ABCMdb

PMID: 17988154

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 [PubMed]

Sentences

No. Mutations Sentence Comment

40 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln 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 ABCB1 p.Glu556Gln 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 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln 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 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln 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 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln 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 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln 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 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln 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 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln 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 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln 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 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln 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 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln 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 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln 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 ABCB1 p.Glu1201Gln ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln ABCB1 p.Glu556Gln 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 261 ABCB3 p.Glu552Gln These studies demonstrated that the E552Q/E1197Q mutant occludes minimal amounts of [14 C]ADP after an extended incubation of 120 min [see Figure 5 in (20)]. Login to comment 262 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln 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 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln 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 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln 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 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln We next compared the occlusion of ATP and ADP into the mutant Pgp (E556Q/E1201Q). Login to comment 297 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln 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 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln 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 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln (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 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln (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 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln (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 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln (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 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln 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 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln 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 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln 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 ABCB1 p.Glu1201Gln ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln ABCB1 p.Glu556Gln (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 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln (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 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln (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 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln 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 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln ABCB3 p.Glu552Ala 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 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln 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 ABCB1 p.Glu1201Gln ABCB1 p.Glu556Gln 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