ABCB1 p.Thr945Arg
| Predicted by SNAP2: | A: N (61%), C: D (59%), D: D (80%), E: D (80%), F: D (75%), G: N (66%), H: D (66%), I: D (75%), K: D (85%), L: D (75%), M: D (66%), N: D (66%), P: D (85%), Q: D (71%), R: D (85%), S: N (97%), V: D (75%), W: D (75%), Y: D (80%), |
| Predicted by PROVEAN: | A: N, C: N, D: N, E: N, F: D, G: N, H: D, I: D, K: N, L: D, M: N, N: N, P: N, Q: N, R: N, S: N, 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
235 To test if intradomain interactions in TMD2 could also act as a rescue mechanism, we examined the T945R suppressor mutation.
X
ABCB1 p.Thr945Arg 21182301:235:98
status: NEW236 The T945R mutation was selected as it was one of the most effective suppressor mutations identified in TMD2 (42).
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ABCB1 p.Thr945Arg 21182301:236:4
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.Thr945Arg 21182301:238:8
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.Thr945Arg 21182301:239:18
status: NEW245 Since both Glu875 and Thr945 are located in TMD2, we tested if the T945R mutation would promote maturation of the truncation mutant lacking the NBDs (TMD1 þ 2).
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ABCB1 p.Thr945Arg 21182301:245:67
status: NEW246 Immunoblot analysis of whole cell extracts of the transfected cells showed that the T945R mutation promoted maturation of TMD1 þ 2 (Figure 7E).
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ABCB1 p.Thr945Arg 21182301:246:84
status: NEW251 To test if T945R would promote or inhibit the alternative topology, it was introduced into C-half P-gp.
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ABCB1 p.Thr945Arg 21182301:251:11
status: NEW254 Expression of C-half/T945R yielded a single protein product corresponding to unglycosylated P-gp (Figure 8C).
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ABCB1 p.Thr945Arg 21182301:254:21
status: NEW255 The C-half/ T945R protein was not glycosylated as it was not sensitive to endoglycosidases (data not shown).
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ABCB1 p.Thr945Arg 21182301:255:12
status: NEW258 (C) Whole cell extracts from cells expressing A52-tagged mutant G251V (none) or G251V/T945R (T945R) with the indicated changes topotential hydrogen bondpartnersinTM10(lane4) orTM12(lanes 7-9) were subjected to immunoblot analysis with monoclonal antibody against A52 or GAPDH.
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ABCB1 p.Thr945Arg 21182301:258:86
status: NEWX
ABCB1 p.Thr945Arg 21182301:258:93
status: NEW262 (E) Whole cell extracts of cells transfected with A52-tagged wild-type or T945R forms of TMD1 þ 2 were subjected to immunoblot analysis with monoclonal antibody against A52 or GAPDH.
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ABCB1 p.Thr945Arg 21182301:262:74
status: NEW264 FIGURE 8: Effect of T945R mutation on expression of C-half P-gp.
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ABCB1 p.Thr945Arg 21182301:264:20
status: NEW267 (C) Whole cell extracts of cells expressing A52-tagged wild-type (None) or T945R forms of C-half P-gp were subjected to immunoblot analysis with monoclonal antibody against A52 or GAPDH.
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ABCB1 p.Thr945Arg 21182301:267:75
status: NEW311 The large effect of the T945A mutation on ATPase activity is consistent with the observation that wild-type P-gp containing W232R exhibits verapamiland rhodamine-stimulated ATPase activity whereas wild-type P-gp containing T945R does not (42).
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ABCB1 p.Thr945Arg 21182301:311:223
status: NEW316 Second, the NBDs are not required for rescue by W232R or T945R.
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ABCB1 p.Thr945Arg 21182301:316:57
status: NEW357 Strong support for the prediction that intradomain interactions between TM segments can modulate topologywas theobservation that T945R inhibited the formation of an alternative topology in C-half P-gp when it was expressed alone (Figure 8C).
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ABCB1 p.Thr945Arg 21182301:357:129
status: NEW[hide] Mapping the Binding Site of the Inhibitor Tariquid... J Biol Chem. 2015 Dec 4;290(49):29389-401. doi: 10.1074/jbc.M115.695171. Epub 2015 Oct 26. Loo TW, Clarke DM
Mapping the Binding Site of the Inhibitor Tariquidar That Stabilizes the First Transmembrane Domain of P-glycoprotein.
J Biol Chem. 2015 Dec 4;290(49):29389-401. doi: 10.1074/jbc.M115.695171. Epub 2015 Oct 26., [PMID:26507655]
Abstract [show]
ABC (ATP-binding cassette) transporters are clinically important because drug pumps like P-glycoprotein (P-gp, ABCB1) confer multidrug resistance and mutant ABC proteins are responsible for many protein-folding diseases such as cystic fibrosis. Identification of the tariquidar-binding site has been the subject of intensive molecular modeling studies because it is the most potent inhibitor and corrector of P-gp. Tariquidar is a unique P-gp inhibitor because it locks the pump in a conformation that blocks drug efflux but activates ATPase activity. In silico docking studies have identified several potential tariquidar-binding sites. Here, we show through cross-linking studies that tariquidar most likely binds to sites within the transmembrane (TM) segments located in one wing or at the interface between the two wings (12 TM segments form 2 divergent wings). We then introduced arginine residues at all positions in the 12 TM segments (223 mutants) of P-gp. The rationale was that a charged residue in the drug-binding pocket would disrupt hydrophobic interaction with tariquidar and inhibit its ability to rescue processing mutants or stimulate ATPase activity. Arginines introduced at 30 positions significantly inhibited tariquidar rescue of a processing mutant and activation of ATPase activity. The results suggest that tariquidar binds to a site within the drug-binding pocket at the interface between the TM segments of both structural wings. Tariquidar differed from other drug substrates, however, as it stabilized the first TM domain. Stabilization of the first TM domain appears to be a key mechanism for high efficiency rescue of ABC processing mutants that cause disease.
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No. Sentence Comment
188 These were in TM7 (Q725R, F728R, and F732R) (Fig. 6A), TM10 (V865R, I868R, and G872R) (Fig. 6D), TM11 (F942R, T945R, Q946R, M949R, Y950R, and Y953R) (Fig. 6E), and TM12 (L975R, F978R, and V982R) (Fig. 6F).
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ABCB1 p.Thr945Arg 26507655:188:110
status: NEW193 It was found that 16 of the 28 mutants resembled the G251V/I868R mutant as expression in the presence of 5 òe;M cyclosporine A yielded mature P-gp as the major product in TM1 (H61R, G64R, L65R, M68R, and M69R), TM5 (F303R, I306R, and S309R), TM7 (Q725R and F728R), TM10 (I868R and G872R), TM11 (F942R, T945R, and Q946R), and TM12 (V982R) (Fig. 7).
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ABCB1 p.Thr945Arg 26507655:193:306
status: NEW212 Seventeen of the 30 G251V/arginine mutants (M68R, M69R, and F72R in TM1; I306R, Y307R, S309R, and Y310R in TM5; F336R in TM6; F728R and F732R in TM7; I868R and G872R in TM10; F942R, T945R, M949R, and S952R in TM11; and V982R in TM12) that could not be rescued with tariquidar showed little or no stimulation of ATPase activity with tariquidar (Fig. 8A).
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ABCB1 p.Thr945Arg 26507655:212:182
status: NEW283 We identified 13 additional arginine mutations (H61R, G64R, L65R, and M68R in TM1; A129R in TM2; I306R and S309R in TM5; F343R in TM6; F942R, T945R, Q946R, and Y950R in TM11; and L975R in TM12) (Fig. 11A) that were not predicted to lie within 4.5-&#c5; of the predicted site 3 tariquidar-binding site (9).
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ABCB1 p.Thr945Arg 26507655:283:142
status: NEW313 Therefore, arginines predicted to lie outside of the tariquidar-binding site in TM1 and TM11 in the docking studies (H61R, G64R, L65R, and M68 in TM1; F942R, T945R, Q946R, and Y950R in TM11) might alter interactions between TM1 and TM11.
X
ABCB1 p.Thr945Arg 26507655:313:158
status: NEW