Apolipoprotein E interacts with amyloid-β oligomers via positively cooperative multivalent binding

Abstract

Interaction of apolipoprotein E (apoE) isoforms with amyloid-{beta} (A{beta}) peptides is considered a critical determinant of the progression of Alzheimers disease. However, molecular mechanism of the apoE-A{beta} interaction is poorly understood. Here we characterize the nature of the apoE-A{beta} complexes and identify the region of apoE that interacts with A{beta}. We have prepared three distinct fragments of apoE4, viz., the N-terminal fragment (NTF), hinge domain fragment (HDF) and C-terminal fragment (CTF) to compare its interactions with A{beta}. Kinetics of aggregation of A{beta} is delayed dramatically in presence of low, substoichiometric concentrations of both NTF and CTF in lipid-free, as well as, in lipidated forms. Effect of HDF is found to be small. Strong inhibition by NTF and CTF at substoichiometric concentrations indicate interactions with the intermediates or the oligomers of A{beta}. Kinetics of Forster Resonance Energy Transfer (FRET) between full-length apoE4 labeled with EDANS at positions 62, 139, 210, 247, and 276 and tetramethylrhodamine (TMR)-labeled A{beta} further support involvement of multiple regions of apoE in the interactions. Since the interactions involve intermediates of A{beta} quantitative evaluation of the binding affinities are not feasible. Hence we employed a competitive binding assay to examine whether the N- and C-terminal domains interact cooperatively. Addition of unlabeled full-length apoE eliminates the FRET between EDANS-NTF + EDANS-CTF and TMR-A{beta} almost completely but not vice versa. Furthermore, full-length apoE but not the equimolar mixture of the fragments could displace the already bound EDANS-apoE molecules from the complexes. Therefore, binding affinity of the A{beta} oligomers to the intact full-length apoE is much higher than the affinity to the domains when mixed together as fragments. Thus, our results indicate that apoE-A{beta} complex formation is mediated by positively cooperative multivalent binding between the multiple sites on apoE and the oligomeric forms of A{beta}.