Helix Orientation of the Functional Domains in Apolipoprotein E in Discoidal High Density Lipoprotein Particles

Abstract

Human apolipoprotein E (apoE) mediates high affinity binding to the low density lipoprotein receptor when present on a lipidated complex. In the absence of lipid, however, apoE does not bind the receptor. Whereas the x-ray structure of lipid-free apoE3 N-terminal (NT) domain is known, the structural organization of its lipidassociated, receptor-active conformation is poorly understood. To study the organization of apoE amphipathic ␣-helices in a lipid-associated state, single tryptophan-containing apoE3 variants were employed in fluorescence quenching studies. Apolipoprotein E (ApoE) is a key regulator of plasma cholesterol homeostasis. Its interactions with the low density lipoprotein receptor (LDLR) 1 family and cell surface heparan sulfate proteoglycans (1-3) are a critical step for the cellular uptake of apoE-containing lipoproteins. Transgenic mice overexpressing apoE manifest decreased plasma cholesterol levels on chow diet and a marked resistance to hypercholesterolemia on a high cholesterol/fat diet (4). On the other hand, apoE-deficient subjects display features of type III hyperlipoproteinemia (5) and apoE null mice exhibit massive accumulation of remnant lipoproteins (6), documenting the direct relevance of apoE in lipoprotein metabolism. However, structural features of lipidassociated apoE responsible for its receptor interaction properties are not understood at the molecular level. ApoE comprises two independently folded structural and functional domains that are linked by a protease-sensitive loop segment. The globular 22 kDa N-terminal (NT) domain houses the LDLR recognition site, whereas the 10 kDa Cterminal (CT) domain bears high affinity lipoprotein-binding and self-association sites (7,8). The NT domain is composed of four elongated amphipathic ␣-helices organized as an upand-down helix bundle in the absence of lipid (9, 10). Helix 4 of the NT domain (residues 131-164) harbors key residues required for interaction with lipoprotein receptors (1). However, lipid association is a requirement for apoE (full-length or NT domain) to display receptor recognition capability, with apoE bound to model lipid particles displaying receptor binding ability comparable with that of native apoE-containing lipoproteins (11). Upon interaction with lipid it has been proposed that the NT domain helix bundle "opens" to expose the hydrophobic faces of its amphipathic helices to potential lipid surface binding sites, thereby achieving a receptor-active conformation (1, 12). Monolayer surface balance studies at the air/water interface provide evidence that the NT domain occupies a larger surface area than can be accounted for by its globular 4-helix bundle conformation (13), consistent with adoption of an "open" conformation. Subsequent fluorescence energy transfer analysis revealed that helical segments in the NT domain realign as a function of the transition from lipid-free helix bundle to lipid-bound state (12,14). Attenuated total reflectance Fourier-transformed infrared spectroscopic studies (15) indicate that ap...

The Adipophilin C Terminus Is a Self-folding Membrane-binding Domain That Is Important for Milk Lipid Secretion

“…The calculated helical content of ADPH(172-425) in aqueous solution was 53%. Its degree of helicity increased to 61% after incubation in 50% trifluoroethanol, suggesting that unstructured regions of the recombinant protein may have additional helix formation potential in a membrane-like environment (36). These initial experiments support our homology model and demonstrate that ADPH(172-425) is an independently folding structure with significant ␣-helical nature.…”

Apolipoprotein E isoform-specific binding to the low-density lipoprotein receptor

“…In addition, complexing with DMPC resulted in formation of discrete lipid particles with diameters in the range of 18 nm. Based on previous studies, it is known that these particles exist as disk-shaped phospholipid bilayers wherein two or more apoE-NT molecules circumscribe the disk perimeter through interaction with phospholipid fatty acyl chains [16,17].…”

Examination of Lipid-bound Conformation of Apolipoprotein E4 by Pyrene Excimer Fluorescence

“…We conclude that excimer fluorescence arose from intramolecular proximity between R61C and E255C in the context of reconstituted lipoprotein particles. In recent investigations, several lines of evidence indicated formation of discoidal phospholipid bilayer complexes of DMPC/apoE3, with the ␣-helices circumscribing the periphery of the particles (30,49,50). Furthermore, examination of VLDL-and HDL-bound pyr-R61C/E255C/apoE4 revealed excimer fluorescence (Fig.…”

“…7). In previous studies, it was suggested that apoE3 undergoes a dramatic conformational alteration upon lipid interaction, involving movement of the two domains away from each other (30,56), accompanied by further opening of the helix bundle (49,57) to yield a fully extended molecule circumscribing the periphery of discoidal bilayer of phospholipids (50). In the case of apoE4, we propose that a similar movement of the two domains about the hinge region occurs; however, spatial proximity between Arg-61 and Glu-255 is re-established upon lipid interaction, yielding a looped-back arrangement of helices.…”