Crosslinking of membrane-embedded cysteines reveals contact points in the EmrE oligomer.
EmrE is a small multidrug transporter that extrudes various drugs in exchange with protons, thereby rendering Escherichia coli cells resistant to these compounds. In this study, relative helix packing in the EmrE oligomer solubilized in detergent was probed by intermonomer crosslinking analysis. Unique cysteine replacements in transmembrane domains were shown to react with organic mercurials but not with sulfhydryl reagents, such as maleimides and methanethiosulfonates. A new protocol was developed based on the use of HgCl(2), a compound known to react rapidly and selectively with sulfhydryl groups. The reaction can bridge vicinal pairs of cysteines and form an intermolecular mercury-linked dimer. To circumvent problems inherent to mercury chemistry, a second crosslinker, hexamethylene diisocyanate, was used. After the HgCl(2) treatment, excess reagent was removed and the oligomers were dissociated with a strong denaturant. Only those previously crosslinked reacted with hexamethylene diisocyanate. Thus, vicinal cysteine-substituted residues in the EmrE oligomer were identified. It was shown that transmembrane domain (TM)-1 and TM4 in one subunit are in contact with the corresponding TM1 and TM4, respectively, in the other subunit. In addition, TM1 is also in close proximity to TM4 of the neighboring subunit, suggesting possible arrangements in the binding and translocation domain of the EmrE oligomer. This method should be useful for other proteins with cysteine residues in a low-dielectric environment.
Soskine
Misha
M
Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem 91904, Israel.
Steiner-Mordoch
Sonia
S
Schuldiner
Shimon
S
D000595:Amino Acid Sequence; D017920:Antiporters / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000737:chemistry; D024901:Drug Resistance, Multiple, Bacterial; D004926:Escherichia coli / Q000737:chemistry / Q000187:drug effects / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
D017920:Antiporters; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; C075436:EmrE protein, E coli; D003545:Cysteine