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.
Dalmas
Olivier
O
Institut de Biologie et Chimie des Protéines, UMR 5086 CNRS-UCBL1 and IFR 128, 7 passage du Vercors, 69367 Lyon Cedex 07, France.
Do Cao
Marie-Ange
MA
Lugo
Miguel R
MR
Sharom
Frances J
FJ
Di Pietro
Attilio
A
Jault
Jean-Michel
JM
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D024881:Drug Resistance, Bacterial; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D011510:Proteolipids / Q000737:chemistry / Q000378:metabolism; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives* / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D011510:Proteolipids; C020485:proteoliposomes; C075041:Bmr protein, Bacillus subtilis; C049748:4-dimethylaminophenylazophenyl-4'-maleimide; D004124:p-Dimethylaminoazobenzene