ABCMdb

ABCC1 p.Asp793Glu

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Publications

PMID: 12882957 [PubMed] Payen LF et al: "Role of carboxylate residues adjacent to the conserved core Walker B motifs in the catalytic cycle of multidrug resistance protein 1 (ABCC1)."

No. Sentence Comment

69 The forward primers for D793E and E1455D were 5Ј-CCTCTTCGATGAGCCCCTCTCAGC-3Ј and 5Ј-CTTGTGTTG- GATGATGCCACGGCAGC-3Ј, respectively. Login to comment
71 The Bsu36I-SphI fragments bearing mutations at NBD1 were isolated from pGEM-NBD1 and used to replace the same region in pFBDual-halves to create pFBDual-halves/D793E. Login to comment
75 In the D793E/E1455D double mutant, the SalI-XbaI fragment with the mutation in NBD1 was isolated and ligated to pFBDual-D45/E1455D, which had been digested with the same enzymes, to generate pFBDual-halves/D793E/E1455D. Login to comment
122 Effect of D793E and E1455D Single and Double Mutations on LTC4 Transport Activity-LTC4 uptake by membrane vesicles from Sf21 cells expressing wild-type and mutant MRP1 half-molecules was determined at 23 °C, as described by Loe et al. (7). Login to comment
124 Uptake at 3 min, by vesicles containing the D793E mutant NH2-terminal fragment and the wild-type COOH-proximal half was ϳ20% of that obtained with vesicles containing two wild-type fragments. Login to comment
125 The transport activity of vesicles containing either the E1455D mutant fragment and the wild-type NH2-terminal half, or co-expressed D793E and E1455D mutant fragments was similar to that of the beta-Gus control (Fig. 2B). Login to comment
126 Thus the effects of the D793E and E1455D mutations on LTC4 transport were similar to previously described mutations of the Walker A motif that eliminate ATP hydrolysis and decrease ATP binding by NBD1 and NBD2, respectively (24, 25). Login to comment
127 Photolabeling with 8-Azido-[␥-32 P]ATP in the Presence of AMP-PNP at 4 °C-To determine whether or not the D793E and E1455D mutations altered ATP binding, studies were carried out at 4 °C using the radioactive photoactivable analog of ATP, 8-azido-[␥-32 P]ATP, hydrolysis of which results in loss of the ␥-32 P label (24, 33). Login to comment
130 ATP labeling of wild-type MRP1, D793E, E1455D single mutant proteins was similar and occurred preferentially at NBD1, whereas it was slightly decreased at NBD1 of the double mutant protein. Login to comment
132 Labeling at NBD2 in the E1455D and D793E/E1455D mutant proteins was strongly and moderately enhanced, respectively, relative to the wild-type NBDs. Login to comment
134 Nucleotide Trapping by Wild-type MRP1 and D793E, E1455D Single and Double Mutant Proteins Using 8-Azido-[␣- 32 P]ATP-To determine whether ATP hydrolysis by the mutant NBDs was altered, ADP trapping experiments were performed at 37 °C using various concentrations of 8-azido-[␣- 32 P]ATP in the presence or absence of vanadate. Login to comment
139 In comparison to wild-type MRP1, ADP trapping was increased at the D793E mutant NBD1 and decreased at the co-expressed wild-type NBD2. Login to comment
140 Increased labeling of the mutant NBD1 was observed at all 8-azido-[␣-32 P]ATP concentrations tested and unlike the wild-type NBD1, was readily detectable at 2.5 ␮M 8-azido-[␣-32 P]ATP with both the D793E and D793E/E1455D mutants (Fig. 3, A and C). Login to comment
143 Effect of D793E and E1455D single and double mutations on ATP-dependent LTC4 transport activity (B) and on ATP binding at 4 °C (C). Login to comment
144 A, membrane proteins (1 ␮g from Sf21 cells expressing both halves of either MRP1 (MRP1 dh), mutant proteins (D793E, E1455D, and D793E/E1455D)) were separated by SDS-PAGE on gradient gels and transferred to Immobilon-P membranes. Login to comment
148 B, membrane vesicles (2 ␮g) containing wild-type MRP1 (f), MRP1 mutants D793E (Œ), E1455D (), D793E/E1455D (q), and control beta-Gus vector (ࡗ) were assayed for ATP-dependent LTC4 transport activity at 23 °C for up to 3 min in transport buffer containing [3 H]LTC4 (50 nM, 0.13 ␮Ci), as described under "Experimental Procedures." Login to comment
151 C, at 4 °C, 8-azido-[␥-32 P]ATP photolabeling by wild-type MRP1 and mutant D793E, E1455D, and D793E/E1455D was carried out. Login to comment
156 Catalytic Cycle of MRP138540 The E1455D mutation dramatically increased photolabeling of the mutant NBD2 and unlike the D793E mutation, increased rather than decreased labeling of the co-expressed wild-type NBD1. Login to comment
159 Despite the striking increase in photolabeling of the E1455D mutant NBD2, in the double D793E/E1455D mutant, labeling occurred predominantly at NBD1 and was markedly diminished at NBD2 relative to the E1455D single mutant. Login to comment
161 Photolabeling with 8-Azido-[␥-32 P]ATP or 8-Azido-[␣-32 P]ADP at 37 °C-The relatively weak vanadate dependence of photolabeling observed when using 8-azido-[␣-32 P]ATP, particularly with the E1455D and D793E/E1455D mutants, raised the possibility that the mutant NBD2 may be capable of tight binding of both ATP and ADP. Login to comment
163 In the presence of 1 mM vanadate, photolabeling of wild-type and the D793E mutant NBD1 by 8-azido-[␥-32 P]ATP was barely detectable, presumably as a result of the hydrolytic loss of the [␥-32 P]PO4 (Fig. 4A). Login to comment
164 These data combined with trapping using 8-azido-[␣-32 P]ATP indicate that the nucleotide "trapped" by the NBDs of the wild-type and D793E mutant proteins was indeed ADP. Login to comment
165 In contrast, both NBDs of the E1455D mutant were strongly labeled by 8-azido-[␥-32 P]ATP, while in the D793E/E1455D double mutant labeling of NBD2 was much reduced and labeling of NBD1 was essentially eliminated (Fig. 4A). Login to comment
167 These experiments suggest that the tight binding of ATP by the E1455D mutant NBD2 stimulates the binding of ATP by the co-expressed wild-type NBD1 and conversely, that the increased trapping of ADP at the D793E mutant NBD1, diminishes ATP binding by both the co-expressed wild-type and E1455D mutant NBD2. Login to comment
169 Nucleotide trapping by D793E, E1455D single and double mutant proteins using 8-azido-[␣-32 P]ATP. Login to comment
170 A-C, at 37 °C, under hydrolytic conditions, the effect of the 8-azido-[␣-32 P]ATP concentration on ADP trapping by wild-type MRP1 and D793E (A), E1455D (B), and D793E/E1455D (C) mutant proteins was evaluated. Membrane vesicles (20 ␮g) were incubated with 8-azido-[␣-32 P]ATP (1-15 ␮M) in the absence (-) or presence (ϩ) of 1 mM vanadate for 15 min in transport buffer containing 5 mM MgCl2. Login to comment
176 Effect of D793E, E1455D single and double mutations on ATP binding and ADP labeling under hydrolytic conditions. Login to comment
177 A, at 37 °C, 8-azido-[␥-32 P]ATP photolabeling by wild-type MRP1 and D793E, E1455D single and double mutations was carried out. Login to comment
181 The position of the labeled MRP1 NH2-half and COOH-half polypeptides are indicated, and endogenous proteins labeled are indicated by E followed by arrows. B, at 37 °C, 8-azido-[␣- 32 P]ADP photolabeling by wild-type MRP1 and mutant D793E, E1455D, and D793E/E1455D proteins was evaluated. Login to comment
190 In comparison to wild-type, ADP labeling of the D793E mutant NBD1 was increased and was almost maximal using 5 ␮M 8-azido-[␣-32 P]ADP (Fig. 4B, lanes 2, 4, and 5). Login to comment
191 In contrast, photolabeling by 15 ␮M 8-azido-[␣- 32 P]ADP was clearly decreased at the wild-type NBD2 co-expressed with the D793E mutant NBD1, when compared with the co-expressed wild-type half-molecules (Fig. 4B, lanes 2-5). Login to comment
193 In the D793E/E1455D double mutant, photolabeling of NBD2 was diminished and labeling of NBD1 was increased relative to the single D793E and E1455D single mutants, so that ADP labeling was similar at both NBDs (Fig. 4B, lanes 2-9). Login to comment
194 Thus as observed with 8-azido-[␥-32 P]ATP, the increased binding of ADP by the E1455D mutant NBD2 appears to stimulate tight binding of ADP at both the wild-type and D793E mutant NBD1. Login to comment
195 In contrast, the increased ADP binding of the D793E mutant NBD1 decreases ADP binding by both the wild-type and E1455D mutant NBD2, as would be predicted by an alternating site model of catalysis. Login to comment
196 Evaluation of ADP Release by Wild-type and D793E, E1455D Single and Double Mutant Proteins-It has been demonstrated that ADP release constitutes the rate-limiting step in the normal catalytic cycle of P-GP (41). Login to comment
200 Consequently, we investigated the influence of D793E, E1455D, and D793E/ E1455D MRP1 mutants on ADP release by each NBD. Login to comment
205 As described above, weak photolabeling of NBD1 in samples cross-linked immediately after washing could be detected with the D793E and D793E/E1455D mutants (Fig. 5, lanes 3 and 5). Login to comment
209 Unlike the single E1455D mutant, very little photolabeling of NBD2 was retained following reincubation of the D793E/E1455D double mutant (Fig. 5, lanes 5 and 11). Login to comment
210 To further investigate the impairment of ADP release by the E1455D and the D793E/E1455D mutants, cold ADP (1 mM) was added 3 min before the end of the initial nucleotide labeling with 8-azido-[␣-32 P]ATP (Fig. 5, lanes 6-8). Login to comment
211 ADP competed completely for 8-azido-␣-32 P-nucleotide trapping at both NBDs of the D793E mutant and at NBD1 of the D793E/E1455D double mutant indicating that the bound nucleotide was readily exchangeable (Fig. 5, lanes 6 and 8). Login to comment
213 In the D793E/E1455D double mutant all labeling at NBD1 was lost and labeling of NBD2 was markedly decreased compared with the E1455D single mutant (Fig. 5, lanes 7 and 8). Login to comment
215 Thus the results indicate that the release of ADP by the E1455D NBD2 is severely impaired and that this also decreases the ability of the co-expressed wild-type NBD1, but not the D793E mutant, to exchange nucleotide. Login to comment
216 LTC4 Photolabeling by Wild-type and D793E, E1455D Single, and D793E/E1455D Double Mutant Proteins Under Hydrolytic and Non-hydrolytic Conditions-We have shown previously that LTC4 can photolabel MRP1 at sites in MSD2 and MSD3 and that photolabeling, particularly of the site in MSD2, is strongly attenuated under ADP trapping conditions. Login to comment
219 To examine this prediction further, we investigated the effect of the D793E and E1455D mutations on the binding of LTC4 because the former, unlike the Walker A mutations, increases ADP trapping at NBD1, whereas the latter increases nucleotide binding and markedly slows down nucleotide release at NBD2. Login to comment
221 Evaluation of ADP release by wild-type and D793E, E1455D single and double mutations using 8-azido-[␣-32 P]ATP. Login to comment
230 Photolabeling of the D793E mutant in the absence of nucleotide was indistinguishable from that of the co-expressed wild-type half-molecules. Login to comment
231 However, in contrast to the previously described Walker A mutations, the D793E mutation essentially abolished the effect of ATP, and ATP plus vanadate on LTC4 binding (Fig. 6A, lanes 7-10). Login to comment
238 The double D793E/E1455D mutant behaved in a manner very similar to that of the D793E single mutation, with the exception that ATP␥S retained some ability to diminish LTC4 labeling (Fig. 6B, lanes 6-11). Login to comment
239 Thus these results together with those of vanadate trapping experiments are again consistent with the suggestion that increased trapping of ADP by the D793E mutant NBD1 decreases the nucleotide binding and hydrolysis by wild-type and E1455D mutant NBD2 and prevents conversion to a low affinity transition state. Login to comment
248 Unlike the D793E mutation, which decreased LTC4 transport activity by 80%, NBD1 mutations (D793S, D793N, and D793Q) had little effect on ATP-dependent LTC4 uptake (Fig. 7B). Login to comment
252 However, NBD1 was still more strongly photolabeled than NBD2, as observed with the wild-type co-expressed halves and, unlike the D793E mutation that diminished labeling of NBD2, labeling was unaffected by substitution with the three polar uncharged residues (Fig. 8A, lanes 3, 5, and 7). Login to comment
255 Effect of ATP␥S or ADP trapping on [3 H]LTC4 photolabeling by wild-type MRP1, D793E, E1455D, and D793E/E1455D mutant proteins. Login to comment
256 Wild-type MRP1, D793E (A) and E1455D, D793E/ E1455D (B) membrane proteins (75 ␮g) were incubated in transport buffer containing 5 mM MgCl2 at 23 °C for 20 min in the absence (-) or presence (ϩ) of ATP (1 mM), vanadate (1 mM), or ATP␥S (4 mM), alone or in combination, prior to addition of [3 H]LTC4 (200 nM, 0.13 ␮Ci). Login to comment
263 The effect of the non-conservative mutations on nucleotide binding and ADP trapping was examined at 37 °C using 5 ␮M 8-azido-[␣-32 P]ATP in the presence or absence of vanadate, exactly as described for the D793E and E1455D mutations. Login to comment
265 This contrasts with the D793E mutation in which photolabeling of NBD1 was readily detectable in the presence of 5 ␮M 8-azido-[␣-32 P]ATP and in the absence of vanadate. Login to comment
285 Based on the behavior of NBD2 of MRP1 and other NBDs containing Glu adjacent to the Walker B motif, we anticipated that the D793E mutation in NBD1 would increase ATP hydrolysis and possibly decrease the affinity for ATP. Login to comment
290 In contrast to the wild-type NBD1, vanadate did not further increase nucleotide trapping by the D793E mutant when photolabeling was carried out with 8-azido-[␣-32 P]ATP at 37 °C (Fig. 3A). Login to comment
292 In addition, the D793E mutation slightly decreased binding of 8-azido-[␥-32 P]ATP at 4 °C by the co-expressed wild-type NBD2 (Fig. 2C) and caused a major decrease in the trapping of ADP at 37 °C in the presence of vanadate (Fig. 3A). Login to comment
294 Consistent with the decrease in ATP binding and ADP trapping at the co-expressed wild-type NBD2, the decrease in LTC4 binding observed with the wild-type protein in the presence of ATP and vanadate was completely abrogated by the D793E mutation (Fig. 6A, lanes 7-10). Login to comment
295 Thus the D793E mutation decreases LTC4 transport by preventing the transition from a high to low affinity binding state. Login to comment
318 Consistent with such a prediction, the D793E mutation increased ADP trapping at the mutant NBD1 and decreased vanadate-dependent trapping by the co-expressed wild-type NBD2. Login to comment
331 Like the single mutations, the D793E/E1455D mutation had no effect on LTC4 binding in the absence of nucleotides. Login to comment
332 However, in the presence of ATP or ATP plus vanadate, the D793E mutation in the double mutant abrogated the shift from a high to a low affinity binding state, despite the potentiating effect observed with the E1455D single mutation. Login to comment
333 Paradoxically, a decrease of LTC4 binding by the D793E/E1455D double mutant protein was still observed in the presence of ATP␥S (Fig. 6B). Login to comment
355 However, a requirement for such a hydrolytic step, as envisaged in an alternating sites model of transport, is very difficult to reconcile with the strong negative effect on transport of mutations that enhance ATPase activity of NBD1, such as the D793E mutation, and the relatively minor effect of mutations at the same location that eliminate the negative side chain at the position of Asp793. Login to comment

PMID: 15737336 [PubMed] Yang R et al: "Nucleotide dissociation from NBD1 promotes solute transport by MRP1."

No. Sentence Comment

42 The definition of D793E means that the D793E mutated N-half is co-expressed with wild-type C-half and E1455Q, the wild-type N-half co-expressed with E1455Q mutated C-half. Login to comment
44 Membrane vesicles were prepared from Sf21 cells infected with viral particles expressing pDual without MRP1 cDNA insertion (lane 1), wild-type N-half+wild-type C-half (Wild-type, lanes 2-4), D793E mutated N-half+wild-type C-half (D793E, lanes 5-7), D793L mutated N-half+wild-type C-half (D793L, lanes 8-10), D793N mutated N-half+wild-type C-half (D793N, lanes 11-13) and wild-type N-half+E1455Q mutated C-half (E1455Q, lanes 14-16). Login to comment
49 The ratios of the band intensities in the same amount of total membrane proteins, for example, 300 ng of wild-type N-half (co-expressed with wild-type C-half) versus 300 ng of the D793E mutated N-half (co-expressed with wild-type C-half), were determined, considering the amount of wild-type N-half (or C-half) as 1.000. Login to comment
50 Since the ratio of N-half, for example, D793E mutated N-half, is similar to that of the C-half co-expressed with D793E mutated N-half, the mean ratios of the protein expressions including N-half and C-half are: 0.993F0.168 (D793E), 0.991F0.073 (D793L), 1.151F0.186 (D793N) and 0.921F0.108 (E1455Q). Login to comment
78 Generation of constructs The oligo-nucleotides to introduce the mutations in MRP1 are: MRP/D793E/forward, 5V-CT GAC ATT TAC CTC TTC GAT GAA CCC CTC TCA GCA GTG GAT GCC-3V; MRP/D793E/reverse, 5V-GGC ATC CAC TGC TGA GAG GGG TTC ATC GAA GAG GTA AAT GTC AG-3V; MRP/D793N/forward, 5V-CT GAC ATT TAC CTC TTC GAT AAT CCC CTC TCA GCA GTG GAT GCC -3V; MRP/D793N/reverse, 5V-GGC ATC CAC TGC TGA GAG GGG ATT ATC GAA GAG GTA AAT GTC AG-3V; MRP/ E1455Q/forward, 5V-G AAG ATC CTT GTG TTG GAT CAG GCC ACG GCA GCC GTG GAC CTG G-3V; MRP/ E1455Q/reverse, 5V-C CAG GTC CAC GGC TGC CGT GGC CTG ATC CAA CAC AAG GAT CTT C-3V. Login to comment
81 The aspartic acid residue at position 793 was mutated to either glutamic acid or asparagine (Fig. 1B, D793E or D793N) by using the forward/reverse primers and the QuikChange site directed mutagenesis kit from Stratagene [28]. Login to comment
92 This strategy was also used to generate constructs expressing D793E, L, and N mutated N-half and E1455Q mutated C-half. Login to comment
93 To make constructs expressing D793E, D793L and D793N mutated N-half and wild-type C-half simultaneously, the KpnI- DraIII fragment from pDual/N-half/C-half and the DraIII- XhoI fragments from pNUT/D793E, pNUT/D793L or pNUT/D793N were cloned into the KipI-XhoI fragment from pDual/N-half/C-half, named as pDual/D793E-N-half/ C-half, pDual/D793L-N-half/C-half or pDual/D793N- N-half/C-half. Login to comment
156 Substitution of the Asp residue with a non-acidic amino acid in NBD1 increased the Km and Vmax values for LTC4 in MRP1 mediated transport In order to test whether these Walker B mutations, D793E, D793L and D793N in NBD1 and E1455Q in R. Yang et al. Login to comment
160 The mutation of the corresponding residue D793 in NBD1 to the longer arm acidic glutamic acid (D793E) changed the kinetics of ATP-dependent LTC4 transport (Fig. 3). Login to comment
161 The transport activity of D793E is lower than that of wild-type at lower LTC4 concentrations, consistent with the previously reported results [43]. Login to comment
163 These might be the consequence of the altered properties of this D793E mutated NBD1 [43]. Login to comment
169 Since the amounts of MRP1 proteins in membrane vesicles containing wild-type, D793E, D793L, D793N and E1455Q are similar (Fig. 1C), the much lower Vmax value of E1455Q than that of the wild-type (Table 1), although the amount of E1455Q (ratio of 0.921) is slightly less than wild-type, indicates a greatly decreased k2 value, which is perhaps directly associated with the greatly diminished ATPase activity at Fig. 3. Login to comment
174 The samples are: wild-type, wild-type N-half+wild-type C-half; D793E, D793E mutated N-half+wild-type C-half; D793L, D793L mutated N-half+wild-type C-half; D793N, D793N mutated N-half+wild-type C-half and E1455Q, wild-type N-half+E1455Q mutated C-half. Login to comment
175 Table 1 Km and Vmax values (LTC4) of wild-type and mutant MRP1s Sample Km (nM LTC4)a Vmax (pmol LTC4 mgÀ1 minÀ1 )N-half C-half Wild-type Wild-type 59F1 287.5F7.5 D793E Wild-type 110F10 365.0F25.0 D793L Wild-type 100F0 560.0F0.0 D793N Wild-type 105F5 575.0F75.0 Wild-type E1455Q 50F0 37.5F0.5 a The Km values (n=2) and Vmax values (n=2) were derived from Fig. 3. Login to comment
185 Combination of D793E, D793L or D793N mutated NBD1 with E1455Q mutated NBD2 does not enhance ATP-dependent LTC4 transport activity The k2 values should be directly associated with the rates of ATP hydrolysis by variant MRP1 mutants. Login to comment
189 In order to test these two possibilities, the D793E, D793L or D793N mutated N-half was co-expressed with the E1455Q mutated C-half. Login to comment
191 The results in Fig. 4B show that all the mutants, including wild-type N-half+E1455Q mutated C-half, D793E mutated N-half+E1455Q mutated C-half, D793L mutated N-half+E1455Q mutated C-half and D793N mutated N-half+E1455Q mutated C-half, have similar ATP-dependent LTC4 transport activities. Login to comment
194 D793E, D793L or D793N mutated NBD1 does not enhance the ATP-dependent LTC4 transport activity of E1455Q mutated NBD2. Login to comment
197 The mean ratios of the protein expressions including N-half and C-half are: 1.33F0.11 (E1455Q), 1.49F0.13 (D793E/E1455Q), 0.98F0.05 (D793L/ E1455Q) and 1.88F0.29 (D793N/E1455Q). Login to comment
204 The samples are: wild-type, wild-type N-half+wild-type C-half; D793E, D793E mutated N-half+wild-type C-half; D793L, D793L mutated N-half+wild-type C-half; D793N, D793N mutated N-half+wild-type C-half and E1455Q, wild-type N-half+E1455Q mutated C-half. Login to comment
205 Table 2 Km values (ATP) of wild-type and mutant MRP1s Sample Km (AM ATP)a N-half C-half Wild-type Wild-type 72.2F1.6 D793E Wild-type 106.0F9.7 D793L Wild-type 107.0F7.5 D793N Wild-type 92.0F12.5 Wild-type E1455Q 55.0F0.0 a Km values (for wild-type, D793E, D793L and D793N, n=5; for E1455Q, n=) were derived from corresponding Michaelis-Menten curves shown in Fig. 5. R. Yang et al. Login to comment
210 The Km (ATP) value of E1455Q, the putative catalytic base mutant in NBD2, is slightly less than that of wild-type (Table 2), whereas the Km (ATP) values of D793E, D793L and D793N are slightly higher than that of wild-type (Table 2). Login to comment
220 Fig. 6A, D, G, J and M show the autoradiograms reflecting [a-32 P]-8-N3ATP labeling of wild-type, D793E, D793L, D793N and E1455Q. Labeling was quantified by Packard Instant Imager and plotted against the concentration of [a-32 P]-8-N3ATP (Fig. 6B, C, E, F, H, I, K, L, N and O). Login to comment
222 The Kd (ATP) value for D793E mutated NBD1, co-expressed with wild-type NBD2, is slightly less than that of wild-type NBD1 (Table 3), implying moderately increased affinity for ATP. Login to comment
223 However, the Kd (ATP) value for wild-type NBD2 co-expressed with D793E mutated NBD1 is slightly higher than that of the wild-type NBD2 co-expressed with wild-type NBD1, implying that the D793E mutated NBD1 has an effect on the wild-type NBD2. Login to comment
237 Fig. 7A, D, G, J and M show the autoradiograms reflecting [g-32 P]-8-N3ATP labeling of wild-type, D793E, D793L, D793N and E1455Q. Labeling was quantified by Packard Instant Imager and plotted against the incubation time (Fig. 7B, C, E, F, H, I, K, L, N and O). Login to comment
247 In the cases of D793E, D793L and D793N, the T1/2 values (for NBD1 and NBD2) are shorter than that of the wild-type and E1455Q (Table 4). Login to comment
263 In addition, the substitution of this putative catalytic residue D793 with a longer spacer-arm negatively charged Glu enhances its hydrolytic capacity [43], but does not increase the ATP-dependent solute transport activity of D793E/E1455Q mutated MRP1 (Fig. 4) or markedly Fig. 6. Login to comment
267 A, D, G, J, and M: Autoradiograms of [a-32 P]-8-N3ATP labeled wild-type N-half co-expressed with wild-type C-half; D793E mutated N-half+wild-type C-half; D793L mutated N-half+wild-type C-half; D793N mutated N-half+wild-type C-half; and wild-type N-half+E1455Q mutated C-half. Login to comment
270 E and F: D793E mutated N-half (E) co-expressed with wild-type C-half (F). Login to comment
274 Table 3 Substitution of D793 with a non-acidic amino acid decreases affinity for ATP Sample Kd of NBD1 (AM ATP)a Kd of NBD2 (AM ATP)N-half C-half Wild-type Wild-type 11.7F2.8 32.7F2.3 D793E Wild-type 7.8F4.1 41.0F8.1 D793L Wild-type 30.5F2.5 32.9F1.9 D793N Wild-type 28.4F4.5 33.7F0.7 Wild-type E1455Q 19.4F3.3 155.8F9.0 a The Kd (AM ATP) values (for wild-type, n=12; D793E, n=9; D793L and E1455Q, n=; D793N, n=8) were derived from Fig. 6. R. Yang et al. Login to comment
301 A, D, G, J, and M: Autoradiograms of [g-32 P]-8-N3ATP labeled wild-type N-half co-expressed with wild-type C-half; D793E mutated N-half+wild-type C-half; D793L mutated N-half+wild-type C-half; D793N mutated N-half+wild-type C-half; and wild-type N-half+E1455Q mutated C-half. Login to comment
306 E and F: D793E mutated N-half (E) co-expressed with wild-type C-half (F). Login to comment
310 Table 4 Release rate of the bound nucleotide at the wild-type and mutated NBDs Sample T1/2 of NBD1 (min)a T1/2 of NBD2 (min)N-half C-half Wild-type Wild-type 5.3F0.3 3.7F2.0 D793E Wild-type 3.0F0.7 3.4F1.0 D793L Wild-type 2.3 2.3 D793N Wild-type 2.5F0.5 2.2F0.7 Wild-type E1455Q 6.1F0.3 25.6F2.4 a The T1/2 value (for wild-type, D793E, D793N and E1455Q, n=3; for D793L, n=1) is the time required to release 50% of the bound nucleotide and was derived from Fig. 7. R. Yang et al. Login to comment

PMID: 15755910 [PubMed] Payen L et al: "Functional interactions between nucleotide binding domains and leukotriene C4 binding sites of multidrug resistance protein 1 (ABCC1)."

No. Sentence Comment

44 A D793E mutation in NBD1 enhanced its hydrolytic capacity but caused occlusion of the resultant ADP by the mutant NBD1 in the absence of vanadate. Login to comment

PMID: 16697012 [PubMed] Ramaen O et al: "Structure of the human multidrug resistance protein 1 nucleotide binding domain 1 bound to Mg2+/ATP reveals a non-productive catalytic site."

No. Sentence Comment

129 The role of the catalytic carboxylate was examined from NBD1- Asp793 and NBD2-Glu1455 mutations: the Asp793Glu mutation in NBD1 enhances its hydrolytic capacity, whereas the Glu1455Asp mutation in NBD2 results in an increased affinity of NBD2 for ATP, but a reduced hydrolytic activity.25 Our model shows that the orientation of His1486 could be sensitive to the conformation of His827, which is constrained by a strong hydrogen bond with Asp793, itself possibly stabilized by the participation of Asp792 to the nucleotide-binding site in the Mg2C /ATP-bound state. Login to comment

PMID: 17295059 [PubMed] Chang XB et al: "A molecular understanding of ATP-dependent solute transport by multidrug resistance-associated protein MRP1."

No. Sentence Comment

245 For example, although ATPγS binding to wild-type or E1455D-mutated MRP1 significantly inhibited LTC4 labeling [62], ATPγS itself did not support the ATP-dependent LTC4 or E217βG transport [33, 47]; ATP can efficiently bind to E1455D, D793E/ E1455D or E1455L [62, 144], but mutation of this putative catalytic residue abolished the ATP-dependent solute transport [62, 144]. Login to comment

PMID: 19063607 [PubMed] Qin L et al: "Residues responsible for the asymmetric function of the nucleotide binding domains of multidrug resistance protein 1."

No. Sentence Comment

7 We found that conversion of Trp653 to Tyr and/or Pro794 to Ala enhanced transport activity of the D793E mutant and the release of ADP from NBS1. Login to comment
9 Molecular dynamic simulations revealed that, while the D793E mutation increased hydrolysis of ATP, the presence of the adjacent Pro794, rather than the more typical Ala, decreased flexibility of the region linking Walker B and the D-loop, markedly diminishing the rate of release of Mg2+ and ADP. Login to comment
10 Overall, these results suggest that the rate of release of ADP by NBD1 in the D793E background may be the rate-limiting step in the transport cycle of MRP1. Login to comment
36 To determine why ADP release was impaired in the D793E mutant and to gain additional insight into the role of NBS1 in the transport cycle, we have examined the effects of replacing other variant amino acid residues in NBD1 with the corresponding amino acids present in NBD2. Login to comment
37 These studies show that conversion of Trp653 to Tyr and/or Pro794 to Ala is sufficient to restore ADP release by the D793E mutant and to increase LTC4 transport activity to the level of the wild-type (wt) protein. Login to comment
41 Thus, the modeling and simulation results provide a possible mechanistic explanation for the ability of the P794A mutation to restore catalytic activity to the D793E mutation and to enhance activity of the wt protein. Login to comment
54 The dh MRP1 construct containing the D793E mutation was also constructed previously and designated dh D793E (26). Login to comment
55 Additional mutations (W653Y, V680T, P794A, V1432G, L1437R, and C1439S) were introduced into the dh D793E vector by site-directed mutagenesis using a QuikChange II kit (Stratagene, La Jolla, CA). Login to comment
58 Briefly, to make W653Y, V680T, and P794A mutations, a BamHI-XbaI fragment from the dh D793E construct was cloned into pBluescript II KS (+) vector (Fermentas International Inc., Burlington, Ontario, Canada), and mutagenesis PCR reactions were performed according to the manufacturer`s manual. Login to comment
60 The isolated Bsu36I-XbaI fragment containing the mutation was used to replace the same region in the pFBdh D793E vector. Login to comment
62 Mutations were validated by sequencing (ACGT Corp., Toronto, Ontario, Canada) and cloned into the pFBdh D793E vector to replace the equivalent KpnI-ClaI DNA sequence. Login to comment
103 Dried gels were then autoradiographed at -70 °C. EValuation of the Effects of D793E and D793E/P794A Mutations by Molecular Modeling and Dynamic Simulation Analysis. Login to comment
104 To evaluate the impact of the D793E mutation on ATP hydrolysis at NBS1 and the influence of the D793E/P794A double mutation on ADP release, a MRP1 NBD1/NBD2 heterodimer model was constructed using the MJ0796 NBD dimer (PDB code 1L2T) as a template, based on a previously published method (27). Login to comment
108 With the available wt NBD dimer model, NBD1-D793E and NBD1-D793E/P794A mutants were generated using the Xtalview program (41). Login to comment
141 The conserved asymmetric residues in NBD1 and NBD2 that were mutated in the background of the previously described D793E mutation are shown in Figure 2, with each of the atypical residues being replaced by the corresponding amino acid from the other NBD. Login to comment
146 As previously reported, the D793E mutation decreased ATP-dependent LTC4 transport by 50-70% relative to the wt construct (26). Login to comment
147 Determination of the effects of the additional mutations revealed that two second mutations, P794A and W653Y, were each able to restore activity of the D793E mutant to levels that were comparable to or somewhat higher than wt MRP1. Login to comment
148 The Walker A V680T mutation and the signature C V1432G mutation in NBD2 were without effect, while the double L1437R/C1439S signature C mutation essentially inactivated the D793E protein (Figure 3B). Login to comment
149 To investigate if the effects of the P794A and W653Y mutations on LTC4 transport activity were additive, both mutations were introduced into the D793E mutant. Login to comment
150 Although LTC4 transport activity was increased in the triple mutant relative to that of dh D793E, it was similar to that of wt dh MRP1 (Figure 3D). Login to comment
151 Thus the positive effect of the individual W653Y and P794A mutations on activity of the D793E mutant MRP1 was not additive to any detectable extent. Login to comment
155 As found previously (26), the D793E mutation did not change the relative levels of photolabeling of either NBD (Figure 4). Login to comment
156 Overall, the effects of the additional mutations on the labeling profile of the D793E mutant were minimal with the possible exception of the W653Y mutation which modestly decreased the photolabeling of NBD1 relative to NBD2. Login to comment
159 Previously, we demonstrated that the D793E mutation markedly increased the extent of tight binding of ADP at NBS1 (26). Login to comment
161 Consequently, we proposed that the decrease in LTC4 transport activity of MRP1 D793E was attributable to tight VO4-independent binding of ADP by the mutant NBS1 and a decrease in its rate of release (26). Login to comment
162 Concomitant with the tight binding of ADP at NBS1 by the D793E mutant protein, we observed a decrease in VO4-dependent trapping of ADP at NBS2. Login to comment
167 The results also confirmed previous studies indicating that the D793E mutation resulted in VO4-independent occlusion of nucleotide at NBS1 and that VO4-dependent trapping at NBS2 was markedly decreased (26). Login to comment
168 In contrast, BeFx markedly increased the extent of trapping at both sites, particularly at NBS2, so that the photolabeling profile of the D793E mutant was similar to that obtained with the wt protein. Login to comment
169 The D793E/V1432G mutation, which did not restore LTC4 transport activity, displayed VO4-independent photolabeling of NBD1 and a decrease in VO4-dependent photolabeling of NBD2 relative to wt MRP1, as observed with the D793E FIGURE 2: Topology of MRP1 showing the positions of conserved asymmetric mutations selected for mutation. Login to comment
172 However, both second mutations that restored LTC4 transport by D793E MRP1, W653Y and P794A, decreased VO4-independent photolabeling of NBD1 and enhanced the extent of VO4-dependent trapping at this site. Login to comment
173 The photolabeling profile of the all of the double mutants in the presence of BeFx was similar to the single D793E mutant and wt protein with NBD2 being the predominantly labeled site. Login to comment
179 One of the striking effects of the D793E mutation is that it prevents FIGURE 3: Expression of wt and mutant dual halves of MRP1 in Sf21 cells and determination of [3 H]LTC4 transport activity. Login to comment
180 (A) Immunoblot of membrane vesicle proteins isolated from Sf21 cells expressing wt or mutant dual halves of MRP1 (dh D793E, dh W653Y/D793E, dh V680T/D793E, dh D793E/P794A, dh D793E/V1432G, and dh D793E/L1437R/C1439S) or Sf21 cells infected with a control vector expressing -gus. Login to comment
181 (C) Immunoblots of the wt and mutant dual halves of MRP1 (dh D793E and dh W653Y/D793E/P794A). Login to comment
192 In view of the effects of the W653Y and P794A mutations on VO4-dependent nucleotide trapping at NBS1, we examined their influence on the ability of MRP1 D793E to shift between high-and low-affinity conformations. Login to comment
193 As previously observed, ATP in the presence of VO4 markedly decreased LTC4 photolabeling of wt MRP1 but had no effect on the apparent affinity of the D793E mutant (26) (Figure 6A). Login to comment
195 Consistent with their effects on VO4-dependent nucleotide trapping by the D793E mutant protein (Figure 5), both the W653Y and P794D second mutations restored the ability of ATP plus VO4 to induce the shift from high- to low-affinity LTC4 binding (Figure 6A). Login to comment
199 However, in contrast to VO4, BeFx plus ATP also markedly decreased LTC4 photolabeling of the single D793E mutant. Login to comment
200 To investigate the possibility that this difference between VO4 and BeFx may have been attributable to the trapping of ATP rather than ADP, we compared the photolabeling of wt and D793E dh of MRP1 under BeFx trapping conditions, using both azido-[γ-32 P]ATP and azido-[R-32 P]ATP (Figure 6B). Login to comment
202 However, essentially no photolabeling of NBS1 of the D793E mutant was detected under similar conditions. Login to comment
211 (B) Photolabeling of wt and D793E mutant dh of MRP1 with 8-azido-[γ-32 P]ATP or 8-azido- [R-32 P]ATP in the presence or absence of BeFx or VO4. Login to comment
212 Membrane vesicles (20 µg of total protein) from Sf21 cells expressing wt or D793E dh of MRP1 were incubated with 8-azido-[γ-32 P]ATP or 8-azido-[R-32 P]ATP in the presence and absence of BeFx or VO4 under trapping conditions, as described in Figure 5. Login to comment
213 (C) Sf21 membrane vesicles (50 µg of total protein) containing D793E or D793E/P794A dh of MRP1 were incubated with 8-azido-[R-32 P]ATP (15 µM) for 15 min at 37 °C. Login to comment
217 In an attempt to directly compare the rate of release of ADP from NBS1 of the D793E and D793E/P794A mutants, both proteins were incubated with azido-[γ-32 P]ATP under hydrolytic conditions in the absence of VO4 and BeFx. Login to comment
221 The rapidity of the loss of nucleotide from NBD1 of the D793E/P794A mutant precluded determination of a time course of release. Login to comment
233 Molecular Dynamics Modeling and Possible Mechanisms Underlying the Effects of the D793E and the Secondary D794A Mutations. Login to comment
239 In this model, the Glu residue in the D793E mutant NBD1 forms a [carboxylate oxygen-hydrolytic water-ATP γ-P] catalytic dyad for ATP hydrolysis (Figure 10). Login to comment
244 However, in the D793E mutant, the distance variation range FIGURE 7: Comparison of [3 H]LTC4 transport by wt dh MRP1 and dh MRP1P794A. Login to comment
250 In the D793E mutant the distance between the carboxylate oxygen of E793 and the catalytic water hydrogen atom is approximately 2.74 Å, which is short enough to form a stable H-bond. Login to comment
252 This provides a plausible explanation for the increase in ATP hydrolysis at NBS1 resulting from the D793E mutation (26). Login to comment
258 The modeling and dynamic simulation data suggest that in D793E MRP1 the -amino atom of K684 in the Walker A motif forms tight and stable H-bonds of ~3.0 Å with the -phosphate oxygen, with the FIGURE 8: Photolabeling of wt dh MRP1 and dh MRP1P794A by 8-azido-[γ-32 P]ATP, 8-azido-[R-32 P]ATP, and [3 H]LTC4. Login to comment
276 Thus in the D793E/P794A mutant, the variation in distances between the K684 -amino nitrogen atom and -Pi is considerably increased compared to the D793E single mutation (Figure 11B,C). Login to comment
277 In addition, in D793E/P794A MRP1 the interaction between Q713 and the Mg2+ ion is also decreased with an abrupt fluctuation in distance from 2 to 6.0-8.0 Å occurring during a 40 ps dynamic simulation (Figure 11B,D). Login to comment
294 Among the mutants tested, two secondary mutations, W653Y and P794A, increased LTC4 transport activity of the D793E mutant to levels equal to or greater than that of the wt protein (Figure 3). Login to comment
301 However, the extent of VO4-dependent ADP trapping at NBS2 in the W653Y/D793E and P794A/D793E mutants was reduced relative to wt MRP1, as observed with the original D793E mutant (26). Login to comment
306 In contrast to VO4, BeFx markedly increased trapping at NBS2 and modestly increased trapping at NBS1 of the D793E single mutant, so that the labeling profile of MRP1D793E was very similar to that of wt protein (Figure 5). Login to comment
328 These effects on transport are consistent with our observations with MRP1D793E, but our results strongly suggest that the increase in activity is attributable to an increase in the release of ADP from the D793E/W653Y mutant protein, that may not have been apparent from studies of wt MRP1. Login to comment
337 This possibility is also strongly supported by direct comparison of the rapidity of the release of ADP from NBS1 of the D793E and D793E/P794A mutant proteins (Figure 6C). Login to comment
347 Overall, the experimental results of this and previous studies, combined with molecular dynamics simulations, strongly suggest that ATP hydrolysis occurs at NBS1, albeit with low efficiency, and that ADP release from this site, at least in the D793E mutant protein, may be rate limiting in the transport cycle of MRP1. Login to comment

PMID: 21143116 [PubMed] He SM et al: "Structural and functional properties of human multidrug resistance protein 1 (MRP1/ABCC1)."

No. Sentence Comment

654 The Asp793Glu mutation in NBD1 enhanced its hydrolytic capacity, whereas the Glu1455Asp mutation in NBD2 resulted in an increased affinity of NBD2 for ATP, but a decreased hydrolytic activity [322]. Login to comment
671 The Asp793Glu mutant of NBD1 showed increased hydrolytic capacity but had occlusion of the resultant ADP in the absence of vanadates [322]. Login to comment