A Structured Loop Modulates Coupling Between the Substrate-Binding and Dimerization Domains in the Multidrug Resistance Transporter EmrE [Protein Structure and Folding]

November 18th, 2014 by Banigan, J. R., Gayen, A., Cho, M.-K., Traaseth, N. J.

Secondary active transporters undergo large conformational changes to facilitate the efflux of substrates across the lipid bilayer. The smallest known transport proteins are within the small multidrug resistance (SMR) family which are comprised 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 E. 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 was 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 loop's structure and length 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.
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