*2.1. Homology Models of Human and Rat ABCC6/Abcc6*

ABC transporters have a common structural core that in ABCC6 and all other ABCC subfamily members consist of transmembrane domain 1 (TMD1, encompassing transmembrane helices (TM) 6 to 11), nucleotide-binding domain 1 (NBD1), TMD2 (TM12 to 17) and NBD2. We built a sequence alignment for TMD1-NBD1 and TMD2-NBD2 (see Supporting Information files TMD1-NBD1\_Alignment.pdf and TMD2-NBD2\_Alignment.pdf), using several orthologues of ABCC1, ABCC6, and ABCC5, for which negatively charged substrates have been reported. From these alignments, we observed a 46% and 48% sequence identity for rAbcc6-bAbcc1 and hABCC6-bAbcc1 TMD1-NBD1, respectively, and 52% and

53% sequence identities for rAbcc6-bAbcc1 and hABCC6-bAbcc1 TMD2-NBD2, respectively. The % sequence identity calculated for TMD1 (rAbcc6 residues 298–608) and TMD2 (rAbcc6 residues 933–1242) are 39% and 48%, respectively, for rAbcc6 and bAbcc1, and 40% for TMD1 and 48% for TMD2 between hABCC6 and bAbcc1. Considering the high sequence identity with bAbcc1, we modeled the structural core for rAbcc6 and hABCC6 in two distinct conformational states (Figure S1), based on the cryoEM structures reported for the LTC4-bound, ATP-free, inward-facing state and on the ATP-bound outward-facing state of bAbcc1 [22,23]. ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐

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Consistent with their relatively high degree of sequence identity, both hABCC6/rAbcc6 and bAbcc1 showed a strong positive potential in the cavity along TMs of both TMD1 and TMD2 (Figure 1). Another common feature of hABCC6/rAbcc6 and bAbcc1 is the presence of a more negative potential on the extracellular end of the TMDs (Figure S2A), which following the conformational change to the ATP-bound, outward-facing state, appears less prominent (Figure S2). ‐ ‐

‐ ‐ ‐ ‐ − **Figure 1.** Electrostatic potential of the inward-facing state of hABCC6/rAbcc6 and bAbcc1. Electrostatic potential mapped on the molecular surface of the ATP-free, inward-facing (**A**) rAbcc6 model, (**B**) hABCC6 model, and (**C**) bAbcc1 cryoEM structure. The isovalue was set at −10 kBT/e for the negative potential (red) and +10 kBT/e for the positive potential (blue). For each transporter, the surface is clipped, and the two halves are shown side by side. The region of the transporters embedded in the membrane is highlighted by the gray slab. TMDs, transmembrane domains; ICLs, intracellular loops, i.e., the intracellular extension of the TMDs; NBD1 and NBD2, nucleotide binding domain 1 and 2.

‐ In the transmembrane cavity of bAbcc1, several residues have been proposed as participating in the recognition of LTC<sup>4</sup> through a network of hydrogen bonds, salt bridges, and van der Waals contacts (Figure S3, Figure 2 and Table 1) [22], although not all of the proposed interactions are supported by biochemical studies [28,29].

**Table 1.** Amino acid residues in rat and human ABCC6, human ABCC1 and human ABCC5 at the same positions proposed to form the LTC<sup>4</sup> binding cavity in bAbcc1. In the last column it is indicated to which transmembrane helix (TM) and transmembrane domain (TMD) the residues belong. bAbcc1, bovine Abcc1; hABCC1, human ABCC1; hABCC6, human ABCC6; rAbcc6, rat Abcc6; hABCC5, human ABCC5.



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**Table 1.** *Cont.*

‐ ‐ ‐ ‐ ‐ **Figure 2.** rAbcc6 residues in the transmembrane cavity corresponding to those in the bAbcc1 cavity surrounding LTC<sup>4</sup> . View from the extracellular side of the transmembrane cavity of the inward-facing, ATP-free (**A**) and outward-facing, ATP-bound (**B**) models of rAbcc6. The residues corresponding to those of the bAbcc1 LTC<sup>4</sup> binding cavity are shown as sticks in light cyan for TMD1 and in teal for TMD2.

‐ ‐ The residues in the cavity are proposed to form a bipartite binding site [30] with a more prominent positive charge on one side (residues K332, H335, L381, F385, Y440, F594 of TMD1, and R1196, N1244, and R1248 of TMD2, namely the P-pocket) to bind the hydrophilic glutathione moiety of LTC<sup>4</sup> and a more hydrophobic pocket (namely, the H-pocket) to accommodate the lipid tail of LTC<sup>4</sup> (residues T550, W553 of TMD1, and M1092, Y1242, W1245 of TMD2) [22]. Our goal was to address if ABCC6 (the corresponding residues shown in Figure 2 for rAbcc6 and in Figure S3 for hABCC6), in both the ATP-free and ATP-bound states, might be involved in ATP interaction and efflux. First, we analyzed how the proposed P-pocket and H-pocket residues are conserved across the sequences taken into account to build the models. The analysis of the sequence alignment (Supplemental information files TMD1-NBD1\_Alignment.pdf and TMD2-NBD2\_Alignment.pdf) showed that bAbcc1 W553 and W1245 in the hydrophobic pocket are conserved among the ABCC6, ABCC1, and ABCC5 sequences, and correspond to rAbcc6 residues F537 (TM10 in TMD1) and W1217 (TM17 in TMD2) (Figure 2). In the P-pocket, the more conserved residues are bAbcc1 R1196, R1248, and N1244, which correspond to rAbcc6 R1168 (TM16 in TMD2), R1220 and Q1216 (TM17 in TMD2) (Figure 2).

The degrees of similarity for the other residues of the bAbcc1 P- and H-pockets vary across the ABCC1, ABCC6, and ABCC5 sequences considered in the alignment. Charged residues that are not conserved are: (1) bAbcc1 K332, which is a leucine in ABCC6 (L316 in rAbcc6) and ABCC5, (2) H335 in hABCC1/bAbcc1, which is a serine in the hABCC6/rAbcc6 sequences, and (3) rAbcc6 E365, which is a glutamate only in ABCC6 sequences and a leucine in hABCC1 (L381 in bAbcc1) and ABCC5. Of note, in hABCC1, K332, and to a lesser extent H335, are indispensable for LTC<sup>4</sup> binding and transport, indicating K332 and H335 are crucial amino acid residues in its LTC4-binding site [29,31]. An additional alignment performed using sequences of human ABC transporters of the C subfamily confirmed these observations and demonstrate exceptionally high conservation of R1168 and R1220 (numbering of rAbcc6) among ABCC proteins (Figure S5).
