ABCA1 is an extracellular phospholipid translocase

Abstract

Production of high density lipoprotein (HDL) requires ATP-binding cassette transporter A1 (ABCA1) to drive phospholipid (PL) from the plasma membrane into extracellular apolipoprotein A-I. Here, we use simulations to show that domains of ABCA1 within the plasma membrane remove PL from the membrane's outer leaflet. In our simulations, after the lipid diffuses into the interior of ABCA1's outward-open cavity, PL extracted by the gateway passes through a ring-shaped domain, the annulus orifice, which forms the base of an elongated hydrophobic tunnel in the transporter's extracellular domain. Engineered mutations in the gateway and annulus strongly inhibit lipid export by ABCA1 without affecting cell-surface expression levels. Our finding that ABCA1 extracts lipid from the outer face of the plasma membrane and forces it through its gateway and annulus into an elongated hydrophobic tunnel contrasts with the alternating access model, which proposes that ABCA1 flops PL substrate from the inner leaflet to the outer leaflet of the membrane. Consistent with our model, ABCA1 lacks the charged amino acid residues in the transmembrane domain found in the floppase members of the ABC transporter family.

Fig. 1. Details of POPC translocation from the outward-facing transmembrane cavity of ABCA1 through the gateway/annulus complex into the elongated hydrophobic cavity.

The outward-facing transmembrane cavity, TMD-1 and TMD-2 (orange), ECD-1 (peach), and ECD-2 (light blue) and the elongated hydrophobic tunnel (green) were identified in the structure of ABCA1. The elongated hydrophobic tunnel in ECD-1 was demarcated using PyMol cavity algorithm. A Gateway (residues 564–592, magenta) and annulus (residues 69, 71–80, 363, 368–379, cyan). The annulus forms the bottom of the elongated hydrophobic tunnel. Shown is the pathway for diffusion of POPC from the outer leaflet of the membrane bilayer (blue lines) into the outward-facing transmembrane cavity (yellow arrow). B Final frame after 2 µs simulation of the ECDs and TMDs in a POPC bilayer (all-atom model). Five POPC molecules (termed the membrane mound, yellow space-filling) diffused from the outer membrane leaflet into the outward-facing transmembrane cavity (yellow arrow, A) and then displaced ~10–12 Å above the plane of the bilayer into the outward-facing transmembrane cavity. The gateway is the 29-residue charged loop of ECD-1 that binds the membrane mound in CGMD simulations. This loop is contiguous with the outward-facing transmembrane cavity on one side and the annulus of the elongated hydrophobic tunnel. C Location in the gateway of four of the eight Tangier disease point mutations identified in the ECD-1 of human ABCA1. D–F SMD Freeze-frames of the translocation of a single POPC molecule up to and through the annulus and partway into the elongated hydrophobic tunnel. The gateway and annulus are colored magenta and cyan, respectively. D The 1.9 μsec all-atom frame from the 10 μsec CGMD simulation of ABCA1 embedded in a POPC bilayer was the starting structure. The annulus orifice (residues 73-75, 77, 78, 371, 375) is shown in white. E The POPC was translocated by SMD to the annulus orifice. F The POPC then was translocated halfway through the annulus orifice. This process required significant energy in the SMD (Supplementary Fig. 4). To be energetically favorable in vivo, we propose that the outward-facing transmembrane cavity would close, likely from an ATP-dependent process, forcing the PL through a modified orifice.

Fig. 2. Structural details of the gateway/annulus complex.

A, B The backbone of the gateway (magenta). A The basic (blue, stick) and acidic (red, stick) amino acid residues of the gateway (residues 564–592, magenta) from the ABCA1 structure determined by cryo-EM. The three amino acid residues in the gateway known to be mutated in Tangier disease (four point mutations in total) are shown in the space-filling mode. B The POPC molecule (yellow stick) forms salt-bridges to residues D571 and K568 (space-filling red and blue, respectively) in the 1.9 µsec frame during coarse-grained molecular dynamics (CGMD) modeling of unmutated ABCA1. All side chains but residues D571 and K568 are magenta stick. C The gateway (magenta)/annulus (cyan) complex. Residues of the elongated hydrophobic tunnel that lie within 10 Å of any gateway residue form the annulus domain (residues 69, 71–80, 363, and 368–379). D The annulus (cyan) viewed from the side opposite the outward-facing transmembrane cavity. Note the small orifice (residues 73-75, 77, 78, 371, 375, colored white) in the middle of the annulus through which magenta-colored residues of the gateway on the opposite side are visible. E The base of the annulus (D) rotated 180° around the y-axis to show the gateway. The view is from the outward-facing transmembrane cavity. F Representation of the annulus with the gateway removed to display the annulus orifice. G The amino acid composition of the gateway/annulus complex. Acidic residues, rose; basic residues, blue; aromatic residues, magenta; hydrophobic residues, orange; prolines, yellow; neutral residues, green; glycines, white.

Fig. 3. Charged amino acids in the gateway promote PL extraction and lipid export by ABCA1.

A Extraction of POPC molecules from the membrane bilayer into the gateway by wild-type and mutated ABCA1. Final frames of the three 10 μsec CGMD simulations showing the gateway (magenta, cartoon), annulus (cyan, cartoon), and annulus orifice (white, space-filling) for different ABCA1 monomers inserted into a POPC bilayer. The ABCA1 monomers are wild-type (WT, left panel), K568A (middle panel), and 11CmutA (mutation of all 11 charged residues to alanine, right panel), respectively. The position of the upper monolayer surface in each image is shown by orange space-filling phosphorus atoms. The single POPC molecule extracted by the ABCA1 monomer of WT and K568A are shown in yellow (space-filling). The 11CmutA monomer failed to extract POPC (yellow, space-filling). Left panel, K568 is space-filling blue, E584 is space-filling red and D581 and D585 are stick red; middle panel, K568A is space-filling white, D571 is space-filling red. B Effects of gateway mutations K568 and 11CmutA on PL and cholesterol efflux by ABCA1. Wild-type, K568A, 11CmutA, D581K/E584K/D585K, F583K/W590E, and Y573F ABCA1 were expressed in BHK cells. PL and cholesterol efflux by the cells were quantified after incubation with APOA1 or an equal weight of L-4F, an APOA1-peptide mimetic. Lipid efflux and ABCA1 expression were quantified as described in Methods. Relative cholesterol and phospholipid efflux are normalized to WT and are presented as mean ± SD of three independent experiments with three replicates per experiment. Source data are provided as a Source Data file.

Fig. 4. Sequential interactions with gateway residues as an extracted POPC molecule translocates from the external transmembrane cavity to the annulus orifice.

A–C Salt-bridge formation of acidic amino acid residues D575, E584, and D585 with the N(CH)₃⁺ headgroup moiety of POPC as the lipid molecule moves from the monolayer toward the annulus during MD simulation. Gateway, green cartoon with attached stick amino acid residues (aromatic residues, magenta); annulus, cyan space-filling; annulus orifice, white space-filling; extracted POPC, yellow space-filling; salt bridged amino acid residues, space-filling; location of outer monolayer, space-filling phosphorus atoms. D–F Changes in the conformation of the Y573/W574 aromatic cluster shield the PO₄⁻ and acyl side chains of POPC during translocation of the lipid molecule from the monolayer toward the annulus during MD simulation. G, H Van der Waals contacts of POPC upon its approach to the annulus orifice during SMD simulation of the translocation of POPC. G Protein in cartoon mode. H Protein in space-filling mode. Color code: annulus orifice, white; gateway—aromatic residues, magenta; basic residues, blue; acidic residues, red; hydrophobic residues, orange; other residues, green.

Fig. 5. PL and cholesterol efflux by WT ABCA1 and ABCA1 with mutations in the annulus and annulus orifice.

WT and mutated ABCA1s (I74K/I371E, I74C/I371C, I371C/L375C, I371C, V304C/V308C) expressed in BHK cells were incubated for 4 h at 37 °C with APOA1 or an equal weight of the APOA1-mimetic peptide, L-4F. PL and cholesterol efflux, and total and cell-surface ABCA1 expression, were determined as described in Methods. Relative cholesterol and phospholipid efflux are normalized to WT and are presented as mean ± SD of three independent experiments with three replicates per experiment. Source data are provided as a Source Data file.

Fig. 6. The structures of ATP-free ABCA1 determined by cryo-EM and ATP-bound ABCA1 based on homology modeling with ABCA4.

A Cartoon of the cryo-EM determined NBDs, cytoplasmic regulatory domains (RDs), TMDs, gateway and lipid annulus of ATP-free ABCA1. POPC has diffused into the outward-facing transmembrane cavity from the outer cell membrane monolayer. B Surface representation of the portion of ABCA1 shown in panel A. PC extracted by the gateway is pulled from the outer leaflet of the plasma membrane. C Surface representation of ATP-bound ABCA1 derived from a homology model of ATP-bound ABCA4 (the power stroke). The outward-facing cavity of ABCA1 is in the outward-closed conformation. Closure of the cavity drives extracted lipid through the annulus into the elongated hydrophobic tunnel. The annulus orifice opens fully during the power stroke initiated by ATP-binding. D–G Structural differences in the annulus and annulus orifice of ATP-free ABCA1 determined by cryo-EM and ATP-bound ABCA based on homology modeling with ABCA4. D, F The annulus, gateway and TMDs in the ATP-free and ATP-bound forms of ABCA1, respectively. Annulus, space-filling cyan; gateway, magenta cartoon; TMD1-2, space-filling green. E, G The annulus orifice in the ATP-free and ATP-bound forms of ABCA1, respectively. Note the difference in size of the orifices in the two forms of ABCA1. H, I Space-filling and cartoon figures, respectively, of the annulus (cyan) and orifice (white) of the 29.0 ns frame of a SMD simulation. Note the 8.1 Å; Cα distance—I74 to I371—between the two helices. J, K Cartoon and space-filling figures, respectively, of the annulus (cyan) and annulus orifice (white) from the outwardly closed ATP-bound ABCA4-homology model. Note the 13.8 Å Cα distance—I74 to I371—between the two helices.

Fig. 7. Structural features of ABCA1 and ABCA4 may contribute to their transporting substrates in opposite directions.

Our model for substrate transport by ABCA1 proposes that the negatively charged phosphate group of PL in the outer leaflet of the membrane initially interacts with the positively charged residue K568. It then moves through the gateway (A, magenta) and into the elongated hydrophobic tunnel. The ATP-free structure of ABCA4 exhibits a structure analogous to the gateway (B, magenta). An extended loop, termed the S-loop (green, space-filling) because it binds N-retinylidene-phosphatidylethanolamine (B, yellow space-filling), resides adjacent to the gateway of ABCA4. The location of the S-loop in the outward-facing transmembrane cavity of ABCA4 may prevent movement of N-retinylidene-phosphatidylethanolamine into the gateway of ABCA4.

Fig. 8. Basic residue distribution in the TMDs of ABCA1 and six established lipid flippases.

The locations of tryptophan residues (space-filling magenta) are used to mark the membrane interfaces. In A, B, lysine residues and arginine residues are pale blue and dark blue, respectively. In C, intramembrane lysine residues and arginine residues are cyan. A ATP-free human ABCA1 (PDB entry 5XJY). B ATP-free S. Typhimurium MsbA (PDB entry 6BL6). C Six aligned structures of MsbA orthologs from three different bacterial species (PDB entries 2HYD, 3B5Z, 3B60, 5TTP, 5TV4, and 6BPP)^–.