ABCB1 p.Trp232Ala
| Predicted by SNAP2: | A: D (53%), C: N (72%), D: D (75%), E: D (71%), F: N (66%), G: D (59%), H: D (53%), I: N (57%), K: D (75%), L: N (57%), M: N (82%), N: D (66%), P: D (85%), Q: D (66%), R: D (75%), S: D (53%), T: D (53%), V: N (57%), Y: N (72%), |
| 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, N: D, P: D, Q: D, R: D, S: D, T: D, V: 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
58 (B) HEK 293 cells were transfected with A52-tagged wild-type P-gp, mutants G251V, G251V/W232R, and G251V/ W232A,orplasmidvector(control).Wholecellextractsweresubjected to immunoblot analysis with monoclonal antibody against A52 or GAPDH.
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ABCB1 p.Trp232Ala 21182301:58:106
status: NEW84 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.Trp232Ala 21182301:84:107
status: NEW114 We tested whether a W232A change would also promote maturation of the G251V mutant.
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ABCB1 p.Trp232Ala 21182301:114:20
status: NEW118 By contrast, immature protein was the major product in mutants G251V (10 ( 4% mature) and G251V/W232A (13 ( 5% mature).
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ABCB1 p.Trp232Ala 21182301:118:96
status: NEW285 Stable BHK cell lines expressing mutants W232A, N296A, E875A, or T945A were generated.
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ABCB1 p.Trp232Ala 21182301:285:41
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.Trp232Ala 21182301:289:38
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.Trp232Ala 21182301:299:144
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.Trp232Ala 21182301:304:54
status: NEW307 By contrast, the maximum activity of mutants W232A, E875A, and T945A were reduced relative to wild-type P-gp.
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ABCB1 p.Trp232Ala 21182301:307:45
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.Trp232Ala 21182301:308:19
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.Trp232Ala 21182301:310:117
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.Trp232Ala 21182301:336:37
status: NEW