scholarly journals A kinetically-driven free exchange mechanism of EmrE antiport sacrifices coupling efficiency in favor of promiscuity

2017 ◽  
Author(s):  
Anne E. Robinson ◽  
Nathan E. Thomas ◽  
Emma A. Morrison ◽  
Bryan Balthazor ◽  
Katherine A. Henzler-Wildman
Keyword(s):  
Multidrug Resistance ◽  
Coupling Efficiency ◽  
Binding Pocket ◽  
Exchange Model ◽  
Drug Uptake ◽  
Drug Efflux ◽  
E Coli ◽  
Small Multidrug Resistance ◽  
Pure Exchange ◽  
Multidrug Resistance Transporter

ABSTRACTEmrE is a small multidrug resistance transporter found in E. 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 new kinetically-driven free exchange model for EmrE antiport that is consistent with these results and recapitulates ΔpH-driven concentrative drug uptake. Our results suggest that EmrE sacrifices coupling efficiency for initial transport speed and multidrug specificity.SIGNIFICANCEEmrE facilitates E. coli multidrug resistance by coupling drug efflux to proton import. This antiport mechanism has been thought to occur via a pure exchange model which achieves coupled antiport by restricting when the single binding pocket can alternate access between opposite sides of the membrane. We test this model using NMR titrations and transport assays and find it cannot account for EmrE antiport activity. We propose a new kinetically-driven free exchange model of antiport with fewer restrictions that better accounts for the highly promiscuous nature of EmrE drug efflux. This model expands our understanding of coupled antiport and has implications for transporter design and drug development.

scholarly journals New free-exchange model of EmrE transport

2017 ◽  
Vol 114 (47) ◽  
pp. E10083-E10091 ◽  
Author(s):  
Anne E. Robinson ◽  
Nathan E. Thomas ◽  
Emma A. Morrison ◽  
Bryan M. Balthazor ◽  
Katherine A. Henzler-Wildman
Keyword(s):  
Nmr Spectra ◽  
Coupling Efficiency ◽  
Exchange Model ◽  
Drug Uptake ◽  
Multidrug Efflux ◽  
Drug Release Rate ◽  
Uptake Kinetic ◽  
Functional Consequence ◽  
Small Multidrug Resistance ◽  
Multidrug Resistance Transporter

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.

scholarly journals Crystal structures of bacterial Small Multidrug Resistance transporter EmrE in complex with structurally diverse substrates

2022 ◽  
Author(s):  
Ali A Kermani ◽  
Olive E. Burata ◽  
B Ben Koff ◽  
Akiko Koide ◽  
Shohei Koide ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Binding Site ◽  
Quaternary Ammonium ◽  
Structural Information ◽  
Structural Description ◽  
Small Multidrug Resistance ◽  
Novel Approach ◽  
Backbone Structure ◽  
Multidrug Resistance Transporter ◽  
Ammonium Compounds

Proteins from the bacterial small multidrug resistance (SMR) family are proton-coupled exporters of diverse antiseptics and antimicrobials, including polyaromatic cations and quaternary ammonium compounds. The transport mechanism of the Escherichia coli transporter, EmrE, has been studied extensively, but a lack of high-resolution structural information has impeded a structural description of its molecular mechanism. Here we apply a novel approach, multipurpose crystallization chaperones, to solve several structures of EmrE, including a 2.9 Å structure at low pH without substrate. We report five additional structures in complex with structurally diverse transported substrates, including quaternary phosphonium, quaternary ammonium, and planar polyaromatic compounds. These structures show that binding site tryptophan and glutamate residues adopt different rotamers to conform to disparate structures without requiring major rearrangements of the backbone structure. Structural and functional comparison to Gdx-Clo, an SMR protein that transports a much narrower spectrum of substrates, suggests that in EmrE, a relatively sparse hydrogen bond network among binding site residues permits increased sidechain flexibility.

scholarly journals Highly coupled transport can be achieved in free-exchange transport models

2019 ◽  
Vol 152 (1) ◽  
Author(s):  
Grant A. Hussey ◽  
Nathan E. Thomas ◽  
Katherine A. Henzler-Wildman
Keyword(s):  
Multidrug Resistance ◽  
Transport Model ◽  
Exchange Model ◽  
Fine Tuning ◽  
Coupling Mechanism ◽  
Coupled Transport ◽  
Multidrug Efflux ◽  
Transport Models ◽  
Small Multidrug Resistance ◽  
Transport Direction

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.

scholarly journals Substrate-bound structure of the E. coli multidrug resistance transporter MdfA

Cell Research ◽  
2015 ◽  
Vol 25 (9) ◽  
pp. 1060-1073 ◽  
Author(s):  
Jie Heng ◽  
Yan Zhao ◽  
Ming Liu ◽  
Yue Liu ◽  
Junping Fan ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
E Coli ◽  
Multidrug Resistance Transporter

scholarly journals Few Conserved Amino Acids in the Small Multidrug Resistance Transporter EmrE Influence Drug Polyselectivity

2018 ◽  
Vol 62 (8) ◽  
Author(s):  
Marwah Saleh ◽  
Denice C. Bay ◽  
Raymond J. Turner
Keyword(s):  
Amino Acids ◽  
Multidrug Resistance ◽  
Drug Binding ◽  
Conserved Residues ◽  
Content Type ◽  
Small Multidrug Resistance ◽  
Transporter Family ◽  
Wide Range ◽  
Multidrug Resistance Transporter ◽  
Conserved Amino Acids

ABSTRACT EmrE is the archetypical member of the small multidrug resistance transporter family and confers resistance to a wide range of disinfectants and dyes known as quaternary cation compounds (QCCs). The aim of this study was to examine which conserved amino acids play an important role in substrate selectivity. On the basis of a previous analysis of EmrE homologues, a total of 33 conserved residues were targeted for cysteine or alanine replacement within E. coli EmrE. The antimicrobial resistance of each EmrE variant expressed in Escherichia coli strain JW0451 (lacking dominant pump acrB) to a collection of 16 different QCCs was tested using agar spot dilution plating to determine MIC values. The results determined that only a few conserved residues were drug polyselective, based on ≥4-fold decreases in MIC values: the active-site residue E14 (E14D and E14A) and 4 additional conserved residues (A10C, F44C, L47C, W63A). EmrE variants I11C, V15C, P32C, I62C, L93C, and S105C enhanced resistance to polyaromatic QCCs, while the remaining EmrE variants reduced resistance to one or more QCCs with shared chemical features: acylation, tri- and tetraphenylation, aromaticity, and dicationic charge. Mapping of EmrE variants onto transmembrane helical wheel projections using the highest resolved EmrE structure suggests that polyselective EmrE variants were located closest to the helical faces surrounding the predicted drug binding pocket, while EmrE variants with greater drug specificity mapped onto distal helical faces. This study reveals that few conserved residues are essential for drug polyselectivity and indicates that aromatic QCC selection involves a greater portion of conserved residues than that in other QCCs.

scholarly journals Identification of a Spermidine Excretion Protein Complex (MdtJI) in Escherichia coli

2007 ◽  
Vol 190 (3) ◽  
pp. 872-878 ◽  
Author(s):  
Kyohei Higashi ◽  
Hiroyuki Ishigure ◽  
Risa Demizu ◽  
Takeshi Uemura ◽  
Kunihiko Nishino ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Multidrug Resistance ◽  
Cell Growth ◽  
Protein Complex ◽  
Cell Toxicity ◽  
E Coli ◽  
Inhibition Of Growth ◽  
Small Multidrug Resistance ◽  
Cells Cultured ◽  
In Cells

ABSTRACT A spermidine excretion protein in Escherichia coli was looked for among 33 putative drug exporters thus far identified. Cell toxicity and inhibition of growth due to overaccumulation of spermidine were examined in an E. coli strain deficient in spermidine acetyltransferase, an enzyme that metabolizes spermidine. Toxicity and inhibition of cell growth by spermidine were recovered in cells transformed with pUCmdtJI or pMWmdtJI, encoding MdtJ and MdtI, which belong to the small multidrug resistance family of drug exporters. Both mdtJ and mdtI are necessary for recovery from the toxicity of overaccumulated spermidine. It was also found that the level of mdtJI mRNA was increased by spermidine. The spermidine content in cells cultured in the presence of 2 mM spermidine was decreased, and excretion of spermidine from cells was enhanced by MdtJI, indicating that the MdtJI complex can catalyze excretion of spermidine from cells. It was found that Tyr4, Trp5, Glu15, Tyr45, Tyr61, and Glu82 in MdtJ and Glu5, Glu19, Asp60, Trp68, and Trp81 in MdtI are involved in the excretion activity of MdtJI.

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