Investigating the Structure of the Drug Transporter EmrE

Abstract

Multidrug resistance in bacteria is a critical challenge in public health and drug discovery. One of the primary mechanisms of resistance is efflux pumps, which couple an energetically favorable process with the export of a drug molecule against its concentration gradient.1 Efflux pumps from the small multidrug resistance protein family are ubiquitous among bacteria. These secondary active transporters couple the efflux of a wide variety of toxic compounds with the proton gradient of the inner membrane.2 To gain insight into this transport process we carried out a series of biophysical experiments, including solution and solid-state NMR spectroscopy using the multidrug transporter EmrE. EmrE is an asymmetric and antiparallel homodimer, which couples the efflux of cationic drugs with the import of protons. Previously, we revealed a large change in structure and dynamics due to the acid/base chemistry at a conserved glutamic acid residue.3 In this work we focus on describing the site-specific structural changes within the substrate binding pocket that drive the large conformational change in EmrE. Specifically, our oriented solid-state NMR and solution NMR experiments reveal backbone and side chain structural perturbations upon binding protons or drugs that reveal a specific mechanism involving aromatic residues in the binding pocket. Finally, we built on our previous findings 3 to show how conformationally biased mutants allow for the unambiguous determination of monomer-specific restraints for structural elucidation of EmrE in lipid bilayers.