NMR investigation of the multidrug transporter EmrE, an integral membrane protein

Schwaiger, Manfred; Lebendiker, Mario; Yerushalmi, Hagit; Coles, Murray; Gröger, Adriane; Schwarz, Christian; Schuldiner, Shimon; Kessler, Horst

doi:10.1046/j.1432-1327.1998.2540610.x

Cited by 90 publications

(101 citation statements)

References 11 publications

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“…Three liquid state NMR studies on SMR proteins (EmrE and Smr) have been reported before (57)(58)(59). All studies used nonnative membrane mimicking conditions such as chloroform/ methanol for monomeric EmrE (59), lysopalmitoyl phosphatidylglycerol micelles for dimeric Smr (58), or biPC (1,2-di-Otetradecyl-and 1,2-di-O-hexyl-sn-glycero-3-phosphocholine spiked with ditetradecanoyl-/dihexanoylphosphatidylserine) bicelles for dimeric SMR (57).…”

Section: Interpretation Of Glu 14 Line Shape and Chemical Shiftsmentioning

confidence: 99%

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

Lehner

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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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Section: Interpretation Of Glu 14 Line Shape and Chemical Shiftsmentioning

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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“…It is a small (110-residue) multidrug transporter that extrudes various positively charged drugs in exchange for protons, thus rendering bacteria resistant to these drugs (1)(2)(3). The protein has been characterized, purified, and reconstituted in a functional form (1,(4)(5)(6). High-affinity substrate binding has been established as a reliable and sensitive assay for activity of the detergent-solubilized transporter (4).…”

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confidence: 99%

Substrate-Induced Tryptophan Fluorescence Changes in EmrE, the Smallest Ion-Coupled Multidrug Transporter

et al. 2005

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Tryptophan residues may play several roles in integral membrane proteins including direct interaction with substrates. In this work we studied the contribution of tryptophan residues to substrate binding in EmrE, a small multidrug transporter of Escherichia coli that extrudes various positively charged drugs across the plasma membrane in exchange with protons. Each of the four tryptophan residues was replaced by site-directed mutagenesis. The only single substitutions that affected the protein's activity were those in position 63. While cysteine and tyrosine replacements yielded a completely inactive protein, the replacement of Trp63 with phenylalanine brought about a protein that, although it could not confer any resistance against the toxicants tested, could bind substrate with an affinity 2 orders of magnitude lower than that of the wild-type protein. Double or multiple cysteine replacements at the other positions generate proteins that are inactive in vivo but regain their activity upon solubilization and reconstitution. The findings suggest a possible role of the tryptophan residues in folding and/or insertion. Substrate binding to the wild-type protein and to a mutant with a single tryptophan residue in position 63 induced a very substantial fluorescence quenching that is not observed in inactive mutants or chemically modified protein.The reaction is dependent on the concentration of the substrate and saturates at a concentration of 2.57 µM with the protein concentration of 5 µM supporting the contention that the functional unit is a dimer. These findings strongly suggest the existence of an interaction between Trp63 and substrate, and the nature of this interaction can now be studied in more detail with the tools developed in this work.

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“…MARS has been tested on 14 proteins ranging in size from the 71-residue Z domain of Staphylococcal protein A to 723-residue malate synthase G (Alattia et al, 2000;Gardner et al, 1998;Garrett et al, 1997;Ikura et al, 1991;Liu et al, 2000;Schwaiger et al, 1998;Tashiro et al, 1997;Tugarinov et al, 2002;Vathyam et al, 1999;Wang et al, 1995). Special focus was put on proteins that are challenging with respect to assignment either by their size or because chemical shifts are missing for a substantial portion of residues (Table 1).…”

Section: Testing Of Marsmentioning

confidence: 99%

Mars – robust automatic backbone assignment of proteins

2004

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MARS a program for robust automatic backbone assignment of 13 C/ 15 N labeled proteins is presented. MARS does not require tight thresholds for establishing sequential connectivity or detailed adjustment of these thresholds and it can work with a wide variety of NMR experiments. Using only 13 C α / 13 C β connectivity information, MARS allows automatic, error-free assignment of 96% of the 370-residue maltose-binding protein. MARS can successfully be used when data are missing for a substantial portion of residues or for proteins with very high chemical shift degeneracy such as partially or fully unfolded proteins. Other sources of information, such as residue specific information or known assignments from a homologues protein, can be included into the assignment process. MARS exports its result in SPARKY format. This allows visual validation and integration of automated and manual assignment.

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NMR investigation of the multidrug transporter EmrE, an integral membrane protein

Cited by 90 publications

References 11 publications

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

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

Substrate-Induced Tryptophan Fluorescence Changes in EmrE, the Smallest Ion-Coupled Multidrug Transporter

Mars – robust automatic backbone assignment of proteins

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