Abstract:
The misfolding of the mammalian prion protein from its α-helix rich cellular isoform to its β-sheet rich infectious isoform is associated with several neurodegenerative diseases. The determination of the structural mechanism by which misfolding commences, still remains an unsolved problem. In the current study, native-state hydrogen exchange coupled with mass spectrometry has revealed that the N state of the mouse prion protein (moPrP) at pH 4 is in dynamic equilibrium with multiple partially unfolded forms (PUFs) capable of initiating misfolding. Mutation of three evolutionarily conserved aromatic residues, Tyr168, Phe174, and Tyr217 present at the interface of the β2-α2 loop and the C-terminal end of α3 in the structured C-terminal domain of moPrP significantly destabilize the native state (N) of the protein. They also reduce the free energy differences between the N state and two PUFs identified as PUF1 and PUF2**. It is shown that PUF2** in which the β2-α2 loop and the C-terminal end of α3 are disordered, has the same stability as the previously identified PUF2*, but to have a very different structure. Misfolding can commence from both PUF1 and PUF2**, as it can from PUF2*. Hence, misfolding can commence and proceed in multiple ways from structurally distinct precursor conformations. The increased extents to which PUF1 and PUF2** are populated at equilibrium in the case of the mutant variants, greatly accelerate their misfolding. The results suggest that the three aromatic residues may have been evolutionarily selected to impede the misfolding of moPrP.