ABCB4 p.Phe728Cys
| Predicted by SNAP2: | A: D (63%), C: N (57%), D: D (85%), E: D (75%), G: D (75%), H: D (71%), I: D (63%), K: D (85%), L: N (53%), M: N (53%), N: D (59%), P: D (80%), Q: D (59%), R: D (80%), S: D (63%), T: D (66%), V: D (63%), W: D (63%), Y: N (72%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: 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, W: D, Y: N, |
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[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.Phe728Cys 17636884:74:200
status: NEW146 Samples were then subjected to immunoblot analysis. Figure 5C shows that in the absence of vinblastine, almost all of the mutant L339C(TM6)/F728C- (TM7) P-gp was cross-linked in the presence of ADP or ATP.
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ABCB4 p.Phe728Cys 17636884:146:140
status: NEW149 In contrast, the cross-linking efficiency of mutant L339C(TM6)/F728C- (TM7) in the absence of vinblastine was reduced in the presence of AMP‚PNP.
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ABCB4 p.Phe728Cys 17636884:149:63
status: NEW156 To further inhibit the ATPase activity of mutant L339C(TM6)/F728C- (TM7), the E556Q(NBD1) and E1201Q(NBD2) catalytic carboxylate mutations were introduced.
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ABCB4 p.Phe728Cys 17636884:156:60
status: NEW192 Cross-linking of mutant L339C(TM6)/F728C- (TM7) (Figure 6) was observed to be ~4-fold faster in the presence of ATP than in the presence of AMP‚PNP.
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ABCB4 p.Phe728Cys 17636884:192:35
status: NEW245 In contrast, we did not observe any detectable difference in the apparent affinity for vinblastine in the presence of ATP (Figures 6 and 7) during cross-linking of mutant L339C(TM6)/F728C- (TM7).
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ABCB4 p.Phe728Cys 17636884:245:182
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
254 For example, covalent labeling of F728C (TM7) (22), L65C (TM1) (57), or I306C (TM5) (24) with a thiol-reactive derivative of verapamil increased basal ATPase activity of P-gp by 7-12-fold.
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ABCB4 p.Phe728Cys 22700974:254:34
status: NEW247 For example, covalent labeling of F728C (TM7) (22), L65C (TM1) (57), or I306C (TM5) (24) with a thiol-reactive derivative of verapamil increased basal ATPase activity of P-gp by 7-12-fold.
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ABCB4 p.Phe728Cys 22700974:247:34
status: NEW