The role of apolipoprotein AI domains in lipid binding

Abstract

Apolipoprotein AI (apoAI) is the principal protein constituent of high density lipoproteins and it plays a key role in human cholesterol homeostasis; however, the structure of apoAI is not clearly understood. To test the hypothesis that apoAI is organized into domains, three deletion mutants of human apoAI expressed in Escherichia coli were studied in solution and in reconstituted high density lipoprotein particles. Each mutant lacked one of three specific regions that together encompass almost the entire 243 aa sequence of native apoAI (apoAI ⌬44-126, apoAI ⌬139-170, and apoAI ⌬190-243). Circular dichroism spectroscopy showed that the ␣-helical content of lipid-free apoAI ⌬44-126 was 27% while the other mutants and native apoAI averaged 55 ؎ 2%, suggesting that the missing N-terminal portion contains most of the ␣-helical structure of lipid-free apoAI. ApoAI ⌬44-126 exhibited the largest increase in ␣-helix upon lipid binding (125% increase versus an average of 25% for the others), confirming the importance of the C-terminal half of apoAI in lipid binding. Denaturation studies showed that the N-terminal half of apoAI is primarily responsible for ␣-helix stability in the lipid-free state, whereas the C terminus is required for ␣-helix stability when lipid-bound. We conclude that the N-terminal half (aa 44-126) of apoAI is responsible for most of the ␣-helical structure and the marginal stability of lipid-free apoAI while the C terminus (aa 139-243) is less organized. The increase in ␣-helical content observed when native apoAI binds lipid results from the formation of ␣-helix primarily in the C-terminal half of the molecule.

Impact of Self-association on Function of Apolipoprotein A-I

“…This result is consistent with the notion that self-association of apoA-I is the structural response of an otherwise unstructured C-terminal domain, wherein hydrophobic residues seek protection from solvent exposure by establishing new intermolecular contacts (15,77). Furthermore, the only two self-associated species present in native ⌬W-apoA-I are significantly larger and more stable than the self-associated species of WT-apoA-I.…”

“…2A). The estimated ␣-helical content of WT[High] (ϳ60%) was similar to previously reported values under the same conditions (15,61), whereas the ϳ82% ␣-helical content of ⌬W[High] was significantly higher and comparable with the ␣-helicity of apoA-I on reconstituted discoidal HDL (15,61,62).…”

“…1B), in accordance with the largely accepted notion that self-association of apoA-I is concentration-dependent (15,38). In fact, the single narrow peak at K av ϭ 0.47 ml in the SEC chromatogram of diluted WT[High] (Fig.…”

“…Early studies indicated that lipid-free apoA-I self-associates to form dimers, tetramers, and octamers in a concentration-dependent manner and that simple dilution to Յ0.1 mg/ml disrupts the self-associated species to monomeric apoA-I (15,38,39). However, the actual distribution of apoA-I self-associated species in solution is not known, and their presence has been reported even at 0.1 mg/ml (40).…”

“…Structural elements implicated in apolipoprotein function may involve tertiary and quaternary interactions, such as the four-helix bundle, a structural motif that is essential for function in apolipoprotein (apo) E (5-7) and apoA-I (8 -10). At the quaternary structure level, propensity for self-association is a widespread feature of apolipoproteins that may also affect function (apoE (11)(12)(13), apoA-I (14,15), apoA-II (14)), apoA-IV (16), and apoCs (17)). In the case of apoE, for instance, wherein the apoE4 allele associates with higher incidence of Alzheimer disease (18), the isoform-specific distribution of self-associated species (tetramers, dimers, and monomers) has been shown to contribute to the different lipid-binding efficiencies of the two major isoforms (apoE3 and apoE4) and has been proposed to participate in the mechanism determining the dysfunctional phenotype associated with apoE4 (19 -21).…”