Predicted consequences of site-directed mutagenesis and the impact of species variation on prion protein misfolding through the N-terminal domain

Variant Creutzfeldt-Jacob disease (vCJD) is considered to afflict humans through the acquisition of variant isomers and misfolding of the normal cellular prion polypeptide, PrPC. Although the exact mechanism of the misfolding is not been yet clearly understood, this paper provides four additional pieces of evidence in support of the hypothesis that misfolding within PrPC involves N-terminal residues, up to and including Asn(178). Structural predictions for N-terminal residues between Leu(4) and Gly(124) revealed that Leu4-Leu19 might adopt a helical conformation. Furthermore, measurement of C-alpha distance variations, as determined from available NMR solution structures of wild type, as well as the biologically significant Val(166), Asn(170) and Lys(220) variants of PrPC, revealed previously unreported global and local conformational differences may occur in PrPC as a result of these amino-acid substitutions. Notably, three regions, His(140)-Tyr(150) and Met(166)-Phe(175) showed deviations greater than 3 angstrom in their C-alpha-coordinates (cf wild type) indicating that the majority of the N-terminal domain is likely to contribute to the misfolding of PrPC. Minor variations in the orientation of amino acids Thr(193)-Glu(200), located towards the C terminus of the protein, were also noted. This most likely indicates the presence of a hinge mechanism, inherent to a Helix-Loop-helix (HLH) motif formed by amino acids within alpha 2, LIII and alpha 3, in order to accommodate reorientation of the motif in response to misalignment of the N-terminal domain. An unexpected 3 angstrom deviation from the coordinates of the wild type polypeptide, absent from either Val(166), Asn(170) variants was observed over the region Arg(154)-Tyr(155) within the Val(166) form of PrPC. This may contribute to the explanation as to why patients carrying the Val(166) isoform of PrPC may be more susceptible to vCJD.