Detailed structural information on human exchangeable apolipoproteins (apo) is required to understand their functions in lipid transport. Using a series of deletion mutants that progressively lacked different regions along the molecule, we probed the structural organization of lipid-free human apoA-I and the role of different domains in lipid binding, making comparisons to apoE, which is a member of the same gene family and known to have two structural domains. Measurements of ␣-helix content by CD in conjunction with tryptophan and 8-anilino-1-naphthalenesulfonic acid fluorescence data demonstrated that deletion of the amino-terminal or central regions disrupts the tertiary organization, whereas deletion of the carboxyl terminus has no effect on stability and induces a more cooperative structure. These data are consistent with the lipid-free apoA-I molecule being organized into two structural domains similar to apoE; the amino-terminal and central parts form a helix bundle, whereas the carboxyl-terminal ␣-helices form a separate, less organized structure. The binding of the apoA-I variants to lipid emulsions is modulated by reorganization of the helix bundle structure, because the rate of release of heat on binding is inversely correlated with the stability of the helix bundle. Based on these observations, we propose that there is a two-step mechanism for lipid binding of apoA-I: apoA-I initially binds to a lipid surface through amphipathic ␣-helices in the carboxyl-terminal domain, followed by opening of the helix bundle in the amino-terminal domain. Because apoE behaves similarly, this mechanism is probably a general feature for lipid interaction of other exchangeable apolipoproteins, such as apoA-IV.

The ATP-binding cassette transporter A1 (ABCA1) plays a critical role in the biogenesis of high density lipoprotein (HDL) particles and in mediating cellular cholesterol efflux. The mechanism by which ABCA1 achieves these effects is not established, despite extensive investigation. Here, we present a model that explains the essential features, especially the effects of ABCA1 activity in inducing apolipoprotein (apo) A-I binding to cells and the compositions of the discoidal HDL particles that are produced. The apo A-I/ABCA1 reaction scheme involves three steps. First, there is binding of a small regulatory pool of apo A-I to ABCA1, thereby enhancing net phospholipid translocation to the plasma membrane exofacial leaflet; this leads to unequal lateral packing densities in the two leaflets of the phospholipid bilayer. Second, the resultant membrane strain is relieved by bending and by creation of exovesiculated lipid domains. The formation of highly curved membrane surface promotes high affinity binding of apo A-I to these domains. Third, this pool of bound apo A-I spontaneously solubilizes the exovesiculated domain to create discoidal nascent HDL particles. These particles contain two, three, or four molecules of apo A-I and a complement of membrane phospholipid classes together with some cholesterol. A key feature of this mechanism is that membrane bending induced by ABCA1 lipid translocase activity creates the conditions required for nascent HDL assembly by apo A-I. Overall, this mechanism is consistent with the known properties of ABCA1 and apo A-I and reconciles many of the apparently discrepant findings in the literature.