ABCMdb

PMID: 19063607

Qin L, Zheng J, Grant CE, Jia Z, Cole SP, Deeley RG
Residues responsible for the asymmetric function of the nucleotide binding domains of multidrug resistance protein 1.
Biochemistry. 2008 Dec 30;47(52):13952-65., 2008-12-30 [PubMed]

Sentences

No. Mutations Sentence Comment

7 ABCC1 p.Asp793Glu ABCC1 p.Trp653Tyr ABCC1 p.Pro794Ala 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 8 ABCC1 p.Pro794Ala Moreover, introduction of the P794A mutation into wild-type MRP1 increased transport of leukotriene C4 approximately 2-fold. Login to comment 9 ABCC1 p.Asp793Glu 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 ABCC1 p.Asp793Glu 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 ABCC1 p.Asp793Glu 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 ABCC1 p.Asp793Glu ABCC1 p.Trp653Tyr ABCC1 p.Pro794Ala 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 39 ABCC1 p.Pro794Ala Molecular modeling of an NBD1/2 dimer, based on the recently published crystal structure of NBD1 of MRP1 (27), combined with molecular dynamics simulations, revealed that introduction of the P794A mutation increases flexibility of the linker region between the Walker B motif and D-loop. Login to comment 41 ABCC1 p.Asp793Glu ABCC1 p.Pro794Ala 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 ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu The dh MRP1 construct containing the D793E mutation was also constructed previously and designated dh D793E (26). Login to comment 55 ABCC1 p.Asp793Glu ABCC1 p.Trp653Tyr ABCC1 p.Pro794Ala ABCC1 p.Leu1437Arg ABCC1 p.Val680Thr ABCC1 p.Cys1439Ser ABCC1 p.Val1432Gly 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 56 ABCC1 p.Trp653Tyr ABCC1 p.Pro794Ala ABCC1 p.Leu1437Arg ABCC1 p.Val680Thr ABCC1 p.Cys1439Ser ABCC1 p.Val1432Gly The forward primers for W653Y, V680T, P794A, V1432G, L1437R, and C1439S were 5'-GCCACATTCACCTATGCCAGGAGC- GAC-3', 5'-GTGGTGGGCCAGACGGGCTGCGGAAAG-3', 5'-TTTA- CCTTCGATGAGGCCCTCTCAGCAGTGGA-3', 5'-AGAAC- CTCAGTGGCGGGCAGCGC-3', and 5'-CAGCGCCAGC- GTGTGAGCCTAGCCCG-3', respectively. Login to comment 58 ABCC1 p.Asp793Glu ABCC1 p.Trp653Tyr ABCC1 p.Pro794Ala ABCC1 p.Val680Thr 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 ABCC1 p.Asp793Glu The isolated Bsu36I-XbaI fragment containing the mutation was used to replace the same region in the pFBdh D793E vector. Login to comment 61 ABCC1 p.Leu1437Arg ABCC1 p.Cys1439Ser ABCC1 p.Val1432Gly To make V1432G, L1437R, and C1439S mutations, a KpnI-ClaI MRP1 DNA fragment was cloned into pBluescript II KS vector, and PCR mutagenesis was performed as described above. Login to comment 62 ABCC1 p.Asp793Glu 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 63 ABCC1 p.Pro794Ala The P794A mutation was also created in wt MRP1. Login to comment 65 ABCC1 p.Pro794Ala The forward primer for the P794A mutation was 5'-TACCTCT- TCGATGATGCCCTCTCAGCAGTGG-3', and the reverse primer was its complement. Login to comment 67 ABCC1 p.Pro794Ala The Bsu36I-XbaI fragment with the P794A mutation was cloned into pFBdh MRP1 to replace the equivalent DNA. Login to comment 103 ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Pro794Ala 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 ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Pro794Ala 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 ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Pro794Ala 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 ABCC1 p.Asp793Glu 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 ABCC1 p.Asp793Glu As previously reported, the D793E mutation decreased ATP-dependent LTC4 transport by 50-70% relative to the wt construct (26). Login to comment 147 ABCC1 p.Asp793Glu ABCC1 p.Trp653Tyr ABCC1 p.Pro794Ala 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 ABCC1 p.Asp793Glu ABCC1 p.Leu1437Arg ABCC1 p.Val680Thr ABCC1 p.Cys1439Ser ABCC1 p.Val1432Gly 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 ABCC1 p.Asp793Glu ABCC1 p.Trp653Tyr ABCC1 p.Pro794Ala 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 ABCC1 p.Asp793Glu 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 ABCC1 p.Asp793Glu ABCC1 p.Trp653Tyr ABCC1 p.Pro794Ala 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 ABCC1 p.Asp793Glu As found previously (26), the D793E mutation did not change the relative levels of photolabeling of either NBD (Figure 4). Login to comment 156 ABCC1 p.Asp793Glu ABCC1 p.Trp653Tyr 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 ABCC1 p.Asp793Glu Previously, we demonstrated that the D793E mutation markedly increased the extent of tight binding of ADP at NBS1 (26). Login to comment 161 ABCC1 p.Asp793Glu 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 ABCC1 p.Asp793Glu 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 ABCC1 p.Asp793Glu 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 ABCC1 p.Asp793Glu 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 ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Val1432Gly 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 ABCC1 p.Asp793Glu ABCC1 p.Trp653Tyr ABCC1 p.Pro794Ala 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 ABCC1 p.Asp793Glu 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 ABCC1 p.Asp793Glu 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 ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Trp653Tyr ABCC1 p.Pro794Ala ABCC1 p.Val680Thr ABCC1 p.Cys1439Ser (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 ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Trp653Tyr ABCC1 p.Pro794Ala (C) Immunoblots of the wt and mutant dual halves of MRP1 (dh D793E and dh W653Y/D793E/P794A). Login to comment 192 ABCC1 p.Asp793Glu ABCC1 p.Trp653Tyr ABCC1 p.Pro794Ala 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 ABCC1 p.Asp793Glu 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 ABCC1 p.Asp793Glu ABCC1 p.Trp653Tyr ABCC1 p.Pro794Asp 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 ABCC1 p.Asp793Glu However, in contrast to VO4, BeFx plus ATP also markedly decreased LTC4 photolabeling of the single D793E mutant. Login to comment 200 ABCC1 p.Asp793Glu 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 ABCC1 p.Asp793Glu However, essentially no photolabeling of NBS1 of the D793E mutant was detected under similar conditions. Login to comment 211 ABCC1 p.Asp793Glu (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 ABCC1 p.Asp793Glu 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 ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Pro794Ala (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 ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Pro794Ala 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 ABCC1 p.Asp793Glu ABCC1 p.Pro794Ala 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 222 ABCC1 p.Pro794Ala Nevertheless, the results are consistent with the P794A mutation increasing the rate of release of ADP from NBS1, as suggested by additional data presented above. Login to comment 223 ABCC1 p.Pro794Ala Effect of the P794A Mutation Alone on LTC4 Transport, ATP Binding, and Nucleotide Trapping. Login to comment 224 ABCC1 p.Trp653Tyr Introduction of a W653Y mutation in wt MRP1 has been reported to have no detectable effect on ATP binding and hydrolysis (49). Login to comment 225 ABCC1 p.Pro794Ala Consequently, we focused on the effect of the P794A mutation on the wt protein, both with respect to LTC4 transport, as well as ATP binding and nucleotide trapping. Login to comment 226 ABCC1 p.Pro794Ala The P794A mutation almost doubled LTC4 transport relative to wt MRP1 (Figure 7B). Login to comment 228 ABCC1 p.Pro794Ala However, the P794A mutation had a modest effect on the decrease in LTC4 binding observed in the presence of ATP- γ-S (Figure 8C). Login to comment 229 ABCC1 p.Pro794Ala Densitometry indicated that, in the presence of ATP-γ-S, photolabeling of the NH2-terminal half of wt MRP1 decreased ~4-fold while labeling of the P794A mutant decreased only ~2-fold. Login to comment 230 ABCC1 p.Pro794Ala To further examine the underlying cause of the increase in transport activity resulting from the P794A mutation, we determined the ATP dependence of LTC4 transport (Figure 9). Login to comment 231 ABCC1 p.Pro794Ala ABCC1 p.Pro794Ala The results of transport assays carried out with vesicles containing wt or P794A dh MRP1 at various concentrations of ATP indicated that the mutation did not affect the Km(ATP), which was determined to be 59 µM for the wt protein and 62 µM for the P794A mutant. Login to comment 233 ABCC1 p.Asp793Glu Molecular Dynamics Modeling and Possible Mechanisms Underlying the Effects of the D793E and the Secondary D794A Mutations. Login to comment 239 ABCC1 p.Asp793Glu 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 ABCC1 p.Asp793Glu 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 ABCC1 p.Asp793Glu 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 ABCC1 p.Asp793Glu This provides a plausible explanation for the increase in ATP hydrolysis at NBS1 resulting from the D793E mutation (26). Login to comment 253 ABCC1 p.Pro794Ala Dynamic Simulation of ADP-Bound MRP1-NBD1 Suggests That the Additional P794A Mutation Affects ADP Binding through the Walker A Motif and the Q-Loop. Login to comment 258 ABCC1 p.Asp793Glu 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 268 ABCC1 p.Pro794Ala FIGURE 9: Determination of the effect of the P794A mutation on the ATP dependence of LTC4 transport. Login to comment 273 ABCC1 p.Pro794Ala However, the additional P794A mutation eliminated the rigid, main-chain kink of P794. Login to comment 276 ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Pro794Ala 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 ABCC1 p.Asp793Glu ABCC1 p.Pro794Ala 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 ABCC1 p.Asp793Glu ABCC1 p.Trp653Tyr ABCC1 p.Pro794Ala 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 295 ABCC1 p.Trp653Tyr ABCC1 p.Pro794Ala Neither of the W653Y or P794A mutations altered azido-ATP binding by MRP1D793E under nonhydrolytic conditions, but both decreased VO4-independent tight binding of ADP at 37 °C by NBD1 (Figure 5). Login to comment 298 ABCC1 p.Trp653Tyr ABCC1 p.Pro794Ala Consistent with this suggestion, the W653Y and P794A mutations increased VO4-dependent nucleotide trapping at NBS1 of MRP1D793E. Login to comment 301 ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Trp653Tyr ABCC1 p.Pro794Ala 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 ABCC1 p.Asp793Glu 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 315 ABCC1 p.Pro794Ala The functional importance of the aromatic residue at the position corresponding to W653 has been studied previously in several ABC transporters, including wt FIGURE 11: Molecular dynamics simulations of ADP binding at NBS1 of MRP1D793E and MRP1D793E/P794A. Login to comment 316 ABCC1 p.Pro794Ala Panels A and B show the spatial relationship of selected key residues from NBD1 interacting with ADP and Mg2+ in MRP1D793E (A) and MRP1D793E/ P794A (B). Login to comment 319 ABCC1 p.Pro794Ala In MRP1D793E/P794A (panel B), the -amino atom of K684 cannot form H-bonds with oxygens of the ADP -phosphate, and the side chain of Q713 will "swing" away, thus weakening its bond to the Mg2+ ion. Login to comment 321 ABCC1 p.Pro794Ala In panel C, the dynamic distance between the K684 -amino nitrogen atom and ADP -Pi in MRP1D793E (0) was compared to the equivalent distance in MRP1D793E/P794A (2). Login to comment 322 ABCC1 p.Pro794Ala Panel D shows a similar comparison of the distance between the Q713 δ-oxygen and the Mg2+ ion (MRP1D793E, 0; MRP1D793E/P794A, 2). Login to comment 328 ABCC1 p.Asp793Glu ABCC1 p.Trp653Tyr 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 331 ABCC1 p.Pro794Ala Consequently, in addition to studying the effect of the P794A mutation on the function of MRP1D793E, we also generated this mutation in the wt protein. Login to comment 334 ABCC1 p.Pro794Ala ABCC1 p.Pro794Ala We also assessed the effect of the P794A mutation on the apparent affinity for ATP by comparing the ATP dependence of LTC4 transport by the P794A mutant and wt proteins. Login to comment 337 ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Pro794Ala 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 338 ABCC1 p.Pro794Ala To obtain more mechanistic insight into the effect of the P794A mutation on nucleotide binding, we also generated a model of the MRP1 NBD dimer and used a molecular dynamics approach to simulate the predicted effects of the Pro to Ala mutation on ADP binding by MRP1D793E. Login to comment 347 ABCC1 p.Asp793Glu 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