Abstract
The β2–α2 loop of PrPC is a key modulator of disease-associated prion protein misfolding. Amino acids that differentiate mouse (Ser169, Asn173) and deer (Asn169, Thr173) PrPC appear to confer dramatically different structural properties in this region and it has been suggested that amino acid sequences associated with structural rigidity of the loop also confer susceptibility to prion disease. Using mouse recombinant PrP, we show that mutating residue 173 from Asn to Thr alters protein stability and misfolding only subtly, whilst changing Ser to Asn at codon 169 causes instability in the protein, promotes oligomer formation and dramatically potentiates fibril formation. The doubly mutated protein exhibits more complex folding and misfolding behaviour than either single mutant, suggestive of differential effects of the β2–α2 loop sequence on both protein stability and on specific misfolding pathways. Molecular dynamics simulation of protein structure suggests a key role for the solvent accessibility of Tyr168 in promoting molecular interactions that may lead to prion protein misfolding. Thus, we conclude that ‘rigidity’ in the β2–α2 loop region of the normal conformer of PrP has less effect on misfolding than other sequence-related effects in this region.
Highlights
Gene in humans and animals have been linked to susceptibility to acquired Transmissible spongiform encephalopathies (TSEs) infections[11,12]
There is a significant body of evidence that suggests that the β 2–α 2 loop plays a key role in the disease-associated misfolding of PrP during prion diseases[21,33,34]
The S169N/N173T substitutions were chosen because they produce a ‘rigid loop’ phenotype in PrPC, such that the loop region is structurally well defined in NMR studies[23], whilst in non-‘rigid loop’ prion proteins, including the murine wildtype sequence, the loop conformation is ill-defined as a result of line-broadening during NMR measurements
Summary
We have previously demonstrated that combined in vitro and in silico studies can be used to understand the structural and functional effects of amino acid changes at codon 164 in murine PrP27,28. Since unfolding intermediates had previously been identified at reduced pH using similar methodology, we included additional data points at pH 4.0 to allow us to discern such intermediates, if they were present At both pH 4.0 and 7.0, all recPrP proteins produced urea denaturation profiles which were essentially sigmoidal, as shown, but which differed in terms of the midpoints of the unfolding transitions. Residue 169 of the other protein variants occupy similar areas of the plot, for MoPrP-S169N the polyproline area (− 60, 120) is occupied at the expense of the β -structure area and the double mutant protein, MoPrP-S169N/N173T, does not occupy the left-handed helix area Overall, these data indicate that residues 168–170 of each protein can adopt specific structures that are either elongated or partially helical and that the ensembles of different structures can be distinguished by the φ –ψ angles of residue 169, depicted on Fig. 7(C–F). When residues 168–170 are in a turn-like conformation (residue 169 φ –ψ angles are − 80, 0) the side chain of Tyr[168] interacts with Phe[174], through hydrophobic ring stacking, and the tyrosine hydroxyl interacts
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