the one that is axially bonded to Cu, the water moves away from the metal ion, yielding a final Cu O distance of 3.561 A˚ (Fig. 3B). These results highlight the important role that explicit water molecules H-bonded to the metal-bound water play in stabilizing axial water coordination to Cu. Interestingly, the extension of this model to the PHGGGWGQ sequence, including six water molecules, leads again ˚ to a very long Cu O distance for the axial water molecule (3.313 A, Fig. 3D), suggesting that the peptide conformation can also alter the water network around the site. However, this effect must be analyzed with caution, as the GWGQ residues constitute a flexible linker that adopts a bent or turn structure when more than one PHGGGWGQ unit are included in MD simulations [79]. The coordination of an axial water molecule has also been challenged by classical and quantum molecular dynamics studies [80–84]. We will refer to the former as force field MD (ffMD) and to the latter as ab initio MD. Ab initio MD simulations using the Car–Parrinello approach and program (CPMD) were performed at 150 K in a Cu–HGGG model with an axial water ligand and showed that water does not remain bonded to the metal ion [81], while the 3NO equatorial coordination is maintained. The authors argue that water coordination in the axial position in the crystal structure can be partially ascribed to crystal packing, and that the Cu–OH2 interaction can be of a dynamical nature. On the other hand, ffMD simulations in a Cu–PHGGGWGQ model including the six water molecules derived from the crystal structure as an initial configuration yield different results depending on the bonding model and charge sets used [83]. Simulations using the non-bonded model (with a charge of 2+ at the Cu center) lead to a complex with two axial water ligands where the Trp residue influences the solvent structure on one side of the copper center, while the axial water defines the distribution of water molecules nearby. In contrast, simulations with a bonded model lead to a very different spatial distribution of solvent molecules around the metal ion. In this case, the Cu ion does not coordinate axial water molecules in a stable manner, and instead, backbone carbonyl groups such as the one from the Trp residue are able to interact axially with the metal ion. It was concluded that the bonded model best describes the Cu(II)–octarepeat interaction. Finally, ffMD simulations in a model containing four linked units of the PHGGGWGQ–Cu(II) complex [84], revealed that the Cu ion displays a cation–␲ interaction with the Trp indole ring without water mediation. This type of

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Fig. 3. Effect of water molecules in the structure of the HGGGW–Cu(II) complex. Geometry optimized structures of the HGGGW–Cu(II) complex with six (A), one (B) or no explicit water molecules (C); and for the PHGGGWGQ–Cu(II) complex with six water molecules (D). The Cu O distance for the water molecule coordinated to the metal ion in the axial position is shown in each structure. This figure was made using model coordinates as reported in the supporting information of [62], with permission of American Chemical Society.

interaction seems to be particularly favored when using models with more than one OR–Cu(II) unit. In summary, electronic structure calculations and MD simulations yield the picture that the equatorial coordination shell of the Cu ion in Component 1 (as depicted in Figs. 2 and 3) is very stable, while the coordination of an axial water ligand is labile, and the structure of the solvent molecules around the complex is very dynamic. However, under certain conditions, such as those for the crystal structure, axial water coordination may be favored by several factors including the peptide conformation, and the H-bonding network that involves the Trp residue and water molecules in the vicinity. These conclusions have significant implications for the theoretical investigation of Cu binding sites in PrPC , as they underscore the importance of including explicit water molecules and different water networks when modeling Cu coordination to the unstructured region of PrPC . 2.1.2. Electronic structure of the OR–Cu(II) complex (Component 1) The electronic configuration for Cu(II) is d9 with one unpaired electron. Consistent with experimental EPR measurements, single occupied molecular orbitals as derived from electronic structure calculations involve the Cu dx2 −y2 orbital (Fig. 4) [57,58]. The dominating ligand–metal interactions occur with the directly coordinated nitrogen atoms from the imidazole ring (NHis ), and the two deprotonated backbone amide groups (Namide ). Electronic structure calculations using models for Component 1 have shown that the interactions with these three equatorial nitrogen donors are non-equivalent (Fig. 4) [57,58]. Indeed, the Cu N bonds with the amide groups are more covalent than the Cu NHis bond. Mulliken atomic spin density distributions show that 3–5% of the spin density is delocalized to the imidazole nitrogen (NHis ), while 14–18% is delocalized to each of the two deprotonated amide nitrogen’s (Namide ); this trend is independent of the functional used for the calculation [58]. In contrast, only a small percent of the spin density (3–4%) is found at the oxygen from a backbone carbonyl that binds to the Cu ion, while the electron density located around the oxygen

atom of the axial water molecule is almost negligible. These results are in line with conclusions from ab initio MD simulations stating that the Cu Namide bonds are stronger than the Cu NHis bond, while the Cu O bond with the backbone carbonyl that completes the equatorial coordination shell is relatively weak [80,81]. The distribution of the electron spin density in Component 1, as depicted in Fig. 4, relates directly to its EPR spectroscopic properties. Experimentally, X- and S-band continuous wave EPR and ESEEM experiments have been used to study Component 1 [41,49,53]. DFT calculations of EPR parameters for this site have also been performed, using different functionals and structural models [57,58,62]. The computed Cu g tensors always yield gz > gy > gx > 2.00, consistent with having a tetragonal Cu(II) with a dx2 −y2 ground state, while computed gx and gy values indicate that the complex is affected by a small rhombic distortion [58]. Calculations of the gz tensor yield values that range from 2.09 to 2.17, which are smaller than the experimental value (2.24) [49,85], indicating that the exchange-correlation density functionals currently available are inadequate for quantitative comparison with experimental data [57,58]. However, calculated g tensors can reproduce experimental trends and may help to distinguish between different coordination geometries [86,87], particularly in combination with calculated Cu hyperfine tensors (Cu A). Some studies show that the calculation of both tensors improves using hybrid functionals instead of pure-GGA functionals [86–92]. Mulliken atomic spin density distributions show that 53–60% of the spin density is located at the Cu center, which relates directly to the Cu electron–nuclear hyperfine interaction, as coupling of the unpaired electron spin (S = 1/2) with the Cu nuclear spin (I = 3/2) leads to a hyperfine splitting. Generally, electron–nuclear metal hyperfine coupling constants (Cu A) have three main contributions: (i) Fermi contact, which is isotropic and relates to the interaction of the unpaired electron spin density of Cu(II) at the nucleus with the nuclear spin; (ii) the spin–dipolar interaction which is anisotropic and involves the electron spin vector dipolar coupling with the nuclear spin on the metal; and (iii) the orbital dipolar or spin–orbit coupling contribution which is also anisotropic [93].

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Fig. 4. Electronic structure of the HGGGW–Cu(II) complex (Component 1). Singly occupied molecular orbital for the HGGG–Cu(II) complex with three (A) and six (B) explicit water molecules. This figure was generated using data reported in Ref. [57], with permission of Society for Biological Inorganic Chemistry.

All three contributions can be calculated in the frame of DFT tools [94–96]. The computed Cu hyperfine tensors Az range from 400 to 550 MHz [57,58], while the best agreement with the experimental result (450–490 MHz) [53,85] is achieved with the B3LYP hybrid functional, using a CP(PPP) basis set [97] for Cu and models that include all six waters in the vicinity of the Cu center. Particularly, the inclusion of a water molecule as an axial ligand improves significantly the agreement with experimental results, supporting the notion that the Cu–water interaction as described in the crystal structure is present in solution (see discussion in Section 2.1.1). As mentioned above, the most covalent metal–ligand interaction in Component 1 is the Cu Namide bond. Understanding the electronic nature of the Cu Namide bond is of particular interest as this kind of Cu(II) ligation is found in other amyloidogenic proteins associated to neurodegenerative diseases, such as the highest affinity Cu binding site in ␣-synuclein [98–100] and one of the Cu(II) coordination modes found in ␤-amyloid at physiological pH [101,102]. The electron pair of a deprotonated amide N atom is in a hybrid orbital with sp2 and sp3 contributions. A deprotonated amide can have two resonant structures: one with the lone pair and negative charge at the Namide atom, and another one where the electron pair is located at the carbonyl oxygen while the Namide forms a double bond with the carbon atom. This resonance allows for some delocalization of the spin density onto the carbonyl oxygen. Indeed, Mulliken atomic spin density distributions for Component 1 show that 1–3% of the spin density is delocalized to the carbonyl oxygen of the ligating amide group [58]. Moreover, the extent of the spin density distribution into the carbonyl oxygen correlates well with the covalency of the Cu Namide bond (Fig. 4). The double bond character of the amide moiety induces the planarity of the peptide linkage and favors a structure where the metal ion is close to the plane formed by the Namide ligands [103]. The strength and covalency of the Cu Namide bond yield distinct spectroscopic effects: (i) a strong superhyperfine interaction between the electron spin and the nuclear spin (I = 1) of the amide nitrogen become evident in the EPR spectrum; (ii) an intense Namide →Cu(II) ligand to metal charge transfer (LMCT) transition is observed in electronic absorption and circular dichroism spectra at 290–320 nm [104–106]; and (iii) the d→d transitions shift to higher energy with the number of Cu Namide bonds present in the complex, indicating an increase in the ligand field strength [103]. Particular emphasis has been placed in the calculation of superhyperfine couplings associated to the Cu Namide bond [57,58,62]. Electron-nuclear superhyperfine interactions, such as the coupling between the unpaired electron of Cu(II) with the nuclear spin (I = 1) at the coordinating nitrogens, can be described with the same contributions as mentioned above. However, it is usually assumed that the spin–orbit contribution to the hyperfine coupling is only important for atoms with large nuclear charge, such as the Cu(II)

ion, while for atoms with small nuclear charge and spin–orbit coupling constants, such as 14 N, the spin–orbit contribution is usually negligible. Additionally, the covalency of the metal–ligand bond will also affect the magnitude of the superhyperfine coupling, as delocalization of the electron spin onto the ligand allows it to directly interact with the nuclear spin of such ligand [93]. The isotropic contribution for the superhyperfine coupling tensor (Aiso ) for the three Cu N bonds in Component 1 has been calculated using several different functionals [57,58]. Only relatively small variations in Aiso are observed as the functional is varied and solvent effects are included, displaying the same trend. Consistent with the electron spin density distribution (Fig. 4), the Aiso values indicate that the most covalent Cu N bond corresponds to the Cu Namide bond that is trans to the equatorial Cu O bond (with Aiso values in the range of 51–60 MHz), followed by the Cu Namide bond that is trans to the Cu NHis bond (with Aiso values in the range of 26–40 MHz). The least covalent Cu N interaction corresponds to the Cu NHis bond, with Aiso values in the range of 16–23 MHz. The calculated average Aiso for the three nitrogens is in the range of 31–39 MHz [57,58,62], which is in good agreement with the reported experimental values (36–42 MHz) [53,85]. However, it should be noted that the notion of having three non-equivalent Cu N interactions in this complex has emerged from electronic structure calculations, as the experimental EPR results were initially interpreted in terms of having three equivalent nitrogen ligands with a potential hydrogen superhyperfine interaction. A recent interpretation and simulations of the experimental EPR data using information derived from electronic structure calculations have demonstrated that the splitting pattern observed in the experimental S-band EPR spectrum is due to three non-equivalent nitrogen atoms with no contribution of a hydrogen hyperfine coupling [57]. In summary, electronic structure studies of Component 1 indicate that the dominating Cu(II) bonding interactions in this complex involve the two deprotonated amide ligands, which yield Cu Namide bonds that are significantly more covalent than the Cu NHis bond, and determine the main spectroscopic features of this site. The calculation of EPR parameters using DFT and their comparison to experimental results has yielded important insights into the electronic structure of this complex. Most notably, the notion that the Cu N bonds are non-equivalent, as derived from calculations, has aided the interpretation of EPR data, thus, highlighting the value of correlating electronic structure calculations to experimental spectroscopic results. 2.1.3. Structural properties of multiple histidine coordination modes As mentioned above, at low Cu:protein ratios, Cu(II) can bind to three or four His residues in the OR region (Component 3, Fig. 2).

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As the geometry optimization of models including the whole OR region to form this binding site would not be feasible for electronic structure calculations using current programs, the His residues for this coordination mode have been modeled by 4-methylimidazoles, neglecting the role of the amino acid sidechains in the vicinity of the His residues and the geometric constraints that the peptide backbone may impose to the complex [107]. Alternatively, multiple His coordination modes have also been studied using molecular dynamics [50,61,80,108–113]. These studies show that Cu coordination via four N␦ atoms with the terminal methyl groups oriented in the same direction increases strain. Overall, Cu coordination by the N␧ atom of the imidazole ring is favored over N␦ coordination [107,109–112]. Also, Cu(II) complexes with four imidazoles are more stable than complexes with three imidazoles and one water molecule [107,109,110]. Thus, the best description for Component 3 is a Cu(His)4 complex with all Cu NHis bonds formed via the N␧ atoms, as shown in Fig. 2. At higher Cu:protein ratios, an intermediate component with a 2N2O equatorial coordination is observed, where Cu(II) is likely bound by two His residues, although different structural models have been proposed based on experimental data. Some authors proposed that Cu coordinates to two His residues, one deprotonated amide and two water molecules (Component 2, Fig. 2), where one of the His residues binds equatorially, while the other one seems to act as an axial ligand [CuHiseq N− amide (H2 O)2 Hisaxial ] [49,52]. A structural model based on the [CuHiseq N− amide (H2 O)2 Hisaxial ] proposal was built using a hybrid DFT/DFT method. The structure was compared to another model where the axial His was removed and replaced by an axial water molecule [110]. The energy difference between the two coordination modes indicates that the axial His is energetically favored over the axial water. Alternatively, a structure for Component 2 involving two His residues and two deprotonated amides has been proposed [Cu(His)2 (N− amide )2 ], based on spectroscopic data and a ffMD simulated annealing protocol [50,51]. A study using CPMD simulations further showed that multiple His coordination can take place even when amide deprotonation occurs [108]. In fact, the most stable structure identified in this study is a [Cu(His)2 (N− amide )2 ] coordination mode with a distorted tetrahedral geometry; the stability for this model is ascribed to the formation of four Cu N bonds. On the other hand, irrespective of the protonation states of the amide groups, the inclusion of a second His residue in the Cu coordination shell has a stabilizing effect on the complex [108]. In fact, it was determined that at zero-temperature, the 3NO coordination mode as in Component 1 (Fig. 2) has the same energy as the [Cu(His)2 (N− amide )2 ] coordination mode. This implies that, if two protons are removed from the backbone and the Cu:protein ratio is relatively low, the formation of the [Cu(His)2 (N− amide )2 ] species would be favorable [108]. Considering the different geometries of these two coordination modes, it was hypothesized that the tetrahedral nature of the [Cu(His)2 (N− amide )2 ] species would be more amenable for Cu(II) reduction to Cu(I) than the square planar 3NO structure, as it would

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reduce the reorganization energy and maximize the reduction rate [108,114]. In summary, experimental and theoretical studies agree that the Cu coordination mode favored at low Cu:protein ratio, namely Component 3, corresponds to a Cu(His)4 complex with all Cu NHis bonds formed via the N␧ atoms, as shown in Fig. 2. In contrast, the structure associated to the intermediate species or medium Cu:protein ratio, namely Component 2, remains elusive. There is consensus in the notion that two His residues participate in Cu(II) coordination, but two different modes have been proposed: [CuHiseq N− amide (H2 O)2 Hisaxial ] or [Cu(His)2 (N− amide )2 ]. Theoretical studies seem to support the latter model, however, a detailed comparison of the electronic structure of these two Cu coordination models with spectroscopic data should help discriminate between these two proposals. 2.2. Cu binding outside the octarepeat region 2.2.1. Cu binding to His96 While the coordination properties of the high Cu(II) occupancy species in the OR region have been revealed by very detailed spectroscopic, crystallographic and theoretical studies, the nature of the Cu(II) coordination outside the octarepeat region has received much less attention. His96 and His111 have been recognized as the primary anchoring residues for Cu(II) binding [43,115–117], and the PrP(92–96) and PrP(106–113) fragments have been identified as the minimal sequences to reproduce Cu(II) binding to each of these His residues [41,42,118]. However, the formation of a Cu(II) complex involving both His residues cannot be ruled out, particularly at low pH and low Cu:protein ratios [119]. Early EPR studies clearly demonstrated that Cu(II) binding to the PrP(92–96) fragment (with sequence GGGTH) involves the His96 imidazole and deprotonated amide groups from the preceding Thr and Gly residues [41]. Cu(II) binding at the His96 site is highly pH-dependent and it displays several protonation equilibria, one of them with a pKa of 7.8 [120]. At physiological pH, two binding modes are relevant, with 3NO and 4N equatorial coordination modes [120,121]. The 3NO mode involves two deprotonated amides, the His96 imidazole ring and a backbone carbonyl, while in the 4N coordination mode, the latter is replaced by a third deprotonated amide (Fig. 5). The electronic structure of the His96 site has been relatively unexplored in theoretical studies [56,107,122]. The lack of a crystal structure for this Cu(II) binding site makes the construction of appropriate structural models much more challenging. Thus, the comparison of calculated spectroscopic parameters with experimental data becomes crucial for validating different coordination models. Unfortunately, little has been done to calculate spectroscopic properties for His96 complexes; only one study has addressed the simulation of CD spectra for Cu(II) complexes with a GGGH model for this site [122]. On the other hand, structural models using the GTH sequence have shown that the Cu(II) complexes are not completely planar, being the 3NO complex the most

Fig. 5. Cu(II) coordination modes for the His96 and His111 binding sites. Cu(II) binding to these sites is pH-dependent with pKa values of 7.8 for the His96 site and 7.5 for the His111, yielding two different coordination modes at physiological pH: 3NO (left) and 4N (right).

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distorted from planar coordination [56]. The same study reports that the Cu(II) binding affinity for the 3NO mode is comparable to that for the Component 1 of the octarepeat region, while the 4N complex shows a larger affinity for Cu(II). An important difference between the His96 and His111 coordination modes (Fig. 5) and Component 1 in the octarepeat region (Fig. 2) is that Cu binding at His96 and His111 involves amide groups that precede the His residue (i.e. binding in the “reverse direction”), while the binding in Component 1 involves amide groups that follow the His residue in the sequence (i.e. binding in the “forward direction”). A detailed theoretical study has established the propensity of Cu(II) to coordinate in the reverse or forward direction relative to the His residue, using a series of tetrapeptide models, namely HGGG, GHGG, GGHG, GGGH, acetylated at the N-terminus and amidated at the C-terminus [122]. The relative free energies

associated to Cu(II) binding to these peptides and sequential loss of protons of the system were determined and compared. For all peptides, the initial binding of the solvated Cu ion [Cu(H2 O)4 ]2+ to yield His-anchored Cu complexes at low pH (i.e. without the loss of protons) is a thermodynamically favored process (G < 0). The first amide to lose a proton is the one that lies trans to the Cu N bond of the His residue, to yield a 2N2O complex [54]; ab initio MD simulations show that a water molecule can extract the proton from this amide nitrogen [81]. For the HGGG and GHGG complexes, after the loss of one proton and three water molecules, the most stable coordination mode is a 2N2O mode in the “forward direction” where the deprotonated amide corresponds to the second Gly that follows the His in the sequence (2N2Oforward in Fig. 6). In contrast, for the GGHG and GGGH models, the first proton is lost from the amide corresponding to the Gly that precedes the His in

Fig. 6. Cu(II) binding to the backbone chain in the “forward” and “reverse” directions. This scheme shows Cu(II) binding to a His containing peptide (HisL) involving amide deprotonation from residues that follow (Y) the His (“forward binding”) as in the case of Component 1 in the OR region, or from residues that precede (X) the His (“forward binding”) as in the His96 or His111 sites. The initial binding yields a Cu complex where the metal ion is bound to the His imidazole group and three water molecules. Upon the loss of a proton and three water molecules, 2N2O complexes are formed, subsequent proton losses yield 3NO and 4N coordination modes.

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the sequence, yielding a 2N2O coordination mode in the “reverse direction” with the deprotonated amide in a trans position to the His (2N2Oreverse in Fig. 6). These results indicate that the choice of Cu binding in the forward or reverse direction is dictated by two factors: (i) the preference of Cu(II) to have the first deprotonated amide coordinating in a trans position with respect to the anchoring His residue, and (ii) the availability of amide groups around the His residue. On the other hand, for all models, the second proton is lost from the intervening amide group to yield a neutral 3NO coordination mode, while the loss of a third proton can lead to a 4N coordination mode with three deprotonated amides (Fig. 6). A comparison of the relative aqueous free energies for the most stable structures with one, two and three deprotonated amide groups yields interesting results [122]. For singly (2N2O) and doubly (3NO) deprotonated structures, coordination modes resulting from binding in the forward direction are more stable than those from reverse binding, indicating that generally binding in the forward direction would be preferred. In contrast, 4N structures resulting from backbone deprotonation in the reverse direction yield a five, five, six ring pattern (4Nreverse in Fig. 6) and are more stable than the five, five, seven ring pattern that results from forward binding (4Nforward in Fig. 6). The stability difference between the 4N structures may simply be due to the difference in stability between the six and seven membered rings formed with the coordination of the His residue and the closest deprotonated amide in each case [54]. However, it is interesting that, even though these rings with the His residue are formed in the doubly deprotonated 3NO structures, the stability trend for those structures is reversed. The second deprotonation yields 3NO structures with a set of five, five and seven membered rings in the forward direction (3NOforward in Fig. 6), while in the reverse direction it yields a set of six, five and seven membered rings (3NOreverse in Fig. 6). Thus, the stability difference between the 3NOreverse and 3NOforward structures could be related to the difference in stability between their five- and six-membered rings. This means that another determining factor for binding in the forward vs. reverse direction is the relative stability of the ring patterns that are formed in the chelate. An important consequence of the relative stabilities for 3NO and 4N structures in the reverse and forward directions is that, when Cu binding occurs in the forward direction, the 3NO mode is stabilized and further loss of a proton to form the 4N complex is less favored (G = 98 kJ mol−1 for the conversion of 3NOforward to 4Nforward in the HGGG system); in contrast, for Cu binding in the reverse direction, the conversion of 3NOreverse to 4Nreverse faces a less positive G (66 kJ mol−1 for the GGGH system) [122]. This effect may explain the difference in pKa values associated to the 3NO to 4N conversion between Component 1 (8.9) [85,123] and the His96 and His111 sites (7.8 and 7.5, respectively) [120,124]. Finally, the nature of the sidechains in the vicinity of the His residue must also play an important role in determining the direction of the binding, although it has not been systematically evaluated. In particular, for Component 1 in the OR region, the Pro residue prevents amide deprotonation in the reverse direction and it induces a turn in the peptide, which would further stabilize forward binding [54]. In the case of the His96 site, having Gly residues preceding the His may favor amide deprotonation in the reverse direction over the forward direction, which involves the bulkier SQW sidechains. The presence of the Trp residue may also play a role in destabilizing forward binding, although this has not been probed. In summary, Cu binding to His96 has been relatively unexplored with theoretical methods. The electronic structure of the coordination modes proposed for this site are likely to be similar to those for Component 1 in the OR region or the 3NO and 4N modes proposed for the His111 site (vide infra). However, an important difference

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between the His96 site and Component 1 is the direction of Cu binding and amide deprotonation. Theoretical studies have shed light into some factors that determine the choice of Cu(II) binding in the forward or reverse direction, including: (i) the preference of Cu(II) to have the first deprotonated amide coordinating in a trans position with respect to the anchoring His residue; (ii) the availability and accessibility of amide groups around the His residue; (iii) the relative stability of the ring pattern that is formed upon chelation; and most likely, (iv) the nature of the sidechains in the vicinity of the His residue, an aspect that must be further evaluated. 2.2.2. Cu binding to His111 In contrast to all other Cu binding sites at the N-terminal of human PrPC , an interesting property of the His111 site is that two Met residues are located in the vicinity of His111, opening the possibility of having Met coordination to Cu. However, the ligating residues involved in Cu(II) binding to His111 have been a matter of great controversy. There is consensus that Cu(II) coordinates to His111 and to a number of deprotonated amide groups from the backbone (one to three) [116,119,125–130], while the participation of the Met residues has been debated. The different proposals for equatorial coordination modes of Cu(II) bound to His111 at physiological pH include: 3NO with [128] or without [119,124] two axial waters, 4N [119,124,126], 3NS [126], and 2N2S and 2NSO with the participation of both Met109 and Met112 residues [129–131]. Given the lack of a crystal structure for this Cu(II) binding site, comparison of spectroscopic parameters as derived from electronic structure calculations to experimental results can be a useful approach to discriminate between different coordination modes. A DFT study modeled Cu(II) binding to His111 with 2NOS, 3NS and 3NO coordination modes, using the short peptides KH and GKH, acetylated at the N-terminus and with the C-terminal carboxylate removed from the His residue, and modeling Met coordination with dimethylthioether [130]. The absorption spectra and EPR parameters were calculated for each model and compared to experimental results. A comparison of the experimental absorption spectrum for the KTNMKHMAG–Cu(II) with the calculated spectra for each model, as derived from time dependent DFT calculations, allowed to discard the 3NS model. The authors considered that the 2NOS model provides the best match between calculated and experimental EPR parameters. Unfortunately, these conclusions are obscured by the fact that the spectroscopic data were collected at pH 7.4, where a mixture of two species is present considering that the pKa for the KTNMKHMAGA–Cu(II) complex is 7.5, as derived from circular dichroism data [124]. A recent spectroscopic study [124] characterized in detail the two protonation states of the KTNMKHMAGA–Cu(II) complex that are relevant at physiological pH, yielding the following conclusions: (i) the equatorial coordination changes from a nitrogen rich environment (4N mode) at pH 8.5 to a 3NO coordination mode at pH 6.5, as shown in Fig. 5; (ii) Cu binding involves amide deprotonation in the reverse direction and the pKa of 7.5 can be ascribed to the backbone amide of Met109; (iii) substitution of the Met residues 109 and 112 do not cause significant changes in the pKa of the complex and the spectroscopic features associated to the two coordination modes. However, the M109A substitution leads to an increased ligand field, suggesting the participation of Met109 as an axial ligand in the 3NO complex. In order to further characterize the nature of the two Cu(II) coordination modes associated to His111, the same study reports electronic structure calculations on a large series of model complexes, using the full peptide KTNMKHMA acetylated at the N-terminus and amidated at the C-terminal, and two different functionals (LDA and PBE) [124]. Three different series of models were evaluated, with 4N, 3N and 2N equatorial coordination, including models that involve Met109 or Met112 coordinated axially or equatorially to yield a total of

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Fig. 7. Electronic structure of the two coordination modes for Cu(II)–KTNMKHMA. Geometry optimized structures and ␤-LUMO spin densities for the Cu(II)–KTNMKHMA complex in 3NO (A) and 4N (B) coordination modes. All hydrogen atoms and some amino acid sidechains are removed for clarity. This figure was prepared using data reported in [124], with permission of American Chemical Society.

13 different equatorial coordination models. Additionally, for each model, explicit water molecules were included as axial ligands. The geometry optimization of 4N models clearly indicated that when the Cu(II) ion coordinates to the His imidazole and three deprotonated amides, it cannot stabilize an axial interaction with Met ˚ The geomresidues, as the smallest Cu S distance found was 4.7 A. etry optimization of the 3N model series yields interesting results: (i) Cu models with a 3NO equatorial coordination mode as shown in Fig. 5 cannot stabilize an axial interaction with Met residues ˚ or water molecules; (ii) (smallest Cu S distance found was 4.7 A) in contrast, 3NS models with either Met112 or Met109 coordinated equatorially optimize well, although they get destabilized upon the addition of explicit water molecules. As a result, the most stable coordination model in the 3N series is 3NO as depicted in Fig. 5 with no axial coordination to water or Met residues. Finally, the results for the 2N series indicates that the most stable 2N coordination model is 2N2O where the equatorial oxygen ligands are provided by a backbone carbonyl and a water molecule, while a second water molecule is coordinated axially. The EPR parameters, namely g and Cu A tensors, were calculated for all models and compared to experimental results. With such a large set of models, it became evident that the Cu hyperfine splitting Cu Az is particularly sensitive to changes in coordination mode. The best match for the experimental data at pH 6.5 (gz = 2.226 and Cu Az = 501 MHz) was achieved with a 3NO equatorial coordination mode with no axial ligands, as depicted in Figs. 5 and 7A. The model includes two explicit water molecules and the closest one to the Cu center is 3.0 A˚ away. On the other hand, the best match for the experimental data at pH 8.5 (gz = 2.199 and Cu Az = 573 MHz) was achieved with a 4N equatorial coordination mode as depicted in Figs. 5 and 7B. The electronic structure of the two coordination modes associated to Cu(II) binding to His111 is shown in Fig. 7. For the 3NO mode, the spin density distribution is fairly similar to that of Component 1 (Section 2.1.2) having 58% of the spin density located at the Cu center, 4% at the ligating imidazole nitrogen NHis , 10–15% at each of the two deprotonated amide nitrogens, and ∼3% at the ligating oxygen from the backbone carbonyl [124]. Similarly, computed superhyperfine N A tensors (including both the Fermi contact and dipolar contributions, vide supra) for the nitrogens indicate that the most covalent Cu N bond corresponds to the Cu Namide bond that is trans to the equatorial Cu O bond (with N A|| = 58 MHz, N A⊥ = 40 MHz), followed by the Cu Namide bond that is trans to the Cu NHis bond (with N A|| = 38 MHz, N A = 25 MHz), while the least covalent Cu N interaction corre⊥ sponds to the Cu NHis bond (with N A|| = 29 MHz, N A⊥ = 23 MHz) [124]. In contrast, the 4N coordination mode has a different spin density distribution, particularly among the nitrogens, as there are three deprotonated amides coordinating the metal ion. Calculated superhyperfine N A tensors indicate that the most covalent Cu N bond is now the Cu Namide that is trans to the Cu NHis bond (with N A|| = 61 MHz, N A⊥ = 40 MHz). This is probably due because the weakest metal–ligand bond in the 4N complex is now

the Cu NHis one (with N A|| = 28 MHz, N A⊥ = 22 MHz). The second strongest Cu N bond corresponds to the newly formed Cu Namide from the Met109 amide (with N A|| = 50 MHz, N A⊥ = 38 MHz), which is trans to the weakest Cu Namide bond from the His111 amide (with N A|| = 38 MHz, N A⊥ = 27 MHz). Interestingly, while the electron spin density is completely redistributed among the Cu Namide bonds upon deprotonation of the complex going from 3NO to 4N, the spin density at the imidazole nitrogen NHis remains the same (∼4%). The redistribution of spin density among the Cu Namide bonds obeys the trend that the strongest Cu Namide interaction is always in a trans position to the weakest equatorial bond: the Cu O bond with a backbone carbonyl in 3NO structures, and the Cu NHis bond in 4N structures. In summary, Cu(II) coordination to His111 yields a mixture of two species at physiological pH, similarly to the case of the His96 site. Electronic structure calculations and their correlation to experimental EPR data have shed light into the nature of these two coordination modes, providing strong evidence for the coordination models 3NO and 4N, as depicted in Fig. 5. The electronic structure of these sites show strong covalent interactions with the deprotonated amide nitrogens, which yield non-equivalent Cu N bonds. The electron spin density gets redistributed among the Cu Namide bonds when the complex goes from 3NO to a 4N coordination mode. An interesting feature of this binding site is the presence of two Met residues in the vicinity of His111; although they do not seem to participate in Cu(II) coordination, they could contribute to Cu(I) binding and the redox activity of the site. Finally, it must be noted that the His111 site is the closest Cu binding site to the amyloidogenic region of human PrPC , underscoring the importance of evaluating the impact of Cu binding at this site in the aggregation properties of the protein. 2.2.3. Cu binding to the C-terminal domain Few studies have addressed Cu binding at the C-terminal structured domain of PrPC , yet there is controversy in this regard. EPR [132], ESEEM and ENDOR [133] experiments have identified potential Cu coordination to three C-terminal His residues (His 140, His 170 and His 187). A study of a series of His to Ser mutations in the PrP(121–231) fragment led to the conclusion that His177 is the anchoring residue for Cu(II) binding in this region [46]. However, this conclusion was obscured by the fact that the H177S and H187S samples presented aggregation during the Cu binding study. On the other hand, Cu(II) binding to the PrP(180–193) fragment has been demonstrated using EPR, CD, UV–vis spectroscopy and ESI mass spectrometry [45], pointing at His187 as the Cu anchoring residue. In contrast, a recent study on PrP(23–231) where the C-terminal His residues were mutated to Tyr, reports no differences in Cu binding relative to the wild type protein, concluding that the relevant high affinity Cu binding sites that relate to PrPC function are restricted to the N-terminal domain of the protein [117]. Cu(II) binding to the globular C-terminal domain of PrP has been evaluated using QM/MM calculations, placing Cu(II) ions near

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His residues 140, 177 and 187, and defining such sites as part of the QM region [48,134]. No stable Cu coordination was identified at His140 and His177 sites, while Cu seems to bind only to His187. Another QM/MM study used a Car–Parinello approach to evaluate Cu(II) coordination at each of the His residues mentioned above in the PrP(124–226) domain. Six possible Cu(II) binding modes were identified [47]. Three coordination modes were distinguished at the His140 binding site, two of them involve coordination of His140 and Asp147, with either Met138 or Asp144; while in a third mode, His140 protonates and moves away from the Cu ion to yield a site with only Asp147, Asp144 and water molecules. On the other hand, the His177 binding site involves Cu(II) coordination by the His imidazole, Asp178 and solvent water molecules. Finally, two possible coordination modes were identified for the His187 binding site, both involving the His187 and water molecules: one mode includes Glu197 while the other mode includes Asp202 and Met206. Given that the experimental EPR parameters (gz = 2.295 and Cu Az = 457 MHz) [132,133] suggest that Cu(II) bound to this region of the protein is square planar with a possible fifth ligand, two of these coordination modes were discarded. EPR parameters, namely gz and Cu Az , for the remaining four possible coordination modes were calculated and compared to experimental values. This comparison clearly determined that none of the coordination modes involving Met residues could be in agreement with experimental EPR results, leaving the His177 and the His187/Glu197 sites as potential candidates [47]. A comparison of computed nitrogen superhyperfine splittings to the experimental value (26 MHz) [133], together with a reinterpretation of experimental proton couplings, favor the His187 site. Thus, the authors conclude that Cu(II) binding to the C-terminal domain of PrP is likely to involve His187 and Glu197 residues [47]. 3. Effects of copper binding to PrPC 3.1. Redox properties of Cu–PrPC complexes As discussed above, PrPC has been established as a Cu binding protein with several binding sites, where the nature of the Cu coordination modes highly depend on pH and Cu:protein ratios. Understanding the redox behavior of the different copper binding modes could shed light into PrPC function. Several different proposals regarding the redox activity and functional implications of Cu–PrPC complexes have been put forward. It has been proposed that PrPC can play a protective antioxidant role, scavenging reactive oxygen species (ROS) before they damage other cellular components [135,136]. Alternatively, the antioxidant activity of PrPC has been interpreted in terms of its SOD-like activity [30]. In contrast, spin trapping studies suggest that PrPC is involved in a metal-mediated oxidative damage mechanism, where Cu–PrPC complexes could generate H2 O2 and ROS, causing oxidative damage in proteins and contributing to oxidative stress [129,137–139]. A recent review discusses these proposals in detail in light of recent findings on the electrochemical properties of Cu–PrPC complexes, giving important insight into their redox activity and functional implications [140]. Electrochemical studies of PrPC fragments have demonstrated that the main Cu(II) complex in the OR region (Component 1) and Cu(II) complexed with a peptide fragment containing His96 and His111 have reduction potentials (172 and 110 mV vs NHE, respectively) that allow them to oxidize ascorbic acid to form their corresponding Cu(I) complexes [141]. Both complexes in their reduced form can also generate hydrogen peroxide in the presence of oxygen; however, the amount of H2 O2 produced by such reaction is considerably lower than that by free Cu(II). In contrast, the multiple His Cu coordination mode present in the OR region at low pH and/or low Cu:protein ratios (Component 3) displays a

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higher reduction potential (323 mV vs. NHE), such that it can oxidize ascorbic acid to yield a Cu(I) complex, but it is not capable of reducing molecular oxygen to generate H2 O2 (E◦ associated to oxygen reduction to H2 O2 is 296 mV vs. NHE). These results clearly indicate that Cu chelation by PrPC can attenuate the redox activity and generation of H2 O2 by cellular free copper. These results place PrPC as an important player that regulates the concentration and redox reactions of cellular free copper [140]. Far from facilitating the generation of massive amounts of H2 O2 or ROS, PrPC displays antioxidant activity by modulating the generation of hydrogen peroxide as a function of pH and Cu:protein ratios: no H2 O2 production when the Cu(His)4 complex (Component 3) is formed at low pH and/or low Cu:protein ratios, and mild H2 O2 production at higher pH or Cu:protein ratios (Fig. 8B and C), which is proposed to trigger cell signaling cascades [140,141]. Provided the distinctive electrochemical behavior of the different Cu coordination modes found in PrPC , interesting questions arise on how the electronic nature of each Cu binding site relates to their redox activity. Few theoretical studies have addressed the redox activity of Cu–PrP complexes [56,107,111,112,142]. However, the notion that Components 1 and 3 (Fig. 2) could have distinctly different redox behavior emerged from DFT studies that compared a Cu(His)4 model for Component 3 with a model for Component 1 using a HGG peptide in a 3NO equatorial coordination as depicted in Fig. 2 [56]. The same study compares the 3NO and 4N coordination modes for the His96 site, as depicted in Fig. 5 and modeled by a GTH peptide. The redox potentials for each species were calculated using the B3LYP functional and the 6-311+G* basis set, including diffuse functions that are needed to improve the estimation of redox properties. Solvation free energies were also calculated using the polarizable continuum model. The Cu(His)4 complex was identified as the species with the greatest potential for reduction among the series; this is attributed to its coordination mode with four His residues in a distorted tetrahedral geometry that helps stabilize the Cu(I) form. The calculated reduction potential for the Cu(II)/Cu(I) couple in this multiple His coordination mode (+380 mV vs. NHE) [56] is in excellent agreement with the experiment (+323 mV vs. NHE) [141]. In contrast, the 3NO and 4N coordination modes with deprotonated amides in the Cu coordination shell yield computed negative reduction potentials that deviate greatly from experimental values, probably due to the high degree of electronic delocalization that characterizes the Cu Namide bonds in this type of coordination modes [56]. However, in spite of the huge discrepancies between computed and experimental redox potentials, a general trend can be established: the reduction potentials associated to the Cu(II)/Cu(I) and Cu(III)/Cu(II) redox couples decrease with the number of deprotonated amide nitrogens that participate in Cu coordination. This trend reflects the fact that deprotonated amide nitrogens behave as hard bases that help stabilize higher oxidation states of Cu. Another study used the MKH peptide to model different coordination modes (4N, 3NO and 3NS) for the His111 Cu binding site [142]. The computed reduction potentials for these species do not correlate well with experimental findings; however, similar trends with respect to the number of deprotonated amides in the coordination shell are observed. Moreover, the coordination modes with softer ligands, such as backbone carbonyl oxygen or Met thioether sulfur, tend to stabilize the reduced form Cu(I), and contribute to increase the reduction potential for the Cu(II)/Cu(I) couple. Undoubtedly, the electronic structure of each distinct Cu coordination mode at the N-terminal of PrPC is intimately related to its redox properties. Particularly, the number of deprotonated amides participating in Cu(II) coordination as a function of pH, and the presence of softer ligands such as Met thioether sulfurs as in the case of the His111 binding site, are key factors that regulate the reduction potential of each site. It is interesting that the computed

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Fig. 8. Schematic summary of the effects of Cu binding to PrPC . At low Cu:protein ratios (A), Cu(II) binds to the OR region to form a [Cu(His)4 ]2+ complex (Component 3) and a more stable conformation for the unstructured N-terminal region. The [Cu(His)4 ]2+ complex can be reduced by ascorbic acid (AA) to yield a Cu(I) complex [Cu(His)4 ]+ that does not react with oxygen and cannot produce hydrogen peroxide. Alternatively, the [Cu(His)4 ]2+ complex can be reduced by a Trp residue to yield a radical cation that leads to ␤-cleavage of the backbone chain. At high Cu:protein ratios (B), 3NO Cu(II) complexes (Component 1) can form ring stacking patterns, yielding a more compact and globular structure at the N-terminal. Component 1 can be reduced by AA and react with oxygen to produce hydrogen peroxide. Cu(II) binding to His96 or His111 sites (C) forms two coordination modes at physiological pH (3NO and 4N), inducing a “turn” in the backbone and favoring ␤-sheet formation. Similarly to component 1, these complexes can react with AA leading to a mild production of hydrogen peroxide. Cu(II) complexes are denoted with blue spheres, Cu(I) in orange spheres. No structural information is available for the reduced forms, but the X ligands may be other His residues or backbone carbonyls; for the His111 binding site, X could also be a Met thioether.

redox potential for the Cu(II)/Cu(I) couple in the Cu(His)4 complex is in excellent agreement with experimental results, while the calculated reduction potentials for all species involving deprotonated amides (in the range of −1.3 to −0.5 V vs. NHE) [56,142] deviate greatly from experimental values (+0.1 to 0.17 V vs. NHE) [140]. This is probably due to the fact that computed reduction potentials assume no changes in the coordination shell upon reduction. However, it is very likely that reduction of these sites is accompanied by a large rearrangement of the Cu binding site. As mentioned above, Cu(I) may prefer to bind to softer ligands such as backbone carbonyl oxygen or Met thioether sulfur as opposed to deprotonated amide nitrogens. Thus, reduction of the site could be coupled to protonation of the coordinating amide nitrogens, ligand exchange and rearrangement of the peptide chain, yielding different coordination and geometric arrangements for the Cu(I) complexes. For example, X-ray absorption spectroscopy studies of Cu(I) bound to the His111 site demonstrated that the two Met residues in the vicinity (Met109 and Met112) participate in Cu(I) coordination [129,143]. In contrast, Cu(II) coordination at this site does not seem to involve Met coordination [124]. If Cu coordination and geometry changes

radically upon reduction, it would result in a large reorganization energy of the site, and thus a slow rate of reduction. Experimental findings seems to support this notion, as the Cu(II)/Cu(I) redox cycling and H2 O2 production is very sluggish for the coordination modes that involve deprotonated amides, reflecting that these sites are not optimized for fast redox cycling. Theoretical studies in correlation to experimental results can definitely help dissect the steps associated to Cu(II) reduction and rearrangement at these sites and their contribution to their reduction potential. For such purpose, structural characterization of the reduced form of all Cu–PrP complexes is a key element that needs to be further assessed. Finally, another aspect of the redox chemistry of Cu–PrP complexes that has been extensively studied is in relation to the metal-catalyzed ␤-cleavage of the protein backbone. ␤-Cleavage occurs at the OR region of the N-terminal with some variability in the site of cleavage [144], and it seems to be dependent on Cu and Trp residues [145]. Thus, it has been proposed that the Cu(His)4 mode can be reduced to Cu(I) using a nearby Trp residue as an electron donor (Fig. 8A). This reaction is supported by theoretical studies, as DFT calculations predict that the formation of Cu(I)

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and a Trp indole-based radical cation is strongly favored [112]. MD simulations with the full OR region in a Cu(His)4 arrange˚ to the Cu ment showed that Trp81 and Trp89 stay close (6–7 A) ion, suggesting electron transfer from redox-active Trp to Cu(II) via the metal-coordination histidine residue. A potential mechanism for the protein ␤-cleavage involving this Trp indole-based radical cation has been proposed [112]. Briefly, this mechanism involves de-protonation of the Trp radical cation (the calculated pKa for this species is 6.7) to produce a neutral indole-based radical, which in turn can abstract an H-atom from an adjacent Gly alpha carbon. H-atom abstraction from a Gly residue by a neutral indole-based radical is predicted to be thermodynamically favored. The Gly alpha carbon radical could react with oxygen to undergo C C bond homolysis, leading to ␤-cleavage of the protein backbone (Fig. 8A) [111]. 3.2. Effects of Cu–PrPC interactions in protein folding and aggregation As most of the Cu binding sites in PrPC are located in its intrinsically unstructured N-terminal region, it is expected that this region would be the most sensitive to metal coordination in terms of folding. A better understanding of the different Cu coordination modes and their redox properties has been achieved in the last decade. An interesting and unresolved question is how these different Cu coordination modes and their redox activity affect PrP aggregation. Yet there is no consensus as to whether copper impedes or promotes prion disease. While some studies have shown that copper inhibits the in vitro formation of fibrils in full-length recombinant PrP [26], others suggest that Cu promotes aggregation and selfassociation [146,147]. Further research in this area should advance our understanding of the effect of copper on PrP aggregation. Given the difficulty of studying the molecular structure of PrPSc by experimental methods, the use of computational models in molecular simulations have gained special attention as a source of atomiclevel information of the processes involved in the conversion from PrPC to PrPSc . Although several theoretical studies have aimed to describe the impact of Cu binding on PrPC structure and aggregation [50,81,83,84,107,108,110–113,118], many questions remain unanswered, particularly with respect to the mechanism of aggregation and conversion of PrPC to PrPSc . This situation is mainly due to the limitations in quantum mechanical methods related to the size of the system affordable in a calculation, as well as the inability of analytic potentials used in protein force fields to reliably describe transition metals in classical molecular mechanics simulations. One of the features described in the local structure of the Cu(II)–octarepeat complex (Component 1) is the conformation of the Trp side chain within this fragment. Using a ffMD simulated annealing protocol, a structural model of the complex showed the indole group from Trp residue in close proximity of the copper ion [148], consistent with other theoretical studies [83,84]. This behavior would have the effects of: (i) reducing the flexibility of the region, (ii) changing the physicochemical properties of this portion of the protein, and (iii) promoting the copper “sequestering” ability of the PrP by hindering the access of solvent and other species to the metal ion. Copper coordination would also produce a consequent reduction of the flexibility of the protein backbone, especially in the binding modes involving deprotonated amides. This effect was observed in ab initio MD simulations with a model anchoring the Cu atom to a His residue without the participation or deprotonation of glycine’s amides [108]. In this study a weak interaction between the protonated nitrogen atoms from Gly residues and Cu bonded to the His residue was suggested to assist the folding of the peptide around the metal [114]. A preferred folding of the backbone in the octarepeat region of PrPC has also been observed in classical and ab initio MD

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simulations using models including two or more tandem repeats bonded to Cu in a high occupancy mode (Component 1, Fig. 2). In simulations of two tandem repeats with and without copper [79], it was observed that the sequence CWGQ connecting the two octarepeats is susceptible to form a turn allowing the neighbor metal centers to collapse, which brings two Cu ions within a distance of ˚ consistent with experimental EPR results [49]. Similar results 4–7 A, were obtained by ab initio simulations for the dimer (HGGG)2 [81], and in classical simulations with the full octarepeat domain (including four tandem repeats in the state of high copper occupancy) [84,113]. The ring structures formed on each Cu binding site tend to interact among each other and with other rings such as Pro residues and indole rings from Trp residues within the same octarepeat domain. These interactions form different competing ring stacking patterns, and have the effect of structuring the N-terminal region of PrPC , yielding more compact and globular structures than in the absence of copper coordination (Fig. 8B) [79,84,110]. It has been hypothesized that these stabilized structures would be less prone to misfolding, and would sequester Cu ions more effectively by isolating them from solvent and other species capable to produce ROS, thus relating to the Cu-induced structuring of the N-terminal to the neuroprotective role of PrPC [33,109]. Additionally, the differences in structure and physicochemical properties of the fully Cu-loaded and Cu-free forms of the N-terminal region could modify the interaction of PrPC with proteins or membranes, and thus, represent a Cu-dependent switch for cellular processes and signaling. The effect of Cu coordination to PrPC in the low Cu occupancy mode has also been studied. A ffMD simulation of the full-length PrPC supported by DFT calculations [110] compared the dynamics of the Cu-free form of PrPC to those for the protein bound to one Cu ion in the octarepeat domain (as Cu(His)4 , Fig. 8). Both, the structured C-terminal and unstructured N-terminal domains, reflected no significant changes in their secondary and global structure in the presence of the metal. However, the N-terminal domain appeared to be more stable with a 17% more prevalence of structural motifs during the simulation. On the other hand, as mentioned in Section 3.1, reduction of the Cu(His)4 mode by a nearby Trp could produce an indole-based radical cation that leads to ␤-cleavage of the protein backbone (Fig. 8A) [111,112]. Although ␤-cleavage occurs in the OR region of PrPC with some variability in the site of cleavage [144], the cleavage always occurs before His96. MD simulations indicate that Cu coordination to His96 and His111 residues in the remaining of the cleaved protein can induce localized ␤sheet structure, particularly when Cu binds to both His residues [111,112]. Structures with Cu(II) bridging between the two His residues generate a ␤-hairpin structure with hydrogen bonds that stabilize the antiparallel ␤-sheet structure. Alternatively, ffMD simulations also show that Cu(II) binding to His96 and His111 involving coordination by deprotonated backbone amides can induce ␤-sheet formation, mainly due to the “turn” in the backbone that Cu(II) coordination to backbone amides causes (Fig. 8C) [107]. These theoretical results are consistent with experimental findings, as the hydrophobic region around His111 has been identified as key for the conversion of PrPC to PrPSc [1,149], while Cu binding to His111 induces ␤-sheet structure in this region, as demonstrated by circular dichroism spectroscopy [150]. These findings have led to the proposal of a Cu-mediated molecular mechanism for ␤-sheet formation and potential nucleation for amyloid structures, involving initially the ␤-cleavage of the protein backbone, followed by Cuinduced ␤-sheet structured in the region of His96 and His111 sites. In summary, the use of MD simulations has been useful in studying dynamic features on the structure of PrPC that are influenced by the presence of copper. In particular, Cu bound to the octarepeat region in a high occupancy mode (Component 1) seems to induce more compact and globular structures at the N-terminal region (Fig. 8B), potentially preventing misfolding. Also, a Cu-mediated

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molecular mechanism for ␤-sheet formation and potential nucleation for amyloid structures has been proposed. Thus, theoretical studies may help to gain insight on how Cu–PrPC interactions relate to its physiological function and/or to PrPC aggregation and the molecular events producing disease. 4. Discussion and closing remarks In the last decade, PrPC has emerged as an important copper binding protein. There is considerable evidence supporting the notion that the interaction of copper with PrPC may play important roles in both, the physiological function of the protein, and in the pathogenesis of prion diseases. As reviewed here, the nature of the different Cu(II) binding sites in PrPC has been explored using electronic structure calculations and molecular dynamics simulations. These theoretical studies have expanded the understanding attained from experimental results and provide important insights into Cu–PrPC interactions. Particular emphasis has been placed in discussing the nature of the electronic structure of these Cu binding sites and their relation to redox properties. For most Cu coordination modes, the dominating Cu(II) bonding interactions involve deprotonated amide nitrogens. The nature of the Cu Namide bond has been discussed in detail, as it is also relevant for understanding Cu(II) coordination to other important amyloidogenic proteins associated to neurodegenerative diseases, such as ␣-synuclein [98–100] and ␤-amyloid [101,102]. Cu Namide bonds are significantly more covalent than the Cu NHis bond, and the distribution of electron spin density among them rearranges as a function of the type of Cu coordination mode. The electronic structure of each distinct Cu coordination mode is intimately related to its redox properties. Thus, the number of deprotonated amides participating in Cu coordination as a function of pH is a key factor that regulates the reduction potential of the site, as the hard nature of these ligands help to stabilize higher oxidation states. Conversely, the presence of softer ligands such as Met thioether sulfurs, as in the case of the His111 binding site, would stabilize the reduced form of the complex. Recently, important advances were made towards understanding the redox behavior of different Cu–PrPC coordination modes [140]. Theoretical studies are successful in calculating redox potentials for Cu binding modes that involve only His residues in the coordination shell, as is the case of the mode Cu(His)4 (Component 3). However, calculated reduction potentials for Cu binding modes involving deprotonated amides deviate greatly from experimental values, probably because calculations thus far have assumed no changes in coordination shell or geometry upon reduction. For Cu binding modes with deprotonated amides, reduction of the site is expected to have a large reorganization energy, as it could be coupled to protonation of the coordinating amide nitrogens, ligand exchange and rearrangement of the peptide chain, yielding different coordination and geometric arrangements for the Cu(I) complexes. Thus, having a good structural description for the reduced form of these Cu binding modes is imperative. Overall, the nature of reduced Cu(I) complexes associated to amyloidogenic proteins, such as ␣-synuclein, ␤-amyloid and PrPC , has been relatively unexplored [37,99,101], in spite of the fact that the redox activity of these Cu–protein complexes has often been invoked as part of mechanisms that link ROS production to protein aggregation and disease. For the case of PrPC , the emerging picture is that the redox activity of Cu–PrPC complexes is a proton and Cu concentration dependent property that modulates mild H2 O2 production (Fig. 8), which could be involved in cell signaling [140]. At the same time, the proposal that reduction of the multiple His Cu coordination mode Cu(His)4 by Trp could lead to ␤-cleavage at the OR region of the protein and relate to ␤-sheet formation, cannot be discarded (Fig. 8). In any case, further experimental and theoretical research efforts must aim at elucidating Cu(I) coordination

to the different binding sites in PrPC , as this knowledge is essential to understand their redox properties and reactivity with oxygen. In contrast to all other Cu binding sites at the N-terminal of human PrPC , an interesting property of the His111 site is that it has Met residues in the vicinity and it is the closest Cu binding site to the amyloidogenic region that is key for PrPC aggregation. For this site, correlation of electronic structure calculations to spectroscopic data has been useful to elucidate the nature of the relevant Cu coordination modes at physiological pH [124]. Now, with a clear picture of the electronic structure of this Cu binding site, some interesting questions can be addressed. In particular, it could be of interest to evaluate the role that Met residues 109 and 112 may play in Cu(I) coordination, redox properties of the site, and how it relates to PrP aggregation. The region encompassing residues 109–112 in the human sequence has been identified as key for the efficiency of prion propagation [151]. Met112 is not conserved among mammalian PrPs, while Met109 is highly conserved, being mouse PrP one of the very few mammalian PrPs that do not have a Met residue at this position [5]. These observations underscore the importance of evaluating the role of these Met residues in the redox properties of the Cu–His111 complexes and their impact in PrPC folding and aggregation. In this review, the impact of Cu–PrPC interactions in the protein structure and the molecular mechanisms that may initiate PrP aggregation were discussed. The use of MD simulations has been useful in studying dynamic features on the structure of PrPC that are influenced by the presence of copper. For instance, Cu binding to the OR region seems to yield more compact and globular structures at the N-terminal region of PrPC than in the Cu-free protein (Fig. 8A and B) [79,84,110]. It has been proposed that the Cu-induced structuring of the N-terminal may render the protein less prone to misfolding, and could modify the interaction of PrPC with proteins or membranes, representing a Cu-dependent switch for cellular processes and signaling [33,109]. On the other hand, based on MD simulations, a mechanism has been proposed for how Cu(II) binding to His96 and His111 may initiate ␤-sheet formation after ␤-cleavage at the OR region of the protein [111,112]. Alternatively, Cu(II) binding to His96 and His111 involving coordination by deprotonated backbone amides could also induce ␤-sheet formation, mainly due to the “turn” in the backbone that Cu(II) coordination to backbone amides causes (Fig. 8C) [107]. An interesting feature of the His96 and His111 coordination modes is that they involve amide groups that precede the His residue (i.e. binding in the “reverse direction”), while the binding in Component 1 involves amide groups that follow the His residue in the sequence (i.e. binding in the “forward direction”). Theoretical studies have shed light into the key factors that determine the direction of Cu(II) binding to backbone amides in the vicinity of the anchoring His. An interesting question to address is how these two different “directions” of Cu binding to the backbone can affect PrPC conformation and folding. In summary, theoretical studies have provided important insights on the nature of the different Cu–PrPC complexes and how their electronic structure relates to their redox activity. It is clear that different Cu–PrPC complexes have diverse redox properties and can have distinct effects in the structure of the protein. How do Cu–PrPC interactions relate to the physiological function of PrPC and/or to protein aggregation and the molecular events producing disease? Answering this question requires further understanding on how Cu–PrPC interactions are related to or modulated by other events, such as: (i) the presence and trafficking of other metal ions in neurons; and (ii) the interaction of Cu and PrPC with other membrane bound proteins and receptors. As zinc has emerged as an important metal ion in neurobiology that reaches concentrations at the synapse comparable to those of Cu ions [152], understanding the interplay of Zn and Cu binding to PrPC becomes of special interest. Considering the coordination preferences of Zn ions, they are

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likely to compete for Cu only in the case of multiple His coordination modes, such as Component 3. Indeed, theoretical studies support this notion, as Cu(II) displays higher affinity for the octarepeat than Zn(II) when considering the structural model for Component 1 [55,64]. In contrast, at low Cu concentrations when Component 3 is favored, Zn can displace Cu from this multiple His binding site, forcing it to bind only one His residue and adopt a Component 1 coordination mode [153]. Thus, Zn binding to PrPC may be an important modulator of Cu–PrPC interactions, and the interplay of such interactions must be explored. Similarly, recent findings on the relationship between Cu and Ca trafficking in neurons [154], along with the notion that Cu–PrPC interactions are important for modulating NMDA receptor activity in neurons [39] open exciting research directions.

Acknowledgments The authors would like to thank Prof. Sarah Larsen for kindly facilitating data for Fig. 4. The authors thank Consejo Nacional de Ciencia y Tecnología (CONACYT) for research grants CB2009128255 (to L.Q.) and CB2009-128369 (to A.V.A.), postdoctoral fellowship to L.R.A., and PhD fellowships to R.G.A., C.Z.G. and T.A.L.

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