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[Preprint]. 2024 Aug 12:2024.08.12.607598.
doi: 10.1101/2024.08.12.607598.

Structural insight into binding site access and ligand recognition by human ABCB1

Devanshu Kurre  1 , Phuoc X Dang  1   2 , Le T M Le  1   3 , Varun V Gadkari  4 , Amer Alam  1
Affiliations

Structural insight into binding site access and ligand recognition by human ABCB1

Devanshu Kurre et al. bioRxiv. .

Update in

Abstract

ABCB1 is a broad-spectrum efflux pump central to cellular drug handling and multidrug resistance in humans. However, its mechanisms of poly-specific substrate recognition and transport remain poorly resolved. Here we present cryo-EM structures of lipid embedded human ABCB1 in its apo, substrate-bound, inhibitor-bound, and nucleotide-trapped states at 3.4-3.9 Å resolution without using stabilizing antibodies or mutations and each revealing a distinct conformation. The substrate binding site is located within one half of the molecule and, in the apo state, is obstructed by transmembrane helix (TM) 4. Substrate and inhibitor binding are distinguished by major differences in TM arrangement and ligand binding chemistry, with TM4 playing a central role in all conformational transitions. Our data offer fundamental new insights into the role structural asymmetry, secondary structure breaks, and lipid interactions play in ABCB1 function and have far-reaching implications for ABCB1 inhibitor design and predicting its substrate binding profiles.

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Conflict of interest statement

Competing Financial Interests The authors declare no competing financial Interests.

Figures

Figure 1
Figure 1. Conformational landscape of lipid embedded human ABCB1.
A Comparison of saposin and nanodisc reconstituted human ABCB1 by nMS B) Comparison of ATPase activity of saposin, MSP1D1 nanodisc, and Liposome reconstituted human ABCB1. n=3 and error bars denote standard deviation. C Structures of human ABCB1 in multiple distinct conformational states. EM density for the two halves is colored differently and that of modeled acyl chains is colored gray.
Figure 2.
Figure 2.. Structure of apo-ABCB1.
A Overall structure with the two halve 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). Blue transparent arrows indicate major movements of select TMs. The gray bars represent the plasma membrane.
Figure 3
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 grey (0.01 contour threshold), respectively. 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 (grey sticks) as transparent pink and gray surfaces, respectively. Domain swapped halves are highlighted demarcated by grey 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) shown
Figure 4
Figure 4. Comparison of Zosuquidar and Taxol binding.
A Overall structure of the 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 with EM density for both Zosuquidar molecules (teal sticks, Z1 and Z2) shown as a transparent teal surface (0.017 contour). C Ligand interaction plot of ABCB1 complexed to Taxol. D Ligand interaction plot of zosuquidar bound ABCB1 with the second Zosuquidar molecule shown in yellow.
Figure 5
Figure 5. Structural Transitions in ABCB1.
A Overlay of full transporter in all 4 conformations with Half 1 and half 2 shown as transparent surfaces (front and back views, respectively) and with individual TMDs and NBDs outlined top and bottom views, respectively. B Pairwise structural alignment of linked TM pairs expected to move together in different type II ABC exporter conformational states.
Figure 6
Figure 6. Substrate and inhibitor interactions in ABCB1
A Overlay of TM4/5 and TM10/11 of all structures reported, highlighting overall conformational changes linked to substrate (Taxol, pink) or inhibitor (zosuquidar, teal) binding and CH2 and CH4 movements B Schematic of working model for substrate transport and inhibition in human ABCB1. With the exception of the OFOPEN state (based on homologous transporters like human ABCD1 and Sav1866). Green circles highlight potential intermediate/alternate states.
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