The transmembrane domains of the ABC multidrug transporter LmrA form a cytoplasmic exposed, aqueous chamber within the membrane

The ABC multidrug transporter LmrA of Lactococcus lactis consists of six putative transmembrane segments (TMS) and a nucleotide binding domain. LmrA functions as a homodimer in which the two membrane domains form the solute translocation path across the membrane. To obtain structural information of LmrA a cysteine scanning accessibility approach was used. Cysteines were introduced in the cysteine-less wild-type LmrA in each hydrophilic loop and in TMS 6, and each membrane-embedded aromatic residue was mutated to cysteine. Of the 41 constructed single cysteine mutants, only one mutant, L301C, was not expressed. Most single-cysteine mutants were capable of drug transport and only three mutants, F37C, M299C, and N300C, were inactive, indicating that none of the aromatic residues in the transmembrane regions of LmrA are crucial for substrate binding or transport. Modification of the active mutants with N-ethylmaleimide blocked the transport activity in five mutants (S132C, L174C, S206C, S234C, and L292C). All cysteine residues in external and internal loops were accessible to fluorescein maleimide. The labeling experiments also showed that this thiol reagent cannot cross the membrane under the conditions used and confirmed the presence of six TMSs in each monomeric half of the transporter. Surprisingly, several single cysteines in the predicted TMSs could also be labeled by the bulky fluorescein maleimide molecule, suggesting unrestricted accessibility via an aqueous pathway. The periodicity of fluorescein maleimide accessibility of residues 291 to 308 in TMS 6 showed that this membrane-spanning a-helix has one face of the helix exposed to an aqueous cavity along its full-length. This finding, together with the solvent accessibility of 11 of 15 membrane-embedded aromatic residues, indicates that the transmembrane domains of the LmrA transporter form, under nonenergized conditions, an aqueous chamber within the membrane, which is open to the intracellular milieu.