Disulfide-crosslink scanning reveals prion-induced conformational changes and prion strain-specific structures of the pathological prion protein PrP.

Prions are composed solely of the pathological isoform (PrP) of the normal cellular prion protein (PrP). Identification of different PrP structures is crucially important for understanding prion biology because the pathogenic properties of prions are hypothesized to be encoded in the structures of PrP However, these structures remain yet to be identified, because of the incompatibility of PrP with conventional high-resolution structural analysis methods. Previously, we reported that the region between the first and the second α-helix (H1∼H2) of PrP might cooperate with the more C-terminal side region for efficient interactions with PrP From this starting point, we created a series of PrP variants with two cysteine substitutions (C;C-PrP) forming a disulfide-crosslink between H1∼H2 and the distal region of the third helix (Ctrm). We then assessed the conversion capabilities of the C;C-PrP variants in N2a cells infected with mouse-adapted scrapie prions (22L-ScN2a). Specifically, Cys substitutions at residues 165, 166, or 168 in H1∼H2 were combined with cysteine scanning along Ctrm residues 220-229. We found that C;C-PrPs are expressed normally with glycosylation patterns and subcellular localization similar to WT PrP, albeit differing in expression levels. Interestingly, some C;C-PrPs converted to protease-resistant isoforms in the 22L-ScN2a cells, but not in Fukuoka1 prion-infected cells. Crosslink patterns of convertible C;C-PrPs indicated a positional change of H1∼H2 toward Ctrm in PrP-induced conformational conversion. Given the properties of the C;C-PrPs reported here, we propose that these PrP variants may be useful tools for investigating prion strain-specific structures and structure-phenotype relationships of PrP.