Cryo-EM structures of human ABCA7 provide insights into its phospholipid translocation mechanisms

Abstract

Phospholipid extrusion by ABC subfamily A (ABCA) exporters is central to cellular physiology, although the specifics of the underlying substrate interactions and transport mechanisms remain poorly resolved at the molecular level. Here we report cryo-EM structures of lipid-embedded human ABCA7 in an open state and in a nucleotide-bound, closed state at resolutions between 3.6 and 4.0 Å. The former reveals an ordered patch of bilayer lipids traversing the transmembrane domain (TMD), while the latter reveals a lipid-free, closed TMD with a small extracellular opening. These structures offer a structural framework for both substrate entry and exit from the ABCA7 TMD and highlight conserved rigid-body motions that underlie the associated conformational transitions. Combined with functional analysis and molecular dynamics (MD) simulations, our data also shed light on lipid partitioning into the ABCA7 TMD and localized membrane perturbations that underlie ABCA7 function and have broader implications for other ABCA family transporters.

Keywords: ABCA7; Alzheimer's disease; cryo-EM; exporter; flippase.

Figure 1. Functional characterization and structure of human ABCA7 incorporated in nanodiscs

1. A, B

   (A) ATPase activity of ABCA7 at different ATP concentrations and (B) different nanodisc phospholipid/cholesterol compositions. Cholesterol (20%) presence is indicated by + or −. Experimental replicates (n) = 3 and error bars represent standard deviation (s.d.).

2. C, D

   (C) Cryo‐EM map of ABCA7_BPL (yellow) at 3.6 Å resolution and (D) ABCA7_PE at 4.0 Å resolution (purple). Density for protein is shown in yellow (0.025 contour) and that for modeled lipid acyl chains (0.035 contour) shown in pink (TMD luminal lipids) and white (peripherally associated lipids).

3. E

   ABCA7_PE shown in ribbon format with half 1 colored yellow and half 2 purple, along with density for TMD lipids (pink 0.025 contour) and bound nucleotide (yellow 0.035 contour). Acyl chains and glycans are shown as pink and gray sticks, respectively.

4. F

   TMD lumen with density for TMD lipids (pink 0.025 contour) viewed from the extracellular side (top), membrane plane (middle) and cytoplasmic side (bottom). C_α atoms for the cytoplasmic gate are shown as black spheres.

5. G

   View of the TMD‐ECD interface with select residues oriented toward lipids (transparent red spheres) shown.

Figure EV1. Variation in local resolution and TMD electrostatics

1. Local resolution maps of ABCA7_BPL and ABCA7_PE structures. Color keys are indicated on the left of each set of maps with numbers representing resolution (Å).

2. Analysis of TMD electrostatics of ABCA7_PE with electrostatic potential maps for TMD1 (left) and TMD2 (right) shown along with modeled lipids (pink sticks).

Figure EV2. Purification and characterization of ABCA7 and ABCA7_AAA

1. SEC chromatograms and SDS‐PAGE analysis of ABCA7 and ABCA7_AAA in DDM/CHS.

2. SDS‐PAGE gel and ATPase activity comparison of ABCA7 and ABCA7_AAA reconstituted in liposomes. Experimental replicates (n) = 6 and error bars represent standard deviation (s.d.). Statistical significance by unpaired, two‐tailed t‐test P‐value of < 0.0001 is indicated by ****.

3. Detection of endogenous lipids co‐purified with ABCA7 and ABCA7_AAA by thin‐layer chromatography (TLC).

Source data are available online for this figure.

Figure 2. Cryo‐EM structure of ABCA7_DIGITONIN

1. Cryo‐EM map of human ABCA7_DIGITONIN at 3.9 Å with density for protein shown in green (0.013 contour) and extraneous density likely belonging to detergent shown in pink (0.013 contour).

2. Cryo‐EM structure of human ABCA7_DIGITONIN shown in ribbon format with each half colored differently (blue and green).

3. TMD lumen of ABCA7_DIGITONIN with density for bulk micelle shown in white at 0.015 contour where detergent density inside TMDs disappears. Sticks for unmodeled detergent molecules are shown for which density is visible at the lower contour of 0.013.

4. TMD lumen of ABCA7_PE with density (0.035 contour) for TMD lipids (pink) and peripherally associated ordered lipids (white) shown.

5. Same as (D) with higher density contour of 0.046 where density of peripherally associated lipids is absent but that of luminal lipids remains.

Figure 3. Cryo‐EM structure of ABCA7_EQ‐ATP

1. Cryo‐EM map of human ABCA7_EQ‐ATP in BPL/Chol nanodiscs at 3.7 Å resolution (orange density, 0.02 contour).

2. Cryo‐EM structure of ABCA7_EQ‐ATP shown in ribbon format with half 1 and half 2 colored yellow and orange, respectively. The transparent orange oval demarcates the observed exit pocket.

3. Central slice of electrostatic surface map showing surface details of exit pocket in two different orientations.

4. 4‐TM bundle forming the exit pocket.

5. NBDs viewed from the extracellular side with density for bound nucleotide (sticks colored by element) shown in blue (0.02 contour). Two ATP molecules are shown with the density at two different contours 0.02 (mesh) and 0.035 (blue) at the bottom.

Figure 4. Comparison of open and closed conformations of ABCA7

1. Overall structural alignment of the three ABCA7 conformations.

2. Overall alignment of the three ABCA7 conformations showing only TMD1 and TMD2 viewed from the extracellular side using the TMD1‐NBD1 pair as an alignment reference. TMs lining the TMD pathway are numbered, and modeled lipid acyl chains are shown as outlined white spheres.

3. Same as panel (B), viewed from the cytoplasmic side with C_α atoms of gate forming residues shown as spheres.

4. Individual alignments of rigid body pairs TMD1‐NBD1, TMD2‐NBD2, and ECD along with the Root mean square deviations (RMSD) of aligned atoms of ABCA7_PE versus ABCA7_EQ‐ATP and ABCA7_DIGITONIN.

Figure EV3. Conservation of ABCA and ABCG family structural elements

1. Ribbon representation of select ABCG family transporter structures and their respective PDB IDs including apo open ABCG2 (red), closed ATP‐bound structure of ABCG2_EQ (black), and ABCG5/G8 (blue and green, respectively).

2. Alignment of NBD‐TMD pairs from open and closed conformations of ABCG2 (left), ABCG2 and ABCG5/G8 (center), and closed ABCG2 with TMD‐NBD pairs from ABCA7 half 1 (gold) and half 2 (orange) along with the root mean square deviations (RMSD) of aligned atoms shown at the bottom.

Figure 5. MD Simulations of ABCA7 in Lipid Bilayers

1. A

   Left: A representative simulation system with four copies of ABCA7 (silver), taken from the POPE/cholesterol (yellow/cyan) lipid patch. Right: the phospholipid belt (blue lines) formed in (top) the POPE/cholesterol (yellow/cyan) and (bottom) POPC/cholesterol (red/cyan) membranes (t = 2 μs).

2. B, C

   Heatmaps representing the average height (z values) of POPC headgroups with respect to the membrane midplane in the extracellular/upper (B) and cytoplasmic/lower (C) leaflets. Phospholipids are observed to climb the protein and form an elevated configuration within the TMD lumen.

3. D

   Snapshot of lipids partitioned in the TMD lumen at t = 2 μs. Polar, nonpolar, basic, and acidic residues are colored green, gray, blue, and red, respectively. TMD2 is hidden for a clearer view of the luminal dome‐like lipid configuration (outlined by blue lines).

Figure EV4. Lipid configuration induced by protein copies in a bilayer and criteria used for identifying luminal lipids in MD simulations

1. A, B

   POPE headgroup height calculated for extracellular (A) and cytoplasmic (B) leaflets.

2. C

   Dimension and position of the box used to calculate the number of phospholipids (yellow) partitioned in the TMD leaflets. The box is centered at the protein center and has dimensions of 50 and 25 Å in x and y directions, respectively. The ABCA7 transporter is hidden for clarity.

3. D

   Contact map, based on the number of POPE headgroups in proximity of each residue throughout the simulation.

4. E

   Accumulation of POPE headgroups (yellow spheres) in close proximity of most frequently contacted residues (blue spheres) throughout the simulation. All protein residues are represented as spheres (C_α atoms).

Figure 6. Proposed model of ABCA7 conformational transitions and lipid interactions

The proposed model displays the fully open conformation (left) with ordered lipids (red and blue) in the TMD, the intermediate open (middle) with the upward protrusion of the upper and lower leaflets with black dash arrows indicating movement of lipids under the influence of TMD conformation, and the closed conformation (right) exhibiting the lipid exit pocket for extrusion to the ECD where they may be sequestered and reside until an acceptor molecule is available. Question marks denote speculated mechanisms. The membrane bilayer leaflets are shown in gray. The surface maps on the upper panel represent three different states of ABCA7 the mechanistic model is derived from. Differences in ECD appearance are meant to show different orientations and not large‐scale structural transitions.

Figure EV5. Structure alignment of ABCA7, ABCA1, ABCA3 and ABCA4

1. A, B

   Superposition of (A) ABCA7 (green, this manuscript), ABCA1 (gray, PDB: 7TC0), ABCA3 (blue, PDB: 7W01), and ABCA4 (red, PDB: 7LKP) structures in detergent and (B) ABCA7 (orange, this manuscript), ABCA1 (gray, PDB: 7TBW), ABCA3 (blue, PDB: 7W02), and ABCA4 (red, PDB: 7E7Q) structures in ATP‐bound states. Color keys are indicated on the left and the RMSD of aligned atoms of ABCA7 versus ABCA1, ABCA3, and ABCA4 are displayed at the bottom of each set of structures.