Modeling by Assembly and Molecular Dynamics Simulations of the Low Cu²⁺ Occupancy Form of the Mammalian Prion Protein Octarepeat Region: Gaining Insight into Cu²⁺-Mediated β-Cleavage

Abstract

The prion protein has garnered considerable interest because of its involvement in prion disease as well as its unresolved cellular function. The octarepeat region in the flexible N-domain is capable of binding copper through multiple coordination modes. Under conditions of low pH and low Cu²⁺ concentration, the four octarepeats (ORs) cooperatively coordinate a single copper ion. Based on the average structure of the PHGG and GWGQ portions of a copper-free OR₂ model from molecular dynamics simulations, the starting structures of the OR₄ complex could be constructed by assembling the repeating structure of PHGG and GWGQ fragments. The resulting model contains a preformed site suitable for Cu²⁺ coordination. Molecular dynamics simulations of Cu²⁺ bound to the assembled OR₄ model (Cu:OR₄) reveal a close association of specific Trp and Gly residues with the Cu²⁺ center. This low Cu²⁺-occupancy form of prion protein is redox-active and can readily initiate cleavage of the OR region, mediated by reactive oxygen species generated by Cu⁺. The OR region is known to be required for β-cleavage, as are the Trp residues within the OR region. The β-cleaved form of the prion protein accumulates in amyloid fibrils. Hence, the close approach of Trp and Gly residues to the Cu²⁺ coordination site in the low Cu²⁺-occupancy form of the OR region may signal an important interaction for the initiation of prion disease.

INTRODUCTION

Prion diseases, or transmissible spongiform encephalopathies, are a family of neurodegenerative diseases that occur in humans and several animals. They may arise spontaneously through inheritance, or may be acquired through alternative routes such as the ingestion of prion-contaminated foodstuffs (1). Much interest in the prion protein (PrP) has been generated from its central role in prion disease. However, to date, the physiological function(s) of the cellular form of the protein (PrP^C) remain unclear. The PrP^C was proposed to function as a copper-buffering protein (2,3), and to play a role in neuronal development (4), neuroprotection (2,4,6,7), and possibly memory (8).

The octarepeat (OR) region is located in the flexible disordered N-domain of human PrP, and consists of four tandem repeats containing the sequence PHGGGWGQ (Fig. 1). Spontaneous and inherited gene mutations that give rise to additional copies of the OR sequence in PrP were implicated as risk factors for Creutzfeldt-Jakob disease (9,10). Mice expressing an OR-deletion form of PrP demonstrated delayed prion-disease onset and altered disease pathology after exposure to infectious prion material (11). The OR region can selectively bind Cu²⁺ (12) through as many as three dissimilar coordination modes, depending on the pH and/or local Cu²⁺ concentration, corresponding to high-occupancy, intermediate-occupancy, or low-occupancy forms (13). In addition, His-96, His-111, and the N-terminal amine in the full-length N-domain can also contribute to Cu²⁺ coordination in the full-length protein (14). Under low Cu²⁺-occupancy conditions, copper coordination to the full-length N-terminal domain results in multiple His-bound isomeric forms, primarily involving the OR region and His-96 (15). The Cu²⁺ binding to isolated fragments of the OR region was studied extensively, using spectroscopic methods (16–18). Multiple forms of copper (II) coordination occur throughout the disordered N-terminal region of the PrP at pH 7.4. However, there is limited information regarding the protein's three-dimensional (3D) structure after Cu²⁺ binding to the OR region. A small-molecule crystal structure of Cu²⁺ bound to the HGGGW residues of a single repeat was described (19), and is indicative of the binding mode of the OR region under conditions of high copper occupancy. This binding mode was recently used in extended molecular-dynamics (MD) simulations to predict how the OR region folds in response to high Cu²⁺ occupancy (7). Models were also proposed for the intermediate form of Cu²⁺ binding, based on spectroscopic data, mostly from electron paramagnetic resonance, circular dichroism, and nuclear magnetic resonance (NMR) spectroscopy (13,18). Electron paramagnetic resonance reveals that Cu²⁺ is bound by several imidazole N-donor ligands in the low-occupancy form, and its spectroscopic fingerprint is indistinguishable from that of Cu²⁺ in the presence of 50-M excess imidazole (13), indicating that the His imidazole side chains from each repeat are the primary ligands involved in coordination. Under low copper occupancy conditions, the metal center was shown to be redox-active, giving rise to the formation of reactive oxygen species (ROS) and ROS-mediated cleavage of PrP^C (20–22). The cleavage site, termed β-cleavage, is around Gly-90, near the end of the OR region.

FIGURE 1.

Sequences of OR₄ and OR₂ peptide models, compared with a portion of human PrP sequence.

Normal proteolytic processing of membrane-anchored PrP^C occurs through cleavage at position 111/112, termed α-cleavage (18), which leaves PrP(112–231) anchored to the cell surface. However, in prion-diseased brains, longer PrP fragments accumulate in amyloid fibrils comprised of PrP(∼90–231), with some variability in the site of cleavage (18). The OR region is necessary for ROS formation, and an OR-deletion form of PrP results in a loss of β-cleavage and increased sensitivity to oxidative stress (23). Loss of β-cleavage was linked to a loss of function of PrP^C (6), and may therefore be an important component in a protective mechanism for clearing oxidants in the extracellular neuronal environment. Two disease-associated PrP mutations failed to undergo β-cleavage, and PrP^−/− cells, as well as cells expressing PrP without the OR region, are more susceptible to oxidative stress (22). Without this protective capacity, PrP is susceptible to oxidative modification (oxidative damage is also a hallmark of prion disease), and such alterations increase the protein's propensity to aggregate (24,25).

In determining how Cu²⁺-mediated ROS formation and subsequent β-cleavage may occur, molecular modeling represents a necessary first step, because the inherent flexibility of this portion of the PrP and the paramagnetic properties of Cu²⁺ make it difficult to apply conventional structure-determination methods such as x-ray crystallography or NMR spectroscopy methods. Predicting the folded structure of a protein a priori is problematic. The backbone of each amino acid in a protein may have at least two stable conformations, and to predict how n-residues could fold together would require sampling as many as 2ⁿ conformations. For the OR region of human PrP, this would involve a minimum of 2³² (∼4.3 billion) conformations. The method we use here for generating a starting structure of the globular form of the metal-free OR₄ region follows an approach that resembles the zipping-and-assembly procedure of Ozkan et al. (26), where the average structure of a smaller metal-free fragment, as previously described (7), is used to assemble a starting conformation of the full-length OR region. The repetitive nature of the OR region (Fig. 1) makes it particularly amenable to such an approach.

Computational procedure

Assembly of OR₄ model

The full-length OR₄ model includes the GGWGQ portion of the non-Cu²⁺-binding sequence that precedes the four ORs in human PrP. Previous metal-free OR₂ simulations (7) showed a preference for the formation of a well-defined bend in the GWGQ region of the OR region, whereas the PHGG portion of the sequences formed flexible loops (Fig. 2). Based on apo-OR₂ simulations, the average φ and ψ angles for each residue in the PHGG loop and GWGQ bend were used to construct an average structure for the full-length OR region (Fig. 2).

FIGURE 2.

Stable structures of PHGG and GWGQ portions of a single octarepeat (OR) were derived from two 130-ns metal-free OR₂ MD simulations and used to assemble the Cu:OR₄ model. RMS-fitted overlays of PHGG and GWGQ portions of the OR₂ model are shown in two orientations. Loops and turns of the PHGG and GWGQ regions are emphasized. The PHGG and GWGQ residues in a representative OR of the assembled Cu:OR₄ model are highlighted. The remaining His residues and the copper center in the Cu:OR₄ model are shown for clarity.

Cu²⁺ binding site

Each of the four His side chains in the assembled model is closely packed and already suitably clustered together to accommodate coordination of a metal ion. The His χ₁ and χ₂ angles were modified to produce suitable starting structures for Cu²⁺ coordination, where either all Nδ1-atoms or Nɛ2-atoms were oriented toward a common center for insertion of a Cu²⁺ atom. In the copper-free OR₂ simulations, close association between His side chains was also evident, as shown in Fig. S7 in Supplementary Material, Data S1.

Density functional theory modeling and derivation of

Small-molecule structure models of the His side chain were modeled as 4-methylimidazole, in two alternate protonation states or Cu²⁺-bound states (Fig. 3). The Cu²⁺ complexes were modeled as [Cu^II(4-methylimidazole)(OH₂)₃]²⁺. All density functional calculations were performed with the Gaussian 03 suite of software (27). Geometry optimizations were performed at the B3LYP/6-31G(d) level, until the difference in energy between subsequent optimization steps was below 0.03 kJ mol⁻¹. This step also included harmonic-frequency calculations, from which zero-point vibrational energies, temperature-dependent enthalpy corrections, and entropies were derived. Single-point energy calculations from B3LYP/6-31G(d) structures were computed, using a larger basis set to obtain more accurate relative energies between structures (B3LYP/6-311+G(2df,2p)). Solvation was taken into account using a polarizable continuum model, IEFPCM (28–30), with the radius for Cu²⁺ as 1.40 Å, and the dielectric constant set at 78.39 (water).

FIGURE 3.

Tautomeric forms of His side chain, modeled as 4-methylimidazole. Theory and experiment both predict the Nɛ2-position as the favored site for protonation. Alternate sites of Cu²⁺ coordination are compared, and modeled as [Cu^II(4-methylimidazole)(OH₂)₃]²⁺ (coordinating solvent not depicted). Relative free energies are in kJ mol⁻¹.

The aqueous free-energy change () is calculated as:

where ΔH_(g) and ΔS_(g) are the energy and entropy difference between product and reactant enthalpies, ΔH_(g) is corrected to 298 K and includes the zero-point energy, and ΔS_(g) is the entropy difference between species, as provided in the frequency-calculation output. The ΔΔG_(solv) term is the difference between the free energy of solvation (ΔG_(solv)) for the product and reactant species.

Initial Cu²⁺-bound OR₄ geometry setup

Short-duration simulations were run with a single Cu²⁺-atom bound through either the Nδ1-atoms or Nɛ2-atoms of all four His residues. These initial models contained Cu²⁺ close to the desired His N-donor atoms, and were energy-minimized by steepest descent in the absence of solvent molecules, using GROMACS (31,32) and the OPLS all-atom force field (33,34). After energy minimization, the Cu₁:OR₄ models were simulated for 100 ps to relax the Cu²⁺-binding region. This procedure generated structures that could be subsequently simulated in the presence of solvent.

MD simulation

After the generation of the starting geometries, each molecule was placed in a 4.5 × 4.5 × 4.5 nm box, surrounded by 2836 SPC solvent molecules (35,36). Two Cl⁻ counterions were also added, to maintain the charge neutrality of the copper-containing system. Each of the systems was energy-minimized in the presence of explicit solvent, and simulated for 200 ns at 300 K, with an initial 5-ns equilibration phase. Simulations were subsequently performed according to previously described methods (7).

Parameterization of Cu²⁺ binding models

Alternate Nδ1-bound and Nɛ2-bound [Cu^II(4-methylimidazole)₄]²⁺ models were geometry optimized at the B3LYP/6-31G(d) level, until the difference in energy between subsequent optimization steps was below 0.03 kJ mol⁻¹. The geometry-optimization step was followed by a harmonic-frequency calculation at the same level of theory, to ensure that geometries were at a stationary point on the potential energy surface, and to derive force constants for Cu-ligand bonds.

The CHelpG charges (37) were calculated for each of the structures at the B3LYP/6-31G(d) level. Calculated parameters were imported into the OPLS all-atom force field, following previously described methods (7). The CHelpG charges for the copper centers in each of the [Cu^II(4-methylimidazole)₄]²⁺ models were averaged to +0.700. The backbone charges for His residues were maintained from the OPLS force field, whereas the imidazole ring and CβH₂ fragments had a net charge of +0.325 each, making the total charge for the copper(His)₄ binding sites +2.000. In the Nδ1-bound Cu:OR₄ simulations, the Cu²⁺-N-donor atom distances were all 1.985 Å, averaged from the associated Nδ1-bound [Cu^II(4-methylimidazole)₄]²⁺ models, and the force constants for each of the Cu-N bonds were 45,082 kJ mol⁻¹ nm⁻². The Nɛ2-bound Cu:OR₄ simulations used a Cu²⁺-N-donor atom distance of 1.914 Å, with force constants of 46,346 kJ mol⁻¹ nm⁻². The Cu²⁺ centers were in a pseudosquare planar geometry, based on the coordination geometries of the calculated [Cu^II(4-methylimidazole)₄]²⁺ models, and were of pseudo-D_2d symmetry. The parameters for restraining the coordination geometries in MD simulations used improper dihedral force constants of 33.472 kJ mol⁻¹ rad⁻².

RESULTS

Generation of starting conformation of OR₄ model

Using the results of previously described MD simulations (7) as well as additional simulations containing modified force-field parameters for tryptophan, a total of 300 ns of simulation time was generated for the copper-free OR₂ model (Fig. 1). Cluster analysis was used to group each of the visited conformations with similar conformations from all simulations, based on a root mean-square deviation (RMSD) cutoff of 0.1 nm, and included all residues in the OR₂ model. The PHGG and GWGQ residues from the major cluster of each simulation, corresponding to the most populated conformation shown in Fig. 2, were then used to generate average φ and ψ angles for each residue.

The mean φ and ψ angles were input for each series of PHGG and GWGQ residues in the OR₄ model. The assembled structure (Fig. 2) contains no other overlapping atoms or side-chain clashes, aside from initially overlapping His imidazole rings (not shown). The His side chains in the OR₂ models were also seen to make prolonged close approaches to one another (Fig. S7 in Data S1). The close packing of His side chains in the assembled model present a preformed site suitable for coordination of a single Cu²⁺ atom. The His side-chain χ₁ and χ₂ angles were subsequently modified, so that either the Nδ1-atoms or Nɛ2-atoms were suitably oriented to coordinate a central Cu²⁺ atom, as depicted in Fig. 2.

Histidine side-chain tautomers and Cu²⁺ coordination sites

The His side chain contains two N-atoms that confer the imidazole ring with multiple protonation states, and at physiological pH, two tautomeric forms of the charge-neutral imidazole ring may exist, as shown in Fig. 3. These tautomeric forms differ in the site of protonatation, at either the Nδ1-atom or Nɛ2-atom. Experimentally, a slight preference was found for protonation at the Nɛ2 position of the imidazole ring at physiological pH, meaning that the Nδ1-atom is most likely to bear the lone electron pair (38–40). The calculated aqueous free-energy difference () for the two tautomeric forms of His, modeled as 4-methylimidazole, is −1.9 kJ mol⁻¹, slightly favoring protonation at the Nɛ2-atom position. This agrees with the experimentally observed equilibrium for His (38,39). The real ΔG_(aq) for the amino acid has further contributions from H-bonds that stabilize this tautomer through the Nδ1-atom lone pair and adjacent backbone amide protons (39,41).

The between the two tautomeric forms for the site of copper coordination, at either the Nδ1-atom or Nɛ2-atom position, modeled as [Cu^II(4-methylimidazole)(OH₂)₃]²⁺, reveals that the Nɛ2-bound form is 3.0 kJ mol⁻¹ more stable than the Nδ1-bound form (Fig. 3). This indicates that coordination through the ɛ N-atom is likely. Miura et al. observed that at pH ∼6.5, the OR₄ region binds Cu²⁺ via the Nɛ2-atoms, whereas at lower pH, the His side chains protonate, making them unavailable for coordination, and as pH is increased, coordination via the Nδ1-atoms is ultimately preferred (42). Thus, coordination of Cu²⁺ via the Nɛ2-atom site appears to be preferred. However, the small free-energy difference for Cu²⁺ coordination, as shown in Fig. 3, led us to model two alternate coordination modes for the Cu²⁺:OR₄ model, with His coordination through either four Nδ1-atoms or four Nɛ2-atoms concurrently.

Average Cu²⁺:OR₄ structures

No stable helical or β-sheet structures formed during MD simulations of the assembled structures, in agreement with the lack of secondary structure evidenced by circular dichroism spectroscopy (43,44). Comparing results from the Nδ1-bound and Nɛ2-bound Cu²⁺:OR₄ models, coordination by all four Nδ1-atoms of the His residues maintains a more compact globular form of the OR₄ region, whereas Nɛ2-coordination tends to fan each OR loop outward (Fig. 4) during a simulation. The Nδ1-bound form also sequesters the metal center more than does the Nɛ2-bound form, which maintains a solvent-exposed metal-binding site (Fig. 4). The RMS-fitted overlays from the final 20 ns of the (PHGGGWGQ)₃PH portions of each Cu²⁺:OR₄ model, as well as each individual repeat (numbered OR1 through OR4) from each simulation, are shown in Fig. 4. Once anchored to the Cu²⁺ center through the His side chains, each repeat deviates little from its initial average conformation. The N-terminal and C-terminal ends of the model comprise the preceding GGWGQ, and the terminating GWGQ residues are disordered and display greater flexibility than the remaining OR₄ regions in the course of simulations. Additional RMSD data are presented in Fig. S8 in Data S1.

FIGURE 4.

The Nδ1- and Nɛ2-bound complexes (A and B, respectively) were each simulated for 200 ns. Overlays of 2000 structures from the final 20 ns of each simulation, shown in two orientations, are RMS-fitted, using (PHGGGWGQ)₃PH backbone heavy atoms. The major conformation of each octarepeat is shown, with Cu²⁺-binding His and GWGQ residues explicitly drawn. Numbering from the human PrP sequence is shown.

Tryptophan and glycine residues in the OR region can interact with bound Cu²⁺

The peptide backbone of much of the Nδ1-bound and Nɛ2-bound models is held away from the metal center because of coordination via the His imidazole side chains. This makes interactions between other portions of the OR region with the metal coordination site less likely. In the Nδ1-bound Cu:OR₄ system (Fig. 4), although the metal center is somewhat sequestered from access by solvent, a portion of the fourth repeat can come into close contact with the Cu²⁺ ion, whereas in the Nɛ2-bound form, a portion of the fourth repeat as well as the third repeat can both make transient close approaches to the metal coordination site.

The Trp residues, located at the turns in the OR loops between the successive Cu²⁺-binding His residues, are always solvent-exposed during simulations. On average, the Trp-Cu²⁺ internuclear separation (measured between the Trp indole Nɛ1-atom and Cu²⁺) for all Trp residues in both the Nɛ2-bound and Nδ1-bound simulations is 10 Å or greater. Specific Trp residues in each simulation, however, demonstrate significantly closer approaches to the metal center, such as the Trp residue from the third repeat in the Nɛ2-bound model and the Trp residue from the fourth repeat in the Nδ1-bound model. In these instances, the Trp-Cu²⁺ separation may be as low as 6–7 Å (Fig. 5 A). A summary of all Trp Nɛ1-Cu²⁺ internuclear separations from each simulation are presented in Fig. S9 in Data S1.

FIGURE 5.

Snapshots of the close approach of Trp residues to the Cu²⁺-binding site in (A) Nɛ2-bound and (B) Nδ1-bound models from MD simulations. Each of the His and Trp side chains is drawn for clarity. Nterm and Cterm denote the N-terminal and C-terminal ends of the sequence, respectively.

Applicability of Cu:OR₄ models

The procedure of constructing copper-binding OR₄ models through assembly has some specific assumptions associated with it. Firstly, the assembled OR₄ structure assumes that all four OR regions will cooperatively fold in the same manner as they do in the copper-free OR₂ models from which they are derived. Secondly, coordination by all four His residues concurrently does not allow for additional conformational flexibility within the OR region. Each of these points is addressed below.

Previous NMR experiments by Yoshida et al. (45) and Zahn (46) revealed that the OR region adopts loop and turn-type structures, which are also formed in our apo-OR₂ models (7). These experimental observations lend indirect support to the conformation of the preformed copper-coordinating structure that we established through the assembly of OR fragments.

The ability of His ligands to become labile and exit the coordination sphere would not only allow those regions of the backbone greater flexibility, but could also allow for further variability in Cu²⁺-coordination when the His imidazole reenters the coordination sphere, with the possibility that an alternate imidazole N-donor atom may coordinate to copper, or alternate backbone conformations may be accessible. This type of exchange in the ligand environment could in principle give rise to 16 alternate four-coordinate geometries (4²), and as many local potential energy surfaces to explore. Further consideration of intermediate coordination geometries, e.g., by only three His residues, could further increase the complexity and number of possible models.

Taking these considerations into account, the models presented here comprise the most tractable initial approach to understanding the low-Cu²⁺-occupancy form of the OR region. The Cu²⁺ coordination via Nɛ2-atoms of the His residues in the Cu₁OR₄ model is in agreement with the previously observed coordination mode of the OR₄ peptide at pH 6.5 by Raman spectroscopy (42).

DISCUSSION

The Trp residues within the OR region were shown to be necessary for the redox competence of the low-occupancy form of Cu²⁺ bound to the OR region of PrP (47), whereas successive binding of Cu²⁺ renders the metal ions redox-inactive. The consequence of Cu²⁺ binding in the low-occupancy, redox-active form is ROS formation and subsequent β-cleavage of the OR region from the remainder of membrane-anchored PrP^C. The Trp residues may act as one-electron donors, facilitating reduction of the single Cu²⁺ center to Cu⁺, within the OR region. Our MD simulations implicate a close association between the bound Cu²⁺ atom and specific Trp residues within the OR region.

The only Trp residues from either series of simulations to make a significantly close approach to the Cu²⁺ ion are Trp-80 and Trp-89. However, other Trp residues may occasionally come to within 10 Å as well. A previous model from Miura et al. indicated that Trp-65 and Trp-73 could interact with Cu²⁺ (42). Because their model was derived by only constraining the coordinating His residues to remain bound to the Cu²⁺ center, without accounting for the folding preference of the sequence, we are inclined to assert that Trp-80 and Trp-89 (from the two C-terminal repeats) are important for β-cleavage. These observations beg the question of whether such interactions may play a role in the ability of the OR region to reduce Cu²⁺ to Cu⁺ during ROS formation. In addition to Cu²⁺ coordination, the four-His coordination environment of the OR region is also compatible with Cu⁺ binding. The OR₄ region is sufficiently flexible to accommodate the tetrahedral coordination geometry required for stabilizing Cu⁺ formation.

As noted earlier, the assembled OR₄ models can accommodate tetrahedral coordination geometry, and are not only compatible with Cu⁺ binding, but with Zn²⁺ coordination as well (data not shown). Copper and zinc binding to the N-terminal domain triggers endocytosis of PrP (48), and these models offer further structural details that may be used to refine structure interactions with neighboring regions of PrP and micelle environments (49), as well as offer insights into the initial events leading to β-cleavage. The structures presented here may provide the first clues that relate Cu²⁺ binding, ROS formation, and the site specificity of β-cleavage.

Because β-cleavage is the result of ROS formation, it is likely that the site at which reactive species are formed is at, or near, the site of cleavage. Generation of ROS at a more remote site would require the formation of diffusible ROS that would ultimately result in less site-specific cleavage, indicating that the site of ROS formation likely involves the initial close approach of the protein backbone to the metal center. Several repeats in each of the series of simulations make such an approach to the metal center, and both the Cα-atoms of Trp (residue 89) and Gly (residue 90) make transient approaches to within <6 Å of the Cu²⁺ center in the Nδ1-bound simulation, as shown in Fig. 5 B. Similar interaction with other repeats in alternate conformations may provide a structural mechanism for ROS-mediated β-cleavage within the OR region.

Our proposed mechanism for copper-mediated ROS formation, shown in Fig. 6, is initiated by close association of Trp with the Cu²⁺ center, followed by subsequent electron transfer from the Trp indole to form Cu⁺. In the presence of H₂O₂, Cu⁺ is readily oxidized back to Cu²⁺, and results in the formation of HO⁻ and HO^·. Hydroxyl radicals are highly reactive, and are likely to react at or near their site of formation: in this instance, through H-atom abstraction from a nearby aliphatic group. Because of the high abundance of Gly residues in the OR region (Fig. 1), the local protein backbone contains multiple potential targets for H-atom abstraction. Hydroxyl radical damage at a Gly residue results in a C-centered radical, and the subsequent reaction of Gly(Cα^·) with O₂ triggers C-N or C-C backbone cleavage within the damaged residue. The most likely Gly residues to be targets of HO^· attack would be those closest to the Cu²⁺ center. The close approach of Trp-89 and each of its adjacent Gly residues, as observed in these simulations, lends support for their proximity to this potential ROS-generating site. The formed Trp indole radical cation (Trp^·+) may undergo subsequent reactions, including deprotonation at the indole N(H) site, as well as cross-linking reactions with other Trp^·+ residues. It may also be possible that Trp^·+ plays a role in directing β-cleavage of the local protein backbone. However, further work would be necessary to establish a mechanistic link.

FIGURE 6.

Mechanism of copper-mediated hydroxyl radical formation and subsequent β-cleavage of protein backbone. Copper coordination environments for Cu²⁺ and Cu⁺ are depicted as their preferred square planar and tetrahedral geometry respectively, both of which can be accommodated by the flexible octarepeat region of PrP.

Previous experiments (50) suggested that copper-binding to PrP can induce a proteinase-resistant form of PrP, possibly through folding in the N-domain. Although it is clear that the OR region can become structured in response to increasing copper loads, recent evidence indicates that copper itself inhibits proteinase-K, and therefore cannot be used to infer the presence of specific structuring within PrP in this manner (51).

These structures provide the first account of the conformation of the OR region under conditions of low-Cu²⁺-occupancy in mammalian PrP. The OR region was recently shown to bind Zn²⁺ in a manner similar to that of Cu²⁺ (52), and these structures are also compatible with Zn²⁺ coordination. These results are a necessary first step toward more elaborate structural models for Cu²⁺-binding to this region of PrP, and toward understanding the associated redox activity of PrP-bound copper and their relationship to prion disease.

SUPPLEMENTARY MATERIAL

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