Antiparallel dimers of the small multidrug resistance protein EmrE are more stable than parallel dimers.
The bacterial multidrug transporter EmrE is a dual-topology membrane protein and as such is able to insert into the membrane in two opposite orientations. The functional form of EmrE is a homodimer; however, the relative orientation of the subunits in the dimer is under debate. Using EmrE variants with fixed, opposite orientations in the membrane, we now show that, although the proteins are able to form parallel dimers, an antiparallel organization of the subunits in the dimer is preferred. Blue-native PAGE analyses of intact oligomers and disulfide cross-linking demonstrate that in membranes, the proteins form parallel dimers only if no oppositely orientated partner is present. Co-expression of oppositely orientated proteins almost exclusively yields antiparallel dimers. Finally, parallel dimers can be disrupted and converted into antiparallel dimers by heating of detergent-solubilized protein. Importantly, in vivo function is correlated clearly to the presence of antiparallel dimers. Our results suggest that an antiparallel arrangement of the subunits in the dimer is more stable than a parallel organization and likely corresponds to the functional form of the protein.
Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University SE-106 91 Stockholm, Sweden.
Pilar
P
Lloris-Garcerá
Frans
F
Bianchi
Joanna S G
JS
Slusky
Susanna
S
Seppälä
Daniel O
DO
Daley
Gunnar
G
von Heijne
D017920:Antiporters / Q000737:chemistry / Q000378:metabolism*; D003553:Cystine / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary
D017920:Antiporters; D029968:Escherichia coli Proteins; C075436:EmrE protein, E coli; D003553:Cystine