Prion disease susceptibility is affected by β-structure folding propensity and local side-chain interactions in PrP

Abstract

Prion diseases occur when the normally α-helical prion protein (PrP) converts to a pathological β-structured state with prion infectivity (PrP^Sc). Exposure to PrP^Sc from other mammals can catalyze this conversion. Evidence from experimental and accidental transmission of prions suggests that mammals vary in their prion disease susceptibility: Hamsters and mice show relatively high susceptibility, whereas rabbits, horses, and dogs show low susceptibility. Using a novel approach to quantify conformational states of PrP by circular dichroism (CD), we find that prion susceptibility tracks with the intrinsic propensity of mammalian PrP to convert from the native, α-helical state to a cytotoxic β-structured state, which exists in a monomer–octamer equilibrium. It has been controversial whether β-structured monomers exist at acidic pH; sedimentation equilibrium and dual-wavelength CD evidence is presented for an equilibrium between a β-structured monomer and octamer in some acidic pH conditions. Our X-ray crystallographic structure of rabbit PrP has identified a key helix-capping motif implicated in the low prion disease susceptibility of rabbits. Removal of this capping motif increases the β-structure folding propensity of rabbit PrP to match that of PrP from mouse, a species more susceptible to prion disease.

Prion diseases are a group of fatal, neurodegenerative diseases that include Creutzfeldt–Jakob disease (CJD), Gerstmann–Sträussler–Scheinker syndrome, fatal familial insomnia, and kuru in humans, scrapie in goats and sheep, bovine spongiform encephalopathy (BSE) in cattle, and chronic wasting disease in elk and deer (1). According to the “protein-only” hypothesis, prion diseases are caused by the misfolding of the prion protein (PrP) into a conformation that is pathogenic and infectious (2, 3). PrP exists normally in a monomeric, mostly α-helical state (), and upon prion infection it refolds into an aggregation-prone, mostly β-structured state that is infectious (PrP^Sc).

Although it is difficult to rank the susceptibility of different species to contract prion disease with the limited data available, certain trends are clear. Previous studies of prion transmission in hamsters indicated that this species is highly susceptible to prion diseases, for hamsters develop prion disease when inoculated with various prion isolates from different animal donors, including humans, cows, sheep, mice, mink, and other hamsters (4–7). Mice show comparable prion disease susceptibility (4, 5, 7–10), but rabbits to date have not succumbed to prion disease despite being inoculated with human, sheep, and mouse prions and can resist prion infection for at least 3 y (4, 11). Furthermore, reports from the BSE crisis in the United Kingdom indicate clear differences in BSE susceptibility among larger mammals. Humans and many feline species, including cheetahs, pumas, and domestic cats, were susceptible to the BSE agent, whereas no cases of prion disease were reported in canine or equine species (12, 13). However, because of a lack of experimental data, it is unclear how dogs or horses would fare when challenged with a variety of prion isolates.

Although understanding the determinants of prion susceptibility would yield insights into the mechanism of pathogenesis in prion diseases, the varying susceptibilities of mammalian species has yet to be explained mechanistically. It has been known for a number of years that PrP can form a β-structured state (β-state) under slightly destabilizing conditions (14–16). Hornemann and Glockshuber (15) proposed that the β-state was a monomeric folding intermediate that lay between the native and unfolded state on the PrP folding pathway. Later, Baskakov et al. (17) provided evidence that the β-state was composed of oligomeric forms of PrP and suggested that these assemble after the PrP molecules completely unfold. Subsequent studies showed the β-structured oligomers to be predominantly octameric for both mouse and hamster PrP (18, 19). Whether β-structured monomers of PrP exist is controversial. Although some studies have reported the existence of β-structured monomers (20), other studies suggest the opposite (17). Results reported in the present study demonstrate that slight changes in solution conditions can greatly influence the degree to which β-structured monomers are populated (vide infra). The proposal that the β-state of PrP in general plays a role in the mechanism underlying prion diseases (21) raises the possibility that the propensity to form the β-state is a major determinant of prion disease susceptibility. To date, a quantitative measure of the propensity of PrPs to populate β-structured monomers and octamers has not been available.

We have developed a two-wavelength method of circular dichroism (CD) analysis to quantify the propensity of PrP to transform into the β-state and have applied this, along with analytical ultracentrifugation experiments reporting on the oligomeric state of PrP, to compare hamster, mouse, rabbit, horse, and dog PrP. Using urea and acid treatment to induce structural transitions, we find that the PrP proteins from these five species under specific conditions can each populate four distinct conformational states: native monomers, unfolded monomers, and a β-structured state (β-state) that can be monomeric or octameric. We find that the β-state propensity of PrP from these species varies with their prion disease susceptibility. Using this two-wavelength CD method in conjunction with X-ray crystallography, we have been able to identify a key helix-capping motif that controls the formation of the β-state, which may govern the susceptibility to prion disease.

Results

Urea Denaturation of Hamster PrP 90-231 Produces α-Helical, β-Structured, and Unfolded States.

Purified, recombinant, hamster PrP (9.5 μM) encompassing residues 90–231 (hamster PrP 90-231) was incubated in urea (0–9 M, pH 4, 25 °C, 5 d) and CD measurements were made at 220 nm. When plotted as a function of urea concentration, the mean residue ellipticity of hamster PrP 90-231 shows a biphasic transition (Fig. 1A). The CD spectrum of hamster PrP 90-231 measured at 0, 2.5, and 7.5 M urea (pH 4), respectively, displays a typical α-helical structure, a β-sheet-rich structure, and an unfolded structure (Fig. 1B). By relating the three unique CD spectra of hamster PrP 90-231 in Fig. 1B to the biphasic unfolding curve in Fig. 1A, it is apparent that urea does not cause unfolding of the native state to a partially folded intermediate state, which then completely unfolds. Instead, moderate amounts of urea at pH 4 cause the native α-helical state of hamster PrP 90-231 to convert to a very different, nonnative, β-sheet-rich conformation, and addition of higher amounts of urea results in complete unfolding of this unique β-sheet-rich state. We denote this β-sheet-rich conformation as β-state PrP.

Fig. 1.

Hamster PrP 90-231 adopts three distinct secondary structural states. (A) Urea-unfolding curve of hamster PrP 90-231 at pH 4 monitored by CD at 220 nm. (B) CD spectra of hamster PrP 90-231 in the native (○), β-state (▪), and unfolded (□) conformations at pH 4.

A Monomer–Octamer Equilibrium best Describes the Oligomerization State of Hamster PrP 90-231 in the β-State.

In usual practice, denaturation curves that display a characteristic biphasic shape are fit to a three-state transition model to determine the thermodynamic stabilities of the three stably populated species. However, these models usually assume that the protein remains monomeric at all denaturant concentrations. Therefore, before fitting our CD data we determined the oligomerization states of native, β-state, and unfolded PrP.

Sedimentation data indicated that native (no urea, pH 4.0) and unfolded PrP (7.5 M urea, pH 4.0) were monomeric with a molecular mass of 16 kDa. However, sedimentation analysis indicated that β-state PrP self-associates in a monomer–octamer equilibrium, which was confirmed by analysis of the residuals of the fits to various monomer–oligomer models (SI Appendix, Fig. S1 A and B). Size exclusion chromatography (SEC) (SI Appendix, Fig. S1C) supports the results from sedimentation equilibrium ultracentrifugation. Thus, β-state PrP, unlike native or unfolded PrP, exists as an equilibrium mixture of two species: monomers and octamers.

A Novel Two-Wavelength Method of CD Analysis Allows Measurement of Fractional Concentrations of β-State PrP.

The finding that β-state PrP exists as a mixture of monomers and octamers indicates that a simple three-state model is inadequate to fit the biphasic, urea unfolding curves of PrP. Analysis of the data using a four-state equilibrium model is also not straightforward because various sequential or branched models are equally probable and cannot be discerned from the urea unfolding curves. Furthermore, the greater complexity of the four-state model, with its higher number of parameters, increases the difficulty in differentiating local goodness-of-fit minima in the parameter landscape from the global minimum. For these reasons, we decided to develop an approach that does not make presumptions about the oligomerization state. Our method takes advantage of the fact that β-state PrP possesses a β-sheet CD spectrum that is distinct from both the α-helical native and random coil-like unfolded PrP spectra (Fig. 1B). Because the CD spectrum is a sum of the CD contributions from all protein molecules present in their particular conformations, our observed CD signal (θ_220,obs) measured from a sample can be expressed as follows:

[1]

In the above, [θ]_220,Native, [θ]_220,β-state, and [θ]_220,Unfolded represent the molar ellipticities of native state, β-state, and unfolded state PrP at 220 nm, respectively. The above expression can be simplified by normalizing the CD data to fraction apparent values where the native state and unfolded state have values of 1 and 0, respectively:

[2]

Here, F_app220 is the observed normalized CD signal, z₂₂₀ is the normalized CD signal for β-state PrP at 220 nm, and F_Native and F_β-state are fractional concentrations of the native and β-state PrP, respectively.

To solve for F_β-state, PrP unfolding may be monitored at a second wavelength, allowing generation of a system of two equations. In order to solve for F_β-state, the normalized CD signal z_λ for the β-state at the second wavelength would need to be different from z₂₂₀ and also distinct from the native and the unfolded CD signals. On the basis of the spectra in Fig. 1B, a wavelength of 229 nm fulfills these criteria, yielding the following second equation:

[3]

Combining Eqs. 2 and 3 and solving for F_β-state yields

[4]

Thus, by monitoring urea unfolding of PrP at both 220 and 229 nm, the relative fraction of β-state PrP can be calculated at any given urea concentration and pH value. Note that this two-wavelength method treats the CD spectrum of the native, β-state, and unfolded PrP as a unique signature of that particular state. Procedures that deconvolve CD spectra into percent helix, percent β-sheet, and percent random coil were not used.

Urea Treatment of PrP Causes Conversion of PrP to the β-State at low pH.

Fig. 2 shows the normalized urea unfolding curves of hamster PrP 90-231 at pH values of 7.0, 5.0, 4.5, and 4.0. The CD signals were monitored at wavelengths of 220 and 229 nm, and the data were normalized to F_app. A relationship is observed between the shape of the urea unfolding curves and the difference between the normalized CD signals at the two wavelengths of 220 and 229 nm. At pH 7.0, the urea unfolding curves are monophasic in shape and the curves at the two different wavelengths are superimposable (Fig. 2A). At pH 5.0, some biphasic shape and some differences between the curves monitored at two wavelengths are detectable (Fig. 2B). At pH values of 4.0 and 4.5, the two wavelengths generate strikingly different biphasic curves (Fig. 2 C and D). Because unfolding curves measured at the two wavelengths do not overlay at intermediate urea concentrations and low pH, this gives a qualitative indication that β-state PrP must be significantly populated. Quantification of the amount of β-state PrP is possible with our two-wavelength method.

Fig. 2.

Urea-unfolding curves of hamster PrP 90-231 monitored by CD at 220 (▴) and 229 nm (○) at pH (A) 7.0, (B) 5.0, (C) 4.5, and (D) 4.0. The lines are intended as a guide to the eye.

The Fractional Concentration of β-State PrP from Hamster, Mouse, Rabbit, Horse, and Dog PrP 90-231 Correlates with Prion Susceptibility.

Mouse, rabbit, horse, and dog PrP 90-231 formed monomer–octamer equilibrium mixtures at pH 3.5–4.0 and 2–6 M urea that were similar to hamster PrP 90-231 (SI Appendix, Fig. S2). Urea-induced unfolding of PrP 90-231 constructs of each of these species at pH values of 7.0, 5.0, 4.5, and 4.0 was monitored by CD measurements at 220 and 229 nm (SI Appendix, Figs. S3 and S4). A clear gradation is observed between the five species. Differences between the two wavelength curves are greatest with hamster followed by mouse and then by rabbit, horse, and dog, thus providing an index of β-state concentrations in the five species. Importantly, refolding experiments of these PrP proteins demonstrate that, at pH values of 7.0 and 4.0, unfolding and refolding of the proteins are completely reversible, indicating that equilibrium has been achieved (SI Appendix, Fig. S5).

The value of z₂₂₀–z₂₂₉ was calculated at pH values that rendered a biphasic curve for the PrP constructs; this value was 0.144 ± 0.020 and was the same, within error, for all PrP proteins examined.

By using a value of 0.144 for z₂₂₀–z₂₂₉, the fractional concentrations of β-state PrP were calculated for each of the five species at various urea concentrations and pH values (Fig. 3). At pH 7.0, none of the five PrP proteins populates the β-state (Fig. 3A). At pH 4.0, all five species populate the β-state fraction to some degree (Fig. 3D). At pH 5.0 (Fig. 3B) and 4.5 (Fig. 3C), they show varying fractional concentrations of the β-state. The propensity to form the β-state is greatest for hamster PrP, followed by mouse PrP, and then by rabbit, horse, and dog PrP. This propensity correlates with prion disease susceptibility in these mammals.

Fig. 3.

The propensity to populate β-state PrP correlates with species susceptibility to prion disease. Comparison of the β-state PrP fraction between hamster (red line), mouse (green line), rabbit (blue line), horse (dark yellow line), and dog PrP 90-231 (purple line) as a function of urea concentration at pH (A) 7.0, (B) 5.0, (C) 4.5, and (D) 4.0. The lines are intended as a guide to the eye.

Comparison of the Fraction Octamer with the β-State Fraction: Detection of a Monomeric, β-State PrP Species.

The fractional concentrations of octamer in the various samples can be determined from sedimentation equilibrium data from hamster, mouse, and rabbit PrP 90-231 by fitting the sedimentation data to a double-exponential equation (SI Appendix, Fig. S6). The fraction octamer was calculated at urea concentrations of 2.5, 3.2, 4.0, and 4.5 M for hamster PrP, at 3.7, 4.3, and 5.0 M urea for mouse PrP, and at 3.0, 3.7, and 4.4 M urea for rabbit PrP 90-231, all at pH 4.0 (SI Appendix, Fig. S7). It is clear that the fractional concentration of β-state PrP is significantly higher than the fractional concentration of the octamer. The maximum fraction octamer reaches a value of 70% for hamster PrP (SI Appendix, Fig. S7A), 60% for mouse PrP (SI Appendix, Fig. S7B), and 50% for rabbit PrP (SI Appendix, Fig. S7C). Under these conditions the fractional concentration of β-state PrP is 100% for each of these species. Because sedimentation equilibrium and SEC analysis indicated that only monomers and octamers are present, we conclude that the β-state PrP fraction is a mixture of β-structure-containing monomers and octamers for hamster, mouse, and rabbit PrP 90-231.

To further demonstrate the existence of β-structured monomers, hamster PrP 90-231 was diluted into 3.6 M urea, pH 4.0 and secondary structure and oligomerization status were examined by CD and SEC, respectively. The data show that the reduction in the levels of monomers and the appearance of octamers occur over a period of hours (SI Appendix, Fig. S8A); however, formation of β-sheet structure is complete at the starting point of the experiment (SI Appendix, Fig. S8 B and C). Thus the monomers that are well-populated at early time points are β-structured. Furthermore, the monomeric peak of hamster PrP 90-231 was collected from the size exclusion column, and the CD spectra of the sample indicated that monomers of hamster PrP 90-231 in 4.1 M urea, pH 4.5 were β-structured (SI Appendix, Fig. S8 D–F). SEC of the protein sample after performing the CD scan indicated that hamster PrP 90-231 remained monomeric during this time period, thus providing direct evidence that the monomers are β-structured (SI Appendix, Fig. S8E).

β-State PrP from Mouse PrP 90-231 Is Cytotoxic.

We performed cytotoxicity studies to confirm that β-state PrP has similar toxicity to oligomeric β-structured PrP examined previously (22, 23). We have observed that, once formed, β-state PrP remains stable in solution for at least one week, when the conditions are changed from 4.0 M urea, pH 4.0 to phosphate buffered saline, pH 7.4. This stability of β-state PrP allowed investigation of its cytotoxic properties using differentiated PC12 cells, a common cell culture model of neurons. Native-state or β-state mouse PrP 90-231 were added to differentiated PC12 cells, and the effects of each on cell survival were monitored (Fig. 4). Bee venom mellitin, a known cytotoxin, was used as a positive control. Whereas native-state PrP did not display any toxic properties, β-state PrP was as cytotoxic as bee venom mellitin on a molar basis. These data indicate that these β-state PrP preparations possess potent cytotoxic activity under physiological conditions.

Fig. 4.

β-state PrP from mouse PrP 90-231 is cytotoxic. Toxicity of β-state PrP when exposed to neuronal-differentiated, PC-12 cells. Absorbance readings were taken at 550 nm after performing the sulforhodamine B (SRB) cytotoxicity assay to measure cell viability of cells exposed to no protein (*), β-state PrP of mouse PrP 90-231 (▴), native PrP (▪), and melittin (○). The SRB assay was performed after 4 d cultivation of cells at 37 °C.

X-Ray Crystal Structure of Rabbit PrP 121-230.

To find out whether the structure of natively folded rabbit PrP contains any unique structural elements that might contribute to its resistance to conversion into the β-state, we solved the X-ray crystal structure of rabbit PrP 121-230 to 1.6-Å resolution (see SI Appendix, Table S1 for data collection and refinement statistics) (Fig. 5). Although numerous structures of PrP from various species have been solved by nuclear magnetic resonance spectroscopy, to date X-ray crystal structures are available only for sheep PrP, human PrP (as a domain-swapped dimer), and human PrP bound to an antibody (24–26). These crystal structures are all determined to resolutions of 2.0 Å or poorer.

Fig. 5.

The crystal structure of rabbit PrP^C 126-230. The observed monomeric fold is similar to previously observed structures of the ordered domain of PrP^C. The β2–α2 loop is highlighted in the Inset box.

The asymmetric unit of the rabbit PrP 121-231 crystal structure contains two molecules. The two molecules associate closely along a twofold-symmetry axis that is almost parallel to helix 2 (SI Appendix, Fig. S9). The rmsd between all backbone atoms in the two molecules is 0.38 Å, indicating that the folds of the two molecules are practically identical. They are also very similar to those of other structurally known PrP proteins; for example, the rmsd between the backbone atoms of rabbit PrP 121-230 and sheep PrP^C is 0.80 Å (24).

The loop between β-strand 2 and helix 2 (residues 166–174), sometimes referred to as the β2–α2 loop or rigid loop (27), has been implicated as a possible amyloidogenic motif that forms a steric zipper (28). In the rabbit PrP 121-230 structure, electron density for residues 165–175 is well defined (SI Appendix, Fig. S10), indicating that the region is clearly ordered. The main-chain carbonyls of P165 and V166 form hydrogen bonds with the amides of Q168 and Y169, respectively, generating a short 3₁₀ helix, as seen in previous structures (27, 29–31). The side chains of D167 and Q168 are surface exposed, but that of V166 forms hydrophobic interactions with Y218 of helix 3, possibly contributing to the stability of this loop (SI Appendix, Fig. S11). The 3₁₀ helix preceding the N terminus of helix 2 produces a small kink in the loop before the 171-NQNS-174 sequence.

Side-Chain Interactions Between Residues 171 and 174 Form a Helix-Capping Motif That Affects β-State Propensity of Rabbit PrP.

The amino acid at position 174 (serine in rabbit PrP and asparagine in mouse PrP) is of particular interest because it is known to affect prion susceptibility (32). In rabbit PrP 121-231, the side-chain carbonyl of N171 forms a hydrogen bond with the backbone amide of S174 and, in turn, the side-chain hydroxyl of S174 binds to the backbone carbonyl of N171 (Fig. 6). These reciprocal side-chain-to-backbone-hydrogen bonds, together with the flanking hydrophobic residues Y169 and F175, form a helix-capping motif similar to the so-called “hydrophobic staple” (33, 34), thereby bestowing additional stability upon the N terminus of helix 2. The S174N mutation in this helix-capping motif causes rabbit PrP 121-231 to populate the β-state fraction in 4 M urea, pH 4.5, whereas the wild-type rabbit PrP 121-231 does not populate the β-state under these conditions (SI Appendix, Fig. S12). Disruption of this helix-capping motif increases the β-state propensity of rabbit PrP to match that of PrP from mouse, a species more susceptible to prion disease.

Fig. 6.

The helix-capping motif in rabbit PrP^C. Comparison of residues 170–174 of the rigid loop from rabbit PrP^C structures and the lowest energy structures from the hamster and mouse PrP^C NMR structure ensembles.

Discussion.

Previous studies have reported that PrP can populate β-structured octameric states (18, 19), though there are conflicting reports on the existence of β-structured monomeric states (17, 20) (SI Appendix, Fig. S8). We believe that this discrepancy is caused by slight differences in solution conditions of the different studies. It is well-established that the conformation of PrP is exquisitely sensitive to pH, ionic strength, and denaturant concentration (17–20). A study by Baskakov et al. (17), which did not detect a β-structured monomer, used the relatively strongly destabilizing condition of 200 mM NaCl, 5 M urea, and pH 3.6. Another study by Gerber et al. (20), which did detect a β-structured monomer, used the less destabilizing condition of 200 mM NaCl, 1 M urea, and pH 3.6. We have found that the β-structured monomer can be populated in 50 mM sodium acetate, 74 mM NaCl, 3.6 M urea, and pH 4.0 (SI Appendix, Fig. S8). It appears that the β-structured monomer can be populated only under moderately destabilizing conditions. We suggest that the β-structured monomer is transiently populated to much lower levels under other conditions.

We have investigated whether the propensity to form these β-structured monomeric and octameric states could underlie susceptibility to prion disease. The work presented here provides a unique method to quantify the populations of β-state equilibrium species under a broad range of conditions and to assess the propensity for PrP from a given animal species to populate the β-state, allowing interspecies comparisons. By using a two-wavelength CD method, combined with analytical ultracentrifugation, we were able to measure the fractional concentrations of all conformational states present at equilibrium for several animal PrP proteins. We found that there was a gradation of propensities to populate the monomeric and octameric β-states, with a descending rank order of hamster, mouse, rabbit, horse, and dog PrP. Because PrP from susceptible species (hamsters and mice) had significantly higher β-state propensity than PrP from apparently resistant species (rabbits, horses, and dogs), this shows that β-state propensity varies with prion disease susceptibility.

The fact that PrP can populate a β-structured, nonnative state has been known for a number of years (14–16), and some have suggested that β-structured, oligomeric forms of PrP represent the more toxic and/or infectious form underlying prion diseases (21, 35). Much has been done to map the pathway of conversion, but historically, it has been difficult to quantify the relative abundances of potentially disease-relevant folding intermediates. It has been shown by stopped-flow techniques that prion disease-causing variants of human PrP have larger populations of partially folded, kinetic intermediates than the wild-type protein (36). Our finding that the β-state propensity at equilibrium tracks with prion disease susceptibility across several animal species lends support to the notion that β-state monomers and/or octamers play a prominent role in the mechanism of prion disease.

Contributions of Sequence Versus Structure to Prion Disease Susceptibility.

Early experimental findings on prion susceptibility suggested that primary sequence similarity between the prions from the donor animal and the PrP from the recipient animal plays an important role in determining the outcome of prion transmission (37). For example, wild-type mice show low susceptibility to hamster prions, yet transgenic mice expressing hamster PrP are much more susceptible (38). However, there have been cases where primary sequence similarity does not determine a mammal’s susceptibility to prion strains. Bank voles are more susceptible to human prions than to hamster or mouse prions, despite the fact that there is greater sequence similarity between bank vole PrP and hamster or mouse PrP than between bank vole PrP and human PrP (39). Also, variant CJD isolates from humans infect wild-type mice more readily than the same isolates infect transgenic mice expressing human PrP (40). Furthermore, the fact that many prion strains exist for a single sequence of PrP, and exhibit varying biochemical, histopathological, and neuropathological characteristics when introduced in the same species of mammal, suggests that prion susceptibility involves more than just primary sequence similarity between donor prions and host PrP (41).

Our finding that PrP proteins from hamsters, mice, rabbits, horses, and dogs display a gradation of resistance to adopting the β-state, which correlates with prion susceptibility in these mammals, suggests that the conformational malleability of PrP, which is encoded in the primary sequence, is a determinant of prion susceptibility.

Structural Features of Native Rabbit PrP That May Impede Conformational Transition into the β-State.

Studies have shown that by exposing scrapie-infected mouse neuroblastoma cells to molecules that stabilize PrP^C, such as antibodies (26) or chemical chaperones like trimethylamine N-oxide and dimethylsulfoxide (42), one hinders the conformational transition into the PrP^Sc form, enabling improved survival when cultured cells are exposed to infectious scrapie material. Thus, stabilizing certain aspects of the native structure of PrP^C hinders the conformational transition into the PrP^Sc form. From our studies it appears that the difference in the β-state propensity of the five species studied resides within the covalent structure of PrP. Therefore, certain atomic interactions in the natively folded rabbit PrP structure may stabilize segments of secondary structure that reduce conversion to the β-state.

Structural studies on PrP^C from a variety of mammalian species indicate that single amino acid changes do not have drastic structural effects on the overall fold. Rather, variation in local interactions in the PrP^C monomer may affect the mechanism of conversion to the infectious form. For example, whereas mouse neuroblastoma cells expressing wild-type PrP are susceptible to scrapie infection, an N174S mutant of mouse PrP (the analogous residue in rabbit PrP) is not (32). In our high-resolution crystal structure of rabbit PrP^C 121-231, N171 and S174 are part of a hydrophobic staple-like helix-capping interaction at the N terminus of α-helix 2 (Fig. 6). Such an arrangement is not present in any other known PrP structure. Our demonstration that disruption of this motif through the S174N mutation increases the β-state propensity of rabbit PrP (SI Appendix, Fig. S12) to match that of mouse PrP implicates this motif as a determinant of prion infectivity.

Concluding Remarks.

Our current findings demonstrate that the propensity to form β-state PrP is a valid marker of prion disease susceptibility in hamsters, mice, rabbits, horses, and dogs. This relationship is likely to apply to other species. β-state propensity measurements were used in conjunction with high-resolution structural techniques to identify key amino acid side-chain interactions that affect the conformational transition between helical and β-structured states of PrP—namely the hydrophobic staple capping motif. The methods employed here have the potential to be used to test the efficacy of compounds that may reduce the effective β-state propensity of PrP, and which could therefore be of therapeutic benefit in prion disease.

Materials and Methods

Details of the protein preparation, CD spectroscopy, analytical ultracentrifugation, cytotoxicity assay, and protein crystallization and structure determination are provided in the SI Appendix.

Urea Unfolding of PrP 90-231 Monitored by CD at 220 and 229 nm.

CD measurements were obtained at wavelengths of both 220 and 229 nm; 100 measurements at a rate of 1 measurement per second were averaged. For each pH, equilibrium curves were normalized according to Santoro and Bolen (43). The fraction β-state PrP at each urea concentration was determined by applying Eq. 4. The z parameters in Eq. 4 represent the normalized CD signal of β-state PrP at wavelengths 220 and 229 nm; the values of these critical points were determined graphically (44).

Determination of the Fraction Octamer.

Sedimentation data collected from hamster, mouse, and rabbit PrP 90-231 at pH 4.0 in urea concentrations ranging from 2.5 to 5.0 M were fit to a double-exponential model, where the absorbance of the PrP sample equals the summation of the absorbance of the monomer and the absorbance of the octamer, as a function of radius:

[5]

[6]

where A and B represent proportionality constants and k = [ω²/(2RT)] ∗ [M ∗ (1 - νρ)]; here, ω represents the angular velocity of the rotor (in radians per second), R represents the gas constant (8.314 ∗ 10⁷ erg/mol ∗ K), T represents the temperature in kelvin, M represents the gram molecular weight of the protein, ν represents the partial specific volume of the protein, and ρ represents the density of the solvent. For each sample, the fraction octamer was determined at the radius value at which the sedimentation absorbance readings at speeds ranging from 10,000 to 15,000 rpm intersected with the absorbance readings at 3,000 rpm. Because the absorbance readings at 3,000 rpm represent the absorbance of the sample before protein sedimentation has occurred, the total PrP concentration at this radius equals the concentration prior to any sedimentation. The fraction octamer was calculated by using 3–6 separate datasets.