ABCB1 p.Met68Cys
| Predicted by SNAP2: | A: D (59%), C: D (53%), D: D (80%), E: D (80%), F: D (53%), G: D (66%), H: D (75%), I: N (53%), K: D (85%), L: N (66%), N: D (66%), P: D (85%), Q: D (63%), R: D (80%), S: D (66%), T: D (71%), V: N (72%), 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: N, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D, |
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[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
130 The activity of M68C, was inhibited by 44%, whereas that of mutant L65C was increased (189%).
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ABCB1 p.Met68Cys 12223492:130:16
status: NEW[hide] ATP hydrolysis promotes interactions between the e... Biochemistry. 2005 Aug 2;44(30):10250-8. Loo TW, Bartlett MC, Clarke DM
ATP hydrolysis promotes interactions between the extracellular ends of transmembrane segments 1 and 11 of human multidrug resistance P-glycoprotein.
Biochemistry. 2005 Aug 2;44(30):10250-8., 2005-08-02 [PMID:16042402]
Abstract [show]
P-glycoprotein (P-gp, ABCB1) actively pumps a broad range of structurally unrelated cytotoxic compounds out of the cell. It has two homologous halves that are joined by a linker region. Each half has a transmembrane (TM) domain containing six TM segments and a nucleotide-binding domain (NBD). Cross-linking studies have shown that the drug-binding pocket is at the interface between the TM domains. The two NBDs interact to form the ATP-binding sites. Coupling of ATP hydrolysis to drug efflux has been postulated to occur by conversion of the binding pocket from a high-affinity to a low-affinity state through alterations in the packing of the TM segments. TM 11 has also been reported to be important for drug binding. Here, we used cysteine-scanning mutagenesis and oxidative cross-linking to test for changes in the packing of TM 11 during ATP hydrolysis. We generated 350 double cysteine mutants that contained one cysteine at the extracellular end of TM11 and another cysteine at the extracellular ends of TMs 1, 3, 4, 5, or 6. The mutants were expressed in HEK293 cells and treated with oxidant in the absence or presence of ATP. Cross-linked product was not detected in SDS-PAGE gels in the absence of ATP. By contrast, cross-linked product was detected in mutants M68C(TM1)/Y950C(TM11), M68C(TM1)/Y953C(TM11), M68C(TM1)/A954C(TM11), M69C(TM1)/A954C(TM11), and M69C(TM1)/ F957C(TM11) in the presence of ATP but not with ADP or AMP.PNP. These results indicate that rearrangement of TM11 may contribute to the release of drug substrate during ATP hydrolysis.
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No. Sentence Comment
97 Figure 2A shows that the slower migrating product in mutants M68C(TM1)/Y950C(TM11), M68C- (TM1)/Y953C(TM11), M68C(TM1)/A954C(TM11), M69C- (TM1)/A954C(TM11), and M69C(TM1)/F957C(TM11) was greatly reduced upon exposure to 10 mM dithiothreitol.
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ABCB1 p.Met68Cys 16042402:97:84
status: NEW134 Membranes were prepared from HEK 293 cells (A), expressing mutants M68C(TM1)/Y950C(TM11), M68C(TM1)/Y953C(TM11), M68C- (TM1)/A954C(TM11), M69C(TM1)/A954C(TM11), M69C(TM1)/ F957C(TM11), or the negative mutant F72C(TM1)/F957C(TM11); (B) the single cysteine mutants M68C(TM1), M69C(TM1), Y950C(TM11), Y953C(TM11), A954C(TM11), or F957C(TM11); or (C) coexpressing the single cysteine mutants M68C(TM1) plus Y950C(TM11), M68C(TM1) plus Y953C(TM11), M68C(TM1) plus A954C(TM11), M69C(TM1) plus A954C(TM11), or M69C- (TM1) plus F957C(TM11).
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ABCB1 p.Met68Cys 16042402:134:113
status: NEW173 Histidine-tagged wild-type, Cys-less, and P-gp mutants M68C- (TM1)/Y950C(TM11), M68C(TM1)/Y953C(TM11), M68C(TM1)/ A954C(TM11), M69C(TM1)/A954C(TM11), or M69C(TM1)/ F957C(TM11) were expressed in HEK 293 cells.
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ABCB1 p.Met68Cys 16042402:173:55
status: NEW[hide] Transmembrane segment 1 of human P-glycoprotein co... Biochem J. 2006 Jun 15;396(3):537-45. Loo TW, Bartlett MC, Clarke DM
Transmembrane segment 1 of human P-glycoprotein contributes to the drug-binding pocket.
Biochem J. 2006 Jun 15;396(3):537-45., 2006-06-15 [PMID:16492138]
Abstract [show]
P-glycoprotein (P-gp; ABCB1) actively transports a broad range of structurally unrelated compounds out of the cell. An important step in the transport cycle is coupling of drug binding with ATP hydrolysis. Drug substrates such as verapamil bind in a common drug-binding pocket at the interface between the TM (transmembrane) domains of P-gp and stimulate ATPase activity. In the present study, we used cysteine-scanning mutagenesis and reaction with an MTS (methanethiosulphonate) thiol-reactive analogue of verapamil (MTS-verapamil) to test whether the first TM segment [TM1 (TM segment 1)] forms part of the drug-binding pocket. One mutant, L65C, showed elevated ATPase activity (10.7-fold higher than an untreated control) after removal of unchanged MTS-verapamil. The elevated ATPase activity was due to covalent attachment of MTS-verapamil to Cys65 because treatment with dithiothreitol returned the ATPase activity to basal levels. Verapamil covalently attached to Cys65 appears to occupy the drug-binding pocket because verapamil protected mutant L65C from modification by MTS-verapamil. The ATPase activity of the MTS-verapamil-modified mutant L65C could not be further stimulated with verapamil, calcein acetoxymethyl ester or demecolcine. The ATPase activity could be inhibited by cyclosporin A but not by trans-(E)-flupentixol. These results suggest that TM1 contributes to the drug-binding pocket.
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No. Sentence Comment
98 Verapamil Colchicine Vinblastine Mutant Vmax (%)* S50 (µM)† Vmax (%) S50 (µM) Vmax (%) S50 (µM) M51C 101 11.0 + - 0.6 96 391 + - 36 94 2.4 + - 0.2 V52C ND ND ND ND ND ND V53C 104 12.0 + - 0.2 101 389 + - 30 102 2.2 + - 0.1 G54C ND ND ND ND ND ND T55C 114 10.3 + - 1.1 95 418 + - 22 91 2.2 + - 0.1 L56C 103 12.2 + - 0.3 87 440 + - 41 95 2.5 + - 0.2 A57C 108 11.3 + - 0.3 98 377 + - 34 92 2.4 + - 0.2 A58C 90 12.5 + - 0.2 94 434 + - 20 95 2.6 + - 0.3 I59C 115 11.2 + - 0.8 95 380 + - 33 114 2.5 + - 0.2 I60C 102 11.1 + - 0.7 91 408 + - 18 110 2.5 + - 0.2 H61C 97 54.0 + - 5.0 61 912 + - 86 105 5.4 + - 0.4 G62C ND ND ND ND ND ND A63C 114 10.5 + - 1.2 99 362 + - 42 105 2.0 + - 0.3 G64C 106 45.0 + - 6.0 88 613 + - 55 60 2.4 + - 0.1 L65C 72 9.3 + - 1.1 112 368 + - 32 78 2.0 + - 0.2 P66C 95 13.0 + - 0.5 86 480 + - 39 97 2.8 + - 0.4 L67C 101 12.3 + - 0.3 106 423 + - 21 100 2.3 + - 0.1 M68C 119 9.7 + - 1.1 105 365 + - 32 92 2.3 + - 0.2 M69C 107 11.8 + - 0.6 110 431 + - 25 108 2.2 + - 0.1 L70C 94 11.4 + - 0.7 90 413 + - 18 98 2.3 + - 0.1 V71C 106 11.9 + - 0.3 90 370 + - 27 102 2.5 + - 0.5 Cys-less 100 12.0 + - 1.0 100 412 + - 48 100 2.2 + - 0.3 * Maximum activity relative to that of Cys-less P-gp.
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ABCB1 p.Met68Cys 16492138:98:905
status: NEW