The ABC transporter BmrA from Bacillus subtilis is a functional dimer when in a detergent-solubilized state.
BmrA from Bacillus subtilis is a half-size ABC (ATP-binding cassette) transporter involved in multidrug resistance. Although its supramolecular organization has been investigated after reconstitution in a lipid bilayer environment, and shows a dimeric and possibly a tetrameric form, the precise quaternary structure in a detergent-solubilized state has never been addressed. In the present study, BmrA was purified from Escherichia coli membranes using an optimized purification protocol and different detergents. Furthermore, the ATPase activity of BmrA and the quantity of bound lipids and detergent were determined, and the oligomeric state was analysed using SEC (size-exclusion chromatography) and analytical ultracentrifugation. The activity and the quaternary structure of BmrA appeared to be strongly influenced by the type and concentration of the detergent used. SEC data showed that BmrA could be purified in a functional form in 0.05 and 0.01% DDM (n-dodecyl-beta-D-maltoside) and was homogeneous and monodisperse with an R(s) (Stokes radius) of 5.6 nm that is compatible with a dimer structure. Sedimentation-velocity and equilibrium experiments unequivocally supported that BmrA purified in DDM is a dimer and excluded the presence of other oligomeric states. These observations, which are discussed in relation to results obtained in proteoliposomes, also constitute an important first step towards crystallographic studies of BmrA structure.
Ravaud
Stéphanie
S
Laboratoire de BioCristallographie, Institut de Biologie et Chimie des Protéines, UMR 5086 CNRS/UCBL, IFR 128 BioSciences Lyon-Gerland, 7 Passage du Vercors, F-69367 Lyon Cedex 07, France.
Do Cao
Marie-Ange
MA
Jidenko
Marie
M
Ebel
Christine
C
Le Maire
Marc
M
Jault
Jean-Michel
JM
Di Pietro
Attilio
A
Haser
Richard
R
Aghajari
Nushin
N
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000378:metabolism; D001412:Bacillus subtilis / Q000737:chemistry*; D002850:Chromatography, Gel; D003902:Detergents / Q000494:pharmacology*; D019281:Dimerization; D005960:Glucosides / Q000494:pharmacology; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D020836:Protein Structure, Quaternary / Q000187:drug effects; D012995:Solubility / Q000187:drug effects; D014461:Ultracentrifugation
D018528:ATP-Binding Cassette Transporters; D003902:Detergents; D005960:Glucosides; D010743:Phospholipids; C040358:dodecyl maltoside