Electrostatic Lock Controlling Structural Transition in the EmrE Polyaromatic Cation Transporter

Abstract

EmrE is a homodimeric bacterial transporter that exploits the proton gradient across the membrane to export polyaromatic cations (e.g., drugs) out of the cell, thereby conferring antibiotic resistance. EmrE is an antiporter, and is expected to undergo structural transitions between the inward-facing (IF) and outward facing (OF) states only when the transporter is loaded with either the substrate or protons. Despite the antiparallel homodimeric configuration of EmrE, which gives it symmetry that makes it ideal for studying complex conformational transitions in membrane transporters, the low resolution of its 3D structures has substantially hampered detailed structural and dynamical examination of the protein, which are necessary for exploring the underlying molecular mechanism of coupled transport. Most notably, previous simulation studies of this small transporter have reported large structural deformations during simulation, signaling the instability of their starting structure. Here we present a substantial refinement procedure applied to the structure using experimental cryoEM data combined with molecular dynamics flexible fitting to generate a stable atomic structural model.From this new starting model we conducted equilibrium and non-equilibrium molecular dynamics simulations of EmrE in different states of the transport cycle, specifically the apo, substrate bound, and protonated forms. These simulations indicate dynamism in structure commensurate with the estimated fluctuations within EmrE. From hydrogen bonding patterns between monomers throughout the transport cycle, we determine that specific interactions between Glu14 and Tyr60 of opposite monomers lock in place the relative conformations of the two monomers, preventing global transition of the transporter in the apo state. When Glu14 is protonated or polyaromatic cations are bound, nonequilibrium simulations show that the energetic barrier conformational transitions can occur more rapidly. These findings substantiate earlier mutagenesis studies indicating an essential role for Tyr60 in the mechanism of EmrE.