Conformational pH dependence of intermediate states during oligomerization of the human prion protein

Intermediate states are key to understanding the molecular mechanisms governing protein misfolding. The human prion protein (PrP) can follow various misfolding pathways, and forms a soluble beta-sheet-rich oligomer under acidic, mildly denaturing, high salt conditions. Here we describe a fast conformational switch from the native alpha-monomer to monomeric intermediate states under oligomer-forming conditions, followed by a slower oligomerization process. We observe a pH dependence of the secondary structure of these intermediate forms, with almost native-like alpha-helical secondary structure at pH 4.1 and predominantly beta-sheet characteristics at pH 3.6. NMR spectroscopy differentiates these intermediate states from the native protein and indicates dynamic rearrangements of secondary structure elements characteristic of a molten globule. The alpha-helical intermediate formed at pH 4.1 can convert to the beta-sheet conformation at pH 3.6 but not vice versa, and neither state can be reconverted to an alpha-monomer. The presence of methionine rather than valine at codon 129 accelerates the rate of oligomer formation from the intermediate state.     

On the mechanism of alpha-helix to beta-sheet transition in the recombinant prion protein

It is believed that the critical event in the pathogenesis of transmissible spongiform encephalopathies is the conversion of the prion protein from an alpha-helical form, PrP(C), to a beta-sheet-rich conformer, PrP(Sc). Recently, we have shown that incubation of the recombinant prion protein under mildly acidic conditions (pH 5 or below) in the presence of low concentrations of guanidine hydrochloride results in a transition to PrP(Sc)-like beta-sheet-rich oligomers that show fibrillar morphology and an increased resistance to proteinase K digestion [Swietnicki, W., Morillas, M, Chen, S., Gambetti, P., and Surewicz, W. K. (2000) Biochemistry 39, 424-431]. To gain insight into the mechanism of this transition, in the present study we have characterized the biophysical properties of the recombinant human prion protein (huPrP) at acidic pH in the presence of urea and salt. Urea alone induces unfolding of the protein but does not result in protein self-association or a conversion to beta-sheet structure. However, a time-dependent transition to beta-sheet structure occurs upon addition of both urea and NaCl to huPrP, even at a sodium chloride concentration as low as 50 mM. This transition occurs concomitantly with oligomerization of the protein. At a given protein and sodium chloride concentration, the rate of monomeric alpha-helix to oligomeric beta-sheet transition is strongly dependent on the concentration of urea. Low and medium concentrations of the denaturant accelerate the reaction, whereas strongly unfolding conditions are not conducive to the conversion of huPrP into an oligomeric beta-sheet-rich structure. The present data strongly suggest that partially unfolded intermediates may be involved in the transition of the monomeric recombinant prion protein into the oligomeric scrapie-like form.     

Pathway complexity of prion protein assembly into amyloid

In vivo under pathological conditions, the normal cellular form of the prion protein, PrP(C) (residues 23-231), misfolds to the pathogenic isoform PrP(Sc), a beta-rich aggregated pathogenic multimer. Proteinase K digestion of PrP(Sc) leads to a proteolytically resistant core, PrP 27-30 (residues 90-231), that can form amyloid fibrils. To study the kinetic pathways of amyloid formation in vitro, we used unglycosylated recombinant PrP corresponding to the proteinase K-resistant core of PrP(Sc) and found that it can adopt two non-native abnormal isoforms, a beta-oligomer and an amyloid fibril. Several lines of kinetic data suggest that the beta-oligomer is not on the pathway to amyloid formation. The preferences for forming either a beta-oligomer or amyloid can be dictated by experimental conditions, with acidic pH similar to that seen in endocytic vesicles favoring the beta-oligomer and neutral pH favoring amyloid. Although both abnormal isoforms have high beta-sheet content and bind 1-anilinonaphthalene-8-sulfonate, they are dissimilar structurally. Multiple pathways of misfolding and the formation of distinct beta-sheet-rich abnormal isoforms may explain the difficulties in refolding PrP(Sc) in vitro, the need for a PrP(Sc) template, and the significant variation in disease presentation and neuropathology.     

Aggregation and fibrillization of the recombinant human prion protein huPrP90-231

According to the "protein-only" hypothesis, the critical step in the pathogenesis of prion diseases is the conformational transition between the normal (PrP(C)) and pathological (PrP(Sc)) isoforms of prion protein. To gain insight into the mechanism of this transition, we have characterized the biophysical properties of the recombinant protein corresponding to residues 90-231 of the human prion protein (huPrP90-231). Incubation of the protein under acidic conditions (pH 3.6-5) in the presence of 1 M guanidine-HCl resulted in a time-dependent transition from an alpha-helical conformation to a beta-sheet structure and oligomerization of huPrP90-231 into large molecular weight aggregates. No stable monomeric beta-sheet-rich folding intermediate of the protein could be detected in the present experiments. Kinetic analysis of the data indicates that the formation of beta-sheet structure and protein oligomerization likely occur concomitantly. The beta-sheet-rich oligomers were characterized by a markedly increased resistance to proteinase K digestion and a fibrillar morphology (i.e., they had the essential physicochemical properties of PrP(Sc)). Contrary to previous suggestions, the conversion of the recombinant prion protein into a PrP(Sc)-like form could be accomplished under nonreducing conditions, without the need to disrupt the disulfide bond. Experiments in urea indicate that, in addition to acidic pH, another critical factor controlling the transition of huPrP90-231 to an oligomeric beta-sheet structure is the presence of salt.     

The role of disulfide bridge in the folding and stability of the recombinant human prion protein

It is believed that the critical step in the pathogenesis of transmissible spongiform encephalopathies is a transition of prion protein (PrP) from an alpha-helical conformation, PrP(C), to a beta-sheet-rich form, PrP(Sc). Native prion protein contains a single disulfide bond linking Cys residues at positions 179 and 214. To elucidate the role of this bridge in the stability and folding of the protein, we studied the reduced form of the recombinant human PrP as well as the variant of PrP in which cysteines were replaced with alanine residues. At neutral pH, the reduced prion protein and the Cys-free mutant were insoluble and formed amorphous aggregates. However, the proteins could be refolded in a monomeric form under the conditions of mildly acidic pH. Spectroscopic experiments indicate that the monomeric Cys-free and reduced PrP have molten globule-like properties, i.e. they are characterized by compromised tertiary interactions, an increased exposure of hydrophobic surfaces, lack of cooperative unfolding transition in urea, and partial loss of native (alpha-helical) secondary structure. In the presence of sodium chloride, these partially unfolded proteins undergo a transition to a beta-sheet-rich structure. However, this transition is invariably associated with protein oligomerization. The present data argue against the notion that reduced prion protein can exist in a stable monomeric form that is rich in beta-sheet structure.     

Conserved and cooperative assembly of membrane-bound alpha-helical states of islet amyloid polypeptide

The conversion of soluble protein into beta-sheet-rich amyloid fibers is the hallmark of a number of serious diseases. Precursors for many of these systems (e.g., Abeta from Alzheimer's disease) reside in close association with a biological membrane. Membrane bilayers are reported to accelerate the rate of amyloid assembly. Furthermore, membrane permeabilization by amyloidogenic peptides can lead to toxicity. Given the beta-sheet-rich nature of mature amyloid, it is seemingly paradoxical that many precursors are either intrinsically alpha-helical or transiently adopt an alpha-helical state upon association with membrane. In this work, we investigate these phenomena in islet amyloid polypeptide (IAPP). IAPP is a 37-residue peptide hormone which forms amyloid fibers in individuals with type II diabetes. Fiber formation by human IAPP (hIAPP) is markedly accelerated by lipid bilayers despite adopting an alpha-helical state on the membrane. We further show that IAPP partitions into monomeric and oligomeric helical assemblies. Importantly, it is this latter state which most strongly correlates to both membrane leakage and accelerated fiber formation. A sequence variant of IAPP from rodents (rIAPP) does not form fibers and is reputed not to permeabilize membranes. Here, we report that rIAPP is capable of permeabilizing membranes under conditions that permit rIAPP membrane binding. Sequence and spectroscopic comparisons of rIAPP and hIAPP enable us to propose a general mechanism for the helical acceleration of amyloid formation in vitro. As rIAPP cannot form amyloid fibers, our results show that fiber formation need not be directly coupled to toxicity.     

Self-assembly of recombinant prion protein of 106 residues

The central event in the pathogenesis of prion diseases is a profound conformational change of the prion protein (PrP) from an alpha-helical (PrP(C)) to a beta-sheet-rich isoform (PrP(Sc)). The elucidation of the mechanism of conformational transition has been complicated by the challenge of collecting high-resolution biophysical data on the relatively insoluble aggregation-prone PrP(Sc) isoform. In an attempt to facilitate the structural analysis of PrP(Sc), a redacted chimeric mouse-hamster PrP of 106 amino acids (MHM2 PrP106) with two deletions (Delta23-88 and Delta141-176) was expressed and purified from Escherichia coli. PrP106 retains the ability to support PrP(Sc) formation in transgenic mice, implying that it contains all regions of PrP that are necessary for the conformational transition into the pathogenic isoform [Supattapone, S., et al. (1999) Cell 96, 869-878]. Unstructured at low concentrations, recombinant unglycosylated PrP106 (rPrP106) undergoes a concentration-dependent conformational transition to a beta-sheet-rich form. Following the conformational transition, rPrP106 possesses properties similar to those of PrP(Sc)106, such as high beta-sheet content, defined tertiary structure, resistance to limited digestion by proteinase K, and high thermodynamic stability. In GdnHCl-induced denaturation studies, a single cooperative conformational transition between the unstructured monomer and the assembled beta-oligomer was observed. After proteinase K digestion, the oligomers retain an intact core with unusually high beta-sheet content (>80%). Using mass spectrometry, we discovered that the region of residues 134-215 of rPrP106 is protected from proteinase K digestion and possesses a solvent-independent propensity to adopt a beta-sheet-rich conformation. In contrast to the PrP(Sc)106 purified from the brains of neurologically impaired animals, multimeric beta-rPrP106 remains soluble, providing opportunities for detailed structural studies.     

Folding of prion protein to its native alpha-helical conformation is under kinetic control

The recombinant mouse prion protein (MoPrP) can be folded either to a monomeric alpha-helical or oligomeric beta-sheet-rich isoform. By using circular dichroism spectroscopy and size-exclusion chromatography, we show that the beta-rich isoform of MoPrP is thermodynamically more stable than the native alpha-helical isoform. The conformational transition from the alpha-helical to beta-rich isoform is separated by a large energetic barrier that is associated with unfolding and with a higher order kinetic process related to oligomerization. Under partially denaturing acidic conditions, MoPrP avoids the kinetic trap posed by the alpha-helical isoform and folds directly to the thermodynamically more stable beta-rich isoform. Our data demonstrate that the folding of the prion protein to its native alpha-helical monomeric conformation is under kinetic control.     

Globular and pre-fibrillar prion aggregates are toxic to neuronal cells and perturb their electrophysiology

Prion diseases are characterised at autopsy by neuronal loss and accumulation of amorphous protein aggregates and/or amyloid fibrils in the brains of humans and animals. These protein deposits result from the conversion of the cellular, mainly alpha-helical prion protein (PrP(C)) to the beta-sheet-rich isoform (PrP(Sc)). Although the pathogenic mechanism of prion diseases is not fully understood, it appears that protein aggregation is itself neurotoxic and not the product of cell death. The precise nature of the neurotoxic species and mechanism of cell death are yet to be determined, although recent studies with other amyloidogenic proteins suggest that ordered pre-fibrillar or oligomeric forms may be responsible for cellular dysfunction. In this study we have refolded recombinant prion protein (rPrP) to two distinct forms rich in beta-sheet structure with an intact disulphide bond. Here we report on the structural properties of globular aggregates and pre-fibrils of rPrP and show that both states are toxic to neuronal cells in culture. We show that exogenous rPrP aggregates are internalised by neuronal cells and found in the cytoplasm. We also measured the changes in electrophysiological properties of cultured neuronal cells on exposure to exogenous prion aggregates and discuss the implications of these findings.     

Mechanism for the alpha-helix to beta-hairpin transition

The aggregation of alpha-helix-rich proteins into beta-sheet-rich amyloid fibrils is associated with fatal diseases, such as Alzheimer's disease and prion disease. During an aggregation process, protein secondary structure elements-alpha-helices-undergo conformational changes to beta-sheets. The fact that proteins with different sequences and structures undergo a similar transition on aggregation suggests that the sequence nonspecific hydrogen bond interaction among protein backbones is an important factor. We perform molecular dynamics simulations of a polyalanine model, which is an alpha-helix in its native state and observe a metastable beta-hairpin intermediate. Although a beta-hairpin has larger potential energy than an alpha-helix, the entropy of a beta-hairpin is larger because of fewer constraints imposed by the hydrogen bonds. In the vicinity of the transition temperature, we observe the interconversion of the alpha-helix and beta-sheet states via a random coil state. We also study the effect of the environment by varying the relative strength of side-chain interactions for a designed peptide-an alpha-helix in its native state. For a certain range of side-chain interaction strengths, we find that the intermediate beta-hairpin state is destabilized and even disappears, suggesting an important role of the environment in the aggregation propensity of a peptide.     

Study of the conformational transition of A beta(1-42) using D-amino acid replacement analogues

A critical event in Alzheimer's disease is the transition of Abeta peptides from their soluble forms into disease-associated beta-sheet-rich conformers. Structural analysis of a complete D-amino acid replacement set of Abeta(1-42) enabled us to localize in the full-length 42-mer peptide the region responsible for the conformational switch into a beta-sheet structure. Although NMR spectroscopy of trifluoroethanol-stabilized monomeric Abeta(1-42) delineated two separated helical domains, only the destabilization of helix I, comprising residues 11-24, caused a transition to a beta-sheet structure. This conformational alpha-to-beta switch was directly accompanied by an aggregation process leading to the formation of amyloid fibrils.     

An unusual soluble beta-turn-rich conformation of prion is involved in fibril formation and toxic to neuronal cells

A key molecular event in prion diseases is the conversion of the prion protein (PrP) from its normal cellular form (PrPC) to the disease-specific form (PrPSc). The transition from PrPC to PrPSc involves a major conformational change, resulting in amorphous protein aggregates and fibrillar amyloid deposits with increased beta-sheet structure. Using recombinant PrP refolded into a beta-sheet-rich form (beta-PrP) we have studied the fibrillization of beta-PrP both in solution and in association with raft membranes. In low ionic strength thick dense fibrils form large networks, which coexist with amorphous aggregates. High ionic strength results in less compact fibrils, that assemble in large sheets packed with globular PrP particles, resembling diffuse aggregates found in ex vivo preparations of PrPSc. Here we report on the finding of a beta-turn-rich conformation involved in prion fibrillization that is toxic to neuronal cells in culture. This is the first account of an intermediate in prion fibril formation that is toxic to neuronal cells. We propose that this unusual beta-turn-rich form of PrP may be a precursor of PrPSc and a candidate for the neurotoxic molecule in prion pathogenesis.     

Structural intermediates in the putative pathway from the cellular prion protein to the pathogenic form

The conversion of the alpha-helical, protease sensitive and noninfectious form of the prion protein (PrP(C)) into an insoluble, protease resistant, predominantly beta-sheeted and infectious form (PrP(Sc)) is the fundamental event in prion formation. In the present work, two soluble and stable intermediate structural states are newly identified for recombinant Syrian hamster PrP(90-231) (recPrP), a dimeric alpha-helical state and a tetra- or oligomeric, beta-sheet rich state. In 0.2% SDS at room temperature, recPrP is soluble and exhibits alpha-helical and random coil secondary structure as determined by circular dichroism. Reduction of the SDS concentration to 0.06% leads first to a small increase in alpha-helical content, whereas further dilution to 0.02% results in the aquisition of beta-sheet structure. The reversible transition curve is sigmoidal within a narrow range of SDS concentrations (0.04 to 0.02%). Size exclusion chromatography and chemical crosslinking revealed that the alpha-helical form is dimeric, while the beta-sheet rich form is tetra- or oligomeric. Both the alpha-helical and beta-sheet rich intermediates are soluble and stable. Thus, they should be accessible to further structural and mechanistic studies. At 0.01% SDS, the oligomeric intermediates aggregated into large, insoluble structures as observed by fluorescence correlation spectroscopy. Our results are discussed with respect to the mechanism of PrP(Sc) formation and the propagation of prions.     

Enhanced stability of human prion proteins with two disulfide bridges

We compare the folding equilibrium of the globular domain of the human prion protein with two variants of this domain, for which an additional disulfide bond was introduced into the location where it is found in the naturally occurring doppel protein. We find that the unfolding transition midpoint of the variants is shifted toward higher denaturant concentration, indicating that the engineered disulfide bond significantly stabilizes the global protein structure. Our results further reveal that the two-disulfide variant proteins, while possessing the same global fold as the wild-type, display marked differences in their folding pathway-in particular, the absence of a characteristic alpha-helix to beta-sheet transition, which is a fundamental feature associated with misfolding of proteins into amyloid fibrils, especially in the context of prion diseases. These surprising characteristics of disulfide mutant prion proteins have important implications for the understanding of the generic aberrant processes leading to amyloid fibril formation and protein aggregation, as well as providing insight into possible therapeutic strategies.     

Scrapie-infected cells, isolated prions, and recombinant prion protein: a comparative study

Fourier -transform infrared microscopic spectra of scrapie-infected nervous tissue measured at high spatial resolution (approximately 6 microm) were compared with those obtained from the purified, partly proteinase K digested scrapie isoform of the prion protein isolated from nervous tissue of hamsters infected with the same scrapie strain (263K) to elucidate similarities/dissimilarities between prion structure investigated in situ and ex vivo. A further comparison is drawn to the recombinant Syrian hamster prion protein SHaPrP(90-232) after in vitro conformational transition from the predominantly alpha-helical isoform to beta-sheet-rich structures. It is shown that prion protein structure can be investigated within tissue and that detectability of regions with elevated beta-sheet content as observed in microspectra of prion-infected tissue strongly depends on spatial resolution of the experiment.     

Amyloid fibrils of mammalian prion protein are highly toxic to cultured cells and primary neurons

A growing body of evidence indicates that small, soluble oligomeric species generated from a variety of proteins and peptides rather than mature amyloid fibrils are inherently highly cytotoxic. Here, we show for the first time that mature amyloid fibrils produced from full-length recombinant mammalian prion protein (rPrP) were highly toxic to cultured cells and primary hippocampal and cerebella neurons. Fibrils induced apoptotic cell death in a time- and dose-dependent manner. The toxic effect of fibrils was comparable with that exhibited by soluble small beta-oligomers generated from the same protein. Fibrils prepared from insulin were not toxic, suggesting that the toxic effect was not solely due to the highly polymeric nature of the fibrillar form. The cell death caused by rPrP fibrils or beta-oligomers was substantially reduced when expression of endogenous PrP(C) was down-regulated by small interfering RNAs. In opposition to the beta-oligomer and amyloid fibrils of rPrP, the monomeric alpha-helical form of rPrP stimulated neurite out-growth and survival of neurons. These studies illustrated that both soluble beta-oligomer and amyloid fibrils of the prion protein are intrinsically toxic and confirmed that endogenously expressed PrP(C) is required for mediating the toxicity of abnormally folded external PrP aggregates.     

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.     

Annealing prion protein amyloid fibrils at high temperature results in extension of a proteinase K-resistant core

Amyloids are highly ordered, rigid beta-sheet-rich structures that appear to have minimal dynamic flexibility in individual polypeptide chains. Here, we demonstrate that substantial conformational rearrangements occur within mature amyloid fibrils produced from full-length mammalian prion protein. The rearrangement results in a substantial extension of a proteinase K-resistant core and is accompanied by an increase in the beta-sheet-rich conformation. The conformational rearrangement was induced in the presence of low concentrations of Triton X-100 either by brief exposure to 80 degrees C or, with less efficacy, by prolonged incubation at 37 degrees C at pH 7.5 and is referred to here as "annealing." Upon annealing, amyloid fibrils acquired a proteinase K-resistant core identical to that found in bovine spongiform encephalopathy-specific scrapie-associated prion protein. Annealing was also observed when amyloid fibrils were exposed to high temperatures in the absence of detergent but in the presence of brain homogenate. These findings suggest that the amyloid fibrils exist in two conformationally distinct states that are separated by a high energy barrier and that yet unknown cellular cofactors may facilitate transition of the fibrils into thermodynamically more stable state. Our studies provide new insight into the complex behavior of prion polymerization and highlight the annealing process, a previously unknown step in the evolution of amyloid structures.     

Scrapie infectivity is independent of amyloid staining properties of the N-terminally truncated prion protein

The prion protein undergoes a profound conformational change when the cellular isoform (PrP(C)) is converted into the disease-causing form (PrP(Sc)). Limited proteolysis of PrP(Sc) produces PrP 27-30, which readily polymerizes into amyloid. To study the relationship between PrP amyloid and infectivity, we employed organic solvents that perturb protein conformation. Hexafluoro-2-propanol (HFIP), which promotes alpha-helix formation, modified the ultrastructure of PrP amyloid and decreased the beta-sheet content as well as prion infectivity. HFIP reversibly decreased the binding of Congo red dye to the PrP amyloid rods while inactivation of prion infectivity was irreversible. In contrast, 1,1,1-trifluoro-2-propanol (TFIP) did not inactivate prion infectivity but like HFIP, TFIP did alter the morphology of the rods and abolished Congo red binding. Solubilization using various solvents and detergents produced monomeric and dimeric PrP that lacked infectivity. Proteinase K resistance of detergent-treated PrP 27-30 showed no correlation with scrapie infectivity. Our results separate prion infectivity from the amyloid properties of PrP 27-30 and underscore the dependence of prion infectivity on PrP(Sc) conformation. These findings also demonstrate that the specific beta-sheet-rich structures required for prion infectivity can be differentiated from those required for amyloid formation.     

Elongated oligomers assemble into mammalian PrP amyloid fibrils

In prion diseases, the mammalian prion protein PrP is converted from a monomeric, mainly alpha-helical state into beta-rich amyloid fibrils. To examine the structure of the misfolded state, amyloid fibrils were grown from a beta form of recombinant mouse PrP (residues 91-231). The beta-PrP precursors assembled slowly into amyloid fibrils with an overall helical twist. The fibrils exhibit immunological reactivity similar to that of ex vivo PrP Sc. Using electron microscopy and image processing, we obtained three-dimensional density maps of two forms of PrP fibrils with slightly different twists. They reveal two intertwined protofilaments with a subunit repeat of approximately 60 A. The repeating unit along each protofilament can be accounted for by elongated oligomers of PrP, suggesting a hierarchical assembly mechanism for the fibrils. The structure reveals flexible crossbridges between the two protofilaments, and subunit contacts along the protofilaments that are likely to reflect specific features of the PrP sequence, in addition to the generic, cross-beta amyloid fold.