Structure of P-glycoprotein reveals a molecular basis for poly-specific drug binding

Abstract

P-glycoprotein (P-gp) detoxifies cells by exporting hundreds of chemically unrelated toxins but has been implicated in multidrug resistance (MDR) in the treatment of cancers. Substrate promiscuity is a hallmark of P-gp activity, thus a structural description of poly-specific drug-binding is important for the rational design of anticancer drugs and MDR inhibitors. The x-ray structure of apo P-gp at 3.8 angstroms reveals an internal cavity of approximately 6000 angstroms cubed with a 30 angstrom separation of the two nucleotide-binding domains. Two additional P-gp structures with cyclic peptide inhibitors demonstrate distinct drug-binding sites in the internal cavity capable of stereoselectivity that is based on hydrophobic and aromatic interactions. Apo and drug-bound P-gp structures have portals open to the cytoplasm and the inner leaflet of the lipid bilayer for drug entry. The inward-facing conformation represents an initial stage of the transport cycle that is competent for drug binding.

Fig. 1

Structure of Pgp. (A) Front and (B) back stereo views of PGP. TM1-12 are labeled. The N- and C-terminal half of the molecule is colored yellow and blue, respectively. TM4-5 and TM10-11 cross over to form intertwined interfaces that stabilize the inward facing conformation. Horizontal bars represent the approximate positioning of the lipid bilayer. The N- and C-termini are labeled in panel A. Transmembrane (TM) domains and nucleotide binding domains (NBD) are also labeled.

Fig. 2

Binding of novel cyclic peptide Pgp inhibitors. Chemical structures of (A) QZ59-RRR and (B) QZ59-SSS. (C) Location of one QZ59-RRR (green spheres) and (D) two QZ59-SSS (blue and cyan spheres) molecules in the Pgp internal cavity. (E-F) Stereo images showing interaction of transmembrane helices with QZ59 compounds viewed from the intracellular side of the protein looking into the internal chamber. In both cases the compound(s) are sandwiched between previously identified drug binding TMs 6 and 12. The location of the QZ59 compounds was verified by anomalous Fourier (Fig. S15B-C) and Fo-Fc maps (Fig. S15D-E and S16-S17).

Fig. 3

Drug binding residues of Pgp. (A) Stereo view of the drug-binding cavity. Cα trace shown in gray. The QZ59-SSS in the “lower” (cyan) and “upper” (blue), as well as QZ59-RRR occupying the “middle” site (green) are superimposed. Residues within ∼4-5 Å of QZ59 compounds are shown as spheres. Spheres colored orange and red represent residues that only contact QZ59-SSS in the “lower” and “upper” site, respectively. Residues in common between QZ59-RRR and QZ59-SSS sites are colored yellow. Four residues (grey spheres) are close to QZ59-RRR but neither QZ59-SSS molecules. (B) Venn diagram of residues in close proximity to QZ59 molecules and residues that are protected from MTS labeling by verapamil binding (Fig. S1) (20, 21). Only residues in contact with the model for QZ59-SSS are displayed. Verapamil-only interacting residues are omitted from (A) for clarity and shown in Fig. S16.

Fig. 4

Model of substrate transport by Pgp. (A) Substrate (magenta) partitions into the bilayer from outside of the cell to the inner leaflet and enters the internal drug-binding pocket through an open portal. The residues in the drug binding pocket (cyan spheres) interact with QZ59 compounds and verapamil in the inward facing conformation. (B) ATP (yellow) binds to the NBDs causing a large conformational change presenting the substrate and drug-binding site(s) to the outer leaflet/extracellular space. In this model of Pgp, which is based on the outward facing conformation of MsbA and Sav1866 (13, 14), exit of the substrate to the inner leaflet is sterically occluded providing unidirectional transport to the outside.