Rigidifying the β2−α2 Loop in the Mouse Prion Protein Slows down Formation of Misfolded Oligomers

Transmissible Spongiform Encephalopathies are fatal neurodegenerative diseases caused by the misfolding of the cellular prion protein (PrP^C) into its pathological isoform (PrP^Sc). Efficient transmission of PrP^Sc occurs within the same species, but a species barrier limits interspecies transmission. While PrP structure is largely conserved among mammals, variations at the β2−α2 loop are observed, and even minor changes in the amino acid sequence of the β2−α2 loop can significantly affect transmission efficiency. The present study shows that the introduction of the elk/deer-specific amino acid substitutions at positions 169 (Ser to Asn) and 173 (Asn to Thr) into the mouse prion protein, which are associated with the structural rigidity of the β2−α2 loop, has a substantial impact on protein dynamics as well as on the misfolding pathways of the protein. Native state hydrogen–deuterium exchange studies coupled with mass spectrometry, show that the rigid loop substitutions stabilize not only the β2−α2 loop but also the C-terminal end of α3, suggesting that molecular interactions between these two segments are strengthened. Moreover, the energy difference between the native state and multiple misfolding-prone partially unfolded forms (PUFs) present at equilibrium, is increased. The decreased accessibility of the PUFs from the native state leads to a slowing down of the misfolding of the protein. The results of this study provide important insights into the early events of conformational conversion of prion protein into β-rich oligomers, and add to the evidence that the β2−α2 loop is a key determinant in prion protein aggregation.