Exploring the role of a unique carboxyl residue in EmrE by mass spectrometry.
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.
Weinglass
Adam B
AB
Howard Hughes Medical Institute, Department of Physiology, Molecular Biology Institute, University of California Los Angeles, 90095-1662, USA.
Soskine
Misha
M
Vazquez-Ibar
Jose-Luis
JL
Whitelegge
Julian P
JP
Faull
Kym F
KF
Kaback
H Ronald
HR
Schuldiner
Shimon
S
D017920:Antiporters / Q000737:chemistry* / Q000648:ultrastructure; D001665:Binding Sites; D001692:Biological Transport; D002234:Carbodiimides / Q000494:pharmacology; D003488:Cyanogen Bromide / Q000737:chemistry; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D018698:Glutamic Acid / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry* / Q000648:ultrastructure; D008954:Models, Biological; D008956:Models, Chemical; D009861:Onium Compounds / Q000737:chemistry; D009943:Organophosphorus Compounds / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011522:Protons; D021241:Spectrometry, Mass, Electrospray Ionization; D013997:Time Factors
D017920:Antiporters; D002234:Carbodiimides; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D009861:Onium Compounds; D009943:Organophosphorus Compounds; D010455:Peptides; D011522:Protons; C075436:EmrE protein, E coli; D018698:Glutamic Acid; C081611:1,3-diisopropylcarbodiimide; D003488:Cyanogen Bromide; C013289:tetraphenylphosphonium