Structure, Dynamics, and Substrate-induced Conformational Changes of the Multidrug Transporter EmrE in Liposomes

Abstract

EmrE, a member of the small multidrug transporters superfamily, extrudes positively charged hydrophobic compounds out of Escherichia coli cytoplasm in exchange for inward movement of protons down their electrochemical gradient. Although its transport mechanism has been thoroughly characterized, the structural basis of energy coupling and the conformational cycle mediating transport have yet to be elucidated. In this study, EmrE structure in liposomes and the substrate-induced conformational changes were investigated by systematic spin labeling and EPR analysis. Spin label mobilities and accessibilities describe a highly dynamic ligand-free (apo) conformation. Dipolar coupling between spin labels across the dimer reveals at least two spin label populations arising from different packing interfaces of the EmrE dimer. One population is consistent with antiparallel arrangement of the monomers, although the EPR parameters suggest deviations from the crystal structure of substrate-bound EmrE. Resolving these discrepancies requires an unusual disposition of TM3 relative to the membrane-water interface and a kink in its backbone that enables bending of its C-terminal part. Binding of the substrate tetraphenylphosphonium changes the environment of spin labels and their proximity in three transmembrane helices. The underlying conformational transition involves repacking of TM1, tilting of TM2, and changes in the backbone configurations of TM3 and the adjacent loop connecting it to TM4. A dynamic apo conformation is necessary for the polyspecificity of EmrE allowing the binding of structurally diverse substrates. The flexibility of TM3 may play a critical role in movement of substrates across the membrane.One mechanism of multidrug resistance involves the active extrusion of toxic molecules out of the cell by dedicated membrane transporters. In prokaryotes, five superfamilies of transporters handle vectorial drug traffic moving energetically uphill against substrate concentration gradients (1). The thermodynamics of the problem are rendered favorable through coupling of substrate movement to the direct use of ATP energy or the discharge of electrochemical ion gradients. Small multidrug resistance transporters are the smallest bacterial transporters with four predicted transmembrane ␣-helices and no significant extramembrane domain (2, 3). They function as dimers coupling translocation of positively charged hydrophobic substrates out of the cell to the inward movement of protons.Much of the current mechanistic understanding of small multidrug resistance transporters has emerged from seminal studies in the laboratory of Schuldiner and co-workers (4 -7)defining fundamental steps in the transport cycle of Escherichia coli EmrE. Protons and substrates share a common binding site organized around the absolutely conserved, membrane-embedded, glutamate 14 in transmembrane (TM) 3 helix 1 (8). Binding of positively charged hydrophobic substrates is coordinated by the Glu-14 side chain and stabilized by interactions with aromatic res...

Structural Basis for Allosteric Coupling at the Membrane-Protein Interface in Gloeobacter violaceus Ligand-gated Ion Channel (GLIC)

“…Purified protein was labeled with a methanethiosulfonate spin probe (1-oxyl-2,2,5,5-tetramethylpyrrolidin-3-yl) methyl methanethiosulfonate) (Toronto Research) at a 10:1 label:protein molar ratio. Mutants exhibiting dipolar coupling were underlabeled by protocols, as described previously (30). Briefly, mutants were labeled with a 0.5 molar excess of methanethiosulfonate label and incubated on ice for 30 min, after which a 20-fold molar excess of diamagnetic spin label (1-acetoxy-2,2,5,5-tetramethyl-⌬3-pyrroline-3-methyl) methane thiosulfonate was added and incubated for 2 h. The labeled protein was purified by size exclusion chromatography on a Superdex 20/200 column (GE Healthcare).…”

Protonation-dependent conformational dynamics of the multidrug transporter EmrE

“…Because the current structural data are exclusively for the TPP-bound state, we will consider it as a reference for the purpose of interpreting the distance distributions in a structural context. Previous work from our laboratory provided a complete view of the accessibility and mobility profile of spin labels in this state (5). The data pointed to extensive disagreement between the crystal structure and the conformation in liposomes.…”

“…The nature of these disagreements and the low resolution of the structure lead to the conclusion that there are substantial issues with the orientation of helices in the crystal structure (5).…”

“…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)(3)(4)(5)(6)(7). Seminal work from the Schuldiner laboratory over the last two decades has unlocked mechanistic principles of substrate-ionantiport (1,(8)(9)(10).…”

Detergent-mediated incorporation of transmembrane proteins in giant unilamellar vesicles with controlled physiological contents

“…11 and 12). Even in the case of highly hydrophobic proteins, short peripheral loops, as for EmrE (17) or for LeuT (18), or five negatively charged amino acids in the carboxylic tail of BR (19), are sufficient to produce a single orientation in the membrane.…”