The model multi-drug efflux pump from Escherichia coli, EmrE, can perform multiple types of transport leading to different biological outcomes, conferring resistance to some drug substrates and enhancing susceptibility to others. While transporters have traditionally been classified as antiporters, symporters, or uniporters, there is growing recognition that some transporters may exhibit mixed modalities. This raises new questions about their regulation and mechanism. Here, we show that the C-terminal tail of EmrE acts as a secondary gate, preventing proton leak in the absence of drug. Substrate binding unlocks this gate, allowing transport to proceed. Truncation of the C-terminal tail (∆107-EmrE) leads to altered pH regulation of alternating access, an important kinetic step in the transport cycle, as measured by NMR. ∆107-EmrE has increased proton leak in proteoliposomes, and bacteria expressing this mutant have reduced growth. Molecular dynamics simulations of ∆107-EmrE show the formation of a water wire from the open face of the transporter to the primary binding site in the core, facilitating proton leak. In WT-EmrE, the C-terminal tail forms specific interactions that block the formation of the water wire. Together, these data strongly support the C-terminus of EmrE acting as a secondary gate that regulates access to the primary binding site.

Multi-drug-resistant bacterial infections pose a major threat to public health. As case numbers continue to rise and new strains are discovered with resistance to existing antibiotics, it is increasingly imperative to develop novel treatments. The recent discovery that the model multi-drug efflux pump from Escherichia coli, EmrE, can perform multiple types of transport suggests this may be a compelling target for therapeutic intervention. Initial studies have identified several small-molecule substrates capable of inducing transporter-dependent susceptibility rather than the well-known antibiotic resistance phenotype. However, many questions regarding the underlying mechanism and regulation of this transporter still remain. Here we use solid-supported membrane electrophysiology to refine our mechanistic understanding of EmrE transport by systematically examining the regulation of transporter activity. Using small molecule substrates with diverse chemical properties in tandem with protein mutagenesis we examine the role of substrate interactions in modulating the net transport through EmrE. Careful manipulation of substrate concentrations and gradient magnitudes allows for examination of net flux under different conditions while repeated washings allow for exchange of internal buffer conditions to enable high-throughput analysis. With a high degree of familial conservation in addition to a suggested role in transporter evolution, mechanistic insight into the transport regulation of EmrE may be broadly applicable across multi-drug resistant efflux pumps. By examining the innate modalities for regulation within the protein and how the molecular features of various substrates interact with these entitles we add to the growing body of knowledge supporting this system as a tractable target for novel antibiotic development.

Small multidrug resistance (SMR) transporters contribute to antibiotic resistance through proton-coupled efflux of toxic compounds. Previous biophysical studies of the E. coli SMR transporter EmrE suggest that it should also be able to perform proton/toxin symport or uniport, leading to toxin susceptibility rather than resistance in vivo. Here we show EmrE does confer susceptibility to several previously uncharacterized small-molecule substrates in E. coli , including harmane. In vitro electrophysiology assays demonstrate that harmane binding triggers uncoupled proton flux through EmrE. Assays in E. coli are consistent with EmrE-mediated dissipation of the transmembrane pH gradient as the mechanism underlying the in vivo phenotype of harmane susceptibility. Furthermore, checkerboard assays show this alternative EmrE transport mode can synergize with some existing antibiotics, such as kanamycin. These results demonstrate that it is possible to not just inhibit multidrug efflux, but to activate alternative transport modes detrimental to bacteria, suggesting a strategy to address antibiotic resistance.

Small multidrug resistance (SMR) transporters perform coupled antiport of protons and toxic substrates, contributing to antibiotic resistance through efflux of these compounds from the bacterial cytoplasm. Extensive biophysical studies of the molecular transport mechanism of the E. coli SMR transporter EmrE indicate that it should also be capable of performing proton/drug symport or uniport, either of which will lead to drug susceptibility rather than drug resistance in vivo. Here we show that EmrE does indeed confer susceptibility to some small molecule substrates in the native E. coli in addition to conferring resistance to known polyaromatic cation substrates. In vitro experiments show that substrate binding at a secondary site triggers uncoupled proton uniport that leads to susceptibility. These results suggest that the SMR transporters provide one avenue for bacterial-selective dissipation of the proton-motive force. This has potential for antibiotic development and disruption of antibiotic resistance due to drug efflux more broadly.

The dimeric transporter, EmrE, effluxes polyaromatic cationic drugs in a proton-coupled manner to confer multidrug resistance in bacteria. Although the protein is known to adopt an antiparallel asymmetric topology, its high-resolution drug-bound structure is so far unknown, limiting our understanding of the molecular basis of promiscuous transport. Here we report an experimental structure of drug-bound EmrE in phospholipid bilayers, determined using 19 F and 1 H solid-state NMR and a fluorinated substrate, tetra(4-fluorophenyl) phosphonium (F 4 -TPP + ). The drug-binding site, constrained by 214 protein-substrate distances, is dominated by aromatic residues such as W63 and Y60, but is sufficiently spacious for the tetrahedral drug to reorient at physiological temperature. F 4 -TPP + lies closer to the proton-binding residue E14 in subunit A than in subunit B, explaining the asymmetric protonation of the protein. The structure gives insight into the molecular mechanism of multidrug recognition by EmrE and establishes the basis for future design of substrate inhibitors to combat antibiotic resistance.

Transport stoichiometry determination can provide great insight into the mechanism and function of ion-coupled transporters. Traditional reversal potential assays are a reliable, general method for determining the transport stoichiometry of ion-coupled transporters, but the time and material costs of this technique hinder investigations of transporter behavior under multiple experimental conditions. Solid-supported membrane electrophysiology (SSME) allows multiple recordings of liposomal or membrane samples adsorbed onto a sensor and is sensitive enough to detect transport currents from moderate-flux transporters that are inaccessible to traditional electrophysiology techniques. Here, we use SSME to develop a new method for measuring transport stoichiometry with greatly improved throughput. Using this technique, we were able to verify the recent report of a fixed 2:1 stoichiometry for the proton:guanidinium antiporter Gdx, reproduce the 1H+:2Cl- antiport stoichiometry of CLC-ec1, and confirm loose proton:nitrate coupling for CLC-ec1. Furthermore, we were able to demonstrate quantitative exchange of internal contents of liposomes adsorbed onto SSME sensors to allow multiple experimental conditions to be tested on a single sample. Our SSME method provides a fast, easy, general method for measuring transport stoichiometry, which will facilitate future mechanistic and functional studies of ion-coupled transporters.

Membrane cotransporters perform essential cellular functions, selectively transporting nutrients as well as maintaining ion concentration gradients. Although careful structural, biochemical, and electrophysiological studies have revealed important elements of transporter mechanisms, numerous aspects remain ambiguous. Generally, the precise order of binding events is unknown, and it remains an open question whether multiple mechanistic pathways are at play. Here we address the identifiability question: Which mechanistic details can be resolved in principle based on both typical and idealized experimental data? Our approach builds on previous work to search and fit models using the entire allowed transporter reaction and state space. We apply our analysis to both synthetic data and solid-supported membrane electrophysiology current traces from a tightly-coupled transporter over a range of conditions.

Transport stoichiometry provides insight into the mechanism and function of ion-coupled transporters, but measuring transport stoichiometry is time-consuming and technically difficult. With the increasing evidence that many ion-coupled transporters employ multiple transport stoichiometries under different conditions, improved methods to determine transport stoichiometry are required to accurately characterize transporter activity. Reversal potential was previously shown to be a reliable, general method for determining the transport stoichiometry of ion-coupled transporters (Fitzgerald & Mindell, 2017). Here, we develop a new technique for measuring transport stoichiometry with greatly improved throughput using solid supported membrane electrophysiology (SSME). Using this technique, we are able to verify the recent report of a fixed 2:1 stoichiometry for the proton:guanidinium antiporter Gdx. Our SSME method requires only small amounts of transporter and provides a fast, easy, general method for measuring transport stoichiometry, which will facilitate future mechanistic and functional studies of ion-coupled transporters.

The Mesh1 class of hydrolases found in bacteria, metazoans and humans was discovered as able to cleave an intact pyrophosphate residue esterified on the 3'hydroxyl of (p)ppGpp in a Mn2+ dependent reaction. Here, thin layer chromatography (TLC) qualitative evidence is presented indicating the substrate specificity of Mesh1 from Drosophila melanogaster and human MESH1 also extends to the (p)ppApp purine analogs. More importantly, we developed real time enzymatic assays, coupling ppNpp hydrolysis to NADH oxidation and pppNpp hydrolysis to NADP+ reduction, which facilitate estimation of kinetic constants. Furthermore, by using this assay technique we confirmed TLC observations and also revealed that purified small alarmone hydrolase (SAHMex) from Methylobacterium extorquens displays a strong hydrolase activity toward (p)ppApp but only negligible activity toward (p)ppGpp. In contrast, the substrate specificity of the hydrolase present in catalytically active N-terminal domain of the RSH protein from Streptococcus equisimilis (RelSeq) includes (p)ppGpp but not (p)ppApp. It is noteworthy that the RSH protein from M. extorquens (RSHMex) has been recently shown to synthesize both (p)ppApp and (p)ppGpp.

Ion-coupled transporters are involved in numerous physiological processes, including nutrient acquisition, ion homeostasis, and toxin excretion, but the mechanistic details of how the energy stored in ion gradients is translated to uphill substrate transport are still poorly understood. This puzzle is especially interesting in the case of multidrug transporters, which must be able to couple ion flux to the transport of chemically diverse substrates. Using nuclear magnetic resonance (NMR) spectroscopy, our lab discovered that the proton-coupled multidrug transporter EmrE from E. coli must be capable of coupling drug and proton with multiple different stoichiometries. Here, we use mass action kinetic simulations and solid supported membrane (SSM) electrophysiology to rethink the bounds of ion-coupled transport. These results suggest the intriguing possibility that it may be possible to reverse the direction of driven transport, hijacking promiscuous transporters for targeted drug delivery. Our work has broad implications for the mechanism, evolution, and design of ion-coupled transporters.

Proteins that perform active transport must alternate the access of a binding site, first to one side of a membrane and then to the other. This conformational change is the key step in moving bound substrates across the membrane from one aqueous compartment to another. We have discovered both EmrE point mutants and EmrE substrates that significantly change the rate of this alternating access process. Charge and hydrophobicity are common features influencing the rate of alternating access and overall transport in both the mutants and the substrates. Kinetic modeling suggests that shifting the rate of alternating access relative to substrate binding and release can influence both net flux and the efficiency of proton coupled transport in this promiscuous multidrug transporter. Together this data provides insight into the molecular details that regulate the rate of conformational transitions within an integral membrane protein, and how changing that rate impacts biological function.

EmrE is a multidrug resistance transport protein from E. coli that couples the efflux of toxic, lipophilic cations to the influx of protons down their electrochemical gradient. Active transporters require alternating access between open‐in (binding pocket accessible to the cytoplasm) and open‐out (binding pocket accessible to the periplasm) conformations to move substrate across the membrane. Strict restrictions on alternating access or substrate binding lead to simplified models that achieve stoichiometric symport (coupling movement of both substrates in same direction) or “pure‐exchange” antiport (coupling movement of both substrates in opposite directions). However, recent data suggests that common strictly‐coupled models of transport may be oversimplified. For example, the EmrE point mutant, W63G, confers resistance to erythromycin, indicating H+‐coupled transport of that substrate, while also conferring susceptibility to polyamines, indicating uniport or H+‐coupled symport of those substrates. We recently developed a “free‐exchange” model of EmrE transport that can account for this behavior as well as NMR data showing that EmrE can simultaneously bind drug and proton and can alternate access when bound to both, one, or no substrates in violation of pure‐exchange models. Here, we develop a free‐exchange mathematical model to explore features that tip the balance between different H+‐coupled transport regimes. We find that under physiological conditions, transport is faster in the free‐exchange model and comparable substrate gradients can be produced. Using the available kinetic data for EmrE and our mathematical model, we show that EmrE can concentrate drug as experimentally observed in liposomal‐flux assays and also show how drug identity can switch directionality of transport, as observed for W63G‐EmrE.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Secondary active transporters couple the transport of an ion species down its concentration gradient to the uphill transport of another substrate. Despite the importance of secondary active transport to multidrug resistance, metabolite transport, and nutrient acquisition, among other biological processes, the microscopic steps of the coupling mechanism are not well understood. Often, transport models illustrate coupling mechanisms through a limited number of "major" conformations or states, yet recent studies have indicated that at least some transporters violate these models. The small multidrug resistance transporter EmrE has been shown to couple proton influx to multidrug efflux via a mechanism that incorporates both "major" and "minor" conformational states and transitions. The resulting free exchange transport model includes multiple leak pathways and theoretically allows for both exchange and cotransport of ion and substrate. To better understand how coupled transport can be achieved in such a model, we numerically simulate a free-exchange model of transport to determine the step-by-step requirements for coupled transport. We find that only moderate biasing of rate constants for key transitions produce highly efficient net transport approaching a perfectly coupled, stoichiometric model. We show how a free-exchange model can enable complex phenotypes, including switching transport direction with changing environmental conditions or substrates. This research has broad implications for synthetic biology, as it demonstrates the utility of free-exchange transport models and the fine tuning required for perfectly coupled transport.

Proteins that perform active transport must alternate the access of a binding site, first to one side of a membrane and then to the other, resulting in the transport of bound substrates across the membrane. To better understand this process, we sought to identify mutants of the small multidrug resistance transporter EmrE with reduced rates of alternating access. We performed extensive scanning mutagenesis by changing every amino acid residue to Val, Ala, or Gly, and then screening the drug resistance phenotypes of the resulting mutants. We identified EmrE mutants that had impaired transport activity but retained the ability to bind substrate and further tested their alternating access rates using NMR. Ultimately, we were able to identify a single mutation, S64V, which significantly reduced the rate of alternating access but did not impair substrate binding. Six other transport-impaired mutants did not have reduced alternating access rates, highlighting the importance of other aspects of the transport cycle to achieve drug resistance activity in vivo. To better understand the transport cycle of EmrE, efforts are now underway to determine a high-resolution structure using the S64V mutant identified here.

Ion-coupled transporters must regulate access of ions and substrates into and out of the binding site to actively transport substrates and minimize dissipative leak of ions. Within the single-site alternating access model, competitive substrate binding forms the foundation of ion-coupled antiport. Strict competition between substrates leads to stoichiometric antiport without slippage. However, recent NMR studies of the bacterial multidrug transporter EmrE have demonstrated that this multidrug transporter can simultaneously bind drug and proton, which will affect the transport stoichiometry and efficiency of coupled antiport. Here, we investigated the nature of substrate competition in EmrE using multiple methods to measure proton release upon the addition of saturating concentrations of drug as a function of pH. The resulting proton-release profile confirmed simultaneous binding of drug and proton, but suggested that a residue outside EmrE's Glu-14 binding site may release protons upon drug binding. Using NMR-monitored pH titrations, we trace this drug-induced deprotonation event to His-110, EmrE's C-terminal residue. Further NMR experiments disclosed that the C-terminal tail is strongly coupled to EmrE's drug-binding domain. Consideration of our results alongside those from previous studies of EmrE suggests that this conserved tail participates in secondary gating of EmrE-mediated proton/drug transport, occluding the binding pocket of fully protonated EmrE in the absence of drug to prevent dissipative proton transport.

EmrE is a small multidrug resistance transporter found in Escherichia coli that confers resistance to toxic polyaromatic cations due to its proton-coupled antiport of these substrates. Here we show that EmrE breaks the rules generally deemed essential for coupled antiport. NMR spectra reveal that EmrE can simultaneously bind and cotransport proton and drug. The functional consequence of this finding is an exceptionally promiscuous transporter: not only can EmrE export diverse drug substrates, it can couple antiport of a drug to either one or two protons, performing both electrogenic and electroneutral transport of a single substrate. We present a free-exchange model for EmrE antiport that is consistent with these results and recapitulates ∆pH-driven concentrative drug uptake. Kinetic modeling suggests that free exchange by EmrE sacrifices coupling efficiency but boosts initial transport speed and drug release rate, which may facilitate efficient multidrug efflux.