ABCB1 p.Gln195Cys
| Predicted by SNAP2: | A: D (66%), C: D (66%), D: D (80%), E: D (59%), F: D (71%), G: D (71%), H: D (59%), I: D (75%), K: D (85%), L: D (80%), M: D (63%), N: D (63%), P: D (85%), R: D (80%), S: D (63%), T: D (63%), V: D (75%), W: D (80%), Y: D (66%), |
| 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, R: D, S: D, T: D, V: D, W: D, Y: D, |
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[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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No. Sentence Comment
77 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.Gln195Cys 10681495:77:717
status: NEW[hide] Location of the rhodamine-binding site in the huma... J Biol Chem. 2002 Nov 15;277(46):44332-8. Epub 2002 Sep 9. Loo TW, Clarke DM
Location of the rhodamine-binding site in the human multidrug resistance P-glycoprotein.
J Biol Chem. 2002 Nov 15;277(46):44332-8. Epub 2002 Sep 9., 2002-11-15 [PMID:12223492]
Abstract [show]
The human multidrug resistance P-glycoprotein (P-gp) pumps a wide variety of structurally diverse compounds out of the cell. It is an ATP-binding cassette transporter with two nucleotide-binding domains and two transmembrane (TM) domains. One class of compounds transported by P-gp is the rhodamine dyes. A P-gp deletion mutant (residues 1-379 plus 681-1025) with only the TM domains retained the ability to bind rhodamine. Therefore, to identify the residues involved in rhodamine binding, 252 mutants containing a cysteine in the predicted TM segments were generated and reacted with a thiol-reactive analog of rhodamine, methanethiosulfonate (MTS)-rhodamine. The activities of 28 mutants (in TMs 2-12) were inhibited by at least 50% after reaction with MTS-rhodamine. The activities of five mutants, I340C(TM6), A841C(TM9), L975C(TM12), V981C(TM12), and V982C(TM12), however, were significantly protected from inhibition by MTS-rhodamine by pretreatment with rhodamine B, indicating that residues in TMs 6, 9, and 12 contribute to the binding of rhodamine dyes. These results, together with those from previous labeling studies with other thiol-reactive compounds, dibromobimane, MTS-verapamil, and MTS-cross-linker substrates, indicate that common residues are involved in the binding of structurally different drug substrates and that P-gp has a common drug-binding site. The results support the "substrate-induced fit" hypothesis for drug binding.
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No. Sentence Comment
133 In TM3, the activities of two mutants (K189C and Q195C) were inhibited 76 and 78%, respectively, by MTS-rhodamine.
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ABCB1 p.Gln195Cys 12223492:133:49
status: NEW[hide] Suppressor mutations in the transmembrane segments... J Biol Chem. 2007 Nov 2;282(44):32043-52. Epub 2007 Sep 11. Loo TW, Bartlett MC, Clarke DM
Suppressor mutations in the transmembrane segments of P-glycoprotein promote maturation of processing mutants and disrupt a subset of drug-binding sites.
J Biol Chem. 2007 Nov 2;282(44):32043-52. Epub 2007 Sep 11., 2007-11-02 [PMID:17848563]
Abstract [show]
Defective folding of cystic fibrosis transmembrane conductance regulator protein missing Phe508 (DeltaF508) is the major cause of cystic fibrosis. The folding defect in DeltaF508 cystic fibrosis transmembrane conductance regulator might be correctable because misfolding of a P-glycoprotein (P-gp; ABCB1) mutant lacking the equivalent residue (DeltaY490) could be corrected with drug substrates or by introduction of an arginine residue into transmembrane (TM) segments 5 (I306R) or 6 (F343R). Possible mechanisms of arginine rescue were that they mimicked some of the effects of drug substrate interactions with P-gp or that they affected global folding such that all drug substrate/modulator interactions with P-gp were altered. To distinguish between these mechanisms, we tested whether arginines introduced into other TMs predicted to line the drug-binding pocket (TM1 or TM3) would affect folding. It was found that mutation of L65R(TM1) or T199R(TM3) promoted maturation of processing mutants. We then tested whether arginine suppressor mutations had local or global effects on P-gp interactions with drug substrates and modulators. The L65R(TM1), T199R(TM3), I306R(TM5), or F343R(TM6) mutations were introduced into the P-gp mutant L339C(TM6)/F728C(TM7), and thiol cross-linking was carried out in the presence of various concentrations of vinblastine, cyclosporin A, or rhodamine B. The presence of arginine residues reduced the apparent affinity of P-gp for vinblastine (L65R, T199R, and I306R), cyclosporin (I306R and F343R), or rhodamine B (F343R) by 4-60-fold. These results show that the arginine mutations affect a subset of drug-binding sites and suggest that they rescue processing mutants by mimicking drug substrate interactions with P-gp.
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No. Sentence Comment
154 The mutants Q195C and T199C, however, exhibited different properties than Cys-less P-gp after treatment with MTS-rhodamine.
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ABCB1 p.Gln195Cys 17848563:154:12
status: NEW155 Whereas mutant Q195C showed a 6.4-fold stimulation of ATPase activity with rhodamine B before treatment with MTS-rhodamine, its activity could only be stimulated 1.3-fold after treatment with MTS-rhodamine.
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ABCB1 p.Gln195Cys 17848563:155:15
status: NEW168 Fold-stimulation 1 2 3 4 5 6 Cys-less I190C G191C M192C F193C F194C Q195C S196C M197C A198C T199C F200C F201C T202C G203C F204C I205C V206C G207C F208C T209C A 7 Fold-stimulation 1 2 3 4 5 6 Cys-less B _ + MTS-rhod Rhod B+ _ + Q195C + + _ + T199C + + _ + _ _+ + + 7 FIGURE 2.
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ABCB1 p.Gln195Cys 17848563:168:68
status: NEWX
ABCB1 p.Gln195Cys 17848563:168:227
status: NEW173 B, Cys-less, Q195C and T199C P-gp mutants were treated with (ϩ) or without (-) 2 mM MTS-rhodamine and histidine-tagged P-gp isolated by nickel-chelate chromatography. Equivalent amounts of P-gp were mixed with lipid, and ATPase activity was determined in the presence (ϩ) or absence (-) of 2 mM rhodamine B (Rhod B).
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ABCB1 p.Gln195Cys 17848563:173:13
status: NEW