Role of Folding Intermediates in Initiating Aggregation of the Prion Protein

Abstract

The spontaneous conformational conversion of the constitutively expressed, non‐infectious, cellular prion protein, PrPC, from its natively‐folded functional state to infectious, mis‐folded, amyloidogenic, PrPSc forms is the primary cause of most cases of transmissible spongiform encephalopathies (TSEs). TSEs are typically characterized by severe motor‐neuronal and cognitive impairments and progressive dementia accompanied by spongiform degeneration of the mammalian brain. Although the mechanism of this spontaneous conformational conversion process is not known, various studies in vitro have implicated that the conformational flexibility of the native (N) state of the prion protein is a crucial factor in the initiation of aggregation.High conformational flexibility of N states of proteins facilitates fluctuations to aberrant high energy intermediate conformations with altered topology and exposure of hydrophobic surfaces capable of initiating aggregation. However, owing to their transient nature, detection and structural characterization of such aggregation‐prone conformations has been difficult. Nevertheless, such conformations have been shown to initiate aggregation in a few pathogenic and non‐pathogenic proteins. This study aims at understanding the molecular basis of initiation of prion misfolding and aggregation by identifying and structurally characterizing the monomeric precursor conformation which initiates aggregation of the prion protein.In this study, mutagenesis in the hydrophobic core of the globular domain of the mouse prion protein is observed to stabilize a monomeric, on‐pathway, unfolding intermediate conformation, which populates significantly at equilibrium. The rate of misfolding of the protein to cytotoxic β‐structured oligomers exhibiting partial proteinase‐K resistance, which is a hall‐mark feature of PrPSc aggregates, is found to correlate well with the extent to which the intermediate populates. Additionally, the oligomerization process is limited by dimerization of this intermediate suggesting that it is the direct monomeric precursor initiating misfolding and oligomerization. Structural and energetic characterization of the intermediate by native‐state hydrogen exchange monitored by mass spectrometry and NMR indicates that the intermediate is a partially unfolded form of the native state of the protein populated under misfolding‐prone solvent conditions, where the α1‐β2 subdomain is separated from the hydrophobic α2‐α3 subdomain thus exposing the aggregation‐prone α2‐α3 helices to solvent. Kinetic folding studies in such misfolding‐prone solvent conditions using rapid mixing (in μs‐timescales) techniques indicate that the prion protein folds through multiple pathways with the aggregation‐prone intermediate being obligatory to the folding of the protein.Thus, a partially unfolded conformation of the prion protein acts as a common monomeric intermediate between the folding and aggregation of the prion protein. Structural characterization of this intermediate carried out in this study may significantly contribute to the design of drugs to prevent the misfolding and aggregation of the prion protein.Support or Funding InformationFunded by Tata Institute of Fundamental Research and by the Department of Science and Technology, Government of India