ABCB1 p.Asn296Ala
| Predicted by SNAP2: | A: D (75%), C: D (75%), D: D (71%), E: D (85%), F: D (85%), G: N (72%), H: D (85%), I: D (85%), K: D (75%), L: D (75%), M: D (85%), P: D (85%), Q: D (63%), R: D (91%), S: D (59%), T: D (80%), V: D (85%), W: D (91%), Y: D (85%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D, |
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[hide] The W232R suppressor mutation promotes maturation ... Biochemistry. 2011 Feb 8;50(5):672-85. Epub 2011 Jan 11. Loo TW, Bartlett MC, Clarke DM
The W232R suppressor mutation promotes maturation of a truncation mutant lacking both nucleotide-binding domains and restores interdomain assembly and activity of P-glycoprotein processing mutants.
Biochemistry. 2011 Feb 8;50(5):672-85. Epub 2011 Jan 11., 2011-02-08 [PMID:21182301]
Abstract [show]
ATP-binding cassette (ABC) proteins contain two nucleotide-binding domains (NBDs) and two transmembrane (TM) domains (TMDs). Interdomain interactions and packing of the TM segments are critical for function, and disruption by genetic mutations contributes to disease. P-glycoprotein (P-gp) is a useful model to identify mechanisms that repair processing defects because numerous arginine suppressor mutations have been identified in the TM segments. Here, we tested the prediction that a mechanism of arginine rescue was to promote intradomain interactions between TM segments and restore interdomain assembly. We found that suppressor W232R(TM4/TMD1) rescued mutants with processing mutations in any domain and restored defective NBD1-NBD2, NBD1-TMD2, and TMD1-TMD2 interactions. W232R also promoted packing of the TM segments because it rescued a truncation mutant lacking both NBDs. The mechanism of W232R rescue likely involved intradomain hydrogen bond interactions with Asn296(TM5) since only N296A abolished rescue by W232R and rescue was only observed when Trp232 was replaced with hydrogen-bonding residues. In TMD2, suppressor T945R(TM11) also promoted packing of the TM segments because it rescued the truncation mutant lacking the NBDs and suppressed formation of alternative topologies. We propose that T945R rescue was mediated by interactions with Glu875(TM10) since T945E/E875R promoted maturation while T945R/E875A did not.
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No. Sentence Comment
84 Baby hamster kidney (BHK) cells expressing A52-tagged wild-type P-gp or mutants G251V, G251V/W232R, W232R, W232A, N296A, E875A, or T945A were generated as described previously (25).
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ABCB1 p.Asn296Ala 21182301:84:114
status: NEW167 The N296A Mutation Abolishes W232R Rescue.
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ABCB1 p.Asn296Ala 21182301:167:4
status: NEW184 It was observed that only one mutation in TM5 (N296A) inhibited maturation of the G251V/W232R mutant when it was expressed in the absence of cyclosporin A (Figure 4C, lane 7).
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ABCB1 p.Asn296Ala 21182301:184:47
status: NEW186 Introduction of the N296A mutation into G251V/W232R(TM4) caused the mutant to behave in a fashion similar to the original FIGURE 3: Effect of W232R on cross-linking between domains of processing mutants.
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ABCB1 p.Asn296Ala 21182301:186:20
status: NEW194 (C) HEK 293 cells transfected with A52-tagged mutant G251V or G251V/ W232R containing alanine mutations in potential hydrogen-bonding residues in TM5 (T294A, N296A, S298A) or G251V/N296A were expressed in the absence (-) or presence (þ) of cyclosporin A (cyclo A).
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ABCB1 p.Asn296Ala 21182301:194:158
status: NEWX
ABCB1 p.Asn296Ala 21182301:194:181
status: NEW200 Like the G251V parent (Figure 4C, lanes 1 and 2), the G251V/W232R/N296A mutant (Figure 4C, lane 7) showed about 15% maturation efficiency when expressed in the absence of drug substrates, and the mature 170 kDa P-gp was the major product when expressed in the presence of cyclosporin A (Figure 4C, lane 8).
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ABCB1 p.Asn296Ala 21182301:200:66
status: NEW201 Therefore, the presence of the N296A mutation prevented the ability of W232R to promote maturation of the G251V mutant.
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ABCB1 p.Asn296Ala 21182301:201:31
status: NEW202 Figure 4A (lanes 11 and 12) shows that the N296A change alone had little effect on the maturation characteristics of the G251V parent because it could still be rescued with cyclosporin A.
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ABCB1 p.Asn296Ala 21182301:202:43
status: NEW203 These results suggest that Arg232(TM4) promotes maturation of the G251V mutant through hydrogen bond interactions with Asn296(TM5) because the N296A mutation inhibited the ability of W232R to promote maturation of G251V (Figure 4C, lane 7).
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ABCB1 p.Asn296Ala 21182301:203:143
status: NEW219 No detectable mature protein was observed in cells expressing TMD1 þ 2 lacking W232R or with mutant TMD1 þ 2(W232R/N296A) (Figure 6A).
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ABCB1 p.Asn296Ala 21182301:219:125
status: NEW229 (A) Whole cell extracts of cells transfected with A52-tagged wild-type, W232R, or W232R/N296A forms of TMD1 þ 2 were subjected to immunoblot analysis with monoclonal antibody against A52 or GAPDH.
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ABCB1 p.Asn296Ala 21182301:229:88
status: NEW285 Stable BHK cell lines expressing mutants W232A, N296A, E875A, or T945A were generated.
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ABCB1 p.Asn296Ala 21182301:285:48
status: NEW289 For example, cells expressing mutants W232A and N296A were more resistant to colchicine (P < 0.05) than cells expressing wild-type P-gp, but their resistance to paclitaxel was reduced (P < 0.05).
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ABCB1 p.Asn296Ala 21182301:289:48
status: NEW299 Each value is average of triplicate assays ( SD. (B) Whole cell extracts of BHK cells expressing vector (control), A52-tagged wild-type, mutant W232A, N296A, E875A, or T945A P-gp were subjected to immunoblot analysis with monoclonal antibody against A52 or GAPDH.
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ABCB1 p.Asn296Ala 21182301:299:151
status: NEW304 The verapamil-stimulated ATPase activities of mutants W232A and N296A, however, resembled that of wild-type enzyme (S50 of 30 and 42 μM, respectively).
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ABCB1 p.Asn296Ala 21182301:304:64
status: NEW306 The maximum activity was increased by 50% in mutant N296A when compared to wild-type P-gp.
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ABCB1 p.Asn296Ala 21182301:306:52
status: NEW308 The S50 of mutants W232A (45 μM) and T945A (200 μM) differed from wild-type P-gp (102 μM) while those of mutants N296A (78 μM) and E875A (86 μM) were similar.
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ABCB1 p.Asn296Ala 21182301:308:131
status: NEW310 In summary, the E875A and T945A mutations altered both verapamiland rhodamine B-stimulated ATPase activity while the W232A and N296A mutations only affected rhodamine B-stimulated ATPase activity.
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ABCB1 p.Asn296Ala 21182301:310:127
status: NEW336 Histidine-tagged wild-type or mutant W232A, N296A, E875A, or T945A P-gp was expressed in HEK 293 cells and isolated by nickel-chelate chromatography.
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ABCB1 p.Asn296Ala 21182301:336:44
status: NEW