Reversible Conversion of Monomeric Human Prion Protein Between Native and Fibrilogenic Conformations

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Science  19 Mar 1999:
Vol. 283, Issue 5409, pp. 1935-1937
DOI: 10.1126/science.283.5409.1935


Prion propagation involves the conversion of cellular prion protein (PrPC) into a disease-specific isomer, PrPSc, shifting from a predominantly α-helical to β-sheet structure. Here, conditions were established in which recombinant human PrP could switch between the native α conformation, characteristic of PrPC, and a compact, highly soluble, monomeric form rich in β structure. The soluble β form (β-PrP) exhibited partial resistance to proteinase K digestion, characteristic of PrPSc, and was a direct precursor of fibrillar structures closely similar to those isolated from diseased brains. The conversion of PrPC to β-PrP in suitable cellular compartments, and its subsequent stabilization by intermolecular association, provide a molecular mechanism for prion propagation.

Prion diseases are associated with the accumulation of a conformational isomer (PrPSc) of host-derived prion protein (PrPC) with an increase in its β-sheet secondary structure content (1) and the acquisition of partial resistance to digestion with proteinase K (PK). According to the protein-only hypothesis, PrPSc is the principal or sole component of transmissible prions (2). Although the structure of PrPC has been determined (3), the insolubility of PrPSc, which is isolated from tissue in a highly aggregated state, has precluded high-resolution structural analysis.

Human PrP91–231 can be expressed in large amounts inEscherichia coli and purified as a highly soluble, monomeric protein with a single intact disulfide bridge (4). Analysis by circular dichroism (CD) spectropolarimetry revealed a structure rich in α-helical content (∼50%) with little β sheet (∼20%) (Fig. 1A). One-dimensional (1D)1H nuclear magnetic resonance (NMR) spectra (Fig. 1C) and two-dimensional 1H-15N correlation NMR spectra (5) of this material showed it to be conformationally similar to the mouse and hamster prion proteins (3,6) and to another human PrP90–231 construct (7).

Figure 1

Secondary and tertiary structure of the two human PrP isoforms (26). (A) Far-UV CD spectra (27). Oxidized human PrP (pH 8.0) (○) displays a typically α-helical spectrum. Reduced human PrP (pH 4.0) (▵) displays a β-sheet spectrum with little or no helix present. (B) Near-UV CD spectra for oxidized human PrP (pH 8.0) (○), reduced human PrP (pH 4.0) (▵), and denatured human PrP (□). The oxidized protein displays extensive tertiary organization, whereas denatured PrP lacks any distinct tertiary interactions. Reduced human PrP displays an intermediate level of tertiary organization. (C)1H NMR spectra of the upfield regions of the α and β forms of human PrP91–231 (28). This includes the resonances at higher field than 0.7 ppm characteristic of strong tertiary interactions between methyl groups and aromatic rings in the α form. (D) Expanded region of a1H-15N HSQC spectrum of the β form of human PrP91–231 showing its chemical shift dispersion, which is much reduced relative to the α form (7).

Reduction of the disulfide bond in human PrP91–231 and lowering the pH to 4.0, in a dilute acetate buffer without additives, generated a highly soluble protein that can be concentrated to at least 12 mg/ml. When subjected to gel filtration, it eluted as a monomeric species (Fig. 2). The CD signal in the amide region of the spectrum (Fig. 1A) suggests that this highly soluble, reduced species adopted a different conformation from the native cellular form of PrP (α-PrP). The native state is characterized by a strong α-helical signal, whereas the reduced form is qualitatively dominated by β sheet. This type of secondary structural transition occurs in proteins that go from a soluble monomeric state to an aggregated fibrous (amyloid) form in which β-structure is stabilized by intermolecular interactions (8). However, to our knowledge, it is unprecedented for a protein to undergo such an extensive β-sheet conversion while remaining in a monomeric state at high protein concentrations and in the absence of denaturants. This is in contrast to the folding intermediates of mouse PrP121–231 (9) and human PrP90–231 (10, 11). We also observe the equilibrium folding intermediate of α-PrP described in (10), but in contrast to β-PrP, this is poorly soluble and has an apparent molecular weight of 40 kD (Fig. 2) indicative of tertiary disorder and expanded molecular volume. In marked contrast, the folded β-PrP we observe is populated, and therefore stable, in the absence of denaturant with an apparent molecular volume indistinguishable from that of α-PrP (Fig. 2).

Figure 2

Determination of the apparent molecular weight of PrP by size-exclusion chromatography (29). (A) Elution profile of standards used to construct a calibration curve of molecular weight (MW) versus elution time. (B) Oxidized human PrP (pH 8.0) elutes with an apparent MW of 18 kD. (C) Reduced human PrP (pH 4.0) also elutes as an 18-kD monomer. (D) Oxidized human PrP (pH 4.0) partially denatured with 1 M GuHCl, which results in aggregation and precipitation. Clarified supernatant contains a denatured form of PrP with an increased molecular volume (apparent MW, 40 kD). (E) β-PrP is more prone than α-PrP to form high-MW aggregates (30). Right-angle light scattering of α-PrP (○) shows that no high-MW aggregates are formed upon addition of GuHCl. In contrast, β-PrP readily forms high-MW aggregates upon addition of low concentrations of GuHCl (▵). Identical results are obtained with other common salts (for instance, NaCl and KCl).

Using amide CD alone, we cannot be certain whether β-PrP is sufficiently condensed to have immobilized side chains characteristic of the native state of orthodox, globular proteins. However, the aromatic CD spectra contained signals from aromatic side chains in asymmetric environments. Compared to the native, oxidized molecule, the β form retained a signal from aromatic residues, but with diminished intensity (Fig. 1B). Thus, although the β conformation contained less extensive, weaker tertiary interactions than those present in α-PrP, some remained. This finding is consistent with the gel filtration behavior of reduced human β-PrP91–231, which revealed the same level of compactness as the α-PrP conformation (Fig. 2).

The availability of the β form of PrP as a monomeric species at 0.75 mM provided the opportunity to examine its physical properties using NMR. The 1D 1H NMR spectrum of native human PrP91–231 exhibited wide chemical shift dispersion characteristic of a fully folded globular protein, whereas the 1D1H and 1H-15N heteronuclear single-quantum coherence (HSQC) spectra of the β form of PrP exhibited considerably less chemical shift dispersion (Fig. 1, C and D). This lack of dispersion is characteristic of the loss of fixed side-chain interactions, which, in conjunction with the aromatic CD results, suggests some similarities with molten globule states (12–14). In addition, proton and nitrogen linewidths of the β form (Fig. 1D) were comparable to those observed in the folded and unfolded regions of the α-PrP conformation. This finding confirms the gel filtration results and indicates that the β form was monomeric at the extremely high concentrations required for NMR. The mobile unstructured regions of β-PrP were assigned from the sharpness and height of the peaks. Residues 91 to 126 and residues 229 and 230 were mobile in β-PrP; moreover, this is the same region that is unstructured in α-PrP (3). Thus, most of the rearrangement from α helix to β sheet has probably occurred within the structured region of α-PrP.

Within the structured region of β-PrP we could identify 57 peaks out of an expected 97 in the HSQC spectrum (Fig. 1D). None of these correlated with peak positions in the α-PrP spectrum, indicating no residual contamination from the oxidized form. The intensity ratio of weak and strong peaks within the HSQC spectrum from the unstructured and structured regions of β-PrP was identical to the ratio seen with α-PrP, indicating that a high degree of heterogeneity was unlikely.

The switch from α to β conformation was reversible. When the reduced β form was exposed to a higher pH (8.0), the native α conformation was restored. However, the rates of conversion in either direction were extremely slow, requiring days for completion (15). This high kinetic barrier, however, could be circumvented by fully denaturing and refolding at the appropriate pH to generate either isoform.

The solubility of the two isoforms was not equivalent. The α form of PrP can be titrated with the denaturant guanidine hydrochloride (GuHCl) to determine equilibrium parameters for the folding pathway (4). However, the β form of PrP was also highly soluble in aqueous buffers, whereas titration with GuHCl led to intermolecular associations and a visible precipitate (Fig. 2E). Physiological concentrations of other salts (150 mM NaCl or 150 mM KCl) also caused aggregation, indicating that this was simply an effect of ionic strength. Salt-precipitated material was initially composed of irregular spherical particles (Fig. 3A) that associated over several hours to form fibrils (Fig. 3B) with a diameter of up to 20 nm and lengths up to 500 nm. The morphology and dimensions of these fibrils are within the range described for scrapie-associated fibrils extracted from brains affected by prion disease (16).

Figure 3

β-PrP assembles into fibrils (31). Two forms of protein aggregates are seen with negative stain electron microscopy. (A) The most common form is small (diameter ∼10 nm), irregularly shaped, and seen in all samples. (B) The other form is fibrillar and its proportion increases with incubation. These fibers intertwine with increased time of incubation. Scale bars, 200 nm. For biosafety reasons, electron microscopy was performed on mouse PrP91–231 treated in an identical manner to human protein. (C) β-PrP displays partial PK resistance in monomeric and aggregated states (32). α-PrP is completely digested by PK at 0.5 μg/ml. Using identical conditions in which β-PrP remains soluble and monomeric (5), soluble β-PrP has partial PK resistance, with the majority undigested at 0.5 μg/ml. Aggregated β-PrP exhibits increased PK resistance, with some surviving intact at 5 μg/ml.

As with native PrPC, α-PrP was extremely sensitive to digestion with PK (Fig. 3C). However, β-PrP showed marked protease resistance. This PK resistance was a function of the structural reorganization of the monomeric β form, with only a moderate further increase associated with aggregation (Fig. 3C). The different patterns of proteolytic cleavage fragments seen upon PK digestion of α-PrP and β-PrP provide further evidence of a major conformational rearrangement in β-PrP. In contrast, the partially structured β-sheet conformation of reduced hamster PrP90–231 is fully sensitive to PK digestion (17).

Unusually for a protein with a predominantly helical fold, most of the residues in PrP91–231 have a preference for β conformation. This suggests that PrP is balanced between radically different folds with a high energy barrier between them—one dictated by local secondary structural propensity (the β conformation) and one requiring the precise docking of side chains (the native α conformation). Such a balance would be influenced by mutations causing inherited prion diseases (18). Individuals homozygous for valine at polymorphic residue 129 of human PrP (where either methionine or valine can be encoded) are more susceptible to iatrogenic Creutzfeldt-Jakob disease (19), and valine has a much higher β propensity than methionine. Our results support this hypothesis, because the molecule is capable of slow conversion between α and β conformations. Furthermore, the β form can be locked by intermolecular association, thus supplying a plausible mechanism of propagation of a rare conformational state. The precise subcellular localization of PrPSc propagation remains controversial, although considerable evidence implicates late endosome–like organelles or lysosomes (20–23). This α-PrP to β-PrP conversion, caused by reduction and mild acidification, may be relevant to the conditions that PrPC would encounter within the cell after its internalization during recycling (24). Reduction and acidification within the endosomal pathway is required for activation of diphtheria toxin (25). Such a mechanism could underlie prion propagation and hence could account for the transmitted, sporadic, and inherited etiologies of prion disease. Initiation of a pathogenic self-propagating conversion reaction, with accumulation of aggregated β-PrP, may be induced by exposure to a “seed” of aggregated β-PrP after prion inoculation, or as a rare stochastic conformational change, or as an inevitable consequence of expression of a pathogenic PrPC mutant that is predisposed to form β-PrP.

  • * To whom correspondence should be addressed. E-mail: J.Collinge{at}


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