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The human apolipoprotein (apo) E4 isoform, which differs from wild-type apoE3 by the single amino acid substitution C112R, is associated with elevated risk of cardiovascular and Alzheimer’s diseases, but the molecular basis for this variation between isoforms is not understood. Human apoE is a two-domain protein comprising an N-terminal helix bundle and a separately folded C-terminal region. Here, we examine the concept that the ability of the protein to bind to lipid surfaces is influenced by the stability (or readiness to unfold) of these domains. The lipid-free structures and abilities to bind to lipid and lipoprotein particles of a series of human and mouse apoE variants with varying domain stabilities and domain–domain interactions are compared. As assessed by urea denaturation, the two domains are more unstable in apoE4 than in apoE3. To distinguish the contributions of the destabilization of each domain to the greater lipid-binding ability of apoE4, the properties of the apoE4 R61T and E255A variants, which have the same helix bundle stabilities but altered C-terminal domain stabilities, are compared. In these cases, the effects on lipid-binding properties are relatively minor, indicating that the destabilization of the helix bundle domain is primarily responsible for the enhanced lipid-binding ability of apoE4. Unlike human apoE, mouse apoE behaves essentially as a single domain, and its lipid-binding characteristics are more similar to those of apoE4. Together, the results show that the overall stability of the entire apoE molecule exerts a major influence on its lipid- and lipoprotein-binding properties.

Although the partitioning of apolipoprotein A-I (apoA-I) molecules in plasma between high-density lipoprotein (HDL)-bound and -unbound states is an integral part of HDL metabolism, the factors that control binding of apoA-I to HDL particles are poorly understood. To address this gap in knowledge, we investigated how the properties of the apoA-I tertiary structure domains and surface characteristics of spherical HDL particles influence apoA-I binding. The abilities of (14)C-labeled human and mouse apoA-I variants to associate with human HDL and lipid emulsion particles were determined using ultracentrifugation to separate free and bound protein. The binding of human apoA-I (243 amino acids) to HDL is largely mediated by its relatively hydrophobic C-terminal domain; the isolated N-terminal helix bundle domain (residues 1-190) binds poorly. Mouse apoA-I, which has a relatively polar C-terminal domain, binds to human HDL to approximately half the level of human apoA-I. The HDL binding abilities of apoA-I variants correlate strongly with their abilities to associate with phospholipid (PL)-stabilized emulsion particles, consistent with apoA-I-PL interactions at the particle surface being important. When equal amounts of HDL2 and HDL3 are present, all of the apoA-I variants partition preferentially to HDL3. Fluorescence polarization measurements using Laurdan-labeled HDL2 and HDL3 indicate that PL molecular packing is looser on the more negatively charged HDL3 particle surface, which promotes apoA-I binding. Overall, it is clear that both apoA-I structural features, especially the hydrophobicity of the C-terminal domain, and HDL surface characteristics such as the availability of free space influence the ability of apoA-I to associate with HDL particles.

Apolipoprotein (apo) E is thought to undergo conformational changes in the N-terminal helix bundle domain upon lipid binding, modulating its receptor binding activity. In this study, site-specific fluorescence labeling of the N-terminal (S94) and C-terminal (W264 or S290) helices in apoE4 by pyrene maleimide or acrylodan was employed to probe the conformational organization and lipid binding behavior of the N- and C-terminal domains. Guanidine denaturation experiments monitored by acrylodan fluorescence demonstrated the less organized, more solvent-exposed structure of the C-terminal helices compared to the N-terminal helix bundle. Pyrene excimer fluorescence together with gel filtration chromatography indicated that there are extensive intermolecular helix-helix contacts through the C-terminal helices of apoE4. Comparison of increases in pyrene fluorescence upon binding of pyrene-labeled apoE4 to egg phosphatidylcholine small unilamellar vesicles suggests a two-step lipid-binding process; apoE4 initially binds to a lipid surface through the C-terminal helices followed by the slower conformational reorganization of the N-terminal helix bundle domain. Consistent with this, fluorescence resonance energy transfer measurements from Trp residues to acrylodan attached at position 94 demonstrated that upon binding to the lipid surface, opening of the N-terminal helix bundle occurs at the same rate as the increase in pyrene fluorescence of the N-terminal domain. Such a two-step mechanism of lipid binding of apoE4 is likely to apply to mostly phospholipid-covered lipoproteins such as VLDL. However, monitoring pyrene fluorescence upon binding to HDL(3) suggests that not only apoE-lipid interactions but also protein-protein interactions are important for apoE4 binding to HDL(3).