title: Structure of the multidrug resistance P-glycoprotein to 2.5 nm resolution determined by electron microscopy and image analysis.
abstract: P-glycoprotein (P-gp) is a member of the ATP binding cassette superfamily of active transporters and can confer multidrug resistance on cells and tumors by pumping chemotherapeutic drugs from the cytoplasm. P-gp was purified from CHrB30 cells and retained the ability to bind substrates and hydrolyze ATP. Labeling of P-gp with lectin-gold particles suggested it is monomeric. An initial structure of purified P-gp was determined to 2.5 nm resolution by electron microscopy and single particle image analysis of both detergent-solubilized and lipid-reconstituted protein. The structure was further refined by three dimensional reconstructions from single particle images and by Fourier projection maps of small two-dimensional crystalline arrays (unit cell parameters: a, 14.2 nm; b, 18.5 nm; and gamma, 91.6 degrees ). When viewed from above the membrane plane the protein is toroidal, with 6-fold symmetry and a diameter of about 10 nm. There is a large central pore of about 5 nm in diameter, which is closed at the inner (cytoplasmic) face of the membrane, forming an aqueous chamber within the membrane. An opening from this chamber to the lipid phase is present. The projection of the protein perpendicular to the membrane is roughly rectangular with a maximum depth of 8 nm and two 3-nm lobes exposed at the cytoplasmic face of the membrane, likely to correspond to the nucleotide binding domains. This study provides the first experimental insight into the three-dimensional architecture of any ATP binding cassette transporter.
authors.affiliation: Department of Biochemistry and Applied Molecular Biology, University of Manchester Institute of Science and Technology, P. O. Box 88, Manchester M60 1QD, United Kingdom.
authors.forename: M F
authors.initials: MF
authors.lastname: Rosenberg
authors.forename: R
authors.initials: R
authors.lastname: Callaghan
authors.forename: R C
authors.initials: RC
authors.lastname: Ford
authors.forename: C F
authors.initials: CF
authors.lastname: Higgins
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D003196:Computer Graphics; D006224:Cricetinae; D018360:Crystallography, X-Ray; D005583:Fourier Analysis; D008854:Microscopy, Electron; D008961:Models, Structural; D008970:Molecular Weight; D011485:Protein Binding; D011487:Protein Conformation; D012984:Software; D014747:Vinblastine / Q000378:metabolism
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D014747:Vinblastine; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases