Abstract
Secondary active transporters undergo large conformational changes to facilitate the efflux of substrates across the lipid bilayer. Among the smallest known transport proteins are members of the small multidrug resistance (SMR) family that are composed of four transmembrane (TM) domains and assemble into dimers. An unanswered question in the SMR field is how the dimerization domain (TM4) is coupled with the substrate-binding chamber (TM1-3). To provide insight for this essential aspect of ion-coupled transport, we carried out a structure-function study on the SMR protein EmrE using solid-state NMR spectroscopy in lipid bilayers and resistance assays in Escherichia coli. The chemical shifts for EmrE were consistent with β-strand secondary structure for the loop connecting TM3 and TM4. Based on these structural results, EmrE mutants were created to ascertain whether a specific loop length and composition were necessary for function. A linker encompassing six extra Gly residues relative to wild-type EmrE failed to give resistance; however, the number of residues in the loop was not the only criterion for a functional efflux pump. Replacement of the central hydrophobic residue with Gly (L83G) also conferred no ethidium resistance phenotype, which supported the conclusion that the structure and length of the loop were both essential for ion-coupled transport. Taken together with a bioinformatics analysis, a structured linker is likely conserved across the SMR family to play an active role in mediating the conformational switch between inward-open and outward-open states necessary for drug efflux. These findings underscore the important role loops can play in mediating efflux.
Highlights
Truncated EmrE Lacking Transmembrane-4 Is Prone to Aggregation—Previous work on small multidrug resistance (SMR) proteins showed that conserved Gly residues in TM4 play a role in dimer stability [16, 50, 51]
Because our interests were to determine the structural basis for domain coupling between the proposed dimerization and substrate-binding regions of EmrE, we designed a simple experiment to test whether the helices involved in drug binding were stable in the absence of TM4
As observed in the plot, wild-type EmrE eluted as a single peak at ϳ12.8 ml, whereas TM1–3 had a large fraction that eluted earlier than the wild-type profile, which was indicative of aggregation and confirmed by gel electrophoresis
Summary
The SMRs are membrane protein transporters with 100 –140 residues and four transmembrane (TM) domains that confer resistance to a wide variety of toxic compounds, including ethidium, dequalinium, methyl viologen, and acriflavine [3,4,5] These proteins are the smallest known efflux pumps, which makes them an excellent model system for understanding ioncoupled active transport as well as molecular recognition mechanisms involved in multidrug resistance [6]. Some loops connecting TM domains seem to possess no specific composition requirement, such as the central linker that connects TM1– 6 with TM7–12 in the MFS family [22] These experiments carried out with lactose permease showed that the loop length is conserved and necessary to act as a temporal delay in membrane insertion rather than a specific functional role in the transporter [22]. The -strand character revealed from NMR chemical shifts and the sequence homology analyses suggest structural conservation across the SMR family, which uncovers important clues into the active role this loop plays in the inward-open to outward-open conformational transition in the dimer
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