• SURFE²R N1

    Easy-to-learn all-in-one device, ideal for teaching and university research
  • SURFE²R N1

    Finally label-free functional assays for transporters available
  • SURFE²R N1

    High signal amplification compared to patch-clamp: transport & binding assays
  • SURFE²R N1

    The only instrument on the market for SSM-based electrophysiology
  • SURFE²R N1

    Turn-key system for efficient transporter protein analysis

2022 - Charge neutralization of the active site glutamates does not limit substrate binding and transport by small multidrug resistance transporter EmrE

 Icon N1   SURFE2R N1 publication in J. Biol. Chem. (2022)

Authors:
Spreacker P., Brousseau M., Hisao G., Soltani M., Davis J.,Henzler-Wildman A.

Journal:
J. Biol. Chem. (2022) doi:10.1016/j.jbc.2022.102805


Abstract: 

EmrE, a small multidrug resistance (SMR) transporter from E. coli, confers broad-spectrum resistance to polyaromatic cations and quaternary ammonium compounds. Previous transport assays demonstrate that EmrE transports a +1 and a +2 substrate with the same stoichiometry of 2 protons:1 cationic substrate. This suggests that EmrE substrate binding capacity is limited to neutralization of the two essential glutamates, E14A and E14B (one from each subunit in the antiparallel homodimer), in the primary binding site. Here we explicitly test this hypothesis, since EmrE has repeatedly broken expectations for membrane protein structure and transport mechanism. We previously showed that EmrE can bind a +1 cationic substrate and proton simultaneously, with cationic substrate strongly associated with one E14 residue while the other remains accessible to bind and transport a proton. Here we demonstrate that EmrE can bind a +2 cation substrate and a proton simultaneously using NMR pH titrations of EmrE saturated with divalent substrates, for a net +1 charge in the transport pore. Further, we find that EmrE can alternate access and transport a +2 substrate and proton at the same time. Together, these results lead us to conclude that E14 charge neutralization does not limit the binding and transport capacity of EmrE.


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