ABCB1 p.Glu875Cys
| Predicted by SNAP2: | A: D (80%), C: D (80%), D: D (80%), F: D (91%), G: D (71%), H: D (63%), I: D (91%), K: D (91%), L: D (91%), M: D (80%), N: D (59%), P: D (95%), Q: N (78%), R: D (75%), S: D (66%), T: D (66%), V: D (85%), W: D (91%), Y: D (91%), |
| Predicted by PROVEAN: | A: D, C: D, D: N, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: N, R: D, S: D, T: D, V: D, W: D, Y: D, |
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[hide] The glycosylation and orientation in the membrane ... Biochemistry. 1999 Apr 20;38(16):5124-9. Loo TW, Clarke DM
The glycosylation and orientation in the membrane of the third cytoplasmic loop of human P-glycoprotein is affected by mutations and substrates.
Biochemistry. 1999 Apr 20;38(16):5124-9., 1999-04-20 [PMID:10213617]
Abstract [show]
Multiple topologies have been detected for the COOH-terminal half of the human multidrug resistance P-glycoprotein (P-gp). In one topology, the predicted third cytoplasmic loop (CL3) is on the cytoplasmic side (P-gp-CL3-cyt) of the membrane. In an alternate topology, CL3 is on the extracellular side of the membrane (P-gp-CL3-ext). It is not known if both forms of P-gp are active because it is difficult to distinguish either topology in the full-length molecule. When the halves of P-gp are expressed as separate polypeptides, the two topologies of the C-Half are readily distinguished on SDS-PAGE, because only the C-Half (CL3-ext) is glycosylated. To test whether both topologies can fold into an active enzyme, we assayed for interaction between the N- and C-Halves of P-gp since functional P-gp requires interaction between both halves. In a mutant P-gp (E875C) that gave about equal amounts of both topologies, only the C-Half (CL3-cyt) could be recovered by nickel chromatography after coexpression with the histidine-tagged N-Half P-gp. The isolated N-Half and E875C C-Half (CL3-cyt) polypeptides, when expressed together, exhibited verapamil- and vinblastine-stimulated ATPase activities that were similar to the wild-type enzyme. We also found that biosynthesis of mutant E875C C-Half in the presence of the N-Half P-gp resulted in enhanced expression of C-Half (CL3-cyt). By contrast, interaction of C-Half (CL3-ext) with N-Half P-gp was not detected. These results show that the topology of the C-Half portion of P-gp greatly influences its interactions with the amino-terminal half of the molecule.
Comments [show]
None has been submitted yet.
No. Sentence Comment
83 During structure-function analysis studies (unpublished observations), we found a single mutant, E875C, that gave increased amounts of glycosylated C-Half polypeptide.
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ABCB1 p.Glu875Cys 10213617:83:97
status: NEW85 As shown in Figure 2 (lane 1), expression of the COOH-half of mutant E875C in HEK 293 cells resulted in the appearance of about equivalent amounts of the glycosylated (C-Half-CHO) and unglycosylated (C-Half) forms of the protein.
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ABCB1 p.Glu875Cys 10213617:85:69
status: NEW95 Mutation E875C is shown as a shaded circle in TM 10.
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ABCB1 p.Glu875Cys 10213617:95:9
status: NEW101 Whole cell extracts of cells expressing the A52-tagged wild-type C-Half, mutant E875C C-Half or mutant T811A C-Half P-gps were treated with (+Endo H) or without (-Endo H) endoglycosidase H and subjected to SDS-PAGE and immunoblot analysis with monoclonal antibody A52.
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ABCB1 p.Glu875Cys 10213617:101:80
status: NEW105 Since mutant E875C gave relatively equal amounts of C-Half molecules with both topologies, it was used for further analysis.
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ABCB1 p.Glu875Cys 10213617:105:13
status: NEW112 To monitor the expression of the C-Half polypeptide, an A52 epitope tag was attached to mutant E875C C-Half molecule, while the N-Half was not tagged.
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ABCB1 p.Glu875Cys 10213617:112:95
status: NEW113 The untagged N-Half molecule of P-gp was coexpressed with the A52-tagged C-Half of mutant E875C in the presence or absence of drug substrate.
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ABCB1 p.Glu875Cys 10213617:113:90
status: NEW114 Figure 3 shows the relative expression levels of glycosylated (C-Half-CHO) or unglycosylated (C-Half) forms of mutant E875C C-Half when expressed in the presence or absence of untagged N-Half molecule and drug substrate (cyclosporin A).
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ABCB1 p.Glu875Cys 10213617:114:118
status: NEW115 When E875C C-Half was expressed alone, the CL3(cyt) and CL3(ext) topologies were present in about equivalent amounts (Figure 3, lane 2).
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ABCB1 p.Glu875Cys 10213617:115:5
status: NEW116 Similar results were obtained when the C-Half of mutant E875C was expressed alone in the presence of drug substrate (lane 3) or when it was coexpressed with the N-Half in the absence of substrate (lane 5).
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ABCB1 p.Glu875Cys 10213617:116:56
status: NEW122 In this assay, a histidine-tagged N-Half molecule was coexpressed with an A52 epitope-tagged E875C C-Half.
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ABCB1 p.Glu875Cys 10213617:122:93
status: NEW125 Histidine-tagged N-Half P-gp (N-Half-His) was coexpressed with the A52-tagged C-Half of mutant E875C (C-Half-A52) in HEK 293 cells, solubilized with n-dodecyl- -D-maltoside, and the solubilized material was subjected to nickel chromatography (18).
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ABCB1 p.Glu875Cys 10213617:125:95
status: NEW133 HEK 293 cells were transfected with A52-tagged mutant E875C C-Half cDNA with or without (+ or -) N-Half P-gp cDNA.
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ABCB1 p.Glu875Cys 10213617:133:54
status: NEW137 HEK 293 cells were cotransfected with histidine-tagged N-Half and A52-tagged mutant E875C C-Half cDNAs, and solubilized with n-dodecyl- -D-maltoside and the extract subjected to nickel-chelate chromatography using 10 mM imidazole in the washing steps.
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ABCB1 p.Glu875Cys 10213617:137:84
status: NEW144 The full-length mutant E875C P-gp also exhibited drug-stimulated ATPase activities that were about 80-90% of the wild-type enzyme (Figure 5).
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ABCB1 p.Glu875Cys 10213617:144:23
status: NEW147 Coexpression of wild-type N-Half-His with mutant E875C C-Half-A52 in the presence of cyclosporin A resulted in the formation of an active complex that had 85-95% of the activity of the wild-type enzyme (Figure 5).
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ABCB1 p.Glu875Cys 10213617:147:49
status: NEW160 When the C-Half of mutant E875C was synthesized to the presence of the N-Half polypeptide and cyclosporin A, only the amount of unglycosylated C-Half was increased.
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ABCB1 p.Glu875Cys 10213617:160:26
status: NEW171 It is surprising that a single mutation, E875C, promotes an alternative topology in P-gp.
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ABCB1 p.Glu875Cys 10213617:171:41
status: NEW173 When expressed in the presence of drug substrates, the mutant E875C showed vinblastineand verapamil-stimulated ATPase activity that was similar to that of wild-type enzyme (Figure 5).
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ABCB1 p.Glu875Cys 10213617:173:62
status: NEW174 In the absence of drug substrate, maturation of mutant E875C was inefficient, such that the majority of the product was a core-glycosylated 150 kDa protein (data not shown).
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ABCB1 p.Glu875Cys 10213617:174:55
status: NEW177 The FIGURE 5: Drug-stimulated ATPase activities of wild-type and mutant E875C P-gps.
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ABCB1 p.Glu875Cys 10213617:177:72
status: NEW179 Equivalent amounts of histidine-tagged full-length wild-type or mutant E875C P-gps isolated by nickel-chelate chromatography, were added to lipid and assayed for ATPase in the absence or presence verapamil (1 mM) or vinblastine (0.05 mM).
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ABCB1 p.Glu875Cys 10213617:179:71
status: NEW180 Histidine-tagged N-Half (N-Half-H) that was expressed alone or coexpressed with A52 epitope-tagged wild-type or mutant E875C C-Half were isolated by nickel-chelate chromatography.
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ABCB1 p.Glu875Cys 10213617:180:119
status: NEW189 In P-gp, it appears that the E875C mutation influences topological folding of the C-terminal domain such that increased amounts of CL-3(ext) are the major product.
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ABCB1 p.Glu875Cys 10213617:189:29
status: NEW192 It is therefore possible that the E875C mutation promotes the formation of P-gp with this topology.
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ABCB1 p.Glu875Cys 10213617:192:34
status: NEW[hide] The packing of the transmembrane segments of human... J Biol Chem. 2000 Feb 25;275(8):5253-6. Loo TW, Clarke DM
The packing of the transmembrane segments of human multidrug resistance P-glycoprotein is revealed by disulfide cross-linking analysis.
J Biol Chem. 2000 Feb 25;275(8):5253-6., 2000-02-25 [PMID:10681495]
Abstract [show]
Residues from several transmembrane (TM) segments of P-glycoprotein (P-gp) likely form the drug-binding site(s). To determine the organization of the TM segments, pairs of cysteine residues were introduced into the predicted TM segments of a Cys-less P-gp, and the mutant protein was subjected to oxidative cross-linking. In SDS gels, the cross-linked product migrated with a slower mobility than the native protein. The cross-linked products were not detected in the presence of dithiothreitol. Cross-linking was observed in 12 of 125 mutants. The pattern of cross-linking suggested that TM6 is close to TMs 10, 11, and 12, while TM12 is close to TMs 4, 5, and 6. In some mutants the presence of drug substrate colchicine, verapamil, cyclosporin A, or vinblastine either enhanced or inhibited cross-linking. Cross-linking was inhibited in the presence of ATP plus vanadate. These results suggest that the TM segments critical for drug binding must be close to each other and exhibit different conformational changes in response to binding of drug substrate or vanadate trapping of nucleotide. Based on these results, we propose a model for the arrangement of the TM segments.
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None has been submitted yet.
No. Sentence Comment
65 Twelve of the 125 P-gp mutants (TM4/TM12 constructs L227C/S993C, V231C/S993C, W232C/S993C, A233C/S993C, I235C/S993C, and L236C/S993C; TM5/TM12 constructs A295C/S993C and I299C/S993C; TM10/TM6 constructs V874C/P350C, E875C/ P350C, and M876C/P350C; and TM11/TM6 construct G939C/ P350C), however, had slower mobilities in SDS-PAGE after treatment with oxidant.
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ABCB1 p.Glu875Cys 10681495:65:216
status: NEW77 In these cross-linking experiments, the amount of oxidant was lowered by 10-fold (0.2 mM), and the minimum temperature required to induce cross-TABLE I Cross-linking analysis of P-gp Cross-linking of S993C (TM12) with residues in the following TM: TM1 TM2 TM3 TM4 TM5 M51C -a Y130C - G185C - G226C - I293C - V52C - I131C - I186C - L227C ϩb T294C - V53C - Q132C - G187C - S228C - A295C ϩ G54C - V133C - D188C - A229C - N296C - T55C - S134C - K189C - A230C - I297C - L56C - F135C - I190C - V231C ϩ S298C - A57C - W136C - G191C - W232C ϩ I299C ϩ A58C - C137C - M192C - A233C ϩ G300C - I59C - L138C - F193C - K234C - A301C - I60C - A139C - F194C - I235C ϩ A302C - H61C - A140C - Q195C - L236C ϩ F303C - G141C - S196C - S237C - L304C - Cross-linking of P350C (TM6) with residues in the following TM: TM7 TM8 TM9 TM10 TM11 F711C - F770C - A828C - I867C - A935C - V712C - F771C - I829C - I868C - H936C - V713C - L772C - G830C - A869C - I937C - G714C - Q773C - S831C - I870C - F938C - V715C - G774C - R832C - A871C - G939C ϩ F716C - F775C - L833C - G872C - I940C - C717C - T776C - A834C - V873C - T941C - A718C - F777C - V835C - V874C ϩ F942C - I719C - G778C - I836C - E875C ϩ S943C - I720C - K779C - T837C - M876C ϩ F944C - N721C - A780C - Q838C - K877C - T945C - G722C - G781C - N839C - M878C - Q946C - G723C - E782C - I840C - L879C - A947C - I783C - a -, no cross-linked product detected in SDS-PAGE. b ϩ, cross-linked product detected in SDS-PAGE.
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ABCB1 p.Glu875Cys 10681495:77:1221
status: NEW82 Cyclosporin A inhibited the cross-linking of mutants L227C(TM4)/S993C(TM12), W232C(TM4)/ S993C(TM12), I299C(TM5)/S993(TM12), E875C (TM10)/ P350C(TM6), and M876C(TM10)/P350C(TM6).
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ABCB1 p.Glu875Cys 10681495:82:125
status: NEW99 Mutants L227C/S993C, V231C/ S993C, W232C/S993C, A233C/S993C, I235C/S993C, L236C/ S993C, A295C/S993C, I299C/S993C, V874C/P350C, E875C/ P350C, M876C/P350C, and G939C/P350C were inhibited by 81, 88, 90, 89, 93, 81, 78, 87, 87, 77, 70, and 78%, respectively.
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ABCB1 p.Glu875Cys 10681495:99:127
status: NEW118 TABLE II Minimum temperature required for cross-linking Residues TM segments 4 °C 21 °C 37 °C L227C/S993C 4/12 -a - ϩ V231C/S993C 4/12 - ϩ ϩ W232C/S993C 4/12 - ϩ ϩ A233C/S993C 4/12 ϩb ϩ ϩ I235C/S993C 4/12 ϩ ϩ ϩ L236C/S993C 4/12 ϩ ϩ ϩ A295C/S993C 5/12 - ϩ ϩ I299C/S993C 5/12 ϩ ϩ ϩ V874C/P350C 10/6 - ϩ ϩ E875C/P350C 10/6 - - ϩ M876C/P350C 10/6 - ϩ ϩ G939C/P350C 11/6 - ϩ ϩ a -, no cross-linked product detected in SDS-PAGE. b ϩ, cross-linked product detected in SDS-PAGE.
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ABCB1 p.Glu875Cys 10681495:118:444
status: NEW[hide] The W232R suppressor mutation promotes maturation ... Biochemistry. 2011 Feb 8;50(5):672-85. Epub 2011 Jan 11. Loo TW, Bartlett MC, Clarke DM
The W232R suppressor mutation promotes maturation of a truncation mutant lacking both nucleotide-binding domains and restores interdomain assembly and activity of P-glycoprotein processing mutants.
Biochemistry. 2011 Feb 8;50(5):672-85. Epub 2011 Jan 11., 2011-02-08 [PMID:21182301]
Abstract [show]
ATP-binding cassette (ABC) proteins contain two nucleotide-binding domains (NBDs) and two transmembrane (TM) domains (TMDs). Interdomain interactions and packing of the TM segments are critical for function, and disruption by genetic mutations contributes to disease. P-glycoprotein (P-gp) is a useful model to identify mechanisms that repair processing defects because numerous arginine suppressor mutations have been identified in the TM segments. Here, we tested the prediction that a mechanism of arginine rescue was to promote intradomain interactions between TM segments and restore interdomain assembly. We found that suppressor W232R(TM4/TMD1) rescued mutants with processing mutations in any domain and restored defective NBD1-NBD2, NBD1-TMD2, and TMD1-TMD2 interactions. W232R also promoted packing of the TM segments because it rescued a truncation mutant lacking both NBDs. The mechanism of W232R rescue likely involved intradomain hydrogen bond interactions with Asn296(TM5) since only N296A abolished rescue by W232R and rescue was only observed when Trp232 was replaced with hydrogen-bonding residues. In TMD2, suppressor T945R(TM11) also promoted packing of the TM segments because it rescued the truncation mutant lacking the NBDs and suppressed formation of alternative topologies. We propose that T945R rescue was mediated by interactions with Glu875(TM10) since T945E/E875R promoted maturation while T945R/E875A did not.
Comments [show]
None has been submitted yet.
No. Sentence Comment
309 It was not surprising that the mutations affected rhodamine B-stimulated ATPase activity because it was previously shown that treatment of mutants W232C, N296C, and T945C (E875C was not tested) with a thiol-reactive analogue of rhodamine inhibited activity (15).
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ABCB1 p.Glu875Cys 21182301:309:172
status: NEW366 It was previously reported that residue Glu875 could contribute to folding of TM segments because the E875C mutation altered the topology of C-half Pgp (45).
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ABCB1 p.Glu875Cys 21182301:366:102
status: NEW