Exploring the role of a unique carboxyl residue in EmrE by mass spectrometry
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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ABSTRACT: Antiporters are ubiquitous membrane proteins that catalyze obligatory exchange between two or more substrates across a membrane in opposite directions. Some utilize proton electrochemical gradients generated by primary pumps by coupling the downhill movement of one or more protons to the movement of a substrate. Since the direction of the proton gradient usually favors proton movement towards the cytoplasm their function results in removal of substrates other than protons from the cytoplasm, either into acidic intracellular compartments or out to the medium. H(+)-coupled antiporters play central roles in living organisms, e.g. storage of neurotransmitter and other small molecules, resistance to antibiotics, homeostasis of ionic content and more. Biochemical and structural data support a general mechanism for H(+)-coupled antiporters whereby the substrate and the protons cannot bind simultaneously to the protein. In several cases, it was shown that the binding sites overlap and therefore there is a direct competition between the protons and the substrate. In others, the "competition" seems to be indirect and it is most likely achieved by allosteric mechanisms. To ensure the feasibility of such a mechanism the pKa of one or more carboxyls in the protein must be tuned appropriately. In this review I discuss in detail the case of EmrE, a multidrug transporter from Escherichia coli and evaluate the information available for other H(+)-coupled antiporters.Journal of Molecular Biology 05/2014; DOI:10.1016/j.jmb.2014.05.020 · 3.96 Impact Factor