ABCA1 and amphipathic apolipoproteins form high-affinity molecular complexes required for cholesterol efflux

Abstract

Apolipoproteins, such as apolipoprotein A-I (apoA-I), can stimulate cholesterol efflux from cells expressing the ATP binding cassette transporter A1 (ABCA1). The nature of the molecular interaction between these cholesterol acceptors and ABCA1 is controversial, and models suggesting a direct protein-protein interaction or indirect association have been proposed. To explore this issue, we performed competition binding and chemical cross-linking assays using six amphipathic plasma proteins and an 18 amino acid amphipathic helical peptide. All seven proteins stimulated lipid efflux and inhibited the cross-linking of apoA-I to ABCA1. Cross-linking of apoA-I to ABCA1 was saturable and occurred at high affinity ( K d of 7.0 ؎ 1.9 nM), as was cross-linking of apoA-II. After binding to ABCA1, apoA-I rapidly dissociated (half-life of 25 min) from the complex and was released back into the medium. A mutant form of ABCA1 (W590S) that avidly binds apoA-I but fails to promote cholesterol efflux released apoA-I with similar kinetics but without transfer of cholesterol to apoA-I. Thus, a highaffinity, saturable, protein-protein interaction occurs between ABCA1 and all of its amphipathic protein ligands. Dissociation of the complex leads to the cellular release of cholesterol and the apolipoprotein. However, dissociation is not dependent on cholesterol transfer, which is a clearly separable event, distinguishable by ABCA1 mutants.

Influence of ApoA-I Structure on the ABCA1-mediated Efflux of Cellular Lipids

“…Summary-Our results with apoA-I mutants support the two-step model for ABCA1-mediated lipid efflux proposed by Freeman, Zannis, and colleagues (24,25) while providing more insight into the lipidation process (see Fig. 8).…”

“…The latter interaction is required for apoA-I to acquire and/or retain PL and FC to create nascent HDL particles. Recent evidence (24,25) indicates that apoA-I can form a high affinity complex with ABCA1 and that this is the first step in a two-step process of FC and PL efflux via ABCA1. The binding to ABCA1 is not very specific and apparently involves interactions of amphipathic helices with a hydrophobic site on the transporter (25).…”

“…Recent evidence (24,25) indicates that apoA-I can form a high affinity complex with ABCA1 and that this is the first step in a two-step process of FC and PL efflux via ABCA1. The binding to ABCA1 is not very specific and apparently involves interactions of amphipathic helices with a hydrophobic site on the transporter (25). The second step involves lipidation of apoA-I and release of the nascent HDL particles into the extracellular medium.…”

“…The ability of apoA-I to bind lipids is likely to be important in both the acquisition of PL and FC via ABCA1 and in the retention of these lipids to form stable HDL particles. Importantly, hydrophobic interactions between apoA-I and ABCA1 are also likely to be significant in the overall process of PL and FC efflux (24,25).…”

ATP-binding Cassette Transporter A1 Contains a Novel C-terminal VFVNFA Motif That Is Required for Its Cholesterol Efflux and ApoA-I Binding Activities

“…We have previously elucidated the ABCA1 transporter's topology and characterized some of its interactions with apoA-I using this approach (11,12). In our ongoing effort to define the structure/function relationships that underlie the ABCA1 efflux mechanism, we focused on a truncation mutant that we had originally identified in a patient with Tangier disease (8).…”