Structure and efflux mechanism of the yeast pleiotropic drug resistance transporter Pdr5

Abstract

Pdr5, a member of the extensive ABC transporter superfamily, is representative of a clinically relevant subgroup involved in pleiotropic drug resistance. Pdr5 and its homologues drive drug efflux through uncoupled hydrolysis of nucleotides, enabling organisms such as baker’s yeast and pathogenic fungi to survive in the presence of chemically diverse antifungal agents. Here, we present the molecular structure of Pdr5 solved with single particle cryo-EM, revealing details of an ATP-driven conformational cycle, which mechanically drives drug translocation through an amphipathic channel, and a clamping switch within a conserved linker loop that acts as a nucleotide sensor. One half of the transporter remains nearly invariant throughout the cycle, while its partner undergoes changes that are transmitted across inter-domain interfaces to support a peristaltic motion of the pumped molecule. The efflux model proposed here rationalises the pleiotropic impact of Pdr5 and opens new avenues for the development of effective antifungal compounds.

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“…Recently, the first PDR transporter structures, namely, Pdr5 in the open and closed conformation, were published ( 52 ). The structures are in complete agreement with the models described above.…”

“…An interesting consequence of this novel “asymmetric” motif is that Pdr5 has only one opening, between TMS1 and TMS11, as the second pseudosymmetric entrance cavity between TMS5 and TMS7 ( Fig. 1C ) was found to be closed in both conformations ( 52 ). This finding strengthens the proposed gate-keeper function for Cdr1-G521 at the center of TMS1 ( Fig.…”

“…Although these structures represent a significant milestone in PDR transporter structure and function elucidation, there are valid concerns as to how accurately these structures reflect Pdr5 in native plasma membranes ( 53 ). The detergent-extracted Pdr5 particles that were reconstituted into peptidiscs had an ∼96% lower ATPase activity (73 nmol/min/mg) than Pdr5 (∼2,000 nmol/min/mg) in native plasma membranes ( 46 ) possibly due to some constriction of the protein by the peptidisc surrounding the transporter ( 52 ). Yeast plasma membranes are highly organized structures with a number of distinct subcompartments ( 54 ) of defined protein and lipid composition that provide a flexible environment for conformational changes of membrane proteins ( 55 ).…”

“…PDR transporters are constitutively active asymmetric ABC transporters. Their characteristic LDs connect TMD1 with NBD2 ( 52 ) so that “gate 2” between TMS5 and TMS7 ( Fig. 1C ) is closed and the noncanonical CNBD1 directly underneath binds, but does not hydrolyze, ATP at all times ( 52 ).…”

“…Their characteristic LDs connect TMD1 with NBD2 ( 52 ) so that “gate 2” between TMS5 and TMS7 ( Fig. 1C ) is closed and the noncanonical CNBD1 directly underneath binds, but does not hydrolyze, ATP at all times ( 52 ). The EDs with their characteristically large EL3 and EL6 are tightly linked to the TMDs and the NBDs so that the canonical CNBD2 underneath the entrance cavity between TMS1 and TMS11 cannot be activated via an induced fit-type mechanism which is the hallmark of ABCB type transporters ( 56 ) lacking such large EDs.…”