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

Prions are composed solely of the pathological isoform (PrP^Sc) of the normal cellular prion protein (PrP^C). Identification of different PrP^Sc structures is crucially important for understanding prion biology because the pathogenic properties of prions are hypothesized to be encoded in the structures of PrP^Sc However, these structures remain yet to be identified, because of the incompatibility of PrP^Sc 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^C might cooperate with the more C-terminal side region for efficient interactions with PrP^Sc 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^Sc-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^Sc.