ABCMdb

ABCB1 p.Phe343Cys

Predicted by SNAP2:	A: D (66%), C: D (63%), D: D (85%), E: D (85%), G: D (75%), H: D (85%), I: N (53%), K: D (85%), L: N (53%), M: N (82%), N: D (80%), P: D (91%), Q: D (66%), R: D (80%), S: D (75%), T: D (71%), V: D (71%), W: D (71%), Y: N (53%),
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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Publications
[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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No. Sentence Comment
140 The activity of mutant F343C, however, was increased (347%) after treatment with MTS-rhodamine. Login to comment
164 Two mutants, L65C and F343C, showed increased activity after treatment with MTS-rhodamine. Login to comment
167 Mutant F343C showed about 3.5-fold increase in activity after treatment with MTS-rhodamine. Login to comment
168 Because mutant F343C had about 60% of the activity of Cys-less P-gp, reaction with MTS-rhodamine essentially causes a 2-fold increase in activity relative to the Cys-less parent. 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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No. Sentence Comment
116 It appears that the presence of verapamil increased the reactivity or proximity of F343C to TMEA. Login to comment
137 Mutant F343C was also treated with various concentrations of TMEA in the absence of verapamil (-Ver). Login to comment

[hide] Methanethiosulfonate derivatives of rhodamine and ... J Biol Chem. 2003 Dec 12;278(50):50136-41. Epub 2003 Oct 1. Loo TW, Bartlett MC, Clarke DM
Methanethiosulfonate derivatives of rhodamine and verapamil activate human P-glycoprotein at different sites.
J Biol Chem. 2003 Dec 12;278(50):50136-41. Epub 2003 Oct 1., 2003-12-12 [PMID:14522974]

Abstract [show]
The human multidrug resistance P-glycoprotein (P-gp, ABCB1) actively extrudes a broad range of potentially cytotoxic compounds out of the cell. Key steps in understanding the transport process are binding of drug substrates in the transmembrane domains, initiation of ATPase activity, and subsequent drug efflux. We used cysteine-scanning mutagenesis of the transmembrane segment residues and reaction with the thiol-reactive drug substrate analog of rhodamine, methane-thiosulfonate-rhodamine (MTS-rhodamine), to test whether P-gp could be trapped in an activated state with high levels of ATPase activity. The presence of such an activated P-gp could be used to further investigate P-gp-drug substrate interactions. Single cysteine mutants (149) were treated with MTS-rhodamine, and ATPase activities were determined after removal of unreacted MTS-rhodamine. One mutant, F343C(TM6), showed a 5.8-fold increase in activity after reaction with MTS-rhodamine. Pre-treatment of mutant F343C with rhodamine B protected it from activation by MTS-rhodamine, indicating that residue Cys-343 contributes to the rhodamine-binding site. The ATPase activity of MTS-rhodamine-treated mutant F343C, however, was not stimulated further by colchicine or calcein-AM. By contrast, verapamil and Hoechst 33342 stimulated and inhibited, respectively, the ATPase activity of the MTS-rhodamine-treated mutant F343C. These results indicate that the MTS-rhodamine binding site overlaps that of colchicine and calcein-AM but not that of verapamil and Hoechst 33342 within the common drug-binding pocket.

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No. Sentence Comment
6 Pre-treatment of mutant F343C with rhodamine B protected it from activation by MTS-rhodamine, indicating that residue Cys-343 contributes to the rhodamine-binding site. Login to comment
7 The ATPase activity of MTS-rhodamine-treated mutant F343C, however, was not stimulated further by colchicine or calcein-AM. Login to comment
8 By contrast, verapamil and Hoechst 33342 stimulated and inhibited, respectively, the ATPase activity of the MTS-rhodamine-treated mutant F343C. Login to comment
89 The covalent attachment of MTS-rhodamine to Cys-343 seems to mimic the interaction of MTS-rhodamine with Cys-less P-gp, because maximal stimulation of mutant F343C by MTS-rhodamine was 5.8-fold, whereas that of Cys-less P-gp was about 6.2-fold. Login to comment
90 Therefore, it is unlikely that labeling of mutant F343C by MTS-rhodamine is coincidental. Login to comment
93 The rationale is that substrates will not affect the activity of the MTS-rhodamine-treated mutant F343C if they occupy the same binding site as rhodamine but will further stimulate or inhibit the activity if their binding sites are distinct from that of rhodamine. Login to comment
94 Accordingly, mutant F343C was purified by nickel-chelate chromatography after incubation with or without 2 mM MTS-rhodamine, mixed with lipid and assayed for ATPase activity in the presence of different drug substrates. Login to comment
95 Fig. 3 shows that in the absence of substrate, covalent modification of F343C resulted in a 5.8-fold increase in activity over that of the untreated mutant F343C. Login to comment
96 The activity of untreated mutant F343C in the presence of calcein-AM or colchicine was stimulated 12or 9.2-fold, respectively. Login to comment
99 In the presence of verapamil, the MTS-rhodamine-labeled and untreated mutants F343C were stimulated 9.9and 10.3-fold, respectively (Fig. 3). Login to comment
101 Therefore, we tested whether Hoechst 33342 would affect the activity of the MTS-rhodamine-labeled F343C mutant. Login to comment
105 We then tested whether drug substrates could protect mutant F343C from modification by MTS-rhodamine. Login to comment
106 The rationale is that the presence of another drug substrate in the rhodamine-binding site will prevent mutant F343C from being labeled by MTS-rhodamine. Login to comment
107 Similarly, if the binding site of the drug substrate is distinct from that of rhodamine, then mutant F343C should still be labeled by MTS-rhodamine in the presence of these substrates. Login to comment
110 Accordingly, mutant F343C was pre-incubated with 10 mM colchicine, 10 mM verapamil, 3 mM rhodamine B, or no drug substrates, treated with or without 1 mM MTS-rhodamine and then isolated by nickel-chelate chromatography. Login to comment
111 The presence of colchicine or rhodamine B prevented MTS-rhodamine activation of mutant F343C (5.8-fold) by 77% (2-fold) and 60% (2.8-fold), respectively. Login to comment
114 Labeling of mutant F343C by MTS-rhodamine, however, showed little protection with verapamil. Login to comment
117 The presence of the reducing agent inhibited activation of mutant F343C by MTS-rhodamine by more than 80% (Fig. 4). Login to comment
118 FIG. 2. Effect of MTS-rhodamine treatment on the basal activity of Cys-less and F343C mutant P-gps. Login to comment
124 FIG. 3. Effect of drug substrates on the ATPase activity of mutant F343C before and after labeling with MTS-rhodamine. Login to comment
129 The characteristics of the ATPase activity of MTS-rhodamine-labeled mutant F343C were also examined. Login to comment
132 Fig. 4 shows that vanadate trapping inhibits the activity of the activated F343C mutant. Login to comment
133 Labeling of mutant F343C did not appear to affect the affinity of the enzyme for ATP. Login to comment
150 It is interesting that residue F343C is on the same face of the TM6 ␣-helix as residue I340C (Fig. 6A). Login to comment
155 Two observations suggest that MTS-rhodamine attached to F343C mimics the interaction of rhodamine compounds with P-gp. Login to comment
156 First, the ATPase activity of MTS-rhodamine-labeled F343C (5.8-fold) is similar to that observed with MTS-rhodamine and Cys-less P-gp (6.2-fold). Login to comment
157 Second, rhodamine B protected mutant F343C from labeling by MTS-rhodamine. Login to comment
158 Calcein-AM and colchicine did not affect the activity of MTS-rhodamine-treated mutant F343C. Login to comment
162 Verapamil, however, was able to further stimulate the activity of the MTS-rhodamine treated mutant F343C indicating that verapamil could still bind to the mutant protein. Login to comment
163 It is unlikely that verapamil increased activity by binding to unlabeled mutant F343C because labeling of mutant F343C by MTS-rhodamine was saturable (Fig. 2). Login to comment
164 We also found that subjecting MTS-rhodamine treated F343C to a second round of labeling with MTS-rhodamine did not increase ATPase activity (data not shown). Login to comment
166 Inhibition of MTS-rhodamine activation of mutant F343C. Login to comment
200 This may explain why both F343C and I340C can be labeled with MTS-rhodamine, but only F343C is activated upon labeling. 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
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 topography of transmembrane segment six is alt... J Biol Chem. 2004 Aug 13;279(33):34913-21. Epub 2004 Jun 10. Rothnie A, Storm J, Campbell J, Linton KJ, Kerr ID, Callaghan R
The topography of transmembrane segment six is altered during the catalytic cycle of P-glycoprotein.
J Biol Chem. 2004 Aug 13;279(33):34913-21. Epub 2004 Jun 10., 2004-08-13 [PMID:15192095]

Abstract [show]
Structural evidence has demonstrated that P-glycoprotein (P-gp) undergoes considerable conformational changes during catalysis, and these alterations are important in drug interaction. Knowledge of which regions in P-gp undergo conformational alterations will provide vital information to elucidate the locations of drug binding sites and the mechanism of coupling. A number of investigations have implicated transmembrane segment six (TM6) in drug-P-gp interactions, and a cysteine-scanning mutagenesis approach was directed to this segment. Introduction of cysteine residues into TM6 did not disturb basal or drug-stimulated ATPase activity per se. Under basal conditions the hydrophobic probe coumarin maleimide readily labeled all introduced cysteine residues, whereas the hydrophilic fluorescein maleimide only labeled residue Cys-343. The amphiphilic BODIPY-maleimide displayed a more complex labeling profile. The extent of labeling with coumarin maleimide did not vary during the catalytic cycle, whereas fluorescein maleimide labeling of F343C was lost after nucleotide binding or hydrolysis. BODIPY-maleimide labeling was markedly altered during the catalytic cycle and indicated that the adenosine 5'-(beta,gamma-imino)triphosphate-bound and ADP/vanadate-trapped intermediates were conformationally distinct. Our data are reconciled with a recent atomic scale model of P-gp and are consistent with a tilting of TM6 in response to nucleotide binding and ATP hydrolysis.

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No. Sentence Comment
130 Values refer to the mean Ϯ S.E. obtained from at least eight independent protein purification preparations. P-gp isoform Substrate affinity , Km Maximal activity, Vmax -Fold stimulationBasal Stimulated Basal Stimulated mM ␮mol Pi min-1 mg protein-1 Cys-less 0.58 Ϯ 0.06 0.38 Ϯ 0.04 0.58 Ϯ 0.15 1.46 Ϯ 0.30 2.9 Ϯ 0.3 V331C 0.50 Ϯ 0.06 0.26 Ϯ 0.02 0.45 Ϯ 0.05 1.54 Ϯ 0.20 3.5 Ϯ 0.3 T333C 0.49 Ϯ 0.05 0.23 Ϯ 0.02 0.35 Ϯ 0.04 1.22 Ϯ 0.15 3.3 Ϯ 0.1 F335C 0.40 Ϯ 0.05 0.24 Ϯ 0.03 0.65 Ϯ 0.15 1.61 Ϯ 0.31 2.2 Ϯ 0.2 S337C 0.53 Ϯ 0.06 0.26 Ϯ 0.04 0.59 Ϯ 0.10 1.67 Ϯ 0.23 3.2 Ϯ 0.4 L339C 0.51 Ϯ 0.07 0.31 Ϯ 0.04 0.57 Ϯ 0.07 1.47 Ϯ 0.15 2.9 Ϯ 0.3 G341C 0.40 Ϯ 0.04 0.24 Ϯ 0.02 0.42 Ϯ 0.03 1.12 Ϯ 0.09 3.1 Ϯ 0.5 F343C 0.41 Ϯ 0.04 0.26 Ϯ 0.03 0.47 Ϯ 0.04 1.17 Ϯ 0.15 2.6 Ϯ 0.3 generate stable covalent bonds with thiol groups under physiological conditions. Login to comment
155 In contrast, F343C in the native protein conformation was accessible to labeling with FM as adjudged by the 81 Ϯ 2% labeling extent (Lext), which was characterized by a half-life (t1/2 ϭ 47 Ϯ 8 min) almost 4 times slower than observed for G324C. Login to comment
166 Values refer to the mean Ϯ S.E. obtained from a minimum of three independent protein purification preparations. P-gp isoform Potency of drug effect Nicardipine, EC50 Vinblastine, EC50 Vanadate, IC50 ␮M ␮M ␮M Cys-less 3.2 Ϯ 0.3 4.2 Ϯ 0.6 4.0 Ϯ 0.4 V331C 3.3 Ϯ 0.4 7.2 Ϯ 1.7 3.2 Ϯ 0.4 T333C 2.3 Ϯ 0.2 4.6 Ϯ 0.4 3.9 Ϯ 0.8 F335C 2.3 Ϯ 0.4 4.2 Ϯ 0.8 5.5 Ϯ 1.1 S337C 2.7 Ϯ 0.5 4.1 Ϯ 1.0 5.8 Ϯ 0.8 L339C 2.1 Ϯ 0.3 5.1 Ϯ 0.8 4.2 Ϯ 0.7 G341C 3.9 Ϯ 0.5 4.0 Ϯ 0.6 6.8 Ϯ 1.3 F343C 2.1 Ϯ 0.3 5.6 Ϯ 2.7 2.7 Ϯ 0.8 FIG. 1. Login to comment
184 Surprisingly, mutant F343C, which was the only isoform labeled by the hydrophilic compound FM, reacted with the amphiphilic probe BM (Lext ϭ 68 Ϯ 11%) with a half-life of only 18 Ϯ 4 min. Login to comment
189 Isoform F343C was the only protein to label with the hydrophilic probe FM, and the labeling was significantly reduced by both AMP-PNP binding (Lext ϭ 29 Ϯ 3%) and vanadate trapping (Lext ϭ 26 Ϯ 4%). Login to comment
191 The residual labeled protein is likely to arise from the proportion of F343C P-gp that was not completely bound with AMP-PNP or vanadate-trapped. Login to comment
192 This was confirmed by similar labeling half-lives compared with nucleotide free F343C P-gp. Login to comment
193 F343C was the only isoform whose labeling with FM was affected by the catalytic cycle because all other mutant P-gp proteins remained inaccessible to labeling with this hydrophilic molecule. Login to comment
200 Isoforms T333C (t1/2 ϭ 16 Ϯ 3 min) and F343C (t1/2 ϭ 18 Ϯ 5 min) labeled with CM with significantly increased rates compared with the values observed in either basal conditions or in the presence of AMP-PNP, reflecting an increased accessibility of the introduced cysteine residues. Login to comment
203 Whereas under basal conditions V331C, L339C, and F343C were accessible to BM, only the latter was labeled (Lext ϭ 90 Ϯ 8%) after AMP-PNP binding to the protein (Fig. 4b). Login to comment
205 This was further confirmed by the observation that the half-life of labeling with BM in F343C was unaffected by the binding of AMP-PNP (t1/2 ϭ 18 Ϯ 1 min) compared with the basal state. Login to comment
206 Vanadate trapping of the F343C isoform also failed to alter either the extent (Lext ϭ 98 Ϯ 3%) or FIG. 4. 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
127 In this study [41], it was found that the ATPase activity of mutant F343C in TM6 could be permanently activated by covalent attachment of MTS-Rhodamine. 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
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
177 Cross-linking was due to linkage between Cys343 and Cys728 because cross-linked product was not detected on SDS/ PAGE when membranes prepared from HEK-293 cells transfected with the F343C or F728C single cysteine mutant cDNAs, or cotransfected with the F343C and F728C mutant cDNAs, were treated with the homobifunctional cross-linkers (results not shown). Login to comment

[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. Login to comment

[hide] Inhibition of multidrug resistance by adamantylgb3... J Biol Chem. 2008 Feb 22;283(8):4501-11. Epub 2007 Nov 13. De Rosa MF, Ackerley C, Wang B, Ito S, Clarke DM, Lingwood C
Inhibition of multidrug resistance by adamantylgb3, a globotriaosylceramide analog.
J Biol Chem. 2008 Feb 22;283(8):4501-11. Epub 2007 Nov 13., 2008-02-22 [PMID:18003606]

Abstract [show]
Multidrug resistance (MDR) via the ABC drug transporter (ABCB1), P-glycoprotein (P-gp/MDR1) overexpression, is a major obstacle in cancer chemotherapy. Many inhibitors reverse MDR but, like cyclosporin A (CsA), have significant toxicities. MDR1 is also a translocase that flips glucosylceramide inside the Golgi to enhance neutral glycosphingolipid (GSL) synthesis. We observed partial MDR1/globotriaosylceramide (Gb3) cell surface co-localization, and GSL removal depleted cell surface MDR1. MDR1 may therefore interact with GSLs. AdamantylGb3, a water-soluble Gb3 mimic, but not other GSL analogs, reversed MDR1-MDCK cell drug resistance. Cell surface MDR1 was up-regulated 1 h after treatment with CsA or adaGb3, but at 72 h, cell surface expression was lost. Intracellular MDR1 accumulated throughout, suggesting long term defects in plasma membrane MDR1 trafficking. AdaGb3 or CsA rapidly reduced rhodamine 123 cellular efflux. MDR1 also mediates gastrointestinal epithelial drug efflux, restricting oral bioavailability. Vinblastine apical-to-basal transport in polarized human intestinal C2BBe1 cells was significantly increased when adaGb3 was added to both sides, or to the apical side only, comparable with verapamil, a standard MDR1 inhibitor. Disulfide cross-linking of mutant MDR1s showed no binding of adaGb3 to the MDR1 verapamil/cyclosporin-binding site between surface proximal helices of transmembrane segments (TM) 6 and TM7, but rather to an adjacent site nearer the center of TM6 and the TM7 extracellular face, i.e. close to the bilayer leaflet interface. Verotoxin-mediated Gb3 endocytosis also up-regulated total MDR1 and inhibited drug efflux. Thus, a functional interplay between membrane Gb3 and MDR1 provides a more physiologically based approach to MDR1 regulation to increase the bioavailability of chemotherapeutic drugs.

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213 In contrast, no inhibition with adaGb3 pretreatment was seen for the F343C containing mutants. Login to comment
292 AdaGb3/MDR1 binding may be similar to colchicine, demecolcine, Hoescht 33342, or flupentixol that similarly do not inhibit TM6 F343C disulfide cross-linking (33). Login to comment

[hide] Cytosolic region of TM6 in P-glycoprotein: topogra... Biochemistry. 2008 Mar 25;47(12):3615-24. Epub 2008 Feb 28. Storm J, Modok S, O'Mara ML, Tieleman DP, Kerr ID, Callaghan R
Cytosolic region of TM6 in P-glycoprotein: topographical analysis and functional perturbation by site directed labeling.
Biochemistry. 2008 Mar 25;47(12):3615-24. Epub 2008 Feb 28., 2008-03-25 [PMID:18303860]

Abstract [show]
Reduced intracellular drug accumulation due to the activity of the drug efflux pump ABC (B1) is a major mechanism in the resistance of cancer cells to chemotherapy. ABC (B1) is a poly specific transporter, and the molecular mechanism of its complex translocation process remains to be elucidated. To understand the process will require information on the regions involved in drug binding and those that couple this event to nucleotide hydrolysis. The present investigation focuses on the cytosolic region of transmembrane helix 6 (TM6), which has been widely attributed with a central role in the translocation process. A series of ABC (B1) isoforms containing a unique cysteine within TM6 was constructed and the resultant proteins purified and reconstituted. Accessibility of the cysteines to covalent modification by maleimide reagents was measured for the basal, ATP bound and vanadate trapped conformations of each isoform. Residues at the two extremes of the TM6 region examined (amino acids 344 to 360) were considerably more accessible than the central segment, the latter of which also failed to undergo significant conformational changes during the catalytic cycle. Covalent modification of the cytosolic segment of TM6 did, however, attenuate drug stimulation of ATP hydrolysis and demonstrates an important role for this segment in coupling drug binding to ATP hydrolysis during translocation.

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239 This lack of change is in contrast to the considerable conformational changes previously observed in the segment between V331C to F343C (42). Login to comment
241 This is in good agreement with the data from Rothnie et al. demonstrating similar high accessibility for the adjacent residue, F343C (42). Login to comment

[hide] Drug-stimulated ATPase activity of human P-glycopr... J Biol Chem. 1997 Aug 22;272(34):20986-9. Loo TW, Clarke DM
Drug-stimulated ATPase activity of human P-glycoprotein requires movement between transmembrane segments 6 and 12.
J Biol Chem. 1997 Aug 22;272(34):20986-9., 1997-08-22 [PMID:9261097]

Abstract [show]
Transmembrane segments (TM) 6 and 12 are directly connected to the ATP-binding domain in each homologous half of P-glycoprotein and are postulated to be important for drug-protein interactions. Cysteines introduced into TM6 (L332C, F343C, G346C, and P350C) were oxidatively cross-linked to cysteines introduced into TM12 (L975C, M986C, G989C, and S993C, respectively). The pattern of cross-linking was consistent with a left-handed coiled coil arrangement of the two helices. To detect conformational changes between the helices during drug-stimulated ATPase activity, we tested the effects of substrates and ATP on cross-linking. Cyclosporin A, verapamil, vinblastine, and colchicine inhibited cross-linking of mutants F343C/M986C, G346C/G989C, and P350C/S993C. By contrast, ATP promoted cross-linking between only L332C/L975C. Enhanced cross-linking between L332C/L975C was due to ATP hydrolysis, since cross-linked product was not observed in the presence of ATP and vanadate, ADP, ADP and vanadate, or AMP-PNP. Cross-linking between P350C/S993C inhibited verapamil-stimulated ATPase activity by about 75%. Drug-stimulated ATPase activity, however, was fully restored in the presence of dithiothreitol. These results show that TM6 and TM12 undergo different conformational changes upon drug binding or during ATP hydrolysis, and that movement between these two helices is essential for drug-stimulated ATPase activity.

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68 To test these predictions, we introduced pairs of cysteines into a Cys-less mutant of P-glycoprotein to create the mutants F336C/S979C, L339C/V982C, F343C/M986C, G346C/G989C, and P350C/S993C. Login to comment
76 Fig. 1D shows that a product with reduced mobility on SDS-PAGE gels was present when mutants F343C/M986C, G346C/G989C, and P350C/ S993C were treated with oxidant. Login to comment
80 We also tested mutants F335C/L976C, L339C/S979C, F343C/F983C, G347C/A987C, and S351C/ V991C for cross-linking since they were predicted to lie on opposing faces of TM6 and TM12 modeled in a right-handed coiled-coil. Login to comment
100 The effect of nucleotides on cross-linking was also tested on mutants F343C/M986C, G346C/G989C, and P350C/S993C. Login to comment
103 To test the effect of drug substrates, cross-linking of mutants L332C/L975C, F343C/M986C, G346C/G989C, and P350C/ S993C was done in the presence of verapamil, cyclosporin A, vinblastine, or colchicine. Login to comment
105 By contrast, all the drug substrates were effective in blocking cross-linking of mutants F343C/M986C and G346C/G989C (Fig. 3, B and C), but were less effective in preventing cross-linking of mutant P350C/S993C (Fig. 3D). Login to comment
108 Effect of Cross-linking on Drug-stimulated ATPase Activity- Mutants L332C/L975C, F343C/M986C, G346C/G989C, and P350C/S993C were still active since they retained about 90, 30, 10, and 70%, respectively, of the verapamil-stimulated ATPase activity of the Cys-less P-glycoprotein. Login to comment
109 Cross-linking of mutants F343C/M986C, G346C/G989C, and P350C/S993C, but not L332C/L975C, was reversed by treatment with dithiothreitol (Fig. 4A). Login to comment
126 Membranes prepared from HEK 293 cells expressing mutants L332C/L975C (A), F343C/M986C (B), G346C/G989C (C), and P350/S993C (D) were treated without (-) or with (ϩ) 2 mM (A) or 0.2 mM (B-D) copper phenanthroline for 10 min at 37 °C in the presence of 5 mM ATP, 5 mM ATP plus 0.2 mM sodium vanadate, 5 mM ADP, 5 mM ADP plus 0.2 mM sodium vanadate, or 5 mM AMP-PNP. Login to comment
132 Membranes prepared from HEK 293 cells expressing mutants L332C/ L975C (A), F343C/M986C (B), G346C/G989C (C), and P350/S993C (D) were treated without (-) or with (ϩ) 2 mM (A) or 0.2 mM (B-D) copper phenanthroline for 10 min at 37 °C in the presence of 1 mM verapamil, 0.1 mM vinblastine, 50 ␮M cyclosporin A, or 5 mM colchicine. Login to comment
144 Drug substrates inhibited cross-linking of mutants F343C/ M986C, G346C/G989C, and P350C/S993C. Login to comment

[hide] Identification of residues in the drug-binding sit... J Biol Chem. 1997 Dec 19;272(51):31945-8. Loo TW, Clarke DM
Identification of residues in the drug-binding site of human P-glycoprotein using a thiol-reactive substrate.
J Biol Chem. 1997 Dec 19;272(51):31945-8., 1997-12-19 [PMID:9405384]

Abstract [show]
We identified a thiol-reactive compound, dibromobimane (dBBn), that was a potent stimulator (8.2-fold) of the ATPase activity of Cys-less P-glycoprotein. We then used this compound together with cysteine-scanning mutagenesis to identify residues in transmembrane segment (TM) 6 and TM12 that are important for function. TM6 and TM12 lie close to each other in the tertiary structure and are postulated to be important for drug-protein interactions. The majority of P-glycoprotein mutants containing a single cysteine residue retained substantial amounts of drug-stimulated ATPase activity and were not inhibited by dBBn. The ATPase activities of mutants L339C, A342C, L975C, V982C, and A985C, however, were markedly inhibited (>60%) by dBBn. The drug substrates verapamil, vinblastine, and colchicine protected these mutants against inhibition by dBBn, suggesting that these residues are important for interaction of substrates with P-glycoprotein. We previously showed that residues Leu339, Ala342, Leu975, Val982, and Ala985 lie along the point of contact between helices TM6 and TM12, when both are aligned in a left-handed coiled coil (Loo, T. W., and Clarke, D. M. (1997) J. Biol. Chem. 272, 20986-20989). Taken together, these results suggest that the interface between TM6 and TM12 likely forms part of the potential drug-binding pocket in P-glycoprotein.

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141 Cross-linking between residues F343C/M986C, G346C/G989C, and P350C/S993C was prevented by the presence of drug substrates. Login to comment

[hide] A new structural model for P-glycoprotein. J Membr Biol. 1998 Nov 15;166(2):133-47. Jones PM, George AM
A new structural model for P-glycoprotein.
J Membr Biol. 1998 Nov 15;166(2):133-47., 1998-11-15 [PMID:9841738]

Abstract [show]
Multidrug resistance to anti-cancer drugs is a major medical problem. Resistance is manifested largely by the product of the human MDR1 gene, P-glycoprotein, an ABC transporter that is an integral membrane protein of 1280 amino acids arranged into two homologous halves, each comprising 6 putative transmembrane alpha-helices and an ATP binding domain. Despite the plethora of data from site-directed, scanning and domain replacement mutagenesis, epitope mapping and photoaffinity labeling, a clear structural model for P-glycoprotein remains largely elusive. In this report, we propose a new model for P-glycoprotein that is supported by the vast body of previous data. The model comprises 2 membrane-embedded 16-strand beta-barrels, attached by short loops to two 6-helix bundles beneath each barrel. Each ATP binding domain contributes 2 beta-strands and 1 alpha-helix to the structure. This model, together with an analysis of the amino acid sequence alignment of P-glycoprotein isoforms, is used to delineate drug binding and translocation sites. We show that the locations of these sites are consistent with mutational, kinetic and labeling data.

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204 Four cross-linked pairs, namely L332C/L975C, F343C/M986C, G346C/G989C and P350C/S993C, were generated in separate mutant molecules. 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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244 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). Login to comment
237 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). 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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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
81 The mutants F343C and F343C/V982C also exhibit normal basal activity. Login to comment
150 doi: 10.1371/journal.pone.0082463.g003 F343C/V982C) show dramatically reduced transport of NBD-CsA. 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

[hide] Equilibrated atomic models of outward-facing P-gly... Sci Rep. 2015 Jan 20;5:7880. doi: 10.1038/srep07880. Pan L, Aller SG
Equilibrated atomic models of outward-facing P-glycoprotein and effect of ATP binding on structural dynamics.
Sci Rep. 2015 Jan 20;5:7880. doi: 10.1038/srep07880., [PMID:25600711]

Abstract [show]
P-glycoprotein (Pgp) is an ATP-binding cassette (ABC) transporter that alternates between inward- and outward-facing conformations to capture and force substrates out of cells like a peristaltic pump. The high degree of similarity in outward-facing structures across evolution of ABC transporters allowed construction of a high-confidence outward-facing Pgp atomic model based on crystal structures of outward-facing Sav1866 and inward-facing Pgp. The model adhered to previous experimentally determined secondary- and tertiary- configurations during all-atom molecular dynamics simulations in the presence or absence of MgATP. Three long lasting (>100 ns) meta-stable states were apparent in the presence of MgATP revealing new insights into alternating access. The two ATP-binding pockets are highly asymmetric resulting in differential control of overall structural dynamics and allosteric regulation of the drug-binding pocket. Equilibrated Pgp has a considerably different electrostatic profile compared to Sav1866 that implicates significant kinetic and thermodynamic differences in transport mechanisms.

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196 Specifically, ATP binding inhibited the crosslink of pairs of human Pgp between TM6 and TM12 at L339C-V982C (mouse L334-V978) and L332C-L975C (mouse L328-L971) but promoted the crosslink of F343C-V982C (mouse F339-V978). Login to comment