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Related Experiment Videos

Conformational changes in the multidrug transporter EmrE associated with substrate binding.

Christopher G Tate1, Iban Ubarretxena-Belandia, Joyce M Baldwin

  • 1MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK. cgt@mrc-lmb.cam.ac.uk

Journal of Molecular Biology
|August 30, 2003
PubMed
Summary

The bacterial multidrug transporter EmrE binds one tetraphenylphosphonium (TPP+) molecule per dimer. This binding event, visualized using cryo-electron microscopy, causes conformational changes in transmembrane alpha-helices.

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Area of Science:

  • Structural biology
  • Molecular microbiology
  • Biochemistry

Background:

  • EmrE is a key bacterial multidrug transporter.
  • It belongs to the small multidrug resistance (SMR) family.
  • EmrE functions via a proton antiport mechanism to expel hydrophobic cations like TPP+.

Purpose of the Study:

  • To determine the stoichiometry of tetraphenylphosphonium (TPP+) binding to EmrE.
  • To elucidate the structural consequences of TPP+ binding to EmrE.

Main Methods:

  • Purification and solubilization of EmrE.
  • Binding assays to determine TPP+ stoichiometry.
  • Reconstitution of EmrE into lipid bilayers.
  • Two-dimensional crystallization.
  • Cryo-electron microscopy (cryo-EM) and image processing to 7A resolution.

Related Experiment Videos

  • Difference mapping between native and TPP+-bound states.

    • Main Results:

    • One TPP+ molecule binds per EmrE dimer.
    • TPP+ binding induces conformational changes, including the movement of at least one transmembrane alpha-helix.
    • Cryo-EM revealed the TPP+ binding site at the monomer-monomer interface within the EmrE dimer.

    Conclusions:

    • The study provides structural insights into the mechanism of TPP+ transport by EmrE.
    • Binding of the substrate TPP+ at the dimer interface triggers conformational changes essential for transporter function.