ABCB1 p.Gly939Cys
| Predicted by SNAP2: | A: D (75%), C: D (80%), D: D (91%), E: D (91%), F: D (91%), H: D (91%), I: D (91%), K: D (95%), L: D (91%), M: D (91%), N: D (85%), P: D (91%), Q: D (91%), R: D (91%), S: D (75%), T: D (85%), V: D (91%), 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] Identification of residues in the drug-binding dom... J Biol Chem. 1999 Dec 10;274(50):35388-92. Loo TW, Clarke DM
Identification of residues in the drug-binding domain of human P-glycoprotein. Analysis of transmembrane segment 11 by cysteine-scanning mutagenesis and inhibition by dibromobimane.
J Biol Chem. 1999 Dec 10;274(50):35388-92., 1999-12-10 [PMID:10585407]
Abstract [show]
The drug-binding domain of the human multidrug resistance P-glycoprotein (P-gp) probably consists of residues from multiple transmembrane (TM) segments. In this study, we tested whether the amino acids in TM11 participate in binding drug substrates. Each residue in TM11 was initially altered by site-directed mutagenesis and assayed for drug-stimulated ATPase activity in the presence of verapamil, vinblastine, or colchicine. Mutants G939V, F942A, T945A, Q946A, A947L, Y953A, A954L, and G955V had altered drug-stimulated ATPase activities. Direct evidence for binding of drug substrate was then determined by cysteine-scanning mutagenesis of the residues in TM11 and inhibition of drug-stimulated ATPase activity by dibromobimane, a thiol-reactive substrate. Dibromobimane inhibited the drug-stimulated ATPase activities of two mutants, F942C and T945C, by more than 75%. These results suggest that residues Phe(942) and Thr(945) in TM11, together with residues previously identified in TM6 (Leu(339) and Ala(342)) and TM12 (Leu(975), Val(982), and Ala(985)) (Loo, T. W., and Clarke, D. M. (1997) J. Biol. Chem. 272, 31945-31948) form part of the drug-binding domain of P-gp.
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No. Sentence Comment
117 Mutant G939C had the lowest activity (70% of that of Cys-less P-gp).
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ABCB1 p.Gly939Cys 10585407:117:7
status: NEW120 Fig. 5 shows that the majority of the mutants, except for G939C, F942C, T945C, and Y953C, were not affected by treatment with dBBn.
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ABCB1 p.Gly939Cys 10585407:120:58
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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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.Gly939Cys 10681495:65:270
status: NEW66 A representative result (mutant G939C/P350C) is shown in Fig. 1A.
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ABCB1 p.Gly939Cys 10681495:66:32
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.Gly939Cys 10681495:77:1057
status: NEW79 A representative result (mutant G939C/P350C) is shown in Fig. 1C.
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ABCB1 p.Gly939Cys 10681495:79:32
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.Gly939Cys 10681495:99:158
status: NEW110 Oxidative cross-linking of P-gp mutant G939C/P350C.
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ABCB1 p.Gly939Cys 10681495:110:39
status: NEW111 A, membranes from HEK 293 cells transfected with vector alone (Control), mutant G939C/P350C, or Cys-less (C-less) P-gp cDNA were treated for 10 min at 37 °C in the presence (ϩ) or absence (-) of 2 mM Cu2ϩ (phenanthroline)3.
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ABCB1 p.Gly939Cys 10681495:111:80
status: NEW115 C, membranes from HEK 293 cells expressing mutant G939C/P350C were treated with 0.2 mM Cu2ϩ (phenanthroline)3 in the presence of no drug (None), 1 mM colchicine (Colch), 0.15 mM verapamil (Ver), 10 M cyclosporin A (Cyclo) or 35 M vinblastine (Vin) or in the presence of no addition (None), 5 mM ATP (ATP), 5 mM ATP and 0.1 mM vanadate (ATP/VO4) or 0.1 mM vanadate (VO4).
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ABCB1 p.Gly939Cys 10681495:115:50
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.Gly939Cys 10681495:118:508
status: NEW[hide] Substrate-induced conformational changes in the tr... J Biol Chem. 2003 Apr 18;278(16):13603-6. Epub 2003 Feb 27. Loo TW, Bartlett MC, Clarke DM
Substrate-induced conformational changes in the transmembrane segments of human P-glycoprotein. Direct evidence for the substrate-induced fit mechanism for drug binding.
J Biol Chem. 2003 Apr 18;278(16):13603-6. Epub 2003 Feb 27., 2003-04-18 [PMID:12609990]
Abstract [show]
The human multidrug resistance P-glycoprotein (P-gp, ABCB1) is quite promiscuous in that it can transport a broad range of structurally diverse compounds out of the cell. We hypothesized that the transmembrane (TM) segments that constitute the drug-binding site are quite mobile such that drug binding occurs through a "substrate-induced fit" mechanism. Here, we used cysteine-scanning mutagenesis and oxidative cross-linking to test for substrate-induced changes in the TM segments. Pairs of cysteines were introduced into a Cys-less P-gp and the mutants treated with oxidant (copper phenanthroline) in the presence or absence of various drug substrates. We show that cyclosporin A promoted cross-linking between residues P350C(TM6)/G939C(TM11), while colchicine and demecolcine promoted cross-linking between residues P350C(TM6)/V991C(TM12). Progesterone promoted cross-linking between residues P350C(TM6)/A935C(TM11), P350C(TM6)/G939C(TM11), as well as between residues P350C(TM6)/V991C(TM12). Other substrates such as vinblastine, verapamil, cis-(Z)-flupenthixol or trans-(E)-flupenthixol did not induce cross-linking at these sites. These results provide direct evidence that the packing of the TM segments in the drug-binding site is changed when P-gp binds to a particular substrate. The induced-fit mechanism explains how P-gp can accommodate a broad range of compounds.
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No. Sentence Comment
91 In mutant P350C(TM6)/G939C- (TM11), progesterone was more efficient in promoting cross-linking than cyclosporin A.
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ABCB1 p.Gly939Cys 12609990:91:21
status: NEW124 In the presence of drug substrate (progesterone), TM segments 11 and 12 undergo rotational and/or lateral movements so that cross-linking can occur between P350C and V991C (black ball) in TM12 and with A935C (red ball) and G939C (turquoise ball) in TM11.
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ABCB1 p.Gly939Cys 12609990:124:223
status: NEW135 The presence of progesterone, however, promoted cross-linking of residue P350C(TM6) with two residues in TM 11 (A935C and G939C) and to residue V991C in TM12.
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ABCB1 p.Gly939Cys 12609990:135:122
status: NEW142 When the residues in TM11 are modeled as an ␣-helical wheel, residues A935 and G939C are found on the same face of the TM segment (Fig. 5B).
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ABCB1 p.Gly939Cys 12609990:142:86
status: NEW144 Cyclosporin A also promoted cross-linking between TM6 and TM11, but only between P350C and G939C.
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ABCB1 p.Gly939Cys 12609990:144:91
status: NEW[hide] Val133 and Cys137 in transmembrane segment 2 are c... J Biol Chem. 2004 Apr 30;279(18):18232-8. Epub 2004 Jan 28. Loo TW, Bartlett MC, Clarke DM
Val133 and Cys137 in transmembrane segment 2 are close to Arg935 and Gly939 in transmembrane segment 11 of human P-glycoprotein.
J Biol Chem. 2004 Apr 30;279(18):18232-8. Epub 2004 Jan 28., 2004-04-30 [PMID:14749322]
Abstract [show]
P-glycoprotein (P-gp; ABCB1) transports a wide variety of structurally diverse compounds out of the cell. The protein has two homologous halves joined by a linker region. Each half consists of a transmembrane (TM) domain with six TM segments and a nucleotide-binding domain. The drug substrate-binding pocket is at the interface between the TM segments in each half of the protein. Preliminary studies suggested that the arrangement of the two halves of P-gp shows rotational symmetry (i.e. "head-to-tail" arrangement). Here, we tested this model by determining whether the cytoplasmic ends of TM2 and TM3 in the N-terminal half are in close contact with TM11 in the C-terminal half. Mutants containing a pair of cysteines in TM2/TM11 or TM3/TM11 were subjected to oxidative cross-linking with copper phenanthroline. Two of the 110 TM2/TM11 mutants, V133C(TM2)/G939C(TM11) and C137C(TM2)/A935C (TM11), were cross-linked at 4 degrees C, when thermal motion is reduced. Cross-linking was specific since no cross-linked product was detected in the 100 double Cys TM3/TM11 mutants. Vanadate trapping of nucleotide or the presence of some drug substrates inhibited cross-linking of mutants V133C(TM2)/G939C(TM11) and C137C(TM2)/A935C(TM11). Cross-linking of TM2 and TM11 also blocked drug-stimulated ATPase activity. The close proximity of TM2/TM11 and TM5/TM8 (Loo, T. W., Bartlett, M. C., and Clarke, D. M. (2004) J. Biol. Chem. 279, 7692-7697) indicates that these regions between the two halves must enclose the drug-binding pocket at the cytoplasmic side of P-gp. They may form the "hinges" required for conformational changes during the transport cycle.
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No. Sentence Comment
115 Disulfide cross-linking of P-gp mutants. Membranes were prepared from HEK 293 cells expressing P-gp mutant V133C(TM2), G939C(TM11), C137C(TM2), A935C(TM11), V133C(TM2)/G939C (TM11), or C137C(TM2)/A935C(TM11).
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ABCB1 p.Gly939Cys 14749322:115:168
status: NEW124 TABLE I Cross-linking between residues in TM2 and TM11 -, no cross-linked product detected on SDS-polyacrylamide gels at 37 °C; ϩ, relatively weak cross-linking (Ͻ50% of P-gp cross-linked) at 37 °C; ϩϩ, relatively strong cross-linking (Ͼ50% of P-gp cross-linked) at 37 °C; *, cross-linked product also detected at 22 °C; **, cross-linked product also detected at 22 and 4 °C. TM2 TM11 A935C H936C I937C F938C G939C I940C T941C F942C S943C F944C A128C - - - - - - - - - - A129C - - - - - - - - - - Y130C - - - - ϩ ϩ - ϩ ϩ - I131C - - - - - - - - - - Q132C - - - - - - - - - - V133C - - - ϩ ϩϩ, ** - - ϩ ϩ - S134C ϩ ϩ - - ϩ ϩ - - - - F135C - - - - - - - - - - W136C - - - - - - - - - - C137C ϩϩ, ** - - - ϩ - - - - - L138C ϩϩ, * - - - - - - - - - TM11 are close to each other at their cytoplasmic ends.
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ABCB1 p.Gly939Cys 14749322:124:464
status: NEW