Flipping and other astonishing transporter dance moves in fungal drug resistance

Abstract

In all domains of life, transmembrane proteins from the ATP‐binding cassette (ABC) transporter family drive the translocation of diverse substances across lipid bilayers. In pathogenic fungi, the ABC transporters of the pleiotropic drug resistance (PDR) subfamily confer antibiotic resistance and so are of interest as therapeutic targets. They also drive the quest for understanding how ABC transporters can generally accommodate such a wide range of substrates. The Pdr5 transporter from baker's yeast is representative of the PDR group and, ever since its discovery more than 30 years ago, has been the subject of extensive functional analyses. A new perspective of these studies has been recently provided in the framework of the first electron cryo‐microscopy structures of Pdr5, as well as emergent applications of machine learning in the field. Taken together, the old and the new developments have been used to propose a mechanism for the transport process in PDR proteins. This mechanism involves a “flippase” step that moves the substrates from one leaflet of the bilayer to the other, as a central element of cellular efflux.

Residues forming the gating regions of asymmetric multidrug transporter Pdr5 also play roles in conformational switching and protein folding

“…This was shown to be directly attributable to drug reflux. In contrast, when we performed a whole-cell transport assay in the direction of the gradient so that R6G was diluted in the extracellular buffer, the mutant phenotype was nearly WT ( 10 ). We tested the F683L mutant in analogous fashion.…”

“…This switch results in a peristaltic motion that squeezes out the bound substrate from its binding site in the IF conformation into the exit channel and releases the substrate at the outer leaflet–extracellular space interface. This suggests that substrates are not released into the aqueous medium but rather repartition into the outer leaflet of the membrane ( 9 , 10 ).…”

Investigations on the substrate binding sites of hemolysin B, an ABC transporter, of a type 1 secretion system

“…Y477 is a residue of the A-loop, which coordinates the adenine ring. Interestingly, Pdr5, the ABC transporter involved in pleiotropic resistance from baker’s yeast, have no aromatic A-loop residue but still able to hydrolyses ATP with high efficiency ( Raschka et al, 2022 ).…”

Catching a fungal multidrug ABC transporter on the flipping act

“…In this issue of BioEssays, the authors subsequently proposed a mechanistic interpretation of these structures, in which the protein translocates its substrates according to a flippase (or perhaps more specifically floppase) mechanism, that is, that the molecules are translocated from the inner leaflet to the outer leaflet of the bilayer. [ 3 ] Although such a mechanism has been described for ABC transporters translocating lipids or lipid‐linked molecules, [ 4 ] it has not been experimentally demonstrated for any MDR transporter, and various other transport models have been derived from the structures of multidrug efflux pumps, such as P‐glycoprotein (ABCB1). [ 5 ] As pointed out by Raschka et al., [ 3 ] demonstrating experimentally the flippase mechanism of Pdr5 will be challenging.…”

“…[ 3 ] Although such a mechanism has been described for ABC transporters translocating lipids or lipid‐linked molecules, [ 4 ] it has not been experimentally demonstrated for any MDR transporter, and various other transport models have been derived from the structures of multidrug efflux pumps, such as P‐glycoprotein (ABCB1). [ 5 ] As pointed out by Raschka et al., [ 3 ] demonstrating experimentally the flippase mechanism of Pdr5 will be challenging. Interestingly, the authors proposed some elegant in vitro fluorescent assays while discussing their current technical limitations and putative experimental weaknesses.…”