ABCMdb

ABCC1 p.Pro794Ala

None has been submitted yet.

Publications

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
8 Moreover, introduction of the P794A mutation into wild-type MRP1 increased transport of leukotriene C4 approximately 2-fold. 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
39 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 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
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
56 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 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
63 The P794A mutation was also created in wt MRP1. Login to comment
65 The forward primer for the P794A mutation was 5'-TACCTCT- TCGATGATGCCCTCTCAGCAGTGG-3', and the reverse primer was its complement. Login to comment
67 The Bsu36I-XbaI fragment with the P794A mutation was cloned into pFBdh MRP1 to replace the equivalent DNA. 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
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
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
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
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
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
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
222 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 Effect of the P794A Mutation Alone on LTC4 Transport, ATP Binding, and Nucleotide Trapping. Login to comment
225 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 The P794A mutation almost doubled LTC4 transport relative to wt MRP1 (Figure 7B). Login to comment
228 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 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 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 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
253 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
268 FIGURE 9: Determination of the effect of the P794A mutation on the ATP dependence of LTC4 transport. Login to comment
273 However, the additional P794A mutation eliminated the rigid, main-chain kink of P794. 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
295 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 Consistent with this suggestion, the W653Y and P794A mutations increased VO4-dependent nucleotide trapping at NBS1 of MRP1D793E. 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
315 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 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 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 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 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
331 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 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 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 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