On Parallel and Antiparallel Topology of a Homodimeric Multidrug Transporter

Abstract

The recently suggested antiparallel topology of EmrE has intriguing implications for many aspects of the biology of ioncoupled transporters. However, it is at odds with biochemical data that demonstrated the same topology for all protomers in the intact cell and with extensive cross-linking studies. To examine this apparent contradiction we chemically cross-linked dimers with a rigid bifunctional maleimide using Cys replacements at positions not permissible by an antiparallel topology. A purified cross-linked dimer binds substrate and transports it in proteoliposomes with kinetic constants similar to those of the non-cross-linked dimer. The cross-linked dimers do not interact with non-cross-linked dimers as judged from the fact that inactive mutants do not affect their activity (negative dominance). The results support the contention that EmrE with parallel topology is fully functional. We show that the detergents used in crystallization increase the fraction of monomers in solution. We suggest that the antiparallel orientation observed is a result of the arrangement of the monomers in the crystal. Functionality of EmrE with the suggested antiparallel orientation of the monomers remains to be characterized.Recent publications describing high resolution structures of transporters are changing a field that has been waiting avidly for such advancements (1-4). This welcome revolution provides fundamental insights for designing new biochemical/biophysical approaches and will deepen understanding of transport mechanisms.However, in some cases, as for the channel-forming peptide Gramicidin, for the ABC transporter MsbA, and for EmrE, an Escherichia coli ion-coupled multidrug transporter, different structures have been reported for the same protein, and it is not evident that these purportedly different conformations are physiologically relevant (5-8). The question is raised whether these are proteins with multiple conformations that fulfill functions yet unknown to us or whether the reported conformations are an experimental artifact created by the different milieu the proteins face when removed from their native environments. This may turn out to be especially critical for membrane proteins where we can only feebly mimic the original conditions after solubilization with detergents. A leading criterion at this stage should be whether the protein has some measurable function in the detergent-solubilized state.Two x-ray structures of EmrE have been published, and they are very different from each other (7,8). The presence of substrate in the second one may be responsible for the large differences between the two structures, although similar substrate-induced conformational changes were not observed in two-dimensional crystals (9, 10). The x-ray structure shows an asymmetric dimer with the protomers in an antiparallel topological orientation. This finding has obvious and exciting similarities to the internal structural repeat found in several membrane proteins such as aquaporins, ClC channel, and the neurotransmitter tran...

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

“…3B). These results are in agreement with cross-linking analysis in this detergent suggesting destabilization of the EmrE dimer (21). They are also consistent with the conclusion of Chen et al (17) that the crystal structure of apo EmrE in this detergent is likely to be misfolded.…”

“…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). Finally, limited structural constraints derived from EPR analysis of spin-labeled EmrE in TM3 were interpreted as consistent with a parallel orientation (19).…”

X-ray structure of EmrE supports dual topology model

“…4). Overall, our measured affinities are in general agreement with published values (6,7,(19)(20)(21) and show that the EmrE proteins used for these studies are functional in terms of substrate binding activity and stoichiometry.…”

“…4 (19). Despite the differences in binding affinity, however, the structure of the transporter is unchanged in these two detergents (discussed below and in Fig.…”

Identification of a Glycine Motif Required for Packing in EmrE, a Multidrug Transporter from Escherichia coli

“…A C␣ model of the transmembrane region was constructed by considering the evolutionary conservation pattern of each helix (34) and was supported by a re-evaluation of the data published in the retracted papers (38). Our own work demonstrates that dimers with parallel topology are functional and the functionality of antiparallel dimers remains to be established (36,39,40). Therefore, we cannot use the available structural information to learn whether TM4s are indeed directly involved in the interdimeric interface.…”