Structural and functional comparison of hexahistidine tagged and untagged forms of small multidrug resistance protein, EmrE

Abstract

EmrE is a member of the small multidrug resistance (SMR) protein family in Escherichia coli. EmrE confers resistance to a wide variety of quaternary cation compounds (QCCs) as an efflux transporter driven by proton motive force. The purification yield of most membrane proteins are challenging because of difficulties in over expressing, isolating and solubilizing them and the addition of an affinity tag often improves purification. The purpose of this study is to compare the structure and function of hexahistidinyl (His6) tagged (T-EmrE) and untagged (UT-EmrE) versions of EmrE. In vivo QCC resistance assays determined that T-EmrE demonstrated reduced resistance as compared to UT-EmrE. We isolated EmrE using the two different purification methods, an organic solvent extraction method used to isolate UT-EmrE and nickel affinity chromatography of T-EmrE. All proteins were solubilized in the same buffered n-dodecyl-β-d-maltopyranoside (DDM) detergent and their conformations were examined in the presence/absence of different QCCs. In vitro analysis of protein multimerization using SDS-Tricine PAGE and dynamic light scattering analysis revealed that both proteins predominated as monomers, but the formation of dimers was more constant and uniform in T-EmrE compared to UT-EmrE. The aromatic residue conformations of both proteins indicate that T-EmrE form is more aqueous exposed than UT-EmrE, but UT-EmrE appeared to have a more dynamic environment surrounding its aromatic residues. Using fluorescence to obtain QCC ligand-binding curves indicated that the two forms had differences in dissociation constants (Kd) and maximum specific one-site binding (Bmax) values for particular QCCs. In vitro analyses of both proteins demonstrated subtle but significant differences in multimerization and QCC binding. In vivo analysis indicates differences caused by the addition of the tag, we also observed differences in vitro that could be a result of the tag and/or the different purification methods.

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

“…Despite this factor, all EmrE variants showed WT levels of resistance to one or more QCCs, highlighting the resilience of the protein to sequence variation. It should be noted that differences in ligand binding, protein folding, and MIC values for affinity-tagged EmrE and untagged EmrE were identified and suggested that untagged EmrE binds ligands (TPP, MV, ET, CPC) more tightly and conferred higher levels of drug resistance in a recent study (17). This highlights the importance of studying unmodified versions of a protein along with tagged constructs and now provides greater insight into the resistance conferred by native EmrE protein.…”

“…In an effort to examine the resistance attributed to conserved residues of unmodified EmrE, all variants were expressed in E. coli ΔacrB strains without an affinity tag. Our goal was to examine the resistance conferred by EmrE which had the least amount of modifications to avoid drug selection biases stemming from the affinity tag itself, as noted in a recent study (17). The vast majority of studies involving EmrE have involved the addition of an affinity tag (as reviewed in reference 1), specifically, a C-terminal myc-His 6 tag, which extends the protein by 30 amino acids and reduces its solubility but not membrane targeting (17).…”

“…Our goal was to examine the resistance conferred by EmrE which had the least amount of modifications to avoid drug selection biases stemming from the affinity tag itself, as noted in a recent study (17). The vast majority of studies involving EmrE have involved the addition of an affinity tag (as reviewed in reference 1), specifically, a C-terminal myc-His 6 tag, which extends the protein by 30 amino acids and reduces its solubility but not membrane targeting (17). Furthermore, each EmrE variant that we examined conferred resistance to at least 2 or more QCCs similar to that for the WT (Fig.…”

“…variant was screened against the wild type (WT) for its QCC resistance using agar spot dilution testing. To avoid drug selection biases caused by the addition of affinity tags previously used to study EmrE, such as hexahistidine (10,17) or hemagglutinin epitopes (18), modifications to untagged wild-type EmrE sequences were made. The MIC values for each variant and drug were statistically assessed, sorted, and visualized using hierarchical agglomerative clustering, which was used to compare fold change differences in the MIC values of different QCCs for the EmrE variants and the strain with wild-type EmrE.…”

Influence of quaternary cation compound on the size of the Escherichia coli small multidrug resistance protein, EmrE

“…In addition to Glu14, aromatic residues have been shown to play a role in defining the active site: for example, Trp63 was demonstrated to be located close to the QCC binding pocket, and a variant of this residue abolished ligand binding [8] , [24] . Two other tryptophans are located within transmembrane segments 2 and 3 and a fourth tryptophan can be found in loop 2, all of which are expected to be involved in ligand interaction and can be used as spectroscopic probes of ligand binding [25] .

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