Importance of the Conserved Walker B Glutamate Residues, 556 and 1201, for the Completion of the Catalytic Cycle of ATP Hydrolysis by Human P-glycoprotein (ABCB1)

Abstract

The human MDR1 (ABCB1) gene product, P-glycoprotein (Pgp), functions as an ATP-dependent efflux pump for a variety of chemotherapeutic drugs. In this study, we assessed the role of conserved glutamate residues in the Walker B domain of the two ATP sites (E556 and E1201, respectively) during the catalytic cycle of human Pgp. The mutant Pgps (E556Q, E556A, E1201Q, E1201A, E556/1201Q, and E556/1201A) were characterized using a vaccinia virus based expression system. Although steady-state ATP hydrolysis and drug transport activities were abrogated in both E556Q and E1201Q mutant Pgps, [alpha-(32)P]-8-azidoADP was trapped in the presence of vanadate (Vi), and the release of trapped [alpha-(32)P]-8-azidoADP occurred to a similar extent as in wild-type Pgp. This indicates that these mutations do not affect either the first hydrolysis event or the ADP release step. Similar results were also obtained when Glu residues were replaced with Ala (E556A and E1201A). Following the first hydrolysis event and release of [alpha-(32)P]-8-azidoADP, both E556Q and E1201Q mutant Pgps failed to undergo another cycle of Vi-induced [alpha-(32)P]-8-azidoADP trapping. Interestingly, the double mutants E556/1201Q and E556/1201A trapped [alpha-(32)P]-8-azidoADP even in the absence of Vi, and the occluded nucleotide was not released after incubation at 37 degrees C for an extended period. In addition, the properties of transition state conformation of the double mutants generated in the absence of Vi were found to be similar to that of the wild-type protein trapped in the presence of Vi (Pgp x [alpha-(32)P]-8-azidoADP xVi). Thus, in contrast to the single mutants, the double mutants appear to be defective in the ADP release step. In aggregate, these data suggest that E556 and E1201 residues in the Walker B domains may not be critical as catalytic carboxylates for the cleavage of the bond between the gamma-P and the beta-P of ATP during hydrolysis but are essential for the second ATP hydrolysis step and completion of the catalytic cycle.

Exploiting Reaction Intermediates of the ATPase Reaction to Elucidate the Mechanism of Transport by P-glycoprotein (ABCB1)

“…Formation of the Pgp⅐ATP (in the mutant Pgp) or Pgp⅐ADP⅐Vi (in the wild-type Pgp) reaction intermediates is a slow process that occurs over several minutes (20,22,30). Moreover, we show that in the temperature range 10 -34°C, the trapping reaction is linear for at least 5 min (e.g.…”

“…State-Several groups have studied the role of the highly conserved glutamates within the Walker B region of the N-and the C-ATP sites of mouse (22) and human (20) Pgp. In human Pgp, the double mutant E556Q/E1201Q does not show steady state hydrolysis but can trap the nucleotide in the transition state (20), and the results were identical with equivalent mutations in mouse Pgp (22).…”

“…The Vi-induced, ADP-trapped state of Pgp has been well characterized (18,19), and is believed to be comparable with the posthydrolysis, Pgp⅐ADP⅐P i reaction intermediate. Moreover, recent studies have found that mutating the highly conserved glutamates (Glu 556 and Glu 1201 in human Pgp) in the Walker B region results in a phenotype wherein the protein shows no transport function and minimal steady-state ATP hydrolysis but can occlude the nucleotide (20). One explanation for this has been that these conserved glutamates constitute the catalytic carboxylate; consequently, the mutant cannot cleave the bond between the ␤-P and the ␥-P, and ATP is "trapped" (21,22).…”

Nucleotide-Binding Sites of the Heterodimeric LmrCD ABC-Multidrug Transporter of Lactococcus lactis Are Asymmetric

“…Furthermore, in LmrC, a glutamine residue replaces the histidine of the H loop that has been proposed to form a hydrogen bond with γ phosphate (reviewed in ref 27). Finally, in LmrD, the first glycine of the C loop, which is also involved in making contacts with γ phosphate (22), is substituted by a valine. Our results demonstrate that glutamate 587 in LmrD is essential for ATP hydrolysis and drug extrusion.…”

The motor domains of ABC-transporters

“…Mutating only one of the Glu residues in the two NBDs actually results in the retention of measurable ATPase activity (Tombline et al 2004b). Moreover, for similar mutations in human Pgp, efficient vanadate trapping could also be obtained (Sauna et al 2002). In contrast to these findings concerning the Glu residue, mutation of the H-loop His resulted in a complete loss of ATPase activity and, in some cases, of transport (Davidson and Sharma 1997;Nikaido and Ames 1999;Shyamala et al 1991;Zaitseva et al 2005b).…”