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Dear Editor, Cobalamin, also known as vitamin B12, can only be biosynthesized by certain bacteria and archaea. As an essential nutrient for humans, it should be obtained from daily food. Exogenous cobalamin is taken up by the cell through an endocytosis process, then released into the lysosome as a free form, and finally pumped out to the cytosol for utilization. The human ATPbinding cassette (ABC) transporter ABCD4 localized on lysosomal membrane is indispensable for the efflux of cobalamin from lysosome to cytosol. Mutations in ABCD4 gene may result in cobalamin deficiency and inborn diseases. The patients suffer from combined symptoms: hypotonia, lethargy, poor feeding, bone marrow suppression, macrocytic anemia, and heart defects. However, the absence of the structure of human ABCD4 limits our understanding of the molecular mechanism of pathogenesis. The 606-aa-long human ABCD4 consists of a transmembrane domain (TMD) with six transmembrane (TM) helices followed by a nucleotide-binding domain (NBD). Based on the phylogenetic distance, ABCD4 was classified into the subfamily D, one of seven subfamilies that consist of 48 members of human ABC proteins. Expression and purification of the recombinant full-length human ABCD4 in HEK 293F cells are described in Supplementary information, Data S1. The protein appeared to be stable and homogeneous after extraction and purification in detergent micelles (Supplementary information, Figs. S1a and 2a). The purified protein samples reconstituted into liposomes were subjected to ATPase activity assay. The maximal ATPase activity (Vmax) of ABCD4 is 11.8 nmol/min/mg (Supplementary information, Fig. S1b), which is comparable to that in the previous report. Using single-particle cryogenic electron microscopy (cryo-EM), we solved the structure of human ABCD4 in the presence of ATP, with the catalytic residue Glu549 substituted by glutamine. After several rounds of iterative 2D and 3D classification and refinement, the overall resolution of the EM map was calculated to 3.6 Å out of 276,557 selected particles according to the gold-standard Fourier shell correlation (FSC) 0.143 criterion (Supplementary information, Figs. S2b–d and 3; Table S1). The EM density exhibits prominent side-chain features in the core region of ABCD4 homodimer that allow us to unambiguously register the residues for all 12 TM helices and the core regions of NBDs (Supplementary information, Figs. S2e and 4). The final model composed of residues Arg13-Lys604, except for a 27-aa linker region (residues Lys357-Ala383), was refined against the EM density to an excellent geometry and statistics (Supplementary information, Fig. S2f and Table S1). The overall structure of ABCD4 revealed a lysosome-open conformation, with two ATP molecules binding to the interface between two NBDs that are localized in the cytosol (Fig. 1a). Remarkably, most human ABC exporters are localized on the cell membrane that allow both the substrate and ATP to access from the cytosolic side, the so-called cis-side of NBD, thus termed cis-acting exporters. In contrast, human ABCD4 represents a unique exporter that effluxes the substrate in a trans-acting manner, different from the classic cis-acting exporters. Each TMD contains six helices with significant cytosolic extensions, a hallmark of type I exporters. The TM helices pack against each other closely at the cytosolic side, and diverge into two discrete “wings” facing the lysosomal matrix, thus exhibiting a lysosomeopen conformation. Due to domain swapping, each “wing” consists of TM1 and TM2 from one subunit packing against helices 3–6 from the other subunit (Fig. 1b). The two NBDs at the cytosolic side form a typical head-to-tail dimer via extensive contacts. At the dimeric interface, we found two ATP molecules, each of which is stabilized by the Walker A motif of one NBD and the ABC signature motif of the other (Fig. 1c). Notably, the magnesium ion was not observed in our structure, similar to a previous report of ABC transporter. The adenine ring of ATP is stacked by two hydrophobic residues Pro397 and Val523, whereas the ribose moiety is stabilized by Arg508 and Glu528 via hydrogen bonds (Fig. 1d). The three phosphate groups mainly interact with the residues from Walker A motif, Q-loop, and signature motif through a network of hydrogen bonds. Remarkably, ABCD4 possesses an ABC signature motif of LSPGE, rather than the canonical LSGGQ. In the case of ABCB1 (PDB code: 6C0V), the small residue Gly1178 at the very N-terminus is key to the long helix to direct a positively charged helical dipole toward the γ-phosphate of ATP. Thus, we propose that replacement of the small residue with a proline in ABCD4 should interfere with the helical dipole interaction (Fig. 1e), which may result in a relatively lower ATPase activity of ABCD4, compared to other ABC exporters. Sequence homology search enabled us to reveal that this proline exists not only in eukaryotic ABCD4, but also in some putative cobalamin transporters from prokaryotes. Multiplesequence alignment further indicated that ABCD4 and homologs possess a distinct proline-containing signature motif (Fig. 1f). Dissection of electrostatic surface potential of the TMDs showed that the transmembrane cavity starts with an entrance composed of hydrophobic residues, extends to the cytosolic side through a narrowing cleft consisting of four pairs of negatively charged residues, and finally gets closed at the boundary of membrane (Fig. 1g). The hydrophobic entrance is constituted of residues Ile57, Val60, Pro64 from TM1, Thr155, Tyr174 from TM3, and F315, Tyr319 from TM6 (Fig. 1h), similar to the substrate entrance at the TMDs of Escherichia coli vitamin B12 ABC importer BtuCD. In fact, an open hydrophobic cavity was proposed to accommodate the corrin ring moiety of vitamin B12. Moreover, a pair of positively charged residues, namely Arg155 in ABCD4, at the bottom of the cavity are complementary in charge to the phosphate groups of lipid bilayer (Fig. 1h), similar to those in the previously reported structures. Some mutations in ABCD4 gene result in cobalamin deficiency and severe diseases. The present structure of ABCD4 enabled us to elucidate the molecular basis of pathogenesis due to these mutations. To date, seven clinical mutations in ABCD4 gene have