**2. Atomic Structures of Eukaryotic Multidrug ABC Efflux Exporters**

Several high-resolution structures, obtained through X-ray crystallography or cryo-EM approaches in the past five years, provided a better understanding of the structure–function relationships of mammalian type I and type II exporters (Table 3). However, despite ever-

increasing structural information, the path from static atomic structures to precise molecular mechanisms has turned out to be a rocky road for scientists and drug discovery. Thus, we are still far from understanding the conformational dynamics as well as the mechanics driving their transport cycles. Each half-molecule of ABCB and ABCC type I exporters harbor at least six transmembrane helices that extend into the intracellular loops linked to their coupling helices, thus connecting to both the proximal NBD and crossing over to the distal NBD [8,31,39,293–296]. In the ATP-free apo state, if it ever exists inside cells, these exporters maintain an inward-facing configuration, whereby both NBDs appear apart to offer access for ATP and possibly substrates (Figure 2A) [297]. Importantly, while the ABCC subfamily shares a highly similar overall fold with ABCB, MRP transporters have an additional N-terminal domain known as TMD0 [20,298,299]. Notably, CFTR also adds the so-called R(egulatory)-domain residing in the core of the channel between the N-terminal and the C-terminal TMDs [21,300,301] (Figure 1B).

**Figure 2.** Atomic structures, folds and evolutionary conservation of MDR exporters. (**A**) Crystal or cryo-EM particle structures of multidrug resistance-related ABC exporters from the type I (ABCB and ABCC) and type II families (ABCG subfamily) using the color codes as shown in Figure 1B. (**B**) Conservation analysis of ABC exporters from panel A. Multiple sequence alignments of amino acid sequences from Tables 1 and 2 were generated for all subfamilies. The degree of conservation was calculated and indicated as a color gradient ranging from low conservation (yellow) to high conservation (blue).

At present, the available atomic structures of type I exporters are as listed in Table 3. For the ABCB1 subfamily, many conformations, including inward-facing, outward-facing, occluded state, substrate-bound, inhibitor-bound and antibody-bound, have been solved, all in all yielding 24 structures in the PDB [296,302–310]. For the ABCC subfamily, four cryo-EM structures are available for bovine ABCC1 in the PDB [311,312]. Furthermore, seven structures are available for CFTR [300,301,313–315], and 12 structures for ABCC8 or SUR1 [316–320]. Strikingly, the first atomic structure of the heterodimeric ABCG subfamily member ABCG5/G8 came as a surprise, as the X-ray crystals revealed an unexpected

compact fold, in which NBDs were located in close proximity to the TMDs. In addition, the molecule held a lid-like structure at the extracellular roof of the translocation pathway. This fold resembled bacterial importers more than a prototypic export pump [38]. This structure paved the way for solving the human ABCG2 transporter, for which now 11 atomic structures are available in the PDB [256–259] (Table 3).


**Table 3.** Atomic structures of eukaryotic MDR ABC transporters.
