ABCMdb

ABCC1 p.Asp793Asn

Predicted by SNAP2:	A: D (95%), C: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (95%), I: D (95%), K: 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, E: D, F: D, G: D, H: D, I: D, K: 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] Role of carboxylate residues adjacent to the conse... J Biol Chem. 2003 Oct 3;278(40):38537-47. Epub 2003 Jul 27. Payen LF, Gao M, Westlake CJ, Cole SP, Deeley RG
Role of carboxylate residues adjacent to the conserved core Walker B motifs in the catalytic cycle of multidrug resistance protein 1 (ABCC1).
J Biol Chem. 2003 Oct 3;278(40):38537-47. Epub 2003 Jul 27., 2003-10-03 [PMID:12882957]

Abstract [show]
MRP1 belongs to subfamily "C" of the ABC transporter superfamily. The nucleotide-binding domains (NBDs) of the C family members are relatively divergent compared with many ABC proteins. They also differ in their ability to bind and hydrolyze ATP. In MRP1, NBD1 binds ATP with high affinity, whereas NBD2 is hydrolytically more active. Furthermore, ATP binding and/or hydrolysis by NBD2 of MRP1, but not NBD1, is required for MRP1 to shift from a high to low affinity substrate binding state. Little is known of the structural basis for these functional differences. One minor structural difference between NBDs is the presence of Asp COOH-terminal to the conserved core Walker B motif in NBD1, rather than the more commonly found Glu present in NBD2. We show that the presence of Asp or Glu following the Walker B motif profoundly affects the ability of the NBDs to bind, hydrolyze, and release nucleotide. An Asp to Glu mutation in NBD1 enhances its hydrolytic capacity and affinity for ADP but markedly decreases transport activity. In contrast, mutations that eliminate the negative charge of the Asp side chain have little effect. The decrease in transport caused by the Asp to Glu mutation in NBD1 is associated with an inability of MRP1 to shift from high to low affinity substrate binding states. In contrast, mutation of Glu to Asp markedly increases the affinity of NBD2 for ATP while decreasing its ability to hydrolyze ATP and to release ADP. This mutation eliminates transport activity but potentiates the conversion from a high to low affinity binding state in the presence of nucleotide. These observations are discussed in the context of catalytic models proposed for MRP1 and other ABC drug transport proteins.

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No. Sentence Comment
78 The forward primers for D793Q, D793N, D793S, E1455Q, E1455N, E1455S, and E1455L were 5Ј-GCTGACATTTACCTCTTCGATCAACCGCTCTC- AGCAGTGGATGCC-3Ј, 5Ј-GCTGACATTTACCTCTTCGATAATCCGC- TCTCAGCAGTGGATGCC-3Ј, 5Ј-GCTGACATTTACCTCTTCGATTCT- CCCCTCTCAGCAGTGGATGCC-3Ј, 5Ј-CGAAGATCCTTGTGTTGGA- TCAGGCCACGGCGGCCGTGGACCTGG-3Ј, 5Ј-CGAAGATCCTTGTG- TTGGATA ACGCCACGGCCGCCGTGGACCTGG-3Ј, 5Ј-CGAAGATCC- TTGTGTTGGATTCGGCCACGGCAGCCGTGGACCTGG-3Ј, 5Ј-CGAA- GATCCTTGTGTTGGATTTGGCCACGGCCGCCGTGGACCTGG-3Ј, respectively. Login to comment
241 To determine whether there may be additional consequences to eliminating the carboxylate side chain, we mutated Asp793 and Glu1455 to Asn, Gln, and Ser as observed in cystic fibrosis transmembrane conductance regulator. 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
264 Similar to wild-type, in the absence of vanadate, D793S, D793Q, and D793N mutant proteins did not display any nucleotide binding at either NBD (Fig. 8B). Login to comment
267 Although labeling at NBD2 was decreased in the D793Q and D793N mutants, this domain remained the predominant site of photolabeling (Fig. 8B, lanes 7 and 9). Login to comment
270 Effect of D793Q, D793N, D793S, E1455S, E1455Q, E1455N, E1455L mutations on [3 H]LTC4 transport activity. Login to comment
271 A, membrane proteins (1 ␮g) from Sf21 cells expressing both halves of either MRP1 (MRP1 dh) or mutant proteins (D793Q, D793N, D793S, E1455S, E1455Q, E1455N, E1455L) were separated by SDS-PAGE on gradient gels and transferred to Immobilon-P membranes. Login to comment
275 B, membrane vesicles (2 ␮g) containing MRP1 dh, D793Q, D793S, D793N, E1455Q, E1455S, E1455N, E1455L, or beta-Gus 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
304 Comparison of nucleotide binding and vanadate trapping by wild-type MRP1 and mutant proteins (D793Q, D793N, D793S, E1455S, E1455Q, E1455N, and E1455L). Login to comment
305 A, at 4 °C, 8-azido- [␣-32 P]ATP photolabeling by wild-type MRP1 and mutant proteins (D793Q, D793N, D793S, E1455S, E1455Q, E1455N, and E1455L) was evaluated. Membrane vesicles (20 ␮g) were incubated with 5 ␮M 8-azido-[␣-32 P]ATP for 5 min on ice in transport buffer containing 5 mM MgCl2. Login to comment
308 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 and C, at 37 °C under trapping conditions, 8-azido-[␣-32 P]ADP trapping by wild-type MRP1 mutant proteins (D793Q, D793N, D793S, E1455S, E1455Q, E1455N, and E1455L) was studied. Login to comment
339 Mutation of Asp793 to Asn, Gln, and also Ser, which is the residue found at the comparable position of cystic fibrosis transmembrane conductance regulator, all decreased slightly LTC4 transport activity, whereas the comparable mutations of Glu1455 inactivated the protein (Fig. 7B). Login to comment

[hide] Nucleotide dissociation from NBD1 promotes solute ... Biochim Biophys Acta. 2005 Mar 1;1668(2):248-61. Yang R, McBride A, Hou YX, Goldberg A, Chang XB
Nucleotide dissociation from NBD1 promotes solute transport by MRP1.
Biochim Biophys Acta. 2005 Mar 1;1668(2):248-61., 2005-03-01 [PMID:15737336]

Abstract [show]
MRP1 transports glutathione-S-conjugated solutes in an ATP-dependent manner by utilizing its two NBDs to bind and hydrolyze ATP. We have found that ATP binding to NBD1 plays a regulatory role whereas ATP hydrolysis at NBD2 plays a dominant role in ATP-dependent LTC4 transport. However, whether ATP hydrolysis at NBD1 is required for the transport was not clear. We now report that ATP hydrolysis at NBD1 may not be essential for transport, but that the dissociation of the NBD1-bound nucleotide facilitates ATP-dependent LTC4 transport. These conclusions are supported by the following results. The substitution of the putative catalytic E1455 with a non-acidic residue in NBD2 greatly decreases the ATPase activity of NBD2 and the ATP-dependent LTC4 transport, indicating that E1455 participates in ATP hydrolysis. The mutation of the corresponding D793 residue in NBD1 to a different acidic residue has little effect on ATP-dependent LTC4 transport. The replacement of D793 with a non-acidic residue, such as D793L or D793N, increases the rate of ATP-dependent LTC4 transport. Along with their higher transport activities, their Michaelis constant Kms (ATP) are also higher than that of wild-type. Coincident with their higher Kms (ATP), their Kds derived from ATP binding are also higher than that of wild-type, implying that the rate of dissociation of the bound nucleotide from the mutated NBD1 is faster than that of wild-type. Therefore, regardless of whether the bound ATP at NBD1 is hydrolyzed or not, the release of the bound nucleotide from NBD1 may bring the molecule back to its original conformation and facilitate the protein to start a new cycle of ATP-dependent solute transport.

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No. Sentence Comment
7 The replacement of D793 with a non-acidic residue, such as D793L or D793N, increases the rate of ATP-dependent LTC4 transport. 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
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
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
164 Interestingly, the substitution of the putative catalytic base D793 in NBD1 with a non-acidic amino acid, such as D793L or D793N, increases ATP-dependent LTC4 transport activity (Fig. 3 and Table 1), implying that ATP hydrolysis at NBD1 might not be essential for the transport. 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
183 / Biochimica et Biophysica Acta 1668 (2005) 248-261 253 the E1455Q mutated NBD2 as shown in Fig. 7M and O; whereas the higher Vmax value (Table 1) of D793L (a ratio of 0.993 indicates that the amount of D793L is slightly less than wild-type) or D793N (ratio of 1.151) indicates a slightly increased k2 value, leading to a higher Km (LTC4) value and a higher rate of ATP-dependent LTC4 transport. 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
187 Whereas the moderately increased k2 values for D793L and D793N could be interpreted in the following two ways: (1) The mutation of the putative catalytic base D793 to a non-acidic amino acid, such as L or N, somehow increases the rate of ATP hydrolysis at the mutated NBD1 and enhances ATP-dependent LTC4 transport; (2) The mutation of the putative catalytic base D793 to a non-acidic amino acid, such as L or N, decreases the affinity for ATP and increases the release rate of the bound nucleotide from the mutated NBD1. 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
224 The Kd (ATP) values for D793L and D793N mutated NBD1 co-expressed with wild-type NBD2 are almost three fold higher than that of wild-type NBD1 (Table 3), implying that the mutation of this acidic D793 residue to a non-acidic amino acid decreased k1 (lower rate of binding) and/or increased kÀ1 (higher rate of releasing), i.e. lower affinity. Login to comment
225 The mutation of D793L or D793N does not have a significant effect on the wild-type NBD2 (Table 3). Login to comment
227 In contrast to the counterpart NBD1 mutants D793N and D793L, the substitution of the E1455 with a non-acidic amino acid has an effect on the wild-type NBD1 (Table 3). Login to comment
229 The substitution of the putative catalytic D793 residue with a non-acidic amino acid increases release rate of the bound ATP The higher Kd (ATP) values of D793L and D793N mutated NBD1 were interpreted as that the binding rate was decreased whereas the release rate of the bound ATP was increased. 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
264 D793L and D793N mutated NBD1s have higher Kd values than that of wild-type. 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
272 K and L: D793N mutated N-half (K) co-expressed with wild-type C-half (L). 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
287 In contrast, interestingly, the substitution of the putative catalytic residue D793 in NBD1 with a non-acidic amino acid, such as D793L or D793N, increased the rate of ATP-dependent LTC4 transport ([29] and Fig. 3). Login to comment
288 This radical substitution increased the Kd (ATP) values of the D793L or D793N mutated NBD1 (Fig. 6 and Table 3), where Kd=kÀ1/k1, since ATP hydrolysis on ice is limited. Login to comment
289 The increased Kd of D793N or D793L means increased kÀ1, in other words, increased releasing rate of the bound ATP, and/or decreased k1, in other words, decreased the rate of ATP binding. Login to comment
290 The time required to lose 50% of the bound ATP from the D793L or D793N mutated NBD1 and the co-expressed wild-type NBD2 is much shorter than that of the wild-type (Fig. 7 and Table 4), implying the increased kÀ1 and/or decreased k1 values. 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
308 K and L: D793N mutated N-half (K) co-expressed with wild-type C-half (L). 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

[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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No. Sentence Comment
262 Interestingly, substitution of the corresponding putative catalytic residue D793 in NBD1 with a non-acidic residue, such as D793L or D793N, increased the rate of ATP-dependent LTC4 transport [139, 144]. Login to comment