Protonation-dependent conformational dynamics of the multidrug transporter EmrE

Abstract

The small multidrug transporter from Escherichia coli, EmrE, couples the energetically uphill extrusion of hydrophobic cations out of the cell to the transport of two protons down their electrochemical gradient. Although principal mechanistic elements of proton/substrate antiport have been described, the structural record is limited to the conformation of the substrate-bound state, which has been shown to undergo isoenergetic alternating access. A central but missing link in the structure/mechanism relationship is a description of the protonbound state, which is an obligatory intermediate in the transport cycle. Here we report a systematic spin labeling and double electron electron resonance (DEER) study that uncovers the conformational changes of EmrE subsequent to protonation of critical acidic residues in the context of a global description of ligand-induced structural rearrangements. We find that protonation of E14 leads to extensive rotation and tilt of transmembrane helices 1-3 in conjunction with repacking of loops, conformational changes that alter the coordination of the bound substrate and modulate its access to the binding site from the lipid bilayer. The transport model that emerges from our data posits a proton-bound, but occluded, resting state. Substrate binding from the inner leaflet of the bilayer releases the protons and triggers alternating access between inward-and outward-facing conformations of the substrate-loaded transporter, thus enabling antiport without dissipation of the proton gradient.EmrE | SMR | EPR | DEER | multidrug transport P owered by the proton electrochemical gradient across the inner membrane of prokaryotes, small multidrug resistance (SMR) transporters extrude a spectrum of cytotoxic molecules that are primarily hydrophobic cations (1). The functional unit is typically a homodimer wherein each protomer consists of four hydrophobic transmembrane helices (TM). TM1-3 cradle a substrate binding pocket, whereas TM4 is involved in the contacts that stabilize the dimer. EmrE, the SMR transporter from Escherichia coli, has been a focal point of structural, spectroscopic, and mechanistic investigations (2-7). Seminal work from the Schuldiner laboratory over the last two decades has unlocked mechanistic principles of substrate-ionantiport (1,(8)(9)(10). EmrE binds hydrophobic substrates in a membrane-embedded chamber coordinated by glutamate 14 (E14), an absolutely conserved, membrane-embedded, acidic residue in the middle of TM1. Coupling between substrate and proton arises from the principle of mutual exclusion between the two ligands at the binding site (8,10).In contrast to the elaborate understanding of the EmrE antiport mechanism, the conformational changes that enable binding and release of ligands have not been elucidated. Although the general framework of antiport is presumed to follow the principles of alternating access, the only structure available is of EmrE bound to the substrate tetraphenylphosphonium (TPP) (3). EM analysis of 2D crystals established an antiparallel ...

The contribution of modern EPR to structural biology

“…Based on the Anfinsen dogma, it became customary to explain function in terms of a single conformation or of well-defined transitions between a few conformations defined at atomic resolution. While this is certainly a reasonable approximation in some cases [75][76][77], availability of distance distributions demonstrates that rather often conformation transitions are coupled to order-disorder transitions or are shifts in disorder equilibria [39,[78][79][80][81][82][83][84][85][86][87][88][89][90][91][92][93][94]. Among the systems addressed by PDS to date, the fraction where at least one state is genuinely disordered is surprisingly large.…”

“…The multidrug transporter EmrE is ordered in its substrate-bound form, but flexible when protonated at a pH of 5 [87]. Likewise, the homodimeric multidrug ABC transporter LmrA undergoes a disorder-order transition from a very broad conformational ensemble to a rather well-defined conformation upon nucleotide binding [80].…”

Stable membrane orientations of small dual-topology membrane proteins

“…The functional form of EmrE is a homodimer, in which the two identical protomers in the dimer have opposite membrane topologies and associate in antiparallel orientations relative to one another (15)(16)(17)(18)(19), although less stable parallel dimers can also form under certain conditions (16,(20)(21)(22)(23). EmrE is the most extensively studied example of the class of dual-topology proteins: single polypeptide chains that are inserted into the membrane in two opposite orientations and form antiparallel homodimers in vivo (15,(24)(25)(26).…”

The Synergetic Effects of Combining Structural Biology and EPR Spectroscopy on Membrane Proteins

“…The small multidrug resistance exporter family (SMR), which supports proton/substrate antiport, is represented by EmrE. Here in particular a study unraveling the protonation-dependent conformational dynamics has contributed significantly to the understanding of the underlying transport mechanism [147]. A combined effort of NMR, EPR and MD simulations shed light on global structural changes in the Cl − /H + exchanger family CLC, which are necessary to form the previously unknown outward-facing open conformation [148].…”