Maltose-binding protein containing an interdomain disulfide bridge confers a dominant-negative phenotype for transport and chemotaxis.
Bacterial substrate-binding proteins exist in an equilibrium among four forms: open/substrate-free, open/substrate-bound, closed/substrate-free, and closed/substrate-bound. Ligands stabilize the closed conformation, whereas the open conformation predominates in the substrate-free species. In its closed form, the NH2-terminal and COOH-terminal domains of maltose-binding protein (MBP) are proposed to be aligned to allow residues in both domains to interact simultaneously with complementary sites on the MalF and MalG proteins of the maltodextrin uptake system or with the Tar chemotactic signal transducer. However, the initial interaction might occur with an open/substrate-bound form of the binding protein, which would then close in contact with MalFG or Tar. Ligand would help stabilize this complex. We introduced cysteines (G69C and S337C) by site-directed mutagenesis into each domain of MBP and found that they formed an interdomain disulfide cross-link that should hold the protein in a closed conformation. This mutant MBP confers a dominant-negative phenotype for growth on maltose, for maltose transport, and for maltose chemotaxis. The growth and transport defects are partially reversed when the cells are exposed to the reducing agent dithiothreitol. We conclude that the cross-linked form of MBP competes with wild-type MBP in vivo for interaction with MalFG and Tar.
Zhang
Y
Y
Department of Biology, Texas A&M University, College Station, Texas 77843-3258, USA.
Mannering
D E
DE
Davidson
A L
AL
Yao
N
N
Manson
M D
MD
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport / Q000502:physiology; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002633:Chemotaxis / Q000502:physiology*; D003545:Cysteine / Q000235:genetics; D004220:Disulfides; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins; D008320:Maltose / Q000378:metabolism*; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D010641:Phenotype; D011487:Protein Conformation
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D004220:Disulfides; D029968:Escherichia coli Proteins; C079123:MalG protein, E coli; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; C461599:maltose transport system, E coli; D008320:Maltose; D003545:Cysteine