ABCB1 p.Leu976Cys
| Predicted by SNAP2: | A: N (53%), C: N (53%), D: D (80%), E: D (80%), F: N (53%), G: D (75%), H: D (75%), I: N (82%), K: D (53%), M: N (82%), N: D (75%), P: D (85%), Q: D (53%), R: N (53%), S: D (66%), T: N (53%), V: N (82%), W: D (75%), Y: D (75%), |
| 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] Transmembrane helix 12 modulates progression of th... Biochemistry. 2009 Jul 7;48(26):6249-58. Crowley E, O'Mara ML, Reynolds C, Tieleman DP, Storm J, Kerr ID, Callaghan R
Transmembrane helix 12 modulates progression of the ATP catalytic cycle in ABCB1.
Biochemistry. 2009 Jul 7;48(26):6249-58., 2009-07-07 [PMID:19456124]
Abstract [show]
Multidrug efflux pumps, such as P-glycoprotein (ABCB1), present major barriers to the success of chemotherapy in a number of clinical settings. Molecular details of the multidrug efflux process by ABCB1 remain elusive, in particular, the interdomain communication associated with bioenergetic coupling. The present investigation has focused on the role of transmembrane helix 12 (TM12) in the multidrug efflux process of ABCB1. Cysteine residues were introduced at various positions within TM12, and their effect on ATPase activity, nucleotide binding, and drug interaction were assessed. Mutation of several residues within TM12 perturbed the maximal ATPase activity of ABCB1, and the underlying cause was a reduction in basal (i.e., drug-free) hydrolysis of the nucleotide. Two of the mutations (L976C and F978C) were found to reduce the binding of [gamma-(32)P]-azido-ATP to ABCB1. In contrast, the A980C mutation within TM12 enhanced the rate of ATP hydrolysis; once again, this was due to modified basal activity. Several residues also caused reductions in the potency of stimulation of ATP hydrolysis by nicardipine and vinblastine, although the effects were independent of changes in drug binding per se. Overall, the results indicate that TM12 plays a key role in the progression of the ATP hydrolytic cycle in ABCB1, even in the absence of the transported substrate.
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No. Sentence Comment
6 Two of the mutations (L976C and F978C) were found to reduce the binding of [γ-32 P]-azido-ATP to ABCB1.
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ABCB1 p.Leu976Cys 19456124:6:22
status: NEW67 This necessitated the centrifugation (100000g for 30 min) of 500 μL Table 1: Mutagenic Oligonucleotide Primers Used To Generate TM12 Mutationsa mutation primer sequence (50 -30 ) diagnostic restriction digest L976C GAGGATGTTCTAtgtGTATTTTCAGCTGTTG -SpeI F978C GTTCTACTAGTATgTTCtGCaGTTGTCTTTGGTG +PstI A980C CTACTAGTATTTTCAtgcGTTGTCTTTGGTGCCATGGCC -PvuII V982C CTAGTATTTTCAGCgGTTtgCTTTGGTGCCATGGCC -PvuII G984C GCTGTTGTCTTTtGTGCtATGGCCGTGG -NcoI M986C GTATTTGGTGCttgtGCtGTGGGGCAAGTC -NcoI V988C GGTGCCATGGCCtgtGGGCAAGTCAGTTC -BstXI G989C CTTTGGTGCCATGGCCGTGtGcCAAGTCAGTTCATTTGC +BstXI Q990C GGCCGTGGGGtgtGTCtcTTCATTTGCTCC +EarI a Primer sequences contain an introduced cysteine residue (bold) and additional silent mutations (lower case), with respect to the coding sequence that generates or removes the indicated restriction site.
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ABCB1 p.Leu976Cys 19456124:67:215
status: NEW118 The most striking alterations were the approximately 5-fold reductions in basal ATPase activity for the L976C (Vmax=26 ( 4 nmol min-1 mg-1 )and F978C (Vmax=27 ( 9 nmol min-1 mg-1 ) mutations compared to the control cysteine-less isoform of ABCB1.
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ABCB1 p.Leu976Cys 19456124:118:104
status: NEW141 L976C (Vmax = 231 ( 80 nmol min-1 mg-1 ), F978C (Vmax = 142 ( 40 nmol min-1 mg-1 ), V988C, G989C, and Q990C all caused statisticallysignificant (p<0.05) reductionsinthe Vmax valuesfor nicardipine-stimulated ATPase activities (Figure 4B).
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ABCB1 p.Leu976Cys 19456124:141:0
status: NEW149 For example, mutations L976C, F978C, V988C, and Q990C all displayed statistically significant reductions in maximal ATPase activity in the presence of vinblastine compared to the control cysteine-less isoform.
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ABCB1 p.Leu976Cys 19456124:149:23
status: NEW155 Table 2: Potency and Degree of Drug Stimulation of ATP Hydrolysis by ABCB1a nicardipine vinblastine EC50 (μM) fold stimulation EC50 (μM) fold stimulation Cys-less 4.1 ( 1.1 4.0 ( 0.6 5.91 ( 2.9 2.2 ( 0.2 L976C 5.2 ( 0.2 7.4 ( 1.4 10.0 ( 0.0 3.5 ( 0.6 F978C 24.1 ( 2.3b 9.5 ( 1.4 42.9 ( 4.3b 2.3 ( 0.5 A980C 3.4 ( 0.3 5.1 ( 0.9 12.3 ( 1.8 3.2 ( 0.8 V982C 5.8 ( 0.9 4.2 ( 0.5 2.0 ( 0.7 1.8 ( 0.2 G984C 37.6 ( 11.2b 16.2 ( 6.6b 6.7 ( 1.7 6.2 ( 2.3 M986C 9.2 ( 0.8 4.7 ( 1.1 15.0 ( 2.0b 2.8 ( 0.7 V988C 3.9 ( 0.6 3.1 ( 0.1 7.3 ( 2.3 1.9 ( 0.2 G989C 13.6 ( 1.5 5.1 ( 1.6 4.9 ( 0.9 2.4 ( 0.3 Q990C 6.9 ( 1.1 3.7 ( 1.0 NDc NDc S992C 4.9 ( 0.5 4.2 ( 0.6 7.1 ( 2.6 2.3 ( 0.4 F994C 1.7 ( 0.4 3.2 ( 0.8 5.9 ( 2.5 1.6 ( 0.3 a ATPase activity was plotted as a function of the drug concentration and potency (EC50) and degree of stimulation obtained by nonlinear regression of the dose-response relationship equation.
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ABCB1 p.Leu976Cys 19456124:155:216
status: NEW172 Figure 5A presents a representative autoradiogram of [γ-32 P]-azido-ATP binding to the mutants L976C, F978C, A980C, V988C, G989C, and Q990C.
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ABCB1 p.Leu976Cys 19456124:172:101
status: NEW174 The results indicate that at a concentration of 10 μM [γ-32 P]-azido-ATP there was a discernible difference between the binding of the ATP analogue to L976C, F978C, and A980C.
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ABCB1 p.Leu976Cys 19456124:174:163
status: NEW175 Mutant isoforms L976C and F978C displayed a 4and 15-fold decrease in binding, whereas A980C showed a 1.8-fold increase in binding at the concentration of [γ-32 P]-azido-ATP used.
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ABCB1 p.Leu976Cys 19456124:175:16
status: NEW178 Figure 5B shows the autoradiography data obtained for [γ-32 P]-azido-ATP photolabeling of the cysteine-less and L976C isoforms of ABCB1.
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ABCB1 p.Leu976Cys 19456124:178:118
status: NEW179 Figure 5C shows the densitometric analysis of the dose-response curve for the cysteine-less, A980C, L976C, and F978C isoforms.
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ABCB1 p.Leu976Cys 19456124:179:100
status: NEW181 In contrast, the mutant isoforms L976C (Bmax = 0.24, KD = 17 μM) and F978C (Bmax = 0.19, KD =17 μM) displayed 4-5-fold reductions in the amount of [γ-32 P]-azido-ATP binding compared to the cysteine-less control.
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ABCB1 p.Leu976Cys 19456124:181:33
status: NEW182 Therefore, the decrease in basal and drug-stimulated ATPase activity observed for the L976C and F978C isoforms can be explained at least in part by impaired ATP binding at the NBDs.
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ABCB1 p.Leu976Cys 19456124:182:86
status: NEW192 (B) Autoradiograms showing the binding of [γ-32 P]-azido-ATP (10-100 μM) to the purified, reconstituted cysteine-less and L976C mutant isoforms.
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ABCB1 p.Leu976Cys 19456124:192:134
status: NEW193 (C) Data obtained for the cysteine-less, L976C, F978C, and A980C isoforms were analyzed by densitometry, and the amount bound was plotted as a function of the [γ-32 P]-azido-ATP concentration.
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ABCB1 p.Leu976Cys 19456124:193:41
status: NEW220 The most dramatic effects in TM12 were observed in residues at the extracellular Table 3: Nucleotide Binding to ABCB1a ABCB1 isoform [32 P]-N3-ATP [32 P]-N3-ATP+ 1 mM ATP [32 P]-N3-ATP+ 1 mM ADP Cys-less 1.00 0.21 ( 0.05 0.23 ( 0.06 L976C 0.21 ( 0.05 0.10 ( 0.02 0.05 ( 0.03 F978C 0.07 ( 0.01 ND ND A980C 1.81 ( 0.71 0.45 ( 0.10 0.15 ( 0.08 V988C 0.53 ( 0.20 ND ND G989C 0.83 ( 0.04 0.10 ( 0.05 0.13 ( 0.06 Q990C 1.05 ( 0.30 0.19 ( 0.11 0.01 ( 0.01 a The ABCB1 isoforms were incubated with 10 μM [γ32 P]-azido-ATP in the presence or absence of excess unlabeled nucleotides (1 mM).
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ABCB1 p.Leu976Cys 19456124:220:233
status: NEW232 (L976C and F978C) and intracellular (V988C and Q990C) ends of the helix.
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ABCB1 p.Leu976Cys 19456124:232:1
status: NEW234 In contrast, the reduced basal activity observed with mutants L976C and F978C appeared to be a consequence of reduced nucleotide binding.
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ABCB1 p.Leu976Cys 19456124:234:62
status: NEW[hide] Transmembrane helix 12 plays a pivotal role in cou... FEBS J. 2010 Oct;277(19):3974-85. doi: 10.1111/j.1742-4658.2010.07789.x. Epub 2010 Aug 20. Crowley E, O'Mara ML, Kerr ID, Callaghan R
Transmembrane helix 12 plays a pivotal role in coupling energy provision and drug binding in ABCB1.
FEBS J. 2010 Oct;277(19):3974-85. doi: 10.1111/j.1742-4658.2010.07789.x. Epub 2010 Aug 20., [PMID:20731718]
Abstract [show]
Describing the molecular details of the multidrug efflux process of ABCB1, in particular the interdomain communication associated with bioenergetic coupling, continues to prove difficult. A number of investigations to date have implicated transmembrane helix 12 (TM12) in mediating communication between the transmembrane domains and nucleotide-binding domains (NBDs) of ABCB1. The present investigation further addressed the role of TM12 in ABCB1 by characterizing its topography during the multidrug efflux process with the use of cysteine-directed mutagenesis. Cysteines were introduced at various positions along TM12 and assessed for their ability to covalently bind thiol-reactive fluorescent probes with differing physiochemical properties. By analysing each isoform in the basal, ATP-bound and posthydrolytic states, it was possible to determine how the local environment of TM12 alters during the catalytic cycle. Labelling with hydrophobic CM and zwitterionic BM was extensive throughout the helix in the basal, prehydrolytic and posthydrolytic states, suggesting that TM12 is in a predominantly hydrophobic environment. Overall, the carboxy region (intracellular half) of TM12 appeared to be more responsive to changes in the catalytic state of the protein than the amino region (extracellular half). Thus, the carboxy region of TM12 is suggested to be responsive to nucleotide binding and hydrolysis at the NBDs and therefore directly involved in interdomain communication. This data can be reconciled with an atomic-scale model of human ABCB1. Taken together, these results indicate that TM12 plays a key role in the progression of the ATP hydrolytic cycle in ABCB1 and, in particular, in coordinating conformational changes between the NBDs and transmembrane domains.
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No. Sentence Comment
63 The lowest labelling observed in the selection of TM12 mutant isoforms was at L976C, with an Lext of 38 ± 5%.
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ABCB1 p.Leu976Cys 20731718:63:78
status: NEW82 The rate of labelling (i.e. t1 / 2) was divided into fast (L986C- G992C, average t1 / 2 $ 8 min) and slow (L976C- G984C, average t1 / 2 $ 25 min) kinetics between the carboxy-half and the amino-half, respectively.
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ABCB1 p.Leu976Cys 20731718:82:107
status: NEW118 Conformational changes - amino region of TM12 As shown in Table 2, the amino region of TM12 (L976C-V982C) was not associated with large alterations in topography.
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ABCB1 p.Leu976Cys 20731718:118:93
status: NEW121 For example, L976C became less accessible to BM, but more accessible to CM, following a shift from the basal to the nucleotide-bound conformation.
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ABCB1 p.Leu976Cys 20731718:121:13
status: NEW139 Mutant CM BM FM Lext (%) t1 / 2 (min) Lext (%) t1 / 2 (min) Lext (%) t1 / 2 (min) L976C 38 ± 5 29 ± 12 66 ± 14 29 ± 18 - - A980C 53 ± 6 34 ± 1 54 ± 8 20 ± 9 - - V982C 98 ± 14 15 ± 6 164 ± 50 27 ± 17 - - G984C 73 ± 14 29 ± 6 84 ± 24 22 ± 7 13 ± 10 ND M986C 89 ± 30 25 ± 10 51 ± 5 3 ± 2 21 ± 2 ND V988C 53 ± 6 37 ± 18 221 ± 63 18 ± 12 - - G989C 64 ± 7 15 ± 6 21 ± 3 9 ± 2 - - S992C 55 ± 4 22 ± 6 51 ± 5 4 ± 1 32 ± 3 25 ± 5 F994C 51 ± 10 11 ± 9 111 ± 35 13 ± 10 129 ± 24 8 ± 3 Conformational changes - central region Two of the residues examined in the central region (G984C and M986C) of TM12 have been shown to accommodate partial labelling with FM, suggestive of aqueous accessibility in the basal state. At M986C, the extent of labelling with the hydrophilic probe was increased following the addition of nonhydrolysable nucleotide.
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ABCB1 p.Leu976Cys 20731718:139:82
status: NEW164 ABCB1 isoform Catalytic intermediate CM BM FM L976C Basal ++ +++ ) AMP-PNP +++ ++ ) Vi trapped +++ +++ ) A980C Basal ++ ++ ) AMP-PNP +++ + ) Vi trapped +++ +++ ) V982C Basal +++ +++ ) AMP-PNP +++ +++ ) Vi trapped +++ +++ ) G984C Basal +++ +++ + AMP-PNP +++ +++ + Vi trapped +++ ++ ) M986C Basal +++ ++ + AMP-PNP ++ +++ ++ Vi trapped +++ ++ ) V988C Basal ++ +++ ) AMP-PNP +++ +++ ) Vi trapped +++ +++ ) G989C Basal ++ + ) AMP-PNP ++ ++ ) Vi trapped ++ + ) S992C Basal ++ ++ + AMP-PNP +++ +++ ++ Vi trapped ++ ++ + F994C Basal ++ +++ +++ AMP-PNP ++ +++ ++ Vi trapped +++ +++ + reflect localization at the membrane-solute interface.
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ABCB1 p.Leu976Cys 20731718:164:46
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
80 We also tested mutants F335C/L976C, L339C/S979C, F343C/F983C, G347C/A987C, and S351C/ V991C for cross-linking since they were predicted to lie on opposing faces of TM6 and TM12 modeled in a right-handed coiled-coil.
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ABCB1 p.Leu976Cys 9261097:80:29
status: NEW