Asymmetric protonation of glutamate residues drives a preferred transport pathway in EmrE

Significance EmrE is a proton-coupled efflux pump that confers multidrug resistance to Escherichia coli . Here, we probed the electrostatic environment surrounding each essential Glu14 residue in the EmrE dimer by determining monomer specific p K a values using NMR spectroscopy. We discovered that acid/base chemistry at one of the two Glu14 residues in the homodimer potentiates a global conformational change within EmrE. Our findings revealed that asymmetric protonation states of EmrE leads to a preferred pathway of substrate transport. These insights led to a model to explain how EmrE accomplishes proton-coupled transport without leaking protons. Since the overall structure of EmrE resembles those of other transporters containing inverted repeat domains, our findings have application to other secondary active transporters.