Drug Binding in Human P-glycoprotein Causes Conformational Changes in Both Nucleotide-binding Domains

The human multidrug resistance P-glycoprotein (P-gp, ABCB1) uses ATP to transport many structurally diverse compounds out of the cell. It is an ABC transporter with two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs). Recently, we showed that the “LSGGQ” motif in one NBD (531LSGGQ535 in NBD1;1176LSGGQ1180 in NBD2) is adjacent to the “Walker A” sequence (1070GSSGCGKS1077 in NBD2;427GNSGCGKS434 in NBD1) in the other NBD (Loo, T. W., Bartlett, M. C., and Clarke, D. M. (2002)J. Biol. Chem. 277, 41303–41306). Drug substrates can stimulate or inhibit the ATPase activity of P-gp. Here, we report the effect of drug binding on cross-linking between the LSGGQ signature and Walker A sites (Cys431(NBD1)/C1176C(NBD2) and Cys1074(NBD2)/L531C(NBD1), respectively). Seven drug substrates (calcein-AM, demecolcine, cis(Z)-flupentixol, verapamil, cyclosporin A, Hoechst 33342, andtrans(E)-flupentixol) were tested for their effect on oxidative cross-linking. Substrates that stimulated the ATPase activity of P-gp (calcein-AM, demecolcine, cis(Z)-flupentixol, and verapamil) increased the rate of cross-linking between Cys431(NBD1-Walker A)/C1176C(NBD2-LSGGQ) and between Cys1074(NBD2-Walker A)/L531C(NBD1-LSGGQ) when compared with cross-linking in the absence of drug substrate. By contrast, substrates that inhibited ATPase activity (cyclosporin A, Hoechst 33342, andtrans(E)-flupentixol) decreased the rate of cross-linking. These results indicate that interaction between the LSGGQ motifs and Walker A sites must be essential for coupling drug binding to ATP hydrolysis. Drug binding in the transmembrane domains can induce long range conformational changes in the NBDs, such that compounds that stimulate or inhibit ATPase activity must decrease and increase, respectively, the distance between the Walker A and LSGGQ sequences. The human multidrug resistance P-glycoprotein (P-gp, ABCB1) uses ATP to transport many structurally diverse compounds out of the cell. It is an ABC transporter with two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs). Recently, we showed that the “LSGGQ” motif in one NBD (531LSGGQ535 in NBD1;1176LSGGQ1180 in NBD2) is adjacent to the “Walker A” sequence (1070GSSGCGKS1077 in NBD2;427GNSGCGKS434 in NBD1) in the other NBD (Loo, T. W., Bartlett, M. C., and Clarke, D. M. (2002)J. Biol. Chem. 277, 41303–41306). Drug substrates can stimulate or inhibit the ATPase activity of P-gp. Here, we report the effect of drug binding on cross-linking between the LSGGQ signature and Walker A sites (Cys431(NBD1)/C1176C(NBD2) and Cys1074(NBD2)/L531C(NBD1), respectively). Seven drug substrates (calcein-AM, demecolcine, cis(Z)-flupentixol, verapamil, cyclosporin A, Hoechst 33342, andtrans(E)-flupentixol) were tested for their effect on oxidative cross-linking. Substrates that stimulated the ATPase activity of P-gp (calcein-AM, demecolcine, cis(Z)-flupentixol, and verapamil) increased the rate of cross-linking between Cys431(NBD1-Walker A)/C1176C(NBD2-LSGGQ) and between Cys1074(NBD2-Walker A)/L531C(NBD1-LSGGQ) when compared with cross-linking in the absence of drug substrate. By contrast, substrates that inhibited ATPase activity (cyclosporin A, Hoechst 33342, andtrans(E)-flupentixol) decreased the rate of cross-linking. These results indicate that interaction between the LSGGQ motifs and Walker A sites must be essential for coupling drug binding to ATP hydrolysis. Drug binding in the transmembrane domains can induce long range conformational changes in the NBDs, such that compounds that stimulate or inhibit ATPase activity must decrease and increase, respectively, the distance between the Walker A and LSGGQ sequences. P-glycoprotein ATP-binding cassette adenosine 5′-(β,γ-imino)triphosphate nucleotide-binding domain NH2-terminal NBD COOH-terminal NBD transmembrane TM domain P-glycoprotein (P-gp)1 is an ATP-dependent drug pump that transports numerous structurally diverse compounds of different sizes 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 (1921) Google Scholar and 2Borst P. Elferink R.O. Annu. Rev. Biochem. 2002; 71: 537-592Crossref PubMed Scopus (1348) Google Scholar). Therefore, P-gp can complicate cancer and AIDS chemotherapy because many therapeutic compounds are substrates of P-gp (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 (460) Google Scholar, 4Krishna R. Mayer L.D. Eur. J. Pharm. Sci. 2000; 11: 265-283Crossref PubMed Scopus (982) Google Scholar). P-gp is a single polypeptide of 1280 amino acids. It is organized as two repeating units of 610 amino acids that are joined by a linker region of about 60 amino acids (5Chen 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 (1717) Google Scholar). Each repeat has six transmembrane (TM) segments and a hydrophilic domain containing an ATP-binding site (6Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 843-848Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 7Kast C. Canfield V. Levenson R. Gros P. J. Biol. Chem. 1996; 271: 9240-9248Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). P-gp functions as a monomer (8Loo 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 to function (9Loo T.W. Clarke D.M. J. Biol. Chem. 1994; 269: 7750-7755Abstract Full Text PDF PubMed Google Scholar,10Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 24759-24765Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). The transmembrane domains alone are sufficient to mediate drug binding (10Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 24759-24765Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), but both ATP-binding sites must be functional for drug efflux activity (11Azzaria M. Schurr E. Gros P. Mol. Cell. Biol. 1989; 9: 5289-5297Crossref PubMed Scopus (270) Google Scholar, 12Doige C.A., Yu, X. Sharom F.J. Biochim. Biophys. Acta. 1992; 1109: 149-160Crossref PubMed Scopus (138) Google Scholar, 13al-Shawi M.K. Urbatsch I.L. Senior A.E. J. Biol. Chem. 1994; 269: 8986-8992Abstract Full Text PDF PubMed Google Scholar, 14Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 22957-22961Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). An important aspect in understanding the mechanism of P-gp is how drug transport is coupled to ATP hydrolysis. The observation that drug binding to P-gp can either stimulate or inhibit ATP hydrolysis suggests that drug binding and ATP hydrolysis must be tightly regulated (12Doige C.A., Yu, X. Sharom F.J. Biochim. Biophys. Acta. 1992; 1109: 149-160Crossref PubMed Scopus (138) Google Scholar, 15Urbatsch I.L. Senior A.E. Arch. Biochem. Biophys. 1995; 316: 135-140Crossref PubMed Scopus (132) Google Scholar,16Ramachandra M. Ambudkar S.V. Chen D. Hrycyna C.A. Dey S. Gottesman M.M. Pastan I. Biochemistry. 1998; 37: 5010-5019Crossref PubMed Scopus (241) Google Scholar). The “signature” sequence (LSGGQ) in each NBD appears to be an important region in P-gp. Although the signature sequences are present in all ABC transporters (17Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3373) Google Scholar), their function is unknown. We recently showed that the LSGGQ in one NBD was close to the Walker A sequence in the other NBD (18Loo T.W. Bartlett M.C. Clarke D.M. J. Biol. Chem. 2002; 277: 41303-41306Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). We postulated that the LSGGQ sequence might play a role in conveying conformational changes from the drug-binding site to the ATP-binding sites. In this study, we examined the effect of drug substrates on cross-linking between the LSGGQ motifs and the Walker A sites. A histidine-tagged Cys-less P-gp was constructed and then used for making mutants containing pairs of cysteines (6Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 843-848Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 19Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 19965-19972Abstract Full Text PDF PubMed Google Scholar, 20Loo T.W. Clarke D.M. J. Biol. Chem. 1996; 271: 27482-27487Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Two mutants that contained a cysteine in the LSGGQ site and another in the Walker A site were constructed (18Loo T.W. Bartlett M.C. Clarke D.M. J. Biol. Chem. 2002; 277: 41303-41306Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). One mutant (L531C/Cys1074) contained a cysteine in the NH2-terminal 531LSGGQ535 site and an endogenous cysteine (Cys1074) in the COOH-terminal Walker A site (1070GSSGCGKC1077). The other mutant (Cys431/L1176C) contained the endogenous Cys431 in the NH2-terminal Walker A site (427GNSGCGKS434) and another cysteine in the COOH-terminal 1176LSGGQ1180 site. The mutant cDNAs were expressed in HEK 293 cells in the presence of cyclosporin A to promote maturation of P-gp (21Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 709-712Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar,22Loo T.W. Clarke D.M. J. Biol. Chem. 1998; 273: 14671-14674Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Membranes were prepared as described previously (19Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 19965-19972Abstract Full Text PDF PubMed Google Scholar, 23Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 21449-21452Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). For disulfide cross-linking analysis, aliquots of membranes were added to equal volumes of Tris-buffered saline (10 mm Tris-HCl, pH 7.4, 150 mm NaCl) containing 1 mmCu2+-(phenanthroline)3. The samples were incubated at 21 or 4 °C for various intervals, and the reactions were stopped by the addition of SDS sample buffer (125 mmTris-HCl, pH 6.8, 20% (v/v) glycerol, and 4% (w/v) SDS) containing 50 mm EDTA and no reducing agent. The reaction mixtures were subjected to SDS-PAGE (7.5% polyacrylamide gels) and immunoblot analysis with a rabbit polyclonal antibody against P-gp (8Loo T.W. Clarke D.M. J. Biol. Chem. 1996; 271: 27488-27492Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). To test the effect of nucleotide or vanadate on cross-linking, the membranes were incubated with an equal volume of Tris-buffered saline containing the following: 1) 12 mm ATP, 24 mmMgCl2 and 0.6 mm sodium orthovanadate; 2) 12 mm ATP and 24 mm MgCl2; 3) 12 mm ATP; 4) 24 mm MgCl2; 5) 0.6 mm sodium orthovanadate; 6) 12 mm ADP; or 7) 12 mm AMP-PNP. Sodium orthovanadate was prepared from Na3VO4, pH 10 (24), and boiled for 2 min to break down polymeric species (25Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 31800-31805Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). The samples were incubated for 10 min at 37 °C and then cooled in an ice bath before treatment with oxidant at 21 °C. At this temperature there is almost complete cross-linking in both mutants (18Loo T.W. Bartlett M.C. Clarke D.M. J. Biol. Chem. 2002; 277: 41303-41306Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). To test the effect of drug substrates on cross-linking, the mutant P-gps were preincubated with drug substrate for 10 min at 21 °C, then chilled at 4 °C for 10 min, and then treated with oxidant. At 4 °C, the rate of cross-linking is also slowed, and this allowed us to detect changes in cross-linking. The purification of histidine-tagged P-gp mutants and an assay of drug-stimulated ATPase activities were done as described previously (23Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 21449-21452Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 26Loo T.W. Clarke D.M. J. Natl. Cancer Inst. 2000; 92: 898-902Crossref PubMed Scopus (96) Google Scholar) except that the isolated samples were mixed withEscherichia coli lipid rather than sheep brain phosphatidylethanolamine. E. coli lipids were used because basal P-gp ATPase activity is higher in these lipids than those with sheep brain phosphatidylethanolamine. This made measuring the inhibition of P-gp ATPase activity much easier. Also, the drug-stimulated ATPase activity of P-gp reconstituted with E. coli lipids is similar to that measured in isolated mammalian plasma membranes that are enriched in P-gp (15Urbatsch I.L. Senior A.E. Arch. Biochem. Biophys. 1995; 316: 135-140Crossref PubMed Scopus (132) Google Scholar). We showed previously by disulfide cross-linking analysis that the contact between the NBDs of P-gp could occur between the LSGGQ signature sequence in one NBD and the Walker A site in the other NBD (18Loo T.W. Bartlett M.C. Clarke D.M. J. Biol. Chem. 2002; 277: 41303-41306Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Mutants in which the leucine residue in the LSGGQ site is replaced with cysteine can be oxidatively cross-linked with the endogenous cysteine in the opposing Walker A sequence ((L531C(NBD1-LSGGQ)/Cys1074(NBD2-Walker A) or Cys431(NBD1-Walker A)/L1176C(NBD2-LSGGQ)). Fig. 1 shows that that cross-linking was almost complete in mutants L531C/Cys1074 and Cys431/L1176C when treated with 0.5 mm copper phenanthroline for 15 min at 21 °C. We previously showed that cross-linking occurred in the active molecule, because cross-linking resulted in an inactive molecule. Activity was restored, however, after the addition of dithiothreitol (18Loo T.W. Bartlett M.C. Clarke D.M. J. Biol. Chem. 2002; 277: 41303-41306Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). To determine whether cross-linking between the NBDs could be disrupted, the mutants were pre-treated with nucleotide or subjected to vanadate trapping. P-gp traps nucleotides in the presence of vanadate plus Mg-ATP and results in a transition state (27Urbatsch I.L. Sankaran B. Weber J. Senior A.E. J. Biol. Chem. 1995; 270: 19383-19390Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar, 28Loo T.W. Clarke D.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3511-3516Crossref PubMed Scopus (73) Google Scholar). Vanadate traps ADP at either NBD by occupying the position of the γ-phosphate adjacent to ADP. Vanadate trapping at one site then inhibits ATP hydrolysis at the second ATP-binding site (27Urbatsch I.L. Sankaran B. Weber J. Senior A.E. J. Biol. Chem. 1995; 270: 19383-19390Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). Fig. 1 shows that inhibition of cross-linking in mutants L531C/Cys1074 and Cys431/L1176C was observed only after treatment with vanadate plus Mg-ATP. Inhibition of cross-linking was not observed when the mutants were pre-treated with ATP, MgCl2, vanadate, Mg-ATP, ADP, or with the non-hydrolyzable ATP analog AMP-PNP. It is unlikely that vanadate trapping of nucleotide denatures the protein, because trapping of nucleotide is reversible (27Urbatsch I.L. Sankaran B. Weber J. Senior A.E. J. Biol. Chem. 1995; 270: 19383-19390Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). We have proposed that the LSGGQ motifs might participate in transmitting conformational changes from the transmembrane domains to the NBDs (18Loo T.W. Bartlett M.C. Clarke D.M. J. Biol. Chem. 2002; 277: 41303-41306Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Mutations in the LSGGQ motifs do not prevent ATP binding or vanadate trapping of nucleotides (29Bakos E. Klein I. Welker E. Szabo K. Muller M. Sarkadi B. Varadi A. Biochem. J. 1997; 323: 777-783Crossref PubMed Scopus (55) Google Scholar, 30Szakacs G. Ozvegy C. Bakos E. Sarkadi B. Varadi A. Biochem. J. 2001; 356: 71-75Crossref PubMed Google Scholar). One way of inducing different conformational changes in the TMDs is to use drug substrates with different structures. P-gp is interesting in that some drug substrates stimulate whereas others inhibit the ATPase activity of P-gp. Therefore, it is possible that the conformational changes in the TMDs may be monitored by changes in the cross-linking patterns in mutants L531C/Cys1074 and Cys431/L1176C. Drug substrates (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 (1921) Google Scholar) that either stimulated (calcein-AM, demecolcine,cis(Z)-flupentixol, and verapamil) or inhibited (cyclosporin A, Hoechst 33342, and trans(E)-flupentixol) the ATPase activity of Cys-less P-gp (data not shown) were identified. The flupentixol isomers are interesting in that thecis(Z)-isomer stimulates, whereas thetrans(E)-isomer inhibits the ATPase activity of wild-type P-gp (31Dey S. Ramachandra M. Pastan I. Gottesman M.M. Ambudkar S.V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10594-10599Crossref PubMed Scopus (357) Google Scholar). These drug substrates were then tested on mutants L531C/Cys1074 and Cys431/L1176C. Fig. 2 shows that the ATPase activity of mutant L531C/Cys1074 was stimulated by calcein-AM, demecolcine, cis(Z)-flupentixol, and verapamil (6.4-, 6.8-, 3.5-, and 4-fold, respectively). Half-maximal stimulation of ATPase activity with calcein-AM, demecolcine, cis(Z)-flupentixol, and verapamil occurred at 30, 163, 29, and 9 μm, respectively. Cyclosporin A, Hoechst 33342, andtrans(E)-flupentixol inhibited the activity of mutant L531C/Cys1074 with 50% inhibition occurring at concentrations of about 0.12, 0.67, and 1.1 mm, respectively. Similar results were obtained with mutant Cys431/L1176C (data not shown). We then tested whether the structurally different stimulatory and inhibitory drug substrates affected cross-linking in the NBDs. Accordingly, membranes from mutant L531C/Cys1074 were preincubated for 10 min at 21 °C with 1 mm calcein-AM, 2 mm demecolcine, 1 mm cis(Z)-flupentixol, 0.2 mm verapamil, 0.5 mm cyclosporin A, 0.5 mm Hoechst 33342, or 1 mm trans(E)-flupentixol. These concentrations were required for maximal stimulation or inhibition of ATPase activity (Fig. 2). The membranes were then treated with oxidant at 4 °C for various intervals. The rationale for doing the cross-linking at 4 °C was that thermal motion in the protein would be reduced and that subtle changes caused by drug substrate binding may be detected. Fig. 3 shows the effects of substrates on cross-linking of mutant L531C/Cys1074. In the absence of drug substrates, ∼50% of the mutant protein was cross-linked by 16 min. In the presence of compounds (calcein-AM, demecolcine,cis(Z)-flupentixol, and verapamil) that stimulate the ATPase activity of P-gp, however, the rate of cross-linking was significantly increased so that 50% cross-linking occurred by 2 min. The presence of the inhibitory compounds (cyclosporin A, Hoechst 33342, andtrans(E)-flupentixol) had the opposite effect. In the presence of these inhibitors, <50% cross-linking was observed at 32 min (Fig. 3). We then tested the effect of drug substrates on cross-linking of mutant Cys431/L1176C. In the absence of drug substrates, 50% of the mutant protein was cross-linked with an oxidant after 16 min (Fig. 4). In the presence of the stimulatory drug substrates (calcein-AM, demecolcine,cis(Z)-flupentixol, and verapamil), the rate of cross-linking was increased, because 50% of the mutant protein was cross-linked by 2–4 min. Drug substrates that inhibited ATPase activity (cyclosporin A, Hoechst 33342, andtrans(E)-flupentixol) of the mutant P-gp also inhibited the cross-linking of the mutant protein. Fig. 4 shows that in the presence of these compounds <50% cross-linking occurred at 32 min. The effect of substrates on mutants L531C/Cys1074 and Cys431/L1176C were very similar. Compounds that stimulated the ATPase activity also stimulated the rate of cross-linking of the mutants, whereas those that inhibited ATPase activity also inhibited the rate of cross-linking. It is unlikely that drug substrates are binding directly to the ATP-binding sites, because it has been shown that drug binding does not alter the affinity for ATP (15Urbatsch I.L. Senior A.E. Arch. Biochem. Biophys. 1995; 316: 135-140Crossref PubMed Scopus (132) Google Scholar, 16Ramachandra M. Ambudkar S.V. Chen D. Hrycyna C.A. Dey S. Gottesman M.M. Pastan I. Biochemistry. 1998; 37: 5010-5019Crossref PubMed Scopus (241) Google Scholar, 32Ambudkar S.V. Lelong I.H. Zhang J. Cardarelli C.O. Gottesman M.M. Pastan I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8472-8476Crossref PubMed Scopus (381) Google Scholar). Disulfide cross-linking between adjacent cysteines in the Walker A sequence of one NBD and the LSGGQ site in the other NBD is a useful approach for monitoring changes in the NBDs. A condition that dramatically affects cross-linking between these two sites occurs after vanadate trapping of a nucleotide (Fig. 1). Hydrolysis of ATP in the presence of vanadate was essential for the inhibition of cross-linking because cross-linking was not inhibited in the presence of the non-hydrolyzable ATP analog AMP-PNP. Similarly, cross-linking was not inhibited when the mutants were pre-incubated with only ATP, MgCl2, ADP, vanadate, or Mg-ATP. These results indicate that the trapped vanadate either occupies the space between the cross-linkable cysteines in the LSGGQ and Walker A sites in the two NBDs or that its presence causes the two NBDs to move apart. The drug-binding site in the TMDs (33Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 39272-39278Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 34Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 14972-14979Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar) and the ATP binding sites in P-gp must be quite far apart. Fluorescence resonance energy transfer studies indicate that the ATP-binding sites are about 40 Å from the drug-binding site (35Qu Q. Sharom F.J. Biochemistry. 2001; 40: 1413-1422Crossref PubMed Scopus (85) Google Scholar). Our results show that binding of drug substrates must induce conformational changes in the drug-binding site that are transmitted distally to the NBDs. Similarly, conformational changes in the NBDs are also transmitted to the drug-binding site in the TMDs (25Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 31800-31805Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 28Loo T.W. Clarke D.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3511-3516Crossref PubMed Scopus (73) Google Scholar). Therefore, there must be continuous “cross-talk” among the domains of P-gp. The LSGGQ sequence can increase or decrease the rate of ATP hydrolysis depending on its distance from the Walker A site as shown in Fig. 5. Inhibitory substrates may cause both of the Walker A and LSGGQ sites to move farther apart and/or reduce the rate of ATP hydrolysis. In this study, the inhibitory substrates reduced the rate of cross-linking, and this may occur by moving the two NBDs apart (Figs. 3 and 4). Drug substrates that stimulate ATPase activity of P-gp must bring the Walker A and LSGGQ sites closer together so that hydrolysis of ATP occurs at a faster rate. There is no detailed crystal structure about ABC crystal structure studies on other ABC transporters such as A. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), 2002; PubMed Scopus Google Scholar), and J.E. J. Mol. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), however, show that the LSGGQ sequences in these are adjacent to the γ-phosphate of It is that as the LSGGQ motif closer to the Walker A the rate of ATP hydrolysis Our results this and may a substrate is a or of P-gp. These results could the for the of of P-gp.

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