ABCB1 p.Val988Cys
| Predicted by SNAP2: | A: N (57%), C: D (53%), D: D (91%), E: D (85%), F: D (75%), G: D (85%), H: D (85%), I: N (78%), K: D (91%), L: N (82%), M: N (66%), N: D (85%), P: D (91%), Q: D (85%), R: D (91%), S: D (80%), T: D (75%), W: D (91%), Y: D (85%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: N, K: D, L: N, M: N, N: D, P: D, Q: D, R: D, S: D, T: 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
67 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.Val988Cys 19456124:67:493
status: NEW119 There were also less dramatic (40%) reductions in the basal activities of the V988C (Vmax = 66 ( 4 nmol min-1 mg-1 ) and Q990C (Vmax=61 ( 12 nmol min-1 mg-1 ) mutations.
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ABCB1 p.Val988Cys 19456124:119:78
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.Val988Cys 19456124:141:84
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.Val988Cys 19456124:149:37
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.Val988Cys 19456124:155:505
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.Val988Cys 19456124:172:122
status: NEW176 In contrast, the V988C, G989C, and Q990C isoforms did not show a statistically significant reduction in the degree of [γ-32 P]-azido-ATP photolabeling, despite their reduced ATPase activity (see above).
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ABCB1 p.Val988Cys 19456124:176:17
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.Val988Cys 19456124:187:243
status: NEW212 In the model, V988 is located on the lipid-accessible surface of the protein in a cleft between TM9 and TM10, where it forms a trimeric β-branched stabilization point with I868 (TM10) and M949 (TM11).MutationtoV988C disruptsthe hydrophobic interaction with I868 from TM9, and the V988C side chain is oriented toward the TM10 residue M949.
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ABCB1 p.Val988Cys 19456124:212:286
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.Val988Cys 19456124:220:341
status: NEW232 (L976C and F978C) and intracellular (V988C and Q990C) ends of the helix.
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ABCB1 p.Val988Cys 19456124:232:37
status: NEW233 The V988C and Q990C mutations reduced basal ATPase activity without any significant change in nucleotide binding per se. This implies that the altered helical properties caused by site-directed mutagenesis modulated the conformational communication route between the transmembrane and nucleotide binding domains.
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ABCB1 p.Val988Cys 19456124:233:4
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
45 Figure 1 (lower panel) shows a representative labelling reaction, in this case a time course for the V988C isoform with coumarin maleimide (CM).
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ABCB1 p.Val988Cys 20731718:45:101
status: NEW62 The C-terminal stretch (V988C-F994C) was also capable of interacting with CM, albeit with lower values of Lext, in the range 50-60%.
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ABCB1 p.Val988Cys 20731718:62:24
status: NEW67 Detection of CM labelling of the V988C isoform.
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ABCB1 p.Val988Cys 20731718:67:33
status: NEW68 SDS / PAGE analysis of the V988C isoform incubated in the presence of CM for 0-300 min.
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ABCB1 p.Val988Cys 20731718:68:27
status: NEW79 A similar stretch of TM12 (namely V982C-V988C) displayed the greatest propensity to be labelled with BM, with only isoform M986C being not completely labelled by the probe.
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ABCB1 p.Val988Cys 20731718:79:40
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.Val988Cys 20731718:139:405
status: NEW144 Conformational changes - proximal to the central region The region immediately proximal to the centre of TM12 (V988C-G989C) showed avid labelling by both of the lipophilic probes (BM and CM) in the basal configurations, and there were no significant alterations in accessibility upon progression of the catalytic cycle.
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ABCB1 p.Val988Cys 20731718:144:111
status: NEW145 Labelling of V988C and G989C with the hydrophilic FM was negligible, regardless of the conformational state.
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ABCB1 p.Val988Cys 20731718:145:13
status: NEW151 The labelling properties of V988C-G989C suggest that this region of TM12 undergoes minimal conformational transition.
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ABCB1 p.Val988Cys 20731718:151:28
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.Val988Cys 20731718:164:342
status: NEW