ABCMdb

ABCB1 p.Glu1201Gln

None has been submitted yet.

Publications

PMID: 12437356 [PubMed] Sauna ZE et al: "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)."

No. Sentence Comment

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
129 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 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
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
179 Similar results were also obtained with the E1201Q mutant Pgp (data not shown). 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

PMID: 14576852 [PubMed] Ambudkar SV et al: "P-glycoprotein: from genomics to mechanism."

No. Sentence Comment

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

PMID: 14596601 [PubMed] Carrier I et al: "Analysis of catalytic carboxylate mutants E552Q and E1197Q suggests asymmetric ATP hydrolysis by the two nucleotide-binding domains of P-glycoprotein."

No. Sentence Comment

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

PMID: 15159388 [PubMed] Tombline G et al: "Combined mutation of catalytic glutamate residues in the two nucleotide binding domains of P-glycoprotein generates a conformation that binds ATP and ADP tightly."

No. Sentence Comment

48 Two such mutants have been reported, namely the E556Q/E1201Q and E556A/E1201A mutants of human Pgp (28). 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

PMID: 16844693 [PubMed] Sauna ZE et al: "Exploiting reaction intermediates of the ATPase reaction to elucidate the mechanism of transport by P-glycoprotein (ABCB1)."

No. Sentence Comment

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

PMID: 17237262 [PubMed] Sauna ZE et al: "About a switch: how P-glycoprotein (ABCB1) harnesses the energy of ATP binding and hydrolysis to do mechanical work."

No. Sentence Comment

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

PMID: 17636884 [PubMed] Loo TW et al: "Nucleotide binding, ATP hydrolysis, and mutation of the catalytic carboxylates of human P-glycoprotein cause distinct conformational changes in the transmembrane segments."

No. Sentence Comment

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
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
102 HEK 293 cells expressing histidine-tagged Cys-less P-gp (Cys-less) or mutants containing the E556Q(NBD1), E1201Q- (NBD2), or E556Q(NBD1)/E1201Q(NBD2) mutations were isolated by nickel chelate chromatography. Login to comment
114 It appeared that the E1201Q- (NBD2) mutation caused a long-range conformational change in the TMDs to allow cross-linking between Cys332(TM6) and Cys975(TM12) in the absence of ATP. 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
116 The TMDs appear to undergo different conformational changes during ATP binding, during ATP hydrolysis, or in the presence of the E1201Q mutation. Login to comment
172 Membranes prepared from cells expressing mutant L339C(TM6)/F728C(TM7)/E556Q(NBD1)/E1201Q- (NBD2) were treated with 5 mM ATP, 10 mM MgCl2, and 0.2 mM sodium vanadate (ATP/VO4) for 10 min at 37 °C and then chilled on ice. Login to comment
199 The conformational changes in the TMDs appear to be quite sensitive to subtle changes in the ATP-binding sites as the conservative E1201Q mutation had a pronounced effect on the cross-linking pattern of mutant L332C(TM6)/ L975C(TM12) (Figure 4). Login to comment
224 The catalytic carboxylate mutations had asymmetric effects on cross-linking of mutant L332C(TM6)/L975C(TM12) as introduction of the E556Q(NBD1) mutation inhibited cross-linking even in the presence of ATP while the E1201Q- (NBD2) mutation caused cross-linking even in the absence of ATP. Login to comment
247 (A) The positions of the cysteine residues [L332C(TM6)/L975C(TM12) and F343C(TM6)/V982C(TM12)] that could be cross-linked and the catalytic carboxylate mutations [E556Q(NBD1) and E1201Q- (NBD2)] are shown. Login to comment
254 The presence of the E1201Q mutation (II), however, appears to mimic some of the changes induced by ATP hydrolysis since Cys332(TM6) could be cross-linked with Cys975(TM12) with copper phenanthroline. Login to comment

PMID: 17988154 [PubMed] Sauna ZE et al: "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."

No. Sentence Comment

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
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

PMID: 18058211 [PubMed] Sauna ZE et al: "Genomics and the mechanism of P-glycoprotein (ABCB1)."

No. Sentence Comment

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

PMID: 22041108 [PubMed] Shukla S et al: "Use of baculovirus BacMam vectors for expression of ABC drug transporters in mammalian cells."

No. Sentence 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

PMID: 22700974 [PubMed] Loo TW et al: "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."

No. Sentence Comment

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

PMID: 24064216 [PubMed] Kapoor K et al: "Mutations in intracellular loops 1 and 3 lead to misfolding of human P-glycoprotein (ABCB1) that can be rescued by cyclosporine A, which reduces its association with chaperone Hsp70."

No. Sentence Comment

157 Cysless-WT and the non-functional mutant E556Q/E1201Q (22) were used as positive and negative controls, respectively. Login to comment
175 The transport function of the three mutant P-gps was compared with cysless-WT (blue, positive control) and E556Q/E1201Q (referred to as EQ)-mutant P-gp (red, negative control), which is known to be completely functionless (22). Login to comment

PMID: 24349290 [PubMed] Chufan EE et al: "Multiple transport-active binding sites are available for a single substrate on human P-glycoprotein (ABCB1)."

No. Sentence Comment

213 The inactive E501Q/E1201Q (EQ) mutant P-gp is used as a control to show completely aborted function as described in Materials and Methods. Login to comment
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

PMID: 25016028 [PubMed] Zolnerciks JK et al: "The Q loops of the human multidrug resistance transporter ABCB1 are necessary to couple drug binding to the ATP catalytic cycle."

No. Sentence Comment

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
122 In contrast, the catalytically inactive Walker B E1201Q mutant was trapped in the inward-closed conformation, which has a low affinity for drug, and the cells accumulated Bodipy-verapamil intracellularly but not in the plasma membrane (bottom panels). 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
151 cells or the cells expressing the Walker B mutant E1201Q of ABCB1 (Fig. 2C). Login to comment
154 Consequently, UIC2 binds more readily to wild-type ABCB1 than the Walker B mutant E1201Q (Fig. 2D) in cells that express equal amounts of these proteins, because the Walker B mutant can bind but cannot hydrolyze ATP (40). Login to comment
164 In contrast, no Bodipy-verapamil was detected bound to the Walker B E1201Q mutant, which is trapped in the inward-closed conformation. 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

PMID: 25053414 [PubMed] Loo TW et al: "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."

No. Sentence Comment

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