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TRPV6 is a calcium-selective ion channel implicated in epithelial Ca2+ uptake. TRPV6 inhibitors are needed for the treatment of a broad range of diseases associated with disturbed calcium homeostasis, including cancers. Here we combine cryo-EM, calcium imaging, and mutagenesis to explore molecular bases of human TRPV6 inhibition by the antifungal drug econazole and the universal ion channel blocker ruthenium red (RR). Econazole binds to an allosteric site at the channel’s periphery, where it replaces a lipid. In contrast, RR inhibits TRPV6 by binding in the middle of the ion channel’s selectivity filter and plugging its pore like a bottle cork. Despite different binding site locations, both inhibitors induce similar conformational changes in the channel resulting in closure of the gate formed by S6 helices bundle crossing. The uncovered molecular mechanisms of TRPV6 inhibition can guide the design of a new generation of clinically useful inhibitors., TRPV6 is a calcium-selective ion channel that is involved in numerous calcium-dependent physiological processes and it is of interest as a potential drug target. Here, the authors present the cryo-EM structures of human TRPV6 with the bound inhibitors ruthenium red and the antifungal drug econazole and discuss their inhibition mechanisms.

Infections arising from multidrug-resistant pathogenic bacteria are spreading rapidly throughout the world and threaten to become untreatable. The origins of resistance are numerous and complex, but one underlying factor is the capacity of bacteria to rapidly export drugs through the intrinsic activity of efflux pumps. In this work, a summary is provided of our current understanding of the structures and molecular mechanisms of multidrug efflux pumps in bacteria (Chapter 1). The emerging picture of the structure, function and regulation of efflux pumps suggests opportunities for countering their activities. Although this thesis primarily explores structure and function, it also elucidates the hidden regulatory mechanism (post-translational) behind the association of a small protein called AcrZ with the tripartite complex AcrAB/TolC, in connection with the lipid environment, and the resulting changes in the latter’s functionality (Chapter 2). A regulatory role of the native membrane lipid environment as well as of small proteins for efflux pump activity have previously been hypothesised. I present the first example of a function-regulating role of the lipid cardiolipin in combination with a small protein binding partner (AcrZ) for the substrate selectivity and transport activity of an efflux pump protein (AcrB). This regulation happens through induced structural changes which have remained unseen so far. Alongside with these results, a nanodisc reconstitution method was experimentally adapted for a structure-function investigation of an efflux pump (complex) using cryo-EM (Chapter 2). Beyond some fundamental regulatory insights, hidden intrinsic transport mechanisms for some transporters have also remained to be explored and studied. The discovery of a mechanism for active influx by a prominent efflux pump model system (Chapter 3) provides hope that this phenomenon is more common amongst multidrug transporters and that it could be utilised for drug discovery purposes. This novel feature explains the contradictory findings on this transporter in the past and raises new questions about the little-known physiological role and evolution of efflux pumps. The development and evolution of antimicrobial resistance has frequently shown to be a multifactorial and fast-moving process. One of these factors is the evolution of pumps itself towards an altered functionality (e.g. towards a broader or altered substrate spectrum or higher efflux rates). Against this background, the role of key carboxylate residues for efflux-energising proton trafficking was investigated for a prominent study model of a secondary-active transporter (Chapter 4). The re-allocation and/or addition of acidic residues was demonstrated to result in the preservation of wild type activity or the generation of hyper-efflux activity, respectively. These findings suggest that rapid emergence of antimicrobial resistance could be enhanced by the ‘plasticity’ in the location of key carboxylate residues with a role in proton coupling. It also demonstrates the necessity of antimicrobial drug design programmes to anticipate possible trajectories of an adaptive evolution of efflux pump. The ‘cryo-EM revolution’ has boosted the pace at which new structural and functional insights into multidrug efflux pumps are gained. Nevertheless, in order to derive the structure of individual pump components or of a full assembly, it is sometimes necessary to identify and characterise homologues and mutants, which would allow the application of cryo-EM for obtaining near-atomic maps. Functional analyses presented in this work helped to characterise a homologue and mutants of the MacAB/TolC tripartite complex to justify the obtained protein structures and strategies for further functional characterisation (Chapter 5). Given (1) the unusual stoichiometry of a MacB dimer in complex with a hexameric membrane-fusion protein (MacA), which leads to a seeming leakiness of the assembly, and (2) the fact that substrate has to pass through a narrow aperture in the membrane-fusion protein for extrusion, it is rather surprising that MacB was previously shown to transport an entire toxin. An experimental approach was developed that could enable the structure determination of a toxin-bound full assembly of MacAB/TolC (Chapter 5). Finally, the role of multidrug efflux pumps for the evolution of multidrug resistance is yet to be studied and better explored. For instance, evolutionary trajectories of pump overexpression, as compared to those of regular expression or no expression, are unknown yet could have the potential to reveal useful insights for spread prevention and drug design. The outline of an experimental design with some preliminary validating data is presented in Chapter 6., Herchel Smith Research Studentship

The MacA–MacB–TolC tripartite complex is a transmembrane machine that spans both plasma membrane and outer membrane and actively extrudes substrates, including macrolide antibiotics, virulence factors, peptides and cell envelope precursors. These transport activities are driven by the ATPase MacB, a member of the ATP-binding cassette (ABC) superfamily. Here, we present the crystal structure of MacB at 3.4-Å resolution. MacB forms a dimer in which each protomer contains a nucleotide-binding domain and four transmembrane helices that protrude in the periplasm into a binding domain for interaction with the membrane fusion protein MacA. MacB represents an ABC transporter in pathogenic microorganisms with unique structural features., The tripartite multidrug efflux pump MacA-MacB-TolC in Gram-negative bacterial pathogens is driven by the ATPase MacB, which belongs to the ATP-binding cassette (ABC) superfamily. Here the authors present the 3.4 Å resolution crystal structure of MacB, and compare it with other known ABC transporter structures.

The first data on repurposing provides support for recent initiatives seeking new uses for failed drugs, but those efforts should not be at the expense of continued investment in basic research.

Efflux pumps are membrane proteins which contribute to multi-drug resistance . In Gram-negative bacteria , some of these pumps form complex tripartite assemblies in association with an outer membrane channel and a periplasmic membrane fusion protein. These tripartite machineries span both membranes and the periplasmic space , and they extrude from the bacterium chemically diverse toxic substrates. In this chapter, we summarise current understanding of the structural architecture, functionality, and regulation of tripartite multi-drug efflux assemblies.

Thousands of biotech companies are developing promising products, but have insufficient resources to complete the clinical testing process, while large, well-funded companies have increasingly focused on the need to access external innovation. As a result, licensing deals are an essential and growing part of this industry. Yet, casting a shadow over the licensing market is the classic Lemons Problem: Does asymmetrical information put licensees at a severe disadvantage, leading to a market dominated by inferior opportunities, with the best products retained for internal development? Our analysis of clinical stage products developed over three decades shows that there is no Lemons Problem. We discuss the results of this first apples-to-apples analysis of the biomedical licensing market, and suggest reasons why the Lemons Problem does not exist where it might be most expected – in a high technology, knowledge-based industry.

The MacA-MacB-TolC assembly of Escherichia coli is a transmembrane machine that spans the cell envelope and actively extrudes substrates, including macrolide antibiotics and polypeptide virulence factors. These transport processes are energized by the ATPase MacB, a member of the ATP-binding cassette (ABC) superfamily. We present an electron cryo-microscopy structure of the ABC-type tripartite assembly at near-atomic resolution. A hexamer of the periplasmic protein MacA bridges between a TolC trimer in the outer membrane and a MacB dimer in the inner membrane, generating a quaternary structure with a central channel for substrate translocation. A gating ring found in MacA is proposed to act as a one-way valve in substrate transport. The MacB structure features an atypical transmembrane domain with a closely packed dimer interface and a periplasmic opening that is the likely portal for substrate entry from the periplasm, with subsequent displacement through an allosteric transport mechanism.

The expression of polyspecific membrane transporters is one important mechanism by which cells can obtain resistance to structurally different antibiotics and cytotoxic agents. These transporters reduce intracellular drug concentrations to subtoxic levels by mediating drug efflux across the cell envelope. The major facilitator superfamily multidrug transporter LmrP from $\textit{Lactococcus}$ lactis catalyses drug efflux in a membrane potential and chemical proton gradient-dependent fashion. To enable the interaction with protons and cationic substrates, LmrP contains catalytic carboxyl residues on the surface of a large interior chamber that is formed by transmembrane helices. These residues co-localise together with polar and aromatic residues, and are predicted to be present in two clusters. To investigate the functional role of the catalytic carboxylates, we generated mutant proteins catalysing membrane potential-independent dye efflux by removing one of the carboxyl residues in Cluster 1. We then relocated this carboxyl residue to six positions on the surface of the interior chamber, and tested for restoration of wildtype energetics. The reinsertion at positions towards Cluster 2 reinstated the membrane potential dependence of dye efflux. Our data uncover a remarkable plasticity in proton interactions in LmrP, which is a consequence of the flexibility in the location of key residues that are responsible for proton/multidrug antiport.

Multidrug transporters mediate the active extrusion of antibiotics and toxic ions from the cell. This reaction is thought to be based on a switch of the transporter between two conformational states, one in which the interior substrate binding cavity is available for substrate binding at the inside of the cell, and another in which the cavity is exposed to the outside of the cell to enable substrate release. Consistent with this model, cysteine cross-linking studies with the Major Facilitator Superfamily drug/proton antiporter LmrP from Lactococcus lactis demonstrated binding of transported benzalkonium to LmrP in its inward-facing state. The fluorescent dye Hoechst 33342 is a substrate for many multidrug transporters and is extruded by efflux pumps in microbial and mammalian cells. Surprisingly, and in contrast to other multidrug transporters, LmrP was found to actively accumulate, rather than extrude, Hoechst 33342 in lactococcal cells. Consistent with this observation, LmrP expression was associated with cellular sensitivity, rather than resistance to Hoechst 33342. Thus, we discovered a hidden "Janus" amongst LmrP substrates that is translocated in reverse direction across the membrane by binding to outward-facing LmrP followed by release from inward-facing LmrP. These findings are in agreement with distance measurements by electron paramagnetic resonance in which Hoechst 33342 binding was found to stabilize LmrP in its outward-facing conformation. Our data have important implications for the use of multidrug exporters in selective targeting of "Hoechst 33342-like" drugs to cells and tissues in which these transporters are expressed.