Structures of P-glycoprotein reveal its conformational flexibility and an epitope on the nucleotide-binding domain

Abstract

P-glycoprotein (P-gp) is one of the best-known mediators of drug efflux-based multidrug resistance in many cancers. This validated therapeutic target is a prototypic, plasma membrane resident ATP-Binding Cassette transporter that pumps xenobiotic compounds out of cells. The large, polyspecific drug-binding pocket of P-gp recognizes a variety of structurally unrelated compounds. The transport of these drugs across the membrane is coincident with changes in the size and shape of this pocket during the course of the transport cycle. Here, we present the crystal structures of three inward-facing conformations of mouse P-gp derived from two different crystal forms. One structure has a nanobody bound to the C-terminal side of the first nucleotide-binding domain. This nanobody strongly inhibits the ATP hydrolysis activity of mouse P-gp by hindering the formation of a dimeric complex between the ATP-binding domains, which is essential for nucleotide hydrolysis. Together, these inward-facing conformational snapshots of P-gp demonstrate a range of flexibility exhibited by this transporter, which is likely an essential feature for the binding and transport of large, diverse substrates. The nanobody-bound structure also reveals a unique epitope on P-gp.

Keywords: ABC transporter; membrane protein structure; nanobody-transporter complex.

Fig. 1.

Structure of inward-facing P-gp. (A) Open inward-facing conformation (crystal 1) with the NBDs far apart. The N-terminal half of the protein (blue) and the C-terminal half (yellow) are connected by a flexible linker region (black dashed line) that is disordered in the structure. (B) Stereo view of superposition of anomalous Fourier mercury peaks validating the position of knocked-in and wild-type cysteine residue positions. Relative peak positions were determined by aligning the corresponding TMD or NBD subdomains of the model derived from the mutant diffraction data (Table S2) with the model derived from crystal 1. The locations of the 24 anomalous mercury peaks (mesh, sigma values) were used to confirm the registration of amino acids in the structure.

Fig. 2.

X-ray structure of P-gp in complex with Nb592. (A) Overview of the entire structure. Nb592 (red) binds to the C terminus of NBD1. There are additional interactions with NBD2 (yellow). The binding site of the nanobody precludes the ABC domains from coming together, explaining its potent ATPase inhibition properties. (B) Close up view of Nb592 binding site on P-gp. The view is rotated 180° from A. The complementarity determining regions (CDR1: residues 25–33 for Nb592, blue; CDR2: residues 51–57 for Nb592, yellow; CDR3: residues 98–107, green) of the nanobody all interact with the C-terminal portion of NBD1. CDR3 inserts into a shallow pocket formed by three helices in NBD1. The walker-A motif (residues 423–430) located on NBD1 is colored in cyan. The conserved H583 is also shown in violet.

Fig. 3.

Nb592 is a strong inhibitor of P-gp’s ATPase activity. (A) Nb592 inhibits verapamil-stimulated ATPase activity with an IC₅₀ of 520 ± 57 nM. Data points indicate the average activity ± SEM from four independent experiments, relative to P-gp’s activity in the absence of Nb592. Lines represent nonlinear regression analysis of the data points; R² value for the fit was 0.95. (B) Inhibition of verapamil-stimulated ATPase activity in the presence of 5 mM DTT, without or with 7.5 µM Nb592 (n = 4). (C) Nb592 prevents vanadate-induced 8-azido-[α³²P]-ADP trapping in P-gp’s catalytic sites. P-gp was incubated with 100 µM verapamil (VER) and 200 µM orthovanadate (Vi) in the 8-azido-[α³²P]-ATP hydrolysis/trapping reaction as indicated above the lanes. Upper, autoradiogram; Lower, Coomassie-stained SDS/PAGE gel showing the presence of P-gp (loading control).

Fig. 4.

Conformational changes by P-gp. (A) Stereoview of the three P-gp structures described in this study (crystal 1, red; crystal 2, green; P-gp–Nb592 complex [crystal 3], blue) aligned by using residues in TMD1 and NBD1 (residues 33–209, 852–961, and 320–626; designated as “Half Aligned”). The rmsd on Cα atoms for the aligned portion was 0.11 Å between crystal 2 and crystal 1 and 0.28 Å between P-gp–Nb592 complex (crystal 3) and crystal 1. The “TM-hinge regions” for this half of the molecule (L3-4 and L5-6 as described in the text) are marked. The relatively large displacement of the other half of the molecule, including NBD2, is clearly shown. The relative position of the Nb592 is marked in interaction with NBD1. It also makes a smaller contact with NBD2. The P-gp–Nb592 complex is the most closed inward-facing conformation described in this study. (B) Same structural alignment as A except turned 180° to show the opposite side of the transporter. The “TM-hinge region” comprised of L9-10 and L11-12 is indicated.