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Figure 7. Dimeric ferrochelatase bridges ABCB7 and ABCB10 homodimers in a molecular complex required for heme biosynthesis. A functional dimeric fer- rochelatase (FECH) bridges ABCB7 and ABCB10 by binding near the nucleotide-binding domains (NDB) of each ATB-binding cassette (ABC) transporter. Two sequences in the C terminus of human ABCB7, regions V450-L463 and G527-D538, were essential for binding to FECH. Amino acid residues 90-115 of FECH, which formed the closing gate of the active site of the enzyme containing protoporphyrin IX (PPIX) were specifically involved in the interaction with ABCB7. The region between lysines 133 and 145 of FECH interacted with both ABCB7 and ABCB10. Likely the 133-145 region of one protomer of FECH interacted with ABCB7 and the corresponding region of the other protomer of the dimeric enzyme interacted with ABCB10. Interestingly, FECH interacted directly with regions of ABCB7 and ABCB10 near the NBD which are enriched in histidines, residues that are known heme-ligating amino acids (see also Online Supplementary Figure S12), raising the possibility that one or both ABC transporters may function as a mitochondrial matrix heme exporter.
tein interactions are fundamental to understanding the mechanisms and regulation of most biological processes, and interacting partners provide insights into biological functions that can be exploited for therapeutic purposes. We found that endogenous Abcb7 formed a complex with Fech and Abcb10 during erythroid differentiation. We per- formed XL-MS to investigate the architecture of the ABCB7/FECH complex, followed by extensive mutational analyses of ABCB7. Our studies identified two sequences in the C-terminal domain of ABCB7, residues V450-V504 and G527-D538, which were major molecular determinants of the interaction with FECH. Interestingly, these regions are adjacent to the Walker A motif of ABCB7, which is essen- tial for nucleotide binding and transport activity.47 The over- all architecture of the ABCB7/FECH/ABCB10 complex is shown in our proposed model (Figure 7), in which a func- tional dimeric FECH bridges ABCB7 and ABCB10 homo- dimers. Interestingly, residues 90-115 of FECH enclose the enzymatic active site containing PPIX and specifically bind ABCB7 near its nucleotide-binding domain. It is tempting to speculate that hydrolysis of ATP by ABCB7 may drive a conformational rearrangement on the region 90-115 of FECH, which would enable opening of the pocket and release of the newly synthesized proto-heme.
A previous model was proposed based on the identifica- tion of a Fech/Abcb10/Mfrn1 complex,31 in which the interaction of Fech with Abcb10 and Mfrn1 was required to integrate mitochondrial iron import with its utilization for heme synthesis. In the same study,31 a separate interac- tion of Fech with Abcb7 was also reported. We did not identify Mfrn1 as part of the Abcb7/Fech/Abcb10 com- plex (Figure 5A and Online Supplementary Table S1).
However, since Mfrn1 was reported to be the direct inter- acting partner of Abcb10,31 it is possible that in our co- immunoprecipitation experiments using an anti-Abcb7 antibody we mainly immunocaptured Abcb7 and its clos- est interacting partner, FECH, which in turn mediated an interaction with Abcb10; thus, we may not have detected other interactions of Abcb10 with Mfrn1 which are more physically distant from the Abcb7 partners. Nonetheless, Mfrn1 upregulation was essential to meet the exceptional- ly high iron demand for heme biosynthesis of erythroid cells during differentiation (Figure 4A-G), and decreased Mfrn1 half-life in the iron-deficient conditions generated by loss of Irp2 caused mitochondrial iron deficiency and impaired heme biosynthesis (Figure 4A-G). Our studies offer a potential molecular mechanism for reported cases of erythropoietic protoporphyria in patients harboring mutations between residues 68 and 220,48 which we found were involved in binding ABCB7. Overall, our studies highlight the importance of ABCB7 for mitochondrial function and provide the biochemical characterization of a functional complex formed of ABCB7, FECH and ABCB10, which is required for cellular iron homeostasis, mitochondrial function and heme biosynthesis. Our work suggests that more definitive experiments deploying coor- dinated activity of the entire complex may aid identifica- tion of its physiological substrates.
Acknowledgments
The authors thank Dr. Manik Ghosh for technical help, mem- bers of the Rouault laboratory for helpful discussion and the Eunice Kennedy Shriver NICHD Intramural Research Program for sup- port.
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haematologica | 2019; 104(9)