Disease-associated F198S Mutation Increases the Propensity of the Recombinant Prion Protein for Conformational Conversion to Scrapie-like Form

Abstract

The critical step in the pathogenesis of transmissible spongiform encephalopathies (prion diseases) is the conversion of a cellular prion protein (PrP c ) into a protease-resistant, ␤-sheet rich form (PrP Sc ). Although the disease transmission normally requires direct interaction between exogenous PrP Sc and endogenous PrP C , the pathogenic process in hereditary prion diseases appears to develop spontaneously (i.e. not requiring infection with exogenous PrP Sc ). To gain insight into the molecular basis of hereditary spongiform encephalopathies, we have characterized the biophysical properties of the recombinant human prion protein variant containing the mutation (Phe 198 3 Ser) associated with familial Gerstmann-Straussler-Scheinker disease. Compared with the wild-type protein, the F198S variant shows a dramatically increased propensity to self-associate into ␤-sheet-rich oligomers. In a guanidine HClcontaining buffer, the transition of the F198S variant from a normal ␣-helical conformation into an oligomeric ␤-sheet structure is about 50 times faster than that of the wild-type protein. Importantly, in contrast to the wildtype PrP, the mutant protein undergoes a spontaneous conversion to oligomeric ␤-sheet structure even in the absence of guanidine HCl or any other denaturants. In addition to ␤-sheet structure, the oligomeric form of the protein is characterized by partial resistance to proteinase K digestion, affinity for amyloid-specific dye, thioflavine T, and fibrillar morphology. The increased propensity of the F198S variant to undergo a conversion to a PrP Sc -like form correlates with a markedly decreased thermodynamic stability of the native ␣-helical conformer of the mutant protein. This correlation supports the notion that partially unfolded intermediates may be involved in conformational conversion of the prion protein.

Human prion proteins with pathogenic mutations share common conformational changes resulting in enhanced binding to glycosaminoglycans

“…Both wild-type and mutant rPrP tend to aggregate in vitro (37,38), and aggregation of rPrP is pH-, concentration-, and agedependent. At 1 g/ml rPrP, the concentration we used in all capture ELISA experiments, no rPrP aggregate is detected by using the aggregation-specific ELISA (at least a dimer) (39) or by native, nondenaturing PAGE (25) (additional results not shown).…”

Molecular Dynamics as an Approach to Study Prion Protein Misfolding and the Effect of Pathogenic Mutations

“…Apart from the fact that PrP Sc has a significantly increased β-sheet content and decreased α-helical content [7,19,20], little is known about its precise conformation from experiment. The conversion from PrP C to PrP Sc appears to be triggered by a decrease in pH [21,22], introduction of mutations [23,24] and by the presence of PrP Sc [25].…”

From conversion to aggregation: Protofibril formation of the prion protein

“…A theoretical approach provides a means to predict the elusive infectious forms of PrP Sc . Because conversion can be triggered by mutations (21,22) and low pH levels (23), molecular dynamics (MD) simulations should be able to model the required environment for conversion. Consequently, we aim to map the molecular basis of conversion by means of realistic simulations of PrP in solution and to develop reasonable models for aggregated forms of PrP Sc based on the MD-generated converted form.…”