apoE3 N-terminal Domain Research Articles

Analysis of Unfolding of Apolipoprotein E Offers Insights into Lipid Binding Mechanism

Apolipoprotein E (apoE) is a cholesterol transport protein that plays a critical role in lipid homeostasis. It is composed of a series of amphipathic α-helices: H1-H4 in the N-terminal (NT), and C1-C3 in the C-terminal (CT) domain. In humans, those bearing the APOE e4 allele are at a higher risk for developing heart disease and Alzheimer's disease than those with APOE e3 allele. The goal is to understand isoform specific differences in behavior between apoE3 and apoE4. The objectives are to compare their unfolding behavior at the sub-domain level, and, to determine the order of helix unfolding. Recombinant apoE bearing single Cys at defined locations were generated for labeling with fluorophores. Chemical-induced unfolding was monitored by circular dichroism and fluorescence spectroscopy. Fluorescence polarization measurements were carried out to determine changes in probe mobility. With apoE3 NT domain, helices H3 and H4 unfold with a significantly lower mid point of denaturation compared to H1 and H2, whereas with the CT domain, helix C3 offers the least resistance to unfolding compared to C1 and C2. Studies with the intact protein suggest a bi-phasic unfolding for apoE3, where the two domains unfold independently, and a cooperative unfolding for apoE4 possibly due to interaction between helices in the NT and CT domains. Taken together, our studies suggest the order of unfolding of helices: C3-C1/C2-H3/H4-H1/H2. We propose that apoE3 follows a similar order of lipid binding, based on the rationale that the ease of unfolding is reflective of the ease of lipid binding, with less structured segments seeking stability by lipid interaction. Our data are significant because understanding isoform-specific differences at the molecular level allows us to obtain insights into the physiological behavior of apoE in lipid metabolism.

Abstract 586: Ordered Opening of the 4-Helix Bundle in the LDL-Receptor Binding Domain of Human Apolipoprotein E3 as Revealed by Fluorescence Polarization Analysis

Apolipoprotein E3 (apoE3) is an anti-atherogenic protein that helps maintain triglycerides and cholesterol levels in the plasma. It is responsible for binding and cellular uptake of plasma lipoproteins via the low-density lipoprotein receptor (LDLr) family of proteins. It is a highly alpha-helical protein that can exist in lipid-free and lipid-bound states, and undergoes a dramatic conformational re-organization when transitioning between the two states. The objective of this study is to understand the mechanism of chemical-induced unfolding of the 4-helix bundle N-terminal (NT) domain housing the LDLr binding sites. The rationale is that the ease of unfolding is a reflection of ease of helix bundle opening during lipid interaction. We tested the hypothesis that the 4-helix bundle undergoes a concerted opening to reveal the hydrophobic interior, analogous to opening an umbrella. Using X-ray and NMR structure guided rational approach, single cysteine-containing constructs were designed; a fluorescent probe was attached to the free -SH groups to serve as a spectroscopic reporter about the microenvironment and mobility of selected segments of the protein: the 4 main helices observed in the X-ray structure (H1 to H4) and the relatively flexible helixes flanking the helix bundle as observed in the NMR structure. We induced unfolding by chemical denaturation and monitored changes in protein secondary structure and tertiary conformation by circular dichroism and fluorescence spectroscopy, respectively. The overall global was relatively unaltered in the labeled variants; however, fluorescence emission and polarization studies revealed that during unfolding, the microenvironment of the probe on H3 and H4 is more polar and that these helices become more mobile at lower denaturant concentrations, compared to probes on H1 and H2. The data suggest that the 4-helix bundle undergoes a sequential unfolding pattern of the main helices in the following order: helix 4/helix 3, helix 2 and lastly helix 1. Our results offer insights into the mechanism of lipid and lipoprotein binding of apoE3 NT domain, with the segment towards the C-terminal end likely triggering lipid interaction.

Characterization of low density lipoprotein receptor ligand interactions by fluorescence resonance energy transfer

The low density lipoprotein receptor (LDLR) is the prototype of a family of cell surface receptors involved in a wide range of biological processes. A soluble low density lipoprotein receptor (sLDLR) and a tryptophan (Trp)-deficient variant human apolipoprotein E3 (apoE3) N-terminal domain (NT) were used in binding studies. The sole cysteine in apoE3-NT was covalently modified with an extrinsic fluorescence probe, N-(iodoacetyl)-N'-(5-sulfo-1-napthyl)ethylenediamine (AEDANS), and the protein was complexed with lipid. Incubation of sLDLR with AEDANS-Trp-null apoE3-NT dimyristoylphosphatidylcholine (DMPC) disks, but not lipid-free AEDANS-apoE, induced an enhancement in AEDANS fluorescence emission intensity (excitation, 280 nm) consistent with intermolecular energy transfer from excited Trp in sLDLR to receptor-bound apoE. Ligand binding to sLDLR required calcium and was saturable. In competition binding assays, unlabeled apoE3-NT DMPC inhibited AEDANS-apoE DMPC binding to sLDLR more effectively than low density lipoprotein. Fluorescence changes in this system reflected pH-dependent ligand binding and release from sLDLR consistent with models derived from the X-ray crystal structure of the receptor at endosomal pH. Intermolecular energy transfer from excited Trp in LDLR family members to fluorescently tagged ligands represents a sensitive and convenient assay for the characterization of the myriad molecular interactions ascribed to this family of receptor.