Apolipoprotein E4 exhibits intermediates with domain interaction

Abstract

ApoE4(C112R) is the strongest risk factor for Alzheimer's disease, while apoE3(C112) is considered normal. The C112R substitution is believed to alter the interactions between the N-terminal (NTD) and the C-terminal domain (CTD) leading to major functional differences. Here we investigate how the molecular property of the residue at position 112 affects domain interaction using an array of C112X substitutions with arginine, alanine, threonine, valine, leucine and isoleucine as 'X'. We attempt to determine the free energy of domain interaction (∆G INT) from stabilities of the NTD (∆G NTD) and CTD (∆G CTD) in the full-length apoE, and the stabilities of fragments of the NTD (∆G NTF) and CTD (∆G CTF), using the relationship, ∆G INT = ∆G NTD + ∆G CTD - ∆G NTF - ∆G CTF. We find that although ∆G NTD is strongly dependent on the C112X substitutions, ∆G NTD - ∆G NTF is small. Furthermore, ∆G CTD remains nearly the same as ∆G CTF. Therefore, ∆G INT is estimated to be small and similar for the apoE isoforms. However, stability of domain interaction monitored by urea dependent changes in interdomain Forster Resonance Energy Transfer (FRET) is found to be strongly dependent on C112X substitutions. ApoE4 exhibits the highest mid-point of denaturation of interdomain FRET. To resolve the apparently contradictory observations, we hypothesize that higher interdomain FRET in apoE4 in urea may involve 'intermediate' states. Enhanced fluorescence of bis-ANS and susceptibility to proteolytic cleavage support that apoE4, specifically, the NTD of apoE4 harbor 'intermediates' in both native and mildly denaturing conditions. The intermediates could hold key to the pathological functions of apoE4.