title: N-terminal helix-cap in α-helix 2 modulates β-state misfolding in rabbit and hamster prion proteins.
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 PrP(C). 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.
authors.affiliation: Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Braden
authors.initials: B
authors.lastname: Sweeting
authors.forename: Eric
authors.initials: E
authors.lastname: Brown
authors.forename: M Qasim
authors.initials: MQ
authors.lastname: Khan
authors.forename: Avijit
authors.initials: A
authors.lastname: Chakrabartty
authors.forename: Emil F
authors.initials: EF
authors.lastname: Pai
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000737:chemistry; D002942:Circular Dichroism; D006224:Cricetinae; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D017096:Prion Diseases / Q000209:etiology; D011328:Prions / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D017510:Protein Folding / Q000187:drug effects; D017433:Protein Structure, Secondary; D011817:Rabbits; D016415:Sequence Alignment; D012694:Serine / Q000737:chemistry; D014508:Urea / Q000494:pharmacology
chemical_list: D011328:Prions; D012694:Serine; D001216:Asparagine; D014508:Urea