ABCB1 p.Ala980Cys
| Predicted by SNAP2: | C: N (53%), D: D (80%), E: D (75%), F: D (85%), G: D (71%), H: D (80%), I: D (75%), K: D (80%), L: D (75%), M: D (71%), N: D (75%), P: D (85%), Q: D (75%), R: D (80%), S: N (66%), T: D (63%), V: D (59%), W: D (85%), Y: D (85%), |
| Predicted by PROVEAN: | C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: N, T: D, V: D, 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
7 In contrast, the A980C mutation within TM12 enhanced the rate of ATP hydrolysis; once again, this was due to modified basal activity.
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ABCB1 p.Ala980Cys 19456124:7:17
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.Ala980Cys 19456124:67:306
status: NEW121 In complete contrast, the A980C mutation in TM12 caused an increase in the basal ATPase activity (Vmax=203 ( 40 nmol min-1 mg-1 ) compared to the cysteine-less isoform.
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ABCB1 p.Ala980Cys 19456124:121:26
status: NEW142 Again, in contrast, the nicardipine-stimulated activity of the A980C isoform was almost 3-fold higher (Vmax =1352 ( 50 nmol min-1 mg-1 ) than observed for the cysteine-less control.
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ABCB1 p.Ala980Cys 19456124:142:63
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.Ala980Cys 19456124:155:313
status: NEW164 Excluding the A980C isoform, which displayed enhanced ATPase activity, the effects were primarily a reduction of ATP hydrolysis.
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ABCB1 p.Ala980Cys 19456124:164:14
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.Ala980Cys 19456124:172:115
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.Ala980Cys 19456124:174:181
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.Ala980Cys 19456124:175:86
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.Ala980Cys 19456124:179:93
status: NEW180 At the range of [γ-32 P]-azido-ATP concentrations studied, there was no discernible difference between the labeling of cysteine-less (Bmax = 1.00, KD = 25 μM) and A980C (Bmax = 1.21, KD = 23 μM) isoforms of ABCB1.
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ABCB1 p.Ala980Cys 19456124:180:175
status: NEW183 However, mutant A980C, which was characterized by a significantly higher ATPase activity, did not show any appreciable changes in nucleotide binding.
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ABCB1 p.Ala980Cys 19456124:183:16
status: NEW187 To investigate whether such differences are a reflection of a physical asymmetry between the helices and their surroundings, we attempted to rationalize three of the functionally perturbed TM12 single cysteine mutants (i.e., F978C, A980C, and V988C) byreference toamolecularmodel ofABCB1.
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ABCB1 p.Ala980Cys 19456124:187:232
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.Ala980Cys 19456124:193:59
status: NEW199 Of the six mutations, only A980C resulted in increased ATPase activity.
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ABCB1 p.Ala980Cys 19456124:199:27
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.Ala980Cys 19456124:220:299
status: NEW229 (B) Mutation to A980C allows for hydrogen bonding to S850 (orange), increasing TM12-TM9 contacts.
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ABCB1 p.Ala980Cys 19456124:229:16
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
123 A980C shifted to a low level of BM accessibility following nucleotide binding by ABCB1, and again, an opposite shift was seen for CM.
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ABCB1 p.Ala980Cys 20731718:123:0
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.Ala980Cys 20731718:139:143
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.Ala980Cys 20731718:164:105
status: NEW