ABCB1 p.Glu875Ala
| Predicted by SNAP2: | A: D (80%), C: D (80%), D: D (80%), F: D (91%), G: D (71%), H: D (63%), I: D (91%), K: D (91%), L: D (91%), M: D (80%), N: D (59%), P: D (95%), Q: N (78%), R: D (75%), S: D (66%), T: D (66%), V: D (85%), W: D (91%), Y: D (91%), |
| Predicted by PROVEAN: | A: D, C: D, D: N, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: N, 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).
X
ABCB1 p.Glu875Ala 21182301:84:121
status: NEW238 A G251V/T945R/E875A mutant was constructed to test if removal of the negative charge would affect maturation.
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ABCB1 p.Glu875Ala 21182301:238:14
status: NEW239 It was found that T945R no longer promoted maturation when the E875A mutation was present (Figure 7C, lane 4).
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ABCB1 p.Glu875Ala 21182301:239:63
status: NEW285 Stable BHK cell lines expressing mutants W232A, N296A, E875A, or T945A were generated.
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ABCB1 p.Glu875Ala 21182301:285:55
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.Glu875Ala 21182301:299:158
status: NEW303 Higher concentrations of verapamil were required for half-maximal activation (S50) of ATPase activity for mutants T945A (370 μM) and E875A (>1 mM) compared to wild-type P-gp (48 μM).
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ABCB1 p.Glu875Ala 21182301:303:139
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.Glu875Ala 21182301:307:52
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.Glu875Ala 21182301:308:155
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.Glu875Ala 21182301:310:16
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.Glu875Ala 21182301:336:51
status: NEW