The mammalian multidrug resistance P-glycoprotein (P-gp) utilises the energy of ATP hydrolysis to pump hydrophobic compounds from cells and plays an important role in the resistance of cancers to chemotherapy.

In order to gain further insight into the mechanism of action of this protein, and by inference other ABC transporters, progress in obtaining structural information will be reported. Using single particle imaging of active, purified protein solubilised or reconstituted into lipid bilayers, a low-resolution structure was obtained (Rosenberg et al. 1997). By 2D cryoelectron microscopy a structure of close to 10 ü resolution has been obtained (Rosenberg et al. 2001). The functional unit of the protein is a monomer. The protein forms an aqueous chamber within the membrane, open to the extracellular milieu but closed at the cytoplasmic face of the membrane. The nucleotide binding domains (NBDs) are tightly apposed to the inner surface of the membrane, and may be partly embedded in the membrane. Cysteine-scanning mutagenesis has shown that the NBDs do not span the membrane (Blott et al. 1999).

Importantly, significant conformational changes are seen when nucleotides are bound to the NBDs: three distinct conformations can be identified, both by structural and biochemical analysis, corresponding to three distinct steps in the ATP hydrolytic cycle (Rosenberg et al. 2001). These changes involve major rearrangements of the putative transmembrane α-helices with respect to each other, and cysteine-scanning mutagenesis suggests helical rotation within the plane of the membrane. Based on these conformational changes and detailed studies of drug binding (Martin et al. 1999, 2000a, b) and ATP hydrolysis by P-gp and the related LmrA protein (van Veen et al. 1998, 2000), a model for the mechanism of transport by P-gp will be presented.

Blott, E., Higgins, C.F. & Linton, K.J (1999). EMBO J. 18, 6800-6808.

Martin, C., Berridge, G., Higgins, C.F., Mistry, P., Charlton, P. & Callaghan, R. (2000a). Mol. Pharmacol. 57, 624-632.

Martin, C., Berridge, G., Mistry, P., Higgins, C.F., Charlton, P. & Callaghan, R. (1999). Br. J. Pharmacol. 128, 765-771.

Martin, C., Berridge, G., Mistry, P., Higgins, C.F., Charlton, P. & Callaghan, R. (2000b). Biochemistry (in the Press).

Rosenberg, M.F., Callaghan, R., Ford, R.C. & Higgins, C.F. (1997). J. Biol. Chem. 272, 10685-10694.

Rosenberg, M.F., Ford, R.C., Callaghan, R., Berridge, G., Kerr, I.D., Schmidlin, A., Linton, K.J., Velarde, G. & Higgins, C.F. (2001). Cell (submitted).

van Veen, H.W., Callaghan, R., Soceneantu, L., Sardini, A., Konings, W.N. & Higgins, C.F. (1998). Nature 391, 291-295.

van Veen, H.W., Margolles, A., Muller, M., Higgins, C.F. & Konings, W.N. (2000). EMBO J. 19, 2503-2514.