ABCB1 p.Gly62Cys
| Predicted by SNAP2: | A: D (75%), C: D (80%), D: D (85%), E: D (91%), F: D (91%), H: D (91%), I: D (85%), K: D (91%), L: D (91%), M: D (85%), N: D (80%), P: D (91%), Q: D (91%), R: D (91%), S: D (66%), T: D (80%), V: D (80%), W: D (91%), Y: D (91%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, H: D, I: D, K: D, L: D, M: D, 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
172 Three cysteine mutants (V52C, G54C, and G62C) in TM1 were not tested because of low expression, indicating that these residues must be important for structure and/or function.
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ABCB1 p.Gly62Cys 12223492:172:40
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.Gly62Cys 16492138:98:626
status: NEW104 The activities of mutants V52C, G54C and G62C were not determined because of very low expression [32].
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ABCB1 p.Gly62Cys 16492138:104:41
status: NEW