Web Release Date: December 4,
Fructose-1,6-bisphosphate Acts Both as an Inducer and as a Structural Cofactor of
the Central Glycolytic Genes Repressor (CggR)
n Rivas, and
INSERM (U554), Montpellier, France, and Centre de Biochimie Structurale, CNRS (UMR5048), Université Montpellier 1, 29, rue de Navacelles, F-34090 Montpellier, France, Microbiologie et Génétique Moléculaire, INRA (UMR1238) and CNRS (UMR2585), Institut National Agronomique Paris-Grignon, F-78850 Thiverval-Grignon, France, and Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain
Received September 4, 2007
Revised Manuscript Received October 11, 2007
Abstract:
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 
M) 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 M. 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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