ABCMdb

ABCB1 p.Ile306Cys

Predicted by SNAP2:	A: D (63%), C: D (53%), D: D (85%), E: D (85%), F: N (53%), G: D (75%), H: D (80%), K: D (85%), L: D (63%), M: N (82%), N: D (71%), P: D (85%), Q: D (80%), R: D (85%), S: D (66%), T: D (63%), V: N (72%), W: D (85%), Y: D (80%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, K: D, L: N, 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] Permanent activation of the human P-glycoprotein b... J Biol Chem. 2003 Jun 6;278(23):20449-52. Epub 2003 Apr 23. Loo TW, Bartlett MC, Clarke DM
Permanent activation of the human P-glycoprotein by covalent modification of a residue in the drug-binding site.
J Biol Chem. 2003 Jun 6;278(23):20449-52. Epub 2003 Apr 23., 2003-06-06 [PMID:12711602]

Abstract [show]
The human multidrug resistance P-glycoprotein (ABCB1) transports a broad range of structurally diverse compounds out of the cell. The transport cycle involves coupling of drug binding in the transmembrane domains with ATP hydrolysis. Compounds such as verapamil stimulate ATPase activity. We used cysteine-scanning mutagenesis of the transmembrane segments and reaction with the thiol-reactive substrate analog of verapamil, methanethiosulfonate (MTS)-verapamil, to test whether it caused permanent activation of ATP hydrolysis. Here we report that one mutant, I306C(TM5) showed increased ATPase activity (8-fold higher than untreated) when treated with MTS-verapamil and isolated by nickel-chelate chromatography. Drug substrates that either enhance (calcein acetoxymethyl ester, demecolcine, and vinblastine) or inhibit (cyclosporin A and trans-(E)-flupentixol) ATPase activity of Cys-less or untreated mutant I306C P-glycoprotein did not affect the activity of MTS-verapamil-treated mutant I306C. Addition of dithiothreitol released the covalently attached verapamil, and ATPase activity returned to basal levels. Pretreatment with substrates such as cyclosporin A, demecolcine, verapamil, vinblastine, or colchicine prevented activation of mutant I306C by MTS-verapamil. The results suggest that MTS-verapamil reacts with I306C in a common drug-binding site. Covalent modification of I306C affects the long range linkage between the drug-binding site and the distal ATP-binding sites. This results in the permanent activation of ATP hydrolysis in the absence of transport. Trapping mutant I306C in a permanently activated state indicates that Ile-306 may be part of the signal to switch on ATP hydrolysis when the drug-binding site is occupied.

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No. Sentence Comment
5 Drug substrates that either enhance (calcein acetoxymethyl ester, demecolcine, and vinblastine) or inhibit (cyclosporin A and trans-(E)-flupentixol) ATPase activity of Cys-less or untreated mutant I306C P-glycoprotein did not affect the activity of MTS-verapamil-treated mutant I306C. Login to comment
7 Pretreatment with substrates such as cyclosporin A, demecolcine, verapamil, vinblastine, or colchicine prevented activation of mutant I306C by MTS-verapamil. Login to comment
8 The results suggest that MTS-verapamil reacts with I306C in a common drug-binding site. Login to comment
9 Covalent modification of I306C affects the long range linkage between the drug-binding site and the distal ATP-binding sites. Login to comment
11 Trapping mutant I306C in a permanently activated state indicates that Ile-306 may be part of the signal to switch on ATP hydrolysis when the drug-binding site is occupied. Login to comment
70 One mutant, I306C, showed an 8-fold increase in ATPase activity after treatment with MTS-verapamil. Login to comment
72 Fig. 1 shows that maximal stimulation occurred after treatment of mutant I306C with 0.1-1 mM MTS-verapamil, and half-maximal stimulation was 39 ␮M. Nickel-chelate chromatography was effective in removing unreacted MTS-verapamil because the activity of Cys-less P-gp remained at basal levels even after pretreatment with 1 mM MTS-verapamil (Fig. 1). Login to comment
73 To test whether all of mutant I306C had been modified by MTS-verapamil, we assayed for stimulation or inhibition of the ATPase activity by other drug substrates. Login to comment
74 The rationale is that if a significant amount of unmodified mutant I306C is present, then the presence of other substrates or inhibitors should affect the activity of the mutant. Login to comment
76 Fig. 2 shows the effect of various stimulators and inhibitors on the ATPase activity of mutant I306C before and after treatment with MTS-verapamil. Login to comment
77 Before treatment with MTS-verapamil, the ATPase activity of mutant I306C was stimulated by demecolcine, calcein-AM, verapamil, and vinblastine by 11.9-, 9.8-, 7-, and 3-fold, respectively. Login to comment
79 When mutant I306C was pretreated with MTS-verapamil, the presence of other stimulators or inhibitors of P-gp had little effect on its ATPase activity (7.7-8.1-fold versus 8-fold increase) (Fig. 2). Login to comment
80 This inability to further stimulate or inhibit the activity of the MTS-verapamil-treated mutant I306C suggests that most (more than 90%) of the mutant I306C P-gp was modified and that covalent attachment of verapamil in the drug-binding site blocks access of other drug substrates to the drug-binding site. Login to comment
82 Activation of mutant I306C after treatment with MTS-verapamil. Login to comment
83 Histidine-tagged Cys-less or mutant I306C P-gps were expressed in HEK 293 cells and solubilized with n-dodecyl beta-D-maltoside. Insoluble material was removed by centrifugation, and equivalent amounts of the supernatant were treated with various concentrations of MTS-verapamil. Login to comment
90 Effect of drug substrates and inhibitors on activity of mutant I306C before and after labeling with MTS-verapamil. Login to comment
91 Histidine-tagged mutant I306C P-gps were expressed in HEK 293 cells, solubilized with n-dodecyl beta-D-maltoside, and treated with 0.3 mM MTS-verapamil (ϩ MTS-verapamil) or without MTS-verapamil (Untreated). Login to comment
96 If modified I306C retained a "native" structure, then it should be possible to restore basal levels of ATPase activity if covalent attachment of MTS-verapamil was removed. Login to comment
97 Accordingly modified I306C was treated with 20 mM dithiothreitol to reduce the disulfide bond between P-gp and MTS-verapamil, and then P-gp was reisolated by nickel-chelate chromatography. Login to comment
98 Fig. 3 shows that reduction of the disulfide bond by dithiothreitol reduced the activity of the mutant as its ATPase activity was only slightly higher (1.5-fold) than the untreated I306C mutant. Login to comment
99 Mutant I306C remained active after removal of the covalently bound MTS-verapamil since it retained the ability to be stimulated by verapamil (6.9-fold). Login to comment
100 The characteristics of the ATPase activity of modified I306C mutant were also examined. Login to comment
103 Fig. 3 shows that mutant I306C that had been labeled with MTS-verapamil was still inhibited by vanadate. Login to comment
104 In addition, the modified mutant I306C had a Km for ATP (1.1 mM) that was very similar to that of Cys-less P-gp (1 mM) (data not shown). Login to comment
105 If MTS-verapamil occupied the drug-binding site in mutant I306C, then pretreatment of the mutant with other drug substrates should protect it from labeling by MTS-verapamil if they shared a common drug-binding site. Login to comment
106 Accordingly mutant I306C was treated with or without the drug substrates calcein-AM, demecolcine, verapamil, vinblastine, cyclosporin A, or colchicine before treatment with MTS-verapamil for 10 min at 22 °C. P-gp was isolated by nickel-chelate chromatography, and ATPase activity was determined. Login to comment
107 Fig. 4 shows that all of the drug substrates protected mutant I306C from labeling with MTS-verapamil since they prevented modification and activation of I306C by MTS-verapamil by 70-85%. Login to comment
109 DISCUSSION Treatment of mutant I306C with MTS-verapamil followed by removal of unreacted MTS-verapamil caused the mutant to FIG. 3. Login to comment
110 Reversal of MTS-verapamil activation of mutant I306C. Login to comment
111 Histidine-tagged mutant I306C P-gps were expressed in HEK 293 cells, solubilized with n-dodecyl beta-D-maltoside, and treated without MTS-verapamil (Untreated) or with 0.3 mM MTS-verapamil (ϩ MTS-verapamil) The proteins were isolated by nickel-chelate chromatography and then incubated on ice with 20 mM dithiothreitol (ϩ DTT) or without dithiothreitol (None) for 20 min. Login to comment
114 The ATPase activity of an equivalent amount of the MTS-verapamil-treated I306C P-gp was determined in the presence of 0.2 mM sodium vanadate (ϩVi). Login to comment
117 Protection of mutant I306C from labeling by MTS-verapamil with substrates and inhibitors. Login to comment
118 Histidine-tagged mutant I306C P-gp was expressed in HEK 293 cells and solubilized with n-dodecyl beta-D-maltoside. Insoluble material was removed by centrifugation. Login to comment
124 Model of interaction between mutant I306C and verapamil or MTS-verapamil. Login to comment
136 Evidence that MTS-verapamil tethered to I306C mimics the interaction of the mutant with verapamil is that both verapamil and MTS-verapamil caused similar activation of ATPase activity (7-8-fold). Login to comment
137 In addition, activation of I306C by MTS-verapamil was reversed by dithiothreitol, and the dithiothreitol-treated mutant could rebind verapamil. Login to comment
138 Both MTS-verapamil and verapamil must bind to the same drug-binding site because the activity of MTS-verapamil-treated I306C was not affected by verapamil or other stimulators and inhibitors of P-gp, and mutant I306C can be protected from labeling by verapamil and the other substrates and inhibitors. Login to comment
150 This study also shows that TM5 must contribute residues to the drug-binding site since other substrates and inhibitors could protect I306C from labeling by MTS-verapamil. Login to comment
151 We had predicted that I306C likely lined the drug-binding site because it could be cross-linked to other cysteines in TMs 10, 11, and 12 with thiol-reactive cross-linker substrates (20). Login to comment

[hide] Simultaneous binding of two different drugs in the... J Biol Chem. 2003 Oct 10;278(41):39706-10. Epub 2003 Aug 7. Loo TW, Bartlett MC, Clarke DM
Simultaneous binding of two different drugs in the binding pocket of the human multidrug resistance P-glycoprotein.
J Biol Chem. 2003 Oct 10;278(41):39706-10. Epub 2003 Aug 7., 2003-10-10 [PMID:12909621]

Abstract [show]
The human multidrug resistance P-glycoprotein (P-gp, ABCB1) transports a wide variety of structurally diverse compounds out of the cell. The drug-binding pocket of P-gp is located in the transmembrane domains. Although occupation of the drug-binding pocket by one molecule is sufficient to activate the ATPase activity of P-gp, the drug-binding pocket may be large enough to accommodate two different substrates at the same time. In this study, we used cysteine-scanning mutagenesis to test whether P-gp could simultaneously interact with the thiol-reactive drug substrate, Tris-(2-maleimidoethyl)amine (TMEA) and a second drug substrate. TMEA is a cross-linker substrate of P-gp that allowed us to test for stimulation of cross-linking by a second substrate such as calcein-acetoxymethyl ester, colchicine, demecolcine, cyclosporin A, rhodamine B, progesterone, and verapamil. We report that verapamil induced TMEA cross-linking of mutant F343C(TM6)/V982C(TM12). By contrast, no cross-linked product was detected in mutants F343C(TM6), V982C(TM12), or F343C(TM6)/V982C(TM12) in the presence of TMEA alone. The verapamil-stimulated ATPase activity of mutant F343C(TM6)/V982C(TM12) in the presence of TMEA decreased with increased cross-linking of the mutant protein. These results show that binding of verapamil must induce changes in the drug-binding pocket (induced-fit mechanism) resulting in exposure of residues F343C(TM6)/V982C(TM12) to TMEA. The results also indicate that the common drug-binding pocket in P-gp is large enough to accommodate both verapamil and TMEA simultaneously and suggests that the substrates must occupy different regions in the common drug-binding pocket.

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Sentences [show]
No. Sentence Comment
180 Residue I306C covalently binds to methanethiosulfonate-verapamil, and the P-gp ATPase activity is permanently activated. Login to comment

[hide] Disulfide cross-linking analysis shows that transm... J Biol Chem. 2004 Feb 27;279(9):7692-7. Epub 2003 Dec 10. Loo TW, Bartlett MC, Clarke DM
Disulfide cross-linking analysis shows that transmembrane segments 5 and 8 of human P-glycoprotein are close together on the cytoplasmic side of the membrane.
J Biol Chem. 2004 Feb 27;279(9):7692-7. Epub 2003 Dec 10., 2004-02-27 [PMID:14670948]

Abstract [show]
Human P-glycoprotein (P-gp) transports a wide variety of structurally diverse compounds out of the cell. Knowledge about the packing of the transmembrane (TM) segments is essential for understanding the mechanism of drug recognition and transport. We used cysteine-scanning mutagenesis and disulfide cross-linking analysis to determine which TM segment in the COOH half of P-gp was close to TMs 5 and 6 since these segments in the NH(2) half are important for drug binding. An active Cys-less P-gp mutant cDNA was used to generate 240 double cysteine mutants that contained 1 cysteine in TMs 5 or 6 and another in TMs 7 or 8. The mutants were subjected to oxidative cross-linking analysis. No disulfide cross-linking was observed in the 140 TM6/TM7 or TM6/TM8 mutants. By contrast, cross-linking was detected in several P-gp TM5/TM8 mutants. At 4 degrees C, when thermal motion is low, P-gp mutants N296C(TM5)/G774C(TM8), I299C(TM5)/F770C(TM8), I299C(TM5)/G774C(TM8), and G300C(TM5)/F770C(TM8) showed extensive cross-linking with oxidant. These mutants retained drug-stimulated ATPase activity, but their activities were inhibited after treatment with oxidant. Similarly, disulfide cross-linking was inhibited by vanadate trapping of nucleotide. These results indicate that significant conformational changes must occur between TMs 5 and 8 during ATP hydrolysis. We revised the rotational symmetry model for TM packing based on our results and by comparison to the crystal structure of MsbA (Chang, G. (2003) J. Mol. Biol. 330, 419-430) such that TM5 is adjacent to TM8, TM2 is adjacent to TM11, and TMs 1 and 7 are next to TMs 6 and 12, respectively.

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No. Sentence Comment
74 Transmembrane segments 5 and 6 are particularly interesting because labeling of mutants I306C (TM5) and F343C(TM6) with methanethiosulfonate (MTS)-verapamil or MTS-rhodamine, respectively, caused permanent activation of P-gp ATPase activity (23, 44). Login to comment
92 Residues that react with the MTS derivatives of drug substrates verapamil (I306C) and rhodamine (F343C) to stimulate ATPase activity are shown as closed circles. Login to comment

[hide] The drug-binding pocket of the human multidrug res... Biochemistry. 2004 Sep 28;43(38):12081-9. Loo TW, Bartlett MC, Clarke DM
The drug-binding pocket of the human multidrug resistance P-glycoprotein is accessible to the aqueous medium.
Biochemistry. 2004 Sep 28;43(38):12081-9., 2004-09-28 [PMID:15379547]

Abstract [show]
P-Glycoprotein (P-gp) is an ATP-dependent drug pump that transports a broad range of compounds out of the cell. Cross-linking studies have shown that the drug-binding pocket is at the interface between the transmembrane (TM) domains and can simultaneously bind two different drug substrates. Here, we determined whether cysteine residues within the drug-binding pocket were accessible to the aqueous medium. Cysteine mutants were tested for their reactivity with the charged thiol-reactive compounds sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) and [2-(trimethylammonium)ethyl)]methanethiosulfonate (MTSET). Residue Ile-306(TM5) is close to the verapamil-binding site. It was changed to cysteine, reacted with MTSES or MTSET, and assayed for verapamil-stimulated ATPase activity. Reaction of mutant I306C(TM5) with either compound reduced its affinity for verapamil. We confirmed that the reduced affinity for verapamil was indeed due to introduction of a charge at position 306 by demonstrating that similar effects were observed when Ile-306 was replaced with arginine or glutamic acid. Mutant I306R showed a 50-fold reduction in affinity for verapamil and very little change in the affinity for rhodamine B or colchicine. MTSES or MTSET modification also affected the cross-linking pattern between pairs of cysteines in the drug-binding pocket. For example, both MTSES and MTSET inhibited cross-linking between I306C(TM5) and I868C(TM10). Inhibition was enhanced by ATP hydrolysis. By contrast, cross-linking of cysteine residues located outside the drug-binding pocket (such as G300C(TM5)/F770C(TM8)) was not affected by MTSES or MTSET. These results indicate that the drug-binding pocket is accessible to water.

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No. Sentence Comment
92 A good candidate is mutant I306C in TM5 (Figure 1A). Login to comment
93 Cys-306 is close to the verapamil-binding site because treatment of mutant I306C with thiol-reactive MTS-verapamil resulted in covalent attachment of the verapamil to Cys-306 (46). Login to comment
102 Histidine-tagged mutant I306C was expressed in HEK 293 cells, isolated by nickel-chelate chromatography, and mixed with lipid. Login to comment
103 Mutant I306C was then treated with 1 mM MTSET or 5 mM MTSES and assayed for verapamil-stimulated ATPase activity after removal of untreated MTSET or MTSES using gel filtration columns. Login to comment
107 Figure 2 shows that treatment of mutant I306C with either MTSET or MTSES profoundly affected the verapamil-stimulated ATPase activity. Login to comment
108 Treatment of mutant I306C with MTSET reduced the apparent affinity of P-gp for verapamil from 24 µM to greater than 600 µM. Treatment of mutant I306C with MTSES caused about a 10-fold reduction in apparent affinity (260 µM) for verapamil, as well as about 50% decrease in ATPase activity. Login to comment
109 The change in activity was apparently due to modification of residue I306C since 1 mM MTSET or 5 mM MTSES had no effect on basal or verapamil-stimulated ATPase activity of cysteine-less P-gp (data not shown). Login to comment
116 Labeling of residue I306C with MTS-verapamil permanently activated P-gp ATPase activity (46). Login to comment
132 Wild-type P-gp showed an 8.2-fold maximal activation and FIGURE 2: Effect of MTSES or MTSET on verapamil-stimulated ATPase activity of mutant I306C. Login to comment
133 Histidine-tagged mutant I306C P-gp was expressed in HEK 293 cells and isolated by nickel-chelate chromatography. Login to comment
196 Accordingly, cells expressing mutants L531C(NBD1)/C1074C(NBD2), I306C- (TM5)/I868C(TM10), L339C(TM6)/F942C(TM12), and S222C(TM4)/G872C(TM10) were treated with MTSEA, MTSES, or MTSET before cross-linking. Login to comment
201 Cross-linking of mutants with cysteines within the drug-binding pocket (mutants I306C- (TM5)/I868C(TM10), L339C(TM6)/F942C(TM12), and S222C(TM4)/G872C(TM10)) was also completely inhibited by MTSEA. Login to comment
212 Samples of mutants L339C/(TM6)/F942C(TM11), I306C- (TM5)/I868C(TM10), and S222C(TM4)/G872C(TM10) were then treated with (+) or without (-) M17M cross-linker for 15 min at 22 °C. Mutant G300C(TM5)/F770C(TM8) was cross-linked with 1 mM copper (phenanthroline)3 (CuP) for 15 min at 22 °C. In panel B, whole cells expressing mutants L531C(NBD1)/C1074C(NBD2), I306C(TM5)/I868C(TM10), L339C(TM6)/F942C(TM11), or S222C- (TM4)/G872C(TM10) were incubated for 10 min at 22 °C in the presence of 2.5 mM MTSEA, 10 mM MTSES, or 1 mM MTSET. Login to comment
229 Modification of mutant I306C by these compounds resulted in a change in affinity for verapamil (Figure 2). Login to comment
272 The mutant, however, could still interact with hydrophobic substrates such as cyclosporin A (Figure 6B) These results are consistent with the labeling studies involving I306C with MTS-rhodamine and MTS-verapamil that show distinct binding sites for the verapamil and rhodamine B (30). Login to comment

[hide] Recent advances in molecular modeling and medicina... Curr Drug Metab. 2006 Feb;7(2):205-17. Srinivas E, Murthy JN, Rao AR, Sastry GN
Recent advances in molecular modeling and medicinal chemistry aspects of phospho-glycoprotein.
Curr Drug Metab. 2006 Feb;7(2):205-17., [PMID:16472108]

Abstract [show]
Phospho-glycoprotein (P-gp) is an efflux transporter expressed in many organs (ex: kidney, lung, liver and spleen) and in hormone producing or responsive tissues (ex: adrenal cortex, testis and placenta). It is involved in many important physiological functions. Among them the major one is extrusion of xenobiotics in order to detoxify the cells. This property of P-gp is associated with multidrug resistance (MDR) for many pathological conditions. While the experimental determination of three-dimensional structure is not yet successful, the transmembrane (TM) 5, 6, 11 and 12 are sensitive to mutations and contain substrate binding sites. Designing of potential and selective inhibitors of P-gp is still hampered by a lack of information upon the three dimensional structure of P-gp. The design of P-gp inhibitors was traditionally driven by quantitative structure activity relationship studies, which is complicated by factors such as different types of assays, multiple drug binding sites and diverse chemical structures. Clearly a conclusive and predictive SAR does not seem to be practical, despite progress in the last few years towards more specific SAR suggesting well defined structural features responsible for activity. Advances made recently in solving the crystal structure of prokaryotic ATP binding cassette proteins (ABC) transporters, Ec-MsbA, Vc-MsbA and BtuCD yielded suitable templates for construction of homology models of P-gp. Few molecular dynamics (MD) simulations aimed at elucidating the functional dynamics of ABC transporters have provided useful insights to their mechanism and structure. The present review aims at the general overview of importance, expression, structure, organization and drug binding sites of P-gp. This review also highlights recent developments in the homology modeling, molecular dynamics simulations of P-gp and progress in QSAR, pharmacophore modeling of P-gp modulators.

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No. Sentence Comment
156 It is also reported that MTS-verapamil is responsible for the permanent activation of ATP-hydrolysis by covalent modification of a residue I306C in the drug-binding site [80]. 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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No. Sentence Comment
72 The locations of residues L65C in TM1, I306C in TM5 and F343C in TM6 are shown. 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
229 Therefore the presence of a permanently bound verapamil molecule at I306C may interfere with movement between TM1 and TM11 during ATP hydrolysis (Figure 1B), resulting in inhibition of ATPase activity. 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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Sentences [show]
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] 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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No. Sentence Comment
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

[hide] Drug rescue distinguishes between different struct... Biochemistry. 2013 Oct 15;52(41):7167-9. doi: 10.1021/bi401269m. Epub 2013 Oct 2. Loo TW, Clarke DM
Drug rescue distinguishes between different structural models of human P-glycoprotein.
Biochemistry. 2013 Oct 15;52(41):7167-9. doi: 10.1021/bi401269m. Epub 2013 Oct 2., [PMID:24083983]

Abstract [show]
There is no high-resolution crystal structure of the human P-glycoprotein (P-gp) drug pump. Homology models of human P-gp based on the crystal structures of mouse or Caenorhabditis elegans P-gps show large differences in the orientation of transmembrane segment 5 (TM5). TM5 is one of the most important transmembrane segments involved in drug-substrate interactions. Drug rescue of P-gp processing mutants containing an arginine at each position in TM5 was used to identify positions facing the lipid or internal aqueous chamber. Only the model based on the C. elegans P-gp structure was compatible with the drug rescue results.

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13 It was found that labeling of the I306C mutant with a thiol-reactive derivative of the substrate verapamil activated ATPase activity ~8-fold12 and labeling was blocked by verapamil. Login to comment