<i>In vitro</i>
            synthesis of fully functional EmrE, a multidrug transporter, and study of its oligomeric state

Elbaz, Yael; Steiner‐Mordoch, Sonia; Danieli, Tsafi; Schuldiner, Shimon

doi:10.1073/pnas.0306533101

Cited by 132 publications

(109 citation statements)

References 26 publications

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“…Studies have shown that the basic functional unit of EmrE is an oligomer, as would be expected for a membrane protein of its small size. It appears established that the basic functional unit of EmrE is a homodimer, as shown by oligomerization assays, substrate binding experiments, negative dominance studies, and crosslinking analyses (3)(4)(5)(6)(7)(8). This conclusion is further supported by the existence of paired SMR proteins, such as YdgE/YdgF of E. coli, and EbrA/EbrB and YkkC/YkkD of Bacillus subtilis.…”

mentioning

confidence: 82%

X-ray structure of EmrE supports dual topology model

Chen

Pornillos

Lieu

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

263

277

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EmrE, a multidrug transporter from Escherichia coli, functions as a homodimer of a small four-transmembrane protein. The membrane insertion topology of the two monomers is controversial. Although the EmrE protein was reported to have a unique orientation in the membrane, models based on electron microscopy and now defunct x-ray structures, as well as recent biochemical studies, posit an antiparallel dimer. We have now reanalyzed our x-ray data on EmrE. The corrected structures in complex with a transport substrate are highly similar to the electron microscopy structure. The first three transmembrane helices from each monomer surround the substrate binding chamber, whereas the fourth helices participate only in dimer formation. Selenomethionine markers clearly indicate an antiparallel orientation for the monomers, supporting a ''dual topology'' model. membrane protein structure ͉ multidrug transport ͉ SMR family O ne of the mechanisms by which cells neutralize the effect of toxic compounds is through the action of membrane transporters. Secondary transporters, such as the EmrE protein of Escherichia coli, couple the efflux of drugs to the inward movement of protons across the cell membrane (refs. 1 and 2, and references therein). EmrE is the prototypical member of the SMR (small multidrug resistance) family and is one of the smallest known transporters in nature, composed of only 110 amino acid residues. Studies have shown that the basic functional unit of EmrE is an oligomer, as would be expected for a membrane protein of its small size. It appears established that the basic functional unit of EmrE is a homodimer, as shown by oligomerization assays, substrate binding experiments, negative dominance studies, and crosslinking analyses (3-8). This conclusion is further supported by the existence of paired SMR proteins, such as YdgE/YdgF of E. coli, and EbrA/EbrB and YkkC/YkkD of Bacillus subtilis. These transporters require coexpression of the two component polypeptides for proper drug efflux activity and presumably form heterodimers analogous to the EmrE homodimer (9-13).Electron microscopy (EM) studies of EmrE in complex with a transport substrate, tetraphenylphosphonium (TPP), and reconstituted in lipid bilayers have revealed the overall architecture of the transporter at 7.5 Å resolution in-plane and 16 Å perpendicular to the membrane (14). EmrE binds TPP as an asymmetric dimer, in a chamber that appears open to one side of the bilayer. Each monomer is composed of four transmembrane (TM) helices. Because of the low resolution, however, the monomers could not be delineated unambiguously, nor the TM segments assigned in a sequence-specific manner based on the experimental map alone.We have previously reported x-ray crystal structures of EmrE in the unbound form and in complex with TPP (15, 16). Regrettably, the electron density maps were calculated in the wrong hand because of an unfortunate change of sign of the anomalous differences (17), and helices were misassigned in the TPP-bound model. Recalculation of the...

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mentioning

confidence: 82%

X-ray structure of EmrE supports dual topology model

Chen

Pornillos

Lieu

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

263

277

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“…This enabled unambiguous chemical shift assignment and simplified data interpretation. The capability of E. coli-based cell-free transcription and translation systems to express wild type EmrE in a functional form with and without detergent was previously shown (17,35). Furthermore, it has been reported that amino acid selective isotope labeling without dilution or scrambling is possible (34).…”

Section: Generalmentioning

confidence: 99%

The Key Residue for Substrate Transport (Glu14) in the EmrE Dimer Is Asymmetric

Lehner

Basting

Meyer

et al. 2008

Journal of Biological Chemistry

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Transport proteins exhibiting broad substrate specificities are major determinants for the phenomenon of multidrug resistance. The Escherichia coli multidrug transporter EmrE, a 4-transmembrane, helical 12-kDa membrane protein, forms a functional dimer to transport a diverse array of aromatic, positively charged substrates in a proton/drug antiport fashion. Here, we report 13 Multidrug drug resistance, in particular bacterial resistance to clinical antibiotics, is a widely known phenomenon. Basic defense mechanisms of bacteria include permeability barriers, inactivation of antimicrobials, modification of antibiotic targets, and active drug efflux (1). Active efflux is conducted by primary and secondary active transport proteins and the latter are found in almost all transporter families (2). The molecular mechanism of the broad substrate specificity of multidrug efflux pumps is not yet fully understood. To this end structural studies are desirable, but currently only 13 three-dimensional structures of different transport proteins are known and not every transport family is represented. The only multidrug transporter with known three-dimensional structure is AcrB (4, 5). A three-dimensional structure of the ABC transporter Sav1866 has also been reported (6), which is assumed to function as a multidrug efflux pump.Here, we are especially interested in Escherichia coli EmrE, a member of the medically relevant SMR 2 protein family (TC number 2.A.7.1 (8, 9)). Due to its small size (12 kDa), EmrE was originally proposed as ideal structure-function paradigm (10). It has attracted significant interest due to its controversial topological organization (11-15), oligomerization state (16 -19), transport cycle steps (12, 20 -23), and unknown three-dimensional structure (24). EmrE transports a diverse array of aromatic, positively charged substrates in exchange for protons (21) via at least one occluded transport cycle intermediate state (25). Other SMR proteins have overlapping but significantly different substrate specificities with measured affinities in the nanomolar to millimolar range (20, 26). All SMR proteins are of similar size (ϳ11-12 kDa), have a 4-transmembrane helix topology and a highly conserved key residue Glu 14 (20,27). It has been shown that Glu 14 is an essential residue and directly involved in drug and proton binding (28 -30). It can reasonably be assumed, that Glu 14 of both protomers in a dimer form a shared binding pocket (31,32).Whether EmrE forms a symmetric or an asymmetric dimer should be reflected in the chemical shifts of residues such as Glu 14 , which are likely to be found at the dimerization interface. We therefore 13 C-labeled Glu 14 in EmrE by utilizing a cell-free expression system. To allow an unambiguous NMR analysis, the nonessential residue Glu 25 was replaced with alanine (EmrE E25A) to create a single glutamate mutant. The protein was reconstituted into E. coli lipids allowing the most native environment possible. The sample was maintained at pH 8.0. Under these conditions, withou...

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“…Not all detergents are suitable for the refolding step but dodecylymaltoside (DDM), dodecylphosphocholine and lyso-phosphoglycerol derivatives (LMPG, LPPG) have been shown to successfully solubilize precipitates [55]. This approach has been successfully applied to the production of EmrE, a multidrug transporter [55,56], and to the human histamine-1 receptor [57]. Second, the addition of detergent directly to is that the membrane protein will be produced in a "native-like" environment which is necessary to obtain a functional protein.…”

Section: / Cell-free Membrane Protein Expressionmentioning

confidence: 99%

Cell-Free Synthesis of Macromolecular Complexes

Botte

Deniaud

Schaffitzel

2016

Advanced Technologies for Protein Complex Production and Characterization

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General rightsThis document is made available in accordance with publisher policies. Please cite only the published version using the reference above. Full terms of use are available: http://www.bristol.ac.uk/pure/about/ebr-terms ABSTRACTCell-free protein synthesis based on E. coli cell extracts has been described for the first time more than fifty years ago. To date, cell-free synthesis is widely used for the preparation of toxic proteins, for studies of the translation process and its regulation as well as for the incorporation of artificial or labelled amino acids into a polypeptide chain. Many efforts have been directed towards establishing cell-free expression as a standard method for gene expression, with limited success. In this chapter we will describe the state-of-theart of cell-free expression, extract preparation methods and recent examples for successful applications of cell-free synthesis of macromolecular complexes.3 1/ Introduction -Motivation and challengesThe capacity of cell extracts to synthesize proteins has been shown in the fifties of the last century [1,2], several years before the identification of ribosomes as proteinsynthesizing machines [3]. The cell-free extract was based on the classical S30 fraction obtained by a 30,000 x g centrifugation step at 4°C for 1 hour. Initially, endogenous mRNA was used for in vitro translation [4]. Subsequently, Nirenberg and Matthaei developed a protocol to degrade endogenous messenger RNA present in the cell extract and to add exogenous mRNA [5,6]. The first cell-free protein synthesis (CFPS) from DNA, using a socalled coupled transcription-translation system was developed in the late sixties by the group of Zubay [7]. They used their coupled transcription-translation system to study the regulation of gene expression by the E. coli lactose operon. Most cell-free extract preparation and in vitro translation protocols are based on this protocol [8,9].Significant improvements with respect to protein yields were achieved in the late eighties, in particular by the group of Spirin, which established the use of phage-specific RNA polymerases, SP6 [10] or T7 RNA polymerases [8]. Using these polymerases a high level of a specific mRNA during the in vitro transcription-translation reaction can be achieved and maintained. Importantly, the Spirin laboratory described the first 'continuous' in vitro translation system. It allows for a continuous exchange of small molecules between a 'feeding compartment' providing energy and substrates (amino acids) for the translation reaction and a 'reaction compartment' from which inhibitory reaction products are removed by dialysis [10,11]. In a continuous set-up, the in vitro translation reaction can continue for several hours or even days, compared to 40-60 minutes using the classical reaction set-up.This allows obtaining significantly increased yields: for instance 6 mg chloramphenicol Cell-free expression has thus several very attractive applications, related to the expression of toxic proteins, rapid production of small q...

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In vitro synthesis of fully functional EmrE, a multidrug transporter, and study of its oligomeric state

Cited by 132 publications

References 26 publications

X-ray structure of EmrE supports dual topology model

X-ray structure of EmrE supports dual topology model

The Key Residue for Substrate Transport (Glu14) in the EmrE Dimer Is Asymmetric

Cell-Free Synthesis of Macromolecular Complexes

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