Contributions of the N- and C-Terminal Helical Segments to the Lipid-Free Structure and Lipid Interaction of Apolipoprotein A-I

• Published in: Biochemistry (Easton) Vol. 45; no. 34; pp. 10351 - 10358
• Main Authors: Tanaka, Masafumi, Dhanasekaran, Padmaja, Nguyen, David, Ohta, Shinya, Lund-Katz, Sissel, Phillips, Michael C, Saito, Hiroyuki
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 29.08.2006
• Subjects: Amino Acid Substitution; Apolipoprotein A-I - chemistry; Apolipoprotein A-I - genetics; Apolipoprotein A-I - metabolism; Humans; Lipid Metabolism - genetics; Lipids - chemistry; Point Mutation; Protein Binding - genetics; Protein Structure, Secondary; Protein Structure, Tertiary - genetics; Structure-Activity Relationship; Thermodynamics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi060726t

The tertiary structure of lipid-free apolipoprotein (apo) A-I in the monomeric state comprises two domains:  a N-terminal α-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 α 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 α-helical segment in this region is part of the helix bundle. Calorimetric and gel-filtration measurements showed that disruption of the C-terminal α helix significantly reduced the enthalpy and free energy of binding of apoA-I to lipids, whereas disruption of the N-terminal α 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 α 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 α-helical segment in the N terminus of apoA-I modulates the lipid-free structure and lipid interaction in concert with the C-terminal domain.