Protein-protein interactions with fructose-1-kinase alter function of the centralEscherichia colitranscription regulator, Cra

Abstract

SummaryIn E. coli, the master transcription regulator Cra regulates >100 genes in central metabolism by binding upstream DNA operator sequences. Genes encoding glycolytic enzymes are repressed, whereas those for gluconeogenesis and the citric acid cycle are activated. Cra-DNA binding is allosterically diminished by binding to either fructose-1-phosphate (F-1-P, generated upon fructose import) or fructose-1,6-bisphosphate (F-1,6-BP). F-1,6-BP is generated from F-1-P by the enzyme fructose-1-kinase (FruK) or from other sugars and is a key intermediate in glycolysis. Here, we report that Cra directly interacts with FruK to form a tight protein-protein complex. Further, growth assays with a fruK knockout strain show that FruK has a broader role in metabolism than its known role in fructose catabolism. Biochemical experiments show that F-1,6-BP binding enhances either the Cra/FruK interaction and/or CRA binding to DNA and that FruK can catalyze the reverse reaction of F-1,6-BP to F-1-P. Results were used to propose a model in which the Cra-FruK complex enhances activation of gluconeogenic genes. Finally, since FruK itself is repressed by Cra, these newly-reported events add layers to the dynamic regulation of E. coli central metabolism that occur in response to changing nutrients.

Assessment of the interaction between the flux‐signaling metabolite fructose‐1,6‐bisphosphate and the bacterial transcription factors CggR and Cra

“…Strikingly, among 47 central metabolites F1P displayed the highest intracellular concentration variance across 23 different growth conditions and a highly significant direct correlation with FBP (Kochanowski et al, ), indicating that F1P is produced in E. coli also in conditions other than growth in fructose as the only carbon source. In this regard, a recent work described that the enzyme FruK can work reversibly and convert FBP into F1P (Singh et al, ). Interestingly enough, the same work reports that FruK interacts with Cra in vivo , and that conversion FBP into F1P regulates Cra/FruK affinity for Cra’s DNA operators in vitro (Singh et al, ).…”

“…In this regard, a recent work described that the enzyme FruK can work reversibly and convert FBP into F1P (Singh et al, ). Interestingly enough, the same work reports that FruK interacts with Cra in vivo , and that conversion FBP into F1P regulates Cra/FruK affinity for Cra’s DNA operators in vitro (Singh et al, ). Thus, these results raise an interesting scenario where, similarly to cAMP or fructose‐2,6‐bisphosphate (in mammals), F1P would act as a second messenger (that is, not involved directly in central metabolism) to relay metabolic fluxes into a physiological response, via regulation of Cra.…”

The strengths and limitations of using biolayer interferometry to monitor equilibrium titrations of biomolecules

“…Since the homodimer is the minimal DNA binding unit for all characterized LacI/GalR proteins (reviewed in Reference ), we presumed that LLhF behaved accordingly. LLhF also binds the hetero‐protein FruK (Supplementary Methods, Table SI ), and this interaction was used as a model system for quantifying the interactions of a heteroprotein binding to a protein‐DNA complex with BLI.…”

“…Note that, as with the operator release experiments, the FruK binding assays did not discriminate whether the midpoint (a) corresponded to K d for FruK binding to the LLhF‐DNA complex or (b) was a composite of FruK binding and altered LLhF‐DNA binding in the presence of this heteroprotein. Interestingly, since FruK also binds to wild‐type FruR (the LLhF parent protein also known as “Cra”), this reasonably strong affinity indicates that FruK might play a physiological role in regulating central E. coli metabolism.…”

The imbroglio of the physiological Cra effector clarified at last

“…The scenario could be indeed the case in view of the recent work of Kochanowski et al () indicating that that F1P can be produced in the absence of fructose as carbon source and that one can establish a correlation between F1P and FBP levels across different conditions. Along the line, it has been recently shown that F1P can be generated from FBP by FruK and that this enzyme interacts with Cra in vivo (Singh et al, ), an issue that deserves further research. It is clear that more studies are still necessary to interconnect all the possible actors and to establish under what conditions and in what way the Cra/F1P system manages to regulate the central metabolism.…”