ABCMdb

ABCB1 p.Val988Cys

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Publications

PMID: 19456124 [PubMed] Crowley E et al: "Transmembrane helix 12 modulates progression of the ATP catalytic cycle in ABCB1."

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. Login to comment
119 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. Login to comment
141 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). Login to comment
149 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. Login to comment
155 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. Login to comment
172 Figure 5A presents a representative autoradiogram of [γ-32 P]-azido-ATP binding to the mutants L976C, F978C, A980C, V988C, G989C, and Q990C. Login to comment
176 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). Login to comment
187 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. Login to comment
212 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. Login to comment
220 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). Login to comment
232 (L976C and F978C) and intracellular (V988C and Q990C) ends of the helix. Login to comment
233 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. Login to comment

PMID: 20731718 [PubMed] Crowley E et al: "Transmembrane helix 12 plays a pivotal role in coupling energy provision and drug binding in ABCB1."

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). Login to comment
62 The C-terminal stretch (V988C-F994C) was also capable of interacting with CM, albeit with lower values of Lext, in the range 50-60%. Login to comment
67 Detection of CM labelling of the V988C isoform. Login to comment
68 SDS / PAGE analysis of the V988C isoform incubated in the presence of CM for 0-300 min. Login to comment
79 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. Login to comment
139 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. Login to comment
144 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. Login to comment
145 Labelling of V988C and G989C with the hydrophilic FM was negligible, regardless of the conformational state. Login to comment
151 The labelling properties of V988C-G989C suggest that this region of TM12 undergoes minimal conformational transition. Login to comment
164 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. Login to comment