title: ATP modulates subunit-subunit interactions in an ATP-binding cassette transporter (MalFGK2) determined by site-directed chemical cross-linking.
abstract: The binding protein-dependent maltose transport system of enterobacteria (MalFGK(2)), a member of the ATP-binding cassette (ABC) transporter superfamily, is composed of two integral membrane proteins, MalF and MalG, and of two copies of an ATPase subunit, MalK, which hydrolyze ATP, thus energizing the translocation process. In addition, an extracellular (periplasmic) substrate-binding protein (MalE) is required for activity. Ligand translocation and ATP hydrolysis are dependent on a signaling mechanism originating from the binding protein and traveling through MalF/MalG. Thus, subunit-subunit interactions in the complex are crucial to the transport process but the chemical nature of residues involved is poorly understood. We have investigated the proximity of residues in a conserved sequence ("EAA" loop) of MalF and MalG to residues in a helical segment of the MalK subunits by means of site-directed chemical cross-linking. To this end, single cysteine residues were introduced into each subunit at several positions and the respective malF and malG alleles were individually co-expressed with each of the malK alleles. Membrane vesicles were prepared from those double mutants that contained a functional transporter in vivo and treated with Cu(1,10-phenanthroline)(2)SO(4) or bifunctional cross-linkers. The results suggest that residues Ala-85, Lys-106, Val-114, and Val-117 in the helical segment of MalK, to different extents, participate in constitution of asymmetric interaction sites with the EAA loops of MalF and MalG. Furthermore, both MalK monomers in the complex are in close contact to each other through Ala-85 and Lys-106. These interactions are strongly modulated by MgATP, indicating a structural rearrangement of the subunits during the transport cycle. These data are discussed with respect to current transport models.
authors.affiliation: Humboldt-Universität zu Berlin, Institut für Biologie/Bakterienphysiologie, Chausseestrasse 117, D-10115 Berlin, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Hunke
authors.forename: M
authors.initials: M
authors.lastname: Mourez
authors.forename: M
authors.initials: M
authors.lastname: Jehanno
authors.forename: E
authors.initials: E
authors.lastname: Dassa
authors.forename: E
authors.initials: E
authors.lastname: Schneider
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D016384:Consensus Sequence; D003432:Cross-Linking Reagents; D003545:Cysteine; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D058619:Maltose-Binding Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D033902:Periplasmic Binding Proteins; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012486:Salmonella typhimurium / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
chemical_list: D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; C461619:MalE protein, E coli; C079123:MalG protein, E coli; C068021:MalK protein, Bacteria; C489398:MalK protein, E coli; C489399:MalK protein, Salmonella typhimurium; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D011994:Recombinant Proteins; C461599:maltose transport system, E coli; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D003545:Cysteine