Prions are composed solely of the pathological isoform (PrPSc) of the normal cellular prion protein (PrPC). Identification of different PrPSc structures is crucially important for understanding prion biology because the pathogenic properties of prions are hypothesized to be encoded in the structures of PrPSc However, these structures remain yet to be identified, because of the incompatibility of PrPSc with conventional high-resolution structural analysis methods. Previously, we reported that the region between the first and the second α-helix (H1∼H2) of PrPC might cooperate with the more C-terminal side region for efficient interactions with PrPSc 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 PrPSc-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 PrPSc.
Keywords: prion; prion conversion; prion disease; prion protein; protein conformation; protein crosslinking; protein misfolding; protein structure.
© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.