Mass Spectrometry-Based Structural Proteomics and Analytical Ultracentrifugation Characterize the Structures of...

Apolipoprotein E4 (ApoE4), one of the three common isoforms of ApoE, differs from ApoE3 and ApoE2 by just one amino acid substitution each. Yet these changes in ApoE4 profoundly impact protein structure and function to make it the strongest genetic risk factor for late-onset Alzheimer's disease (AD). ApoE4 exhibits a tendency to form oligomers (e.g., tetramers) that complicate high-resolution structural characterization in aqueous environments. Very little is known about the self-associated oligomeric structures. In this study, we employed ultracentrifugation, native mass spectrometry (MS), and fast photochemical oxidation of proteins (FPOP), a mass spectrometry-based protein footprinting approach, to investigate conformational differences among wild-type ApoE4 and two of its mutant variants ApoE4(V236E) (Jacksonville, JV) and ApoE4(V236E/L268E) (VL). These substitutions shift the oligomeric equilibrium toward dimeric and monomeric states, respectively, and allow the tetramer state to be compared with the dimer and monomer. Comparative footprinting reveals that conformational changes driven by these mutations result in altered solvent accessibility in key regions of the protein, suggesting disruption of interfaces responsible for oligomerization and altered access of lipid-binding regions. Given ApoE4's central role in lipid metabolism and its altered interaction with lipid surfaces compared to other isoforms, these structural changes likely modulate lipid binding affinity and may contribute to its pathological role in AD. Our findings highlight the importance of conformational states in ApoE4 function and provide new insights into the structural basis of its disease-associated behavior.