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

ATP-binding cassette (ABC) multidrug transporters are large, polytopic membrane proteins that exhibit astonishing promiscuity for their transport substrates. These transporters unidirectionally efflux thousands of structurally and functionally distinct compounds. To preclude the reentry of xenobiotic molecules via the drug-binding pocket, these proteins contain a highly conserved molecular gate, essentially allowing the transporters to function as molecular diodes. However, the structure-function relationship of these conserved gates and gating regions are not well characterized. In this study, we combine recent single-molecule, cryo-EM data with genetic and biochemical analyses of residues in the gating region of the yeast multidrug transporter Pdr5, the founding member of a large group of clinically relevant asymmetric ABC efflux pumps. Unlike the symmetric ABCG2 efflux gate, the Pdr5 counterpart is highly asymmetric, with only four (instead of six) residues comprising the gate proper. However, other residues in the near vicinity are essential for the gating activity. Furthermore, we demonstrate that residues in the gate and in the gating regions have multiple functions. For example, we show that Ile-685 and Val-1372 are required not only for successful efflux but also for allosteric inhibition of Pdr5 ATPase activity. Our investigations reveal that the gating region residues of Pdr5, and possibly other ABCG transporters, play a role not only in molecular gating but also in allosteric regulation, conformational switching, and protein folding.

An A666G mutation in transmembrane helix 5 of the yeast multidrug transporter Pdr5 increases drug efflux by enhancing cooperativity between transport sites.

Resistance to antimicrobial and chemotherapeutic agents is a significant clinical problem. Overexpression of multidrug efflux pumps often creates broad-spectrum resistance in cancers and pathogens. We describe a mutation, A666G, in the yeast ABC transporter Pdr5 that shows greater resistance to most of the tested compounds than does an isogenic wild-type strain. This mutant exhibited enhanced resistance without increasing either the amount of protein in the plasma membrane or the ATPase activity. In fluorescence quenching transport assays with rhodamine 6G in purified plasma membrane vesicles, the initial rates of rhodamine 6G fluorescence quenching of both the wild type and mutant showed a strong dependence on the ATP concentration, but were about twice as high in the latter. Plots of the initial rate of fluorescence quenching versus ATP concentration exhibited strong cooperativity that was further enhanced in the A666G mutant. Resistance to imazalil sulfate was about 3-4x as great in the A666G mutant strain as in the wild type. When this transport substrate was used to inhibit the rhodamine 6G transport, the A666G mutant inhibition curves also showed greater cooperativity than the wild-type strain. Our results suggest a novel and important mechanism: under selection, Pdr5 mutants can increase drug resistance by improving cooperative interactions between drug transport sites.

The deviant ATP-binding site of the multidrug efflux pump Pdr5 plays an active role in the transport cycle.

Pdr5 is the founding member of a large subfamily of evolutionarily distinct, clinically important fungal ABC transporters containing a characteristic, deviant ATP-binding site with altered Walker A, Walker B, Signature (C-loop), and Q-loop residues. In contrast to these motifs, the D-loops of the two ATP-binding sites have similar sequences, including a completely conserved aspartate residue. Alanine substitution mutants in the deviant Walker A and Signature motifs retain significant, albeit reduced, ATPase activity and drug resistance. The D-loop residue mutants D340A and D1042A showed a striking reduction in plasma membrane transporter levels. The D1042N mutation localized properly had nearly WT ATPase activity but was defective in transport and was profoundly hypersensitive to Pdr5 substrates. Therefore, there was a strong uncoupling of ATPase activity and drug efflux. Taken together, the properties of the mutants suggest an additional, critical intradomain signaling role for deviant ATP-binding sites.