ABCMdb

ABCB1 p.Leu339Cys

Predicted by SNAP2:	A: D (59%), C: N (61%), D: D (85%), E: D (80%), F: D (59%), G: D (80%), H: D (80%), I: N (87%), K: D (85%), M: N (82%), N: D (75%), P: D (85%), Q: D (75%), R: D (80%), S: D (71%), T: D (66%), V: N (78%), W: D (63%), Y: D (71%),
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] Repacking of the transmembrane domains of P-glycop... EMBO J. 2001 Oct 15;20(20):5615-25. Rosenberg MF, Velarde G, Ford RC, Martin C, Berridge G, Kerr ID, Callaghan R, Schmidlin A, Wooding C, Linton KJ, Higgins CF
Repacking of the transmembrane domains of P-glycoprotein during the transport ATPase cycle.
EMBO J. 2001 Oct 15;20(20):5615-25., 2001-10-15 [PMID:11598005]

Abstract [show]
P-glycoprotein (P-gp) is an ABC (ATP-binding cassette) transporter, which hydrolyses ATP and extrudes cytotoxic drugs from mammalian cells. P-gp consists of two transmembrane domains (TMDs) that span the membrane multiple times, and two cytoplasmic nucleotide-binding domains (NBDs). We have determined projection structures of P-gp trapped at different steps of the transport cycle and correlated these structures with function. In the absence of nucleotide, an approximately 10 A resolution structure was determined by electron cryo-microscopy of two-dimensional crystals. The TMDs form a chamber within the membrane that appears to be open to the extracellular milieu, and may also be accessible from the lipid phase at the interfaces between the two TMDs. Nucleotide binding causes a repacking of the TMDs and reduction in drug binding affinity. Thus, ATP binding, not hydrolysis, drives the major conformational change associated with solute translocation. A third distinct conformation of the protein was observed in the post-hydrolytic transition state prior to release of ADP/P(i). Biochemical data suggest that these rearrangements may involve rotation of transmembrane alpha-helices. A mechanism for transport is suggested.

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136 The mutant proteins were active, with a drug-stimulated ATPase activity comparable to that of cysteine-less P-gp (except for L329C and L339C, which were discounted) (data not shown). Login to comment

[hide] Evidence for the locations of distinct steroid and... Mol Pharmacol. 2002 Nov;62(5):1238-48. Gruol DJ, King MN, Kuehne ME
Evidence for the locations of distinct steroid and Vinca alkaloid interaction domains within the murine mdr1b P-glycoprotein.
Mol Pharmacol. 2002 Nov;62(5):1238-48., [PMID:12391288]

Abstract [show]
P-glycoproteins (P-gp) cause the efflux of a wide variety of unrelated hydrophobic compounds out of cells. However, the locations of the sites at which different classes of molecules initially interact with the protein are not well defined. A unique system was developed to search for P-gp drug-interaction domains using mutational analysis. The strategy is based upon identifying mutations that cause a decrease in the activity of P-gp inhibitors, which are structurally related to chemotherapeutic drugs transported by P-gps. Evidence of distinct steroid and taxane interaction domains has already been presented. The work reported here extends the study of the steroid interaction domain and presents evidence for a separate vinblastine interaction domain. A total of 10 steroid-related mutations, involving seven amino acids that are confined within transmembrane segments (TMS) 4 to 6, have been characterized. The location of these mutations indicates that steroids interact with the transporter within the inner leaflet of the plasma membrane. Four previously unidentified, Vinca-related mutations, involving three amino acids, have also been found. Unexpectedly, these mutations are clustered within an eight-amino acid segment proximal to the TMS-4 region. This portion of the protein is thought to be within the cytoplasmic compartment of the cell. Thus, the results suggest that at least part of the initial interaction between P-gp and Vinca alkaloids occurs in the cytoplasm. The steroid interaction domain does not extend into this region of the protein. However, this cytoplasmic section of the protein is likely to play an important role in promoting steroid transport.

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172 However, the human L339C mutation produced a nearly complete loss of the ability of methanethiosulfonate-verapamil to induce ATPase activity. 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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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
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
160 Only the L339C isoform was significantly different with more rapid reaction kinetics (t1/2 ϭ 23 Ϯ 1 min, p Ͻ 0.05, n ϭ 3). 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
183 L339C was also efficiently labeled with BM (Lext ϭ 71 Ϯ 2%); however, the reaction kinetics were faster (t1/2 ϭ 49 Ϯ 7 min) than observed with V331C. Login to comment
195 Similarly, the kinetics of the reaction were also unchanged (t1/2 values of 45-65 min) with the exception of isoform L339C. Login to comment
201 In the vanadate-trapped state, L339C labeling (t1/2 ϭ 28 Ϯ 3 min) regained the rapid reaction kinetics with CM that were observed in the basal state but lost in the nucleotide-bound protein. 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
215 The effects on L339C accessibility to BM paralleled the observations with CM. Login to comment

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

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

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212 Samples of mutants L339C/(TM6)/F942C(TM11), I306C- (TM5)/I868C(TM10), and S222C(TM4)/G872C(TM10) were then treated with (+) or without (-) M17M cross-linker for 15 min at 22 °C. Mutant G300C(TM5)/F770C(TM8) was cross-linked with 1 mM copper (phenanthroline)3 (CuP) for 15 min at 22 °C. In panel B, whole cells expressing mutants L531C(NBD1)/C1074C(NBD2), I306C(TM5)/I868C(TM10), L339C(TM6)/F942C(TM11), or S222C- (TM4)/G872C(TM10) were incubated for 10 min at 22 °C in the presence of 2.5 mM MTSEA, 10 mM MTSES, or 1 mM MTSET. Login to comment

[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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55 In the presence of ATP, however, mutant F343C(TM6)/V982C(TM12), but not mutant L339C- (TM6)/V982C(TM12), was cross-linked with TMEA (31). Login to 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
81 Cross-linking with TMEA was inhibited by the presence of ATP in both mutants L339C(TM6)/V982C(TM12) and L339C- (TM6)/V982C(TM12)/E556Q(NBD1)/E1201Q(NBD2). Login to comment
106 Membranes were prepared from HEK 293 cells expressing P-gp mutants L339C- (TM6)/V982C(TM12) or L339C(TM6)/V982C(TM12)/E556Q- (NBD1)/E1201Q(NBD2) (A) and F343C(TM6)/V982C(TM12) or F343C(TM6)/V982C(TM12)/E556Q(NBD1)/E1201Q(NBD2) (B). Login to comment
167 Samples were then subjected to immunoblot analysis. Figure 7C shows that there was little difference in the concentration-dependent cross-linking of mutant L339C- (TM6)/F728C(TM7)/E556Q(NBD1)/E1201Q(NBD2) in the presence or absence of ATP. Login to comment
171 Therefore, we confirmed that mutant L339C- (TM6)/F728C(TM7)/E556Q(NBD1)/E1201Q(NBD2) did not exhibit ATP hydrolysis by subjecting it to vanadate trapping of nucleotide. Login to comment

[hide] Residue G346 in transmembrane segment six is invol... Biochemistry. 2007 Sep 4;46(35):9899-910. Epub 2007 Aug 14. Storm J, O'Mara ML, Crowley EH, Peall J, Tieleman DP, Kerr ID, Callaghan R
Residue G346 in transmembrane segment six is involved in inter-domain communication in P-glycoprotein.
Biochemistry. 2007 Sep 4;46(35):9899-910. Epub 2007 Aug 14., 2007-09-04 [PMID:17696319]

Abstract [show]
Multidrug transporters such as P-glycoprotein require considerable inter-domain communication to couple energy utilization with substrate translocation. Elucidation of the regions or residues involved in these communication pathways is a key step in the eventual molecular description of multidrug transport. We used cysteine-scanning mutagenesis to probe the functional involvement of residues along the cytoplasmic half of transmembrane segment 6 (TM6) and its extension toward the nucleotide binding domain. The mutation of one residue (G346C) in this segment adversely affected drug transport in cells. Further investigation using purified protein revealed that the underlying biochemical effect was a reduction in basal ATP hydrolysis. This G346C mutation also affected the stimulation of ATPase activity in a drug dependent manner but had no effect on drug binding, ATP binding, or ADP release. Homology modeling of P-glycoprotein indicated that the G346C mutation caused a steric interaction between TM5 and TM6, thereby precluding a helical movement required to support ATP hydrolysis.

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293 A similar conclusion was demonstrated for the Q347C and S349C isoforms (Tables 3 and 4) and was also shown for the L339C isoform (29). Login to comment

[hide] Suppressor mutations in the transmembrane segments... J Biol Chem. 2007 Nov 2;282(44):32043-52. Epub 2007 Sep 11. Loo TW, Bartlett MC, Clarke DM
Suppressor mutations in the transmembrane segments of P-glycoprotein promote maturation of processing mutants and disrupt a subset of drug-binding sites.
J Biol Chem. 2007 Nov 2;282(44):32043-52. Epub 2007 Sep 11., 2007-11-02 [PMID:17848563]

Abstract [show]
Defective folding of cystic fibrosis transmembrane conductance regulator protein missing Phe508 (DeltaF508) is the major cause of cystic fibrosis. The folding defect in DeltaF508 cystic fibrosis transmembrane conductance regulator might be correctable because misfolding of a P-glycoprotein (P-gp; ABCB1) mutant lacking the equivalent residue (DeltaY490) could be corrected with drug substrates or by introduction of an arginine residue into transmembrane (TM) segments 5 (I306R) or 6 (F343R). Possible mechanisms of arginine rescue were that they mimicked some of the effects of drug substrate interactions with P-gp or that they affected global folding such that all drug substrate/modulator interactions with P-gp were altered. To distinguish between these mechanisms, we tested whether arginines introduced into other TMs predicted to line the drug-binding pocket (TM1 or TM3) would affect folding. It was found that mutation of L65R(TM1) or T199R(TM3) promoted maturation of processing mutants. We then tested whether arginine suppressor mutations had local or global effects on P-gp interactions with drug substrates and modulators. The L65R(TM1), T199R(TM3), I306R(TM5), or F343R(TM6) mutations were introduced into the P-gp mutant L339C(TM6)/F728C(TM7), and thiol cross-linking was carried out in the presence of various concentrations of vinblastine, cyclosporin A, or rhodamine B. The presence of arginine residues reduced the apparent affinity of P-gp for vinblastine (L65R, T199R, and I306R), cyclosporin (I306R and F343R), or rhodamine B (F343R) by 4-60-fold. These results show that the arginine mutations affect a subset of drug-binding sites and suggest that they rescue processing mutants by mimicking drug substrate interactions with P-gp.

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86 Disulfide Cross-linking Analysis-The double cysteine mutants L339C(TM6)/F728C(TM7), L65R(TM1)/L339C(TM6)/ F728C(TM7), T199R(TM3)/L339C(TM6)/F728C(TM7), I306R (TM5)/L339C(TM6)/F728C(TM7), or F343R(TM6)/L339C (TM6)/F728C(TM7) were transiently expressed in HEK 293 cells (32). Login to comment
193 Accordingly, mutants L65R(TM1)/L339C(TM6)/F728C(TM7), T199R(TM3)/L339C (TM6)/F728C(TM7), I306R(TM5)/L339C(TM6)/F728C(TM7), and F343R(TM6)/L339C(TM6)/F728C(TM7) were constructed and expressed in HEK 293 cells. 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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270 C, panel i, membranes prepared from HEK 293 cells expressing mutant L339C/F728C were preincubated for 15 min at 22 °C with different concentrations of adaGb3 (0-500 ␮M) (lanes 2-6). Login to comment
291 Modification of the L339C residue altered signal transduction from several distinct MDR1 drug-binding sites (73), suggesting adaGb3 should prove effective against many MDR1 substrates. Login to comment

[hide] Arginines in the first transmembrane segment promo... J Biol Chem. 2008 Sep 5;283(36):24860-70. Epub 2008 Jul 2. Loo TW, Bartlett MC, Clarke DM
Arginines in the first transmembrane segment promote maturation of a P-glycoprotein processing mutant by hydrogen bond interactions with tyrosines in transmembrane segment 11.
J Biol Chem. 2008 Sep 5;283(36):24860-70. Epub 2008 Jul 2., 2008-09-05 [PMID:18596043]

Abstract [show]
A key goal is to correct defective folding of mutant ATP binding cassette (ABC) transporters, as they cause diseases such as cystic fibrosis. P-glycoprotein (ABCB1) is a useful model system because introduction of an arginine at position 65 of the first transmembrane (TM) segment could repair folding defects. To determine the mechanism of arginine rescue, we first tested the effects of introducing arginines at other positions in TM1 (residues 52-72) of a P-glycoprotein processing mutant (G251V) that is defective in folding and trafficking to the cell surface (20% maturation efficiency). We found that arginines introduced into one face of the TM1 helix (positions 52, 55, 56, 59, 60, 62, 63, 66, and 67) inhibited maturation, whereas arginines on the opposite face of the helix promoted (positions 64, 65, 68, and 71) or had little effect (positions 61, and 69) on maturation. Arginines at positions 61, 64, 65, and 68 appeared to lie close to the drug binding sites as they reduced the apparent affinity for drug substrates such as vinblastine and verapamil. Therefore, arginines that promoted maturation may face an aqueous drug translocation pathway, whereas those that inhibited maturation may face the lipid bilayer. The highest maturation efficiencies (60-85%) were observed with the Arg-65 and Arg-68 mutants. Mutations that removed hydrogen bond acceptors (Y950F/Y950A or Y953F/Y953A) in TM11 predicted to lie close to Arg-65 or Arg-68 inhibited maturation but did not affect maturation of the G251V parent. Therefore, arginine may rescue defective folding by promoting packing of the TM segments through hydrogen bond interactions.

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194 TABLE 2 Concentrations of drug substrates required to inhibit cross-linking by 50% Mutant Vinblastine Cyclosporin A Rhodamine B ␮M ␮M ␮M L339C/F728C 0.5 Ϯ 0.2a 0.8 Ϯ 0.3 52 Ϯ 14 G64R/L339C/F728C 53 Ϯ 15b 1.5 Ϯ 0.4 61 Ϯ 10 M68R/L339C/F728C 266 Ϯ 62b 1.3 Ϯ 0.3 57 Ϯ 7 V71R/L339C/F728C 0.6 Ϯ 0.2 0.8 Ϯ 0.2 57 Ϯ 10 a Each value is the mean Ϯ S.D. (n ϭ 3-4 separate cross-linking experiments). 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
78 No cross-linked product was observed for mutants F336C/S979C and L339C/V982C. 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
107 Mutants S979C/F336C or L339C/V982C did not yield any cross-linked product even in the presence of ATP or drug substrates (data not shown). Login to comment
124 Cross-linking was not observed between F336C/S979C or L339C/V982C, even in the presence of ATP or drug substrates FIG. 2. 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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21 We show that the drug-stimulated ATPase activities of mutants L339C and A342C (TM6) and L975C, V982C, and A985C (TM12) were particularly sensitive to inhibition by dBBn and that the inhibition was prevented by various drug substrates. Login to comment
98 In contrast, mutants L339C, A342C, L975C, V982C, and A985C were significantly inhibited by dBBn, because they retained only 10, 40, 13, 25, and 32% of their activities, respectively. Login to comment
99 The concentration of dBBn required to give 50% inhibition of ATPase activity for mutants L339C, L975C, V982C, A985C, and A342C were 90, 112, 320, 480, and 700 ␮M, respectively. Login to comment
111 The P-glycoproteins(His)10 of Cys-less and mutants L339C, A342C, L975C, V982C, and A985C were mixed with lipid and then preincubated for 15 min at 4 °C without drug or in the presence of 2 mM verapamil (Ver. Login to comment
124 Similarly, mutants L339C, L975C, and V982C were also protected from dBBn inactivation by various drug substrates. Login to comment
126 Colchicine was also very effective in protecting mutant L339C from dBBn inactivation because it retained about 80% of its colchicine-stimulated ATPase activity. Login to comment
129 It offered little or no protection for mutant V982C and only moderately protected mutants L339C and L975C. 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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211 In contrast, two other potential pairs that lie between the first and second of the four cross-linked pairs within TMs 6 and 12 (Loo & Clarke, 1997), namely F336C/S979C and L339C/V982C, failed to form cross-links. 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] New light on multidrug binding by an ATP-binding-c... Trends Pharmacol Sci. 2006 Apr;27(4):195-203. Epub 2006 Mar 20. Shilling RA, Venter H, Velamakanni S, Bapna A, Woebking B, Shahi S, van Veen HW
New light on multidrug binding by an ATP-binding-cassette transporter.
Trends Pharmacol Sci. 2006 Apr;27(4):195-203. Epub 2006 Mar 20., [PMID:16545467]

Abstract [show]
ATP-binding-cassette (ABC) multidrug transporters confer multidrug resistance to pathogenic microorganisms and human tumour cells by mediating the extrusion of structurally unrelated chemotherapeutic drugs from the cell. The molecular basis by which ABC multidrug transporters bind and transport drugs is far from clear. Genetic analyses during the past 14 years reveal that the replacement of many individual amino acids in mammalian multidrug resistance P-glycoproteins can affect cellular resistance to drugs, but these studies have failed to identify specific regions in the primary amino acid sequence that are part of a defined drug-binding pocket. The recent publication of an X-ray crystallographic structure of the bacterial P-glycoprotein homologue MsbA and an MsbA-based homology model of human P-glycoprotein creates an opportunity to compare the original mutagenesis data with the three-dimensional structures of transporters. Our comparisons reveal that mutations that alter specificity are present in three-dimensional 'hotspot' regions in the membrane domains of P-glycoprotein.

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78 Single-cysteine mutants in human P-glycoprotein that are protected from cross-linking to cysteine-reactive MTS substrate analogues by the non-reactive substratea P-glycoprotein residueb Corresponding residue in V. cholera MsbA Cysteine-reactive substrate I340C (6) G293 MTS-rhodamine A841C (9) A151 MTS-rhodamine L975C (12) T285 MTS-rhodamine V981C (12) M291 MTS-rhodamine V982C (12) F292 MTS-rhodamine S222C (4) A175 MTS-verapamil L339C (6) M291 MTS-verapamil A342C (6) M295 MTS-verapamil I868C (10) F180 MTS-verapamil F942C (11) Q256 MTS-verapamil T945C (11) A259 MTS-verapamil G984C (12) L294 MTS-verapamil a Data adapted from [24,2]. Login to comment
76 Single-cysteine mutants in human P-glycoprotein that are protected from cross-linking to cysteine-reactive MTS substrate analogues by the non-reactive substratea P-glycoprotein residueb Corresponding residue in V. cholera MsbA Cysteine-reactive substrate I340C (6) G293 MTS-rhodamine A841C (9) A151 MTS-rhodamine L975C (12) T285 MTS-rhodamine V981C (12) M291 MTS-rhodamine V982C (12) F292 MTS-rhodamine S222C (4) A175 MTS-verapamil L339C (6) M291 MTS-verapamil A342C (6) M295 MTS-verapamil I868C (10) F180 MTS-verapamil F942C (11) Q256 MTS-verapamil T945C (11) A259 MTS-verapamil G984C (12) L294 MTS-verapamil a Data adapted from [24,25]. Login to comment

[hide] The coupling mechanism of P-glycoprotein involves ... FEBS Lett. 2005 Jul 18;579(18):3984-90. Rothnie A, Storm J, McMahon R, Taylor A, Kerr ID, Callaghan R
The coupling mechanism of P-glycoprotein involves residue L339 in the sixth membrane spanning segment.
FEBS Lett. 2005 Jul 18;579(18):3984-90., [PMID:16004994]

Abstract [show]
The transmembrane (TM) domains in P-glycoprotein (P-gp) contain the drug binding sites and undergo conformational changes driven by nucleotide catalysis to effect translocation. However, our understanding of exactly which regions are involved in such events remains unclear. A site-directed labelling approach was used to attach thiol-reactive probes to cysteines introduced into transmembrane segment 6 (TM6) in order to perturb function and infer involvement of specific residues in drug binding and/or interdomain communication. Covalent attachment of coumarin-maleimide at residue 339C within TM6 resulted in impaired ATP hydrolysis by P-gp. The nature of the effect was to reduce the characteristic modulation of basal activity caused by transported substrates, modulators and the potent inhibitor XR9576. Photoaffinity labelling of P-gp with [(3)H]-azidopine indicated that residue 339C does not alter drug binding per se. However, covalent modification of this residue appears to prevent conformational changes that lead to drug stimulation of ATP hydrolysis.

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75 [3 H]-Azidopine labelling of P-glycoprotein Preparations of both cys-less and L339C isoforms of P-gp were labelled to an extent of 90% with CM as described [22]. Login to comment
91 The time-course shown in Fig. 1 was obtained for mutant L339C, although similar profiles were generated for each P-gp isoform. Login to comment
94 The half-lives for reaction of introduced cysteine residues with CM varied from 23 ± 1 min obtained for L339C to 52 ± 3 min for the F335C isoform. Login to comment
95 The half-life for L339C was significantly shorter (P < 0.05) than that obtained for the other TM6 mutant isoforms. Login to comment
110 The Vmax of hydrolysis in the presence of vinblastine was reduced to 41 ± 5% (P < 0.01) of that observed in the absence of CM for the L339C isoform. Login to comment
112 Full Michaelis-Menten plots for the unlabelled and CM-modified L339C isoform of P-gp are shown in Fig. 3. Login to comment
113 The data demonstrate that the vinblastine mediated reduction in Vmax was not accompanied by a shift in affinity (Km(ATP)) of the CM labelled L339C (Km = 0.40 ± 0.05 mM) compared to native protein (Km=0.32 ± 0.05 mM). Login to comment
114 Similarly, of the TM6 mutants examined, only L339C displayed a significant reduction in the nicardipine stimulated Vmax (46 ± 4%, P < 0.01) following labelling with CM (Fig. 2(c)). Login to comment
115 In the presence of nicardipine, the Km of unmodified L339C (Km = 0.33 ± 0.03 mM) was not significantly altered (Km = 0.29 ± 0.05 mM) following covalent modification with CM (Fig. 3). Login to comment
118 Coumarin-maleimide labelled 339C P-gp displays modified TMD fi NBD coupling To further examine the perturbed drug stimulation of ATPase activity, full dose-response analyses were generated for native and CM labelled L339C P-gp for several drugs (Fig. 4). Login to comment
123 Stimulation by vinblastine was characterized by a 2.3 ± 0.1 fold increase in basal activity for unlabelled L339C and at a potency of EC50 = 6.8 ± 0.5 lM (Fig. 4(a)). Login to comment
124 However, for the CM labelled L339C P-gp vinblastine did not stimulate activity, even at concentrations as high as 100 lM. Login to comment
125 The stimulation of unlabelled L339C P-gp (2.3 ± 0.2 fold-basal) by nicardipine was described with a potency of EC50 = 2.3 ± 0.4 lM Fig. 1. Login to comment
126 Time course for the labelling of L339C P-gp with CM. Login to comment
129 (b) The time-course of labelling L339C P-gp with CM obtained from three independent purified protein preparations. Login to comment
131 In contrast to the effect seen with vinblastine, the labelled L339C isoform retained stimulation of ATP hydrolysis by nicardipine although the overall extent was reduced by 60% to 1.3 ± 0.1 fold-basal (P < 0.05, n = 4). Login to comment
133 The effect of paclitaxel on ATP hydrolysis in the CM labelled L339C isoform of P-gp was similar to that observed for vinblastine. Login to comment
134 The ATPase activity of unlabelled L339C-P-gp was stimulated 2.1 ± 0.1 fold by paclitaxel (EC50 = 8.6 ± 3.7 lM) and this was abrogated following the attachment of CM (Fig. 4(c)). Login to comment
136 Following the reaction of L339C with CM, XR9576 retained the ability to inhibit ATP hydrolysis to a level of 0.33 ± 0.03 fold-basal. Login to comment
139 Altered drug stimulated ATPase activity of CM labelled L339C P-gp is not due to impaired drug binding The effect of labelling 339C on drug modulation of ATPase activity was not identical for the four established [3] distinct drug binding sites, with changes observed in potency or fold stimulation. Login to comment
142 The extent of [3 H]- azidopine binding to the CM labelled L339C isoform of P-gp was identical to unlabelled protein. Login to comment
152 The effect of CM labelling of L339C on the Michaelis-Menten parameters describing ATP hydrolysis. Login to comment
155 Activity was normalized such that the Vmax obtained for unlabelled L339C P-gp in the presence of nicardipine was 100%. Login to comment
167 Dose-dependent drug stimulation of ATP hydrolysis by native and CM labelled L339C. Login to comment
171 Native protein is shown as filled circles (d), whilst labelled L339C is depicted with open circles (s). Login to comment
174 [3 H]-Azidopine photoaffinity labelling of native and CM labelled L339C. Login to comment
178 (a) Autoradiograms obtained for native and unlabelled L339C in the absence (lane i), or presence of vinblastine (ii), nicardipine (iii), paclitaxel (iv) or XR9576 (v). Login to comment
74 [3 H]-Azidopine labelling of P-glycoprotein Preparations of both cys-less and L339C isoforms of P-gp were labelled to an extent of 90% with CM as described [22]. Login to comment
90 The time-course shown in Fig. 1 was obtained for mutant L339C, although similar profiles were generated for each P-gp isoform. Login to comment
93 The half-lives for reaction of introduced cysteine residues with CM varied from 23 &#b1; 1 min obtained for L339C to 52 &#b1; 3 min for the F335C isoform. Login to comment
109 The Vmax of hydrolysis in the presence of vinblastine was reduced to 41 &#b1; 5% (P < 0.01) of that observed in the absence of CM for the L339C isoform. Login to comment
111 Full Michaelis-Menten plots for the unlabelled and CM-modified L339C isoform of P-gp are shown in Fig. 3. Login to comment
117 Coumarin-maleimide labelled 339C P-gp displays modified TMD fi NBD coupling To further examine the perturbed drug stimulation of ATPase activity, full dose-response analyses were generated for native and CM labelled L339C P-gp for several drugs (Fig. 4). Login to comment
122 Stimulation by vinblastine was characterized by a 2.3 &#b1; 0.1 fold increase in basal activity for unlabelled L339C and at a potency of EC50 = 6.8 &#b1; 0.5 lM (Fig. 4(a)). Login to comment
128 (b) The time-course of labelling L339C P-gp with CM obtained from three independent purified protein preparations. Login to comment
130 In contrast to the effect seen with vinblastine, the labelled L339C isoform retained stimulation of ATP hydrolysis by nicardipine although the overall extent was reduced by 60% to 1.3 &#b1; 0.1 fold-basal (P < 0.05, n = 4). Login to comment
132 The effect of paclitaxel on ATP hydrolysis in the CM labelled L339C isoform of P-gp was similar to that observed for vinblastine. Login to comment
135 Following the reaction of L339C with CM, XR9576 retained the ability to inhibit ATP hydrolysis to a level of 0.33 &#b1; 0.03 fold-basal. Login to comment
138 Altered drug stimulated ATPase activity of CM labelled L339C P-gp is not due to impaired drug binding The effect of labelling 339C on drug modulation of ATPase activity was not identical for the four established [3] distinct drug binding sites, with changes observed in potency or fold stimulation. Login to comment
141 The extent of [3 H]- azidopine binding to the CM labelled L339C isoform of P-gp was identical to unlabelled protein. Login to comment
151 The effect of CM labelling of L339C on the Michaelis-Menten parameters describing ATP hydrolysis. Login to comment
154 Activity was normalized such that the Vmax obtained for unlabelled L339C P-gp in the presence of nicardipine was 100%. Login to comment
166 Dose-dependent drug stimulation of ATP hydrolysis by native and CM labelled L339C. Login to comment
170 Native protein is shown as filled circles (d), whilst labelled L339C is depicted with open circles (s). Login to comment
173 [3 H]-Azidopine photoaffinity labelling of native and CM labelled L339C. Login to comment
177 (a) Autoradiograms obtained for native and unlabelled L339C in the absence (lane i), or presence of vinblastine (ii), nicardipine (iii), paclitaxel (iv) or XR9576 (v). 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