Seeded conversion of recombinant prion protein to a disulfide-bonded oligomer by a reduction-oxidation process

The infectious form of prion protein, PrP(Sc), self-propagates by its conversion of the normal, cellular prion protein molecule PrP(C) to another PrP(Sc) molecule. It has not yet been demonstrated that recombinant prion protein can convert prion protein molecules from PrP(C) to PrP(Sc). Here we show that recombinant hamster prion protein is converted to a second form, PrP(RDX), by a redox process in vitro and that this PrP(RDX) form seeds the conversion of other PrP(C) molecules to the PrP(RDX) form. The converted form shows properties of oligomerization and seeded conversion that are characteristic of PrP(Sc). We also find that the oligomerization can be reversed in vitro. X-ray fiber diffraction suggests an amyloid-like structure for the oligomerized prion protein. A domain-swapping model involving intermolecular disulfide bonds can account for the stability and coexistence of two molecular forms of prion protein and the capacity of the second form for self-propagation.     

High-level expression and characterization of a glycosylated covalently linked dimer of the prion protein

There is evidence that prion protein dimers may be involved in the formation of the scrapie prion protein, PrP(Sc), from its normal (cellular) form, PrP(c). Recently, the crystal structure of the human prion protein in a dimeric form was reported. Here we report for the first time the overexpression of a human PrP dimer covalently linked by a FLAG peptide (PrP::FLAG::PrP) in the methylotrophic yeast Pichia pastoris. FLAG-tagged human PrP (aa1-aa253) (huPrP::FLAG) was also expressed in the same system. Treatment with tunicamycin and endoglycosidase H showed that both fusion proteins are expressed as various glycoforms. Both PrP proteins were completely digested by proteinase K (PK), suggesting that the proteins do not have a PrP(Sc) structure and are not infectious. Plasma membrane fractionation revealed that both proteins are transported to the plasma membrane of the cell. The glycosylated proteins might act as powerful tools for crystallization trials, PrP(c)/PrP(Sc) conversion studies and other applications in the life cycle of prions.     

Preventing misfolding of the prion protein by trimethylamine N-oxide

Transmissible spongiform encephalopathies are a class of fatal neurodegenerative diseases linked to the prion protein. The prion protein normally exists in a soluble, globular state (PrP(C)) that appears to participate in copper metabolism in the central nervous system and/or signal transduction. Infection or disease occurs when an alternatively folded form of the prion protein (PrP(Sc)) converts soluble and predominantly alpha-helical PrP(C) into aggregates rich in beta-structure. The structurally disordered N-terminus adopts beta-structure upon conversion to PrP(Sc) at low pH. Chemical chaperones, such as trimethylamine N-oxide (TMAO), can prevent formation of PrP(Sc) in scrapie-infected mouse neuroblastoma cells [Tatzelt, J., et al. (1996) EMBO J. 15, 6363-6373]. To explore the mechanism of TMAO protection of PrP(C) at the atomic level, molecular dynamics simulations were performed under conditions normally leading to conversion (low pH) with and without 1 M TMAO. In PrP(C) simulations at low pH, the helix content drops and the N-terminus is brought into the small native beta-sheet, yielding a PrP(Sc)-like state. Addition of 1 M TMAO leads to a decreased radius of gyration, a greater number of protein-protein hydrogen bonds, and a greater number of tertiary contacts due to the N-terminus forming an Omega-loop and packing against the structured core of the protein, not due to an increase in the level of extended structure as with the PrP(C) to PrP(Sc) simulation. In simulations beginning with the "PrP(Sc)-like" structure (derived from PrP(C) simulated at low pH in pure water) in 1 M TMAO, similar structural reorganization at the N-terminus occurred, disrupting the extended sheet. The mechanism of protection by TMAO appears to be exclusionary in nature, consistent with previous theoretical and experimental studies. The TMAO-induced N-terminal conformational change prevents residues that are important in the conversion of PrP(C) to PrP(Sc) from assuming extended sheet structure at low pH.     

Structural instability of the prion protein upon M205S/R mutations revealed by molecular dynamics simulations

The point mutations M205S and M205R have been demonstrated to severely disturb the folding and maturation process of the cellular prion protein (PrP(C)). These disturbances have been interpreted as consequences of mutation-induced structural changes in PrP, which are suggested to involve helix 1 and its attachment to helix 3, because the mutated residue M205 of helix 3 is located at the interface of these two helices. Furthermore, current models of the prion protein scrapie (PrP(Sc)), which is the pathogenic isoform of PrP(C) in prion diseases, imply that helix 1 disappears during refolding of PrP(C) into PrP(Sc). Based on molecular-dynamics simulations of wild-type and mutant PrP(C) in aqueous solution, we show here that the native PrP(C) structure becomes strongly distorted within a few nanoseconds, once the point mutations M205S and M205R have been applied. In the case of M205R, this distortion is characterized by a motion of helix 1 away from the hydrophobic core into the aqueous environment and a subsequent structural decay. Together with experimental evidence on model peptides, this decay suggests that the hydrophobic attachment of helix 1 to helix 3 at M205 is required for its correct folding into its stable native structure.     

Molecular dynamics simulation of dimeric and monomeric forms of human prion protein: insight into dynamics and properties

A central theme in prion protein research is the detection of the process that underlies the conformational transition from the normal cellular prion form (PrP(C)) to its pathogenic isoform (PrP(Sc)). Although the three-dimensional structures of monomeric and dimeric human prion protein (HuPrP) have been revealed by NMR spectroscopy and x-ray crystallography, the process underlying the conformational change from PrP(C) to PrP(Sc) and the dynamics and functions of PrP(C) remain unknown. The dimeric form is thought to play an important role in the conformational transition. In this study, we performed molecular dynamics (MD) simulations on monomeric and dimeric HuPrP at 300 K and 500 K for 10 ns to investigate the differences in the properties of the monomer and the dimer from the perspective of dynamic and structural behaviors. Simulations were also undertaken with Asp178Asn and acidic pH, which is known as a disease-associated factor. Our results indicate that the dynamics of the dimer and monomer were similar (e.g., denaturation of helices and elongation of the beta-sheet). However, additional secondary structure elements formed in the dimer might result in showing the differences in dynamics and properties between the monomer and dimer (e.g., the greater retention of dimeric than monomeric tertiary structure).     

Disease-associated F198S mutation increases the propensity of the recombinant prion protein for conformational conversion to scrapie-like form

The critical step in the pathogenesis of transmissible spongiform encephalopathies (prion diseases) is the conversion of a cellular prion protein (PrP(c)) into a protease-resistant, beta-sheet rich form (PrP(Sc)). Although the disease transmission normally requires direct interaction between exogenous PrP(Sc) and endogenous PrP(C), the pathogenic process in hereditary prion diseases appears to develop spontaneously (i.e. not requiring infection with exogenous PrP(Sc)). To gain insight into the molecular basis of hereditary spongiform encephalopathies, we have characterized the biophysical properties of the recombinant human prion protein variant containing the mutation (Phe(198) --> Ser) associated with familial Gerstmann-Straussler-Scheinker disease. Compared with the wild-type protein, the F198S variant shows a dramatically increased propensity to self-associate into beta-sheet-rich oligomers. In a guanidine HCl-containing buffer, the transition of the F198S variant from a normal alpha-helical conformation into an oligomeric beta-sheet structure is about 50 times faster than that of the wild-type protein. Importantly, in contrast to the wild-type PrP, the mutant protein undergoes a spontaneous conversion to oligomeric beta-sheet structure even in the absence of guanidine HCl or any other denaturants. In addition to beta-sheet structure, the oligomeric form of the protein is characterized by partial resistance to proteinase K digestion, affinity for amyloid-specific dye, thioflavine T, and fibrillar morphology. The increased propensity of the F198S variant to undergo a conversion to a PrP(Sc)-like form correlates with a markedly decreased thermodynamic stability of the native alpha-helical conformer of the mutant protein. This correlation supports the notion that partially unfolded intermediates may be involved in conformational conversion of the prion protein.     

Post-translational hydroxylation at the N-terminus of the prion protein reveals presence of PPII structure in vivo

The transmissible spongiform encephalopathies are characterized by conversion of a host protein, PrP(C) (cellular prion protein), to a protease-resistant isoform, PrP(Sc) (prion protein scrapie isoform). The importance of the highly flexible, N-terminal region of PrP has recently become more widely appreciated, particularly the biological activities associated with its metal ion-binding domain and its potential to form a poly(L-proline) II (PPII) helix. Circular dichroism spectroscopy of an N-terminal peptide, PrP(37-53), showed that the PPII helix is formed in aqueous buffer; as it also contains an Xaa-Pro-Gly consensus sequence, it may act as a substrate for the collagen-modifying enzyme prolyl 4-hydroxylase. Direct evidence for this modification was obtained by mass spectrometry and Edman sequencing in recombinant mouse PrP secreted from stably transfected Chinese hamster ovary cells. Almost complete conversion of proline to 4-hydroxyproline occurs specifically at residue Pro44 of this murine protein; the same hydroxylated residue was detected, at lower levels, in PrP(Sc) from the brains of scrapie-infected mice. Cation binding and/or post-translational hydroxylation of this region of PrP may regulate its role in the physiology and pathobiology of the cell.     

The charge structure of helix 1 in the prion protein regulates conversion to pathogenic PrPSc

The prion diseases are transmissible neurodegenerative disorders linked to a pathogenic conformer (PrP(Sc)) of the normal prion protein (PrP(C)). Accumulation of PrP(Sc) occurs via a poorly defined process in which PrP(Sc) complexes with and converts endogenous PrP(C) to nascent PrP(Sc). Recent experiments have focused on the highly charged first alpha helix (H1) of PrP. It has been proposed that two putative asparagine-to-arginine intrahelical salt bridges stabilize H1 in PrP(C) yet form intermolecular ionic bonds with adjacent PrP molecules during conversion of PrP(C) to PrP(Sc) (M. P. Morrissey and E. I. Shakhnovich, Proc. Natl. Acad. Sci. USA 96:11293-11298, 1999). Subsequent work (J. O. Speare et al., J. Biol. Chem. 278:12522-12529, 2003 using a cell-free assay of PrP(Sc) conversion suggested that rather than promoting conversion, the salt bridges stabilize PrP(C) against it. However, the role of individual H1 charges in PrP(Sc) generation has not yet been investigated. To approach this question, we systematically reversed or neutralized each charged residue in H1 and tested the effect on conversion to PrP(Sc) in scrapie-infected murine neuroblastoma (ScN2a) cells. We find that replacements of charged H1 residues with like charges permit conversion, while charge reversals hinder it. Neutralization of charges in the N-terminal (amino acids 143 to 146) but not the C-terminal (amino acids 147 to 151) half of H1 permits conversion, while complete reversal of charge orientation of the putative salt bridges produces a nonconvertible PrP. Circular dichroism spectroscopy studies and confocal microscopy immunofluorescence localization studies indicated that charge substitutions did not alter the secondary structure or cell surface expression of PrP(C). These data support the necessity of specific charge orientations in H1 for a productive PrP(Sc)-PrP(C) complex.     

Scrapie infection of prion protein-deficient cell line upon ectopic expression of mutant prion proteins

Expression of the cellular prion protein (PrP(C)) is crucial for susceptibility to prions. In vivo, ectopic expression of PrP(C) restores susceptibility to prions and transgenic mice that express heterologous PrP on a PrP knock-out background have been used extensively to study the role of PrP alterations for prion transmission and species barriers. Here we report that prion protein knock-out cells can be rendered permissive to scrapie infection by the ectopic expression of PrP. The system was used to study the influence of sheep PrP-specific residues in mouse PrP on the infection process with mouse adapted scrapie. These studies reveal several critical residues previously not associated with species barriers and demonstrate that amino acid residue alterations at positions known to have an impact on the susceptibility of sheep to sheep scrapie also drastically influence PrP(Sc) formation by mouse-adapted scrapie strain 22L. Furthermore, our data suggest that amino acid polymorphisms located on the outer surfaces of helix 2 and 3 drastically impact conversion efficiency. In conclusion, this system allows for the fast generation of mutant PrP(Sc) that is entirely composed of transgenic PrP and is, thus, ideally suited for testing if artificial PrP molecules can affect prion replication. Transmission of infectivity generated in HpL3-4 cells expressing altered PrP molecules to mice could also help to unravel the potential influence of mutant PrP(Sc) on host cell tropism and strain characteristics in vivo.     

Novel differences between two human prion strains revealed by two-dimensional gel electrophoresis

The phenotype of human sporadic prion diseases is affected by patient genotype at codon 129 of the prion protein (PrP) gene, the site of a common methionine/valine polymorphism, and by the type of the scrapie PrP (PrP(Sc)), which likely reflects the prion strain. However, two distinct disease phenotypes, identified as sporadic Creutzfeldt-Jakob disease (M/M2 sCJD) and sporadic fatal insomnia (sFI), share methionine homozygosity at codon 129 and PrP(Sc) type 2. One-dimensional gel electrophoresis and immunoblotting reveal no difference between the M/M2 sCJD and sFI species of PrP(Sc) in gel mobility and glycoform ratio. In contrast, the two-dimensional immunoblot demonstrates that in M/M2 sCJD the full-length PrP(Sc) form is overrepresented and carries glycans that are different from those present in the PrP(Sc) of sFI. Because the altered glycans are detectable only in the PrP(Sc) and not in the normal or cellular PrP (PrP(C)), they are likely to result from preferential conversion to PrP(Sc) of rare PrP(C) glycoforms. This is the first evidence that a qualitative difference in glycans contributes to prion diversity.     

The role of rafts in the fibrillization and aggregation of prions

A key molecular event in prion diseases is the conversion of the prion protein (PrP) from its normal cellular form (PrP(C)) to the disease-specific form (PrP(Sc)). The transition from PrP(C) to PrP(Sc) involves a major conformational change, resulting in amorphous aggregates and/or fibrillar amyloid deposits. Here several lines of evidence implicating membranes in the conversion of PrP are reviewed with a particular emphasis on the role of lipid rafts in the conformational transition of prion proteins. New correlations between in vitro biophysical studies and findings from cell biology work on the role of rafts in prion conversion are highlighted and a mechanism for the role of rafts in prion conversion is proposed.     

Solution structure of the E200K variant of human prion protein. Implications for the mechanism of pathogenesis in familial prion diseases

Prion propagation in transmissible spongiform encephalopathies involves the conversion of cellular prion protein, PrP(C), into a pathogenic conformer, PrP(Sc). Hereditary forms of the disease are linked to specific mutations in the gene coding for the prion protein. To gain insight into the molecular basis of these disorders, the solution structure of the familial Creutzfeldt-Jakob disease-related E200K variant of human prion protein was determined by multi-dimensional nuclear magnetic resonance spectroscopy. Remarkably, apart from minor differences in flexible regions, the backbone tertiary structure of the E200K variant is nearly identical to that reported for the wild-type human prion protein. The only major consequence of the mutation is the perturbation of surface electrostatic potential. The present structural data strongly suggest that protein surface defects leading to abnormalities in the interaction of prion protein with auxiliary proteins/chaperones or cellular membranes should be considered key determinants of a spontaneous PrP(C) --> PrP(Sc) conversion in the E200K form of hereditary prion disease.     

Identifying key components of the PrPC-PrPSc replicative interface

In prion disease, direct interaction between the cellular prion protein (PrP(C)) and its misfolded disease-associated conformer PrP(Sc) is a crucial, although poorly understood step promoting the formation of nascent PrP(Sc) and prion infectivity. Recently, we hypothesized that three regions of PrP (corresponding to amino acid residues 23-33, 98-110, and 136-158) interacting specifically and robustly with PrP(Sc), likely represent peptidic components of one flank of the prion replicative interface. In this study, we created epitope-tagged mouse PrP(C) molecules in which the PrP sequences 23-33, 98-110, and 136-158 were modified. These novel PrP molecules were individually expressed in the prion-infected neuroblastoma cell line (ScN2a) and the conversion of each mutated mouse PrP(C) substrate to PrP(Sc) compared with that of the epitope-tagged wild-type mouse PrP(C). Mutations within PrP 98-110, substituting all 4 wild-type lysine residues with alanine residues, prevented conversion to PrP(Sc). Furthermore, when residues within PrP 136-140 were collectively scrambled, changed to alanines, or amino acids at positions 136, 137, and 139 individually replaced by alanine, conversion to PrP(Sc) was similarly halted. However, other PrP molecules containing mutations within regions 23-33 and 101-104 were able to readily convert to PrP(Sc). These results suggest that PrP sequence comprising residues 98-110 and 136-140 not only participates in the specific binding interaction between PrP(C) and PrP(Sc), but also in the process leading to conversion of PrP(Sc)-sequestered PrP(C) into its disease-associated form.     

Strain-specific effects of reducing agents on the cell-free conversion of recombinant prion protein into a protease-resistant form

The pathogenic isoform (PrP(Sc) ) of the host-encoded normal cellular prion protein (PrP(C) ) is believed to be the infectious agent of transmissible spongiform encephalopathies. Spontaneous conversion of α-helix-rich recombinant PrP into the PrP(Sc) -like β-sheet-rich form or aggregation of cytosolic PrP has been found to be accelerated under reducing conditions. However, the effect of reducing conditions on PrP(Sc) -mediated conversion of PrP(C) into PrP(Sc) has remained unknown. In this study, the effect of reducing conditions on the binding of bacterial recombinant mouse PrP (MoPrP) with PrP(Sc) and the conversion of MoPrP into proteinase K-resistant PrP (PrP(res) ) using a cell-free conversion assay was investigated. High concentrations of dithiothreitol did not inhibit either the binding or conversion reactions of PrP(Sc) from five prion strains. Indeed, dithiothreitol significantly accelerated mouse-adapted BSE-seeded conversion. These data suggest that conversion of PrP(Sc) derived from a subset of prion strains is accelerated under reducing conditions, as has previously been shown for spontaneous conversion. Furthermore, the five prion strains used could be classified into three groups according to their efficiency at binding and conversion of MoPrP and cysteine-less mutants under both reducing and nonreducing conditions. The resulting classification is similar to that derived from biological and biochemical strain-specific features.     

The elusive intermediate on the folding pathway of the prion protein

A key molecular event in prion diseases is the conversion of the cellular conformation of the prion protein (PrP(C)) to an altered disease-associated form, generally denoted as scrapie isoform (PrP(Sc)). The molecular details of this conformational transition are not fully understood, but it has been suggested that an intermediate on the folding pathway of PrP(C) may be recruited to form PrP(Sc). In order to investigate the folding pathway of PrP we designed and expressed two mutants, each possessing a single strategically located tryptophan residue. The secondary structure and folding properties of the mutants were examined. Using conventional analyses of folding transition data determined by fluorescence and CD, and novel phase-diagram analyses, we present compelling evidence for the presence of an intermediate species on the folding pathway of PrP. The potential role of this intermediate in prion conversion is discussed.     

Conversion efficiency of bank vole prion protein in vitro is determined by residues 155 and 170, but does not correlate with the high susceptibility of bank voles to sheep scrapie in vivo

The misfolded infectious isoform of the prion protein (PrP(Sc)) is thought to replicate in an autocatalytic manner by converting the cellular form (PrP(C)) into its pathogenic folding variant. The similarity in the amino acid sequence of PrP(C) and PrP(Sc) influences the conversion efficiency and is considered as the major determinant for the species barrier. We performed in vitro conversion reactions on wild-type and mutated PrP(C) to determine the role of the primary sequence for the high susceptibility of bank voles to scrapie. Different conversion efficiencies obtained with bank vole and mouse PrP(C) in reactions with several prion strains were due to differences at amino acid residues 155 and 170. However, the conversion efficiencies obtained with mouse and vole PrP(C) in reactions with sheep scrapie did not correlate with the susceptibility of the respective species to this prion strain. This discrepancy between in vitro and in vivo data may indicate that at least in the case of scrapie transmission to bank voles additional host factors can strongly modulate the species barrier. Furthermore, in vitro conversion reactions with different prion strains revealed that the degree of alteration of the conversion efficiency induced by amino acid exchanges was varying according to the prion strain. These results support the assumption that the repertoire of conformations adopted by a certain PrP(C) primary sequence is decisive for its convertibility to the strain-specific PrP(Sc) conformation.     

Prion protein helix1 promotes aggregation but is not converted into beta-sheet

Prion diseases are caused by the aggregation of the native alpha-helical prion protein PrP(C) into its pathological beta-sheet-rich isoform PrP(Sc). In current models of PrP(Sc), helix1 is assumed to be preferentially converted into beta-sheet during aggregation of PrP(C). This was supported by the NMR structure of PrP(C) since, in contrast to the isolated helix1, helix2 and helix3 are connected by a small loop and are additionally stabilized by an interhelical disulfide bond. However, helix1 is extremely hydrophilic and has a high helix propensity. This prompted us to investigate the role of helix1 in prion aggregation using humPrP(23-159) including helix1 (144-156) compared with the C-terminal-truncated isoform humPrP(23-144) corresponding to the pathological human stop mutations Q160Stop and Y145Stop, respectively. Most unexpectedly, humPrP(23-159) aggregated significantly faster compared with the truncated fragment humPrP(23-144), clearly demonstrating that helix1 is involved in the aggregation process. However, helix1 is not resistant to digestion with proteinase K in fibrillar humPrP(23-159), suggesting that helix1 is not converted to beta-sheet. This is confirmed by Fourier transformation infrared spectroscopy since there is almost no difference in beta-sheet content of humPrP(23-159) fibrils compared with humPrP(23-144). In conclusion, we provide strong direct evidence that in contrast to earlier assumptions helix1 is not converted into beta-sheet during aggregation of PrP(C) to PrP(Sc).     

Mouse prion protein (PrP) segment 100 to 104 regulates conversion of PrP(C) to PrP(Sc) in prion-infected neuroblastoma cells

Prion diseases are characterized by the replicative propagation of disease-associated forms of prion protein (PrP(Sc); PrP refers to prion protein). The propagation is believed to proceed via two steps; the initial binding of the normal form of PrP (PrP(C)) to PrP(Sc) and the subsequent conversion of PrP(C) to PrP(Sc). We have explored the two-step model in prion-infected mouse neuroblastoma (ScN2a) cells by focusing on the mouse PrP (MoPrP) segment 92-GGTHNQWNKPSKPKTN-107, which is within a region previously suggested to be part of the binding interface or shown to differ in its accessibility to anti-PrP antibodies between PrP(C) and PrP(Sc). Exchanging the MoPrP segment with the corresponding chicken PrP segment (106-GGSYHNQKPWKPPKTN-121) revealed the necessity of MoPrP residues 99 to 104 for the chimeras to achieve the PrP(Sc) state, while segment 95 to 98 was replaceable with the chicken sequence. An alanine substitution at position 100, 102, 103, or 104 of MoPrP gave rise to nonconvertible mutants that associated with MoPrP(Sc) and interfered with the conversion of endogenous MoPrP(C). The interference was not evoked by a chimera (designated MCM2) in which MoPrP segment 95 to 104 was changed to the chicken sequence, though MCM2 associated with MoPrP(Sc). Incubation of the cells with a synthetic peptide composed of MoPrP residues 93 to 107 or alanine-substituted cognates did not inhibit the conversion, whereas an anti-P8 antibody recognizing the above sequence in PrP(C) reduced the accumulation of PrP(Sc) after 10 days of incubation of the cells. These results suggest the segment 100 to 104 of MoPrP(C) plays a key role in conversion after binding to MoPrP(Sc).     

The highways and byways of prion protein trafficking

Prions are defined as infectious agents that comprise only proteins and are responsible for transmissible spongiform encephalopathies (TSEs)--fatal neurodegenerative diseases that affect humans and other mammals and include Creutzfeldt-Jacob disease in humans, scrapie in sheep and bovine spongiform encephalopathy in cattle. Prions have been proposed to arise from the conformational conversion of the cellular prion protein PrP(C) to a misfolded form termed PrP(Sc) that precipitates into aggregates and fibrils. The conversion process might be triggered by interaction of the infectious form with the cellular form or it might result from a mutation in the gene encoding PrP(C). Exactly how and where in the cell the interaction and the conversion of PrP(C) to PrP(Sc) occur, however, remain controversial. Recent studies have shed light on the intracellular trafficking of PrP(C), the role of protein mis-sorting and the cellular factors that are thought to be required for the conformational conversion of prion proteins.     

Globular domain of the prion protein needs to be unlocked by domain swapping to support prion protein conversion

Prion diseases are fatal transmissible neurodegenerative diseases affecting many mammalian species. The normal prion protein (PrP) converts into a pathological aggregated form, PrPSc, which is enriched in the β-sheet structure. Although the high resolution structure of the normal PrP was determined, the structure of the converted form of PrP remains inaccessible to high resolution techniques. To map the PrP conversion process we introduced disulfide bridges into different positions within the globular domain of PrP, tethering selected secondary structure elements. The majority of tethered PrP mutants exhibited increased thermodynamic stability, nevertheless, they converted efficiently. Only the disulfides that tether subdomain B1-H1-B2 to subdomain H2-H3 prevented PrP conversion in vitro and in prion-infected cell cultures. Reduction of disulfides recovered the ability of these mutants to convert, demonstrating that the separation of subdomains is an essential step in conversion. Formation of disulfide-linked proteinase K-resistant dimers in fibrils composed of a pair of single cysteine mutants supports the model based on domain-swapped dimers as the building blocks of prion fibrils. In contrast to previously proposed structural models of PrPSc suggesting conversion of large secondary structural segments, we provide evidence for the conservation of secondary structural elements of the globular domain upon PrP conversion. Previous studies already showed that dimerization is the rate-limiting step in PrP conversion. We show that separation and swapping of subdomains of the globular domain is necessary for conversion. Therefore, we propose that the domain-swapped dimer of PrP precedes amyloid formation and represents a potential target for therapeutic intervention.