title: Negative Stain Single-particle EM of the Maltose Transporter in Nanodiscs Reveals Asymmetric Closure of MalK2and Catalytic Roles of ATP, MalE, and Maltose.
abstract: TheEscherichia coliMalE-MalFGK2complex is one of the best characterized members of the large and ubiquitous family of ATP-binding cassette (ABC) transporters. It is composed of a membrane-spanning heterodimer, MalF-MalG; a homodimeric ATPase, MalK2; and a periplasmic maltose receptor, MalE. Opening and closure of MalK2is coupled to conformational changes in MalF-MalG and the alternate exposition of the substrate-binding site to either side of the membrane. To further define this alternate access mechanism and the impact of ATP, MalE, and maltose on the conformation of the transporter during the transport cycle, we have reconstituted MalFGK2in nanodiscs and analyzed its conformations under 10 different biochemical conditions using negative stain single-particle EM. EM map results (at 15-25 Å resolution) indicate that binding of ATP to MalK2promotes an asymmetric, semi-closed conformation in accordance with the low ATPase activity of MalFGK2In the presence of MalE, the MalK dimer becomes fully closed, gaining the ability to hydrolyze ATP. In the presence of ADP or maltose, MalE·MalFGK2remains essentially in a semi-closed symmetric conformation, indicating that release of these ligands is required for the return to the initial state. Taken together, this structural information provides a rationale for the stimulation of MalK ATPase activity by MalE as well as by maltose.
authors.affiliation: From the Department of Anatomy and Cell Biology, Groupe de Recherche Axé sur la Structure des Protéines, Groupe d'Étude des Protéines Membranaires, McGill University, Montreal, Quebec H3A 2B2, Canada and.
authors.forename: Lucien
authors.initials: L
authors.lastname: Fabre
authors.affiliation: the Department of Biochemistry and Molecular Biology, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
authors.forename: Huan
authors.initials: H
authors.lastname: Bao
authors.affiliation: From the Department of Anatomy and Cell Biology, Groupe de Recherche Axé sur la Structure des Protéines, Groupe d'Étude des Protéines Membranaires, McGill University, Montreal, Quebec H3A 2B2, Canada and.
authors.forename: James
authors.initials: J
authors.lastname: Innes
authors.affiliation: the Department of Biochemistry and Molecular Biology, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada fduong@mail.ubc.ca.
authors.forename: Franck
authors.initials: F
authors.lastname: Duong
authors.affiliation: From the Department of Anatomy and Cell Biology, Groupe de Recherche Axé sur la Structure des Protéines, Groupe d'Étude des Protéines Membranaires, McGill University, Montreal, Quebec H3A 2B2, Canada and isabelle.rouiller@mcgill.ca.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Rouiller
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D008024:Ligands; D008854:Microscopy, Electron / Q000379:methods*; D011487:Protein Conformation
chemical_list: D018528:ATP-Binding Cassette Transporters; D029968:Escherichia coli Proteins; D008024:Ligands; C461599:maltose transport system, E coli; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases
keywords: ABC transporter; electron microscopy (EM); membrane protein; membrane transporter reconstitution; single-particle analysis; translocation; transport