Fig. 4: Close-up view of the substrate-binding site.

a Zoom-in view of the Cd(II):GSH-binding site. Bound Cd(II) ion and GSHs are represented by green sphere and cyan stick models, respectively. Cryo-EM maps (gray mesh) of Cd(II) and GSHs are contoured at the 9 and 4 σ level, respectively. The W546 residue is omitted for simplicity (see also Supplementary Fig. 13). The view is rotated by 90 degrees along the vertical axis from Fig. 2b. b Interactions between hABCB6^core and the Cd(II):GSH complex analyzed by LigPlot+ software. Residues involved in nonpolar and van der Waals interactions within 4 Å are depicted as red semicircles. c Zoom-in view of bound Cd(II) ion and PC2s. d Interactions between hABCB6^core and the Cd(II):PC2 complex analyzed by LigPlot+ software. e ATPase activities of mutants affecting GSH binding. Activity was measured in the presence and absence of 800 μM Cd(II) and 1 mM GSH. Data points represent mean ± standard error of the mean (SEM) of at least three measurements using two different batches of purified protein. The symbol *** and ns denote significant differences at p < 0.001 and not statistically significant, respectively, with p-values calculated using a two-sided unpaired t-test and Welch’s correction. f Sequence alignment of TM helices 9 to 11 of ABCB6 and its orthologs involved in Cd(II):GSH-binding. Highly conserved residues among 15 different species are highlighted in red. Conservation of the capping residues between species is highlighted in orange (see also Fig. 5). Hs, Homo sapiens; Bt, Bos taurus; Ua, Ursus americanus; Mm, Mus musculus; Ma, Mesocricetus auratus; Rn, Rattus norvegicus; Xt, Xenopus tropicalis; Dr, Danio rerio; Dm, Drosophila melanogaster; Ce, Caenorhabditis elegans; Sp, Schizosaccharomyces pombe; Sc, Saccharomyces cerevisiae; At, Arabidopsis thaliana; Ct, Thermochaetoides thermophila; Na, Novosphingobium aromaticivorans.