Exploring the Role of a Unique Carboxyl Residue in EmrE by Mass Spectrometry

Department of Biological Chemistry, Hebrew University of Jerusalem, Yerushalayim, Jerusalem, Israel
Journal of Biological Chemistry (Impact Factor: 4.57). 04/2005; 280(9):7487-92. DOI: 10.1074/jbc.M413555200
Source: PubMed


EmrE is a small multidrug transporter in Escherichia coli that extrudes various positively charged drugs across the plasma membrane in exchange with protons, thereby rendering cells resistant to these compounds. Biochemical experiments indicate that the basic functional unit of EmrE is a dimer where the common binding site for protons and substrate is formed by the interaction of an essential charged residue (Glu-14) from both EmrE monomers. Carbodiimide modification of EmrE has been studied using functional assays, and the evidence suggests that Glu-14 is the target of the reaction. Here we exploited electrospray ionization mass spectrometry to directly monitor the reaction with each monomer rather than following inactivation of the functional unit. A cyanogen bromide peptide containing Glu-14 allows the extent of modification by the carboxyl-specific modification reagent diisopropylcarbodiimide (DiPC) to be monitored and reveals that peptide 2NPYIYLGGAILAEVIGTTLM(21) is approximately 80% modified in a time-dependent fashion, indicating that each Glu-14 residue in the oligomer is accessible to DiPC. Furthermore, preincubation with tetraphenylphosphonium reduces the reaction of Glu-14 with DiPC by up to 80%. Taken together with other biochemical data, the findings support a "time sharing" mechanism in which both Glu-14 residues in a dimer are involved in tetraphenylphosphonium and H(+) binding.

Download full-text


Available from: José Luis Vázquez-Ibar, Nov 18, 2015
  • Source
    • "Thus, using electrospray ionization mass spectrometry it was directly demonstrated that the two essential Glu-14 in the dimer are quantitatively (N80 %) modified by carbodiimides in a way that suggested that they are both in a similarly hydrophobic environment [40]. Moreover, tetraphenylphosphonium (TPP + ), a substrate that binds with high affinity, reduces modification of Glu- 14 by ∼ 80%, indicating that both Glu-14 residues in the functional unit are close enough to the binding site that TPP + prevents their modification or that they might be equivalently protected in an allosteric manner by TPP + [40]. The results suggest that the Glu-14 residues in the EmrE dimer are functionally equivalent. "
    [Show abstract] [Hide abstract]
    ABSTRACT: EmrE is a small (110 residues) SMR transporter from Escherichia coli that extrudes positively charged aromatic drugs in exchange for two protons, thus rendering bacteria resistant to a variety of toxic compounds. Due to its size, stability and retention of its function upon solubilization in detergent, EmrE provides a unique experimental paradigm for the biochemical and biophysical studies of membrane based ion-coupled transporters. In addition, EmrE has been in center stage in the past two years because it provides also a paradigm for the study of the evolution of membrane proteins. Controversy around this topic is still going on and some novel concepts are surfacing that may contribute to our understanding of evolution of topology of membrane proteins. Furthermore, based on the findings that the cell multidrug transporters interact functionally we introduce the concept of a cell Resistosome.
    Preview · Article · May 2009 · Biochimica et Biophysica Acta
  • [Show abstract] [Hide abstract]
    ABSTRACT: This paper considers the problem of extracting the signal powers and steering vectors from the signal covariance matrix without the knowledge of array manifold. Under the assumption of narrowband and uncorrelated signals, it is shown that it is necessary and sufficient for the signal powers and steering vectors to satisfy two conditions, termed the signal subspace and orthogonality conditions, in order for them to match the signal covariance matrix asymptotically. Based upon these two conditions, an algorithm is derived to iteratively search for the signal powers and steering vectors which closely match the signal covariance matrix estimated from the observed data. A real-data example is presented to illustrate the robustness of the proposed algorithm
    No preview · Conference Paper · Dec 1993
  • Source
    [Show abstract] [Hide abstract]
    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 microM with the protein concentration of 5 microM 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.
    Full-text · Article · Jun 2005 · Biochemistry
Show more