Identifying Key Components of the PrPC-PrPSc Replicative Interface

In prion disease, direct interaction between the cellular prion protein (PrPC) and its misfolded disease-associated conformer PrPSc is a crucial, although poorly understood step promoting the formation of nascent PrPSc and prion infectivity. Recently, we hypothesized that three regions of PrP (corresponding to amino acid residues 23–33, 98–110, and 136–158) interacting specifically and robustly with PrPSc, likely represent peptidic components of one flank of the prion replicative interface. In this study, we created epitope-tagged mouse PrPC molecules in which the PrP sequences 23–33, 98–110, and 136–158 were modified. These novel PrP molecules were individually expressed in the prion-infected neuroblastoma cell line (ScN2a) and the conversion of each mutated mouse PrPC substrate to PrPSc compared with that of the epitope-tagged wild-type mouse PrPC. Mutations within PrP 98–110, substituting all 4 wild-type lysine residues with alanine residues, prevented conversion to PrPSc. Furthermore, when residues within PrP 136–140 were collectively scrambled, changed to alanines, or amino acids at positions 136, 137, and 139 individually replaced by alanine, conversion to PrPSc was similarly halted. However, other PrP molecules containing mutations within regions 23–33 and 101–104 were able to readily convert to PrPSc. These results suggest that PrP sequence comprising residues 98–110 and 136–140 not only participates in the specific binding interaction between PrPC and PrPSc, but also in the process leading to conversion of PrPSc-sequestered PrPC into its disease-associated form.