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• W2017236738 abstract "The human multidrug resistance P-glycoprotein (P-gp) interacts with a broad range of compounds with diverse structures and sizes. There is considerable evidence indicating that residues in transmembrane segments 4–6 and 10–12 form the drug-binding site. We attempted to measure the size of the drug-binding site by using thiol-specific methanethiosulfonate (MTS) cross-linkers containing spacer arms of 2 to 17 atoms. The majority of these cross-linkers were also substrates of P-gp, because they stimulated ATPase activity (2.5- to 10.1-fold). 36 P-gp mutants with pairs of cysteine residues introduced into transmembrane segments 4–6 and 10–12 were analyzed after reaction with 0.2 mm MTS cross-linker at 4 °C. The cross-linked product migrated with lower mobility than native P-gp in SDS gels. 13 P-gp mutants were cross-linked by MTS cross-linkers with spacer arms of 9–25 Å. Vinblastine and cyclosporin A inhibited cross-linking. The emerging picture from these results and other studies is that the drug-binding domain is large enough to accommodate compounds of different sizes and that the drug-binding domain is “funnel” shaped, narrow at the cytoplasmic side, at least 9–25 Å in the middle, and wider still at the extracellular surface. The human multidrug resistance P-glycoprotein (P-gp) interacts with a broad range of compounds with diverse structures and sizes. There is considerable evidence indicating that residues in transmembrane segments 4–6 and 10–12 form the drug-binding site. We attempted to measure the size of the drug-binding site by using thiol-specific methanethiosulfonate (MTS) cross-linkers containing spacer arms of 2 to 17 atoms. The majority of these cross-linkers were also substrates of P-gp, because they stimulated ATPase activity (2.5- to 10.1-fold). 36 P-gp mutants with pairs of cysteine residues introduced into transmembrane segments 4–6 and 10–12 were analyzed after reaction with 0.2 mm MTS cross-linker at 4 °C. The cross-linked product migrated with lower mobility than native P-gp in SDS gels. 13 P-gp mutants were cross-linked by MTS cross-linkers with spacer arms of 9–25 Å. Vinblastine and cyclosporin A inhibited cross-linking. The emerging picture from these results and other studies is that the drug-binding domain is large enough to accommodate compounds of different sizes and that the drug-binding domain is “funnel” shaped, narrow at the cytoplasmic side, at least 9–25 Å in the middle, and wider still at the extracellular surface. P-glycoprotein methanethiosulfonate transmembrane polyacrylamide gel electrophoresis human embryonic kidney 1,2-ethanediyl bismethanethiosulfonate 1,3-propanediyl bismethanethiosulfonate 1,4-butanediyl bismethanethiosulfonate 1,5-pentanediyl bismethanethiosulfonate 1,6-hexanediyl bismethanethiosulfonate 3,6-dioxaoctane-1,8-diyl bismethanethiosulfonate 3,6,9-trioxaundecane-1,11-diyl bismethanethiosulfonate 3,6,9,12-tetraoxatetradecane-1,14-diyl bismethanethiosulfonate 3,6,9,12,15-pentaoxaheptadecane-1,17-diyl bismethanethiosulfonate The human multidrug resistance P-glycoprotein (P-gp)1 uses ATP to pump out of the cell a wide variety of structurally diverse compounds (recently reviewed in Ref. 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 (1911) Google Scholar). Many of these compounds are clinically important in cancer and AIDS chemotherapy (2Kim R.B. Fromm M.F. Wandel C. Leake B. Wood A.J. Roden D.M. Wilkinson G.R. J. Clin. Invest. 1998; 101: 289-294Crossref PubMed Scopus (1029) Google Scholar, 3Lee C.G. Gottesman M.M. Cardarelli C.O. Ramachandra M. Jeang K.T. Ambudkar S.V. Pastan I. Dey S. Biochemistry. 1998; 37: 3594-3601Crossref PubMed Scopus (457) Google Scholar, 4Robert J. Eur. J. Clin. Invest. 1999; 29: 536-545Crossref PubMed Scopus (71) Google Scholar). Therefore, overexpression of P-gp often leads to multidrug resistance. The pattern of expression in tissues and studies on P-gp knock-out mice indicate that its physiological role may be to protect the organism from toxins in the environment and in the diet (5Thiebaut F. Tsuruo T. Hamada H. Gottesman M.M. Pastan I. Willingham M.C. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7735-7738Crossref PubMed Scopus (2550) Google Scholar, 6Cordon-Cardo C. O'Brien J.P. Casals D. Rittman-Grauer L. Biedler J.L. Melamed M.R. Bertino J.R. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 695-698Crossref PubMed Scopus (1587) Google Scholar, 7Schinkel A.H. Smit J.J. van Tellingen O. Beijnen J.H. Wagenaar E. van Deemter L. Mol C.A. van der Valk M.A. Robanus-Maandag E.C. te Riele H.P. Berns A.J.M. Borst P. Cell. 1994; 77: 491-502Abstract Full Text PDF PubMed Scopus (2055) Google Scholar). P-gp is a member of the ABC (ATP-bindingcassette) family of transporters (8Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3351) Google Scholar). Its 1280 amino acids are organized in two repeating units of 610 amino acids that are joined by a linker region of about 60 amino acids (9Chen 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 (1713) Google Scholar). Each repeat has six transmembrane (TM) segments and a hydrophilic domain containing an ATP-binding site (10Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 843-848Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 11Kast C. Canfield V. Levenson R. Gros P. J. Biol. Chem. 1996; 271: 9240-9248Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). An important goal in determining the mechanism of P-gp is to understand how P-gp can bind so many different compounds and how ATP hydrolysis causes drug transport. The minimal functional unit in P-gp is a monomer (12Loo T.W. Clarke D.M. J. Biol. Chem. 1996; 271: 27488-27492Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Both nucleotide-binding sites are required for function, because P-gp is inactive when ATP hydrolysis at either site in blocked by mutation or chemical modification (13Azzaria M. Schurr E. Gros P. Mol. Cell. Biol. 1989; 9: 5289-5297Crossref PubMed Scopus (270) Google Scholar, 14Loo T.W. Clarke D.M. J. Biol. Chem. 1994; 269: 7750-7755Abstract Full Text PDF PubMed Google Scholar, 15Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 22957-22961Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 16Urbatsch I.L. Sankaran B. Bhagat S. Senior A.E. J. Biol. Chem. 1995; 270: 26956-26961Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 17Hrycyna C.A. Ramachandra M. Ambudkar S.V. Ko Y.H. Pedersen P.L. Pastan I. Gottesman M.M. J. Biol. Chem. 1998; 273: 16631-16634Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 18Loo T.W. Clarke D.M. J. Natl. Cancer Inst. 2000; 92: 898-902Crossref PubMed Scopus (94) Google Scholar). The nucleotide-binding domains may function in an alternating mechanism (19Senior A.E. Gadsby D.C. Semin Cancer Biol. 1997; 8: 143-150Crossref PubMed Scopus (129) Google Scholar, 20Walmsley A.R. Zhou T. Borges-Walmsley M.I. Rosen B.P. J. Biol. Chem. 2001; 276: 6378-6391Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Photolabeling and mutational studies indicate that the drug-binding domain is within the TM domains (21Bruggemann E.P. Currier S.J. Gottesman M.M. Pastan I. J. Biol. Chem. 1992; 267: 21020-21026Abstract Full Text PDF PubMed Google Scholar, 22Greenberger L.M. J. Biol. Chem. 1993; 268: 11417-11425Abstract Full Text PDF PubMed Google Scholar, 23Morris D.I. Greenberger L.M. Bruggemann E.P. Cardarelli C. Gottesman M.M. Pastan I. Seamon K.B. Mol. Pharmacol. 1994; 46: 329-337PubMed Google Scholar, 24Zhang X. Collins K.I. Greenberger L.M. J. Biol. Chem. 1995; 270: 5441-5448Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 25Demmer A. Thole H. Kubesch P. Brandt T. Raida M. Fislage R. Tummler B. J. Biol. Chem. 1997; 272: 20913-20919Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 26Demeule M. Laplante A. Murphy G.F. Wenger R.M. Beliveau R. Biochemistry. 1998; 37: 18110-18118Crossref PubMed Scopus (46) Google Scholar, 27Gros P. Dhir R. Croop J. Talbot F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7289-7293Crossref PubMed Scopus (190) Google Scholar, 28Kajiji S. Talbot F. Grizzuti K. Van Dyke-Phillips V. Agresti M. Safa A.R. Gros P. Biochemistry. 1993; 32: 4185-4194Crossref PubMed Scopus (90) Google Scholar, 29Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 3143-3149Abstract Full Text PDF PubMed Google Scholar, 30Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 19965-19972Abstract Full Text PDF PubMed Google Scholar, 31Loo T.W. Clarke D.M. Biochemistry. 1994; 33: 14049-14057Crossref PubMed Scopus (125) Google Scholar). This is supported by the finding that a deletion mutant lacking both nucleotide-binding domains can still bind drug substrate (32Loo 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 both halves of the TM domains, because drug-stimulated ATPase activity is only observed when both halves are coexpressed (14Loo T.W. Clarke D.M. J. Biol. Chem. 1994; 269: 7750-7755Abstract Full Text PDF PubMed Google Scholar). Disulfide cross-linking studies have provided considerable insight into the structure of membrane proteins (33Falke J.J. Koshland Jr., D.E. Science. 1987; 237: 1596-1600Crossref PubMed Scopus (228) Google Scholar, 34Yu H. Kono M. McKee T.D. Oprian D.D. Biochemistry. 1995; 34: 14963-14969Crossref PubMed Scopus (119) Google Scholar, 35Frillingos S. Sahin-Toth M. Wu J. Kaback H.R. FASEB J. 1998; 12: 1281-1299Crossref PubMed Scopus (319) Google Scholar, 36Bass R.B. Falke J.J. Structure Fold Des. 1999; 7: 829-840Abstract Full Text Full Text PDF Scopus (59) Google Scholar, 37Jiang W. Fillingame R.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6607-6612Crossref PubMed Scopus (150) Google Scholar). Recently, we identified residues in TMs 4, 5, 6, 10, 11, and 12 that contribute to the drug-binding domain (38Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 31945-31948Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 39Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 35388-35392Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 40Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 39272-39278Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 41Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 14972-14979Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). In this study, we used a series of thiol-specific cross-linkers with spacer arms of various lengths to measure distances between these residues. There are seven endogenous cysteines at positions 137, 431, 717, 956, 1074, 1125, and 1227 in wild-type P-gp. None of the cysteines are needed for activity, because mutation of all cysteines to alanine (Cys-less P-gp) resulted in an active molecule (10Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 843-848Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). The Cys-less P-gp cDNA was also modified to code for ten histidine residues at the COOH end of the molecule (Cys-less P-gp(His)10). The histidine tag facilitated purification of the Cys-less P-gp by nickel-chelate chromatography (42Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 21449-21452Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Cysteine residues were then introduced into the Cys-less P-gp(His)10 as described previously (40Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 39272-39278Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). The integrity of the mutated cDNA was confirmed by sequencing the entire cDNA (43Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52505) Google Scholar). Expression and purification of histidine-tagged P-gp mutants were described previously (42Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 21449-21452Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Briefly, 50 10-cm diameter culture plates of HEK 293 cells were transfected with the mutant cDNA. After 24 h, the medium was replaced with fresh medium containing 10 μm cyclosporin A. Cyclosporin A is a substrate of P-gp and is a powerful chemical chaperone for promoting maturation of P-gp (44Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 709-712Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). The transfected cells were harvested 24 h later, solubilized with 1% (w/v)n-dodecyl-β-d-maltoside, and the mutant P-gp was isolated by nickel-chelate chromatography (Ni-NTA columns; Qiagen, Inc., Mississauga, Ontario, Canada). The P-gp-(His)10 mutants were eluted from the column and mixed with an equal volume of 10 mg/ml sheep brain phosphatidylethanolamine (Type II-S; Sigma-Aldrich) that was washed and suspended in 10 mm Tris-HCl, pH 7.5, and 150 mm NaCl. The P-gp:lipid mixture was then sonicated for 45 s at 4 °C. An aliquot of the 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 2 mm MTS cross-linker. The samples were incubated for 30 min at 37 °C, and the amount of inorganic phosphate liberated was determined (45Chifflet S. Torriglia A. Chiesa R. Tolosa S. Anal. Biochem. 1988; 168: 1-4Crossref PubMed Scopus (414) Google Scholar). The mutant P-gps were expressed in HEK 293 cells in the presence of 10 μm cyclosporin A. Membranes were prepared from transfected cells and suspended in Tris-buffered saline (10 mm Tris-HCl, pH 7.4, 150 mm NaCl) and treated with 0.2 mm cross-linker (Toronto Research Chemicals, Toronto, Ontario, Canada; see Fig. 1) for 15 min at 4 °C. At this concentration, the MTS cross-linkers stimulated the ATPase activity of Cys-less P-gp by about 50%. The reactions were stopped by addition of 2× SDS sample buffer containing 10 mm N-ethylmaleimide. In the protection experiments, the membranes were pretreated for 10 min at 4 °C in the presence of 1 mm vinblastine or 1 mmcyclosporin A (saturating conditions). The samples were subjected to SDS-PAGE on 7.5% acrylamide gels and immunoblot analysis with rabbit polyclonal antibody (12Loo T.W. Clarke D.M. J. Biol. Chem. 1996; 271: 27488-27492Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). To measure distances between residues in the NH2 and COOH halves of the drug-binding domain, we constructed P-gp mutants that had a pair of cysteine residues, one in the NH2 half and the other in the COOH half (Table I). The mutants were then tested for cross-linking with thiol-specific cross-linkers. In attempting to measure distances between residues, it would be most useful to use cross-linkers that had similar spacer arms and reactive groups to minimize differences in chemical reactivity with cysteines. A set of thiol-specific cross-linkers with these properties is the MTS cross-linkers shown in Fig.1. Alkylthiosulfonates react selectively with cysteines in a protein resulting in a disulfide attachment of the spacer arm and release of a sulfonic acid byproduct (46Kenyon G.L. Bruice T.W. Methods Enzymol. 1977; 47: 407-430Crossref PubMed Scopus (171) Google Scholar, 47Bruice T.W. Kenyon G.L. J. Protein Chem. 1982; 1: 47-58Crossref Scopus (94) Google Scholar). The MTS compounds are generally more reactive with cysteines than other thiol-specific compounds such as maleimides or iodoacetates (47Bruice T.W. Kenyon G.L. J. Protein Chem. 1982; 1: 47-58Crossref Scopus (94) Google Scholar).Table ICross-linking with MTS cross-linkersS222C(TM4)I306C(TM5)L339C(TM6)A342C(TM6)I868C (TM10)5, 6, 8, 171-aCross-linked product detected in SDS-PAGE using MTS cross-linkers shown in Fig. 1. The number represents the length of the spacer arm of the MTS cross-linker.8, 178, 11, 14, 17–1-bNo cross-linked product detected with any MTS cross-linker.A871C (TM10)––––G872C (TM10)5, 6, 178, 178, 11, 14, 17–F942C (TM11)––17–T945C (TM11)–8, 11, 1714, 17–L975C (TM12)––––V982C (TM12)–8, 11, 14, 1711, 14, 17–G984C (TM12)–8, 11, 14, 17––A985C (TM12)––14, 17–1-a Cross-linked product detected in SDS-PAGE using MTS cross-linkers shown in Fig. 1. The number represents the length of the spacer arm of the MTS cross-linker.1-b No cross-linked product detected with any MTS cross-linker. Open table in a new tab In using cross-linkers to map distances in the drug-binding domain, it is important that the compounds can actually occupy the drug-binding site. It is best to be able to measure P-gp-mediated transport of these MTS compounds. This is technically not feasible, because radioactive forms are not available commercially. Another complication is the short half-lives of MTS compounds in aqueous media (46Kenyon G.L. Bruice T.W. Methods Enzymol. 1977; 47: 407-430Crossref PubMed Scopus (171) Google Scholar), and they are also not fluorescent. One way around these problems is to measure stimulation of ATPase activity. Binding of most substrates to P-gp stimulates its ATPase activity, and there is good correlation between drug-stimulated ATPase activity and drug transport (48Ambudkar S.V. Cardarelli C.O. Pashinsky I. Stein W.D. J. Biol. Chem. 1997; 272: 21160-21166Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Accordingly, all the MTS cross-linkers (Fig. 1) were assayed for their ability to stimulate Cys-less P-gp ATPase activity. Cys-less P-gp has all seven endogenous cysteines replaced with alanine and is nearly as active (drug transport and drug-stimulated ATPase activity) as wild-type P-gp (10Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 843-848Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 42Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 21449-21452Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Fig. 2 shows the stimulation of Cys-less P-gp ATPase activity in the presence of saturating concentrations (2 mm) of each MTS cross-linker. All the compounds, except M3M, stimulated the ATPase activity of Cys-less P-gp. The most potent stimulators of activity were M6M and M8M (about 10-fold). Relatively lower levels of stimulation were observed with M5M, M11M, M14M, and M17M (6- to 8-fold), whereas M2M and M4M stimulated activity 2.5- to 3.5-fold. In general, the best stimulators of activity were compounds with spacer arms of intermediate lengths (10 to 13 Å). For comparison, verapamil, the best stimulator of P-gp activity, stimulates Cys-less P-gp activity about 14-fold, whereas the substrate vinblastine stimulates activity about 6-fold (41Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 14972-14979Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). The results show that most of the MTS cross-linkers can occupy the drug-binding site of P-gp. Recently, we used dibromobimane (38Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 31945-31948Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 39Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 35388-35392Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 40Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 39272-39278Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar) and MTS-verapamil (41Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 14972-14979Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar) to show that cysteines introduced at positions 222(TM4), 306(TM5), 339(TM6), and 342(TM6) in the NH2 half of P-gp and at 868(TM10), 871(TM10), 872(TM10), 942(TM11), 945(TM11), 975(TM12), 982(TM12), 984(TM12), and 985(TM12) in the COOH half of P-gp contribute to the drug-binding domain (40Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 39272-39278Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). To test for cross-linking, mutants with one cysteine in the NH2 half (positions 222, 306, 339, and 342) and another in the COOH half (positions 868, 871, 872, 942, 945, 975, 982, 984, and 985) were made (Table I). The mutants were first expressed in HEK 293 cells and were found to be processed to the fully mature (170 kDa) form of P-gp (data not shown). The 36 mutants (Table I) were then subjected to cross-linking by the MTS cross-linkers (Fig. 1). Membranes were prepared from transfected cells and treated with different cross-linkers (0.2 mm) at 4 °C. The reactions were done at 4 °C to reduce molecular motions in the protein. The reactions were stopped by addition of SDS sample buffer containing N-ethylmaleimide, and the mixture was analyzed by SDS-PAGE. In previous studies we had shown the cross-linking between residues in the NH2 and COOH halves of P-gp resulted in the cross-linked product migrating with lower mobility in SDS gels (49Loo T.W. Clarke D.M. J. Biol. Chem. 1996; 271: 27482-27487Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 50Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 20986-20989Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Similarly, cross-linking by MTS cross-linkers resulted in decreased mobility of the cross-linked protein. Cross-linked product was detected in 13 mutants. Representative positive (mutant I306C/V982C) and negative (mutant I306C/A871C) results are shown in Fig. 3. In mutant I306C/V982C, strongest cross-linking was observed with M14M (20.8 Å) and M17M (24.7 Å). Cross-linking was also observed with M8M (13 Å) and M11M (16.9 Å). No evidence of cross-linking was observed with the smaller cross-linkers M2M to M6M (5.2 to 10.4 Å). Cross-linking of mutant I306C/V982C was reversible, because the cross-linked product was not detected after treatment with 5 mm dithiothreitol (data not shown). We then tested whether drug substrates could inhibit cross-linking. If cross-linking occurred between residues in the drug-binding domain, then the presence of substrates such as cyclosporin A and vinblastine should inhibit cross-linking. Cyclosporin A is an inhibitor of P-gp-medicated drug resistance (51Chan H.S. Grogan T.M. DeBoer G. Haddad G. Gallie B.L. Ling V. Eur. J. Cancer. 1996; 6: 1051-1061Abstract Full Text PDF Scopus (29) Google Scholar) whereas vinblastine is a cytotoxic drug substrate (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 (1911) Google Scholar). The membranes containing P-gp mutant I306C/V982C were preincubated with 1 mmcyclosporin A or 1 mm vinblastine for 10 min at 4 °C. The samples were then treated with M17M and subjected to Western blot analysis. Fig. 3 shows that cyclosporin A and vinblastine blocked cross-linking of mutant I306C/V982C by M17M. An example of a blot showing no detectable cross-linking is seen in mutant I306C/A871C (Fig.3, lower panel). The cross-linking results of the other mutants (data not shown) are summarized in Table I. Two of the nine S222C(TM4) mutants were cross-linked. Mutant S222C(TM4)/I868C(TM10) was cross-linked with M5M, M6M, M8M, and M17M, whereas mutant S222C(TM4)/G872C(TM10) was cross-linked with M5M, M6M, and M17M. Only five of the nine I306C(TM5) mutants could be cross-linked. Mutants I306C(TM5)/I868C(TM10) and I306C(TM5)/G872C(TM10) were cross-linked with M8M and M17M. Mutant I306C(TM5)/T945C(TM11) was cross-linked with M8M, M11M, and M17M. Mutants I306C(TM5)/V982C(TM12) and I306C(TM5)/G984C(TM12) were cross-linked with M8M, M11M, M14M, and M17M. Six L339C(TM6) mutants were cross-linked. Mutant L339C(TM6)/F942C(TM11) was cross-linked with only M17M. Mutants L339C(TM6)/T945C(TM12) and L339C(TM6)/A985C(TM12) were cross-linked with M14M and M17M, mutant L339C(TM6)/V982C(TM12) was cross-linked with M11M, M14M, and M17M, whereas mutants L339C(TM6)/I868C(TM10) and L339C(TM6)/G872C(TM10) were cross-linked with M8M, M11M, M14M, and M17M. No cross-linking was detected with the A342C(TM6) mutants. All cross-linking was blocked when the membranes were preincubated with 1 mm vinblastine, 1 mm cyclosporin A. Similarly, no cross-linked product was detected after treatment with dithiothreitol (data not shown). The cross-linking results in Fig.4A show that residues 222(TM4), 306(TM5), 339(TM6), 868(TM10), 872(TM10), 942C(TM11), 945(TM11), 982(TM12), 984(TM12), and 985(TM12) must line a common drug-binding site. Residues in TMs 5 and 6 were cross-linked to residues in TMs 10, 11, and 12, whereas residue S222C(TM4) was cross-linked with two residues (I868C and G872C) in TM10. In all cases, cross-linking was blocked by substrates such as vinblastine or cyclosporin A. Vinblastine and cyclosporin are quite large molecules. Their crystal structures show that they are about 20–25 Å in length at their widest point (52Bau, R., and Jin, K. K. (2000) J. Chem. Soc. Perkin Trans. I, 2079–2082Google Scholar, 53Jegorov A. Cvak L. Husek A. Simek P. Heydova A. Ondracek J. Pakhomova S. Husak M. Kratochvil B. Sedmera P. Havlicek V. Collect. Czech. Chem. Commun. 2000; 65: 1317-1326Crossref Scopus (13) Google Scholar). The cross-linking results indicate that the drug-binding site would be large enough to accommodate vinblastine or cyclosporin A. For example, mutants L339C(TM6)/F942C(TM11), L339C(TM6)/T945C(TM11), and L339C(TM6)/A985C(TM12) are only cross-linked with MTS compounds with spacer arms of more than 20 Å. The smallest distance between TMs 4, 5, and 6 and TM 10, 11, and 12 as measured by these cross-linkers is about 9 Å and occurs between TMs 4 and 10, because mutants S222C(TM4)/I868C(TM10) and S222C(TM4)/G872C(TM10) could be cross-linked with M5M. It is interesting to note that none of the 36 mutants (Table I) showed any detectable cross-linking with a zero-length cross-linker (copper phenanthroline) at either 22 or 4 °C (data not shown). It was surprising to find mutants such as the S222C mutants that were cross-linked with smaller reagents (e.g. M8M) were also cross-linked with the larger M17M cross-linker. One explanation is that the TMs of P-gp are quite flexible (54Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 5253-5256Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar) and therefore, can expand or contract to accommodate different sizes of substrate. This mobility of the helices in accommodating different substrates would inherently expose different residues to the drug-binding site and thereby dictate the affinity of P-gp for a particular substrate. It is also possible that the reactive cysteines may be in a sterically favored position to react with cross-linker of a certain size. This may indeed be the case. Table I show that some but not all of the mutants react with the same combination of MTS cross-linkers. Similarly, no cross-linking was detected in 23 of the mutants. Another possibility is that M17M may be a particularly flexible reagent. Recently, Green et al. (55Green N.S. Reisler E. Houk K.N. Protein. Sci. 2001; 10: 1293-1304Crossref PubMed Scopus (200) Google Scholar) reported that a potential problem with using homobifunctional protein cross-linking agents as molecular rulers is that they can be quite flexible. These compounds can adopt a range of conformations due to rotation of the bonds that form the linker arm. There are several C-O bonds in the spacer arm of M17M that could adopt gauche conformations, thereby bringing the two reactive ends closer together. Thus, it is possible that some mutants are reacting with M17M when it is in conformations that bring the reactive groups closer together, whereas other mutants would react only with the M17M when it is in the extended conformation. The residues that have been identified to contribute to the drug-binding site are in the middle of the predicted TM segments and to be 9–25 Å apart (Fig. 4B). Previous studies have shown that the cytoplasmic side of the TMs (4–6 and 10–12) predicted to line the drug-binding site are closer than this, because disulfide cross-linking occurred between residues in these TMs with a zero-length cross-linking agent (copper phenanthroline) (54Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 5253-5256Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The TM segments on the extracellular surface, however, must be quite far apart. A low resolution crystal structure of P-gp shows the presence of a pore of about 50 Å in diameter on the extracellular surface (56Rosenberg M.F. Callaghan R. Ford R.C. Higgins C.F. J. Biol. Chem. 1997; 272: 10685-10694Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). The emerging picture of P-gp is shown in a cross-sectional view in Fig.4B. In this model, the drug-binding domain appears to be a “funnel” shape, with the widest point at the extracellular side and the narrowest at the cytoplasmic side. Drug binding would occur close to the center of the pore. ATP hydrolysis would change the affinity of P-gp for substrate through conformational changes in the drug-binding domain by rotation (57Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 31800-31805Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) and possibly large movements of helices (50Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 20986-20989Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). ATPase activity is inhibited if conformational changes are prevented by cross-linking of the TM segments (50Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 20986-20989Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 57Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 31800-31805Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). These movements would result in expulsion of the drug substrate and closing of the extracellular side of the pore. We thank Dr. Randal Kaufman (Boston, MA) for pMT21 and Claire Bartlett for assistance with tissue culture." @default.
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• W2017236738 title "Determining the Dimensions of the Drug-binding Domain of Human P-glycoprotein Using Thiol Cross-linking Compounds as Molecular Rulers" @default.
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