Abstract
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.
Highlights
Pathogenesis in prion disease involves refolding of the host protein, PrP, from the monomeric, primarily a-helical cellular form (PrPC) to a b-sheet enriched, aggregated infectious form (PrPSc) [1]
PrP from hamsters, a species quite susceptible to prion disease, has asparagines at residues 170 and 174 of its amino acid sequence, whereas rabbits, which are less susceptible to prion disease, incorporate serine at the equivalent positions
Space group Cell constants a (A ) b (A ) c (A ) Resolution (A ) Overall reflections Unique reflections Redundancy Completeness (%) R mergeb,I./sI Refinement Statistics Final Rcryst (%) Rfree (%) Solvent (%) No of protein molecules No of all atoms No of water molecules No of sodium ions No of chloride ions Average B-factor (A 2) Ramachandran plot Most favorable R.M.S.D. from ideal geometry Lengths (A ) Angles (u) aValues in parenthesis are for the outer shell. bR = ghklgi|Ii(hkl) – [I(hkl)]|//ghklgiI(hkl), where I(hkl) is the intensity of reflection hkl, ghkl is the sum over all reflections and gi is the sum over i measurements of reflection hkl. cR = ghkl||Fobs| – |Fcalc||/ghkl|Fobs|, where Fobs and Fcalc are the observed and calculated structure-factor amplitutes, respectively
Summary
Pathogenesis in prion disease involves refolding of the host protein, PrP, from the monomeric, primarily a-helical cellular form (PrPC) to a b-sheet enriched, aggregated infectious form (PrPSc) [1]. Mutations in PrP could affect susceptibility and transmission of prion disease by causing changes in the structure of PrPC and/or the mechanism of its conversion to PrPSc. The structure of PrPC from many different species has been determined by X-ray crystallography and NMR spectroscopy [12,13,14,15,16,17], revealing that it is highly conserved between species and the differences in amino acid sequence have little effect on the overall fold. At pH 4.5, all three hamster PrP mutants reach 100% b-state but there are slight differences in the urea concentration at which the b-state begins to form indicating differences in the stability of the PrPC state of these mutants or their ability to form the b-state (Fig. 4D) These results demonstrate that single site mutations in this helix cap motif in PrP affect the b-state propensity PrP, and the mutations have an additive effect
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
