APOLIPOPROTEIN-A-I CONFORMATION IN RECONSTITUTED DISCOIDAL LIPOPROTEINS VARYING IN PHOSPHOLIPID AND CHOLESTEROL CONTENT

The effects of the size and cholesterol content on the conformation of apolipoprotein A-I (apoA-I) have been studied in reconstituted discoidal lipoproteins containing two apoA-I per particle (Lp2A-I), The immunoreactivity of a series of 13 epitopes distributed along the apoA-I sequence has been evaluated in Lp2A-I with a phospholipid/apoA-I molar ratio ranging from 31 to 156 and in Lp2A-I with constant phospholipids but varying in cholesterol content from 0 to 22 molecules, The results are compatible with a three domain structure in apoA-I in which the central domain is located between residues 99 and 143 and postulated to be a hinged domain that responds differentially to changes in phospholipid and cholesterol contents, Increasing the phospholipid content results in significant changes of epitope immunoreactivity throughout the N-terminal and central domains of apoA-I with fewer modifications in the C-terminal domain, In contrast, increasing Lp2A-I cholesterol content modifies only the immunoreactivity of two central epitopes, All (residues 99-132) and 5F6 (residues 118-148), and an extreme N-terminal epitope, 4H1 (residues 2-8), Interestingly, the effects of increasing cholesterol or phospholipids on these epitopes are opposite, This suggests a specific effect of cholesterol on the central domain tertiary structure between residues 99 and 143, Competition binding assays among pairs of antibodies binding to apoA-I on Lp2A-I are best explained by invoking inter as well as intramolecular competitions. The specificity of the intermolecular competitions suggests an N to C termini arrangement of the two apoA-I molecules around the disc, Increasing the phospholipid content of Lp2A-I mainly increases the competitions between 3G10 and antibodies binding to most adjacent epitopes, Simultaneously as Lp2A-I enlarges, several of these antibodies also enhance the binding of 3G10. This has been interpreted as evidence of a structural rearrangement of apoA-I as a result of the size increase where the a-helix (residues 99-121) that contains the 3G10 epitope is increasingly interacting with lipids resulting in the enhanced expression of this epitope, The increasing interactions of apoA-I helices with lipids in the enlarging discs are compatible with