Parallel topology of genetically fused EmrE homodimers

Steiner‐Mordoch, Sonia; Soskine, Misha; Solomon, Dalia; Rotem, Dvir; Gold, Ayala; Yechieli, Michal; Adam, Yoav; Schuldiner, Shimon

doi:10.1038/sj.emboj.7601951

Cited by 47 publications

(42 citation statements)

References 34 publications

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“…minus of the first to N terminus of the second monomer (tail to head). This was achieved by means of defined linkers that are not compatible with an antiparallel topology either because they are too hydrophilic or too short (9). In these studies we showed that all the constructs are functional in vivo because they confer resistance to EmrE substrates, and they catalyze energy-dependent ethidium efflux.…”

Section: Forcing a Homodimer With Identical Protomers Into An Antiparmentioning

confidence: 98%

“…The segment inserted was based on the sequence of a mutant of human glycophorin A (GpA) that does not dimerize (28). Such a fusion, with an odd number of TMs, would have the N and C termini at opposite sides of the membrane, contrasting with the previously generated eight TM proteins with hydrophilic linkers of variable length (9). Fusions with several sequences of GpA and several linkers were constructed (supplemental Table S1), and they all yielded functional proteins.…”

Section: Forcing a Homodimer With Identical Protomers Into An Antiparmentioning

confidence: 99%

“…This was achieved by means of defined linkers that are not compatible with an antiparallel topology either because they are too hydrophilic or too short (9).…”

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confidence: 99%

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Topologically Random Insertion of EmrE Supports a Pathway for Evolution of Inverted Repeats in Ion-coupled Transporters

Nasie¹,

Steiner‐Mordoch²,

Gold

et al. 2010

Journal of Biological Chemistry

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Inverted repeats in ion-coupled transporters have evolved independently in many unrelated families. It has been suggested that this inverted symmetry is an essential element of the mechanism that allows for the conformational transitions in transporters. We show here that small multidrug transporters offer a model for the evolution of such repeats. This family includes both homodimers and closely related heterodimers. In the former, the topology determinants, evidently identical in each protomer, are weak, and we show that for EmrE, an homodimer from Escherichia coli, the insertion into the membrane is random, and dimers are functional whether they insert into the cytoplasmic membrane with the N-and C-terminal domains facing the inside or the outside of the cell. Also, mutants designed to insert with biased topology are functional regardless of the topology. In the case of EbrAB, a heterodimer homologue supposed to interact antiparallel, we show that one of the subunits, EbrB, can also function as a homodimer, most likely in a parallel mode. In addition, the EmrE homodimer can be forced to an antiparallel topology by fusion of an additional transmembrane segment. The simplicity of the mechanism of coupling ion and substrate transport and the few requirements for substrate recognition provide the robustness necessary to tolerate such a unique and unprecedented ambiguity in the interaction of the subunits and in the dimer topology relative to the membrane. The results suggest that the small multidrug transporters are at an evolutionary junction and provide a model for the evolution of structure of transport proteins.Evolution of large transporter genes is thought to have started from a small single one that duplicated, evolved independently, and then fused to the original one to generate a new gene coding for the large polytopic protein (1). Inverted repeats in ion-coupled transporters have now been observed in transporters from many different families (2, 3). It has been suggested that this inverted symmetry is an essential element of the mechanism that allows for the conformational transitions in transporters (3).A possible mode for evolution of these inverted repeats has been suggested based on the supposedly inverted topology of EmrE, a small multidrug transporter from Escherichia coli. A claim for an antiparallel topology of the monomers in the EmrE homodimer was made, but this is still a controversial issue and, in our view, is still inconsistent with the biochemical data. The reasoning that the topology of the monomers in the EmrE dimer is parallel and N in -C in was supported by data from our laboratory that demonstrated the same topology for all protomers in the intact cell and in membrane vesicles (4), by biochemical studies that established the equivalence of the residues in the protomers (reviewed in Ref. 5 and 6), and by extensive cross-linking studies that suggested the proximity of equivalent residues in the two monomers (7). Moreover, dimers cross-linked at positions not compatible with antiparalle...

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Section: Forcing a Homodimer With Identical Protomers Into An Antiparmentioning

confidence: 98%

Section: Forcing a Homodimer With Identical Protomers Into An Antiparmentioning

confidence: 99%

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Topologically Random Insertion of EmrE Supports a Pathway for Evolution of Inverted Repeats in Ion-coupled Transporters

Nasie¹,

Steiner‐Mordoch²,

Gold

et al. 2010

Journal of Biological Chemistry

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“…These include the design and construction of functional EmrE chimeras where monomers are linked by short polar loops not favored energetically to cross the bilayer (20). Furthermore, residues predicted to be on opposite sides of the membrane in the antiparallel dimer model can be cross-linked without significant perturbation of transport (21).…”

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confidence: 99%

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

Amadi¹,

Koteiche²,

Mishra

et al. 2010

Journal of Biological Chemistry

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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...

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“…The structure and topology of the homodimeric carrier protein remained controversial (Ninio et al, 2004;McHaourab et al, 2008;Steiner-Mordoch et al, 2008;Korkhov et al, 2009;Henzler-Wildman, 2012) until a joint NMR and single-molecule FRET investigation has recently revealed an asymmetric antiparallel arrangement with inward-and outward-facing subunits related by a pseudo-twofold symmetry axis parallel to the membrane plane (Morrison et al, 2012). Furthermore, solid state NMR and cryo-EM have demonstrated asymmetry at the ligand-binding pocket (Lehner et al, 2008), whereas spin labelling and EPR have revealed asymmetry in the TM helices .…”

Section: E Primary and Secondary Transportersmentioning

confidence: 99%

Asymmetric perturbations of signalling oligomers

Maksay

Tőke

2014

Progress in Biophysics and Molecular Biology

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This review focuses on rapid and reversible noncovalent interactions for symmetric oligomers of tetramers (voltage-gated cation channels, ionotropic glutamate receptor, CNG and CHN channels); pentameric ligand-gated and mechanosensitive channels; higher order oligomers (gap junction channel, chaperonins, proteasome, virus capsid); as well as primary and secondary transporters. In conclusion, asymmetric perturbations seem to play important functional roles in a broad range of communicating networks.

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Parallel topology of genetically fused EmrE homodimers

Cited by 47 publications

References 34 publications

Topologically Random Insertion of EmrE Supports a Pathway for Evolution of Inverted Repeats in Ion-coupled Transporters

Topologically Random Insertion of EmrE Supports a Pathway for Evolution of Inverted Repeats in Ion-coupled Transporters

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

Asymmetric perturbations of signalling oligomers

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