Three-dimensional structure by cryo-electron microscopy of YvcC, an homodimeric ATP-binding cassette transporter from Bacillus subtilis.
YvcC, a multidrug transporter from Bacillus subtilis, is a member of the ATP-binding cassette superfamily, highly homologous to each half of human multidrug-resistance P-glycoprotein and to several other bacterial half-ABC transporters. Here, the purified recombinant histidine-tagged YvcC has been reconstituted into a lipid bilayer. Controlled and partial detergent removal from YvcC-lipid micelles allowed the production of particularly interesting lipid-detergent-YvcC ring-shaped particles, about 40 nm in diameter, well suited for single particle analysis by cryo-electron microscopy. Furthermore, binding of these histidine-tagged ring-shaped particles to lipid layers functionalized with a Ni(2+)-chelating head group generated a preferential perpendicular orientation, eliminating the missing cone in the final three-dimensional reconstruction. From such analysis, a computed volume has been determined to 2.5 nm resolution giving a detailed insight into the structural organization of this half-ABC transporter within a membrane. The repetitive unit in the ring-shaped particles is consistent with a homodimeric organization of YvcC. Each subunit was composed of three domains: a 5 nm height transmembrane region, a stalk of about 4 nm in height and 2 nm in diameter, and a cytoplasmic lobe of about 5-6 nm in diameter. The latest domain, which fitted with the reported X-ray structure of HisP, was identified as the nucleotide-binding domain (NBD). The 3D reconstruction of the YvcC homodimer well compared with the very recent X-ray crystallographic data on the MsbA homodimer from Escherichia coli, supporting the existence of a central open chamber between the two subunits constituting the homodimer. In addition, the 3D reconstruction of YvcC embedded in a membrane revealed an asymmetric organization of the two NBDs sites within the homodimer, as well as a dimeric interaction between two homodimers.
Chami
Mohamed
M
Institut Curie Section Recherche, UMR-CNRS 168 et LRC-CEA 8, 11 Rue Pierre et Marie Curie, Paris Cedex 05, 75231, France.
Steinfels
Emmanuelle
E
Orelle
Cédric
C
Jault
Jean-Michel
JM
Di Pietro
Attilio
A
Rigaud
Jean-Louis
JL
Marco
Sergio
S
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure*; D026923:Amino Acid Transport Systems, Basic / Q000737:chemistry; D001412:Bacillus subtilis / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure*; D020285:Cryoelectron Microscopy; D018360:Crystallography, X-Ray; D003902:Detergents / Q000378:metabolism; D019281:Dimerization; D029968:Escherichia coli Proteins / Q000737:chemistry; D008051:Lipid Bilayers / Q000378:metabolism; D050356:Lipid Metabolism; D008565:Membrane Proteins / Q000737:chemistry; D008823:Micelles; D008958:Models, Molecular; D009532:Nickel / Q000378:metabolism; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure
D018528:ATP-Binding Cassette Transporters; D026923:Amino Acid Transport Systems, Basic; D001426:Bacterial Proteins; D003902:Detergents; D029968:Escherichia coli Proteins; D008051:Lipid Bilayers; D008565:Membrane Proteins; D008823:Micelles; C079726:MsbA protein, Bacteria; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; C043828:histidine permease, Bacteria; D009532:Nickel