Studies on the transmission of human (Hu) prions to transgenic (Tg) mice suggested that another molecule provisionally designated protein X participates in the formation of nascent scrapie isoform of prion protein (PrP Sc ). We report the identification of the site at which protein X binds to the cellular isoform of PrP (PrP C ) using scrapieinfected mouse (Mo) neuroblastoma cells transfected with chimeric Hu͞MoPrP genes even though protein X has not yet been isolated. Substitution of a Hu residue at position 214 or 218 prevented PrP Sc formation. The side chains of these residues protrude from the same surface of the C-terminal ␣-helix and form a discontinuous epitope with residues 167 and 171 in an adjacent loop. Substitution of a basic residue at positions 167, 171, or 218 also prevented PrP Sc formation: at a mechanistic level, these mutant PrPs appear to act as ''dominant negatives'' by binding protein X and rendering it unavailable for prion propagation. Our findings seem to explain the protective effects of basic polymorphic residues in PrP of humans and sheep and suggest therapeutic and prophylactic approaches to prion diseases.

The scrapie prion protein (PrP Sc ) is the major, and possibly the only, component of the infectious prion; it is generated from the cellular isoform (PrP C ) by a conformational change. N-terminal truncation of PrP Sc by limited proteolysis produces a protein of Ϸ142 residues designated PrP 27-30, which retains infectivity. A recombinant protein (rPrP) corresponding to Syrian hamster PrP 27-30 was expressed in Escherichia coli and purified. After refolding rPrP into an ␣-helical form resembling PrP C , the structure was solved by multidimensional heteronuclear NMR, revealing many structural features of rPrP that were not found in two shorter PrP fragments studied previously. Extensive side-chain interactions for residues 113-125 characterize a hydrophobic cluster, which packs against an irregular ␤-sheet, whereas residues 90-112 exhibit little defined structure. Although identifiable secondary structure is largely lacking in the N terminus of rPrP, paradoxically this N terminus increases the amount of secondary structure in the remainder of rPrP. The surface of a long helix (residues 200-227) and a structured loop (residues 165-171) form a discontinuous epitope for binding of a protein that facilitates PrP Sc formation. Polymorphic residues within this epitope seem to modulate susceptibility of sheep and humans to prion disease. Conformational heterogeneity of rPrP at the N terminus may be key to the transformation of PrP C into PrP Sc , whereas the discontinuous epitope near the C terminus controls this transition.

ABSTRACT

Transgenic (Tg) mouse lines that express chimeric mouse-human prion protein (PrP), designated MHu2M, are susceptible to prions from patients with sporadic Creutzfeldt-Jakob disease (sCJD). With the aim of decreasing the incubation time to fewer than 200 days, we constructed transgenes in which one or more of the nine human residues in MHu2M were changed to mouse. The construct with murine residues at positions 165 and 167 was expressed in Tg(MHu2M,M165V,E167Q) mice and resulted in shortening the incubation time to Ϸ110 days for prions from sCJD patients. The construct with a murine residue at position 96 resulted in lengthening the incubation time to more than 280 days for sCJD prions. When murine residues 96, 165, and 167 were expressed, the abbreviated incubation times for sCJD prions were abolished. Variant CJD prions showed prolonged incubation times between 300 and 700 days in Tg(MHu2M) mice on first passage and incubation times of Ϸ350 days in Tg(MHu2M,M165V,E167Q) mice. On second and third passages of variant CJD prions in Tg(MHu2M) mice, multiple strains of prions were detected based on incubation times and the sizes of the proteaseresistant, deglycosylated PrP Sc fragments. Our discovery of a previously undescribed chimeric transgene with abbreviated incubation times for sCJD prions should facilitate studies on the prion species barrier and human prion diversity.