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 Overexpression of the Escherichia coli sugE Gene Confers Resistance to a Narrow Range of Quaternary Ammonium Compounds

2002 ◽  
Vol 184 (9) ◽  
pp. 2543-2545 ◽  
Author(s):  
Yong Joon Chung ◽  
Milton H. Saier
Keyword(s):  
Escherichia Coli ◽  
Drug Resistance ◽  
Multidrug Resistance ◽  
Quaternary Ammonium ◽  
Narrow Range ◽  
Quaternary Ammonium Compounds ◽  
High Level Expression ◽  
Small Multidrug Resistance ◽  
High Level ◽  
Ammonium Compounds

ABSTRACT SugE of Escherichia coli, first identified as a suppressor of groEL mutations but a member of the small multidrug resistance family, has not previously been shown to confer a drug resistance phenotype. We show that high-level expression of sugE leads to resistance to a subset of toxic quaternary ammonium compounds.

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 Genetic basis of resistance to quaternary ammonium compounds – the qac genes and their role: a review

2012 ◽  
Vol 57 (No. 6) ◽  
pp. 275-281 ◽  
Author(s):  
Z. Jaglic ◽  
D. Cervinkova
Keyword(s):  
Quaternary Ammonium ◽  
Genetic Basis ◽  
Current Knowledge ◽  
Quaternary Ammonium Compounds ◽  
Practical Significance ◽  
Multidrug Resistance Proteins ◽  
Resistance Proteins ◽  
Small Multidrug Resistance ◽  
Cationic Compounds ◽  
Ammonium Compounds

 Although the qac genes are named after one of their main substrates (i.e., quaternary ammonium compounds), these genes also code for resistance to a broad spectrum of other cationic compounds such as intercalating dyes, diamidines and biguanides. The various Qac proteins are involved in relatively low specific efflux-based multidrug pumps and belong to a family of small multidrug resistance proteins. Even though the practical significance of qac-mediated resistance lies mainly in resistance to antiseptics, contradictory findings on this issue are still reported. Therefore, the aim of this review is to summarise the current knowledge on qac-mediated resistance with special emphasis on resistance to antiseptics and its relevance for practice.  

scholarly journals Structures of KcsA in Complex with Symmetrical Quaternary Ammonium Compounds Reveal a Hydrophobic Binding Site

Biochemistry ◽  
2014 ◽  
Vol 53 (32) ◽  
pp. 5365-5373 ◽  
Author(s):  
Michael J. Lenaeus ◽  
Dylan Burdette ◽  
Tobias Wagner ◽  
Pamela J. Focia ◽  
Adrian Gross
Keyword(s):  
Binding Site ◽  
Quaternary Ammonium ◽  
Quaternary Ammonium Compounds ◽  
Hydrophobic Binding ◽  
Ammonium Compounds

Examination of EmrE conformational differences in various membrane mimetic environments

2003 ◽  
Vol 81 (2) ◽  
pp. 61-70 ◽  
Author(s):  
Sandra L Federkeil ◽  
Tara L Winstone ◽  
Glen Jickling ◽  
Raymond J Turner
Keyword(s):  
Multidrug Resistance ◽  
Structural Information ◽  
Sodium Dodecyl ◽  
Tryptophan Fluorescence ◽  
Acrylamide Quenching ◽  
Small Multidrug Resistance ◽  
Membrane Mimetic ◽  
Tryptophan Residues ◽  
Helical Content ◽  
Lipid Environment

Ethidium multidrug resistance protein (EmrE) is a member of the small multidrug resistance family of proteins and is responsible for resistance in Escherichia coli to a diverse group of lipophilic cations. Research is beginning to elucidate structural information as well as substrate binding and extrusion mechanisms for this protein. However, the choice of membrane mimetic environment to perform structural studies needs to be made. In this study EmrE was solubilized in different membrane mimetic environments to investigate the influence of environment on the structure and dynamics of the protein by comparing the fluorescence properties of emission maxima, peak shifts, relative intensities, acrylamide quenching constants, and polarization. Taken together, the different fluorescence observations on EmrE in the various membrane mimetic systems tested suggest that the tryptophan residues in EmrE are present in the most flexible and exposed state when solubilized in methanol, followed by sodium dodecyl sulfate and urea. The two detergents N-dodecyl-β-D-maltoside (DM) and polyoxyethylene(8)dodecyl ether, for the most part, only display subtle differences between the spectral properties with DM best representing the lipid environment. The conformation of EmrE is clearly more open and dynamic in detergent relative to being reconstituted in small unilamellar vesicles. The fluorescence observations of EmrE solubilized in trifluoroethanol shows an environment that is similar to that of EmrE solubilized in detergents. Additionally, secondary structure was monitored by circular dichroism (CD). The CD spectra were similar among the different solubilizing conditions, suggesting little difference in α-helical content. This work establishes groundwork for the choice of solubilizing conditions for future structural, folding, and ligand binding studies.Key words: SMR, EmrE, tryptophan fluorescence, membrane proteins, detergent solubilization, membrane mimetic.

scholarly journals Riboswitch-Associated Guanidinium-Selective Efflux Pumps Frequently Transmitted on Proteobacterial Plasmids Increase Escherichia coli Biofilm Tolerance to Disinfectants

2020 ◽  
Vol 202 (23) ◽  
Author(s):  
Carmine J. Slipski ◽  
Taylor R. Jamieson ◽  
George G. Zhanel ◽  
Denice C. Bay
Keyword(s):  
Escherichia Coli ◽  
Multidrug Resistance ◽  
Quaternary Ammonium ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Cloning And Expression ◽  
Upstream Region ◽  
Ammonium Compound ◽  
Content Type ◽  
Small Multidrug Resistance

ABSTRACT Members of the small multidrug resistance (SMR) efflux pump family known as SugE (recently renamed Gdx) are known for their narrow substrate selectivity to small guanidinium (Gdm+) compounds and disinfectant quaternary ammonium compounds (QACs). Gdx members have been identified on multidrug resistance plasmids in Gram-negative bacilli, but their functional role remains unclear, as few have been characterized. Here, we conducted a survey of sequenced proteobacterial plasmids that encoded one or more SugE/Gdx sequences in an effort to (i) identify the most frequently represented Gdx member(s) on these plasmids and their sequence diversity, (ii) verify if Gdx sequences possess a Gdm+ riboswitch that regulates their translation similarly to chromosomally encoded Gdx members, and (iii) determine the antimicrobial susceptibility profile of the most predominate Gdx member to various QACs and antibiotics in Escherichia coli strains BW25113 and KAM32. The results of this study determined 14 unique SugE sequences, but only one Gdx sequence, annotated as “SugE(p),” predominated among the >140 plasmids we surveyed. Enterobacterales plasmids carrying sugE(p) possessed a guanidine II riboswitch similar to the upstream region of E. coli gdx. Cloning and expression of sugE(p), gdx, and emrE sequences into a low-copy-number expression vector (pMS119EH) revealed significant increases in QAC resistance to a limited range of detergent-like QACs only when gdx and sugE(p) transformants were grown as biofilms. These findings suggest that sugE(p) presence on proteobacterial plasmids may be driven by species that frequently encounter Gdm+ and QAC exposure. IMPORTANCE This study characterized the function of antimicrobial-resistant phenotypes attributed to plasmid-encoded guanidinium-selective small multidrug resistance (Gdm/SugE) efflux pumps. These sequences are frequently monitored as biocide resistance markers in antimicrobial resistance surveillance studies. Our findings reveal that enterobacterial gdm sequences transmitted on plasmids possess a guanidine II riboswitch, which restricts transcript translation in the presence of guanidinium. Cloning and overexpression of this gdm sequence revealed that it confers higher resistance to quaternary ammonium compound (QAC) disinfectants (which possess guanidium moieties) when grown as biofilms. Since biofilms are commonly eradicated with QAC-containing compounds, the presence of this gene on plasmids and its biofilm-specific resistance are a growing concern for clinical and food safety prevention measures.

The structure of the Aquifex aeolicus MATE family multidrug resistance transporter and sequence comparisons suggest the existence of a new subfamily

2021 ◽  
Vol 118 (46) ◽  
pp. e2107335118
Author(s):  
Jiangfeng Zhao ◽  
Hao Xie ◽  
Ahmad Reza Mehdipour ◽  
Schara Safarian ◽  
Ulrich Ermler ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Multidrug Resistance ◽  
Binding Site ◽  
Site Directed Mutagenesis ◽  
Aquifex Aeolicus ◽  
Sequence Comparisons ◽  
Transporter Family ◽  
Domains Of Life ◽  
Multidrug Resistance Transporter ◽  
Mate Transporter

Multidrug and toxic compound extrusion (MATE) transporters are widespread in all domains of life. Bacterial MATE transporters confer multidrug resistance by utilizing an electrochemical gradient of H+ or Na+ to export xenobiotics across the membrane. Despite the availability of X-ray structures of several MATE transporters, a detailed understanding of the transport mechanism has remained elusive. Here we report the crystal structure of a MATE transporter from Aquifex aeolicus at 2.0-Å resolution. In light of its phylogenetic placement outside of the diversity of hitherto-described MATE transporters and the lack of conserved acidic residues, this protein may represent a subfamily of prokaryotic MATE transporters, which was proven by phylogenetic analysis. Furthermore, the crystal structure and substrate docking results indicate that the substrate binding site is located in the N bundle. The importance of residues surrounding this binding site was demonstrated by structure-based site-directed mutagenesis. We suggest that Aq_128 is functionally similar but structurally diverse from DinF subfamily transporters. Our results provide structural insights into the MATE transporter, which further advances our global understanding of this important transporter family.

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.

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