Crosslinking of membrane-embedded cysteines reveals contact points in the EmrE oligomer

Abstract

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.

Fig 1.

Secondary structure model of EmrE-His. The residues in the square are the Myc-His tag.

Fig 2.

(A) Denaturation prevents intermonomer crosslinking in EmrE. ³⁵S-labeled CL-EmrE-His-K22C (lanes 1 and 2) or CL-EmrE-His (lanes 3 and 4) membranes were treated with 100 μM o-PDM or 0.2% HMDC reagent, respectively, in 0.8% DDM-Na buffer (lanes 1 and 3) or SDS-urea buffer (lanes 2 and 4). (B) HMDC crosslinking of DDM-solubilized EmrE mutants. DDM-solubilized membranes from the following mutants radiolabeled with ³⁵S were analyzed after treatment with 0.2% HMDC. Lane 1, EmrE-K22C; lane 2, CLA-EmrE-His K22C; lane 3, EmrE-His K22R; lane 4, EmrE-K22R; lane 5, EmrE.

Fig 3.

HgCl₂ crosslinking of DDM-solubilized protein followed by denaturation and HMDC crosslinking. (A) Experimental scheme flow sheet: [³⁵S]EmrE unique cysteine replacements were solubilized in 0.8% DDM and treated with 50 μM HgCl₂. After removal of the unreacted HgCl₂, denaturation in SDS-urea buffer dissociated oligomers that did not crosslink. The second crosslinking was performed with 0.2% HMDC. Samples were reduced with 1.5% 2-mercaptoethanol and assayed by SDS/PAGE. (B) DDM-solubilized membranes of CL-95C (lanes 1–3) or CL-39C (lanes 4–6) were crosslinked with HgCl₂, denatured with SDS-urea buffer, and further treated with HMDC (lanes 3 and 6, respectively) as described in Experimental Procedures and schematically in A. Controls with HMDC alone are shown in lanes 1 and 4. Controls where the HgCl₂ crosslinking was performed under denaturing conditions are shown in lanes 2 and 5.

Fig 4.

Intermonomer crosslinking between different unique cysteine replacements of EmrE. The indicated ³⁵S-untagged unique cysteine replacements were crosslinked with 100 μM o-PDM without (lanes 1–4) or with mixing with [³⁵S]E14C EmrE-His (lanes 6–9). Lane 5 shows the crosslinking in the [³⁵S]E14C EmrE-His oligomer. U, untagged; T, tagged.

Fig 5.

(A) Helical wheel projection of TM4. Positions that produced dimer on crosslinking with HgCl₂ are labeled in gray. Those that did not form dimer are labeled black. Residues in white were not tested. (B) Summary of the experimental constraints determined by crosslinking in this work. The TMs are represented by circles and the distances are from o-PDM (8 Å) or from Hg²⁺ (4 Å) crosslinking. On the right side, the results with the hetero-oligomers are summarized. Negative crosslinking results indicate a distance larger than 8 Å. (C) Helix packing model of the EmrE dimer. Two monomers are shown where the TMs are represented with circles and the loops with curved lines. The helical contacts determined in this work are marked with straight thick lines.