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J. Biol. Chem., Vol. 279, Issue 7, 5363-5373, February 13, 2004

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Synergy between Conserved ABC Signature Ser Residues in P-glycoprotein Catalysis^*

Gregory Tombline, Lori Bartholomew, Khursheed Gimi, Grace A. Tyndall, and Alan E. Senior

From the Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642

Functional roles of the two ABC signature sequences ("LSGGQ") in the N- and C-terminal nucleotide binding domains of P-glycoprotein were studied by mutating the conserved Ser residues to Ala. The two single mutants (S528A; S1173A) each impaired ATPase activity mildly, and showed generally symmetrical effects on function, consistent with equivalent mechanistic roles of the two nucleotide sites. Synergy between the two mutations when combined was remarkable and resulted in strong catalytic impairment. The Ser residues are not involved significantly in MgATP- or MgADP-binding or in interdomain communication between catalytic sites and drug binding sites. Retention of product MgADP is not the cause of reduced turnover. Mutation of Ser to Ala reduced the strength of interaction with the chemical transition state specifically, as shown by vanadate-ADP and beryllium fluoride-ADP trapping experiments. Therefore, the two conserved ABC signature motif Ser residues of P-glycoprotein cooperatively accelerate ATP hydrolysis via chemical transition state interaction. Because the transition state complex is currently believed to form in the dimerized state of the nucleotide binding domains, one may also conclude that both Ser-OH are necessary for correct formation of the dimer state.

Received for publication, October 31, 2003 , and in revised form, November 19, 2003.

^* This work was supported by National Institutes of Health Grants GM50156 (to A. E. S.) and T32 CA09363 (postdoctoral training support to G. T.). 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.

To whom correspondence should be addressed. Tel.: 585-275-2777; Fax: 585-271-2683; E-mail: alan_senior{at}urmc.rochester.edu.

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