Large disk intermediate precedes formation of apolipoprotein A-I-dimyristoylphosphatidylcholine small disks.

Apolipoprotein A-I (apoA-I) is the major protein component of high-density lipoproteins (HDL). The well-known antiatherogenic properties of HDL can be partially attributed to its ability to remove cholesterol from peripheral tissues for transport to liver for excretion and metabolism, in the reverse cholesterol transport pathway (1;2). ApoA-I is reported to adopt three physiological distinct conformational states: lipid-free, lipid-poor, and lipid-bound (3) . Lipid-free and lipid-poor apoA-I exist in plasma, where they play a physiological role in both cellular cholesterol efflux and lecithin cholesterol acyl transferase activity (4). In vivo, the lipidation of apoA-I is dependent upon cellular lipid efflux mediated by ABCA1 that leads to the formation of nascent discoidal HDL, which can undergo maturation into spherical HDL through additional cellular and plasma activities (5;6). The molecular mechanism by which ABCA1 transforms lipid-free apoA-I into a nascent lipoprotein is not known. A large body of work exists concerning the in vitro formation and structure of discoidal apoA-I phospholipid complexes. The cholate dialysis method can be used to make reconstituted high-density lipoprotein (rHDL) discs, and this method works for various lipids including the physiologically common phospholipids and phospholipid/cholesterol mixtures (7;8). ApoA-I can also spontaneously form stable lipoprotein discoidal complexes in vitro when incubated with dimyristoyl phosphatidylcholine (DMPC) dispersions or liposomes, but not when incubated with the physiologically prominent longer acyl chain phospholipids (9;10). Despite the limitation of this process to the non physiological DMPC, this is the only model that exists in which apoA-I can readily extract phospholipids from a lipid bilayer to form discoid particles that are similar to nascent HDL. The interaction of apoA-I and DMPC to produce nascent HDL-like particles has been studied extensively (7). This includes the interaction of apoA-I with different forms of DMPC, such as multilamellar dispersions (10–13), giant unilamellar vesicles (14), and small unilamellar vesicles (15;16). The nature and the size of apoA-I/DMPC complexes depend on the DMPC to apoA-I ratio, ranging from large vesicles (1000:1) to large discoidal complexes (300:1) and small discoidal complexes (≤100:1), with the large and small discs containing three and two apoA-I molecules, respectively (17). The binding of apoA-I to DMPC dispersions is a very rapid process which takes only a few minutes, while the formation of disc takes several hours at 25°C (16). Analysis of the kinetic data has led to the suggestion that the association of apoA-I with DMPC is thermodynamically stable only near the temperature of lipid phase transition, whereas beyond this region the stability of the complex is determined by kinetic factors (18). Jonas reported that no intermediates were observed when they investigated the interaction of apoA-I with DMPC vesicles, at molar ratios of lipid to protein from 4000:1 down to 50:1 (15). The goal of this study was to investigate the mechanism by which apoA-I binds to and transforms lipid bilayers into discrete apoA-I lipid complexes. To accomplish this we used DMPC large unilamellar vesicles (LUVs) as our model system, since apoA-I cannot transform more physiological lipid bilayers into complexes in vitro without the use of detergents that destroy the lipid bilayer. We discovered the existence of an intermediate large disc that precedes the appearance of small discs. The intermediate ~11 nm large disc fraction was isolated by gel filtration and found to contain three apoA-I molecules. These large discs are fairly stable, but upon incubation with additional apoA-I, they mature into the small ~8.5 nm discs with two molecules of apoA-I. The reaction of apoA-I with DMPC LUVs was monitored by fluorescence resonance energy transfer, and two phases were observed, supporting the formation of an intermediate during the generation of small discs. The lipid dynamics of LUVs and discs were assayed by of diphenylhexatriene (DPH) anisotropy and pyrene excimer formation, and suggest that apoA-I binding to the LUVs constrains the lipid in domains, and that lipid fluidity decreases as large discs mature into small discs. Our data support the model that the formation of apoA-I lipid discs can proceed through remodeling of a larger intermediate into a smaller disc, which may provide insight into the mechanism of HDL biogenesis in vivo.