ABCMdb

ABCC1 p.Glu1455Asp

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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
72 The EcoRI-KpnI fragments with mutations at NBD2 were isolated from pGEM-NBD2 and used to replace the equivalent region in pBS-Asp45 to generate pBS-D45/E1455D. Login to comment
73 This was then digested with NcoI and KpnI and the NcoI-KpnI fragment was used to replace the equivalent region of pFBDual-Asp45 to give pFBDual-D45/E1455D. Login to comment
74 Finally, the SalI-XbaI fragment of pFBDual-halves was isolated and cloned into pFBDual-D45/E1455D, which had been digested with the same enzymes, to generate pFBDual-halves/E1455D. 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
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
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
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
166 In contrast to the ATP binding observed at 4 °C, photolabeling at 37 °C of the E1455D mutant NBD2 was stronger than the co-expressed wild-type NBD1 and was only slightly competed by 1 mM AMP-PNP. 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
192 ADP binding by the E1455D single mutant was strongly increased at both NBDs relative to the co-expressed wild-type fragments although the majority of photolabeling occurred at the mutant NBD2. 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
207 In contrast, the E1455D mutation displayed very strong labeling of the mutant NBD2 that persisted following the reincubation period (Fig. 5, lanes 4 and 10). 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
212 However, relatively strong photolabeling of both NBDs was retained in the E1455D mutant. 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
234 The LTC4 labeling profile of the E1455D mutant in the absence of nucleotide was also similar to that observed for wild-type MRP1 (Fig. 6B, lanes 2 and 11). 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
249 In contrast, the NBD2 mutations (E1455S, E1455N, E1455Q, and E1455L) like E1455D completely abolished LTC4 transport (Fig. 7B). 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
268 Like the E1455D mutant, the E1455S, E1455N, and E1455L mutations resulted in strong vanadate-independent photolabeling of NBD2 and increased vanadate-dependent photolabeling of NBD1 (Fig. 8C, lanes 3, 4, and 7-10). Login to comment
296 The E1455D mutation essentially eliminated transport of LTC4 as observed previously with mutations that inactivate NBD2 (Fig. 2B). Login to comment
302 Thus the E1455D mutation substantially increased affinity for both ATP and ADP. Login to comment
312 The E1455D mutation also strongly enhanced nucleotide binding and vanadate-dependent trapping at the co-expressed wild-type NBD1 (Fig. 3, B and C). Login to comment
319 Contrary to expectation, the markedly increased 8-azido-[␣-32 P]ADP binding by the E1455D mutant NBD2 was accompanied by increased rather than decreased ADP photolabeling of the co-expressed wild-type NBD1, suggesting that both NBDs could be simultaneously occupied by ADP. Login to comment
322 We also found that the E1455D mutation drastically decreased nucleotide release at 37 °C, as evidenced by an inability to displace either prebound 8-azido-[␣- 32 P]ATP (Fig. 5) or 8-azido-[␣-32 P]ADP (data not shown) with a large excess of cold nucleotide. Login to comment
324 In the absence of nucleotides, LTC4 photolabeling was unaffected by the E1455D mutation. Login to comment
327 This is consistent with stable vanadate-independent binding of nucleotide by the E1455D mutant NBD2 (Fig. 3B). Login to comment
328 In addition, ATP␥S was much more effective at reducing binding by the E1455D mutant when compared with the wild-type protein. Login to comment
330 Thus the loss of LTC4 transport activity appears to be attributable to the impaired ability of the E1455D mutant to release nucleotide from NBD2 and to re-establish a high affinity binding state. 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
342 Like the E1455D mutation, the substitution of Glu1455 with Gln, Asn, Leu, and Ser completely abolished LTC4 transport activity (Fig. 7B). Login to comment
343 However, in contrast to the E1455D mutation, which at 4 °C increased ATP binding at NBD2, binding was decreased by the Gln, Ser, and Leu mutations, whereas the Asn mutation had little or no effect relative to wild-type (Fig. 8A, lanes 4, 6, 8, and 9). Login to comment
345 At 37 °C, Ser, Asn, and Leu mutations of Glu1455 strongly increased nucleotide binding and trapping at both NBDs in the presence and absence of vanadate, as observed with the E1455D mutation, whereas binding by the Gln mutant protein, although increased relative to wild-type MRP1, remained strongly vanadate-dependent (Fig. 8C). Login to comment
346 This suggests that the Gln mutant, unlike the other mutant proteins including the E1455D mutant, is able to hydrolyze ATP and probably release ADP in the absence of vanadate. 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

46 The reciprocal E1455D mutation of NBD2 increased the affinity of NBD2 for both azido-ATP and -ADP, resulting in prolonged binding of both nucleotides. 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

242 In contrast, mutation of the putative catalytic residue E1455 to a short chain D residue, E1455D, markedly increased the affinity of the mutated NBD2 for ATP while decreasing its ability to hydrolyze ATP [62], leading to significantly increased α-32 P-ATP labeling regardless of whether Vi is present or not [62]. Login to comment
243 Binding of ATP, ATP + Vi, or ATPγS to E1455D significantly inhibited the LTC4 labeling [62], further supporting the above hypothesis that occupancy of both NBD1 and NBD2 by nucleotide binding without hydrolysis may be sufficient to transport the bound substrate across membrane bilayer. Login to 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
261 This conclusion is further supported by mutation of the putative catalytic residue E1455 in NBD2 that all the mutants, including E1455S, E1455Q, E1455N, E1455L and E1455D, lost their abilities to transport LTC4 across membrane bilayer [62, 144]. Login to comment

PMID: 19949927 [PubMed] Chang XB et al: "Molecular mechanism of ATP-dependent solute transport by multidrug resistance-associated protein 1."

No. Sentence Comment

151 However, binding of poorly hydrolysable ATP analog ATPgS to wt MRP1 significantly inhibits the 3 H-LTC4 labeling (99, 100), implying that ATPgS binding might be sufficient to transport the bound LTC4 from high to low affinity site. This conclusion was further supported by the fact that ATPgS or ATP binding to the incompetent E1455D or E1455Q mutants, which were unable to hydrolyze the bound ATP, significantly inhibited the 3 H-LTC4 labeling (100, 156, 157). Login to comment
158 Conversely, mutation of the putative catalytic residue E1455 to a short chain D residue, E1455D, markedly increased the affinity of the mutated NBD2 for ATP while decreased its ability to hydrolyze ATP (100), leading to significantly increase the a-32 P-ATP labeling regardless of whether Vi is present or not (100). Login to comment
159 Binding of ATP, ATP + Vi, or ATPgS to E1455D significantly inhibited the LTC4 labeling (100), further supporting the above hypothesis that occupancies of both NBD1 and NBD2 by nucleotide binding without hydrolysis may be sufficient to transport the bound substrate across membrane bilayer. 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
673 The Glu1455Asp mutant with a reciprocal mutation of NBD2 exhibited an increased affinity for both azido-ATP and -ADP. Login to comment