Structural Analysis of Missense Mutations on the Stability of APOE3 and APOE4.

Background/Objectives: Apolipoprotein E (APOE) plays a central role in lipid transport and neuronal cholesterol metabolism. Among its three major isoforms (APOE2, APOE3, and APOE4), the APOE4 variant is the strongest genetic risk factor for late-onset Alzheimer's disease (AD). However, the structural consequences of specific APOE mutations on protein stability remain poorly understood. Methods: Here, we performed computational saturation mutagenesis and molecular dynamics simulations on the non-lipidated N-terminal fragments of APOE3 and APOE4 to examine how missense mutations affect their structural stability. Results: Based on the folding energy (ΔΔG) calculations, mutations G165W and L155W were particularly destabilizing in APOE4. Molecular dynamics analyses showed that these mutations altered local flexibility and compactness, particularly within the helix 4 region, a key structural element for maintaining APOE's structural integrity. Conclusions: Our findings, which are state dependent and hypothesis generating, highlight isoform-dependent differences in protein stability and identify regions of structural vulnerability within APOE. These insights enhance our understanding of APOE's conformational dynamics and may inform future studies on its role in neurodegenerative disease mechanisms.