title: Interaction of extracellular domain 2 of the human retina-specific ATP-binding cassette transporter (ABCA4) with all-trans-retinal.
abstract: The retina-specific ATP-binding cassette (ABC) transporter, ABCA4, is essential for transport of all-trans-retinal from the rod outer segment discs in the retina and is associated with a broad range of inherited retinal diseases, including Stargardt disease, autosomal recessive cone rod dystrophy, and fundus flavimaculatus. A unique feature of the ABCA subfamily of ABC transporters is the presence of highly conserved, long extracellular loops or domains (ECDs) with unknown function. The high degree of sequence conservation and mapped disease-associated mutations in these domains suggests an important physiological significance. Conformational analysis using CD spectroscopy of purified, recombinant ECD2 protein demonstrated that it has an ordered and stable structure composed of 27 +/- 3% alpha-helix, 20 +/- 3% beta-pleated sheet, and 53 +/- 3% coil. Significant conformational changes were observed in disease-associated mutant proteins. Using intrinsic tryptophan fluorescence emission spectrum of ECD2 polypeptide and fluorescence anisotropy, we have demonstrated that this domain specifically interacts with all-trans-retinal. Furthermore, the retinal interaction appeared preferential for the all-trans-isomer and was directly measurable through fluorescence anisotropy analysis. Our results demonstrate that the three macular degeneration-associated mutations lead to significant changes in the secondary structure of the ECD2 domain of ABCA4, as well as in its interaction with all-trans-retinal.
authors.affiliation: Department of Bioscience Technologies, Jefferson School of Health Professions, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA. esther.biswas@jefferson.edu
authors.forename: Esther E
authors.initials: EE
authors.lastname: Biswas-Fiss
authors.forename: Deepa S
authors.initials: DS
authors.lastname: Kurpad
authors.forename: Kinjalben
authors.initials: K
authors.lastname: Joshi
authors.forename: Subhasis B
authors.initials: SB
authors.lastname: Biswas
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D016880:Anisotropy; D002942:Circular Dichroism; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012160:Retina / Q000378:metabolism; D012172:Retinaldehyde / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D014785:Vision, Ocular
chemical_list: C056475:ABCA4 protein, human; D018528:ATP-Binding Cassette Transporters; D000255:Adenosine Triphosphate; D012172:Retinaldehyde
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