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J. Biol. Chem., Vol. 281, Issue 16, 10769-10777, April 21, 2006

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Allosteric Modulation Bypasses the Requirement for ATP Hydrolysis in Regenerating Low Affinity Transition State Conformation of Human P-glycoprotein^*

From the Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, F. Edward Hébert School of Medicine, Bethesda, Maryland 20814-4799

ATP-dependent drug transport by human P-glycoprotein (Pgp, ABCB1) involves a coordinated communication between its drug-binding site (substrate site) and the nucleotide binding/hydrolysis domain (ATP sites). It has been demonstrated that the two ATP sites of Pgp play distinct roles within a single catalytic turnover; whereas ATP binding or/and hydrolysis by one drives substrate translocation and dissociation, the hydrolytic activity of the other resets the transporter for the subsequent cycle (Sauna, Z. E., and Ambudkar, S. V. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 2515–2520; Sauna, Z. E., and Ambudkar, S. V. (2001) J. Biol. Chem. 276, 11653–11661). Trapping of ADP (or 8-azido-ADP) and vanadate (ADP·Vi or 8-azido-ADP·Vi) at the catalytic site, following nucleotide hydrolysis, markedly reduces the affinity of Pgp for its transport substrate [¹²⁵I]iodoarylazidoprazosin ([¹²⁵I]IAAP), resulting in dissociation of the latter. Regeneration of the [¹²⁵I]IAAP site requires an additional round of nucleotide hydrolysis. In this study, we demonstrate that certain thioxanthene-based allosteric modulators, such as cis-(Z)-flupentixol and its closely related analogs, induce regeneration of [¹²⁵I]IAAP binding to vanadate-trapped (or fluoroaluminate-trapped) Pgp without any further nucleotide hydrolysis. Regeneration was facilitated by dissociation of the trapped nucleotide and vanadate. Once regenerated, the substrate site remains accessible to [¹²⁵I]IAAP even after removal of the modulator from the medium, suggesting a modulator-induced relaxation of a constrained transition state conformation. Consistent with this, limited trypsin digestion of vanadate-trapped Pgp shows protection by cis-(Z)-flupentixol of two Pgp fragments (60 kDa) recognizable by a polyclonal antiserum specific for the NH₂-terminal half. No regeneration was observed in the Pgp mutant F983A that is impaired in modulation by flupentixols, indicating involvement of the allosteric modulator site in the phenomenon. In summary, the data demonstrate that in the nucleotide-trapped low affinity state of Pgp, the allosteric site remains accessible and responsive to modulation by flupentixol (and its closely related analogs), which can reset the high affinity state for [¹²⁵I]IAAP binding without any further nucleotide hydrolysis.

Received for publication, November 23, 2005 , and in revised form, February 21, 2006.

^* This work was supported by United States Public Health Services Grant GM067926 and Uniformed Services University of the Health Sciences Intramural Grant C071FU. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Uniformed Services University of Health Sciences, F. Edward Hébert School of Medicine, 4301 Jones Bridge Rd., Bethesda, MD 20814-4799; Tel.: 301-295-3449; Fax: 301-295-3512; E-mail: sdey{at}usuhs.mil.

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