P-glycoprotein is an ATP-binding cassette transporter that is associated with multidrug resistance and the failure of chemotherapy in human patients. We have previously shown, based on two-dimensional projection maps, that P-glycoprotein undergoes conformational changes upon binding of nucleotide to the intracellular nucleotide binding domains. Here we present the threedimensional structures of P-glycoprotein in the presence and absence of nucleotide, at a resolution limit of ϳ2 nm, determined by electron crystallography of negatively stained crystals. The data reveal a major reorganization of the transmembrane domains throughout the entire depth of the membrane upon binding of nucleotide. In the absence of nucleotide, the two transmembrane domains form a single barrel 5-6 nm in diameter and about 5 nm deep with a central pore that is open to the extracellular surface and spans much of the membrane depth. Upon binding nucleotide, the transmembrane domains reorganize into three compact domains that are each 2-3 nm in diameter and 5-6 nm deep. This reorganization opens the central pore along its length in a manner that could allow access of hydrophobic drugs (transport substrates) directly from the lipid bilayer to the central pore of the transporter.
ATP Binding Cassette (ABC)1 transporters are an extended family of membrane proteins defined by a highly conserved domain, the ATP binding cassette (1); they mediate the ATP-dependent transport of a wide variety of compounds across cellular membranes (2, 3). The core ABC transporter consists of two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs). The NBDs are peripherally located at the cytoplasmic face of the membrane, bind ATP, and couple ATP hydrolysis to the transport process. All NBDs whose structures have been determined have very similar tertiary folds (4 -8). The TMDs bind the transported substrate and form the pathway through which it crosses the membrane. In contrast to the NBDs, the TMDs of different ABC transporters share little primary sequence similarity, except between closely related members of a subfamily; this may be because of the variety of substrates transported by different ABC proteins. Little is known about the structures of the TMDs of ABC transporters or how the binding/ hydrolysis of ATP by the NBDs is coupled to transmembrane transport of solute. Hydrophobicity plots typically predict six transmembrane ␣-helices per TMD, but there are notable exceptions with additional predicted transmembrane ␣-helices (9, 10). The structures of two complete bacterial ABC transporters (9, 11) have confirmed that the membrane-spanning segments are indeed ␣-helical, although the packing of these ␣-helices within the membrane differs markedly between the two structures.P-glycoprotein (P-gp) is a mammalian ABC transporter that pumps hydrophobic drugs across the cell membrane and can confer multidrug resistance on cells and tumors. P-gp is probably the best characterized ABC transporter, and much is known about the ATP hydrolytic cycle (1...