Structural features of the ABCA4 transporter. (a) A linear diagram showing the full transporter arranged as two tandem halves. Each half consists of six membrane spanning segments which together make up the transmembrane domain (TMD). A large exocytoplasmic (extracellular/lumen) domain (ECD) separates the first membrane spanning segment from the remaining five membrane spanning segments in each half. The nucleotide binding domain (NBD) lies downstream of the multiple membrane spanning segments. (b) Topological model showing the organization of ABCA4 in the membrane. Each ECD contains multiple N-linked oligosaccharide chains (hexagons) exposed to the lumen side of the disc membrane [14, 15]. A six amino acid motif (VFVNFA) present in both ABCA4 and ABCA1 is close to the C-terminus [38, 44]. Other ABCA family members have a similar topological organization.

Role of ABCA4 in the visual cycle. Light isomerizes 11-cis retinal to all-trans retinal in rhodopsin. All-trans retinal dissociates from opsin and is reduced to all-trans retinol by all-trans retinol dehydrogenase (RDH8). All-trans retinol is esterified by lecithin:retinol acyl transferase (LRAT), isomerized to 11-cis retinol by Rpe65-isomerase, and oxidized to 11-cis retinal by 11 cis retinol dehydrogenases. 11-cis retinal is transported back to the outer segment where it can recombine with opsin to produce rhodopsin. All-trans retinal released from rhodopsin can also react with phosphatidylethanolamine (PE) to produce N-retinylidene-PE which is in equilibrium with its protonated form. In this model ABCA4 transports N-retinylidene-PE from the lumen to the cytoplasmic side of the disc membrane where all-trans retinal can be reduced to all-trans retinol by RDH8 as part of the visual cycle.

A simplified model for the transport of N-retinylidene-PE across the disc membrane by ABCA4. In the absence of ATP, N-retinylidene-PE binds to a high affinity site within the transmembrane domain (TMD) of ABCA4 altering the affinity of the nucleotide binding domains (NBDs) for ATP. The binding of ATP to one or both NBDs causes a dimerization of the NBDs and a conformation change in the TMD that converts the high affinity N-retinylidene-PE substrate binding site to a low affinity site and access to the cytoplasmic side of the disc membrane. Dissociation of N-retinylidene-PE from ABCA4 on the cytoplasmic side of the lipid bilayer together with ATP hydrolysis and dissociation of ADP returns ABCA4 to its initial state .

(a). Alignment of the nucleotide binding domains of ABCA4. Conserved motifs including the A-loop, Walker A, signature, Walker B, Q-loop, and the H-loop are in bold. Disease mutations, which are substituted in Stargardt disease, are shown in red italics – NBD1 (N965S, T971N, A1038V, S1071V, E1087K, R1108C); NBD2 (G1961E, L1971R, G1977S, L2027F, R2038W, R2077W, R2106C, R2107H). (b) Homology model of the NBD1 of ABCA4 shows Walker A (purple) and B (blue) sites with the Q-loop (yellow) and signature motif (orange). The α/β domain and α-helical domain are demarcated by an arbitrary line representing Arms I and II. This model was generated on the basis of the coordinates from the crystal structure of an ATP binding domain from an ATP binding domain (35% identity; 55% similarity; PDB: 1VPL) using Modeller 7v7 [Sali and Blundell, 1993] by making a structural alignment of NBD1, NBD2, and 1VPL. 100 models of NBD1 were generated. Five models with the lowest objective function value were selected for energy minimization in GROMACS 3.3. Energy minimization was done by steepest gradient descent, with an initial step size of 0.01 and a maximum of 1000 steps; each of the models converged and the energy minimized models were evaluated for stereochemical quality by PROCHECK and ranked according to score. For the purpose of demonstration, the highest-ranking model was used.

Reactions involved in the formation of A2E due to loss in ABCA4 activity. All-trans retinal reacts with phosphatidylethanolamine (PE) to form N-retinylidene-PE which can condense with all-trans retinal to produce the di-retinoid pyridinium-PE compound known as A2PE in outer segments. Upon phagocytosis, A2PE can be hydrolyzed by lysosomal enzymes in the RPE cells to A2E and phosphatidic acid (PA). The reduction of N-retinylidene-PE to N-retinyl-PE by sodium borohydride is also shown.

Model for the ABCA4-mediated photoreceptor degeneration in Stargardt disease. Partial or complete loss of ABCA4 function due to disease-causing mutations (ABCA4 – star) results in the accumulation of all-trans retinal and N-retinylidene-PE (N-Ret-PE) in outer segment disc membrane following the light activation of rhodopsin. All-trans retinal condenses with N-retinylidene-PE to produce A2PE. Outer segments undergo phagocytosis by adjacent RPE cells and degradation by lysosomal enzymes. As part of this process A2PE is hydrolyzed to A2E which then accumulates as lipofuscin deposits in the RPE cells. A2E can act as a detergent or alternatively it can be converted to toxic epoxides by the action of short wavelength light. These compounds can compromise RPE cell function leading to apoptosis. Loss in RPE cells will result in photoreceptor cell death and a loss in vision.