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

Abstract

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.

Conformation-dependent influences of hydrophobic amino acids in two in-register parallel β-sheet amyloids, an α-synuclein amyloid and a local structural model of PrPSc

“…For example, the region between the first and the second α -helices of PrP (residues 155– 177), which is likely an interface between the substrate PrP C and template PrP Sc on the C-terminal side, 43 may also form a β -arch. In a disulfide-crosslink scanning experiment, the conversion efficiencies of the series of mutant mouse PrPs into PrP Sc forms by RML/Chandler scrapie suggest that the side chains of residues 165 and 166 (in mouse PrP numbering) are directed outward from a β -arch, forming a loop; 44,45 this supports our view. However, because hydrophobic residues are sparse while there are many asparagine residues in the region, hydrogen bonds might be more important for reinforcement of the β -arch, rather than for hydrophobic effects.…”

Analysis of the electrophoretic profiles of prion protein in carcinous and pericarcinous lysates of six different types of cancers

“…PrP dimer is observable in normal brain tissues, in some cultured mammalian cells and even in the prokaryotic lysates expressing recombinant PrP [11]. As the smallest aggregate, PrP dimer can be disulfide-bonded or non-disulfide linked [12]. Based on the recombinant human PrP proteins, they even illustrate that three motifs with PrP peptide modulate PrP dimerization, the negative motif of residues 36-42, and positive ones of residues 90-125 and residues 195-212 [13].…”

Prion Protein Strain Diversity and Disease Pathology

“…Although significant advancements have been made in comprehending the phenomenon of prion strains, many pieces of information are still missing, most important among them is the definitive evidence for the structural differences between prion strains and the relationship between the strain-specific properties of PrPSc and the resulting phenotype of disease [48,49].…”