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 fou r forms: open/substrate-free, open/substrate-bound, closed/substrate-f ree, and closed/substrate-bound. Ligands stabilize the closed conforma tion, whereas the open conformation predominates in the substrate-free species, In its closed form, the NH2-terminal and COOH-terminal domai ns of maltose-binding protein (MBP) are proposed to be aligned to allo w residues in both domains to interact simultaneously with complementa ry sites on the MalF and MalG proteins of the maltodextrin uptake syst em or with the Tar chemotactic signal transducer. However, the initial interaction might occur with an open/substrate-bound form of the bind ing protein, which would then close in contact with MalFG or Tar, Liga nd would help stabilize this complex, We introduced cysteines (G69C an d S337C) by site-directed mutagenesis into each domain of MBP and foun d that they formed an interdomain disulfide cross-link that should hol d the protein in a closed conformation, This mutant MEP confers a domi nant-negative phenotype for growth on maltose, for maltose transport, and for maltose chemotaxis. The growth and transport defects are parti ally reversed when the cells are exposed to the reducing agent dithiot hreitol. We conclude that the cross-linked form of MBP competes with w ild-type MBP in vivo for interaction with MalFG and Tar.