ABCMdb

PMID: 12437356

Sauna ZE, Muller M, Peng XH, Ambudkar SV
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).
Biochemistry. 2002 Nov 26;41(47):13989-4000., 2002-11-26 [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln ABCB1 p.Glu1201Ala ABCB1 p.Glu556Ala The mutant Pgps (E556Q, E556A, E1201Q, E1201A, E556/1201Q, and E556/1201A) were characterized using a vaccinia virus based expression system. Login to comment 3 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln Although steady-state ATP hydrolysis and drug transport activities were abrogated in both E556Q and E1201Q mutant Pgps, [R-32P]-8-azidoADP was trapped in the presence of vanadate (Vi), and the release of trapped [R-32P]- 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. Login to comment 4 ABCB1 p.Glu1201Ala ABCB1 p.Glu556Ala Similar results were also obtained when Glu residues were replaced with Ala (E556A and E1201A). Login to comment 5 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln Following the first hydrolysis event and release of [R-32P]-8-azidoADP, both E556Q and E1201Q mutant Pgps failed to undergo another cycle of Vi-induced [R-32P]-8-azidoADP trapping. Login to comment 32 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln ABCB1 p.Glu1201Ala ABCB1 p.Glu556Ala In this study, in addition to the mutants E556Q and E1201Q we have also characterized the E556A and E1201A as well as the double (E556/1201Q and E556/1201A) mutant Pgps. Login to comment 37 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln The results of this study show that the E556Q and E1201Q mutant Pgps hydrolyze ATP and allow normal release of ADP during the first step but are defective in the second ATP hydrolysis event, and as a result of which, both steady-state ATP hydrolysis and drug transport activities are abrogated [part of this work has appeared in abstract form (24)]. Login to comment 54 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln ABCB1 p.Glu1201Ala ABCB1 p.Glu556Ala The coding sequence for the E556Q mutant primer was 5'-ATC CTC CTG CTG GAT CAG GCC ACG TCA GCC TTG-3'; for the E1201Q mutant primer, 5'-ATT TTG CTT TTG GAT CAA GCC ACG TCA GCT CTG-3'; for the E556A mutant primer, 5'-ATC CTC CTG CTG GAT GCG GCC ACG TCA GCC TTG-3'; and for the E1201A primer, 5'-ATT TTG CTT TTG GAT GCA GCC ACG TCA GCT CTG-3'. Login to comment 65 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln ABCB1 p.Glu1201Ala ABCB1 p.Glu556Ala Crude membranes were prepared from vTF7-3 infected HeLa cells transfected with vector pTM1-MDR1 wild type, pTM1-MDR1-E556Q, pTM1-MDR1-E1201Q, pTM1-MDR1-E556/1201Q, pTM1-MDR1-E556A, pTM1-MDR1-E1201A, and pTM1-MDR1- E556/1201A as described previously (28, 31) and stored at -70 °C. Total protein was quantified by the Amido Black protein estimation method as previously described (32), and Pgp expression level was determined by immunoblot analysis using the monoclonal antibody C219 (30, 33). Login to comment 95 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln We generated the single mutants E556Q and E1201Q and the double mutant E556/1201Q of human Pgp and characterized them in a Vaccinia virus based expression system. Login to comment 99 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln However, cells expressing equivalent amounts of E556Q, E1201Q, and E556/1201Q mutant Pgps accumulate high levels of calcein comparable to the HeLa cells infected with the control pTM1 vector. Login to comment 104 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln Wild-Type, E556Q, E1201Q, and E556/1201Q Pgps Exhibit Differences in the Trapping of [R-32 P]-8-AzidoADP. Login to comment 114 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln Both of the mutant Pgps, E556Q and E1201Q, exhibit enhanced trapping of [R-32 P]-8-azidoADP in the presence of Vi. Login to comment 115 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln The mutant E1201Q Pgp shows a higher level of trapping compared to the E556Q protein in the absence of Vi. Login to comment 117 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln Phosphorimager analysis of the gel depicted as an autoradiogram in Figure 1C shows that the mutant Pgps E556Q, E1201Q, and E556/1201Q trapped 26%, 43%, and 96% 8-azidoADP in the absence of Vi compared to that observed in the presence of Vi. Login to comment 119 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln The results with the single mutants E556Q and E1201Q are consistent with those obtained with the equivalent mutants (E552Q and E1197Q) in mouse Mdr3 (10). Login to comment 121 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln The E556Q and E1201Q Mutant Pgps Are DefectiVe in Repeated Cycles of Vi-Induced [R-32 P]-8-AzidoADP Trapping and Release. Login to comment 122 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln Although both the wild-type and mutant (E556Q and E1201Q) Pgps exhibit Vi-induced [R-32 P]-8-azidoADP trapping, the mutant Pgps show a complete loss of steady-state ATP hydrolysis and transport function. Login to comment 126 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln The experiment in Figure 2A (panel I) demonstrates that within 10 min wild-type Pgp and the mutants E556Q and E1201Q show a saturating and similar level of Vi-induced trapping of [R-32 P]-8-azidoADP. Login to comment 128 ABCB1 p.Glu556Gln The results of this experiment depicted in Figure 2A (panel II) demonstrate that in the mutant Pgp, E556Q, the release [R-32 P]-8-azidoADP occurs at the same rate as the wild-type Pgp. Login to comment 129 ABCB1 p.Glu1201Gln In the mutant E1201Q Pgp, the release of nucleoside diphosphate is slightly slower, but the extent of release is comparable to wild-type Pgp. Login to comment 134 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln We observed (Figure 2A, panel III) that while the wild-type Pgp exhibits a second Vi-induced trapping of [R-32 P]- 8-azidoADP comparable to the first Vi-induced trapping FIGURE 1: Cell surface expression, substrate transport, and Vi-induced trapping in wild-type and mutant Pgps, E556Q, E1201Q, and E556/1201Q. Login to comment 138 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln The lanes represent (from left to right) wild-type Pgp and the mutant Pgps E556Q, E1201Q, and E556/1201Q, respectively. Login to comment 141 ABCB1 p.Glu1201Gln ABCB1 p.Glu1201Gln ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln ABCB1 p.Glu556Gln ABCB1 p.Glu556Gln Panels A and B show HeLa cells infected with vTF7-3 and transfected with vector pTM1 (control) or pTM1-MDR1 wild type (WT), pTM1-MDR1-E556Q (E556Q), pTM1-MDR1-E1201Q (E1201Q), and pTM1-MDR1-E556/1201Q (E556Q/ E1201Q, double mutant). Login to comment 148 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln event (Figure 2A, panel I), the mutants E556Q and E1201Q exhibit drastically reduced Vi-induced [R-32 P]-8-azidoADP trapping (<10% compared to wild-type Pgp). Login to comment 149 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln These observations suggest that the defect in the mutants (E556Q and E1201Q) arises from their inability to initiate a second ATP hydrolysis event after the release of the nucleoside diphosphate. Login to comment 151 ABCB1 p.Glu556Gln To address this question, we monitored [R-32 P]-8-azidoATP binding at 4 °C to wild-type and E556Q Pgp before and after trapping with 8-azidoADP and Vi (Figure 2B). Login to comment 152 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln We then washed off excess 8-azidoATP and Vi by centrifugation and incubated the samples at 37 °C for 15 min to release the 8-azidoADP, brought the FIGURE 2: Analyses of the various steps in the catalytic cycle of wild-type and E556Q and E1201Q mutant Pgps. Login to comment 153 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln (A) Time course of Vi-induced trapping, release, and retrapping of [R-32P]-8-azidoADP into wild-type, E556Q, and E1201Q Pgps. Login to comment 164 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln Key for panels I, II, and III: (b) Pgp wild-type, (2) Pgp-E556Q, and (9) Pgp-E1201Q. Login to comment 165 ABCB1 p.Glu556Gln (B) Binding of [R-32P]-8-azidoATP to wild-type and E556Q Pgp at various steps in the catalytic cycle. Login to comment 172 ABCB1 p.Glu556Gln (C) Binding of IAAP to wild-type and E556Q Pgp during different steps in the catalytic cycle. Login to comment 175 ABCB1 p.Glu556Gln In (B) and (C) the empty bars represent wild-type Pgp and the filled bars the E556Q Pgp. Login to comment 177 ABCB1 p.Glu556Gln Both the wild-type and mutant E556Q Pgp show a marked decrease in the incorporation of [R-32 P]-8-azidoATP into Pgp in the Vi-trapped state (Figure 2B, step 2), consistent with our previous work (23), which is restored to normal levels after dissociation of 8-azidoADP (Figure 2B, step 3). Login to comment 178 ABCB1 p.Glu556Gln Thus, the binding of nucleotide is normal, and it is the second ATP hydrolysis event per se that appears to be impaired in the mutant E556Q. Login to comment 179 ABCB1 p.Glu1201Gln Similar results were also obtained with the E1201Q mutant Pgp (data not shown). Login to comment 183 ABCB1 p.Glu556Gln The mutant (E556Q) Pgp also shows reduced binding of IAAP in the Vi-trapped state. Login to comment 184 ABCB1 p.Glu556Gln However, in contrast to wild-type protein, IAAP binding to the E556Q mutant Pgp is not recovered after incubation with 1 mM ATP, under hydrolysis conditions (Figure 2C, steps 2 and 3). Login to comment 186 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln The experiment in Figure 1C shows that the double mutant E556/1201Q Pgp is able to trap [R-32 P]-8-azidoADP to the same extent in the absence or presence of Vi, a phenomenon that is distinct from that exhibited by wild-type Pgp or the single mutants (E556Q and E1201Q). Login to comment 188 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln However, unlike the wild type and E556Q or E1201Q mutant, the double mutant failed to release [R-32 P]-8-azidoADP trapped in both the presence and absence of Vi (compare Figure 2A, panel II, and Figure 3). Login to comment 217 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln Similarly, the distribution of the trapped [R-32 P]-8-azidoADP was also found to be more or less equal in both the Nand the C-terminal ATP sites in the single mutant Pgps, E556Q and E1201Q (data not given). Login to comment 241 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln We find that the requirement for cations, during trapping of [R-32 P]-8-azidoADP, is similar for the wild-type and mutant Pgps E556Q, E1201Q, and E556/ 1201Q. Login to comment 261 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln Intact HeLa Cells Expressing the Mutant (E556Q, E1201Q, and E556/1201Q) Pgps Show Reduced Binding of the Drug Substrate, IAAP. Login to comment 267 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln To test this hypothesis, intact HeLa cells overexpressing the wild-type or the mutant (E556Q, E1201Q, E556/1201Q) Pgps were incubated with IAAP and photocross-linked. Login to comment 275 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln and Figure 6 demonstrates that wild-type Pgp shows binding of IAAP (which is sensitive to the Pgp modulator cyclosporin A), whereas the mutant Pgps (E556Q, E1201Q, and E556/ 1201Q) show significantly reduced binding of IAAP. Login to comment 278 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln The experiments described above indicate that substitution of E556, in the N-terminal ATP site, or its equivalent residue (E1201) with Q, in the C-terminal ATP site, or both (E556Q, E1201Q, or E556/ 1201Q) does not have any significant effect on the cleavage of -γ-phosphate bond of ATP per se but affects subsequent steps in the catalytic cycle of Pgp. This is particularly significant because E556 or E1201 (and its homologues in other ABC transporters as well as other ATPases) has been implicated as the catalytic carboxylate (10, 13, 57). Login to comment 279 ABCB1 p.Glu1201Ala ABCB1 p.Glu556Ala To assess whether a Glu to Ala substitution will affect the cleavage of the -γ-phosphate bond of ATP, we generated the mutant Pgps where Glu was replaced with Ala (E556A, E1201A, and E556/1201A). Login to comment 280 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln ABCB1 p.Glu1201Ala ABCB1 p.Glu556Ala Characterization of these mutants showed that (1) they exhibited comparable cell surface expression, (2) similar to E f Q (see Figure 1A), E f A substitutions also resulted in loss of transport activity, and (3) the single mutants E556A and E1201A, similar to E556Q and E1201Q (see Figure 1C), showed some trapping of [R-32 P]-8-azidoADP in the absence of Vi, which was enhanced in the presence of 0.25 mM Vi (data not shown). Login to comment 294 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln ABCB1 p.Glu1201Ala ABCB1 p.Glu1201Ala ABCB1 p.Glu556Ala ABCB1 p.Glu556Ala Human Pgp mutants of the conserved glutamate residue in the Walker B region (E556Q, E556A, E1201Q, E1201A, E556/1201Q, and E556/1201A) showed cell surface expression levels comparable to that of the wild-type protein, but the transport function was abrogated in all of the mutant Pgps (Figure 1A,B; data for E556A, E1201A, and E556/1201A not shown). Login to comment 295 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln Moreover, our results are in agreement with the findings in Mdr3 that show that replacement of Glu 556 or Glu 1201 with Gln causes a loss of steady-state ATPase activity but permits Vi-induced trapping of [R-32 P]-8-azidoADP (Figure 1C and ref 10). Login to comment 297 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln But, whether the glutamate residue is necessary for ATP hydrolysis cannot be addressed on the basis of this evidence as only one of the two ATP sites is modified and there is the confounding influence of the FIGURE 6: Photoaffinity labeling of wild-type and mutant (E556Q, E1201Q, and E556/1201Q) Pgps in intact HeLa cells. Login to comment 333 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln ABCB1 p.Glu1201Ala ABCB1 p.Glu1201Ala ABCB1 p.Glu556Ala ABCB1 p.Glu556Ala The single mutants (E556Q, E556A, E1201Q, and E1201A), on the other hand, show normal release of ADP and can bind ATP during next step but exhibit greatly reduced ability to hydrolyze it (see Figure 2A, panels I-III; data with E556A and E1201A not shown). Login to comment 341 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln The Pgp mutants E556Q and E1201Q might be expected to be fully functional as both NBDs hydrolyze ATP and release ADP to the same extent as wild-type Pgp. Login to comment 346 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln Indeed, the data given in Figure 6 indicate that the binding of IAAP is significantly reduced in the intact cells expressing the mutant Pgps (E556Q, E1201Q, and E556/1201Q, respectively) compared to wild-type Pgp. Login to comment