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 inDrosophilaand 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 inDrosophilaassays 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.