ABCB1 p.Leu332Cys
| Predicted by SNAP2: | A: D (59%), C: N (53%), D: D (85%), E: D (80%), F: N (53%), G: D (80%), H: D (80%), I: N (82%), K: D (85%), M: N (87%), N: D (75%), P: D (85%), Q: D (71%), R: D (80%), S: D (71%), T: D (63%), V: N (57%), W: D (66%), Y: D (71%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: N, K: D, M: N, N: D, P: D, Q: D, R: D, S: D, T: D, V: N, W: D, Y: D, |
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[hide] The human multidrug resistance P-glycoprotein is i... FASEB J. 1999 Oct;13(13):1724-32. Loo TW, Clarke DM
The human multidrug resistance P-glycoprotein is inactive when its maturation is inhibited: potential for a role in cancer chemotherapy.
FASEB J. 1999 Oct;13(13):1724-32., [PMID:10506575]
Abstract [show]
The human multidrug resistance P-glycoprotein (P-gp) contributes to the phenomenon of multidrug resistance during cancer and AIDS chemotherapy. A potential novel strategy to circumvent the effects of P-gp during chemotherapy is to prevent maturation of P-gp during biosynthesis so that the transporter does not reach the cell surface. Here we report that immature, core-glycosylated P-gp that is prevented from reaching the cell surface by processing mutations or by proteasome inhibitors such as lactacystin or MG-132 exhibited no detectable drug-stimulated ATPase activity. Disulfide cross-linking analysis also showed that the immature P-gp did not exhibit ATP-induced conformational changes as found in the mature enzyme. In addition, the immature P-gp was more sensitive to trypsin than the mature enzyme. These results suggest that P-gp is unlikely to be functional immediately after synthesis. These differences in the structural and enzymatic properties of the mature and core-glycosylated, immature P-gp could potentially be used during chemotherapy, and should result in the search for compounds that can specifically inhibit the maturation of P-gp.
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No. Sentence Comment
42 The Cys-less mutant was also modified to contain mutations L332C in TM6 and L975C in TM12 (27).
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ABCB1 p.Leu332Cys 10506575:42:59
status: NEW120 Cys-less P-gp containing the mutations L332C and L975C was expressed with or without MG-132 and cross-linked in the presence or absence of Mg⅐ATP.
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ABCB1 p.Leu332Cys 10506575:120:39
status: NEW121 Figure 5 shows that ATP induces cross-linking in mutant L332C/L975C when expressed in the absence of MG-132.
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ABCB1 p.Leu332Cys 10506575:121:56
status: NEW123 In the presence of MG-132, however, the 150 kDa core-glycosylated mutant L332C/L975C was not cross-linked by oxidant in the absence or presence of ATP (Fig. 5, lanes 6 and 8).
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ABCB1 p.Leu332Cys 10506575:123:73
status: NEW139 HEK 293 cells were transfected with Cys-less P-gp cDNA containing mutations L332C/L975C and then incubated with (ϩMG-132) or without (-MG-132) MG-132 for 24 h. Membranes were then prepared and treated with (ϩ) or without (-) copper phenanthrolene (CuPhen) for 5 min at 37°C in the presence (ϩATP) or absence (-ATP) of 10 mM ATP.
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ABCB1 p.Leu332Cys 10506575:139:76
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
81 It has been shown that TM12 (L975C) approaches TM6 (L332C) on the extracellular side during ATP hydrolysis (27).
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ABCB1 p.Leu332Cys 16042402:81:52
status: NEW171 (B) Membranes prepared from HEK 293 cells expressing mutant M69C(TM1)/A954C(TM11) or mutant L332C- (TM6)/L975C(TM12) were preincubated with 10 mM colchicine (Colch), 0.05 mM cyclosporin A (Cyclo), 1 mM verapamil (Ver), 0.1 mM vinblastine (Vin), or no drug substrate (None) for 15 min at 20 °C. The samples were then incubated with buffer containing 5 mM ATP and with (+) or without (-) oxidant for 10 min at 37 °C. The reactions were stopped by addition of SDS sample buffer containing EDTA and subjected to immunoblot analysis.
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ABCB1 p.Leu332Cys 16042402:171:92
status: NEW[hide] Nucleotide binding, ATP hydrolysis, and mutation o... Biochemistry. 2007 Aug 14;46(32):9328-36. Epub 2007 Jul 18. Loo TW, Bartlett MC, Clarke DM
Nucleotide binding, ATP hydrolysis, and mutation of the catalytic carboxylates of human P-glycoprotein cause distinct conformational changes in the transmembrane segments.
Biochemistry. 2007 Aug 14;46(32):9328-36. Epub 2007 Jul 18., 2007-08-14 [PMID:17636884]
Abstract [show]
P-Glycoprotein (P-gp, ABCB1) transports a variety of structurally unrelated cytotoxic compounds out of the cell. Each homologous half of P-gp has a transmembrane (TM) domain containing six TM segments and a nucleotide-binding domain (NBD) and is joined by a linker region. It has been postulated that binding of two ATP molecules at the NBD interface to form a "nucleotide sandwich" induces drug efflux by altering packing of the TM segments that make up the drug-binding pocket. To test if ATP binding alone could alter packing of the TM segments, we introduced catalytic carboxylate mutations (E556Q in NBD1 and E1201Q in NBD2) into double-cysteine mutants that exhibited ATP-dependent cross-linking so that the mutants could bind but not hydrolyze ATP. It was found that ATP binding alone could alter disulfide cross-linking between the TM segments. For example, ATP inhibited cross-linking of mutant L339C(TM6)/V982C(TM12)/E556Q(NBD1)/E1201Q(NBD2) but promoted cross-linking of mutant F343C(TM6)/V982C(TM12)/E556Q(NBD1)/E1201Q(NBD2). Cross-linking of some mutants, however, appeared to require ATP hydrolysis as introduction of the catalytic carboxylate mutations into mutant L332C(TM6)/L975C(TM12) inhibited ATP-dependent cross-linking. Cross-linking between cysteines in the TM segments also could be altered via introduction of a single catalytic carboxylate mutation into mutant L332C(TM6)/L975C(TM12) or by using the nonhydrolyzable ATP analogue, AMP.PNP. The results show that the TM segments are quite sensitive to changes within the ATP-binding sites because different conformations could be detected in the presence of ATP, AMP.PNP, during ATP hydrolysis or through mutation of the catalytic carboxylates.
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No. Sentence Comment
39 Construction of mutants containing pairs of cysteines that exhibited ATP-sensitive cross-linking in the presence of copper phenanthroline [L332C- (TM6)/L975C(TM12)] (33), 3,6,9,12-tetraoxatetradecane-1,14-diyl bismethanethiosulfonate (M14M) [L339C(TM6)/ F728C(TM7)] (34), or tris(2-maleimidoethyl)amine (TMEA) [L339C(TM6)/V982C(TM12) and F343C(TM6)/V982C- (TM12)] (31) were described previously.
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ABCB1 p.Leu332Cys 17636884:39:139
status: NEW74 The positions of the catalytic carboxylate mutations (E556Q in NBD1 and E1201Q in NBD2) and the cysteine mutations in the TM segments used in the disulfide cross-linking studies (L332C, L339C, F343C, F728C, L975C, and V982C) are shown.
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ABCB1 p.Leu332Cys 17636884:74:179
status: NEW89 Mutant L332C(TM6)/L975C(TM12) contains one cysteine residue at the extracellular end of TM6 (L332C) and another at the extracellular end of TM12 (L975C) (Figure 1).
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ABCB1 p.Leu332Cys 17636884:89:93
status: NEW125 Membranes prepared from HEK 293 cells expressing mutants L332C(TM6)/L975C(TM12), L332C- (TM6)/L975C(TM12)/E556Q(NBD1)/E1201Q(NBD2), L332C- (TM6)/L975C(TM12)/E556Q(NBD1), or L332C(TM6)/L975C- (TM12)/E1201Q(NBD2) were treated with (+) or without (-) 1 mM copper phenanthroline (CuP) for 10 min at 37 °C in the absence (None) or presence of 5 mM ATP (ATP).
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ABCB1 p.Leu332Cys 17636884:125:81
status: NEWX
ABCB1 p.Leu332Cys 17636884:125:132
status: NEW[hide] The structure and functions of P-glycoprotein. Curr Med Chem. 2010;17(8):786-800. Li Y, Yuan H, Yang K, Xu W, Tang W, Li X
The structure and functions of P-glycoprotein.
Curr Med Chem. 2010;17(8):786-800., [PMID:20088754]
Abstract [show]
P-glycoprotein (P-gp) is an ATP-driven transmembrane transporter capable of effluxing a wide variety of structurally diverse and functionally unrelated hydrophobic compounds out of the cell. Multidrug resistance (MDR), often associated with the over-expression of P-gp, has been implicated as a major obstacle to effective chemotherapy for cancer, parasitic diseases, AIDS, and other diseases. Drug efflux mediated by P-gp is also involved in decreasing the oral bioavailability of drugs by limiting intestinal absorption. Our appreciation of the structural and functional aspects of P-gp has definitely improved in recent years, benefiting from the deciphering of the structure of some bacterial transporters that paved the way for construction of homology models for more complex transporters. Here, we will review the recent advances in the studies of the structure and functional characteristics of P-gp with the hopes of facilitating rational drug design in developing novel potent MDR modulators.
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No. Sentence Comment
165 In 2002, Loo & Clarke found that with hydrolysis of the first molecule of ATP there is a further conformational change such as an -helix rotation between TM6 and TM12, which can be detected by disulfide cross-linking between cysteines (residue L332C in TM6 and L975C in TM12) [66].
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ABCB1 p.Leu332Cys 20088754:165:244
status: NEW[hide] Inhibition of oxidative cross-linking between engi... J Biol Chem. 1996 Nov 1;271(44):27482-7. Loo TW, Clarke DM
Inhibition of oxidative cross-linking between engineered cysteine residues at positions 332 in predicted transmembrane segments (TM) 6 and 975 in predicted TM12 of human P-glycoprotein by drug substrates.
J Biol Chem. 1996 Nov 1;271(44):27482-7., 1996-11-01 [PMID:8910331]
Abstract [show]
Each homologous half of P-glycoprotein consists of a transmembrane domain with six potential transmembrane segments and an ATP-binding domain. Labeling studies with photoactive drug analogs show that labeling occurs within or close to predicted transmembrane segments (TM) 6 (residues 331-351) and TM12 (residues 974-994). To test if these segments are in near-proximity we generated 42 different P-glycoprotein mutants in which we re-introduced a pair of cysteine residues into a Cys-less P-glycoprotein, one within TM6 (residues 332-338) and one within TM12 (residues 975-980) and assayed for cross-linking between the cysteines. All the mutants retained verapamil-stimulated ATPase activity. We found that only the mutant containing Cys-332 and Cys-975 was cross-linked in the presence of oxidant as judged by its decreased mobility on SDS gels. Similar results were obtained when the same mutations were introduced into Cys-less NH2-terminal and COOH-terminal half-molecules of P-glycoprotein followed by coexpression and treatment with oxidant. Cross-linking between Cys-332 and Cys-975, however, was inhibited by verapamil or vinblastine but not by colchicine. These results suggest that residues Cys-332 and Cys-975, which occupy equivalent positions when TM6 and TM12 are aligned, are close to each other in the tertiary structure of P-glycoprotein.
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No. Sentence Comment
116 B, the above experiment was repeated with Cys-less P-glycoprotein(His)10, mutants containing only one cysteine (L332C or L975C), or a mutant containing both cysteines (L332C ϩ L975C).
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ABCB1 p.Leu332Cys 8910331:116:112
status: NEWX
ABCB1 p.Leu332Cys 8910331:116:168
status: NEW117 C, mutant L332C ϩ L975C was treated with (lane 2) or without (lane 1) copper phenanthroline for 15 min at 37 °C and then solubilized with SDS sample buffer.
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ABCB1 p.Leu332Cys 8910331:117:10
status: NEW151 To test for this possibility, the cDNAs coding for Cys-less P-glycoprotein and mutant L332C ϩ L975C were transfected into NIH 3T3 cells, and drug-resistant colonies were selected in the presence of 45 nM colchicine or 5 nM vinblastine.
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ABCB1 p.Leu332Cys 8910331:151:86
status: NEW156 A, membranes prepared from cells transfected with vector alone (control) or cotransfected with cDNA for mutant L332C in the Cys-less NH2-terminal half-molecule A52 and the cDNA for mutant L975C in the Cys-less COOH-terminal half-molecule A52 or with cDNA for mutant F335C in the NH2-terminal half-molecule A52 and the cDNA for mutant F978C in the COOH-terminal half-molecule A52 were treated with oxidant for various intervals and at different temperatures.
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ABCB1 p.Leu332Cys 8910331:156:111
status: NEW158 B, HEK 293 cells were co-transfected with cDNAs encoding Cys-less NH2-and COOH-terminal half-molecules A52; the cDNAs of mutant L332C in the NH2-terminal half-molecule A52 and the Cys-less COOH-terminal half-molecule A52; the cDNAs of Cys-less NH2-terminal half-molecule-A52 and mutant L975C in the Cys-less COOH-terminal half-molecule A52 or with the cDNAs of the mutant L335C in the NH2-terminal half-molecule A52 and mutant L975C in the COOH-terminal half-molecule A52.
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ABCB1 p.Leu332Cys 8910331:158:128
status: NEW167 As shown in Fig. 5, the ATPase activities of both mutants (Cys-less and L332C ϩ L975C) were similar in the presence of 5 mM colchicine, 1 mM verapamil, and 100 M vinblastine.
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ABCB1 p.Leu332Cys 8910331:167:72
status: NEW168 Compared with the wild-type enzyme, both Cys-less and L332C ϩ L975C mutants had lower drug-stimulated ATPase activities.
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ABCB1 p.Leu332Cys 8910331:168:54
status: NEW[hide] Drug-stimulated ATPase activity of human P-glycopr... J Biol Chem. 1997 Aug 22;272(34):20986-9. Loo TW, Clarke DM
Drug-stimulated ATPase activity of human P-glycoprotein requires movement between transmembrane segments 6 and 12.
J Biol Chem. 1997 Aug 22;272(34):20986-9., 1997-08-22 [PMID:9261097]
Abstract [show]
Transmembrane segments (TM) 6 and 12 are directly connected to the ATP-binding domain in each homologous half of P-glycoprotein and are postulated to be important for drug-protein interactions. Cysteines introduced into TM6 (L332C, F343C, G346C, and P350C) were oxidatively cross-linked to cysteines introduced into TM12 (L975C, M986C, G989C, and S993C, respectively). The pattern of cross-linking was consistent with a left-handed coiled coil arrangement of the two helices. To detect conformational changes between the helices during drug-stimulated ATPase activity, we tested the effects of substrates and ATP on cross-linking. Cyclosporin A, verapamil, vinblastine, and colchicine inhibited cross-linking of mutants F343C/M986C, G346C/G989C, and P350C/S993C. By contrast, ATP promoted cross-linking between only L332C/L975C. Enhanced cross-linking between L332C/L975C was due to ATP hydrolysis, since cross-linked product was not observed in the presence of ATP and vanadate, ADP, ADP and vanadate, or AMP-PNP. Cross-linking between P350C/S993C inhibited verapamil-stimulated ATPase activity by about 75%. Drug-stimulated ATPase activity, however, was fully restored in the presence of dithiothreitol. These results show that TM6 and TM12 undergo different conformational changes upon drug binding or during ATP hydrolysis, and that movement between these two helices is essential for drug-stimulated ATPase activity.
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No. Sentence Comment
82 Effect of Nucleotides and Drug Substrates on Cross-linking-An interesting observation was that the amount of cross-linking seen in mutant L332C/L975C in whole cells (9) varied with the metabolic state of the transfected cells.
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ABCB1 p.Leu332Cys 9261097:82:138
status: NEW84 A possible explanation was that cross-linking of mutant L332C/L975C was promoted by the presence of ATP.
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ABCB1 p.Leu332Cys 9261097:84:56
status: NEW96 pressing mutant L332C/L975C were cross-linked in the presence of nucleotides.
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ABCB1 p.Leu332Cys 9261097:96:16
status: NEW99 These results suggest that cross-linking between L332C and L975C occurred during ATP hydrolysis.
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ABCB1 p.Leu332Cys 9261097:99:49
status: NEW103 To test the effect of drug substrates, cross-linking of mutants L332C/L975C, F343C/M986C, G346C/G989C, and P350C/ S993C was done in the presence of verapamil, cyclosporin A, vinblastine, or colchicine.
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ABCB1 p.Leu332Cys 9261097:103:64
status: NEW104 No cross-linked product was observed for mutant L332C/L975C (Fig. 3A).
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ABCB1 p.Leu332Cys 9261097:104:48
status: NEW108 Effect of Cross-linking on Drug-stimulated ATPase Activity- Mutants L332C/L975C, F343C/M986C, G346C/G989C, and P350C/S993C were still active since they retained about 90, 30, 10, and 70%, respectively, of the verapamil-stimulated ATPase activity of the Cys-less P-glycoprotein.
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ABCB1 p.Leu332Cys 9261097:108:68
status: NEW109 Cross-linking of mutants F343C/M986C, G346C/G989C, and P350C/S993C, but not L332C/L975C, was reversed by treatment with dithiothreitol (Fig. 4A).
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ABCB1 p.Leu332Cys 9261097:109:76
status: NEW126 Membranes prepared from HEK 293 cells expressing mutants L332C/L975C (A), F343C/M986C (B), G346C/G989C (C), and P350/S993C (D) were treated without (-) or with (ϩ) 2 mM (A) or 0.2 mM (B-D) copper phenanthroline for 10 min at 37 °C in the presence of 5 mM ATP, 5 mM ATP plus 0.2 mM sodium vanadate, 5 mM ADP, 5 mM ADP plus 0.2 mM sodium vanadate, or 5 mM AMP-PNP.
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ABCB1 p.Leu332Cys 9261097:126:57
status: NEW132 Membranes prepared from HEK 293 cells expressing mutants L332C/ L975C (A), F343C/M986C (B), G346C/G989C (C), and P350/S993C (D) were treated without (-) or with (ϩ) 2 mM (A) or 0.2 mM (B-D) copper phenanthroline for 10 min at 37 °C in the presence of 1 mM verapamil, 0.1 mM vinblastine, 50 M cyclosporin A, or 5 mM colchicine.
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ABCB1 p.Leu332Cys 9261097:132:57
status: NEW140 ATP hydrolysis rather than nucleotide binding was responsible for cross-linking between L332C/ L975C.
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ABCB1 p.Leu332Cys 9261097:140:88
status: NEW142 The observation that ATP hydrolysis promoted cross-linking between L332C/L975C suggests that inhibition of cross-linking of L332C/L975C in whole cells by verapamil or vinblastine occurred indirectly through depletion of intracellular ATP.
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ABCB1 p.Leu332Cys 9261097:142:67
status: NEWX
ABCB1 p.Leu332Cys 9261097:142:124
status: NEW[hide] A new structural model for P-glycoprotein. J Membr Biol. 1998 Nov 15;166(2):133-47. Jones PM, George AM
A new structural model for P-glycoprotein.
J Membr Biol. 1998 Nov 15;166(2):133-47., 1998-11-15 [PMID:9841738]
Abstract [show]
Multidrug resistance to anti-cancer drugs is a major medical problem. Resistance is manifested largely by the product of the human MDR1 gene, P-glycoprotein, an ABC transporter that is an integral membrane protein of 1280 amino acids arranged into two homologous halves, each comprising 6 putative transmembrane alpha-helices and an ATP binding domain. Despite the plethora of data from site-directed, scanning and domain replacement mutagenesis, epitope mapping and photoaffinity labeling, a clear structural model for P-glycoprotein remains largely elusive. In this report, we propose a new model for P-glycoprotein that is supported by the vast body of previous data. The model comprises 2 membrane-embedded 16-strand beta-barrels, attached by short loops to two 6-helix bundles beneath each barrel. Each ATP binding domain contributes 2 beta-strands and 1 alpha-helix to the structure. This model, together with an analysis of the amino acid sequence alignment of P-glycoprotein isoforms, is used to delineate drug binding and translocation sites. We show that the locations of these sites are consistent with mutational, kinetic and labeling data.
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No. Sentence Comment
204 Four cross-linked pairs, namely L332C/L975C, F343C/M986C, G346C/G989C and P350C/S993C, were generated in separate mutant molecules.
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ABCB1 p.Leu332Cys 9841738:204:32
status: NEW209 The cross-linking of the first pair (L332C/L975C) required the presence of ATP, was unaffected by drug substrates, and could not be reversed by treatment with dithiothreitol.
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ABCB1 p.Leu332Cys 9841738:209:37
status: NEW[hide] Equilibrated atomic models of outward-facing P-gly... Sci Rep. 2015 Jan 20;5:7880. doi: 10.1038/srep07880. Pan L, Aller SG
Equilibrated atomic models of outward-facing P-glycoprotein and effect of ATP binding on structural dynamics.
Sci Rep. 2015 Jan 20;5:7880. doi: 10.1038/srep07880., [PMID:25600711]
Abstract [show]
P-glycoprotein (Pgp) is an ATP-binding cassette (ABC) transporter that alternates between inward- and outward-facing conformations to capture and force substrates out of cells like a peristaltic pump. The high degree of similarity in outward-facing structures across evolution of ABC transporters allowed construction of a high-confidence outward-facing Pgp atomic model based on crystal structures of outward-facing Sav1866 and inward-facing Pgp. The model adhered to previous experimentally determined secondary- and tertiary- configurations during all-atom molecular dynamics simulations in the presence or absence of MgATP. Three long lasting (>100 ns) meta-stable states were apparent in the presence of MgATP revealing new insights into alternating access. The two ATP-binding pockets are highly asymmetric resulting in differential control of overall structural dynamics and allosteric regulation of the drug-binding pocket. Equilibrated Pgp has a considerably different electrostatic profile compared to Sav1866 that implicates significant kinetic and thermodynamic differences in transport mechanisms.
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No. Sentence Comment
196 Specifically, ATP binding inhibited the crosslink of pairs of human Pgp between TM6 and TM12 at L339C-V982C (mouse L334-V978) and L332C-L975C (mouse L328-L971) but promoted the crosslink of F343C-V982C (mouse F339-V978).
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ABCB1 p.Leu332Cys 25600711:196:130
status: NEW