Probing the conformation of the resting state of a bacterial multidrug ABC transporter, BmrA, by a site-directed spin labeling approach.

Previously published 3-D structures of a prototypic ATP-binding cassette (ABC) transporter, MsbA, have been recently corrected revealing large rigid-body motions possibly linked to its catalytic cycle. Here, a closely related multidrug bacterial ABC transporter, BmrA, was studied using site-directed spin labeling by focusing on a region connecting the transmembrane domain and the nucleotide-binding domain (NBD). Electron paramagnetic resonance (EPR) spectra of single spin-labeled cysteine mutants suggests that, in the resting state, this sub-domain essentially adopts a partially extended conformation, which is consistent with the crystal structures of MsbA and Sav1866. Interestingly, one of the single point mutants (Q333C) yielded an immobilized EPR spectrum that could arise from a direct interaction with a vicinal tyrosine residue. Inspection of different BmrA models pointed to Y408, within the NBD, as the putative interacting partner, and its mutation to a Phe residue indeed dramatically modified the EPR spectra of the spin labeled Q333C. Moreover, unlike the Y408F mutation, the Y408A mutation abolished both ATPase activity and drug transport of BmrA, suggesting that a nonpolar bulky residue is required at this position. The spatial proximity of Q333 and Y408 was also confirmed by formation of a disulfide bond when both Q333 and T407 (or S409) were replaced jointly by a cysteine residue. Overall, these results indicate that the two regions surrounding Q333 and Y408 are close together in the 3-D structure of BmrA and that residues within these two sub-domains are essential for proper functioning of this transporter.

Time-resolved fluorescence resonance energy transfer shows that the bacterial multidrug ABC half-transporter BmrA functions as a homodimer.

Members of the ATP-binding cassette (ABC) transporters share the same basic architecture, with a four-core domain made of two transmembrane plus two nucleotide-binding domains. However, a supramolecular organization has been detected in some ABC transporters, which might be relevant to physiological regulation of substrate transport. Here, the oligomerization status of a bacterial half-ABC multidrug transporter, BmrA, was investigated. Each BmrA monomer containing a single cysteine residue introduced close to either the Walker A or the ABC signature motifs was labeled using two probes, 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid (fluorescence donor) or 4-dimethylaminophenylazophenyl-4'-maleimide (fluorescence acceptor). Reconstitution into proteoliposomes of BmrA monomers labeled separately with either the fluorescence donor or the fluorescence acceptor allowed measurement of time-resolved fluorescence resonance energy transfer between the two probes, showing that efficient reassociation of the singly labeled BmrA monomers occurred upon reconstitution. The efficiency of energy transfer studied as a function of increasing concentration of BmrA-labeled with the fluorescence acceptor argues for a dimeric association of BmrA instead of a tetrameric one. Furthermore, the efficiency of energy transfer allowed estimation of the distances between the two bound probes. Results suggest that, in the resting state, BmrA in a lipid bilayer environment preferentially adopts a closed conformation similar to that found in the BtuCD crystal structure and that the presence of different effectors does not substantially modify its global conformation.