The Role of Apolipoprotein A-I Helix 10 in Apolipoprotein-mediated Cholesterol Efflux via the ATP-binding Cassette Transporter ABCA1

Abstract

Recent studies of Tangier disease have shown that the ATP-binding cassette transporter A1 (ABCA1)/apolipoprotein A-I (apoA-I) interaction is critical for high density lipoprotein particle formation, apoA-I integrity, and proper reverse cholesterol transport. However, the specifics of this interaction are unknown. It has been suggested that amphipathic helices of apoA-I bind to a lipid domain created by the ABCA1 transporter. Alternatively, apoA-I may bind directly to ABCA1 itself. To better understand this interaction, we created several truncation mutants of apoA-I and then followed up with more specific point mutants and helix translocation mutants to identify and characterize the locations of apoA-I required for ABCA1-mediated cholesterol efflux. We found that deletion of residues 221-243 (helix 10) abolished ABCA1-mediated cholesterol efflux from cultured RAW mouse macrophages treated with 8-bromo-cAMP. Point mutations in helix 10 that affected the helical charge distribution reduced ABCA1-mediated cholesterol efflux versus the wild type. We noted a strong positive correlation between cholesterol efflux and the lipid binding characteristics of apoA-I when mutations were made in helix 10. However, there was no such correlation for helix translocations in other areas of the protein as long as helix 10 remained intact at the C terminus. From these observations, we propose an alternative model for apolipoprotein-mediated efflux.

Identification of an Apolipoprotein A-I Structural Element That Mediates Cellular Cholesterol Efflux and Stabilizes ATP Binding Cassette Transporter A1

“…Recent studies indicate that deletion of helix 10 (⌬220 -243) and/or helices 9 plus 10 (⌬209 -243) from full-length apoA-I greatly diminishes (ϳ80%) ABCA1-dependent cholesterol efflux (23,24). These published observations are consistent with our findings that the 9/10 helical combination represents a key structure capable of mediating cholesterol efflux via ABCA1.…”

“…These published observations are consistent with our findings that the 9/10 helical combination represents a key structure capable of mediating cholesterol efflux via ABCA1. Extensively truncated forms of apoA-I including deletion mutants ⌬165-220 (deletion of helices 7, 8, and 9) as well as ⌬1-41/⌬185-243 (deletion of the N terminus and helices 8, 9 and 10) exhibit normal cholesterol efflux capability (23,24,27,41). There are at least two possible explanations for these findings.…”

“…This could explain why various helix-deletion mutants of apoA-I stimulate cholesterol efflux in an ABCA1-dependent manner (23).…”

“…Apolipoprotein A-I is a 243-amino acid protein possessing distinct structural domains (21,22). The C-terminal domain (aa 44 -243) of apoA-I is able to mediate cholesterol efflux, indicating that it contains the helical segments responsible for interacting with the ABCA1 transporter (23,24). The amphipathic ␣-helices that define the architecture of the C-terminal domain of apoA-I consist of 11 and 22 amino acids arranged in series and separated by proline residues (21,25).…”

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

“…First, it could form part of the active site that binds apoA-I and transfers lipid, steps that are thought to occur in the lipid bilayer and on the exoplasmic face of the transporter (17). Considering the crystal structures of a variety of bacterial ABC transporters in which the ATP-binding cassettes have been found to fold into similar core structures, this possibility seems remote.…”