Molecular models of human P-glycoprotein in two different catalytic states

Abstract

Background: P-glycoprotein belongs to the family of ATP-binding cassette proteins which hydrolyze ATP to catalyse the translocation of their substrates through membranes. This protein extrudes a large range of components out of cells, especially therapeutic agents causing a phenomenon known as multidrug resistance. Because of its clinical interest, its activity and transport function have been largely characterized by various biochemical studies. In the absence of a high-resolution structure of P-glycoprotein, homology modeling is a useful tool to help interpretation of experimental data and potentially guide experimental studies.

Results: We present here three-dimensional models of two different catalytic states of P-glycoprotein that were developed based on the crystal structures of two bacterial multidrug transporters. Our models are supported by a large body of biochemical data. Measured inter-residue distances correlate well with distances derived from cross-linking data. The nucleotide-free model features a large cavity detected in the protein core into which ligands of different size were successfully docked. The locations of docked ligands compare favorably with those suggested by drug binding site mutants.

Conclusion: Our models can interpret the effects of several mutants in the nucleotide-binding domains (NBDs), within the transmembrane domains (TMDs) or at the NBD:TMD interface. The docking results suggest that the protein has multiple binding sites in agreement with experimental evidence. The nucleotide-bound models are exploited to propose different pathways of signal transmission upon ATP binding/hydrolysis which could lead to the elaboration of conformational changes needed for substrate translocation. We identified a cluster of aromatic residues located at the interface between the NBD and the TMD in opposite halves of the molecule which may contribute to this signal transmission. Our models may characterize different steps in the catalytic cycle and may be important tools to understand the structure-function relationship of P-glycoprotein.

Figure 1

Multiple sequence alignment. Multiple sequence alignment of V. cholera, S. typhimurium, E. coli MsbA, SAV1866 and human P-gp used to generate the 3D models of P-gp. The predicted trans-membrane regions are grayed.

Figure 2

A. Molecular surface representation of the 3D models. (a) Nucleotide-bound model built with the SAV1866 template. (b) Nucleotide-bound model built with the S. typhimurium MsbA template. (c) Closed and (d) Open nucleotide-free models built with the V. cholerae MsbA and the E. coli MsbA templates respectively. The surface of hydrophobic residues (Ala, Leu, Val, Ile, Pro, Phe and Met) are colored in yellow. Other residues are colored in blue. The two red lines indicate the position of the lipid polar heads in the cellular membrane. B. View of a nucleotide-bound conformation of P-gp. The N-terminal half is highlighted: the three extracellular loops (ECL) are colored in pink (ECL1 is truncated (see text)), the two long intracellular loops are colored in yellow with the small coupling helices in pale green. The 6 trans-membrane helices are colored in blue (TM1), red (TM2), gray (TM3), orange (TM4), cyan (TM5) and green (TM6) and the nucleotide binding domain (NBD) is colored in magenta. The intracellular segment of TM1 and TM6 are depicted in light blue. All these segments are labeled from the N-terminus to the C-terminus.

Figure 3

NBD closed dimmer. Ribbon representation (top view from the membrane) of the NBD closed dimer of one of the nucleotide-bound models in presence of two ATP molecules depicted as sticks. The N-terminal NBD (light blue) and the C-terminal NBD (light pink) are associated in a head to tail fashion. Each ATP binding pocket is formed by the A-loop (yellow), the Walker A motif (dark blue), the Walker B motif (black) and the Gln of the Q-loop depicted in orange from one NBD and is closed by the Signature motif colored in red from the other NBD.

Figure 4

Top: Drug binding site of the closed nucleotide-free model. Tube representation of the transmembrane region of the closed nucleotide-free model. The residues experimentally identified to alter drug specificity are represented in balls and sticks: H61, G64, L65, Y118, V125, M197, T199, S222, I306, A311, V331, T333, F335, S337, V338, L339, I340, G341, A342, F343, Q725, F728, A729, S766, T769, I840, A841, N842, I864, I867, I868, A871, G872, A935, F938, F942, S943, T945, Q946, Y950, F951, S952, Y953, F957, L975, F978, V981, V982, F983, G984 and A985. The two red lines indicate the position of the lipid polar heads in the cellular membrane. Bottom: One predicted position for each docked ligand. Ligands are depicted in purple and the residues experimentally identified to alter their specificity are colored according to their chemical type (carbon in cyan; oxygen in red; nitrogen in blue). (a) colchicine, (b) rhodamineB, (c) verapamil and (d) vinblastine.

Figure 5

NBD:TMD communication pathways. For sake of clarity only the residues identified starting from the N-terminal NBD are indicated. First pathway: From adenine → directly or through NBD aromatic residues → ICL1 → ICL4. Second pathway: From ATP phosphates → directly to Q-loop of the same NBD or through ABC signature from the ATP of the facing NBD to the Q-loop → ICL4. The colors of the links between the residues depict the physical nature of their interactions: yellow: hydrophobic, magenta: aromatic-aromatic, cyan: hydrogen bond and blue: electrostatic.

Figure 6

Residues potentially involved in the hinge bending motion of TM3–TM4 and TM5–TM6 pairs. Ribbon representation of TM3–TM4 (in blue) and TM5–TM6 (in green). The conserved residues identified by ConSurf (see text) with a potential role in the hinge conformational change upon ATP binding are depicted as red spheres.