ABCMdb

ABCB1 p.Leu65Cys

Predicted by SNAP2:	A: N (72%), C: N (66%), D: N (53%), E: N (61%), F: N (93%), G: N (53%), H: N (61%), I: N (87%), K: N (57%), M: N (93%), N: N (72%), P: N (57%), Q: N (93%), R: N (66%), S: N (78%), T: N (87%), V: N (87%), W: N (82%), Y: N (78%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: N, 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] Location of the rhodamine-binding site in the huma... J Biol Chem. 2002 Nov 15;277(46):44332-8. Epub 2002 Sep 9. Loo TW, Clarke DM
Location of the rhodamine-binding site in the human multidrug resistance P-glycoprotein.
J Biol Chem. 2002 Nov 15;277(46):44332-8. Epub 2002 Sep 9., 2002-11-15 [PMID:12223492]

Abstract [show]
The human multidrug resistance P-glycoprotein (P-gp) pumps a wide variety of structurally diverse compounds out of the cell. It is an ATP-binding cassette transporter with two nucleotide-binding domains and two transmembrane (TM) domains. One class of compounds transported by P-gp is the rhodamine dyes. A P-gp deletion mutant (residues 1-379 plus 681-1025) with only the TM domains retained the ability to bind rhodamine. Therefore, to identify the residues involved in rhodamine binding, 252 mutants containing a cysteine in the predicted TM segments were generated and reacted with a thiol-reactive analog of rhodamine, methanethiosulfonate (MTS)-rhodamine. The activities of 28 mutants (in TMs 2-12) were inhibited by at least 50% after reaction with MTS-rhodamine. The activities of five mutants, I340C(TM6), A841C(TM9), L975C(TM12), V981C(TM12), and V982C(TM12), however, were significantly protected from inhibition by MTS-rhodamine by pretreatment with rhodamine B, indicating that residues in TMs 6, 9, and 12 contribute to the binding of rhodamine dyes. These results, together with those from previous labeling studies with other thiol-reactive compounds, dibromobimane, MTS-verapamil, and MTS-cross-linker substrates, indicate that common residues are involved in the binding of structurally different drug substrates and that P-gp has a common drug-binding site. The results support the "substrate-induced fit" hypothesis for drug binding.

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130 The activity of M68C, was inhibited by 44%, whereas that of mutant L65C was increased (189%). Login to comment
164 Two mutants, L65C and F343C, showed increased activity after treatment with MTS-rhodamine. Login to comment
165 Because mutant L65C had only 51% of the verapamil-stimulated ATPase activity relative to that of the Cys-less parent (22), treatment with MTS treatment essentially restored the activity of the mutant to that of the Cys-less P-gp. Login to comment

[hide] Transmembrane segment 1 of human P-glycoprotein co... Biochem J. 2006 Jun 15;396(3):537-45. Loo TW, Bartlett MC, Clarke DM
Transmembrane segment 1 of human P-glycoprotein contributes to the drug-binding pocket.
Biochem J. 2006 Jun 15;396(3):537-45., 2006-06-15 [PMID:16492138]

Abstract [show]
P-glycoprotein (P-gp; ABCB1) actively transports a broad range of structurally unrelated compounds out of the cell. An important step in the transport cycle is coupling of drug binding with ATP hydrolysis. Drug substrates such as verapamil bind in a common drug-binding pocket at the interface between the TM (transmembrane) domains of P-gp and stimulate ATPase activity. In the present study, we used cysteine-scanning mutagenesis and reaction with an MTS (methanethiosulphonate) thiol-reactive analogue of verapamil (MTS-verapamil) to test whether the first TM segment [TM1 (TM segment 1)] forms part of the drug-binding pocket. One mutant, L65C, showed elevated ATPase activity (10.7-fold higher than an untreated control) after removal of unchanged MTS-verapamil. The elevated ATPase activity was due to covalent attachment of MTS-verapamil to Cys65 because treatment with dithiothreitol returned the ATPase activity to basal levels. Verapamil covalently attached to Cys65 appears to occupy the drug-binding pocket because verapamil protected mutant L65C from modification by MTS-verapamil. The ATPase activity of the MTS-verapamil-modified mutant L65C could not be further stimulated with verapamil, calcein acetoxymethyl ester or demecolcine. The ATPase activity could be inhibited by cyclosporin A but not by trans-(E)-flupentixol. These results suggest that TM1 contributes to the drug-binding pocket.

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4 One mutant, L65C, showed elevated ATPase activity (10.7-fold higher than an untreated control) after removal of unchanged MTS-verapamil. Login to comment
6 Verapamil covalently attached to Cys65 appears to occupy the drug-binding pocket because verapamil protected mutant L65C from modification by MTS-verapamil. Login to comment
7 The ATPase activity of the MTS-verapamil-modified mutant L65C could not be further stimulated with verapamil, calcein acetoxymethyl ester or demecolcine. Login to comment
41 A series of double cysteine mutants containing L65C in TM1 with another cysteine in TMD2 (C-terminal TMD containing TM7-TM12) predicted to line the drug-binding pocket [34] (i.e. F942C or T945C in TM11 and L975C, V981C, V982C, G984C or A985C in TM12) were also constructed for cross-linking analysis. Login to comment
44 The mutant L65C was also stably expressed in BHK cells (baby hamster kidney cells). Login to comment
45 Briefly, BHK cells were transfected with the His-tagged mutant L65C cDNA in pMT21 as described previously [35]. Login to comment
47 BHK or HEK-293 cells expressing mutant L65C were also grown in the presence of 10 µM cyclosporin A for 24 h because it acts as a pharmacological/specific chemical chaperone to increase the yield of mature enzyme [37]. Login to comment
49 Reaction with MTS-verapamil and measurement of ATPase activity HEK-293 or BHK cells expressing His-tagged mutant L65C from 20 (10 cm diameter) plates were washed three times with PBS (10 mM sodium phosphate, pH 7.4, and 150 mM NaCl) and then suspended in a total volume of 1.5 ml of TBS (Tris-buffered saline; 10 mM Tris/HCl, pH 8.0, and 150 mM NaCl). Login to comment
60 Disulphide cross-linking analysis Mutants L65C, F942C, T945C, L975C, V981C, V982C, G984C, A985C, L65C/F942C, L65C/T945C, L65C/975C, L65C/V981C, L65C/V982C, L65C/G984C and L65C/A985C were transiently expressed in HEK-293 cells. Login to comment
72 The locations of residues L65C in TM1, I306C in TM5 and F343C in TM6 are shown. Login to comment
86 A potential complicating factor in our earlier study to screen for mutants Figure 2 Reaction and ATPase activity of mutant L65C treated with MTS-verapamil (A) Schematic reaction of a thiol group with MTS-verapamil and attachment of verapamil to the protein via a disulphide bond. Login to comment
91 (C) His-tagged mutant L65C expressed in HEK-293 cells was solubilized with n-dodecyl-β-D-maltoside, treated with various concentrations of MTS-verapamil and isolated by nickel-chelate chromatography. Login to comment
98 Verapamil Colchicine Vinblastine Mutant Vmax (%)* S50 (µM)† Vmax (%) S50 (µM) Vmax (%) S50 (µM) M51C 101 11.0 + - 0.6 96 391 + - 36 94 2.4 + - 0.2 V52C ND ND ND ND ND ND V53C 104 12.0 + - 0.2 101 389 + - 30 102 2.2 + - 0.1 G54C ND ND ND ND ND ND T55C 114 10.3 + - 1.1 95 418 + - 22 91 2.2 + - 0.1 L56C 103 12.2 + - 0.3 87 440 + - 41 95 2.5 + - 0.2 A57C 108 11.3 + - 0.3 98 377 + - 34 92 2.4 + - 0.2 A58C 90 12.5 + - 0.2 94 434 + - 20 95 2.6 + - 0.3 I59C 115 11.2 + - 0.8 95 380 + - 33 114 2.5 + - 0.2 I60C 102 11.1 + - 0.7 91 408 + - 18 110 2.5 + - 0.2 H61C 97 54.0 + - 5.0 61 912 + - 86 105 5.4 + - 0.4 G62C ND ND ND ND ND ND A63C 114 10.5 + - 1.2 99 362 + - 42 105 2.0 + - 0.3 G64C 106 45.0 + - 6.0 88 613 + - 55 60 2.4 + - 0.1 L65C 72 9.3 + - 1.1 112 368 + - 32 78 2.0 + - 0.2 P66C 95 13.0 + - 0.5 86 480 + - 39 97 2.8 + - 0.4 L67C 101 12.3 + - 0.3 106 423 + - 21 100 2.3 + - 0.1 M68C 119 9.7 + - 1.1 105 365 + - 32 92 2.3 + - 0.2 M69C 107 11.8 + - 0.6 110 431 + - 25 108 2.2 + - 0.1 L70C 94 11.4 + - 0.7 90 413 + - 18 98 2.3 + - 0.1 V71C 106 11.9 + - 0.3 90 370 + - 27 102 2.5 + - 0.5 Cys-less 100 12.0 + - 1.0 100 412 + - 48 100 2.2 + - 0.3 * Maximum activity relative to that of Cys-less P-gp. Login to comment
111 We found that the activity of only one cysteine mutant in TM1 (L65C) was permanently activated after treatment with MTS-verapamil (Figure 2B). Login to comment
112 Mutant L65C showed a 10.7-fold increase in activity after modification with 0.3-1 mM MTS-verapamil compared with an untreated control. Login to comment
115 To test whether activation of mutant L65C by MTS-verapamil was due to covalent attachment of verapamil, we treated the modified mutant with 20 mM of the reducing agent DTT (dithiothreitol) and then measured ATPase activity. Figure 3 shows that the presence of DTT almost completely abolished the enhanced ATPase activity of mutant L65C. Login to comment
116 After treatment with DTT, the activity of MTS-verapamil-treated mutant L65C Figure 3 Effect of DTT on mutant L65C modified with MTS-verapamil His-tagged mutant L65C was expressed in HEK-293 cells. Login to comment
123 We then tested whether labelling of mutant L65C with MTS-verapamil could be inhibited by the drug substrate verapamil. Login to comment
124 The rationale for these experiments was that if residue L65C contributed to binding of MTS-verapamil, then the presence of verapamil should protect mutant L65C from being modified. Login to comment
125 We also tested whether Rhodamine B had any effect on labelling of mutant L65C by MTS-verapamil. Login to comment
130 Accordingly, BHK cells stably expressing mutant L65C were used for these studies because they do not show any variation in P-gp expression as observed with transient expression in HEK293 cells. Login to comment
133 BHK cells stably expressing His-tagged L65C were harvested, solubilized with n-dodecyl-β-D-maltoside and then incubated with saturating levels of verapamil (2 mM) or Rhodamine B (3 mM) for 10 min at 20◦ C. The samples were then reacted for Figure 4 MTS-verapamil labelling of mutant L65C is inhibited by verapamil BHK cells stably expressing His-tagged mutant L65C were solubilized with n-dodecyl-β- D-maltoside. Insoluble material was removed by centrifugation. Equivalent amounts of supernatant were incubated for 10 min at 20◦C in the presence of no drug (No Drug), 2 mM verapamil (Ver) or 3 mM Rhodamine B (Rhod). Login to comment
136 10 min at 20◦ C in the absence or presence of 0.1 mM MTS-verapamil, as this was the minimum concentration that gave almost complete modification of mutant L65C (Figure 2C). Login to comment
139 The eluted samples were mixed with lipid, sonicated and assayed for ATPase activity. Figure 4 shows that the presence of verapamil reduced the labelling efficiency of mutant L65C by MTS-verapamil by more than 80%. Login to comment
140 In contrast, the presence of Rhodamine B during labelling with MTS-verapamil caused very little (<10%) reduction in labelling of mutant L65C (Figure 4). Login to comment
142 We then tested whether other drug substrates could still interact with mutant L65C that had been covalently labelled with MTS-verapamil. Login to comment
143 The rationale was that substrates will not affect the ATPase activity of MTS-verapamil-modified mutant L65C if they occupy the same binding site as verapamil or if their binding site significantly overlaps that of verapamil, but will further stimulate or inhibit the activity if their binding sites are different from that of verapamil. Login to comment
145 BHK cells stably expressing His-tagged mutant L65C were solubilized with n-dodecyl-β-D-maltoside and then treated with or without 0.3 mM MTS-verapamil. Login to comment
146 His-tagged P-gp was then isolated by nickel-chelate chromatography, mixed with lipids from E. coli, sonicated and assayed for ATPase Figure 5 Effect of drug substrates and inhibitors on the ATPase activity of mutant L65C before and after labelling with MTS-verapamil BHK cells stably expressing His-tagged mutant L65C were solubilized with n-dodecyl-β- D-maltoside and then incubated in the absence (Untreated; A) or presence (+MTS-Verapamil; B) of 0.3 mM MTS-verapamil. Login to comment
152 We used E. coli lipids because the higher basal ATPase activity made it easier for us to detect for inhibition of activity. Figure 5(A) shows that in unmodified mutant L65C, verapamil stimulated the ATPase activity 4.6-fold, whereas calcein-AM and demecolcine stimulated the ATPase activity 7.0and 5.9-fold respectively. Login to comment
154 When mutant L65C was modified with MTS-verapamil however, its basal activity was increased 4.6-fold compared with an untreated sample (Figure 5B). Login to comment
157 These results suggest that modification of mutant L65C by MTS-verapamil blocks interaction of P-gp with drug substrates calcein-AM, demecolcine and trans-(E)-flupentixol, but not its interaction with cyclosporin. Login to comment
158 The characteristics of the mutant L65C after labelling with MTS-verapamil suggested that Cys65 lined the drug-binding pocket. Another method to test for this possibility is to test whether Cys65 can be cross-linked to cysteine residues in the TMs of TMD2 that were previously shown to be involved in drug binding [15,17]. Login to comment
160 Accordingly, Figure 6 Disulphide cross-linking of P-gp mutants (A) Membranes were prepared from HEK-293 cells (A) expressing mutants L65C, L65C/T945C, L65C/V982C, L65C/G984C or L65C/A985C. Login to comment
162 (B) Membranes prepared from HEK-293 cells expressing mutants L65C, V982C or L65C/V982C were treated with 0.2 mM M11M for various times at 4◦C. The reactions were stopped by addition of SDS sample buffer containing EDTA and subjected to immunoblot analysis on SDS/7.5% polyacrylamide gels. Login to comment
186 Mutant L65C(TM1)/ I306C(TM5) showed only a 2.8-fold increase in activity after treatment with MTS-verapamil (Figure 7), whereas mutant L65C showed >10-fold increase in activity (Figure 2B). Login to comment
189 DISCUSSION Only one mutant in TM1, L65C, was able to adopt a conformation that permanently hydrolysed ATP after modification with MTS-verapamil. Login to comment
191 Attachment of MTS-verapamil to Cys65 seems to mimic interaction of P-gp with verapamil because the ATPase activity of the MTS-verapamil-treated mutant L65C was very similar to that of untreated mutant L65C in the presence of saturating levels of verapamil. Login to comment
200 Arrangement of the residues in TM1 in a cylindrical helix (Figure 8A) shows that residues that react with MTS-verapamil (L65C; the present study) show alterations in substrate specificity when mutated (H61C, G644C and L65C; [44,45]) or show ATP-dependent cross-linking (M68 and M69; [30]) and occupy one face of the helix. Login to comment
204 Since the present study has shown that L65C is involved in binding of verapamil, then cross-linking studies between TM1 and TM11 during ATP hydrolysis [30] would indicate that conformational changes in TM1 and TM11 probably contribute to the release of drug substrate during ATP hydrolysis. Login to comment
212 While the ATPase activities of both mutants L65C and I306C modified by MTS-verapamil could not be further stimulated by calcein-AM or demecolcine, the inhibition of their activities were different. Login to comment
213 The ATPase activity of mutant I306C modified by MTS-verapamil could not be inhibited by cyclosporin A or trans-(E)-flupentixol [40], whereas that of MTS-verapamil- modified mutant L65C was inhibited only by cyclosporin A (Figure 5). Login to comment
214 This difference in sensitivity to inhibition by cyclosporin A suggests that both verapamil and cyclosporin A could bind simultaneously to mutant L65C, whereas covalent Figure 8 Arrangement of TM1 residues in a cylindrical helix and MTS-verapamil in the drug-binding pocket (A) The residues of TM1 are arranged in a cylindrical helix. Login to comment
215 Residues that show alterations in substrate specificity when mutated (H61C, G644C and L65C, circled) or show ATP-dependent cross-linking (Met68 and Met69 , boxed) with residues in TM11 are shown as occupying one face of the helix. Login to comment
227 Labelling of mutant L65C (TM1) (the present study) or Ile306 (TM5) [40] with MTS-verapamil stimulated the ATPase activity of P-gp by more than 8-10-fold but labelling of the double cysteine mutant L65C(TM1)/I306C(TM5) with MTS-verapamil reduced the verapamil-stimulated ATPase activity of the enzyme (Figure 7). Login to comment

[hide] Transmembrane segment 7 of human P-glycoprotein fo... Biochem J. 2006 Oct 15;399(2):351-9. Loo TW, Bartlett MC, Clarke DM
Transmembrane segment 7 of human P-glycoprotein forms part of the drug-binding pocket.
Biochem J. 2006 Oct 15;399(2):351-9., 2006-10-15 [PMID:16813563]

Abstract [show]
P-gp (P-glycoprotein; ABCB1) protects us by transporting a broad range of structurally unrelated compounds out of the cell. Identifying the regions of P-gp that make up the drug-binding pocket is important for understanding the mechanism of transport. The common drug-binding pocket is at the interface between the transmembrane domains of the two homologous halves of P-gp. It has been shown in a previous study [Loo, Bartlett and Clarke (2006) Biochem. J. 396, 537-545] that the first transmembrane segment (TM1) contributed to the drug-binding pocket. In the present study, we used cysteine-scanning mutagenesis, reaction with an MTS (methanethiosulfonate) thiol-reactive analogue of verapamil (termed MTS-verapamil) and cross-linking analysis to test whether the equivalent transmembrane segment (TM7) in the C-terminal-half of P-gp also contributed to drug binding. Mutation of Phe728 to cysteine caused a 4-fold decrease in apparent affinity for the drug substrate verapamil. Mutant F728C also showed elevated ATPase activity (11.5-fold higher than untreated controls) after covalent modification with MTS-verapamil. The activity returned to basal levels after treatment with dithiothreitol. The substrates, verapamil and cyclosporin A, protected the mutant from labelling with MTS-verapamil. Mutant F728C could be cross-linked with a homobifunctional thiol-reactive cross-linker to cysteines I306C(TM5) and F343C(TM6) that are predicted to line the drug-binding pocket. Disulfide cross-linking was inhibited by some drug substrates such as Rhodamine B, calcein acetoxymethyl ester, cyclosporin, verapamil and vinblastine or by vanadate trapping of nucleotides. These results indicate that TM7 forms part of the drug-binding pocket of P-gp.

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No. Sentence Comment
90 We have previously shown that modification of specific cysteines in TM1 (L65C) and in TM5 (I306C) with MTS-verapamil caused permanent activation of P-gp ATPase activity (an 8to 11-fold increase in activity compared with untreated P-gp) [27,36]. Login to comment
161 cysteines facing the drug-binding pocket that may be cross-linked with F728C are L65C(TM1), I306C(TM5) and F343C(TM6) because they were covalently modified with MTS-verapamil (L65C and I306C) or with MTS-Rhodamine (F343C). Login to comment

[hide] Arginines in the first transmembrane segment promo... J Biol Chem. 2008 Sep 5;283(36):24860-70. Epub 2008 Jul 2. Loo TW, Bartlett MC, Clarke DM
Arginines in the first transmembrane segment promote maturation of a P-glycoprotein processing mutant by hydrogen bond interactions with tyrosines in transmembrane segment 11.
J Biol Chem. 2008 Sep 5;283(36):24860-70. Epub 2008 Jul 2., 2008-09-05 [PMID:18596043]

Abstract [show]
A key goal is to correct defective folding of mutant ATP binding cassette (ABC) transporters, as they cause diseases such as cystic fibrosis. P-glycoprotein (ABCB1) is a useful model system because introduction of an arginine at position 65 of the first transmembrane (TM) segment could repair folding defects. To determine the mechanism of arginine rescue, we first tested the effects of introducing arginines at other positions in TM1 (residues 52-72) of a P-glycoprotein processing mutant (G251V) that is defective in folding and trafficking to the cell surface (20% maturation efficiency). We found that arginines introduced into one face of the TM1 helix (positions 52, 55, 56, 59, 60, 62, 63, 66, and 67) inhibited maturation, whereas arginines on the opposite face of the helix promoted (positions 64, 65, 68, and 71) or had little effect (positions 61, and 69) on maturation. Arginines at positions 61, 64, 65, and 68 appeared to lie close to the drug binding sites as they reduced the apparent affinity for drug substrates such as vinblastine and verapamil. Therefore, arginines that promoted maturation may face an aqueous drug translocation pathway, whereas those that inhibited maturation may face the lipid bilayer. The highest maturation efficiencies (60-85%) were observed with the Arg-65 and Arg-68 mutants. Mutations that removed hydrogen bond acceptors (Y950F/Y950A or Y953F/Y953A) in TM11 predicted to lie close to Arg-65 or Arg-68 inhibited maturation but did not affect maturation of the G251V parent. Therefore, arginine may rescue defective folding by promoting packing of the TM segments through hydrogen bond interactions.

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185 It was shown that mutant L65C could be labeled with a thiol-reactive derivative of verapamil to cause permanent activation of ATPase activity. Login to comment

[hide] Novel cGMP efflux inhibitors identified by virtual... J Med Chem. 2012 Apr 12;55(7):3049-57. Epub 2012 Mar 20. Sager G, Orvoll EO, Lysaa RA, Kufareva I, Abagyan R, Ravna AW
Novel cGMP efflux inhibitors identified by virtual ligand screening (VLS) and confirmed by experimental studies.
J Med Chem. 2012 Apr 12;55(7):3049-57. Epub 2012 Mar 20., [PMID:22380603]

Abstract [show]
Elevated intracellular levels of cyclic guanosine monophosphate (cGMP) may induce apoptosis, and at least some cancer cells seem to escape this effect by increased efflux of cGMP, as clinical studies have shown that extracellular cGMP levels are elevated in various types of cancer. The human ATP binding cassette (ABC) transporter ABCC5 transports cGMP out of cells, and inhibition of ABCC5 may have cytotoxic effects. Sildenafil inhibits cGMP efflux by binding to ABCC5, and in order to search for potential novel ABCC5 inhibitors, we have identified sildenafil derivates using structural and computational guidance and tested them for the cGMP efflux effect. Eleven compounds from virtual ligand screening (VLS) were tested in vitro, using inside-out vesicles (IOV), for inhibition of cGMP efflux. Seven of 11 compounds predicted by VLS to bind to ABCC5 were more potent than sildenafil, and the two most potent showed K(i) of 50-100 nM.

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85 This is in accordance with a study on ABCB1 where cysteine-scanning mutagenesis and reaction with a methanethiosulfonate (MTS) thiol reactive analogue of verapamil (MTS verapamil) showed that mutants Leu65Cys (TMH1) and Ile306Cys (TMH5) modified with MTS verapamil have slightly different characteristics, indicating that the bound verapamil molecules in these mutants have different orientations and that the protein can function quite well with the substrate bound in different orientations.28 Theoretically, even though 10 different conformations of each ligand were evaluated by ICM during VLS, focusing on only the ligand orientation with best score may lead to missing out better inhibitors oriented in a pose yielding a poorer score. Login to comment

[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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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. Login to comment
247 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. Login to comment