NMR studies of human prion protein mutants in solution

Transmissible spongiform encephalopathies (TSEs) are a rare group of rapidly progressive, invariably fatal neurodegenerative diseases affecting humans and animals. A hallmark of TSEs is the conformational conversion of the physiological cellular prion protein, PrPC, into a disease-associated form known as prion or PrPSc. A major focus in prion biology is unraveling the molecular mechanism leading to the structural conversion of PrPC to its pathological form PrPSc. In our recent studies, we tried to understand the early events of the conformational changes leading to PrPSc using as investigative tools point mutations clustered in the open reading frame of the human PrP gene and linked to genetic forms of human prion diseases. We have determined high-resolution solution-state NMR structures of the truncated recombinant human (Hu) PrP (residues 90-231) with pathological Q212P and V210I mutations linked to Gerstmann-Sträussler-Scheinker (GSS) syndrome and genetic Creutzfeldt–Jakob disease (CJD), respectively. In addition, we examined the NMR structure of the recombinant HuPrP (residues 90-231) carrying naturally occurring E219K polymorphism considered to protect against sporadic CJD (sCJD). 3D structures of HuPrP(Q212P), HuPrP(V210I) and HuPrP(E219K) proteins share similar global architecture with the WT HuPrP consisting of highly flexible N-terminal tail and a well-structured C-terminal domain, the latter containing three alpha-helices and a short antiparallel beta-sheet. Detailed inspection revealed that pathological Q212P and V210I mutations introduce some local structural variations with respect to the WT HuPrP which are mostly clustered at the alpha2-alpha3 interhelical interface and in the beta2-alpha2 loop region. Alteration of conformation of the beta2-alpha2 loop region and subsequent higher exposure of hydrophobic residues to solvent may facilitate intermolecular interactions involved in spontaneous generation of PrPSc in genetic TSEs. On contrary to the HuPrP(Q212P) and HuPrP(V210I), the structure of HuPrP(E219K) does not point to interruption of aromatic and hydrophobic interactions in the beta2-alpha2 loop region. Noteworthy, it seems that the beta2-alpha2 loop in HuPrP(E219K) is somewhat better defined in comparison to the same region in the WT protein and proteins with pathological Q212P and V210I mutations. In addition to such subtle structural rearrangements localized within the epitopes critical for prion conversion, replacement of Glu219 by Lys induces significant alteration of surface charge distribution with respect to the WT protein what may further account for protective influence of E219K polymorphism against sCJD.