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

Abstract

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.

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

“…Purification was performed essentially as in Ref. 7. Reconstitution was performed as described (28).…”

“…Trp-63 has been shown to be essential and even a conservative replacement by an aromatic residue yielded an inactive transporter (7). Tyr-40 and Tyr-60 can be only replaced with hydroxyamino acids (8).…”

Transforming a drug/H + antiporter into a polyamine importer by a single mutation

“…Trp63 is completely conserved in the SMR family, and we previously showed that it is essential for EmrE function (7). However, because we did not previously use a Gly replacement, we investigated the W63G mutant.…”

“…The new mutant gained a new specificity but also conserved its ability to transport most of the original substrates. On the other hand, the mutation conferring resistance to erythromycin was a replacement of Trp63, a fully conserved and essential amino acid (7). Interestingly, this mutant completely lost the capacity to recognize and remove the classical substrates of EmrE but gained, in addition to its ability to confer resistance to erythromycin, a completely new functionthe ability to import polyamines into the cell.…”

Probing of the rates of alternating access in LacY with Trp fluorescence

“…However, functionally important Trp residues are found in the binding sites of different transport proteins where they are involved in stacking or -interactions (23)(24)(25)(26)(27)(28)(29). Soluble sugar-binding proteins (30)(31)(32), as well as two sugar transport proteins, contain Trp (8) or Tyr (33) residues that undergo aromatic interactions with sugar in the binding site.…”