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E.E. Biswas–Fiss, S. Biswas, K. Frempong, J. LeGates; Analysis of Intra–Protein Interactions in the Retina Specific ABC Transporter, ABCR . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1702.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:The human retina specific ATP binding cassette transporter, ABCR, is known to function in the translocation of retinal derivatives. Mutations in the ABCR gene have been linked to several inherited macular degenerations, including Stargardt disease, Age Related Macular Degeneration, and Cone–Rod Dystrophy. A key feature of many ABC ATPases is the interaction between the two nucleotide–binding domains. The intra–protein interactions in the ABCR protein remains poorly understood. In the present study, we analyzed the intra–protein interactions by fluorescence anisotropy. Methods: Using recombinant DNA technology, individual polypeptides corresponding to the nucleotide binding domains of ABCR were cloned, expressed and purified. Using fluorescence anisotropy NBD1–NBD2 domain–domain interaction was measured. Analyses were carried out in the presence of various nucleotides and with mutant NBD’s harboring disease associated mutations. Results:We have quantitatively analyzed the interactions in various nucleotide bound states and have demonstrated that the affinity is greatest in the ATP bound state, and that it decreases in the ADP bound state. We also demonstrate alterations in the nucleotide binding domain affinity as a result of disease specific mutations. Homology based molecular modeling provided a structural basis for NBD1–NBD2 interaction in ABCR, in which the two nucleotide–binding domains form a composite catalytic center, with ATP buried in the domain–domain interface. Conclusions: The conclusions derived from our studies are (1) ATP hydrolysis to ADP negatively modulates the domain–domain interaction; (2) whereas ATP binding promotes the domain–domain interaction; and (3) certain disease–associated mutations in these domains significantly alter the domain–domain interaction.
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