title: A structured loop modulates coupling between the substrate-binding and dimerization domains in the multidrug resistance transporter EmrE.
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.
authors.affiliation: From the Department of Chemistry, New York University, New York, New York 10003.
authors.forename: James R
authors.initials: JR
authors.lastname: Banigan
authors.affiliation: From the Department of Chemistry, New York University, New York, New York 10003.
authors.forename: Anindita
authors.initials: A
authors.lastname: Gayen
authors.affiliation: From the Department of Chemistry, New York University, New York, New York 10003.
authors.forename: Min-Kyu
authors.initials: MK
authors.lastname: Cho
authors.affiliation: From the Department of Chemistry, New York University, New York, New York 10003 traaseth@nyu.edu.
authors.forename: Nathaniel J
authors.initials: NJ
authors.lastname: Traaseth
mesh_terms: D017920:Antiporters / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D001665:Binding Sites; D018432:Drug Resistance, Multiple / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary / Q000235:genetics; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D017920:Antiporters; D029968:Escherichia coli Proteins; D008051:Lipid Bilayers; C075436:EmrE protein, E coli
keywords: Drug Resistance; EmrE; Membrane Protein; Membrane Transporter; Nuclear Magnetic Resonance (NMR); Protein Structure; Secondary Active Transport; Small Multidrug Resistance Family; Solid State NMR