ABCB1 p.Thr199Cys
| Predicted by SNAP2: | A: D (59%), C: D (66%), D: D (80%), E: D (85%), F: D (85%), G: D (53%), H: D (71%), I: D (63%), K: D (91%), L: D (66%), M: N (66%), N: D (75%), P: D (91%), Q: D (66%), R: D (71%), S: N (72%), V: D (80%), W: D (85%), Y: D (75%), |
| 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: N, V: D, W: D, Y: D, |
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[hide] Suppressor mutations in the transmembrane segments... J Biol Chem. 2007 Nov 2;282(44):32043-52. Epub 2007 Sep 11. Loo TW, Bartlett MC, Clarke DM
Suppressor mutations in the transmembrane segments of P-glycoprotein promote maturation of processing mutants and disrupt a subset of drug-binding sites.
J Biol Chem. 2007 Nov 2;282(44):32043-52. Epub 2007 Sep 11., 2007-11-02 [PMID:17848563]
Abstract [show]
Defective folding of cystic fibrosis transmembrane conductance regulator protein missing Phe508 (DeltaF508) is the major cause of cystic fibrosis. The folding defect in DeltaF508 cystic fibrosis transmembrane conductance regulator might be correctable because misfolding of a P-glycoprotein (P-gp; ABCB1) mutant lacking the equivalent residue (DeltaY490) could be corrected with drug substrates or by introduction of an arginine residue into transmembrane (TM) segments 5 (I306R) or 6 (F343R). Possible mechanisms of arginine rescue were that they mimicked some of the effects of drug substrate interactions with P-gp or that they affected global folding such that all drug substrate/modulator interactions with P-gp were altered. To distinguish between these mechanisms, we tested whether arginines introduced into other TMs predicted to line the drug-binding pocket (TM1 or TM3) would affect folding. It was found that mutation of L65R(TM1) or T199R(TM3) promoted maturation of processing mutants. We then tested whether arginine suppressor mutations had local or global effects on P-gp interactions with drug substrates and modulators. The L65R(TM1), T199R(TM3), I306R(TM5), or F343R(TM6) mutations were introduced into the P-gp mutant L339C(TM6)/F728C(TM7), and thiol cross-linking was carried out in the presence of various concentrations of vinblastine, cyclosporin A, or rhodamine B. The presence of arginine residues reduced the apparent affinity of P-gp for vinblastine (L65R, T199R, and I306R), cyclosporin (I306R and F343R), or rhodamine B (F343R) by 4-60-fold. These results show that the arginine mutations affect a subset of drug-binding sites and suggest that they rescue processing mutants by mimicking drug substrate interactions with P-gp.
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No. Sentence Comment
147 Fig. 2A shows that all of the mutants except T199C had activities similar to that of Cys-less P-gp (less than 1.5-fold increase).
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ABCB1 p.Thr199Cys 17848563:147:45
status: NEW148 Mutant T199C showed a 6.7-fold increase in activity after treatment with MTS-rhodamine.
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ABCB1 p.Thr199Cys 17848563:148:7
status: NEW154 The mutants Q195C and T199C, however, exhibited different properties than Cys-less P-gp after treatment with MTS-rhodamine.
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ABCB1 p.Thr199Cys 17848563:154:22
status: NEW157 By contrast, covalent modification of mutant T199C with MTS-rhodamine fully activated its ATPase activity such that rhodamine B had no further effect on its activity.
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ABCB1 p.Thr199Cys 17848563:157:45
status: NEW158 If MTS-rhodamine activates P-gp ATPase activity because it occupies the rhodamine-binding site when it is attached to Cys199 , then labeling of mutant T199C should be protectable with rhodamine B.
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ABCB1 p.Thr199Cys 17848563:158:151
status: NEW160 Histidine-tagged mutant T199C was expressed in HEK 293 cells, solubilized with detergent, and reacted with various concentrations of MTS-rhodamine.
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ABCB1 p.Thr199Cys 17848563:160:24
status: NEW162 To test whether rhodamine B protected mutant T199C from labeling, HEK 293 cells expressing the histidine-tagged mutant were solubilized with detergent and treated with 1 mM MTS-rhodamine in the presence or absence of 5 mM rhodamine B for 10 min at 20 °C.
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ABCB1 p.Thr199Cys 17848563:162:45
status: NEW163 The P-gps were isolated by nickel-chelate chromatography and mixed with lipids, and ATPase activities were determined. Fig. 3B shows that ATP hydrolysis was reduced by 78% when mutant T199C was reacted with MTS-rhodamine in the presence of 5 mM rhodamine B.
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ABCB1 p.Thr199Cys 17848563:163:184
status: NEW168 Fold-stimulation 1 2 3 4 5 6 Cys-less I190C G191C M192C F193C F194C Q195C S196C M197C A198C T199C F200C F201C T202C G203C F204C I205C V206C G207C F208C T209C A 7 Fold-stimulation 1 2 3 4 5 6 Cys-less B _ + MTS-rhod Rhod B+ _ + Q195C + + _ + T199C + + _ + _ _+ + + 7 FIGURE 2.
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ABCB1 p.Thr199Cys 17848563:168:92
status: NEWX
ABCB1 p.Thr199Cys 17848563:168:241
status: NEW173 B, Cys-less, Q195C and T199C P-gp mutants were treated with (ϩ) or without (-) 2 mM MTS-rhodamine and histidine-tagged P-gp isolated by nickel-chelate chromatography. Equivalent amounts of P-gp were mixed with lipid, and ATPase activity was determined in the presence (ϩ) or absence (-) of 2 mM rhodamine B (Rhod B).
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ABCB1 p.Thr199Cys 17848563:173:23
status: NEW177 Labeling of mutant T199C with increasing concentrations of MTS-rhodamine and protection by rhodamine B.
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ABCB1 p.Thr199Cys 17848563:177:19
status: NEW178 A, HEK 293 cells expressing histidine-tagged mutant T199C were solubilized with n-dodecyl-beta-D-maltoside, and insoluble material was removed by centrifugation.
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ABCB1 p.Thr199Cys 17848563:178:52
status: NEW269 The labeling of Cys199 also appeared to be specific for MTS-rhodamine because the ATPase activity of mutant T199C was not activated by MTS-verapamil.
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ABCB1 p.Thr199Cys 17848563:269:108
status: NEW[hide] The ATPase activity of the P-glycoprotein drug pum... J Biol Chem. 2012 Aug 3;287(32):26806-16. doi: 10.1074/jbc.M112.376202. Epub 2012 Jun 14. Loo TW, Bartlett MC, Detty MR, Clarke DM
The ATPase activity of the P-glycoprotein drug pump is highly activated when the N-terminal and central regions of the nucleotide-binding domains are linked closely together.
J Biol Chem. 2012 Aug 3;287(32):26806-16. doi: 10.1074/jbc.M112.376202. Epub 2012 Jun 14., [PMID:22700974]
Abstract [show]
The P-glycoprotein (P-gp, ABCB1) drug pump protects us from toxic compounds and confers multidrug resistance. Each of the homologous halves of P-gp is composed of a transmembrane domain (TMD) with 6 TM segments followed by a nucleotide-binding domain (NBD). The predicted drug- and ATP-binding sites reside at the interface between the TMDs and NBDs, respectively. Crystal structures and EM projection images suggest that the two halves of P-gp are separated by a central cavity that closes upon binding of nucleotide. Binding of drug substrates may induce further structural rearrangements because they stimulate ATPase activity. Here, we used disulfide cross-linking with short (8 A) or long (22 A) cross-linkers to identify domain-domain interactions that activate ATPase activity. It was found that cross-linking of cysteines that lie close to the LSGGQ (P517C) and Walker A (I1050C) sites of NBD1 and NBD2, respectively, as well as the cytoplasmic extensions of TM segments 3 (D177C or L175C) and 9 (N820C) with a short cross-linker activated ATPase activity over 10-fold. A pyrylium compound that inhibits ATPase activity blocked cross-linking at these sites. Cross-linking between the NBDs was not inhibited by tariquidar, a drug transport inhibitor that stimulates P-gp ATPase activity but is not transported. Cross-linking between extracellular cysteines (T333C/L975C) predicted to lock P-gp into a conformation that prevents close NBD association inhibited ATPase activity. The results suggest that trapping P-gp in a conformation in which the NBDs are closely associated likely mimics the structural rearrangements caused by binding of drug substrates that stimulate ATPase activity.
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No. Sentence Comment
255 Covalent labeling of T199C (TM3) with a thiol-reactive derivative of rhodamine increased basal activity 7-fold (58).
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ABCB1 p.Thr199Cys 22700974:255:21
status: NEW248 Covalent labeling of T199C (TM3) with a thiol-reactive derivative of rhodamine increased basal activity 7-fold (58).
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ABCB1 p.Thr199Cys 22700974:248:21
status: NEW