Rigidifying\nthe β2−α2 Loop in the\nMouse Prion Protein Slows down Formation of Misfolded Oligomers

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