Abstract
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 ((531)LSGGQ(535) in NBD1; (1176)LSGGQ(1180) in NBD2) is adjacent to the "Walker A" sequence ((1070)GSSGCGKS(1077) in NBD2; (427)GNSGCGKS(434) 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 (Cys(431)(NBD1)/C1176C(NBD2) and Cys(1074)(NBD2)/L531C(NBD1), respectively). Seven drug substrates (calcein-AM, demecolcine, cis(Z)-flupentixol, verapamil, cyclosporin A, Hoechst 33342, and trans(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 Cys(431)(NBD1-Walker A)/C1176C(NBD2-LSGGQ) and between Cys(1074)(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, and trans(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.
Highlights
P-glycoprotein (P-gp)1 is an ATP-dependent drug pump that transports numerous structurally diverse compounds of different sizes out of the cell
Substrates that inhibited ATPase activity (cyclosporin A, Hoechst 33342, and trans(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
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(NBD2LSGGQ))
Summary
P-glycoprotein (P-gp) is an ATP-dependent drug pump that transports numerous structurally diverse compounds of different sizes out of the cell (recently reviewed in Refs. 1 and 2). Each repeat has six transmembrane (TM) segments and a hydrophilic domain containing an ATP-binding site [6, 7]. The transmembrane domains alone are sufficient to mediate drug binding [10], but both ATP-binding sites must be functional for drug efflux activity [11,12,13,14]. The “signature” sequence (LSGGQ) in each NBD appears to be an important region in P-gp. We recently showed that the LSGGQ in one NBD was close to the Walker A sequence in the other NBD [18]. We postulated that the LSGGQ sequence might play a role in conveying conformational changes from the drug-binding site to the ATP-binding sites. We examined the effect of drug substrates on cross-linking between the LSGGQ motifs and the Walker A sites
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
