ABCMdb

ABCB1 p.Gln725Cys

Predicted by SNAP2:	A: N (66%), C: N (66%), D: N (72%), E: N (66%), F: N (87%), G: N (72%), H: N (93%), I: N (57%), K: N (53%), L: N (72%), M: N (87%), N: N (87%), P: N (57%), R: D (53%), S: N (82%), T: N (82%), V: N (61%), W: N (66%), Y: N (93%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: N, F: D, G: D, H: N, I: D, K: N, L: D, M: D, N: N, P: D, R: N, S: D, T: D, V: D, W: D, Y: D,

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[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
74 Mutant Verapamil S50 (µM) Vinblastine S50 (µM) Colchicine S50 (µM) F711C 9.8 + - 1.1 2.3 + - 0.2 410 + - 20 V712C 10.0 + - 0.8 1.9 + - 0.2 320 + - 40 V713C 9.9 + - 1.3 1.8 + - 0.2 340 + - 40 G714C 14.1 + - 2.1 1.9 + - 0.3 410 + - 30 V715C 9.8 + - 1.3 1.8 + - 0.3 330 + - 40 F716C 10.1 + - 1.0 2.1 + - 0.1 340 + - 30 C717C 10.0 + - 1.5 2.3 + - 0.3 390 + - 20 A718C 10.5 + - 1.3 2.0 + - 0.1 330 + - 30 I719C 10.5 + - 1.5 1.9 + - 0.2 370 + - 30 I720C 12.4 + - 0.7 2.4 + - 0.2 390 + - 10 N721C 13.0 + - 1.2 1.7 + - 0.2 380 + - 40 G722C ND ND ND G723C 11.1 + - 0.4 2.0 + - 0.3 410 + - 20 L724C 12.0 + - 1.5 2.4 + - 0.3 340 + - 30 Q725C 11.7 + - 1.1 2.0 + - 0.1 390 + - 20 P726C 11.9 + - 1.0 1.9 + - 0.2 450 + - 30 A727C 21.2 + - 3.1 1.9 + - 0.2 480 + - 40 F728C 48.7 + - 2.2 2.7 + - 0.3 830 + - 90 A729C 34.3 + - 3.0 2.3 + - 0.2 760 + - 80 I730C 12.0 + - 1.1 2.0 + - 0.1 420 + - 30 I731C 13.5 + - 1.9 1.9 + - 0.2 410 + - 30 Cys-less 11.6 + - 1.3 2.1 + - 0.3 400 + - 40 into the endoplasmic reticulum during synthesis of the protein. Login to comment
97 Samples of the isolated P-gp were mixed with lipid and assayed for ATPase activity and compared with untreated P-gp. All of the mutants, except Q725C, P726C, F728C and A729C, were unaffected by treatment with MTS-verapamil (Figure 2A). Login to comment
99 By contrast, the ATPase activity of mutants Q725C, F728C and A729C increased 2.6-, 4.7and 4.5-fold respectively, compared with untreated mutant P-gp. Login to comment
100 Although mutants Q725C, F728C and A729C showed increased ATPase activity after treatment with MTS-verapamil, the activities were less than 50% of the verapamil-stimulated ATPase activity of Cys-less P-gp (maximum 11.1-fold stimulation; Figure 2B). Login to comment
101 We have shown previously that mutants Q725C, F728C and A729C were stimulated more than 10-fold with verapamil [37]. Login to comment
103 Therefore it was possible that reaction of MTS-verapamil with mutants Q725C, F728C and A729C was incomplete or that they were completely modified but the bound verapamil was in a sub-optimal conformation for activation of ATPase activity. Login to comment
104 To distinguish between these two possibilities, mutants Q725C, F728C and A729C (activated by 0.3 mM MTS-verapamil) were treated with or without 3 mM MTS-verapamil. Login to comment
109 In contrast, labelling of mutants Q725C or F728C with 3 mM MTS-verapamil increased their ATPase activities further Figure 2 Effect of MTS-verapamil on basal ATPase activity of TM7 cysteine mutants (A) His-tagged Cys-less or mutants F711C/I731C P-gps were expressed in HEK-293 cells and solubilized with n-dodecyl-β-D-maltoside. Insoluble material was removed by centrifugation and the supernatants were treated with or without 0.3 mM MTS-verapamil. Login to comment
112 (B) Cys-less, Q725C, F728C and A729C P-gp mutants were treated with or without 3 mM MTS-verapamil and His-tagged P-gp isolated by nickel-chelate chromatography. Equivalent amounts of P-gp were mixed with lipid and ATPase activity determined in the presence (+) or absence (-) of 1 mM verapamil (Ver). Login to comment
117 Therefore mutants Q725C and F728C were chosen for further investigation because their labelling with MTS-verapamil appeared to mimic the interaction of P-gp with unmodified verapamil. Login to comment
118 We then tested whether drug substrates could protect mutants Q725C and F728C from being labelled by MTS-verapamil. Login to comment
119 HEK-293 cells expressing mutants Q725C or F728C were solubilized with n-dodecyl-β-D-maltoside and then treated with or without saturating levels of verapamil (3 mM), cyclosporin A (0.2 mM), colchicine (10 mM) or trans-(E)-flupentixol (0.6 mM). Login to comment
123 Figure 3 shows that the substrates verapamil and cyclosporin A protected mutant F728C, but not mutant Q725C, from labelling with MTS-verapamil. Login to comment
132 Exposure of the MTS-verapamil labelled F728C mutant to DTT caused a 75% decrease in ATPase activity (see Figure 3 Inhibition of MTS-verapamil labelling of mutants Q725C and F728C by substrates HEK-293 cells expressing mutants Q725C or F728C were solubilized with n-dodecyl- β-D-maltoside. Insoluble material was removed by centrifugation. Login to comment
140 Mutant F728C was treated with or without Figure 4 Effect of drug substrates and inhibitors on the ATPase activity of mutants F728C and Q725C before and after labelling with MTS-verapamil HEK-293 cells expressing His-tagged mutants F728C or Q725C were solubilized with n-dodecyl-β-D-maltoside and then incubated in the absence (A and C) or presence (B and D) of 3 mM MTS-verapamil. Login to comment
154 To distinguish between these possibilities, we compared the properties of mutant Q725C with mutant F728C. Login to comment
155 The activity of mutant Q725C was very similar to that of mutant F728C when assayed in the presence of calcein-AM, demecolcine, verapamil, cyclosporin A or trans-(E)-flupentixol before and after labelling with MTS-verapamil (Figures 4C and 4D). Login to comment
187 For comparison, we tested the cross-linking characteristics of mutant Q725C with a second cysteine residue introduced at positions Leu65 , Ile306 or Phe343 . Login to comment
228 Residue Gln725 did not appear to directly participate in drug binding since the mutant Q725C did not show any changes in apparent affinity for verapamil, vinblastine or colchicine (Table 1). Login to comment

[hide] Multiple transport-active binding sites are availa... PLoS One. 2013 Dec 5;8(12):e82463. doi: 10.1371/journal.pone.0082463. eCollection 2013. Chufan EE, Kapoor K, Sim HM, Singh S, Talele TT, Durell SR, Ambudkar SV
Multiple transport-active binding sites are available for a single substrate on human P-glycoprotein (ABCB1).
PLoS One. 2013 Dec 5;8(12):e82463. doi: 10.1371/journal.pone.0082463. eCollection 2013., [PMID:24349290]

Abstract [show]
P-glycoprotein (Pgp, ABCB1) is an ATP-Binding Cassette (ABC) transporter that is associated with the development of multidrug resistance in cancer cells. Pgp transports a variety of chemically dissimilar amphipathic compounds using the energy from ATP hydrolysis. In the present study, to elucidate the binding sites on Pgp for substrates and modulators, we employed site-directed mutagenesis, cell- and membrane-based assays, molecular modeling and docking. We generated single, double and triple mutants with substitutions of the Y307, F343, Q725, F728, F978 and V982 residues at the proposed drug-binding site with cys in a cysless Pgp, and expressed them in insect and mammalian cells using a baculovirus expression system. All the mutant proteins were expressed at the cell surface to the same extent as the cysless wild-type Pgp. With substitution of three residues of the pocket (Y307, Q725 and V982) with cysteine in a cysless Pgp, QZ59S-SSS, cyclosporine A, tariquidar, valinomycin and FSBA lose the ability to inhibit the labeling of Pgp with a transport substrate, [(125)I]-Iodoarylazidoprazosin, indicating these drugs cannot bind at their primary binding sites. However, the drugs can modulate the ATP hydrolysis of the mutant Pgps, demonstrating that they bind at secondary sites. In addition, the transport of six fluorescent substrates in HeLa cells expressing triple mutant (Y307C/Q725C/V982C) Pgp is also not significantly altered, showing that substrates bound at secondary sites are still transported. The homology modeling of human Pgp and substrate and modulator docking studies support the biochemical and transport data. In aggregate, our results demonstrate that a large flexible pocket in the Pgp transmembrane domains is able to bind chemically diverse compounds. When residues of the primary drug-binding site are mutated, substrates and modulators bind to secondary sites on the transporter and more than one transport-active binding site is available for each substrate.

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No. Sentence Comment
7 In addition, the transport of six fluorescent substrates in HeLa cells expressing triple mutant (Y307C/Q725C/V982C) Pgp is also not significantly altered, showing that substrates bound at secondary sites are still transported. Login to comment
55 For biochemical studies, crude membranes were prepared from High-Five insect cells infected with baculovirus coding for cysless WT, single mutants Y307C, F343C, Q725C, F728C, F978C, V982C, double mutants Y307C/V982C, F343C/V982C, Q725C/V982C, F728C/V982C, and a triple mutant Y307C/Q725C/V982C. Login to comment
61 However, CsA, tariquidar and valinomycin lose almost completely this ability to inhibit IAAP photo-labeling when residues Y307, Q725 and V982 are mutated to cysteine (i.e., Y307C/Q725C/V982C mutant). Login to comment
73 Inhibition of IAAP labeling for single mutants Q725C, Y307C, F728C and V982C (upper graphs) and for double Q725C/V982C, Y307C/V982C, F728C/V982C, and triple Y307C/Q725C/V982C (lower graphs) mutants at different concentrations of (A) CsA and (B) tariquidar, are shown. Inhibition of IAAP labeling for cysless WT is included in all graphs, as a reference. Login to comment
74 Autoradiograms corresponding to cysless, V982C and Y307C/Q725C/V982C, as representative examples of complete inhibition, partial inhibition and no inhibition of IAAP-labeling, respectively, are shown at the top of the figure. Login to comment
83 The rest of the mutants, Q725C, Y307C (and their corresponding double and triple mutants) and F728 show intermediate levels of basal ATPase activity. Login to comment
90 Mutation(s) CsA Tariquidar Max Inhibition (%) IC50 (&#b5;M) Max Inhibition (%) IC50 (&#b5;M) Cysless WT 86 &#b1; 3 0.05 &#b1; 0.01 97 &#b1; 4 0.14 &#b1; 0.03 Q725C 24 &#b1; 4 -- 37 -- Q725C/V982C 11 -- 22 -- Y307C 35 &#b1; 2 -- ND -- Y307C/V982C 46 -- ND -- F728C 48 -- 40 -- F728C/V982C 49 -- ND -- V982C 56 0.40 64 0.70 Y307C/Q725C/ V982C 12 -- 23 -- F978C 86 0.54 73 3.6 Mean values with standard errors are reported when more than two experiments were carried out; otherwise only average values are reported. Login to comment
93 Valinomycin also stimulated ATP hydrolysis of all mutants except V982C and Q725C/V982C. Login to comment
94 It is interesting to observe that the effect of the V982C mutation is not dominant in the rest of the double mutants and even in the triple mutant Y307C/Q725C/V982C, in which case valinomycin does stimulate ATP hydrolysis. Login to comment
95 FSBA also stimulates the ATP hydrolysis of most of the mutants, with the exception of the double F728C/V982C and the triple Y307C/Q725C/V982C mutant. Login to comment
108 In Figure 4A, representative histograms show the cell surface expression for single (Y307C), double (Y307C/V982C) and triple (Y307C/ Q725C/V982C) mutants. Login to comment
114 Even with the triple (Y307C/Q725C/V982C) mutant the efflux of none of the above-mentioned substrates is completely abolished, although many of these substrates are transported at lower levels when compared to the cysless WT Pgp (Table 2). Login to comment
116 Figure 5A shows the transport of rhodamine 123 (Rh123), which is normal for the single (Y307C) and double (Y307C/V982C) mutants but is decreased considerably for the triple (Y307C/Q725C/V982C) mutant. Login to comment
123 NBD-CsA loses the ability to inhibit the IAAP labeling of the triple (Y307C/Q725C/V982C) Figure 2. Login to comment
141 As expected, the corresponding double mutants (Q725C/V982C; F728C/V982C; Figure 3. Login to comment
151 Nonetheless, Y307C/V982C and the triple mutant Y307C/ Q725C/V982C show some rescue of the NBD-CsA transport. Login to comment
160 The middle panel shows the same for the double mutant Y307C/V982C, and the right panel for the triple mutant Y307C/Q725C/V982C. Login to comment
162 The conformation sensitivity towards CsA of single (Y307C), double (Y307C/V982C) and triple (Y307C/Q725C/V982C) mutants was similar to cysless WT Pgp, as shown in the three panels, respectively. Login to comment
175 Mutation(s) Cell surface expression Transport function CalAM BD-PRA NBD-CsA Rh123 Dauno BD-PAC Y307C 100 90-100 80-90 90-100 90-100 90-100 90-100 Q725C 100 90-100 90-100 90-100 90-100 90-100 90-100 F728C 100 90-100 80-90 90-100 90-100 90-100 90-100 V982C 100 90-100 80-100 <10 90-100 90-100 90-100 F343C 50-60 90-100 80-100 90-100 90-100 90-100 90-100 F978C 100 90-100 90-100 90-100 90-100 90-100 90-100 Y307C/ V982C 100 90-100 50-60 50-60 90-100 90-100 90-100 Q725C/ V982C 100 90-100 80-90 <20 90-100 90-100 90-100 F728C/ V982C 30-40 55-65 30-40 <20 70-80 50-60 90-100 F343C/ V982C 70-80 90-100 80-90 <20 90-100 90-100 90-100 Y307/ Q725C/ V982C 100 90-100 30-40 50-60 70-80 60-70 90-100 For cell surface expression, the cells were incubated with MRK-16 antibody for 30 min followed by FITC-labeled anti-mouse secondary antibody for 30 min. Login to comment
211 The transport functions of single (Y307C), double (Y307/V982C) and triple (Y307C/Q725C/V982C) mutants are differentially affected. Login to comment
238 Here, we report QZ59-SSS, CsA, tariquidar, valinomycin and FSBA lost the ability to inhibit IAAP-labeling of Q725C to the same extent as cysless WT Pgp. Login to comment
240 The partial inhibition of IAAP labeling observed for single mutants (Y307C, Q725C, F728C, and V982C) and even double mutants (Y307C/V982C, Q725C/V982C, F728C/V982C) is indicative of some drug interaction with Pgp (Figure 1). Login to comment
251 In all mutants, even the triple Y307C/Q725C/V982C, ATP hydrolysis is inhibited by both CsA and tariquidar. Login to comment
257 In steady-state conditions, calcein-AM and bodipy-placitaxel are transported by the triple (Y307C/Q725C/V982C) mutant to the same extent as cysless WT Pgp. Login to comment
261 Therefore, NBD-CsA does not bind to its primary site on Y307C/Q725C/V982C, but to a secondary site, where it is transported at 50-60% of the rate of cysless WT Pgp. Login to comment
328 Effect of QZ59-SSS-sulfur on the photocrosslinking of cysless WT and mutant Pgpswith IAAP. Inhibition of IAAP-labeling for cysless WT and for triple mutant Y307C/Q725C/V982C at different concentrations of QZ59-SSS-sulfur are shown (graph). Login to comment
333 Inhibition of IAAP-labeling for single mutants Q725C, Y307C and V982C (upper graph) and for double (Q725C/V982C and Y307C/ V982C) and triple (Y307C/Q725C/V982C) mutants (lower graph) at different concentrations of valinomycin are shown. Inhibition of IAAP-labeling of cysless WT is included in both graphs, as a reference. Login to comment
338 Effect of FSBA on the photocrosslinking of cysless WT and mutant Pgps with IAAP. Inhibition of IAAP-labeling of cysless WT and triple (Y307C/Q725C/V982C) mutant at different concentrations of FSBA are shown (graph). Login to comment