Structural insights into binding-site access and ligand recognition by human ABCB1

Abstract

ABCB1 is a broad-spectrum efflux pump central to cellular drug handling and multidrug resistance in humans. However, how it is able to recognize and transport a wide range of diverse substrates remains poorly understood. Here we present cryo-EM structures of lipid-embedded human ABCB1 in conformationally distinct apo-, substrate-bound, inhibitor-bound, and nucleotide-trapped states at 3.4-3.9 Å resolution, in the absence of stabilizing antibodies or mutations. The substrate-binding site is located within one half of the molecule and, in the apo state, is obstructed by the transmembrane helix (TM) 4. Substrate and inhibitor binding are distinguished by major TM rearrangements and their ligand binding chemistry, with TM4 playing a central role in all conformational transitions. Furthermore, our data identify secondary structure-breaking residues that impart localized TM flexibility and asymmetry between the two transmembrane domains. The resulting structural changes and lipid interactions that are induced by substrate and inhibitor binding can predict substrate-binding profiles and may direct ABCB1 inhibitor design.

Keywords: ABC Transporter; ABCB1/MDR1/ p-glycoprotein; Multidrug Resistance; Structural Biology; cryo-EM.

Figure 1. Conformational landscape of lipid-embedded human ABCB1.

(A) Comparison of saposin A and nanodisc reconstituted human ABCB1 by nMS. Normalized SEC chromatograms of both are shown in the top right corner. (B) Comparison of ATPase activity of saposin A, MSP1D1 nanodisc, and Liposome reconstituted human ABCB1 in the presence of inhibitor, Zosuquidar (solid shapes and lines) and Taxol (clear shapes and dashed lines), basal ATPase rates are shown in black dashed box. Data are presented as mean of experimental replicates (N = 3) and error bars denote standard deviation (center = mean). (C) Structures of human ABCB1 in multiple distinct conformational states. EM density for the two halves is colored differently with N-terminal half (half1) in lighter shade and C-terminal half (half2) in darker shade and that of modeled acyl chains is colored gray. Source data are available online for this figure.

Figure 2. Structure of apo-ABCB1.

(A) Overall structure with the two halves colored as different shades of red and density modeled as lipid acyl chains (gray sticks) shown as transparent gray surfaces. (B) 3TM bundle formation by TM4, TM6, and TM12. TM4 sub-helical segments. The yellow dashed triangle highlights the central 3TM bundle in top and bottom views. (C) Comparison of the cryo-EM structure of apo-ABCB1, colored as in (A), and its alphafold-predicted structure (transparent cartoon). Black arrows indicate major movements of select TMs. The gray bars represent the plasma membrane.

Figure 3. Structure of ABCB1 bound to Taxol.

(A) Overall structure with first and second halves (primary structure based) colored green and white, respectively, and distinguished from domain-swapped (DS) halves. Density for Taxol and lipids is shown in pink and gray (0.01 contour threshold), respectively. The weaker density for the NBD1 nucleotide is shown in blue (0.008 contour threshold). The zoom panel shows Taxol (pink sticks) density along with associated density features modeled as a lipid acyl chain (gray sticks) as transparent pink and gray surfaces, respectively. Domain-swapped halves are highlighted and demarcated by gray and green semicircles. (B) Overall comparison of apo and Taxol complexes of ABCB1 (transparent brown and green cartoons, respectively) with 3TM forming helices (solid tube helices) and Taxol (pink spheres) is shown.

Figure 4. Comparison of Zosuquidar and Taxol binding.

(A) Overall structure of ABCB1 bound to zosuquidar. Zosuquidar and ATP density is shown (0.0175 contour) as teal and blue surfaces, respectively. (B) Zoomed view of the occluded TMD cavity with TM4 and TM10 shown. EM density for both zosuquidar molecules (teal sticks, Z1 and Z2) is shown as a transparent teal surface (0.017 contour). (C) Ligand interaction plot of ABCB1 complexed to Taxol. (D) Ligand interaction plot of zosuquidar (z) bound ABCB1 with the second zosuquidar molecule is shown in yellow.

Figure 5. Structural transitions in ABCB1.

(A) Overlay of TM4/5 and TM10/11 of all ABCB1 structures reported, highlighting overall conformational changes linked to substrate (Taxol, pink surface) or inhibitor (zosuquidar, teal surface) binding and CH2 and CH4 movements (bottom) with distances between selected Cα pairs shown. (B) Pairwise structural alignment of linked TM pairs expected to move together in different type II ABC exporter conformational states with TM4/5 and TM10/11 pairs boxed to highlight their greater conformational flexibility in the four conformations reported.

Figure 6. Mechanism of ABCB1 transport function.

Schematic of working model for substrate transport and inhibition in human ABCB1: In the apo state an IF_CLOSED state dominates with TM4 blocking the substrate-binding site. Substrate (Taxol, green star) binding promotes transition to the IF_OPEN state, favoring ATP binding that leads to a transition to the OF_OPEN state for substrate release through a preceding IF_OCCLUDED state. Unlike substrates, inhibitors like zosuquidar (red L-shape) stabilize the IF_OCCLUDED state, effectively trapping the transporter and arresting the transport cycle. Substrate release leads to the formation of an OF_CLOSED state and ATP hydrolysis resets the transport cycle. With the exception of the OF_OPEN state (based on homologous transporters like human ABCD1(16) and Sav1866(17)), all other states are based on experimentally determined structures. Select TMs driving conformational transitions are highlighted. Green circles highlight potential intermediate/alternate states. D = ADP, T = ATP. Pi inorganic phosphate. Dashed blue lines = ATP-binding elements.

Figure EV1. Secondary structure (SS) breaks in apo-ABCB1.

Gly and Pro residues colored teal and blue, respectively, and predicted SS breaks shown as spheres. An ECL3 and ECL6 sequence alignment is also shown with residues colored similarly and predicted SS-breaking residues underlined. TM4/5 and TM10/11 pairs are colored red. Acyl chains for prospective lipid/sterol molecules are shown as transparent spheres.

Figure EV2. Mismatch between TMD1 and TMD2 cavities for Taxol binding.

(A) Overlay of domain-swapped (DS) halves of ABCB1. The Taxol molecule bound to TMD2_DS is shown as transparent pink spheres. The Zoom panel shows electrostatic potential map of the TMD2_DS cavity (left) and its TMD1_DS cavity equivalent (right) showing electrostatic and steric clashes with Taxol. (B) TMD1_DS equivalent residues of TMD2_DS residues (Blue sticks) within 5 Angstroms of bound Taxol (transparent sticks), with residue labels colored similarly.

Figure EV3. Overlay of different conformational states of ABCB1.

Overall structural alignments between each conformation. R.m.s.d. values are also shown for total and aligned C alpha pairs.