ABCMdb

ABCC1 p.Asp1084Asn

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] Functional importance of polar and charged amino a... J Biol Chem. 2003 Nov 14;278(46):46052-63. Epub 2003 Sep 3. Zhang DW, Gu HM, Situ D, Haimeur A, Cole SP, Deeley RG
Functional importance of polar and charged amino acid residues in transmembrane helix 14 of multidrug resistance protein 1 (MRP1/ABCC1): identification of an aspartate residue critical for conversion from a high to low affinity substrate binding state.
J Biol Chem. 2003 Nov 14;278(46):46052-63. Epub 2003 Sep 3., 2003-11-14 [PMID:12954620]

Abstract [show]
Human multidrug resistance protein 1 (MRP1) confers resistance to many chemotherapeutic agents and transports diverse conjugated organic anions. We previously demonstrated that Glu1089 in transmembrane (TM) 14 is critical for the protein to confer anthracycline resistance. We have now assessed the functional importance of all polar and charged amino acids in this TM helix. Asn1100, Ser1097, and Lys1092, which are all predicted to be on the same face of the helix as to Glu1089, are involved in determining the substrate specificity of the protein. Notably, elimination of the positively charged side chain of Lys1092, increased resistance to the cationic drugs vincristine and doxorubicin, but not the electroneutral drug etoposide (VP-16). In addition, mutations S1097A and N1100A selectively decreased transport of 17beta-estradiol 17-(beta-d-glucuronide) (E217betaG) but not cysteinyl leukotriene 4 (LTC4), demonstrating the importance of multiple residues in this helix in determining substrate specificity. In contrast, mutations of Asp1084 that eliminate the carboxylate side chain markedly decreased resistance to all drugs tested, as well as transport of both E217betaG and LTC4, despite the fact that LTC4 binding was unaffected. We show that these mutations prevent the ATP-dependent transition of the protein from a high to low affinity substrate binding state and drastically diminish ADP trapping at nucleotide binding domain 2. Based on results presented here and crystal structures of prokaryotic ATP binding cassette transporters, Asp1084 may be critical for interaction between the cytoplasmic loop connecting TM13 and TM14 and a region of nucleotide binding domain 2 between the conserved Walker A and ABC signature motifs.

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No. Sentence Comment
51 They are as follows: T1082A (5Ј-C TCC AAG GAG CTC GAC GCA GTG GAC TCC-3Ј), D1084N (5Ј-CTG GAC ACA GTG AAT TCC ATG ATC CCG-3Ј), D1084A (5Ј-CTG GAC ACA GTG AAA TCG ATG ATC CCG-3Ј), D1084E (5Ј-CTG GAC ACA GTG GAC TCG ATG ATC CCG-3Ј), D1084V (5Ј-CTG GAC ACA GTG GTA TCG ATG ATC CCG-3Ј), S1085A (5Ј-CTG GAC ACA GTC GAC GCC ATG ATC CCG G-3Ј), K1092M (5Ј-C CCG GAG GTC ATC ATG ATG TTC ATG GGC-3Ј), K1092A (5Ј-C CCG GAG GTC ATC GCG ATG TTC ATG GGC-3Ј), K1092E (5Ј-C CCG GAG GTC ATC GAG ATG TTC ATG GGC-3Ј), K1092R (5Ј-C CCG GAG GTC ATC AGG ATG TTC ATG GGC-3Ј), S1097A (5Ј-G ATG TTC ATG GGC GCC CTG TTC AAC-3Ј), N1100A (5Ј-TTC ATG GGC TCG CTC TTC GCC GTC ATT GGT G-3Ј), N1100S (5Ј-TTC ATG GGC TCG CTC TTC AGT GTC ATT GGT G-3Ј). Login to comment
81 To generate MRP1D1084N -pFASTBAC Dual vector, pCEBV7-MRP1 containing mutation D1084N was digested with BstEII, and an ϳ600-bp fragment comprising nucleotides 3369-3913 of MRP1D1084N was isolated. Login to comment
118 RESULTS Expression of Mutant MRP1 in Stably Transfected HEK293 Cells-To examine the functional importance of charged and polar residues in TM14 of MRP1, we generated a series of six mutant proteins in which Thr1082 , Ser1085 , Ser1097 , and Asn1100 were replaced with Ala, Asp1084 was replaced with Asn, and Lys1092 was mutated to Met (Fig. 1). Login to comment
137 The only mutation that affected LTC4 transport was replacement of Asp1084 by Asn, which almost completely eliminated transport (Fig. 3, A-C). Login to comment
138 ATP-dependent transport of [3 H]E217betaG was unaffected by the T1082A, S1085A and K1092M mutations (Fig. 3, D-F), but substitution of Ser1097 with Ala and conversion of Asp1084 to Asn both dramatically decreased transport. Login to comment
140 Thus, mutations S1097A and N1100A only affected transport of the estrogen conjugate, whereas mutation D1084N decreased the ability of MRP1 to transport both LTC4 and E217betaG. Login to comment
143 Similarly, mutation D1084N also affected the drug resistance profile of MRP1. Login to comment
144 The D1084N mutant protein essentially lost its ability to confer resistance to vincristine and doxorubicin, and retained only ϳ30% of the ability of the wild type protein to increase VP-16 resistance. Login to comment
147 Transport of [3 H]GSH by Wild Type and Mutant MRP1-The studies described above indicated that mutations S1097A and D1084N affected the ability of MRP1 to confer drug resistance and to transport conjugated organic anions, whereas the K1092M mutation influenced only the drug resistance profile of MRP1. Login to comment
163 As observed when examining LTC4 transport, only replacement of Asp1084 by Asn influenced the ability of MRP1 to transport GSH, and this mutation essentially eliminated transport activity. Login to comment
166 Kinetic Parameters of [3 H]LTC4 and [3 H]E217betaG Transport by Wild Type and Mutant MRP1-Because mutation D1084N FIG. 3. Login to comment
177 For E217betaG transport, the normalized Vmax values for mutations D1084N, S1097A, and N1100A were lower than that for wild type MRP1 (3207 pmol/mg/min for wild type MRP1, versus 151, 2279, and 1852 pmol/mg/min for mutations D1084N, S1097A and N1100A, respectively) (Fig. 5A and Table II). Login to comment
179 However, replacement of Asp1084 with Asn decreased the Km value ϳ3-fold (0.5 ␮M). Login to comment
181 Thus, although mutations D1084N, S1097A, and N1100A all decreased the conjugated estrogen transport, the three mutations had different effects on the apparent Km values for E217betaG uptake. Login to comment
183 However, replacement of Asp1084 with Asn decreased the values of normalized Vmax and the apparent Km for LTC4 transport ϳ8- and 2-fold, respectively (30 pmol/mg/min, and 77 nM for mutation D1084N) (Fig. 5B and Table II). Login to comment
184 Thus, similar to the results obtained with E217betaG as a substrate, the D1084N mutation appeared to increase the affinity of MRP1 for substrate while decreasing the Vmax. Login to comment
201 Effect of Mutation D1084N on Photolabeling of MRP1 with [3 H]LTC4 and 8-Azido-[␣-32 P]ATP-Because transport studies indicated that mutation D1084N markedly decreased the Vmax for LTC4 transport while modestly decreasing the apparent Km, we confirmed the ability of the mutant protein to bind [3 H]LTC4 by photolabeling studies. Login to comment
202 Based on densitometry of several preparations of membrane proteins, substitution of Asp1084 with Asn had no significant effect on photolabeling with [3 H]LTC4 (Fig. 6B). Login to comment
206 To investigate how mutation D1084N abrogated the effect of ATP on the binding of LTC4, we examined the ability of the wild type and mutant proteins to be photolabeled with 8-azido-[␣-32 P]ATP both at 4 °C to minimize hydrolysis and under vanadate-induced ADP trapping conditions at 37 °C. As shown in Fig. 6C, mutation D1084N had no marked effect on the binding of the ATP analog at 4 °C. However, the D1084N mutation virtually abolished the vanadate-dependent trapping of 8-azido-[␣-32 P]ADP by MRP1 at 37 °C (Fig. 6D), suggesting that it substantially decreased ATP hydrolysis by MRP1. Login to comment
208 To investigate the effect of mutation D1084N on the trapping of ATP more precisely, we took advantage of a pFASTBAC Dual vector, in which the COOH-proximal MRP1 fragment (amino acids 932-1531) was modified to contain mutation D1084N, and co-expressed with a wild type NH2-proximal fragment (amino acids 1-932). Login to comment
212 As observed with results obtained from analyses of membrane vesicles prepared from HEK293 cells stably transfected with full-length mutant MRP1D1084N , replacement of Asp1084 by Asn essentially eliminated the transport activity. Login to comment
217 Vanadate-induced trapping experiments revealed that replacement of Asp1084 by Asn dramatically reduced trapping of 8-azido-ADP by both NBD1 and NBD2 of MRP1 (Fig. 7E), strongly suggesting that it decreased the ability of both NBDs to hydrolyze ATP. Login to comment
218 Effects of Mutations D1084A, D1084E, D1084V, D1084R, K1092A, K1092E, K1092R, and N1100S on Transport of [3 H]LTC4 and [3 H]E217betaG by Wild Type MRP1-Because mutation D1084N dramatically affected all of the MRP1 functions tested, we also mutated Asp1084 to Ala, Glu, Arg, and Val. Login to comment
244 Thus, similar to the results obtained with photolabeling of mutant D1084N with LTC4, substitution of Asp1084 by Arg or Glu had no significant effect on the photolabeling. Login to comment
249 Substitution of Asp1084 by Ala and Val, as observed with mutation D1084N, significantly reduced resistance to vincristine, VP-16, and doxorubicin. Login to comment
322 Photolabeling studies with [3 H]LTC4 also showed that the replacement of Asp1084 by Asn and Glu had no effect on binding of the substrate to the protein. Login to comment
326 In the present study, kinetic analyses of the residual transport activity of the D1084N mutant protein yielded a lower Km for both LTC4 and E217betaG than the wild type protein, suggesting that the affinity for both substrates was actually increased. Login to comment
330 Consistent with this suggestion, we found that LTC4 binding by the D1084N mutation did not decrease in the presence of ATP even with the addition of vanadate. Login to comment
331 This finding together with the effect of mutation D1084N on the overall activity of MRP1 raised the interesting possibility that the mutation might affect the binding and/or hydrolysis of ATP, so that substrate could not be translocated and/or released from the mutant protein. Login to comment
333 At 37 °C, the D1084N mutation drastically decreased trapping of 8-azido-ADP at NBD2 when photolabeling was carried out in the absence or presence of vanadate. Login to comment
334 These findings indicate that the mutation D1084N decreased the ability of NBD2 to hydrolyze ATP. Login to comment

[hide] Mutational analysis of ionizable residues proximal... J Biol Chem. 2004 Sep 10;279(37):38871-80. Epub 2004 Jun 18. Situ D, Haimeur A, Conseil G, Sparks KE, Zhang D, Deeley RG, Cole SP
Mutational analysis of ionizable residues proximal to the cytoplasmic interface of membrane spanning domain 3 of the multidrug resistance protein, MRP1 (ABCC1): glutamate 1204 is important for both the expression and catalytic activity of the transporter.
J Biol Chem. 2004 Sep 10;279(37):38871-80. Epub 2004 Jun 18., 2004-09-10 [PMID:15208328]

Abstract [show]
The multidrug resistance protein MRP1 is an ATP-dependent transporter of organic anions and chemotherapeutic agents. A significant number of ionizable amino acids are found in or proximal to the 17 transmembrane (TM) helices of MRP1, and we have investigated 6 of these at the cytoplasmic interface of TM13-17 for their role in MRP1 expression and transport activity. Opposite charge substitutions of TM13 Arg(1046) and TM15 Arg(1131) did not alter MRP1 expression nor did they substantially affect activity. In contrast, opposite charge substitutions of TM16 Arg(1202) and Glu(1204) reduced protein expression by >80%; however, MRP1 expression was not affected when Arg(1202) and Glu(1204) were replaced with neutral or same-charge residues. In addition, organic anion transport levels of the R1202L, R1202G, and R1202K mutants were comparable with wild-type MRP1. In contrast, organic anion transport by E1204L was substantially reduced, whereas transport by E1204D was comparable with wild-type MRP1, with the notable exception of GSH. Opposite charge substitutions of TM16 Arg(1197) and TM17 Arg(1249) did not affect MRP1 expression but substantially reduced transport. Mutants containing like-charge substitutions of Arg(1197) or Arg(1249) were also transport-inactive and no longer bound leukotriene C(4). In contrast, substrate binding by the transport-compromised E1204L mutant remained intact. Furthermore, vanadate-induced trapping of azido-ADP by E1204L was dramatically increased, indicating that this mutation may cause a partial uncoupling of the catalytic and transport activities of MRP1. Thus, Glu(1204) serves a dual role in membrane expression of MRP1 and a step in its catalytic cycle subsequent to initial substrate binding.

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No. Sentence Comment
98 sitely charged amino acid caused a global and almost complete loss of transport activity. We reported previously (27) that substitution of Asp1084 with Asn, Ala, or Val also eliminated LTC4 and E217betaG transport by MRP1. Login to comment

[hide] Transmembrane helix 11 of multidrug resistance pro... Biochemistry. 2004 Jul 27;43(29):9413-25. Zhang DW, Nunoya K, Vasa M, Gu HM, Theis A, Cole SP, Deeley RG
Transmembrane helix 11 of multidrug resistance protein 1 (MRP1/ABCC1): identification of polar amino acids important for substrate specificity and binding of ATP at nucleotide binding domain 1.
Biochemistry. 2004 Jul 27;43(29):9413-25., 2004-07-27 [PMID:15260484]

Abstract [show]
Human multidrug resistance protein 1 (MRP1) is an ATP binding cassette (ABC) transporter that confers resistance to many natural product chemotherapeutic agents and can transport structurally diverse conjugated organic anions. MRP1 has three polytopic transmembrane domains (TMDs) and a total of 17 TM helices. Photolabeling and mutagenesis studies of MRP1 indicate that TM11, the last helix in the second TMD, may form part of the protein's substrate binding pocket. We have demonstrated that certain polar residues within a number of TM helices, including Arg(593) in TM11, are determinants of MRP1 substrate specificity or overall activity. We have now extended these analyses to assess the functional consequences of mutating the remaining seven polar residues within and near TM11. Mutations Q580A, T581A, and S585A in the predicted outer leaflet region of the helix had no detectable effect on function, while mutation of three residues close to the membrane/cytoplasm interface altered substrate specificity. Two of these mutations affected only drug resistance. N597A increased and decreased resistance to vincristine and VP-16, respectively, while S605A decreased resistance to vincristine, VP-16 and doxorubicin. The third, S604A, selectively increased 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG) transport. In contrast, elimination of the polar character of the residue at position 590 (Asn in the wild-type protein) uniformly impaired the ability of MRP1 to transport potential physiological substrates and to confer resistance to three different classes of natural product drugs. Kinetic and photolabeling studies revealed that mutation N590A not only decreased the affinity of MRP1 for cysteinyl leukotriene 4 (LTC(4)) but also substantially reduced the binding of ATP to nucleotide binding domain 1 (NBD1). Thus, polar interactions involving residues in TM11 influence not only the substrate specificity of MRP1 but also an early step in the proposed catalytic cycle of the protein.

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No. Sentence Comment
312 The D1084N mutation essentially eliminated the transport of LTC4 and prevented the transition of the protein from a high- to low-affinity substrate binding state in the presence of ATP (33). Login to comment