Crystal structure of the multidrug transporter P-glycoprotein from Caenorhabditis elegans

P-glycoprotein (P-gp) is an ATP-binding cassette transporter that confers multidrug resistance in cancer cells. It also affects the absorption, distribution and clearance of cancer-unrelated drugs and xenobiotics. For these reasons, the structure and function of P-gp have been studied extensively for decades. Here we present biochemical characterization of P-gp from Caenorhabditis elegans and its crystal structure at a resolution of 3.4 ångströms. We find that the apparent affinities of P-gp for anticancer drugs actinomycin D and paclitaxel are approximately 4,000 and 100 times higher, respectively, in the membrane bilayer than in detergent. This affinity enhancement highlights the importance of membrane partitioning when a drug accesses the transporter in the membrane. Furthermore, the transporter in the crystal structure opens its drug pathway at the level of the membrane’s inner leaflet. In the helices flanking the opening to the membrane, we observe extended loops that may mediate drug binding, function as hinges to gate the pathway or both. We also find that the interface between the transmembrane and nucleotide-binding domains, which couples ATP hydrolysis to transport, contains a ball-and-socket joint and salt bridges similar to the ATP-binding cassette importers, suggesting that ATP-binding cassette exporters and importers may use similar mechanisms to achieve alternating access for transport. Finally, a model of human P-gp derived from the structure of C. elegans P-gp not only is compatible with decades of biochemical analysis, but also helps to explain perplexing functional data regarding the Phe335Ala mutant. These results increase our understanding of the structure and function of this important molecule. P-glycoprotein uses the energy from ATP hydrolysis to pump substrates across the cell membrane. Drug transport depends on ATP hydrolysis, and the ATPase activity of P-gp is stimulated by the transported drugs. Caenorhabditis elegans P-gp is 46% identical to human P-gp. To ensure that this level of sequence identity translates into functional similarity, we tested whether overexpression of C. elegans P-gp confers cellular resistance to cytotoxic drugs known to be transported by human P-gp. Two potent anticancer drugs, actinomycin D and paclitaxel (Taxol) kill Spodoptera frugiperda (Sf9) cells at concentrations greater than 0.1mM (Fig. 1a). In comparison, cells infected by recombinant baculoviruses carrying the C. elegans P-gp gene are resistant to these drugs in concentrations more than 1,000-fold higher (Fig. 1a and Supplementary Fig. 1a). Using detergent-purified protein, we measured whether substrates of human P-gp also stimulate the ATPase activity of C. elegans P-gp. Among the 30 compounds we screened, actinomycin D, paclitaxel, progesterone, dipyridamole, and valinomycin increased the ATPase activity of C. elegans P-gp more than fivefold (Fig. 1b and Supplementary Fig. 1b). As expected for an ATP-binding cassette (ABC) transporter, both the basal and drug-stimulated levels of ATPase activity are inhibited by vanadate (Fig. 1b). The ATPase activity plotted as a function of drug concentration shows the characteristic biphasic response: with increasing drug concentration, ATPase activity increases to a maximum value and then decreases (Fig. 1c). This behaviour has been studied extensively with human P-gp. Like the P-glycoproteins identified in other species, the substrate profile of C. elegans P-gp only partly overlaps with that of human P-gp. Nevertheless, the similarities between their amino-acid sequences and functional properties suggest that the structure of C. elegans P-gp would be a reasonable starting point for a mechanistic understanding of how human P-gp functions as a multidrug pump. P-glycoprotein is a single polypeptide with two homologous halves, each containing a transmembrane domain (TMD) and a cytoplasmic nucleotide-binding domain (NBD) (Fig. 2a). Crystals of C. elegans P-gp were obtained in the absence of nucleotides and transport substrates. The structure was determined at a resolution of 3.4 Å and the amino-acid assignments were confirmed by 34 selenium-labelled methionines and three mercury-labelled cysteines (Supplementary Fig. 2 and Supplementary Table 1). Compared with the crystal structure of mouse P-gp determined at a resolution of 3.8 Å (ref. 23), that of C. elegans P-gp shows a similar