Prion protein (PrP) misfolding is the key trigger in the devastating prion diseases. Yet the sequence and structural determinants of PrP conformation and toxicity are not known in detail. Here, we describe the impact of replacing Y225 in human PrP with A225 from rabbit PrP, an animal highly resistant to prion diseases. We first examined human PrP-Y225A by molecular dynamics simulations. We next introduced human PrP in Drosophila and compared the toxicity of human PrP-WT and Y225A in the eye and in brain neurons. Y225A stabilizes the β2-α2 loop into a 3₁₀-helix from six different conformations identified in WT and lowers hydrophobic exposure. Transgenic flies expressing PrP-Y225A exhibit less toxicity in the eye and in brain neurons and less accumulation of insoluble PrP. Overall, we determined that Y225A lowers toxicity in Drosophila assays by promoting a structured loop conformation that increases the stability of the globular domain. These findings are significant because they shed light on the key role of distal α-helix 3 on the dynamics of the loop and the entire globular domain.

朊病毒蛋白（PrP）错误折叠是毁灭性朊病毒疾病的关键触发因素。然而，PrP 构象和毒性的序列和结构决定因素尚不清楚。在这里，我们描述了用兔 PrP（一种对朊病毒疾病具有高度抵抗力的动物）中的 A225 替换人 PrP 中的 Y225 的影响。我们首先通过分子动力学模拟检查了人类 PrP-Y225A。接下来，我们将人 PrP 引入果蝇中，并比较人 PrP-WT 和 Y225A 在眼睛和脑神经元中的毒性。Y225A 将WT 中鉴定的六种不同构象的β2-α2 环稳定为 3 _{10螺旋，并降低疏水性暴露。}表达 PrP-Y225A 的转基因果蝇在眼睛和脑神经元中表现出较低的毒性，并且不溶性 PrP 的积累较少。总的来说，我们确定 Y225A通过促进结构化环构象来降低果蝇测定中的毒性，从而增加球状结构域的稳定性。这些发现意义重大，因为它们揭示了远端 α 螺旋 3 对环和整个球状结构域动力学的关键作用。