ABCMdb

ABCB1 p.Glu556Gln

Predicted by SNAP2:	A: D (85%), C: D (80%), D: N (61%), F: D (91%), G: D (91%), H: D (85%), I: D (85%), K: D (91%), L: D (91%), M: N (72%), N: D (85%), P: D (91%), Q: N (53%), R: D (91%), S: D (85%), T: D (85%), V: D (85%), W: D (91%), Y: D (91%),
Predicted by PROVEAN:	A: D, C: D, D: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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[hide] Importance of the conserved Walker B glutamate res... Biochemistry. 2002 Nov 26;41(47):13989-4000. 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 [PMID:12437356]

Abstract [show]
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.

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2 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 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
5 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 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 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 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 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 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 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 Wild-Type, E556Q, E1201Q, and E556/1201Q Pgps Exhibit Differences in the Trapping of [R-32 P]-8-AzidoADP. Login to comment
114 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 The mutant E1201Q Pgp shows a higher level of trapping compared to the E556Q protein in the absence of Vi. Login to comment
117 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 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 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 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 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 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
134 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 The lanes represent (from left to right) wild-type Pgp and the mutant Pgps E556Q, E1201Q, and E556/1201Q, respectively. Login to comment
141 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 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 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 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 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 (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 Key for panels I, II, and III: (b) Pgp wild-type, (2) Pgp-E556Q, and (9) Pgp-E1201Q. Login to comment
165 (B) Binding of [R-32P]-8-azidoATP to wild-type and E556Q Pgp at various steps in the catalytic cycle. Login to comment
172 (C) Binding of IAAP to wild-type and E556Q Pgp during different steps in the catalytic cycle. Login to comment
175 In (B) and (C) the empty bars represent wild-type Pgp and the filled bars the E556Q Pgp. Login to comment
177 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 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
183 The mutant (E556Q) Pgp also shows reduced binding of IAAP in the Vi-trapped state. Login to comment
184 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 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 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 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 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 Intact HeLa Cells Expressing the Mutant (E556Q, E1201Q, and E556/1201Q) Pgps Show Reduced Binding of the Drug Substrate, IAAP. Login to comment
267 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 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 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
280 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 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 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 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 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 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 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

[hide] P-glycoprotein: from genomics to mechanism. Oncogene. 2003 Oct 20;22(47):7468-85. Ambudkar SV, Kimchi-Sarfaty C, Sauna ZE, Gottesman MM
P-glycoprotein: from genomics to mechanism.
Oncogene. 2003 Oct 20;22(47):7468-85., 2003-10-20 [PMID:14576852]

Abstract [show]
Resistance to chemically different natural product anti-cancer drugs (multidrug resistance, or MDR) results from decreased drug accumulation, resulting from expression of one or more ATP-dependent efflux pumps. The first of these to be identified was P-glycoprotein (P-gp), the product of the human MDR1 gene, localized to chromosome 7q21. P-gp is a member of the large ATP-binding cassette (ABC) family of proteins. Although its crystallographic 3-D structure is yet to be determined, sequence analysis and comparison to other ABC family members suggest a structure consisting of two transmembrane (TM) domains, each with six TM segments, and two nucleotide-binding domains. In the epithelial cells of the gastrointestinal tract, liver, and kidney, and capillaries of the brain, testes, and ovaries, P-gp acts as a barrier to the uptake of xenobiotics, and promotes their excretion in the bile and urine. Polymorphisms in the MDR1 gene may affect the pharmacokinetics of many commonly used drugs, including anticancer drugs. Substrate recognition of many different drugs occurs within the TM domains in multiple-overlapping binding sites. We have proposed a model for how ATP energizes transfer of substrates from these binding sites on P-gp to the outside of the cell, which accounts for the apparent stoichiometry of two ATPs hydrolysed per molecule of drug transported. Understanding of the biology, genetics, and biochemistry of P-gp promises to improve the treatment of cancer and explain the pharmacokinetics of many commonly used drugs.

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276 Adapted from Sauna et al. (2001a, b) Furthermore, the equivalent mutations in human P-gp, E556Q, E556A, E1201Q, E1201A, and the double mutants E556Q/E1201Q and E556A/E1201A, all allow for normal levels of Vi-dependent [a-32 P]8-azidoADP trapping, and the trapped nucleotide has been demonstrated to be [a-32 P]8-azidoADP (Sauna et al., 2002). Login to comment
277 Thus, the substitutions of the residues E556 and E1201 with Q or A in human P-gp do not block hydrolysis of ATP per se. Interestingly, the double mutants E556Q/ E1201Q and E556A/E1201A show trapping of [a-32 P]8-azidoADP even in the absence of Vi. Login to comment
279 These double mutants of P-gps (E556Q/E1201Q and E556A/E1201A) thus provide an independent validation that the Vi-trapped transition state of P-gp is indeed a 'true` transition state, and they provide an interesting system where one can obtain the transition state of P-gp in the absence of agents such as Vi or beryllium fluoride. Login to comment
326 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 are unable to hydrolyse it. Login to comment

[hide] Analysis of catalytic carboxylate mutants E552Q an... Biochemistry. 2003 Nov 11;42(44):12875-85. Carrier I, Julien M, Gros P
Analysis of catalytic carboxylate mutants E552Q and E1197Q suggests asymmetric ATP hydrolysis by the two nucleotide-binding domains of P-glycoprotein.
Biochemistry. 2003 Nov 11;42(44):12875-85., 2003-11-11 [PMID:14596601]

Abstract [show]
In the nucleotide-binding domains (NBDs) of ABC transporters, such as mouse Mdr3 P-glycoprotein (P-gp), an invariant carboxylate residue (E552 in NBD1; E1197 in NBD2) immediately follows the Walker B motif (hyd(4)DE/D). Removal of the negative charge in mutants E552Q and E1197Q abolishes drug-stimulated ATPase activity measured by P(i) release. Surprisingly, drug-stimulated trapping of 8-azido-[alpha-(32)P]ATP is still observed in the mutants in both the presence and absence of the transition-state analogue vanadate (V(i)), and ADP can be recovered from the trapped enzymes. The E552Q and E1197Q mutants show characteristics similar to those of the wild-type (WT) enzyme with respect to 8-azido-[alpha-(32)P]ATP binding and 8-azido-[alpha-(32)P]nucleotide trapping, with the latter being both Mg(2+) and temperature dependent. Importantly, drug-stimulated nucleotide trapping in E552Q is stimulated by V(i) and resembles the WT enzyme, while it is almost completely V(i) insensitive in E1197Q. Similar nucleotide trapping properties are observed when aluminum fluoride or beryllium fluoride is used as an alternate transition-state analogue. Partial proteolytic cleavage of photolabeled enzymes indicates that, in the absence of V(i), nucleotide trapping occurs exclusively at the mutant NBD, whereas in the presence of V(i), nucleotide trapping occurs at both NBDs. Together, these results suggest that there is single-site turnover occurring in the E552Q and E1197Q mutants and that ADP release from the mutant site, or another catalytic step, is impaired in these mutants. Furthermore, our results support a model in which the two NBDs of P-gp are not functionally equivalent.

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229 Moreover, in their recent work, Sauna and colleagues also demonstrated using Rand γ-labeled 8-azido-[R-32 P]ATP that mutants at the equivalent positions of the human MDR1 protein (E556Q and E556A, E1201Q and E1201A, and the double mutants) are indeed capable of ATP hydrolysis and single-site catalysis (59). Login to comment

[hide] Combined mutation of catalytic glutamate residues ... J Biol Chem. 2004 Jul 23;279(30):31212-20. Epub 2004 May 24. Tombline G, Bartholomew LA, Urbatsch IL, Senior AE
Combined mutation of catalytic glutamate residues in the two nucleotide binding domains of P-glycoprotein generates a conformation that binds ATP and ADP tightly.
J Biol Chem. 2004 Jul 23;279(30):31212-20. Epub 2004 May 24., 2004-07-23 [PMID:15159388]

Abstract [show]
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., Muller, 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.

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48 Two such mutants have been reported, namely the E556Q/E1201Q and E556A/E1201A mutants of human Pgp (28). Login to comment
85 We have found however that the mutations E556Q, E552A, or E1197Q in mouse MDR3 Pgp have no effect on the ability of the protein to undergo repeated cycles of Vi-induced trapping of ADP and release (G. Tombline, L. A. Bartholomew, I. L. Urbatsch, and A. E. Senior, manuscript in preparation). Login to comment
277 It may also be mentioned that Sauna et al. (28) found by UV-photolabeling and trypsin hydrolysis that the 8-azido-ADP occluded by the E556Q/E1201Q mutant of human Pgp after preincubation with 8-azido-ATP was distributed about equally in Nand C-halves of the protein, implying binding in both NBDs (the stoichiometry of bound nucleotide was not determined). Login to comment

[hide] Exploiting reaction intermediates of the ATPase re... J Biol Chem. 2006 Sep 8;281(36):26501-11. Epub 2006 Jul 14. Sauna ZE, Nandigama K, Ambudkar SV
Exploiting reaction intermediates of the ATPase reaction to elucidate the mechanism of transport by P-glycoprotein (ABCB1).
J Biol Chem. 2006 Sep 8;281(36):26501-11. Epub 2006 Jul 14., 2006-09-08 [PMID:16844693]

Abstract [show]
The transport cycle of ABC transporters in general and P-glycoprotein in particular has been extensively studied, but the molecular mechanism remains controversial. We identify stable reaction intermediates in the progression of the P-glycoprotein-mediated ATPase reaction equivalent to the enzyme-substrate (E.S, P-glycoprotein.ATP) and enzyme-product (E.P, P-glycoprotein.ADP.P(i)) reaction intermediates. These have been characterized using the photoaffinity analog 8-azido-[alpha-32P]ATP as well as under equilibrium conditions using [alpha-32P]ATP, in which a cross-linking step is not involved. Similar results were obtained when 8-azido-[alpha-32P]ATP or [alpha-32P]ATP was used. The reaction intermediates were characterized based on their kinetic properties and the nature (triphosphate/diphosphate) of the trapped nucleotide. Using this defined framework and the Walker B E556Q/E1201Q mutant that traps nucleotide in the absence of vanadate or beryllium fluoride, the high to low affinity switch in the transport substrate binding site can be attributed to the formation of the E.S reaction intermediate of the ATPase reaction. Importantly, the posthydrolysis E.P state continues to have low affinity for substrate, suggesting that conformational changes that form the E.S complex are coupled to the conformational change at the transport substrate site to do mechanical work. Thus, the formation of E.S reaction intermediate during a single turnover of the catalytic cycle appears to provide the initial power stroke for movement of drug substrate from inner leaflet to outer leaflet of lipid bilayer. This novel approach applies transition state theory to elucidate the mechanism of P-glycoprotein and other ABC transporters and has wider applications in testing cause-effect hypotheses in coupled systems.

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5 Using this defined framework and the Walker B E556Q/E1201Q mutant that traps nucleotide in the absence of vanadate or beryllium fluoride, the high to low affinity switch in the transport substrate binding site can be attributed to the formation of the E⅐S reaction intermediate of the ATPase reaction. Login to comment
33 If this were so, the double mutant (E556Q/ E1201Q) may, in the presence of nucleotide, represent a prehydrolysis transition state of the Pgp-catalyzed ATP hydrolysis reaction. Such a view is consistent with several recent structures of the NBDs of ABC proteins that suggest that ATP acts as molecular glue bringing together the two NBDs to form the ATP sandwich dimer. Login to comment
37 We also show strong coupling between the NBDs and the transport substrate site(s) in purified and reconstituted wild-type and mutant (E556Q/E1201Q) Pgps. Login to comment
40 We estimated the activation energies for the formation of the nucleotide triphosphate-trapped (prehydrolysis) state using the mutant (E556Q/E1201Q) Pgp as well as the nucleotide diphosphate-trapped (posthydrolysis) state. Login to comment
48 Preparation of Crude Membranes from High Five Insect Cells Infected with Recombinant Baculovirus Carrying the Wild-type and Mutant Human MDR1 Gene-High Five insect cells (Invitrogen) were infected with the recombinant baculovirus carrying the human MDR1 cDNA (either wild type or the mutant, E556Q/E1201Q) with a His6 tag at the C-terminal end as described (9). Login to comment
73 [␣-32 P]ATP or [␣-32 P]ADP Trapping-Purified wild type and E556Q/E1201Q mutant Pgps reconstituted into liposomes were incubated with 200 ␮M [␣-32 P]ATP for 5 min in the ATPase buffer (see above). Login to comment
103 RESULTS The Mutant Pgp E556Q/E1201Q Exhibits Very Low Levels of ATP Hydrolysis but Occludes [␣-32 P]ATP in the Transition State-Several groups have studied the role of the highly conserved glutamates within the Walker B region of the Nand the C-ATP sites of mouse (22) and human (20) Pgp. Login to comment
104 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). Login to comment
108 Since the latter is the more direct method, we overexpressed human Pgp (both wild-type and the E556Q/E1201Q mutant) in High Five insect cells, prepared crude membranes, purified the Pgp, and reconstituted into proteoliposomes as described under "Experimental Procedures." Login to comment
110 The mutant Pgp (E556Q/E1201Q), on the other hand, shows negligible ATP hydrolysis, and Vi has no effect on the reaction. Login to comment
121 The E556Q/E1201Q Mutant and Wild-type Pgps Show Comparable Affinities for Nucleotides-As shown in Fig. 1, the mutant Pgp occludes [␣-32 P]ATP such that it cannot be exchanged by a 50-fold excess of cold ATP. Login to comment
124 Hydrolysis of [␣-32 P]ATP and occlusion of [␣-32 P]ATP/ADP by wild-type and the E556Q/E1201Q mutant Pgp. Login to comment
138 Determination of the affinities of nucleotides for wild-type and the E556Q/E1201Q mutant Pgps. A, the apparent Kd (8-azido-ATP) was estimated by determining the photolabeling of reconstituted purified Pgps in the presence of increasing concentrations of 8-azido-[␣-32 P]ATP at 4 °C, as described under "ExperimentalProcedures. Login to comment
148 The graphs depict incorporation of nucleotide in wild-type Pgp (F) and the E556Q/E1201Q mutant Pgp (E). Login to comment
154 We compared the apparent Kd (8-azido-ATP) for the binding of 8-azido-[␣-32 P]ATP at 4 °C. Fig. 2A shows that both wild-type and the E556Q/E1201Q mutant Pgps have comparable affinities (11.07 Ϯ 0.4 ␮M and 10.7 Ϯ 0.28 ␮M, respectively). Login to comment
163 The Energetics of Nucleotide Trapping in the E556Q/E1201Q Mutant and Wild-type Pgps-The data in Fig. 2 suggest that the Glu 3 Gln mutant Pgp attains the ATP-trapped state via a conformational change and is not a consequence of inherent greater affinity for nucleotide(s). Login to comment
166 Whereas it is impossible to trap the Pgp⅐MgATP transition state of the wild-type protein in the absence of Vi or other transition state analogs of Pi, the mutant Pgp, E556Q/E1201Q, by preventing cleavage of the ␥-phosphate, permits the experimental characterization of the Pgp (E556Q/E1201Q)⅐MgATP reaction intermediate. Login to comment
172 Thermal titrations of nucleotide occlusion in wild-type and the E556Q/E1201Q mutant Pgps. A, purified wild-type and mutant Pgps (100 ␮g of protein/ml) reconstituted into proteoliposomes were incubated with 50␮M 8-azido[␣-32 P]ATPintheabsenceorpresenceofVifor5minatdifferent temperatures in the range of 4-37 °C. Login to comment
193 Moreover, these data support the proposition that the mutant Pgp occludes ATP as a reaction intermediate, Pgp(E556Q/E1201Q)⅐MgATP, which may be equivalent to the Pgp⅐MgATP (or the E⅐S) state in the catalytic cycle of Pgp. Login to comment
210 Kinetics of Nucleotide Occlusion and Inhibition of [125 I]IAAP Binding in the E556Q/E1201Q Mutant Pgp-We have demonstrated above that the formation of the E⅐S and E⅐P states of the ATPase reaction and the concomitant decrease in binding of TABLE 1 Activation energy (Eact) values for the formation of different reaction intermediates during Pgp-mediated ATP hydrolysis Activation energy (Eact) ϭ -(slope) 2.3R. Login to comment
212 Formation of the Pgp⅐ATP (in the E556Q/E1201Q mutant Pgp) or Pgp⅐ADP⅐Vi (in the wild-type Pgp) reaction intermediates is a slow process that occurs over several minutes (see supplemental Fig. S2). Login to comment
218 Reaction Nucleotide trappinga Inhibition of IAAP bindingb Azido-ATP/ADP ATP/ADP kJ/mol Vi-induced nucleotide trapping in wild-type Pgpc 65.3 Ϯ 9.7 43 Ϯ 7.9 49.3 Ϯ 8.8 BeFx-induced nucleotide trapping in wild-type Pgpd 62.4 Ϯ 10.3 33.4 Ϯ 5.9 44.1 Ϯ 6.1 Vior BeFx-independent nucleotide trapping in mutant Pgp (E556Q/E1201Q)e 63.4 Ϯ 8.3 37 Ϯ 4.3 44.1 Ϯ 5.7 a Nucleotide trapping was initiated by using either 8-azido-͓␣-32 P͔ATP or ͓␣-32 P͔ATP. Login to comment
220 The incubation with wild-type Pgp was carried out in the presence of Vi and in the absence of Vi for the E556Q/E1201Q mutant Pgp. Login to comment
231 For purification and reconstitution of the wild type and E556Q/E1201Q double mutant, we used protocol that allows Ͼ90% recovery of verapamil-stimulated ATPase activity (23, 25, 26). Login to comment
233 In addition, when we fit the curves taking into account these values, we found that the maximal stoichiometry FIGURE4.Thermaltitrationsoftransportsubstrate,[125 I]IAAP,bindingto Pgp following occlusion of nucleotides in wild-type and mutant Pgps. A, purified Pgps, wild-type (E) and the E556Q/E1201Q mutant (‚) reconstituted intoproteoliposomes(100␮gofprotein/ml),wereincubatedfor5min(under equilibrium conditions for nucleotide trapping) with 1 mM ATP at different temperaturesintherangeof4-37 °C.Thewild-typePgpwasincubatedinthe presence of 250 ␮M Vi, and the mutant Pgp was incubated in the absence of Vi. Login to comment
246 Kinetics of nucleotide occlusion and inhibition of [125 I]IAAP binding in wild-type and E556Q/E1201Q mutant Pgps. A, purified Pgps (wild type and the E556Q/E1201Q mutant) reconstituted into proteoliposomes (100 ␮g/ml) were incubated with increasing concentrations of [␣-32 P]ATP at 34 °C for 5 min. Login to comment
251 Incorporation of [␣-32 P]ADP/ATP into wild type (f) and mutant Pgps (F) is depicted. B, purified mutant Pgp (E556Q/E1201Q) reconstituted into proteoliposomes (100 ␮g/ml) was incubated with increasing concentrations of ATP for 5 min at 34 °C and then transferred to ice. Login to comment
269 Additionally, Table 1 shows that the activation energy for BeFx-induced trapping (33.4 kJ/mol) is comparable with that obtained for Vi-independent trapping in the E556Q/E1201Q mutant Pgp (37 kJ/mol). Login to comment
289 The E556Q/E1201Q mutant Pgp can trap [␣-32 P]ATP in the absence of Vi (Fig. 1B) (20, 22), and we demonstrate in this study that this is not due to altered affinities for nucleotides (Fig. 2). Login to comment
313 The Pgp⅐ATP intermediate is equivalent to the E⅐S complex and captured by the experimental stratagem of mutations (e.g. E556Q/E1201Q mutant) in the ATP sites of Pgp that drastically reduces the hydrolysis of the ␥-phosphate or by BeFx-mediated trapping of nucleotide in wild-type protein. Login to comment

[hide] About a switch: how P-glycoprotein (ABCB1) harness... Mol Cancer Ther. 2007 Jan;6(1):13-23. Sauna ZE, Ambudkar SV
About a switch: how P-glycoprotein (ABCB1) harnesses the energy of ATP binding and hydrolysis to do mechanical work.
Mol Cancer Ther. 2007 Jan;6(1):13-23., [PMID:17237262]

Abstract [show]
The efflux of drugs by the multidrug transporter P-glycoprotein (Pgp; ABCB1) is one of the principal means by which cancer cells evade chemotherapy and exhibit multidrug resistance. Mechanistic studies of Pgp-mediated transport, however, transcend the importance of this protein per se as they help us understand the transport pathway of the ATP-binding cassette proteins in general. The ATP-binding cassette proteins comprise one of the largest protein families, are central to cellular physiology, and constitute important drug targets. The functional unit of Pgp consists of two nucleotide-binding domains (NBD) and two transmembrane domains that are involved in the transport of drug substrates. Early studies postulated that conformational changes as a result of ATP hydrolysis were transmitted to the transmembrane domains bringing about drug transport. More recent structural and biochemical studies on the other hand suggested that ATP binds at the interface of the two NBDs and induces the formation of a closed dimer, and it has been hypothesized that this dimerization and subsequent ATP hydrolysis powers transport. Based on the mutational and biochemical work on Pgp and structural studies with isolated NBDs, we review proposed schemes for the catalytic cycle of ATP hydrolysis and the transport pathway.

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90 In human Pgp, the double-mutant E556Q/E1201Q does not show steady-state hydrolysis but can occlude the nucleoside triphosphate in a reaction intermediate (21, 55). Login to comment
106 Figure 1 shows likely transition states during the formation of the occluded conformation, which occurs in mutants of Pgp where both ''catalytic carboxylates`` have been altered (E556Q/E1201Q for human Pgp). Login to comment
112 By using the E599Q mutant (equivalent to the human Pgp mutant E556Q/ E1201Q described above) and site-specific labeling with a fluorophore, they were able to monitor the kinetics of the occlusion in real time and obtain initial rates. Login to comment
198 However, it is not clear from these data whether the nonhydrolyzable analogues of ATP represent the ''nucleotide bound`` state or the ''occluded nucleotide`` state of Pgp. Recent work from our laboratory (58) suggests that the formation of the ATP-occluded conformation of the mutant human Pgp E556Q/E1201Q is energy dependent with an activation energy of f70 kJ/mol, a value that is comparable with that obtained for ATP-driven dimerization of an equivalent mutant of Mdl1 (50). Login to comment
200 We have furthermore shown that occlusion of ATP in the mutant human Pgp E556Q/E1201Q is accompanied by a decreased binding of transport substrate. Login to comment
254 Conformational changes in the drug-binding sites during the different reaction intermediates of the ATPase reaction mediated by Pgp Reaction intermediate Trapped intermediate Effect on drug-binding site References E + S Pgp + ATP No effect on IAAP binding (34, 70) E.S Pgp (E556Q/E1201Q).ATP Reduced affinity for IAAP (55, 58) Pgp.AMPPNP Reduced affinity for vinblastine (67, 69) Structural changes in transmembrane domains observed by electron microscopy (67, 78) Conformational changes using the antibody UIC2 (67) Pgp.ATP-g-S Reduced affinity for vinblastine (68) and * E.P Pgp.ADP.Vi Reduced affinity for IAAP (34, 35, 37, 55, 70) Reduced affinity for vinblastine (67-69) Structural changes in transmembrane domains observed by electron microscopy (67, 78) Conformational changes using the antibody UIC2 (67, 79, 80) Rotation of helices TM6 and TM12 (76) E + P Pgp + ADP + Vi (Pi) Reduced affinity for IAAP (34, 35) *Sauna and Ambudkar, unpublished data. Login to comment

[hide] Evidence for a Sav1866-like architecture for the h... FASEB J. 2007 Dec;21(14):3937-48. Epub 2007 Jul 12. Zolnerciks JK, Wooding C, Linton KJ
Evidence for a Sav1866-like architecture for the human multidrug transporter P-glycoprotein.
FASEB J. 2007 Dec;21(14):3937-48. Epub 2007 Jul 12., [PMID:17627029]

Abstract [show]
The recently reported structures of the bacterial multidrug exporter Sav1866 suggest a domain architecture in which both nucleotide-binding domains (NBDs) of this ATP binding cassette (ABC) transporter contact both transmembrane domains (TMDs). Such a domain arrangement is particularly unexpected because it is not found in the structures of three solute importers BtuCD, HI1470/1, and ModBC from the same protein family. There is also no precedent for such an arrangement from biochemical studies with any ABC transporter. Sav1866 is homologous with the clinically relevant human P-glycoprotein (ABCB1). If the structure proposed for Sav1866 is physiologically relevant, the long intracellular loops of P-glycoprotein TMD2 should contact NBD1. We have tested this by using cysteine mutagenesis and chemical cross-linking to verify proximal relationships of the introduced sulfhydryls across the proposed interdomain interface. We report the first biochemical evidence in support of the domain arrangement proposed for the multidrug resistance class of ABC transporters. With a domain arrangement distinctly different from the three solute importers it seems likely that the TMDs of ABC importers and exporters have evolved different mechanisms to couple to common conformational changes at conserved NBDs.

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87 The E556Q mutant was generated by mutation of the wild-type P-glycoprotein using the oligonucleotide E556Q, 5Ј- C C C C A A G A T C C T C C T G C T T G A T C A G G C C A C G - TCAGCCTTGG-3Ј. Login to comment
151 Untransfected and mock-transfected cells and cells expressing an inactive form of P-glycoprotein [the E556Q mutant (36)] are indistinguishable from each other and accumulate drug at a high rate, demonstrating that the efflux observed in the cells transfected with pMDR-wt is due to P-gp activity. Login to comment
189 In (A) the initial rates of Bodipy-taxol uptake by cells expressing E556Q and mock-transfected cells (dashed, blue and green lines, respectively) are described by the right-hand ordinate. Login to comment
207 Efflux activity of cysteine mutants wt Pgp-cys- L443C S909C L443CϩS909C S474C R905C S474CϩR905C E556Q Mock Mean % activity 100 100.8 100.7 99.9 100.6 99.5 96.9 101.2 8.6 0.0 (Ϯse) Ϯ 0.6 Ϯ 0.8 Ϯ 0.4 Ϯ 0.5 Ϯ 1.4 Ϯ1.7 Ϯ0.9 Ϯ1.2 Ϯ 11.9 Ϯ 8.9 n 6 6 2 2 4 2 4 2 6 4 The efflux activity of the mutants generated in this study is presented as a percentage of the drug efflux activity of wild-type P-glycoprotein (wt), as described in Materials and Methods. Login to comment

[hide] Nucleotide binding, ATP hydrolysis, and mutation o... Biochemistry. 2007 Aug 14;46(32):9328-36. Epub 2007 Jul 18. Loo TW, Bartlett MC, Clarke DM
Nucleotide binding, ATP hydrolysis, and mutation of the catalytic carboxylates of human P-glycoprotein cause distinct conformational changes in the transmembrane segments.
Biochemistry. 2007 Aug 14;46(32):9328-36. Epub 2007 Jul 18., 2007-08-14 [PMID:17636884]

Abstract [show]
P-Glycoprotein (P-gp, ABCB1) transports a variety of structurally unrelated cytotoxic compounds out of the cell. Each homologous half of P-gp has a transmembrane (TM) domain containing six TM segments and a nucleotide-binding domain (NBD) and is joined by a linker region. It has been postulated that binding of two ATP molecules at the NBD interface to form a "nucleotide sandwich" induces drug efflux by altering packing of the TM segments that make up the drug-binding pocket. To test if ATP binding alone could alter packing of the TM segments, we introduced catalytic carboxylate mutations (E556Q in NBD1 and E1201Q in NBD2) into double-cysteine mutants that exhibited ATP-dependent cross-linking so that the mutants could bind but not hydrolyze ATP. It was found that ATP binding alone could alter disulfide cross-linking between the TM segments. For example, ATP inhibited cross-linking of mutant L339C(TM6)/V982C(TM12)/E556Q(NBD1)/E1201Q(NBD2) but promoted cross-linking of mutant F343C(TM6)/V982C(TM12)/E556Q(NBD1)/E1201Q(NBD2). Cross-linking of some mutants, however, appeared to require ATP hydrolysis as introduction of the catalytic carboxylate mutations into mutant L332C(TM6)/L975C(TM12) inhibited ATP-dependent cross-linking. Cross-linking between cysteines in the TM segments also could be altered via introduction of a single catalytic carboxylate mutation into mutant L332C(TM6)/L975C(TM12) or by using the nonhydrolyzable ATP analogue, AMP.PNP. The results show that the TM segments are quite sensitive to changes within the ATP-binding sites because different conformations could be detected in the presence of ATP, AMP.PNP, during ATP hydrolysis or through mutation of the catalytic carboxylates.

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3 To test if ATP binding alone could alter packing of the TM segments, we introduced catalytic carboxylate mutations (E556Q in NBD1 and E1201Q in NBD2) into double-cysteine mutants that exhibited ATP-dependent cross-linking so that the mutants could bind but not hydrolyze ATP. Login to comment
36 Recently, it was shown that introduction of the E556Q- (NBD1) and E1201Q(NBD2) mutations into P-gp yielded a mutant that did not hydrolyze ATP but exhibited normal affinity for ATP (32). Login to comment
40 Modifications to the P-gp cDNAs to introduce the catalytic carboxylate mutations, E556Q or E1201Q, were performed by oligonucleotide-directed mutagenesis (35). Login to comment
74 The positions of the catalytic carboxylate mutations (E556Q in NBD1 and E1201Q in NBD2) and the cysteine mutations in the TM segments used in the disulfide cross-linking studies (L332C, L339C, F343C, F728C, L975C, and V982C) are shown. Login to comment
106 Membranes were prepared from HEK 293 cells expressing P-gp mutants L339C- (TM6)/V982C(TM12) or L339C(TM6)/V982C(TM12)/E556Q- (NBD1)/E1201Q(NBD2) (A) and F343C(TM6)/V982C(TM12) or F343C(TM6)/V982C(TM12)/E556Q(NBD1)/E1201Q(NBD2) (B). Login to comment
111 Since the E556Q mutation inhibited the verapamil-stimulated ATPase activity of Cys-less P-gp (>90%), the results are consistent with finding that cross-linking between Cys332- (TM6) and Cys975(TM12) requires ATP hydrolysis. Login to comment
115 The results suggest that the conformation of the TM segments is quite sensitive to changes in the ATP-binding site and that the E556Q and E1201Q mutations induce asymmetric changes in the TMDs. Login to comment

[hide] Catalytic cycle of ATP hydrolysis by P-glycoprotei... Biochemistry. 2007 Dec 4;46(48):13787-99. Epub 2007 Nov 8. Sauna ZE, Kim IW, Nandigama K, Kopp S, Chiba P, Ambudkar SV
Catalytic cycle of ATP hydrolysis by P-glycoprotein: evidence for formation of the E.S reaction intermediate with ATP-gamma-S, a nonhydrolyzable analogue of ATP.
Biochemistry. 2007 Dec 4;46(48):13787-99. Epub 2007 Nov 8., 2007-12-04 [PMID:17988154]

Abstract [show]
Structural and biochemical studies of ATP-binding cassette (ABC) transporters suggest that an ATP-driven dimerization of the nucleotide-binding domains (NBDs) is an important reaction intermediate of the transport cycle. Moreover, an asymmetric occlusion of ATP at one of the two ATP sites of P-glycoprotein (Pgp) may follow the formation of the symmetric dimer. It has also been postulated that ADP drives the dissociation of the dimer. In this study, we show that the E.S conformation of Pgp (previously demonstrated in the E556Q/E1201Q mutant Pgp) can be obtained with the wild-type protein by use of the nonhydrolyzable ATP analogue ATP-gamma-S. ATP-gamma-S is occluded into the Pgp NBDs at 34 degrees C but not at 4 degrees C, whereas ATP is not occluded at either temperature. Using purified Pgp incorporated into proteoliposomes and ATP-gamma-35S, we demonstrate that the occlusion of ATP-gamma-35S has an Eact of 60 kJ/mol and the stoichiometry of ATP-gamma-35S:Pgp is 1:1 (mol/mol). Additionally, in the conserved Walker B mutant (E556Q/E1201Q) of Pgp, we find occlusion of the nucleoside triphosphate but not the nucleoside diphosphate. Furthermore, Pgp in the occluded nucleotide conformation has reduced affinity for transport substrates. These data provide evidence for the ATP-driven dimerization and ADP-driven dissociation of the NBDs, and although two ATP molecules may initiate dimerization, only one is driven to an occluded pre-hydrolysis intermediate state. Thus, in a full-length ABC transporter like Pgp, it is unlikely that there is complete association and disassociation of NBDs and the occluded nucleotide conformation at one of the NBDs provides the power-stroke at the transport-substrate site.

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40 The E556Q/E1201Q and the E552A/ E1197A double mutants of human and mouse Pgps, respectively, have been extensively characterized in recent years (16, 19-22). Login to comment
56 The kinetics and thermodynamics of ATP-γ-S occlusion in wild-type Pgp are comparable to those for the occlusion of ATP in the mutant (E556Q/Q1201Q) Pgp, described previously (16). Login to comment
57 Finally, we show, using the Walker B glutamate (E556Q/E1201Q) mutant of Pgp, that the occluded nucleotide conformation could be generated with 8-azido-[R-32 P]ATP but not 8-azido-[R-32 P]ADP, which supports the hypothesis that the γ-phosphate plays a critical role in driving the closure of the NBDs (14). Login to comment
84 Trapping of 8-Azido-[R-32 P]ATP into Mutant (E556Q/ E1201Q) Pgp. Crude membranes of High Five insect cells (100 µg) or purified and reconstituted protein (5-10 µg) were incubated in the ATP assay buffer (see above) containing 50 µM 8-azido-[R-32 P]ATP (5 µCi/nmol) in the dark at 34 °C for 5 min. Login to comment
92 However, we found in a previous study that occlusion of ATP in the E556Q/E1201Q mutant Pgp is maximal between 30 and 34 °C, and there is a substantial (~20%) decrease at 37 °C (16). Login to comment
134 Purified wild-type and E556Q/E1201Q mutant Pgps reconstituted into liposomes were incubated with 200 µM [R-32 P]ATP or [R-32 P]- ADP for 20 min in ATPase buffer (see above). Login to comment
180 In addition, we have demonstrated that the double mutant of human Pgp, E556Q/E1201Q, occludes ATP in the absence of Vi and this conformation of Pgp also shows reduced binding of [125 I]IAAP (16, 19). Login to comment
182 The temperature-dependent occlusion of ATP-γ-S shown in Figure 1 is analogous to the Vi-independent occlusion of ATP in the Walker B glutamate (E556Q/E1201Q) mutant of Pgp, a pre-hydrolysis reaction intermediate. Login to comment
234 Previous work has shown that, unlike ATP binding, Vi-induced trapping of nucleoside diphosphate and Vi-independent occlusion of nucleoside triphosphate in the Pgp mutant E556Q/E1201Q are both strongly temperature-dependent (16, 29). Login to comment
240 Our previous work with the E556Q/ E1201Q mutant human Pgp (16) and the data presented in this study support the view that if ATP hydrolysis cannot occur, the transporter can be locked in the ATP-bound conformation. Login to comment
241 To test this hypothesis, we compared the occlusion of 8-azido-[R-32 P]ATP and 8-azido-[R-32 P]ADP in wild-type and mutant (E556Q/E1201Q) Pgps. Login to comment
253 The mutant Pgp (E556Q/ E1201Q), on the other hand, occludes nucleotide even in the absence of Vi or BeFx when it is incubated with 8-azido- [R-32 P]ATP at 34 °C (Figure 4A, lanes 7-9). Login to comment
258 Our previous studies using crude membranes from HeLa cells infected with vTF7-3 and transfected with vector pTM1-MDR1 (wild type or mutants) suggested that the double mutant (E556Q/E1201Q) occludes either 8-azido-[R-32 P]ATP or 8-azido-[R-32 P]ADP (19). Login to comment
262 We therefore compared the occlusion of [R-32 P]ATP and [R-32 P]ADP into wild-type Pgp and the mutant E556Q/E1201Q using purified human Pgp reconstituted into proteoliposomes. Login to comment
264 The mutant Pgp, E556Q/E1201Q, occludes [R-32 P]ATP in the absence of Vi but shows minimal occlusion of [R-32 P]ADP. Login to comment
269 Finally we show (Figure 5) that the relative affinities of nucleoside triphosphate and nucleoside diphosphate for the mutant (E556Q/E1201Q) Pgp do not provide the explanation for the lack of occlusion of ADP. Login to comment
272 We next compared the occlusion of ATP and ADP into the mutant Pgp (E556Q/E1201Q). Login to comment
297 The occluded nucleotide conformation (16, 22) has been characterized by using mutants of the conserved glutamate in Walker B (E556Q/ E1201Q in human Pgp) but not by using nonhydrolyzable ATP analogues. Login to comment
299 This is particularly important in understanding the coupling between conformational changes at the TMDs during different steps of the ATPase reaction, where the mutant Pgp could have subtle FIGURE 4: Occlusion of nucleoside triphosphates and nucleoside diphosphates in mutant (E556Q/E1201Q) Pgp. Login to comment
300 (A) Purified Pgps (wild type and E556Q/E1201Q mutant) reconstituted into proteoliposomes (100 µg/mL) were incubated with 50 µM 8-azido-[R-32P]ATP or 50 µM 8-azido-[R-32P]ADP either alone or in the presence of Vi or BeFx at 34 °C for 5 min. Login to comment
303 (B) Purified Pgps (wild type and E556Q/E1201Q mutant) reconstituted into proteoliposomes (25-30 µg/assay) were incubated with 200 µM [R-32P]ATP or [R-32P]ADP either alone (solid bars) or in the presence of Vi (open bars) at 34 °C for 5 min. Login to comment
308 (C) Occlusion of [R-32P]ATP or [R-32P]ADP into the E556Q/E1201Q mutant Pgp was monitored as a function of time as described for panel B. Data were normalized for protein, and the radioactivity at time (t ) 0) was subtracted from the data set. Login to comment
310 (D) Occlusion of [R-32P]ATP (b) or [R-32P]ADP (9) into the E556Q/E1201Q mutant Pgp was monitored as a function of temperature as described for panel B. Data were normalized for the amount of protein recovered in each sample at the end of the assay. Login to comment
324 Previous work with the E556Q/E1201Q mutant of Pgp showed that occlusion of the nucleoside triphosphate has an activation energy of approximately 50 kJ/mol (16). Login to comment
332 We reported earlier that the mutant Pgp, E556Q/E1201Q, in the occluded nucleotide conformation brings about the high-affinity to low-affinity switch (16). Login to comment
335 A comparable decrease in the cross-linking of [125 I]IAAP occurs when Pgp is incubated with increasing concentrations of ATP in the presence of 0.25 mM Vi at 34 °C. Moreover, the IC50 values for the inhibition of [125 I]IAAP binding and occlusion of ATP-γ-S are comparable (compare Figures 1B FIGURE 5: Determination of the affinities of nucleotides for the E556Q/E1201Q mutant Pgp. Login to comment
336 (A) Apparent Kd(8-azido-ATP/8-azido-ADP) for Pgp was estimated by determining the photolabeling of reconstituted purified mutant Pgp (E556Q/E1201Q) in the presence of increasing concentrations of 8-azido-[R-32P]ATP (b) or 8-azido-[R-32P]ADP (O) at 4 °C, as described under Experimental Procedures. Login to comment
337 (B) Apparent affinities of ATP and ADP were estimated by incubating increasing concentrations of either ATP (b) or ADP (O) with purified reconstituted mutant (E556Q/E1201Q) Pgp (5-10 µg of protein) and 10 µM 8-azido-[R-32P]ATP (8-10 µCi/nmol) at 4 °C, followed by irradiation at 365 nm on ice for 5 min. Login to comment
339 (C) Purified reconstituted mutant (E556Q/E1201Q) Pgp (25-35 µg of protein) was incubated with increasing concentrations of either ATP (b) or ADP (O) at 34 °C for 5 min. Login to comment
374 The occluded nucleotide (E‚S) can be obtained either by use of wild-type Pgp and ATP-γ-S (Figures 1-3) or by use of the Walker B, E556Q/E1201Q double mutant of human Pgp (16, 22, 23). Login to comment
375 with Walker B glutamate mutants of Pgp (E556Q/E1201Q in human and E552A/E1197A in mouse) have characterized the occluded nucleotide conformation (16, 19-22). Login to comment
377 We show in Figure 3A, using ATP-γ-35 S, that occlusion is strongly temperature-dependent, comparable to the occlusion of [R-32 P]ATP in the mutant human Pgp, E556Q/E1201Q. Login to comment
388 Similarly, we find that the Pgp mutant (E556Q/ E1201Q) occludes 8-azido-[R-32 P]ATP in the absence of Vi or BeFx, although the mutant Pgp cannot occlude 8-azido- [R-32 P]ADP or [R-32 P]ADP (Figure 4). Login to comment

[hide] Genomics and the mechanism of P-glycoprotein (ABCB... J Bioenerg Biomembr. 2007 Dec;39(5-6):481-7. Sauna ZE, Kim IW, Ambudkar SV
Genomics and the mechanism of P-glycoprotein (ABCB1).
J Bioenerg Biomembr. 2007 Dec;39(5-6):481-7., [PMID:18058211]

Abstract [show]
The development of effective clinical interventions against multidrug resistance (MDR) in cancer remains a significant challenge. Single nucleotide polymorphisms (SNPs) contribute to wide variations in how individuals respond to medications and there are several SNPs in human P-glycoprotein (P-gp) that may influence the interactions of drug-substrates with the transporter. Interestingly, even some of the synonymous SNPs have functional consequences for P-gp. It is also becoming increasingly evident that an understanding of the transport pathway of P-gp may be necessary to design effective modulators. In this review we discuss: (1) The potential importance of SNPs (both synonymous and non-synonymous) in MDR and (2) How new concepts that have emerged from structural studies with isolated nucleotide binding domains of bacterial ABC transporters have prompted biochemical studies on P-gp, leading to a better understanding of the mechanism of P-gp mediated transport. Our results suggest that the power-stroke is provided only after formation of the pre-hydrolysis transition-like (E.S) state during ATP hydrolysis.

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108 In addition, we characterized the double mutant (E556Q/E1201Q) where the glutamates in both NBDs were simultaneously mutated (Sauna et al. 2002). Login to comment
110 Additional studies from our laboratory (using the E556Q/E1201Q mutant of human P-gp) demonstrated that the occlusion of ATP is strongly temperature-dependent and that the occluded nucleotide conformation shows reduced binding of the transport-substrate [125 I]iodoarylazidoprazosin ([125 I]IAAP) (Sauna et al. 2006). Login to comment
115 Double mutants of the conserved glutamate, however, are hydrolysis deficient and cannot generate ADP and occlude ATP in a pre-hydrolysis intermediate, P-gp(E556Q/E1201Q)·MgATP (see Fig. 2) and represent the enzyme-substrate (E·S) state. Login to comment
117 Both the Senior group and we have demonstrated that unlike the ATP-driven dimers obtained with isolated NBDs, which exhibit a stoichiometry of 2 mol ATP per dimer (see for example, Smith et al. 2002), P-gp shows a maximal P-gp + MgATP P-gp•MgATP P-gp•MgADP•Pi P-gp•MgADP•Vi Vi Pi Wild-type Mutant E556/1201Q P-gp + MgATP P-gp•MgATP P-gp•MgADP•Pi E•P E•S ATP in exchangeable form Trapped ADP in non-exchangeable form "Vi-trapped state" Occluded ATP in non-exchangeable form "Occluded-nucleotide conformation" Fig. 2 Schematic showing the ATPase reaction in wild-type and mutant (E556Q/E1201Q) P-gps. Login to comment
119 The E556Q/E1201Q mutant P-gp shows minimal ATP hydrolysis and "occludes" MgATP in a non-exchangeable form. Login to comment
132 Our studies with the P-gp mutant (E556Q/E1201Q) also allow us to address the question of what role, if any, the occluded nucleotide conformation plays in the transport pathway. Login to comment
136 The formation of the occluded nucleotide conformation of the mutant human P-gp, E556Q/E1201Q, has an activation energy of ~60 kJ/mol and the activation energies for nucleotide trapping and decrease in drug binding are equivalent (Sauna et al. 2006). Login to comment
139 Conclusions A substantive body of work now exists that suggests that SNPs in the human MDR1 gene (both synonymous and non-synonymous) can affect protein levels as well as 0 5 10 15 20 25 30 35 40 0 50 100 150 200 250 0.00 0.05 0.10 0.15 0.20 0.25 0.30 [125I]IAAPincorporated (arbitraryunits) [α-32P]ATPincorporated (arbitraryunits) ATP ATP NC NC ATP ATP Temperature, oC Fig. 3 Occlusion of ATP in the mutant P-gp (E556Q/E1201Q) is accompanied by a high-affinity to low affinity switch at the TMDs. Login to comment
140 The E556Q/E1201Q mutant P-gp occludes [α-32 P]ATP in a temperature-dependent manner (black squares). Login to comment

[hide] Use of baculovirus BacMam vectors for expression o... Drug Metab Dispos. 2012 Feb;40(2):304-12. Epub 2011 Oct 31. Shukla S, Schwartz C, Kapoor K, Kouanda A, Ambudkar SV
Use of baculovirus BacMam vectors for expression of ABC drug transporters in mammalian cells.
Drug Metab Dispos. 2012 Feb;40(2):304-12. Epub 2011 Oct 31., [PMID:22041108]

Abstract [show]
ATP-binding cassette (ABC) drug transporters ABCB1 [P-glycoprotein (Pgp)] and ABCG2 are expressed in many tissues including those of the intestines, the liver, the kidney and the brain and are known to influence the pharmacokinetics and toxicity of therapeutic drugs. In vitro studies involving their functional characteristics provide important information that allows improvements in drug delivery or drug design. In this study, we report use of the BacMam (baculovirus-based expression in mammalian cells) expression system to express and characterize the function of Pgp and ABCG2 in mammalian cell lines. BacMam-Pgp and BacMam-ABCG2 baculovirus-transduced cell lines showed similar cell surface expression (as detected by monoclonal antibodies with an external epitope) and transport function of these transporters compared to drug-resistant cell lines that overexpress the two transporters. Transient expression of Pgp was maintained in HeLa cells for up to 72 h after transduction (48 h after removal of the BacMam virus). These BacMam-baculovirus-transduced mammalian cells expressing Pgp or ABCG2 were used for assessing the functional activity of these transporters. Crude membranes isolated from these cells were further used to study the activity of these transporters by biochemical techniques such as photo-cross-linking with transport substrate and adenosine triphosphatase assays. In addition, we show that the BacMam expression system can be exploited to coexpress both Pgp and ABCG2 in mammalian cells to determine their contribution to the transport of a common anticancer drug substrate. Collectively, these data demonstrate that the BacMam-baculovirus-based expression system can be used to simultaneously study the transport function and biochemical properties of ABC transporters.

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24 ABBREVIATIONS: ABC, ATP-binding cassette; Pgp, P-glycoprotein; Pgp-EQ, E556Q/E5561206Q nonfunctional Pgp mutant; BacMam, baculovirus-based expression in mammalian cells; FTC, fumitremorgin C; IAAP, iodoarylazidoprazosin; DMEM, Dulbecco`s modified Eagle`s medium; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; ATPase, adenosine triphosphatase; VBL, vinblastine; HEK, human embryonic kidney. Login to comment
75 In brief, BacMam-Pgp (wild-type) or BacMam-Pgp-EQ (E556Q/E1206Q Pgp mutant that is defective in ATP hydrolysis)-transduced cells (2.5 ϫ 105 cells/well) were grown in a monolayer in a 24-well tissue culture plate at 37°C. Login to comment
110 In addition, we also transduced HeLa cells with BacMam-Pgp-EQ virus, which expresses a nonfunctional mutant Pgp (E556Q/ E1201Q) with the glutamate residue in the Walker B motif of each nucleotide-binding domain that is changed to glutamine (Sauna et al., 2002). Login to comment

[hide] The ATPase activity of the P-glycoprotein drug pum... J Biol Chem. 2012 Aug 3;287(32):26806-16. doi: 10.1074/jbc.M112.376202. Epub 2012 Jun 14. Loo TW, Bartlett MC, Detty MR, Clarke DM
The ATPase activity of the P-glycoprotein drug pump is highly activated when the N-terminal and central regions of the nucleotide-binding domains are linked closely together.
J Biol Chem. 2012 Aug 3;287(32):26806-16. doi: 10.1074/jbc.M112.376202. Epub 2012 Jun 14., [PMID:22700974]

Abstract [show]
The P-glycoprotein (P-gp, ABCB1) drug pump protects us from toxic compounds and confers multidrug resistance. Each of the homologous halves of P-gp is composed of a transmembrane domain (TMD) with 6 TM segments followed by a nucleotide-binding domain (NBD). The predicted drug- and ATP-binding sites reside at the interface between the TMDs and NBDs, respectively. Crystal structures and EM projection images suggest that the two halves of P-gp are separated by a central cavity that closes upon binding of nucleotide. Binding of drug substrates may induce further structural rearrangements because they stimulate ATPase activity. Here, we used disulfide cross-linking with short (8 A) or long (22 A) cross-linkers to identify domain-domain interactions that activate ATPase activity. It was found that cross-linking of cysteines that lie close to the LSGGQ (P517C) and Walker A (I1050C) sites of NBD1 and NBD2, respectively, as well as the cytoplasmic extensions of TM segments 3 (D177C or L175C) and 9 (N820C) with a short cross-linker activated ATPase activity over 10-fold. A pyrylium compound that inhibits ATPase activity blocked cross-linking at these sites. Cross-linking between the NBDs was not inhibited by tariquidar, a drug transport inhibitor that stimulates P-gp ATPase activity but is not transported. Cross-linking between extracellular cysteines (T333C/L975C) predicted to lock P-gp into a conformation that prevents close NBD association inhibited ATPase activity. The results suggest that trapping P-gp in a conformation in which the NBDs are closely associated likely mimics the structural rearrangements caused by binding of drug substrates that stimulate ATPase activity.

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263 In support of this prediction, we observed that E556Q and E1201Q mutations to the catalytic carboxylates had different effects on cross-linking between TM segments 6 and 12 (59). Login to comment
256 In support of this prediction, we observed that E556Q and E1201Q mutations to the catalytic carboxylates had different effects on cross-linking between TM segments 6 and 12 (59). Login to comment

[hide] Multiple transport-active binding sites are availa... PLoS One. 2013 Dec 5;8(12):e82463. doi: 10.1371/journal.pone.0082463. eCollection 2013. Chufan EE, Kapoor K, Sim HM, Singh S, Talele TT, Durell SR, Ambudkar SV
Multiple transport-active binding sites are available for a single substrate on human P-glycoprotein (ABCB1).
PLoS One. 2013 Dec 5;8(12):e82463. doi: 10.1371/journal.pone.0082463. eCollection 2013., [PMID:24349290]

Abstract [show]
P-glycoprotein (Pgp, ABCB1) is an ATP-Binding Cassette (ABC) transporter that is associated with the development of multidrug resistance in cancer cells. Pgp transports a variety of chemically dissimilar amphipathic compounds using the energy from ATP hydrolysis. In the present study, to elucidate the binding sites on Pgp for substrates and modulators, we employed site-directed mutagenesis, cell- and membrane-based assays, molecular modeling and docking. We generated single, double and triple mutants with substitutions of the Y307, F343, Q725, F728, F978 and V982 residues at the proposed drug-binding site with cys in a cysless Pgp, and expressed them in insect and mammalian cells using a baculovirus expression system. All the mutant proteins were expressed at the cell surface to the same extent as the cysless wild-type Pgp. With substitution of three residues of the pocket (Y307, Q725 and V982) with cysteine in a cysless Pgp, QZ59S-SSS, cyclosporine A, tariquidar, valinomycin and FSBA lose the ability to inhibit the labeling of Pgp with a transport substrate, [(125)I]-Iodoarylazidoprazosin, indicating these drugs cannot bind at their primary binding sites. However, the drugs can modulate the ATP hydrolysis of the mutant Pgps, demonstrating that they bind at secondary sites. In addition, the transport of six fluorescent substrates in HeLa cells expressing triple mutant (Y307C/Q725C/V982C) Pgp is also not significantly altered, showing that substrates bound at secondary sites are still transported. The homology modeling of human Pgp and substrate and modulator docking studies support the biochemical and transport data. In aggregate, our results demonstrate that a large flexible pocket in the Pgp transmembrane domains is able to bind chemically diverse compounds. When residues of the primary drug-binding site are mutated, substrates and modulators bind to secondary sites on the transporter and more than one transport-active binding site is available for each substrate.

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290 The accumulation of substrates was calculated with respect to the accumulation in E556Q/E1201Q mutant Pgp, which is completely inactive [48], and is treated as a negative control for all transport experiments. Login to comment

[hide] The Q loops of the human multidrug resistance tran... FASEB J. 2014 Oct;28(10):4335-46. doi: 10.1096/fj.13-245639. Epub 2014 Jul 11. Zolnerciks JK, Akkaya BG, Snippe M, Chiba P, Seelig A, Linton KJ
The Q loops of the human multidrug resistance transporter ABCB1 are necessary to couple drug binding to the ATP catalytic cycle.
FASEB J. 2014 Oct;28(10):4335-46. doi: 10.1096/fj.13-245639. Epub 2014 Jul 11., [PMID:25016028]

Abstract [show]
For a primary active pump, such as the human ATP-binding-cassette (ABC) transporter ABCB1, coupling of drug-binding by the two transmembrane domains (TMDs) to the ATP catalytic cycle of the two nucleotide-binding domains (NBDs) is fundamental to the transport mechanism, but is poorly understood at the biochemical level. Structure data suggest that signals are transduced through intracellular loops of the TMDs that slot into grooves on the NBDs. At the base of these grooves is the Q loop. We therefore mutated the eponymous glutamine in one or both NBD Q loops and measured the effect on conformation and function by using a conformation-sensitive antibody (UIC2) and a fluorescent drug (Bodipy-verapamil), respectively. We showed that the double mutant is trapped in the inward-open state, which binds the drug, but cannot couple to the ATPase cycle. Our data also describe marked redundancy within the transport mechanism, because single-Q-loop mutants are functional for Bodipy-verapamil transport. This result allowed us to elucidate transduction pathways from twin drug-binding cavities to the Q loops using point mutations to favor one cavity over the other. Together, the data show that the Q loop is the central flexion point where the aspect of the drug-binding cavities is coupled to the ATP catalytic cycle.

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74 Plasmids Mutations were introduced into a plasmid encoding human ABCB1 with a C-terminal hexahistidine tag (pCIneo-wtABCB1-6His; ref. 25) by site-directed mutagenesis (QuikChange XL; Stratagene, La Jolla, CA, USA) using the following oligonucleotides: Q132A, 5=-GGTTGCTGCTTACATCGCGGTTTCATTTTGGTGC- 3=; Q132R, 5=-GGTTGCTGCTTACATTCGAGTTTCATTTTG- GTGC-3=; Q475A, 5=-GGGAAATCATTGGTGTGGTGAGTGCT- GAGCCTGTATTGTTTGCCACCACG-3=; Q773A, 5=-GGAATTA- TTTCTTTTATTACATTTTTCCTTGCGGGTTTCACATTTG- GCAAAGCTGG-3=; Q773R, 5=-GGAATTATTTCTTTTATTA- CATTTTTCCTTCGAGGTTTCACATTTGGCAAAGCTGG-3=; Q1118A, 5=-GGGCATCGTGTCCGCGGAACCCATCCTGTTTG-3=; E556Q, 5=-CCCCAAGATCCTCCTGCTTGATCAGGCCACGT- CAGCCTTGG-3=; and E1201Q, 5=-CAGCCTCATATTTTGCTTCT- TGATCAGGCCACGTCAGCTCTGGATAC-3=. Login to comment
133 As controls, mutations E556Q and E1201Q in the Walker B motifs of each NBD, which render the transporter catalytically inactive, were also generated. Login to comment
211 The catalytically inactive Walker B mutants NBD1-E556Q and NBD2-E1201Q (46) were also tested for comparison and did not efflux Bodipy-verapamil. Login to comment
251 Catalytically inactive Walker B mutants NBD1-E556Q and NBD2-E1201Q served as negative controls. Login to comment

[hide] Cysteines introduced into extracellular loops 1 an... J Biol Chem. 2014 Sep 5;289(36):24749-58. doi: 10.1074/jbc.M114.583021. Epub 2014 Jul 22. Loo TW, Clarke DM
Cysteines introduced into extracellular loops 1 and 4 of human P-glycoprotein that are close only in the open conformation spontaneously form a disulfide bond that inhibits drug efflux and ATPase activity.
J Biol Chem. 2014 Sep 5;289(36):24749-58. doi: 10.1074/jbc.M114.583021. Epub 2014 Jul 22., [PMID:25053414]

Abstract [show]
P-glycoprotein (P-gp) is an ATP-binding cassette drug pump that protects us from toxic compounds and confers multidrug resistance. The protein is organized into two halves. The halves contain a transmembrane domain (TMD) with six transmembrane segments and a nucleotide-binding domain (NBD). The drug- and ATP-binding sites reside at the TMD1/TMD2 and NBD1/NBD2 interfaces, respectively. ATP-dependent drug efflux involves changes between the open inward-facing (NBDs apart, extracellular loops (ECLs) close together) and the closed outward-facing (NBDs close together, ECLs apart) conformations. It is controversial, however, whether the open conformation only exists transiently in intact cells because of the presence of high levels of ATP. To test for the presence of an open conformation in intact cells, reporter cysteines were placed in extracellular loops 1 (A80C, N half) and 4 (R741C, C half). The rationale was that cysteines A80C/R741C would only come close enough to form a disulfide bond in an open conformation (6.9 A apart) because they are separated widely (30.4 A apart) in the closed conformation. It was observed that the mutant A80C/R741C cross-linked spontaneously (>90%) when expressed in cells. In contrast to previous reports showing that trapping P-gp in a closed conformation highly activated ATPase activity, here we show that A80C/R741C cross-linking inhibited ATPase activity and drug efflux. Both activities were restored when the cross-linked mutant was treated with a thiol-reducing agent. The results show that an open conformation can be readily detected in cells and that cross-linking of cysteines placed in ECLs 1 and 4 inhibits activity.

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212 These catalytic carboxylates have been shown previously to be essential for ATP hydrolysis because mutating both Glu-556 and Glu-1201 to glutamine completely abolished ATPase activity (48, 49). Login to comment
213 HEK 293 cells were then transfected with A52-tagged wild-type P-gp, mutant A80C/R741C (in a wild-type background), or A80C/R741C/E556Q/E1201Q (in a wild-type background). Login to comment
215 It was observed that the presence of E556Q/E1201Q mutations did not inhibit cross-linking (Fig. 9A). Login to comment
216 Both the A80C/R741C and A80C/R741C/E556Q/E1201Q mutants yielded cross-linked P-gp as the major product. Login to comment
219 We then determined whether spontaneous cross-linking of A80C/R741C and A80C/R741C/E556Q/E1201Q mutants was inefficient by testing the effect of an oxidant (copper phenanthroline) to catalyze formation of the disulfide bond at a reduced temperature. Login to comment
220 HEK 293 cells were transfected with the A52-tagged mutants A80C/R741C and A80C/R741C/E556Q/ E1201Q (in a wild-type background). Login to comment
238 P-gp Is Inhibited by Extracellular Loop Cross-linking 24754 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289ߦNUMBER stopped by addition of SDS sample buffer containing 50 mM EDTA and no reducing agent. Samples were then subjected to immunoblot analysis. Fig. 9B shows that treatment of DTT-treated cells with oxidant almost completely cross-linked the mature 170-kDa protein in both mutants, A80C/R741C and A80C/R741C/E556Q/E1201Q, within 1 min. Login to comment
261 A, HEK 293 cells were transfected with A52-tagged wild-type P-gp, mutants A80C/R741C (in a wild-type background), or A80C/ R741C/E556Q/E1201Q. Login to comment
263 Samples were then subjected to immunoblot analysis. B, HEK 293 cells expressing A52-tagged mutants A80C/R741C or A80C/R741C/E556Q/E1201Q (in a wild-type background) were treated with 10 mM DTT for 3 min. Login to comment

[hide] Expression of the multidrug transporter P-glycopro... Exp Cell Res. 2015 Aug 15;336(2):318-28. doi: 10.1016/j.yexcr.2015.06.005. Epub 2015 Jun 19. Souza PS, Madigan JP, Gillet JP, Kapoor K, Ambudkar SV, Maia RC, Gottesman MM, Leung Fung K
Expression of the multidrug transporter P-glycoprotein is inversely related to that of apoptosis-associated endogenous TRAIL.
Exp Cell Res. 2015 Aug 15;336(2):318-28. doi: 10.1016/j.yexcr.2015.06.005. Epub 2015 Jun 19., [PMID:26101157]

Abstract [show]
Multidrug resistance (MDR) has been associated with expression of ABC transporter genes including P-glycoprotein (Pgp, MDR1, ABCB1). However, deregulation of apoptotic pathways also renders cells resistant to chemotherapy. To discover apoptosis-related genes affected by Pgp expression, we used the HeLa MDR-off system. We found that using doxycycline to control Pgp expression has a significant advantage over tetracycline, in that doxycycline caused less endogenous gene expression modification/perturbation, and was more potent than tetracycline in suppressing Pgp expression. Cells overexpressing Pgp have lower TNFSF10 (TRAIL) expression than their parental cells. Controlled downregulation of Pgp increased endogenous TRAIL protein expression. Also, ectopic overexpression of TRAIL in Pgp-positive cells was associated with a reduction in Pgp levels. However, cells expressing a functionally defective mutant Pgp showed an increase in TRAIL expression, suggesting that Pgp function is required for TRAIL suppression. Cells in which Pgp is knocked down by upregulation of TRAIL expression are less susceptible to TRAIL ligand (sTRAIL)-induced apoptosis. Our findings reveal an inverse correlation between functional Pgp and endogenous TRAIL expression. Pgp function plays an important role in the TRAIL-mediated apoptosis pathway by regulating endogenous TRAIL expression and the TRAIL-mediated apoptosis pathway in MDR cancer cells.

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82 HeLa cell transductions with BacMam-Pgp virus wild-type and the E556Q/E1206Q mutant were performed as described previously [26,27]. Login to comment
175 To further confirm the link between Pgp function and TRAIL expression, we compared TRAIL expression in HeLa cells transfected with wild-type ABCB1 and with a functional mutant ABCB1 (E556Q/E1206Q). Login to comment