Molecular basis of the polyspecificity of P-glycoprotein (ABCB1): recent biochemical and structural studies

Abstract

ABCB1 (P-glycoprotein/P-gp) is an ATP-binding cassette transporter well known for its association with multidrug resistance in cancer cells. Powered by the hydrolysis of ATP, it effluxes structurally diverse compounds. In this chapter, we discuss current views on the molecular basis of the substrate polyspecificity of P-gp. One of the features that accounts for this property is the structural flexibility observed in P-gp. Several X-ray crystal structures of mouse P-gp have been published recently in the absence of nucleotide, with and without bound inhibitors. All the structures are in an inward-facing conformation exhibiting different degrees of domain separation, thus revealing a highly flexible protein. Biochemical and biophysical studies also demonstrate this flexibility in mouse as well as human P-gp. Site-directed mutagenesis has revealed the existence of multiple transport-active binding sites in P-gp for a single substrate. Thus, drugs can bind at either primary or secondary sites. Biochemical, molecular modeling, and structure-activity relationship studies suggest a large, common drug-binding pocket with overlapping sites for different substrates. We propose that in addition to the structural flexibility, the molecular or chemical flexibility also contributes to the binding of substrates to multiple sites forming the basis of polyspecificity.

Keywords: ABC transporter; Drug-binding sites; Multidrug resistance; P-glycoprotein; Polyspecificity; Structural flexibility.

Figure 1

Comparison of the separation of the two nucleotide-binding domains (NBDs) in human, mouse, and C. elegans P-gps based on cross-linking studies and X-ray crystallography. The first panel to the left shows a schematic representation of the human P-gp structure based on cross-linking studies (Sim, Bhatnagar, Chufan, Kapoor, & Ambudkar, 2013), while the rest of the panels gather the crystal structures of mouse and C. elegans P-gps as cartoon models (4F4C.pdb, Jin et al., 2012; 4M1M.pdb, Li, Jaimes, & Aller, 2014; 4KSC.pdb, Ward et al., 2013). The figure is ordered from the data that show the NBD domains least separated to the most separated. The distance in Angstroms indicates the separation between the cysteine residues of the Walker A motif (431-1074 in human; 427-1070 in mouse; 455-1116 in C. elegans). The double arrow symbol denotes the distance between residues C431-C1074 in human P-gp. The X-ray structures are colored in the following manner: green and cyan, yellow and blue, and magenta and gray for the N-terminal and C-terminal halves of mouse P-gp 4M1M. pdb, 4KSC.pdb and C. elegans P-gp 4F4C.pdb, respectively. Both Walker A cysteine residues are shown as black balls in the X-ray crystal structures, and the black line represents the distance between them.

Figure 2

The proposed drug-binding sites on P-gp. Panel A: The sites where the cyclic peptides QZ59-RRR (red) and QZ59-SSS (blue) bind mouse P-gp as determined by X-ray crystallography (4M2S.pdb and 4M2T.pdb, respectively) are shown in ball models. Panel B: The proposed location of the R and H sites are shown in the homology model of human P-gp based on the mouse P-gp structure (Martinez et al., 2014; Pajeva et al., 2013). Panel C: The residues of the rhodamine B binding site determined by cysteine-scanning mutagenesis (Loo & Clarke, 2002) are shown as balls (at the α-carbon position) and compared with the proposed location of the R site. Panel D: The residues of the verapamil-binding site determined by cysteine-scanning mutagenesis are shown as balls (at the α-carbon position) and compared with the R and H sites. A group of residues (61-64-65, 118-125, 942-945, 868-871-872) suggest the existence of a verapamil-binding site different from the proposed R and H; this site is demarcated as a red oval and indicated with a question mark. In all the panels, the structure of P-gp is shown as a ribbon model in green (N-terminal) and cyan (C-terminal). The approximate location of the plasma membrane is demarcated with dashed lines in panels A and B. Stick models of the QZ59 molecules (RRR-isomer in red and SSS-isomer in blue) are shown in panels B, C, and D for reference. The four panels are shown as stereo images. The figures were prepared with PyMOL 1.5.0.5.

Figure 3

Sequence alignment of transmembrane domains 1 and 2 of human P-gp. Residues 52–390 of domain 1 and 712–1033 of domain 2 were selected for alignment. Using the homology model of human P-gp based on mouse P-gp X-ray structure 4M1M.pdb, the residues facing the central cavity were selected and are shown in red color. The symbol “+” denotes similarity between residues.

Figure 4

Register shift at transmembrane helix 12 between the refined structure of mouse P-gp published by Li et al. (2014) (4M1M.pdb) and the new crystal structure of mouse P-gp published by Ward et al. (2013) (4KSB.pdb). The structures were aligned in PyMOL 1.5.0.5 and only the TM12s are shown for clarity. The helices are shown as cartoon models in green (4M1M.pdb) and magenta (4KSB.pdb). Residues facing the central cavity are shown as stick models: S979, F983, and A987 of 4M1M.pdb and F978, V982, and M986 of 4KSB.pdb (residue numbers denote the human P-gp sequence).