ABCMdb

PMID: 19456124

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 [PubMed]

Sentences

No. Mutations Sentence Comment

6 ABCB1 p.Phe978Cys ABCB1 p.Leu976Cys Two of the mutations (L976C and F978C) were found to reduce the binding of [γ-32 P]-azido-ATP to ABCB1. Login to comment 7 ABCB1 p.Ala980Cys In contrast, the A980C mutation within TM12 enhanced the rate of ATP hydrolysis; once again, this was due to modified basal activity. Login to comment 67 ABCB1 p.Gly989Cys ABCB1 p.Val982Cys ABCB1 p.Gly984Cys ABCB1 p.Phe978Cys ABCB1 p.Leu976Cys ABCB1 p.Ala980Cys ABCB1 p.Met986Cys ABCB1 p.Gln990Cys ABCB1 p.Val988Cys 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 108 ABCB1 p.Met986Cys Panels B and C of Figure 2 show a time course of proteolytic digestion of the purified, reconstituted Cys-less and M986C proteins. Login to comment 118 ABCB1 p.Phe978Cys ABCB1 p.Leu976Cys 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. Login to comment 119 ABCB1 p.Gln990Cys ABCB1 p.Val988Cys 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 121 ABCB1 p.Ala980Cys 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. Login to comment 127 ABCB1 p.Met986Cys Proteolysis of purified, reconstituted Cys-less (B) and M986C (C) was carried out in the presence of 0.5 μg of bovine pancreatic trypsin. Login to comment 141 ABCB1 p.Gly989Cys ABCB1 p.Phe978Cys ABCB1 p.Leu976Cys ABCB1 p.Gln990Cys ABCB1 p.Val988Cys 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 142 ABCB1 p.Ala980Cys 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. Login to comment 146 ABCB1 p.Phe978Cys However, the F978C isoform did display an altered nicardipine-ABCB1 interaction; that is, the potency of nicardipine was reduced from 4.1 ( 1.1 to 24.1 ( 2.3 μM. Vinblastine also stimulates the ATPase activity of ABCB1. Login to comment 149 ABCB1 p.Phe978Cys ABCB1 p.Leu976Cys ABCB1 p.Gln990Cys ABCB1 p.Val988Cys 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 150 ABCB1 p.Phe978Cys However, of these, only the F978C isoform again displayed a reduction in the potency for stimulation of its ATPase activity by vinblastine, although the degree of stimulation remained unaffected. Login to comment 151 ABCB1 p.Gly984Cys There was, however, a drug-dependent difference on the stimulation of ATPase activity observed with the G984C and FIGURE 4: Maximal ATPase activity of TM12 mutant isoforms. Login to comment 155 ABCB1 p.Gly989Cys ABCB1 p.Val982Cys ABCB1 p.Gly984Cys ABCB1 p.Phe978Cys ABCB1 p.Leu976Cys ABCB1 p.Ala980Cys ABCB1 p.Met986Cys ABCB1 p.Gln990Cys ABCB1 p.Val988Cys ABCB1 p.Phe994Cys ABCB1 p.Ser992Cys 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 159 ABCB1 p.Met986Cys M986C isoforms (Table 2). Login to comment 160 ABCB1 p.Gly984Cys For G984C, nicardipine was associated with a reduction in potency and degree of stimulation, whereas the influence of vinblastine was similar to that in cysteine-less ABCB1. Login to comment 161 ABCB1 p.Met986Cys In contrast, for the M986C isoform, the effects of nicardipine were similar to cysteine-less ABCB1, while the potency of vinblastine was significantly (p<0.05) reduced. Login to comment 164 ABCB1 p.Ala980Cys Excluding the A980C isoform, which displayed enhanced ATPase activity, the effects were primarily a reduction of ATP hydrolysis. Login to comment 172 ABCB1 p.Gly989Cys ABCB1 p.Phe978Cys ABCB1 p.Leu976Cys ABCB1 p.Ala980Cys ABCB1 p.Gln990Cys ABCB1 p.Val988Cys 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 174 ABCB1 p.Phe978Cys ABCB1 p.Leu976Cys ABCB1 p.Ala980Cys 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. Login to comment 175 ABCB1 p.Phe978Cys ABCB1 p.Leu976Cys ABCB1 p.Ala980Cys ABCB1 p.Ala980Cys 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. Login to comment 176 ABCB1 p.Gly989Cys ABCB1 p.Gln990Cys ABCB1 p.Val988Cys 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 178 ABCB1 p.Leu976Cys Figure 5B shows the autoradiography data obtained for [γ-32 P]-azido-ATP photolabeling of the cysteine-less and L976C isoforms of ABCB1. Login to comment 179 ABCB1 p.Phe978Cys ABCB1 p.Leu976Cys ABCB1 p.Ala980Cys Figure 5C shows the densitometric analysis of the dose-response curve for the cysteine-less, A980C, L976C, and F978C isoforms. Login to comment 180 ABCB1 p.Ala980Cys 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. Login to comment 181 ABCB1 p.Phe978Cys ABCB1 p.Leu976Cys 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. Login to comment 182 ABCB1 p.Phe978Cys ABCB1 p.Leu976Cys 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. Login to comment 183 ABCB1 p.Ala980Cys However, mutant A980C, which was characterized by a significantly higher ATPase activity, did not show any appreciable changes in nucleotide binding. Login to comment 187 ABCB1 p.Phe978Cys ABCB1 p.Ala980Cys ABCB1 p.Val988Cys 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 192 ABCB1 p.Leu976Cys (B) Autoradiograms showing the binding of [γ-32 P]-azido-ATP (10-100 μM) to the purified, reconstituted cysteine-less and L976C mutant isoforms. Login to comment 193 ABCB1 p.Phe978Cys ABCB1 p.Leu976Cys ABCB1 p.Ala980Cys (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. Login to comment 199 ABCB1 p.Ala980Cys Of the six mutations, only A980C resulted in increased ATPase activity. Login to comment 203 ABCB1 p.Phe978Cys The F978C mutation, located near the extracellular protein-lipid interface in the membrane-embedded region of TM12, is part of a conserved sequence motif present in TM12 and TM6. Login to comment 207 ABCB1 p.Phe335Cys ABCB1 p.Phe978Cys However, unlike the TM12 F978C mutation, the TM6 F335C mutation does not cause a significant reduction in ATPase activity (15). Login to comment 208 ABCB1 p.Phe978Cys In TM12, mutation to the smaller 978C increases the distance between F978C and F72, preventing hydrophobic interactions (Figure 6D) and decreasing the number of TM12-TM1 contact points. Login to comment 210 ABCB1 p.Phe978Cys Interestingly, the modeled mutation also alters the orientation of the F978C side chain, resulting in the complete exposure of the cysteine side chain into a pore below L975, which has been implicated in dibromobimane binding (47). Login to comment 212 ABCB1 p.Val988Cys 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 219 ABCB1 p.Gly346Cys A similar study with TM6 revealed significantly fewer positions at which site-directed mutagenesis to cysteine caused functional perturbation in the basal state (notably mutant isoform G346C). Login to comment 220 ABCB1 p.Gly989Cys ABCB1 p.Phe978Cys ABCB1 p.Leu976Cys ABCB1 p.Ala980Cys ABCB1 p.Gln990Cys ABCB1 p.Val988Cys 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 229 ABCB1 p.Ala980Cys (B) Mutation to A980C allows for hydrogen bonding to S850 (orange), increasing TM12-TM9 contacts. Login to comment 231 ABCB1 p.Phe978Cys (D) Mutation to F978C (CPK) prevents hydrogen bonding to F72. Login to comment 232 ABCB1 p.Phe978Cys ABCB1 p.Leu976Cys ABCB1 p.Gln990Cys ABCB1 p.Val988Cys (L976C and F978C) and intracellular (V988C and Q990C) ends of the helix. Login to comment 233 ABCB1 p.Gln990Cys ABCB1 p.Val988Cys 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 234 ABCB1 p.Phe978Cys ABCB1 p.Leu976Cys In contrast, the reduced basal activity observed with mutants L976C and F978C appeared to be a consequence of reduced nucleotide binding. Login to comment 239 ABCB1 p.Gly989Cys Mutant G989C did not affect vinblastine-stimulated activity but markedly reduced the degree and potency of stimulation by nicardipine. Login to comment 240 ABCB1 p.Gly989Cys Both nicardipine and vinblastine have previously been shown to interact at pharmacologically distinct sites (9, 53), suggesting that mutant G989C is involved in communicating nicardipine but not vinblastine binding to the NBDs. Login to comment 241 ABCB1 p.Gly346Cys In contrast, mutation of the equivalent residue (G346C) in TM6 was implicated in communication between the vinblastinebinding siteand the NBDs(37).The datacontinuetosupport our assertion that drugs communicate to the NBDs via distinct pathways and that allosteric communication between TMD1 and TMD2 to NBD1 and NBD2 is not symmetrical in ABCB1. Login to comment