Combined Mutation of Catalytic Glutamate Residues in the Two Nucleotide Binding Domains of P-glycoprotein Generates a Conformation That Binds ATP and ADP Tightly

Gregory Tombline,Lori A Bartholomew,Ina L Urbatsch,Alan E Senior

doi:10.1074/jbc.m404689200

Gregory Tombline, Lori A Bartholomew + Show 2 more

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https://doi.org/10.1074/jbc.m404689200

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Abstract

Combined mutation of "catalytic carboxylates" in both nucleotide binding domains (NBDs) of P-glycoprotein generates a conformation capable of tight binding of 8-azido-ADP (Sauna, Z. E., Müller, M., Peng, X. H., and Ambudkar, S. V. (2002) Biochemistry 41, 13989-14000). Here we characterized this conformation using pure mouse MDR3 P-glycoprotein and natural MgATP and MgADP. Mutants E552A/E1197A, E552Q/E1197Q, E552D/E1197D, and E552K/E1197K had low but real ATPase activity in the order Ala > Gln > Asp > Lys, emphasizing the requirement for Glu stereochemistry. Mutant E552A/E1197A bound MgATP and MgADP (1 mol/mol) with K(d) 9.2 and 92 microm, showed strong temperature sensitivity of MgATP binding and equal dissociation rates for MgATP and MgADP. With MgATP as the added ligand, 80% of bound nucleotide was in the form of ATP. None of these parameters was vanadate-sensitive. The other mutants showed lower stoichiometry of MgATP and MgADP binding, in the order Ala > Gln > Asp > Lys. We conclude that the E552A/E1197A mutation arrests the enzyme in a conformation, likely a stabilized NBD dimer, which occludes nucleotide, shows preferential binding of ATP, does not progress to a normal vanadate-sensitive transition state, but hydrolyzes ATP and releases ADP slowly. Impairment of turnover is primarily due to inability to form the normal transition state rather than to slow ADP release. The Gln, Asp, and Lys mutants are less effective at stabilizing the occluded nucleotide, putative dimeric NBD, conformation. We envisage that in wild-type the occluded nucleotide conformation occurs transiently after MgATP binds to both NBDs with associated dimerization, and before progression to the transition state.

Highlights

The numerous compounds transported by Pgp, and there is the realization that a substantial number of future new drug candidates will be transport substrates, so much current interest centers on strategies aimed at disabling or circumventing Pgp
We conclude that the E552A/E1197A mutation arrests the enzyme in a conformation, likely a stabilized nucleotide binding domains (NBDs) dimer, which occludes nucleotide, shows preferential binding of ATP, does not progress to a normal vanadate-sensitive transition state, but hydrolyzes ATP and releases ADP slowly
Pgp [25] and other ABC transporters [26, 27] has been seen to impair ATP hydrolysis and it appears to function as a “catalytic carboxylate.”3 X-ray crystallography of the dimer formed by the mutant MJ0796 NBD subunits [29] revealed structural details demonstrating two NaATP molecules sandwiched at the dimer interface between the Walker A motif in one NBD subunit and the LSGGQ ABC transporter signature motif of the other

Summary

Introduction

The numerous compounds transported by Pgp, and there is the realization that a substantial number of future new drug candidates will be transport substrates, so much current interest centers on strategies aimed at disabling or circumventing Pgp. Earlier work had established that Pgp showed drug-stimulated ATPase activity [9, 10] and that hydrolysis of ATP occurred in both NBDs [11] It was clear from studies with inhibitors N-ethylmaleimide and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole [10, 12,13,14], mutations in the catalytic sites [15, 16], and vanadate-trapping experiments [17] that the two NBDs cooperated strongly and mandatorily for hydrolysis, and an alternating sites mechanism was proposed in 1995 [18]. This work potentially opens up an avenue to studies of an intermediate conformational state in the Pgp mechanism, namely the closed dimer state with nucleotide bound

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