ABCMdb

ABCC1 p.Glu1455Gln

Predicted by SNAP2:	A: D (95%), C: D (95%), D: 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, D: 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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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
245 The exception was the NH2-proximal of the E1455Q mutant that was expressed at a level 1.75 times higher than its COOH-proximal half (Fig. 7A). Login to comment
246 Because the ATP-dependent uptake of LTC4 relies upon association of both halves of the protein and the levels of all COOH-proximal fragments were comparable, no adjustment was made to compensate for the increased expression of the NH2-proximal half of E1455Q (Fig. 7A). 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
261 Labeling of the wild-type NBD1 co-expressed with the E1455Q mutation appears to be increased, but we cannot completely exclude the possibility that this is attributable at least in part to the 1.75-fold higher level of expression of this fragment. Login to comment
269 The E1455Q FIG. 7. 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
280 These observations are consistent with NBD2 of the E1455Q mutant being occupied predominantly by ADP in the presence of vanadate, whereas in the other mutations that display significant levels of vanadate independent binding, the photolabeling of NBD2 may be the result of occupancy by either ATP or ADP. 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
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
349 Thus the behavior of the MRP1 Glu1455 to Gln mutation is similar to that of the comparable inactivating mutation in human P-GP, whereas the other mutations appear to block ADP release, as reported in studies of murine mdr3 (32, 33). 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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42 The definition of D793E means that the D793E mutated N-half is co-expressed with wild-type C-half and E1455Q, the wild-type N-half co-expressed with E1455Q mutated C-half. 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
64 This mutation in NBD2 (E1455Q) almost completely eliminates ATP-dependent LTC4 transport. 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
83 E1455Q was also introduced into the cDNA by the same strategy. Login to comment
92 This strategy was also used to generate constructs expressing D793E, L, and N mutated N-half and E1455Q mutated C-half. Login to comment
94 To make a construct expressing wild-type N-half and E1455Q mutated C-half simultaneously, the ClaI-BspEI fragment derived from pNUT/E1455Q and the RsrII-ClaI fragment from pDual/N-half/C-half were cloned into the RsrII-BspEI fragment derived from pDual/N-half/ C-half, named as pDual/N-half/E1455Q-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
158 Fig. 3 shows that E1455Q is almost completely inactivated, consistent with the previously reported result [43]. 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
186 The greatly decreased k2 value for E1455Q is interpreted as that mutation of the putative catalytic E1455 residue to a non-acidic amino acid greatly diminishes the rate of ATP hydrolysis at the mutated NBD2. 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
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
226 The Kd for E1455Q mutated NBD2 co-expressed with wild-type NBD1 is almost five fold higher than that of wild-type NBD2 (Table 3), implying that the substitution of the putative catalytic E1455 residue with 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
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
240 / Biochimica et Biophysica Acta 1668 (2005) 248-261 255 expected, most of the labeling disappeared within a short time, except for the E1455Q mutated NBD2 (Fig. 7M and O). Login to comment
241 The labeling with [g-32 P]-8-N3ATP at the C-half containing E1455Q mutated NBD2 is increased almost three fold within 2 min incubation at 37 8C and the T1/2 of this mutated NBD2 is much longer than that of wild-type (Table 4). Login to comment
243 / Biochimica et Biophysica Acta 1668 (2005) 248-261256 decreased Kd) at the E1455Q mutated NBD2 at 37 8C, consistent with our previous finding [51]; (2) the bound ATP at the E1455Q mutated NBD2 cannot be efficiently hydrolyzed due to the substitution of the putative catalytic acidic residue with a non-acidic amino acid (greatly decreased k2); (3) the release rate of the bound ATP from this mutated NBD2 is much lower (decreased kÀ1) than that of wild-type, leading to a longer T1/2 value (Table 4). Login to comment
245 In addition, the T1/2 value for the wild-type NBD1 co-expressed with E1455Q mutated NBD2 is slightly longer than that of wild-type N-half co-expressed with the wild-type C-half (Table 4), perhaps reflecting the stabilization effect of the bound nucleotide at NBD1 by the occluded nucleotide at NBD2 [28]. Login to comment
246 These results are consistent with the lower Km (ATP) value of E1455Q than that of the wild-type (Table 2) and the low ability to transport LTC4 (Figs. 3 and 5). 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
259 The substitution of the counterpart putative catalytic residue E1455 with a non-acidic amino acid (E1455Q) almost completely abolished the ATP-dependent LTC4 transport (Figs. 3 and 5), presumably due to the greatly decreased ATPase activity of this E1455Q mutated NBD2 (Fig. 7M and O). Login to comment
263 In addition, the substitution of this putative catalytic residue D793 with a longer spacer-arm negatively charged Glu enhances its hydrolytic capacity [43], but does not increase the ATP-dependent solute transport activity of D793E/E1455Q mutated MRP1 (Fig. 4) or markedly Fig. 6. Login to comment
273 N and O: Wild-type N-half (N) co-expressed with E1455Q mutated C-half (O). 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
283 The substitution of the putative catalytic E1455 with a non-acidic amino acid Q greatly increased the Kd (ATP) of the E1455Q mutated NBD2 (Fig. 6 and Table 3), meaning increased kÀ1 and decreased k1 on ice. Login to comment
284 This E1455Q mutation at NBD2 also has a negative effect on the co-expressed wild-type NBD1, leading to the increased Kd (Table 3), indicating the increased kÀ1 and decreased k1 on ice. Login to comment
285 However, the properties of the E1455Q mutated protein at 37 8C are different from that on ice. Login to comment
286 The [g-32 P]-8-N3ATP bound to the E1455Q mutated NBD2 cannot be efficiently hydrolyzed (greatly decreased k2) but occluded there (decreased kÀ1) (Fig. 7M and Table 4); in the meantime, the occluded [g-32 P]-8-N3ATP at the E1455Q mutated NBD2 stabilized the bound [g-32 P]-8-N3ATP [28] at the wild-type NBD1 co-expressed with the E1455Q mutated NBD2 (Fig. 7N), meaning decreased kÀ1; all these changes lead to a decreased Km (ATP) value and an inability to transport LTC4 (Table 1 and 2). Login to comment
295 E1455Q mutated NBD2 greatly diminishes the rate of ATP hydrolysis at the mutated NBD2. Login to comment
309 N and O: Wild-type N-half (N) co-expressed with E1455Q mutated C-half (O). 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] 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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220 Considering the data accumulated from other NBD2 mutants, such as Y1302C, E1455Q, H1486F and H1486L, the conformational alterations caused by the K1333L mutation may not be the only reason preventing ATP hydrolysis at the mutated site. 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
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

[hide] Interaction between the bound Mg.ATP and the Walke... Biochemistry. 2008 Aug 12;47(32):8456-64. Epub 2008 Jul 18. Yang R, Scavetta R, Chang XB
Interaction between the bound Mg.ATP and the Walker A serine residue in NBD2 of multidrug resistance-associated protein MRP1 plays a crucial role for the ATP-dependent leukotriene C4 transport.
Biochemistry. 2008 Aug 12;47(32):8456-64. Epub 2008 Jul 18., 2008-08-12 [PMID:18636743]

Abstract [show]
Structural analysis of human MRP1-NBD1 revealed that the Walker A S685 forms a hydrogen bond with the Walker B D792 and interacts with the Mg (2+) cofactor and the beta-phosphate of the bound Mg.ATP. We have found that substitution of the S685 with an amino acid that potentially prevents the formation of the hydrogen bond resulted in misfolding of the protein and significantly affect the ATP-dependent leukotriene C4 (LTC4) transport. In this report we tested whether the corresponding substitution in NBD2 would also result in misfolding of the protein. In contrast to the NBD1 mutations, none of the mutations in NBD2, including S1334A, S1334C, S1334D, S1334H, S1334N, and S1334T, caused misfolding of the protein. However, elimination of the hydroxyl group at S1334 in mutations including S1334A, S1334C, S1334D, S1334H, and S1334N drastically reduced the ATP binding and the ATP-enhanced ADP trapping at the mutated NBD2. Due to this low efficient ATP binding at the mutated NBD2, the inhibitory effect of ATP on the LTC4 binding is significantly decreased. Furthermore, ATP bound to the mutated NBD2 cannot be efficiently hydrolyzed, leading to almost completely abolishing the ATP-dependent LTC4 transport. In contrast, S1334T mutation, which retained the hydroxyl group at this position, exerts higher LTC4 transport activity than the wild-type MRP1, indicating that the hydroxyl group at this position plays a crucial role for ATP binding/hydrolysis and ATP-dependent solute transport.

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160 In order to test this hypothesis, membrane vesicles containing these mutants were used to label them with 3 H-LTC4 in the presence or absence of 1 mM ATP and vanadate. As shown in Figure 7, the 3 H-LTC4 labeling on wild type MRP1 and S1334T as well as E1455Q is almost completely inhibited by the presence of ATP and vanadate. Login to comment
183 This inhibitory effect is directly related to nucleotide binding, but not hydrolysis, at NBD1 and NBD2 since the E1455Q mutation, which abolished ATP hydrolysis but not binding (12), exerted similar ability as wild type MRP1 to inhibit the LTC4 binding (Figure 7). 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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4 Additionally, the E1455Q mutant was found to be nonfunctional. Login to comment
33 In order to introduce the E1455Q mutation into NBD2 of the pET21a-NBD1-GST-NBD2 construct, primers 50 -GAA GAT CCT TGT GTT GGA TCA GGC CAC GGC AGC CGT GGA CCT GG-30 and 50 -CCA GGT CCA CGG CTG CCG TGG CCT GAT CCA ACA CAA GGA TCT TC-3` were used, and pET21a-NBD1-GST-NBD2 construct was used as template in the mutagenesis using QuikChange site directed mutagenesis kit (Stratagene). Login to comment
114 To this end, we constructed a NBD1-GST-NBD1 fusion protein and an E1455Q mutant of the NBD1-GST-NBD2 fusion protein. Login to comment
116 Similar to isolated NBD1 and NBD2, neither NBD1-GST-NBD1 nor NBD1-GST-NBD2/E1455Q showed any detectable ATPase activity (Fig. 4B). Login to comment
138 (B) ATPase activities of the chimeric fusion protein NBD1-GST-NBD2, the NBD1(+)NBD2 mixture, the NBD1(+)GST-NBD2 mixture, NBD1-GST-NBD1, and NBD1-GST-NBD2/E1455Q, all purified from Superdex 200 columns. Login to comment
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
164 The results clearly showed that the NBD1-GST-NBD2/ E1455Q mutant was not functional, consistent with a previous report on this null-mutant in the intact MRP1 [22]. Login to comment

[hide] Molecular mechanism of ATP-dependent solute transp... Methods Mol Biol. 2010;596:223-49. Chang XB
Molecular mechanism of ATP-dependent solute transport by multidrug resistance-associated protein 1.
Methods Mol Biol. 2010;596:223-49., [PMID:19949927]

Abstract [show]
Millions of new cancer patients are diagnosed each year and over half of these patients die from this devastating disease. Thus, cancer causes a major public health problem 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. Over-expression of ATP-binding cassette transporters, such as P-glycoprotein, breast cancer resistance protein and/or multidrug resistance-associated protein 1 (MRP1), confers an acquired MDR due to their capabilities of transporting a broad range of chemically diverse anticancer drugs across the cell membrane barrier. In this review, the molecular mechanism of ATP-dependent solute transport by MRP1 will be addressed.

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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