The tertiary structure of lipid-free apolipoprotein (apo) A-I in the monomeric state comprises two domains: a N-terminal alpha-helix bundle and a less organized C-terminal domain. This study examined how the N- and C-terminal segments of apoA-I (residues 1-43 and 223-243), which contain the most hydrophobic regions in the molecule and are located in opposite structural domains, contribute to the lipid-free conformation and lipid interaction. Measurements of circular dichroism in conjunction with tryptophan and 8-anilino-1-naphthalenesulfonic acid fluorescence data demonstrated that single (L230P) or triple (L230P/L233P/Y236P) proline insertions into the C-terminal alpha helix disrupted the organization of the C-terminal domain without affecting the stability of the N-terminal helix bundle. In contrast, proline insertion into the N terminus (Y18P) disrupted the bundle structure in the N-terminal domain, indicating that the alpha-helical segment in this region is part of the helix bundle. Calorimetric and gel-filtration measurements showed that disruption of the C-terminal alpha helix significantly reduced the enthalpy and free energy of binding of apoA-I to lipids, whereas disruption of the N-terminal alpha helix had only a small effect on lipid binding. Significantly, the presence of the Y18P mutation offset the negative effects of disruption/removal of the C-terminal helical domain on lipid binding, suggesting that the alpha helix around Y18 concealed a potential lipid-binding region in the N-terminal domain, which was exposed by the disruption of the helix-bundle structure. When these results are taken together, they indicate that the alpha-helical segment in the N terminus of apoA-I modulates the lipid-free structure and lipid interaction in concert with the C-terminal domain.

Although the N-terminal region in human apolipoprotein (apo) A-I is thought to stabilize the lipid-free structure of the protein, its role in lipid binding is unknown. Using synthetic fragment peptides, we examined the lipid-binding properties of the first 43 residues (1-43) of apoA-I in comparison with residues 44-65 and 220-241, which have strong lipid affinity in the molecule. Circular dichroism measurements demonstrated that peptides corresponding to each segment have potential propensity to form alpha-helical structure in trifluoroethanol. Spectroscopic and thermodynamic measurements revealed that apoA-I (1-43) peptide has the strong ability to bind to lipid vesicles and to form alpha-helical structure comparable to apoA-I (220-241) peptide. Substitution of Tyr-18 located at the center of the most hydrophobic region in residues 1-43 with a helix-breaking proline resulted in the impaired lipid binding, indicating that the alpha-helical structure in this region is required to trigger the lipid binding. In contrast, apoA-I (44-65) peptide exhibited a lower propensity to form alpha-helical structure upon binding to lipid, and apoA-I (44-65/S55P) peptide exhibited diminished, but not completely impaired, lipid binding, suggesting that the central region of residues 44-65 is not pivotally involved in the formation of the alpha-helical structure and lipid binding. These results indicate that the most N-terminal region of apoA-I molecule, residues 1-43, contributes to the lipid interaction of apoA-I through the hydrophobic helical residues.