| Abstract
| - We report here a novel regulation of the ATPase activity of the human retina specific ATPbinding cassette transporter (ABC), ABCR, by nucleotide binding domain interactions. We also presentevidence that recombinant nucleotide binding domains of ABCR interact in vitro in the complete absenceof transmembrane domains (TMDs). Although similar domain−domain interactions have been describedin other ABC transporters, the roles of such interactions on the enzymatic mechanisms of these transportershave not been demonstrated experimentally. A quantitative analysis of the in vitro interactions as a functionof the nucleotide-bound state demonstrated that the interaction takes place in the absence of nucleotide aswell as in the presence of ATP and that it only attenuates in the ADP-bound state. Analysis of the ATPaseactivities of these proteins in free and complex states indicated that the NBD1−NBD2 interactionsignificantly influences the ATPase activity. Further investigation, using site-specific mutants, showedthat mutations in NBD2 but not NBD1 led to the alteration of the ATPase activity of the NBD1·NBD2complex and residue Arg 2038 is critical to this regulation. These data indicate that changes in the oligomericstate of the nucleotide binding domains of ABCR are coupled to ATP hydrolysis and might represent apossible signal for the TMDs of ABCR to export the bound substrate. Furthermore, the data support amechanistic model in which, upon binding of NBD2, NBD1 binds ATP but does not hydrolyze it or doesso with a significantly reduced rate.
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