ABCB1 p.Val53Cys
| Predicted by SNAP2: | A: N (87%), C: N (97%), D: D (66%), E: D (59%), F: N (93%), G: N (82%), H: N (72%), I: N (97%), K: D (63%), L: N (97%), M: N (93%), N: D (59%), P: D (66%), Q: D (59%), R: D (59%), S: N (82%), T: N (87%), W: D (63%), Y: N (72%), |
| Predicted by PROVEAN: | A: N, C: N, D: D, E: D, F: N, G: D, H: D, I: N, K: D, L: N, M: N, N: D, P: D, Q: D, R: D, S: N, T: N, W: D, Y: N, |
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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.Val53Cys 10681495:77:354
status: NEW[hide] Biochemical characterization of domains in the mem... Biochemistry. 2005 Feb 22;44(7):2661-70. Kaur P, Rao DK, Gandlur SM
Biochemical characterization of domains in the membrane subunit DrrB that interact with the ABC subunit DrrA: identification of a conserved motif.
Biochemistry. 2005 Feb 22;44(7):2661-70., 2005-02-22 [PMID:15709779]
Abstract [show]
DrrA and DrrB proteins confer resistance to the commonly used anticancer agents daunorubicin and doxorubicin in the producer organism Streptomyces peucetius. The drrAB locus has previously been cloned in Escherichia coli, and the proteins have been found to be functional in this host. DrrA, a soluble protein, belongs to the ABC family of proteins. It forms a complex with the integral membrane protein DrrB. Previous studies suggest that the function and stability of DrrA and DrrB are biochemically coupled. Thus, DrrA binds ATP only when it is in a complex with DrrB in the membrane. Further, DrrB is completely degraded if DrrA is absent. In the present study, we have characterized domains in DrrB that may be directly involved in interaction with DrrA. Several single-cysteine substitutions in DrrB were made. Interaction between DrrA and DrrB was studied by using a cysteine to amine chemical cross-linker that specifically cross-links a free sulfhydryl group in one protein (DrrB) to an amine in another (DrrA). We show here that DrrA cross-links with both the N- and the C-terminal ends of the DrrB protein, implying that they may be involved in interaction. Furthermore, this study identifies a motif within the N-terminal cytoplasmic tail of DrrB, which is similar to a motif recently shown by crystal structure analysis in BtuC and previously shown by sequence analysis to be also present in exporters, including MDR1. We propose that the motif present in DrrB and other exporters is actually a modified version of the EAA motif, which was originally believed to be present only in the importers of the ABC family. The present work is the first report where domains of interaction in the membrane component of an ABC drug exporter have been biochemically characterized.
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No. Sentence Comment
77 The mutants were named S4C, S15C, S23C, S35C, A44C, C260S up: 5'-GGCCTGGTCCTGTCCGTGTCGGCAGGG-3' C260S dn: 5'-CCCTGCCGACACGGACAGGACCAGGCC-3' S23C up: 5'- CGGACGGTGCTGTGCGCGGGTGAACGG-3' S23C dn: 5'- CCGTTCACCCGCGCACAGCACCGTCCG-3' V53C, T70C, S80C, V92C, S107C, V116C, A129C, T149C, A160C, V173C, S213C, S236C, T249C, A270C, and A282C, respectively.
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ABCB1 p.Val53Cys 15709779:77:230
status: NEW168 substitutions, including V53C, T70C, V92C, A129C, S236C, and S249C, showed a decrease in resistance to doxorubicin (Table 1).
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ABCB1 p.Val53Cys 15709779:168:25
status: NEW187 (B) A44C, V53C, T70C, V92C, S107C, and V116C.
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ABCB1 p.Val53Cys 15709779:187:10
status: NEW196 Table 1: Doxorubicin Resistance of E. coli N43 Cells Expressing Wild Type DrrA with DrrB Containing Different Cysteine Substitutionsa amt of dox, µg/mLdomain of DrrB location of cysteine 0 4 6 8 (wild type) C260 +++ +++ +++ ++ N-terminus S15C +++ +++ +++ ++ N-terminus S23C +++ +++ +++ ++ N-terminus S35C +++ +++ ++ ++ N-terminus A44C +++ +++ ++ + TM 1 V53C +++ ++ ++ - TM 1 T70C +++ + + + P 1 S80C +++ ++ + - TM 2 V92C +++ ( - - TM 2 S107C +++ +++ ++ ++ C 1 V116C +++ +++ +++ ++ TM 3 A129C +++ ++ + - TM 4 T149C +++ ++ ++ + C 2 A160C +++ +++ ++ + TM 5 V173C +++ +++ ++ + TM 6 S213C +++ +++ ++ + C 3 S236C +++ ++ + - TM 7 S249C +++ + + - TM 8 A270C +++ +++ ++ ++ C-terminus A282C +++ +++ ++ ++ cysteine-less DrrB C260S +++ +++ +++ +++ vector only pSU2718 ++++ ( - - a Legend: +++, very good growth; ++, good growth; +, some growth; -, no growth.
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ABCB1 p.Val53Cys 15709779:196:358
status: NEW214 Analysis of the mutants in TM1 (V53C and T70C) and TM2 (V92C and S107C) also showed the 60 kDa cross-linked species containing DrrA and DrrB (Figure 4B).
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ABCB1 p.Val53Cys 15709779:214:32
status: NEW278 Six (V53C, T70C, V92C, A129C, S236C, S249C) out of 20 substitutions created in this study, however, showed varying levels of doxorubicin-sensitive phenotype (Table 1).
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ABCB1 p.Val53Cys 15709779:278:5
status: NEW281 Secondary structure analysis by the Chou-Fasman method showed that five (V53C, T70C, V92C, A129C, S249C) of these six mutants, which resulted in doxorubicin sensitivity, showed no change at all in their predicted secondary structure, the exception being S236C, which showed a split in the helical stretch predicted between residues 226 and 248 in the C terminus of DrrB.
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ABCB1 p.Val53Cys 15709779:281:73
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.Val53Cys 16492138:98:192
status: NEW