ABCMdb

ABCC1 p.Tyr568Ala

Predicted by SNAP2:	A: D (75%), C: D (66%), D: D (91%), E: D (91%), F: N (61%), G: D (80%), H: D (66%), I: D (75%), K: D (91%), L: D (80%), M: D (80%), N: D (85%), P: D (91%), Q: D (85%), R: D (91%), S: D (80%), T: D (80%), V: D (80%), W: D (85%),
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, W: D,

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Publications
[hide] Mutational analysis of polar amino acid residues w... Drug Metab Dispos. 2006 Apr;34(4):539-46. Epub 2006 Jan 13. Zhang DW, Nunoya K, Vasa M, Gu HM, Cole SP, Deeley RG
Mutational analysis of polar amino acid residues within predicted transmembrane helices 10 and 16 of multidrug resistance protein 1 (ABCC1): effect on substrate specificity.
Drug Metab Dispos. 2006 Apr;34(4):539-46. Epub 2006 Jan 13., [PMID:16415113]

Abstract [show]
Human multidrug resistance protein 1 (MRP1) has a total of 17 transmembrane (TM) helices arranged in three membrane-spanning domains, MSD0, MSD1, and MSD2, with a 5 + 6 + 6 TM configuration. Photolabeling studies indicate that TMs 10 and 11 in MSD1 and 16 and 17 in MSD2 contribute to the substrate binding pocket of the protein. Previous mutational analyses of charged and polar amino acids in predicted TM helices 11, 16, and 17 support this suggestion. Mutation of Trp(553) in TM10 also affects substrate specificity. To extend this analysis, we mutated six additional polar residues within TM10 and the remaining uncharacterized polar residue in TM16, Asn(1208). Although mutation of Asn(1208) was without effect, two of six mutations in TM10, T550A and T556A, modulated the drug resistance profile of MRP1 without affecting transport of leukotriene C4, 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG), and glutathione. Mutation T550A increased vincristine resistance but decreased doxorubicin resistance, whereas mutation T556A decreased resistance to etoposide (VP-16) and doxorubicin. Although conservative mutation of Tyr(568) in TM10 to Phe or Trp had no apparent effect on substrate specificity, substitution with Ala decreased the affinity of MRP1 for E(2)17betaG without affecting drug resistance or the transport of other substrates tested. These analyses confirm that several amino acids in TM10 selectively alter the substrate specificity of MRP1, suggesting that they interact directly with certain substrates. The location of these and other functionally important residues in TM helices 11, 16, and 17 is discussed in the context of an energy-minimized model of the membrane-spanning domains of MRP1.

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No. Sentence Comment
34 In TM10, Thr550 , Thr552 , Thr556 , Thr564 , and Thr570 were individually mutated to Ala, and Tyr568 was mutated to Ala, Ser, Phe, and Trp. Login to comment
48 Mutations T550A, T552A, T556A, Y568A, Y568S, Y568F, Y568W, T570A, and N1208A were generated using the Quikchange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA). Login to comment
50 Oligonucleotides bearing mismatched bases at the residues to be mutated (underlined) were synthesized by ACGT Corp. with the following sequences: T550A, 5Ј-GTCAGCCGTGGG- CGCCTTCACCTGGGT-3Ј; T552A, 5Ј-CGTGGGCACCTTCGCCTGGGTC- TGCAC-3Ј; T556A, 5Ј-CACCTGGGTCTGCGCGCCCTTTCTGGT-3Ј; Y568A, 5Ј-TGCACATTTGCCGTCGCCGTGACCATTGACGA-3Ј; Y568S, 5Ј-TGCACATTTGCCGTCTCCGTGACCATTGACGA-3Ј; Y568F, 5Ј-TGC- ACATTTGCCGTCTTCGTGACCATTGACGA-3Ј; Y568W, 5Ј-TGCACAT- TTGCCGTCTGGGTGACCATTGACGA-3Ј; T570A, (5Ј-TGCCGTCTACG- TGGCCATTGACGAGAACAAC-3Ј; and N1208A, 5Ј-GCCGTGCGGCTG- GAGTGTGTGGGCGCC-3Ј. Login to comment
120 ATP-dependent transport of [3 H]E217betaG was also examined (Fig. 3, D to F), only replacement of Tyr568 with Ala decreased the transport efficiency by approximately 60%, indicating that the polar and/or the bulky aromatic side chain of the residue at position 568 is important for the ability of MRP1 to transport E217betaG. Login to comment
122 Because replacement of Tyr568 by Ala selectively decreased transport of E217betaG, this residue was also mutated to Ser, Phe, and Trp. Login to comment
126 Like mutation Y568A, substitution of Tyr568 with Ser did not affect the transport of LTC4 but decreased E217betaG transport. Login to comment
129 Kinetic Parameters of [3 H]LTC4 and [3 H]E217betaG Transport by Wild-Type and Y568A Mutant MRP1. Login to comment
130 To more precisely determine the influence of mutation Y568A on the ability of MRP1 to transport E217betaG, we compared kinetic parameters for the wild-type and mutant proteins (Fig. 5). Login to comment
131 For wild-type MRP1 and Y568A, the Km and normalized Vmax values for LTC4 uptake were essentially identical. Login to comment
132 Linear regression using a Hanes-Woolf transformation yielded values of 115 nM and 143 nM, and 76.8 pmol/mg/min and 64 pmol/mg/min, for the Km and Vmax values of wild-type and Y568A proteins, respectively (Fig. 5, B and C). Login to comment
151 We have shown that mutations T550A and T556A both affect the ability of MRP1 to confer drug resistance, whereas mutation Y568A only influenced the transport of E217betaG. Login to comment
233 However, kinetic analysis showed that the nonconservative mutation Y568A increased the apparent Km for E217betaG approximately 5-fold, without altering Vmax. 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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Sentences [show]
No. Sentence Comment
61 Mutations resulting in the replacement of two of its threonine residues (T550A and T556A) modulated the drug-resistance profile of hMRP1 and a mutation affecting the tyrosine residue (Y568A) reduced the affinity of hMRP1 for E217bG (Zhang et al. 2006). Login to comment