TY - JOUR
T1 - Crosslinking of membrane-embedded cysteines reveals contact points in the EmrE oligomer
AU - Soskine, Misha
AU - Steiner-Mordoch, Sonia
AU - Schuldiner, Shimon
PY - 2002/9/17
Y1 - 2002/9/17
N2 - 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 HgCl2, 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 HgCl2 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.
AB - 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 HgCl2, 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 HgCl2 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.
KW - Drug resistance
KW - Helix packing
KW - Ion-coupled transporter
KW - Mercurials
KW - o-PDM
UR - http://www.scopus.com/inward/record.url?scp=0037126033&partnerID=8YFLogxK
U2 - 10.1073/pnas.192392899
DO - 10.1073/pnas.192392899
M3 - ???researchoutput.researchoutputtypes.contributiontojournal.article???
C2 - 12221291
AN - SCOPUS:0037126033
SN - 0027-8424
VL - 99
SP - 12043
EP - 12048
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 19
ER -