A structured loop modulates coupling between the substrate-binding and dimerization domains in the multidrug resistance transporter EmrE

James R. Banigan, Anindita Gayen, Min Kyu Cho, Nathaniel Traaseth

Research output: Contribution to journalArticle

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 substratebinding chamber (TM1-3). To provide insight for this essential aspect of ion-coupled transport, we carried out a structurefunction study on the SMR protein EmrE using solid-stateNMR 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.Alinker 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.

Original languageEnglish (US)
Pages (from-to)805-814
Number of pages10
JournalJournal of Biological Chemistry
Volume290
Issue number2
DOIs
StatePublished - Jan 9 2015

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Lipid bilayers
Dimerization
Multiple Drug Resistance
Ion Transport
Lipid Bilayers
Ions
P-Glycoproteins
Ethidium
Chemical shift
Substrates
Bioinformatics
Dimers
Escherichia coli
Assays
Carrier Proteins
Switches
Pumps
Spectroscopy
Computational Biology
Spectrum Analysis

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

A structured loop modulates coupling between the substrate-binding and dimerization domains in the multidrug resistance transporter EmrE. / Banigan, James R.; Gayen, Anindita; Cho, Min Kyu; Traaseth, Nathaniel.

In: Journal of Biological Chemistry, Vol. 290, No. 2, 09.01.2015, p. 805-814.

Research output: Contribution to journalArticle

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