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Small is Mighty: EmrE, a Multidrug Transporter as an Experimental Paradigm

Shimon Schuldiner, Dorit Granot, Sonia Steiner Mordoch, Shira Ninio, Dvir Rotem, Michael Soskin, Christopher G. Tate and Hagit Yerushalmi

S. Schuldiner, D. Granot, S. Steiner Mordoch, S. Ninio, D. Rotem, M. Soskin, and H. Yerushalmi are at the Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel. C. G. Tate is at the MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.

	Abstract

 
EmrE is a multidrug transporter from Escherichia coli that functions as a homooligomer and is unique in its small size. In each monomer there are four tightly packed transmembrane segments and one membrane-embedded charged residue. This residue provides the basis for the coupling mechanism as part of a binding site "time shared" by substrates and protons.

	Introduction

Top Introduction EmrE as an experimental... Oligomeric structure of EmrE The substrate pathway: a... A model for coupling... Physiological role of MDTs References

 
Multidrug transporters (MDTs) recognize a broad range of substrates with relatively high affinity and actively move them away from the cytoplasm. Since at times the substrates are toxic, these transporters have been associated with resistance to the effects of multiple drugs, antibiotics, and antineoplastic agents (4, 8). Multidrug resistance is a major concern in medical and agricultural diseases. In medicine, the emergence of resistance to multiple drugs is a significant obstacle in the treatment of several tumors as well as many infectious diseases. In agriculture, controlling the resistance of plant pathogens is of major economic importance. Because of the clinical relevance of these proteins and because of their apparently paradoxical ability of high affinity multidrug recognition, MDTs have been the topic of extensive studies.

A family comprising the smallest multidrug transporters (SMR) has been identified on the basis of their primary amino acid sequence similarity (9). In those cases in which they have been studied (5, 15), they were shown to extrude various drugs in exchange with protons, thereby rendering bacteria resistant to these compounds. All of the genes identified thus far as coding for SMR proteins are restricted to the eubacterial kingdom, both in Gram-negative and -positive organisms. SMRs have been identified in many pathogens (Fig. 1).

View larger version (43K): [in this window] [in a new window]   FIGURE 1. Model of EmrE. The 4 transmembrane regions are predicted by hydropathy plots and experimentally confirmed as described in the text. A partial list of the organisms in which at least one gene coding for a smallest multidrug transporter protein was identified is shown at bottom. TMS, transmembrane segment.

The EmrE protein has been characterized, purified, and reconstituted in a functional form (15, 18). EmrE-mediated transport is driven by a proton electrochemical gradient both in intact cells and in proteoliposomes (15). It behaves as an MDT capable of recognizing a wide range of substrates and inhibitors, including methyl viologen, ethidium, acriflavine, benzalkonium, and others (11, 15, 18).

	EmrE as an experimental paradigm: some unique properties

Top Introduction EmrE as an experimental... Oligomeric structure of EmrE The substrate pathway: a... A model for coupling... Physiological role of MDTs References

 
EmrE provides a unique model for the study of structure-function aspects of transport reactions in ion-coupled processes. Its small size offers a considerable advantage for mutagenesis studies and for studies that use unique replacements of specific residues. Solubility in organic solvents has provided a quick and quite efficient purification procedure for the wild-type protein and an opportunity to study its secondary structure by using high resolution heteronuclear nuclear magnetic resonance (12). After tagging the protein with a polyhistidine tail, it was possible to immobilize the detergent-solubilized and purified protein on Ni²⁺-nitrolotriacetic acid (NTA) beads and develop a very convenient binding assay of a high affinity substrate (6). This assay has provided a very quick tool to screen for appropriate detergents and for studying the stability of the protein (unpublished results). Some of the more salient stability properties are summarized in Fig. 2. EmrE tagged with six His residues (EmrE-His) can be solubilized with detergent and purified (6). When the purification is carried out under denaturing conditions in SDS (1%) and urea (6 M), as expected, no activity is detected (Fig. 2). However, after removal of the above reagents, activity was fully regained (Fig. 2). In addition, the stability to temperature is quite striking. EmrE retains the ability to bind a high affinity ligand, tetraphenylphosphonium (TPP⁺), even after prolonged incubations at 60 and 80C (Fig. 2) (D. Rotem, N. Salman, and S. Schuldiner, unpublished observations). This unusually high stability has proved very useful in our work with EmrE, allowing, for example, the development of an in vitro oligomerization assay described below.

View larger version (24K): [in this window] [in a new window]   FIGURE 2. Stability of EmrE activity to various treatments. Top: tetraphenylphosphonium (TPP+) binding activity (6) was measured for EmrE solubilized in dodecylmaltoside (control), exposed to SDS (1%) and urea (6 M), or washed with dodecylmaltoside after exposure to the latter agents. Bottom: TPP+ binding activity (6) was measured after exposure of the detergent-solubilized protein to the indicated temperatures for various times (D. Rotem, N. Salman, and S. Schuldiner, unpublished observations).

	Oligomeric structure of EmrE

Top Introduction EmrE as an experimental... Oligomeric structure of EmrE The substrate pathway: a... A model for coupling... Physiological role of MDTs References

The oligomeric state of EmrE was also investigated using detergent-solubilized EmrE, which binds the substrate TPP⁺ with high affinity. The stoichiometry of TPP⁺ binding suggests the existence of one binding site per trimer or tetramer (6).

For EmrE, we have now developed an in vitro method to study its oligomerization properties directly (D. Rotem, N. Salman, and S. Schuldiner, unpublished observations). In this method, membranes from cells with [³⁵S]Met-labeled EmrE and membranes from cells that overexpress EmrE-His (6) are solubilized with the detergent dodecylmaltoside and mixed. The amount of ³⁵S-labeled EmrE associated with Ni²⁺-NTA beads is used to assess the degree of oligomerization. The formation of the mixed oligomers (³⁵S-labeled EmrE + EmrE-His) is achieved after prior dissociation at relatively high temperature (~80°C). Wild-type EmrE and mutants of EmrE compete with ³⁵S-labeled EmrE for oligomerization with EmrE-His, whereas homologues of EmrE (EM109 and EM121) and other unrelated proteins do not. These results show that the formation of the homooligomers of EmrE is highly specific.

Recently, EmrE was crystallized in two dimensions and a projection structure was calculated to 7-Å resolution (Fig. 3) (14). The minimal structural unit of EmrE shown in Fig. 3 is composed of an arc of four tilted -helices separating two -helices nearly perpendicular to the membrane from another -helix nearly perpendicular to the membrane and a highly tilted -helix. The minimal structural unit thus has sufficient density to represent eight -helices, suggesting that EmrE is a dimer. One surprising feature of this structure is that it is asymmetric. An asymmetric projection map could arise from a symmetric EmrE homodimer if the two-fold axis is highly tilted with respect to the membrane normal, which would make it very hard to visualize. The other intriguing possibility is that the two EmrE monomers may have slightly different structures. A consequence of an asymmetric dimer would be that the two Glu¹⁴ residues that are essential for proton and drug transport could be structurally nonequivalent, raising interesting possibilities for the transport mechanism. More experimentation is now necessary to determine whether the asymmetric dimer associates in vivo to form a tetramer or higher oligomeric structures.

View larger version (57K): [in this window] [in a new window]   FIGURE 3. Projection structure of EmrE. The electron density map is shown at 7Å resolution. -Helices that are nearly perpendicular to the membrane are labeled P, and highly tilted -helices are labeled T. The arc of 4 potential -helices is labeled 4xT.

	The substrate pathway: a "hydrophobic channel"

Top Introduction EmrE as an experimental... Oligomeric structure of EmrE The substrate pathway: a... A model for coupling... Physiological role of MDTs References

In the case of EmrE, the substrates are quite hydrophobic, and therefore it may be energetically more favorable to interact directly with the protein rather than permeate through a hydrophilic pathway. Movement of substrates through a tightly packed protein must require disruption and reorganization of the existing structure.

	A model for coupling of H+ and substrate fluxes based on "time sharing" of a common binding site

Top Introduction EmrE as an experimental... Oligomeric structure of EmrE The substrate pathway: a... A model for coupling... Physiological role of MDTs References

 
Glu¹⁴ is the only charged residue in the putative membrane domain of EmrE. This residue has an unusually high pK and is an essential part of the binding domain shared by substrates and protons. The occupancy of the binding domain is mutually exclusive, and, as such, this provides the molecular basis for the obligatory exchange catalyzed by EmrE. In our view of the alternate access model for EmrE, we postulate that a substrate molecule is bound in a hydrophobic pocket via an interaction with Glu¹⁴ (17, 18). Glu¹⁴ residues in each monomer presumably participate in the binding, forming a charged cluster in which the charged Glu¹⁴ residues could be neutralized by protons. The binding interaction of the substrate with different parts of the protein, and the electrostatic interactions with the Glu cluster, influence the latter in such a way that it induces release of the protons. Following this, the binding site, now occupied by the substrate, becomes modified so that it is accessible to the other face of the membrane. The interaction of the delocalized charge in the substrate with the charged Glu¹⁴ residues in the protein is likely to be strong in the hydrophobic environment of the putative binding site. Such a stable complex can be efficiently dissociated only when renewed proton binding to the cluster occurs. This assumption is experimentally supported by the finding that low pH accelerates TPP⁺ release from the protein. Therefore, we suggest that the ternary complex H⁺-EmrE-substrate is very short lived. After protonation and substrate release, the binding site relaxes back to the other face of the membrane so that a new cycle can start.

The role of carboxylic residues in substrate binding and H⁺ translocation has been postulated in the mechanism proposed for the lac permease. (3, 10). A common feature of both transporters is that carboxyl residues with unusually high pK play a central role. Changes in the occupancy of the substrate binding site induce changes in the protein that modify the pK_a of one or more of these residues. This results in protonation or deprotonation of the residues followed by conformational changes enabling vectorial proton translocation. Residues with an unusually high pK_a are also found in other membrane proteins, such as bacteriorhodopsin and the F₀F₁ ATPase subunit c. Both Asp⁹⁶ in bacteriorhodopsin and Asp⁶¹ in subunit c exhibit very high pK_a and are critical for proton translocation in these proteins.

Several major differences exist between lac permease and EmrE besides their size: in the lac permease movement of the substrate and the coupling ion is in the same direction (cotransport or symport), whereas in EmrE it is in opposite directions (antiport). In addition, the lac permease has been shown to be very flexible and probably contains water-filled cavities (10), but this is not the case for EmrE. Finally, the nature of the substrate, the H⁺/substrate stoichiometry, and the specificity clearly differ. In the lac permease, substrate exchange can occur without H⁺ release because sugar is released before protons (reviewed in Ref. 10). In EmrE, on the other hand, both binding and release of substrate can occur only on the corresponding release or binding of protons. For EmrE, these findings suggest a direct mechanism of coupling based on the mutually exclusive occupancy of a single binding site. In the lac permease, the two sites are suggested to be distinct, and they interact with each other through conformational changes of the protein. Although EmrE shows the simplest mode of coupling and demonstrates the advantage of this transporter as a model system, it is most likely that in the larger transporters the more complex modes of coupling have evolved to provide additional flexibility, modes of regulation, and functions still unknown to us.

	Physiological role of MDTs

Top Introduction EmrE as an experimental... Oligomeric structure of EmrE The substrate pathway: a... A model for coupling... Physiological role of MDTs References

 
The resistance associated with the activity of MDTs poses a serious problem in medicine and agriculture. However, these traits existed for aeons before the drugs and antibodies were discovered by humans. Resistance to a wide range of cytotoxic compounds is a common phenomenon observed in many organisms throughout the evolutionary scale and probably developed to cope with the variety of toxic compounds that are part of the natural environment in which living cells dwell. Only those organisms that have developed through evolution the ability to cope with a wide variety of compounds have been able to survive. One of the strategies that evolved is removal of toxic substances by MDTs. A question commonly asked about MDTs is what their "real" function is (for a more detailed discussion and references see, for example, Ref. 7). Are these proteins functioning solely for protection of the organism against toxic compounds, or do they have a very specific function and, just accidentally, happen to be polyspecific as well? The answer seems to be a complex one: clearly in proteins functioning in the blood brain barrier or in the kidney, there is little doubt that they are protecting the organism against toxic compounds by removing them from the organism or by preventing their passage to the brain. In the case of bacterial proteins, whose expression is regulated by multiple xenobiotics, it also seems reasonable that they still have a major role in protection of the cell, as judged from their regulation (7). Yet, in other cases, it seems that some proteins have evolved to perform specific roles other than multidrug resistance, i.e., the vesicular neurotransmitter transporters, lipid translocators, and bacterial amino acid and sugar transporters from the ATP binding cassette family (7).

Whatever their role, MDTs are interesting proteins that can handle recognition and transport of a wide variety of toxicants. A basic question, which can be asked of all known transporters, pumps, and channels, remains unanswered: what is the molecular mechanism mediating substrate recognition and translocation? In addition, in the case of MDTs, what is the basis for recognition of a wide variety of substrates with high affinity? We believe that EmrE, because of its size and its unique properties, provides an excellent experimental paradigm to approach the above questions.

	Acknowledgments

 
Work in our laboratory is supported by grants from the Deutsche-Israeli Program (BMBF International Bureau at the DLR) and from The Israel Science Foundation.

For correspondence, contact S. Schuldiner (shimon.schuldiner{at}huji.ac.il).

	References

Top Introduction EmrE as an experimental... Oligomeric structure of EmrE The substrate pathway: a... A model for coupling... Physiological role of MDTs References

 

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