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.

A New Twist in ABC Transporter Mediated Multidrug Resistance - Pdr5 is a Drug/proton Co-transporter.

The two major efflux pump systems that are involved in multidrug resistance (MDR) are (i) ATP binding cassette (ABC) transporters and (ii) secondary transporters. While the former use binding and hydrolysis of ATP to facilitate export of cytotoxic compounds, the latter utilize electrochemical gradients to expel their substrates. Pdr5 from Saccharomyces cerevisiae is a prominent member of eukaryotic ATP binding cassette (ABC) transporters that are involved in multidrug resistance (MDR) and used as a frequently studied model system. Although investigated for decades, the underlying molecular mechanisms of drug transport and substrate specificity remain elusive. Here, we provide electrophysiological data on the reconstituted Pdr5 demonstrating that this MDR efflux pump does not only actively translocate its substrates across the lipid bilayer, but at the same time generates a proton motif force in the presence of Mg2+-ATP and substrates by acting as a proton/drug co-transporter. Importantly, a strictly substrate dependent co-transport of protons was also observed in in vitro transport studies using Pdr5-enriched plasma membranes. We conclude from these results that the mechanism of MDR conferred by Pdr5 and likely other transporters is more complex than the sole extrusion of cytotoxic compounds and involves secondary coupled processes suitable to increase the effectiveness.

Flipping and other astonishing transporter dance moves in fungal drug resistance.

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.

A Model for Allosteric Communication in Drug Transport by the AcrAB-TolC Tripartite Efflux Pump.

RND family efflux pumps are complex macromolecular machines involved in multidrug resistance by extruding antibiotics from the cell. While structural studies and molecular dynamics simulations have provided insights into the architecture and conformational states of the pumps, the path followed by conformational changes from the inner membrane protein (IMP) to the periplasmic membrane fusion protein (MFP) and to the outer membrane protein (OMP) in tripartite efflux assemblies is not fully understood. Here, we investigated AcrAB-TolC efflux pump's allostery by comparing resting and transport states using difference distance matrices supplemented with evolutionary couplings data and buried surface area measurements. Our analysis indicated that substrate binding by the IMP triggers quaternary level conformational changes in the MFP, which induce OMP to switch from the closed state to the open state, accompanied by a considerable increase in the interface area between the MFP subunits and between the OMPs and MFPs. This suggests that the pump's transport-ready state is at a more favourable energy level than the resting state, but raises the puzzle of how the pump does not become stably trapped in a transport-intermediate state. We propose a model for pump allostery that includes a downhill energetic transition process from a proposed 'activated' transport state back to the resting pump.

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

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.

Interactions of a Bacterial RND Transporter with a Transmembrane Small Protein in a Lipid Environment.

The small protein AcrZ in Escherichia coli interacts with the transmembrane portion of the multidrug efflux pump AcrB and increases resistance of the bacterium to a subset of the antibiotic substrates of that transporter. It is not clear how the physical association of the two proteins selectively changes activity of the pump for defined substrates. Here, we report cryo-EM structures of AcrB and the AcrBZ complex in lipid environments, and comparisons suggest that conformational changes occur in the drug-binding pocket as a result of AcrZ binding. Simulations indicate that cardiolipin preferentially interacts with the AcrBZ complex, due to increased contact surface, and we observe that chloramphenicol sensitivity of bacteria lacking AcrZ is exacerbated when combined with cardiolipin deficiency. Taken together, the data suggest that AcrZ and lipid cooperate to allosterically modulate AcrB activity. This mode of regulation by a small protein and lipid may occur for other membrane proteins.

Cryo-EM structure of the MinCD copolymeric filament from Pseudomonas aeruginosa at 3.1 Å resolution.

Positioning of the division site in many bacterial species relies on the MinCDE system, which prevents the cytokinetic Z-ring from assembling anywhere but the mid-cell, through an oscillatory diffusion-reaction mechanism. MinD dimers bind to membranes and, via their partner MinC, inhibit the polymerization of cell division protein FtsZ into the Z-ring. MinC and MinD form polymeric assemblies in solution and on cell membranes. Here, we report the high-resolution cryo-EM structure of the copolymeric filaments of Pseudomonas aeruginosa MinCD. The filaments consist of three protofilaments made of alternating MinC and MinD dimers. The MinCD protofilaments are almost completely straight and assemble as single protofilaments on lipid membranes, which we also visualized by cryo-EM.

Cryo-EM reconstruction of AlfA from Bacillus subtilis reveals the structure of a simplified actin-like filament at 3.4-Å resolution.

Low copy-number plasmid pLS32 of Bacillus subtilis subsp. natto contains a partitioning system that ensures segregation of plasmid copies during cell division. The partitioning locus comprises actin-like protein AlfA, adaptor protein AlfB, and the centromeric sequence parN Similar to the ParMRC partitioning system from Escherichia coli plasmid R1, AlfA filaments form actin-like double helical filaments that arrange into an antiparallel bipolar spindle, which attaches its growing ends to sister plasmids through interactions with AlfB and parN Because, compared with ParM and other actin-like proteins, AlfA is highly diverged in sequence, we determined the atomic structure of nonbundling AlfA filaments to 3.4-Å resolution by cryo-EM. The structure reveals how the deletion of subdomain IIB of the canonical actin fold has been accommodated by unique longitudinal and lateral contacts, while still enabling formation of left-handed, double helical, polar and staggered filaments that are architecturally similar to ParM. Through cryo-EM reconstruction of bundling AlfA filaments, we obtained a pseudoatomic model of AlfA doublets: the assembly of two filaments. The filaments are antiparallel, as required by the segregation mechanism, and exactly antiphasic with near eightfold helical symmetry, to enable efficient doublet formation. The structure of AlfA filaments and doublets shows, in atomic detail, how deletion of an entire domain of the actin fold is compensated by changes to all interfaces so that the required properties of polymerization, nucleotide hydrolysis, and antiparallel doublet formation are retained to fulfill the system's biological raison d'être.

SnapShot: The Bacterial Cytoskeleton.

Most bacteria and archaea contain filamentous proteins and filament systems that are collectively known as the bacterial cytoskeleton, though not all of them are cytoskeletal, affect cell shape, or maintain intracellular organization. To view this SnapShot, open or download the PDF.