ABCMdb

ABCC1 p.Trp553Ala

Predicted by SNAP2:	A: D (75%), C: D (71%), D: D (91%), E: D (91%), F: N (53%), G: D (91%), H: D (91%), I: D (80%), K: D (91%), L: D (71%), M: D (63%), N: D (80%), P: D (95%), Q: D (91%), R: D (91%), S: D (80%), T: D (91%), V: D (75%), Y: D (66%),
Predicted by PROVEAN:	A: D, C: D, D: 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, Y: D,

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[hide] Multiple membrane-associated tryptophan residues c... J Biol Chem. 2002 Dec 20;277(51):49495-503. Epub 2002 Oct 17. Koike K, Oleschuk CJ, Haimeur A, Olsen SL, Deeley RG, Cole SP
Multiple membrane-associated tryptophan residues contribute to the transport activity and substrate specificity of the human multidrug resistance protein, MRP1.
J Biol Chem. 2002 Dec 20;277(51):49495-503. Epub 2002 Oct 17., 2002-12-20 [PMID:12388549]

Abstract [show]
The multidrug resistance protein, MRP1, is a clinically important ATP-binding cassette transporter in which the three membrane-spanning domains (MSDs), which contain up to 17 transmembrane (TM) helices, and two nucleotide binding domains (NBDs) are configured MSD1-MSD2-NBD1-MSD3-NBD2. In tumor cells, MRP1 confers resistance to a broad spectrum of drugs, but in normal cells, it functions as a primary active transporter of organic anions such as leukotriene C(4) and 17beta-estradiol 17beta-(D-glucuronide). We have previously shown that mutation of TM17-Trp(1246) eliminates 17beta-estradiol 17beta-(D-glucuronide) transport and drug resistance conferred by MRP1 while leaving leukotriene C(4) transport intact. By mutating the 11 remaining Trp residues that are in predicted TM segments of MRP1, we have now determined that five of them are also major determinants of MRP1 function. Ala substitution of three of these residues, Trp(445) (TM8), Trp(553) (TM10), and Trp(1198) (TM16), eliminated or substantially reduced transport levels of five organic anion substrates of MRP1. In contrast, Ala substitutions of Trp(361) (TM7) and Trp(459) (TM9) caused a more moderate and substrate-selective reduction in MRP1 function. More conservative substitutions (Tyr and Phe) of the Trp(445), Trp(553), and Trp(1198) mutants resulted in substrate selective retention of transport in some cases (Trp(445) and Trp(1198)) but not others (Trp(553)). Our findings suggest that the bulky polar aromatic indole side chain of each of these five Trp residues contributes significantly to the transport activity and substrate specificity of MRP1.

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48 Tryptophan substitutions were generated in the pGEM-3Z and pBluescriptSK(ϩ) plasmids above according to the manufacturer`s instructions with the following mutagenic primers (substituted nucleotides are underlined): MSD1 Trp mutants W40A (5Ј-G GTC CTC GTG GCC GTG CCT TG-3Ј, W47A (5Ј-GT TTT TAC CTC GCC GCC TGT TTC CCC-3Ј), W82A (5Ј-C TTG GGA TTT TTG CTG GCG ATC GTC TGC TGG GC-3Ј), W86A (5Ј-G CTG TGG ATC GTC TGC GCT GCA GAC CTC TTC TAC TC-3Ј), W94A (5Ј-C CTC TTC TAC TCT TTC GCG GAA AGA AGT CGG GGC-3Ј), W142A (5Ј-GGG ATC ATG CTC ACT TTC GCA CTG GTA GCC CTA ATG TG-3Ј), W142F (5Ј-G CTC ACT TTT TTC CTG GTA GCC C-3Ј); MSD2 Trp mutants W361A (5Ј-C ACG AAG GCC CCA GAT GCG CAG GGC TAC TTC TAC-3Ј), W445A (5Ј-G TAC ATT AAC ATG ATC GCG TCA GCC CCC CTG CAA G-3Ј), W445F (5Ј-CG TAC ATT AAC ATG ATC TTC TCA GCC CCC CTG CAA GTC-3Ј), W445Y (5Ј-CC ACG TAC ATT AAC ATG ATC TAC TCA GCG CCC CTG CAA GTC-3Ј), W459A (5Ј-GCT CTC TAC CTC CTG GCG CTG AAT CTG GGC CC-3Ј), W553A (5Ј-G GGC ACC TTC ACC GCG GTC TGC ACG CCC-3Ј), W553F (5Ј-G GGC ACC TTC ACC TTC GTC TGC ACG CCC-3Ј), W553Y (5Ј-GCC CTG GGC ACC TTC ACA TAT GTC TGC ACG CCC-3Ј; and MSD3 Trp mutants W1198A (5Ј-C GTG GCC AAC AGG GCG CTG GCC GTG CGG C-3Ј), W1198F (5Ј-GTG GCC AAC AGG TTC CTG GCC GTG CGG C-3Ј), W1198Y (5Ј-GTG GCC AAC AGG TAC CTG GCC GTG CGG C-3Ј). Login to comment
50 Construction of MRP1-GFP Fusion Proteins-Constructs encoding GFP fusion proteins of selected MRP1 Trp mutations were generated by exchanging the 1.3-kb ClaI/AflII fragment of a pcDNA3.1(-)-MRP1-GFP construct with the comparable fragments containing the W445A, W553A, and W1198A mutations generated above and designated pcDNA3.1-W445A/MRP1-GFP, pcDNA3.1-W553A/MRP1-GFP, and pcDNA3.1-W1198A/MRP1-GFP, respectively (39). Login to comment
108 All four mutants generated (W361A, W445A, W459A, and W553A) were expressed at levels 60-90% those of wild-type MRP1, indicating that none of the mutations had a major effect on the expression levels of the protein. Login to comment
110 After 1 min, ATP-dependent [3 H]LTC4 uptake by the W445A and W553A MRP1 mutants was reduced by ϳ75 and 50%, respectively, whereas uptake by the W361A and W459A mutants was comparable with wild-type MRP1 (Fig. 3B). Login to comment
113 In contrast, after 1 min, [3 H]E217betaG uptake by the W361A, W445A, and W553A MRP1 mutants was ϳ50, 25, and 10%, respectively, of wild-type MRP1 (Fig. 3D). Login to comment
119 Similarly, GSH-stimulated E13SO4 uptake levels by the W445A and W553A mutants were just 30 and Ͻ10% of wild-type MRP1 levels, respectively, whereas uptake by the W361A mutant was similar to wild-type MRP1, and uptake by W459A MRP1 was reduced by just 25% (Fig. 4B). Login to comment
121 A, ATP-dependent uptake of [3 H]LTC4 was measured in membrane vesicles prepared from HEK293T cells transfected with empty vector pcDNA3.1(-) (E) and vectors containing wild-type MRP1 (f) and MSD2 Trp-Ala mutant MRP1 cDNAs (W361A, Œ; W445A, ; W459A, ࡗ; and W553A, q). Login to comment
124 C, the time course of ATP-dependent uptake of [3 H]E217betaG by wild-type MRP1 and MSD2 mutants W361A, W445A, W459A, and W553A was measured as described for A. D, relative levels of [3 H]E217betaG uptake at 1 min are shown and were determined from the time course shown in C as described for B. Login to comment
126 Finally, [3 H]MTX uptake by the W445A and W553A MRP1 mutants, as observed for GSH and E13SO4 uptake, was dramatically reduced by more than 80% (Fig. 4C). Login to comment
128 Thus, substitution of either Trp445 or Trp553 by an Ala residue resulted in a general loss of MRP1 organic anion transport activity, whereas Ala substitution of Trp361 and Trp459 had a much more moderate and selective effect on the substrate specificity of the protein. Login to comment
148 In contrast to the W445A and W1198A mutants, more conservative substitutions of the Ala-substituted Trp553 mutant W553A were much less effective in restoring MRP1 transport activity. Login to comment
153 Relative uptake levels of 3 H-labeled organic anions by membrane vesicles enriched for wild-type MRP1 (solid bar) and W361A, W445A, W459A, and W553A mutant MRP1 proteins (shaded bars) were determined as described under "Experimental Procedures." Login to comment
183 Similarly, the transport of four MRP1 substrates by the W553A mutant was decreased by at least 80%, and transport of the fifth, LTC4, was reduced by ϳ50%. Login to comment
189 Ala substitution of Trp1198 in MSD3 also resulted in a broad and profound decrease in MRP1 transport activity except for FIG. 6. ATP-dependent organic anion transport activity of wild-type and mutant MRP1 containing conservative Phe and Tyr substitutions of Trp1198 in TM16 of MSD3. A, immunoblot of membrane vesicles prepared from HEK293T cells transfected with empty vector (pcDNA3.1(-)), wild-type (WT-MRP1), and mutant (W445A, W445F, and W445Y; W553A, W553F and W553Y; W1198A, W1198F, and W1198Y) MRP1 cDNAs. MRP1 proteins were detected with monoclonal antibody QCRL-1, and relative levels of expression shown under the blot were estimated by densitometry. Login to comment
191 Ala-substituted mutants (W445A, W553A, and W1198A; shaded bars) were included for comparison. Login to comment
194 The Ala-substituted Trp1246 mutant, like W553A and W1198A, also showed significantly reduced transport of multiple MRP1 substrates with the exception of LTC4 (39). Login to comment
224 HEK293T cells were transfected with pcDNA3.1(-)MRP1K-GFP (WT-MRP1-GFP) (A), pcDNA3.1-W445A/MRP1-GFP (B), pcDNA3.1-W553A/MRP1-GFP (C), and pcDNA3.1-W1198A/MRP1-GFP (D), and 48 h later, cells were processed for confocal fluorescence microscopy. Login to comment

[hide] Transverse and tangential orientation of predicted... Eur Biophys J. 2011 Sep;40(9):1043-60. Epub 2011 Jun 24. de Foresta B, Vincent M, Garrigos M, Gallay J
Transverse and tangential orientation of predicted transmembrane fragments 4 and 10 from the human multidrug resistance protein (hMRP1/ABCC1) in membrane mimics.
Eur Biophys J. 2011 Sep;40(9):1043-60. Epub 2011 Jun 24., [PMID:21701864]

Abstract [show]
The human multidrug-resistance-associated protein 1 (hMRP1/ABCC1) belongs to the large ATP-binding cassette transporter superfamily. In normal tissues, hMRP1 is involved in tissue defense, whereas, in cancer cells, it is overproduced and contributes to resistance to chemotherapy. We previously investigated the folding properties of the predicted transmembrane fragments (TM) TM16, and TM17 from membrane-spanning domain 2 (MSD2). These TMs folded only partially as an alpha-helix and were located in the polar headgroup region of detergent micelles used as membrane mimics (Vincent et al. in Biochim Biophys Acta 1768:538-552, 2007; de Foresta et al. in Biochim Biophys Acta 1798:401-414, 2010). We have now extended these studies to TM4 and TM10, from MSD0 and MSD1, respectively. TM10 may be involved in the substrate translocation pathway whereas the role of TM4 is less predictable, because few studies have focused on MSD0, a domain present in some hMRP1 homologs only. Each TM contained a single Trp residue (W142 or W553) acting as an intrinsic fluorescent probe. The location and dynamics of the TMs in dodecylphosphocholine (DPC) or n-dodecyl-beta-D: -maltoside (DDM) micelles were studied by Trp steady-state and time-resolved fluorescence, including quenching experiments. Overall TM structure was analyzed by far-UV circular dichroism studies in detergent micelles and TFE. TM10 behaved similarly to TM16 and TM17, with an interfacial location in micelles consistent with a possible role in lining the transport pore. By contrast, TM4 behaved like a classical TM fragment with a high alpha-helical content, and its transmembrane insertion did not require its interaction with other TMs.

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62 In addition, mutations affecting the single proline (P557A) or the single Trp (W553A) residue decreased the transport of various organic anion substrates (Koike et al. 2002, 2004). Login to comment

[hide] Bindings of hMRP1 transmembrane peptides with dode... Biochim Biophys Acta. 2014 Jan;1838(1 Pt B):493-509. doi: 10.1016/j.bbamem.2013.10.012. Epub 2013 Oct 21. Abel S, Lorieau A, de Foresta B, Dupradeau FY, Marchi M
Bindings of hMRP1 transmembrane peptides with dodecylphosphocholine and dodecyl-beta-d-maltoside micelles: a molecular dynamics simulation study.
Biochim Biophys Acta. 2014 Jan;1838(1 Pt B):493-509. doi: 10.1016/j.bbamem.2013.10.012. Epub 2013 Oct 21., [PMID:24157718]

Abstract [show]
In this paper, we describe molecular dynamics simulation results of the interactions between four peptides (mTM10, mTM16, TM17 and KTM17) with micelles of dodecylphosphocholine (DPC) and dodecyl-beta-d-maltoside (DDM). These peptides represent three transmembrane fragments (TM10, 16 and 17) from the MSD1 and MSD2 membrane-spanning domains of an ABC membrane protein (hMRP1), which play roles in the protein functions. The peptide-micelle complex structures, including the tryptophan accessibility and dynamics were compared to circular dichroism and fluorescence studies obtained in water, trifluoroethanol and with micelles. Our work provides additional results not directly accessible by experiments that give further support to the fact that these peptides adopt an interfacial conformation within the micelles. We also show that the peptides are more buried in DDM than in DPC, and consequently, that they have a larger surface exposure to water in DPC than in DDM. As noted previously by simulations and experiments we have also observed formation of cation-pi bonds between the phosphocholine DPC headgroup and Trp peptide residue. Concerning the peptide secondary structures (SS), we find that in TFE their initial helical conformations are maintained during the simulation, whereas in water their initial SS are lost after few nanoseconds of simulation. An intermediate situation is observed with micelles, where the peptides remain partially folded and more structured in DDM than in DPC. Finally, our results show no sign of beta-strand structure formation as invoked by far-UV CD experiments even when three identical peptides are simulated either in water or with micelles.

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36 For example, mutations of two threonine (T550A and T556A), a tryptophan (W553A), and a proline (P557A) in TM10 modify the drug-resistance profile of the protein or decrease the transport of various organic substrates [32-35]. Login to comment