Mapping Conformational Changes Associated with the Catalytic Cycle of Human P-Glycoprotein (ABCB1)

Abstract

P-glycoprotein (Pgp, ABCB1) is an ABC transporter that uses the energy of ATP hydrolysis to efflux a variety of amphipathic substrates including anti-cancer agents from the cell. In this study, we have used disulfide crosslinking and Pulsed Dipolar Electron Spin Resonance Spectroscopy (PDS) to elucidate the conformational changes associated with the catalytic cycle. We generated a library of double cysteine mutants in a cys-less background and expressed in baculovirus-insect and mammalian cell expression systems. All the mutants expressed at the cell surface in HeLa cells and were also functionally active. Disulfide crosslinking was performed in crude membranes and each double cysteine mutant of Pgp was tested for its ability to crosslink with ten crosslinkers of varied spacer arm length. We observed crosslinking in four double cysteine mutants with Cys residues in TM3/TM9 (T173C/T816C), ICL2/NBD2 (A266C/I1115C), NBD1/ICL4 (A481C/S909C) and NBD1/NBD2 (A431C/A1074C). All the mutants showed modulation of crosslinking with change in protein conformation from APO to ADP-trapped. In the mutant T173C/T816C, the transmission of signal from the ATP-binding sites to TM3 and TM9 may occur via intracellular loops ICL1 and ICL3. This is highlighted by the fact that substitution of conserved residues D164 and D805 with Cys in ICL1 and ICL3, respectively reduced the basal ATPase activity and was unaffected by addition of inhibitors such as tariquidar and cyclosporine A. In addition, Cys sites in the Walker A domain of both NBD's cross linked in apo conformation to 20-25 A long crosslinkers. This suggests that the ATP sites are much closer than predicted by the homology model of human Pgp (36 A) based on the mouse Pgp structure. In aggregate, the broad distance measurements from PDS and disulfide crosslinking suggest high degree of flexibility in Pgp.