Abstract
The recently suggested antiparallel topology of EmrE has intriguing implications for many aspects of the biology of ion-coupled 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.
Highlights
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 [5,6,7,8]
Single Cysteine Replacements Chosen to Probe Relative Arrangement of the EmrE transmembrane 4 (TM4) Segments within the Dimer— EmrE contains four transmembrane helices (TM), connected by relatively short loops
To reevaluate our data obtained with the use of various cross-linking reagents we focused on two single cysteine replacements at the N and C termini of the TM4, D84C and T108C
Summary
The recently suggested antiparallel topology of EmrE has intriguing implications for many aspects of the biology of ioncoupled transporters 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. 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 transporter homologue LeuT [11,12,13] It is at odds with biochemical data that demonstrated the same topology for all protomers in the intact cell and in membrane vesicles [14] and with extensive cross-linking studies [15]. We suggest that the monomers generated are the ones that crystallize and the antiparallel arrangement in the crystal is, most likely, a crystallographic dimer
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