title: In vitro reassembly of the ribose ATP-binding cassette transporter reveals a distinct set of transport complexes.
abstract: Bacterial ATP-binding cassette (ABC) importers are primary active transporters that are critical for nutrient uptake. Based on structural and functional studies, ABC importers can be divided into two distinct classes, type I and type II. Type I importers follow a strict alternating access mechanism that is driven by the presence of the substrate. Type II importers accept substrates in a nucleotide-free state, with hydrolysis driving an inward facing conformation. The ribose transporter in Escherichia coli is a tripartite complex consisting of a cytoplasmic ATP-binding cassette protein, RbsA, with fused nucleotide binding domains; a transmembrane domain homodimer, RbsC2; and a periplasmic substrate binding protein, RbsB. To investigate the transport mechanism of the complex RbsABC2, we probed intersubunit interactions by varying the presence of the substrate ribose and the hydrolysis cofactors, ATP/ADP and Mg(2+). We were able to purify a full complex, RbsABC2, in the presence of stable, transition state mimics (ATP, Mg(2+), and VO4); a RbsAC complex in the presence of ADP and Mg(2+); and a heretofore unobserved RbsBC complex in the absence of cofactors. The presence of excess ribose also destabilized complex formation between RbsB and RbsC. These observations suggest that RbsABC2 shares functional traits with both type I and type II importers, as well as possessing unique features, and employs a distinct mechanism relative to other ABC transporters.
authors.affiliation: From the Department of Biological Sciences and the Purdue Center for Cancer Research and.
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Clifton
authors.affiliation: From the Department of Biological Sciences and the Purdue Center for Cancer Research and.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Simon
authors.affiliation: From the Department of Biological Sciences and the Purdue Center for Cancer Research and.
authors.forename: Satchal K
authors.initials: SK
authors.lastname: Erramilli
authors.affiliation: the Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
authors.forename: Huide
authors.initials: H
authors.lastname: Zhang
authors.affiliation: the Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
authors.forename: Jelena
authors.initials: J
authors.lastname: Zaitseva
authors.affiliation: the Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Hermodson
authors.affiliation: From the Department of Biological Sciences and the Purdue Center for Cancer Research and cstauffa@purdue.edu.
authors.forename: Cynthia V
authors.initials: CV
authors.lastname: Stauffacher
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001692:Biological Transport / Q000235:genetics; D015153:Blotting, Western; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D033902:Periplasmic Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D012266:Ribose / Q000378:metabolism*
chemical_list: D018528:ATP-Binding Cassette Transporters; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D046912:Multiprotein Complexes; D033902:Periplasmic Binding Proteins; D021122:Protein Subunits; C102053:RbsA protein, E coli; C028003:RbsB protein, E coli; C102054:RbsC protein, E coli; D000244:Adenosine Diphosphate; D012266:Ribose; D000255:Adenosine Triphosphate; D008274:Magnesium
keywords: ABC Transporter; ATPase; Bacterial Metabolism; Electron Paramagnetic Resonance (EPR); Membrane Transport; Membrane Transporter Reconstitution; Ribose Transport