Only the Two End Helixes of Eight Tandem Amphipathic Helical Domains of Human Apo A-I Have Significant Lipid Affinity

Abstract

Human apolipoprotein A-I (apo A-I) possesses multiple tandem repeating 22-mer amphipathic alpha-helixes. Computer analysis and studies of model synthetic peptides and recombinant protein-lipid complexes of phospholipids have suggested that apo A-I interacts with HDL surface lipids through cooperation among its individual amphipathic helical domains. To delineate the overall lipid-associating properties of apo A-I, the first step is to understand the lipid-associating properties of individual amphipathic helical domains. To this end, we synthesized and studied each of the eight tandem repeating 22-mer domains of apo A-I: residues 44-65, 66-87, 99-120, 121-142, 143-164, 165-186, 187-208, and 220-241. Among the 22-mers, only the N- and C-terminal peptides (44-65 and 220-241) were effective in clarifying multilamellar vesicles (MLVs) of dimyristoylphosphatidylcholine (DMPC). These two peptides also exhibited the highest partition coefficient into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine liposomes, the highest exclusion pressure for penetration into an egg yolk phosphatidylcholine monolayer, and the greatest reduction in the enthalpy of the gel-to-liquid crystalline phase transition of DMPC MLVs. These results suggest that the strong, lipid-associating properties of apo A-I are localized to the N- and C-terminal amphipathic domains. Although each of the eight peptides studied has an amphipathic structure, models based on changes in residual effective amino acid hydrophobicity resulting from differing depths of helix penetration into the lipid are best able to explain the high lipid affinity possessed by the two terminal domains. Differential scanning calorimetry (DSC) studies showed that on a molar basis, apo A-I is about 10 times more effective than the most effective peptide analyzed in reducing the enthalpy of the gel-to-liquid crystalline phase transition of DMPC MLVs. Because previous proteolysis experiments coupled with the present DSC results suggest that the lipid-associating domains of apo A-I are distributed throughout the length of the 243 amino acid residues, we propose that the terminal amphipathic helical domains are involved in the initial binding of apo A-I to the lipid surface to form HDL particles, followed by cooperative binding of the middle six amphipathic helical domains, perhaps aided by salt-bridge formation between adjacent helixes arranged in an antiparallel orientation.

The extreme N‐terminal region of human apolipoprotein A‐I has a strong propensity to form amyloid fibrils

“…The first amyloidogenic segment (residues [14][15][16][17][18][19][20][21][22] in the N-terminal region contains very hydrophobic amino acids (LATVYVDVL), overlapping with the hydrophobic helix-forming region upon lipid binding of apoA-I [26,27]. The deletion of the extreme N-terminal residues 1-43 was shown to destabilize the N-terminal helix bundle structure and induce a large reduction in the enthalpy of lipid binding of full-length apoA-I [17,28], indicating critical roles of the 1-43 segment in the lipid-free conformation and lipid interaction of apoA-I.…”

Conformation and Lipid Binding of a C-Terminal (198−243) Peptide of Human Apolipoprotein A-I

“…A continuous helix repeat assignment describes 10 tandemly repeated helices punctuated mainly by prolines. However, although this continuous helical model is the most widely used one, assignment of the precise secondary structural distribution throughout the apoA-I sequence is far from agreement by different approaches [11,[15][16][17][18][19][20][21][22] Thus, studies of peptides [15,[23][24][25] that model different regions of apoA-I can provide important information on the roles of specific residues and segments, and their interactions in the structure and stability of the parent apolipoprotein. Analysis of the consensus sequences of the A and B repeat derived from the sequences of multiple exchangeable apolipoproteins [7], together with studies of 44-residue consensus sequence peptides, showed ~ 90% and 5 0% α-helical contents for peptides with ABAB [26] and ABBA [27] sequence ordering respectively.…”

Cysteine mutants of human apolipoprotein A-I: a study of secondary structural and functional properties

“…Studies have revealed that the N-terminal region of apoA-I plays an important role in initiating lipid binding and the formation of nascent HDL (3). The central region (residues 144-186) was the key domain for the activation of LCAT (4,5) and was also important for the maturation and stability of HDL (6).…”