Article

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

ABSTRACT

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.

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Available from: José Luis Vázquez-Ibar, Nov 18, 2015
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    • "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. "
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