Single-molecule spectroscopy of Apolipoprotein E reveals a complex conformational ensemble.

Abstract

The ε4-allele variant of Apolipoprotein E (ApoE4) is the strongest genetic risk factor for Alzheimer's disease, though it only differs from its neutral counterpart ApoE3 by a single amino acid substitution. While ApoE4 influences the formation of plaques and neurofibrillary tangles, the structural determinants of its pathogenicity remain undetermined due to limited structural information. Previous studies have led to conflicting models of the C-terminal region positioning with respect to the N-terminal domain across isoforms, largely due to the potential contribution of data representing heterogenous oligomers in solution. Here, we apply a combination of single-molecule spectroscopy and molecular dynamics simulations to construct an atomically-detailed model of monomeric ApoE4 and probe the effect of lipid association. Importantly, our approach overcomes previous limitations by allowing us to work at picomolar concentrations where only the monomer is present. Our data reveal that ApoE4 is far more disordered and extended than previously thought and retains significant conformational heterogeneity after binding lipids. We further investigate the impact of key mutations by inserting serine residues that mimic the ApoE3 and ApoE2 isoform sequence differences, C112S and C112S C158S, respectively. Comparing the proximity of the N- and C-terminal domains across the three major isoforms (ApoE4, ApoE3, and ApoE2) suggests that all maintain heterogenous conformations in their monomeric form, with ApoE2 adopting a slightly more compact ensemble. Inspection of local conformational changes across different domains, in the context of the full-length protein, reveals that the major difference across isoforms occurs in the conformations adopted by the C-terminal tail. Overall, these data provide a new foundation for understanding how ApoE differs from non-pathogenic and protective variants of the protein.