Structural Insights into Porphyrin Recognition by the Human ATP-Binding Cassette Transporter ABCB6

Abstract

Human ABCB6 is an ATP-binding cassette transporter that regulates heme biosynthesis by translocating various porphyrins from the cytoplasm into the mitochondria. Here we report the cryo-electron microscopy (cryo-EM) structures of human ABCB6 with its substrates, coproporphyrin III (CPIII) and hemin, at 3.5 and 3.7 Å resolution, respectively. Metalfree porphyrin CPIII binds to ABCB6 within the central cavity, where its propionic acids form hydrogen bonds with the highly conserved Y550. The resulting structure has an overall fold similar to the inward-facing apo structure, but the two nucleotide-binding domains (NBDs) are slightly closer to each other. In contrast, when ABCB6 binds a metal-centered porphyrin hemin in complex with two glutathione molecules (1 hemin: 2 glutathione), the two NBDs end up much closer together, aligning them to bind and hydrolyze ATP more efficiently. In our structures, a glycine-rich and highly flexible "bulge" loop on TM helix 7 undergoes significant conformational changes associated with substrate binding. Our findings suggest that ABCB6 utilizes at least two distinct mechanisms to fine-tune substrate specificity and transport efficiency.

Keywords: ABCB6; ATP-binding cassette transporter; cryoelectron microscopy; glutathione; porphyrin.

Fig. 1. Functional characterization.

(A) The chemical structures of various porphyrins used in this study. (B) NADH-coupled ATPase activity of the ABCB6-∆TMD0 as a function of PPIX concentration. The Km of PPIX was found to be 4.5 ± 1.2 µM, and the maximal ATPase activity was 69 ± 1.5 nmol/mg/min (or 9.7 min^–1). The dashed line indicates the basal ATPase activity in the absence of substrate. Data points represent mean ± SD of triplicate measurements. (C) ATPase activity of the ABCB6-∆TMD0 as a function of CPIII concentration. The Km of CPIII was found to be 19 ± 3.7 µM, and the maximal ATPase activity was 175 ± 7.5 nmol/mg/min (or 25 min^–1). (D) ATPase activity of ABCB6-∆TMD0 as a function of hemin concentration with and without 1 mM GSH. The Km for hemin in conjunction with GSH was determined to be 3 ± 0.3 µM and the maximal ATPase activity was 251 ± 7.8 nmol/mg/min (or 36 min^–1). (E) ATPase activity of ABCB6-∆TMD0 as a function of cobalt (III) PPIX (Co-PPIX) concentration with and without 1 mM GSH. The Km for Co-PPIX in conjunction with GSH was determined to be 3.4 ± 0.5 µM and the maximal ATPase activity was 276 ± 8.0 nmol/mg/min (or 39 min^–1).

Fig. 2. Structure of CPIII-bound ABCB6-∆TMD0.

(A) Schematic representation of the human ABCB6 monomer. The N-terminal TMD0 was not included in the construct, and is drawn with a dashed line. (B) The molecular structure of CPIII-bound ABCB6-∆TMD0. The monomers are shown in pink and teal, respectively. The CPIII is shown as purple spheres. The density corresponding to CPIII (front view) is shown at 4 σ level. The elbow helix was not built due to poor electron density. Single apostrophes are used for the residues (or helices) of one monomer to differentiate them from those of the other monomer. (C) Structure comparison of apo (PDB ID 7D7R) and CPIII-bound ABCB6-∆TMD0. The Cα distance between the conserved G626 of the Walker A motif and the S728 of the signature motif is indicated. Black arrows indicate movements of the NBDs.

Fig. 3. Close-up view of the CPIII-bound site.

(A) The view is rotated by 105 degrees along the vertical axis from Fig. 2B. Dashed lines indicate hydrogen bonds between CPIII and Y550. (B) Structure comparison of the substrate binding sites with and without CPIII. Structural changes are marked by black arrows. (C) Sequence alignments of TM 7 of the human ABCB6 homologues. The residues in the TM 7 bulge loop are highlighted in red. (D) ATPase activities of various mutants with 100 µM CPIII. The basal activity of ABCB6-∆TMD0 is set to 100%. Values are mean ± SD of three replicates. (E) CPIII transport activities of the ABCB6-∆TMD0 and other mutants in liposomes. The ATPase-defective mutant E752Q was used as a negative control. Data for protein-free liposomes in the presence of 200 µM CPIII and 2 mM ATP was taken as 100%. Values represent the mean ± SD of triplicate measurements.

Fig. 4. Structure of ABCB6- ∆TMD0 in complex with hemin and GSH.

(A) Overall structure of ABCB6-∆TMD0 in complex with hemin and GSH. Hemin and GSH are shown in purple sphere and cyan stick representation, respectively. The Cα distance between the conserved G626 of the Walker A motif and the S728 of the signature motif is shown at bottom. Zoom-in view of the boxed region is presented in Supplementary Fig. S13 (see also Supplementary Discussion). (B) Structure comparison of the substrate binding site of ABCB6-∆TMD0. The CPIII- and hemin:GSH-bound structures are aligned.

Fig. 5. Close-up view of the hemin:GSH binding site.

(A) This view is rotated by 105 degrees along the vertical axis with respect to Fig. 4A. The EM densities of hemin and neighboring residues are shown at 4 σ level. Dashed lines indicate hydrogen bond between hemin and Y550. The GSHs are omitted for simplicity. (B) ATPase activities of various mutants at 10 µM hemin and 1 mM GSH. The basal activity of ABCB6-∆TMD0 is set at 100%. Data points represent mean ± SD of triplicate measurements. (C) Structure of the hemin:GSH binding site in ABCB6-∆TMD0. The view is rotated by 90 degrees along the vertical axis from Fig. 4A. (D) The EM density map of hemin:GSH with neighboring residues is contoured at 4 σ. (E) ATPase activities of the mutants affecting GSH binding. The activity was measured in the presence of 10 µM hemin and 1 mM GSH.

Fig. 6. Proposed transport mechanism of the ABCB6 transporter.

(A) Binding of metal-free porphyrins does not place the NBDs close enough to allow effective ATP hydrolysis. Hence, ABCB6 activity is only modestly stimulated by the presence of metal-free porphyrins. (B) In conjunction with GSH, metal porphyrins prime ABCB6 for effective ATP hydrolysis and substrate translocation. TMD0 is omitted for simplicity.