Substrate-induced Conformational Changes in the Transmembrane Segments of Human P-glycoprotein

The human multidrug resistance P-glycoprotein (P-gp, ABCB1) is quite promiscuous in that it can transport a broad range of structurally diverse compounds out of the cell. We hypothesized that the transmembrane (TM) segments that constitute the drug-binding site are quite mobile such that drug binding occurs through a “substrate-induced fit” mechanism. Here, we used cysteine-scanning mutagenesis and oxidative cross-linking to test for substrate-induced changes in the TM segments. Pairs of cysteines were introduced into a Cys-less P-gp and the mutants treated with oxidant (copper phenanthroline) in the presence or absence of various drug substrates. We show that cyclosporin A promoted cross-linking between residues P350C(TM6)/G939C(TM11), while colchicine and demecolcine promoted cross-linking between residues P350C(TM6)/V991C(TM12). Progesterone promoted cross-linking between residues P350C(TM6)/A935C(TM11), P350C(TM6)/G939C(TM11), as well as between residues P350C(TM6)/V991C(TM12). Other substrates such as vinblastine, verapamil, cis-(Z)-flupenthixol ortrans-(E)-flupenthixol did not induce cross-linking at these sites. These results provide direct evidence that the packing of the TM segments in the drug-binding site is changed when P-gp binds to a particular substrate. The induced-fit mechanism explains how P-gp can accommodate a broad range of compounds. The human multidrug resistance P-glycoprotein (P-gp, ABCB1) is quite promiscuous in that it can transport a broad range of structurally diverse compounds out of the cell. We hypothesized that the transmembrane (TM) segments that constitute the drug-binding site are quite mobile such that drug binding occurs through a “substrate-induced fit” mechanism. Here, we used cysteine-scanning mutagenesis and oxidative cross-linking to test for substrate-induced changes in the TM segments. Pairs of cysteines were introduced into a Cys-less P-gp and the mutants treated with oxidant (copper phenanthroline) in the presence or absence of various drug substrates. We show that cyclosporin A promoted cross-linking between residues P350C(TM6)/G939C(TM11), while colchicine and demecolcine promoted cross-linking between residues P350C(TM6)/V991C(TM12). Progesterone promoted cross-linking between residues P350C(TM6)/A935C(TM11), P350C(TM6)/G939C(TM11), as well as between residues P350C(TM6)/V991C(TM12). Other substrates such as vinblastine, verapamil, cis-(Z)-flupenthixol ortrans-(E)-flupenthixol did not induce cross-linking at these sites. These results provide direct evidence that the packing of the TM segments in the drug-binding site is changed when P-gp binds to a particular substrate. The induced-fit mechanism explains how P-gp can accommodate a broad range of compounds. P-glycoprotein (P-gp) 1The abbreviations used are: P-gpP-glycoproteinTMtransmembraneHEKhuman embryonic kidneyTMD1+2residues 1–379 + residues 681–1025 of P-gp1The abbreviations used are: P-gpP-glycoproteinTMtransmembraneHEKhuman embryonic kidneyTMD1+2residues 1–379 + residues 681–1025 of P-gp is an ATP-dependent drug pump that is capable of transporting structurally diverse compounds out of the cell (recently reviewed in Refs. 1Ambudkar S.V. Dey S. Hrycyna C.A. Ramachandra M. Pastan I. Gottesman M.M. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 361-398Crossref PubMed Scopus (1897) Google Scholar and 2Borst P. Elferink R.O. Annu. Rev. Biochem. 2002; 71: 537-592Crossref PubMed Scopus (1318) Google Scholar). P-gp is a member of theATP-Binding Cassette (ABC) family of transporters. The 1280 amino acids of P-gp are arranged as two repeating units of 610 amino acids that are joined by a linker region of about 60 amino acids (3Chen C.J. Chin J.E. Ueda K. Clark D.P. Pastan I. Gottesman M.M. Roninson I.B. Cell. 1986; 47: 381-389Abstract Full Text PDF PubMed Scopus (1708) Google Scholar). Each repeat has six transmembrane (TM) segments and a hydrophilic domain containing an ATP-binding site (4Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 843-848Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 5Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 22957-22961Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 6Kast C. Canfield V. Levenson R. Gros P. Biochemistry. 1995; 34: 4402-4411Crossref PubMed Scopus (87) Google Scholar). P-gp functions as a monomer (7Loo T.W. Clarke D.M. J. Biol. Chem. 1996; 271: 27488-27492Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), but the two halves of the molecule do not have to be covalently linked for function (8Loo T.W. Clarke D.M. J. Biol. Chem. 1994; 269: 7750-7755Abstract Full Text PDF PubMed Google Scholar, 9Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 24759-24765Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Studies on deletion mutants have shown that the TM domains alone are sufficient to mediate drug binding (9Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 24759-24765Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Drug binding requires the contribution of the six NH2-terminal TM segments and the six COOH-terminal TM segments (10Loo T.W. Clarke D.M. J. Biol. Chem. 1998; 273: 14671-14674Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). P-glycoprotein transmembrane human embryonic kidney residues 1–379 + residues 681–1025 of P-gp P-glycoprotein transmembrane human embryonic kidney residues 1–379 + residues 681–1025 of P-gp An important goal in understanding the mechanism of P-gp is to determine how P-gp recognizes so many structurally diverse compounds. Studies on cysteine mutants and their reactivity with different thiol-reactive substrate analogs indicate that the drug-binding site is lined with residues from multiple TM segments (11Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 31945-31948Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 12Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 35388-35392Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 13Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 39272-39278Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 14Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 14972-14979Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 15Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 36877-36880Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 16Loo T.W. Clarke D.M. J. Biol. Chem. 2002; 277: 44332-44338Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). Therefore, it has been proposed that P-gp binds these substrates through an induced-fit mechanism where the size and shape of the substrate changes packing of the TM segments. In this study we used cysteine-scanning mutagenesis and oxidative cross-linking to test for rearrangement of the TM segments in the presence of different substrates. A histidine-tagged Cys-less P-gp cDNA was constructed (4Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 843-848Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 17Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 19965-19972Abstract Full Text PDF PubMed Google Scholar). Pairs of cysteines were then introduced into the Cys-less P-gp (18Loo T.W. Clarke D.M. J. Biol. Chem. 1996; 271: 27482-27487Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). The presence of the histidine tag facilitated purification of the mutant P-gps by nickel-chelate chromatography (19Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 21449-21452Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). A cDNA coding for the TM domains only (TMD1+2, residues 1–379 + residues 681–1025) and tagged with the epitope for monoclonal antibody A52 was constructed as described previously (9Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 24759-24765Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar,20Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 3143-3149Abstract Full Text PDF PubMed Google Scholar). HEK 293 cells were transfected with the mutant cDNAs. After 24 h, the medium was replaced with fresh media and the cells grown for 72 h at 27 °C. Membranes were prepared as described previously (17Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 19965-19972Abstract Full Text PDF PubMed Google Scholar, 19Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 21449-21452Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar) and used for disulfide cross-linking analysis (21Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 5253-5256Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) by incubation with Cu2+(phenanthroline)3 (0.2 mm final concentration) under the conditions described in the figure legends. The histidine-tagged P-gp mutants were expressed in HEK 293 cells, purified, and mixed with lipid as described previously (19Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 21449-21452Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). An aliquot of the P-gp:lipid mixture was assayed for drug-stimulated ATPase activity by addition of an equal volume of buffer containing 100 mm Tris-HCl, pH 7.5, 100 mm NaCl, 20 mm MgCl2, 10 mm ATP and 10 mm colchicine, 2 mmdemecolcine, 0.4 mm progesterone, 0.2 mmcis-(Z)-flupenthixol, 1 mm verapamil, or 20 μm vinblastine. These concentrations gave maximal stimulation of the mutant P-gp mutant P350C. The samples were incubated for 30 min at 37 °C, and the amount of inorganic phosphate liberated was determined (22Chifflet S. Torriglia A. Chiesa R. Tolosa S. Anal. Biochem. 1988; 168: 1-4Crossref PubMed Scopus (407) Google Scholar). We had proposed that substrate binding occurred through a “substrate-induced fit” mechanism (14Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 14972-14979Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). The TM segments contributing residues to the drug-binding site are predicted to be quite mobile so that drug binding causes rearrangement of the TM segments. Cysteine-scanning mutagenesis and oxidative cross-linking studies are ideal techniques for detecting altered packing of the TM segments in the presence of drug substrates. The TM segments in the resting state are arranged in a “funnel shape” in which the cytoplasmic ends are close together (15Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 36877-36880Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 21Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 5253-5256Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). The cytoplasmic side of TM6 and TM12 is relatively close together, because mutant P350C(TM6)/S993C(TM12) could be cross-linked. We showed that cross-linking of this mutant inhibited drug-stimulated ATPase activity, but activity was restored after addition of a thiol reducing agent (23Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 20986-20989Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Therefore, it may be possible to detect changes in the cross-linking pattern if P350C(TM6) or S993C(TM12) is paired with another cysteine at the cytoplasmic end of other TM segments. Accordingly, 143 double cysteine mutants that were constructed previously were used in this study (21Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 5253-5256Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). These mutants contained P350C(TM6) and another cysteine on the cytoplasmic half of TMs 7–12 (i.e. P350C + another cysteine at positions: 711–723 (TM7), 770–783 (TM8), 828–840 (TM9), 867–879 (TM10), 935–947 (TM11), and 986–994 (TM12)) or S993C(TM12) with another cysteine in the cytoplasmic half of TMs 1–6 (i.e. S993C + another cysteine at positions: 51–61 (TM1), 130–141 (TM2), 185–196 (TM3), 226–237 (TM4), 293–304 (TM5), and 343–351 (TM6)). We had shown that most of the double cysteine mutants retained at least 70% of the verapamil-stimulated ATPase activity of the Cys-less P-gp (21Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 5253-5256Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). We then tested compounds of different shapes and sizes (colchicine, demecolcine, verapamil, vinblastine, cyclosporin A, progesterone,cis-(Z)-flupenthixol andtrans-(E)-flupenthixol) for their ability to promote cross-linking. Vinblastine and colchicine are classic transport substrates of P-gp (24Horio M. Chin K.V. Currier S.J. Goldenberg S. Williams C. Pastan I. Gottesman M.M. Handler J. J. Biol. Chem. 1989; 264: 14880-14884Abstract Full Text PDF PubMed Google Scholar, 25Choi K. Frommel T.O. Stern R.K. Perez C.F. Kriegler M. Tsuruo T. Roninson I.B. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7386-7390Crossref PubMed Scopus (81) Google Scholar). Demecolcine and verapamil induce large increases in ATPase activity of P-gp (26Loo T.W. Bartlett M.C. Clarke D.M. J. Biol. Chem. 2003; 278: 1575-1578Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Cyclosporin A is a large cyclic peptide substrate (27Tamai I. Safa A.R. J. Biol. Chem. 1991; 266: 16796-16800Abstract Full Text PDF PubMed Google Scholar), while the flupenthixol isomers have opposite effects on the ATPase activity of P-gp (28Dey S. Hafkemeyer P. Pastan I. Gottesman M.M. Biochemistry. 1999; 38: 6630-6693Crossref PubMed Scopus (57) Google Scholar). Progesterone is a steroid substrate of P-gp (29Yang C.P. Cohen D. Greenberger L.M. Hsu S.I. Horwitz S.B. J. Biol. Chem. 1990; 265: 10282-10288Abstract Full Text PDF PubMed Google Scholar, 30Vo Q.D. Gruol D.J. J. Biol. Chem. 1999; 274: 20318-20327Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). The mutants were expressed transiently in HEK 293 cells. Membranes were prepared and preincubated at 21 °C for 10 min with or without saturating levels of colchicine (5 mm), demecolcine (1 mm), verapamil (1 mm), vinblastine (0.1 mm), cyclosporin A (0.1 mm), cis-(Z)-flupenthixol (2 mm), trans-(E)-flupenthixol (2 mm), or progesterone (2 mm). The membranes were treated with 0.2 mm copper phenanthroline (oxidant) for 10 min at 21 °C. The reactions were stopped by addition of EDTA and the samples subjected to SDS-PAGE and immunoblot analysis. Fig.1 shows that in the absence of oxidant, mature P-gp migrates with an apparent mass of 170 kDa. Cross-linked P-gp, however, migrates with a slower mobility in SDS-PAGE (18Loo T.W. Clarke D.M. J. Biol. Chem. 1996; 271: 27482-27487Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 31Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 19435-19438Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar,32Urbatsch I.L. Gimi K. Wilke-Mounts S. Lerner-Marmarosh N. Rousseau M.E. Gros P. Senior A.E. J. Biol. Chem. 2001; 276: 26980-26987Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). In most mutants, cross-linked product was not detected in the presence of drug substrates. An example is mutant P350C(TM6)/A869C(TM10) (Fig. 1). Three mutants (P350C(TM6)/A935C(TM11), P350C(TM6)/G939C(TM11) and P350C(TM6)/V991C(TM12)), however, showed drug-induced cross-linking (Fig. 1). Mutant P350C(TM6)/A935C(TM11) was cross-linked only in the presence of progesterone. Cross-linking of mutant P350C(TM6)/G939C(TM11) was enhanced in the presence of cyclosporin A and progesterone, while mutant P350C(TM6)/V991C(TM12) showed enhanced cross-linking with colchicine, demecolcine, and progesterone (Fig. 1). It was important to express the mutant P-gps in the absence of drug substrate or to thoroughly wash the membranes several times with tris-buffered saline if they were prepared from cells that had been exposed to a drug substrate. This is because residual substrate in the membrane could potentially influence the cross-linking pattern. An example was mutant P350C(TM6)/G939C(TM11). When this mutant was expressed in the absence of cyclosporin A, it showed no cross-linking at 21 °C (Fig. 1), but some cross-linking was detected when membranes prepared from cyclosporin A-treated cells were treated with oxidant (21Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 5253-5256Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Therefore, in this study, membranes were prepared from transfected cells that were not treated with any substrate. To compare the effectiveness of the drug substrates in promoting cross-linking, time-dependent cross-linking studies were done on P350C(TM6)/A935C(TM11), P350C(TM6)/G939C(TM11), and P350C(TM6)/V991C(TM12). Fig. 2 shows that in the absence of drug substrate, no cross-linked product was detected in any of the mutants. Progesterone, however, promoted cross-linking in all three mutants and was the only drug substrate that promoted cross-linking of mutant P350C(TM6)/A935C(TM11). In mutant P350C(TM6)/G939C(TM11), progesterone was more efficient in promoting cross-linking than cyclosporin A. In mutant P350C(TM6)/V991C(TM12), the drug substrates colchicine, demecolcine, and progesterone were equally effective in promoting cross-linking. Cross-linking promoted by the presence of drug substrates cyclosporin A, colchicine, demecolcine, or progesterone could be explained on the basis that drug binding to the TM domain alters TM segment packing. It has been reported, however, that some drug substrates such as progesterone bind to mouse P-gp in a region of the nucleotide-binding domain that is in close proximity to the ATP site (33Dayan G. Baubichon-Cortay H. Jault J.M. Cortay J.C. Deleage G. Di Pietro A. J. Biol. Chem. 1996; 271: 11652-11658Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). To test whether cyclosporin A, colchicine, demecolcine, or progesterone can bind to the TM domains, we used a “drug rescue” assay involving a P-gp mutant that lacked the NBDs (TMD1+2, residues 1–379 + residues 681–1025). The rationale for the drug rescue assay is that the TMD1+2 mutant is misfolded when transiently expressed in the absence of drug substrate and is retained within the cell as a 80-kDa core-glycosylated protein (34Loo T.W. Clarke D.M. J. Biol. Chem. 1994; 269: 28683-28689Abstract Full Text PDF PubMed Google Scholar). Expression of the mutant TMD1+2 in the presence of drug substrate, however, induces the mutant protein to fold properly into a 100-kDa protein endoglycosidase H-resistant form that is transported to the cell surface (9Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 24759-24765Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). It appears that the drug substrate diffuses into the cell and acts as a specific chemical chaperone to bind and stabilize the newly synthesized misfolded P-gp that is present transiently and thereby induce proper folding and trafficking of the protein (10Loo T.W. Clarke D.M. J. Biol. Chem. 1998; 273: 14671-14674Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 35Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 709-712Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). The TMD1+2 deletion mutant was expressed in HEK 293 cells with or without demecolcine, colchicine, or progesterone. The cells were solubilized with SDS buffer and subjected to immunoblot analysis. Fig.3 shows that mutant TMD1+2 is expressed as an 80-kDa protein in the absence of substrate. In the presence of demecolcine, colchicine, or progesterone, however, the presence of a 100-kDa protein is detected. Cyclosporin A also induced proper folding of mutant TMD1+2 (9Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 24759-24765Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Therefore, these drug substrates can interact with only the TM domains. We then tested whether the mutants could interact with colchicine, demecolcine, progesterone, cis-(Z)-flupenthixol, verapamil, and vinblastine, because only some of the drugs affected the cross-linking pattern of the mutants. It was important to determine that the drugs that had no effect on cross-linking could still bind to the mutant P-gps. There is good correlation between drug-stimulated ATPase activity and drug transport (36Ambudkar S.V. Cardarelli C.O. Pashinsky I. Stein W.D. J. Biol. Chem. 1997; 272: 21160-21166Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar) (37Doige C.A. Yu X. Sharom F.J. Biochim. Biophys. Acta. 1992; 1109: 149-160Crossref PubMed Scopus (137) Google Scholar). Accordingly, we tested whether the drug substrates colchicine, demecolcine, progesterone,cis-(Z)-flupenthixol, verapamil, or vinblastine stimulated the ATPase activities of histidine-tagged mutants P350C(TM6)/A935C(TM11), P350C(TM6)/G939C(TM11), and P350C(TM6)/V991C(TM12). Their activities were compared with that of mutant P350C (parent). We previously showed that mutant P350C exhibited high levels of drug-stimulated ATPase activity (11Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 31945-31948Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Fig.4 shows that the ATPase activities of mutants P350C(TM6)/A935C(TM11), P350C(TM6)/G939C(TM11), and P350C(TM6)/V991C(TM12) were stimulated by colchicine, demecolcine, progesterone, cis-(Z)-flupenthixol, verapamil, or vinblastine. The ATPase activities of the mutants were inhibited by cyclosporin A and trans-(Z)-flupenthixol (data not shown). This is consistent with previous observations that saturating concentrations of cyclosporin A inhibits P-gp ATPase activity, while the cis-(Z)- andtrans-(E)-isomers of flupenthixol stimulate and inhibit, respectively, the ATPase activity of P-gp (26Loo T.W. Bartlett M.C. Clarke D.M. J. Biol. Chem. 2003; 278: 1575-1578Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 38Sarkadi B. Muller M. Semin. Cancer Biol. 1997; 8: 171-182Crossref PubMed Scopus (59) Google Scholar, 39Dey S. Ramachandra M. Pastan I. Gottesman M.M. Ambudkar S.V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10594-10599Crossref PubMed Scopus (355) Google Scholar). These results indicate that the mutant P-gps can still bind drug substrates. Cross-linking of mutant P350C(TM6)/S993(TM12) is inhibited by drug substrates (23Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 20986-20989Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). In the present study, we show that substrates such as progesterone are effective in promoting new cross-links with P350C (Fig. 2). In the absence of drug substrate, residue P350C in TM6 can be cross-linked to S993C in TM12. The presence of progesterone, however, promoted cross-linking of residue P350C(TM6) with two residues in TM 11 (A935C and G939C) and to residue V991C in TM12. One way to explain these results is through a model (Fig.5). Based on the model of the TM segments (15Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 36877-36880Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar), the cytoplasmic ends of TMs and are together than the ends In the absence of substrate, it is possible to Therefore, the presence of drug substrate causes a of TM12 to TM6 such that residue V991C to P350C. and demecolcine induced drug substrates also promoted cross-linking of mutant P350C(TM6)/V991C(TM12). TM12 is to any or in TM12 in When the residues in are as an residues and are on the of the TM segment (Fig. In the presence of progesterone residues close to P350C(TM6) to be cross-linked Cyclosporin A also promoted cross-linking between TM6 and but only between P350C and It is possible that cyclosporin A altered the or between TM6 and such that only but not were close to P350C to be cross-linked. The ability of a substrate to the cross-linking pattern that the TM segments can their shape to accommodate structurally different compounds. in any TM segment could in of residues contributing to the drug-binding A substrate with specific in the different TM segments for binding Therefore, it that residues could be in the binding of different substrates. This for the ability of P-gp to bind structurally diverse compounds.