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 carboxylspecific modification reagent diisopropylcarbodiimide (DiPC) to be monitored and reveals that peptide 2 NPY-IYLGGAILAEVIGTTLM 21 is ϳ80% 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.EmrE, 1 an Escherichia coli multidrug resistance protein, provides a unique model for the study of polytopic membrane proteins. It is a small (110-residue) multidrug transporter that extrudes various positively charged drugs in exchange for protons, thus rendering bacteria resistant to these drugs (Fig. 1, A and B) (1-3). The protein has been characterized, purified, and reconstituted in a functional form (1, 4 -6). High affinity substrate binding has been established as a reliable and sensitive assay for activity of the detergent-solubilized transporter (4). Structural and biochemical evidence suggests that the basic EmrE oligomer is a dimer (7-10). EmrE has only one membrane-embedded charged residue, Glu-14, which is conserved in more than 100 homologous proteins (5). Acidic side chains embedded in the membrane have been shown to be important for activity in various ion-coupled transporters (for a review, see Ref. 11). In some cases, these acidic side chains are involved in substrate recognition and binding; in others, they comprise part of the coupling ion translocation pathway. EmrE is unique in that the same acidic side chain (the carboxyl group of Glu-14) is involved in recognition of both substrate and the coupling ion. Furthermore, because replacement of the two other acidic side chains in EmrE (E25C/D84C) 2 does not detrimentally effect the activity of the protein, it is concluded that the only carboxyl group required for catalysis is that of 13).Within the small multidrug resistance family of transporters, a comparative analysis reveals that the face of transmembrane domain 1 containing Glu-14 is conserved, displaying a helica...

Exploring the Binding Domain of EmrE, the Smallest Multidrug Transporter

“…four helices from each monomer, with density for TPP ϩ in a binding chamber formed from six out of the eight ␣-helices, confirming the suggestion that TPP ϩ binds near the center of the dimer (9). Our studies described here and previously with Glu 14 (34) suggest that residues in TM1 in the functional unit of EmrE are functionally equivalent, playing a direct role in a single binding site shared by protons and substrate. It is tempting to speculate that TM1 contributes two of the six helices forming the binding chamber.…”

Quasi-symmetry in the Cryo-EM Structure of EmrE Provides the Key to Modeling its Transmembrane Domain

“…12 (ii) Several key residues that were shown to be critical for substrate binding are not in a position to bind substrate in the structure. For instance, it was demonstrated by different experimental approaches that Glu14 residues from both monomers are crucial for translocation, participate in substrate and proton binding, [14][15][16][17][18][19] and are in proximity to one another. 20 By contrast, the X-ray structure shows that Glu14 from only one monomer forms partial contact with substrate and the two glutamate residues are over 20 Å apart.…”

The fast release of sticky protons: Kinetics of substrate binding and proton release in a multidrug transporter

“…EmrE provides a unique experimental paradigm not only because of its size and stability but also because, under proper conditions, the detergent-solubilized protein binds the substrate and releases protons in a mode that reflects, with high fidelity, its catalytic activity in the membrane; this has enabled a detailed study of the molecular basis of substrate recognition and the coupling between protons and substrate (4)(5)(6)(7)(8)(9)(10). EmrE has only one membrane-embedded charged residue, Glu-14, which is also conserved in Ͼ200 homologous proteins in bacteria and archaea (11,12).…”