title: Diverse effects of mutation on the activity of the Escherichia coli export chaperone SecB.
abstract: The Escherichia coli SecB protein binds newly synthesized precursor maltose-binding protein (preMBP) and promotes its rapid export from the cytoplasm. Site-directed mutagenesis of two regions of SecB was carried out to better understand factors governing the SecB.preMBP interaction. 30 aminoacyl substitution mutants were analyzed, revealing two distinct classes of secB mutants. Substitutions at the alternating positions Phe-74, Cys-76, Val-78, or Gln-80 reduced the ability of SecB to form stable complexes with preMBP, but caused only mild defects in the rate of MBP export from living cells. The pattern revealed by this class of mutants suggests that a primary binding site for preMBP is hydrophobic and contains beta-sheet secondary structure. In contrast, substitutions at Asp-20, Glu-24, Leu-75, or Glu-77 caused a severe slowing in the rate of MBP export but did not disrupt SecB.preMBP complex formation. These largely acidic residues may function to regulate the opening of a preprotein binding site, allowing both high affinity preprotein binding and rapid dissociation of SecB.preprotein complexes at the membrane translocation site.
authors.affiliation: Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
authors.forename: H H
authors.initials: HH
authors.lastname: Kimsey
authors.forename: M D
authors.initials: MD
authors.lastname: Dagarag
authors.forename: C A
authors.initials: CA
authors.lastname: Kumamoto
mesh_terms: D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D007700:Kinetics; D008320:Maltose / Q000378:metabolism; D058619:Maltose-Binding Proteins; D018832:Molecular Chaperones / Q000378:metabolism*; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D017354:Point Mutation; D011498:Protein Precursors / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism
chemical_list: D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D018832:Molecular Chaperones; D009004:Monosaccharide Transport Proteins; D009838:Oligodeoxyribonucleotides; D011498:Protein Precursors; D011994:Recombinant Proteins; C057842:SecB protein, Bacteria; C461599:maltose transport system, E coli; D008320:Maltose