ABCMdb

ABCC1 p.Lys684Leu

Predicted by SNAP2:	A: D (95%), C: D (95%), D: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (95%), I: D (95%), L: D (95%), M: D (95%), N: D (95%), P: D (95%), Q: D (95%), R: D (95%), S: D (95%), T: D (95%), V: D (95%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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[hide] Allosteric interactions between the two non-equiva... J Biol Chem. 2000 Jul 7;275(27):20280-7. Hou Y, Cui L, Riordan JR, Chang X
Allosteric interactions between the two non-equivalent nucleotide binding domains of multidrug resistance protein MRP1.
J Biol Chem. 2000 Jul 7;275(27):20280-7., 2000-07-07 [PMID:10781583]

Abstract [show]
Membrane transporters of the adenine nucleotide binding cassette (ABC) superfamily utilize two either identical or homologous nucleotide binding domains (NBDs). Although the hydrolysis of ATP by these domains is believed to drive transport of solute, it is unknown why two rather than a single NBD is required. In the well studied P-glycoprotein multidrug transporter, the two appear to be functionally equivalent, and a strongly supported model proposes that ATP hydrolysis occurs alternately at each NBD (Senior, A. E., al-Shawi, M. K., and Urbatsch, I. L. (1995) FEBS Lett 377, 285-289). To assess how applicable this model may be to other ABC transporters, we have examined adenine nucleotide interactions with the multidrug resistance protein, MRP1, a member of a different ABC family that transports conjugated organic anions and in which sequences of the two NBDs are much less similar than in P-glycoprotein. Photoaffinity labeling experiments with 8-azido-ATP, which strongly supports transport revealed ATP binding exclusively at NBD1 and ADP trapping predominantly at NBD2. Despite this apparent asymmetry in the two domains, they are entirely interdependent as substitution of key lysine residues in the Walker A motif of either impaired both ATP binding and ADP trapping. Furthermore, the interaction of ADP at NBD2 appears to allosterically enhance the binding of ATP at NBD1. Glutathione, which supports drug transport by the protein, does not enhance ATP binding but stimulates the trapping of ADP. Thus MRP1 may employ a more complex mechanism of coupling ATP utilization to the export of agents from cells than P-glycoprotein.

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33 Stable cell lines expressing wild-type and mutant MRP1s, K684L, D792L/D793L, K1333L, and D1454L/E1455L, were generated by using procedures described previously (11). Login to comment
205 Fig. 5C shows that although labeling by N3[␣-32 P]ATP was not abolished by the mutations, it was greatly reduced: K684L was ϳ10% of wild-type; K1333L, ϳ5%; D1454L/D1455L, ϳ15%. Login to comment
207 Fig. 5D demonstrates that labeling of K684L by N3[␥-32 P]ATP was almost eliminated and labeling of K1333L and D1454L/E1455L were decreased to ϳ10% and ϳ15% of the wild-type levels, respectively. Login to comment
213 The K1333L mutation in NBD2 nearly abolished ATP-dependent uptake as did the NBD2 Walker B substitution, whereas the K684L substitution reduced it by approximately half. Login to comment
232 Lane 1, 10 ␮g of wild-type MRP1; lane 2, 20 ␮g of K684L; lane 3, 10 ␮g of K1333L; lane 4, 10 ␮g of D1454L/ E1455L. Login to comment
234 Lane 1, 10 ␮g of wild-type MRP1; lane 2, 20 ␮g of K684L; lane 3, 10 ␮g of K1333L; lane 4, 10 ␮g of D1454L/ E1455L. Login to comment
235 E, ATP-dependent LTC4 uptake by membrane vesicles containing wild-type (closed diamonds) and mutant MRPs: NBD1 Walker A lysine mutant K684L (open circles), NBD2 Walker A lysine mutant K1333L (open square), NBD2 Walker B aspartate mutant D1454L/ E1455L (closed circles). Login to comment

[hide] ATP binding to the first nucleotide-binding domain... J Biol Chem. 2002 Feb 15;277(7):5110-9. Epub 2001 Dec 7. Hou YX, Cui L, Riordan JR, Chang XB
ATP binding to the first nucleotide-binding domain of multidrug resistance protein MRP1 increases binding and hydrolysis of ATP and trapping of ADP at the second domain.
J Biol Chem. 2002 Feb 15;277(7):5110-9. Epub 2001 Dec 7., 2002-02-15 [PMID:11741902]

Abstract [show]
Multidrug resistance protein (MRP1) utilizes two non-equivalent nucleotide-binding domains (NBDs) to bind and hydrolyze ATP. ATP hydrolysis by either one or both NBDs is essential to drive transport of solute. Mutations of either NBD1 or NBD2 reduce solute transport, but do not abolish it completely. How events at these two domains are coordinated during the transport cycle have not been fully elucidated. Earlier reports (Gao, M., Cui, H. R., Loe, D. W., Grant, C. E., Almquist, K. C., Cole, S. P., and Deeley, R. G. (2000) J. Biol. Chem. 275, 13098-13108; Hou, Y., Cui, L., Riordan, J. R., and Chang, X. (2000) J. Biol. Chem. 275, 20280-20287) indicate that intact ATP is observed bound at NBD1, whereas trapping of the ATP hydrolysis product, ADP, occurs predominantly at NBD2 and that trapping of ADP at NBD2 enhances ATP binding at NBD1 severalfold. This suggested transmission of a positive allosteric interaction from NBD2 to NBD1. To assess whether ATP binding at NBD1 can enhance the trapping of ADP at NBD2, photoaffinity labeling experiments with [alpha-(32)P]8-N(3)ADP were performed and revealed that when presented with this compound labeling of MRP1 occurred at both NBDs. However, upon addition of ATP, this labeling was enhanced 4-fold mainly at NBD2. Furthermore, the nonhydrolyzable ATP analogue, 5'-adenylylimidodiphosphate (AMP-PNP), bound preferentially to NBD1, but upon addition of a low concentration of 8-N(3)ATP, the binding at NBD2 increased severalfold. This suggested that the positive allosteric stimulation from NBD1 actually involves an increase in ATP binding at NBD2 and hydrolysis there leading to the trapping of ADP. Mutations of Walker A or B motifs in either NBD greatly reduced their ability to be labeled by [alpha-(32)P]8-N(3)ADP as well as by either [alpha-(32)P]- or [gamma-(32)P]8-N(3)ATP (Hou et al. (2000), see above). These mutations also strongly diminished the enhancement by ATP of [alpha-(32)P]8-N(3)ADP labeling and the transport activity of the protein. Taken together, these results demonstrate directly that events at NBD1 positively influence those at NBD2. The interactions between the two asymmetric NBDs of MRP1 protein may enhance the catalytic efficiency of the MRP1 protein and hence of its ATP-dependent transport of conjugated anions out of cells.

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50 Stable cell lines expressing wild-type and mutant MRP1s, K684L, D792A, K1333L, and D1454L/E1455L were established previously (2, 31). Login to comment
117 Essentially the stimulation was much reduced in the NBD1 mutants, K684L and D792A (Fig. 4, C and D), and in the NBD2 mutants, K1333L and D1454L/E1455L (Fig. 4, E and F), and the stimulation effects were shifted to higher ATP concentrations (Fig. 4, C-F). Login to comment
173 Lane 1, 10 ␮g of wild-type MRP1; lane 2, 15 ␮g of K684L; lane 3, 20 ␮g of D792A; lane 4, 10 ␮g of K1333L; lane 5, 10 ␮g of D1454L/E1455L. Login to comment
175 The results for K684L and D792A are the average of three independent experiments and for K1333L and D1454L/E1455L are the average of two independent experiments. C, influence of ATP on the [␣-32 P]8-N3ADP labeling of K684L. Login to comment
176 15 ␮g of K684L was labeled in the presence of varying amounts of ATP indicated above each lane. D, influence of ATP on the [␣-32 P]8-N3ADP labeling of D792A. Login to comment

[hide] ATP binding, not hydrolysis, at the first nucleoti... J Biol Chem. 2003 Feb 7;278(6):3599-605. Epub 2002 Nov 27. Hou YX, Riordan JR, Chang XB
ATP binding, not hydrolysis, at the first nucleotide-binding domain of multidrug resistance-associated protein MRP1 enhances ADP.Vi trapping at the second domain.
J Biol Chem. 2003 Feb 7;278(6):3599-605. Epub 2002 Nov 27., 2003-02-07 [PMID:12458196]

Abstract [show]
Multidrug resistance-associated protein (MRP1) transports solutes in an ATP-dependent manner by utilizing its two nonequivalent nucleotide binding domains (NBDs) to bind and hydrolyze ATP. We found that ATP binding to the first NBD of MRP1 increases binding and trapping of ADP at the second domain (Hou, Y., Cui, L., Riordan, J. R., and Chang, X. (2002) J. Biol. Chem. 277, 5110-5119). These results were interpreted as indicating that the binding of ATP at NBD1 causes a conformational change in the molecule and increases the affinity for ATP at NBD2. However, we did not distinguish between the possibilities that the enhancement of ADP trapping might be caused by either ATP binding alone or hydrolysis. We now report the following. 1) ATP has a much lesser effect at 0 degrees C than at 37 degrees C. 2) After hexokinase treatment, the nonhydrolyzable ATP analogue, adenyl 5'-(yl iminodiphosphate), does not enhance ADP trapping. 3) Another nonhydrolyzable ATP analogue, adenosine 5'-(beta,gamma-methylene)triphosphate, whether hexokinase-treated or not, causes a slight enhancement. 4) In contrast, the hexokinase-treated poorly hydrolyzable ATP analogue, adenosine 5'-O-(thiotriphosphate) (ATPgammaS), enhances ADP trapping to a similar extent as ATP under conditions in which ATPgammaS should not be hydrolyzed. We conclude that: 1) ATP hydrolysis is not required to enhance ADP trapping by MRP1 protein; 2) with nucleotides having appropriate structure such as ATP or ATPgammaS, binding alone can enhance ADP trapping by MRP1; 3) the stimulatory effect on ADP trapping is greatly diminished when the MRP1 protein is in a "frozen state" (0 degrees C); and 4) the steric structure of the nucleotide gamma-phosphate is crucial in determining whether binding of the nucleotide to NBD1 of MRP1 protein can induce the conformational change that influences nucleotide trapping at NBD2.

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226 Indeed, mutations of K684L and D792A greatly diminish the ATP enhancing effect on ADP trapping (34). Login to comment

[hide] Replacement of the positively charged Walker A lys... Biochem J. 2006 Jul 1;397(1):121-30. Buyse F, Hou YX, Vigano C, Zhao Q, Ruysschaert JM, Chang XB
Replacement of the positively charged Walker A lysine residue with a hydrophobic leucine residue and conformational alterations caused by this mutation in MRP1 impair ATP binding and hydrolysis.
Biochem J. 2006 Jul 1;397(1):121-30., 2006-07-01 [PMID:16551273]

Abstract [show]
MRP1 (multidrug resistance protein 1) couples ATP binding/hydrolysis at its two non-equivalent NBDs (nucleotide-binding domains) with solute transport. Some of the NBD1 mutants, such as W653C, decreased affinity for ATP at the mutated site, but increased the rate of ATP-dependent solute transport. In contrast, other NBD1 mutants, such as K684L, had decreased ATP binding and rate of solute transport. We now report that mutations of the Walker A lysine residue, K684L and K1333L, significantly alter the tertiary structure of the protein. Due to elimination of the positively charged group and conformational alterations, the K684L mutation greatly decreases the affinity for ATP at the mutated NBD1 and affects ATP binding at the unmutated NBD2. Although K684L-mutated NBD1 can bind ATP at higher concentrations, the bound nucleotide at that site is not efficiently hydrolysed. All these alterations result in decreased ATP-dependent solute transport to approx. 40% of the wild-type. In contrast, the K1333L mutation affects ATP binding and hydrolysis at the mutated NBD2 only, leading to decreased ATP-dependent solute transport to approx. 11% of the wild-type. Consistent with their relative transport activities, the amount of vincristine accumulated in cells is in the order of K1333L> or =CFTR (cystic fibrosis transmembrane conductance regulator)>K684L>>>wild-type MRP1. Although these mutants retain partial solute transport activities, the cells expressing them are not multidrug-resistant owing to inefficient export of the anticancer drugs by these mutants. This indicates that even partial inhibition of transport activity of MRP1 can reverse the multidrug resistance caused by this drug transporter.

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3 In contrast, other NBD1 mutants, such as K684L, had decreased ATP binding and rate of solute transport. We now report that mutations of the Walker A lysine residue, K684L and K1333L, significantly alter the tertiary structure of the protein. Login to comment
4 Due to elimination of the positively charged group and conformational alterations, the K684L mutation greatly decreases the affinity for ATP at the mutated NBD1 and affects ATP binding at the unmutated NBD2. Login to comment
5 Although K684L-mutated NBD1 can bind ATP at higher concentrations, the bound nucleotide at that site is not efficiently hydrolysed. Login to comment
10 Consistent with their relative transport activities, the amount of vincristine accumulated in cells is in the order of K1333L CFTR (cystic fibrosis transmembrane conductance regulator) > K684L wild-type MRP1. Login to comment
40 However, this speculation cannot explain why the K684L mutation decreases affinity for ATP at the mutated NBD1, in other words, increases the release rate from the mutated NBD1, but does not increase the rate of ATP-dependent solute transport. We have now found that replacement of the Walker A lysine residue with a leucine residue in either NBD1 (K684L) or NBD2 (K1333L) significantly alters the tertiary structure of the protein and affects ATP binding/hydrolysis and ATP-dependent solute transport. Login to comment
66 Reconstitution of wild-type, K684L- and K1333L-mutated MRP1 was achieved by employing SM-2 Bio-Beads to remove detergent from the protein/detergent/ lipid mixture as described previously [28]. Login to comment
70 Sample preparation Reconstituted wild-type, K684L- or K1333L-mutated MRP1s (20 µg) was mixed with either ATP + Vi or AMP-PNP (the molar ratio of protein to ATP is 1:6). Login to comment
105 RESULTS Mutation of NBD2 has a greater effect on the ATP-dependent LTC4 transport than the corresponding mutation of NBD1 We have found that the K1333L-mutated MRP1 almost completely abolished ATP-dependent solute uptake, whereas the corresponding mutation in NBD1, K684L, reduced it by approximately half [16]. Login to comment
107 0.564 (K684L) or approx. Login to comment
109 The ATP-dependent LTC4 transport activities of K684L- and K1333L-mutated MRP1, after adjusting the amount of MRP1 in the membrane vesicles to a similar amount with membrane vesicles containing CFTR, are approx. Login to comment
110 40% (K684L) and 11% (K1333L) of the wild-type MRP1 (Figure 1B), indicating that mutation of NBD2 has a larger effect on the ATP-dependent LTC4 transport than the corresponding mutation of NBD1. Login to comment
112 8% of the wild-type MRP1, implying that Figure 1 Expression and functional analysis of wild-type and Walker A lysine mutants (A) Expression of wild-type (WT), K684L- and K1333L-mutated MRP1 in BHK cells. Login to comment
113 Membrane vesicles were prepared from BHK cells expressing wild-type, K684L- or K1333L-mutated MRP1 and used in Western-blot analyses. Login to comment
117 The ratios of the band intensities are: 1.000 (wild-type MRP1), 0.564 + - 0.093 (K684L, n = 3) and 1.608 + - 0.175 (K1333L, n = 3). Login to comment
118 (B) Relative LTC4 transport activity by membrane vesicles containing wild-type, K684L- and K1333L-mutated MRP1. Login to comment
120 Since the amounts of MRP1 proteins in the membrane vesicles containing wild-type, K684L- and K1333L-mutated MRP1 were different, they were adjusted to a similar amount of MRP1 with membrane vesicles containing CFTR (1.692 µg of wild-type MRP1 + 1.308 µg of CFTR; 3 µg of K684L; 1.05 µg of K1333L + 1.95 µg of CFTR; 3 µg of CFTR) to determine the ATP-dependent LTC4 transport activity. Login to comment
123 Thus the ATP-dependent LTC4 transport activity of K684L- or K1333L-mutated MRP1 should be less than 40% or 11% of the wild-type MRP1. Login to comment
125 To test this hypothesis, membrane vesicles containing wild-type, K684L- and K1333L-mutated MRP1s were labelled with various concentrations of [α-32 P]8-N3ATP on ice (Figures 2A-2C). Login to comment
127 Figures 2(A) and 2(C) show that wild-type and K1333L-mutated MRP1 are heavily labelled by 16 µM [α-32 P]8-N3ATP, whereas K684L is not heavily labelled even at 256 µM (Figure 2B), implying that ATP binding to K684L-mutated NBD1 and unmutated NBD2 is significantly decreased. Login to comment
130 Using the same rationale, the labelling of K684L-mutated MRP1 should mainly occur at the unmutated NBD2. Login to comment
132 However, the Kd value of K684L is increased to 56 µM, which is much higher than that of wild-type MRP1 (Table 1) or the Kd value of wild-type NBD2 determined by digesting the labelled wild-type MRP1 with trypsin [20], implying that K684L mutation impairs the regulatory effect of ATP binding to NBD1 [19]. Login to comment
134 We then decided to use the non-hydrolysable ATP analogue, [α-32 P]8-N3-AMP-PNP, to label the MRP1 protein at 37◦ C. Figures 2(D-F) (wild-type MRP1, K684L and K1333L respectively) show that the labelling patterns of these MRP1 proteins with [α-32 P]8-N3-AMP-PNP at 37◦ C are similar to those performed on ice with [α-32 P]8-N3ATP (Figures 2A-2C). Login to comment
135 For example, the labelling intensity of K684L is much weaker than either wild-type or K1333L-mutated MRP1. Login to comment
136 Due to very low levels of labelling, the Kd (AMP-PNP) of K684L-mutated MRP1 cannot be accurately determined. Login to comment
139 To test this hypothesis, wild-type, K684L- and K1333L-mutated MRP1s were labelled with 8 µM of either [α-32 P]8-N3ATP or [γ -32 P]8-N3ATP at 37◦ C in the presence of Vi (Figure 3A). Login to comment
142 83.9% (K684L) and approx. Login to comment
143 109.3% (K1333L), implying that the ATP bound to K684L is more efficiently hydrolysed than the ATP bound to K1333L and less efficiently hydrolysed than the ATP bound to wild-type MRP1. Login to comment
144 These conclusions are supported by the results in Figure 3(B), i.e. (i) both [α-32 P]8-N3ATP and [γ -32 P]8-N3ATP labelled the unmutated NBD1 of K1333L-mutated MRP1 with similar intensity; (ii) [α-32 P]8-N3ATP mainly labelled the unmutated Figure 2 Nucleotide binding to wild-type and mutant MRP1s Samples were mixed in 10 µl of a solution containing 10 µg of wild-type MRP1 (A), K684L (B) or K1333L (C) membrane proteins and various concentrations of [α-32 P]8-N3ATP, incubated on ice for 1 min and UV-irradiated on ice for 2 min. Login to comment
145 Due to high background at a higher concentration of [α-32 P]8-N3ATP, the labelled K684L and K1333L proteins were immunoprecipitated with MRP1-specific antibodies 42.4 and 897.2 [16]. Login to comment
149 Samples were mixed in 10 µl of a solution containing 10 µg of wild-type MRP1 (D), K684L (E) or K1333L (F) and various concentrations of [α-32 P]8-N3-AMP-PNP, incubated at 37◦C for 10 min and UV-irradiated after washing with 500 µl of ice-cold Tris/EGTA buffer (0.1 mM EGTA and 40 mM Tris/HCl, pH 7.5) [16]. Login to comment
150 In order to prove that the labelling at K684L- or K1333L-mutated MRP1 mainly occurs at the unmutated NBD, the labelled wild-type (WT), K684L- and K1333L-mutated MRP1 were digested with trypsin (G). Login to comment
152 NBD2 of K684L, whereas [γ -32 P]8-N3ATP barely labelled NBD1 or NBD2 of K684L; (iii) [α-32 P]8-N3ATP labelled both NBD1 and NBD2 of wild-type MRP1, whereas [γ -32 P]8-N3ATP mainly labelled NBD1 of wild-type MRP1. Login to comment
153 At a higher concentration of ATP (128 µM in Figure 3C), K684L-mutated NBD1 and K1333L-mutated NBD2 are clearly labelled by either [α-32 P]8-N3ATP or [γ -32 P]8-N3ATP. Login to comment
154 Interestingly, the [γ -32 P]8-N3ATP labelling intensity of the unmutated NBD2 of K684L-mutated MRP1 is much weaker than the [α-32 P]8-N3ATP labelling, whereas the [γ -32 P]8-N3ATP labelling intensity of the K684L-mutated NBD1 is similar to that of [α-32 P]8-N3ATP labelling, implying that ATP bound to the K684L-mutated NBD1 is not efficiently hydrolysed. Login to comment
156 Table 1 Mean Kd (ATP) of wild-type and mutant MRP1 Protein Kd (µM ATP)* Wild-type MRP1 32.3 + - 10.9 K684L 55.9 + - 16.5 K1333L 10.8 + - 5.3 * The Kd values of wild-type MRP1 (n = 6), K684L (n = 6) and K1333L (n = 6) were derived from Figures 2(A)-2(C). Login to comment
167 The kinetics of H/2 H exchange of wild-type, K684L- and K1333L-mutated MRP1s in the absence of nucleotide were different from each other, with 37, 21 and 43% of amide hydrogen remaining unexchanged after a 2 h exposure to 2 H2O (Figure 5A), implying that replacement of the lysine residue with leucine altered the tertiary structure of the protein. Login to comment
169 Thus the protein with the K684L mutation becomes more 'relaxed` (greater extent of water accessibility) than that of the wild-type MRP1, whereas the protein with the K1333L mutation becomes slightly more 'compact` (lesser extent of water accessibility) than that of the wild-type MRP1. Login to comment
170 Conformational changes induced by nucleotide binding/hydrolysis at NBD1 are different from that at NBD2 Since binding of ATP to NBD1 induced conformational changes of the protein and enhanced ATP binding to NBD2 [19], we expected that conformational changes induced by ATP binding/ Figure 3 Walker A lysine mutations affect ATP binding and hydrolysis Samples were mixed in 10 µl of a solution containing wild-type (10 µg), K684L- (15 µg) or K1333L-mutated MRP1 (10 µg), 800 µM Vi and 8 µM of either [α-32 P]8-N3ATP or [γ -32 P]8-N3ATP,incubatedat37◦Cfor4 minandUV-irradiatedonicefor2 min(A).Thelabelled samples were subjected to SDS/PAGE (7% gel) and electroblotted on to a nitrocellulose membrane. Login to comment
171 The amounts of radioactivity incorporated into MRP1 were determined by using a Packard instant imager, yielding a ratio ([γ -32 P]8-N3ATP labelling versus [α-32 P]8-N3ATP labelling) of 60.4 + - 16.6% (wild-type, n = 7); 83.9 + - 17.5% (K684L, n = 4); and 109.3 + - 20.6% (K1333L, n = 5). Login to comment
172 Samples were mixed in 10 µl of a solution containing wild-type (10 µg), K684L- (15 µg) or K1333L-mutated MRP1 (10 µg), 800 µM Vi and 8 µM (B) or 128 µM (C) of either [α-32 P]8-N3ATP or [γ -32 P]8-N3ATP, incubated at 37◦C for 10 min, UV-irradiated on ice for 2 min, washed with 500 µl of ice-cold Tris-EGTA buffer and digested with trypsin (trypsin/protein ratio = 1:16). Login to comment
180 Table 2 Secondary structure analysis of wild-type, K684L- and K1333L-mutated MRP1 in the presence or in the absence of nucleotide Proportion (%) Protein and substrate α-Helix β-Sheet β-Turn Random coil MRP1 42 + - 3 29 + - 3 12 + - 4 17 + - 6 MRP1 + AMP-PNP 40 + - 2 28 + - 2 11 + - 3 21 + - 4 MRP1 + ATP + Vi 43 + - 2 27 + - 3 12 + - 3 18 + - 3 K684L 41 + - 2 31 + - 4 9 + - 4 19 + - 3 K684L + AMP-PNP 39 + - 3 30 + - 4 10 + - 3 21 + - 5 K684L + ATP + Vi 40 + - 2 32 + - 3 11 + - 3 17 + - 4 K1333L 42 + - 2 28 + - 4 11 + - 3 19 + - 3 K1333L + AMP-PNP 41 + - 3 29 + - 2 12 + - 2 18 + - 4 K1333L + ATP + Vi 39 + - 3 30 + - 3 13 + - 4 18 + - 4 (Figure 5B), indicating that ATP binding, hydrolysis and trapping of the hydrolysis product ADP by Vi induced conformational changes slightly different from those observed after AMP-PNP binding. Login to comment
181 For the K684L mutant, non-hydrolysable ATP analogue AMP-PNP binding increased the amount of unexchanged amide hydrogen from 21 to 25% (Figure 5C), whereas binding of hydrolysable ATP in the presence of Vi increased the amount of unexchanged amide hydrogen from 21 to 31% (Figure 5C). Login to comment
182 These results indicate that although nucleotide binding to the K684L-mutated NBD1 is significantly reduced (Figure 2), AMP-PNP binding made the protein more 'compact` than the original state, and ATP binding, hydrolysis and trapping of ADP (probably at the unmutated NBD2) led the protein to an even more 'compact` structure (Figure 5C). Login to comment
187 40, 11 and 8% of the wild-type MRP1, we expected that cells expressing K684L-mutated MRP1 should be partially resistant to anticancer drugs, whereas the cells expressing K1333L-mutated MRP1 should not. Login to comment
189 However, the cells expressing K684L-mutated MRP1 are also not resistant to daunomycin (Figure 6A), colchicine (Figure 6B) or vincristine (Figure 6C). Login to comment
190 The amount of vincristine that accumulated in cells expressing K1333L-mutated MRP1 is similar to that in cells expressing CFTR (Figure 7), whereas the accumulation of vincristine in cells expressing either wild-type or K684L-mutated MRP1 is slightly less than in cells expressing K1333L-mutated MRP1 or CFTR within 15 min incubation at 37◦ C (Figure 7), implying that either wild-type or K684L-mutated MRP1 has greater ability to transport vincristine out of the cells than the K1333L-mutated MRP1. Login to comment
191 The amount of vincristine that accumulated in cells expressing K684L-mutated MRP1 is more than in cells expressing wild-type MRP1, but less than in cells expressing either K1333L-mutated MRP1 or CFTR after 30, 60 or 120 min incubation at 37◦ C (Figure 7), implying that K684L-mutated MRP1 is more active than K1333L-mutated MRP1, but less active than wild-type MRP1. Login to comment
192 This lower transport activity resulted in the cells expressing K684L-mutated MRP1 not being resistant to vincristine (Figure 6C). Login to comment
193 Cells expressing either K684L- or K1333L-mutated MRP1s are not hypersensitive to verapamil We have found that cells expressing wild-type MRP1 are hypersensitive to verapamil [38]. Login to comment
194 Since K684L- or K1333L-mutated MRP1 has approx. Login to comment
195 40 or 11% of the wild-type MRP1 transport activity (Figure 1B), we expected that cells expressing K1333L-mutated MRP1 should have similar sensitivity to verapamil as the cells without MRP1 expression, whereas the cells expressing K684L-mutated MRP1s might be more sensitive to verapamil than the cells without MRP1 expression. Login to comment
197 However, the cells expressing K684L-mutated MRP1s are not more sensitive to verapamil than its parental BHK cells (Figure 6D), implying that the intracellular glutathione is not efficiently co-transported out with verapamil by the K684L-mutated MRP1. Login to comment
199 Both NBDs of MRP1 can bind nucleotide and contribute to solute transport Figure 5 Evolution of the proportion of unexchanged amide hydrogen in wild-type and mutant MRP1 as a function of the deuteration time The data were derived from Figure 4 by using wild-type (A, B), K684L- (A, C) or K1333L-mutated (A, D) MRP1s in the absence or presence of AMP-PNP or ATP + Vi. Login to comment
206 This asymmetry is visualized by IR spectroscopy that shows distinct conformational alterations for the Walker A lysine mutant K684L in NBD1, and the corresponding NBD2 mutant K1333L (Figure 5A), suggesting that the original tertiary structures of the two NBDs are different. Login to comment
212 In contrast, replacement of the positively charged Walker A Lys684 in NBD1 with a hydrophobic leucine greatly decreased the ATP binding at the mutated NBD1 (Figure 2) and significantly diminished the enhancing effect of ATP on the [α-32 P]8-N3ADP trapping [19] and the ATP-dependent LTC4 transport [15,16], probably due to the elimination of a positively charged group and the conformational alterations caused by the K684L mutation (Figure 5A). Login to comment
217 The results in Figure 3(C) indicate that the ATP bound to either K684L-mutated NBD1 or K1333L-mutated NBD2 is not efficiently hydrolysed. Login to comment
222 In contrast, replacement of the putative catalytic base Glu1455 , Figure 6 BHK cells expressing K684L- or K1333L-mutated MRP1 are neither multidrug-resistant nor hypersensitive to verapamil Cell survival experiments were performed according to the chemosensitivity assay as described in the Materials and methods section. Login to comment
223 Various concentrations of daunomycin (A), colchicine (B), vincristine (C) and verapamil (D) were applied to 96-well plates containing parental, wild-type MRP1-, K684L- or K1333L-transfected BHK cells. Login to comment
226 Figure 7 Vincristine accumulation in cells expressing wild-type, K684L- and K1333L-mutated MRP1 Intracellular accumulation of 3 H-labelled vincristine was carried out according to the vincristine accumulation method as described in the Materials and methods section. Login to comment
227 3 H-labelled vincristine (1 µM) was applied to the 24-well plate containing CFTR-, MRP1-, K684L- or K1333L-transfected BHK cells. Login to comment

[hide] A molecular understanding of ATP-dependent solute ... Cancer Metastasis Rev. 2007 Mar;26(1):15-37. Chang XB
A molecular understanding of ATP-dependent solute transport by multidrug resistance-associated protein MRP1.
Cancer Metastasis Rev. 2007 Mar;26(1):15-37., [PMID:17295059]

Abstract [show]
Over a million new cases of cancers are diagnosed each year in the United States and over half of these patients die from these devastating diseases. Thus, cancers cause a major public health problem in the United States and worldwide. Chemotherapy remains the principal mode to treat many metastatic cancers. However, occurrence of cellular multidrug resistance (MDR) prevents efficient killing of cancer cells, leading to chemotherapeutic treatment failure. Numerous mechanisms of MDR exist in cancer cells, such as intrinsic or acquired MDR. Overexpression of ATP-binding cassette (ABC) drug transporters, such as P-glycoprotein (P-gp or ABCB1), breast cancer resistance protein (BCRP or ABCG2) and/or multidrug resistance-associated protein (MRP1 or ABCC1), confers an acquired MDR due to their capabilities of transporting a broad range of chemically diverse anticancer drugs. In addition to their roles in MDR, there is substantial evidence suggesting that these drug transporters have functions in tissue defense. Basically, these drug transporters are expressed in tissues important for absorption, such as in lung and gut, and for metabolism and elimination, such as in liver and kidney. In addition, these drug transporters play an important role in maintaining the barrier function of many tissues including blood-brain barrier, blood-cerebral spinal fluid barrier, blood-testis barrier and the maternal-fetal barrier. Thus, these ATP-dependent drug transporters play an important role in the absorption, disposition and elimination of the structurally diverse array of the endobiotics and xenobiotics. In this review, the molecular mechanism of ATP-dependent solute transport by MRP1 will be addressed.

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256 Mutation of the Walker A motif K684 residue in NBD1, such as K684L [40, 141, 148], K684M [16, 63, 118], K684R [61] or K684E [61], significantly reduced ATP binding (at 4°C) at the mutated NBD1 and the intact NBD2 and Vi dependent ADP trapping at 37°C, but never completely abolished ATP-dependent solute transport. Login to comment

[hide] Characterization of the ATPase activity of a novel... Arch Biochem Biophys. 2009 May 15;485(2):102-8. Epub 2009 Mar 11. Wan L, Liang X, Huang Y
Characterization of the ATPase activity of a novel chimeric fusion protein consisting of the two nucleotide binding domains of MRP1.
Arch Biochem Biophys. 2009 May 15;485(2):102-8. Epub 2009 Mar 11., 2009-05-15 [PMID:19285030]

Abstract [show]
Nucleotide Binding Domains (NBDs) are responsible for the ATPase activity of the multidrug resistance protein 1 (MRP1). A series of NBD1-linker-NBD2 chimeric fusion proteins were constructed, expressed and purified, and their ATPase activities were analyzed. We report here that a GST linked NBD1(642-890)-GST-NBD2(1286-1531) was able to hydrolyze ATP at a rate of about 4.6 nmol/mg/min (K(m)=2.17 mM, V(max)=12.36 nmol/mg/min), which was comparable to the purified and reconstituted MRP1. In contrast, neither a mixture of NBD1 and GST-NBD2 nor the NBD1-GST-NBD1 fusion protein showed detectable ATPase activity. Additionally, the E1455Q mutant was found to be nonfunctional. Measurements by both MIANS labeling and circular dichroism spectroscopy revealed significant conformational differences in the NBD1-GST-NBD2 chimeric fusion protein compared to the mixture of NBD1 and GST-NBD2. The results suggest a direct interaction mediated by GST between the two NBDs of MRP1 leading to conformational changes which would enhance its ATPase activity.

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163 Among the NBD1-GST-NBD2 mutants, K684L in Walker A of NBD1, K1333L in Walker A of NBD2, and D1454L/E1455L in Walker B of NBD2 were expressed mainly as inclusion bodies in E. coli, and only the E1455Q mutant was expressed in a sufficient quantity of soluble protein to allow activity analysis. Login to comment

[hide] Mutations of the Walker B motif in the first nucle... Arch Biochem Biophys. 2001 Aug 1;392(1):153-61. Cui L, Hou YX, Riordan JR, Chang XB
Mutations of the Walker B motif in the first nucleotide binding domain of multidrug resistance protein MRP1 prevent conformational maturation.
Arch Biochem Biophys. 2001 Aug 1;392(1):153-61., [PMID:11469806]

Abstract [show]
ATP-binding cassette (ABC) transporters couple the binding and hydrolysis of ATP to the translocation of solutes across biological membranes. The so-called "Walker motifs" in each of the nucleotide binding domains (NBDs) of these proteins contribute directly to the binding and the catalytic site for the MgATP substrate. Hence mutagenesis of residues in these motifs may interfere with function. This is the case with the MRP1 multidrug transporter. However, interpretation of the effect of mutation in the Walker B motif of NBD1 (D792L/D793L) was confused by the fact that it prevented biosynthetic maturation of the protein. We have determined now that this latter effect is entirely due to the D792L substitution. This variant is unable to mature conformationally as evidenced by its remaining more sensitive to trypsin digestion in vitro than the mature wild-type protein. In vivo, the core-glycosylated form of that mutant is retained in the endoplasmic reticulum and degraded by the proteasome. A different substitution of the same residue (D792A) had a less severe effect enabling accumulation of approximately equal amounts of mature and immature MRP1 proteins in the membrane vesicles but still resulted in defective nucleotide interaction and organic anion transport, indicating that nucleotide hydrolysis at NBD1 is essential to MRP1 function.

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42 Stable cell lines expressing wild-type and mutant MRP1s, K684L, D792L/D793L, K1333L, and D1454L/E1455L were established previously (8). Login to comment
147 This was also true in the case of D793L (Fig. 5E) and mutations of the Walker A lysine residues in both NBDs (Fig. 5B, K684L, and Fig. 5G, K1333L), where the protein matured normally as did a variant in which the Walker B aspartate in NBD2 was mutated (Fig. 5H, D1454L/ E1455L). Login to comment
203 (B) K684L, 0.6 ␮g protein in each lane. Login to comment
146 This was also true in the case of D793L (Fig. 5E) and mutations of the Walker A lysine residues in both NBDs (Fig. 5B, K684L, and Fig. 5G, K1333L), where the protein matured normally as did a variant in which the Walker B aspartate in NBD2 was mutated (Fig. 5H, D1454L/ E1455L). Login to comment
202 (B) K684L, 0.6 òe;g protein in each lane. Login to comment