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E.E. Biswas–Fiss, S.B. Biswas; Interaction of the Nucleotide Binding Domains and Regulation of the ATPase Activity of the Human Retina Specific the Human Retina Specific ABC Transporter, ABCR . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2023.
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© ARVO (1962-2015); The Authors (2016-present)
We report here a novel regulation of the ATPase activity of human retina specific ATP binding cassette transporter (ABC), ABCR, by interactions of its nucleotide binding domains. ATP hydrolysis is a major event in ABCR mediated transport. These studies were aimed at further understanding the functional implications, in terms of nucleotidase activity, of nucleotide binding domain interaction in ABCR.
We have used purified recombinant NBD1 and NBD2 polypeptides of ABCR, enzymological analyses and fluorescence anisotropy to assess the functional consequences of NBD1/NBD2 interaction.
A quantitative analysis of the in vitro interactions as a function of nucleotide bound states demonstrated that the interaction takes place in the absence of nucleotide as well as in the presence of ATP, and it only attenuates in the ADP bound state. Analysis of the ATPase activities of these proteins in free and complex states indicated that the NBD1•NBD2 interaction significantly influences the ATPase activity. Further investigation, using site–specific mutants, showed that mutations in NBD2, but not NBD1 led to the alteration of the ATPase activity of the NBD1•NBD2 complex, and the residue, Arg 2038, is critical to this regulation.
These data indicate that changes in the oligomeric state of the nucleotide binding domains of ABCR are coupled to ATP hydrolysis as well as that their interactions represent possible signals for the TMDs of ABCR to export the bound substrate. Furthermore, the data support a mechanistic model in which upon binding NBD2, NBD1 binds ATP, but does not hydrolyze it, or does so with a significantly reduced rate.
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