Hoechst 33342 Is a Hidden “Janus” amongst Substrates for the Multidrug Efflux Pump LmrP

RESEARCH ARTICLE Hoechst 33342 Is a Hidden “Janus” amongst Substrates for the Multidrug Efflux Pump LmrP Arthur Neuberger, Hendrik W. van Veen* Department of Pharmacology, University of Cambridge, Cambridge, United Kingdom * Abstract OPEN ACCESS Citation: Neuberger A, van Veen HW (2015) Hoechst 33342 Is a Hidden “Janus” amongst Substrates for the Multidrug Efflux Pump LmrP. PLoS ONE 10(11): e0141991. doi:10.1371/journal. pone.0141991 Editor: Gergely Szakacs, Hungarian Academy of Sciences, HUNGARY Received: June 25, 2015 Accepted: October 15, 2015 Published: November 5, 2015 Copyright: © 2015 Neuberger, van Veen. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 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 inwardfacing 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. Data Availability Statement: All relevant data are within the paper. Funding: AN is a recipient of a Herchel-Smith Scholarship. Research in the Van Veen group is also supported by the Biotechnology and Biological Sciences Research Council (BBSRC), the Medical Research Council (MRC), the Human Frontier Science Program (HFSP), the British Society for Antimicrobial Chemotherapy (BSAC), and the Commonwealth Trust. Competing Interests: The authors have declared that no competing interests exist. Introduction Drug transporters play a vital role in the intrinsic and acquired resistance of microorganisms against antibiotics and cytotoxic agents [1–3]. Some of these drug transporters are rather specific for a drug or class of drugs, whereas others recognize a wide range of structurally diverse substrates. Bacterial multidrug transporters fall into 5 protein families of which the Major Facilitator Superfamily (MFS) is the largest [4]. The 408-amino-acid MFS member LmrP from Lactococcus lactis functions as a drug-proton antiporter that utilizes both the membrane PLOS ONE | DOI:10.1371/journal.pone.0141991 November 5, 2015 1 / 11 Active Hoechst 33342 Uptake by LmrP potential (interior negative) and the chemical proton gradient (interior alkaline) of the protonmotive force to mediate the efflux of amphiphilic substrates from the cell [5–8]. Evidence for the proton-motive force-dependent transport of drugs, independent of any other accessory proteins, was provided in studies in proteoliposomes containing purified and functionally reconstituted protein in which ion gradients were imposed artificially [9,10]. One typical LmrP substrate is the fluorescent dye Hoechst 33342. In aqueous solution, Hoechst 33342 is essentially non-fluorescent, whereas its partitioning in the membrane and binding to DNA are associated with a remarkable enhancement of the fluorescence intensity [11]. LmrP-mediated Hoechst 33342 transport has mostly been studied in inside-out membrane vesicles in which transport from the membrane to the aqueous buffer is associated with a decrease in fluorescence [12]. The transport reaction in membrane vesicles is inhibited by other LmrP substrates: competitively in the presence of quinine and verapamil, non-competitively by nicardipin and vinblastin, and un-competitively by tetraphenylphosphonium ions [12]. This finding suggested that LmrP has multiple drug binding sites, which would explain its ability to transport drugs belonging to different classes of antibiotics including lincosamides, macrolides, streptogramins, and tetracyclines [13]. LmrP is predicted to contain a large internal substrate-binding cavity that contains catalytic carboxylates on its surface: D235 and E327 are located in the apex, and D142 is located at a side [6]. All three carboxylates can function as proton binding sites, whereas D235 and E327 have also been implicated in the binding of the divalent cationic substrates Ca2+ and propidium [7,9]. Residues Asp 128 and Asp 68, located in cytosolic loops outside the internal cavity were also shown to be involved in the proton motive force-mediated change of LmrP’s accessibility for substrates [14]. Using cysteine cross-linking techniques with a cysteine-less LmrP mutant containing I34C in the N-terminal half and V240C in the C-terminal half, evidence was obtained that the protein can switch between at least two major conformational states that are interconverted by the movement of both halves of the transporter, i.e., the inward-facing conformation in which I34C and V240C are in close proximity and can interact with each other, and the outward-facing conformation in which these cysteines are too far apart for cross-linking [8]. Using this method, it was found that the binding of the transport substrate benzalkonium to LmrP stabilizes the protein in the inward-facing conformation in which the binding cavity is opened to the inside of the cell. However, more recently, measurements of intramolecular distances in LmrP by electron paramagnetic resonance (EPR) techniques suggested that the binding of Hoechst 33342 to LmrP stabilizes the protein in the outward-facing conformation, and questioned the relevance of an inward-facing conformation for drug binding in the transport cycle of LmrP [15]. The paradigm in these and related studies has always been that fluorescent dyes such as ethidium, propidium and Hoechst 33342 are expelled from the cell by LmrP by active efflux. Here, w (...truncated)