Octarepeat Region Research Articles

Functional implications of multistage copper binding to the prion protein

The prion protein (PrP) is responsible for a group of neurodegenerative diseases called the transmissible spongiform encephalopathies. The normal function of PrP has not yet been discovered, but indirect evidence suggests a linkage to its ability to bind copper. In this article, low-copper-concentration bindings of Cu(2+) to PrP are investigated by using a recently developed hybrid density functional theory (DFT)/DFT method. It is found that at the lowest copper concentrations, the binding site consists of 4 histidine residues coordinating the copper through epsilon imidazole nitrogens. At higher concentrations, 2 histidines are involved in the binding, one of them in the axial position. These results are in good agreement with existing experimental data. Comparison of free energies for all modes of coordination shows that when enough copper is available, the binding sites will spontaneously rearrange to accommodate more copper ions, despite the fact that binding energy per copper ion decreases with concentration. These findings support the hypothesis that PrP acts as a copper buffer in vivo, protecting other proteins from the attachment of copper ions. Using large-scale classical molecular dynamics, we also probe the structure of full-length copper-bound PrP, including its unfolded N-terminal domain. The results show that copper attachment leads to rearrangement of the structure of the Cu-bonded octarepeat region and to development of turns in areas separating copper-bound residues. These turns make the flexible N-terminal domain more rigid and thus more resistant to misfolding. The last result suggests that copper binding plays a beneficial role in the initial stages of prion diseases.

Difference in redox behaviors between copper-binding octarepeat and nonoctarepeat sites in prion protein

We studied the redox behavior of copper-binding sites in prion protein (PrP) to clarify copper's role in the pathological mechanism underlying prion diseases. We investigated the coordination structures, binding affinities, and redox potentials of copper-binding peptide fragments derived from the N-terminal domain of PrP by density functional theory calculations. We used four models for copper-binding moieties in PrP(60-96): two were derived from the PHGGGWGQ octapeptide repeat region of PrP(60-91), and the others were tripeptide Gly-Thr-His fragments derived from the copper-binding nonoctarepeat site around His96. We found that such PrP-derived copper-binding complexes exhibit conformationally dependent redox behavior; for example, the copper-binding complex derived from the octarepeat region tends to possess high reduction potential for the Cu(II)/Cu(I) couple, exceeding 0 V versus the standard hydrogen electrode, whereas the copper-binding nonoctarepeat model around His96 tends to possess high oxidation potential for the Cu(II)/Cu(III) couple and stabilize the higher-valent Cu(III) state. It is possible that such distinct redox activities of a copper-binding PrP are involved in the mechanism underlying prion diseases.

Prion protein region 23–32 interacts with tubulin and inhibits microtubule assembly

In previous studies we have demonstrated that prion protein (PrP) binds directly to tubulin and this interaction leads to the inhibition of microtubule formation by inducement of tubulin oligomerization. This report is aimed at mapping the regions of PrP and tubulin involved in the interaction and identification of PrP domains responsible for tubulin oligomerization. Preliminary studies focused our attention to the N-terminal flexible part of PrP encompassing residues 23-110. Using a panel of deletion mutants of PrP, we identified two microtubule-binding motifs at both ends of this part of the molecule. We found that residues 23-32 constitute a major site of interaction, whereas residues 101-110 represent a weak binding site. The crucial role of the 23-32 sequence in the interaction with tubulin was confirmed employing chymotryptic fragments of PrP. Surprisingly, the octarepeat region linking the above motifs plays only a supporting role in the interaction. The binding of Cu(2+) to PrP did not affect the interaction. We also demonstrate that PrP deletion mutants lacking residues 23-32 exhibit very low efficiency in the inducement of tubulin oligomerization. Moreover, a synthetic peptide corresponding to this sequence, but not that identical with fragment 101-110, mimics the effects of the full-length protein on tubulin oligomerization and microtubule assembly. At the cellular level, peptide composed of the PrP motive 23-30 and signal sequence (1-22) disrupted the microtubular cytoskeleton. Using tryptic and chymotryptic fragments of alpha- and beta-tubulin, we mapped the docking sites for PrP within the C-terminal domains constituting the outer surface of microtubule.

Thermodynamic and Voltammetric Characterization of the Metal Binding to the Prion Protein: Insights into pH Dependence and Redox Chemistry

The prion protein is a high-affinity copper binding protein that plays a role in the neurodegenerative prion diseases when it is converted into an altered isoform. The function of the protein remains controversial, but its relationship to its metallochemistry has prompted further investigation. While many researchers continue to use short peptide models for binding studies, the clear discrepancy between data obtained with such models when compared to those of full-length recombinant proteins requires clarification with this more appropriate model. Isothermal titration calorimetry was used to assess metal affinity for PrP. Using both full-length native and recombinant prion protein, we have demonstrated that the prion protein binds copper but has little affinity for other metals. Metal binding is highly pH sensitive, being optimal at pH 7.5 for copper, nickel, and zinc and at pH 5.5 for iron. Metal binding affinity for PrP was not altered by protein glycosylation. The use of suitable thermodynamic modeling reveals complex and cooperative copper binding, with evidence of negative cooperativity within the octarepeat region. Cyclic voltammetry was utilized to assess the electrochemistry of copper-charged prion protein, and we show that mPrP has a redox potential of 0.03 +/- 0.01 V versus the saturated calomel electrode at pH 7. The analysis also indicated that PrP is able to undergo reversible redox cycling with equal oxidative and reductive charges that are largely dependent on the copper bound to the octarepeat. The fifth site provides a small contribution to this redox activity, but only when the octarepeat is present. These results show conclusively that PrP can utilize copper for electron transfer, which would be expected for a radical detoxifying enzyme, and that the octarepeat region is the functional domain.

Can copper binding to the prion protein generate a misfolded form of the protein?

The native prion protein (PrP) has a two domain structure, with a globular folded alpha-helical C-terminal domain and a flexible extended N-terminal region. The latter can selectively bind Cu(2+) via four His residues in the octarepeat (OR) region, as well as two sites (His96 and His111) outside this region. In the disease state, the folded C-terminal domain of PrP undergoes a conformational change, forming amorphous aggregates high in beta-sheet content. Cu(2+) bound to the ORs can be redox active and has been shown to induce cleavage within the OR region, a process requiring conserved Trp residues. Using computational modeling, we have observed that electron transfer from Trp residues to copper can be favorable. These models also reveal that an indole-based radical cation or Cu(+) can initiate reactions leading to protein backbone cleavage. We have also demonstrated, by molecular dynamics simulations, that Cu(2+) binding to the His96 and His111 residues in the remaining PrP N-terminal fragment can induce localized beta-sheet structure, allowing us to suggest a potential mechanism for the initiation of beta-sheet misfolding in the C-terminal domain by Cu(2+).

The POM Monoclonals: A Comprehensive Set of Antibodies to Non-Overlapping Prion Protein Epitopes

PrPSc, a misfolded and aggregated form of the cellular prion protein PrPC, is the only defined constituent of the transmissible agent causing prion diseases. Expression of PrPC in the host organism is necessary for prion replication and for prion neurotoxicity. Understanding prion diseases necessitates detailed structural insights into PrPC and PrPSc. Towards this goal, we have developed a comprehensive collection of monoclonal antibodies denoted POM1 to POM19 and directed against many different epitopes of mouse PrPC. Three epitopes are located within the N-terminal octarepeat region, one is situated within the central unstructured region, and four epitopes are discontinuous within the globular C-proximal domain of PrPC. Some of these antibodies recognize epitopes that are resilient to protease digestion in PrPSc. Other antibodies immunoprecipitate PrPC, but not PrPSc. A third group was found to immunoprecipitate both PrP isoforms. Some of the latter antibodies could be blocked with epitope-mimicking peptides, and incubation with an excess of these peptides allowed for immunochromatography of PrPC and PrPSc. Amino-proximal antibodies were found to react with repetitive PrPC epitopes, thereby vastly increasing their avidity. We have also created functional single-chain miniantibodies from selected POMs, which retained the binding characteristics despite their low molecular mass. The POM collection, thus, represents a unique set of reagents allowing for studies with a variety of techniques, including western blotting, ELISA, immunoprecipitation, conformation-dependent immunoassays, and plasmon surface plasmon resonance-based assays.

Modeling the interplay of glycine protonation and multiple histidine binding of copper in the prion protein octarepeat subdomains

The octarepeat region of the prion protein can bind Cu(2+) ions up to full occupancy (one ion per octarepeat) at neutral pH. While crystallographic data show that the HGGG octarepeat subdomain is the basic binding unit, multiple histidine coordination at lower Cu occupancy has been reported by X-ray absorption spectroscopy, EPR, and potentiometric experiments. In this paper we investigate, with first principles Car-Parrinello simulations, the first step for the formation of the Cu low-level binding mode, where four histidine side chains are coordinated to the same Cu(2+) ion. This step involves the further binding of a second histidine to an already HGGG domain bonded Cu(2+) ion. The influence of the pH on the ability of Cu to bind two histidine side chains was taken into account by simulating different protonation states of the amide N atoms of the two glycines lying nearest to the first histidine. Multiple histidine coordination is also seen to occur when glycine deprotonation occurs and the presence of the extra histidine stabilizes the Cu-peptide complex. Though the stabilization effect slightly decreases with the number of deprotonated glycines (reaching a minimum when both N atoms of the two nearest glycines are available as Cu ligands), the system is still capable of binding the second histidine in a 4N tetrahedral (though slightly distorted) coordination, whose energy is very near to that of the crystallographic square-planar 3N1O coordination. This result suggests that at low metal concentration the reorganization energy associated with Cu(II)/Cu(I) reduction is small also at pH approximately 7, when glycines are deprotonated.

Modeling by Assembly and Molecular Dynamics Simulations of the Low Cu 2+ Occupancy Form of the Mammalian Prion Protein Octarepeat Region: Gaining Insight into Cu 2+-Mediated β-Cleavage

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 Cu 2+ 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 OR 2 model from molecular dynamics simulations, the starting structures of the OR 4 complex could be constructed by assembling the repeating structure of PHGG and GWGQ fragments. The resulting model contains a preformed site suitable for Cu 2+ coordination. Molecular dynamics simulations of Cu 2+ bound to the assembled OR 4 model (Cu:OR 4) reveal a close association of specific Trp and Gly residues with the Cu 2+ center. This low Cu 2+-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 Cu 2+ coordination site in the low Cu 2+-occupancy form of the OR region may signal an important interaction for the initiation of prion disease.

Prion Proteins Leading to Neurodegeneration

Prion diseases are fatal neurodegenerative disorders related to the conformational alteration of the prion protein (PrP C) into a pathogenic and protease-resistant isoform PrP(Sc). PrP(C) is a cell surface glycoprotein expressed mainly in the central nervous system and despite numerous efforts to elucidate its physiological role, the exact biological function remains unknown. Many lines of evidences indicate that prion is a copper binding protein and thus involved in the copper metabolism. Prion protein is not expressed only in mammals but also in other species such as birds, reptiles and fishes. However, it is noteworthy to point out that prion diseases are only observed in mammals while they seem to be spared to other species. The chicken prion protein (chPrP C) shares about 30% of identity in its primary sequence with mammal PrP C. Both types of proteins have an N-terminal domain endowed with tandem amino acid repeats (PHNPGY in the avian protein, PHGGGWQ in mammals), followed by a highly conserved hydrophobic core. Furthermore, NMR studies have highlighted a similar globular domain containing three alpha-helices, one short 3(10)-helix and a short antiparallel beta-sheet. Despite this structural similarity, it should be noted that the normal isoform of mammalian PrP C is totally degraded by proteinase K, while avian PrP C is not, thereby producing N-terminal domain peptide fragments stable to further proteolysis. Notably, the hexarepeat domain is considered essential for protein endocytosis, and it is supposed to be the analogous copper-binding octarepeat region of mammalian prion proteins. The number of copper binding sites, the affinity and the coordination environment of metal ions are still matter of discussion for both mammal and avian proteins. In this review, we summarize the similarities and the differences between mammalian and avian prion proteins, as revealed by studies carried out on the entire protein and related peptide fragments, using a range of experimental and computational approaches. In addition, we report the metal-driven conformational alteration, copper binding modes and the superoxide dismutase-like (SOD-like) activity of the related copper(II) complexes.

Copper and the structural biology of the prion protein

PrP (prion-related protein) is a cell-surface Cu(2+)-binding glycoprotein which, when misfolded, is responsible for a number of transmissible spongiform encephalopathies. The co-ordination geometry, stoichiometry and affinity of Cu(2+) for PrP are the subject of much debate. In the present paper, we review the recent progress we have made in these areas. As many as six Cu(2+) ions bind to PrP with submicromolar affinity. Initially, two Cu(2+) ions bind to full-length PrP in the amyloidogenic region, between the octarepeats and the structured domain, at His(95) and His(110). Only subsequent Cu(2+) ions bind to single histidine residues within the octarepeat region. Competitive chelators have been used to determine the affinity of the first molar equivalent of Cu(2+) bound to full-length PrP; this approach places the affinity in the nanomolar range. The affinity and number of Cu(2+)-binding sites support the suggestion that PrP could act as an antioxidant by binding potentially harmful Cu(2+) ions and sacrificially quenching of free radicals generated as a result of copper redox cycling. Finally, the effect of Cu(2+) on the prion structure and misassembly into oligomers and fibres is discussed.

The octarepeat region of prion protein, but not the TM1 domain, is important for the antioxidant effect of prion protein

The cellular prion protein (PrP c) plays a crucial role in the pathogenesis of prion diseases, but its physiological function is far from understood. Several candidate functions have been proposed including binding and internalization of metal ions, a superoxide dismutase-like activity, regulation of cellular antioxidant activities, and signal transduction. The transmembrane (TM1) region of PrP c (residues 110–135) is particularly interesting because of its very high evolutionary conservation. We investigated a possible role of TM1 in the antioxidant defense, by assessing the impact of overexpressing wt-PrP or deletion mutants in N 2A mouse neuroblastoma cells on intracellular reactive oxygen species (ROS) levels. Under conditions of oxidative stress, intracellular ROS levels were significantly lowered in cells overexpressing either wild-type PrP c (wt-PrP) or a deletion mutant affecting TM1 (Δ8TM1-PrP), but, as expected, not in cultures overexpressing a deletion mutant lacking the octapeptide region (Δocta-PrP). Overexpression of wt-PrP, Δ8TM1-PrP, or Δocta-PrP did not affect basal ROS levels. Interestingly, the mitochondrial membrane potential was significantly lowered in Δocta-PrP-transfected cultures in the absence of oxidative stress. We conclude that the protective effect of PrP c against oxidative stress involves the octarepeat region but not the TM1 domain nor the high-affinity copper binding site described for human residues His96/His111.

Atypical BSE (BASE) Transmitted from Asymptomatic Aging Cattle to a Primate

BackgroundHuman variant Creutzfeldt-Jakob Disease (vCJD) results from foodborne transmission of prions from slaughtered cattle with classical Bovine Spongiform Encephalopathy (cBSE). Atypical forms of BSE, which remain mostly asymptomatic in aging cattle, were recently identified at slaughterhouses throughout Europe and North America, raising a question about human susceptibility to these new prion strains.Methodology/Principal FindingsBrain homogenates from cattle with classical BSE and atypical (BASE) infections were inoculated intracerebrally into cynomolgus monkeys (Macacca fascicularis), a non-human primate model previously demonstrated to be susceptible to the original strain of cBSE. The resulting diseases were compared in terms of clinical signs, histology and biochemistry of the abnormal prion protein (PrPres). The single monkey infected with BASE had a shorter survival, and a different clinical evolution, histopathology, and prion protein (PrPres) pattern than was observed for either classical BSE or vCJD-inoculated animals. Also, the biochemical signature of PrPres in the BASE-inoculated animal was found to have a higher proteinase K sensitivity of the octa-repeat region. We found the same biochemical signature in three of four human patients with sporadic CJD and an MM type 2 PrP genotype who lived in the same country as the infected bovine.Conclusion/SignificanceOur results point to a possibly higher degree of pathogenicity of BASE than classical BSE in primates and also raise a question about a possible link to one uncommon subset of cases of apparently sporadic CJD. Thus, despite the waning epidemic of classical BSE, the occurrence of atypical strains should temper the urge to relax measures currently in place to protect public health from accidental contamination by BSE-contaminated products.

PrP mutants with different numbers of octarepeat sequences are more susceptible to the oxidative stress

One of the physiological functions of cellular prion protein (PrP(C)) is believed to work as a cellular resistance to oxidative stress, in which the octarepeats region within PrP plays an important role. However, the detailed mechanism is less clear. In this study, the expressing plasmids of wild-type PrP (PrP-PG5) and various PrP mutants containing 0 (PrP-PG0), 9 (PrP-PG9) and 12 (PrP-PG12) octarepeats were generated and PrP proteins were expressed both in E. coli and in mammalian cells. Protein aggregation and formation of carbonyl groups were clearly seen in the recombinant PrPs expressed from E. coli after treatment of H(2)O(2). MTT and trypan blue staining assays revealed that the cells expressing the mutated PrPs within octarepeats are less viable than the cells expressing wild-type PrP. Statistically significant high levels of intracellular free radicals and low levels of glutathione peroxidase were observed in the cells transfected with plasmids containing deleted or inserted octarepeats. Remarkably more productions of carbonyl groups were detected in the cells expressing PrPs with deleted and inserted octarepeats after exposing to H(2)O(2). Furthermore, cells expressing wild-type PrP showed stronger resistant activity to the challenge of H(2)O(2) at certain extent than the mutated PrPs and mock. These data provided the evidences that the octarepeats number within PrP is critical for maintaining its activity of antioxidation. Loss of its protective function against oxidative stress may be one of the possible pathways for the mutated PrPs to involve in the pathogenesis of familial Creutzfeldt-Jacob diseases.

Octarepeat changes of prion protein in Parkinson's disease

Polymorphism in prion protein (PrP) is related to different phenotypes of spongiform encephalopathies and some mental illnesses. The octarepeat region of PrP, encompassing the codon 51 through 91, is related to cellular anti-oxidation function and may play a role in genetic contribution of PrP polymorphism to neurodegeneration, such as Parkinson's disease (PD). We analyzed the genomic patterns of PrP gene from 528 subjects and found a predominance of Met/Met variant at codon 129 of PD subjects without significant difference (97.3%, and 96.5% in controls). But among PD subjects there were one with heterozygosity of silent nucleotide substitution (NS) on octarepeats (R1–2–3g–3–4/R1–2–2–3–4) and three with heterozygosity of single copy deletion (CD) on octarepeats (R1–2–3–4/R1–2–2–3–4). Consistent genomic DNA and cDNA sequences were found in a PD subject without any octarepeat changes and the one with NS, but R1–2–3g–3–4/R1–2–2–3–4 of cDNA pattern occurred in the one with genomic CD. This is the first report of the polymorphic PrP octarepeat change among those with parkinsonism. We proposed a hypothesis about an initial secondary hairpin structure of the template strand followed by the transcript “shift backward” due to the high homology of the sequences between R2 and R3 motifs while synthesizing RNA. This phenomenon may be a key step of neurodegeneration resulting from PrP polymorphism and require further studies.

Deconvoluting the Cu2+ Binding Modes of Full-length Prion Protein

The prion protein (PrP) is a cell-surface Cu(2+)-binding glycoprotein that when misfolded is responsible for a number of transmissible spongiform encephalopathies. Full-length PrP-(23-231) and constructs in which the octarepeat region has been removed, or His(95) and His(110) is replaced by alanine residues, have been used to elucidate the order and mode of Cu(2+) coordination to PrP-(23-231). We have built on our understanding of the appearance of visible CD spectra and EPR for various PrP fragments to characterize Cu(2+) coordination to full-length PrP. At physiological pH, Cu(2+) initially binds to full-length PrP in the amyloidogenic region between the octarepeats and the structured domain at His(95) and His(110). Only subsequent Cu(2+) ions bind to single histidine residues within the octarepeat region. Ni(2+) ions are used to further probe metal binding and, like Cu(2+), Ni(2+) will bind individually to His(95) and His(110), involving preceding main chain amides. Competitive chelators are used to determine the affinity of the first mole equivalent of Cu(2+) bound to full-length PrP; this approach places the affinity in the nanomolar range. The affinity and number of Cu(2+) binding sites support the suggestion that PrP could act as a sacrificial quencher of free radicals generated by copper redox cycling.

Vaccine Approaches to Prevent and Treat Prion Infection

Prion diseases are transmissible neurodegenerative diseases of humans and animals. The prion agent consists of a misfolded protein, PrPSc (prion protein, scrapie form), of a glycosylphosphatidylinositol-anchored host protein, PrPC (PrP cellular form) of unknown function. During prion replication, PrPSc induces host PrPC to adopt its pathogenic conformation. Some PrPSc may aggregate to microscopically visible, extracellular prion plaques that stain for amyloid. The development of antiprion vaccines presents some challenges. While there is strong self-tolerance to an endogenous antibody response to PrPC and PrPSc, highly potent monoclonal antibodies (mAbs) have been raised in mice in which the prion protein gene has been deleted by gene targeting. These mAbs have been demonstrated to be antiprion-active in permanently scrapie-infected neuroblastoma (ScN2a) cells, primarily when bound to one of four epitopes (the octarepeat region, the region around codons 90-110, helix 1 region codons 145-160, and the extreme C-terminal codons 210-220). The mAbs directed against codon regions 90-110 or 145-160 are also antiprion-active in vivo, but only after intraperitoneal infection with prions, not intracerebral infection, suggesting their blood-brain barrier (BBB) impermeability. The challenge will be to make antibodies, or recombinant derivatives thereof, BBB permeable; this is preferably achieved by monovalent antibody fragments since divalent ones were found to be neurotoxic. Self-tolerance of wild-type animals to PrP immunizations was found to be of extrathymic origin. Even though antibodies raised in wild-type mice were found to display antiprion activity in ScN2a cells, these mice did not have significant extensions of incubation times when challenged intraperitoneally with prions. A general low affinity of these antibody responses to native surface-bound PrPC may account for this. Since wild-type mice were found to develop sufficient T-cell responses to codon regions 145-160 and 210-220, we believe that there is a theoretical chance of a successful vaccination therapy. The influence of the way the immunogen is presented has already been shown to be of major importance for the ensuing immune response, in that presentation of PrP with CpG oligodeoxynucleotides as adjuvant or viral packaging improved antibody responses. Major progress for active immunizations may therefore be expected in this field. Eradication programs will be one of the most important uses of active immunization protocols. For this purpose, vaccines will have to be inexpensive, easy to handle, and effective. In the short term, passive immunizations will likely be most promising for therapy of prion disease, including for human medical interventions. Active immunization protocols are less likely to succeed quickly, and will take years if not decades to be validated for domestic or free-ranging animals.

A DFT study of EPR parameters in Cu(ii) complexes of the octarepeat region of the prion protein

The present paper shows how theoretically determined electron paramagnetic resonance (EPR) parameters can help in assigning the most favorable structure of Cu(ii) complexes in octarepeat (OR) regions (PHGGGWGQ) of the prion protein (PrP). This could contribute to a better understanding of the molecular structure of the Cu(OR) complexes, as some features of such species in solution are still unclear. The present theoretical investigation on [Cu(ii)(HGGG)] and [Cu(ii)(HGGGW)] complexes was carried out to confirm the stability of relevant isomers, and in particular, to evaluate the hyperfine coupling constants (hcc) with (63)Cu, (14)N and (17)O nuclei, as well as the g values. The hcc (and to a lesser extent the g components) are useful probes for checking whether the computed EPR parameters for specific isomers fit the experimental data, thus permitting the association of the observed spectra with a specific complex structure. The results obtained suggest that the Cu(ii) ion in the [Cu(HGGG)] isomers prefers a square pyramidal coordination with three nitrogen atoms of the peptide and one carbonyl oxygen atom in the basal plane. Also the Cu(ii) ion in the [Cu(HGGGW)] complex is penta-coordinated. The penta-coordination does not actually involve the tryptophan residue but an additional water molecule, forced to occupy the axial coordination position by a rather extended hydrogen-bond network, promoted by the tryptophan residue. The comparison between the calculated and experimental values of EPR parameters allows one to suggest the assignment of the coordination mode of the Cu(ii) ion in the considered peptide ligands. The computed values of the g components seem to be little affected by a particular coordination mode. In particular, the g( parallel) component is always underestimated by about 0.1 with respect to the experiment. The calculated values of the hcc, in contrast, are in acceptable agreement with the experimental values, in spite of the fact that the large size of the species under consideration forced us to accept a certain level of approximation in the computational procedure. Nevertheless, the present study must be considered as one of the first examples of truly ab initio calculations of EPR parameters for systems as large as the Cu(OR)-type complexes.

Molecular Dynamics Simulations of Two Tandem Octarepeats from the Mammalian Prion Protein: Fully Cu 2+-bound and Metal-Free Forms

Molecular dynamics simulations have been conducted on a model fragment (Ac-PHGGGWGQPHGGGW-NH 2) of the prion protein octarepeat domain, both in the Cu 2+-bound and metal-free forms. The copper-bound models are based on the consensus structure of the core Cu 2+-binding site of an individual octarepeat, relevant to the fully Cu 2+-occupied prion protein octarepeat region. The model peptides contain Cu 2+ bound through a His imidazole ring and two deprotonated amide N-atoms in the peptide backbone supplied by the following two Gly residues. Both the copper-bound and metal-free models have been simulated with the OPLS all-atom force field with the GROMACS molecular dynamics package. These simulations, with two tandem copper-binding sites, represent the minimum model necessary to observe potential structuring between the copper-binding sites in the octarepeat region. The GWGQ residues constitute a flexible linker region that predominantly adopts a turn, serving to bring adjacent His residues into close proximity. The consequent formation of stable structures demonstrates that the copper-bound octarepeat region allows the copper-coordinating sites to come into van der Waals contact, packing into particular orientations to further stabilize the bend in the GWGQ linker region.

The prion protein is a combined zinc and copper binding protein: Zn2+ alters the distribution of Cu2+ coordination modes.

PrP binds copper in the highly conserved, unstructured N-terminal half of the protein. The octarepeat region consists of four tandem repeats of PHGGGWGQ and binds 4 equiv of copper at full occupancy. Adjacent to the octarepeats are two additional histidines that may also bind copper. We recently showed that, when the octarepeat region is titrated with Cu2+, the copper binding mode depends on the number of equivalents of copper bound. In addition to copper, other metals have been associated with PrP; however, zinc is the only metal other than copper that induces PrP endocytosis, inhibits fibril formation, and promotes intermolecular interactions. In this work, we show that even large excesses of zinc (>1 mM) are unable to displace copper from either the octarepeat region or the full-length protein. However, EPR reveals that physiologically relevant levels of zinc significantly alter the distribution of copper among the available binding modes. Diethyl pyrocarbonate (DEPC) modification and mass spectrometry...

Probing the Role of PrP Repeats in Conformational Conversion and Amyloid Assembly of Chimeric Yeast Prions

Oligopeptide repeats appear in many proteins that undergo conformational conversions to form amyloid, including the mammalian prion protein PrP and the yeast prion protein Sup35. Whereas the repeats in PrP have been studied more exhaustively, interpretation of these studies is confounded by the fact that many details of the PrP prion conformational conversion are not well understood. On the other hand, there is now a relatively good understanding of the factors that guide the conformational conversion of the Sup35 prion protein. To provide a general model for studying the role of oligopeptide repeats in prion conformational conversion and amyloid formation, we have substituted various numbers of the PrP octarepeats for the endogenous Sup35 repeats. The resulting chimeric proteins can adopt the [PSI+] prion state in yeast, and the stability of the prion state depends on the number of repeats. In vitro, these chimeric proteins form amyloid fibers, with more repeats leading to shorter lag phases and faster assembly rates. Both pH and the presence of metal ions modulate assembly kinetics of the chimeric proteins, and the extent of modulation is highly sensitive to the number of PrP repeats. This work offers new insight into the properties of the PrP octarepeats in amyloid assembly and prion formation. It also reveals new features of the yeast prion protein, and provides a level of control over yeast prion assembly that will be useful for future structural studies and for creating amyloid-based biomaterials.