Agarwal Sonya, Döring Kristina, Gierusz Leszek A, Iyer Pooja, Lane Fiona M, Graham James F, Goldmann Wilfred, Pinheiro Teresa J T, Gill Andrew C
The Roslin Institute and R(D)SVS, University of Edinburgh, Easter Bush Veterinary Centre, Roslin, Edinburgh EH25 9RG, UK.
School of Life Sciences, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, UK.
Sci Rep. 2015 Oct 22;5:15528. doi: 10.1038/srep15528.
The β2-α2 loop of PrP(C) is a key modulator of disease-associated prion protein misfolding. Amino acids that differentiate mouse (Ser169, Asn173) and deer (Asn169, Thr173) PrP(C) 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.
朊蛋白（PrP）野生型（PrP(C)）的β2-α2环是疾病相关朊病毒蛋白错误折叠的关键调节因子。区分小鼠（Ser169、Asn173）和鹿（Asn169、Thr173）PrP(C)的氨基酸在该区域似乎具有显著不同的结构特性，并且有人提出与环的结构刚性相关的氨基酸序列也赋予了对朊病毒疾病的易感性。使用小鼠重组朊蛋白，我们发现将173位残基从Asn突变为Thr只会轻微改变蛋白质稳定性和错误折叠，而在密码子169处将Ser变为Asn会导致蛋白质不稳定，促进寡聚体形成并显著增强纤维形成。与任一单突变体相比，双突变蛋白表现出更复杂的折叠和错误折叠行为，提示β2-α2环序列对蛋白质稳定性和特定错误折叠途径具有不同的影响。蛋白质结构的分子动力学模拟表明，Tyr168的溶剂可及性在促进可能导致朊病毒蛋白错误折叠的分子相互作用中起关键作用。因此，我们得出结论，PrP正常构象的β2-α2环区域中的“刚性”对错误折叠的影响小于该区域中其他与序列相关的影响。
