Fructose-1,6-bisphosphate acts both as an inducer and as a structural cofactor of the central glycolytic genes repressor (CggR).
2007
Zorrilla, Silvia | Chaix, Denis | Ortega, Alvaro | Alfonso, Carlos | Doan, Thierry | Margeat, Emmanuel | Rivas, Germán | Aymerich, Stephan | Declerck, Nathalie | Royer, Catherine A
CggR is the transcriptional repressor of the gapA operon encoding central glycolytic enzymes in Bacillus subtilis. Recently, a detailed mechanistic characterization of gapA induction revealed that the binding of fructose-1,6-bisphosphate (FBP) to a low affinity site on CggR (Kd > 100 microM) is responsible for repressor release from the DNA. In addition, this prior work demonstrated that FBP binds to a second high affinity site on the repressor, causing a conformational change in the CggR/DNA complexes, but with no consequence on CggR affinity for its operator DNA. In the present study we have thoroughly analyzed the structural and thermodynamic consequences of FBP binding to CggR. Results of fluorescence anisotropy titrations, calorimetry and limited proteolysis confirm the existence in CggR of a high affinity site for FBP, with a Kd of around 6 microM. Using analytical size-exclusion chromatography, ultracentrifugation as well as fluorescence correlation spectroscopy (FCS) and pressure perturbation, we show that FBP binding at this site reduces the size of the CggR oligomers and induces conformational changes that stabilize the dimer against denaturation. Hence, FBP has a dual role on CggR structure and regulatory function. In addition to acting as an inducer of transcription at the low affinity site, FBP bound to the high affinity site acts as a structural cofactor for the repressor, with profound effects on its quaternary structure as well as on its conformational dynamics and stability. This high affinity FBP site apparently evolved from the sugar substrate binding site of homologous enzymes.
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