ABCB1 p.Ser909Cys
| Predicted by SNAP2: | A: N (66%), C: D (53%), D: D (75%), E: D (75%), F: D (71%), G: N (78%), H: D (75%), I: D (71%), K: D (80%), L: D (75%), M: D (63%), N: D (63%), P: D (85%), Q: D (75%), R: D (80%), T: N (53%), V: D (75%), W: D (85%), Y: D (71%), |
| 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, T: D, V: D, W: D, Y: D, |
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[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
86 AGTTGTTTCTTTGAC-3Ј; and S909C, 5Ј-CCGAACCGTTG- TTTGTCTCACCCAGGAGCAGAAGTTTG-3Ј.
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ABCB1 p.Ser909Cys 17627029:86:30
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.Ser909Cys 17627029:139:156
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.Ser909Cys 17627029:155:74
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.Ser909Cys 17627029:169:80
status: NEW173 However, to rule out the possibility that the gel retardation is the product of intermolecular cross linking between, for example, L443C on one P-glycoprotein with S909C on a second molecule, we cotransfected HEK293T cells with plasmids encoding both the single cysteine forms of the double mutants.
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ABCB1 p.Ser909Cys 17627029:173:164
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.Ser909Cys 17627029:180:135
status: NEW183 Both treatments caused a reproducible reduction in the level of cross-linking between L443C and S909C (fewer slow migrating P-glycoprotein species and more noncrosslinked species) with each of the three chemical cross-linkers, although the effect seems more pronounced with BMH (Fig. 7A).
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ABCB1 p.Ser909Cys 17627029:183:96
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.Ser909Cys 17627029:207:54
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.Ser909Cys 17627029:212:140
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.Ser909Cys 17627029:219:51
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.Ser909Cys 17627029:220:167
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.Ser909Cys 17627029:245:112
status: NEW257 Evidence in support of the interaction between ICL4 and NBD1 being a site of energy transduction through which the ATP and drug binding sites are coupled allosterically is provided by the modulation of the cross-linking efficiency between L443C and S909C by the nonhydrolysable ATP analog AMP-PNP, and also by ADP in the presence of vanadate (ADP.Vi) (Fig. 7A).
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ABCB1 p.Ser909Cys 17627029:257:249
status: NEW264 While it is unlikely that all the P-glycoprotein molecules in a membrane fraction would be accessible to nucleotide (the NBDs of some in the fraction are likely to be intravesicular), the data suggest that in the presence of AMP-PNP or ADP.Vi the L443C and S909C cysteines are less available for cross-linking.
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ABCB1 p.Ser909Cys 17627029:264:257
status: NEW[hide] The ATPase activity of the P-glycoprotein drug pum... J Biol Chem. 2012 Aug 3;287(32):26806-16. doi: 10.1074/jbc.M112.376202. Epub 2012 Jun 14. Loo TW, Bartlett MC, Detty MR, Clarke DM
The ATPase activity of the P-glycoprotein drug pump is highly activated when the N-terminal and central regions of the nucleotide-binding domains are linked closely together.
J Biol Chem. 2012 Aug 3;287(32):26806-16. doi: 10.1074/jbc.M112.376202. Epub 2012 Jun 14., [PMID:22700974]
Abstract [show]
The P-glycoprotein (P-gp, ABCB1) drug pump protects us from toxic compounds and confers multidrug resistance. Each of the homologous halves of P-gp is composed of a transmembrane domain (TMD) with 6 TM segments followed by a nucleotide-binding domain (NBD). The predicted drug- and ATP-binding sites reside at the interface between the TMDs and NBDs, respectively. Crystal structures and EM projection images suggest that the two halves of P-gp are separated by a central cavity that closes upon binding of nucleotide. Binding of drug substrates may induce further structural rearrangements because they stimulate ATPase activity. Here, we used disulfide cross-linking with short (8 A) or long (22 A) cross-linkers to identify domain-domain interactions that activate ATPase activity. It was found that cross-linking of cysteines that lie close to the LSGGQ (P517C) and Walker A (I1050C) sites of NBD1 and NBD2, respectively, as well as the cytoplasmic extensions of TM segments 3 (D177C or L175C) and 9 (N820C) with a short cross-linker activated ATPase activity over 10-fold. A pyrylium compound that inhibits ATPase activity blocked cross-linking at these sites. Cross-linking between the NBDs was not inhibited by tariquidar, a drug transport inhibitor that stimulates P-gp ATPase activity but is not transported. Cross-linking between extracellular cysteines (T333C/L975C) predicted to lock P-gp into a conformation that prevents close NBD association inhibited ATPase activity. The results suggest that trapping P-gp in a conformation in which the NBDs are closely associated likely mimics the structural rearrangements caused by binding of drug substrates that stimulate ATPase activity.
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No. Sentence Comment
106 The location of residues that were mutated to cysteine to test for the effect of cross-linking between NBD1 and NBD2 (P517C/I1050C), NBD1 and TMD2 (L443C/S909C), ICL1 and ICL3 (D177C/N820C), TM segments 2 and 11 (C137/A935C) or 6 and 12 (T333C/L975C) are shown.
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ABCB1 p.Ser909Cys 22700974:106:154
status: NEW152 The ATPase activity of M17M cross-linked L175C/N820C could still be highly activated when assayed in the presence of verapamil.
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ABCB1 p.Ser909Cys 22700974:152:13
status: NEWX
ABCB1 p.Ser909Cys 22700974:152:70
status: NEW154 ATPase Activity Is Not Activated when the Homologous Halves Are Cross-linked at Locations Predicted Not to Undergo Large Distance Changes during the Open to Closed Conformational Change-Mutant C137/A935C contains natural Cys-137 in TM segment 2 (TMD1) and the A935C mutation in TM segment 11 (TMD2).
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ABCB1 p.Ser909Cys 22700974:154:70
status: NEWX
ABCB1 p.Ser909Cys 22700974:154:220
status: NEW156 Mutant L443C/S909C contains a cysteine (L443) in NBD1 and a cysteine (S909C) in the ICL4 loop that connects the cytoplasmic extensions of TM segments 10 and 11.
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ABCB1 p.Ser909Cys 22700974:156:13
status: NEWX
ABCB1 p.Ser909Cys 22700974:156:70
status: NEW158 In previous studies we showed that mutants C137/A935C (39) and L443C/ S909C (27) had verapamil-stimulated ATPase activities similar to Cys-less P-gp.
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ABCB1 p.Ser909Cys 22700974:158:70
status: NEW159 Membranes prepared from cells expressing mutants C137/ A935C or L443C/S909C were treated with M4M cross-linker.
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ABCB1 p.Ser909Cys 22700974:159:70
status: NEW163 These results indicate that stimulation of ATPase activity observed in M4M cross-linked mutants P517C/I1050C (Fig. 2B), D177C/N820C, or L175C/N820C (Fig. 3C) was likely a specific effect of trapping the protein in the closed conformation.
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ABCB1 p.Ser909Cys 22700974:163:124
status: NEW166 A, membranes prepared from HEK 293 cells expressing the NBD1/TMD2 mutant L443C/ S909C or the TM2/TM11 mutant C137/A935C were treated with (ϩ) or without (-) the M4M cross-linker.
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ABCB1 p.Ser909Cys 22700974:166:80
status: NEW168 The positions of the cross-linked (X-link) and mature (170 kDa) P-gps are indicated. B, membranes expressing mutants L443C/ S909C or C137/A935C were treated with or without (None) M4M cross-linker and histidine-tagged P-gp isolated by nickel-chelate chromatography.
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ABCB1 p.Ser909Cys 22700974:168:124
status: NEW287 One explanation is that some movement is required at the C137/A935C and L443C/S909C interfaces during the catalytic cycle.
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ABCB1 p.Ser909Cys 22700974:287:78
status: NEW301 Direct cross-linking of cysteines in the mutants would trap C137/A935C or L443C/S909C at a distance of about 5.6 Å, the average distance between ␣ carbons in a disulfide bond (64).
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ABCB1 p.Ser909Cys 22700974:301:80
status: NEW304 Another plausible explanation as to why direct cross-linking of mutants C137/A935C or L443C/S909C inhibited activity is that cross-linking may have trapped the protein in an inactive conformation with the sites closer than normal.
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ABCB1 p.Ser909Cys 22700974:304:92
status: NEW104 The location of residues that were mutated to cysteine to test for the effect of cross-linking between NBD1 and NBD2 (P517C/I1050C), NBD1 and TMD2 (L443C/S909C), ICL1 and ICL3 (D177C/N820C), TM segments 2 and 11 (C137/A935C) or 6 and 12 (T333C/L975C) are shown.
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ABCB1 p.Ser909Cys 22700974:104:154
status: NEW161 A, membranes prepared from HEK 293 cells expressing the NBD1/TMD2 mutant L443C/ S909C or the TM2/TM11 mutant C137/A935C were treated with (af9;) or without (afa;) the M4M cross-linker.
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ABCB1 p.Ser909Cys 22700974:161:80
status: NEW280 One explanation is that some movement is required at the C137/A935C and L443C/S909C interfaces during the catalytic cycle.
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ABCB1 p.Ser909Cys 22700974:280:78
status: NEW294 Direct cross-linking of cysteines in the mutants would trap C137/A935C or L443C/S909C at a distance of about 5.6 &#c5;, the average distance between ॷ carbons in a disulfide bond (64).
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ABCB1 p.Ser909Cys 22700974:294:80
status: NEW297 Another plausible explanation as to why direct cross-linking of mutants C137/A935C or L443C/S909C inhibited activity is that cross-linking may have trapped the protein in an inactive conformation with the sites closer than normal.
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ABCB1 p.Ser909Cys 22700974:297:92
status: NEW