The Packing of the Transmembrane Segments of Human Multidrug Resistance P-glycoprotein Is Revealed by Disulfide Cross-linking Analysis

Residues from several transmembrane (TM) segments of P-glycoprotein (P-gp) likely form the drug-binding site(s). To determine the organization of the TM segments, pairs of cysteine residues were introduced into the predicted TM segments of a Cys-less P-gp, and the mutant protein was subjected to oxidative cross-linking. In SDS gels, the cross-linked product migrated with a slower mobility than the native protein. The cross-linked products were not detected in the presence of dithiothreitol. Cross-linking was observed in 12 of 125 mutants. The pattern of cross-linking suggested that TM6 is close to TMs 10, 11, and 12, while TM12 is close to TMs 4, 5, and 6. In some mutants the presence of drug substrate colchicine, verapamil, cyclosporin A, or vinblastine either enhanced or inhibited cross-linking. Cross-linking was inhibited in the presence of ATP plus vanadate. These results suggest that the TM segments critical for drug binding must be close to each other and exhibit different conformational changes in response to binding of drug substrate or vanadate trapping of nucleotide. Based on these results, we propose a model for the arrangement of the TM segments. Residues from several transmembrane (TM) segments of P-glycoprotein (P-gp) likely form the drug-binding site(s). To determine the organization of the TM segments, pairs of cysteine residues were introduced into the predicted TM segments of a Cys-less P-gp, and the mutant protein was subjected to oxidative cross-linking. In SDS gels, the cross-linked product migrated with a slower mobility than the native protein. The cross-linked products were not detected in the presence of dithiothreitol. Cross-linking was observed in 12 of 125 mutants. The pattern of cross-linking suggested that TM6 is close to TMs 10, 11, and 12, while TM12 is close to TMs 4, 5, and 6. In some mutants the presence of drug substrate colchicine, verapamil, cyclosporin A, or vinblastine either enhanced or inhibited cross-linking. Cross-linking was inhibited in the presence of ATP plus vanadate. These results suggest that the TM segments critical for drug binding must be close to each other and exhibit different conformational changes in response to binding of drug substrate or vanadate trapping of nucleotide. Based on these results, we propose a model for the arrangement of the TM segments. P-glycoprotein transmembrane polyacrylamide gel electrophoresis The multidrug resistance P-glycoprotein (P-gp1; product of the human MDR1 gene) uses energy from ATP hydrolysis to pump a broad range of cytotoxic compounds out of the cell. It is found in the plasma membrane of cells lining the gastrointestinal tract, the brush border of renal proximal tubules, on the biliary face of hepatocytes (1.Thiebaut 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 (2545) Google Scholar), or on the luminal surface of capillary endothelial cells of the brain and testes (1.Thiebaut 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 (2545) Google Scholar, 2.Thiebaut F. Tsuruo T. Hamada H. Gottesman M.M. Pastan I. Willingham M.C. J. Histochem. Cytochem. 1989; 37: 159-164Crossref PubMed Scopus (552) Google Scholar). The location of P-gp in tissues, together with studies on P-gp “knock-out” mice suggest that P-gp likely protects the organism from toxic xenobiotics (3.Schinkel A.H. Semin. Cancer Biol. 1997; 8: 161-170Crossref PubMed Scopus (439) Google Scholar).P-gp is clinically important because it contributes to the phenomenon of multidrug resistance during AIDS (4.Kim 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 (1027) Google Scholar) and cancer chemotherapies (reviewed in Ref. 5.Ambudkar S.V. Dey S. Hrycyna C.A. Ramachandra M. Pastan I. Gottesman M.M. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 361-398Crossref PubMed Scopus (1907) Google Scholar). The protein is a member of the ATP-binding cassette family of transporters (6.Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3346) Google Scholar). The 1280 amino acids of P-gp are organized in two tandem repeats of 610 amino acids that are joined by a linker region of 60 amino acids (7.Chen 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 (1709) Google Scholar, 9.Kast C. Canfield V. Levenson R. Gros P. J. Biol. Chem. 1996; 271: 9240-9248Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar).The mechanism that allows P-gp to recognize such a broad range of compounds is unknown. Both halves of P-gp are required for substrate-stimulated ATPase activity (10.Loo T.W. Clarke D.M. J. Biol. Chem. 1994; 269: 7750-7755Abstract Full Text PDF PubMed Google Scholar) and for drug binding (11.Loo T.W. Clarke D.M. J. Biol. Chem. 1998; 273: 14671-14674Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). It was recently shown that the TM domains alone could mediate drug binding (12.Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 24759-24765Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). A deletion mutant lacking both nucleotide-binding domains could still interact with drug substrates. The drug-binding site(s) in P-gp likely consists of residues from multiple TM segments (13.Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 35388-35392Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Understanding the mechanism of drug recognition by P-gp will require knowledge about the packing of the TM segments and their response to the presence of different substrates.In this study, the packing and flexibility of the TMs of P-gp were examined by disulfide cross-linking analysis. Pairs of cysteine residues were introduced into the predicted TM segments of a mutant Cys-less P-gp. The mutant proteins were assayed for disulfide cross-linking.DISCUSSIONThe results from this and previous studies (14.Loo T.W. Clarke D.M. J. Biol. Chem. 1996; 271: 27482-27487Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 15.Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 20986-20989Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar) suggest that TMs 4, 5, and 6 in the NH2-terminal half are close to TMs 10, 11, and 12 in the COOH-terminal half of P-gp. Disulfide cross-linking can occur between residues in TM6 and TMs 10, 11, and 12 and between residues in TM12 and TMs 4, 5, and 6.Our results suggest that binding of substrate induces conformational changes in the TM segments. Colchicine, verapamil, cyclosporin A, and vinblastine had different effects on the cross-linking pattern. A transporter with restricted substrate specificity would be expected to show limited conformational changes upon substrate binding. The capacity of P-gp to accommodate such structurally different substrates could partly be due to relative flexibility and fluidity of TM domains. This is consistent with the finding that there is considerable variability in the ability of colchicine, verapamil, cyclosporin A, or vinblastine to stimulate the ATPase activity of P-gp (13.Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 35388-35392Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar) or to induce conformational changes in the nucleotide-binding sites (22.Liu R. Sharom F.J. Biochemistry. 1997; 36: 2836-2843Crossref PubMed Scopus (83) Google Scholar). The fluid nature of the TM segments is suggested by the finding that only 4 of the 12 mutants could be cross-linked at 4 °C (Table II), while all were cross-linked at 37 °C.Vanadate plus ATP inhibited cross-linking of the mutants by locking-in the nucleotide-binding domain(s) of P-gp in a fixed conformation (Table III). The rigid nature of the locked-in conformation is then transmitted to the TM domains. This supports the notion that there is “cross-talk” between the nucleotide-binding domains and the TM domains and that ATPase activity is directly linked to drug-P-gp interaction.We have proposed a working model of the arrangement of the TM segments of P-gp (Fig. 2). An interesting feature about the model is that the TMs involved in cross-linking have been reported to be important for P-gp-drug interactions. Cysteine-scanning mutagenesis of the TM segments and labeling with a thiol-reactive substrate, dibromobimane, provided direct evidence for interaction of substrate with residues in TMs 6, 11, and 12 (13.Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 35388-35392Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 20.Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 31945-31948Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Dibromobimane reacted with specific cysteine residues in TMs 6, 11, and 12 and inhibited the activity of P-gp. The presence of substrates such as colchicine, verapamil, or vinblastine inhibited labeling by dibromobimane. Many mutations in TMs 4, 5, and 6, and in TMs 10, 11, and 12, have been reported to alter the substrate specificity or activity of the transporter (23.Devine S.E. Ling V. Melera P.W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4564-4568Crossref PubMed Scopus (127) Google Scholar, 24.Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 3143-3149Abstract Full Text PDF PubMed Google Scholar, 25.Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 19965-19972Abstract Full Text PDF PubMed Google Scholar, 26.Loo T.W. Clarke D.M. Biochemistry. 1994; 33: 14049-14057Crossref PubMed Scopus (125) Google Scholar, 27.Kajiji 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, 28.Chen G. Duran G.E. Steger K.A. Lacayo N.J. Jaffrezou J.P. Dumontet C. Sikic B.I. J. Biol. Chem. 1997; 272: 5974-5982Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 29.Hanna M. Brault M. Kwan T. Kast C. Gros P. Biochemistry. 1996; 35: 3625-3635Crossref PubMed Scopus (55) Google Scholar, 30.Shoshani T. Zhang S. Dey S. Pastan I. Gottesman M.M. Mol. Pharmacol. 1998; 54: 623-630PubMed Google Scholar, 31.Hafkemeyer P. Dey S. Ambudkar S.V. Hrycyna C.A. Pastan I. Gottesman M.M. Biochemistry. 1998; 37: 16400-16409Crossref PubMed Scopus (80) Google Scholar). Labeling studies with photoactive analogs of P-gp substrates also suggest that TMs 4, 5, and 6 and TMs 10, 11, and 12 may participate in drug-protein interactions (32.Bruggemann E.P. Germann U.A. Gottesman M.M. Pastan I. J. Biol. Chem. 1989; 264: 15483-15488Abstract Full Text PDF PubMed Google Scholar, 33.Bruggemann E.P. Currier S.J. Gottesman M.M. Pastan I. J. Biol. Chem. 1992; 267: 21020-21026Abstract Full Text PDF PubMed Google Scholar, 34.Greenberger L.M. J. Biol. Chem. 1993; 268: 11417-11425Abstract Full Text PDF PubMed Google Scholar, 35.Morris 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, 36.Demmer 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). The model in Fig. 2 also takes into consideration that no cross-linked products were detected between TMs 1 to 3 and TMs 7 to 9 when cysteine residues were introduced into the full-length Cys-less P-gp or into the Cys-less half-molecules (data not shown).In summary, the TM segments postulated to be involved in drug binding are quite close to each other on the cytoplasmic side of the membrane. The TM segments and nucleotide-binding domains appear to be conformationally flexible to be able to accommodate structurally different substrates. The multidrug resistance P-glycoprotein (P-gp1; product of the human MDR1 gene) uses energy from ATP hydrolysis to pump a broad range of cytotoxic compounds out of the cell. It is found in the plasma membrane of cells lining the gastrointestinal tract, the brush border of renal proximal tubules, on the biliary face of hepatocytes (1.Thiebaut 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 (2545) Google Scholar), or on the luminal surface of capillary endothelial cells of the brain and testes (1.Thiebaut 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 (2545) Google Scholar, 2.Thiebaut F. Tsuruo T. Hamada H. Gottesman M.M. Pastan I. Willingham M.C. J. Histochem. Cytochem. 1989; 37: 159-164Crossref PubMed Scopus (552) Google Scholar). The location of P-gp in tissues, together with studies on P-gp “knock-out” mice suggest that P-gp likely protects the organism from toxic xenobiotics (3.Schinkel A.H. Semin. Cancer Biol. 1997; 8: 161-170Crossref PubMed Scopus (439) Google Scholar). P-gp is clinically important because it contributes to the phenomenon of multidrug resistance during AIDS (4.Kim 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 (1027) Google Scholar) and cancer chemotherapies (reviewed in Ref. 5.Ambudkar S.V. Dey S. Hrycyna C.A. Ramachandra M. Pastan I. Gottesman M.M. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 361-398Crossref PubMed Scopus (1907) Google Scholar). The protein is a member of the ATP-binding cassette family of transporters (6.Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3346) Google Scholar). The 1280 amino acids of P-gp are organized in two tandem repeats of 610 amino acids that are joined by a linker region of 60 amino acids (7.Chen 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 (1709) Google Scholar, 9.Kast C. Canfield V. Levenson R. Gros P. J. Biol. Chem. 1996; 271: 9240-9248Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). The mechanism that allows P-gp to recognize such a broad range of compounds is unknown. Both halves of P-gp are required for substrate-stimulated ATPase activity (10.Loo T.W. Clarke D.M. J. Biol. Chem. 1994; 269: 7750-7755Abstract Full Text PDF PubMed Google Scholar) and for drug binding (11.Loo T.W. Clarke D.M. J. Biol. Chem. 1998; 273: 14671-14674Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). It was recently shown that the TM domains alone could mediate drug binding (12.Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 24759-24765Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). A deletion mutant lacking both nucleotide-binding domains could still interact with drug substrates. The drug-binding site(s) in P-gp likely consists of residues from multiple TM segments (13.Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 35388-35392Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Understanding the mechanism of drug recognition by P-gp will require knowledge about the packing of the TM segments and their response to the presence of different substrates. In this study, the packing and flexibility of the TMs of P-gp were examined by disulfide cross-linking analysis. Pairs of cysteine residues were introduced into the predicted TM segments of a mutant Cys-less P-gp. The mutant proteins were assayed for disulfide cross-linking. DISCUSSIONThe results from this and previous studies (14.Loo T.W. Clarke D.M. J. Biol. Chem. 1996; 271: 27482-27487Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 15.Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 20986-20989Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar) suggest that TMs 4, 5, and 6 in the NH2-terminal half are close to TMs 10, 11, and 12 in the COOH-terminal half of P-gp. Disulfide cross-linking can occur between residues in TM6 and TMs 10, 11, and 12 and between residues in TM12 and TMs 4, 5, and 6.Our results suggest that binding of substrate induces conformational changes in the TM segments. Colchicine, verapamil, cyclosporin A, and vinblastine had different effects on the cross-linking pattern. A transporter with restricted substrate specificity would be expected to show limited conformational changes upon substrate binding. The capacity of P-gp to accommodate such structurally different substrates could partly be due to relative flexibility and fluidity of TM domains. This is consistent with the finding that there is considerable variability in the ability of colchicine, verapamil, cyclosporin A, or vinblastine to stimulate the ATPase activity of P-gp (13.Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 35388-35392Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar) or to induce conformational changes in the nucleotide-binding sites (22.Liu R. Sharom F.J. Biochemistry. 1997; 36: 2836-2843Crossref PubMed Scopus (83) Google Scholar). The fluid nature of the TM segments is suggested by the finding that only 4 of the 12 mutants could be cross-linked at 4 °C (Table II), while all were cross-linked at 37 °C.Vanadate plus ATP inhibited cross-linking of the mutants by locking-in the nucleotide-binding domain(s) of P-gp in a fixed conformation (Table III). The rigid nature of the locked-in conformation is then transmitted to the TM domains. This supports the notion that there is “cross-talk” between the nucleotide-binding domains and the TM domains and that ATPase activity is directly linked to drug-P-gp interaction.We have proposed a working model of the arrangement of the TM segments of P-gp (Fig. 2). An interesting feature about the model is that the TMs involved in cross-linking have been reported to be important for P-gp-drug interactions. Cysteine-scanning mutagenesis of the TM segments and labeling with a thiol-reactive substrate, dibromobimane, provided direct evidence for interaction of substrate with residues in TMs 6, 11, and 12 (13.Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 35388-35392Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 20.Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 31945-31948Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Dibromobimane reacted with specific cysteine residues in TMs 6, 11, and 12 and inhibited the activity of P-gp. The presence of substrates such as colchicine, verapamil, or vinblastine inhibited labeling by dibromobimane. Many mutations in TMs 4, 5, and 6, and in TMs 10, 11, and 12, have been reported to alter the substrate specificity or activity of the transporter (23.Devine S.E. Ling V. Melera P.W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4564-4568Crossref PubMed Scopus (127) Google Scholar, 24.Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 3143-3149Abstract Full Text PDF PubMed Google Scholar, 25.Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 19965-19972Abstract Full Text PDF PubMed Google Scholar, 26.Loo T.W. Clarke D.M. Biochemistry. 1994; 33: 14049-14057Crossref PubMed Scopus (125) Google Scholar, 27.Kajiji 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, 28.Chen G. Duran G.E. Steger K.A. Lacayo N.J. Jaffrezou J.P. Dumontet C. Sikic B.I. J. Biol. Chem. 1997; 272: 5974-5982Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 29.Hanna M. Brault M. Kwan T. Kast C. Gros P. Biochemistry. 1996; 35: 3625-3635Crossref PubMed Scopus (55) Google Scholar, 30.Shoshani T. Zhang S. Dey S. Pastan I. Gottesman M.M. Mol. Pharmacol. 1998; 54: 623-630PubMed Google Scholar, 31.Hafkemeyer P. Dey S. Ambudkar S.V. Hrycyna C.A. Pastan I. Gottesman M.M. Biochemistry. 1998; 37: 16400-16409Crossref PubMed Scopus (80) Google Scholar). Labeling studies with photoactive analogs of P-gp substrates also suggest that TMs 4, 5, and 6 and TMs 10, 11, and 12 may participate in drug-protein interactions (32.Bruggemann E.P. Germann U.A. Gottesman M.M. Pastan I. J. Biol. Chem. 1989; 264: 15483-15488Abstract Full Text PDF PubMed Google Scholar, 33.Bruggemann E.P. Currier S.J. Gottesman M.M. Pastan I. J. Biol. Chem. 1992; 267: 21020-21026Abstract Full Text PDF PubMed Google Scholar, 34.Greenberger L.M. J. Biol. Chem. 1993; 268: 11417-11425Abstract Full Text PDF PubMed Google Scholar, 35.Morris 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, 36.Demmer 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). The model in Fig. 2 also takes into consideration that no cross-linked products were detected between TMs 1 to 3 and TMs 7 to 9 when cysteine residues were introduced into the full-length Cys-less P-gp or into the Cys-less half-molecules (data not shown).In summary, the TM segments postulated to be involved in drug binding are quite close to each other on the cytoplasmic side of the membrane. The TM segments and nucleotide-binding domains appear to be conformationally flexible to be able to accommodate structurally different substrates. The results from this and previous studies (14.Loo T.W. Clarke D.M. J. Biol. Chem. 1996; 271: 27482-27487Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 15.Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 20986-20989Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar) suggest that TMs 4, 5, and 6 in the NH2-terminal half are close to TMs 10, 11, and 12 in the COOH-terminal half of P-gp. Disulfide cross-linking can occur between residues in TM6 and TMs 10, 11, and 12 and between residues in TM12 and TMs 4, 5, and 6. Our results suggest that binding of substrate induces conformational changes in the TM segments. Colchicine, verapamil, cyclosporin A, and vinblastine had different effects on the cross-linking pattern. A transporter with restricted substrate specificity would be expected to show limited conformational changes upon substrate binding. The capacity of P-gp to accommodate such structurally different substrates could partly be due to relative flexibility and fluidity of TM domains. This is consistent with the finding that there is considerable variability in the ability of colchicine, verapamil, cyclosporin A, or vinblastine to stimulate the ATPase activity of P-gp (13.Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 35388-35392Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar) or to induce conformational changes in the nucleotide-binding sites (22.Liu R. Sharom F.J. Biochemistry. 1997; 36: 2836-2843Crossref PubMed Scopus (83) Google Scholar). The fluid nature of the TM segments is suggested by the finding that only 4 of the 12 mutants could be cross-linked at 4 °C (Table II), while all were cross-linked at 37 °C. Vanadate plus ATP inhibited cross-linking of the mutants by locking-in the nucleotide-binding domain(s) of P-gp in a fixed conformation (Table III). The rigid nature of the locked-in conformation is then transmitted to the TM domains. This supports the notion that there is “cross-talk” between the nucleotide-binding domains and the TM domains and that ATPase activity is directly linked to drug-P-gp interaction. We have proposed a working model of the arrangement of the TM segments of P-gp (Fig. 2). An interesting feature about the model is that the TMs involved in cross-linking have been reported to be important for P-gp-drug interactions. Cysteine-scanning mutagenesis of the TM segments and labeling with a thiol-reactive substrate, dibromobimane, provided direct evidence for interaction of substrate with residues in TMs 6, 11, and 12 (13.Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 35388-35392Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 20.Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 31945-31948Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Dibromobimane reacted with specific cysteine residues in TMs 6, 11, and 12 and inhibited the activity of P-gp. The presence of substrates such as colchicine, verapamil, or vinblastine inhibited labeling by dibromobimane. Many mutations in TMs 4, 5, and 6, and in TMs 10, 11, and 12, have been reported to alter the substrate specificity or activity of the transporter (23.Devine S.E. Ling V. Melera P.W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4564-4568Crossref PubMed Scopus (127) Google Scholar, 24.Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 3143-3149Abstract Full Text PDF PubMed Google Scholar, 25.Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 19965-19972Abstract Full Text PDF PubMed Google Scholar, 26.Loo T.W. Clarke D.M. Biochemistry. 1994; 33: 14049-14057Crossref PubMed Scopus (125) Google Scholar, 27.Kajiji 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, 28.Chen G. Duran G.E. Steger K.A. Lacayo N.J. Jaffrezou J.P. Dumontet C. Sikic B.I. J. Biol. Chem. 1997; 272: 5974-5982Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 29.Hanna M. Brault M. Kwan T. Kast C. Gros P. Biochemistry. 1996; 35: 3625-3635Crossref PubMed Scopus (55) Google Scholar, 30.Shoshani T. Zhang S. Dey S. Pastan I. Gottesman M.M. Mol. Pharmacol. 1998; 54: 623-630PubMed Google Scholar, 31.Hafkemeyer P. Dey S. Ambudkar S.V. Hrycyna C.A. Pastan I. Gottesman M.M. Biochemistry. 1998; 37: 16400-16409Crossref PubMed Scopus (80) Google Scholar). Labeling studies with photoactive analogs of P-gp substrates also suggest that TMs 4, 5, and 6 and TMs 10, 11, and 12 may participate in drug-protein interactions (32.Bruggemann E.P. Germann U.A. Gottesman M.M. Pastan I. J. Biol. Chem. 1989; 264: 15483-15488Abstract Full Text PDF PubMed Google Scholar, 33.Bruggemann E.P. Currier S.J. Gottesman M.M. Pastan I. J. Biol. Chem. 1992; 267: 21020-21026Abstract Full Text PDF PubMed Google Scholar, 34.Greenberger L.M. J. Biol. Chem. 1993; 268: 11417-11425Abstract Full Text PDF PubMed Google Scholar, 35.Morris 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, 36.Demmer 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). The model in Fig. 2 also takes into consideration that no cross-linked products were detected between TMs 1 to 3 and TMs 7 to 9 when cysteine residues were introduced into the full-length Cys-less P-gp or into the Cys-less half-molecules (data not shown). In summary, the TM segments postulated to be involved in drug binding are quite close to each other on the cytoplasmic side of the membrane. The TM segments and nucleotide-binding domains appear to be conformationally flexible to be able to accommodate structurally different substrates.

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