Apolipoprotein E3 (apoE3) is an anti-atherogenic apolipoprotein with the ability to exist in lipid-free and lipoprotein-associated states. During atherosclerosis, its function in promoting cholesterol efflux from macrophages via the ATP-binding cassette transporter A1 (ABCA1) takes a prominent role, leading to generation of nascent high density lipoprotein (nHDL) particles. The objective of this study is to understand the conformation adopted by apoE3 in macrophage-generated nHDL using a fluorescence spectroscopic approach involving pyrene. Pyrene-labeled recombinant human apoE3 displayed a robust ability to stimulate ABCA1-mediated cholesterol efflux from cholesterol-loaded J774 macrophages (which do not express apoE), comparable to that elicited by unlabeled apoE3. The nHDL recovered from the conditioned medium revealed the presence of apoE3 by immunoblot analysis. A heterogeneous population of nHDL bearing exogenously added apoE3 was generated with particle size varying from ~ 12 to ~19 nm in diameter, corresponding to molecular mass of ~ 450 to ~700 kDa. The lipid: apoE3 ratio varied from ~60:1 to 10:1. A significant extent of pyrene excimer emission was noted in nHDL, indicative of spatial proximity between Cys112 on neighboring apoE3 molecules similar to that noted in reconstituted HDL. Cross-linking analysis using Cys-specific cross-linkers revealed the predominant presence of dimers. Taken together the data indicate a double belt arrangement of apoE molecules on nHDL. A similar organization of the C-terminal tail of apoE on nHDL was noted when pyrene-apoEA277C(201-299) was used as the cholesterol acceptor. These studies open up the possibility of using exogenously labeled apoE3 to generate nHDL for structural and conformational analysis.

Apolipoprotein E (apoE) (299 residues) is a highly helical protein that plays a critical role in cholesterol homeostasis. It comprises a four-helix bundle N-terminal (NT) and a C-terminal (CT) domain that can exist in lipid-free and lipid-associated states. In humans, there are two major apoE isoforms, apoE3 and apoE4, which differ in a single residue in the NT domain, with apoE4 strongly increasing risk of Alzheimer’s disease (AD) and cardiovascular diseases (CVD). It has been proposed that the CT domain initiates rapid lipid binding, followed by a slower NT domain helix bundle opening and lipid binding to yield discoidal reconstituted high density lipoprotein (rHDL). However, the contribution of the NT domain on the CT domain organization in HDL remains poorly understood. To understand this, we employed Cys-specific cross-linking and spatially-sensitive fluorophores in the NT and CT domains of apoE3 and apoE4, and in isolated CT domain. We noted that the helices in isolated CT domain are oriented parallel to those in the neighboring molecule in rHDL, whereas full length apoE3 and apoE4 adopt either an anti-parallel or hairpin-like organization. It appears that the bulky NT domain determines the spatial organization of its CT domain in rHDL, a finding that has significance for apoE4, which is more susceptible to proteolytic cleavage in AD brains, showing increased accumulation of neurotoxic NT and CT fragments. We envisage that the structural organization of HDL apoE would have profound functional consequences in its ability to regulate cholesterol homeostasis in AD and CVD.

Synthetic model peptides such as GWALP23 (acetyl-GGALW 5 LALALALA LALALW 19 LAGA-amide)provide a useful host framework for investigations of the influence of polar amino acids, for example histidine residues, within the hydrophobic core of a transmembrane helix. Importantly, membranespanning GWALP23 is quite sensitive to single-residue replacements, in part because the transmembrane helix exhibits only limited dynamic averaging of solid-state NMR observables such as the 2 H quadrupolar splitting (Biophys. J. 101, 2939). We inserted His residues into position 12 and/or 13 of GWALP23 (replacing either L12 or A13) and incorporated specific 2 H-Ala labels within the helical core sequence. Solid-state 2 H NMR spectra of GWALP23-H12 reveal a marked difference in peptide behavior between acidic and neutral pH conditions. At neutral pH, GWALP23-H12 exhibits a well-defined tilted transmembrane orientation in both DOPC and DLPC bilayer membranes. To