ABCMdb

ABCB1 p.Leu175Cys

Predicted by SNAP2:	A: D (66%), C: N (57%), D: D (91%), E: D (91%), F: N (66%), G: D (85%), H: D (85%), I: N (87%), K: D (91%), M: N (87%), N: D (85%), P: D (91%), Q: D (85%), R: D (91%), S: D (80%), T: D (80%), V: N (61%), W: D (80%), Y: D (80%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: N, K: D, M: N, N: D, P: D, Q: D, R: D, S: D, T: D, V: N, W: D, Y: D,

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[hide] Exhaustive sampling of docking poses reveals bindi... PLoS Comput Biol. 2011 May;7(5):e1002036. Epub 2011 May 12. Klepsch F, Chiba P, Ecker GF
Exhaustive sampling of docking poses reveals binding hypotheses for propafenone type inhibitors of P-glycoprotein.
PLoS Comput Biol. 2011 May;7(5):e1002036. Epub 2011 May 12., [PMID:21589945]

Abstract [show]
Overexpression of the xenotoxin transporter P-glycoprotein (P-gp) represents one major reason for the development of multidrug resistance (MDR), leading to the failure of antibiotic and cancer therapies. Inhibitors of P-gp have thus been advocated as promising candidates for overcoming the problem of MDR. However, due to lack of a high-resolution structure the concrete mode of interaction of both substrates and inhibitors is still not known. Therefore, structure-based design studies have to rely on protein homology models. In order to identify binding hypotheses for propafenone-type P-gp inhibitors, five different propafenone derivatives with known structure-activity relationship (SAR) pattern were docked into homology models of the apo and the nucleotide-bound conformation of the transporter. To circumvent the uncertainty of scoring functions, we exhaustively sampled the pose space and analyzed the poses by combining information retrieved from SAR studies with common scaffold clustering. The results suggest propafenone binding at the transmembrane helices 5, 6, 7 and 8 in both models, with the amino acid residue Y307 playing a crucial role. The identified binding site in the non-energized state is overlapping with, but not identical to, known binding areas of cyclic P-gp inhibitors and verapamil. These findings support the idea of several small binding sites forming one large binding cavity. Furthermore, the binding hypotheses for both catalytic states were analyzed and showed only small differences in their protein-ligand interaction fingerprints, which indicates only small movements of the ligand during the catalytic cycle.

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138 In addition, a recent cross-linking study further strengthened this by showing that an M1M cross-link between L175C and N820C did not prevent verapamil and rhodamine B to be transported [46]. 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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10 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. Login to comment
129 A, model showing predicted orientations of Leu175 , Asp177 , andAsn820 intheclosedconformation.B,membranespreparedfromHEK293 cells expressing the ICL1/ICL3 mutants D177C/N820C or L175C/N820C were treated with cross-linkers M1M, M4M, or M17M or no cross-linker (Control). Login to comment
132 C, membranes expressing mutants D177C/N820C or L175C/N820C were treated with or without (None) cross-linker and histidine-tagged P-gp isolated by nickel-chelate chromatography. Login to comment
143 In this study, we also observed that the majority of mutant L175C/N820C was cross-linked when the mutant was treated with 0.02 mM M1M at 20 °C while only a low level of cross-linking (about 25%) was observed when D177C/N820C was treated under the same conditions. Login to comment
144 Since D177C is predicted to have a more favorable orientation toward N820C compared with L175C (Fig. 3A), it would be expected that cross-linking of D177C/N820C would be more efficient than L175C/N820C. Login to comment
146 We previously observed that mutant L175C/N820 only showed efficient cross-linking with a narrow concentration range of M1M (about 10-20 ␮M). Login to comment
150 We tested the effects of M4M and M17M cross-linking on the ATPase activity of mutant L175C/N820C and found that the effects were similar to those observed with mutant D177C/ N820C (Fig. 3C). Login to comment
151 Cross-linking of L175C/N820C with M4M highly activated the basal ATPase activity of the mutant while cross-linking with M17M caused little activation. Login to comment
152 The ATPase activity of M17M cross-linked L175C/N820C could still be highly activated when assayed in the presence of verapamil. Login to comment
158 In previous studies we showed that mutants C137/A935C (39) and L443C/ S909C (27) had verapamil-stimulated ATPase activities similar to Cys-less P-gp. Login to comment
163 These results indicate that stimulation of ATPase activity observed in M4M cross-linked mutants P517C/I1050C (Fig. 2B), D177C/N820C, or L175C/N820C (Fig. 3C) was likely a specific effect of trapping the protein in the closed conformation. Login to comment
236 The observation that cross-linked mutants P517C/I1050C, D177C/N820C, and L175C/N820C showed high levels of ATPase activity indicates that the protein does not have to adopt an inward-facing conformation with separation of the NBDs for ATP hydrolysis to continue after the first two hydrolysis steps. Login to comment
264 We predict that covalent attachment of thiol-reactive drug substrates to cysteines in the TMDs (or M4M cross-linking of mutants P517C/I1050C, D177C/N820C, or L175C/N820C) traps P-gp in states IV-VI (Fig. 8) that show a high rate of ATP hydrolysis. Login to comment
126 A, model showing predicted orientations of Leu175 , Asp177 , andAsn820 intheclosedconformation.B,membranespreparedfromHEK293 cells expressing the ICL1/ICL3 mutants D177C/N820C or L175C/N820C were treated with cross-linkers M1M, M4M, or M17M or no cross-linker (Control). Login to comment
140 In this study, we also observed that the majority of mutant L175C/N820C was cross-linked when the mutant was treated with 0.02 mM M1M at 20 &#b0;C while only a low level of cross-linking (about 25%) was observed when D177C/N820C was treated under the same conditions. Login to comment
141 Since D177C is predicted to have a more favorable orientation toward N820C compared with L175C (Fig. 3A), it would be expected that cross-linking of D177C/N820C would be more efficient than L175C/N820C. Login to comment
147 Cross-linking of L175C/N820C with M4M highly activated the basal ATPase activity of the mutant while cross-linking with M17M caused little activation. Login to comment
148 The ATPase activity of M17M cross-linked L175C/N820C could still be highly activated when assayed in the presence of verapamil. Login to comment
229 The observation that cross-linked mutants P517C/I1050C, D177C/N820C, and L175C/N820C showed high levels of ATPase activity indicates that the protein does not have to adopt an inward-facing conformation with separation of the NBDs for ATP hydrolysis to continue after the first two hydrolysis steps. Login to comment
257 We predict that covalent attachment of thiol-reactive drug substrates to cysteines in the TMDs (or M4M cross-linking of mutants P517C/I1050C, D177C/N820C, or L175C/N820C) traps P-gp in states IV-VI (Fig. 8) that show a high rate of ATP hydrolysis. Login to comment

[hide] Human P-glycoprotein is active when the two halves... Biochem Biophys Res Commun. 2010 May 7;395(3):436-40. Epub 2010 Apr 13. Loo TW, Bartlett MC, Clarke DM
Human P-glycoprotein is active when the two halves are clamped together in the closed conformation.
Biochem Biophys Res Commun. 2010 May 7;395(3):436-40. Epub 2010 Apr 13., [PMID:20394729]

Abstract [show]
The P-glycoprotein (P-gp, ABCB1) drug pump protects us from toxic compounds and confers multidrug resistance. Each of the two homologous halves of P-gp is composed of a transmembrane domain (TMD) with six TM segments followed by a nucleotide-binding domain (NBD). The drug- and ATP-binding sites reside at the interface between the TMDs and NBDs, respectively. Crystal structures show drug pumps in the open and closed conformations, where the drug-binding pocket and NBDs are open or closed at the cytoplasmic side, respectively. Although it has been postulated that drug substrates enter the drug-binding pocket in the open conformation, it is unknown if they can enter in the closed conformation. To determine this, we introduced cysteines into regions of TM3 (residues 175-178) and TM9 (residues 820-822) that extend into the cytoplasm and are 4 A and 20 A apart in the closed and open conformations, respectively. The 12 double cysteine mutants were then cross-linked with a short cross-linker, M1M (4 A) at 0 degrees C to reduce thermal motion in the protein. Only mutant L175C/N820C was cross-linked. Cross-linking was not increased in the presence of ATP or drug substrates. Cross-linking increased its basal ATPase activity about 3-fold. Activity could be increased further by drug substrates such as verapamil and rhodamine B. These results suggest that P-gp in the membrane is in the closed conformation that has a high affinity for drug substrates.

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7 Only mutant L175C/N820C was cross-linked. Login to comment
58 The majority of mutant L175C/N820C was cross-linked after treatment with M1M cross-linker (Fig. 2A). Login to comment
59 Cross-linked product was not observed in the other 11 mutants (Fig. 2A) or in the single cysteine mutants L175C and N820C (data not shown). Login to comment
60 When mutant L175C/N820C was treated with various concentrations of M1M, it was found that the highest amount of cross-linked product was obtained in the presence of about 0.01-0.02 mM cross-linker and decreased with higher concentrations of cross-linker (Fig. 2B). Login to comment
62 To test whether mutant L175C/N820C was still active, the histidine-tagged version of mutant L175C/N820C was isolated by nickel-chelate chromatography and assayed for drug-stimulated ATPase activity. Login to comment
64 It was found that activation and apparent affinity of mutant L175C/N820C for the drug substrates verapamil, vinblastine, rhodamine B, and colchicine were not significantly different from the Cys-less parent (data not shown). Login to comment
65 For example, mutant L175C/N820C showed 50% stimulation of ATPase activity (S50) in the presence of 12.6 ± 1.0 lM verapamil, 44 ± 7 lM rhodamine B, 450 ± 40 lM colchicine, and 2.2 ± 0.4 lM vinblastine. Login to comment
67 Therefore, the mutant was selected for further analysis. Cross-linking of mutant L175C/N820C with M1M (Fig. 2A) suggested that these residues were in close proximity. Login to comment

[hide] Identification of the distance between the homolog... J Biol Chem. 2014 Mar 21;289(12):8484-92. doi: 10.1074/jbc.M114.552075. Epub 2014 Feb 12. Loo TW, Clarke DM
Identification of the distance between the homologous halves of P-glycoprotein that triggers the high/low ATPase activity switch.
J Biol Chem. 2014 Mar 21;289(12):8484-92. doi: 10.1074/jbc.M114.552075. Epub 2014 Feb 12., [PMID:24523403]

Abstract [show]
P-glycoprotein (P-gp, ABCB1) is an ATP-binding cassette drug pump that protects us from toxic compounds and confers multidrug resistance. Each homologous half contains a transmembrane domain with six transmembrane segments followed by a nucleotide-binding domain (NBD). The drug- and ATP-binding sites reside at the interface between the transmembrane domain and NBDs, respectively. Drug binding activates ATPase activity by an unknown mechanism. There is no high resolution structure of human P-gp, but homology models based on the crystal structures of bacterial, mouse, and Caenorhabditis elegans ATP-binding cassette drug pumps yield both open (NBDs apart) and closed (NBDs together) conformations. Molecular dynamics simulations predict that the NBDs can be separated over a range of distances (over 20 A). To determine the distance that show high or low ATPase activity, we cross-linked reporter cysteines L175C (N-half) and N820C (C-half) with cross-linkers of various lengths that separated the halves between 6 and 30 A (alpha-carbons). We observed that ATPase activity increased over 10-fold when the cysteines were cross-linked at distances between 6 and 19 A, although cross-linking at distances greater than 20 A yielded basal levels of activity. The results suggest that the ATPase activation switch appears to be turned on or off when L175C/N820 are clamped at distances less than or greater than 20 A, respectively. We predict that the high/low ATPase activity switch may occur at a distance where the NBDs are predicted in molecular dynamic simulations to undergo pronounced twisting as they approach each other (Wise, J. G. (2012) Biochemistry 51, 5125-5141).

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No. Sentence Comment
10 To determine the distance that show high or low ATPase activity, we cross-linked reporter cysteines L175C (N-half) and N820C (C-half) with cross-linkers of various lengths that separated the halves between 6 and 30 &#c5; (ॷ-carbons). Login to comment
12 The results suggest that the ATPase activation switch appears to be turned on or off when L175C/N820 are clamped at distances less than or greater than 20 &#c5;, respectively. Login to comment
47 To address these questions, we used cross-linkers of various lengths to cross-link a P-gp mutant that contained reporter cysteines at positions L175C (N-half) and N820 (C-half) in a Cys-less background. Login to comment
48 The L175C and N820C reporter cysteines were selected because they are located at positions predicted to undergo large changes in distance between the closed (12 &#c5; between ॷ-carbons) and open (about 30 &#c5; between ॷ-carbons) conformations. Login to comment
49 In addition, modifications to L175C and N820C are not expected to have a major impact on activity because they reside outside the drug- and ATP-binding sites. Login to comment
50 We previously showed that L175C/N820C could be efficiently cross-linked with the short cross-linker 1,4-butanediyl bismethanethiosulfonate (M4M) that can span a distance of 7.5 &#c5; (about 13 &#c5; between ॷ-carbons) to activate ATPase activity (40). Login to comment
51 Here, we tested the effects of cross-linking mutant L175C/ N820C at 10 different distances between the halves. Login to comment
52 It was found that linkage of L175C to N820C with cross-linkers predicted to span a distance of less than 20 &#c5; highly activated P-gp ATPase activity (over 10-fold), but cross-linking with longer cross-linkers yielded P-gps with low ATPase activity. Login to comment
55 Mutations L175C and N820C were introduced into Cys-less P-gp as described previously (42). Login to comment
60 Disulfide Cross-linking Analysis-The double cysteine mutant L175C/N820C was transiently expressed in HEK 293 cells. Login to comment
66 Intramolecular disulfide cross-linking between L175C and N820C can be detected because the cross-linked product migrates with a slower mobility on SDS-polyacrylamide gels (47). Login to comment
68 Cross-linking of L175C/N820C with M1M or oxidant (copper phenanthroline) was performed using P-gp isolated by nickel-chelate chromatography. Login to comment
81 P-gp ATPase Activity Is Highly Activated When Residues L175C and N820C Are Cross-linked in Close Proximity-In a previous study, we found that cross-linking the two homologous halves through formation of a direct disulfide bond between A259C in intracellular helix 2 (IH2) and W803C in IH3 inhibited activity (49). Login to comment
83 Because cysteines at positions L175C and N820C are located away from the IH TMD/NBD contact points (Fig. 1), cross-linking L175C and N820C in close proximity might not disrupt TMD/NBD interactions. Login to comment
84 The L175C or N820C mutations did not appear to affect activity of P-gp because the drug-stimulated ATPase activity of the L175C/N820C mutant was very similar to the Cys-less parent (42). Login to comment
86 Cross-linking of mutant L175C/N820C was low (b0d;50% cross-linking) when membranes prepared from transfected cells were treated with oxidant (copper phenanthroline) even at 37 &#b0;C (data not shown). Login to comment
88 Accordingly, histidine-tagged mutant L175C/N820C was expressed in HEK 293 cells, isolated by nickel chelate chromatography and treated with oxidant. Login to comment
89 In the presence of dodecyl maltoside detergent, mutant L175C/ N820C could be efficiently cross-linked with copper phenanthroline (Fig. 2, A and B). Login to comment
101 Cross-linking of L175C and N820C in close proximity highly activates P-gp basal ATPase activity. Login to comment
102 A, purified histidine-tagged mutant L175C/N820C was treated with (af9;) or without (afa;) copper phenanthroline (CuP) or M1M cross-linker. Login to comment
105 B, amount of cross-linked protein relative to total P-gp was quantitated from three different transfections afe; S.D. C, ATPase activities of mutant L175C/N820C were determined in the absence (afa;) or presence (af9;) of verapamil (Ver) before (None) and after cross-linking with copper phenanthroline or M1M. Login to comment
107 To test for the effects of cross-linking on activity, isolated L175C/N820C was treated with or without 0.5 mM copper phenanthroline or 0.009 mM M1M. Login to comment
112 Activation of mutant L175C/N820C basal ATPase activity with M1M was higher than previously observed using membranes (42) suggesting that the efficiency of cross-linking was improved using purified P-gp. Login to comment
115 P-gp ATPase Activity Is Not Highly Activated When Residues L175C and N820C Are Cross-linked with Long Cross-linkers- The next step was to test the effects of cross-linking with MTS cross-linkers that could span longer distances such as M2M (5.2 &#c5;), M4M (7.8 &#c5;), M5M (8.8 &#c5;), M6M (10.4 &#c5;), M8M (13 &#c5;), M11M (16.9 &#c5;), M14M (19 &#c5;) and M17M (22 &#c5;) (45). Login to comment
117 For example, we used 0.009 mM M1M to treat L175C/N820C because we previously showed that cross-linking efficiency was sharply reduced at lower concentrations (0.003 mM) or higher concentrations (0.027 mM) (42). Login to comment
118 Accordingly, membranes prepared from cells expressing mutant L175C/N820C were treated with various concentrations of MTS cross-linkers with various lengths of spacer arms for 10 min at 20 &#b0;C, and samples were subjected to immunoblot analysis. Login to comment
127 Membranes prepared from cells expressing mutant L175C/ N820C were first treated for 10 min at 20 &#b0;C in the absence or presence of 0.027 mM MTS cross-linkers of different lengths. Login to comment
129 Because mutant L175C/N820C was efficiently cross-linked with the MTS cross-linkers, we tested whether cross-linking affected activity. Login to comment
131 Cross-linking of L175C/N820C P-gp with various concentrations of M2M and M17M cross-linker. Login to comment
132 Membranes prepared from HEK 293 cells expressing mutant L175C/N820C were treated with various concentrations (0-2.2 mM) of M2M (A and B) or M17M (C and D) cross-linker. Login to comment
136 Activation Switch for P-gp ATPase Activity MARCH 21, 2014ߦVOLUME 289ߦNUMBER 12 JOURNAL OF BIOLOGICAL CHEMISTRY 8487 membranes and assayed for ATPase activity in the presence or absence of saturating concentrations (0.4 mM) of verapamil. Fig. 5 shows that untreated mutant L175C/N820C isolated from membranes exhibited about a 12-fold increase in verapamil-stimulated ATPase activity. Login to comment
139 By contrast, treatment of mutant L175C/ N820C with the intermediate size MTS cross-linkers (M2M, M4M, M5M, M6M, and M8M) that span maximum distances of about 5.2-13 &#c5; (distance of about 10.6-18.6 &#c5; between the ॷ-carbons) highly activated ATPase activity in the absence of verapamil. Login to comment
142 It appeared that cross-linking of mutant L175C/N820C with the intermediate size cross-linkers locked P-gp in an activated state resulting in high levels of ATPase activity in the absence or presence of drug substrates. Login to comment
143 A quite different effect was observed when mutant L175C/ N820C was treated with the long cross-linkers M11M, M14M, and M17M that could span maximum distances of about 16.9, 19, and 22 &#c5; in length, respectively (span distances of about 22 to 28 &#c5; between the ॷ-carbons) (Fig. 5). Login to comment
145 Cross-linking of mutant L175C/N820C with the intermediate size MTS cross-linkers like M2M increased the basal ATPase activity of the mutant (Fig. 5). Login to comment
147 To test these possibilities, a series of control experiments were carried out with L175C/N820C using M2M. Login to comment
150 We compared the effects of treating mutant L175C/N820C with low (0.027 mM; promotes cross-linking) or high (3 mM; likely labels each cysteine with a separate M2M molecule) concentrations of M2M on ATPase activity. Login to comment
154 Next, mutant histidine-tagged P-gps were constructed that contained only the L175C or N820C mutation alone to test if modification of only one cysteine had an impact on ATPase activity. Login to comment
159 Cross-linking of mutant L175C/N820C with cross-linkers of various lengths. Login to comment
160 A, membranes prepared from HEK 293 cells expressing mutant L175C/N820C were treated without (None) or with MTS cross-linkers of various lengths (MXM). Login to comment
164 ATPase activity of mutant L175C/N820C cross-linked with various MXM cross-linkers. Login to comment
165 Membranes were prepared from HEK 293 cells expressing mutant histidine-tagged L175C/N820C and cross-linked with various MXM cross-linkers (X afd; 2-17). Login to comment
169 These results show that activation of mutant L175C/N820C basal ATPase activity by M2M was due to cross-linking between the two cysteines. Login to comment
173 In agreement with the EPR and simulation studies (55), we found that the presence of ATP did not promote the formation of a more closed structure as it did not promote L175C/N820C cross-linking (42). Login to comment
174 We also found that verapamil did not promote or inhibit cross-linking of L175C/N820C (42). Login to comment
179 The experiments presented here show that human L175C/ N820C P-gp remains active when it is trapped in conformations with a wide range of separations between the halves (about 6-30 &#c5;). Login to comment
192 Activity of L175C/N820C, Cys-less, L175C, and N820C P-gp treated with various concentrations of M2M. Login to comment
193 A, histidine-tagged mutant L175C/N820C was isolated from membranes treated was treated without (None), low (0.027 mM), or high (3 mM) concentrations of M2M and ATPase activity determined in the absence (afa;) or presence (af9;) of verapamil (Ver). Login to comment
194 B, membranes expressing histidine-tagged mutant L175C/N820C, Cys-less, L175C, or N820C P-gp were treated without (afa;) or with (af9;) 0.027 mM M2M. Login to comment
197 Correlation of ATPase activity of L175C/N820C treated with cross-linkers of various lengths and the distance between the ॷ-carbons of L175C and N820C. Login to comment
198 The activity of mutant L175C/N820C cross-linked with MXM cross-linker (M1M (2), M2M (3), M4M (4), M5M (5), M6M (6), M8M (7), M11M (8), M14M (9), or M17M (10)) was compared with that treated with copper phenanthroline (1). Login to comment
211 Could P-gp still transport drug substrates when L175C/ N820C is cross-linked with short (M2M to M8M) cross-linkers? Login to comment
212 Unfortunately, it was not possible to use whole cell transport or drug resistance assays because cross-linking could not be detected when cells expressing the L175C/N820C mutant were treated with the MTS cross-linkers.3 The lack of cross-linking in whole cells could be due to the presence of intracellular thiol reductants such as glutathione that would not only react with the MTS cross-linkers but also reduce any disulfide bond. Login to comment
214 We predict, however, that P-gp cross-linked at L175C/ N820C with short cross-linkers should be able to bind substrates with relatively high affinity because it was reported that P-gp trapped in the closed vanadate-trapped transition state had similar affinity for the drug substrates verapamil, Hoechst 33342, cyclosporin A, vinblastine, colchicine, rhodamine B, and doxorubicin as in the relaxed (open) state (66). Login to comment

[hide] Cysteines introduced into extracellular loops 1 an... J Biol Chem. 2014 Sep 5;289(36):24749-58. doi: 10.1074/jbc.M114.583021. Epub 2014 Jul 22. Loo TW, Clarke DM
Cysteines introduced into extracellular loops 1 and 4 of human P-glycoprotein that are close only in the open conformation spontaneously form a disulfide bond that inhibits drug efflux and ATPase activity.
J Biol Chem. 2014 Sep 5;289(36):24749-58. doi: 10.1074/jbc.M114.583021. Epub 2014 Jul 22., [PMID:25053414]

Abstract [show]
P-glycoprotein (P-gp) is an ATP-binding cassette drug pump that protects us from toxic compounds and confers multidrug resistance. The protein is organized into two halves. The halves contain a transmembrane domain (TMD) with six transmembrane segments and a nucleotide-binding domain (NBD). The drug- and ATP-binding sites reside at the TMD1/TMD2 and NBD1/NBD2 interfaces, respectively. ATP-dependent drug efflux involves changes between the open inward-facing (NBDs apart, extracellular loops (ECLs) close together) and the closed outward-facing (NBDs close together, ECLs apart) conformations. It is controversial, however, whether the open conformation only exists transiently in intact cells because of the presence of high levels of ATP. To test for the presence of an open conformation in intact cells, reporter cysteines were placed in extracellular loops 1 (A80C, N half) and 4 (R741C, C half). The rationale was that cysteines A80C/R741C would only come close enough to form a disulfide bond in an open conformation (6.9 A apart) because they are separated widely (30.4 A apart) in the closed conformation. It was observed that the mutant A80C/R741C cross-linked spontaneously (>90%) when expressed in cells. In contrast to previous reports showing that trapping P-gp in a closed conformation highly activated ATPase activity, here we show that A80C/R741C cross-linking inhibited ATPase activity and drug efflux. Both activities were restored when the cross-linked mutant was treated with a thiol-reducing agent. The results show that an open conformation can be readily detected in cells and that cross-linking of cysteines placed in ECLs 1 and 4 inhibits activity.

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No. Sentence Comment
257 Direct cross-linking of the cysteines L175C and N820C (23) or cross-linking of mutant P571C/I1050C with the short 1,4-butanediyl bismethanethiosulfonate cross-linker (7.8 &#c5;) (24) highly activated P-gp ATPase activity over 10-fold in the absence of drug substrates. Login to comment
258 Cysteines L175C/N820C are located in the intracellular loops (Fig. 1, C and D), whereas cysteines P517C/I1050C are located in the NBDs (Fig. 1, C and D). Login to comment
259 An explanation for the difference is that linkage of the homologous halves at the L175C/N820C or P517C/I1050C sites would tend to favor the closed conformation (Fig. 1C) to FIGURE 9. Login to comment