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.
Susceptibility of a particular species to prion disease is affected by small differences in the sequence of PrP and correlates with the propensity of its PrP to assume the β-state. A helix-cap motif in the β2−α2-loop of native α-helical rabbit PrP, a resistant species, contains sequence differences that influence intra- and interspecies transmission. To determine the effect the helix-cap motif on β-state refolding propensity, we mutated S170N, S174N, and S170N/S174N of the rabbit PrP helix-cap to resemble that of hamster PrP and conversely, N170S, N174S, and N170S/N174S of hamster PrP to resemble the helix-cap of rabbit PrP. High-resolution crystal structures (1.45–1.6 Å) revealed that these mutations ablate hydrogen-bonding interactions within the helix-cap motif in rabbit PrPC. They also alter the β-state-misfolding propensity of PrP; the serine mutations in hamster PrP decrease the propensity up to 35%, whereas the asparagine mutations in rabbit PrP increase it up to 42%. Rapid dilution of rabbit and hamster into β-state buffer conditions causes quick conversion to β-state monomers. Kinetic monitoring using size-exclusion chromatography showed that the monomer population decreases exponentially mirrored by an increase in an octameric species. The monomer-octamer transition rates are faster for hamster than for rabbit PrP. The N170S/N174S mutant of hamster PrP has a smaller octamer component at the endpoint compared to the wild-type, whereas the kinetics of octamer formation in mutant and wild-type rabbit PrP are comparable. These findings demonstrate that the sequence of the β2−α2 helix-cap affects refolding to the β-state and subsequently, may influence susceptibility to prion disease.
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