title: A putative helical domain in the MalK subunit of the ATP-binding-cassette transport system for maltose of Salmonella typhimurium (MalFGK2) is crucial for interaction with MalF and MalG. A study using the LacK protein of Agrobacterium radiobacter as a tool.
abstract: The ATP-binding-cassette (ABC) protein LacK of Agrobacterium radiobacter displays high sequence similarity to the MalK subunit of the Salmonella typhimurium maltose-transport system (MalFGK2). We have used LacK as a tool to identify sites of interaction of MalK with the membrane-integral components MalF and MalG. Small amounts of LacK, resulting from the expression of the plasmid-borne lacK gene, proved to be sufficient for partial restoration of growth of a malK strain of S. typhimurium on maltose. LacK failed to substitute for MalK in regulating the expression of maltose-inducible genes but the hybrid complex MalFGLacK2 was sensitive to inducer exclusion. The lacK gene also complemented a ugpC mutant of Escherichia coli to growth on sn-glycerol-3-phosphate as the phosphate source. Partially purified LacK exhibited a spontaneous ATPase activity comparable to that of MalK. A MalK"-'LacK chimeric protein was isolated (by in vivo recombination) in which the N-terminal 140 amino acids of MalK are fused to residues 141-363 of LacK. The protein substituted for MalK in maltose transport considerably better than LacK. Furthermore, random mutagenesis of the plasmid-borne lacK gene yielded three clones that were superior to wild-type lacK in complementing a malK mutation. Single mutations (V114M or L123F) substantially improved the growth of a malK strain on maltose, whereas a double mutation (L123F, S295N) resulted in growth and transport rates that were indistinguishable from those obtained with MalK. In contrast, the introduction of the single change S295N into LacK had no effect but combination with the V114M mutation led to a further twofold increase in transport activity. These results indicate that a putative helical domain in MalK, encompassing residues 89-140, is crucial for a functional, high-affinity interaction with MalF and MalG.
authors.affiliation: Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Wilken
authors.forename: G
authors.initials: G
authors.lastname: Schmees
authors.forename: E
authors.initials: E
authors.lastname: Schneider
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005994:Glycerophosphates / Q000378:metabolism; D058619:Maltose-Binding Proteins; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012231:Rhizobium / Q000378:metabolism*; D012486:Salmonella typhimurium / Q000235:genetics / Q000378:metabolism*
chemical_list: D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D005994:Glycerophosphates; C079123:MalG protein, E coli; C068021:MalK protein, Bacteria; C489398:MalK protein, E coli; C489399:MalK protein, Salmonella typhimurium; D058619:Maltose-Binding Proteins; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; C083717:UgpC protein, E coli; C461599:maltose transport system, E coli; C029620:alpha-glycerophosphoric acid; D000251:Adenosine Triphosphatases