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

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 (K_d) and maximum specific one-site binding (B_max) 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.     

Multimeric forms of the small multidrug resistance protein EmrE in anionic detergent

Escherichia coli multidrug resistance protein E (EmrE) is a four transmembrane α-helix protein, and a member of the small multidrug resistance protein family that confers resistance to a broad range of quaternary cation compounds (QCC) via proton motive force. The multimeric states of EmrE protein during transport or ligand binding are variable and specific to the conditions of study. To explore EmrE multimerization further, EmrE extracted from E. coli membranes was solubilized in anionic detergent, sodium dodecyl sulphate (SDS), at varying protein concentrations. At low concentrations (≤ 1 μM) in SDS-EmrE is monomeric, but upon increasing EmrE concentration, a variety of multimeric states can be observed by SDS-Tricine polyacrylamide gel electrophoresis (PAGE). Addition of the (QCC), tetraphenyl phosphonium (TPP), to SDS-EmrE samples enhanced EmrE multimer formation using SDS-Tricine PAGE. The relative shapes of EmrE multimers in SDS with or without TPP addition were determined by small angle neutron scattering (SANS) analysis and revealed that EmrE dimers altered in conformation depending on the SDS concentration. SANS analysis also revealed that relative shapes of larger EmrE multimers (≥ 100 nm sizes) altered in the presence of TPP. Circular dichroism spectropolarimetry displayed no differences in secondary structure under the conditions studied. Fluorescence spectroscopy of SDS-EmrE protein demonstrated that aromatic residues, Trp and Tyr, are more susceptible to SDS concentration than TPP addition, but both residues exhibit enhanced quenching at high ligand concentrations. Hence, EmrE forms various multimers in SDS that are influenced by detergent concentration and TPP substrate addition.     

Functional response of the small multidrug resistance protein EmrE to mutations in transmembrane helix 2

Escherichia coli EmrE is a small multidrug resistance protein encompassing four transmembrane (TM) sequences that oligomerizes to confer resistance to antimicrobials. Here we examined the effects on in vivo protein accumulation and ethidium resistance activity of single residue substitutions at conserved and variable positions in EmrE transmembrane segment 2 (TM2). We found that activity was reduced when conserved residues localized to one TM2 surface were replaced. Our findings suggest that conserved TM2 positions tolerate greater residue diversity than conserved sites in other EmrE TM sequences, potentially reflecting a source of substrate polyspecificity.