Cryo-EM structures of the ABCA4 importer reveal mechanisms underlying substrate binding and Stargardt disease

Abstract

ABCA4 is an ATP-binding cassette (ABC) transporter that flips N-retinylidene-phosphatidylethanolamine (N-Ret-PE) from the lumen to the cytoplasmic leaflet of photoreceptor membranes. Loss-of-function mutations cause Stargardt disease (STGD1), a macular dystrophy associated with severe vision loss. To define the mechanisms underlying substrate binding and STGD1, we determine the cryo-EM structure of ABCA4 in its substrate-free and bound states. The two structures are similar and delineate an elongated protein with the two transmembrane domains (TMD) forming an outward facing conformation, extended and twisted exocytoplasmic domains (ECD), and closely opposed nucleotide binding domains. N-Ret-PE is wedged between the two TMDs and a loop from ECD1 within the lumen leaflet consistent with a lateral access mechanism and is stabilized through hydrophobic and ionic interactions with residues from the TMDs and ECDs. Our studies provide a framework for further elucidating the molecular mechanism associated with lipid transport and disease and developing promising disease interventions.

Fig. 1. Localization of ABCA4 in photoreceptor rod outer segments and its role in the visual cycle.

a Schematic showing part of a rod outer segment with a stack of disc membrane surrounded by a plasma membrane. ABCA4 is located along the rim and incisures of the disc membrane. b Reversible formation of N-retinylidene-phosphatidylethanolamine (N-Ret-PE) from all-trans retinal (ATR) and phosphatidylethanolamine (PE). c Diagram showing the role of ABCA4 in the transport of N-Ret-PE across the disc membrane. Excess 11-cis-retinal (11-cis-ral) not required for the regeneration of rhodopsin reversibly reacts to PE to produce N-Ret-PE (cis). ABCA4 flips N-Ret-PE (cis) from the lumen side to the cytoplasmic side of the disc membrane. Chemical isomerization converts N-Ret-PE (cis) to its trans isomer. Dissociation of N-ret-PE (trans) to ATR and PE enables ATR to be reduced to all-trans retinol (all-trans-rol) by retinol dehydrogenase 8 (RDH8) for entry into the visual cycle. ATR produced by photobleaching of rhodopsin reversibly reacts with PE to form N-Ret-PE (trans) which is flipped to the cytoplasmic leaflet by ABCA4. After dissociation, ATR is reduced to all-trans retinol by RDH8 for entry into the visual cycle.

Fig. 2. Molecular characterization and structural features of ABCA4.

a Representative ATPase activity as a function of ATP for purified ABCA4 in the presence of phosphatidylethanolamine (PE) alone (Basal activity) and in the presence of PE and all-trans retinal (ATR) to generate N-retinylidene-PE (N-Ret-PE). Data expressed as a mean ± SD for three replicate measurements. Three independent experiments gave similar results The ATPase-deficient variant (MM-ABCA4) in which a lysine residue in each Walker A motif is replaced with a methionine is shown as a control. Similar curves were generated in three independent experiments. b Topological model of ABCA4 showing the N-linked glycosylation sites (blue hexagons) and disulfide bridges in the exocytoplasmic domains (ECD). Nucleotide binding domains (NBD) together with the regulatory domains (RD) are on the cytoplasmic side of the membranes. Helices in the transmembrane domains (TMD) are presented as cylinders with TMD1 in dark green and TMD2 in light green. Each TMD contains two internal transverse helices (IH) and two external helices (EH) as part of a α-helix-turn-α-helix structure. c Overall structure of ABCA4 in the unbound state. The structure is represented as cartoon with the N- and C- terminal halves colored dark and light green, respectively. N-linked glycans are represented as pink sticks. d Schematic showing the dimensions of two stacked discs. ABCA4 only can be accommodated in the rim region due its elongated ECDs. Localization of rhodopsin in the flat part of the disc is shown for comparison.

Fig. 3. Closeup view of the TMD and ECD.

a–c Surface and ribbon representations of the transmembrane domains (TMD). EM densities of the lipids are indicated with an arrow. a EM density resembling a lipid is located in between TM1/2/11. b Orthogonal view. c Most probable orientation of the lipid (black) between TMD1 and TMD2. N- and C- halves of ABCA4 are colored as dark green and light green, respectively. d–f Surface and ribbon representations of the exocytoplasmic domains, showing (d) the tunnel that is accessible from the lumen side. e EM density was also found on the opposite side of the exocytoplasmic domain (ECD), indicated as purple mesh. f Orthogonal view of ECD showing the EM densities (arrow).

Fig. 4. Cartoon representation of exocytoplasmic domains (ECD) with cysteines involved in disulfide bridges represented as sticks.

a, b Disulfide bonds are located within ECD1 and ECD2 and one disulfide bond connects ECD1 and ECD2 (C641-C1490). N- and C- halves are colored as dark green and light green, respectively. The associated EM density is shown as purple mesh with σ = 5.0.

Fig. 5. N-retinylidene-phosphatidylethanolamine (N-Ret-PE) bound to ABCA4.

a UV-Vis spectra of ABCA4 in its unbound (black line) and bound state (gray line) normalized at 280 nm. The peak absorbance (λ_MAX = 362 nm) corresponds to N-Ret-PE bound to ABCA4. b N-Ret-PE associated electron microscope (EM) density is shown as blue mesh, with σ = 6.0, and displays the substrate in a transverse position, wedged between the transmembrane domains (TMD) and exocytoplasmic domain (ECD). c View from the lumen side of the membrane showing N-Ret-PE with the β-ionone group of the all-trans-retinal moiety close to TM8/11 and the phosphate group close to TM2/5. d View from the cytoplasmic side of the membrane showing bound substrate close to the B-loop of ECD1.

Fig. 6. Residues involved in the substrate binding pocket.

a N-Ret-PE is wedged between transmembrane domains TMD1 and TMD2 and B-loop. The residues that interact with the substrate are indicated as purple sticks. R653 (TM2) and R587 (ECD1) form ionic interactions with the phosphate group of N-Ret-PE. The interactions include several aromatic residues in B-loop (W339, Y340, F348). The β-ionone group interacts with Y345 (B-loop), L1674 (TM8), S1677 (TM8), L1812 (TM11) and L1815 (TM11). b Orthogonal view of the binding pocket showing the residues involved in the binding to phosphate and residues belonging to TMD2. c Residues in the B-loop as viewed from the exocytoplasmic domain.

Fig. 7. Residues involved in the substrate binding pocket through the acyl chains of PE.

a Residues that make up the N-Ret-PE binding site are indicated as purple sticks. Both acyl chains appear to be associated with L42 (TM1), I649 (TM2) I650 (TM2) and F1397 (loop between TM7 and ECD2) through hydrophobic interactions. The main chain of I649 interacts with the side chain of Y588 (ECD1 loop). b Closeup view of the residues involved in the acyl chain coordination.

Fig. 8. Purification and functional characterization of ABCA4 variants with amino acid substitutions in residues involved in substrate binding.

a Representative coomassie blue stained gel of wild-type ABCA4 (WT) and variants expressed in HEK293T cells and purified by immunoaffinity chromatography. Data reproduced in three independent experiments. b ATPase activity of the ABCA4 variants in the absence (Basal) and presence of 40 µM all-trans retinal (ATR) to generate N-Ret-PE. Activity, normalized to WT basal activity, is expressed as the mean ± SD for n ≥ 3 independent experiments. P values between basal and ATR ATPase activities: WT = 0.001 (n = 5); W339G = 0.09 (n = 5); R587A = 0.009 (n = 5); Y345A = 0.07 (n = 3); Y345C = 0.82 (n = 5); R653C = 0.125 (n = 3) (two-tailed, paired Student T test). Data for R653C is from Garces et al. c % ATP-dependent transport of N-Ret-PE for samples treated with 1 mM ATP for 1 h. Data expressed as a mean ± SD for n = 3 (WT, Y345C) and n = 4 (W339G), and a range of values for n = 2 (R587A, R653C) independent experiments. d Binding of N-Ret-PE as a function of ATR concentration. WT ABCA4 had an apparent Kd of 1.7 ± 0.3 µM, R587A had an apparent Kd = 16.8 ± 24.6 µM, Y345C had a Kd of 12.6 ± 7.3 µM, and W339G had a Kd of 4.0 ± 0.5. Source data are provided as a Source Data file. e Location of key residues surrounding of the binding pocket. Protein is shown as ribbon and key residues as spheres. N-Ret-PE is shown as yellow sticks. Purple: Reported disease-associated variants in the vicinity of the binding pocket that do not directly interact with N-Ret-PE. Black: Disease-associated mutants that directly interact with N-Ret-PE. Green: Residues shown in our studies to interact with N-Ret-PE, but have not yet been reported to cause STGD1.

Fig. 9. Proposed mechanism of N-Ret-PE transport from the lumen side to the cytoplasmic side of the disc membrane by ABCA4.

State 1: ABCA4 is in an outward-facing conformation in the resting apo-state. N-Ret-PE binds to ABCA4 through a lateral access mechanism from the lumen leaflet of the membrane as shown in State 2. Substrate binding (substrate – yellow) is stabilized by hydrophobic interactions within transmembrane domains 1 and 2 (TMD1 and TMD2) and exocytoplasmic domain 1 (ECD1) at the level of the lumen leaflet of the membrane. The phosphate group is stabilized by ionic interactions with two arginine residues. Two ATP molecules bind in the nucleotide binding domains 1 and 2 (NBD1 and NBD2) leading to State 3. The structure of ABCA4 containing bound ATP was reported by the Chen group (PDB ID 7LKZ);. Nucleotide binding leads to NBD dimerization, followed by the close association of the TMDs and a rotation in the ECD. The conformational change induced by ATP-binding results in collapse of the substrate binding site, the transport of N-Ret-PE across the membrane, possibly along a crevice on the external surface of ABCA4, and its release into the cytoplasmic leaflet of the disc membrane. Finally, ATP hydrolysis brings the protein back to its original State 1 allowing for a new cycle of transport.