ABCB1 p.Arg905Cys
| Predicted by SNAP2: | A: D (75%), C: D (75%), D: D (91%), E: D (85%), F: D (85%), G: D (85%), H: D (80%), I: D (80%), K: D (63%), L: D (85%), M: D (75%), N: D (80%), P: D (91%), Q: D (75%), S: D (71%), T: D (75%), V: D (85%), W: D (91%), Y: 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, S: D, T: D, V: D, W: D, Y: D, |
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[hide] Processing mutations disrupt interactions between ... J Biol Chem. 2008 Oct 17;283(42):28190-7. Epub 2008 Aug 16. Loo TW, Bartlett MC, Clarke DM
Processing mutations disrupt interactions between the nucleotide binding and transmembrane domains of P-glycoprotein and the cystic fibrosis transmembrane conductance regulator (CFTR).
J Biol Chem. 2008 Oct 17;283(42):28190-7. Epub 2008 Aug 16., 2008-10-17 [PMID:18708637]
Abstract [show]
P-glycoprotein (P-gp, ABCB1) is an ATP-dependent drug pump. Each of its two homologous halves contains a transmembrane domain (TMD) that has six transmembrane (TM) segments and a nucleotide-binding domain (NBD). Determining how the two halves interact may provide insight into the folding of P-gp as the drug-binding pocket and nucleotide-binding sites are predicted to be at the interface between the two halves. Here, we present evidence for NBD1-TMD2 and NBD2-TMD1 interactions. We also show that TMD-NBD interactions in immature and mature P-gp can be affected by the presence of a processing mutation. We found that the NBD-TMD mutants L443C(NBD1)/S909C(TMD2) and A266C(TMD1)/F1086C(NBD2) could be cross-linked at 0 degrees C with oxidant (copper phenanthroline). Cross-linking was inhibited by vanadate-trapping of nucleotide. The presence of a processing mutation (G268V/L443C(NBD1)/S909C(TMD2); L1260A/A266C(TMD1)/F1086C(NBD2)) resulted in the synthesis of the immature (150 kDa) protein as the major product and the mutants could not be cross-linked with copper phenanthroline. Expression of the processing mutants in the presence of a pharmacological chaperone (cyclosporin A), however, resulted in the expression of mature (170 kDa) protein at the cell surface that could be cross-linked. Similarly, CFTR mutants A274C(TMD1)/L1260C(NBD2) and V510C(NBD1)/A1067C(TMD2) could be cross-linked at 0 degrees C with copper phenanthroline. Introduction of DeltaF508 mutation in these mutants, however, resulted in the synthesis of immature CFTR that could not be cross-linked. These results suggest that establishment of NBD interactions with the opposite TMD is a key step in folding of ABC transporters.
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No. Sentence Comment
107 The locations of positions equivalent to cysteines S473C and R905C in the other half of P-gp (A266C/F1086C) are indicated.
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ABCB1 p.Arg905Cys 18708637:107:61
status: NEW[hide] Evidence for a Sav1866-like architecture for the h... FASEB J. 2007 Dec;21(14):3937-48. Epub 2007 Jul 12. Zolnerciks JK, Wooding C, Linton KJ
Evidence for a Sav1866-like architecture for the human multidrug transporter P-glycoprotein.
FASEB J. 2007 Dec;21(14):3937-48. Epub 2007 Jul 12., [PMID:17627029]
Abstract [show]
The recently reported structures of the bacterial multidrug exporter Sav1866 suggest a domain architecture in which both nucleotide-binding domains (NBDs) of this ATP binding cassette (ABC) transporter contact both transmembrane domains (TMDs). Such a domain arrangement is particularly unexpected because it is not found in the structures of three solute importers BtuCD, HI1470/1, and ModBC from the same protein family. There is also no precedent for such an arrangement from biochemical studies with any ABC transporter. Sav1866 is homologous with the clinically relevant human P-glycoprotein (ABCB1). If the structure proposed for Sav1866 is physiologically relevant, the long intracellular loops of P-glycoprotein TMD2 should contact NBD1. We have tested this by using cysteine mutagenesis and chemical cross-linking to verify proximal relationships of the introduced sulfhydryls across the proposed interdomain interface. We report the first biochemical evidence in support of the domain arrangement proposed for the multidrug resistance class of ABC transporters. With a domain arrangement distinctly different from the three solute importers it seems likely that the TMDs of ABC importers and exporters have evolved different mechanisms to couple to common conformational changes at conserved NBDs.
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No. Sentence Comment
54 Novel cysteine codons were introduced into pMDR-cys- by site-directed mutagenesis (QuikChange multisite; Stratagene, La Jolla, CA, USA) using the oligonucleotides L443C, 5Ј-GTCCAGCTGATGCAGCGCTGCTATGACCCCACAG-3Ј; S474C, 5Ј-CTACGGGAAATCATTGGCGTCGTGTGTCAGGA- ACCTGTATTG-3Ј; R905C, 5Ј-GCAATAGAAAACTTCTGTAC- Figure 1.
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ABCB1 p.Arg905Cys 17627029:54:295
status: NEW139 The residues in ICL4 and NBD1 predicted to be in close proximity were replaced individually and also in the following pairwise combinations; Leu443Cys with Ser909Cys (L443CϩS909C), and Ser474Cys with Arg905Cys (S474CϩR905C).
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ABCB1 p.Arg905Cys 17627029:139:206
status: NEW155 Each of the single cysteine substitution mutants L443C, S474C, R905C, and S909C retain significant levels of drug transport activity, as do the L443CϩS909C and S474CϩR905C double mutants (Fig. 3B, C).
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ABCB1 p.Arg905Cys 17627029:155:63
status: NEW169 B) Histogram of cell surface expression of P-glycoproteins L443C, S474C, R905C, S909C and double mutants L443CϩS909C and S474CϩR905C are shown as indicated.
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ABCB1 p.Arg905Cys 17627029:169:73
status: NEW180 Taken together with the results of the cross-linking, the simplest interpretation of these data is that L443C is in close proximity to S909C and that S474C is in close proximity to R905C in the tertiary structure of monomeric P-glycoprotein, entirely consistent with the hypothesis based on the crystal structure of Sav1866.
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ABCB1 p.Arg905Cys 17627029:180:181
status: NEW184 Repetition with the mutant P-glycoprotein with a cysteine pair at positions S474C and R905C showed no evidence of conformational change (Fig. 7B), providing a convenient negative control for non-specific effects of the added nucleotides.
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ABCB1 p.Arg905Cys 17627029:184:86
status: NEW207 Efflux activity of cysteine mutants wt Pgp-cys- L443C S909C L443CϩS909C S474C R905C S474CϩR905C E556Q Mock Mean % activity 100 100.8 100.7 99.9 100.6 99.5 96.9 101.2 8.6 0.0 (Ϯse) Ϯ 0.6 Ϯ 0.8 Ϯ 0.4 Ϯ 0.5 Ϯ 1.4 Ϯ1.7 Ϯ0.9 Ϯ1.2 Ϯ 11.9 Ϯ 8.9 n 6 6 2 2 4 2 4 2 6 4 The efflux activity of the mutants generated in this study is presented as a percentage of the drug efflux activity of wild-type P-glycoprotein (wt), as described in Materials and Methods.
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ABCB1 p.Arg905Cys 17627029:207:84
status: NEW212 SDS-PAGE and Western analyses of P-glycoproteins with novel cysteines introduced into NBD1 (L443C and S474C) and/or ICL4 of TMD2 (R905C and S909C), as indicated.
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ABCB1 p.Arg905Cys 17627029:212:130
status: NEW219 L443C P-glycoprotein was coexpressed in cells with S909C P-glycoprotein, and likewise S474C P-glycoprotein was coexpressed with R905C P-glycoprotein.
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ABCB1 p.Arg905Cys 17627029:219:128
status: NEW220 SDS-PAGE and Western analyses showed no evidence of intermolecular cross-linking (lack of a slow mobility P-glycoprotein species) between the L443C on one protein and S909C on another, or between S474C and R905C on different P-glycoproteins.
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ABCB1 p.Arg905Cys 17627029:220:206
status: NEW245 SDS-PAGE and Western analyses of P-glycoprotein double mutants with novel cysteines introduced at (A) L443C and S909C, or (B) S474C and R905C.
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ABCB1 p.Arg905Cys 17627029:245:136
status: NEW266 The differential response of the two cysteine pairs (cross-linking in the S474C and R905C double mutant appears insensitive to added nucleotide), suggests that S474 and R905 may move in tandem during the ATP catalytic cycle, if they move at all.
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ABCB1 p.Arg905Cys 17627029:266:84
status: NEW