Influence of N-terminal helix bundle stability on the lipid-binding properties of human apolipoprotein A-I.

As the principal component of high-density lipoprotein (HDL), apolipoprotein (apo) A-I plays essential roles in lipid transport and metabolism. Because of its intrinsic conformational plasticity and flexibility, the molecular details of the tertiary structure of lipid-free apoA-I have not been fully elucidated. Previously, we demonstrated that the stability of the N-terminal helix bundle structure is modulated by proline substitution at the most hydrophobic region (residues around Y18) in the N-terminal domain. Here we examine the effect of proline substitution at S55 located in another relatively hydrophobic region compared to most of the helix bundle domain to elucidate the influences on the helix bundle structure and lipid interaction. Fluorescence measurements revealed that the S55P mutation had a modest effect on the stability of the bundle structure, indicating that residues around S55 are not pivotally involved in the helix bundle formation, in contrast to the insertion of proline at position 18. Although truncation of the C-terminal domain (Δ190-243) diminishes the lipid binding of apoA-I molecule, the mutation S55P in addition to the C-terminal truncation (S55P/Δ190-243) restored the lipid binding, suggesting that the S55P mutation causes a partial unfolding of the helix bundle to facilitate lipid binding. Furthermore, additional proline substitution at Y18 (Y18P/S55P/Δ190-243), which leads to a drastic unfolding of the helix bundle structure, yielded a greater lipid binding ability. Thus, proline substitutions in the N-terminal domain of apoA-I that destabilized the helix bundle promoted lipid solubilization. These results suggest that not only the hydrophobic C-terminal helical domain but also the stability of the N-terminal helix bundle in apoA-I are important modulators of the spontaneous solubilization of membrane lipids by apoA-I, a process that leads to the generation of nascent HDL particles.