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

Abstract

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.

Figure 1. C. elegans P-gp is a multidrug transporter

a, Cytotoxicity assay. Sf9 cells expressing P-gp were cultured with various concentrations of actinomycin D (blue line) or paclitaxel (red line). Uninfected cells were cultured in the presence of the same drugs as controls (dashed lines). b, ATPase activity in the presence and absence of 1 mM orthovanadate. c, ATPase activity as a function of substrate concentration. The protein concentration was kept at 0.15 μM for all measurements. Data points represent the means ± standard deviation (S.D.) of triplicate measurements from the same preparation.

Figure 2. The molecular architecture of P-gp

a, The secondary structure. b, Ribbon presentation. c, The TM cavity (green mesh) open to the cytosol and continuous with the membrane inner leaflet. The loops in TM10 and TM12 are colored in red. d, Drug-stimulated ATPase activities in isolated membranes. The differences measured in the absence and presence of vanadate (1 mM) are plotted. Membranes from untransfected cells were used as controls (dashed lines). The data points show the means and S.D. of three determinations from the same preparation. These results have been reproduced from different protein preparations.

Figure 3. Interactions between the TMDs and NBDs

a, the NBD/TMD interface of the maltose importer. MalF is the TM subunit and MalK is the NBD. E401 is the conserved glutamate residue in the “EAA” loop. b, and c, the NBD1/TMD (b) and NBD2/TMD (c) interfaces in P-gp. Dash lines indicate the conserved salt-bridges and hydrogen bonds. The Walker A and Walker B motifs are labeled as WA and WB, respectively.

Figure 4. A model of human P-gp

a, The overall structure. b,c, Pairs of residues in TMDs that formed disulfide bonds (green line) when mutated to cysteines^,. d,e, Pairs of residues at the NBD/TMD interfaces that were crosslinked^,. f, Stereo view of the drug transport pathway. Drug-interacting residues^– are labeled and shown as magenta balls. The non-protected residues, Y118, V125, V133, C137, Q195, N296, G300, Y310, F314, A729, F759, S766, G774, N842, A871, S943 and F957, are shown in grey. g, F335 in TM6 (red) interacts with residues in TM4 and TM5 (yellow sticks), thereby stabilizing the inward-facing conformation.