Functional Analysis of Novel Multidrug Transporters from Human Pathogens

Ninio, Shira; Rotem, Dvir; Schuldiner, Shimon

doi:10.1074/jbc.m108231200

Cited by 61 publications

(73 citation statements)

References 27 publications

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“…EmrE, Hsmr, BPsmr, PAsmr, and TBsmr) and heterodimers (e.g. EbrAB) have been identified (26,27,32,33). Membrane protein topology is largely governed by the positive-inside rule, i.e.…”

Section: Discussionmentioning

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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“…EmrE, Hsmr, BPsmr, PAsmr, and TBsmr) and heterodimers (e.g. EbrAB) have been identified (26,27,32,33). Membrane protein topology is largely governed by the positive-inside rule, i.e.…”

Section: Discussionmentioning

confidence: 99%

“…The interaction with EmrE is a nonproductive one that may result in the formation of a heterodimer with low or no activity. We have previously shown that EmrE can interact with a variety of homologues forming heterodimers with various affinities (32).…”

Section: Discussionmentioning

confidence: 99%

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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“…The Glu14 residue is conserved in the SMR family multidrug transporters. 22) It has been reported with EmrE of E. coli that Glu14 is involved in the binding of substrate. 23) Thus, it is likely that Glu14 in the SsmE is also involved in substrate binding.…”

Section: Discussionmentioning

confidence: 99%

Functional Gene Cloning and Characterization of the SsmE Multidrug Efflux Pump from Serratia marcescens

Minato

Shahcheraghi

Ogawa

et al. 2008

Biological & Pharmaceutical Bulletin

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We cloned a gene responsible for multidrug resistance from chromosomal DNA of Serratia marcescens, and determined the nucleotide sequence. We designated the gene as ssmE. The deduced amino acid sequence of SsmE showed high similarity with the small multidrug resistance (SMR)-type multidrug efflux pumps. Cells of Escherichia coli KAM32, a drug hyper-susceptible mutant, transformed with a plasmid pESM437 carrying the ssmE gene showed elevated minimum inhibitory concentrations of structurally unrelated antimicrobial agents. E. coli KAM32/pESM437 showed elevated energy dependent efflux of ethidium. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed that ssmE was expressed in cells of S. marcescens.

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“…Structural and biochemical evidence suggests that the basic EmrE oligomer is a dimer (7)(8)(9)(10). EmrE has only one membraneembedded charged residue, Glu14, that is conserved in more than 100 homologous proteins (5,11) and plays a central role in substrate and proton binding (12)(13)(14)(15). Deprotonation of Glu14 is required for substrate binding (4,14).…”

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confidence: 99%

“…It is a small (110-residue) multidrug transporter that extrudes various positively charged drugs in exchange for protons, thus rendering bacteria resistant to these drugs (1)(2)(3). The protein has been characterized, purified, and reconstituted in a functional form (1,(4)(5)(6). High-affinity substrate binding has been established as a reliable and sensitive assay for activity of the detergent-solubilized transporter (4).…”

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confidence: 99%

Substrate-Induced Tryptophan Fluorescence Changes in EmrE, the Smallest Ion-Coupled Multidrug Transporter

et al. 2005

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Tryptophan residues may play several roles in integral membrane proteins including direct interaction with substrates. In this work we studied the contribution of tryptophan residues to substrate binding in EmrE, a small multidrug transporter of Escherichia coli that extrudes various positively charged drugs across the plasma membrane in exchange with protons. Each of the four tryptophan residues was replaced by site-directed mutagenesis. The only single substitutions that affected the protein's activity were those in position 63. While cysteine and tyrosine replacements yielded a completely inactive protein, the replacement of Trp63 with phenylalanine brought about a protein that, although it could not confer any resistance against the toxicants tested, could bind substrate with an affinity 2 orders of magnitude lower than that of the wild-type protein. Double or multiple cysteine replacements at the other positions generate proteins that are inactive in vivo but regain their activity upon solubilization and reconstitution. The findings suggest a possible role of the tryptophan residues in folding and/or insertion. Substrate binding to the wild-type protein and to a mutant with a single tryptophan residue in position 63 induced a very substantial fluorescence quenching that is not observed in inactive mutants or chemically modified protein.The reaction is dependent on the concentration of the substrate and saturates at a concentration of 2.57 µM with the protein concentration of 5 µM supporting the contention that the functional unit is a dimer. These findings strongly suggest the existence of an interaction between Trp63 and substrate, and the nature of this interaction can now be studied in more detail with the tools developed in this work.

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Functional Analysis of Novel Multidrug Transporters from Human Pathogens

Cited by 61 publications

References 27 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

Functional Gene Cloning and Characterization of the SsmE Multidrug Efflux Pump from Serratia marcescens

Substrate-Induced Tryptophan Fluorescence Changes in EmrE, the Smallest Ion-Coupled Multidrug Transporter

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