ABSTRACT
The BglG protein positively regulates expression of the bgl operon in Escherichia coli by binding as a dimer to the bgl transcript and preventing premature termination of transcription in the presence of β-glucosides. BglG activity is negatively controlled by BglF, the β-glucoside phosphotransferase, which reversibly phosphorylates BglG according to β-glucoside availability, thus modulating its dimeric state. BglG consists of an RNA-binding domain and two homologous domains, PRD1 and PRD2. Based on structural studies of a BglG homologue, the two PRDs fold similarly, and the interactions within the dimer are PRD1-PRD1 and PRD2-PRD2. We have recently shown that the affinity between PRD1 and PRD2 of BglG is high, and a fraction of the BglG monomers folds in the cell into a compact conformation, in which PRD1 and PRD2 are in close proximity. We show here that both BglG forms, the compact and noncompact, bind to the active site-containing domain of BglF, IIBbgl, in vitro. The interaction of BglG with IIBbgl or BglF is mediated by PRD2. Both BglG forms are detected as phosphorylated proteins after in vitro phosphorylation with IIBbgl and are dephosphorylated by BglF in vitro in the presence of β-glucosides. Nevertheless, genetic evidence indicates that the interaction of IIBbgl and BglF with the compact form is seemingly less favorable. Using in vivo cross-linking, we show that BglF enhances folding of BglG into a compact conformation, whereas the addition of β-glucosides reduces the amount of this form. Based on these results we suggest a model for the modulation of BglG conformation and activity by BglF.
Hydroxo-Bridged Active Site of Flavodiiron NO Reductase Revealed by Spectroscopy and Computations
