ABCMdb

ABCB1 p.Thr333Cys

Predicted by SNAP2:	A: N (57%), C: D (59%), D: D (75%), E: D (80%), F: D (80%), G: D (66%), H: D (75%), I: N (57%), K: D (80%), L: D (63%), M: D (66%), N: D (53%), P: D (85%), Q: N (57%), R: D (85%), S: N (57%), V: N (57%), W: D (80%), Y: D (80%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, V: N, W: D, Y: D,

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[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
141 Typical time courses for labeling of T333C with FM and G341C with CM are shown in Fig 1, b and c, respectively. Login to comment
142 Panel b shows a time-dependent increase in the labeling of T333C P-gp (lanes i-vii). Login to comment
154 Isoforms V331C, T333C, F335C, S337C, L339C, and G341C displayed labeling extents in the range 7-12%, and none was significantly different from the Cys-less isoform (ANOVA). Login to comment
163 Isoforms T333C, F335C, S337C, and G341C did not label with BM since the Lext values (19-27%) were not signif- TABLE II Potency of drugs that affect the ATPase activity of purified reconstituted single cysteine mutants of P-gp Pure, reconstituted P-gp (0.3 ␮g) was incubated in the presence of ATP (2 mM) and varying concentrations of nicardipine, vinblastine, or vanadate. 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
168 Structures of maleimide-containing probes (a) and the fluorescence profiles of SDS-PAGE gels obtained for time courses of fluorescein maleimide labeling of T333C (b) and coumarin maleimide labeling of G341C (c). 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
212 Isoform T333C, which was inaccessible to BM under basal conditions, remained so after nucleotide binding and only labeled to a significant extent in the vanadate-trapped state (Lext ϭ 85 Ϯ 2%). 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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No. Sentence Comment
13 Cross-linking between extracellular cysteines (T333C/L975C) predicted to lock P-gp into a conformation that prevents close NBD association inhibited ATPase activity. Login to comment
106 The location of residues that were mutated to cysteine to test for the effect of cross-linking between NBD1 and NBD2 (P517C/I1050C), NBD1 and TMD2 (L443C/S909C), ICL1 and ICL3 (D177C/N820C), TM segments 2 and 11 (C137/A935C) or 6 and 12 (T333C/L975C) are shown. Login to comment
201 Accordingly, we introduced cysteines in regions of TM segments 6 (T333C) and 12 (L975C) predicted to lie at or close to the extracellular surface of the cell (Fig. 1). Login to comment
202 The T333C and L975C mutations were selected because we previously showed that they had little effect on P-gp activity and treatment of the single cysteine mutants with a thiol-reactive derivative of verapamil had little effect on activity (43). Login to comment
203 Since the T333C and L975C mutations were predicted to reside at or close to the extracellular surface of the cell, we performed cross-linking analysis on intact cells expressing the mutant. Login to comment
206 Fig. 6A shows that P-gp mutant T333C/L975C was efficiently cross-linked (Ͼ 90% efficiency) when intact cells were treated with BMOE. Login to comment
207 To test for the effect of cross-linking on activity, histidine-tagged T333C/L975C P-gp was isolated by nickel-chelate chromatography before and after cross-linking with BMOE. Login to comment
210 We then tested the effect of the stimulators (tariquidar, P12) and inhibitor (P10) of ATPase activity on cross-linking of mutant T333C/L975C. Login to comment
212 The results suggest that the stimulators may promote the closed conformation where the T333C and L975C residues are far apart. Login to comment
225 C, cells expressing mutant T333C/L975C were pretreated with 0.1 mM P10, 0.1 mM P12, 0.03 mM tariquidar (Tar), or no compound (None) and then incubated in the absence (-) or presence (ϩ) of 0.5 mM BMOE. Samples were subjected to immunoblot analysis. Login to comment
104 The location of residues that were mutated to cysteine to test for the effect of cross-linking between NBD1 and NBD2 (P517C/I1050C), NBD1 and TMD2 (L443C/S909C), ICL1 and ICL3 (D177C/N820C), TM segments 2 and 11 (C137/A935C) or 6 and 12 (T333C/L975C) are shown. Login to comment
195 Accordingly, we introduced cysteines in regions of TM segments 6 (T333C) and 12 (L975C) predicted to lie at or close to the extracellular surface of the cell (Fig. 1). Login to comment
196 The T333C and L975C mutations were selected because we previously showed that they had little effect on P-gp activity and treatment of the single cysteine mutants with a thiol-reactive derivative of verapamil had little effect on activity (43). Login to comment
197 Since the T333C and L975C mutations were predicted to reside at or close to the extracellular surface of the cell, we performed cross-linking analysis on intact cells expressing the mutant. Login to comment
200 Fig. 6A shows that P-gp mutant T333C/L975C was efficiently cross-linked (b0e; 90% efficiency) when intact cells were treated with BMOE. Login to comment
204 We then tested the effect of the stimulators (tariquidar, P12) and inhibitor (P10) of ATPase activity on cross-linking of mutant T333C/L975C. Login to comment
218 C, cells expressing mutant T333C/L975C were pretreated with 0.1 mM P10, 0.1 mM P12, 0.03 mM tariquidar (Tar), or no compound (None) and then incubated in the absence (afa;) or presence (af9;) of 0.5 mM BMOE. Samples were subjected to immunoblot analysis. Login to comment

[hide] Human P-glycoprotein contains a greasy ball-and-so... J Biol Chem. 2013 Jul 12;288(28):20326-33. doi: 10.1074/jbc.M113.484550. Epub 2013 Jun 3. Loo TW, Bartlett MC, Clarke DM
Human P-glycoprotein contains a greasy ball-and-socket joint at the second transmission interface.
J Biol Chem. 2013 Jul 12;288(28):20326-33. doi: 10.1074/jbc.M113.484550. Epub 2013 Jun 3., [PMID:23733192]

Abstract [show]
The P-glycoprotein drug pump protects us from toxins. Drug-binding sites in the transmembrane (TM) domains (TMDs) are connected to the nucleotide-binding domains (NBDs) by intracellular helices (IHs). TMD-NBD cross-talk is a key step in the transport mechanism because drug binding stimulates ATP hydrolysis followed by drug efflux. Here, we tested whether the IHs are critical for maturation and TMD-NBD coupling by characterizing the effects of mutations to the IH1 and IH2 interfaces. Although IH1 mutations had little effect, most mutations at the IH2-NBD2 interface inhibited maturation or activity. For example, the F1086A mutation at the IH2-NBD2 interface abolished drug-stimulated ATPase activity. The mutant F1086A, however, retained the ability to bind ATP and drug substrates. The mutant was defective in mediating ATP-dependent conformational changes in the TMDs because binding of ATP no longer promoted cross-linking between cysteines located at the extracellular ends of TM segments 6 and 12. Replacement of Phe-1086 (in NBD2) with hydrophobic but not charged residues yielded active mutants. The activity of the F1086A mutant could be restored when the nearby residue Ala-266 (in IH2) was replaced with aromatic residues. These results suggest that Ala-266/Phe-1086 lies in a hydrophobic IH2-NBD2 "ball-and-socket" joint.

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No. Sentence Comment
82 IH2 Mediates TMD/NBD Coupling in the P-gp Drug Pump JULY 12, 2013ߦVOLUME 288ߦNUMBER 28 JOURNAL OF BIOLOGICAL CHEMISTRY 20327 Effect of Mutations on ATP-dependent Cross-linking of P-gp Extracellular Segments-Mutant T333C/L975C contains cysteines predicted to reside at the extracellular ends of TM segments 6 and 12, respectively (30). Login to comment
83 The double cysteine T333C/ L975C constructs (with or without the F1086A or A266F mutations) were transiently expressed in HEK 293 cells. Login to comment
162 Accordingly, cross-linking of mutant T333C/L975C was used to test the effect of F1086A on coupling. Login to comment
163 The T333C and L975C mutations are located at the extracellular ends of TM segments 6 and 12, respectively (Fig. 3, A and B). Login to comment
164 Mutant T333C/L975C can be cross-linked when intact cells expressing the mutant are treated with BMOE cross-linker (30). Login to comment
165 Mutant T333C/L975C, however, does not show cross-linking if membranes containing the mutant are treated only with BMOE (Fig. 4A). Login to comment
170 To determine whether the F1086A mutation affected NBD/ TMD coupling, it was introduced into mutant T333C/L975C and subjected to cross-linking. Login to comment
171 It was observed that the F1086A mutation inhibited ATP-dependent cross-linking of the mutant T333C/L975C (Fig. 4B). Login to comment
187 A and B, membranes prepared from cells expressing mutant T333C/L975C (A) or T333C/L975C/F1086A (B) were treated with BMOE in the absence (None) or presence of nucleotides (ATP, AMP-PNP, or ADP). Login to comment
197 Accordingly, the A266F mutation was introduced into mutant F1086A/T333C/L975C to test whether it affected cross-linking between the TMDs. Login to comment
221 D, membranes prepared from cells expressing mutants T333C/L975C (None), T333C/L975C/F1086A, or T333C/L975C/F1086A/A266F were treated with BMOE in the presence (af9;) or absence (afa;) of ATP. Login to comment

[hide] Mapping the Binding Site of the Inhibitor Tariquid... J Biol Chem. 2015 Dec 4;290(49):29389-401. doi: 10.1074/jbc.M115.695171. Epub 2015 Oct 26. Loo TW, Clarke DM
Mapping the Binding Site of the Inhibitor Tariquidar That Stabilizes the First Transmembrane Domain of P-glycoprotein.
J Biol Chem. 2015 Dec 4;290(49):29389-401. doi: 10.1074/jbc.M115.695171. Epub 2015 Oct 26., [PMID:26507655]

Abstract [show]
ABC (ATP-binding cassette) transporters are clinically important because drug pumps like P-glycoprotein (P-gp, ABCB1) confer multidrug resistance and mutant ABC proteins are responsible for many protein-folding diseases such as cystic fibrosis. Identification of the tariquidar-binding site has been the subject of intensive molecular modeling studies because it is the most potent inhibitor and corrector of P-gp. Tariquidar is a unique P-gp inhibitor because it locks the pump in a conformation that blocks drug efflux but activates ATPase activity. In silico docking studies have identified several potential tariquidar-binding sites. Here, we show through cross-linking studies that tariquidar most likely binds to sites within the transmembrane (TM) segments located in one wing or at the interface between the two wings (12 TM segments form 2 divergent wings). We then introduced arginine residues at all positions in the 12 TM segments (223 mutants) of P-gp. The rationale was that a charged residue in the drug-binding pocket would disrupt hydrophobic interaction with tariquidar and inhibit its ability to rescue processing mutants or stimulate ATPase activity. Arginines introduced at 30 positions significantly inhibited tariquidar rescue of a processing mutant and activation of ATPase activity. The results suggest that tariquidar binds to a site within the drug-binding pocket at the interface between the TM segments of both structural wings. Tariquidar differed from other drug substrates, however, as it stabilized the first TM domain. Stabilization of the first TM domain appears to be a key mechanism for high efficiency rescue of ABC processing mutants that cause disease.

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66 Mutant T333C/L975C was cross-linked in whole cells at 20 &#b0;C with 0.5 mM BMOE (bismaleimidoethane) cross-linker (Thermo Fisher Scientific, Burlington, ON) in the absence or presence of 0.25 òe;M tariquidar (30). Login to comment
118 The conditions for cross-linking mutants T333C/L975C and P517C/I1050C, however, were different. Login to comment
119 Mutant T333C/L975C was cross-linked with BMOE using intact cells because ATP hydrolysis is required for cross-linking (41). Login to comment