**6. Conclusions and Future Perspectives**

The increase in atomic structures of ABC transporters, and data from extensive biochemical and genetic experiments, have been propelling the field, since they have yielded novel insights and a better understanding of ABC transporter mechanisms. At the same time, while atomic structures have been invaluable, they have to be interpreted with caution, especially when biochemistry or genetics are not in line with structural data [265] or when biological relevance appears doubtful. Indeed, the painful history of ABC transporter structures [207,265], shows that even higher resolution structures suffer from their static nature that only reflects snapshots of a catalytic cycle. Thus, we need atomic structures reflecting more than a single conformation and possibly many transition states [352], as well as extensive validation by biochemistry and genetics, to validate their biological relevance and define catalytic cycles. Furthermore, there is an unmet need for more interdisciplinary collaborations that also engage alternative structural approaches like NMR [353–356] as well as complementary biophysical methods [265,357–359] to expand our mechanistic views of ABC transporters in all living kingdoms.

The past few years challenge previous notions that a unified transport mechanism exists for MDR exporters from all three major classes, such as ABCB1/MDR1/P-gp [35,293, 295,302,303,307,310], ABCC1/MRP1 [298,311,312] and ABCG2/BCRP [256–259] (Table 3). While early homology modeling attempts of the ABCG/PDR family yielded incorrect folds due to using type I exporter templates rather than exporter type II [360–363], the use of proper coordinates have now validated the usefulness of modeling for dissecting mechanisms of ABC transport cycles [40,256]. Of note, we have taken a "reverse" approach here, since we exploited human ABCG2 and ABCG5/G8 structures to model evolutionarily conserved fungal PDR transporters. This not only yielded new testable homology models, but also hinted that catalytic cycles may have been conserved at least in orthologous families such as human ABCG and fungal PDR. Of note, four cryo-EM structures of yeast Pdr5 [339] at atomic resolutions from 2.8 to 3.5 Å just emerged [339]. Most remarkably, as we show here for Cdr1, the paradigm yeast Pdr5 efflux pump shows similar transitional movements during the catalytic cycle [339], strongly supporting the proposed catalytic cycle for PDR/ABCG transporters operating as uncoupled peristaltic pumps [339]. *Twist* and *Squeeze* may be used by different types of transporters, and these driving mechanisms may appear related or even similar when looking at it from the mechanics, as either *Twist* or *Squeeze* or a combination of both can result in peristaltic pressure during the switch. While there are many challenges remaining ahead of us, the reversal of clinical MDR phenomena in fungal pathogens in infectious disease settings have regained attention, especially since the catalytic cycle of human ABCG2 likely reflects the mode of action of fungal PDR transporters implicated in anti-infective drug resistance.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/ijms22094806/s1. Figure S1. Amino acid sequence alignment of fungal ABC transporters (PDR subfamily) and mammalian ABCG subfamily.

**Author Contributions:** N.K. performed all analysis and prepared figures. N.K. and K.K. interpreted the data and wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Austrian Science Fund (FWF-SFB-035-20 to K.K.).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The authors confirm that all data are fully available without restrictions. All relevant data are provided in the manuscript and in the Supplementary Materials.

**Data and Analysis:** Figures and phylogenetic analyses were generated by BioEdit, ClustalX2 and MEGA-X. The homology model was generated using SWISS-MODEL using a Cdr1 sequence from UniProt (accession no. P43071), using the human ABCG2 as a template (PDB ID 6VXF). The molecular visualizations were performed using PyMOL, v1.8.4.

**Acknowledgments:** Open Access Funding by the Austrian Science Fund (FWF). This work was supported by grants from the Austrian Science Fund FWF-SFB-035-20 to K.K. We are very much indebted to Ian D. Kerr, Thomas Stockner and Yuan Eric Lee for constructive and helpful discussions. We also appreciate the constructive and helpful comments from reviewer 2.

**Conflicts of Interest:** The authors have no competing financial interests.
