A lipid-dependent link between activity and oligomerization state of the M. tuberculosis SMR protein TBsmr

Abstract

TBsmr is a secondary active multidrug transporter from Mycobacterium tuberculosis that transports a plethora of compounds including antibiotics and fluorescent dyes. It belongs to the small multidrug resistance (SMR) superfamily and is structurally and functionally related to E. coli EmrE. Of particular importance is the link between protein function, oligomeric state and lipid composition. By freeze fracture EM, we found three different size distributions in three different lipid environments for TBsmr indicating different oligomeric states. The link of these states with protein activity has been probed by fluorescence spectroscopy revealing significant differences. The drug binding site has been probed further by (19)F-MAS NMR through chemical labeling of native cysteine residues showing a water accessible environment in agreement with the alternating access model.

Plasma Membrane Cholesterol Regulates the Allosteric Binding of 1-Methyl-4-Phenylpyridinium to Organic Cation Transporter 2 (SLC22A2)

“…Oligomerization is an important determinant of correct localization, function, and regulation of different transporter proteins, and membrane lipid composition can affect the oligomerization state of the transmembrane proteins embedded therein (Mörs et al, 2013;Alguel et al, 2016;Gupta et al, 2017). Because the removal of cholesterol from OCT2-HEK293 cells did not seem to alter the equilibria between the different states of oligomerization observed for this transporter, it is plausible to hypothesize that the allosterism is a feature of OCT2 binding pockets regardless of the oligomerization state.…”

EmrE dimerization depends on membrane environment

“…It is likely that dimerization affinity is further affected by the exact composition of the lipid membrane. When the EmrE homolog, TBsmr, is reconstituted into liposomes, ethidium transport activity depends on the lipid composition and increased activity is correlated with increased oligomerization [22]. EmrE transport activity in liposomes is itself dependent on the lipid composition, with significant differences in transport rate when phosphatidylglycerol (PG) or phosphatidylethanolamine (PE) lipids are included in the liposome [20, 21].…”

“…Even in the most native-like environment, lipid bilayers, the substrate binding affinity and transport rate of EmrE vary with lipid composition [20, 21]. Recently, the oligomeric state of the close homolog TBsmr has been investigated using freeze fracture electron miscroscopy in different lipid environments and the oligomeric state correlated with differences in TBsmr activity [22]. Here we measure the dimerization affinity of EmrE in bicelles and liposomes to better understand the effect of environment on the functionally important dimerization of this unique and well-studied integral membrane protein.…”

Functional Role of YnfA, an Efflux Transporter in Resistance to Antimicrobial Agents in Shigella flexneri

“…Numerous SMR proteins from each subfamily have been identified in bacterial pathogens, as they provide increased levels of resistance to clinically used antibiotics such as cephalosporins, β-lactams, aminoglycosides, inhibitors of dihydrofolate, and various antiseptics ( 34 , 35 ). Some of the well-characterized representatives of the SMR family include the EmrE transporter in E. coli ( 37 ); SugE in Citrobacter and E. coli ( 41 , 42 ); QacE transporter in S. aureus and Klebsiella ( 43 – 45 ); EbrAB and YkkCD transporters in Bacillus subtilis ( 46 , 47 ); AbeS transporter in Acinetobacter baumanni ( 48 ); and TBsmr transporter in Mycobacterium tuberculosis ( 49 ).…”