ABCB1 p.Ser474Cys
| Predicted by SNAP2: | A: N (61%), C: D (66%), D: D (75%), E: N (72%), F: D (80%), G: N (78%), H: D (71%), I: D (80%), K: D (80%), L: D (75%), M: D (71%), N: N (82%), P: D (80%), Q: N (82%), R: N (57%), T: D (63%), V: D (80%), W: D (85%), Y: D (80%), |
| Predicted by PROVEAN: | A: N, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: N, P: D, Q: D, R: D, T: N, V: D, W: D, Y: D, |
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[hide] Cysteine-scanning mutagenesis provides no evidence... EMBO J. 1999 Dec 1;18(23):6800-8. Blott EJ, Higgins CF, Linton KJ
Cysteine-scanning mutagenesis provides no evidence for the extracellular accessibility of the nucleotide-binding domains of the multidrug resistance transporter P-glycoprotein.
EMBO J. 1999 Dec 1;18(23):6800-8., 1999-12-01 [PMID:10581253]
Abstract [show]
Multidrug resistance of cancer cells is, at least in part, conferred by overexpression of P-glycoprotein (P-gp), a member of the ATP-binding cassette (ABC) superfamily of active transporters. P-gp actively extrudes chemotherapeutic drugs from cells, thus reducing their efficacy. As a typical ABC transporter, P-gp has four domains: two transmembrane domains, which form a pathway through the membrane through which substrates are transported, and two hydrophilic nucleotide-binding domains (NBDs), located on the cytoplasmic side of the membrane, which couple the energy of ATP hydrolysis to substrate translocation. It has been proposed that the NBDs of ABC transporters, including the histidine permease of Salmonella typhimurium and the cystic fibrosis transmembrane conductance regulator, are accessible from the extracellular surface of the cell, spanning the membrane directly or potentially contributing to the transmembrane pore. Such organization would have significant implications for the transport mechanism. We determined to establish whether the NBDs of P-gp are exposed extracellularly and which amino acids are accessible, using cysteine-scanning mutagenesis and limited proteolysis. In contrast to other transporters, the data provided no evidence that the P-gp NBDs are exposed to the cell surface. The implications for the structure and mechanism of P-gp and other ABC transporters are discussed.
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No. Sentence Comment
190 Oligonucleotides used for site-directed mutagenesis pSC-name Diagnostic restriction site Mutagenic oligonucleotide sequence 5Ј-3Ј N280C ϩAvaI GGTACAACAAATGTCTCGAGGAAGCTAAAAG G324C -AflIII CCTTGGTCTTATCATGTGAATATTCTATTGG S403C ϩPstI CTTCAGTTACCCCTGCAGAAAAGAAGTTAAG S419C ϩEco57I GAACCTGAAAGTGCAGTGTGGGCAGACG Q456C ϩBstEI GTTGATGGATGCGATATCCGGACCATAAATG F465C ϩBanI ATGTAAGGTGCCTACGGGAA I469C ϩNsiI CTACGGGAATGCATTGGTGT S474C ϩHpaII GGTGTCGTGTGTCAGGAACCGGTATTGTTT T482C ϩBsrGI GTATTGTTTGCCTGTACAATAGCTGAAAAC I488C ϩEclXI GCTGAAAACTGTCGCTACGGCCGTGAAAATG Y490C none ACATTCGCTGTGGCCGTGA V495C ϩBsrGI GGCCGTGAAAATTGTACAATGGATGAGATTG D498C -NcoI GTCACCATGTGTGAGATTGAG I500C ϩBsmI GTCACCATGGATGAATGCGAGAAAGCTGTC K502C ϩBsmI GGATGAGATTGAATGCGCTGTCAAGGAAG N508C ϩFspI GTCAAGGAAGCATGCGCATATGACTTTATC D511C ϩAflIII GGAAGCCAACGCGTATTGCTTTATCATG K515C ϩAflIII GAAGCCAACGCGTATGACTTTATCATGTGCCTGCCTCAT K519C ϩStyI GAAACTGCCTCATTGCTTTGACACCTTGGTTGGAGAGAGAG A529C ϩBanI GAGAGAGGGTGCCAGTTGAG K536C ϩBlpI GAGAGAGAGGCGCCCAGTTGAGTGGTGGGCAGTGCCAGAGGATCG R547C ϩBssSI GCACGTGCCCTCGTGTGCAACCCCAAG P549C ϩSspI TGGTTCGCAACTGCAAAATATTCCTGCTGGA L554C ϩSspI CGCAACCCCAAAATATTGCTGTGCGATGAGGCCACG S565C ϩBsmI GACACAGAATGCGAAGCAG V569C ϩBsmI GACACAGAATGCGAAGCAGTGTGTCAGGTGG K578C ϩBsiEI GATAAGGCCAGATGCGGCCGGACCACC R580C -MslI GCCACAAAAGGTTGCACGACCATTGTGATA T581C ϩApaLI GAAAAGGTCGGTGCACCATTGTG E638C ϩEclXI GAAGTTGAATTATGCAATGCGGCCGATGAATC 'ϩ` represents the introduction of a new restriction endonuclease site; '-` represents the removal of an existing site.
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ABCB1 p.Ser474Cys 10581253:190:466
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.Ser474Cys 17627029:54:224
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.Ser474Cys 17627029:139:191
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.Ser474Cys 17627029:155:56
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.Ser474Cys 17627029:169:66
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.Ser474Cys 17627029:180:150
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.Ser474Cys 17627029:184:76
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.Ser474Cys 17627029:207:78
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.Ser474Cys 17627029:212:102
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.Ser474Cys 17627029:219:86
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.Ser474Cys 17627029:220:196
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.Ser474Cys 17627029:245:126
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.Ser474Cys 17627029:266:74
status: NEW