Probing early misfolding events in prion protein mutants by NMR spectroscopy

Abstract

The post-translational conversion of the ubiquitously expressed cellular form of the prion protein, PrPC, into its misfolded and pathogenic isoform, known as prion or PrPSc, plays a key role in prion diseases. These maladies are denoted transmissible spongiform encephalopathies (TSEs) and affect both humans and animals. A prerequisite for understanding TSEs is unraveling the molecular mechanism leading to the conversion process whereby most α-helical motifs are replaced by β-sheet secondary structures. Importantly, most point mutations linked to inherited prion diseases are clustered in the C-terminal domain region of PrPC and cause spontaneous conversion to PrPSc. Structural studies with PrP variants promise new clues regarding the proposed conversion mechanism and may help identify "hot spots" in PrPC involved in the pathogenic conversion. These investigations may also shed light on the early structural rearrangements occurring in some PrPC epitopes thought to be involved in modulating prion susceptibility. Here we present a detailed overview of our solution-state NMR studies on human prion protein carrying different pathological point mutations and the implications that such findings may have for the future of prion research.

Figure 1

(a) Secondary structure of the immature HuPrP with all the currently identified disease-associated mutations and polymorphisms (unclassified phenotype in gray; fCJD in black; GSS in red; PrP-CAA underlined; FFI in blue; and polymorphisms in green). The mature HuPrP consists of residues 23–231, while the N-terminal and C-terminal signal peptides are cleaved during protein maturation. In the lower panel, the frequency of the mutations and polymorphisms along the PRNP sequence is represented by colored rectangles. (b) Cartoon of the full-length HuPrP with the sequence-based representation of the disordered N-terminus and the structured C-terminal domain from PDB code 2LSB [28]. Up to four Cu²⁺ ions are bound to the OR (residues 60–92) and one Cu²⁺ ions is bound to the non-OR (residues 93–111). (c) Schematic representation of the secondary elements in the C-terminal HuPrP structured domain. The stabilizing long-range interactions were inferred from structural analysis and published data [31,32,33]. Residues involved in disease-influential mutations or polymorphisms are labeled.

Figure 2

Cartoon representations and backbone heavy atom overlays of the different WT and mutants HuPrP folded domains (residues 126–231): WT at pH 5.5 in red (a, PDB code 2LSB), E219K in blue (b, PDB code 2LFT), E200K in light gray (c, PDB code 1FO7), Q212P in black (d, PDB code 2KUN), V210I at pH 5.5 in gray (e, PDB code 2LEJ) and (f) V210I at pH 7.2 in magenta (PDB code 2LV1) superimposed with the WT at pH 7 in green (PDB code 1HJN). The red frame on the WT (a) highlights the β₂-α₂ loop region. Black arrows point at the location of each mutation.

Figure 3

(a) Distances in Å between residues Met166 and Tyr218 within the β₂-α₂ loop and of α₃-helix in different WT and mutant HuPrP structures. Distances were calculated using VMD software [138]. (b) Cartoon representations and backbone heavy atoms overlays of WT at pH 5.5, E219K and V210I at pH 7.2. This peculiar view highlights the different spacing observed among the HuPrP structures between the β₂-α₂ loop and those of α₃-helix. (c) Average values of solvent accessibility (%) for selected residues in different WT and mutant HuPrP structures calculated using GETAREA software [139]. (d) Cartoon representations and backbone heavy atom overlays of WT and Q212P structures. Selected hydrophobic and aromatic residues are highlighted.