W546 stacking disruption traps the human porphyrin transporter ABCB6 in an outward-facing transient state

Abstract

Human ATP-binding cassette transporter subfamily B6 (ABCB6) is a mitochondrial ATP-driven pump that translocates porphyrins from the cytoplasm into mitochondria for heme biosynthesis. Within the transport pathway, a conserved aromatic residue W546 located in each monomer plays a pivotal role in stabilizing the occluded conformation via π-stacking interactions. Herein, we employed cryo-electron microscopy to investigate the structural consequences of a single W546A mutation in ABCB6, both in detergent micelles and nanodiscs. The results demonstrate that the W546A mutation alters the conformational dynamics of detergent-purified ABCB6, leading to entrapment of the transporter in an outward-facing transient state. However, in the nanodisc system, we observed a direct interaction between the transporter and a phospholipid molecule that compensates for the absence of the W546 residue, thereby facilitating the normal conformational transition of the transporter toward the occluded state following ATP hydrolysis. The findings also reveal that adoption of the outward-facing conformation causes charge repulsion between ABCB6 and the bound substrate, and rearrangement of key interacting residues at the substrate-binding site. Consequently, the affinity for the substrate is significantly reduced, facilitating its release from the transporter.

Fig. 1. Inhibition of the functional activity of hABCB6^core by ATP analogs.

a ATPase activities of detergent-purified hABCB6^core and the E752Q mutant with and without porphyrin substrates (20 μM CPIII or 10 μM hemin: 1 mM GSH) in the presence of 10 mM Mg²⁺ and 2 mM ATP plus VO₄^3-, AlF₄^-, or SO₄^2-. Results are means ± standard deviation (SD) of more than three independent experiments. b Inhibition of the CPIII transport activity of hABCB6^core (or its variants) by different ATP analogs. Liposome-based transport activity was measured in the presence of 50 μM CPIII, 10 mM Mg²⁺, and 4 mM ATP (or its derivatives). The activity of protein-free liposomes was set at 100%. Results are means ± SD of at least five independent measurements from two different liposome preparations.

Fig. 2. The occluded state of hABCB6^core is highly stabilized by W546-W546’ interaction.

a, b Representative reference-free 2D class averages (a) and 3D classification (b) of hABCB6^core in the presence of CPIII and Mg²⁺/ADP·VO₄. The bottom numbers in (b) refer to the number of particles used to compute each 3D class. See also Supplementary Fig. 3. c Structural comparison of hABCB6^core in inward-facing (Class 1 from Fig. 2b) and occluded (Class 2 from Fig. 2b) states viewed from the plane of the membrane (left) and the extracellular side (right). The upper and central cavities are drawn as cyan and blue mesh, respectively. Mg²⁺/ ADP·VO₄ molecules in the occluded structure are omitted for simplicity. The EM density (blue mesh) of the W546 residue is contoured at the 4 σ level. Note that the residues (or helices) of one monomer are numbered with single primes to differentiate them from those of the other monomer. d Sequence alignment of TM 11 of ABCB6 homologs. Conserved tryptophan residues (or phenylalanine residues in bacterial orthologs) are highlighted in red. e Residues of TM 11 involved in stabilization of the pre- (PDB ID 7EKL) and post-occluded conformations are shown as sticks.

Fig. 3. ATPase activity of hABCB6^core and W546A proteins in detergent micelles or various nanodiscs.

a Comparison of the ATPase activities as a function of ATP concentration. Results are expressed as means ± standard deviation (SD) from at least four separate experiments using the two different protein preparations. b At saturating concentration (2 mM ATP), maximal basal activities are represented as fold increase. The activity of detergent-purified hABCB6^core was set at 100% as the reference. See also Supplementary Table 1.

Fig. 4. Comparison of cryo-EM maps of the hABCB6^core-W546A mutant in detergent micelles and nanodiscs.

a, b Cryo-EM analysis of the W546A mutant in detergent micelles, showing representative 2D class averages (a) and 3D classification (b). The bottom numbers in (b) refer to the number of particles used to compute each 3D class. See also Supplementary Fig. 6. c, d Cryo-EM analysis of the W546A mutant reconstituted into nanodiscs, showing selected 2D class averages (c) and 3D classification (d). The bottom numbers in (d) refer to the number of particles used to compute each 3D class. See also Supplementary Fig. 9.

Fig. 5. The post-occluded conformation of the hABCB6^core-W546A mutant is stabilized by a phospholipid embedded within the hydrophobic groove of the TMD surface.

a Overall structure of the post-occluded W546A mutant in nanodiscs. The two monomers are colored blue and green, respectively. ADP·VO₄ is shown as sticks and Mg²⁺ is shown as a magenta sphere. Bound POPE molecules are shown as yellow spheres. The area enlarged in (b) and (c) is boxed. b Close-up view of protein-POPE interactions. The POPE molecule is shown as yellow sticks and its EM density map (4 σ) is displayed as blue mesh. c Surface representation of the lipid-binding groove with the color scheme based on atom type. d Sequence alignment of ABCB6 homologs highlighting residue conservation (red) within the lipid-binding groove.

Fig. 6. Cryo-EM structure of the hABCB6^core-W546A mutant in an outward-facing state.

a Overall structure of the W546A mutant in the outward-facing state. The two monomers are shown in blue and green, respectively. ADP·VO₄ is shown as sticks (orange and yellow) and Mg²⁺ is shown as a sphere (magenta). b Surface slab view. c Comparison of the conformation of transmembrane helices between outward-facing and post-occluded states. d Bottom view of the NBD dimer. Mg²⁺ and ADP·VO₄ are represented by spheres (magenta) and sticks (orange and yellow), respectively. The area enlarged in (e) is boxed. e Zoomed-in view of the ADP·VO₄-binding site. Side chains involved in ADP·VO₄ binding are shown as sticks. Cryo-EM map (gray mesh) of ADP·VO₄ is contoured at the 4 σ level. f Close-up view from the extracellular side into the transport pathway. The surface electrostatic potential, contoured from -5 kT/e (negative, red) to +5 kT/e (positive, blue) was represented using the APBS-PDB2PQR software suite, assuming a pH of 7.0 (http://www.poissonboltzmann.org). Overlaid structures of acidic residues are shown as sticks.

Fig. 7. Conformational changes at the substrate-binding site during the transport cycle.

a, b Close-up views of the CPIII- (a) and hemin:GSH- b binding sites of each conformation viewed from within the plane of the membrane. Bound substrates and key residues that interact with substrates are represented by sticks and colored by heteroatom. The structures of CPIII and hemin:GSH superimposed on the outward-facing hABCB6^core are drawn as empty black sticks.

Fig. 8. Schematic diagrams of conformational equilibria between the outward-facing and post-occluded configurations of hABCB6^core and the W546A mutant.

a–c Subunits are colored blue and cyan. Mg²⁺/ATP is depicted as an orange oval. Phospholipids are presented as brown heads and yellow tails. Dark-colored states indicate ‘preferred’ forms, while pale-colored states represent ‘unfavorable’ forms.

Fig. 9. Role of conserved aromatic residues in stabilizing the occluded conformation of the ABC transporter family.

a−d Cylindrical (left) and cartoon (right) representations of representative ABC transporters. Residues involved in stabilization of the occluded conformation are shown as sticks. a C. thermophilum Atm1 (PDB ID 7PR1). b A. thaliana Atm3 (PDB ID 7N5A). c Zebrafish CFTR (ABCC7, PDB ID 5W81). d Human P-glycoprotein (ABCB1, PDB ID 6C0V).