title: 3D cryo-electron reconstruction of BmrA, a bacterial multidrug ABC transporter in an inward-facing conformation and in a lipidic environment.
abstract: ABC (ATP-binding cassette) membrane exporters are efflux transporters of a wide diversity of molecule across the membrane at the expense of ATP. A key issue regarding their catalytic cycle is whether or not their nucleotide-binding domains (NBDs) are physically disengaged in the resting state. To settle this controversy, we obtained structural data on BmrA, a bacterial multidrug homodimeric ABC transporter, in a membrane-embedded state. BmrA in the apostate was reconstituted in lipid bilayers forming a mixture of ring-shaped structures of 24 or 39 homodimers. Three-dimensional models of the ring-shaped structures of 24 or 39 homodimers were calculated at 2.3 nm and 2.5 nm resolution from cryo-electron microscopy, respectively. In these structures, BmrA adopts an inward-facing open conformation similar to that found in mouse P-glycoprotein structure with the NBDs separated by 3 nm. Both lipidic leaflets delimiting the transmembrane domains of BmrA were clearly resolved. In planar membrane sheets, the NBDs were even more separated. BmrA in an ATP-bound conformation was determined from two-dimensional crystals grown in the presence of ATP and vanadate. A projection map calculated at 1.6 nm resolution shows an open outward-facing conformation. Overall, the data are consistent with a mechanism of drug transport involving large conformational changes of BmrA and show that a bacterial ABC exporter can adopt at least two open inward conformations in lipid membrane.
authors.affiliation: Institut Curie, Centre de Recherche, 26 rue d'Ulm, 75231 Paris, France; CNRS, UMR 168, 11 rue Pierre et Marie Curie, 75231 Paris, France. Electronic address: canapif@gmail.com.
authors.forename: Pierre Frederic
authors.initials: PF
authors.lastname: Fribourg
authors.affiliation: Center for Cellular Imaging and NanoAnalytics, Biozentrum, University Basel, Mattenstrasse 26, CH-4058 Basel, Switzerland. Electronic address: mohamed.chami@unibas.ch.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Chami
authors.affiliation: Centro Nacional de Biotecnología, Unidad de Bioinformática, Campus Universidad Autónoma (Cantoblanco), 28049 Madrid, Spain. Electronic address: coss@cnb.csic.es.
authors.forename: Carlos Oscar S
authors.initials: CO
authors.lastname: Sorzano
authors.affiliation: Institut Curie, Centre de Recherche, 26 rue d'Ulm, 75231 Paris, France; CNRS, UMR 168, 11 rue Pierre et Marie Curie, 75231 Paris, France. Electronic address: francesca.gubellini@pasteur.fr.
authors.forename: Francesca
authors.initials: F
authors.lastname: Gubellini
authors.affiliation: Centro Nacional de Biotecnología, Unidad de Bioinformática, Campus Universidad Autónoma (Cantoblanco), 28049 Madrid, Spain. Electronic address: roberto.marabini@uam.es.
authors.forename: Roberto
authors.initials: R
authors.lastname: Marabini
authors.affiliation: Institut Curie, Centre de Recherche, 26 rue d'Ulm, 75231 Paris, France; Institut Curie, Centre de Recherche, Inserm, U759, Centre Universitaire d'Orsay, Bâtiment 112, Orsay 91405, France. Electronic address: Sergio.marco@curie.fr.
authors.forename: Sergio
authors.initials: S
authors.lastname: Marco
authors.affiliation: Institut de Biologie Structurale, UMR 5075, Université Joseph Fourier/CEA/CNRS, 41 rue Jules Horowitz, 38027 Grenoble Cedex 1, France. Electronic address: jean-michel.jault@ibcp.fr.
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Jault
authors.affiliation: Institut Curie, Centre de Recherche, 26 rue d'Ulm, 75231 Paris, France; CNRS, UMR 168, 11 rue Pierre et Marie Curie, 75231 Paris, France; The BioImaging Cell and Tissue Core Facility, Institut Curie, 75231 Paris, France. Electronic address: daniel.levy@curie.fr.
authors.forename: Daniel
authors.initials: D
authors.lastname: Lévy
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D016764:Cell Polarity / Q000502:physiology; D020285:Cryoelectron Microscopy / Q000379:methods*; D018360:Crystallography, X-Ray; D021621:Imaging, Three-Dimensional; D008051:Lipid Bilayers / Q000737:chemistry*; D026901:Membrane Transport Proteins / Q000737:chemistry*; D008958:Models, Molecular; D011487:Protein Conformation; D013237:Stereoisomerism
chemical_list: D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D008051:Lipid Bilayers; D026901:Membrane Transport Proteins; C075041:Bmr protein, Bacillus subtilis
keywords: ABC transporter; BmrA; conformation in lipid bilayer; electron microscopy; inward-facing conformation