title: Substrate binding stabilizes a pre-translocation intermediate in the ATP-binding cassette transport protein MsbA.
abstract: ATP-binding cassette (ABC) transporters belong to one of the largest protein superfamilies that expands from prokaryotes to man. Recent x-ray crystal structures of bacterial and mammalian ABC exporters suggest a common alternating access mechanism of substrate transport, which has also been biochemically substantiated. However, the current model does not yet explain the coupling between substrate binding and ATP hydrolysis that underlies ATP-dependent substrate transport. In our studies on the homodimeric multidrug/lipid A ABC exporter MsbA from Escherichia coli, we performed cysteine cross-linking, fluorescence energy transfer, and cysteine accessibility studies on two reporter positions, near the nucleotide-binding domains and in the membrane domains, for transporter embedded in a biological membrane. Our results suggest for the first time that substrate binding by MsbA stimulates the maximum rate of ATP hydrolysis by facilitating the dimerization of nucleotide-binding domains in a state, which is markedly distinct from the previously described nucleotide-free, inward-facing and nucleotide-bound, outward-facing conformations of ABC exporters and which binds ATP.
authors.affiliation: Department of Pharmacology, University of Cambridge, Cambridge CB2 1PD, United Kingdom.
authors.forename: Rupak
authors.initials: R
authors.lastname: Doshi
authors.forename: Hendrik W
authors.initials: HW
authors.lastname: van Veen
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001562:Benzimidazoles / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004996:Ethidium / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006868:Hydrolysis; D009154:Mutation; D011485:Protein Binding; D055503:Protein Multimerization; D013379:Substrate Specificity; D014700:Verapamil / Q000378:metabolism
chemical_list: D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D001562:Benzimidazoles; C079726:MsbA protein, Bacteria; D000255:Adenosine Triphosphate; D014700:Verapamil; D000251:Adenosine Triphosphatases; D004996:Ethidium; D003545:Cysteine; C017807:bisbenzimide ethoxide trihydrochloride
keywords: ABC Transporter; Lipid Transport; Membrane Transport; Multidrug Transporters; Protein Conformation; Substrate-stimulated ATPase