**3. Key Residues and Motifs Are Conserved in Multidrug Transporters ABCG/PDR**

The mammalian ABCGs are the closest orthologues of yeast PDR transporters [216,245, 250,251,321], especially Pdr5 and Cdr1 [166,322,323] (Supplementary Figure S1). To identify conserved regions of fungal ABC proteins based on mammalian ABC structures, we determined a conservation score for the fungal PDR family, and mapped conserved residues into the atomic structures of mammalian orthologues (Figure 2B). We subjected the fungal ABCG/PDR subfamily to multiple sequence alignments with the mammalian ABCGs (Supplementary Figure S1), showing that NBDs hold several highly conserved motifs related to ATP consumption. Although TMDs are usually more diverse, several regions are also preserved between PDR and ABCG, implying that these domains are pivotal for an evolutionarily related catalytic cycle.

Conservation in the NBD. First, the NBDs in fungal PDRs share highly conserved motifs as well as residues with ABCG required for ATP-binding and hydrolysis [45,153,324–326], including Walker A, Q-loop, Hot spot helix, Signature motif, Pro-loop, Walker B, D-loop and H-loop, respectively [327]. The alignment indicates that fungal NBDs adopt a RecAlike structure, an ATPase-containing fold that was first seen in RecA, which is involved in DNA recombination, and was later found in many ATPases [328] (Supplementary Figure S1 and Table 4). There are only minor differences though, as ABCG2 and ABCG5/G8 are half-transporters that require homo- or hetero-dimerization, while all fungal PDRs are fullsize transporters, some of which with asymmetric deviant ATP-binding sites (Table 4). In the first NBD of PDR transporters, the glutamine (Q) in the Q-loop is replaced by glutamate (E), the Pro-loop disappeared and histidine (H) in the H-loop is substituted by tyrosine (Y). Hence, the notion emerged that fungal PDRs would follow an asymmetric catalytic cycle [173,216,306,329,330].

Conservation in the TMD region. Second, the general architecture and configuration of TMDs are maintained, as each TMD in ABCG/PDR transporters contains six putative membrane-spanning helices, and a rather short first intracellular loop contains the coupling helix. The large ECL is part of the lid architecture in the extracellular region. Interestingly, two putative helices residing at both lipid bilayer leaflets, which are important for function, as they restrict dynamic movements during transport cycle, are only found in the ABCG subfamily [40,42]. The amino acid alignments reveal the consensus sites present in conserved motifs and domains (Figure 1B and Supplementary Figure S1).



\* Some PDRs may consume GTP as well [186,331]. \*\* Substitution with another residue.

The elbow helix is a 15-residue amphipathic helix sharing highly conserved residues among all ABCG/PDR members. This elbow helix is an intrinsic part of the triple helical bundle (THB), which also engages the hot spot helix from the NBD and the coupling helix from the first intracellular loop (ICL1) [332]. Importantly, the center of the elbow helix contains a highly conserved arginine (R), which is essential for a salt bridge interaction with a glutamate (E) residue in ICL1 to stabilize the THB [40].

The transmembrane helix 1 (TMH1) is a prototypic 20-residue membrane-spanning helix, following the elbow helix in all ABCG/PDR exporters. The small extracellular loop 1 (ECL1) is a short linker connecting TMH1 and TMH2 at the cell surface. Notably, ECL1 in the first half holds a conserved arginine (R), which is important for salt bridge formation with a conserved glutamate (E) in the re-entry helix of the same molecule [42]. TMH2 is thought to be a part of the substrate/inhibitor binding zone in human ABCG2 [333]. A conserved phenylalanine (F) in the middle of TMH2 may be a recognition site for both substrates and inhibitors [333].

The intracellular loop 1 (ICL1) spans over 30 residues in the ABCG/PDR subfamily and holds a critical U-turn motif. ICL1 operates as the coupling helix for the NBD–TMD communication by participating in a triple helical bundle (THB). The entire THB functions as a molecular spring that controls the catalytic cycle by regulating the conformational switch. Indeed, two conserved negative residues within ICL1 of human ABCG2, E451 and E585, are thought to control the intracellular gating mechanism and engage in salt bridges with the elbow helix, respectively. Remarkably, ICL1 holds an essential tyrosine residue that is conserved in all ABCG/PDR transporters. This Y464 contributes a salt bridge interaction within the THB complex for stabilization of the transmission interface.

TMH3 also has several conserved residues within the ABCG/PDR group such as negative residues (E or D), proline, as well as positive amino acids (K or R). D477 at the start of TMH3 in human ABCG2 is on top of the transmission interface and may contribute to an intracellular gate. ECL2 is again rather short, containing only eight residues with a kink, connecting TMH3 and TMH4 at the cell surface.

The hydrophobic valve is a kinked domain just after TMH5, consisting of the conserved "glycine-Φ-Φ" (Φ is hydrophobic residue) motif that subtends the extracellular bilayer leaflet. The valve is pivotal for controlling substrate transport through a putative translocation pathway whose precise nature and dynamics remains obscure. The re-entry helix is the counterpart of the elbow helix and is unique, establishing a kinked hallmark motif in the ABCG/PDR subfamily with several conserved residues. In addition, ECL3 is the only large extracellular loop, and a main part of the extracellular roof architecture. Interestingly, ECL3 in human ABCG2 has three cysteines, which form intra- and inter-molecular disulfide formations, and perhaps facilitate dimerization and drug release [334,335]. Likewise, conserved cysteines in fungal PDR transporters may engage in an intra-molecular disulfide bond to stabilize the PDR biogenesis. Finally, TMH6 is followed by a very small but highly conserved C-terminus that contains several positive charges at the C-terminus.
