ABCB4 p.Leu975Cys
| Predicted by SNAP2: | A: N (61%), C: N (57%), D: D (75%), E: D (71%), F: N (53%), G: D (75%), H: D (75%), I: N (82%), K: N (53%), M: N (87%), N: D (75%), P: D (80%), Q: N (66%), R: N (57%), S: D (59%), T: N (78%), V: N (87%), W: D (75%), Y: D (71%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: N, K: D, M: N, N: D, P: D, Q: D, R: D, S: D, T: D, V: N, W: D, Y: D, |
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[hide] Recent progress in understanding the mechanism of ... J Membr Biol. 2005 Aug;206(3):173-85. Loo TW, Clarke DM
Recent progress in understanding the mechanism of P-glycoprotein-mediated drug efflux.
J Membr Biol. 2005 Aug;206(3):173-85., [PMID:16456713]
Abstract [show]
P-glycoprotein (P-gp) is an ATP-dependent drug pump that can transport a broad range of hydrophobic compounds out of the cell. The protein is clinically important because of its contribution to the phenomenon of multidrug resistance during AIDS/HIV and cancer chemotherapy. P-gp is a member of the ATP-binding cassette (ABC) family of proteins. It is a single polypeptide that contains two repeats joined by a linker region. Each repeat has a transmembrane domain consisting of six transmembrane segments followed by a hydrophilic domain containing the nucleotide-binding domain. In this mini-review, we discuss recent progress in determining the structure and mechanism of human P-glycoprotein.
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No. Sentence Comment
200 During ATP hydrolysis, conformational changes between TM6 and TM12 were detected by disulfide cross-linking between cysteines introduced at the extracellular ends of these TMs (residue L332C in TM6 and L975C in TM12) (Loo & Clarke, 1996a, 1997b).
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ABCB4 p.Leu975Cys 16456713:200:202
status: NEW201 Cross-linking between L332C/L975C prevented conformational changes during ATP hydrolysis, resulting in inhibition of ATPase activity.
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ABCB4 p.Leu975Cys 16456713:201:28
status: NEW[hide] Nucleotide binding, ATP hydrolysis, and mutation o... Biochemistry. 2007 Aug 14;46(32):9328-36. Epub 2007 Jul 18. Loo TW, Bartlett MC, Clarke DM
Nucleotide binding, ATP hydrolysis, and mutation of the catalytic carboxylates of human P-glycoprotein cause distinct conformational changes in the transmembrane segments.
Biochemistry. 2007 Aug 14;46(32):9328-36. Epub 2007 Jul 18., 2007-08-14 [PMID:17636884]
Abstract [show]
P-Glycoprotein (P-gp, ABCB1) transports a variety of structurally unrelated cytotoxic compounds out of the cell. Each homologous half of P-gp has a transmembrane (TM) domain containing six TM segments and a nucleotide-binding domain (NBD) and is joined by a linker region. It has been postulated that binding of two ATP molecules at the NBD interface to form a "nucleotide sandwich" induces drug efflux by altering packing of the TM segments that make up the drug-binding pocket. To test if ATP binding alone could alter packing of the TM segments, we introduced catalytic carboxylate mutations (E556Q in NBD1 and E1201Q in NBD2) into double-cysteine mutants that exhibited ATP-dependent cross-linking so that the mutants could bind but not hydrolyze ATP. It was found that ATP binding alone could alter disulfide cross-linking between the TM segments. For example, ATP inhibited cross-linking of mutant L339C(TM6)/V982C(TM12)/E556Q(NBD1)/E1201Q(NBD2) but promoted cross-linking of mutant F343C(TM6)/V982C(TM12)/E556Q(NBD1)/E1201Q(NBD2). Cross-linking of some mutants, however, appeared to require ATP hydrolysis as introduction of the catalytic carboxylate mutations into mutant L332C(TM6)/L975C(TM12) inhibited ATP-dependent cross-linking. Cross-linking between cysteines in the TM segments also could be altered via introduction of a single catalytic carboxylate mutation into mutant L332C(TM6)/L975C(TM12) or by using the nonhydrolyzable ATP analogue, AMP.PNP. The results show that the TM segments are quite sensitive to changes within the ATP-binding sites because different conformations could be detected in the presence of ATP, AMP.PNP, during ATP hydrolysis or through mutation of the catalytic carboxylates.
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No. Sentence Comment
74 The positions of the catalytic carboxylate mutations (E556Q in NBD1 and E1201Q in NBD2) and the cysteine mutations in the TM segments used in the disulfide cross-linking studies (L332C, L339C, F343C, F728C, L975C, and V982C) are shown.
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ABCB4 p.Leu975Cys 17636884:74:207
status: NEW89 Mutant L332C(TM6)/L975C(TM12) contains one cysteine residue at the extracellular end of TM6 (L332C) and another at the extracellular end of TM12 (L975C) (Figure 1).
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ABCB4 p.Leu975Cys 17636884:89:146
status: NEW125 Membranes prepared from HEK 293 cells expressing mutants L332C(TM6)/L975C(TM12), L332C- (TM6)/L975C(TM12)/E556Q(NBD1)/E1201Q(NBD2), L332C- (TM6)/L975C(TM12)/E556Q(NBD1), or L332C(TM6)/L975C- (TM12)/E1201Q(NBD2) were treated with (+) or without (-) 1 mM copper phenanthroline (CuP) for 10 min at 37 °C in the absence (None) or presence of 5 mM ATP (ATP).
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ABCB4 p.Leu975Cys 17636884:125:184
status: NEW94 The mutant L332C(TM6)/L975C- (TM12) exhibited extensive cross-linking only in the presence of ATP (Figure 4).
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ABCB4 p.Leu975Cys 17636884:94:22
status: NEW200 Mutant L332C(TM6)/L975C- (TM12) normally exhibits cross-linking only in the presence of ATP.
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ABCB4 p.Leu975Cys 17636884:200:18
status: NEW234 The cross-linking pattern of mutant L332C(TM6)/L975C- (TM12)/E556Q(NBD1)/E1201Q(NBD2) indicates that ATP hydrolysis induces conformational changes in the TMDs that are distinct from those involving ATP binding.
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ABCB4 p.Leu975Cys 17636884:234:47
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
106 The location of residues that were mutated to cysteine to test for the effect of cross-linking between NBD1 and NBD2 (P517C/I1050C), NBD1 and TMD2 (L443C/S909C), ICL1 and ICL3 (D177C/N820C), TM segments 2 and 11 (C137/A935C) or 6 and 12 (T333C/L975C) are shown.
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ABCB4 p.Leu975Cys 22700974:106:244
status: NEW201 Accordingly, we introduced cysteines in regions of TM segments 6 (T333C) and 12 (L975C) predicted to lie at or close to the extracellular surface of the cell (Fig. 1).
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ABCB4 p.Leu975Cys 22700974:201:76
status: NEW202 The T333C and L975C mutations were selected because we previously showed that they had little effect on P-gp activity and treatment of the single cysteine mutants with a thiol-reactive derivative of verapamil had little effect on activity (43).
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ABCB4 p.Leu975Cys 22700974:202:14
status: NEW203 Since the T333C and L975C mutations were predicted to reside at or close to the extracellular surface of the cell, we performed cross-linking analysis on intact cells expressing the mutant.
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ABCB4 p.Leu975Cys 22700974:203:20
status: NEW206 Fig. 6A shows that P-gp mutant T333C/L975C was efficiently cross-linked (Ͼ 90% efficiency) when intact cells were treated with BMOE.
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ABCB4 p.Leu975Cys 22700974:206:37
status: NEWX
ABCB4 p.Leu975Cys 22700974:206:97
status: NEW207 To test for the effect of cross-linking on activity, histidine-tagged T333C/L975C P-gp was isolated by nickel-chelate chromatography before and after cross-linking with BMOE.
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ABCB4 p.Leu975Cys 22700974:207:76
status: NEW210 We then tested the effect of the stimulators (tariquidar, P12) and inhibitor (P10) of ATPase activity on cross-linking of mutant T333C/L975C.
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ABCB4 p.Leu975Cys 22700974:210:135
status: NEW212 The results suggest that the stimulators may promote the closed conformation where the T333C and L975C residues are far apart.
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ABCB4 p.Leu975Cys 22700974:212:97
status: NEW225 C, cells expressing mutant T333C/L975C were pretreated with 0.1 mM P10, 0.1 mM P12, 0.03 mM tariquidar (Tar), or no compound (None) and then incubated in the absence (-) or presence (ϩ) of 0.5 mM BMOE. Samples were subjected to immunoblot analysis.
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ABCB4 p.Leu975Cys 22700974:225:33
status: NEW244 Evidence that ATP hydrolysis appears to cause lateral movement or rotation of the helices were the observations that ATP hydrolysis was required for cross-linking of mutant L332C(TM6)/L975C (50) and ATP hydrolysis shifted cross-linking of V982C in TM12 from L339C to F343C in TM6 (51).
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ABCB4 p.Leu975Cys 22700974:244:184
status: NEW104 The location of residues that were mutated to cysteine to test for the effect of cross-linking between NBD1 and NBD2 (P517C/I1050C), NBD1 and TMD2 (L443C/S909C), ICL1 and ICL3 (D177C/N820C), TM segments 2 and 11 (C137/A935C) or 6 and 12 (T333C/L975C) are shown.
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ABCB4 p.Leu975Cys 22700974:104:244
status: NEW195 Accordingly, we introduced cysteines in regions of TM segments 6 (T333C) and 12 (L975C) predicted to lie at or close to the extracellular surface of the cell (Fig. 1).
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ABCB4 p.Leu975Cys 22700974:195:81
status: NEW196 The T333C and L975C mutations were selected because we previously showed that they had little effect on P-gp activity and treatment of the single cysteine mutants with a thiol-reactive derivative of verapamil had little effect on activity (43).
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ABCB4 p.Leu975Cys 22700974:196:14
status: NEW197 Since the T333C and L975C mutations were predicted to reside at or close to the extracellular surface of the cell, we performed cross-linking analysis on intact cells expressing the mutant.
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ABCB4 p.Leu975Cys 22700974:197:20
status: NEW200 Fig. 6A shows that P-gp mutant T333C/L975C was efficiently cross-linked (b0e; 90% efficiency) when intact cells were treated with BMOE.
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ABCB4 p.Leu975Cys 22700974:200:37
status: NEW204 We then tested the effect of the stimulators (tariquidar, P12) and inhibitor (P10) of ATPase activity on cross-linking of mutant T333C/L975C.
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ABCB4 p.Leu975Cys 22700974:204:135
status: NEW218 C, cells expressing mutant T333C/L975C were pretreated with 0.1 mM P10, 0.1 mM P12, 0.03 mM tariquidar (Tar), or no compound (None) and then incubated in the absence (afa;) or presence (af9;) of 0.5 mM BMOE. Samples were subjected to immunoblot analysis.
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ABCB4 p.Leu975Cys 22700974:218:33
status: NEW237 Evidence that ATP hydrolysis appears to cause lateral movement or rotation of the helices were the observations that ATP hydrolysis was required for cross-linking of mutant L332C(TM6)/L975C (50) and ATP hydrolysis shifted cross-linking of V982C in TM12 from L339C to F343C in TM6 (51).
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ABCB4 p.Leu975Cys 22700974:237:184
status: NEW